Bio-hydrogen and Methane Production from Lignocellulosic Materials DOI: http://dx.doi.org/10.5772/intechopen.85138

#### Table 1.

2. Pretreatment of lignocellulosic material

Biomass for Bioenergy - Recent Trends and Future Challenges

included.

2.1 Physical pretreatment

an industrial scale process due to its cost.

2.2 Chemical pretreatment

106

Lignocellulosic biomass is abundantly available, relatively low-cost, and is a good feedstock for the production of biofuels due to their compositions (cellulose, hemicellulose, and lignin). The natural microorganisms cannot directly ferment lignocellulosic biomass into biofuels. The pretreatment step is required to overcome the recalcitrance attributed to the structural characteristic of lignocellulosic biomass

pretreatment methods must meet the following requirements: (1) increase the sugar production or ability to afterward form sugar by enzymatic hydrolysis, (2) minimize the formation of inhibitors that affect the hydrolysis and fermentation process, (3) avoid the loss of carbohydrates, and (4) be cost-effective. The present section summarizes the performance of various pretreatment technologies, including physical, chemical, physicochemical, and biological processes. Furthermore, the advantages and disadvantages of different pretreatment technologies are also

Physical pretreatment involves an increase in the accessible surface area of

disrupting their crystalline structures. The physical pretreatment methods such as chipping, milling, and grinding are applied to pretreat several lignocellulosic materials [15]. Chipping and grinding are used to reduce a huge lignocellulosic material into small pieces. Thus, milling is required to mill lignocellulosic material into fine particles. Among these physical methods, milling can significantly reduce the degree of crystallinity and particle size and consequently improve their enzymatic hydrolysis [16]. The energy requirement for physical pretreatment methods depends on the particle size and the reduction of crystallinity in lignocellulosic material. In fact, the required energy is higher than the theoretical energy content available in the biomass [15]. As aforementioned, these methods cannot be used in

Microwave irradiation is another physical pretreatment method. It is a heating method which directly applies an electromagnetic field to the molecular structure. Microwaves are nonionizing electromagnetic radiation with the wavelengths ranging from 1 mm to 1 m. The electromagnetic spectrums are located between 300 and 300,000 MHz. The application of microwave pretreatment causes swelling and fragmentation of lignocellulosic biomass. The study of Shahzadi et al. [17] indicates that the use of microwave irradiation can enhance the digestibility of lignocellulosic material. In order to enhance the hydrolysis efficiency, microwave pretreatment assisted with catalysts such as acid and alkaline are applied [18]. The advantages and disadvantages of physical pretreatment method are tabulated in Table 1.

Acid, alkaline, ionic liquid, and organic solvent (organosolv) are used as catalysts in the chemical pretreatment methods. Since 1819, acids including sulfuric and hydrochloric are applied to pretreat lignocellulosic materials [19]. After the discovery, various concentrated and diluted acids have been used to pretreat various lignocellulosic materials [20, 21]. The concentrated acid pretreatments can degrade

lignocellulosic materials to enzymes by breaking down the particle size or

and hydrolyze the lignocellulose biomass into fermentation sugars. Various pretreatment technologies have been proposed, challenging the complexity of bio-

mass structure and attempting to recover high fermentable sugars. The

Summary of advantages and disadvantages of each pretreatment methods [22–24].

cellulose and produce a high concentration of inhibitors, such as furfural and 5 hydroxymethylfurfural (5-HMF). In addition, the utilization of concentrated acid causes corrosion of equipment, making the process less attractive [21]. Dilute acid is an attractive method due to its ability to hydrolyze both hemicellulose and cellulose. As results, pentose sugars (xylose and arabinose) and hexose (glucose) sugar are obtained in the hydrolysate. Moreover, this process minimizes the inhibitor formation compared with concentrated acid pretreatment. However, both concentrated and diluted acids slightly degrade lignin.

formation, and enzymatic hydrolysis improvement [21]. Liquid hot water pretreatment process is quite similar to steam explosion pretreatment, but it uses water instead of steam. This leads to less formation of inhibitors at the high

Bio-hydrogen and Methane Production from Lignocellulosic Materials

DOI: http://dx.doi.org/10.5772/intechopen.85138

In the ammonia-based or ammonia fiber explosion (AFEX) process, the lignocellulosic biomass is subjected to liquid ammonia at a high pressure (250–300 psi) and a temperature around 60–100°C for a few minutes. After that, the pressure is immediately released [28]. Liquid ammonia can cause the swelling of lignocellulose structure, resulting in an increase in the enzymatic hydrolysis efficiency. The immediate release of the pressure causes the physical disruption in the crystalline cellulose, resulting in a decrease in the crystallinity of lignocellulosic biomass. However, the lignin and hemicellulose degradation efficiency is low. AFEX process has advantages such as mild reaction temperature and low formation of inhibitors. SPORL pretreatment process consists of two steps. First, the lignocellulosic materials are treated with magnesium sulfite or calcium sulfite in order to remove the lignin and hemicellulose fractions. Second, the mechanical disk miller is used to reduce the particle size of pretreated lignocellulosic material. This method is efficient to pretreat various lignocellulosic materials [21]. The amounts of HMF and furfural generated from SPORL pretreatment are less than those obtained using acid pretreatment. This is attributed to the fact that at the same acid charge, higher amount of bisulfite leads to higher pH which reduces the decomposition of sugar to

In the biological pretreatment, microorganisms and enzymes are the key points used to pretreat lignocellulosic materials before enzymatic hydrolysis [22, 28]. Main biological process is delignification and saccharification process. Microorganisms, such as brown, white, and soft rot fungi, have been used to degrade lignocellulosic materials. White and soft rot fungi mainly degrade lignin and hemicellulose while brown rot fungi are used to degrade cellulose [22, 28]. White rot fungi such as Cyathus stercoreus, Phanerochaete chrysosporium, Ceriporia lacerata, Ceriporiopsis subvermispora, Pycnoporus cinnabarinus, and Pleurotus ostreatus are frequently applied to degrade lignin because these species contain lignin degradation enzymes, including peroxidase and laccase [22, 28]. Also, Basidiomycetes species, such as Bjerkandera adusta, Irpex lacteus, Fomes fomentarius, and Trametes versicolor are studied for breaking down lignocellulosic materials [11, 12]. Recently, cellulose hydrolyzing bacteria such as Clostridia and Actinomycetes are widely used to pretreat lignocellulosic materials. Clostridia and Actinomycetes grow and degrade lignocellulose under anaerobic and aerobic conditions, respectively [29]. Clostridia have an extracellular complex enzyme system called "cellulosome" that can degrade

lignocellulosic materials. This system contains various enzymes, such as endoglucanases, exoglucanases, hemicellulases, chitinases, pectin lyases, and

As for enzymes used in biological pretreatment, both commercial and extracted enzymes from microbes are used. Commercial cellulase and xylanase are commonly used to degrade lignocellulosic materials such as sugarcane bagasse [31], rice straw [32], napier grass [33], etc. Extracted lignin degradation enzymes, including lignin peroxidase, manganese peroxidase, and laccase, from white rot fungi, are also used to degrade lignin from lignocellulosic materials [28]. The study of Taniguchi et al. [34] found that pretreating rice straw with Pleurotus ostreatus enhanced the degradation of lignin and hemicellulose to 41 and 48% degradation efficiency, respectively. The lignin and hemicellulose degradation by Pleurotus ostreatus occurs

temperatures.

HMF and furfural [21].

lichenases [30].

109

2.4 Biological pretreatment

Alkaline pretreatment is the most commonly used to degrade lignin in lignocellulosic material. Alkaline reagents, such as sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium hydroxide (Ca(OH)2), aqueous ammonia (NH4OH), and oxidative alkaline, are mainly used to cleave the ester linkages in lignin and hemicellulose structures. The cleavages of these linkages significantly enhance the solubilization of lignin and hemicellulose, resulting in a higher cellulose hydrolysis to fermentable sugar by microorganisms or enzymatic hydrolysis.

Ionic liquids (ILs) are salts composed of cations and anions. These liquids have melting point lower than 100°C and low vapor pressure [25]. Anions and cations in ILs form hydrogen bonds with cellulose hydroxyl groups, resulting in a cellulose precipitation. In addition, lignin can be dissolved in the ILs [25]. This reaction occurs in mild conditions with the ease of cellulose recovery, as well as the ILs, with no toxic or odor emission. However, the utilization of ILs to pretreat lignocellulosic materials is not favorable due to its cost. In comparison to other chemical pretreatments, ILs have the advantages of low toxicity, high solvation power, low volatility, thermal stability, as well as inflammability.

In the organosolv process, organic solvents are mainly used to cleave the linkage of lignin and hemicellulose which can increase the pore volume and accessible surface area of cellulose. The resulting lignin is dissolved in the organic solvent phase, while cellulose is recovered as the solid. Many organic solvents such as ethanol, methanol, acetone, organic acids, and ethylene glycol have been utilized to pretreat various lignocellulosic materials. Among these, ethanol is the most favorable solvent due to its low toxicity and its ease of recovery. This process can occur in the presence or absence of catalysts (acid or base) [26]. Comparing with other chemical pretreatments, organosolv process has many advantages such as easy to recover solvent by distillation, low environmental impact, and recovery of highquality lignin as by-product. Contrastingly, high price of organic solvent and potential hazard of handling large volume of organic solvents limit the utilization of organosolv process. The overall advantages and disadvantages of chemical pretreatments are shown in Table 1.

#### 2.3 Physicochemical pretreatment

Physicochemical pretreatment is a combination between physical and chemical pretreatments, which aims to enhance lignin removal and increase the hydrolysis efficiency. Several successful physicochemical pretreatments, such as steam explosion, liquid hot water, wet oxidation, ammonia-based, and sulfite pretreatment (SPORL), are applied to various lignocellulosic materials.

Steam explosion is a combined method between thermo-mechano-chemical treatments. In this process, biomass is exposed to a high pressure (0.69–4.83 MPa) with a saturated steam at a high temperature (160–260°C) for a few seconds [21, 27]. The steam penetrates into the biomass and swells the cell wall of the fibers before the explosion and partial hydrolysis. During pretreatment, the hydrolysis of hemicellulose into hexose and pentose sugars is accomplished by the action of acetic acid produced from the acetyl groups of hemicellulose. This process is called "autohydrolysis." The efficiency of steam explosion can be enhanced by adding the catalyst such as sulfuric acid (H2SO4), SO2, or CO2. Among these catalysts, acid is the best in terms of sugar recovery, minimization of the inhibition compound

formation, and enzymatic hydrolysis improvement [21]. Liquid hot water pretreatment process is quite similar to steam explosion pretreatment, but it uses water instead of steam. This leads to less formation of inhibitors at the high temperatures.

In the ammonia-based or ammonia fiber explosion (AFEX) process, the lignocellulosic biomass is subjected to liquid ammonia at a high pressure (250–300 psi) and a temperature around 60–100°C for a few minutes. After that, the pressure is immediately released [28]. Liquid ammonia can cause the swelling of lignocellulose structure, resulting in an increase in the enzymatic hydrolysis efficiency. The immediate release of the pressure causes the physical disruption in the crystalline cellulose, resulting in a decrease in the crystallinity of lignocellulosic biomass. However, the lignin and hemicellulose degradation efficiency is low. AFEX process has advantages such as mild reaction temperature and low formation of inhibitors.

SPORL pretreatment process consists of two steps. First, the lignocellulosic materials are treated with magnesium sulfite or calcium sulfite in order to remove the lignin and hemicellulose fractions. Second, the mechanical disk miller is used to reduce the particle size of pretreated lignocellulosic material. This method is efficient to pretreat various lignocellulosic materials [21]. The amounts of HMF and furfural generated from SPORL pretreatment are less than those obtained using acid pretreatment. This is attributed to the fact that at the same acid charge, higher amount of bisulfite leads to higher pH which reduces the decomposition of sugar to HMF and furfural [21].

#### 2.4 Biological pretreatment

an attractive method due to its ability to hydrolyze both hemicellulose and cellulose. As results, pentose sugars (xylose and arabinose) and hexose (glucose) sugar are obtained in the hydrolysate. Moreover, this process minimizes the inhibitor formation compared with concentrated acid pretreatment. However, both concentrated

Alkaline pretreatment is the most commonly used to degrade lignin in lignocellulosic material. Alkaline reagents, such as sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium hydroxide (Ca(OH)2), aqueous ammonia (NH4OH), and oxidative alkaline, are mainly used to cleave the ester linkages in lignin and hemicellulose structures. The cleavages of these linkages significantly enhance the solubilization of lignin and hemicellulose, resulting in a higher cellulose hydrolysis

Ionic liquids (ILs) are salts composed of cations and anions. These liquids have melting point lower than 100°C and low vapor pressure [25]. Anions and cations in ILs form hydrogen bonds with cellulose hydroxyl groups, resulting in a cellulose precipitation. In addition, lignin can be dissolved in the ILs [25]. This reaction occurs in mild conditions with the ease of cellulose recovery, as well as the ILs, with no toxic or odor emission. However, the utilization of ILs to pretreat lignocellulosic materials is not favorable due to its cost. In comparison to other chemical pretreatments, ILs have the advantages of low toxicity, high solvation power, low volatility,

In the organosolv process, organic solvents are mainly used to cleave the linkage

Physicochemical pretreatment is a combination between physical and chemical pretreatments, which aims to enhance lignin removal and increase the hydrolysis efficiency. Several successful physicochemical pretreatments, such as steam explosion, liquid hot water, wet oxidation, ammonia-based, and sulfite pretreatment

Steam explosion is a combined method between thermo-mechano-chemical treatments. In this process, biomass is exposed to a high pressure (0.69–4.83 MPa) with a saturated steam at a high temperature (160–260°C) for a few seconds [21, 27]. The steam penetrates into the biomass and swells the cell wall of the fibers before the explosion and partial hydrolysis. During pretreatment, the hydrolysis of hemicellulose into hexose and pentose sugars is accomplished by the action of acetic acid produced from the acetyl groups of hemicellulose. This process is called "autohydrolysis." The efficiency of steam explosion can be enhanced by adding the catalyst such as sulfuric acid (H2SO4), SO2, or CO2. Among these catalysts, acid is the best in terms of sugar recovery, minimization of the inhibition compound

of lignin and hemicellulose which can increase the pore volume and accessible surface area of cellulose. The resulting lignin is dissolved in the organic solvent phase, while cellulose is recovered as the solid. Many organic solvents such as ethanol, methanol, acetone, organic acids, and ethylene glycol have been utilized to pretreat various lignocellulosic materials. Among these, ethanol is the most favorable solvent due to its low toxicity and its ease of recovery. This process can occur in the presence or absence of catalysts (acid or base) [26]. Comparing with other chemical pretreatments, organosolv process has many advantages such as easy to recover solvent by distillation, low environmental impact, and recovery of highquality lignin as by-product. Contrastingly, high price of organic solvent and potential hazard of handling large volume of organic solvents limit the utilization of organosolv process. The overall advantages and disadvantages of chemical

to fermentable sugar by microorganisms or enzymatic hydrolysis.

and diluted acids slightly degrade lignin.

Biomass for Bioenergy - Recent Trends and Future Challenges

thermal stability, as well as inflammability.

pretreatments are shown in Table 1.

2.3 Physicochemical pretreatment

108

(SPORL), are applied to various lignocellulosic materials.

In the biological pretreatment, microorganisms and enzymes are the key points used to pretreat lignocellulosic materials before enzymatic hydrolysis [22, 28]. Main biological process is delignification and saccharification process. Microorganisms, such as brown, white, and soft rot fungi, have been used to degrade lignocellulosic materials. White and soft rot fungi mainly degrade lignin and hemicellulose while brown rot fungi are used to degrade cellulose [22, 28]. White rot fungi such as Cyathus stercoreus, Phanerochaete chrysosporium, Ceriporia lacerata, Ceriporiopsis subvermispora, Pycnoporus cinnabarinus, and Pleurotus ostreatus are frequently applied to degrade lignin because these species contain lignin degradation enzymes, including peroxidase and laccase [22, 28]. Also, Basidiomycetes species, such as Bjerkandera adusta, Irpex lacteus, Fomes fomentarius, and Trametes versicolor are studied for breaking down lignocellulosic materials [11, 12]. Recently, cellulose hydrolyzing bacteria such as Clostridia and Actinomycetes are widely used to pretreat lignocellulosic materials. Clostridia and Actinomycetes grow and degrade lignocellulose under anaerobic and aerobic conditions, respectively [29]. Clostridia have an extracellular complex enzyme system called "cellulosome" that can degrade lignocellulosic materials. This system contains various enzymes, such as endoglucanases, exoglucanases, hemicellulases, chitinases, pectin lyases, and lichenases [30].

As for enzymes used in biological pretreatment, both commercial and extracted enzymes from microbes are used. Commercial cellulase and xylanase are commonly used to degrade lignocellulosic materials such as sugarcane bagasse [31], rice straw [32], napier grass [33], etc. Extracted lignin degradation enzymes, including lignin peroxidase, manganese peroxidase, and laccase, from white rot fungi, are also used to degrade lignin from lignocellulosic materials [28]. The study of Taniguchi et al. [34] found that pretreating rice straw with Pleurotus ostreatus enhanced the degradation of lignin and hemicellulose to 41 and 48% degradation efficiency, respectively. The lignin and hemicellulose degradation by Pleurotus ostreatus occurs

through the action of peroxidase and laccase. Biological pretreatment is environmentally friendly as no chemicals and lower energy are used compared with other pretreatment methods. The advantages and disadvantages of biological pretreatment methods are given in Table 1.

its low yield due to the large quantity of side products formed. The substrates for photo-fermentative bacteria are limited to simple sugars and organic acids, and the hydrogen production rate by photo-fermentation is usually lower than dark fermentation [44]. However, with the high substrate conversion efficiency and high hydrogen yield (HY), the photo-fermentation is also considered a promising technology for bio-hydrogen production [45]. In addition, recent research reported the sequential dark-photo-fermentation as an efficient bio-hydrogen production process. The VFAs from dark fermentation are further utilized for hydrogen production in photo-fermentation, thus the HY and substrate conversion efficiency can be improved via sequential dark-photo-fermentation [45–47]. Typical reactions for dark fermentation with acetic acid formation and photo-fermentation can be stated

Bio-hydrogen and Methane Production from Lignocellulosic Materials

DOI: http://dx.doi.org/10.5772/intechopen.85138

Dark fermentation : C6H12O6 ! 2CH3COOH þ 2CO2 þ 4H2 (1)

Photo-fermentation : 2CH3COOH þ 4H2O þ Light ! 4CO2 þ 8H2 (2) Despite the continuing research at the laboratory scale, the biological hydrogen production from lignocellulosic biomass at pilot and industrial scales is still limited. Various kinds of lignocellulosic feedstock have been investigated for bio-hydrogen production by different microorganisms. Typically, the feedstocks are pretreated prior to fermentation in order to enhance hydrogen production efficiency.

Pretreatment of the biomass can be conducted by physical (such as size reduction), physicochemical (such as steam, ammonia fiber, and carbon dioxide explosion, hot water, and microwave pretreatment), chemical (such as alkaline, diluted acid, and hydrogen peroxide pretreatment), and biological (such as enzymatic pretreatment)

depending on the types of substrates, pretreatment methods and microorganisms

The pretreated lignocellulosic biomass (in solid form) can also be directly fermented to hydrogen. Alkaline-pretreated sugarcane bagasse fermentation

51.9 mL-H2/L-substrate was obtained by fermenting corn stover obtained after steam explosion using mixed cultures of C. celluloblyticum and Citrobacter amalonaticus [54]. The pretreated solid biomass could also be used as feedstocks for hydrogen production via simultaneous saccharification and fermentation (SSF) process. The cellulolytic enzymes mostly perform well under thermophilic condition (50–60°C).

However, hydrogen production by SSF under mesophilic condition had been investigated by some researchers based on the optimal temperature for growth and activity of hydrogen producers. Hydrogen yield of 72 mL-H2/g-substrate was obtained from acetic acid steam-exploded corn straw by SSF with Ethanoligenens harbinense at 37°C [55]. A lower yield of 68 mL-H2/g-substrate was obtained from steam-exploded corn

Fermentation under thermophilic condition (50–65°C) was reported to improve dark fermentative hydrogen production via enhancing substrate degradation rate. Various thermophilic hydrogen producers, such as Thermoanaerobacterium thermosaccharolyticum [57, 58], C. thermosaccharolyticum, and C. thermocellum [59, 60], as well as thermophilic mixed cultures [61], were applied for hydrogen

methods. The yield of hydrogen from lignocellulosic feedstocks is diverse

used. Under mesophilic condition, dark fermentation of untreated water hyacinth by mixed culture of Enterobacter sp. and Clostridium sp. resulted in 119.6 mL-H2/g-VS [48]. Enzymatic hydrolysates of agave bagasse yielded 1.53–3.40 mol-H2/mol-substrate by anaerobic mixed cultures [49, 50]. Higher hydrogen production from acid hydrolysate of sugarcane bagasse (6980 mL-H2/Lsubstrate) was observed with mixed cultures compared to the pure culture of

Enterobacter aerogenes (1000 mL-H2/L-substrate) [51, 52].

straw by SSF with C. bytyricum AS1 at 35°C [56].

111

by C. beijerinckii yielded 0.733 mmol-H2/g-substrate [53]. The HY of

as follows:

Currently, the combined physical, chemical, and biological pretreatment process is investigated for enhancing the degradation efficiency [21]. The combined process is more effective as compared to a single process. Yu et al. [35] combined physical, chemical, and biological pretreatment process to pretreat rice husk. Results indicate that the combination of chemical (2% H2SO4) and biological (P. ostreatus) pretreatments leads to a higher lignin degradation than single-step pretreatments. The combined pretreatment of napier grass carried out using 2% NaOH along with cellulase enzyme was found to be more effective as compared with single alkaline pretreatment, in which a 3.97 time higher methane production (MP) was obtained [36].
