Single-cell analysis of S. cerevisiae growth recovery after a sublethal heat-stress applied during an alcoholic fermentation

Abstract

Interest in bioethanol production has experienced a resurgence in the last few years. Poor temperature control in industrial fermentation tanks exposes the yeast cells used for this production to intermittent heat stress which impairs fermentation efficiency. Therefore, there is a need for yeast strains with improved tolerance, able to recover from such temperature variations. Accordingly, this paper reports the development of methods for the characterization of Saccharomyces cerevisiae growth recovery after a sublethal heat stress. Single-cell measurements were carried out in order to detect cell-to-cell variability. Alcoholic batch fermentations were performed on a defined medium in a 2 l instrumented bioreactor. A rapid temperature shift from 33 to 43 °C was applied when ethanol concentration reached 50 g l⁻¹. Samples were collected at different times after the temperature shift. Single cell growth capability, lag-time and initial growth rate were determined by monitoring the growth of a statistically significant number of cells after agar medium plating. The rapid temperature shift resulted in an immediate arrest of growth and triggered a progressive loss of cultivability from 100 to 0.0001% within 8 h. Heat-injured cells were able to recover their growth capability on agar medium after a lag phase. Lag-time was longer and more widely distributed as the time of heat exposure increased. Thus, lag-time distribution gives an insight into strain sensitivity to heat-stress, and could be helpful for the selection of yeast strains of technological interest.

Yeast fermentation of sugarcane for ethanol production: Can it be monitored by using in situ microscopy?

“…In addition, it was demonstrated that the ISM resolved different morphologies in an industrially used yeast strain, thereby providing a basis for future studies of morphological features related to reagent-free cell viability classification (Wei et al, 2007;Tibayrenc et al, 2011). Morphological features have also been previously successfully exploited to assess the viability of animal cells (Wiedemann et al, 2011).…”

“…Indeed, also previous work on image analysis applied to laboratory yeast samples is based on a homogeneous image background (Costello and Monk, 1985;Huls et al, 1992;Pons et al, 1993;Thomas and Paul, 1996;Tibayrenc et al, 2011). This precondition facilitates the application of standard image-processing operations as for example a threshold operation.…”

Evolutionary engineering of Saccharomyces cerevisiae for improved industrially important properties

“…They observed heterogeneity in the strength of stress response in the clonal population tested and stated that this heterogeneity was mainly physiologically based, resulting mainly from asynchronous growth in batch cultures (Attfield et al, 2001). Other recent reports focus on comparative genomic analysis of wild-type yeast strains to identify the major type of genome variability that confers genetic diversity to natural yeast populations (Carreto et al, 2008), the development of single-cell analysis methods to detect cell-to-cell variability upon stress exposure (Tibayrenc et al, 2011), and the contribution of positive and negative epistasis to adaptation and reproductive isolation of S. cerevisiae in low-glucose and high-salt environments (Parreiras et al, 2011). For this purpose, survival of stress-sensitive S. cerevisiae mutants was tested and compared with that of the wild type.…”

“…Similarly, the high heterogeneity of our final evolved populations may also provide a survival advantage at high-stress levels. Other recent reports focus on comparative genomic analysis of wild-type yeast strains to identify the major type of genome variability that confers genetic diversity to natural yeast populations (Carreto et al, 2008), the development of single-cell analysis methods to detect cell-to-cell variability upon stress exposure (Tibayrenc et al, 2011), and the contribution of positive and negative epistasis to adaptation and reproductive isolation of S. cerevisiae in low-glucose and high-salt environments (Parreiras et al, 2011). The high level of heterogeneity of the final evolving populations in our studies might also have resulted from the negative epistasis of different mutations, which individually confer an increased fitness in those evolving populations.…”

Quantitative Flow Cytometry to Understand Population Heterogeneity in Response to Changes in Substrate Availability in Escherichia coli and Saccharomyces cerevisiae Chemostats

“…Apart from affecting the cells' metabolism, dynamic cultivation conditions impose metabolic stresses on cells and create inherent population heterogeneity (Carlquist et al, 2012; Ackermann, 2015). Consequently, averaged response values fail to describe the influence of different subpopulations and may even mask important characteristics of single cells in a bioprocess (Díaz et al, 2010; Fernandes et al, 2011; Tibayrenc et al, 2011; Gonzalez-Cabaleiro et al, 2017).…”