The focus of this review has been twofold. First, to present a summary of the economic and socio-political landscape that has fueled the resurgence in bioethanol as a biofuel, and consequently, the general adoption of industrial biotechnology as a cost-effective, sustainable, and preferred alternative to traditional petrochemical processing. Second, to offer the hypothesis that sig­nificant scientific achievements in metabolic engineering and systems biology have been applied to bioethanol and other chemical products for successful commercialization, suggesting a graduation of the field to industrial systems biology. If we revert back to Fig. 1, we have focused most of our attention on process economics, with some indirect discussion of environmental im­pact and sustainability/self-sufficiency. Within the discussion of industrial systems biology we have focused only on the upstream bioprocessing steps schematically shown in Fig. 2, namely, systems biology used for enhance­ment of metabolic engineering. An area that we have not discussed, but is addressed in the chapter co-authored by Warren E. Mabee in this volume, and suggested in Fig. 1, is public perception and policy.

In the July 2006 issue of Nature Biotechnology there were a series of edito­rials and commentaries written exploring bioethanol as a biofuel [146-150]. Amongst these articles was a discussion of the various public perception is­sues facing bioethanol, ranging from statements of support, such as, “At least three major factors — rapidly increasing atmospheric CO2 levels, dwindling fossil fuel reserves and their rising costs — suggest that we now need to ac­celerate research plans to make greater use of plant-based biomass for energy production and as a chemical feedstock as part of a sustainable energy econ­omy” [149], to highly critical statements, such as, “At present, ethanol is not price competitive by any stretch of the imagination — even with the absurd and decidedly anti-free-market 54-cent per gallon tariff Washington imposes upon Brazilian ethanol” [147]. Both in the scientific peer-review and main­stream literature, there is still debate as to whether bioethanol for biofuel makes sense. This debate has prompted the development of new methods for analysis of whether bioethanol is economically feasible, and more impor­tantly, sustainable over the long-term. A general classification often used to evaluate process feasibility is life-cycle analysis.

Life-cycle energy analysis is a methodology used to answer the bottom­line question: is more energy contained in the fuel than is used in the production of that fuel? Life-cycle energy analysis, much like the tools em­ployed in functional genomics, is a systems approach to evaluate all aspects of the production process from feedstock processing, availability, and trans­portation, to opportunities for recycling of energy and mass pre-, during, and post-production [151-153]. Life-cycle energy analysis, unlike earlier ap­proaches, has suggested that process integration, energy recycling, and care­ful selection of raw materials and unit operations can yield bioethanol pro­cesses that are energetically favorable [151-153]. Consequently, biorefineries are viewed as a natural extension of bioethanol production facilities given the opportunities for integration, recycling, and production of higher value chemicals.

Holistic approaches that take a systems level approach must be refined, im­proved, and presented to policy makers and key stakeholders to finally put an end to the question — does bioethanol make sense? The question should be revised to ask under which conditions does bioethanol makes sense, and what is required to commercialize those conditions. Bioethanol, biorefineries, and industrial biotechnology will not be successful and expand to novel areas if we do not focus on engineering public perception and policy. Bioethanol has become a commercial vehicle for industrial systems biology applied to in­dustrial biotechnology. Now, it needs to become the commercial vehicle for reaching public, government, and corporate support, and again, it will take systems level data to get there.

Acknowledgements J. M. Otero is a Merck Doctoral Fellow and acknowledges financial support from the Division of Bioprocess Research & Development, Merck Research Labs, Merck & Co., Inc.

G. Panagiotou, PhD, acknowledges financial support from the Villum Kann Rasmussen foundation.

There are numerous organisms that rely on biomass degradation for their survival, often existing in the natural environment as a complex consortia of fungi, bacteria, and protozoa, working synergistically to decay the plant cell wall. All of these organisms are potential sources of enzyme discovery, but current commercial products for biomass treatment are derived from fungi because these organisms produce a complex mix of enzymes at high produc­tivity and catalytic efficiency, both of which are required for low-cost enzyme supply. Unlike most bacteria, which express complexes of many carbohydrate­degrading activities arrayed on molecular scaffolds physically attached to the bacterial cell wall, fungal cellulases are typically secreted into the growth medium, allowing cost-efficient separation of the active enzymes in a liquid form suitable for delivery to a hydrolysis reactor.

4.1

From the investigations presented above it has been concluded that the max­imum yields of mannose, the main hemicellulose sugar in softwood, and of glucose are not obtained at the same degree of severity. The optimal yield of mannose is obtained at a lower severity than that required for maximum di­gestibility of the cellulose in the subsequent enzymatic hydrolysis step. This suggests two-stage steam pretreatment, in which the first stage is performed at low severity to hydrolyse the hemicellulose, and a second stage, at a higher degree of severity, in which the solid material from the first step is pretreated again [82].

Although there are several studies on two-stage acid hydrolysis of soft­wood, the number of studies on two-stage steam pretreatment is scarcer. Soderstrom et al. [85-87] performed a thorough investigation on the two — stage pretreatment of spruce using either SO2 impregnation or H2SO4 im­pregnation in both steps, as well as H2SO4 in the first stage and SO2 in the second. The highest sugar yields were achieved for two-step pretreat­ment with either SO2 impregnation or H2SO4 impregnation in both steps (see Fig. 2). A wide range of pretreatment conditions resulted in similar sugar yields of about 50 g per 100 g raw material.

The highest sugar yield was 51.7 g per 100 g, corresponding to 80% of the theoretical, obtained for pretreatment conditions of 190 °C for 2 min and 210 °C for 5 min. This yield (in %) is slightly lower than that reported by Nguyen et al. [ 14]. However, the amount of sugar obtained expressed as grams per 100 g dry raw material is higher. Ngyuen et al. stated that they obtained a sugar yield of 82%, which, in their case, corresponds to 46 g/100 g dry raw

material. They used a cellulase activity of 60 FPU/g cellulose, which is more than twice the amount that was used in the study shown in Fig. 2. The max­imum overall sugar yield obtained with two-step pretreatment using H2SO4 in both stages was only slightly lower, 77% of theoretical.

Besides overall sugar yield it is also of importance to investigate the fer — mentability of the pretreated materials. Impregnation with dilute H2SO4 fol­lowed by pretreatment at a high combined severity (i. e. high temperature and/or long residence time) resulted in materials that were not fermentable. Impregnation with SO2, however, was successful in creating fermentable ma­terials for all investigated pretreatment severities.

The two-step pretreatment results in a higher ethanol yield than does the one-step pretreatment, and it has also the advantage of lower requirement of enzymes and water in the SSF step. Major drawbacks are, however, the higher capital cost and the higher energy consumption. In a study by Wingren et al. [90] the overall ethanol production cost was shown to be very much de­pendent on the way the two pretreatment steps are performed, especially if the pressure is released or not between the steps, and also on the dry matter (WIS) content in the second step. The lowest cost estimated for the two-stage process, 3.90 SEK/L, which was about 6% lower than that for the one-stage process, requires a high ethanol yield, high concentration of WIS in the fil­ter cake between the steps, and that the sugars being fed to the second step will not become degraded. The higher yield has been demonstrated experi­mentally, but the two other assumptions still need to be verified on the pilot scale.

5

Conclusions

In conclusion, a large number of pretreatment methods have been investi­gated and developed during the last 10 years, resulting in high recovery of sugars and rather high overall ethanol yields. However, most of the results were obtained in studies using batch-operating equipment on a rather small scale. Enzymatic hydrolysis has also, in most cases, been assessed at low sub­strate concentration.

One problem with the data produced so far is the difficulty in comparing methods, as the assessment is performed in different ways. In most cases the pretreatment is not assessed under realistic process conditions. The whole process must be considered as the various pretreatment methods give dif­ferent types of materials: hemicellulose sugars can be obtained either in the liquid as monomer or oligomer sugars, or in the solid material to various ex­tents; lignin can be either in the liquid or remain in the solid; the composition and amount/concentration of possible inhibitory compounds also vary. This will affect how the enzymatic hydrolysis should be performed (e. g. with or without hemicellulases), how the lignin is recovered and also the use of the lignin co-product.

For agricultural residues a large number of pretreatment methods result in high sugar yields while for wood, and especially softwood, the number of feasible methods is smaller. Acid hydrolysis and steam pretreatment with acid catalyst seem to be the methods that can be used for all types of raw ma­terials, but the drawback is the high equipment cost and the formation of inhibitors. This requires further improvement and also a better integration with the enzymatic hydrolysis development, as improved enzyme mixtures may lead to less severe pretreatment conditions and thereby lower cost and reduce formation of inhibitory compounds.

To verify the technology the next step is to implement all of these improve­ments in a pilot-scale process with all steps integrated into a continuous pilot plant. This will provide better data for assessment and for scale-up to a demo­or full-scale process. It will also give better information on how various pre­treatment conditions will affect all the other process steps, i. e. enzymatic hydrolysis, fermentation, downstream processing and wastewater treatment, as well as product and co-product quality.

Today, industrial biotechnologists are no longer discussing whether a single product can be produced via biotechnology, but are rather considering di­verse portfolios of products leveraging expertise and resources. The various systems biology toolboxes applied to bioethanol production are now being exploited to develop integrated processes that will form “biorefineries”. The concept of the biorefinery was first defined in 1999, when it was postulated that lignocellulosic raw materials may be converted to numerous biocom­modities via integrated unit processes, and offer competitive performance to traditional petrochemical refineries [27]. Several chapters in this volume will more closely examine the biorefinery as a model and platform for future bio-based processes in terms of policy issues and process integration.

If the biorefinery platform model is to evolve from academic conception to industrial reality it will require two key components. First, the economic and socio-political landscape must support and warrant the significant financial investment, favorable legislative policy, and consumer-driven demand that will be required. Second, the advances and tools developed within systems biology for metabolic engineering must be successfully applied in commer­cial environments. Bioethanol is the first industrial biotechnology product to demonstrate that if these two elements are co-supported, then numer­ous bio-based processes can be developed and integrated into a biomass economy.

2

The assignment of specific substrate factors that render a substrate recalci­trant to cellulase hydrolysis is a controversial subject. Crystallinity, DP and specific surface area have all been thought to undergo significant changes during pretreatment, consequently influencing subsequent hydrolysis [2]. The original work prior to the 1990s, focusing on the physical character­ization of substrates, has been presented in previous reviews [2,7]. More recently, our group and others have published work correlating the physical properties of wood pulp fibers employed in papermaking to their hydrolyz — ability, as cellulases have been explored as a potential means to improve both the drainage of recycled pulps and to enhance pulp fiber properties [101]. Admittedly, pulps are not identical to substrates pretreated specifically for subsequent hydrolysis by cellulases. However, it should be noted that, sim­ilarly to pretreated substrates, pulp fibers also represent a lignocellulosic matrix and that the general principles gained from examining the physical properties of pulp fibers that affect hydrolysis can be cautiously extrapolated to substrates pretreated for bioconversion.

5.1

Mats Galbe • Guido Zacchi (И)

Dept. of Chemical Engineering, Lund University, P. O. Box 124, 221 00 Lund, Sweden Guido. Zacchi@chemeng. lth. se

1 Introduction……………………………………………………………………………………………….. 42

2 Assessment of Pretreatment…………………………………………………………………………. 44

3 Pretreatment Methods…………………………………………………………………………………. 47

3.1 Physical Methods………………………………………………………………………………………… 48

3.2 Chemical Methods………………………………………………………………………………………. 48

3.3 Physicochemical Methods…………………………………………………………………………….. 49

3.4 Biological Methods………………………………………………………………………………………. 51

4 Results from Pretreatment Studies………………………………………………………………. 51

4.1 Corn Stover…………………………………………………………………………………………………. 52

4.2 Softwood Species………………………………………………………………………………………… 57

4.3 Two-Stage Pretreatment………………………………………………………………………………. 60

5 Conclusions………………………………………………………………………………………………… 62

References……………………………………………………………………………………………………. 62

Abstract Second-generation bioethanol produced from various lignocellulosic materials, such as wood, agricultural or forest residues, has the potential to be a valuable substitute for, or a complement to, gasoline. One of the crucial steps in the ethanol production is the hydrolysis of the hemicellulose and cellulose to monomer sugars. The most promising method for hydrolysis of cellulose to glucose is by use of enzymes, i. e. cellulases. However, in order to make the raw material accessible to the enzymes some kind of pretreatment is necessary. During the last few years a large number of pretreatment methods have been developed, comprising methods working at low pH, i. e. acid based, medium pH (without addition of catalysts), and high pH, i. e. with a base as catalyst. Many methods have been shown to result in high sugar yields, above 90% of theoretical for agricultural residues, especially for corn stover. For more recalcitrant materials, e. g. softwood, acid hydrolysis and steam pretreatment with acid catalyst seem to be the methods that can be used to obtain high sugar and ethanol yields. However, for more accurate comparison of differ­ent pretreatment methods it is necessary to improve the assessment methods under real process conditions. The whole process must be considered when a performance evalua­tion is to be made, as the various pretreatment methods give different types of materials. (Hemicellulose sugars can be obtained either in the liquid as monomer or oligomer sug­ars, or in the solid material to various extents; lignin can be either in the liquid or remain in the solid part; the composition and amount/concentration of possible inhibitory com­pounds also vary.) This will affect how the enzymatic hydrolysis should be performed

(e. g. with or without hemicellulases), how the lignin is recovered and also the use of the lignin co-product.

Keywords Assessment • Enzymatic hydrolysis • Lignocellulose • Pretreatment • Review

1

Introduction

Replacement of gasoline by liquid fuels produced from renewable sources is a high-priority goal in many countries worldwide. It is driven by the aims of a secure and sustainable energy supply and a desire to diminish the green­house effect. The transportation sector in the European Union (EU) is totally dependent on imported fossil fuels, and thus extremely vulnerable to market disturbances. It is also the sector responsible for the main part of the increase in CO2 emissions. The use of biofuels in the EU is encouraged by a Directive that set a target of 2% substitution of gasoline and diesel with biofuels in 2005 on an energy basis, which should have increased to 5.75% by 2010 [1].

Bioethanol is projected to be one of the dominating renewable biofuels in the transportation sector within the coming 20 years, and has already been introduced on a large scale in Brazil, the USA and some European countries. The advantages of bioethanol are that it can be produced from a variety of raw materials, it is non-toxic and is easily introduced into the existing in­frastructure, either as a low blend with gasoline (e. g. E5 and E10) or used in flexi-fuel vehicles at a high concentration (e. g. E85) or as a neat fuel in dedi­cated engines. However, almost all bioethanol today is produced from sugar — or starch-based agricultural crops, using so-called first-generation technolo­gies. Although this ethanol is produced at a competitive cost, the raw material supply will not be sufficient to meet the increasing demand for fuel ethanol, and also the reduction of greenhouse gases resulting from the use of sugar — or starch-based ethanol is not as high as desirable.

One of the most promising options to meet this challenge is the production of bioethanol from lignocellulose feedstocks, such as agricultural residues (e. g. wheat straw, sugar cane bagasse, corn stover) and forest residues (e. g. sawdust, thinning rests), as well as from dedicated crops (salix, switch grass) using second-generation technologies. These raw materials are sufficiently abundant and generate very low net greenhouse gas emissions, reducing en­vironmental impacts.

However, to compete with gasoline the production cost must be substan­tially lowered. Today, raw material and enzyme production are two of the main contributors to the overall production cost [2,3]. Efficient use of the whole crop is required, i. e. high overall yield of ethanol produced by hydro­lysis and fermentation of the carbohydrate fraction (hemicellulose and cel­lulose), as well as a high yield of the main co-product (lignin). However,
producing monomer sugars from cellulose and hemicellulose at high yields is far more difficult than deriving sugars from sugar — or starch-containing crops, e. g. sugar cane or corn.

Ethanol production from lignocellulosic materials comprises the following main steps: hydrolysis of hemicellulose; hydrolysis of cellulose; fermenta­tion; separation of lignin residue; recovery and concentration of ethanol; and wastewater handling. Figure 1 shows a simplified process flowsheet for ethanol production from lignocellulosic materials based on enzymatic hydro­lysis. Some of the most important factors to reduce the production cost are: an efficient utilization of the raw material by having high ethanol yields, high productivity, high ethanol concentration in the distillation feed, and also by employing process integration in order to reduce capital cost and energy de­mand. Part of the lignin can be burnt to provide heat and electricity for the process, and the surplus is sold as a co-product for heat and power appli­cations, which will increase the energy efficiency of the whole system. It is thus necessary to minimize the internal energy demand and to maximize the production of the solid fuel. The two conversion steps can be considered key processes: hydrolysis of the hemicellulose and cellulose to sugars, and fer­mentation of all the sugars.

The enzymatic process is regarded as the most attractive way to degrade cellulose to glucose [4-6]. However, enzyme-catalysed conversion of cellulose to glucose is very slow unless the biomass has been subjected to some form of pretreatment, as native cellulose is well protected by a matrix of hemicel­lulose and lignin. Pretreatment of the raw material is perhaps the single most crucial step as it has a large impact on all the other steps in the process, e. g. enzymatic hydrolysis, fermentation, downstream processing and wastewater handling, in terms of digestibility of the cellulose, fermentation toxicity, stir­ring power requirements, energy demand in the downstream processes and wastewater treatment demands.

An effective pretreatment should have a number of features. It has to:

• Result in high recovery of all carbohydrates.

• Result in high digestibility of the cellulose in the subsequent enzymatic hydrolysis.

• Produce no or very limited amounts of sugar and lignin degradation prod­ucts [7]. The pretreatment liquid should be possible to ferment without detoxification.

• Result in high solids concentration as well as high concentrations of liber­ated sugars in the liquid fraction.

• Have a low energy demand or be performed in a way so that the energy can be reused in other process steps as secondary heat.

• Have a low capital and operational cost.

Additional positive features would be if hemicellulose sugars were obtained in the liquid as monomer sugars, as this would help to avoid the use of hemi — cellulases, and/or if the lignin—without being oxidized—was separated from the cellulose, as this would alleviate the unproductive binding of cellulases on lignin in the enzymatic hydrolysis step.

This chapter will focus on pretreatment of lignocellulosic raw materials. Some of the most common methods that have been investigated will be pre­sented and to some extent compared for various raw materials.

2

The most widespread, commercial enzyme products currently available for biomass hydrolysis are produced by submerged fermentation of the sapro­phytic mesophilic fungus T. reesei [30]. This organism, first isolated over 60 years ago from decaying cotton tents during World War II [31] is a prolific producer of secreted cellulases. Since its initial isolation, numerous mutants have been isolated that increase the productivity of the strain by over 20­fold [28,32,33]. Three enzymes classes form the core of the T. reesei cellulase system: exoglucanases comprised of two primary cellobiohydrolases, a num­ber of endoglucanases, and в-glucosidases (Fig. 5). There are two types of

cellobiohydrolases, CBH I and CBH II, that constitute roughly 60% and 20% of the secreted protein mix and are critical to the efficient hydrolysis of cel­lulose [34]. The CBH I and II hydrolyze the cellulose chain processively from the reducing and non-reducing ends of cellulose chains, respectively, releas­ing the glucose disaccharide cellobiose. Endoglucanases (EG I-IV) constitute roughly 15% of the secreted protein and hydrolyze в-1,4 linkages within the cellulose chains, creating new reducing and non-reducing ends that can then be attacked by the CBHs. в-Glucosidases (BGL I and II), constituting roughly 0.5% of the secreted protein mix, and hydrolyze cellobiose and some other short-chain cellodextrins into glucose.

4.2

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

Bioethanol was introduced into the transportation fuel supply chain as early as the 1970s with the introduction of the PROALCOOL program by the Brazil­ian government in an original effort to stabilize the international price of sugarcane, which was highly sensitive to subsidies by other domestic produc­ers. In 1979, the Brazilian government strengthened the program by spon­soring development of a fleet of ethanol-fueled vehicles [42]. Although the history of bioethanol in Brazil is quite tumultuous with significant govern­ment sponsorship, tax incentives, and subsidies, Brazil has emerged as the second largest producer of bioethanol (4.3 billion gallons/year in 2005) re­quiring 25% ethanol blends in transportation fuel, and has become energy self-sufficient by supplementing internal petroleum supplies and refining cap­acity with bioethanol production [43]. In 2005, total Brazilian petroleum production was estimated at 2 million bbl/day with consumption estimated at 1.6 million bbl/day, in contrast to the USA which produced 7.6 billion bbl/day, yet consumed 20 billion bbl/day [35].

Bioethanol may serve both as an additive or complete replacement for petroleum-derived transportation fuels, particularly gasoline in spark igni­tion (SI) engines. The volumetric energy fraction of ethanol is approximately 66% that of gasoline, suggesting a one-third reduction in the total kilometers per volume of ethanol consumed. However, review of the comparative phys­ical chemistry data provides insight into why ethanol combustion results in a 15% higher efficiency [44]. Ethanol (C2H5OH, 34.7 wt % oxygen) is a par­tially oxidized fuel compared to gasoline (C4-C12, 0 wt % oxygen), resulting in a lower stoichiometric air-to-fuel ratio. Therefore, a larger mass or volume of ethanol compared to gasoline is required to yield the same caloric output from combustion. However, ethanol also has a higher octane number, per­mitting the fuel to be burned at a higher compression ratio (defined as the ratio of the volume between the piston and cylinder head before and after a compression stroke). A higher compression ratio results in higher power output, efficiency, and consequently favorable fuel economy [45]. Compared to gasoline, there is only a 20-25% reduction in kilometer efficiency [44]. Fur­thermore, as a result of the significantly higher latent heat of vaporization for ethanol (1177 kJ/kg compared to 348 kJ/kg at 60 °C) there is an effective engine cooling and leaner operation. This leads to significant reductions in CO(g) and NOx,(g) emissions, with 85% ethanol blends of gasoline (referred to as E-85) yielding NOx,(g) emission reductions of 20% compared to pure gasoline. However, the emission of reactive aldehydes, including acetalde­hyde and formaldehyde, is increased [46,47]. Several studies on the effect of ethanol-gasoline blends (up to 60% ethanol) on engine performance and exhaust emissions have suggested that proper fine tuning of engine parame­ters can lead to excellent performance with significantly reduced hydrocarbon and CO(g) emissions [46-48].

In 1990 the USA enacted the Clear Air Act Amendments, mandating that oxygenated additives (methyl-tertiary-butyl ether, MTBE; ethyl-tertiary-butyl ester, ETBE; or ethanol) be included at 2 wt % oxygen to decrease hazardous emissions. In 1999, 21 million tons of MTBE were produced globally, primar­ily in the USA, although Europe produced approximately 3.3 million tons.

In the USA, it is among the most frequently found groundwater contami­nants with over 400 000 underground storage tanks identified to be leaking by the US Environmental Protection Agency (EPA) since 1988 [49]. Although there is still debate in the public health community as to the toxicity level and health risk that MTBE human consumption poses, a number of US states have banned the use of MTBE as a fuel additive. Furthermore, many European nations, including the UK, Germany, and Switzerland have identified MTBE — contaminated sites and are transitioning to ethanol enrichment [50,51]. As a result, ethanol has been the favored fuel additive for increasing oxygenation.

In August 2005, the Energy Policy Act (EPACT) was enacted into US law creating the national Renewable Fuels Standard (RFS). The RFS calls for

15.1 billion L of renewable fuels (primarily ethanol but may include alterna­tive fuels such as biodiesel) to be used by 2006, increasing by 2.6 billion L/year until 2011 when a final volume of 28.4 billion L will be called for in 2012 [43].

The USA is neither alone nor first with actively passing legislation that requires and promotes the integration of biofuels into the transportation economy. As previously discussed, Brazil presently requires a 25% ethanol blend of all gasoline, and continues to provide preferential tax treatment for ethanol producers and consumers. Argentina is requiring a 5% ethanol blend over the next 5 years. Thailand requires that all gasoline sold in Bangkok must be composed of 10% ethanol. India is requiring 5% ethanol gasoline blends. Canada has provided tax benefits for ethanol producers and consumers since 1992 [43].

The European Union (EU) has also taken an aggressive stance in reshap­ing its transportation fuel and energy supply chain, in addition to promoting industrial biotechnology as a sustainable and cost-effective alternative to petrochemical processes. In December 2005, the EU adopted the Biomass and Biofuels Action Plan. This plan encompasses more then 20 specific ac­tion items, including creation of the Biofuels Technology Platform with the explicit purpose of advancing research into the use of forestry, agricultural, and woody crops for energy purposes. In February 2006, the EU adopted the Strategy for Biofuels, which set out three objectives: (1) to promote biofuels in both the EU and developing countries, (2) to prepare for large-scale use of biofuels by improving their cost-competitiveness and increasing research into second-generation fuels, and (3) to support developing countries where biofuel production could stimulate sustainable economic growth [52].

Furthermore, the EU has established quantitative targets for incorporation of biofuels into a broader and emerging bio-based economy. The EU trans­port sector accounts for more than 30% of the total energy consumption, with a 98% dependency on fossil fuels. There is also significant pressure for the EU to comply with the Kyoto Protocol, an agreement under the United Nations Framework Convention on Climate Change, ratified by 160 countries to sig­nificantly reduce greenhouse gas emissions, specifically CO2,(g). The EU has failed to meet the Kyoto targets with 90% of the increases in CO2,(g) emissions between 1990 and 2010 attributable to transportation fuel usage. Therefore, significant reform in transportation fuel usage is required. There are three specific legislative actions in place [53]:

• Promotion of renewable energy-based electricity generation from 14% in 1997 to 21% by 2010 for the EU 25 (22.1% for EU 15) (Directive 2001/77/EC)

• Promotion of biofuels for transport applications by replacing diesel and petrol to the level of 5.75% by 2010 (Directive 2003/30 EC) accompanied by detaxation of biofuels

• Promotion of cogeneration of heat and electricity (Directive 2004/8/EC)

It is clear, however, that the EU is not meeting the objectives set forth. Spe­cifically, the current production of liquid biofuels in the EU is 2 million tons of oil equivalent (Mtoe), less than 1% of the market. The EU policy target for 2010 was 18 Mtoe in the transportation sector alone. Although it is un­likely the target will be met, it should be noted that between 4 and 13% of the total agricultural land in the EU would be required to meet the above target. Therefore, through the creation of the various plans and platforms highlighted before, the EU has established, “An ambitious and realistic vi­sion for 2030 is that up to one-fourth of the EU’s transport fuel needs could be meet by clean and CO2-efficient biofuels” [53]. Although it remains to be seen whether the appropriate resources will be allocated to meet this goal, it is certainly clear that industrial biotechnology, in particular the concept of a bio-based economy with biorefineries at its core, has taken center stage in the EU meeting its energy needs and environmental targets.

2.3

As has been demonstrated previously with pretreated substrates [30], the rates and extents of hydrolysis of pulp fibers have also been directly corre­lated to their initial specific surface area. This is not surprising, since the very existence of a substrate pretreatment step stems from the necessity to increase the accessibility of reaction sites on substrates to cellulases, as lig — nocellulosic substrates such as wood possess limited reactive surface area available to cellulases prior to pretreatment. Coincidentally, both the chem­ical and mechanical pulping processes that have been applied to produce pulp for the formation of paper also result in an increase in accessibility to cellulases compared to the starting lignocellulosic furnish. During pulping, wood chips are subjected to either physical or physiochemical action, re­sulting in the breakdown of the lignocellulosic matrix into fiber cells [48]. Consequently, the breakdown of the wood yields fibers with various phys­ical attributes such as length, coarseness, width, kink and curl [102,103]. Surface area of pulp fibers can be divided into exterior surface area affected mainly by fiber length and width, or interior surface area, which is gov­erned by the size of the lumen and the number of fiber pores and cracks. The varying fiber lengths and widths produced during pulping can be viewed in a similar manner as the array of particle sizes produced during the pre­treatment oflignocellulosic substrates for bioconversion. The specific surface area of a mixture of particles is inversely proportional to their average diam­eter, therefore, a smaller average particle size results in an increase in surface area. Indeed, cellulases have been shown to act on the surface of pulp fibers, resulting in a “peeling effect” [104]. Therefore, smaller particle sizes with a greater amount of specific surface area would be expected to hydrolyze at a faster rate.

In an investigation assessing the hydrolysis of Douglas-fir Kraft and me­chanical pulps, Mooney et al. showed that at equal lignin contents, the “fines” (small particles) of a delignified mechanical pulp were hydrolyzed faster than the longer fibers (large particles) of the Kraft pulp [17]. When each fiber length fraction was hydrolyzed separately, it was shown that the isolated long fiber fraction hydrolyzed slower and consequently adsorbed a lower amount of cellulases than the whole pulp [17]. The increased hydrolysis rate of the whole pulp was attributed to the greater amount of specific surface available for the adsorption of cellulases provided by the pulp fines and short fibers. Although it is apparent that particle size has a significant effect on cellulose hydrolyzability, it has also been shown that the fiber delamination and en­hanced swelling that results from mechanical treatment of Kraft and recycled pulp fibers has a greater effect on hydrolysis by cellulases than does a de­crease in particle size [105]. Since recycled pulps originate from fiber sources that undergo irreversible changes in their structure upon drying [106], their swelling properties must be regenerated by employing a mechanical treat­ment referred to as “refining” or “beating”. The swelling properties of pulps can be measured using the water retention value measurement [107]. After beating, the pulp sample usually drains at an inadequate rate to be used on a high-speed paper machine. Consequently, cellulases have been shown to improve the drainage of recycled pulps. Oksanen et al. [108] applied sepa­rate EG1, EG2 and CBH1 cellulase components to pulps during each recycling round. As each pulp was beaten after recycling, the water retention value (WRV) increased and the pulp became more responsive to cellulases, espe­cially EG1 and EG2. Although the particle size and swelling properties of pulps have been shown to be related to the ease of hydrolysis of lignocellulosic substrates, it has been shown that a greater amount of information related to the action of cellulases can be obtained from measurements of the pores or “interior” surface area of pulp fibers available for penetration by cellulases.

Direct correlations have been found between the initial pore volume or interior surface area of lignocellulosic substrates and their extent of hydro­lysis [30,83,90]. It has been proposed that the efficacy of cellulose hydrolysis is enhanced when the pores of the substrate are large enough to accommo­date both large and small enzyme components to maintain the synergistic action of the cellulase enzyme system [11]. From extensive studies, Grethlein et al. [83] and others [109-111] have found that the rate-limiting pore size for the hydrolysis of lignocellulosic substrates was 5.1 nm, thus the solute exclu­sion technique utilizing molecular probes in this size range has been shown to be effective for assessing the pore volume of substrates. Mooney et al. [68] utilized dextran molecular probes in the solute exclusion method to measure the pore volume of refiner mechanical pulp (RMP), sulfonated RMP, sodium chlorite delignified RMP and Kraft pulp from Douglas-fir to assess the ease of these pulps to subsequent hydrolysis by cellulases. As mentioned earlier, the delignification of the RMP resulted in a greater rate and extent of hydrolysis than the Kraft pulp sample, which may be attributed to the smaller particle size of the RMP. The sulfonation of the RMP dramatically increased swelling. Unlike delignification, however, this did not translate into either enhanced access to the 5.1 nm probe or hydrolysis performance. The most feasible ex­planation for these results is that the lignin content of the sulfonated pulp (30.9%) inhibited hydrolysis, regardless of the greater swelling of the pores, thus demonstrating the detrimental effect of substrate lignin on hydrolysis as mentioned earlier.

Since it is well known that the pore volumes of pulps undergo significant reductions upon drying [106], Esteghlalian et al. [112] innovatively applied the Simons’ stain technique to measure changes in pore volume imparted by air, oven and freeze drying prior to enzymatic hydrolysis. As expected, drying significantly reduced the number of larger pores in the pulp sample, which most likely restricted the access of the fibers to cellulases and thus de­creased hydrolysis rate over 12 h [112]. Although the specific surface area of the substrate provided by decreased particle size and increased swelling and pore volume plays a significant role in facilitating hydrolysis by cellulases, the interconnecting role of other substrate factors such as crystallinity and DP should also be considered.

5.2