Glycerol is present in animal fats and oils in the form of triglycerides. These triglycerides consist of three fatty acids linked to three hydroxyl groups of glycerol through an ester bond. In most industrial applications, glycerol is recovered from the triglyceride molecules by breaking the ester bonds through various chemical or biological processes. Fatty acids present in triglycerides are directly converted into another material such as soap or biodiesel by saponification or transesterification, respectively (Figure 6.1; Yazdani and Gonzalez 2007). Glycerol is obtained from these processes as crude solution with various contaminants.

Biofuels from Agricultural Wastes and Byproducts Edited by Hans P. Blaschek, Thaddeus C. Ezeji and Ju rgen Scheffran 97 © 2010 Blackwell Publishing. ISBN: 978-0-813-80252-7

Biodiesel industries are the major sources of crude glycerol accumulation. During biodiesel production, fatty acid molecules are detached from the glycerol moiety of triglyceride by the action of a catalyst, and alcohol (methanol or ethanol) binds in its place through transesteri­fication reaction (Figure 6.2). The catalysts commonly used for this purpose are sodium and potassium hydroxide. Reaction could take place at room temperature, albeit slowly. Heating triglyceride at 55°C helps completion of the reaction in 1-3 hours. The mixture is kept at room temperature for 1-2 days for the separation of glycerol and biodiesel to occur. Glycerol is heavier than ester and settles at the bottom of the container (Figure 6.3). For every 100 lb of biodiesel produced by the transesterification of triglycerides, 101b of crude glycerol is generated.

Pure glycerol is primarily used in the production of various foods, beverages, pharmaceu­ticals, cosmetics, and so on. The cost of purifying crude glycerol is high and uneconomical. Pure glycerol is currently sold at a relatively high price, which is in the range of $0.60-$0.90/ lb. The high cost associated with the purification of crude glycerol makes it unattractive for biodiesel companies to invest in the purification process. The number of companies producing biodiesel has grown since the early 1990s due to increased demand for biodiesel fuel. This has led to a huge glut of crude glycerol. Crude glycerol used to be a desired coproduct that

Glycerol Biodiesel

Figure 6.2. The production of biodiesel. The alcohol reacts with the fatty acid component of triglyc­eride (fat or oil) in the presence of a catalyst such as NaOH, KOH, H2 SO4, and so on to form alkyl ester (biodiesel) and glycerol.

Biodiesel

Glycerol

Figure 6.3. Biodiesel stored in a bottle upon completion of transesterification reaction. Glycerol is heavier than biodiesel and settles at the bottom of the flask.

contributes to the economics of biodiesel production but has now become an unwanted waste stream with disposal cost associated with it.

Companies that produce bioethanol are another well-known source of crude glycerol accu­mulation (Figure 6.12 . During sugar fermentation to ethanol, yeast produces a substantial amount of glycerol in response to external osmotic stress (Figure 6.42 . In a conventional fermentation, 4g of glycerol is generated for every 48 g of ethanol produced and 100 g of reducing sugar consumed (http://www. freepatentsonline. com/5177008.html). There are more than 170 ethanol production facilities in the United States with a total annual capacity of more than 10 billion gallons of ethanol (http://www. ethanol. org/index. php? id=77), resulting in several million gallons of crude glycerol production annually.

The surplus glycerol generated by the biodiesel and bioethanol industries will not only reduce the price of crude glycerol, but its disposal will also become a major issue. The

development of processes that can convert crude glycerol into high-value products is expected to make biofuels economically viable.

Lignocellulosic plant biomass like corn stover and switchgrass is harvested and packaged in bales that are delivered in flatbed trucks. Unlike granular feedstocks that have a high density and are free flowing, bales have low density, typically 160-200kg/m3 . They come in bulky packages of biomass that are either round, square, or rectangular, and require man-operated handling equipment like forklifts and telescopic loaders to unload and load them. Weighing stations with weigh bridges for truck commonly available at elevators and ethanol plant can be used for biomass. Weighing stations would need to have the necessary handling equipment such as forklifts to unload bales in a minimum amount of time. However, it must be noted that the logistical cost for handling are higher with bales than with grain, which can use automated handling conveyors. The development of innovative concepts for automatic unloading of bales from trucks and their stacking in a storage building is needed for commercial utilization of biomass on a large scale.

Handling of Preprocessed Feedstock

Preprocessing biomass into bulk particulate solids will increase the feedstock density and thus enable a large quantity of feedstock hauled per truck to the facility. It also enables the use of automated handling equipment for granular feedstock that is commonly available in the industry. Densifying operations have already been discussed extensively in the Biomass preprocessing section and the Operations to produce dense biomass section, and so will not be discussed here. Trucking cost to the plant and inbound logistics cost will be reduced by densification. As was noted before, the cost of densification can be very high. While the economics of densification had looked less favorable under low world petroleum prices and relaxed environmental policy, recent high prices of petroleum fuel and efforts to reduce global carbon emissions make the use of densification a viable option to reduce feedstock delivery cost. Improvements in densification and reductions in cost will make this approach a more viable option than using bales directly.

Proteins are macromolecules formed from simpler compounds, a-amino acids. An a-amino acid is a carboxylic acid that has an amino group bonded to the carbon atom next to the carboxyl group. The designation a denotes the position of the amino group. The carbon atom adjacent to the carboxyl group is called the a-carbon atom. The general formula for an amino acid compound is shown in Figure 10.8 . In the protein structure, amino acids are linked together by peptide bonds forming the long chains. These bonds are easily broken at high temperatures resulting in the formation of amino acids. Animal manure and food processing waste are rich in protein contents. For example, proteins comprise about 25% of the total solids in swine manure.

The radical, R, in the formula shown in Figure 10.8 differentiates the types of amino acids. The R may be simple hydrocarbons, ring compounds, additional amino or carboxylic groups, or — SH or hydroxyl groups. Alanine, leucine, aspartic acid (aspartate), and glutamic acid (glutamate) account for half of the total amino acids in swine manure (Figure 10.9). Proteins and amino acids are the major sources of organic nitrogen present in swine manure. Organic sulfur is mainly from two particular amino acids, cysteine (R = — CH2-SH) and methionine (R = — CH2CH2- S-CH3).

O

Stearic acid (CH3(CH2)14COOH)

Figure 10.10. Structures of dominant fatty acids in swine manure.

Lipids

Lipids are substances that can be dissolved away from biological material by solvents that are nonpolar or slightly polar. Since the classification is based on solubility, not structure, a wide variety of compounds fall under lipids. Fatty acids are long straight-chain carboxylic acids some of which are saturated and some of which contain one or more double bonds. Almost all fatty acids isolated from natural sources contain an even number of carbon atoms. Among the fatty acids identified in swine manure, stearic acid and palmitic acid are most dominant (Figure 10.10).

One of the key steps in the conversion of lignocellulosic biomass to fermentable sugars is pretreatment. The goal of pretreatment is to alter the biomass macroscopic and micro­scopic size and structure as well as its submicroscopic chemical composition so that enzymatic hydrolysis of the carbohydrate fraction to monomeric sugars can be achieved with greater yield (Figure 3.1; Mosier et a

could also be conducted to disrupt and separate lignin from the hemicellulose component of the lignocellulosic biomass. For pretreatment to be effective, it must meet the fol­lowing criteria: (1) simple to operate and inexpensive; (2) has the ability to expose the cellulose component of biomass and increase its vulnerability to enzymatic attack;

(3) does not degrade hydrolyzed sugars; (4) generates little or no microbial inhibitory products; and (5) must be environmentally compatible. Unfortunately, during pretreatment and hydrolysis of lignocellulosic biomass, degradation and hydrolysis products such as furfural, HMF, syringaldehyde, glucuronic acid, p-coumaric acid, ferulic acid, syringic acid, levulinic acid, and other phenolic compounds may be generated (Figure 3.2b; Martinez et al. 2001; Ezeji et al. 2007a, b). These compounds can inhibit growth of microbes including fermenting microorganisms (Martinez et al. 2001; Ezeji et al. 2007a, b; Ezeji and Blaschek 2008a). During an investigation on the effect of some of the lignocellulosic hydrolysates inhibitors on growth and ABE production by Clostridium beijerinckii 592, ferulic, and p-coumaric acids were found to be potent inhibitors of growth and ABE production (Figures 3.3 and 3.4). Interestingly, glucuronic acid and HMF were not inhibi­tory to the C. beijerinckii 592, but rather were stimulatory to growth and ABE production at concentrations up to 2.0 g/L (Figures 3.3 and 3.4; Ezeji et al. 2007b; Ezeji and Blaschek 2008a). Similar results were obtained when C. beijerinckii BA101 (Ezeji et al. 2007b) and other solventogenic Clostridium species (Ezeji and Blaschek 2008a) were used.

It is important to note the number and amount of inhibitors likely to be generated during pretreatment of biomass may depend on the type and intensity of pretreatment applied. Some of the processes currently used in the pretreatment of lignocellulosic biomass are subse­quently described in this chapter.

-A-Furfura -©-HMF

Syringaldehyde

-0-Coumaricacid

Glucuronicacid

Ferulic acid

Control

0.5

Inhibitors concentration (g/L)

Figure 3.3. Effect of representative lignocellulosic biomass degradation products on the cell growth of Clostridium beijerinckii 592.

Size Reduction

Size reduction is a mechanical pretreatment of lignocellulosic biomass with the objective being reduction of particle size of biomass by a combination of chipping and milling. During size reduction operation, lignocellulosic biomass material such as corn stover is reduced to 5 to 20 mm in size in a straw chopping machine. Following the chopping process is milling, which reduces the particle size further to <0.5 to 2.0 mm diameters using a hammer, ball, or pin mill. The reduction in particle size results to increase in biomass density and improves handling during subsequent pretreatments. More importantly, it leads to an increase of surface area of lignocellulosic biomass, which improves the contact of polymers and enzymes and ultimately, hydrolysis of biomass to fermentable sugars. A particle size reduction below 40 mesh (0.42 mm) has little or no effect on the hydrolysis of or sugar yield from lignocellulosic biomass (Chang and Holtzapple 2000).

Anaerobic digestion of agricultural wastes is a mature technology with numerous full-scale digesters located all over the world. Noteworthy is the recent interest in digesting or co­digesting of bioenergy crops in the EU because of the relative high-energy efficiency of methanogenic food webs (no side product formation and product inhibition). Even though the level of maturity is high, research on reactor stability is necessary, especially when new applications are pursued. It is generally accepted by scientists and engineers that new prob­lems and unanticipated challenges keep coming up. The complex microbial community and food web is the culprit, with more powerful techniques, such as metagenomics and stable — i sotope probing, starting to shed light on the required mechanistic understanding of the microbial community and interactions (Lfibken et al. 2007; Schlfifer et al. 2008; Li et al. 2009 ).

For wastes from agriculture with complex nutrient and water cycles, anaerobic digestion should be seen in the larger context of an integrated system in which nutrients and water from digester effluent are continuously recycled. During the treatment of animal wastes with anaerobic digesters, for example, nutrients, such as ammonia and phosphate, are freed from biomass and are accumulated in solution. These nutrients must be recycled back to agricul­tural production in an environmentally friendly and sustainable way. In North Carolina (United States) an integrated system that recycled digester effluent to a greenhouse for tomato production (Cheng et al. 2004) was studied. Others are optimizing struvite precipitation to recover N and P (Borgerding 1972; Ohlinger et al. 1998; Schuiling and Andrade 1999; Battistoni et al. 2000; Mfinch and Barr 2001) . In addition, upgrading the energy carrier methane to more valuable products may be necessary to guarantee economical viability. In Utah (United States) biogas from a swine waste digester was cleaned, steam reformed into synthesis gas (i. e., syngas), converted into methanol through a thermochemical process, and combined with triglyceride to produce biodiesel through a trans-esterification reaction process (Dugba 2003).

With the need for co-digestion, an opportunity exists to link agriculture, rural communi­ties, and industry for sustainable rural community development. A U. S. example for this system approach is BioTown in Richmond, Indiana, where the goal is to create an energy self-sufficient community of about 500 persons using an anaerobic digester as an integrated technology to create biogas from animal manures, food wastes, organic municipal wastes, and crop residues (BioTown 2008). Communities such as these illustrate that anaerobic digestion is a significant and practical technology with relatively high — energy efficiencies and that agricultural wastes play an essential role.

Acknowledgements

LTA was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2004-35504-14896. In addition, The New York State Energy Research and Development Authority is gratefully acknowl­edged for partial support of anaerobic digestion studies at Cornell University. Finally, we thank Rodrigo Labatut and Nick Scalfone for generating Figures 4.1 and 4.2, respectively.

Identification of pathways and environmental conditions affecting the metabolism of glycerol under anaerobic conditions by wild-type E. coli provide opportunities to manipulate the microorganism for enhancement of ethanol yield and productivity. Fermentation of glycerol to either ethanol and H2 or ethanol and formate is one of the most effective ways of exploiting the reduced property of glycerol for bioproducts production.

Ethanol is predominantly produced following glycerol fermentation by E. coli. The product mixture, however, contains succinate, acetate, and formate. Succinate and acetate are com­peting byproducts because some substrates that would have been used for the production of ethanol are diverted toward the production of succinate and acetate (Murarka et al. 2008- , resulting in decreased ethanol yield. Blocking pathways for succinate and acetate synthesis are possible ways to enhance ethanol yield (Figure 6.7

For the ethanol-H2 production pathway, we carried out two mutations in E. coli to disrupt genes that encode fumarate reductase (FRD) and phosphotransacetylase (PTA; Figure 6.7). FRD and PTA are two key enzymes involved in the production of succinate and acetate, respectively. The resultant strain, SY03, produced an almost equimolar amount of ethanol and hydrogen and yield comparable to theoretical maximum of 1 mol of each product per mole of glycerol (Yazdani and Gonzalez 2008). To facilitate coproduction of formate and ethanol, another mutation was introduced in the gene (fdhF) encoding component of FHL in the strain SY03 (Figure 6.7). FHL is responsible for the oxidation of formate into H2 and CO2 . This triple mutant strain, called SY04, produced exclusively ethanol and formate at yields of 92%-96 % of the theoretical maximum.

The strategies used in the generation of these mutants led to a decrease in the growth rates of E. coli mutants (Yazdani and Gonzalez 2008). The overexpression of GldA and dihydroxy — acetone kinase (DHAK), which are responsible for converting glycerol into glycolytic intermediate DHAP, was assessed for improving the growth rate of E. coli mutants. We overexpressed GldA and DHAK separately and in combination with plasmids pZSgldA, expressing GldA, pZSKLM expressing DHAK, and pZSKLMgldA expressing both GldA and DHAK to improve the rate of glycerol fermentation (Yazdani and Gonzalez 2008) . Transformation of these plasmids in E. coli MG1655 led to a 10-20-fold increase in GldA and a 5-6-fold increase in DHAK activities. When the effect of overexpression of GldA and DHAK genes in E. coli MG1655 was tested on glycerol fermentation, individual overexpres­sion of either GldA or DHAK did not have a significant effect on glycerol fermentation. Simultaneous overexpression of GldA and DHAK from plasmid pZSKLMgldA led to a 3.4- fold increase in the amount of glycerol fermented in E. coli MG1655. Overexpression of GldA and DHAK in the host SY04 with mutations in three genes, f rdA, pta, and f dhF, for the coproduction of ethanol-formate led to the production of ethanol and formate at maximum volumetric rates of 3.58 and 3.18 mmol/L/h, respectively.

Further experiments involving various mutants confirmed role of both respiratory and fermentative pathways of glycerol utilization under microaerobic condition (Durnin et al. 2009). Enzymes involved in the respiratory pathway of glycerol metabolism, glycerol kinase (GlpK), and glycerol-3 .phosphate dehydrogenase (GlpD) exhibited higher activities in the initial aerobic phase of glycerol utilization. The transition to microaerobic conditions, char­acterized by undetectable amounts of dissolved oxygen, resulted in a 2.5- fold increase in glycerol-3-phosphate dehydrogenase activity and a 10-fold increase in both GldA and DHAK activities. The GlpK-GlpD pathway predominated during the early phase of fermentation, while the GldA-DHAK pathway predominated during the later stage of cultivation. Under microaerobic conditions, the engineered strains were able to utilize glycerol to produce ethanol and hydrogen, or ethanol and formate in basal media.

Conclusions and Future Outlook

Considering the worldwide surplus of crude glycerol and the need to find new uses for this cheap abundant carbon source, the use of anaerobic fermentation to convert low-value crude glycerol streams generated during the production of biodiesel to value-added products represents a promising step toward achieving an economically viable biodiesel industry. A number of organisms are able to ferment glycerol to different bioproducts with a wide range of applications. The success of these strategies will depend on the use of robust microorganisms that are amenable to industrial applications.

Grain is unloaded by positioning the hopper-bottom trailer over a unload pit and opening the hopper to allow the grain to flow into the pit. Unload time is about 5 minutes. The grain harvest season is so short that all farmers harvest the maximum amount each day to get their crop harvested before they are slowed by late fall weather delays. Often a queue of trucks forms waiting to be unloaded, thus it is typical for the unload time to average 30 minutes or more. There is a trade-off between the additional investment for a larger unload capacity and the farmer wait time. If the grain storage company invests more in the receiving facility, they have to charge the farmer more to store the grain to recover their cost.

The receiving facility typically does not delay operations at the cotton gin. Some gins haul in all modules they have under contract before they begin the ginning season. Most gins, however, begin ginning as soon as they have an inventory of modules, perhaps 100-500 modules depending on their gin capacity (number of modules ginned per day). The module haulers run continuously to keep the gin supplied. Sometimes they cannot keep up and the gin runs out of material. When this happens, the gin shuts down until the “at-gin” inventory is built back up.

The cotton gin is a mechanical process; it can be started and stopped with the throw of a switch. (This is a little bit of a simplification, but it can be stopped and restarted with less cost penalty than a sugar mill or bioenergy plant.) Gin owners want to gin the maximum number of bales per year, thus their goal is continuous operation.

Module haulers unload onto the ground in the storage yard, thus there is no waiting in a queue unless two trucks arrive at the scale at the same time. Even then, the delay is minimal. Operations at the gin receiving facility set the standard for all other biomass hauling opera­tions. The key disadvantage of the gin system is that the truck only hauls one module, thus the load is only about 6 dry t.

The sugarcane receiving facility, because of the large number of trucks unloaded per day (typically about 1000 loads per day at one sugar mill in South Florida), is an excellent example of an optimized receiving facility operation. If the unloading station is clear (no truck unloading) when a truck arrives, it side-dumps the bins and immediately returns to the field. The typical time to weigh in, unload, and weigh out is about 3 minutes. If the truck proceeds to the storage area where the full bins are removed and empty bins are placed on the truck, this operation typically takes 3-4 additional minutes.

Unfavorable oxidation-reduction potential (ORP) has also been cited as a cause of poor fer­mentability (Leonard and Hajny 1945). Collingsworth and Reid (1935) found that the addition of reducing agents to media improved their fermentability. Three methods have been pro­posed for overcoming unfavorable ORP in fermentation media: phytochemical reduction by large amounts of yeast; use of reducing agents; and production of reducing substances from sugars by either caramelization or alkali degradation.

When Na2SO3, NaHSO2, Na3SO3.5H2O, Na2S2O3, Na2S2O5, KHSO3, Na2S, sulfite waste liquor, alkali-decomposed sugar, ascorbic acid, cysteine, or reduced iron filings were added to wort hydrolysates, an improved fermentation was observed, which underscored the effect of ORP (Leonard and Hajny 1945; Leonard and Peterson 1947). Diethanolamine, triethanol­amine, pyridine, aniline, dimethylaniline, and similar substances also showed favorable action toward fermentation under the same conditions. The amount of reducing agent required is dependent upon the length and temperature of heat treatment period (Leonard and Hajny 1945; Leonard and Peterson 1947). The mechanism of detoxification by reducing agents is not clear. However, researchers have found that toxic and oxidizing compounds such as furfural and HMF would be reduced to their less inhibitory alcohol forms inside yeast cells associated with oxidation of NAD(P)H, and redirect yeast energy to fixing the damage caused by furans and by intracellular reduced NAD(P)H and ATP levels (Nilsson et al. 2005; Almeida et al. 2007).

About 77.1 million metric tons of wheat straw is produced in the United States annually, and this quantity has the potential to produce 54 million metric tons of fermentable sugars (http:// www. biofuelscenter. org). Wheat straw, which is composed of 67% of cellulose and hemicel — lulose (Table 3.1), can serve as a cheap substrate for butanol production by solventogenic Clostridium species. Qureshi et al. (2007, 2008b, c) have pioneered research on potential use of wheat straw as substrate for ABE production. During one investigation, five different processes were evaluated for ABE from wheat straw by Clostridium beijerinckii P260 (Qureshi et al. 2008b). The five processes were fermentation of pretreated wheat straw (Process I), separate hydrolysis and fermentation of wheat straw to ABE without removing sediments (Process II), simultaneous hydrolysis and fermentation of wheat straw without agitation (Process III), simultaneous hydrolysis and fermentation with additional sugar sup­plementation (Process IV), and simultaneous hydrolysis and fermentation with butanol recov­ery by gas stripping (Process V). In these studies, process IV and V achieved maximum ABE concentrations of 17.92 g/L and 22.42 g/L, resulting in ABE productivities of 0.19 and 0.31 g/L/h, respectively. In the control experiment (glucose), an ABE productivity of 0.30 g/L/h was achieved (Qureshi et al. 2008b).

To improve C. beijerinckii 260 tolerance to toxic compounds generated during pretreat­ment and hydrolysis of wheat straw biomass and enhance fermentability of acid pretreated wheat straw, Qureshi et al. (2008d) used alkaline peroxide and electrodialysis treatment methods to detoxify acid pretreated wheat straw hydrolysates prior to ABE fermentation. A maximum ABE concentration of 22.17g/L with reactor productivity of 0.55 g/L/h was pro­duced, compared to ABE concentration of 21.37 g/L with reactor productivity of 0.30 g/L/h produced by the control (glucose). To improve ABE productivity further, Qureshi et al. (2008c) used C. beijerinckii P260 to produce butanol from wheat straw hydrolysates in a fed-batch reactor with simultaneous ABE recovery by gas stripping. A total of 192.0g ABE was produced in a 2.5 L bioreactor (1 L reaction volume) at the end of fermentation, resulting in an ABE productivity of 0.77g/L/h and ABE yield of 0.44. Simultaneous hydrolysis and fermentation of wheat straw to butanol and/or fed-batch fermentation of wheat straw hydro­lysates and simultaneous butanol recovery by gas stripping are, therefore, attractive options with great potential of replacing glucose for butanol production.

Conclusion

With continuous world population growth and energy demand, current production of liquid biofuel (ethanol) from food crops such as corn and sugar cane is unsustainable. The use of agricultural residues to produce liquid biofuels—(n particular, the production of butanol— holds great interest as a means for generating sustainable transportation fuel and feedstock chemicals. The possibility of using agricultural residues such as corn fiber, corn stover, wheat straw, as well as energy crops such as switchgrass, Miscanthus, and so on for butanol production and incorporating gas stripping in an in-line product recovery process has gener­ated considerable interest. Simultaneous butanol fermentation and recovery has dramatically improved production of butanol from wheat straw. Considerable progress has been made in the development of biomass pretreatment and hydrolysis technologies. Progress has also been made on the improvement of some technologies that were previously developed so that use is more environmentally compatible and generating little or no lignocellulosic deg­radation compounds that are toxic to fermenting microorganisms. Availability of efficient biomass pretreatment technology will help in utilizing bio-based conversion of agricultural residues into value-added products to become attractive. By employing in-line recovery systems during butanol fermentation, substrate inhibition and butanol toxicity to the culture are drastically reduced, with ABE productivity dramatically increased. Given that butanol is an excellent potential fuel and the United States is rich in lignocellulosic biomass, butanol production from agricultural residues is crucial in future energy production endeavors.

Acknowledgm ents

This work was supported by funding from Northeast Sungrant (Cornell University) Award/

Contract # GRT00012344, National Research Initiative of the USDA Cooperative State

Research, Education and Extension Service, grant number 2006-35504-17419, and Seed grant

from Ohio Agricultural Research and Development Center (OARDC), Wooster.

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In contrast to the free enzyme systems, some enzymes are attached directly to the cell wall. This is frequently accomplished in Gram-positive bacteria via a specialized type of module, the S-layer homology (SLH) module, previously shown to be associated with the cell surface of Gram-positive bacteria (Lupas et al. 1994). Attachment of enzymes to the cell wall may have evolved to provide a more efficient assimilation of the soluble sugars produced due to their proximity to the cell surface. This arrangement would serve to reduce competition with other bacteria for the soluble products.

Examples of putative cell surface enzymes that contain an SLH module include a GH5 cellulase and GH13 amylase-pullulanase from Bacillus, a GH10 xylanase from Caldicellulosiruptor (Saul et al. 1990), a GH5 endoglucanase from Clostridium josui, a GH16 lichenase and GH10 xylanase from Clostridium thermocellum (Jung et al. 1998), and a variety of enzymes (GH10 xylanases, a GH5 mannanase, and a GH13 amylase-pullulanase) from different species of Thermoanaerobacter (Matuschek et al. 1996). The modular architecture of these enzymes may be particularly intricate, containing numerous different modules in a single polypeptide chain, thus forming extremely large enzymes.