The Effect of Water Activity on the Growth and Respiration of Pseudomonas fluorescens

The growth rate of Pseudomonas fluorescens was greater and continued at lower water activity ( a _w) values when glycerol controlled the a _w of glucose minimal medium than when the a _w was controlled by NaCl and sucrose. Growth was not observed below 0·945, 0·970 and 0·964 a _w when glycerol, sucrose and NaCl respectively controlled the a _w. The catabolism of glucose, Na lactate and DL-arginine as measured by respirometry was completely inhibited at a _w values greater than the minimum for growth when the a _w was controlled with NaCl. When the a _w was controlled with glycerol, catabolism of the three substrates continued at a _w values significantly below the a _w for growth on glucose. Catabolism of glucose in the presence of sucrose occurred at a level below the minimum growth a _w but catabolism of the other two substrates ceased at a _w values greater than the minimum growth a _w. Arrhenius plots between 10° and 34°C of the growth rate in glucose minimal medium at 0·98 a _w showed that the order of inhibition was sucrose > NaCl > glycerol. The order of inhibition differed when Arrhenius plots of catabolism of glucose was examined between 10° and 34 °C, namely NaCl > sucrose > glycerol. The mechanism of action of solutes controlling a _w is discussed.     

Water Relations of Glucose-catabolizing Enzymes in Pseudomonas fluorescens

Examination of the catabolism of glucose via the Entner-Doudoroff pathway by standard enzyme assays showed that the activity of glucose-6-phosphate dehydrogenase, glucokinase and 2-ketoglu-conokinase plus phosphoketogluconate reductase was completely inhibited at a _w values less than 0.965, 0.98 and 0.96 respectively when NaCl was used to adjust the a _w. The other glucose-catabolizing enzymes were inhibited to a lesser degree. When sucrose was used to control a _w, glucokinase and glucose-6-phosphate dehydrogenase were inhibited at 0.92 a _w but the other enzymes remained active below 0.86 a _w. Enzymes were relatively active at reduced a _w when adjusted with glycerol and most remained active even at 0.80 a _w. When a _w was controlled by potassium glutamate, the activity of glucokinase and glucose-6-phosphate dehydrogenase was markedly less inhibited than by NaCl at similar a _w. Possible reasons for the variation in activity by glucose-catabolizing enzymes in response to a _w controlled by various solutes could be location of the enzyme in the cell, ability of the solute to penetrate the cell and ability to withstand high salt and sucrose concentrations. When the a _w of the growth medium was reduced to 0.98 by glycerol, NaCl and polyethylene glycol 400, levels of glucokinase were significantly reduced while higher levels of glucose dehydrogenase and gluconate dehydrogenase were induced. This suggests that reduction in a _w could regulate the routes of catabolism in the Entner-Doudoroff pathway. When sucrose was used to control a _w of the growth medium high levels of most enzymes were induced, suggesting catabolism of the sucrose by the organism.     

Effect of water activity and temperature on growth and fumonisin B1 and B2 production by Fusarium proliferatum and F. moniliforme on maize grain

S. MARIN, V. SANCHIS, I. VINAS, R. CANELA AND N. MAGAN. 1995. The effect of different water activities ( a _w, 0.968, 0.956, 0.944, 0.925) and temperature (25°C and 30°C) on colonization and production of fumonisin B₁ (FB₁) and B₂ (FB₂) on sterile layers of maize by Fusarium proliferatum and F. moniliforme isolates was determined over periods of 6 weeks. Generally, both F. moniliforme and F. proliferatum grew faster with increasing a _w and best at 30°C. All three isolates produced more FB 1 than FB₂ regardless of a _w or temperature. Very little FB₁ and FB₂ were produced at 0.925 a _w, with maximum produced at 0.956 and 0.968 a _w at both temperatures tested. Most FB₁ and FB₂ were produced by F. moniliforme (25N), followed by F. proliferatum isolates (73N and 131N). At all a _w levels and both temperatures there was an increase in FB₁ and FB₂ concentration with time. Statistical analyses of a _w, temperature, time, two- and three-way interactions showed some significant differences between isolates and FB₁ and FB₂ production.     

Interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii

The interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii were investigated. Growth rates in media supplemented with glucose, sucrose or NaCl to a _w 0.93 were more rapid than in unsupplemented media ( a _w 0.99). Although growth in unsupplemented medium was lower at 35°C, incubation at 21°C or 35°C had little effect on growth in media supplemented with glucose and sucrose. The addition of 300 μg potassium sorbate/ml to media resulted in reduced growth rates, particularly at 35°C. Heat resistance of Z. rouxii was substantially greater in cultures previously incubated at 35°C than in cultures incubated at 21° in media both with and without 300 μg potassium sorbate/ml. Zygosaccharomyces rouxii was tolerant to freezing at - 18°C for up to 120 d in all test media supplemented with glucose, sucrose or NaCl. The addition of 300 μg potassium sorbate/ml to sucrose-supplemented media resulted in increased resistance to freezing in cultures previously incubated at 21°C. Sensitivity to freezing increased when cultures were incubated at 21°C in media not supplemented with solutes. Glucose and sucrose provided the best protection against inactivation by heating and freezing, regardless of the presence of potassium sorbate in growth media.     

Growth of Fusarium moniliforme and its biosynthesis of fumonisin B1 on maize grain as a function of different water activities

The production of fumonisin by Fusarium moniliforme during its growth on maize depends on extrinsic factors. In particular, experiments on maize grain at different water activities ( a _w)(1, 0.95, 0.90, 0.85) have demonstrated the influence of a _w on fumonisin biosynthesis, and on fungal growth defined by measurement of ergosterol levels. Fumonisin levels dropped threefold when a _w was lowered by 5%, but growth rate was unchanged. A 10% reduction in a _w from 1 to 0.90 resulted in a 20-fold drop in fungal growth, and fumonisin production was reduced 300-fold. At a threshold a _w of 0.85–0.86, F. moniliforme exhibited virtually no measurable metabolic activity, and hence no fumonisin production.     

Determination of optimal growth parameters for the bioincising fungus Physisporinus vitreus by means of response surface methodology

Aim:  To evaluate the influence of water activity ( a _w), temperature and pH on the radial growth and lag phase of Physisporinus vitreus (E-642), a basidiomycete was used in the biotechnological process of bioincising.
Methods and Results:  Radial growth was monitored for 20 days on malt extract agar medium. Five levels of a _w (0·998, 0·982, 0·955, 0·928, 0·892) were combined with three incubation temperatures (10, 15, 20°C) and three pH values (4, 5, 6). Data analyses showed a highly significant effect of a _w and temperature ( P <  0·0001) and a significant effect of pH ( P <  0·05). The radial growth rate and lag phase of P. vitreus were very sensitive to a _w reduction. Although P. vitreus was able to grow at all the selected temperatures and pH values, the lag phase increased with decreasing a _w and growth became inhibited at a _w = 0·955. Optimal conditions for growth of P. vitreus were a _w = 0·998, 20°C and pH 5. The response surface model provided reliable estimates of these growth parameters and confirmed a greater dependence on a _w than on temperature or pH under in vitro conditions.
Conclusions:  Low levels of a _w can prevent growth of P. vitreus , so wood moisture content should be adjusted accordingly.
Significance and Impact of the Study:  Implementation of these results should contribute towards the optimization and efficiency of bioincising.     

Comparison of combinations of diluents and media for enumerating Zygosaccharomyces rouxii in intermediate water activity foods

Combinations of five diluents (0.1% peptone, 40 and 50% glucose, and 18 and 26% glycerol) and three enumeration media (tryptone glucose yeast extract, dichloran 18% glycerol and malt extract yeast extract 50% glucose (MY50G) agars) were evaluated for recovering a xerotolerant yeast, Zygosaccharomyces rouxii , from foods with intermediate water activity ( a _w). Combinations of 40% ( a _w, 0.936) or 50% ( a _w 0.898) glucose diluent and MY50G agar ( a _w 0.890) were superior in recovering highest populations. The type of solute in the diluent, as well as a reduced a _w, influences efficiency of recovering viable cells.     

Growth-induced water potentials may mobilize internal water for growth

Abstract. Wphen there is no external source of water, plants can grow by mobilizing internal water from nongrowing tissues. We investigated how this internal water moves by measuring continuously and simultaneously the water potential (ψ_w) of soybean ( Glycine max L. Merr.) seedlings in the upper, growing stem tissues and the lower, non-growing stem tissues. When external water was available to the roots, the stems grew rapidly and the ψ_w of the growing tissue was continually below that of the nongrowing tissue and the medium around the roots. This indicated that a growth-induced gradient in ψ_w favoured water movement from the external source to the growing cells. When the external source was removed, the ψ_w of the growing tissue remained constant for a time and the ψ_w of the nongrowing tissue decreased somewhat. Growth took place slowly as water was withdrawn from the nongrowing tissue but ψ_w gradients continued to favour water transport to the growing cells. On the other hand, if this internal source was removed by excision, growth ceased abruptly. In this case, the cell walls relaxed and the ψ_w of the growing tissue decreased by about 0.1 MPa instead of remaining constant. The ψ_w of the detached nongrowing tissues remained constant instead of decreasing. This indicates not only that water mobilization required attached nongrowing or slowly growing tissues but also that mobilization affected wall relaxation. Thus, ψ_w differences may mobilize internal water, may explain the continued growth of plants and plant parts removed from external sources of water, and may account for discrepancies in measurements of cell wall properties in growing tissues.     

Modelling the growth of Weissella cibaria as a function of fermentation conditions

Aims:  To investigate the effect of pH, water activity ( a _w) and temperature on the growth of Weissella cibaria DBPZ1006, a lactic acid bacterium isolated from sourdoughs.
Methods and Results:  The kinetics of growth of W. cibaria DBPZ1006 was investigated during batch fermentations as a function of pH (4·0–8·0), a _w (0·935–0·994) and temperature (10–45°C) in a rich medium. The growth curve parameters (lag time, growth rate and asymptote) were estimated using the dynamic model of Baranyi and Roberts (1994. A dynamic approach to predicting bacterial growth in food. Int J Food Microbiol 23, 277–294). The effect of pH, a _w and temperature on maximum specific growth rate (μ_max) were estimated by fitting a cardinal model. μ_max under optimal conditions (pH = 6·6, a _w = 0·994, T  = 36·3°C) was estimated to be 0·93 h⁻¹. Minimum and maximum estimated pH and temperature for growth were 3·6 and 8·15, and 9·0°C and 47·8°C, respectively, while minimum a _w was 0·918 (equivalent to 12·2% w/v NaCl).
Conclusions:  Weissella cibaria DBPZ1006 is a fast-growing heterofermentative strain, which could be used in a mixed starter culture for making bread.
Significance and Impact of the Study:  This is the first study reporting the modelling of the growth of W. cibaria , a species that is increasingly being used as a starter in sourdough and vegetable fermentations.     

The survival of Salmonella enterica serovar Typhimurium DT104 and total viable counts on beef surfaces at different relative humidities and temperatures

Aims:  The aim of this study was to investigate changes in Salmonella and total viable count (TVC) survival on beef carcass surfaces stored for 72 h under different combinations of relative humidity (i.e. RH 75% or 96%) and temperature (5°C or 10°C).
Methods and Results:  The influence of low water activity ( a _w) and temperature on the survival and growth of Salmonella enterica serovar Typhimurium DT104 and the aerobic mesophilic flora on meat pieces from different sites on beef carcasses was investigated, under controlled conditions (75% or 96% RH; 5 or 10°C) in an environmental cabinet. Salmonella counts declined during storage at low a _w (75% RH) conditions at 5°C or 10°C. Salmonella counts increased during storage at high a _w (96% RH) at 10°C only. At 5°C, TVCs increased during storage at high a _w, but not at low a _w. TVCs increased on all samples from carcasses stored at high or low a _w at 10°C, except those samples taken from areas of surface fat.
Conclusions:  This suggests that substrate composition dictates growth rates under low a _w conditions. The results are discussed in terms of the possible protective effects of substrate osmolyte accumulation in bacterial survival and/or growth.
Significance and Impact of the Study:  The data obtained in this study provides useful insights on the influence of a _w and temperature on pathogen survival on meat surfaces at chill temperature.     

Microbial growth at reduced water activities: studies of aw prediction in solutions of compatible solutes

Studies were made of the prediction of intracellular water activity ( a _w) of microorganisms grown at reduced a _w. These included the determination and prediction of a _w of potassium L-glutamate solutions and the prediction of a _w in mixed solutions of various compatible solutes and macromolecules. Compatible solutes studied were L-proline, glycerol, potassium chloride and D-arabitol. The results indicate that a _w predictions of such mixtures may be done with acceptable accuracy by neglecting solute-solute interactions.     

The Effect of Water Activity and the aw Controlling Solute on Spore Germination of Bacillus stearothermophilus

The germination of spores of Bacillus stearothermophilus was studied in nutrient broth in relation to the water activity ( a _w) of the medium, the nature of the a _w controlling solutes glycerol, sucrose, KCl, and NaCl, and temperature. Quantitation of germination was based on the change of the phase-bright spore to phase-dark. Activation of spores was by exposure to 100°C/10 min in a medium of the same composition as that used for germination.
Of the four solutes used, sucrose proved most inhibitory to germination, especially in the upper part of the temperature range 38-75°C, glycerol was the most favourable whereas KCl and NaCl, whose effect was almost identical, occupied an intermediate place. The glycerol effect became more pronounced as the a _w of the medium decreased towards 0.960, becoming inhibitory thereafter.
The solute effect on spore germination followed a pattern that related to the class of solute, i.e. electrolyte or non-electrolyte, and its cell penetration characteristics.
Solute penetration during heat activation and germination was considered as the major germination factor and was associated with the osmoregulation mechanism within the spore proposed recently as the basis of spore dormancy and resistance.     

The Intracellular Water Activity of Bacteria in Relation to the Water Activity of the Growth Medium

Intracellular water activities ( a _w) calculated from the solute composition of various bacterial cells, are in good agreement with values derived from intracellular freezing point data. Further, and confirming literature results based on freezing points, the intracellular a _w was found to be generally equal to or lower than that of the growth medium.     

Radiation Resistance of Spores of Bacillus subtilis and B. stearothermophilus at Various Water Activities

S ummary . The radiation resistance of spores of Bacillus subtilis and B. stearothermophilus was determined under anoxic conditions in water vapour and in aqueous solutions of glucose and glycerol at various water activities. In water vapour, resistance of both kinds of spores increased slightly with decreasing water activity ( a _w). In glycerol solutions, a phase of rapid increase in resistance with decreasing a _w was followed by a slower increase on further reduction of a _w. This second phase was almost parallel to the water vapour curve, which, however, showed considerably lower resistance. The initial phase of rapid increase in resistance was also observed in glucose solutions.     

Mould growth and conidiation in cereal grains as affected by water activity and temperature

The effects of different water activities ( a _w) and temperatures on growth of storage moulds ( Aspergillus candidus and Penicillium implicatum ) on maize and paddy rice grains were measured using ergosterol content and dilution plating. The results showed that the mode of fungal growth depends on a _w. In all cases, conidiation occurred more readily at low a _w, whereas mycelial growth is more marked at higher a _w. The xerotolerant, thermophilic characteristics of A. candidus were more clearly revealed by the ergosterol content, which reflected mycelial development, than by enumeration of spores formed. The ergosterol content proved to be a better index of technological quality of cereals (assessed by acidity) than the number of fungal propagules. A 10-fold increase in the number of spores corresponded to a doubling of the ergosterol content.     

Relations between carbohydrate, water status and adventitious root formation in leafy pea cuttings rooted under various levels of atmospheric CO2 and relative humidity

Three levels of atmospheric CO₂ and 2 levels of relative humidity (RH) during the rooting period were tested for their effect on several factors presumed to influence adventitious root formation in leafy pea ( Pisum sativum L. cv. Alaska) cuttings. Compared to normal CO₂ levels (350 μl l⁻¹), neither 1800 nor 675 μl l⁻¹ CO₂ affected the rooting percentage or the number of roots per cutting. However, 1800 μl l⁻¹ CO₂ increased root and shoot dry weight, root length, carbohydrate levels in the base of the cuttings and water potential (Ψ_w) of cuttings compared to normal levels of CO₂. Compared to 87% RH. 55% RH decreased all of the above parameters, including the number of roots per cutting. A polyvinyl chloride antitranspirant (which partially blocks stomata and slows photosynthesis) applied simultaneously with 87% RH increased Ψ_w and root length but lowered all of the other above parameters, compared to 87% RH without antitranspirant. Increasing current photosynthate (products of photosynthetic activity after excision), carbohydrate, or Ψ_w either alone or together was associated with increased root system size but not necessarily with increased rooting percentage or root number. The data are consistent with a hypothesis that the number of roots per cutting increased with increasing current photosynthate and carbohydrate until some other factor became limiting. Also, the effect of Ψ_w on rooting percentage and root number was mediated through its effect on current photosynthate and carbohydrate.     

Predictive assessment of ochratoxin A accumulation in grape juice based-medium by Aspergillus carbonarius using neural networks

Aims:  To study the ability of multi-layer perceptron artificial neural networks (MLP-ANN) and radial-basis function networks (RBFNs) to predict ochratoxin A (OTA) concentration over time in grape-based cultures of Aspergillus carbonarius under different conditions of temperature, water activity ( a _w) and sub-inhibitory doses of the fungicide carbendazim.
Methods and Results:  A strain of A. carbonarius was cultured in a red grape juice-based medium. The input variables to the network were temperature (20–28°C), a _w (0·94–0·98), carbendazim level (0–450 ng ml⁻¹) and time (3–15 days after the lag phase). The output of the ANNs was OTA level determined by liquid chromatography. Three algorithms were comparatively tested for MLP. The lowest error was obtained by MLP without validation. Performance decreased when hold-out validation was accomplished but the risk of over-fitting is also lower. The best MLP architecture was determined. RBFNs provided similar performances but a substantially higher number of hidden nodes were needed.
Conclusions:  ANNs are useful to predict OTA level in grape juice cultures of A. carbonarius over a range of a _w, temperature and carbendazim doses.
Significance and Impact of the Study:  This is a pioneering study on the application of ANNs to forecast OTA accumulation in food based substrates. These models can be similarly applied to other mycotoxins and fungal species.     

Enhanced dopaminergic differentiation of human neural stem cells by synergistic effect of Bcl-xL and reduced oxygen tension

Neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. Here we investigated the effect of the anti-apoptotic protein Bcl-x_L and oxygen tension on dopaminergic differentiation and survival of a human ventral mesencephalic stem cell line (hVM1). hVM1 cells and a Bcl-x_L over-expressing subline (hVMbcl-x_L) were differentiated by sequential treatment with fibroblast growth factor-8, forskolin, sonic hedgehog, and glial cell line-derived neurotrophic factor. After 10 days at 20% oxygen, hVMbcl-x_L cultures contained proportionally more tyrosine hydroxylase(TH)-positive cells than hVM1 control cultures. This difference was significantly potentiated from 11 ± 0.8% to 17.2 ± 0.2% of total cells when the oxygen tension was lowered to 3%. Immunocytochemistry and Q-PCR-analysis revealed expression of several dopaminergic markers besides of TH just as dopamine was detected in the culture medium by HPLC analysis. Although Bcl-x_L-over-expression reduced cell death in the cultures, it did not alter the relative content of GABAergic, neurons, while the content of astroglial cells was reduced in hVMbcl-x_L cell cultures compared with control. We conclude that Bcl-x_L and lowered oxygen tension act in concert to enhance dopaminergic differentiation and survival of human neural stem cells.     

Microbial growth at reduced water activities: some physicochemical properties of compatible solutes

C hirife , J., F avetto , G. & F erro F ontán, c. 1984. Microbial growth at reduced water activities: some physicochemical properties of compatible solutes. Journal of Applied Bacteriology 56 , 259–268.
The water activities ( a _w) of solutions of D-arabitol (a major compatible solute in yeasts grown at reduced a _w) and also of related polyols (L-arabitol, xylitol and ribitol) were determined at 25C. The a _w of saturated solutions of L-proline and monopotassium L-glutamate (solutes which accumulate in bacterial cells at reduced a _w) were also determined. The viscosities of L-proline, monopotassium L-glutamate and D-arabitol solutions were measured at 25, 30, 35 and 40C for various concentrations up to high molalities. The relative viscosity curves were described using a theoretical model for the concentration dependence of viscosity. No relationship of viscosity of the electrolytes to effectiveness as 'compatible solutes' were evident, neither were the compatible solutes particularly effective 'structure makers' or 'structure breakers.'     

Carbohydrate partitioning, photosynthesis and growth in Lemna gibba G3. II. Effects of phosphorus limitation

The relationship between CO₂ assimilation rate, growth and partitioning of carbon among starch, sucrose, glucose and fructose were studied in phosphorus (P_i)-limited Lemna gibba L. G3. Two experimental models were used: 1) Cultures were grown at various stable, suboptimal rates regulated by the supply of P_i; 2) cultures growing at optimal rates were transferred to P_i-free medium. The response to a P_i deficiency can be divided into two phases. Phase I is characterized by hyperactivity of the sucrose synthesis pathway, leading to high levels of glucose and fructose. Phase II is characterized by starch accumulation associated with a decrease in the cytoplasmic pools of soluble sugars owing to inhibition of carbon export from the chloroplast. A strong negative correlation was found between the CO₂ assimilation rate and starch levels. No significant correlation was found between assimilation and ATP levels and decrease in relative growth rate did not significantly affect the adenylate energy charge (EC). The regulatory aspects of the partitioning of carbon among soluble sugars and starch as well as the negative correlation between carbohydrate levels and CO₂ assimilation at P_i-limited growth are discussed.