A major problem associated with the fermentation of hydrolysates of lignocellulosic biomass is the presence of a broad range of degradation and products (nonsugar) of hemicellulose hydrolysis that inhibit fermenting microorganisms (Ezeji et al. 2007a, b). These inhibitors can be divided into three groups (Figure 3.2a, b) on the basis of origin: compounds released from the hemicellulose component of lignocellulosic biomass (e. g., acetic, ferulic, glucuronic acid); lignin degradation products (e. g., syringaldehyde, syringic acid, and phenolic com­pounds); and sugar degradation products (e. g., furfural, HMF, formic, and levulinic acid; Ezeji et al. 2007a, b). The type, number, and concentrations of these inhibitory compounds vary depending on the type of lignocellulosic biomass and pretreatment conditions. For effi­cient utilization of lignocellulosic hydrolysates by fermenting microorganisms, hydrolysates must be detoxified to rid or reduce concentrations of these inhibitors to tolerant amounts prior to fermentation. Villarreal et al. in 2006 evaluated five different detoxification procedures
to remove microbial inhibitors from eucalyptus hemicellulose hydrolysates prior to xylitol production by Candida guilliermondii. The detoxification methods employed in the investiga­tion include treatment with activated charcoal and four different resins (cationic and anionic) connected in sequence. Among the detoxification methods employed, ion exchange resins were more efficient in the removal of all three major groups of inhibitory compounds without sugar loss than activated charcoal (Villarreal et al. 2006). Marchal et al. (1986) found hydro­lysates obtained by enzymatic saccharification of wheat straw or corn stover pretreated by steam explosion in acidic conditions could not be fermented into ABE. A simple treatment involving heating the hydrolysates in the presence of calcium or magnesium compounds such as Ca(OH)2 or MgCO3 at neutral pH values restored normal fermentation of these hydroly­sates to ABE. Qureshi et al. (2008a) , during evaluation for use of corn fiber as a substrate for ABE production by C. beijerinckii BA101, reported a maximum cell concentration of 0.65g/L when dilute acid pretreated corn fiber hydrolysates (54.3g/L total sugar) were the substrate compared to a maximum cell concentration of 3.37g/L obtained from the control (pure mixed sugars). C. beijerinckii BA101 grown in dilute acid pretreated corn fiber hydro­lysates experienced a longer lag phase than the control, and the culture was unable to make a transition from the acidogenic to solventogenic ABE production phase, consistent with it being inhibited by lignocellulosic degradation products. When dilute acid pretreated corn fiber hydrolysates were treated with XAD-4 resin prior to fermentation, growth and ABE production by C. beijerinckii BA101 increased by approximately 300% and 500%, respec­tively. Soni et al. (1982), in addition, reported that bagasse and rice straw hydrolysates were inhibitory to Clostridium saccharoperbutylacetonicum.

A fungus, Coniochaeta ligniaria NRRL30616, which metabolizes furfural, 5-HMF, alde­hydes, aromatic, and aliphatic acids, was recently isolated (Nichols et al. 2008). The inves­tigators found that C. ligniaria NRRL30616 grew in corn stover dilute-acid hydrolysates, and compounds representing all of the three groups (aromatic and aliphatic acids, aldehydes, and phenolic compounds) of inhibitory products were removed during the course of fungal growth. Fungal laccase and peroxidase enzymes, in addition, have been used to detoxify wood hydrolysates (Jonsson et al. 1998- Martin et al. 2002). Furthermore, Larsson et al. (2001) expressed laccase in Saccharomyces cerevisiae and the mutant had increased resistance to phenolic compounds. These developments show that biological inhibitor abatement for reduc­ing or eliminating inhibitory compounds from biomass hydrolysates appears to be a promising method for detoxification but drawbacks in terms of cost-effectiveness and competition for substrate could be a problem. The development of fermenting strains that can tolerate greater concentrations of inhibitory compounds generated during acid pretreatment and hydrolysis of lignocellulosic biomass remains a priority.

The cellulases and hemicellulases belong to a large group of enzymes called glycoside hydro­lases, which hydrolyze the glycosidic bond between two or more carbohydrates or between a carbohydrate and a noncarbohydrate moiety. Previous classification schemes have been based usually on the substrate specificities of the enzyme, but such classification is largely inappropriate for the glycoside hydrolases, because single protein folds are known to harbor a diversity of substrate specificities. A better classification scheme has been instituted over a decade ago by Bernard Henrissat and colleagues (Coutinho and Henrissat 1999; Henrissat and Davies 2000) Cantarel et al. 2009) , which is based on the amino acid sequence and consequent fold of the protein. The various glycoside hydrolases are thus divided into fami­lies, which currently number 114. This scheme serves to provide comparative structural features of the enzymes within a family, their evolutionary relationships, and their mechanism of action. A compendium of the glycoside hydrolases and related carbohydrate-active enzymes (CAZymes) can be found on the CAZy website (http://www. cazy. org/).

The members of most of the glycoside hydrolase families, relevant to this chapter, exhibit multiple types of activities on either cellulosic and/or hemicellulosic substrates—independent of the fold, although some of the families are restricted to a certain type of activity. The specificity of these enzymes is thus a function of the architecture of the active site, the car­bohydrate binding modules(s), and the linker peptide(s); not necessarily dictated by the overall structure of the enzyme.

The enzymes of some families occur mainly or exclusively in fungi, for example, GH7, GH45, and GH61. Conversely, members of some other families occur mainly or exclusively in bacteria, for example, GH8, GH44, and GH48. The major glycoside hydrolases and their key substrate activities are listed in Table 5.1.

Indeed, the glycoside hydrolase is usually only part of the story, albeit the definitive “busi­ness” part of the protein where the actual bond cleavage of the target carbohydrate is per­formed. Nevertheless, hydrolysis is generally modulated by the action of additional ancillary components of the enzymes, usually in the form of modules—an independently folding sequence within the intact polypeptide. The glycoside hydrolase itself forms the major cata­lytic module of the enzyme. Various other modules may often be present. Some of these ancillary modules may have enzymatic activity, such as the carbohydrate esterases, notably the feruloyl and coumaroyl esterases and the acetyl xylan esterases, all of which are commonly found in association with xylanase catalytic modules. Moreover, some glycoside hydrolases comprise two or more catalytic modules (multiple GHs from one or more fami­lies). In view of the multi-modular nature of these enzymes, they sometimes reach extremely large molecular proportions.

Preprocessing of biomass is an important step in preparing and supplying biomass to a bio­refinery. Literature indicates that the particles used for hydrolysis and subsequent fermenta­tion should be in the range of 2 mm. For pulping applications size ranges should not be less than 20 mm and not more than 40 mm. For pyrolysis, particle size influences the speed of the pyrolysis. For fast pyrolysis where bio-oil are produced, the smaller the particle is the more efficient the process becomes because of high rate of heat transfer. For slow pyrolysis like gasification and charcoal making, the size of particle can be as big as 50 mm where the process of heat treatment is very slow. For pelletization of biomass to small diameter pellets, a particle size in the range of 1-2 mm is preferred. For larger cube sizes (10-30 mm) the size of particles can increase.

Loose-cut biomass has a low bulk density ranging from 50 to 120kg/m( depending on the particle size (Table 7.3). In case of chopped and ground biomass, the bulk density can be increased substantially (~25%) by vibrating the biomass holder (e. g., truck box, con­tainer). To increase density, the biomass must be mechanically compacted (Sokhansanj et al. 1999( . When biomass is densified to briquettes, cubes, or pellets, densities in the range of 300-700kg/m3 can be obtained.

Pellets are usually in the form of a hardened biomass cylinder, 4.8-19.1 mm in diameter, with a length of 12.7-25.4 mm. Pellets are made by extruding ground biomass through round or square cross — sectional dies. The unit density of pellets (density of a single pellet) is 960-1120kg/m3. Bulk density of pellets may be as high as 750kg/m-. Cubes have a lower density than pellets. Typical bulk density of cubes range from 450 to 550kg/m- depending upon the size of cubes.

Recovery of oil from ethanol processing byproducts offers some very promising opportunities for dry-mill plants to expand products and markets. DDGS has become a very valuable live­stock feed in recent years but possesses some characteristics that limit its use in some appli­cations. DDGS contains about 10.5% oil, which is about three times the level found in most corn. This relatively high oil content limits its use as a feed supplement particularly with respect to swine (Plain 2006). Additionally, the high oil content reduces DDGS longevity with respect to storage and shipping because the oil can become rancid, rendering the DDGS unsuitable for use as feed.

A 56-lb bushel of corn produces 2.72gallons of ethanol and approximately 171b of dis­tillers ’ grains in various forms (Renewable Fuels Association 20081. It is estimated that approximately 2.6 billion bushels of corn will be used to produce ethanol per year by 2010 (Baker and Zahniser 2006). About 2/3 of that corn will be used in dry-milling operations (Murthy et al. 2004). Consequently, an anticipated 29 billion pounds of DDGS will be produced from dry — mill plants. The DDGS contains about 10.5% oil, and the oil weighs about 7.6 lb per gallon, so about 400 million gallons of oil is potentially available for alternative uses if it could be separated from the DDGS (about 0.085 gallons of oil for every gallon of ethanol produced). For comparison purposes, a single dry-mill ethanol plant that produces 100 million gallons of ethanol annually would have about 8.5 million gallons of oil available for separation and/or extraction.

The oil can be removed from fractionated germ prior to fermentation, in which case it is suitable for human consumption. Alternatively, the oil can be removed from DDGS after fermentation. In this case, the oil would be unfit for human consumption because of its rela­tively high levels of free fatty acids (about 8%-9%). However, the oil could be used as animal feed or as feedstock in the production of biodiesel (methyl ester). Given that biodiesel pro­duction was 495 million gallons in 2007, the oil contained in DDGS offers considerable potential as a feedstock for biodiesel production without committing additional acreage to biofuel crops (ICM 2009).

Corn kernels are composed of four primary parts. The endosperm is the largest component (about 82%) of the kernel and is made up primarily of starch and protein. This starch fraction is the portion that is fermented into ethanol. The germ is the next largest fraction at 12% of the kernel and is the primary source of corn oil. The pericarp is the seed hull and composed 5% of the kernel, while the tip cap is where the seed was attached to the cob and makes up about 1% of the kernel. Wet-milling processes can efficiently fractionate the kernels and remove the oil-containing germ from the fermentable fraction prior to producing ethanol, thus allowing for the extraction of food — grade oil from the germ. Dry — mill plants can also be retrofitted to remove the germ prior to fermentation. This is accomplished by either (1) quick germ (and quick germ quick fiber) methods or (2) enzymatic milling. In the quick germ process, the whole corn is soaked in water for 3-12 hours at 60°C. Soaking the ground corn in water with enzymes increases specific gravity such that the germ and fiber float prior to fermentation. After soaking, the corn, a conventional Bauer mill, is used for degermination, similar to the methods used in the wet-milling process. The germ is recovered using germ hydroclones, and the rest of the corn is ground and processed through the dry-mill process (Singh and Eckhoff 1997).

In enzymatic milling, soaking is followed by incubating with protease and starch-degrading enzymes for 2-4 hours. After incubation, quick germ processes are used to recover germ and pericarp fibers. The remaining slurry is screened on a 200-mesh sieve to recover endosperm fibers. In either case, separation of the germ allows for the recovery of high-value food-grade corn oil from the germ (Singh et al. 2005). This can be accomplished through the use of an expeller and/or solvent extraction. Removal of endosperm fibers will further increase fermen­tation capacity and reduce fibers in the DDGS and increase the protein content of the DDGS, making the DDGS more suitable for a wider variety of livestock applications. Also, these modified milling processes can produce additional ethanol per batch because non-fermenta — bles (germ, pericarp fibers, and endosperm fibers) are removed. These non-fermentables can be replaced by a more fermentable substrate. Plants performing these modified milling pro­cesses can potentially increase the amount of corn processed and therefore produce more ethanol per batch compared with conventional dry-mill process (Singh et al. 2005).

Retrofitting an existing dry-mill plant to remove the germ prior to fermentation using either the quick germ or enzymatic milling processes is a relatively involved and costly endeavor requiring significant modifications and capital investment in equipment. However, the improvements associated with ethanol production, along with the high value of coproducts such as the food-grade corn oil produced and the improved quality of the DDGS, can justify the investment. Consequently, ethanol production technology providers such as Mercer Energy, FWS, FCStone Carbon, and ICM now offer fractionation technology to their clients with dry-mill plants.

Two primary options, centrifugation and solvent extraction, are available to dry-mill ethanol producers that can facilitate recovery of oil from either individual process streams or the combined DDGS byproduct. The centrifugation method is relatively straightforward, requiring a relatively low capital investment ($500,000 to $1 million to implement for an average ethanol plant). Solvent extraction methods are considerably more involved, require considerable capital investment, and create hazards that require careful consideration prior to implementation.

During the distillation process, solids comprising the grain and added yeast, as well as liquid from the water added during the process, accumulate in the bottom of the distillation tanks (ICM 2009) . The solids are processed through centrifuges for separation into thin stillage (a liquid with 5%-10% solids) and wet distillers’ grain. Some of the thin stillage is routed back upstream in the process for use as makeup water, reducing the amount of fresh water required. The rest is sent through a multiple-effect evaporation system where it is concentrated into syrup containing 25%-50% solids. This syrup, which is also high in protein and oil content, is then mixed back in with the distillers’ grain and further processed to create animal feed. In an effort to recover the valuable oil, some facilities have added a centrifugation step to the syrup prior to mixing it with distillers’ grain (ICM 2009).

The thin stillage syrup contains about 3.5% to 7% oil, depending on moisture content. Centrifugation will remove about 1/3 of the oil contained in the syrup. Therefore, a 60 gallon per minute centrifuge (the size implemented by ethanol plants that produce 50 to 100 million gallons of ethanol per year) could realistically produce about 500,000 to 1 million gallons of oil per year. Implementation of centrifugation to remove oil from the syrup is a relatively inexpensive and nondisruptive process change. However, centrifugation of the thin stillage syrup would only recover about 10% to 15% of the total oil available from the entire plant. The remaining oil is present in the DDGS and requires extraction with a solvent-based process.

Solvent extraction systems are much more efficient methods with respect to removing nearly all of the available oil from either the germ or DDGS. However, they tend to be complex and hazardous to operate because of the flammable solvents used for extraction. With solvent extraction processes, the germ or DDGS is fed into an extractor, where the material forms a uniform shallow bed and is washed with a solvent such as hexane as it is conveyed across the upper, horizontal section of the extractor, counter current to the solvent. The concentrated oil-solvent mixture discharges from the extractor through a hydroclone. The hydroclone “scrubs” the fines from the oil-solvent mix before being pumped further to a distillation system. On a typical extractor, there are seven stages of oil-solvent mixture, ranging from about 2% oil concentration to approximately 25%. From the extractor, the concentrated oil-solvent mixture is sent to a distillation system, where the oil and solvent are separated. The solvent is recycled back into the extraction process and the oil is available for sale or for conversion into other products. De-oiled DDGS would be available for markets that need less oil for the livestock it will be fed to, or for applications that require long-term storage or shipping.

Most conventional large- s cale biodiesel plants use base — catalyzed transesterification to convert triglyceride in the form of vegetable oil to methyl ester (Steinbach 2007).

Base

Triglyceride + Methanol —Catalyst > Methyl esters + Glycerol

This process alone is not suitable for processing oil derived from DDGS because of the relatively high levels of free fatty acids present in the oil. The base catalyst converts the free fatty acids to soap, thus reducing the yield of methyl ester. Consequently, the base-catalyzed process must be supplemented with a pretreatment step with an acid catalyst to convert the fatty acids to methyl ester. Following the pretreatment step, the remaining triglycerides can be converted to methyl ester using the base-catalyzed process.

Acid

Fatty Acid + Methanol —Catalyst > Fatty Acid Methyl Esters + Water

Biodiesel is now well accepted as a petroleum diesel fuel alternative offering multiple advantages such as renewability, energy security, and superior environmental performance. It is anticipated that feedstocks for biodiesel production will be increasingly in short supply in coming years. The number of biodiesel producers increased by more than 400% from 2004 to 2007. The total biodiesel production capacity available in the United States, as of January 2008, was 2.2 billion gallons from 171 plants, of which 137 came on line in 2004 (Steinbach 2007). Less than 1/2 billion gallons (less than 25% capacity) was actually produced in 2007 partly due to issues associated with feedstock availability (particularly soybean oil) and price. Developing alternative sources of feedstocks will become increasingly important as these plants attempt to increase their production to levels near their capacity.

Low-Rate versus High-Rate Treatment of Animal Waste

Low-Rate Animal Waste Digestion

Many full — scale animal waste digester systems are low-rate anaerobic digesters, such as covered anaerobic lagoons, plug-flow digesters, and CSTRs. A typical VS loading rate (VSLR) for an unheated covered anaerobic lagoon is 0.24 g/L/day with a hydraulic retention time (HRT) of 65 days (Cheng et al. 1999). By feeding dairy waste at a VS concentration of 5g/L in a 4.5-L low-rate bioreactor, we achieved a maximum VSLR in a heated (34°C) and completely stirred anaerobic digester (semi-batch fed system) of 3.5 g/L/day and a minimum HRT and sludge retention time of 15 days (Hoffmann et al. 2008). A typical VSLR and HRT for a heated (35°C) CSTR-fed swine waste at a concentration of 20gVS/L are 1.4g/L/day and 15 days, respectively. Design parameters for a completely stirred system are dependent on the VS concentration in the influent, the operating temperatures, and composition of the biomass in the waste. Cow and swine wastes have a considerably different composition and require different operating conditions to achieve a stable and sufficient treatment perfor­mance. In addition, each farm is different, for example, in how the choice of bedding material for dairy cows will affect the required operating conditions (NRCS 1996).

I n recent years, the concept of designer cellulosomes has become a popular notion for a prospective solution to improving cellulase action, an approach first proposed over a decade ago (Bayer and Lamed 1992; Bayer et al. 1994; Ohmiya et al. 2003). But what is wrong with the native cellulosome systems? Indeed, they are considered to be the most efficient natural enzyme systems for cellulosic biomass, but the cellulosome-producing bacteria produce them in relatively small quantities, compared to the free cellulase systems produced by the aerobic fungi and bacteria. The current idea, therefore, would be to produce large quantities of arti­ficial cellulosomes or cellulosomal components in an appropriate host cell system, which would then be cost-effective for industrial use.

Designer cellulosomes are artificial cellulosome complexes of defined composition, con­taining different recombinant enzyme components (Figure 5.5). The exact position of a given enzyme in the cellulosome can be predetermined by producing chimaeric scaffoldins that contain divergent cohesins of defined specificity and by attaching to the enzymes dockerins of matching specificity. Using this approach, the desired cellulosomes will self — assemble according to our initial design. In this manner, we can control the enzyme content

Chimaeric Scaffoldin Dockerin-containing Enzymes

I

Designer Cellulosomes

Figure 5.5. Construction of a designer cellulosome. A chimeric scaffoldin is designed containing a substrate-targeting CBM and multiple divergent cohesins (enumerated) for selective incorporation of the enzymes (A, B, and C). The recombinant enzyme hybrids contain a dockerin module selected for its matching specificity with one of the cohesins of the chimeric scaffoldin.

and position of the enzyme within the complex. Thus, any given complement of enzymes can be incorporated into such cellulosome complexes. These designer cellulosomes can be utilized as tools for both understanding cellulosome action and for future application in waste management (Bayer et al. 2007) and for production of biofuels (Bayer et al. 2008a, c).

The production of designer cellulosomes has been shown to be experimentally feasible. The first proof of concept involved the preparation of an exhaustive array of small bi­functional artificial cellulosomes, whose activity on specific substrates was examined (Fierobe et al. 2001, 2002). Small chimaeric scaffoldins were prepared that contained two divergent cohesins and matching dockerin-bearing cellulases, and their synergistic action on recalcitrant cellulosic substrates was demonstrated. Additional studies involved the action on crude native substrates, such as wheat straw, whereby designer cellulosomes containing a xylanase together with potent cellulases were demonstrated to dramatically enhance the degradation of complex lignocellulosic substrates (Fierobe et al. 2005) . Later studies demonstrated that enzymes foreign to the native cellulosomes can also be included in the active state into designer cel- lulosomes (Caspi et al. 2006, 2008; Mingardon et al. 2007a), and radical designer cellulosome architectures can be produced at will, as long as the appropriate pre-design and experimental expertise are applied (Mingardon et al. 2007b).

To date, designer cellulosomes have been fabricated on a small scale using only two or three enzymes in complex and with only very modest gains in synergistic activity. Future work with designer cellulosomes should determine whether more impressive results can be obtained with larger numbers of enzymes within the individual designer cellulo — somes, as we extend the size of designer cellulosomes to approach that of the native cellulosome systems. Another distinctive difference between the artificial and natural systems is that designer cellulosomes are uniform in composition, containing stoichiometric ratios of the desired enzymes whereas the native cellulosomes are heterogeneous in content and dispersion of its enzymes. The possible consequences of these differences vis-a-vis efficiency of deconstruction of cellulosic biomass are yet to be determined experimentally.

Typically cobs are not handled in country elevators. The system described here is taken from Mukunda -2007) and also based on the authors ’ visit to this facility. Corn cobs are delivered by trucks from seed processors to the facility where they are weighed and blown

Elevator Cob processing Ethanol plant

(Dry mill)

Figure 7.8. Grain and bulk (cob) handling operations in an elevator and in dry-grind com ethanol plant.

pneumatically unto a pile several meters high. Trucks are weighed before being unloaded but no grading is done to the cargo. The cob pile is stored uncovered and reclaimed daily for processing at the cob mill about 400 m from the cob pile by using front bucket loaders. Cobs delivered to the processing plant are first dried with a rotary drum dryer and stored in steel silos from which they are fed into the plant and processed to various dried granular bulk products that are packaged in 50-lb bags or large 2000-lb bulk bags.

The inbound receipt of cob delivery trucks appear not to be a bottleneck at this facility, because the inventory of cobs at the facility stored in the outdoor pile is large to meet many months of production. Because the topmost layer of cobs on the pile protects the cobs beneath the pile from deterioration above the threshold quality limit for use, the outdoor storage can be used as a low management system in this operation. Outdoor storage can be applied to cobs because of their rigid solid form, which does not drastically deteriorate as a result of spoilage from inclement weather, making handling very difficult. However, spontaneous combustion and fires that smolder for days occur in the cob pile at this plant once every several years. Therefore, the management approach of outdoor storage at this plant, while low in operational cost, can be expensive if all the pile is lost in a fire. Outdoor storage might not be feasible for all biomass types and so the effect of storage on subsequent han­dling must be considered on a biomass-type basis. For example, the mechanical strength of lignocellulosic plant biomass, like wheat and rice straw or switchgrass, would degrade much faster in outdoor storage than corn cobs, and this would affect their subsequent handling and processing such as grinding and flow through material-handling equipment.

HTL, also called hydrous pyrolysis, is a process for the reduction of complex organic materi­als such as bio-waste or biomass into crude oil and other chemicals. It mimics the natural geological processes thought to be involved in the production of fossil fuels. HTL is one of the processes of a general term of TCC which includes gasification, liquefaction, HTL, and pyrolysis. There is a general consensus that all fossil fuels found in nature—petroleum, natural gas, and coal, based on biogenic hypothesis—are formed through processes of TCC from biomass buried beneath the ground and subjected to millions of years of high tempera­ture and pressure. In particular, existing theories attribute that petroleum is from diatoms (algae) and deceased creatures and coal is from deposited plants. Gasification of biomass produces a mixture of hydrogen and carbon monoxide, commonly called syngas. The syngas is then reformed into liquid oil with the presence of a catalyst. Pyrolysis is a heating process of dried biomass to directly produce syngas and oil. Both gasification and pyrolysis require dried biomass as feedstock, and the processes occur in an environment higher than 600oC. HTL involves direct liquefaction of biomass, with the presence of water and perhaps some catalysts, to directly convert biomass into liquid oil, with a reacting temperature of less than 400oC.

HTL has different pathways for the biomass feedstock. Unlike biological treatment such as anaerobic digestion, HTL converts feedstock into oil rather than gases or alcohol. There are some unique features of the HTL process and its product compared with other biological processes. First, the end product is crude oil which has a much higher energy content than syngas or alcohol. And second, if the feedstock contains a lot of water, HTL does not require drying as gasification or pyrolysis. The drying process typically takes large quantities of energy and time. The energy used to heat up the feedstock in the HTL process could be recovered effectively with the existing technology.

HTL may have two pathways from biomass to biofuel: (1) direct conversion of biomass or (2) pretreatment of biomass and then fermentation. For the biomass with little lignocellulosic fraction—such as waste streams from animal, human, and food processing—it can be directly converted into biofuel thermochemically. Pretreatment is currently a bottleneck in the conversion of cellulosic feedstock. HTL may hold a sub­stantially greater potential to shorten the fermentation time of lignocellulose. Traditionally, acid hydrolysis was commonly used to convert lignocellulosic materials to monosac­charides, but the high concentration of acids used in hydrolysis requires extensive waste treatment or recovery costs.

Companies in the business of using biomass sugars for fermentation can rely on a sustainable source of low — cost material. This alternative source of sugars can be exploited to fill the growing demand for transportation biofuels to supplement the need for crude oil and secure a domestic (and international) supply of liquid transportation fuel. The cellulosic microorgan­isms can provide a platform in which to make other beneficial proteins and products for the developing cellulosic ethanol industry. Existing corn starch in ethanol facilities provide loca­tions into which pilot lignocellulosic biorefineries can be bolted. This alternative biomass feedstock is expected to provide a great resource for cellulosic fermentation to ethanol.

Ethanol produced from cellulosic biomass has the potential as a large-scale transportation fuel. Desirable features include ethanol’s fuel properties as well as benefits with respect to air quality, global climate change, balance of trade, and energy security. Energy balance, feedstock supply, and environmental impact considerations are not seen as significant obsta­cles to the widespread use of fuel ethanol derived from cellulosic biomass. Profitability of the conversion is the major challenge, however.

Biomass is the only known, large — scale, renewable resource that can be converted into liquid fuels for transportation. Cellulosic ethanol is particularly promising because it can capitalize on microbial engineering and the power of biotechnology to reduce costs, is derived from low-cost and plentiful feedstocks, can achieve high yields, has high octane and other desirable fuel properties, and is environmentally friendly. Lignocellulosic feedstocks, such as switchgrass, woody plants, mixtures of prairie grasses, agricultural residues, and municipal waste, have been proposed to offer environmental and economic advantages over current biofuel sources, because these biomass feedstocks require fewer agricultural inputs than annual crops and can be grown on agriculturally marginal lands.

As a result of the above-mentioned advantages and developments made with respect to the production of liquid biofuels from cellulosic biomass, numerous companies have initi­ated programs to use this substrate and commercialize ethanol or butanol production. The companies that have initiated programs on production of biofuels from biomass in the United States include Coskata (Warrenvile, IL), Poet Inc. (Soiux City, SD), RangeFuels (Broomfield, CO), Amyris (Emeryville, CA), Mascoma (New Hampshire), the DuPont Danisco BP venture (Wilmington, DE), BlueFire Ethanol (Irvine, CA), Qteros (Marlborough, MA), Verenium (Cambridge, MA), Valero (Sioux Falls, SD), ExxonMobil and Synthetic Genomics, Inc. (LaJolla, CA), KL Energy (Rapid City, SD), INEOS (Fayetteville, AR), Osage Bioenergy, Inc. (Hopewell, VA), Cobalt Biofuels (Mountain View, CA), Tetravitae Biosciences (Chicago, IL), and Gevo (Colorado). There are numerous other international companies that have started working on the production of biofuels from biomass, including Petrobras (Sao Paulo, Brazil), Iogen (Ottawa, Ontario, Canada), SEKAB & Taurus Energy (Ornskoldsvik, Sweden), Abengoa Bioenergy (Salamanca, Spain), and Praj Industries (Pune, India).

In Europe, the greatest adoption of anaerobic digestion has taken place in Germany. The Committee on Agriculture and Rural Development of the European Parliament (2004-2009) developed a report on Sustainable Agriculture and Biogas, which stated that “A Need for Review of EU-Legislation acknowledges biogas as a vital energy resource to contribute to sustainable economic, agricultural and rural development and environmental protection with the strong recommendation that the EU exploit the very large potential for biogas” (EU 2008). A recent German report claimed that Germany could produce more biogas by 2020 than all of EU’s current natural gas imports from Russia (Burgermeister 2008).

At the end of 2007, Germany had -3700 biogas plants with a total electric capacity of 1.25 GW in operation. Most of the new biogas plants have an electrical capacity between 400-800 kW, which was estimated by the German Energy Agency (DENA 2008). Energy crops make up a substantial portion of the substrate mixture with manure substrate at 50% or less. Germany is growing energy crops on -1.3 million ha (-11.4 % of its arable land). Although there are a number of large biogas digesters at wastewater treatment plants, landfill installations, and industrial bio — waste processing plants, the greatest volume of biogas is produced on farms and large co-digestion biogas plants. Noteworthy is that in Germany (and also in Austria where a similar biogas revolution has taken place) some automobiles run on biogas.

In Germany almost all agro-biogas plants use co-digestion with over 30 different organic byproducts and wastes. The drivers for the extensive biogas plants and electrical generating capacity are the implementation of: (1) the Renewable Energy Resources Act; (2) guaran­teed purchase of electricity produced from biogas at preferential rates for 20 years; (3) bonuses for electricity produced from renewable resources; (4) CHP systems; and (5) new technologies.

The confluence of renewable energy and economic and rural development has led to biogas-driven energy for sustainable rural communities. A good example is the village of Jfihnde, where a biogas plant of 700kW provides all of the required electricity and most of the thermal energy for ~750 residents. Home heating is supplied via a district hot water grid of ~3.3 miles (Fangmeier 2008). The feedstock for the digester is derived from manure of nine farms and from energy crops (i. e., maize, grasses, and wheat).