Ethanol production from AFEX pretreated corn fiber by recombinant bacteria

Summary Fermentation of an enzymatic hydrolyzate of ammonia fiber explosion (AFEX) pretreated corn fiber (containing a mixture of different sugars including glucose, xylose, arabinose, and galactose) by genetically-engineered Escherichia coli strain SL40 and KO11 and Klebsiella oxytoca strain P2 was investigated under pH-controlled conditions. Both E. coli strains (SL40 and KO11) efficiently utilized most of the sugars contained in the hydrolyzate and produced a maximum of 26.6 and 27.1 g/l ethanol, respectively, equivalent to 90 and 92% of the theoretical yield. Very little difference was observed in cell growth and ethanol production between fermentations of the enzymatic hydrolyzate and mixtures of pure sugars, simulating the hydrolyzate. These results confirm the fermentability of the AFEX-treated corn fiber hydrolyzate by ethanologenic E. coli. K.oxytoca strain P2, on the other hand, showed comparatively poor growth and ethanol production (maximum 20 g/l) from both enzymatic hydrolyzate and simulated sugar mixtures under the same fermentation conditions.     

Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol

Use of agricultural biomass, other than corn-starch, to produce fuel ethanol requires a microorganism that can ferment the mixture of sugars derived from hemicellulose. Escherichia coli metabolizes a wide range of substrates and has been engineered to produce ethanol in high yield from sugar mixtures. E. coli metabolizes glucose in preference to other sugars and, as a result, utilization of the pentoses in hemicellulose-derived sugar mixtures is delayed and may be incomplete. Residual sugar lowers the ethanol yield and is problematic for downstream processing of fermentation products. Therefore, a catabolite repression mutant that simultaneously utilizes glucose and pentoses would be useful for fermentation of complex substrate mixtures. We constructed ethanologenic E. coli strains with a glucose phosphotransferase (ptsG) mutation and used the mutants to ferment glucose, arabinose, and xylose, singly and in mixtures, to ethanol. Yields were 87-94% of theoretical for both the wild type and mutants, but the mutants had an altered pattern of mixed sugar utilization. Phosphotransferase mutants metabolized the pentoses simultaneously with glucose, rather than sequentially. Based upon fermentations of sugar mixtures, a catabolite-repression mutant of ethanologenic E. coli is expected to provide more efficient fermentation of hemicellulose hydrolysates by allowing direct utilization of pentoses.     

A genetic overhaul of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 424A(LNH-ST) to improve xylose fermentation

Robust microorganisms are necessary for economical bioethanol production. However, such organisms must be able to effectively ferment both hexose and pentose sugars present in lignocellulosic hydrolysate to ethanol. Wild type Saccharomyces cerevisiae can rapidly ferment hexose, but cannot ferment pentose sugars. Considerable efforts were made to genetically engineer S. cerevisiae to ferment xylose. Our genetically engineered S cerevisiae yeast, 424A(LNH-ST), expresses NADPH/NADH xylose reductase (XR) that prefer NADPH and NAD⁺-dependent xylitol dehydrogenase (XD) from Pichia stipitis, and overexpresses endogenous xylulokinase (XK). This strain is able to ferment glucose and xylose, as well as other hexose sugars, to ethanol. However, the preference for different cofactors by XR and XD might lead to redox imbalance, xylitol excretion, and thus might reduce ethanol yield and productivity. In the present study, genes responsible for the conversion of xylose to xylulose with different cofactor specificity (1) XR from N. crassa (NADPH-dependent) and C. parapsilosis (NADH-dependent), and (2) mutant XD from P. stipitis (containing three mutations D207A/I208R/F209S) were overexpressed in wild type yeast. To increase the NADPH pool, the fungal GAPDH enzyme from Kluyveromyces lactis was overexpressed in the 424A(LNH-ST) strain. Four pentose phosphate pathway (PPP) genes, TKL1, TAL1, RKI1 and RPE1 from S. cerevisiae, were also overexpressed in 424A(LNH-ST). Overexpression of GAPDH lowered xylitol production by more than 40%. However, other strains carrying different combinations of XR and XD, as well as new strains containing the overexpressed PPP genes, did not yield any significant improvement in xylose fermentation.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Ethanol production from corn cob hydrolysates by <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Corn cob hydrolysates, with xylose as the dominant sugar, were fermented to ethanol by recombinant Escherichia coli KO11. When inoculum was grown on LB medium containing glucose, fermentation of the hydrolysate was completed in 163 h and ethanol yield was 0.50 g ethanol/g sugar. When inoculum was grown on xylose, ethanol yield dropped, but fermentation was faster (113 h). Hydrolysate containing 72.0 g/l xylose and supplemented with 20.0 g/l rice bran was readily fermented, producing 36.0 g/l ethanol within 70 h. Maximum ethanol concentrations were not higher for fermentations using higher cellular concentration inocula. A simulation of an industrial process integrating pentose fermentation by E. coli and hexose fermentation by yeast was carried out. At the first step, E. coli fermented the hydrolysate containing 85.0 g/l xylose, producing 40.0 g/l ethanol in 94 h. Baker's yeast and sucrose (150.0 g/l) were then added to the spent fermentation broth. After 8 h of yeast fermentation, the ethanol concentration reached 104.0 g/l. This two-stage fermentation can render the bioconversion of lignocellulose to ethanol more attractive due to increased final alcohol concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 124–128 doi:10.1038/sj.jim.7000287 Received 20 February 2002/ Accepted in revised form 04 June 2002     

Metabolic evolution of non-transgenic <Emphasis Type="Italic">Escherichia coli</Emphasis> SZ420 for enhanced homoethanol fermentation from xylose

Efficient utilization of pentose sugars (xylose and arabinose) is an essential requirement for economically viable ethanol production from cellulosic biomass. The desirable pentose-fermenting ethanologenic biocatalysts are the native microorganisms or the engineered derivatives without recruited exogenous gene(s). We have used a metabolic evolution (adaptive selection) approach to improve a non-transgenic homoethanol Escherichia coli SZ420 (ldhA pflB ackA frdBC pdhR::pflBp6-aceEF-lpd) for xylose fermentation. An improved mutant, E. coli KC01, was evolved through a 3 month metabolic evolution process. This evolved mutant increased pyruvate dehydrogenase activity by 100%, cell growth rate (h⁻¹) by 23%, volumetric ethanol productivity by 65% and ethanol tolerance by 200%. These improvements enabled KC01 to complete 50 g xylose l⁻¹ fermentations with an ethanol titer of 23 g l⁻¹ and a yield of 90%. The improved cell growth and ethanol production of KC01 are likely attributed to its three fold increased ethanol tolerance.     

Adaptive evolution of nontransgenic <Emphasis Type="Italic">Escherichia coli</Emphasis> KC01 for improved ethanol tolerance and homoethanol fermentation from xylose

Due to its excellent capability to ferment five-carbon sugars, Escherichia coli has been considered one of the platform organisms to be engineered for production of cellulosic ethanol. Nevertheless, genetically engineered ethanologenic E. coli lacks the essential trait of alcohol tolerance. Development of ethanol tolerance is required for cost-effective ethanol fermentation. In this study, we improved alcohol tolerance of a nontransgenic E. coli KC01 (ldhA pflB ackA frdBC pdhR::pflBp6-aceEF-lpd) through adaptive evolution. During ~350 generations of adaptive evolution, a gradually increased concentration of ethanol was used as a selection pressure to enrich ethanol-tolerant mutants. The evolved mutant, E. coli SZ470, was able to grow anaerobically at 40 g l⁻¹ ethanol, a twofold improvement over parent KC01. When compared with KC01 for small-scale (500 ml) xylose (50 g l⁻¹) fermentation, SZ470 achieved 67% higher cell mass, 48% faster volumetric ethanol productivity, and 50% shorter time to complete fermentation with ethanol titer of 23.5 g l⁻¹ and yield of 94%. These results demonstrate that an industry-oriented nontransgenic E. coli strain could be developed through incremental improvements of desired traits by a combination of molecular biology and traditional microbiology techniques.     

Fermentation of galacturonic acid and other sugars in orange peel hydrolysates by the ethanologenic strain of Escherichia coli

Summary Enzymatic hydrolysates of orange peel contain relatively high levels of galacturonic acid and arabinose which are not fermentable to ethanol by yeasts. We observed complete utilization of both sugars during fermentation of peel hydrolysates by the ethanologenic construct of E. coli KO11. The bacterium exhibits a novel pattern of galacturonic acid fermentation producing equimolar amounts of acetate and ethanol accompanied by carbon dioxide.     

Kinetics of ethanol production by recombinant Escherichia coli KO11

The fermentation kinetics for separate as well as simultaneous glucose and xylose fermentation with recombinant ethanologenic Escherichia coli KO11 are presented. Glucose and xylose were consumed simultaneously and exhibited mutual inhibition. The glucose exhibited 15 times stronger inhibition in xyclose fermentation than vice versa. The fermentation of condensate from steampretreated willow (Salix) was investigated. The kinetics were studied in detoxified as well as in nondetoxified condensate. The fermentation of the condensate followed two phases: First the glucose and some of the pentoses (xylose in addition to small amounts of arabinose) were fermented simultaneously, and then the remaining part of the pentoses were fermented. The rate of the first phase was independent of the detoxification method used, whereas the rate of the second phase was found to be strongly dependent. When the condensate was detoxified with overliming in combination with sulfite, which was the best detoxification method investigated, the sugars in the condensate, 9 g/L, were fermented in 11 h. The same fermentation took 150 h in nondetoxified condensate. The experimental data were used to develop an empirical model, describing the batch fermentation of recombinant E. coli KO11 in the condensate. The model is based on Monod kinetics including substrate and product inhibition and the sum of the inhibition exerted by the rest of the inhibitors, lumped together. (c) 1995 John Wiley & Sons, Inc.     

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

Background

Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose.

Results

The specific aerobic arabinose growth rate was identical, 0.03 h^-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h^-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)^-1 h^-1 than the xylose isomerase strain, which only reached 0.03 h^-1 and 0.02 g (g cells)^-1h^-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g^-1 compared with 0.18 g g^-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g^-1 compared with 0.32 g g^-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l^-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l^-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)^-1 h^-1 compared with 0.01 g (g cells)^-1 h^-1 for the xylose reductase/xylitol dehydrogenase strain and the xylose isomerase strain, respectively.

Conclusion

The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.     

Continuous ethanol production from wheat straw hydrolysate by recombinant ethanologenic <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> strain FBR5

Continuous production of ethanol from alkaline peroxide pretreated and enzymatically saccharified wheat straw hydrolysate by ethanologenic recombinant Escherichia coli strain FBR5 was investigated under various conditions at controlled pH 6.5 and 35°C. The strain FBR5 was chosen because of its ability to ferment both hexose and pentose sugars under semi-anaerobic conditions without using antibiotics. The average ethanol produced from the available sugars (21.9–47.8 g/L) ranged from 8.8 to 17.3 g/L (0.28–0.45 g/g available sugars, 0.31–0.48 g/g sugar consumed) with ethanol productivity of 0.27–0.78 g l⁻¹ h⁻¹ in a set of 14 continuous culture (CC) runs (16–105 days). During these CC runs, no loss of ethanol productivity was observed. This is the first report on the continuous production of ethanol by the recombinant bacterium from a lignocellulosic hydrolysate.     

宿主遗传背景对重组酿酒酵母木糖共发酵影响的研究进展

木糖是纤维素原料水解液中最主要的五碳糖成分,由于野生的酿酒酵母缺乏有效的木糖利用途径,将外源木糖代谢途径整合至酿酒酵母中使其具有发酵木糖生产乙醇的能力是构建纤维素乙醇发酵菌株的关键。国内外学者的研究表明,同一木糖代谢途径导入不同酿酒酵母菌株中,所得到的重组菌发酵性能存在明显差异,表明宿主的遗传背景对菌株利用木糖能力和发酵性能具有重要的影响。就酿酒酵母宿主对重组菌株的木糖发酵性能的影响进行了综述,分析了产生宿主差异的内在机理,为进一步选育高效木糖共发酵菌种提供借鉴。     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Correlation of genomic and physiological traits of thermoanaerobacter species with biofuel yields

Thermophilic anaerobic noncellulolytic Thermoanaerobacter species are of great biotechnological importance in cellulosic ethanol production due to their ability to produce high ethanol yields by simultaneous fermentation of hexose and pentose. Understanding the genome structure of these species is critical to improving and implementing these bacteria for possible biotechnological use in consolidated bioprocessing schemes (CBP) for cellulosic ethanol production. Here we describe a comparative genome analysis of two ethanologenic bacteria, Thermoanaerobacter sp. X514 and Thermoanaerobacter pseudethanolicus 39E. Compared to 39E, X514 has several unique key characteristics important to cellulosic biotechnology, including additional alcohol dehydrogenases and xylose transporters, modifications to pentose metabolism, and a complete vitamin B₁₂ biosynthesis pathway. Experimental results from growth, metabolic flux, and microarray gene expression analyses support genome sequencing-based predictions which help to explain the distinct differences in ethanol production between these strains. The availability of whole-genome sequence and comparative genomic analyses will aid in engineering and optimizing Thermoanaerobacter strains for viable CBP strategies.     

Genetic Engineering of Enterobacter asburiae Strain JDR-1 for Efficient Production of Ethanol from Hemicellulose Hydrolysates

Dilute acid pretreatment is an established method for hydrolyzing the methylglucuronoxylans of hemicellulose to release fermentable xylose. In addition to xylose, this process releases the aldouronate methylglucuronoxylose, which cannot be metabolized by current ethanologenic biocatalysts. Enterobacter asburiae JDR-1, isolated from colonized wood, was found to efficiently ferment both methylglucuronoxylose and xylose in acid hydrolysates of sweet gum xylan, producing predominantly ethanol and acetate. Transformation of E. asburiae JDR-1 with pLOI555 or pLOI297, each containing the PET operon containing pyruvate decarboxylase (pdc) and alcohol dehydrogenase B (adhB) genes derived from Zymomonas mobilis, replaced mixed-acid fermentation with homoethanol fermentation. Deletion of the pyruvate formate lyase (pflB) gene further increased the ethanol yield, resulting in a stable E. asburiae E1(pLOI555) strain that efficiently utilized both xylose and methylglucuronoxylose in dilute acid hydrolysates of sweet gum xylan. Ethanol was produced from xylan hydrolysate by E. asburiae E1(pLOI555) with a yield that was 99% of the theoretical maximum yield and at a rate of 0.11 g ethanol/g (dry weight) cells/h, which was 1.57 times the yield and 1.48 times the rate obtained with the ethanologenic strain Escherichia coli KO11. This engineered derivative of E. asburiae JDR-1 that is able to ferment the predominant hexoses and pentoses derived from both hemicellulose and cellulose fractions is a promising subject for development as an ethanologenic biocatalyst for production of fuels and chemicals from agricultural residues and energy crops.Lignocellulosic resources, including forest and agricultural residues and evolving energy crops, offer benign alternatives to petroleum-based resources for production of fuels and chemicals. As renewable resources, these lignocellulosic materials are expected to decrease dependence on exhaustible supplies of petroleum and mitigate the net release of carbon dioxide into the atmosphere. The development of economically acceptable bioconversion processes requires pretreatments that release the maximal quantities of hexoses (predominantly glucose released from cellulose) and pentoses (arabinose and xylose) from hemicelluloses and also requires microbial biocatalysts that efficiently convert these compounds to a single targeted product.As one of three main components of lignocellulosics, hemicellulose contains polysaccharides comprised of pentoses, hexoses and sugar acids that account for 20 to 35% of the total biomass from different sources (21). Methylglucuronoxylans (MeGAX_n), consisting of long chains of as many as 70 β-xylopyranose residues linked by β-1,4-glycosidic bonds (25), are the predominant components in the hemicellulose fractions of agricultural residues and energy crops, including corn stover, sugarcane bagasse, poplar, and switchgrass (7, 18, 23, 24). In hardwood and softwood xylans, a 4-O-methylglucuronic acid is attached at the 2′ position of every sixth to eighth xylose residue (12, 15). Dilute acid hydrolysis is commonly used to make the monosaccharides comprising hemicellulose accessible for fermentation (7, 22). However, the α-1,2 glucuronosyl linkage in xylan is resistant to dilute acid hydrolysis, which results in the release of methylglucuronoxylose (MeGAX) along with xylose and other monosaccharides. MeGAX is not fermented by bacterial biocatalysts currently used to convert hemicellulose to ethanol, such as Escherichia coli KO11 (2, 6). In sweet gum xylan, as much as 27% of the carbohydrate may be in this unfermentable fraction after dilute acid pretreatment (2, 20). Complete utilization of all hemicellulosic sugars can improve the efficiency of conversion of lignocellulosic materials to fuel ethanol and other value-added products.Our previous research on the processing of hemicelluloses for fermentation led to isolation of Enterobacter asburiae strain JDR-1. This isolate performed mixed-acid fermentation of the principal hexoses and pentoses that can be derived from cellulose and hemicellulose fractions of lignocellulosic biomass and exhibited a novel metabolic potential based on its ability to ferment MeGAX and xylose to ethanol and acetate as major fermentation products from sweet gum MeGAX_n hydrolysates generated by dilute acid pretreatment (2). This strain has been genetically modified to produce d-(−)-lactate as the predominant product from acid hydrolysates of MeGAX_n (3).In this study, the PET operon containing the pdc, adhA, and adhB genes from Zymomonas mobilis (10, 11) was incorporated into a pflB E. asburiae JDR-1 isolate by plasmid transformation to construct homoethanologenic strains. The resulting recombinant strains were compared with E. asburiae wild-type strain JDR-1 and the ethanologenic strain E. coli KO11 to evaluate their efficiencies of production of ethanol from dilute acid hydrolysates of sweet gum MeGAX_n.     

Metabolite labelling reveals hierarchies in Clostridium acetobutylicum that selectively channel carbons from sugar mixtures towards biofuel precursors

Clostridial fermentation of cellulose and hemicellulose relies on the cellular physiology controlling the metabolism of the cellulosic hexose sugar (glucose) with respect to the hemicellulosic pentose sugars (xylose and arabinose) and the hemicellulosic hexose sugars (galactose and mannose). Here, liquid chromatography–mass spectrometry and stable isotope tracers in Clostridium acetobutylicum were applied to investigate the metabolic hierarchy of glucose relative to the different hemicellulosic sugars towards two important biofuel precursors, acetyl‐coenzyme A and butyryl‐coenzyme A. The findings revealed constitutive metabolic hierarchies in C. acetobutylicum that facilitate (i) selective investment of hemicellulosic pentoses towards ribonucleotide biosynthesis without substantial investment into biofuel production and (ii) selective contribution of hemicellulosic hexoses through the glycolytic pathway towards biofuel precursors. Long‐term isotopic enrichment demonstrated incorporation of both pentose sugars into pentose‐phosphates and ribonucleotides in the presence of glucose. Kinetic labelling data, however, showed that xylose was not routed towards the biofuel precursors but there was minor contribution from arabinose. Glucose hierarchy over the hemicellulosic hexoses was substrate‐dependent. Kinetic labelling of hexose‐phosphates and triose‐phosphates indicated that mannose was assimilated but not galactose. Labelling of both biofuel precursors confirmed this metabolic preference. These results highlight important metabolic considerations in the accounting of clostridial mixed‐sugar utilization.     

Novel two‐stage fermentation process for bioethanol production using Saccharomyces pastorianus

Bioethanol produced from lignocellulosic materials has the potential to be economically feasible, if both glucose and xylose released from cellulose and hemicellulose can be efficiently converted to ethanol. Saccharomyces spp. can efficiently convert glucose to ethanol; however, xylose conversion to ethanol is a major hurdle due to lack of xylose‐metabolizing pathways. In this study, a novel two‐stage fermentation process was investigated to improve bioethanol productivity. In this process, xylose is converted into biomass via non‐Saccharomyces microorganism and coupled to a glucose‐utilizing Saccharomyces fermentation. Escherichia coli was determined to efficiently convert xylose to biomass, which was then killed to produce E. coli extract. Since earlier studies with Saccharomyces pastorianus demonstrated that xylose isomerase increased ethanol productivities on pure sugars, the addition of both E. coli extract and xylose isomerase to S. pastorianus fermentations on pure sugars and corn stover hydrolysates were investigated. It was determined that the xylose isomerase addition increased ethanol productivities on pure sugars but was not as effective alone on the corn stover hydrolysates. It was observed that the E. coli extract addition increased ethanol productivities on both corn stover hydrolysates and pure sugars. The ethanol productivities observed on the corn stover hydrolysates with the E. coli extract addition was the same as observed on pure sugars with both E. coli extract and xylose isomerase additions. These results indicate that the two‐stage fermentation process has the capability to be a competitive alternative to recombinant Saccharomyces cerevisiae‐based fermentations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:300–310, 2014     

Ethanol production from pentoses and sugar-cane bagasse hemicellulose hydrolysate by Mucor and Fusarium species

The fermentation of carbohydrates and hemicellulose hydrolysate by Mucor and Fusarium species has been investigated, with the following results. Both Mucor and Fusarium species are able to ferment various sugars and alditols, including d-glucose, pentoses and xylitol, to ethanol. Mucor is able to ferment sugar-cane bagasse hemicellulose hydrolysate to ethanol. Fusarium F5 is not able to ferment sugar-cane bagasse hemicellulose hydrolysate to ethanol. During fermentation of hemicellulose hydrolysates, d-glucose was utilized first, followed by d-xylose and l-arabinose. Small amounts of xylitol were produced by Mucor from d-xylose through oxidoreduction reactions, presumably mediated by the enzyme aldose reductase¹ (alditol: NADP⁺ 1-oxidoreductase, EC 1.1.1.21). For pentose fermentation, d-xylose was the preferred substrate. Only small amounts of ethanol were produced from l-arabinose and d-arabitol. No ethanol was produced from l-xylose, d-arabinose or l-arabitol.