Bioethanol
Introduction
Currently the United States consumes approximately 20 million barrels of crude oil daily of which about 60% is imported. 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 billion gal of renewable fuels into gasoline by 2012. In addition, many states have passed renewable fuels standards that require the sale of 10% and 20% blends (E10 and E20) by certain dates [1]. By far the most common renewable fuel is ethanol, and annual ethanol production in the United States recently surpassed 4 billion gal, with global production twice that. In the United States, the major raw material for ethanol is corn grain (starch). The US has the capacity to produce 13 billion gallons per year from corn alone and will probably reach the 7.5 billion gal per year goal much sooner than expected. However, any further increases in ethanol production will have to come from feedstocks other than corn grain because of limitations in supply. These feedstocks are typically grouped under the heading of ‘biomass’ and include agricultural residues, wood, municipal solid waste and dedicated energy crops. The US Department of Agriculture and Department of Energy have estimated that the US has the resource potential to produce over 1 billion tons of biomass annually [2], thus accounting for close to 30% displacement 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%), hemicellulose (25–35%) and lignin (15–20%). Starch processing is a fairly mature technology utilizing enzymatic liquefaction and saccharification, which produces a relatively clean glucose stream that is then fermented to ethanol by Saccharomyces yeasts. Recent advances in starch processing have improved the economics and efficiency of the process. One example has been the development of low pH α-amylases that simplify the process and reduce chemical costs as well as improving ethanol yield [3]. The other major advance is the development of enzymes that function on raw, uncooked starch, thereby improving overall process economics [4, 5].
Starch is a storage compound consisting of glucose linked via α-1,4 and α-1,6 glycosidic linkages (amylose and amylopectin), whereas cellulose is a structural compound composed exclusively of glucose linked via β-1,4 glycosidic bonds. Because of the β-1,4 linkage, cellulose is highly crystalline and compact making it very resistant to biological attack. In general, hemicellulose consists of a main chain xylan backbone (β-1,4 linkages) with various branches of mannose, arabinose, galactose, glucuronic acid, etc (Figure 1). The degree of branching and identity of the minor sugars in hemicellulose tends to vary depending upon the type of plant. Furthermore, lignin can be covalently linked to hemicellulose via ferulic acid ester linkages. The compactness and complexity of lignocellulose makes it much more difficult than starch to enzymatically degrade to fermentable sugars. Hence, the cost of producing a gallon of ethanol from biomass is higher than production from starch [6]. In order to be cost competitive with grain-derived ethanol, the enzymes used for biomass hydrolysis must become more efficient and far less expensive. In addition, the presence of non-glucose sugars in the feedstock complicates the fermentation process because conversion of pentose sugars into ethanol is less efficient than conversion of the hexose sugars.
In this review, we focus on advances over the past several years in the development of processes to more effectively and efficiently convert lignocellulosic materials into ethanol. There are three major steps in the conversion process (Figure 2): first, thermochemical pretreatment — a preprocessing step that improves enzyme access to the cellulose; second, enzymatic saccharification — use of cellulases and on some occasions hemicellulases; and thirdly, fermentation of the released sugars by specialized organisms.
Section snippets
Thermochemical pretreatment
Raw, untreated biomass is extremely recalcitrant to enzymatic digestion. Therefore, a number of thermochemical pretreatment methods have been developed to improve digestibility [7••]. Pretreatment disrupts the plant cell wall and improves enzymatic access to the polysaccharides. Studies have shown a direct correlation between the removal of lignin and hemicellulose and the digestibility of cellulose [8]. Pretreatment chemistries vary from very acidic to quite alkaline, thereby having different
Enzymatic depolymerization of cellulose and hemicellulose
Cellulases comprise three types of enzymes: endoglucanases (EC 3.2.1.4), which cleave internal β-1,4-glucosidic bonds; exoglucanases (EC 3.2.1.91), which processively act on the reducing and non-reducing ends of cellulose chains to release short-chain cello-oligosaccharides; and β-glucosidases (EC 3.2.1.21), which hydrolyze soluble cellooligosaccharides (e.g. cellobiose) to glucose. Hemicellulases include enzymes that break down both β-1,4-xylan (xylanases EC 3.2.1.8 and β-xylosidases EC
Cellulase engineering
Several approaches have been utilized to improve cellulase performance and decrease the amount of enzyme needed to saccharify biomass substrates. The primary target for cellulase engineering has been the cellobiohydrolases, as they tend to constitute 60–80% of natural cellulase systems [26]. Teter et al. [27••] used a combination of site-directed mutagenesis, site-saturation mutagenesis, error-prone PCR and DNA shuffling to generate variants of T. reesei Cel7A. The mutants were expressed in S.
Enzyme–substrate interaction
Cellulases often contain carbohydrate-binding modules (CBMs) to facilitate the interaction between the enzyme and the substrate surface. Similar to catalytic domains of glycosyl hydrolases, CBMs are divided into families on the basis of amino acid similarities and crystal structures (http://afmb.cnrs-mrs.fr/CAZY). Subtle differences in the structure of CBMs can lead to very different ligand specificity [32••]. Through binding to the surface of crystalline cellulose, CBMs target their cognate
Hemicellulases
The efficient degradation of hemicellulose requires the synergistic action of many enzymes. More importantly, hemicellulases facilitate cellulose hydrolysis by exposing the cellulose fibers, thus making them more accessible [21•]. Commercial development for hemicellulases in lignocellulose hydrolysis is not as advanced as cellulases because current commercial preparations have been primarily developed on dilute-acid pretreated biomass where hemicellulose is removed before saccharification.
Hexose and pentose fermentation
Production of ethanol from sugar derived from starch and sucrose has been commercially dominated by the yeast S. cerevisiae [42]. However, sugar derived from biomass is a mixture of hexoses (primarily glucose) and pentoses (primarily xylose) and most wild-type strains of S. cerevisiae do not metabolize xylose. Researchers have basically taken two approaches to increase fermentation yields of ethanol derived from biomass sugars. The first approach has been to add to yeast and other natural
Conclusions
Significant progress has been made in the past several years in all aspects of lignocellulosic conversion to ethanol. The key to the establishment of a commercial process is a reduction in capital and operating costs of each of the unit operations. There is a much better understanding of the capital costs associated with pretreatment, suggesting ways to further reduce costs without compromising performance. Similarly enzyme costs have been reduced by a combination of protein engineering and
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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