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:BackgroundThe cost-effective production of second-generation bioethanol, which is made from lignocellulosic materials, has to face the following two problems: co-fermenting xylose with glucose and enhancing the strain's tolerance to lignocellulosic inhibitors. Based on our previous study, the wild-type diploid Saccharomyces cerevisiae strain BSIF with robustness and good xylose metabolism genetic background was used as a chassis for constructing efficient xylose-fermenting industrial strains. The performance of the resulting strains in the fermentation of media with sugars and hydrolysates was investigated.ResultsThe following two novel heterologous genes were integrated into the genome of the chassis cell: the mutant MGT05196N360F, which encodes a xylose-specific, glucose-insensitive transporter and is derived from the Meyerozyma guilliermondii transporter gene MGT05196, and Ru-xylA (where Ru represents the rumen), which encodes a xylose isomerase (XI) with higher activity in S. cerevisiae. Additionally, endogenous modifications were also performed, including the overproduction of the xylulokinase Xks1p and the non-oxidative PPP (pentose phosphate pathway), and the inactivation of the aldose reductase Gre3p and the alkaline phosphatase Pho13p. These rationally designed genetic modifications, combined with alternating adaptive evolutions in xylose and SECS liquor (the leach liquor of steam-exploding corn stover), resulted in a final strain, LF1, with excellent xylose fermentation and enhanced inhibitor resistance. The specific xylose consumption rate of LF1 reached as high as 1.089 g g-1 h-1 with xylose as the sole carbon source. Moreover, its highly synchronized utilization of xylose and glucose was particularly significant; 77.6% of xylose was consumed along with glucose within 12 h, and the ethanol yield was 0.475 g g-1, which is more than 93% of the theoretical yield. Additionally, LF1 performed well in fermentations with two different lignocellulosic hydrolysates.ConclusionThe strain LF1 co-ferments glucose and xylose efficiently and synchronously. This result highlights the great potential of LF1 for the practical production of second-generation bioethanol.

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:BackgroundDespite its semi-commercial status, ethanol production from lignocellulosics presents many complexities not yet fully solved. Since the pretreatment stage has been recognized as a complex and yield-determining step, it has been extensively studied. However, economic success of the production process also requires optimization of the biochemical conversion stage. This work addresses the search of bioreactor configurations with improved residence times for continuous enzymatic saccharification and fermentation operations. Instead of analyzing each possible configuration through simulation, we apply graphical methods to optimize the residence time of reactor networks composed of steady-state reactors. Although this can be easily made for processes described by a single kinetic expression, reactions under analysis do not exhibit this feature. Hence, the attainable region method, able to handle multiple species and its reactions, was applied for continuous reactors. Additionally, the effects of the sugars contained in the pretreatment liquor over the enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) were assessed.ResultsWe obtained candidate attainable regions for separate enzymatic hydrolysis and fermentation (SHF) and SSF operations, both fed with pretreated corn stover. Results show that, despite the complexity of the reaction networks and underlying kinetics, the reactor networks that minimize the residence time can be constructed by using plug flow reactors and continuous stirred tank reactors. Regarding the effect of soluble solids in the feed stream to the reactor network, for SHF higher glucose concentration and yield are achieved for enzymatic hydrolysis with washed solids. Similarly, for SSF, higher yields and bioethanol titers are obtained using this substrate.ConclusionsIn this work, we demonstrated the capabilities of the attainable region analysis as a tool to assess the optimal reactor network with minimum residence time applied to the SHF and SSF operations for lignocellulosic ethanol production. The methodology can be readily modified to evaluate other kinetic models of different substrates, enzymes and microorganisms when available. From the obtained results, the most suitable reactor configuration considering residence time and rheological aspects is a continuous stirred tank reactor followed by a plug flow reactor (both in SSF mode) using washed solids as substrate.

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.

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Flocculation causes inhibitor tolerance in Saccharomyces cerevisiae for second-generation bioethanol production.

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Flocculation causes inhibitor tolerance in Saccharomyces cerevisiae for second-generation bioethanol production.

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