Overexpression of vacuolar H+‐ATPase‐related genes in bottom‐fermenting yeast enhances ethanol tolerance and fermentation rates during high‐gravity fermentation

Vacuolar H⁺‐ATPase (V‐ATPase) is thought to play a role in stress tolerance. In this study it was found that bottom‐fermenting yeast strains, in which the V‐ATPase‐related genes DBF2, VMA41/CYS4/NHS5 and RAV2 were overexpressed, exhibited stronger ethanol tolerance than the parent strain and showed increased fermentation rates in a high‐sugar medium simulating high‐gravity fermentation. Among the strains examined, the DBF2‐overexpressing bottom‐fermenting yeast strain exhibited the highest ethanol tolerance and fermentation rate in YPM20 medium. Using this strain, high‐gravity fermentation was performed by adding sugar to the wort, which led to increased fermentation rates and yeast viability compared with the parent strain. These findings indicate that V‐ATPase is a stress target in high‐gravity fermentation and suggests that enhancing the V‐ATPase activity increases the ethanol tolerance of bottom‐fermenting yeast, thereby improving the fermentation rate and cell viability under high‐gravity conditions. Copyright © 2012 The Institute of Brewing & Distilling     

Altered Patterns of Maltose and Glucose Fermentation by Brewing and Wine Yeasts Influenced by the Complexity of Nitrogen Source

Maltose and glucose fermentations by industrial brewing and wine yeasts strains were strongly affected by the structural complexity of the nitrogen source. In this study, four Saccharomyces cerevisiae strains, two brewing and two wine yeasts, were grown in a medium containing maltose or glucose supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low sugar concentration for brewing and wine strains, independent of nitrogen supplementation, and the type of sugar. At high sugar concentrations altered patterns of sugar fermentation were observed, and biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for brewing and wine strains. In maltose, high biomass production was observed under peptone and casamino acids for the brewing and wine strains, however efficient maltose utilization and high ethanol production was only observed in the presence of casamino acids for one brewing and one wine strain studied. Conversely, peptone and casamino acids induced higher biomass and ethanol production for the two other brewing and wine strains studied. With glucose, in general, peptone induced higher fermentation performance for all strains, and one brewing and wine strain produced the same amount of ethanol with peptone and casamino acids supplementation. Ammonium salts always induced poor yeast performance. The results described in this paper suggest that the complex nitrogen composition of the cultivation medium may create conditions resembling those responsible for inducing sluggish/stuck fermentation, and indicate that the kind and concentration of sugar, the complexity of nitrogen source and the yeast genetic background influence optimal industrial yeast fermentation performance.     

A novel alcoholic beverage developed from shiitake stipe extract and cane sugar with various Saccharomyces strains

The stipe of shiitake, a disposable byproduct in mushroom industry, was applied as a nitrogen source in alcoholic fermentation. Four yeast strains were screened by evaluating their fermentative efficiency and ethanol tolerance. The results showed that shiitake stipe extract could enable yeast cell proliferation and be sequent to good performance of sugar utilization and ethanol production, depended on strains. The ethanol production (mL/100 mL) of 13.7 ± 0.8, 14.1 ± 0.9, 11.9 ± 0.1 and 10.9 ± 0.3 was obtained from strains 20262, 22236, 21686 and 21992, respectively, at the end of fermentation. Ethanol tolerance of strain in term of glucose utilization rate was more consistent with the extent of ethanol production during fermentation than that of survival rate. Therefore, the evaluation of glucose utilization rate in external ethanol condition might help establish a simple and quick method to screen the strains capable of high ethanol production. The shiitake stipe alcoholic beverage would also provide comparable consumer acceptances with a commercial Taiwanese white wine.     

Isolation and Characterisation of Sri Lankan Yeast Germplasm and Its Evaluation for Alcohol Production

Use of inferior yeast cultures represents one of the reasons for low fermentation efficiencies in Sri Lankan alcohol distilleries that use sugarcane molasses. The present study isolated and characterised yeast strains found in natural environments in Sri Lanka and evaluated their performance under laboratory conditions in an effort to select superior strains for industrial fermentations. Yeasts were characterised based on morphological and physiological features such as sugar fermentation and nitrate assimilation. Ethanol production, alcohol tolerance and growth rate of the most promising strains were monitored following laboratory fermentations of molasses. Over a thousand yeast cultures were collected and screened for fermentative activity and a total of 83 yeast isolates were characterised as higher ethanol producers. Most of these belonged to the genus Saccharomyces. Certain strains produced over 10% (v/v) alcohol in molasses media during 72 h laboratory fermentations. Only two strains, SL‐SRI‐C‐102 and 111, showed an appreciable fermentation efficiency of about 90%. The latter strain produced the highest level of ethanol, 11% (v/v) within a 48 h fermentation and exhibited improved alcohol tolerance when compared with the baker's yeast strains currently used in Sri Lankan alcohol distilleries. This study highlights the benefits of exploiting indigenous yeasts for industrial fermentation processes.     

THE EFFECTS OF INCREASED MAGNESIUM AND CALCIUM CONCENTRATIONS ON YEAST FERMENTATION PERFORMANCE IN HIGH GRAVITY WORTS*

The response of e number of Saccharomyces cerevisiae (ale) and Saccharomyces uvarum (caris-bargenaia) (lager) strains to altered starting levels of magnesium and calcium in 12°P (1048 original gravity) or 20°P (1080 original gravity) wort were investigated. In general, the same trends were observed in all 6 strains, however the extent of the response to adjusted levels of magnesium and calcium were found to be strain dependent. The results indicate that an increased ratio of magnesium to calcium causes an increase in the initial fermentation rate, the rate and yield of ethanol produced and an increase in vitality at the end of fermentation, in all strains employed. Upon increasing the calcium to magnesium ratio it was found that the initial fermentation rate was decreased, resulting in an increased attenuation time in the case of the lager strains. It was also noted that increasing the calcium to magnesium ratio led to a decreased ethanol production, maltotriose uptake, and in the case of the lager strains, maltose uptake was also adversely affected under these conditions. Altering the calcium and magnesium levels had no effect on the viability of the yeast or on glycogen levels .     

Sucrose Fermentation by Brazilian Ethanol Production Yeasts in Media Containing Structurally Complex Nitrogen Sources

Four Saccharomyces cerevisiae Brazilian industrial ethanol production strains were grown, under shaken and static conditions, in media containing 22% (w/v) sucrose supplemented with nitrogen sources varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Sucrose fermentations by Brazilian industrial ethanol production yeasts strains were strongly affected by both the structural complexity of the nitrogen source and the availability of oxygen. Data suggest that yeast strains vary in their response to the nitrogen source's complex structure and to oxygen availability. In addition, the amount of trehalose produced could be correlated with the fermentation performance of the different yeasts, suggesting that efficient fuel ethanol production depends on finding conditions which are appropriate for a particular strain, considering demand and dependence on available nitrogen sources in the fermentation medium.     

自选酵母菌株草莓酒发酵特性比较

从草莓自然发酵液中分离筛选出酵母菌株D、E、H,以酿酒酵母A 为对照,采用气相色谱法分析不同酵母发酵草莓酒的主要香气成分,并通过降糖速率、产酒精能力、产酸量及感官质量评价,比较不同菌株的发酵特性。结果表明,菌株A 产酒精能力较强,菌株H 和菌株D 产香效果较好,菌株E 各项指标均较差。其中菌株H 发酵性能优良,还原糖含量可降至18.3g/L,酒精体积分数可达到12.92%,异丁醇、异戊醇、乳酸乙酯和β- 苯乙醇等香气成分均明显高于其他菌株,且酒体色泽澄清透亮,口味纯正,具有草莓酒的典型风味。     

Optimization of Citric Acid Production from a New Strain and Mutant of Aspergillus niger Using Solid State Fermentation

A new strain of Aspergillus niger isolated from soil and its mutant were used for citric acid production from carob under solid-state fermentation conditions. The parental strain produced 30 g/kg citric acid, while the mutant G4, selected after four rounds of gamma ray irradiation, produced 60 g/kg. Maximum citric acid production was obtained after 7 days of incubation, as the acid production was 34 and 64 g/kg for parental and mutant strains, respectively. The addition of 2% methanol increased citric acid production from the parental strain to 42 and the mutant G4 to 65 g/kg. Trace elements, namely Cu, Fe, and Zn, promoted the production of citric acid as the acid production from the parental strain increased to 46 g/kg and for mutant G4 increased to 73 g/kg after their addition. The optimum spore inoculum concentration for acid production was 10⁷ ml^-1, and the optimum pH was 5 for both parental and mutant strains.     

Impact of ammonium additions on volatile acidity, ethanol, and aromatic compound production by different Saccharomyces cerevisiae strains during fermentation in controlled synthetic media

Variations in ethanol, volatile acidity, and aromatic compounds produced by different Saccharomyces cerevisiae strains were studied in controlled synthetic medium (CSM). Different amounts of assimilable nitrogen (in the form of ammonium sulfate) were added at two fermentation stages (i.e. the beginning of fermentation and the halfway point). There were significant differences in the amount of ethanol produced when ammonia was added to the CSM, although this depended on the yeast strain used. When assimilable nitrogen was added, ethanol production either increased (with Fermicru AR2 and Stellevin NT116 yeast strains) or decreased (with LW LVCB CT1+ yeast strain). The degree of variation also depended on the time that the ammonia was added, with differences of up to 0.7% (v/v). Adding ammonium to the CSM always resulted in lower volatile acidity in the fermentation product. Different yeast strains ( P < 0.0001) and varying amounts of ammonium produced significant differences. Maximum impact – up to 70% less volatile acidity – was obtained using the Stellevin NT116 yeast strain and adding 280 mgN/L ammonium at the halfway point in fermentation. Acetoin production increased at higher concentrations of added ammonium, most markedly when it was added halfway through fermentation. The total amount of esters increased when 140 mgN/L was added to all three yeasts strains tested, irrespective of the fermentation stage. Smaller amounts of higher alcohols were produced following larger ammonium additions, especially at the beginning of fermentation.     

The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains

Disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase (G6PDH) has been shown to reduce the xylitol yield and the xylose consumption in the xylose-utilizing recombinant Saccharomyces cerevisiae strain TMB3255. In the present investigation we have studied the influence of different production levels of G6PDH on xylose fermentation. We used a synthetic promoter library and the copper-regulated CUP1 promoter to generate G6PDH-activities between 0% and 179% of the wild-type level. G6PDH-activities of 1% and 6% of the wild-type level resulted in 2.8- and 5.1-fold increase in specific xylose consumption, respectively, compared with the ZWF1-disrupted strain. Both strains exhibited decreased xylitol yields (0.13 and 0.19 g/g xylose) and enhanced ethanol yields (0.36 and 0.34 g/g xylose) compared with the control strain TMB3001 (0.29 g xylitol/g xylose, 0.31 g ethanol/g xylose). Cytoplasmic transhydrogenase (TH) from Azotobacter vinelandii has previously been shown to transfer NADPH and NAD(+) into NADP(+) and NADH, and TH-overproduction resulted in lower xylitol yield and enhanced glycerol yield during xylose utilization. Strains with low G6PDH-activity grew slower in a lignocellulose hydrolysate than the strain with wild-type G6PDH-activity, which suggested that the availability of intracellular NADPH correlated with tolerance towards lignocellulose-derived inhibitors. Low G6PDH-activity strains were also more sensitive to H(2)O(2) than the control strain TMB3001.     

优良耐酸酿酒酵母的筛选及发酵特性研究

该研究采用酸性选择培养基、氯化三苯基四氮唑（TTC）显色法、杜氏小管发酵法从实验室保藏的87株酿酒酵母（Saccharomyces cerevisiae）中筛选耐酸性强的优良菌株，并测定其最适生长条件、耐受性及发酵性能。结果表明，最终筛选出一株耐受性强及发酵性能优良的菌株SC-62，其最适生长温度为28 ℃、最适pH为4.0，可耐受乙醇体积分数14%、NaCl 100 g/L、葡萄糖500 g/L，具有发酵力[5.93 g CO2/（100 mL·24 h）]强，酒精产量（7.55%vol）高，延滞期（12 h）短、适应性强等优点。该菌株的成功筛选为后续高效生产酒精提供了良好的菌种来源。     

Optimization of Citric Acid Production from a New Strain and Mutant of Aspergillus niger Using Solid State Fermentation

A new strain of Aspergillus niger isolated from soil and its mutant were used for citric acid production from carob under solid-state fermentation conditions. The parental strain produced 30 g/kg citric acid, while the mutant G4, selected after four rounds of gamma ray irradiation, produced 60 g/kg. Maximum citric acid production was obtained after 7 days of incubation, as the acid production was 34 and 64 g/kg for parental and mutant strains, respectively. The addition of 2% methanol increased citric acid production from the parental strain to 42 and the mutant G4 to 65 g/kg. Trace elements, namely Cu, Fe, and Zn, promoted the production of citric acid as the acid production from the parental strain increased to 46 g/kg and for mutant G4 increased to 73 g/kg after their addition. The optimum spore inoculum concentration for acid production was 10⁷ ml^?1, and the optimum pH was 5 for both parental and mutant strains.     

运动发酵单胞菌发酵产乙醇的中试研究

从6株Zymomonas.mobilis基因工程菌中筛选出一株乙醇高产菌Z4,在500ml摇瓶小试的基础上利用5L全自动发酵罐进行了中试放大。结果表明Z4菌株的最适初糖浓度为18%;pH6.0,35℃,发酵48h乙醇最高含量达67.9g·L-1,得率达77.0%。补料分批培养实验结果表明,补料时机以发酵起始后24h为佳,发酵48h后乙醇含量达最高值93.1g·L-1,得率为77.6%。     

CAMBRIDGE PRIZE LECTURE IMPROVING YEAST FERMENTATION PERFORMANCE

A number of factors affecting yeast fermentation performance have been investigated. These include the mode of substrate feeding, nutrient supplementation, temperature, osmotic pressure, oxygen, intracellular ethanol accumulation, and yeast ethanol tolerance. Nutrient supplementation was observed to play a key role in yeast fermentations employing high gravity media and at high temperatures. Furthermore, complete attenuation of high gravity wort (25°P) could be achieved by optimizing the yeast pitching rate, fermentation temperature, and level of wort oxygenation. Genetic manipulation techniques, such as spheroplast fusion, were successfully employed to obtain ethanol and osmotolerant yeast strains. In addition, a number of stable 2-deoxy-D-glucose resistant mutants, isolated from brewing and non-brewing yeast strains, were observed to rapidly utilize maltose and maltotriose in the presence of high concentrations of glucose. Fermentation and ethanol production rates were increased in these strains. Therefore, employing strategies of optimized fermentation conditions and strain development have resulted in improvements in yeast fermentation performance.     

Regulation of alcoholic fermentation in batch and chemostat cultures of Kluyveromyces lactis CBS 2359

Kluyveromyces lactis is an important industrial yeast, as well as a popular laboratory model. There is currently no consensus in the literature on the physiology of this yeast, in particular with respect to aerobic alcoholic fermentation (‘Crabtree effect’). This study deals with regulation of alcoholic fermentation in K. lactis CBS 2359, a proposed reference strain for molecular studies. In aerobic, glucose-limited chemostat cultures (D=0·05–0·40 h⁻¹) growth was entirely respiratory, without significant accumulation of ethanol or other metabolites. Alcoholic fermentation occurred in glucose-grown shake-flask cultures, but was absent during batch cultivation on glucose in fermenters under strictly aerobic conditions. This indicated that ethanol formation in the shake-flask cultures resulted from oxygen limitation. Indeed, when the oxygen feed to steady-state chemostat cultures (D=0·10 h⁻¹) was lowered, a mixed respirofermentative metabolism only occurred at very low dissolved oxygen concentrations (less than 1% of air saturation). The onset of respirofermentative metabolism as a result of oxygen limitation was accompanied by an increase of the levels of pyruvate decarboxylase and alcohol dehydrogenase. When aerobic, glucose-limited chemostat cultures (D=0·10 h⁻¹) were pulsed with excess glucose, ethanol production did not occur during the first 40 min after the pulse. However, a slow aerobic ethanol formation was invariably observed after this period. Since alcoholic fermentation did not occur in aerobic batch cultures this is probably a transient response, caused by an imbalanced adjustment of enzyme levels during the transition from steady-state growth at μ=0·10 h⁻¹ to growth at μ_max. It is concluded that in K. lactis, as in other Crabtree-negative yeasts, the primary environmental trigger for occurrence of alcoholic fermentation is oxygen limitation. © 1998 John Wiley & Sons, Ltd.     

马克斯克鲁维酵母木薯乙醇发酵工艺研究

通过单因素试验对一株耐高温马克斯克鲁维酵母（Kluyveromyces marxianus）HY32的木薯乙醇发酵工艺进行了研究。结果表明,HY32利用木薯发酵乙醇的最佳工艺条件为料水比1∶5（g∶mL）,发酵时间96 h,接种量11%,发酵温度40 ℃,液化时间1 h,液化温度95 ℃,液化酶添加量为20 U/g淀粉,糖化酶添加量为150 U/g淀粉,硫酸铵添加量6 g/L,初始pH=5.0。在此条件下,HY32发酵木薯酒精度可达8.90%vol,淀粉利用率与淀粉出酒率分别为87.120%和49.48%,残糖量为0.03 g/L。与未优化的初始发酵条件相比,发酵醪的酒精度提高了16.65%。     

Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene

Glycerol is a major fermentation product of Saccharomyces cerevisiae that contributes to the sensory character of wine. Diverting sugar to glycerol overproduction and away from ethanol production by overexpressing the glycerol 3-phosphate dehydrogenase gene,GPD2, caused S. cerevisiae to produce more than twice as much acetic acid as the wild-type strain (S288C background) in anaerobic cell culture. Deletion of the aldehyde dehydrogenase gene, ALD6, in wild-type and GPD2 overexpressing strains (GPD2-OP) decreased acetic acid production by three- and four-fold, respectively. In conjunction with reduced acetic acid production, the GPD2-OP ald6Delta strain produced more glycerol and less ethanol than the wild-type. The growth rate and fermentation rate were similar for the modified and wild-type strains, although the fermentation rate for the GPD2 ald6Delta strain was slightly less than that of the other strains from 24h onwards. Analysis of the metabolome of the mutants revealed that genetic modification affected the production of some secondary metabolites of fermentation, including acids, esters, aldehydes and higher alcohols, many of which are flavour-active in wine. Modification of GPD2 and ALD6 expression represents an effective strategy to increase the glycerol and decrease the ethanol concentration during fermentation, and alters the chemical composition of the medium such that, potentially, novel flavour diversity is possible. The implications for the use of these modifications in commercial wine production require further investigation in wine yeast strains.     

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