Ethanol-tolerant Strains Research Articles

Ethanol Cellular Defense Induce Unfolded Protein Response in Yeast.

Ethanol is a valuable industrial product and a common metabolite used by many cell types. However, this molecule produces high levels of cytotoxicity affecting cellular performance at several levels. In the presence of ethanol, cells must adjust some of their components, such as the membrane lipids to maintain homeostasis. In the case of microorganism as Saccharomyces cerevisiae, ethanol is one of the principal products of their metabolism and is the main stress factor during fermentation. Although, many efforts have been made, mechanisms of ethanol tolerance are not fully understood and very little evidence is available to date for specific signaling by ethanol in the cell. This work studied two S. cerevisiae strains, CECT10094, and Temohaya-MI26, isolated from flor wine and agave fermentation (a traditional fermentation from Mexico) respectively, which differ in ethanol tolerance, in order to understand the molecular mechanisms underlying the ethanol stress response and the reasons for different ethanol tolerance. The transcriptome was analyzed after ethanol stress and, among others, an increased activation of genes related with the unfolded protein response (UPR) and its transcription factor, Hac1p, was observed in the tolerant strain CECT10094. We observed that this strain also resist more UPR agents than Temohaya-MI26 and the UPR-ethanol stress correlation was corroborated observing growth of 15 more strains and discarding UPR correlation with other stresses as thermal or oxidative stress. Furthermore, higher activation of UPR pathway in the tolerant strain CECT10094 was observed using a UPR mCherry reporter. Finally, we observed UPR activation in response to ethanol stress in other S. cerevisiae ethanol tolerant strains as the wine strains T73 and EC1118. This work demonstrates that the UPR pathway is activated under ethanol stress occurring in a standard fermentation and links this response to an enhanced ethanol tolerance. Thus, our data suggest that there is a room for ethanol tolerance improvement by enhancing UPR response.

Using global transcription machinery engineering (gTME) to improve ethanol tolerance of Zymomonas mobilis.

BackgroundWith the increasing global crude oil crisis and resulting environmental concerns, the production of biofuels from renewable resources has become increasingly important. One of the major challenges faced during the process of biofuel production is the low tolerance of the microbial host towards increasing biofuel concentrations.ResultsHere, we demonstrate that the ethanol tolerance of Zymomonas mobilis can be greatly enhanced through the random mutagenesis of global transcription factor RpoD protein, (σ70). Using an enrichment screening, four mutants with elevated ethanol tolerance were isolated from error-prone PCR libraries. All mutants showed significant growth improvement in the presence of ethanol stress when compared to the control strain. After an ethanol (9 %) stress exposure lasting 22 h, the rate of glucose consumption was approximately 1.77, 1.78 and 1.39 g L−1 h−1 in the best ethanol-tolerant strain ZM4-mrpoD4, its rebuilt mutant strain ZM4-imrpoD and the control strain, respectively. Our results indicated that both ZM4-mrpoD4 and ZM4-imrpoD consumed glucose at a faster rate after the initial 9 % (v/v) ethanol stress, as nearly 0.64 % of the initial glucose remained after 54 h incubation versus approximately 5.43 % for the control strain. At 9 % ethanol stress, the net ethanol productions by ZM4-mrpoD4 and ZM4-imrpoD during the 30–54 h were 13.0–14.1 g/l versus only 6.6–7.7 g/l for the control strain. The pyruvate decarboxylase activity of ZM4-mrpoD4 was 62.23 and 68.42 U/g at 24 and 48 h, respectively, which were 2.6 and 1.6 times higher than the control strain. After 24 and 48 h of 9 % ethanol stress, the alcohol dehydrogenase activities of ZM4-mrpoD4 were also augmented, showing an approximate 1.4 and 1.3 times increase, respectively, when compared to the control strain. Subsequent quantitative real-time PCR analysis under these stress conditions revealed that the relative expression of pdc in cultured (6 and 24 h) ZM4-mrpoD4 increased by 9.0- and 12.7-fold when compared to control strain.ConclusionsCollectively, these results demonstrate that the RpoD mutation can enhance ethanol tolerance in Z. mobilis. Our results also suggested that RpoD may play an important role in resisting high ethanol concentration in Z. mobilis and manipulating RpoD via global transcription machinery engineering (gTME) can provide an alternative and useful approach for strain improvement for complex phenotypes.

Isolation and Identification of Local Ethanol Tolerant Yeast Populating Distillation and Milling Sites in Nigeria

Two yeast strains referred to as OY and SY were isolated and characterised from local distillery and cereal milling sites. Isolation was done using potato dextrose media supplemented with 0.2% chloramphenicol. Morphological and biochemical results together with the rDNA internal transcribed spacer region (ITS) were identified as <i>Pichia kudriavzevii</i> strains GY1 and L9 respectively. OY and SY are ethanol tolerant strains, withstanding ethanol concentrations of up to 20% (v/v) in yeast extract, peptone, malt extract, glucose media. OY and SY displayed good growth in ethanol supplemented medium with pH ranging from 4.8-5.5 at 30°C. Growth measurements were determined by measuring optical density of the cells in broth using spectrophotometer at 570nm. The results obtained suggested that OY and SY demonstrated good parameters as ideal candidates for bioethanol production.

Correlation between ethanol resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria

The aim of this study was to understand the correlation between ethanol resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria. Two ethanol-tolerant strains, FY-24 and DY-5, identified as Acetobacter pasteurianus were isolated from Chinese vinegar Pei. FY-24 and DY-5 maintained the growth and fermentation ability with 12 % (v/v) ethanol at 37 °C, but A. pasteurianus AS1.41 (widely used in China) could only be resistant to 10 % ethanol. ADH activities of FY-24 (6.13 U/mg) and DY-5 (5.41 U/mg) were much higher than that of AS1.41 (1.57 U/mg) at 8 % ethanol. Using purified ADH, it was shown that the ADHs from FY-24 and DY-5 exhibited a higher ethanol tolerance as ethanol increasing from 10 to 40 %. The optimum ethanol concentration for ADHs of FY-24 and DY-5 was 10 %, but was 5 % for that of AS1.41. Additionally, ADHs from FY-24 and DY-5 exhibited a higher resistance to temperature and acetic acid than that of AS1.41. From the amino acid sequence of ADH subunits, a certain amount of residues were found in the ADHs of FY-24 and DY-5 (411th D and 731th N in subunit I, 170th D in subunit II and 40th G in subunit III), which were different from that of AS1.41. The difference in amino acid residues might partly relate to the ethanol tolerance. Our results suggested that FY-24 and DY-5 could be developed for high ethanol acetic acid fermentation.

Physiology, Genomics, and Pathway Engineering of an Ethanol-Tolerant Strain of Clostridium phytofermentans.

Novel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bacterium Clostridium phytofermentans was adapted to increased ethanol concentrations. The resulting ethanol-tolerant (ET) strain has nearly doubled ethanol tolerance relative to the wild-type level but also reduced ethanol yield and growth at low ethanol concentrations. The genome of the ET strain has coding changes in proteins involved in membrane biosynthesis, the Rnf complex, cation homeostasis, gene regulation, and ethanol production. In particular, purification of the mutant bifunctional acetaldehyde coenzyme A (CoA)/alcohol dehydrogenase showed that a G609D variant abolished its activities, including ethanol formation. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase in the ET strain increased cellulose consumption and restored ethanol production, demonstrating how metabolic engineering can be used to overcome disadvantageous mutations incurred during adaptation to ethanol. We discuss how genetic changes in the ET strain reveal novel potential strategies for improving microbial solvent tolerance.

Differential expression of selected Oenococcus oeni genes for adaptation in wine-like media and red wine

The differential expression of five genes related to adaptation to wine conditions in Oenococcus oeni was evaluated by studying transcriptional changes in wine-like media and in real wine during malolactic fermentation. Two strains isolated from high ethanol wine and another, less tolerant, strain were used. Expression of the selected genes (mleA, citE, hsp18, clpP and gsh) was studied by means of RT-qPCR. A higher level of gene expression was found for the more ethanol-tolerant strains, especially for citE, hsp18 and clpP. Thus, these three genes might be good markers for the more stress-resistant strains of O. oeni. In most cases, the results of expression in wine-like medium with 14 % ethanol had a similar trend to those in real wine, suggesting that the wine-like medium can be a good model for studying the stress response in O. oeni. Different expression between strains was well correlated with their ability to perform malolactic fermentation. In this way, the identified gene markers can be used in the selection of new strains as starters for malolactic fermentation.

Simultaneous saccharification and fermentation of steam-exploded corn stover at high glucan loading and high temperature.

BackgroundSimultaneous saccharification and fermentation (SSF) is a promising process for bioconversion of lignocellulosic biomass. High glucan loading for hydrolysis and fermentation is an efficient approach to reduce the capital costs for bio-based products production. The SSF of steam-exploded corn stover (SECS) for ethanol production at high glucan loading and high temperature was investigated in this study.ResultsGlucan conversion of corn stover biomass pretreated by steam explosion was maintained at approximately 71 to 79% at an enzyme loading of 30 filter paper units (FPU)/g glucan, and 74 to 82% at an enzyme loading of 60 FPU/g glucan, with glucan loading varying from 3 to 12%. Glucan conversion decreased obviously with glucan loading beyond 15%. The results indicated that the mixture was most efficient in enzymatic hydrolysis of SECS at 3 to 12% glucan loading. The optimal SSF conditions of SECS using a novel Saccharomyces cerevisiae were inoculation optical density (OD)600 = 4.0, initial pH 4.8, 50% nutrients added, 36 hours pre-hydrolysis time, 39°C, and 12% glucan loading (20% solid loading). With the addition of 2% Tween 20, glucan conversion, ethanol yield, final ethanol concentration reached 78.6%, 77.2%, and 59.8 g/L, respectively, under the optimal conditions. The results suggested that the solid and degradation products’ inhibitory effect on the hydrolysis and fermentation of SECS were also not obvious at high glucan loading. Additionally, glucan conversion and final ethanol concentration in SSF of SECS increased by 13.6% and 18.7%, respectively, compared with separate hydrolysis and fermentation (SHF).ConclusionsOur research suggested that high glucan loading (6 to 12% glucan loading) and high temperature (39°C) significantly improved the SSF performance of SECS using a thermal- and ethanol-tolerant strain of S. cerevisiae due to the removal of degradation products, sugar feedback, and solid’s inhibitory effects. Furthermore, the surfactant addition obviously increased ethanol yield in SSF process of SECS.

Increase in Ethanol Yield via Elimination of Lactate Production in an Ethanol-Tolerant Mutant of Clostridium thermocellum

Large-scale production of lignocellulosic biofuel is a potential solution to sustainably meet global energy needs. One-step consolidated bioprocessing (CBP) is a potentially advantageous approach for the production of biofuels, but requires an organism capable of hydrolyzing biomass to sugars and fermenting the sugars to ethanol at commercially viable titers and yields. Clostridium thermocellum, a thermophilic anaerobe, can ferment cellulosic biomass to ethanol and organic acids, but low yield, low titer, and ethanol sensitivity remain barriers to industrial production. Here, we deleted the hypoxanthine phosphoribosyltransferase gene in ethanol tolerant strain of C. thermocellum adhE*(EA) in order to allow use of previously developed gene deletion tools, then deleted lactate dehydrogenase (ldh) to redirect carbon flux towards ethanol. Upon deletion of ldh, the adhE*(EA) Δldh strain produced 30% more ethanol than wild type on minimal medium. The adhE*(EA) Δldh strain retained tolerance to 5% v/v ethanol, resulting in an ethanol tolerant platform strain of C. thermocellum for future metabolic engineering efforts.

Metabolic Adaption of Ethanol-Tolerant Clostridium thermocellum

Clostridium thermocellum is a major candidate for bioethanol production via consolidated bioprocessing. However, the low ethanol tolerance of the organism dramatically impedes its usage in industry. To explore the mechanism of ethanol tolerance in this microorganism, systematic metabolomics was adopted to analyse the metabolic phenotypes of a C. thermocellum wild-type (WT) strain and an ethanol-tolerant strain cultivated without (ET0) or with (ET3) 3% (v/v) exogenous ethanol. Metabolomics analysis elucidated that the levels of numerous metabolites in different pathways were changed for the metabolic adaption of ethanol-tolerant C. thermocellum. The most interesting phenomenon was that cellodextrin was significantly more accumulated in the ethanol-tolerant strain compared with the WT strain, although cellobiose was completely consumed in both the ethanol-tolerant and wild-type strains. These results suggest that the cellodextrin synthesis was active, which might be a potential mechanism for stress resistance. Moreover, the overflow of many intermediate metabolites, which indicates the metabolic imbalance, in the ET0 cultivation was more significant than in the WT and ET3 cultivations. This indicates that the metabolic balance of the ethanol-tolerant strain was adapted better to the condition of ethanol stress. This study provides additional insight into the mechanism of ethanol tolerance and is valuable for further metabolic engineering aimed at higher bioethanol production.

Isolation and characterization of ethanol tolerant yeast strains.

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains.

Isolation, selection, and characterization of highly ethanol-tolerant strains of Oenococcus oeni from south Catalonia.

Twenty-one strains of Oenococcus oeni were isolated during the malolactic fermentation of wines from south Catalonia. Due to their high ethanol tolerance (14 %, or more), these strains may serve as promising starters. The strains were screened by assays in a wine-like medium and by their co-inoculation in wine, resulting in the selection of well-performing strains, subsequently shown not to produce the main biogenic amines and lacking the genes involved in their synthesis. The genetic diversity of the isolates was studied by multilocus sequence typing (MLST), in which seven housekeeping genes were sequenced. Although the concatenated allelic profile of some strains was the same, the profiles obtained by random amplification of polymorphic DNA together with the variable number of tandem repeats at several loci showed that none of the strains were identical. A phylogenetic tree was constructed based on MLST with the seven genes and clearly showed two phylogroups, in accordance with previous studies. The best-performing strains occurred in members of both subgroups, suggesting that the grouping of housekeeping genes is not directly related to adaptation and ethanol tolerance.

Adaptive evolution of the lager brewing yeastSaccharomyces pastorianusfor improved growth under hyperosmotic conditions and its influence on fermentation performance

An adaptive evolution method to obtain stable Saccharomyces pastorianus brewing yeast variants with improved fermentation capacity is described. The procedure involved selection for rapid growth resumption at high osmotic strength. It was applied to a lager strain and to a previously isolated ethanol-tolerant strain. Fermentation performance of strains was compared at 15°P wort strength. A selected osmotolerant variant of the ethanol-tolerant strain showed significantly shorter fermentation time than the parent strain, producing 6.45% alcohol by volume beer in 4-5days with mostly similar organoleptic properties to the original strain. Diacetyl and pentanedione contents were 50-75% and 3-methylbutyl acetate and 2-phenylethyl acetate 50% higher than with the original strain, leading to a small flavour change. The variant contained significantly less intracellular trehalose and glycogen than the parent. Transcriptional analysis of selected genes at 24h revealed reduced transcription of hexose transport genes and increased transcription of the MALx1 and MALx2genes, responsible for α-glucoside uptake and metabolism. It is suggested that an attenuated stress response contributes to the improved fermentation performance. Results show that sequential selection for both ethanol tolerance and rapid growth at high osmotic strength can provide strains with enhanced fermentation speed with acceptable product quality.

Improving Ethanol Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)

A major challenge in bioethanol fermentation is the low tolerance of the microbial host towards the end product bioethanol. Here we report to improve the ethanol tolerance of E. coli from the transcriptional level by engineering its global transcription factor cAMP receptor protein (CRP), which is known to regulate over 400 genes in E. coli. Three ethanol tolerant CRP mutants (E1– E3) were identified from error-prone PCR libraries. The best ethanol-tolerant strain E2 (M59T) had the growth rate of 0.08 h−1 in 62 g/L ethanol, higher than that of the control at 0.06 h−1. The M59T mutation was then integrated into the genome to create variant iE2. When exposed to 150 g/l ethanol, the survival of iE2 after 15 min was about 12%, while that of BW25113 was <0.01%. Quantitative real-time reverse transcription PCR analysis (RT-PCR) on 444 CRP-regulated genes using OpenArray® technology revealed that 203 genes were differentially expressed in iE2 in the absence of ethanol, whereas 92 displayed differential expression when facing ethanol stress. These genes belong to various functional groups, including central intermediary metabolism (aceE, acnA, sdhD, sucA), iron ion transport (entH, entD, fecA, fecB), and general stress response (osmY, rpoS). Six up-regulated and twelve down-regulated common genes were found in both iE2 and E2 under ethanol stress, whereas over one hundred common genes showed differential expression in the absence of ethanol. Based on the RT-PCR results, entA, marA or bhsA was knocked out in iE2 and the resulting strains became more sensitive towards ethanol.

Influence of Beer Characteristics on Yeast Refermentation Performance during Bottle Conditioning of Belgian Beers

Although bottle conditioning of beer seems a rather simple process, problems often arise in industrial practice. Lack of process control can result in variable batch-to-batch quality, while constant product quality should be maintained anytime. In the present study, the refermentability of several Belgian beers was investigated to reveal important beer-related compounds influencing overall yeast fermentation performance during bottle conditioning. Bottled beer was inoculated with freshly propagated yeast and fermentable extract. Growth and refermentation performance of a less ethanol-tolerant yeast strain was more affected by high initial beer alcohol levels compared with a more ethanol tolerant strain. Yeast growth seemed no prerequisite for the completion of the refermentation process but could be beneficial, especially when using a less ethanol-tolerant yeast strain. A decrease of yeast activity during bottle conditioning resulted in delayed or stuck refermentations as well as an altered beer flavor profile with off-flavors like acetaldehyde. Initial beer alcohol content greatly determined yeast refermentation performance, though other beer characteristics or process related compounds should be considered, as well as a synergistic effect of various adverse factors. When bottle conditioning was successfully completed, a CO2 saturated beer with slightly increased alcohol content and enhanced fruity flavor was achieved.

Genome-scale identification and characterization of ethanol tolerance genes in Escherichia coli

The identification of relevant gene targets for engineering a desired trait is a key step in combinatorial strain engineering. Here, we applied the multi-Scalar Analysis of Library Enrichments (SCALEs) approach to map ethanol tolerance onto 1,000,000 genomic-library clones in Escherichia coli. We assigned fitness scores to each of the ∼4,300 genes in E. coli, and through follow-up confirmatory studies identified 9 novel genetic targets (12 genes total) that increase E. coli ethanol tolerance (up to 6-fold improved growth). These genetic targets are involved in the processes related to cell membrane composition, translation, serine biosynthesis, and transcription regulation. Transcriptional profiling of the ethanol stress response in 5 of these ethanol-tolerant clones revealed a total of 700 genes with significantly altered expression (mapped to 615 significantly enriched gene ontology terms) across all five clones, with similar overall changes in global gene expression between two clone clusters. All ethanol-tolerant clones analyzed shared 6% of the overexpressed genes and showed enrichment for transcription regulation-related GO terms. iTRAQ-based proteomic analysis of ethanol-tolerant strains identified upregulation of proteins related to ROS mitigation, fatty acid biosynthesis, and vitamin biosynthesis as compared to the parent strain's ethanol response. The approach we outline here will be useful for engineering a variety of other traits and further improvements in alcohol tolerance.

Impact of dried, creamed and cake supply formats on the genetic variation and ethanol tolerance of three Saccharomyces cerevisiae distilling strains

Scotch whisky fermentations typically employ high-gravity fermentation practices to maximize product formation and to minimize both energy and water inputs. This approach increases ethanol concentrations at the end of fermentation, creating stressful conditions for the yeast. In this work we examined the relative tolerance of four Saccharomyces cerevisiae distilling yeast strains, supplied in dried, creamed, cake or slurry format, to ethanol under CO2-induced anaerobic conditions. The cells were assessed for their capacity to recover and grow on inhibition spot plates and to maintain cell viability in ethanol-dosed suspensions. Variations in ethanol tolerance were observed between strains and between the same strain supplied in different formats. The creamed yeast format typically exhibited a higher tolerance to ethanol. One possible explanation for this observation is that cells surviving the dehydration and rehydration process might incur sub-lethal genome damage. Thus the genetic integrity of the most ethanol-tolerant strain was assessed as a function of supply format (two dried and one creamed). The mitochondrial DNA was examined using mitochondrial restriction fragment length polymorphism and the chromosomal DNA using pulsed field gel electrophoresis and polymerase chain reaction with both ITS and delta-specific primers. In one dried yeast sample, genetic integrity was compromised, highlighting the requirement for yeast intake quality assurance programmes. Copyright © 2012 The Institute of Brewing & Distilling

Ethanol reduces mitochondrial membrane integrity and thereby impacts carbon metabolism of Saccharomyces cerevisiae

Saccharomyces cerevisiae is an excellent ethanol producer, but is rather sensitive to high concentration of ethanol. Here, influences of ethanol on cellular membrane integrity and carbon metabolism of S.cerevisiae were investigated to rationalize mechanism involved in ethanol toxicity. Addition of 5% (v/v) ethanol did neither significantly change the permeability of the cytoplasmic membrane of the reference strain S.cerevisiae BY4741 nor of the ethanol-tolerant strain iETS3. However, the addition of ethanol resulted in a marked decrease in the mitochondrial membrane potential and in increased concentrations of intracellular reactive oxygen species (ROS). The carbon flux was redistributed under these conditions from mainly ethanol production to the TCA cycle. This redistribution was possibly a result of increased energy demand for cell maintenance that increased from about zero to 20-40mmolATP(g(CDW) h)(-1) . This increase in maintenance energy might be explained by the ethanol-induced reduction of the proton motive force and the required removal of ROS. Thus, the stability of the mitochondrial membrane and subsequently the capacity to keep ROS levels low could be important factors to improve tolerance of S.cerevisiae against ethanol.

English

The fermentation process of sugar feedstock materials at industrial scale requires the utilization of microorganisms capable of working at high ethanol concentration and high temperatures. The selection of&nbsp;Saccharomyces cerevisiae&nbsp;strains, able to ferment sugars obtained from different material at temperatures above 35&deg;C with high ethanol yield, has become a necessity. Three yeast strains were irradiated with gamma ray and screened for their ability to grow and ferment molasses in a temperature range of 35-45&deg;C. The yeasts were placed in a liquid medium, and irradiated&nbsp;at different doses (0.1, 1, 2, 3, 4, 5 and 10 KGy/h). Although all the isolated strains had growth (in agar plates) at 35 and 40&deg;C, but just two strains showed growth at 42&deg;C, and there was no growth at 45&deg;C. Two pure yeast strains were isolated (PTCC5269&nbsp;M3&nbsp;and&nbsp;Areni M7). The efficiency of temperature and high concentrations of ethanol tolerant strains were more than double of ethanol production compared with using the&nbsp;initial strains of&nbsp;yeast.&nbsp;All resistant strains were tested on liquid medium of molasses, and nutrients with 30% (v/v) ethanol had significant difference (P&gt;0.01) for growth intensity at same condition with&nbsp;initial strains. &nbsp; Key words:&nbsp;Bioethanol, gamma radiation,&nbsp;Saccharomyces cerevisiae, thermotolerant.

Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum

Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1-2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.

Isolation of ethanol-tolerant Saccharomyces cerevisiae strains by using a novel mutagenesis method

Isolation of ethanol-tolerant Saccharomyces cerevisiae strains by using a novel mutagenesis method