R. P. Chandra1 • R. Bura2 • W. E. Mabee1 • A. Berlin1 • X. Pan3 •

J. N. Saddler1 (И)

faculty of Forestry, University of British Columbia, 2424 Main Mall,

Vancouver, British Columbia V6T 1Z4, Canada jack. saddler@ubc. ca

2College of Forest Resources, University of Washington, Box 352100,

Seattle, WA 98195-2100, USA

3Biological Systems Engineering, University of Wisconsin-Madison,

460 Henry Mall, Madison, WI 53706, USA

1 Background………………………………………………………………………………………………… 68

1.1 Summary of Pretreatment Processes…………………………………………………………….. 69

1.2 Steam Pretreatment of Biomass…………………………………………………………………… 71

2 Substrate Characteristics of Steam-Pretreated Wood…………………………………….. 73

3 Substrate Lignin………………………………………………………………………………………….. 75

3.1 The Effects of Pretreatment on Lignin Content……………………………………………….. 76

3.2 The Effects of Substrate Lignin on Enzymatic Hydrolysis……………………………….. 78

4 Substrate Hemicelluloses……………………………………………………………………………… 80

4.1 The Effect of Pretreatment on Hemicellulose Content……………………………………… 81

4.2 The Effect of Substrate Hemicellulose Content on Hydrolysis…………………………. 83

5 Physical Properties Affecting the Hydrolysis of Substrates by Cellulases. 84

5.1 Specific Surface Area…………………………………………………………………………………… 85

5.2 Cellulose Crystallinity and Degree of Polymerization…………………………………….. 87

6 Conclusions………………………………………………………………………………………………… 88

References……………………………………………………………………………………………………. 90

Abstract Although the structure and function of cellulase systems continue to be the subject of intense research, it is widely acknowledged that the rate and extent of the cellulolytic hydrolysis of lignocellulosic substrates is influenced not only by the effective­ness of the enzymes but also by the chemical, physical and morphological characteristics of the heterogeneous lignocellulosic substrates. Although strategies such as site-directed mutagenesis or directed evolution have been successfully employed to improve cellulase properties such as binding affinity, catalytic activity and thermostability, complemen­tary goals that we and other groups have studied have been the determination of which substrate characteristics are responsible for limiting hydrolysis and the development of pretreatment methods that maximize substrate accessibility to the cellulase complex. Over the last few years we have looked at the various lignocellulosic substrate characteristics

at the fiber, fibril and microfibril level that have been modified during pretreatment and subsequent hydrolysis. The initial characteristics of the woody biomass and the effect of subsequent pretreatment play a significant role on the development of substrate prop­erties, which in turn govern the efficacy of enzymatic hydrolysis. Focusing particularly on steam pretreatment, this review examines the influence that pretreatment conditions have on substrate characteristics such as lignin and hemicellulose content, crystallinity, degree of polymerization and specific surface, and the resulting implications for effective hydrolysis by cellulases.

Keywords Biomass • Cellulose • Cellulases • Hemicellulose • Hydrolysis • Lignin •

Steam pretreatment

1

Background

There have been several recent reviews [1-7] that have considered the various enzymatic factors that influence the efficiency of hydrolysis of lignocellu — losic substrates. However, it is apparent that the physical and chemical nature of lignocellulosic substrates imparted by different pretreatment procedures are just as complex and influential as the enzyme systems used to break­down the various components that comprise a lignocellulosic substrate into fermentable monosaccharides and other industrially relevant chemical com­pounds. Despite intensive research over the last 30 years or so, obtaining the rapid, complete and efficient conversion of cellulosic substrates by enzy­matic hydrolysis remains a challenging goal. Up until about 5 or 6 years ago, various technoeconomic models had indicated that the enzyme production step of the overall biomass-to-ethanol process was one of the most expensive. Recent efforts by some of the world’s leading industrial enzyme manufac­turers have resulted in an approximate 20- to 30-fold reduction in the cost of cellulases utilized for the hydrolysis of pretreated corn stover [8]. How­ever, it is acknowledged that the nature of the substrate and pretreatment method used continue to influence the effectiveness of the enzyme mix em­ployed [9]. The significant decreases in the cost of the enzyme hydrolysis step have highlighted how the cost and nature of the biomass feedstock and the pretreatment method used to enhance both overall product recovery and enzymatic hydrolysis of the cellulosic and hemicellulosic components are sig­nificant technical and economic considerations.

Typically, after an initial rapid phase, the hydrolysis rate decreases rapidly during the saccharification process, resulting in lower glucose yields and longer processing times and, in most cases, the accumulation of a recalcitrant residue due to incomplete hydrolysis of the substrate. When a typical progress curve for enzymatic hydrolysis of cellulose is plotted, the reaction rate usually remains constant during the first few hours. However, the reaction rate even­tually slows down and it has been suggested that the decrease in reaction rate can be attributed to both enzyme — and substrate-related factors [2-4,6]. Var­ious substrate-related factors that affect hydrolysis include: how the presence of extraneous materials such as lignin and hemicellulose impede the action of cellulases, the influence of cellulose crystallinity and degree of polymeriza­tion (DP), and the amount of accessible surface area available to react with cellulases [2]. Enzyme-related factors that have been studied include: shear or thermally induced deactivation [10] occurring during mixing or exposure to high temperatures, the separation of enzyme components by the physical characteristics of the substrate resulting in a loss of synergism [11], as well as product inhibition due to an accumulation of cellobiose and glucose in the re­action medium. It is known that both enzyme — and substrate-related factors influence the efficiency of enzymatic hydrolysis [2]. However, depending on the nature of the substrate and pretreatment used, one factor could be more influential than another.

As mentioned earlier, an effective pretreatment method should be cheap (both capital and operating costs), effective on a wide range of lignocellulosic materials, require minimum preparation/handling steps prior to pretreat­ment, ensure recovery of all of the lignocellulosic components in a useable form, and provide a cellulosic stream that can be efficiently hydrolyzed with low concentrations of enzymes. With regard to the latter requirement, it would be beneficial if the pretreatment process could degrade the cell-wall structure by reducing the cellulose crystallinity, DP and particle size, while removing hemicellulose and lignin and increasing pore volume such that the cellulosic and hemicellulosic surface area available to the enzymes is greatly increased. However, as will be discussed in more detail, no one currently available pretreatment process can provide all of these desired outcomes on all lignocellulosic materials and it is the nature of the compromised condi­tions that will be described in this review.

1.1