Pierre-André Marechal

The effect of osmotic pressure on the membrane fluidity of Saccharomyces cerevisiae at different physiological temperatures

Abstract. Membrane fluidity in whole cells of Saccharomyces cerevisiaeW303-1A was estimated from fluorescence polarization measurements using the membrane probe, 1,6-diphenyl-1,3,5-hexatriene, over a wide range of temperatures (6–35xa0°C) and at seven levels of osmotic pressure between 1.38xa0MPa and 133.1xa0MPa. An increase in phase transition temperatures was observed with increasing osmotic pressure. At 1.38xa0MPa, a phase transition temperature of 12±2xa0°C was observed, which increased to 17±4xa0°C at 43.7xa0MPa, 21±7xa0°C at 61.8xa0MPa, and 24±9xa0°C at an osmotic pressure of 133.1xa0MPa. From these results we infer that, with increases in osmotic pressure, the change in phospholipid conformation occurs over a larger temperature range. These results allow the representation of membrane fluidity as a function of temperature and osmotic pressure. Osmotic shocks were applied at two levels of osmotic pressure and at nine temperatures, in order to relate membrane conformation to cell viability.

Rates of chilling to 0°C: implications for the survival of microorganisms and relationship with membrane fluidity modifications

The effects of slow chilling (2°C min−1) and rapid chilling (2,000°C min−1) were investigated on the survival and membrane fluidity of Escherichia coli, of Bacillus subtilis, and of Saccharomyces cerevisiae. Cell death was found to be dependent on the physiological state of cell cultures and on the rate of temperature downshift. Slow temperature decrease allowed cell stabilization, whereas the rapid chilling induced an immediate loss of viability of up to more than 90 and 70% for the exponentially growing cells of E. coli and B. subtilis, respectively. To relate the results of viability with changes in membrane physical state, membrane anisotropy variation was monitored during thermal stress using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene. No variation in the membrane fluidity of all the three microorganisms was found after the slow chilling. It is interesting to note that fluorescence measurements showed an irreversible rigidification of the membrane of exponentially growing cells of E. coli and B. subtilis after the instantaneous cold shock, which was not observed with S. cerevisiae. This irreversible effect of the rapid cold shock on the membrane correlated well with high rates of cell inactivation. Thus, membrane alteration seems to be the principal cause of the cold shock injury.

New insights into the effect of medium-chain-length lactones on yeast membranes. Importance of the culture medium

In hydrophobic compounds biotechnology, medium-chain-length metabolites often perturb cell activity. Their effect is usually studied in model conditions of growth in glucose media. Here, we study whether culture on lipids has an impact on the resistance of Yarrowia lipolytica to such compounds: Cells were cultured on glucose or oleate and submitted to γ-dodecalactone. After a 60-min exposure to 3xa0g L−1, about 80% of the glucose-grown cells (yeast extract peptone dextrose (YPD) cells) had lost their cultivability, 38% their membrane integrity, and 31% their reducing capacity as shown with propidium iodide and methylene blue, respectively. For oleate-grown cells, treatment at 6xa0g L−1 did not alter cultivability despite some transient loss of membrane integrity from 3xa0g L−1. It was shown with diphenylhexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene that oleate-grown cells had membranes more fluid and less sensitive to the lactone-induced fluidization. Analyses revealed also higher contents of ergosterol but, for YPD- and minimum-oleate-grown cells (YNBO cells), the addition of lactone provoked a decrease in the concentration of ergosterol in a way similar to the depletion by methyl-β-cyclodextrin and an important membrane fluidization. Ergosterol depletion or incorporation increased or decreased, respectively, cell sensitivity to lactone. This study shows that the embedment of oleate moieties into membranes as well as higher concentrations of sterol play a role in the higher resistance to lactone of oleate-grown cells (YPO cells). Similar oleate-induced increase in resistance was also observed for Rhodotorula and Candida strains able to grow on oleate as the sole carbon source whereas Saccharomyces and Sporidiobolus cells were more sensitive after induction.

Synergistic action of rapid chilling and nisin on the inactivation of Escherichia coli

The effect of rapid and slow chilling on survival and nisin sensitivity was investigated in Escherichia coli. Membrane permeabilization induced by cold shock was assessed by uptake of the fluorescent dye 1-N-phenylnapthylamine. Slow chilling (2°C min−1) did not induce transient susceptibility to nisin. Combining rapid chilling (2,000°C min−1) and nisin causes a dose-dependent reduction in the population of cells in both exponential and stationary growth phases. A reduction of 6xa0log of exponentially growing cells was achieved with rapid chilling in the presence of 100xa0IU ml−1 nisin. Cells were more sensitive if nisin was present during stress. Nevertheless, addition of nisin to cell suspension after the rapid chilling produced up to 5xa0log of cell inactivation for exponentially growing cells and 1xa0log for stationary growing cells. This suggests that the rapid chilling strongly damaged the cell membrane by disrupting the outer membrane barrier, allowing the sensitization of E. coli to nisin post-rapid chilling. Measurements of membrane permeabilization showed a good correlation between the membrane alteration and nisin sensitivity. Application involving the simultaneous treatment with nisin and rapid cold shock could thus be of value in controlling Gram negatives, enhancing microbiological safety and stability.

Fluorescent probes to evaluate the physiological state and activity of microbial biocatalysts: A guide for prokaryotic and eukaryotic investigation

Many fluorescent techniques are employed to evaluate the viability and activity of microbial cells used in biotechnology. These techniques are sometimes complex and the interpretation of results opened to misunderstanding. Moreover, new developments are constantly proposed especially concerning a more accurate evaluation of the state of the cells including eukaryotic microorganisms. This paper aims at presenting to biotechnologists unfamiliar with fluorescence the principles of these methods and the related possible pitfalls. It focuses on probes of the physical (integrity and fluidity) and energetical (intracellular pH and membrane potential) state of the cell membrane (bacterial and yeast cells) and presents also other probes (nucleic acids, respiration...) and new technical trends. The specificities of Gram‐negative bacterial cells are also discussed.

Cottage Level Cassava Starch Processing Systems in Colombia and Vietnam

In the tropics, cassava starch is produced at artisanal and industrial scales. This paper focuses on a new methodology enabling the technoeconomical comparison of small-scale cassava starch manufacturing process (1–5xa0t of starch/day) in two markedly different contexts (Colombia and Vietnam). Measurements were conducted during trial runs for each unit operation (washing/pealing, rasping, extraction and separation). Starch mass balance was calculated from sample composition (moisture, starch and crude fiber and ash content). Production capacity, water consumption, electric requirements and capital–labor costs were also measured. The manufacturing processes differed mainly on starch recovery from starch present in washed roots (65 vs. 76%), extraction capacity (0.3 vs. 0.9xa0t of washed roots/h), water consumption (45 vs. 21xa0m3/t of dry starch), energy consumption (59 vs. 55 kWh/t of starch) and production costs (1,156 vs. 162 US

A shift to 50°C provokes death in distinct ways for glucose- and oleate-grown cells of Yarrowia lipolytica

/t of starch) for Colombia and Vietnam, respectively. Moreover, the effectiveness of the starch extraction process could largely be attributed to the differences in the extent of root disintegration achieved with different rasping equipment.

Modeling small-scale cassava starch extraction. Simulation of the reduction of water consumption through a recycling process

Based on the observation that shocks provoked by heat or amphiphilic compounds present some similarities, this work aims at studying whether cells grown on oleate (amphiphilic pre-stress) acquire a tolerance to heat shock. In rich media, changing glucose for oleate significantly enhanced the cell resistance to the shock, however, cells grown on a minimal oleate medium lost their ability to grow on agar with the same kinetic than glucose-grown cells (more than 7-log decrease in 18xa0min compared with 3-log for oleate-grown cells). Despite this difference in kinetics, the sequence of events was similar for oleate-grown cells maintained at 50°C with a (1) loss of ability to form colonies at 27°C, (2) loss of membrane integrity and (3) lysis (observed only for some minimal-oleate-grown cells). Glucose-grown cells underwent different changes. Their membranes, which were less fluid, lost their integrity as well and cells were rapidly inactivated. But, surprisingly, their nuclear DNA was not stained by propidium iodide and other cationic fluorescent DNA-specific probes but became stainable by hydrophobic ones. Moreover, they underwent a dramatic increase in membrane viscosity. The evolution of lipid bodies during the heat shock depended also on the growth medium. In glucose-grown cells, they seemed to coalesce with the nuclear membrane whereas for oleate-grown cells, they coalesced together forming big droplets which could be released in the medium. In some rare cases of oleate-grown cells, lipid bodies were fragmented and occupied all the cell volume. These results show that heat triggers programmed cell death with uncommon hallmarks for glucose-grown cells and necrosis for methyl-oleate-grown cells.