Halophilic Archaea cultured from ancient halite,Death Valley,California

Halophilic Archaea cultured from ancient fluid inclusions in a 90‐m‐long (0‐ to 100 000‐year‐old) salt core from Death Valley, California, demonstrate survival of bacterial cells in subsurface halite for up to 34 000 years. Five enrichment cultures, representing three genera of halophilic Archaea (Halorubrum, Natronomonas and Haloterrigena), were obtained from five surface‐sterilized halite crystals exclusively in one section of the core (13.0–17.8 m; 22 000–34 000 years old) containing perennial saline lake deposits. Prokaryote cells were observed microscopically in situ within fluid inclusions from every layer that produced culturable cells. Another 876 crystals analysed from depths of 8.1–86.7 m (10 000–100 000 years old) failed to yield live halophilic Archaea. Considering the number of halite crystals tested (culturing success of 0.6%), microbial survival in fluid inclusions in halite is rare and related to the paleoenvironment, which controls the distribution and abundance of trapped microorganisms. Two cultures from two crystals at 17.8 m that yielded identical 16S rRNA sequences (genus: Haloterrigena) demonstrate intra‐laboratory reproducibility. Inter‐laboratory reproducibility is shown by two halophilic Archaea (genus: Natronomonas), with 99.3% similarity of 16S rRNA sequences, cultured from the same core interval, but at separate laboratories.     

Fluid-deposited graphite and its geobiological implications in early Archean gneiss from Akilia, Greenland

Graphite, interpreted as altered bioorganic matter in an early Archean, ca. 3.83‐Ga‐old quartz‐amphibole‐pyroxene gneiss on Akilia Island, Greenland, has previously been claimed to be the earliest trace of life on Earth. Our petrographic and Raman spectroscopy data from this gneiss reveal the occurrence of graphitic material with the structure of nano‐crystalline to crystalline graphite in trails and clusters of CO₂, CH₄ and H₂O bearing fluid inclusions. Irregular particles of graphitic material without a fluid phase, representing decrepitated fluid inclusions are common in such trails too, but occur also as dispersed individual or clustered particles. The occurrence of graphitic material associated with carbonic fluid inclusions is consistent with an abiologic, fluid deposited origin during a poly‐metamorphic history. The evidence for fluid‐deposited graphitic material greatly complicates any claim about remnants of early life in the Akilia rock.     

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.     

Salmonella spp. are affected by different levels of water activity in closed microcosms

Controlling water activity (a _w) can significantly impact the growth of Salmonella in poultry litter and manure — a phenomenon that was studied quantitatively using two common serotypes of Salmonella. The quantitative effect of changes in levels of a _w on Salmonella populations was determined using inoculated, frosted glass rectangles placed in closed chambers (microcosms). Glass rectangles with known concentrations of Salmonella enteritidis and S. brandenburg were placed in microcosms maintained at an a _w level of 0.893 for 24 h at room temperature (RT) and then transferred to other microcosms maintained at the same temperature but with higher a _w levels (0.932 and 0.987). Salmonella populations on the slides were quantified at 4, 18, 24, and 48 h. Slightly elevated levels of a _w (<0.1, i.e., 10% equilibrium relative humidity) for 24 h resulted in a 100-fold increase in counts of Salmonella. The data also suggested that in vitro adaptation to dry environments may occur when the organisms are exposed to alternating levels of relatively high and low (0.987 and 0.893) levels of a _w. Any increased tolerance of Salmonella to reduced levels of a _w could be the result of physico-chemical changes in the organism due to selective environmental pressure, formation of a protective biofilm, and/or entry into a dormant state. Results from this study are compatible with those from previously reported on-farm surveys, reinforcing the contention that maintaining a _w below 0.85 in and around litter/manure surfaces in poultry or livestock bedding areas may be a critical factor in safe production of food. Journal of Industrial Microbiology & Biotechnology (2001) 26, 222–225. Received 18 May 2000/ Accepted in revised form 24 January 2001     

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.     

Modelling the effect of water activity and temperature on growth rate and aflatoxin production by two isolates of Aspergillus flavus on paddy

Aims: This study was conducted to characterize the growth of and aflatoxin production by Aspergillus flavus on paddy and to develop kinetic models describing the growth rate as a function of water activity (a_w) and temperature. Methods and Results: The growth of A. flavus on paddy and aflatoxin production were studied following a full factorial design with seven a_w levels within the range of 0·82–0·99 and seven temperatures between 10 and 43°C. The growth of the fungi, expressed as colony diameter (mm), was measured daily, and the aflatoxins were analysed using HPLC with a fluorescence detector. The maximum colony growth rates of both isolates were estimated by fitting the primary model of Baranyi to growth data. Three potentially suitable secondary models, Rosso, polynomial and Davey, were assessed for their ability to describe the radial growth rate as a function of temperature and a_w. Both strains failed to grow at the marginal temperatures (10 and 43°C), regardless of the a_w studied, and at the a_w level of 0·82, regardless of temperature. Despite that the predictions of all studied models showed good agreement with the observed growth rates, Davey model proved to be the best predictor of the experimental data. The cardinal parameters as estimated by Rosso model were comparable to those reported in previous studies. Toxins were detected in the range of 0·86–0·99 a_w with optimal a_w of 0·98 and optimal temperature in the range of 25–30°C. Conclusions: The influences of a_w and temperature on the growth of A. flavus and aflatoxin production were successfully characterized, and the models developed were found to be capable of providing good, related estimates of the growth rates. Significance and Impact of the Study: The results of this study could be effectively implemented in minimizing the risk of aflatoxin contamination of the paddy at postharvest.     

Water activity‐temperature state diagrams of freeze‐dried Lactobacillus acidophilus (La‐5): Influence of physical state on bacterial survival during storage

Water activity‐temperature state diagrams for Lactobacillus acidophilus freeze‐dried in a sucrose or a lactose matrix were established based on determination of stabilized glass transition temperatures by differential scanning calorimetry during equilibration with respect to water activity at fixed temperatures. The bacteria in the lactose matrix had higher stabilized glass transition temperatures for all a_w investigated. The survival of Lactobacillus acidophilus determined as colony forming units for up to 10 weeks of storage at 20°C for (i) a_w = 0.11 with both freeze‐dried matrices in the glassy state, (ii) a_w = 0.23 with the bacteria in the lactose matrix in a glassy state but with the bacteria in sucrose matrix in the nonglassy state, and (iii) a_w = 0.43 with both freeze‐dried matrices in a nonglassy state showed that the nature of the sugar was more important for storage stability than the physical state of the matrix with the nonreducing sucrose providing better stability than the reducing lactose. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Dynamics of total surface bacteria and bacterial species counts during desiccation in the Malaysian sea lettuce, Ulva reticulata (Ulvales, Chlorophyta)

The present study illustrates the dynamics of surface bacteria during post‐harvest desiccation of Ulva reticulata Forsskal. Algal fronds were subjected to desiccation for 31 days. The total surface bacteria and bacterial species counts were monitored for moisture content and water activity index (a_w). There was an 86% decrease in total algal moisture content. However, a_w showed a more gradual decrease. The total bacterial count increased in the first week, reaching a maximum on day 7. After this, there was a drastic drop in the total bacterial count until day 14, and then a more gradual decline towards the end of the process. Six species of bacteria were isolated throughout this process: Azomonas sp., Aeromonas hydrophila, Vibrio alginolyti‐cus, Escherichia coli, Proteus vulgaris and Vibrio para‐haemolyticus. The dynamics of each of these bacterial species exhibited trends similar to the total bacterial count. Based on these findings, the drastic decrease in the total bacterial count after seventh day of desiccation could not be attributed to the a_w or salinity. Therefore, the possible exposure of these bacteria to the algal internal fluid upon the rupture of the thallus cells was seen as the most likely reason for the drop in the bacterial population. Scanning electron microscope micrographs taken after the tenth day of desiccation showed the presence of cracks and areas where the bacteria were exposed to the algal internal fluid. In vitro antibacterial tests of three different solvent extracts of Ulva reticulata were also carried out against these surface bacteria to verify the antibacterial potential of its internal fluid. It was apparent that all these surface bacteria were inhibited by at least one of the three extracts, and there were indications of the possible presence of multicompound antibacterial potential, since extracts of different polarity showed bacteria‐specific activity. Hence, it is possible that Ulva reticulata has the potential to protect itself against the opportunistic bacteria present on its surface and in its environment.     

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.     

Effects of reduction in beef surface water activity on the survival of Salmonella Typhimurium DT104 during heating

Aim: To investigate the influence of reducing beef surface water activity (a_w) on the survival of Salmonella Typhimurium DT104 during heating. Methods and Results: Beef discs were surface inoculated with S. Typhimurium DT104 and either untreated or dried to achieve surface a_w values of 0·95, 0·85 and 0·70. The samples were vacuum packed, heat‐treated at 60°C and removed at predetermined times. The inactivation curves were influenced by a_w and treatment time. Biphasic inactivation curves were observed for S. Typhimurium DT104 heat‐treated on beef samples with altered a_w values, which were characterized by an initial decline in cell numbers at commencement of heating followed by a much slower rate of inactivation during the remaining treatment period. Point estimates of the heating time required to achieve a 1 log reduction on beef surfaces with a_w of 0·99, 0·95, 0·85 and 0·70 were 0·5, 1·55, 11·25 and 17·79 min, respectively. Conclusions: A decrease in beef surface a_w can substantially enhance the survival of S. Typhimurium DT104 after heating. Significance and Impact of the Study: Caution needs to be taken using dry air as a decontamination method as this may rapidly decrease product surface and pathogen a_w values resulting in enhanced survival.     

Elevated CO2 affects photosynthetic responses in canopy pine and subcanopy deciduous trees over 10 years: a synthesis from Duke FACE

Leaf responses to elevated atmospheric CO₂ concentration (C_a) are central to models of forest CO₂ exchange with the atmosphere and constrain the magnitude of the future carbon sink. Estimating the magnitude of primary productivity enhancement of forests in elevated C_a requires an understanding of how photosynthesis is regulated by diffusional and biochemical components and up‐scaled to entire canopies. To test the sensitivity of leaf photosynthesis and stomatal conductance to elevated C_a in time and space, we compiled a comprehensive dataset measured over 10 years for a temperate pine forest of Pinus taeda, but also including deciduous species, primarily Liquidambar styraciflua. We combined over one thousand controlled‐response curves of photosynthesis as a function of environmental drivers (light, air C_a and temperature) measured at canopy heights up to 20 m over 11 years (1996–2006) to generate parameterizations for leaf‐scale models for the Duke free‐air CO₂ enrichment (FACE) experiment. The enhancement of leaf net photosynthesis (A_net) in P. taeda by elevated C_a of +200 μmol mol^?1 was 67% for current‐year needles in the upper crown in summer conditions over 10 years. Photosynthetic enhancement of P. taeda at the leaf‐scale increased by two‐fold from the driest to wettest growing seasons. Current‐year pine foliage A_net was sensitive to temporal variation, whereas previous‐year foliage A_net was less responsive and overall showed less enhancement (+30%). Photosynthetic downregulation in overwintering upper canopy pine needles was small at average leaf N (N_area), but statistically significant. In contrast, co‐dominant and subcanopy L. styraciflua trees showed A_net enhancement of 62% and no A_net–N_area adjustments. Various understory deciduous tree species showed an average A_net enhancement of 42%. Differences in photosynthetic responses between overwintering pine needles and subcanopy deciduous leaves suggest that increased C_a has the potential to enhance the mixed‐species composition of planted pine stands and, by extension, naturally regenerating pine‐dominated stands.     

Proteins as potential bacterial growth inhibitors in foods: Suppression of the activity of water by proteins as determined by NMR relaxation methods

Summary Food microbiologists have long known that suppression of the activity of water,a _w, can retard microbial growth in food systems. Traditionally,a _w, suppression has been achieved by addition of salts or humectants to foods. To limit the amount of preservatives added to food products, studies were initiated to assess the feasibility of using proteins to suppressa _w to a practical value for retarding bacterial growth and to determine the optimum environmental condition for maximizing this effect for milk proteins. New expressions were developed relating observed longitudinal and transverse NMR relaxation rates, in the absence of cross-relaxation, to protein hydration , to the protein activity coefficient, _p, and to the correlation time of the bound water, _c. From _p, the second virial coefficient of the protein,B _o, can be found. By use of andB _o,a _w could then be directly evaluated at any protein concentration. Resulting expressions were tested by²H-NMR relaxation measurements made as a function of protein concentration, for: -lactoglobulin A (the major whey protein) under nonassociating (pH 6.0) and associating (pH 4.65) conditions; and for casein (the major milk protein) in the micellar (with added Ca²⁺) and submicellar (without Ca²⁺) forms. Values ofa _w calculated from these²H-NMR data show that casein, at all the concentrations and temperatures examined, suppressesa _w more than does -lactoglobulin A because of a largerB _o. In turn, micellar casein suppressesa _w to a larger extent than does submicellar casein because of a larger . Extrapolation ofa _w at 4°C to a concentration ten times that in normal milk yields a value, ofa _w of less than 0.95, at whichSalmonella and some strains ofClostridium botulinum no longer grow. These results are in agreement with what is known about storageability of condensed milk. Generalizations regarding the types of proteins and cosolutes to be used for suppressinga _w will be discussed. Structural information on these proteins calculated from _c will also be presented.     

Physicochemical parameters of partially hydrolyzed S. salivarius levan fractions

The fractions obtained from the partially hydrolyzed branched Streptococcus salivarius levan were examined in solution. Sedimentation coefficients, S⁰, intrinsic viscosities, [η], weight-average molecular weights, M _w, and radii of gyration were obtained from sedimentation velocity, viscosity, and light-scattering measurements. Double logarithmic plots of [η] vs M _w and S⁰ vs M _w each yielded two linear segments intersecting at M _w ≈ 10⁵. Hydrodynamic data suggest that fractions of M _w > 10⁵ behave as compact spheres, whereas for M _w < 10⁵, the particles are best characterized as linear random coils. Calculations based on theories of random coils and spheres support the above observations.     

Stomatal responses to humidity and temperature in darkness

Stomatal responses to leaf temperature (T_l) and to the mole fractions of water vapour in the ambient air (w_a) and the leaf intercellular air spaces (w_i) were determined in darkness to remove the potential effects of changes in photosynthesis and intercellular CO₂ concentration. Both the steady‐state and kinetic responses of stomatal conductance (g_s) to w_a in darkness were found to be indistinguishable from those in the light. g_s showed a steep response to the difference (Δw) between w_a and w_i when w_a was varied. The response was much less steep when w_i was varied. Although stomatal apertures responded steeply to T_l when Δw was held constant at 17 mmol mol^?1, the response was much less steep when Δw was held constant at about zero. Similar results were obtained in the light for Δw = 15 mmol mol^?1 and Δw ≈ 0 mmol mol^?1. These results are discussed in the context of mechanisms for the stomatal response to humidity.     

Beta diversity of geometrid moths (Lepidoptera: Geometridae) in an Andean montane rainforest

Abstract. Turnover in species composition of the extremely species‐rich family Geometridae (Lepidoptera) was investigated along an elevational gradient ranging from 1040 m to 2677 m above sea level. Moths were sampled using weak light traps (30 W) in three field periods in 1999 and 2000 in an Andean montane rainforest in the province of Zamora‐Chinchipe in southern Ecuador. A total of 13 938 specimens representing 1010 species were analysed. Similarities of ensembles of all geometrid moths and of the subfamilies Ennominae and Larentiinae were calculated using the NESS index (with m_max). Ordinations performed using nonmetric multidimensional scaling (NMDS) and correspondence analysis depicted a gradual change of the ensembles along the altitudinal gradient. Extracted ordination scores significantly correlate with altitude (?0.97 ≤ r ≤ ?0.95, P < 0.001) and with ambient air temperature (0.93 ≤ r ≤ 0.97, P < 0.001). Temperature is therefore assumed to be the most important abiotic determinant responsible for the species turnover among the moths. Matrix correlation tests were performed in order to compare faunal matrices with matrices derived from available environmental factors. Both tree diversity and vegetation structure significantly correlate with faunal data, but tree diversity explains considerably more of the data variability (range: Mantel r = 0.81–0.83, P < 0.001) than vegetation structure (range: Mantel r = 0.35, P < 0.005 to r = 0.43, P < 0.001). Tree diversity also changes gradually and scores of the first NMDS dimension are highly significantly correlated with altitude (r = 0.98, P < 0.001). A common underlying factor such as ambient temperature might also be responsible for such vegetation changes. Additionally, simulated model data was developed that assumed a constant turnover of moth species and equal elevational ranges of all species involved. Despite the simplicity of the models, they fit empirical data very well (Mantel r > 0.80 and P < 0.001 in all models).     

Effect of temperature and water activity on growth and ochratoxin A production boundaries of two Aspergillus carbonarius isolates on a simulated grape juice medium

Aims: To develop and validate a logistic regression model to predict the growth and ochratoxin A (OTA) production boundaries of two Aspergillus carbonarius isolates on a synthetic grape juice medium as a function of temperature and water activity (a_w). Methods and Results: A full factorial design was followed between the factors considered. The a_w levels assayed were 0·850, 0·880, 0·900, 0·920, 0·940, 0·960, 0·980 and the incubation temperatures were 10, 15, 20, 25, 30, 35 and 40°C. Growth and OTA production responses were evaluated for a period of 25 days. Regarding growth boundaries, the degree of agreement between predictions and observations was >99% concordant for both isolates. The erroneously predicted growth cases were 3·4–4·1% false‐positives and 0·7–1·4% false‐negatives. No growth was observed at 10°C and 40°C for all a_w levels assayed, with the exception of 0·980 a_w/40°C, where weak growth was observed. Similarly, OTA production was correctly predicted with a concordance rate >98% for the two isolates with 0·7–1·4% accounting for false‐positives and 2·0–2·7% false‐negatives. No OTA production was detected at 10°C or 40°C regardless of a_w, and at 0·850 a_w at all incubation temperatures. With respect to time, the OTA production boundary shifted to lower temperatures (15–20°C) as opposed to the growth boundary that shifted to higher temperature levels (25–30°C). Using two literature datasets for growth and OTA production of A. carbonarius on the same growth medium, the logistic model gave one false‐positive and three false‐negative predictions out of 68 growth cases and 13 false‐positive predictions out of 45 OTA production cases. Conclusions: The results of this study suggest that the logistic regression model can be successfully used to predict growth and OTA production interfaces for A. carbonarius in relation to temperature and a_w. Significance and Impact of the Study: The proposed modelling approach helps the understanding of fungal‐food ecosystem relations and it could be employed in risk analysis implementation plans to predict the risk of contamination of grapes and grape products by A. carbonarius.     

Clostridium botulinum Type A Growth and Toxin Production in Media and Process Cheese Spread

We found that Clostridium botulinum type A grew well and produced toxin in media with a water activity (a_w) of 0.972 or 0.965 and a pH of 5.7, but no growth or toxin production was observed at or below an a_w of 0.949 during incubation at 30°C for 52 to 59 days. a_w and pH values of media were adjusted to those of cheese spreads commercially produced. Solutes used to adjust a_w included combinations of NaCl, cheese whey powder, emulsifying salt, sodium tripolyphosphate, and glycerol. In agreement with results obtained for media, toxin was produced in samples of cheese spread (a_w, 0.970; pH, 5.7) at 30 to 70 days of incubation at 30°C.     

Variation in the significant environmental factors affecting larval abundance of four major Chinese carp species: fish spawning response to the Three Gorges Dam

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

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