2.5.1 A Multiplicity of Hemicellulases

Mirroring the variety of polysaccharides containing pentoses, hexoses, or both (with or without sugar hydroxyl group modifications) collectively described as hemicelluloses (figure 1.23), hemicellulolytic organisms are known across many species and genera:15 [16]

Table 2.8 summarizes major classes of hemicellulases, their general sites of action, and the released products. Microorganisms capable of degrading hemicelluloses have, however, multiple genes encoding many individual hemicellulases; for exam­ple, Bacillus subtilis has in its completely sequenced genome at least 16 separate genes for enzymes involved in hemicellulose degradation.127

TABLE 2.8

Major Hemicellulases, Their Enzymic Sites of Action and Their Products

Hemicellulase EC number Site(s) of action Released products

Hemicelluloses comprise both linear and branched heteropolysaccharides. Endoxylanases fragment the xylan backbone, and xylosidases cleave the resulting xylan oligosaccharides into xylose; removal of the side chains is catalyzed by glucuron­idases, arabinofuranosidases, and acetylesterases — the action of these enzymes can limit the overall rate of hemicellulose saccharification because endoacting enzymes cannot bind to and cleave xylan polymers close to sites of chain attachment.128

Much of the fine detail of hemicellulase catalytic action is beginning to emerge and will be vital for directed molecular evolution of improved hemicellulase biocatalysts.129,130 Already, however, a thermostable arabinofuranosidase has been identified and shown to have a unique selectivity in being able to degrade both branched and debranched arabinans.131 Synergistic interactions among different microbial arabinofuranosidases have also been demonstrated to result in a more extensive degradation of wheat arabinoxylan than found with individual enzymes.132 The activity of biotech companies in patenting novel hemicellulase activities is evi­dent (table 2.9) in exploring hemicellulases from unconventional microbial sources, and deep-sea thermophilic bacteria from the Pacific have been shown to synthesize thermotolerant xylanases, to be active over a wide pH range, and to degrade cereal hemicelluloses.133

BIOFUEL: PRESENT STATUS AND FUTURE PROSPECTS

The developments of sugar-derived ethanol as a major transportation fuel in Brazil and corn-derived ethanol as a niche (but rapidly growing) market in the United States were conditioned by different mixes of economic and environmental imperatives from the early 1970s to the present day. Together and separately, they have been criticized as unsuitable for sustainable alternatives to gasoline in the absence of tax incentives; highly integrated production processes, with maximum use of coproducts and high degrees of energy efficiency, are mandated to achieve full economic competitiveness. Although technically proven as a technology, the scope of ethanol production from food crops (primarily sugar and corn) is limited by agricultural and geographical factors, and only cellulosic sources offer the quantita­tive availability to significantly substitute for gasoline on a national or global basis. Even for that goal, however, bioethanol is only one player in a diverse repertoire of strategic and tactical ploys and mechanisms — from carbon capture and storage to hydrogen-utilizing fuel cells — with which to change the oil economy and reduce dependence on fossil fuels.103

Both corn — and sugarcane-derived ethanol have — unarguably but not irre­versibly — emerged as industrial realities.104 Even detailed questions about their NEBs and the implications of their use in mitigating greenhouse gas emissions have become unavoidable on Web site discussion forums. A major article in the October issue of National Geographic magazine accepted the marginal energy gain in corn ethanol production and the disappointingly small reduction of total CO2 emissions in the total production/use cycle but noted the much clearer benefits of sugarcane ethanol.105 Two quotes encapsulate the ambiguous reactions that are increasingly evident as the public debate over biofuels spreads away from scientific interest groups to embrace environmentalist and other “lobbies”:

It’s easy to lose faith in biofuels if corn ethanol is all you know, and

If alcohol is a “clean” fuel, the process of making it is very dirty… especially the

burning of cane and the exploitation of cane workers.

All forms of ethanol produced from plant substrates are, however, best viewed as “first-generation” biofuels, operating within parameters determined by (in varying allocated orders): the internal combustion engine, mass personal transport, and the global oil industry.105 Together with biodiesel (i. e., chemically esterified forms of the fatty acids present in vegetable oils — see chapter 6, section 6.1), they have proven potential for extending the availability of gasoline blends and are affordable within the budgets accepted by consumers in the late twentieth and early twenty — first centuries while helping to reduce greenhouse gas emissions and (with some technologies, at least) other atmospheric pollutants. It is, however, likely that different countries and supranational economic groupings (e. g., the European Union) will value differently energy security, economic price, and ecological factors in the face of fluctuating oil prices and uncertain mid — to long-term availabilities of fossil fuels.106 Fermentation products other than ethanol (e. g., glycerol, butanol) and their thermochemical conversion to synthesis gas mixtures to power electricity generation are second-generation technologies, whereas biohydrogen, fuel cells, and microbial fuel cells represent more radical options with longer lead times.107108

Sugar — and corn-derived ethanol production processes were simply extrapolated from preexisting technologies, and to varying extents, they reflect the limitations of “add-on” manufacturing strategies.109110 As one example of the National Geographic writer’s anticipated “breakthrough or two,” lignocellulosic ethanol only emerged as a commercial biofuel reality in 2004, and biotechnology is crucial to its establishment as a major industry.105111 For that reason, and because of the uniquely global potential of this nonfood sector of the bioethanol supply chain, the microbial biotechnology of cellulosic ethanol will now be considered at length, to provide not only a snapshot of commercially relevant contemporary science but also to extrapolate existing trends in the development of the scientific base.

The importance of including hemicellulosic sugars in the conversion of lignocellulosic feedstocks to ethanol to ensure process efficiency and an economic base for biofuel production has often been emphasized.134135 Given that hemicellulosic sugars constitute a fermentable resource equal to approximately 50% of the glucose residues present in the cellulose in most plant species (table 1.5), this is an unsurprising conclusion.

Thermochemical and acid-catalyzed pretreatments of lignocellulosic biomass materials extensively degrade hemicelluloses (see above, section 2.3.2); depending

on the pretreatment method and the feedstock. However, hemicellulose solubilization may be as high as 100% or as low as 10%, and the hemicellulose sugars may be pres­ent primarily as monomers (xylose, arabinose, etc.) or as oligomers.60 A detailed investigation of destarched corn fiber as a starting material, initially extracted with hot water, showed that 75% of the total hemicellulose could be easily extracted; if enzyme preparations generated by growing Hypocrea jecorina and Aspergillus niger (containing xylanase, P-xylosidase, and feruloyl esterase as well as cellulase) were then used, the recovery of total xylan as xylose increased greatly (from <15% to >50%), and the arabinose yield also improved, although from a much higher starting point of more than 50%; total recoveries of hemicellulosic sugars such as arabinose and xylose reached 80% when supplementations with commercial glucoamylase, P-glucosidase, and feruloyl esterase were included.136 Similarly, a xylose yield of 88% of the theoretical — as the free monosaccharide xylose — was achieved from pretreated corn stover by supplementing cellulase with xylanase, pectinase, and P-glucosidase activities.122 Commercial cellulase preparations contain variable but often high activities of hemicellulases; this may have adventitiously contributed to the production of hemicellulose sugars in lignocellulosic materials processed for ethanol production.137,138

2.1 BIOMASS AS AN ENERGY SOURCE:

TRADITIONAL AND MODERN VIEWS

Biomass energy in its traditional sense is vegetation (mostly woody plants but also sun-dried grasses) and, extrapolating further up the food chain, animal manure, combusted as a direct source of heat for cooking and heating. For commercial purposes, wood was also the major energy substrate before the rapid development of coal extraction in the late eighteenth and nineteenth centuries ushered in the Industrial Revolution. Even in the early twenty-first century, traditional biomass still accounts for 7% of the total global energy demand, amounting to 765 million tonnes of oil equivalents (Mtoe) in 2002, and this is projected to increase to 907 Mtoe by

2030.1 Especially in Sub-Saharan Africa, much of this primary energy demand is unsustainable, as population growth outstrips the biological capacity of increasingly drought — and crisis-damaged ecosystems to replace continuous harvestings of firewood.

Biomass has, however, a much more modern face in the form of substrates for power generation, especially in combined heat and power production in OECD regions. For example, biomass-based electricity was 14% in Finland and 3% in Austria in 2002, and as discussed in chapter 1, the burning of sugarcane bagasse in Brazil is a significant energy source, 3% of national electricity in 2002.1 The use of biomass for power generation is increasingly attractive as a decentralized mechanism of supplying electricity locally or for isolated communities as well as cofiring with coal to reduce CO2 emissions. In contrast, even taken together, hydroelectricity, solar, geothermal, wind, tide, and wave energy may account for 4% or less of total global energy demand by 2030 (figure 2.1).

Thermal conversion (combustion, burning) of lignocellulosic biomass is an ancient but inefficient means of liberating the energy content of the biological material. Compared with solid and liquid fossil fuels, traditionally used biomass has only 0.33-0.50 of their energy densities (e. g., as expressed by the higher heat value).2 The higher the water content, the lower the energy density becomes and the more difficult is the task of extracting the total calorific equivalent, as the gas — phase flames are relatively cool. In a well-oxygenated process, the final products of biomass combustion are CO2, water, and ash (i. e., the inorganic components and salts); intermediate reactions, however, proceed by a complex group of compounds including carbon monoxide (CO), molecular hydrogen, and a wide range of

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Biomass utilization has generally been regarded as a low-technology solution for renewable energy and slow to generate public enthusiasm or investor funding for projects in most OECD countries. Nevertheless, energy analysis calculations have shown that combustion, pyrolytic, and gasification technologies are competitive in their energy conversion efficiencies with biotechnological production of fuel ethanol or biogas, that is, methane (figure 2.2).4 Such biomass-utilizing facilities are highly suitable for inputs that can be broadly described as “waste”; these materials include sawmill residues and forestry, herbaceous agriculture, and construction/ demolition waste that might otherwise be simply burned off or dumped in landfill sites. The appropriateness of thermochemical energy production is well illustrated by the commercialization of the SilvaGas process, originally demonstrated in a 10-ton/day plant at the Battelle Memorial Institute (Columbus, Ohio) and subsequently at a 200-ton/day plant in Vermont. The company Biomass Gas & Electric, LLC (Atlanta, Georgia, www. biggreenenergy. com) announced an agreement in October 2006 to construct a 35-MW plant capable of supplying electricity to 8% of residential properties in Tallahassee, Florida.5 By 2010, this plant will convert 750 tons/day of timber, yard trimmings, and clean construction debris to a medium-energy producer gas by a patented advanced gasification process. The product can substitute for natural gas in most industrial applications, and the process can, therefore, either generate electricity in situ in gas turbines or be injected into the natural gas distribution network. A second (20-MW) facility is under construction northeast

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FIGURE 2.2 Bioconversion efficiencies of thermochemical and microbiological processes for biomass. (Data from Lewis.4)

of Atlanta, adjacent to a landfill site that will divert construction and woody plant material — compared with untreated material, the SilvaGas process reduces solid waste by up to 99%.

To underline the contemporary nature of biomass fuels, U. S. Patent 7,241,321 awarded to Ecoem, LLC (Greenwich, Connecticut) in July 2007 describes an innovative procedure for the production of biomass fuel briquettes: finely ground wood chips, bark, sawdust, or wood charcoal powder can, after drying, be mixed with a vegetable oil to produce a material capable of being pressed into briquettes or ingots. This “organically clean biomass fuel” provides a clean burning, nontoxic fuel, presumably a superior fuel for domestic heating (as well as for igniting the charcoal on barbecues) and offers an alternative route for using vegetable oils as biofuels (chapter 6, section 6.1).

“Biomass” extends well beyond the collection of firewood. Between the late 1960s and the mid-1980s, a program in the United States explored seaweeds as rivals to terrestrial biomass plants because seaweeds have high growth rates, probably more than 100 dry tons/hectare/year, that is, comparable with the highest rates determined for sugarcane and such invasive/problem plant species as water hyacinth.6 The ocean margins are filled with such highly productive species, including the following:

• The giant kelp (Macrocystis pyrifera), whose massive fronds can be iepeatedly harvested and, when managed, a “plantation” might require reseeding only every five to ten years, if at all

• Other kelps, including the oriental kelp (Laminaria japonica), already harvested in tens of thousand of tons annually as food in China and Japan (both of which countries actively have considered this species as a possible biomass source)

• Other brown algae, including Sargassum spp., some of which harbor nitrogen-fixing bacteria and require little artificial fertilizer application

• Red algae such as Gracilaria tikvahiae, once considered as a possible summer crop in Long Island Sound and capable of growth without any attachment in closed or semiclosed cultures

• Green algae, many of which have very high biomass production rates

A feature common to many of these species is that anaerobic digestion with methane­forming bacteria is particularly efficient at converting their biomass to a combustible, widely used fuel gas.6

Research in China and Japan has continued until the present day, exploring the potential for the development of “marine bioreactors.”7 Genetic transformation techniques have also been adapted from the established body of knowledge acquired with terrestrial plants for indoor cultivation systems with such transgenic kelp.