Direct Ethanol Fermentation Research Articles

Technical assessment of natural deep eutectic solvent (NADES) mediated biorefinery process: A case study

Comprehensive evaluation of an integrated process for cellulosic ethanol production from lactic acid+choline chloride+water (LA-CC-H2O) mixture based natural deep eutectic solvent (NADES) pretreated rice straw; solvent recovery and its reusability; extraction of value added products viz., lignin and xylan; enzymatic saccharification and fermentation were studied. Four separate processes viz. pretreatment at 5% (w/v) and 10% (w/v) solids loading and enzymatic hydrolysis at 10% (w/v) and 25% (w/v) solids loading were investigated. No significant difference was observed in holocellulose recovery and lignin solubility in all the tested processes. Hydrolysis at 10% (w/v) and 25% (w/v) solids loading produced maximum glucose yields of 38.6±1.2g/L and 90.3±2.3g/L, respectively. Additional concentration step of sugars is needed from 10% (w/v) hydrolysis for direct ethanol fermentation using Clavispora NRRL Y-50464, while this could be voided with 25% (w/v) hydrolysis sugars. Maximum ethanol yield of 36.7g/L was obtained with 9% (w/v) glucose with a theoretical conversion efficiency of 79.9% which estimated to produce ~129L of ethanol per ton of raw biomass. Alongside, NADES pretreatment resulted in solubilization of 72-75% (w/v) of biomass bound lignin from which 86–90% (w/v) of lignin was recovered. Additionally, >98% (v/v) of the treated NADES reagent was recovered and reused in three consecutive biomass pretreatment cycles with no loss in pretreatment efficiency. This study clearly demonstrated that pretreatment at 10% (w/v) solids loading followed by enzymatic hydrolysis at 25% (w/v) solids loading is the most effective integrated biorefinery process using NADES pretreated rice straw.

Direct Ethanol Production from Ionic Liquid-Pretreated Lignocellulosic Biomass by Cellulase-Displaying Yeasts.

Among the many types of lignocellulosic biomass pretreatment methods, the use of ionic liquids (ILs) is regarded as one of the most promising strategies. In this study, the effects of four kinds of ILs for pretreatment of lignocellulosic biomass such as bagasse, eucalyptus, and cedar were evaluated. In direct ethanol fermentation from biomass incorporated with ILs by cellulase-displaying yeast, 1-butyl-3-methylimidazolium acetate ([Bmim][OAc]) was the most effective IL. The ethanol production and yield from [Bmim][OAc]-pretreated bagasse reached 0.81g/L and 73.4% of the theoretical yield after fermentation for 96h. The results prove the initial concept, in which the direct fermentation from lignocellulosic biomass effectively promoted by the pretreatment with IL.

Direct ethanol fermentation of the algal storage polysaccharide laminarin with an optimized combination of engineered yeasts

Laminarin is the algal storage glucan and represents up to 35% of the dry weight of brown macroalgae. In this study, a novel laminarinase, Gly5M, was first found using focused proteome analysis of a laminarin-assimilating marine bacterium, Saccharophagus degradans, and the encoding gene was isolated. A Gly5M-displaying yeast strain was prepared with the cell surface display system using Saccharomyces cerevisiae. It showed a laminarin-degrading activity on the cell surface and caused the dominant accumulation of gentiobiose. The obtained gentiobiose was converted into glucose and could be assimilated by an Aspergillus aculeatus β-glucosidase (BG)-displaying yeast strain. When Gly5M- and BG-displaying yeasts were anaerobically cultivated together in fermentation medium containing 20g/L laminarin as a sole carbon source, the coculture system with the combination of optimized ratios of the 2 yeast strains directly produced 5.2g/L ethanol. This coculture system of the 2 engineered yeast strains would be a platform for the use of laminarin and contribute to the complete utilization of brown macroalgae.

Bioethanol production using the sodium hydroxide pretreated sweet sorghum bagasse without washing

Sweet sorghum, with its multi-platform of resources of starch, sugar and cellulose, is considered a very promising energy crop to produce bioethanol. In this study, bioethanol production of sodium hydroxide (NaOH) pretreated sweet sorghum bagasse without solid substrate washing was investigated. The results showed that direct enzymatic saccharification and ethanol fermentation of NaOH pretreated solid substrate without washing significantly improved the fermentable sugars conversion and ethanol theoretical yield from 44.06±0.93% and 44.85±1.15% to 65.14±0.19% and 61.81±1.28%, respectively. Experiments where NaOH pretreated liquor (SL) was added into the thoroughly washed NaOH pretreated solid substrate showed the fermentable sugars conversion yield improved significantly. Furthermore, analysis of enzymes activities during enzymatic saccharification showed that SL maintained cellulase activity significantly, especially for that of xylanase activity. These results suggested that the NaOH pretreated lignocellulosic solid can be employed for direct enzymatic saccharification and subsequent ethanol fermentation without washing. This process would improve the sustainability and economic viability of bioethanol production.

Direct Ethanol Production from Steam-Exploded Corn Stover using a Synthetic Diploid Cellulase-displaying Yeast Consortium

The assembly of functional cellulolytic enzymes was displayed using a synthetic, cell-surface engineered diploid yeast consortium. Trichoderma reesei endoglucanase II (EGII), cellobiohydrolase II (CBHII), and Aspergillus aculeatus β-glucosidase I (BGLI) were displayed as fusion proteins with the AGA2p C-terminus of a-agglutinin on the cell surface of the diploid yeast strain Saccharomyces cerevisiae Y5. The cell-surface immobilization of each enzyme was confirmed by immunofluorescence microscopy. This type of yeast consortium allowed convenient optimization of ethanol production by adjusting the combination ratios of each cell type for inducing synergy in cellulose hydrolysis. Next, the direct ethanol fermentation from steam-exploded corn stover was investigated. The optimized cellulase-displaying consortium produced 20.4 g/L ethanol from 48.4 g cellulose per liter after 72 h in the presence of a small amount of cellulase reagent (0.9 FPU/mL). These results suggested the feasibility of the cellulase-displaying yeast consortium for simultaneous saccharification and fermentation from insoluble cellulosic materials.

Ethanol production from Jerusalem artichoke tubers at high temperature by newly isolated thermotolerant inulin-utilizing yeast Kluyveromyces marxianus using consolidated bioprocessing.

Thermotolerant inulin-utilizing yeast strains were successfully isolated in this study. Among the isolated strains, Kluyveromyces marxianus DBKKU Y-102 was found to be the most effective strain for direct ethanol fermentation at high temperature from fresh Jerusalem artichoke (JA) tubers without inulin hydrolysis under consolidated bioprocessing (CBP). The maximum ethanol concentrations produced by this strain under the optimum culture conditions were 104.83 and 97.46 g L(-1) at 37 and 40 °C, respectively. Data from this study clearly demonstrated that the use of thermotolerant inulin-utilizing yeast K. marxianus for ethanol production from fresh JA tubers in the CBP process not only provided high levels of ethanol, but also could eliminate the addition of external enzyme for inulin hydrolysis, which might lead to the reduction of operating costs. The expression of genes involved in carbohydrate metabolism in K. marxianus DBKKU Y-102 during ethanol fermentation was investigated by real-time RT-PCR, and the results revealed that expression levels were distinctive depending on the growth phase and growth conditions. However, among the genes tested, adh4 and tdh2 were highly expressed under high temperature conditions in both exponential- and stationary-growth phases, suggesting that these genes might play a crucial role in acquiring thermotolerance ability in this organism under stress conditions.

Improvement of direct ethanol fermentation from woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low temperature condition

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 can quite uniquely ferment various sugars under low temperature conditions. When strain SK-4 fermented lignocellulosic biomass using the direct ethanol fermentation (DEF) technique, approximately 30% to 65% of the theoretical ethanol yield was obtained without and with the addition of the non-ionic surfactant Tween 80, respectively. Therefore, DEF from lignocellulosic biomass with M. blollopis SK-4 requires the addition of a non-ionic surfactant to improve fermentation efficiency. DEF with lipase converted Eucalyptus and Japanese cedar to 12.6g/l, and 14.6g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80 and 5U/g-dry substrate lipase, ethanol concentration increased about 1.4- to 2.4-fold compared to that without Tween 80 and lipase. We therefore consider that the combination of M. blollopis SK-4 and DEF with Tween 80 and lipase has good potential for ethanol fermentation in cold environments.

Direct fermentation of amorphous cellulose to ethanol by engineered Saccharomyces cerevisiae coexpressing Trichoderma viride EG3 and BGL1.

Direct ethanol fermentation from amorphous cellulose was achieved using an engineered industrial Saccharomyces cerevisiae strain. Two cellulase genes endoglucanase (eg3) and β-glucosidase (bgl1) were obtained from Trichoderma viride and integrated into the genome of S. cerevisiae. These two cellulases could be constitutively coexpressed and secreted by the recombinant strain S. cerevisiae-eb. The enzyme activities were analyzed in the culture supernatants, with the highest endoglucanase activity of 2.34 units/ml and β-glucosidase activity of 0.95 units/ml. The effects of pH, temperature and metal ions on enzyme activities were analyzed. The coexpression strain S. cerevisiae-eb could grow in carboxymethyl cellulose (CMC) and utilize it as the single carbon source. The 20 g/L CMC as a model substrate of amorphous cellulose was used in fermentation. The ethanol production reached 4.63 g/L in 24 h, with the conversion ratio of 64.2% compared with the theoretical concentration. This study demonstrated that the engineered industrial strain S. cerevisiae-eb could convert amorphous cellulose to ethanol simultaneously and achieve consolidated bioprocessing (CBP) directly.

Synergetic effect of yeast cell-surface expression of cellulase and expansin-like protein on direct ethanol production from cellulose

BackgroundNumerous studies have examined the direct fermentation of cellulosic materials by cellulase-expressing yeast; however, ethanol productivity in these systems has not yet reached an industrial level. Certain microorganisms, such as the cellulolytic fungus Trichoderma reesei, produce expansin-like proteins, which have a cellulose-loosening effect that may increase the breakdown of cellulose. Here, to improve the direct conversion of cellulose to ethanol, yeast Saccharomyces cerevisiae co-displaying cellulase and expansin-like protein on the cell surface were constructed and examined for direct ethanol fermentation performance.ResultsThe cellulase and expansin-like protein co-expressing strain showed 246 mU/g-wet cell of phosphoric acid swollen cellulose (PASC) degradation activity, which corresponded to 2.9-fold higher activity than that of a cellulase-expressing strain. This result clearly demonstrated that yeast cell-surface expressed cellulase and expansin-like protein act synergistically to breakdown cellulose. In fermentation experiments examining direct ethanol production from PASC, the cellulase and expansin-like protein co-expressing strain produced 3.4 g/L ethanol after 96 h of fermentation, a concentration that was 1.4-fold higher than that achieved by the cellulase-expressing strain (2.5 g/L).ConclusionsThe PASC degradation and fermentation ability of an engineered yeast strain was markedly improved by co-expressing cellulase and expansin-like protein on the cell surface. To our knowledge, this is the first report to demonstrate the synergetic effect of co-expressing cellulase and expansin-like protein on a yeast cell surface, which may be a promising strategy for constructing direct ethanol fermenting yeast from cellulose.

Direct ethanol fermentation from lignocellulosic biomass by Antarctic basidiomycetous yeast Mrakia blollopis under a low temperature condition

Antarctic basidiomycetous yeast Mrakia blollopis SK-4 has unique fermentability for various sugars under a low temperature condition. Hence, this yeast was used for ethanol fermentation from glucose and also for direct ethanol fermentation (DEF) from cellulosic biomass without/with Tween 80 at 10°C. Maximally, 48.2g/l ethanol was formed from 12% (w/v) glucose. DEF converted filter paper, Japanese cedar and Eucalyptus to 12.2g/l, 12.5g/l and 7.2g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80, ethanol concentration increased by about 1.1–1.6-fold compared to that without Tween 80. This is the first report on DEF using cryophilic fungi under a low temperature condition. We consider that M. blollopis SK-4 has a good potential for ethanol fermentation in cold environments.

Efficient direct ethanol production from cellulose by cellulase- and cellodextrin transporter-co-expressing Saccharomyces cerevisiae

Efficient degradation of cellulosic biomass requires the synergistic action of the cellulolytic enzymes endoglucanase, cellobiohydrolase, and β-glucosidase. Although there are many reports describing consolidation of hydrolysis and fermentation steps using recombinant Saccharomyces cerevisiae that express cellulolytic enzymes, the efficiency of cellulose degradation has not been sufficiently improved. Although the yeast S. cerevisiae cannot take up cellooligosaccharide, some fungi can take up and assimilate cellooligosaccharide through a cellodextrin transporter. In this study, a S. cerevisiae strain co-expressing genes for several cell surface display cellulases and the cellodextrin transporter was constructed for the purpose of improving the efficiency of direct ethanol fermentation from phosphoric acid swollen cellulose (PASC). The cellulase/cellodextrin transporter-coexpressing strain produced 1.7-fold more ethanol (4.3 g/L) from PASC during a 72-h fermentation than did a strain expressing cellulase only (2.5 g/L). Direct ethanol production from PASC by the recombinant S. cerevisiae strain was improved by co-expression of cellulase display and cellodextrin transporter genes. These results suggest that cellulase- and cellodextrin transporter-co-expressing S. cerevisiae could be a promising technology for efficient direct ethanol production from cellulose.

Ethanol production from high cellulose concentration by the basidiomycete fungus Flammulina velutipes

Ethanol production by Flammulina velutipes from high substrate concentrations was evaluated. F. velutipes produces approximately 40–60 g l−1 ethanol from 15 % (w/v) d-glucose, d-fructose, d-mannose, sucrose, maltose, and cellobiose, with the highest conversion rate of 83 % observed using cellobiose as a carbon source. We also attempted to assess direct ethanol fermentation from sugarcane bagasse cellulose (SCBC) by F. velutipes. The hydrolysis rate of 15 % (w/v) SCBC with commercial cellulase was approximately 20 %. In contrast, F. velutipes was able to produce a significant amount of ethanol from 15 % SCBC with the production of β-glucosidase, cellobohydrolase, and cellulase, although the addition of a small amount of commercial cellulase to the culture was required for the conversion. When 9 mg g−1 biomass of commercial cellulase was added to cultures, 0.36 g of ethanol was produced from 1 g of cellulose, corresponding to an ethanol conversion rate of 69.6 %. These results indicate that F. velutipes would be useful for consolidated bioprocessing of lignocellulosic biomass to bioethanol.

Construction of a novel selection system for endoglucanases exhibiting carbohydrate-binding modules optimized for biomass using yeast cell-surface engineering

To permit direct cellulose degradation and ethanol fermentation, Saccharomyces cerevisiae BY4741 (Δsed1) codisplaying 3 cellulases (Trichoderma reesei endoglucanase II [EG], T. reesei cellobiohydrolase II [CBH], and Aspergillus aculeatus β-glucosidase I [BG]) was constructed by yeast cell-surface engineering. The EG used in this study consists of a family 1 carbohydrate-binding module (CBM) and a catalytic module. A comparison with family 1 CBMs revealed conserved amino acid residues and flexible amino acid residues. The flexible amino acid residues were at positions 18, 23, 26, and 27, through which the degrading activity for various cellulose structures in each biomass may have been optimized. To select the optimal combination of CBMs of EGs, a yeast mixture with comprehensively mutated CBM was constructed. The mixture consisted of yeasts codisplaying EG with mutated CBMs, in which 4 flexible residues were comprehensively mutated, CBH, and BG. The yeast mixture was inoculated in selection medium with newspaper as the sole carbon source. The surviving yeast consisted of RTSH yeast (the mutant sequence of CBM: N18R, S23T, S26S, and T27H) and wild-type yeast (CBM was the original) in a ratio of 1:46. The mixture (1 RTSH yeast and 46 wild-type yeasts) had a fermentation activity that was 1.5-fold higher than that of wild-type yeast alone in the early phase of saccharification and fermentation, which indicates that the yeast mixture with comprehensively mutated CBM could be used to select the optimal combination of CBMs suitable for the cellulose of each biomass.

Direct saccharification and ethanol fermentation of cello-oligosaccharides with recombinant yeast

Ethanol was produced at good rates by direct saccharification and fermentation of cello-oligosaccharides with pYBGA1 yeast, a recombinant laboratory yeast expressing β-glucosidase. Cellobiose in the concentration of 50g/L was directly fermented for 60h with 1×108cells/mL of pYBGA1 yeast at 30°C to give ethanol at an 80% theoretical conversion rate and a concentration of more than 20g/L of concentration. Conversion to ethanol increased with increasing cellobiose concentration in the feed. When cellobiose was used at the concentration of 100g/L, ethanol conversion and concentration increased to 85% and 45g/L, respectively, in 96h incubation. Other cello-oligosaccharides, cellotriose, cellotetraose, and cellopentaose at the concentration of 50g/L, respectively, were also fermented directly for 72h with 1×108cells/mL of pYBGA1 yeast to produce ethanol in the conversion rates and concentrations of 71–73% and 18.0–18.5g/L, respectively. The direct saccharification and fermentation mechanism of cello-oligosaccharides with pYBGA1 yeast, as revealed by HPLC measurements, suggesting that cellotetraose, for example, was saccharificated to cellotriose, cellobiose, and glucose and then fermented to give ethanol. These results suggest that the direct saccharification and fermentation of cello-oligosaccharides with pYBGA1 has several advantages as a simple procedure and for time, cost, and energy consumptions.

Kluyveromyces marxianus-based platform for direct ethanol fermentation and recovery from cellulosic materials under air-ventilated conditions

Consolidated bioprocessing represents an attractive approach to converting cellulosic materials into bioethanol, yet is practically unavailable. We developed a ventilation-mediated, simultaneous ethanol fermentation and recovery system. Running the system under air-supplied conditions, apparently pure ethanol (28g) was recovered from cellobiose (100g) by growing recombinant Kluyveromyces marxianus expressing β-glucosidase.

Nutrient and alkalinity removal by corn grain, stover and cob harvest in Upper Midwest USA

Abstract In the USA, most corn stover currently remains in fields as crop residue that provides soil erosion control and maintains soil organic carbon levels. This stover is a potential biofuel feedstock for direct combustion, pyrolysis, and ethanol fermentation. At a research site in south central Wisconsin, the northern edge of the US Corn Belt, corn grain harvest averaged 9.8 Mg ha −1 DM over a 6-year period, 1997 to 2002. Removal of all stover could recover an additional 7.2 Mg ha −1 y −1 DM and, in the process, remove an additional 47, 6, 81 and 197 kg ha −1 y −1 of N, P, K and calcium carbonate equivalent, respectively. The fertilizer replacement cost for stover removal is 32 $ Mg −1 DM, which is 95% of the fertilizer value of the grain. However, most of the N, P, K and alkalinity of the stover is found in the leaves, stalk, and husks, not in the cob. At our study site, complete stover removal would export 235 $ ha −1 y −1 of fertilizer and limestone, mainly as K, while cob export would be worth 20 $ ha −1 y −1 in nutrient equivalents. Based on this research, removal of cobs only is equivalent to 16.6% of total stover removal but with a greatly reduced fertilizer replacement cost of 17 $ Mg −1 DM and the same energy density.

Ethanol fermentation from Jerusalem artichoke powder using Saccharomyces cerevisiae KCCM50549 without pretreatment for inulin hydrolysis

A strain of Saccharomyces cerevisiae, KCCM50549, was found to efficiently ferment the inulin-containing carbohydrates in Jerusalem artichoke without acidic or enzymatic pretreatment prior to fermentation. S. cerevisiae KCCM50549 could utilize almost completely the fructo-oligosaccharides present in Jerusalem artichoke (up to degree of polymerization (DP) of 15), in contrast to the other S. cerevisiae strain such as NCYC625 that fermented the fructo-oligosaccharides with DP of up to around six. Inulin-fermenting S. cerevisiae KCCM50549 produced c.a. 1.6 times more ethanol from Jerusalem artichoke compared with S. cerevisiae NCYC625. Direct ethanol fermentation of Jerusalem artichoke flour at 180 g/L without any supplements or pretreatments by S. cerevisiae KCCM50549 in a 5 L jar fermentor yielded 36.2 g/L of ethanol within 36 h. The conversion efficiency of inulin-type sugars to ethanol was 70% of the theoretical ethanol yield.

Ethanol production from cellulosic materials using cellulase‐expressing yeast

We demonstrate direct ethanol fermentation from amorphous cellulose using cellulase-co-expressing yeast. Endoglucanases (EG) and cellobiohydrolases (CBH) from Trichoderma reesei, and beta-glucosidases (BGL) from Aspergillus aculeatus were integrated into genomes of the yeast strain Saccharomyces cerevisiae MT8-1. BGL was displayed on the yeast cell surface and both EG and CBH were secreted or displayed on the cell surface. All enzymes were successfully expressed on the cell surface or in culture supernatants in their active forms, and cellulose degradation was increased 3- to 5-fold by co-expressing EG and CBH. Direct ethanol fermentation from 10 g/L phosphoric acid swollen cellulose (PASC) was also carried out using EG-, CBH-, and BGL-co-expressing yeast. The ethanol yield was 2.1 g/L for EG-, CBH-, and BGL-displaying yeast, which was higher than that of EG- and CBH-secreting yeast (1.6 g/L ethanol). Our results show that cell surface display is more suitable for direct ethanol fermentation from cellulose.

In this issue: Biotechnology Journal 5/2010

AbstractBaker's yeast for biofuelYanase et al., Biotechnol. J. 2010, 5, 449–455Environmental concerns and the depletion of oil reserves result in governmental incentives to establish greater energy independence. At present, research on environmentally friendly and sustainable biofuels focusses on ethanol. The yeast S. cerevisiae is a highly suitable microorganism for bioethanol production because of its high ethanol productivity, high ethanol tolerance, high endurance in industrial processes, and the simplicity of genetic engineering. However, S. cerevisiae cannot utilize cellulosic materials, and a cellulose saccharification process to produce glucose is necessary before fermentation. Scientists from Kobe (Japan) now demonstrate direct ethanol fermentation from amorphous cellulose using cellulase‐co‐expressing yeast. Cellulose degradation is increased 3‐ to 5‐fold and the ethanol yield was 2.1 g/L, showing that cell surface display of cellulase is highly suitable for direct ethanol fermentation from cellulose.PEGylation and chromatographyAbe et al., Biotechnol. J. 2010, 5, 477–483Although various technologies have been extensively studied as drug delivery systems, PEGylation, the covalent attachment of polyethylene glycol (PEG) chains to protein, is quite attractive for development of protein or peptide drugs. The retention and binding mechanisms in electrostatic interaction‐based chromatography (ion‐exchange chromatography) of PEGylated proteins is here investigated by researchers from Ube, Japan. Their findings indicate that when a protein is mono‐PEGylated, the binding site is not affected and the elution volume reduces due to the steric hindrance between PEGylated protein and ion‐exchange ligand.Automatable bilayer chipsPoulos et al., Biotechnol. J. 2010, 5, 511–514As a step towards an automated and operator‐free ion channel measurement platform, scientists from Incheon, Korea, have previously demonstrated a solution formulation for artificial lipid bilayers that enable indefinite storage and shipping of frozen bilayer precursors. Here, they have adapted pin tools to deposit the bilayer precursor solutions onto multi‐element arrays, which is a popular method for microarray solution deposition. The pin tools enable the deposited volume to be applied in a repeatable and highly controlled manner. This results in reduction of bilayer formation times to <1 h. The pin tools are also compatible with computerized motion control platforms, enabling automated and high throughput production. This technology can be applied to produce high density bilayer arrays for high throughput measurement of ion channels incorporated into artificial bilayers.

Cellulosic alcoholic fermentation using recombinantSaccharomyces cerevisiaeengineered for the production ofClostridium cellulovoransendoglucanase andSaccharomycopsis fibuligeraβ-glucosidase

In this study, Saccharomyces cerevisiae was engineered for simultaneous saccharification and fermentation of cellulose by the overexpression of the endoglucanase D (EngD) from Clostridium cellulovorans and the beta-glucosidase (Bgl1) from Saccharomycopsis fibuligera. To promote secretion of the two enzymes, the genes were fused to the secretion signal of the S. cerevisiaealpha mating factor gene. The recombinant developed yeast could produce ethanol through simultaneous production of sufficient extracellular endoglucanase and beta-glucosidase. When direct ethanol fermentation from 20 g L(-1)beta-glucan as a substrate was performed with our recombinant strains, the ethanol concentration reached 9.15 g L(-1) after 50 h of fermentation. The conversion ratio of ethanol from beta-glucan was 80.3% of the theoretical ethanol concentration produced from 20 g L(-1)beta-glucan. In conclusion, we have demonstrated the construction of a yeast strain capable of conversion of a cellulosic substrate to ethanol, representing significant progress towards the realization of processing of cellulosic biomass in a consolidated bioprocessing configuration.