**Meet the editor**

Prof. Eduardo Jacob-Lopes is currently an associate professor at the Department of Food Technology and Science, Federal University of Santa Maria. He graduated with a master's degree in Food Engineering in Federal University of Rio Grande do Sul, doctorate degree in Chemical Engineering from the State University of Campinas, and postdoctoral at the State University of

Campinas. He has more than 15 years of teaching and research experience. He is a technical and scientific consultant of several companies, agencies, and scientific journals. He has 360 publications/communications which include 3 books, 17 book chapters, 60 original research papers, and 280 research communications in international and national conferences and has registered 7 patents. His research interest includes environmental biotechnology with emphasis on microalgal biotechnology.

Prof. Leila Queiroz Zepka is currently an associate professor at the Department of Food Technology and Science, Federal University of Santa Maria. She graduated with a master's degree in Food Engineering in Federal University of Rio Grande do Sul and a doctorate degree in Food Science from the State University of Campinas. She has more than 15 years of teaching and research ex-

perience. She is a technical and scientific consultant of several companies, agencies, and scientific journals. She has 360 publications/communications which include 3 books, 14 book chapters, 50 original research papers, and 200 research communications in international and national conferences, and has registered 4 patents. Her research interest includes microalgal biotechnology with emphasis on biomolecules.

## Contents

### **Preface XIII**



#### Contents **X**



## Preface

Human society will faceenormous problems in the near future in order to cover the increas‐ ing demandsof energy. The currentways these demandsare covered bysociety are not sus‐ tainable and result in unacceptable changes in our environment.

 To this end, this book aims to make a contribution to further exploring this area of bioener‐ gy and biofuel research and development in the form of a compilation of topics covering the characterization, production, and uses of bioenergy, biofuels, and coproducts, summarizing a range of useful products and technologies applied to energy production.

 We are convinced that this book will be an important resource for anyone who is interested in bioenergy and biofuels, and we express the hope that this book will stimulate and help re‐ searchers and industry professionals to move this field into new and improved applications.

> **Eduardo Jacob-Lopes**  Federal University of Santa Maria, Brasil

> **Leila Queiroz Zepka**  Federal University of Santa Maria, Brasil

## **Introductory Chapter: Life Cycle Assessment as a Fundamental Tool to Define the Biofuel Performance**

Mariany Costa Deprá, Leila Queiroz Zepka and Eduardo Jacob-Lopes

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64677

 Thewidespreadavailabilityofinexpensivepetroleumduringthetwentiethcentury,growing- concernsoffossilfueldepletion,aswellasstricteremissionregulationsandthesearchfor- alternativesourcesandeconomicallyviablesubstrateshasbeenthemainfocusofresearchers- seekingtoovercometheeconomicandenvironmentalbarrierstotherenewableenergysector.- Theidealsourceforproductionofbiofuelsmainlydependsonitsavailabilityandcost.-Thus,a- needarisestoaddressthecurrentenergyandenvironmentalissuestoproducebiofuels-[1,-2].-

 Biofuelshavebecomeanalternativesourceoverthetraditionalenergysources.-Therefore,the- progressofknowledgethroughtheestablishmentofmorerobustmethodsofanalysis,suchas- thelifecycleassessment-(LCA),highlightstheweaknessesofthesystems,pressingtheprocess- engineeringtodevelopsustainablesolutionsforapplicationinproductionchains-[3].-

The life cycle assessment is a methodology to quantify the input and output streams of- materials and energy throughout the production chain. Moreover, it is a useful tool to assess- resource use and environmental burdens related to systems. According to **Figure 1**, four stages- are used for conducting an LCA: (i) objective and scope definition;-(ii) inventory analysis (LCI);- (iii) impact assessment (LCIA); and (iv) interpretation [4].-

 Thegoalandscopedefinitionstageincludestheintendedapplication,thereasonstocarry- outthestudy,theintendedaudience,andtheuseoftheresults.-Inaddition,thesystem- boundaryandthefunctionalunitshouldalsobeclearlydefined.-Thisstageisincludedinall- thepapersanalyzed,althoughnotalwayswiththesamelevelofdetail.-Thesystemboundary- definestheprocessestobeincludedintheanalysis.-Thelifecycleinventory-(LCI)stage- involvesthecompilationandquantificationofinputsandoutputsforeachprocessincluded- withinthesystemboundary.-Theimpactassessmentcategoriesarechosentohaveanoverview- oftheinventorydata:energybalance,waterfootprint,globalwarmingpotential-(GWP),-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 potentialofacidification,andeutrophication.-Theinterpretationevaluatestheinventory- analysisresultsandimpactanalysistoselectthefavoriteproductorprocess,withaclear- understandingoftheuncertaintiesandassumptionsusedtogenerateresults.-

**Figure 1.** Stages for conducting an LCA.-

 Theenergyratio-(NER)isdefinedastheratiooftotalenergyproduced-(feedstockenergy- potential)overtheenergycontentofconstructionandmaterial,plusenergyrequiredforall- plantoperations.-Incaseoftheenergybalance,thestartingpointfortheeconomicandenvi‐ ronmental viability of processes is the consolidation of a favorable energy balance (NER > 1) [5].-

Moreover, water footprint (WF) of an enclosed area or process is determined by the sum of the- water footprints of all processes. The blue WF refers to the amount of water incorporated in- the product, which is determined by the evaporation rate plus incorporation and return flow.- The green WF refers to the volume of water consumed in a production process, plus the water- incorporated into the finish.-The sum of all processes is determined per a volume of water per- unit time [6].-

Across the globe, there are two main public policy objectives driving the development of- biofuel industries improving energy security and reducing global warming. Absorption- capacity, concentration, and residence time of gases are used to evaluate the so‐called global- warming potential (GWP). The environmental impact generated by greenhouse gases, as well- as the potential for acidificationand eutrophication, in general can be quantifiedby the sum- of the masses of the substances of gases (CO2, CH4, NO*x*), multiplied by the characterization- factors of these same substances. Once each of the factors will be differentwhen related to the- impactful gas to be measured [7].-

Finally, including the life cycle assessment as a fundamental tool to definebiofuel performance- is a decision making that provides an understanding of the environmental impacts, and- impacts on human health have traditionally not considered when selecting a product. This- valuable tool should be used to expand the knowledge base of productive systems and their- relationship with the environment, once can increase the efficiencyof its processes, reduce the- costs, and further promote marketing their products in such a sustainable way.-

#### **Author details-**

Mariany Costa Deprá, Leila Queiroz Zepka and Eduardo Jacob‐Lopes\*-

 \*Addressallcorrespondenceto:jacoblopes@pq.cnpq.br-

 Food-Science-Technology-Department,-Federal-Universityof-Santa-Maria,-UFSM,-Santa-Maria,- RS,-Brazil-

### **References-**


## **Cell Wall Proteomics as a Means to Identify Target Genes to Improve Second‐Generation Biofuel Production**

Maria J. Calderan‐Rodrigues, Juliana G. Fonseca, Carlos A. Labate and Elisabeth Jamet

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66189

#### **Abstract-**

 Second‐generationbiofuels-(B2G)generallyusesresiduescomposedoflignocellulosic- materialstoproducerenewableenergy-(potentiallyupto-50%),withoutincreasingthe- plantedareas.-However,thehighcostofenzymesrequiredforcellwalldisassembly- priortothesaccharificationmakesthe-B2Gproductionmoreexpensiveyet,compared- tothefirst‐generationbiofuels.-Designingplantswithlesslignin,abarrierto-B2G- production,orfacilitatingcellwalldisassemblybysearchingfortheplantmechanisms- canbethewaytoobtain-B2Gfeasibility.-Therewith,plantcellwallproteomicsprovides- valuableinformationconcerningthemaincellwallproteins-(CWPs)involvedinits- biosynthesisandrearrangements.-Essentially,twoplants ofthegrassfamilyhavebeen- studied:sugarcaneasacropamenabletosecond‐generationethanol-(E2G)production;- and-*Brachypodiumdistachyon*asamodelplantamenabletogenetictransformation.-Cell- wallproteomicshasallowedtheidentificationofnumerous-CWPsaswellastheirfine- profiling in differentorgansandatvariousdevelopmentalstages.-Proteinsactingon- carbohydrates,mostlyglycosylhydrolases,andoxidoreductases,includingclass-III- peroxidasesandlaccases,canbehighlighted.-Bothkindsof-CWPsareassumedto- contributetotheremodellingofcellwallpolysaccharidesbyenzymaticornon‐ enzymaticmechanisms.-CWPspresentingrowingorganscouldalsobeattractive- candidatessincetheygreatlycontributetocellwallplasticity.-

**Keywords:** *Brachypodium distachyon*, cell wall protein, grass, second generation etha‐ nol, sugarcane-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction-**

 Second‐generationbiofuels-(B2G)areapromisingrenewablealternativetosupplyenergy- demandoffossilfuelsworldwide,whoseadvantageismostlyduetotheloweremissionof- greenhousegasesandthepossibilitytoincreasetheproductionwithoutwideningtheplanted- area.-However,wearestillfarfromproducing-B2Gataneconomicallycompetitivewayand- reasonableamounttoreplacefossilfuels.-B2Guseslignocellulosicmaterialassubstrates.-Since- sugarcanehasbeenconsideredoneofthebestcropstoproducebioethanol,itsbagasseandstraw- havebeenstudiedasoneofthemaincomplementarysourcesof-C6and-C5sugarsfor-B2G.-One- ofthemainconstrainstoitseconomicfeasibilityreliesontherateofsuccessoftheenzymatic- saccharificationenablingtheconversionoftheplantcellwallsugarsintobioethanol-[1].- Saccharificationofthecellwallistheprocessofhydrolysisbywhichacomplexcarbohydrate,- suchascellulosecanbebrokenintomonosaccharides.-Thus,theproductionrequiresapre‐ treatmentofthebiomasspriortoexposethewallcarbohydratestosubstantialamountsof- expensiveenzymesintheindustrialprocess.-

Several strategies have been recently used to improve saccharification,mostly using microor‐ ganism enzymes. Differentenzymes with cell wall polysaccharide degradation activity have- been prospected from several organisms such as seaweed [2], termite stomach [3] and fungi- [4]. However, even presenting some advances [5, 6], the cost of E2G is not competitive for first‐ generation ethanol production from sugarcane.-

 Newapproachesareemergingfromtheplant'sperspectiveitself,whichtogethermaybethe- "eureka"tosolvethispuzzle.-Presentlyappliedresearchhasbeenfocusingonloweringor- modifyingthelignincontenttoallowitsremovalintheindustrialproductionandthusin‐ creasingtheaccessofcarbohydratestosaccharification-[7].-Indeed,ligninisfrequentlythe- majorreasonforbiomassrecalcitrance.-However,severalstrategiesthatfocusedondimin‐ ishingthelignincontent,andthusleadingtoimprovedsaccharification,resultedindeleteri‐ ouseffectonplantdevelopment-[8].-Adifferentpoint‐of‐viewbasedonligninmodification- maybemoreeffective,sinceevenincreasedlignincontentshowedimprovedsaccharifica‐ tionin-*Brachypodiumdistachyon-*[9].-Thereby,theexpressionofabacterialenzymeinto-*Arabi‐ dopsisthaliana*alteredligninandimprovedsaccharification,withoutloweringthelignin- content-[10].-

 Another strategy is to engineer the plant cell wall genes in order to enable the plant itself to- produce easier breakable sugars. By producing cellulose with more adequate characteristics- to allow a more efficientsaccharification,such as cristallinity, the plant material showed to- have improved saccharificationefficiency in *A. thaliana-*[11]. Genetic engineered rice and wheat- also showed increased enzymatic saccharificationwhen cell wall proteins (CWPs) acting on- polysaccharides had their expression changed [1, 12].-

The plant cell wall represents 50% of the organic carbon present on earth [13]. Cellulose is a- major cell wall polysaccharide and the major second‐generation ethanol (E2G) source. The- biosynthesis of wall polymers and all the processes that occur in the plant cell wall are mediated- by CWPs among which numerous enzymes. Prospective and directed studies to increase theknowledge on CWPs both in model species and in plants of agricultural interest provide- valuable information on target‐proteins in order to direct the plant pathways and produce- plant carbohydrates easily saccharified.-Accordingly, the high potential of this research can be- the key to B2G industrial production.-

#### **2. Plant cell wall proteomics-**

### **2.1. The plant cell wall-**

The plant cell wall was once considered as a static structure, but since the 1990s, it has been- addressed as a dynamic part of the cell, more similar to an extracellular compartment [14]. It- has to be strong and flexibleat the same time to enable its several roles such as mechanical- stability, osmotic control, signalling and defence against differenttypes of stresses. Its com‐ position varies according to the stage of development, cell types and environmental cues. As- an example, epidermis cells have to be better prepared for water loss than inner cells [15].-

Cell walls can be classifiedinto two types: primary and secondary. The former is found in- growing tissues, and thus extendable; and the lattertype is formed after the end of cell growth.- It can allow cells to resist to compression forces [16]. Cell wall composition includes cellulose,- hemicelluloses, pectins, proteins [17] and lignin in some cell types [18].-

Cellulose is a cell wall polysaccharide with a high molecular mass, formed by long linear chains- of β‐1,4‐linked glucose residues forming microfibrils [19]. Primary walls contain around- 20‐30% cellulose, and secondary walls up to 50% [20]. Hemicelluloses are composed of β‐1,4‐ linked monosaccharides with side chains [19]. The most present hemicelluloses in dicots and- grasses are xyloglucan (XG) and β‐(1,3‐1,4)‐mixed linked glucans, respectively. XG is probably- involved in forming cross‐links between cellulose microfibrils [21]. Pectic polysaccharides are- formed by structures enriched by galacturonic acid with complex side chain structures [22].- Sugarcane and other grass family species cell walls present specific characteristics such as being- poor in pectins and having no XG interlocking the cellulose microfibrilsin dividing cells; this- role is performed by glucuronoarabinoxylans (GAXs) [14]. Lignin is a phenolic polymer and- confers rigidity to cellulose microfibrils, and thus, to the cell wall [23].-

Cell wall biosynthesis seems to be specificfor each cell type [21]. During this process, cellulose- is synthesized at the level of the plasma membrane by specificprotein complexes. Conversely,- non‐cellulosic polysaccharides, such as hemicelluloses and pectins, are synthesized in the- secretion pathway and secreted to the apoplast, where they form the wall networks together- with cellulose [24]. Cell expansion occurs with enzymatic or non‐enzymatic cleavages of cell- wall polymers and the osmotic pressure separating the microfibrils.-Polymers are then- deposited in the internal part of the cell wall, forming the new cross‐linked network [14].- Several phytohormones are involved in cell expansion, acting specifically at the reorientation- of the microtubules, which may reorient the cellulose deposition [21].-

As widely known, sugarcane is the raw material for one of the largest bioethanol production.- E2G production uses lignocellulosic material to convert into ethanol through the steps ofpretreatment (to expose the cell wall polysaccharides to the enzymes), hydrolysis of the- cellulosic and hemicellulosic polysaccharides into monomers and finallyfermentation of these- sugars into ethanol [25].-

Over the years, the information regarding cell wall components from the chemical point‐of‐ view has increased, enabling us to think about strategies to modulate the cell wall structure.- There is knowledge available related to cellulose and hemicelluloses biochemical properties- and to the pectic polysaccharides biochemistry [26]. However, less is known about the overall- architecture of the cell wall. This knowledge should be enlarged to provide clues to engineer- walls. Indeed, since the cell wall is constantly being modifiedeither to respond to internal and- external stimuli, this self‐regulatory mechanism could be modulated to respond to commercial- interests.-

### **2.2. The plant cell wall proteome-**

The concept of CWPs includes not only the proteins present inside the cell wall structure but- also those present in the apoplast. CWPs are essential to the wall functions such as modification- of the cell wall components, its structure, signalling, interaction with the plasma membrane- and response to stresses [27]. Several factors can modify the cell wall proteome content, such- as development [28–31] and biotic or abiotic stresses [32, 33].-

CWPs share three common characteristics: a signal peptide to be targeted to the secretory- pathway, no intracellular retention motif and the absence of hydrophobic trans‐membrane- domains. The signal peptide presents a positive charge at its N‐terminus, a hydrophobic central- region and a polar C‐terminus [34]. One of the best‐described intracellular retention motif is- the C‐terminal H/KDEL, which maintains proteins inside the endoplasmic reticulum [35]. On- the contrary, other sorting determinants are more complex. For example, vacuolar targeting- routes are diverse and there seems to be differenttypes of vacuole sorting determinants [36].- Bioinformatic programs can help predicting the subcellular location of proteins through- protein amino acid sequences, but they rely on experimental evidence which can be incom‐ plete [37].-

Three types of CWPs can be considered according to their interaction with the cell wall matrix- [27]. The labile proteins have littleor no interaction with the cell wall polysaccharides and- circulate in the extracellular matrix. They can be recovered by vacuum infiltrationof tissues- [38]. The weakly bound proteins can be linked to the wall components through Van der Waals- interaction, hydrogen bonds, or ionic links and can be recovered with salt solutions. Strongly- bound proteins such as structural proteins (SPs) are resistant to salt extractions and can be- linked together or to polysaccharides by covalent bonds [39]. Regarding functions, CWPs can- be divided into nine functional classes including a class of miscellaneous proteins (MPs) and- a class of proteins yet unknown function (PUFs) [40]. As all classifications, this one has some- drawbacks like the difficultyto classify proteins with dual functions such as protease possibly- involved in protein turnover or in signalling, but it allows gettingan overview of cell wall- proteomes [41].-

Proteins acting on carbohydrates (PACs) mostly comprise glycosyl hydrolases (GHs) and are- involved in cell wall polysaccharides remodelling [42]. PACs belong to the most represented- classes in cell wall proteomes. Cellulases and glucanases are examples of proteins that can be- found in this family. These enzymes are used in enzymatic hydrolysis cocktails used in E2G- production, so they could be targets for manipulation in the plant species. Oxidoreductases- (ORs) are mostly class III peroxidases (Prxs). Prx activities are diverse, they can break cell wall- polysaccharides in a non‐enzymatic way and facilitate cell wall extension but they can also- favour the cross‐linking of cell wall components such as monolignols and SPs [43]. Proteins- related to lipid metabolism (PLMs) are almost all lipid transfer proteins and lipases. Some of- them could be involved in cell wall loosening through the bind of lipids to their hydrophobic- cavity [44]. Proteases (Ps) can play roles in protein turnover, protein maturation, signalling or- defence [45]. SPs, such as hydroxyproline‐rich glycoproteins, proline‐rich proteins and- glycine‐rich proteins can be cross‐linked in cell walls and contribute to its architecture [46,- 47]. Proteins with interaction domains with proteins or polysaccharides (PIDs) comprise lectins- and enzyme inhibitors. There is a lack of knowledge regarding the role of lectins in plant cell- walls [48]. Enzyme inhibitors play a critical role in the regulation of enzymatic activities. As- an example, there is a subtle interplay between pectin methylesterase and pectin methylester‐ ase inhibitors [49]. Proteins possibly involved in signalling (PSs) include arabinogalactan- proteins which have been assumed to play diverse roles during plant development, and- particularly in calcium signalling [50]. The miscellaneous proteins (MPs) contain many protein- families which are not numerous enough to form a distinct class. The roles of proteins with- domains of unknown function (PUFs) are mostly unknown, but this functional class offers- potential for future research. Among PUFs, the DUF642 proteins have been shown to interact- with cellulose *in vitro-*[51]. They could also be involved in pectin methylesterificationor in- defence [52, 53].-

 Isolating and identifying CWPs is particularly challenging. Indeed, the difficulty begins with- the extraction procedure. The cell wall is an open compartment and the polysaccharidic- network can be a trap for intracellular contaminants. Either destructive (DP) or non‐destructive- (NDP) protocols have been used. DPs rely on grinding the tissues to isolate cell walls prior to- the extraction of proteins with salt solutions [54]. The purificationof cell walls relies on the fact- that it is the denser cell compartment [55]. NDPs, using vacuum infiltrationof tissues with- mannitol or salt solutions, do not harm the cells and allow extraction of apoplastic proteins- [56]. Usually, the salts used in the extraction protocols are CaCl2and LiCl. CaCl2extract CWPs- through a competition mechanism [40] since pectins strongly chelate calcium ions [57]. An- illustration of the effectsof CaCl2has been provided by plasmolysis experiments performed- on leaf tissues transiently expressing a CWP fused to the fluorescent TagRFP (red fluorescent- protein) [38]. The fusion protein in displaced from the cell wall to the apoplastic space upon- CaCl2application. On the other hand, LiCl is able to extract hydroxyproline‐rich glycopro‐ teins [58]. The use of both types of protocols to extract CWPs can be a good strategy to increase- the coverage of the cell wall proteome [30]. However, some CWPs still escape because they are- strongly bound to cell wall components [38]. At present, the cell wall proteomes are poor in- SPs such as hydroxyproline‐rich glycoproteins or proline‐rich proteins. In addition, since some- CWPs are heavily glycosylated, these post‐translational modificationscan be a problem forprotein identificationby mass spectrometry. Finally, proteomics studies of species that do not- have a fully sequenced genome present an additional bottleneckbecause the precise identifi‐ cation of proteins cannot be achieved.-

Even carefully performing all these protocols, the identification of proteins that are not secreted- through the classical secretory pathway has been reported. These proteins can be predicted to- belong to differentcell compartments such as cytoplasm, nucleus, mitochondria, chloroplasts- or vacuoles. The question of the existence of alternative routes of secretion is still a matter of- debate [41].-

#### **3. A focus on** *B. distachyon* **and sugarcane cell wall proteomes-**

After designing several protocols to analyse the cell wall proteome of *A. thaliana*as a test case,- around 700 CWPs have been identifiedin differentorgans such as leaves, stems, roots and- etiolated hypocotyls as well as in cell suspension cultures, i.e. about one‐third of the expected- total number [59]. In order to widen the knowledge regarding CWPs targeted to findcandidate- routes to improve E2G production from the plant perspective, two additional species were- studied: (i) *B. distachyon*as a model for grass species from temperate areas, amenable to genetic- transformation and having a fully sequenced genome [60]; and (ii) sugarcane, only having a- large EST collection, but being one of the major sources for E2G production.-

#### **3.1. Plant material-**

 For-*B.distachyon*,threetypesoforganswereused:leaves,internodesandgrains-(**Figures-1A**,- **B**).-Two‐month‐oldplantswereusedandthe-CWPextractionswereperformedinyoungor-

**Figure 1.** *B. distachyon*and sugarcane plants used for proteomics studies: 2‐month‐old sugarcane plants (A), 4‐month‐ old sugarcane plants (B, C), and 2‐month‐old *B. distachyon*plants (D, E). f (young leaves), g (mature leaves), h (apical- internodes), and i (basal internodes).-

 matureleavesandapicalorbasalinternodes-[29].-Theseorganswerestudiedinordertocom‐ parethedifferencesbetweenorgansandtolookforproteinspossiblyinvolvedincellwall- extensionandgrowtharrest.-Grainswerecollectedatdifferenttimesafterflowering-(9,-13or- 19 days)-[31,-61].-Theaimofthestudywastounderstandthemodificationsofcellwallpolysac‐ charidesduringgraindevelopmentandfillingbecausetheyarekeydeterminantofthesize- and mass of thegrain.-

In the case of sugarcane, three types of materials have been studied (**Figures 1C**–**E**): 11‐day‐ old cell suspension cultures [62], 2‐month‐old stems [30], and 4‐month‐old young or mature- leaves and apical or basal internodes [63]. The aim was to identify among CWPs possible- targets for cell wall modification in order to facilitate E2G production.-

### **3.2. Methods-**

#### *3.2.1. Extraction procedures-*

In these experiments, differentextraction techniques were used. For *B. distachyon*, a DP was- used for all the materials [54]. It started with mixing the tissue in a 5mM sodium acetate buffer,- pH 4.6, 0.4-M sucrose and protease inhibitor cocktail. After that, the mixture had to be ground- in a blender at full speed for about 15min. PVPP was added to the homogenate, and it was- stirred for 30min at 4°C. To isolate cell walls, the mixture was submittedto several successive- centrifugations (1000×*g*) in a solution of increasing sucrose concentration (0.6‐1.0-M). The pellet- was then extensively washed through a Nylon net (25µm) to remove sucrose. The cell wall- fraction was ground in liquid nitrogen. Then, proteins were extracted by differentsalt buffers- prepared in 5mM sodium acetate, pH-4.6: twice in 0.2-M CaCl2, followed by twice in 2-M LiCl.- Cell walls were resuspended in these buffersand centrifuged at high speed (40,000×*g*/15 min/- 4°C). The four supernatants were pooled.-

    The same DP with minor modificationswas used for sugarcane cell suspension cultures and- 2‐month‐old stems [30, 62]. Another extraction method was tested with young or mature leaves- and basal or apical internodes. This method was based on vacuum infiltration [56], which is a- NDP requiring working with fresh material only. The plant organs were cut to fit in a beaker- and completely immersed in a solution of 3.0-M mannitol and 0.2-M CaCl2in a dessicator- connected to a vacuum pump. The tissues were vacuum‐infiltratedfor 5min. Plant organs- were centrifuged in a swinging bucket rotor (200×*g*/15 min/20°C). The apoplastic fluids- (released at the bottomof the tube) were collected and stored at low temperature. This- procedure was repeated once with the same solution. Additional two rounds of vacuum- infiltrationwere performed in a solution with 2-M LiCl instead of 0.2-M CaCl2. All four extracts- were pooled.-

Samples resulted from DP and NDP were desalted, freeze‐dried to concentrate proteins and- then used in 1D‐electrophoresis (1D‐E) to check the quality of the protein extracts.-

It should be mentioned that all the experiments have been repeated twice or thrice to take into- account biological variation. Only CWPs identifiedin at least two biological replicates have- been validated. A detailed description of these protocols can be found in Refs. [29–31, 61–63].-

### *3.2.2. Identification of proteins by mass spectrometry and bioinformatic analyses-*

Then, proteins were identifiedby mass spectrometry (LC‐MS/MS) and bioinformatics after- tryptic digestion performed at 4°C, after separation by 1D‐E or in solution. A detailed de‐ scription of the parameters used for MS analysis can be found in [29–31, 61–63]. For *B.- distachyon*, the genomic sequence data were used [64, 65]. For sugarcane, the SUCEST trans‐ lated EST database was used [66]. The amino acid sequences of the identifiedproteins were- systematically compared to those of *Sorghum bicolor*, the closest related species having a fully- sequenced genome [64]. In case of partial EST sequence, this comparison allowed the bioin‐ formatics prediction of sub‐cellular localization and functional domains.-

For both plant species, the bioinformatics analysis of the identifiedproteins was carried out- *de novo*in the same way regarding the prediction of their subcellular localization and of- functional domains using the ProtAnnDB annotation pipeline [67, 68]. All the experimental- data were collected in the WallProtDB database [59, 69]. The Venn diagrams used in this chapter- were made with the Venny online software [70].-

#### *3.2.3. A comparative survey of B. distachyon and sugarcane cell wall proteomes-*

As a key indicator of the quality of the protein extract, the percentage of proteins predicted to- be secreted and not retained in an intracellular compartment can be calculated (**Figure 2**). The- other proteins can be considered as intracellular contaminants. The highest proportion of- proteins predicted to be intracellular has been found in sugarcane cell suspension cultures- (82%). This could be explained by two facts: a DP was used thus increasing the chance for- intracellular proteins to be trapped in the cell wall polysaccharidic matrix; and/or cell suspen‐ sion cultures contain a certain proportion of dead cells whose content is released in the culture- medium, so that intracellular proteins can interact with the cell walls of living cells. Such result- has also been obtained with cell suspension cultures of *A. thaliana-*[71]. Apart from this sample,- the proportion of proteins predicted to be intracellular is above 40%. The highest proportion- of CWPs was obtained with basal internodes of *B. distachyon*. In that case, we noticed that the-

**Figure 2.** Percentage of CWPs and proteins predicted to be intracellular in each proteome. *B. distachyon*proteomes are- in black and white, whereas sugarcane proteomes are in green and white. AI: apical internodes; BI: basal internodes; C:- cell suspension cultures; G: grains; ML: mature leaves; YL: young leaves; 2MS: 2‐month‐old stems.-

sedimentation of cell wall fragments were particularly easy for this sample, thus facilitating- its purification [29].-

Altogether, 567 and 273 different-CWPs were identifiedin all mentioned experiments for *B.- distachyon*and sugarcane, respectively. At present, these species, together with *Oryza sativa-* (270 CWPs), have the largest cell wall proteomes among monocots [59].-

The specificproteins found in each experiment, and the common ones are shown in **Figure 3-** for both species. A firstcomparison can be made between the cell wall proteomes of the aerial- parts of *B. distachyon*and sugarcane, the most amenable to E2G production. Sixty‐three out of- the 314 CWPs (20.1%) identified in *B. distachyon*leaves and internodes were common to both- organs taken at two different stages of development (**Figure 3A**). The percentage of common- proteins two by two was also homogenous, varying from 27% to 39%. This proportion was- very differentfor sugarcane cell wall proteomes, with only 3.0% of the proteins common to all- samples, i.e. 6 of 201 CWPs (**Figure 3C**). The comparison two by two reached a result similar- to that obtained with *B. distachyon*only for CWPs present in apical and basal internodes (37.4%).- The other duos have between 4.0% and 14.0% of common CWPs. This is probably related to- the smaller size of the sugarcane cell wall proteomes of compared to those of *B. distachyon*and- to the very different number of CWPs identified in leaves in comparison to stems for sugarcane.- Using 2‐month‐old leaves, the difficultyin extracting proteins from cell walls was also- observed (unpublished results). This might be inherent to the leather type of sugarcane leaves- requiring a differentextraction strategy. Another explanation could rely on the hexa‐ to- octaploid genetic basis of sugarcane [72], which could lead to the expression of different sets- of multigene family members at different developmental stages and in different organs.-

**Figure 3.** Venn diagrams showing common and specific-CWPs for each experiment performed with *B. distachyon-*(A- and B) or sugarcane (C and D). AI: apical internodes; BI: basal internodes; C: cell suspension cultures; G: grains; ML:- mature leaves; YL: young leaves; 2MS: 2‐month‐old stems.-

Including the cell wall proteomes of *B. distachyon*grains, 25% of the CWPs were common to- all organs (**Figure 3B**). It should be noted that the largest cell wall proteome was that of grains- comprising 481 CWPs and that 45% of its CWPs were specific to this organ.-

Now, looking at all the known cell wall proteomes of sugarcane, cell suspension cultures,- leaves, 2‐ and 4‐month‐old stems only showed two common CWPs (**Figure 3D**). Eighty two of- 273 CWPs (30.4%) were specific to 4‐month‐old basal and apical internodes.-

These comparisons are of special interest because they allow identifying both CWPs specific- to organ or developmental stages and CWPs common to all organs which may belong to a set- of housekeeping CWPs essential for cell wall maintenance. For example, the set of proteins- common to the 8 cell wall proteomes of *B. distachyon*comprises 42 CWPs among which 10 GHs,- 4 Prxs, 8 proteases, 1 lipid transfer protein (LTP), 2 GDSL lipases and 1 DUF642 protein. In- sugarcane, six CWPs were found to be common to 4‐month‐old leaves and internodes- (**Figure 3C**): one GH, two Prxs, two proteinase inhibitors, and one subtilisin, whereas two- CWPs were common to all six cell wall proteomes (**Figure 3D**): a protein of unknown function- and a cys‐protease. These CWPs would deserve functional studies to better understand their- functions. The case of sugarcane seems more complex than that of *B. distachyon*with less- putative housekeeping CWPs identified up to now.-

Now, cell wall proteomes can be considered from the functional point of view. As explained- above, it is possible to group proteins according to the prediction of functional domains [27,- 56]. **Table 1**shows the distribution of *B. distachyon*and sugarcane CWPs into functional classes- in the differentcell wall proteomes. Some specificfeatures can be noticed in *B. distachyon*: (i)- PACs are less represented in basal internodes; (ii) ORs are more represented in internodes; (iii)- PLMs are less represented in mature leaves; (iv) Ps are more represented in leaves; and (v)- PIDs are less represented in mature leaves. Finally, SPs have been only found in grains with- two leucine‐rich extensins identified.-In sugarcane, the situation is very different:-(i) PACs are- less represented in cell suspension cultures and in leaves; (ii) ORs are more represented in cell- suspension cultures and in mature leaves; (iii) PLMs are less represented in cell suspension- cultures and in internodes of 4‐month‐old plants; (iv) Ps are less represented in cell suspension- cultures, but more in 4‐month‐old stems; (v) PIDs are poorly represented in 2‐month‐old stems,- but more represented in cell suspension cultures and in mature leaves; and (vi) PSs are less- represented in cell suspension cultures, 2‐month‐old stems, and in young leaves. In both plants,- there are also variations in the contribution of MPs and PUFs to all cell wall proteomes.-

 This overview allows gettinga profilingof the cell wall proteomes and to focus on specific- functional classes of CWPs. Because of the variations observed in the contribution of each- functional class to the whole proteomes, it also shows that each plant and each organ has to- be studied in detail before choosing a strategy to modify its cell walls. For example, ORs- includes mostly Prxs, but also blue copper‐binding proteins, and multicopper oxidases. Prxs- are involved in diverse physiological processes, such as signalling [43], lignification [73], and- cross‐linking of SPs [74]. Their roles in cell wall polysaccharide and protein network rear‐ rangements could be the reason why they are more represented in *B. distachyon*stems.- Curiously, the sugarcane cell wall proteomes exhibit the highest proportions of ORs compared-


to other plants. Such CWPs are interesting targets whose genes could be engineered for E2G- production optimization.-

Results are expressed as percentages of the number of CWPs identified in each proteome.-

Values in bold are average values calculated with all proteomes data and values much differentfrom these average values.-

MPs: miscellaneous proteins; PLMs: proteins related to lipid metabolism; ORs: oxidoreductases; PACs: proteins acting- on carbohydrates; PIDs: proteins with interaction domains; Ps: proteases; PSs: proteins involved in signalling; PUFs:- proteins of unknown function; SPs: structural proteins; AI: apical internodes; BI: basal internodes; C: cell suspension- cultures; G: grains; ML: mature leaves of 4‐month‐old plants; YL: young leaves of 4‐month‐old plants; 2MS: 2‐month‐old- stems.-

**Table 1.** Distribution of the CWPs found in each cell wall proteome of *B. distachyon* and sugarcane into functional- classes.-

PLMs are mostly represented by LTPs and GDSL lipases. LTPs exact biological roles are yet- unknown, but they have been related to cell wall loosening and extension [44], pathogen- response, and cutin assembly [75]. Since sugarcane at young developmental stages are similar- to rolled leaves, this may explain the high proportion of LTPs, probably playing roles in cutin- assembly of both sides leather‐like leaves. Nevertheless, the betterunderstanding of the- mechanisms under this protein class may lead to the design of new strategies to increase- biomass production.-

The low percentage of PACs in sugarcane cell suspension cultures and leaves is also puzzling.- PACs mostly include GHs, such as β‐xylosidase, β‐galactosidase and have been associated- with cell wall loosening and expansion [76]. GH3, GH35, GH27, and GH51 can be of special- interest since they show homology to enzymes of interest used for E2G production [3].-

The two studied plant species, *B. distachyon*and sugarcane, appear to be complementary to- identify CWPs and look for their functions. Although both plants are monocots and have- similar cell wall composition, they seem to have differentstrategies to modulate cell wallstructure during development. Combining genetics and biochemical approaches should allow- getting insight in those mechanisms.-

#### **3.3. Perspectives and targets for E2G production-**

 Changes in lignin composition have led to a subtle improved saccharificationwith no relevant- deleterious effect [77]. However, for the cell wall polysaccharides, the challenge is still bigger- since there is less knowledge regarding their synthesis. The main players able to modify cell- wall polysaccharides are (i) the transcription factors that control the initial steps of gene- expression and (ii) the enzymes and proteins involved in the biosynthesis of cell wall compo‐ nents and in their modifications*in muro-*[78]. By altering transcription factors in *A. thaliana*, it- was possible both to increase cellulose and decrease lignin content [79] and improve secondary- cell wall synthesis in fibrecells [80]. In addition, the golden pot may be near; transgenic *A.- thaliana*expressing microbial hydrolases showed no visible changes in phenotype and- increased wall degradability [81]. An alternative to decrease the transgenic debate and perhaps- optimize efficiencycould be altering the expression of the own plant enzymes generating a- genetically modifiedplant, but not a transgenic one. Besides hydrolases, another possibility is- to consider the potential of the plant cell wall as a sensor to perceive changes and direct cell- wall polysaccharides synthesis, such as in microorganisms [78]. Then, attentionshould be paid- to the fasciclin arabinogalactan proteins, wall‐associated kinases and other membrane- proteins. Expressing carbohydrate‐binding proteins such as expansins could facilitate cell- loosening, and it may be a possibility to improve saccharification as well [82].-

As can be seen, modulation of CWPs expression offersa wide range of possibilities to achieve- a plant cell wall more cost‐effective in terms of E2G production. Since some CWPs have been- reported to act on cell wall remodelling or expansion, and we observed a different proportion- of them in the several organs and developmental stages, we suggest focusing studies on some- CWP families such as Prxs, GHs and LTPs, mostly those found in young and growing organs.- By targeting the level of expression of these proteins or their spatial distribution, it may be- possible to design plants with cell walls easily saccharifiedto E2G production. In order to- achieve this goal, it is recommended to use tissue‐specificand spatial regulation of gene- expression using precise gene promotors, so that there will be no deleterious effectto the living- plant. Notwithstanding, we highlight that more information on the modificationsoccurring- on cell wall polysaccharides has to be collected in order to provide the basis for applied results.-

### **Acknowledgements-**

This research was partially funded by USP‐COFECUB (Proc. 10.1.1947.11.9), BIOEN/PRONEX- FAPESP (Proc. 2008/56100‐5), INCT do Bioetanol, CNPQ (Proc.: 142784/2007‐9), and FAPESP- (Proc. 2007/59327‐8 and 2012/1212521‐2). The authors are thankful to Dr. R Pont‐Lezica who- was at the origin of the projects. They also thank Dr MC Falco and CTC for providing the- sugarcane plants and cells. They thank Dr GM Souza and Dr M Nishiyama for providing the- SUCEST sequences. MJR, JGF, and CAL are thankful to the Max Feffer-Laboratory of PlantGenetics team, especially TR Cataldi and S Guidettifor handling the mass spectrometer and- FE Morais and LM Franceschini for the bioinformatics assistance. EJ wishes to thank the CNRS,- the Paul Sabatier‐Toulouse III University and ANR (Grant Génoplante/PCS‐08‐KBBE‐003/- CELLWALL) for supporting her research.-

### **Nomenclatures-**


XG:- Xyloglucan-

#### **Author details-**

Maria J. Calderan‐Rodrigues1 , Juliana G. Fonseca2 , Carlos A. Labate2 and Elisabeth Jamet3\*-

 \*Addressallcorrespondenceto:jamet@lrsv.ups‐tlse.fr-

 1-Brazilian-Bioethanol-Scienceand-Technology-Laboratory-(CTBE)/Brazilian-Centerof- Researchin-Energyand-Materials-(CNPEM),-Campinas,-Brazil-

2-Department of Genetics/Laboratory Max Fefferof Plant Genetics/Higher School of Agri‐ culture "Luiz de Queiroz"/University of São Paulo, Piracicaba, Brazil-

3-Plant Science Research Laboratory (LRSV)/University of Toulouse/CNRS/UPS, Auzeville,- France-

### **References**


lignin polymerization and enhance saccharification.-Plant Biotechnol J. 2012;10(5):609–- 620. doi:10.1111/j.1467‐7652.2012.00692.x-


 sugarcanecellsuspensioncultures.-Proteomics.-2014;14(6):738–749.doi:10.1002/- pmic.201300132-


of saccharum and sorghum chromosomes: comparative organization of closely related- diploid and polyploid genomes. Genetics. 1998;150(4):1663–1682. doi:PMC1460436-


## **Advances in the Application of Spectroscopic Techniques in the Biofuel Area over the Last Few Decades**

João Cajaiba Da Silva, Alex Queiroz, Alline Oliveira and Vinícius Kartnaller

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65552

#### **Abstract-**

 Guided by the instability of the oil market, as well as limited availability of and, especially,- theenvironmentalimpactsoffossilfuels,theneedsofthemarketforenvironmentalfriendlyenergysourceshaveincreased.-However,aswithanyotherproductthatis- intendedtoplaceonthemarket,itisessentialtoensurethequalityofthefuelforsuccessful- marketingandacceptancebyconsumers.-Spectroscopictechniqueshavebeenwidely- usedfordifferentpurposesintheliteratureforthepastdecades,frombiological- applicationstothemeasurementoftheelementalcompositionofplanets.-Fromstudies- focusedonbiodiesel,bioethanol,biomassandbiofuelingeneral,differentspectroscopic- techniqueshavealsobeenappliedinthearea.-Thefocusofthischapteristoelucidate- whathasbeenpublishedinthelastfewdecadesoverthesubject,detailingthebasic- conceptsofthemainspectroscopictechniquesappliedandshowingtheresultsand- developmentsoverbiofuel.-Theaimofthechapteristoachieveasetofinformationthat- canbeusedasabiggercompileofinformationofthestateoftheartregardingthetheme.-

**Keywords:** biofuel, spectroscopy, infrared, Raman, NMR, UV/Vis, image analysis,- ICP-OES-

### **1. Introduction-**

 Spectroscopy is a general term for the science that deals with the interaction between the various- typesofradiationandmatter-[1].-Therefore,thesetypesofmeasuresareonlypossibleifthe- interactionbetweenphotonsandmattercausessomekindofchangeinoneormoreproperties-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 ofthesample.-Theelectromagneticspectrumcanbedividedintoregionsaccordingtothe- wavelengthoftheradiation,asshownin-**Figure-1**.-

**Figure 1.** The electromagnetic spectrum showing the wavelength limits corresponding to each type of electromagnetic- wave. The colored area shows the visible region.-

According to the region of the electromagnetic spectrum, differenttypes of spectroscopic- techniques can be used, and the phenomena explored by them are of totally a differentnature.- This fact explains the applicability of each technique for specificcases. **Table 1**shows the- spectroscopic methods according to the spectral region and the transition type.-

Because of its versatility, the spectroscopic methods have been widely used for different- purposes in literature for the past few decades, from biological applications [3, 4] to the- measurement of elemental abundance in astronomical objects [5, 6]. Since the beginning of the- century, the number of publications involving such techniques has grown exponentially.-

At the same time, guided by the instability of the oil market, as well as limited availability and- especially the environmental impacts of fossil fuels [7], the needs of the market for environmental-friendly energy sources have increased. However, as with any other product that is- intended to place on the market, it is essential to ensure the quality of the fuel for successful- marketing and acceptance by consumers [8]. For this reason, many countries have specific- legislation that establishes desirable characteristics to minimize problems arising from the use- of these compounds. Quality control, not only for biofuels but also for their increasingly used- blends, is of paramount importance. The type (the chain length, degree of unsaturation, and- presence of other chemical functions) and the concentration of the fattyacids used in the- synthesis of biodiesel, for example, can influencethe properties of the finalproduct and its- prerequisites in terms of storage and their tendency to oxidize [9]. The raw materials used and- the process of obtaining biofuel can be decisive in terms of the introduction of some harmful- contaminants in the finalproduct [7]. Therefore, the monitoring of reactions is critical for- quality control [10], and the monitoring of minor components, such as glycerol, water, residual- alcohol, and heavy metals, is very relevant [11].-

Advances in the Application of Spectroscopic Techniques in the Biofuel Area over the Last Few Decades 27 http://dx.doi.org/10.5772/65552


**Table 1.** Spectroscopic methods based on electromagnetic radiation\* .-

Chromatographic and spectroscopic methods are the most popular ways for biofuel analysis- [11]. Spectroscopic techniques, however, have attributesthat make them ideal for use in these- kinds of analytical measurements. These attributes include the following [12]:-


For the above reasons, it is relevant to examine the recent advances provided by the introduction of spectroscopic methods for biofuel analysis. This chapter aims to provide the theoretical- basis of the most widely used techniques for this purpose and to analyze the impact of their- use on this area.-

#### **2. Vibrational spectroscopy: infrared and Raman spectroscopy for biofuel- evaluations-**

The notion that systems comprised by molecules can undergo vibrational motions, in which- their atoms are in constant movement around their equilibrium position, can be explained by- vibrational spectroscopy [13]. Every molecule contains a ground state energy (including- vibrational energy), which can be described as the sum of different components. This energy- definesthe minimum energy with which atoms in a molecule can move in a periodic motion,- described mainly by the six movements shown in **Figure 2**.-

**Figure 2.** Main vibrational modes for molecules.-

The principle of this spectroscopy type is that electromagnetic radiation can interact with- molecules, leading them from their ground state energy to a vibrational excited state energyin which the atoms' movements have higher energy. The state transition follows quantum- mechanical rules and obeys quantized energy levels; for a harmonic oscillator view of the- vibration, the energy that takes a molecule from a vibrational state to an immediately higher- state is ( 1 2ℎ), where *<sup>h</sup>* is the Planck constant and *<sup>v</sup>* is the frequency of the vibration. Hence,- the photon that can interact with a molecule must have an energy that equals this value in- order to change its vibrational state; otherwise, the transition is not achieved. The portion of- the electromagnetic spectrum that can lead to these transitions is the infrared region. This- region can be divided into three differentparts, with differentapplications for spectroscopic- study: near infrared (NIR), 14,000–4000 cm−1, allows the study of overtones and harmonic or- combination vibrations; mid infrared (MIR), 4000–400 cm−1, allows the study of the fundamental vibrations and the rotation-vibration structure of small molecules; and far infrared- (FIR), 400–10 cm−1, allows the study of low-heavy atom vibrations [14]. A polyatomic molecule- can undergo many types of vibration, depending on their number of atoms and their degree- of freedom. For nonlinear molecules, the number of possible vibrations can be defined by the- 3*N* – 6 rule, where *N* is the total number of atoms. Hence, differentmolecules undergo different- types of vibration, leading to differentpossible transitions by absorbing infrared radiation.- This is the premise of this type of spectroscopy, since a spectrum achieved by such technique- can be seen as the fingerprintof a molecule and can be used specially for functional group- analysis [15].-

 Thetwomajortechniquestostudythesevibrationsareinfraredand-Ramanspectroscopy,and- theyhavebeenstudiedtoagreaterdegreethantheothertechniquesforapplicationssincethe- middleofthetwentiethcentury.-Althoughthetheoreticalknowledgewasdevelopedduring- thepriorcentury,itwasnotuntil-World-War-IIthatinfraredspectroscopyhadabreakthrough,- withthenumberofinstrumentsgoingfromlessthan-20priortothewarto-700by-1947-[16,- 17].-Infact,several-UK/USprogramsduringthewarpromptedresearchanddevelopmentof- theearlycommerciallyavailableinfraredinstruments,suchasthoseusedinpetroleum- analysis-(e.g.,totracetheoriginofgasolineusedbythe-Germanairforce),inqualitycontrol- fortheproductionofsyntheticrubber,andinresolutionofthestructureofpenicillin-[16–18].- Examplesofearlypublicationsrangefromindustrialapplicationstomoreacademicstudies- Forexample,-Downingetal.-[19]usedinfraredspectroscopytodifferentiateisomersofthe- moleculedichlorodiphenyltrichloroethaneandtoqualitativelydetectthepresenceof- impuritiesinitssamples,and-Pfannetal.-[20]usedinfraredspectroscopytoevaluatethe- presenceofmolecularfragmentsoftheinitiatorsusedtocatalyzeapolymerizationreaction- inthestructureofthefinalpolymer.-Regardingthebiofuelsarea,sinceitisamorerecent- subject,thefirstdescribedapplicationsofinfraredspectroscopystartedtoappearonlybythe- endofthe-1990s.-In-1996,-Adjayeetal.-[21]usedittocharacterizedifferentmixturesofsilicaaluminaand-HZSM-5ascatalystsforthecatalyticconversionofabiofuelfromthethermal- processingofmaplewoodtoliquidhydrocarbons;inthesameyear,-Sandersonetal.-[22]used- thetechniqueforthecompositionalanalysisofbiomassfeedstocks.-Ramanspectroscopy,on- theotherhand,wasappliedforbiofuelstudiesalmostadecadelaterthaninfrared- spectroscopy,asintheworkof-Oliveiraetal.-[23].-Theyusedthetechniquetodeterminethe- adulterationofdiesel/biodieselblendsbytheadditionofvegetableoil.-Inthisstudy,both-

 Ramanandnear-infraredspectroscopywereappliedforthedetermination,andbothgavea- finalsimilaraccuracyresultforthequantificationoftheadulteration,dependentonthe- algorithmusedforthecalculation.-

The two techniques, although related to the vibrational modes of the molecules, have differentprinciples and are complementary. Infrared spectroscopy is based on radiation absorption,while Raman spectroscopy is based on the interaction via inelastic collisions. In infraredspectroscopy, an infrared photon of certain energy (with a frequency *v*), can be absorbed by amolecule if that energy is exactly the same as the energy differencebetween a vibrationalground state and a vibrational excited state; using a simplifiedharmonic model, the energydifferencebetween the two states is ( 1 2ℎ), as mentioned before in the text. Hence, the photonenergy (*Ep*) needed so that a transition is possible is defined by:-

$$E\_p = \Delta E = \frac{1}{2}h\nu.\tag{1}$$

However, even if a photon has such energy, this state transition may not exist due to the mainselection rule of infrared spectroscopy: for absorption to occur (hence, transition), the vibrationneeds to lead to a change in the dipole moment. If no change occurs, then that transition isconsidered "infrared forbidden" [13]. The intensity in which the transitions occur also dependson the molecular bonds related to the vibration, since it is proportional to the square of thisdipole moment [24].-

For Raman spectroscopy, the dipole moment change by the vibration is not the cuttingrule,and vibration transitions that are not allowed in infrared spectroscopy may be allowed in-Raman spectroscopy. This is due to the differentphenomena taking place in this spectroscopy;as said, the technique takes advantage of an inelastic collision between the photon and themolecule, leading to the scatteringof the electromagnetic radiation with different wavelengththan the irradiated. The electric fieldin the electromagnetic radiation can interact with themolecule, creating an additional induced dipole moment as a response of the electrons andnuclei moving in opposite directions of the field, in accordance with Coulomb's law [25]. Thedependency in this case relates to the ability of the molecule to be polarized, measured by itspolarizability, that is, the deformability of the electron cloud around the molecule by anexternal electric field.In fact, the selection rule for a vibration transition in Raman spectroscopyis that it causes a change in the polarizability of the molecule. The incident photon is thenmomentarily absorbed by the molecule, leading to a transition from the ground state into avirtual state; a new photon is created and scatteredby a transition from this virtual state to alower energy vibrational state, as shown in **Figure 3**. Most of the scatteredlight is the samefrequency as the initial light, as described by Rayleigh scattering,which does not contain anyinformation regarding the molecule. The information comes from the Stokes and anti-Stokesscattering,in which the molecule goes from a ground state to a virtual state or from an excitedstate to a virtual state; in the firstcase, the scatteredlight has a frequency smaller than the initiallight, while for the second, the scatteredlight has a frequency higher than the initial light.- Hence, the Raman spectra can be divided by both types of scattering spectra, and the frequency- difference is equivalent to the one in infrared spectroscopy.-

**Figure 3.** Energy-level diagram for the possible transitions in infrared and Raman spectroscopy.-

For both techniques, characterization is one of the main possibilities, since the frequencies of- the molecular vibrations depend on the masses of the atoms, their geometric arrangement, and- the strength of their chemical bonds. The spectra provide information on the molecular- structure, dynamics, and environment [24].-

In terms of application, both techniques have differentapparatus schemes, which will not be- dealt by the authors here, but can be easily found elsewhere. Regarding the differencebetween- the techniques for comparison purposes, Peter Larkin has described it in his book "*Infrared and- Raman Spectroscopy: Principles and Spectral Interpretation*," as shown in **Table 2**.-


**Table 2.** Comparison of Raman, mid-IR and near-IR spectroscopy, taken from [24].-

The differencebetween the spectra of samples achieved by infrared spectroscopy and Raman- spectroscopy is illustrated by Corsettiet al. [26], in which both techniques were used to- quantitative measurements in systematically varied blends of ethanol and a gasoline, a subject- of interest due to the production of bioethanol. **Figure 4**shows a comparison between the two- techniques for the mixture and for pure ethanol and gasoline.-

**Figure 4.** IR and Raman spectra of pure ethanol (red), pure gasoline (dashed blue), and blends (black), presented in-[26].-

A comparison between the spectra can be very direct, in which bands that are very intense for-IR spectroscopy have low intensity in Raman spectroscopy, and vice versa. The vibrationalbands related to alcohol molecules, which are the stretches of the O─H (3600–3000 cm−1) and- the C─O (1000–1100 cm−1) bands, are intense in the IR spectra due to the electronegativity thatcauses great change in the dipole moment; however, these vibrations are not very polarizable,so they are shown with low intensity in the Raman spectra. On the other hand, the bandsrelated to the stretches and bending of the C─H bands (3000–2800 cm−1and 1600–1200 cm−1)do not cause much change in the dipole moment and are not very intense in the IR spectra,while they tend to be more polarizable and have greater intensity in the Raman spectra.-

As mentioned previously, the firstapplications of the two techniques in the biofuel area were- for the characterization and quantificationof components/properties of biofuels. These types- of applications are very common, and a great amount of work from the past two decades can- be found in the literature regarding it. Characterization is the principal feature of these- techniques, which have been applied in differentsituations. One of them is the evaluation of- catalysts, as in the study of heterogeneous biocatalysts from sucrose, saw dust, and chicken- egg shells for biodiesel production by Wembabazi et al. [27]. Another study investigated the- production of hydrocarbon biofuels through hydroprocessing of carinata oil [28], while other- looked at the production of bioadditive fuels from bioglycerol under eco-friendly conditions- [29]. Many other works can be found that deal with this theme [21, 30–32] Characterization- has been applied not only to the catalysts used for biofuel processes but also to the biofuel- itself, such as the structural analysis of bio-oils from the subcritical and supercritical hydrothermal liquefaction of a plant from Turkey in a study by Durak and Aysu [33], or the characterization of insoluble material isolated from Colombian palm oil biodiesel in Plata et al. [34].- Also, Nanda et al. evaluated horse manure through catalytic supercritical water gasification- as a possible next generation feedstock for biofuel production [35]. Many others have used this- approach [36–42].-

Another great feature of these techniques is their concentration dependency. The intensity of- the signals is related to the characteristic of the band vibration and to the number of molecules- needed to make the state transition (i.e., molecular concentration). This dependence can be- definedby the Lambert-Beer law, which states that there is a linear relationship between signal- and concentration. Hence, this can be used for the estimation of the concentration of components present in mixtures or for other properties in differentsystems. As commented earlier,- one of the firstarticles published using infrared spectroscopy aimed to achieve a compositional- analysis of biomass feedstock, where they determined the chemical composition of several- woody and herbaceous feedstocks (121 samples in total) using NIR spectroscopy. Samples were- analyzed for ethanol extractives, ash, lignin, uronic acids, arabinose, xylose, mannose,- galactose, glucose, and C, H, N, and O, and all those responses were quantifiedusing spectroscopy. The results showed great correlations for most responses, as depicted in **Figure 5**,- suggesting that infrared spectroscopy was able to quantify components related to biomass- feedstocks with high accuracy responses.-

For quantificationor classificationpurposes, many chemometric algorithms can be applied to- vibrational data to transform the data to more relevant quantitative information. In the case- of the Sanderson's work presented above, partial least squares regression (PLSR) was used to- convert the spectra data to the responses. Several other works in the biofuel area have used- differentalgorithms for the quantitative evaluation of differentresponses or for quality control- and classification,such as multivariate linear regression (MLR), principal component regression (PCR), support vector machines (SVM), artificialneural network (ANN), partial least- squares-discriminant analysis (PLS-DA), principal component analysis (PCA), K-nearest- neighbors (KNN), linear discriminant analysis (LDA), quadratic discriminant analysis (QDA),- regularized discriminant analysis (RDA), amongst others [39, 43–60] (see **Figure 6**).-

**Figure 5.** Graph of predicted vs. observed values of the responses studied in [22] for the quantificationof componentspresent in biomass feedstock.-

**Figure 6.** Scheme of the biodiesel production reaction made by [79].-

Other applications in the area using the vibrational techniques are the evaluation of biofueldegradation [61–64] and process evaluation [65–70]. Regarding the latter subject, a newstrategy for process optimization and control emerged in the last decade, namely, the processanalytical technology (PAT) initiative [71]. PAT can be definedas the optimal application oftechnologies in feedback process-control strategies, information management tools, and/orproduct-process optimization strategies, as described by Chadwick et al. [72]. To meet itsobjectives, real-time sensing techniques are employed to control and monitor parametersthroughout the processing chain, achieving a great amount of data. In the literature, PAT has- been applied to follow reaction paths, monitor conversion over time, and even quantitativelyevaluate catalyst efficienciesfor biodiesel production [73–79]. As an example, from ourresearch group, Kartnaller et al. have used infrared spectroscopy and chemometrics to followthe esterificationreaction of a free fattyacid to biodiesel, quantifying each component in the- mixture and evaluating differentenzymatic catalysts [79]. **Figure 7a**shows the change in the- spectra of the reaction mixture in which the bands related to different components change as- the reaction progress. As discussed, the spectroscopic signal is concentration dependent per- the Lambert-Beer law; hence, if a product in a mixture changes in its concentration, so will the- intensity of its bands. With the advance of technology, the infrared equipment has changed in- the past few decades and new types have been shown in the market. This has allowed the- equipment to acquire more information in less time and in an automated manner, which may- generate a great deal of information and allow easy monitoring of reaction paths, as shown in- **Figure 7b**.-

**Figure 7.** (a) Change in the intensity of infrared bands from different components in a mixture due to reaction and- (b) quantitative monitoring of the different components of the reaction over time, calculated by the Lambert-Beer- law, from [79].-

Hence, it is easy to conclude that vibrational spectroscopy has a large range of applications in- general, and the biofuel area has used it for many types of goals in the past years. With the- increase of technological advances, both from the technical perspective and the biofuel- research, the use of the technique is bound to increase even more.-

#### **3. Nuclear magnetic resonance and biofuel applications-**

The theory of nuclear magnetic resonance (NMR) spectroscopy was proposed by Pauli in 1924.- He suggested that when certain atomic nuclei have spin and a nuclear magnetic moment, inthe presence of an applied magnetic field-(Bo), they could be induced to absorb energy and- change their spin orientation with respect to this applied field [1, 15].-

The independent works of Felix Block and Eduard Purcell in 1946 experimentally showed that- by absorbing electromagnetic radiation in the presence of an intense magnetic field,the nucleus- experiences an unfolding of its spin state energy levels, as shown in **Figure 8**, which is- dependent on the spin itself. In addition, the energy absorbed must equal the energy difference- between the two states involved; in this case, the energy absorption is a quantized process [15].- The stronger the applied magnetic field,the greater the energy differencebetween the two- inverse spin states, as shown in **Figure 9**.-

**Figure 8.** Energy levels for a nucleus with spin quantum number; (a) *I* = 1/2 and (b) *I* = 3/2 in the absence and in the- presence of an applied magnetic field (Bo).-

**Figure 9.** Spin state energy separation as a function of the increase in the magnetic field.-

NMR spectroscopy is based on the measurement of the absorption of electromagnetic radiation- in the radiofrequency region (4–900 MHz). The applied magnetic field differentiatesthe energyof the two inverse spins of the nuclei that was previously degenerated, allowing the state- transition to be achieved by the absorption of radiation and generating the signal. Hence, in- this technique, the nuclei of the atoms are the main parts involved in the absorption process,- while there is a great dependence on the applied magnetic field.-

After the studies performed by Block and Purcell, other scientists discovered that the frequency- of absorbed radiation by certain nuclei is strongly affectedby their chemical environment,- which is influencedby their electrons and nuclei neighbors. Hence, this phenomenon can- associate the absorption spectra with the molecular structure. This discovery showed the great- utility of NMR, since it is based on spectrum information, it is possible to obtain the structural- elucidation of a molecule.-

**Figure 10**shows the predicted 1 H NMR spectra for ethanol, in which the nucleus evaluated is- of hydrogen-1. Since the hydrogen nucleus is composed solely on a proton, the spectroscopy- is commonly referred to as proton NMR. As was said before, not all nuclei in a molecule have- a transition resonance at the same frequency, since the nuclei in a molecule are surrounded by- electrons and exist in slightly differentelectronic environments. For the molecule of ethanol,- there are three differentchemical environments, which generate small differencesin the- absorption frequency of the nuclei. This is definedby the chemical shift (*δ*). The chemical shift- can be used to identify functional groups and can help in the elucidation of the structure- arrangements of groups. These applications are based on empiric correlations between- structure and shift. Several tables have been published with values of the chemical shifts related- to functional groups and molecules [15, 80]. In addition, the exact values of chemical shift- depend on the solvent type and solution concentration. For the proton NMR spectra, the- chemical shift values are defined in relation to an internal standard molecule, tetramethylsilane- (TMS), and they vary between 0 at 20 ppm, while TMS is defined as zero.-

**Figure 10.** Predicted 1 H NMR spectra of the ethanol molecule.-

Nowadays, two types of spectrometers are commercially available: the continuous-wave (CW)- and the pulsed or Fourier transform spectrometer (FT-NMR). Historically, CW instrumentswere mainly used for spectroscopic studies. Today, FT-NMR instruments dominate the market.- For the CW instruments, the absorption sign is monitored while the frequency source is varied- slowly. In some instruments, the frequency source stays constant, and the intensity of the- magnetic fieldis varied. For the pulsed instruments, the sample is irradiated with a periodic- energy pulse of radiofrequency, which leads to a signal in the time domain that decays in the- interval between the pulses. This decay is known as the free induction decay (FID), and the- signal is converted to a sign frequency domain using the Fourier transform (a mathematical- operation), resulting in a spectrum, as shown in **Figure 11**.-

**Figure 11.** (a) Time domain. FID Curve for 1 H NMR for acetone; (b) spectra created using the Fourier transformation to- convert the time domain to a frequency domain.-

The integrated peak areas of 1 H NMR are proportional to the number of nuclei present in the- molecule; that means, for the molecule of ethanol shown in **Figure 10**, the three differenttypes- of hydrogen have signals in which the areas are proportional to 3:2:1. However, for 13C NMR- spectra (another very common NMR technique in which the nuclei analyzed are from the- carbon-13), the signals are not proportional to the number of nuclei present, and they cannot- be used for this type of proportional characterization. Other applications use proton NMR for- the determination of functional groups within the molecules, such as hydroxyl in alcohols and- phenols, aldehydes, carboxylic acids, olefins,amines, and amides [1]. NMR can also be- correlated to the concentration of the molecule itself, not only to the amount of nuclei within- it. The technique can be used for the quantificationof compounds in mixtures, especially when- there are different types of functional groups [81, 82].-

13C NMR, as with 1 H NMR, can be used for the determination of organic and biochemical- structures. These determinations are based on the chemical shifts and, as for the 1 H spectra,- these values are relative to tetramethylsilane, with values between 0 and 200 ppm. In general,- the effectsof the environment are analogous to the ones observed for the 1 H NMR. Theapplications of Fourier transform methodology and broadband decoupling of protons have- accelerated the development of, and enhanced the interest in, 13C NMR spectroscopy. Techniques such as distortionless enhancement by polarization transfer (DEPT) and 1 H-13C COSY- (HETCOR) have further endeared 13C NMR spectroscopy to organic chemists. In general, 13C- NMR spectroscopy allows chemists to directly observe the carbon framework of organic- moieties and to make (collaborative) inferences about active nuclei attachedto the carbon- backbone. The technique is particularly valuable in research on natural products, pharmaceutical drugs, and biochemical processes, where complex cyclic species are common and often- difficult to identify with only 1 H NMR [82].-

This section illustrates the utility and application of NMR spectroscopy as a probative tool in- the fieldof biomass, bio-oils and biofuels. Examples of its application include describing the- characterization of the pyrolysis of biomass to achieve bio-oils, analyzing oil quality using- quantification methodologies, and real-time monitoring of reaction paths for biofuel production [83–87].-

Many reports in the literature describe the techniques used to obtain liquid fuels from biomass,- including fractionation, liquefaction, pyrolysis, hydrolysis, fermentation, and gasification.- Biomass pyrolysis is the cheapest way to obtain these products [88], and therefore it is the most- studied methodology for such conversion [89], either in terms of physical properties or- characterization of the resulting components [84, 90–92]. Compounds such as acids, esters,- alcohols, ketones, aldehydes, anhydrosugars, furans, and phenols are usually found in bio-oil- [93, 94], and its exact composition is strongly dependent on a number of factors, including the- biomass origin and composition, the reaction temperature and heating rates to which the- biomass is submitted,and the residence time on reactors [95]. During the pyrolysis process,- the oil fractions are condensed, collected in a sample tube, and posteriorly analyzed. NMR- spectroscopy is a suitable method for biomass pyrolysis studies. Differentfrom other spectroscopic techniques, which can be limited for only the qualitative analysis of bio-oil samples,- 1 H and 13C NMR can be used as a way to obtain approximate ratios of the chemical environments of protons and carbon atoms. Besides, it can be useful to determine the approximate- aromatic/aliphatic ratios. Another advantage is that NMR is a nondestructive technique, and- the sample fractions can normally be recovered after the procedure.-

Mullen et al., for example, described a method using 1 H and 13C NMR to characterize bio-oils- fast pyrolysis from numerous types of biomass [95]. Based on the proton NMR spectra of six- differentbio-oils and the integral values of selected regions, the 1 H and 13C NMR spectra were- used to determine the functional groups present in each oil. Using the DEPT spectra that were- enabled, it was possible to evaluate the extent of branching in the molecules. These procedures- provided important structural data about the analyzed samples. The work of Ben and- Ragauskas [96] described an in-situ investigation of the relationship between the structures of- pyrolysis oil at various time points during the accelerated aging process at 80°C, using 1 H and- 13C NMR. It was possible to detect a reduction in the percentage of aldehyde, carboxylic acid,- and ether compounds, while the level of aromatic, alkene, and alkane compounds increased.- In other words, it was possible to use NMR spectroscopy in the investigation of structural- changes of pyrolysis oil compounds before and after an accelerated aging process.-

The NMR technique has an advantage with respect to other common methods, such as infrared- spectroscopy or chromatographic techniques, as it has high resolution compared to the others,- and it also offersthe possibility of obtaining valuable structural information. Besides that, the- accelerated aging process of pyrolysis oil is carried out inside the NMR equipment, so the- analysis is made in situ and in real time [96].-

NMR spectroscopy can also be used for quality control either of the vegetable oils used as raw- material for biofuel production or the finalobtained product [38]. The quality of the vegetable- oil influencesmany properties in the produced biodiesel, such as viscosity, lubricity oxidative- stability, cold flow,ignition quality, and the heat of combustion. The type of fattyacid also- influences the final product. Monounsaturated and saturated fatty acids are more stable than- highly unsaturated ones, although they show higher viscosity and a bigger tendency to solidify- in cold weather.-

Analytical techniques for fattyacid determination, such as gas chromatography and nearinfrared spectroscopy, need calibration models using some standards with similar chemical- composition related to the analyzed sample [38]. High-resolution NMR has an advantage- above the others because calibration models are not necessary. It is possible to quantify- compounds in the sample through observing the integrated area of peaks with the chemical- shift corresponding to a specifictype of fattyacid [97]. Usually, the acquisition time for this- type of analysis is quite long. However, Prestes et al. developed a method that is useful in- determining the fattyacid profilein intact seeds in a fast and automated way using lowresolution NMR. It was possible to analyze more than a thousand samples per hour, thus- working as a powerful tool to speed up the selection of oilseeds that are suitable for biodiesel- applications [98]. Garro Linck et al. successfully analyzed biodiesel obtained via the transesterification of different raw materials using low-field spectrometers [99].-

 Another-NMRapplicationisasthe-PATtool-(asdescribedforinfraredspectroscopy),which- canbeusedforthe*in-situ* monitoringofthebiodieselproductionprocess,wheresampling- isnotnecessary-[85].-Aswasdiscussed,-1 H-NMRspectroscopycanbearobust,rapid,and- quantitativemethodthatcanbeappliedfordeterminingthepresenceofmultiplecomponentsduetospecificchemicalshiftsinthespectrum,andreactionmonitoringcanbeappliedovertime,basedontheintegrationofindividualprotonsignals.-Anexampleofthis- applicationistheworkof-Andersonand-Franz,wheretheyusedhigh-resolution-NMR- equipmentforthemonitoringofthebiodieselproductionreaction,evaluatingthetransesterificationoftriacylglycerol-(TAG)andtheresultingproducts,includingdiacylglycerol- (DAG),monoacylglycerol-(MAG),glycerol,andfattyacidmethylesters-(FAME)-[100].-Due- todifferentmolecularstructuresanddifferentenvironmentsanddifferentformsofhydrogen,itispossibletodifferentiatethesignalsfromeachmoleculeandevaluatethemover- time,asshownin-**Figure-12**.-

 Andersonand-Franzwereabletosee,then,thedecreaseofthe-TAGbandsduetothebiodieselproductionandalsotheisomersofintermediates,suchas-DAG,throughoutthereaction.-Thisisastatementofthesensibilityandsensitivityofthe-NMRspectroscopy- techniqueinachievinginformationfromthemolecules,whichcouldnotbeachievedby- othertechniques.-

Advances in the Application of Spectroscopic Techniques in the Biofuel Area over the Last Few Decades 41 http://dx.doi.org/10.5772/65552

**Figure 12.** <sup>1</sup> H NMR stacked spectra over time of transesterificationfor biodiesel production, where Y indicates the TAG- glyceryl methine at 5.27 ppm, Y1 indicates 1,2-DAG glyceryl methine at 5.08 ppm, Y2 indicates TAG glyceryl methylene at 4.29 ppm and Y3 indicates 1,3-DAG glyceryl methine at 4.09 ppm [100].-

#### **4. UV/Vis spectroscopy and image analysis for the quantification and- classification of biofuels-**

The absorption of radiation in the ultraviolet/visible (UV/Vis; 200–700 nm) region results from- the excitation of bounding electrons. The UV/Vis radiation has enough energy to promote- electronic transitions, and this is the main principle investigated by UV/Vis absorption- spectroscopy. This technique is very useful in identifying functional groups in a molecule- because a correlation between the absorption bands and the functional group can be done.- While inorganic compounds normally absorb light in the visible part of the spectrum, organic- molecules usually present some functional groups capable of absorbing radiation from UV- light sources. These functional groups must be unsaturated or have a heteroatom with nonbonding electrons, such as oxygen, sulfur, or halogens. **Table 3**shows some functional groups- and the radiation wavelength absorbed [1].-

According to the Lambert-Beer law, the intensity of the absorption of radiation by the species- present in the sample is directly proportional to its concentration in the system. Thus, quantitative determination of compounds containing absorbing groups can be easily made. UV/Vis- spectroscopy is widely used for many applications, including in the biofuel area, since it is low- cost and allows the analyst to perform qualitative and quantitative analysis in a fast and reliable- way.-


**Table 3.** Ultraviolet absorption of some organic molecules.-

UV/Vis spectroscopy is very useful, for example, for the evaluation and quantificationof the- blend level in biodiesel/diesel mixtures. The utilization of blends between biodiesel and- conventional diesel is very common, and the blend level can vary. Usually, pure biodiesel is- referred to as B100, while some blends are classifiedaccording to its percentage of biodiesel,- such as B20, B5, and B2 (respectively presenting 20, 5, and 2% of biodiesel in conventional- diesel). The blend level directly influencessome important characteristics of the fuel, such as- its lubricity and tail pipe emissions [101]. Thus, it is very important to develop adequate- methods for such applications. For example, Zawadzki et al. studied different types of biodiesel- blends, employing an UV-Vis spectrometer as a low-cost detector to identify the range of UV- frequencies suitable for detecting the biodiesel blend level [102]. The proposed method is- robust, even with changes in the biodiesel feedstock and the fuel diesel origin.-

UV/Vis detectors are also widely employed in high-performance liquid chromatography- (HPLC) [103–105]. Many papers describing the use of HPLC-UV/Vis for biofuel analysis can- be found in the literature. An interesting example is a statistical study performed by Foglia et- al. [106], in which the precision and accuracy of HPLC-UV/Vis were evaluated with good- results. This system was employed to determinate the level of simple alkyl esters of fats and- oils (biodiesel) blended in petroleum diesel and to validate this method for this kind of analysis- using different biodiesel feedstocks.-

Another interesting application involving biodiesel/diesel blends is the detection of adulterations using vegetable oil. Commonly, near/mid infrared spectroscopy and spectrofluorimetry- are used for this kind of analysis together with chemometric tools for multivariate classification/calibration [104]. These are expensive methods, so it would be interesting to evaluate the- possibility of detecting adulterated blends using UV/Vis spectroscopy, which is simpler andcheaper. A study performed by Fernandes et al. employed UV/Vis and chemometric tools to- detect soybean oil in biodiesel/diesel blends, focusing on biodiesel/diesel blend (B5) adulterations with soybean oil percentages from 0.5 to 2.5% (v/v) [105].-

Another application that is indirectly correlated to the concept of visible light spectroscopy- and that has been applied throughout the past few years is image analysis. The simplest- definition for the word *image* can be understood as an optical replica of a luminous or illuminated object formed by a mirror of lens. Therefore, the object that gives rise to an image does- so through an interaction with electromagnetic radiation, either by emission or absorption- processes. Over the years, a wide variety of analytical methods involving image analysis as- the main tool have been introduced, due to the ease with which they are carried out in a fast,- noninvasive, and inexpensive way compared to more advanced spectroscopic techniques.- New fieldswere opened with the introduction of chemometric methods to analyze image- analysis results, such as exploratory image analysis, multivariate statistical process monitoring, multivariate regression, and image resolution [107]. The versatility of this technique has- been explored in several areas, from flamedetermination of elements [108] to biochemistry- analysis [109], providing fast and cheap results in a simple way.-

In the field of renewable fuels, image analysis has been also explored for applications in various- stages of production. For example, for the application of microalgae for biofuel production, an- area that has attractedincreasing attentionrecently, digital image processing has been- successfully used as a way for monitoring and quantifying the amount of biomass present on- photobioreactors, using the RGB color system [110], and also to build the light distribution- profilein microalgae cultivation [111], a key factor for mobile productivity. Another interesting- application related to microorganisms with potential use in biodiesel production is the- determination of the intracellular accumulation level of lipids in yeast cells, which can be done- in a dynamic and nondestructive manner via high-content images [112].-

Because biodiesel quality control is becoming increasingly important, given the high market- demand for renewable energy sources, methods such as image analysis have emerged as fast- and reliable alternatives for the evaluation of some parameters. RGB/gray scale histograms- obtained from digital images have been used together with statistical methods in biodiesel- classificationas an efficientway to determine the feedstock used in its production, since this- influencesthe properties of the finalproduct [113] without, however, employing timeconsuming techniques, such as chromatographic methods. Another example is the application- of image analysis together with thin layer chromatography, a very simple method, for glycerol- detection in biodiesel [114]. This organic compound is a byproduct of the manufacturing- process and is considered a contaminant in the finalproduct. Similarly to fossil fuels, the- burning of biodiesel, for example, causes the emission of pollutants, such as NOx species.- Image analysis has proved useful in the prediction of the level of these emissions by the use- of flame radical imaging to monitor the biomass combustion process [115].-

In the coming years, researchers expect that image analysis will be explored in other stages of- the production process because of its potential for easily obtaining relevant results for the- biofuel area.-

#### **5. Atomic absorption and emission: trace element determination in- biofuel samples-**

The determination of trace elements in biofuels is a key parameter for their use, and the- importance of conducting such analyses reliably and quickly grows as the global demand for- renewable energy sources becomes greater. The main spectroscopic techniques currently used- for trace elements determination in biofuels are based on the phenomena of emission and- absorption of electromagnetic radiation by atoms or elementary ions. In the case of the atomic- absorption principle, electronic excitation occurs in atoms from the ground state to more- energetic electronic levels because of energy absorption at specificwavelengths that are- characteristic of each element. The excited electrons tend to quickly return to the ground state,- releasing energy at wavelengths also characteristic of each element. This is the concept- explored by atomic emission methods. Both of the concepts are shown in **Figure 13**.-

**Figure 13.** Atomic energy absorption/emission. The blue circles represent electrons, and the blue vacancies representthe finalelectron state after the absorption/emission occurs; h is the Planck constant and λrepresents the wavelengthin which the transition takes place.-

The most common techniques are inductively coupled plasma-optical emission spectrometry- (ICP-OES) and atomic absorption spectrometry (AAS) [116]. ICP-OES employs a highly- energetic plasma, formed by an electrically neutral gas, usually argon, converted to positive- ions and electrons. Such plasma has enough energy to atomize, ionize, and virtually excite all- the elements of the periodic table to more energetic electronic states [117]. These species tend- to return rapidly to the ground state, releasing energy at characteristic wavelengths depending- on the elements present in the analyzed sample, and the radiation intensity is directly proportional to the concentration of the element in the sample [1]. AAS also employs atomization- methods as well as emission techniques. Commonly, this process is carried out by means of a- flame,in which desolvation, evaporation, and dissociation of the molecules into atoms take- place [1]. Another common atomizing method is electrothermal atomization, in which the- energy for volatilization and atomization is provided by means of an electric current applied- to a graphite furnace [118]. Two more specificatomization techniques are hydride atomization- by heating a quartztube, a very common method for the analysis of some metals, such asarsenic, and the cold vapor technique, which is widely used for mercury determination. After- atomization, the analytes are submittedto radiation from a source, whether a single emission- line source (e.g., hollow cathode lamps, multielement lamps or electrodeless discharge lamps)- or a continuous source (e.g., halogen or deuterium lamps) that needs an auxiliary monochromator system to select the desired lines [119]. The analytes in the ground state absorb energy- at specificwavelengths corresponding to their more favorable electronic transitions, thus- generating an absorption spectrum whose intensity depends on the population of the atoms- in the ground state, directly proportional to their concentration in the sample.-

Trace elements may cause significant problems with serious implications for the use of biofuels,- such as biodiesel and bioethanol. Such elements can be present in original vegetable oils- subjected to transesterification processes due to absorption from the soil by the plant used as- a feedstock, or they may even be incorporated in the vegetable matrix by means of the catalysts- used in the biofuel synthesis or extraction/refiningprocedures [120, 121]. Sodium and potassium, for example, are used in the form of hydroxides in the synthesis of biodiesel, and together- with Al, Ca, Fe, Mg, and Ti, they form a group of elements that tend to form a large amount- of ash (metal oxides) after the fuel is burned [122, 123]. It causes difficulties for the operation- of the gears, reducing the longevity of the engines. Furthermore, these elements are also- involved in corrosion processes [121]. Some transition metals, such as Cu, Pb, Cd, and Zn, can- cause biodiesel oxidation [124], resulting in residues that may be deposited in the engines.- Moreover, they can contribute to air pollution and cause environmental damage due to their- toxicity [116]. The sulfur level in the produced biofuels also must be carefully monitored- because of emission legislation. Sulfur has been related to the depletion of the Earth's ozone- layer, acid rain incidence, and chronic respiratory diseases. Low sulfur levels are also needed- for good performance in modern engines [125, 126]. Iron and vanadium can act as catalyst- poisoners and may reduce the efficiencyof advanced catalysts that are commonly used in gears- [127]. Some additives that are added to biofuels to improve physical or burning characteristics- also contain metals, and their levels must be monitored. Because of these problems, strict laws- have been adopted for the maximum level of metal contaminants in commercial biodiesel.- **Table 4** presents the tolerated level of some metals established by ASTM standards [128].-


**Table 4.** Maximum concentrations allowed for some trace elements in biofuels (ASTM standard).-

It has become essential to develop precise, accurate, and sensitive methods that are applicable- for monitoring metal contaminants to qualify biofuels according to the established standards- for their use. The quantification of metals in bioethanol and biodiesel, for example, is associated- with a number of difficulties,such as their low concentration in the organic matrix (in the range- of µg/L), the limited number of certified standards, the sample complexity, and the dependence- of the final product on the raw material used [128].-

Despite ICP-OES and AAS being the most widely used techniques, the details involved in- the determination of metals in biofuel samples must be observed. In general, organic matter- decomposition procedures (often microwave assisted) can be applied to convert the matter- to a simple aqueous matrix, therefore reducing the chances of spectral interference with the- measurements and avoiding difficultiesin the injection of these samples [129–134]. This- process is also necessary because of the difficultiesin introducing the samples in atomizers- and in selecting appropriating calibration standards for the analyzed system [135].-

In some cases, however, direct injection of samples of biodiesel, bioethanol or fuel ethanol- may be advantageous in order to reduce the number of steps of the analysis procedure. This- operation brings some drastic consequences. The injection, for example, can be greatly hindered due to the physical-chemical characteristics of the sample, such as viscosity and surface tension, thus modifying the ease of suction of the components. Both in the case of the- ICP-OES technique (in which a nebulizer is used for generating an aerosol) [128] and in the- case of the sample introduction systems used in flameatomic absorption equipment, problems in the injection of organic samples affectthe efficiencyand reproducibility of the analytical methods. Burning organic compounds normally generates instability in the plasma- with ICP-OES [136], and the generated pyrolysis products cause some spectral interference.- Another problem may be the deposition of material originating in the pyrolysis processes on- the torch or other spectrometer facilities [128, 137]. When using a graphite furnace as an- atomization method for AAS, organic samples usually cause excessive material spreading- during volatilization [116].-

Due to the importance of determining trace elements in biofuels, and taking into account the- difficultiesmentioned above, several research groups have been working on the development of new methods for this purpose. Some interesting approaches have been described in- the past few years. Dilution with an organic solvent or water can be an interesting way to- minimize the errors observed in the analysis of biodiesel samples [127, 138–142]. Methods- involving pre-emulsificationof the sample using surfactants have also achieved good reproducibility [121, 123, 143–148]. Furthermore, the extraction of analytes from a sample for further analysis provides good results in some cases [149–151].-

Surely, the spectroscopic methods, whether based on atomic emission or absorption principles, are the most popular for elemental determination in biofuel samples due to their high- sensibility and reliability, despite the specificcharacteristics of the analytical methods involving organic samples.-

### **6. Summary-**

To conclude, biofuels have been presented in the last few decades as alternatives or substitutes- for fossil fuels to decrease the amount of fossil fuels used. With the increase in awareness of- the need to develop more sustainable ways to support the earth's energy system in the future,- much study is still needed, mainly in the areas of production, characterization, and quality- control. This chapter described several methods that involve spectroscopic techniques,- including infrared, Raman, NMR, UV/Vis absorption, image analysis, ICP-OES, and AAS. With- these techniques, applications can be performed to characterize organic components in- biofuels, to study the development of methods for controlling quality (e.g., the validation of- biodiesel blends or detection of adulterations), to develop methods for discerning trace- elements in biofuel, and even to monitor the production of biofuels in real time.-

The referenced works in this chapter represent only a brief summary of the uses of spectroscopic techniques, depicting their importance in terms of fostering new developments in the- biofuels area.-

#### **Author details-**

João Cajaiba Da Silva, Alex Queiroz, Alline Oliveira and Vinícius Kartnaller\*-

 \*Addressallcorrespondenceto:vkartnaller@yahoo.com.br-

 Federal-Universityof-Riode-Janeiro,-Chemistry-Institute,-Cidade-Universitária,-Riode-Janeiro,- RJ,-Brazil-

### **References-**


## **Liquid Scintillation Spectrometry as a Tool of Biofuel Quantification**

Romana Krištof and Jasmina Kožar Logar

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65549

### **Abstract-**

 Biofuelsarecommonadditionorsubstituteforfossilfuels,appliedasanattemptto- decreasetheimpactoftransportontheenvironment.-Becauseofalargevarietyof- alreadyknownbiofuelsandintensiveresearchinthefield,thereisahighdemandfor- analyticaltechniquesfortheirquantificationinfuels.-Liquidscintillationcounting-(LSC)- isoneoftheidealcandidatesforthiskindofmeasurementsbecausethemeasured- substanceisradiocarbonfoundinallbiofuels.-Thischapterdescribesthefundamental- featureof-LSCmeasurementsandpossiblesamplepreparationsteps.-Oneofthe- methods-(direct-LSCmethod)ishighlighted.-Oneofthemethod'sadvantagesissimple- sample preparation, thus suitabilityfor every LSC laboratory. Calibration and validation- resultsofthreetypesofbiocomponents,i.e.,bioethanol,syntheticbiodiesel-[hydrogen‐ atedvegetableoil-(HVO)],andconventionalbiodiesel-[fattyacidmethylesters-(FAME)],- arepresented.-Allresultsshowthatthedescribedmethodissuitableforroutineanalysis- ofvariousbiocomponents.-

**Keywords:** LSC, liquid scintillation spectrometry, 14C, diesel, bioethanol, synthetic die‐ sel, fossil fuels-

### **1. Introduction-**

 Recentlyobservedenvironmentalchangesand increasedexploitationoffuelsleadtoaconcern- andemphasisofsubstitutingfossilfuelsbybiofuels.-Withnumeroustypesofrenewableswith- verydifferentcharacteristics,frombioethanol,-ETBE-(ethyltetrabutylether),biodieselor- FAME-(fattyacidmethylesters),syntheticdieselor-HVO-(hydrogenatedvegetableoil),- Fischer‐Tropschproducts,etc.,theneedforseveralmethodsforbiofuelquantificationhas- aroused-[1].-Severalanalyticaltechniquesareinuse,someofthemhaveastatusofstandar‐

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 dizedmethods.-Forinstance,in-EN-14078-[2],near‐infraredspectrometrywithtrans‐reflec‐ tancefiberopticsfordeterminationofmethylestersismentionedwhileoxygenates,i.e.,ethanoland-ETBEaredeterminedusinganoxygenateflameionizationdetector-(EN-1601)orgaschromatographyusingcolumnswitching-(EN-13132)-[3,-4].-

For differentiation between ethanol and bioethanol, common analytical techniques cannot be- used since both components are chemically identical. Measurement of 14C activity of such fuel- mixture presents a solution because radiocarbon is only present in recently grown components- (biocomponent) while in fossil component all 14C has already decayed with radionuclide's half‐ life (5760 years). Radiocarbon or 14C is a cosmogenic radionuclide produced in the atmosphere- by neutron capture from 14N. Since production and decay of 14C in the atmosphere is in- equilibrium, living organisms who uptake 14C via photosynthesis, ingestion or inhalation have- closely related activity as its environment [5]. The principle of radiocarbon dating is therefore- usable also for quantificationof 14C in fuels. Genuine standard ASTM D6866 describes the- accelerator mass spectrometry (AMS) and liquid scintillation counting (LSC) methods. Sample- treatment is needed for these two methods. Additionally, a so‐called direct LSC method that- does not require special sample preparation before measurement is included in standard DIN- 51637 [6]. It is only mentioned for limited types of biocomponents.-

The following section explains the detection principle of the LSC system. Possible samplepreparation techniques and their differences will be explained. Finally, the chapter shows somebiofuel measurements using the direct LSC method. Results of ethanol, synthetic biofuel, and-FAME measurements as well as validation parameters are presented and discussed.-

### **2. Liquid scintillation counting (LSC)-**

Liquid scintillation spectrometry is one of the techniques for radioactivity measurements;especially suitable for the detection of β‐emittingisotopes such as 14C. As previously stated,isotope 14C is naturally produced in the atmosphere by neutron capture. When radiocarbondecays it releases electron and antinevtrino (see Eq. (1)), produced energy is distributed amongcreated particles what makes distinguishing continuous spectra of the β particle.-

$$\mathbf{^{14}C} \xrightarrow{\text{decay}} \mathbf{^{14}N} + \boldsymbol{\beta}^{-} + \overline{\mathbf{v}} \tag{1}$$

where: is Antinevtrino.-

In order to detect βdecay by means of LSC, a scintillation cocktail consisted of three types ofchemicals: solvent, emulsifier,and scintillator (fluorescentmaterial) has to be mixed withsample. Decay energy is absorbed in the emulsifierand via solvent transferred to the scintil‐ lator. The scintillator then emits energy in the form of light and so produced scintillations aredetected by the photomultiplier tubes (PMT) in which conversion to an electrical pulse occurs-(see **Figure 1**). If measurement is conducted without interferences, the measured count rate isdirectly proportional to the activity of the sample. In the case of biofuels, counting efficiency- should be taken into account for proper transformation of counts to activity because of- substantial effects of quenching.-

**Figure 1.** Quenching processes [7].-

The quenching occurs when energy transfer between the radioactive isotope and the scintil‐ lation cocktail is disturbed. Reduced photon production results in a reduced number of counts- as well as shifted spectrum toward lower energies. There are several types of quenching but- two the most important ones have also an effecton biofuel measurements, i.e., chemical and- color quench. As shown in **Figure 1**, chemical quench disturbs energy transmission between- solvent and the scintillator while color quench affectstransmission of energy between- scintillator and PMT by attenuation of produced light in the sample.-

A level of quenching can be evaluated through quench indication parameters describing the- path by which they were obtained, i.e., the spectral quench parameter of the external standard- (SQP(E)), spectral index of the sample (SIS), and spectral quench parameter of the internal- standard (SQP(I)). A calibration curve describes the relation between the quenching parameter- and counting efficiency.-The most common approach to obtain the calibration curve goes- through the preparation of a spiked set of samples in which various quantities of quenchers- such as nitromethane is added. Efficiencyis calculated by easy division of a count rate with- known activity of the sample. There is also a possibility of quench set made from samples- which by their nature have a variety of quenching; biodiesel blends have various quench levels- (depending on feedstock and quantity of the biofuel) what can be useful in quench curve- sample preparation.-

#### **2.1. Sample preparation-**

There are several sample preparation methods in combination with the LSC technique. In the- so‐called LSC‐A method, also known as the CO2or carbamate method, organic carbon in the- sample has to be converted to CO2. This process is conducted in a special apparatus where- sample is combusted to the form of CO2under a controlled environment. The gas is trapped- or absorbed on an absorbent or on one of the components of specially designed scintillationcocktails. In the LSC community, various mixtures are known [8, 9]. In the LSC‐B method, the- combusted sample in the form of CO2is further carbonized to benzene [10]. Benzene synthesis- consists of three steps: forming of carbide (usually lithium carbide), hydrolyzation to acetylene- and trimerization into benzene [8]. The LSC‐A and LSC‐B methods are used for environmental- sample measurements for the purposes of age determination, monitoring of nuclear site, etc.- The third option for the sample preparation is the direct LSC‐C method that does not need any- sample pretreatment. The liquid scintillation (LS) sample is prepared by simple mixing of the- sample with a suitable scintillation cocktail. Since there is a lack of sample pre‐treatment,- various matrixes and thus rather more complicated calibration with careful quench correction- are needed. In the case of biofuels, extensive quench is induced by samples' yellow color.- Namely, yellowish samples are actually very unfavorable for LSC due to complementarity to- blue, which is the typical scintillation color. Some authors have reported attempts to degrade- and limit the color of the LS sample [11, 12]. Biodegradation and low oxidative stability of- biocomponent presents another difficultyin calibration process. The most important features- of all three mentioned LSC methods are summarized in **Table 1**.-


**Table 1.** Comparisons of different LSC methods for quantification of biocomponents in fuels.-

### **3. Biofuel measurements with direct LSC-**

#### **3.1. Bioethanol measurements-**

Bioethanol is used as an additive to gasoline or substitution of ethanol. Several authors have- already reported measurements of bioethanol using various LSC methods [8, 13–17]. Since- ethanol/bioethanol matrices are colorless, the calibration can easily be made also directly froma count rate. This is a so‐called one‐step calibration curve, conducted as a correlation between- the count rate and the biofuel content. In blends of ethanol/bioethanol/gasoline, a two‐step- calibration is advised. The two‐step calibration consists of efficiency correction due to matrix- variation and expected chemical quench.-

Calibration samples can easily be made by blending certifiedfossil ethanol (in our case Fisher- Scientific)or gasoline (provided by local petroleum industry) with certifiedbioethanol (like- Carbo Elba ethanol). In each matrix, at least 10 LS samples were made and analysis was- conducted. The obtained spectra of the sample can be seen in **Figure 2**. We found that it is- useful if blends were made in accordance with market demands; that is several blends in the- ranges from 0–to 10% and 80 to 100% and the rest of LS samples in blends in between.- Measurements show that the differencein counting efficiencyamong ethanol/bioethanol- Blends does not exceed 1% what is within uncertainty of a typical quench curve. However, in- ethanol/bioethanol/gasoline blends, the differenceamong counting efficienciescan be up to- 10% (between 82 and 72%); therefore, quench affectsthe counting efficiencyand it has to be- taken into account.-

**Figure 2.** The set of bioethanol/ethanol blend spectra.-

#### **3.2. Synthetic biodiesel (HVO) measurements-**

Synthetic biodiesel or hydrotreated vegetable oil has recently been introduced into fuel market- as a substitution of classic biodiesel (FAME) due to its oxidative stability and similarity to fossil- diesel. The fuel itself can be mixed with diesel in various quantities or can be even substitution- of fossil fuel. The latteris a problem with FAME since changes in automotive engine has to be- made, while HVO can be used without any consequences to engine. Several authors have- reported measurements of HVO blends in the range currently reasonable for fuel market,- which is up to 20% [14, 18, 19]. In the same range, also a standardized method with direct LSC- and FT‐IR methods is available [6].-

The HVO is colorless so one can expect only chemical quench when blends with diesel are- made. Furthermore, our measurements have shown that HVO can work as a slight reducer of- quench (see **Figure 3**) and counting efficiencieswere higher (from 74 to 83%). As in the case of- bioethanol blends, blends varying from fossil diesel up to 100% HVO were made and analyzed.- Since there is significantdifference among counting efficienciesof various blends, a two‐step- calibration is advised. Counting efficiencycan be evaluated with the same quench curve as- bioethanol blends in the case of the same range. HVO can be produced from various feedstocks,- but according to our results and experiences, the activity of various blends is similar regardless- of the initial feedstock or preparation procedure of HVO.-

**Figure 3.** The set of HVO blend spectra.-

#### **3.3. Fatty acid methyl ester (FAME) measurements-**

As one of the most important parts of diesel's biofuels, FAME is referred to as biodiesel.- Characteristics such as chemical form, color and oxidative stability depends on feedstock oils,- what can affectanalysis by the direct LSC method. As was explained in the sample preparation- (Section 2.1.), the color of biodiesel has an effecton counting and thus besides chemical also- color quench can be observed. Biodiesel aging or oxidative instability is one of the drawbacks- that affectthe use of biocomponent in fuel market, but as research had shown, it has positive- effectson the LSC measurement [12]. That occurs due to decomposition of fattyacid esters- while radiocarbon is still present in the sample (see **Figure 4**). However, forced oxidation was- not shown as a promising step in sample treatment because of big differences among biodiesels- from various feedstocks.-

Several authors have reported measurements of biodiesel in various blends but mainly in level- up to 20% of biodiesel what is limit of current fuel market [19–21]. However, our researchshows that reliable analyzes of biodiesel in quantities up to 100% of biocomponent can be- conducted as well. Some changes of preprepared setups of LS counter Quantulus™ (Perki‐ nElmer) and counting protocol gave promising results, together with careful and precise- calibration [14]. Hence, changes in coincidence circuit enabled analysis of various feedstock- biodiesels in the whole range (from 0 to 100% biodiesel). **Figure 5**presents spectra that were- obtained measuring various feedstock biodiesel ranging in quench levels from 360 up to 850,- where the FAME from waste oil presents the sample with the lowest quenching number and- thus the highest observed quenching while sample with the lowest observed quenching was- made from sunflower oil.-

**Figure 4.** Biodiesel blend spectra. The spectra set of the firstmeasurement (left) belongs to the fresh biocomponent,- while the set of second measurement (right) represents the spectra of already oxidized biocomponent.-

**Figure 5.** Biodiesel spectra after protocol changes. Legend: BG: background; SFE: sunflower-(Spain), SFSI: sunflower-(Slovenia).-

#### **3.4. Validation parameters-**

All analytical methods need to go through validation in order to use them in routine analysis.- Uncertainty, detection limit, linearity, repeatability, and sensitivity were evaluated. Law of- uncertainty propagation was followed while contributions of variable parameters were- evaluated using GUM guidelines [22].-

It was found that the uncertainty results are directly affectedby the uncertainty of the balance,- sample and background count rate, counting efficiency,and uncertainty of calibration. The- indirect contributions are represented with uncertainty of pipette,temperature variation, and- luminescence of the LS samples. It was found that uncertainties of sample and background- count rate represent the largest contribution in the measurements near detection limits. In- analysis of blends with biocomponents quantity higher than 10%, the largest contribution of- uncertainty causes counting efficiencydetermination. In both cases, the uncertainty of the- balance presents negligible part of the uncertainty budget.-

In recent years, a new approach in determination of limits of detection is taken; the standard- ISO 11929 applies a null measurements uncertainty [23]. Besides background count rate, the- background and sample counting time present variables of limit of detection calculation.- Although a long‐term average of background is taken in our routine analysis, the detection- limits were evaluated conservatively, and thus 1000 min of background and 500 min of sample- counting time were taken. Obtained limits of detection (see **Table 2**) are comparable to those- obtained by other laboratories and methods [2–4, 6, 8, 13, 15–21].-


**Table 2.** Validation parameters summary.-

 Linearity of calibration curves is demonstrated using the least‐square method and the- correlation coefficient-*R*2while uncertainties of individual calibration curve parameters were- used for calculation of calibration uncertainty. As can be seen in **Table 2**, excellent linearity of- calibration curves was obtained in all biocomponents measurements. It has to be noted that- the individual calibration curve is a result of at least 20 blends analysis.-

Repeatability was tested by several measurements (at least 10) of the same sample; standard- deviation of analysis results was calculated. A test was conducted on various blends in the fullrange of the calibration. As can be seen in **Table 2**, results of the analysis with the direct LSC- method are repeatable within 1 standard deviation of results; furthermore, it never exceeds- 0.54% that was achieved with bioethanol calibration.-

Steepness of the calibration curve slope was taken as a parameter for determination of- sensitivity; factor *k* from linear regression line was compared. The most sensitive calibration- was shown to be for bioethanol analysis (17.5) while the least sensitive was the HVO calibration- curve with 9.156, respectively.-

#### **4. Conclusions-**

Biocomponents in world fuel market differin their chemical characteristics. As a consequence,- several analytical techniques have to be applied for their quantificationin fuel blends. Their- maintenance needs a lot of human and financial sources, especially in the form of equipment- and knowledge. Liquid scintillation spectrometry is a good alternative. Namely, the measured- quantity is always the same. Radiocarbon, 14C is found in all biocomponents regardless the- type of biofuel.-

Three approaches can be applied as the sample preparation step before LSC analysis. Two of- them, LSC‐A and LSC‐B consist of several sample preparation phases, from raw fuel to CO2- production and benzene synthesis. The finalresult of sample preparation is the same matrix- regardless the initial biocomponent. On the other hand, the LS sample in the LSC‐C method- can appear in many differentforms. The matrix is not constant and predictable. Calibration- and validation processes of the method are therefore extended and need expert knowledge.- But, it should be done only once.-

The maintenance of the already calibrated and validated LSC‐C direct method is simple. It- demands only periodical and simple check‐ups of calibration parameters. The method does- not need any special sample preparation steps. Routine analysis with this method is therefore- very fast, cheap and does not need highly trained experts.-

### **Author details-**

Romana Krištof1,2\* and Jasmina Kožar Logar1,2-


### **References**


## **Chromatographic Methods Applied to the Characterization of Bio-Oil from the Pyrolysis of Agro-Industrial Biomasses**

Maria Silvana A. Moraes, Débora Tomasini, Juliana M. da Silva, Maria Elisabete Machado, Laíza C. Krause, Claudia A. Zini, Rosângela A. Jacques and Elina B. Caramão

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66326

#### **Abstract**

Biomass conversion into solid, liquid and gaseous products by pyrolytic technology is one of the most promising alternative to convert the biomass into useful products and energy. The total characterization of the products from the pyrolysis of biomass is one of the great challenges in this field, mainly due to their molecular complexity. Pyrolysis is a process that causes degradation of biomass in a non‐oxidative atmosphere, at relatively high temperatures, producing a solid residue rich in carbon and mineral matter, gases and bio-oil. The yield and properties of the products depend on the nature of the biomass and the type of the pyrolysis process (type of reactor, temperature, gas flow, catalyst). Due to the high molecular complexity of bio‐oil, many different technical had been developed to their complete characterization. This chapter describes the principles of the techniques and main application of chromatographic methods (GC, LC, GC × GC, LC × LC, Nano‐LC) in the analysis of bio‐oils derived from thermo‐degradation of biomasses. Especial attention is carried out to two‐dimensional techniques that represent the state of the art in terms of separation, sensibility, selectivity and velocity of data acquisition for characterization of complex organic mixtures. For proper use of bio‐oil in the chemical industry, it is essential the identification and unambiguous determination of its major constituents. Only then, it is possible to propose a recovery route of some of these components for the development of an industry dedicated to a bio‐refinery. For this, chromatographic methods, especially GC × GC/MS, are fundamental because they allow analysis with high sensitivity and accuracy in identifying each constituent of the bio‐oil.-

**Keywords:** Chromatography, bio-oil, biomass, pyrolysis

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

The current world energy scenario shows a tendency to decrease the use of mineral resources source, considering its environmental impact, the socioeconomical and market problems if compared to renewable sources-[1–3]. In addition, the diversification of energy sources is necessary in order to meet the growing energy demand. In this context, it arises the biomass coming from different sources of natural resources, which is a renewable alternative, abundant and suitable for competition with conventional fossil fuels [3, 4]. The biomass conversion into solid, liquid and gaseous products by pyrolytic technology is a promising alternative to convert the biomass into useful products and energy [5–7]. The total characterization of the products from the pyrolysis of biomass is one of the great challenges in this field, mainly due to their molecular complexity [8].

Pyrolysis is a process that causes degradation of biomass in a non‐oxidative atmosphere, at relatively high temperatures, producing a solid residue rich in carbon and mineral matter, gases and- bio‐oil [6, 7, 9, 10]. The yield and properties of the products depend on the nature of the biomass and the type of the pyrolysis process (type of reactor, temperature, gas flow, catalyst) [7, 11–13].

One of the main products of biomass pyrolysis is the bio-oil and, due to its high molecular complexity, it has been subject of several characterization studies in order to identify its compounds and indicate their best uses [8, 10, 14, 15].

This chapter describes the principles of the techniques and main application of chromatographic methods (GC, LC, GC × GC, LC × LC and Nano‐LC) in the analysis of bio‐oils derived from thermo‐degradation of biomasses. Especially, attention is done to two‐dimensional techniques that represent the state of the art in terms of separation, sensibility, selectivity and velocity of data acquisition for characterization of complex organic mixtures.-

### **2. Pyrolysis of biomass**

Energy production in the twentieth century was dominated by fossil fuels (coal, oil and gas) that represented, still in the beginning of the century, about 80% of all energy produced in the world. Nuclear power, hydropower and renewable energy sources (solar, wind, geothermal and small hydropower plants), which are the most attractive from an environmental point of view, represented only 1.5% of world production in those years [1]. Currently, the depletion of natural resources of coal and oil, together with the greenhouse effect, has attracted great interest for the production of sustainable energy [2]. The search for alternative fuels has led some countries to opt for biofuels, which in turn increased interest in energy from biomass [3].

The conversion of biomass into fuels and chemicals can economize fossil reserves and thus boost research, social and economic activities, especially in countries where the biomass is an abundant raw material in the agro‐industrial sector [4]. The use of renewable sources for obtaining chemicals such as bio-plastics, bio-fertilizers and bio-polyesters can help reducing the demand of insumes from petrochemical origin.

The biomass can be converted into energy using thermal, biological, mechanical or physical processes. The biological processing (biological catalysis) is very selective and produces a discrete number of products in high yield, but it requires a raw material containing- sugar or carbohydrates and a water content exceeding 40% [5]. The thermochemical methods are more suited to dry biomass (moisture content <10%) and rich in lignin (which- is less suitable for biochemical conversion, since it is hardly broken through enzymatic activity), such as wood and agricultural waste. Furthermore, the thermal conversion process provides often multiple products in a short reaction time, typically using inorganic catalyst to improve the quality of the product. The main processes used for the thermal conversion of biomass into a more useful form of energy are combustion, gasification and- pyrolysis [5, 6].

Pyrolysis is defined as a thermal decomposition of the biomass that occurs in the absence of oxygen at temperatures between 350 and 700°C, producing gases, liquid and solid products [5, 7]. Processes using lower temperatures and long residence time favor the production of charcoal, while processes using higher temperatures and long residence time increase the conversion of biomass into gas. A great production of liquids is favored using processes at moderate temperature and short residence time [9, 10]. It should be noted that the three products are always generated, but the proportions may be varied by adjusting the process parameters such as heating rate, gas flow, pressure, particle size of biomass and residence time of the biomass in the reactor [6, 11]. Also in accordance with these parameters, the pyrolysis can be classified as slow, fast, flash, catalytic or vacuum [12, 13].

The slow pyrolysis, or carbonization, employs low temperatures and long residence times, favoring the production of charcoal. In the case of fast pyrolysis, the heating occurs at higher rates (above 20°C min-1), while in the flash pyrolysis, heating rates are between 500 and 1000°C s-1 [14].

Fast pyrolysis produces 60–75% by weight of bio‐oil (liquid) 15–25% by weight of biochar (solid) and 10–20% by weight of noncondensable gases, depending on conditions and the biomass used. The solid residue from this process can be charcoal or ash only, depending on the final temperature and on the content of mineral matter originally present in the biomass used. This residue can be used as fuel or as a soil additive or still for the production of ceramic material. The gas produced can be recycled back to the process and facilitating cleavage reactions of the original biomass [14]. The main objective of fast pyrolysis is to prevent the breakdown of primary products in small noncondensable gas molecules, or even prevent them from being recombined and polymerized. In any of these cases, it obtains a smaller yield of bio‐oil [10].

The order of the reactions that occur during the pyrolysis process and the yield of obtained compounds will depend on parameters such as the heating rate, the pyrolysis temperature, pretreatment of biomass and catalyst effects. The study of these reactions and the effect of these parameters are important for obtaining high yields of the desired products [5]. To perform the pyrolysis, it can be used different types of reactors, according to the main purpose of the process.

### **2.1. Pyrolysis reactors**

### *2.1.1. Fixed bed reactors (FxBR)*

In this type of pyrolysis reactor, the fluid phase (gas) passes through the particulate solid phase (biomass). This FxBR aims to promote intimate contact between the phases involved in the process—the gaseous fluid phase with the stationary phase (particles of biomass) [15–22]. The FxBR is constituted by tubular structures of stainless steel or quartz, being used as an oven or grill support during the controlled heating of the system. The inert gas passes through the compartment with the biomass (in steady state) carrying the products out of the reaction bed during the pyrolysis [15]. Typically, units for feeding biomass, ash removal and outlet or collecting gases are added to these reactors. Such reactors operate with high residence time of the biomass in the oven and low inert gas velocities [16–22]. In this way, they are considered suitable for research in laboratory scale or bench. Some units operating in China can convert up to 600 kg/h of biomass to bio‐oil [23].

Pyrolysis in FxBR is very used to the slow pyrolysis, with the main purpose of producing coal or ashes.

### *2.1.2. Fluidized bed reactors (FzBR)*

In this type of reactor, a fluid (gas) is passed through a granular solid material at high speed, sufficient to suspend the solid material and cause it to behave like a fluid. This process known as fluidization provides extensive use in studies on the pyrolysis of biomass and is ideal for the technique of fast pyrolysis because it can achieve the necessary requirements for its realization and is virtually the only ones used in the world on a commercial scale for the production of biofuels and chemicals [24–27].

The FzBR operates with suspended solids by the action of rising gases, which are introduced from the bottomof the reactor. To promote more effective heat transfer, there is used a bed of solid particles, generally consisting of sand finely dispersed in the biomass itself [23, 28].

The rapid exchange of heat between the heat source and the biomass is one of the most important points in the pyrolytic process. In this context, in FzBR, the dried and comminuted biomass is introduced into the reactor, wherein, in the heating zone, the material remains in a continuous movement, promoted by the carrier gas flow (inert gas), which maintaining the reactor with a low oxygen content as it is heated to high temperatures (500–900°C) using a heating rate between 100 and 500°C min-1 [23, 29, 30].

Among the many advantages of this model, we can mention its ease of construction and operation, good temperature control, the operation at atmospheric pressure, the easy scale-up and operation possibility with fine particle size biomass, which is common in agriculture, in forestry and industry. Beside this, the reactor has an excellent heat exchange between the heat source and the biomass, and an efficient gas‐solid contact due to the movement of the particle bed. This effective contact simulates an isotherm condition, which implies an operation more secure and with optimum performance, generating yields of liquid products of approximately 70–75% by weight (dry basis), minimizing side reactions [24, 26].

But, the heterogeneity of the residence times in the reactor due to the agitation of the solids in the bed can compromise the uniformity of the products. Moreover, the wear caused by the- moving particles and agglomeration of ash produced with the inert bed material may lead to- loss of fluidization and therefore the pyrolytic process [23, 25, 28]. Therefore, the successful application of this technology depends on understanding and overcoming their disadvantages and thus the development of a reactor that meets the needs of the pyrolysis process in a whole.-

Several research groups in the development of fast pyrolysis use the fluidized bed reactor. Among the many examples, one can cite: the Union Fenosa located in Meirama, Spain, which has a pilot plant with biomass feed capacity of approximately 250 kg h-1. Still in Europe, the Wellman Company in the UK, which has a pilot plant with a capacity of 200 kg h-1, in Canada, the DynaMotive, which employs a pilot plant with capacity of 8000 kg h-1 [23, 24].

### *2.1.3. Continuously feeding reactors (CFRs)*

Continuously feeding reactors (CFRs) are those which operate at all times with an input a specific substrate(s) and output of product(s). These types of reactors are widely used in industrial scale [31].

In general, CFRs offer reduced fixed and operating costs and improve heat exchange capability [31, 32]. Furthermore, they provide an increase in quality of the final product, since the variations that exist between batches are eliminated [33]. Continuous processes are still able to reduce losses caused by operational problems during the process, and it is not necessary to interrupt the production line [33] (start‐up and shutdown). The applications of these reactors are aimed to minimize the difficulties encountered in the process on an industrial level using reactors in pilot scale [31].

### **2.2. Catalytic pyrolysis**

Studies of the composition of bio‐oils obtained by pyrolysis of various biomasses (rice husk, coconut husk fiber, core tropical fruits, straw sugarcane, wood residues, etc.) found that the volatile fractions of bio‐oils consist of a complex mixture of different classes of compounds such as, ketones, phenols, aldehydes, hydrocarbons [8, 15, 34–40]. These bio-oils have physicochemical characteristics that avoid their direct use as fuel, without any treatment. One of the reasons is its high oxygen content, thus leading to a high chemical instability during storage hindering their production on an industrial scale [41].

For industrial use of bio‐oil, some enhancement process is needed (upgrading). The catalytic pyrolysis has emerged as an alternative for improving the quality of liquid products of pyrolysis acting as an upgrading process of bio-oil. The main variable of this process is the type of catalyst used, in particular those able to considerably reduce its oxygen content [41].

A method used worldwide is the hydrodeoxygenation (HDO), which converts and fragments heavy molecules in the biomass into hydrocarbons with lower oxygen content by the catalytic addition of hydrogen [42]. This process has been studied with the aim of producing a liquid mixture of hydrocarbons that do not have the undesirable properties of bio‐oil and thus can be used as fuel. This method is considered the most efficient for the upgrading of bio‐oils [42]. Several catalysts can be applied for HDO, being necessary the use of high temperatures and pressures, under an atmosphere of H<sup>2</sup> . The most used are those consisting of cobalt and nickel and may be supported on alumina (Al2 O3 ), silica (SiO<sup>2</sup> ), carbon, zeolites (ZSM‐5), among others [43, 44].

The H‐ZSM‐5 catalysts, for example, have strongly acidic active sites, which can supply hydrogen for the pyrolytic reactions, favoring the deoxygenation of bio‐oil. Thus, the great advantage of the use of zeolites for production of bio‐oils is the possibility of working on hydrogen-free atmosphere and the use of ambient pressure during the catalytic pyrolysis process [45].

Zeolites have been shown to be highly effective for converting lignocellulosic biomass into aromatics through catalytic pyrolysis. Whereas this type of biomass has low amounts of hydrogen in their composition (low H/C ratio), the maximum yield of hydrocarbons that can be obtained in the absence of H2 as external reagent is limited. Thus, an upgrading using zeolites generally results in yields of aromatic hydrocarbons and olefinic near 30% [46, 47]. In addition, in the catalytic pyrolysis occurs greater coke formation when compared with conventional pyrolysis [48].

Catalysts conventional hydrotreating as Co‐Mo and Ni‐Mo and supported noble metal catalysts have been studied to produce stable products with high calorific value from pyrolysis [49], however, require high pressures of H<sup>2</sup> that lead to hydrogenation of the aromatic ring, resulting in reduction in calorific value and increase in H<sup>2</sup> consumption [50].

### *2.2.1. Catalytical pyrolysis in-situ versus ex-situ*

Depending on the method of contact of catalyst and vapors of the pyrolysis, catalytic pyrolysis can be classified as in situ or ex situ.-

In the in situ catalytic pyrolysis, the catalyst is mixed with the biomass to be pyrolyzed. Thus, the most suitable type of reactor for this is the fluidized bed reactor since this biomass is directly mixed with the catalyst. As the catalyst is exposed to a concentrated stream of vapors generated by depolymerization of the biomass components, the catalysis reactions are facilitated [51–54].

For the ex situ catalytic pyrolysis, biomass is pyrolyzed in a separate compartment of the catalyst. The pyrolysis vapors generated in the first compartment are diluted in an inert gas which is inserted between the two compartments being transported into a second compartment which is filled with the catalyst. As the second gas flow between the compartments, the contact time with the catalyst decreases [51]. An advantage of this technique is the use of different temperatures for both the pyrolysis reactor and for the catalysis reactor, thus allowing the use of catalysts which are sensitive to high temperatures [51–55].

### **2.3. Microwave assisted pyrolysis (MWAP)**

The different pyrolysis processes have various conventional and unconventional heating methods. The development of an efficient heating method, with a precise control of heating parameters and with a reduction of adverse effects on the quality of the product is one of the challenges to be overcome in the development of efficient pyrolytic processes [56]. The use of microwave can be an efficient way of heating the biomass in a thermochemical conversion processes. Tech‐En Ltd (UK) did the first study of the use of microwave pyrolysis in early 1990 [57, 58]. This is a technology which can be a very effective alternative for pyrolysis of biomass, presenting several advantages such as reduction in waste volume, fast heating, better chemical reactivity, ease of control, energy saving, overall cost‐effectiveness, portability of equipment and processes, a cleaner source of energy compared to conventional systems [59].

Microwave heating allows a more careful control of the parameters of the pyrolysis process, enabling the maximization of the production of liquids or gases, once these parameters may induce or modify specific chemical reactions resulting in different product profiles. The process can be modulated, aiming the product optimization in accordance with conditions of temperature, power and residence time used [60].

The biggest advantage in the use of microwave heating as compared with the conventional process is the significant reduction in temperature and consequent energy gain in the pyrolysis process [61].

Another important aspect of the heating by microwave is capability to obtain basically the volatile organic compounds at lower temperatures when compared to conventional heating. Moreover, obtaining bio‐oil and gases is nearly synchronized. This heat and mass transfer characteristics of the process are related to the selective heating of the components to absorb the microwave with more intensity [60].

Obtaining a greater heating uniformity in the process may be possible if the temperature is homogenized at some point during the process, so that, without this, different product compositions are obtained due to the temperature profile formed [60].

Furthermore, the temperature selection depends on the desired product. Processes at low temperatures provide greater bio‐oil yields and lower energy cost. The determination and use of the appropriate power to microwave are also important. Lower powers, with lower heating rates favor formation of biochar, whereas higher power with higher heating rates favors the gasification reaction. In both cases, there is a reduction in the yield of bio‐oil [60, 61].

The use of microwaves also has some challenges for the applying in the pyrolysis such as the need for different systems for measuring the temperature [62], the limitation of usable materials for propagation of microwave [63] and obtaining heating equipment compatible with the process and with an efficient scale [64].

### **2.4. Pilot plants of pyrolysis**

The pilot plant consists of many components (steps/processes) that together form a unique process, which enables testing technologies to evaluate the quantity and quality of products desired [65, 66]. According to RESEM®, a Sino‐American Company specialized in equipment for pyrolysis [61], there are different sizes of pilot plants, reactors differentiated according to the type and number of samples to be processed or products to be produced [67, 68].

The development of new technologies for the production from clean energy sources [69, 70], are associated with the emergence of pilot plants for the production of bio‐oil through pyrolysis of biomass in both, laboratory and industrial scale, mainly because it is a simple, reproductive and fast process [71].

Pilot plants are equipments that consist of a closed or open system with physical, chemical and thermodynamic operation, in order to perform a technological process on a small scale. So, a prototype, designed for industrial processes, can be installed either in research laboratories or in industries [66]. In these prototypes, new and different technologies, shapes, sizes and physical structures for generating and processing information for use in pyrolytic systems can be tested before the scale‐up [67].

According to **Figure 1** that shows one graphic with the research scenario related to global patent on the theme in pyrolysis plant, from 1940 to this year, they were deposited 673 patents worldwide [72, 73].

**Figure 1.** Distribution of patents related to pyrolysis pilot plants in the last 80 years (according https://patentes.google.- com and https://patentscope.wipo.int [72, 73]).

Few of these patents actually generated commercial pyrolysis plant, indicating the need a lot of investment in this regard, especially in countries with great potential for use of biomass and lower oil reserves. As already described, in recent decades, there has been a growing concern about the processing of biomass through pyrolysis due to the interest in their products, both biochar [74], as bio‐oil. In this sense, grow‐related searches to biomass conversion technologies studies from laboratory scale to the development of bio‐refinery [75] involving processes for the production of fuels and chemicals [76, 77].

The experimental setup on a pilot scale is based on the adjustment process through the system variables, and especially in the reactor used technology.

The reactors in pilot plants can be classified in two systems [78]:-


With the technological advancement of pyrolysis technique, some models of reactors have been exploited to optimize the process, cost and quality of the products generated, and the main ones are the fluidized bed (bubbling and circulating), in addition to these, some others can be found like fixed bed, ablative, vacuum, rotary cone, plasma, microwave and solar, among others [79–99].

### *2.4.1. Fixed bed pilot reactor*

The pilot fixed bed system, similar to the bench reactors (Section 2.1.1), consists in fixed reactor with an inert carrier gas, similar to the bench scale. The technology of the fixed bed reactor is considered simple, reliable [100], and efficient for the production of bio‐oil in the pilot plant. In these reactors, the solids move down and collide in counter current with a heated inert gas. They are used in small‐scale productions [101]. The cooling and cleaning gas system consist in a filtration and in the use of separators cyclones. The biggest problem is associated with the removal of bio-oil and losses involved in this process.

The fixed‐bed pilot plants developed from the late 1980s, with a final capacity of 5 tons per day are considered small, from the point of view of the transfer for industry, considering that these technologies are still in development stages for commercial applications.

### *2.4.2. Fluidized bed pilot reactor*

As mentioned in Section 2.1.2, in the pilot fluidized bed reactor, a mixture of fluid and solid is obtained by introducing a pressurized fluid through the solid particles with smaller diameter [102, 103]. The general scheme of this type of reactor is very similar to that used in fluidized bed in bench scale.

Two main types of fluidized bed reactors are used in pilot plant:-

*Bubbling fluidized bed reactor*: According to the literature [104], the bubbling bed reactors are simple to construct and operate. They provide a better ability to control the temperature, fluid‐solid contact, heat transfer and capacity of storage of solids. Heated sand is used as a bed solid phase, rapidly heating the biomass in an oxygen‐absent atmosphere. The biomass is decomposed into coal, steam, gas and aerosols. The fluidized gas stream entrains the compounds produced out of the reactor [105].

After the pyrolytic reaction, biochar is removed by a cyclone separator and stored. An important factor for the full operation of these reactors is that the biomass needs to be in small particles (less than 2–3 mm) to achieve high heating rates in the oven.-

*Circulating fluidized bed reactor*: It has similar characteristics to the reactor in bubbling fluidized bed except for the shorter residence time. This design results in higher speed and higher yield of bio‐oil compared to fluidized bed reactors [106]. The reactor moves around its main axis. One advantage is its high performance even with a more complex hydrodynamics.-

### **2.5. Bio-oil from pyrolysis of biomass**

The bio‐oil, or pyrolysis oil, is a dark brown color liquid with a characteristic smell and comprising a complex mixture of hydrocarbons and oxygenated compounds with an appreciable amount of water, originated from the natural moisture of the biomass as well as a product of reactions that occurred during the pyrolysis process [26, 41, 107].

The literature registers that the bio‐oil can contain more than 400 different chemical compounds from different chemicalclasses, varying among organic acids, aldehydes, ketones, alcohols, esters, furans, sugar derivatives, phenols, among others [108, 109]. In addition to the oxygenated compounds, many aliphatic and aromatic hydrocarbons can be found [8].

The anhydrous‐sugar levoglucosan (1, 6‐anhydro‐β‐d-glucopyranose) is the main component of bio-oil, being derived from the thermal depolymerization of the cellulose and from it, many other sugar derivatives can be formed. The yield of these anhydrous‐sugars is affected by the source of biomass and by the experimental pyrolysis conditions. The increase in pyrolysis temperature reduces the concentration of levoglucosan, in contrast to other products because it stimulates breakdown of this molecule [110].

Mixtures of compounds as phenols, cresols and catechols (monomers and oligomers) are formed from the lignin [111, 112]. Phenolic compounds found in bio‐oils are composed mainly of simple phenols with a hydroxyl and which may contain other substituent group on the benzene ring, forming mixed functions (carbonyl, carboxyl, alkyl or aryl radical) [108].

Due to the presence of acids, especially acetic and formic, bio‐oil may show pH values in the range 2–4 [41, 113] that constitutes a problem, since it will affect storage conditions (equipment, transport) and its processing [113, 114].

The oxygen content in bio‐oil is approximately 35–40% by weight. The specific composition- depends mainly on the type of biomass used, the pyrolysis conditions (temperature, residence time and heating rate) and the storage conditions of bio‐oil [10, 41, 113]. The high oxygen concentration results in a low energy density that is less than 50% of the value for conventional oils. It- also affects the bio‐oil miscibility with other petroleum fuels and the stability of bio‐oil [41, 114].

The water constitutes 10–30% bio‐oil, and their quantity depends on the original biomass, and the pyrolysis conditions, since the moisture is coming from biomass and also from dehydration reactions taking place during pyrolysis [41, 108, 113]. Depending on the feedstock and process conditions, the ratio of aqueous and oil phase can vary from 50:50 to 30:70, and the presence of two phases can hinder the application of bio‐oil. This high water content may cause problems in the ignition engines by reducing the rate of vaporization of the oil, which prevents its application directly as fuel [115]. In many instances, drying the biomass prior to the pyrolysis is sufficient to reduce this problem.-

The instability of bio-oil is mainly due to the presence of highly reactive organic compounds (ketones, aldehydes, organic acids), which can undergo reactions to form ethers, acetals or hemiacetals [41, 116]. This kind of reactions may increase the average molecular weight oil, water content and its viscosity, resulting in a low quality oil and that, when stored, results in phase separation. However, the addition of polar solvents such as methanol or acetone can significantly reduce the viscosity of the bio‐oil [41].

The ash content of the bio-oil can also cause problems in some applications. The composition of the ash is dominated by alkali metals (potassium and sodium) that are responsible for severe corrosion and deposition turbines on heating surfaces during combustion [115, 117].

However, the crude bio‐oil, before use as fuel, must be chemically modified through complex processes such as hydrodeoxygenation, hydrocracking, decarbonylation or decarboxylation to reduce oxygen content, which is the main unwanted constituent in the bio‐oil for energy purposes [113]. Another alternative improving (upgrading) of the bio-oil is catalytic pyrolysis using zeolites alumina or metals as catalysts [114].

For the use and recovery of chemicals from bio‐oil can be used conventional separation techniques, such as solvent extraction, column chromatography and distillation (single, fractionated, or under vacuum). Solvents commonly used for extraction of compounds of interest in bio‐oil include water, alcohols, ethyl acetate, hydrocarbons such as toluene and mixtures thereof [108].

Fractionation in open or pressurized column has also been used as a pretreatment for the separation of compounds from bio‐oil [118–120].

Among its uses, bio-oils are potential fuels to diesel engines, gas turbines and boilers. They can be used also as raw materials for obtaining hydrocarbons by catalytic conversion or hydrotreating [121]. Considering its phenolic fraction, bio-oil appears as a substituent for petrochemical phenol in the production of phenolic resins (phenol-formaldehyde) or can be used in pharmaceutical, food or fine chemicals industries [108, 122]. Furthermore, bio‐oil may be fractionated to obtain many other products of commercial interest, such as abrasives, filter elements, battery separators, electric components, refractory materials, adhesives for wood, paints, varnishes, enamels, etc. [8, 123].

The reaction of bio‐oil with ammonia, urea or other amino compounds produces amides and amines stable, nontoxic to plants and can be used as organic fertilizer. The bio‐oils derived from wood residues can be commercially applied in smoking food [10]. In case of bio-oils with high concentrations of hydrolyzable sugars, it may be favorable to the production of ethanol by fermentation, whereas bio‐oils with high phenol content are mentioned as attractive starting material for the production of adhesive [123].

As application examples of someof the most important bio‐oil compounds can be mentioned: the levoglucosan (food additive, pharmaceutical industry); the levoglucosenone (synthesis of antibiotics and rare sugars); furfural (pharmaceuticals, pesticides); acetic acid (chemical industry); formic acid (wood preservatives, antibacterial agents); and hydroxyacetaldehyde (pharmaceuticals, fragrances) [124].

### *2.5.1. Aqueous phase of bio-oil*

As mentioned above, the amount of aqueous phase in the bio‐oil will depend on the original biomass composition, its original moisture and the pyrolysis conditions [108, 113]. However, it cannot be removed by conventional methods such as distillation [10]. The phase separation will occur when the amount of water exceeds the maximum level in bio‐oil (usually above 30–45%) or by extraction methods [125].

The addition of water allows to readily separating the bio‐oil into organic and aqueous phase. The aqueous phase contains mainly components of higher polarity, such as levoglucosan and other anhydrous‐sugars, furan, furfural, organic acids of low molecular weight, hydroxyacetone, hydroxyacetaldehyde and guaiacol [126–128]. The separation of the aqueous phase of the bio-oil is also commonly performed using dichloromethane and sodium bicarbonate solution, obtaining an acid extract [129, 130]. Although solvent extraction is widely used in phase separation of bio‐oil, it can affect the qualitative and quantitative composition of the extracted sample due to the different affinity of the solvent for each class of chemical compounds present in the sample [131].

The aqueous phase of the bio‐oil cannot be directly discarded as wastewater, since some compounds may be above discharge limits. Different processes may be applied for the treatment of wastewater together with the pyrolysis process or subsequently in a wastewater treatment plant [132, 133].

Currently, several studies have been conducted for the treatment of aqueous phase and the application of its compounds as industrial raw material. Upgrading processes such as the hydrodeoxygenation and moderate catalytic cracking allow the production of hydrogen, hydrocarbons, alcohols and olefins from the aqueous phase [127, 130, 134].

Due to high amount of oxygenated compounds from C2 to C6 such as aldehydes, ketones, acids, and carbohydrates, several gasoline additives, alcohols and diols can be produced from the aqueous phase by hydrogenation processes [135, 136]. Light alkanes C1 to C6 and liquid alkanes C7 to C15 may also be produced from the aqueous phase carbohydrates through upgrading processes as dehydration and hydrogenation [137].

The sugars present in the aqueous phase (levoglucosan, pentoses and hexoses) are recognized as key compounds for the production of furan derivatives, such as furfural and 5‐hydroxymethylfurfural [126]. The conversion of sugars into furan derivatives can improve the economic perspective for using the aqueous phase as source of raw material for a wide variety of chemicals [124, 126].

The acidity of the aqueous phase, as already mentioned [127], can cause corrosion in equipments (based on carbon steel); however, these organic acids may also be valuable byproducts that can be used in industry as solvents and wood preservatives [124, 127].

### **2.6. Analytical techniques applied to bio-oil and aqueous phase**

The full chemical characterization of pyrolysis oils is very complex, mainly because they are- formed by the degradation of carbohydrates and lignin, that have an abundant content of water- and a great variety of organic functions associated a small amount of inorganic material [138].

The composition of bio‐oils can be divided into four distinct fractions: moderately polar monomers, detectables by GC (40%); polar monomers directly detectables by HPLC or by GC after derivatization (12%); water (28%); lignin and pyrolytic materials (20%) [139] not detectables by GC.

Gas chromatography is the most widely used technique in chemical analysis of bio‐oils and- other complex mixtures. Despite its wide application, the GC [7, 107, 110, 111] has some limitations when applied to mixtures with a great variety of compounds and different concentration- ranges. Co‐elution is a major impediment for the separation and unambiguous identification of a- compound. In this sense, there was developed the comprehensive two‐dimensional gas chromatography (GC × GC) which is being applied to this kind of sample [8, 15, 119, 140], which greatly- reduces the number of co‐elutions in a chromatogram, through two dimensions separations.-

Known since the 90s, the GC × GC is an analytical tool that differs from other techniques due to the sequential use of two chromatographic columns, which allows a significant increase in selectivity, favoring the structuring of the peaks in the chromatographic space. Regarding the dimensional gas chromatography, GC × GC shows most significant increase in the sensitivity and resolution, allowing a higher peak capacity, that is, a higher number of peaks separated and identified [141, 142].

Studies employing liquid chromatography for characterization of bio‐oils have also been recently undertaken [143–149]. In view of the complexity of the samples, the comprehensive two‐dimensional liquid chromatography (LC × LC) [150] in the same way as it happens for GC × GC becomes an important tool for characterizing bio‐oils, because there is a large increase in the resolving power when compared to one‐dimensional methods. Another great alternative is the use of micro HPLC columns that allow very small mobile phase flows, facilitating the coupling to more efficient detectors than conventional ones [151–153].

### **3. Gas chromatographic methods applied to the analysis of bio-oils**

### **3.1. One-dimensional gas chromatography (1D-GC)**

One‐dimensional gas chromatography (1D‐GC) coupled to mass spectrometry (GC/MS) or- flame ionization detector (GC‐FID) is an important analytical tool that provides chemical profile- information of bio-oil, aiming its correct destination as fuel or in the chemical industry. There are several studies in the literature about the use of 1D-GC to chemical characterization of bio-oils from pyrolysis of various biomasses [12, 154, 155]. The large Brazilian biodiversity contributes to many options of biomasses, significantly increasing the total number of identified chemical- compounds and the potential use of these materials. There are several studies using 1D-GC to analysis of bio‐oil from Brazilian biomasses such as straw and sugarcane bagasse (*Saccharum officinarum*) [38, 70, 156], eucalyptus sawdust (*Eucalyptus globulus*) [70, 156], Amazon tucumã (*Astrocaryum aculeatum*) [157], mangaba seed (*Hancornia speciosa*) [39], coconut fiber (*Cocos nucifera*) [34], fruit of palm (*Arecaceae*) and pine wood *(Pinus*) [158], among others. A large part of this work has employed the GC/MS to identify families and major compounds, usually containing oxygen in its constitution, as phenols, furans, alcohols, ketones, aldehydes, esters and carboxylic acids, regardless of the biomass used. As an illustration of this type of analysis, **Figure 2** 

shows the chromatogram of a sample of sugarcane straw bio‐oil using the GC/MS system with- DB‐5 column (60 m × 0.25 mm × 0.25 μm), developed in our laboratory. In this work, 208 and 336- compounds were detected in the analyzed bio‐oil sample directly and by SPME with PDMS- fiber, respectively. Among these, 33 and 35 compounds were identified in two cases.-

**Figure 2.** Total ion chromatogram (GC/MS) for bio‐oil (A) and SPME (B) of the pyrolysis of straw sugarcane. Chromatographic Conditions: DB‐5 column (60 m × 0.25 mm × 0.25 μm); oven temperature: initial oven temperature was 40°C, hold for 2 min, heating to 280°C at a 5°C/min, where it stayed for 2 min.-

In general, phenolics compounds are the most family in the majority of bio‐oils. Phenols are widely used in fine chemical industry, food processing, pharmaceutical and production of phenolics resins [34, 108, 158].

Hydrocarbons (saturated, unsaturated and aromatic) are also identified by GC/MS in most- bio‐oils; however, their percentage is very small compared to other components, with fewexceptions. In the study of Santos et al. [39] were identified various hydrocarbons (saturated,- unsaturated and aromatic) through GC/MS in the bio‐oil from pyrolysis of mangaba seed- (*Hancornia speciosa*). The total relative area of peaks which is one of the ways used to estimate the quantitative composition of bio-oil ranged from 8.1 to 13.8% of total compounds tentatively identified in this bio‐oil. In this same work, carboxylic acids were the family- of major compounds found (from 72.5 to 84.4%). Carboxylic acids, although lower quality becomes the bio-oil, are possible hydrocarbon precursors, enabling further study of the bio-oil upgrading.

Patel et al. [54] characterized by GC/MS the crude bio‐oil from sugarcane bagasse produced by fast pyrolysis in a fluidized bed and evaluated the efficiency of Mo<sup>2</sup> C/Al<sup>2</sup> O3 catalyst in the deoxygenation and quality of bio‐oil. The catalyst resulted in an increase in the content of phenolics and furans, which arouse great industrial interest.-

Spilã et al. [159] developed a method for separation and analysis of the aqueous fraction of bio‐oil by adding water to raw bio‐oil followed by extraction with ion exchange resin and ethyl ether. The ether soluble compounds were analyzed by GC/MS, since the insoluble fraction was evaporated and solubilized in methanol for analysis (GC/MS, CHN and pyrolysis‐ GC/MS). The characterization method was applied to bio‐oils derived from wood, scots pine and wheat straw. Further studies have applied the same separation method to different biomass such as forest waste [160] and wood [161] with subsequent chromatographic analysis (GC/MS and GC‐FID).-

Wiggers et al. [162] performed a study on pyrolysis of soybean oil at pilot scale, in continuous system aiming higher yield of bio-oil. The authors conducted a prior distillation to separate light bio‐oil fractions (LBO) and heavy bio‐oil fractions (HBO). Various hydrocarbons were found in bio‐oil and benzene, toluene, ethylbenzene, p‐xylene, o‐xylene and linear hydrocarbons (C7 to C12) being the majority for LBO; while for HBO the majority were fatty acids, toluene, ethylbenzene and linear hydrocarbons (C8 to C17).

Owing to the industrial importance of phenols and furfural, the author highlights the refining of the aqueous phase can extract these aromatic compounds of high value to the industry.-

GC/MS can be also coupled directly to one pyrolyzer system since it is required to just check the potential of the chosen biomass to generate bio-oil, since the amount of sample used in this case is very short not allowing further analysis with the bio‐oil produced. This technique is known as analytic pyrolysis and represented by Py‐GC/MS.-

### **3.2. Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS)**

Analytical pyrolysis (Py‐GC/MS) is an important characterization tool of the products generated by the pyrolysis of lignocellulosic material, in addition to allowing a better understanding of the function and effects of catalysts for the production of hydrocarbons or other desirable compounds prior to the pyrolysis in analytical laboratory scale. Direct analysis of condensable gases is held in a pyrolyzer coupled to a gas chromatograph with mass spectrometry detection (Py‐GC/MS). In the pyrolyzer, a small amount of biomass is subjected to a heat treatment and condensable gases are simultaneously separated and identified by their mass spectra and retention times [163–165].

The Py‐GC/MS have advantages, such as the use of small quantities of biomass, which allows the realization of several pyrolysis under various operating conditions (ratio catalyst/biomass, temperature, heating rate, etc.), in a simple and fast way [166, 167]. Through Py‐GC/- MS can also predict what kinds of industrial interest compounds are produced during the process, which the effect of catalysts, and if inhibition occurs, etc.-

Liaw et al. [165] studied the optimal pyrolysis temperature for two standards cellulose and two different types of wood by Py‐GC/MS, and yields variations were evaluated by PCA (principal component analysis). The PCA showed that pyrolysis products can be divided into groups and are strongly influenced by the nature of the raw material studied. However, the levoglucosan represents those few compounds that are influenced by temperature because as the temperature increases, its concentration decreases.

Barbosa et al. [168] developed method of determining the ratio between syringyl and guaiacyl content (S/G) in lignins from eucalyptus wood by Py‐GC/MS using different markers of syringyl and guaiacyl units. Traditional methods, which involve chemical degradation, generally are laborious, time consuming and require large sample amounts. However, the method developed by the authors is fast, uses very small amount of sample and highly sensitive.

The analysis of macauba fruit (*Acrocomia sclerocarpa M.*) [169] by Py‐GC/MS revealed the formation of alkanes, alkenes, dienes and cycloalkanes, being the main products the 2-propenal and acrolein (triglycerides derived). In the bio-oil from the oil pulp, most of the compounds found (71–79%) were aldehydes, cycloalkanes, alkenes and dienes. Through this method, it was possible to observe the differences in composition of biomass and its influence on the quality of bio-oil formed.

Py‐GC/MS enables understanding of formation of fatty acids obtained by pyrolysis of seed and babassu oil, studying their composition and fragmentation mechanisms of the compounds produced by thermal degradation of the samples [170].

The evaluation of the use of different catalysts (ZnO, CaO, ZnCl2 and MgCl<sup>2</sup> ) to obtain bio-oil from several biomasses [171–173] was also assessed by Py‐GC/MS.-

Despite numerous studies reported in the literature, 1D‐GC has limitations when applied to the characterization of complex sample, such as low resolution and co‐elution, which lead to incorrect identification of some analytes [112, 174, 175]. A wide range of chemical classes present in the matrix of bio‐oil also complicates the choice of chromatographic columns proper polarity at all [165]. Therefore, currently, most jobs that show a characterization of bio‐oil by one-dimensional chromatography also make use of the characterization by chromatography comprehensive two‐dimensional gas (GC × GC).-

#### **3.3. Comprehensive two‐dimensional gas chromatography (GC × GC)-**

The comprehensive two‐dimensional gas chromatography (GC × GC) is a multi‐dimensional chromatographic technique that emerged in the early 90s [176]. Due to its high separation power [177], GC × GC has been widely used use in the analyzes of pyrolytic bio‐oils, once these are complex mixtures and their composition can present more than 400 organic compounds belonging to a great number of distinct chemical classes. The bio-oil composition may vary according to the pyrolysis conditions and the kind of biomass chosen [178, 179].

This technique provides many advantages in relation to 1D-GC in the elucidation of the composition of complex samples [177]. Among these advantages one can be highlighted the increase in peak capacity, which leads to a better separation, not only between analytes, but also between them and the matrix. An increase in detectability, due to the narrow- chromatographic bands resulting from the modulation, may also be considered another advantage.

Furthermore, the technique GC × GC compared to conventional gas chromatography also provides an increase in sensitivity and a generation of structured diagrams that facilitate the identification of unknown compounds [180–183].

The two‐dimensional system consists of two chromatographic columns connected in series, one with a standard size (normally 30 or 60 m length and 0.25 mm internal diameter) and other shorter and with smaller diameter (∼2 m length and 0.15 mm i.d.). A column set commonly referred as conventional consists of a nonpolar column in the first dimension (<sup>1</sup> D) and a polar or with intermediate polarity in the second dimension (<sup>2</sup> D). The two columns have different separation mechanisms (orthogonal separation), that is, the first column performs separation of compounds according to molecular weight or boiling point and second column by polarity, providing a major breakthrough in the separation of complex samples [141, 177, 184–188].

**Figure 3** shows a GC × GC system where is possible to observe the modulator between both columns and some details of the peak processing. The modulators have the main function of to continuously collect fractions from the first column, re‐concentered them and to inject in the second one [190–192]. This procedure is responsible for the increase in the signal-to-noise and the decreasing in detection limits, if compared to 1D‐GC [188].

In the graphical representation of GC × GC, the register of the detector signal versus retention time is a continuous sequence of short chromatograms of each eluted fraction from the second column. After this, tridimensional graphics can be constructed considering the detector signal and the retention times in the first and second dimensions (<sup>1</sup> t R and 2 t R) [191, 192].

Data acquisition from 2D‐GC can be better explained viewing **Figure 3b**. In this figure, one co‐ eluted peak corresponding to three analytes) eluted from the first dimension pass through the modulator being fractionated and eluted from the second column. The crude chromatogram originated corresponds to the sum of all the chromatograms obtained on 2 D. The second step (**Figure 2c** is the transformation (by and adequate software) of these data in a two‐dimensional diagram (1 t R × <sup>2</sup> t <sup>R</sup>). The last step is the tri-dimensional visualization of the results (**Figure 2c**  that was also performed by adequate software [182].

There are two main kinds of commercial modulators: thermal and with valves [193–196]. The thermal modulators (as by heating or by freezing) present more utilization in GC × GC [193, 195]. The cryogenic modulators can use liquid nitrogen or liquid CO2 and act as a cold trap for the analytes [196].

 **Figure 3.** (a) GC × GC system; (b) three peaks co‐eluted in the first dimension (<sup>1</sup> D); (c) transformation of raw data into a two‐dimensional chromatogram (<sup>1</sup> t R × <sup>2</sup> t <sup>R</sup>); (d) reconstruction process of chromatographic peaks forming the color diagrams (two‐dimensional and three‐dimensional) [189].

GC × GC allows the utilization of several detectors, with few modifications to adapt them to the low volume and high velocity of data processing. One can be highlight the flame ionization detector (FID), time‐of‐flight mass spectrometry (TOF‐MS) and quadrupole mass spectrometry (qMS) as the more used [197–203].

FID has been associated to GC × GC since the first works found in the literature, including the initial researches in the bio‐oil characterization [204–207].

GC × GC promotes high speed separations, providing very narrow peaks, requiring detectors with equally rapid acquisition rates to get a sufficient number of points per peak [208, 209] to permit quantitation. The detector scan should be short and its internal volume has to be small [194]. The high acquisition rate of the FID (up to 200 Hz) [210] allied to a good response for almost all organic compounds and its good performance in quantitative analysis justifies the wide use of this detector in two‐dimensional chromatographic analysis. The main difficulty in the FID employment is that it does not provide structural information about the separate peaks. Thus, detectors with mass analyzers gain space for identification and confirmation of the separate compounds.

The TOFMS is particularly effective for GC × GC, since it presents acquisition rates between- 50 and 500 Hz [180, 182, 197–203], making it the preferred choice among researchers for these studies. The coupling of separation GC × GC with efficient detection TOFMS has anadditional advantage, which is its higher sensitivity than the full scan mode over conventional mass spectrometry detectors with quadrupole analyzer (qMS). Consequently,- TOFMS outperforms the other detectors in qualitative and quantitative analysis. The disadvantages of systems of this type analyzer are its relatively high cost, the need for proper training and specific operating conditions for daily operation, especially due to its high- sensitivity [191].

The GC × GC‐qMS system is also showing its potential to analyze complex samples [211–213]. Initially, quadrupole mass spectrometers showed very low data acquisition rates (up to- 20 Hz) which made them too slow to use in GC × GC systems [214]. However, decreasing the- mass range investigated or monitoring only a few ions during the run, it is possible to obtain- a higher scan rate [191]. This has been the subject of research of several research groups [180, 208, 214, 215], that is, to develop quadrupole mass analyzers faster and comparable to TOFMS.- From this, it was introduced on the market a fast quadrupole system, which allows achieving- data acquisition rates above 50 Hz allowing their use coupled to GC × GC system [209, 216].

In recent years, it is possible to find a lot of research in the literature applying GC × GC in the analysis of the chemical composition of bio-oils.

Among the initial studies of bio‐oils through GC × GC, it can be highlighted the Works- of Marsman [205–207] and Sfetsas [174]. Marsman et al. [206, 207] evaluated the compounds presented in the bio‐oil from beech using GC × GC with FID and TOFMS detectors.- Authors used GC × GC‐FID and GC × GC/TOFMS for the identification of approximately- 248 and 368 compounds, respectively, with concentration higher than 0.3% in beech- (*Fagussylvatica*) hydrodesoxygenated (HDO). In these studies, it was also made a classification for these compounds, according to their chemical class into nine groups (acids,- aldehydes, ketones, furans, guaiacols, syringols, sugars, alkyl phenols, alkyl-benzenes). The major compounds found in beech bio‐oil were levoglucosan, hydroxymethyl furfural, furanone, furfural, mequinol and butanediol. Similarly, Sfetsas et al. [174] used the GC × GC technique for analyzing three oils pyrolysis, in which were tentatively identified, approximately 96 compounds with concentration higher than 0.3%, classified in acids,- esters, aldehydes and ketones, hydrocarbons, aromatic hydrocarbons, phenols, sugars and other compounds not classified. Acetic acid, levoglucosan and hydroxy‐propanone were- the majority compounds.

Bio-oils derived from the fast pyrolysis of several Brazilian residual biomasses as orange bagasse [217], peach core [15, 189], rice husk [15] and sugarcane straw [8] were recently characterized by GC × GC. The analysis of bio‐oil from orange bagasse [15], without any pretreatment, by GC × GC/FID and GC × GC/TOFMS was compared. The last one showed better results and 167 compounds were identified, belonging to acids, aldehydes, ketones, phenols, esters, ethers and some nitrogen‐compounds. From these, 26 compounds appeared in concentration above 1%.

Bio‐oils from peach core and rice husk analyzed by GC × GC/TOFMS in a conventional set of columns showed the presence of ketones, phenols, alcohols, ethers, acids, aldehydes sugar derivatives and hydrocarbons, with around 500 peaks in each sample [15].

Studying the bio‐oil from peach core, by comparison with 1D‐GC/qMS and GC × GC/TOFMS, Migliorini et al. [189] observed the superiority of the multidimensional technique. Another observation was the spatial structuration of the GC × GC color diagram, which allowed the identification of all the isomers of C1 to C4 alkyl phenols.-

This group of researchers also applies another tool for the identification: the dispersion graphics (DG). These graphics, constructed using ExcelTMsoftware clarifying the distribution of compounds and allow to preview the presence of others homologues in a series of compounds, like alkyl substitutes in phenols or in aromatic hydrocarbons. **Figure 4** shows examples of DG for the separation of alkyl phenols. The results from GC/MS and GC × GC/- TOFMS showed, for the same sample, 51 and 220 components, respectively. The chemical classes found were alcohols, aldehydes, anhydrides, ketones, esters, ethers and phenols and were observed by both techniques employed. However, using GC × GC were also found carboxylic acids, hydrocarbons and sugar derivatives such as levoglucosan [189].

The analysis of the aqueous extract of peach core pyrolysis is illustrated in **Figure 5**. This figure has been an example of the spatial distribution of the constituents in a GC × GC/- TOFMS (**Figure 5A**) and an illustration of separation capacity offered by the second dimension (**Figure 5B**). In this last one, it is observed a separation of four peaks that co-eluted in the first dimension and is adequately separated in the second one, giving mass spectra of- high purity.

 **Figure 4.** Dispersion graphic of the separation of phenols in the bio‐oil from pyrolysis of peach core. Legend: Cx represent the side alkyl chain on the aromatic main chain of the phenols were x is equal to the number of carbon in the side chain. Based on Ref. [91].

Chromatographic Methods Applied to the Characterization of Bio-Oil from the Pyrolysis of Agro-Industrial Biomasses 91 http://dx.doi.org/10.5772/66326

 **Figure 5.** (A) Color two‐dimensional diagram for analysis by GC × GC/TOFMS of the organic extract of aqueous fraction of the pyrolysis of peach core. (B) Example of chromatographic separation by GC × GC using the total ion current chromatogram (TIC) and the extracted ion chromatogram (EIC) for the following compounds (a) C2‐cyclopentenone, (b) benzene acetaldehyde, (c) ethyl furandione and (d) ethyl furanone and their respective mass spectra. Chromatographic conditions: column set: DB5 (60 m × 0.25 mm × 0.25 μm) and DB17 (1.94 m × 0.18 mm × 0.18 μm); heating ramp: 40.0°C (5.0 min)–3°C/min–270°C.-

In the study of fast pyrolysis of sugarcane straw, Moraes and coworkers [8] used GC × GC/- TOFMS, infrared spectroscopy with Fourier transform (FTIR) and scanning electron microscopy- (SEM) to fully characterizing the products from the pyrolysis of sugarcane straw. The results of- the analysis of bio‐oils have demonstrated efficiency in the combination of techniques, especially,- GC × GC/TOFMS, showed the presence of 123 compounds belonging, mostly, to the aldehydes- and carboxylic acids. Maciel et al. [218] also studied the fast pyrolysis of sugarcane straw by- GC × GC/TOFMS, but researching the aqueous phase of this process. They found that this phase is- very similar to the bio-oil but enriched more soluble phenols, such as *ortho, meta* and *para* cresols.

The technique using GC × GC with detector quadrupole mass detector (qMS) is growing and- showing the efficiency of this detector coupled to two‐dimensional gas chromatography. In- work carried out by Da Cunha et al. [119] and Schneider et al. [219], using a set of conventional speakers have shown the potential of the technique to evaluate the straw pyrolysis product of- sugar cane and forest wood sawdust (lignocel). The bio‐oil from pyrolysis of sugarcane straw- was fractionated on a silica column with pressurized liquid, being separated hydrocarbons of- other polar fractions. In this sample, 166 compounds was to find including carboxylic acids, aldehydes, ketones, esters, phenols, ethers, alcohols and sugar derivatives in the polar fraction, and, the nonpolar fraction, were formed from aromatic, aliphatic, cyclic and olefinic hydrocarbons- [119]. The polar compounds of the bio‐oil from lignocel sample (forestry wood sawdust) [219] were extracted with alkaline solution before chromatographic analysis, and 130 compounds- were identified by GC × GC/qMS among phenols, ethers, ketones, aldehydes, carboxylic acids,- alcohols and aromatic hydrocarbons. This analysis is illustrated in **Figure 6** demonstrating the quality of data generated using a mass spectrometry detector with quadrupole analyzer [119].

 **Figure 6.** Color diagram (GC × GC/qMS) of the bio‐oil from sugarcane straw. Chromatographic conditions: set of columns: OV–5 (60 m × 0.25 mm × 0.25 μm) and DB‐17ms (2.15 m × 0.18 mm × 0.18 μm). Heating ramp: 40°C–3°C/min to 120°C, then 2°C/min to 200°C [119].

In recent years, the literature has been publishing a wide range of papers in the pyrolysis of many biomasseswith lignocellulosic or residual origin. Eucalyptus [220], mango seed [35], coconut fiber [221], residual cake of crambe seed [37] and castor seed [222], waste of forest industry [36], fruits of palm and pine wood chips [223], sugarcane bagasse [61] sugarcane straw [119] and forest wood sawdust (lignocel) [219] were some of these. These biomasses were submitted to different kinds of pyrolysis (slow [220, 222], fast [35, 37, 177, 221, 223], intermediate [36] and catalyst [34, 218]).

Many other analytical methods can be used to improve the quality of bio‐oil and to facilitate its characterization. In this context, extraction methods have been used to isolated fractions and better analyze their components. Some examples of application of extraction techniques are solid phase micro extraction (SPME) [220], liquid‐liquid extraction (LLE) [35, 219, 221], mechanical press extraction [37], soxhlet extraction [37], pressurized fluid extraction (PFE) [37] and pressurized liquid fractionation (PLF) [119] among others.

The use of another analytical method beside GC or GC × GC is also a good choice for a more complete characterization of these products. According to reports in the literature GC × GC, especially with TOFMS detector, has been widely used in association with other techniques like GC/qMS [36, 221], FTIR [35], FT‐ICR MS [223] and 1H NMR [46] for determining the composition of the biomass pyrolysis products.

The results of the characterization of bio‐oil only by GC × GC and the association thereof with other techniques show that different classes of chemical compounds form these samples. The differences in the pyrolysis conditions [14, 224] or in the chemical composition of the biomass can influence the constitution of the composition of bio‐oils [178]. The main compounds belonging to the chemical class of phenols, esters, ethers, acids, aldehydes, ketones, alcohols, hydrocarbons (saturates, olefinics and aromatics), sugar derivatives, N‐compounds (nitriles, anilines, quinolines, pyridines, indoles, pyrazines, pyrroles, carbazoles and acridines) and sulfur compounds (disulfides and thiophenes) [35–37, 46, 119, 218–223].

As with any other chemical analysis, no single technique is sufficient for complete identification of bio‐oil samples. However, the GC × GC has demonstrated its high potential for use combined with other techniques such as infrared spectroscopy, elemental analysis and liquid chromatography with the use of mass detectors among others. The development of rapid chromatographic processes (columns in micro scale) and multidimensional systems (especially comprehensive) allow full characterization of the samples for both constituents in greater proportion as for those at trace levels.

### **4. Liquid chromatographic methods applied to the analysis of bio-oils**

Liquid chromatography (LC) techniques are important tools for the separation and identification of compounds present in bio-oil fractions that are not analyzable by gas chromatography (GC). The high performance liquid chromatography (HPLC) analysis is widely used in different types of samples mainly polar and thermally labile compounds [155]. The main advantage of the LC techniques is the possibility of direct injection of aqueous phases obtained without extraction and sample preparation step. Studies employing such techniques for the characterization of bio-oils and aqueous phases have been recently reported in the literature and are briefly summarized in this chapter.-

Similarly as for the GC, the development of LC has been, especially, toward miniaturization and improved chromatographic resolution. Then, one can classify the liquid chromatographic methods in one‐dimensional LC and two‐dimensional LC.-

### **4.1. One-dimension high performance liquid chromatography**

The system can use many detectors, but, the main used for pyrolysis products are UV, RID and MS.-

HPLC‐UV uses a simple UV detector or diode array detectors (DAD detector or more specifically HPLC PDA detector) especially in the determination of polar compounds containing carbonyl, carboxyl and hydroxyl groups in aqueous samples derived from pyrolysis. The identification of aldehydes in biomass derivatives is the main application of this technique because these compounds are very soluble in water and the recovery of their extraction using conventional techniques such as liquid‐liquid extraction or solid phase extraction is very low.-

Successful applications of HPLC‐UV in the identification of furfural and hydroxymethyl‐ furfural (HMF) in the aqueous phase of the bio‐oil obtained by pyrolysis of agroindustrial biomasses have been described recently [126, 143, 225]. In some cases [225], aldehydes were confirmed and quantified in bio‐oil using a GC/MS system.-

*The* HPLC‐RID is considered standard for analysis of sugars in aqueous samples. The RID detector is used for detection of compounds that do not absorb in the UV or visible because it is based on measurement of the difference in refractive index between the pure mobile phase and the eluent coming out of the column containing the sample components. As some biomasses (like sugarcane bagasse and sugarcane straw) contain a great amount of sugars, this technique can be important in the characterization of compounds formed during the pyrolysis of these biomasses.

The HPLC‐RID has been used, normally, as a complementary technique The bio‐oil obtained from red oak fast pyrolysis in a fluidized bed reactor was characterized and sugar derivatives were identified in the water‐soluble fraction [148]. In this work, levoglucosan, maltosan celobiosan, xylose and cellobiose were quantified. Johnston and Brown [147] also analyzed glucose and xylose in switchgrass bio‐oil samples using HPLC‐RID.-

HPLC‐MS is a technique for the analysis of polar or thermolabile fractions not analyzable by GC on samples of bio‐oil and water fraction derived from pyrolysis of biomass. The main difference between the MS in a GC and the MS in a LC system is that LC‐MS performs the ionization of analytes in atmospheric pressure (API) with a low‐energy fragmentation ("soft"), allowing identification of the molecular ion. The fragmentation can only be done for selected ions and there is no library for identification of compounds. The analyzer must use standard compounds and has to study the entire fragmentation pattern for each peak. HPLC‐MS technique is compatible for volatiles and nonvolatiles in a wide range of polarity [226, 227].

On Py‐HPLC‐MS (online system of pyrolysis and liquid chromatography‐mass spectrometry) was recently developed [228] and applied to the analysis of lignin isolated from forest waste sample. In MS, compounds were detected with molecular masses in the range of 250–500 Daltons. The major compounds identified were syringol and resorcinol derivatives.-

A method based on the pyrolysis online with HPLC‐UV was developed for analyzing the bio‐oil derived from polymers [229]. The bio‐oil was fractionated and the resulting fractions were analyzed by mass spectrometry ionization and laser desorption matrix assisted (MALDI‐MS) system and finally HPLC‐MS using electrospray ionization (ESI). This system is mainly appropriate for the measurements of oligomeric products, being tested in polymer samples forming less volatile pyrolyzates such as poly(butylene terephthalate) and poly(2, 6 dimethyl‐1, 4‐phenylene ether). Another utility of this method was in the characterization of the cross-linking sequences in some polymeric resins.

HPLC‐MS technique in combination with GC‐FID and GC/MS was used for characterization of bio‐oils from different forest residues [149], showing a wide range of compounds with masses of between 100 and 400 Da. The major compounds identified in all bio‐oils were cyclohexane carboxylic acid, 1, 2, 4‐trimethoxy benzene and 2, 6‐dimethyl phenol.-

#### **4.2. Two‐dimensional LC (heart‐cutting (LC‐LC)) and comprehensive two‐dimensional liquid chromatography (LC × LC)-**

Two‐dimensional liquid chromatography techniques involve two distinct separations, which can be classified as heart‐cutting (LC‐LC) or comprehensive (LC × LC). In heart‐cutting 2D‐ LC, only relevant parts of the effluent, containing the target compounds, are directed to the second dimension. The main applications of LC‐LC are the analytes purification, improvement of the separation efficiency and the sensitivity of analysis [150]. Bio-oil obtained from pyrolysis of pine sawdust was analyzed by LC‐LC after the gel permeation chromatography (which made a lean up of the high molecular weight lignin derivatives) allowing the separation of phenolic fraction [144]. The results were compared with earlier analyzes by GC/MS. Among the phenols quantified in this work, the major compounds were guaiacol, vanillin, o‐cresol e catechol. LC‐LC technique proved to be a faster analysis, with a minimal sample preparation and with less loss of analytes than GC/MS.-

Similarly to GC, LC techniques with a higher resolution power are also required due to the complexity of the bio oil and aqueous phase samples, and therefore the comprehensive two‐dimensional liquid chromatography (LC × LC) becomes also important for the characterization of this kind of sample. This technique involves the coupling of two independent mechanisms of separation, through a high‐pressure switching valve, and it is able to provide a complete separation of the whole sample, since all fractions eluting from the first dimension are subjected to a second separation [150, 230–239]. The use of LC × LC solves co‐eluting problems due to increased peak capacity, which results in a larger number of identified compounds. This technique represents a great improvement in the analysis of organic samples mainly due to enhanced of the separation power and the resolution. Carr and Stoll [239] wrote an excellent chapter, edited by Agilent, with theory, instrumental and applications of 2D‐LC.-

Le Masle et al. [151] used LC × LC with detection by photodiode array (PDA) for the separation of compounds from the aqueous phase formed during the pyrolysis of oak. Using a standard solution with 38 compounds (phenols, acids, ketones, aldehydes, alcohols and furans), the authors developed a separation method, evaluating the peak capacity and the orthogonality of different sets of columns. However, the compounds in the aqueous phase samples have not been identified, since a more informative detector as MS would be required for this. This work was after compared with the LC × SFC technique (on‐line comprehensive liquid chromatography × supercritical fluid chromatography) for the analysis of the aqueous phase samples by the same researchers, with the aim of evaluating the two‐dimensional system. The new method showed a larger peak capacity in comparison with the previous method [152].

Tomasini et al. [153] described a method for the characterization of aqueous phases from bio‐oils of coconut fiber, sugarcane straw and sugarcane bagasse using comprehensive two‐ dimensional liquid chromatography with detection by diode array followed by mass spectrometry with atmospheric pressure chemical ionization. Using this system, it was identified- 26 compounds belonging to the classes of phenols, ketones, furans and alcohols. Phenol and- 2‐hydroxy‐3‐methyl‐2‐cyclopenten‐1‐one were found in greater abundance for all samples.- Belonging to furans, the furfural was detected in higher concentrations in the aqueous phases- of coconut fiber and sugarcane bagasse and the 1‐(2‐furanyl)‐ethanone was detected in higher- concentrations in the aqueous phase of the sugarcane straw. Belonging to alcohols, the phenyl- propanol was detected in higher concentrations in the aqueous phase of coconut fiber, while for- samples of sugarcane (straw and bagasse), the coniferyl alcohol had the highest concentration.-

### **4.3. NanoLC**

Among the innovations that LC has been showing in last years, it stands out techniques that- substantially reduce the volume of solvent employed [240, 241]. The NanoLC is one of these- innovations. It consists in the use of a column with micro‐dimensions and low solvent flow,- producing separations in short time but with high performance [242]. As the number of columns manufactured for LC techniques using micro to nano flows is very small (compared to- conventional HPLC), there is a limited number of studies described in the literature on this- subject [242, 243]. However, due to the positive results obtained, the NanoLC has been successfully applied in many fields such as biomedical, pharmaceutical, agrochemical and food [244].

The low flow used due to the nano‐dimensions allows better coupling to mass detectors, including those with electron impact ionization, normally used for GC [153, 244]. In the case of coupling a NanoLC with a mass spectrometer by electron impact (EI‐MS), there is the advantage of compounds identification be performed by direct comparison with mass spectral libraries [153, 244, 245].

As a complementary part to the analysis by two‐dimensional liquid chromatography of aqueous phases obtained from pyrolysis from Brazilian biomasses, Tomasini et al. [153] applied the NanoLC‐EI‐MS to confirm the identification of compounds through the mass available libraries in the same samples before cited. The analysis showed a similar composition, with compounds belonging to the classes of ketones, phenols, and furans. The aqueous phase (AP) from coconut fiber presented the phenol as major compound, while the AP from sugarcane bagasse presented a higher amount of furfural and AP from sugarcane straw presented a lower concentration of almost all the compounds.-

The use of a mass spectrometry detector with ionization by electron impact (EI‐MS) shows an enrichment of information for characterization of the samples, since the obtained mass spectra can be identified by comparison to spectra available in libraries of software used. In addition to the advantages presented by the detection of NanoLC system, it is important to emphasize the "green chemistry" approach due to the reduced volume of solvent used in the analysis of this technique.

In general, the LC is necessary for the characterization of polar compounds remain in the aqueous phase, which normally would be discarded. This discard would cause environmental and economic damage, since the identified compounds can have some kind of industrial application. Furthermore, the injection of aqueous samples is not possible without a prior step of extraction, which may have different yields due to the presence of compounds belonging to different chemical classes or can result in contamination of the sample, besides the higher use of materials and longer time.

Although there is a still reduced number of works applying LC for the analysis of bio‐oil compared to GC, it should be considered the importance of using new analytical techniques for a complete characterization of bio-oils and aqueous phases from pyrolysis of biomass.

### **5. Conclusions**

The initially suggested use for bio‐oil was as an alternative biofuel to diesel and petroleum. However, this route has proved to be nonviable, due to the high oxygen content of bio‐oils and operating cost of deoxygenation. Currently, the major studies indicate the use of bio‐oil in the chemical industry, particularly the chemistry of phenols, furfural and levoglucosan. The bio‐oil can be considered an alternative for crude oil for fine chemical industry.-

For proper use of bio‐oil in the chemical industry, it is essential the identification and unambiguous determination of its major constituents. Only then, it is possible to propose a recovery route of some of these components for the development of an industry dedicated to a bio‐refinery. For this, chromatographic methods, especially GC × GC/MS, are fundamental because they- allow analysis with high sensitivity and accuracy in identifying each constituent of the bio‐oil.-

### **Acknowledgements**

Authors would like to thank to CNPq, CAPES, FINEP and Petrobras for the financial support to this work through scholarships, grants and direct funding.-

### **Author details**

Maria Silvana A. Moraes1, 2, Débora Tomasini1 , Juliana M. da Silva1, 3, Maria Elisabete Machado1, 4, Laíza C. Krause4, 5, Claudia A. Zini1, 5, Rosângela A. Jacques1, 5 and Elina B. Caramão1, 4, 5\*

\*Address all correspondence to: elina@ufrgs.br-

1 University of Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brazil-

2 Pampa University, Rio Grande do Sul, Brazil-

3 Liberato Salzano Vieira da Cunha, Federal Technical School, Novo Hamburgo, Rio Grande do Sul, Brazil-

4 Tiradentes University, Aracaju, Sergipe, Brazil-

5 INCT‐E&A, Brazil-

### **References**


## **Perceptions on Internal and External Factors Impacting the U.S. Nonfood Advanced Biofuel Industry**

Henry Jose Quesada‐Pineda, Jeremy Withers and Robert Smith

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64611

#### **Abstract-**

 Thegoalofthischapteristointroduceanddiscussinternalandexternalbarriers- impactingthenonfoodadvancedbiofuelindustryinthe-United-States.-Since-2005when- the-EPActwascreated,-59cellulosicbiofuelprojectshavebeenattemptedinthe-U.S.- withlittlecommercialsuccess.-Aninitiallistofinternalandexternalbarrierswas- extractedfromsecondarysourcesusingqualitativeanalysistechniquessuchas- groundedtheory.-Oncethelistwasvalidated,asurveywassenttothebiofuelindustry- memberstogainmoreknowledgeandclarificationontheinitiallistofbarriers.- Statisticalanalysisrevealeddifferencesinperceptionsfromindustrymemberswhen- barrierswerecomparedbyprojectstatus,technology,andtypeofproject.-Inaddition,- barriersformarketinganddistributionofadvancedbiofuel'scoproductsandby‐ productswereidentifiedandrankedbyindustrymembers,academicians,andother- stakeholders.-

**Keywords:** biofuel, cellulosic biofuel, internal and external barriers, coproducts, by‐ products-

### **1. Introduction-**

 Thedevelopmentofanenvironmentalbioeconomyisnecessaryinthe-U.S.toreducefossilfuel- energy dependency. The term energy is classified into three main categories: fossil, nuclear, and- renewable.-Themainfossilfuelsarepetroleum,coal,naturalgas,andnuclearmaterial.-They- arecurrentlynonrenewableandcontributetotheaccumulationofgreenhousegases-(GHGs),- oneofthecausesofclimatechange.-Fossilfuels,namely,petroleumfortransportationfuel,are-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 beingconsumedatanincreasingratefromdiminishingfinitereserves.-Onemodelestimates- that,atthecurrentusagerates,fossilfuelreservesofoil,coal,andgaswilllastapproximately- 35,107,and137years,respectively[1].Otherresearchershaveestimatedthatfossilfueldepletion- willoccurbetweentheyears-2100and-2200-[2].-

There are three primary methods to create liquid advanced biofuel (AB) and its coproducts:- direct microbial conversion (DMC‐biochemical), simultaneous saccharificationand fermenta‐ tion (SSF‐thermochemical), or a hybrid of these techniques [3]. These two main approaches are- further broken down into six secondary options for developing cellulosic biofuel: (1) catalytic- pyrolysis and hydrotreating to hydrocarbons; (2) gasificationand Fischer‐Tropsch synthesis- to hydrocarbons; (3) gasificationand methanol‐to‐gasoline synthesis; (4) dilute acid hydroly‐ sis, fermentation to acetic acid, and chemical synthesis to ethanol; (5) enzymatic hydrolysis to- ethanol; and (6) consolidated bioprocessing (single‐step enzyme production, hydrolysis, and- fermentation) to biofuel [3].-

Liquid biofuel is one such renewable energy source. Biofuel is a fuel additive capable of- increasing octane levels by blending it into the U.S. fuel supply, or can be used as a fuel in- internal combustion engines [4]. The total renewable biofuel sector is currently diversifiedinto- first-(1G)‐, second (2G)‐, and third (3G)‐generation lignocellulosic biomass forms of energy.- For example, 1G is derived from corn and sugarcane, 2G advanced biofuel is derived from- wood, grasses, municipal wastes, and crop residues, and 3G is derived from algae. Biomass is- considered as living or nonliving agricultural vegetation such as wood and grass crops. In this- case, biomass is typically differentiatedby dedicated wood and grass energy crops, and- unmerchantable timber and forest waste. Lignocellulosic feedstock's price currently ranges- from \$50 to 80/ton of biomass [5]. These feedstocks could be from unmerchantable timber,- forest thinnings (slash), sawdust, waste paper, mill residues, paper mill sludge, grasses, and- grass variety residues. All biomass feedstock differsin moisture content and may have- differentcosts. Dedicated energy crops are considered for energy use only. In this study,- dedicated energy crops are categorized and differentiatedas herbaceous crops (grasses) and- wood‐based crops. Herbaceous grass crops are harvested annually, with only the roots- surviving the nongrowth cold seasons (e.g., switchgrass, *Miscanthus*). Wood‐based crops,- including fast‐growing trees such as poplar, are harvested on a 3‐ to 12‐year rotation cycle;- harvest rotation cycles for slower growing trees may be as long as 25 years.-

For this study, nonfood lignocellulosic biomass consisted specificallyof biomass from wood- and from grass varieties for the current purpose of substituting fossil petroleum‐based fuels- with renewable biofuel. Advanced biofuel is a contemporary liquid fuel for transportation- produced primarily from cellulose and hemicellulose of renewable lignocellulosic biomass. It- is derived from lignocellulose, which consists of three major components: cellulose, hemicel‐ lulose, and lignin. The cellulose and hemicellulose portions are the desired components for- producing the highest value‐added biofuel coproducts. Lignocellulosic biofuel currently has- the greatest potential for energy, being the most abundant and rapidly renewable resource- produced by photosynthesis [6]. The lignin portion typically becomes a process by‐product,- but recently was considered a coproduct when blended as filler for wood products.-

This study presents results on an investigation conducted between 2014 and 2016 related to- the status of AB projects in the U.S. market. It was found that the majority of AB projects never- achieved a commercialization stage. Therefore, the research team was interested in learning- more about the barriers and factors that have prevented the AB industry to reach commercial- state, including impact not only on the AB production itself but also in coproducts and by‐ products of the AB industry.-

#### **2. Factors affecting the advanced biofuel industry-**

#### **2.1. Biofuel policy-**

There are a multitude of government policies using a push‐type strategy to bring the bioecon‐ omy technology to the marketplace. The Environmental Protection Agency (EPA), U.S.- Department of Agriculture (USDA), Energy Information Administration (EIA), Department- of Energy (DOE), and Department of Defense (DOD) have jointly developed these policies to- drive the bioeconomy. According to Reidy [7], the major goals and policy incentive's objectives- driving the bioeconomy marketplace are the following:-

#### **1.-To reduce GHG emissions and sequester carbon-**


#### **2.- To achieve greater energy efficiency-**

	- **•-**Transportation fuel and biofuels: Rural Energy for America Program (REAP)-
	- **•-**Federal Transit Administration (FTA) Clean Fuels (DOT Grants)-
	- **•-**Clean coal‐to‐liquid or gaseous fuel technologies grant program (NSF Grants)-

Six main policies were created in the United States to bolster, develop, and implement the four- incentives driving the bioeconomy. Sequentially, they are: (1) Clean Air Act 1970—throughcurrent amendments [8], (2) Energy Policy Act of 2005 (EPAct) [9, 10], (3) Advanced Energy- Initiative 2006 [11], (4) Renewable Fuels Standards (RFS) of Energy Independence and Security- Act of 2007 (EISA) [12–14]), (5) California Low Carbon Fuel Standard (LCFS) [15], and (6) Food,- Conservation, and Energy Act of 2008 [16].-

As of 2015, there were six policies driving the inception of advanced biofuels, and EISA carried- the most focus toward developing biofuel projects while removing market share from the fossil- industry. There are a host of incentives for industry development of advanced biofuels (AB),- such as the 2005 EPAct creating the Renewable Fuel Standard, and its modification with 2007- EISA, and new components of RFS2: Renewable Volume Obligations (RVO), Renewable- Identification-Number (RIN), and Code of Federal Regulations (CFR). These policies provided- production tax credits and research and development (R+D) funding to promote the RFS- concept of replacing 35 billion gallons of fossil fuel with drop‐in biofuel blends. The policy- subsidies and incentives were the drivers leading to advanced biofuel (AB) project attempts- from 2005 to 2015.-

Biofuel projects are divided into three generations by feedstock type: firstgeneration is ethanol- —corn and sugarcane; second generation (2G) is advanced biofuel—wood, grass, and crop- residues; and third generation (3G) is algae and butanol. Those feedstocks are in the \$50–80 p/- ton range. This chapter is focused on 2G wood and grass advanced biofuel. Wood and grass- feedstock (lignocellulose) is typically separated by its major components in order of value:- cellulose, hemicellulose, and lignin.-

#### **2.2. Advanced biofuel project status-**

The U.S. total renewable biofuels (TRFs) projects are classifiedas pilot with costs ranging \$9- million or less, demonstration project costs ranging \$100 million or less, or commercial projects- costs ranging \$100–500 million [17–19]. These three project types are further divided into five- operational status categories: cancelled, shutdown, under construction, planning, and- operating. Cancelled projects are considered terminal. Shutdown projects were stopped and- put on hold, but potentially could be restarted at a later time. Under construction projects are- currently being built, and planning projects are in the research and development phase, prior- to construction. For operating projects, construction was completed and attemptsat biofuel- production have begun. References [18] and [19] provided the only accessible publication- covering a large portion of wood‐based biofuel projects, separated by location, type, and status,- from their Forisk‐Wood Bioenergy U.S. (WBUS) database. They indicated 36 cancelled projects,- 4 shutdown projects, and 12 projects in planning or construction stages, stating that 75% have- failed to advance [18, 19].-

 Currently,fewadvancedbiofuelprojectsareproducingbiofuel,withnonereachingsustain‐ ablecommercialproductioneconomiesofscalewherebiofuelprojectsizetoproduce- commercial‐levelbiofuelwasgreaterthancosts.-Somedocumentsintheliteratureidentified- barriers,buttheauthorsonlyfocusedonbroadcategories.-Themostinclusivedocuments- providedapartiallistofwood‐basedbiofuelprojectsbytypeandstatus-[18,-19].-Inexamining- literatureonbarrierstoadvancedbiofuelprojects,thefollowing-10mainbarrierswere- determined:-(1)highcapitalrisks,-(2)-Organizationofthe-Petroleum-Exporting-Countries-

 (OPEC)‐basedpricedistortions,-(3)constrainedblendingmarkets,-(4)policyfluctuations,-(5)- financing,-(6)productioncosts,-(7)globalfinancialsituation,-(8)economichurdles,-(9)- efficiency,effectiveness,andscalingtechnology,and-(10)toomanytechnologypaths.-

### **2.3. Factors impacting the advanced biofuel industry-**

Prior to 2005 EPAct, the corn ethanol industry was preestablished to close in 40 years, moving- away from utilizing government subsidizes and close to achieving commercial production- economies of scale. This subsidized preestablishment was the firstbarrier to advanced biofuel- and 3G biofuel technologies. The EPAct led to a second barrier: different subsidy and expect‐ ation levels among the renewable fuel types. The EPAct created the RFS that forced the fossil- fuel industry to relinquish approximately 10% yearly of the production output over the next- 17 years until 2022. This created another barrier: a line drawn in the sand between OPEC‐ backed fossil fuel companies and government support of the emerging bioeconomy. Addi‐ tionally, methyl tertiary butyl ether (MTBE) was increasingly being banned for environmental- and health‐related concerns, but fossil fuel companies needed the MTBE to increase the octane- content of diesel and gasoline. MTBE was able to be transported in fossil fuel's current- infrastructure, but biofuel has to be transported separately to the refineryand was more- expensive. This was a third blow to the fossil fuel industry: reduction of their monopoly with- market share percentage loss over time, MTBE could become banned with potential lawsuits,- and unable to maximize delivery economies of scale without expensive upgrades to infra‐ structure for ethanol. These led to initial fossil infrastructure upgrades and supporting biofuel- as a lubricant and octane enhancer with the 2005 EPAct.-

The 2007 Energy Independence and Security Act and its modified-RFS (EISA‐RFS2) brought- more specificity, policy incentive type drivers, and, subsequently, more barriers. The fossil fuel- industry opposed the new RFS‐2 and, to date, mounts continual media attacksto repeal the- RFS. By 2007, the steady decline of fossil fuel consumption should have triggered more concern- with the near‐term potential for constrained blending markets. In 2012, the blend wall arrived;- the advanced biofuel projects saturated market demand, with nowhere to put their fuel for- blending above their mandate since D6 (RIN code for renewable fuel based on corn ethanol)- by itself was fillingmore fuel capacity than available. The blend wall led to the next major- barrier: political involvement in an attemptto create demand. The government was forced to- balance the fallout of subsidizing and building an industry with diminishing room to put their- products as they strive to meet mandated production economies of scale.-

Lack of infrastructure and lack of factual knowledge are the main barriers to the public not- having enough flexfuel vehicles and ethanol pumps to maintain low gas prices. The main- barrier to all groups is time. Transportation fuel stations are willing to upgrade infrastructure- [20] when the vehicles have upgraded technology. Republicans will not budge until the- demand increases. Democrats cannot increase the infrastructure demand until they have- control of the House and Senate. The vehicle demand will not increase until the vehicle- infrastructure for higher blends is affordable.-Advanced biofuel projects will have to receive- subsidies until that happens. The public would not support another tax (i.e., carbon tax), whilepetroleum and gas prices are low [21]. Therefore, time is the overarching barrier with certainty,- in an uncertain climate.-

 Theknowledgegapsfromthebroadbarriercategoriesarenotpreciseenoughtofullyaidin- developinganindustry.-Furthermore,-75%of-ABprojectshavebeenlostsinceinception-[18,- 19].-Noarticleswerefoundanalyzingif-ABlocation,status,ortechnologytypewasabarrier.- The-Renewable-Fuel-Standard-(RFS)appearstoworkforsomeandnotforothers.-Examining- thebarriersacrossmultiplebioeconomygroups,suchasacademia,government,biofuel- publishers,advancedbiofuelprojects,andtheremainderofthebioeconomy,waspivotalto- determineaprogressionofbarriersandhowthelevelofunderstandingchangeswhenmoving- outwardsfromtheproprietaryinnerworkingsofcompaniestothebroaderbioeconomy.-No- consolidatedlistswerefoundofcoproductsandby‐productsfrom-2G-ABcompanies.-The- focuswasmainlyplacedontheirfundingandtechnologyissues,asiftheyarenotutilizing- theirsecondaryproducts.-

Therefore, this study was deemed necessary due to the perceived advanced biofuel investment- risk, investment potential in the bioeconomy, infrastructure need, and 75% loss of projects in- less than 8 years. Additionally, a simplifiedunderstanding of internal and external barriers- across and within industry stakeholders groups and market and distribution barriers of their- products was needed to drive faster return on investment from reducing risk, as conditioned- bioeconomy reinforcement. Determination of these knowledge gaps in a singular document- will more quickly aid in bioeconomy collaboration maximizing the RFS‐2 potential.-

### **3. Methods-**

This research was conducted in two phases. Phase one identifiedall wood and grass (nonfood)- AB projects that have been attemptedby their status, location, feedstock, and technology type- in the U.S. During phase two, a survey was conducted requesting industry members to rank- internal and external barriers for the AB industry. In addition, industry members, academi‐ cians, government representatives, and other stakeholders were also asked to rank marketa‐ bility and distribution barriers of biofuel's coproducts and by‐products. After compiling- survey responses, interviews with a selected group of industry members were conducted to- discuss and gain more insights on the specific barriers.-

The geographical location, operational status, and demographics information for each project- were determined by examining secondary sources of information such as technical reports,- peer‐reviewed papers, trade journals, and newspapers. These were based on the biofuel- industry terminology used in the Wood Bioenergy U.S. database according to Forisk Consult‐ ing [22] along with acquired secondary sourced data from the literature review. The data were- used to individually classify and code categories directly associated with advanced biofuel- projects as follows: type (pilot, demonstration, and commercial), operational status (cancelled,- shutdown, operating, planning, and under construction), demographic (project, name, and- location), feedstock type used, and contact information.-

Grounded theory was used to examine peer‐reviewed papers, industry reports, technical- reports, trade journals, and newspapers to detect barriers impacting the AB industry. The goal- of the grounded theory analytical technique is to classify and categorize information based on- higher level categories. The technique starts with an initial open coding involving labeling,- data segmentation, conceptualizing, and developing categories. Higher level grouping and- categorization includes axial coding to analyze the most significantand frequent data from the- initial coding, thus relating categories to subcategories [23]. Following the extraction of- barriers, a list of the most common by‐products, and coproducts were also extracted from- secondary sources.-

 The outputs of grounded theory (list of barriers) were used to design a questionnaire to have- biofuel industry members provide their perceptions on the list of barriers impacting the AB- industry separated by internal, external, and marketing and distribution of coproducts and- by‐products. In addition, discussions with a sample of the biofuel industry experts were- conducted to clarify survey results and gain additional insights. Industry members were- chosen by direct requests from the projects identifiedin the firstphase. The survey included- Likert‐type questions, open‐ended questions, and close‐ended questions. The Likert‐scale- questions were developed for nine differentconstructs that were identifiedduring the- literature review. A scale from 1 to 5 was used, where 1 was strongly disagree and 5 was- strongly agree.-

#### **4. Results-**

#### **4.1. Project status-**

 A total of 59 AB projects were identifiedand classifiedby project status (**Figure 1**). The- geographical distribution visually indicated that there was a relationship by region and project- status for the Eastern part of the U.S. and in Mississippi. The geographic location analysis- indicated that most of the advanced biofuel projects are located in the Eastern region, but the- proportion rates of projects when comparing the Eastern and the Western regions does not- show any significantdifferencebetween regions. Mississippi seems to have state policies- designed to attractthe industry. Other projects seem to be uniformly scatteredin the Eastern- region. In total, 19 projects were cancelled or shutdown. Of the 59 projects started since 2007,- only 13 are operating in 2015.-

A contingency table analysis indicated that the majority of projects have been started in the- Eastern region (*n* = 41, 82%). Given that there could be a relationship between the regions and- the status of projects, a test was conducted to test if the proportion of status of projects was the- same for both regions. The results of the Chi‐square test indicated that there was no significant- relationship between regions and status of projects (*p* = 0.3260).-

There are fivestages of technology development for advanced biofuel projects (**Figure 2**). Each- stage is representative of the feasibility of planning, financial constraints, proving conceptualdesign, and intellectual rights. Finally, repeat the success. The average pilot plant typically- costs \$10 million or less, the average demonstration plant cost is less than \$100 million, and a- commercial plant cost varies from \$100 to \$500 million. **Figure 2**shows the number of indi‐ vidual projects by technology status achieved from 2005 to current.-

**Figure 1.** Map of all advanced biofuel projects since 2005 (Withers [23]).-

**Figure 2.** Project stages of technology development and percentage status where Shtdn = shutdown, Cancld = canceled,-Dem = demonstration, and Comm = commercial (Withers 2016 [23]).-

#### **4.2. Perception of industry members on internal and external barriers-**

### *4.2.1. Internal barriers-*

A total of 16 industry members participated in the initial survey. Participants generally agreed- that internal barriers include technology yield per ton (56%), technology conversion (50%),- and lack of continuous project growth (44%). Participants did not view the following categories- as barriers: coproducts marketing (69%), coproducts distribution (56%), by‐products market‐ ing (63%), by‐products distribution (63%), strategy (56%), management (50%), and product- development (44%).-

**Table 1**shows the median responses on internal barriers by project type, project status, and- project technology. All participants had to indicate project type, project status, and technology- type. Each of these categories was further divided in subcategories as shown in **Table 1**.- Responses across project status are very similar and do not show a clear distinction between- the subcategories. In the case of the category project type, it seems that industry members- classifiedas pilot have a higher perception on barriers than the ones identifiedas open and- closed. Also, in the technology type category, industry members classifiedas biochemical seem- to have a stronger perception of internal barriers than the other technology types.-


**Table 1.** Median values of internal barriers by type, status, and technology.-

A contingency analysis was conducted to compare the differenceswithin each category or- group. It was found that there were no differenceswithin type (commercial, demonstration,- and pilot) and status (closed, open, and planning). However, the contingency analysis by the- technology group (biochemical, hybrid, and thermochemical) yielded a significantdifference- on internal barriers by‐products distribution and coproducts marketing on the biochemical- technology type. Given that the number of counts by cells was less than fivein some cases, a- Fisher's exact test was then performed on these categories; the Fisher's test determined thatby‐products distribution (*p-*= 0.074) and coproducts marketing (*p-*= 0.028) were significant- barriers for a significance level of 0.1.-

#### *4.2.2. External barriers-*

In the case of external barriers, biofuel industry members agreed that funding (100%),- renewable volume obligation (75%), EPA pathway process (75%), and RFS and RINs (56%)- were external barriers. Noticeable uncertainty was placed in DOE pathway process and waiver- credits. The categories of competitors, energy costs, suppliers, and third‐party relationships- yielded fairly similar disagreement.-

 The sample was also divided in categories, similar to the internal barriers analysis. The median- responses on external barriers by project type, project status, and technology type were also- examined (**Table 2**). Overall, the data show that industry members in all categories have a- higher perception of external barriers than internal barriers. A contingency analysis was- performed to compare the subcategories by project type, project status, and technology type- to determine if the differenceswithin each subcategory were significant. It was found that there- were no differencesin the category project status (closed, open, and planning are the same) on- the perception of barriers. However, significantdifferenceswere found in the project type and- technology type categories. By project type, differenceswere found on the perception of- barriers competitors (demonstration and pilot differentthan commercial) and energy costs- (pilot differentthan commercial and demonstration). And differenceswere found on the- perceptions of barriers competitors (biochemical is different),energy costs (biochemical and- hybrid different),and third‐party relationships (biochemical is differentto the other two). In- all cases, an exact Fisher's test was conducted with a significance level of 0.1.-

#### **4.3. Marketability and distribution barriers for coproducts and by‐products from advanced- biofuel industries-**

In this part of the study, a ranking and classificationof barriers impacting the marketability- and distribution of lignocellulosic biofuel's coproducts and by‐products were conducted.- Coproducts and by‐products are an important component of the AB industry business model.- Without the proper marketing and commercialization strategies, coproducts and by‐products- cannot be commercialized. As it is today, AB industry needs to have revenue from its copro‐ ducts and by‐products in order to remain competitive.-

The advanced biofuel production process yields by‐products and further processing generates- subsequent coproducts. The list showed in **Table 3**was obtained through research from- secondary sources. Combining or improving by‐products can lead to desired coproducts.- Unused by‐products increase expenses [25], since they require disposal; as a result, increasing- the value from by‐products and coproducts could help sustain a biofuel project [26]. Viveka‐ nandhan [26] suggests that many of the biofuel industry small‐scale projects do not generally- collect coproducts due to high opex (ongoing) costs foregoing added profitpotential, while- the opposite is true for commercial scale projects. The coproducts and by‐products are more- valuable to reduce energy costs when burned for biofuel projects are placed in landfillas waste- [28]. Therefore, understanding harmful by‐product waste streams is economically and-

 environmentally beneficialwhen planning scaling projects to reduce harmful impact [25, 29].- According to Doherty et al. [29] and Gellerstedt et al. [30] providing value‐added coproducts- may lead to improved biorefineryfinancialsuccess, and some coproducts could actually be- more valuable than the biofuel itself [25].-


**Table 2.** External median quantiles by type, status, and technology.-





**Table 3.** List of potential coproducts and by‐products from AB industry.-

Many initial biofuel projects as in early in 2005, did not focus on these secondary products,- but instead focused on more pressing technology and funding issues. Forty‐two percent of- all projects included in this study are pilot and demonstration plants designed for testing- purposes, with reduced focus on secondary outputs. The commercial facilities are realizingthe value of their coproducts and are restrategizing. For example, Virent Biogasoline, a com‐ mercial biofuel company impacted by the blend wall, changed its website to list available- quantities of various coproducts they produce. Discussing survey results with the industry- indicated there are at least 44 coproducts produced, nearly twice the number identified from- the literature. This increase was based on companies currently stymied by blend‐wall limita‐ tions that reduce demand to fund production economies of scale. These limitations drive- stakeholders to consider new markets beyond biofuel to meet shareholder financial expecta‐ tions. Advanced biofuel companies are currently focused on shifting to platform technolo‐ gies, targeting higher value coproducts and the available funding arena [32, 33].-

In addition to the perception of AB industry members, perceptions of other AB industry- stakeholders such as academicians, government representatives, and journalists are included- to rank AB's barriers for by‐product and coproducts. Altogether, a total of 44 responses were- obtained from all stakeholders. Out of the 44 responses, 28 respondents provided usable data- to this section, identifying barriers to coproduct marketability (*N* = 27) and distribution (*N* =- 28), as well as by‐product marketability (*N* = 28) and distribution (*N* = 22), see **Tables 4**and **5**.- Cost, financing,and public awareness were the main barriers across the four classifications.- There are many similarities of response between the four categories of coproducts and by‐ products marketability and distribution barriers, such as infrastructure, fossil industry control,- public perception, and policy. Some responses are very similar to the internal and external- barriers analyzed in the previous section; however, many are unique to this study, such as sole- source risk, heated rail car shortage, and flooding a niche market.-

The perceived need of coproducts and by‐products' infrastructure to support the already- subsidized industry was not expected. Nor did the industry expect to be stymied by the blend- wall, the fossil fuel industry buying cellulosic waiver credits (CWCs) and lobbying against- them, politics, or a slowly developing infrastructure. It would seem the advanced biofuel- industry initially did not examine the end‐user market demand and capabilities for additional- by‐products and coproducts. The survey results indicated that by‐product and coproducts- infrastructure are a niche market and saturated in the short term, since the industry was already- shifting toward platform technologies. According to, there was a 9% growth in premium- renewable biochemicals in 2015, which implies that the shift to platform technology would- potentially become a barrier, as well, in a niche market. Reidy [32] stated the industry is moving- to produce and sell premium products. Selling premium products would imply the niche- market barrier may only affectthose in competition with advanced biofuels that already- produce nonrenewable premium chemicals, such as the fossil fuel industry. The shift in this- industry to compete at a multiproduct platform level other than biofuel in new markets was- an attemptto avoid sole source risk and maximize by‐products potential and funding. Rural- economic development was one of the three primary objectives established by the government.- The survey results indicated that some projects face lack of heated distribution channels from- declining rural rail systems. In the short term, premium coproducts, such as waxes, will have- to be developed to offsetthe cost of changing perceived risk to increase demand for the- revitalization of the heated rural rail infrastructure.-


**Table 4.** Coproducts marketability and distribution barriers.-



**Table 5.** By‐products marketability and distribution barriers.-

### **5. Conclusions-**

The barrier analysis indicated the perspectives on barriers to production of advanced biofuel- are differentby project type, status, and technology. The barrier impact changed across time- and type of project. The closed projects faced the same barriers; however, fewer barriers than- the current projects now that the blend wall is a permanent factor. Discussions with bioecon‐ omy industry representatives about the implications of the blend wall led to an improved RFS- model and improved understanding of the system.-

Overall, timing is the main barrier to advanced biofuel projects. If the decline in fuel con‐ sumption was realized by all parties, the advanced biofuel group may not currently exist.- However, the outcome of timing has created the realization that the remaining advanced- biofuel projects are now rapidly moving to become advanced biochemical platform technology- companies, quickly and annually claiming market share of global premium coproducts. They- are well poised to either blend higher levels of biofuel and/or premium coproducts, dependent- upon the full spectrum of petroleum barrel price and demand. Additionally, they are unifying- their effortsto become a household lifestyle premium brand. Will the petroleum industryrealize its marketing myopia and grow with the bioeconomy global brand, or will it inadver‐ tently continue as the increasingly undesired environmentally unfriendly brand? A review of- the literature did not distinguish any lists of barriers to the marketability and distribution of- coproducts and by‐products. However, through the survey and interviews in this study, an- extensive list of barriers was developed, including 27 coproducts marketability and 28- coproducts distribution barriers, and 28 by‐products marketability and 22 by‐products- distribution barriers. The main barriers were cost, funding, fossil industry control of market,- and public awareness-

To move the bioeconomy forward faster, developing an incremental greenhouse gas (GHG)- carbon tax is needed on an incremental level to fund the developing infrastructure, public- education, and factual perception to bolster the demand for biofuel and biochemicals. The- funding is privately earmarked, ready, and in bearish stance, awaiting public demand. The- information compiled in this study can aid the biofuel industry and the bioeconomy in future- pursuits; it can provide guidance to inform R+D to reduce costs and improve perceived risk,- increasing investment viability.-

#### **Author details-**

Henry Jose Quesada‐Pineda\* , Jeremy Withers and Robert Smith-

 \*Addressallcorrespondenceto:quesada@vt.edu-

 Virginia-Tech,-Blacksburg,-United-States-

#### **References-**


**The Biofuel Crops in Global Warming Challenge: Carbon Capture by Corn, Sweet Sorghum and Switchgrass Biomass Grown for Biofuel Production in the USA** 

Roland Ahouélété Yaovi Holou and Valentin Missiakô Kindomihou

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65690

#### **Abstract-**

 Thisresearchevaluatespotentialcarboncaptureofsweetsorghum,switchgrass,and- corngrownin-Portageville,-Missouri,from-2007to-2009.-Ourresultsshowedthatcorn- grain-Ccontentaveraged-43%,whereas-Cgraincapturedwas-1.3–4.7-Mg-C ha−1- dependingonyearand-Nrate.-Nfertilizationsignificantlyincreased-Ccapture,butnot- Ccontentofgrain.-Ccapturebyswitchgrassdependedoncultivarsandharvestdate.- Switchgrasscv.-Alamobiomasscontained-46%-Ccomparedto-44%-Cfor-Blackwell's.- Alamomaximum-Ccapturedependedonyear,being-9.8-Mg-C ha−1in-2008and-13.4-Mg- C ha−1in-2009.-Cisequivalentto-32.3–49.6-Mg-CO2 ha−1,while-Blackwellcaptured-3.7–- 4.4-Mg-C ha−1.-Cinsweetsorghumbiomassrangedfrom-42to-45%,whereastotal-C- capturerangedfrom-3.2to-13.8-Mg ha−1accordingtoyear,soil,and-Nrate.-Thehighest- Ccaptureappearedinloam.-Sweetsorghumabovegroundbiomassshowed-82%-C- capturedinthestalk.-Whenconvertedinto-CO2,-Ccapturedbysweetsorghumwas- equivalentto-12–51-Mg-CO2 ha−1.-Inadditiontotheirbiofuelpotential,corn,switchgrass,- andsweetsorghumcan substantiallycontribute toenvironmentalcleaningbycapturing- a significantamountof-CO2.-

**Keywords:** carbon, corn, sweet sorghum, switchgrass, global warming, CO2-

### **1. Introduction-**

 Formanydecades,reportshaveincreasinglyproventhattheclimateofourplanetischanging- mostlybecauseofanthropogeniceffectsthatareincreasingtheglobalwarming-[1–3].-The-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 increaseoftheconcentrationofcertainmoleculessuchascarbondioxide-(CO2),methane,- nitrousoxide,chlorofluorocarbons,aerosol,andsulfurintheatmosphereisoneoftheleading- causesofthetemperatureincrease.-Actionsarebeingtakentoreducetheconcentrationof- greenhousegasesintheatmosphereandtobetterprotecttheozonelayer-[4];oneofthese- actionsistheuseofbiofuelinengines-[5–7];-[3].-

Interestingly, plants have a unique ability to uptake CO2from the air through their stomata- and use it for photosynthesis [8]. Therefore, plants help clean the environment by capturing- the CO2-[9, 11], and integrating it into the metabolic systems that make up carbohydrates and- other carbon‐containing compounds. When they die, plants return the carbon they sequestered- in their roots and leaves to the soil [4]. The mechanism by which plants capture the atmospheric- carbon and move it to a soil has been reviewed [12]. The previous author showed that grasses- transfer more carbon to the soil than trees. However, because of the biological activity of- microorganisms and some anthropogenic reasons, not all the carbon captured by plants is- sequestered in the soil [12, 13]. Some carbon captured by plants returns to the atmosphere in- the form of CO2-[14, 8]. Although there is controversy about the impact of biofuel crops on the- climate, the majority of the papers that have evaluated the environmental impact of these crops- showed that each crop can significantlydecrease the emission of greenhouse gases [15]. For- instance, the greenhouse gas emittedby the cellulosic ethanol produced from switchgrass is- 94% smaller than that from gasoline, so the production of ethanol from switchgrass is much- cleaner than the production of gasoline [16].-

 Rootsarethemaincarbondepositorinthesoil,andtheydecomposeveryslowly-[17,-8].- Unfortunately,itisdifficulttostudycarbonsequestrationinroots-[8].-Nevertheless,-Andress- [18]provedthattheamountof-CO2sequesteredbyswitchgrassinthesoilis-138.1kgof- CO2/Mgofabovegroundbiomass.-Inthenorthernplainsofthe-US,switchgrassgrownfor- biofuelproductionreturnedtothesoilupto-4.42-Mg-C ha‐1year‐1-[19].-Sorghumcancapture- 3.4–7.2-Mgof-CO2perhectare-[20].-Inaddition,thesepreviousauthorsfoundthatsorghum- rootscanaccumulateupto-14%ofthetotalcarboncaptureintheaboveandunderground- biomass.-Ingeneral,rootscancontributefrom-7to-43%ofthetotal-Csequesteredbyaplant- [21].-Inthecaseofcorn,theamountof-Csequesteredintherootsis-60%morethanthatof- thestover-[22].-Residuesfromcropsareasignificantwaytoimprove-Csequestrationinto- asoil-[20].-Usually,plantsthatproducealotofbiomasscapturethemost-C.-Oneofthe- strategiescurrentlyusedistopayfarmersacarboncredittoenticethemtogrowbetter- cropsthatcanhelpreduceglobalwarming.-Ingeneral,carbondioxidecosts-\$100permetric- ton-[23,-24].-

Our research proved that sweet sorghum and switchgrass can produce a significant amount- of biomass, suggesting that their C capture will be high. Unfortunately, very littleis known- about the carbon capture potential of these crops in Missouri. Nevertheless, this kind of- information is needed to make a complete economic and environmental assessment of these- biofuel crops. The objectives of this research have been (1) to determine the carbon capture by- corn grain, switchgrass, and sweet sorghum biomass, and (2) to determine the impact of- nitrogen (N) application and the soil type on carbon capture. For sweet sorghum, we also- studied the partitioning of the carbon capture between the leaves and the stalk. Our generalgoal is to show that in addition to producing biofuel, sweet sorghum, switchgrass, and corn- have additional advantages in their ability to clean the environment and therefore help resolve- the global warming challenge.-

### **2. Methods-**

The research was carried out in South‐eastern Missouri over a 2‐year period (2008–2009) on- Tiptonville silt loam soil (fine‐silty, mixed, superactive, thermic Oxyaquic Argiudolls). The test- was located at Portageville (36°24*'*N, 89°41'W) in 2008 and Hayward (36°23'N, 89°39'W) in- 2009. Weather data were obtained from Missouri Historical Agricultural Weather Database- (www.agebb.missouri.edu/weather/history). Electronic weather stations (Campbell Scientific- Inc., Logan, UT) were established at each test site to measure hourly air temperature, relative- humidity, wind direction and speed, solar radiation, and rainfall.-

The experimental design [25–27] was a four‐replicate, randomized complete block. Each block- consisted of seven N treatments. Each N treatment corresponded to a plot. Each plot was 8.30- m long and 3.05 m wide (four rows spaced 76.2 cm apart).-

 Corn (*Zea mays*cv. P33N58) was planted mid‐ to late April at 79,071 seeds ha‐1on a silt loam- soil type and the sweet sorghum (*Sorghum bicolor*cv. M81E), in May at 296,516 seeds ha‐1on- loamy, clayed, and sandy soils. The nitrogen rates applied were 0, 45, 90, 134, 179, 224, and 269- kg N ha‐1on the corn while 0, 22, 45, 67, 90, 112, and 135 kg N ha‐1on the sweet sorghum. Less- than 2 weeks after planting, the fieldreceived two applications of atrazine (2‐chloro‐4‐ ethylamine‐6‐isopropyl amino‐S‐triazine) at 1.1 kg ha‐1active ingredient to control weeds.- Additionally, the fieldwas regularly hoed as needed to reduce weeds not controlled by the- herbicides. Three weeks after planting, the appropriate N rate was broadcast by hand on each- plot using ammonium nitrate (17% nitrate‐N, 17% ammonium‐N). Ammonium nitrate was- chosen because it does not have urea's potential for ammonia volatilization, simplifying the- test by minimizing and removing the uncertainty of ammonia losses. The field was irrigated- as needed. The corn fieldwas fivefurrow irrigated with 76‐mm water applications per year.- At maturity, the two middle rows in each plot were harvested using a plot combine. The grain- yield was calculated per plot based on 15% moisture content. By the sorghum side, fivefurrow- irrigations (76‐mm water applications) per year were made on the loam and clay compared- with six to eight sprinklers (25‐mm applications) per year on the sand. The sandy soil was- irrigated with linear move sprinkler irrigation, whereas the loam and the clay soils were furrow- irrigated. The sweet sorghum heads were removed a month before the harvest of the stalk.- This was done to maximize sugar in the stalk. Four and a half months after germination- (**Table 1**), sweet sorghum was harvested using a hay sickle mower. The fieldsused in 2007 and- 2008 had been previously planted in cottonand soybean, respectively. In 2009, the sorghum- planted on the clay soil followed soybean, the sorghum planted on the loam followed corn,- and the sorghum planted on the sand followed cotton.-


†: Numbers followed by different letters are statistically different within the same column at p≤0.05.-

**Table 1.** Mean separation of the carbon capture in corn grain.-

 Switchgrass (*Panicum virgatum*cv. Alamo) was drill planted in 6‐m wide strips (east to west),- spaced parallel 100 m apart and 450 m long by a cottonfieldnear Portageville (MO, USA;- 36.4253°N, 89.6994°W) [28, 29]. The main soil in the field was a Bosket finesandy loam (bosket,- fineloamy, mixed active, thermic, mollic, and Typic *Hapludales*) soil. The fieldwas located in- the upper Mississippi River Delta region where the topography is nearly flatand southwest- winds in May and June sometimes cause damage to young crops. The farmer planted the- switchgrass as a wind break to minimize blowing sand injury to cottonseedlings. An added- benefitof the strips is habitat for birds and rabbits. The fieldis burned every 4 years in April- and mowed annually in September or October, and switchgrass strips have not received any- lime, pesticides, N, P, or K since establishment in 1990. This crop required fewer nutrients- probably because it fixedthe atmospheric N and had mycorrhizae activity. The optimal N- fertilization rate applied in the US is not clear but ranged from 120 to 224 kg N ha‐1 [29].-

In 2008 and 2009, switchgrass biomass was monthly harvested from May to November. For- each sampling date, four plots in the fieldwere evaluated. The biomass was harvested from- the center of the strips by hand in a floristicallyhomogeneous subplot of 1.67 m2using a hay- sickle mower. Total fresh biomass weight was separated into leaves, stem, and head and oven- dried for constant weight. Switchgrass biomass was determined by extrapolation from the- biomass obtained in the 1.67‐m2 subplot.-

The biomass of four samples of each N treatment was dried in an oven and analyzed for carbon- using the LECO SC 44 Carbon Analyser (Leco Corp, St Joseph, MI) adapted from NRCS [30].- The carbon content (%) was directly read from the machine. The carbon capture in the biomass- was calculated as follows:-

$$\text{Carbon capture (kg ha}^{-1}\text{)} = \text{dired Biomass (kg ha}^{-1}\text{)} \times \text{Carbon content (\%)}\tag{1}$$

The equivalent captured CO2 was calculated based on the oxidation reaction of carbon:-

The Biofuel Crops in Global Warming Challenge: Carbon Capture by Corn, Sweet Sorghum and Switchgrass... 143 http://dx.doi.org/10.5772/65690

$$\rm{C} + \rm{O}\_{2} \rightarrow \rm{CO}\_{2} \tag{2}$$

Based on the molecular weight of carbon (14 g) and that of CO2-(44 g), each gram of carbon- sequestered by a plant is equivalent to 3.14 g of CO2uptake. The equivalent amounts of CO2- captured by the plants were extrapolated considering that ratio.-

 The equivalent CO2‐sequestered switchgrass soil was extrapolated based on a previous study- done by Andress [18] who proved that switchgrass sequestered 138.1 kg of CO2/Mg of- aboveground biomass. The data were analyzed using the Proc mixed model in SAS 9.2 (SAS- Institute Inc., Cary, NC). Significant differences were assumed for *p* ≤ 0.05. The year of the study,- the soil type, and the N rate were considered the main fixedfactors, whereas the block (repeat)- was classifiedas a random variable. For the Proc mixed model, the estimation method was the- restricted maximum likelihood (REML). Means were separated and grouped by letterby using- the macro developed by Saxton [31]. Significantdifferences are assumed for *p* < 0.05.-

#### **3. Results-**

#### **3.1. Carbon capture in corn grain-**

 In general, the carbon content (%) and the amount of carbon captured (kg ha‐1) in corn grain- depended on the year (*p* < 0.0001). In 2008, 43.8% of the grain was carbon compared to 42.9%- in 2009. Additionally, N fertilization significantlyaffectedthe amount of C sequestered in the- grain (*p* <0.0001), but not the carbon content of the grain (*p* = 0.4051). Usually, the carbon capture- in the grain increased as the N rate went up (**Table 1**).-

Moreover, the impact of the N rate on the C capture in the grain was more pronounced in 2009-(*p* < 0.0001) than in 2008 (*p* = 0.03) (**Figure 1**).-

**Figure 1.** Carbon content of switchgrass biomass.-

This confirmedthe N need that the corn had on the loam in 2009, which was planted after corn- in the rotation system. The corn was then unable to capture as much carbon from the air when- compared to the previous year.-

### **3.2. Carbon capture in switchgrass biomass-**

The carbon content in switchgrass biomass depended on the year of the study and the variety.- Switchgrass var. Alamo contained more C (45.73%) than the Blackwell variety (44%). The- carbon content of the underground biomass was similar to that of the aboveground. The- amount of carbon sequestered in the aboveground biomass depended on the switchgrass- variety, the year of the study (*p* = 0.002), and the date (*p* < 0.0001). The amount of C captured- by the Alamo variety was more than three times that of the Blackwell one (**Table 2**).-

    Usually, the maximum carbon captured by the Alamo variety was reached in October (**Table 2**).- In 2008, the Alamo variety captured a maximum of 9.8 Mg ha‐1compared to 13.4 Mg ha‐1in- 2009. By contrast, the Blackwell variety captured 3.7 Mg ha‐1in 2008 as opposed to 4.4 Mgha‐1- in 2009. When the maximum carbon captured by the Alamo variety was converted into CO2,- it was equivalent to 32.3 and 49.6 Mgha‐1in 2008 and 2009, respectively (**Table 2**). By contrast,- the Blackwell variety captured 13.6 Mg CO2 ha‐1in 2008 compared to 16.2 Mg CO2 ha‐1in 2009.- The amount of CO2 sequestered to the soil by Alamo ranged from 3.03 to 4.02 Mgha‐1 according- to the year (**Table 2**) compared to 1.17–1.34 Mg ha‐1 for the Blackwell variety (**Table 2**).-


**Table 2.** Carbon capture in switchgrass cv. Alamo.-

#### **3.3. Sweet sorghum carbon capture-**

In addition to its biofuel potential, sweet sorghum captured 3.2–13.8 Mg ha‐1according to the- year, the soil, and the N rate (**Table 3**). The highest carbon capture was recorded in the loam.- Usually, the carbon content of the aboveground biomass ranged from 41.9 to 44.6% (**Table 3**).- Unlike in the loam and the sand, the carbon in the clay was not affectedby the N rate (**Table 4**).- This suggested that the lack of available N in the soil reduced the ability of sweet sorghum to- assimilate atmospheric CO2into its metabolic systems to build organic compounds. The factthat in 2009 (year where sweet sorghum was planted after corn in the loam) the impact of the- N rate on the C content was significantonly in the loam confirmedthat sweet sorghum had- difficultytaking up the amount of N it needed from the soil to perform photosynthesis. In- general, the application of N improved the C content of the biomass.-


† Numbers followed by differentletters are statistically different within the same column at *p* ≤ 0.05.-

**Table 3.** Carbon content and capture by sweet sorghum aboveground biomass.-


† The values (i.e., symbols) in the table are the probability associated with the test of the impact of *N*; \**p* < 0.05; \*\**p* < 0.01,- and \*\*\**p* < 0.001.-

**Table 4.** Impact of *N* on the carbon content and capture in sweet sorghum aboveground biomass.-

However, the impact of N on the biomass C content depended on the organ. Indeed, in both- years, the C content of the leaves did not depend on the N rate (**Table 4**). However, in 2009,- the impact of the N rate on the carbon content of the stalk was significant (*p* = 0.0135). These- results suggested that the accumulation of organic compounds in the stalk was affectedby the- lack of N. The predominant organic molecules in sweet sorghum stalks are sugars. Therefore,- the decrease of the sugar content in the stalk may explain why its carbon content decreased as- N is lacking. In other words, the lower C content means less C available to make the sugars so- less sugar.-

Nitrogen fertilization affectedthe C capture in sweet sorghum biomass depending on the soil- and the year. Unlike the carbon content, the carbon capture in the clay was significantly affected- by the N rate (**Table 4**). These results showed that the application of N is required in the clay- if an increase of N capture by sweet sorghum is persuaded. Similarly, on the loam, when sweet- sorghum is grown after corn, its ability to sequester the atmospheric carbon is limited by N (*p* = 0.03) (**Table 4**). Except in the loam 2009 (*p* = 0.016), the N rate did not affectthe amount of- carbon captured in the leaves (**Table 2**). By contrast, the N fertilization improved the seques‐ tration of the C in the stalk in the clay in both years and in the loam in 2009. The C accumulation- in the stalk and in the sand was never affectedby the N rate (**Figure 2**). These results suggested- that the significantimpact of the N rate on the total C capture in the sand was due to its effect- on the leaves. Therefore, in sand, the leaves are more sensitive to the C capture than the stalk- in cases when the N is lacking. By contrast, in the clay and in the loam, when N is deficient,- the carbon accumulation in the stalk is highly affected.-In general, 82% of the carbon captured- in the biomass is found in the stalk (**Figure 2**). When converted into equivalent CO2, the amount- of C captured by sweet sorghum was 11.9–51.1 Mg ha‐1according to the soil type and the N- rate (**Table 3**).-

The Biofuel Crops in Global Warming Challenge: Carbon Capture by Corn, Sweet Sorghum and Switchgrass... 147 http://dx.doi.org/10.5772/65690

**Figure 2.** Impact of the soil type, the year, and the N rate on the partitioning of the C capture in sweet sorghum leaves- and stalk.-

### **4. Discussion-**

Our results proved that the carbon content ranged from 42.9 to 43.8% in corn grain, 44–45.7%- in switchgrass and 41.9–44.6% for sweet sorghum biomass. Generally, the carbon capture by- corn depended on the N rate. The maximum C capture by corn grain was recorded with the- application of 134 kg N ha‐1in 2008 (4.7 Mg C kg ha‐1) compared to 2.9 Mg C ha‐1 in 2009 with- 224 kg N ha‐1.-

  We observed that the maximum C capture by switchgrass cv. Alamo was reached in October,- ranging from 9.8 Mg C ha‐1-(in 2008) to 14.4 Mg C ha‐1-(in 2009). That is three to four times- higher than what corn put in its grain. However, the C capture is smaller with switchgrass var.- Blackwell (3.7 Mg ha‐1in 2008 compared to 4.4 Mg Cha‐1in 2009) which is the same range as- what corn had in its grain. Furthermore, sweet sorghum captured 3.2–13.8 Mg C ha‐1-(**Table 3**),- suggesting that switchgrass can sequester more C than sweet sorghum. The amount of C- sequestered by sweet sorghum in our study was higher than the 3–7 Mg of CO2per hectare- recorded by [20].-

The high ability of switchgrass to capture atmospheric carbon was also recorded [32]. In most- cases, sweet sorghum captured two to three times the amount of C that corn put in its grain.- We found that switchgrass captured more carbon than sweet sorghum and corn grain. These- results showed the additional environmental impact that switchgrass and sorghum may have- over corn grain. Because of their high C capture, sweet sorghum and switchgrass can clean theenvironment from CO2as has been shown feasible with other crops [9–11]. The C sequestered- in the soil will also significantlyincrease the positive environmental effectsof these crops.- Other authors also pointed to the potential C sequestration in crop roots as an important- component of the fightagainst climate change. The theoretical maximum C sequestered in the- soil by switchgrass in our study was about 4 Mg ha‐1, a little less than the 4.42 Mg C ha‐1year‐1- observed by previous authors in the northern plains of the US [19].-

Finally, farmers that grow these crops should be compensated with carbon credit. However, if- not well managed, the captured carbon can return to the air. To avoid that scenario, farming- techniques that disturb the soil less should be encouraged [12, 13]. Farming techniques that- minimize soil disturbance (e.g., non‐tillage and use of cover crops) can help sequester C in the- soil, and consequently reduce the effectsof global warming [20]. While non‐tillage is ideal, it- is also impractical. Still, it is important to point out that in contrast to tillage that increases the- rate of soil C mineralization, non‐tillage improves the storage of soil C [33, 34, 21].-

#### **5. Conclusion-**

Our results showed that since the carbon capture by sweet sorghum, switchgrass, and corn are- statistically significant,these crops can help reduce the concentration of CO2in the environ‐ ment and therefore contribute to the reduction of global warming.-

### **Acknowledgements-**

Research for this chapter was funded by financialsupport from the Missouri Life Science- Research Board, and Missouri Fertilizer and Ag. Lime Board (USA).-

### **Author details-**

Roland Ahouélété Yaovi Holou1,3 and Valentin Missiakô Kindomihou2\*-

 \*Addressallcorrespondenceto:vkindomihou@yahoo.fr-

 1-Universityof-Missouri‐Delta-Center,-Portageville,-MO,-USA-

2-Laboratory of Applied Ecology, Faculty of Agronomic Sciences, University of Abomey‐ Calavi, Benin Republic, West Africa-

3 DiasporaEngager, Augusta, GA, USA-

### **References**


## **Theoretical Considerations for Economics of Secondand Third-Generation Biofuels**

Fouzia Tabssum and Javed Iqbal Qazi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65735

#### **Abstract-**

 Groundisbeingpreparedallovertheworldforinstallationofbiofuelplantswhichcan- governthesustainablesupplyofcleanerfuelsataffordablepricesandpredictable- amounts.-Atthedawnofthiscenturybiofuelsidentifiedlowcostfeedstocks,their- diversepretreatments,differentmethodsofsaccharificationsandfermentationsand- thoseforcultivationofbiodieselyieldingorganisms.-Bioalcohols,biohydrogenand- biogasrepresentthebiofuelswhicharederivedfrommicrobialworkonthebiowasteresources.-Extensionsinthissectorhavefocusedthesolarenergycapturedbythe- microalgaefromwhichoilscanbeextractedforbiodiesel.-Undoubtedly,allformsof- availableenergies on this planet earthhad/have been derived,directly or indirectly,from- thesolarinputs.-Inthischapterpivotalroleofsolarinsolationwillbediscussedalbeit- forregenerationaswellasprocessingoflignocellulosicbiomassforobtainingbiofuels.- Conclusively,biofuels'sustainablesupplies,roleofsolarenergyhasbeendreamtat- variousstepsoftheprocess;fromthecollectionofbiowasteresourcesthroughstepsof- pretreatment,saccharification-/fermentationandpurificationoftheproduct.-This- chapterdiscussesthesubjectmatterintotwomajorsub-headings:-1)-Biofuelsfrom- lignocellulosic-/foodindustrialwastesand-2)-Cultivationofmicrobesforbiodiesel.-

**Keywords:** biodiesel, bioethanol, biohydrogen, economizing biofuel production,- lignofuels, second-generation bioenergy, third-generation bioenergy-

### **1. Introduction-**

 Theform of life we humansknow andunderstand tosomeextend is impossible without the- sun.-Thebiospherewillnotbesustainablewithoutcontinuousrainofsunenergy.-Recalling- ourbasicinformationonenergyconversioninandbetweendifferentsystems,efficienciescan-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 neverattain-100%levels.-Sameistrueforbioticcomponentsofthebiosphere.-Foodtransfer- throughdifferenttrophiclevelsandmetabolites'circulationandtheirtransferthrough- biochemicalpathwaysallpassthroughthislimitation.-Thus,sunenergyispivotaltothe- existenceandsustenanceofallformsofenergyinthebiosphere.-Thefossilsfuelsonwhom,- atpresentwearedependentto-"noreturn"levelhadbeenderivedfromancientorganisms- whosepresenceatthattimewasalsodependentonthesunenergy.-Nowwhenthefossil- fuels'reservoirsaredepletingandfurthertheyhavepollutedourair,soil,andwater,wemust- lookforalternatives,whichmustbesustainableandenvironmentfriendlytoo.-Energyfrom- biomasscanbecondensedintolowvolumehighefficiencyfuelsthroughmicrobialfermentationand/orotherroutesofbioconversionofabundantlyavailablematter.-However,food- competingsourcesofcarbohydratescannotbedivertedtobiofuels'refineriesbyignoringthe- everincreasinghumanpopulation.-Whereasfeedstockforsecond- andthird-generations'- biofuelsareabundantlyavailableatlittleornocost.-Biowasteshaveagreatpotentialinthis- regard.-Forinstance,ithasbeenestimatedthatupto-442billionlitersperyearofbioethanol- couldbeproducedfromlignocellulosicmaterials-[1].-Buttheirpretreatments,saccharification,andthenfermentationstillneedalotofworktobedone.-Low-costandenvironment- compatiblestrategiestorenderlignocellulosicbiomassaccessibleforenzymaticsaccharificationshavetobedeveloped.-Furtherforlignocellulosicwastesofdifferentplantorigins,- differentmethodsmayberequiredforoptimumoutputs.-Liberationofundesiredcompounds- followingpretreatmentsandsaccharificationisanotherareademandingmoreinputs.- Inhibitors'resistantsaccharifyingand/orfermentingmicrobesandenzymeshavetobe- isolated,developed,characterized,andoptimizedforselectfeedstocksandlocations.- Jumpingtothird-generationbiofuels,biowasteshavetobeidentifiedtosupportrapidgrowth- ofmicroalgaeandfastfixationof-CO2.-Thischapterdescribessometheoreticalconsiderations- whichneedtobeconsideredthroughexperimentalverificationtoeconomizethebiofuels'- production.-Althoughthescienceofbioenergy,especiallyforsecond- andthird-generation- biofuelsisstillatitsinfancy,buttakingintoaccountthedrasticallyexhaustedfossilfuels'- reservoirs,thealreadypollutedbiosphere,everincreasinghumanpopulation,andits- elevatingdemandsformorecomforts,whichneedmoresupplyofenergy,weareleftwith- limitedchoicesincludingthefascinatingfutureofbiofuels.-

#### **2. Biofuels from lignocellulosic/agri/food industrial wastes-**

Fossil sources of energy are depleting at a very rapid rate and alternative sources of renewable- energy are being searched. Agricultural wastes are produced daily and get accumulated into- differentenvironments. They pose special problems of solid waste management in urban- environments and industrial locations. On the other hand, they are potential source of- fermentable carbohydrates. These wastes could be identifiedas low-cost feedstocks for- biofuels' production. For example, bioethanol can be fermented from diverse feedstocks- including corn, sugarcane, wood, and fruit and vegetable wastes such as pawpaw and sweet- potatoes, etc. In addition to the production of biofuels, utilization of lignocellulosic agriwastes- will concomitantly solve the solid waste disposal problem, as the fermentation residues canbe used as solid fertilizers. A number of physical and chemical treatments methods have been- reported for the production of bioethanol from diverse categories of plant biomass. However,- researchers are continuously describing new methods and techniques for economic yields of- bioethanol [2–7]. From the biofuels headings, bioethanol is becoming increasingly popular as- fossil fuel additive and for reducing the stress of decline in crude oil availability. Bioethanol- productions require sustainable supplies of fermentable sugars, efficientfermenting microbes,- a few nutrients (depending upon the nature of feedstock as well as microbes), and optimized- culture conditions. Resultantly, bioethanol productions have been described from diverse- waste resources such as market vegetable waste, carrot discard, hydrolyzed agricultural- wastes, banana peels, and pulp and peels of mango. [8–13].-

Interests in the area of bioethanol production from organic waste materials emerged in the late- 1980s. Since then lignocellulosic material extraction and enzymatic hydrolysis have been- reported extensively, however, for development of technically feasible and economically viable- large-scale enzyme-based biomass to ethanol conversion processes addressing diverse waste- resources and fermentation conditions a lot of work still has to be done to cover different- regions of the world. The success of cellulose to ethanol conversion processes has been- described as a function of cellulose fiberpretreatment, enzyme selection, and operating- conditions [14]. Nigam and Singh [15] have discussed that researchers have been redirecting- their interests in biomass-based fuels for sustainable development in the content of economical- and environmental considerations. These researchers further stressed that renewable bioresources are available globally in the form of residual agricultural biomass and wastes.- However, much research has to be done for the development of an effective, economical, and- efficient conversion process.-

 Estimatesforbioconversionofgloballywastedcropappearpromising.-Forexample,-Kimand- Dale-[16]havedescribedthatbyemployingthewastedcropsforbioenergyproductionconflict- betweenhumanfoodandindustrialusecanbeavoided.-Crops'residuesandindustrial- lignocellulosicwastescanbeconsideredfeedstocksforbioethanolproductions.-Wastedcrops- haveapotentialtoproduceupto-49.1-GLyear−1ofbioethanol.-Theseauthorsfurtherdiscussed- thatfromtheresiduesandwastedcropspotentially-49.1-GLyear−1,ethanolproductionis- possible.-Accordingly,bioethanolcouldreplace-353-GLofgasolinewhenusedin-E85fuel.-

Following unprecedented growth of human population and industrialization, ethanol demand- is increasing continuously. Conventional crops of bioethanol production are unable to meet- the demand due to their primary value for food and feed. Lignocellulosic substances including- agricultural wastes have therefore become attractivefeedstocks for bioethanol production.- They are cost effective,renewable, and abundant. The promising technology of bioethanol- from waste resources has several challenges and limitations such as biomass transport and- handling, and efficient pretreatment methods for total delignification of lignocellulosics. Novel- pretreatment methods may increase yields of fermentable sugars after enzymatic saccharification [17].-

Voluminous work regarding the diversity of lignocellulosic resources dictate for development- of diverse methods of pretreatment and to economize further recognition of different categories of saccharifying and fermenting microbes, which might be required for different sorts ofsubstrates to be processed at differentlocations in a country. Local sociocultural and economic- situations can influence the overall efficiencyof biofuels' productions from waste biomass. In- this context, cost of transport of raw material from its natural origin or industrial processing- units must be considered and if it is profitablethen the pretreatment and subsequent concentration/detoxificationprocesses be accomplished at the firstplace. Alternatively, the small- pretreatment as well as fermentation plants might be installed at or near locations of the- biowaste resources. Heat for the pretreatment should be secured directly/indirectly from sun- rays. Efficientacidic or alkaline pretreatments for differentlignocellulosic substrates have to- be identified.-To reduce the expenditure of neutralizing a substrate pretreated with acid/alkali,- respective category of acidophilic or alkaliphilic cellulolytic, and/or ethanologenic microbes- must be searched and recruited accordingly. Similarly, a lignocellulosic or a food industrial- waste known for higher amounts of certain inhibitors' yield during the initial processes might- be attacked with the inhibitor(s)' resistant and/degrading microbes, which must be cellulolytic/- ethanologenic too or their enzymes thereof. The points highlighted above will definitely add- to the economics of biofuels production from biowaste resources. A layout of schematic- thoughts in this regard has been depicted in **Figure 1**, while consideration of different- lignocellulosic substrates is summarized in **Table 1**.-

**Figure 1.** An overview of differentsteps of theoretical considerations to promote economics of second-generation biofuels deriving from solid wastes.-

As can be seen from **Figure 1**that overall conversion of lignocellulosic substrates to low volume high-energy content biofuels involves expenditure of energy at various steps. The raw- material processing including washing with hot water, decontaminating or sterilizing, fermentative, and product purificationsteps need thermal inputs of energy. At least four steps- of overall process depicted in **Figure 1**can be accomplished by solar energy inputs. Some- other steps to improve economics of lignofuels and their application at present time when- the fossil fuels still represent efficient fuels for transport sector, at least, are given below.-



**Table 1.** Compatibility of different pretreatments and fermentative microbes for economic production of biofuels from- diverse and abundantly available lignocellulosic and other biowastes.-

### **2.1. Transport of feedstocks-**

Lignocellulosic feedstocks from agricultural fieldsand relevant industrial units are required- to be transported to pretreatment and/or biofuel's production units. This involves an obvious- expenditure; and at present will necessitate consumption of fossil fuels to run the transporting- trucks. Important to consider is that biofuels are being attempted/generatedto lessen the- present burden of fossil fuels' consumption. To reduce the cost of biofuels and render them- compatible at present with the fossil fuels' supplies, every step is required to be optimized. In- this regard it might prove quite fruitful if the lignocellulosic biowaste feedstocks are pretreated- at or near to their generation locations, so that relatively low-volume/mass pretreated material- ready for saccharification or biofermentation can be transported at affordable expenses.-

To further reduce the feedstock transportation costs, processes of pretreatments and/or- saccharificationcan be accomplished near to the lignocellulosic biowaste origins and the sugar- streams can be transported dynamically employing pipe system. Alternatively, sugars syrups- can be concentrated into low-volume thick fluidsto reduce the cost by usual transport systems.- In a country like Pakistan, processes of pretreatments and sugars syrups' concentration can be- accomplished by direct solar energy. Another strategy would be installation of those industrial- units in one locality whose wastes can be coutilized cost effectively.-

#### **2.2. Concept of cluster(s) of industrial units having wastes of mutual interest-**

Man can achieve maximum efficiencyand sustainability of his effortsaddressing construction- of production units approaching a level and design not superseding natural system(s). In this- regard man has been imitating the nature subconsciously in the past. While conferring to the- cumulative maturity time has come to recognize/realize that the nature tailored models are- best to follow. Exploration of biogeochemical cycles within and among differentecosystems- has taught us that sustainability relies upon the utilization of waste of one unit as resource by- the other and vice versa. Thus, to save cost presently we have to pay to treat industrial effluents/- solidwastes or to transport them to other locations for treatment to utilize them as biowaste- resource, industrial units can be managed in the form of different consortia in which for a given- set of industrial units, waste(s) from one unit can serve feedstock for another. For instance,- sugar industries in Pakistan produce millions of tons of sugarcane bagasse (SCB). Chemical- pretreatment of SCB requires the application of dilute acid(s) or alkali. Many other industrial- effluentsare characterized with considerably high or low pH and are not accompanied with- further process interfering chemicals. Coinstallation of such relevant industrial units can solve- many problems including low cost pretreatment of SCB and value-added consumption of the- otherwise negative-valued acidic/alkaline industrial effluents, for instance.-

#### **2.3. Tax relaxation for biofuels' consumers-**

New trends whose immediate impact on modern human minds reflectsreduction in efficiency- of desired task(s) are difficultto get acceleration. Their acceptance in the society must be- escalated with some incentive. Bioethanol-driven motor cars, especially at the start, might not- run with speed comparable to petrol-driven engines. Whereas the environmental friendlyemission from biofuel-driven motor cars might be strengthen with reduction in the motor- vehicle taxes.-

Waste biomass for biofuels' generation will have to be utilized from differentsources for- sustainability of the process. The wastes, in general, represent solid wastes while organic- content-rich food industrial effluentscan also be utilized for the fermentation of differenttypes- of biofuels. An overview of differentlignocellulosic wastes, their needs of pretreatments, and- microbes along with potential biofuels and additional benefits is given in **Table 1**.-

Regarding the fluidwastes, continuous and sustainable resources are represented by food,- industrial, and domestic sewages, which may contain varying levels of fermentable organic- loads. Schemes have been developed and shown in **Figures 2**and **3**, for upgrading the waste- effluents into biofuels' generation (**Figures 2**and **3**). Responding to above theoretical notions- for improving economics of second-generation liquid biofuels, it appears pertinent here refer- to the work of Nigam and Singh [15]. These authors concluded that four challenges are- imperative for sustainable biofuel productions:-


**Figure 2.** Rebust bioreactor for reducing agri/food industrial effluents'-BOD with concomitant efficientyield of microalgae for biodiesel production.-

Nigam and Singh [15] further added that there is much potential for biofuel market and now- it is a matterof time before they compete with petroleum-based fuels. Technological developments, in future, will enhance energy balance and reduce emissions and production cost of- biofuels.-

**Figure 3.** A workable multistep plane for sustainable treatment of domestic/organic content-rich wastewaters with concomitant microalgal yield.-

#### **3. Cultivations of microbes for biodiesel (third-generation biofuels)-**

First-generation biofuels, primarily produced from food crops and mostly oil seeds, will- remain far behind to achieve targets of biofuel production, climate change mitigation, and- economic growth. Whereas second-generation biofuels from lignocellulosic wastes are still in- their infancy. Meanwhile, another bioenergy sector derives from nonfood feedstocks such as- microalgae. Differenttypes of photobioreactors and open ponds to cultivate microalgae with- additional benefitsof wastewater treatment and as food additive for human health and for- aquaculture have been described [18].-

In an agri-based country like Pakistan, a vast diversity of nutritionally enriched effluentsfrom- food industries like corn steep liquor, molasses, and whey are produced in abundant amounts.- These can be semipretreated with solar insolation derived heat and fermented for the generation of CO2and less BOD effluents.-Thus, processed effluentscan then be employed for rapid- growth of microalgae for biodiesel production. For this particular notion a robust bioreactor- has been designed (**Figure 2**). The food/other industrial effluentspretreated in the bioreactor- (**Figure 2**) and made optimum through dilutions and/or by incorporating essential nutrients- for the cultivation of microalgae can then be routed to **Figure 3**for obtaining biofuel and- ultimate treatment of the effluents.-

Domestic sewage can be biotreated specificallydesigned for the generation of effluents suitable- for cultivation of microalgae. Such practices would incur economy of the process as treatment- of domestic/industrial effluentswill be achieved in a profitablemanner. Whereas in the case- of water shortage, the fresh water sources will not be affordablefor diverse biotechnological- processes without involving additional costs. In this context, biofuels' generation from- biowastes/effluentswould be an appealing fieldof development for sustainable supplies of- biofuels and water for irrigation/other processes (**Figure 3**).-

Mata et al. [18] concluded that for algal biodiesel development of strategies for large-scale- cultivation and harvesting would be required. Growth conditions and provision of affordable- nutrients for large-scale algal cultivations have to be identifiedfor optimum yields. For this oil- extraction strategies, provision of light, CO2, and nutrients and turbulence, temperature, and- O2 levels must be optimized for a given location for conserving efficient oil content and biomass- yield. These workers further indicated that using sources of CO2,nutrient-rich wastewaters, or- inexpensive fertilizers are expected to increase algal yields. In addition to biofuel production,- considering microalgae biomass for differentapplications, such as food, agriculture, and- medicine, will contribute to the sustainability and market competitiveness of the microalgal- industry [18].-

 Anotherstreamofbiodieselmightoriginatefromtheidentificationofsuchbiowastes,- whosepretreatments/hydrolysescanleadtothesuccessfulcultivationofoleaginousyeasts.- Even-CO2hasbeenclaimedasawasteresourceforcultivatingmicroalgaetoextractbiodiesel-(**Table-1**).-Manyotherwasteresourceswillhopefullybesoonimaginedasresourcesfor- biotechnologicalproductionsofvalue-addedproductsinthenearfuture.-

### **4. Conclusion-**

After excessive exploitation of the fossil fuels' reservoirs and rendering the biosphere highly- polluted, humans are left to extract/ferment fuels from biomass as we had been obtaining foods- directly and/or indirectly from firsttrophic level. Parts of the plant biomass we preferably- consume as food cannot be affordedlonger for biofuels' production. Although the lignocellulosic residues and agriwastes represent potential sources of bioenergy productions. Accordingly, low/no cost feedstocks have been identifiedproperly in differentcountries. Now the- challenges of processing the feedstocks in terms of their economical and environmentally- sustainable pretreatments, saccharification,and energy productions have been accepted by- scientists of differentdisciplines. It is hoped that collaborative effortsof mechanical engineers,- chemical engineers, biologists, and other scientists will bring the human wisdom to the hub- of biotechnology for sustainable production of biofuels in the near future. Time is not too far- when the humans will intentionally be cultivating specificspecies of plants and harvesting- them or their parts presently considered as wastes to supply the biorefineries. Another business- sector will represent biofuel productions from the lignocellulosic residues and industrial- wastes.-

#### **Author details-**

Fouzia Tabssum and Javed Iqbal Qazi\*-

 \*Addressallcorrespondenceto:qazi.zool@pu.edu.pk-

 Microbial-Biotechnology-Laboratory,-Departmentof-Zoology,-Universityofthe-Punjab,- Lahore,-Pakistan-

### **References-**


## **Emerging Green Technologies for Biodiesel Production**

Hanifa Taher and Sulaiman Al-Zuhair

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66187

#### **Abstract-**

 Thecurrentglobalenergydemandismetbyburningthenon-renewablefossilfuels.- Asthedemandisescalating,resourcesandreservesarediminishing.-Inaddition,the- environmentisthreatenedbythecontinuousemissionofgreenhousegases;mainly- CO2,whichisworrying.-Therefore,searchingforalternativesisinevitable.-Biodiesel- receivedaconsiderableattentiontopotentiallyreplacepetroleum-basedfuels.-Itcan- beproducedfromoil-richfeedstocksthroughseveralmethodsusingdifferent- technologies,includingtransesterification.-Althoughalkalicatalyzedbiodieselprocess- iscommerciallyviable,severalchallengeswereraised.-Inthischapter,anoverviewof- thecurrentstatusofbiodieselproductionapproachesisdiscussedandtheemerging- technologiesarehighlighted.-Thechapterrewardstheattentionofusinggreen- processes,wheretheeffectivenessofusing;microalgaebiomassasagreenfeedstock- (comparedtoconventionalcrop-basedseeds),lipasesasgreencatalysts-(comparedto- conventionalchemicalcatalysts),andgreenandtunablesolvents,suchasneoteric- solventsandsupercriticalfluids-(comparedtoconventionalvolatileorganicsolvents)- areaddressed.-

**Keywords:** biodiesel, microalgae, lipase, green solvents, microwave-

#### **1. Biodiesel-**

 Thecontinuousdwindlingfossilfuelssupplyandincreasingatmosphericcarbondioxide- emissionhaveputthepressureonfindinganddevelopingsustainablealternativefuels.- Biodiesel,whichisamixtureoffattyacidsalkylesters,istheproposedalternativethatcan- replacetotheconventionalpetroleumdiesel.-Thephysicalpropertiesofbiodieselaresimilar- topetroleumdieselandcanbeusedwithoutanymodificationsintheengine-[1,-2].-In- addition,biodieselisarenewable,non-toxicandbiodegradablefuelthatcandecreasesthe-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 relianceonfossilfuelsandreducesharmfulgassesemissions-[3–8].-Furthermore,biodiesel- haslowersulfurandaromaticcontents.-Accordingtothe-U.S.-Departmentof-Energystatistics- andanalysis-[9],in-2015,theproductionofbiodieselinthe-United-Statesalonereached-1.2-×- 109gallons.-Thestatisticsalsoindicatedthatthecurrentbiodieselproductioninfirst-6months- of-2016isabout-21%higherthanthatobtainedin-2015,duringsameduration.-

#### **2. Feedstocks-**

#### **2.1. Conventional feedstocks-**

Triglycerides from oil-rich feedstock's, such as soybean, rapeseed, canola, sunflower, and palm,- have been commonly used due to their abundant availability [10–16]. The firstinitiation was- by the diesel engine inventor, Rudolf Diesel, who tested the use of peanut oil. However, natural- oils are viscose with inappropriate cetane number. Thus, the idea was not accepted and- vegetable oils were replaced by petroleum oil [7, 17–19]. The recent concern about limited oil- reservoirs and oil explosion increased activities recalled the attentionto use oil-rich feedstocks.- Oils dilution with solvent, thermal cracking, pyrolysis, micro emulsions and transesterification- has been suggested to overcome this viscosity limitation [20, 21]. Among these, transesterificationwith short chain alcohols, such as methanol and ethanol, in the existence of proper- catalyst is the preferred and commonly used approach.-

Although vegetable oils are available in large quantities, biodiesel production from these- vegetable oils competes with their use as food source, which results in increasing their prices- and affectfood market. In addition, cultivating oil-rich crops requires lands and freshwater. It- was reported that vegetable oil accounts for more than 60% of biodiesel overall production- cost [5, 22]. From that prospective, non-edible oils such as those from non-edible plants that- are not used in nutrition and can grow in the unfertile lands were suggested. However,- freshwater requirement still exist. The use of waste oils and fats have been recommended,- where there use is a waste management process [23], but contains large amount of free fatty- acids and water which increases the production cost. Furthermore, cannot satisfy the everincreasing global demands of diesel [24–26].-

### **2.2. Green feedstock-**

Choosing an inexpensive and more sustainable oil feedstock is the critical step to get costeffective biodiesel. Currently, microalgae, which are micro-organisms, received a promising- attention.-Due to their high oil content and growth rate, they have been considered as potential- feedstock that can replace the conventional diesel [27–31]. Furthermore, cultivation of microalgae cells does not require land development neither freshwater. Several algae strains were- found to grow in seawater and wastewater. The oil contents of such feedstocks are usually- between 20% and 50% and reach be in some strains to 80% in dry basis. The strain may also- change its composition by altering the growth conditions, such as light, nutrients, and- temperature. The stressful environment usually results to higher oil productivity.-

More interestingly, microalgae cells contain of protein, carbohydrates and lipids, which extend- the application domains of produced biomass from food to biofuel. Microalgae cells are also- used for CO2mitigation. However, to use microalgae biomass for biodiesel production, several- steps have to be carried out, which are strain selection, biomass production and harvesting,- and oil extraction and conversion.-

#### **3. Production technologies-**

Oils derivatization by transesterificationis the most common approach used commercially.- Typically, transesterificationis a reaction between the oil and a short-chain alcohol that results- to form an esters mixture and glycerol, as side product. The reaction is commonly take place- in the presence of catalyst that can speed up the reaction, where three moles of alcohols are- needed to react with 1 mol of the oil. Higher alcohol to oil molar ratios than the stoichiometry,- however, is usually employed to produce more biodiesel.-

### **3.1. Conventional catalysts-**

As mentioned earlier, transesterificationreactions are chemically catalyzed, which can be- either base or acid catalysts, depending on the oil quality free fattyacids (FFAs) and water- contents). Alkali catalysts, such as sodium hydroxide (NaOH) and potassium hydroxide- (KOH), are the commonly used, due to their low cost and high achievable yields of more than- 98% within a hour at reasonable temperature of 60°C [7, 32]. The reaction starts by preparing- the alkoxide solution and charging it to the reactor with oils. The reaction is then heated to the- reaction temperature for few hours. Products are then separated by gravity and crude biodiesel- is obtained, which needs further washing to recover unreacted oils and alcohols. In addition- to neutralization to receive the catalyst used.-

Although the process is simple and commercially used, it is not practical with feedstocks- containing high free fatty acids (FFAs) and water contents such as those from non-edible and- waste oil due to soap formation that lowers the overall production yield and require large- amount of catalysts [33, 34]. Pretreatment of oil prior transesterification by acid esterification- have been suggested, where sulfuric acid (H2SO4) is commonly adopted. Although it could be- beneficialin enhancing oil quality, the process very slow and requires large amount of alcohols.- In addition, acids are corrosive [5, 17, 35].-

#### **3.2. Green catalysts-**

The use of enzymes, which are green catalysts, has been suggested. Among the several- available enzymes, lipases (EC 3.1.1.3), which are of hydrolytic enzymes received an increasing- attentionin biodiesel production. Lipase-catalyzed biodiesel production due to their ability to- act on ester bonds, at mild temperatures with less energy needs [35].-

Lipases, namely the non-specificone, can convert oils from differentsources, including from- hose cooking, without any pre-treatment needs with easy product separation and no soapformation. Among the several studied, lipases from *Candida antartica-*[36–44], *Pseudomonas- fluorescens-*[45, 46], *Pseudomonas cepacia* [47], *Candida rugosa* [48–50] and *Rizhomucor miehei* [51,- 52] are commonly used.-

Although lipases are superior compared to chemical catalysts, accumulation of glycerol which- is a by-product negatively affectthe enzyme activity and reaction yield. Glycerol accumulation- increases reaction mixture viscosity and forms hydrophilic layer around rhe enzyme, preventing the reaction substrate to reach enzyme active site [53, 54]. The highest glycerol- inhibition effectwas found when silica, which has the highest micro-pores structure, was used- in the immobilization protocol of the lipase. Continuous removal of produced glycerol from- reaction mixture and/or using *tert*-butanol as solvent was proposed [55, 56]. The used of silica- gel that can absorb glycerol is also advantageous in such case [57].-

The activity of the enzyme was also found to decrease when more than 1.5 molar equivalents- of alcohol is used. This is because at certain concentration, alcohols which are hydrophilic- becomes insoluble in oils and tends to strip-offthe hydration layer of water from the lipase.- Therefore, inhibition and lose in activity [2, 58–60]. Numerous solutions have been proposed- to overcome short-chain alcohols inhibition limitation. These include step-wise alcohols- addition [38, 61], use of acetates as acceptors [57, 58, 62], lipase pretreatment and activity- enhancement [39], use of genetically modifiedmethanol-tolerant lipase and improving the- polarity of the reaction medium using organic solvents. The latteris commonly adopted- method.-

On the other hand, enzymatic biodiesel production is not yet commercialized due to enzymes- high costs. Immobilization of the lipase is usually considered to re-use the enzyme in several- cycles. Immobilization can also enhance the stability. For example, Novozym®435, which is- an immobilized enzyme form of *Candida antartica,*was reused for 12 continuous cycles without- any detectable loss in the activity [57] when the non-edible oil from Jatropha was transesterified- with methanol. Whereas, when *tert*-butanol was used as reaction media Novozym®435 activity- was maintained for 200 cycles [63].-

#### **4. Reaction medium-**

The solvent-free reaction systems are always the preferable one in enzyme catalyzed processes,- however when the reaction is catalyzed by a lipase the use of solvents is essential to prevent- the inhibition. By introducing hydrophobic solvents to the reaction, the solubility of reaction- substrates increases resulting in reduced inhibition effect of hydrophilic substrates/products.- In addition, the viscosity and transport limitations of reaction mixture to enzyme active sites- decreases, which results in increased reaction yield [64, 65].-

### **4.1. Conventional organic solvents-**

Numerous organic solvents have been used in biodiesel production, where the hydrophobicity- was considered as main factor in selecting the proper solvent [66]. It was found that thebiodiesel production rate increases with the increase in the hydrophobicity of the solvent used,- and hydrophilic solvents resulted tend to strip‐offthe bound water from the enzyme surface- is used [39, 67–70]. Generally, the stripping was reported to take place when an organic solvent- with Log P (hydrophobicity) <2 used. *n*‐Hexane, which has log *P* = 3.5, has been commonly- used, where its effecton enhancing the production yield, compared to solvent‐free system,- was observed in several studies. These includes the work of Nelson et al. [36], who tested the- effect of using *n*‐hexane in tallow fats transesterification with methanol at 3:1 methanol to oil- molar ratio when catalyzed by *Mucor miehei*lipase. High yield reaching 95%, compared to 19%- in solvent free, was obtained.-

*tert-Butanol*is another solvent used in lipase catalyzed biodiesel processes. It has been selected- as a capable alternative to *n*‐hexane that cannot dissolve glycerol and minimize its inhibition- effect [1, 13, 71–74]. A high methanol to oil molar ratio of 6:1 could be reached in soybean oils- transesterificationwith Novozym®435, resulting in 60% yield, compared to only 10% in- solvent‐free system.-

#### **4.2. Green solvents-**

Although organic solvents enhance the production yield, an additional downstream unit is- required to separate the solvent from the products, resulting in an additional production cost.- Moreover, organic solvents are toxic and volatile and their use could pose several environ‐ mental issues that should be minimized. Efforts have been made to find alternative non‐toxic- and environmental benign solvents. In this regard, supercritical CO2and ionic liquids (ILs)- have been suggested.-

### *4.2.1. Supercritical carbon dioxide-*

 Supercritical fluidsare fluidsat temperatures and pressures above their critical points. They- have been used in several applications. Among the differentfluids,supercritical carbon- dioxide (SC—CO2) and is been the most commonly used. Supercritical CO2is a non‐toxic and- cheap fluid that appear in abundant with moderate critical parameters [75]. Compared to- organic solvents, SC—CO2has liquid solubilization capacity and gas diffusivityand viscosity,- where small changes in the conditions can lead to a significantincrease in the properties. These- unique physiochemical properties allow it to be used in several applications, including- separation and reaction [76–78]. Moreover, easy products separation can be achieved using SC- —CO2.-

Although SC—CO2has been commonly used in esters transesterificationin the presence of- lipase, its employment in biodiesel production is still new [79]. The compatibility of SC—CO2- with lipases is well recognized, and by using it in biodiesel production, the mass transfer of- reaction substrates into enzyme active sites would be enhanced. In spite of the high pressure- uses, it was clearly verifiedthat it has minimal effecton enzyme inhibition at pressure less than- 200 bars [80, 81]. Comparable yields to organic solvent were achieved when palm kernel and- Jatropha oils were transesterifiedin the presence of Novozym®435 in SC—CO2-[75, 82–84].- Higher yield of 80% was obtained when SC—CO2was used in microalgae lipids transesterification in the presence of same lipase [42].-

Supercritical CO2has been also used to extract oils for biodiesel production, such as those oils- from vegetable crops [76, 77], microalgae cells [85–90] and fats from animal meat. The use of- SC—CO2is adopted to minimize the use of toxic solvents and utilize the leftover, after- extractions, in other applications such as in food and pharmaceutical industries, unlike *n*hexane which is toxic and its use is an energy intensive process. Its effectiveness depends on- the selected extraction conditions; namely the temperature, pressure and flowrate, where- increasing the pressure increases SC—CO2density and the extraction yield whereas the- temperature has two opposite effectthat become equal at crossover pressure. By increasing- the extraction temperature, SC—CO2density decreases and reduces its capability to solubilize- the desired solute, while solute vapor pressure increases resulting in more solutes extraction.- Several studies had considered the effectivenessof using SC—CO2. For example, similar yields- performance of *n*-hexane were reported for oils extraction from *Spirulina platensis-*[86], *Spirulina- maxima-*[87] and *Pavlova*sp. [89] microalgae cells. A higher efficiency was reported in extracting oils from *Chlorococum* sp. and *Nannochloropsis* sp. [90, 91].-

As mentioned earlier, SC—CO2has many advantages. However, high pressure is needed for- pumping and reaching the supercritical state of CO2, making the process costly. Depressurization to separate the biodiesel from enriched SC—CO2 could negatively affectlipase structural- confirmation,therefore its stability. To minimize the effect,continuous operation has been also- considered. It has been successfully employed for soybean [92], corn oil [93, 94], microalgae- and sunfloweroils [95]. Taher et al. [55, 56] had stated that the feasibility of using SC—CO2for- energy production is not evident, but combining oil extraction conversions to biodiesel in SC- —CO2in one integrated system would be feasible and the additional pumping cost for energy- production could be justifiedand make the overall process more feasible [42, 44, 96]. On the- other hand, the presence of water could results to carbonic acid formation that change the- reaction pH and denaturant the lipase. CO2 may also react with the amine groups on the surface- of lipase to form carbamates [97].-

### *4.2.2. Ionic liquids-*

 Ionic liquids (ILs) are liquids of low crystallization tendency. They are composed of cations- and anions and distinguished from conventional solvents in their non-vapor pressure feature.- Thus, known to as "designer solvents." They have developed as green alternative solvents to- replace the conventional volatile solvents in several process, including biodiesel production.- The firstattemptto use them in lipase catalyzed reactions was with [bmim][PF6] and [bmim]- [BF4], which were used in several reactions, including transesterification [98]. The selection the- proper IL depends on its effectto enhance the enzyme activity reaction substrates/products- solubility [99–103].-

Typically, by judicious selection of the alkali chain on the cation and anion group, the physiochemical properties of designed IL can be tuned. For example, symmetric and shorter alkyl- chains cations in the IL result in a higher melting temperature than those with asymmetric- cations [104, 105], and increasing chain branching results in an increased the melting point[106, 107]. However, it decreases with the increase in anion size. On the other hand, ILs with- symmetric and fluorinatedanions, have high viscosity, which is not preferable in enzyme- catalyzed reactions. Ionic liquids based on cations with aromatic phenyl ring also have high- viscosity as well [108].-

The miscibility of the reaction substrates with the IL and IL hydrophobicity are main factor- affectingthe overall reaction yield, where the high solubility of reaction substrates to enhance- reaction rate and low solubility of the biodiesel in the IL are the desired features in biodiesel- production. The hydrophobicity of the IL depends mainly on the anions used. For example,- [PF6 − ] and [Tf2N− ] anions, which are hydrophobic results in making hydrophobic ILs, where- those with hydrophilic anions, such as [Cl− ], [Br− ], [I− ], [NO3 − ], [CH3COO− ] and [CF3COO− ] for- hydrophilic ILs. The hydrophobicity of ILs can also be affectedby the length of the alkyl chain- on the cation, in which longer alkyl chain results in a more hydrophobic IL [109–111]. Similar- to of organic solvents, hydrophobic ILs are preferable in enzyme catalyzed biodiesel process,- wherein hydrophilic ILs may strip‐off the essential hydration layer and deactivate the lipase.- Moreover, the nucleophilicity of the anion used in the IL combination affectlipase activity and- stability, where high nucleophilicity of an IL may affectlipase structure activity interacting- with the positively charged sites in lipase [112].-

Among the several tested ILs in biodiesel production, [PF6 ‐ ] and [NTf2 ‐ ] based ILs are com‐ monly used. For example, [bmim][PF6] and [emim][PF6] where used in sunflower oil transes‐ terification-Novozyme®435. High yield, reaching 98%, was achieved in [emim][PF6] due to its- higher hydrophobicity, however, insignificantproducts were obtained when [BF4 − ]‐based ILs- were tested [113]. The high yield obtained in [emim][PF6] compared to [bmim][PF6] is due to- the ability of long‐chain cation‐based ILs to dissolve reaction substrates, thus the reaction take- place in a two‐phase system resulting in moderate efficiency.-

In addition to ILs uses in lipase catalyzed reaction, they have been employed as green catalyst- to overcome the reaction complication and product purificationissues in chemical catalyzed- reactions. The brønsted acidic ILs [PY(CH2)4SO3H][HSO4] and [CyN1,1PrSO3H][Tos] was found- to be effective in transesterifying cottonseed-(92% yield) and coconut (98% yield) oils, respec‐ tively, where comparable to that using concentrated sulfuric acid were obtained [114, 115].- Similar yield was also obtained from esterificationof long‐chain free fatty acids in [NMP]- [CH3SO3] [116]. In addition, biodiesel yield of 87 and 97% were also achieved using the basic- IL [bmim][OH] [117] and [hmm][OH], respectively [118].-

Ionic liquids have been used to extract oils, commonly from microalgae cells as they can be- used with wet cells without the need cell walls disruption. In such processes, hydrophilic ILs- are used where they have the capability to dissolve algal cell components leaving the oils- insoluble and float. The extraction from wet cells of *Chlorella vulgaris* was tested using IL [emim]- [DEP], where 40% higher than *n*‐hexane‐methanol (7:3 v/v) mixture was obtained [119]*.* The- effectof adding a polar solvent with the IL was also evaluated [120]. For example, a mixture- of [emim][CH3SO4] and methanol was tested with *Chlorella sp.* cells containing 70% water, and- an yield of 75% was achieved at 1:1.2 (w/w) solvents ratio.-

The main challenge of employing ILs at industrial scale is in their high costs. Therefore, the- recycling step is important. In addition, when long alkyl chain on cation-based ILs is used, the- separation step is not easy and continuous recovery of biodiesel from reaction mixture as they- produced is vital. Combination ILs with SC—CO2-(IL-SC—CO2) has been recently suggested,- where biodiesel can be recovered using SC—CO2in an effective manner. Such system was- tested for biodiesel production from triolein using Novozym®435 in different-ILs and high- yields reaching 98% was obtained after 6 h [121].-

#### **5. Conclusions-**

Biodiesel production using chemical catalysts and solvents from received the attentionto- replace conventional diesel fuel. However, the process is not commercialized due to many- shortcomings raised. The employment of green catalysts and solvents, either by SC—CO2or- by ILs has been suggested to several technical restrictions. The use of integrated processes that- combine the use of differentgreen catalysts and solvents in a one process to enhance product- separation and solvent recover is discussed.-

#### **Author details-**

Hanifa Taher1\* and Sulaiman Al-Zuhair2-


### **References-**


## **Biogas, Biodiesel and Bioethanol as Multifunctional Renewable Fuels and Raw Materials**

Venko Beschkov

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65734

### **Abstract-**

 Nowadaystheworldeconomyisbasedmainlyonpetrolasanenergysourceandraw- materialforchemicalproducts.-Theglobaleconomicgrowthinthepastcenturyhasled- tohighenergyconsumption,mainlyfromfossilfuels,suchascoal,oil,andnaturalgas.- The extensive use of fossil fuelsformed and stored underground formillions of years has- madeimpossibleforthepresentvegetationon-Earthtotreattheemittedcarbondioxide- byphotosynthesis,leadingtostrongemissionsofcarbondioxideandgreenhouseeffect- withtheconsequentclimatechanges.-Oneofthewaystocopewiththisglobalproblem- istoclosethecarboncycleinnaturebytheuseofrenewablebiofuelsenablingrecycling- thesourcesofbiologicaloriginbyenergyproductionandconsumptionoftheresulting- carbonbyphotosynthesis.-Someofthesebiofuelsarebiogas-(amixtureofmethaneand- carbondioxide)generatedfromorganicwaste;ethanol,producedbyfermentationof- carbohydrates;andbiodiesel,producedbytransesterificationoflipids.-Anotherfeature- ofthisapproachistheutilizationoforganicwasteasenergy,thusleadingtomultiple- benefitsfortheenvironment:wastetreatmentwithenergyproduction,closingthenatural- carboncycle,andsavingoffossilfuels.-Biofuelswiththeirfeedstockalsoserveasraw- materialsfornewtechnologiesforchemicalsbeingnowproducedfrompetrol,natural- gas,andcoal.-

**Keywords:** biomass, renewable fuels, biogas, biodiesel, ethanol, carbon dioxide recy‐ cling-

### **1. Introduction-**

 Duringthetwentiethcentury,theworldeconomywasmainlybasedonpetrolasanenergy- sourceandrawmaterialforchemicalproducts.-Energyconsumptionhasincreasedsteadily-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 overthelastcenturyduetotheworldpopulationgrowthandthetechnicalprogressdevel‐ opment.-Theenormouseconomicgrowthonaglobalscaleinthepastcenturyhasledtothe- extensiveuseoffossilfuels,suchascoal,oil,andnaturalgas.-

A reason of concern was the extensive use of fossil fuels formed and stored underground for- millions of years. It has made impossible for the present vegetation on Earth to treat the emitted- carbon dioxide by photosynthesis. The result was greenhouse effectwith the consequent- climate changes. The climate changes assigned to the increased emissions of greenhouse gases- forced humanity to develop alternative energy sources, one of them being biomass, either fresh- or residual.-

Another reason for humanity to turn to the renewable energy resources is the concern of- depletion of the overall oil reserves [1], because the eight great economies (except Brazil) and- many other nations depend on oil, the consequences of inadequate oil availability could be- severe. Therefore, there are great incentives in exploring alternative energy sources.-

One of the ways to cope with this global problem is to close the carbon cycle in nature by the- use of renewable fuels enabling recycling the sources of biological origin by energy production- and consumption of the resulting carbon by photosynthesis. Such biofuels are biogas (a- mixture of methane and carbon dioxide) generated by anaerobic digestion of organic waste,- ethanol, produced by fermentation of carbohydrates, and biodiesel, produced by transesteri‐ fication of lipids.-

The main feature of these approaches is the utilization of organic waste as energy, thus leading- to multiple benefitsfor the environment: waste treatment with energy production, closing the- natural carbon cycle, and saving of fossil fuels. Moreover, there are options to utilize the- biofuels and their derivatives and residues as sources for chemical production.-

#### **2. Biogas-**

The anaerobic digestion of organic waste is a well spread process in nature. The huge amounts- of natural gas collected underground are formed by this process during millions of years. The- result is gas, containing about 95% methane with some contaminations. Nowadays, this- process is used for agricultural waste treatment producing biogas with satisfactory heating- capacity. Biogas is a mixture of methane and carbon dioxide with some contaminations of- hydrogen sulfide,mercaptans, ethane, etc. The methane content varies from 55 to 90% volume- depending on the substrate nature and content, the method of digestion, etc. The gas contain‐ ing less than 50% methane is not combustible.-

Biogas is broadly distributed in countries with developed agriculture (like India, China, Brazil,- etc.), being a cheap and environmentally friendly option for the simultaneous solution of waste- treatment problems and energy demand. Anaerobic digestion is also a convenient technology- for activated sludge utilization and waste treatment in the food industry, pulp and paper- industry, in household waste treatment, etc.-

The anaerobic digestion with biogas production is a complicated process of consequent- hydrolysis of organic macromolecules (carbohydrates and proteins) to oligosaccharides and- peptides, acidogenesis to volatile fatty acids (mainly formic, acetic, and propionic), acetogen‐ esis, and methanogenesis [2]. The overall process is shown in the scheme in **Figure 1**.-

**Figure 1.** Four steps in biogas production [2].-

The gross chemical reaction describing the first step, i.e., hydrolysis is:-

$$\text{n(C}\_6\text{H}\_{10}\text{O}\_5)\text{n} + \text{nH}\_2\text{O} = \text{nC}\_6\text{H}\_{12}\text{O}\_6\tag{1}$$

In the second step, acidogenic bacteria convert the products of hydrolysis into simple organic- compounds, mostly short‐chain carboxylic acids, ketones and alcohols.-

The chemical reactions are shown below. Glucose is parallely converted into ethanol and- propionic acid:-

$$\rm C\_6H\_{12}O\_6 \leftrightarrow \rm 2CH\_3CH\_2OH + 2CO\_2 \tag{2}$$

$$\rm C\_6H\_{12}O\_6 + 2H\_2 \leftrightarrow 2CH\_3CH\_2COOH + 2H\_2O \tag{3}$$

The acetogenic reactions are-

$$\rm H\_3CH\_2COO^- + \rm 3H\_2O \leftrightarrow CH\_3COO^- + H^+ + HCO\_3^- + \rm 3H\_2 \tag{4}$$

$$\rm C\_6H\_{12}O\_6 + 2H\_2O \rightleftharpoons 2CH\_3COOH + 2CO\_2 + 4H\_2 \tag{5}$$

$$\text{CH}\_3\text{CH}\_2\text{OH} + \text{O}\_2 \rightleftharpoons \text{CH}\_3\text{COOH} + \text{H}\_2\text{O} \tag{6}$$

$$\text{H}\_2\text{HCO}\_3^- + 4\text{H}\_2 + \text{H}^+ \equiv \text{CH}\_3\text{COO}^- + 4\text{H}\_2\text{O} \tag{7}$$

In the last step, methanogenic bacteria convert acetic acid into CH4 and CO2:-

$$\text{CH}\_3\text{COOH} = \text{CH}\_4 + \text{CO}\_2 \tag{8}$$

There is another parallel pathway to produce methane by reduction of carbon dioxide resultingfrom formic acid degradation:-

$$\text{CHCOOH} \leftrightarrow \text{CO}\_2 + \text{H}\_2 \tag{9}$$

$$\text{CH}\_2 + 4\text{H}\_2 \leftrightarrow \text{CH}\_4 + 2\text{H}\_2\text{O} \tag{10}$$

The activity of the digesting bacteria and biogas production of gas is most rapid in two- temperature ranges: between 29°C and 41°C (fermentation is known as mesophilic) or between-49°C and 60°C (the thermophilic range). The mesophilic regime between 32°C and 35°C is morereliable for stable and continuous production of methane. Biogas produced outside thistemperature range is rich of carbon dioxide, it is not combustible and that is why it has nocalorificvalue. The thermophilic regime gives higher yield of biogas, but with less net energy- efficiency because of the energy losses for high temperature maintenance.-

Differentmethanogenic strains are responsible for these parallel and competitive processes.-The bacteria from the genus *Methanosarcina* are capable to grow during the catabolism ofacetate to CO2and CH4 [3], whereas the strains *Methanobacterium* and *Methanobrevibacter* convert carbon dioxide by reduction of hydrogen to methane [4–8].-

The balance between decarboxylation of acetic acid and carbon dioxide reduction is importantfor the methane content in the resulting biogas. If only decarboxylation of acetic acid occurs,- methane content will be 50% only. The high methane content in the biogas means that carbon- dioxide reduction prevails.-

Anyway, all methanogenic strain are vital in neutral media, i.e., for pH values between 6 and- 8. Big deviations either in the acid domain or in the alkaline one lead to strong inhibition and- even to death. Acidogenesis is one of the inevitable steps in biogas production. On one hand,- methanogenesis is favorized by fattyacid formation, but on the other hand, it could be strongly- inhibited by their accumulation due to the pH drop. In such cases, the produced gas is very- rich in carbon dioxide and it is not combustible. That is why, one must be very careful in the- feeding strategy by substrate and in the selection of bioreactor and flow organization.-

One suitable way to minimize the effectof acid accumulation on the biogas formation is to- distribute spatially the consecutive processes of biogas formation and to carry them out- simultaneously. Such a construction is the baffledbioreactor separated into consecutive- compartments fed from the one end with outflowat the other one, cf. **Figure 2**. It is known- that such reactors are stable toward disturbances in feed, pH oscillations, temperature- variations, etc. [9].-

**Figure 2.** Multistage bioreactor for biogas production.-

The main advantage of this type of reactor in the considered case is the distribution of the- differentconsecutive processes (hydrolysis, acidogenesis, acetogenesis, and methanogenesis)- in differentreactor compartments. Due to its one‐way feeding, the intermediate products in- one compartment passed as substrates to the next one. Because of this feeding organization,- differentbacteria are spontaneously cultivated, specialized to transform different intermedi‐ ates of the overall methanogenesis.-

There are successful applications of such bioreactor for biogas production by residual stillage- from ethanol distillation as a feed [10]. The intermediate profileand the microbial distribution- in eight compartment bioreactor for this process are illustrated in **Figure 3**by author's- experimental data. Acetic and propionic acids prevail in the firstthree compartments wherereducing sugars are present, due to the hydrolysis of carbohydrates. Obviously, the first three- steps of biogas production, i.e., hydrolysis, acidogenesis, and acetogenesis predominantly take- place in the firstthree compartments where sugars are present and the concentration of- methanogenic bacteria is very low or negligible. Methanogenics prevails in the next compart‐ ments 5–7, which corresponds to the very low acid concentration. There are few methanogenic- bacteria in compartment 8 which corresponds to the negligible acid concentration. The biogas- productivity rate for those experiments was ca. 4 vol.biogas/vol.reactor/day.-

**Figure 3.** Acid and microbial profiles along the compartments in the bioreactor (own data).-

The biogas produced by anaerobic digestion findsapplications in differentarea. First, it couldbe used directly for heating purposes. Next, after some processing to remove carbon dioxideand sulfur‐containing compounds the biogas could replace partially the natural gas for localapplications. This biogas could be supplied directly in the pipelines, it could be used for thepublic transport and for electricity production by cogeneration.-

Another promising application is the direct electricity production in fuel cells [11]. For thispurpose, biogas should be scrubbed for carbon dioxide and sulfur compounds removal andthen the purified methane could be fed to solid oxide fuel cells (SOFC) and molten carbonatefuel cells (MCFC). In this case, methane is directly converted to hydrogen and carbon monoxideby a catalyst in the anodic space. Another approach is to convert methane into carbon mon‐ oxide by steam reforming (SR) or partial oxidation reforming (POX) and consequent water‐ gas shift reaction to isolate hydrogen, supplied to a fuel cell:-

$$\text{CH}\_4 + \text{H}\_2\text{O} = \text{CO} + \text{3H}\_2\text{(SR)}\tag{11}$$

$$\text{CH}\_4 + \%\text{O}\_2 = \text{CO} + 2\text{H}\_2(\text{POX}) \tag{12}$$

### **2.1. Biogas as a source of organic fuels-**

There are some papers claiming to utilize biogas as a source for other organic fuel production- by catalytic auto‐thermal reforming [12, 13]. Another approach is to use biogas being a mixture- of methane and carbon dioxide to produce synthesis gas (mixture of carbon monoxide and- hydrogen) [14]:-

$$\text{CH}\_4 + \text{CO}\_2 = 2\text{CO} + 2\text{H}\_2\tag{13}$$

Furthermore, the synthesis gas could be converted into light hydrocarbons by the Fischer‐ Tropsch process.-

### **3. Biodiesel-**

Biodiesel consists of methyl or ethyl esters of fattyacids produced by transesterificationof- natural lipids. Differentnatural fats are used as raw materials, namely, rapeseed, soybean,- processed residual sunfloweroil, animal fats, and some kinds of algae. The latterare attractive- because they can utilize the carbon dioxide from fluegases by photosynthesis thus reducing- the emissions of greenhouse gases [15].-

The energy content of biodiesel is within 37 and 40 MJ/L compared to 46 MJ/L of the traditional- diesel fuel. Biodiesel does not contain sulfur compounds.-

The idea for the use of vegetable oils as fuel for diesel engines is more than 100 years old [16,- 17]. Just in the 1970s, the petrol crises and the enhanced environmental conscience in the- modern societies have led to the secondary discovery of this possible alternative to the- hydrocarbon‐based fossil fuels. However, the direct use of vegetable oil as a fuel is not- convenient, because of its very high viscosity, high flame point, trend to polymerization, etc.,- all leading to engine damage [18].-

Transesterificationwith low alcohols is the best modificationof natural oil for the biodiesel- purposes.-

Today, biodiesel is in commercial use throughout the world. It is used as a single fuel or blended- with traditional diesel (with 30–36%).-

Biodiesel is produced in the European Union since 1992. The world production attained-3.8- mln tons in 2005 to reach 3.7 mln tons only in the USA in 2007. The total world production for- 2016 is about 15 mln tons.-

#### **3.1. Benefits of biodiesel use-**

Biodiesel does not contain sulfur and aromatic compounds and its use in the conventional- engines leads to reduction of emissions of noncombusted hydrocarbons and carbon monoxide.- Comparison of the emissions resulting by the use of biodiesel and traditional one is shown in- **Table 1**.-


**Table 1.** Comparison of emissions released by biodiesel and conventional diesel fuel [19].-

#### **3.2. Problems in biodiesel production and use-**

The main disadvantage at biodiesel production and use is the uncertain standardization- depending on the source of lipids. It reflectsthe differentcetane number and the variable- temperature of gelatinization depending on the esters and the raw material type.-

Another severe problem is crude glycerol, released as byproduct after transesterificationof- lipids. Its amount is about 10% of the substrate and it is almost equal of the methanol used.- This residual glycerol is contaminated by potassium hydroxide, water, some nonreacted lipids,- some soaps, and monoglycerides and diglycerides. The low quality of this product makes it- impossible for direct practical application.-

Provided the annual world production of biodiesel is about 15 mln metric tons, one could- expect that 1.5 mln metric tons of crude glycerol would be released. It is an enormous amount- and it poses the necessity for its application and processing.-

Pure glycerol has various practical applications but it could be hardly replaced by the residual- crude glycerol after biodiesel production. That is why new application should be sought.-

 Recentstudiesshowtheopportunityforcrudeglycerolutilizationassyngasbysteamre‐ forming [20–22], cf. Eq. (9). Other applications are proposed,for example, hydrogen produc‐ tionbyphoto‐fermentation-[23,-24],orasafuelinfuelcellsandmicrobialfuelcells-[25,-26].- However, in these cases the contamination by methanol is not recommended [27, 28].-

#### **3.3. Glycerol utilization for hydrogen and other chemicals production-**

The large amounts of residual crude glycerol prompted to the search of simultaneous waste- treatment and for new applications as a raw material, alternative to the petrol for the traditional- organic synthesis [29–31]. Such effortsare directed toward production of chemicals of broad- industrial importance, e.g., polyols as precursors of plastics (2, 3‐butanediol, 1, 3‐propandiol)- [32–35], propionic acid [36, 37], succinic acid [38], or hydrocarbons by catalytic reforming [39],- for epichlorohydrin, some ethers [40], polyesters, etc.-

Among the potential applications of waste glycerol are the production of biodegradable- polymers for packaging [31, 32, 41, 42], as antifreezing agents [43] as substrate for microbial- syntheses, etc. Crude glycerol has been used as carbon source in the nutrition media for- biopolymer production by the species *Bacillus* and *Pseudomonas* [44–46].-

#### **3.4. Microbial conversion of glycerol into chemical products-**

The bacterium *Rhodopseudomonas palustris*is capable for photo‐fermentative conversion of- crude glycerol to hydrogen [23]. The conversion rates and the yields depend on the concen‐ trations of the added nitrogen containing compounds. Higher yields of hydrogen and also- ethanol are registered at the use of *Enterobacter aerogenes-*HU‐101 [47]. There are works on the- production of different chemicals from glycerol in microbial processes. Different bacteria (from- the genera *Klebsiella, Clostridium*, and *Enterobacter*) are capable to convert glycerol, producing- basic chemicals, differingby the intermediate reactions and products [30]. The metabolic- pathway of glycerol conversion by bacteria from the genus *Klebsiella*was proposed and- discussed by Saxena et al. [48] and Zhang et al. [49]. It is shown in **Figure 4**. It is seen that two- diols (1,3‐propandiol and 2,3‐butanediol) are produced by two competitive mechanisms.- Those two diols are interesting as precursors for polymer production, e.g., polypropylene and- butadiene. Besides succinic and lactic acids are produced, ethanol too.-

**Figure 4.** Metabolic pathway for glycerol digestion by bacteria from the genus *Klebsiella* [48, 49].-

The studies of glycerol conversion at the metabolism of bacteria from the genus *Clostridium* show similar processes like in the previous case [50–52]. At *Clostridium* mainly 1,3‐propanediol,- organic acids (formic, acetic, butyric, and lactic) as well as n‐butanol are produced.-

Additionally, formic and acetic acids are also produced. These two carboxylic acids are very- important for the consequent production of biogas being a mixture of methane and carbon- dioxide.-

The studies of the metabolism of *Enterobacter* bacteria show predominant formation of ethanol- and hydrogen [53–55].-

### **3.5. Glycerol for biogas production-**

The microbial production of acetic and formic acids from glycerol is interesting with the- relationship of biogas production by anaerobic digestion. The two main pathways for biogas- production by methanogenic bacteria are based on acetate decarboxylation, or carbon dioxide- reduction by hydrogen, both produced from formic acid decomposition, cf. Eqs. (8) and (10).-

Conversion of glycerol into biogas by anaerobic fermentation is an interesting option to- produce renewable energy together with waste glycerol treatment [56–58]. It is reported that- glycerol considerably enhances biogas formation by properly selected microbial population- [59]. There are also many studies for the glycerol impact on biogas yield from various sub‐ strates, such as cattledung [60–62], pig manure [57, 62, 63], activated sludge [64–66], as well- as at more complicated mixtures of cellulose and household waste [57, 58, 67].-

In any case, the results are considerable enhancement of biogas yield from 180 to 400% with- respect to the reference substrate. It is typical, however, that the amounts of the added glycerol- are restricted to 1–4% wt. from the main substrate. Addition of bigger amounts of glycerol- leads to strong acidificationof the broth and inhibition of methanogenesis [39, 56]. It means- that no considerable amounts of crude glycerol could be utilized as biogas.-

However, it was reported recently that crude glycerol may serve as a single substrate for biogas- production with pretty high yield, i.e., 0.345 L biogas/g COD [68].-

For attainmentof maximum efficientbiogas production differentschemes of bioreactor feed- are studied, as well as choice of the reactor construction, flow organization, etc.-

### **4. Ethanol-**

Ethanol is a renewable energy source produced through fermentation of sugars. Ethanol is- widely used as a partial gasoline replacement worldwide. Fuel ethanol that is produced from- corn has been used in gasohol or oxygenated fuels since the 1980s. These gasoline fuels contain- up to 10% ethanol by volume. As a result, the US transportation sector now consumes about- 4540 million liters of ethanol annually, about 1% of the total consumption of gasoline. Recently,- US automobile manufacturers have announced plans to produce significantnumbers of- flexible‐fueledvehicles that can use an ethanol blend with 85% ethanol and 15% gasoline byvolume—alone or in combination with gasoline. Using ethanol‐blended fuel for automobiles- can significantly reduce petroleum use and exhaust greenhouse gas emission.-

Ethanol is also a safer alternative to methyl tertiary butyl ether (MTBE), the most common- additive to gasoline used to provide cleaner combustion.-

However, the cost of ethanol as an energy source is relatively high compared to fossil fuels. A- dramatic increase in ethanol production using the current corn starch‐based technology (or- other cereals) may not be practical for small countries because corn production for ethanol will- compete for the limited agricultural land needed for food and feed production. Additional- drawback is the increasing prices of cereals used extensively as substrate for ethanol produc‐ tion by fermentation due to the enhanced demand and thus puttingthe third‐world countries- in disadvantaged position.-

An alternative potential source for low‐cost ethanol production is to utilize lignocellulosic- biomass (LCB) such as straw, stems, cobs, grass, sawdust, wood chips, and forestry waste. This- approach is known as "second‐generation" ethanol production. Extensive research has been- completed on conversion of lingo‐cellulose to ethanol in the past two decades [69–74].-

The conversion includes two main steps: pretreatment with hydrolysis of cellulose in the LCB- to fermentable reducing sugars and fermentation of the sugars to ethanol [75].-

 The purpose of pretreatment is to remove lignin and hemicellulose, reduce cellulose crystal‐ linity, and increase the porosity of the materials. Physical, physical‐chemical, chemical, and- biological processes have been used for pretreatment of LCB. The hydrolysis is usually- catalyzed by cellulolytic enzymes, and the fermentation is carried out by yeasts or bacteria.- The factors that have been identifiedto affectthe hydrolysis of cellulose include porosity- (accessible surface area) of the waste materials, cellulose fibercrystallinity, and lignin and- hemicellulose content [2, 75]. The presence of lignin and hemicellulose impedes the access of- cellulases to cellulose, thus reducing the efficiencyof the hydrolysis. Removal of lignin and- hemicellulose, reduction of cellulose crystallinity, and increase of porosity in pretreatment- processes can significantly improve the hydrolysis [76, 77].-

Another disadvantage of the enzymatic hydrolysis of cellulose is the strong product inhibition- of glucose and therefore the low‐product concentrations and the low‐process rate. The easier- but not environmentally friendly way is to use acid hydrolysis by sulfuric acid [78]. In this- case, higher concentrations of fermentable sugars and oligosaccharides are produced. The- usual approach is to employ a two‐step dilute acid hydrolysis, where the hemicellulose is- hydrolyzed to xylose and recovered in the firststage and a more vigorous second‐stage- hydrolysis is employed for conversion of cellulose to glucose [79].-

Several differentorganisms have been proposed for convert fermenting sugars into ethanol.- The mostly spread ones are the yeast, *Saccharomyces cerevisiae*, due to its robust growth rate- and high ethanol tolerance, up to 23% [80, 81]. The effortto use thermostable yeast has shown- that they suffer from low ethanol tolerance [82].-

The bacterium, *Zymomonas mobilis*, has been shown to produce higher ethanol yields but with- lower ethanol tolerance [82].-

After fermentation ends the "beer" containing 2–12% ethanol is subjected to distillation to- produce the azeotropic mixture of 96% (vol.) ethanol and 4% water. This mixture is not- appropriate for blending with gasoline for the water separation, particularly at low tempera‐ tures. That is why additional drying is required to reach water content of less than 1%.-

The classical methods are extractive distillation by adding solvents like benzene, cyclohexane,- or ether to break the azeotrope.-

Most advantageous is the molecular sieve drying technology, where the azeotrope is passed- through a bed of synthetic zeolite with uniform pore sizes which preferentially adsorb water- molecules. After the bed becomes saturated, it is regenerated by heating or evacuating the bed- to remove the adsorbed water. The most efficienttechnology is the vapor‐phase "pressure- swing" adsorption molecular sieve process [83]. Nowadays, this process is preferred to the- classical extractive distillation due to the clean process and the lack of side chemical products- due to extraction and distillation.-

The problem to be solved is the stillage processing. Stillage is the waste after ethanol distillation- and it contains a lot of cellulose residues, nonfermented oligosaccharides, proteins, etc., with- COD reaching 70 kg/m3 . The stillage amounts are between 1 L/kg feedstock for cereals and 20- L/kg for cellulosic substrate from coniferous origin.-

There are differentways to treat this waste. One of them is to use it as animal feed after- evaporation and concentration. Another option is to use it as substrate for single cell protein- production with the subsequent use as animal fed of the residue. The simplest and the- straightforward method is to use stillage for biogas production by anaerobic digestion [84,- 85]. According to our experience, the produced biogas has over 70% (vol.) methane content.- The COD was decreased from 70 to 1 g/L (over 98% efficiency)and after some additional- treatment the wastewater could safely discharged or used for irrigation.-

There is a relatively new proposal for consolidated bioprocessing (CBP) of lingo‐cellulosic- materials consisting in cellulase production, substrate hydrolysis, and fermentation accom‐ plished in a single process by cellulolytic microbes [86].-

CBP offers the potential for lower biofuel production costs due to simpler substrate processing,- low energy inputs, and higher conversion efficienciescompared to separate hydrolysis and- fermentation processes. It is an economically attractive goal for "third‐generation" biofuel- production.-

#### **4.1. Ethanol applications as fuel and raw materials-**

The use of ethanol as fuel depends on the oil prices on the global market and the local- regulations in the different states. However, they are other options to use the "bioethanol" as- a substrate and a raw material for chemical purposes. Besides the well‐known applications as- commodity product, chemical reactant, and solvent, ethanol may serve as a source of hydrogen- production by steam reforming [87, 88] or chemical products, like ethylacetate [89, 90].-

#### **4.2. Problems and drawbacks-**

The main problems associated to ethanol production for fuel purposes are either from- economical or environmental point of view. The economic problems are related to the prices- of cereals competing its application as food. Therefore, the extensive use of cereals for- industrial or fuel purposes may be unfavorable for their alimental needs. Next, the demand of- new area for crop growing may lead to deforestation and disturbing the biodiversity and- environmental balance.-

The use of second‐generation raw materials for ethanol production (cellulose‐based waste) is- restricted due to environmental reasons. Not all of the waste lingo‐cellulosic biomass could be- safely converted into ethanol without disturbing the natural ecological processes. The- extensive use of lingo‐cellulose for ethanol production could lead to deforestation and threat- on biodiversity in large area of land. That is why decision making on the size and the rate of- lingo‐cellulosic waste use for this and for any other purpose should be very carefully, after- precise and thorough environmental analysis.-

#### **5. Carbon dioxide utilization-**

Unfortunately, each of the described processes of biomass utilization ends with the inevitable- release of carbon dioxide, resulting of the fuel combustion. Next, crops growing in industrial- scale require considerable input of energy, most frequently taken from oil‐based fuels. That is- why abiotic carbon dioxide utilization and conversion is a major task for the future research- and technology of biomass‐based renewable fuels. There are two trends for this challenge:- utilization photosynthesis by vegetation and recycling the biomass by chemical or electro‐ chemical reduction to organic fuels, like methane.-

The firstone is to pass fluegases containing carbon dioxide through greenhouse area contain‐ ing algae capable to produce lipids and other organics being biofuels. Algae can be grown in- open ponds, closed‐loop systems, and photo‐bioreactors. Algae are capable of much higher- yields with lower resource inputs than other feedstock and that is why they are moved to an- own category. The following biofuels could be produced by algae: biodiesel, butanol, gasoline,- methane, ethanol, and kerosene [91, 92].-

There are some problems associated with the efficiencyof photosynthesis for industrial- purposes, the utilization rate of carbon dioxide, etc. Another drawback regarding algae is that- biofuel produced from them are less chemically stable than biodiesel produced from other- sources because the biofuels have unsaturated bonds in their molecules and they are subjected- to spontaneous polymerization.-

Another approach is to utilize carbon dioxide as a raw material for various chemical products,- like ethers, dimethylcarbonate, as antiknocking additive, monomers for plastics production,- acyl carbonates [93]. All of these products are currently produced from petrol and that is why- carbon dioxide recycling is important to greenhouse emissions but also to the reduced use of- oil as a whole.-

On the other hand, carbon dioxide is irreplaceable tool for supercritical extraction of biologi‐ cally active and thermo‐instable substances from natural products.-

An attractive approach is to reduce electrochemically carbon dioxide to methane in presence- of methanogenic bacteria [94–97] or to ethanol and acetic acid [98].-

#### **6. Conclusions-**

The present review demonstrates differentoptions for biomass utilization to replace, at least- partially, the use of fossil fuels and thus to reduce the pressure of greenhouse gases emissions- and to close the carbon cycle in nature within the present times. The biomass and the waste of- biofuel production could be also utilized as raw material for various chemical manufacturing- currently produced from oil. However, in some cases this option may additionally influence- the environment and create secondary pollution. It could happen in cases of bioethanol and- biodiesel production when large area of land is required for crop growth and much energy for- crop production is requested. That is why local solutions about the use of renewable fuels- based on biomass should be made very careful after thorough environmental analysis.-

More attractive option is biogas production and utilization, because it is always related to- simultaneous waste processing and energy production with closing the carbon cycle at local- level.-

Of course, the most promising research is dedicated to carbon dioxide recycling turning it to- fuels or value‐added chemicals.-

#### **Acknowledgements-**

This work was supported by the National Science Fund of Bulgaria under the grant DFNI‐ E02/16, 2014.-

### **Author details-**

Venko Beschkov-

 Addressallcorrespondenceto:vbeschkov@yahoo.com-

 Instituteof-Chemical-Engineering,-Bulgarian-Academyof-Sciences,-Sofia,-Bulgaria-

### **References-**

 [1]-Campbell,-C.-J.and-Laherrere,-H.-1998,-Theendofcheapoil,-Scientific-American,- 3,-78–83.-


obtained from biodisel production plant as sole carbon source. Journal of Scientificand-Industrial Research (India), 71, 06, 396–406.-


from silomais, grain mare, rapeseed cake and pig manure (in German). Institut für- Landtechnik, Universität für Bodenkultur Wien. Source: http://www.nas.boku.ac.at/- fileadmin/\_/H93/H931/AmonPublikationen/SEEGEndbericht.pdf;- 2004 [accessed- 25.05.09].-


## **Potential of Cellulosic Ethanol to Overcome Energy Crisis in Pakistan**

Saima Mirza, Habib ur Rehman, Waqar Mahmood and Javed Iqbal Qazi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66534

### **Abstract**

Liquid biofuel industry in Pakistan may become a promising source for saving our foreign exchange and environment. Currently, bioethanol production is dependent on cane molasses, a product of sugar industry. Harnessing of more bioethanol from lignocellulosic waste crop residue has potential to respond to the fuel scarcity. Lignocellulose exists in nature as a polymer and serves as the largest sink for fixed global carbon and could be used both as a carbon source for microbial growth-assisted bioethanol production and for fabricating enzymes for more energetic simultaneous production to represent an important segment of the renewable energy sector. An exciting aspect of this research is the development of new biorefining techniques that facilitate the extraction of sugarderived biofuel by processing of waste crop residues by employing novel nature inspired lignolytic enzyme. Further research will explore more avenues for stabilization of system in terms of process parameters for optimum bioethanol yield from enzymatically hydrolyzed lignin waste streams. The chapter can be considered as an anticipatory work and exploration of new dimensions for promotion of nature-inspired enzyme-assisted lignocellulose-based bioethanol production industry, which maximizes sustainable development opportunities especially in energy sector.

**Keywords:** crop residue conversion into biofuel, agriculture waste bioethanol, enzymatic ethanol, lignin biofuel, sustainable ethanol

### **1. Introduction**

There has been a universal consensus that greenhouse gases (GHG) such as methane (CH), carbon dioxide (CO) and nitrous oxide (NO) are the main cause of global warming. This extreme apprehension forced many nations of the world to reach treaty on Japanese Protocol.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Pakistan signed the Kyoto Protocol on Climate Change in 2004 and accredited the scientific invention's potential as a possible way to control the emissions of GHG [1]. Transportation consumes approximately 27% of primary energy [2]. In EU25 countries, transportation consumes about 28% of the energy and more than 80% is for lane transport [3]. The current oil requirement is around 12 million tons a day and there is forecasting that it may increase to about 16 million tons per day in 2030. The 30% of the world oil production is used to fulfill the fuel requirements for transport. In the current energy blend in Pakistan, the share of petroleum products is about 40%. Its use has grown up suddenly, mainly by fuel oil and the gasoline [4]. The immediate focus of this chapter is to review the current developments in the dimensions of waste crop residue-based bioethanol production and applications of natureinspired enzymes efficient production of the liquid fuel in Pakistan.-

Pakistan is facing severe energy crisis in these days which is resulting in many social and economic problems. Solution of this crisis might come from alternative(s), addressing cheap and eco-friendly fuel sources. This utmost and urgent requirement is likely to be achieved by biomass resources that have been mainly ignored previously, while they are accessible in enough quantities to solve the energy crisis in the country. Agriculture has remained the basis of Pakistan's economy as it provides employment to 45% of the population and provides feedstocks to agro-based industry. Clean energy supply is critical in agricultural areas in Pakistan where biofuels are currently not an option because of the lack of cost-effective and efficient biofuel production technologies, although villagers depend on conventional diesel for powering different agriculture machinery and gasoline for transporting agricultural goods from farm/market to end consumers. Fuel shortage has also led to a cut in electricity production. It is thus clear that the major limiting factor is energy which creates barriers for developing economies. Careful estimates show that by 2050, Pakistan's energy needs will increase three times without a concurrent increase in supply. Pakistan plans to cut natural gas supply by around 4,247,527 m3 /d to fertilizer plants and compressed natural gas (CNG) pumps to increase electricity supply to cities facing daily rolling blackouts. In 2012, Pakistan's natural gas supply had about 15 billion m3 /year deficit with increasing tendency. Large biofuel production plants that can contribute significant amounts of sustainable fuel are the only solution to supplement the power shortage in the country. Rise in conventional fuel price and its continuous depletion naturally brings great demand for the innovative biofuel production technologies as a Clean Energy Solution in Pakistan. The valuable progress starts with the beneficial use of the waste material and crop residues as feedstocks which otherwise represent environmental liabilities. The development in this sector will further provide opportunities to create multiple symbiotic partnerships among the farmers and the business community.

Ethanol is an important energy source which has the huge potential to lessen the sole dependency on fossil-derived fuels and alleviate hazards to our environment. Additionally, it is an ideal precursor molecule because of its promising potential as fuel, beverage, feedstock, antiseptic and industrial solvent. Currently, it is replacing around 3% of the gasoline that is derived from fossils throughout the world which is compatible with petroleum and recommended for transportation in both blended and pure forms. The consumption of ethanol is around 1.6 million tons and consumption of fuel ethanol can be increased up to 160,000 tons by 10% blending of bioethanol in petrol. According to Chris Somerville, director of the Energy Biosciences Institute, USA, annual production of ethanol from corn is around 13–14 billion gallons in the United States, which is equal to 10% of gasoline use. In Brazil, 40% of liquid transportation fuel is bioethanol and 15% of the nation's electricity is deriving from it. Therefore, it is assumed that current technology is healthy enough to produce ethanol as an alternative fuel to some extent for immediate partial replacement of oil. However, corn ethanol which is already in use has several drawbacks as corn is a food crop. Furthermore, when cost-to-benefit ratio regarding equipment and processing involved in planting, harvesting and transporting corn ethanol are considered, it becomes incomparable with gasoline. Therefore, hardy, fast-growing plants, like switchgrass, elephant grass and miscanthus, are more favorable feedstocks options. These grasses can grow up to 10 feet height, thrive on marginal land and can survive even with little or no fertilizer [5]. Moreover, cellulose-rich waste material of paper and other industries and waste crop residues can also be considered attractive options for cost-effective and even zero cost bioethanol productions by using nature inspired enzymatic processes.

### **2. Biomass: a cheap and sustainable biofuel resource**

Pakistan is an agricultural country and huge capacity of biomass in the form of waste crop residues such as rice straw, wheat straw, cotton stalks, maize stalks, sugarcane tops and so on are available for bioethanol production. Pakistan annually generates around 69 million tons of field-based crop residues. Field-based crop residues are generally considered useless and it has been estimated that 50 million tons of residue/waste is produced every year from major crops (including 6.88 million tons of sugarcane bagasse). These are either burned in the fields/- homes or buried in the land. Direct burning of this field base left over emits carbon dioxide and smoke which are hazardous for health and a source of ozone with risks to the atmosphere. Excluding domestic consumption and commercial usage, the net available resource potential of four crops, that is (wheat, cotton, rice and corn) for biomass power generation, is estimated to be about 10.942 million tons [6]. These estimations showed that Pakistan is endowed with abundant availability of biomass resources, which can be economically exploited for developing a sustainable biomass energy system, because the country has been perennially facing power demand-supply gap, which is currently estimated at 4500–5500 MW [7]. The system is being maintained by resorting to load shedding, often extending up to 12–16 h. Pakistan has strategies to add 9700 MW of electricity generation capacity by 2030 as per the Medium Term Development Framework [8], which would partially take care of current fuel scarcities. In this context, power generation through biomass can also play an important role in bridging the overall demand-supply gap. It would be essential to expand and diversify the resource base, particularly in the context of continuous access to electricity in all regions of the country. Large numbers of industries in Pakistan are currently dependent on liquid fuels for meeting their captive demand for electricity and heat. The situation is therefore ideally suited for promoting biomass-based liquid biofuel production as a sustainable and renewable alternative for the industrial sector as well. If only field left over crop residues are used for production of bioethanol, even then a sufficient amount of bioethanol can be produced to cut oil import and improve the profitability of the farming sector.-

It is known that some developed countries like the United States and Brazil are largest bioethanol producers and ethanol production in these countries is achieved by fermentation of corn glucose [9]. The production of ethanol from molasses is not new, but some areas need to be researched for enhanced yield. Until now, in Pakistan, sugar mills distilleries are operational for ethanol production using molasses, but in order to utilize molasses fully and get maximum benefit, it is important to increase the number of distillery units on one hand and assurance of possible involvement of efficient enzymes and engineered microbes on the other hand.- Furthermore, application of mathematical imitations would be used to explore efficient way- for optimized yield without intervention of any pilot plant [10, 11]. The major steps for largescale microbial production of ethanol are fermentation of sugars, distillation and dehydration.

### **3. Microbial fermentation**

Different ethanologenic microbes have been known to have promising qualities like limited- growth requirements, genetically amenable, higher sugar and ethanol tolerance. Bioethanol production by two strains (mutant and wild) of yeast *Kluyveromyces marxianus* have been documented [12]. Wild strain showed maximum specific growth rate at 40°C while mutant- showed maximum specific growth and ethanol formation rates at 45°C from fermentation of- diluted molasses. Results of this study anticipated that large-scale production may also be economically feasible by employing these microbes. Yeast-assisted bioethanol production process is more common and commercially applicable method in Pakistan [13]. *Zymomonas mobilis* is also attracting more attention for bioethanol production due to less process limitations [14, 15]. Different experimental studies in this regard revealed that optimum ethanol production up to- 55.8 g L−1 can be achieved by *Zymomonas mobilis* at 30°C after 48 h of retention time [16, 17].

Sugar beet, molasses and sugarcane juice are one of the most vital and easily accessible raw materials for the fermentative production of alcohol. The increased cost of molasses has triggered many distilleries to search alternate sources of feed stocks for the production of bioethanol in Pakistan. In starch industry, a by-product called enzose hydrol contains 5, 12, 56 and 5% of oligosaccharides, maltose, glucose and maltotriose, respectively, and is a cheap and good source of fermentable sugars. Mostly oligo-saccharides and maltotriose are not completely consumed by ethanologenic microbes and therefore need pretreatment [18]. Similarly, bioconversion of cellulose into ethanol can be accompanied by various microbes as well as by some filamentous fungi, including *Neurospora crassa* [19, 20] *Zymomonas* [21], *Trichoderma viride* [22], *Paecilomyces* sp. [23], *Zygosaccharomyces rouxii* [24] and *Aspergillus* sp. [25], termites' gut enzymes, genetically engineered bacteria such as *Escherichia coli* [26] and thermophilic, anaerobic bacteria, such as *Clostridium thermocellum* [27]. Thus, certain possible methods need to be designed for economical production of ethanol from agricultural farm residues by employing most effective microbes [28]. Among such agro-based wastes, wheat straw is one of the most plentiful crop residues which has broadly been studied and is abundantly available too [29].

Current investigations are focusing on pretreatment of the hard biomass, that is, lignocellulosic sugarcane bagasse, rice straw and wheat straw and subsequent production of ethanol from the pretreated biomass using ethanologenic microorganism. Different fungal species have promisingpotential for breaking down lignin and therefore may be applied for efficient ethanol fermentation. Hypothetical yield of ethanol is 0.511 g per gram of glucose consumed. Practically, this yield cannot be achieved because part of fermentable glucose is consumed for cell maintenance, for synthesis of by-products like glycerol and lactic acid and therefore is not completely converted into ethanol. Nevertheless, at the manufacturing level, under ideal conditions, it remains 90–95% of the hypothetical yield [30]. Ethanol formation represents a specific loop of the general- cellular metabolism; however, its general production route is shown in **Figure 1** [31].

Generalized bioethanol production is as follows [31].

**Figure 1.** Part of the fermentative metabolism directly involved in the ethanol production.

### **4. Bioethanol production potential of industrial sector in Pakistan**

 Few industries in Pakistan are already involved in bioethanol production from by-products or industrial effluents, but it is necessary to develop nature inspired bioethanol production on a- large scale that may not only provide a solution to Pakistan's power shortages but can also be profitable enough to render their viability in local conditions. The biomass like rice straw, sugarcane molasses, bagasse and wheat stubble are the chief resources of lignocellulose feedstocks worldwide [32]. One of the largest available biomass is rice straw which is about 7.31 × 1014 rice stubbles per year in world and 90% of its annual global production comes from the Asia [33]. Another abundantly available biomass, a by-product of sugarcane processing, is the sugarcane bagasse which represents important source for fuel generation systems and ethanol production due to its high easily accessible sugar contents for fermentation [34].

Sugar industry is the biggest agro-industry in Pakistan after textile and has been playing key role in the production of ethanol. There are about 76 sugar mills in Pakistan already which are producing seasonal ethanol from around 2.5 million metric tons (MMT) of molasses. However, being an agricultural country, the best option is second-generation ethanol. However, for this, the complex lignin-cellulose-hemicelluloses matrix of the biomass has to be broken and the carbohydrate polymers need to undergo hydrolyses to yield fermentable sugars. The important source of the livelihood of farmers is sugar industry and their 70% population is dependent on it. The yield of sugar in Pakistan is about 85.95 kg per 100 kg of sugarcane. The molasses production from sugarcane is approximately 40 kg per ton of cane from which ethanol production is approximately10 L. There is 270,000 tonnes per annum current production capacity for ethanol of fuel grade in our country which can readily be increased up to 400,000 tonnes per annum through the rise in employments and feedstock like waste crop residue [18, 35]. This molasses-to-bioethanol conversion process is conducted in distilleries. But most of the distilleries are located onsite in sugar mills which make the production cycle an integrated one. The mills, after processing sugarcane, store the molasses in storage tanks on-site and then pass it on to the distilleries for bioethanol production. Simple molecular sieve technology is used for bioethanol production in most of these mills which requires 1.5 million USD capital expenditure and can be completed in 5–6 months.

AL-Abbas sugar mill production plant is situated exactly in the center of one of the huge sugarcane growing areas of province Sindh at Mirwah Gorchani. This area is also known to be the most fruitful regarding sugarcane cultivation in Pakistan, assuring the supply of good crop of sugarcane throughout the entire season for the sugarcane plant. The plant is linked to the national highway by means of a mile of metal led road and is also accessible by a web of many other roads from different directions which facilitate transport of sugarcane from the plantation sites to the sugarcane plant. Total crushing potential of this sugarcane crushing plant is about 7500 M ton per day. AL-Abbas sugar mill established the largest ethanol distillery plant in 1999. The plant design is equipped with highly advanced French technology using multieffect vacuum distillation. The ethanol production capacity of unit-I is approximately 87,500 L per day. The growing demand of ethanol has urged the management to set up unit-II with the same capacity of ethanol production. The bioethanol-based power plant of AL-Abbas sugar mill has 15 MW electricity production capacity.

Shakarganj sugar mill is located in Jhang, Pakistan. They are producing three different types of- ethanol, that is, concentrated ethyl alcohol, denatured spirit and methylated spirit for industrial and alternate source of energy usage. The mill is exporting approximately 90% of its total ethyl alcohol and is a four-time award winner for the highest export of ethyl alcohol. The unit produces anhydrous alcohol employing eco-friendly dry dehydration technology. The denatured and methylated spirit is in high demand in local wood product and paint industries.

Another bioethanol-producing sugar mill is located in Nankana, Sheikhupura, Pakistan. The ethanol production potential of this distillery is 125,000 L/day. Besides this capacity, the distillery also produces ethanol of fuel grade with 99.8% purity from the mill molasses. Stateof-the-art distributed control system (DCS) which not only promises for increased steadiness and but also approachability of plant is used. The distillery system is established with fewer number of devices and lesson wiring. The distillery can cut in half the costs related to applying and sustaining the loops by incorporating the transmitter controllers into the process and by opting not to tie any critical loops back into the DCS. The distillery is equipped with ultramodern machinery and is working on International Standard Operating processes to carry them to produce high-quality products and is meeting the demands of end users.

Crystalline Chemical Industries (Pvt.) Ltd (CCI) also practices sugarcane molasses fermentation for ethanol production, located in Sargodha, Pakistan. This unit of distillation exports about 90% of its ethanol produce.

Habib sugar mills Ltd. has industrial alcohol production capacity up to 142,500 L/day. Pinnacle distilleries (Pvt.) Ltd. is producing rectified spirit for portable applications, technical alcohol, anhydrous ethanol 99.7% minimum for manufacturing use. The fuel grade alcohol is produced up to 30,000 tons per year.

Almost all sugar industries in Pakistan are producing bioethanol mainly from molasses containing feasible level of fermentable sugars. Presently, the biomass proportions which can be economically converted into ethanol are sugar (sugarcane) and starch (e.g. corn). In future, there will be plentiful industrial scale progress in the subject of lignoethanol where the hard part of a plant (cellulose) will be converted into fermentable sugars and consequently converted to bioethanol. After microbial fermentation, the produce is subjected to distillation, dehydration and then is condensed for quality improvement and water and other impurities removal. However, due to high cost in the form of energy input, this traditional process is replaced with some energy saving processes (molecular sieve) mainly to avoid distillation completely for dehydration. This process involves the use of ethanol vapors under pressure and allows these vapors to pass through molecular sieve beads bed. The energy saving by this technology of dehydration accounts for 3000 btus/gallon (840 kJ/L) than that of azeotropic distillation.

If all raw sugarcane molasses is converted to bioethanol, then it has the potential to substitute 5–7% consumption of gasoline. This will be a very important contribution in future to lessen the burdens on Pakistan economy. The government of Pakistan should make policy to endorse the blending of ethanol in transportation fuels as early as it becomes conceivable [18]. With the production of bioethanol from Pakistan's own raw molasses, about 600 million of precious foreign exchange can be saved [36]. Besides this, other advantages of ethanol usage are good engine performance and better yields; it burns more efficiently and keeps our environment clean and more easily biodegradable, as well as consistent with the global focus on biofuel. No doubt this is a most effective way for production of bioethanol from raw/- waste material; however, involvement of a variety of waste biomass or crop residue will be more optimistic for solution of energy issues. The main factor in ethanol production is the content of lignocellulose present in substrates which will be hydrolyzed by different hydrolyzing agents to provide fermentable glucose [37, 38]. The nature-inspired enzymes from wood fungus and termite may be used as an extra bonus in the presence of exiting bioethanol production technologies, which can convert the long chains of polysaccharides into monosaccharaides. Different industries like forestry, pulp and paper, agriculture and food processing including municipal solid waste (MSW) and animal wastes are major producers of lignocellulosic waste materials [39, 40].

### **5. Present challenges for bioethanol production from lignocellulosic feedstocks**

Currently, lignocellulolytic enzymes are derived from fungus, gut of termite and certain bacteria [41]. Established technology for bioethanol in Pakistan is relatively of low-tech approach to meet some needs by employing molasses and some selective biomass. Such limitations with biomass make the process and yield profit limited. At the same time, the farmers and agribusinesses cannot access recent technologies that may greatly expand the use of bioethanol to meet the demand for power in many applications.

The current energy scenario warrants the demand for research and development of biomassbased biofuel production systems. Biomass, due to its renewable nature and abundance, is becoming an increasingly attractive fuel source. Lignin, the second most abundant biomass constituting aromatic biopolymer on Earth, is highly recalcitrant to depolymerization. Lignin serves as bonding for hemicellulose and cellulose and creates an obstacle for penetration of any solution or enzyme to lignocellulosic structure which is the major structural component of all plants and can be depolymerized to fermentable sugars. Microbes enhance the conversion of lignin into fermentable sugars but there are some hurdles which need to be removed first. Recalcitrant nature of lignin could be tackled through different biocatalysts due to their nonhazardous and eco-friendly nature. Therefore, lignocellulolytic microorganisms like fungi and some bacteria are considered as promising biomass degraders especially for large-scale applications due to their potential yields of extracellular synergistically acting enzymes into the environment. These enzymes can contribute significantly in degradation of lignocellulosic material by converting long chain polysaccharides into their 5- and 6-carbon sugar components [42, 43]. Although lignin resists attack by most microorganisms, basidiomycetes, whiterot fungi are able to degrade lignin efficiently [44, 45]. Lignocellulolytic enzymes-producing fungi are widespread and include species from the ascomycetes (e.g. *Trichoderma reesei)* and basidiomycetes phyla such as white-rot (e.g. *Phanerochaete chrysosporium)* and brown-rot fungi (e.g. *Fomitopsis palustris)* [46, 47]. Few basidiomycetes, for example, *P. eryngii, P. chrysosporium* and *T. versicolor* can act as biocatalysts for ethanol production by having potential for lignin degradation/depolymerization. Ethanol fermentation requires high concentration of sugar solutions; therefore, biocatalytic conversion of lignocellulosic material into hydrolysate containing high concentration of sugar will be incentive for decreasing production cost. Therefore, variety of lignocellulytic material (wheat straw, rice straw and rice husk) could be degraded by basidiomycetes and subject to ethanologenic fermentation for ethanol production cost-effectively. However, some strains of white-rot fungi have promising potential to degrade lignin by simultaneous attack on lignin, hemicellulose and cellulose, whereas few can selectively work just on lignin. It is pertinent here to note that synergistic biocatalytic ability of white rot fungi would be source of efficient depolymerization method and will be helpful in proving that the heteropolymer lignin represents an untapped resource of renewable aromatic chemicals [48, 49]. Lignocellulosic biofuel production is not yet economically competitive with fossil fuels; therefore, to ensure successful utilization of all sugars is important for improving the overall economy especially in terms of maximum theoretical yield. Xylose is one of the most abundant sugars in lignocellulosic hydrolysate. Therefore, over expression of xylose isomerase will facilitate complete utilization of xylose present in hydrolysate which otherwise remains to varying extent in spent culture [18, 50]. Another matter of concerns regarding lignin depolymerization and its conversion into biofuels/bioethanol is repolymerization of lignin-derived low molecular weight sp. into high molecular weight molecules which are not easy to be degraded by microbes. Repolymerization is observed to occur within few hours after onset of lignin volarization. For this purpose, organization of most effective microbial sink for immediate utilization of low molecular species for bioethanol production is the most appealing option [51, 52].

For overcoming this bottleneck, microbial sink/consortium of different microbes with xylose- overexpression is an offered strategy. Preventing repolymerization of low molecular weight- lignin species into high molecular weight lignin compounds and ensuring the complete utilization and conversion of available sugars into bioethanol can make the bioprocess costeffective. The description in this chapter will lead to development of technologies that can- be helpful in efficient depolymerization of lignin and its simultaneous conversion into highvalued microbial-assisted advanced biofuel. The chapter represents need for development of road map for advanced level of biofuel production from waste crop residues. Nature-inspired enzymes' involvement is the most effective way for enhanced bioethanol production from- biomass. The enzymes convert the long chains of polysaccharides into monosaccharaides. Currently, lignocellulytic enzymes used for ethanol production from cellulosic biomass are obtained from fungus, gut of termite and certain bacteria [40]. Present restrictions of enzymatic breakdown of lignocellulose-based biomass are mostly due to concern of enzymatic steadiness and vulnerability to inhibitors or by-products [53, 54]. Continuous bioengineering efforts and prospecting should provide novel enzymes with lower susceptibility to inhibitors- and relatively higher specific activity [55]. Few insects such as termites have very efficient- approaches to break the lignocellulose-based substrates as potential mean of bioenergy [56]. In case of lower termites, activity (cellulolytic) is normally dependent on enzymes produced by endosymbiotic, flagellated protists [57], while in case of higher termites, their guts contain lignocellulytic enzymes which combine with cellulases secreted by certain endosymbiotic gut bacteria [58, 59].

Hence, establishment of large-scale bioethanol production plant by treating waste crop residues with such novel enzyme will enhance the production and can successfully provide support to deteriorating economy of Pakistan (**Figure 2**). The resulting cleaner environment is another benefit that has monetary values that the government may be financially and ethically interested- in. Such multiple positive benefits will attract different interested parties to involve in replication of process, each with resources and benefits to sustain and multiply. Additionally, the- greenhouse gas emission will be reduced by burning bioethanol, as the net CO2 emission is zero because the amount of CO2 emitted on burning is equal to the amount of it, which is absorbed- from the atmosphere by the process of plant photosynthesis which will be used for production of bioethanol [60]. In Pakistan, the current domestic production of raw oil presently satisfies- only almost 25% of the country's consumption and remaining demands are met by importing fuels from abroad. This make Pakistan's economy vulnerable to different social and economic- issues; however, incorporation of biofuel/bioethanol will reduce burden on country's economy significantly.-

Federal Cabinet, Economic Coordination Committee (ECC) of Pakistan has decided to permit marketing of Ethanol 10 as motor vehicle fuel on the trial basis. Anhydrous ethanol can be blended with gasoline in different proportions having less than 1% water content. Many of the motor vehicles having gasoline engines operate well with ethanol blend of 10% in their fuels (E 10). The Government of Pakistan enacted a 15% duty on export of molasses to prefer the use of molasses for ethanol production rather than export [61]. The government of Pakistan should make policy to enforce the blending of ethanol in transportation fuels as early as it becomes conceivable [18]. Successful implementation of large-scale waste crop residue-based bioethanol production concept will attract private sector investment and company-farm partnership to accelerate the development and commercialization of new bioenergy solution to improve emerging economies and transform the lives of at least small farmers. The concept is readily adoptable by different agricultural regions as the essential supply of the feedstock is available in the form of agricultural residues that are sustainable and typically available abundantly and locally.

**Figure 2.** Schematic representation of nature inspired enzyme assisted bioethanol production process.

### **6. Concluding remarks**

To import conventional energy resources, Pakistan is spending around 7 US\$ billion equivalent to 40% of total imports. Careful estimates show that by 2050 Pakistan's energy needs will be increased three times while the supplies are not very inspiring. In 2012, Pakistan natural gas supply had about 15 billion-m3 /year deficit with increasing tendency. Rise in- natural gas price (0.51 \$/m3 ) brings great potential to promote biomass-based biofuel production in Pakistan.

Pakistan being an agricultural country produces wheat, sugarcane and potatoes as one of its biggest crops [62–64]. Consequently, large amounts of wheat straw and sugarcane bagasse are obtained as by waste products. Due to high ambient temperature in most part of the year, poor post-harvest processing and storage of thousands of tons of biomass are wasted each year. In short access of promising process for the initiation of sustainable strategies for waste crop residue-based bioethanol production while consuming starch, cellulosic and lignin loads of effluents of respective origins in the country, bestowed with suitable biomass- and temperature optima for successful cultivation of ethanologenic microbes, is expected to provide sustainable supplies of biofuels. Additionally, more than three billion acres worldwide which are not suitable for agriculture purpose due to dryness could be utilized for growing drought-hardy plants for biofuels production. The only disadvantage of using these crops is that they contain lignocellulose, a hard plant material, that needs more treatment than either corn or sugarcane to be converted into alcohol. Therefore, search for ways to make the overall process more efficient by reusing materials, changing the fermenting agent- and searching for better and nature-inspired enzymes will be milestone in this regard. The- process development of nature inspired enzyme-assisted conversion of agricultural and food waste into bioethanol that can be used as clean biofuel is demand of time. Adoption of these new dimensions for bioethanol production will definitely reduce pressure on energy and- transportation sector, entire dependence on conventional fuels and can triumph fight against- climate change.

### **Author details**

Saima Mirza1 , Habib ur Rehman2 , Waqar Mahmood3 and Javed Iqbal Qazi4 \*

\*Address all correspondence to: qazi.zool@pu.edu.pk

1 Energy System Engineering, Punjab Bioenergy Institute, University of Agriculture, Faisalabad, Pakistan

2 Punjab Bioenergy Institute, University of Agriculture, Faisalabad, Pakistan

3 Centre for Energy Research and Development, University of Engineering and Technology, Lahore, Pakistan

4 Microbial Biotechnology Laboratory, Department of Zoology, University of the Punjab, Lahore, Pakistan

### **References**


## **Jatropha Biofuel Industry: The Challenges**

M. Moniruzzaman, Zahira Yaakob, M. Shahinuzzaman, Rahima Khatun and A.K.M. Aminul Islam

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64979

#### **Abstract-**

 Consideringenvironmentalissuesandtoreducedependencyonfossilfuelmany- countrieshavepoliticizedtoreplenishfossilfueldemandfromrenewablesources.- Citingthepotentialof-Jatrophamostlywithoutanyscientificandtechnological- backup,itisbelievedtobeoneofthemostsuitablebiofuelcandidates.-Hugegrants- werereleasedbymanyprojectsforhugeplantationof-Jatropha-(millionsofhectares).- Unfortunately,therehasbeennosignificantprogress,and-Jatrophadidnotcontribute- muchintheenergyscenario.-Unavailabilityofhigh-yieldingcultivar,large-scale- plantationwithouttheevaluationoftheplantingmaterials,knowledgegapandbasic- researchgapseemtobethemainreasonsforfailure.-Thus,theproductionof-Jatropha- asabiofuelhasbeenconfrontedwithvariouschallengessuchasproduction,oil- extraction,conversionandalsoitsuseasasustainablebiofuel.-Inthischapter,we- disclosethechallengesandpossibleremedyforthecontributioninthebiofuel- industry.-

**Keywords:** *Jatropha curcas*, biofuel industry, renewable energy, challenges, solutions-

#### **1. Introduction-**

 Jatrophabelongstothefamily-Euphorbiaceaeandhas-175species.-Ithasoriginatedfrom- tropical-Americaandhasspreadalloverthetropicsandsubtropicsof-Asiaand-Africa-[1].- Throughouttheworld,morethan-1,000,000haof-Jatrophahavebeenpropagated.-Majority- (85%)ofthemareinthe-Asiancountries,i.e.,-India,-Chinaand-Myanmar;theremaining,-12%- in-Africaand-2%in-Latin-America-(Braziland-Mexico).-Indiaisthelargestcultivatorof-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 Jatropha-[2].-Intheancienttimes,-Jatrophahasbeenusedinvariousfields,suchasstorm- protection,soilerosioncontrol,firewood,hedgesandtraditionalmedicines-[3–6].-Theseed- oilof-Jatrophaisalsousedaslampfuel,soapmanufacturingingredient,paintsandasa- lubricant-[4,-7,-8].-Thecharacteristicsof-Jatrophaseedoilmatchwithcharacteristicsofdiesel- [9–11],thusitiscalledabiodieselplant-[12].-Jatrophagrowsondiversewastelandwithout- anyagriculturalimpute-(irrigationandfertilization)andhas-40–60%oilcontent-[12,-13].-Easy- propagation,rapidgrowth,droughttolerance,pestresistance,higheroilcontentthanother- oilcrops,adaptationtoawiderangeofenvironmentalconditions,smallgestationperiod,- andoptimumplantsizeandarchitecture-(whichmaketheseedcollectionmoreconvenient;- actuallyinconvenient-[14])aresomecharacteristicsof-Jatropha-[15],whichmakesita- promisingcropforbiofuel-[16].-Although-Jatropharankedbehindpalm-(palm->-*Calophylum inophyllum>* Cocussp.->-Jatropha)accordingtoannualoilyield/hectare,itisfavouredasa- non-ediblefeedstock-[17,-18].-Anumberofearlierreports,proceedings,expectationsand- assumptionspredictedthattheseedyieldof-Jatropharangefrom-2to-5-Mg/haandeven-7.8- to-12-Mg/ha- withoutanyscientificandtechnologicalbackup-[19].-

There is a complete mismatch between theoretical expectation and actual seed production of- Jatropha in fieldconditions [9, 20–22]. The research on Jatropha opened the floodgateto the- scientificcommunity to grab funds and publish papers in high impact journals because seed- oil of Jatropha has characteristics of biodiesel and this crop was non-native of arid, semiarid- and subtropical regions. Singh and co-authors [19] depict the expectation and contribution- picture from Jatropha policy (**Figure 1**). The reported yields of Jatropha in field conditions in- India, Belgium, South Africa and Tanzania, are 0.5–1.4 mg/ha/yr, 0.5 mg/ha/yr, 0.35 mg/ha/yr- and 2 mg/ha/yr, respectively [23]. The less productivity is because of unavailability of suitable- high yielding varieties, large-scale plantation without evaluating the genetic potential of- planted materials, consideration of Jatropha as no/low impute crop and lack of knowledge on- agronomy. In this chapter, we present the challenges confronted by Jatropha as a biofuel and- recommended solutions.-

**Figure 1.** Expectation and contribution from Jatropha biofuel industry.-

#### **2. Potentials of Jatropha-**

 Jatrophahasmultipleuses-(**Figure-2**).-Jatrophaseedoilpossessesbiodieselandjetfuel- production potentials. Its wood, leaves and fruits have beenusing asfirewood in rural areas.- Italsohasindustrialapplications.-Preparationofsoapandcosmetics,anddyeingclothesand- fishingnetsaresomeofitscommonapplications.-Traditionally,-Jatrophahasbeenknownas- amedicinalplant.-Thetherapeuticcompoundsfrom-Jatrophacanbeusedasanti-microbial,- anti-inflammatory,healing,homeostatic,anti-cholinesterase,anti-diarrheal,anti-hypertensive- andanti-canceragentsinmodernpharmaceuticalindustry.-Asitcontainstoxins,beforeusing- Jatrophaand/oritsderivativesasatherapeuticagent,toxicologicalstudiesmustbeconducted.- Jatrophaseedcakecansupplementanimalfeedandorganicfertilisersasitbearshigher- percentageofproteinandothernutrients.-Soilerosioncontrolandusedashedgesare- prehistoricusesof-Jatropha.-

**Figure 2.** Multipurpose uses of *Jatropha curcas*.-

#### **2.1. Jatropha as energy source-**

Various features like, ease of production, sustainability and environmentally friendly nature- of biomass draw attentionas a potential renewable energy to replenish fossil fuel demand.- Among the crops identifiedas energy crops for firstgeneration biofuels, *Jatropha curcas-*L. (JCL)- has been acknowledged as one of the promising candidates [24].-

Parts of Jatropha plant, like wood, fruit shells, seed husks and kernels [25], are used to produce- energy. Raw oil is the major resource obtained from Jatropha. Depending on the variety/- cultivars, decorticated seeds contain 40–60% oil [26–31]. The oil is utilised for many purposes,- such as lighting, lubricating, making soap [32] and most importantly as biodiesel. Biodiesel- from Jatropha comply with European biodiesel standards (**Table 1**).-


**Table 1.** Chemical and physical properties of Jatropha oil.-

There are approximately 24.60%, 47.25% and 5.54% of crude protein, crude fat and moisture,- respectively, in Jatropha oil [33]. Both saturated and unsaturated fattyacids are present in the- oil. The major saturated fattyacids are Palmitic acid (16:0) at 14.1% and stearic acid (18:0) at- 6.7%, oleic acid (18:1) at 47.0% and linoleic acid (18:2) at 31.6%. The usefulness of Jatropha oil- and its esters instead of petro-diesel has been reported [34]. The energy value of Jatropha seed- oil (39MJ kg-1) is higher than anthracite coal and is comparable to crude oil [35].-

By mass, the shells bears about 35–40% of the dry fruit, that is, 60–65% of the seed weight [36].- There are approximately 42% husk and 58% kernel in a seed [25]. The gross energy value of- Jatropha seed is 24 MJ kg−1which is higher than lignite coal and cattle manure and is comparable to corn cobs [37].-

The firststep of oil extraction is the mechanical removal of the shell from the fruit. Jatropha- seed shell contains cellulose (34%), hemicelluloses (10%) and lignin (12%) [25]. Approximately- 11.1 MJ energy is driven from one (1) kg seed shell of Jatropha [35].Ash (4%), volatile matter- (71%) and fixedcarbon (25%) are the components of seed husk. Approximately, 16 MJ energy- is driven from one (1) kg seed husk [36], which is comparable to wood.-

Less energy expenditures and the prospect of using a cheap substrate make hydrogen (H2) gas- a lucrative source of future renewable energy. Lignocellulose biohydrogen can be produced- by the fermentation of de-oiled Jatropha solid waste (DJSW) and Jatropha seed cake that- contains lignocellulose [38–41]. Kuma and co-workers reported highest achievable cumulative- hydrogen production (CHP) of 296 mL H2 by the fermentation of de-oiled Jatropha waste under- optimum conditions. The reported optimum conditions are; substrate concentration 211 g/L,- pH 6.5 and temperature 55.4°C [40]. Lopes and co-workers produced 68.2 mL H2/gVSiJSC- biohydrogen by dark fermentation of seed cake by a pure strain of the bacteria *Enterobacter aerogenes* without pretreatment of the substrate [41]. In the viewpoint of energy saving, it is- significant.-

#### **2.2. Industrial use-**

The common use of viscous oil from Jatropha seed is for making soap [31]. The manufacturing- of soft and durable soap from Jatropha oil becomes easy because of the high palmitic acid- content and its hydrophobic nature. People in West Africa, Zambia, Tanzania and Zimbabwe- are familiar with the use of Jatropha soap [42]. The presence of glycerine in Jatropha oil soap- makes the white soap good for skin. It also have very good foaming [43] properties. Jatropha- soap can be used for various skin diseases because of its medicinal properties [44]. The 36%- linoleic acid (C18:2) content in Jatropha kernel oil is good for skin care [42, 45]. The oil is also- an ingredient in hair conditioners [46].-

Jatropha is rich in many phytochemical constituents. Alkaloids, coumarins, flavonoids,- lignoids, phenols, saponins, steroids, tannins and terpenoids are found in differentparts of- this plant [47]. These components show anti-microbial [48], anti-inflammatory [49–51], healing,- homeostatic [52], anti-cholinesterase [53, 54], anti-diarrheal [55–57], anti-hypertensive activities [58] and are anti-cancer agents [59, 60]. It is necessary to study the toxicity associated with- these phytochemicals. The toxic effects could decrease its medicinal value.-

The strong purgative activity of oil helps to cure skin diseases and to soothe rheumatic pain.- It is also used in pesticides. The people in the Philippines have been using the dye obtained- from Jatropha bark for colouring finishingnets, cloths and lines [61]. Another application of- seed oil is in eczema treatment [62]. Manufacturing of soap and cosmetics from Jatropha- derivatives (as an alternative to Karitee butter) is a non-energy application of Jatropha.-

### **2.3. Other uses-**

From prehistoric days, Jatropha is used to make hedges. The advantage is that animals do not- feed on it. Seed germinating plants bears taproots along with surface roots; Jatropha is a seed- germinating plant, and it protects soil against erosion. It also act as a nutrient pump because- the roots can uptake the leached down minerals and return them in the form of leaf fall, fruit- debris and other organic remains.-

The higher protein content (58.1% by weight) of Jatropha seed cake, after detoxification,when- compared to that of soy meal (48%), makes it a valuable animal feed protein supplement. As- it possesses most of the minerals nutrients— nitrogen, potassium, calcium, magnesium,- sulphur, iron, phosphorus, zinc, copper and manganese, Jatropha seed cake is considered to- be an excellent organic fertiliser [63, 64].-

#### **3. Limitation of Jatropha as a biofuel crop-**

– A good commercial variety with a higher yield and disease resistance is still lacking.-

– High fluctuation of yield among trees.-

– It requires proper irrigation and nutrients for fruiting, though it can survive on insufficient- irrigation and nutrients.-


#### **4. Jatropha production challenges-**

### **4.1. Poor seed yield-**

 Relatedexpertssuggestthatthe-Jatrophaseedyieldof-4–5-Mg/ha/yrisneededforthe- commercialviabilityoftheindustry.-Iftheusualseedyieldof-3.75-Mg/hawith-30–35%oil- contentor-1.2-Mg/haoilyieldonlythen-Jatrophawouldcompetewithsoybeans-(USA-0.38- Mgoil/ha)andrapeseed-(Europe-1.0-Mgoil/ha)-[65].-However,thereishighflocculationof- theunitseedyieldandseedoilcontentof-Jatropha.-Anumberofauthorsreportedthatthe- lowseedyieldandthelowseedoilcontentaretheoneofthemostimportantbarrierfor-

 commercialviabilityof-Jatrophabiodieselindustry-[19,-66–69].-In-India,adifferentlocation- trialatdiverseagro-climaticregionswasconductedandtheaverageseedyieldwasrecorded- as-0.5–1.4-Mg/ha/yrafter-5yearsofplantation-[14].-Asimilarresultwasobservedfrom- plantationof-24eliteaccessionswithgoodplantarchitecture-(heightandbranchingpattern)- insodicsoil-[20].-In-Belgium,theaverageseedyieldwasreportedas-0.5-Mgseed/haafter-4- yearsofplantation,usingthebestknownproductiontechniques-[70].-Recentassessment- revealedthatgloballytheaverageseedproductivityof-Jatrophais-1.6-Mg/hawhichis- equivalentto-0.475-Mg/ha/yrbiodieselproductivity,whichisnotasafepositionforthe- industrytobeeconomicallyfeasible.-In-South-Africa,thehighestseedyieldwas-0.35-Mg/ha- after-5yearsofplantgrowth-[21].-A-Jatrophasilvi-pastoralproductionsystemincentral-west- Brazilwherehybridseedswereused,however,itcouldnotensureanysignificantseedyield,- againsttheexpectationof-2.4kg/plant-[22].-In-Tanzania,anegligiblegainat-US\$-9ha−1with- yieldsof-3-Mg/haandalossof-US\$-65ha−1onlandswithyieldsof-2-Mg/haofseedsafter-5 yearinvestment-[23]wereobtained.-In-Panzhihua,-China,-Jatrophacouldnotchangelocal- energyscenarioandtheindustryhasbeenconfrontedbyanumberofriskfactors-[71].-

Developing of a higher yielding and more oil containing variety is one of the main effective- solutions. However, a good commercial variety is still missing [72]. Zhang et al. [67] and Yu et- al. [68] also point out that variety breeding is one of the main hurdles for Jatropha planting.-

Actually, the current Jatropha breeding program is limited to conventional breeding and- surveying of germplasm resources of wild Jatropha plants [67]. However, the study of modern- biotechnology application on Jatropha improvement is limited [72]. Particularly, studies on- cloning, expression and biological function annotation for Jatropha genes, which are responsible for economical traits, are largely absent.-

The enhancement of unit seed yield of Jatropha for commercial use should be the main- objective of cultivation. Therefore, the techniques of Jatropha cultivation refers to many field- practices such as propagation, site preparation, tree density and canopy control, insects and- diseases control, fertilization and irrigation management, cropping treatments [67, 68, 73]. Few- studies on planting techniques and poor management for planting base limit large-scale- plantation of Jatropha [68, 74]. However, there is limited research to demonstrate precisely and- scientificallythe impact of fieldoperation on the seed yield of Jatropha. Moreover, there are- no/a few detailed reports on field observation on the seed yield under different treatments of- cultivation techniques. For example, data on tree density for Jatropha cultivation, canopy- pruning intensity and frequency, insecticide effectas well as fertilization and irrigation- efficiency are largely absent in the literature.-

#### **4.2. Consider as low impute crop-**

 *J.curcas*isbelievedasalowinputcropbecauseofitsabilitytogrowonbarrenland.-However,- itneedsadequatenutrientsasfertilizerandrainfallorirrigationforgrowingasaproductive- crop.-Ontheotherhand,excessivefertilizationandirrigationmaycausevegetativegrowth- (biomassproduction)atthecostoffruitproduction.-Moistureandnutrientshavelarger- influenceontheseedyieldandoilproductivityfromtheplantationonmarginallands.-The- plantgrowthandtheseedyieldof-*J.curcas*weresignificantlyincreasedunderirrigated-

 conditionsascomparedtonon-irrigatedconditions-[9,-14].-Itwasobservedthattherewas- 750kg/hayieldunderirrigatedconditionsatthesametimeonly-450kg/hawasrecorded- underrainfedconditionsfrom-3-year-oldplantations-[75].-Applicationofnitrogenand- phosphorusincreasedthegrowth,seedyieldandoilyieldof-*J.curcas-*[76].-Furthermore,- anotherreportby-BAIF-Development-Research-Foundationshowedthattherewasabout-500- kg/haseedyieldunderrainfedconditionsinthefifthyearofplantation.-However,after- regularirrigationofthesameplantation,innextyeartheseedyieldwasrecordedabout-1200- kg/ha-[77].-

The systematic studies for yield improvement, the agronomy (especially the irrigation and- nutritional requirements) in differentagro climatic conditions have not been adequately- addressed, despite advocacy for large-scale plantation of *J. curcas* [78].-

#### **4.3. Pest and disease susceptibility-**

Control of insects and diseases is particularly one of the most important technical issues which- could seriously shape Jatropha cultivation (**Figure 3**). Though it was claimed that Jatropha is- free of pests and diseases, the current study do not support the claim. Recent studies reported- that the plants were susceptible to viral infection (Cucumber mosaic virus), insect attack,- rodents, powdery mildew, leaf spots, insect defoliations and fungal diseases of the soil [14,- 21]. In Belgium, leaf miner *Stomphastis thraustica*, the leaf and stem miner *Pempelia morosalis-* and the shield-backed bug *Calidea panaethiopica*are the major pests affecting Jatropha [79]. Fruit- sap sucking predators *Scutellera perplexa-*[80] and *Maconellicoccus hirsutus*have recently been- investigated in India [81]. These infections caused approximately 60–80% damage to the- standing Jatropha crop at different study sites [14, 79–81].-

**Figure 3.** The photographs (A and B) show viral incidence in Jatropha.-

Moreover, monocropping could result in the spreading of insects and diseases. In Panzhihua,- China Yu and co-workers found 24 species of insects and diseases that are affecting Jatropha[82]. Wu and co-authors reported eight diseases and seven species of insects on Jatropha in the- dry-hot valley of Yunnan Province [83]. Jatropha monoculture expansion may spread insects- and diseases.-

### **4.4. Jatropha breeding objectives [72]-**


#### **5. Oil extraction challenges-**

Jatropha seeds contain 40–60% of oil depending on the variety [18, 84]. The firststep of oil- extraction is the removal of shells from the seeds after collecting the ripe fruits from trees. Seed- oil can be extracted manually, mechanically, chemically and enzymatically. The oil extraction- process is shown in **Figure 4**. Oil can be extracted by mechanical pressure, solvent extraction- and enzymatic degradation of kernel. Mechanical extraction yields about 90% of total oil from- the seed [85]. Solvent and enzymatic extraction yield almost 100% of oil from the seed.- However, these are complex processes and take long time. Solvent extraction involves handling- of large volume dangerous chemicals. Commercially suitable enzyme(s) is still not available- for enzymatic extraction of oil from seed kernel [86, 87] till date.-

In the mechanical process, a machine is used to exert pressure on seeds for the removal of oil.- After cleaning and checking, the seeds are fed into the hopper of the machine. For Jatropha- seed 0.41 L of oil is extracted from 1 kg. Mechanical parameters and pretreatment of seeds- affectoil yields. The effectsof treatment and physical parameters on the oil extraction are- shown on **Figure 5**. The amount of oil that can be recovered from the seeds is affected by:-

**•-***Throughput*: It is the amount of seed crashed per hour (kg/h). The higher throughput recovers- less amount of oil per kg of seeds, because of short time exposure of seeds to pressure. It- can be regulated by altering the turning pace of the screw throughput.-


**Figure 4.** Oil extraction steps and use.-


**Figure 5.** Pre-treatment and mechanical factors effect on seed oil recovery.-

The mechanical method is easier and less expensive but produces less oil (8–9%). Heat is- generated during the process that affectsthe quality of biodiesel. A high efficient oil recovery- (90–98%) technique, solvent extraction, is the most widely used. However, high energy input- and toxicity of solvent used are major disadvantage of this technique. Enzyme-based techniques may be the solution [88]. For extraction of oil from *Jatropha* seeds, aqueous enzymatic oil- extraction (AEOE) is a promising technique. Plant cell walls are composed of a complex- chemical structure. Enzymes that present in the system break cell walls and oil bodies and- accelerate oil recovery. This eco-friendly process does not produce volatile organic compounds- as atmospheric pollutants. Prolonged reaction time is the major disadvantage associated with- AEOE. Moreover, suitable commercial enzyme is not available till date. Winkler et al. [87]- studied enzyme supported oil extraction. They used alkaline protease, protease in combination- with hemicelluloses and/or cellulose. Alkaline protease treatment produces 86% oil.-

Ahmad et al. [89] isolated a bacteria marked MB4, which produces xylanases that enhanced- the extraction yield of Jatropha oil. The advantages offeredby this process are: protein in the- residue can be further processed for other applications, no purifiedenzyme preparation is- needed, and the resulting oil can be used for biodiesel production. Immobilization of lipase- has gained immense potential in the biofuel industry mainly to reduce the production costs- and to make the method more economical [90].-

#### **6. Conversion challenges-**

There are two types of methods, which are generally used for conversion of hydrocarbon fuels- from renewable feedstock. One is the thermochemical process and another is the biochemical- process. The thermochemical process is the conversion of biomass to hydrocarbons in the- presence of high temperature and pressure. In the biochemical conversion process, biomass is- converted into carbohydrates over some steps by the method of fermentation using enzymes- or micro-organisms [91]. The thermochemical conversion can be carried out mainly by- transesterification,pyrolysis, microemulsion, esterification,gasification,etc. Among these- processes, pyrolysis and transesterificationare the promising methods, which are used to- produce Jatropha biofuel, mainly biodiesel and bio-jet-fuel. Details of these processes are- discussed below.-

#### **6.1. Transesterification-**

Transesterification,also called alcoholysis, is the reaction where the oil converts into its- corresponding fattyester [92, 93]. This is a similar process to hydrolysis but here, alcohol is- used instead of water. So, transesterificationis the organic reaction where one ester transfer- into another ester by interchanging the alkoxy moiety. The basic reaction involved in transesterificationis shown in **Figure 6**. This reaction is used to decrease the high viscosity of- triglyceride. Due to the reversible nature of this reaction, extra alcohol is used to move the- equilibrium towards the product. A catalyst is used to promote the reaction rate and the- product yield [94]. Two types of catalysts are used in the transesterificationreaction. The acidcatalyst makes the carbonyl group more reactive by donating a proton while the base catalyst- remove a proton from alcohol to make it more reactive [93].-

**Figure 6.** Catalytic transesterification of triglyceride.-

The transesterificationprocess of Jatropha oil produces mono fatty acid alkyl esters and- glycerol as the by-product. In this process, methanol is the alcohol used due to its low price,- low temperature reaction, minimum reaction time and high yield of fattyacid methyl esters- [95]. This reaction is affected by several factors, such as molar ratio of glycerides and alcohol,- reaction temperature, time, catalyst and also the free fattyacid content and moisture content- in the Jatropha seed oil. Generally, the homogeneous base catalysts, NaOH and KOH, are used- because of their higher yield and quality fatty acid methyl esters (FAMEs) [96]. However,- homogeneous base catalyst for transesterificationof Jatropha oil associates some problems. It- is very difficultto separate the catalyst from the product and the purification step produces a- large amount of alkaline wastewater. Treatment of this water also increases the production- cost [97]. Because of the presence of the higher free fatty acid content and the moisture content- in Jatropha oil, the base catalyst induce saponificationreaction which decrease the production- yield [98]. To overcome this problem, an acid catalyst can be used in transesterificationof- Jatropha vegetable oil, but with the acid catalyst, the reaction requires more oil-methanol molar- ratios and the reaction will be very slow [99]. Another possible solution to overcome this- problem is a two-step procedure for the treatment of Jatropha oil. First step is esterification of- free fattyacid and the second step is transesterificationof Jatropha oil triglyceride [100]. But- this is also not cost effective. Instead of a homogeneous catalyst, a heterogeneous catalyst is a- betteroption for transesterificationof higher FFA containing vegetable oil because it can result- in good conversion and a high yield of FAME with optimum reaction conditions [101]. Many- researchers recommended the heterogeneous catalysts for transesterificationof vegetable oil- in their investigation [99, 102–111].-

#### **6.2. Pyrolysis/thermal cracking-**

Pyrolysis or cracking of vegetable oil is one of the promising routes to produce biofuel- (biodiesel and bio-jet-fuel) because of the straight chain alkanes and high cetane number of- the product [112–114]. Pyrolysis is definedas the thermal conversion of vegetable oils by heat- in absence of air in favour of a catalyst into alkanes, alkenes, aromatics, carboxylic acids and- littleamounts of gaseous products [115]. When compared with transesterification of Jatrophavegetable oil for producing biofuel, the hydrocracking process needs a higher energy and- temperature (280–300°C) [116] but the pyrolysed products have a higher cetane number and- oxidation stability. Catalytic pyrolysis increases the yield of product by breaking large- molecules, and also improves the quality of the product (biofuel).-

Catalytic cracking of vegetable oil is a three-step mechanism. First one is the removal of oxygen- by C═O bond hydrogenation, then C─O bond rupture and finally C─C bond breaking with- the aid of a catalyst. The cracking reaction may occur by different routes such as: hydrodeoxygenation, decarboxylation and decarbonylation, which are shown in **Figure 7**. Each route- produces shorter and straight chain hydrocarbons with the removal of water, CO, CO2, etc.- Catalytic cracking of Jatropha oil in the presence of differentheterogeneous catalysts shows- better result. The activities of different catalysts in cracking of Jatropha oil for producing biofuel- are investigated under different conditions.-

**Figure 7.** Catalytic cracking of triglyceride.-

Today, the conventional catalysts which are used to crack vegetable oil are mainly sulphided- silica, alumina-supported Ni-Mo, Co-Mo or Ni-W [117–122]. So using these catalysts needs the- addition of sulphur containing compounds which are responsible for the production of- sulphur residue with the end product and causes greenhouse emission like H2S and also- corrosion problem. Moreover, the noble metal catalysts are more active but the disadvantages- with them are high price and rarity, as well as they are responsible for catalyst poisoning and- impurities [123]. Recently some novel metal catalysts have been developed for hydrocracking- of Jatropha oil. PtPd/Al2O3and sulphided NiMoP/Al2O3at 330–390°C temperature and 3 MPa- pressure [124]; Ni/H-ZSM-5 [125]; sulphided form of Co-Mo/Al2O3, Ni-W/SiO2–Al2O3; and Ni–- Mo/Al2O3have been developed for producing biofuel from Jatropha oil. Among the other- catalytic systems, the homogeneous solid base catalyst is more beneficialfor hydroprocessing- Jatropha oil because of its reusability, low cost and high selectivity [126]. But the base catalyst- produces soap with FFA and it needs a high purity Jatropha oil which is the main obstacle [127].- The major problems with Jatropha bio-jet-fuel are its freezing point and low yield. The freezing- point of Jatropha hydrocarbon produced by catalytic cracking is higher than zero degrees- whereas the freezing point of conventional jet fuel is less than −40o C [128–130]. To overcome- this problem, a new catalyst system has to be designed for hydroprocessing Jatropha oil. There- are many advantages of using metal supported on microporous zeolite catalysts for hydrocracking Jatropha oil due to the versatile characteristics of zeolite [131]. Zeolite catalysts have- ion-exchange abilities with high porosity, broad surface area and concurrent-base character- [132]. It can solve the diffusion limitation and increase the production yield due to its unique- structure. For cracking reactions, high temperature (280–300°C) and pressure are necessary,- which increase the production cost. So it is most important to select and improve the nonsulphided metal supported zeolite catalyst as well as the selection of optimum conditions- (temperature, pressure and reaction time) for hydrocracking of Jatropha oil to produce diesel- and jet-fuel range hydrocarbons.-

The mostly used zeolites for cracking reaction are Zeolite Y, Meso-Y, H/ZSM-5, Na/ZSM-5, Ni/- ZSM-5, Ru/ZSM-5, Zeolite β, SAPO-11, SAPO-34, Ni/SAPO-11, ultrastable-Y zeolite (USYZ),- rare earth-Y zeolite (REY), Bentonite P-140, SBA-15, MCM-41, etc. [133–141]. The catalytic- activity of several zeolites depends on its structure, shape and size of the substrate, polarity- and the reaction parameters such as temperature, pressure, time, etc. A high reaction temperature shows the high activity of zeolite. Some investigations showed the cracking result of- differentvegetable oils with differentsupported zeolite catalysts. The cracking of sunflower- oil with the CaO/SBA-14 catalyst under 160°C temperature and 5 hours reaction time showed- 95% biodiesel yield [142], whereas SAPO-11 showed 83–90% yield on the treatment of palm- oil under 7 wt% Ni loading, 493°C temperature and 2 MPa H2pressure for 6 hours [117]. On- the other hand, Mesoporous-Y zeolite showed 40.5% bio-jet-fuel yield at 400°C temperature- for 3 h cracking [138].-

#### **7. Use challenges-**

### **7.1. Crude oil use-**

Non-edible Jatropha oil is the promising alternative as bio-energy for diesel and jet engine.- But, due to the high viscosity, large molecular mass and chemical structure of Jatropha oil, it- cannot be used directly to the compression ignition (C.I.) engines for long time. From the study- it is very clear that using Jatropha oil directly can cause some problems to the engine [143–- 145]. The main problems are pumping, burning and atomization with the injector system of- compression ignition engine. Unburned Jatropha oil can distort the injector nozzle, stick to the- ring and damage the cylinder of the diesel engine [146, 147]. It also makes the emission of- particulate substance such as smoke, unburned hydrocarbon and carbon which affect human- health and pollute the environment [146]. Hence, the better way to use Jatropha oil directly to- the diesel engine is by the reduction of its viscosity by means of blending *Jatropha curcas*oil- with diesel oil in different proportions.-

 Therehavebeensomeinvestigationstotheuseof-Jatrophaoilblendsindieselengines.-At- differentproportionsof-Jatrophaoilanddieselblends,itshowsdifferentoilpropertiesand- engineperformance.-**Table-2-**[143,-146]showsthedifferentpropertiesofdieselanddiesel/oil- blendsindifferentproportions.-Fromthedataitisclearthattheviscosityanddensity- graduallydecreasedbydecreasingtheamountofcrude-Jatrophaoilinthediesel/oilblend.- Itisobservedthattheoilblendcontainingmorethan-20%-Jatrophaoilhavehighviscosity- comparedtothemaximumviscositylimitofthedieselengine.-So,theviscosityneedstobe- reducedmoretomaketheblendusablefordieselengine.-Wherethepermeablelimitoffuel- viscosityfordieselengineismaximum-870kg/m3 ,theviscosityofoil/dieselblendfor-2.6:97.4- proportionis-868kg/m3whichisveryneartothemaximumlimit.-But,additionof-Jatropha- oilwithdieseldecreasestheexhaustgastemperature.-So,onlyasmallportion-(about-2.6%)- of-Jatrophaoilcanbeusedwithdieselfuelastheignition-acceleratoradditives.-


**Table 2.** Different properties of Jatropha oil-diesel blends.-

### **7.2. By-product: seed cake and glycerine-**

*Jatropha curcas*seed oil is the most offering alternative source of feedstock for biofuel industries.- From Jatropha biofuel plant, some by-products are produced. The main by-products are seed- cake and glycerine. To make sustainable and economically viable industry, it is necessary to- use by-products properly. But there are some problems to recover and use these by-products- directly. Jatropha seed oil and seed cake contain 58–64% protein with high nutritional value- [148]. But, they also contain some toxic ingredients such as: phorbol esters, lectins, trypsin- inhibitors, phytate, saponins, tannins, etc., which make them non-edible for human, fish, goat- and mice. Also, the process to recover glycerine from biofuel is not easy. So, proper steps should- be taken for using these by-products.-

*Seed cake*: Due to the presence of toxic components, Jatropha seed cake cannot be used as a feed- meal for human, fish,goat, chicken and rat. Phorbol ester is responsible for cancer, skin- irritation, tumour promotion and purgation [149]. Lectins cause haemorrhagic spot and- trypsin inhibitor causes adverse effect in monogastrics [150]. So, before using seed cake as an- animal feed it needs to be detoxified.-Lectins and trypsin inhibitors are heat sensitive and they- decrease during biofuel processing at about 160°C. But phorbol ester decreases only 5% at hightemperature. By increasing the digestible organic matterand metabolizable energy, heat- treatment increases the nutritive value of *Jatropha curcas*seed meal [151]. So, the main issue is- to neutralize the toxic phorbol ester from Jatropha seed meal before use. **Table 3-**[151, 152]- shows the different toxic components present in Jatropha and soybean meal.-


**Table 3.** Toxic components present in Jatropha curcas oil and seed cake.-

There have been several methods to detoxify the toxic phorbol ester from Jatropha oil and seed- kernel. Some of them are fungal isolation, γ-irradiation, adsorption, plasma application,- ozonation, etc. During the Jatropha oil refining and purificationprocesses about 55% phorbol- ester can be removed by bleaching and de-acidificationstep but degumming and deodorizing- step cannot remove phorbol ester [151]. About 44% phorbol ester can be removed by chemical- treatment by using NaHCO3, whereas the combination of chemical treatment and heat- treatment can remove 56% and the combination of chemical treatment and ozonation can- remove up to 75% phorbol ester from *Jatropha curcas*seed oil and seed meal [153]. Gamma- irradiation can remove 71.35% of phorbol esters at 50 kGy absorption dose. But this method- takes long time, high temperature and high dose of gamma irradiation which is not economic.- On the other hand, fungal isolation can remove 97.8% of phorbol esters from Jatropha seed- and oil [154]. Among all the processes, the adsorption process is more effective to detoxify- Jatropha oil phorbol ester and this process can remove up to 99.5% phorbol ester present in- the Jatropha seed oil. Un-detoxifiedoil contains 2.70 mg/g phorbol ester. After detoxification- by the adsorption process, Jatropha oil contains 0.02 mg/g phorbol ester, which is lower than- permissible limit (0.09 mg/g) [152, 155].-

 In the adsorptionprocess,theone-timeadsorption carriedout with 3.2% (w/v) bentonite 200- astheadsorbentat-32°Ctemperature,-100rpmstirringrateand-15minadsorptiontimecan- remove-98%phorbolester.-Two-timeadsorptionwith-0.8%-(w/v)bentonite-200undersame- conditionscanremove-99.50%phorbolester-[152].-

*Glycerine*: Glycerine is the major by-product of Jatropha biofuel processing plant. So, the- recovery and proper use of this product is more beneficialfor the Jatropha biofuel project. But,- the recovery of glycerine from biofuel is not an easy process. Traditionally, glycerine is- recovered from biofuel by washing with water. In this process, water is mixed with the- biodiesel, agitating the mixture gently, allowing the mixture to separate the several phases and- finallyglycerine is extracted from the water phase [156]. But, this process is not favourablebecause water causes many problems when used to wash biofuel. Washing of biofuel needs- the use of deionized water, produce large amount of wastewater. This water can degrade the- biofuel by hydrolysis and it can increase the processing time with multiple drying, multiple- washes and water-biodiesel separation steps [157]. The suitable alternative to recovery- glycerine from the biofuel is the adsorption with a bed of ion exchange resin [158]. But this- process is not established yet. Recovery of glycerine from biofuel by ion exchange resin is the- combination of four steps: filtration,physical adsorption, ion exchange and removal of soap- by glycerine affinity.-

#### **8. Alternative of Jatropha-**

There are many potential non-edible oil-rich plants in almost every country (mostly tropical- and subtropical). Mostly they are wild and naturally growing. They may or may not have yet- been explored for oil producing potential. In India, 11 tree species (*Garcinia indica, Azadirachta- indica, Hevea brasiliensis, Calophylluum inophyllum, Madhuca indica, Mesua ferrea, Mallotus- philippines, Ricinus communis, Pongamia glabra, Salvadora*and *Shorea robusta*) are largely distributed that have biodiesel producing potentials [17]. *Camelina sativa, Gossypium hirsutum, Cynara- cardunculus, Abutilon muticum, Simmondsia chinensis, Passifloraedulis, Aleurites moluccana,- Carnegiea gigantea, Pachira glabra, Croton megalocarpus*and *Terminalia bellirica*have high content- of non-edible oil in their seeds [159]. These plants may also be explored for their suitability to- meet the blending requirements rather than focusing on a single candidate (Jatropha).-

#### **9. Lifecycle assessment (LCA) of the Jatropha biofuel-**

Environmental impact of biofuels is determined by lifecycle assessment [160, 161]. The- environmental flowsthroughout the lifecycle stages of a product or services are evaluated by- LCA [162]. The four interacting individual phases—scoping, inventory analysis, impact- assessment and interpretation—are the basic of LCA study (ISO, 2006). International Organization for Standardization (ISO) regulates it as 14040:2006 and 14044:2006 standards.-

Jatropha cultivation, oil extraction, conversion of seed oil into biodiesel and biodiesel use are- four major phases of the Jatropha biodiesel system. **Figure 8**shows the system boundaries of- the Jatropha biodiesel. The flowprocesses, inputs and outputs in the Jatropha biodiesel system- are summarized in **Table 4**. Energy balance, global warming potential (GWP), and land-use- impact, net energy gain (NEG), net energy ratio (NER), ecosystem structural quality (ESQ) and- ecosystem functional quality (EFQ) are most relevant impact categories of LCA systems [161,- 163]. The data on LCA of Jatropha biodiesel is not sufficientthough there are some reports on- the LCA methodology [163–167] and LCA [168, 169] for Jatropha biodiesel.-

**Figure 8.** System boundaries of the Jatropha biodiesel system.-


**Table 4.** Flow processes, inputs and outputs of the Jatropha biodiesel system.-

Jatropha is believed to be a suitable crop for wastelands with low inputs. However, an industry- cannot be established depending on unreliable feedstock supply based on low-input agriculture. Thus, fertilizers, irrigation and pesticide use will be unavoidable in commercial Jatropha- production. Furthermore, transesterification,the major conversion technology, is the majorcontributor to GHG emissions and energy consumption. To be a suitable alternative of petrodiesel in terms of mitigation of climate change, Jatropha biodiesel needs to be supported by- lifecycle data. The environmental benefitsof Jatropha biodiesel in comparison to petro-diesel- is in doubt [169, 170]. Following measures can improve lifecycle performance of Jatropha- biodiesel:-


#### **10. Contribution and expectation from Jatropha-**

 Withenormouspotentialsonsocial,agricultural,environment,sustainableenergyproduction- andindustrialfronts,-Jatrophaisattractinginterestfromresearchersandpolicymakers.- Researchshowedthat-Jatrophaseedhas-40–60%oilcontentwithaproductivityof-0.1–12tons- perha-[32,-63].-Yieldsofbothfruitsandoildependonspecies,accession,soil,climateand- agronomy-[66,-171].-Aseedyieldof-4–5mg/ha/yrisexpectedforthecommercialviabilityof- the-Jatropha-basedbiofuelprogram.-With-30–35%oilcontentandanaverageseedyieldof- 3.75mgha/yr-Jatrophaiseconomicallymorebeneficialtotheaverageyieldprofileofsoybeans- andrapeseed-[19].-

However, the actual seed production of Jatropha in fieldconditions was poor than expected- [20–22]. The reported yields of Jatropha under fieldconditions in India, Belgium, South Africa- and Tanzania are 0.5–1.4 mg/ha/yr, 0.5 mg/ha/yr, 0.35 mg/ha/yr and 2 mg/ha/yr, respectively- [23]. The less productivity is because of unavailability of suitable high yielding varieties, largescale plantation without evaluating the genetic potential of planted materials, consideration- of Jatropha as no/low input crop and lack of knowledge on agronomy.-

Jatropha seed cake is an excellent source of protein. To add commercial value it is expected to- utilize the press cake as an animal feed protein supplement. The presence of toxic compounds- hinders its utilization for this purpose. The discovery of non-toxic Jatropha varieties and the- detoxificationprocess of toxins are an advantage. Many biological active chemical compounds- are extracted from bark, leaves and roots that are expected to be used in the pharmaceutical- industry. However, toxicity must be studied before the use of Jatropha products as therapeutic- agents or medicines.-

Oil from Jatropha has been used as cooking fuel and in soap and cosmetic manufacturing in- ancient times. It is reported that Jatropha oil can be utilized as a fuel with diesel engine directly- or with slight modificationof the engine. Jatropha oil can be converted to biodiesel by chemical- reaction called transesterification.-This biodiesel can be used by blending with petro-diesel.- High viscosity is the major problem of using Jatropha seed oil as fuel.-

#### **11. Technological intervention for Jatropha improvement-**

Jatropha bears multi-dimensional potentials [172]. But it is still behind to compete in commercialization. High yielding Jatropha varieties are not found yet. Lack of agronomic knowledge,- unawareness or knowledge gap of farmers and the common belief that no impute crop renders- Jatropha's productivity [2, 173]. It requires extensive searching for natural germplasms and- systematic breeding programs for genetic improvement. The success of breeding depends on- the availability of diverse germplasms [62]. However, some study showed narrow genetic- diversity among the worldwide population [174–179]. It limits the success of conventional- breeding. Inter-species and inter-generic hybridization, haploid breeding (anther, pollen,- ovary culture), somaclonal and germaclonal variation and mutation breeding are biological- techniques that have proven records for variation creation and breeding of many important- crops. For Jatropha there are some reports on organogenesis only. Other technologies are still- unexplored. Genetic resources technology, i.e. marker-assisted selection (MAS), molecular- breeding, genomic selection, genome-wide association studies and genetic engineering have- been used with confidentfor many crop breeding programs. Jatropha genomic technology- research is still far behind in comparison to other important crops though some reports are- available on that [180–186]. Now it is time to explore full potentials of Jatropha by using modern- technologies.-

#### **12. Conclusions-**

It is very crucial to findreliable renewable energy sources for healthy economy and environment. Jatropha is a promising candidate of renewable energy as it has interesting characteristics- and non-competition with food. A number of big projects has been launched and completed.- However, the result is disappointing. Unavailability of good commercial variety, considering- low impute and disease resistance crop, knowledge gap, lack of basic research and theoretical- assumption mostly without scientificand technological backup are the major reasons of the- failure. Thus, Jatropha biofuel industry is confronting a number of challenges.-

Efficientoil extraction methods form Jatropha seeds need to be explored. Mechanical pressing- is commonly used but it is poor yielding and also affectsthe oil quality. Solvent extraction- needs to use many hazardous solvents. Enzymatic process is good but has a slow reaction rate.- It needs to find/develop suitable enzymes and to increase the reaction rate. Conversion of crude- Jatropha oil to biofuel (biodiesel or jet fuel) is another challenge. Transesterificationand- thermal cracking are commonly used. It needs to develop environmentally friendly catalyst- with high conversion efficiency. By-product utilization can reduce waste management cost and- also add economic value. Seed oil cake and glycerine are the main by-products of the Jatropha- industry. It needs appropriate method of glycerine recovery and detoxificationmethod of seed- cake for safe use.-

Plant breeding in application of biotechnology is the gateway of crop improvement (yield and- quality). Diverse germplasm is the basis of a breeding program. Accumulation and utilizationof specialised but scatteredknowledge is important for Jatropha improvement. Major Jatropha- cultivating countries—India, China, Malaysia, Indonesia, Brazil, Mexico and South Africa—- can establish an international organization. To design a strategic breeding program for- Jatropha improvement, the researchers can share their learning gained by several years of- experience.-

Biotechnology application in Jatropha breeding is far behind as compared to some other crop.- Somaclonal and germaclonal variation are created by *in vitro*mutagenesis, *in vitro*micropropagation, anther and microspore culture, ovary and ovule culture, protoplast culture, nucleolus culture, endosperm culture, and somatic embryogenesis. It needs to explore them because- these techniques have proven record for crop improvement. There are a few works on Jatropha- genome though; it is still far behind in comparison to other agricultural systems.-

Though there are some biotechnology studies, however, Jatropha genome work is far behind- than the model and other agricultural systems. Researchers require a high density linkage map- for the determination of the association of markers with high oil yield. The *J. curcas*genome is- very small (ca.400 Mb). Thus, marker-assisted selection, genome-wide association studies- (GWAS) and genomic selection (GS) could be even more attractive. A well-assembled reference- genome of Jatropha is indispensable for these applications. Increasing female flowersin- inflorescence, reducing toxins and increasing resistance are on priority.-

#### **Author details-**

M. Moniruzzaman1\*, Zahira Yaakob1 , M. Shahinuzzaman1 , Rahima Khatun1 and- A.K.M. Aminul Islam2-

 \*Addressallcorrespondenceto:monirbge@gmail.com-

 1-Departmentof-Chemicaland-Process-Engineering,-Facultyof-Engineeringand-Built- Environment,-Universiti-Kebangsaan-Malaysia,-Selangor,-Malaysia-

2 Department of Genetics and Plant Breeding, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh-

#### **References-**


## **Review of Continuous Fermentative Hydrogen-Producing Bioreactors from Complex Wastewater**

Paula Rúbia Ferreira Rosa and Edson Luiz Silva

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/110416

#### **Abstract**

In recent years, the production of hydrogen through dark fermentation has become increasingly popular because it is a sustainable approach to produce clean energy. Thus, an evaluation of studies reported on hydrogen production from different complex wastewaters will be of immense importance in economizing production technologies. This work presents a review of the advances in the bioreactor and bioprocess design for biohydrogen production from different complex wastewaters. The biohydrogen production is discussed emphasizing the production metabolic pathways, bioreactor configuration and operation, organic loading rate (OLR), pretreatment of wastewater, as well as microbial diversity. Also, in this review, various bioreactor configurations and performance parameters including H2 yield (HY) and hydrogen production rate (HPR) are evaluated and presented. The work concludes with challenges and prospects of biohydrogen production and claims for more systematic and comprehensive studies on the subject.

**Keywords:** biogas, global warming, dark fermentation, bioreactor, process parameters-

### **1. Introduction**

According to the IPCC [1] (Intergovernmental Panel on Climate Change), global warming of more than 2°C would have serious consequences, such as an increase in the number of extreme climate events. In Copenhagen in 2009, the countries stated their determination to limit global warming to 2°C between 2015 and 2100. To reach this target, climate experts estimate that global greenhouse gas (GHG) emissions need to be reduced by 40–70% by 2050 and that carbon neutrality (zero emissions) needs to be reached by the end of the century at the latest. To reduce global warming, substantial effort is being made at a global scale to explore renewable energy sources that could replace fossil fuels.-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Hydrogen gas can be an ideal sustainable energy carrier, which can reduce the over-reliance on fossil fuels. Some of the advantages of hydrogen can be listed as follows: (i) high energy conversion efficiencies, (ii) production from water with no emissions and (iii) abundance [2].-

Dark fermentation is a biological approach commonly used to produce H2 in the absence of light. It is driven by anaerobic bacteria that can produce hydrogen from wastewaters- [3]. Thistechnology has attracted attention because it can use a versatile range of substrates, particularly renewable resources that are organically rich such as stillage, sludge,- leachate, pomace, stalks and bagasse [4]. Wastewaters generated from various industrial- processes are considered to be the ideal substrates because they contain high levels of easily degradable organic material, which results in a net positive energy or economic balance [5]. From the anaerobic digestion process, complex wastewater can be converted into- hydrogen, while promoting the treatment of these wastewaters, providing environmental- sustainability.

Studies on batch, semi-continuous and continuous hydrogen-producing bioreactors have been conducted. Batch hydrogen fermentation normally brings about lower hydrogen production rates (HPRs) in comparison with its semi-continuous or continuous counterpart. Besides the extensively studied continuous stirred tank reactor (CSTR), numerous biohydrogen bioreactor processes such as anaerobic sequencing batch reactor (ASBR), fixed-bed bioreactor, fluidized-bed bioreactor and upflow anaerobic sludge blanket (UASB) bioreactor have been developed with high production yields and output [6].-

Inevitably, performance of hydrogen-producing bioreactor systems and operation are determined by various factors that are associated with environmental conditions, process operating conditions and chemical conditions, such as inoculum, nutrients, hydrogen partial pressure, temperature, hydraulic retention time (HRT) and substrate concentration [6]. Variations in these factors result in different microbial communities, resulting in different hydrogen yields. In this context, this review summarizes the above factors that influence hydrogen production by dark fermentation from different complex wastewaters.-

### **2. Microbiology of hydrogen production: metabolic pathways**

Hydrogen can be produced through different metabolic pathways that can be broadly grouped into two distinct categories—light-dependent and light-independent processes. Light-dependent processes include direct or indirect photolysis and photo-fermentation, whereas dark fermentation is a major light-independent process [7]. According to Sinha and Pandey [8], compared to the photosynthetic processes of hydrogen production, fermentation processes have the advantage of a rapid rate of hydrogen production and simplicity of operation.

The anaerobic digestion process generally consists of the four stages, i.e. hydrolysis, fermentation, acetogenesis and methanogenesis (**Figure 1**). In the first two stages, dark fermentation is involved in the production of hydrogen. Various microorganisms are involved in each step and cooperated with each other to achieve carbohydrates that are converted into hydrogen gas, VFAs and alcohols, which are organic pollutants and energy carriers.-

Review of Continuous Fermentative Hydrogen-Producing Bioreactors from Complex Wastewater 259 http://dx.doi.org/10.5772/110416

**Figure 1.** The steps involved in anaerobic digestion [9].-

According to Levin et al. [10], carbohydrates are the preferred substrates for the production of- hydrogen. Different complex wastewaters have different hydrogen yield per mole of glucose,- depending on the metabolic pathway of the final product. When acetic acid is the final product,- the maximum theoretical yield is 4 mol/mol glucose (**Table 1**, Eq. (1)). However, when butyrate is the final product, the maximum theoretical yield is 2 mol/mol glucose (**Table 1**, Eq. (2)).-

The absence of propionic acid, valeric acid and caproate production ensures higher hydrogen production due to no demand for H2 formation of this acid (**Table 1**, Eqs. (3) and (11)–(15)). Lactic acid is produced from glucose through three metabolic pathways (**Table 1**, Eqs. (4)–(6)), and in all three metabolic pathways, hydrogen is neither consumed nor produced. The same is true for ethanol production, where the balance of hydrogen is zero (**Table 1**, Eq. (7)).-

Hydrogen can be produced simultaneously with ethanol (Eq. (8)) [13, 14]. In addition, there may be a joint production of organic acids (Eqs. (9) and (10)).-

Siriwongrungson et al. [15] show that the acetate formed in the acetogenesis may be a consumer of hydrogen. The reducing reaction of hydrogen with carbon dioxide acetate is called homoacetogênese (Eq. (16)). This in turn becomes an important factor in the production of hydrogen, since there is a drop in consumption and performance, through the accumulation of acetate in the medium.


**Table 1.** Reactions during acetogenic hydrogen fermentative.

In the step of acetogenesis, the hydrogen could be formatted from lactic acid, ethanol, propionic acid and butyric acid (Eqs. (17)–(20)).-

Hydrogen could be consumed for archaea hydrogenotrophic (Eq. (21)). Approximately 70% of all the methane produced in anaerobic digestion process stems from Eq. (22). Furthermore, methane is formed from acetate, butyrate, formate, ethanol and methanol (Eqs. (22)–(26)).-

Among the fermentative anaerobes, *Clostridia* have been well known and studied extensively, not for their hydrogen production capability but for their role in the industrial solvent production from various carbohydrates [7]. Hydrogen production by these bacteria is highly dependent on the process conditions such as pH, hydraulic retention time (HRT), and gas partial pressure, which affect metabolic balance. Thus, fermentation end-products produced by microorganism depend on the environmental conditions in which it grows [10].-

There are also some bacteria that consumed hydrogen, such as *Lactobacillus* spp. and *Bifidobacterium* spp. [4]. Moreover, the major H2 -consuming microorganisms other than hydrogenotrophic methanogens are homoacetogens, such as *Methanobacterium*.

Depending on the pathway, the theoretical biogas composition is around 67% of H<sup>2</sup> (acetate pathway) or 50% of H2 (butyrate pathway). The various metabolic pathways that may establish can either be promoted or inhibited, depending on the adopted operating conditions, which govern the production of specific volatile fatty acids (VFAs) and alcohols including acetate, propionate, butyrate, lactate and ethanol [16].-

### **3. Dark fermentation from complex wastewaters**

Dark fermentation is a biological approach commonly used to produce H2 in the absence of light and hence the configuration of the bioreactor is simpler and cheaper. Hydrogen production by dark fermentation has several other advantages such as the ability to produce hydrogen from organic waste and therefore control and stabilize biological waste which has a potential danger of contamination. For instance, dark fermentation can be integrated into wastewater treatment systems to produce H2 from wastewater. Producing hydrogen from organic waste has a potential to reduce hydrogen production costs since organic waste (including wastewater) is cheap and easily available [2]. Moreover, the regulatory need of treatment of wastewater prior to disposal is making them an ideal commodity to produce biohydrogen from the anaerobic treatment [17].-

The main source for the fermentative H2 production is complex wastewater containing carbohydrate substances. Crucial points for improving the efficiency of hydrogen production- that are frequently emphasized throughout literature are associated with facilitated access- to cheap wastewater, such as vinasse, cassava wastewater, cheese whey, glycerol, sago- wastewater and textile wastewater. There are several articles in the literature that demonstrate thehydrogen production from these wastewaters, indicated in **Table 2**, including the process parameters such as substrate concentration, pH, temperature, HRT, reactor type and seed sludge.

Numerous works have been focused on vinasse. This wastewater, one of the major by-products- of the ethanol production process with nearly 14 L of vinasse produced per litre of ethanol,- can cause extensive pollution due to its high organic load (up to 40 g COD/L) [18]. Cassava- wastewater and cheese whey, main components of agro industrial processes, are considered- as highly polluting due to their high organic load and the volume generated, representing a significant environmental impact for the agro-industry [19, 20]. They were also used for the- successful H2 production [21–25].-





**Table 2.** Studies of anaerobic biohydrogen production processes using complex wastewater.- The performance parameters were hydrogen production yield (HY) and hydrogen production rate (HPR). The process parameters including pH [22, 26], hydraulic retention time [22, 27–32], temperature [33–35], substrate concentration [30, 31, 36–38], different sludge [21], support materials [39], pretreatment of wastewater [24, 25, 40–42], use of co-substrate [21, 25, 30, 43, 44], inoculum pretreatment [22, 45, 46], addition of nutrients [44, 47], reactor configuration [45, 48] and effects of some heavy metal ions [49] have already been evaluated. Several types of wastewaters listed in this review could produce hydrogen with a HY range of 0.74–5.3 mmol-H2 /g-COD and a HPR range of 0.03–14.04 L/L/d. Among the wastewaters studied, one of highest HY of 5.3 mmol/g COD was obtained using continuously stirred anaerobic bioreactor (CSABR) from condensed molasses; it was successfully operated for 300 days [48].-

### **4. Simultaneous hydrogen and methane production in a single-stage biosystem**

The key point in the fermentative production of hydrogen is the inhibition of the methanogenic step so that the formed hydrogen is not consumed for the formation of methane. Two routes can lead to the formation of methane: the route acetoclastic from acetic acid and methanol (methanogenic acetoclastic or acetotrophic microorganism (**Table 1**, Eqs. (22) and (25)) and the hydrogenotrophic route from H2 (hydrogen consumers microorganisms or hydrogenotrophic (**Table 1**, Eq. (21)).-

Among the forms of control, methanogenic activity can be used to maintain the acidic pH medium for cultivation and processing of the inoculum in order to inactivate methanogens [50].- Furthermore, for continuous reactors, the reduction of HRT and consequently higher organic- loading rate (OLR), they can avoid the use of H2 as a substrate for methanogenesis.

Recently produced hythane (H2 + methane) in a single-stage biosystem, using complex wastewaters with low pH, shorts HRT and high concentration of organic matter suggested that some archaea can survive at conditions that do not favour methanogens [22, 25, 27, 30, 33, 36]. The hythane production has also received much commercial attention in the transportation sector [51], and a production in a single stage has the advantage of being economically more viable due to economic financial, energy and manpower, than the hythane production by two-stage fermentation [52].-

Kim et al. [53] conducted a study on the influence of pH on the activity of users consuming hydrogen methanogens. According to the authors, the formed methane left in consumers of hydrogen archaeas, which are commonly inhibited at pH below 5.0, proved to be more tolerant of acidic conditions than other methane-producing microorganisms.

Carrillo-Reyes et al. [22] evaluated the reduction of pH (5.63–4.5) in UASB reactors fed cheese- whey, with a HRT of 6 h and OLR of 20 kg COD/m<sup>3</sup> .d (**Table 2**). The authors reported that the strategy of reducing the pH to 4.5 to avoid methane production was not efficient. This fact did- not favour the hydrogen production and even caused a sharp drop in the total gas production. Similar results were found by Taconi et al. [54], who found a 30% increase in methanogenic- activity when the pH was decreased from 7 to 4.5.-

As maintaining the pH in acidic conditions does not guarantee the inactivation of methanogens, the heat treatment of the inoculum is not conclusive. For instance, the acetoclásticas- microorganisms can survive thermal shock, leading to the consumption of hydrogen to acetic acid formation [50]. The formed acetic acid is then converted into methane (**Table 1**, Eq.-(23)).-

This fact is reported by Luo et al. [55] who used cassava stillage as the substrate for hydrogen- production and found that thermal pretreatment of inoculum does not improve the yield of hydrogen in continuous reactors under mesophilic temperatures. The study analysed the effect- of different pretreatments of the inoculum such as acid treatment, heat treatment and shock- load in repeated batch tests, demonstrated that inoculum pretreatment could not permanently inhibit methanogenesis either. According to the authors, the methane inhibition only occurs by proper control of fermentation, pH and temperature.

Given the resistance of methanogenic archaea of pretreatment of inoculum, Carrillo-Reyes et al. [22] showed that repeated heat treatment of the granular sludge was the only strategy that completely inhibited methane production, leading to high volumetric hydrogen production rates (1.67 L H<sup>2</sup> /L-d). In the same study, the authors use a strategy to decrease methane production: the shock loads (from 20 to 30 g COD/L-d) was a more effective strategy to decrease the methane production rate (75%) and to increase the hydrogen production rate (172%), without stopping reactor operation.-

Methanogens were detected in different hydrogen-producing reactors operated at low pH (values between 4.0 and 5.63) and with high organic loading rate (**Table 2**) revealing that they can survive under these extreme conditions.-

 In-ASBRs,-Buitrón and Carvajal [33] reported the production of methane (35–44%) concomitant with the production of H2 when employed with HRT of 24 h. They found that the higher the concentration of vinasse, the greater the percentage of methane achieved. According to the authors, methanogens could be already present in vinasse and before the source of organic acids, and H2 produced by the reactor found an environment conducive to development on the other hand, using similar wastewater, and even reactor. Searmsirimongkol et al. [36] evaluated the effect of concentration (20, 30, 40 g/L) on the hydrogen production. The highest methane yields (approximately 40% methane content of the biogas) were found at lower concentration (20 g/L); however, concentration higher than 60 g/L did not verify the presence of methane. Serious methanogeneses were reported in high rate reactors, such as UASB [22] and AFBR [25, 30].-

The hydraulic retention time (HRT) is also an important parameter in the fermentation processes. Higher rates of volumetric hydrogen production and increased percentages of hydrogen in biogas can be obtained by decreasing the HRT and thus increasing the organic loading rate (OLR) [56]. In addition, low HRT could suppress methane producers and inhibit- methanogenesis. However, in many complex wastewaters, this behaviour is not checked.- Exemplifying, Rosa et al. [57] evaluated the effects of different hydraulic retention times- (HRTs) of 4, 2 and 1 h and varying sources of inoculum (sludge from swine and sludge from- poultry) on the hydrogen production in two AFBRs from cheese whey. When the HRT wasreduced, methane was produced concurrently with hydrogen in both reactors, with maximum methane production of 0.68 L CH<sup>4</sup> /h/L with an applied HRT of 1 h. Carrillo-Reyes et al.- [56] found that the application of a OLR of 20 g COD/L/d and a gradual decrease of HRT from- 24 to 6 h led to a decrease in H2 production from 0.03 to 0.015 LH<sup>2</sup> /L/h, due to the presence of- methane. According to the authors, the delay in the production of methane from this reactor, when compared to other reactors in their study, was due to the application of high substrate- concentrations. The maximum methane yields of 0.02 L/h/L were obtained in reactors with the- application of HRT of 6 h, and OLR from 5 to 20 g COD/L. Other studies have also found the- simultaneous hydrogen and methane production in short HRTs, from different wastewaters,- such as stillage [30, 33], rich in starch wastewater [25, 27].-

These results indicated that the low HRT in different configurations of reactors might reduce microbial richness through the washout of microbes and increase microbial diversity through accelerating the proliferation of non-hydrogen-producing microorganism. So, methanogens could adapt to the conditions imposed in hydrogen-producing reactors (low pH, high OLR and low HRT). In spite of the negative effect of these organisms in hydrogen production, they may have an important application in the production of hythane (H2 and CH4 ) using wastewaters with low pH and high concentration of organic matter.-

### **5. Bioreactor configuration-**

The reactor configuration and the improvement of operating parameters is essential to obtain best hydrogen production rates, indicating that the system performance is largely influenced by the retention of biomass reactor [58]. The batch modes of operation and continuity have been reported in the literature for producing hydrogen. Most batch studies have the advantage of being easily operated, flexible, generating a series of studies with different wastes to produce hydrogen [9]. However, these reactors provide lower H<sup>2</sup> production rates as compared to continuous systems.

Continuous stirred tank reactors (CSTR) are the most common continuous system used for hydrogen production by dark fermentation from olive milk wastewater, cheese whey and- condensed molasses (**Table 2**). Reactors upflow anaerobic sludge blanket (UASB), anaerobic- fluidized bed (AFBR) and anaerobic packed bed reactor (APBR) also are used for the production of hydrogen in different complex wastewater. The advantages and disadvantages of- different types of bioreactors for H2 production are listed in **Table 3** [59, 60].-

Glycerol [49], sago wastewater [46] and brewery wastewater [35] were proved to be feasible substrates by batch tests showing the maximum HY of 2.2 mmol/L, 323.4 mL/g starch and 158 mL/g COD, respectively. In continuous H2 production, the main reactor used was AFBR [25, 30, 43], UASB [22, 23, 28, 32, 37, 38] and CSTR [29, 34, 45].-

In fermentative hydrogen production, the HRT, and in turn the OLR, affect the substrate conversion efficiency, the type of active microbial population as well as the metabolic pathways established in the system [16]. In the following sections, a discussion of literature findings about the influence of these parameters is presented.-


**Table 3.** Bioreactors for H2 production: advantages and drawbacks.-

#### **5.1. Influence of OLR-**

The parameters that constitute the OLR are the concentration of organic matter and HRT. For it is a design variable which determines the capacity and the reactor operating conditions. Changes in OLR have a considerable influence on the diversity of the microbial population and on the metabolism pathways of bacteria that may favour hydrogen production [61].-

According to De Gioannis et al. [16], there is a discrepancy in the literature regarding the effect- of OLR and HY. According to these authors, the OLR is affected by the accumulation of acid, pH- changes and variations in the composition that subsequently change the metabolic pathways.-

### *5.1.1. Substrate concentration*

The substrate concentration should be selected in order to meet the needs of microbial growth and hydrogen production and its increase can ensure a stable production of hydrogen in high yield [43]. However, concentrations of organic matter in excess decrease substrate conversion and the yield of hydrogen due to the accumulation of inhibitory compounds in the medium, reducing the competitiveness of hydrogen producers for other microorganisms [3, 43].

In the batch tests, optimal substrate concentration varied and was deeply influenced by other operational parameters such as the pH. When the pH was not controlled, HY usually decreased with increasing substrate concentration due to low pH condition. In contrast, finding the optimal substrate concentration in continuous operation mode is more meaningful and practical, since the batch mode does not take into consideration the hydrodynamic effect, steady state of the substrate concentration and pH condition for bacterial growth [62].-

Higher feeding concentrations of the substrate could increase H2 production [22, 26]; however,- excessive substrate concentrations may decrease this capacity [25, 28, 31, 34, 36, 37]. Chu et al.- [48] in a suspended sludge bioreactor producing H2 fed with condensed molasses fermentation soluble, increased the H2 production rate by 2.3 times by elevating the substrate concentration from 40 to 60 g COD/L at a HRT of 2 h. Already, in continuous mixed immobilized sludge- reactor from molasses wastewater, Han et al. [26] increased the HPR 3.36 times by elevating- the substrate concentration from 2 to 6 g COD/L at a HRT of 6 h. In contrast, when varying thetofu-processing wastewater concentration from 10 to 40 g COD/L in a batch reactor, Lay et al.- [34] found that 20 g COD/L was the optimum concentration for H2 production.

 Most studies reported that hydrogen production from complex wastewaters had substrate- concentrations lower than 40 g COD/L (**Table 2**). Often it is noted that higher concentration of any substrate leads to a drop in HY [34]. Moreover, it has been reported that in some cases hydrogen production can be inhibited by the toxicity of the complex wastewaters. This fact is- noted by Searmsirimongkol et al. [36] who then diluted alcohol distillery wastewater to obtain- various feed COD values of 20,000, 40,000 and 60,000 mg/L. The highest concentrations of- hydrogen production resulted in inhibition due to the presence of high potassium concentration. Already Liu et al. [31] showed that SO<sup>3</sup> 2−affected the hydrogen production at the substrate- concentration of 10 g total sugar/L process, when the performance of hydrogen production- decreases, HPR was reduced from 34.59 to 6.50 L/L/d, yield was reduced from 0.92 hexose to- 0.08 mol H<sup>2</sup> /mol hexose, when SO<sup>3</sup> 2−iincreased from 0 to 80 mg/L. Sulphate-reducing bacteria- (SRBs) causes hydrogen gas converting hydrogen sulphide become less efficient in hydrogen- production.

A maximum hydrogen production of 11.39 L/d/L was obtained at HRT 1.0 h (a concentration of 10 g) from washing wastewater of beverage production process with continuously- stirred anaerobic bioreactor [31]. The authors suggested that the hydrogen-producing bacteria (HPBs) were adaptive to the system.-

High substrate concentration allows more energy-efficient operation but product inhibition is likely to set the upper limit. Certain level of metabolic products in the dark fermentation may inhibit H2 producing pathway as well as microbial activity [58].-

### *5.1.2. HRT*

HRT indicates the time that the organic matter remains in the reactor. This time depends on the metabolism rate of organic matter by microbial community and may vary according to the process. HRT can be used to select a producer of hydrogen community depending on the substrate used.

HRT is also an important parameter in the fermentation process. Higher rates of volumetric hydrogen production and increased percentages of hydrogen in biogas can be obtained by decreasing the HRT and thus increasing the organic loading rate (OLR) [56]. Shortening hydraulic retention times (HRTs) is a well-used and effective operation strategy to enhance hydrogen production from organic wastewater and solid wastes because of its ability to exclude methanogens which have longer generation time.-

In most studies on continuously dark fermentative hydrogen production, continuous systems are expected to operate at a low HRT 36–12 h [27, 33, 34, 37, 39] and very low of HRT 12–2 h [21, 22, 25, 26, 28–30, 43, 45, 48] for obtaining a high biohydrogen production that can be operated at extremely low HRT 2–0.5 h [30, 47, 48] with immobilized cell in the biohydrogen.-

As shown in **Table 2**, the range of organic loading rate (OLR) was 16–320 kg/m<sup>3</sup> /d equivalent by a gradual decrease in HRT from 32 to 0.5 h. When considering the variation in hydrogen production with respect to the HRT, it can be seen that the HRT greatly affected microbial activity and metabolic products, leading to variations in gas production rate, gas composition and hydrogen production rate [36].-

With regard to the microbial community, short HRT is also preferred from beverage wastewater- [29, 31], sugarcane stillage and glucose [43] and crude glycerol [28]. In contrast, most studies had- a drop in hydrogen production because: of too low mixing and poor contact of glycerol with the- microorganisms [28]; of the occurrence of OLR shock from tapioca wastewater [27]; of longer- reaction time, which allowed for more time to metabolize the Tequila vinasses [33]; microbial- cells were washed out from the system as a result from the toxicity of VFA accumulation from- alcohol wastewater [32] and of lactate accumulation from tofu-processing wastewater [34].-

A maximum hydrogen production of 55 L/d/L was obtained at HRT 1.5 h (an OLR of 320 g/L-d hexose equivalent) from beverage industry wastewater (20 g/L hexose equivalent) with CSTR [29]. This HPR value is much higher than those of other complex wastewaters employed in fermentative hydrogen production.

Therefore, it is essential to define a range of OLR, which will enable to achieve constant efficiency in the biological reactor, or an optimum OLR value for maximum H<sup>2</sup> yield. As a result, the fermentative routes and final metabolites products may be modified due to the OLR applied, as well as the conversion efficiency of the substrate and the microbial community established in the system [16].-

### **6. Strategies for improved hydrogen production**

Fermentative hydrogen production is a very complex process and is influenced by many factors- such as inoculum, substrate, reactor type, temperature and pH. The effects of these factors on- hydrogen production have been reported by a great number of studies throughout the world- in the last few years.-

Wang and Wan [63] showed that there usually existed some disagreements on the optimal condition of a given factor for fermentative hydrogen production, thus more researches in this respect are recommended.

### **6.1. Pretreatment of complex wastewater**

To enhance the fermentations of some complex wastewaters, such as cassava wastewater,- tofu-processing wastewater, corn starch wastewater and textile wastewater, pretreatment- must be done to make the process feasible and sustainable. These processes include various combinations of biological, physical and chemical treatment processes [24, 25, 34, 40–42]. Each- of these pretreatment methods has a unique purpose and will depend on the wastewater used.-

Starch can be hydrolyzed into glucose and maltose by acid or enzymatic hydrolysis followed by- biological conversion of the carbohydrates into organic acids and then into hydrogen gas [64].- Moreover, mixed culture could produce more various hydrolases which could utilize complex- substrates present in wastewater than pure culture [65]. Rosa et al. [25] used the technique ofacid hydrolysis with sulphuric acid and heated the cassava wastewater at 120°C for 30 min- before being used as a substrate. A maximum hydrogen yield of 2.0 mmol/g COD was achieved- with OLRs of 10 kg COD/m<sup>3</sup> /d.-

The heat treatment of complex wastewater rich in starch (corn starch wastewaters, rice mill- wastewater, cassava wastewater) is common, with the purpose to remove the mixed population- of microorganism in the wastewater, which could either compete with biohydrogen producers- or inhibit their growth [24, 42, 44]. In these studies, the heat treatment was made in 120°C with- times between 15 and 25 min. This pretreatment was also used for tofu-processing wastewater,- but at temperature 70°C for 30 min to inhibit the hydrogen-consuming bacteria [34].-

Lactic acid bacteria (LABs) are members of the autochthonous microbiota of cassava and are responsible for the fermentation of the root; furthermore, LAB reduces cassava toxicity and- prevents post-harvest deterioration [66]. However, in hydrogen-producing reactors, a few LAB- strains may have an inhibitory effect due to their bacteriocins, which are antimicrobial peptides- that have a deleterious effect on H<sup>2</sup> -producing bacteria [67]. Gomes et al. [24] conducted pretreatment heat (121°C; 15 min) in order to eliminate probable negative effects of the presence- of LAB in the cassava wastewater used. The bacteriocins as well as their degradation products- were detected in both the raw and heat-treated cassava wastewater samples. Their presence- suggests that the poor results of hydrogen production observed in all assays could be attributed to these compounds, and demonstrated that the heat treatment of wastewaters may not- completely deactivate bacteriocins. In contrast, Seo et al. [41] evaluated the effect of different- pretreatment of cheese whey for hydrogen production (heat pretreatment; sonication pretreatment; and hydrodynamic cavitation). All the treated samples exhibited H<sup>2</sup> production activity, suggesting the fact that LABs, which exist predominantly in the raw cheese whey and produce- lactic acid, were effectively suppressed. The maximum H2 yield of 1.89 mol H<sup>2</sup> /mol lactose was- obtained from the cheese whey pretreated with hydrodynamic cavitation for 15 min.-

The production of bio-H2 , particularly from more complex wastewater, such as textile wastewater, has been treated with activated carbon. This technique is available for wastewater industries, solvent recovery, chemical catalyst, gold extraction, gas separation and liquid adsorption. Li et al. [40] used the textile wastewater, hydrolyzed by α-amylase with a concentration of 0.2 mL/L for 20 min. After α-amylase hydrolysis, the hydrolysate was pretreated with activated carbon and cation exchange resin with a concentration of 1% w/w for 30 min. The removal efficiency of ion concentration was 95.85%. After that, the hydrolysate was fed into the batch reactor and the best hydrogen yield was 1.37 mol H<sup>2</sup> /mol, reducing sugar.-

The application of pretreatment to the complex wastewater was tested in an attempt to overcome eventual limitations these wastewater. The selection of suitable hydrolysis method and/or- control of inhibitors production will improve the fermentation, resulting in a positive effect and- improving the degradability of the complex wastewater during the biological process [24, 25, 42].-

### **6.2. Nutrients**

Excluding the main substrate, carbohydrate materials, dark fermentative hydrogen production requires nutrients for bacterial activity like all biological treatment processes. The nutrientsinclude nitrogen (N), phosphorous (P), ferrous (Fe) and some trace metals. Among the many- kinds of nutrients, N is the most essential one for bacterial growth. Optimal C/N ratio is 47 according to Lin and Lay [68]. P and Fe concentrations affect the metabolic pathway of *Clostridium* sp., and hydrogen production potential decreases when their concentrations are limited.-

Appropriate ratios of carbon and nitrogen, carbon and phosphorus, and between carbon and- sulphate increase bioproduction of hydrogen by modifying metabolic pathways associated- with the nutritional requirement of microorganisms [69]. Argun et al. [70] observed that an- adequate nitrogen concentration depends on the phosphorus concentration in the medium.- That is, systems with a low phosphorus concentration require a low nitrogen concentration- and vice versa. However, in their research, the best hydrogen yield of 281mL H<sup>2</sup> /g starch- was obtained at a C/N ratio of 200 and a C/P ratio of 1000, namely, for lower concentrations- of nutrients. High nitrogen and phosphorous concentrations could inhibit hydrogen formation by dark fermentation, which likely alters the metabolic pathway [47]. In contrast, low- at C/N and the pH values below 3.5 suggests that surplus carbon source could cause rapid- acidification and influence the metabolism and growth of microorganism [44].-

Some complex wastewater, such as cheese whey [21, 22], Tequila vinasses [33], cassava wastewater [24, 25], soft-drink wastewater [47] and corn starch wastewaters [44], added nutrients to ensure that all the required components were present.-

Peixoto et al. [47] showed a similar example when added urea (COD:N of 100:0.7) was used as the nitrogen source in one of their upflow fixed-bed reactors. Under that condition, the hydrogen production ceased completely after 8 days of operation. In contrast, the reactor with a COD:N ratio of 100:0.3 produced hydrogen continuously for 70 days with an average hydrogen yield of 3.5 mol H<sup>2</sup> /mol substrate. These authors suggested that the excessive cell growth caused by the addition of nutrients affected the reactor's hydrodynamic pattern, hindering the liquid-gas transfer mass of hydrogen. In addition, the decrease of the HRT increased the production of non-reduced compounds.

Searmsirimongkol et al. [36] evaluated hydrogen production using as source substrate wastewater from ethanol processing produced from sugarcane in anaerobic sequencing batch reactor- (ASBR). Through concentration of 40 g/L, OLR of 60 kg COD/m<sup>3</sup> /d, HRY of 16 h and pH 5.5, at- 37°, reached 3320 mL H<sup>2</sup> /L/d and 172 mL H<sup>2</sup> /g COD removed. The high concentrations of potassium and sulphate observed in raw stillage (with COD of 150 g/L), 8.8 and 7.0 g/L, respectively,- show the need to dilute the affluent to avoid toxic effect to the hydrogen-producing bacteria. At- concentrations above 40 g COD/L, system performance decreased in terms of hydrogen production due to higher concentrations of PO4-3 and SO4-2. Gomes et al. [24] showed that hydrogen- production from cassava wastewater quickly decreased and terminated even in the presence of- the heat-treated wastewater with or without nutrient supplementation. The authors suggested- that the problems were not due to lack of nutrients, but due to the presence of lactic bacteria.-

### **6.3. Temperature**

Temperature affects the growth rate and the metabolic pathways of microorganisms, and is considered one of the most important operating parameters which affect fermentative production of hydrogen. Microorganisms are capable of producing hydrogen at a temperature ranging from 15 to 85°C. Fermentative reactions for hydrogen production are mainly conducted at mesophilic (25–40°C) and thermophilic (40–65°C) temperatures, while few studies have been conducted in hyperthermophilic temperatures (above 80°C) [8].-

As shown in **Table 1**, most of the studies were conducted under mesophilic conditions- (25–40°C). Only a few studies [35, 43] were conducted under thermophilic conditions- (45–55°C). High temperature can promote hydrolysis and simplify microbial diversity in- a manner favourable to H2 production, but it can also bring about monotonous microbial diversity, resulting in incomplete substrate degradation, especially in the treatment of actual waste. Also, operation at high temperature places an economic burden, as it- requires a tight and closed structure and immense energy to heat and maintain the temperature of the reactor. Therefore, the temperature effect must be thoroughly investigated- considering not only the H2 fermentation performance but also substrate degradation and economic factors [62].-

Few studies have evaluated the effect of temperature, and the substrates during the investigation of the effect of temperature on fermentative hydrogen production were tofu-processing wastewater [34], brewery wastewater [35] and Tequila vinasses [33].-

It should be noted that fermentative processes operating under thermophilic conditions have some advantages over mesophilic processes. This is due to: (i) higher temperature has lower solubility gas (Henry's law); (ii) the hydrogen synthesis pathways are less affected by the partial pressure of hydrogen (pH2 ) [10] and (iii) the rates of chemical and enzymatic reactions are higher [71]. However, according to Mohan et al. [72], the optimal temperature for the production of hydrogen depends on the nature of the biocatalyst and the type of wastewater to be used as a substrate.

The effect of the temperature (25 and 3))5°C) on hydrogen production from Tequila vinasse was studied using a sequencing batch reactor, with HRT of 24 h [33]. A maximum HPR of 50.5 mL H<sup>2</sup> /h/L and an average hydrogen content in the biogas of 29.2 ± 8.8% were obtained when the reactor was fed with 3 g COD/L, at 35 °C and 12-h HRT. It is 6.2 times greater than the temperature of 25°C under the same conditions.-

Lay et al. [34] used two different temperatures (35 and 55°C) and two different seed sludges to evaluate the hydrogen production performance and obtain the best criteria for maximum production from tofu-processing wastewater. The temperature variation did not affect the HY significantly. The maximum HY of 61.2 mL/g COD was obtained at 35°C. Similar values were obtained with the 55°C ) under the same conditions (HY of 58.8 mL/g COD).-

### **6.4. Use of co-substrates**

The use of co-substrates is motivated by other objectives being pursued concomitantly, including (a) combined treatment of different waste streams, (b) ability to treat residues otherwise difficult to manage individually, (c) dilution of potentially toxic/inhibitory compounds, (d) optimization of the conditions for hydrogen production and (e) optimization of the carbohydrate/protein ratio [16].-

The literature also reports that simple substrates, such as glucose, have been used in mixtures with other complex substrates in the search for optimal conditions for hydrogen production: mixture of sugarcane stillage and glucose [30, 43]; glucose and cheese whey [21];- glucose and cassava wastewater [25]; and corn starch wastewaters [44]. The strategy of- using mixed substrates demonstrates the high interest among researchers in evaluating the- feasibility of hydrogen production through waste fermentation in the presence of glucose.-

Wang et al. [44] using corn starch wastewaters exhibited an efficient H2 yield which was found to be 76.0 and 31.7% higher than that of using corn starch and cassava starch, respectively. Moreover, in the study of Ferreira Rosa et al. [21] showed that the use of mixed substrates also favoured the production of hydrogen, when compared using glucose as an individual substrate. The co-fermentation of the cheese whey and glucose mixture was favourable for the concomitant production of hydrogen and ethanol, with yields of up to 1.7 mmol H<sup>2</sup> /g COD and 3.45 mol EtOH/g COD in AFBR.-

 Most studies of co-fermentation focused on the performance of hydrogen production in AFBRs. It is interesting to note that there was a variation in biogas composition when- the carbon source was changed from a mixture of glucose/wastewater [21, 30, 43]. Even- with different operating conditions and wastewater, the same pattern of behaviour was- observed, indicating that the substrate mixtures are a preferable carbon source compared- with glucose.-

Chen et al. [73] reported the inhibition of anaerobic processes, suggesting that to effect a better- adaptation of microbial community, prior to use more complex substrate is placed on a simpler carbon source until their total consumption. Acclimation of anaerobic microorganisms both increases their tolerance to the toxicants shock and enhances toxicant biodegradability.-

Co-fermentation from wastewaters with glucose and adaptation of microorganisms to inhibitory- substances can significantly improve the wastewater treatment efficiency and hydrogen production. Possibly a favourable environment for the development of microorganisms has been created, with the presence of simple substrates and nutrients. However, the costs for pure carbohydrate sources are high for practical-scale hydrogen production, which can only be viable- when based on renewable and low cost sources [6]. Studies to analyse the nutrients of different- wastewaters, in order to get a better rate C:N and C:P, could make viable the combination of two- complex wastewaters. This would make it feasible to process hydrogen production, due to the- lower cost of substrates.-

### **7. Microbial diversity**

**Table 4** shows that a limited number of reports co-exist on microbial communities producing hydrogen from complex wastewaters. These studies analysed the composition of microbial communities by cloning and sequencing the 16s rRNA from sugarcane vinasse, 454-pyrosequencing data analysis from sugarcane vinasse, fluorescent *in situ* hybridization (FISH) from glycerol and affiliation of band sequence from denaturing gradient gel electrophoresis (DGGE) from beverage wastewater.-



**Table 4.** Microbial diversity from complex wastewaters.-

Hydrogen can be efficiently and economically obtained from dark fermentation by hydrogen-producing bacteria (HPB) [74]. *Clostridium* and *Enterobacter* were the most widely used microorganisms for fermentative hydrogen production in mesophilic conditions, and *Thermoanaerobacterium* genus under thermophilic conditions [75]. The members of genus *Clostridium* are Gram-positive, and contain endospore-forming rods that produce hydrogen. Already, *Enterobacter* are Gram-negative, rod-shaped and facultative anaerobes [63].-

Among the fermentative anaerobes, *Clostridia* have been well known and studied extensively, not for their hydrogen production capability but for their role in the production of industrial solvent from various carbohydrates [7]. This is a common sense, and numerous- studies have already been conducted considering the investigation and identification of- *Clostridium* sp. with hydrogen yield productive capacity. However, in the production of- hydrogen from complex wastewater it has been shown that it is possible to produce hydrogen from other bacteria beyond the genus *Clostridium*. Ferraz Júnior et al. [39] showed by- 454-pyrosequence analysis, organisms affiliated with the *Clostridiu*m and *Pectinatus* genera were dominant in the sample associated with hydrogen production from sugarcane- vinasse. In contrast, from the same wastewater, Reis et al. [30] showed by cloning and- sequencing the 16s rRNA that 55% belonged to the phylum Bacteroidetes and uncultured- Prevotella, and 28% belonged to the phylum Firmicutes genus Megasphaera. Also, the- presence of 3% of uncultured *Clostridia* also belonged to the phylum Firmicutes. Under- thermophilic conditions, both *Thermoanaerobacterium* sp. and *Clostridium* sp. were efficient- hydrogen producers [43].

Many studies reported in the literature that evaluated the microbial community did not report a direct association between microorganisms found and hydrogen production. The- likely cause for this is due to the diversity of other organisms found, different *Clostridium*. In contrast, Sivagurunathan et al. [29] reported that the *Clostridium* species dynamics were- not significantly affected, but total microbial community structure changed with respect- to HRT variation as evident from PCR-DGGE analyses. Moreover, the appearance of *Selenomonas* spp. in a CSTR at low OLR improved the HY, whereas the disappearance of- *Selenomonas* spp. at high OLR improved the HPR, but gave a drop in HY from beverage industry wastewaters.-

Other organisms have also been found in complex wastewater, such as *Klebsiella oxytoca* and *Enterobacter* sp., indicating the presence of predominant hydrogen-producing bacteria from textile wastewater and glycerol [38, 40].-

### **8. Conclusions and perspectives**

The analysis of over 35 literature references on fermentative hydrogen production from- complex wastewater has shown that numerous process parameters have the potential of- affecting the evolution of the metabolic pathways involved, in turn affecting the process- kinetics and the conversion yield.

The production of hydrogen from wastewaters should contribute technologically to the- fate of some wastewater, opening the possibility for them to be used as raw material to- produce bioenergy. Thus, the discovery of new raw materials for the production of a sustainable fuel contributes to the consolidation of the sector. However, this review showed- that there usually existed some disagreements on the optimal condition of a given factor- for fermentative hydrogen production from complex wastewaters, thus more researches in- this respect are recommended.

### **Author details**

Paula Rúbia Ferreira Rosa and Edson Luiz Silva\*-

\*Address all correspondence to: edsilva@ufscar.br-

Department of Chemical Engineering, Federal University of São Carlos, São Carlos, São Paulo, Brazil-

### **References**


reactor. International Journal of Hydrogen Energy. 2015;**40**:8498–8509. DOI: 10.1016/j.- ijhydene.2015.04.136-


## **Bifunctional Heterogeneous Catalysts for Biodiesel Production using Low Cost Feedstocks: A Future Perspective**

Anita Ramli, Muhammad Farooq, Abdul Naeem, Saleem Khan, Muhammad Hummayun, Azhar Iqbal, Sohail Ahmed and Liaqat Ali Shah

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65553

#### **Abstract-**

 Currently,thefossilfuelsourcesarethemajorcontributorstotheworld'senergymix.- However,theseconventionalenergysourcesaredepletingveryfastduetotheirfinite- natureandextensiveuses.-Anadditiontotheirfinitenature,environmentalproblems- relatedtotheirusesaregettingprogressivelyworseandworse,initiatingchallenging- debatesforscientificcommunities.-Biodiesel,arenewablefuel,hasshownpromising- prospectsduetoitsstrongsocioeconomicbenefitsandmotivationsinmostofthe- countriesoftheworld.-Bifunctionalheterogeneouscatalystsarestronglyrecommended- forbiodieselproductionfromdifferentfeedstockstosimplifytheprocess.-Thisreview- highlightsthechallengesandopportunitiesassociatedwiththeheterogeneouscatalysts- andsomerecommendationstodesignanefficientbifunctionalheterogeneouscatalyst- foreconomicalbiodieselproductionfromwastecookingoil.-

**Keywords:** biodiesel, bifunctional heterogeneous catalyst, recommendation-

### **1. Introduction-**

 Deepconcernsregardingfastdepletionofconventionalenergyresourcesandtheirassociated- environmentalissuesarehotdebatesforbothdevelopedanddevelopingcountries.-Moreover,- theseconventionalenergysourcesarelocatedinpoliticallyunstableregions,creatingissues- aboutscarcityofsuppliesandinstabilitiesintheinternationaloilprices-[1].-Itis,therefore-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 essentialtoexploresomealternative,potential,renewable,andsustainablesourceofenergy- thatwouldbesecure,economical,andenvironmentfriendly-[2–4].-Amongexploredbiofuels,- biodiesel has received a great deal of attention as compared to other biofuels due to its domestic- and renewable origin,nontoxicnature,biodegradability, environmentalbenefits,andexcellent- lubricity-[5,-6].-

A variety of oleo‐chemical feedstocks such as vegetable oils (edible and non‐edible oils), waste- cooking oils (WCOs), animal fats, and greases have been identifiedas potential raw oils for- biodiesel production in differentcountries (**Figure 1**) [7]. At present, biodiesel is mainly- produced from edible oils all over the world (more than 95%) [8, 9]. However, the extensive- use of edible oils for biodiesel production might lead to chronic food crisis in poor and- developing countries. Therefore, to avoid the food problems, World Food Organization (WFO)- has also legislated strict regulations over the use of edible oils for fuel purpose. The use of non‐ edible oils or waste cooking oils is therefore considered to be the possible solution to overcome- the food issues as well as lower the biodiesel production cost‐effectively [10–14]. In addition,- the use of waste cooking will further solve the environmental problems that arise from its- disposal [15, 16].-

**Figure 1.** Production of biodiesel from different feed stocks [7].-

Commercially, biodiesel production is carried out from vegetable oils or animal fats using- homogeneous catalysts all over the world [17, 18]. Homogeneous base catalysts possess high- catalytic activity under mild reaction conditions (from 40 to 65°C in normal atmospheric- pressure) [19]. Similarly, homogeneous acid catalysts are also used in the biodiesel production- from high free fattyacids feedstocks. However, homogeneous catalysts bear some technical- issues such as soap formation, reactor corrosion, difficultto recover, and the production of- large amount of polluted water, which in turn increase the overall biodiesel production cost- and hazards [20, 21]. Therefore, special attentionis being paid to the involvement of the- heterogeneous catalyst for biodiesel production due to their green and recyclable catalyticactivities. Heterogeneous catalysis has the ability to mitigate the various challenges encoun‐ tered with the use of homogeneous catalysts for biodiesel production from low cost feedstocks.- Heterogeneous catalysts bear several technical advantages such as easy separation and- purificationof the reaction products, reduced reactor corrosion, low sensitivity towards free- fattyacids and moisture contents [22, 23]. In general, solid base catalysts show high activity,- relatively shorter reaction times, and require lower reaction temperatures as compared to solid- acid catalysts [24]. However, solid acid/ base catalysts have several limitations such as- feedstock specification,deactivation due to leaching, and reusability for sustainable biodiesel- production.-

 Recently, the concept of bifunctional heterogeneous catalysts has been introduced in the- biodiesel technology for efficientbiodiesel production from differentfeedstocks. Several- studies have been carried out on the use of the bifunctional heterogeneous catalyst for biodiesel- production from low cost feedstocks [25–28]. Bifunctional heterogeneous catalysts exhibit both- acid and base character, therefore can simultaneously carry out esterificationof free fattyacids- and transesterificationof triglycerides to develop cleaner and economical processes for- biodiesel production. More importantly, a bifunctional heterogeneous catalyst can easily be- modifiedto introduce the desired physicochemical properties so that the presence of free fatty- acids or water does not adversely affectthe reaction steps during the transesterification- process.-

This study aims to review the role of bifunctional heterogeneous catalysts in biodiesel- production from differentfeedstocks for a sustainable energy process. In addition, the possible- recommendations will also be discussed in the light of existing problems associated with the- current biodiesel technology to design a robust catalyst for a sustainable biodiesel technology.-

#### **2. Biodiesel and its production scenario-**

 Accordingto-American-Societyfor-Testingand-Materials-(ASTM),biodieselisamixtureof- mono‐alkylestersoflong‐chainfattyacidsderivedfromvegetableoils-(edibleandnon‐edi‐ bleorigin)oranimalfats.-Biodieselisregardedasacleanfuel,emittingnegligibleamount- ofpollutantsintotheenvironmentandsufficientlyreducestheemissionofpollutantswhen- blendedwithdiesel-[29–31].-Anumberofmethodsarecurrentlyusedforbiodieselproduc‐ tionfromdifferentfeedstockstoovercomethehighviscosityofthevegetableoilsasafuel.- However,therearefourmainprocessesemployedforbiodieselproduction-(**Figure-2**):di‐ rectuseandblendingofrawoils-[32,-33],micro‐emulsions-[34,-35],thermalcracking-[36,- 37],andtransesterification-[38–40].-Amongthem,themostcommonlyusedmethodfor- convertingoilsintobiodieselistransesterificationbecausethefuelproducedbythismeth‐ odshowsgoodcompatibilitywiththeexistingengine-[41,-42].-Thevegetableoilsandfats- areextremelyviscous-(10–17timesviscousthanpetroleumdieselfuel),thereforeposing- severalproblemsasalternativeenginefuels-[43].-Themainobjectiveofthetransesterifica‐ tionprocessistolowertheviscosityoftheoilclosetothatofpetro‐diesel.-Intransesterifi‐ cation,vegetableoilsoranimalfatsarechemicallyconvertedintotheircorrespondingfatty- acidestersinthepresenceofalcoholusingasuitablecatalyst-[44,-45].-

**Figure 2.** Methods used for biodiesel production.-

 Thecostofbiodieseliswidelyregardedasaprincipalbarrierinthedevelopmentofthe- sustainableenergyprocess.-Ithasbeenreportedthatthefeedstockcostisthemajorfactor- contributingtothefinalbiodieselcostandthiscostrepresentsapproximately-70–95%of- thetotalcostofbiodieselproduction-[46–48].-Therefore,severaleffortshavebeendevoted- toexploreandselectanidealfeedstockforeconomicallyviablebiodieselproduction.-Asa- result,somecountriesoftheworldhavefocusedontheusenon‐edibleoilsasafeedstock- forbiodieselproductiontoavoidfoodproblems.-However,theuseofnon‐edibleoilsfor- biodieselproductioncannotbetheultimatesolutionforasustainableenergyprocessas- largeplantationlandareaswouldberequiredforlarge‐scalenon‐edibleoilsproduction.- Thismaydisturbtheentireanimalandplantecosystems-[45,-47].-Therefore,itisimportant- tosearchforcosteffectivebiodieselfeedstocksuchaswastecookingoil,whichischeap- andeasilyavailableallovertheworld.-Theutilizationofwastecookingoilasafueleffec‐ tivelyresolvestheenvironmentalproblemsassociatedwithitsdirectdischargeintothe- drainagesystem-[49].-

Similarly, another important factor contributing to the final cost of biodiesel is the technological- challenge, involving exploration of highly effective catalysts and their corresponding processes- for a sustainable biodiesel technology. A wide range of catalysts (homogeneous/heterogene‐ ous) can be employed for biodiesel production from differentfeedstocks as shown in **Fig‐ ure 3**. Heterogeneous catalysis has been considered to be the best choice for the biodiesel- technology in the near future. Heterogeneous catalysts can easily be recovered, recycled, and- have environmental friendly behavior. Recently, bifunctional heterogeneous catalysts have- gained worldwide interest for biodiesel production due to their excellent performance in- biodiesel production from low cost feedstocks. A bifunctional heterogeneous catalyst has the- ability to carry out simultaneous esterificationof free fattyacids and transesterificationof- triglycerides present in the waste cooking oil efficiently.-

Bifunctional Heterogeneous Catalysts for Biodiesel Production using Low Cost Feedstocks: A Future Perspective 289 http://dx.doi.org/10.5772/65553

**Figure 3.** Catalysts used for biodiesel production.-

### **3. Feedstocks for biodiesel production-**

Biodiesel is mainly produced by tranesterifying vegetable oils or animal fats commercially.- However, compared to petroleum‐derived diesel, the high cost of biodiesel is a major obstacle- to its commercialization, which is 1.5–3 times higher than petroleum derived diesel [14]. The- feedstocks mainly contribute to a major portion of the overall biodiesel production cost.- Therefore, it is essential to select a feedstock for biodiesel synthesis, which is cheap, domesti‐ cally available, and not compete with the food materials.-

The choice of feedstocks for biodiesel production depends on two main factors, its availability- and cost. Biodiesel is produced from differentbiological raw materials such as vegetable oils- (edible and non‐edible oils), animal fats, algal lipids, waste cooking oil, etc. [50–52]. Edible- vegetable oils such as canola oil and soybean oil in USA, palm oil in Malaysia, rapeseed oil in- Europe, and corn oil have been used as feedstocks for biodiesel production and found to be- good diesel substitutes. Non‐edible vegetable oils, such as oil from *Pongamia pinnata-*(Karanja- or Honge), *Jatropha curcas-*(Jatropha or Ratanjyote) and *Madhuca indica-*(Mahua) have also been- found to be suitable feedstocks for biodiesel production [53, 54].-

Similarly, algal lipids as feedstocks for biodiesel production are also gaining interest all over- the world [55]. Algae convert carbon dioxide into sugar and proteins in the presence of- sunlight. However, in the absence of nitrogen they mainly produce oil. A microalgae *Chlorella- protothecoides*, has been grown under autotrophic and heterotrophic conditions to obtain lipids- as a raw material for biodiesel industries. The lipid content in the heterotrophic cells reached- 55.20% as compared to 14.57% in autotrophic cells [56].-

Vegetable oils (edible and non‐edible oils) are the predominant raw materials for the produc‐ tion of biodiesel, because they are renewable, potentially an inexhaustible source of energy,- possess environmentally friendly characters, and can be produced on a large scale [16]. More- than 95% of the biodiesel production is made from edible oils in differentcountries. It has been- reported that approximately 70–95% of the total biodiesel production cost is related to the cost- of the raw materials (vegetable oil or animal fats) [11, 21, 22]. According to reports of Food and- Agriculture Organization (FAO), esculent plants containing oil are used for the production of- biodiesel among which about 84% of the biodiesel is from rapeseed oil (RSO) and the remaining- is from sunflower (13%), palm oil (1%), soybean, and others (2%) [2].-

Moreover, the use of edible oils for fuel production is puzzling as more and more of the global- food demand rises. There are also issues of deforestation and ecological imbalance while- diverting the virgin forests and arable lands to large‐scale biofuel production feedstocks [57].- In other words, the sustainability of edible oils as a biodiesel feed is under threat. Thus, the- biodiesel production technology faces several challenges that must be overcome to make it- sustainable.-

The use of waste cooking oils for biodiesel production instead of edible oils can be a promising- choice to enhance the economic viability of biodiesel production on a large scale. It has been- reported that the biodiesel production cost can be reduced effectivelyfrom 60 to 70% by using- waste cooking oil [24]. Since waste oil is easily available at a relatively low price, therefore can- be a workable feedstock for biodiesel production to make the biodiesel competitive in price- with petroleum‐based diesel.-

Huge amounts of waste cooking oils are produced all over the world every day, especially in- the developed countries. Such a large amount of waste cooking oil production can lead to- several disposal problems and contamination to water and land resources and ultimately to- environmental pollution. Therefore, we need to search a green utilization of waste cooking oil- to avoid the disposal problems. The utilization of waste cooking oil, as feedstock for biodiesel- production is not only economical and guarantees food safety but will also minimize the- environmental problems associated with their disposal [58]. The estimated waste cooking oil- production in the selected countries is depicted in **Table 1**. Recently, several studies have been- made to investigate the biodiesel production from waste cooking oil using heterogeneous- catalysts for sustainable energy production. The details of these studies are given in **Table 2**.-


**Table 1.** Annual production of waste cooking oil in the selected countries [59].-

Bifunctional Heterogeneous Catalysts for Biodiesel Production using Low Cost Feedstocks: A Future Perspective 291 http://dx.doi.org/10.5772/65553



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**Table 2.** Summary of various heterogeneous catalysts used in the transesterification reaction of waste cooking oil.-

The catalysts mentioned in **Table 2**, show good catalytic activity in biodiesel production from- low cost waste cooking oil feedstocks. However, several problems such as separation, recy‐ cling, soap formation, leaching, reactor corrosion, etc., are associated with these catalysts while- using for biodiesel production from low cost feedstocks containing high free fattyacids and- water contents. In order to overcome these problems, bifunctional heterogeneous catalysts- have been formulated to produce biodiesel from low cost feedstocks for a sustainable energy- process. Bifunctional heterogeneous catalysts show very good efficiencyin low cost feedstock- conversion into biodiesel by carrying transesterificationof triglycerides and esterificationof- free fattyacids present in the feedstock without any substantial loss in their activity due to the- water content present.-

#### **4. Bifunctional heterogeneous catalysts for biodiesel production-**

Several studies have been carried out to address the technical challenges associated with the- biodiesel technology for large‐scale and profitablebiodiesel production. First, the high cost of- edible vegetable oil as the source of triglycerides plays a key role in process profitability.- Therefore, to reduce the biodiesel production costs and make it competitive with petroleum- based diesel, low cost feedstocks, such as non‐edible oils, waste frying oils, and animal fats,- may be utilized as raw materials. However, the major problem associated with such feedstock- is higher amounts of free fattyacids and water, resulting in soap formation in the presence of- an alkali catalyst. Thus, additional steps are required in order to remove any water and either- the free fatty acids or soap from the reaction mixture.-

Therefore, synthesis of novel heterogeneous catalysts with desired physical and chemical- properties for biodiesel production form low‐grade feedstocks is one of the focuses of the- catalytic scientist. In this context, the bifunctional heterogeneous catalyst with acid‐base- character has attaineda great focus for various organic reactions over the past decade.- Bifunctional heterogeneous catalysts carry out simultaneous esterificationof free fattyacidsand transesterificationof TG present in the oil effectively without being affected by the water- content present or produced during the biodiesel formation due to the presence of both active- acids and bases sites on the surface of catalyst, thereby effectivelyreducing the biodiesel- production cost. Moreover, bifunctional heterogeneous catalysts can easily be tuned to include- desired catalyst properties so that the presence of free fatty acids or water does not adversely- affectthe reaction steps and the biodiesel yield during the transesterification of triglycerides.- The general mechanism for bifunctional heterogeneous catalysts is presented in **Figure 4**.-

**Figure 4.** General mechanism for simultaneous esterificationand transesterificationreactions on bifunctional heteroge‐ neous catalyst [107].-

Several attemptshave been made to investigate the catalytic activity of this type of catalysts- in the transesterificationreaction of low‐grade feedstocks for biodiesel production. These- catalysts show good catalytic performance during the biodiesel production from different- feedstocks. The serious problem associated with the heterogeneous catalyst is its deactivation,- which in turn reduce the catalyst reusability and chemical stability. These two factors play a- key role in the biodiesel production cost, which is the main hurdle for its commercialization.- In the view of the literature presented, it shows that the biodiesel technology is still not so- mature to contribute more for overcoming the energy needs. Therefore, effortsare still in- progress all over the world to develop an efficient bifunctional catalyst for biodiesel productionto overcome the serious disadvantages associated with the present biodiesel production- technology. The bifunctional heterogeneous catalysts developed in the last few years are- studied in detail below.-

 Yanetal.-[64]synthesizednovelzincandlanthanummixedoxidesasheterogeneouscata‐ lystsforbiodieselproductionfromunrefinedorwasteoils.-Theyalsostudiedthephysico‐ chemicalpropertiesofthesynthesizedcatalysts,effectsofthemetaloxidemolarratio,free- fattyacidsandwatercontentsinfeedstock,molarratioofmethanolandoil,andreaction- temperature,ontheyieldofbiodiesel.-Theauthorsfoundthatastronginteractionbetween- the-Znand-Laspeciesexistedwithenhancedcatalystactivities.-Moreover,lanthanumen‐ hancedthezincoxidedistribution,thereby,increasedthesurfaceacidandbasesiteswhich- inturnenhancedthecatalystabilityforsimultaneoustransesterificationandesterification- reactions.-Theyreportedthatthecatalystwitha-3:1ratioofzinctolanthanumcouldeffec‐ tivelycatalyzebothtransesterification/esterificationreactions,andwithnegligiblehydroly‐ sisactivityinoilaswellasinbiodiesel.-Thebiodieselyieldof-96%offattyacidmethyl- esters-(FAME)wasrecordedwithin-3husingwasteoilsinreactiontemperaturerangeof- 170–220°C.-

 Macarioetal.-[108]producedbiodieselfromwasteoilseedfruitswithmethanolinthe- presenceofheterogeneous/homogeneoussystemsusingacidandbasiccatalysts.-Theau‐ thorssynthesizedcatalystswithstrongacidsites-(USY,-BEA,-FAU‐X)andcatalystswith- weakacidsites-(MCM‐41and-ITQ‐6)bythehydrothermalprocess.-Potassiumwasintro‐ ducedintodifferentmaterialsbyionicexchangesuchas-K‐MCM‐41and-K‐ITQ‐6topre‐ parebifunctionalcatalysts-(acid–basecatalysts).-Theyfoundthathighesttriglyceride- conversionandbiodieselyieldvaluescouldbeachievedusing-K‐ITQ‐6catalystsinreac‐ tiontimeof-24hat-180°Ctemperature.-Theyfurtherdemonstratedthatdeactivationofthe- catalystoccurredduetopotassiumleaching.-Thecatalystexhibitedgoodregenerationvia‐ bilityandcouldbereusedforbiodieselproductionfromlow‐qualityoilforcheaperbio‐ dieselproduction.-

 Cannilla et al. [109] investigated the catalytic performance of the MnCeO*<sup>x</sup>* type bifunctional- catalyst using refinedsunfloweroil for biodiesel production with methanol. They synthesized- a series of manganese‐ceria catalysts with the Mn/Ce atomic ratio ranging between 0.4 and 3.4- by the redox‐precipitation method. NH3‐TPD and CO2‐TPD were utilized for acid‐base active- sites measurements on the surface of MnCeO*<sup>x</sup>* catalysts. It was found that MnCeO*<sup>x</sup>* catalytic- systems exhibited high surface area, high chemical and thermal stability and high Mn- dispersion, thereby resulted in high biodiesel production by the transesterificationreaction of- sunfloweroil with methanol at a temperature of 140°C in 5 h of reaction time at low- catalyst/oil ratio (1 wt.%). Moreover, the catalyst showed excellent catalytic stability and the- deactivation phenomenon was found to be lower than that employed for acid catalytic- reactions. They concluded that catalyst performance was the result of a synergic role played- by both the surface acid/base character and textural porosity.-

Wen et al. [110] prepared a TiO2–MgO bifunctional mixed oxide catalyst by the sol–gel method- to carry out simultaneous transesterificationand esterificationreactions in waste cooking oil- into biodiesel. They found that the catalyst withone molar ratio of Ti and calcined at 923 K- exhibited high activity and stability in the biodiesel reaction. They suggested that titanium- improved the stability of the catalyst as a result of defects induced by the substitution of Ti- ions for Mg ions in the magnesia lattice.-Furthermore, they found that the MT‐1‐923 catalyst- provided maximum biodiesel yield of 92.3% in the firstuse and increased after reuse of fourth- time. The increase in the catalytic activity was attributedto increase in the specificsurface area- and average pore diameter after regeneration. The TiO2‐MgO mixed oxide catalyst showed- good potential in large‐scale biodiesel production from waste cooking oil.-

 Borges et al. [111] studied biodiesel production from sunfloweroil and frying oil with a- bifunctional heterogeneous catalyst derived from natural porous silica, pumice. The bifunc‐ tional activity of the catalyst was enhanced by introduction of K into the catalyst. They further- investigated the dependence of the reaction variables such as temperature, reaction time,- catalyst loading, and methanol/oil molar ratio on biodiesel yield using sunfloweroil and waste- oil as feedstocks. The natural material, pumice exchanged with a KOH aqueous solution- demonstrated to be an efficientbifunctional heterogeneous particulate catalyst for simultane‐ ous transesterificationof triglycerides and esterificationof free fattyacids present in sunflower- oil and waste frying oil at low temperature (55°C). Moreover, the reusability studies showed- that there was no considerable change in the activity of the catalyst even after five regenera‐ tions, demonstrating it to be a stable material.-

Misra et al. [112] synthesized bifunctional heterogeneous catalysts by the conventional sol gel- method for biodiesel production from different feedstocks such as soy, canola, coffee and waste- vegetable oils containing variable amounts of free fattyacids (0–30 wt%). The authors reported- that the synthesized bifunctional Quntinite‐3T catalyst could effectivelyconverted the free- fattyacids and triglycerides (TGs) simultaneously into biodiesel in a single step of batch- reactor. Similarly, Quntinite‐3T also showed substantial reusability up to fivetimes with more- than 95% catalytic activity.-

 Omaretal.-[107]studiedthecatalyticactivityofalkalinemodifiedzirconia-(Mg/ZrO2,- Ca/ZrO2,-Sr/ZrO2,and-Ba/ZrO2)toidentifyanefficientbifunctionalheterogeneouscatalyst- forbiodieselproductionfromwastecookingoil-(WCO).-Physicochemicalpropertiesofthe- synthesizedcatalystswereanalyzedby-BETsurfacearea,-XRD,-FESEMand-CO2‐NH3‐TPD.- Theauthorsreportedthatamongdifferentsynthesizedcatalysts,-Sr/ZrO2exhibitedhigher- catalyticactivityduetothepresenceofoptimalactivebasic/acidicsites,facilitatingsimul‐ taneoustransesterificationandesterificationreactionsin-WCO.-Theresultsdemonstrated- thatthemethylester-(ME)yieldof-79.7%couldbeobtainedbyusing-2.7wt%catalyst- loading-(Sr/ZrO2),-29:1methanoltooilmolarratio,-169minofreactiontimeand-115.5°C- temperature.-

 Jiménez‐Lópezetal.-[113]reportedthatincorporationofthe-WO*x* speciesintoamesopo‐ rouszirconium‐dopedsilicacouldleadtoaneffectivecatalystformulationthatcouldcata‐ lyzebothtransesterificationandesterificationreactionsintheusedoil.-Duringtheir- investigations,theyfoundthatthecatalystcalcinedat-700°Cwith-15wt%-WO3loading- providedthebiodieselyieldhigherthan-80%after-2.5hreactiontimeatreactiontempera‐ tureof-200°Cinthepresenceofmethanol.-Moreover,thecatalyticactivitycouldbemain‐

 tainedeveninthepresenceof-5wt%ofwaterandafterthreecyclesofre‐utilization,- withoutanyfurthertreatmentofthecatalyst.-

Salinas et al. [114] investigated the catalytic performance of potassium supported on titania as- a catalyst for the production of biodiesel from canola oil. The authors reported that low- loadings of potassium lead to the formation of weak basic sites on the acid support (titania),- therefore exhibited bifunctional behavior to produce biodiesel from low cost feedstocks.- Furthermore, they added that the synthesized catalyst presented interesting activities with- robust character since there was no need for in situ pre‐treatment or inert reaction environment.-

Farooq et al. [115] employed mixed oxide supported bifunctional heterogeneous catalysts in- biodiesel production from waste cooking oil. The author reported that the synthesized catalysts- show improved transesterificationactivities and provided the maximum biodiesel yield of- 91.4% in reaction time of 4 h at a reaction temperature of 100°C, methanol to oil molar ratio of- 27:1 and an agitation speed of 500 rpm. Moreover, the synthesized catalyst showed substantial- chemical stability and could be reused for at least eight times without major loss in its catalytic- activity. The catalyst deactivation in the higher run was attributedto strongly adsorbed organic- molecules onto the active sites and leaching of the various active metals during biodiesel- production from waste cooking oil. They further concluded that the physicochemical proper‐ ties of the biodiesel produced from waste cooking oil comply with the international standard- specifications.-

 Similarly, Taufiq and his research team [116] modified La2O3 with Bi2O3 (1–7 wt%) to develop- an efficientcatalyst for simultaneous esterification and transesterificationreactions at atmos‐ pheric pressure. The catalysts were characterized by X‐ray diffraction-(XRD), BET surface area,- desorption of CO2-(TPD‐CO2) and NH3-(TPD‐NH3). The authors found that the bismuth- concentration significantly affected the performance of the catalyst and La2O3‐Bi2O3 mixed with- 5 wt% bismuth exhibited excellent transesterificationactivity for biodiesel production from- jatropha oil. The bifunctional catalyst, under optimal reaction condition of methanol/oil molar- ratio of 15:1, 2 wt% of the catalyst amount, reaction temperature of 150°C and reaction time of- 4 h, provided the highest conversion of 93% from jatropha oil. This catalyst maintained 87%- of FAME conversion after three successive recycling experiments. The decrease in the catalytic- activity of the La2O3‐Bi2O3catalyst was related to the decrease in the concentration of Bi and- Li metals in the catalyst when used in transesterification of jatropha oil.-

Taufiq [28] and his research group also reported a CaO‐La2O3mixed oxide based bifunctional- heterogeneous catalyst for biodiesel production from jatropha oil. They studied the stability- of the CaO‐La2O3binary system in detail for the sustainable biodiesel process. The authors- mentioned that the metal–metal oxide network between Ca and La resulted in well dispersion- of CaO on the composite surface and thereby, increased the number of active acidic and basic- sites as compared to that of bulk CaO and La2O3metal oxide which in turns enhanced the- catalytic activity. Furthermore, the biodiesel conversion increased with the increase in the Ca/- La atomic ratio up to 8.0, where the stability of the CaO‐La2O3binary system decreased at the- Ca/La atomic ratio of 10.0 due to highly saturation of CaO on the catalyst surface. The authors- reported highest biodiesel yield of 98.76% at 160°C, 3 h, 25 methanol/oil molar ratio and 3 wt%. In addition, the CaO‐La2O3binary system was stable even after four cycles with negligible- leaching of Ca2+ ion into the reaction medium.-

Alhassan et al. [117] reported Fe2O3‐MnO‐SO4 2‐/ZrO2nano sized bifunctional heterogeneous- catalysts for biodiesel production from low‐grade waste cooking oil. The physicochemical- properties of the synthesized catalysts were studied by using X‐ray diffraction-(XRD), Tem‐ perature Programmed Desorption of NH3-(TPD‐NH3/CO2), Thermal gravimetric analysis- (TGA), Fourier Transform Infrared Spectroscopy (FT‐IR), Brunner–Emmett–Teller-(BET)- surface area measurement, Energy Dispersive X‐ray Spectroscopy (EDS), Transmission- Electron Microscopy (TEM) and X‐ray Photoelectron Spectroscopy (XPS). The authors- reported that the catalyst could be reused six times without any substantial loss in its catalytic- activity with the maximum yield of 96.5 ± 0.02% at the optimized conditions of the reaction- temperature of 180°C; stirring speed of 600 rpm, 1:20 M ratio of oil to alcohol and 3 wt/wt%- catalyst loading.-

Recently, zinc oxide (ZnO) nanostar, synthesized by the microwave‐assisted surfactant free- hydrolysis method was applied as the catalyst for biodiesel synthesis through one‐step- simultaneous esterificationand transesterificationfrom high free fattyacid by Kwong et al.- [118]. The author reported that the ZnO nanostar catalyst showed high stability and robustness- at the end of the reaction, providing a biodiesel yield of 97.3% via simultaneous esterification- and transesterificationin low grade feedstock like waste cooking oil and crude plant oils. They- pointed out that the high efficiencyof the synthesized catalyst could be attributedto the in‐ situ formation of the ZnOl intermediate and the ZnGly deposited on the catalyst surface to- form a new co‐catalyst.-

In the view of above discussion, bifunctional heterogeneous catalysts seem to be a promising- technology for biodiesel production from low‐grade feedstocks, but after several time usage- their catalytic activity topple down due to deactivation by strong adsorption of organics and- leaching of the various active metals during transesterificationof low‐cost feedstocks. Hence,- still there is enough gap for improvement to develop a robust bifunctional heterogeneous- catalyst and incorporate desired physicochemical properties with enhanced catalytic activity,- selectivity, and stability for economical biodiesel production from low cost feedstocks.-

#### **5. Recommendation-**

Environmentally friendly and efficientheterogeneous catalysts for a sustainable biodiesel- technology are gettingmore and more attentionamong the research communities. The- invention of the novel heterogeneous catalysts that have desirable physical and chemical- properties, more stable, durable, and efficientunder ambient conditions is one of the primary- goals in focus of many research communities. The following steps may be recommended to- design an efficient catalyst for sustainable biodiesel production from different biomass.-

**1.-** An appropriate catalytic configurationand preparation method may be adopted to design- an efficientbifunctional catalyst with desired physical and chemical properties, which in- terms help to achieve good thermal and mechanical stability.-


Currently, our research group is also working on the modificationof naturally available clay- for biodiesel production from waste cooking oil. We have introduced differentmetals and- groups into selected clays by the modifiedimpregnation method to develop bifunctional- activity of the catalyst with enhanced catalyst stability and activity for sustainable biodiesel- technology.-

#### **6. Conclusion-**

The use of bifunctional heterogeneous catalysts for biodiesel production seems to be a- promising technology for the efficient biodiesel production from low cost feedstocks, such as- waste cooking oil for sustainable energy production. Therefore, it is essential to design a- bifunctional heterogeneous catalyst with desired physical and chemical properties, which- show potential ability to carry simultaneous esterification and transesterification reactions in- low grade feedstocks for sustainable biodiesel production. The catalyst structure‐activity- correlation should be properly studied to modify the physicochemical properties of the- synthesized catalyst to design an effectiveheterogeneous catalyst for biodiesel production.- Moreover, the use of waste cooking oils as feedstocks and waste materials for catalyst prepa‐ ration is very interesting, and may further make the biodiesel production process economically- feasible and will greatly decrease the production cost of biodiesel.-

#### **Acknowledgements-**

The authors would like to express their sincere gratitude to Universiti Teknologi PETRONAS,- Malaysia.-

### **Author details-**

Anita Ramli1\*, Muhammad Farooq1,2\*, Abdul Naeem2 , Saleem Khan2 ,- Muhammad Hummayun2 , Azhar Iqbal2 , Sohail Ahmed1 and Liaqat Ali Shah2-

 \*Addressallcorrespondenceto:anita\_ramli@petronas.com.myand- farooq\_khann@yahoo.com-

1-Department of Fundamental and Applied Sciences, Universiti Teknologi PETRONAS- (UTP), Tronoh, Perak, Malaysia-

2-National Centre of Excellence in Physical Chemistry, University of Peshawar, Peshawar,- Pakistan-

#### **References-**


## **Role of Mass-Transfer Interfacial Area in the Biodiesel Production Performance of Acid-Catalyzed Esterification**

Devjyoti Nath, Adisorn Aroonwilas and Amornvadee Veawab

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65657

#### **Abstract-**

 Thisworkinvestigatedtheroleofmass-transferinterfacialareainthebiodiesel- productionusingtheacid-catalyzedesterificationprocess.-Theinterfacialareabetween- alcoholandoilfeedstockwasdeterminedbyconductingacid-catalyzedesterification- experimentsusingmethanolandoleicacid-(asfreefattyacid)underrangesoffive- processparameters:reactiontemperature-(45–65°C),agitationspeed-(200–400rpm),- methanol-to-oilratio-(3:1–9:1mol/mol),catalystconcentration-(0.5–2.0%),andconcentrationoffreefattyacid-(5–30%).-Effectsoftheseparametersonthebiodieselconversion- rateandtheinterfacialareawerequantified.-Anempiricalcorrelationfortheinterfacial- areawasdevelopedasafunctionofprocessparameters.-Resultsshowthatthe- enhancementofbiodieselproductionrateisattributedtoreactionkineticsand/or- interfacialarea.-Theinterfacialareaisthesolecontributortotheincreaseinbiodiesel- productionrateduetotheincreaseinmethanol-to-oilratioandagitationspeed.-Both- kineticsandinterfacialareacontributetotheincreaseinbiodieselproductionratedue- tothereactiontemperatureandcatalystconcentration.-Theinterfacialareaplays- negligibleroleinthechangeinbiodieselproductionrateduetothefreefattyacid- content.-

**Keywords:** biodiesel, esterification,mass-transfer interfacial area, reactor design, hydrodynamics, parametric effects-

### **1. Introduction-**

 Energy use is considered to be the most fundamental requirement for various human activities,- especiallyintheindustrial,transportation,andagriculturalsectors.-Amongdifferentkindsof-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 fuels,petroleumconstitutesthemajorityoftheworld'senergysupply.-However,petroleumis- a finiteandnonrenewableenergysource,whichhasalreadycausedseriousenvironmental- pollution.-Therefore,asustainable,affordable,andenvironmentallyfriendlyalternativeto- petroleumisurgentlyneeded.-

Biodiesel is considered to be an important alternative to conventional petroleum-based diesel- [1]. It is nontoxic, and biodegradable, offerslower emissions of a number of air pollutants, can- be used in typical diesel engines without any major modifications,and has greater lubricity- than conventional diesel, thus reducing corrosion in diesel engines [2]. Biodiesel can be used- in its pure form called B100 (100% biodiesel) or in a blend with differentproportions of- conventional diesel fuels. Common blends include B20 (20% biodiesel and 80% conventional- diesel), which are much closer to diesel fuel properties than B100 and B5 (5% biodiesel and- 95% conventional diesel).-

 Inthetypicalbiodieselproductionprocess,thecatalyzedtransesterificationoresterification- reactioniscarriedoutinamultiphasereactorwherethetwoimmisciblechemicalreactants- (oilfeedstockandalcohol-containingcatalyst)arebroughtintocontactusingdifferentagitationormixingmechanisms.-Becausethecostofbiodieselproductiondependsheavilyonthe- costofrawmaterials,usinglow-qualityfeedstockssuchaswastecookingoilsornonedible- oilsinsteadofhigh-qualityfeedstockswillsignificantlyreducethebiodieselproduction- cost.-However,low-qualityfeedstockshavehighcontentoffreefattyacids-(FFAs),which- canreactwiththealkalinecatalystandproducesoaps.-Thissidereactioninthealkali-catalyzedtransesterificationprocesswillreducethecatalystefficiencyandthebiodieselconversionrate.-Additionally,theformationofsoapswillmakethelaterpurificationprocess- difficult.-Asaresult,theundesiredsidereactionscausedby-FFAswillincreasethecostof- biodieselproduction.-Therefore,whenusinglow-qualityfeedstocksforbiodieselproduction,thecontentof-FFAsmustbereducedtoanacceptablelevel-(typicallybelow-1%accordingtoreferences-[3–5])beforethealkali-catalyzedtransesterificationprocess.-Oneefficient- methodforremovingthe-FFAsfromfeedstocksisesterification,wherethe-FFAsreactwith- alcoholtoformesterandwaterasproducts.-

Due to the nature of the multiphase reaction, the efficiency or rate of biodiesel production relies- heavily on two primary factors: (i) the kinetics of catalyzed transesterificationor esterification- reactions and (ii) the hydrodynamics of liquid-liquid mixing promoted by reactor design and- operation. In order to arrive at a high-efficiencyand optimized biodiesel reactor, these two- fundamental features must be understood. To date, a large quantity of biodiesel research works- has been carried out in many differentaspects, such as production rate and the quality of- biodiesel products derived from differentfeedstocks, kinetic studies to findoptimal reaction- conditions for achieving higher yields, and use of enzyme and heterogeneous catalysts as an- alternative to the conventional homogeneous catalysts [6–11]. Most kinetic works reported- biodiesel conversion profilesas a function of reaction time under specific reaction conditions- and for specifictypes of reactor design and operation. As such, the reported kinetic data- essentially reflect the combined performance of both reaction kinetics and hydrodynamics of- liquid-liquid reaction systems.-

Despite its importance to the development of high-performance reactors, the knowledge of- hydrodynamics or mass-transfer interfacial area (*ae*) between the two immiscible reactants- during biodiesel reaction is very limited. Only one study by Stamenkovic et al. [12] relates to- the interfacial area in the biodiesel production process. In their work, the effectof agitation- intensity during the base-catalyzed transesterificationof sunfloweroil was investigated under- a specificreaction condition, that is, 20°C and alcohol-to-oil ratio of 6:1. There are no other- studies reporting the interfacial area for the acid-catalyzed reaction system.-

Therefore, the objectives of this work are: (i) to extend knowledge of interfacial area formed- between immiscible reactants during the acid-catalyzed esterificationreaction which can be- used for the design of a high-efficiencyreactor, (ii) to investigate the role of process parameters- on interfacial area in the esterificationprocess, and (iii) to develop an empirical correlation for- interfacial area estimation as a function of process parameters. To achieve these objectives, a- series of esterificationexperiments were performed using a stirred reactor operated under- variable ranges of reaction conditions (**Table 1**). The experimental results were obtained in- forms of free fattyacid (FFA) conversion profileswhich were subsequently used for determining the interfacial area values.-


**Table 1.** Summary of test conditions for esterification experiments.-

### **2. Methods-**

### **2.1. Materials-**

Two sets of chemicals were used in the experiments: (i) reactants and an acid catalyst for the- esterificationreaction and (ii) supporting chemicals for liquid sample analysis. For esterificationexperiments, canola oil was used as the base ingredient of oil feedstock. Oleic acid (90%)- from Sigma-Aldrich (Oakville, Ontario) was used as the representative of free fatty acids (FFAs)- commonly found in the feedstock. A predetermined amount of oleic acid was added to the- base canola oil in order to simulate low-quality feedstock. Sulfuric acid (98%) was used as the- acid catalyst, and methanol (99.98%) was chosen to represent the alcohol reactant. Both sulfuric- acid and methanol were purchased from Fisher Scientific-(Ottawa, Ontario). For liquid sampleanalysis, toluene (99.9%), isopropyl alcohol (99.9%), and potassium hydroxide (0.1 N) were- used for titrations to determine the acid number or FFA content of the oil phase.-

### **2.2. Experimental setup-**

The mass-transfer interfacial area (*ae*) and reaction kinetics between oil feedstock and methanol- were determined by carrying out esterification experiments in a bench-scale reaction system.- As shown in **Figure 1**, the reaction system consists of a 500-mL glass reactor that is jacketed- for heating/cooling (Ace Glass Inc., USA), a mechanical agitator powered by a variable-speed- drive (Cole-Parmer, Canada), and a water bath with a temperature controller/circulator (Cole-Parmer, Canada). The reactor was designed for operating pressures and temperatures of up to- 35 psig and 100°C, respectively. The reactor head has three connecting ports: one for the- mechanical agitator, one for sampling collection, and one for temperature measurement. A- glass bearing with PTFE coupling was connected to the reactor head to accommodate the- agitator. The sampling port was equipped with a silicone rubber septum, thus making possible- the collection of liquid samples without interrupting the reaction progress. A K-type thermocouple connected to a handheld meter was used for monitoring reaction temperature. During- the experiments, a heating medium (i.e., water from the temperature-controlled water bath)- was circulated through the reactor jacket in order to keep reaction temperature constant.-

**Figure 1.** A schematic diagram of the bench-scale reaction system for esterification experiments.-

#### **2.3. Experimental procedures-**

The experiments were conducted in two differentmodes: (i) esterificationtests with a welldefinedinterfacial area between oil feedstock and methanol, and (ii) esterificationtests with- the complete mixing between the two reactants. The firstmode of experiments provided thetrue kinetic features of the esterificationreaction, while the second gave the reaction performance that integrates both kinetic and hydrodynamic effects of the reaction system.-

For the experiments with a fixedinterfacial area (firstmode), the canola oil was mixed with- oleic acid to simulate a low-quality feedstock containing differentlevels of FFA. A 250 mL of- the prepared feedstock was then transferred into the 500-mL glass reactor and maintained at- a desired reaction temperature. An impeller or agitator was placed in the middle of this oil- phase and set at a particular mixing speed in order to keep the oil phase homogenized but yet- the oil-surface undisturbed. Meanwhile, a predetermined amount of H2SO4-(catalyst) was- mixed with methanol to form a catalyst/methanol mixture with a desired catalyst concentration. For each experimental run, a 93 mL of catalyst/methanol mixture was used to ensure an- excessive amount of methanol (more than 40 mol/mol ratio) available for reacting with FFA in- the oil phase. Prior to the reaction, the catalyst/methanol mixture was heated to the desired- reaction temperature in a water bath. Once the reaction temperature was reached, the methanol- mixture was transferred into the glass reactor to start the esterificationreaction. In order to- keep the interface between the oil phase and the methanol phase undisturbed, a separating- funnel was used to smoothly transfer the preheated catalyst/methanol mixture into the reactor.- For each experiment, the reaction temperature was controlled by the water bath. The reaction- was timed until it reached its equilibrium. During the experiment, a series of samples were- collected from the oil phase at differenttime intervals. Each sample was transferred into a test- tube and then immersed in cold water at 4°C to quench the reaction immediately. For better- separation of the finalmixture, the samples were centrifuged for 5 min at 3000 rpm, and then,- the top layer sample was collected and sent for analysis.-

For the experiments with the complete mixing (second mode), each esterification experiment- also began with the preparation of low-quality feedstock by mixing canola oil and oleic acid- at a specificratio. The FFA content of the prepared feedstock was analyzed in terms of acid- number in accordance with the ASTM D974-04 standard, the details of which are provided- in the next subsection. Following the preparation, a known amount of feedstock was charged to the reactor and heated to the desired reaction temperature with an accuracy of ±1°C.- The feedstock was also stirred by the agitator at a fixedspeed. Once the reaction temperature was reached, a predetermined amount of methanol/sulfuric acid mixture (with a given- catalyst concentration) was rapidly injected into the reactor to start the esterificationreaction. Prior to injection, this alcohol/catalyst mixture was preheated to the reaction temperature in order to avoid unwanted fluctuationin reaction temperature, especially at the- beginning of the test. Each experimental run was carried out for at least 70 min at the desired temperature and agitation speed. A series of liquid samples (3 mL) were collected from- the reactor at a regular time interval during the experiment. These liquid samples were then- analyzed for their acid number so as to determine the depletion of FFA as a function of time.-

#### **2.4. Sample analysis-**

A 3-mL liquid sample collected from the reactor was transferred to a test tube where 6 mL of- de-ionized water was added. The tube was then capped and shaken vigorously to promote- complete contact between water and the sample. This allowed the methanol and catalyst tocombine with water, thus separating them from the sample. After being shaken, the test tubewas placed in a centrifuge operating at 4000 rpm for 10 min. The centrifugal force helpeddevelop two liquid layers, that is, the top layer for oil and the bottomlayer for a mixture ofwater, methanol, and catalyst. The top layer was then withdrawn from the test tube for FFAcontent analysis by ASTM D974-04. A 2-mL sample was taken from the oil phase, weighed forits mass, and then dissolved in a 100-mL titration solvent (a mixture of toluene, water, andisopropyl alcohol with a volumetric mixing ratio of 100:1:99). Then, p-naphtholbenzein (thetitration indicator) was added into the sample which was eventually titrated with 0.1 Npotassium hydroxide (KOH) solution. Results from titration were then used for calculating theacid number (in mg KOH/g oil) based on the following equation:-

$$\text{Acid number} = \left(\frac{\left(A - B\right) \times M \times 56.1}{W}\right) \tag{1}$$

where *A* is the volume of KOH solution required for the titration of the sample in mL, *B* is thevolume of KOH solution required for the titration of 100 mL of titration solvent in mL, *M* isthe molarity of the KOH solution, and *W* is the weight of the sample in grams. The acid numberwas then converted to a FFA content value.-

#### **2.5. Data analysis-**

Data obtained from each esterificationexperiment were composed of a set of FFA contentvalues (or acid numbers) taken at differentreaction times. These data were subsequently usedfor determining mass-transfer interfacial area (*ae*) formed during esterificationreaction. Thefollowing demonstrates how kinetic and mass-fluxequations were used for the analysis of *ae*.-

The rate of esterificationreaction is essentially the rate of FFA conversion into fattyacid methylester (FAME). With the stoichiometric ratio of 1:1, the conversion rate can be expressed as afunction of reactant concentrations (i.e., *CFFA* for free fatty acid and *CAlc* for alcohol):-

$$rate = -\frac{dC\_{FFA}}{dt} = kC\_{FFA}C\_{Alc} \tag{2}$$

where *k* is the reaction rate constant varying with reaction temperature. Because an excessamount of alcohol for reaction was used in this experimental study, the conversion rate can berewritten in the pseudo–first-order form:-

$$rate = -\frac{dC\_{FFA}}{dt} = k'C\_{FFA} \tag{3}$$

where ′ is the pseudo–first-orderconstant (). It should be noted that, in an immiscible- reaction system (i.e., oil and alcohol), the reaction rate also depends upon the measure of- dispersion or interfacial contact between two immiscible reactants. Due to the involvement of- the interface between oil and alcohol, the rate of FFA conversion can also be expressed in terms- of the mass-transfer flux of FFA (*NFFA*):-

$$-\frac{dC\_{FFA}}{dt} = a\_{\text{e}}N\_{FFA} \tag{4}$$

where *ae* is the interfacial area per unit volume of the reaction system. By combining Eqs. (3)and (4), the mass-transfer flux can be written as a function of FFA concentration:-

$$N\_{FFA} = \left(\frac{k\text{\textquotedblleft}}{a\_e}\right) C\_{FFA} \tag{5}$$

Because the magnitude of constant ′ is proportional to the degree of contact between oil and- ′ alcohol, the ratio in Eq. (5) can be considered to be a constant value, suggesting that mass transfer flux,-*NFFA*, at a given *CFFA* concentration should have a fixedvalue. Then, Eq. (5) can be- rewritten as:-

$$N\_{FFA} = \left(\frac{k\,\,\,\,}{a\_e}\right) C\_{FFA} = \left(\frac{k\,\,\,\,}{a\_e}\right)\_{Ref} C\_{FFA} = \text{constant} \tag{6}$$

 where ′ is the ratio derived from the reference esterificationexperiments with the well- definedinterfacial area (the firstmode experiments). With a known *NFFA* flux,-Eq. (4) can be- rewritten as:-

$$1 - \frac{dC\_{FFA}}{dt} = a\_e \left(\frac{k}{a\_e}\right)\_{Ref} C\_{FFA} \tag{7}$$

Integrating the above equation results in the following equation:-

$$\ln\left(\frac{C\_{FFA,0}}{C\_{FFA}}\right) = a\_e \left(\frac{k^\*}{a\_e}\right)\_{Ref} t \tag{8}$$

where *CFFA,0*is the initial FFA concentration. To determine *ae*under a given reaction condition,- a plot between , 0 and reaction time (*t*) was developed using the experimental data.- ′ The values of the ratio were obtained as a function of reaction temperature and catalyst- concentration and reported in a separate work. [13]-

### **3. Results and discussion-**

#### **3.1. Parametric effects on FFA conversion rate and mass-transfer interfacial area-**

### *3.1.1. Effect of reaction temperature-*

 The effectof reaction temperature was observed from the experiments carried out at three- differenttemperatures: 45°C, 55°C, and 65°C and for oil feedstock containing 5%, 15%, and- 30% FFA. Other experimental conditions were fixedat 0.5 wt% H2SO4catalyst, 6:1 methanolto-oil ratio, and 300 rpm agitation speed. Results in **Figure 2a**, **b**show that the conversion of- FFA proceeded rapidly at the beginning of the reaction period. As much as 80% conversion- (based on initial FFA concentration) was observed within the first-20 min. Then, the conversion- rate diminished significantlywhen FFA conversion approached the plateau. Both figures also- show that the FFA conversion rate (or slope of FFA conversion profilesat the firstreaction- period) increased with reaction temperature regardless of the initial FFA concentration. The- increasing conversion rate was quantifiedand presented in terms of percent improvement- compared to the conversion rate at 45°C, as shown in **Figure 3**. It appears that the conversion- rate could be enhanced as much as 160% when the reaction temperature was raised from 45- to 65°C. Both kinetic and hydrodynamic factors (*ae*) contribute to the rate improvement.- Between the two factors, the kinetics plays the major role in controlling the conversion of FFA.-

As for the role of temperature on *ae*, results in **Figure 2c**show that *ae*increases with temperature.- The *ae*could increase approximately 30 - 60% when the temperature increases from 45°C to- 65°C. This is due to the decrease in liquid density and viscosity with increasing temperature.- The dependence of density and viscosity of oil on temperature was previously reported by [14,- 15]. According to [16], the rate of any reactions in an immiscible liquid-liquid system is- controlled by the mass transfer of chemical species across the interface between the two liquids.- For the FFA esterification,mass-transfer interfacial area is dependent upon the dispersion level- of methanol in the oil feedstock, which is usually controlled by mixing characteristics (e.g.,- flow,shear, and turbulence). Such mixing characteristics are ultimately dependent upon- physical properties, especially the density and viscosity of liquids. This is supported by the- fact that Reynolds number (*Re*) is a function of density and viscosity [17]. Therefore, an increase- in reaction temperature causes the density and viscosity of liquids to drop, thus allowing- methanol to easily disperse in oil.-

**Figure 2.** Effectof temperature on esterificationperformance: (a) FFA conversion profilesfor initial FFA concentration- of 5%; (b) FFA conversion profilesfor initial FFA concentration of 30%; and (c) interfacial area at different temperatures- (300 rpm agitation speed, 0.5 wt% of catalyst, 6:1 methanol-to-oil ratio).-

**Figure 3.** Hydrodynamic and kinetic contributions for effectof reaction temperature on FFA conversion rate (300 rpm- agitation speed, 0.5 wt% of H2SO4, and 6:1 mol/mol methanol-to-oil ratio).-

### *3.1.2. Effect of methanol-to-oil ratio-*

The effectof methanol-to-oil ratio was investigated under 0.5 wt% H2SO4, 300 rpm agitation- speed, 45°C and 65°C reaction temperature, for three different-FFA concentrations (5%, 15%,- and 30%). It was found that methanol-to-oil ratio has a significantimpact on FFA conversion- performance. An increase in methanol-to-oil ratio enhances the conversion rate for all test- conditions. From **Figure 4a**, **b-**FFA conversion rate could be improved by as much as 30 - 35%- when methanol-to-oil ratio increases from 3:1 to 9:1. The increasing conversion rate is due to- a significantincrease in interfacial area *ae*. As shown in **Figure 4c**, **d**, the area *ae*increases by 2.1- 5.3 times when methanol-to-oil ratio increases from 3:1 to 9:1. This is due to the greater amount- of methanol available for dispersion in the oil phase.-

Based on the analysis shown in **Figure 5**, the improvement in FFA conversion rate due to- increasing methanol-to-oil ratio is primarily caused by *ae*, not reaction kinetics. This is because the increasing methanol-to-oil ratio leads to more dispersion of methanol, which in- turn provides a greater interfacial area for esterificationreaction. On the contrary, increasing- the amount of methanol in oil does not result in any changes in concentration of methanol at- the reaction interface; thus, the reaction kinetics is unaffected.-

Role of Mass-Transfer Interfacial Area in the Biodiesel Production Performance of Acid-Catalyzed Esterification 319 http://dx.doi.org/10.5772/65657

**Figure 4.** Effectof methanol-to-oil ratio on esterificationperformance: (a) FFA conversion profilesat 45°C for initial-FFA concentration of 30%; (b) FFA conversion profilesat 65°C for initial FFA concentration of 30%; (c) interfacial areaplottedagainst methanol-to-oil ratio at 45°C; and (d) interfacial area plottedagainst methanol-to-oil ratio at 65°C (300rpm agitation speed, 0.5 wt% of catalyst).-

#### *3.1.3. Effect of agitation speed-*

The effectof agitation speed on FFA conversion was investigated by varying the agitationspeed from 200 to 300 rpm and further to 400 rpm. The investigation was done for threedifferent-FFA concentrations (5%, 15%, and 30%) at 0.5 wt% H2SO4, 6:1 methanol-to-oil ratio,and 45°C and 65°C. Results show that agitation speed has an impact on FFA conversion performance. As shown in **Figure 6a**, **b**, increasing agitation speed from 200 to 300 rpm leads toa significantincrease in the conversion rate. For instance, the rate could be improved by-150% at the reaction temperature of 45°C for oil feedstock containing 5% FFA (**Figure 7**).-However, it should be noted that raising agitation speed further from 300 to 400 rpm leadsto only a small increase in the rate of FFA conversion. It is apparent that the improvementunder fixedreaction conditions (excluding agitation speed) was solely caused by an increasein *ae*, not reaction kinetics. Raising agitation speed induces more turbulence, thereby creating smaller size methanol droplets in oil and in turn providing a greater *ae*for esterificationreaction. The increase in *ae* is evidenced in **Figure 6c**, **d**.-

**Figure 5.** Hydrodynamic and kinetic contributions for the effectof methanol-to-oil ratio on FFA conversion rate: (a)reaction temperature of 45°C; (b) reaction temperature of 65°C (test conditions = 300 rpm agitation speed and 0.5 wt%of H2SO4).-

It should be noted that the degree of rate improvement also depends on reaction temperature.-This exhibits an interaction effectbetween agitation speed and temperature. The effectofagitation speed at a lower reaction temperature (45°C) is much greater than the effectat thehigher temperature (65°C). This behavior can be explained by comparing the magnitude ofinterfacial area formed at these two temperatures. From **Figure 6c**, **d**it can be seen that thehigher temperature (65°C) tends to offera greater area, *ae*, than the lower temperature (45°C)- does. This is due to the reduction in density and viscosity of liquid mixtures with an increase- in temperature. Therefore, increasing agitation speed at 65°C, where the higher *ae* is already- established, does not yield a much greater improvement in conversion rate.-

**Figure 6.** Effectof agitation speed on esterificationperformance: (a) FFA conversion profilesat 45°C for initial FFA concentration of 5%; (b) FFA conversion profilesat 65°C for initial FFA concentration of 30%; (c) interfacial area plotted- against agitation speed at 45°C; and (d) interfacial area plottedagainst agitation speed at 65°C (6:1 methanol-to-oil ratio, 0.5 wt% of catalyst).-

As mentioned previously, raising agitation speed beyond 300 rpm does not have much impact- on the conversion rate of FFA. This can be explained by considering the conventional power- correlation for agitated reaction. According to McCabe et al. [18], the power number, *NP*, for- the typical stirred reactor (i.e., an index that reflects friction preventing the impeller rotation)- tends to decrease with the Reynolds number (*Re*), especially at low and moderate turbulence- regions, while it remains virtually unaffectedby the Reynolds number under highly turbulent- conditions. This suggests that the effectof agitation speed should be gradually diminished- with the increasing level of system turbulence. This behavior was observed in this work. The- *ae* increases considerably due to the significantreduction in friction on the impeller when- agitation speed increases from 200 to 300 rpm. However, when agitation speed increases from- 300 to 400 rpm, despite the increase in turbulence, the friction on the impeller does not diminish- much further. This indicates that the friction may reach its minimum for a given systemgeometry that accounts for the design and dimensions of the reaction system as well as the- type of fluid in the reactor. As such, the degree of mixing does not improve, causing the- interfacial area, *ae*, to remain unchanged. This in turn results in the stabilization of the FFA- conversion rate.-

**Figure 7.** Hydrodynamic and kinetic contributions for the effectof agitation speed on FFA conversion rate (0.5 wt% of- H2SO4 and 6:1 methanol-to-oil ratio).-

#### *3.1.4. Effect of catalyst concentration-*

 Theeffectofcatalystconcentrationwasstudiedbyvarying-H2SO4concentrationfrom-0.5to- 2.0wt%.-Theeffectwasexaminedforthree-FFAconcentrations-(5%,-15%,and-30%)andtwo- reactiontemperatures-(45°Cand-65°C)at-6:1methanol-to-oilratioand-300rpmagitation- speed.-Resultsin-**Figure-8a**, **b**showthatanincreasein-H2SO4concentrationleadstoanenhancementof-FFAconversionperformanceforalltestconditions.-Forinstance,theconversionratecanbeimprovedby-70%when-H2SO4 concentrationincreasesfrom-0.5to-2.0wt%- at-45°C.-Bothhydrodynamicsandkineticswerefoundtocontributetosuchimprovementas- shownin-**Figure-9**.-Thehydrodynamiccontribution-(oranincreasein*ae*)resultsfromthe- reductioninliquidviscosity.-Notethatthehydrodynamiccontributionisnotassignificant- asthekineticcontributionatahighertemperature-(i.e.,-65°C).-Thisissupportedbytheresultsin-**Figure-8c**, **d**whichshowthatthechangein*ae*with-H2SO4concentrationisrelatively- smallatthehighertemperature.-

**Figure 8.** Effectof catalyst concentration on esterificationperformance: (a) FFA conversion profilesat 45°C for initial- FFA concentration of 30%; (b) FFA conversion profilesat 65°C for initial FFA concentration of 30%; (c) interfacial area- plottedagainst catalyst concentration at 45°C; and (d) interfacial area plottedagainst catalyst concentration at 65°C- (300 rpm agitation speed, 6:1 methanol-to-oil ratio).-

#### *3.1.5. Effect of FFA concentration in oil feedstock-*

 The effectof FFA concentration was examined over ranges of operating conditions, that is, 45- - 65°C reaction temperature, 200 - 400 rpm agitation speed, 3:1 - 6:1 methanol-to-oil ratio, and- 0.5 - 2.0 wt% catalyst concentration. The results in **Figure 10**show that FFA concentration plays- an important role in the FFA conversion performance. An increase in FFA concentration causes- the conversion rate to decrease. However, it should be noted that the hydrodynamics of the- reaction system in this case does not contribute to the changes in FFA conversion rate since the- interfacial area, *ae*, does not vary with FFA concentration in oil (**Figure 11**). It seems that the- unaffected*ae*is a result of the invariable physical properties of oil feedstock. According to- Kulkarni et al. [19] and Zhou et al. [20], the viscosity and density of canola oil (base ingredient- of oil feedstock) and oleic acid (FFA) are in similar ranges. The density of canola oil and oleic- acid is 0.912 and 0.90 g/mL, while the viscosity of canola oil and oleic acid is 33.4 and 34.8 cP,- respectively. Due to the similar properties of the two ingredients, increasing FFA concentration- from 5 to 30% does not considerably alter the viscosity and density of the oil mixture. The- unchanged oil properties help establish the stable turbulence level within the reaction system,- thus keeping the interfacial area, *ae*, relatively unchanged.-

**Figure 9.** Hydrodynamic and kinetic contributions for the effectof catalyst concentration on FFA conversion rate: (a)- reaction temperature of 45°C; (b) reaction temperature of 65°C (test conditions = 300 rpm agitation speed and 6:1 methanol-to-oil ratio).-

Role of Mass-Transfer Interfacial Area in the Biodiesel Production Performance of Acid-Catalyzed Esterification 325 http://dx.doi.org/10.5772/65657

**Figure 10.** Effectof FFA concentration on esterificationperformance: (a) based on temperature data series; (b) based onagitation speed data series; (c) based on catalyst concentration data series; and (d) based on methanol-to-oil ratio dataseries.-

#### **3.2. Empirical correlation for mass-transfer interfacial area-**

The effectsof process parameters on the interfacial area reported earlier were correlated in theform of an empirical equation that would facilitate the design of a biodiesel reactor. Development of the correlation was focused primarily on four important parameters controlling theinterfacial area between methanol and oil feedstock, that is, reaction temperature, agitationspeed, methanol-to-oil ratio, and catalyst concentration. Firstly, the effectof each processparameter was regressed individually to arrive at the best mathematical expression offeringsimplicity and the lowest data deviation. Four types of mathematical expressions wereconsidered in this screening step: linear, exponential, logarithmic, and power forms. It wasfound that most parametric effectscan be described by linear expressions, except for the effectof agitation speed, the nonlinear behavior of which can be expressed well by the logarithmicequation. Values of average absolute deviation (%AAD) and R2derived from individualregressions are summarized in **Table 2**.-

Based on the selected equations in the screening step, an overall empirical correlation that- combines all four parametric effects was formulated and expressed in the following form:-

$$a\_e = k\_l T + k\_2 \ln\left(n - k\_3\right) + k\_4 R + k\_5 c + k\_6 \tag{9}$$

 where*k1* to*k6* arecorrelationconstants,-*T* isreactiontemperaturein-K,*n* isagitationspeedinrpm,-*R* ismethanol-to-oilratioinmol/mol,and*c* iscatalystconcentrationinwt%.-Thecalculatedinterfacialarea-(*ae*)ispresentedinm2 /m3units.-Basedonallexperimentaldataobtainedinthisstudy,acomputer-softwarepackagecalled-"*NLREG*" wasusedforregressiontoarriveatvaluesofcorrelationconstants-(*k1* to*k6*)aslistedin-**Table-3**.-Itshouldbenotedthatthisempiricalcorrelationiscapableofpredictingmethanol-oilinterfacialareawithanaverageabsolutedeviation-(AAD)of-12%.-Agoodagreementbetweenthecalculated*ae* valuesandexperimentaldatacanbeobservedfromaparityplotin-**Figure-12**,whichshowsa-R2valueof-0.88.-

**Figure 11.** Effectof FFA concentration on mass-transfer interfacial area: (a) based on temperature data series; (b) basedon agitation speed data series; (c) based on catalyst concentration data series; and (d) based on methanol-to-oil ratiodata series.-


**Table 2.** Results of individual regressions for parametric effects.-

Role of Mass-Transfer Interfacial Area in the Biodiesel Production Performance of Acid-Catalyzed Esterification 327 http://dx.doi.org/10.5772/65657


**Table 3.** Correlation constants for Eq. (9).-

**Figure 12.** Parity plot between experimental data and calculated interfacial area.-

### **4. Conclusions-**

Mass-transfer interfacial area plays an important role in the performance of acid-catalyzedesterification-basedbiodiesel production. Increasing the interfacial area enhances rate ofbiodiesel production (or rate of free fattyacid conversion). The magnitude of the interfacialarea varies with process parameters, except free fattyacid content in oil feedstock. Theinterfacial area increases with increasing reaction temperature, agitation speed, methanol-tooil ratio, and catalyst concentration, thus resulting in the increase in biodiesel production rate.-

The increase in the biodiesel production rate may or may not be solely attributedto theavailable interfacial area. It can be attributedto both reaction kinetics and interfacial area. Theinterfacial area is the exclusive contributor to the increase in the biodiesel production rate whenthe agitation speed or the methanol-to-oil ratio increases. Both interfacial area and kineticscontribute to the enhancement of biodiesel production rate when the reaction temperature or the catalyst concentration increases.-

### **Acknowledgements-**

The authors would like to thank the Natural Sciences and Engineering Research Council of Canada (NSERC) and the City of Regina for their financial support and collaboration.-

### **Author details-**

Devjyoti Nath, Adisorn Aroonwilas and Amornvadee Veawab\*-

 \*Addressallcorrespondenceto:veawab@uregina.ca-

 Energy-Technology-Laboratory,-Facultyof-Engineeringand-Applied-Science,-Universityof Regina,-Regina,-Saskatchewan,-Canada-

### **References-**


## **Biodiesel Compatibility with Elastomers and Steel**

Salete Martins Alves, Valdicleide Silva e Mello and

Franklin Kaic Dutra-Pereira

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65551

#### **Abstract-**

 Thischapterdescribesthecompatibilityofbiodieselwithautomotivecomponents,such- as metallic and polymeric materials. It consists of a survey of literature as well as research- resultsobtainedbytheauthors.-Aspectsaswear,corrosion,anddegradationmaterials- arediscussed.-

**Keywords:** biodiesel, biofuel, elastomers, steel, corrosion, lubricity-

**1. Introduction-**

 Thegreatattention isattributedtobiodieselinrecentyears,duetoitsrenewablecharacterand- sustainableuse thatcan minimize damageto theenvironment. Theycan reduce emissions of- pollutantsgasandparticulatematerialscomparedtodieselfrompetroleum.-Also,theyhave- biodegradableandnontoxiccharacter.-Inordertomeetmarketrequirements,inrecentyearsa- significantadvanceininstalledcapacityofthebiodieselindustrywasobserved.-Theuseofthis- fuelanditsincreaseintheenergeticmatriximpliesinconstantresearchanddevelopmentof- technology to ensure its safety use. In the Brazilian energymatrix, the biodiesel use isregulated- in 7% biodiesel blended with diesel. However, in this year, the biodiesel proportion will increase- to10%.Associatedwithgrowthinbiodieseldemand,thefuelqualitycontrolisagreaterconcern,- because of its natural process of degradation, corrosion or tampering, and consequently of their- blendswithdiesel.-

On the other hand, due to the unsaturated molecules present in your chemical composition,- some adverse effectswere reported by various authors [1–6]. Most of them are focused on the- corrosive character because it is more oxidative and causes enhanced corrosion and material-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

degradation. However, at low concentrations, no serial problems were reported to parts of the- engine.-

The correct material selection minimizes the corrosion problem presented by biodiesel. For- example, the material used to biodiesel transportation and storage is commonly stainless steel- because it has a good corrosion resistance, as well as benefitscost relation. It had been cited- that they have an excellent compatibility with corrosive fluids.-Some metallic substrates are- used in automotive systems like tanks and carbon steel plates (covered or not by zinc), ironzinc alloys, aluminum-zinc or nickel-zinc, lead, and tin [7–9].-

Studies about the biodiesel compatibility with the other kinds of materials are some important,- especially because of their injection process in the automotive application. In this step, it gets- in contact with different materials such as metallic, ferrous, and even elastomeric.-

The chapter describes studies of the biodiesel compatibility with some components of fuel- injection system and materials used to storage and transporting of this fuel, focusing on- elastomers and metals degradation after biodiesel and diesel contact.-

#### **2. Fuel and biofuel compatibility with seals of injection systems of diesel- engine-**

Some studies had shown that when biodiesel was used as fuel in diesel engines, the injection- system has been sufferingsome damages, such as swelling in the elastomeric seals in injection- distribution, which may result in leakage of fuel [10].-

Swelling results showed an incompatibility between the elastomer and the fuel, which cause- a substantial loss of elastomeric properties and thus loss of sealing ability. Faced with this issue,- some questions arise associated with the use of blends of biodiesel vehicle: How to evaluate- the deterioration of elastomers applied to the injection system of a diesel engine caused by the- use of biofuel? How the addition of the biodiesel in diesel influencesthe mechanical properties- loss and swelling of elastomers? How are biodiesel compatibility and their blends with- elastomers?-

Searching answers for these questions, many authors have exposed the elastomeric materials,- which are used in sealing automotive, in contact with fuel. The compatibility methods are- described in the following sections of this chapter, as well as the results of degradation and- compatibility of some fuels with these stamps.-

#### **2.1. Conventional compatibility test-**

The compatibility evaluation is commonly performed by immersion test, which consists of- immersed sample in a suitable solvent, analyzing the damage by swelling by gravimetric- analysis. Hasseb et al. studied the degradation of differentelastomers in contact with palm- biodiesel [9]. After immersion test, they found that some properties such as tensile strength,- elongation and toughness were significantlyreduced for both the nitrile rubber (NBR) andchloroprene rubber (CR), while minor changes were found to fluoro-VITON. Bessee and Fey- assessed the influenceof methylic soybean biodiesel blends in the mechanical properties of- the elastomer, such as hardness, tensile strength, elongation, and swell [11]. They observed- changes for nitrile, nylon 6/6, and high-density polypropylene exhibited in the mechanical- properties listed above. On the other hand, this behavior is not observed for VITON. Trakarnpruk and Porntangjitlikit investigated the impact of biodiesel on the properties of six types of- elastomers commonly found in fuel systems (NBR, HNBR, NBR/PVC, rubber, acrylic copolymer FKM, and FKM terpolymer). Biodiesel is mixed with diesel to prepare B10 (10% mixed- with diesel) [12]. The study showed littleimpact on properties for the polymer FKM and FKM- terpolymer, ensuring consumer confidence in the use of B10 for contact with these polymers.-

Michal Dubovsky reviewed the changes in the physical and mechanical properties of rubber- mixtures (produced from NBR) immersed in blends of biodiesel-diesel, B10, B50, B75, and B100- at room temperature (23°C) for 3000 h and at 100°C for 500 h [13]. After immersion tests, the- greatest degradation of samples exposed to a higher concentration of biodiesel (B100) at 100°C- was observed. So rate used the SAE J1748 standard for assessing the compatibility of natural- rubber, nylon, and EPDM (monomer ethylene-propylene diene) immersed in high oil biodiesel- FFA (high content of free fatty acids) for 500 h at 55 ± 2°C. EPDM and nylon change significantly- after immersion tests, but no changes were observed to natural rubber [14].-

Lei Zhu studied the NBR compatibility with nine differentfuels: diesel, PME, WCOME, PME,- (C12:0), (C16:0), (C18:0), (C18:1 M), and (C18:1 E). After 168 h of immersion test at ambient- temperature (25°C), the changes in mass, volume, and mechanical properties of NBR samples- showed that biodiesel has a higher solvent power than diesel fuel. Mei Sze Loo investigated- the effectof fatigue on the nitrile rubber [15]. The elastomers were immersed in the conventional diesel engine for 3 months and palm biodiesel for 10 days, and the same degree of- swelling was obtained before the application of uniaxial fatigue loading. The authors through- the stretch-N curves found that the swollen rubbers B100 had a shorter life when compared- with swollen rubbers diesel [16].-

#### **2.2. Compressive immersion and pressurized tests-**

In order to analyze the changes in shaft seals sufferedby fuel contact, some authors conducted- tests and simulated that the real conditions of contact in these shaft seals are submitted.-

In their investigations, Chai et al. [17] submitted two kinds of rubbers (NBR and CR) to a test- tensioned by a set of plates that pressed the seal. The device consisted of four rectangular- stainless steel plates with spacer bars between them. The spacer bars are designed to introduce- pre-compression on the rubber specimens while they are immersed into biodiesel. Then, the- device with rubbers was immersed in differentpalm biodiesel blends (B0, B25, B75, and B100)- for 30 and 90 days, respectively. After the tests, it was found that for the types of rubber used,- there was an increase in mass as well as a change in volume when the exposure time is increased- from 30 to 90 days, especially when using the higher percentage of biodiesel content (B100).- However, it is also noted that the pre-compression stress applied in the test reduced the amount- of swelling, as compared to rubber without application of tension. That is, the swelling of NBR- and CR increases with increasing biodiesel content and decreases with increasing precompressive stress [15].-

Chai et al. and Mello [17–19] were performed immersion tests in innovative pressurized fluid- device fluid,to simulate and investigate the degradation of a rubber seal in a fuel injection- pump. She studied two elastomers NBR and VITON, as a sealing material. Pressurized fluids- used were: diesel, biodiesel soybean, sunflowerand palm oil, and its mixture with diesel (B5- and B20). The system is available in BR 10 2014 028966 6 patent.-

In this study, the elastomeric seals degradation because fuel contact was investigated by testing- for static and pressurized immersion. Thus it is possible to identify changes in the seal- degradation under compression conditions. Below, schematic drawing device has been- described.-

The compression was employed using m steel cylinder by applying a preload 2500N, calculated by the required torque to maintain the closed cylinder during the expansion (**Figure 1**).- O-rings (NBR and VITON) were compressed within the cylinder. All equipment was subjected- to the pressure of fluid-200 bar for 5 h. The pressure amounted to 80% of the less-resistant- elastomer pressurized NBR. The fluidsused were diesel and biodiesel of soybean, sunflower- and palm oil, and its mixture with diesel (B5 and B20).-

**Figure 1.** Simulation of the fuel injection system compression.-

#### **2.3. Comparative analysis of the compatibility of seals with fuel by the static immersion- and pressurized method-**

Samples of elastomeric seals, NBR (nitrile rubber) and VITON (fluorine-carbon rubber), were- studied by two exposure methods in order to verify the influenceof the two approaches in the- compatibility of the seals with fuel and degradation mechanismssuffered by seals.-

In comparison purposes with the norm, the seals were also exposed to the solvent provided- in the standard (toluene) with the same temperature ASTM D3616-95, but for 100 h. After the- samples were weighed in the precision balance, the samples were weighed before and after- the immersion time to determinate the weight loss and were dried in an oven at 108°C for 24- h, and weighed again. The evaluation of the compatibility of the elastomer with the fuel occurs- based on the analysis of mechanical properties changes, swelling degree and morphological- analysis of the seals after exposure to fluids.-

### *2.3.1. Swelling rate-*

The **Figure 2**concerns the comparison of elastomers tested, regarding their changes in weight- after static immersion (ASTM D3616-95) in soybean, sunflower,and palm biodiesel, and its- blends.-

**Figure 2.** Change in weight of elastomers after static display of biodiesel.-

The increase in weight for NBR shows an exponential trend to increased biodiesel concentration for all tested biodiesels. For VITON, moderate increase is observed for all fuels. These- results are similar to those found in studies [10].-

The nitrile elastomer (NBR) showed significantweight changes in contact with pure biodiesel- (B100) due to the swelling ability that increased the absorption of fluidcompared to soluble- elastomer components. Differentbiofuels (soybean and palm biodiesel) did not show a big- differencein the swelling degree of elastomers. Thus, it can be concluded that differencesin- the biodiesel composition do not affectin elastomer damage. However, the degree of swelling- evaluated for sunflowershows a significantincrease due to the moisture content in the- biodiesel. **Figure 3**gives the change in the weight of elastomers in contact with pressurized- fluids.-

**Figure 3.** Change in weight of the elastomer after exposure of pressurized biodiesel.-

Analogous to static swelling test, the volume change shows the same behavior for the elastomers in the pressure test, even with a time lesser than the static test exposure. Similarly, the- VITON elastomer showed substantially constant, this being more compatible with the fuel- investigated. This indicates that the pressure contact role should be considered when analyzing- systems in which this parameter is present. Based on these results, we found the significant- influenceof the pressurized contact in the elastomer properties, despite the short-time- exposure.-

### *2.3.2. Mechanical properties-*

The results in the hardness changes for the elastomers are shown in **Figure 4**. For the NBR, the- hardness in the condition B0 and B100 (only soybean and sunflowerbiodiesel) decreases- compared to the hardness of the untested material. To contact the palm biodiesel, a reduction- in this property for all blends (B0, B5, B20, and B100) occurs. For VITON, significant changes- in this property with the use of all biodiesels and their blends are not observed.-

The elastomers based on carbon and silica with materials used as fillersmay serve to improve- the hardness properties, abrasion resistance, tear resistance, and tensile strength. Also, the- physical properties of elastomeric materials are determinate by addition of curing agents and- accelerators, because they promote cross-linking between polymer chains or backbone.-

Trakarnpruk and Porntangjitlikit [12] explain that biodiesel can be absorbed by the polymer- that swells and therefore reduces entanglement of the polymer chain. Haseeb et al. [10] suggest- an interaction between biodiesel with fillersand curing systems used in the production of- elastomers.-

**Figure 4.** Change in hardness of the elastomers.-

According to Haseeb et al. [10], after exposure of differentelastomers biodiesel, these crosslinking agents and/or fillappear to react with various components of biodiesel and thus,- deteriorate the mechanical properties.-

The results of tensile strength tests are shown in **Figure 5**. After the pressure test, there was a- greater loss in tensile strength compared to NBR nonpressurized material. Similarly, they- observed differentbehaviors for various fuels, such as palm oil biodiesel, and it, in its pure- form, showed a littleloss in tensile strength compared to soybean biodiesel, in particular, for- the conditions with B20 and B100. This loss was even more pronounced for biodiesel sunflower- and all their blends.-

**Figure 5.** Tensile strength change.-

The study concludes that the compatibility of the elastomer with biodiesels showed greater- losses in mechanical properties, for investigated elastomers (NBR and VITON), even this- displaying lower mechanical properties in conditions not tested.-

 It also concludes that testing of pressurized biodiesel contact with elastomers showed- significantinfluenceson changes in materials, despite the short-time exposure. This demonstrates the importance of pressure in studies of degradation of elastomers.-

#### **3. Corrosion caused by biofuel-**

The damage resulting from corrosive processes includes not only the need to replace metal- parts in industries but also environmental problems with improper waste disposal of waste- from these processes. Few studies report about corrosion of metallic materials in organic- media, although of broad applications.-

Biodiesel is a hygroscopic fuel, with greater capacity of water absorption than petroleum diesel.- Studies reported by Kovács et al. [4] demonstrated that biodiesel is 30 times more hygroscopic- than regular diesel. This feature depends on the feedstock, which favors the oxidation- reactions. According to Fazal et al. [20], these reactions change the fuel properties and increase- its destructive potential. The water absorbed can act directly on the corrosion of materials; it- can cause biodiesel hydrolysis reactions, increasing, therefore, the metal corrosion, and- promote microbial growth and, consequently, microbial corrosion.-

According to Kovács et al. [4] biodiesel must avoid to its aging and/or oxidation during storage- and should not deteriorate of the storage tank materials. Commercial automotive fuels contain- additives carefully formulated to meet the stability of the product requirements-

Ferrous and nonferrous metal parts after contact with biodiesel corrode through the chemical- and electrochemical attack.-According to Singh et al. [3], corrosion and wear are caused by- contact of metallic materials with biodiesel. However, differentmaterials present different- corrosion behaviors in biodiesel. Ferrous alloys have better compatibility with biodiesel than- nonferrous alloys. On the other hand, the copper alloys are more susceptible to corrosion than- ferrous alloys. The fluorocarbons, a new group of compounds, have a high corrosion resistance.-

The use of biodiesel plays considerable economic importance to national and global energy- matrix. The corrosion knowledge related to fuel is a relevant issue for investigation, due the- damage and cost caused by corrosive processes. Thus, the challenge is to findreliable methods- to quantify the biodiesel corrosiveness, materials compatibility, and way to prevent corrosion.-

The Brazilian National Agency of Petroleum (ANP) recommends the use of the ASTM D130-04- test standard to evaluate the fuel corrosiveness. This standard consists in the immersion of- copper strip (clean and polished) in fuel for 1 h at 37.8°C. After this time, the piece is removed- and compared against a color chart. In Refs. [4, 21–23] assess the corrosion of metals by- gravimetric techniques such as ASTM G1 rules (2003) and ASTM G31 (2004) and by electrochemical techniques such as potentiodynamic polarization, linear polarization resistance, and- electrochemical impedance spectroscopy (EIS).-

### **3.1. Evaluation of corrosion by gravimetric techniques-**

The immersion tests or gravimetric technique is an almost used method to determinate- corrosion rate, corrosion speed, and thickness loss when it is important to investigate the- influenceof organic or inorganic fluidon metallic materials. ASTM G3172 describes this test- (2004) and consists of some steps, such as sample preparation (sanding and polishing),- immersion time (hours), pickling (metal immersion in HCl solution 0.2 mol/L for 120 s), and- weighing (before and after immersion test).-

The degradation of different automotive materials such as copper, brass, aluminum, and cast- iron was evaluated by Fazal et al. [23]. These materials were immersed in palm biodiesel and- diesel at room temperature for 2880 h. The results showed that biodiesel is more corrosive than- diesel, once the corrosion rate of copper (Cu), brass (BS), aluminum (Al), and cast iron (CI)- increased when immersed in biodiesel. Also, corrosion rate in biodiesel for the studied- materials was: copper (0.39278 mpy), brass (0.209898 mpy), aluminum (0.173055 mpy), and- cast iron (0.112232 mpy). Also, the surface damage was analyzed by scanning electron- microscopy. They concluded that the corrosion attackson biodiesel-exposed metal surfaces are- greater than diesel. The surface damage of aluminum was less than copper, brass, and cast- iron. The corrosion was uniform on the metal surface.-

The temperature influenceon biodiesel corrosiveness was studied by [1]. Mild steel coupons- were immersed in diesel (B0), blend palm biodiesel and diesel (B50) and palm biodiesel (B100)- at 27, 50, and 80°C. They observed that corrosion rate increases with an increase in temperature- for all analyzed fuel. This fact can be attributedto fast dissolution of corrosion products. EDS- analysis showed the presence of oxygen on coupons surfaces indicating the formation of iron- oxides or iron carbide. The metal surface was degraded by subsequent formation of other- oxides and their dissolution in fuel. The biodiesel attacksmore the metal surface than diesel- fuel.-

Dutra-Pereira [6], his master thesis, investigated the stainless AISI 316 corrosion when in- contact with differentbiodiesel. These biodiesels were synthesized from vegetable oils- (soybean, sunflower,and castor) and methanol/ethanol. The influenceof alcohol used in- biodiesel formation on corrosion was studied. The experimental procedure followed ASTM- G3172 (2004) and time immersion of 2160 h. **Figure 6**shows the mass loss of stainless samples- by corrosion related to biodiesel. The nomenclature of biodiesel was adopted considering the- precursor oil and alcohol used in transesterification, p.e. soybean methylic (the biodiesel was- synthesized from soybean oil and methanol), and B7 is a commercial fuel in Brazil (a mix of- 7% of biodiesel and 93% of diesel).-

From **Figure 6**, it is possible to verify that the loss mass is a littlebigger when ethanol was used- in biodiesel synthesis, and it can be justifiedfor two carbons in the carbon chain. Considering- the precursor oil, it is noted that soybean biodiesel promotes less mass loss than other biodiesel.- The B7 has more mass loss indicating that the mix of diesel and biodiesel increases the- corrosiveness of fuel, once diesel doesn't promote corrosion in stainless [22].-

**Figure 6.** Loss mass due to the corrosion process [6].-

The corrosion attackon stainless steel surface was analyzed by Ref. [6] by scanning electronic- micrographs (**Figure 7**). Comparing with as-received steel and post-corrosion samples, it is- possible to see little damage in surfaces. Basically, the corrosion occurs in pitting form.-

**Figure 7.** Scanning electronic micrographs of stainless steel after 2160 h of immersion time in biodiesel [6].-

In **Figure 8**, it is clear that all biofuels corrode the metal surfaces, and the corrosion rate- increases with time immersion. Although soy biodiesel and sunflowerhave similar molecular- structure, the corrosion rate is different, being higher for sunflower biodiesel.-

**Figure 8.** Corrosion rate in function of immersion time [6].-

According to Fazal et al. [24], some factors influencethe biodiesel corrosiveness: (i) biodiesel- composition (ester and its polarity, unsaturated components, presence of oxygen, and contaminants), (ii) environment (temperature, air, light, moisture, and exposure to different- metals), and nature of components (auto-oxidation, hygroscopic nature, microbial growth, and- affinity of exposed metal).-

The corrosion involves chemical and electrochemical reactions, which can accelerate the metal- wear and promote corrosion. However, there are few studies in the literature about corrosion- of automotive materials in contact with biodiesel [20, 22, 25].-

As mentioned before, there are no specifictechniques to analyze corrosive aspects of contact- between biodiesel and metallic materials. Thus, it is important to study new methods that can- evaluate the biodiesel corrosiveness, such as electrochemical techniques.-

### **3.2. Evaluation of corrosion by electrochemical methods-**

As mentioned before, the most common method to assess the biodiesel corrosiveness in- literature is gravimetric by immersion tests. However, this approach is only qualitative and- did not characterize the trend and mechanism of corrosion of metals in contact with fluids.- Also, it is important to consider that, usually, the metals corrosion is described as an electrochemical mechanism. Based on this, some electrochemical techniques and results are presented- in this topic.-

According to Aquino [2], the evaluation of nonaqueous fluid,as biodiesel, through electrochemical techniques is a challenge because of the high resistivity (or low conductivity) of media- making difficultthe determination of quantitative parameters of corrosion. The most usual- electrochemical techniques are potentiodynamic polarization and electrochemical impedance- spectroscopy (EIS).-

Electrochemical impedance spectroscopy (EIS) is a nondestructive technique and consists "in- a transient method where an excitation is applied to the system and the response (as a function- of frequency) is observed" [26]. In corrosion studies, the diagram more used to analyze the- information from EIS test is the Nyquist diagram (**Figure 9**), where the real part of impedance- is plottedon the x-axis and the imaginary part in the y-axis of a chart. In this plot, the y-axis- is negative, and each point on the Nyquist plot is the impedance Z at one frequency. The- Nyquist diagram is useful to recognize the process type.-

**Figure 9.** An example of Nyquist plot.-

The potentiodynamic polarization is a technique that obtains polarization curve and scanned- the electrode potential continuously. This curve (**Figure 10**) gives some important characteristics of the electrochemical behavior of metal in contact withfluids, like biodiesel.-

**Figure 10.** Typical polarization curve.-

Few works are found in literature about the use of the electrochemical technique to evaluate- corrosiveness of biofuel. In Diaz-Ballote [27] studied the corrosive biodiesel effect on aluminum- by conventional electrochemical techniques. They verifiedthat the corrosion current density- and free corrosion potential depend on the purity of the biodiesel. Also, the biodiesel quality- was determined by the provided electrochemical data.-

As corrosion of steel equipment in biodiesel factory is a major problem, Torres et al. [5]- analyzed AISI 316L steel corrosion in a biodiesel plant using electrical resistance probes at- strategic points in the process. This procedure was developed to monitor the corrosion.-

The biodiesel corrosiveness was studied by Aquino [2] and also gravimetric and electrochemical techniques. The electrochemical characterization was performed by electrochemical- impedance spectroscopy (EIS) in order to evaluate the corrosion behavior of metal in biodiesel,- without the addition of supporting electrolyte. The results of electrochemical characterization- by EIS indicated that it could be used as an interesting tool to evaluate the biodiesel quality as- well as corrosion behavior of metal in biodiesel, but the EIS for this purpose must be investigated more.-

Dutra-Pereira [6] studied the corrosion of stainless steel AISI 304 in biodiesel using two- electrochemical techniques: potentiodynamic polarization and electrochemical impedance- spectroscopy. The experimental setup is shown in **Figure 11**. The potentiodynamic polarization- curves were obtained with a scanning velocity of 1 mV s−1, with the curves was possible to- know the corrosion potential. EIS diagrams were achieved in frequency interval from 100 kHz- to 0.004 Hz with 0.01 V of amplitude.-

According to Dutra-Pereira [6], the results of electrochemical tests demonstrated that the- stainless steels are incompatible with the biofuels because they oxidize in the presence of theorganic medium. The diagrams of Nyquist allowed observing well-definedcapacitive arcs,- presenting behavior second order. The values of Rp (polarization resistance) prove that the- immersion test changes the metal properties. The Rp value is a good number to compare fuel- corrosiveness, less Rp means high corrosion rate. Also, polarization curves showed that- occurred the passivation breaking, allowing the metals dissolution, such as chromium. This- fact is evidenced by high potential values.-

**Figure 11.** Experimental setup to analyze corrosion in biodiesel (a) electrochemical cell, (b) potentiostat/galvanostat, (c)- microcomputer with AUTOLAB software (GPES-4 and FRA) [6].-

#### **4. Biodiesel lubricity-**

The diesel engines require the fuel to have lubricating properties, avoiding the direct contact- between pieces in movement. Biodiesel presents superior lubricity than diesel, becoming an- alternative to replace the diesel.-

In their studies, Mello [18] evaluated the effect of the low-sulfur diesel (LSD) and high-sulfur- diesel (HSD) on diesel lubricity. Also, they studied the biodiesel addition in diesel. A lower- lubricity was detected for diesel with low-sulfur than with high-sulfur content. For blends- from soybean and sunflowerbiodiesel, the wear scar diameters (WSD) were lower showing- greater lubricity.-

Another parameter that affectsthe lubricity is the temperature. Wadumesthrige et al. [28]- observed that the lubricity decreases with increasing temperature between 20 and 70°C, for- blends of 2% of biodiesel in LSD. However, for high temperature (80–90°C), lubricity increases.- The positive effecton lubricity at high temperatures is due to the increased molecular motion- of polar components, allowing their betterdistribution on the metal sometimes positively or- sometimes negatively.-

The influenceof temperature, concentration, and oil precursor type (using in biodiesel- synthesis) was investigated by Mello [18]. The researcher used Box-Behnken statistical tool as- a method to evaluate these variables and their combination on biodiesel lubricity. In her- experimental design were assessed the input parameters: the type of the biodiesel (soybean,- sunflowerand palm), the concentration (5, 20, and 100%), and the temperature of contact (25,- 40, and 60°C). The analyzed output parameters were the percentage of filmformation, the- coefficientof friction, and wear scar diameter (WSD) of the ball, and these output parameters- were obtained by tribometer HFRR. Levels and factors used as test parameters are shown in- **Figure 12**, with their real and coded values.-


**Figure 12.** Provision levels and actual and coded factors [18].-

The trend to the coefficientof friction, in function of variables, is shown in **Figure 13**. In the- contour surface generated for the fuel, the lower results of coefficientof friction for higher- concentration levels were observed, as proposed in the literature. This fact occurs due to the- oxygen present in the ester molecule and the presence of carboxylic acids which improve the- lubricity. In fuels, higher coefficientsof friction were observed for sunflowerbiodiesel,- probably due to the moisture present in this fuel. According to Fazal et al. [29], the high- moisture absorption seems to act as a factor that potentiates the corrosiveness of biodiesel.-

In addition, it is possible to observe the decrease in coefficient of friction at high-temperature- level; at the higher temperature, molecular motion for polar components increases enough,- enabling these to be more evenly distributed on the metal surfaces and, therefore, enhancing- lubricity. Also, the chemical adsorption of polar compounds on the metal surface is greater at- high temperatures.-

The response surface generated for WSD (**Figure 14**) confirmsthe influenceof concentration- and moisture of biodiesel in the lubricity. Small WSD values were found for all fuels in the- upper level of concentration, as observed in friction coefficientand higher WSD values to- sunflower biofuel due to the most moisture level.-

**Figure 13.** The coefficient of friction response [18].-

**Figure 14.** Wear scar diameter response [18].-

The effectof fuel temperature observed in WSD confirmsthe results obtained by Wadumesthrige et al. [28], and betterlubricity ability was noted for fuel at high temperatures. **Figure 14**shows that lubricity increases at the upper-level fuel temperature.-

 **Figure 15**presents the response surface generated for a percentage of filmformation. It is- possible to verify that high rate of filmformation is reached for the concentration levels above- of center and high point. The biodiesel lubricity is due to the presence of a polarity-imparting- heteroatom, the oxygen, and the presence of a carbonyl moiety. The influence of fuel temperature on percentage of filmformation presents a more uniform response surface, showing that- the percentage of the filmhas its optimum performance for analyzed temperature. For these- variables, there is not a significantinfluenceon the concentration in the lubricity of the higher- to an intermediate point. However, there is a negative impact of the synergism by lowest levels- of temperature and concentration. This synergism may compromise the lubrication system. It- is a definitepoint because the working temperature of the engine is high than studied in this- work.-

**Figure 15.** Percentage of film response [18].-

#### **5. Final considerations-**

The biodiesel use is consolidated in some countries like Brazil. Thus, it is essential to research- in technologies to produce this fuel and to minimize the damage during its use in a diesel- engine. In this issue, investigation about compatibility between biodiesel and materials engine- is crucial to avoid premature wear and maintenance in this engine.-

As addressed in this chapter, development of methodologies that described betteror simulated- the real contacts between biofuel and differentmaterials is mentioned, as per example device- that represents the biodiesel in contact with the elastomer of the injection system.-

The corrosion has demonstrated a big challenge to biodiesel use because of its nature that- attackssome metals of diesel automobiles. In this context to know, the corrosion behavior of- metal in biodiesel is necessary, and thus, electrochemical techniques appear a promissory- method to evaluate corrosion mechanism and quantify biodiesel corrosiveness.-

Another important aspect of biodiesel is its lubricity that decreases the wear in the injection- system, besides it can restore lubricity of low-sulfur diesel. Thus, it is possible to conclude that- with some research to adequate the diesel engine to biodiesel and to understand betterits- characteristics, the biofuel is suitable to replace diesel fuel.-

### **Author details-**

Salete Martins Alves1\*, Valdicleide Silva e Mello2 and Franklin Kaic Dutra-Pereira2-

 \*Addressallcorrespondenceto:saletealves@ect.ufrn.br-

 1-Schoolof-Sciencesand-Technology,-Federal-Universityof-Rio-Grandedo-Norte,-Natal,-Brazil-

2 Federal University of Rio Grande do Norte-UFRN, Natal, RN, Brazil-

### **References**


## **Biofuel Additives: Conversion of Glycerol with Benzyl Alcohol over SBA‐15 with Sulfonic Acid Groups**

Pedro Canhão and Jose E. Castanheiro

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65800

#### **Abstract-**

 Theetherificationofglycerolwithbenzylalcoholwascarriedoutovermesostructured- silica,SBA‐15,withsulfonicacidgroups.Theproductsofglyceroletherificationareethers- (glycerolmono‐ether,glyceroldi‐etherandtri‐glycerolether).-Itwaspreparedwith- differentcatalysts,consistingof-SBA‐15withdifferentamounts ofsulfonic groups-(SBA‐ 15,-[SO3H]1‐SBA‐15,-[SO3H]2‐SBA‐15,and-[SO3H]3‐SBA‐15).-Itwasobservedthatthe- activityincreasedwiththeamountofsulfonicacidgroupson-SBA‐15untilamaximum- ([SO3H]2‐SBA‐15).-However,withhighamountofacidgroups,adecreaseincatalytic- activitywasobserved.-Theeffectofdifferentparameters,suchascatalystsloading,- temperature,andinitialconcentrationofglycerol,wasstudiedinordertooptimizethe- reactionconditions.-Catalyst-[SO3H]2‐SBA‐15showedgoodactivityafterfouruses.-

**Keywords:** biodiesel, glycerol, benzyl alcohol, SBA‐15‐SO3H, etherification-

#### **1. Introduction-**

 Biodieselisdefinedasmono‐alkylestersoffattyacids,whichcanbeobtainedfromdifferent- feedstocks-(animalfatsandvegetableoils)-[1–7].-Biodieselisarenewable,biodegradablefuel- withlowersulfurcontent,environmentallylesstoxicity,andbetterlubrication.-

Biodiesel production can be carried out by transesterification of triglycerides or by esterifica‐ tionof free fattyacids with methanol or ethanol in the presence of base or acid catalysts [1–7].- **Figure 1** represents the transesterification of triglycerides with different alcohols.-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** Representation of transesterification of triglycerides with alcohols into biodiesel and glycerol.-

In the previous years, an increase in biodiesel production has been observed, and consequently,- a large amount of glycerol has been produced. So, it is imperative to develop different processes- to produce glycerol as a product with high commercial value. The main use of glycerol is in- personal care and cosmetics, but its use as a valuable feedstock for new products and processes- is growing in importance [8–13]. Glycerol ethers have many potential uses, such as fuel- additives, solvents, and cryogenics. Reactions of glycerol with isobutene or *tert*‐butanol under- acid catalysis conditions afford*tert*‐butyl‐glycerol ethers, which have potential for blending- with diesel [12].-

Traditionally, strong homogeneous acid catalysts have been used. In order to become the- process a "green process," the homogenous catalysts have been replaced by heterogeneous- ones [13].-

Gu et al. [14] reported the etherification of glycerol with different alcohols catalyzed by acid‐ functionalized silica. They reported yields varying from 61 to 96% of the mono and di‐glycerol- ethers, using batch reaction conditions. These results prompted us to report some preliminary- data of glycerol benzylation with benzyl alcohol, using different types of heterogeneous acid- catalysts, aiming to produce mono, di, and tribenzyl glycerol ethers (**Figure 2**).-

**Figure 2.** Etherification of glycerol with benzyl alcohol.-

Etherificationof glycerol with benzyl alcohol was carried out in the presence of zirconia- modified with sulfuric acid (1 and 2 mol dm−3). The products were mono‐ and dibenzyl glycerol- ethers. The catalytic tests were carried out at differenttemperatures and initial reactant massratios. The catalyst prepared with the highest sulfuric acid concentration showed the highest- activity [15].-

Due to high surface areas and controlled pore sizes, mesoporous materials, such as SBA‐15,- PMO, and MCM‐41, have been used in heterogeneous catalysis as catalyst supports. This kind- of materials can be functionalized with differentorganic groups on the surface. There are- differenttechniques to change the materials: by grafting or co‐condensation. These modified- materials can be used as catalysts in different chemical reactions [16–18].-

In the present work, we studied the etherificationof glycerol with benzyl alcohol over SBA‐15- with sulfonic acid groups.-

#### **2. Experimental-**

### **2.1. Preparation of catalysts-**

The catalyst samples were prepared according to Grieken et al. [19].-

#### **2.2. Catalysts characterization-**

Micromeritics ASAP 2010 apparatus was used to determine the nitrogen adsorption isotherm- at 77 K.-

A CHNS Elemental Analyser 1112 series Thermo Finnigan instrument was used to determine- the amount of sulfur present in SBA‐15.-

Cation‐exchange capacities of catalysts were determined by potentiometrical titration. An- aqueous solution of sodium chloride (NaCl, 2M) was used as a cationic‐exchange agent.-

X‐ray diffraction-(XRD) patterns of the catalysts were obtained by using a Rigaku powder- diffractometer.-

#### **2.3. Catalytic experiment-**

The catalytic experiments were carried out in a stirred batch reactor at 80°C. In a typical- experiment, the reactor was loaded with 10 mL of benzyl alcohol, 4 g of glycerol, and 0.2 g of- catalyst.-

The catalytic stability of [SO3H]2‐SBA‐15 was carried out in the same conditions with the same- sample. The catalyst was separated from reaction mixture by centrifugation. After this- operation, the catalyst was washed with acetone, and it was dried at 80°C overnight.-

Undecano was used as the internal standard. Samples were taken periodically and analyzed- by GC, using a Hewlett Packard instrument equipped with a 30 m × 0.25 mm HP‐5 column.-

#### **3. Results and discussion-**

#### **3.1. Catalyst characterization-**

 SBA‐15 and SBA‐15 with sulfonic acid groups show a typical IV adsorption isotherm, accord‐ ing to the IUPAC classification.-**Table 1**reports the physicochemical characterization of- materials. It was observed that the surface area (*S*BET) and the pore volume decreased with the- amount of sulfonic acid groups immobilized on SBA‐15. It can be also observed that the amount- of sulfur, determined by elemental analysis, is similar to the amount of sulfonic acid groups,- determined by acid‐base titration (**Table 1**).-


**Table 1.** Physicochemical characterization of materials.-

**Figure 3**shows the powder X‐ray diffractionpatterns of SBA‐15 and SBA‐15 with sulfonic acid.- It can be observed that the materials with sulfonic acid exhibit a hexagonal pore structure,- characteristic of SBA‐15 materials:-

**Figure 3.** X‐ray diffraction of materials. (A) SBA‐15; (B) [SO3H]1‐SBA‐15; (C) [SO3H]2‐SBA‐15; and (D) [SO3H]3‐SBA‐15.-

### **3.2. Catalytic experiments-**

**Figure 2**shows the products obtained in the glycerol etherificationwith benzyl alcohol. The- products resulting from glycerol etherificationreaction are mono‐glycerol ether, di‐glycerol- ether, and tri‐glycerol ether.-

**Figure 4**shows the initial activity of the catalysts in the glycerol etherificationreaction with- benzyl alcohol. It is observed that the catalytic activity increases from the material SBA‐15 to- the catalyst sample [SO3H]2‐SBA‐15, which can be explained by the amount of sulfonic acid- groups present on the SBA‐15 surface (**Table 1**). However, when the amount of sulfonic acid- groups increases (from catalyst sample [SO3H]2‐SBA‐15 to [SO3H]3‐SBA‐15) the catalytic- activity decreases, which can be explained by the decrease of accessibility to the active centers.- In fact, a decrease in *S*BETand total pore volume with the amount of sulfonic acid groups were- observed (**Table 1**).-

**Figure 4.** Etherification of glycerol with benzyl alcohol over [SO3H]‐SBA‐15 catalysts.-

**Table 2**shows the values of glycerol conversion and selectivity of catalysts for the different- products obtained from glycerol etherificationwith benzyl alcohol, after 7 h of reaction. It is- observed that the sample of [SO3H]2‐SBA‐15 got the highest conversion.-

The selectivity mono‐ether ranges from 72%, in the presence of SBA‐15, to 78%, in the presence- of the catalyst [SO3H]1‐SBA‐15. It is observed that the selectivity for tri‐ether is reduced. This- behavior of this compound may be explained by obtaining consecutive reactions.-


**Table 2.** Conversion of glycerol and selectivity to the glycerol etherification products over sulfonic acid groups- presents on SBA‐15 surface.-

The effectof differentparameters (catalyst loading, initial glycerol concentration, and tem‐ perature) in etherification reaction with [SO3H]2‐SBA‐15 catalyst was also studied in order to- optimize the reaction.-

### *3.2.1. Effect catalyst loading-*

In order to study the effectof catalyst loading [SO3H]2‐SBA‐15 in glycerol conversion, at 80°C,- differentexperiments were carried out. The amount of catalyst ranges from 0.05 to 0.20 g. The- initial concentration of glycerol (2.9mol dm−3) was kept constant. **Figure 5**shows the conver‐ sion of glycerol versus time. It was observed that when the catalyst loading increases, equili‐ brium conversion can be obtained faster. This behavior could be explained by the total number- of active sites, with the increase in the amount of catalyst used in the reaction. However, when- the catalyst amount increases from 0.1 to 0.2 g, only a slight increase in the glycerol conversion- was observed.-

**Figure 5.** Etherificationof glycerol with benzyl alcohol in the presence of SBA‐15 with sulfonic acid groups (catalyst- [SO3H]2‐SBA‐15). Effect of catalyst amount. Conversion (%) versus time (h).-

It was also observed that the increase in catalyst loading has no effecton the equilibrium- conversion (**Figure 5**).-

The amount of effectof catalyst [SO3H]2‐SBA‐15 on selectivity to the differentcompounds wasalso studied. In all catalyst tests with the sample [SO3H]2‐SBA‐15, similar values of selectivityto mono‐ether (70%, at 70% of glycerol conversion) were observed.-

### *3.2.2. Effect of the initial glycerol concentration-*

The initial concentration of glycerol ranged between 1.7 and 2.9 mol dm−3, while the reaction- temperature (*T-*= 80°C) and the catalyst amount (*m-*= 0.10 g) were kept constant. The etherifi‐ cationreaction of glycerol was performed with the catalyst [SO3H]2‐SBA‐15. The results are- shown in **Figure 6**. It was found that the conversion of glycerol increases with the increase in- the initial glycerol concentration under the same reaction conditions. This behavior may be- explained by the increased reaction rate with the concentration of glycerol.-

**Figure 6.** Etherificationof glycerol with benzyl alcohol in the presence of SBA‐15 with sulfonic acid groups (catalyst- [SO3H]2‐SBA‐15). Effect of catalyst amount. Conversion (%) versus time (h).-

 The effect of glycerol initial concentration for the different compounds was also studied. Similar- valuesofselectivitytomono‐ether-(about-72%,-70%glycerolconversion)wereobserved.-

### *3.2.3. Effect of temperature-*

In this work, we also studied the effectof temperature on the glycerol etherification. Catalytic- tests with the catalyst [SO3H]2‐SBA‐15 were performed at differenttemperatures, whereas the- initial concentration of glycerol (2.9 mol dm−3) and the catalyst amount (*m-*= 0.10 g) were kept- constant. **Figure 7**shows the effectof temperature on the glycerol conversion. An increase in- glycerol conversion with temperature was observed.-

The effectof temperature on catalyst selectivity [SO3H]2‐SBA‐15 for the different compounds- was also studied. Increasing the temperature resulted in a decrease in the selectivity to mono‐ ether (about 81%, 70% glycerol conversion (*T* = 55°C) to 60%, the glycerol conversion (*T* =- 110°C)). This behavior can be explained by the increase in the reaction rate.-

The catalytic stability of [SO3H]2‐SBA‐15 catalyst was also studied. Different catalytic experi‐ ments were also carried out. A slight decrease in catalytic activity was observed after the second- run (**Figure 8**).-

**Figure 7.** Etherificationof glycerol with benzyl alcohol in the presence of SBA‐15 with sulfonic acid groups (catalyst- [SO3H]2‐SBA‐15). Effect of temperature. Conversion (%) versus time (h).-

**Figure 8.** Catalytic stability of [SO3H]2‐SBA‐15 catalyst in etherification of glycerol with benzyl alcohol.-

#### **4. Conclusions-**

The etherificationof glycerol with benzyl alcohol can be achieved with the use of heterogene‐ ous acid catalysts, consisting in SBA‐15‐SO3H. The products of the glycerol etherification- reaction are mono‐glycerol ether, di‐glycerol ether, and tri‐glycerol ether.-

The catalytic activity increased with the amount of sulfonic acid groups on SBA‐15 surface,- until a maximum. After this value, the catalytic activity decreases.-

Differentreaction parameters were optimized. It was observed that increasing the catalyst- loading allows faster equilibrium conversion. However, the increases in catalyst loading do- not affectthe equilibrium conversion. Another conclusion is that the glycerol conversion- increased with increase in the initial glycerol concentration. An increase in the glycerol- conversion with temperature was observed.-

After the second use ([SO3H]2‐SBA‐15), the catalyst tends to stabilize.-

#### **Acknowledgements-**

The study was supported by Project PTDC/CTM‐POL/114579/2009 and is gratefully acknowl‐ edged.-

#### **Author details-**

Pedro Canhão and Jose E. Castanheiro\*-

 \*Addressallcorrespondenceto:jefc@uevora.pt-

 Evora-Chemistry-Centre,-Chemistry-Department,-Evora-University,-Évora,-Portugal-

#### **References-**


## **Thermodynamic Properties of Propanol and Butanol as Oxygenate Additives to Biofuels**

Eduardo A. Montero, Fernando Aguilar, Natalia Muñoz-Rujas and Fatima E. M. Alaoui

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66297

### **Abstract-**

 Alternativeandrenewableenergytechnologiesarebeingsoughtthroughouttheworld- toreducepollutantemissionsandincreasetheefficiencyofenergyuse.-Oxygenate- second-generationbiofuelsfuelsleadtoareductioninpollutantemissionsandtheir- thermodynamicandtransportpropertiesallowthatthefacilitiesfortransport,storage- anddistributionoffuelscouldbeusedwithoutmodification.-Higheralcohols,like- propanolandbutanol,enhancetheoctanenumber,boostingtheanti-knockeffectin- gasoline.-Thenthecompressionratiooftheenginescanbeincreasedwithoutriskof- knocking,leadingtohigherdeliveryofpower.-Fromthecombustionpointofview,the- productionofcarbonmonoxideandvolatilehydrocarbonsfromthecombustionof- alcoholsislessthantheoneofgasoline.-Thischaptercoversmixturesofbutanoland- propanolwithhydrocarbons.-Thepropertiesreviewedareexcessvolumeordensity- (VE),vapour-liquidequilibrium-(VLE),andheatcapacity-(Cp).-

**Keywords:** butanol, propanol, biofuel, density, enthalpy, phase equilibrium, heat- capacity-

### **1. Introduction-**

 Biofuels, as environmental friendly fluids, have been paid much attention over the last decades.- Theycontributetodiminishthegreenhousegasemissionsduetoitsneutralcarbondioxide- balance.-Moreover,someoxygenatedcompoundsareusedasbiofueladditivesastheyleadto-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 areductioninpollutantemissionsandtoanincreaseintheenergyefficiencyofvehicleengines- [1,-2].-

Some alcohols and ethers, as oxygenated compounds additives, are added to present gasoline- with the aim of reducing the emission of gases that produce environmental impact. The- advantages of these oxygenates can be classifiedinto several categories. First, they can be- obtained from renewable, agricultural and raw materials, reducing the dependence of fossil- sources [3]. Second, they enhance the octane number, boosting the anti-knock effect in gasoline.- Then, the compression ratio of the engines can be increased without risk of knocking, leading- to higher delivery of power. From the combustion point of view, the production of carbon- monoxide and volatile hydrocarbons from the combustion of alcohols is less than the one of- gasoline. Amongst the thermodynamic properties, the heat of vaporization of alcohols is high- and leads to a reduction in the peak temperature of combustion, which means lower emissions- of nitrogen oxides.-

Alternative and renewable energy technologies are being sought to reduce pollutant emissions- and increase the efficiencyof energy use. Propanol and butanol have been proposed as an- alternative to conventional gasoline and diesel fuels [4, 5]. They are higher member of the series- of alcohols with each molecule containing three or four carbon atoms rather than two as in- ethanol. The EN standards of the European Union (EU) and the World-Wide Fuel Charter- (WWFC) for gasoline include, for example, 2-propanol, 2-methyl-2 propanol (also known as- tert-butyl alcohol, TBA), and 2-methyl-1 propanol [6, 7] as gasoline components.-

The traditional production and consumption of bioethanol have found an alternative with the- second-generation biofuels, such as biobutanol. For example, 85% ethanol, E85, needs some- modificationof the internal combustion engines specifications,unlike butanols, which can- work directly in present engines. The energy content per volume unit of butanol is similar to- the one of gasoline, and higher than the same for ethanol. Concerning the contribution to the- anti-knocking effect,butanol behaves almost the same as other alcohols like methanol or- ethanol. And in the presence of water, the mixture butanol/gasoline shows lesser tendency to- separation of phases than the mixture ethanol/gasoline. Then, all the facilities for transport,- storage and distribution of fuels can be used without modification.-Butanol, which can be- synthesized chemically or biologically, is an alternative transportation fuel since it has- properties that would allow its use in existing engines with minor hardware modifications [5].- For practical purposes, ASTM D7862 [8] gives specifications for blends of butanol with gasoline- at 1–12.5% in volume for automotive spark ignition engines. Three butanol isomers are covered- by the specification,-1-butanol, 2-butanol, and 2-methyl-1-propanol, while specificallyexcludes 2-methyl-2-propanol (TBA).-

Besides its use as fuel component, its industrial uses covers a broad range of applications as- solvent or as reactive for the production of other chemicals. Applications, chemicals and- products that use butanol include solvents, plasticizers, coatings, chemical intermediate or raw- material, textiles, cleaners, cosmetics, drugs and antibiotics, hormones, and vitamins.-

Since the 1950s, most butanol is obtained from fossil sources [6]. 1-butanol and/or 2-butanol- could be obtained from reduction of butyraldehyde with hydrogen, which is previouslyobtained by hydroformulation reaction of propene (propyelene). Meanwhile, propylene oxide- production leads to isobutene, from which TBA could be derived. Butanol from biomass is- called biobutanol [9], and it can be used in unmodifiedgasoline engines. Biobutanol can be- produced by fermentation of biomass by the ABE process [9, 10]. The process uses the- bacterium *Clostridium acetobutylicum*, the bacterium for the production of acetone from starch.- The butanol was a by-product of this fermentation. Other by-products as acetic, lactic and- propionic acids, isopropanol and ethanol, as well as a certain amount of H2, are generated by- the process. *Ralstonia eutropha*can also be used to produce biobutanol, by means of an electrobioreactor and the input of carbon dioxide and electricity.-

According to DuPont [11], existing bioethanol plants can be converted to biobutanol production with low economic cost. The main modification could affect to the fermentation process,- with minor changes in distillation, as both alcohols use the same stocks: food energy crops- (sugar beets, sugar cane, corn grain, wheat, etc.), non-food energy crops (switchgrass, cellulose,- etc.) and agricultural by-products (straw, corn stalks, etc.).-

Biopropanol is a rarely discussed biofuel. Tough propanol is included as regular component- of gasolines [6], its frequent use as chemical solvent makes it rare to consider it as a fuel.- Biopropanol could be produced from microbial fermentation of biomass (cellulose), but the- process is extremely inefficient [12]. The issues with microbial production of biopropanol are- analogous to the issues with microbial production of biobutanol, so if biobutanol becomes a- more practical biofuel to produce, then biopropanol will also become more feasible.-

This paper concerns thermodynamic properties of 1-propanol, 2-propanol, 1-butanol, 2 butanol and TBA. Accurate experimental data on thermodynamic properties should be- available to check and develop predictive empirical equations, models and simulation- programs. Industrial processes as storage, transport, separation and mixing processes also- need reliable data for its design. As a result, the experimental literature reviews on properties- of pure compounds and its mixtures with characteristic hydrocarbons can provide valuable- information about the fluid behaviour under various temperature and pressure conditions.-

The paper presents the literature review of available data on thermodynamic properties- (density, vapour-liquid equilibrium, specificheat,) of the mixtures of 1-propanol, 1-butanol,- TBA and its mixtures with hydrocarbons representatives of gasoline. Density has to do with- the volumetric behaviour of the mixtures under pressure and temperature conditions and is- the primary data to check equations of state. The vapour-liquid equilibria, which allows the- calculation of the Gibbs function, deal with the equilibrium between the liquid and vapour- phase under fixedpressure and temperature conditions. And the heat capacity gives information related to the sensible energy storage of the liquids. The review includes only the- interval of temperature and pressure of every property reported. The wider is the range of- pressure and temperature of the measured properties, so it would be the reliability of the- applications of predictive and equations and models. Discussion of further data (uncertainties,- experimental apparatus, etc.) would require more space than available. Interested readers- should access the literature references to check these issues.-

#### **2. The literature review-**

Thermodynamic properties of liquid propanol and butanol and its liquid mixtures with some- hydrocarbon have been obtained from the literature search using online library databases (Web- of Science©, Scopus©, NIST©-Standard Reference Database) and high impact electronic journals.-

Special attentionis given to alcohol + hydrocarbon mixtures. As stated, 1-propanol, 1-butanol- and TBA have been selected as alcohols. As representative of hydrocarbons, n-heptane, 2,2,4- trimethylpentane (iso-octane), cyclohexane, methyl-cyclohexane, benzene, toluene and 1 hexene have been chosen. They represent linear, branched and cyclic alkanes, aromatics, as- well as olefins,which are regular components of gasoline. **Table 1**presents the list of selected- compounds.-


**Table 1.** Selected alcohols and hydrocarbons.-

 Concerning properties, there is a huge amount of available thermodynamic data for pure- compounds. With respect to the mixtures, density data are shown in **Table 2**for binary- mixtures alcohol (1) + hydrocarbon (2). **Tables 3**and **4**present the vapour-liquid equilibria- selected for mixtures alcohol (1) + hydrocarbon (2) and alcohol (1) + hydrocarbon (2) +- hydrocarbon (3). Finally, heat capacity data for binary mixtures alcohol (1) + hydrocarbon (2)- are provided in **Table 5**.-


Thermodynamic Properties of Propanol and Butanol as Oxygenate Additives to Biofuels 367 http://dx.doi.org/10.5772/66297




**Table 2.** Reported density (g cm−3) for binary mixtures alcohol (1) + hydrocarbon (2).-


Thermodynamic Properties of Propanol and Butanol as Oxygenate Additives to Biofuels 371 http://dx.doi.org/10.5772/66297



**Table 3.** Reported vapour-liquid equilibria for binary mixtures alcohol (1) + hydrocarbon (2).-


**Table 4.** Reported vapour-liquid equilibria for ternary mixtures alcohol (1) + hydrocarbon (2) + hydrocarbon (3).-


**Table 5.** Reported heat capacity for binary mixtures alcohol (1) + hydrocarbon (2).-

### **3. Discussion-**

#### **3.1. Density of mixtures 1-propanol, or 1-butanol, + hydrocarbon-**

**Table 2**presents density data for the selected mixtures alcohol (1) + hydrocarbon (2). Fifty-nine- references correspond to mixtures 1-propanol (1) + hydrocarbon (2) and 51 to the one 1-butanol- (1) + hydrocarbon (2), while only 16 references have been found for TBA (1) + hydrocarbon (2).-

For 1-propanol (1) + hydrocarbon (2), only atmospheric pressure density data have been found- for the binary mixtures, except Refs. [16, 56] that are above 5 MPa. The highest pressure, 30- MPa, is reported by Zeberg-Mikkelsen and Andersen [56]. Temperatures above 350 K are only- measured by Zawisza and Vejrosta [16]. Concerning 1-butanol (1) + hydrocarbon (2), Refs. [67,- 76] report pressure above the atmospheric pressure. Hundred Megapascal is the maximum- pressure measured in Ref. [76]. Reference [67] also reports temperature above 350 K. Finally,- mixtures TBA (1) + hydrocarbon are reported only at atmospheric pressure and moderate- temperatures, being 323.15 K the highest measured temperature [87]. No data were found for- the mixture TBA (1) + 1-hexene (2).-

#### **3.2. Vapour-liquid equilibrium of mixtures 1-propanol, or 1-butanol, + hydrocarbon-**

With respect to the binary mixtures, **Table 3**shows 43 references for VLE data on 1-propanol- (1) + hydrocarbon (2), 47 for 1-butanol (1) + hydrocarbon (2) and 24 for TBA (1) + hydrocarbon- (2). No references for the mixtures 1-propanol (1) + 1-hexene (2) and TBA (1) + 1-hexene (2)- were found, while [131] was the only one for 1-butanol (1) + 1-hexene (2). Most references were- found for pressures lower or equal to atmospheric pressure. Studies done in Refs. [97, 122,- 132] were measured at moderate pressures, below 1.0 MPa, and only Ref. [114] reports pressure- close to 5 MPa for both mixtures 1-propanol (1), or 1-butanol (1), + benzene (2).-

Concerning temperature, most measurements were performed at low and moderate temperatures. Within the interval 350–400 K, we found a limited number of 27 set of data [51, 63, 84,- 98, 105, 109, 111–113, 119, 121, 123, 125, 127, 128, 132, 134–136, 142, 143, 146–150, 155]. Only- Refs. [16, 97, 114, 122, 139] report temperatures between 400 and 573 K.-

 Onlythreereferenceswerefoundreporting-VLEdataofternarymixtures,asshownin-**Table-4**,- atatmosphericorlowerpressures.-Temperaturesweremoderate,withmaximumat-344-K- measuredin Ref.-[98]. Noternarymixture with-1-propanolwas found.-

#### **3.3. Heat capacity of mixtures 1-propanol, or 1-butanol, + hydrocarbon-**

Only eight references reporting heat capacity of binary mixtures alcohol (1) + hydrocarbon (2)- are cited. Three of them correspond to the binary mixture 1-propanol (1) + heptane (2) at- atmospheric pressure and at moderate temperatures (up to 300 K). No other mixture of 1 propanol with the any of selected hydrocarbons was found.-

While the heat capacity of 1-butanol with heptane, 2,2,4 trimethylpentane, cyclohexane,- toluene and 1-hexane was measured by several authors. It must be pointed out that some- measurements [86, 160, 161] have been performed at pressures up to 25 MPa and temperature- of 313 K.-

#### **4. Conclusion-**

The literature review on thermodynamic properties of liquid mixtures of 1-propanol, 1-butanol- and TBA with representative hydrocarbons has been reported. Seven hydrocarbons (linear,- branched and cyclic alkanes, aromatics, and olefins)have been selected as representative of- present and future unleaded gasoline. The review covers density, vapour-liquid equilibrium- and heat capacity of mixtures.-

The review of density data shows a big amount of data at low pressure and moderate temperatures. Only two references report data above 30 MPa at a maximum temperature of 333- K. And at temperatures above 450 K, the maximum pressure is 5.5 MPa. With respect to the- vapour-liquid equilibrium, only one reference shows measurements over 555 K at 5 MPa. Heat- capacity data of mixtures are very scarce, tough some high pressure and high temperature- data can be found for some alcohol + hydrocarbon mixtures.-

The performance of fuels and biofuels in engines and other devices shows a trend of increasing- pressure and temperature, which leads to the need of more reliable predictive models for- complex mixtures at such conditions. Availability of high pressure and high temperature- thermodynamic properties is then a requisite for the implementation of these equation and- models. The review shows a lack of reliable data at high pressure and high temperature- thermodynamic data, which serve as a basis for the development of predictive equations and- models.-

#### **Author details-**

Eduardo A. Montero1\*, Fernando Aguilar1 , Natalia Muñoz-Rujas1 and Fatima E. M. Alaoui2-

 \*Addressallcorrespondenceto:emontero@ubu.es-


### **References-**


alkan-2-ols + cyclohexane and alkan-1-ols + methylcyclohexane and theoretical- interpretation. Fluid Phase Equilibria. 2004; 218: 131–140. doi:10.1016/j.fluid.- 2003.11.012-


+ toluene + methylcyclohexane at 298.15 K. Fluid Phase Equilibria. 1999; 166: 53–65. doi:-10.1016/S0378-3812(99)00284-8-


tography. Journal of Chemical and Engineering Data. 1982; 27: 399–405. doi:10.1021/- je00030a010-


systems containing either 1-butanol or 2-butanol + 1-hexene + methylbenzene at 313.15- K. Fluid Phase Equilibria. 2015; 386: 1–6. doi:10.1016/j.fluid.2014.11.010-


## **Photocatalytic Reforming of Lignocelluloses, Glycerol, and Chlorella to Hydrogen**

Masahide Yasuda

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64901

#### **Abstract-**

 Bioethanol,biodiesel,andbiogashavegainedmuchattentionassustainableenergy- alternativestopetroleum‐basedfuels.-Bioethanolproductionisthemosttypicalmethod- toprovideliquidfuel.-Recentlycellulosicmaterialshavebeenrecognizedasoneofthe- promisingsourcesforbioethanol,sincetheyarenotdirectlyincompetitionwithfood- sources.-However,ethanolconcentrationisusuallytoolowtoseparatebydistillationat- alow‐energycost.-Gaseous-H2isspontaneouslyisolatedwithoutoperationtoseparate.- Therefore,-H2productionisaneconomicalapproachtobiofuels.-Photocatalytic-H2- productionovera-Pt‐loaded-TiO2isinitiatedbythechargeseparation.-Electronsreduce- watertogenerate-H2,whileholesoxidizehydroxidetohydroxylradicals.-Generally,the- useofsacrificialagentsremarkablyacceleratesthe-H2productionsincethehydroxyl- radicalisconsumedbythem.-Thischapterdealswiththephotocatalytic-H2production- (PR)usingsacrificialwater‐solublematerialsderivedfromlignocelluloses,lipids,and- Chlorella.-Lignocellulosic-Italianryegrass-(2.00g)wasturnedinto-H2-(78.7mg)through- alkalitreatment,hydrolysis,and-PRprocesses.-The-PRprocessofglycerol-(10.4g)and- methanol-(11.3g),whichwereby‐productsinbiodieselsynthesis,formed-H2-(3.10g).- Dried-Chlorella-(10g)wasturnedinto-H2-(578mg)byproteasehydrolysisand-PR.-

**Keywords:** TiO2, sacrificial agents, lignocelluloses, BDF, Chlorella-

### **1. Introduction-**

 Plantscollectsunlightenergythroughphotosynthesisandstoreitasavarietyofpolymeric- saccharides.-Polymericsaccharidesareconvertedintomonomericsaccharides,whicharethen- convertedintoenergyinalllivingorganisms.-Thus,saccharidesareenergy‐storagesubstances- whichareproducedfrom-CO2andeasilyconvertedtoenergyalongwith-CO2emission.-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 Therefore,saccharideshavehighlypotentialresourcestoproducerenewableenergy.-Bioetha‐ nol production fromstarch of maize, sugarcane,and sugar sorghumisthe mosttypical method- toproviderenewableliquidfuel-[1,-2].-Recentlyinordertoavoidthedirectcompetitionwith- foodsources,cellulosicmaterialshavebeenwidelyrecognizedasoneofthepromising- sustainableresourcestoproducesecond‐generationbioethanol-[3].-However,theethanol- concentrations-(<5.0-%)werestilltoolowtoseparatebydistillationatalow‐energycost-[4].- Ontheotherhand,gaseous-H2isspontaneouslyisolatedfromreactionmixtureswithout- operationstoseparate.-Therefore,-H2productionfromsaccharidesandbiomass‐derived- materialsisoneoftheeconomicalapproachestobiofuels-[5].-

In this chapter, I will show the photocatalytic reforming over titanium dioxide (TiO2) using- saccharides, glycerol, and amino acids, which are derived by hydrolysis of lignocelluloses,- lipids, and Chlorella, respectively. This will lead to construct the sustainable energy system- alternatives to petroleum‐based fuels.-

#### **2. Outline of photocatalytic biomass reforming-**

A general procedure of biomass reforming is started by the production of water‐soluble- materials from biomass through biological treatment as well as chemical reaction. The resulting- water‐soluble materials are converted to biofuels such as ethanol, methane, and hydrogen- through various catalytic reactions involving methane fermentation and steam reforming. It- was demonstrated that the photocatalytic H2production from biomass‐derived materials had- an advantage compared with other thermal catalytic reforming by Shimura and Yoshida in- their review in 2011 [6].-

Our biomass reforming was performed in aqueous solution through enzymatic and chemical- hydrolysis of biomass (lignocelluloses, lipids, and Chlorella) followed by photocatalytic- reaction of water‐soluble materials (saccharides, glycerol, and amino acids) over TiO2under- UV‐irradiation (**Figure 1**). Saccharides were produced by enzymatic hydrolysis of lignocellu‐ loses using cellulase and xylanase. Glycerol was obtained by transesterificationof lipid with- methanol. Amino acids were obtained from hydrolysis of Chlorella by protease. These water‐ soluble materials were served as sacrificialagents for the photocatalytic H2production in- aqueous solution. Details of each process were described in the following sections.-

**Figure 1.** Outline of photocatalytic reforming of biomass.-

#### **3. Biological reactions-**

For biological reaction, a cellulase from *Acremonium cellulolyticus-*(Acremozyme KM, Kyowa- Kasei, Osaka, Japan) [7] was selected among commercially available cellulases. A xylanase- from *Trichoderma longibrachiatum-*(reesei) (Sumizyme X, Shin Nihon Chemicals, Anjyo, Japan)- was selected from commercially available enzymes. Proteins were hydrolyzed by protease- (protease A AmanoSD, Amano enzyme, Nagoya) at 50°C in a phosphate buffer-(0.1 M, pH 7.6)- which was prepared by dissolving Na2HPO4-(2.469 g) and NaH2PO4-(0.312 g) in 100 mL of- water.-

The cell suspension of *Saccharomyces cerevisiae*was prepared as follows. *S. cerevisiae-*NBRC 2044- was grown at 30°C for 24 h in a basal medium consisting of glucose (20.0 g/L), bactotryptone- (1.0 g/L, Difco), yeast extract (1.0 g/L), MaSO4-(3.0 g/L), and NaHPO4-(1.0 g/L) at initial pH 5.5- [7].-

Cellulose and hemicellulose (holocellulose), which were composed of glucan and xylan, were- hydrolyzed to glucose and xylose by the enzymatic saccharification-(SA, Eq. 1). The powdered- and pre‐treated lignocellulose (4.0 g) was dispersed in an acetate buffersolution (80 mL, pH- 5.0, 0.1 M) which was prepared by mixing 0.808 g acetic acid and 3.05 g sodium acetate in 500- mL of water. Cellulase (200 mg) and xylanase (200 mg) were added to the suspension of- lignocellulose. The SA was performed by stirring the solution vigorously with a magnetic- stirrer at 45°C for 120 h. After centrifugation of reaction mixture, the supernatant solution- involving glucose and xylose was analyzed by HPLC and used as sacrificialagents in the- following photocatalytic reaction.-

$$\text{Xylan} + \text{Glucan} \xrightarrow[\text{water}]{\text{Cellulase, Xylanase}} \text{Xylose} + \text{Glucose} \tag{1}$$

Also, lignocellulose could be turned into ethanol and xylose through simultaneous sacchari‐ ficationand fermentation (SSF, Eq. 2) using cellulase and xylanase as well as *S. cerevisiae*as- follows [8]. An acetate buffersolution (10 mL, pH 5.0, 0.1 M) was added to pre‐treated- lignocelluloses (3.0 g) in the reaction vessel. The reaction vessel was autoclaved at 120°C for- 20 min. After cooling, cellulase (180 mg) and xylanase (120 mg) in an acetate buffersolution- (8.0 mL) and the cell suspension of *S. cerevisiae-*(0.36 mL) were introduced into the reaction- vessel. After air was purged with N2stream for 15 min, the SSF was performed at 34°C under- stirring vigorously with a magnetic stirrer. The evolved CO2was collected by a measuring- cylinder to monitor the volume of CO2gas. The SSF reaction was continued for about 96 h until- CO2evolution ceased. After unreacted biomass was removed from the reaction mixture by- centrifugation, the supernatant solution was analyzed by gas chromatography (GC) and high‐ performance liquid chromatography (HPLC) to determine the concentrations of ethanol and- saccharides, respectively. Ethanol was collected from the SSF solution by evaporation under- reduced pressure while the residual xylose was subjected to the photocatalytic reaction.-

$$\text{Xydan} + \text{Glucan} \xrightarrow[S.\text{Ceverisine}]{\text{Cellulase, Xylanase}} \text{Xylose} + \text{CO}\_2 + \text{C}\_2\text{H}\_3\text{OH} \tag{2}$$

$$\text{water}$$

Another process to convert lignocellulose to ethanol is simultaneous saccharification and co‐ fermentation (SSCF). A recombinant *Escherichia coli-*KO11 which can ferment xylose was used.-Glucan and xylan in lignocellulose are turned to ethanol by SSCF using cellulase, xylanase,yeast, and *E. coli-*KO11. An example is an SSCF process of the low‐moisture anhydrousammonia (LMAA)‐treated Italian ryegrass (Section 6.1), which produced ethanol in 84.6%yield [9]. In this case, it was not necessary to undergo the photocatalytic process.-

$$\text{CJyan} + \text{Glucan} \xrightarrow[\text{S.creveisine}]{\text{Cellulase, Xylanase}} \text{CO}\_2 + \text{C}\_2\text{H}\_2\text{OH} \tag{3}$$

$$\text{water}$$

### **4. Photocatalytic H2 production-**

### **4.1. Titanium dioxide (TiO2) as the photocatalyst-**

TiO2is a white powder material which is thermally stable, non‐flammable,and no health- hazards. Therefore, TiO2has been used for many years in industrial and consumer goods,- including paints, coated fabrics and textiles, cosmetics, and so on. The photocatalytic H2- production was performed by use of an anatase‐type TiO2. It has a semi‐conductor structure- whose band gap is known to be 3.20 eV, which corresponds to 385 nm. Therefore, TiO2can be- excited by 366 nm‐emission from a high‐pressure mercury lamp. Irradiation induces charge- separation into electrons and holes on the TiO2-[10]. Electrons (e‐ ) reduce water to generate- H2, while holes (h+ ) oxidize hydroxide anions to hydroxyl radicals (**Figure 2**) [11]. In most cases,- noble metals (Pt, Pd, and Au) were loaded on TiO2to accelerate the reduction of water by- electrons. We used a Pt‐loaded TiO2 (Pt/TiO2) throughout the present investigation.-

**Figure 2.** Hydrogen evolution on Pt/TiO2 under irradiation.-

Moreover, it was well known that the use of sacrificialagents remarkably accelerates H2- production because the hydroxyl radical is consumed by them. Especially, we have elucidated- that sacrificialagents with all of the carbon attachedheteroatoms (O and N) are superior- sacrificialagents because they continued to serve as electron sources until their sacrificial- ability was exhausted [12, 13]. Glucose, xylose, glycerol, and glycine meet this requirement.- The photocatalytic H2production using sacrificialagents is called "sacrificial H2production."-

#### **4.2. Preparation of Pt‐loaded TiO2 photocatalyst-**

For photocatalytic reaction, almost researches have continued to use a P25 (Degussa Co. Ltd,- Germany) and a ST01 (Ishihara Sangyo Co. Ltd., Japan). The P25 is prepared through hydrol‐ ysis of TiCl4and composed of 75% of anatase and 25% of rutile, while the ST01 was prepared- through hydrolysis of TiOSO4 and composed of 100% of anatase.-

The Pt‐loaded TiO2 (Pt/TiO2) was prepared by the method reported by Kennedy and Datye [14]- as follows. An aqueous solution (400 mL) containing TiO2-(4.0 g, ST01, particle size 7 nm and- surface area 300 m2 g‐1), K2PtCl6-(200 mg), and 2‐propanol (3.06 mL) was introduced into a- reaction vessel which is illustrated in Section 4.3. After O2was purged by N2gas, the solution- was irradiated by a high‐pressure mercury lamp with stirring for 24 h when the gas evolution- reached over 100 mL. After the irradiation, water was entirely evaporated. The resulting gray- precipitate was moved on a filter and washed with water and then dried and ground to produce- Pt/TiO2powder. The Pt‐content on TiO2was optimized to be 2.0 wt% from the photocatalytic- H2evolution by various Pt‐content TiO2using glucose as a sacrificialreagent. Identification- of Pt/TiO2was usually performed by an XRD patternand TEM image [15]. **Figure 3**shows a- TEM image and an X‐ray diffraction pattern of a P/TiO2 (2.0 wt% of Pt content).-

 **Figure 3.** (A) TEM images of Pt/TiO2-(2.0 wt% of Pt content). (B) X‐ray diffractionof a P/TiO2-(2.0 wt% of Pt content).- Mark \* was the peak for Pt. Mark # was the peak for impurity of Teflon removed from the stirrer chip.-

### **4.3. Experimental method-**

The photocatalytic H2production was performed using a photo‐irradiation apparatus- (**Figure 4**). The catalyst (100 mg) and the given amounts of aqueous solution of sacrificialagent- were introduced into a reaction vessel. The volume of the reaction solution was adjusted to- 150 mL with water. The reaction vessel was connected with a measuring cylinder through a- gas‐impermeable fluororubbertube to collect the evolved gas. A high‐pressure mercury lamp- (100 W, UVL‐100HA, Riko, Japan) was inserted into the reaction vessel, which was set in a- water bath to keep it at a constant temperature (usually 20°C). After O2was purged from the- reaction vessel by N2gas for 15 min, the reaction mixture was irradiated with vigorous stirring- using a magnetic stirrer until the gas evolution ceased. The evolved gas was collected by a- measuring cylinder to measure the total volume of the evolved gas. The evolved gas (0.5 mL)- was obtained using a syringe and was subjected to the quantitative analysis of H2, N2, and- CO2, which were performed on a Shimadzu GC‐8A equipped with a TCD detector at a- temperature raised from 40 to 180°C using a stainless column (3 mmΦ, 6 m) packed with a- SHINCARBON ST (Shimadzu). In the absence of sacrificial agents, the H2 evolution from water- was small (<2 mL).-

**Figure 4.** Apparatus for photocatalytic reaction.-

#### **4.4. Analysis of photocatalytic reaction-**

Theoretically, the photocatalytic reaction can convert glucose and xylose to 12 and 10 equiva‐ lents of H2-(Eq. 4). Indeed, the photocatalytic reaction using glucose and xylose produced 11.8- and 10.0 mol of H2 from 1 mol of glucose [15] and xylose [16], respectively.-

Photocatalytic Reforming of Lignocelluloses, Glycerol, and Chlorella to Hydrogen 397 http://dx.doi.org/10.5772/64901

$$\mathrm{C}\_{n}\mathrm{H}\_{2n}\mathrm{O}\_{n} + \mathrm{nH}\_{2}\mathrm{O} \frac{\mathrm{UV}}{\mathrm{Pt/TiO}\_{2}} \rightarrow \mathrm{nCO}\_{2} + 2\mathrm{nH}\_{2} \tag{4}$$

 I show a method to determine the amounts of H2evolved from 1 mol of sacrificialagent. Atypical example is the photocatalytic H2production using saccharides obtained from enzy‐ matic saccharificationof Napier grass. Although the saccharides contained not only xylose butalso glucose, the evolved H2and CO2were plottedagainst the moles of xylose in a mixture ofxylose and glucose, as shown in **Figure 5A**. Gas volumes of H2and CO2increased with theincrease of xylose. However, the molar ratios of H2to xylose (H2/xylose) were not constant tothe amount of xylose used. It was speculated that the colored material in the solution and thecarboxylic acids formed during the photocatalytic reaction may lower the activity of photo‐ catalyst. Therefore, the H2/xylose ratio was plottedagainst the molar ratio of xylose to catalyst-(xylose/catalyst), as shown in **Figure 5B**. As the xylose/catalyst ratios decreased, the H2/xylosemax ratios increased. The intercept of the plots was equaled to *H*2- , which is the limiting moleamount of H2produced from one mole of xylose (sacrificialagent) at an infiniteamount ofcatalyst [17]. Thus, the total molar amount of H2was calculated by the equation: *H*<sup>2</sup> max-× (molesof sacrificial agent).-

**Figure 5.** The TiO2‐photocatalytic H2production using a mixture of xylose and glucose obtained from the enzymatic- saccharification of Napier grass. (A) Dependence of volumes of H2 (-) and CO2 (◇) against the mole of xylose. (B) Plots- of H2/xylose (-) and CO2/xylose (◇) ) against xylose/catalyst.-

max- Similar plots of *CO*2/xylose against the xylose/catalyst were performed, giving the *CO*2- values from the intercept of the plots. Other gasses such as methane and CO were not detected- in evolved gas.-

### **5. Energy recovery efficiency (***E***ff)-**

 Total energy recovery efficiency (*E*ff) from biomass to biofuels was calculated using combustion- energy: *E*ff-= 100*H*F/*H*0where *H*0and *H*Fwere the combustion energies of biomass and biofuels,- respectively. The combustion energies of sacrificialagents such as glucose, xylose, and glycerol- are 2803 [18], 2342 [19], and 1654 kJ/mol [18], respectively. The combustion energies of biofuels- such as ethanol and H2are 285 and 1367 kJ/mol [18], respectively. In the case of lignocellulose,- the *H*0value was combustion energy of glucose and xylose at the complete hydrolysis of glucan- and xylan which were determined by the National Renewable Energy Laboratory (NREL) [20].-

#### **6. Practical photocatalytic biomass reforming-**

#### **6.1. Lignocelluloses-**

Lignocellulosic biomass was composed of cellulose, hemicellulose, lignin, and other compo‐ nents. The components of glucan, xylane, lignin, ash, and others in non‐treated lignocelluloses- are summarized in **Table 1**. Since the contents of cellulosic components in lignocelluloses were- in the range of 41.0–66.5 wt%, only a half of lignocelluloses were utilized for production of- H2. The method to determine the content of each component was shown as follows.-


a The values in parenthesis are the contents of glucan and xylan in holocellulose.-

**Table 1.** Components of non‐treated lignocelluloses.-

Lignocelluloses were cut by a cutterand dried at 70°C for 72 h. The dried matterwas powdered- by a blender until the powder passed through a sieve with 150 μm of mesh. The powdered- lignocellulose (30 g) was treated with a 1% aqueous solution of NaOH (400 mL) at 95°C for 1- h. The reaction mixture was centrifuged and filtered to isolate the holocellulose (a mixture of- cellulose and hemicellulose) as a pale yellow precipitate. The supernatant solution was made- acidic (pH 5.0) with a dilute HCl solution to isolate dark brown precipitate which was identified- as lignin. The precipitate was collected via centrifugation at 10,000 rpm for 10 min.-

The contents of saccharides in holocellulose were analyzed according to the methods published- by NREL [20]. Sulfuric acid (72 wt%, 3.0 mL) was added slowly to holocellulose (300 mg) in a- reaction vessel and kept at 30°C for 1 h. Water (84 mL) was added to the reaction vessel so that- the concentration of sulfuric acid became 4.0 wt%. Acid hydrolysis was performed by auto‐ claving at 121°C for 1 h in an autoclave. The treated solution was neutralized with CaCO3and- was centrifuged. The supernatant solution (ca. 87 mL) was concentrated to 30 mL by evapo‐ ration. The solution was analyzed by HPLC to determine the amounts of glucose and xylose.- The amounts of glucan and xylan were determined from the amounts of glucose and xylose.- The ash component in lignocellulose was obtained by the burning of the lignocellulose (2.0 g)- in an electric furnace (KBF784N1, Koyo, Nara, Japan) for 2 h at 850°C.-

The pre‐treatments to promote an enzymatic digestibility of the cellulosic components and to- remove the lignin component were usually performed. Alkali (AL) treatment is a popular- method to remove lignin from lignocelluloses [21]. A powdered lignocellulose (30 g) was- added to a 1% aqueous solution of NaOH (400 mL). The mixture was heated under stirring at- 95°C for 1 h. The reaction mixture was subjected to centrifugation at 10,000 rpm for 10 min.- The lignin remained in the supernatant solution. The holocellulose, which is a mixture of- cellulose and hemicellulose, is isolated as a pale yellow precipitate, which was washed by- dispersion in water to remove the contaminated lignin. After the pH adjustment to 7.0, the- washed precipitate was collected by centrifugation and dried. Thus, lignin‐removed holocel‐ lulose was obtained. The AL treatment is effective for saccharification of the lignocellulose with- higher lignin contents. However, in the case of lignocelluloses with low lignin content such as- Napier grass, the AL treatment retarded the yeast‐fermentation rate because AL treatment- removed not only lignin but also nutrients to help yeast fermentation [22].-

Another useful pretreatment of lignocelluloses is LMAA (low‐moisture anhydrous ammonia- pretreatment), described as follows [23]. Dry powdered lignocelluloses (100 g, volume 320 mL)- were mixed homogeneously with water (100 g) in a flask-(1 L). The flaskcontaining wet- lignocellulose was evacuated with a pump and then gaseous NH3was introduced into the- flaskrepeatedly until the atmosphere inside the flaskwas entirely replaced with NH3gas. The- moist powdered lignocellulose was kept under an NH3gas atmosphere at room temperature- for 28 days. After NH3was removed with an evaporator, the treated lignocellulose was washed- with water to liberate the brownish aqueous alkali solution of the lignin. This washing- operation was continued until the pH became below 7.7. The treated lignocellulose was dried- at 60°C. Here, NH3served for transformation of the cellulose crystal phase to a highly reactive- structure toward enzymatic degradation rather than the removal of lignin [24]. As a special- pretreatment method, TiO2‐photocatalytic pretreatment was developed by our group [25].-

 Thephotocatalyticreformingwasappliedtolignocellulosessuchas-Italianryegrass-[26],- Napiergrass-[26],bamboo-[27],ricestraw-[27],andsilvergrass-[27].-Theresultsaresum‐ marizedin-**Table 2**.-The-SA→PR method isa process throughtheenzymaticsaccharification- (SA)ofthepretreatedlignocellulosesintoglucoseandxylosewhichwerethenusedas- sacrificialagentsforthephotocatalytic-H2productionover-Pt/TiO2-(PR).-Forexample,the- dried-Italianryegrass-(2.00g)wassubjectedtothe-ALtreatmenttogivethe-AL‐treated-Italian- ryegrass-(1.00g)whichwasturnedinto-554mgofglucoseand-193mgofxyloseby-SA.-The-

 SAofxylanwasmoreinefficientthanthatofglucan.-Glucoseandxylosewereturnedinto- H2-(78.7mg)by-PR.-Asaresult,thetotalenergyrecoveryefficiency-(*E*ff)from-AL‐treated- Italianryegrassto-H2wascalculatedtobe-71.9%-(**Figure-6**).-Inthecaseof-Napiergrass,dried- Napiergrass-(2.075g)wassubjectedtothe-ALtreatmenttogivethe-AL‐treated-Napiergrass- (1.00g)whichwasturnedinto-487mgofglucoseand-197mgofxyloseby-SA.-The-PRof- glucoseandxylosegave-84.0mgof-H2,whichcorrespondedto-77.0%of-*E*ff.-


a SSF = simultaneous saccharification and fermentation using cellulase and yeast. SA = enzymatic saccharification. PR =- photocatalytic H2 production over Pt/TiO2. SSCF= Simultaneous saccharification and co‐fermentation using cellulase,- yeast, and recombinant *E. coli* KO11. Referred from reference [9].-

b PT, pretreatment; LMAA, low moisture anhydrous ammonia pretreatment; AL, alkali pretreatment.-

c *W*G and *W*X were the amounts of glucan (G) and xylan (X) per 1 g of the pretreated lignocellulose.-

dThe total combustion energies (*H*0) of xylose and glucose theoretically derived from 1.0 g of the pretreatedlignocelluloses were calculated according to the following equation: *H*0 =2803 × *W*G/162 + 2342 ×*W*X/132.- e Total combustion energy (*H*F) of biofuels (ethanol and hydrogen).-

f Energy recovery efficiency (*E*ff) = 100 ×*H*F/*H*0.-

**Table 2.** Biofuel production from lignocelluloses.-

**Figure 6.** AL→SA→PR process of Italian ryegrass.-

In the case of the SSF→PR method, the LMAA treatment of the dried Italian ryegrass (1.458 g)- gave the LMAA‐treated Italian ryegrass (1.0 g) which was turned into ethanol (250 mg), xylose(121 mg), and glucose (19 mg) by SSF process. Ethanol was removed from SSF solution,- whereas the residual xylose and glucose were converted to H2-(17.3 mg) by PR. The *E*ffvalue- of H2combined with ethanol was 82.7% from the LMAA‐treated Italian ryegrass. We have- reported the ethanol production through an SSCF process of Italian ryegrass [9]. The *E*ffvalue- was 82.7%. These *E*ffvalues showed similar values. In the cases of Napier grass, the LMAA- treatment of the dried Napier grass (1.637 g) gave the LMAA‐treated Napier grass (1.0 g) which- was turned into ethanol (177 mg), xylose (167 mg), and glucose (13 mg) by SSF process. After- ethanol was removed from SSF solution, the residual xylose and glucose were converted to- H2-(21.0 mg) by PR. The *E*ffvalue of H2combined with ethanol was 77.2% from the LMAA‐ treated Napier grass. In the cases of bamboo, rice straw, and silver grass, the AL treatment of- bamboo (1.656 g), rice straw (2.092), and silver grass (2.439 g) produced the AL‐treated- lignocelluloses (1.00 g). They were turned into ethanol and H2by the SSF→PR process with *E*ff- of over 73.4%.-

#### **6.2. Glycerol-**

Biodiesel (BDF) is one of new sustainable energy alternatives to petroleum‐based fuels. BDF- market has significantlyincreased in Europe to adhere energy and climate policies [28]. BDF- (methyl alkanoate) is produced by transesterificationof vegetable oil or animal fats with- methanol under basic conditions [29]. However, glycerol as co‐production and unreacted- methanol was not utilized and went to waste. Glycerol has a potential to produce H2in- maximum theoretical yield of seven equivalents (Eq. 5). Also methanol can produce three- equivalents of H2. Hydrogen transformation of glycerol and unreacted methanol isolated from- the BDF synthesis was performed by sacrificial H2 production over a Pt/TiO2 [30].-

 CHO (glycerol) <sup>+</sup> <sup>3</sup>HO UV ® 3CO + 7H 383*<sup>n</sup>* 2 2 2 (5)-Pt / TiO<sup>2</sup>

 Asstartingmaterial,weusedvegetableoilwhichwasmainlycomposedofoleicacid- (C17H33CO2H)triglyceride.-Theaveragemolecularweightofvegetableoilwasthoughttobe- 884g/mol.-Vegetableoil-(150mL,-136.5g,-0.154mol)wassetinareactionvessel.-Methanol- (30mL,-23.8g,-0.743mol)wasmixedwith-NaOH-(0.485g,-0.012mol).-Abouthalfofthemixture- ofmethanoland-NaOHwaspouredintoareactionvesselandthenkeptat-61°Cfor-1h.- Moreover,theremainingmixtureofmethanoland-NaOHwasaddedintothereactionvessel- andthereactionmixturewaskeptat-61°Cforanother-1h.-Aftercooling,thereactionmixtures- wereseparatedintoalowerlayerandanupperlayer.-Theprocedureofthefollow‐upprocess- isshownin-**Figure-7**.-Thelowerlayer-(GLlayer)containedglycerol-(GL,-0.113mol)and- methanol-(0.214mol).-Theupperlayer-(BDFlayer)waswashedwithwater-(300mL)togive- BDF-(114.5g,-0.387mol)andtheaqueouswashingsolutionwhichcontained-0.137molof- methanol.-Thetotalrecoveryyieldofunreactedmethanolwas-47.5%.-Theyieldsof-GLand- BDFwere-73.3and-83.7%,respectively.-

**Figure 7.** Outline for preparation of BDF and the follow‐up process.-

 The photocatalytic reaction was performed by irradiation of aqueous solution containing- Pt/TiO2powder (100 mg, 1.25 mmol) and GL layer, which was added to the reaction vessel so- that the amounts of GL became 0.25, 0.50, 0.75, 1.00, and 1.25 mmol. The limiting mole amount- of H2-(*H*<sup>2</sup> max) per 1 mol of GL was obtained from the plots of the H2/GL against the GL/catalyst.- Similarly the photocatalytic reaction was performed for the washing solution, which contained- methanol. Using *H*<sup>2</sup> maxvalues, it was calculated that 2.82 and 0.28 g of H2was obtained from- the GL layer and washing solution, respectively. The *E*ffvalue of H2was determined to be- 100.8% using *H*Fof H2-(444 kJ) and the sum of combustion energy of glycerol (*H*0-= 187 kJ) and- unreacted methanol (*H*0 = 255 kJ).-

#### **6.3. Chlorella-**

Chlorella is single‐cell green algae with 2–10 μm diameter and multiplies rapidly, requiring- only carbon dioxide, water, sunlight, and a small amount of minerals [31]. Chlorella is mostly- composed of proteins (45%), lipids (20%), saccharides (20%), and minerals (10%). Thus, the- content of saccharides is low, suggesting that ethanol production is inefficient.-

We examined the photocatalytic H2production from Chlorella [32]. The frozen Chlorella was- thawed and dried in a drying machine and then ground. Gas evolution did not occur from the- non–enzymatic‐treated solution, which was prepared by magnetic stirring of the Chlorella- powder (10 g) in a phosphate buffer-(60 mL) for 48 h at 50°C. Therefore, the enzymatic- hydrolysis of Chlorella powder (10 g) was performed using protease (1.0 g) in a phosphate- buffer-(0.1 M, pH 7.6, 60 mL) under stirring at 50°C for 48 h to give the enzymatic hydrolyzed- solution. The solution was subjected to centrifugation to remove the precipitate. The super‐ natant solution (EH solution) was collected. The EH solution was subjected to freezing‐drying- in order to weigh the water‐soluble components in the EH solution. It was determined to be- 117 g/L. Since the weight of the solid was 167 g/L before hydrolysis, more than 70% of the solidwas hydrolyzed into water‐soluble components. The EH solution was composed of 98.0 g/L of- amino acids and 18.3 g/L of glucose which were determined by colorimetric analysis using- ninhydrin and by HPLC analysis, respectively.-

The photocatalytic H2production was performed using the EH solution (0.10 – 0.50 mL) over- a Pt/TiO2-(100 mg) in 150 mL of water. The limiting volume of H2per 1 mL of the EH solution- (*H*<sup>2</sup> max) was determined to be 119 mL/mL from the plots of the H2/(EH solution) against the- (EH solution)/catalyst. We successfully produced 579 mg of H2from 10.0 g of dry Chlorella- (**Figure 8**). This yield is higher than 394 mg for the H2production through AL treatment,- saccharification,and photocatalytic H2production from non‐treated Italian ryegrass (10.0 g)- [25, 27]. Thus, the photocatalytic reforming is applicable to not only saccharides but also amino- acids.-


**Figure 8.** Mass balance for the H2 production from Chlorella.-

Chlorella includes colored materials such as chlorophyll which may disturb the light absorp‐ tion by the catalyst. Therefore, dried Chlorella (20 g) was subjected to refluxingin ethanol (100- mL) for 6 h to remove the colored materials. Almost all amount of colored materials remained- in the ethanol solution. However, the decolorization did not affectthe amount of H2but could- shorten the irradiation time.-

#### **7. Conclusions-**

We examined photocatalytic H2production using sacrificial saccharides, glycerol, and amino- acid derived from lignocelluloses, lipids, and Chlorella. As a conclusion, the photocatalytic- reforming of biomass has the following features:-


In this chapter, biohydrogen production was discussed from the viewpoints of feedstock and- methodology to transform biomass to fuels. This will help life recycle assessment (LCA) to- evaluate CO2emission during cultivation, transportation, and manufacturing, as performed- for bioethanol from cellulose [33].-

### **Acknowledgements-**

This study was supported by a Grant‐in‐Aid for Scientific-Research (C) No 24610055 from the- Ministry of Education, Culture, Sports, Science, and Technology of Japan.-

#### **Author details-**

Masahide Yasuda-

 Addressallcorrespondenceto:yasuda@cc.miyazaki‐u.ac.jp-

 Departmentof-Applied-Chemistry,-Facultyof-Engineering,-Universityof-Miyazaki,-Gakuen‐ Kibanadai-Nishi,-Miyazaki,-Japan-

### **References-**


and fermentation process followed by a pentose fermentation with *Escherichia coli-*KO11. J Biosci Bioeng. 2012;114:188–192.-


## **Renewable Hydrocarbons from Triglyceride's Thermal Cracking**

Vinicyus R. Wiggers, Ramon F. Beims, Laércio Ender, Edésio L. Simionatto and Henry F. Meier

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65498

### **Abstract-**

 This chapter gives anoverviewof renewable hydrocarbon production throughtriglycer‐ ide's thermal‐cracking process. The influence of feedstock characteristics and availability- is discussed.It also presents issues about the reaction, the effect of operational conditions,- andcatalysts.-Aschemeofthereactionispresentedanddiscussed.-Thecompositionand- properties of bio‐oil is presented for both thermal and catalytic cracking. The high content- of olefins and the high acid index are drawbacks thatrequire downstream processes. The- reactordesign,kinetics,andscale‐upareopportunitiesforfuturestudies.-However,the- similarityofbio‐oilwithoilturnsthisprocessattractive.-

**Keywords:** waste fatty acids, triglyceride, pyrolysis, biofuels, green chemicals-

### **1. Introduction-**

 Nowadays,thesearchforprocessesthataimstoreducetheuseandthedependenceoffossil- fuelsisimperative.-Decreaseintheemissionofgreenhousegasesmightbeaglobaleffort.-In- thisway,thebiomassappearstobethelogicalchoicetoproducesolid,liquid,andgaseous- fuels,onceitisabundantandavailableallovertheworld-[1].-Therearemanytechnological- processesappliedtodifferentkindsofbiomassbeingstudiedandproposedbyscientific- community-[2].-Onethingis for sure,there will notbeonlyonetechnologythat will solveall- theissues,butdifferenttechnologicalroutestakingintoaccountthespecificcharacteristicsof- thesourceregionandthefeedstock.-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Besides the fact that these new technologies to produce biofuels must be environmentally- friendly, they are facing some obstacles to overcome economic and technical viability, high- scale, and stable production. Specificallyon liquid biofuels, another technical barrier is the fact- that almost every machine and vehicle was designed for fossil fuels usage. These fossil fuels- have several regulations and quality parameters that must be attendedfor commercialization.- In this way, it is a "sine‐qua‐non" condition that the new generation on liquid biofuels shall be- compatible with actual standard of the engines. The modern electronic fuel injection systems- make possible the use of differentfuels maintaining a good combustion in the engine.- However, how higher the similarity of the biofuel with fossil fuels, higher is the possibility for- its commercial application. In this way, the organic liquid product produced by thermal- cracking of vegetable oils and waste fats appears with high potential of oil substitute in the- refineries [3].-

The objective of this chapter is to provide a brief overview about the thermal conversion of- triglycerides into a liquid fraction, called bio‐oil, rich in hydrocarbons, presenting its proper‐ ties.-

#### **2. Thermal cracking of triglycerides-**

 Theproductionofbio‐oilthroughthermalcrackingofbiomassiseasilyfoundinliterature- [4–32].-Thebio‐oilisdefinedasadarkbrownviscouscorrosivefuelobtainedfrombiomass- pyrolysis-[33],butitisveryimportanttohighlightthatthebio‐oilhasdifferentproperties- accordingtothefeedstock.-Ifitisproducedfromlignocellulosicmaterials,thebio‐oilhas- significantamountofwaterandoxygencontent,decreasingitsgrosscalorificvalueandits- stability-[34,-35].-Ontheotherhand,ifthefeedstockistriglycerides,theoxygenandwater- contentislowandthehighheatingvalueiscomparabletothefossilfuels-[6,-36].-Another- importantcharacteristicisthesimilarchemicalcomposition,basedonhydrocarbons-[37].-So,- basedontheseissues,thebio‐oilproducedfromtriglyceridesappearslikeoneofthemost- promisingtechnologiesforbiofuelsproduction-[38].-

### **2.1. Feedstock-**

The triacylglycerol, also known as triglyceride (TAG) is an ester derived from glycerol and- three fattyacids [38]. It can be found in edible and nonedible vegetable oils, animal fats, and- used oils. The most abundant vegetable oils are soybean, palm, canola, sunflower, rapeseed,- among others. From animals, the main sources are pork lard, poultry fat, fishoil, and beef- tallow [39]. Waste greases or tap greases are found in cooking oils and sewage scum [40].-

In general, they have similar physical properties and chemical structure. They differin the- composition of the fatty acids, in the acidity and content of saturated fatty acids [39]. The acidity- of the oil is evaluated through the acid index determination (ASTM D974) which gives the free- fatty acids (FFAs) content in the oil. Waste oils are classifiedin yellow and brown greases- according to the content of FFA. Oils with lower than 15% (w/w) are classifiedas yellow- greases, while if it has more than 15% it is brown greases.-

The iodine index (pr EN 14111) provides the number of double bonds in the fatty acids. Oils- with high content of unsaturated acids are liquid in ambient conditions; however, oils with- high content of saturated acids are solid or semisolid in the same conditions.-

The fatty acid composition is provided by the fatty acid methyl ester (FAME) determination- [41]. It is a chromatographic analysis, which is a well‐accepted method for its determination.- The fatty acids composition of various TAGs can be found in the literature [34, 41, 42].-

One fact that must be pondered over, when one talks about biofuels production using TAGs,- is the feedstock availability [42]. In this way, we have two subjects to consider: the use of edible- oils and the logistic to join the wasted ones.-

In the firstcase, we need to consider the food versus fuels issues. The main concern is based- on the assumption that biofuel feedstocks tend to be more profitablethan food feedstocks,- which may lead to food shortages. Thus, it must be carefully pondered in order to efficiently- attend both markets [43].-

In the second case, it is possible to consider the waste‐cooking oils, the animal fats, and the- sewage scum. From cooking oils, its generation varies to each country, as it depends on the- vegetable oil consumption. The estimated generation in the European Union (EU) is about- 700,000–1,000,000 tons/year [44]. Only the UK generates an amount of approximately 250,000- tons per year [45]. Canada produces around 135,000 tons of yellow grease every year [46].- Mexico's generation is about 840,000 tons every year, similar to Malaysia. Japan produces- around 450,000–570,000 tons/year [47]. Hong Kong generates approximately 20,000 tons/year- [48]. The USA's generation is about 1,000,000 tons/year [47]. Even so, it is estimated that the- general worldwide generation is around 4.1 kg per habitant per year [49].-

Animal fats availability is also related to the region. It is well known that China, the USA, and- Brazil are large producers of meat. Only in 2013, the US industry processed 180,000 tons of- meat and poultry [50]. The fishindustry also plays an important role. In 2014, the world fish- production was about 146 million tons of fish [51]. As the amount of oil ranges from 40 to 65%- [52], it represents around 70.8 million tons of waste fish oil.-

Thus, these numbers show that it is possible to use biofuels production as a final destination- to these wastes. It is important to highlight the complex logistic to use it and that these amounts- will not replace the oil, but they can be a viable alternative.-

#### **2.2. Process and reaction-**

The thermal‐cracking reaction is definedas thermal decomposition of the organic chains by- heat in an atmosphere free of oxygen, with or without the aid of a catalyst. **Figure 1**presents- a basic scheme of the triglycerides thermal‐cracking process. As one can see in the scheme, the- reaction will generate always a solid fraction, generally called coke, a liquid product named- as bio‐oil, and a gaseous stream known as biogas.-

This reaction is affectedby the feedstock characteristics and the pair temperature‐residence- time [34]. The higher the temperature and the residence time, the higher the yield of the gas- product. Lower temperatures and higher residence times improve the coke formation.- Moderate temperatures with short residence times yield the liquid product. This last opera‐ tional condition is called fast pyrolysis [5]. The fast pyrolysis process is gaining attention due- to the possibility to obtain high amounts of bio‐oil, which can be used as fuel. **Figure 1**shows- that independent of the operational conditions, the solid fraction called coke will appear, and- this product will not be easily removed from the reactor. In general, this product formation is- associated with clogging [53]. One possibility to remove it is to proceed a controlled burning- in the heated reactor through feeding air instead of biomass, for a certain period of time,- promoting the combustion of the coke.-

**Figure 1.** A general scheme of thermal‐cracking process.-

The reactor design is the heart of the process [54]. Differentconfigurationshave been proposed- in the literature for several researches. It is possible to findbatch [9, 10, 12, 16, 21, 22, 24, 31,- 32] and continuous configurations [4–8, 11, 13, 17–20, 23, 25–27, 55]. In general, the batch- reactors are used to evaluate the reaction mechanism, kinetics, yields, and chemical charac‐ terization. As it works with lower capacities, they are not appropriated for industrial applica‐ tions. The continuous ones are in a higher sizes, bench or pilot, testing differentreactor designs- and operational conditions, evaluating the kinetics, yields, characterization, energy consump‐ tion, and economic evaluation, aiming the scale‐up studies [26].-

The irreversible reaction is highly endothermic and requires high heat transfer rates. The- possibility to run the process in an autothermal operation promotes an advantage over other- processes. This condition can be reached burning a fraction of the products to produce the- thermal energy required for the reaction. An energy balance of the TAGs thermal cracking was- presented by [5].-

Due to the complexity of the organic reactions, there is no complete knowledge about all the- reactions involved, just proposals for the principal ones. A simplifiedreaction step for the- thermal cracking of triglycerides is presented in **Figure 2**. The reaction starts with the decom‐ position of the triglyceride molecule forming heavy oxygenated hydrocarbons. Some of the- saturated fattyacids formed may not sufferany subsequent breaking. The decarboxylationand decarbonylation reactions (2) are favored by unsaturations and compete with the C‐C bond- cleavage reaction (3). The CO and CO2are formed by the deoxygenation reactions in (2) and- (4). The isomerization, polymerization, dehydrogenation, and cyclization are responsible for- dienes, acetylenes, cycloparaffins,and polyolefins-(5). The Diels‐Alder addition of dienes to- olefinsalso produce cyclo‐olefins-(8) resulting in hydrogen formation. The hydrogenation of- cyclo‐olefinsto cycloparaffinsand the reverse reaction occurs in steps (6) and (7). Hydrogen- also comes from steps (9) and (10). The solid product coke is produced directly from trigly‐ ceride (12), by the polycondensation of heavy hydrocarbons and saturated fatty acids (11) and- aromatics (10). The polymerization of olefinscan also lead to coke (13). Considering the- reaction scheme in **Figure 2**, it is very important to advance the cracking at least to the point- which deoxygenation reaction occurs, eliminating the oxygen by CO and CO2. It is also- important avoid coke formation (steps 10 and 13 in the **Figure 2**). As a firstconclusion, for- thermal cracking, the temperature‐residence time is the key factor for this process.-

**Figure 2.** Proposed reaction scheme for the thermal cracking of vegetable oil and animal fats (triglyceride). Adapted- from [13, 26, 38, 56]. (1) Initial cracking, thermolysis of triglyceride molecule ester bond; (2) decarboxylation/decarbon‐ ylation of long‐chain oxygenated hydrocarbons; (3) C‐C bond cleavage of unsaturated oxygenated hydrocarbons; (4)- decarboxylation/decarbonylation of short‐chain oxygenated hydrocarbons; (5) isomerization, polymerization/dehydro‐ genation, cyclization to form dienes, acetylenes, cycloparaffins, and polyolefins;-(6) dehydrogenations of cycloparaffins- to form cyclo‐olefins;-(7) hydrogenations of cyclo‐olefinsto form cycloparaffins;-(8) Diels‐Alder addition of dienes to- olefinsto form cyclo‐olefins;-(9) aromatization of cyclo‐olefinsto form aromatics and polyaromatics hydrocarbons; (10)- Coking from polyaromatics; (11) coking by polycondensation of oxygenated hydrocarbons; (12) coking by polyconden‐ sation of triglyceride molecule; (13) polymerization of olefinsto form coke; (14) direct route for C1‐C5 hydrocarbon- formation from triglyceride molecule.-

The use of catalysts aims to aid the reaction and increases the products' quality [57]. As the- composition of the products may vary due to catalyst material, size, and shape [58], several- works evaluate the use of many types of catalysts. **Table 1**shows the different catalysts used- for the cracking of triglycerides. One of the concerns involving catalysts use relies on their- stability and reutilization, which directly affectthe cost of the process [31]. In general, the coke-


formation limits the use of heterogeneous catalysts, due to the deactivation, and this phenom‐ enon requires a regeneration process for its reuse, making the entire process for the conversion- complex. A scheme reaction for catalytic cracking was proposed by [59].-

**Table 1.** Main catalysts used.-

### **2.3. Yields, properties and characterization-**

The yields of the products are strongly affected by the operational conditions. **Table 2**shows- the range of temperature and residence time applied in published papers, presenting the- average product yields obtained in thermal [4–6, 9, 10, 13–15, 17, 18, 22, 24, 26, 31, 55] and- catalytic cracking [7, 9, 11, 15, 17, 18, 20, 21–23, 25, 27, 31, 55]. In thermal‐cracking processes,- the temperature range is higher than catalytic. One can also note that the yield of liquid and-


gas products tends to be a little higher in thermal cracking. On the other hand, the coke- formation is more favorable in the catalytic cracking.-

**Table 2.** Average products yielding obtained with thermal and catalytic cracking of triglycerides.-

The liquid fuels have fundamental importance in finalenergy consumption, especially due to- its energy density. So, in this way, most of the researches are being conducted in the way to- maximize the organic liquid product. No less important are the properties and the character‐ ization of this product. **Table 3**presents average properties of the bio‐oil presented in the- literature for thermal [4, 5, 9, 10, 12–15, 18, 19, 22, 24, 31, 55] and catalytic cracking [7–9, 11, 15,- 17, 18, 20–22, 55]. The elementary chemical composition for bio‐oil does not vary so much and- the sulfur content is low. The high heating value (HHV) is also comparable to the fossil fuels.- The acidity of the bio‐oil is higher for the thermal cracking compared to catalytic, but in both- cases, the bio‐oil requires a reduction in this property for processing and usage. The esterifi‐ cationreaction and reactive distillation were performed by [11] and [60] to reduce the acid- index.-

The content of olefinsin the liquid can also be problematic, once its content is associated with- poor stability, which may lead to gum or insoluble materials formation. To saturate the double- bonds, the hydrorefiningprocess can be applied [61]. The direct hydrocracking also can be an- option [62–64].-

 **Figure-3**presentstypicalchromatogramsfromtwosamplesofbio‐oilproducedthroughfast- pyrolysisofsoybeanoilandwaste‐cookingoil.-Forcomparison,thechromatogramsofan- n‐alkanesampleandanoilsampleareshowntogether.-Thesampleswereinjectedatthe- sameconditions.-Theoilandbio‐oilsamplesarecomplexmixturescontaininghundredsof- compoundsandthisturnsdifficulttodeterminethecompletecompositionandphysico‐ chemicalproperties.-


**Table 3.** Average properties of bio‐oil.-

One way of characterizing these liquid fuels is the distillation curve, used to plot the true- boiling point (TBP) versus distilled volume fraction. In general, a simple distillation is- performed according to ASTM D86 and ASTM D1160 methods and data obtained are con‐ verted to TBP according to correlations outlined in [65]. Process simulators also can be used- for this conversion and to predict the thermophysical properties of the oil and its fractions [66].- The bio‐oil characterization using distillation curves applying the oil correlations was pre‐ sented by [34]. The authors showed that it is possible to use this method, but it requires more- studies to confirm the results.-

A chemical characterization was performed by [37] in the distilled fractions of the bio‐oil- produced by [4]. The purifiedproducts, light bio‐oil and heavy bio‐oil, were obtained in the- range of the gasoline and diesel oil, respectively. The detailed hydrocarbon analysis (DHA)- performed in light fraction showed that it was composed by aromatics (16.86%), i‐paraffins- (8.31%), naphthenes (6.07%), olefins-(26.56%), paraffins (4.48%), C14+ (5.3%), oxygenates- (0.06%), and unclassified-(32.38%). The main composition of heavy bio‐oil was formed by- olefins,aromatics, and carboxylic acid residues. In a continuation of the study [60], samples of- the bio‐oil were submittedto a reactive distillation process to produce light and heavy bio‐oil- cuts, with lower acid index.-

**Figure 3.** GC‐FID chromatogram of n‐alkanes sample, an oil sample, bio‐oil from soybean oil, and a bio‐oil from waste‐ cooking oil.-

The gaseous products have great importance as liquids, once it has short hydrocarbons and a- high HHV and it can be fuel source for the thermal energy required by the endothermic- reaction. **Table 4**presents the average composition of biogas from thermal [5, 10, 13, 55] and- catalytic cracking [7, 8, 17, 23, 55]. Using this average composition, the HHV is estimated in- 46.6 MJ/kg (thermal cracking) and 46.3 MJ/kg (catalytic cracking). The high content of ethene- also makes this product interesting for petrochemical industries.-


**Table 4.** Average composition of the biogas produced from thermal and catalytic cracking.-

### **2.4. Kinetics-**

 Oneofthetechnicaldifficultiestoscaleuptheprocessisthedeterminationofthereaction- kinetics.-Oncetheprocesshashundreds,maybethousandsofreactions,itisverydifficultto- determineanaccuratekineticmechanism.-Inthesecases,thefirststepistousethelumping- methodtoproposesimplifiedmechanisms.-Thelumpingstrategyconsistsinjoingroupsof- productsaccordingtosomesimilarproperty,theboilingrange,forexample.-Theworksof- [67–69]presentedthefirstkineticlumpedmodelsfor-TAG'sthermalcracking.-**Table-5**shows- thekineticmodelsproposedintheliterature.-Themodelproposedby-[67]issimplerthanthe- othermodelsonceithasfewerlumps,butitcanpredictthesolidfraction.-Thestudyofthe- kineticofcrackingof-TAGsisincreasingandsoonmoremodelsshallappear.-

#### **2.5. Challenges-**

The continuous availability of the feedstock is an issue that requires a complex logistic to solve- the high‐scale collection. In certain regions, staying close to animal‐rendering facilities can be- an option [70].-

The industrial application of the thermal/catalytic‐cracking technology has some obstacles to- overcome [71]. The firstis related to reactor design and scale‐up. With the improvement of the- kinetics, the simulation using computational fluiddynamics shall help to deal with this issue.- A short work presented by [72] deals with the simulation of TAG's thermal‐cracking reactor- aiming scale‐up studies.-


Renewable Hydrocarbons from Triglyceride's Thermal Cracking 417 http://dx.doi.org/10.5772/65498

The products upgrading is required also, especially to deal with the acid index and olefins- content. The acidity reductions, mainly caused by carboxylic acids, using the esterification- reaction and neutralization, are opportunities for this issue. The reduction of alkenes content- can be done through hydrotreatment reactions, widely used in oil refineries.-The use of actual- sites for oil refining can be suitable for this biofuel production, once most of polishing processes- are present.-

#### **3. Conclusions-**

The thermal and/or catalytic‐cracking processes are a promising technique to produce- renewable source for hydrocarbon production. The product similarity with fossil fuels turns- its usage and development attractive.-However, some obstacles such as feedstock availability,- reactor design, scale‐up, and products upgrading require more studies. The thermal/catalytic- cracking of triglycerides will not completely substitute the oil, but it can reduce our depend‐ ence and be a suitable environmental option.-

#### **Acknowledgements-**

The authors are grateful to the University of Blumenau (FURB), FAPESC, and the Brazilian- governmental agencies ANP, CAPES, CNPq, and FINEP for the financial support.-

#### **Author details-**

Vinicyus R. Wiggers1\*, Ramon F. Beims1 , Laércio Ender1 , Edésio L. Simionatto2 and- Henry F. Meier1-

 \*Addressallcorrespondenceto:vwiggers@furb.br-

 1-Chemical-Engineering-Department,-Universityof-Blumenau,-Blumenau,-Santa-Catarina,- Brazil-

2 Chemistry Department, University of Blumenau, Blumenau, Santa Catarina, Brazil-

#### **References-**

[1]-Herbert G.M.J., Krishnan A.U. Quantifying environmental performance of biomass- energy. Renewable and Sustainable Energy Reviews. 2016;59:292–308. DOI: 10.1016/- j.rser.2015.12.254.-


ment of catalyst lifetime by ion exchange. Fuel. 2016;172:228–237. DOI: 10.1016/j.fuel.-2015.12.059.-


## **Biogasification of Horse Dung Using a Cylindrical Surface Batch Biodigester**

Patrick Mukumba, Golden Makaka and Sampson Mamphweli

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65373

### **Abstract-**

 Anaerobicdigestionofanimaldungoffersseveralbenefitssuchasreductionofodors,- pathogens,andproductionofrenewableenergybiogas.-Inthisstudy,a-1m3 -surface- batchbiogasdigesterwasdesigned,constructed,andinsulatedwithsawdustto- minimizetemperaturefluctuationswithinthedigester.-Thehorsedungwascollected- fromthe-Universityof-Fort-Hare-Honeydalefarmandfedintothebatchbiogasdigester.- Thehorsedungwasanalyzedfortotalsolids-(TS),volatilesolids-(VS),totalalkalinity- (TA),calorificvalue-(CV),pH,chemicaloxygendemand-(COD),andammoniumnitrogen-(NH4-N).-Theoptimumtotalalkalinity,ammonium-nitrogen,andchemical- oxygendemandwere-6235,-901,and-24230mg/L,respectively.-Thestudyfoundthat- horsedungproducedbiogasyieldwithanaveragemethaneyieldof-51%without- codigestingitwithotherwastes.-Therefore,horsedungisagoodsubstrateforbiogas- production,anditsuseinbiogasdigesterscanreducegreenhousegasemissionsinto- theatmosphereleadingtoclimatechange.-

**Keywords:** biogasification, digester, horse dung, biogas, methane-

### **1. Introduction-**

 South-Africalikeanydevelopingcountryisoverdependentonconventionalenergysources- such as coal and firewood. Coal is a fossil fuel and is the main source of electricity in the country.- Fossilfuelshavemanynegativeimpactsontheenvironment,whichincludeenvironmental- degradation, climate change, and human health problems [1]. Biogas production would benefit-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 mainlytheruralpopulationbyprovidingacleanfuelfromrenewablesubstratesandhelpto- endenergypoverty.-

After the ratificationof the United Nations Framework Convention on Climate Change- (UNFCCC) and Kyoto Protocol in August 1997 and July 2002, respectively, the South African- government embarked on numerous projects related to climate change, including projects that- have been intended as measures to reduce greenhouse gases. Furthermore, the high volatility- in oil prices in the recent past resulting in turbulence in energy markets has also compelled- the country to look for alternative sources of energy such as from biogasification. As a result,- the South Africa Integrated Resource Plan (SAIRP) approved by the parliament in 2010 sets a- target of 40% renewable energy contribution by the year 2030 [2].-

Biogasificationis a method by which biogas is produced from organic material through the- action of microbes. It is therefore a biological process in which organic materials break down- in an environment that is sufficientlywarm and oxygen-free. The end product of biogasificationis biogas, mainly methane, carbon dioxide, and digestion sludge. The sludge can be used- as a fertilizer. Biogas is a combustible gas, which is generally used as a source for cogeneration,- for producing electricity and heat by means of gas or dual fuel engines [3].-

 Biogas from anaerobic digestion (AD) can be viewed as one of the vehicles to reduce rural- poverty and could lead to rural development. The process produces less greenhouse gases- than waste treatment processes such as composting [4] and land filling [5]. It can substitute- fossil fuels and can decrease environmental pollution including acid rains and global warming [6]. Therefore, AD has wide flexibilityand can be modifiedto fulfillthe precise requirements in management of agricultural farms [7]. When compared to other renewable energy- sources, such as solar and wind power, the methane component of biogas can be easily stored- in bio-bags. Furthermore, the biogas digesters are not prone to theft unlike solar panels and- wind turbines. In addition, biogas production would benefitmainly the rural population by- providing a clean fuel and reduce energy poverty.-

According to Ref. [8], biogas is an overlooked source of fuel in spite of the excitement surrounding the use of biofuels as an alternative source of energy. The use of biogas digesters can- improve the lives of the people in rural areas in many ways; it reduces deforestation, reduces- greenhouse emissions, and controls unpleasant odors from human or animal wastes and- reduction of workload and marginalization of women who collect firewood.-

The most common types of biogas digesters are fixeddome digester, balloon-type digester,- and floating drum digester. The two most familiar types in developing countries are fixed dome- and floatingdrum digesters. The three main digesters are discussed and their advantages and- disadvantages are also given.-

The fixeddome digester is the most popular digester; its archetype was developed in China- as early as 1936. The fixed dome digester is shown in **Figure 1**.-

It is a closed dome shape digester with an immovable, rigid gas-holder and a displacement pit- (compensating tank). The biogas produced by methanogenic bacteria in the biogas digester is- captured in the gas holder and the slurry is displaced in the compensating tank. When gas isconsumed, slurry enters back into the digester from the overflowtank. As a result of these- movements, a certain degree of mixing is obtained. The more the gas is produced, the higher- the level at the slurry outlet [10].-

**Figure 1.** Chinese fixed dome digester, adapted with permission from [9].-

The fixeddome digester has some advantages that include: relatively cheap and durable, no- moving parts, and well insulated [11]. However, the fixeddome digester has disadvantages- that include: high technical skills are required for a gas tight construction, special sealant is- required for the gasholder, difficultto construct in high water table areas, requires more- excavation work, and enormous structural strength is required for construction [11, 12].-

A balloon digester (bag digester) is a plastic or rubber bag combining the gas holder and- digester. This is a plug‐flow‐typereactor. This design was developed in the 1960s in Taiwan.- Gas is collected in the upper part and manure in the lower part. The inlet and outlet are attached- to the skin of the bag. The pressure of the biogas is adjustable by laying stones on the bag [13].- **Figure 2** shows a balloon digester. The biogas is collected in the balloon.-

**Figure 2.** Balloon digester, adapted with permission from [10].-

The advantages of the bag digesters include: low cost, simple technology, and easy to clean.- However, the disadvantages include: short lifespan, susceptible to physical damage, hard to- repair, need high quality plastic, and difficult to insulate [14].-

Floating drum digesters are common in India. The digesters have a moving floating gas‐holder,- or drum. The gas holder floatseither directly in the fermenting slurry or in a separate water- jacket. The drum in which the biogas collects has an internal or external guide frame thatprovides stability and keeps the drum upright. When the biogas is produced the drum moves- up, and when the gas is consumed, the gas holder sinks back.-

The floatingdrum digesters have advantages which include: the operation of the plant is easy- to understand, the gas drum is air tight, and there is constant gas pressure as a result of the- weight of the drum [15]. However, it does also have disadvantages which are: steel drum is- relatively expensive and needs regular maintenance (priming, painting, and coating) and the- effectof low temperature during winter is high [11]. A floatingdrum digester is shown in- **Figure 3**.-

**Figure 3.** Indian-type digester, adapted with permission from [9].-

The main aim of the project was to measure methane content in horse dung using a designed- and constructed 1 m3 cylindrical batch biogas digester.-

#### **2. Objectives-**

The research was carried out with the following objectives in mind:-


#### **3. Methodology-**

#### **3.1. Design of the digester-**

Biogas digester design plays a crucial role in digester performance and a number of considerations are taken into account. The following aspects were considered during the design- process: durability, air tightness, availability of local materials and easy operation. The design- parameters included:-

### *3.1.1. Total solid (TS) contents of organic materials-*

The total solid (TS) contained in a substrate is usually used as the material unit to indicate the- biogas production rate of the materials. The most favorable TS value is 8% for betterbiogas- production [16].-

#### *3.1.2. Favorable temperature, pH value, and carbon/nitrogen ratio for good fermentation-*

The mesophilic temperature between 25 and 35°C was chosen in the design. The digester was- insulated to keep the temperature within the lattermesophilic range to optimize mesophilic- bacterial activity. The pH value selected ranged from 6.8 to 7.8 because the methanogens prefer- a neutral atmosphere with pH between 6.8 and 7.5.-

The carbon/nitrogen ratio considered ranged from 20:1 to 30:1. Carbon and nitrogen are the- main nutrients required by microorganisms. Therefore, a C/N ratio of 20–30:1 was considered- for optimum anaerobic digestion, based on biodegradable organic carbon [17, 18]. Hence,- codigestion experiments were done to maintain the carbon/nitrogen ratio within the desired- range.-

#### *3.1.3. Hydraulic retention time (HRT)-*

For mesophilic digestion where temperature varies from 25 to 35°C, the HRT was greater than- 20 days. In the thermophilic environment, HRT is usually less than 10 days [19]. Shortening- retention time can lead to increase in the volatile fattyacids (VFA) [20], and this is why- mesophilic digestion was considered. A surface cylindrical biogas digester was chosen because- it was easy to feed, insulate, clean, and easy to construct and remove slurry after every- hydraulic retention period. In addition, the batch digester was easy to agitate. **Figure 4**shows- the cylindrical batch digester body with all the calculated values indicated and **Table 1**shows- calculated volume and geometrical dimensions of the batch biogas digester.-

**Figure 4.** Geometrical dimensions of the cylindrical shaped biogas digester body. KEY: Volume of gas collecting chamber = *V*GA; Volume of gas storage chamber = *V*GB; Volume of fermentation chamber = *V*GC; Volume of the sludge layer =- *V*GD; Total volume of the digester, *V* = *V*GA + *V*GB+ *V*GC + *V*GD.-


**Table 1.** Assumptions for volume and geometrical dimensions [21].-

**Figure 5**shows a more detailed diagram of the batch biogas digester with the monitoring- sensors' positions.-

**Figure 5.** Detailed diagram of the designed batch biogas digester with various sensors positions.-

#### **3.2. Construction of the biogas digester-**

A number of issues were considered during the construction of the biogas digester to ensure- nonleakages and minimization of influenceof ambient temperatures on the substrate temperature. The construction of the digester was done in the following stages:-


The main building materials for the biogas digester included clinker bricks, sand, concrete- stones, and Portland cement. The concrete stones were free of soil and organic material. Furthermore, the sand used was clean in order to increase the strength of the digester.-

The klinker bricks were firstsoaked in clean water for 5 minutes in order to remove dust- and to prevent the bricks from sucking moisture from the mortar thus allowing a strong- bonding. Clinker bricks were used in the construction because they offered the following advantages;-


Portland cement was used because it has a low thermal conductivity of 0.29 W/(m.K) compared to masonry cement with a thermal conductivity of 0.5 W/(m.K) and epoxy was used- for painting the inside of the batch biogas digester because it has a high water proofingand- low thermal conductivity of 0.30 W/(m.K). Sawdust was selected for insulation because of its- availability in the area and low thermal conductivity of 0.08 W/(m.K) compared to dry sand,- which has a thermal conductivity of 0.15–0.25 W/(m.K). Therefore, the heat transfer in materials with low thermal conductivity, for example, sawdust, is very low.-

The biogas digester was constructed and reinforced concrete dome was placed on top of the- plastered biogas digester with mortar smeared on its top surface to ascertain tight air seal as- shown in **Figure 6**.-

**Figure 6.** Biogas digester with a reinforced concrete dome.-

#### **3.3. Second wall construction of the biogas digester and insulation of the biogas digester-**

An outer wall of 1 inch (115 mm) was constructed with bricks to make the biogas digester two- walled as shown in **Figure 7**. The separation gap for the two walls was 200 mm. Sawdust, an- insulating material, was then put after the plastering of the outer wall.-

Sawdust was selected for insulation because of its availability in the area and low thermal- conductivity of 0.08 W/(m.K) compared to dry sand which has a thermal conductivity of 0.15–- 0.25 W/(m.K).-

**Figure 7.** Constructed second wall of the biogas digester.-

### **3.4. Source of substrate and mixing proportions-**

Fresh horse dung was collected from University of Fort Hare Honey dale farm. The horse dung,- before fed into the 1 m3biogas digester, was chopped with a compost chopper to accelerate- biogasification.-

#### **3.5. Substrate parameters-**

The following parameters in horse dung were determined: pH, total solids (TS), volatile solids- (VS), ammonium-nitrogen (NH4-N), total alkalinity (TA), temperature (T), and caloric value- (CV). All the analytical determinations were performed according to the standard methods for- examination of water and wastewater [22].-

#### **3.6. Biogas analysis-**

The biogas composition was analyzed using the biogas analyzer consisting of nondispersive- infrared (NDIR) sensor for sensing methane and carbon dioxide and palladium/nickel (Pd/Ni)- sensor for sensing hydrogen and hydrogen sulfide.-The data for biogas composition was- recorded by a CR1000 data logger at a time interval of 2 minutes. The biogas analyzer and the- CR1000 data logger were powdered by a 12V DC batterythat was connected to a 20 W- photovoltaic module. The slurry and ambient temperatures were measured using type K- thermocouples connected to the same CR1000 data logger as the biogas sensors. The data- logger was interfaced to a computer.-

The data acquisition system which consisted of a palladium-nickel and nondispersive infrared- sensors is shown in **Figure 8**.-

**Figure 8.** The data acquisition system.-

### **4. Results and discussion-**

The substrate characteristics of the horse dung is shown in **Table 2**, namely, total solids (TS),- volatile solids (VS), total alkalinity (TA), pH, caloric value (CV) and ammonium-nitrogen- (NH4-N).-


**Table 2.** Substrate characteristics of horse dung.-

The average slurry temperature of the batch biogas digester after the insulation was 30°C.- Therefore, it can be concluded that the insulation of the batch biogas digester was advantageous because the desired temperature for optimum biogas production was achieved. **Figure 9-** shows the biogas yield from horse dung. The biogas production increased until it reached the- peak and then began to decline.-

**Figure 9.** Biogas yield for horse dung.-

The horse dung had a peak biogas yield of 0.51 m3on day 18. The linear biogas production of- horse dung from day 12 (0.11 m3 ) to day 17 (0.54 m3 ) can be approximated by the equation:-

$$1Y = 0.0686t - 0.6643\qquad\text{for } 12 \le t \le 17\tag{1}$$

The decay of biogas production for horse dung from the peak (day 17) to day 22 is representedby:-

$$Y = -0.977t - 2.188 \qquad \qquad \text{for } 17 \le t \le 22 \tag{2}$$

The relationship between biogas yield and pH in horse dung is shown in **Figure 10**. Themaximum biogas yield of 0.54 m3was produced at pH 6.9. The initial pH of was 7.9 and thepH was observed to decline with time, attaininga minimum value of 6.9 where an optimumbiogas production of 0.54 m3 was achieved. The decline is due to the conversion of the substrateto acids during acidogenesis and acetogenesis stages of methane production. As from day 17,the pH was seen to increase as the acids produced were converted to methane by the methanogens.-

Relationship between biogas yield and chemical oxygen demand (COD) in horse dung isshown in **Figure 11**. The highest biogas yield of 0.54 m3was produced on day 17 where the-COD value was 24,230 mg/L as shown in **Figure 11**.-

**Figure 10.** Relationship between biogas yield and pH in horse dung.-

**Figure 11.** Relationship between biogas yield and COD in horse dung.-

The initial COD for horse dung was 37,110 mg/L and the final-COD was 22,110 mg/L. The- highest COD destruction was between days 15 and 19. The concentration of COD destroyed- from day 22 to day 28 was very low indicating a low biogas yield and a higher COD destruction- means a high biogas yield.-

The relationship between biogas yield and NH4-N in horse dung is shown **Figure 12**. The- highest biogas yield of 0.54 m3was produced at NH4-N concentrations of 901 mg/L. The initial- NH4-N concentration was 1196 mg/L and the final NH4-N concentration was 962 mg/L. It was- observed that the concentrations NH4-N for horse dung were between 850 and 1196 mg/L.- However, there was no inhibitory effect of the ammonium ion because the NH4-N concentrations were below 1500 mg/L. In the experiment, it was observed that higher NH4-N values- corresponded with lower biogas production-

The inhibiting concentrations of NH4-N are reported to be above 1500 mg/L [23–25]. The- stability of NH4-N levels in the substrate improved biogasification.-Relationship between- biogas yield and total alkalinity is shown in **Figure 13**.-

From the graph it was observed that the total alkalinity for horse dung was between 6190 and- 6256 mg/L. The higher the alkalinity, the greater the bufferingcapacity in the anaerobic- digestion process which in turn promoted a stable pH value (**Figure 10**) and this resulted in- an increase in the biogas yield. It was observed that total alkalinity changes were directly- proportional to changes in the pH.-

**Figure 12.** Relationship between biogas yield and NH4-N in horse dung.-

**Figure 13.** Relationship between biogas yield and total alkalinity.-

The composition of biogas in horse dung is shown in **Table 3**. The methane content horse dung- was 51% and the carbon dioxide content was 43%. Theoretically, horse dung produces more- biogas than cow dung because of its carbon/nitrogen ratio of 25:1 [10].-


**Table 3.** Composition of biogas in horse dung.-

The biogas from horse dung with methane content of above 50% can be used by rural communities for electricity production and as fuel for stoves, refrigerators, and generators, thereby- replacing liquid petroleum gas (LPG) as fuel. In addition, the total lifecycle environmental- impacts of the produced biogas are decreased via anaerobic digestion, since methane from- biogas can be used as fuel for diesel or petrol engines. However, for biogas from horse dung- to be used as fuel for vehicles, it should follow processes such as purification,upgrading,- compression, and storage. The digestate of horse dung from the biogas digester is subsequently- collected and used mainly to replace mineral fertilizers. Any overflowof the effluentfrom the- storage tanks is discharged directly into the aquatic environment, as nutrient for vegetable- crops. From **Table 3**, biogas from horse dung has a hydrogen content of 6%. The hydrogen- from anaerobic digestion of horse dung can be used as fuel in hydrogen fuel cells. Hydrogen- fuel cells have the following benefits:have a higher efficiencythan diesel or gas engines,- operate silently compared to internal combustion engines, fuel cells have no "memory effect"- when they are getting refueled, and finally, the maintenance of fuel cells is simple since there- are few moving parts in the system.-

### **5. Conclusion-**

The 1 m3batch biogas digester was successfully designed, constructed, and insulated with- sawdust and fed with horse dung. The batch biogas digester designed, constructed, and- insulated with sawdust was easy to feed and clean as compared to underground fixed dome- digesters which are not easy to clean and stir to agitate biogas production. Therefore, the- designed biogas digester could ease energy problems if installed in rural communities of South- Africa that have energy crisis. The results of the batch anaerobic digestion experiment show- that horse dung is a good substrate for biogas production. The total methane potential of the- horse dung was 51%, while the carbon dioxide content was 43%. In the experiment, it was- observed that the methane yield from the horse dung increased exponentially with time and- ceased after certain days. Furthermore, the study found that the optimum total alkalinity,- ammonium-nitrogen, and chemical oxygen demand for horse dung are 6235, 901, and 24,230- mg/L, respectively.-

It can also be concluded that anaerobic digestion of horse dung and other biogas digester- substrates in the country would improve the country's service delivery and serve as a local- solution to the world energy crisis caused by deletion of fossil fuels. From the research findings,- no previous experiments to measure methane content in horse dung were done using fieldscale digesters. The current study would be the firststudy to operate with a large digester- insulated with sawdust in an outdoor setting operating at an average temperature of 30°C.-

#### **6. Acknowledgements-**

The authors would like to thank ESKOM for funding this project and University of Fort Hare- for providing facilities for this project.-

#### **7. Author contributions-**

Patrick Mukumba designed, constructed, and fed the biogas digester with horse dung.- Golden Makaka and Sampson Mamphweli supervised the research project. All the authors- contributed to preparing and approving the final manuscript.-

#### **Author details-**

Patrick Mukumba1\*, Golden Makaka1 and Sampson Mamphweli2-


### **References-**


## **Refractory Materials for Biofuel Boilers**

Valentin Antonovič, Jacek Szczerba, Jadvyga Keriene, Rimvydas Stonys and Renata Boris

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65550

#### **Abstract-**

 Theenergyequipmentusableforsolidbiofuelincinerationusuallyoperatesupon- aggressiveconditions.-Theinternalstructures-(lining)oftheequipmentaremadeof- refractorymaterialsthatareaffectedbycombinedloads:thermal,mechanicaland- chemical-(i.e.hightemperature–upto-1200°C,chemicalimpactofalkalinecompounds- andslag,repeatingthermalshocks,abrasiveeffectcausedbysolidparticlesandsoon).- A majorityof traditional refractories usable for lining in such equipment are notdurable.- Uponcertainconditionsofuse-(suchashighlocaltemperatures,influenceofalkaline- biofuelcombustionproductsandsoon),durabilityofthetraditionalmaterialsis-1–2- yearsonly.-Theopportunitiesofnewrefractorymaterialsapplicationshouldbesetupon- takingintoaccounttheconditionsofoperationforbiofuelboilersofspecifictypes.-In- thissection-- thedataonthepeculiaritiesofusingrefractorymaterialsinbiofuelboilers- arereviewed,andtheimpactofaggressiveoperatingconditionsofsuchthermal- equipmentonthepropertiesofrefractorymaterialsisdiscussed.-Inaddition,the- investigationsresultsofrefractorycastablesalkaliresistanceanditsexplosivespalling- arediscussed.-Therecommendationsforuseofrefractorymaterialsinbiofuelboilers- arealsopresented.-

**Keywords:** biofuel boiler, refractory materials, refractory castables, alkali resistance-

### **1. Introduction-**

 Biofuelboilersareusedinmodernfuelcombustionsystems,withthefunctiontoensurehigh- energyconversionefficiencyandcomplywithenvironmentalstandardsappliedonsuch- devices.-Despitethefactthatenergyequipmentmayhavedifferentdesigns,theiroperationis- basedonastandardthree-levelprocessdiagram-(**Figure-1**)-[1].-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** Solid biofuel incinerator scheme: 1—primary combustion chamber with fuel feeding system; 2—secondary- combustion chamber; 3—exhaust gas and the heating system [1].-

Refractory materials are used for biofuel boilers' internal structures (lining). These materials- are non-metallic inorganic materials, which do not melt and do not decompose at high- temperatures (600–2000°C). Main elements of lining (**Figure 2**) are made of shaped (bricks,- blocks, etc.) and unshaped (concrete, mortar, coatings, etc.) refractories. They can be classified- by general (chemical or mineral composition, refractoriness, porosity, etc.) and specific (type- of binder and main raw material, forming method, etc.) features.-

**Figure 2.** Biofuel boiler lining made of (a) fired bricks and (b) castables.-

One of the most important groups in the unshaped materials classificationis refractory- castables. These are mixtures of refractory aggregates and bond(s), mainly supplied dry andused after addition and mixing with water or another liquid. They are placed by casting with- vibration, by casting without vibration (self‐flowing),by rodding, by shotcreting or when- necessary by tamping. Based on the standard EN ISO 1927‐1:2012, they can be dense or- insulating and divided into chemically and hydraulically bounded; the latterare then divided- into regular and deflocculated.-The above‐mentioned standard contains reference castable- classificationscheme (**Figure 3**), which can be used when reviewing the variety of refractory- castables.-

**Figure 3.** General classification scheme of dense or insulating castables according to standard EN ISO 1927‐1:2012.-

In **Figure 3**, refractory castables with hydraulic binders (calcium aluminate cement) are- divided into groups according to the scheme depending on the amount of cement. Regular- castable (RC) group includes castables with calcium aluminate cement content of up to 15–- 30%, medium cement content (MCC) group—8–15%, castables with low amount of cement- (LCC) group—4–8%, castables group with ultra‐low content of cement (ULCC)—1–3% and no- cement group includes cast castables without cement (NCC). MCC, LCC and ULCC types of- castables, compared with RC, contain special ultra‐fineparticles (less than 1 µm), and various- deflocculants-(soluble compound (usually an electrolyte) which, when added even in very- small quantities, will reduce the water content in castable). Hydraulically bonded castables set- and harden at ambient temperatures.-

The manufacture of modern LCC or ULCC refractory castables with very low cement content- often involves a number of process difficulties.-Among them the problem of the loss of- workability of castables, since these castables are sensitive to environmental temperature- changes during manufacture, water quantity and quality, mixing parameters and other factors.- For example, increase in water content by 2% in LCC and ULCC type of castable with chamotte- fillerreduces the cold crushing strength (CCS) of those castables after drying at 110°C and- firingat the temperature of 1100°C from 80 and 90 MPa to 60 and 20 MPa, respectively [2].- MCC type refractory castables with performance characteristics much betterthan the traditional concrete are not as "sensitive" to the conditions of production; they are attractive to use- in a variety of thermal equipment linings.-

Not only castables with hydraulic binders, but also with other types of binders as well as- gunning and ramming materials can be used in biofuel boilers. Another bond may be:-


When choosing refractory linings for biofuel boiler, it is necessary to know the effect of loads- attributableto the material during operation and to adapt it to the service properties of the- materials [3] (**Table 1**). Before selecting the material, it is also important to check the material- bond, in order to know about the need of special heat treatment. This will help to avoid- discussions about settingtimes, progress of work, and the date of taking the boiler into- operation [4].-


**Table 1.** Important service properties of refractory materials [3].-

Information about properties of refractory materials and their determination methods can be- found in various works that are subject to EN or ASTM standards [4, 5]. Some of these- information will be presented in this chapter.-

#### **2. Influence of operation conditions of biofuel boilers on durability of- refractory materials-**

It is noted that thermal equipment that use solid biofuel experience significantincrease in- thermal and mechanical loads and chemical effectson lining and refractory materials. In some- cases, sudden spalling of lining is observed in biofuel boilers as soon as after 1–2 years of use.- The observation of the lining and the investigations of refractory materials used in various- biofuel boilers show that the cause of poor durability of refractory materials is a combined- impact of negative factors, such as high temperatures, an aggressive chemical effectof alkali- compounds, an abrasive effectcaused by solid particles, repeating thermal shocks and- mechanical loads. It is noted that the risk of failure of materials highly increases with the- increase in alkali when changing the type of fuel.-

In biofuel boilers, depending on the type and sort of firewoodused, differentlevels of ash and- alkali metals (Na, K) are produced during combustion that adversely affectsrefractory- materials. **Table 2**shows the content of alkaline oxides in differenttypes of wood ashes [6].- Wood ash contains much more potassium than sodium; moreover, potassium diffusionto- refractory material is faster than that of sodium. Therefore, while investigating the lining- materials that spall in solid biofuel combustion devices, potassium compounds are found in- corrosion products.-


**Table 2.** Various types of wood ash chemical composition (of total dry mass of wood) [6].-

Although ash content in wood fuel or other solid fuels is low (up to several per cent), ash fusion- characteristics have an impact on the properties of refractory materials because ash melt easily- penetrates to the structure of the material. Ash melting behaviour depends on the type of fuel.- With the ASTM D1857 standard, the changes in the shape of a standard ash cone by burning- it in acidifying (oxidizing) environment are defined:initial deformation—IT; softening- temperature—ST; the point of hemisphere formation—HT; and flow temperature—YP.-

**Table 3**presents fusibility characteristics of ash of some wood types [7]. As we can see, pine- sawdust ash may adhere to the material of lining when the boiler's operating temperature- reaches the ash ST of around 1180°C. Such ash begins to melt and flowat temperature of- 1225°C. This means that pine sawdust fuel greatly increases the chemical effectson therefractory materials given the boiler operation if the local temperature (e.g. in the secondary- combustion chamber) is around 1200°C.-


**Table 3.** Fusibility characteristics of wood ash [7].-

### **2.1. Alkali effects at high temperatures-**

In the combustion chamber, under reducing environment alkali metals react with the refractory lining material. There are two differenttypes of alkaline reactions with refractory- materials: in dry conditions under the influenceof alkali vapour or in humid environment,- when melt ash is formed on the surface of the refractory material.-

Potassium or sodium released during combustion reacts with CO gas [8]:-

$$\text{C}2\text{K} + \text{CO} \rightarrow \text{K}\_2\text{O} + \text{C}\tag{1}$$

$$\text{2K} + \text{3CO} \rightarrow \text{K}\_2\text{CO}\_3 + \text{2C} \tag{2}$$

$$\rm K\_2CO\_3 + CO \rightarrow 2K + \rm 2CO\_2 \text{(}>930°C\text{; potassium} - \text{vapour}\text{)}\tag{3}$$

Potassium vapour over time can penetrate into the refractory material to the depth of morethan 100 mm [8].-

When refractory material is exposed to alkali vapour or melt, it may form the following- compounds: kalsilite (K2O Al2O3- 2SiO2), leucite (K2O Al2O3- 4SiO2), feldspar (KAlSi3O8,- NaAlSi3O8, CaAl2Si2O8) and others [9]. Formation of this type of minerals in the refractory- material increases its volume by 15–30% and sometimes even 55%, compared to the initial- volume of the material. This promotes the formation of porous structure (**Figure 4**) [10], microcracks in refractory material and the spalling degradation due to alkali effects.-

Under wet conditions when the melt forms on the surface of the refractory material, it can leadto reactions that reduce the temperature of melt formation [11]:-

$$\rm K\_2CO\_3 + SiO\_2 \rightarrow K\_2O \cdot SiO\_2 \text{(liquid)} \; + \; CO\_2 \text{(first melt} < 1000^{\circ}C\text{)}\tag{4}$$

$$\rm{K\_2CO\_3 + 3Al\_2O\_3 \cdot 2SiO\_2 \rightarrow K\_2O \cdot Al\_2O\_3 \cdot 2SiO\_2 + 2Al\_2O\_3 + CO\_2 \text{(first melt > 850°C)}}\tag{5}$$

Further, 4K2O CaO 10SiO2first melt <950°C.-

Thus, spalling of refractory material due to the effectof alkali may be intensive even with- reduced operating temperature of the boiler.-

**Figure 4.** Mullite brick structure: (a) undamaged layer and (b) porous layer affected by alkali [10].-

 It should be noted that the possibilities of the melt penetration into the structure of the material- depend on the porosity of refractory materials, effective potential of pores and capillaries, etc.- Thermal expansion coefficientof melt is significantlydifferentfrom the thermal expansion- coefficientof the refractory material. Therefore, with cooling material (e.g. when the boiler is- stopped), expansion differencesof unaffectedrefractory materials and its areas saturated with- melt, cause stresses leading to the layering, crumbling and destruction of the product. **Figure 5-** shows characteristic nature of disintegration of shaped refractories when affectedby ash melt- [12].-

Resistance of refractory materials to alkaline compounds is often measured with the crucible- method [9, 13]. When analysing, the alkali resistance of castables or fired bricks with test- samples with a cylindrical cavity are made. The cylindrical cavity is filledwith certain alkali- salt (K2CO3, K2SO4, etc.), and the samples are heated for some time at the temperature of- ≥1000°C. After multiple tests (each time anew by adding a fixedamount of salt), the samples- are visually inspected, capturing the occurrence of micro-cracks. Some of the specimens are- cut along the cylindrical axis into two parts, and the depth of the material affectedby alkaline- substances is evaluated.-

**Figure 5.** Typical fragmentation of bricks affectedby ash melt (a) and scheme of a bricks' step-by-step degradation (b):- 1,2,3—steps of degradation.-

#### **2.2. Resistance to the impact of carbon monoxide (CO)-**

Incompletely burned carbon compound products, the main of which is carbon monoxide (CO)- can penetrate (diffuse) in the material and react with refractory materials containing iron oxide.- In such a case, four-step reaction occurs in which one of the end-products is Fe3C [3]:-

$$\text{3Fe}\_2\text{O}\_3 + \text{CO} \rightarrow 2\text{Fe}\_3\text{O}\_4 + \text{CO}\_2 \tag{6}$$

$$\text{Fe}\_3\text{O}\_4 + \text{CO} \rightarrow \text{3FeO} + \text{CO}\_2\tag{7}$$

$$\text{FeO} + \text{CO} \rightarrow \text{Fe} + \text{CO}\_2 \tag{8}$$

$$\text{3Fe} + 2\text{CO} \rightarrow \text{Fe}\_3\text{C} + \text{CO}\_2 \tag{9}$$

Fe3C can react with CO:-

$$\text{20Fe}\_3\text{C} + \text{14CO} \rightarrow \text{3Fe}\_{20}\text{C}\_9 + \text{7CO}\_2 \tag{10}$$

$$2\text{Fe}\_{20}\text{C}\_{\text{9}} \rightarrow 20\text{Fe}\_{3}\text{C} + 7\text{C} \tag{11}$$

 If the refractory material contains metallic iron and/or iron oxides, CO in the temperatureinterval of 400–800°C produces carbon: 2CO → CO2-+ C. Mechanical stresses caused bycrystallization of carbon deposited in local areas may cause complete disintegration of thematerial. It has been found [14] that degradation of certain types of aluminosilicates due to CO- exposure is a result of two interrelated processes: reduction in iron oxides and volume changes- and carbon formation and its accumulation in the material structure.-

It has been observed that when excessive CO has been formed in the boiler for extended periods- (disrupted boiler operational mode), disintegration of refractory castable with high iron oxide- content (>4.4%) due to the general effectof CO and alkali occurred already after 8 months of- operation (**Figure 6**) [15].-

**Figure 6.** Cracking (a) and degradation of aluminosilicate materials due to the formation of new compounds (carbon- and leucite) in its structure (b) [15].-

Risks of refractories degradation due to CO can be reduced by using materials with as low- amount of Fe2O3 as possible (<1%).-

#### **2.3. Thermal shock resistance-**

This indicator shows the ability of refractory materials to resist thermal stresses in its structure- from temperature gradients. Such temperature gradients cause degradation of refractory- materials when boiler is often stopped (material is cooled) and start to operate (material is- heating up). Burning of solid biofuels generates a lot of fly ash that cause fouling of heat transfer- surfaces. As a result, boilers must be frequently stopped for cleaning and therefore linings- experience repeated thermal shocks. Differentcountries apply differentmethods [16] to- determine thermal shock resistance (number of cycles) of refractory materials, which vary by- sample size, heating temperature and sample cooling method (water, air, water-cooled panels).- It is noted that the thermal shock resistance of the refractory material may differdepending- on the selected method [17]. Where it is difficultto evaluate test results obtained in one or- another method, thermal shock resistance criteria R4 and Rst are calculated [18, 19].-

The thermal shock resistance of refractories can be evaluated not only by calculating the- thermal shock resistance criteria, but also by the refractory material surface appearance after- thermal shocks—test sample heating and cooling cycles. In the case of a low thermal shock- resistance refractory castable, a network of long cracks appears on the surface (**Figure 7a**).- Meanwhile, in the case of high thermal shock resistance refractory castable, a network of short- cracks is formed (**Figure 7b**). Such fragmental structure of the castable compensates its thermal- extensions and relaxes its stress. Therefore, when the number of cycles was increased the cracks- slightly widened but the castable did not collapse.-

**Figure 7.** Surface of castable before break up of sample: (a) which withstood 9 cycles (water—800°C) [20] and (b) 45- cycles [21].-

Refractory materials that have structures with built-in micro-cracks show betterthermal shock- resistance than rigid systems. In some refractory materials, the bond possesses micro-structural defects or cracks that provide better thermal shock resistance [4].-

#### **2.4. Abrasion resistance-**

Abrasion resistance is a feature of material to it surface that resists external mechanical effect- when solid particles fly at a high speed and mechanically rubs the material surface.-

Refractory materials used in chemical and cement plants, when process products intensively- circulate and rubs the surface of refractory material, must have a high abrasion resistance.- Abrasion resistance is determined according to standard ASTM C-704:1999. The abrasion- resistance rate of materials used under the above-mentioned conditions must not esceed 5–6- cm3 .-

In biofuel combustion plants, abrasion resistance of refractory materials is relevant when the- fluidizedbed system (movement of a mixture of sand and fuel) is used in the technology and- also when during boiler pipe blowing off-(clean procedure) ash particles flyat a high speed.- **Figure 8**shows a fragment of cross section of a fireclay brick where the surface in the bottom- part of the picture has been exposed to high speed particles flyingat the direction marked with- the arrow.-

Abrasion resistance and compressive strength are correlated with each other: the higher the- compressive strength, the greater its abrasion resistance. In this regard, strength characteristics- of refractory materials used in biofuel combustion equipment must be maximally high.-

**Figure 8.** Sectional fragment of fireclaybrick after 3 months of exploitation in solid biofuel combustion lining. Particles- flying at the direction marked with the arrow mechanically affected the brick surface in the bottom part of the picture.-

#### **2.5. Carbonation of calcium aluminate cement‐bonded regular refractory castable-**

 The observations showed that the lining of domestic boilers made by using regular castables- do not have long durability. Having been exploited for some time, it destructs. One of the- reasons that cause this destruction might be the so-called "carbonation" of calcium aluminate- cement hydration products. It is known that the main hydration products formed during the- reaction between calcium aluminate cement and water are as follow: CAH10-(forms at the- temperature <21°C), C2AH8and AH3-(21–35°C) and C3AH6and AH3-(>35°C) [22]. The carbonation of calcium aluminate cement hydration products is thought to occur by the following- reactions [23]:-

$$\rm{CaH}\_{10} + \rm{CO}\_{2} + \rm{xH}\_{2}\rm{O} \rightarrow \rm{CaCO}\_{3} + \rm{Al}\_{2}\rm{O}\_{3} \times \rm{yH}\_{2}\rm{O} + \rm{(10 + x-y)H}\_{2}\rm{O} \tag{12}$$

$$\rm C\_2Al\_3 + 2CO\_2 + xH\_2O \rightarrow 2CaCO\_3 + Al\_2O\_3 \times yH\_2O + (8 + x - y)H\_2O \tag{13}$$

$$\rm{^3C\_3AH\_6} + \rm{3CO\_2} + xH\_2O \rightarrow \rm{\mathfrak{XCaCO\_3}} + Al\_2O\_3 \times yH\_2O + (6+x-y)H\_2O \tag{14}$$

Carbonation causes a large-scale destruction of calcium aluminate cement materials [24] when-Na+ , K+ions participate in the so-called "alkaline hydrolysis" [25]. CO2, alkalis and H2Oenvironment is typical for domestic boilers during often stopping and starting of operations.-After the calcium aluminate cement hydration products dehydration at the temperature of- 500–800°C, C12A7is formed, which, after heating at 1000°C is converted to CA, CA2. If theoperation temperature is less than 1000°C (usually in domestic boiler), C12A7in humidenvironment (in moment of stopping and starting of boiler operation) is repeatedly hydrated.- Then carbonation of hydrates occurs (**Figure 9**), and the destruction of castable will start. It- was established that the additive of micro-silica (SiO2) in regular castable increases its resistance to the carbonation [25].-

**Figure 9.** C3AH6 hydrates (a) and its carbonation products (CP) (b).-

#### **2.6. Destruction of SiC‐based refractory materials-**

 Studies have shown that castables with SiC fillerresist much betterthe effectsof alkali- compounds than those with aluminosilicate filler-(fireclayand mullite) [13]. It should be noted,- however, that in the oxidizing atmosphere at >900°C SiC castable fillercan oxidise resulting in- the formation of SiO2and higher volume of minerals. The reaction takes place according to the- following scheme [26]:-

$$2\text{SiC} + 3\text{O}\_2 \rightarrow 2\text{SiO}\_2 + 2\text{CO} \tag{15}$$

Reverse reaction in castable with SiC may occur under reducing environment [11]:-

$$\text{SiO}\_2 + \text{3H}\_2 + \text{CO} \leftrightarrow \text{SiC} + \text{3H}\_2\text{O} \tag{16}$$

Because of mineralogical changes of structural elements of refractory material with SiC, the- strength is critically reduced.-

#### **3. Materials for working layer of linings of biofuel boilers and itsinvestigations-**

Over the last decade it has been noted that the use of shaped products is reducing, while the- use of unshaped materials such as refractory castables is constantly growing. This is due to- the shortcomings of shaped products: long duration of installation of the lining in thermal- equipment, complex repairs, complex design and manufacturing technologies of thermal- equipment from shaped products and higher cost of production of shaped products.-

Research shows [9, 13] that in alkali-resistant castables, under the influenceof alkali on the- surface of the material, a layer of glass of high viscosity is formed, which prevents further- penetration of alkali into the material.-

The aim of investigations [26] was to evaluate the resistance to potassium compounds' attack- on refractory castables, modifiedand unmodified,by additive of milled quartzsand (SiO2).- The findings are presented below.-

 Unmodifiedcommercial fireclaycastable (B0) and unmodifiedclinker castable (B1) and- modifiedclinker castables (B2, B3), in which ground quartzsand was used to increase alkali- resistance, were tested. Chemical composition (mass %) of castables B0, B1, B2, B3 was as- follows: B0—Al2O3-45.7; SiO2-43.6; CaO 7.6; Fe2O3-1.50; B1—Al2O3-42.9; SiO2-25.5; CaO 27.3;- Fe2O3-1.77; B2—Al2O3-41.8; SiO2-27.3; (2.5% of this quantity has ground quartz sand additive);- CaO 26.6; Fe2O3-1.79; B3—Al2O3-40.9; SiO2-29.0 (5.0% of this quantity has quartzsand additive);- CaO 26.0; Fe2O3-1.80 [26]. **Table 4**presents technical characteristics of castables used in alkali- tests with potassium carbonate salt by crucible method.-


**Table 4.** Technical characteristics of fireclay and clinker refractory castables after firing at the temperature of 1100°C- [26].-

Macroscopic assessment of samples is presented in **Table 5**. It was found that the samples of- commercial fireclaycastable B0, affected by K2CO3, cracked after 1 cycle (**Figure 10a**) and after- 2 cycles split into multiple fragments. The analysis of the surface view of the sample cut along- the cylinder bore axis (**Figure 10b**) shows changed zones because of alkaline impact (penetration depth ∼11 mm) [26].-

Clinker-based castable B1 without additives during the alkali test split into separate fragments- after 3 cycles (**Table 5**), while clinker castable with ground quartzsand additive (B2, B3),- depending to its quantity, split after 6–8 cycles.-

The analysis of the surfaces of sawn samples of clinker castable B1 without additives and B3- with ground quartz sand additive (**Figure 11**) shows that decomposition products of potassium- carbonate salt already in the firstcycle are easily penetrated into the structure of castable- without additive (similar as with commercial fireclaycastable, **Figure 10b**). Potassium- carbonate salt decomposition products penetrated the structure of the castable B3, modified- with ground quartzsand additive, with more difficulty.-After 3 cycles, a protective layer of 2–- 3 mm was observed (in some places up to 8 mm), capturing the penetration of potassium- carbonate salt decomposition products into the material (deeper) (**Figure 11b**). This increased- the resistance of castable samples to alkaline compounds—samples cracked just after 8 cycles- [26].-


**Table 5.** Macroscopic assessment of fire clay and clinker refractory castables, affected by K2CO3 [26].-

**Figure 10.** The view of specimens of commercial fireclaycastable after the tests with alkali compounds: (a) appearance- of over 0.4 mm wide cracks and (b) the section view of the specimens after one cycle [26].-

**Figure 11.** The view of sections of castable specimens after firingat the temperature of 1100°C with K2CO3: (a) B1 after- 1 cycle and (b) B3 after 3 cycles [26].-

The phase composition of substances formed during the reaction with K2CO3was found with- the tablet method [13]. The results are provided in **Table 6**. For comparison, the table also- contains the phase composition of products formed in firedcastables at the temperature of- 1100°C in the absence of the effect of K2CO3. These data show that commercial fireclaycastable- B0 contains the following minerals after firingat the temperature of 1100°C: gehlenite (C2AS),- mullite(3Al2O3-2SiO2) and quartz (SiO2). The resistance test to alkaline compounds allowed to- identify new products in this castable—feldspars and leucite. In clinker castables, without- additive (B1) and with ground quartzsand additive (B2, B3), a new product leucite was also- identified.-Test results of the tablet method suggest that in all cases, both in absence and- presence of quartzsand additive in clinker castable, during the reaction of its compounds with- K2CO3decomposition products, leucite is formed. However, the tablet method, which allows- to identify the chemical composition of compounds occurring from the reaction, does not allow- to assess a very important factor of castable corrosion—diffusionrate of corrosion-causing- substances deeper into the castable. So, a comparison of the penetration depth of fireclay- castable B0 without ground quartzsand additives (**Figure 10b**) and clinker B1 (**Figure 11a**),- with the penetration depth of castable with quartzadditives B3 (**Figure 11b**) shows that in the- case of castable B3, diffusionwas stopped. Apparently, the reaction of grounded quartz with- decomposition products of K2CO3resulted in a viscous layer of this reaction product inhibiting- the penetration of alkaline compounds deeper into the sample. Therefore, a destruction and- disintegration of the specimens caused by formation of corrosion products and different- thermal expansion coefficientsof the initial material and zone saturated with the melt in the- castable with ground quartz sand additive appeared considerably later.-


**Table 6.** The phase composition of fireclay and clinker refractory concretes before and after test with K2CO3 upon- applying the tablet method and firing at the temperature of 1100°C [26].-

The above test results show that often traditional fireclaymaterials used in biofuel boilers are- not resistant to the effectsof alkaline compounds. Refractory materials recommended for- biofuel boiler lining should be examined in laboratories to evaluate the alkaline salt penetration- into the material.-

### **3.1. Explosive spalling of refractory castable-**

Calcium aluminate cement-based refractory castable should be dried and heated up after- curing for moisture removal. In the process of heating, the temperature is gradually raised- until the operational temperature (1000–1200°C) of the boiler is achieved. During heating up- of the castable, chemical and physical processes causing the removal of chemically bound- water and formation of new crystalline phases take place. All these processes also cause great- changes in the micro-structure of a castable and pose a threat of its explosive spalling [27, 28].- In **Figure 12**, part of the structure of the heating unit used in oil refinery,damaged by explosive- spalling, is shown.-

**Figure 12.** The part of the combustion zone structure in the heating unit used in oil refineriesdamaged by explosive- spalling (metal anchors can be seen on the photograph) [29].-

Explosive spalling is usually caused by water vapour pressure, which builds up when- chemically bound water is turned into free water. The risk of explosion of the structure is- greatly increased, if the following types of castable are used: MCC, LCC and ULCC. To avoid- explosive spalling of refractory castable due to the pressure of water vapours developed at the- initial stage of heating, new produced linings of thermal equipment are dried and heated up- for the firsttime in a very careful way [29]. But in the case of biofuel boilers, in practice, it is- hardly technically possible to perform the procedure of castable drying accurately. Therefore,- in order to reduce a risk of explosive spalling, when castable drying and the initial heating- modes are not rigorously controlled, various additives (e.g. aluminium powder, polymer fiber,- etc.), which increase castable permeability by forming a capillary system for removing water- vapour without damaging the castable, are used. Aluminium powder reacts with water in the- alkaline medium, releasing hydrogen, which causes the formation of open porosity in castable- and makes it more easily permeable to water vapours. However, though the addition of- aluminium powder increases castable permeability to water vapours, a loose structure is- formed; therefore, the mechanical properties of the castable is decreased.-

It has been found that the additive of polypropylene fibres-(PPF) (**Figure 13a**) is well suited- for decreasing the risk of explosive spalling of refractory castables [29]. A positive effectof this- additive, with regard to its ability to decrease the risk of explosive spalling, is explained by the- fact that PPF disintegrates at the temperature of 150–180°C, leading to the formation of microchannels (**Figure 13b**), allowing water vapours to pass through, and help to avoid a dangerous- rise of pressure.-

The testing of cylindrical MCC-type castable specimens, for their resistance to explosive- spalling [29], has shown that the MCC-type specimen without of PPF additive explode at the- temperature of 600°C under the conditions when temperature is raised to 1000°C at a rate of- 40°C/min (**Figure 14a**). The specimen with PPF additive does not explode when the temperature is raised at the same range in heating up to 1000°C (**Figure 14b**).-

**Figure 13.** SEM micrographs of the PPF (a) and the micro-structure of refractory material with burned PPF after heating at 170°C (b) [29].-

**Figure 14.** Castable specimens tested for explosive spalling, when the temperature was raised at the rate of 40°C/min:- (a) castable sample without PPF additive exploded, when the temperature was raised to 600°C and (b) castable sample- with PPF additive that did not explode, when the temperature was raised to 1000°C.-

In order to simplify the drying and the firstheating procedure, and to reduce the risk of- explosive spalling, expensive NCC-type castables [30] are used. Such castables considerably- reduce the time of drying and the first heating procedure.-

#### **3.2. Recommendations for use of refractory materials in biofuel boilers-**

Due to the aggressive operating conditions in biofuel incineration plants, manufacturers of- refractory materials use the following specificshaped and unshaped materials: fireclaywith- a small amount of iron oxide, silicon carbide (SiC), mullite, zirconia, andalusite and chrome- (**Table 7**) [11]. Fireclay, mullite and andalusite materials belong to the Al2O3–SiO2-(aluminosilicate) system.-


CO resistance ASTM C 288 always A, not relevant for alumina-chrome materials.-

**Table 7.** Lining recommendations for biomass combustion furnaces [11].-

It is stated [11] that refractory materials, suitable for use in biofuel boilers installations, should- be dense (>2200 kg/m3 ), with CCS of at least 50 MPa, thermal shock resistance >30 cycles (under- DIN 51068-1:1976 standard) and iron oxide content less than 1%.-

However, it should be noted that the recommended high-grade materials with zirconium and- chromium fillersare considerably more expensive than with fireclayand andalusite fillers.- These materials are very dense (≥3000 kg/m3 ), their heat transfer coefficientis high and reaches- up to 1.6–2.5 W/(m K); therefore, the lining increases the need for insulating materials. After- the lining operation time, refractory materials containing chromium oxide must be disposed- of in hazardous waste landfills because their processing is complicated.-

 Inpractice,uptillnowthemostwidelyusedmaterialsinsolidbiofuelcombustionplant- liningsarebricksandcastableswithaluminosilicatefillerssuchasfireclayandandalusite.- Selectionofthistypeofmaterialforbiofuelboilerliningsmustbelongtothealkaliresistanceclassofmaterialsandtheirgeneralcharacteristicsshouldbenolessthanthatspecified- in-**Table-7**.-In-**Figure-15(a)**,aviewofliningmadeofnon-alkaliresistancefireclaybricks,- damagedbyalkaliattackafter-6monthsofboileroperation,isshown,andin-**Figure-15(b)**,- aviewofnon-damagedliningmadeofalkaliresistancefireclaybricksafter-8monthsof- boileroperationisshown.-Themainreasonofthedifferencebetweenresistanceofthese- liningswasthedifferentalkaliresistanceclassofthematerialsusedfortheirproduction.-

**Figure 15.** View of lining made of not alkali resistance fireclay bricks after 6 months of boiler operation (a) and made- of alkali resistance fire clay bricks after 8 months of boiler operation (b).-

It is also necessary to note that not only quality, but also suitability for high durability lining- of used materials is of great importance. The correct installation of lining is very important as- well. Especially in the case of installation of monolithic lining such key quality control elements- such as installation monitoring, as-installed testing, pre-dryout inspection, dryout monitoring- and post-dryout inspection are necessary. Some standards [31, 32] can be useful for organization of quality control for the installation of biofuel boilers lining.-

### **Author details-**

Valentin Antonovič1\*, Jacek Szczerba2 , Jadvyga Keriene1 , Rimvydas Stonys1 and-Renata Boris1-


#### **References-**


**Chapter 23** 

### **Power Form Agripellets**

Claudia Santibáñez Varnero and Marcela Vargas Urrutia

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/110404

#### **Abstract-**

 Currently,theproductionofthermalenergybybiomasshasshownacleartrendtoward- densifiedbiofuels-(pellets).-Thisisduetotheirconsistentsizeandshapethatcanbe- moreeasilydeliveredtohomes,businesses,andpowerplantsandcanbeautomatically- fedintoadvancedpelletboilersinacontrolledandcalibratedway.-Theuseofdensified- biofuelsalsoreducesthecostsassociatedwithhandlingandtransportation,duetothe- increaseindensityinvolvedbydensificationprocess.-Demandforwoodpelletsis- currentlygrowingatafasterratethansupplyin-Europe.-Itisestimatedthatpellet- marketisgrowingto-50-Mtyear−1by-2025;however,mostwoodwasteisalready- committedforpressedwoodproductsandpellets,thereforemoresupplyofraw- materialsareneeded.-Withthepossibleshortageofwoodyrawmaterialsforpellet- productionandconsideringthelowforestryresiduespotentialinseveralcountries,- agriculturalresiduescouldbelargelyusedinthefutureforfuelpelletsmanufacturing.- Agriculturalpellets,aswellknownas-"agripellets",areemergingandpromising.- However,theyhavecertaindifferencescomparedtoconventionalwoodpellets.-

**Keywords:** agripellets, biomass, solid biofuels, agricultural residues-

#### **1. Introduction-**

 Thegrowingdomesticandindustrialdemandofbiomassforheatandpowerproductionin- Canada,-United-States,-Europe,and-Chinahasresultedinastronggrowingglobalpellet- markedduringthelastdecades,andcontinuousgrowthofthemarketispredictedforthenext- years-[1].-Itisestimatedthatthedemandforpelletswillbetriplesfrom-2012to-2020,rising- from-16to-46millionmetrictonsperyear-[2].-Inthepelletproduction,thereisashortageof- woodyrawmaterials,andthepriceofthewoodrawmaterialincreases.-Consideringthatonly-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 woodypelletsfromforestryresidueshavealreadysuccessfullyestablishedtechnologiesand- markets for production and consumption in these countries, it is necessary to focus on studying- thepelletizationofnewsourcesofrawmaterials.-Agriculturalresiduescanbeoneofthe- potentialalternativefeedstocksinceitisabundantlyavailableandatlowcost.-Inthenear- future,agriculturalresidueshaveatremendouspotentialinbiomasspelletsindustry.-Itis- thereforeofgreatinteresttostudythecharacteristicsofthisnewcategoryofrawmaterial,- payingspecialattentiontotheproblemsthattheymaytriggerbothatproductionand- utilizationlevel.-Atatechnicallevel,themaindifferencebetweenwoodpelletsandagripellets- isthesomehowhigherfriability,theslightlylowerenergycontentandthehigherashcontent- ofthelatter.-

The future of agripellets as a carrier of energy from biomass appears to be promising. This is- influencedby many factors. Popularity of biomass is motivated by aspects of various types,- such as:-


#### **2. Agricultural residues for energy purposes: agripellets-**

Nowadays, fuel pellets are mainly made from sawdust, wood chips, and wood shavings. The- supply of wood materials can be limited because producers of fiberboard, particleboard, and- oriented strand board compete for the same forestry and mill residues as pellet producers [3].- This competition and the current increased demand for wood pellets, both for residential and- industrial use, have led to a shortage of sawdust and wood shavings. If demand and prices- continue to rise, other biomass wastes than sawdust, wood chips, and wood shavings should- be considered for pellet production. Agricultural residues are among those future new raw- materials. Agricultural residues refer all the organic materials which are produced as by‐ products from harvesting and processing of agricultural crops. These residues can be further- categorized into primary residues and secondary residues. Primary or field‐basedresidues are- those generated in the fieldat the time of harvest (e.g., straw, stalks, and leaves that are left- over after harvest), whereas those coproduced during processing are called secondary or- processing‐based residues (e.g., sugar beet pulps, cotton mill wastes, peanut shells).-

Availability of primary residues for energy application is usually low since collection is- difficult,and they have other uses as fertilizer, soil conservation, animal feeding, and litter. The- amount of secondary residues varies widely depending on the crop and processing methods- used [4].-

Currently, large amounts of agricultural residues are left in the fieldto rot or are burned in the- open air, ultimately releasing carbon dioxide to the atmosphere. This biomass could be used- to produce pellets which are a form of solid fuel. The most important reason for using- agricultural residues for energy purposes is that it is carbon neutral, that is, the carbon emitted- during their combustion is taken up in the regrowth of the biomass used to produce them and- therefore does not add to greenhouse gas emissions. Further, any consumption of fossil fuels- replaced by biomass will lower CO2 emissions.-

Agricultural residues are available in large quantities and can be utilized for sustainable heat- and power production, when used as fuel. However, they have low energy density (MJ m−3)- and low yield per unit area (dry tons ha−1) [5]. Often, long distances have to be bridged between- the biomass place of origin and the place of its utilization, resulting in expensive handling and- transportation. Transportation costs of low‐density and high‐moisture agricultural residues- which increase the total biomass‐processing cost are a major constraint to their use as an energy- source [6]. To increase the density of the biomass, it can be compressed into pellets using a- mechanical process in which pressure is applied to the biomass to crush its cellular structure,- and thereby increasing its density. Densifiedbiomass, especially pellets, has drawn attention- due to its superiority over raw biomass in terms of its physical and combustion characteristics- [7]. Many materials originated by agriculture could be used for the production of densified- biomass fuels: straw, grain hull waste, tree pruning, fruit stones, dry fruit waste, grain, cork,- cotton, and other wastes.-

The main characteristics of some selected agripellets are summarized in **Table 1**. The higher- heating value (HHV) of agripellets is high, being even higher than pine sawdust pellets (in the- case of pellets made of olive pomace and tomato peels and seed). The ash content confirmsthe- necessity of blending agricultural residues with sawdust or other woody material. In fact, the- ash contents for agripellets (3.3–12%) are significantlyhigher comparing to pine sawdust- (2.5%).-

According to Colley [14], pellets durability is regarded as high if exceeding 80%, medium if- measured as 70–80%, and low if values do not reach 70%. Therefore, agripellets show high- quality in terms of durability.-

#### **2.1. Olive mill residues-**

The olive oil industry produces significantquantities of solid olive residues (pieces of skin,- pulp, and stones) which due to their characteristics can be utilized for the production of cleaner- energy [15]. According to Barbanera et al. [16], it can be assumed that 1 ha of olive tree produces- about 2500 kg of olives and about 35 kg of olive pomace. Several studies have been conducted- during the last two decades that examined the thermochemical characteristics and perform‐ ance of solid olive residues [17–21]. The results obtained from these studies suggest that these-


solid residues constitute a promising biomass resource because their thermochemical charac‐ teristics provide the opportunity for their potential utilization for energy purposes, offering at- the same time a solution to the management problems [20].-

**Table 1.** Characteristics of some agripellets.-

The solid fraction combustion of olive residues indicates good combustion behavior of olive- kernels and the residual olive pomace, with suitable efficiencyand a reduced presence of- unburned fraction. However, lower combustion efficienciesare observed during pulp proc‐ essing [10]. The olive pomace, once it has been subjected to a drying process, can be used as a- fuel. However, their oleaginous characteristics limit the densificationduring the pelletizing- process. Moreover, their high concentrations of certain components such as nitrogen and ashes- exceed the specificationsgiven by pellets quality standards [20]. Therefore, it is necessary to- blend the olive oil by‐products with other biomass residues that must present suitable- characteristics for an ideal pelletization [22]. Barbarena et al. [16] reported that adding olive- tree pruning to olive pomace, the chemical composition of pellet blends respects the standard- requirements in terms of mechanical durability and N and Cu content. In addition, the bulk- density was enhanced allowing a reduction of transport and storage economic cost. On the- other hand, Brlek et al. [23] suggest that limitations regarding combustion of olive pomace- pellets can be established due to elevated nitrogen content and higher percent of abrasion.- These constrains can possibly be diminished by adding wood biomass to pelletization blend.-

### **2.2. Vine residues-**

The wine industry produces huge amounts of residues every year. Marculescu and Ciuta [24]- estimate that for every kilogram of grape processed for wine, more than 20% is residue. The- current use of the wood residues produced by the annual pruning activity is generally- eliminated through crushing in the vineyard and then spread along the soil, in order to reduce- erosion and recycle nutrients that can be incorporated into the soil [25].-

Another important residue generated in the production of wine is grape marc. This residue is- the skins and pips that remain after the grapes have been crushed. These residues are highly- wet (more than 60% of moisture content on wet basis) and have low pH. Also, they present- high contents of phosphorous (P), potassium (K), organic matter,phytotoxic, and antibacterial- phenolic substances, which make them resistant to biological degradation. These wastes have- high contents of lignin and tannin. Hence, they are not appropriate as a nutritional supplement- for animals [26]. In addition, due to their high C/N ratio, their recovery as soil fertilizer presents- difficulties.-Another important constraint related to the management and disposal of wineries- and distilleries processing industries is the generation of large quantities in which discharges- are usually centralized and seasonal in a short period of the year (3–4 months).-

Fernandéz‐Puratich et al. [8] studied the use of vine wastes for the production of pellets and- concluded that the use of these biomass residues is a viable alternative option in terms of- economy as well as energy.-

Marculescu and Ciuta [24] studied the thermal degradation of grape marc in a laboratory- furnace. They found that grape marc has high energy content (19.7 kJ kg−1) and they have- recommended for energy production.-

Kraiem et al. [12] recommend blending these residues with pine sawdust. They found that a- blend with sawdust leads to the decrease of ash contents while densificationleads to the- increase of the energy densities. Combustion tests of pellets prepared from these residues- indicate that boiler and combustion efficienciesare comparable to wood pellets. However,- gaseous and particulate emissions are higher and are strongly affectedby the operating- parameters of the domestic boilers.-

#### **2.3. Industrial tomato residues-**

The industrial processing of tomato leads to a great variety of output products. Large volumes- of residual biomass (mainly peels and seeds) are generated by tomato industrial processing- plants. Currently, industrial tomato residues do not generate so many benefits for industries,- in particular for storage and preservation issues. The accumulation of these residues, predom‐ inantly in the warm periods, promotes uncontrolled anaerobic fermentations leading to- environmental problems [27]. In this way, fast consumption is advised in order to prevent- fermentation processes, which are favored by high temperatures during the industrial- processing period. Those residues have a high moisture content, which leads to some storage- difficulties,and are generated in large quantities in which discharges are usually centralized- and seasonal in a short period of the year. Also, important costs are derived from transport of- wastes with significantmoisture content, thus impeding their reasonable use. To avoid added- costs related to disposal process, tomato manufacturing companies often give their production- residues for free to other companies that generally use them for feeding livestock [11] or as- soil amendment [28]. However, Rossini et al. [29] found that tomato waste could be suitable- for combustion, but the relatively higher nitrogen content can generate environmental- problems in terms of NOXemissions. In addition, the high chlorine and sulfur contents may- lead to the corrosion of the combustion systems. Therefore, as a solution, the authors proposed- to separate tomato waste into peels for combustion and seeds for vegetable oil production.- González et al. [30] studied the tomato waste combustion in a mural boiler. They found that- tomato residues give higher boiler efficiency than other biomasses (forest residues, sorghum,- almond pruning, and reed). Ruiz‐Celma et al. [11] studied tomato seeds and peels pellets. They- reported a high heating value of these pellets and an energy density (approaching 8 GJ/m3 )- similar to that of other biomass pellets, regardless their low bulk density values.-

#### **3. Techniques for biomass densification-**

Biomass is densifiedvia two main processes: pelletizing (mechanical densification)and- torrefaction. Pelletizing involves applying pressure to mechanically densify the material, while- torrefaction involves heating the biomass in the absence of oxygen.-

#### **3.1. Pelletizing-**

The low density of agricultural residues poses a challenge for the handling, transportation,- storage, and combustion processes. Those problems are mainly related to the high bulk- volume, which results in high transportation costs and demands for large storage capacities,- and to the high moisture content which results in freezing and blocking the in‐plant transpor‐ tation systems, as well as in biological degradation. In addition, variations in moisture content- make difficultoptimal plant operation and process control. All these problems may be- addressed through densification,a process that produces solid fuel with denser and more- uniform properties than the raw biomass [31].-

The main advantages of densified compared to non‐densified fuels are the following:-


All these factors make pellets one of the more attractive forms of biomass‐based energy. The- major disadvantage to biomass densificationtechnologies is the relative high energy cost for- the pelleting process, increasing the price of the end product. In addition, it is important to- have in mind that agricultural residues are highly dispersed and may be over long distancesfrom the pelleting facilities. An appropriate solution could be to carry the pelletizing mill to- the raw material. A few mobile pelletizing mills already exist. Such a pelletizing mill can meet- a specificdemand, for instance several farmers wanting to share the investment cost of a- pelleting equipment which after can be moved from a place to another [32].-

The process of pellet manufacturing was firstdeveloped for the livestock feed industry. The- process consists of a few basic sub‐processes: comminuting of the raw material, drying,- pelletizing, and cooling. The raw material is firstcleaned of contaminant such as rocks, metals- and other foreign material, and then grinded in a hammer mill or a chipping machine. The- particle size is adjusted to a uniform maximum dimension and should have proper size and- be consistent. The moisture content in the raw material can be considerably high and are- usually up to 50–60% which should be reduced. Rotary drum dryer is the most common- equipment used for this purpose, where the moisture content of the uniformly dimensioned- particles is reduced to about 10–15% (w.b.). Drying increases the efficiency of biomass, and it- produces almost no smoke on combustion. The feedstock should not be over dried, as a small- amount of moisture helps in binding the biomass particles. The drying process is the most- energy intensive process and accounts for about 70% of the total energy used in the pelletiza‐ tion process. Thereafter, raw material can be conditioned according to legal specifications-(i.e.,- steam or organic binding agents can be added). The particles are then moved by conveyor to- a pellet mill, where the pellets are compressed against a heated metal plate (known as die)- using a roller. Due to the high pressure, frictional forces increase, leading to a considerable rise- in temperature (90–100°C), and are immediately air quenched down to 25°C. High temperature- causes the lignin, and resins present in biomass to soften which acts as a binding agent between- the biomass fibers. This sets up the lignin and hardens the product, and contributes to maintain- its quality during storage and handling. On the outer side of the latter, a knife cut offthe pellets- at the desired length. Residual moisture in the feedstock turns to steam during compression- and helps to lubricate the compression die [33].-

Finally, the pellets are packed into bags using an overhead hopper and a conveyor belt. Pellets- are then ready for storage (in a silo) or for automatic packing (in 25 kg bags or big bags—1 to- 1.5 m3 ). Commercial pellet mills and other pelletizing equipment are widely available world‐ wide.-

### **3.2. Torrefaction-**

Torrefaction is a very promising technology for improving the fuel properties of solid biomass- (e.g. pellets). This technology is a version of slow pyrolysis processes that comprise the heating- of biomass in the absence of oxygen and air [30, 34] in which the goal is to dry, embrittle, and- waterproof the biomass. This is accomplished by heating the biomass in an inert environment- at temperatures of 200–320°C. During the treatment, biomass starts to decompose and releases- combustible volatile matter, mainly composed by organic compounds, together with moisture.- Biomass loses most of the low‐energy content material in the form of gaseous and condensable- liquids. Common events that occur during torrefaction include drying, depolymerization and- recondensation, limited devolatilization and carbonization, and extensive devolatilization and- carbonization [35]. Several studies have been conducted to evaluate a combined torrefaction‐

pelletization process possible in a commercial scale. Reed and Bryant [36] first considered the- combination of torrefaction and pelletization to produce a new type of high energy density- and water‐resistant pellets. Bergman [37] proposed and demonstrated a combined torrefaction- and pelletization process for the production of high energy density wood pellets. The addition- of pelletization to torrefaction would potentially create a bio‐based fuel with similar energy- density to coal, prompting the adoption of this product for replacing coal in heat and power- facilities. Currently, a number of torrefaction pilot plants have been designed, under construc‐ tion, or publicly announced [38]. Carapeda [35] reports that if the biomass is torrefied before- being densified,the energy consumption during the pelleting process is reduced by a factor- of 2 and the throughput is increased, also by a factor of 2.-

The main advantages of torrefaction of raw biomass feedstock include:-


The main disadvantage of torrefaction of raw biomass feedstock includes additional cost,- energy, and equipment required for processing. In addition, ash content is not removed so ash- content would likely increase per unit of weight [35]. Therefore, torrefaction can be considered- as one of the major pretreatment technologies for improving the properties of agricultural- residues, in order to deal with such problems as high bulk volume, high moisture content, and- poor grindability.-

### **4. Combustion of agripellets-**

Physical and chemical properties vary significantly within and between the different agricul‐ tural raw materials. Depending on the type of application, these variations may be critical and- may affectthe performance of the system. Physical properties, such as bulk density, moisture- content, particle size and distribution, and durability, are important for the choice of processes- and equipment. On the other hand, chemical properties are of great importance for the energy- efficiency, environmental pollution, and ash‐related operating problems.-

Agripellets combustion triggers several major obstacles regarding emissions (gas, dust, and- aerosols), deposit formation (slagging, fouling), and corrosion. Another problem is that theash content of agripellets is higher than wood pellets (about 2–10 times higher than that of- wood pellets). All those problems not only depend on the fuel characteristics but also on the- design of the combustion equipment and the way it is operated. Recently, Kraiem et al. [12]- reported that silicon (Si), potassium (K), calcium (Ca), magnesium (Mg), phosphorous (P), and- aluminum (Al) are the major elements of agripellets. Compared to wood pellets, a typical- feature of agripellets is their higher content in nitrogen (N), sulfur (S), chlorine (Cl), and K,- increased by the use of fertilizers and pesticides/herbicides in agriculture. The presence of- those elements leads to relatively important emissions of NOx, SOx, and HCl compared to- wood pellets. In addition, K influencesboth particulates emission and slagging (by lowering- the softening temperature of the fuel) of an increased ash volume. Besides, a high Cl content- results both in corrosion problem on the surfaces of the boiler and in formation of dioxins and- furans. Finally, for a large‐scale use, in relation with the high ash content and the low melting- point, it has been stated that straw pellets could present betterresults with grate combustion- or fluidized bed systems. Those problems can be overcome by the use of multi‐fuel boilers in- the range of 10–60 kW which is more suitable for burning agripellets; co‐firing of agricultural- residues with fossil fuel; cleaning the agricultural residues before pelletizing them into- agripellets to make them with less ash content; and to add in specificanti‐slagging agents (e.g.,- kaolin) or mix in some sawdust to change the fuel characteristic.-

Environmental and technical features of combustion technologies indicate that pellets made- from agricultural residues should be used primarily in large‐scale combustion plants equipped- with sophisticated combustion control systems and fluegas cleaning systems, whereas wood- pellets should be preferred for small‐scale heating systems. In the future, the main technical- challenges regarding agripellets are the production of a high‐quality fuel, and technological- improvement for small‐scale combustion devices.-

### **4.1. Emissions-**

During combustion, N, S, and Cl in the fuel (present in higher proportion in agripellets than- in wood pellets) may lead to atmospheric pollutants such as nitrogen oxides (NOx), sulfur- dioxide (SO2), hydrogen chlorine (HCl), and chlorinated hydrocarbons. Moreover, Cl favors- the formation of dioxins and furans. The incomplete combustion of agripellets is mainly the- result of low combustion temperatures, short residence times, oxygen shortage, or combina‐ tions of these effects.-Incomplete combustion results in emissions of carbon monoxide (CO)- and volatile organic compounds (VOC), particles, tar, and polycyclic aromatic hydrocarbons- (PAH). Zeng et al. [39] demonstrated that the emission of NOx, SO2, HCl, and total particulate- mattercan be reduced by blending agricultural raw materials with woody biomass though- substantial reduction potential was only observed for blends with at least 50 wt% wood.-

 In addition, ashes formed during agripellets combustion can generally be divided into bottom- ashes, coarse flyashes, and aerosols (fineflyash). These fractions differsignificantly concern‐ ing their particle size and chemical composition as well as their formation mechanisms.-

The bottomash is the ash fraction remaining in the furnace after combustion of the fuel and is- then removed by the de‐ashing system. Coarse flyashes are particles entrained from the fuel- bed with the fluegas. They mainly consist of refractory species (such as Ca, Mg, Si as well assmall amounts of K, Na, and Al), and their particle sizes can vary between some µm and 100- µm. Particles that are small enough to follow the flue gas on its way through the furnace and- the boiler finally form the coarse flyash emission at the boiler outlet. Aerosols are formed by- gas‐to‐particle conversion processes in the furnace and in the boiler. Some of the aerosol- particles coagulate with coarse fly ashes due to collisions [33]. During combustion of agripel‐ lets, part of the volatile compounds is released from the fuel to the gas phase: aerosols are then- formed by condensation or nucleation of these volatiles compounds. Aerosols are much- smaller than coarse flyash (typical particle size significantly <1 µm). Aerosol emissions present- high concentrations of heavy metals and sometimes of organic compounds. By their dimen‐ sion, they can remain suspended in the air for long period of time and enter into the inner parts- of lungs. In small‐scale pellet furnaces and boilers, the main ash fraction is bottomash. Furnace- and boiler ash form the major share of coarse flyash, which is usually precipitated and mixed- with the bottom ash. A small amount of course fly ash is emitted with the flue gas [33].-

### **4.2. Deposit formation-**

Biomass boiler issues regarding slagging, fouling, and corrosion are related to alkali species- present in agricultural residues. These alkali species are released as gaseous alkali chlorides,- hydroxides, and/or sulfates during combustion. Alkali chlorides/sulfates later condense on- cold boiler surfaces enhancing fouling and corrosion. This is referred to as slagging when the- deposits are in a molten or highly viscous state, or fouling when the deposits are built up- largely by species that have vaporized and then condensed. Slagging is often found in the- radiant section of the furnace, while fouling occurs in the cooler furnace regions where the- heat exchanger equipment is located [40]. The negative effectsof slagging and fouling are high- furnace material wear, heat transfer efficiencyreduction with pressure drop, and increased- corrosion of the boiler.-

Potassium and sodium compounds are present in all agricultural residues. During combustion,- these alkali compounds combine with silica and causes slagging and fouling problems in- conventional combustion equipment designed for burning wood at higher temperatures.- Volatile alkali also lowers the fusion temperature of ash; combustion of agricultural residue- causes slagging and deposits on heat transfer surfaces. In order to overcome this problem,- special boilers have been designed with lower furnace exit temperatures or low operation- temperature. These designs can reduce slagging and fouling from combustion of agripellets.-

Hence, this underlines the necessity of a careful treatment of raw materials so as to avoid- mineral contamination. Deposit formation related problems affectingagripellets deserve a- special attentionbecause they lead to reduced accessibility of the appliances, and also to bad- publicity for the agripellet market.-

### **4.3. Corrosion-**

The presence of even a small concentration of Cl in fuel will result in the formation of alkaline- chloride compounds on boiler surfaces. Chlorine can influencethe corrosion of superheater- tubes in many ways. Gases containing Cl2, HCl, NaCl, and KCl may cause a direct corrosionby accelerating the oxidation of the metal alloys. Such gases may also influence the corrosion- caused by other mechanisms, such as molten alkali sulfate corrosion of superheater alloys and- sulfidation of water walls. In addition, Cl may also deposit on superheater tubes and thereby- influenceits corrosion [41]. Chlorine corrosion could be prevented by co-firingaluminum- silicates containing fuel, such as coal or peat. When those fuels are co-firedwith agricultural- residues, chloride formation can be avoided. In addition, a parameter that has been often- referred to is the sulfur-to-chlorine atomic ratio (S/Cl) in fuel or fuel blend. It has been- suggested that if this ratio in fuel is less than two, there is a high risk for superheater corrosion.- When the ratio is at least four, the blend could be regarded as noncorrosive. However, the best- way to prevent the molten phase corrosion is to keep superheater metal temperature below- the firstmelting temperature of deposits, in practice below 500°C when firingagripellets [32].-

Several fieldstudies have shown that the main contributor to superheater corrosion in boilers- is Cl, in particular alkali chlorides (NaCl, KCl). The relatively low sulfur content in most- agricultural residues may introduce corrosion problems in the superheaters.-

#### **4.4. Ash recycling for agricultural applications-**

The rapidly growing number of pellet heating installations illustrates an increased interest in- environmentally friendly heating systems. The problems associated with the use of agripellets- are essentially linked to the ash management. Thus, the recycling or storage of agripellets ash- deserves a special attention.-

Ash is the inorganic uncombustible part of fuel left after complete combustion and contains- the bulk of the mineral fraction of the original biomass [42]. In wood pellets, ash represents- less than 2%, while in agripellets, it can be 5–10% and up to 30–40% in rice husks and milfoil- [43].-

The firstand direct consequence for small scale stoves and boilers of the increased ash residue- with agripellets is that there the ash storage under the furnace will have to be emptied more- frequently, which is quite negative as far as the convenience of users is concerned. Considering- that the ash storage should normally be emptied once every 5–15 days with wood pellets- depending on the consumption, agripellets would oblige to remove ashes more frequently- (almost daily). On the other hand, James et al. [43] suggest that inefficienciesin boilers and- furnaces result in high percentages of unburned organic matterin ash. This carbon content- may be recycled to the boiler or furnace to improve energy output and increase the process- efficiency.-

Part of the ash is taken out in the bottomof the boiler and is called bottomash while the- remainder is composed of the fineparticles that are driven out of the boiler with the fluegases.- This part of the ash is called flyash. Each ash fraction has different composition. Filter fly ash- tends to accumulate the largest part of heavy metals. Ashes from agripellets are produced in- higher quantities, but the content in heavy metals for each fraction seems to be lower.-

 The collected bottomash and flyash from the combustion of agripellets should be disposed- of in a safe way. These ashes contain nutrients, primarily potassium, and other soil-fertilizing- elements like magnesium, phosphorus, and calcium and can therefore be applied in agricultureas fertilizer. It seems that ash is strongly alkaline (pH of 11–12) and could cause sharp increase- of pH and ion concentration in the soil after spreading [32]. Thus, ash should not be used unless- a soil pH test has been done. Such a phenomenon would be harmful with respect to plant- growth. Consequently, ash could be treated (e.g., granulated) in some way to reduce impact- on soil. In regard to acidic soil correction, agripellets ashes as a garden amendment are a much- more convenient means than the traditionally used ground limestone, bearing in mind that it- is an absolutely costless resource.-

In order to utilize the nutrients from the flyash, a utility owned plant has developed a method- for washing them leaving heavy metals behind in a fraction to be stored. The product is a- valuable fertilizer, and the process could be carried out centrally using flyash from both utility- owned plants and district heating plants.-

According to Gomez‐Barea et al. [44], the utilization of ash has also seen its application in the- construction industry. Fly ash can be used as a cement replacement in concrete, for soil- stabilization, as a road base, structural fillerin asphalt and asphalt base products, lightweight- bricks and synthetic aggregate.-

#### **5. Pellets quality standards-**

The combustion of densifiedbiomass fuels in fully automatic heating systems for residential- sector and small‐scale furnaces requires high fuel quality. However, high quality is not- necessary if these fuels are used in larger industrial furnaces because they are equipped with- more sophisticated fluegas cleaning, combustion, and process control systems. Pellets are a- standardized fuel, which simplifiesconstruction and operation of burners. For pellets,- producers are very important to have quality standardization because it increases the customer- confidence.-However, the quality of the pellets should be definedin terms of the heating- technology, since differentheating systems require differentfuel qualities. For example, large- heating plants are not demanding in terms of pellets durability or amount of fines.-In contrast,- pellets stoves require an extremely durable pellet, which does not produce too much dust in- the storage bunker, and do not cause technical problems in the feeding and combustion unit.- From this perspective, the differentquality of agripellets would suggest a differentuse,- preferentially in large‐scale systems [45].-

In the present, the development of quality standards for wood pellets is set on fivedifferent- levels in Europe: (1) European Commission and European Committeefor Standardization; (2)- EU member state governments; (3) European Biomass Association and European Pellet- Council (which represent the European biomass sector); (4) Wood Pellet Buyers Initiative- (which represents end users of biomass); and (5) standards developed by individual private- companies [46]. Standards exist on national (e.g., DIN), European (EN), and on international- level (ISO).-

Usually, standards developed by standards organizations are voluntary but can become- mandatory if adopted by a government or business contract.-

According to European Standard ENplus which is related to wood pellets for nonindustrial- use and which will gradually supersede all national standards (e.g., ÖNORM M1735, or- DINplus), wood pellets can be classifiedinto three basic categories: ENplus A1, ENplus A2,- and ENplus B. The quality requirements of ENplus are based on an international standard:- ISO 17225‐2. This international standard has replaced the European Standard EN 14961‐2. The- ENplus certificaterequires stricter quality criteria. This quality seal stands for low emissions- and trouble‐free heating with high energy value.-

Class A1 includes wood pellets originating from stem wood, without chemical additives and- with low ash and Cl content. Class A2 considers wood pellets with slightly higher ash and/or- Cl content. Wood pellets derived from reused wood, residues, or bark are included into class- B. **Table 2**shows the specificationsof the three wood pellet classes according to ENplus in- comparison with German (DIN‐plus) and Austrian (ÖNORM M7135) standards.-


**Table 2.** Comparison of regulations related to pellet quality.-

Generally, limit values for ash content, moisture, net calorificvalue, and chlorine are fairly- similar. All standards prohibit the use of binding agents. Austrian and German standards do- not mention the amount of fines,while in Enplus, finesmust not be more than 0.5–1.0%.- German and Austrian standards do not definedurability or mechanical stability despite the- importance of these attributes.-This is because during transport in tankers and the pneumatic- fillingof storage bunkers, mechanical strain on pellets is high. In addition, pellets with poor- mechanical stability produce large amounts of dust. Hence, small heating systems require very- high pellet quality. In these systems, the amount of fines in fuel pellets is of special importance.- In contrast, combustion units in large heating systems are not affectedby the amount of fines.- The differentrequirements of small and large combustion systems make necessary definition- of different groups of standards [32].-

### **6. Conclusions-**

The increasing competition for solid biomass, such as wood pellets, will create space for- relatively novel biomass sources to enter the market, among which agricultural residues have- the greatest potential. In comparison with wood, agricultural residues present high ash, N, K,- and Cl content. The underlying problems are higher related emissions, deposit formation and- corrosion. Many techniques are currently used, while others are under improvement stage to- overcome the inherent drawbacks of agripellets composition. These techniques include- agricultural practices, fuel preparation, combustion technologies, fluegas cleaning systems,- and the possibility of co‐combustion of agripellets with solid fossil fuels. ENplus quality- certificationis a major step toward establishing biomass pellets as a widely used energy source.- However, the high nitrogen and ash contents strongly limit the certificationof pure agripellets.- In this regard, several studies have shown that blending agricultural residues with sawdust- before pelleting could help to meet ENplus certifications.-

Consequently, the use of agripellets in the residential heating sector cannot be recommended- at present, because small‐scale pellet furnaces are not specially designed for this kind of fuel.- Therefore, for small‐scale heating systems, which require high‐quality fuels, the use of wood- pellets is recommended. Agripellets should be used primarily in large‐scale combustion plants- equipped with sophisticated combustion control systems and flue gas cleaning systems.-

#### **Author details-**

Claudia Santibáñez Varnero\* and Marcela Vargas Urrutia-

 \*Addressallcorrespondenceto:claudia.santibanez@umayor.cl-

 Departmentof-Agronomy,-Facultyof-Sciences,-Univesidad-Mayor,-Santiago,-Chile-

### **References-**


## **SWOT Analysis Applied to Wheat Straw Utilization as a Biofuel in Mexico**

Gisela Montero, Conrado García, Marcos A. Coronado, Lydia Toscano, Margarita Stoytcheva, Ricardo Torres, Ana M. Vázquez and Daniela G. Montes

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65496

#### **Abstract-**

 Wheatisoneofthemaincropsworldwidewithaproductionof-733millionoftonsby- 2015.-By-2013,thewheatgrainproductionin-Mexicowas-3,357,307t.-Wheatstrawis- generatedasabiomasswasteoncethewheatisharvested.-However,theagricultural- biomasswastehasacquiredinternationalrelevanceasasourceofbioenergy.-The- utilizationofbioenergyhassignificantenvironmentalbenefits,andalsoeconomic- benefitsbecausethebiomasswasteisvalorizedasbiofuel.-Theuseofwheatstrawas- rawmaterialforanyproductiveprocesspresentsdiversefactorsthatmustbeconsid‐ ered.-Amongthosefactorsarethelowdensityofbiomass,handlingandhightranspor‐ tationcost,anattractiveheatingvalue,andthephysicochemicalcharacterization.- Therefore,theaimofthisworkwastoapplythe-SWOTanalysistowheatstraw- utilizationasabiofuelin-Mexico.-Themainfindingshighlightedanestimationof- 4,612,950.23tofwheatstrawgenerated.-Theexperimentalresultsofproximateanalysis- were-64.42%volatilematter,-19.49%fixedcarbonand-16.09%ash.-Thehigherheating- was-14.86-MJ/kg.-Anenergypotentialof-69-PJperagriculturalcyclewascalculated,- equivalentto-19%ofthebiomassenergysharereportedin-Mexico's-National-Energy- Balance,by-2014.-

**Keywords:** biofuel, SWOT analysis, wheat straw, biomass, agricultural waste-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction-**

 Wheatisoneofthemaincropsoftheworldwithanannualproductionof-733milliontons- reportedin-2015-[1].-In-Mexico,itwasranked-7thamongallthecropsin-2013,withaharvested- surfaceof-683,044.42ha-[2].-Themostcommonharvestedwheatvarietiesare-*Triticumaestivum-* and-*Triticumdurum*.-Notethat-90%ofwheatproductionisobtainedinthefall‐winterseason,- andtheremaining-10%correspondstothespring‐summerseason.-Theharvestseasonisdone- mainly in May and June [3]. The Northwest region is the largest area for wheat crop production.- Hence,themajorquantityofwheatstrawisgeneratedinthatregion.-Itwasestimatedthat- 4,612,950.23tofwheatstrawwasgeneratedin-Mexico.-Approximately,-85%ofthatbiomass- waste is burned as a traditional practice performed by farmers at the end of the agricultural cycle- [4]. The burning of agricultural waste biomass is regulated by NOM‐015‐SEMARNAT/SAGAR‐ PA‐2007thatestablishesthetechnicalspecificationsofmethodsontheuseoffireinforestland- andagriculturallanduse,inordertopreventandreduceforestfires.-However,itisnotapplied- becauseofthelackofhumanresourcetoinspectandsupervisethoseevents-[5].-In-**Figure 1**,the- burningpracticesandtheirimpactontheenvironmentaredepicted.-Generally,theburning- practiceisdoneinuncontrolledandunsafeconditionsthatcauseairpollution,soilorganic- nutrients loss, elimination of microorganisms, and pH soilmodification. However, wheat straw- isabiomassresourcethatcanbevalorizedandusedasabiofuelbecauseithasanattractive- heatingvalue.-

**Figure 1.** Wheat straw burning practices in Mexico [source: by the authors].-

Using wheat straw as a raw material for any productive process presents diverse factors that- must be considered. Among those factors are the constant supply of wheat straw, the low- density, handling and high transportation cost, the higher heating value, and the physico‐ chemical characterization. Due to the type of the differentfactors involved, the SWOT- (strengths, weaknesses, opportunities, and threats) methodology is a useful tool for analyzing- such factors. Therefore, the aim of this work was to apply the SWOT analysis to wheat straw- utilization as a biofuel in Mexico.-

### **1.1. Power generation through wheat straw-**

Traditionally, biomass has been used for heating in open fireplacesor stoves. Currently, the- biomass utilization as fuel for electricity generation has gained more importance internation‐ ally. It is a productive alternative to the exploitation of waste biomass generated in the- agriculture and forestry, annually.-

With the purpose of reducing greenhouse gases (GHG) emissions and depletion of nonre‐ newable energy sources, there is an increase in the share of renewables worldwide. Bioenergy- plays a vital role in the reduction of GHG and climate change.-

In 2012, the installed biomass power generation capacity reached 83 gigawatt‐electric (GWe),- equivalent to 1.5% of global power generation capacity [6]. Denmark is a pioneer in developing- power plants using agricultural wastes; the firstcommercial straw power plant, Haslev, has- been developed since 1989. Four power plants were developed and operated with wheat straw- as the sole fuel. Moreover, large‐scale straw power plants also have been commissioned in the- United Kingdom (38 MW, Ely in 2002) and Spain (25 MW, Sangüesa in 2002) [7]. The biggest- advantage of using straw in the energy sector is that it is a CO2neutral fuel, which does not- contribute to an increase of the atmosphere's content of greenhouse gases.-

#### **1.2. Biomass share in the Mexican energy matrix-**

By 2014, the biomass share in the energy matrix of Mexico was 4.07%, and it represented the- highest among all the renewable energy sources [8]. The biomass considered by the National- Energy Balance was only firewoodand sugarcane bagasse. The energetic use of biomass in- Mexico is limited to food cooking processes in rural places and as a fuel in power generation- plants in sugar refineries.-The biomass electricity generation has a total capacity of 634 MW [9].-

The current economic situation of the energy sector of Mexico is leading to many opportunities- to increase the renewable energy share in the energy matrix. Renewable energy is an alternative- to a petroleum‐based economy that in the recent years has shown high prices fluctuations of- crude oil. The regulatory framework was already established, and it is comprised in the laws- of energy transition, promotion and development of bioenergy, and renewable energy from- Baja California. However, there is the need to create mandates accompanied with the right- energy policy to encourage and increase the renewable energy market in Mexico.-

### **1.3. Renewable energy regulation in Mexico-**

In 2014, the energy situation in Mexico had experienced a radical change with the approval of- the energy reform. Its aim to maintain the energy security of the country and economic- connectivity and make energy as a mottoof the Mexican economy to create jobs and attract- investments and technology. The main structural changes are established in the reform, such- as the opening of the electricity market. These changes are reflectedin the modifications- performed to the articles 27 and 28 of the Mexico's Constitution [10]. The article 27 establishes- that the planning and control of the national electricity system, energy transmission, and- distribution are exclusive functions of the nation. It is forbidden to provide concessions to- private companies related to the mentioned functions. However, it allows the State to have- contracts with the private sector on behalf of the nation, to carry out financing, maintenance,- management, operation, and expansion of the necessary infrastructure to provide the public- service of transmission and distribution of electricity. The elimination of the exclusivity to- generate electricity by the State was the main modification to the article 28. Nevertheless, the- planning and control of the national electricity system and the public service of electricity- transmission and distribution are exclusive areas of the State.-

The energy reform allows the private electricity producers to sell energy, not only in the self‐ supply modality as previously, but openly. Also, it removed entry barriers of the energy sector,- allowing greater flexibilityfor private sector investment and promoting equitable and- competitive conditions for all private generations including the Federal Electricity Commis‐ sion. This reform represents an opportunity for the increment of the biomass share in the- national energy matrix.-

In 2015, the Law of Energy Transition was enacted. The purpose of this law is to regulate the- sustainable use of energy as well as the obligations of clean energy and reduction of pollutant- emissions from the electricity industry while maintaining the competitiveness of the produc‐ tive sectors [11]. In this law, it is established that power consumption is met by a portfolio of- alternatives that include energy efficiencyand an increasing proportion of clean energy- generation in conditions of economic viability. Through clean energy and energy efficiency- goals, the Secretariat of Energy will encourage electricity generation from clean energy sources- to reach the levels established in the Mexican General Law on Climate Change. The Secretariat- should consider the biggest boost to energy efficiencyand clean energy generation that can be- supported in a sustainable way under the economic conditions and the electricity market in- the country. Policies and measures to boost energy efficiencyand renewable resources to- replace fossil fuels in final consumption will be considered.-

#### **2. Wheat straw generation in Mexico-**

#### **2.1. Wheat producers in Mexico-**

**Table 1**shows the wheat producers in Mexico, the wheat harvested area, and the wheat straw- generated. Sonora and Baja California were responsible for the production of 61.69% of wheat- straw.-


**Table 1.** Wheat producer's states in Mexico in 2013 [12].-

The states of Mexico that produce wheat were ranked and localized geographically through- the analysis of the statistical information system of crops. **Figure 2**illustrates the location of- the states from Mexico that produced wheat in 2013.-

Based on data reported by the Secretariat of Energy, a generation index of 7.3 t/ha [13] of wheat- straw was used for the estimation of the wheat straw availability. The experimental determi‐ nations were performed to *Triticum aestivum*that is one of the most common wheat varieties- that is harvested in Mexico. The proximate analysis and higher heating value determinations- were applied to the wheat straw. The proximate analysis was conducted according to ASTM- E870‐82, and the heating value was determined following the ASTM E711. Based on the wheat- straw estimation and the experimental results, the SWOT methodology was applied to- evaluate the internal and external factors affectingthe utilization of wheat straw as biofuel in- Mexico.-

**Figure 2.** Location of wheat producer's states in Mexico.-

#### **2.2. Wheat straw experimental determinations-**

The proximate analysis and higher heating value determinations were applied to the wheat- straw. The analysis procedures were conducted according to ASTM E870‐82 (2006), and the- heating value was determined according to ASTM E711 [14, 15].-

#### **2.3. Proximate analysis-**

Among the analysis for physicochemical characterization of the biomass, the proximate- analysis is the one with less complexity. It does not require sophisticated laboratory equipment.- The proximate analysis allows determining the weight percentages of moisture (M), volatile- matter-(VM), fixedcarbon (FC), and ash of the biomass. With the results obtained from this- analysis, it is possible to definethe most suitable biomass conversion process, e.g., biological- or thermochemical processes. It also permits establishing fuel quality criteria, among others- [16].-

#### **2.4. Higher heating value-**

The heating value is an important parameter that must be determined in the evaluation of any- fuel and to analyze and design bioenergy systems [17]. It is a measure of the amount of energy- that can be released per unit mass, through an oxidation reaction. It is one of the most importantcharacteristics to definethe suitability of a solid biomass as a fuel. The heating value was- determined experimentally by employing an adiabatic calorimetric bomb IKA WERKE; model- C2000 basic.-

#### **2.5. SWOT analysis-**

The SWOT analysis evaluates the strengths, weaknesses, opportunities, and threats related to- the development of a project. The strengths and weaknesses of the project are internal- characteristics and are controllable while opportunities and threats are external factors but can- react at a determining moment in their favor [18]-

The implementation of the SWOT analysis allows understanding the strengths of a project and- to exploit its opportunities and plan based on them. Also, it contributes to recognize treat or- avoid the weaknesses and protect against any threat known [19]. The SWOT methodology was- applied to evaluate the internal and external factors affectingthe utilization of wheat straw as- biofuel in Mexico.-

#### **3. Results-**

The main findingshighlighted an estimation of 4,612,950.23 t of wheat straw generated in- Mexico. The states of Sonora and Baja California were responsible for 61.69% of the wheat- straw generation.-

The results of proximate analysis experimentally obtained were 64.42% volatile matter,-19.49%- fixed carbon, and 16.09% ash.-

The experimental higher heating of wheat straw determined was 14.86 MJ/kg. Based on these- results, an energy potential of 69 PJ per agricultural cycle was calculated, equivalent to 19% of- the biomass energy share reported in Mexico's National Energy Balance, in 2014.-

**Table 2**depicts the results of the SWOT analysis applied to evaluate the internal and external- factors affecting the utilization of wheat straw as biofuel in Mexico.-

The main strength identifiedfor the use of wheat straw as biofuel in Mexico was its higher- heating value and high intensive activity in the agricultural sector, specifically, wheat harvest‐ ing. The higher heating value of the wheat straw is an attractive and the most important- characteristic from the energy point of view. The amount of wheat straw generated annually- in the Mexican agriculture is considerable and highlights high resource availability. It is an- important aspect because it can contribute to ensuring the biomass supply. The valorization- of wheat straw for energy applications can foster the economic development of the agricultural- sector of Mexico, provide to energy security, and reduce the fossil fuel use. It is a sustainable- alternative that helps to control and reduce the pollutant emissions by avoiding the open- burning practices of waste agricultural biomass. In the global market, there are proven- technologies for the utilization of agricultural waste as biofuel. In Mexico, there is experience- in power generation by waste biomass from the agriculture. It is an advance regarding the- learning curve.-

About the weaknesses found, the wheat straw has a low density. It is an issue that requires- physical conditioning and densificationof the biomass to facilitate its collection, handling,- transportation, and storage. The addition of these preprocessings increases the costs due to- the implementation of specialized equipment, labor, and fuel consumption. The wheat straw- is not concentrated in one place. Therefore, the long distances involved between the wheat- straw generation fields represent a challenge to collect it.-


**Table 2.** SWOT analysis results.-

The current situation in the energy sector of Mexico provides opportunities for the use of wheat- straw as biofuel. The recent energy reform, the Law of Energy Transition, and the goals to- increase the participation of renewable energy in Mexico are settingthe platform to favor and- to encourage the exploitation of waste biomass for energy applications.-

Among the main threats analyzed are the biomass supply ensuring the annual crop harvested- surface variations, the price of residual biomass, and the lack of public policy that promotes- the valorization of waste biomass. There is another one, related to a sociocultural aspect, and- it is the traditional burning practices of wheat straw performed by farmers across the country.- Therefore, it is necessary to gain the social acceptability of farmers and the rural communities- strategically to avoid burning of wheat straw and to assure the constant supply of wheat straw.-

### **4. Conclusion-**

Due to the current situation that Mexico is facing concerning energy security, decreasing of- dependence to conventional energetics, as well as the reduction of greenhouse gases emissions,- it is necessary to findalternatives to diversify the energy sources. For that reason the Mexican- government committedto sustainability has empowered the Secretariat of Energy based on- international trends that postulate changing patternsof production and use of energy, to- develop a national strategy for the energetic transition due to environmental, social, and- economic issues. The energetic transition involves major changes, including the promotion of- renewable energy sources, e.g., solar, wind, biomass, hydraulic, and the rational use of energy- as key strategic actions. The main goal of the Law of Energy Transition is to increase the share- of clean energy production to 25% by 2018, 30% by 2021, 35% by 2024, and 40% by 2035. The- biomass and waste biomass can play a key role in the energy transition because the high- intensive activity in the agriculture sector. The wheat straw standout as an abundant biomass- residue generated in Mexico and it has an important energy potential estimated at 69 PJ per- agricultural cycle. The valorization and utilization of wheat straw for bioenergy purposes is- equivalent to 19% of the biomass energy share reported in Mexico's National Energy Balance,- in 2014.-

The results of the SWOT analysis applied to evaluate the internal and external factors affecting- the utilization of wheat straw as biofuel in Mexico depicted nine strengths, seven weaknesses,- fiveopportunities, and fivethreats. The development of the SWOT analysis provided the- consideration of the main factors for the utilization of wheat straw as a biofuel in Mexico. The- actual conditions in Mexico are favorable for the exploitation of wheat straw as a biofuel.-

### **Author details-**

Gisela Montero1\*, Conrado García1 , Marcos A. Coronado1 , Lydia Toscano2 ,- Margarita Stoytcheva1 , Ricardo Torres1 , Ana M. Vázquez3 and Daniela G. Montes1-

 \*Addressallcorrespondenceto:gmontero@uabc.edu.mx-

 1-Universidad-Autónomade-Baja-California,-Institutode-Ingeniería,-Mexicali,-Baja-California,- México-

2 Academia de Química y Bioquímica, Instituto Tecnológico de Mexicali, Mexicali, Baja Cali‐ fornia, México-

3 Universidad Autónoma de Baja California, Escuela de Ingeniería y Negocios Guadalupe- Victoria, Mexicali, Baja California, México-

### **References**


## **Use of Corn Dried Distillers Grains (DDGS) in Feeding of Ruminants**

Ewa Pecka-Kiełb, Andrzej Zachwieja, Dorota Miśta, Wojciech Zawadzki and Anna Zielak-Steciwko

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66357

#### **Abstract**

Bioethanol is the product of fermentation of starch contained in renewable resources, such as corn, wheat, rye and rice. Depending on the technology used for its production, dried distillers decoction may exist in different forms: dried distillers grain (DDG); dried distillers grain with solubles (DDGS) and high-protein dried distillers grains (HPDDG), as well as wet distillers grain (WDG), wet distillers grain with solubles (WDGS), and high-protein wet distillers grains HPWDG). Research conducted in recent years has demonstrated the possibilities of corn DDG as feed for livestock due to its high content of valuable protein, high calorific value and bioelements. Distillers grain has been used as feed for beef and dairy cattle, sheep, swine and poultry. In case of ruminants, it is important that distillers grain is foodstuff high in ruminal undegradable protein, with beneficial fibre content that does not cause rumen acidosis. DDGS has positive influence on milk yield and its fat and protein content. Research on rumen fermentation has proven that DDGS positively affecs processes in forestomachs: methanogenesis, ammonia emission and volatile fatty acids profile. Reprocessing of agri-food industry by-products may well be an alternative for traditional methods of feeding animals and utilizing valuable nutrients that they contain.

**Keywords:** dried distillers grains with solubles (DDGS), corn, ruminants, animal production

### **1. Introduction**

The prospective exhaustion of non-renewable energy sources and the negative influence their burning has on the environment enforces the search for alternative fuels coming from the

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

plant biomass. Nowadays, in an attempt of replacing conventional fuels, bioethanol coming from the plant-based biofuels is used [1]. Bioethanol is obtained from the fermentation of starch contained in renewable material, such as, corn, wheat, rye, rice, and similar things. Its production involves fermentation of raw material and its distillation followed by dehydration. The byproduct of ethanol production is a decoction.

### **2. Bioethanol production process and types of distillation decoctions**

The chemical formula for ethyl ethanol is C2 H5 OH and its calorific value equals 19.6-MJ/L.- There exist two technological processes for obtaining ethanol from corn: the wet and the dry- ones [2, 3] (**Figure 1**). The processes are alike, but they result in different byproducts. Dry- grinding allows for greater volume of bioethanol, but the only other product is the animal- feed (WDGS, DDGS). When the wet technology is used, apart from ethanol and animal feed- corn oil, corn syrup and gluten are obtained. The production of ethanol requires a two-stage- fermentation of starch: the first stage is the decomposition of starch into glucose and maltose,- the second stage is yeast fermentation during which disaccharides and monosaccharides are converted into ethanol. The polymeric structure of starch is destroyed by enzymes and temperature. Two enzymes are used in the industrial process. The first one is α-amylase, whose- function is to hydrolyze polymers to produce shorter chains (dextrins), which remain in the- solution: this is the condensation stage. Then, due to the activity of glucoamylase in the saccharification stage, dextrins convert to simple sugars, glucose and maltose (dimer of α-1–4- glucose) [2]. The obtained solution undergoes yeast fermentation (*Saccharomyces cerevisiae*): one- molecule of glucose is converted to two molecules of carbon dioxide. Post fermentation liquid- is then distilled and the decoction is separated into the solid and liquid phases with the use of- decanters. The liquid phase is then evaporated and condensed into syrup, which is mixed with- the solid phase from the decanters. The resulting decoction is centrifuged, dried and finally- granulated.

Depending on the technology used for ethanol production, different types of decoctions- may be obtained [4]: dried distillers grains (DDG) obtained from distilling ethanol from- yeast production; dried distillers grains with soluble (DDGS)—the most widely used—- obtained from wet corn residues (DG) mixed with condensed liquid phase in the form of- syrup (CDS) and dried; and high-protein dried distillers grains (HPDDG)—bran and germ- (rich in fiber and fat) are removed before distillation allowing for the production of dried- decoction with high-protein content [5]. Foodstuffs in hydrated form containing dry mass- between 5% and 8% (WDG, WDGS, and HPWDG) are cheaper but difficult to transport and- to store. That is the reason why dried distillers grain is the most widely used product of this kind [1].

The output of bioethanol depends on the plant used (**Table 1**). Yield per hectare, soil and weather conditions make corn the main resource for ethanol production. According to Food and Agricultural Policy Research Institute [6], in 2021, the production of ethanol will reach 141,28 metric tons in the USA and 7174 in the UE (**Figure 2**).-

**Figure 1.** Ethanol production from corn: (A) Dry mill process and (B) wet mill process [3].


**Table 1.** Alcohol production rates from different raw materials [7].

According to Lee and Bressan [7], it is possible to produce 308 l of ethanol and 329 kg of DDGS from 1 ton of corn [8]. The numbers suggest that in 2021, the production of DDGS will be 146,784 metric tons in the United States and 7453 metric tons in the EU.-

More and more restrictive laws concerning the disposal of biofuel byproducts make it necessary to utilize the decoctions in an alternative way: using them as feed for livestock is a good solution. Additionally, corn dried distillers grain may reduce prices of nutritive fodder used for feeding animals [9]. In July 2006, according to IndexMundi [10], the price of a ton of corn in the USA was \$ 119.69, soybean—\$ 349.33, oats—\$ 161.6, barley—\$ 140.19, and DDGS—\$ 120.00. Price analysis suggests that replacing 20% of soy would reduce the cost of feed by 13%.-

**Figure 2.** Production of ethanol from corn in the United States and the European Union. Food and Agricultural Policy Research Institute (FAPRI) [6].

### **3. Nutritional characteristics of DDGS**

Corn is one of the most frequently grown agricultural crops in the world and increasingly- important not only in food but also in chemical and energy industries. The earliest research into dried distillers grain as possible sources of protein in feeding livestock took place in 1945 [11]. In recent years, the possibility of using dried distillers grains as a feed for farm animals, especially cattle (both beef cattle and dairy cattle), pigs, and poultry, has been demonstrated [12, 13].

Corn dried distillers grain is high-protein feed: on average it contains 28–36% total protein (BO) in dry matter [14] which is characterized by the low rate of decomposition in the rumen, resulting in high content of ungradable fraction (RUP)—from 47% to 63% BO (55% on average) [4]. The presence of dead yeast cells gives the protein better amino acids composition and very good nutritive value.-

Because of the high content of insoluble fibre, DDGS has positive influence on digestion and lowers the pH in the digestive system. This results in the reduction of pathogen population and diminishes the occurrence of diarrhoea in young animals. DDGS is also a good source of protein and energy for lactating cows [15].

DDGS has lower level of energy than the soybean meal (by 4%), barley (by 17%), and wheat (by 25%), but higher than the rapeseed meal (20–40%). Energy values for different feeds in swine, poultry, and ruminants are shown in **Table 2** [16]. The tabular content of energy for corn DDGS, except for gross energy (heat of combustion), is lower than in grains. Technological improvements in the ethanol production have made it possible for the net energy of lactation in the decoction to equal the concentration of energy in the grains.-


**Table 2.** Wheat DDGS in comparison to other major types of feed [16].

The main source of energy in corn grain is starch, which is almost completely fermented in the process of biofuel production. In DDGS, the main carrier of energy is fat and neutral detergent fibre (NDF). Ether extract constitutes 8.2–11.7% of dry matter, with nonsaturated acids accounting for 80% of fatty acids [14]. Therefore, appropriate introduction of DDGS to feed rations assumes not only the concentration of energy coming from fat but also the improvement of fat composition in milk (enrichment in nonsaturated acids). On the other hand, 5% is the upper limit of fat concentration in the dry mass of feed ratio and it should not be exceeded because of DDGS. Neutral detergent fibre (NDF), which constitutes 40–45% of dry matter, has low content of lignin and easily ferments in the rumen to produce volatile fatty acids. So, DDGS is a foodstuff that does not threaten the rumen acidosis.-However, because of large fragmentation of the decoction, it cannot be treated as a source of physically efficient NDF.-

The content of amino acids in DDGS is higher than in corn (**Table 3**). It is worth stressing that corn DDGS has the lowest content of lysine, since it is produced from the grain which is poor in this amino acid. Yeast present in the decoction not only improves the composition of amino acids in its protein but also the taste. The ingestion of TMR with DDGS content is usually higher in cows [17]. Nevertheless, it is necessary to balance lysine and methionine, in poultry mainly. The content of protein and fat in DDGS is relatively high, but its composition is slightly different depending on the source (**Table 4**).-


**Table 3.** Comparison of essential amino acid content (g/100 g of dry matter) in DDGS and corn [17].


**Table 4.** Composition of DDGS.-

Corn dried distillers grain is rich in phosphorus (0.43–0.83% of dry matter) and its level depends mostly on the content of condensed syrup (CDS)—the carrier of phosphorus compounds. In our research [22], it has been determined that high producing dairy cows may show symptoms of subclinical hypophosphatemia, which is often accompanied by postpartum paralysis [23]. Very low level of phosphorus in cows' blood serum is probably underrated in diagnosing postpartum paresis (milk fever). For cows, DDGS may be a valuable source of phosphorus in postpartum period preventing hypophosphatemia.-

Another element whose concentration in DDGS is visibly higher than in other foodstuffs is sulphur. According to Shurson [24], corn decoction may contain from 0.31% to 1.93% S in dry matter. Its high concentration is partly due to sulphates from sulphuric acid used for cleaning brewery installations. High content of sulphur in DDGS is yet another argument for utilizing it in the postpartum period. The decoction may provide sulphur anion reducing cation-anion balance of feed ration (DCAB), which is recommended for preventing postpartum paralysis. It is essential that feed ratio does not contain more than 0.4% of dry matter (NCR, 2001). Feeding diets with higher concentration of this element may result in disorders of the nervous system, and disturb absorption and metabolism of copper and selenium (the so-called antagonism of elements syndrome)-

### **4. Influence of corn dried distillers grains on health and productivity of animals**

Literature abides in research results concerning the addition of wet (WDGS) [25] and dried (DDGS) [26] distillers grains to TMR. Distillers grains is used as a substitute for the postextraction of soy meal, or as an additive to TMR mixture in the ratio of 10% to 20% [25]. According to Janicek et al. [21], this ratio of DDGS in compound feeds for cattle influences the growth of milk yield and the content of fat and protein in it. Powers et al. [26] showed that the use of DDGS and WDGS in feeding high producing dairy cows gives positive result irrespective of the type of decoction, i.e., dried and wet.

The percentage of fat in milk increases slightly in livestock fed TMR with the addition of DDGS and WDGS. However, feeding the wet decoction causessubstantial growth of FAT percentage in milk, probably due to access to fibre in WDGS.-

Other authors [25, 26] demonstrated that the use of mixtures with dried distillers grains decreases the ratio of n6/n3 fatty acids in milk, which improves its dietary properties.-

One of the possible reasons may be the reactions of lipolysis, hydrogenation, and synthesis of fatty acids in the rumen, so their volume depends on the ratio and changes in the profile during fermentation. Analyzing conversions of fatty acids in cow and sheep rumen and their flow to duodenum, Beam et al. [27] and Jenkins [28], assert the amount of fat obtained from the feed. The compositions of DDGS show high levels of nonsaturated fatty acids (**Table 5**), which has a beneficial influence on their profile in the rumen digesta. The level of C18:1n9c and C18:2n6c acids in the rumen digesta in the *in vitro* examination increases with the addition of DDGS. However, the levels of C15:0, C16:0, and C20:0 saturated fatty acids and nonsaturated C14:1 in the rumen digesta during *in vitro* fermentation does not change [29].


**Table 5.** Percentage of fatty acids in DDGS [29].

According to Al-Suwaiegh et al. [25] and Anderson et al. [30], the percentage of protein and lactose in milk in cows fed DDGS is similar to those fed WDGS. The growth in milk production and the percentage of casein fraction after the inclusion of DDGS in the feed ration of milking cows in the early stages of lactation have been shown (**Figure 3**) [20].

The use of 10–15% of DDGS dry feed in cows in the postpartum period increases the general protein and immunoglobulin level in colostrum. DDGS has no impact on the content of amino acids in colostrum per 1 g of protein. However, with increased DDGS content in cow diet, some physico-chemical properties of colostrum deterirate (decrease in thermal stability and shortening of coagulation time under the influence of rennet). Yet, despite the deterioration of the values of technological properties, DDGS demonstrates the beneficial use of colostrum components, and in consequence the level of total protein and immunoglobulin in the serum of calves [29].

In sheep, 10–20% of DDGS in the feed does not have negative effect on the production of milk. It slightly lowers the protein, dry matter, and the fat content [31]. In sheep and goats, the use of DDGS may increase the level of PUFA acids in kefir produced from their milk [32]. Available- literature does not present research on the influence of DDGS on the composition and quality- of goat's milk. It might be the consequence of a small number of these animals as compared to- the dairy cows. Studies by other authors show that the DDGS and WDGS have limited usage- in optimizing feeding of livestock. Heavy doses are harmful because of the high level of fat,- which reduces the digestibility of feed ratios. About 20–25% of DDGS in compound feeds is- considered to be safe and optimal for dairy cows, whereas for beef cows it is safe up to 50%.

**Figure 3.** Changes in the percentage of whey protein and casein in cow's milk [20].

In feeding sheep, DDGS has no impact on the condition of animals or milk productivity [33]. About 21.20% of DDGS in dry matter of feed for ewes decreasesglucose level in blood and increases the level of insulin. The authors have also confirmed that DDGS is a good source of nutrient for sheep, which has a positive influence on the mass growth of newborn lambs, and has no impact on their mortality [34].

Şahin et al. [35] demonstrated that the inclusion of DDGS in the diet of 3-month-old lambs did not have negative impact on their growth, forage consumption or rumen parameters. According to the authors, only when DDGS constitute 20% of forage, it can be considered as a good source of protein in lambs' diet, as digestibility is considerably lower when DDGS constitutes 10% of the feed. Schauer et al. [36] asserted that 60% inclusion of DDGS in the diet does not show considerable impact on the productivity in growing slaughter lambs (**Table 6**). In goats, however, DDGS may completely replace the soybean meal and up to 31% of corn in addition to the dry matter of the diet. Also, other authors did not observe negative influence of DDGS on productivity in slaughter animals, carcass components, or fatty acids profile in sheep meat [37]. Literature of the subject does not contain research on the influence of DDGS on the quality of goat meat. As in the case of goat's milk, it may be linked with small number of animals and insignificant influence of goat meat on the world economy.-

In beef cows, 35% content of DDGS in feed ration results in the growth of PUFA and CLA acids in meat [38]. Other authors asserted that 25% content of dried distillers grain does not harm the quality of meat (**Table 7**) [39].

Recent research has demonstrated the impact of DDGS on fermentation in the rumen. It- increases the pH amplitude of ruminal fluid and extends the time in which the pH falls- below 5.8 [15], and the concentration of acetate and its proportions to propionate decrease [40]. Corn dried distillers grain results in linear decrease of methane production in the rumen of cows in proportion to the growth in DDGS contents in the diet *in vivo* [41] as well as *in vitro* [42].


1 Control = 0% replacement of barley with dried distillers grains; 20% = 20% dried distillers grains in ration replacing barley; 60% = 60% dried distillers grains in ration replacing barley.-

**Table 6.** The influence of dried distillers grains on feedlot lamb [36].


**Table 7.** The influence of corn dried distillers grains on feedlot beef cattle [39].

In sheep, a 3% decrease in the production of methane in the group fed 30% of DDGS is observed. This decrease in the rumen fermentation is considered good for animals. The emission of methane is a waste of energy, which may result in drop in milk productivity of ruminants [18, 43]. In cows, the growing proportion of DDGS in the substrate in the *in vitro* study causes a significantly reduced ammonia production in the rumen digesta; after 24 hours of fermentation, the amount of ammonia is more than five times lower with DDGS (22.4 mmol/l) in comparison to control, where TMR was the substrate (124.6 mmol/l). The i*n vitro* studies showed that the use of DDGS reduced the acetate and propionate levels in lambs [44]. In sheep and cows, the contents of DDGS in forage reduce the production of acetate in the rumen and increases the ratio of propionate [18, 42].

In the *in vivo* conditions, DDGS does not change SCFA concentration in the ruminal fluid of- cows, but it lowers the content of acetate in SCFA in groups of animals fed DDGS (57.4mol%- in the group of 10% DDGS in dry matter ration, 53.1 in 15% DDGS in dry matter ration, and- 63.5 in 30% DDGS in dry matter ration) as compared to control group where traditional TMR- (65.7mol%) was used. DDGS increases the levels of propionate. The SCFA utilization factor- expressed as the ratio of nonglycogenic to glycogenic SCFA acids (that is NGR) decreases in- animals fed DDGS [45].

The obtained results show beneficial impact of DDGS on the content of the most important volatile acids in the rumen digesta. The use of DDGS as a substrate in the *in vitro* fermentation of the rumen digesta in cows as well as sheep changed the levels of butyric and isovaleric acids: their levels were decreasing with the augmented ration of DDGS-

The consequence of the drop in production of isoacids reduced the decomposition of protein in the rumen, which is desirable in this group of animals [18, 42].

Research results suggest the possibility of using corn dried distillers grain as an addition or a substitute for other compound feeds in feeding lactating dairy cows. In recent years, studies of corn DDGS in feed rations for cows in the dry period showed that it may be included in TMR in this phase of the production cycle. The dry period is connected with the significant physiological, metabolic, and nutritional changes. Feeding cows determine possible problems in the postpartum period, define their metabolic status, and in consequence their health condition, fertility, value of functional traits, which affect the efficiency of milk production. Proper inclusion of DDGS in the feed ration allows for the assumption that not only the concentration of energy from fat, but also the improvement of milk composition (it will be richer in non-saturated fats). DDGS does not threaten the incidence of rumen acidosis. It may be an important source of phosphorus for cows in postpartum period and its use may prevent hypophosphatemia. High content of sulphur in DDGS is yet another argument for its use around the calving period. The decoction is an important source of sulphur anion which diminishes the cation-anion balance of the feed ration (DCAD). When DDGS was used in the last three weeks before calving as 10%, 15%, and 20% of the dry matter of feed ration, respectively, the experiment results showed a drop of DCAD of the feed ration from 189 (TMR without the addition of DDGS) to 10 mEq/kg when 20% of DDGS was used [29].

When 10% DDGS is used, in cows immediately after calving, the level of liver enzyme aspartate aminotransferase type (AST) grows and the level of triglycerides drops, which suggests the development of subclinical ketosis. However, the 15–20% DDGS content does not increase ketogenesis or alkalosis. Large doses of 20% DDGS cause excessive increase of AST activity after calving. Feeding 20% DDGS to cows in the postpartum period favourably influences the content of Ca and P in the serum after calving, and physiological hypocalcaemia is not observed in this period. The decrease of the total protein and G type immunoglobulins in the blood of cows receiving larger amounts of DDGS in their feed rations simultaneously causes a slight decreasein the level of albumins. It may indicate the possibility of more intense transmission of immunoglobulins to mammary gland before calving [29].

The results of the study in their overwhelming majority confirm the possibility of safe and efficient inclusion of DDGS in nutritional programme of ruminants, but—as in the case of all types of feed—standard precautions are necessary. Changeable/varying content of particular nutritional components, physical and chemical properties connected with the method of fermentation in the production of bioethanol or storing of the decoction may pose problems.

The process of drying DDGS is of significant importance for the production of DDGS—too fast and in too high temperature causes negative changes in protein: denaturation of protein takes place and products of Maillard's reaction are created, which results in the growth of nitrogen insoluble in acidic detergent (ADIN), indigestible fraction of the total protein [24]. In the studies by Cromwell et al. (1993) [46], the percentage of ADIN in total nitrogen of DDGS is between 8.8% and 36.9%. It shows that with inappropriate drying of the decoction, almost 40% of the total protein may have no nutritional value. Colour is a good marker of a correct/- accurate drying process of DDGS—good quality decoction should be light orange in colour.-

Another problem encountered when using DDGS is a big variability in the molecular size.- American studies determined that the average size of decoction molecules is 1282μm and the- range was from 612 to 2125μm. Such large differences in the molecular size cause spontaneous- stratification of DDGS components during the transport and storage of the feedstuff. The smallest precipitated fraction has strong caking properties and may result in dangerous suspension- of hard mass in storing silos. Additional factors enlarging the problem is high temperature- (summer period), increased water content (secondary moisture), and fat concentration [24].

Distillers grains may also be a source of mycotoxin. If bioethanol is produced from low-quality mould-infected grain, it may pose great threat to animal health and determine the quality of animal products. The concentration of mycotoxins in DDGS is on average three times higher than in the mould-infected grain from which it comes. In Austrian research [19], where 89 samples of DDGS (70% from the USA, 30% from Asia) were tested, it was shown that the biggest/most serious problem was the presence of zearalenone (ZON), B1 and B2fumonisins (FUM), and deoxynivalenol (DON). Mycotoxins were discovered in 91, 85, and 57% of samples. Aflatoxin B1 (AFB1) and T-2 toxins were smaller threats. Their highest mean and maximum levels of concentration were observed for DON (1405 and 12000 g/kg) and FUM (935 and over g/kg). It was also demonstrated that only 1% of samples were free from any mycotoxins. Because of the confirmed threat of toxic compounds, it is recommended that every batch of DDGS reaching farmsteads is examined in reference laboratories (with the use of chromatographic techniques) for the presence of mycotoxins.-

One of the great advantages of DDGS is the possibility of storing it even for a year; WDGS may be stored for 3–7 days. However, there may be difficulties in balancing the diet combining different components of the feed rations with distillers grains [37]. DDGS contains relatively large amounts of elements such as sulphur, phosphorus, and nitrogen, which enter the environment as a consequence of excretion process. High content of phosphorus in the organisms of ruminants may lead to disturbances in phosphate**-**calcium balance between phosphate and calcium [33, 45, 47]. Because of the need to balance DDGS as a feed additive containing different proportions of nutrients, every batch of DDGS requires standard chemical analyses performed on all compound feeds by the manufacturers [37, 45]. **Figure 4** shows the recommended chemical analyses of distillers grains [2].

**Figure 4.** Diagram of biomass analysis [2].

### **5. Conclusions**

Existing research results suggest the effective use of dry and wet distillers grains in livestock nutrition and especially the inclusion of corn dried distillers grains (DDGS) in feed rations for cows, sheep, swine, poultry, and even rabbits. Reprocessing the byproducts of agriculture and food industry is likely an alternative for traditional nutrition of animals. It is also a good way of utilizing the valuable nutrients that these byproducts contain. Compared to other feeds, DDGS is cheaper but its use poses problems, as it is a changeable composition, which requires technological procedures to standardize it.-

The growing demand for renewable sources of energy will parallel the supply of biofuels, whose byproducts are the alternative feed materials, rich in energy and protein. The production of corn dried distillers grains (DDGS) acquired in the process of bioethanol making is relatively large and it may lead to problems utilizing it without negative impact on the environment. One of the eco-friendly alternatives for using dried distillers grains is feeding the livestock. Corn DDGS is particularly a valuable feed for dairy cows in the postpartum period, when its use prevents the postpartum paralysis (it is a good source of phosphorus and sulphur), diminishes the negative balance of energy (large fat content) and the threat of rumen acidosis (favourable composition fibre fraction NDF), as well as improves the feed intake (yeast content). The decoction may partly substitute the soybean meal in feed rations for high producing lactating cows. Optimum addition of DDGS for dairy cows is 10–15% of dry matter in the feed ration. One of the beneficial effects of DDGS as a component in feed rations is the decrease in methane production. Another one may be lowering the costs of feed for animals as it is relatively cheap. Propagation of DDGS reprocessing as animal feed will significantly reduce potential threats for the environment.-

### **Author details**

Ewa Pecka-Kiełb1\*, Andrzej Zachwieja<sup>2</sup> , Dorota Miśta<sup>1</sup> , Wojciech Zawadzki<sup>1</sup>and Anna Zielak-Steciwko<sup>1</sup>

\*Address all correspondence to: ewa.pecka@up.wroc.pl-

1 Department of Biostructure and Animal Physiology, Wroclaw University of Environmental Life Sciences, Wroclaw, Poland-

2 Department of Cattle Breeding and Milk Production, Wroclaw University of Environmental Life Sciences, Wrocław, Poland-

### **References**


## *Edited by Eduardo Jacob-Lopes and Leila Queiroz Zepka*

Frontiers in Bioenergy and Biofuels presents an authoritative and comprehensive overview of the possibilities for production and use of bioenergy, biofuels, and coproducts.

Issues related to environment, food, and energy present serious challenges to the success and stability of nations. Te challenge to provide energy to a rapidly increasing global population has made it imperative to fnd new technological routes to increase production of energy while also considering the biosphere's ability to regenerate resources. Te bioenergy and biofuels are resources that may provide solutions to these critical challenges.

Divided into 25 discreet parts, the book covers topics on characterization, production, and uses of bioenergy, biofuels, and coproducts.

Frontiers in Bioenergy and Biofuels provides an insight into future developments in each feld and extensive bibliography. It will be an essential resource for researchers and academic and industry professionals in the energy feld.

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