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.

Evaluation of ITS PCR and RFLP for Differentiation and Identification of Brewing Yeast and Brewery 'Wild' Yeast Contaminants.

A reference library of ITS PCR/RFLP profiles was collated and augmented to evaluate its potential for routine identification of domestic brewing yeast and known 'wild' yeast contaminants associated with wort, beer and brewing processes. This library contains information on band sizes generated by restriction digestion of the ribosomal RNA-encoding DNA (rDNA) internal transcribed spacer (ITS) region consisting of the 5.8 rRNA gene and two flanking regions (ITS1 and ITS2) with the endonucleases CfoI, HaeIII, HinfI and includes strains from 39 non-Saccharomyces yeast species as well as for brewing and non-brewing strains of Saccharomyces. The efficacy of the technique was assessed by isolation of 59 wild yeasts from industrial fermentation vessels and conditioning tanks and by matching their ITS amplicon sizes and RFLP profiles with those of the constructed library. Five separate, non-introduced yeast taxa were putatively identified. These included Pichia species, which were associated with conditioning tanks and Saccharomyces species isolated from fermentation vessels. Strains of the lager yeast S. pastorianus could be reliably identified as belonging to either the Saaz or Frohberg hybrid group by restriction digestion of the ITS amplicon with the enzyme HaeIII. Frohberg group strains could be further sub-grouped depending on restriction profiles generated with HinfI.

Petite mutation in aged and oxidatively stressed ale and lager brewing yeast.

AIMS: To determine the role of oxidative stress and chronological ageing on the propensity of brewing yeast strains to form respiratory deficient 'petites'. METHODS AND RESULTS: Four industrial yeast strains (two ale and two lager strains) were exposed to oxidative stress in the form of H(2)O(2) (5 mmol l(-1)) for two hours. Cell viability and occurrence of petites were determined by the slide culture and TTC-overlay techniques, respectively. Increases in petite frequency were observed but only in those strains sensitive to oxidative stress. Chronological ageing under aerobic conditions led to an increase in petites in strains sensitive to oxidative stress. No such increase was observed under anaerobic conditions. CONCLUSION: Ageing may contribute to mitochondrial DNA damage and increase the propensity of brewing yeast cells to become respiratory deficient. Tolerant strains may be less likely to generate petites as a result of serial re-pitching. SIGNIFICANCE AND IMPACT OF THE STUDY: Continuous re-use of brewing yeast is associated with an increase in the frequency of petites within brewery yeast slurries, a phenomenon resulting in reduced fermentative capacity. The cause of petite generation during brewery handling is unknown. We show that endogenous oxidative stress has the potential to generate petites within brewing yeast populations.

The impact of catalase expression on the replicative lifespan of Saccharomyces cerevisiae.

The role of catalase on Saccharomyces cerevisiae replicative lifespan was investigated using a wild-type haploid laboratory yeast W303a, a catalase A mutant, a catalase T mutant and an acatalasaemic mutant. Lifespan analysis was performed in two different environmental conditions. Under repressing conditions, on glucose media, catalase T activity, but not catalase A activity was necessary to assure longevity. However, under derepressing conditions, on ethanol media, both catalases were required for longevity assurance. Although catalase activity and carbon source influence yeast lifespan, the relationship between oxidative defence and replicative senescence is complex.

The impact of media composition and petite mutation on the longevity of a polyploid brewing yeast strain.

Ageing in Saccharomyces cerevisiae is a finite phenomenon, determined by replicative, rather than chronological lifespan. Yeast physiological condition is known to influence industrial fermentation performance, however, until recently cellular senescence has not been considered as a brewing yeast stress factor. A polyploid lager yeast (BB11) and a brewery isolate, exhibiting petite mutation were analysed for longevity. It was observed that mitochondrial deficiency induced a reduction in lifespan. In addition, replicative capacity was perceived to be dependent on environmental conditions.

Effect of Cu,Zn superoxide dismutase disruption mutation on replicative senescence in Saccharomyces cerevisiae.

The role of oxidative damage in determining the replicative lifespan of Saccharomyces cerevisiae was investigated using a wild-type haploid laboratory yeast and a Cu,Zn superoxide dismutase (sod1) mutant derivative on glucose, ethanol, glycerol and galactose media. SOD1 expression was necessary to ensure longevity on all carbon sources tested. Whilst carbon source and SOD1 gene expression do influence yeast lifespan, the relationship between the two factors is complex.

The importance of surface charge and hydrophobicity for the flocculation of chain-forming brewing yeast strains and resistance of these parameters to acid washing.

The cell surface charge and hydrophobicity of the brewing yeast cell surface influences flocculation. Physiological stress, such as starvation, affects the capacity of some strains to flocculate due to the reorganisation of the cell wall and the modification of the surface physical properties. On completion of a brewery fermentation, yeast is removed from the beer, stored and inoculated (repitched) into a subsequent fermentation. Prior to repitching, brewing yeast slurries may be acid washed to remove any contaminating bacteria. This treatment has been shown to cause yeast cell surface "blistering". Acid washing treatment was used to examine the susceptibility of the physical properties of two chain-forming brewing yeast strains to stress. Although acid-washing affected the cell surface charge and hydrophobicity in both strains, the flocculation response was strain dependant. It is suggested that surface charge and the non-separation of progeny from mother cells rather than hydrophobicity influences the flocculation of chain-forming brewing yeast.