Effect of Water Potential on Microbial Growth and Activity

Publisher Summary The response of a fungus to osmotic stress involves the integrated function of many components of cell metabolism. The dehydration stress is countered by an important mechanism that entails accumulation of polyols, primarily glycerol, to achieve an internal environment that is conducive for enzyme function and growth under water stress. The changes in the composition of the cytoplasm are controlled by systems for biosynthesis of polyols and for transport of inorganic ions. The intracellular retention of glycerol is controlled at the level of glycerol efflux and by systems for glycerol uptake. The osmoregulatory processes require energy to drive transport and carbon supply for polyol formation. The capacity for substrate uptake under osmotic stress and the efficiency operating the osmoregulatory processes are important in setting the limits for growth at low water potentials. The fungal response to changes in the external water potential must involve sensing as well as transduction of the received signal. Combined genetic and physiological analysis is required for a deeper understanding of fungus-water relations. Analysis at this level has revealed sequential induction of osmotically controlled genes in enteric bacteria and given exciting insights in signal transduction and regulation of the process.

Physiology of osmotolerance in fungi

Salt stress is primarily osmotic stress, and halophilic/halotolerant microorganisms have evolved two basic mechanisms of osmoadaplation: the KCI-type and the compatible-solute type, the latter representing a very flexible mode of adaptation making use of distinct stabilizing properties of compatible solutes. A comprehensive survey, using HPLC and NMR methods, has revealed the full diversity of euhacterial compatible solutes found in nature. With the exception of proline (a proteinogenic amino acid) they are characterized as amino acid derivatives of the following types: betaines, ectoines, N-acetylated diamino acids and N-derivatized carboxamides of glutamine. From our present knowledge of hiosynthetic pathways it appears that, apart from glycine betaine, all nitrogen-containing compatible solutes originate from two major pathways (the aspartate branch and the glutamate branch). Uptake of compatible solutes from the growth medium (environment) seems to have preference over de novo synthesis. Therefore in the natural ecosystem the solutes of primary producers (mainly glycine betaine), which are readily excreted upon dilution stress, certainly play an important role as a ‘preferred’ solute source for heterolrophic organisms, and as a ‘vital’ source for organisms unable to synthesize their own compatible solutes.

Microbial behaviour in salt‐stressed ecosystems

A low water activity (alpha omega) medium (0.95 alpha omega) containing 18% (wt/wt) glycerol and 2 micrograms of dichloran per ml was developed for enumerating the fungal flora of dried and semidried foods. The medium, designated DG18, was shown to be significantly better than Christensen malt salt agar when both media were tested with foodstuffs and with pure culture inocula. The need for a medium of reduced alpha omega for enumerating xerophilic fungi from low-moisture foods was demonstrated by comparing fungal counts obtained on both high-alpha omega and low-alpha omega media.

Dichloran-glycerol medium for enumeration of xerophilic fungi from low-moisture foods.

An effective selective medium for the enumeration of Aspergillus flavus and Aspergillus parasiticus has been developed by modification of Bothast and Fennell's Aspergillus Differential Medium. Results can be obtained with the new medium, Aspergillus flavus and parasiticus Agar (AFPA), after 42 h incubation at 30°C. The medium is thus suitable for use in quality control as a guide to the presence of A. flavus and, potentially, of aflatoxins. AFPA has been extensively tested on peanuts and soils. Results were reproducible and comparable with those on standard fungal enumeration media incubated for much longer periods. A very low percentage of false positives or negatives was found.

An improved medium for the detection of Aspergillus flavus and A. parasiticus

Germination and growth of six xerophilic fungi, Aspergillus flavus, Aspergillus ochraceus, Eurotium chevalieri, Chrysosphorium fastidium, Wallemia sebi and Xeromyces bisporus were examined on media of a wide range of water activities (aw). The influence of three solutes, NaCl, glycerol and a glucose/fructose mixture, was studied at pH 4-0 and pH 6-5 using a plate-slide technique. Germination times and growth rates were affected by solute type, but the influence of pH was less marked. Except for Wallemia sebi, the fungi grew most strongly on glucose/fructose and were partially or completely inhibited by NaCl. The results showed that a universal isolation medium for xerophilic fungi could be based on glycerol or glucose/fructose but not on NaCl as aw-limiting solute.

Influence of solute and hydrogen ion concentration on the water relations of some xerophilic fungi.

Summary: Natural-abundance 13C nuclear magnetic resonance spectroscopy was used to study the patterns of accumulation of osmotically active internal solutes in five different fungi. Four xerophilic fungi (Penicillium janczewskii, Eurotium chevalieri, Xeromyces bisporus and Wallemia sebi), and one non-xerophilic fungal species (P. digitatum) were grown at three different water activities (a
w) on media containing sorbitol, glucose/fructose or NaCl as the controlling solute. Under all conditions studied, the major internal solutes detected in aqueous ethanol extracts of these fungi were simple polyhydric alcohols: glycerol, erythritol, arabitol and mannitol. The most important osmoregulatory solute accumulated by all species was glycerol. On the sorbitol and the glucose/fructose media, all five fungi were able to accumulate glycerol. However, when NaCl was used to control a
w, only one species, W. sebi, was able to accumulate glycerol below 0.92 a
w. Significant quantities of the controlling solutes were also present in the extracts.
When intact mycelia of P. janczewskii were examined by NMR, resonances of all the major internal solutes were clearly discernible, although they were not as well resolved as those from the fungal extracts. Relaxation measurements showed that the solutes were relatively mobile inside the cells.

http://mic.sgmjournals.org/content/129/9/2915.full.pdf

Natural-abundance 13C nuclear magnetic resonance studies on the internal solutes of xerophilic fungi

Summary: One non-xerophilic fungus, Penicillium digitatum, and four xerophilic fungi, Penicillium janczewskii, Eurotium chevalieri, Wallemia sebi and Polypaecilum pisce, were grown at six different water activities (a
w) on media containing various concentrations of sodium chloride. Each species was sampled as soon as visible growth appeared and up to six times thereafter during various stages of the growth cycle. The fungal mycelium was extracted and assayed for glycerol using a specific enzymic method. At the highest a
w, 0.997, only small amounts of glycerol were present in the fungi. At lower a
w values, glycerol concentrations rose rapidly at first, then declined as the cultures aged. There appeared to be a correlation between the amount of glycerol accumulated, and the complexity of the spore-bearing structures. Glycerol depletion appeared to be related to the formation of spores and their maturation.

Ailsa D. Hocking

Papers

Influence of solute and hydrogen ion concentration on the water relations of some xerophilic fungi.

Natural-abundance 13C nuclear magnetic resonance studies on the internal solutes of xerophilic fungi

Effects of water activity and culture age on the glycerol accumulation patterns of five fungi

Food spoilage fungi, 2. Heat resistant fungi [fruit-based products]