The role of an active transport mechanism in glycerol accumulation during osmoregulation by Zygosaccharomyces rouxii

Summary At water activities (a _w) of 0.998 (no osmoticum) and 0.960 a _w(NaCl), the affinity (K _m) of glycerol transport by Zygosaccharomyces rouxii was 25.6 and 6.4 mmol/l respectively. The maximum uptake rate (V _max) was ca. 2.3 mol/g/min at both a _w's. However, at an a _wof 0.960 using polyethylene glycol (PEG) 400 the K _mand V _max for glycerol transport increased to 61.1 mmol/l and 32.2 mol/g per minute respectively. This suggests that different glycerol transport mechanisms operate during stress by the two osmotica. The addition of uncouplers (2,4-dinitrophenol or carbonylcyanide-m-chlorophenylhydrazine) resulted in the outflow of accumulated [^14C]glycerol from Z. rouxii after on osmotic upshock indicating that an active transport mechanism was operative. The transport mechanism was specific for glycerol since other polyols (mannitol, meso-erythritol and arabitol) had no effect on the uptake rate. During upshock from 0.998 to 0.960 a _w(NaCl), a transient increases in the rate of [¹⁴C]glycerol uptake was observed. However, if PEG 400 was used as osmoticum, the rate of glycerol uptake failed to increase.Offprint requests to: P. J. van Zyl     

Regulation of glycerol metabolism in Zygosaccharomyces rouxii in response to osmotic stress

Summary Enzyme analyses indicated that the metabolism of glycerol by Zygosaccharomyces rouxii occurred via either glycerol-3-phosphate (G3P) or dihydroxyacetone (DHA). The route via DHA is significant in osmoregulation. The specific activities of glycerol dehydrogenase (GDHG) and DHA kinase, which metabolize glycerol via DHA, increased nine- and fourfold respectively during osmotic stress [0.960 water activity (a_w) adjusted with NaCl] when compared to non-stressed conditions (0.998 a_w). Both pathways are under metabolic regulation. Glycerol kinase, mitochondrial G3P dehydrogenase and DHA kinase are induced by glycerol while the latter is also repressed by glucose. Cells treated with cycloheximide prior to osmotic upshock showed significantly lower DHA kinase and GDHG levels and lower intracellular glycerol concentrations when compared to untreated control cells. Thus protein synthesis is essential for osmotic adaptation. Offprint requests to: B. A. Prior     

Adaptation ofZygosaccharomyces rouxii to changes in water activity in transient continuous culture

Summary The polyols glycerol and arabitol were accumulated against an increasing concentration ratio in response to a_W reduction. Glycerol was the main osmoregulatory solute accumulated and arabitol accumulation only occurred during the initial transitory phase. The polyols were accumulated to concentrations less than that required to maintain equilibrium across the membrane.     

Physiological responses of Zygosaccharomyces rouxii to osmotic stress

When cell suspensions of Zygosaccharomyces rouxii were subjected to osmotic shock with NaCl, the cell volume decreased sharply and plasmolysis was observed. The cell subsequently recovered and volumes similar to those of cells growing at the respective water activity (a_w) values were found. Cycloheximide prevented cell recovery, indicating the involvement of protein synthesis in the recovery process. The intracellular glycerol concentration of Z. rouxii incubated in the presence of [¹⁴C]glycerol increased from 13 to 96 mmol/l during the initial 20 min after an upshock from 0.998 a_w to 0.96 a_w. All the intracellular glycerol was labelled and therefore derived from the medium. Labelled glycerol was subsequently utilized and replaced by unlabelled glycerol produced by the cell within 90 min. The initial increase in glycerol concentration following the upshock was confirmed by ¹³C-nuclear magnetic resonance (NMR) spectroscopic studies of cell extracts. The combined dihydroxyacetone and dihydroxyacetone phosphate concentrations fluctuated during this period, whereas glycerol-3-phosphate initially increased and then remained constant. This indicates that the production of glycerol is regulated. Decreases in ATP and polyphosphate levels were observed following osmotic upshock and may reflect a greater demand for ATP during the period of adjustment to decreased a_w. The changes in cell volume and in ATP concentration following osmotic upshock may serve as osmoregulatory signals in Z. rouxii, as suggested previously for other microorganisms. Correspondence to: S. G. Kilian     

The effect of water activity on growth and end-product formation of two Lactobacillus spp. and Brochothrix thermosphacta ATCC 11509T

Summary The effect of water activity (a_w) on the growth and end-product formation of Lactobacillus viridescens SMRICC 174, Lactobacillus SMRICC 173 (homofermentative) and Brochothrix thermosphacta ATCC 11509^T was studied. All strains orginated from meat or meat products. The a_w was adjusted in the range 0.94–0.99 with NaCl or glycerol. A greater reduction in growth rates was found for L. viridescens and B. thermosphacta when a_w was regulated with NaCl rather than with glycerol, the opposite was true for Lactobacillus 173. L. viridescens grew at a_w >-0.94. At 0.94 a_w B. thermosphacta was totally inhibited when NaCl was the solute and Lactobacillus 173 when glycerol was the solute. Only minor variations in the end-product formation of the Lactobacillus spp. were found at different a_w values. In aerobic culture B. thermosphacta produced less l-lactic acid and more acetic acid as the a_w was decreased with NaCl, while the yields were unaffected when glycerol was used.     

Effects of osmotic and matric potential on radial growth and accumulation of endogenous reserves in three isolates of Pochonia chlamydosporia

For the first time, the effects of varying osmotic and matric potential on fungal radial growth and accumulation of polyols were studied in three isolates of Pochonia chlamydosporia. Fungal radial growth was measured on potato dextrose agar modified osmotically using potassium chloride or glycerol. PEG 8000 was used to modify matric potential. When plotted, the radii of the colonies were found to grow linearly with time, and regression was applied to estimate the radial growth rate (mm day^?1). Samples of fresh mycelia from 25-day-old cultures were collected and the quantity (mg g^?1 fresh biomass) of four polyols (glycerol, erythritol, arabitol and mannitol) and one sugar (glucose) was determined using HPLC. Results revealed that fungal radial growth rates decreased with increased osmotic or matric stress. Statistically significant differences in radial growth were found between isolates in response to matric stress (P<0.006) but not in response to osmotic stress (P=0.759). Similarly, differences in the total amounts of polyols accumulated by the fungus were found between isolates in response to matric stress (P<0.001), but not in response to osmotic stress (P=0.952). Under water stress, the fungus accumulated a combination of different polyols important in osmoregulation, which depended on the solute used to generate the stress. Arabitol and glycerol were the main polyols accumulated in osmotically modified media, whereas erythritol was the main polyol that was accumulated in media amended with PEG. The results found that Pochonia chlamydosporia may use different osmoregulation mechanisms to overcome osmotic and matric stresses.     

Effect of water activity adjusted with different solutes on growth and lactic acid production byLactobacillus helveticus

Effect of water activity (a _w) adjusted with NaCl or glycerol on the growth and metabolism ofLactobacillus helveticus var.pragensis at 40°C was demonstrated by growth curves (generation time) and changes in the degree of acidity of a milk medium expressed as lactic acid content. NaCl-regulateda _w of 0.970 inhibited growth and acid production completely. The same, glycerol-regulateda _w of 0.970 increased the generation time only from 33 min (a _w=0.994) to 67 min which was less than the generation time at the highera _w of 0.982 adjusted with NaCl (103 min). Glycerol-regulateda _w of 0.970 did not decrease the acid production (2.6% lactic acid). Ata _w of 0.951, the acid production was decreased by 39% compared with the values found in milk media with the original, unadjusteda _w of 0.994, after the same time of incubation. Media witha _w adjusted to the same values with NaCl or glycerol do not influence the growth and acid production ofL. helveticus in the same way. In contrast to glycerol, NaCl has a strong effect.     

Solute stresses affect growth patterns, endogenous water potentials and accumulation of sugars and sugar alcohols in cells of the biocontrol yeast Candida sake

AIM: To evaluate the effect of modifications of water activity (aw 0. 996-0.92) of a molasses medium with different solutes (glycerol, glucose, NaCl, proline or sorbitol) on growth, intracellular water potentials (psi(c)) and endogenous accumulation of polyols/sugars in the biocontrol yeast Candida sake. METHODS AND RESULTS: Modification of solute stress significantly influenced growth, psi(c) and accumulation of sugars (glucose/trehalose) and polyols (glycerol, erythritol, arabitol and mannitol) in the yeast cells. Regardless of the solute used to modify aw, growth was always decreased as water stress increased. Candida sake cells grew better in glycerol- and proline-amended media, but were sensitive to NaCl. The psi(c) measured using psychrometry showed a significant effect of solutes, aw and time. Cells from the 0.96 aw NaCl treatment presented the lowest psic value (- 5.20 MPa) while cells from unmodified media (aw = 0. 996) had the highest value (- 0.30 MPa). In unmodified medium, glycerol was the predominant reserve accumulated. Glycerol and arabitol were the major compounds accumulated in media modified with glucose or NaCl. In proline media, the concentration of arabitol increased. In glycerol- and sorbitol-amended media, the concentration of glycerol rose. Some correlations were obtained between compatible solutes and psi(c). CONCLUSIONS AND SIGNIFICANCE: This study demonstrates that subtle changes in physiological parameters significantly affect the endogenous contents of C. sake cells. It may be possible to utilize such physiological information to develop biocontrol inocula with improved quality.     

Temporal accumulation of mannitol and arabitol in Geotrichum candidum

The temporal depletion and accumulation of polyols were investigated in the fungus Geotrichum candidum. The major intracellular polyols were tentatively identified by paper chromatography as mannitol and arabitol. Inositol was also present in small quantities, and trehalose was also detected in appreciable concentrations.Germination and vegetative growth depended on the type and concentration of the sole exogenous carbon source. Mannitol occurred in arthrospores at 9.4% of the dry weight after several days growth in 2% (w/v) glucose solid medium, and became depleted during germination and vegetative growth in liquid medium containing 2% (w/v) glucose, 2% (w/v) sodium acetate or 25% (w/v) glucose as sole carbon source. This hexitol latter accumulated during arthrosporulation. The depletion and accumulation of ethanol-soluble carbohydrate believed to be primarily trehalose was temporally similar to that of mannitol. Arabitol accumulated intracellularly during germination and vegetative growth in sodium acetate medium and 25% glucose medium. This pentitol was not detected intracellularly at any culture age during growth in 2% glucose medium.Prolonged incubation of the culture in 25% glucose medium after stationary phase was reached resulted in the gradual disappearance of arabitol from the arthrospores simultaneously with an increase in intracellular mannitol. In comparison, ethanol-soluble carbohydrate did not change with prolonged incubation in this medium.     

Control of arabitol formation in Schizophyllum commune development

Summary Dikaryotic cells of S. commune synthesized polyols throughout the life cycle when grown on glucose, cellobiose, or cellulose. Basidiospores contained arabitol and mannitol which were depleted during germination. The mannitol content of the young germlings rose to normal levels within a day; arabitol accumulation remained depressed for 5 to 7 days and then returned to normal levels characteristic of vegetative cells. Individual homokaryons differed in their production of intracellular polyols, which, unlike germlings, remained constant with cultural age. Homokaryon (str. 699) produced low levels of arabitol but high levels of glycerol while another homokaryon (str. 845) was the reverse. Mixtures of these homokaryons as well as the dikaryon (699×845) produced arabitol and glycerol levels intermediate between the parent homokaryons. High concentrations of glucose did not change the nature of the polyols produced. Arabitol formation could be induced prematurely in germlings or elevated in the dikaryon by growth on acetate or ethanol. Both homokaryons responded to growth on acetate with elevated arabitol production; acetate induction of arabitol formation was repressed in all types of cells if glucose were added simultaneously with acetate. Maltose, cellobiose, and trehalose also stimulated arabitol formation in young germlings, suggesting that glucose repression was the cause of decreased arabitol formation in basidiospore germlings. There was no correlation between the formation of arabitol and the derepression of isocitrate lyase or change in specific activities of alkaline and acid phosphatase in germlings grown on various carbon sources.     

Improving water stress tolerance of the biocontrol yeast Candida sake grown in molasses-based media by physiological manipulation

The biocontrol agent Candida sake was cultured on either an unmodified molasses-based medium (water activity, a(w) 0.996) or on water stressed media produced by the addition of glycerol, glucose, NaCl, sorbitol, or proline to 0.98, and 0.96 a(w) for 24, 48, and 72 h, to study their impact on subsequent cell viability, and on concentrations of endogenous sugars (trehalose and glucose) and polyols (glycerol, erythritol, arabitol, and mannitol). The viability of cells of different ages cultured on these media was evaluated on NYDA medium with freely available water (a(w) 0.995), and on medium modified with polyethylene glycol to a(w) 0.95. Regardless of solute used, viable counts of cells grown on molasses-based medium (a(w) 0.98) were equal to or higher than those obtained from the medium with water freely available. The amino acid proline stimulated growth at 10% concentration. In contrast, water stress induced by addition of NaCl, glucose, or sorbitol at a(w) 0.96 caused a significant reduction in viable counts. Older cultures were more resistant to water stress. Glycerol and arabitol were the main solutes accumulated by C. sake cells in response to lowered a(w). Intracellular concentration of these polyols depended more on the solute used to adjust the a(w) than on the a(w) itself. Candida sake was more resistant to water stress with higher intracellular concentration of glycerol and erythritol.     

Short communication: Effect of water activity on cell growth and phenol oxidase production by Streptomyces cyaneus var. viridochromogenes in surface culture

In surface cultures of Streptomyces cyaneus var. viridochromogenes, NaCl depressed water activity (a _w) without supporting growth. Reducing a _w from 0.987 to 0.951 led to 3- and 4-fold increases in intracellular and extracellular phenol oxidase activities, respectively.     

Xerotolerance in fission yeasts and the role of glycerol as compatible solute

The effect on growth of reducing the water activity (a _w) of a medium with various solutes has been investigated for 27 strains of fission yeasts (Schizosaccharomyces). The minimum-tolerated a _w (MTA) was dependent on both the nature of the solute and the species. When the strains of each species were grouped together, the lowest mean MTA values were found with glucose, fructose or glycerol as stressing solutes, being in the range 0.89–0.90 for S. pombe, S. malidevorans, S. octosporus and S. slooffiae, but in the range 0.92–0.94 for S. japonicus. With the non-metabolizable sugars sorbose and xylose and the salts NH₄Cl, KCl, and NaCl, the mean MTA values were in the range 0.96–0.985, except for (1) the single strain of S. slooffiae, which was more tolerant of NH₄Cl and KCl with values of 0.95 and 0.94, respectively, and (2) the strains of S. pombe, S. malidevorans and S. japonicus, which were less tolerant of NaCl with mean values of about 0.99. One strain of each species was examined for intracellular solutes when actively growing in the presence of near-limiting concentrations of stressing solute. With glucose, fructose or glycerol, all five strains contained substantial amounts of glycerol but no other polyol; with the other solutes no glycerol or other polyol was found, except for small amounts of glycerol in strains of S. octosporus and S. slooffiae stressed with NH₄Cl, KCl, or NaCl.Abbreviations MTA Minimum-tolerated water activity - a _w water activity - YEPG yeast extract, phosphate, glucose medium     

Production of arabitol from glycerol: strain screening and study of factors affecting production yield

Glycerol is a major by-product from biodiesel production, and developing new uses for glycerol is imperative to overall economics and sustainability of the biodiesel industry. With the aim of producing xylitol and/or arabitol as the value-added products from glycerol, 214 yeast strains, many osmotolerant, were first screened in this study. No strains were found to produce large amounts of xylitol as the dominant metabolite. Some produced polyol mixtures that might present difficulties to downstream separation and purification. Several Debaryomyces hansenii strains produced arabitol as the predominant metabolite with high yields, and D. hansenii strain SBP-1 (NRRL Y-7483) was chosen for further study on the effects of several growth conditions. The optimal temperature was found to be 30°C. Very low dissolved oxygen concentrations or anaerobic conditions inhibited polyol yields. Arabitol yield improved with increasing initial glycerol concentrations, reaching approximately 50% (w/w) with 150 g/L initial glycerol. However, the osmotic stress created by high salt concentrations (≥50 g/L) negatively affected arabitol production. Addition of glucose and xylose improved arabitol production while addition of sorbitol reduced production. Results from this work show that arabitol is a promising value-added product from glycerol using D. hansenii SBP-1 as the producing strain.     

Physiological and genetic characterisation of osmosensitive mutants of Saccharomyes cerevisiae

The screening of 20,000 Saccharomyces cerevisiae random mutants to identify genes involved in the osmotic stress response yielded 14 mutants whose growth was poor in the presence of elevated concentrations of NaCl and glucose. Most of the mutant strains were more sensitive to NaCl than to glucose at the equivalent water activity (a_w) and were classified as salt-sensitive rather than osmosensitive. These mutants fell into 11 genetic complementation groups and were designated osr1–osr11 (osmotic stress response). All mutations were recessive and showed a clear 2⁺ : 2^– segregation of the salt-stress phenotype upon tetrad analysis when crossed to a wild-type strain. The complementation groups osr1, osr5 and osr11 were allelic to the genes PBS2, GPD1 and KAR3, respectively. Whereas intracellular and extracellular levels of glycerol increased in the wild-type strains when exposed to NaCl, all mutants demonstrated some increase in extracellular glycerol production upon salt stress, but a number of the mutants showed little or no increase in intracellular glycerol concentrations. The mutants had levels of glycerol-3-phosphate dehydrogenase, an enzyme induced by osmotic stress, either lower than or similar to those of the parent wild-type strain in the absence of osmotic stress. In the presence of NaCl, the increase in glycerol-3-phosphate dehydrogenase activity in the mutants did not match that of the parent wild-type strain. None of the mutants had defective ATPases or were sensitive to heat stress. It is evident from this study and from others that a wide spectrum of genes is involved in the osmotic stress response in S. cerevisiae. Received: 5 January 1998 / Accepted: 24 March 1998     

The salt relations of Dunaliella

Dunaliella tertiolecta (marine) and D. viridis (halophilic) were each trained by serial transfer to growth at salt concentrations previously regarded as the other's domain. D. viridis then had a salt optimum at 1.0–1.5 M sodium chloride whereas that for D. tertiolecta was less than 0–2 M. Nevertheless D. tertiolecta grew faster than the halophil at all salt concentrations up to 3.5 M, the highest at which they were compared.Both species accumulate glycerol, which is necessary for growth at elevated salinities and which responds in its content to water activity (a _w ) rather than specifically to salt concentration. Variation in glycerol content is a metabolic process which occurs in the dark from accumulated starch as well as photosynthetically. Regulation of glycerol content by a _w does not require protein synthesis. The NADP-specific glycerol dehydrogenase of each of the algae is likely to be directly involved in the regulation of glycerol content. Kinetic studies, together with those described in an earlier publication, show that the enzyme has regulatory properties, and that both glycerol and dihydroxyacetone act as effectors as well as reactants. A mechanism of the reaction is tentatively proposed.     

Production of the fungal biocontrol agent Epicoccum nigrum by solid substrate fermentation: effect of water activity on accumulation of compatible solutes

Epicoccum nigrum conidia were produced by solid fermentation on wheat grains (cv. Rendeveaux and Brigadier) at different water activities (a_w). Conidial production was highest at high a_w(0.996) than at reduced a_w (0.98). However, conidial production at reduced a_w was improved when the a_w of the substrate was adjusted with a mixture of glycerol and water. Maximum levels ofconidiation were 7–11 × 10⁶ conidia g⁻¹ grain. The a_w of the solid substrate affected the pattern of accumulation of compatible solutes in the conidia. Mannitol was the main polyol in all conidialtypes. However, the amounts of mannitol were higher in conidia produced at high a_w. At reduced a_w the conidia of E. nigrum accumulated moreglycerol, which is more efficient in the osmorregulation proccess than mannitol. Arabitol accumulated in low amounts, specifically in conidia produced at the lower a_w, on cv. Rendeveaux but not on cv. Brigadier. Trehalose was detected in higher amounts in cv. Rendeveaux than in cv. Brigadier, andthe amounts were higher in conidia produced at high a_w. A significant amount of endogenous solutes was detected in the washing liquid used for the separation of the conidia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of water activity on exo-pectinase production byAspergillus niger CH4 on solid state fermentation

Summary Ethylene glycol, sorbitol and glycerol were used as water activity depressors to study the effect of water activity on pectinase production byAspergillus niger CH4. Ethylene glycol depressed a_w without supporting growth nor strongly affecting pectinase production in petri dish cultures. This depressor was used to evaluate the influence of water activity on exo-pectinase production by SSF. It was found that although pectinase production decreased at low a_w values, this activity was present at a_w values as low as 0.90. The specific activity increased up to 4.5 fold by reducing a_w from 0.98 to 0.90. The reducing groups accumulated extracellularly suggesting sugar transport limitation as a consequence of a_w depression.     

Osmotic adaptation in halotolerant yeast, Debaryomyces nepalensis NCYC 3413: role of osmolytes and cation transport

Debaryomyces nepalensis NCYC 3413, a food spoiling yeast isolated from rotten apple, has been previously demonstrated as halotolerant yeast. In the present study, we assessed its growth, change in cell size, and measured the intracellular polyol and cations (Na⁺ or K⁺) accumulated during growth in the absence and presence of different concentrations of salts (NaCl and KCl). Cells could tolerate 2 M NaCl and KCl in defined medium. Scanning electron microscopic results showed linear decrease in mean cell diameter with increase in medium salinity. Cells accumulated high amounts of K⁺ during growth at high concentrations of KCl. However, it accumulated low amounts of Na⁺ and high amounts of K⁺ when grown in the presence of NaCl. Cells grown in the absence of salt showed rapid influx of Na⁺/K⁺ on incubation with high salt. On incubation with 2 M KCl, cells grown at 2 M NaCl showed an immediate efflux of Na⁺ and rapid uptake of K⁺ and vice versa. To withstand the salt stress, osmotic adjustment of intracellular cation was accompanied by intracellular accumulation of polyol (glycerol, arabitol, and sorbitol). Based on our result, we hypothesize that there exists a balanced efflux and synthesis of osmolytes when D. nepalensis was exposed to hypoosmotic and hyperosmotic stress conditions, respectively. Our findings suggest that D. nepalensis is an Na⁺ excluder yeast and it has an efficient transport system for sodium extrusion.     

Moisture Requirements for Growth and Metabolite Production by Lactic Acid Bacteria

The effect of water activity (a_w) reduction on growth and acid and diacetyl production by three lactic streptococci was studied. In addition, the influence of low moisture conditions on several bacteria of significance in the fermentation of sauerkraut was examined. The minimal a_w supporting growth of dairy lactics was 0.93 in a medium adjusted with glycerol. Media adjusted with sucrose generally were more inhibitory than those in which glycerol was the humectant. Titratable acidity, although not related to the type of humectant, did depend on the a_w of the medium and was directly related to the extent of growth. Diacetyl concentration increased in cultures of reduced a_w when the media were adjusted with both humectants; however, the effect was greatest with glycerol. A lactic strain associated with sauerkraut fermentation appeared to grow at a lower minimal a_w in a glycerol-adjusted medium than in a system adjusted with NaCl; however, none of the sauerkraut organisms grew at a_w levels of <0.95 when NaCl was the solute. Acid production appeared to be related to the presence and extent of growth at all of the a_w levels studied.