Ethanol-tolerant Saccharomyces cerevisiae strains isolated under selective conditions by over-expression of a proofreading-deficient DNA polymerase δ

Ethanol damages the cell membrane and functional proteins, gradually reducing cell viability, and leading to cell death during fermentation which impairs effective bioethanol production by budding yeast Saccharomyces cerevisiae. To obtain more suitable strains for bioethanol production and to gain a better understanding of ethanol tolerance, ethanol-tolerant mutants were isolated using the novel mutagenesis technique based on the disparity theory of evolution. According to this theory evolution can be accelerated by affecting the lagging-strand synthesis in which DNA polymerase δ is involved. Expression of the pol3-01 gene, a proofreading-deficient of DNA polymerase δ, in S. cerevisiae W303-1A grown under conditions of increasing ethanol concentration resulted in three ethanol-tolerant mutants (YFY1, YFY2 and YFY3), which could grow in medium containing 13% ethanol. Ethanol productivity also increased in YFY strains compared to the wild-type strain in medium containing 25% glucose. Cell morphology of YFY strain cells was normal even in the presence of 8% ethanol, whereas W303-1A cells were expanded by a big vacuole. Furthermore, two of these mutants were also resistant to high-temperature, Calcofluor white and NaCl. Expression levels of TPS1 and TSL1, which are responsible for trehalose biosynthesis, were higher in YFY strains relative to W303-1A, resulting in high levels of intracellular trehalose in YFY strains. This contributed to the multiple-stress tolerance that makes YFY strains suitable for the production of bioethanol.     

Disruption of URA7 and GAL6 improves the ethanol tolerance and fermentation capacity of Saccharomyces cerevisiae

Screening of the homozygous diploid yeast deletion pool of 4741 non-essential genes identified two null mutants (Deltaura7 and Deltagal6) that grew faster than the wild-type strain in medium containing 8% v/v ethanol. The survival rate of the gal6 disruptant in 10% ethanol was higher than that of the wild-type strain. On the other hand, the glucose consumption rate of the ura7 disruptant was better than that of the wild-type strain in buffer containing ethanol. Both disruptants were more resistant to zymolyase, a yeast lytic enzyme containing mainly beta-1,3-glucanase, indicating that the integrity of the cell wall became more resistance to ethanol stress. The gal6 disruptant was also more resistant to Calcofluor white, but the ura7 disruptant was more sensitive to Calcofluor white than the wild-type strain. Furthermore, the mutant strains had a higher content of oleic acid (C18 : 1) in the presence of ethanol compared to the wild-type strain, suggesting that the disruptants cope with ethanol stress not only by modifying the cell wall integrity but also the membrane fluidity. When the cells were grown in medium containing 5% ethanol at 15 degrees C, the gal6 and ura7 disruptants showed 40% and 14% increases in the glucose consumption rate, respectively.     

Rapid differentiation of Candida albicans from other Candida species using its unique germ tube formation at 39 degrees C

In this study, we found that no Candida species other than C. albicans is able to form germ tubes at 39 degrees C in serum-free YEPD (1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) dextrose) media, which makes it easy to identify C. albicans from other Candida species. When cultivated in rabbit serum for at least 2 h at 37 degrees C, more than 60% of C. albicans cells generated germ tubes. In YEPD, however, germ tubes began to appear from C. albicans cells within 30 min at 39 degrees C, and more than 60% of C. albicans cells formed the germ tubes after 1 h at 39 degrees C. Standard Candida strains (ATCC, CBS), three C. albicans and two C. dubliniensis strains were cultured in serum at 37 degrees C for 2 h and in YEPD at 39 degrees C for 1 h. All of the three C. albicans formed germ tubes at 39 degrees C. The two C. dubliniensis strains formed germ tubes in serum at 37 degrees C, but grew as a yeast form in YEPD at 39 degrees C. All of the clinically isolated C. albicans strains in our laboratory formed germ tubes in YEPD at 39 degrees C for 1 h, and none of the clinically isolated Candida species other than C. albicans generated germ tubes in YEPD at 39 degrees C. Thus, the unique germ tube formation of C. albicans induced by high temperature (39 degrees C) in YEPD could be applied to a protocol for the rapid and convenient identification of C. albicans in clinical laboratories.     

Increasing alcohol yield by selected yeast fermentation of sweet sorghum. II. Isolation and evaluation of mutants and wild types for ethanol production

Three yeast strains, which gave over 93% sugar conversion efficiency (SCE) in sweet sorghum juice containing 20% total sugar in a previous study, were submitted to 3 and 6% ethylmethanesulfonate (EMS) to induce mutations. Several mutants produced by the EMS treatment and their respective wild types grew well in media containing up to 40% sugar. Some mutants tolerated 2·5% more alcohol than their respective wild types. Saccharomyces cerevisiae IZ 1716 Mutant 10 produced a significantly greater yield of ethanol from sweet sorghum juice containing 26% sugar than the wild type. Under large-scale fermentation, this mutant had an SCE of 89·4% after 36h in sweet sorghum juice containing 28% sugar. The final alcohol concentration reached 13·28% (w/v) after 48h, corresponding to an SCE of 93·57%.     

Efficient very‐high‐gravity fermentation of sugarcane molasses by industrial yeast strains

In this study industrial strains were inoculated, in successive cycles, at high cell density into a sugarcane‐based juice containing normal (22%, w/v) and very high sucrose (30%, w/v) levels and supplemented with peptone as a nitrogen source. At 30 °C, in shaken cultures, in the normal gravity situation, efficient sucrose utilization occurred in both the supplemented and unsupplemented medium. At higher sugar concentrations, supplementation with peptone induced a more efficient fermentation compared with the unsupplemented medium, with higher biomass accumulation and maintenance of cell viability, and ethanol levels as high as 16% (v/v). Trehalose was also high during the cycles, probably as a necessary response of the yeast to the high stress fermentation conditions. This suggests that it is possible to increase ethanol production by using very‐high‐fermentation technology and that nutritional supplementation would have a positive effect on the fermentation, allowing for efficient sugar consumption and cell viability maintenance. Copyright © 2016 The Institute of Brewing & Distilling     

Cellular mechanism for the improvement of multiple stress tolerance in brewer's yeast by potassium ion supplementation

The ethanol fermentation efficiency was affected by multiple stress tolerance of yeast during brewing and bioethanol industry. The effect of KCl on the multiple stress tolerance of yeast cells was examined. Results showed that KCl addition significantly enhanced the tolerance of yeast cells to osmotic and ethanol stress, which correlated with the decreased membrane permeability, the increased intracellular ergosterol and ATP content, and the improved activity of complex II and complex III in yeast cells. Biomass and viability of yeast cells under osmotic and ethanol stress were increased significantly by KCl addition. Supplementation of 4 and 10 g L⁻¹ KCl exhibited the best promotion activity for yeast cells present in medium with 500 g L⁻¹ sucrose and 10% (v v⁻¹) ethanol, respectively. These results suggested that exogenous potassium addition might be an effective strategy to improve yeast tolerance and fermentation efficiency during industrial very-high-gravity (VHG) fermentation.     

Release of cell wall polysaccharides from Saccharomyces cerevisiae thermosensitive autolytic mutants during alcoholic fermentation

In order to increase the release of cell wall polysaccharides during alcoholic fermentation, a wine strain of Saccharomyces cerevisiae was subjected to UV mutagenesis to obtain thermosensitive autolytic mutants affected in cell wall integrity. Five mutants and the parental strain were utilized in fermentation trials conducted at 28, 32 and 34 degrees C. Results showed that at all temperatures the mutant strains released into the medium a higher polysaccharide quantity than the parental strain. In particular, at 28 degrees C there was a doubling of these macromolecules. At the end of alcoholic fermentation, all strains showed at 28 degrees C elevated and similar levels of viable cells; at 32 degrees C this parameter remained high for mutant strains ts16 and ts39 and the parental strain; at 34 degrees C all strains underwent a drop in cell viability, which was less intense in the case of strain ts16. As a relationship between cell viability and the quantity of polysaccharides released by the yeast strain was not found, it can be assumed that the mutation led to cells with a less stable wall and thus an easier release of macromolecules into the medium.     

小麦秸秆同步糖化发酵制取燃料乙醇

利用酿酒酵母Saccharomyces cerevisiae BY4742对小麦秸秆同步糖化发酵(simultaneously saccharification and fermentation,SSF)生产燃料乙醇的条件进行了研究,系统考察和研究了温度、固体含量、纤维素酶投加量、酵母菌浓度对SSF过程中乙醇浓度和产率的影响,并对以上参数做了初步优化,以提高最终乙醇浓度和产率。结果表明,小麦秸秆同步糖化发酵乙醇的最优条件为:温度38℃,固体含量16.0%(m/V),纤维素酶投加量35FPU/g底物,酵母菌浓度8 g/L。在此条件下,NaOH预处理后的小麦经过120 h同步糖化发酵,乙醇浓度达到最大值,为38.32 g/L,产率达理论产率的71.71%,木糖浓度为12.94 g/L。     

Characteristics of yeast flora in Chinese strong‐flavoured liquor fermentation in the Yibin region of China

The yeast community in the Chinese strong‐flavoured liquor region of Yibin was investigated and the ethanol producing abilities and extracellular enzymes activities of the isolates were tested. A total of 110 yeast were isolated on Wallerstein Laboratory medium and through 26S rRNA D1/D2 region sequence analysis identified as 13 yeast species. These were Wickerhamomyces anomalus, Debaryomyces hansenii, Issatchenkia orientalis, Lodderomyces elongisporus, Clavispora lusitaniae, Saccharomyces cerevisiae, Pichia fermentans, Pichia manshurica, Pichia membranifaciens, Torulaspora delbrueckii, Trichosporon insectorum, Trichosporonoides megachiliensis, Zygosaccharomyces bailii, and one uncertain species. These yeast species, composed of various strains, formed the special yeast community in the Yibin region. Approximately 73.6% of the strains belong to the four dominant species: W. anomalus, D. hansenii, I. orientalis and L. elongisporus. The 110 yeast strains produced 0.6–9.0% (v/v) alcohol (average of 5.4%, v/v) in a grain medium, and 0.2–7.2% (v/v) alcohol (average value of 2.9%, v/v) in a yeast extract–peptone–dextrose medium. Furthermore, the 49 strains that produced pectinase, lipase, cellulase, amylase or protease generally showed better ethanol‐producing ability than those strains that do not produce extracellular enzymes. This work profiles the ethanol‐producing ability and the organic matter utilization of the yeast community in Chinese strong‐flavoured liquor produced in the Yibin region and provides a better understanding of Chinese strong‐flavoured liquor fermentation. Copyright © 2016 The Institute of Brewing & Distilling     

VITALITY OF CIDER YEAST GROWN MICRO-AEROBICALLY WITH ADDED ETHANOL,BUTAN-J-OL OR ISO-BUTANOL

Vitality and viability of an alcohol-tolerant wine yeast, used in cider production, were assessed after exposure to alkanol(s) during growth. Criteria employed were: methylene blue reduction, ability to form colonies on yeast extract-peptone-glucose agar medium, glucose driven proton efflux rates (“acidification power”), fermentative (CO₂ output) rates and adenylate energy charge values. We also monitored the maintenance of transmembrane electrochemical potential across the plasma membrane as measured by flow cytometry and by scanning confocal laser microscopy of oxonol dye exclusion. Growth rates were diminished by a third by 7.5% (v/v) added ethanol, 1% butan-1-ol or 1.4% iso-butanol. Exposure to 10% (v/v) ethanol gave 16% loss of “viability”, as measured by methylene blue reduction, during the first 20 h of growth. For 1% butan-1-ol, 50% loss of “viability” occurred over 40 h, whereas a similar effect of iso-butanol took 55 h. Adenylate charge values were high (<0.8) in growing cultures, remained high in early stationary phase but declined to 0.4 after 115 h. These values were hardly affected by 5 or 7.5% (v/v) ethanol whereas 10% or 15% (v/v) ethanol gave values of 0.58 and 0.16 after only 5 h exposure. 1% butan-1-ol or iso-butanol decreased adenylate charge values to a greater extent than 10% (v/v) ethanol, with the straight chain alcohol the more potent. Oxonol exclusion indicated that the vast majority of colls with greatly diminished vitality have maintained the plasma membrane potential values required to retain viability, despite extensive exposure to alkanol(s). Thus loss of ability to reduce methylene blue indicates diminished vitality but is not a reliable index of loss of viability. “Acidification power” was a more sensitive indicator of vitality than adenylate charge values. When mixtures of C₂ + C₄ alcohols were employed effects were generally additive rather than synergistic.     

Robust industrial Saccharomyces cerevisiae strains for very high gravity bio-ethanol fermentations

The application and physiological background of two industrial Saccharomyces cerevisiae strains, isolated from harsh industrial environments, were studied in Very High Gravity (VHG) bio-ethanol fermentations. VHG laboratory fermentations, mimicking industrially relevant conditions, were performed with PE-2 and CA1185 industrial strains and the CEN.PK113-7D laboratory strain. The industrial isolates produced remarkable high ethanol titres (>19%, v/v) and accumulated an increased content of sterols (2 to 5-fold), glycogen (2 to 4-fold) and trehalose (1.1-fold), relatively to laboratory strain. For laboratory and industrial strains, a sharp decrease in the viability and trehalose concentration was observed above 90 g l?1 and 140 g l?1 ethanol, respectively. PE-2 and CA1185 industrial strains presented important physiological differences relatively to CEN.PK113-7D strain and showed to be more prepared to cope with VHG stresses. The identification of a critical ethanol concentration above which viability and trehalose concentration decrease significantly is of great importance to guide VHG process engineering strategies. This study contributes to the improvement of VHG processes by identifying yeast isolates and gathering yeast physiological information during the intensified fermentation process, which, besides elucidating important differences between these industrial and laboratory strains, can drive further process optimization.     

Effect of overexpression of LAS17 on stress tolerance and the stability of extrachromosomal DNA in Saccharomyces cerevisiae

The effects of the overexpression of LAS17/BEE1, which encodes a yeast protein exhibiting sequence homology to the Wiscott-Aldrich syndrome protein, on the cell growth of Saccharomyces cerevisiae were examined. Sake yeast strain UT-1 grows at a faster rate as a result of the overexpression of LAS17 than control cultures under various stresses such as high temperature, high ethanol concentration, and oxidative stress, and the tolerance to these stresses was increased compared with the control. Moreover, a high cell survival rate was attained with overexpression of LAS17, when cells in the stationary phase of the growth cycle were subjected to heat killing (48 degrees C) or ethanol killing (20% v/v). In addition, the rate of induction of rho- was markedly reduced by overexpression of LAS17 when serine, tyrosine, and aspartic acid were used as N sources and the yeast was cultured at 35 degrees C, while rho- strains in control cultures were induced at a high frequency. After the incubation of cells harboring a multicopy vector in YPD or synthetic complete medium, almost all of the cells inherited the vector at about 15 copies per cell as a result of the overexpression of LAS17, whereas the cells harboring the control vector accounted for only 15% of the total number of cells. These results suggest that Las17p might be a multifunctional protein involved in cell growth regulation, extrachromosomal DNA transportation and stress responses.     

高耐性酿酒酵母的筛选及其耐受性研究

优良的耐逆性菌株的添加能有效提高酱油生产效率和产品品质风味,该研究筛选出两株耐高温、耐高渗和耐高酸的酵母菌株用于酱油发酵。通过对酵母菌株高温热激之后稀释点板,对比各稀释度的菌落数量和形态,以及通过在高渗板和高酸板上各个菌的生长情况和在抗性培养基中菌的生长曲线测定来对比各菌株的耐受性。稀释点板实验以及生长曲线结果都显示,酿酒酵母L-19和L-38在55 ℃热激条件下以及在分别含有6%NaCl、0.6%乙酸和5%乳酸固体平板上菌落形态和大小都优于酱油酵母,而且在含有高盐和高酸的液体培养基中生长速率均高于酱油酵母。因此,成功筛选出两株具有高耐性的酿酒酵母。     

一株产乙酸乙酯酵母C42的分离与鉴定

从采自沧州良种枣繁育基地的200份枣果实样品中利用含有10%乙醇的YPD平板分离、筛选得到了36株耐酒精酵母菌株,其中一株酵母能产生愉悦的水果香味,命名为C-42。通过形态学,生理生化实验和18S rDNA序列同源性比对,证明该株酵母菌是异威克汉逊酵母（Wickerhamomyces anomalus）。气相色谱分析表明酵母菌株C-42在麦芽汁培养基中发酵一周后可产生乙酸乙酯,浓度可达154mg/L,说明该菌株在发酵工业中具有应用潜力。      

天山冻土中嗜冷酵母菌生物多样性

采用麦芽浸膏富集平板涂布天山冻土中可培养酵母菌,通过麦芽浸膏分离培养基筛选菌株.采用酵母菌常规生理生化实验、最适生长温度、最适pH对分离菌株的生理学进行研究.以NL1和NL4为PCR扩增引物,通过26S rRNA基因D1/D2区序列同源性分析,初步确定酵母菌株的系统进化地位.分离筛选到4株嗜低温酵母菌,分属于隐球酵母(Cystofilobasidium infirmominiatum)、掷孢酵母(Sporobolomyces patagonicus)、棒孢酵母(Clavispora lusitaniae)和毕赤酵母(Pichia kluyveri).     

Cultivation of the carotenoid-hyperproducing mutant 2A2N of the red yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) with molasses

The carotenoid-hyperproducing mutant 2A2N of the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) was cultivated using sugar beet blackstrap molasses. This molasses was composed of 70% (w/v) total solid and 50% (w/v) total sugar. Biomass yield (biomass/carbohydrate) significantly decreased at >5% (v/v) molasses. Atomic emission spectrometry revealed that Na and P were the limiting nutrients when molasses was used. Molasses (5%, v/v) containing urea (30 g/l molasses) and sodium phosphate (NaH2PO4, 5 g/l molasses) was formulated for biomass production by the mutant. The optimal pH for carotenoid production was 4.9 during the growth phase and 2.6-3.5 during the stationary phase. The three main sugars in molasses (sucrose, glucose, and fructose) were assimilated by the mutant but fructose was consumed slowly. When the formulated medium with pH 4.5-5.5 was used, the maximal biomass yield was 36 g/l (0.18 g of yeast l(-1)h(-1) and 40 mg of carotenoid l(-1)) in fed-batch pilot-scale 100-l cultivation.     

Functionality of selected strains of moulds and yeasts from Vietnamese rice wine starters

The role of starch-degrading mycelial fungi, and the alcohol production and ethanol tolerance of the yeasts isolated from selected Vietnamese traditional rice wine starters were examined, and optimum conditions for these essential steps in rice wine fermentation were determined. Of pure isolates from Vietnamese rice wine starters, mould strains identified as Amylomyces rouxii, Amylomyces aff. rouxii, Rhizopus oligosporus and Rhizopus oryzae, were superior in starch degradation, glucose production and amyloglucosidase activity during the saccharification of purple glutinous rice. A. rouxii was able to produce up to 25%w/w glucose with an amyloglucosidase activity up to 0.6 Ug(-1) of fermented moulded mass. Five yeast isolates identified as Saccharomyces cerevisiae were selected for their superior alcohol productivity. They were able to deplete a relatively high initial percentage of glucose (20% w/v), forming 8.8% w/v ethanol. The ethanol tolerance of S. cerevisiae in challenge tests was 9-10% w/v, and 13.4% w/v as measured in fed-batch fermentations. Optimum conditions for the saccharification were: incubation for 2 d at 34 degrees C, of steamed rice inoculated with 5 log cfu g(-1); for the alcoholic fermentation 4 d at 28.3 degrees C, of saccharified rice liquid inoculated with 5.5 log cfu mL(-1).     

Production of menaquinone (vitamin K2)-7 by Bacillus subtilis

Menaquinone-7 (MK-7) is a highly bioactive homologue of vitamin K. We obtained a diphenylamine-resistant mutant strain D200-41 from Bacillus subtilis strain MH-1 which was isolated from fermented soybeans, natto. The mutant strain exhibited decreased production of MK-6. Using strain D200-41, efficient production of MK-7 was achieved. We found that, compared with an agitated and aerated culture, production of MK-7 was increased by static culture. The sporulation of the cells progressed more slowly in a static culture than in an agitated culture. The maximum concentration of MK reached about 60 mg/l in a medium containing 10% soybean extract, 5% glycerol, 0.5% yeast extract and 0.05% K2HPO4 (pH 7.3) when D200-41 cells as well as MH-1 cells were statically cultured at 45 degrees C for 5 d after being cultured with shaking at 37 degrees C for 1 d.     

Very high gravity sucrose fermentation by Brazilian industrial yeast strains: effect of nitrogen supplementation

In this study three industrial strains were inoculated into natural sugar cane juice containing 22, 30 and 35% (w/v) sucrose supplemented with nitrogen sources with differing structural complexities, which varied from a single ammonium salt (ammonium sulphate) to peptides (peptone), under aerobic and anaerobic conditions. At 30 °C, in shaken cultures, it was found that efficient sucrose utilization occurred only in media supplemented with a nitrogen source. In general, under agitation, supplementation with peptone led to more efficient fermentation compared with ammonium sulphate supplementation, with higher biomass accumulation and maintenance of cell viability. In a 35% (w/v) sucrose fermentation, under conditions with an inoculation of low cell density, nitrogen supplementation was required to obtain complete sucrose utilization, suggesting the possibility of producing wines with higher amounts of ethanol under working conditions that approach the limit of yeast alcohol tolerance. The results in this study have industrial relevance and they indicate that, under appropriate environmental and nutritional conditions, the commercial Brazilian yeast strains studied can efficiently use sugar, with high cell viability, even during very high gravity sucrose fermentation conditions. Copyright © 2012 The Institute of Brewing & Distilling