Characterization of the growth and sporulation behavior of Penicillium roquefortii in solid substrate fermentation by material and bioenergetic balances

When Pencillium roquefortii is grown on two kinds of buckwheat, exhibiting a low [0.85 g water/g dry matter (DM), buckwheat A] and a high [1.5 g water/g initial dry matter (IDM), buckwheat B] water content, a marked difference in the mode of development of the fungus is observed. Material balances for buckwheat A show that growth does not stop because of nutrient exhaustion. Analysis of water balance shows that active growth proceeds with a permanent limitation by the turgor potential which disappears when the water activity of the substrate is close to 0.96, thus arresting growth. This limitation causes intensive water excretion from the system due to the lowering of the water activity of the substrate. The water content of the mycelium thus decreases from 79% at the beginning of the cultivation to 74% when the growth stops. This is linked to a substantial oxidative metabolism and a high sporulation efficiency, close to 0.85. The spores obtained have a low dry weight and a reduced nitrogen content. In the case of buckwheat B, the active growth is shown to stop because of available mineral nitrogen depletion. No significant decrease in the water activity of the substrate is found during the protein synthesis, and the turgor potential remains high at the end of this period. Culture proceeds with new wall synthesis; the sporulation efficiency remains high and the spores obtained exhibit a high dry weight and a high nitrogen content. The bioenergetic balances show that the P/O ratio varies with the kind of substrate used; its value is close to 1.56 for the low water medium and to 2.21 for the high one. The ATP yield Z is always close to 1, and fungal development occurs with limitations of both anabolism and catabolism on buckwheat B and only of anabolism and catabolism on buckwheat B and only of anabolism on buckwheat A.     

Water and water activity in the solid state fermentation of cassava starch by Aspergillus niger

Summary During the solid state fermentation (SSF) of cassava starch by Aspergillus niger estimations were made of total water, consumed water and the residual water remaining in small quantities after 23 h. A theoretical calculation based on the Ross equation showed that the water activity (a _w) of the substrate decreased to 0.85 towards the end of the culture. Such low values were assumed to be inhibitory to growth. The a _w of the substrate was increased when sugarcane bagasse was used as a high water retention capacity support. Higher growth rates and substrate conversion to biomass were obtained with this system, confirming that water availability is a critical factor in the SSF of starch substrates.Abbreviations A, B Experimental constants - a _w Water activity - H₂O_c Consumed water - H₂O_R Residual water - H₂O_T Total water - IDW Initial dry weight - IMC Initial moisture content - OUR Oxygen uptake rate - S Substrate dry weight - Sc Substrate conversion: consumed substrate/initial substrate - S _H Amount of sugars hydrolysed - SSF Solid state fermentation - X Biomass dry weight - W ^* Amount of solids/g of water     

Survival of alginate-entrapped cells of Azospirillum lipoferum during dehydration and storage in relation to water properties

Survival of alginate-entrapped cells of Azospirillum lipoferum was studied during dehydration using a dry air stream and during prolonged storage at various constant water activity values (a_w). During the drying operation, the viability loss remained almost constant from the initial water content to 0.35 g water/g dry weight (DW) corresponding to a 98.5% water removal, strongly increased until a water content of 0.25 g/g DW and then stopped until the end of the drying operational (final a_w 0.18). A water content of 0.25 g/g DW (a_w=0.55) corresponded to the critical point of the moisture sorption isotherm curve from which water became restricted to the dry material. A high drying rate (5 g/g DW per hour) was shown to be more detrimental for cell viability than a low drying rate (1.18 g/g DW per hour). When the product was stored in a closed chamber with a regulated a_w (0.23), the number of living cells decreased during a short period (less than 15 days) corresponding to the product a_w stabilization, and then remained constant for more than 150 days. In addition, cell survival during storage was not affected by a_w values in the range 0–0.55. Above a_w=0.55, the higher the a_w and the storage duration, the lower the residual survival percentage. The influence of the drying and storage conditions on the cell death rate are discussed with regard to both the mechanisms generally involved in viability loss and the hydration properties of water. Correspondence to: A. Pareilleux     

Effect of water activity and relative air humidity on the growth of Penicillium chrysogenum thom, Aspergillus repens (Corda) Sacc., and Trichoderma viride Pers. isolated from living spaces

Original data on the growth parameters of the fungi Penicillium chrysogenum Thom, Aspergillus repens (Corda) Sacc., and Trichoderma viride Pers. isolated from living spaces in Moscow are presented. Spore germination, fungal growth, and the radial growth rate of the colonies were investigated upon cultivation on agarized nutrient media with different water activity (a_w) values. Spore germination and fungal growth were studied in house dust under laboratory conditions at different relative air humidity (RH). It was shown that, at decreased a_w and RH, the spore germination time increased, as did the period from germination to mycelium and conidia formation, while the radial growth rate of colonies decreased. House dust was found to be a suitable growth substrate for A. repens and P. chrysogenum, supporting their complete life cycle. It was suggested that house dust is unsuitable as a substrate for the growth of T. viride. The a_w and RH ranges for development of these micromycetes were determined. On this basis, the A. repens, P. chrysogenum, and T. viride strains isolated from living spaces were identified as xerophilic, xerotolerant, and hygrophilic ones, respectively.     

Optimization of the spore production of Penicillium roqueforti in solid substrate fermentation on buckwheat seeds

Summary The effect of substrate (buckwheat seeds) pretreatment on the growth and the sporulation behaviour of Penicillium roqueforti is presented. When a saccharifying enzyme (-amylase) is added to a medium which exhibits a low water content (0.46 g water/g initial dry matter, IDM), a more rapid internal colonization of the seeds occurs, but the final spore production does not increase and remains close to 8.10⁹ spores/g dry matter (DM) at 500 h. No carbon source limitation is then observed. The addition of casein hydrolysate to this medium gives rise to a great increase of the sporulation, since 14.5 10⁹ spores/g DM are obtained after 600 h. This result is attained by a better spore yield from the mycelium, the substrate colonization being unchanged. High water content (0.60 g water/g IDM) of buckwheat seeds induces a shorter cultivation time along with a higher biomass production. However, the spore content of the medium remains close to the low water content one, but 60% total spores are external against 30% to 35% in the other media.     

Changes in protein secretion of Aspergillus niger caused by the reduction of the water activity by potassium chloride

The effect of the rapid reduction of the water activity (a_w) on the extracellular protein and amylolytic activity of Aspergillus niger was studied. An a_w value gradient from 0.90 to 0.99 in KCl solutions was applied for the mycelium treatment. It was found that the a_w reduction considerably influenced the protein secretion. This phenomenon was dependent on the age of the treated mycelium and the range of the a_w gradient. The highest protein and enzyme secretion yields were obtained at a_w = 0.98 using a 72-h old mycelium. In comparison with the non-treated mycelium, the increase in the secretion amounted to about 60% for the amylolytic activity and 37% for the soluble protein, respectively. It was shown that the mycelium incubated in KCl solutions of an a_w value from 0.90 to 0.99 had the ability for regeneration in fresh CZAPEK-DOX medium. The effect of the osmotic shock on the protein secretion was limited only for the treated cell population and declined in the mycelium which was regenerated after the transfer into the culture medium.     

Determination of the strength of the cell walls of vegetative cells ofBacillus megaterium andBacillus stearothermophilus

The tensile strength of the cell walls ofBacillus megaterium andBacillus stearothermophilus was found to be about 2.4×10⁷ N/m². The internal pressure and water activity of the cells were 14 atm, 0.99 a_w forB. megaterium and 28 atm, 0.98 a_w forB. stearothermophilus. The greater strength ofB. stearothermophilus cells, considered as pressure vessels, restricts absorption of water by the protoplasm so that the water content on a dry weight basis is 3.4 g/g forB. megaterium cells in water but only 1.8 g/g forB. stearothermophilus.     

Survival of Bacteria and Fungi in Relation to Water Activity and the Solvent Properties of Water in Biopolymer Gels

Survival of bacteria (Rhizobium, Agrobacterium, and Arthrobacter spp.), fungal spores (Penicillium sp.), and yeasts (Saccharomyces sp.) was studied in relation to water activity (a_w) and the presence of nutritive solutes. The cells were entrapped in polysaccharide gels, as is done to immobilize cells or enzymes, and then dehydrated. The number of living cells (10¹⁰ g of dry polymer⁻¹) remained constant for periods of storage of >3 years at 28°C when the inocula were kept at an a_w of <0.069. At a_w values between 0.069 and 0.83 the number of survivors diminished more and more rapidly as the a_w was raised. For a given a_w and organism, there were large differences in survival rate as a function of the nutritive solutes used to culture the microorganisms. Low-molecular-weight compounds (with three or five carbon atoms) had a deleterious effect on survival, whereas compounds of higher molecular weight (C₆ to C₁₂) had a protecting effect. Thus, the a_w alone was not a sufficient explanation for the deterioration of the inocula. Survival seemed to be more directly related to some properties of the water in the biopolymer. New concepts such as the discontinuity of properties of water and the point of mobilization of solutes, already proposed by Duckworth and Kelly (J. Food Technol. 8:105-113, 1973) and Seow (J. Sci. Food Agric., 26:535-536, 1975), have been taken into consideration to explain the interactions of water with the biopolymer and their specific effects on the microorganisms.     

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.     

Continuous fermentation to produce fungal protein. Effect of growth rate on the biomass yield and chemical composition of Fusarium moniliforme

Fusarium moniliforme was grown on a carob aqueous extract in a chemostat for fungal protein production. The substrate was adjusted to provide 0.5% carob sugars supplemented with inorganic salts. The dilution rate varied from 0.086 to 0.227 hr^?1 under constant conditions of temperature (30°C), pH (4.5), and oxygen saturation (60–80%). A yield of 0.709 g dry mycelium/g consumed carob sugar and a productivity value of 0.687 g dry mycelium/liter hr^?1 were obtained at μ = 0.205 hr^?1. The maintenance coefficient was 0.077 g carob sugar/g dry mycelium hr^?1. While the carbohydrate and purine content of dry mycelium increased at μ values from 0.114 to 0.205 hr^?1 both true (Lowry) and crude (N × 6.25) protein contents decreased at the same μ range. Maximum values of 36.3% true and 47.9% crude protein of dry mycelium were obtained at μ = 0.114 hr^?1, whereas a minimum purine content of 99.8 μmol/g corresponding to 6.42% nucleic acids was recorded at μ = 0.086 hr^?1. It was concluded that a continuous fermentation of carob aqueous extract using F. moniliforme should be operated at growth rates of approximately 0.205 hr^?1 in order to maximize protein production.     

Factors determining the microflora of stored barley grain

Colonisation of barley grain has been studied during storage at different water contents and with and without restriction of the air supply to simulate conditions in unsealed silos. Grain stored with a water activity >0·9 a_w (20% water content) heated spontaneously when aeration was unrestricted, the maximum temperature attained increasing with a_w to 58 °C at 1·0 a_w (39% water content). The presence of many unripe grains increased the tendency to heat at a given mean water content. Although heating was prevented by sheeting to restrict the air supply, it could occur subsequently when the sheeting was removed. Both heating and restriction of the air supply were associated with increased carbon dioxide (to >25%) and decreased oxygen concentrations (to <5%). Germination of grain after 6·9 months storage was correlated with a_w; germination levels approaching 100% were retained only at about 0·7 a_w (13·5% water content). Colonisation by Aspergillus species was correlated with a_w and temperature and similar correlations with Penicillium species were also found, with P. verrucosum var. cyclopium abundant at 0·85-0·90 a_w (17·20% water content) and P. piceum, P. funiculosum and P. capsulatum at 0·90-0·95 a_w (20–25% water content). In sealed containers P. roquefortii occurred at 1·00 a_w (39% water content) and P. hordei at 0·90-0·92 a_w (20–22% water content). Spores of fungi and actinomycetes formed during spontaneous heating of grain survived 6 months sealed storage although Absidia corymbifera and Micropolyspora faeni may have declined in numbers. Fungicides applied to the ripening grain had only limited effect on the colonisation of the grain during storage.     

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.     

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 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.     

Water balance of over-wintering beetles in relation to strategies for cold tolerance

The vapour pressure of the haemolymph of a supercooled insect is higher than the vapour pressure of the haemolymph of a frozen insect at the same temperature. The aim of the study was to see whether this may affect the water loss of freeze-avoiding and freezetolerant, over-wintering beetles. The rates of water loss were determined on freeze-tolerant Pytho depressus larvae and Upis ceramboides adults. Within each species one group was kept supercooled whereas another group was frozen. All groups were incubated at-5°C. Both species displayed significantly lower rates of water loss when they were frozen than when they were supercooled. Values of respiratory rate and water loss of freeze-avoiding and freeze-tolerant species were compared to corresponding values of desert beetles. The results indicate that the freeze-avoiding species have lower rates of cuticular water loss than freeze-tolerant species. This indicates that the freeze-avoiding species have developed more efficient water-saving mechanisms than freeze-tolerant species. The reason for this may be that the haemolymph in frozen animals will be in vapour pressure equlibrium with the ice in the hibernaculum and thus there is no danger of desiccation during winter. The supercooled insects will have a vapour pressure of the haemolymph that is higher than the vapour pressure of water in the surrounding air and will thus lose water.Abbreviations BW body weight - BW_i initial body weight - BW_t body weight at time t - P vapour pressure difference between the water in the haemolymph and the water in the air - DWL_t dry weight loss at time t - M _w rate of metabolic water production - MF_w mol fraction of water, in the haemolymph - MO₂ rate of oxygen consumption - Osm osmolality of the haemolymph - P _a vapour pressure of water in the air - P _h vapour pressure of water in the haemolymph - P _w vapour pressure of pure water - Q a constant (2,02 1 oxygen per g fat metabolized) relating oxygen consumption to dry weight loss when fat is metabolized - R a constant (1,89 1 oxygen per g water produced) relating metabolic water production to oxygen consumption when fat is metabolized - R _dwl rate of dry weight loss - RH relative humidity of the air - RWC_i initial relative water content measured by weighing - RWC_t relative water content at time t - STP standard temperature and pressure     

Relation of the Heat Resistance of Salmonellae to the Water Activity of the Environment

The effect of water activity (a_w) on the heat resistance of eight strains of Salmonella was studied. Heat resistance of the organisms increased as the a_w of the heating menstruum was reduced. Sucrose afforded the cells a greater degree of protection than did fructose, glycerol, and sorbitol. A direct correlation between a_w and heat resistance could not be established over the range of a_w levels tested in this study. There was variation among the strains of salmonellae in the magnitude of the increase in heat resistance as the a_w level was reduced. All strains of Salmonella tested showed a greater increase in heat resistance than S. senftenberg 775W as the environment became drier. Washed cells had D values 25 to 75% lower than unwashed cells. Prior growth of the organisms in media with a reduced a_w increased the heat resistance of the organisms when glycerol, but not when sucrose, was the controlling substance.     

Pressure treatment of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in low-moisture environments

We investigated the influence of cell hydration on the ability of Saccharomyces cerevisiae CBS 1171 to withstand extreme hydrostatic pressure in order to determine the mechanisms involved in cell resistance. Hydration conditions were modified in two different ways. We first modulated the chemical potential of water by adding glycerol in cell suspensions. Another procedure consisted in dehydrating cells aerobically and immersing them in perfluorooctane, an innocuous hydrophobic liquid used as a pressure-transmitting medium, prior to pressure treatments. This original method made it possible to transmit isostatic pressure to yeast powders without changing the initial water activity (a _w) level at which cells had been equilibrated. The a _w ranged between 0.11 and 0.99. Pressure treatments were applied at levels of up to 600 MPa for 10 min, 24 h, and 6 days. The dehydration of cells was found to strongly limit, or even prevent, cell inactivation under pressure. Notably, cells suspended in a water–glycerol mixture with a _w levels of 0.71 or below were completely protected against all pressure treatments. Moreover, cells dehydrated aerobically survived for 6 days at 600 MPa even when a _w levels were relatively high (up to 0.94). We highlighted the crucial role of water content in determining cellular damage under pressure. When water is available in a sufficient amount, high pressure induces membrane permeabilization, causing uncontrolled mass transfers that could lead to death during a prolonged holding under pressure. Possible mechanisms of membrane permeabilization are discussed.     

Influence of water activity on the enzyme biosynthesis and enzyme activities produced by Trichoderma viride TS in solid-state fermentation

Influence of water activity (a_w) on biosynthesis of polygalacturonase, d-xylanase and β-glucosidase in solid culture system of Trichoderma viride TS was studied. It was found that the production of enzymes was strongly affected by water activity of substrate and nature of a_w depressor used. The polygalacturonase and d-xylanase production were maximized at a_w = 0.995 whereas β-glucosidase formation was favored at a_w = 0.96–0.98. The influence of water activity on catalytic effect of enzymes using sodium chloride, glycerol and sorbitol as a_w depressor was also investigated. It was observed that sorbitol improved the thermal stability of polygalacturonase and d-xylanase.     

Influence of age of mycelium and water activity of the medium on aroma production by Trichoderma viride grown on solid substrate

The production of 2-heptanone (cheese aroma) by Trichoderma viride TS cultivated on agar media was evaluated using headspace gas chromatographic analysis. The radial growth rate of the filamentous fungi increased with high water activity values, but the mycelial density was higher for lower water activity. Maximum aroma production of a culture was obtained at a_w = 0.96. An apparatus intended to measure the aroma production of different areas of a mycelial colony was set up. The study of the aroma production of these areas showed that the production values were greatly different and were evolving with time. It was shown that the mycelium aroma production was maximum when mycelia were about 3.5 to 6.5 d old.     

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.