INTRODUCTION Fermentation (heterotrophic metabolism), requires an organic compound as a terminal electron (or hydrogen) acceptor. In fermentations, simple organic end products are formed from the anaerobic dissimilation of glucose (or some other compound). Energy (ATP) is generated through the dehydrogenation reactions that occur as glucose is broken down enzymatically. Fermentation microbiology and industrial biotechnology revolutionizes the production of our energy and the chemicals needed by other industries. Bioethanol production currently relies predominantly on the utilization of dextrose • derived from corn starch (United States) or sucrose from sugar cane (Brazil). However, the relatively high value and increasing demand of these feedstocks for food production is a major manufacturing cost that renders current bioethanol production uncompetitive with gasoline. Consequently, several economic studies have shown that successful fermentation of sugars derived from lignocellulosic wastes is crucial for achieving commercial success. The conversion of cellulose and hemicellulose for production of fuel ethanol is being studied extensively with a view to develop a technically and economically viable bioprocess. Ethanol’s high octane and high heat of vaporization values make it more efficient than gasoline. Furthermore, ethanol is low in toxicity, volatility, and photochemical reactivity, resulting in reduced ozone emission and smog formation compared with conventional fuels. PENTOSE FERMENTATION The ideal organism should be able to ferment all monosaccharides present and withstand potential inhibitors in the hydrolysate. Pentose-fermenting microorganisms are found among bacteria, yeasts, and fungi, with the yeasts Pichia stipitis, Candida shehatae, and Pachysolen tannophilus being the most promising naturally occurring microorganisms. Yeasts produce ethanol efficiently from hexoses by the pyruvate decarboxylase–alcohol dehydrogenase (PDC-ADH) system. However, during xylose fermentation the byproduct xylitol accumulates, thereby reducing the yield of ethanol. Furthermore, yeasts are reported to ferment l-arabinose only very weakly. Only a handful of bacterial species are known that do possess the important PDC-ADH pathway to ethanol. Among these, Zymomonas mobilis has the most active PDC-ADH system; however, it is incapable of dissimilating pentose sugars. In general, microorganisms metabolize xylose to xylulose through two separate routes (Figure). The one-step pathway catalyzed by xylose isomerase is typical in bacteria, whereas the two-step reaction involving xylose reductase (XR) and xylitol dehydrogenase (XDH) is usually found in yeast. Xylulose is subsequently phosphorylated with xylulokinase (XK) to xylulose-5- hosphate that can be further catabolized via the pentose phosphate pathway and the Embden-Meyerhof-Parnas (EMP) pathway or the Entner–Doudoroff (ED) pathway in organisms such as Z. mobilis. Empowered with the modern tools of genetic engineering and high throughput screening, several groups have been pursuing the construction of yeast or bacterial organisms that can efficiently convert most of the sugars present in biomass derived hydrolysates to useful products. 1. The approaches can be divided in two groups: Engineering organisms with an expanded substrate spectrum It has focused on good ethanologenic organisms, such as S. cerevisiae or Z. mobilis, with the aim of introducing the pathways for xylose or arabinose metabolism. 2. Engineering organisms with enhanced abilities of converting key intermediates of central carbon metabolism (e.g., pyruvate) to useful compounds such as ethanol, lactate, or Succinate The second approach starts with organisms that have a wide sugar substrate range (C6 and C5), such as E. coli, and introduce pathways for converting these sugars to various fermentation products, including ethanol, lactate, acetate, pyruvate, or succinate. ZYMOMONAS MOBILIS FERMENTATION Xylose is a useful carbon source for the ethanol producer Z. mobilis. Zymomonas mobilis is natural fermentative agent in alcoholic beverage production and has been shown to have ethanol productivity superior to that of yeast. Overall, it demonstrates many of the desirable traits sought in an ideal biocatalyst for ethanol, such as high ethanol yield, selectivity and specific productivity, as well as low pH and high ethanol tolerance. In glucose medium, Z. mobilis can achieve ethanol levels of at least 12% (w/v). When compared with yeast, Z. mobilis exhibits 5–10% higher yields and up to 5-fold greater volumetric productivities. As a matter of fact, Zymomonas is the only genus identified to date that exclusively utilizes the ED pathway anaerobically. The stoichiometry of ethanol production in this recombinant organism can be summarized as follows [neglecting the NAD(P)H balances: 3 Xylose + 3 ADP + 3 Pi → 5 Ethanol + 5 CO2 + 3 ATP + 3 H2O Thus, the theoretical yield on ethanol is 0.51 g of ethanol/g of xylose (1.67 mol mol–1). It is important to note that the metabolically engineered pathway yields only 1 mol of ATP from 1 mol of xylose, compared with the 5/3 moles typically produced through a combination of the pentose phosphate and EMP pathways. When converting glucose to ethanol, this organism produces only 1 mol of ATP per mole of glucose through the ED pathway compared with 2 moles produced via the more common EMP pathway. Furthermore, glucose can readily cross the cell membrane of this organism by facilitated diffusion, efficiently be converted to ethanol by an overactive PDC-ADH system, and is a generally recognized as a safe (GRAS) organism for use as an animal feed. As discussed earlier, the main drawback of this microorganism is that it can only utilize glucose, fructose, and sucrose and thus is unable to ferment the widely available pentose sugars. SACCHAROMYCES CEREVISIAE FERMENTATION Saccharomyces spp. are the safest and most effective microorganisms for fermenting sugars to ethanol and traditionally have been used in industry to ferment glucose (or hexose sugar)-based agricultural products to ethanol. Cellulosic biomass, which includes agriculture residues, paper wastes, wood chips, etc., is an ideal inexpensive, renewable, abundantly available source of sugars for fermentation to ethanol, particularly ethanol used as a liquid fuel for transportation. However, Saccharomyces spp. are not suitable for fermenting sugars derived from cellulosic biomass because Saccharomyces spp., including S. cerevisiae, are not able to ferment xylose to ethanol or to use this pentose sugar for aerobic growth. Although Saccharomyces spp. are not able to metabolize xylose aerobically and anaerobically, there are other yeasts, such as P. stipitis and C. shehatae, that are able to ferment xylose to ethanol and to use xylose for aerobic growth. However, these naturally occurring xylose-fermenting yeasts are not effective fermentative microorganisms, and they have a relatively low ethanol tolerance. Many attempts to introduce the one-step pathway by cloning the gene coding for xylose isomerase from E. coli or Bacillus subtilis in S. cerevisiae were unsuccessful because of the inactivity of the heterologous protein in the recombinant host cell. One of the limitations of xylose utilization in S. cerevisiae was attributed to the inefficient capacity of the nonoxidative pentose phosphate pathway, as indicated by the accumulation of sedoheptulose-7-phosphate MICROBES PRODUCING ETHANOL FROM LIGNOCELLULOSE It would be desirable if microbes producing ethanol from lignocellulose also had means to depolymerize cellulose, hemicellulose, and associated carbohydrates. Many plant pathogenic bacteria (soft-rot bacteria), such as Erwinia carotovora has evolved sophisticated systems of hydrolases and lyases that aid the solubilization of lignocellulose and allow them to break up and penetrate plant tissue Genetic engineering of these bacteria for ethanol production represents an attractive alternative to the solubilization of lignocellulosic biomass by chemical or enzymatic means. E. carotovora SR38 was genetically engineered and shown to produce ethanol and CO2 efficiently as primary fermentation products from cellobiose and glucose HEMICELLULOSIC FEEDSTOCKS FOR THERMOPHILIC ETHANOL FERMENTATION Optimal feedstocks are lignocellulosic residues arising from processing of already harvested crops in which all agricultural and transport costs are borne by the primary product. Such residues are generally dried and burned as plant fuel or sold as low-grade animal feed, but they are sometimes composted and returned to the soil. Mild acid or enzyme hydrolysis releases most of the hemicellulosic sugars for bioethanol production and can actually add value to the insoluble lignocellulosic fibers by reducing drying costs and/or supplementing the animal feed with protein-rich spent thermophile cells, which resemble fishmeal in containing lysine and methionine and as such have similar animal feed value. Hence, the cost of the C5 and C6 sugar feedstock is essentially only the hydrolysis cost. Some examples are discussed as follow: Sugar cane Baggase is the residual crushed cane that contains mainly the lignocellulosic rind and significant amounts of hemicelluloses and residual cane sugars arising from the pith The bagasse is normally air-dried, which allows fermentation of the residual sugars by adventitious microorganisms, including thermophiles, to increase calorific value. Sugar beet However, the whole crop is rich in hemicelluloses and pectins that could potentially greatly lower the production cost as in the above example. Only the roots are currently harvested and the tops are used for fodder or ploughed in. These could be combined with the beet pulp and vinasse that are major byproducts of beet sugar production to produce yet more bioethanol from thermophilic fermentations. Because that bioethanol would be produced from agricultural wastes it would not carry a significant carbon footprint. Palm oil residues The oil and presscake are increasingly being exported for biodiesel and animal feed production, respectively, but this has engendered a “fuel versus food” debate. It would make economic and environmental sense to combine the oil extraction with biodiesel production on-site and to use the palm-oil residues and glycerol to produce bioethanol and animal feed by the Biocaldol process. A portion of the ethanol could be used to enhance oil extraction from the kernels and presscake and to recycle the extract for use in transesterification in the biodiesel process.
