For enzymatic hydrolysis and fermentation, different strategies have been explored including separate hydrolysis and fermentation (SHF), SSF nonisothermal simultaneous saccharification and fermentation (NSSF), simultaneous saccharification and cofermentation (SSCF), or consolidated bioprocessing (CBP) (Lynd et al., 2002; Taherzadeh and Karimi, 2007). Each process has advantages and disadvantages.

For SHF, the main advantage is the possibility to separately optimize hydrolysis and fermentation steps and the main drawback is the inhibition of cellulase activ­ity by the released sugars, mainly cellobiose and glucose (Taherzadeh and Karimi, 2007). SSF, different from SHF, combines the enzymatic hydrolysis and fermentation in one step, thus minimizing the product inhibition of cellulase enzymes as the released sugars are immediately consumed by the microorganism. In addition, cellulase production and fermentation of hemicellulose hydrolysis products occur in two additional, discrete process steps. This process has many advantages over SHF such as increased ethanol yield, decreased enzyme loading, decreased contamination, and lower capital cost. The dis­advantages are differences between optimum tempera­tures for enzyme hydrolysis and fermentation and inhibition of cellulase by the produced ethanol (Lynd et al., 2002; Olofsson et al., 2008).

To solve the issue of temperature difference, the NSSF process was proposed (Wu and Lee, 1998) in which saccharification and fermentation occur simultaneously but in two separate reactors, each operated at its own optimum temperature. Compared to SSF, NSSF increased ethanol yield and productivity with a reduced overall enzyme loading of 30—40%. The disadvantage is increased capital cost for extra equipment.

In SSCF, enzymatic biomass hydrolysis and fermen­tation of both cellulose and hemicellulose hydrolysis products all occur in a single bioreactor with a single microorganism (Teixeira et al., 2000). It is considered an improved process compared to SSF, which requires two bioreactors with two different microorganisms and two different biomass production setups (Hame — linck et al., 2005; McMillan, 1997; McMillan et al.,

1999) . However, SSCF usually requires a metabolically engineered microorganism that can robustly coferment both glucose and xylose (Teixeira et al., 2000) without synthesis of side products. For example, when a natu­rally occurring strain, Lactobacillus pentosus (American Type Culture Collection, ATCC 8041), was used in an SSCF process using pretreated corn stover as substrate and the commercial cellulase Spezyme-CP for hydroly­sis, the maximum yield of lactic acid was >90% of the theoretical maximum on the basis of all available fermentable sugars. However, acetic acid was also pro­duced through a different metabolic pathway that as­similates pentoses (xylose and arabinose). Another drawback of the process is the difficulty in improving lactic acid concentration due to end-product inhibition of the nonengineered strain (Zhu et al., 2007).

All the above-mentioned processes require a separate enzyme production step or an external supply of en­zymes for biomass hydrolysis. In CBP, enzyme produc­tion, biomass hydrolysis, and fermentation of pentoses and hexoses are accomplished in a single reactor by mono — or cocultures of microorganisms (Lynd et al.,

2002) . The obvious advantages of CBP are decreased cap­ital costs and no extra cost for enzyme production or pur­chasing (Hamelinck et al., 2005; Lynd et al., 2005). However, since naturally occurring microorganisms cannot simultaneously synthesize enough of the neces­sary saccharolytic enzymes and convert released sugars into the desired end products, the CBP configuration requires the development of engineered microorganisms (Hasunuma and Kondo, 2012a; Xu et al., 2009). Such "superbugs" need to not only secrete high titer, robust en­zymes, but also efficiently produce ethanol and other bio­products at high yields under harsh environments containing toxic compounds. CBP is gaining increasing recognition as a potential breakthrough for low-cost biomass processing (Hasunuma and Kondo, 2012a; van Zyl et al., 2007). The company Mascoma Corporation claims to have successfully engineered microorganisms for industrial CBP application (http://www. mascoma. com/).

Commonly Used Microorganisms in Biomass Conversion and Some Application Examples

A large number of microorganisms are capable of degrading plant cell walls including bacteria and fungi. With few exceptions, two distinct cellulolytic strategies have been adapted by the aerobic and anaerobic groups. While aerobic bacteria and fungi produce numerous individual, extracellular enzymes with many of them acting in synergy for effective hydrolysis, anaerobic bacteria and fungi possess a unique extracellular multi­enzyme complex, termed the cellulosome, that can efficiently hydrolyze crystalline cellulose (Bayer et al., 2004, 1998; Doi and Kosugi, 2004; Fontes and Gilbert, 2010; Lamed et al., 1983; Lynd et al., 2002; Schwarz, 2001; Shoham et al., 1999; Steenbakkers et al., 2003). Metabolic utilization of the monomeric sugars from hydrolyzed biomass leads to the natural production of biofuels and bioproducts, mostly as side products by different microorganisms. For ethanol fermentation of lignocellulosic biomass, most frequently considered microorganisms include the bacteria E. coli, Z. mobilis and Clostridium phytofermentans; themophilic bacteria such as Clostridium thermocellum; yeasts such as S. cerevi — siaeand Pichia stipitis; and filamentous fungi (Amore and Faraco, 2012; Hahn-Hagerdal et al., 2007; Weber et al., 2010; Xu et al., 2009).

Like ethanol, the majority of other potential biofuels and bioproducts are naturally produced by various mi­croorganisms as side products. The viability of a fermentation process for industrial application depends on its cost-competitiveness. As listed in Table 5.1, most microorganisms cannot use polymeric carbohydrates directly as fermentation substrates; therefore, biomass has to be broken down into monomeric sugars to be used as fermentation substrates. For an economically viable manufacturing process of biofuels from ligno — cellulosic biomass, pentose utilization is essential. Therefore, an optimal microorganism should be able to simultaneously ferment both hexose and pentose sugars and give rise to high productivities and yields. In addition, it should have high tolerance to fermenta­tion inhibitors and end products and resist microbial

contamination, e. g. bacteriophage infections (Weber et al., 2010).

No naturally occurring microorganism has all the required features. Promising means to develop a microorganism for sustainable bioethanol/bioproduct production include breeding technologies, genetic en­gineering and the search for undiscovered species (Weber et al., 2010). For production of a particular product from a specific biomass, native organisms can be selected from a group of different species of mi­crobes based on their fermentation performance, such as substrate utilization efficiency, inhibitor resistance, and productivity (Rumbold et al., 2010, 2009). The yeast S. cerevisiae is by far the most widely used organ­ism in the existing fermentation industry. To improve its application in bioethanol fermentation from biomass, targeted evolution strategy has been applied to obtain inhibitor-tolerant S. cerevisiae that can resist
individual or multiple inhibitors (Ding et al., 2012; Heer and Sauer, 2008; Liu, 2006). When adaptation and selection processes were applied to the parental fungus Rhizopus oryzae, a new strain was obtained that exhibited significantly improved efficiency of sub­strate utilization and enhanced production of l-(+)- lactic acid from corncob hydrolysate. The final product concentration, yield, and volumetric productivity more than doubled compared with its parental strain (Bai et al., 2008).

Applications of thermotolerant mesophilic microor­ganisms in the fermentation process have considerable potential for cost-effective ethanol and other bioproduct production. The thermotolerant yeast Kluyveromyces marxianus grows well at temperatures as high as 45—52 °C and can efficiently ferment ethanol at temper­atures of between 38 and 45 °C. A 5 °C increase in the fermentation temperature can greatly decrease fuel

ethanol production costs (Babiker et al., 2010). Results from solid state fermentation of sweet sorghum stalk to ethanol with the thermotolerant yeast strain Issatchen — kia orientalis IPE 100A showed great potential for its practical application in large-scale, deep-bed solid state fermentation (Kwon et al., 2011).

The thermotolerant Bacillus coagulans strain 36D1 can ferment both hexoses and pentoses from enzymatically hydrolyzed biomass at 50—55 °C and pH 5.0 producing l (+)-lactic acid as the primary fermentation product. Since such conditions are closer to the optimum fungal enzyme functioning requirements, the amount of enzyme required for cellulose conversion is signifi­cantly reduced in comparison with yeast or lactic acid bacteria currently used by the industry as microbial biocatalysts. In addition, both biomass conversion efficiency and product yield are greatly increased with a dramatically decreased fermentation time, thus reducing the cost of both the process and final product (Ou et al., 2009).

The anaerobic mesophilic bacterium C. phytofermen- tans (ATCC 700,394) is a promising native microor­ganism for biomass conversion since its genome encodes the highest number of enzymes for degradation of lignocellulosic material among sequenced Clostridial genomes (Warnick et al., 2002; Weber et al., 2010). It se­cretes noncomplex, individual enzymes to hydrolyze both cellulose and hemicelluloses to both hexose and pentose sugars, which are mostly directly consumed, producing ethanol and acetate as the major products (Warnick et al., 2002; Weber et al., 2010). When used in the CBP process with pretreated corn stover as sub­strate, at optimal conditions with low solid loading (0.5% w/w), C. phytofermentans hydrolyzed 76% of glucan and 88.6% of xylan in 10 days. These values reach 87% and 102% of those obtained by SSCF process using commercial enzymes and S. cerevisiae 424A with an ethanol titer of 2.8 g/l corresponding to 71.8% of that yielded by SSCF (3.9 g/l) (Jin et al., 2011a). Howev­er, using a similar process with high solid loading (4% w/w), the side product acetate became a major product (Jin et al., 2012).

Even though C. thermocellum seems a good candidate for ethanol fermentation from cellulosic biomass, there are a few disadvantages as listed in Table 5.1. Despite its ability to degrade lignocellulosic waste to both hex — ose and pentose sugars, it can only utilize hexose sugars from cellulose and not the pentose sugars derived from hemicellulose (Lynd et al., 2002; Taylor et al., 2009). This drawback could be solved by the use of mixed cultures for the degradation and fermentation of all sugars derived from lignocellulosic materials. For example, the anaerobic thermophile Thermoanaerobacterium saccha- rolyticum, which can ferment xylan and almost all soluble biomass sugars, would be a good candidate for coculture with C. thermocellum. A twofold reduction of the bioethanol production cost from lignocellulose could be achieved when using thermophilic anaerobic mixed cultures (Demain et al., 2005; Lynd et al., 2002). Since there is currently no perfect CBP microbe that can degrade lignocellulosic biomass efficiently and at the same time utilize all the sugars released from biomass to produce mostly ethanol, coculture or community/ mixed fermentation may be a suitable option (Barnard et al., 2010; Demain, 2009; Jin et al., 2011a). Chen reviewed 35 coculture systems for ethanol production by cofermentation of glucose and xylose and concluded that even though still in its infancy, this strategy is prom­ising as it can increase ethanol yield and productivity, shorten fermentation time, and reduce process costs (Chen, 2011).

FUTURE PERSPECTIVES

For a particular product made from lignocellulosic biomass fermentation, it will be difficult to predict which particular microorganism should be finally used in commercial production. For different processes, it is possible that different species may be required. For bioethanol production, S. cerevisiae has some advan­tages since it is already widely used in large-scale, first-generation bioethanol production with well- established processes and technology. An ideal biomass sugar fermentation process needs to reach high product yield by fermenting all biomass sugars including glucose, xylose, arabinose, mannose, and galactose with an optimal microorganism that is resistant to toxic materials/chemicals in biomass hydrolysates such as acids, phenolics, salts, and sugar oligomers. In addi­tion, the microorganism should be robust, resistant to contamination and environmental stresses, with mini­mal metabolic by-product production. To achieve these goals, metabolic engineering, or extensive physiolog­ical reprogramming of the producing organisms may provide solutions.