Ethanol production from biomass: technology and commercialization status

324 Ethanol production from biomass: technology and commercialization status Jonathan R Mielenz Owing to technical improvements in the processes used to produce ethanol from biomass, construction of at least two waste-to-ethanol production plants in the United States is expected to start this year. Although there are a number of robust fermentation microorganisms available, initial pretreatment of the biomass and costly cellulase enzymes remain critical targets for process and cost improvements. A highly efficient, very low-acid pretreatment process is approaching pilot testing, while research on cellulases for ethanol production is expanding at both enzyme and organism level. Addresses White Cliff Biosystems Co, 107 Lake Meadow Drive, Johnson City, Tennessee 37615, USA; e-mail: jmielenz92@aol.com Current Opinion in Microbiology 2001, 4 :324–329 1369-5274/01/$ —see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations SSCF simultaneous saccharification and cofermentation SSF simultaneous saccharification and fermentation Introduction With the inevitable depletion of the world’s petroleum supply [1], there has been an increasing worldwide interest in alternative, non-petroleum-based sources of energy. As petroleum supplies 97% of the energy consumed for transportation [2], industry and governments worldwide have been actively identifying, developing and commercializing technology for alternative transportation fuels over the past 20 years [3]. A growing, yet controversial, source of transportation fuel in the United States is fermentation-derived ethanol whose production cost requires significant government subsidy to permit producers to remain in business. Nearly all fuel ethanol is produced by fermentation of corn glucose in the United States or sucrose in Brazil [4], but any country with a significant agronomic-based economy can use current technology for fuel ethanol production. This is possible because, during the last two decades, technology for ethanol production from non-food-plant sources has been developed to the point at which large-scale production will be a reality in the next few years. Therefore, agronomic residues such as corn stover (corn cobs and stalks), sugar cane waste, wheat or rice straw, forestry and paper mill discards, the paper potion of municipal waste, and dedicated energy crops — collectively termed ‘biomass’ — can be converted to fuel ethanol. This review will focus on the current status of biomass-ethanol, or ‘bioethanol’, technology, especially processes that liberate monomeric sugars from biomass carbohydrates, termed ‘pretreatment’, and the commercialization of bioethanol production. Functional, environmental and strategic benefits of ethanol All automobile manufacturers produce vehicles that can readily use 10% ethanol or 85% ethanol (E85) blends for fuel, and ethanol can replace diesel in heavy vehicles as well. About 12.5 billion liters of ethanol is produced from cane sugar in Brazil, and is used as either 22% blends with gasoline or as neat ethanol fuels containing 100% ethanol. The United States produces about 5 billion liters from starch crops, mainly corn, and has 111 fueling stations for E85 ethanol (Alternative Fuels Data Center Refueling Sites URL http://www.afdc.doe.gov/refueling.html). Many of these stations are in the Midwest or corn belt of the United States, and limit availability of E85 ethanol in spite of Ford and Chrysler selling flexible fuel vehicles with no increase in price [5]. Expansion of the market for ethanol to as much as 38–53 billion liters per year, or nearly enough for a 10% ethanol blend of gasoline used in the United States, could occur if all available agricultural residues were converted to ethanol [5]. Blending oxygenates such as ethanol and methyl tertiary butyl ether (MTBE) are well recognized for causing reduced carbon monoxide levels by improving overall combustion (oxidation) of the fuel. However, MTBE is being phased out of use in California because it contaminates approximately 1% of the domestic wells in that state [6]. Ethanol is being evaluated as a replacement [7] and may be readily accepted because of California's environmental consciousness, their huge diverse agricultural economy, and the lack of any clear alternative oxygenate. However, the fact that ethanol has approximately 65–69% of the energy density of hydrocarbon fuels [8] must be considered. On the other hand, while the reality of global warming continues to be discussed [3], the use of fuel ethanol will significantly reduce net carbon dioxide emissions when it replaces fossil fuels, because fermentation-derived ethanol is already part of the global carbon cycle [9]. Finally, as the current price of gasoline fluctuates at the whim of Oil Producing and Exporting Countries (OPEC), the development of a domestically produced renewable transportation fuel can have important strategic value [3]. Biomass ethanol production process The primary difficulty for commercialization of ethanol produced by fermentation is its high cost of production relative to the local cost of gasoline. Recent increases in the wholesale price of crude oil appear to be helping to close the cost gap between ethanol and gasoline. The cost of ethanol is linked, in part, to the inescapable loss of half of the carbon during fermentation of sugars by microorganisms. Although the production of ethanol from cane sugar is a relatively simple process, complexity increases when Ethanol production from biomass: technology and commercialization status Mielenz 325 Figure 1 Simplified diagram showing possible processes for ethanol production. The process depicted along the top of the figure is the concentrated acid process that uses sulfuric acid to hydrolyze the biomass prior to fermentation to ethanol. Very efficient acid recycling is required for the process to be cost-effective. The process depicted along the bottom of the figure is the SSF/SSCF process that requires pretreatment of biomass for both ethanol fermentation and enzyme production for SSF/SSCF. The decision to use the SSF/SSCF process is influenced by whether or not the fermentation organism can utilize pentose sugars along with glucose. The slanting line in the bottom process indicates that enzyme treatment can be sequential to fermentation (it is more costly, however). Conc acid, concentrated acid; temp, temperature. Biomass milling and blending Conc acid hydrolysis Acid recovery Sugar fermentation to ethanol Ethanol recovery Biomass milling and blending High temp pretreatment Low temp cellulase treatment Sugar fermentation to ethanol Ethanol recovery Biomass ethanol is produced from corn or wheat starch, as these processes require enzymes to hydrolyze starch to glucose prior to fermentation. Production of ethanol from biomass requires even more extensive processing to release the polymeric sugars in cellulose and hemicellulose that account for 23%–53% and 20%–35% of plant material, respectively. Cellulose is a beta-linked glucose polymer, whereas hemicellulose is a highly branched chain of xylose and arabinose that also contains glucose, mannose and galactose [10]. Hydrolysis of these carbohydrate polymers is usually accomplished by exposure to acid (contributed either by the biomass or added externally) and by enzymes, as shown in the bottom part of Figure 1. The process shown in the upper part of Figure 1 uses concentrated sulfuric acid to hydrolyze the biomass carbohydrates. After hydrolysis, the acid is separated from the sugars, which are fermented to ethanol. However, the emphasis here will be on the SSF/SSCF process for bioethanol, whose evolution over the last 20 years is due to many improvements in biotechnology and process improvements [11]. Enzyme production Current Opinion in Microbiology but result in a more dilute sugar solution [13]. A new counter-current flow-through process that yields very high levels of hydrolyzed cellulose and hemicellulose while using only 0.07% sulfuric acid is being developed [14]. This counter-current process results in shrinkage of the biomass, this being responsible for the critical maintenance of relatively high sugar concentrations. Eighty-two percent hydrolysis of cellulose and near-total depolymerization of xylose have been reported to yield a solution containing approximately 4% sugar [15••,16••]. Lee et al. [16••] report that a pilot reactor is being constructed for testing later this year. With these exceptional results, they speculate that it may be possible to eliminate or greatly reduce the need for cellulase in the bioethanol process. However, they acknowledge that detoxification of acid-hydrolyzed lignin and other ‘extractables’ in the sugar hydrolysate will present additional costs for the total hydrolysis process, costs that could be avoided entirely if a fully enzymatic process (yet to be developed) is implemented instead. The role of cellulases Pretreatment of biomass In addition to polymeric carbohydrates, plant matter contains varying amounts of polyphenolic lignin and other ‘extractables’. The pretreatment process aims to separate the carbohydrates from the lignin matrix while minimizing chemical destruction of fermentation sugars required for ethanol production. Development of an ideal pretreatment process is difficult, given that ‘biomass’ includes such sources as hardwood and softwood trees, agricultural residues such as corn stover, and non-recyclable paper waste. These diverse feedstocks have caused researchers to test numerous pretreatment processes ranging from hot water and steam explosion treatments, to alkaline and solvent pretreatments, to many useful versions of acid pretreatment [12]. The kinetics and yields of the various acid-based batch and flow-through processes have been compared recently, and it is clear that flow-through processes provide higher sugar yields and cause less sugar destruction, The pretreatment process shown at the bottom of Figure 1 is designed only to initiate the breakdown of the biomass structure and partially hydrolyze the carbohydrate polymers, making them accessible to enzymatic attack [17]. Therefore, enzymatic degradation of cellulose and hemicellulose has been extensively studied over the past 40 years, with significant progress. As hemicellulose is readily hydrolyzed by mild-acid conditions, the recalcitrant semicrystalline cellulose has been the target of most biomass enzyme research [18,19]. Historically, cellulases were first applied in a sequential process (pretreatment → cellulase hydrolysis → ethanol fermentation). However, the simultaneous saccharification and fermentation (SSF) process provides significant cost reduction because cellulase hydrolysis occurs during fermentation of the glucose [17]. The processes currently used include fermentation of all biomass sugars in a simultaneous saccharification cofermentation (SSCF) process (see below). 326 Ecology and industrial microbiology Even after over 40 years of research on cellulases [5], the costs of these enzymes have remained high, in comparison to the costs of proteases and amylases, for a few key reasons. Largely owing to their complex, insoluble semicrystalline substrates, cellulases are relatively slow catalysts [20•]. Also, maximal activity requires multiple, related enzyme activities acting synergistically. Specifically, complete hydrolysis of cellulose requires exoglucanases, endoglucanases and beta-glucosidases to fully hydrolyze cellulose to glucose [21•]. Analyses pinpointing areas in bioethanol production that require most research have identified four critical areas related to cellulase research: increased thermostability, improved cellulase binding, increased specific activity, and reduced nonspecific binding to lignin [22]. To improve cellulase activity, mixtures of bacterial and fungal enzyme were used to determine the ideal interaction of these enzyme activities, but this superior blend is not available in a single organism (yet) [23,24]. With the help of modern genetic technology, production of a ‘superior’ blend by a single organism is certainly possible. The production and usage of cellulase enzymes from Trichoderma and other organisms are being improved continuously. Recent research appears to address many of the aforementioned critical needs [25•–31•]. One of the most active cellulases known, endoglucanase E1 from Acidothermus cellulolyticus, has been expressed in tobacco and potatoes [32••,33••], providing a potential source of this enzyme. Recently, the United States Department of Energy has added more resources to this effort by awarding two research contracts to the world’s leading industrial enzyme producers: Novozymes (www.novozymes.com) and Genencor International (www.genencor.com) are to improve cellulases for use in biomass hydrolysis in bioethanol production. To be successful, their efforts should not just address classical enzyme improvements (such as yield, stability and specific activity), but must also meet the end user’s need for a robust, synergistic enzyme mixture that will remain active even in harsh environments generated by, for example, acid pretreatment [34]. The ideal bioethanol producer would ferment all biomass sugars, possess good resistance to lignin monomers, acetate and other inhibitory byproducts, and produce a synergistic combination of cellulases needed for full cellulose hydrolysis. Ingram and colleagues took steps in this direction by cloning two Erwinia endoglucanase genes into an ethanolproducing Klebsiella species [44,45••]. The resulting organism produced up to 22% more ethanol when fermenting crystalline cellulose synergistically with added fungal cellulases. Lactobacillus is a legendary persistent and resistant contaminant in ethanol fermentation. Recent genetic engineering of this organism has added the genes for xylose utilization with the aim of enabling it to produce lactic acid from biomass [46] and, eventually, for ethanol production (see Office of Fuels Development Biofuels Program, Bioethanol Technology URL www.ott.doe.gov/biofuels/ bioethanol.html). Others have also successfully introduced cellulase genes into Lactobacillus [47], although not necessarily for fermentation. Furthermore, various thermophilic Clostridium and Thermoanaerobium species have been investigated for their potential as ethanol producers, but have been consistently found to suffer from end-product inhibition and membrane damage [48,49]. Recently, salt accumulation during pH-controlled fermentations has been determined to be an additional or even overriding barrier to production of ethanol in, Thermoanaerobacterium thermosaccharolyticum, a thermophilic bacterium that is otherwise tolerant to higher levels of ethanol [50•]. The authors suggest that using genetic engineering to eliminate acid production in ethanol fermentations could yield a highly productive, anaerobic, ethanol-producing strain (LL Lynd, personal communication). Like Klebsiella and Lactobacillus, such an organism could be improved further to produce multiple cellulases for direct microbial conversion of biomass to ethanol, although careful selection of the enzymes to be added is recommended for all these instances in order to avoid genetically fine tuning production of a ‘second rate’ enzyme(s). Also, the overall metabolic energy burden of both ethanol fermentation and cellulase production in an energy-frugal anaerobe is just beginning to be evaluated [51,52]. Process economics Fermentation to ethanol Fermentation of biomass involves significantly greater challenges, owing to the necessity of converting pentose as well as multiple hexose sugars to ethanol in a SSCF step. As with pretreatment, fermentation microorganisms have undergone continuous improvement, especially with the application of genetic engineering. Both yeasts (such as Saccharomyces and Pichia species) and bacteria (such as Escherichia coli, Klebsiella and Zymomonas) have been genetically engineered to ferment glucose, xylose and arabinose sugars [35–42]. Commercially, BC International Corporation (www.bcintlcorp.com) is using genetically engineered E. coli that produces ethanol from biomass sugars [43], and Arkenol Inc. (www.arkenol.com) is evaluating Zymomonas for use in its concentrated-acid process. Current bioethanol research is driven by the need to reduce the cost of production. Historically, the projected cost of bioethanol has dropped from about US$1.22 per liter to about US$0.31 per liter on the basis of continuous improvement in pretreatment, enzyme application and fermentation [17]. Further economic analysis of the bioethanol process has yielded a projected cost of as low as US$0.20 per liter in 2015 if enzymatic processing and biomass improvement targets are met [22]. A lower cost projections of approximately US$0.13 per liter has been proposed by Lynd [8]. A similar estimate using corn stover as feedstock yielded a projected cost of US$0.12 per liter [53•]. Therefore, there is real reason to be optimistic about the future of using bioethanol instead of gasoline without governmental price support. Ethanol production from biomass: technology and commercialization status Mielenz Biomass ethanol coproducts Biomass sugars are valuable fermentation feedstocks for many other products that can be manufactured along with bioethanol [54]. Likely coproducts include organic acids and other organic alcohols [55–57], 1,2-propandiol [58] and aromatic chemical intermediates [59]. Additionally, Holtzapple will shortly be piloting a process that converts biomass to mixed alcohols [60]. It is critical in commercialization of any coproduct that the market is not flooded with single products produced on a grand scale simply because they are tied to large bioethanol plants. Therefore, the careful selection of coproducts to be produced in a bioethanol plant is an important consideration. Commercialization Either in 2001 or 2002, the construction of the first dedicated large-scale plant for the conversion of waste biomass to ethanol will probably be undertaken by two entities: BC International Corporation and Masada Resource Group (www.masada.com). BC International Corporation will be constructing and modifying a facility for the manufacture of ethanol from sugar cane waste and other biomass sources. The plant will produce 20 million gallons of ethanol using BCI’s proprietary biotechnology based on research by Ingram et al. ([43,61]; J Doyle, personal communication). Masada Resource Group will build a municipal solid-wasteto-ethanol facility. The plant will produce approximately 9.5 million gallons of ethanol using Masada’s patented CES OxyNoltm concentrated acid hydrolysis technology and will recover other recyclable solid materials using waste from from 24 surrounding municipalities (T Judge, personal communication; see also Update). Success with these innovative production plants is critical to expanding financial and governmental support to this new industry. addition of Ca(OH)2 to the acidic hydrolysate until a pH of 9–10 is reached, followed by neutralization to pH 7 for fermentation, is 'overliming' with calcium hydroxide. Recent work [62] has determined that titration of a hydrolysate with sodium hydroxide, which is much easier to use, to either pH 11 or pH 7 is a direct predictor of overliming requirements for bioethanol fermentation. This should be very useful to large-scale fermentation operations. Recently, Ingram and coworkers [62,63••] have undertaken the dissection of the carbon flux and gene expression during ethanol production using gene array technology. Xylose catabolism genes in the engineered E. coli KO11 were compared to the parent strain during xylose-to-ethanol fermentation and found to be overexpressed by 1.5 to eight times, with xylose isomerase and xylulose kinase gene expression and enzyme activity increasing two- to threefold. Iogen Corporation (www.iogen.com), a leading producer of cellulases, has completed a 40 ton per day biomass-toethanol demonstration facility that is currently in its start-up phase (P Foody Jr, personal communication). Acknowledgements The author thanks those who provided helpful comments and copies of their latest publications. 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 1. Kerr RA: The next oil crisis looms large —and possibly close. Science 1998, 281 :1128-1131. 2. Putsche V, Sandor D: Strategic, economic, and environmental issues for transportation fuels. In Handbook on Bioethanol: Production and Utilization. Edited by Wyman CE. Washington DC: Taylor and Francis; 1996:21-35. 3. Sheehan J: Bioconversion for production of renewable transportation fuels in the United States: a strategic perspective. In Enzymatic Conversion for Biomass Fuels Production. Edited by Himmel M, Baker J, Overend R. Washington DC: American Chemical Society; 1994:1-52. 4. Rosillo-Calle F, Cortez L: Towards proalcohol II: a review of the Brazilian bioethanol programme. Biomass Bioenergy 1998, 14 :115-124. 5. Sheehan J: The road to bioethanol: a strategic perspective of the US Department of Energy’s national ethanol program. In Glycosyl Hydrolases for Biomass C onversion. Edited by Himmel M, Baker JO, Saddler JN. Washington DC: American Chemical Society; 2001:2-25. 6. Blackburn B, MacDonald T, McCormack M, Perez P, Tiengco V: Evaluation of biomass-to-ethanol fuel potential in California. California Energy Commission Docket 1999-12-22-500-99-022, 1999. 7. Unnasch S, Kaahaaina N, Kassoy E, Vehkatesh S, Rury P, Counts R: Costs and benefits of a biomass-to-ethanol production industry in California. California Energy Commission Docket P500-01-002, 2001. 8. Lynd L: Overview and evaluation of fuel ethanol from cellulosic biomass: technology, economics, and policy. Annu Rev Energy Environ 1996, 21 :403-465. 9. Wyman C: Alternative fuels from biomass and their impact on carbon dioxide accumulation. Appl Biochem Biotechnol 1994, 45–46 :897-915. Conclusions After over 20 years of research and development, commercial plants for the production of bioethanol are becoming a reality. The success of these novel production plants is likely to stimulate more commercial projects and additional research and development, echoing the rise of the corn and cane ethanol industry over the past 15 years. If the cost estimates of the shrinking bed pretreatment process are favorable, this process may offer a short-term alternative for the next bioethanol plants to use while the difficult challenges of developing a fully enzymatic or organismal bioethanol process are met. Eventually, production of a variety of chemicals is anticipated to take place in bioethanol plants, owing to improved financial returns and added flexibility in the marketplace. Therefore, during the next decade, bioethanol technology is likely to be commercialized in biorefineries throughout the world in a commercially viable response to the need for domestically sourced transportation fuels. Update The classic method to remove toxic compounds from acidic hydrolysates of biomass, which involves careful 327 328 Ecology and industrial microbiology 10. Weislogel A, Tyson S, Johnson D: Biomass feedstock resources and composition. In Handbook on Bioethanol: Production and Utilization. Edited by Wyman C. Washington DC: Taylor and Francis; 1996:105-118. 11. Wyman C: Twenty years of trials, tribulations and research progress on bioethanol technology. Appl Biochem Biotechnol 2001, in press. 12. Hsu T: Pretreatment of biomass. In Handbook on Bioethanol: Production and Utilization. Edited by Wyman C. Washington DC: Taylor and Francis; 1996:179-212. 13. Jacobson SE, Wyman CE: Cellulose and hemicellulose hydrolysis models for application to current and novel pretreatment processes. Appl Biochem Biotechnol 2000, 84–86 :81-96 14. Lee YY, Wu Z, Torget R: Modeling of countercurrent shrinking-bed reactor in dilute-acid total-hydrolysis of lignocellulosic biomass. Bioresource Technol 2000, 71 :29-39. 15. Torget R, Kim J, Lee YY: Fundamental aspects of dilute acid •• hydrolysis/ fractionation kinetics of hardwood carbohydrates. 1. Cellulose hydrolysis. Ind Eng Chem Res 2000, 39 :2817-2825. See annotation to [16 ••]. 16. Lee YY, Iyer P, Torget R: Dilute-acid hydrolysis of lignocellulosic •• biomass. Adv Biochem Eng Biotechnol 1999, 65 :93-115. This paper and [15 ••] present both theoretical and experimental results that clearly outline the differences and advantages of the counter-current reactor. Additionally, the authors provide the status of ongoing scale-up activities, and a cost comparison to enzymatic hydrolysis processes. 17 . Wyman C: Biomass ethanol: technical progress, opportunities, and commercial challenges. Annu Rev Energy Environ 1999, 24 :189-226. 18. Paech C: Approaches to cellulase purification. In Enzymatic Conversion of Biomass for Fuels Production. Edited by Himmel M, Baker JO, Overend RP. Washington DC: American Chemical Society; 1994: 130-178. 19. Himmel M, Adney WS, Baker JO, Nieves RE, Thomas SR: Cellulases: structure, function, and applications. In Handbook on Bioethanol: Production and Utilization. Edited by Wyman C. Washington DC: Taylor and Francis; 1996:143-161. 20. Esteghlalian AR, Srivastava V, Gilkes N, Gregg NJ, Saddler J: An • overview of factors influencing the enzymatic hydrolysis of lignocellulosic feedstocks. In Glycosyl Hydrolases for Biomass Conversion. Edited by Himmel M, Baker JO, Saddler JN. Washington DC: American Chemical Society; 2001:100-111. The challenges of cellulase hydrolysis of ligncellulose in biomass are clearly explained and illustrated as being far more complex than just enzyme-substrate activity and accessibility. 21. Himmel ME, Ruth MF, Wyman CE: Cellulase for commodity • products from cellulosic biomass. Curr Opin Biotechnol 1999, 10 :358-364. This paper provides a valuable historical overview, and evaluates the current technical status of the application of cellulases specifically to biomass-to-ethanol processes. 22. Wooley R, Ruth M, Glassner D, Sheehan J: Process design and costing of bioethanol technology: a tool for determining the status and direction of research and development. Biotechnol Prog 1999, 15 :794-803. 27. • Brun E, Johnson PE, Creagh AL, Tomme P, Webster P, Haynes CA, McIntosh LP: Structure and binding specificity of the second N-terminal cellulose-binding domain for Cellulomonaas fimi endoglucanase C. Biochem 2000, 39 :2445-2458. See annotation to [25 •]. 28. Boisset C, Fraschini C, Schulein M, Henrissat Chanzy H: Imaging the • enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase Cel6A from Humicola insolens and its mode of synergy with cellobiohydrolaseCel7A. Appl Environ Micro 2000, 66 :1444-1452. See annotation to [25 •]. 29. Boer H, Teeri TT, Koivula A: Characterization of Trichoderma • reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters. Biotechnol Bioeng 2000, 69 :486-494. See annotation to [25 •]. 30. Quixley KW, Reid SJ: Construction of a reporter gene vector for • Clostridium beijerinckii using a Clostridium endoglucanase gene. J Mol Microbiol Biotechnol 2000, 2 :53-57. See annotation to [25 •]. 31. Hazell BW, Te’o VS, Bradner JR, Bergquist PL, Navalainen KM: Rapid • transformation of high cellulase-producing mutant strains of Trichoderman reesei by microprojectile bombardment. Lett Appl Microbiol 2000, 30 :282-286. See annotation to [25 •]. 32. Dai Z, Hooker BS, Anderson DB, Thomas SR: Expression of •• Acidothermus cellulolyticus endoglucanase E1 in transgenic tobacco: biochemical characteristics and physiological effects. Transgenic Res 2000, 9 :43-54. The importance of this paper and [33 ••] is that they describe not only the potential for low cost cellulases, but also that a bioethanol plant can blend transgenic plants directly into the SSCF fermentor. If traditional drying methods (i.e. for burley tobacco) can maintain cellulase activity, a year-round supply could be available from existing tobacco farms surrounding the authors region (Tennessee, Virginia, North Carolina, etc.) 33. Hooker BS Dai Z Anderson DB Quesenberry RD Ruth MF •• Thomas SR. Production of microbial cellulases in transgeniccrop plants. In Glycosyl Hydrolases for Biomass Conversion. Edited by Himmel M, Baker JO, Saddler JN. Washington DC: American Chemical Society; 2001:55-90. See annotation to [32 ••]. 34. McMillian JM, Dowe N, Mohagheghi A, Newman M: Assessment of efficacy of cellulase enzyme preparations under simultaneous saccharification and fermentation process conditions. In Glycosyl Hydrolases for Biomass Conversion. Edited by Himmel M, Baker JO, Saddler JN. Washington DC: American Chemical Society; 2001:144-166. 35. Picataggio S, Zhang M: Biocatalyst development for bioethanol production from hydrolysates. In Handbook of Bioethanol: Production and Utilization. Edited by Wyman CE. Washington, DC: Taylor and Francis; 1996:163-178. 36. Bothast RJ, Nichols NN, Dien BS: Fermentations with new recombinant organisms. Biotechnol Prog 1999, 15 :867-875. 37. Ingram LO, Aldrich HC, Borges AC, Causey TB, Martinez A, Morales F, Saleh A, Underwood SA, Yomano LP, York SW et al.: Enteric bacterial catalysts for fuel ethanol production. Biotechnol Prog 1999, 15 :855-866. 23. Baker JO, Adney WS, Thomas SR, Nieves RA, Chou YC, Vinzant TB, Tucker MP, Laymon RA, Himmel ME: Synergism between purified bacterial and fungal cellulases. Edited by Himmel ME, Saddler JN. In Enzymatic Degradation of Insoluble Carbohydrates, ACS Symposium Series 618 . Washington DC: American Chemical Society; 1995:113-141. 38. Ho NW, Chen Z, Brainard AP, Sedlak M: Successful design and development of genetically engineered Saccharomyces yeasts for effective cofermentation of glucose and xylose from cellulosic biomass to fuel ethanol. Adv Biochem Eng Biotechnol 1999, 65 :163-192. 24. Baker JO, Ehrman C, Adney WS, Thomas SR, Himmel ME: Hydrolysis of cellulose using ternary mixtures of purified cellulases. Appl Biochem Biotechnol 1998, 70–71 :395-403. 39. Nigam JN: Development of xylose-fermenting yeast Pichia stipitis for ethanol production through adaptation on hardwood hemicellulose acid prehydrolysate. J Appl Microbiol 2001, 90 :208-215. 25. Irwin DC, Zhang S, Wilson DB: Cloning, expression and • characterization of a family 48 exocellulase, Cel48A, from Thermobifida fusca . Eur J Biochem 2000, 267 :4988-4997. This and [26 •–31 •] address various aspects of the production and improvement of activity of some of the better cellulase enzymes available today. 26. Zhang S, Irwin DC, Wilson DB: Site-directed mutation of • noncatalytic residues of Thermobifida fusca exocellulase Cel6B. Eur J Biochem 2000, 267 :3101-3115. See annotation to [25 •]. 40. Nissen TL, Kielland-Brandt MC, Nielsen J, Villadsen J: Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation. Metab Eng 2000, 2 :69-77. 41. Joachimsthal EL, Rogers PL: Characterization of a high-productivity recombinant strain of Zymomonas mobilis for ethanol production from glucose/ xylose mixtures. Appl Biochem Biotechnol 2000, 8486 :343-356. Ethanol production from biomass: technology and commercialization status Mielenz 329 43. Ingram LO, Conway T, Clark DP, Sewell DW, Preston JF: Genetic engineering of ethanol production in Escherichia coli. Appl Envrion Microbiol 1987, 35 :2420-2425. 53. Arntzen C, Dale B, Beachy R, Bemiller J, Burgess R, Gallagher P, • Hardy R, Johnson D, Kirk T, Krishore G et al.: Case study of lignocellulose ethanol processing, appendix A. In Biobased Industrial Products. Edited by the National Research Council, Washington DC: National Academy Press; 2000:137-143. A valuable overview of potential options for new agricultural products and technology, written by a panel of experts. 44. Zhou S, Ingram LO: Engineering of endoglucanase-secreting strains of Klebiella oxytoca P2. J Indust Microbiol 1999, 22 :600-607. 54. Lynd L, Wyman C, Gerngross T: Biocommodity engineering. Biotechnol Prog 1999, 15 :777-793. 45. Zhou S, Davis FC, Ingram LO. Gene integration and expression and •• extracellular secretion of Erwinia chrysanthemi endoglucanase CelY ( celY) and CelZ ( celZ) in ethanologenic Klebsiella oxytoca P2. Appl Environ Microbiol 2001, 67 :6-14. This approach is critical for the next-generation, total-hydrolysis microorganism that will produce synergistic mixtures of cellulases needed to hydrolyze cellulose, and be able to produce ethanol at high levels. 55. Tsao G, Cao N, Du G, Gong C: Production of multifunctional organic acids from renewable resources. Adv Biochem Eng Biotechnol 1999, 65 :243-280. 42. Aristidou A, Penttial M: Metabolic engineering applications to renewable resource utilization. Curr Opin Biotechnol 2000, 11 :187-198. 46. Picataggio SK, Zhang M, Franden MA, McMillan J, Finkelstein M: Recombinant Lactobacillus for fermentation of xylose to lactic acid and lactate. US Patent 5798237, 1998. 47. Cho JS, Choi YL, Chung DK: Expression of Clostridium thermocellum endoglucanase gene in Lactobacillus gasseri and Lactobacillus johnsonii and characterization of the genetically modified probiotic lactobacilli. Curr Microbiol 2000, 40 :257-263. 48. Ingram LO: Ethanol tolerance in bacteria. CRC Crit Rev Biotechnol 1990, 9 :305-319. 49. Jones RP: Biological principles for the effects of ethanol. Enz Microb Technol 1989, 11 :130-151. 50. Lynd LR, Baskaran S, Casten S: Salt accumulation resulting from • base added for pH control, and not ethanol, limits growth of Thermoanaerobacterium thermosaccharolyticum HG-8 at elevated feed xylose concentrations in continuous culture. Biotechnol Prog 2001, 17 :118-125. Ethanol fermentation of elevated temperatures (e.g. >55°C) would facilitate product recovery, but thermophilic bacteria are poor ethanol producers. If metabolic inhibition by the salt accumulation is a generalized phenomenon, interest in thermophiles for ethanol and other volatile products could be rekindled. 51. van Walsum PG, Lynd LL: Allocation of ATP to synthesis of cells and hydrolytic enzymes in cellulolytic fermentative microorganisms: bioenergetics, kinetics, and bioprocessing. Biotech Bioeng 1997, 58 :316-320. 52. Desvaux M, Guedon E, Petitdemange H: Carbon flux distribution and kinetics of cellulose fermentation in steady-state continous cultures of Clostridium cellulolyticium on a chemically defined medium. J Bacteriol 2001, 183 :119-130. 56. Iyer PV, Thomas S, Lee YY: High-yield fermentation of pentoses into lactic acid. Appl Biochem Biotech 2000, 84–86 :665-677. 57. Borden JR, Lee YY, Yoon HH: Simultaneous saccharification and fermentation of cellulosic biomass to acetic acid. Appl Biochem Biotechnol 2000, 84–86 :963-970. 58. Altaras NE, Etzel MR, Cameron DC: Conversion of Sugars to 1,2-Propanediol by Thermoanaerobacterium thermosaccharolyticum HG-8. Biotechnol Prog 2001, 17 :52-56. 59. Li K, Frost JW : Microbial synthesis of 3-dehydroshikimic acid: a comparative analysis of D-xylose, L-arabinose, and D-glucose carbon sources. Biotechnol Prog 1999, 15 :876-883. 60. Holtzapple M, Davidson R, Ross M, Aldrett-Lee S, Nagwani M, Lee C-W, Lee C, Adelson S, Kaar E, Gaskin D et al.: Biomass conversion to mixed alcohol fuels using the MixAlco process. Appl Biochem Biotechnol 1999, 77–79 :609-631. 61. Ingram LO, Conway T, Alterthum F: Ethanol production by Escherichia coli strains co-expressing Zymomonas PDC and ADH genes. United States Patent 5000000, 1991. 62. Martinez A, Rodriguez ME, Wells ML,York SW, Preston JF, Ingram LO: Detoxification of dilute acid hydrolysates of lignocellulose with lime. Biotechnol Prog 2001, 17 :287-293. 63. Tao H, Gonzalez R, Martinez A, Rodriguez M, Ingram LO, Preston JF, •• Shanmugam KT: Engineering a homoethanol pathway in Escherichia coli: increased glycolytic flux and levels of expression of glycolytic genes during xylose fermentation. J Bacteriol 2001, 185 :2979-2988. This paper utilizes gene array technology to evaluate direct gene responses to the fermentation environment, in this case xylose-to-ethanol, which will soon become a standard tool of industry as companies complete the genome sequencing of their production strain needed for microarray assay development.