BioethanolIntroductionThermochemical pretreatmentEnzymatic depolymerization of cellulose and hemicelluloseCellulase engineeringEnzyme-substrate interactionHemicellulasesHexose and pentose fermentationConclusionsReferences and recommended readingBioethanolKevin A Gray1, Lishan Zhao1and Mark Emptage2Alternatives to petroleum-derived fuels are being sought inorder to reduce the world’s dependence on non-renewableresources. The most common renewable fuel today is ethanolderived from corn grain (starch) and sugar cane (sucrose). It isexpected that there will be limits to the supply of these rawmaterials in the near future, therefore lignocellulosic biomass isseen as an attractive feedstock for future supplies of ethanol.However, there are technical and economical impediments tothe development of a commercial processes utilizing biomass.Technologies are being developed that will allow cost-effectiveconversion of biomass into fuels and chemicals. Thesetechnologies include low-cost thermochemical pretreatment,highly effective cellulases and hemicellulases and efficient androbust fermentative microorganisms. Many advances havebeen made over the past few years that makecommercialization more promising.Addresses1Diversa Corporation, 4955 Directors Place, San Diego CA, 92121,USA2DuPont Central Research & Development, Experimental Station –E328, Wilmington, DE 19880, USACorresponding author: Gray, Kevin A ([email protected])Current Opinion in Chemical Biology 2006, 10:141–146This review comes from a themed issue onBiocatalysis and biotransformationEdited by Ben Davis and Grace DeSantisAvailable online 7th March 20061367-5931/$ – see front matter# 2006 Elsevier Ltd. All rights reserved.DOI 10.1016/j.cbpa.2006.02.035IntroductionCurrently the United States consumes approximately 20million barrels of crude oil daily of which about 60% isimported. Liquid transportation fuels including gasoline,diesel and jet fuel account for almost 70% of the total.The US Energy Policy Act of 2005 (http://www.ferc.gov)states that the oil industry is required to blend 7.5 billiongal of renewable fuels into gasoline by 2012. In addition,many states have passed renewable fuels standards thatrequire the sale of 10% and 20% blends (E10 and E20) bycertain dates [1]. By far the most common renewable fuelis ethanol, and annual ethanol production in the UnitedStates recently surpassed 4 billion gal, with global pro-duction twice that. In the United States, the major rawmaterial for ethanol is corn grain (starch). The US has thecapacity to produce 13 billion gallons per year from cornalone and will probably reach the 7.5 billion gal per yeargoal much sooner than expected. However, any furtherincreases in ethanol production will have to come fromfeedstocks other than corn grain because of limitations insupply. These feedstocks are typically grouped under theheading of ‘biomass’ and include agricultural residues,wood, municipal solid waste and dedicated energy crops.The US Department of Agriculture and Department ofEnergy have estimated that the US has the resourcepotential to produce over 1 billion tons of biomassannually [2], thus accounting for close to 30% displace-ment of current fossil fuel usage (about 80 billion gal).Unlike corn grain where the major carbohydrate is starch,biomass is composed of cellulose (40–50%), hemicellu-lose (25–35%) and lignin (15–20%). Starch processing is afairly mature technology utilizing enzymatic liquefactionand saccharification, which produces a relatively cleanglucose stream that is then fermented to ethanol bySaccharomyces yeasts. Recent advances in starch proces-sing have improved the economics and efficiency of theprocess. One example has been the development of lowpH a-amylases that simplify the process and reducechemical costs as well as improving ethanol yield [3].The other major advance is the development of enzymesthat function on raw, uncooked starch, thereby improvingoverall process economics [4,5].Starch is a storage compound consisting of glucose linkedvia a-1,4 and a-1,6 glycosidic linkages (amylose and amy-lopectin), whereas cellulose is a structural compoundcomposed exclusively of glucose linked via b-1,4 glycosi-dic bonds. Because of the b-1,4 linkage, cellulose is highlycrystalline and compact making it very resistant to biolo-gical attack. In general, hemicellulose consists of a mainchain xylan backbone (b-1,4 linkages) with variousbranches of mannose, arabinose, galactose, glucuronicacid, etc (Figure 1). The degree of branching and identityof the minor sugars in hemicellulose tends to vary depend-ing upon the type of plant. Furthermore, lignin can becovalently linked to hemicellulose via ferulic acid esterlinkages. The compactness and complexity of lignocellu-lose makes it much more difficult than starch to enzyma-tically degrade to fermentable sugars. Hence, the cost ofproducing a gallon of ethanol from biomass is higher thanproduction from starch [6]. In order to be cost competitivewith grain-derived ethanol, the enzymes used for biomasshydrolysis must become more efficient and far less expen-sive. In addition, the presence of non-glucose sugars in thefeedstock complicates the fermentation process becauseconversion of pentose sugars into ethanol is less efficientthan conversion of the hexose sugars.www.sciencedirect.com Current Opinion in Chemical Biology 2006, 10:141–146In this review, we focus on advances over the past severalyears in the development of processes to more effectivelyand efficiently convert lignocellulosic materials into etha-nol. There are three major steps in the conversion process(Figure 2): first, thermochemical pretreatment — a pre-processing step that improves enzyme access to thecellulose; second, enzymatic saccharification — use ofcellulases and on some occasions hemicellulases; andthirdly, fermentation of the released sugars by specializedorganisms.Thermochemical pretreatmentRaw, untreated biomass is extremely recalcitrant toenzymatic digestion. Therefore, a number of thermoche-mical pretreatment methods have been developed toimprove digestibility [7]. Pretreatment disrupts theplant cell wall and improves enzymatic access to thepolysaccharides. Studies have shown a direct correlationbetween the removal of lignin and hemicellulose and thedigestibility of cellulose [8]. Pretreatment chemistriesvary from very acidic to quite alkaline, thereby havingdifferent effects upon the major constituents in biomass.For