Edward A. Bayer, Raphael Lamed, Bryan A. White, Shi-You Ding, and Michael E. Himmel

Abstract

Some forms of agricultural residues represent an attractive resource for lignocellulosic biomass in our quest to reduce the dependence of the Western World on fossil fuels. After crops have been harvested, the residues usually represent relatively large amounts of cellu — losic material that could be returned to the soil for its future enrichment in carbon and nutri­ents. However, today, many believe that a substantial portion of these residues could be made available for further conversion to biofuels. Likewise, animal wastes, particularly from her­bivores and notably from ruminants, are high in cellulose content and can also be converted to liquid biofuels. Although such agricultural byproducts cannot compensate completely for the large volumes of liquid fuels required to sustain our transportation energy requirements, they can play a decisive local and regional role to fill these needs.

However, in this case, nature and mankind have different agendas. The challenge regarding cellulosic biomass is that cellulose plays a critical structural role in the terrestrial plant cell wall. Glucose, the most desirable plant sugar for fermentation, is incorporated within the cellulose microfibrils that make up the complex plant cell wall. The most successful future bioconversion processes for the production of biofuels from lignocellulose may indeed ulti­mately mimic the concerted action of the cellulolytic microbes, the bacteria, and fungi that have evolved to produce cellulases and cellulosomes. It is now very clear that the major bottleneck in this process—both from a biochemical and economical point of view—is the deconstruction of the plant cell wall, liberating both C6 and C5 sugars. Nature has evolved microbes and their enzymes to deal primarily with damaged and decaying vegetation. Ultimately, much of this plant matter is again converted to a form that can be incorporated into living plant tissue. Nature thus has the time needed to manage the plant biosphere with low-energy consuming processes that can be less than ideal. We, on the other hand, must deploy rapid, efficient, and most importantly, cost-effective conversion processes that will meet our future energy needs.

The present chapter deals with the current status of our knowledge regarding the function of cellulases and cellulosomes, and how we might use them in processes for biomass

Biofuels from Agricultural Wastes and Byproducts Edited by Hans P. Blaschek, Thaddeus C. Ezeji and Ju rgen Scheffran 67 © 2010 Blackwell Publishing. ISBN: 978-0-813-80252-7

conversion to biofuels. This includes a description of various types of cellulosic biomass in agricultural wastes and the pretreatment strategies required to enhance enzymatic attack and to avoid toxic byproducts that would interfere with enzyme action and fermentation. The effects of treatment with free cellulases versus treatment with cellulosomes are also detailed. The natural cellulases and cellulosomes, their various families, modular, and subunit archi­tectures, are all documented. The search for novel enzymes, and strategies for mutation and modification of cellulases and cellulosomes for future application to bioenergy initiatives are considered as well. We address some of the bottlenecks and pitfalls that await us in our current and future efforts to provide efficient processes for conversion of cellulosic biomass to usable sugars for biofuel production.

Introduction

Lignocellulosic biomass has long been recognized as a potential low-cost renewable source of mixed sugars for fermentation to fuel ethanol (Lynd et al. 1991; Wheals et al. 1999; Lynd et al. 2002; Dien et al. 2003; Demain et al. 2005 ; Ragauskas et al. 2006; Schubert 2006; Himmel et al. 2007; Wall et al. 2008). One approach would be to degrade plant cell wall cellulose and hemicellulose to soluble sugars using severe chemistries, prior to conversion to ethanol, but economic and environmental issues preclude such strategies. The more accepted alternative is to employ microbial cellulases and related enzymes to cell walls that have been conditioned thermally and chemically in a milder process, known as “pretreatment.”

Several technologies have been developed over the past century that allow this conversion process to occur (Figure 5.1) , yet the clear objective now is to make this process cost- competitive in today’s markets (Bayer et al. 2007). Perhaps the major bottleneck for

Agricultural

Residues

Figure 5.1. Major steps in conversion of plant cell wall biomass to biofuels. Following growth and harvesting of crops, the agricultural wastes are collected and transferred to a central processing facility. The pretreated plant cell wall material is used to grow cellulolytic fungal or bacterial cell cultures to produce large amounts of free sugars. Cellulolytic enzymes are also produced from the cells and are used to hydrolyze the pretreated biomass directly. Ethanogenic microbes (e. g., yeast or appropriate bacterium) are grown on the resultant sugars (glucose and other simple sugars), which results in the production of ethanol (or other fuels). The enzymatic breakdown of cellulose is the major bottleneck in the design of a cost-effective process.

conversion of biomass to ethanol is the high cost and low efficiency of the enzymes, the cellulases, and other glycoside hydrolases, which are capable of degrading crystalline cel­lulose and related plant cell wall polysaccharides. Efficient hydrolysis is impeded by limited accessibility of the enzymes and the recalcitrance of cellulose, owing to its extremely stable “microcrystalline” arrangement of the cellulose chains in the cell wall microfibrils.

The rate — l imiting step in the hydrolysis of cellulose is not the catalytic cleavage of the P-1,4 bond, but the disruption of a single chain of the substrate from its native crystalline matrix, thereby rendering it accessible to the active site of the enzyme. Single cellulolytic enzymes alone are generally incapable of efficient cellulose hydrolysis. The mode of action of the various cellulases is different, and they are known now to act synergistically. Consequently, the secret to potent enzymatic degradation of the recalcitrant substrate is embedded in the knowledge of how these different types of enzymes work together.

This chapter describes the status of cellulases and cellulosomes en route to the efficient degradation of cellulosic biomass for the production of biofuels. We discuss the nature of cellulosic biomass in agricultural residues, various pretreatment strategies, and their effects on the microorganisms. We also discuss cellulolytic microorganisms, the various enzyme systems for biomass deconstruction, and future approaches for agricultural biomass decon­struction, while focusing on the production of soluble sugars. In doing so, we deem other topics as beyond the scope of the present chapter, notably microbial fermentation of soluble sugars to biofuels (Jeffries 2006; Hahn-Hagerdal et al. 2007a), metabolic engineering of bacteria, fungi, or yeast (Zhang et al. 1995; Hahn-Hagerdal et al. 2007b), and consolidated bioprocessing of cellulosic biomass directly to biofuels (Lynd et al. 2005; van Zyl et al. 2007; Lynd et al. 2008).