Patrick Gervais

The role of water in solid-state fermentation

Abstract Solute diffusion as well as cell absorption occurs in aqueous medium. Thermodynamics and kinetic parameters relative to water motion in solid media are successively examined with regard to their consequence on the solid-state fermentation (SSF). Specific attention is focused on fungal physiology: • at microscopic level through the influence of cell water flux on the maintenance of a constant cell turgor pressure, through the constant branching orientation and hyphal growth rate; • at macroscopic level through the influence of the hydration on the maintenance of a radial growth rate, on the evolution of germination, sporulation and metabolic activity of a fungal colony. Interactions between water status, oxygen supply and heat removing from SSF are finally pointed out.

Cell Size and Water Permeability as Determining Factors for Cell Viability after Freezing at Different Cooling Rates

ABSTRACT This work studied the viabilities of five types of cells (two yeast cells, Saccharomyces cerevisiae CBS 1171 and Candida utilis; two bacterial strains, Escherichia coli and Lactobacillus plantarum; and one human leukemia K562 cell) as a function of cooling rate during freezing. The range of investigated cooling rates extended from 5 to 30,000°C/min. Cell viability was classified into three ranges: (i) high viability for low cooling rates (5 to 180°C/min), which allow cell water outflow to occur completely and do not allow any intracellular crystallization; (ii) low viability for rapid cooling rates (180 to 5,000°C/min), which allow the heat flow to prevail over water outflow (in this case, cell water crystallization would occur as water was flowing out of the cell); (iii) high viability for very high cooling rates (>5,000°C/min), which allow the heat flow to be very rapid and induce intracellular crystallization and/or vitrification before any water outflow from the cell. Finally, an assumption relating cell death to the cell water crystallization as water is flowing out of the cell is made. In addition, this general cell behavior is different for each type of cell and seems to be moderated by the cell size, the water permeability properties, and the presence of a cell wall.

Saccharomyces cerevisiae viability is strongly dependant on rehydration kinetics and the temperature of dried cells

The effects of rehydration kinetics and temperature on the viability of Saccharomyces cerevisiae dehydrated by drying were studied. During rehydration, a water activity range of 0·117–0·455 must be crossed slowly in order to maintain cell viability. If this range is crossed rapidly, cell viability can be preserved if rehydration takes place at 50 °C. Several hypotheses have been proposed to explain previous results. One hypothesis, which relates cell mortality after rapid rehydration to water flow through the membrane in phase transition, is the more plausible and requires further investigation.

Viability of Escherichia coli after combined osmotic and thermal treatment: a plasma membrane implication

This study investigates the influence of temperature (T) and osmotic pressure (Pi) on the viability of Escherichia coli K12 during an osmotic treatment. Osmotic shock (dehydration and rehydration within 1 s) in liquid media at different temperatures (4, 10, 30 and 37 degrees C) and different levels of osmotic pressure (26, 30, 35, 40, 82 and 133 MPa) were realized. Results show that a sudden dehydration, below 40 MPa, destroyed up to 80% of the bacterial population for each tested temperature, whereas viability was greater than 90% for an osmotic pressure less than 26 MPa. The influence of T and Pi on the membranes physical structure is finally considered to explain the results in light of FTIR and electron microscopy study of the influence of temperature and osmotic pressure on E. coli membrane phospholipids conformation.

ERGOSTEROL BIOSYNTHESIS: A FUNGAL PATHWAY FOR LIFE ON LAND?

Sterols, essential lipids of most eukaryotic cells, ensure important structural and signaling functions. The selection pressure that has led to different dominant sterols in the three eukaryotic kingdoms remains unknown. Here, we investigated the influence of the progression in the different steps of the ergosterol biosynthetic pathway (EBP) on the yeast resistance to transitions from aqueous to aerial media, typical perturbations of the higher fungi habitats. Five mutants of the EBP (ergΔ), accumulating different sterol intermediates in the EBP, and the wild‐type (WT) strain were exposed to drying under atmospheric air or nitrogen and wetting. Results show that the progression in the EBP parallels an increase in the yeast resistance to air‐drying with a maximal survival rate for the WT strain. When drying/wetting was performed under nitrogen, yeast survival was higher, particularly for the earlier mutants of the EBP. Thus, ergosterol, through its protective role against mechanical and oxidative stress, might have been selected by the pressure induced by drying/wetting cycles occurring in the fungi habitats. These results support the Bloch hypothesis, which postulates that the properties of sterols are gradually optimized for function along the biosynthetic pathway and provide a response to the enduring question “why ergosterol in fungi?”.

Yeast cell mortality related to a high-pressure shift: occurrence of cell membrane permeabilization

The shrinkage of yeast cells caused by high‐pressure treatment (250 MPa, 15 min) was investigated using direct microscopic observation. A viable staining method after treatment allowed the volume variation of two populations to be distinguished: an irreversible volume decrease (about 35% of the initial volume) of pressure‐inactivated cells during pressure holding time, and viable cells, which were less affected. A mass transfer was then induced during high‐pressure treatment. Causes of this transfer seem to be related to a pressure‐induced membrane permeabilization, allowing a subsequent leakage of internal solutes, where three ions (Na+, K+ and Ca2+), plus endogenous glycerol, were verified. This glycerol leakage was found to occur after yeast pressurization in a medium having low water activity, although the yeast was not inactivated. All these observations lead to the hypothesis that pressure‐induced cell permeabilization could be the cause of yeast inactivation under pressure.

A new design intended to relate high pressure treatment to yeast cell mass transfer

A new optical device has been developed to allow the observation of microorganisms during a high pressure treatment up to 700 MPa. To measure cell volume variation during the high pressure application, an image analysis system was connected with the light microscope. With this device, growth of Saccharomyces cerevisiae was studied at moderate pressure (10 MPa) through the observation of individual cell budding. Cell volume variations were also measured on the yeast Saccharomycopsis fibuligera on fixed cells as well on a population sample and a shrinkage in average cell volume was observed consequently to a pressure increase of 250 MPa. The observed compression rate (25%) under pressure and the partial irreversibility of cell compression (10%) after return to atmospheric pressure lead to the conclusion that a mass transfer between cell and cultivation medium occurred. The causes of this transfer could be explained by a modification of membrane properties, i.e., disruption or increase in permeability.

Yeast viability related to water potential variation: influence of the transient phase

The decrease rate of the water potential was found to have a great effect on yeasts submitted to hypertonic shifts. The application of slow and linear decreases of the water potential of the medium to cells of Saccharomyces cerevisiae demonstrated that the cells could survive (90 to 100% of viability) very low levels of water potential (Ψ=−101 MPa). Resistance of the cells was examined through viability measurements and cell volume changes. The kinetics of cell volume variation were measured continuously using an on-line image analysis system coupled to a microscope. No biological response of the cells occurred, due to the lack of a usable carbon-energy source in the medium. The viability level was found to be a function of the water exit flow rate from the cells. The denaturation of the membrane structure was assumed to be involved in such phenomena.

Lateral reorganization of plasma membrane is involved in the yeast resistance to severe dehydration.

In this study, we investigated the kinetic and the magnitude of dehydrations on yeast plasma membrane (PM) modifications because this parameter is crucial to cell survival. Functional (permeability) and structural (morphology, ultrastructure, and distribution of the protein Sur7-GFP contained in sterol-rich membrane microdomains) PM modifications were investigated by confocal and electron microscopy after progressive (non-lethal) and rapid (lethal) hyperosmotic perturbations. Rapid cell dehydration induced the formation of many PM invaginations followed by membrane internalization of low sterol content PM regions with time. Permeabilization of the plasma membrane occurred during the rehydration stage because of inadequacies in the membrane surface and led to cell death. Progressive dehydration conducted to the formation of some big PM pleats without membrane internalization. It also led to the modification of the distribution of the Sur7-GFP microdomains, suggesting that a lateral rearrangement of membrane components occurred. This event is a function of time and is involved in the particular deformations of the PM during a progressive perturbation. The maintenance of the repartition of the microdomains during rapid perturbations consolidates this assumption. These findings highlight that the perturbation kinetic influences the evolution of the PM organization and indicate the crucial role of PM lateral reorganization in cell survival to hydric perturbations.

Damage in Escherichia coli Cells Treated with a Combination of High Hydrostatic Pressure and Subzero Temperature

ABSTRACT The relationship between membrane permeability, changes in ultrastructure, and inactivation in Escherichia coli strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the cell suspension in situ under high hydrostatic pressure (HHP) and subzero-temperature conditions made it possible to show the partial reversibility of pressure-induced nucleoid condensation. However, based on visual examination of TEM micrographs, protein aggregation did not seem to be reversible. Reversible cell membrane permeabilization was noticeable, particularly for HHP treatments at subzero temperature. A correlation between membrane permeabilization and cell inactivation was established, suggesting different mechanisms at room and subzero temperatures. We propose that the inactivation of E. coli cells under combined HHP and subzero temperature occurs mainly during their transiently permeabilized state, whereas HHP inactivation at room temperature is related to a balance of transient and permanent permeabilization. The correlation between TEM results and cell inactivation was not absolute. Further work is required to elucidate the effects of pressure-induced damage on nucleoids and proteins during cell inactivation.