The Production of Volatile Compounds by Yeasts Isolated from Small Brazilian cachaça distilleries

Summary The production of volatile compounds by 24 strains of Saccharomyces cerevisiae and one strain each of Candida apicola, C. famata, C. guilliermondii, Hanseniaspora occidentalis, Pichia subpelicullosa and Schizosaccharomyces pombe was evaluated with respect to the production of cacha?a. They were isolated from small cacha?a distilleries (27), industrial cacha?a distilleries (2) and one sugarcane alcohol distillery, and tested in synthetic medium for the production of acetaldehyde, ethyl acetate, propanol, isobutanol, isoamyl alcohol, acetic acid and glycerol. The Saccharomyces strains showed a narrow range of variation in the production of such compounds, near 50% of the average of each volatile compound concentration. Principal component analysis showed the separation of the strains into six groups, and acetic acid production was the variable of greatest impact in the differentiation of the strains. The strains of S. pombe formed a distinct group (Group 2), and the strains of C. apicola and H. occidentalis formed a joint group (Group 6) as did Sc13 and Sc4 (Group 4). Group 1 was formed exclusively of S. cerevisiae. The closest non-Saccharomyces strains were C. apicola and H. occidentalis, with a similarity index of about 0.95. The strain P. subpelliculosa showed general characteristics more similar to those of the S. cerevisiae strains than to the non-Saccharomyces strains.     

Flocculation of industrial and laboratory strains ofSaccharomyces cerevisiae

Summary A comparative study has been made of different laboratory and industrial wild-type strains ofSaccharomyces cerevisiae in relation to their flocculation behavior. All strains were inhibited by mannose and only one by maltose. In regard to the stability of these characters in the presence of proteases and high salt concentrations, a relevant degree of variation was found among the strains. This was to such an extent that it did not allow their inclusion in the Flol or NewFlo phenotypes. Genetic characterization of one wild-type strain revealed that the flocculation-governing gene was allelic toFLO1 found in genetic strains.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

酿酒酵母L610利用菊糖生产乙醇发酵条件的研究

以1株能够直接利用菊糖产乙醇的酿酒酵母L610为出发菌株,对其利用菊糖生产乙醇的发酵条件进行了一系列研究。结果表明,L610最适乙醇发酵温度为37℃,且40℃高温发酵对其产乙醇能力无显著影响;L610对酸性发酵环境有良好的耐受性,当发酵液p H值降至3.5时,其糖醇转化率及乙醇产量仍保持较高水平;以0.025~0.10 vvm的通气量通气12 h有利于L610发酵菊糖产乙醇;L610对350 g/L的高浓度菊糖有良好的转化率,乙醇浓度和生产强度分别达到129 g/L和1.35 g/(L·h);当直接以300 g/L菊芋粗粉为唯一底物进行发酵时,L610发酵产乙醇浓度达到89.6 g/L,为理论产量的78.1%。本研究所取得的成果为酿酒酵母一步法发酵菊芋生产乙醇的工业化发展提供参考。     

Establishment of a xylose metabolic pathway in an industrial strain of Saccharomyces cerevisiae

To produce an industrial strain of Saccharomyces cerevisiae that metabolizes xylose, we constructed a rDNA integration vector and YIp integration vector, containing the xylose-utilizing genes, XYL1 and XYL2, which encode xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis, and XKS1, which encodes xylulokinase (XK) from S. cerevisiae, with the G418 resistance gene KanMX as a dominant selectable marker. The rDNA results in integration of multiple copies of the target genes. The industrial stain of S. cerevisiae NAN-27 was transformed with the two integration vectors to produce two recombinant strains, S. cerevisiae NAN-127 and NAN-123. Upon transformation, multiple copies of the xylose-utilizing genes were integrated into the genome rDNA locus of S. cerevisiae. Strain NAN-127 consumed twice as much xylose and produced 39% more ethanol than the parent strain, while NAN-123 consumed 10% more xylose and produced 10% more ethanol than the parent strain over 94 h.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains associated with the production of cachaça: identification and characterization by traditional and molecular methods (PCR,PFGE and mtDNA-RFLP)

A study of the genetic diversity of populations of Saccharomyces cerevisiae was conducted in ten different cachaça producers (alambiques) in the southern state of Minas Gerais, Brazil. A total of 106 isolates were identified by PCR using the primer SCREC114, specific to S. cerevisiae, by pulsed-field gel electrophoresis (PFGE) and by restriction fragment polymorphism of mitochondrial DNA analysis (RFLP-mtDNA). PCR showed a product of amplification to 61 isolates, enabling a rapid identification of S. cerevisiae in different alambiques. Nine different profiles were found by PFGE; all the yeasts identified as S. cerevisiae by PCR had profiles similar to that of the marker S. cerevisiae, highlighting the specificity of primer SCREC114. RFLP-mtDNA, using four different enzymes, enabled the grouping of strains of S. cerevisiae, with 80%–100% similarity. Some alambiques that had a higher frequency of S. cerevisiae characterized by PCR and PFGE, had a lower level of genetic diversity determined by RFLP-mtDNA, indicating the ability of these strains to lead the fermentative process.     

Cloning of the two essential yeast genes, PRP6 and PRP9, and their rapid mapping, disruption and partial sequencing using a linker insertion strategy.

Summary In the yeast Saccharomyces cerevisiae, some thermosensitive (ts) mutants have been shown to be impaired in pre-mRNA splicing (prp mutants). From a yeast genomic library, we have isolated plasmids that complement prp6 or prp9 is mutations. These plasmids also complement the is growth defect of additional independent mutants identified as new prp6 and prp9 is alleles, indicating that the cloned DNAs encode PRP6 and PRP9 genes, respectively. Here, we describe the restriction maps of these loci which are localized on chromosome II and IV, respectively. The limits of open reading frames (ORFs) within the cloned inserts have been determined using a linker insertion strategy combined with the is complementation assay. Double-strand DNA sequencing was also performed directly on the yeast expression vector from the inserted linkers. Gene disruption experiments demonstrate that both genes are essential for viability.     

An optimized protocol for the production of interdelta markers in Saccharomyces cerevisiae by using capillary electrophoresis

The amplification of genomic sequence blocks flanked by delta elements of retrotransposon origin has proved to be a very convenient method for molecular characterization of Saccharomyces cerevisiae strains. Fluorescent automated capillary electrophoresis (CE) was used to detect interdelta marker (IDM) patterns in S. cerevisiae, using the ABI Prism 3130 Genetic Analyzer. Main experimental parameters were studied and the optimal conditions for IDM amplification and samples run on the CE apparatus were determined. Fingerprints from fluorescent-labelled IDM produced using CE with the same sample analyzed by agarose electrophoresis (AE) were compared. The CE analysis was able to distinguish 43 different IDM profiles among 45 S. cerevisiae isolates with a discriminating capacity of 99.8%, whereas the AE analysis of the same samples allowed the identification of 27 different patterns (discriminatory power equal to 96%). Detection of fluorescent IDM was fast and reliable, and it facilitated data comparison. For the first time in our knowledge, the fluorescent CE proved to be well suited for IDM fingerprinting. Moreover, it could be routinely applied for the molecular differentiation of S. cerevisiae strains.     

Comparative proteome analysis of robust <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> insights into industrial continuous and batch fermentation

A robust Saccharomyces cerevisiae strain has been widely applied in continuous and batch/fed-batch industrial fermentation. However, little is known about the molecular basis of fermentative behavior of this strain in the two realistic fermentation processes. In this paper, we presented comparative proteomic profiling of the industrial yeast in the industrial fermentation processes. The expression levels of most identified protein were closely interrelated with the different stages of fermentation processes. Our results indicate that, among the 47 identified protein spots, 17 of them belonging to 12 enzymes were involved in pentose phosphate, glycolysis, and gluconeogenesis pathways and glycerol biosynthetic process, indicating that a number of pathways will need to be inactivated to improve ethanol production. The differential expressions of eight oxidative response and heat-shock proteins were also identified, suggesting that it is necessary to keep the correct cellular redox or osmotic state in the two industrial fermentation processes. Moreover, there are significant differences in changes of protein levels between the two industrial fermentation processes, especially these proteins associated with the glycolysis and gluconeogenesis pathways. These findings provide a molecular understanding of physiological adaptation of industrial strain for optimizing the performance of industrial bioethanol fermentation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Immobilization and characterization of Saccharomyces cerevisiae in crosslinked,prepolymerized polyacrylamide-hydrazide

Summary Baker's yeast (Saccharomyces cerevisiae) was immobilized in gels made of prepolymerized, linear, water soluble polyacrylamide, partially substituted with acylhydrazide groups. Gelation was effected by the addition of controlled amounts of dialdehydes (e.g. glyoxal). The immobilized yeasts retained full glycolytic activity. Moreover, the entrapped cells were able to grow inside the chemically corsslinked gel during continuous alcohol production. Glyoxal was found to be the most favourable crosslinking agent for this system. the system employed allowed for the free exchange of substrate and products. The gel surrounding the entrapped cells had no effect on temperature stability profile. On the other hand, substantial enhancement in survival of cells in presence of high ethanol concentrations was recorded for the entrapped yeast. The capability of the immobilized yeast to carry out continuous conversion of glucose to ethanol was demonstrated.     

商业酵母在不同品种葡萄酒工业化生产中的定殖差异

[背景]酵母菌在葡萄酒酿造中起到重要的作用,接种商业活性干酵母(active dry yeast,ADY)进行葡萄酒酿造在国内较为普遍,然而商业酿酒酵母(Saccharomyces cerevisiae)对我国本土酵母菌资源的影响及二者竞争关系的相关报道不多.[目的]比较商业酿酒酵母在不同品种葡萄酒工业化生产中的定殖差...     

The influence of pH on sulphite formation by yeasts

The influence of the initial pH of the substrate on the sulphite formation of three low-sulphite-and five high-sulphite-forming yeasts is described. Four distinctly different groups become apparent. The need for better evaluation of pure culture wine yeasts is stressed.     

走出青藏高原：拉格啤酒酵母的起源与演化

采用低温底层发酵的拉格(lager)啤酒15世纪开始在德国巴伐利亚地区出现,19世纪初流行至全世界,目前已成为全球产量最高的酒精饮料。目前已阐明,拉格啤酒发酵酵母为巴斯德酿酒酵母(Saccharomyces pastorianus),该种是一个杂交种,由艾尔(ale)啤酒酵母(Saccharomyces cerevisiae)与野生真贝氏酿酒酵母(Saccharomyces eubayanus)杂交而成,后者赋予了拉格啤酒酵母的耐低温能力。近年的群体遗传学和群体基因组学研究表明,拉格啤酒酵母的野生亲本S.eubayanus起源于青藏高原,可能通过丝绸之路传播到了欧洲。比较基因组学研究表明,拉格啤酒酵母包含2个株系,即Ⅰ系/Saaz系和Ⅱ系/Frohberg系,早期分别流行于中欧和西欧地区。前者为近似异源3倍体,后者为近似异源4倍体。2个株系在耐低温、麦芽三糖利用和风味物质产生能力等方面具有明显差异。在中国普通微生物菌种保藏管理中心(China General Microbiological Culture Collection Center,CGMCC)保藏的S.pastorianus...     

Acceleration of high gravity yeast fermentations by acetaldehyde addition

In high gravity Saccharomyces cerevisiae fermentations containing 300 g glucose l^–1, daily addition of acetaldehyde to a total of 93 mM shortened the time required to ferment the first 250 g glucose l^–1 from 790 h to 585 h. Acetaldehyde feeding had no effect on the ethanol yield but increased by 135%, 78% and 77% the final concentrations of 2,3-butanediol, 2-methylpropanol and acetate, while decreasing that of glycerol by 14%. Controlled acetaldehyde feeding has potential as a technique for accelerating high gravity fuel or industrial ethanol fermentations and may be useful in preventing incomplete fermentations.     

Prevalence and susceptibility of Saccharomyces cerevisiae causing vaginitis in Greek women

Saccharomyces cerevisiae is an ascomycetous yeast, that is traditionally used in wine bread and beer production. Vaginitis caused by S. cerevisiae is rare.The aim of this study was to evaluate the frequency of S. cerevisiae isolation from the vagina in two groups of women and determined the in vitro susceptibility of this fungus.

Subjects and methods

Vaginal samples were collected from a total of262 (asymptomaticandsymptomatic) women with vaginitis attending the centre of family planning of General hospital ofPiraeus. All blastomycetes that isolated from the vaginal samples were examined for microscopic morphological tests and identified by conventional methods: By API 20 C AUX and ID 32 C (Biomerieux). Antifungal susceptility testing for amphotericin B,fluconazole itraconazole,voriconazole, posaconazole and caspofungin was performed by E -test (Ab BIODIKS SWEDEN) against S. cerevisiae.

Results

A total of 16 isolates of S. cerevisiae derived from vaginal sample of the referred women, average 6.10%. Susceptibility of 16 isolates of S. cerevisiae to a variety of antimycotic agents were obtained. So all isolates of S. cerevisiae were resistant to fluconazole, posaconazole and intraconazole, but they were sensitive to voriconazole caspofungin and Amphotericin B which were found sensitive (except 1/16 strains). None of the 16 patients had a history of occupational domestic use of baker’s yeast.

Conclusions

Vaginitis caused by S. cerevisiae occur, is rising and cannot be ignored. Treatment of Saccharomyces vaginitis constitutes a major challenge and may require selected and often prolonged therapy.     

Use of a Novel Immobilization Yeast System for Winemaking

Penicillium was used to immobilize Saccharomyces cerevisiae, without using physico-chemical external supports, to form yeast biocapsules. The biocapsules, once the Penicillium was killed by the ethanol produced, were used in a grape must fermentation. Must fermentation was carried out for 160 h with the biocapsules and for 300 h with free yeast cells. Acetaldehyde (84 vs. 63 mg/l), isobutanol (217 vs. 194 mg/l), L-proline (7.7 vs. 6.5 mM) and aspartic acid (0.42 vs. 0 mM) in final wine were higher with the biocapsules than with free cells.     

Crabtree-negative characteristics of recombinant xylose-utilizing Saccharomyces cerevisiae

For recombinant xylose-utilizing Saccharomyces cerevisiae, ethanol yield and productivity is substantially lower on xylose than on glucose. In contrast to glucose, xylose is a novel substrate for S. cerevisiae and it is not known how this substrate is recognized on a molecular level. Failure to activate appropriate genes during xylose-utilization has the potential to result in sub-optimal metabolism and decreased substrate uptake. Certain differences in fermentative performance between the two substrates have thus been ascribed to variations in regulatory response. In this study differences in substrate utilization of glucose and xylose was analyzed in the recombinant S. cerevisiae strain TMB3400. Continuous cultures were performed with glucose and xylose under carbon- and nitrogen-limited conditions. Whereas biomass yield and substrate uptake rate were similar during carbon-limited conditions, the metabolic profile was highly substrate dependent under nitrogen-limited conditions. While glycerol production occurred in both cases, ethanol production was only observed for glucose cultures. Addition of acetate and 2-deoxyglucose pulses to a xylose-limited culture was able to stimulate transient overflow metabolism and ethanol production. Application of glucose pulses enhanced xylose uptake rate under restricted co-substrate concentrations. Results are discussed in relation to regulation of sugar metabolism in Crabtree-positive and -negative yeast.     

In situ monitoring by quantitative Raman spectroscopy of alcoholic fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under high pressure

We monitored alcoholic fermentation in Saccharomyces cerevisiae as a function of high hydrostatic pressure. Ethanol production from 0.15 M glucose was measured by Raman spectroscopy in situ in a diamond-anvil cell. At 10 MPa, fermentation proceeds three times faster than at ambient pressure and the fermentation yield is enhanced by 5% after 24 h. Above 20 MPa, the reaction kinetics slows down with increasing pressure. The pressure above which no more ethanol is produced is calculated to be 87 ± 7 MPa. These results indicate that the activity of one or several enzymes of the glycolytic pathway is enhanced at low pressure up to 10 MPa. At higher pressures, they become progressively repressed, and they are completely inhibited above 87 MPa. Although fermentation was predicted to stop at ca. 50 MPa, due to the loss of activity of phosphofructokinase, the present study demonstrates that there is still an activity of ca. 30% of that measured at ambient pressure at 65 MPa. This study also validates the use of Raman spectroscopy for monitoring the metabolism of living microorganisms.     

Genetic identification of naturalSaccharomyces sensu stricto yeasts from Finland,Holland and Slovakia

Genetic and karyotypic studies of naturalSaccharomyces sensu stricto yeasts from Finland, Holland and Slovakia revealed three wild sibling-species:Saccharomyces cerevisiae, Saccaromyces bayanus andSaccharomyces paradoxus.