Production of bioethanol from organic whey using <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Ethanol production by K. marxianus in whey from organic cheese production was examined in batch and continuous mode. The results showed that no pasteurization or freezing of the whey was necessary and that K. marxianus was able to compete with the lactic acid bacteria added during cheese production. The results also showed that, even though some lactic acid fermentation had taken place prior to ethanol fermentation, K. marxianus was able to take over and produce ethanol from the remaining lactose, since a significant amount of lactic acid was not produced (1–2 g/l). Batch fermentations showed high ethanol yield (~0.50 g ethanol/g lactose) at both 30°C and 40°C using low pH (4.5) or no pH control. Continuous fermentation of nonsterilized whey was performed using Ca-alginate-immobilized K. marxianus. High ethanol productivity (2.5–4.5 g/l/h) was achieved at dilution rate of 0.2/h, and it was concluded that K. marxianus is very suitable for industrial ethanol production from whey.     

Production of ethanol by the thermotolerant yeastKluyveromyces marxianus IMB3 during growth on lactose-containing media

Summary The thermotolerant yeast strain,Kluyveromyces marxianus IMB3 was shown to be capable of growth and ethanol production on lactose containing media at 45°C. On media containing 4% (w/v) lactose, ethanol production increased to 6.0g/l within 50h and this represented 29% of theoretical yield. During growth on lactose containing media the organism was shown to produce a cell-associated β-galactosidase and no significant enzyme could be detected in the extracellular culture filtrate. Addition of β-galactosidase, released fromKluyveromyces marxianus IMB3 cells, to active fermentations, resulted in increasing ethanol production to 53% of theoretical yield at 45°C.     

Studies on the use of a thermotolerant strain of Kluyveromyces marxianus in simultaneous saccharification and ethanol formation from cellulose

 The thermotolerant yeast strain, Kluyveromyces marxianus IMB3, was found to be capable of ethanol production during growth at 45°C on media containing milled paper and exogenously added commercial cellulase. At maximum achievable cellulose concentrations in shake-flask cultures, ethanol production increased to 6.6 g/l at 45°C, representing an overall level of conversion of 21% of the maximum theoretical yield. Subsequent studies involving variations in added cellulase concentrations to the batch systems demonstrated that ethanol yields could be increased to 10 g/l at 45°C, which represented 39% of the maximum theoretical yield. As a result of ethanol production at 45°C in the systems examined, we suggest that the thermotolerant ethanol-producing yeast strain K. marxianus represents a novel candidate for use in simultaneous saccharification and conversion of the resulting substrates to ethanol. Received: 9 June 1994/Received revision: 8 August 1994/Accepted: 12 August 1994     

Production of thermotolerant and alkalotolerant cellulolytic enzymes by isolated <Emphasis Type="Italic">Nocardiopsis</Emphasis> sp. KNU

A novel cellulolytic bacterium was isolated from the forest soil of KNU University campus. Through 16S rRNA sequence matching and morphological observation it was identified as Nocardiopsis sp. KNU. This strain can utilize a broad range of cellulosic substrates including: carboxymethyl cellulose (CMC), avicel, xylan, cellobiose, filter paper and rice straw by producing a large amount of thermoalkalotolerant endoglucanase, exoglucanase, xylanase and glucoamylase. Optimal culture conditions (Dubos medium, 37°C, pH 6.5 and static condition) for the maximal production of the cellulolytic enzymes were determined. The activity of cellulolytic and hemicelluloytic enzymes produced by this strain was mainly present extracellularly and the enzyme production was dependent on the cellulosic substrates used for the growth. Effect of physicochemical conditions and metal additives on the cellulolytic enzymes production were systematically investigated. The cellulases produced by Nocardiopsis sp. KNU have an optimal temperature of 40°C and pH of 5.0. These cellulases also have high thermotolerance as evidenced by retaining 55–70% activity at 80°C and pH of 5.0 and alkalotolerance by retaining >55% of the activity at pH 10 and 40°C after 1 h. The efficiency of fermentative conversion of the hydrolyzed rice straw by Saccharomyces cerevisiae (KCTC-7296) resulted in 64% of theoretical ethanol yield.     

Glutamate production from β-glucan using endoglucanase-secreting <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

We demonstrate glutamate production from β-glucan using endoglucanase (EG)-expressing Corynebacterium glutamicum. The signal sequence torA derived from Escherichia coli K12, which belongs to the Tat pathway, was suitable for secreting EG of Clostridium thermocellum using C. glutamicum as a host. Using the torA signal sequence, endoglucanase from Clostridium cellulovorans 743B was successfully expressed, and the secreted EG produced 123 mg of reducing sugar from 5 g of β-glucan at 30 °C for 72 h, which is the optimal condition for C. glutamicum growth. Subsequently, glutamate fermentation from β-glucan was carried out with the addition of Aspergillus aculeatus β-glucosidase produced by recombinant Aspergillus oryzae. Using EG-secreting C. glutamicum, 178 mg/l of glutamate was produced from 15 g of β-glucan. This is the first report of glutamate fermentation from β-glucan using endoglucanase-secreting C. glutamicum.     

Characterization of the catalytic domains of <Emphasis Type="Italic">Trichoderma reesei</Emphasis> endoglucanase I,II, and III,expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The genes encoding the catalytic domains (CD) of the three endoglucanases (EG I; Cel7B, EG II; Cel5A, and EG III; Cel12A) from Trichoderma reesei QM9414 were expressed in Escherichia coli strains Rosetta-gami B (DE3) pLacI or Origami B (DE3) pLacI and were found to produce functional intracellular proteins. Protein production by the three endoglucanase transformants was evaluated as a function of growth temperature. Maximal productivity of EG I-CD at 15°C, EG II-CD at 20°C and EG III at 37°C resulted in yields of 6.9, 72, and 50 mg/l, respectively. The endoglucanases were purified using a simple purification method based on removing E. coli proteins by isoelectric point precipitation. Specific activity toward carboxymethyl cellulose was found to be 65, 49, and 15 U/mg for EG I-CD, EG II-CD, and EG III, respectively. EG II-CD was able to cleave 1,3–1,4-β-d-glucan and soluble cellulose derivatives. EG III was found to be active against cellulose, 1,3–1,4-β-d-glucan and xyloglucan, while EG I-CD was active against cellulose, 1,3–1,4-β-d-glucan, xyloglucan, xylan, and mannan.     

Identification and characterization of fermentation inhibitors formed during hydrothermal treatment and following SSF of wheat straw

A pilot plant for hydrothermal treatment of wheat straw was compared in reactor systems of two steps (first, 80°C; second, 190–205°C) and of three steps (first, 80°C; second, 170–180°C; third, 195°C). Fermentation (SSF) with Sacharomyces cerevisiae of the pretreated fibers and hydrolysate from the two-step system gave higher ethanol yield (64–75%) than that obtained from the three-step system (61–65%), due to higher enzymatic cellulose convertibility. At the optimal conditions (two steps, 195°C for 6 min), 69% of available C6-sugar could be fermented into ethanol with a high hemicellulose recovery (65%). The concentration of furfural obtained during the pretreatment process increased versus temperature from 50 mg/l at 190°C to 1,200 mg/l at 205°C as a result of xylose degradation. S. cerevisiae detoxified the hydrolysates by degradation of several toxic compounds such as 90–99% furfural and 80–100% phenolic aldehydes, which extended the lag phase to 5 h. Acetic acid concentration increased by 0.2–1 g/l during enzymatic hydrolysis and 0–3.4 g/l during fermentation due to hydrolysis of acetyl groups and minor xylose degradation. Formic acid concentration increased by 0.5–1.5 g/l probably due to degradation of furfural. Phenolic aldehydes were oxidized to the corresponding acids during fermentation reducing the inhibition level.     

Ethanol fermentation with Kluyveromyces marxianus from Jerusalem artichoke grown in salina and irrigated with a mixture of seawater and freshwater

Aims: To study fuel ethanol fermentation with Kluyveromyces marxianus ATCC8554 from Jerusalem artichoke (Helianthus tuberosus) grown in salina and irrigated with a mixture of seawater and freshwater. Methods and Results: The growth and ethanol fermentation of K. marxianus ATCC8554 were studied using inulin as substrate. The activity of inulinase, which attributes to the hydrolysis of inulin, the main carbohydrate in Jerusalem artichoke, was monitored. The optimum temperatures were 38°C for growth and inulinase production, and 35°C for ethanol fermentation. Aeration was not necessary for ethanol fermentation with the K. marxianus from inulin. Then, the fresh Jerusalem artichoke tubers grown in salina and irrigated with 25% and 50% seawater were further examined for ethanol fermentation with the K. marxianus, and a higher ethanol yield was achieved for the Jerusalem artichoke tuber irrigated with 25% seawater. Furthermore, the dry meal of the Jerusalem artichoke tubers irrigated with 25% seawater was examined for ethanol fermentation at three solid concentrations of 200, 225 and 250 g l^?1, and the highest ethanol yield of 0·467, or 91·5% of the theoretical value of 0·511, was achieved for the slurry with a solid concentration of 200 g l^?1. Conclusions: Halophilic Jerusalem artichoke can be used for fuel ethanol production. Significance and Impact of the Study: Halophilic Jerusalem artichoke, not competing with grain crops for arable land, is a sustainable feedstock for fuel ethanol production.     

Gene cloning and expression of a new acidic family 7 endo-β-1,3-1,4-glucanase from the acidophilic fungus Bispora sp. MEY-1

Most reported microbial β-1,3-1,4-glucanases belong to the glycoside hydrolase family 16. Here, we report a new acidic family 7 endo-β-1,3-1,4-glucanase (Bgl7A) from the acidophilic fungus Bispora sp. MEY-1. The cDNA of Bgl7A was isolated and over-expressed in Pichia pastoris, with a yield of about 1,000 U ml^–1 in a 3.7-l fermentor. The purified recombinant Bgl7A had three activity peaks at pH 1.5, 3.5, and 5.0 (maximum), respectively, and a temperature optimum at 60°C. The enzyme was stable at pH 1.0–8.0 and highly resistant to both pepsin and trypsin. Belonging to the group of non-specific endoglucanase, Bgl7A can hydrolyze not only β-glucan and cellulose but also laminarin and oat spelt xylan. The specific activity of Bgl7A against barley β-glucan and lichenan (4,040 and 2,740 U mg^–1) was higher than toward carboxymethyl cellulose sodium (395 U mg^–1), which was different from other family 7 endo-β-glucanases.     

Physiological and fermentation properties of <Emphasis Type="Italic">Bacillus coagulans</Emphasis> and a mutant lacking fermentative lactate dehydrogenase activity

Bacillus coagulans, a sporogenic lactic acid bacterium, grows optimally at 50–55°C and produces lactic acid as the primary fermentation product from both hexoses and pentoses. The amount of fungal cellulases required for simultaneous saccharification and fermentation (SSF) at 55°C was previously reported to be three to four times lower than for SSF at the optimum growth temperature for Saccharomyces cerevisiae of 35°C. An ethanologenic B. coagulans is expected to lower the cellulase loading and production cost of cellulosic ethanol due to SSF at 55°C. As a first step towards developing B. coagulans as an ethanologenic microbial biocatalyst, activity of the primary fermentation enzyme L-lactate dehydrogenase was removed by mutation (strain Suy27). Strain Suy27 produced ethanol as the main fermentation product from glucose during growth at pH 7.0 (0.33 g ethanol per g glucose fermented). Pyruvate dehydrogenase (PDH) and alcohol dehydrogenase (ADH) acting in series contributed to about 55% of the ethanol produced by this mutant while pyruvate formate lyase and ADH were responsible for the remainder. Due to the absence of PDH activity in B. coagulans during fermentative growth at pH 5.0, the l-ldh mutant failed to grow anaerobically at pH 5.0. Strain Suy27-13, a derivative of the l-ldh mutant strain Suy27, that produced PDH activity during anaerobic growth at pH 5.0 grew at this pH and also produced ethanol as the fermentation product (0.39 g per g glucose). These results show that construction of an ethanologenic B. coagulans requires optimal expression of PDH activity in addition to the removal of the LDH activity to support growth and ethanol production.     

Direct and Efficient Production of Ethanol from Cellulosic Material with a Yeast Strain Displaying Cellulolytic Enzymes

For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulose-degrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on α-agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His₆) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII-α-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley β-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and β-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing β-glucan as the sole carbon source and could directly ferment 45 g of β-glucan per liter to produce 16.5 g of ethanol per liter within about 50 h. The yield in terms of grams of ethanol produced per gram of carbohydrate utilized was 0.48 g/g, which corresponds to 93.3% of the theoretical yield. This result indicates that efficient simultaneous saccharification and fermentation of cellulose to ethanol are carried out by a recombinant yeast cells displaying cellulolytic enzymes.     

Biohydrogen production from cellulosic hydrolysate produced via temperature-shift-enhanced bacterial cellulose hydrolysis

A “temperature-shift” strategy was developed to improve reducing sugar production from bacterial hydrolysis of cellulosic materials. In this strategy, production of cellulolytic enzymes with Cellulomonas uda E3-01 was promoted at a preferable temperature (35 °C), while more efficient enzymatic cellulose hydrolysis was achieved under an elevated culture temperature (45 °C), at which cell growth was inhibited to avoid consumption of reducing sugar. This temperature-shift strategy was shown to markedly increase the reducing sugar (especially, monosaccharide and disaccharide) concentration in the hydrolysate while hydrolyzing pure (carboxymethyl-cellulose, xylan, avicel and cellobiose) and natural (rice husk, rice straw, bagasse and Napier-grass) cellulosic materials. The cellulosic hydrolysates from CMC and xylan were successfully converted to H₂ via dark fermentation with Clostridium butyricum CGS5, attaining a maximum hydrogen yield of 4.79 mmol H₂/g reducing sugar.     

d-lactic acid production from cellooligosaccharides and β-glucan using l-LDH gene-deficient and endoglucanase-secreting Lactobacillus plantarum

In order to achieve direct fermentation of an optically pure d-lactic acid from cellulosic materials, an endoglucanase from a Clostridium thermocellum (CelA)-secreting plasmid was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (∆ldhL1) bacterial strain. CelA expression and its degradation of β-glucan was confirmed by western blot analysis and enzyme assay, respectively. Although the CelA-secreting ∆ldhL1 assimilated cellooligosaccharides up to cellohexaose (although not cellotetraose), the main end product was acetic acid, not lactic acid, due to the conversion of lactic acid to acetic acid. Cultivation under anaerobic conditions partially suppressed this conversion resulting in the production of 1.27 g/l of D-lactic acid with a high optical purity of 99.5% from a medium containing 2 g/l of cellohexaose. Subsequently, D-lactic acid fermentation from barley β-glucan was carried out with the addition of Aspergillus aculeatus β-glucosidase produced by recombinant Aspergillus oryzae and 1.47 g/l of D-lactic was produced with a high optical purity of 99.7%. This is the first report of direct lactic acid fermentation from β-glucan and a cellooligosaccharide that is a more highly polymerized sugar than cellotriose.     

Cellulosic alcoholic fermentation using recombinant Saccharomyces cerevisiae engineered for the production of Clostridium cellulovorans endoglucanase and Saccharomycopsis 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 β-glucosidase (Bgl1) from Saccharomycopsis fibuligera . To promote secretion of the two enzymes, the genes were fused to the secretion signal of the S. cerevisiae α mating factor gene. The recombinant developed yeast could produce ethanol through simultaneous production of sufficient extracellular endoglucanase and β-glucosidase. When direct ethanol fermentation from 20 g L⁻¹β-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 β-glucan was 80.3% of the theoretical ethanol concentration produced from 20 g L⁻¹β-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.     

Comparison of separate hydrolysis and fermentation and simultaneous saccharification and fermentation processes for ethanol production from wheat straw by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> strain FBR5

Ethanol production by recombinant Escherichia coli strain FBR5 from dilute acid pretreated wheat straw (WS) by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid (0.5% H₂SO₄) pretreated (160 °C, 10 min) and enzymatically saccharified (pH 5.0, 45 °C, 72 h) WS (86 g/l) was 50.0 ± 1.4 g/l. The hydrolyzate contained 1,184 ± 19 mg furfural and 161 ± 1 mg hydroxymethyl furfural per liter. The recombinant E. coli FBR5 could not grow at all at pH controlled at 4.5 to 6.5 in the non-abated wheat straw hydrolyzate (WSH) at 35 °C. However, it produced 21.9 ± 0.3 g ethanol from non-abated WSH (total sugars, 44.1 ± 0.4 g/l) in 90 h including the lag time of 24 h at controlled pH 7.0 and 35 °C. The bioabatement of WS was performed by growing Coniochaeta ligniaria NRRL 30616 in the liquid portion of the pretreated WS aerobically at pH 6.5 and 30 °C for 15 h. The bacterium produced 21.6 ± 0.5 g ethanol per liter in 40 h from the bioabated enzymatically saccharified WSH (total sugars, 44.1 ± 0.4 g) at pH 6.0. It produced 24.9 ± 0.3 g ethanol in 96 h and 26.7 ± 0.0 g ethanol in 72 h per liter from bioabated WSH by batch SSF and fed-batch SSF, respectively. SSF offered a distinct advantage over SHF with respect to reducing total time required to produce ethanol from the bioabated WS. Also, fed-batch SSF performed better than the batch SSF with respect to shortening the time requirement and increase in ethanol yield.     

Biochemical characterization of Magnaporthe oryzae β-glucosidases for efficient β-glucan hydrolysis

β-Glucosidases designated MoCel3A and MoCel3B were successfully overexpressed in Magnaporthe oryzae. MoCel3A and MoCel3B showed optimal activity at 50 °C and pH 5.0–5.5. MoCel3A exhibited higher activity on higher degree of polymerization (DP) oligosaccharides and on β-1,3-linked oligosaccharides than on β-1,4-linked oligosaccharides. Furthermore, MoCel3A could liberate glucose from polysaccharides such as laminarin, 1,3-1,4-β-glucan, phosphoric acid-swollen cellulose, and pustulan, of which laminarin was the most suitable substrate. Conversely, MoCel3B preferentially hydrolyzed lower DP oligosaccharides such as cellobiose, cellotriose, and laminaribiose. Furthermore, the synergistic effects of combining enzymes including MoCel3A and MoCel3B were investigated. Depolymerization of 1,3-1,4-β-glucan by M. oryzae cellobiohydrolase (MoCel6A) enhanced the production of glucose by the actions of MoCel3A and MoCel3B. In these reactions, MoCel3A hydrolyzed higher DP oligosaccharides, resulting in the release of glucose and cellobiose, and MoCel3B preferentially hydrolyzed lower DP oligosaccharides including cellobiose. On the other hand, MoCel3A alone produced glucose from laminarin at levels equivalent to 80% of maximal hydrolysis obtained by the combined action of MoCel3A, MoCel3B, and endo-1,3-β-glucanase. Therefore, MoCel3A and MoCel3B activities yield glucose from not only cellulosic materials but also hemicellulosic polysaccharides.     

Production of bacterial cellulose from <Emphasis Type="Italic">Enterobacter amnigenus</Emphasis> GH-1 isolated from rotten apple

Bacterial cellulose finds novel applications in biomedical, biosensor, food, textile and other industries. The optimum fermentation conditions for the production of cellulose by newly isolated Enterobacter amnigenus GH-1 were investigated. The strain was able to produce cellulose at temperature 25–35°C with a maximum at 28°C. Cellulose production occurred at pH 4.0–7.0 with a maximum at 6.5. After 14 days of incubation, the strain produced 2.5 g cellulose/l in standard medium whereas cellulose yield in the improved medium was found to be 4.1 g/l. The improved medium consisted of 4% (w/v) fructose, 0.6% (w/v) casein hydrolysate, 0.5% (w/v) yeast extract, 0.4% (w/v) disodium phosphate, and 0.115% (w/v) citrate. Addition of metal ions like zinc, magnesium, and calcium and solvents like methanol and ethanol were found to be stimulatory for cellulose production by the strain. The strain used natural carbon sources like molasses, starch hydrolysate, sugar cane juice, coconut water, coconut milk, pineapple juice, orange juice, and pomegranate juice for growth and cellulose production. Fruit juices can play important role in commercial exploitation of bacterial cellulose by lowering the cost of the production medium.     

Feasibility of ethanol production from coffee husks

The objective of this work was to evaluate the feasibility of ethanol production by fermentation of coffee husks by Saccharomyces cerevisiae. Batch fermentation studies were performed employing whole and ground coffee husks, and aqueous extract from ground coffee husks. It was observed that fermentation yield decreased with an increase in yeast concentration. The best results were obtained for the following conditions: whole coffee husks, 3 g yeast/l substrate, temperature of 30°C. Under these conditions ethanol production was 8.49 ± 0.29 g/100 g dry basis (13.6 ± 0.5 g ethanol/l), a satisfactory value in comparison to literature data for other residues such as corn stalks, barley straw and hydrolyzed wheat stillage (5–11 g ethanol/l). Such results indicate that coffee husks present excellent potential for residue-based ethanol production.     

Cashew apple bagasse as a source of sugars for ethanol production by Kluyveromyces marxianus CE025

The potential of cashew apple bagasse as a source of sugars for ethanol production by Kluyveromyces marxianus CE025 was evaluated in this work. This strain was preliminarily cultivated in a synthetic medium containing glucose and xylose and was able to produce ethanol and xylitol at pH 4.5. Next, cashew apple bagasse hydrolysate (CABH) was prepared by a diluted sulfuric acid pretreatment and used as fermentation media. This hydrolysate is rich in glucose, xylose, and arabinose and contains traces of formic acid and acetic acid. In batch fermentations of CABH at pH 4.5, the strain produced only ethanol. The effects of temperature on the kinetic parameters of ethanol fermentation by K. marxianus CE025 using CABH were also evaluated. Maximum specific growth rate (μ_max), overall yields of ethanol based on glucose consumption Y_{P \mathord}

Direct ethanol production from cellulosic materials by Zymobacter palmae carrying Cellulomonas endoglucanase and Ruminococcus β-glucosidase genes

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we conferred the ability to ferment cellulosic materials directly on Zymobacter palmae by co-expressing foreign endoglucanase and β-glucosidase genes. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, the six genes encoding the cellulolytic enzymes (CenA, CenB, CenD, CbhA, CbhB, and Cex) from Cellulomonas fimi were introduced and expressed in Z. palmae. Of these cellulolytic enzyme genes cloned, CenA degraded carboxymethylcellulose and phosphoric acid-swollen cellulose (PASC) efficiently. The extracellular CenA catalyzed the hydrolysis of barley β-glucan and PASC to liberate soluble cello-oligosaccharides, indicating that CenA is the most suitable enzyme for cellulose degradation among those cellulolytic enzymes expressed in Z. palmae. Furthermore, the cenA gene and β-glucosidase gene (bgl) from Ruminococcus albus were co-expressed in Z. palmae. Of the total endoglucanase and β-glucosidase activities, 57.1 and 18.1 % were localized in the culture medium of the strain. The genetically engineered strain completely saccharified and fermented 20 g/l barley β-glucan to ethanol within 84 h, producing 79.5 % of the theoretical yield. Thus, the production and secretion of CenA and BGL enabled Z. palmae to efficiently ferment a water-soluble cellulosic polysaccharide to ethanol.