**Table 3.**

*Hexose, pentose and lignin contents in different types of biomass.*

There is a strong incentive to develop a process to perform simultaneous saccharification and fermentation (SSF) as it reduces investment costs by reducing the number of vessels and has the potential to become the preferred approach. In SSF, the problem of end-product feedback inhibition is largely eliminated because glucose molecules are fermented immediately by the fermentative microbes as it is produced from hydrolysis of cellulose [85]. However, the benefits come with a major downside which is an inherent mismatch between the optimal temperatures for the enzymes (fungal cellulases and hemicellulose's) on the one hand, and yeast biocatalysts on the other. The temperature optima for saccharifying enzymes (50–55°C for cellulase) are higher than those for fermenting mesophilic culture.

The optimal temperature for yeasts is below 35°C. Mesophilic yeasts (that thrive best in a moderate temperature) exhibit slower growth rates at higher temperatures. Currently, SSF must run at temperatures between the optimum temperature for cellulase and the optimum temperature for fermentative organisms. The compromise results in higher cellulase loading and an increase in enzyme costs. Efficient bioethanol production by SSF requires the use of thermotolerant ethanologenic yeast. It is a hot topic for research to genetically modify microorganisms with the ability to ferment at higher temperatures [43]. Some isolated yeasts, including *Pichia*, *Candida*, *Saccharomyces* and *Wickerhamomyces*, are found to grow at temperatures of 40°C and ferment sugars at higher temperatures [41]. To make SSF process highly efficient in ethanol production, the pentose metabolic pathway is been engineered into microorganisms to enables the use of C5 sugars by microbes that do not ferment them earlier [86].

Reduction in enzyme cost is been sought by searching for new organisms with cellulolytic and hemicellulytic activities [87], lowering the enzyme dosage through protein engineering [86, 88], and improving cellulase thermostability for performing hydrolysis at elevated temperatures to increase the efficiency of cellulose hydrolysis [89]. Cellulase enzyme cost reductions are challenging as cellulase costs need to be significantly lower than those of amylase enzymes on a unit-of-protein basis. The high price of the enzymes encouraged research into solutions to the problem of glucose inhibition and to the deactivation caused by lignin by-products [90].

Further integration of enzyme production with SSF leads to a new technology of consolidated bioprocessing (CBP). One area of research is aimed at engineering all three capabilities (saccharification, hexose fermentation and pentose fermentation) into a single strain for the CBP process [91, 92]. Cellulase-encoding genes may be introduced into specific species during recombination [63] to eliminate the need for exogenous cellulases in the process of SSF and decrease the capital costs of processing. CBP technology promises to eliminate costs associated with enzyme production and additional infrastructure/vessels [93].

#### **9.4 Other opportunities for cost reduction**

Working with a high dry matter (DM) concentration is also potentially an effective way to reduce the hydrolytic enzyme costs. However, high DM content causes an increase in viscosity, inadequate mass and heat transfer within the bioreactor, and, consequently, a strong reduction in the conversion of cellulose/hemicellulose to fermentable sugars. This problem could be overcome by adopting various fedbatch strategies or coprocessing substrates with different degrees of porosity [94].

A variation of SSF, simultaneous saccharification and co-fermentation (SSCF), in which a starch material is co-fermented, has been adopted to address low ethanol concentration issue in lignocellulosic ethanol production. SSCF can reduce ethanol production cost by increasing ethanol concentration and thus reducing distillation cost [95].

Recycling yeasts and enzymes is also an effective way to reduce the cost of ethanol production. The remaining unhydrolyzed solids with some enzymes adsorbed are collected by filtration or centrifuge and are recycled to the next cycle for further hydrolysis. In one study, the enzyme loading was reduced from 36 to 22.3 and 25.8 mg protein per gram glucan, respectively, for separate hydrolysis and fermentation (SHF) and for SSCF on AFEX™ pretreated corn stover [96]. Enzyme adsorption to the residual solids is probably inhibited at high sugar concentrations in the fast SHF process [97] and hence affected enzyme recycling. The fast SSCF process removed most of the sugars by fermentation but produced ethanol whose effect on enzyme adsorption is unclear.
