Identification of Saccharomyces cerevisiae strains for alcoholic fermentation by discriminant factorial analysis on electronic nose signals

An electronic nose (E-nose) coupled to gas chromatography was tested to monitor alcoholic fermentation by Saccharomyces cerevisiae ICV-K1 and Saccharomyces cerevisiae T306, two strains well-known for their use in oenology. The biomass and ethanol concentrations and conductance changes were measured during cultivations and allowed to observe the standard growth phases for both yeast strains. The two strains were characterized by a very similar tendency in biomass or ethanol production during the fermentation. E-nose was able to establish a kinetic of the production of aroma compounds production and which was then easy to associate with the fermentation phases. Principal Component Analysis (PCA) showed that the data collected by E-nose during the fermentation mainly contained cultivation course information. Discriminant factorial analysis (DFA) was able to clearly identify differences between the two strains using the four main principal components of PCA as input data. Nevertheless, the electronic nose responses being mainly influenced by cultivation course, a specific data treatment limiting the time influence on data was carried out and permitted to achieve an overall performance of 83.5%

Measuring cell states within a yeast population (application to studying S. cerevisiae response to various stress related to bioethanol production)

Dans une démarche d'optimisation et de maîtrise d'un bioprocédé, une préoccupation importante concerne la mesure et le suivi de l'état des cellules. Au cours de cette thèse, des mesures in situ et/ou permettant de mettre en évidence une variabilité phénotypique au sein d'une population de levures ont été recherchées. La spectroscopie d'impédance, basée sur la capacité des cellules viables à se polariser sous l'effet d'un champ électrique, a été retenue pour estimer l'état des cellules en-ligne. Pour effectuer des mesures de morphologie et de viabilité à l'échelle de la cellule, un système complet de microscopie et d'analyse d'image automatisées a été développé, en parallèle de l'utilisation d'un compteur de cellules de type Coulter. Enfin, des suivis individuels de croissance sur milieu gélosé ont été réalisés afin de caractériser la population en termes de temps de latence et de vitesse de croissance. Le procédé modèle de cette étude est la production de bioéthanol, qui expose les levures utilisées (S. cerevisiae) à d'importantes contraintes physicochimiques (température, acétate, furfural,...) qui affectent leur état physiologique et limitent l'efficacité de l'étape de fermentation. La population, homogène sur le plan cinétique dans des conditions de culture non stressantes, devient hétérogène lorsqu'une perturbation est appliquée. La mesure de cette hétérogénéité peut être utilisée comme marqueur de la sévérité du stress subi. Au cours des phases de déclin, la mort s'accompagne d'une diminution de la taille des cellules et d'une modification de leur aspect en microscopie. Ces changements permettent d'estimer la proportion de cellules viables à partir des distributions de taille obtenues avec le compteur d'une part, et l'analyse des images de microscopie d'autre part. La spectroscopie d'impédance donne une estimation fiable de la fraction volumique de cellules viables et permet de mesurer la capacitance membranaire Cm, ainsi que la conductivité intracellulaire sin, des paramètres liés à l'état de la membrane et du cytoplasme. Cm, constante tant que les cellules sont viables, s'annule à leur mort, tandis que sin varie selon la phase de culture et en réponse aux stress.For bioprocess control and optimization, biomass monitoring and physiological state evaluation is an important issue. During this work, in situ and at-line measurements have been used to evaluate cell state and detect a phenotypic variability within a yeast population. Dielectric spectroscopy, based on the polarization of viable cell membranes exposed to an electrical field, has been selected to infer cell state on-line. In parallel with the use of a Coulter-type cell counter, a dedicated system of automated microscopy and image analysis has been developed to measure cell morphology and viability. Single-cell growth on agar medium was monitored to characterize individual cells with regard to lag-time and initial growth rate. Bioethanol production with S. cerevisiae has been chosen as a model process since the yeast cells are exposed to strong physicochemical stresses (temperature, acetate, furfural,?) which affect their physiological state and impair fermentation efficiency. The cell population, kinetically homogeneous during stress-free fermentations, became heterogeneous when a perturbation was applied. The mean and the variance of lag-time distributions were related to the stress severity. During the decline phase, cell death went along with a decrease in cell size and changes of their microscopy aspect. These changes were significant enough to infer the proportion of viable cells directly from the size distributions obtained with the cell counter or from microscopy image analysis. Dielectric spectroscopy gave reliable estimates of the viable cell volume fraction and enabled the measurements of membrane capacitance Cm and intracellular conductivity sin, parameters related to membrane and cytoplasm states. The Cm value remained constant as long as the cells were viable and dropped to zero at cell death, while sin varied significantly depending on the growth phase and in response to stress.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Stabilité du couplage fermentation alcoolique-floculation (aspects technologiques et microbiologiques)

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Equilibres gaz-liquide de composés d'arôme en solutions hydro-alcooliques (capteurs d'arômes et maîtrise des procédés biologiques)

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

On-line monitoring of dielectrical properties of yeast cells during a stress-model alcoholic fermentation

International audienc

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

International audienc

Determination of yeast viability during a stress-model alcoholic fermentation using reagent-free microscopy image analysis

International audienc

Assessing yeast viability from cell size measurements?

International audienceDuring microbial cell cultures, environmental conditions affect cell physiology and subsequently process efficiency. Physiological changes result in changing cell morphology, such as cell size variations. The aim of this work was to study cell size evolution of a Saccharomyces cerevisiae population exposed to various stresses during alcoholic batch fermentations, and to evaluate the potential use of cell size measurements to infer cell viability. During a reference culture, without perturbation, viability as assessed by propidium iodide staining (PI) remained 100% and mean cell diameter was found to be above 5µm. A rapid temperature shift from 33 to 43 ◦C at 50gl−1 of ethanol resulted in an immediate arrest of growth and triggered a progressive loss of viability from 100% to 0% and a decrease of mean cell diameter from 5.2 to 3.7µm. Cell size distribution curves obtained with a cell counter showed an increasing subpopulation of significantly smaller cells. At single-cell level, combined microscopy size measurements and PI staining showed that this subpopulation was exclusively composed of dead cells. Similar results were obtained after acetic acid or furfural additions. Accordingly, a multivariate data analysis was achieved to estimate the ratio of dead cells from cell size distributions obtained using the cell counter

Identification of Saccharomyces cerevisiae strains for alcoholic fermentation by discriminant factorial analysis on electronic nose signals

Axe 2 Structuration sous contraintes des agropolymères et Réactivité des poudres . Available from Internet: http://www.ejbiotechnology.cl/content/vol13/issue4/full/11/index.html. ISSN 0717-3458. Contact: [email protected] audienceAn electronic nose (E-nose) coupled to gas chromatography was tested to monitor alcoholic fermentation by Saccharomyces cerevisiae ICV-K1 and Saccharomyces cerevisiae T306, two strains well-known for their use in oenology. The biomass and ethanol concentrations and conductance changes were measured during cultivations and allowed to observe the standard growth phases for both yeast strains. The two strains were characterized by a very similar tendency in biomass or ethanol production during the fermentation. E-nose was able to establish a kinetic of the production of aroma compounds production and which was then easy to associate with the fermentation phases. Principal Component Analysis (PCA) showed that the data collected by E-nose during the fermentation mainly contained cultivation course information. Discriminant factorial analysis (DFA) was able to clearly identify differences between the two strains using the four main principal components of PCA as input data. Nevertheless, the electronic nose responses being mainly influenced by cultivation course, a specific data treatment limiting the time influence on data was carried out and permitted to achieve an overall performance of 83.5