Biochemical conversion of pretreated lignocellulose into biofuels generally consists of four steps—glycosyl hydrolase production, enzymatic hydrolysis of cellulose and hemicellulose, hexose fermentation, and pentose fermentation (Lynd et al. 2002). Figure 5 shows the spectrum of processing platforms, which range from separately accomplishing each step to combining all steps, which when optimized is generally found to improve efficiency (Lynd et al. 2002). The more steps involved in the process, the more time required to complete a fermentation cycle and money used for capital equipment.

Separate enzyme hydrolysis and fermentation (SHF) completes each step as an independent unit. This process allows enzymatic hydrolysis and microorgnism-based fermentation to be carried out each at their own optimal conditions. However, a major problem accompanying separate hydrolysis is the inhibitory effects on cellulase activity caused by accumulated products, like glucose and cellobiose (Philippidis et al. 1993; Gruno et al. 2004). For instance, glucose at 3 g per L reduces p-glucosidase activity by 75% (Philippidis et al. 1993). To release the inhibitory effect, microorgnisms can be added to immediately ferment these products and maintain them at a low concentration.

This combination of enzymatic hydrolysis and fermentation to release enzymatic product inhibition generates another platform, simultaneous saccharification and fermentation (SSF) (Olofsson et al. 2008). This process has been successfully applied to convert lignocellulose to enthanol with higher yield, lower enzyme doses and less capital equipments than SHF (Olsson et al. 2006; Saha et al. 2011; Zhu et al. 2012). In a comparison with ethanol production by Saccharomyces cerevisiae from unpretreated cassava pulp under a variety of feeding regimes, SSF yielded ~50% more ethanol than the similar SHF process (Zhu et al. 2012). On the other hand, other reports showed that the accumulation of enthanol reversibly inhibits cellulase activity (Podkaminer et al. 2012). To maximize the fermentation efficiency of SSF, the key is to select hydrolases and fermenting microorgnisms with similar optimum temperature and pH. However, most microorgnisms need a lower optimum temperature than hydrolases, which makes
saccharification a limiting factor to SSF. One method that researchers trying to overcome this difficulty have designed is nonisothermal simultaneous saccharification and fermentation (NSSF), in which saccharification and fermentation occur simultaneously but in two separate bioreactors at different temperatures, coupled with recirculation of fermentation broth between these two bioreactors (Wu et al. 1998; Oh et al. 2000). So far, this system presents several advantages compared with SSF, including higher ethanol yields, shorter residence time and lower enzyme inputs.

For processing hemicellulose-rich biomass, the aforementioned bioconversion processes all require an additional, separate pentose fermentation step with different microorgnisms in another bioreactor. Simultaneous saccharification and cofermentation (SSCF) aims to improve on SSF by fermenting both pentoses and hexoses in a single bioreactor, with a selected or engineered microorganism or community (Lynd et al. 2002). Recently SSCF has been applied to ferment commercial furfural, corn kernels, and pretreated wheat straw (McMillan et al. 1999; Zhu et al. 2007; Olofsson et al. 2010; Tang et al. 2011). As with other platforms, process details appear to have a large impact. In one recent example, Olofsson and associates were able to increase the xylose conversion from 40% to 50% with both enzyme and substrate continuous feeding compared to only substrate feeding (Olofsson et al. 2010). (Feeding refers to the addition of materials to the reactor.)

Cellulase purchase is a common and significant cost in all of the preceeding processes (Lynd et al. 2008). The newest concept in bioconversion processes is referred to as consolidated bioprocessing (CBP), which employs a microbial consortium for hydrolase production, saccharification and fermentation in a single step in a single bioreactor (Lynd et al. 2002). CBP offers the potential to lower production costs due to simpler conversion processing, lower energy and capital inputs and higher conversion efficiencies than other processes. However, the key challenge of CBP is that there are not yet ideal CBP-enbaled microorganism capable of efficient cellulose hydrolysis and biofuel synthesis. Lynd and colleagues have promoted two alternative strategies to enable CBP as follows: (i) engineering naturally occurring cellulolytic microorganisms to improve biofuel-related properties such as yield and titer, and (ii) engineering non-cellulolytic organisms that exhibit high biofuel yields and titers to express heterologous lignocelluolytic enzyme, enabling cellulose utilization (Lynd et al. 2005). For strategy (i), Clostridium species with native lignocellulose-degrading abilities have been metabolically engineered to synthesize a variety of biofuels, such as hydrogen, isopropanol, butanol and ethanol (Higashide et al. 2011; Lutke — Eversloh et al. 2011; Lee et al. 2012). So far for strategy (ii), E. coli and yeast have been engineered to directly convert cellulose and xylan to ethanol and biodiesel (Steen et al. 2010; Bokinsky et al. 2011; Goyal et al. 2011; Fan et al. 2012). To further promote biomass conversion efficiency, researchers have promoted the idea of co-cultures, in which non-biofuel products generated by one microorgnism could be further converted to biofuels by another organism, or in which the metabolism of one microorgnism could be boosted by the presence of another one. For instance, the co-culture of hyper-cellulase producer, Acremonium cellulolyticus C-1, and an ethanol producer, S. cerevisiae, realized one-pot bioethanol production (Park et al.

2012) . In another example, a co-culture of Clostridium strains produced H2 at higher rates and with similar yields compared to the pure strains (Masset et al. 2012). Nonetheless, numerous challenges need to be met prior to industrial application of CBP. Among the challenges are low fuel yields, and fuel inhibition of microbial growth.