**Metabolic Engineering of Hydrocarbon Biosynthesis for Biofuel Production**

Anne M. Ruffing

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52050

#### **1. Introduction**

The world's supply of petroleum hydrocarbons, which serve as feedstock for the fuel and chemical industries, is rapidly diminishing to satisfy the global demand for energy and consumer goods. In response to this increasing demand and limited supply, the cost of crude oil has risen to over \$100 per barrel in 2012, a 10-fold increase compared to prices in the late 1990s [1]. As fossil fuels are nonrenewable resources, the price of oil is only expected to increase in the future. This unavoidable reality necessitates the development of renewable energy sources in order to maintain the current standard of living. Among the alternative energy options under development, biofuels are anticipated to supplement and eventually replace the petroleum-based fuels that supply the transportation and chemical industries. Currently, first generation biofuels like corn-based ethanol are blended into conventional petroleum fuels, with biofuels supplying 2.7% of the world's transportation fuel in 2010 [2]. It appears that biofuels are on their way to becoming a viable renewable energy source, yet technological and biological advancements are necessary for sustainable and economical biofuel production at the scales necessary to support the world's energy needs.

The current practice of using food crops, like corn or soybean, as feedstocks for biofuel production is not a viable, long-term solution to the energy crisis. In fact, to replace our current petroleum usage with crop-based ethanol production, the entire surface area of land on Earth would be needed for corn production [3]. In addition to this shortcoming, first generation biofuels compete with food production for arable land, require significant nutrient resources (fertilizer and fresh water), and typically have low net energy yields due to the low energy density of the product fuel (i.e. ethanol) and the energy input required to harvest the feedstock and convert it into fuel [4]. Second and third generation biofuels address these limitations. Second generation biofuels use lignocellulosic biomass as the feedstock for fuel production.

© 2013 Ruffing; licensee InTech. This is an open access article 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. © 2013 Ruffi ng; licensee InTech. This is a paper 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.

Lignocellulose, the main component of plant biomass, is the most abundant form of renewable carbon on the Earth, making it an ideal feedstock for renewable hydrocarbon production. The cellulose and hemicellulose components of lignocellulose can be degraded into fermentable sugars to serve as the carbon source for microbial-based fuel production. The carbon feedstocks for both first and second generation biofuels are ultimately derived from carbon dioxide (CO2) fixation through the process of photosynthesis. Third generation biofuels use photo‐ synthetic microorganisms (i.e. microalgae) to directly convert CO2 into fuel molecules or fuel precursors, eliminating the biomass intermediate (Figure 1). While both second and third generation biofuels require land, nutrients, and energy investment for harvesting and fuel production, the fuel production yields from these processes are predicted to be capable of meeting energy needs. However, these technologies have yet to be demonstrated at scale and still require further improvement before they can be economically competitive with fossil fuels.

discuss the metabolic pathways for hydrocarbon fuel production and common metabolic engineering strategies for improving fuel synthesis. Because second and third generation biofuel processes rely on different carbon sources, sugars and CO2 respectively, the remaining sections will focus on the use of organic carbon (heterotrophy) and inorganic carbon (auto‐ trophy) as feedstocks for biofuel production. This division, based on carbon source, is impor‐ tant from both the biofuel production and metabolic engineering perspectives. The chapter will conclude with a discussion of the future outlook for microbial-based, hydrocarbon fuel

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**Figure 2.** Chemical structures of hydrocarbon-based biofuels and fuel precursors. (A) Fuels derived from fatty acid bio‐ synthesis and (B) fuels derived from isoprenoid biosynthesis, including (1) hemiterpene, (2) monoterpenes, and (3) ses‐

synthesis.

quiterpenes.

**Figure 1.** Process steps for (A) second (i.e. lignocellulosic feedstock) and (B) third (i.e. inorganic carbon feedstock) gen‐ eration biofuels.

Both second and third generation biofuels rely on microbes to convert the carbon feedstock into the desired hydrocarbon fuels. Microorganisms have been identified that are capable of producing a range of fuel molecules and fuel precursors, yet the natural rates of microbial fuel synthesis are typically too low to support industrial-scale production. Metabolic engineering is a powerful tool to improve microbial fuel production, either through engineering the metabolic pathways within the native microorganism to encourage high fuel synthesis or though transferring the fuel production pathway into a model organism for optimization. This chapter will focus on the application of metabolic engineering to increase hydrocarbon fuel production. Within this chapter, hydrocarbon-based fuels are defined to include oxygencontaining fuel molecules with long hydrocarbon chains, such as fatty alcohols and fatty acid ethyl esters (FAEE), in addition to pure hydrocarbons like alkanes, alkenes, and isoprenoidbased molecules: hemiterpene (C5), monoterpenes (C10), and sesquiterpenes (C15). Hydro‐ carbon-based fuel precursors will also be considered, including free fatty acids (FFAs) and triacylglycerol (TAG). The structures of these hydrocarbon-based fuels and precursors are illustrated in Figure 2. Hydrocarbon-based fuels and precursors can be produced by both second and third generation biofuel processes. Therefore, the first section in this chapter will discuss the metabolic pathways for hydrocarbon fuel production and common metabolic engineering strategies for improving fuel synthesis. Because second and third generation biofuel processes rely on different carbon sources, sugars and CO2 respectively, the remaining sections will focus on the use of organic carbon (heterotrophy) and inorganic carbon (auto‐ trophy) as feedstocks for biofuel production. This division, based on carbon source, is impor‐ tant from both the biofuel production and metabolic engineering perspectives. The chapter will conclude with a discussion of the future outlook for microbial-based, hydrocarbon fuel synthesis.

**Figure 2.** Chemical structures of hydrocarbon-based biofuels and fuel precursors. (A) Fuels derived from fatty acid bio‐ synthesis and (B) fuels derived from isoprenoid biosynthesis, including (1) hemiterpene, (2) monoterpenes, and (3) ses‐ quiterpenes.

#### **2. Engineering hydrocarbon biosynthesis pathways**

The hydrocarbon-based biofuels considered in this chapter (Figure 2) are all derived from two metabolites: fatty acids and isoprenoids. Thus, the two metabolic pathways commonly targeted by metabolic engineering strategies are the fatty acid biosynthesis pathway and the two pathways for isoprenoid production (Figure 3).

**2.1. Fatty acid derived biofuels**

hydrocarbon fuels or fuel precursors (Figure 3).

mechanisms.

As shown in Figure 3, fatty acid biosynthesis interfaces with the primary metabolism at the acetyl-CoA node. Fatty acid biosynthesis is initiated by the formation of acetoacetyl-ACP, the substrate for fatty acid chain elongation. The conversion of acetyl-CoA to acetoacetyl-ACP includes two key enzymatic steps: (1) the conversion of acetyl-CoA to malonyl-CoA, catalyzed by acetyl-CoA carboxylase (ACC) and (2) the conversion of malonyl-ACP to acetoacetyl-ACP via β-ketoacyl-ACP synthase III (KASIII). These two enzymes are common metabolic engi‐ neering targets for improving fatty acid biosynthesis. In fact, ACC has been shown to be a ratelimiting step of fatty acid synthesis in *Escherichia coli*, and overexpression of ACC has been shown to yield more than a 5-fold increase in FFA production [5]. Overexpression of KASIII in *E. coli* also improved FFA synthesis, increasing lipid production by 20-60% [6]. After acetoacetyl-ACP formation, fatty acid chain elongation proceeds by an iterative process, whereby the hydrocarbon chain is elongated in increments of 2 carbons. Once the elongation process terminates, the final acyl-ACP is divided among three possible paths: one leading to membrane biosynthesis, an essential pathway for cell growth, and the other two yielding

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To produce biofuels with an even-numbered carbon chain, the acyl-ACP is cleaved by a thioesterase (TE), releasing the FFA. The TE is yet another key target for metabolic engineering. The final fuel properties, including viscosity, cloud point, flash point, oxidative stability, ignition delay, and combustion quality, are largely determined by the hydrocarbon chain length and degree of saturation [7]. Accordingly, numerous TEs have been cloned and characterized, predominantly from plant sources, to control the carbon chain length of the FFAs. Engineering strategies often exploit this collection of TEs to tailor the biofuel product. Favored TEs include a truncated TE ('*tesA*) from *E. coli* and acyl-ACP TEs from *Umbellularia californica* and *Cuphea hookeriana*, producing FFAs with carbon lengths of 16:0, 12:0, and 10:0 and 8:0, respectively [8-10]. The FFAs themselves can be extracted as fuel precursors and converted into biodiesel (FAMEs or FAEEs) using acid-catalyzed chemical processes [11]. To allow for FFA accumulation, the β-oxidation pathway and free fatty acid recycling are often eliminated by gene knockout of acyl-CoA synthetase (*acs*) and acyl-ACP synthetase (*aas*) [12]. An alternative strategy was recently demonstrated, whereby FFAs were synthesized through an engineered reversal of the β-oxidation cycle [13]. In this strategy, acetyl-CoA is used directly for fatty acid chain elongation, allowing for improved carbon and energy efficiency compared to the fatty acid biosynthesis pathway which requires activation of acetyl-CoA to malonyl-CoA. Engineering a reversed β-oxidation cycle required modification of multiple regulatory mechanisms, knockout of other fermentative pathways, expression of a TE or other fuel producing enzyme, and overexpression of key enzymes in the β-oxidation pathway [13]. While this strategy yielded the highest reported concentration of FFAs in *E. coli* (7 g/L), its application to other host organisms may be restricted by inadequate knowledge of the native regulatory

With an intact *acs*, FFAs can be converted into acyl-CoA, a precursor for other fuel products including the biodiesel precursor, TAG, and fuels such as FAEEs and fatty alcohols (Figure 3). The conversion of acyl-CoA to TAG requires the provision of 1,2-diacylglycerol and a

**Figure 3.** Hydrocarbon biosynthesis pathways for the production of biofuels, with the fatty acid biosynthesis pathway in blue, isoprenoid pathway in red, mevalonate pathway in green, and methylerythritol phosphate pathway in purple. Biofuels and biofuel precursors are highlighted in the colored boxes. Enyzmes are in italics. Solid arrows represent a single enzymatic step, while dashed arrows represent multiple enzymatic steps. Abbreviations for metabolites and en‐ zymes are listed at the end of the chapter.

#### **2.1. Fatty acid derived biofuels**

As shown in Figure 3, fatty acid biosynthesis interfaces with the primary metabolism at the acetyl-CoA node. Fatty acid biosynthesis is initiated by the formation of acetoacetyl-ACP, the substrate for fatty acid chain elongation. The conversion of acetyl-CoA to acetoacetyl-ACP includes two key enzymatic steps: (1) the conversion of acetyl-CoA to malonyl-CoA, catalyzed by acetyl-CoA carboxylase (ACC) and (2) the conversion of malonyl-ACP to acetoacetyl-ACP via β-ketoacyl-ACP synthase III (KASIII). These two enzymes are common metabolic engi‐ neering targets for improving fatty acid biosynthesis. In fact, ACC has been shown to be a ratelimiting step of fatty acid synthesis in *Escherichia coli*, and overexpression of ACC has been shown to yield more than a 5-fold increase in FFA production [5]. Overexpression of KASIII in *E. coli* also improved FFA synthesis, increasing lipid production by 20-60% [6]. After acetoacetyl-ACP formation, fatty acid chain elongation proceeds by an iterative process, whereby the hydrocarbon chain is elongated in increments of 2 carbons. Once the elongation process terminates, the final acyl-ACP is divided among three possible paths: one leading to membrane biosynthesis, an essential pathway for cell growth, and the other two yielding hydrocarbon fuels or fuel precursors (Figure 3).

To produce biofuels with an even-numbered carbon chain, the acyl-ACP is cleaved by a thioesterase (TE), releasing the FFA. The TE is yet another key target for metabolic engineering. The final fuel properties, including viscosity, cloud point, flash point, oxidative stability, ignition delay, and combustion quality, are largely determined by the hydrocarbon chain length and degree of saturation [7]. Accordingly, numerous TEs have been cloned and characterized, predominantly from plant sources, to control the carbon chain length of the FFAs. Engineering strategies often exploit this collection of TEs to tailor the biofuel product. Favored TEs include a truncated TE ('*tesA*) from *E. coli* and acyl-ACP TEs from *Umbellularia californica* and *Cuphea hookeriana*, producing FFAs with carbon lengths of 16:0, 12:0, and 10:0 and 8:0, respectively [8-10]. The FFAs themselves can be extracted as fuel precursors and converted into biodiesel (FAMEs or FAEEs) using acid-catalyzed chemical processes [11]. To allow for FFA accumulation, the β-oxidation pathway and free fatty acid recycling are often eliminated by gene knockout of acyl-CoA synthetase (*acs*) and acyl-ACP synthetase (*aas*) [12]. An alternative strategy was recently demonstrated, whereby FFAs were synthesized through an engineered reversal of the β-oxidation cycle [13]. In this strategy, acetyl-CoA is used directly for fatty acid chain elongation, allowing for improved carbon and energy efficiency compared to the fatty acid biosynthesis pathway which requires activation of acetyl-CoA to malonyl-CoA. Engineering a reversed β-oxidation cycle required modification of multiple regulatory mechanisms, knockout of other fermentative pathways, expression of a TE or other fuel producing enzyme, and overexpression of key enzymes in the β-oxidation pathway [13]. While this strategy yielded the highest reported concentration of FFAs in *E. coli* (7 g/L), its application to other host organisms may be restricted by inadequate knowledge of the native regulatory mechanisms.

With an intact *acs*, FFAs can be converted into acyl-CoA, a precursor for other fuel products including the biodiesel precursor, TAG, and fuels such as FAEEs and fatty alcohols (Figure 3). The conversion of acyl-CoA to TAG requires the provision of 1,2-diacylglycerol and a

diacylglycerol acyltransferase (DGAT) to catalyze transfer of the acyl chain. While DGAT has been overexpressed to improve TAG production in plants [14], the utility of this strategy still remains to be tested in microorganisms. Most metabolic engineering strategies for microbial TAG synthesis focus on improving the supply of the precursors: FFA and glycerol-3-phosphate (G3P) [15, 16]. Microbial production of FAEEs typically involves heterologous expression of both the pathway for ethanol production and an acyltransferase (AT) [17-19]. Selection of the two genes required for ethanol synthesis, pyruvate decarboxylase (*pdc*) and alcohol dehydro‐ genase (*adh*), will largely depend on the host organism, but generally, efforts involving prokaryotic hosts such as *E. coli* and cyanobacteria will use *pdc* and *adh* from *Zymomonas mobilis* due to their capacity for high ethanol production [20]. To date, only one AT has been heterologously expressed for FAEE production: the wax synthase gene (*aftA*) from *Acineto‐ bacter baylyi* ADP1 [17-19]. A third biofuel product derived from acyl-CoA is fatty alcohols. The enzymatic conversion of acyl-CoA to a fatty alcohol is dependent upon whether the fatty acyl-CoA reductase (*far*) is of prokaryotic or eukaryotic origin. Most prokaryotic FARs reduce acyl-CoA to a fatty aldehyde, requiring another enzyme, fatty aldehyde reductase (ALR), for conversion to the fatty alcohol product. On the other hand, eukaryotic FARs catalyze the direct conversion of acyl-CoA to fatty alcohol without release of an aldehyde intermediate [21]. Metabolic engineering strategies for fatty alcohol production include: expression of a pro‐ karyotic FAR, *acr1* from *Acinetobacter calcoaceticus* BD413, with reliance on native fatty alde‐ hyde reductases for fatty alcohol synthesis [19]; expression of 5 different eukaryotic FAR homologs from the model plant organism *Arabidopsis thaliana* [22]; and expression of a eukaryotic FAR, *far1* from mouse [23]. The recent discovery of a prokaryotic FAR from *Marinobacter aquaeolei* VT8, capable of catalyzing the direct conversion of acyl-CoA to fatty alcohol, may be a beneficial alternative to the use of eukaryotic FARs for fatty alcohol pro‐ duction in prokaryotic hosts such as *E. coli* and cyanobacteria [24]. An alternative strategy used by Dellomonaco and colleagues identifies surrogates for *far* and *adh* in the native *E. coli* genome based on sequence homology [13]. With the numerous biofuel products derived from acyl-CoA and the natural enzymatic diversity for these conversions, we have only just begun to explore and develop the metabolic engineering tools essential to enable large-scale synthesis.

**2.2. Isoprenoid-based biofuels**

phosphate (GAP):

The chemical composition of petroleum-based fuels: gasoline, diesel, and jet fuel, includes linear, branched, and cyclic alkanes, aromatics, and chemical additives [28]. Isoprenoid-based biofuels have the structural diversity to mimic these petroleum compounds, with up to 50,000 known isoprenoid structures including branched and cyclic hydrocarbons with varying degrees of unsaturation [29, 30]. Isoprenoids reported to be potential fuel candidates include: the hemiterpene (C5) isoprene; monoterpenes (C10): terpinene, pinene, limonene, and sabinene; the sesquiterpene (C15) farnesene, and their associated alcohols: isopentenol, terpineol, geraniol, and farnesol [12, 31]. Two metabolic pathways are capable of producing the isoprenoid building blocks isopentenyl pyrophosphate (IPP) and dimethylallyl diphos‐ phate (DMAPP): the mevalonate (MVA) pathway [32] and the methylerythritol phosphate (MEP) pathway, also known as the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway and the non-mevalonate pathway (Figure 3) [33]. In general, the MVA pathway is found in eukaryotes and archaea while the MEP pathway is utilized by prokaryotes. In agreement with the proposed evolutionary origin of plants, they contain both isoprenoid pathways with the MEP pathway localized in the plastid and the MVA pathway in the cytosol [34]. The MVA and MEP pathways differ with respect to their requirement for carbon, energy, and reducing equiva‐ lents; this is illustrated by the net balances for IPP biosynthesis from glyceraldehyde-3-

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( ) ( ) <sup>+</sup> MVA:3 GAP + 3 ADP + 4 NAD P + 2 P IPP + 4 CO + 3 ATP + 4 NAD P H i 2 ® (1)

Based on these balances, IPP production via the MEP pathway is more efficient at carbon utilization, as only 2 GAPs are required and 1 CO2 is emitted, compared to 3 GAPs and 4 CO2 for the MVA pathway. On the other hand, IPP production via the MVA pathway is more energy efficient overall, resulting in ATP generation and yielding a net gain in reducing equivalents (NAD(P)H). These carbon, energy, and reducing equivalent requirements should be considered when designing a metabolic engineering strategy for isoprenoid biosynthesis.

The MVA pathway interfaces with the primary metabolism at the acetyl-CoA node (Figure 3), and it can be divided into two parts: the top, which involves 3 enzymatic steps to convert acetyl-CoA to MVA, and the 3 enzymatic conversions of the bottom portion to produce IPP from MVA. One novel metabolic engineering strategy compared the efficiencies of the top and bottom portions of the MVA pathway in *E. coli* using heterologously expressed pathways from 5 different eukaryotic sources. The most efficient top and bottom portions were combined to maximize the yield of isoprenoid building blocks [35]. Accumulation of an intermediate metabolite, 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA), is a known bottleneck in the top MVA pathway, and HMG-CoA was also shown to inhibit cell growth in *E. coli* [36]. Thus, overexpression of the HMG-CoA reductase (HMGCR) increased MEV production and synthesis of subsequent FPP-derivatives in both *E. coli* and *S. cerevisiae*[36-38]. Whole pathway

MEP:2 GAP + ADP + CTP + P IPP + CO + ATP + CMP + PP i2 i ® (2)

In addition to oxygen-containing biofuels, acyl-ACP can also be converted into pure hydro‐ carbon fuels in the form of alkanes and alkenes (Figure 3). In 2010, the discovery of an alkane synthesis pathway in cyanobacteria provided the genetic knowledge necessary for engineering microbial alkane production [25]. The pathway consists of two enzymatic steps: (1) reduction of acyl-ACP to a fatty aldehyde by means of an acyl-ACP reductase (AAR) and (2) decarbon‐ ylation of the aldehyde to an alkane or alkene, catalyzed by an aldehyde decarbonylase (ADC). Due to the recent discovery of this pathway, few metabolic engineering strategies have been applied for alkane production. Some strategies focus on improving supply of the acyl-ACP precursor, relying on the native cyanobacterial pathway for alkane synthesis [23], while others have simply transferred the alkane pathway (AAR and ADC) into another host organism [25-27]. With the rapidly growing database of genome sequence information, numerous homologs of AAR and ADC have been identified [26, 27], representing a diverse range of targets for metabolic engineering. Future optimization of the alkane biosynthesis pathway may result in the high alkane yields needed for biofuel production.

#### **2.2. Isoprenoid-based biofuels**

The chemical composition of petroleum-based fuels: gasoline, diesel, and jet fuel, includes linear, branched, and cyclic alkanes, aromatics, and chemical additives [28]. Isoprenoid-based biofuels have the structural diversity to mimic these petroleum compounds, with up to 50,000 known isoprenoid structures including branched and cyclic hydrocarbons with varying degrees of unsaturation [29, 30]. Isoprenoids reported to be potential fuel candidates include: the hemiterpene (C5) isoprene; monoterpenes (C10): terpinene, pinene, limonene, and sabinene; the sesquiterpene (C15) farnesene, and their associated alcohols: isopentenol, terpineol, geraniol, and farnesol [12, 31]. Two metabolic pathways are capable of producing the isoprenoid building blocks isopentenyl pyrophosphate (IPP) and dimethylallyl diphos‐ phate (DMAPP): the mevalonate (MVA) pathway [32] and the methylerythritol phosphate (MEP) pathway, also known as the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway and the non-mevalonate pathway (Figure 3) [33]. In general, the MVA pathway is found in eukaryotes and archaea while the MEP pathway is utilized by prokaryotes. In agreement with the proposed evolutionary origin of plants, they contain both isoprenoid pathways with the MEP pathway localized in the plastid and the MVA pathway in the cytosol [34]. The MVA and MEP pathways differ with respect to their requirement for carbon, energy, and reducing equiva‐ lents; this is illustrated by the net balances for IPP biosynthesis from glyceraldehyde-3 phosphate (GAP):

$$\text{NAVA:}\\\text{3GAP} + \text{3 ADP} + \text{4 NAD} \text{(P)}^{\text{+}} + \text{2 P}\_{\text{l}} \rightarrow \text{IPP} + \text{4 CO}\_2 + \text{3 ATP} + \text{4NAD(P)H} \text{(H)}\tag{1}$$

$$\text{MEP-2 GAP} + \text{ADP} + \text{CTP} + \text{P}\_{\text{i}} \rightarrow \text{IPP} + \text{CO}\_2 + \text{ATP} + \text{CMP} + \text{PP}\_{\text{i}} \tag{2}$$

Based on these balances, IPP production via the MEP pathway is more efficient at carbon utilization, as only 2 GAPs are required and 1 CO2 is emitted, compared to 3 GAPs and 4 CO2 for the MVA pathway. On the other hand, IPP production via the MVA pathway is more energy efficient overall, resulting in ATP generation and yielding a net gain in reducing equivalents (NAD(P)H). These carbon, energy, and reducing equivalent requirements should be considered when designing a metabolic engineering strategy for isoprenoid biosynthesis.

The MVA pathway interfaces with the primary metabolism at the acetyl-CoA node (Figure 3), and it can be divided into two parts: the top, which involves 3 enzymatic steps to convert acetyl-CoA to MVA, and the 3 enzymatic conversions of the bottom portion to produce IPP from MVA. One novel metabolic engineering strategy compared the efficiencies of the top and bottom portions of the MVA pathway in *E. coli* using heterologously expressed pathways from 5 different eukaryotic sources. The most efficient top and bottom portions were combined to maximize the yield of isoprenoid building blocks [35]. Accumulation of an intermediate metabolite, 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA), is a known bottleneck in the top MVA pathway, and HMG-CoA was also shown to inhibit cell growth in *E. coli* [36]. Thus, overexpression of the HMG-CoA reductase (HMGCR) increased MEV production and synthesis of subsequent FPP-derivatives in both *E. coli* and *S. cerevisiae*[36-38]. Whole pathway expression and elimination of the HMGCR bottleneck have proven to be successful techniques for enhancing the metabolic throughput of the MVA pathway.

the precursor for isoprenoid production via the MVA pathway. Overexpression of acetalde‐ hyde dehydrogenase (ALDH) and acetyl-CoA synthetase (ACS), both of which produce acetyl-CoA, increased the acetyl-CoA supply and subsequently isoprenoid biosynthesis in *S. cerevisiae*[52]. On the other hand, the MEP pathway requires two precursors from the glycolysis pathway: PYR and GAP. The supply of these metabolites is complicated by the fact that PYR is derived from GAP, and consequently, the PYR/GAP balance is an important metabolic engineering target. The supply of GAP was shown to be limiting in *E. coli*, as modifying the conversion between PEP and PYR to redistribute the flux toward GAP synthesis increased isoprenoid production [53]. In addition to the carbon precursors, co-factors in the form of energy (ATP, CTP) and reducing equivalents (NADPH) are also required for isoprenoid synthesis. Co-factor supply is often overlooked in strategies for isoprenoid production, yet by improving the availability of NADPH in *S. cerevisiae*, isoprenoid synthesis through the MVA pathway increased by 85% [54]. This result emphasizes the importance of co-factor availability. Despite optimizing production of the isoprenoid building blocks, the downstream efficiency of assembling the final fuel product may still limit the overall yield. Successful strategies for improving downstream efficiency include overexpression of GPP and FPP synthases [47], overexpression and codon optimization of hemiterpene, monoterpene, and sesquiterpene synthases [41, 47, 48], fusion proteins to localize FPP synthesis and its conversion to sesqui‐ terpene [47], and downregulation of competing products like squalene [37, 48]. The optimized production of isoprenoid-based fuels requires strategies to address limitations throughout the

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metabolic pathway, from precursor and co-factor supply to end product synthesis.

**3. Influence of feedstock on hydrocarbon-based biofuel production**

Alternatively, the fixation of inorganic carbon feedstock (CO2/HCO3

While hydrocarbon-based biofuel production relies on the biosynthetic pathways discussed in the previous section, the source of feedstock plays an important role in the overall produc‐ tion process. As discussed in the Introduction to this chapter, there are two main feedstocks for biofuel production: lignocellulosic biomass and gaseous CO2, supporting the production of second and third generation biofuels, respectively (Figure 1). Both processes ultimately rely on CO2 and sunlight as the carbon and energy source, but the microbial conversion processes are distinctly different between the two feedstocks. Lignocellulosic biomass deconstruction produces organic carbon, mostly in the form of hexoses and pentoses (C5 and C6 sugars); this feedstock requires heterotrophic microorganisms to convert the organic carbon into biofuel.

upon autotrophic microbes. The heterotroph vs. autotroph requirement of the respective feedstocks is an important distinction from both the metabolic engineering and biofuel production perspectives. Only a few model microorganisms are capable of both heterotrophy and autotrophy, resulting in different host candidates for second and third generation biofuel production. The feedstock will also influence the metabolic engineering targets, as hetero‐ trophs utilize glycolysis and oxidative phosphorylation pathways for carbon consumption and energy production while oxygen-generating autotrophs utilize the Calvin-Benson-Bassham cycle and photosynthesis under light conditions (Figure 4). This section will discuss the host


) into biofuel is reliant

The MEP pathway requires two primary metabolites as precursors: GAP and pyruvate (PYR) (Figure 3). Compared to the 6 enzymatic steps of the MVA pathway, the MEP pathway is comprised of 7 steps. Metabolic engineering strategies for the MEP pathway have primarily focused on the first two enzymatic steps. Overexpression of 1-deoxy-D-xylulose-5-phosphate synthase (*dxs*), catalyzing the conversion of GAP and PYR to 1-deoxy-D-xylulose-5-phosphate (DXP), resulted in 6-10-fold increases in the final isoprenoid product [39, 40]. Targeting the next enzymatic step through overexpression of DXP reductoisomerase (*dxr*) was shown to have little effect on isoprenoid production using the native gene; however, expression of *dxs* and *dxr* from *Bacillus subtilis* improved isoprenoid production 2.3-fold in *E. coli* [41]. The final step of the MEP pathway was also shown to be rate-limiting, as heterologous expression of IPP isomerases (IPPI) enhanced isoprenoid production in *E. coli* [42]. Based on its rate-limiting steps, the MEP pathway is a prime candidate for a push-pull metabolic engineering strategy, whereby overexpression of the first step 'pushes' carbon flux into the MEP pathway and overexpression of the final step 'pulls' the metabolic flux towards the end product. This strategy yielded nearly 2-fold improvements in isoprenoid production in *E. coli* [43, 44]. Lastly, overexpression of the entire MEP pathway can increase isoprenoid biosynthesis. In fact, Leonard and colleagues demonstrated that 5 additional copies of the MEP pathway genes yielded the highest production, while further increasing the gene copy number to 10 produced lower titers [45].

While targeted gene overexpression may alleviate pathway bottlenecks, the pathway is still subject to native regulatory mechanisms which may limit isoprenoid biosynthesis from either the MVA or MEP pathways. A highly successful strategy for overcoming regulatory limitations is overexpression of the non-native isoprenoid pathway. Expression of the MVA pathway from *Saccharomyces cerevisiae* in *E. coli* has enabled higher levels of isoprenoid synthesis compared to engineering the native MEP pathway as the sole isoprenoid pathway [46-50]. The success of this strategy has made it a favorite among metabolic engineers seeking to improve isopre‐ noid biosynthesis. Farmer and Liao presented a clever approach for regulating the carbon flux into an engineered MEP pathway in *E. coli* [51]. In this work, a native regulatory circuit was used to control the carbon flux into and through the MEP pathway by regulating expression of two key enzymes: phosphoenolpyruvate synthase (PPS) and isopentenyl diphosphate isomerase (IPPI). Under excess carbon flux, expression of *pps* and *idi* was activated using the regulatory circuit, redirecting carbon flux into and through the MEP pathway, yet when the carbon flux was growth limiting, expression of these genes was reduced. This strategy allows for high isoprenoid production without negatively impacting cell growth. As evidence, the regulated pathway improved isoprenoid titers by 50%, while simply placing *pps* and *ippi* under control of strong *tac* promoters resulted in growth inhibition [51]. Native regulatory mecha‐ nisms are often obstacles limiting isoprenoid biosynthesis, yet they can also be exploited to optimize the flux balance to support both cell growth and isoprenoid production.

Additional targets for improving isoprenoid-based fuel production include precursor supply, cofactor supply, and optimization of the downstream fuel synthesis pathway. Acetyl-CoA is the precursor for isoprenoid production via the MVA pathway. Overexpression of acetalde‐ hyde dehydrogenase (ALDH) and acetyl-CoA synthetase (ACS), both of which produce acetyl-CoA, increased the acetyl-CoA supply and subsequently isoprenoid biosynthesis in *S. cerevisiae*[52]. On the other hand, the MEP pathway requires two precursors from the glycolysis pathway: PYR and GAP. The supply of these metabolites is complicated by the fact that PYR is derived from GAP, and consequently, the PYR/GAP balance is an important metabolic engineering target. The supply of GAP was shown to be limiting in *E. coli*, as modifying the conversion between PEP and PYR to redistribute the flux toward GAP synthesis increased isoprenoid production [53]. In addition to the carbon precursors, co-factors in the form of energy (ATP, CTP) and reducing equivalents (NADPH) are also required for isoprenoid synthesis. Co-factor supply is often overlooked in strategies for isoprenoid production, yet by improving the availability of NADPH in *S. cerevisiae*, isoprenoid synthesis through the MVA pathway increased by 85% [54]. This result emphasizes the importance of co-factor availability. Despite optimizing production of the isoprenoid building blocks, the downstream efficiency of assembling the final fuel product may still limit the overall yield. Successful strategies for improving downstream efficiency include overexpression of GPP and FPP synthases [47], overexpression and codon optimization of hemiterpene, monoterpene, and sesquiterpene synthases [41, 47, 48], fusion proteins to localize FPP synthesis and its conversion to sesqui‐ terpene [47], and downregulation of competing products like squalene [37, 48]. The optimized production of isoprenoid-based fuels requires strategies to address limitations throughout the metabolic pathway, from precursor and co-factor supply to end product synthesis.

#### **3. Influence of feedstock on hydrocarbon-based biofuel production**

While hydrocarbon-based biofuel production relies on the biosynthetic pathways discussed in the previous section, the source of feedstock plays an important role in the overall produc‐ tion process. As discussed in the Introduction to this chapter, there are two main feedstocks for biofuel production: lignocellulosic biomass and gaseous CO2, supporting the production of second and third generation biofuels, respectively (Figure 1). Both processes ultimately rely on CO2 and sunlight as the carbon and energy source, but the microbial conversion processes are distinctly different between the two feedstocks. Lignocellulosic biomass deconstruction produces organic carbon, mostly in the form of hexoses and pentoses (C5 and C6 sugars); this feedstock requires heterotrophic microorganisms to convert the organic carbon into biofuel. Alternatively, the fixation of inorganic carbon feedstock (CO2/HCO3 - ) into biofuel is reliant upon autotrophic microbes. The heterotroph vs. autotroph requirement of the respective feedstocks is an important distinction from both the metabolic engineering and biofuel production perspectives. Only a few model microorganisms are capable of both heterotrophy and autotrophy, resulting in different host candidates for second and third generation biofuel production. The feedstock will also influence the metabolic engineering targets, as hetero‐ trophs utilize glycolysis and oxidative phosphorylation pathways for carbon consumption and energy production while oxygen-generating autotrophs utilize the Calvin-Benson-Bassham cycle and photosynthesis under light conditions (Figure 4). This section will discuss the host organisms, engineering strategies, and biofuel production processes specific to each carbon feedstock.

**3.1. Hydrocarbon biofuel production from organic carbon feedstocks**

be developed for other organisms with desirable fuel production traits.

20 - 32.6% dcw, 0.12

**Concentration Range Microbial Hosts References**

0.5 – 7 g/L *Escherichia coli* [5, 12, 13, 19, 59,

g/L *Chlamydomonas reinhardtii* [56-58]

0.4 – 0.7 g/L *Saccharomyces cerevisiae* [63, 64]

0.07 – 1.5 g/L *Escherichia coli* [18, 19, 65-67] N/A *Saccharomyces cerevisiae* [17]

0.002 – 1 g/L *Escherichia coli* [35, 39, 42, 45, 50,

0.01 g/L *Saccharomyces cerevisiae* [37, 52]

0.024 – 0.2 g/L *Saccharomyces cerevisiae* [61, 62]

60]

66, 68]

69]

*Heterotrophic Production*

**Fatty alcohols** 0.001 – 1.67 g/L *Escherichia coli* [13, 19, 22, 27, 59,

**Alkanes/Alkenes** 0.042 – 0.32 g/L *Escherichia coli* [25, 27]

**Hydrocarbon Fuel/ Fuel Precursor**

**FFA**

**TAG**

**FAEE**

**Other Isoprenoids (lycopene, β-carotene, amorphadiene,**

The release of C5 and C6 sugars from lignocellulosic biomass deconstruction supports the growth of heterotrophic microorganisms and the metabolic conversion of sugars into biofuel. Representative hydrocarbon-based fuel titers produced by engineered, heterotrophic hosts are listed in Table 1. The most common heterotrophic hosts for biofuel production are the model organisms *Escherichia coli* and *Saccharomyces cerevisiae*. These hosts are attractive candidates for fuel production due to their fast growth rates, well-known genetics and regulation, advanced molecular tools for genetic engineering, and established use in the industrial setting. Neither *E. coli* nor *S. cerevisiae* naturally produce significant amounts of hydrocarbon-based fuels, necessitating the application of metabolic engineering techniques. Heterotrophic organisms that naturally produce hydrocarbon-based fuels are also potential hosts for large-scale biofuel production. For example, *Bacillus subtilis* naturally produces higher concentrations of isoprene than other commonly known bacteria like *E. coli* [55]. *B. subtilis* is also a model organism for Gram-positive bacteria with established tools for genetic modification, advancing its appeal as a host for isoprene production. Similarly, heterotrophic algae can produce significant quantities of TAG. This has motivated some preliminary investigation into engineering the model green alga, *Chlamydomonas reinhardtii*, for TAG production [56-58]. While most meta‐ bolic engineering efforts have focused on these model heterotrophic hosts, genetic tools can

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**Figure 4.** Heterotrophic (A) and autotrophic (B) pathways for carbon utilization, with the Embden-Meyerhof-Parnas (EMP) pathway (glycolysis) in black, the pentose phosphate pathway (PPP) in blue, pentose utilization pathways in red, glycerol metabolism in purple, and the Calvin-Benson-Bassham cycle in green. Abbreviations for metabolites and en‐ zymes are listed at the end of the chapter.

#### **3.1. Hydrocarbon biofuel production from organic carbon feedstocks**

The release of C5 and C6 sugars from lignocellulosic biomass deconstruction supports the growth of heterotrophic microorganisms and the metabolic conversion of sugars into biofuel. Representative hydrocarbon-based fuel titers produced by engineered, heterotrophic hosts are listed in Table 1. The most common heterotrophic hosts for biofuel production are the model organisms *Escherichia coli* and *Saccharomyces cerevisiae*. These hosts are attractive candidates for fuel production due to their fast growth rates, well-known genetics and regulation, advanced molecular tools for genetic engineering, and established use in the industrial setting. Neither *E. coli* nor *S. cerevisiae* naturally produce significant amounts of hydrocarbon-based fuels, necessitating the application of metabolic engineering techniques. Heterotrophic organisms that naturally produce hydrocarbon-based fuels are also potential hosts for large-scale biofuel production. For example, *Bacillus subtilis* naturally produces higher concentrations of isoprene than other commonly known bacteria like *E. coli* [55]. *B. subtilis* is also a model organism for Gram-positive bacteria with established tools for genetic modification, advancing its appeal as a host for isoprene production. Similarly, heterotrophic algae can produce significant quantities of TAG. This has motivated some preliminary investigation into engineering the model green alga, *Chlamydomonas reinhardtii*, for TAG production [56-58]. While most meta‐ bolic engineering efforts have focused on these model heterotrophic hosts, genetic tools can be developed for other organisms with desirable fuel production traits.



in these two xylose utilization pathways include the inhibition of XI by xylitol (Xol) and the reducing equivalents required by XR and XDH [80]. Successful strategies for engineering xylose utilization in *S. cerevisiae* include expression of a fungal XI from *Piromyces* sp. E2 along with overexpression of the non-oxidative PPP pathway [84] and expression of XR and XDH from the xylose-fermenting yeast *Pichia stipitis* [85]. Two pathways have also been expressed in *S. cerevisiae* for arabinose utilization [86, 87]. The bacterial pathway for arabinose catabolism consists of 3 enzymatic steps, while the fungal pathway involves 5 enzymatic steps, 4 of which require cofactors of NADPH or NAD+ (Figure 4). Efficient arabinose utilization in *S. cerevi‐ siae* has been achieved through heterologous expression of a bacterial arabinose catabolism pathway along with overexpression of the non-oxidative PPP and evolutionary engineering [88]. While most of these metabolic engineering examples focus on utilizing sugars for fermentation to ethanol, the strategies for engineering carbon utilization can also be applied

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Unlike *S. cerevisiae*, *E. coli* can utilize the hexoses and pentoses derived from lignocellulose; however, the carbon catabolite repression (CCR) system in *E. coli* leads to inefficient, diauxic growth [89]. Through CCR, *E. coli* sequentially consumes different sources of organic carbon based on substrate preference, leading to delayed and often incomplete utilization of unpre‐ ferred sugars like xylose and arabinose. This translates into lower productivities and yields along with downstream complications due to the presence of unmetabolized sugars [80]. As a result, CCR is often targeted by metabolic engineering to alleviate these undesired effects. A common engineering strategy is to use mutants of the transcriptional activator CRP (cyclic AMP receptor protein) which have been modified to eliminate the allosteric requirement for cAMP, thereby leading to expression of the pentose catabolizing pathways in the presence of the preferred substrate, glucose [90]. The phosphotransferase system (PTS), responsible for the preferential uptake of glucose, has also been deleted to encourage simultaneous utilization of mixed sugars [91]. Lastly, deletion of methylglyoxyal synthase was shown to improve the cometabolism of sugars, ostensibly due to elimination of methylglyoxyal, an inhibitor of sugar metabolism [92]. Through modifying the components of CCR, *E. coli* can be engineered to efficiently utilize the organic carbon mixture resulting from lignocellulose degradation.

In addition to the hexoses and pentoses derived from lignocellulosic biomass, glycerol may soon become an inexpensive organic carbon source for fuel production. Glycerol is a byproduct of the conversion of TAG into biodiesel during algal biofuel processing, and thus, large quantities of glycerol may be available for use as an organic carbon source. The main pathway for aerobic glycerol utilization involves a two-step conversion to produce the glycolytic metabolite DHAP [93]. The glycerol utilization pathway is not a common target for metabolic engineering, yet glycerol has been reported as a supplementary carbon source for the produc‐ tion of isoprenoid-based fuels, farnesol and α-farnesene [47, 48]. Future metabolic engineering

efforts may focus more on glycerol utilization as the availability of glycerol increases.

Second generation biofuel production still remains to be demonstrated at large scales, yet the overall process is easily integrated with current technologies. Equipment and practices used for agricultural harvesting can be directly applied to harvesting lignocellulosic biomass. In fact, some agricultural processes already produce biomass waste streams that can be utilized

for hydrocarbon-based fuel production.

**Table 1.** Hydrocarbon fuels and fuel precursors produced by genetically engineered microorganisms.

Most heterotrophic hosts for biofuel production utilize the Embden-Meyerhof-Parnas (EMP) pathway for sugar catabolism (Figure 4). The EMP pathway has evolved for efficient carbon utilization and is typically not rate-limiting for fuel production. As such, EMP pathway enzymes are not often targeted for genetic manipulation. However, the organic feedstock from lignocellulose deconstruction is comprised of a range of sugars, including hexoses: glucose, mannose, and galactose, and pentoses: xylose and arabinose [79]. A major concern in convert‐ ing these sugars into fuel is the efficient utilization of all available hexoses and pentoses. While some organisms like *E. coli* can naturally metabolize these different forms of sugar, others, like *S. cerevisiae*, can only utilize specific forms [80]. *S. cerevisiae* does not naturally express path‐ ways for catabolizing pentoses. There are two known pathways for xylose catabolism, both of which have been expressed in *S. cerevisiae* [81-83]. Xylose can be converted into xylulose-5 phosphate (Xu5P), an intermediate in the pentose phosphate pathway (PPP), through expres‐ sion of a xylose isomerase (XI) and xylulose kinase (XK) [82]. Alternatively, the XI can be replaced by a xylose reductase (XR) and xylitol dehydrogenase (XDH) [81, 82]. Complications

in these two xylose utilization pathways include the inhibition of XI by xylitol (Xol) and the reducing equivalents required by XR and XDH [80]. Successful strategies for engineering xylose utilization in *S. cerevisiae* include expression of a fungal XI from *Piromyces* sp. E2 along with overexpression of the non-oxidative PPP pathway [84] and expression of XR and XDH from the xylose-fermenting yeast *Pichia stipitis* [85]. Two pathways have also been expressed in *S. cerevisiae* for arabinose utilization [86, 87]. The bacterial pathway for arabinose catabolism consists of 3 enzymatic steps, while the fungal pathway involves 5 enzymatic steps, 4 of which require cofactors of NADPH or NAD+ (Figure 4). Efficient arabinose utilization in *S. cerevi‐ siae* has been achieved through heterologous expression of a bacterial arabinose catabolism pathway along with overexpression of the non-oxidative PPP and evolutionary engineering [88]. While most of these metabolic engineering examples focus on utilizing sugars for fermentation to ethanol, the strategies for engineering carbon utilization can also be applied for hydrocarbon-based fuel production.

Unlike *S. cerevisiae*, *E. coli* can utilize the hexoses and pentoses derived from lignocellulose; however, the carbon catabolite repression (CCR) system in *E. coli* leads to inefficient, diauxic growth [89]. Through CCR, *E. coli* sequentially consumes different sources of organic carbon based on substrate preference, leading to delayed and often incomplete utilization of unpre‐ ferred sugars like xylose and arabinose. This translates into lower productivities and yields along with downstream complications due to the presence of unmetabolized sugars [80]. As a result, CCR is often targeted by metabolic engineering to alleviate these undesired effects. A common engineering strategy is to use mutants of the transcriptional activator CRP (cyclic AMP receptor protein) which have been modified to eliminate the allosteric requirement for cAMP, thereby leading to expression of the pentose catabolizing pathways in the presence of the preferred substrate, glucose [90]. The phosphotransferase system (PTS), responsible for the preferential uptake of glucose, has also been deleted to encourage simultaneous utilization of mixed sugars [91]. Lastly, deletion of methylglyoxyal synthase was shown to improve the cometabolism of sugars, ostensibly due to elimination of methylglyoxyal, an inhibitor of sugar metabolism [92]. Through modifying the components of CCR, *E. coli* can be engineered to efficiently utilize the organic carbon mixture resulting from lignocellulose degradation.

In addition to the hexoses and pentoses derived from lignocellulosic biomass, glycerol may soon become an inexpensive organic carbon source for fuel production. Glycerol is a byproduct of the conversion of TAG into biodiesel during algal biofuel processing, and thus, large quantities of glycerol may be available for use as an organic carbon source. The main pathway for aerobic glycerol utilization involves a two-step conversion to produce the glycolytic metabolite DHAP [93]. The glycerol utilization pathway is not a common target for metabolic engineering, yet glycerol has been reported as a supplementary carbon source for the produc‐ tion of isoprenoid-based fuels, farnesol and α-farnesene [47, 48]. Future metabolic engineering efforts may focus more on glycerol utilization as the availability of glycerol increases.

Second generation biofuel production still remains to be demonstrated at large scales, yet the overall process is easily integrated with current technologies. Equipment and practices used for agricultural harvesting can be directly applied to harvesting lignocellulosic biomass. In fact, some agricultural processes already produce biomass waste streams that can be utilized for feedstock, such as corn stover. Moreover, commercial fermenters can be employed as bioreactors for the microbial fuel conversion. The main technical difficulties in large-scale lignocellulosic fuel production center on provision of the carbon source. The quantities of biomass needed to support industrial-scale fuel production will require a significant invest‐ ment of land and nutrient resources, and the supply will be subject to varying climate conditions. A supply chain infrastructure must also be constructed to harvest the biomass and transport it to the production facilities. A primary technical focus of current research on lignocellulosic-derived fuels is the deconstruction of biomass into useable sugars. The thermal, chemical, and enzymatic processes for biomass deconstruction have been a limiting factor for economical second generation biofuel production [94, 95]. As the cost of biomass deconstruc‐ tion is reduced with new technology, the large-scale production of second generation biofuels will begin to contribute to the world's supply of renewable energy.

[99]. Along with this diversity of habitat, algae have evolved diverse cellular physiologies and genetics, resulting in a wealth of potential hosts and genetic sources for engineering fuel production. Many types of algae are currently under consideration for fuel production due to their natural TAG synthesis, including diatoms, green algae, eustigmatophytes, prymnesio‐ phytes, and red algae [100]. While many types of algae produce the fuel precursor TAG, few algal species have well-developed genetic tools available for engineering improved lipid production [101, 102]. Consequently, there are only a few reported examples of engineering

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To date, the only genetic mutation shown to improve lipid production in algae is the elimina‐ tion of starch biosynthesis, a competing carbon sink. The generation of mutants with impaired starch synthesis using random mutagenesis techniques resulted in up to a 10-fold increase in cellular lipid production in *C. reinhardtii* [56-58, 103]. Other targeted metabolic engineering attempts, such as overexpression of ACC in the diatoms *Cyclotella cryptic* and *Navicula saprophila*, failed to improve TAG biosynthesis [15, 96]. In addition to targeting overall TAG production, metabolic engineering strategies have been applied to influence the chemical composition of the fatty acid side chains. By expressing two heterologous TEs, the diatom *Phaeodactylum tricornutum* produced TAG with increased levels of lauric acid (C12:0) and myristic acid (C14:0) [104]. These shorter chain length fatty acids are more desirable for fuel production, and this demonstrates the potential to control the chemical composition of the fuel product and its associated properties with metabolic engineering. While examples of engi‐ neering algal TAG production are sparse, many engineering strategies have proven successful at improving the fatty acid content in plants. These strategies include expression of ACC and KASIII involved in fatty acid biosynthesis, expression of G3P dehydrogenase (GPD) for production of the glycerol backbone of TAG, expression of ATs such as DGAT, expression of TEs to release FFAs, and deletion of desaturases to alter the fatty acid composition [105]. Similar

strategies may also be successful at improving TAG production in algae.

The metabolic engineering of algae is complicated by several factors. Most algae have a rigid cell wall structure that makes transformation difficult. A common transformation technique uses glass beads (or silicon carbide whiskers) along with a cell wall-deficient algal strain [106]. The cell wall can be removed using enzymatic techniques or through genetic mutation. Alternatively, a microparticle bombardment technique has been applied successfully to transform many different algal species [107]. In this technique, the recombinant DNA is coated onto a metal microparticle and 'shot' into the algal cell using a helium-powered 'gun'. Other transformation methods include electroporation and the traditional plant transformation technique of *Agrobacterium tumefaciens* T-DNA-mediated transfer [107]. Once the recombinant DNA enters the cell, it must integrate into one of 3 algal genomes: nuclear, chloroplast, or mitochondrial (assuming the transformed DNA is not a stably maintained plasmid). DNA has been successfully integrated into the chloroplast genome via homologous recombination, whereby the recombinant gene and marker are flanked by homologous (i.e. matching) regions of the targeted chloroplast DNA, and the recombinant DNA replaces the matching region in the chloroplast. Unfortunately, homologous recombination does not occur in the nuclear genomes of many algae [108], and instead, the recombinant DNA is randomly integrated into

algae for biofuel production.

#### **3.2. Hydrocarbon biofuel production from inorganic carbon feedstocks**

The direct conversion of CO2 into hydrocarbon-based fuels could greatly simplify the overall production process and reduce the cost of biofuel production (Figure 1). The search for autotrophic microorganisms capable of performing this CO2-to-fuel conversion started in the late 1970's with the U.S. Department of Energy's Aquatic Species Program (ASP) [96]. The ASP isolated and screened over 3,000 species of microalgae from a diverse range of environmental habitats. The program focused mainly on eukaryotic algae, as they naturally produce signifi‐ cant amounts of TAG. During the course of the program, the recombinant DNA technology used in metabolic engineering was developed, yet due to the infancy of this technology, it was not applied to microalgae for fuel applications until near the end of the ASP [15]. With the development of recombinant DNA technology, prokaryotic microalgae (i.e. cyanobacteria, previously known as blue-green algae) were recognized as potential hosts for fuel production, and the successful engineering of cyanobacteria for ethanol production confirmed their potential [97]. Unfortunately, research funding for microalgal fuel production waned as crude oil prices fell in the 1990's. However, in the late 2000's, the cost of crude oil soared, spurring a resurgence of interest in microalgae for fuel production and in the application of metabolic engineering to enhance fuel yields. In general, both eukaryotic microalgae (referred to as algae in the subsequent text) and prokaryotic microalgae (referred to as cyanobacteria in the subsequent text) utilize photosynthesis for energy generation and the Calvin-Benson-Bassham cycle for CO2 fixation (Figure 4). However, due to the cellular differences between algae and cyanobacteria, the strategies for engineering autotrophic fuel production will be discussed based on this host division.

#### *3.2.1. Engineering algae for biofuel production*

Algae are predicted to have first appeared approximately 1.5 billion years ago from an endosymbiotic event in which a eukaryotic cell engulfed a cyanobacterium [98]. The cyano‐ bacterium evolved into the modern day chloroplast, the algal organelle responsible for photosynthesis and carbon fixation. Today, algae can be found in a wide-range of environ‐ mental habitats from freshwater lakes and oceans to deserts and even the snow of the Antarctic [99]. Along with this diversity of habitat, algae have evolved diverse cellular physiologies and genetics, resulting in a wealth of potential hosts and genetic sources for engineering fuel production. Many types of algae are currently under consideration for fuel production due to their natural TAG synthesis, including diatoms, green algae, eustigmatophytes, prymnesio‐ phytes, and red algae [100]. While many types of algae produce the fuel precursor TAG, few algal species have well-developed genetic tools available for engineering improved lipid production [101, 102]. Consequently, there are only a few reported examples of engineering algae for biofuel production.

To date, the only genetic mutation shown to improve lipid production in algae is the elimina‐ tion of starch biosynthesis, a competing carbon sink. The generation of mutants with impaired starch synthesis using random mutagenesis techniques resulted in up to a 10-fold increase in cellular lipid production in *C. reinhardtii* [56-58, 103]. Other targeted metabolic engineering attempts, such as overexpression of ACC in the diatoms *Cyclotella cryptic* and *Navicula saprophila*, failed to improve TAG biosynthesis [15, 96]. In addition to targeting overall TAG production, metabolic engineering strategies have been applied to influence the chemical composition of the fatty acid side chains. By expressing two heterologous TEs, the diatom *Phaeodactylum tricornutum* produced TAG with increased levels of lauric acid (C12:0) and myristic acid (C14:0) [104]. These shorter chain length fatty acids are more desirable for fuel production, and this demonstrates the potential to control the chemical composition of the fuel product and its associated properties with metabolic engineering. While examples of engi‐ neering algal TAG production are sparse, many engineering strategies have proven successful at improving the fatty acid content in plants. These strategies include expression of ACC and KASIII involved in fatty acid biosynthesis, expression of G3P dehydrogenase (GPD) for production of the glycerol backbone of TAG, expression of ATs such as DGAT, expression of TEs to release FFAs, and deletion of desaturases to alter the fatty acid composition [105]. Similar strategies may also be successful at improving TAG production in algae.

The metabolic engineering of algae is complicated by several factors. Most algae have a rigid cell wall structure that makes transformation difficult. A common transformation technique uses glass beads (or silicon carbide whiskers) along with a cell wall-deficient algal strain [106]. The cell wall can be removed using enzymatic techniques or through genetic mutation. Alternatively, a microparticle bombardment technique has been applied successfully to transform many different algal species [107]. In this technique, the recombinant DNA is coated onto a metal microparticle and 'shot' into the algal cell using a helium-powered 'gun'. Other transformation methods include electroporation and the traditional plant transformation technique of *Agrobacterium tumefaciens* T-DNA-mediated transfer [107]. Once the recombinant DNA enters the cell, it must integrate into one of 3 algal genomes: nuclear, chloroplast, or mitochondrial (assuming the transformed DNA is not a stably maintained plasmid). DNA has been successfully integrated into the chloroplast genome via homologous recombination, whereby the recombinant gene and marker are flanked by homologous (i.e. matching) regions of the targeted chloroplast DNA, and the recombinant DNA replaces the matching region in the chloroplast. Unfortunately, homologous recombination does not occur in the nuclear genomes of many algae [108], and instead, the recombinant DNA is randomly integrated into the nuclear genome. This complicates metabolic engineering strategies due to the possibility of detrimental genetic effects resulting from the random integration and the lack of a technique for targeted gene knockout. Lastly, algal engineering attempts are often plagued by low gene expression. It has been discovered that many algae, like the model alga *C. reinhardtii*, employ RNA-mediated gene silencing [109]. Numerous strategies have been applied to combat the low gene expression brought about by gene silencing in algae, including codon optimization, the use of 5' and 3' untranslated regions which may participate in regulatory functions, and the inclusion of native intron sequences [108]. Knowledge of the gene silencing mechanisms in algae has led to the development of RNA interference (RNAi) technology for gene knock‐ down. RNAi exploits the native cellular machinery for gene silencing to reduce the expression of target genes [109]. As we continue to expand our knowledge of algal genetics, the list of engineered algae will rapidly increase. As evidence, the biofuel-relevant alga, *Nannochlorop‐ sis* sp., was recently shown to have a high efficiency of homologous recombination in the nuclear genome [110]. This will simplify future strategies for genetic engineering in *Nanno‐ chloropsis* sp. Another promising development is the construction of a plasmid for gene expression in *C. reinhardtii* that is now commercially available through Life Technologies [111]. The greater availability and standardization of tools for the genetic manipulation of algae will move algal engineering towards the advanced stages currently seen with other industrial organisms like *E. coli* and *S. cerevisiae*.

After the initial demonstration of engineering cyanobacteria for ethanol production [97], the production of hydrocarbon-based fuels in engineered cyanobacteria has expanded to include isoprene, FFAs, FAEEs, fatty alcohols, and alkanes/alkenes (Table 1). Isoprene biosynthesis was established in the model cyanobacterium, *Synechocystis* sp. PCC 6803, through expression of the isoprene synthase (*ispS*) from kudzu [78]. Codon optimization of *ispS* and the use of a strong promoter (*psbA2*) increased isoprene production. Engineering strategies targeting the upstream MEP pathway for isoprenoid biosynthesis, as described in Section 2.2 of this chapter, will likely further improve isoprene productivity. The remaining four hydrocarbon-based fuels are all derived from the fatty acid biosynthesis pathway. Common strategies for im‐ proving FFA production (see Section 2.1) have proven successful in cyanobacteria [74-76]. Eliminating non-essential, competing pathways such as polyhydroxybutyrate (PHB), cyano‐ phycin, and acetate biosynthesis also improved FFA production [74]. Liu and colleagues engineered a more permeable peptidoglycan layer to improve FFA excretion in *Synechocystis* sp., yet this weakened cell membrane resulted in slower growth rates and may also make the engineered cyanobacterium more susceptible to external predators and toxins that may be present in large-scale cultivations. Initial engineering attempts for fatty alcohol and alkane/ alkene production entail expression of a heterologous FAR and overexpression of AAR and ADC, respectively [23, 26]. Alkane/alkene synthesis was also observed with ACC overexpres‐ sion and native AAR and ADC activities in cyanobacteria [23]. Despite being derived from fatty acids, the synthesis of fatty alcohols and alkanes/alkenes is up to 1000-fold lower than that observed with FFA production (Table 1), suggesting that the conversion of acyl-ACP to the final fuel product is rate limiting. These inaugural proof-of-concept reports illustrate the potential of cyanobacteria as hosts for autotrophic biofuel production, but additional metabolic engineering will be required to achieve the fuel titers necessary for large-scale synthesis.

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The selection of organic or inorganic carbon feedstock for biofuel production has downstream ramifications on host selection, product yields, and process requirements. Clearly, the feedstock choice will determine whether a heterotrophic or autotrophic host is required, and in turn, this will influence the metabolic engineering strategy. In general, heterotrophic hosts have generated higher fuel titers than autotrophic hosts, with more than 10-fold higher concentrations of FFAs, FAEEs, fatty alcohols, and alkanes/alkenes (Table 1). This does not imply that heterotrophic production is more advantageous than autotrophic production, for the entire production process must be considered (Figure 1). The sugars from lignocellulosic biomass deconstruction (heterotrophic feedstock) have a higher energy content compared to inorganic carbon (autotrophic feedstock). The overall balances for obtaining one molecule of

GAP from heterotrophic and autotrophic metabolisms provide evidence for this:

Heterotrophic: ½ Glc + ATP GAP + ADP ® (3)

<sup>+</sup> Autotrophic: 3 CO + 9 ATP + 6 NADPH + 5 H O GAP + 9 ADP + 6 NADP + 8 P 2 2 ® <sup>i</sup> (4)

**3.3. Heterotrophic vs. autotrophic biofuel production**

#### *3.2.2. Engineering cyanobacteria for biofuel production*

Cyanobacteria are predicted to be the first microorganisms to develop the capability of oxygenic photosynthesis, some 2.7 billion years ago [112]. Similar to algae, cyanobacteria have a great range of diverse morphologies, cellular functions, and genetics, presumably due to their long evolutionary history and their diverse habitats. As discussed previously, the ASP initially deemed cyanobacteria unfit for fuel production due to their lack of natural TAG accumulation. Since they are amenable to genetic manipulation, however, cyanobacteria can be engineered to produce a range of biofuel products (Table 1). As prokaryotes, cyanobacteria are subject to the traditional methods employed for engineering other well-developed bacterial hosts like *E. coli*. Some strains of cyanobacteria are even naturally transformable, uptaking exogenous DNA from their environment without the use of cell permeablization techniques [113]. As progenitors of the algal chloroplast, cyanobacteria also integrate DNA into their chromosomes using homologous recombination. Moreover, cyanobacteria do not possess the cellular components for gene silencing. The genetic tools for engineering some model strains of cyanobacteria are well developed and have been used to genetically modify cyanobacteria for several decades [113]. Another advantage of using cyanobacteria as the microbial host for hydrocarbon-based fuel production is that they have been shown to excrete potential fuel precursors such as FFAs [73]. Fuel excretion enables a continuous production process, eliminating the cost associated with harvesting the algal biomass and the time and nutrients needed to repeatedly grow new batches of algae for fuel production. The advantages of straightforward genetic manipulation and fuel excretion make cyanobacteria contenders for large-scale biofuel production despite the disadvantage of low natural lipid yields.

After the initial demonstration of engineering cyanobacteria for ethanol production [97], the production of hydrocarbon-based fuels in engineered cyanobacteria has expanded to include isoprene, FFAs, FAEEs, fatty alcohols, and alkanes/alkenes (Table 1). Isoprene biosynthesis was established in the model cyanobacterium, *Synechocystis* sp. PCC 6803, through expression of the isoprene synthase (*ispS*) from kudzu [78]. Codon optimization of *ispS* and the use of a strong promoter (*psbA2*) increased isoprene production. Engineering strategies targeting the upstream MEP pathway for isoprenoid biosynthesis, as described in Section 2.2 of this chapter, will likely further improve isoprene productivity. The remaining four hydrocarbon-based fuels are all derived from the fatty acid biosynthesis pathway. Common strategies for im‐ proving FFA production (see Section 2.1) have proven successful in cyanobacteria [74-76]. Eliminating non-essential, competing pathways such as polyhydroxybutyrate (PHB), cyano‐ phycin, and acetate biosynthesis also improved FFA production [74]. Liu and colleagues engineered a more permeable peptidoglycan layer to improve FFA excretion in *Synechocystis* sp., yet this weakened cell membrane resulted in slower growth rates and may also make the engineered cyanobacterium more susceptible to external predators and toxins that may be present in large-scale cultivations. Initial engineering attempts for fatty alcohol and alkane/ alkene production entail expression of a heterologous FAR and overexpression of AAR and ADC, respectively [23, 26]. Alkane/alkene synthesis was also observed with ACC overexpres‐ sion and native AAR and ADC activities in cyanobacteria [23]. Despite being derived from fatty acids, the synthesis of fatty alcohols and alkanes/alkenes is up to 1000-fold lower than that observed with FFA production (Table 1), suggesting that the conversion of acyl-ACP to the final fuel product is rate limiting. These inaugural proof-of-concept reports illustrate the potential of cyanobacteria as hosts for autotrophic biofuel production, but additional metabolic engineering will be required to achieve the fuel titers necessary for large-scale synthesis.

#### **3.3. Heterotrophic vs. autotrophic biofuel production**

The selection of organic or inorganic carbon feedstock for biofuel production has downstream ramifications on host selection, product yields, and process requirements. Clearly, the feedstock choice will determine whether a heterotrophic or autotrophic host is required, and in turn, this will influence the metabolic engineering strategy. In general, heterotrophic hosts have generated higher fuel titers than autotrophic hosts, with more than 10-fold higher concentrations of FFAs, FAEEs, fatty alcohols, and alkanes/alkenes (Table 1). This does not imply that heterotrophic production is more advantageous than autotrophic production, for the entire production process must be considered (Figure 1). The sugars from lignocellulosic biomass deconstruction (heterotrophic feedstock) have a higher energy content compared to inorganic carbon (autotrophic feedstock). The overall balances for obtaining one molecule of GAP from heterotrophic and autotrophic metabolisms provide evidence for this:

$$\text{Heterotropic} \colon \mathbb{M}\text{Glc} + \text{ATP} \to \text{GAP} + \text{ADP} \tag{3}$$

$$\text{Autotropic:}\ 3\text{ CO}\_2 + 9\text{ ATP} + 6\text{ NADH} + 5\text{ H}\_2\text{O} \rightarrow \text{GAP} + 9\text{ ADP} + 6\text{ NADH}^\dagger + 8\text{ P}\_{\text{l}}\tag{4}$$

While autotrophic GAP generation requires a significant investment of energy (9 ATP) and reducing equivalents (6 NADPH), heterotrophic GAP production only requires one energy equivalent. However, if a life cycle perspective is considered, the carbon from lignocellulosic feedstocks is ultimately derived from photosynthesis, requiring the same energy and reducing equivalent input as autotrophic microorganisms. Overlooking this fact will bias a direct comparison between heterotrophic and autotrophic fuel production.

back almost a century [118], and we can capitalize on this wealth of information to engineer improved product tolerance in microbial hosts. Most fatty acid derived fuel molecules have shown some antimicrobial activity. FFAs, with a diverse range of carbon chain lengths and degrees of unsaturation, impart inhibitory effects on organisms including algae, Gramnegative and Gram-positive bacteria, fungi, protozoans, and various cell types of multicellular organisms [119]. Medium chain fatty alcohols such as pentanol, hexanol, heptanol, and octanol inhibited the biological activity of several algal and cyanobacterial strains, including fuelrelevant hosts *C. reinhardtii* and *Dunaliella salina* [120]. Interestingly, long-chain fatty alcohols (>C14) did not exhibit inhibitory effects on yeasts, suggesting that targeting longer chain fatty alcohols may eliminate the toxicity concern [121]. Similarly, medium-length alkanes (hexane, heptane, and isooctane) were toxic to microalgae while long-chain alkanes (C12-C16) elicited no effect [120, 121]. Microbial TAG and FAEE toxicities have not been reported. However, the phospholipid membrane surrounding algal TAGs may mask potential inhibitory effects, and FAEE production has been linked to the toxic effects of alcohol consumption in humans [122]. Isoprenoid-based fuel molecules have also illustrated inhibitory effects. Cyclic terpenes, such as pinene and limonene (Figure 2), inhibited the growth of bacteria and *S. cerevisiae* [123, 124], while branched isoprenoids, such as farnesyl hexanoate and geranyl acetate, were shown to be toxic to *E. coli* [125]. In fact, *E. coli*'s tolerance to isoprenoid-derived biodiesels and bioavi‐ ation fuels only ranged from 0.025 – 1% (v/v) [125]. Based on these previous studies, product toxicity is a major limiting factor and should be integrated into the metabolic engineering

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A variety of strategies can be adopted to address product toxicity. The easiest way to avoid complications from product toxicity is to select non-toxic fuel targets. Toxicity studies can be conducted for each potential host organism, and generally, fatty alcohols longer than C14, alkanes longer than C9, and alkenes longer than C12 have shown minimal microbial inhibition [120, 121]. Alternatively, metabolic engineering techniques can be applied to allow for a more diverse range of hydrocarbon fuel targets. Many cellular modifications have been shown to improve microbial solvent tolerance: changes in membrane lipid composition; altered enzymatic activities of membrane repair and energy transduction enzymes; solvent expulsion via efflux pump activity; and cellular stress responses including heat shock, phage shock, and general stress responses [118, 125, 126]. These natural mechanisms offer a range of engineering targets: expression of a cis-trans isomerase to alter lipid composition; overexpression of enzymes involved in membrane repair and energy transduction; expression of efflux pumps such as *tolC*, *mar*, *rob*, *soxS*, and *acrAB*; and overexpression of stress-induced enzymes such as phage shock protein, heat shock proteins, catalases, and superoxide dismutases [125, 126]. While few metabolic engineering efforts have focused on enhancing product tolerance, a recent study explored improving hydrocarbon-based fuel tolerance in *E. coli* by testing a library of 43 efflux pumps [127]. This work identified efflux pumps that improved tolerance to five potential isoprenoid derived fuels. This preliminary success at engineering solvent tolerance should inspire additional efforts to improve the microbial production of both fatty acid and

strategy.

isoprenoid derived fuels.

One major difference between heterotrophic and autotrophic fuel production is the design considerations for the bioreactor. Heterotrophic microbes, such as *E. coli* and *S. cerevisiae*, are traditional industrial microorganisms with well-established, large-scale cultivation practices and bioreactors. On the other hand, autotrophic hosts like algae and cyanobacteria require light as the energy source to drive photosynthesis and inorganic carbon fixation. This can have a dramatic effect on bioreactor design. Transparent materials can be used with traditional bioreactor designs to allow for light penetration. Light availability, however, will ultimately limit the cell densities of photosynthetic microalgae, and the surface area of light exposure with traditional bioreactor designs is not optimal. Some have proposed to use fiber-optics within the liquid culture to improve light availability [114], but a costly solution such as this is not feasible for a low-value, commodity product like fuel. A wide-range of photobioreactor (PBR) designs have been proposed [115], yet generally, PBRs are characterized by the use of transparent materials, high surface area to volume ratios, and a relatively short pathlength for light. Other PBR design factors include a mechanism for air/CO2 delivery, dissipation of radiative heat, and removal of inhibitory O2 [115]. Due to the low value of fuel products, PBRs for fuel synthesis favor low-tech designs and inexpensive materials to reduce both capital and operating costs. In fact, NASA has proposed to float plastic bags of algal cultures in wastewater to allow for nutrient exchange [116]. Alternatively, open pond systems, traditionally a raceway configuration with a paddle-wheel for mixing, have proven successful for cultivating micro‐ algae at scale [117]. Unlike PBRs, ponds are open to the environment, allowing for evaporative water loss and pond crash due to contamination by predators and competitors. However, the low capital cost of an open pond system makes this design a contender for fuel production. Clearly, the large-scale cultivation techniques for autotrophic fuel production still require additional development and optimization compared to heterotrophic cultivation.

### **4. Other metabolic engineering strategies for industrial production of hydrocarbon fuels**

In addition to improving hydrocarbon-based fuel synthesis, metabolic engineering strategies can also be applied to address other factors affecting large-scale production. Two main issues will be addressed in this section: product toxicity and industrial strain robustness.

Product toxicity was shown to be a limiting factor in the production of first generation biofuels like ethanol. Since the interest in hydrocarbon-based fuels has developed only during the past decade, the toxicities of these fuels have not been fully explored, particularly with respect to autotrophic hosts. Fortunately, interest in hydrocarbon inhibition of microbial growth dates back almost a century [118], and we can capitalize on this wealth of information to engineer improved product tolerance in microbial hosts. Most fatty acid derived fuel molecules have shown some antimicrobial activity. FFAs, with a diverse range of carbon chain lengths and degrees of unsaturation, impart inhibitory effects on organisms including algae, Gramnegative and Gram-positive bacteria, fungi, protozoans, and various cell types of multicellular organisms [119]. Medium chain fatty alcohols such as pentanol, hexanol, heptanol, and octanol inhibited the biological activity of several algal and cyanobacterial strains, including fuelrelevant hosts *C. reinhardtii* and *Dunaliella salina* [120]. Interestingly, long-chain fatty alcohols (>C14) did not exhibit inhibitory effects on yeasts, suggesting that targeting longer chain fatty alcohols may eliminate the toxicity concern [121]. Similarly, medium-length alkanes (hexane, heptane, and isooctane) were toxic to microalgae while long-chain alkanes (C12-C16) elicited no effect [120, 121]. Microbial TAG and FAEE toxicities have not been reported. However, the phospholipid membrane surrounding algal TAGs may mask potential inhibitory effects, and FAEE production has been linked to the toxic effects of alcohol consumption in humans [122]. Isoprenoid-based fuel molecules have also illustrated inhibitory effects. Cyclic terpenes, such as pinene and limonene (Figure 2), inhibited the growth of bacteria and *S. cerevisiae* [123, 124], while branched isoprenoids, such as farnesyl hexanoate and geranyl acetate, were shown to be toxic to *E. coli* [125]. In fact, *E. coli*'s tolerance to isoprenoid-derived biodiesels and bioavi‐ ation fuels only ranged from 0.025 – 1% (v/v) [125]. Based on these previous studies, product toxicity is a major limiting factor and should be integrated into the metabolic engineering strategy.

A variety of strategies can be adopted to address product toxicity. The easiest way to avoid complications from product toxicity is to select non-toxic fuel targets. Toxicity studies can be conducted for each potential host organism, and generally, fatty alcohols longer than C14, alkanes longer than C9, and alkenes longer than C12 have shown minimal microbial inhibition [120, 121]. Alternatively, metabolic engineering techniques can be applied to allow for a more diverse range of hydrocarbon fuel targets. Many cellular modifications have been shown to improve microbial solvent tolerance: changes in membrane lipid composition; altered enzymatic activities of membrane repair and energy transduction enzymes; solvent expulsion via efflux pump activity; and cellular stress responses including heat shock, phage shock, and general stress responses [118, 125, 126]. These natural mechanisms offer a range of engineering targets: expression of a cis-trans isomerase to alter lipid composition; overexpression of enzymes involved in membrane repair and energy transduction; expression of efflux pumps such as *tolC*, *mar*, *rob*, *soxS*, and *acrAB*; and overexpression of stress-induced enzymes such as phage shock protein, heat shock proteins, catalases, and superoxide dismutases [125, 126]. While few metabolic engineering efforts have focused on enhancing product tolerance, a recent study explored improving hydrocarbon-based fuel tolerance in *E. coli* by testing a library of 43 efflux pumps [127]. This work identified efflux pumps that improved tolerance to five potential isoprenoid derived fuels. This preliminary success at engineering solvent tolerance should inspire additional efforts to improve the microbial production of both fatty acid and isoprenoid derived fuels.

In addition to product tolerance, other host traits are desirable for industrial biofuel production, particularly for autotrophic microorganisms. As discussed in the previous section, light availability is often a growth limiting factor in microalgal cultures. Microal‐ gae construct light harvesting complexes (LHC) to capture the available light for use in photosynthesis, and natural species actually absorb more light than is needed for photosyn‐ thesis under light intensities > 400 µmol photons m-2 s-1 [128]. As the sun can generate light intensities as high as 2,000 µmol photons m-2 s-1 during peak hours, it is estimated that as much as 80% of light absorbed by microalgae is 'wasted' as re-emitted fluorescence and heat [129]. In addition to this loss of energy, the excess energy can also cause cellular damage, known as photoinhibition [128]. In nature, this over-absorption of light will give the microalga a competitive advantage, but from a biofuel production perspective, this excess light harvesting will lead to lower culture cell densities and therefore lower biofuel productivities. Thus, there have been many attempts to engineer microalgae to absorb only the amount of light needed for photosynthesis. These efforts target genes of the light harvesting antenna complexes. Most LHC mutants were generated using random mutagen‐ esis techniques including chemical, UV, and transposon mutagenesis [128, 130-134]. Many of these studies focus on the model alga *C. reinhardtii*, but other microalgal species, such as the diatom *Cyclotella* sp. and the cyanobacterium *Synechocystis* sp., have been mutated to reduce the size of their photosynthetic antennae [130, 133]. Several recent works have applied RNAi technology in *C. reinhardtii* to reduce the expression of targeted LHC genes in a more controlled manner [129]. In general, the antenna mutants have shown im‐ proved photosynthetic quantum yields, reduced photoinhibition, enhanced productivity under high light conditions, and increased light penetration within the culture [128, 129, 131-134]. While these results are promising, several questions remain to be addressed: Are the photosynthetic antenna mutants genetically stable, or will they revert back to their more competitive and less efficient forms over time? And are these mutants less fit and there‐ fore more susceptible to predators and competitors in open pond systems?

ant microalgae, such as those isolated from marine or even hypersaline environments, may be selected as host for biofuel production, or efficient fuel-producing hosts can be engi‐ neered for increased salt tolerance. For example, the cyanobacterium *Synechococcus elongatus* PCC 7942, modified with expression of a Δ12 acyl-lipid desaturase (*desA*), showed improved resistance to salt and osmotic stress compared to the wildtype [136]. Lastly, pond crash due to microalgal predators like rotifers and chytrids is a major problem for open pond biofuel production systems. While there have not been any reported attempts at engineering predator-resistant microalgae, there have been reports of natural defense mechanisms such as palmelloid formation by *C. reinhardtii*, which produces non-motile cell aggregates that are simply too large to be consumed by grazing rotifers [137]. Once the genetic mecha‐ nism responsible for palmelloid formation is deciphered, it may be possible to transfer this resistance mechanism to other microalgae using genetic engineering techniques. When devising a metabolic engineering strategy for biofuel production, it is essential to consid‐ er the entire genomic landscape and the natural diversity of genetically-driven traits to

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The microbial production of drop-in replacement fuels faces unprecedented challenges. The sheer quantity of hydrocarbon product required to meet the world's ever increasing demand for energy dwarfs the supply of any current microbially synthesized product. Moreover, both second (lignocellulosic feedstock) and third (microalgal feedstock) generation bio‐ fuels ultimately rely on sunlight and photosynthesis to supply the energy and carbon feedstocks necessary for production. This requires the development of new technology and infrastructure to facilitate the construction of this new supply chain. Finally, the low value of the final fuel product places additional financial restrictions on the development of largescale biofuel production processes. For example, previous reports include the addition of exogenous metabolic precursors like mevalonate for isoprenoid production or FFA for FAEE biosynthesis [18, 50]. While these exogenous metabolites boost production of the desired hydrocarbon-based product, this practice is too expensive for large-scale biofuel applica‐ tions. These challenges currently limit the industrial production of second and third

Fortunately, new biological and technological tools are rapidly being developed and applied to overcome the obstacles in biofuel production. In addition to the metabolic engineering strategies previously described in this chapter, new global strategies are being applied to engineer microbes for biofuel production. With the affordability of next-generation DNA sequencing technologies, new microbial genomes are being reported at an unprecedented rate, and this information can be used to generate metabolic models for biofuel-producing hosts. In turn, these models can be leveraged to analyze proposed metabolic engineering strategies *in silico*, reducing the number of costly and time-intensive strain constructions and experi‐ ments. This technique was shown to be successful at increasing lycopene production, an isoprenoid derivative, in *E. coli* [69, 138]. The advancement of synthetic DNA technology

design the optimal host for the specific industrial constraints.

**5. Conclusions and future outlook**

generation biofuels.

Open pond systems are subject to a variety of changing environmental conditions, and as such, the optimal autotrophic host will have the necessary cellular mechanisms to adapt to these changing conditions. Desirable host traits may include temperature tolerance, salt tolerance, and resistance to predators. Open ponds are exposed to both daily and season‐ al temperature fluctuations which often exceed the normal temperature ranges for opti‐ mal cell growth and may even cause cell death. Engineering efforts have successfully altered the temperature tolerance of cyanobacteria though either gene knockout or heterologous overexpression of desaturases which influence the viscosity of both the cell and photosyn‐ thetic membranes [135]. Alternatively, microalgae with different temperature optima can be rotated seasonally in the open ponds, similar to seasonal crop rotations in agricultural practices. As mentioned previously, open pond systems are complicated by evaporative water loss, particularly for the sunny, arid regions that are ideal for microalgal biofuel production. Evaporation can lead to fluctuations in the salt concentration within the pond culture, and many have proposed to utilize marine or brackish water sources to reduce the cost associated with freshwater systems. Moreover, high salt and saturated salt systems will have lower evaporative water loss compared to freshwater cultures. Naturally salt-toler‐

ant microalgae, such as those isolated from marine or even hypersaline environments, may be selected as host for biofuel production, or efficient fuel-producing hosts can be engi‐ neered for increased salt tolerance. For example, the cyanobacterium *Synechococcus elongatus* PCC 7942, modified with expression of a Δ12 acyl-lipid desaturase (*desA*), showed improved resistance to salt and osmotic stress compared to the wildtype [136]. Lastly, pond crash due to microalgal predators like rotifers and chytrids is a major problem for open pond biofuel production systems. While there have not been any reported attempts at engineering predator-resistant microalgae, there have been reports of natural defense mechanisms such as palmelloid formation by *C. reinhardtii*, which produces non-motile cell aggregates that are simply too large to be consumed by grazing rotifers [137]. Once the genetic mecha‐ nism responsible for palmelloid formation is deciphered, it may be possible to transfer this resistance mechanism to other microalgae using genetic engineering techniques. When devising a metabolic engineering strategy for biofuel production, it is essential to consid‐ er the entire genomic landscape and the natural diversity of genetically-driven traits to design the optimal host for the specific industrial constraints.

#### **5. Conclusions and future outlook**

The microbial production of drop-in replacement fuels faces unprecedented challenges. The sheer quantity of hydrocarbon product required to meet the world's ever increasing demand for energy dwarfs the supply of any current microbially synthesized product. Moreover, both second (lignocellulosic feedstock) and third (microalgal feedstock) generation bio‐ fuels ultimately rely on sunlight and photosynthesis to supply the energy and carbon feedstocks necessary for production. This requires the development of new technology and infrastructure to facilitate the construction of this new supply chain. Finally, the low value of the final fuel product places additional financial restrictions on the development of largescale biofuel production processes. For example, previous reports include the addition of exogenous metabolic precursors like mevalonate for isoprenoid production or FFA for FAEE biosynthesis [18, 50]. While these exogenous metabolites boost production of the desired hydrocarbon-based product, this practice is too expensive for large-scale biofuel applica‐ tions. These challenges currently limit the industrial production of second and third generation biofuels.

Fortunately, new biological and technological tools are rapidly being developed and applied to overcome the obstacles in biofuel production. In addition to the metabolic engineering strategies previously described in this chapter, new global strategies are being applied to engineer microbes for biofuel production. With the affordability of next-generation DNA sequencing technologies, new microbial genomes are being reported at an unprecedented rate, and this information can be used to generate metabolic models for biofuel-producing hosts. In turn, these models can be leveraged to analyze proposed metabolic engineering strategies *in silico*, reducing the number of costly and time-intensive strain constructions and experi‐ ments. This technique was shown to be successful at increasing lycopene production, an isoprenoid derivative, in *E. coli* [69, 138]. The advancement of synthetic DNA technology enables new engineering approaches such as multiplex automated genome engineering (MAGE) [139]. In MAGE, synthetic oligomers, consisting of degenerate DNA sequences flanked by regions homologous to the target sequences, are simultaneously transformed into *E. coli*, and the modified strains are screened for improvements. MAGE was used to target ribosome binding sites, for optimization of protein translation, and to inactivate genes by inserting nonsense mutations; this technique can also be applied to target promoters for improved gene transcription and enzyme active sites for enhanced activities. The technique does have some limitations, however. MAGE will likely require modification of the host organism to allow for efficient integration of the single-stranded oligonucleotides, and a highthroughput screening method is essential for screening the billions of genetic variants that are generated with MAGE. Global or systems-level technologies can also be applied to advance our fundamental understanding of genetic and regulatory mechanisms within a microbial host; this is vital to host development of non-model organisms and newly isolated strains. Omics technologies including genomics, transcriptomics, metabolomics, and proteomics provide global insight at the cellular level, which can be compared across different conditions or time points to identify the native mechanisms that control the cell metabolism. Integration of omics data can identify bottlenecks at the transcriptional, translational, and protein levels, and as such, can be applied to inform the metabolic engineering strategy for biofuel production [34]. Systems-level tools for engineering microbial hosts, including metabolic modeling, MAGE, and omics technologies, will be integral to the successful development of hosts for biofuel production.

**Abbreviations**

1,3-BPG 1,3-bisphosphoglycerate GGPP geranylgeranyl pyrophosphate

AAS acyl-ACP synthetase GPD glycerol-3-phosphate dehydrogease

ACS acetyl-CoA synthetase HMG-CoA 3-hydroxy-3-methyl- glutaryl-CoA

ADP adenosine diphosphate IPPI isopentenyl diphosphate isomerase

ACC acetyl-CoA carboxylase GPP geranyl pyrophosphate

ADC aldehyde decarbonylase HMGCR HMG-CoA reductase

AH aldehyde *ispS* isoprene synthase ALDH acetaldehyde dehydrogenase KASIII β-ketoacyl-ACP synthase ALR aldehyde reductase LHC light harvesting complex AMP adenosine monophosphate L-Ru5P L-ribulose-5-phosphate AOL arabitol L-Xu5P L-xylulose-5-phosphate

ARA arabinose L-Xul L-xylulose

AT acyltransferase MVA mevalonate

CO2 carbon dioxide PBR photobioreactor

CTP cytosine triphosphate Pi phosphate *desA* Δ12 acyl-lipid desaturase PPi pyrophosphate

CoA coenzyme A PDC pyruvate decarboxylase CRP cyclic AMP receptor protein PEP phosphoenolpyruvate

DGAT diacylglycerol acyltransferase PPP pentose phosphate pathway DHAP dihydroxyacetone phosphate PPS phosphoenolpyruvate synthase DMAPP dimethylallyl diphosphate PTS phosphotransferase system

ADH alcohol dehydrogenase IPP isopentenyl Pyrophosphate

ASP aquatic species program MEP methylerythritol phosphate

ATP adenosine triphosphate NAD+ nicotinamide adenine dinucleotide (oxidized) cAMP cyclic AMP NADH nicotinamide adenine dinucleotide (reduced) CCR carbon catabolite repression NADP+ nicotinamide adenine dinucleotide phosphate

CMP cytosine monophosphate NADPH nicotinamide adenine dinucleotide phosphate

(oxidized)

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(reduced)

3-PGA 3-phosphoglycerate Glc glucose AAR acyl-ACP reductase Gly glycerol

ACP acyl carrier protein HCO3 - bicarbonate

Commercial interest in the production of second and third generation biofuels has developed rapidly in the past decade. As evidence of this, there has been a flurry of activity in patent applications regarding microbial hydrocarbon production. Companies invested in heterotro‐ phic hydrocarbon-based fuel production include LS9 [27, 59, 65, 66, 140, 141] and Amyris Biotechnologies [72, 142], which focus mainly on *E. coli* as the host, and Solazyme [143, 144], which initially focused on fuels derived from algae but has since moved toward more highvalue markets, such as cosmetics and nutraceuticals. Most companies interested in algae and cyanobacteria are focused on autotrophically-produced hydrocarbon fuels. Notable compa‐ nies in this industry include Sapphire Energy [145, 146], Joule Unlimited [26, 77, 147], and Synthetic Genomics [68, 75]. The hydrocarbon-based fuels targeted by these companies span the entire gamut of fatty acid and isoprenoid derived fuel products. Despite this commercial interest, hydrocarbon biofuel production still remains to be demonstrated at scale and in a sustainable manner.

This chapter has described the challenges in microbial hydrocarbon production and presented metabolic engineering strategies to resolve these issues. As is evident from this discussion, microbial-based fuel production is only in the initial stages of exploration, and additional research and innovation is necessary to enable large-scale biofuel production. New metabolic engineering tools and techniques are currently being developed for engineering untraditional hosts like eukaryotic algae and cyanobacteria, and as our understanding of these new hosts matures, significant improvement in hydrocarbon yields is anticipated.

#### **Abbreviations**



**References**

126-131.

261-268.

355-359.

275(37):28593-28598.

J. Bacteriol. 176(10):2814-2821.

Annu. Rev. Genet. 44(1):53-69.

[1] Williams JL. (2011) Oil Price History and Analysis. WTRG Economics. Available: http://

Metabolic Engineering of Hydrocarbon Biosynthesis for Biofuel Production

http://dx.doi.org/10.5772/52050

287

[2] Sawin JL, Martinot E, Barnes D, McCrone A, Roussell J, Sims R, et al. (2011) Renewables 2011 Global Status Report. Renewable Energy Policy Network for the 21st Century.

[3] Rittmann BE. (2008) Opportunities for Renewable Bioenergy Using Microorganisms.

[4] Christi Y. (2008) Biodiesel from Microalgae Beats Bioethanol. Trends Biotechnol. 26(3):

[5] Davis MS, Solbiati J, Cronan JE. (2000) Overproduction of Acetyl-CoA Carboxylase Activity Increases the Rate of Fatty Acid Biosynthesis in *Escherichia coli*. J. Biol. Chem.

[6] Lee S, Jeon E, Yun H, Lee J. (2011) Improvement of Fatty Acid Biosynthesis by Engi‐

[7] Ramos MJ, Fernández CM, Casas A, Rodríguez L, Pérez Á. (2009) Influence of Fatty Acid Composition of Raw Materials on Biodiesel Properties. Bioresour. Technol. 100(1):

[8] Dehesh K, Jones A, Knutzon DS, Voelker TA. (1996) Production of High Levels of 8:0 and 10:0 Fatty Acids in Transgenic Canola by Overexpression of Ch FatB2, a Thioes‐

[9] Jiang P, Cronan JE. (1994) Inhibition of Fatty Acid Synthesis in *Escherichia coli* in the Absence of Phospholipid Synthesis and Release of Inhibition by Thioesterase Action.

[10] Voelker TA, Davies HM. (1994) Alteration of the Specificity and Regulation of Fatty Acid Synthesis of *Escherichia coli* by Expression of a Plant Medium-Chain Acyl-Acyl

[11] Lotero E, Liu Y, Lopez DE, Suwannakarn K, Bruce DA, Goodwin JG. (2005) Synthesis

[12] Liu T, Khosla C. (2010) Genetic Engineering of *Escherichia coli* for Biofuel Production.

[13] Dellomonaco C, Clomburg JM, Miller EN, Gonzalez R. (2011) Engineered Reversal of the β-Oxidation Cycle for the Synthesis of Fuels and Chemicals. Nature. 476(7360):

of Biodiesel via Acid Catalysis. Ind. Eng. Chem. Res. 44(14):5353-5363.

neered Recombinant *Escherichia coli*. Biotech. Biopro. Eng. 16(4):706-713.

terase cDNA from *Cuphea hookeriana*. Plant J. 9(2):167-172.

Carrier Protein Thioesterase. J. Bacteriol. 176(23):7320-7327.

www.wtrg.com/prices.htm. Accessed 2012 April 16.

Biotechnol. Bioeng. 100(2):203-212.

#### **Acknowledgements**

This work was supported by the Harry S. Truman Fellowship in National Security Science and Engineering and the Laboratory Directed Research and Development program at Sandia National Laboratories. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corpo‐ ration, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

#### **Author details**

Anne M. Ruffing

Sandia National Laboratories, Department of Bioenergy and Defense Technologies, Albu‐ querque, NM, USA

#### **References**


[14] Jako C, Kumar A, Wei Y, Zou J, Barton DL, Giblin EM, et al. (2001) Seed-Specific Over-Expression of an *Arabidopsis* cDNA Encoding a Diacylglycerol Acyltransferase Enhan‐ ces Seed Oil Content and Seed Weight. Plant Physiol. 126(2):861-874.

[27] Schirmer A, Rude MA, Brubaker S, inventors; LS9, Inc., assignee. (2010) Methods and

Metabolic Engineering of Hydrocarbon Biosynthesis for Biofuel Production

http://dx.doi.org/10.5772/52050

289

[28] Lee SK, Chou H, Ham TS, Lee TS, Keasling JD. (2008) Metabolic Engineering of Microorganisms for Biofuels Production: From Bugs to Synthetic Biology to Fuels. Curr.

[29] Chandran SS, Kealey JT, Reeves CD. (2011) Microbial Production of Isoprenoids.

[30] Fortman JL, Chhabra S, Mukhopadhyay A, Chou H, Lee TS, Steen E, et al. (2008) Biofuel Alternatives to Ethanol: Pumping the Microbial Well. Trends Biotechnol. 26(7):375-381.

[31] Rude MA, Schirmer A. (2009) New Microbial Fuels: A Biotech Perspective. Curr. Opin.

[32] Rilling H, K B. (1959) On the Mechanism of Sqalene Biogenesis from Mevalonic Acid.

[33] Rohmer M, Knani M, Simonin P, Sutter B, Sahm H. (1993) Isoprenoid Biosynthesis in Bacteria: A Novel Pathway for the Early Steps Leading to Isopentenyl Diphosphate.

[34] Muntendam R, Melillo E, Ryden A, Kayser O. (2009) Perspectives and Limits of Engineering the Isoprenoid Metabolism in Heterologous Hosts. Appl. Microbiol.

[35] Yoon S-H, Lee S-H, Das A, Ryu H-K, Jang H-J, Kim J-Y, et al. (2009) Combinatorial Expression of Bacterial Whole Mevalonate Pathway for the Production of β-Carotene

[36] Pitera DJ, Paddon CJ, Newman JD, Keasling JD. (2007) Balancing a Heterologous Mevalonate Pathway for Improved Isoprenoid Production in *Escherichia coli*. Metab.

[37] Asadollahi MA, Maury J, Schalk M, Clark A, Nielsen J. (2010) Enhancement of Farnesyl Diphosphate Pool as Direct Precursor of Sesquiterpenes Through Metabolic Engineer‐ ing of the Mevalonate Pathway in *Saccharomyces cerevisiae*. Biotechnol. Bioeng. 106(1):

[38] Ohto C, Muramatsu M, Obata S, Sakuradani E, Shimizu S. (2009) Overexpression of the Gene Encoding HMG-CoA Reductase in *Saccharomyces cerevisiae* for Production of

[39] Kim S-W, Keasling JD. (2001) Metabolic Engineering of the Nonmevalonate Isopentenyl Diphosphate Synthesis Pathway in *Escherichia coli* Enhances Lycopene Production.

Prenyl Alcohols. Appl. Microbiol. Biotechnol. 82(5):837-845.

Compositions for Producing Hydrocarbons. patent US 2010/0249470.

Opin. Biotechnol. 19(6):556-563.

Process Biochem. 46(9):1703-1710.

Microbiol. 12(3):274-281.

Biochem. J. 295:517-524.

Biotechnol. 84(6):1003-1019.

Eng. 9(2):193-207.

86-96.

in *E. coli*. J. Biotechnol. 140(3–4):218-226.

Biotechnol. Bioeng. 72(4):408-415.

J. Biol. Chem. 234(6):1424-1432.


[40] Matthews PD, Wurtzel ET. (2000) Metabolic Engineering of Carotenoid Accumulation in *Escherichia coli* by Modulation of the Isoprenoid Precursor Pool with Expression of Deoxyxylulose Phosphate Synthase. Appl. Microbiol. Biotechnol. 53(4):396-400.

[52] Shiba Y, Paradise EM, Kirby J, Ro D-K, Keasling JD. (2007) Engineering of the Pyruvate Dehydrogenase Bypass in *Saccharomyces cerevisiae* for High-Level Production of

Metabolic Engineering of Hydrocarbon Biosynthesis for Biofuel Production

http://dx.doi.org/10.5772/52050

291

[53] Farmer WR, Liao JC. (2001) Precursor Balancing for Metabolic Engineering of Lycopene

[54] Asadollahi MA, Maury J, Patil KR, Schalk M, Clark A, Nielsen J. (2009) Enhancing Sesquiterpene Production in *Saccharomyces cerevisiae* Through in Silico Driven Meta‐

[55] Xue J, Ahring BK. (2011) Enhancing Isoprene Production by Genetic Modification of the 1-Deoxy-d-Xylulose-5-Phosphate Pathway in *Bacillus subtilis*. Appl. Environ.

[56] Li Y, Han D, Hu G, Dauvillee D, Sommerfeld M, Ball S, et al. (2010) *Chlamydomonas* Starchless Mutant Defective in ADP-Glucose Pyrophosphorylase Hyper-Accumulates

[57] Li Y, Han D, Hu G, Sommerfeld M, Hu Q. (2010) Inhibition of Starch Synthesis Results in Overproduction of Lipids in *Chlamydomonas reinhardtii*. Biotechnol. Bioeng. 107(2):

[58] Work VH, Radakovits R, Jinkerson RE, Meuser JE, Elliott LG, Vinyard DJ, et al. (2010) Increased Lipid Accumulation in the *Chlamydomonas reinhardtii* sta7-10 Starchless Isoamylase Mutant and Increased Carbohydrate Synthesis in Complemented Strains.

[59] Hu Z, Valle F, inventors; LS9, Inc, assignee. (2011) Enhanced Production of Fatty Acid

[60] Lu X, Vora H, Khosla C. (2008) Overproduction of Free Fatty Acids in *E. coli*: Implica‐

[61] Michinaka Y, Shimauchi T, Aki T, Nakajima T, Kawamoto S, Shigeta S, et al. (2003) Extracellular Secretion of Free Fatty Acids by Disruption of a Fatty Acyl-CoA Synthe‐

[62] Scharnewski M, Pongdontri P, Mora G, Hoppert M, Fulda M. (2008) Mutants of *Saccharomyces cerevisiae* Deficient in Acyl-CoA Synthetases Secrete Fatty Acids due to

[63] Kamisaka Y, Tomita N, Kimura K, Kainou K, Uemura H. (2007) *DGA1* (Diacylglycerol Acyltransferase Gene) Overexpression and Leucine Biosynthesis Significantly Increase Lipid Accumulation in the Δ*snf2* disruptant of *Saccharomyces cerevisiae*. Biochem. J.

Production in *Escherichia coli*. Biotechnol. Prog. 17(1):57-61.

Isoprenoids. Metab. Eng. 9(2):160-168.

bolic Engineering. Metab. Eng. 11(6):328-334.

Triacylglycerol. Metab. Eng. 12(4):387-391.

Microbiol. 77(7):2399-2405.

Eukaryot. Cell. 9(8):1251-1261.

Derivatives. patent US 2011/0256599.

tions for Biodiesel Production. Metab. Eng. 10(6):333-339.

Interrupted Fatty Acid Recycling. FEBS J. 275(11):2765-2778.

tase Gene in *Saccharomyces cerevisiae*. J. Biosci. Bioeng. 95(5):435-440.

258-268.

408:61-68.


[64] Nojima Y, Kibayashi A, Matsuzaki H, Hatano T, Fukui S. (1999) Isolation and Charac‐ terization of Triacylglycerol-Secreting Mutant Strain from Yeast, *Saccharomyces cerevisiae*. The Journal of General and Applied Microbiology. 45(1):1-6.

[77] Berry DA, Afeyan NB, Skraly FA, Ridley CP, Robertson DE, Wilpiszeski R, et al., inventors; Joule Unlimited, Inc., assignee. (2011) Methods and Compositions for the

Metabolic Engineering of Hydrocarbon Biosynthesis for Biofuel Production

http://dx.doi.org/10.5772/52050

293

[78] Lindberg P, Park S, Melis A. (2010) Engineering a Platform for Photosynthetic Isoprene Production in Cyanobacteria, Using *Synechocystis* as the Model Organism. Metab. Eng.

[79] Hahn-Hägerdal B. (1996) Ethanolic Fermentation of Lignocellulose Hydrolysates.

[80] Dellomonaco C, Fava F, Gonzalez R. (2010) The Path to Next Generation Biofuels: Successes and Challenges in the Era of Synthetic Biology. Microb. Cell Fact. 9(1):3.

[81] Jin Y, Lee T, Choi Y, Ryu Y, Seo J. (2000) Conversion of Xylose to Ethanol by Recombi‐ nant *Saccharomyces cerevisiae* Containing Genes for Xylose Reductase and Xylitol

[82] van Maris A, Winkler A, Kuyper M, de Laat W, van Dijken J, Pronk J. (2007) Develop‐ ment of Efficient Xylose Fermentation in *Saccharomyces cerevisiae:* Xylose Isomerase as

[83] Walfridsson M, Anderlund M, Bao X, Hahn-Hägerdal B. (1997) Expression of Different Levels of Enzymes from the *Pichia stipitis* XYL1 and XYL2 Genes in *Saccharomyces cerevisiae* and its Effects on Product Formation During Xylose Utilisation. Appl.

[84] Kuyper M, Hartog MMP, Toirkens MJ, Almering MJH, Winkler AA, van Dijken JP, et al. (2005) Metabolic Engineering of a Xylose-Isomerase-Expressing *Saccharomyces cerevisiae* Strain for Rapid Anaerobic Xylose Fermentation. FEMS Yeast Res. 5(4-5):

[85] Walfridsson M, Hallborn J, Penttilä M, Keränen S, Hahn-Hägerdal B. (1995) Xylose-Metabolizing *Saccharomyces cerevisiae* Strains Overexpressing the TKL1 and TAL1 Genes Encoding the Pentose Phosphate Pathway Enzymes Transketolase and Trans‐

[86] Becker J, Boles E. (2003) A Modified *Saccharomyces cerevisiae* Strain That Consumes l-Arabinose and Produces Ethanol. Appl. Environ. Microbiol. 69(7):4144-4150.

[87] Richard P, Verho R, Putkonen M, Londesborough J, Penttilä M. (2003) Production of Ethanol from L-Arabinose by *Saccharomyces cerevisiae* Containing a Fungal L-Arabinose

[88] Wisselink HW, Toirkens MJ, del Rosario Franco Berriel M, Winkler AA, van Dijken JP, Pronk JT, et al. (2007) Engineering of *Saccharomyces cerevisiae* for Efficient Anaerobic Alcoholic Fermentation of L-Arabinose. Appl. Environ. Microbiol. 73(15):4881-4891.

Dehydrogenase from *Pichia stipitis*. J. Microbiol. Biotech. 10(4):564-567.

a Key Component Biofuels. Adv Biochem Eng Biotechnol. 108:179-204.

Recombinant Biosynthesis of Fatty Acids and Esters. patent US 2011/0111470.

12(1):70-79.

399-409.

Appl. Biochem. Biotechnol. 57-58(1):195-199.

Microbiol. Biotechnol. 48(2):218-224.

aldolase. Appl. Environ. Microbiol. 61(12):4184-4190.

Pathway. FEMS Yeast Res. 3(2):185-189.


[89] Stülke J, Hillen W. (1999) Carbon Catabolite Repression in Bacteria. Curr. Opin. Microbiol. 2(2):195-201.

[103] Wang ZT, Ullrich N, Joo S, Waffenschmidt S, Goodenough U. (2009) Algal Lipid Bodies: Stress Induction, Purification, and Biochemical Characterization in Wild-Type and

Metabolic Engineering of Hydrocarbon Biosynthesis for Biofuel Production

http://dx.doi.org/10.5772/52050

295

[104] Radakovits R, Eduafo PM, Posewitz MC. (2011) Genetic Engineering of Fatty Acid

[105] Yu W-L, Ansari W, Schoepp N, Hannon M, Mayfield S, Burkart M. (2011) Modifications of the Metabolic Pathways of Lipid and Triacylglycerol Production in Microalgae.

[106] Stevens DR, Purton S. (1997) Genetic Engineering of Eukaryotic Algae: Progress and

[107] Rosenberg JN, Oyler GA, Wilkinson L, Betenbaugh MJ. (2008) A Green Light for Engineered Algae: Redirecting Metabolism to Fuel a Biotechnology Revolution. Curr.

[108] Radakovits R, Jinkerson RE, Darzins A, Posewitz MC. (2010) Genetic Engineering of

[109] Cerutti H, Ma X, Msanne J, Repas T. (2011) RNA-Mediated Silencing in Algae: Biolog‐ ical Roles and Tools for Analysis of Gene Function. Eukaryot. Cell. 10(9):1164-1172.

[110] Kilian O, Benemann CSE, Niyogi KK, Vick B. (2011) High-Efficiency Homologous Recombination in the Oil-Producing Alga *Nannochloropsis* sp. Proceedings of the

[111] (2012) *Chlamydomonas* Engineering Kits. Life Technologies Corporation. Available: https://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/ Protein-Expression-and-Analysis/Protein-Expression/algae-engineering-kits/chlamy‐

[112] Rasmussen B, Fletcher IR, Brocks JJ, Kilburn MR. (2008) Reassessing the First Appear‐

[113] Koksharova OA, Wolk CP. (2002) Genetic Tools for Cyanobacteria. Appl. Microbiol.

[114] Ogbonna JC, Soejima T, Tanaka H. (1999) An Integrated Solar and Artificial Light System for Internal Illumination of Photobioreactors. J. Biotechnol. 70(1–3):289-297.

[115] Kunjapur AM, Eldridge RB. (2010) Photobioreactor Design for Commercial Biofuel

[116] Bullis K. (2012) NASA Wants to Launch Floating Algae Farms. Technology Review.

[117] Chaumont D. (1993) Biotechnology of Algal Biomass Production: A Review of Systems

ance of Eukaryotes and Cyanobacteria. Nature. 455(7216):1101-1104.

Production from Microalgae. Ind. Eng. Chem. Res. 49(8):3516-3526.

for Outdoor Mass Culture. J. Appl. Phycol. 5(6):593-604.

Algae for Enhanced Biofuel Production. Eukaryot. Cell. 9(4):486-501.

National Academy of Sciences. 108(52):21265-21269.

domonas-engineering-kits.html. Accessed 2012 April 16.

Starchless *Chlamydomonas reinhardtii*. Eukaryot. Cell. 8(12):1856-1868.

Chain Length in *Phaeodactylum tricornutum*. Metab. Eng. 13(1):89-95.

Microb. Cell Fact. 10(1):91.

Prospects. J. Phycol. 33(5):713-722.

Opin. Biotechnol. 19(5):430-436.

Biotechnol. 58(2):123-137.


[118] Sikkema J, de Bont JA, Poolman B. (1995) Mechanisms of Membrane Toxicity of Hydrocarbons. Microbiol. Rev. 59(2):201-222.

[133] Nakajima Y, Ueda R. (1997) Improvement of Photosynthesis in Dense Microalgal Suspension by Reduction of Light Harvesting Pigments. J. Appl. Phycol. 9(6):503-510.

Metabolic Engineering of Hydrocarbon Biosynthesis for Biofuel Production

http://dx.doi.org/10.5772/52050

297

[134] Polle JEW, Kanakagiri S-D, Melis A. (2003) *tla1*; a DNA Insertional Transformant of the Green Alga *Chlamydomonas reinhardtii* with a Truncated Light-Harvesting Chlorophyll

[135] Gombos Z, Murata N. (2004) Genetic Engineering of the Unsaturation of Membrane Glycerolipid: Effects on the Ability of the Photosynthetic Machinery to Tolerate Temperature Stress Lipids in Photosynthesis: Structure, Function and Genetics. In:

[136] Allakhverdiev SI, Kinoshita M, Inaba M, Suzuki I, Murata N. (2001) Unsaturated Fatty Acids in Membrane Lipids Protect the Photosynthetic Machinery against Salt-Induced

[137] Lurling M, Beekman W. (2006) Palmelloids Formation in *Chlamydomonas reinhardtii*: Defence Against Rotifer Predators? Ann. Limnol. - Int. J. Limnol. 42:65-72.

[138] Jin Y-S, Stephanopoulos G. (2007) Multi-Dimensional Gene Target Search for Improv‐

[139] Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, et al. (2009) Programming Cells by Multiplex Genome Engineering and Accelerated Evolution. Nature. 460(7257):

[140] Friedman L, Rude MA, inventors; LS9, Inc., assignee. (2008) Process for Producing Low Molecular Weight Hydrocarbons from Renewable Resources. patent WO/2008/113041.

[141] Friedman L, da Costa B, inventors; LS9, Inc., assignee. (2008) Hydrocarbon-Producing

[142] Renninger NS, Ryder JA, Fisher KJ, inventors; Amyris Biotechnologies, Inc., assignee. (2008) Jet Fuel Compositions and Methods of Making and Using Same. patent WO/

[143] Trimbur DE, Im C-S, Dillon HF, Day AG, Franklin S, Coragliotti A, inventors; Solazyme, Inc., assignee. (2009) Lipid Pathway Modification in Oil-Bearing Microorganisms.

[144] Trimbur D, Im C-S, Dillon HF, Day AG, Franklin S, Coragliotti A, inventors; Solazyme, Inc., assignee. (2009) Use of Cellulosic Materials for Cultivation of Microorganisms.

[145] Mendez M, Fang S-C, Richard S, inventors; Sapphire Energy, Inc., assignee. (2010) Engineering Salt Tolerance in Photosynthetic Microorganisms. patent WO 2010/105095.

[146] Heaps NA, Behnke CA, Molina D, inventors; Sapphire Energy, Inc., assignee. (2010) Biofuel Production in Prokaryotes and Eukaryotes. patent WO/2010/104763.

ing Lycopene Biosynthesis in *Escherichia coli*. Metab. Eng. 9(4):337-347.

Paul-André S, Norio M, editors.: Springer Netherlands; p. 249-262.

Damage in *Synechococcus*. Plant Physiol. 125(4):1842-1853.

Genes and Methods of Their Use. patent WO/2008/147781.

Antenna Size. Planta. 217(1):49-59.

894-898.

2008/140492.

patent US 2009/0061493.

patent US 2009/0011480.


[147] Berry DA, Robertson DE, Skraly FA, Green BD, Ridley CP, Kosuri S, et al., inventors; Joule Unlimited, Inc., assignee. (2011) Engineered CO2 Fixing Microorganisms Produc‐ ing Carbon-Based Products of Interest. patent US 2011/0008861.

**Chapter 9**

**Catalytic Hydroprocessing of**

Additional information is available at the end of the chapter

Stella Bezergianni

**1. Introduction**

subject of this chapter.

http://dx.doi.org/10.5772/52649

**Liquid Biomass for Biofuels Production**

The depletion of world petroleum reserves and the increased concern on climate change has stimulated the recent interest in biofuels. The most common biofuels are based on energy crops and their products, i.e. vegetable oil for Fatty Acid Methyl Esters (FAME) biodiesel [1] and sugars/starch for bioethanol. However these first generation biofuels and associated production technologies face several considerations related to their economic and social im‐ plications regarding energy crops cultivation, by-products disposal, necessity for large in‐

As a result, **second generation biofuel technologies** have been developed to overcome the limitations of first generation biofuels production [2]. The goal of second generation biofuel processes is to extend biofuel production capacity by incorporating residual biomass while increasing sustainability. This residual biomass consists of the non-food parts of food crops (such as stems, leaves and husks) as well as other non-food crops (such as switch grass, ja‐ tropha, miscanthus and cereals that bear little grain). Furthermore the residual biomass po‐ tential is further augmented by industrial and municipal organic waste such as skins and pulp from fruit pressing, waste cooking oil etc. One such technology is **catalytic hydropro‐ cessing**, which is an alternative conversion technology of liquid biomass to biofuels that is lately raising a lot of interest in both the academic and industrial world and is the proposed

Catalytic hydroprocessingis a key process in petrochemical industry for over a century ena‐ bling heteroatom (sulfur, nitrogen, oxygen, metals) removal, saturation of olefins and aro‐ matics, as well as isomerization and cracking [3]. Due to the numerous applications of catalytic hydroprocessing, there are several catalytic hydroprocessing units in a typical re‐ finery including distillate hydrotreaters and hydrocrackers (see Figure 1). As a result several

> © 2013 Bezergianni; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Bezergianni; licensee InTech. This is a paper 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.

distribution, and reproduction in any medium, provided the original work is properly cited.

vestments to ensure competitiveness and the "food versus fuel" debate.

**Chapter 9**

## **Catalytic Hydroprocessing of Liquid Biomass for Biofuels Production**

Stella Bezergianni

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52649

#### **1. Introduction**

The depletion of world petroleum reserves and the increased concern on climate change has stimulated the recent interest in biofuels. The most common biofuels are based on energy crops and their products, i.e. vegetable oil for Fatty Acid Methyl Esters (FAME) biodiesel [1] and sugars/starch for bioethanol. However these first generation biofuels and associated production technologies face several considerations related to their economic and social im‐ plications regarding energy crops cultivation, by-products disposal, necessity for large in‐ vestments to ensure competitiveness and the "food versus fuel" debate.

As a result, **second generation biofuel technologies** have been developed to overcome the limitations of first generation biofuels production [2]. The goal of second generation biofuel processes is to extend biofuel production capacity by incorporating residual biomass while increasing sustainability. This residual biomass consists of the non-food parts of food crops (such as stems, leaves and husks) as well as other non-food crops (such as switch grass, ja‐ tropha, miscanthus and cereals that bear little grain). Furthermore the residual biomass po‐ tential is further augmented by industrial and municipal organic waste such as skins and pulp from fruit pressing, waste cooking oil etc. One such technology is **catalytic hydropro‐ cessing**, which is an alternative conversion technology of liquid biomass to biofuels that is lately raising a lot of interest in both the academic and industrial world and is the proposed subject of this chapter.

Catalytic hydroprocessingis a key process in petrochemical industry for over a century ena‐ bling heteroatom (sulfur, nitrogen, oxygen, metals) removal, saturation of olefins and aro‐ matics, as well as isomerization and cracking [3]. Due to the numerous applications of catalytic hydroprocessing, there are several catalytic hydroprocessing units in a typical re‐ finery including distillate hydrotreaters and hydrocrackers (see Figure 1). As a result several

© 2013 Bezergianni; licensee InTech. This is an open access article 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. © 2013 Bezergianni; licensee InTech. This is a paper 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.

refinery streams are treated with hydrogen in order to improve final product quality includ‐ ing straight-run naphtha, diesel, gas-oils etc. The catalytic hydroprocessing technology is evolving through the new catalytic materials that are being developed. Even though hydro‐ processing catalysts development is well established [4], the growing demand of petroleum products and their specifications, which are continuously becoming stricter, have created new horizons in the catalyst development in order to convert heavier and lower quality feedstocks [5]. Furthermore the expansion of the technology to bio-based feedstocks has also broadened the R&D spam of catalytic hydrotreatment.

technology for upgrading intermediate products of solid biomass conversion technologies such as **pyrolysis oils** and **Fischer-Tropsch wax** (Figure 2). The growing interest and invest‐ ments of the petrochemical, automotive and aviation industries to the biomass catalytic hy‐ droprocessing technology shows that this technology will play an important role in the

*Wax*

In the sections that follow, the basic technical characteristics of catalytic hydrotreatment are presented including a description of the process, reactions, operating parameters and feed‐ stock characteristics. Furthermore key applications of catalytic hydroprocessing of liquid bi‐ omass are outlined based on different feedstocks including raw vegetable oils, waste cooking oils, pyrolysis oils, Fischer-Tropsch wax and algal oil, and some successful demon‐

The catalytic hydrotreatment of liquid biomass converts the contained triglycerides/lipids into hydrocarbons at high temperatures and pressures over catalytic material under excess hydrogen atmosphere. The catalytic hydrotreatment of liquid biomass process is quite simi‐ lar to the typical process applied to petroleum streams, as shown in Figure 3. A typical cata‐ lytic hydrotreatment unit consists of four basic sections: a) feed preparation, b) reaction, c)

In the feed preparation section the liquid biomass feedstock is mixed with the high pressure hydrogen (mainly from gas recycle with some additional fresh make-up hydrogen) and is preheated before it enters the reactor section. The reactor section consists normally of two hydrotreating reactors, a first guard mild hydrotreating reactor and a second one where the main hydrotreating reactions take place. Each reactor contains two or more catalytic beds in order to maintain constant temperature profile throughout the reactor length. Within the re‐ actor section all associated reactions take place, which will be presented in more detail at a

**Hydroprocessing**

Catalytic Hydroprocessing of Liquid Biomass for Biofuels Production

**Gasoline Diesel**

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301

*Solid Biomass Pyrolysis oil* **Pyrolysis Catalytic**

**Fischer-Tropsch Synthesis**

**Figure 2.** Catalytic hydroprocessing for biomass conversion and upgrading towards fuels production

**2. Technical characteristics of catalytichydrotreatment**

*Liquid Biomass*

biofuels field in the immediate future.

**Gasification**

stration activities are also presented.

product separation and d) fractionation.

later paragraph.

**Figure 1.** Catalytic hydroprocessing units within a refinery, including distillate hydrotreating and hydrocracking

Catalytic hydroprocessing of liquid biomass is a technology that offers great flexibility to the continuously increasing demands of the biofuels market, as it can convert a wide variety of liquid biomass including **raw vegetable oils**, **waste cooking oils**, **animal fats** as well as **al‐ gal oils** into biofuels with high conversion yields. In general this catalytic process technolo‐ gy allows the conversion of triglycerides and lipids into paraffins and iso-paraffins within the naphtha, kerosene and diesel ranges. The products of this technology have improved characteristics as compared to both their fossil counterparts and the conventional biofuels including high heating value and cetane number, increased oxidation stability, negligible acidity and increased saturation level. Besides the application of this catalytic technology for the production of high quality paraffinic fuels, catalytic hydroprocessing is also an effective technology for upgrading intermediate products of solid biomass conversion technologies such as **pyrolysis oils** and **Fischer-Tropsch wax** (Figure 2). The growing interest and invest‐ ments of the petrochemical, automotive and aviation industries to the biomass catalytic hy‐ droprocessing technology shows that this technology will play an important role in the biofuels field in the immediate future.

**Figure 2.** Catalytic hydroprocessing for biomass conversion and upgrading towards fuels production

In the sections that follow, the basic technical characteristics of catalytic hydrotreatment are presented including a description of the process, reactions, operating parameters and feed‐ stock characteristics. Furthermore key applications of catalytic hydroprocessing of liquid bi‐ omass are outlined based on different feedstocks including raw vegetable oils, waste cooking oils, pyrolysis oils, Fischer-Tropsch wax and algal oil, and some successful demon‐ stration activities are also presented.

#### **2. Technical characteristics of catalytichydrotreatment**

The catalytic hydrotreatment of liquid biomass converts the contained triglycerides/lipids into hydrocarbons at high temperatures and pressures over catalytic material under excess hydrogen atmosphere. The catalytic hydrotreatment of liquid biomass process is quite simi‐ lar to the typical process applied to petroleum streams, as shown in Figure 3. A typical cata‐ lytic hydrotreatment unit consists of four basic sections: a) feed preparation, b) reaction, c) product separation and d) fractionation.

In the feed preparation section the liquid biomass feedstock is mixed with the high pressure hydrogen (mainly from gas recycle with some additional fresh make-up hydrogen) and is preheated before it enters the reactor section. The reactor section consists normally of two hydrotreating reactors, a first guard mild hydrotreating reactor and a second one where the main hydrotreating reactions take place. Each reactor contains two or more catalytic beds in order to maintain constant temperature profile throughout the reactor length. Within the re‐ actor section all associated reactions take place, which will be presented in more detail at a later paragraph.

The reactor product then enters the separator section where, after it is cooled down, it enters the high pressure separator (HPS) flash drum in which the largest portion of the gas and liq‐ uid product molecules are separated. The gas product of the HPS includes the excess hydro‐ gen that has not reacted within the reactor section as well as the side products of the reactions including CO, CO2, H2S, NH3 and H2O. The liquid product of the HPS is lead to a second flash drum, the low pressure separator (LPS), for removing any residual gas con‐ tained in the liquid product, and subsequently is fed to a fractionator section. The fractiona‐ tor section provides the final product separation into the different boiling point fractions that yield the desired products including off-gas, naphtha, kerosene and diesel. The heaviest molecules return from the bottom of the fractionator into the reactor section as a liquid recy‐ cle stream.

*2.1.1. Cracking*

As the molecules included in the various types of liquid biomass can be relatively large and complicated, cracking reactions are desired to convert them into molecules of the size and boiling point range of conventional fuels, mainly gasoline, kerosene and diesel. A character‐ istic reaction that occurs during catalytic hydrotreating of oils / fats is the cracking of trigly‐ cerides into its consisting fatty acids (carboxylic acids) and propane as shown in Scheme 1 [5][6]. This reaction is critical as it converts the initial large triglycerides molecules of boiling

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Other cracking reactions may take place however such as those described in Schemes 2 and 3, depending on the type of molecules present in the feedstock. For example Scheme 2 is a cracking reaction which may occur during catalytic hydrotreatment of pyrolysis oil which includes polyaromatic and aromatic compounds. Alternatively Scheme 3 may follow deoxy‐ genation of carboxylic acids on the produced long chain paraffinic molecules, leading to

+ 2·H2 3·R-CH2COOH + CH3-CH2-CH3

Triglyceride carboxylic acid propane

R - R' + H R - R' + H2 R - H + R' - H

Saturation reactions are strongly associated with catalytic hydrotreating as the introduction of excess hydrogen allows the breakage of double C-C bonds and their conversion to single

point over 600°C into mid-distillate range molecules (naphtha, kerosene and diesel).

smaller chain paraffins, during the upgrading of Fischer-Tropsch wax.

CH2-O-C-R

O

CH-O-CO-R

CH2-O-C-R

**Scheme 1.**

**Scheme 2.**

**Scheme 3.**

*2.1.2. Saturation*

O

In order to improve the overall efficiency, a liquid recycle stream is also incorporated, which in essence consists of the heavy molecules that were not converted. The gas product from the HPS and LPS, after being treated to remove the excess NH3, H2S, CO and CO2, is com‐ pressed and fed back to the reactor section as a gas recycle stream in order to maintain a high pressure hydrogen atmosphere within the reactor section.

**Figure 3.** A typical process diagram of catalytic hydrotreatment of liquid biomass

#### **2.1. Reaction mechanisms**

Several types of reactions take place during catalytic hydrotreatment of liquid biomass, based on the type of biomass processed, operating conditions and catalyst employed. The types of reactions that liquid biomass undergoes during catalytic hydroprocessing include: a) cracking, b) saturation, c) heteroatom removal and d) isomerization, which are described in more detail in the following section.

#### *2.1.1. Cracking*

As the molecules included in the various types of liquid biomass can be relatively large and complicated, cracking reactions are desired to convert them into molecules of the size and boiling point range of conventional fuels, mainly gasoline, kerosene and diesel. A character‐ istic reaction that occurs during catalytic hydrotreating of oils / fats is the cracking of trigly‐ cerides into its consisting fatty acids (carboxylic acids) and propane as shown in Scheme 1 [5][6]. This reaction is critical as it converts the initial large triglycerides molecules of boiling point over 600°C into mid-distillate range molecules (naphtha, kerosene and diesel).

Other cracking reactions may take place however such as those described in Schemes 2 and 3, depending on the type of molecules present in the feedstock. For example Scheme 2 is a cracking reaction which may occur during catalytic hydrotreatment of pyrolysis oil which includes polyaromatic and aromatic compounds. Alternatively Scheme 3 may follow deoxy‐ genation of carboxylic acids on the produced long chain paraffinic molecules, leading to smaller chain paraffins, during the upgrading of Fischer-Tropsch wax.

#### **Scheme 3.**

#### *2.1.2. Saturation*

Saturation reactions are strongly associated with catalytic hydrotreating as the introduction of excess hydrogen allows the breakage of double C-C bonds and their conversion to single bonds, as shown in the following reactions. In particular the saturation of unsaturated car‐ boxylic acids into saturated ones depicted in Scheme 4, is a key reaction occurring in lipid feedstocks. Furthermore other saturation reactions lead to the formation of naphthenes by converting unsaturated cyclic compounds and aromatic compounds as in Scheme 5 and 6, which are likely to occur during upgrading of pyrolysis oils.

+ 3·H2 R-CH2CH3 R-CH2COOH + 2·H2O

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R-CH2COOH + H2 R-CH3 + CO + H2O

R-CH2COOH + H2 R-CH3 + CO2

The other heteroatoms, i.e. S and N are removed according to the classic heteroatom remov‐

The straight chain paraffinic molecules resulting from the aforementioned reactions, even though they offer increased cetane number, heating value and oxidation stability in the bio‐ fuels which contain them, they also degrade their cold flow properties. In order to improve the cold flow properties, isomerization reactions are also required, which normally take place during a second step/reactor as they require a different catalyst. Some examples of iso‐

al mechanisms of the fossil fuels in the form of gaseous H2S and NH3 respectively.

R-CH2 R-CH-CH3 -CH2-CH3

Catalytic hydroprocessing of liquid biomass is a technology currently under developed and there is a lot of room for optimization. For example there are not many commercial catalysts specifically designed and developed for such applications, while conventional commercial cat‐ alysts, employed for catalytic hydroprocessing of refinery streams, are used instead. Common

CH3

merization reactions are given in Schemes 10 and 11.

**Scheme 7.**

**Scheme 8.**

**Scheme 9.**

**Scheme 10.**

**Scheme 11.**

**2.2. Hydroprocessing catalysts**

*2.1.4. Isomerization*

RCH = CH-COOH + H H2 RCH2CH2COOH **Scheme 4. Scheme 5.**

#### **Scheme 6.**

As a result of this reaction the produced saturated molecules are less active and less prone to polymerization and oxidation reactions, mitigating the sediment formation and corrosion phenomena appearing in engines.

#### *2.1.3. Heteroatom removal*

Heteroatoms are atoms other than carbon (C) and hydrogen (H) and are often encountered into bio- and fossil- based feedstocks. They include sulfur (S), nitrogen (N) and in the case of bio-based feedstocks oxygen (O). In particular oxygen removal is of outmost importance as the presence of oxygen reduces oxidation stability (due to carboxylic and carbonylic double bonds), increases acidity and corrosivity (due to the presence of water) and even reduces the heating value of the final biofuels. The main deoxygenation reactions that take place include deoxygenation, decarbonylation and decarboxylation presented in Schemes 7, 8 and 9 re‐ spectively [7]. The main products of deoxygenation reactions include n-paraffins, while H2O, CO2 and CO are also produced, but can be removed with the excess hydrogen within the flash drums of the product separation section. It should be noted however that these particular reactions give the paraffinic nature of the produced biofuels, and for this reason the hydrotreated products are often referred to as paraffinic fuels (e.g. paraffinic jet, paraf‐ finic diesel etc)

$$\text{R-C\#}\_2\text{COOH} \quad + \text{3-H}\_2 \xrightarrow{\text{H-C\#}\_2\text{CH}\_3} + 2\cdot\text{H}\_2\text{O}$$

**Scheme 7.**

$$\begin{array}{cccc} \mathsf{R-C\mathsf{H}\_{2}\mathsf{COOH}} & + & \mathsf{H}\_{2} & \longrightarrow & \mathsf{R-C\mathsf{H}\_{3}} + \mathsf{CO} + \mathsf{H}\_{2}\mathsf{O} \end{array}$$

**Scheme 8.**

$$\begin{array}{cccc} \mathsf{R-C}\mathsf{H}\_{2}\mathsf{COOH} & + \mathsf{H}\_{2} & \longrightarrow & \mathsf{R-C}\mathsf{H}\_{3} + \mathsf{CO}\_{2} \end{array}$$

#### **Scheme 9.**

The other heteroatoms, i.e. S and N are removed according to the classic heteroatom remov‐ al mechanisms of the fossil fuels in the form of gaseous H2S and NH3 respectively.

#### *2.1.4. Isomerization*

The straight chain paraffinic molecules resulting from the aforementioned reactions, even though they offer increased cetane number, heating value and oxidation stability in the bio‐ fuels which contain them, they also degrade their cold flow properties. In order to improve the cold flow properties, isomerization reactions are also required, which normally take place during a second step/reactor as they require a different catalyst. Some examples of iso‐ merization reactions are given in Schemes 10 and 11.

**Scheme 10.**

**Scheme 11.**

#### **2.2. Hydroprocessing catalysts**

Catalytic hydroprocessing of liquid biomass is a technology currently under developed and there is a lot of room for optimization. For example there are not many commercial catalysts specifically designed and developed for such applications, while conventional commercial cat‐ alysts, employed for catalytic hydroprocessing of refinery streams, are used instead. Common hydrotreating catalysts employed contain active metals on alumina substrate with increased surface area. The most known commercial catalysts employ Cobalt and Molybdenum (CoMo) or Nikel and Molybdenum (NiMo) in alumina substrate (Al2O3) as shown in Figure 4.

wards developing special hydrotreating catalysts for converting/upgrading liquid biomass

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Gasoline Diesel

**Figure 5.** Catalyst comparison based on gasoline and diesel yields for WCO hydrotreating [8]. (Reprinted from Fuel, 93, S. Bezergianni, A. Kalogianni, A. Dimitriadis, Catalyst evaluation for waste cooking oil hydroprocessing, 638-641,

As it was mentioned earlier, the choice of catalyst and operating parameters affect the reac‐ tions that take place within the hydroprocessing reactor. The key operating parameters of hydroprocessing include the reactor temperature, hydrogen partial pressure, liquid hourly

Most catalytic hydrotreating and hydrocracking reactors operate between 290-450°C. The temperature range is selected according the type of catalyst and feedstock type to be proc‐ essed. In the first stages of the catalyst life (after its loading in the reactor) the temperature is normally kept low as the catalyst activity is already very high. However as time progresses and the catalyst deactivates and cokes, the temperature is gradually increased to overcome

Hydrogen partial pressure affects significantly the hydrotreating reactions as well as the cat‐ alyst deactivation. The catalyst deactivation rate is inverse proportional to the hydrogen

the loss of catalyst activity and to maintain the desired product yield and quality.

to biofuels [9-12].

100

0

2012, with permission from Elsevier).

**2.3. Operating parameters**

*2.3.2. Hydrogen partial pressure*

*2.3.1. Temperature*

space velocity and hydrogen feed-rate.

20

40

60

**Gasoline/Diesel yields (%)**

80

Catalyst A: HDT Catalyst B: MID-HDC Catalyst C: HDC

**Figure 4.** Typical hydrotreating catalysts (a) before use and (b) after use

Hydrotreating catalysts are dual action catalytic material, triggering both hydrogenation and cracking/isomerization reactions. On one hand hydrogenation takes place on the active metals (Mo, Ni, Co, Pd, Pt) which catalyze the feedstock molecules rendering them more ac‐ tive when subject to cracking and heteroatom removal, while limiting coke formation on the catalyst. Furthermore hydrogenation supports cracking by forming an active olefinic inter‐ mediate molecule via dehydrogenation. On the other hand both cracking and isomerization reactions take place in acidic environment such as amorphous oxides (SiO2 – Al2O3) or crys‐ talline zeolites (mainly z-zeolites) or mixtures of zeolites with amorphous oxides.

During the first contact of the feedstock molecules with the catalyst, a temperature increase is likely to develop due to the exothermic reactions that occur. However, during the continu‐ ous utilization of the catalyst and coke deposition, the catalyst activity eventually reduces from 1/3 to 1/2 of its initial one. The catalyst deactivation rate mainly depends on tempera‐ ture and hydrogen partial pressure. Increased temperatures accelerate catalyst deactivation while high hydrogen partial pressure tends to mitigate catalyst deactivation rate. Most of the catalyst activity can be recovered by catalyst regeneration.

The selection of a suitable hydroprocessing catalyst is a critical step defining the hydro‐ processing product yield and quality as well as the operating cycle time of the process in petroleum industry [5]. However the hydrotreating catalyst selection for biomass applica‐ tions is particularly crucial and challenging for two reasons: a) catalyst activity varies sig‐ nificantly, as commercial catalysts are designed for different feedstocks, i.e. feedstocks with high sulfur concentration, heavy feedstocks (containing large molecules), feedstocks with high oxygen concentration etc, and b) there are currently no commercial hydropro‐ cessing catalysts available for lipid feedstocks and other intermediate products of biomass conversion processes (e.g. pyrolysis biooil), and thus commercial hydrotreating catalysts need to be explored and evaluated as different catalyst have different yields (Figure 5) and different degradation rate [8]. Nevertheless, significant efforts have been directed to‐ wards developing special hydrotreating catalysts for converting/upgrading liquid biomass to biofuels [9-12].

**Figure 5.** Catalyst comparison based on gasoline and diesel yields for WCO hydrotreating [8]. (Reprinted from Fuel, 93, S. Bezergianni, A. Kalogianni, A. Dimitriadis, Catalyst evaluation for waste cooking oil hydroprocessing, 638-641, 2012, with permission from Elsevier).

#### **2.3. Operating parameters**

As it was mentioned earlier, the choice of catalyst and operating parameters affect the reac‐ tions that take place within the hydroprocessing reactor. The key operating parameters of hydroprocessing include the reactor temperature, hydrogen partial pressure, liquid hourly space velocity and hydrogen feed-rate.

#### *2.3.1. Temperature*

Most catalytic hydrotreating and hydrocracking reactors operate between 290-450°C. The temperature range is selected according the type of catalyst and feedstock type to be proc‐ essed. In the first stages of the catalyst life (after its loading in the reactor) the temperature is normally kept low as the catalyst activity is already very high. However as time progresses and the catalyst deactivates and cokes, the temperature is gradually increased to overcome the loss of catalyst activity and to maintain the desired product yield and quality.

#### *2.3.2. Hydrogen partial pressure*

Hydrogen partial pressure affects significantly the hydrotreating reactions as well as the cat‐ alyst deactivation. The catalyst deactivation rate is inverse proportional to the hydrogen partial pressure and to hydrogen feed-rate. However high hydrogen partial pressures corre‐ spond to high operational costs, which rise even higher for high olefinic feedstocks that ex‐ hibit higher hydrogen consumption due to the saturation reactions. Therefore hydrogen partial pressure should be balanced with the catalyst activity and catalyst life expectancy in order to optimize the overall process.

double bonds that enriches the H/C analogy. The oxygen content (including the oxygen contained in the water) from over 15%wt can be decreased down to 5wppm. Actually the deep deoxygenation achieved via catalytic hydrotreatment is the most significant contribu‐ tion of this biomass conversion technology, as it improves significantly the oxidation stabil‐ ity of the final biofuels. Furthermore significant improvement is also observed in the biomass density, which is never below 0.9 kg/l while after hydrotreatment it reduces to val‐

> **Liquid biomass (unprocessed)**

**Oxygen content (%wt)** 15 - 40 10-4 – 3 **Density (kg/l)** 0.9 – 1.05 0.75 – 0.8

**Table 1.** Effect of catalytic hydrotreatment on the liquid biomass characteristics

**H/C ratio** 0.08 – 0.1 0.13 – 0.18

Catalytic hydroprocessing has been proven as the most efficient technology for the upgrad‐ ing of liquid biomass as it achieves to increase the H/C ratio and to remove oxygen and wa‐ ter. However the effectiveness of this technology is also shown in other parameters. For example the distillation curve of raw liquid biomass shows that over 90% of its molecules have boiling points exceeding 600°C and only 5% are within diesel range (220-360°C), while after catalytic hydrotreatment upgrading most of 90% of the product molecules are within

0 10 20 30 40 50 60 70 80 90 100

Product recovery (% v/v)

**Figure 6.** Distillation curves of untreated WCO (dashed) and catalytically hydrotreated WCO (solid)

**Hydrotreated liquid biomass and produced biofuels**

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Catalytic Hydroprocessing of Liquid Biomass for Biofuels Production

Diesel range

ues less than 0.8 kg/l

diesel range [13].

Temperature (°C)

0

100

200

300

400

500

600

700

#### *2.3.3. Liquid hourly space velocity*

Liquid hourly space velocity (LHSV) is defined as the ratio of the liquid mass feed-rate (gr/h) over the catalyst mass (gr) and as a result is expressed in hr-1. In fact the inverse of LHSV is proportional to the residence time of the liquid feed in the reactor. In essence the higher the liquid hourly space velocity, the less time is available for the contact of the feed molecules of the reaction mixture with the catalyst, thus the less the conversion. However, maintaining large LHSV imposes a faster degradation of the catalyst therefore in industrial applications the LHSV is maintained in as high values as it is practically possible.

#### *2.3.4. Hydrogen feed-rate*

The hydrogen feed-rate is another important parameter as it also defines hydrogen partial pressure depending on the hydrogen consumption of each application. It actually favours both heteroatom removal and saturation reaction rates. However, as hydrogen cost defines the overall unit operating cost, hydrogen feed-rate is normally optimized depending on the system requirements. Furthermore the use of renewable energy sources for hydrogen pro‐ duction is also envisioned as a potential cost improvement option.

#### **3. Feed sand products**

Even though liquid biomass is currently being exploited as a renewable feedstock for fuels pro‐ duction, its characteristics are far beyond suitable for its use as fuel. More specifically liquid bi‐ omass, just as other types of biomass, has a small H/C ratio and high oxygen content, lowering its heating value and increasing CO and CO2 emissions during its combustion. Moreover liq‐ uid biomass contains water, which can cause corrosion in the downstream processing units if it's not completely removed, or even in the engine parts where its final products are utilized. In addition to the above, liquid biomass has an increased concentration in oxygenated com‐ pounds, mainly acids, aldehydes, ketones etc, which not only reduce the heating value, but al‐ so decrease the oxidation stability and increase the acidity of the produced biofuels. For all the aforementioned reasons it is imperative that liquid biomass should be upgraded and specifi‐ cally that its H/C should be increased while the water and oxygen removed.

The effectiveness of catalytic hydroprocessing towards improving these problematic char‐ acteristics of liquid biomass is presented in Table 1, where the H/C ratio, the oxygen con‐ tent and density before and after catalytic hydrotreatment of basic liquid biomass types are given. The H/C ratio exhibits a significant increase that exceeds 50% in all cases. This is due to the substitution of the heteroatoms by hydrogen atoms as well as in the saturation of double bonds that enriches the H/C analogy. The oxygen content (including the oxygen contained in the water) from over 15%wt can be decreased down to 5wppm. Actually the deep deoxygenation achieved via catalytic hydrotreatment is the most significant contribu‐ tion of this biomass conversion technology, as it improves significantly the oxidation stabil‐ ity of the final biofuels. Furthermore significant improvement is also observed in the biomass density, which is never below 0.9 kg/l while after hydrotreatment it reduces to val‐ ues less than 0.8 kg/l


**Table 1.** Effect of catalytic hydrotreatment on the liquid biomass characteristics

Catalytic hydroprocessing has been proven as the most efficient technology for the upgrad‐ ing of liquid biomass as it achieves to increase the H/C ratio and to remove oxygen and wa‐ ter. However the effectiveness of this technology is also shown in other parameters. For example the distillation curve of raw liquid biomass shows that over 90% of its molecules have boiling points exceeding 600°C and only 5% are within diesel range (220-360°C), while after catalytic hydrotreatment upgrading most of 90% of the product molecules are within diesel range [13].

**Figure 6.** Distillation curves of untreated WCO (dashed) and catalytically hydrotreated WCO (solid)

In the following sections the basic types of liquid biomass and their corresponding products via catalytic hydrotreatment are presented.

**3.2. Waste cooking oils conversion to paraffinic biofuels**

which is estimated as the average demand of a single person for 14 years.

ture, indicating the necessity of a pre-treatment step.

isomerization reactions are favoured by temperature.

Even though vegetable oils are the main feedstock for the production of first generation bio‐ fuels, soon their production has troubled the public opinion due to their abated sustainabili‐ ty and to their association with the food vs. fuel debate. As a result the technology has shifted towards the exploitation of both **solid and liquid residual biomass**. Waste Cooking Oils (WCOs) is a type of residual biomass resulting from frying with typical vegetable fry‐ ing oils (e.g. soybean-oil, corn-oil, olive-oil, sesame-oil etc). WCOs have particular problems regarding their disposal. In particular grease may result in coating of pipelines within the residential sewage system and is one of the most common causes of clogs and sewage spills. Furthermore, in the cases that sewage leaks into the environment, WCOs can cause human and environmental health problems because of the pathogens contained. It has been estimat‐ ed that by disposing 1 lit of WCO, over 1,000,000 of liters of water can be contaminated,

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Catalytic hydroprocessing of WCO was studied as an alternative approach of producing 2nd generation biofuels [20-24]. Initially **catalytic hydrocracking** was investigated over commer‐ cial hydrocracking catalysts leading not only to biodiesel but also to lighter products such as biogasoline [20], employing a continuous-flow catalytic hydroprocessing pilot-plant with a fixed-bed reactor. During this study several parameters were considered including hydro‐ cracking temperature (350-390°C) and liquid hourly space velocity or LHSV (0.5-2.5 hr-1) un‐ der high pressure (140 bar), revealing that the conversion is favoured by high reaction temperature and low LHSV. Lower and medium temperatures, however, were more suita‐ ble for biodiesel production while higher temperatures offered better selectivity for biogaso‐ line production. Furthermore, heteroatom removal (S, N and particularly O) was increased while saturation of double bonds was decreased with increasing hydrocracking tempera‐

However **catalytic hydrotreatment** was later examined in more detail as a more promising technology particularly for paraffinic biodiesel production (Figure 1). The same team has studied the effect of temperature (330-398°C) on the product yields and heteroatom removal [21]. The study was conducted in the same pilot plant utilizing a commercial NiMo/Al2O3 hydrotreating catalyst over lower pressure (80 bar). According to this study, the hydrotreat‐ ing temperature is the key operating parameter which defines the catalyst effectiveness and life. In fact lower temperatures (330°C) favour diesel production and selectivity. Sulfur and nitrogen removal were equally effective at all temperatures, while oxygen removal and satu‐ ration of double bonds were favoured by hydrotreating temperature. The same team also studied the effect of the other three operating parameters i.e. pressure, LHSV and H2/WCO ratio [22]. Moreover they also studied the hydrocarbon content of the products [23] qualita‐ tively via two-dimensional chromatography and quantitatively via Gas Chromatography with Flame Ionization Detector (GC-FID), which indicated the presence of C15-C18 paraf‐ fins. Interestingly this study showed that as hydrotreating temperature increases, the con‐ tent of normal paraffins decreases while of iso-paraffins increases, revealing that

#### **3.1. Raw vegetable oils conversion to paraffinic biofuels**

Vegetable oils are the main feedstock for the production of first generation biofuels, which can offer several CO2 benefits and limit the consumption of fossil fuels. Raw vegetable oils consist of fatty acid triglycerides, the consistency of which depends on their origin (i.e. plant type) as shown in Table 2. Their production, however, is competing for the cultivated areas that were originally dedicated for the production of food and feed crops. As a result the pro‐ duction and utilization of vegetable oils for biofuels production has instigated the "food vs. fuel" debate. For this reason traditional energy crops (soy, cotton, etc) with low oil yield per hectare are being substituted by new energy crops (eg. jatropha, palm, castor etc).


**Table 2.** Fatty acid composition of most common vegetable oils [14][15]

Catalytic hydrotreatment was explored for conversion of vegetable oils in the early 90's. The investigation of the hydrogenolysis of various vegetable oils such as maracuja, buritimtucha and babassu oils over a Ni–Mo/γ-Al2O3 catalyst as well as the effect of temperature and pressure on its effectiveness was firstly investigated [16][17]. The reaction products included a gas product rich in the excess hydrogen, carbon monoxide, carbon dioxide and light hy‐ drocarbons as well as a liquid organic product of paraffinic nature. In more detail these studies showed the conversion of triglycerides into carboxyl oxides and then to high quality hydrocarbons via decarboxylation and decarbonylation reactions. Rapeseed oil hydropro‐ cessing was also studied in lab-scale reactor for temperatures 310° and 360°C and hydrogen pressures of 7 and 15 MPa using three different Ni–Mo/alumina catalysts [18]. These prod‐ ucts contained mostly n-heptadecane and n-octadecane accompanied by low concentrations of other n-alkanes and i-alkanes [19].

#### **3.2. Waste cooking oils conversion to paraffinic biofuels**

Even though vegetable oils are the main feedstock for the production of first generation bio‐ fuels, soon their production has troubled the public opinion due to their abated sustainabili‐ ty and to their association with the food vs. fuel debate. As a result the technology has shifted towards the exploitation of both **solid and liquid residual biomass**. Waste Cooking Oils (WCOs) is a type of residual biomass resulting from frying with typical vegetable fry‐ ing oils (e.g. soybean-oil, corn-oil, olive-oil, sesame-oil etc). WCOs have particular problems regarding their disposal. In particular grease may result in coating of pipelines within the residential sewage system and is one of the most common causes of clogs and sewage spills. Furthermore, in the cases that sewage leaks into the environment, WCOs can cause human and environmental health problems because of the pathogens contained. It has been estimat‐ ed that by disposing 1 lit of WCO, over 1,000,000 of liters of water can be contaminated, which is estimated as the average demand of a single person for 14 years.

Catalytic hydroprocessing of WCO was studied as an alternative approach of producing 2nd generation biofuels [20-24]. Initially **catalytic hydrocracking** was investigated over commer‐ cial hydrocracking catalysts leading not only to biodiesel but also to lighter products such as biogasoline [20], employing a continuous-flow catalytic hydroprocessing pilot-plant with a fixed-bed reactor. During this study several parameters were considered including hydro‐ cracking temperature (350-390°C) and liquid hourly space velocity or LHSV (0.5-2.5 hr-1) un‐ der high pressure (140 bar), revealing that the conversion is favoured by high reaction temperature and low LHSV. Lower and medium temperatures, however, were more suita‐ ble for biodiesel production while higher temperatures offered better selectivity for biogaso‐ line production. Furthermore, heteroatom removal (S, N and particularly O) was increased while saturation of double bonds was decreased with increasing hydrocracking tempera‐ ture, indicating the necessity of a pre-treatment step.

However **catalytic hydrotreatment** was later examined in more detail as a more promising technology particularly for paraffinic biodiesel production (Figure 1). The same team has studied the effect of temperature (330-398°C) on the product yields and heteroatom removal [21]. The study was conducted in the same pilot plant utilizing a commercial NiMo/Al2O3 hydrotreating catalyst over lower pressure (80 bar). According to this study, the hydrotreat‐ ing temperature is the key operating parameter which defines the catalyst effectiveness and life. In fact lower temperatures (330°C) favour diesel production and selectivity. Sulfur and nitrogen removal were equally effective at all temperatures, while oxygen removal and satu‐ ration of double bonds were favoured by hydrotreating temperature. The same team also studied the effect of the other three operating parameters i.e. pressure, LHSV and H2/WCO ratio [22]. Moreover they also studied the hydrocarbon content of the products [23] qualita‐ tively via two-dimensional chromatography and quantitatively via Gas Chromatography with Flame Ionization Detector (GC-FID), which indicated the presence of C15-C18 paraf‐ fins. Interestingly this study showed that as hydrotreating temperature increases, the con‐ tent of normal paraffins decreases while of iso-paraffins increases, revealing that isomerization reactions are favoured by temperature.

**3.3. Pyrolysis oil upgrading**

Pyrolysis oil is the product of fast pyrolysis of biomass, a process that allows the decomposi‐ tion of large organic compounds of biomass such as lignin at medium temperatures in the presence of oxygen. Pyrolysis, that is in essence thermal cracking of biomass, is a well estab‐ lished process for producing bio-oil, the quality of which however is far too poor for direct use as transportation fuel. The product yields and chemical composition of pyrolysis oils de‐ pend on the biomass type and size as well as on the operating parameters of the fast pyroly‐ sis. However, a major distinction between pyrolysis oils is based on whether catalyst is employed for the fast pyrolysis reactions or not. Non-catalytic pyrolysis oils have a higher water content than catalytic pyrolysis oils, rendering the downstream upgrading process a

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Untreated pyrolysis oil is a dark brown, free-flowing liquid with about 20-30% water that cannot be easily separated. It is a complex mixture of oxygenated compounds including wa‐ ter solubles (acids, alcohols, ethers) and water insolubles (n-hexane, di-chloor-methane), which is unstable in long-term storage and is not miscible with conventional hydrocarbonbased fuels. It should be noted that due to its nature pyrolysis oil can be employed for the production of a wide range of chemicals and solvents. However, if pyrolysis oil is to be used as a fuel for heating or transportation, it requires upgrading leading to its stabilization and conversion to a conventional hydrocarbon fuel by removing the oxygen through catalytic hydrotreating. For this reason, a lot of research effort is focused on catalytic hydrotreating of pyrolysis oil, as it is a process enabling oxygen removal and conversion of the highly corro‐

For non-catalytic pyrolysis oils, the catalytic hydrotreating upgrading process involves con‐ tact of pyrolysis oil molecules with hydrogen under pressure and at moderate temperatures (<400°C) over fixed bed catalytic reactors. Single-stage hydrotreating has proved to be diffi‐ cult, producing a heavy, tar-like product. Dual-stage processing, where mild hydrotreating is followed by more severe hydrotreating has been found to overcome the reactivity of the bio-oil. Overall, the pyrolysis oil is almost completely deoxygenated by a combination of hy‐ dro deoxygenation and decarboxylation. In fact less than 2% oxygen remains in the treated, stable oil, while water and off-gas are also produced as byproducts. The water phase con‐ tains some dissolved organics, while the off-gas contains light hydrocarbons, excess hydro‐ gen, and carbon dioxide. Once the stabilized oil is produced it can be further processed into conventional fuels or sent to a refinery. Table 1 shows the properties of some common cata‐

Catalytic pyrolysis oils have been reported to getting upgraded via single step hydropro‐ cessing, most of the times utilizing conventional CoMo and NiMo catalysts. During the sin‐ gle step hydroprocessing, the catalytic pyrolysis oil feedstock is pumped to high pressure, then mixed with compressed hydrogen and enters the hydroprocessing reactor. In Table 5 the typical operating parameters for single stage hydroprocessing and associated deoxyge‐

more challenging one for the case of the non-catalytic pyrolysis oils.

sive oxygen compounds into aromatic and paraffinic hydrocarbons.

nation achievements are given according to literature [29;33-38].

lytic pyrolysis oils according to literature.

**Figure 7.** Catalytic hydrotreatment of WCO to 2nd generation biodiesel

The total liquid product of WCO catalytic hydrotreatment was further investigated in terms of its percentage that contains paraffins within the diesel boiling point range (220-360°C) [24]. The properties of WCO, hydrotreated WCO (total liquid product) and the diesel frac‐ tion of the hydrotreated WCO are presented in Table 3. Based on this study the overall yield of the WCO catalytic hydrotreatment technology was estimated over 92%v/v. The properties of the new 2nd generation paraffinic diesel product indicated a high-quality diesel with high heating value (49MJ/kg) and high cetane index (77) which is double of the one of fossil die‐ sel. An additional advantage of the new biodiesel is its oxidation stability (exceeding 22hrs) and negligible acidity, rendering it as a safe biofuel, suitable for use in all engines. The prop‐ erties and potential of the new biodiesel were further studied [25], for evaluating different fractions of the total liquid product and their suitability as an alternative diesel fuel.


**Table 3.** Basic properties of waste cooking oil, hydrotreated waste cooking oil and final biodiesel

#### **3.3. Pyrolysis oil upgrading**

Pyrolysis oil is the product of fast pyrolysis of biomass, a process that allows the decomposi‐ tion of large organic compounds of biomass such as lignin at medium temperatures in the presence of oxygen. Pyrolysis, that is in essence thermal cracking of biomass, is a well estab‐ lished process for producing bio-oil, the quality of which however is far too poor for direct use as transportation fuel. The product yields and chemical composition of pyrolysis oils de‐ pend on the biomass type and size as well as on the operating parameters of the fast pyroly‐ sis. However, a major distinction between pyrolysis oils is based on whether catalyst is employed for the fast pyrolysis reactions or not. Non-catalytic pyrolysis oils have a higher water content than catalytic pyrolysis oils, rendering the downstream upgrading process a more challenging one for the case of the non-catalytic pyrolysis oils.

Untreated pyrolysis oil is a dark brown, free-flowing liquid with about 20-30% water that cannot be easily separated. It is a complex mixture of oxygenated compounds including wa‐ ter solubles (acids, alcohols, ethers) and water insolubles (n-hexane, di-chloor-methane), which is unstable in long-term storage and is not miscible with conventional hydrocarbonbased fuels. It should be noted that due to its nature pyrolysis oil can be employed for the production of a wide range of chemicals and solvents. However, if pyrolysis oil is to be used as a fuel for heating or transportation, it requires upgrading leading to its stabilization and conversion to a conventional hydrocarbon fuel by removing the oxygen through catalytic hydrotreating. For this reason, a lot of research effort is focused on catalytic hydrotreating of pyrolysis oil, as it is a process enabling oxygen removal and conversion of the highly corro‐ sive oxygen compounds into aromatic and paraffinic hydrocarbons.

For non-catalytic pyrolysis oils, the catalytic hydrotreating upgrading process involves con‐ tact of pyrolysis oil molecules with hydrogen under pressure and at moderate temperatures (<400°C) over fixed bed catalytic reactors. Single-stage hydrotreating has proved to be diffi‐ cult, producing a heavy, tar-like product. Dual-stage processing, where mild hydrotreating is followed by more severe hydrotreating has been found to overcome the reactivity of the bio-oil. Overall, the pyrolysis oil is almost completely deoxygenated by a combination of hy‐ dro deoxygenation and decarboxylation. In fact less than 2% oxygen remains in the treated, stable oil, while water and off-gas are also produced as byproducts. The water phase con‐ tains some dissolved organics, while the off-gas contains light hydrocarbons, excess hydro‐ gen, and carbon dioxide. Once the stabilized oil is produced it can be further processed into conventional fuels or sent to a refinery. Table 1 shows the properties of some common cata‐ lytic pyrolysis oils according to literature.

Catalytic pyrolysis oils have been reported to getting upgraded via single step hydropro‐ cessing, most of the times utilizing conventional CoMo and NiMo catalysts. During the sin‐ gle step hydroprocessing, the catalytic pyrolysis oil feedstock is pumped to high pressure, then mixed with compressed hydrogen and enters the hydroprocessing reactor. In Table 5 the typical operating parameters for single stage hydroprocessing and associated deoxyge‐ nation achievements are given according to literature [29;33-38].


include hydrotreating or hydrotreating and hydrocracking reactions. In the first stage the catalytic hydrotreatment reactor stabilizes the pyrolysis oil by mild hydrotreatment over Co‐ Mo or NiMo hydrotreating catalyst [32;40-42]. The first stage product is then further proc‐ essed in the second-stage hydrotreater, which operates at higher temperatures and lower space velocities than the first stage hydrotreater, employing also CoMo or NiMo catalysts within the reactor. The 2nd stage product is separated into an organic-phase product, waste‐ water, and off-gas streams. In the literature [41], even a 3rdstage hydroprocessing has been used for the heavy fraction (which boils above 350°C) of the 2ndstage product, where hydro‐ cracking reactions take place for converting the heavy product molecules into gasoline and

**Feed 1st stage 2nd stage 3rd stage**

**Temperature (C°)** 150-240 225-370 350-427 **Pressure (psig)** 1000-2000 2015 1280

Biofuels production via the Fischer-Tropsch technology is a conversion process of solid bio‐ mass into liquid fuels (Biomass-To-Liquid or BTL) as it is depicted in Figure 2. More specifi‐ cally the solid biomass is gasified in the presence of air and the produced biogas rich in CO and H2 (synthesis gas), after being pretreated to remove coke residues and sulfur com‐ pounds, enters the Fischer-Tropsch reactor. The Fischer-Tropsch reactions allow the catalytic conversion of the synthesis gas into a mixture of paraffinic hydrocarbons consisting of light (C1-C4), naphtha (C5-C11), diesel (C12-C20) and heavier hydrocarbons (>C20). Even though the Fischer-Tropsch reactions yields depend on the catalyst and operating parameters employed [43-45], the liquid product (naphtha, diesel and heavier hydrocarbons) yield is high (~95%). The produced synthetic naphtha and diesel fuels can be used similarly to their fossil coun‐ terparts. The heavier product however, which is called as Fischer-Tropsch wax, due to its waxy/paraffinic nature should get upgraded via catalytic hydrocracking to get converted to

The conversion of Fischer-Tropsch wax into mainly diesel was studied in virtue of the Euro‐ pean Project RENEW [46]. During this project Fischer-Tropsch wax with high paraffinic con‐ tent of C20-C45 was converted into a total liquid product consisting of naphtha, kerosene and diesel fractions via catalytic hydrocracking. However the total liquid product content of die‐ sel molecules was the highest and the diesel fraction was further separated and character‐

CoMo[32][40]NiMo[32][42],

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others [39] CoMo[4141]

diesel blend components.

**Catalyst** CoMo[32][40],NiMo[32][42],

others[39]

**LHSV (hr-1)** 0.28-1 0.05-0.14

**Deoxygenation (wt%)** 60-98.6

**Table 6.** Multiple-step pyrolysis oil hydroprocessing operating parameters

**3.4. Fischer-Tropsch wax upgrading**

mid-distillate fuels (naphtha and diesel).

**Table 4.** Properties of different pyrolysis oils according to literature


**Table 5.** Single-stage pyrolysis oil hydroprocessing operating parameters

However, in the case of non-catalytic pyrolysis oils or for achieving better quality products, multiple-stage hydroprocessing can be employed for upgrading pyrolysis oils. Multiplestage hydroprocessing utilizes at least two different stages of hydroprocessing, which may include hydrotreating or hydrotreating and hydrocracking reactions. In the first stage the catalytic hydrotreatment reactor stabilizes the pyrolysis oil by mild hydrotreatment over Co‐ Mo or NiMo hydrotreating catalyst [32;40-42]. The first stage product is then further proc‐ essed in the second-stage hydrotreater, which operates at higher temperatures and lower space velocities than the first stage hydrotreater, employing also CoMo or NiMo catalysts within the reactor. The 2nd stage product is separated into an organic-phase product, waste‐ water, and off-gas streams. In the literature [41], even a 3rdstage hydroprocessing has been used for the heavy fraction (which boils above 350°C) of the 2ndstage product, where hydro‐ cracking reactions take place for converting the heavy product molecules into gasoline and diesel blend components.


**Table 6.** Multiple-step pyrolysis oil hydroprocessing operating parameters

#### **3.4. Fischer-Tropsch wax upgrading**

Biofuels production via the Fischer-Tropsch technology is a conversion process of solid bio‐ mass into liquid fuels (Biomass-To-Liquid or BTL) as it is depicted in Figure 2. More specifi‐ cally the solid biomass is gasified in the presence of air and the produced biogas rich in CO and H2 (synthesis gas), after being pretreated to remove coke residues and sulfur com‐ pounds, enters the Fischer-Tropsch reactor. The Fischer-Tropsch reactions allow the catalytic conversion of the synthesis gas into a mixture of paraffinic hydrocarbons consisting of light (C1-C4), naphtha (C5-C11), diesel (C12-C20) and heavier hydrocarbons (>C20). Even though the Fischer-Tropsch reactions yields depend on the catalyst and operating parameters employed [43-45], the liquid product (naphtha, diesel and heavier hydrocarbons) yield is high (~95%). The produced synthetic naphtha and diesel fuels can be used similarly to their fossil coun‐ terparts. The heavier product however, which is called as Fischer-Tropsch wax, due to its waxy/paraffinic nature should get upgraded via catalytic hydrocracking to get converted to mid-distillate fuels (naphtha and diesel).

The conversion of Fischer-Tropsch wax into mainly diesel was studied in virtue of the Euro‐ pean Project RENEW [46]. During this project Fischer-Tropsch wax with high paraffinic con‐ tent of C20-C45 was converted into a total liquid product consisting of naphtha, kerosene and diesel fractions via catalytic hydrocracking. However the total liquid product content of die‐ sel molecules was the highest and the diesel fraction was further separated and character‐ ized having density of 0.78gr/ml and cetane index of 76 [47]. The schematic of the BTL process with actual images of the feedstock, Fischer-Tropsch wax and synthetic diesel are given in Figure 8.

**3.6. Co-hydroprocessing**

tivity.

The effectiveness of catalytic hydroprocessing was also explored for co-processing of lipid feedstocks with petroleum fractions as catalytic hydroprocessing units are available in al‐ most all refineries. The first co-processing study involved experiments of catalytic hydro‐ treating of sunflower oil mixtures with heavy petroleum fractions aiming to produce high quality diesel [55]. The experiments were conducted in a continuous fixed-bed reactor over a wide range of temperatures 300-450°C employing a typical NiMo/Al2O3 hydrotreating cata‐ lyst. The study was focused on the hydrogenation of double C-C bonds and the subsequent paraffin formation via the three different reactions routes: decarbonylation, decarboxylation and deoxygenation. Furthermore the large carbon-chain paraffins can also undergo isomeri‐ zation and cracking leading to the formation of smaller paraffins. This study concluded that the selectivity of products on decarboxylation and decarbonylation is increasing as the tem‐

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In a similar study catalytic hydrocracking over sunflower oil and heavy vacuum gas oil mix‐ tures was investigated [56]. The experiments were conducted in a continuous-flow hydro‐ processing pilot-plant over a range of temperatures (350-390°) and pressures (70-140bar). Three different hydrocracking catalysts were compared under the same conditions and four different feedstocks were employed, incorporating for 10% and 30%v/v of lipid bio-based feedstock and considering non-pretreated and pretreated sunflower oil as a bio-based feed‐ stock. The results indicated that a prior mild hydrogenation step of sunflower oil is necessa‐ ry before hydrocracking. Furthermore, conversion was increased with increasing sunflower oil ratio in the feedstock and increasing temperature, while the later decreased diesel selec‐

The effect of the process parameters and the vegetable oil content of the feedstocks on the yield, physical properties, chemical properties and application properties during co-hydro‐ treating of sunflower oil and gas-oil mixtures utilizing a typical NiMo/Al2O3hydrotreating catalyst was also studied [57]. The experimental results of this study indicated that catalytic co-hydrogenation of gas oil containing sunflower oil in different percentages allowed both vegetable oil conversion reactions (saturation, deoxygenation) and the gas oil quality im‐ provement reactions (hetero atom removal, aromatic reduction). The optimal operating con‐

of feed) resulted in a final diesel product with favorable properties (e.g. less than 10 wppm sulfur, ~20% aromatics) but poor cold flow properties (CFPP=3°C). The study also indicated that for sunflower content in the feedstock higher than 15% reduced the desulfurization effi‐ ciency. Furthermore, the authors also concluded that the presence of sunflower oil in the feedstock has augmented the normal and iso-paraffins content of the final product and as a result has increased the cetane number but degraded the cold flow properties, indicating

The issue of catalyst development suitable for co-hydrotreating and co-hydrocracking of gas-oil and vegetable oil mixtures was recently addressed [10], as there are no commercial hydroprocessing catalysts available for lipid feedstocks. New sulfided Ni–W/SiO2–Al2O3 and sulfided Ni–Mo/Al2O3 catalysts were tested for hydrocracking and hydrotreating of gas-

/m3

and 15% sunflower oil content

perature and vegetable oil content in the feedstock increase [55].

ditions (360-380°C, P=80 bar, LHSV=1.0h-1, H2/oil=600 Nm3

that an isomerization step is required as an additional step.

**Figure 8.** Biomass-to-Liquid production of synthetic diesel

#### **3.5. Micro-algal oil conversion to biofuels**

The rapid development of the biofuels production technologies from different biomass types has given rise to the biomass and food markets as it was aforementioned. Besides the use of residual biomass, research and in particular biotechnology has moved forward into seeking alternative biomass production technologies that will offer higher yields per hectare as well as lipids and carbohydrates, which are not part of the human and animal food-chain, avoid‐ ing competition between food/feed and energy crops. Targeted research efforts have offered a promising solution by the selection of unicellular microorganisms for the production of bi‐ ofuels [48][49]. **Micro-algae** are photosynthetic microorganisms that can produce lipids, pro‐ teins and carbohydrates in large amounts over short periods of time.

Micro-algae are currently considered a prominent source of fatty acids, which offers large yields per hectare with various fatty acid foot-prints from each strain. In fact, there are cer‐ tain strains that offer fatty acids of increased saturation (small content of unsaturated fatty acids) and of smaller carbon-chain length such as *Dunaliellasalina*, *Chlorella minutissima*, *Spir‐ ulina maxima*, *Synechococcus sp.*[50] etc. Another advantage of algal oils is that their fatty acid content can be directed to small carbon-chain molecules either genetically or by manipulat‐ ing the aquaculture conditions such as light source and intensity [51], nitrogen starvation period [52], nutrients and CO2 feeding profiles [53].

Micro-algae and their products formulated the so called 3rd generation biofuels, as they in‐ corporate various characteristics, which render them superior over other biofuels and bio‐ mass types. Micro-algae can also be produced in sea water [54] or even waste water, while they are biodegradable and relatively harmless during an eventual spill. Furthermore, their yield per hectare can reach 3785-5678lit, which is 20-700 higher over the conventional energy crops yield (soy, rape and palm). The lipids contained in most micro-algal oils have a similar synthesis with that of soy-bean oil, while they also contain some poly-saturated fatty acids with four double bonds. As a result catalytic hydrotreating of micro-algal oil is the most promising technology for converting it into biofuels.

#### **3.6. Co-hydroprocessing**

The effectiveness of catalytic hydroprocessing was also explored for co-processing of lipid feedstocks with petroleum fractions as catalytic hydroprocessing units are available in al‐ most all refineries. The first co-processing study involved experiments of catalytic hydro‐ treating of sunflower oil mixtures with heavy petroleum fractions aiming to produce high quality diesel [55]. The experiments were conducted in a continuous fixed-bed reactor over a wide range of temperatures 300-450°C employing a typical NiMo/Al2O3 hydrotreating cata‐ lyst. The study was focused on the hydrogenation of double C-C bonds and the subsequent paraffin formation via the three different reactions routes: decarbonylation, decarboxylation and deoxygenation. Furthermore the large carbon-chain paraffins can also undergo isomeri‐ zation and cracking leading to the formation of smaller paraffins. This study concluded that the selectivity of products on decarboxylation and decarbonylation is increasing as the tem‐ perature and vegetable oil content in the feedstock increase [55].

In a similar study catalytic hydrocracking over sunflower oil and heavy vacuum gas oil mix‐ tures was investigated [56]. The experiments were conducted in a continuous-flow hydro‐ processing pilot-plant over a range of temperatures (350-390°) and pressures (70-140bar). Three different hydrocracking catalysts were compared under the same conditions and four different feedstocks were employed, incorporating for 10% and 30%v/v of lipid bio-based feedstock and considering non-pretreated and pretreated sunflower oil as a bio-based feed‐ stock. The results indicated that a prior mild hydrogenation step of sunflower oil is necessa‐ ry before hydrocracking. Furthermore, conversion was increased with increasing sunflower oil ratio in the feedstock and increasing temperature, while the later decreased diesel selec‐ tivity.

The effect of the process parameters and the vegetable oil content of the feedstocks on the yield, physical properties, chemical properties and application properties during co-hydro‐ treating of sunflower oil and gas-oil mixtures utilizing a typical NiMo/Al2O3hydrotreating catalyst was also studied [57]. The experimental results of this study indicated that catalytic co-hydrogenation of gas oil containing sunflower oil in different percentages allowed both vegetable oil conversion reactions (saturation, deoxygenation) and the gas oil quality im‐ provement reactions (hetero atom removal, aromatic reduction). The optimal operating con‐ ditions (360-380°C, P=80 bar, LHSV=1.0h-1, H2/oil=600 Nm3 /m3 and 15% sunflower oil content of feed) resulted in a final diesel product with favorable properties (e.g. less than 10 wppm sulfur, ~20% aromatics) but poor cold flow properties (CFPP=3°C). The study also indicated that for sunflower content in the feedstock higher than 15% reduced the desulfurization effi‐ ciency. Furthermore, the authors also concluded that the presence of sunflower oil in the feedstock has augmented the normal and iso-paraffins content of the final product and as a result has increased the cetane number but degraded the cold flow properties, indicating that an isomerization step is required as an additional step.

The issue of catalyst development suitable for co-hydrotreating and co-hydrocracking of gas-oil and vegetable oil mixtures was recently addressed [10], as there are no commercial hydroprocessing catalysts available for lipid feedstocks. New sulfided Ni–W/SiO2–Al2O3 and sulfided Ni–Mo/Al2O3 catalysts were tested for hydrocracking and hydrotreating of gasoil and vegetable oil mixtures respectively. The results indicated that the hydrocracking cat‐ alyst was more selective for the kerosene hydrocarbons (140–250°C), while the less acidic hydrotreating catalyst was more selective for the diesel hydrocarbons (250–380°C). The study additionally showed that the deoxygenation reactions are more favored over the hy‐ drotreating catalyst, while the decarboxylation and decarbonylation reactions are favored over the hydrocracking catalyst.

funded by the European Program LIFE". In this project WCO was collected from associated restaurants and the produced 2nd generation bio-diesel, to be called "white diesel" was em‐ ployed. For the demonstration of the new technology, 2 tons of "white diesel' were pro‐ duced via catalytic hydrotreatment of WCO based on the large-scale pilot units available in CERTH. The production process simplified diagram is given in Figure 10. The new fuel will be applied to a garbage truck in a 50-50 mixture with conventional diesel in August 2012, aiming to promote the new technology as it exhibits overall yields exceeding 92% v/v.

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In the USA the Dynamic Fuels company [60] has constructed in Baton Rouge a catalytic hy‐ drotreating unit dedicated to oils and animal fats with 285 Mlit capacity. The unit employs the Syntroleum technology based on Fischer-Tropsch for the production of synthetic 2nd gen‐ eration Biodiesel while it also produces bio-naphtha and bio-LPG. The Bio-Synfining tech‐ nology of Syntroleum converts the triglycerides of fats and oils into n- and iso-paraffins via catalytic hydrogenation, thermal cracking and isomerization as it is applied in the Fischer-

Gas

Liquid Product

*Sediment vessel*

**Figure 10.** Pilot-scale demo application of WCO catalytic hydrotreatment during the BIOFUELS-2G project [59]

Water 7% vol

> Heavy product (C25+) 7-8% vol

*Fractionator*

Biodiesel-2G (C16-C25) > 92-93% vol

Light hydrocarbons (C5-C15) < 1% vol

Tropsch wax upgrading to renewable diesel (R-2) and renewable jet (R-8) fuel.

Hydrogen ( 2)

WCO

*Reactor*

*High pressure separator*

#### **4. Demo and industrial applications**

As catalytic hydrotreating of liquid biomass has given promising results, the industrial world has given enough confidence to apply it in pilot and industrial scale. The NesteOil Corporation has developed the NExBTL technology for converting vegetable oil (primarily palm oil) into a renewable diesel also known as "green" diesel (Figure 9). Based on this tech‐ nology the first catalytic hydrotreatment of vegetable oils unit was constructed in Finland in 2007, within the existing Poorvo refinery of NesteOil, with a capacity of 170 kton/hr. The pri‐ mary feedstock is palm oil, while it can also process rapeseed oil and even waste cooking oil. The same company has constructed a second unit within the same refinery while it has also planned to construct two new units, one in Singapore and one in Rotterdam, with the capacity of 800 kton/yr each.

**Figure 9.** NExBTL catalytic hydrotreating of oils/fats technology for biodiesel production [58]

The catalytic hydrotreatment technology of 100% waste cooking oil for biodiesel production was developed in the Centre for Research and Technology Hellas (CERTH) in Thessaloniki, Greece [21-24] and later demonstrated via the BIOFUELS-2G project [59], which was cofunded by the European Program LIFE". In this project WCO was collected from associated restaurants and the produced 2nd generation bio-diesel, to be called "white diesel" was em‐ ployed. For the demonstration of the new technology, 2 tons of "white diesel' were pro‐ duced via catalytic hydrotreatment of WCO based on the large-scale pilot units available in CERTH. The production process simplified diagram is given in Figure 10. The new fuel will be applied to a garbage truck in a 50-50 mixture with conventional diesel in August 2012, aiming to promote the new technology as it exhibits overall yields exceeding 92% v/v.

In the USA the Dynamic Fuels company [60] has constructed in Baton Rouge a catalytic hy‐ drotreating unit dedicated to oils and animal fats with 285 Mlit capacity. The unit employs the Syntroleum technology based on Fischer-Tropsch for the production of synthetic 2nd gen‐ eration Biodiesel while it also produces bio-naphtha and bio-LPG. The Bio-Synfining tech‐ nology of Syntroleum converts the triglycerides of fats and oils into n- and iso-paraffins via catalytic hydrogenation, thermal cracking and isomerization as it is applied in the Fischer-Tropsch wax upgrading to renewable diesel (R-2) and renewable jet (R-8) fuel.

**Figure 10.** Pilot-scale demo application of WCO catalytic hydrotreatment during the BIOFUELS-2G project [59]

#### **5. Future perspectives**

Catalytic hydrotreating of liquid biomass is continuously gaining ground as the most effec‐ tive technology for liquid biomass conversion to both ground- and air-transportation fuels. The UOP company of Honeywell, via the technology it has developed for catalytic hydro‐ treating of liquid biomass (Figure 11), has announced imminent collaboration with oil and airline companies such as Petrochina, Air China and Boeing for the demonstration of the sustainable air-transport in China. This initiative will lead a strategic collaboration between the National Energy Agency of china with the Commerce and Development Agency of USA leading to the development of the new biofuels market in China.

**Airline Aircraft Partners**

Virgin Atlantic B747-400 Boeing, GE Aviation

Air New Zealand B747-400 Boeing, Rolls-Royce

JAL B747-300 Boeing, Pratt&Whitney,

KLM B747-400 GE, Honeywell UOP

TAM A320 Airbus, CFM

**Table 7.** Pilot flights with biofuels [62]

B737-800 Boeing, GE Aviation, CFM,

Honeywell UOP

Honeywell UOP

The highest interest is exhibited by oil companies around the catalytic hydrotreatment of liq‐ uid biomass technology for the production of biofuels and particularly to its application to oil from micro-algae. ExxonMobil has invested 600M\$ in the Synthetic Genomics company of the pioneer scientist Craig Ventner aiming to research of converting micro-algae to bio‐ fuels with minimal cost. BP has also invested 10M\$ for collaboration with Martek for the production of biofuels from micro-algae for air-, train-, ground- and marine transportation

Catalytic hydrotreatment of liquid biomass is the only proven technology that can overcome its limitations as a feedstock for fuel production (low H/C ratio, high oxygen and water con‐ tent). Even though it has recently started to be investigated as an alternative technology for biofuels production, it fastly gains ground due to the encouraging experimental results and successful pilot/demo and industrial applications. Catalytic hydrotreatment of liquid bio‐ mass leads to a wide range of new alterative fuels including bio-naphtha, bio-jet and biodiesel, are paraffinic in nature and as a result exhibiting high heating values, increased oxidation stability and negligible acidity and corrosivity. As a result it is not over-optimistic to claim that this technology will broaden the biofuels market into scales capable to actually

Contintental Airlines

applications.

**6. Conclusion**

mitigate the climate change problems.

**Biofuel (lipid sources)**

Catalytic Hydroprocessing of Liquid Biomass for Biofuels Production

FAME (coconut & palm)

> HRJ (Jatropha)

HRJ (Jatropha&algea)

HRJ (Camelina, Jatropha& algae)

> HRJ (Camelina)

HRJ (Jatropha) **Blend\***

321

http://dx.doi.org/10.5772/52649

20%

50%

50%

50%

50%

50%

**Figure 11.** Vegetable oil and animal fats conversion technology to renewable fuels of UOP [61]

In the EU airline companies collaborate with universities, research centers and biofuels com‐ panies in order to confront their extensive contribution to CO2 emissions. Since 2008 most airline companies promote the use of biofuels in selected flights as shown in Table 7 [62]. As it is obvious most pilot flights have taken place with Hydrotreated Renewable Jet (HRJ), which is kerosene/jet produced via catalytic hydrotreatment of liquid biomass. Moreover, Lufthansa has also completed a 6-month exploration program of employing HRJ in a 50/50 mixture with fossil kerosene in one of the 4 cylinders of a plane employed for the flight be‐ tween Hamburg-Frankfurt-Hamburg with excellent results [63].

Besides the future applications for air-transportation, the automotive industry is also exhib‐ iting increased interest for the broad use of biofuels resulting from catalytic hydrotreatment of liquid biomass. In fact these paraffinic biofuels can be employed in higher than 7%v/v blending ratio (which is the maximum limit for FAME) as they exhibit high cetane number and have significant oxidation stability [64]


**Table 7.** Pilot flights with biofuels [62]

The highest interest is exhibited by oil companies around the catalytic hydrotreatment of liq‐ uid biomass technology for the production of biofuels and particularly to its application to oil from micro-algae. ExxonMobil has invested 600M\$ in the Synthetic Genomics company of the pioneer scientist Craig Ventner aiming to research of converting micro-algae to bio‐ fuels with minimal cost. BP has also invested 10M\$ for collaboration with Martek for the production of biofuels from micro-algae for air-, train-, ground- and marine transportation applications.

#### **6. Conclusion**

Catalytic hydrotreatment of liquid biomass is the only proven technology that can overcome its limitations as a feedstock for fuel production (low H/C ratio, high oxygen and water con‐ tent). Even though it has recently started to be investigated as an alternative technology for biofuels production, it fastly gains ground due to the encouraging experimental results and successful pilot/demo and industrial applications. Catalytic hydrotreatment of liquid bio‐ mass leads to a wide range of new alterative fuels including bio-naphtha, bio-jet and biodiesel, are paraffinic in nature and as a result exhibiting high heating values, increased oxidation stability and negligible acidity and corrosivity. As a result it is not over-optimistic to claim that this technology will broaden the biofuels market into scales capable to actually mitigate the climate change problems.

#### **Acknowledgements**

The author would like to thank Ms Iva Simcic and InTech Europe for enabling her to publish this book chapter, while she is grateful to Mr Athanasios Dimitriadis who provided support, offered comments, proofreading and design. Finally she would like to express her apprecia‐ tion for the financial support provided by the EU project BIOFUELS-2G which is co-fi‐ nanced by the European Program LIFE+.

[8] Bezergianni S, Kalogianni A, Dimitriadis A, Catalyst Evaluation for Waste Cooking

Catalytic Hydroprocessing of Liquid Biomass for Biofuels Production

http://dx.doi.org/10.5772/52649

323

[9] Funimsky E. Catalytic Hydrodeoxygenation. Applied Catalysis A: General

[10] Tiwari R, Rana BS, Kumar R, Verma D, Kumar R, Joshi RK, Garg MO, Sinha AK. Hy‐ drotreating and hydrocracking catalysts for processing of waste soya-oil and refin‐

[11] Lavrenov AV, Bogdanets EN, Chumachenko YA, Likholobov VA. Catalytic processes for the production of hydrocarbon biofuels from oil and fatty raw materials: Contem‐

[12] Bulushev DA, Ross JRH. Catalysis for conversion of biomass to fuels via pyrolysis

[13] Bezergianni S, Dimitriadis A, Kalogianni A., Pilavachi PA. Hydrotreating of waste cooking oil for biodiesel production. Part I: Effect of temperature on product yields

[14] Tyson KS. Biodiesel Handling and Use Guidelines. National Renewable Energy Lab‐

[15] Winayanuwattikun P, Kaewpiboon C, Piriyakananon K, Tantong S, Thakernkarnkit W, Chulalaksananukul W, Yongvanich T. Potential plant oil feedstock for lipase-cata‐ lyzed biodiesel production in Thailand. Biomass & Bioenergy 2008;321279–1286. [16] Da Rocha Filho GN, Brodzki D, Djega-Mariadassou G. Formation of alkanes alkylcy‐ kloalkanes and alkylbenzenes during the catalytic hydrocracking of vegetable oils.

[17] Gusmao J, Brodzki D, Djega-Mariadassou G, Frety R. Utilization of vegetable oils as an alternative source for diesel-type fuel: Hydrocracking on reduced Ni/ SiO2 and

[18] Simacek P, Kubicka D, Sebor G, Pospisil M. Fuel properties of hydroprocessed rape‐

[19] Simacek P, Kubicka D, Sebor G, Pospisil M. Hydroprocessed rapeseed oil as a source

[20] Bezergianni S, Kalogianni A. Hydrocracking of used cooking oil for biofuels produc‐

[21] Bezergianni S, Dimitriadis A, Kalogianni A, Pilavachi PA. Hydrotreating of waste cooking oil for biodiesel production. Part I: Effect of temperature on product yields

[22] Bezergianni S, Dimitriadis A, Kalogianni A, Knudsen KG. Toward Hydrotreating of Waste Cooking Oil for Biodiesel Production. Effect of Pressure, H2/Oil Ratio, and

and heteroatom removal. Bioresource Technology 2010;101 6651-6656.

and heteroatom removal. Bioresource Technology 2010;101 6651–6656.

ery-oil mixtures. Catalysis Communications 2011;12 559-562.

porary approaches. Catalysis in Industry 2011;3(3) 250-259.

and gasification: A review. Catalysis Today 2011; 171(1) 1-13.

sulphided Ni–Mo/c-Al2O3. Cat Today 1989;5 533–544.

of hydrocarbon-based Biodiesel. Fuel 2009;88456–60.

tion. Bioresource Technology 2009;100(17) 3927-3932.

Oil Hydroprocessing, Fuel 2012;93 638-647.

oratory 2001, NREL/TP-580-30004

Fuel 1993;72 543–549.

seed oil. Fuel 2010;89611–615.

2000;199147-190.

#### **Author details**

Stella Bezergianni\*

Address all correspondence to: sbezerg@cperi.certh.gr

Chemical Processes & Energy Resources Institute (CPERI), Centre for Research & Technolo‐ gy Hellas (CERTH), Thermi-Thessaloniki, Greece

#### **References**


Liquid Hourly Space Velocity. Industrial Engineering Chemistry Research 2011;50(7) 3874-3879.

[37] de Miguel Mercader F, Groeneveld MJ, Kersten SRA, Way NWY, Schaverien CJ, Ho‐ gendoorn JA. Production of advanced biofuels: Co-processing of upgraded pyrolysis oil instandard refinery units. Applied Catalysis B: Environmental 2010;96 57–66. [38] Oasmaa A, Kuoppala E, Ardiyanti A, Venderbosch RH, Heeres HJ. Characterization

Catalytic Hydroprocessing of Liquid Biomass for Biofuels Production

http://dx.doi.org/10.5772/52649

325

[39] Venderbosch RH, Heeres HJ. Pyrolysis Oil Stabilisation by Catalytic Hydrotreat‐

[40] Jones SB, Holladay JE, Valkenburg C, Stevens DJ, Walton C, Kinchin C, Elliott DC, Czernik S. Production of Gasoline and Diesel from Biomass via Fast Pyrolysis, Hy‐ drotreating and Hydrocracking: A Design Case. Prepared for the U.S. Department of

[41] Pindoria RV, Megaritis A, Herod AA, Kandiyoti R. A two-stage fixed-bed reactor for direct hydrotreatment of volatiles from the hydropyrolysis of biomass: effect of cata‐ lyst temperature, pressure and catalyst ageing timeon product characteristics. Fuel

[42] Conti L, Scano G, Boufala J, Mascia S. Experiments of Bio-oil Hydrotreating in a Con‐ tinuous Bench-Scale Plant. In: Bridgwater, A.V.; Hogan, E.N., editors. Bio-Oil Pro‐

[43] Sharma RK, Bakhshi NN. Catalytic Upgrading of Pyrolysis Oil. Energy & Fuels

[44] Subiranas M, Schaub A. Combining Fischer-Tropsch (FT) and hydrocarbon reactions under FT reaction conditions - Catalyst and reactor studies with Co or Fe and Pt/

[45] Rosyadi E, Priyanto U, Suprapto, Roesyadi A, Nurunnabi M, Hanaoka T, Miyazawa T, Sakanishi K, Biofuel production by hydrocracking of biomass FT wax over NiMo/ Al2O3-SiO2 catalyst, Journal of the Japan Institute of Energy 2011;90(12) 1171-1176.

[47] Lappas AA, Voutetakis SS, Drakaki N, Papapetrou M., Vasalos IA. Production of Transportation Biofuels through Mild-Hydrocracking and catalytic cracking of wax‐ es produced from Biomass to Liquids (BTL) Process. Proceedings of the 14thEuro‐

[48] Posten C, Schaub G. Microalgae and terrestrial biomass as source for fuels—a process

[49] Schenk PM, Thomas-Hall SR, Stephens E, Marx UC, Mussgnug JH, Posten C, Kruse O, Hankamer B. Second Generation Biofuels: High-Efficiency Microalgaefor Biodie‐

[50] Patil V, Källqvist T, Olsen E, Vogt G, Gislerød HR. Fatty acid composition of 12 mi‐ croalgae for possible usein aquaculture feed. Aquaculture International 2007;151-9.

ZSM-5, International Journal of Chemical Reactor Engineering 2007; 5(A78).

of Hydrotreated Fast Pyrolysis Liquids. Energy Fuels 2010;24 5264–5272.

ment. Biofuel's Engineering Process Technology.

1998;77(15) 1715–1726.

1993;7 306-314.

Energyunder Contract DE-AC05-76RL01830. February 2009.

duction and Utilization. CPL Press, Newbury Berks, 1996, 198

[46] www.renew-fuel.com (accessed 8 August 2012)

pean Biomass Conference, Paris France; 2005

view. J.Biotechnol. 2009;142 64–69.

sel Production. Bioenerg Res 2008;120-43


[51] Kachroo D, Jolly SMS, Ramamurthy V. Modulation of unsaturated fatty acids content in algae Spirulinaplatensis and Chlorella minutissima in response to herbicide SAN 9785. Electronic Journal of Biotechnology 2006;9(4)386-390.

**Chapter 10**

**Hydrotreating Catalytic Processes for Oxygen Removal**

In a future sustainable scenario a progressive transition by the chemical and energy indus‐ tries towards renewable feedstock will become compulsory. Energy demand is expected to grow by more than 50% by 2035 [1], with most of this increase in demand emerging from developing nations. Clearly, increasing demand from finite petroleum resources cannot be a satisfactory policy for the long term. The transition to a more renewable production system is now underway; however, this transition needs more research and investment in new tech‐

Biomass appears as the only renewable source for liquid fuels and most commodity chemi‐ cals [2]. This is the reason why, in the near future, bio-refineries in which biomass is catalyti‐ cally converted to pharmaceuticals, agricultural chemicals, plastics and transportation fuels will take the place of petrochemical plants [3]. Indeed, biomass represents 77.4% of global renewable energy supply [4]. Current technologies to produce liquid fuels from biomass are typically multistep and energy-intensive processes, including the production of ethanol by fermentation of biomass derived glucose [5],bio-oils by fast pyrolysis or high pressure lique‐ faction of biomass [6,7], polyols and alkanes from hydrogenolysis of biomass derived sorbi‐ tol [8],and biodiesel from vegetable oils [9].Biomass can also be gasified to produce CO and H2(synthesis gas), which can be further processed to produce methanol or liquid alkanes

The so-called "First Generation" biofuels, such as sugarcane ethanol in Brazil, corn ethanol in US, oilseed rape biodiesel in Germany, and palm oil biodiesel in Malaysia,already present mature commercial markets and well developed technologies. Nonetheless, there is a world‐ wide increasing awareness against the use of edible oils and seeds to generate transporta‐ tion fuels, and critical voices have aroused questioning the actual sustainability of these

> © 2013 Gandarias and Arias; licensee InTech. This is an open access article 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.

© 2013 Gandarias and Arias; licensee InTech. This is a paper 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.

**in the Upgrading of Bio-Oils and Bio-Chemicals**

Iñaki Gandarias and Pedro Luis Arias

http://dx.doi.org/10.5772/52581

**1. Introduction**

nologies to be feasible.

through Fischer–Tropsch synthesis [10].

Additional information is available at the end of the chapter

