Project description:UnlabelledBackgroundWhile the ethanol production from biomass by consolidated bioprocess (CBP) is considered to be the most ideal process, simultaneous saccharification and fermentation (SSF) is the most appropriate strategy in practice. In this study, one-pot bioethanol production, including cellulase production, saccharification of cellulose, and ethanol production, was investigated for the conversion of biomass to biofuel by co-culture of two different microorganisms such as a hyper cellulase producer, Acremonium cellulolyticus C-1 and an ethanol producer Saccharomyces cerevisiae. Furthermore, the operational conditions of the one-pot process were evaluated for maximizing ethanol concentration from cellulose in a single reactor.ResultsEthanol production from cellulose was carried out in one-pot bioethanol production process. A. cellulolyticus C-1 and S. cerevisiae were co-cultured in a single reactor. Cellulase producing-medium supplemented with 2.5 g/l of yeast extract was used for productions of both cellulase and ethanol. Cellulase production was achieved by A. cellulolyticus C-1 using Solka-Floc (SF) as a cellulase-inducing substrate. Subsequently, ethanol was produced with addition of both 10%(v/v) of S. cerevisiae inoculum and SF at the culture time of 60 h. Dissolved oxygen levels were adjusted at higher than 20% during cellulase producing phase and at lower than 10% during ethanol producing phase. Cellulase activity remained 8-12 FPU/ml throughout the one-pot process. When 50-300 g SF/l was used in 500 ml Erlenmeyer flask scale, the ethanol concentration and yield based on initial SF were as 8.7-46.3 g/l and 0.15-0.18 (g ethanol/g SF), respectively. In 3-l fermentor with 50-300 g SF/l, the ethanol concentration and yield were 9.5-35.1 g/l with their yields of 0.12-0.19 (g/g) respectively, demonstrating that the one-pot bioethanol production is a reproducible process in a scale-up bioconversion of cellulose to ethanol.ConclusionA. cellulolyticus cells produce cellulase using SF. Subsequently, the produced cellulase saccharifies the SF, and then liberated reducing sugars are converted to ethanol by S. cerevisiae. These reactions were carried out in the one-pot process with two different microorganisms in a single reactor, which does require neither an addition of extraneous cellulase nor any pretreatment of cellulose. Collectively, the one-pot bioethanol production process with two different microorganisms could be an alternative strategy for a practical bioethanol production using biomass.

Project description:BackgroundRobust yeasts with high inhibitor, temperature, and osmotic tolerance remain a crucial requirement for the sustainable production of lignocellulosic bioethanol. These stress factors are known to severely hinder culture growth and fermentation performance.ResultsGrape marc was selected as an extreme environment to search for innately robust yeasts because of its limited nutrients, exposure to solar radiation, temperature fluctuations, weak acid and ethanol content. Forty newly isolated Saccharomyces cerevisiae strains gave high ethanol yields at 40°C when inoculated in minimal media at high sugar concentrations of up to 200 g/l glucose. In addition, the isolates displayed distinct inhibitor tolerance in defined broth supplemented with increasing levels of single inhibitors or with a cocktail containing several inhibitory compounds. Both the fermentation ability and inhibitor resistance of these strains were greater than those of established industrial and commercial S. cerevisiae yeasts used as control strains in this study. Liquor from steam-pretreated sugarcane bagasse was used as a key selective condition during the isolation of robust yeasts for industrial ethanol production, thus simulating the industrial environment. The isolate Fm17 produced the highest ethanol concentration (43.4 g/l) from the hydrolysate, despite relatively high concentrations of weak acids, furans, and phenolics. This strain also exhibited a significantly greater conversion rate of inhibitory furaldehydes compared with the reference strain S. cerevisiae 27P. To our knowledge, this is the first report describing a strain of S. cerevisiae able to produce an ethanol yield equal to 89% of theoretical maximum yield in the presence of high concentrations of inhibitors from sugarcane bagasse.ConclusionsThis study showed that yeasts with high tolerance to multiple stress factors can be obtained from unconventional ecological niches. Grape marc appeared to be an unexplored and promising substrate for the isolation of S. cerevisiae strains showing enhanced inhibitor, temperature, and osmotic tolerance compared with established industrial strains. This integrated approach of selecting multiple resistant yeasts from a single source demonstrates the potential of obtaining yeasts that are able to withstand a number of fermentation-related stresses. The yeast strains isolated and selected in this study represent strong candidates for bioethanol production from lignocellulosic hydrolysates.

Project description:In the search for renewable sources of energy, bioethanol stands out as a benchmark biofuel because its production is based on a proven technological platform. Bioethanol is produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (~2 SNPs per kilobase), and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, and appear to reflect ectopic homologous recombination between repetitive DNA sequences. Despite the complex karyotype of JAY270, this diploid, when sporulated, had a high frequency of viable spores (~93%). Crosses of haploids derived from JAY270 to a haploid of the unrelated laboratory strain S288c also resulted in diploids that had good spore viability (75-95%). Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis and spore viability, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures that may be associated with a fitness benefit. We also explore features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high cell mass production and fermentation kinetics, high temperature growth and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies.

Project description:In the search for renewable sources of energy, bioethanol stands out as a benchmark biofuel because its production is based on a proven technological platform. Bioethanol is produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (~2 SNPs per kilobase), and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, and appear to reflect ectopic homologous recombination between repetitive DNA sequences. Despite the complex karyotype of JAY270, this diploid, when sporulated, had a high frequency of viable spores (~93%). Crosses of haploids derived from JAY270 to a haploid of the unrelated laboratory strain S288c also resulted in diploids that had good spore viability (75-95%). Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis and spore viability, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures that may be associated with a fitness benefit. We also explore features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high cell mass production and fermentation kinetics, high temperature growth and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies. This microarray experiment was used to compare the relative gene expression levels between two unrelated S. cerevisiae strain backgrounds: JAY270 and JAY309. Total RNA from each strain was prepared and used to synthesize differentially labeled cDNAs (Cy5 and C3 respectively). A positive Log2 (Red/Green) ratio indicates transcripts more abundant in JAY270, while a negative Log2 (Red/Green) ration indicates transcripts more abundant in JAY309.

Dataset Information

Bioethanol Production from Cellulose-Rich Corncob Residue by the Thermotolerant Saccharomyces cerevisiae TC-5.

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