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.     

Osmoregulatory responses of bacteria isolated from fresh or composted,olive-mill waste-waters

Halotolerant bacteria isolated from raw, olive-mill waste-waters (alpechin) and from composted alpechin could grow on solid medium containing up to 10% (w/v) NaCl. Most (70%) of these halotolerant isolates could also grow in liquid minimal medium with the same NaCl concentration and three isolates from this group were chosen for further study. When grown in tryptic soy broth (TSB), two isolates responded to lowering of water activity (a _w) by addition of NaCl or sucrose in the expected manner: by increasing the proportion of membrane anionic lipids diphosphatidylglycerol or phosphatidylglycerol. Some solute-specific differences were observed. In contrast, the third isolate did not alter its membrane phospholipid composition significantly in response to growth in NaCl, whereas in sucrose there was an increase in phosphatidylethanolamine. This response is contrary to the accepted interpretation of the function of such a _w-dependent changes, as being a mechanism for preserving the membrane lipid-bilayer phase. When all three isolates were grown in the presence of alpechin, there was a decrease in the proportion of phosphatidylglycerol and a rise in the level of phosphatidylethanolamine. Quantitative and qualitative differences in compatible solute composition were observed when the three isolates were grown in TSB with NaCl or sucrose added to lower a _w. The major compatible solutes in two of the isolates were proline and betaine, whereas in the third they were proline, betaine and ectoine; one isolate also contained some trehalose when NaCl but not sucrose was the osmolyte.     

Chemical Modification Causes Similar Change in Dependence on Water Activity of Chymotrypsin Hydration and Catalysis in Hexane

Amino groups in alpha-chymotrypsin were reacted with pyromellitic anhydride, introducing 17 to 32 additional carboxyl groups. This modification causes a major change in the water adsorption isotherm of the lyophilized protein powder. Little water is bound by the modified enzyme at water activity (a_w) below 0.35, but it shows increased water binding at a_w over 0.5. This correlates with a similar change in the a_w dependence of the catalytic activity of the enzyme powder suspended in hexane, with a much steeper increase in activity of the modified chymotrypsin.     

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.     

Effect of water activity on exo-pectinase production byAspergillus niger CH4 on solid state fermentation

Summary Ethylene glycol, sorbitol and glycerol were used as water activity depressors to study the effect of water activity on pectinase production byAspergillus niger CH4. Ethylene glycol depressed a_w without supporting growth nor strongly affecting pectinase production in petri dish cultures. This depressor was used to evaluate the influence of water activity on exo-pectinase production by SSF. It was found that although pectinase production decreased at low a_w values, this activity was present at a_w values as low as 0.90. The specific activity increased up to 4.5 fold by reducing a_w from 0.98 to 0.90. The reducing groups accumulated extracellularly suggesting sugar transport limitation as a consequence of a_w depression.     

Effects of water activity on Vmax and KM of lipase catalyzed transesterification in organic media

Summary The V_max and K_M of various forms of lipase from Pseudomonas cepacia (powder, adsorbed onto Celite or covalently linked to polyethylene glycol) were determined in organic solvents preequilibrated to water activities (a _w) from <0.1 to 0.84. The model reaction was the transesterification between n-octanol and vinyl butyrate. It was found that K_M for the nucleophile increased with increasing a _w for all three lipase forms. V_max increased with increasing a _w for polyethylene glycol-lipase, whereas there was an optimum at intermediate a _w values (0.11 – 0.38) for lipase powder and Celite-immobilized lipase.     

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 water activity on the mechanical glass transition and dynamical transition of bacteria

The purpose of this study was to clarify the glass-transition behavior of bacteria (Cronobacter sakazakii) as a function of water activity (a_w). From the water sorption isotherm (298 K) for C. sakazakii, monolayer water content and monolayer a_w were determined to be 0.0724 g/g-dry matter and 0.252, respectively. Mechanical relaxation was investigated at 298 K. In a higher a_w range of over 0.529, the degree of mechanical relaxation increased with an increase in a_w. From the effect of a_w on the degree of mechanical relaxation, the mechanical a_wc (a_w at which mechanical glass transition occurs at 298 K) was determined to be 0.667. Mean-square displacement of atoms in the bacteria was investigated by incoherent elastic neutron scattering. The mean-square displacement increased gradually with an increase in temperature depending on the a_w of samples. From the linear fitting, two or three dynamical transition temperatures (low, middle, and high T_ds) were determined at each a_w. The low-T_d values (142–158 K) were almost independent from a_w. There was a minor effect of a_w on the middle T_d (214–234 K) except for the anhydrous sample (261 K). The high T_d (252–322 K) largely increased with the decrease in a_w. From the a_w dependence of the high T_d, the dynamical a_wc was determined to be 0.675, which was almost equivalent to the mechanical a_wc. The high T_d was assumed to be the glass-transition temperature (T_g), and anhydrous T_g was estimated to be 409 K. In addition, molecular relaxation time (τ) of the bacteria was calculated as a function of a_w. From the result, it is suggested that the progress of metabolism in the bacterial system requires a lower τ than approximately 6 × 10^?5 s.     

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 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.     

Stepped water activity control for efficient enzymatic interesterification

The benefits of controlling water activity, a _w, during enzymatically catalysed synthesis reactions, such as reverse-hydrolytic reactions promoted by lipases, are now well recognized. Numerous techniques for controlling a _w in the laboratory and their implementation in continuous reactors have been discussed in the published literature. However, in enzymatic interesterification reactions, such as acidolysis and transesterification, it is not appropriate merely to maintain the a _w of the reaction system at one value since the two stages of the reaction, namely the cleavage of the original acyl bond and the formation of a new one, are best carried out at different levels of water activity – the former at a high a _w and the latter at a lower one. The use of a continuous packed-bed hollow-fibre reactor has been described in this article for carrying out solvent-free acidolysis of ethyl laurate with octanoic acid with in situ a _w control, using air that has been pre-equilibrated with saturated salt solutions to the desired a _w. At a single optimum (a _w = 0.54), the highest steady-state conversion to ethyl octanoate was 32%. However, it is possible to obtain a steady-state conversion of 46% by operating the reactor with a step change in the water activity, from an initial value of unity to 0.23. Received: 10 February 1998 / Received revision: 2 June 1998 / Accepted: 7 June 1998     

Effects of nitrate treatment on a mixed species, oil field microbial biofilm

Biofilms of bacteria, indigenous to oil field produced water, were grown in square section, glass capillary flow cells at 45 °C. Initially, in situ image analysis microscopy revealed predominantly coccoid bacteria (length-to-width ratio measurements (l _c:w _c) of bacterial cells gave a mean value of 1.1), while chemical measurements confirmed sulphate reduction and sulphide production. After nitrate ion addition at 100 and 80 mg/l, in the two repeat experiments respectively, the dominance of rod-shaped bacteria (mean l _c:w _c = 2.8) was observed. This coincided with the occurrence of nitrate reduction in the treated flow cells. Beneficially, no significant increase in biofilm cover was observed after the addition of nitrate. The dominant culturable nitrate-reducing bacterium was Marinobacter aquaeolei. The l _c:w _c ratio measured here concurs with previously reported cell dimensions for this organism. Several Marinobacter strains were also isolated from different oil fields in the North Sea where nitrate treatment has been applied to successfully treat reservoir souring, implying that this genus may play an important role in nitrate treatment.     

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.     

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 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.     

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     

Increased xerotolerance of Saccharomyces cerevisiae during an osmotic pressure ramp over several generations

Although mechanisms involved in response of Saccharomyces cerevisiae to osmotic challenge are well described for low and sudden stresses, little is known about how cells respond to a gradual increase of the osmotic pressure (reduced water activity; a_w) over several generations as it could encounter during drying in nature or in food processes. Using glycerol as a stressor, we propagated S. cerevisiae through a ramp of the osmotic pressure (up to high molar concentrations to achieve testing-to-destruction) at the rate of 1.5 MPa day^-1 from 1.38 to 58.5 MPa (0.990–0.635 a_w). Cultivability (measured at 1.38 MPa and at the harvest osmotic pressure) and glucose consumption compared with the corresponding sudden stress showed that yeasts were able to grow until about 10.5 MPa (0.926 a_w) and to survive until about 58.5 MPa, whereas glucose consumption occurred until 13.5 MPa (about 0.915 a_w). Nevertheless, the ramp conferred an advantage since yeasts harvested at 10.5 and 34.5 MPa (0.778 a_w) showed a greater cultivability than glycerol-shocked cells after a subsequent shock at 200 MPa (0.234 a_w) for 2 days. FTIR analysis revealed structural changes in wall and proteins in the range 1.38–10.5 MPa, which would be likely to be involved in the resistance at extreme osmotic pressure.     

Spherical particles of halophilic archaea correlate with exposure to low water activity - implications for microbial survival in fluid inclusions of ancient halite

Viable extremely halophilic archaea (haloarchaea) have been isolated from million‐year‐old salt deposits around the world; however, an explanation of their supposed longevity remains a fundamental challenge. Recently small roundish particles in fluid inclusions of 22 000‐ to 34 000‐year‐old halite were identified as haloarchaea capable of proliferation (Schubert BA, Lowenstein TK, Timofeeff MN, Parker MA, 2010, Environmental Microbiology, 12, 440–454). Searching for a method to produce such particles in the laboratory, we exposed rod‐shaped cells of Halobacterium species to reduced external water activity (a_w). Gradual formation of spheres of about 0.4 μm diameter occurred in 4 m NaCl buffer of a_w ≤ 0.75, but exposure to buffered 4 m LiCl (a_w ≤ 0.73) split cells into spheres within seconds, with concomitant release of several proteins. From one rod, three or four spheres emerged, which re‐grew to normal rods in nutrient media. Biochemical properties of rods and spheres were similar, except for a markedly reduced ATP content (about 50‐fold) and an increased lag phase of spheres, as is known from dormant bacteria. The presence of viable particles of similar sizes in ancient fluid inclusions suggested that spheres might represent dormant states of haloarchaea. The easy production of spheres by lowering a_w should facilitate their investigation and could help to understand the mechanisms for microbial survival over geological times.     

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.