A flow-cytometric method for determination of yeast viability and cell number in a brewery

BOYD, A; GUNASEKERA, T; Attfield, Paul V.; SIMIC, K; VINCENT, S; VEAL, D

doi:10.1016/s1567-1356(02)00125-3

Cited by 27 publications

(16 citation statements)

References 13 publications

(22 reference statements)

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“…Ethanol was quantified by near infrared spectroscopy (Alcolyzer, Anton Paar). Cell viability was assessed by oxonol staining followed by flow cytometry analysis [42]. The ethanol yield (g of ethanol produced per g of glucose consumed) was calculated by dividing the ethanol produced with the glucose consumed (initial glucose concentration minus glucose leftover).…”

Section: Methodsmentioning

confidence: 99%

Comparative Polygenic Analysis of Maximal Ethanol Accumulation Capacity and Tolerance to High Ethanol Levels of Cell Proliferation in Yeast

et al. 2013

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The yeast Saccharomyces cerevisiae is able to accumulate ≥17% ethanol (v/v) by fermentation in the absence of cell proliferation. The genetic basis of this unique capacity is unknown. Up to now, all research has focused on tolerance of yeast cell proliferation to high ethanol levels. Comparison of maximal ethanol accumulation capacity and ethanol tolerance of cell proliferation in 68 yeast strains showed a poor correlation, but higher ethanol tolerance of cell proliferation clearly increased the likelihood of superior maximal ethanol accumulation capacity. We have applied pooled-segregant whole-genome sequence analysis to identify the polygenic basis of these two complex traits using segregants from a cross of a haploid derivative of the sake strain CBS1585 and the lab strain BY. From a total of 301 segregants, 22 superior segregants accumulating ≥17% ethanol in small-scale fermentations and 32 superior segregants growing in the presence of 18% ethanol, were separately pooled and sequenced. Plotting SNP variant frequency against chromosomal position revealed eleven and eight Quantitative Trait Loci (QTLs) for the two traits, respectively, and showed that the genetic basis of the two traits is partially different. Fine-mapping and Reciprocal Hemizygosity Analysis identified ADE1, URA3, and KIN3, encoding a protein kinase involved in DNA damage repair, as specific causative genes for maximal ethanol accumulation capacity. These genes, as well as the previously identified MKT1 gene, were not linked in this genetic background to tolerance of cell proliferation to high ethanol levels. The superior KIN3 allele contained two SNPs, which are absent in all yeast strains sequenced up to now. This work provides the first insight in the genetic basis of maximal ethanol accumulation capacity in yeast and reveals for the first time the importance of DNA damage repair in yeast ethanol tolerance.

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Section: Methodsmentioning

confidence: 99%

Comparative Polygenic Analysis of Maximal Ethanol Accumulation Capacity and Tolerance to High Ethanol Levels of Cell Proliferation in Yeast

et al. 2013

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“…Flow cytometry was originally established for characterizing and sorting mammalian cells but won recently more and more importance applied at microbial processes [1,2], medical applications [3-5], dairy industry [6,7], alcoholic beverage production [8] and environmental and water systems [9]. In industrial production processes, single cell analysis may give high resolution insights into whole cell cultures concerning the cell status of viability, metabolic activity or even productivity [1,10].…”

Section: Introductionmentioning

confidence: 99%

Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools

et al. 2011

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BackgroundSingle cell analysis for bioprocess monitoring is an important tool to gain deeper insights into particular cell behavior and population dynamics of production processes and can be very useful for discrimination of the real bottleneck between product biosynthesis and secretion, respectively.ResultsHere different dyes for viability estimation considering membrane potential (DiOC2(3), DiBAC4(3), DiOC6(3)) and cell integrity (DiBAC4(3)/PI, Syto9/PI) were successfully evaluated for Bacillus megaterium cell characterization. It was possible to establish an appropriate assay to measure the production intensities of single cells revealing certain product secretion dynamics. Methods were tested regarding their sensitivity by evaluating fluorescence surface density and fluorescent specific concentration in relation to the electronic cell volume. The assays established were applied at different stages of a bioprocess where the antibody fragment D1.3 scFv production and secretion by B. megaterium was studied.ConclusionsIt was possible to distinguish between live, metabolic active, depolarized, dormant, and dead cells and to discriminate between high and low productive cells. The methods were shown to be suitable tools for process monitoring at single cell level allowing a better process understanding, increasing robustness and forming a firm basis for physiology-based analysis and optimization with the general application for bioprocess development.

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“…2001, 2005; Thornton et al. 2002; Boyd et al. 2003) was used to monitor live and dead yeast cell concentrations during fermentation.…”

Section: Introductionmentioning

confidence: 99%

“…Two tanks of each grape variety were diluted to 22-24°Brix prior to fermentation. Flow cytometry (Bruetschy et al 1994;Bouix and Leveau 2001;Malacrino et al 2001Malacrino et al , 2005Thornton et al 2002;Boyd et al 2003) was used to monitor live and dead yeast cell concentrations during fermentation. Chemical changes in these fermentations were followed using Fourier transform infrared spectroscopy.…”

Section: Introductionmentioning

confidence: 99%

Managing high-density commercial scale wine fermentations

et al. 2006

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Aims: To investigate the effects of grape juice dilution and different temperature/nitrogen addition regimes on commercial‐scale, high‐density Shiraz and Chardonnay fermentations. Methods and Results: Duplicated fermentations (30 hl) were conducted at two temperatures for Shiraz and for Chardonnay. Two additional tanks of Chardonnay and Shiraz were diluted. Nitrogen was added once at inoculation or in aliquots over several days. Yeast concentration and viability was determined by flow cytometry. Fermentation chemistry was monitored by Fourier transform infrared spectroscopy. Fermentations arrested in both of the undiluted, higher temperature duplicate tanks of Shiraz. Different fermentation temperature resulted in sensorially different Shiraz, but not Chardonnay, wines made from undiluted musts. The converse was observed for wines made from diluted musts. Conclusions: High‐density musts can be fermented completely using reduced fermentation temperature coupled with incremental nitrogen addition. Significance and Impact of the Study: This is the first study in duplicated, commercial‐scale, high‐density grape juice fermentations to address temperature, nitrogen addition, and juice dilution effects on stuck fermentation potential and wine sensory properties.

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A flow-cytometric method for determination of yeast viability and cell number in a brewery

Cited by 27 publications

References 13 publications

Comparative Polygenic Analysis of Maximal Ethanol Accumulation Capacity and Tolerance to High Ethanol Levels of Cell Proliferation in Yeast

Comparative Polygenic Analysis of Maximal Ethanol Accumulation Capacity and Tolerance to High Ethanol Levels of Cell Proliferation in Yeast

Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools

Managing high-density commercial scale wine fermentations

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