Given the refractory nature of native lignocelluloses, it is not surprising that chemical processing techniques using acids or alkalis and elevated temperatures have been essential for their use as industrial materials. Conventionally, the starting point has been feedstock material such as wood chips, sawdust, and chopped stalks and stems from herbaceous plants.19 Mechanical size reduction is unavoidable and, therefore, has both economic and energy costs unless fragmented waste or by-products (e. g., sawdust) is the starting material.20

Diverse techniques have been explored and described for the pretreatment of size-reduced biomass materials with the aim of producing substrates that can be more rapidly and efficiently hydrolyzed — by either chemical or biological (enzymic) means — to yield mixtures of fermentable sugars. Physical and thermochemical methods described in the literature are summarized in table 2.1. These approaches have in common the use of conditions and procedures to greatly increase the surface area to which aqueous reactants and/or enzymes have access, in particular, the percentage of the major cellulosic materials that are opened up to attack and thereby reduced to glucose and oligosaccharides on hydrolysis within feasible time limits in batch or continuous processes.

Milling has been little favored because the fibrous nature of lignocellulosic materials requires lengthy processing times and unacceptably high energy inputs; only compression milling has been taken to a testing scale beyond the laboratory. Nevertheless, several studies have concluded that milling can greatly increase the susceptibility to enzymic depolymerization of cellulose.21 Irradiation with gamma rays and electron beams was a research topic from the 1950s to the 1980s; fragmentation of polysaccharides and lignin was demonstrated to increase the rates of hydrolysis of

cellulose when subsequently treated with acids or enzymes, but contradictory results, differential responses when using different wood species, and high investment costs meant that no irradiation technique progressed to pilot-scale evaluation.

Both milling and irradiation give single product streams with only minor degradation of lignocellulosic polymers. Thermochemical methods, in particular those using steam explosion,[12] can result in extensive degradation of hemicelluloses.22 Potentially, therefore, a twin-product stream process can be devised by separating solid and liquid phases, the former containing the bulk of the cellulose and the l atter the pentose and hexose components of hemicelluloses, although these may be predominantly present in oligosaccharides.23 At temperatures close to 200°C, even short (10-minute) pretreatment times have major impacts on surface area and enzyme accessibility (figure 2.4). Lignin-carbohydrate bonds are disrupted, some of the l ignin is depolymerized, and much of the morphological coherence of the lignified plant cell wall is destroyed.22 In addition, aqueous extraction at elevated temperatures removes much of the inorganic salts — this is of particular importance with feedstocks such as wheat straw whose combustion (or combustion with coal, etc.) is impeded by their high salt content and the consequent corrosion problems.2425

Chemical pretreatment methods have usually implied hydrolytic techniques using acids and alkalis, although oxidizing agents have also been considered (table 2.2). The use of such chemical reactants introduces a much higher degree of polysaccharide breakdown and greater opportunities for separately utilizing the various potential substrates in lignocellulosic materials. In fact, chemical fractionation procedures for plant cell walls have often been described and have been of inestimable value in the separation and structural elucidation of the cell wall polymers in plant cells and plant organs.26 With wheat straw, for example, sequential treatments (figure 2.5) with

FIGURE 2.4 Efficacy of steaming pretreatments with birch wood. (Data from Puls et al.23)

an aqueous methanol, sodium chlorite, and alkali yield distinct extractives, lignin, cellulose, and hemicellulose fractions.27 Similarly, sequential treatments with alkali (lignin removal) and dilute acid (hemicellulose hydrolysis) to leave a highly enriched cellulosic residue have been devised for a variety of feedstocks including switchgrass, corn cob, and aspen woodchip.28 Because the usual intention is to utilize the sugars present in the polysaccharides as fermentation substrates, however, the developments for bioindustrial applications have invariably focused on faster, simpler, and more advanced engineering options, including some that have been progressed to the pilot- plant scale. Pretreatments involving acids (including SO2 steam explosion) primarily solubilize the hemicellulose component of the feedstock; the use of organic solvents and alkalis tends, on the other hand, to cosolubilize lignin and hemicelluloses. As with thermochemical methods, the product streams can be separated into liquid and solid (cellulosic) phases; if no separation is included in the process, inevitably, a complex mixture of hexoses and pentoses will be carried forward to the fermentation step.

Combinations of physical, thermochemical, and chemical pretreatments have often been advocated to maximize cellulose digestibility by subsequent chemical or

enzymic treatments; this usually involves higher capital and processing costs, and the potential economic benefits of increased substrate accessibility have seldom been assessed in detail. Table 2.3 presents a historical sequence of pilot plants in North America, Japan, New Zealand, Europe, and Scandinavia developed for the processing of lignocellulosic feedstocks to illustrate different approaches to pretreatment choices and cellulose processing; not all of these initiatives included fermentation steps producing ethanol, but every one of them was the result of intentions to generate sugar solutions suitable for subsequent fermentative treatments. Different biomass feedstocks may require different technologies for optimized upstream processing; for example, ammonia-based pretreatments (ammonia fiber explosion and ammonia — recycled percolation) are more effective with agricultural residues (including corn stover and corn straw) than with woody materials.28 Hardwoods yield higher degrees of saccharification after steam explosion than do softwoods.22 An organic base, я-butylamine, has been recommended for pretreatment of rice straw on the evidence of efficient delignification, highly enhanced cellulose hydrolysis by cellulase, the ease of recovery of the amine, and the almost complete reprecipitation of the solubi­lized lignin when the butylamine is removed by distillation.2930

Many accounts of pretreatment optimizations have been published; over a decade ago, a report commissioned by the Energy Research and Development Corpora­tion of Australia estimated that “several thousand” articles dealt with physical and chemical pretreatment methods for lignocellulosics (including by-products from the paper and pulping industries).31 There are several reasons for this sustained research effort, including the large number of lignocellulosics of potential industrial use, the multiplicity of pretreatment methods (tables 2.1 and 2.2) singly and in combinations, uncoordinated funding from national and international agencies, and the various scales, from the laboratory bench up to demonstration units with the capacity of processing two tonnes/hr of feedstocks (table 2.3). A multiauthor review in 2005 of four thermochemical methods, two pretreatments with acid, two with ammonia, and one with lime (calcium hydroxide) as an alkali — all described as “promising tech­nologies” — concluded that although all nine approaches gave positive outcomes on increasing accessible surface area and solubilizing hemicelluloses and although all but one altered lignin structure, only five could reduce the lignin content, and only two (the ammonia-based methods) decrystallized cellulose.32 Exceptions and caveats were, however, noted; for example, ammonia fiber/freeze explosion worked well with herbaceous plants and agricultural residues and moderately well with hard­woods, but poorly with softwoods.20

Detailed comparisons of different pretreatment methods in controlled, side-by­side studies of multiple technologies using single feedstocks are very rare. A collabo­ration between the National Renewable Energy Laboratory and six universities in the United States compared ammonia explosion, aqueous ammonia recycle, controlled pH, dilute acid, flow-through with compressed hot water and lime approaches to prepare corn stover for subsequent biological conversion to sugars; material balances and energy balances were estimated for the processes, and the digestibilities of the solids were assessed by a standardized cellulase procedure.33-39 With this feedstock (already a major “waste” product resulting from the corn ethanol industry), all six pretreatment options resulted in high yields of glucose from cellulose by subsequent treatment with cellulase; in addition, the use of high-pH methods offered potential

Pilot Plants Developed for the Saccharification of Lignocellulosic Biomass

b Direct microbial conversion by cellulase-secreting ethanol producer

for reducing cellulase amounts required in cellulose hydrolysis.40 Differences were, however, observed in the kinetics of sugar release that were sufficient to influence the choice of process, enzymes, and fermentative organisms. This conclusion was foreshadowed by Swedish research on steam pretreatment of fast-growing willow (Salix) with or without SO2 impregnation that showed that, while glucose yields of more than 90% and overall xylose yields of more than 80% could be obtained both with and without SO2, the most favorable pretreatment conditions for the separate yields of glucose and xylose were closest when using SO2-impregnated wood chips.41 To a large extent, therefore, all pretreatment strategies are likely to include a partial compromise because of the very different susceptibilities to hydrolytic breakdown and solubilization of cellulose and hemicelluloses; highly efficient industrial solutions will require biotechnological approaches to provide fermenting organisms capable of using both hexoses and pentoses and both monomeric and oligomeric (and possibly polymeric) carbohydrates (this is discussed in detail in chapter 3). Even for a single choice of pretreatment method, variation in the biological material (the feedstock) will inevitably occur in, for example, the water content that will either necessitate a flexible technology or extra cost outlay to standardize and micromanage the inflow of biomass material.42

The most recent development in pretreatment technologies has been the dem­onstration that microcrystalline cellulose can be readily solubilized and recovered using a class of chemicals called “ionic liquids.” These are salts that are liquids at room temperature and stable up to 300°C; their extreme nonvolatility would also have minimal environmental impact.43 With one such ionic liquid (an я-butyl-methy- limidazolium chloride), cellulose could be solubilized by comparatively short (<3-hr) treatments at 300°C, the cellulose could then be recovered by the addition of “anti­solvents” such as water, methanol, and ethanol, and the resulting cellulose was 50­fold more susceptible to enzyme-catalyzed hydrolysis as compared with untreated cellulose.42 Chinese researchers have shown that ionic liquids can successfully pre­treat materials such as wheat straw; full commercialization still requires economic synthesis routes and toxicological assessments.44,45