Dry and wet mill ethanol production from corn starch is regarded as an essentially mature technology for producing bioethanol. Currently, dry-grind ethanol plants produce the majority of ethanol fuel (ca. 60%) in the United States. With concerns regarding net energy balance and food compared to fuel debate, ethanol production from corn is expected to stabilize (von Braun 2007). Some incremental increases in energy efficiency of this process, however, can be expected as coproduct (DDGS) utilization is incorporated into next — generation plants. Currently, DDGS from corn ethanol production is used as animal feed. Seven million metric tons of DDGS were estimated to have been produced from corn ethanol processing in the United States at the end of 2008 (Blaschek et al. 2010). DDGS conversion into liquid fuels and incorporation of DDGS into the ethanol biorefinery industry will increase the efficiency and profitability of corn ethanol plants (Blaschek et al. 2010).

Ezeji and Blaschek (2008a) conducted a comprehensive study evaluating fermentation capability of dilute acid, LHW, or AFEX pretreated DDGS hydrolysates by solventogenic Clostridium species. The ABE production profiles over the course of a 120-hour fermentation of detoxified dilute acid pretreated DDGS hydrolysates elucidated that C. beijerinckii 260, Clostridium acetobutylicum 824, Clostridium saccharobutylicum 262, C. beijerinckii 592, and C. beijerinckii BA101 produced maximum ABE concentrations of 8.1, 4.9, 12.1, 7.5, and 6.8 g/L, respectively, and total residual acids of 4.7, 4.2, 5.2, 4.0, and 4.1 g/L, respectively (Table 3.2). During the 72-hour fermentation of LHW and AFEX pretreated DDGS and con­tained 48.8 and 41.4 g/L total sugars, C. beijerinckii 260, C. acetobutylicum 824, C. saccharo- butylicum 262, C. beijerinckii 592, and C. beijerinckii BA101 produced maximum ABE concentrations of 12.8, 11.4, 10.5, 12.9, and 11.5 g/L, respectively, and total residual acids of 4.0, 5.3, 4.9, 7.4, and 4.4 g/L, respectively. For AFEX pretreated DDGS medium, C. beijer — inckii 260, C. acetobutylicum 824, C. saccharobutylicum 262, C. beijerinckii 592, and C. bei- jerinckii BA101 produced maximum ABE concentrations of 10.2, 9.0, 7.9, 11.6, and 10.4 g/L, respectively, and total residual acids of 5.4, 4.0, 5.2, 4.3, and 5.1 g/L. Importantly, LHW

and AFEX pretreated DDGS hydrolysates were not subjected to any detoxification process prior to fermentation and there was no significant difference between total ABE produced from DDGS and that of the control (mixed glucose-mannose-arabinose-xylose [GMAX]) with cor­responding amounts of GMAX. Wang et al. (2009a), in addition, investigated the effect of total solids loading on pretreatment of DDGS by electrolyzed water and ABE fermentation. DDGS samples pretreated at 30% solids loading with alkaline electrolyzed water (ALEW) produced 16.9 g/L total ABE in 72 hours upon fermentation by C. beijerinckii P260.

To improve C. beijerinckii BA 101 tolerance to toxic compound generated during pretreat­ment and hydrolysis of lignocellulosic biomass and enhance fermentation capability of AEW — pretreated DDGS, Wang et al. (2009b) conducted microorganism adaptation studies where C. beijerinckii BA 101 was treated with increasing amounts of acid-pretreated DDGS hydro­lysates containing lignocellulosic degradation compounds that are inhibitory to solventogenic Clostridium species. Hydrolysates — adapted C. beijerinckii BA101 strains were able to adjust to the inhibitory environment in less than 20 hours and produce approximately the same amount of ABE at a comparable time to that of the control fermentation.

Cellulolytic bacteria and fungi produce a variety of different cellulases and related glycoside hydrolases, which together convert plant cell wall polysaccharides to simple fermentable sugars. Cellulolytic bacteria and fungi employ different strategies for the degradation of the

Table 5.1. Major glycoside hydrolase families and their enzymatic activities. The glycoside hydrolase families (GHn) in which some members exhibit standard cellulase activities are shown in bold. GH families that include cellulases exclusively are followed by an asterisk (*). See CAZy website for more details: http://www. cazy. org/.

GH Family Enzymes

GH1 Numerous activities, including p-glucosidase, p-galactosidase, p-mannosidase, and

P-glucuronidase; but not p-xylosidase activity

GH2 Numerous activities, including p-galactosidase, p-mannosidase, and p-glucuronidase;

but neither p-glucosidase nor p-xylosidase activities GH3 Numerous activities, notably not only p-glucosidase and p-xylosidase activities, but also

glucan 1,3-p-glucosidase, glucan 1,4-p-glucosidase, and exo-1,3(4)-glucanase activities

GH5 Broad spectrum of cellulase and hemicellulase activities, including cellulase, xylanase,

1,3-p-mannanase; p-mannosidase, glucan 1,3-p-glucosidase, licheninase, glucan endo-1,6-p-glucosidase, mannan endo-1,4-p-mannosidase, endo-1,6-p-galactanase, and xyloglucan-specific endo-1,4-p-glucanase activities GH6* Cellulase activities in both aerobic bacteria and fungi (not found in archaea): both endo — and exo-glucanase (cellobiohydrolase) activities GH7* Cellulase activities exclusive to the fungi: both endo — and exo-glucanase (cellobiohydrolase) activities

GH8 Cellulase, lichenanase, xylanase activities; exclusive to bacteria

GH9* Endo-, processive endo-, and exo-glucanase (cellobiohydrolase) activities in bacteria, plants, and fungi (but not in archaea)

GH10 Endo-1,4-p-xylanase and endo-1,3-p-xylanase activities in bacteria and fungi

GH11 Xylanase activities in bacteria and fungi

GH12 Endoglucanase, xyloglucanase, and 1,3(4)-p-glucanase in the three domains of life GH16 Endo-1,3-p-glucanase, endo-1,3(4)-p-glucanase, lichenanase, and xyloglucanase activities

GH17 Glucan 1,3-p-glucosidase and lichenanase activities GH18 Chitinases

GH19 Chitinases

GH26 p-Mannanase and 1,3-p-xylanase activities

GH30 1,6-p-Glucanase and p-xylosidase activities

GH39 p-Xylosidase activity

GH42 p-Galactosidase activity

GH43 Broad spectrum of hemicellulase activities, including xylanase, arabinanase, p-

arabinofuranosidase, p-xylosidase, and galactan 1,3-p-galactosidase activities in bacteria and fungi

GH44 Endoglucanase and xyloglucanase activities, mainly in bacteria GH45* Endoglucanase activity, mainly in fungi (some bacteria)

GH47 a-Mannosidase activity, mainly in fungi

GH48* Cellobiohydrolases and endo-processive cellulases; mainly in bacteria; an important enzyme in all cellulosomes and in some noncellulosomal systems GH51 a-L-Arabinofuranosidase and endoglucanase activities

GH52 p-Xylosidase activity

GH53 Endo-1,4-p-galactanase activity

GH54 a-L-Arabinofuranosidase and p-xylosidase activities, mainly in fungi

GH55 Exo — and endo-1,3-glucanase activities, mainly in fungi

GH61 Exclusive to fungi. In some cases, annotated as endoglucanases, but probably disrupt cellulose structure rather than cleaving glucoside bonds.

GH62 a-L-Arabinofuranosidase activity

GH64 1,3-p-Glucanase activities; mainly in bacteria

GH67 a-Glucuronidase and xylan a-1,2-glucuronosidase activities

GH74 Xyloglucanase and endoglucanase activities

GH81 1,3-p-Glucanase activity

plant cell wall polysaccharide substrates, which reflect the complement and type(s) of enzymes produced by a given microbe. The resultant “cellulase system” may be characterized by free enzymes, cell-bound enzymes, multifunctional enzymes, cellulosomes, or any combination of the latter (Bayer et al. 2006; Wilson 2008).

Numerous factors influence the size and mode of transportation. A few of these factors that the authors believe are most important are listed as follows:

• The maximum rate of biomass supply to biorefinery (t/hour)

• Form and bulk density of biomass (t/m3)

• The distance biomass has to travel to reach to biorefinery (km)

• Transportation infrastructure (equipment, roadways, waterways, railways) available between the points of biomass dispatch and biorefinery

Transport Equipment

Transport equipment is primarily concerned with loading and unloading operations and transferring biomass from storage and preprocessing depots to a biorefinery. Transport modes include truck, train, barge, ship (ocean freighter) and pipeline. Moving feedstocks from one location to another might involve more than one of these modes of transport. The above factors determine which one of these modes or combinations of modes will suit a particular biorefinery. Truck transport and for a few cases train transport may be the only modes of transport. Barge, ship, and pipeline transport, and often train transport require truck transport as well. Trucks interface with trains at the loading and unloading facilities of a depot or processing facility. Barge, ship, and pipeline require interfacing with train and/or truck transport at major facilities either on land or at the shores.

Physical form and quality of biomass has the greatest influence on the selection of equip­ment for the lowest delivered cost possible. In many transport instances, the rates are fixed for a distance and for a size of container independent of mass to be transported. A higher bulk density will allow more mass of material to be transported per unit distance. Truck transport is generally well developed and is usually the cheapest mode of transport but it becomes expensive as travel distance increases.

Pipeline transport is the least known technology for transport of biomass feedstocks and may prove to be the cheapest and safest mode of transport—but perhaps in a distant future. It is envisioned that biomass could be transported by pipeline in the form of a slurry mixture. Upstream equipment includes receiving, slurry making, and initial pumping. The elements along the pipeline are the booster pumps and, at the end, the equipment for draining the biomass from the carrier liquid (Kumar et al. 2005). Unlike truck and train transport, there is an economy of scale for pipeline transport. A larger diameter pipe has a lower friction and thus lower pumping cost. It is also proposed to make dense granules of biomass impervious to water or other liquids for efficient loading and long-distance transport.

Biological conversion of cellulosic biomass to ethanol is summarized here to provide a context for understanding some of the key features of agricultural residues and their impact on producing fuels, with more details available elsewhere (Valkanas et al. 1977; Fan et al. 1981; Gould 1984; Gusakov et al. 1992; Saddler 1993; Asghari et al. 1996; Baghaei-Yazdi et al. 1996; Belkacemi et al. 1997; Walsh et al. 1998). Typically, cellulosic biomass is com­posed of about 40%-50% cellulose, 20%-30% hemicellulose, 10%-25% lignin, and lesser amounts of minerals, oils, free sugars, starches, and other compounds (Wyman 1996). Enzymes or acids can catalyze the reaction of hemicellulose with water to release sugars, typically arabinose, galactose, glucose, mannose, and xylose, for fermentation to ethanol or other products. Enzymes or acids can also hydrolyze cellulose into glucose in a similar fashion. However, although dilute sulfuric acid can recover sugars from hemicellulose with high yields, yields from dilute acid hydrolysis of cellulose are much lower because higher temperatures are needed to overcome its high crystallinity, resulting in glucose degradation (Grohmann et al. 1985; McMillan 1994; Hsu 1996). Because of their high selectivity, enzymes realize the high yields important to competiveness (Wyman 2007). Concentrated acids can also realize high yields, but costs to recover the high loadings of acid needed are high (Hsu 1996; Yang and Wyman 2008).

Because enzymes cannot penetrate the complex structure of most types of biomass well, a pretreatment step is essential to high yields, and one approach is to employ dilute sulfuric acid to remove much of the hemicellulose and open up the structure for effective sugar release from cellulose by enzymes. In this case, temperatures of about 140-180°C can be employed at acid concentrations of about 0.5%-2.0% and residence times on the order of 10-30 minutes to recover about 80%-90% of the sugars in hemicellulose during pretreatment plus some of the glucose from cellulose. The resulting insoluble solids are enriched in cellulose and lignin, and a small fraction can be used to support growth of the fungus Trichoderma reesei or other aerobic organism that produces enzymes known as cellulase to depolymerize cellulose into glucose and hemicellulases to break down hemicellulose not removed by pretreatment into its component sugars. These enzymes are then added to the pretreated solids to release most of the sugars left in the solids, and an organism can be added to the same vessel to ferment the sugars released to ethanol in a configuration known as the simultaneous saccharification and fermentation (SSF) process, an approach that enhances rates, yields, and concentrations by reducing inhibition by the sugars released and also lowers containment costs (Spindler et al. 1991; Wyman et al. 1992; Katzen and Fowler 1994; Ingram and Zhou 2002) . Sugars released in pretreatment, mostly from hemicellulose, are fermented to ethanol with an organ­ism that has been genetically modified to achieve high yields from the five carbon sugars arabinose and xylose that native organisms could not effectively ferment to ethanol (Ho and Tsao 1995; Zhang et al. 1995; Ingram et al. 1997). The hemicellulose sugar stream could also be left with the pretreated solids and fermented to ethanol in the same vessel in a con­figuration known as simultaneous saccharification and co-fermentation (SSCF). The broth from fermentation is sent to a distillation and dehydration system to remove the ethanol while leaving the unconverted solids (mostly lignin), water, and other leftovers in the column bottoms. Contrary to many incorrect statements, water is not removed from the ethanol, and ethanol recovery is not very energy intensive in a well-engineered process. Rather, we can take advantage of the high volatility of ethanol compared to water to remove high-purity ethanol from a messy fermentation broth and concentrate and burn the residues left in the water to provide more than enough energy to meet all the heat and power needs for the process with significant amounts of electricity left over to export (Wooley et al. 1999a ; Wyman 1999b, 2007).

The pathway outlined above represents the configuration often considered currently. However, as will be discussed later, enzymes are very expensive and are the major showstopper to commercialization. In particular, enzymes suffer from two limitations: (1) they are expensive to make due to the aerobic fermentations used and (2) large doses of enzyme are required to produce sugars with adequate yields (Lynd et al. 1996, 2002; Aden et al. 2002) . An alternate configuration is to employ a single organism or consor­tium of organisms that can both make enzymes and ferment the sugars they produce to ethanol. Several advantages result from this approach. First, less equipment is needed, and fewer transfers are required. In addition, the fermentative organisms are anaerobes, thereby avoiding the high power requirements for making enzymes using fungal systems such as T. reesei. This consolidated bioprocessing (CBP) approach could thus lower costs by reducing capital investments and energy costs. However, although native fermentative organisms can produce enzymes anaerobically, they have a low selectivity for ethanol, resulting in too low yields, and several groups are now working to eliminate competitive pathways so that high yields of ethanol are achieved (Lynd et al. 2005). In addition, these organisms must be hardened to withstand an industrial environment and realize high ethanol concentrations.

Many other pretreatment approaches have been trialed over the years other than dilute acid, and a few such as ammonia fiber expansion (AFEX), controlled pH, ammonia recycle percolation (ARP), lime, and sulfur dioxide technologies can be effective (Mosier et al. 2005b). Those at low pH such as use of sulfur dioxide remove hemicelluloses in the same manner as outlined above, with the primary difference being in the ability to recover and recycle the sulfur dioxide (Schell et al. 1991) . Such low pH pretreatments also produce predominately monomeric sugars that many organisms can ferment to ethanol (Wyman et al. 2007). Use of only water or addition of buffers to maintain the pH nearer to neutral will preserve most of the sugars as short chains that dissolve in water, with the goal of reducing their degradation (Mosier et al. 2005a) . On the other hand, the overall yields of sugars and oligomers are somewhat lower than for dilute acids, and either organisms must be used that can ferment the soluble oligomer chains or additional steps are needed to break them down into fermentable monomers (Eggeman and Elander 2005). Pretreatment through addition of a base such as lime or sodium hydroxide opens up the cellulose to enzymes by removing lignin but can take longer to react or cost more (McMillan 1994) . Ammonia can also be employed to lower pH, but its release for recovery and recycle when the pressure is dropped following pretreatment results in no visible removal of lignin or hemicelluloses (Dale et al. 1996). Yet the resulting solids can be highly susceptible to sugar release by enzymes, and the product stream does not form strong inhibitors of fer­mentation or enzymatic hydrolysis (Wyman et al. 2007). Much more experience has been developed with dilute sulfuric acid than for the other options because of historical limits in funding for such research, but the others can have important advantages and synergies that should be explored.

The demand for energy is rising and given that energy demand is projected to keep rising with constrained oil supplies, oil prices seem unlikely to fall significantly in the near future. Because 60% of U. S. petroleum supplies are imported, there is a need to develop alternative fuel supplies for future energy demands. Bioenergy has become a subject of increasing atten­tion around the world. But the use of crop biomass such as grains, roots, and tubers as a raw material for bioenergy production may compete with food and feed supplies. U. S. fuel ethanol and biodiesel production is at an all-time high, but the industry is also facing a significant problem on how to deal with byproducts and wastes such as corn fiber, dried distillers’ grains and solubles (DDGS), glycerin, food, and animal wastes. For instance, production of 10 tb of diesel results in 1 lb of glycerin and for every bushel of corn converted into ethanol (2.7 gallons), 18 lb of DDGS is generated. Waste, despite being one of the leading environmental problems, has the potential to become one of the largest bioenergy resources. Livestock production worldwide has grown rapidly in light of increased demand, and this has environ­mental implications especially in the area of waste management. In New York State alone, the dairy cow population is about 700,000, generating a significant amount of manure. At 40 lb of waste per cow per day, the energy potential is great. By eliminating the animal waste on a farm, a farmer alleviates or eliminates environmental problems, such as odor and water pollution, and may be able to increase the size of his herd. Animal waste digestion offers many economic benefits (biogas and fertilizer production). Therefore, finding new energy sources from livestock waste streams will be a major strategy to treat the waste and sustain the growth of the livestock industry.

Currently, there is no book on the market that is focused on the production of liquid biofuels and biogas from agricultural byproducts and wastes. This book will provide a comprehensive text on the science of production of liquid biofuels (ethanol and butanol) and biogas (methane) from agricultural byproducts as well as animal and food industry wastes. The book is intended for university researchers (professors, students, libraries), industry scientists (large company QA/QC management, bioenergy companies, start-up companies, microbiologists), as well as engineers and microbiologists from government agencies. This book should serve as an up — to-date reference resource for university and industry scientists in the area of biofuel research, waste treatment, and integrated farm management.

Our lab has experience with treatment of swine waste with ASBR technology, and therefore we will use this technology as an example of a high-rate anaerobic digester system on the farm (Garcia and Angenent 2009). The ASBR is a single-vessel system that requires no feed — distribution and gas-solids-separation systems, which simplify its design (Figure 4.2). However, intermittent mixing is needed to provide sufficient contact between substrate and biomass, distribute heat, and prevent scaling. A settling step before decanting effluent is instrumental to facilitate high biomass levels and long sludge retention times (internal settling; Sung and Dague 1995). Developing a well-settling biomass, while not disturbing bioflocculation, was accomplished by mixing ASBRs intermittently (e. g., 2 minutes of mixing every hour for a laboratory ~ scale bioreactor; Sung and Dague 1995; Zhang et al. 1997; . Development of a well-settling biomass from poorly settling inocula, however, requires long startup periods. Several laboratory; scale studies and one full ; scale study have been per­formed to investigate swine waste treatment with ASBRs (Dague and Pidaparti 1992; Zhang et al. 1997; Angenent et al. 2002b, 2008). Indeed, for efficient swine waste treatment at the design volumetric loading rates, the start-up period was longer than 6 months.

The choice of inoculum influences the start-up period of ASBRs. This became apparent in a study with two 5-L ASBRs that treated diluted swine waste with a VS level of 20 gVS/L (Angenent et al. 2002a). When swine lagoon sludge with a very well-settling ability (sludge volume index [SVI] < 10mL/g total solids [TS]) was used as an inoculum, the period to achieve a design VSLR of 4 g/L/day was 250 days due to the fact that biomass levels in the bioreactor remained around 20gVS/L during the initial 50 days of the operating period (Figure 4.3a, b). The operating period to achieve this design loading rate was 100 days longer when poorly settling anaerobic digester sludge from a CSTR treating waste activated sludge was used as an inoculum (SVI was ~56mL/gTS). Biomass washout in the ASBR with this sludge decreased the biomass levels in the reactor from 20 to 10gVS/L during the first 50 days of the operating period (Figure 4.3b). At the end of the study after a 1-year operating period, the settleability of the biomass in both reactors was identical (17mL/gTS), showing that ASBRs select for a well-settling biomass (Angenent et al. 2002a).

Besides developing a well-settling biomass, the concentration of methanogens in the reactor biomass may also need to be increased to obtain high volumetric loading rates. Timur and Ozturk (1999) reported that an ASBR showed favorable performance during the treatment of landfill leachate. In addition, these researchers reported that the volumetric methane pro­duction rate (VMPR) increased linearly with the volumetric loading rate to achieve a constant methane yield over the operating period during which a stable performance was reported (Timur and Ozturk 1999), which we also observed for animal wastes (Angenent et al. 2002b; Angenent et al. 2008; Hoffmann et al. 2008; Garcia and Angenent 2009). Timur and Ozturk (1999) observed an enrichment of methanogens over the operating period, with an increasing specific methane production rate (in L of methane per g volatile suspended solids [VSS] per day) to keep up with the increase in the food to microorganism (F/M) ratio. This showed that the biomass in the ASBR became more active in terms of methane production per amount of biomass present over the operating time and that a considerable start-up period was required before the ASBR could manage volumetric loading rates of up to 10 gCOD/L/day (Timur and Ozturk 1997) . We have shown higher concentrations of methanogenic small — subunit ribosomal RNA (rRNA) in ASBRs treating swine waste at the higher VMPR under stable reactor performance compared with the lower methane production rate (Figure 4.4). Therefore, anaerobic digesters select for a biomass that is more active in methane production to sustain higher loading rates (Angenent et al. 2002a).

In summary, both the development of a well-settling biomass and an increase in the con­centration of methanogens necessitates a long start-up period for the ASBR. In our work with ASBR treatment of diluted swine waste at 25-35°C, the investment of a relatively long start­up period, however, ensured an operation at a much shorter HRT of 5 days compared with a 15-day HRT for low-rate anaerobic digesters (Angenent et al. 2008; Garcia and Angenent 2009). This shorter HRT directly results in a 66% reduction in reactor volume. At the rela­tively low HRT of 5 days for a 5-L ASBR treating swine waste at a concentration of 2 gVS/L and 25°C, the methane yield was 0.47L methane per g VS fed with an ~52% VS removal efficiency at a VSLR of 4 g/L/day (Angenent et al. 2008). This yield was close to the ultimate methane yield (i. e., methane yield at an infinite HRT), which was estimated to be between 0.44 and 0.52L CH4/g VS fed for waste from swine fed a corn-based diet (Chen 1983), and shows that the ASBR performance is suitable even under relatively low HRTs.

In a subsequent study, we operated the same ASBRs under similar environmental condi­tions, but with swine waste from a different farm. We achieved a lower methane yield of 0.31 L methane/g VS fed (Garcia and Angenent 2009), and this was not caused by an inferior ASBR performance compared with the study described earlier (a 51% and 52% VS removal efficiency at a VSLR of 4 g/L/day for the Angenent et al. [2008] and Garcia and Angenent [2009] studies, respectively). Instead, the large difference in methane yields for both studies was probably due to variations in the composition of swine waste. This shows that it is very difficult to predict the methane yields due to the large variations in conditions at the

farm, including farm practices, such as animal feed composition, antibiotic use, manure collection, manure storage period before transfer into the digester, water consumption, and cleaning habits.

Syed Shams Yazdani, Anu Jose Mattam, and Ramon Gonzalez

Abstract

Glycerol (or glycerin) is a byproduct of biodiesel, oleo-chemical, and bioethanol production processes. Due to the tremendous growth of the biofuel industry, glycerol is now regarded as a waste product, often with a disposal cost associated with it. Glycerol is abundant and inexpensive, and it is also a highly reduced molecule, which offers the opportunity to produce fuels and reduced chemicals at yields higher than those obtained using common sugars. Few microorganisms are able to utilize glycerol in the absence of external electron acceptors to produce high-value chemicals such as 1,3-propanediol, succinic acid, propionic acid, and biosurfactants. However, microorganisms that are amenable to industrial applications, such as Escherichia coli and Saccharomyces cerevisiae, were thought to metabolize glycerol only via respiration. We showed recently that E. coli can fermentatively metabolize glycerol, and we established pathways, mechanisms, and conditions of this metabolic process. Our findings have opened up a new platform for engineering E. coli for the production of several fuels and chemicals. This chapter will focus on the production of ethanol with coproducts hydrogen and formate from glycerol and highlight ways of improving yields and productivities of these products.

The design of inbound logistics of feedstock delivery must consist of the following three unit operations: a sampling station, an unloading station, and storage space. The design layout and accompanying equipment will depend on the type of feedstock to be processed. For lignocellulosic feedstocks such as plant biomass or woods, the form in which the feedstock is delivered will determine how the layout is designed and what types of equipment will be in the operations.

Feedstock Sampling

Feedstock sampling of bales can be quite challenging as well, especially when receiving large volumes of feedstocks. Because of the lack of commercial production of biofuels for cellu — losic biomass feedstocks, automated sampling equipment for bales might not be available. Additionally, it is not certain how feedstock quality would affect conversion performance to fuels for biochemical processes. However, other handling operations such as size reduction and feeding will be affected when their physical properties change due to deterioration. The growth in commercial production of biofuels from cellulosic feedstock and the future trade of biomass as a commodity for energy will determine the value premium processors are willing to pay for a superior-quality feedstock and how the feedstock is graded.

Agricultural biomass and biowaste include crop residues and wood, food processing waste, animal manure, and algae. Crop residues and wood primarily contain lignocellulose, while animal and food processing waste contains lipids, protein, and usually small amounts of lignocellulose (except ruminant animal manure). In this section, the primary compounds and structure in these feedstocks will be summarized and the potential functions in hydro­thermal are explored, in the hope that it provides some bases to understand the interaction of carbohydrates with water (H+ or H * radicals).

Lignocellulose

Lignocellulosic compounds belong to the carbohydrates group of organic compounds. Carbohydrates are hydroxy aldehydes, hydroxy ketones, or substances derived from them. It is the principal substance that composes plants. The carbohydrates in swine manure come from both digested and undigested feed, and food processing waste contains carbohydrates food and lignocellulocic sources.

Glucose is one of the simplest monosaccharides. The isomers of glucose are shown in Figure 10.3. There are four chiral carbon atoms in the molecule. The carbon atoms at the end of the molecule do not hold four different groups and are not chiral. D-glucose forms a cyclic molecule by an addition reaction involving the carbonyl group and a hydroxyl group. The ring formation produces a new chiral center, and two isomers of D-glucose exist that differ in the orientation of the new OH group (Figure 10.4). Notice the a form of the OH group of the extreme right right-hand carbon atom is on the same side of the ring as the OH group of the adjacent carbon atom. In aqueous solution, a and в forms of the D-glucose exist in equilibrium, together with a low concentration of the open-chain form.

H

C==O

Figure 10.4. Ring forms of glucose.

Cellulose is a polysaccharide that only yields D-glucose upon hydrolysis. The number of D-glucose units in the molecular structures is estimated to be as high as several thousands. The D-glucose units of cellulose are linked in long chains in в combination shown in Figure 10.5. The structure is stabilized by hydrogen bonds between adjacent D-glucose units in the same strand. Cellulose occurs in fibrils brought about by hydrogen bonds among different strands.

Hemicelluloses are polysaccharides that are chemically related to cellulose, having back­bones of 1,4-P-linked major sugar units, and being morphologically strongly associated with cellulose in the plant cell walls as well as to lignin in lignified cell walls. These polysac­charides are generally heterogeneous, built up of different hexoses (C6-sugars) and pentoses (C5-sugars), sometimes in addition to uronic acids. They have a lower degree of polymer­ization than cellulose (100-200 units), are largely soluble in alkali, and also more easily hydrolyzed (Figure 10.6).

The chemical structure of lignin is more complex than cellulose and hemicellulose. It resembles a network of aromatic compounds linked together in a more random fashion. The structure varies depending on source. To illustrate, the structure of a possible lignin molecule is shown in Figure 10.7. Lignin has high carbon content typically more than 60% and about 30% oxygen. Although in smaller amounts than cellulose, lignin represents about half of the available combustible energy in naturally occurring sources (Glasser 1985). Thermal decom­position of lignin occurs above 280°C depending on the source of lignin (Chornet and Overend 1985).

Figure 10.8. General structure of amino acid compounds.

Thaddeus C. Ezeji and Hans P. Blaschek

Abstract

As the worldwide demand for fuels and chemicals surges and petroleum deposits are depleted, producers of ethanol fuel are increasingly looking beyond corn, potatoes, and other starchy crops for substrates for ethanol fuel production. Ethanol is currently the most important renewable liquid biofuel, but it has problems ranging from lesser energy content than gasoline, blending limitations with gasoline, potential for corrosion of pipes, and subsequent inability to be transported using existing pipeline infrastructure, to requir­ing modification of car engines with increasing ethanol concentrations such as E85 or 100% ethanol. Attempts are underway to produce alternative renewable liquid biofuels and chemical feedstocks that are superior to ethanol. Butanol is one such biofuel because it has greater energy content, is more miscible with diesel, is less corrosive, and has a lesser vapor pressure and flash point than does ethanol. Butanol can also be used at greater blend amounts with gasoline or even at 100% concentration in car engines with little or no engine modification, and because of its solubility characteristics, it can be transported in existing fuel pipelines and tanks. One of the major problems associated with bio-based production of butanol is the cost of substrate. The cost of substrate has led to recent interest in the production of butanol from alternative, inexpensive materials. However, much of the proposed alternative substrates, such as corn stover, corn fiber, wheat straw, rice straw, or dedicated energy crops such as switchgrass and Miscanthus, present challenges that need to be overcome before they can be used as commercial substrates for butanol production. This chapter, therefore, details the (1) pretreatment and hydrolysis of various lignocellulosic biomass; (2) generation of lignocellulosic degradation products during pretreatment of biomass; (3) effects of degradation products on growth and butanol production by fermenting microorganisms; and (4) strategies for improving lignocellulosic hydrolysates utilization for butanol production.

Biofuels from Agricultural Wastes and Byproducts Edited by Hans P. Blaschek, Thaddeus C. Ezeji and Ju rgen Scheffran 19 © 2010 Blackwell Publishing. ISBN: 978-0-813-80252-7

Introduction

Increasing energy demand worldwide coupled with a limited supply of fossil fuels and the fluctuating price of oil has generated a strong interest in the bioconversion of agricultural biomass and coproducts into fuels and chemical feedstocks. Biofuels are currently produced mainly from carbohydrates (corn, potato, sugar cane, sugar beets, etc.) and oil (soybean, rapeseed, etc.)-rich crops. Ethanol was once considered to be a replacement for gasoline, a fossil fuel currently used in car engines. A study conducted by the U. S. Department of Agriculture (USDA) in 2007 projected that the average corn price will peak at $3.75 per bushel during the 2009-2010 marketing year and will decline before stabilizing at approxi­mately $3.30 through 2016 (USDA-ERS 2007). An expansion of the ethanol and biodiesel industry has influenced the prices of corn and soybeans in the United States. In 2008, approximately 9.0 billion gallons of ethanol was produced from corn in the United States for use as a fuel supplement (Renewable Fuel Association 2008), which represents approxi­mately 4.2% of total gasoline consumption (150 billion gallons; 1 gallon ethanol = 0.7 gallons of gasoline). An increase in ethanol production from corn would require additional acreage and would potentially have an impact on land available for the production of food crops. Therefore, a further increase in ethanol production will require the use of agricultural materials not directly tied to food, especially lignocellulosic biomass such as corn stover, corn fiber, wheat straw, rice straw, or other energy crops such as switchgrass and Miscanthus. Lignocellulosic biomass, which may contain xylan, arabinan, galactan, glucuronic, acetic, ferulic, and coumaric acids, is the most abundant renewable resource on the planet (Koukiekolo et al. 2005) and has great potential as a substrate for butanol production (Ezeji et al. 2007a, b).

Butanol is a four — carbon alcohol with some very interesting attributes with respect to its use as a fuel and fuel extender that are greater than ethanol (Ezeji et al. 2004b; Ezeji and Blaschek 2007). Prior to 1950, the AB (acetone butanol) or acetone butanol ethanol (ABE) fermentation using corn and molasses as substrates, ranked second only to the ethanol fermentation in its importance and scale of production, but subsequently declined due to increasing substrate (sugars and molasses) costs and availability of much cheaper petrochemically derived butanol (Ezeji and Blaschek 2007; Schwarz et al. 2007). Substrate cost has long been recognized as having a dramatic influence on butanol price (Qureshi and Blaschek 2000). Most bacteria use glucose as a preferred carbon source for growth, and only when glucose is limiting are the pentose sugars utilized, making fermentation of complex mixture of sugars in lignocellulosic hydrolysates challenging (Ezeji et al. 2007a). The solventogenic ABE-producing clostridia have an added advantage over many other cultures in that they can utilize both hexose and pentose sugars, which are released from wood and agricultural residues upon hydrolysis to produce ABE (Ezeji et al. 2007b) .

The solvent-producing clostridia are not able to hydrolyze fiber-rich agricultural residues or lignocellulosic biomass. The lignocellulosic biomass must be pretreated and hydrolyzed to simple sugars using physical (size reduction), chemical, and enzymatic methods. Unfortunately, these treatments can result in the formation of a complex mixture of microbial inhibitors that are detrimental to the growth of fermenting microorganisms (Ezeji and Blaschek 2008a). Examples of inhibitory compounds produced include furfural, hydroxymethylfurfural (HMF), syringaldehyde, and acetic, ferulic, glucuronic, p-coumaric, syringic, levulinic acids, and so on (Zaldivar et al. 1999) Zaldivar and Ingram 1999) Varga et al. 2004) Ezeji et al. 2007b). The reduction or elimination of lignocellulosic degradation products during the pretreatment of biomass, removal of inhibitors from lignocellulosic hydrolysates prior to fermentation, adaptation of strains (to these inhibitors) via the development of inhibitor toler­ant mutants, or a combination of the above approaches have been touted to be the panacea for successful production of biofuels from lignocellulosic biomass. Among these options, the development of inhibitor-tolerant mutants via culture adaptation appears to be most viable approach from an economic standpoint. Many laboratories, including those of the authors, are currently involved in research directed toward development of inhibitor-tolerant butanol- producing microorganisms.