Kinetic Analyses of Desulfurization of Dibenzothiophene by Rhodococcus erythropolis in Continuous Cultures

Rhodococcus erythropolis N1-36, a desulfurization strain, was grown in continuous culture at 10 different dilution rates with 50 (mu)M dibenzothiophene sulfone (DBTO(inf2)) as the growth-limiting nutrient. The steady-state biomass, concentrations of substrate (DBTO(inf2)) and product (monohydroxybiphenyl), saturation constant (0.39 (mu)M DBTO(inf2)), and cell yield coefficient (9 mg of biomass(middot)(mu)M(sup-1) DBTO(inf2)) were measured. Continuous cultures at five temperatures allowed calculation of activation energy (0.84 kcal(middot)mol(sup-1) [ca. 3.5 kJ(middot)mol(sup-1)]) near the optimal temperature (30(deg)C) for growth. A washout technique was used to calculate the maximum specific growth rate (0.235 h(sup-1)), a value equivalent to a minimum generation time of 2.95 h.     

Measurement of Growth at Very Low Rates ((mu) >= 0), an Approach To Study the Energy Requirement for the Survival of Alcaligenes eutrophus JMP 134

Alcaligenes eutrophus JMP 134 was grown in a recycling-mode fermenter with 100% biomass retention on 2,4-dichlorophenoxyacetic acid (2,4-D), phenol, and fructose. The growth pattern obtained given a constant supply of substrates exhibited three phases of linear growth on all three substrates. The transition from phase 1 to phase 2, considered to correspond to the onset of stringent (growth) control as indicated by a significant increase in guanosine 5(prm1)-bisphosphate 3(prm1)-bisphosphate (ppGpp), took place at 0.016 h(sup-1) with 2,4-D and at about 0.02 h(sup-1) with phenol and fructose. In the final phase, phase 4, which was achieved after the growth rate on the respective substrates fell below 0.003 to 0.001 h(sup-1), a constant level of biomass was obtained irrespective of further feeding of substrate at the same rate. The yield coefficients decreased by 70 to 80% from phase 1 to phase 3 and were 0 in phase 4. The stationary substrate concentrations s(infmin) in phase 4, calculated from the kinetic constants of the strain, were 1.23, 0.34, and 0.23 (mu)M for 2,4-D, phenol, and fructose, respectively. These figures characterize the minimum stationary substrate concentrations required in a dynamic system to keep A. eutrophus alive. This is caused by a substrate flux which enables growth at a rate >=0 due to the provision of energy to an extent at least satisfying maintenance requirements. According to the constant feed rates of the substrates and the final and stable biomass concentrations, this maintenance energy amounts to 14.4, 4.0, and 2.4 (mu)mol of ATP (middot) mg of dry mass(sup-1) h(sup-1) for 2,4-D, phenol, and fructose, respectively, after correction for the fraction of living cells. The increased energy expenditure in the case of 2,4-D is discussed with respect to uncoupling.     

Transformation of Low Concentrations of 3-Chlorobenzoate by Pseudomonas sp. Strain B13: Kinetics and Residual Concentrations

The transformation of 3-chlorobenzoate (3CB) and acetate at initial concentrations in the wide range of 10 nM to 16 mM was studied in batch experiments with Pseudomonas sp. strain B13. Transformation rates of 3CB at millimolar concentrations could be described by Michaelis-Menten kinetics (K(infm), 0.13 mM; V(infmax), 24 nmol (middot) mg of protein(sup-1) (middot) min(sup-1)). Experiments with nanomolar and low micromolar concentrations of 3CB indicated the possible existence of two different transformation systems for 3CB. The first transformation system operated above 1 (mu)M 3CB, with an apparent threshold concentration of 0.50 (plusmn) 0.11 (mu)M. A second transformation system operated below 1 (mu)M 3CB and showed first-order kinetics (rate constant, 0.076 liter (middot) g of protein(sup-1) (middot) min(sup-1)), with no threshold concentration in the nanomolar range. A residual substrate concentration, as has been reported for some other Pseudomonas strains, could not be detected for 3CB (detection limit, 1.0 nM) in batch incubations with Pseudomonas sp. strain B13. The addition of various concentrations of acetate as a second, easily degradable substrate neither affected the transformation kinetics of 3CB nor induced a detectable residual substrate concentration. Acetate alone also showed no residual concentration (detection limit, 0.5 nM). The results presented indicate that the concentration limits for substrate conversion obtained by extrapolation from kinetic data at higher substrate concentrations may underestimate the true conversion capacity of a microbial culture.     

Measurement of Growth at Very Low Rates ((mu) >= 0), an Approach To Study the Energy Requirement for the Survival of Alcaligenes eutrophus JMP 134

Alcaligenes eutrophus JMP 134 was grown in a recycling-mode fermenter with 100% biomass retention on 2,4-dichlorophenoxyacetic acid (2,4-D), phenol, and fructose. The growth pattern obtained given a constant supply of substrates exhibited three phases of linear growth on all three substrates. The transition from phase 1 to phase 2, considered to correspond to the onset of stringent (growth) control as indicated by a significant increase in guanosine 5(prm1)-bisphosphate 3(prm1)-bisphosphate (ppGpp), took place at 0.016 h(sup-1) with 2,4-D and at about 0.02 h(sup-1) with phenol and fructose. In the final phase, phase 4, which was achieved after the growth rate on the respective substrates fell below 0.003 to 0.001 h(sup-1), a constant level of biomass was obtained irrespective of further feeding of substrate at the same rate. The yield coefficients decreased by 70 to 80% from phase 1 to phase 3 and were 0 in phase 4. The stationary substrate concentrations s(infmin) in phase 4, calculated from the kinetic constants of the strain, were 1.23, 0.34, and 0.23 (mu)M for 2,4-D, phenol, and fructose, respectively. These figures characterize the minimum stationary substrate concentrations required in a dynamic system to keep A. eutrophus alive. This is caused by a substrate flux which enables growth at a rate >=0 due to the provision of energy to an extent at least satisfying maintenance requirements. According to the constant feed rates of the substrates and the final and stable biomass concentrations, this maintenance energy amounts to 14.4, 4.0, and 2.4 (mu)mol of ATP (middot) mg of dry mass(sup-1) h(sup-1) for 2,4-D, phenol, and fructose, respectively, after correction for the fraction of living cells. The increased energy expenditure in the case of 2,4-D is discussed with respect to uncoupling.     

Effect of pH on Bacillus thermoamylovorans Growth and Glucose Fermentation

The effect of pH on the growth and physiology of Bacillus thermoamylovorans, a new moderately thermophilic and non-spore-forming bacterium isolated from palm wine, was studied. Growth occurred from pH 5.4 to 8.5, with optimum growth at 7.0. During the exponential growth phase at optimum pH, glucose was consumed at the maximum rate (qs), 17.87 mmol g(sup-1) h(sup-1), and was mainly fermented into acetate, ethanol, and formate (76.5% of metabolites produced). In acidic or alkaline conditions, glucose specific consumption rates were considerably reduced (qs = 8.06 mmol g(sup-1) h(sup-1) at pH 5.6 and 2.85 mmol g(sup-1) h(sup-1) at pH 8.4), and a switch in glucose metabolism toward lactate production (62.6% of metabolites produced at pH 5.6 and 41.2% of those produced at pH 8.4) was observed. Moreover, optimum cellular yield (Y(infx/ATP)), 14.8 g mol(sup-1), and optimum energy yield (Y(infATP/s)), 2.65 mol mol(sup-1), were observed at neutrality. The results of this study were compared with published data about lactic acid bacteria; this comparison allowed us to complement our previous taxonomic study of B. thermoamylovorans and to identify additional phenotypic differences between B. thermoamylovorans and lactobacilli.     

Effects of Nitrate Availability and the Presence of Glyceria maxima on the Composition and Activity of the Dissimilatory Nitrate-Reducing Bacterial Community

The effects of nitrate availability and the presence of Glyceria maxima on the composition and activity of the dissimilatory nitrate-reducing bacterial community were studied in the laboratory. Four different concentrations of NO(inf3)(sup-), 0, 533, 1434, and 2,905 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1), were added to pots containing freshwater sediment, and the pots were then incubated for a period of 69 days. Upon harvest, NH(inf4)(sup+) was not detectable in sediment that received 0 or 533 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1). Nitrate concentrations in these pots ranged from 0 to 8 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1) at harvest. In pots that received 1,434 or 2,905 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1), final concentrations varied between 10 and 48 (mu)g of NH(inf4)(sup+)-N g of dry sediment(sup-1) and between 200 and 1,600 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1), respectively. Higher input levels of NO(inf3)(sup-) resulted in increased numbers of potential nitrate-reducing bacteria and higher potential nitrate-reducing activity in the rhizosphere. In sediment samples from the rhizosphere, the contribution of denitrification to the potential nitrate-reducing capacity varied from 8% under NO(inf3)(sup-)-limiting conditions to 58% when NO(inf3)(sup-) was in ample supply. In bulk sediment with excess NO(inf3)(sup-), this percentage was 44%. The nitrate-reducing community consisted almost entirely of NO(inf2)(sup-)-accumulating or NH(inf4)(sup+)-producing gram-positive species when NO(inf3)(sup-) was not added to the sediment. The addition of NO(inf3)(sup-) resulted in an increase of denitrifying Pseudomonas and Moraxella strains. The factor controlling the composition of the nitrate-reducing community when NO(inf3)(sup-) is limited is the presence of G. maxima. In sediment with excess NO(inf3)(sup-), nitrate availability determines the composition of the nitrate-reducing community.     

Activity and Distribution of Methane-Oxidizing Bacteria in Flooded Rice Soil Microcosms and in Rice Plants (Oryza sativa)

The activity and distribution of CH(inf4)-oxidizing bacteria (MOB) in flooded rice (Oryza sativa) soil microcosms was investigated. CH(inf4) oxidation was shown to occur in undisturbed microcosms by using (sup14)CH(inf4), and model calculations indicated that almost 90% of the oxidation measured had taken place at a depth where only roots could provide the O(inf2) necessary. Slurry from soil planted with rice had an apparent K(infm) for CH(inf4) of 4 (mu)M and a V(infmax) of 0.1 (mu)mol g (dry weight)(sup-1) h(sup-1). At a depth of 1 to 2 cm, there was no significant difference (P > 0.05) in numbers of MOB between soil from planted and nonplanted microcosms (mean, 7.7 x 10(sup5) g [fresh weight](sup-1)). Thus, the densely rooted soil at 1 to 2 cm deep did not represent rhizospheric soil with respect to the number of MOB. A significantly increased number of MOB was found only in soil immediately around the roots (1.2 x 10(sup6) g [fresh weight](sup-1)), corresponding to a layer of 0.1 to 0.2 mm. Plant-associated CH(inf4) oxidation was shown in a double chamber with carefully washed intact rice plants. Up to 90% of the CH(inf4) supplied to the root compartment was oxidized in the plants. CH(inf4) oxidation on isolated roots was higher and had a larger variability than that in soil slurries. Roots had an apparent K(infm) for CH(inf4) of 6 (mu)M and a V(infmax) of 5 (mu)mol g (dry weight)(sup-1) h(sup-1). The average number of MOB in homogenized roots was larger than on the rhizoplane and increased with plant age. MOB also were found in surface-sterilized roots and basal culms, indicating the ability of these bacteria to colonize the interior of roots and culms.     

Capacity for Methane Oxidation in Landfill Cover Soils Measured in Laboratory-Scale Soil Microcosms

Laboratory-scale soil microcosms containing different soils were permeated with CH(inf4) for up to 6 months to investigate their capacity to develop a methanotrophic community. Methane emissions were monitored continuously until steady states were established. The porous, coarse sand soil developed the greatest methanotrophic capacity (10.4 mol of CH(inf4) (middot) m(sup-2) (middot) day(sup-1)), the greatest yet reported in the literature. Vertical profiles of O(inf2), CH(inf4), and methanotrophic potential in the soils were determined at steady state. Methane oxidation potentials were greatest where the vertical profiles of O(inf2) and CH(inf4) overlapped. A significant increase in the organic matter content of the soil, presumably derived from methanotroph biomass, occurred where CH(inf4) oxidation was greatest. Methane oxidation kinetics showed that a soil community with a low methanotrophic capacity (V(infmax) of 258 nmol (middot) g of soil(sup-1) (middot) h(sup-1)) but relatively high affinity (k(infapp) of 1.6 (mu)M) remained in N(inf2)-purged control microcosms, even after 6 months without CH(inf4). We attribute this to a facultative, possibly mixotrophic, methanotrophic microbial community. When purged with CH(inf4), a different methanotrophic community developed which had a lower affinity (k(infapp) of 31.7 (mu)M) for CH(inf4) but a greater capacity (V(infmax) of 998 nmol (middot) g of soil(sup-1) (middot) h(sup-1)) for CH(inf4) oxidation, reflecting the enrichment of an active high-capacity methanotrophic community. Compared with the unamended control soil, amendment of the coarse sand with sewage sludge enhanced CH(inf4) oxidation capacity by 26%; K(inf2)HPO(inf4) amendment had no significant effect, while amendment with NH(inf4)NO(inf3) reduced the CH(inf4) oxidation capacity by 64%. In vitro experiments suggested that NH(inf4)NO(inf3) additions (10 and 71 (mu)mol (middot) g of soil(sup-1)) inhibited CH(inf4) oxidation by a nonspecific ionic effect rather than by specific inhibition by NH(inf4)(sup+).     

In Vitro Studies on Reductive Vinyl Chloride Dehalogenation by an Anaerobic Mixed Culture

Reductive dehalogenation of vinyl chloride (VC) was studied in an anaerobic mixed bacterial culture. In growth experiments, ethene formation from VC increased exponentially at a rate of about 0.019 h(sup-1). Reductive VC dehalogenation was measured in vitro by using cell extracts of the mixed culture. The apparent K(infm) for VC was determined to be about 76 (mu)M; the V(infmax) was about 28 nmol (middot) min(sup-1) (middot) mg of protein(sup-1). The VC-dehalogenating activity was membrane associated. Propyl iodide had an inhibitory effect on the VC-dehalogenating activity in the in vitro assay. However, this inhibition could not be reversed by illumination. Cell extracts also catalyzed the reductive dehalogenation of cis-1,2-dichloroethene (cis-DCE) and, at a lower rate, of trichloroethene (TCE). Tetrachloroethene (PCE) was not transformed. The results indicate that the reductive dehalogenation of VC and cis-DCE described here is different from previously reported reductive dehalogenation of PCE and TCE.     

Highly Active Microbial Communities in the Ice and Snow Cover of High Mountain Lakes

An exploratory study carried out in Pyrenean and Alpine lakes shows that a rich, active microbial community lives in the slush layers of the winter cover of such lakes in spite of the low temperature and the seasonal occurrence of the habitat. Bacteria were very diverse in morphology, with filaments reaching up to 100 (mu)m long; flagellates, both autotrophic (chrysophytes, cryptophytes, dinoflagellates, and volvocales) and heterotrophic, and ciliates were abundant, reaching biovolume values up to 2.7 x 10(sup6) (mu)m(sup3) ml(sup-1). Species composition was very variable, with dominance depending on date and depth. Although many species were typical of lake plankton communities, some were restricted to the slush, for instance the predatory ciliates Dileptus sp. and Lacrymaria sp., and others were restricted to the surface pools, such as the snow algae Chlamydomonas nivalis. Microbial biomasses and usually bacterial and algal activities were greater in the slush layers than in the lake water. Photosynthesis rate in the upper cover layers reached values up to 0.5 (mu)g of C liter(sup-1) h(sup-1), and high bacterial activities up to 226 pmol of leucine incorporated liter(sup-1) h(sup-1) and 25 pmol of thymidine incorporated liter(sup-1) h(sup-1) were measured. For most species, lake water flooding the ice and snow cover could provide an inoculum. Differential growth depending on the environmental conditions (nutrients, organic matter, light) of a particular slush layer could provide dominance of different groups or species. However, there was no obvious colonizing mechanism for those species not appearing either in plankton or in communities on top of the snowpack.     

The Low Biomass Yields of the Acetic Acid Bacterium Acetobacter pasteurianus Are Due to a Low Stoichiometry of Respiration-Coupled Proton Translocation

Growth energetics of the acetic acid bacterium Acetobacter pasteurianus were studied with aerobic, ethanol-limited chemostat cultures. In these cultures, production of acetate was negligible. Carbon limitation and energy limitation were also evident from the observation that biomass concentrations in the cultures were proportional to the concentration of ethanol in the reservoir media. Nevertheless, low concentrations of a few organic metabolites (glycolate, citrate, and mannitol) were detected in culture supernatants. From a series of chemostat cultures grown at different dilution rates, the maintenance energy requirements for ethanol and oxygen were estimated at 4.1 mmol of ethanol (middot) g of biomass(sup-1) (middot) h(sup-1) and 11.7 mmol of O(inf2) (middot) g of biomass(sup-1) (middot) h(sup-1), respectively. When biomass yields were corrected for these maintenance requirements, the Y(infmax) values on ethanol and oxygen were 13.1 g of biomass (middot) mol of ethanol(sup-1) and 5.6 g of biomass (middot) mol of O(inf2)(sup-1), respectively. These biomass yields are very low in comparison with those of other microorganisms grown under comparable conditions. To investigate whether the low growth efficiency of A. pasteurianus might be due to a low gain of metabolic energy from respiratory dissimilation, (symbl)H(sup+)/O stoichiometries were estimated during acetate oxidation by cell suspensions. These experiments indicated an (symbl)H(sup+)/O stoichiometry for acetate oxidation of 1.9 (plusmn) 0.1 mol of H(sup+)/mol of O. Theoretical calculations of growth energetics showed that this low (symbl)H(sup+)/O ratio adequately explained the low biomass yield of A. pasteurianus in ethanol-limited cultures.     

The Importance of Hydrogen in Landfill Fermentations

Forty-two samples taken from two landfills were monitored for CH(inf4) production and apparent steady-state H(inf2) concentration. The rates of methanogenesis in these samples ranged from below the detection limit to 1,900 (mu)mol kg (dry weight)(sup-1) day(sup-1), and the median steady-state hydrogen concentration was 1.4 (mu)M in one landfill and 5.2 (mu)M in the other. To further investigate the relationship between hydrogen concentration and methanogenesis, a subset of seven landfill samples was selected on basis of their rates of CH(inf4) production, H(inf2) concentrations, sample pHs, and moisture contents. Samples with H(inf2) concentrations of <20 nM had relatively small amounts of volatile fatty acids (VFAs) (undetectable to 18.6 mmol of VFA kg [dry weight](sup-1)), while samples with H(inf2) concentrations of >100 nM had relatively high VFA levels (133 to 389 mmol of VFA kg [dry weight](sup-1)). Samples with high H(inf2) and VFA contents had relatively low pH values (<=6.3). However, methanogenic and syntrophic bacteria were present in all samples, so the lack of methanogenesis in some samples was not due to a lack of suitable inocula. The low rates of methanogenesis in these samples were probably due to inhibitory effects of low pH and VFA accumulation, resulting from a thermodynamic uncoupling of fatty acid oxidation. As in other anaerobic ecosystems, H(inf2) is a critical intermediate that may be used to monitor the status of landfill fermentations.     

Long Lag Times and High Velocities in the Motility of Natural Assemblages of Marine Bacteria

The motility characteristics of natural assemblages of coastal marine bacteria were examined. Initially, less than 10% of the bacteria were motile. A single addition of tryptic soy broth caused an increase in the motile fraction of cells but only after 7 to 12 h. Motility peaked at 15 to 30 h, when more than 80% of cells were motile. These results support the proposal that energy limits motility in the marine environment. Cell speeds changed more than an order of magnitude on timescales of milliseconds and hours. The maximum community speed was 144 (mu)m s(sup-1), and the maximum individual burst velocity was 407 (mu)m s(sup-1). In uniform medium, speed was an inverse function of tryptic soy broth concentration, declining linearly over 0.001 to 1.0%. In media where concentration gradients existed, the mean speed was a function of position in a spatial gradient, changing from 69 to 144 (mu)m s(sup-1) over as little as 15 to 30 (mu)m. The results suggest that marine bacteria are capable of previously undescribed quick shifts in speed that may permit the bacteria to rapidly detect and keep up with positional changes in small nutrient sources. These high speeds and quick shifts may reflect the requirements for useful motility in a turbulent ocean.     

Isolation and Characterization of a Facultatively Aerobic Bacterium That Reductively Dehalogenates Tetrachloroethene to cis-1,2-Dichloroethene

A rapidly-growing facultatively aerobic bacterium that transforms tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-1,2-DCE) at high rates in a defined medium was isolated from a contaminated site. Metabolic characterization, cellular fatty acid analysis, and partial sequence analysis of 16S rRNA showed that the new isolate, strain MS-1, has characteristics matching those of the members of the family Enterobacteriaceae. Strain MS-1 can oxidize about 58 substrates including many carbohydrates, short-chain fatty acids, amino acids, purines, and pyrimidines. It can transform up to 1 mM PCE (aqueous) at a rate of about 0.5 (mu)mol of PCE(middot) h(sup-1)(middot)mg (dry weight) of cell(sup-1). PCE transformation occurs following growth on or with the addition of single carbon sources such as glucose, pyruvate, formate, lactate, or acetate or with complex nutrient sources such as yeast extract or a mixture of amino acids. PCE dehalogenation requires the absence of oxygen, nitrate, and high concentrations of fermentable compounds such as glucose. Enterobacter agglomerans biogroup 5 (ATCC 27993), a known facultative bacterium that is closely related to strain MS-1, also reductively dehalogenated PCE to cis-1,2-DCE. To our knowledge, this is the first report on isolation of a facultative bacterium that can reductively transform PCE to cis-1,2-DCE under defined physiological conditions. Also, this is the first report of the ability of E. agglomerans to dehalogenate PCE.     

Evidence for Intracellular and Extracellular Dimethylsulfoniopropionate (DMSP) Lyases and DMSP Uptake Sites in Two Species of Marine Bacteria

The kinetics of dimethylsulfoniopropionate (DMSP) uptake and dimethylsulfide (DMS) production from DMSP in two bacterial species, Alcaligenes sp. strain M3A, an isolate from estuarine surface sediments, and Pseudomonas doudoroffii, from seawater, were investigated. In Alcaligenes cells induced for DMSP lyase (DL) activity, DMS production occurred without DMSP uptake. In DL-induced suspensions of P. doudoroffii, uptake of DMSP preceded the production of DMS, indicating an intracellular location of DL; intracellular DMSP levels reached ca. 7 mM. DMSP uptake rates in noninduced cells showed saturation at three concentrations (K(inft) [transport] values, 3.4, 127, and 500 (mu)M). In DL-induced cells of P. doudoroffii, DMSP uptake rates increased ca. threefold (V(infmax), 0.022 versus 0.065 (mu)mol of DMSP taken up min(sup-1) mg of cell protein(sup-1)), suggesting that the uptake binding proteins were inducible. DMSP uptake and DL activity in P. doudoroffii were both inhibited by CN(sup-), 2,4-dinitrophenol, and membrane-impermeable thiol-binding reagents, further indicating active uptake of DMSP by cell surface components. The respiratory inhibitors had limited or no effect on DL activity by the Alcaligenes sp. Of the structural analogs of DMSP tested for their effect on DMSP metabolism, glycine betaine (GBT), but not methyl-3-mercaptopropionic acid (MMPA), inhibited DMSP uptake by P. doudoroffii, suggesting that GBT shares a binding protein with DMSP and that MMPA is taken up at a separate site. Two models of DMSP uptake, induction, and DL location found in marine bacteria are presented.     

Glutathione-S-Transferase Activity and Metabolism of Glutathione Conjugates by Rhizosphere Bacteria

Glutathione-S-transferase (GST) activity was determined in 36 species of rhizosphere bacteria with the substrate 1-chloro-2,4-dinitrobenzene (CDNB) and in 18 strains with the herbicide alachlor. Highest levels of CDNB-GST activity (60 to 222 nmol (middot) h(sup-1) (middot) mg(sup-1)) were found in gram-negative bacteria: Enterobacter cloacae, Citrobacter diversus, Klebsiella planticola, Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, and Xanthomonas campestris. There was very low CDNB-GST activity in the gram-positive strains. Rapid metabolism of CDNB-glutathione conjugates, attributable to high levels of (gamma)-glutamyltranspeptidase, also occurred in the gram-negative bacteria, especially pseudomonads. Alachlor-GST activity detected in cell extracts and whole-cell suspensions of some strains of the families Enterobacteriaceae and Pseudomonaceae was 50- to 100-fold lower than CDNB-GST activity (0.5 to 2.5 nmol (middot) h(sup-1) (middot) mg(sup-1)) and was, for the most part, constitutive. The glutathione-alachlor conjugate was rarely detected. Cysteineglycine and/or cysteine conjugates were the major products of alachlor-GST metabolism. Whole-cell suspensions of certain Pseudomonas spp. dechlorinated from 20 to 75% of 100 (mu)M alachlor in 24 h. Results indicate that rhizosphere bacteria, especially fluorescent pseudomonads, may play an important role in the degradation of xenobiotics such as alachlor via GST-mediated reactions.     

Use of Radiolabelled Thymidine and Leucine To Estimate Bacterial Production in Soils from Continental Antarctica

Tritiated thymidine incorporation (TTI) into DNA was used to examine bacterial production in two soil types from the Robertskollen group of nunataks in northwestern Dronning Maud Land, providing the first estimates of bacterial production in soil habitats on the Antarctic continent. Although estimates of bacterial productivity in soils near to bird nests (344 (plusmn) 422 ng of C g [dry weight](sup-1) h(sup-1)) were higher than those for soils from beneath mosses (175 (plusmn) 90 ng of C g [dry weight](sup-1) h(sup-1); measured by TTI at 10(deg)C), these differences were not significant because of patchiness of bacterial activity (P > 0.05). TTI- and [(sup14)C]leucine ([(sup14)C]Leu)-derived estimates of bacterial production were similar when incubations of 3 h were used, although incubations as short as 1 h were sufficient for measurable uptake of radiolabel. Dual-label incorporation of [(sup3)H]thymidine ([(sup3)H]TdR) into DNA and [(sup14)C]Leu into protein indicated that TTI did not reflect bacterial production of in situ assemblages when incubations were longer than 3 h. Isotope dilution analysis indicated that dilution of the specific activity of exogenously supplied [(sup3)H]TdR by de novo synthesis of TdR precursor could be limited by additions of [(sup3)H]TdR at a concentration of 1 nmol per ca. 115 mg of soil. TTI exhibited a psychrotrophic response to variation in temperature, with a temperature optimum of ca. 15(deg)C and a Q(inf10) value for 0 to 10(deg)C of 2.41.     

Iron Stress and Pyoverdin Production by a Fluorescent Pseudomonad in the Rhizosphere of White Lupine (Lupinus albus L.) and Barley (Hordeum vulgare L.)

Induction of high-affinity iron transport during root colonization by Pseudomonas fluorescens Pf-5 (pvd-inaZ) was examined in lupine and barley growing in microcosms. P. fluorescens Pf-5 (pvd-inaZ) contains a plasmid carrying pvd-inaZ; thus, in this strain, ice nucleation activity is regulated by pyoverdin production. Lupine or barley plants were grown for 18 or 8 days, respectively, in soil amended with 2% calcium carbonate and inoculated with P. fluorescens Pf-5 (pvd-inaZ) at a density of 4 x 10(sup8) CFU g (dry weight) of soil(sup-1). A filter paper blotting technique was used to sample cells from the rhizosphere in different root zones, and then the cells were resuspended for enumeration and measurement of ice nucleation activity. The population density of P. fluorescens Pf-5 (pvd-inaZ) in the rhizosphere decreased by one order of magnitude in both lupine and barley over time. The ice nucleation activity ranged from -3.4 to -3.0 log ice nuclei CFU(sup-1) for lupine and -3.0 to -2.8 log ice nuclei CFU(sup-1) for barley, was similar in all root zones, and did not change over time. An in vitro experiment was conducted to determine the relationship between ice nucleation activity and pyoverdin production in P. fluorescens Pf-5 (pvd-inaZ). An ice nucleation activity of approximately -3.0 log ice nuclei CFU(sup-1) was measured in the in vitro experiment at 25 to 50 (mu)M FeCl(inf3). By using the regression between ice nucleation activity and pyoverdin production determined in vitro and assuming a P. fluorescens Pf-5 (pvd-inaZ) population density of 10(sup8) CFU g of root(sup-1), the maximum possible pyoverdin accumulation by P. fluorescens Pf-5 (pvd-inaZ) in the rhizosphere was estimated to be 0.5 and 0.8 nmol g of root(sup-1) for lupine and barley, respectively. The low ice nucleation activity measured in the rhizosphere suggests that nutritional competition for iron in the rhizosphere may not be a major factor influencing root colonization by P. fluorescens Pf-5 (pvd-inaZ).     

A Nitrite Microsensor for Profiling Environmental Biofilms

A highly selective liquid membrane nitrite microsensor based on the hydrophobic ion-carrier aquocyanocobalt(III)-hepta(2-phenylethyl)-cobrynate is described. The sensor has a tip diameter of 10 to 15 (mu)m. The response is log-linear in freshwater down to 1 (mu)M NO(inf2)(sup-) and in seawater to 10 (mu)M NO(inf2)(sup-). A method is described for preparation of relatively large polyvinyl chloride (PVC)-gelled liquid membrane microsensors with a tip diameter of 5 to 15 (mu)m, having a hydrophilic coating on the tip. The coating and increased tip diameter resulted in more sturdy sensors, with a lower detection limit and a more stable signal than uncoated nitrite sensors with a tip diameter of 1 to 3 (mu)m. The coating protects the sensor membrane from detrimental direct contact with biomass and can be used for all PVC-gelled liquid membrane sensors meant for profiling microbial mats, biofilms, and sediments. Thanks to these improvements, liquid membrane sensors can now be used in complex environmental samples and in situ, e.g., in operating bioreactors. Examples of measurements in denitrifying, nitrifying, and nitrifying/denitrifying biofilms from wastewater treatment plants are shown. In all of these biofilms high nitrite concentrations were found in narrow zones of less than 1 mm.     

Effects of Temperature, Water Activity, and Incubation Time on Production of Aflatoxins and Cyclopiazonic Acid by an Isolate of Aspergillus flavus in Surface Agar Culture

An experiment with a full factorial design was used to study the effects of and interactions among temperature, water activity (a(infw)), incubation period, and substrate on coproduction of aflatoxins (AF) and cyclopiazonic acid (CPA) by an isolate of Aspergillus flavus. Analysis of variance showed that there was a complex interaction among all of these factors and that this influenced the relative concentrations of the mycotoxins produced. The optimum temperatures for the production of AF and CPA were 30(deg)C and 25(deg)C, respectively. Both mycotoxins were maximally produced (0.306 to 0.330 (mu)g of AF(middot)ml of medium(sup-1), 4.040 to 6.256 (mu)g of CPA(middot)ml of medium(sup-1)) at an a(infw) of 0.996 and after 15 days of incubation. No AF were produced in either yeast extract agar or Czapek yeast autolysate agar medium at an a(infw) of 0.90 at 20 or 37(deg)C after 15 days (minimum conditions), while 0.077 to 0.439 (mu)g of CPA(middot)ml of medium(sup-1) was produced under the same conditions. Yeast extract agar favored maximum AF production, and Czapek yeast autolysate agar favored maximum CPA production.