Parisutham Vinuselvi1 and Sung Kuk Lee12 1School of Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 2School of Urban and Environmental Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan Republic of Korea

1. Introduction

Lignocellulose, starch, sucrose, and macroalgal biomass are different forms of plant biomass that have been exploited for bioethanol production. Among them, lignocellulose, found in both agricultural and forest waste, has attracted great attention because of its relative abundance in nature (Lynd et al. 2002). Lignocellulose is a complex polymer made up of cellulose, hemicellulose, and lignin. Efficient conversion of lignocellulose into bioethanol involves a series of steps, namely, the collection of biomass; pretreatment to dissolve lignin; size reduction to reduce the number of recalcitrant hydrogen bonds; enzymatic saccharification to yield simple sugars; and, finally, fermentation of the sugars to ethanol. The main hurdle in this process is the lack of low-cost technology to overcome the recalcitrance associated with lignocellulose (Lynd et al. 2002; Himmel et al. 2007; Xu et al. 2009). Pretreatment is needed to dissolve the lignin, and enzymes such as xylanases are needed to hydrolyze the hemicellulosic fraction that otherwise would prevent cellulases from accessing the cellulose (Wen et al. 2009) (Fig 1A). The half-life of crystalline cellulose at neutral pH is estimated to be one hundred million years (Wilson 2008). A cocktail of saccharification enzymes—with endoglucanases, exoglucanases and ^-glucosidases forming the major portion—is needed to disrupt the chemical stability of cellulose. The physical stability of lignocellulose, rendered by hydrogen bonds formed between adjacent cellulose polymers, is still a major obstacle to the efficient hydrolysis of cellulose. An additional challenge in cellulose hydrolysis is the relatively poor kinetics exhibited by cellulases (Himmel et al. 2007). Cellulases have lower specific activities than do other hydrolytic enzymes, because their substrate (cellulose) is insoluble, crystalline, and heterogeneous (Fig 1B) (Zhang and Lynd 2004; Wilson 2008). Activity of each of the cellulases in complex enzyme cocktails is inhibited by intermediates —such as cello-oligosaccharides and cellobiose, produced during cellulose hydrolysis —leading to discontinuity in the process. For example, exoglucanase action yields cellobiose, which inhibits endoglucanase (Fig 1C) (Lee et al. 2010).

Despite these hurdles, several species of Clostridium, Trichoderma, and Aspergillus can efficiently degrade cellulose. Exploitation of the innate potential of the microbial world might be an economical alternative to overcome the recalcitrance associated with lignocellulose (Alper and Stephanopoulos 2009). Two major strategies have been employed to hydrolyze lignocellulose by using microbial consortia. In the first strategy, native cellulolytic organisms like Clostridium spp. are engineered to produce bioethanol. In another approach, cellulolytic ability is imposed on efficient ethanol producers such as Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis (Xu et al. 2009). This chapter focuses mainly on the cellulolytic systems that have been engineered into recombinant microorganisms.