8.2.1 The rationale for CBP

Bioenergy use is propagated by incentives and successful policy interventions that have been enacted to select and encourage industrial development of renewable energy sources. Bioethanol continues to be the dominant biofuel with increasing use as a transportation fuel in Brazil, USA, Canada, and the European Union (EU). The routine ways (SHF, SSF, and SSCF) for industrial ethanol production all involve a dedicated cellulase production step. Avoiding this step has been noted to be the largest potential energy-saving step. For a given amount of pretreated biomass, it has been shown that CBP offers cost savings that are not associated with the routine methods, even at the lowest amount of cellulase loading required to produce fermentation products. However, typical CBP yields of ethanol and other end-products are much lower than obtained via traditional processes (Lynd et al., 2005, 2008).

Hydrogen (H₂), on the other hand, is considered a clean energy, having a gravimetric energy density of 122 KJ g^− 1 and water as the only by-product of combustion. Currently H₂ is produced via energy intensive, environmentally harmful processes such as catalytic steam reformation of methane, nuclear or fossil fuel-mediated electrolysis of water, or by coal gasification (Levin et al., 2004; Carere et al., 2008). Biological H₂ production via anaerobic fermentation using cellulosic substrates is an attractive process for the following reasons: (i) it is less energy intensive, (ii) it utilizes a simple process design and feedstock processing, and (iii) it has the potential to utilize agricultural or agri-industrial by-product streams (Sparling et al., 1997; Valdez-Vazquez et al., 2005; Levin et al., 2006). However, rates and yields of H₂ by direct microbial conversion are low, and increasing the rates and yields remains a challenge for biological H₂ production by CBP (Levin et al., 2009).

8.3.1 Ruminant or natural CBP

Many animals and insects have evolved to feed on and digest raw biomass. Some well-known cellulolytic bacteria have been isolated from these organisms as their natural habitat, e.g. Ruminococcusalbus and Clostridium termitidis inhabit the gut of ruminants and termites, respectively. By taking a close look at the highly developed ruminal fermentation of cattle (i.e., ruminant CBP = rCBP), Weimer et al. (2009) proposed that breakthroughs developed by ruminants and other already existing anaerobic systems with cellulosic biomass conversion can guide future improvements in engineered CBP (eCBP) systems. Comparing the journey of the feed through the bovine digestive tract to the transformation process of a cellulosic feedstock in a biorefinery, Weimer et al. (2009) suggest that the sliding, longitudinal movement of bovine rumination is a better physical pretreatment than conventional grinding. This is because it results in substantial increase in surface area of the plant material available for microbial attack resulting in ‘effective fiber’ properties similar to burr mills. Burr mills consume two-thirds of the energy required by hammer mills, and although they have been considered less efficient in the grinding of grain, they could be efficient in the milling of lignocellulosic biomass for CBP as a result of the ‘effective fiber’ properties generated (Weimer et al., 2009).

A great amount of effort is invested chewing the feed into a fine physically pretreated state. The feed is masticated while it is moist, another strategy supported by recent studies which show that milling after chemical treatment will significantly reduce energy consumption, reduce cost of solid–liquid separation requirements, and reduce the energy required for mixing pretreated slurries (J. Y. Zhu et al., 2009; Zhu and Pan, 2010).

Another similarity is the fact that ruminal microflora (R. albus, R. flavefaciens, and fibrobactersuccinogens) found in high numbers in the rumen are capable of rapid growth on cellulose using cellulosomal complexes similar to the well-characterized cellulosomes of Clostridium thermocellum or C. phytofermentans that are being investigated for eCBP (Lynd et al., 2002; Weimer et al., 2009). Thus, the limitation of rCBP could be explored to develop better operating parameters for eCBP. In summary, it appears that ruminants have developed an efficient and elegant physical pretreatment process that could provide insight in developing a physical pretreatment process tailored for industrial CBP, as well as serve as a model for eCBP.

8.3.2 Engineered CBP

Unlike rCBP, eCBP seeks to utilize pure cultures of specialist native cellulolytic bacteria, or recombinant cellulolytic bacteria, to convert cellulose via direct fermentation to value-added end-products. Aerobic or anaerobic microorganisms could be used for eCBP; however, the use of a separate aerobic step for cell growth is not envisioned because it is not a characteristic feature of CBP. In eCBP, much attention is dedicated to the strategic development of cellulolytic and hemicellulolytic microorganisms both for substrate utilization and end-product formation. Among the many bacteria that have been considered as CBP-enabling microorganisms, anaerobic bacteria such as C. thermocellulum, C. phytofermentans, and the aerobic yeast Saccharomyces cerevisiae have been the most investigated as potential eCBP-enabling microorganisms. Figure 8.3 compares natural and engineered bioprocessing by differentiating the different unit operations.

8.4.1 CBP microorganisms

Based on substrate utilization, carbohydrate hydrolyzing species represent a wide range of specialist and non-specialist microbes, and specialized microbes capable of utilizing cellulose or hemicellulose-derived sugars are preferentially selected as CBP-enabling microorganisms. Cellulolytic bacteria belong to the phyla Actinobacteria, Proteobacteria, Spirochates, Thermotogae, Fibrobacteres, Bacteriodes, and Firmicutes, but approximately 80% of the cellulolytic bacteria are found within the Firmicutes and Actinobacteria (Bergquist et al., 1999). Many of these bacteria isolated from soil, insects, ruminants, compost, and sewage have the natural ability to hydrolyze cellulose and/or hemicellulose with the majority of the reported bacteria belonging to the phylum Firmicutes, and are within the class Clostridia and the genus Clostridium. For example, Clostridium thermocellum is a Gram positive, acetogenic, obligate anaerobe with the highest known growth rate on crystalline cellulose and the most investigated as a potential CBP-enabling bacteria (Lynd et al., 2002; Xu et al., 2009).

Although cellulolytic bacteria belong to aerobic or anaerobic groups of bacteria, for large-scale CBP, anaerobiosis is advantageous because of oxygen transfer limitations that are avoided when using anaerobic bacteria (Demain et al., 2005). However, because of the increasing tendency to consolidate steps for bioethanol production, Saccharomyces cerevisiae and the fungus Tricoderma reesei are being investigated as CBP-enabling candidates for bioethanol production (van Zyl et al., 2007; Xu et al., 2009). Other candidates into which saccharolytic systems have been engineered for CBP include Zymomonas mobilis, Escherichia coli, and Klebsiella oxytoca (van Zyl et al., 2007).

8.4.2 Carbohydrate active enzyme systems

To utilize plant biomass for growth, microorganisms produce multiple enzymes that hydrolyze the cellulose, hemicellulose, and pectin polymers found in plant cell walls (Warren, 1996). As a class, these carbohydrate active enzymes are referred to as glycoside hydrolases (GHs). Extracellular GHs can be secreted freely into the environment surrounding the cell (non-complexGH systems) or they can be cell-associated in large enzyme complexes (cellulosomes). Gycoside hydrolases that specifically target cellulose include:

The ability of cellulases to hydrolyse ß-1,4-glycosidic bonds between glucosyl residues distinguishes cellulase from other glycoside hydrolases (Lynd et al., 2002).

8.4.3 Non-complex glycoside hydrolase systems

Non-complex systems consist of secreted glycoside hydrolases and generally involve fewer enzymes. Aerobic fungi of the genera Trichoderma and Aspergillus have been the focus of research for non-complex cellulase systems. Trichoderma reesei, which is the most researched non-complex cellulase system, produces at least two exoglucanases (CBH I and CBH II), five endoglucanases (EG I, EG II, EG III, EG IV, and EG V) and two ß-glucosidases (BGL I and BGL II) that act synergistically in the hydrolysis of polysaccharides. However, CBH I and CBH II are the principal components of the T. reesei cellulase system representing 60% and 20%, respectively, on a mass basis of the total protein produced (Lynd et al., 2002). The ability to produce and secrete over 100 g of cellulase per liter of culture has established T. reesei as a commercial source of cellulase enzymes (Xu et al., 2009).

8.4.4 Complex glycoside hydrolase systems

Some anaerobic cellulolytic microorganisms possess a specialized macromolecular complex of carbohydrate active enzymes known as the cellulosome. First described for C. thermocellum by Lamed et al. (1983), the cellulosome is an exocellular, multicomponent complex of GHs that mediates binding to lignocellulosic biomass and subsequent hydrolysis of the cellulose and hemicellulose polymers (Lamed et al., 1983; Carere et al., 2008). Functionally, cellulosomes are assembled on the cell walls of bacteria and enable concerted enzyme activity by minimizing distances of enzyme substrate interactions and optimizing synergies among the catalytic components, thus enabling efficient hydrolysis of the polymers and uptake of the hydrolysis products (Lynd et al., 2002).

Although cellulosome compositions can differ in the number and variety of GHs from one species to another, they generally consist of catalytic components attached to a glycosylated, non-catalytic scaffold protein that is anchored to the cell wall. In C. thermocellum, the anchor protein, known as the cellulose integrating protein (CipA), or ‘scalffoldin’, is a large (1,850 amino acid long and 2–16 MDa) polypeptide, which is anchored to the cell wall via type II cohensin domains. The C. thermocellum cellulosome contains nine GHs with endoglucanase activity (CelA, CelB, CelD, CelE, CelF, CelG, CelH, CelN, and CelP), four GHs which exhibit exoglucanase activity (CbhA, CelK, CelO, CelS), five or six GHs which exhibit xylanase activity (XynA, XynB, XynV, XynY, XynZ), one enzyme with chitinase activity (ManA), and one or two with lichenase activity (LicB). CelS, the major exoglucanase, and CelA, the major endoglucanase associated with the C. thermocellum cellulosome generate oligocellulodextrins containing two (cellobiose) to five (cellopentose) glucose residues (Lynd et al., 2002; Demain et al., 2005). The cellulosomes of some strains of C. thermocellum have been shown to degrade pectin probably via pectin lyase, polygalacturonate hydrolase, or pectin methylesterase activities. Other minor activities include ß-xylosidase, ß-galactosidase, and ß-mannosidase (Lynd et al., 2002; Demain et al., 2005). These modules have dockerin moieties that can associate with the cohesins of the scaffoldin to form the cellulosome (Fig. 8.4).

Cellobiosephosphorylase, which hydrolyzes cellobiose and longer chain oligocellulodextrins to glucose and glucose-1-phosphate via substrate level phosphorylation, and cellodextrinphosphorylase, which phosphorylates ß-1,4-oligoglucans via phosphorolytic cleavage, have also been associated with the C. thermocellum cellulase system. Unlike the fungal cellulases, the celllosome of C. thermocellum is able to completely solubilize crystalline cellulose such as Avicel, a characteristic referred to as Avicelase or ‘true cellulase’ activity (Demain et al., 2005). Moreover, the C. thermocellum cellulase system results in the oligosaccharide hydrolysis products that are different from those of aerobic cellulolytic fungi like T. reesei, which generates cellobiose as the primary hydrolysis product (Zhang and Lynd, 2005).

8.4.5 Mode of action

Polysaccharide hydrolyzing enzymes such as cellulases and xylanases are modular proteins consisting of at least two domains: the catalytic module, and the carbohydrate binding module (Gilkes et al., 1991; Horn et al., 2012). Common features of most GH systems that effect binding to cellulose surface and facilitate hydrolysis are the carbohydrate binding modules (CBMs), which are known to have the following functions:

CBMs specific for insoluble cellulose are categorized as Type A CBMs, which interact with crystalline cellulose and Type B, which interact with non-crystalline cellulose (Arantes and Saddler, 2010a).

Recent studies that have focused on bacterial and fungal GHs have identified two GH families that have flat substrate-binding surfaces with the capability of cleaving crystalline polysaccharides via an oxidative reaction mechanism that depends on the presence of divalent metal ions and an electron donor. These two families are the Family 33 carbohydrate binding module (CBM33) proteins identified in bacteria and Family 61 glycoside hydrolases (GH61) from fungi (Vaaje-Kolstad et al., 2010; Horn et al., 2012). CBM33 and GH61 GHs bind to cellulose via their flat CBM substrate binding sites, which disrupt the orderly packing of the crystalline cellulose chains, creating accessible points by both introducing cuts in the polymer chains and by generating charged groups at the cut sites.

Endoglucanases and exoglucanases act synergistically. The endoglucanases generate new reducing and non-reducing ends for exoglucanases, which in turn release soluble cellodextrins and cellobiose that are converted to glucose by ß-glucosidase (Wood and McCrae, 1979; Horn et al., 2012; Kostylev and Wilson, 2012). However, for GHs to efficiently hydrolyze cellulosic biomass, they must first be able to access the cellulose chains that are tightly packed in microfibrils trapped within a heteropolymer matrix (Arantes and Saddler, 2010a; Horn et al., 2012). Factors that increase accessibility have been identified and intensely investigated (Reese, 1956; Jeoh et al., 2007; Arantes and Saddler, 2010a, 2010b; Horn et al., 2012).

8.4.6 Bioenergetics of CBP

The bioenergetics of CBP will differ with the type (aerobic, aerotolerant, or anaerobic) and number (pure, co-, or mixed-cultures) of microorganisms being considered as CBP-enabling agents. This was thought to be even more challenging for anaerobes from a bioenergetic standpoint given that ATP available from catabolism is used to support both cell growth and cellulase production (Lynd et al., 2002). An assessment of the bioenergetic benefits associated with growth on cellulosic substrates in terms of net cellular energy currency (ATP, ADP, or AMP) available for growth and cellulase production is vital for eCBP-enabling microorganisms.

A comprehensive bioenergtic model validating the bioenergetic feasibility of employing C. thermocellum on crystalline cellulose was reported by Zhang and Lynd (2005), who determined that C. thermocellum assimilates oligo-cellodextrins (G2–G6) of mean chain length of n ≈ 4 (where n = degree of polymerization of glucose (G) moieties). The oligocellulodextrins are imported into the cell and then cleaved by substrate level phosphorylation by cellodextrin- and cellobiose-phosphorylases. Phosphorylation results in cleavage of ß-glucosidic bonds releasing glucose and glucose-6-phosphate, that undergo glycolysis via the Emden–Meyerhoff pathway to generate ATP. Assimilation of oligo-cellodextrins with an average of 4.2 glucose units more than compensates for higherATP expended on cellulase synthesis when C. thermocellum is grown on cellulose compared to cellobiose (Zhang and Lynd, 2005). Thus, the anaerobic fermentation of cellulose using C. thermocellum as a CBP-enabling microorganism is bioenergetically feasible, without the need for added saccharolytic enzymes.

8.5.1 Metabolic engineering

Bailey (1991) defined metabolic engineering (ME) as the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with recombinant DNA technology. Metabolic engineering tools being used in the quest for the development of ethanologenic and currently hydrogenic microorganisms include the following:

8.5.2 Natural versus engineered GH systems

The development of bacteria and fungi for CBP has focused mostly on the use of microorganisms that naturally express GH systems to hydrolyze cellulose/hemicellulose and synthesize products of interest from the hydrolysis products. Metabolic engineering of anaerobic cellulolytic bacteria has been the primary approach for enhancing the yields of the desired products so that they can meet the requirements of an industrially consolidated bioprocess. Gene transfers systems, electrotransformation protocols, and recombinant strains with enhanced product synthesis profiles have been described for both C. cellulolyticum and C. thermocellum.

Previous studies have shown that cellulose utilization by the mesophilic C. cellulolyticum is strongly dependent on initial cellulose concentration, which ultimately affects carbon flow distribution leading to end products. And the cessation of early growth was as a result of pyruvate overflow during high carbon flux (Guedon et al., 1999; Desvaux et al., 2000). Increased levels of less reduced metabolite, ethanol and lactate were observed with high levels of carbon flux, whereas at a low carbon flux, pyruvate is oxidized preferentially to acetate and lactate, thus showing an innate capability to balance carbon and electron flow or generation of reducing equivalents (Guedon et al., 1999). However, a decrease in the accumulation of pyruvate at high carbon flux was achieved by heterologous expression of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) from Zymomonas mobilis in a shuttle vector pMG8. Growth of recombinant strain resulted in a 150% increase in cellulose utilization, 180% increase in dry cell weight, 48% decrease in lactate production, 93% increase in acetate and 53% increase in ethanol over the wild C. cellulolyticum, proving that genetically engineered strains could be used to greatly increase yields of cellulose fermentation by C. cellulolyticum (Guedon et al., 2002).

To show the potential of using C. thermocellum as robust platform organism for CBP, Argyros et al. (2011) constructed a mutant with novel genetic engineering tools that allow for the creation of unmarked mutations while using a replicating plasmid. A counter selection strategy was used to delete genes for lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta) resulting in a stable strain with 40:1 ethanol selectivity and a 4.2-fold increase in ethanol yield over the wild-type strain (Argyros et al., 2011).

Expression of heterologous cellulases in non-cellulolytic microorganisms that are known to possess desired product formation characteristics, such as faster sugar consumption, higher ethanol yield and high resistance to ethanol and fermentation inhibitors, has also been accomplished (Hasunuma and Kondo, 2012). For example, genes encoding endoglucanse II (EG II) cellobiohydrolase II (CB II) from T. reesei and beta-glucosidase BGL 1 from Aspergilus aculeatus were integrated into the chromosome of wine yeast strain using a single vector conferring resistance to antibiotics G418. The mutant strain was able to hydrolyze corn stover cellulose and produced ethanol without the addition of exogenous saccharolytic enzymes (Khramtsov et al., 2011). Significant advances related to recombinant enzyme expression support the potential of S. cerevisiae as CBP host, and the number of genes expressed is not probably as important as the metabolic burden and stress responses associated with such high-level expression (van Zyl et al., 2007).

Heterologous expression of cellulolytic enzymes for the development of a cell surface which provides display of cellulolytic enzymes or cellulases that are secreted is currently being investigated in other non-cellulolytic, ethanologenic bacteria such as E. coli, Zymomonas mobilis and Klebsiella oxytoca to enable growth and fermentation of pretreated lignocellulosic biomass (Jarboe et al., 2007; van Zyl et al., 2007).