Spore germination and mycelial growth of streptomycetes at different humidity levels

This study is the first to show the ability of streptomycetes to develop at a very low humidity level. All of the streptomycetes studied produced growth at low humidity (aw 0.86 and 0.67). This capacity was most markedly pronounced in Streptomyces odorifer, whose spores were capable of germinating, and mycelial germs increased in length, at the air humidity aw 0.50. The formation of lateral branches (mycelium branching) at this humidity was noted only in single S. odorifer germs and only after 72 h of incubation. Study of streptomycete growth on an agarized medium with different osmotic pressures, created by various glycerol concentrations in the medium, showed that, at aw 0.67, the spores of all the streptomycetes studied germinate, producing mycelial germs but not microcolonies. The ecological significance of mycelial prokaryotes in soil microbial communities that develop and function under conditions of extremely low humidity is discussed.     

Dynamics of spore germination and mycelial growth of streptomycetes under low humidity conditions

At extremely low values of moisture pressure (?96.4 MPa; a_w 0.50), the spores of xerotolerant streptomycetes (Streptomyces odorifer and S. rubiginosohelvolus) germinated, their germ lengths increased, and lateral branching of the mycelium was observed after 5 days of incubation in a thin layer of agarized nutrient medium. At ?22.6 MPa (a_w 0.86), the mycelium begins to branch after a 2-day incubation; over a 5-day incubation at ?2.8 MPa (a_w 0.98), it goes through a reproduction cycle, which culminates in spore formation. The mathematical model approach enabled us to elucidate the behavioral patterns of Streptomyces spores in a thin layer of agarized nutrient medium at low humidity levels. The dynamics of spore germination is governed by the exponential law, which allows calculation of the average duration of the period a before spore germination, as well as the time needed for 50% of viable spores to germinate.     

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.     

The Influence of Water Activity and Temperature on Germination,Growth and Sporulation of <Emphasis Type="Italic">Stachybotrys chartarum</Emphasis> Strains

The objectives were to determine the influence of water activity (a_w, 0.997–0.92) and temperature (10–37°C) and their interactions on conidial germination, mycelial growth and sporulation of two strains of Stachybotrys chartarum in vitro on a potato dextrose medium. Studies were carried out by modifying the medium with glycerol and either spread plating with conidia to evaluate germination and germ tube extension or centrally inoculating treatment media for measuring mycelial growth rates and harvesting whole colonies for determining sporulation. Overall, germination of conidia was significantly influenced by a_w and temperature and was fastest at 0.997–0.98 a_w between 15 and 30°C with complete germination within 24 h. Germ tube extension was found to be most rapid at similar a_w levels and 25–30°C. Mycelial growth rates of both strains were optimal at 0.997 a_w between 25 and 30°C, with very little growth at 37°C. Sporulation was optimum at 30°C at 0.997 a_w. However, under drier conditions, this was optimum at 25°C. This shows that there are differences in the ranges of a_w x temperature for germination and growth and for sporulation. This may help in understanding the role of this fungal species in damp buildings and conditions under which immune-compromised patients may be at risk when exposed to such contaminants in the indoor air environment.     

Effect of Water Activity on Enterotoxin B Production and Growth of Staphylococcus aureus

Staphylococcus aureus C-243, an enterotoxin B-producing strain, was cultured on media adjusted to various water activity (a_w) levels by means of two different solute systems. Total numbers and rate of growth were diminished at low a_w levels, and enterotoxin synthesis was extremely sensitive to reduction in a_w. A reduction of a_w from 0.99 to 0.98 in one medium and from 0.99 to 0.97 in the other medium resulted in extremely low levels of enterotoxin in spent culture media despite the attainment of high numbers of staphylococci.     

Effect of temperature and water activity on spore germination and mycelial growth of three fungal biocontrol agents against water hyacinth (Eichhornia crassipes)

Aims: To determine the effect of water activity (a_w = 0·880–0·960) and temperature (15–35°C) on the percentage of viable conidia and mycelial growth of three biocontrol agents effective against water hyacinth in Mali: Alternaria sp. isolate Mlb684, Fusarium sacchari isolate Mln799 and Cadophora malorum isolate Mln715. Methods and Results: The fungi were grown in vitro on plates containing potato dextrose agar medium at different a_w values (glycerol being added to adjust the a_w). The percentage of viable conidia and radial growth rate decreased with decreasing water activity. Statistical analysis showed a significant effect of a_w, temperature and the a_w × temperature interaction on mycelial growth (P < 0·0001). Water activity emerged as the factor exerting the greatest influence. Differences were observed between the fungi tested, the C. malorum appearing more tolerant to low a_w and the F. sacchari more tolerant to high temperature (35°C). Growth models predicting the combined effect of a_w and temperature were developed and response surfaces generated, showing fairly good agreement with the experimental values. Conclusions: Our results confirm the previous finding that a_w has a greater influence than temperature on fungal growth. Under most conditions, variation of environmental factors has a detrimental influence on the percentage of viable conidia and mycelial growth rate of fungal isolates. Significance and Impact of the Study: The developed models may contribute to predicting the best environmental conditions for use of these fungi as effective biocontrol agents against water hyacinth.     

Effect of water activity on production and activity of Rhizopus oligosporus polysaccharidases

Summary During tempeh fermentation, Rhizopus oligosporus produced polysaccharidases to degrade soya bean cell walls; the maximum activity for all polysaccharidases tested occurred 20–30 h after inoculation. R. oligosporus was also grown in a soya bean extract model medium to which glycerol was added to control water activity (a _w). The overall activities of the major enzymes produced by the fungus, polygalacturonase, endocellulase and xylanase, appeared to be strongly influenced by a _w. The production of enzymes as well as their specific activities were affected by a _w. The optimum a _w for polygalacturonase and xylanase activity coincided with that for mycelial growth, namely 0.99–1.00. In contrast, the optimum a _w for (endo)cellulase was 0.98, at which mycelial growth was significantly reduced. Correspondence to: M. Sarrette     

The water content/water activity relationship of cured tobacco and water relations of associated spoilage fungi

The relationship between moisture content and water activity (a_w) in cured tobacco was significantly influenced by sugar content. Overall, high sugar tobaccos such as Oriental and Virginia had a higher moisture content at any given water activity compared to low sugar tobaccos such as Burley. Virginia and Burley were both predominantly colonised by Aspergillus and Penicillium spp. Of these, about 80% of isolates could germinate at between 0·75 and 0·85 a_w, equivalent to moisture contents of between 18% and 24% in Burley and between 22% and 31% in Virginia. Growth of the dominant Aspergillus and Penicillium spp. was much slower on Virginia and Burley tobacco extract than on malt extract agars over the range 0.85 to 0.98 a_w. For some species the optimum a_w for growth on tobacco extract medium was altered from that on the richer malt extract agar and for some there was also a significant difference in growth between Virginia and Burley extract agars. The mould-free storage periods for five different tobacco types was influenced by a_w. Visible moulding occurred within 7–14 days at 0.85–0.90 a_w but only after about six months at 0.70–0.75 a_w. There were some differences in rate of moulding between tobacco types as well as in the range of fungi isolated at different a_w storage levels.     

Changes in fatty acid composition and degree of unsaturation of (brady)rhizobia as a response to phases of growth, reduced water activities and mild desiccation

The effects of growth phase, reductions in the water activity (a _w) of the growth medium and mild desiccation on the composition and the degree of unsaturation of cellular fatty acids (CFA) of Sinorhizobium meliloti, Bradyrhizobium elkaniiand Bradyrhizobium japonicum were studied. During the course of growth, an interchange of cis-vaccenic with lactobacillic acid and a slight increase in palmitic acid were observed while other fatty acids remained constant. The degree of unsaturation was significantly higher in the exponential phase of growth. Reductions in the a _w of the medium led to an increase in lag phase, a reduction in growth rate and maximal optical densities (OD) in stationary phase cells. A decrease in the degree of unsaturation of CFA was also observed as the a _w was reduced from 0.999 to 0.969 and after desiccation to 83.5% relative humidity (R.H.). The changes in the degree of unsaturation of CFA observed after growth at reduced a _w may be one of the pre-adaptation steps to endure more severe desiccation.     

Comparison of growth and aflatoxin B1 production profiles of Aspergillus flavus strains on conventional and isogenic GM-maize-based nutritional matrices

Maize grown in both North and South America are now predominantly genetically modified (GM) cultivars with some resistance to herbicide, pesticide, or both. There is little information on the relative colonisation and aflatoxin B₁ (AFB₁) production with maize meal-based nutritional matrices based on kernels of non-GM maize and isogenic GM-ones by strains of Aspergillus flavus. The objectives were to examine the effect of interacting conditions of temperature (25–35 °C) and water availability (0.99–0.90 water activity, a_w) on (a) mycelial growth, (b) AFB₁ production and (c) develop contour maps of optimum and marginal conditions of these parameters for four strains of A. flavus on three different non-GM and isogenic GM-maize based nutritional media. The growth of the four strains of A. flavus (three aflatoxigenic; one non-aflatoxigenic) was relatively similar in relation to the temperature × a_w conditions examined on both non-GM and GM-based matrices. Optimum growth overall was at 30–35 °C and 0.99 a_w for all four strains. Under water stress (0.90 a_w) growth was optimum at 35 °C. Statistically: non-GM, GM cultivars, temperature and a_w all significantly affected growth rates. For AFB₁ production, all single and interacting factors were statistically significant except for non-GM × GM cultivar. In conclusion, colonisation of GM- and non-GM nutritional sources was similar for the different A. flavus strains examined. The contour maps will be very useful for understanding the ecological niches for both toxigenic and non-toxigenic strains in the context of the competitive exclusion of those producing aflatoxins.     

Adaptation of Pseudomonas fluorescens to Low Levels of Water Activity Produced by Different Solutes

A culture of Pseudomonas fluorescens which grew at a minimal a_w of 0.97 in a glucose-salts broth adjusted to that level with NaCl, KCl, or glucose, and at a_w 0.9725 with Na₂SO₄, was adapted to grow at lower a_w levels produced by these solutes. Adaptation to growth at a low a_w produced by means of one solute did not necessarily result in growth at that a_w when another solute was employed to lower the a_w. The culture adapted to grow at a_w 0.9650 produced by addition of glucose could grow equally well in broth adjusted to that a_w with NaCl or KCl, but not Na₂SO₄. NaCl and KCl permitted growth at the lowest a_w level (0.9625), and the culture adapted in KCl did equally well in NaCl.     

Ecophysiological differences between saprotrophic and clinical strains of the microscopic fungus Aspergillus sydowii (Bainier & Sartory) Thom & Church

A number of ecophysiological differences were shown for saprotrophic and clinical strains of the potentially pathogenic microscopic fungus Aspergillus sydowii. The colony growth rates were determined for four saprotrophic and five clinical fungus strains on Czapek medium within the ranges of temperature (5, 10, 15, 20, 30, 35, 37, 40, 42°C) and humidity (0.8, 0.85, 0.9, 0.95, 099 a_w), as well as on media with other sources of organic matter (Sabouraud medium, Hutchinson medium with cellulose, and water agar). The capacity for growth of A. sydowii strains on a broad spectrum of organic substrates was determined with the EKOLOG method for multisubstrate testing. The clinical and saprotrophic strains of A. sydowii differed in the colony growth rates under the same temperature and humidity combinations, as well as in the capacity for growth on different organic substrates. At decreased water activity (0.90–0.85 a_w), the temperature interval for growth of the saprotrophic strains was narrower (30 ± 2°C) than for the clinical strains (25–30°C). Comparison of growth on different media revealed the highest growth rates of the clinical strains on Sabouraud protein-containing medium. The method of multisubstrate testing showed that the saprotrophic strains grew on sugars better than the clinical ones.     

Comparison of growth,nutritional utilisation patterns,and niche overlap indices of toxigenic and atoxigenic Aspergillus flavus strains

The effects of temperatures (20–30 °C) and water activity (0.90–0.99 a_w) on the lag phase duration, mycelial growth, and nutritional utilisation patterns of two toxigenic (AFL1⁺ & AFL2⁺) and three atoxigenic (AFL1⁻, AFL2⁻, & AFL3⁻) Aspergillus flavus strains were evaluated in vitro. Both temperature and a_w and their interactions had a significant influence on the growth and nutritional utilisation patterns (p < 0.05). There were no significant differences between toxigenic and atoxigenic strains in terms of lag phase prior to growth and mycelial growth rates. Based on carbon source (CS) utilisation patterns, toxigenic and atoxigenic strains' niche size was greater at higher temperatures and in wetter conditions. Additionally, based on niche overlap indices (NOIs), regardless of temperature, when water was freely available, atoxigenic and toxigenic strains co-existed. However, under moisture stress, the nutritional competitiveness was variable. Temporal carbon utilisation sequences (TCUS) of toxigenic and atoxigenic strains were compared. At 0.99 a_w most CS sources were utilised by the strains and the time to detection (TTD) of each strain was shortest on monosaccharides at the same level of a_w. Conversely, under moisture stress the least number of CS was utilised. The current study has demonstrated that carbon utilisation patterns are equally important as are other determinants of competitiveness and that growth rate alone is not a key attribute which determines competitiveness.     

Effect of water activity and protective solutes on growth and subsequent survival to air-drying of Lactobacillus and Bifidobacterium cultures

Probiotic cultures of Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei and Lactobacillus acidophilus were grown in media having water activities (a _w) adjusted between 0.99 and 0.94 with NaCl or with a mixture of glycerol and sucrose in order to find conditions of osmotic stress which would still allow for good growth. Cultures grown at a _w?=?0.96 or 0.99 were then recovered by centrifugation, added to a sucrose–phosphate medium and air-dried. In some assays, a 2-h osmotic stress was applied to the cell concentrate prior to air-drying. Assays were also carried out where betaine, glutamate and proline (BGP) supplements were added as protective compounds to the growth or drying media. For most strains, evidence of osmotic stress and benefits of BGP supplementation on growth occurred at a _w?=?0.96. Growing the cells in complex media adjusted at a _w?=?0.96 did not enhance their subsequent survival to air-drying, but applying the 2-h osmotic stress did. Addition of the BGP supplements to the growth medium or in the 2-h stress medium did not enhance survival to air-drying. Furthermore, addition of BGP to a sucrose–phosphate drying medium reduced survival of the cultures to air-drying. This study provides preliminary data for producers of probiotics who wish to use air-drying in replacement of freeze-drying for the stabilization of cultures.     

Water relations of ethanol fermentation by Saccharomyces cerevisiae: Glycerol production under solute stress

The effect of water activity (a_w) upon glycerol production during ethanol fermentation by Saccharomyces cerevisiae was studied in continuous culture using sorbitol to adjust the a_w to 0.971. A reduction of the a_w of the medium resulted in more glycerol being produced. The glycerol yield was 3–4-fold greater at 0.971 a_w than 0.994 a_w. Ethanol yield and specific rate of ethanol production was slightly greater at 0.971 than 0.994 a_w whereas the biomass concentration was lower. Reduced a_w increased the specific rate of glucose uptake and the maintenance requirements of the organism. The results suggest that the effect of a_w on glycerol production can be reduced by controlled feeding of substrate during fermentation.     

Effects of Temperature, Water Activity, and Syrup Film Composition on the Growth of Wallemia sebi: Development and Assessment of a Model Predicting Growth Lags in Syrup Agar and Crystalline Sugar

We investigated the effects of temperature, water activity (a_w), and syrup film composition on the CFU growth of Wallemia sebi in crystalline sugar. At a high a_w (0.82) at both high (20°C) and low (10°C) temperatures, the CFU growth of W. sebi in both white and extrawhite sugar could be described using a modified Gompertz model. At a low a_w (0.76), however, the modified Gompertz model could not be fitted to the CFU data obtained with the two sugars due to long CFU growth lags and low maximum specific CFU growth rates of W. sebi at 20°C and due to the fact that growth did not occur at 10°C. At an a_w of 0.82, regardless of the temperature, the carrying capacity (i.e., the cell concentration at t = ∞) of extrawhite sugar was lower than that of white sugar. Together with the fact that the syrup film of extrawhite sugar contained less amino-nitrogen relative to other macronutrients than the syrup film of white sugar, these results suggest that CFU growth of W. sebi in extrawhite sugar may be nitrogen limited. We developed a secondary growth model which is able to predict colony growth lags of W. sebi on syrup agar as a function of temperature and a_w. The ability of this model to predict CFU growth lags of W. sebi in crystalline sugar was assessed.     

Water relations of xerophilic fungi isolated from prunes

The predominant spoilage fungi of dried and high-moisture prunes were members of the Aspergillus glaucus group and Xeromyces bisporus. Chrysosporium spp. were also important. At the mean pH of prune flesh (3.8) and at 25 C, X. bisporus grew at water activities (a_w) down to 0.605, and Chrysosporium fastidium grew to 0.686. Germination was always followed by growth, but within the 120-day incubation period, the minimum a_w permitting asexual sporulation was usually higher than that permitting germination. Sexual sporulation often required an even higher a_w. The water requirements of aspergilli were appreciably greater at this pH than near neutrality, no species germinating below 0.738 a_w. This was probably a consequence of a high spore-death rate during incubation at low a_w and pH.     

Effect of the sugar-beet pulp water activity on the solid-state culture of Trichoderma viride TS

Summary The growth and the sporulation of Trichoderma viride TS in relation to water activity (a _w) of sugar-beet pulp medium was studied. It was found that the maximum growth, monitored by protein production, substrate utilization and pH alteration, appeared at a _w=0.990–0.992. Optimal water activity of the medium for sporogenesis was 0.980. It was observed that both physiological phenomena appeared in narrow ranges of water activity which caused the rigorous a _w control in solid-state fermentation to be postulated.     

Effects of temperature,water activity and gas atmosphere on mycelial growth of tempe fungi Rhizopus microsporus var. microsporus and R. microsporus var. oligosporus

Rhizopus microsporus var. microsporus and var. oligosporus are used in the manufacture of various Asian fermented foods (tempe, black oncom, sufu). In view of solid-substrate fermentation (SSF) control, mycelial growth of strains of both varieties was tested for sensitivity to fluctuations of temperature, water activity and interstitial gas composition. This was achieved by measuring radial growth as well as biomass dry weight of pre-germinated microcolonies on defined media. The optimum conditions were temperature 40 °C, a _w 0.995 and a gas composition of air for the growth of both strains on a model medium. Whereas radial growth rates of var. microsporus and var. oligosporus were similar, biomass growth rates of var. oligosporus were higher than those of var. microsporus under optimum conditions. The temperature-dependent growth of Rhizopus spp. at a _w > 0.98 could be described by the Ratkowsky Equation. Carbon dioxide (5–10% v/v) inhibited the growth of Rhizopus spp. at non-limiting levels of oxygen. The two strains were able to grow at low (0.5% v/v) oxygen levels, but the mycelial density was rather low. No interrelation of water activity and gas composition was observed, but at high water activity the fungi were more sensitive to changes of temperature. The implications for process control are discussed. This revised version was published online in November 2006 with corrections to the Cover Date.     

Survival and Filamentation of Salmonella enterica Serovar Enteritidis PT4 and Salmonella enterica Serovar Typhimurium DT104 at Low Water Activity

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a_w). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a_w for long periods, but minimum humectant concentrations of 8% NaCl (a_w, 0.95), 96% sucrose (a_w, 0.94), and 32% glycerol (a_w, 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a_w, incubation at 37°C resulted in more rapid loss of viability than incubation at 21°C. At a_w values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 μm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a_w conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a_w highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a_w (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a_w storage. If Salmonella strains form filaments in food products that have low a_w values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.