Functionality of a <Emphasis Type="Italic">Bacillus cereus</Emphasis> biological agent in response to physiological variables encountered in aquaculture

The potential of a Bacillus cereus isolate (NRRL 100132) as a biological agent for aquaculture has been demonstrated in vitro and in vivo. The functionality of this isolate across a range of physiological conditions, including salinity, pH and temperature, based on rearing of high-value ornamental Cyprinus carpio, was investigated. Temperature had a significant influence on germination, specific growth rate and increase in cell number of B. cereus in shake-flask cultures, whilst salinity and pH did not have a measurable effect on growth. Controlled studies in bioreactors and modelling of the data to the Arrhenius function indicated the existence of high and low growth temperature domains. The rates of pathogenic Aeromonas hydrophila suppression and decrease in waste ion concentrations (ammonium, nitrite, nitrate and phosphate) were translated into a linear predictive indicator of efficacy of the B. cereus isolate at different temperatures. The present study confirmed the robustness of the B. cereus isolate (NRRL 100132) as a putative biological agent for aquaculture and further demonstrated a novel method for the assessment of in vitro biological efficacy as a function of temperature.     

Modelling faecal <Emphasis Type="Italic">streptococci</Emphasis> mortality in constructed wetlands implanted with <Emphasis Type="Italic">Eichhornia crassipes</Emphasis>

Faecal streptococci mortality was investigated in a water hyacinth (Eichhornia crassipes) constructed wetland pond. The wetland was 7.5 m long, 1.5 m wide and 1.0 m deep, and was implanted with E. crassipes. In order to assess the performance of the system towards bacterial mortality, a mathematical model, based on plug flow philosophy was developed. The model incorporated the role of factors, namely solar intensity, pH, dissolved oxygen, temperature, sedimentation, and root attached growth. Model analysis strongly suggests that bacterial mortality rate constant was largely influenced by two factors, namely solar intensity and root biofilm attachment, with both contributing approximately 70.5% of removal. The contribution of other factors like temperature, dissolved oxygen, pH and sedimentation on bacterial mortality rate were less significant. For example, dissolved oxygen, pH and sedimentation contributed 5%, 8% and 0.82%, respectively. Thus, the sedimentation factor was omitted from the model because of its insignificant contribution. The same was done for temperature, due to low ambient temperature range (3.1°C) in the study area. The overall model bacterial removal efficiency was 83%.     

A predictive model for the growth rate of Bacillus cereus in broth by response surface methodology

A response surface methodology (RSM) was developed for predicting the growth rate of Bacillus cereus in a tryptic soy broth medium as a function of temperature (10 to 40°C), pH (5.5 to 8.5), and the NaCl concentration (0 to 8%). The primary model showed a good fit (r² = 0.920 to 0.999) to a Gompertz equation to obtain growth rates each condition. The quadratic polynomial model was found to be significant (p < 0.0001) and predicted values were found to be in good agreement with experimental values (R² value of 0.9486). The evaluation of RSM for describing the growth rate of B. cereus used the bias factor (B_f) and the accuracy factor (A_f). Both the B_f value (1.11) and the A_f value (1.50) were within acceptable ranges. This model was provided an efficient and accurate method for predicting the growth of B. cereus as a function of the controlling factors.     

Effect of temperature and pH onCandida blankii in chemostat culture

The growth characteristics ofCandida blankii as a function of temperature and pH in a simulated bagasse hemicellulose hydrolysate were determined in chemostat culture. The highest maximum specific growth rate of 0.44h^–1 was reached at 38°C and at pH 5.5, with a sharp decrease in growth rate on either side of this temperature. Growth occurred at 46°C but not at 48°C. The protein and cell yields varied little below 40°C and the respective values were 0.22 and 0.5 g/g at 38°C. At the lower pH values, a severe linear decrease in cell and protein yields occurred, whereas a small increase in these yields at decreasing pH values was found when acetic acid was omitted from the medium. In the presence of acetic acid, a very sharp decrease in the growth rate at pH values below pH 4.5 was noted, despite the very low residual acetic acid concentrations, of less than 50 mg/l, in the culture.     

Biodegradation of p-nitrophenol by methyl parathion-degrading Ochrobactrum sp. B2

Ochrobactrum sp. B2, a methyl parathion-degrading bacterium, was proved to be capable of using p-nitrophenol (PNP) as carbon and energy source. The effect of factors, such as temperature, pH value, and nutrition, on the growth of Ochrobactrum sp. B2 and its ability to degrade p-nitrophenol (PNP) at a higher concentration (100 mg l⁻¹) was investigated in this study.The greatest growth of B2 was observed at a temperature of 30 °C and alkaline pH (pH 9–10). pH condition was proved to be a crucial factor affecting PNP degradation. Enhanced growth of B2 or PNP degradation was consistent with the increase of pH in the minimal medium, and acidic pH (6.0) did not support PNP degradation. Addition of glucose (0.05%, 0.1%) decreased the rate of PNP degradation even if increased cell growth occurred. Addition of supplemental inorganic nitrogen (ammonium chloride or ammonium sulphate) inhibited PNP degradation, whereas organic nitrogen (peptone, yeast extract, urea) accelerated degradation.     

Solubilization of organic and inorganic phosphates by three highly efficient soil bacterial isolates

Screening soil samples collected from a diverse range of slightly alkaline soil types, we have isolated 22 competent phosphate solubilizing bacteria (PSB). Three isolates identified as Pantoea agglomerans strain P5, Microbacterium laevaniformans strain P7 and Pseudomonas putida strain P13 hydrolyzed inorganic and organic phosphate compounds effectively. Bacterial growth rates and phosphate solubilization activities were measured quantitatively under various environmental conditions. In general, a close association was evident between phosphate solubilizing ability and growth rate which is an indicator of active metabolism. All three PSB were able to withstand temperature as high as 42°C, high concentration of NaCl upto 5% and a wide range of initial pH from 5 to 11 while hydrolyzing phosphate compounds actively. Such criteria make these isolates superior candidates for biofertilizers that are capable of utilizing both organic and mineral phosphate substrates to release absorbable phosphate ion for plants.     

Comparison of conditions for mycelial growth of <Emphasis Type="Italic">Lepista sordida</Emphasis> causing fairy rings on <Emphasis Type="Italic">Zoysia matrella</Emphasis> turf to those on <Emphasis Type="Italic">Agrostis palustris</Emphasis> turf

Symptoms of fairy rings caused by Lepista sordida have been reported on Zoysiagrass (Zoysia spp.) turf maintained at fairway height (2 cm), but not on bentgrass (Agrostis spp.) maintained at putting green height (0.5 cm). The mycelia of this fungus inhabit primarily the upper 0–2 cm layer of the soil extending into the thatch. To compare conditions for the mycelial growth in Z. matrella turf to those in A. palustris turf, we examined the effects of nutrients, temperature, water potential, and pH in the field as well as in the laboratory. Greater growth of the mycelia was observed in medium that included hot water extracts from soil of the 0–1 cm zone in Z. matrella turf compared to that from A. palustris. The upper soil layer in Z. matrella turf contained more organic matter from clippings than that in A. palustris. The temperature and water potential of the 0–2 cm soil zone in Z. matrella turf were also more favorable for the mycelial growth. The soil pH values of this zone in Z. matrella turf were less favorable compared to A. palustris but within the range for accelerating mycelial growth. Part of this study was presented orally at the 46th meeting of the Mycological Society of Japan in 2002     

Distribution,occurrence and characterization of entomopathogenic fungi in agricultural soil in the Palestinian area

The occurrence of entomopathogenic fungi was investigated in irrigated vegetable fields and citrus orchards soils, over a nine-month period (April-December 1999),using the Galleria bait method (GBM). Entomopathogenic fungi were found to occur in 33.6% of the soil samples studied, with positive samples yielding 70 fungal isolates, belonging to 20 species from 13 genera. Conidiobolus coronatus was the most frequent and abundant entomopathogenic species recovered, comprising 31.4% of the total number of isolates. Soil pH, soil moisture content and the geographical location had minor or no effect on the isolation of entomopathogenic fungi in the fields studied. On the other hand, organic matter content of soil, and vegetation type were found to significantly affect the occurrence of entomopathogenic fungi in soil habitats, with orchard fields yielding larger numbers of isolates than vegetable fields. Using Koch's postulates the pathogenicity of fungal isolates to Galleria larvae was found to range from 16–100% (mortality rate). Isolates of C. coronatus proved to be the most virulent isolates recovered. The effect of media and temperature on mycelial growth rate, conidial production and conidial germination of six entomopathogenic fungal species (C. coronatus, Entomophaga grylli, Erynia castrans, Hirsutella jonesii, Paecilomyces farinosus and Sporodiniella umbellata) was also studied. Mycelial growth rate, spore production and spore germination were significantly affected by media, temperature and isolates. In view of the present results, C. coronatus appears to be a good candidate for pest control in agricultural soils, as it has a wide tolerance to agricultural practices, has frequently been isolated from both vegetable and orchard fields, and is characterized by high mycelial growth rate, conidial production and conidial germination.This revised version was published online in October 2005 with corrections to the Cover Date.     

Estimates of Frankia growth under various pH and temperature regimes

Summary The growth of Frankia isolates was monitored by dry weight, total protein and total ATP measurements under different temperature and pH regimes. Significant correlations (P<0.01) were found among all growth measures which meant that similar general conclusions were reached irrespective of the study method involved. The assessment of protein was the method of choice for regular assessments of Frankia growth due to its facility and relatively high sensitivity. The optimum temperature for growth of isolate LDAgp1 and AvcI1 was about 30°C while for CpI1 it lay between 30° and 35°C. No growth was observed at 40°C but some growth was observed at 10°C with isolate CpI1 and LDAgp1 over an extended growth period of 39 days. The range of pH favouring growth lay between 6 and 8. The optimum for LDAgp1 lay between 6.5 and 7, that for AvcI1 and CpI1 is close to 6.5. The pH response was medium dependent. Increases in biomass were observed for some isolates at 4.6 and above 8.0 on some media.     

Conductance measurements for data generation in predictive modeling

Summary The electrical resistance of a growth medium inoculated with bacteria may be automatically recorded throughout an incubation period without the necessity for sampling. The rate of change in conductance is dependent on the bacteria studied, the medium composition and the prevailing growth conditions.The effect of growth medium composition, growth conditions and inoculum level on the conductance response was studied forYersinia enterocolitica O:3. A large number of combinations of factors affecting the growth/activity of the bacteria could be studied simultaneously due to the large instrumental capacity of the Malthus 2000. A polynomial model based on conductance measurements was developed forY. enterocolitica describing the effect of temperature, pH andl-lactate level on conductance response curve parameters. The model was used for predicting growth rates. Growth rates calculated from bacterial counts ofY. enterocolitica growing in minced pork corresponded to growth rates predicted using the polynomial conductance models.     

Molecular and Biochemical Characterization of an Endochitinase (ChiA-HD73) from Bacillus thuringiensis subsp. kurstaki HD-73

An endochitinase gene (chiA-HD73) from the insecticidal bacterium Bacillus thuringiensis subsp. kurstaki HD-73 was cloned, sequenced, and expressed in Escherichia coli DH5αF′. The chitinase activity of the encoded protein was studied in assays with different fluorogenic substrates. The chiA-HD73 gene contained an open-reading frame that encoded an endochitinase with a deduced molecular weight and an isoelectric point of, respectively, 74.5 kDa and 5.75. A putative signal peptide with cleavage sites for both Gram-positive and Gram-negative bacteria was identified. Comparison of ChiA-HD73 with other chitinases revealed a modular structure composed of a catalytic domain and a putative chitin-binding domain. ChiA-HD73 hydrolyzed both tetrameric and trimeric fluorogenic substrates, but not a chitobiose analog substrate, suggesting that the activity of ChiA-HD73 is mainly endochitinolytic. In addition, ChiA-HD73 showed high enzymatic activity within a broad pH range (pH 4–10), with a peak activity at pH 6.5. The optimal temperature for enzymatic activity was observed at 55°C. Its activity in a broad range of temperatures and pH suggests ChiA-HD73 could have biotechnological applications in insect control, particularly in synergizing the insecticidal crystal protein toxins of B. thuringiensis.     

Characterization and optimization of extracellular alkaline lipase production by Alcaligenes sp. Using stearic acid as carbon source

The aim of this study was to improve the production of an extracellular alkaline lipase from Alcaligenes sp. (ATCC 31371) by optimization of the culture medium, for economic production of biodiesel from waste vegetable oil. A number of carbon sources including different types of starch, sugar, sugar alcohol, organic acids, and surfactants were investigated. Polyoxyethylene (20) sorbitan tristearate, whose side chain is stearic acid, was the most effective carbon source for lipase production. Box-Behnken experimental design was used for three factors (soy protein, sodium nitrate, and stearic acid) and the optimal composition for maximum lipase production (1.7-fold enhancement) was established as soy protein 4.07%, sodium nitrate 0.17%, and stearic acid 0.28% at 28°C with an agitation rate of 220 rpm for 24 h. The enzyme was purified to homogeneity and the recovery of the lipase activity was 7.8% with a 30-fold purification. The estimated molecular size of the protein determined by SDS-PAGE was 33 kDa. The optimum pH and temperature of the purified lipase was 8.5 and 40°C, respectively. The purified enzyme was stable in the pH range of 6.0 and 9.5 and in the temperature range of 20 and 50°C.     

The trade-off between growth rate and yield in microbial communities and the consequences for under-snow soil respiration in a high elevation coniferous forest

Soil microbial respiration is a critical component of the global carbon cycle, but it is uncertain how properties of microbes affect this process. Previous studies have noted a thermodynamic trade-off between the rate and efficiency of growth in heterotrophic organisms. Growth rate and yield determine the biomass-specific respiration rate of growing microbial populations, but these traits have not previously been used to scale from microbial communities to ecosystems. Here we report seasonal variation in microbial growth kinetics and temperature responses (Q₁₀) in a coniferous forest soil, relate these properties to cultured and uncultured soil microbes, and model the effects of shifting growth kinetics on soil heterotrophic respiration (R_h). Soil microbial communities from under-snow had higher growth rates and lower growth yields than the summer and fall communities from exposed soils, causing higher biomass-specific respiration rates. Growth rate and yield were strongly negatively correlated. Based on experiments using specific growth inhibitors, bacteria had higher growth rates and lower yields than fungi, overall, suggesting a more important role for bacteria in determining R_h. The dominant bacteria from laboratory-incubated soil differed seasonally: faster-growing, cold-adapted Janthinobacterium species dominated in winter and slower-growing, mesophilic Burkholderia and Variovorax species dominated in summer. Modeled R_h was sensitive to microbial kinetics and Q₁₀: a sixfold lower annual R_h resulted from using kinetic parameters from summer versus winter communities. Under the most realistic scenario using seasonally changing communities, the model estimated R_h at 22.67 mol m⁻² year⁻¹, or 47.0% of annual total ecosystem respiration (R_e) for this forest.     

Identifying Environmental Conditions to Promote Species Coexistence: An Example with the Native Barrens Topminnow and Invasive Western Mosquitofish

Coexistence of a native and invasive species may be possible at certain conditions along an environmental gradient where the individual responses of each species are maximally apart. Water temperature may differentially affect the growth of a native cool-water species like the Barrens topminnow, Fundulus julisia, and an originally warm-water adapted western mosquitofish, Gambusia affinis, who is a recent invader in Barrens Plateau region of middle Tennessee. We measured the specific growth rate (SGR) of the two species separately in laboratory aquaria at 10, 15, 20 and 25 °C, representing a range of temperatures that occur in topminnow habitats throughout the year. Both species grew faster with increasing temperature and SGRs were highest at 25 °C. The interspecific difference in SGR was maximized at 15 °C. At this temperature, mean growth rate of topminnows was 0.78% per day, more than twice that of mosquitofish (0.38% per day). These results suggest that cool springhead habitats with a near-constant thermal environment of 15 °C throughout the year may provide a growth advantage to the Barrens topminnow over the mosquitofish. Other environmental, density-dependent, or behavioral factors not examined here may act along with temperature to mediate the coexistence of the topminnow and mosquitofish.     

Study of physicochemical factors limiting the growth ofKluyveromyces marxianus

Summary The effects of various physicochemical parameters on the growth of twoKluyveromyces marxianus strains were investigated, including: pH values, sodium chloride, water activity in the medium and temperature. Both yeast strains were unaffected by pH changes. Optimal pH for growth was found to be 4 with both strains, but they were able to develop within the pH 3–8 range. Suitable growth was obtained at temperatures of 4–44°C and the optimal temperature for growth was 36°C for both strains. Modelling of this latter parameter is described. Growth of both microorganisms was considerably modified by increased NaCl or decreased water activity in the medium.     

Na<Superscript>+</Superscript> and flagella-dependent swimming of alkaliphilic <Emphasis Type="Italic">Bacillus pseudofirmus</Emphasis> OF4: a basis for poor motility at low pH and enhancement in viscous media in an “up-motile” variant

Flagella-based motility of extremely alkaliphilic Bacillus species is completely dependent upon Na⁺. Little motility is observed at pH values < ∼8.0. Here we examine the number of flagella/cell as a function of growth pH in the facultative alkaliphile Bacillus pseudofirmus OF4 and a derivative selected for increased motility on soft agar plates. Flagella were produced by both strains during growth in a pH range from 7.5 to 10.3. The number of flagella/cell and flagellin levels of cells were not strongly dependent on growth pH over this range in either strain although both of these parameters were higher in the up-motile strain. Assays of the swimming speed indicated no motility at pH < 8 with 10 mM Na⁺, but significant motility at pH 7 at much higher Na⁺ concentrations. At pH 8–10, the swimming speed increased with the increase of Na⁺ concentration up to 230 mM, with fastest swimming at pH 10. Motility of the up-motile strain was greatly increased relative to wild-type on soft agar at alkaline pH but not in liquid except when polyvinylpyrrolidone was added to increase viscosity. The up-motile phenotype, with increased flagella/cell may support bundle formation that particularly enhances motility under a subset of conditions with specific challenges.     

Methanogenium marinum sp. nov., a H2-using methanogen from Skan Bay,Alaska, and kinetics of H2 utilization

A methanogen, strain AK-1, was isolated from permanently cold marine sediments, 38- to 45-cm below the sediment surface at Skan Bay, Alaska. The cells were highly irregular, nonmotile coccoids (diameter, 1 to 1.2 μm), occurring singly. Cells grew by reducing CO₂ with H₂ or formate as electron donor. Growth on formate was much slower than that on H₂. Acetate, methanol, ethanol, 1- or 2-propanol, 1- or 2-butanol and trimethylamine were not catabolized. The cells required acetate, thiamine, riboflavin, a high concentration of vitamin B₁₂, and peptones for growth; yeast extract stimulated growth but was not required. The cells grew fastest at 25 °C (range 5 °C to 25 °C), at a pH of 6.0 – 6.6 (growth range, pH 5.5 – 7.5), and at a salinity of 0.25 – 1.25 M Na⁺. Cells of this and other H₂-using methanogens from saline environments metabolized H₂ to a very low threshold pressure (less than 1 Pa) that was dependent on the methane partial pressure. We propose that the threshold pressure may be limited by the energetics of catabolism. The sequence of the 16S rDNA gene of strain AK-1 was most similar (98%) to the sequences of Methanogenium cariaci JR-1 and Methanogenium frigidum Ace-2. DNA–DNA hybridization between strain AK-1 and these two strains showed only 34.9% similarity to strain JR-1 and 56.5% similarity to strain Ace-2. These analyses indicated strain AK-1 should be classified as a new species within the genus Methanogenium. Phenotypic differences between strain AK-1 and these strains (including growth temperature, salinity range, pH range, and nutrient requirements) support this. Therefore, a new species, Methanogenium marinum, is proposed with strain AK-1 as type strain. This revised version was published online in August 2006 with corrections to the Cover Date.     

Gracilaria conferta and its epiphytes: (2) Interrelationship between the red seaweed and Ulva cf. lactuca

Ulva cf. lactuca has been a disturbing competitor of experimental Gracilaria conferta outdoor cultures in Israel. The effect of environmental conditions on the competitive ability of Ulva versus Gracilaria, and the main limiting factors for which these seaweeds compete, were studied here. Single and biculture experiments of both seaweeds showed that Ulva outgrew and damaged Gracilaria under all irradiance and temperature combinations. The higher competitive ability of Ulva cf. lactuca in bicultures was not a result of responses to shading or nitrogen shortage, but rather to a shortage of available inorganic carbon, an increase in pH and apparent excretions of Ulva which inhibited the growth of Gracilaria.     

Some physiological properties of citrus and noncitrus races ofGeotrichum candidum isolated from soil in Japan

Some physiological properties of citrus and noncitrus races ofGeotrichum candidum isolated from soils of citrus groves and noncitrus fields in Japan were studied. Of 358 isolated of citrus race, 260 isolates required pyridoxine for growth on asparagine-glucose agar, while 98 isolates could grow in the absence of pyridoxine. On the other hand, of 11 isolates of noncitrus race, 10 isolates could grow without pyridoxine, while 1 isolate absolutely required pyridoxine for growth. The citrus race grew well in autoclaved lemon juice at pH 2.2, while the noncitrus race failed to grow. The radial growth rate of the citrus race on potato-dextrose agar was higher than that of the noncitrus race. The two races grew well within the same range of temperature and pH, the optimal temperature ranging between 25 and 30°C, and optimal pH being 6.0. Both races utilized glucose, galactose, xylose, sorbose, sorbitol and mannitol as carbon sources, but could not utilize arabinose, lactose, maltose, rhamnose and sucrose. Arginine and asparaghine were the best nitrogen sources for both races, but KNO₃ could not be utilized.     

Temperature and nutrient availability control growth rate and fatty acid composition of facultatively psychrophilic <Emphasis Type="Italic">Cobetia marina</Emphasis> strain L-2

A facultative psychrophilic bacterium, strain L-2, that grows at 0 and 5°C as minimum growth temperatures in complex and defined media, respectively, was isolated. On the basis of taxonomic studies, strain L-2 was identified as Cobetia marina. The adaptability of strain L-2 to cold temperature was higher than that of the type strain and of other reported strains of the same species. When the bacterium was grown at 5–15°C in a defined medium, it produced a high amount of trans-unsaturated fatty acids. By contrast, in a complex medium in the same temperature range it produced a low amount of trans-unsaturated fatty acids. In the complex medium at 5°C, the bacterium exhibited a three-fold higher growth rate than that obtained in the defined medium. Following a temperature shift from 11 to 5°C, strain L-2 grew better in complex than in defined medium. Furthermore, when the growth temperature was shifted from 0 to 5°C both the growth rate and the yield of strain L-2 growing in complex medium was markedly enhanced. These phenomena suggest that an upshift of the growth temperature had a positive effect on metabolism. The effects of adding complex medium components to the defined medium on bacterial growth rate and fatty acid composition at 5°C were also studied. The addition of yeast extract followed by peptone was effective in promoting rapid growth, while glutamate addition was less effective, resulting in a cis-unsaturated fatty acid ratio similar to that of cells grown in the complex medium. These results suggest that the rapid growth of strain L-2 at low temperatures requires a high content of various amino acids rather than the presence of a high ratio of cis-unsaturated fatty acids in the cell membrane.