Abstract
Organisms that can withstand anhydrobiosis possess the unique ability to temporarily and reversibly suspend their metabolism for the periods when they live in a dehydrated state. However, the mechanisms underlying the cell’s ability to tolerate dehydration are far from being fully understood. The objective of this study was to highlight, for the first time, the cellular damage to Yarrowia lipolytica as a result of dehydration induced by drying/rehydration and freezing/thawing. Cellular response was evaluated through cell cultivability determined by plate counts, esterase activity and membrane integrity assessed by flow cytometry, and the biochemical composition of cells as determined by FT-IR spectroscopy. The effects of the harvesting time (in the log or stationary phase) and of the addition of a protective molecule, trehalose, were investigated. All freshly harvested cells exhibited esterase activity and no alteration of membrane integrity. Cells freshly harvested in the stationary phase presented spectral contributions suggesting lower nucleic acid content and thicker cell walls, as well as longer lipid chains than cells harvested in the log phase. Moreover, it was found that drying/rehydration induced cell plasma membrane permeabilization, loss of esterase activity with concomitant protein denaturation, wall damage and oxidation of nucleic acids. Plasma membrane permeabilization and loss of esterase activity could be reduced by harvesting in the stationary phase and/or with trehalose addition. Protein denaturation and wall damage could be reduced by harvesting in the stationary phase. In addition, it was shown that measurements of loss of membrane integrity and preservation of esterase activity were suitable indicators of loss and preservation of cultivability, respectively. Conversely, no clear effect of freezing/thawing could be observed, probably because of the favorable operating conditions applied. These results give insights into Y. lipolytica mechanisms of cellular response to dehydration and provide a basis to better understand its ability to tolerate anhydrobiosis.
Highlights
In their natural habitats, most living organisms may be periodically subjected to quite intense dehydration, resulting in the state of anhydrobiosis
Cellular response was evaluated through cell cultivability determined by plate counts, esterase activity and membrane integrity assessed by flow cytometry and the biochemical composition of cells as determined by Fourier Transformed Infra-Red (FT-IR) spectroscopy
Cultivability loss of cells dried in saline water was 1.1 log (CFU/ mL) and 0.8 log (CFU/mL) when harvested in the log and stationary phase, respectively, and cultivability loss of cells dried in trehalose was 0.6 log (CFU/mL) and 0.4 log (CFU/mL), respectively
Summary
Most living organisms may be periodically subjected to quite intense dehydration, resulting in the state of anhydrobiosis. Organisms that can withstand anhydrobiosis possess the unique ability to temporarily and reversibly suspend their metabolism for periods when environmental conditions are unfavorable [1] This ability is widely used, mainly in food-related and biotechnology processes that produce or use starters (stabilized microorganisms) that must be efficiently reactivated and functional upon rehydration. The mechanisms underlying the cell’s ability to deal with dehydration are far from being fully understood. From both the genetic and physiological point of view, yeast is a preferred organism for molecular cell biologists because it provides information that is useful in food and applied biotechnology but that is relevant for other eukaryotes such as mammalian and plant cells [2]. Dehydration is thought to cause damage to DNA and nucleic acids, probably by oxidation reactions [3]
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