Is there a common water-activity limit for the three domains of life?

Archaea and Bacteria constitute a majority of life systems on Earth but have long been considered inferior to Eukarya in terms of solute tolerance. Whereas the most halophilic prokaryotes are known for an ability to multiply at saturated NaCl (water activity (a(w)) 0.755) some xerophilic fungi can germinate, usually at high-sugar concentrations, at values as low as 0.650-0.605 a(w). Here, we present evidence that halophilic prokayotes can grow down to water activities of <0.755 for Halanaerobium lacusrosei (0.748), Halobacterium strain 004.1 (0.728), Halobacterium sp. NRC-1 and Halococcus morrhuae (0.717), Haloquadratum walsbyi (0.709), Halococcus salifodinae (0.693), Halobacterium noricense (0.687), Natrinema pallidum (0.681) and haloarchaeal strains GN-2 and GN-5 (0.635 a(w)). Furthermore, extrapolation of growth curves (prone to giving conservative estimates) indicated theoretical minima down to 0.611 aw for extreme, obligately halophilic Archaea and Bacteria. These were compared with minima for the most solute-tolerant Bacteria in high-sugar (or other non-saline) media (Mycobacterium spp., Tetragenococcus halophilus, Saccharibacter floricola, Staphylococcus aureus and so on) and eukaryotic microbes in saline (Wallemia spp., Basipetospora halophila, Dunaliella spp. and so on) and high-sugar substrates (for example, Xeromyces bisporus, Zygosaccharomyces rouxii, Aspergillus and Eurotium spp.). We also manipulated the balance of chaotropic and kosmotropic stressors for the extreme, xerophilic fungi Aspergillus penicilloides and X. bisporus and, via this approach, their established water-activity limits for mycelial growth (∼0.65) were reduced to 0.640. Furthermore, extrapolations indicated theoretical limits of 0.632 and 0.636 a(w) for A. penicilloides and X. bisporus, respectively. Collectively, these findings suggest that there is a common water-activity limit that is determined by physicochemical constraints for the three domains of life.

The extreme xerophilic mould Xeromyces bisporus--growth and competition at various water activities.

Little is known about the mould, Xeromyces bisporus, unique in its strong xerophilicity and ability to grow at water activity (a(w)) 0.62, lower than for any other known organism. The linear growth rates of one fast and one slow-growing strain of X. bisporus were assessed at 20, 25, 30 and 37 °C on solid agar media containing a mixture of glucose and fructose to reduce a(w) to 0.94, 0.88, 0.84, 0.80, 0.76 and 0.66. Growth rates of xerophilic species closely related to X. bisporus, viz. Chrysosporium inops, C. xerophilum and Monascus eremophilus, were also assessed. Optimal conditions for growth of both X. bisporus strains were approx. 0.84 a(w) and 30°C, despite FRR 2347 growing two- to five-fold faster than CBS 185.75. X. bisporus FRR 2347 even grew well at 0.66 a(w) (0.48 mm/day). C. inops and C. xerophilum were more tolerant of high a(w) than X. bisporus, and could be differentiated from each other based on: the faster growth of C. xerophilum; its preference for temperatures ≥ 30 °C and a(w) ≥ 0.94 (c.f.≤ 25 °C and ~0.88 a(w) for C. inops); and its ability to grow at 0.66 a(w), which is the lowest a(w) reported to date for this species. M. eremophilus grew slowly (max. 0.4mm/day) even in its optimal conditions of ~0.88 a(w) and 25 °C. To investigate the competitive characteristics of X. bisporus at low a(w), both X. bisporus strains were grown in dual-culture with xerotolerant species Aspergillus flavus and Penicillium roqueforti, and xerophilic species A. penicillioides, C. inops, C. xerophilum and Eurotium chevalieri, on glucose-fructose agar plates at 0.94, 0.84, 0.80 and 0.76 a(w) and at 25 °C. Growth rates and types of interactions were assessed. Excretion of inhibitory substances acting over a long-range was not observed by any species; inhibitors acting over a short-range that temporarily slowed competitors' growth or produced a protective zone around the colony were occasionally observed for A. penicillioides, C. inops and C. xerophilum. Instead, rapid growth relative to the competitor was the most common means of dominance. The xerotolerant species, A. flavus and P. roqueforti were dominant over X. bisporus at 0.94 a(w). E. chevalieri was often dominant due to its rapid growth over the entire a(w) range. At a(w)<0.80, X. bisporus was competitive because it grew faster than the other species examined. This supports the concept that its ideal environmental niche is sugary foods with low a(w).

Effect of reduced water activity and reduced matric potential on the germination of xerophilic and non-xerophilic fungi.

Reduction in water activity (a(w)) is used as a microbiological hurdle to prevent food spoilage. To minimize the levels of salt and sugar, which are commonly used to reduce a(w), the potential of food structure as a microbiological hurdle needs to be assessed. The concept of matric potential (Psi(m)) is used to measure the effect of food structure on water movement. This study reports the effect of reduced a(w) and reduced Psi(m) on the germination of xerophilic fungi (represented by Eurotium herbariorum) and non-xerophilic fungi (represented by Aspergillus niger) on model glycerol agar media. Germination curves were plotted with the percentage of germinated spores against time. The germination time (t(G)), which is defined as the time at which 50% of the total viable spores have germinated, was estimated using the Gompertz model. Total viable spores was defined as those spores that were able to germinate under the optimum a(w) and Psi(m) conditions for each species, i.e. 0.95 a(w) and 2.5% agar for E. herbariorum and 0.98 a(w) and 2.5% agar for A. niger. As a(w) decreased from 0.90 to 0.85 a(w), t(G) increased significantly for both the xerophilic fungi and non-xerophilic species at equivalent matric potential values. When matric potential was reduced from -12 kPa (2.5% agar) to -38 kPa (12.5% agar), t(G) of A. niger was significantly extended at 0.90 a(w); however, t(G) remained the same for A. niger at 0.85 a(w), and for E. herbariorum at 0.80, 0.85 and 0.90 a(w). This study demonstrated that the germination time for non-xerophilic and xerophilic fungi was extended by reduced a(w), however the effect of reduced Psi(m) was limited.

Evaluation of the efficacy of four weak acids as antifungal preservatives in low-acid intermediate moisture model food systems.

The potential efficacy of four weak acids as preservatives in low-acid intermediate moisture foods was assessed using a glycerol based agar medium. The minimum inhibitory concentrations (MIC, % wt./wt.) of each acid was determined at two pH values (pH 5.0, pH 6.0) and two a(w) values (0.85, 0.90) for five food spoilage fungi, Eurotium herbariorum, Eurotium rubrum, Aspergillus niger, Aspergillus flavus and Penicillium roqueforti. Sorbic acid, a preservative commonly used to control fungal growth in low-acid intermediate moisture foods, was included as a reference. The MIC values of the four acids were lower at pH 5.0 than pH 6.0 at equivalent a(w) values, and lower at 0.85 a(w) than 0.90 a(w) at equivalent pH values. By comparison with the MIC values of sorbic acid, those of caprylic acid and dehydroacetic acid were generally lower, whereas those for caproic acid were generally higher. No general observation could be made in the case of capric acid. The antifungal activities of all five weak acids appeared related not only to the undissociated form, but also the dissociated form, of each acid.

Effect of agar concentration on the matric potential of glycerol agar media and the germination and growth of xerophilic and non-xerophilic fungi.

The concept of water activity (a(w)) does not differentiate between water status resulting from the interaction of water with solutes, and that from interaction of water with matrices, which is termed matric potential (psi(m)). This study reports the effect of agar concentration (1.5, 3.0, 4.5, 6.0, and 7.5%, w/w) on matric potential of glycerol agar media (GA) and the germination and growth of xerophilic fungi (Eurotium herbariorum and E. rubrum) and non-xerophilic fungi (Aspergillus niger, A. flavus, and Penicillium roqueforti) on GA with or without sorbic acid (0.1-0.4%, w/w) at 0.90 a(w) and 0.95 a(w). The matric potential of GA decreased when the agar concentration increased from 1.5 to 7.5%. When the agar concentration increased at each a(w), the radial growth rate of the xerophilic fungi generally increased but the biomass density (biomass per unit area) decreased, whereas the radial growth rate of the non-xerophilic fungi generally decreased but the biomass density was unchanged. In the absence of sorbic acid, the time to germination of each species was similar for all agar concentrations. In the presence of sorbic acid, the time to germination of some species was significantly longer at higher agar concentrations (4.5%-7.5%) than 1.5% agar. This study demonstrated the inhibition effect on germination and growth of non-xerophilic fungi and xerophilic fungi by decreased matric potential resulting from increased agar concentrations, and the different responses of non-xerophilic fungi and xerophilic fungi to water stress from solutes and matrices. The concept of matric potential may be useful in food microbiology to provide a better understanding of fungal growth in complex food matrices.

Inactivation of food spoilage fungi by ultra violet (UVC) irradiation.

The effect of ultraviolet irradiation (254 nm, UVC) on Aspergillus flavus, Aspergillus niger, Penicillium corylophilum and Eurotium rubrum was investigated using three different exposure techniques. Survival was determined for spores suspended in liquid medium after 1, 2 and 3 min UVC exposure at 4644 J/m(2)/min. The same UVC dose was applied to spores on the surface of agar plates for 5, 10, 15, 30, 60 and 120 s. Spores of A. niger were dried onto a membrane filter, then exposed to UVC treatment. In the liquid medium, treatments from 1-3 min significantly (P<0.001) reduce the number of viable spores. On the surface of agar plates, after a 15 s exposure, a 80-99% reduction of viable spores was observed for all species except A. niger, for which the reduction was only 62%. For spores dried onto filter membranes, a 3.5 log(10) reduction was achieved for A. niger after 180 s exposure. These observations suggest that UVC irradiation can effectively inactivate spores of A. flavus, P. corylophilum, E. rubrum and A. niger but the efficacy of UVC radiation against fungal spores varies significantly according to methods of exposure to the irradiation, and among genera.

Utility of microsatellite markers and amplified fragment length polymorphism in the study of potentially ochratoxigenic black aspergilli.

Microsatellite markers and the results of amplified fragment length polymorphism (AFLP) were compared in the characterization of 68 Aspergillus carbonarius and A. niger aggregate strains of differing ochratoxin-producing ability and from different geographic areas, isolated mainly from grapes and soil. AFLP was applied to both A. carbonarius and A. niger aggregate strains, and it clearly differentiated these species. Microsatellite markers were only applied to A. niger aggregate strains because of the species-specific nature of these markers. Both AFLP and microsatellite marker analyses were able to divide A. niger aggregate strains into the two recognized internal transcribed spacer (ITS)-5.8S rDNA RFLP types, N and T. Clustering of A. niger aggregate strains was similar in both AFLP and microsatellite analyses, yielding an additional separation of N type strains into two groups. Both microsatellite marker and AFLP analyses showed high levels of polymorphism in the A. niger aggregate (index of discriminatory power 0.991 and 1.0, respectively). Of the two techniques, microsatellite marker analysis was quicker and more straightforward to perform. In addition, microsatellite marker analysis is more reproducible, and the results can be expressed as quantitative data, making microsatellite markers a good candidate for use in large-scale studies of genetic diversity in A. niger aggregate species.

Fungi and mycotoxins in vineyards and grape products.

Many fungi may occur on grapes during growth in the vineyard, but the main concern from the viewpoint of mycotoxin contamination is the black Aspergilli, Aspergillus carbonarius and A. niger. These fungi are capable of producing ochratoxin A (OA) which may contaminate grapes and grape products such as wine, grape juice and dried vine fruit. Understanding the ecology and physiology of the black Aspergilli can provide tools for management of OA at all stages of grape production and processing. In the vineyard, careful management of cultivation, irrigation and pruning can assist in minimising the levels of black Aspergilli in the soil, which in turn, can minimise contamination of grapes by these fungi. Minimising damage to grapes on the vine by the use of open vine canopies, grape varieties with resistance to rain damage and by the management of insect pests and fungal diseases (e.g., mildew, Botrytis bunch rot) can reduce the incidence of Aspergillus rot in mature berries. The risk of OA in table grapes can be minimised by careful visual inspection to avoid damaged and discoloured berries. In wine, harvesting grapes with minimal damage, rapid processing and good sanitation practices in the winery assist in minimising OA. During vinification, pressing of grapes, and clarification steps which remove grape solids, grape proteins and spent yeast can also remove a significant proportion of OA. For dried vine fruit production, avoiding berry damage, rapid drying, and final cleaning and sorting to remove dark berries can reduce overall OA levels in finished products.

Mycotoxins in Australia: biocontrol of aflatoxin in peanuts.

The major mycotoxin problem in Australia is the formation of aflatoxins in peanuts by Aspergillus flavus and A. parasiticus. This is controlled by good farm management practice, segregation into grades on aflatoxin content at intake to shelling facilities, colour sorting and aflatoxin assays. A second problem is the potential presence of ochratoxin A in grapes and grape products, resulting from infection by Aspergillus carbonarius. Good quality control before and during wine making ensures ochratoxin A is kept to very low levels, but in dried vine fruit, ochratoxin A levels may be higher. Biocontrol by competitive exclusion has been developed as the most promising means of controlling aflatoxins in peanuts. Some details of the process are given, including some basic laboratory experiments.

Fate of ochratoxin A during vinification of Semillon and Shiraz grapes.

Semillon and Shiraz grapes containing ochratoxin A (OA) were obtained by inoculation of bunches on the vine with Aspergillus carbonarius. Citric acid content was greater in the inoculated grapes than in healthy grapes. Samples were collected throughout vinification of these grapes and the OA content was quantified using a stable isotope dilution liquid chromatographic-tandem mass spectrometric method. The mass of processed and waste streams during vinification was also noted. Reduction in the amount of OA in juice and wine occurred at every solid-liquid separation stage. The OA concentration (microg/kg) in white and red wine after racking was 4% and 9%, respectively, of that in crushed grapes. This corresponds to 1% and 6% of the total OA content that was initially present in the inoculated grapes. The OA content was divided between solid and liquid phases at each stage of vinification. OA did not appear to be transformed either chemically or biologically by yeast during fermentation, rather was discarded with the marc, juice lees, and gross lees.

Effect of temperature and water activity on growth and ochratoxin A production by Australian Aspergillus carbonarius and A. niger isolates on a simulated grape juice medium.

The effect of water activity (0.92, 0.95, 0.965 and 0.98) and temperature (15 degrees C, 25 degrees C, 30 degrees C and 35 degrees C) on growth rate and ochratoxin A (OA) production by five strains of Aspergillus carbonarius and two strains of A. niger isolated from Australian vineyards was characterised on a synthetic grape juice medium. Maximum growth for A. carbonarius occurred at ca 0.965 aw and 30 degrees C, and for A. niger, at ca 0.98 aw and 35 degrees C. The optimum temperature for OA production was 15 degrees C and little was produced above 25 degrees C. The optimum aw for toxin production was 0.95-0.98 for A. carbonarius and 0.95 for A. niger. Toxin was produced in young colonies after and, typically, did not continue to accumulate the entire surface area of the plate was colonised. Rather, the amount decreased as colonies aged. Trends for growth and OA production were similar among Australian isolates and those from European grapes, as reported in the literature.

Survival and growth of Aspergillus carbonarius on wine grapes before harvest.

Aspergillus carbonarius, the primary OTA-producing species in Australia, was inoculated onto the surface of Chardonnay and Shiraz bunches at pre-bunch closure, veraison and pre-harvest during the 2002-03 and 2003-04 seasons. Mean A. carbonarius counts decreased between pre-bunch closure and veraison, and increased between veraison and pre-harvest. Increases in A. carbonarius counts from veraison onwards were most marked in Chardonnay bunches during 2003-04; such bunches comprised more berries and were heavier than in 2002-03. Bunches with no berry damage yielded low A. carbonarius counts at pre-harvest and harvest. Exposure to direct sunlight over several days reduced viability of A. carbonarius spores supported on filter membranes by 10(5), despite the spores having thick, heavily melanised walls. The estimated cumulative UV exposure for that period was 10 mWh. Thus, UV radiation may be a contributory factor to the decline of A. carbonarius spores on berry surfaces, particularly in the early stages of berry development.

Diversity of the Trichocomaceae in the Katandra Nature Reserve, Central Coast, NSW, Australia.

The diversity of the family Trichocomaceae, which includes the major anamorph genera Aspergillus and Penicillium, was studied in the Katandra Nature Reserve, Central Coast, NSW, Australia. Soil, living leaves, leaf litter and detritus were examined by both direct and dilution plating techniques. Fungi were isolated on dichloran Rose Bengal chloramphenicol agar, and dichloran 18 % glycerol agar, media suitable for cultivation of many species within this family. Species of Trichocomaceae were isolated from all sites and all substrates examined. A high diversity was found, with more than 50 known species identified, and an equal number of undescribed species detected. More species of Penicillium were recovered than other genera, with Aspergillus species the next most common. Most of the species recovered were anamorphs, though 16 known and unknown ascosporic species were also isolated from heated and unheated soil. Soils, leaf litter, a scat from a native herbivore and leaves of living native plants yielded higher diversity than insects, worms or introduced plants. More species belonging to the family were isolated from soil in dry sclerophyll forest than in rainforest. Conversely, native rainforest plants harboured more diversity than the dry sclerophyll forest plants examined.

Tremor syndrome associated with a fungal toxin: sequelae of food contamination.

We report on an elderly couple who presented with a syndrome that included severe generalised tremor and incoordination after eating soup from a damaged can. Black mould contaminating the can was subcultured and the fungus Penicillium crustosum was identified. This fungus usually produces a potent neurotoxin called penitrem A. The couple displayed symptoms consistent with penitrem A ingestion, all of which resolved fully. Penitrem A intoxication has been well documented in animals, but not in humans.

Microbiology of wheat and flour milling in Australia.

A survey was undertaken to determine the microbiological status of Australian wheat and the distribution of microorganisms in the flour milling fractions and end products. A total of 650 milling process and end product samples was obtained from nine flour mills located in New South Wales (4), Queensland (2), Victoria (2) and Western Australia (1) during the 1997-1998 and 1998-1999 wheat seasons. Most frequent (modal) counts in wheat and flour were, respectively, as follows: aerobic mesophilic plate count, 10(5) and 10(2) colony forming units/gram (cfu/g); coliforms, 10 and 1 most probable number/gram (MPN/g); Bacillus spp., 10(4) and 10(2) cfu/g; B. cereus, 1 and 0.1 MPN/g; mesophilic aerobic spores, 10 and 1 cfu/g; aerobic thermophiles, both 10 cfu/g; yeasts, 10(3) and 10(2) cfu/g, and moulds, 10(3) and 10(2) cfu/g. Bacillus spp., coliforms, yeasts and moulds were the most frequently detected microorganisms throughout the survey. The most common moulds isolated were Aspergillus, Penicillium, Cladosporium and Eurotium spp. Environmental serovars of Salmonella were isolated from two samples. Escherichia coli and B. cereus were present at very low levels, a majority of positive samples being at the minimum level of detection (3 and 0.3 MPN/g, respectively). As wheat grain layers are separated, surface-adhering contaminants are concentrated in end product bran, wheat germ and pollard, which comprise the outer layers of the grain. Consequently, the inner endosperm fraction contains lower microbial counts, and flour is the cleanest end product of the milling process. Higher microbiological counts midstream in the milling process indicate that equipment contamination may contribute to microbiological contamination; however, the microbiological quality of incoming wheat has a strong influence on the ultimate quality of milling end products.

Influence of temperature, water activity and pH on growth of some xerophilic fungi.

The combined effects of water activity (aw), pH and temperature on the germination and growth of seven xerophilic fungi important in the spoilage of baked goods and confectionery were examined. Eurotium rubrum, E. repens, Wallemia sebi, Aspergillus penicillioides, Penicillium roqueforti, Chrysosporium xerophilum and Xeromyces bisporus were grown at 25, 30 and 37 degrees C on media with pH values of 4.5, 5.5, 6.5 and 7.5 and a range of water activities (aw) from 0.92 to 0.70. The aw of the media was controlled with a mixture of equal parts of glucose and fructose. Temperature affected the minimum aw for germination for most species. For example, P. roqueforti germinated at 0.82 aw at 25 degrees C, 0.86 aw at 30 degrees C and was unable to germinate at 37 degrees C. E. repens germinated at 0.70 aw at 30 degrees C, but at 25 and 37 degrees C, its minimum aw for germination was 0.74. C. xerophilum and X. bisporus germinated at 0.70 aw at all three temperatures. The optimum growth occurred at 25 degrees C for P. roqueforti and W. sebi, at 30 degrees C for Eurotium species, A. penicillioides and X. bisporus and at 37 degrees C for C. xerophilum. These fungi all grew faster under acidic than neutral pH conditions. The data presented here provide a matrix that will be used in the development of a mathematical model for the prediction of the shelf life of baked goods and confectionery.

Some Considerations When Analyzing Foods for the Presence of Xerophilic Fungi.

Foodborne fungi capable of growing at reduced water activity (aw) are described as xerophilic. Some xerophilic fungi will not grow or grow very slowly at aw values characteristic of media traditionally used to enumerate yeasts and molds in foods. Populations of xerophiles may therefore be underestimated or go undetected. A brief review of the habitats and physiology of xerophiles, followed by a description of media and methods available for detection, enumeration, and a key for identification are presented.