Saccharomyces cerevisiae Cells Lacking the Zinc Vacuolar Transporter Zrt3 Display Improved Ethanol Productivity in Lignocellulosic Hydrolysates

Yeast-based bioethanol production from lignocellulosic hydrolysates (LH) is an attractive and sustainable alternative for biofuel production. However, the presence of acetic acid (AA) in LH is still a major problem. Indeed, above certain concentrations, AA inhibits yeast fermentation and triggers a regulated cell death (RCD) process mediated by the mitochondria and vacuole. Understanding the mechanisms involved in AA-induced RCD (AA-RCD) may thus help select robust fermentative yeast strains, providing novel insights to improve lignocellulosic ethanol (LE) production. Herein, we hypothesized that zinc vacuolar transporters are involved in vacuole-mediated AA-RCD, since zinc enhances ethanol production and zinc-dependent catalase and superoxide dismutase protect from AA-RCD. In this work, zinc limitation sensitized wild-type cells to AA-RCD, while zinc supplementation resulted in a small protective effect. Cells lacking the vacuolar zinc transporter Zrt3 were highly resistant to AA-RCD, exhibiting reduced vacuolar dysfunction. Moreover, zrt3Δ cells displayed higher ethanol productivity than their wild-type counterparts, both when cultivated in rich medium with AA (0.29 g L−1 h−1 versus 0.11 g L−1 h−1) and in an LH (0.73 g L−1 h−1 versus 0.55 g L−1 h−1). Overall, the deletion of ZRT3 emerges as a promising strategy to increase strain robustness in LE industrial production.

Cellulosic ethanol production by consortia of Scheffersomyces stipitis and engineered Zymomonas mobilis

Background As one of the clean and sustainable energies, lignocellulosic ethanol has achieved much attention around the world. The production of lignocellulosic ethanol does not compete with people for food, while the consumption of ethanol could contribute to the carbon dioxide emission reduction. However, the simultaneous transformation of glucose and xylose to ethanol is one of the key technologies for attaining cost-efficient lignocellulosic ethanol production at an industrial scale. Genetic modification of strains and constructing consortia were two approaches to resolve this issue. Compared with strain improvement, the synergistic interaction of consortia in metabolic pathways should be more useful than using each one separately. Results In this study, the consortia consisting of suspended Scheffersomyces stipitis CICC1960 and Zymomonas mobilis 8b were cultivated to successfully depress carbon catabolite repression (CCR) in artificially simulated 80G40XRM. With this strategy, a 5.52% more xylose consumption and a 6.52% higher ethanol titer were achieved by the consortium, in which the inoculation ratio between S. stipitis and Z. mobilis was 1:3, compared with the Z. mobilis 8b mono-fermentation. Subsequently, one copy of the xylose metabolic genes was inserted into the Z. mobilis 8b genome to construct Z. mobilis FR2, leading to the xylose final-consumption amount and ethanol titer improvement by 15.36% and 6.81%, respectively. Finally, various corn stover hydrolysates with different sugar concentrations (glucose and xylose 60, 90, 120 g/L), were used to evaluate the fermentation performance of the consortium consisting of S. stipitis CICC1960 and Z. mobilis FR2. Fermentation results showed that a 1.56–4.59% higher ethanol titer was achieved by the consortium compared with the Z. mobilis FR2 mono-fermentation, and a 46.12–102.14% higher ethanol titer was observed in the consortium fermentation when compared with the S. stipitis CICC1960 mono-fermentation. Furthermore, qRT-PCR analysis of xylose/glucose transporter and other genes responsible for CCR explained the reason why the initial ratio inoculation of 1:3 in artificially simulated 80G40XRM had the best fermentation performance in the consortium. Conclusions The fermentation strategy used in this study, i.e., using a genetically modified consortium, had a superior performance in ethanol production, as compared with the S. stipitis CICC1960 mono-fermentation and the Z. mobilis FR2 mono-fermentation alone. This result showed that this strategy has potential for future lignocellulosic ethanol production.

Cellulosic Ethanol Production by Engineered Consortia of Scheffersomyces Stipitis and Zymomonas Mobilis

Background: As one of the clean and sustainable energies, lignocellulosic ethanol has achieved much attention around the world. The production of lignocellulosic ethanol does not compete with people for food, while the consumption of ethanol could contribute to the carbon dioxide emission reduction. Two of the conditions that are needed to attain cost-efficient lignocellulosic ethanol production at an industrial scale are the simultaneous transformation of glucose and xylose to ethanol and a highly efficient ethanol fermentation process. Results: In this study, the consortia consisting of suspended Scheffersomyces stipitis CICC1960 and Zymomonas mobilis 8b were cultivated to successfully depress carbon catabolite repression (CCR) in 80G40XRM. With this strategy, a 5.52% more xylose consumption and a 6.52% higher ethanol titer were achieved by the consortium, in which the inoculation ratio between S. stipitis and Z. mobilis was 1:3, at the end of fermentation compared with the Z. mobilis 8b mono-fermentation. Subsequently, one copy of the xylose metabolic genes was inserted into the Z. mobilis 8b genome to construct Z. mobilis FR2, leading to the xylose final-consumption amount and ethanol titer improvement by 15.36% and 6.81%, respectively. Finally, various concentrations of corn stover hydrolysates, in which the sum of glucose and xylose concentrations in the hydrolysates were 60, 90, and 120 g/L respectively, were used to evaluate the fermentation performance of the consortium consisting of S. stipitis CICC1960 and Z. mobilis FR2. Fermentation results showed that a 1.56% - 4.59% higher ethanol titer was achieved by the consortium compared with the Z. mobilis FR2 mono-fermentation, and a 46.12% - 102.14% higher ethanol titer was observed in the consortium fermentation when compared with the S. stipitis CICC1960 mono-fermentation. Conclusions: The fermentation strategy used in this study, i.e., using a genetically modified consortium, had a superior performance in ethanol production, as compared with the S. stipitis CICC1960 mono-fermentation and the Z. mobilis FR2 mono-fermentation alone. Thus, this strategy has potential for future lignocellulosic ethanol production.

Production of enzymatic complex from agro-industrial biomass and its application in combustible ethanol

Waste biomass and agro-industrial by-products, for production ethanol, will meet much of the great demand for this product. To reduce costs and optimize production, this study investigated solid-state fermentation (SSF) to obtain crude enzyme complex (CEC) from different agro-industrial biomasses (sugarcane bagasse, corn peel bran, rice straw bran and roasting and ground coffee residue) using cellulolytic fungi. The most promising CEC were evaluated in simultaneous hydrolysis and fermentation (SHF) for ethanol production by Saccharomyces cerevisiae in a culture broth containing sugarcane bagasse treated by steam explosion, and roast and ground coffee residue. In SSF with bioreactor volume of 0.25 L, containing 40 g of the biomass mixture and 40 g of sterile water with resuspended cells (1.0 x108 spores/g of solid medium) and temperature of 30±2 ºC, the strains Trichoderma reesei and Penicilium oxalicum provided the best enzyme activity. The CEC of T. reesei provided a concentration of 7.5 g L-1 of ethanol in a substrate containing treated sugarcane bagasse (60%) and roast and ground coffee residue (40%), under SHF conditions (pH 4.5, 35±2 °C, 48 h). The results obtained in this study show a promising alternative for correct disposal and use of residues and agro-industrial by-products by use in the production of enzymes and lignocellulosic ethanol.

The Performance Of Clostridium Phytofermentans For Biofuels Production From Lignocellulosic Biomass

Ethanol produced from lignocellulosic biomass is an alternative transportation fuel with the potential to lower greenhouse gas emissions and increase energy security. Source-separated organic waste (SSO) from the city of Toronto was investigated for feasibility as a lignocellulosic ethanol feedstock. Clostridium phytofermentans is a mesophilic, cellulolytic and ethanologenic species with potential application for ethanol production from lignocullulosic biomass. C. phytofermentans was considered for biofuels production through experiments on a variety of substrates including soluble sugars and pure celluloses. Results from this study found that C. phytofermentans produced 73% of the theoretical ethanol yield on cellobiose but grew poorly on glucose and xylose. In addition, C. phytofermentans grew marginally on microcrystalline and ball-milled cellulose, but with supplemental enzymes produced 55% of the theoretical ethanol yield.

The Performance Of Clostridium Phytofermentans For Biofuels Production From Lignocellulosic Biomass

Ethanol produced from lignocellulosic biomass is an alternative transportation fuel with the potential to lower greenhouse gas emissions and increase energy security. Source-separated organic waste (SSO) from the city of Toronto was investigated for feasibility as a lignocellulosic ethanol feedstock. Clostridium phytofermentans is a mesophilic, cellulolytic and ethanologenic species with potential application for ethanol production from lignocullulosic biomass. C. phytofermentans was considered for biofuels production through experiments on a variety of substrates including soluble sugars and pure celluloses. Results from this study found that C. phytofermentans produced 73% of the theoretical ethanol yield on cellobiose but grew poorly on glucose and xylose. In addition, C. phytofermentans grew marginally on microcrystalline and ball-milled cellulose, but with supplemental enzymes produced 55% of the theoretical ethanol yield.