Tolerant Yeast Research Articles

Insights into cell robustness against lignocellulosic inhibitors and insoluble solids in bioethanol production processes

Increasing yeast robustness against lignocellulosic-derived inhibitors and insoluble solids in bioethanol production is essential for the transition to a bio-based economy. This work evaluates the effect exerted by insoluble solids on yeast tolerance to inhibitory compounds, which is crucial in high gravity processes. Adaptive laboratory evolution (ALE) was applied on a xylose-fermenting Saccharomyces cerevisiae strain to simultaneously increase the tolerance to lignocellulosic inhibitors and insoluble solids. The evolved strain gave rise to a fivefold increase in bioethanol yield in fermentation experiments with high concentration of inhibitors and 10% (w/v) of water insoluble solids. This strain also produced 5% (P > 0.01) more ethanol than the parental in simultaneous saccharification and fermentation of steam-exploded wheat straw, mainly due to an increased xylose consumption. In response to the stress conditions (solids and inhibitors) imposed in ALE, cells induced the expression of genes related to cell wall integrity (SRL1, CWP2, WSC2 and WSC4) and general stress response (e.g., CDC5, DUN1, CTT1, GRE1), simultaneously repressing genes related to protein synthesis and iron transport and homeostasis (e.g., FTR1, ARN1, FRE1), ultimately leading to the improved phenotype. These results contribute towards understanding molecular mechanisms that cells might use to convert lignocellulosic substrates effectively.

GPCR-FEX: A Fluoride-Based Selection System for Rapid GPCR Screening and Engineering.

The directed evolution of proteins comprises a search of sequence space for variants that improve a target phenotype, yet identification of desirable variants is inherently limited by library size and screening ability. Selections that couple protein phenotype to cell viability accelerate identification of promising variants by depleting libraries of undesirable variants en masse. Here, we introduce GPCR-FEX, a stringent selection platform that couples G-protein coupled receptor (GPCR) signaling to expression of a fluoride ion exporter (FEX)-GFP fusion gene and concomitant cellular fluoride tolerance in yeast. The GPCR-FEX platform works to deplete inactive GPCR variants from the library prior to high-throughput fluorescence-based cell sorting for rapid, inexpensive screening of receptor libraries that sample an expanded sequence space. Using this system, FEX1 was placed under the control of either PFUS1 or PFIG1, promoters activated upon agonist binding by the native yeast GPCRs, Ste2p or Ste3p. Addition of a C-terminal degron to FEX1p enhanced the dynamic range of cell growth between agonist-treated and untreated cells. Using deep sequencing to enumerate population members, we show rapid selection of a previously engineered Ste2p receptor mutant strain over wild-type Ste2p in a model library enrichment experiment. Overall, the GPCR-FEX platform provides a mechanism to rapidly engineer GPCRs, which are important cellular sensors for synthetic biology.

The multiple roles of lipid metabolism in yeast physiology during beer fermentation.

The ability of brewing yeasts (Saccharomyces cerevisiae and Saccharomyces pastorianus) to cope with the toxic effects of ethanol during beer fermentation depends on the modulation of lipid and lipid droplets (LDs) biosynthesis, which affects membrane fluidity. However, it has been demonstrated that lipids and LDs can modulate different biological mechanisms associated to ethanol tolerance, including proteostasis and autophagy, leading to the hypothesis that lipid and LDs biosynthesis are integrative processes necessary for ethanol tolerance in yeast. Supporting this hypothesis, a transcriptome and systems biology analyses indicated the upregulation of autophagy, lipid biosynthesis, and proteostasis (ALP)-associated genes in lager yeast during beer fermentation, whose respective proteins interact in a shortest-pathway ALP network. These results indicated a cross-communication between various pathways linked to inter-organelle autophagy, lipid metabolism, and proteostasis (ALP) during lager beer fermentation, thus highlighting the importance of lipids for beer fermentation.

Overexpression of a leucine transfer RNA gene tL(CAA)K improves the acetic acid tolerance of Saccharomyces cerevisiae

Acetic acid is a common inhibitor present in lignocellulosic hydrolysate. Development of acetic acid tolerant strains may improve the production of biofuels and bio-based chemicals using lignocellulosic biomass as raw materials. Current studies on stress tolerance of yeast Saccharomyces cerevisiae have mainly focused on transcription control, but the role of transfer RNA (tRNA) was rarely investigated. We found that some tRNA genes showed elevated transcription levels in a stress tolerant yeast strain. In this study, we further investigated the effects of overexpressing an arginine transfer RNA gene tR(ACG)D and a leucine transfer RNA gene tL(CAA)K on cell growth and ethanol production of S. cerevisiae BY4741 under acetic acid stress. The tL(CAA)K overexpression strain showed a better growth and a 29.41% higher ethanol productivity than that of the control strain. However, overexpression of tR(ACG)D showed negative influence on cell growth and ethanol production. Further studies revealed that the transcriptional levels of HAA1, MSN2, and MSN4, which encode transcription regulators related to stress tolerance, were up-regulated in tL(CAA)K overexpressed strain. This study provides an alternative strategy to develop robust yeast strains for cellulosic biorefinery, and also provides a basis for investigating how yeast stress tolerance is regulated by tRNA genes.

Evolutionary and reverse engineering to increase Saccharomyces cerevisiae tolerance to acetic acid, acidic pH, and high temperature.

Saccharomyces cerevisiae scarcely grows on minimal media with acetic acid, acidic pH, and high temperatures. In this study, the adaptive laboratory evolution (ALE), whole-genome analysis, and reverse engineering approaches were used to generate strains tolerant to these conditions. The thermotolerant strain TTY23 and its parental S288C were evolved through 1year, in increasing concentrations of acetic acid up to 12g/L, keeping the pH ≤ 4. Of the 18 isolated strains, 9 from each ancestor, we selected the thermo-acid tolerant TAT12, derived from TTY23, and the acid tolerant AT22, derived from S288C. Both grew in minimal media with 12g/L of acetic acid, pH 4, and 30°C, and produced ethanol up to 29.25 ± 6mmol/gDCW/h-neither of the ancestors thrived in these conditions. Furthermore, only the TAT12 grew on 2g/L of acetic acid, pH 3, and 37°C, and accumulated 16.5 ± 0.5mmol/gDCW/h of ethanol. Whole-genome sequencing and transcriptomic analysis of this strain showed changes in the genetic sequence and transcription of key genes involved in the RAS-cAMP-PKA signaling pathway (RAS2, GPA2, and IRA2), the heat shock transcription factor (HSF1), and the positive regulator of replication initiation (SUM1), among others. By reverse engineering, the relevance of the combined mutations in the genes RAS2, HSF1, and SUM1 to the tolerance for acetic acid, low pH, and high temperature was confirmed. Alone, the RAS2 mutation yielded acid tolerance and HSF1 nutation thermotolerance. Increasing the thermo-acidic niche and acetic acid tolerance of S. cerevisiae can contribute to improve economic ethanol production. KEY POINTS: • Thermo-acid tolerant (TAT) yeast strains were generated by adaptive laboratory evolution. • The strain TAT12 thrived on non-native, thermo-acidic harmful conditions. • Mutations in RAS2, HSF1, and SUM1 genes rendered yeast thermo and acid tolerant.

Effective factors for the performance of a co-generation system for bioethanol and electricity production via microbial fuel cell technology

A co-generation system was designed to extract the redundant electrons generated in the biofermentation process to produce electricity and bioethanol simultaneously by using a microbial fuel cell (MFC) device. The effects of process operating parameters–methylene blue (MB) concentration, substrate concentration, temperature, and anodic pH on a co-generation system of ethanol and electricity were investigated to improve energy efficiency during ethanol fermentation. The results showed that a positive correlation between the power density of the co-generation system with substrate concentration and temperature was found over the range of 20–100 g/L and 25–35 ℃, respectively, in which ethanol was maintained at a high production level. The neutral or acid anodic solution was preferred for the co-generation system rather than the alkaline one when considering both power out and ethanol production. By assisting MFC technology, the total energy efficiency of fermentation was improved from 91.01% to 94.21% where 0.98% additional valuable electric energy was recovered. Yeast domestication is a promising way to enhance the power performance by increasing the tolerance of yeast to MB without reducing ethanol production. The proposed co-generation system indicates that the potential application of MFC in the production of biofuel or bioproducts efficiently and sustainably.

Identification of StAP2/ERF genes of potato (Solanum tuberosum) and their multiple functions in detoxification and accumulation of cadmium in yest: Implication for Genetic-based phytoremediation

The discovery of genes responsible for the tolerance to heavy metals is critical for genome-based phytotechnologies. In this study, we exposed potato (Solanum tuberosum L.) to Cd/Pb/Zn/Ni/Cu as an approach to explore the potential genes associated with stress tolerance. Using genome-wide analysis, we identified 181 potential StAP2/ERF genes that were classified into three subgroups. These StAP2/ERF genes were significantly related to heavy metal stress and are more specifically related to Cd tolerance in yeast. Yeast complementation tests showed that the StAP2/ERF129/139 genes (Subgroup 1) decreased Cd accumulation (Cd reduction-type), whilst the StAP2/ERF044/180 genes (Subgroup 2) promoted Cd accumulation in yeast which showed inhibited growth (Cd accumulation-type). The StAP2/ERF075/077/126 genes (Subgroup 3) promoted Cd accumulation and yeast growth (Cd detoxification-type). We used phylogenetic analysis to classify the 181 genes into three Cd tolerant types defined above in which the numbers of Cd reduction, accumulation, and detoxification type genes were 81, 65 and 35 respectively. Also, we performed tandem duplication, phylogenetic, and conserved motifs analysis to characterization the StAP2/ERF genes and results supported their functions in Cd tolerance. Our study showed that StAP2/ERFs is indispensable in Cd uptake and tolerance, and may be useful towards designing gene-modified plants with improved Cd tolerances.

Copper Tolerance Mechanism of the Novel Marine Multi-Stress Tolerant Yeast Meyerozyma guilliermondii GXDK6 as Revealed by Integrated Omics Analysis.

Various agricultural products used in food fermentation are polluted by heavy metals, especially copper, which seriously endangers human health. Methods to remove copper with microbial strategies have gained interests. A novel Meyerozyma guilliermondii GXDK6 could survive independently under high stress of copper (1400 ppm). The copper tolerance mechanism of GXDK6 was revealed by integrated omics in this work. Whole-genome analysis showed that nine genes (i.e., CCC2, CTR3, FRE2, GGT, GST, CAT, SOD2, PXMP4, and HSP82) were related to GXDK6 copper tolerance. Copper stress elevated glutathione metabolism-related gene expression, glutathione content, and glutathione sulfur transferase activity, suggesting enhanced copper conjugation and detoxification in cells. The inhibited copper uptake by Ctr3 and enhanced copper efflux by Ccc2 contributed to the decrease in intracellular copper concentration. The improved expression of antioxidant enzyme genes (PXMP4, SOD2, and CAT), accompanied by the enhanced activities of antioxidant enzymes (peroxidase, superoxide dismutase, and catalase), decreased copper-induced reactive oxygen species production, protein carbonylation, lipid peroxidation, and cell death. The metabolite D-mannose against harsh stress conditions was beneficial to improving copper tolerance. This study contributed to understanding the copper tolerance mechanism of M. guilliermondii and its application in removing copper during fermentation.

Potentials of multi-stress tolerant yeasts, Saccharomyces cerevisiae and Pichia kudriavzevii for fuel ethanol production from industrial cassava wastes

Cassava processing wastes are suitable substrates for bioethanol production due to their high starch contents. However, these cassava wastes often contain various substances that inhibit cell growth and ethanol production. The abilities of multi-stress tolerant Saccharomyces cerevisiae LC269108 and Pichia kudriavzevii LC375240 to produce ethanol from cassava industrial wastes were evaluated. Analysis of cassava peels and pulps from two cassava starch factories showed that the cassava peels contained high cyanide (KCN) concentrations while cassava pulps contained high concentrations of sodium dodecyl benzene sulfonate (SDBS) used to improve starch extraction in the factory. Although S. cerevisiae LC269108 produced ethanol efficiently from the cassava peel, its growth and ethanol production were inhibited in the cassava pulp. On the other hand, the growth and ethanol production by P. kudriavzevii LC375240 were inhibited slightly in the cassava peel but not in the cassava pulp. Based on the intracellular pyruvate concentrations in the presence of KCN and SDBS, it was inferred that their inhibition site is on the pyruvate synthetic pathways. With a co-culture of the two strains, ethanol production from the cassava peel increased significantly but there was no significant improvement in ethanol production from the cassava pulp.

Development of multiple inhibitor tolerant yeast via adaptive laboratory evolution for sustainable bioethanol production

The present research work aimed at developing robust yeast cell factory via adaptive laboratory evolution (ALE) for improved cellulosic bioethanol production. Kluyveromyces marxianus JKH5, a newly isolated thermotolerant ethanologenic yeast, was engineered by serial passaging for 60 generations in medium supplemented with gradually higher concentration of inhibitors (acetic acid, furfural, and vanillin) that are generated during dilute acid pretreatment. The improved strain K. marxianus JKH5 C60, showed 3.3-fold higher specific growth rate, 56% reduced lag phase and 80% enhanced fermentation efficiency at 42 °C in comparison to parent strain in inhibitor cocktail comprising medium. Bioethanol production by simultaneous saccharification and fermentation of sequential dilute acid-alkali pretreated sugarcane bagasse in presence of inhibitors, resulted in ethanol titre and yield, respectively, 54.8 ± 0.9 g/L and 0.40 g/g. The adapted yeast can be used to ferment unwashed pretreated biomass, thereby, reducing overall cost, time, and wastewater generation, hence making bioethanol production sustainable.

Isolation of thermo-tolerant and ethanol-tolerant yeast from local fermented foods and their potential as bioethanol producers

Bioethanol is a liquid chemical produced from sugar-, starch-or lignocellulosic-based biomass through fermentation by ethanol-producing microbes. Ethanol-producing yeast generally has limited tolerance to ethanol and has limitation to high temperatures above 40°C. High-temperature tolerant yeast is required because it potentially reduces the risk of contamination and it also reduces the cost of the cooling process. This study aims to determine ethanol-producing yeasts that have tolerance to ethanol and high temperatures from local fermented food products. This study uses a descriptive method conducted in three stages. Isolation and selection of yeast were performed from 18 local fermented foods in Indonesia. Temperature and ethanol tolerance of selected yeast were performed by using a spot test method. The ethanol content was tested using Gas Chromatography (GC). The results exhibited that isolate F08b had the highest tolerance to ethanol and temperature. The isolate was able to grow up to a temperature of 50°C and a concentration of 18% ethanol. Meanwhile, isolate F10 was able to produce the highest ethanol concentration at 3.37% (v/v) in 48th-hour fermentation.

Honey Mead Fermentation from Thai Stingless Bee (Tetragonula leaviceps) Honey using Ethanol Tolerant Yeast

Honey mead is a well-known conventional alcoholic beverage made by microbial fermentation of diluted honey. The selection of prospective yeasts for inoculation of honey-must with regard to honey mead quality determines the quality of mead production. The yeast consortium tolerant to ethanol stress was selected for this study using an enrichment technique. The activity of the invertase enzyme and the level of ethanol tolerance have been investigated. Thai stingless bee honey was used as a substrate, and the selected ethanol tolerant yeast consortium was used for mead fermentation. The results revealed that the PP03 had the highest invertase activity of 75.13±9.16 U/mL and the highest ethanol tolerance level of 12%. This is the first study using an ethanol tolerant yeast consortium to ferment honey mead from Thai stingless bee honey.

Antiaging Properties of the Ethanol Fractions of Clove (Syzygium aromaticum L.) Bud and Leaf at the Cellular Levels: Study in Yeast Schizosaccharomyces pombe

The exposure of reactive oxygen species is one of the aging triggers at cellular level. The antioxidants have been used as strategic efforts in overcoming the accumulation of ROS. Previous research using crude extracts of clove bud and leaves showed its potential as an antioxidant agent. However, no data were available regarding the antioxidant and antiaging activities of subsequent fractions of clove extracts. Therefore, this study aimed to analyze the antioxidant and antiaging activities of the n-hexane and ethanol fractions from clove bud and leaves. Antioxidant and antiaging activities were tested at the cellular level using the yeast model Schizosaccharomyces pombe. The highest flavonoid content was shown by clove leaf n-hexane fraction (25.6 mgQE·g−1). However, ethanol fraction of clove bud (FEB) showed the highest antioxidant activity based on TBA and antiglycation assays. FEB (8 μg·mL−1) and leaf ethanol fraction (FEL) (10 μg·mL−1) were able to induce yeast tolerance against oxidative stress. In addition, FEB could induce mitochondrial activity and delay the G1 phase of the cell cycle. FEB was found to be rich in gallic acid and (15Z)-9,12,13-trihydroxy-15-octadecenoic. Based on the data, FEB shows the potential antiaging activity, which is promising for further development as biopharmaceutical product formulations.

Screening and identification of salt-tolerance genes in Sophora alopecuroides and functional verification of SaAQP.

Overexpression of SaAQP can improve the salt tolerance of transgenic soybean hairy roots and A. thaliana. Salt stress severely affects crop yield and food security. There is a need to improve the salt tolerance of crops, but the discovery and utilization of salt-tolerance genes remains limited. Owing to its strong stress tolerance, Sophora alopecuroides is ideal for the identification of salt-tolerance genes. Therefore, we aimed to screen and identify the salt-tolerance genes in S. alopecuroides. With a yeast expression library of seedlings, salt-tolerant genes were screened using a salt-containing medium to simulate salt stress. By combining salt-treatment screening and transcriptome sequencing, 11 candidate genes related to salt tolerance were identified, including genes for peroxidase, inositol methyltransferase, aquaporin, cysteine synthase, pectinesterase, and WRKY. The expression dynamics of candidate genes were analyzed after salt treatment of S. alopecuroides, and salt tolerance was verified in yeast BY4743. The candidate genes participated in the salt-stress response in S. alopecuroides, and their overexpression significantly improved the salt tolerance of yeast. Salt tolerance mediated by SaAQP was further verified in soybean hairy roots and Arabidopsis thaliana, and it was found that SaAQP might enhance the salt tolerance of A. thaliana by participating in a reactive oxygen species scavenging mechanism. This result provides new genetic resources in plant breeding for salt resistance.

Short-term Adaptation Strategy Improved Xylitol Production by Candida guilliermondii on Sugarcane Bagasse Hemicellulosic Hydrolysate

In this work, we proposed a short-term adaptation strategy to improve xylitol production on sugarcane bagasse hemicellulosic hydrolysate through the maximization of Candida guilliermondii FTI 20037 tolerance to inhibitors. Hemicellulosic hydrolysate obtained by diluted acid hydrolysis (1.0% (wv−1) H2SO4, 1:10 solid/liquid ratio, 121 °C, 10 min) was concentrated up to fivefold to obtain hydrolysates with different concentration factors. Yeast was cultivated in each hydrolysate for 24-h and consecutively transferred to the subsequent more concentrated hydrolysate to obtain different adaptation degrees. Adapted cells were used as inoculum in fermentations with the same hydrolysate in which they were adapted. The performance of adapted and non-adapted yeast was compared to validate the adaptation strategy employed. The beneficial effects of adaptation were more pronounced in the hydrolysates with higher inhibitor concentration (twofold concentrated and non-treated, H2N; and fivefold concentrated and treated, H5). It improved xylose assimilation and xylitol production as well as xylitol yield and xylitol volumetric productivity in both hydrolysates. A 62.5% increase in productivity (0.24 to 0.39 gL−1 h−1) and a 15.7% increase in yield (0.51 to 0.59 gg−1) were observed for H5 hydrolysate, while for H2N hydrolysate these increases were 54.5 and 29.6%, respectively. Yeast adaptation also improved arabinose consumption and reduced glycerol production. The reduction in glycerol production indicates a greater tolerance of adapted cells to the inhibitors present in hydrolysates. Short-term adaptation proved to be an efficient strategy to improve yeast tolerance as well as its fermentative performance on sugarcane bagasse hemicellulosic hydrolysate.

Bioprospecting thermotolerant yeasts from distillery effluent and molasses for high-temperature ethanol production.

Isolation, characterization and assessment of inhibitor tolerance of thermotolerant yeasts associated with distillery effluent and molasses, and their use in high-temperature ethanol production from alkali-treated rice straw. A total of 92 thermotolerant yeasts were isolated from seven different distillery effluent and molasses samples. Based on MSP-PCR, 34 yeasts were selected and identified by sequencing the D1/D2 domain of LSU rDNA. These yeasts belonged to eight genera and nine different species. We assessed the inhibitor tolerance of these 34 well-characterized yeasts against various pre-treatment-generated inhibitors (furfural, 5-hydroxymethyl furfural and acetic acid) and also evaluated their ethanol yields at 40, 45 and 50℃. Among selected strains, Pichia kudriavzevii DSA3.2 exhibited the highest ethanol production (24.5gl-1 ) with an efficiency of 95.7% at 40℃ using 5% glucose. At 45℃, P. kudriavzevii DSA3.2 and Kluyveromyces marxianus MSS6.3 yielded maximum ethanol titres; 22.3 and 23gl-1 with 87.4% and 90% efficiency, respectively. While using alkali-treated RS at 45℃, K. marxianus MSS6.3 produced 10.5gl-1 of ethanol with 84.5% fermentation efficiency via separate hydrolysis and fermentation, and 10.9gl-1 of ethanol with 85% efficiency via simultaneous saccharification and fermentation. Pichia kudriavzevii DSA3.2, DSA3.1 and K. marxianus MSS6.3 also exhibited significant tolerance against multiple inhibitors. Yeast isolates P. kudriavzevii DSA3.2 and K. marxianus MSS6.3 exhibited significant inhibitor tolerance and proved to be suitable for high-temperature ethanol fermentation. After additional optimization and scale-up experiments, these isolates can be exemplary candidates for industrial-scale ethanol production from lignocellulosic biomass. Our study recognizes distillery effluents and molasses as specialized niches for yeasts with a broad substrate range, capable of tolerating multiple inhibitors and yielding high levels of ethanol at elevated temperatures. These yeasts can further be exploited for bioethanol production through SSF/SHF at a larger scale.

Overview of yeast environmental stress response pathways and the development of tolerant yeasts

Yeast is widely used for industrial production of various types of products, such as ethanol and enzymes. However, its fermentation efficiency is strongly reduced by harmful environmental stresses. Specifically, harmful environmental stresses damage important cellular components, such as cell wall, cell membrane, proteins, etc. Then, these damages cause cellular metabolic disorders or even death. In the past decades, there has been a portfolio of studies on the environmental stress tolerance of yeasts, which mainly aimed at cell damages caused by different environmental stresses, different ways to improve yeast environmental stress tolerance or a tolerance mechanism for certain environmental stress. However, a comprehensive overview of how yeasts respond to environmental stresses is lacking, and the correlation of tolerance mechanism between different environmental stresses is unclear. In this review, we summarized the general damages induced by most of environmental stresses, the existing major mechanisms of environmental stress tolerance from the perspective of key signalling pathways, and the common ways to improve the resistance to environmental stresses in yeast cells. The tolerance mechanisms of yeast cells to different environmental stresses are diverse, but sometimes they share the same signalling pathway. Cells use sensors on the cell surface to recognize environmental stresses and transmit signals to the nucleus to cause changes in gene expression. By summarizing the main signalling pathways, including MAPK pathway, cAMP/PKA pathway, YAP1/SKN7 pathway, it will provide a powerful reference for future efforts to promote yeast environmental stress tolerance and study yeast tolerance mechanisms.

Transporter engineering promotes the co-utilization of glucose and xylose by Candida glycerinogenes for d-xylonate production

Sugar transporter-mediated sugar transport is the first rate-limiting step in the control of yeast metabolism. Almost all known wild-type sugar transporters do not have the ability to co-transport glucose and xylose, which makes yeast less available to mixed sugar substrates (e.g. lignocellulose hydrolysate) and leads to lower product titers. Here, we achieved co-utilization of glucose and xylose by the tolerant yeast Candida glycerinogenes and facilitated D-xylonate production through rational modification of the hexose transporter (transporter engineering). Specifically, a hexose transporter mutant CgHxt4.2A.S (CgHxt4K9A, N351S, K538A) with high membrane localization stability (MLS) and co-transport of glucose and xylose was obtained by functional characterization, homology modeling, molecular docking and rational mutagenesis. Ultimately, this mutant enhanced the utilization of sugars (especially xylose) by C. glycerinogenes and increased the titer of D-xylonate in sugarcane bagasse hydrolysate by 49.3%. Our study provides a promising strategy for the future efficient utilization of cheaper lignocellulosic hydrolysates by recombinant yeast for the production of high-value chemicals.

Studies on Bioethanol Production with Thermo Tolerant Yeast Isolates and their Co-Cultures using African Wild Cocoyam as Feedstock

In this work different ways of optimally producing bioethanol at various pH with thermotolerant yeasts and their cocultures using a non-human edible starchy food as feedstock was examined. African wild cocoyam, Xanthosoma roseum, sourced from abandoned farmlands in Obukpa, Nsukka, Nigeria was used as the substrate, while strains of Kluyveromyces marxianus and Pichia stipitis were used to ferment them. First the tubers were gelatinized by boiling under pressure above 100oC before hydrolysis with concentrated H2SO4. The hydrolysates were then fermented at 35oC with the thermotolerant yeasts for five days at different pH. Results obtained showed that gelatinized sample of the substrate gave optimum glucose yield when hydrolysed with 1M H2SO4 for 60 minutes. Kluyveromyces marxianus produced more ethanol than Pichia stipitis at all the four fermentation pH values tested. However, optimum ethanol production was obtained when the two yeast strains were used as coculture at pH 4.5. The peak time for ethanol production was 96 hours for the individual yeast cultures while that of their coculture was 72 hours. The results of the study indicated that wild cocoyam is an excellent feedstock for bioethanol production with many advantages including being non-edible, thereby eliminating concerns for food security, and containing high amount of carbohydrate. The study also revealed that fermenting sugar hydrolysates with a coculture of microorganisms during bioethanol production is a more efficient process than using individual cultures.

Устойчивость дрожжей Saccharomyces cerevisiae к экстремальным условиям

The resistance of the yeast Saccharomyces cerevisiae DAW-3а and Y-503 to the conditions of extreme values of pH, NaCl, temperature has been studied. Under different cultivation modes, the rounded shape of DAW-3a cells is pre-served and this parameter Y-503 changes. Under conditions of different pH values and 30°C, the maximum sizes of cells and giant colonies of the polyploid Y-503 strain were found in comparison with the haploid DAW-3a; at 37°C, the advantage of Y-503 was not observed and the colony sizes of both strains were practically the same. The reaction of the strains to critical concentrations of NaCl in the medium was identical: a decrease in the size of cells and colonies was found; change in the shape, surface, color and structure of colonies. Under conditions of the simultaneous influence of an elevated temperature of 37°C, a wide range of pH and 5 % NaCl, the cell size decreased slightly; under neutral and alkaline conditions of cultivation, a slightly greater tolerance of yeast to salt stress was established; a decrease in the size of giant colonies was found, while the maximum size of the studied strains was noted at pH 11.0, the minimum at pH 3.0. The study of the tolerance of the yeast S. cerevisiae DAW-3a and Y-503 to extreme factors is of particular interest in connection with the possibility of using stress-resistant strains in biotechnological processes.