Chapter 7 Science of Alternative Feedstocks Hans P. Blaschek and Thaddeus C. Ezeji 21

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1 Chapter 7 Science of Alternative Feedstocks Hans P. Blaschek and Thaddeus C. Ezeji 21 Introduction Biomass, which represents both cellulosic and non-cellulosic materials, contains the most abundant source of fermentable carbohydrates that can be fermented into fuels and chemicals. According to the Department of Energy (DOE) Roadmap for Biomass Technology in the United States, biobased transportation fuels are projected to increase from the 0.5% of U.S. consumption in 2001 to 4% in 2010, 10% in 2020, and further to 20-30% in 2030, or about 60 billion gallons of gasoline equivalent per year. The production of ethanol from low-cost lignocellulosic biomass that does not compete with food crops may be the key to meeting the DOE target. This approach would be consistent with ethanol as an economically viable and sustainable energy source. As a result, interest in the cultivation of lignocellulosic crops such as switchgrass and Miscanthus for subsequent conversion into fermentable sugars is receiving considerable attention. In addition, industrial and agricultural co-products such as corn fiber, corn stover, dried distillers grains with solubles (DDGS), wheat straw, rice straw, soybean residues, as well as various types of agricultural and industrial wastes are presently considered as potential feedstocks for the production of fermentable sugars. The depolymerization of these renewable and abundant resources to fermentable sugars represents a challenge for microbiologists and chemical engineers due to their recalcitrant nature. One of the initial steps in the lignocellulosic biomass-to-fermentable sugars conversion process is pretreatment. The purpose of pretreatment is to alter the biomass macroscopic and microscopic structure as well as its sub-microscopic chemical composition, thereby allowing cellulase enzymes to access the cellulose with greater ease in order to obtain a greater yield of sugars [Mosier et al., 2005]. Depending on the ethanol production route, pretreatment may or may not be a factor in the conversion of lignocellulosic biomass to ethanol. Pretreatment adds approximately 30% to the cost of processing of the biomass. Various strategies have been developed to utilize alternative substrates for conventional and unconventional ethanol fermentations. This report reviews recent approaches for utilization of alternative feedstocks for ethanol production, including different ethanol production routes, as well as for selecting and manipulating the fermenting microorganisms in order to achieve better product specificity and yield. 21 Professor and Research Assistant, respectively, Department of Food Science and Human Nutrition, University of Illinois, Urbana-Champaign. 112

2 Terminology To facilitate the understanding of some of the terms used in this chapter, below are a few key definitions: Butanol is a four carbon alcohol. It can be produced via clostridial fermentation. Cellulose is a polymer of glucose. Unlike starch, the glucose monomers of cellulose are linked together through β-1-4 glycosidic bonds by condensation resulting in tightly packed and highly crystalline structures that are resistant to hydrolysis. Distillation is a method of separating chemical compounds based on their differences in volatility; and volatility is a measure of the speed at which a chemical compound evaporates. Esterification is a general name for a chemical reaction between alcohols and acids (carboxylic acids, mineral acids, and acid chlorides) to form compounds called esters. Fermentation is microbial conversion of carbohydrates into alcohols or acids by microorganisms. The most common type of fermentation involves product production in the absence of oxygen. Gasification involves a group of processes that turn biomass into combustible gas by breaking apart the biomass using heat and pressure to produce a combustible gas, volatiles, char, and ash. The gases can then be used as a fuel or feedstock chemical. Glucan is the anhydrous form of D-glucose as found within a polysaccharide such as starch or cellulose that has 1 molecule of water (18 g/mol) less mass due to a condensation reaction forming the polymer,c 6 H 10 O 5 Hemicellulose is a highly branched and substituted polymer comprised mainly of xylose and arabinose, with minor amounts of galactose and glucose. In the plant cell walls, hemicellulose holds crystalline microfibers of cellulose in place. Hydrogenolysis is the process of cleaving a molecule or compound with the addition of hydrogen atoms. Hydrolysis is the breaking of a glycosidic bond (formed through a condensation reaction) within a polysaccharide chain through the addition of water. Saccharification is the process of hydrolyzing a complex carbohydrate into a simple soluble fermentable sugar. Starch or oligosaccharides can be saccharified to produce glucose using glucoamylase enzyme. The solubles in DDGS contain residual oligosaccharides, organic acids, and non-volatile 113

3 metabolic by-products of the yeast-based ethanol fermentation. Biomass Composition and Depolymerization Typically, lignocellulosic biomass contains 56-72% fermentable carbohydrates (cellulose and hemicellulose) by dry weight (Figure 1; Table 1). Due to the nature of lignocellulosic biomass, it can be found virtually everywhere in our environment ranging from plants to municipal wastes. Given the right conditions, these different biomass sources can be fermented to ethanol and other liquid fuels (Figure 2). Cellulose, the major constituent of lignocellulosic biomass, is a polymer of glucose. Unlike starch, the glucose monomers of cellulose are linked together through β-1-4 glycosidic bonds resulting in tightly packed and highly crystalline structures that are resistant to hydrolysis. Cellulose fibers are embedded in a lignin-hemicellulose matrix and this property contributes to the recalcitrance of lignocellulosic biomass to hydrolysis. Therefore, pretreatment of lignocellulosic biomass before enzymatic hydrolysis is a vital step. Lignin 15-27% Hemicellulose 22-30% Cellulose 35-48% Figure 1. Typical Lignocellulosic Biomass Composition 114

4 Table 1. Composition of Representative Potential Lignocellulosic Raw Materials For Ethanol Production Hexan Glucan Galactan Mannan Total Pentan Xylan Arabinan Total Total fermentable Lignin % Dry weight basis Corn 2.4 a 71.7 b 5.5 c Corn fiber NA Corn stover DDGS NA Wheat straw Sugarcane bagasse NA Switchgras s Poplar NA: not available. Data on switchgrass and poplar from [Chung et al., 2005]. Data on wheat straw and sugarcane bagasse from [Lee, 1997]. Data on corn stover from [Laureano-Perez et al., 2005]. Data on corn fiber and dried distillers grains and solubles (DDGS) from author s laboratory [unpublished]. Data on corn from [Gulati et al., 1996]. a: cellulose; b: starch; c: xylan + arabinan Types of Biomass Sugar - based: sugarcane, sugar beets Starch - based: corn, potatoes, barley, wheat, etc Lignocellulosic: Switchgrass, Miscanthus, corn stover, corn fiber, DDGS, etc Animal waste: cow, swine, and poultry Food waste Municipal waste Industrial (especially food industry) waste (Bio)conversion Liquid biofuel Methanol CH 3 OH Ethanol C 2 H 5 OH Butanol C 4 H 9 OH Feedstock chemical e.g. acetic acid Figure 2. Biomass Conversion to Liquid Biofuels and Feedstock Chemicals 115

5 Hemicellulose, the second major constituent of lignocellulosic biomass, contributes significantly to the total fermentable sugars of the lignocellulosic biomass (Table 1 & Figure 1). Unlike cellulose, hemicellulose is chemically heterogeneous and easily hydrolyzed to its constituent monosaccharides. Depending on the plant source, these monosaccharides may include hexoses (glucose, galactose, mannose, rhamnose) and pentoses (xylose, arabinose). Acetate and uronic acids (glucuronic and galacturonic acids) are also constituents of the hemicellulosic component of lignocellulosic biomass. These compounds have been found to be a potential source of microbial inhibitors in lignocellulosic hydrolysates (Ezeji et al., 2007). Lignin is a condensate of products obtained from lignin monomers such as p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol (Klinke et al., 2004). While cellulose and hemicellulose contribute to the amount of fermentable sugars for ethanol production, products of lignin degradation are recognized as a potential source of microbial inhibitors (Ezeji et al., 2007). It was proposed over two decades ago that the lignin fraction of biomass be used as an ash-free solid fuel for the generation of energy. To date, there is no known study on the feasibility and scalability of this approach. But can we harness the energy content of lignin through an indirect ethanol production route. This will be discussed later in the chapter. Ethanol Production from Alternative Feedstocks Feedstock costs make up a significant portion of the ethanol production costs and the selection of readily available and cheap feedstock is vital to the economics of the ethanol fermentation. Extensive research is currently being carried out on the various processes that can be used to produce ethanol from heterogeneous feedstock such as lignocellulosic biomass. We therefore contend that the choice of feedstock will determine the ethanol production route (whether conventional or unconventional). In addition to the conventional ethanol production route, we have identified two alternative ethanol production routes using alternative feedstocks which we believe have great potential for scale-up and commercialization. Conventional Ethanol Production In conventional ethanol production, starches and sugars are converted into ethanol in a few steps involving a series of enzymes. Thermostable α-amylase is used in the presence of water and heat to liquefy the starch, followed by glucoamylase which saccharifies the liquefied starch to sugars. Subsequently, a biocatalyst is added in the form of yeast that ferments the sugars to ethanol. This is followed by distillation of the beer to produce pure ethanol (Figure 3). The overall reaction shows the conversion of glucose to ethanol and CO 2, which can be represented stoichiometrically as follows: Starch C 6 H 12 O 6 2C 2 H 5 OH + 2CO 2 + ATP + Amylolytic enzymes + Yeast 116

6 This process is a mature technology that is carried out industrially. When lignocellulose is the substrate for ethanol production, there may be some drawbacks, however. These drawbacks relate to the complexity of the lignocellulosic cell wall components that make up the biomass. Furthermore, hydrolysis of the hemicellulose portion of the lignocellulosic biomass generates significant amounts of pentose sugars and potential microbial inhibitors such as uronic, ferulic and acetic acids. The presence of these compounds in the hydrolysates exerts undue stress on the fermenting microorganism, leading to poor cell growth and low ethanol titers and productivity. Unconventional Ethanol Production Gasification and Fermentation One unconventional ethanol production route involves gasification of the biomass, such as switch grass, Miscanthus, crop residues, etc. to generate producer gas. The producer gas, composed primarily of CO, CO 2, H 2, and N 2, is fermented by a biocatalyst to ethanol and acids. Recent work by Datar et al., 2004 and Ahmed & Lewis, 2007, demonstrated the integration of a fluidized-bed gasifier with a bioreactor to utilize biomass-syngas for producing ethanol. The process is illustrated in Figure 4. The combined technologies of gasification and fermentation have the potential to produce ethanol from recalcitrant lignocellulosic biomass. One major advantage of this process is that the carbon-rich lignin fraction (which contains 50% more carbon than the fermentable carbohydrates) is converted to producer gas and then to ethanol. Pretreatment and hydrolysis of lignocellulosic biomass to produce fermentation sugars is totally eliminated when ethanol is produced via the gasification and fermentation route. Fermentation Esterification Hydrogenolysis Process A second unconventional ethanol production route is a radically new approach for producing fuel ethanol from biomass. The process involves fermentation, esterification, and hydrogenation. During the first step, monosaccharides are fermented to acetic acid at near 100% carbon yield by homoacetogenic microorganisms. In the second step, the acetic acid is esterified in the presence of an alcohol to produce an ester, while in the third step; the ester undergoes hydrogenolysis to produce ethanol (ZeaChem, Inc.). The process is stoichiometrically illustrated in the following reactions: Fermentation Esterification Hydrogenolysis Ethanol Fermentation: C 6 H 12 O 6 O 3CH 3 COH [Acetic acid] O Esterification: 3CH 3 COH +3ROH 3CH 3 COR [Acetate ester]+ 3H 2 O Hydrogenolysis: 3CH 3 COR + 6H 2 3C 2 H 5 OH + 3ROH [alcohol] Net: C 6 H 12 O 6 + 6H 2 3CH 3 CH 2 OH + 3H 2 O 117

7 In this process, 3 moles of ethanol are produced from 1 mole of glucose resulting in a 50% improvement over the conventional route where 2 moles of ethanol are produced from 1 mole of glucose. The energy for the third mole of ethanol is supplied by hydrogen (ZeaChem, Inc.), which can be generated by the gasification of the lignocellulosic biomass. We therefore suggest that in order to accelerate the development of an efficient ethanol production process using alternative feedstocks, research in this area should be diversified to include unconventional ethanol production routes. Alternative Feedstocks The substrate commonly used in the United States today for fuel ethanol production is starch from agricultural crops, primarily corn. In order to meet the DOE projected bioenergy target in the year 2030, the use of alternative feedstocks for fuel ethanol production must increase dramatically. A joint study by the USDA and the U.S. Department of Energy concluded that at least 1 billion tons of biomass in the form of corn stover, wheat straw, wood wastes, etc. could be collected and processed in the U.S. each year which would be independent of the food supply. These feedstocks represent an equivalent of over 65 billion gallons of ethanol, with a potential for replacing 30% of the gasoline consumption in the U.S. (U.S. Department of Energy Biofuels: 30% by 2030 Website). Of course there are significant regional differences in the availability of lignocellulosic feedstocks which must be considered. While corn stover is abundant in the Midwestern States of U.S., rice straw will be an important feedstock source in California and Texas and soft woods will dominate in the Southeastern U.S. Dried Distillers Grain and Solubles (DDGS) and Corn Fiber Dried distillers grain with solubles (DDGS) are residues (proteins, fiber, and oils) obtained following the yeast fermentation of saccharified whole corn grain. DDGS is produced by blending corn distillers liquid solubles with wet corn distillers grains and the mixture is dried. The number of dry grind ethanol plants is growing rapidly in the United States. The boom in construction of drymill based ethanol plants is evidence of the biobased opportunities in this area. For every bushel of corn being converted into ethanol, 18 lb of DDGS is generated, which can be further converted to approximately 6.2 lb of fermentable sugars. In a concerted effort undertaken by the Midwest Consortium for Sustainable Biobased Products and Energy to address the proliferation of low value DDGS, a team of scientists from Purdue University, Michigan State University, University of Illinois, Iowa State University, Ames Laboratory, and USDA NCAUR is conducting research to further process DDGS into fermentable sugars for the production of ethanol and butanol (a secondgeneration liquid fuel), while leaving a solid that is reduced in weight and rich in protein. Furthermore, corn fiber, a co-product from the corn wet milling industry represents another readily available alternative feedstock for fuel ethanol production. Approximately 13.9 billion pounds of corn fiber is produced annually in the U.S. Corn fiber contains about 70% fermentable sugars, of which approximately 20, 14, and 35% come from starch, cellulose, and hemicellulose, respectively. Currently, corn fiber is marketed as a low cost animal feed ingredient. The further utilization of corn fiber and DDGS to produce value-added products such as ethanol and butanol is consistent with the National Renewable Energy Laboratory (NREL) sugar-platform biorefinery model for future dry-grind plants. Such an integrated approach is expected to contribute to biofuel 118

8 profitability and efficiency. Corn Stover Corn stover represents about 50% of the corn plant. Approximately one ton of corn stover is generated from one ton of corn grain produced. Corn stover is made up of about 50% stalks, 23% leaves, 15% cob, and 12% husks. Corn stover contains about 66% fermentable sugars, of which approximately 38 and 28% come from cellulose, and hemicellulose, respectively. Producing ethanol from corn stover hydrolysates will increase the possibility of ethanol production on a larger scale. How much of the corn stover that can be collected without detrimental effect on the soil is a complex question whose answer is beyond the scope of this report. However, one school of thought suggests that about 85% of corn stover residue rots on the ground which releases CO 2 into the atmosphere. The remaining 15% is incorporated in soil as organic matter. Another estimate suggests that 50% of the corn stover left after harvest can be collected without negative impact on the soil carbon. Other critical factors such as erosion control, soil moisture retention and regional climate must also be considered when harvesting corn stover. Switchgrass and Miscanthus Interest in the cultivation of lignocellulosic crops such as switchgrass and Miscanthus for subsequent conversion into fermentable sugars is receiving considerable attention. Switchgrass contains about 56.6% fermentable sugars and 23.2% lignin (Table 1). On the other hand, Miscanthus contains about 68% fermentable sugars, of which approximately 44 and 24% come from cellulose, and hemicellulose, respectively ( factsheet.html). In addition, Miscanthus contains about 17% lignin. By applying the appropriate processing conditions, these fermentable sugars and the lignin should be convertible to ethanol. Miscellaneous (Wheat and Rice Straws, Sugarcane Bagasse, Wood, Etc.) For decades, farmers in the rice-growing region north of Sacramento, California have collected left over rice straw into heaps and burned them after harvest. However, in the early 1990s, California lawmakers passed a law and put in place a program for rice farmers to gradually discontinue routine burning in order to reduce the associated production of smoke and its impact on urban areas. As a result, rice farmers have been looking for alternative ways to utilize or dispose of rice straw. Table 2 shows different lignocellulosic biomass materials which have potential for ethanol fermentation. If these feedstocks are broken down into sugar monomers, the released sugars can be used to make ethanol. This lignocellulosic ethanol can also be purified using the same technology as corn-based ethanol production (Figure 3). 119

9 Table 2. Percentage of Cellulose, Hemicellulose and Lignin Content in Common Agricultural Residues and Wastes Agricultural residue Cellulose Hemicellulose Lignin Hardwood Softwood Corn cobs Grasses Wheat straw Rice straw Source: Prasad et al. (2007), Kaur et al. (1998), and McKendry (2002) Figure 3. Process of Making Ethanol From Whole Corn (Source: Genencor, with Modification) 120

10 Lignocellulosic Biomass Pretreatment For Ethanol Production About 98% of the ethanol produced in the United States today is produced by dry or wet milling processes using corn. These processes are relatively simple: corn grinding (and starch separation in the case of wet milling), liquefaction, saccharification, fermentation, distillation, and dehydration (Figure 3). Alternatively, biomass is composed of lignin, cellulose, and hemicellulose. One of the primary functions of lignin is to provide structural support for the plant; and unfortunately from the standpoint of biofuel production, it also encloses the cellulose and hemicellulose molecules. In addition to the structural characteristics of biomass feedstock, the encapsulation of cellulose by lignin makes the lignocellulosic biomass nearly inaccessible to hydrolytic enzymes, and as a result more difficult to hydrolyze than more traditional starchy materials. Therefore, one of the key steps in the lignocellulosic biomass-to-fermentable sugars conversion is pretreatment. The purpose of pretreatment (Figure 5) is to break the lignin-hemicellulose matrix in order to facilitate cellulase enzymes access to the cellulosic portion of the biomass for subsequent hydrolysis to glucose. The common methods that have been employed to make lignocellulosic biomass more accessible to hydrolysis are dilute acid, alkaline, hot water, and ammonia pretreatments. Due to the recalcitrant nature of these lignocellulosic feedstocks, their pretreatment often requires a combination of physical, chemical, and heat treatments to disrupt the structure and convert it into a more hydrolysable form. The complete depolymerization of these renewable feedstocks in a cost-effective manner with minimal formation of degradation products represents a significant challenge for microbiologists and chemical engineers. Figure 5. Schematic of Goals of Pretreatment on Lignocellulosic Material (Adapted From Hsu et al., 1980). Dilute sulfuric acid pretreatment can be applied to agricultural residues to bring about hydrolysis. This is the oldest technology for converting lignocellulosic feedstock to fermentable sugars with subsequent fermentation to ethanol. Unfortunately, during acid hydrolysis, a complex mixture of microbial inhibitors is generated. Lignins are oxidized or degraded to form phenolic compounds and parts of the sugars that are released during hydrolysis are also degraded into products that inhibit cell growth and fermentation. Examples of the inhibitory compounds 121

11 that may be produced include furfural, hydroxymethyl furfural (HMF), and acetic, ferulic, glucuronic, ρ-coumaric acids (Zaldivar et al., 1999; Ezeji et al., 2007a). These inhibitors can be divided into three groups based on their origin: (1) compounds released from the hemicellulose structure, e.g. acetic, ferulic, glucuronic, ρ-coumaric acids, etc; (2) lignin degradation products, e.g. syringaldehyde; and (3) sugar degradation products, e.g. furfural and hydroxymethylfurfural. Using a single feedstock (corn stover), common analytical protocols, and consistent data interpretation, five research teams documented the technical and economical feasibility of selected pretreatment techniques (Wyman et al., 2005; Eggeman and Elander, 2005). They found that, among the dilute acid (Lloyd and Wyman, 2005), hot water (Mosier et al., 2005b), ammonia fiber expansion (AFEX) (Teymouri et al., 2005), ammonia recycle percolation (ARP) (Kim and Lee, 2005), and lime (Kim and Holtzapple, 2005) pretreatments, low cost pretreatment reactors are often counterbalanced by the higher costs associated with either pretreatment catalyst recovery or down-stream processing. A summary of the pretreatment methods and their limitations is given in Table 3. Table 3. Summary of Biomass Pretreatment Methods For Ethanol Production Pretreatment Increases Decrystalizes Removes Removes surface cellulose hemicellulose lignin area Limitations Corrosion, neutralization, Dilute acid 1 Yes Yes formation of inhibitors, and disposal of neutralization salts Hot water 2 Yes Yes High temperature, need to add alkaline to control ph, and relatively high cost in product recovery AFEX 3 Yes Yes Yes Cost of ammonia ARP 4 Yes Yes Yes Cost of ammonia and relatively Lime 5 Yes ND Yes low conversion of xylose Long treatment time, relatively low conversion of xylose, and relatively low solids a Cumulative soluble sugars as (oligomers + monomers)/monomers. Single number = just monomers. ND: Not determined. b e-hydrolysis = enzymatic hydrolysis. 1 Lloyd and Wyman (2005); 2 Mosier et al. (2005b); 3 Teymouri et al. (2005); 4 Kim and Lee (2005); 5 Kim and Holtzapple (2005). The complete hydrolysis of hemicellulose requires xylanase, β-xylosidase, and several other complimentary enzymes such as acetylxylan esterase, α-arabinofuranosidase, α- glucuronidase, α-galactosidase, ferulic and/or p-coumaric acid esterase (Ezeji et al., 2007b). The activities of these enzymes, in addition to the activities of cellulases on the cellulose component of the biomass, result in the generation of complex mixture of acids (ferulic, p-coumaric, acetic, glucuronic) in addition to monomeric sugars such as glucose, galactose, xylose, and arabinose in the biomass hydrolysates. Acids such as ferulic and p-coumaric have been found to be inhibitory to the solventogenic clostridia (used for producing butanol and acetone) at concentrations as low as 0.3 g/l [Ezeji et al., 2007a]. Therefore, for complete depolymerization of lignocellulosic 122

12 biomass, it is difficult to totally avoid the generation of inhibitory compounds irrespective of the pre-treatment and hydrolysis method utilized. A closer look at Table 3 reveals that the acidic pretreatment methods (including hot water) are effective in removing hemicellulose while the alkali methods (AFEX, ARP, and lime) function to remove lignin and decrystallize the cellulose. Both acidic and alkaline pretreatments are known to improve enzymatic digestibility. In addition, economic analysis of the pretreatment methods has shown that the relatively high costs associated with ethanol production from lignocellulosic biomass arise mainly from three factors: a) harsh pretreatment conditions (high temperature, use of acids or bases, etc.), b) use of costly enzymes, and c) recovery of end products (Eggeman and Elander, 2005). Technologies that lead to improvement in any of the above areas will help to improve the profitability of a biofuel production operation (Dr. Hao Feng, University of Illinois Urbana-Champaign, personal communication). Strain Selection and Improvement For Alternative Feedstock Utilization The fermentation of lignocellulosic biomass to ethanol is both timely and challenging. The ability to utilize all the sugars present in the lignocellulosic feedstock is necessary for efficient production of ethanol. It is not clear which of the three major microbial platforms (yeast, anaerobic Gram-positive bacteria such as clostridia, or Gram-negative microbes such as E. coli, Zymomonas mobilis) for biofuel production will be the primary production microbes of the future. Yeast remains the traditional microorganism of choice for ethanol production due to its high tolerance for ethanol during fermentation. The use of yeast involves fermentation of glucose and sucrose to ethanol. Efficient fermentation of lignocellulosics to ethanol by yeast is difficult due to the heterogeneity (pentoses and hexoses) of the feedstock. Metabolic engineering research is being carried out to develop new industrial yeast strains with the ability to efficiently convert lignocellulosic hydrolysates to ethanol. However, engineering Saccharomyces cerevisiae to co-ferment hexose and pentose sugars is constrained by the stoichiometric feasibility of the enzymatic activities of the introduced genes and the physiology of the yeast. Recombinant S. cerevisiae developed in several laboratories have used xylose oxidatively as opposed to fermentative utilization of glucose for ethanol production (Jin and Jeffries, 2004). Since xylose is a major constituent of lignocellulosic hydrolysate, ethanol production from xylose (with comparable ethanol yield from glucose) is essential for successful utilization of this feedstock for ethanol production. Dr. Lee Lynd has examined simultaneous hydrolysis and fermentation using the anaerobic bacterium Clostridium thermocellum to make ethanol from cellulosic feedstocks (Lynd, 1989; Lynd, 1996; Lynd et al., 1991), but there is a problem. C. thermocellum is capable of hydrolyzing cellulose and xylan to glucose and xylose, respectively. However, this microorganism can only utilize hexoses (and not the pentose sugars) generated from cellulose and hemicellulose (Demain et al., 2005). On the other hand C. thermosaccharolyticum (Saddler et al. 1984, Venkateswaren& Demain, 1986), C. thermohydrosulfuricum (Germain et al., 1986), and Thermoanaerobacter ethanolicus (Wiegel & Ljungdahl, 1981) are capable of utilizing hexose sugars as well as pentose sugars. As a result, the use of mixed culture systems where 123

13 cellulose is broken down by the cellulase complex of C. thermocellum to sugars and the fermentation of these sugars by this microorganism in combination with pentose utilizing microorganisms to ethanol is of great interest (Demain et al., 2005). One such mixed culture system involved C. thermocellum and C. thermosaccharolyticum for ethanol production when using cellulosic feedstock as a carbon source (Duong et al., 1983). However, the formation of co-products such as acetate and lactate that decrease the yield of ethanol and can act as weak uncouplers and inhibit cell growth (Herrero et al. 1985) have been a problem. More work is needed to address this problem. One potential solution is the elimination of metabolic pathways for co-products formation in order to make ethanol as the sole or major fermentation product, thereby enhancing ethanol yield. One obvious approach is to knock out the genes (encoding acetate kinase and/or phosphotransacetylase and lactate dehydrogenase) that are responsible for the branched metabolic pathways (Demain et al., 2005). The effect of this approach on the physiology of the microorganisms needs to be investigated. The question of which microbial platform will dominate as biocatalyst in the quest for alternative feedstock utilization for ethanol production will depend on which area (yeast, Grampositive and Gram-negative bacteria) of research is most successful. When strains that can efficiently utilize mixed pentose and hexose sugars for ethanol production are developed, the process will require that these microbes also have a high tolerance for degradation products such as acetic acid, furfural, HMF, ferulic acid, etc. Conclusions The rationale behind seeking alternative feedstocks for ethanol production is the continued increase in energy demand worldwide, notably in China, India, United States, and the United Kingdom. The use of alternative feedstocks such as lignocellulosic biomass for ethanol production holds great promise due to its widespread availability and abundance. This feedstock is available in the form of agricultural and forestry residues, and industrial and municipal wastes. Due to the recalcitrant nature of lignocellulosic biomass to enzymatic hydrolysis, several investigations have been carried out by agronomists and plant geneticists to improve biomass characteristics. These include development of plants that self-produce cellulases, plants with low lignin content, high polysaccharide content, and higher overall plant biomass yield. Significant progress has been made in this area, but fermentation of lignocellulosic biomass to ethanol still remains a challenge due to the necessity of converting both hexose and pentose sugars (constituents of the hydrolysates) obtained from this feedstock to ethanol in a single fermentation step. In addition, the generation of degradation and microbial inhibitory products during pretreatment and hydrolysis of biomass results in poor growth and ethanol productivity by the fermentation microorganisms. To circumvent these problems, more research effort needs to be directed toward unconventional methods for ethanol production. For instance, lignin and other non-fermentable materials account for nearly 40% of the energy content of the lignocellulosic biomass. This portion can be harnessed and utilized for ethanol production via the simultaneous gasification and fermentation process (Figure 4). 124

14 Biomass: Energy crops, Crop residues, Animal waste, Food waste, etc Gasifier: Conversion of biomass to producer gas (H 2, CO 2, CH 4, CO, N 2 ) Bioreactor: Fermentation of producer gas to ethanol (and other value-added products) Ethanol Clostridium ljungdahlii, C. autoethanogenum, C. carboxidivorans P7 T, etc Figure 4. Process For Ethanol Production By Simultaneous Gasification and Fermentation 125

15 References Ahmed A, Lewis RS: Fermentation of biomass-generated synthesis gas: Effect of nitric oxide. Biotechnol Bioeng 2007 (in press). Chung Y-C, Bakalinsky A, Penner MH: Enzymatic saccharification and fermentation of xyloseoptimized dilute acid-treated lignocellulosics. Appl Biochem Biotechnol 2005, : Datar RP, Shenkman RM, Cateni BG, Huhnke RL, Lewis RS: Fermentation of biomassgenerated producer gas to ethanol. Biotechnol Bioeng 2004, 86: Demain AL, Newcomb M, Wu JHD Cellulase, clostridia, and ethanol. Microbiol Mol Rev. 69: Duong CTV, Johnson EA, Demain AL Thermophilic, anaerobic and cellulolytic bacteria. Enzyme Ferm. Biotechnol. 7: Ezeji TC, Qureshi N, Blaschek HP: Butanol production from agricultural residues: Impact of degradation products on Clostridium beijerinckii growth and butanol fermentation. Biotechnol. Bioeng 2007a, (in press). Ezeji TC, Qureshi N, Blaschek, HP. Bioproduction of butanol from biomass: from genes to bioreactors. Current Opinion in Biotechnology 2007b (in press). Germain P, Toukourou F, Donaduzzi L Ethanol production by anaerobic thermophilic bacteria: regulation of lactate dehydrogenase activity in Clostridium thermohydrosulfuricum. Appl. Microbiol. Biotechnol. 24: Gulati M, Kohlmann K, Ladisch MR, Hespell R, Bothast RJ: Assessment of ethanol production options for corn products. Bioresour Technol 1996, 58: Herrero, AA, Gomez RF, Snedecor B, Tolman CJ, Roberts MF Growth inhibition of Clostridium thermocellum by carboxylic acids: a mechanism based on uncoupling by weak acids. Appl. Microbiol. Biotechnol. 22: Hsu TA, Ladisch MR, Tsao GT, Alcohol from cellulose. Chemical Technology 10: Jin YS, Jeffries TW Stoichiometric network constraints on xylose metabolism by recombinant Saccharomyces cerevisiae. Metabol Eng 6: Kaur PP, Arneja JS, Singh J Enzymatic hydrolysis of rice straw by crude cellulose from Trichoderma reesei. Bioresour Technol 66:

16 Kim, S., Holtzapple, M.T., Lime pretreatment and enzymatic hydrolysis of corn stover, Bioresource Technol., 96: Kim, T.H., Lee, Y.Y., Pretreatment and fractionation of corn stover by ammonia recycle percolation (ARP) process, Bioresource Technol., 96: Klinke HB, Thomsen AB, Ahring BK: Inhibition of ethanol-producing yeast and bacteria by degradation products during pre-treatment of biomass. Appl Microbiol Biotechnol 2004, 66: Laureano-Perez L, Teymouri F, Alizadeh H, Dale BE: Understanding factors that limit enzymatic hydrolysis of biomass. Appl Biochem Biotechnol 2005, : Lee J: Biological conversion of lignocellulosic biomass to ethanol. J Biotechnol 1997, 56: Lloyd and Wyman, 2005 Lloyd, T.A., Wyman, C.E., Combined sugar yields for dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids, Bioresource Technol., 96: Lynd LR Production of ethanol from lignocellulosic material using thermophilic bacteria: critical evaluation of potential and review. Adv. Biochem. Eng. Biotechnol. 38:1 52. Lynd LR Overview and evaluation of fuel ethanol from cellulosic biomass: technology, economics, the environment, and policy. Annu. Rev. Energy Environ. 21: Lynd LR, Cushman JH, Nichols RJ, Wyman CE Fuel ethanol from cellulosic biomass. Science 251: McKendry P Energy production from biomass (part I): overview of biomass. Bioresour Technol 83: Mosier, N., Hendrickson, R., Ho, N., Sedlak, M., Ladisch, M.R., 2005b. Optimization of ph controlled liquid hot water pretreatment of corn stover, Bioresource Technol., 96: Mosier N, Wyman C, Dale BE, Elander R, Lee YY, Holtzapple M, Ladisch MR: Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 2005, 96: Prasad S., Singh A., Joshi HC Ethanol as an alternative fuel from agricultural, industrial and urban residues. Resources, Conservation and Recycling 50: Saddler JN, Chan MK-H Conversion of pretreated lignocellulosic substrates to ethanol by Clostridium thermosaccharolyticum and Clostridium thermohydro sulphuricum. Can. J. Microbiol. 30:

17 Teymouri, F., Laureano-Perez, L., Alizadeh, H., Dale, B.E., Optimization of the ammonia fiber explosion (AFEX) treatment parameters for enzymatic hydrolysis of corn stover, Bioresource Technol., 96: Venkateswaren S, Demain AL The Clostridium thermocellum-clostridium thermosaccharolyticum ethanol production process: nutritional studies and scale-down. Chem. Eng. Commun. 45: Wiegel, J., and L. G. Ljungdahl Thermoanaerobacter ethanolicus gen. nov., a new extreme thermophilic, anaerobic bacterium. Arch. Microbiol. 128: Wiselogel A, Tyson S, Johnson D. Biomass Feedstock Resources and Composition, pp in Handbook on Bioethanol: Production and Utilization (Applied Energy Technology Series), ed. C. E. Wyman, Taylor and Francis (1996). Zaldivar J, Martinez A, Ingram, LO Effect of selected aldehydes on the growth and fermentation of ethanologenic Escherichia coli. Biotechnol Bioeng 65: