The phenotypic characterization of yeast strains to stresses inherent to wine fermentation in warm climates.

AIMS: Climate change is exerting an increasingly profound effect on grape composition, microbiology, chemistry and the sensory aspects of wine. Identification of autochthonous yeasts tolerant to stress could help to alleviate this effect. METHODS AND RESULTS: Tolerance to osmotic pressure, ethanol and pH of 94 Saccharomyces cerevisiae strains and 29 strains non-Saccharomyces from the warm climate region DO 'Vinos de Madrid' (Spain) using phenotypic microarray and their fermentative behaviour were studied. The screening highlighted 12 strains of S. cerevisiae isolated from organic cellars with improved tolerance to osmotic stress, high ethanol concentrations and suitable fermentative properties. Screening of non-Saccharomyces spp. such as Lanchacea thermotolerans, Torulaspora delbrueckii, Schizosaccharomyces pombe and Mestchnikowia pulcherrima also highlighted tolerance to these stress conditions. CONCLUSIONS: This study confirmed the adaptation of native strains to the climatic conditions in each area of production and correlated these adaptations with good fermentation properties. Screening has revealed that identifying yeast strains adapted to fermentation stresses is an important approach for making quality wines in very warm areas. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have special relevance because it is a pioneering study that has approached the problem of climate change for wines from a microbiological aspect and has analysed the situation in situ in wineries from a warm climate zone. Resistant strains were found with good biological properties; studying these strains for their stress response mechanisms during fermentation will be of interest to the wine making industry.

Development of a phenotypic assay for characterisation of ethanologenic yeast strain sensitivity to inhibitors released from lignocellulosic feedstocks.

Inhibitors released by the breakdown of plant cell walls prevent efficient conversion of sugar into ethanol. The aim of this study was to develop a fast and reliable inhibitor sensitivity assay for ethanologenic yeast strains. The assay comprised bespoke 96-well plates containing inhibitors in isolation or combination in a format that was compatible with the Phenotypic Microarray Omnilog reader (Biolog, hayward, CA, USA). A redox reporter within the assay permits analysis of inhibitor sensitivity in aerobic and/or anaerobic conditions. Results from the assay were verified using growth on spot plates and tolerance assays in which maintenance of viability was assessed. The assay allows for individual and synergistic effects of inhibitors to be determined. It was observed that the presence of both acetic and formic acid significantly inhibited the yeast strains assessed, although this impact could be partially mitigated by buffering to neutral pH. Scheffersomyces stipitis, Candida spp., and Pichia guilliermondii demonstrated increased sensitivity to short chain weak acids at concentrations typically present in lignocellulosic hydrolysates. S. cerevisiae exhibited robustness to short chain weak acids at these concentrations. However, S. stipitis, Candida spp., and P. guilliermondii displayed increased tolerance to HMF when compared to that observed for S. cerevisiae. The results demonstrate that the phenotypic microarray assay developed in the current study is a valuable tool that can be used to identify yeast strains with desirable resistance to inhibitory compounds found in lignocellulosic hydrolysates.

Microbial contamination of fuel ethanol fermentations.

Microbial contamination is a pervasive problem in any ethanol fermentation system. These infections can at minimum affect the efficiency of the fermentation and at their worse lead to stuck fermentations causing plants to shut down for cleaning before beginning anew. These delays can result in costly loss of time as well as lead to an increased cost of the final product. Lactic acid bacteria (LAB) are the most common bacterial contaminants found in ethanol production facilities and have been linked to decreased ethanol production during fermentation. Lactobacillus sp. generally predominant as these bacteria are well adapted for survival under high ethanol, low pH and low oxygen conditions found during fermentation. It has been generally accepted that lactobacilli cause inhibition of Saccharomyces sp. and limit ethanol production through two basic methods; either production of lactic and acetic acids or through competition for nutrients. However, a number of researchers have demonstrated that these mechanisms may not completely account for the amount of loss observed and have suggested other means by which bacteria can inhibit yeast growth and ethanol production. While LAB are the primary contaminates of concern in industrial ethanol fermentations, wild yeast may also affect the productivity of these fermentations. Though many yeast species have the ability to thrive in a fermentation environment, Dekkera bruxellensis has been repeatedly targeted and cited as one of the main contaminant yeasts in ethanol production. Though widely studied for its detrimental effects on wine, the specific species-species interactions between D. bruxellensis and S. cerevisiae are still poorly understood.

Detection and enumeration of Dekkera anomala in beer, cola, and cider using real-time PCR.

AIMS: In this article, a quantitative real-time PCR assay for detection and enumeration of the spoilage yeast Dekkera anomala in beer, cola, apple cider, and brewing wort is presented as an improvement upon existing detection methods, which are very time-consuming and not always accurate. METHODS AND RESULTS: Primers were designed to exclude other organisms common in these beverages, and the assay was linear over 6 log units of cell concentrations. The addition of large amounts of non-target yeast DNA did not affect the efficiency of this assay. A standard curve of known DNA was established by plotting the C(t) values obtained from the QPCR against the log of plate counts on yeast peptone dextrose medium and unknowns showed exceptional correlation when tested against this standard curve. The assay was found to detect D. anomala at levels of 10-14 CFU ml⁻¹ in either cola or beer and at levels of 9·4-25·0 CFU ml⁻¹ in apple cider. The assay was also used to follow the growth of D. anomala in brewing wort. CONCLUSIONS: The results indicate that real-time PCR is an effective tool for rapid, accurate detection and quantitation of D. anomala in beer, cola and apple cider. SIGNIFICANCE AND IMPACT OF THE STUDY: This method gives a faster and more efficient technique to screen beer, cola, and cider samples and reduce spoilage by D. anomala. Faster screening may allow for significant reduction in economic loss because of reduced spoilage.

The presence of Saccharomyces cerevisiae DNA in various media used to propagate yeasts and its removal by ethidium monoazide.

AIMS: In this study we demonstrate the interference of yeast extract in enumeration of Saccharomyces cerevisiae using real-time PCR and develop a method for its removal from the media using ethidium monoazide (EMA). METHODS AND RESULTS: Using real-time PCR and primers to S. cerevisiae we demonstrate the presence of yeast DNA in various media as well as the media impact on S. cerevisiae real-time PCR standard curves. By pretreatment with EMA, we were able to remove this interference. CONCLUSIONS: Saccharomyces cerevisiae DNA can be found in a number of common laboratory media and may impact the enumeration of this yeast by real-time PCR. However, pretreatment with EMA eliminates this concern. SIGNIFICANCE AND IMPACT OF THE STUDY: We have developed a method for removal of contaminating DNA in yeast growth media.

An origin of transfer (oriT) on the conjugative element pRS01 from Lactococcus lactis subsp. lactis ML3.

Previous analysis of the Tra1 region of the conjugative element pRS01 from Lactococcus lactis subsp. lactis ML3 suggested that an origin of transfer (oriT) was present. Deletion derivatives of this cloned Tra1 region were assayed for mobilization in the presence of the wild-type pRS01 element in trans. The pRS01 oriT was localized to a 446-nucleotide segment in the intergenic region between open reading frames ltrD and ltrE. Sequence analysis of this region revealed a cluster of direct and inverted repeat structures characteristic of oriT regions associated with other conjugative systems.