Sucrose functions in plants as a highly water-soluble and readily transported product of carbon fixation in leaves, although it can also accumulate in storage organs (e. g., sugarbeets). Starch is mainly a storage polymer in, for example, cereal grains. Cellulose, on the other hand, is essentially a structural polymer in plants (figure 1.23), highly insoluble, organized into crystalline macroscopic fibers, mixed with other polysaccharides (e. g., hemicelluloses), and protected from enzymic attack in native woods by the physical presence of lignin (figure 1.24). Lignins are polyphenolic polymers generated by enzyme-catalyzed free radi­cal reactions from phenylpropanoid alcohols. Unlike nucleic acids and proteins, they have no informational content but are neither inert to enzyme-catalyzed degradation nor incapable of being converted (by hydrogenolysis or oxidative breakdown) to useful chemical intermediates, for example, in the manufacture of synthetic resins, perfume, dyes, and pharmaceuticals.48 As wood chemicals,

Patent Applications and Patents Awarded for Corn Ethanol Technologies

TABLE 1.4 Date of filing or award Title Applicant/assignee Patent/application January 21, 2003 Process for producing ethanol ZeaChem, Inc., Golden, CO US 6509180 October 1, 2004 Improved process for the preparation of ethanol from cereals Etea S. r.l., Savigliano, Italy EP 1 536 016 A! March 9, 2004 Method for producing fermentation-based products from high oil corn Renesson LLC, Bannockburn, IL US 6703227 May 25, 2004 Fermentation-based products from corn and method Renesson LLC, Bannockburn, IL US 6740508 October 20, 2005 Methods and systems for producing ethanol using raw starch and fractionation Bruin and Associates, Inc., Sioux Falls, SD US 2005/0233030 A1 October 27, 2005 Continuous process for producing ethanol using raw starch Bruin and Associates, Inc., Sioux Falls, SD US 2005/0239181 A1 February 23, 2006 Removal of fiber from grain products including distillers dried grains with solubles R. Srinivasan and V. Singh US 2006/0040024 A1 May 2, 2006 Heterologous expression of an Aspergillus kawachi acid — stable alpha amylase… Genencor International, Palo Alto, CA US 7037704 July 11, 2006 Process for producing ethanol from corn dry milling ZeaChem, Inc., Golden, CO US 7074603 August 1, 2006 Method for producing fermentation-based products from corn Renesson LLC, Deerfield, IL US 7083954 September 5, 2006 Alcohol production using sonication UltraForce Technology LLC, Ames, IA US 7101691 November 16, 2006 Hybrid enzymes Novozymes A/S, Bagsvaerd, Denmark US 2006/0257984 A1

extracted celluloses are, however, far more widely known, especially in the paper industry and also (as acetylated, nitrated, and other derivatives) find applications as varied as components of explosives, cigarette filters, cosmetics, and medical products such as gauze and bandages. Among tree species, hardwoods and soft­woods differ in their compositions, hardwoods having (perhaps, paradoxically) less lignin (figure 1.25). Detailed data for more than 120 tree species lists cel­lulose contents as high as 57% (and as low as 38%), with lignin in the 17 to 37% range (by weight).48 This plasticity of biomass chemical composition suggests that plant breeding programs and genetic technologies can accelerate the evolution of “bioenergy” plants as novel cultivars.

Share of Total Ethanol Capacity (%) FIGURE 1.22 Contributors to U. S. fuel ethanol production. (Data from the Renewable Fuel Association.)

TABLE 1.5 Industrial Sites for Bioethanol Production from Cereals and Sugar in Europe and Asia Capacity Manufacturer Location Substrate (million liters per year) Agroethanol AB Norrkoping, Wheat grain 50 ( planned expansion Sweden to 200 in 2008) Biowanze SA Wanze, Belgium Wheat grain, sugar beet 300 (by 2008) Suedzucker Bioethanol Zeitz, Germany Sugar beet 270 GmbH AB Energy France Lacq, France Corn 150 (by 2008) Abengoa Bioenergia 3 sites in Spain Wheat and barley grain 550 HSB Agro Industries Ltd. Ringus, Rajasthan, Rice and sorghum grain 11 India Jilin Fuel Ethanol Co. Ltd. Jilin, China Corn 900 Harbin Winery Harbin, China Corn 4

Hemicelluloses are a diverse group of polysaccharides, with different plant species elaborating structures with two or three types of sugar (sometimes further modified by O-methylation or O-acetylation) and a sugar acid; the major sugar monomers are the pentoses xylose and arabinose and the hexoses glucose, galac­tose, and mannose (figure 1.23). Most plant species contain xylans (1,4-linked poly­mers of xylose); in addition, hardwood and softwood trees contain copolymers of glucose and mannose (glucomannans) — larchwoods are unusual in having a core polymer of galactose. Table 1.6 summarizes compositional data based on sugar

FIGURE 1.23 Chemical structures of cellulose and sugar components of hemicelluloses.

type, taken from analyses presented by the National Renewable Energy Laboratory (Golden, Colorado) for a range of tree species, paper products for recycling, cere­als, and grasses.49

In principle, a bioprocess for producing ethanol from a lignocellulosic substrate could be modeled on those developed for cornstarch (figures 1.20 and 1.21). If only cellulosic glucose is considered as a substrate, the essential stages are

• Milling/grinding of the plant material to reduce particle size

• Chemical and/or physical pretreatment of the plant material to increase the exposure of the cellulose to enzyme (cellulase) attack

• Separation of soluble sugars and oligosaccharides

• Addition of either cellulase or a microorganism capable of secreting active cellulase and utilizing the released sugars for ethanol production by fermentation (simultaneous saccharification and fermentation) or direct microbial conversion

If the hemicellulosic sugars are also to be utilized, then either hemicellulases need to be added or a mixture of organisms used in cofermentations or sequential fermenta­tions. As more of the total potential substrate is included in the fermentation step, the biology inevitably becomes more complex — more so if variable feedstocks are to be used during the year or growing season — as the total available carbohydrate input to the biological fermentation step will alter significantly (table 1.6).

In 1996, several years’ experience with pilot plants worldwide using either enzyme conversion or acid-catalyzed hydrolysis of candidate cellulosic feedstocks inspired the prediction that technologies for the conversion of lignocellulosic bio­mass to ethanol would be rapidly commercialized.50 A decade on, “generic” tech­nologies have failed to emerge on large-scale production sites; in April 2004, Iogen

w о

FIGURE 1.24 Outline of lignin biosynthesis.

Compositional Analyses of Tree Species, Paper Recyclates, Cereal Wastes, and Grasses (% Dry Weight Basis)

TABLE 1.6 Hexose sugars Pentose sugars Lignin Water and alcohol extractives Ash Glucans Galactans Mannans Xylans Arabinans Tree 46.2 1.0 3.3 16.0 2.4 24.2 4.0 0.8 speciesa Paper 62.3 6.7 10.4 20.7 recyclatesb Cereal 37.4 0.9 0.4 20.5 2.3 20.5 8.2 7.6 wastesc Cane sugar 40.2 0.7 0.3 21.1 1.9 25.2 4.4 4.0 bagassed Grassese 31.8 1.1 0.2 18.3 2.8 17.3 18.1 6.3 a Mean of 18 softwood and hardwood species b Municipal solid waste and office paper c Corn stover, wheat straw and rice straw d Cane sugar after removal of cane juice e Four species including switchgrass

(Ottawa, Ontario, Canada) opened a demonstration facility capable of processing 40 tonnes of feedstock/day and producing 3 million liters of ethanol annually from wheat, oat, and barley straw, corn cobs, and corn stalk.51 Iogen was founded in 1974 and has received CAN$91.8 million in research funding from the government of Canada, Petro-Canada, and Shell Global Solutions International BV. Iogen and its

European partners are studying the feasibility of producing cellulosic ethanol in Germany and, in May 2006, attracted CAN$30 million from Wall Street investors Goldman Sachs.52-54 The unique status of Iogen indicated that the technical hurdles to be overcome for cellulosic materials were considerably higher than for corn­starch, and it is highly likely that major advances in biotechnology will be required to tailor enzymes for the fledgling industry as well as providing novel biocatalysts for fermentative steps and optimizing plant species as “energy crops.”55 Signifi­cantly, Iogen is also an industrial producer of enzymes used in textiles, pulp and paper, and animal feed.[7]

Since 2006, a pilot facility in Jennings, Louisiana, has operated for the production of cellulosic ethanol; in February 2007, construction commenced on an adjacent demonstration facility designed to utilize regionally available feedstocks, including sugarcane bagasse, and the technology has also been licensed to Japanese companies to develop a project facility at Osaka to produce 1.3 million liters of ethanol annu­ally from demolition wood waste (www. verenium. com). After developing facilities to produce more than a million tons of ethanol from corn and wheat grain by 1995, Chinese scientists at Shandong University devised a bioethanol production process from corn cob, and a 50,000 ton/year plant for ethanol and xylose-derived products is planned to be constructed at Yucheng.56

The “promise” of lignocellulosic bioethanol remains quantitatively persuasive. Estimates of land area available for biomass energy crops and of the utilization of wood industry, agricultural, and municipal solid waste total 1.3-2.3 billion tons of cellulosic biomass as potential annual inputs to bioethanol production, poten­tially equivalent to a biofuel supply matching 30-50% of current U. S. gasoline consumption.3757 In stark contrast, even if all U. S. corn production were to be dedi­cated to ethanol, only 12% of the gasoline demand would be met.58 Data from Can­ada show similar scenarios, i. e., the total 2004 demand for fuel ethanol was met from 2,025 million liters of wheat, barley, corn, and potatoes, but the available nonfood crop supplies then already amounted to nearly 11,500 million liters as corn stover, straw, wood residues, and forest residues.59

The 2005 Energy Policy Act (http://www. ferc. gov) continued the influential role of legislation on renewable energy sources with initiatives to

• Increase cellulosic ethanol production to 250 million gallons/year

• Establish loan guarantees for new facilities

• Create an advanced biofuels technologies program

Continued interest in novel biotechnological solutions to the problems of lignocellulosic bioethanol are highly likely to be maintained over the next decade. The scientific aspects of present developments and future requirements are discussed in chapters 2 and 3.