Pure Culture Experiments Research Articles

Activated sludge deflocculation in response to chlorine addition: the potassium connection.

Chlorination is often used to control filamentous bulking in activated sludge systems. Pure culture and mixed-liquor experiments showed that soluble potassium (K+) concentrations increased by 2.4 mg/L (80%) and 1.5 to 3.6 mg/L (11 to 30%) in the bulk liquid phase of pure and activated sludge cultures that were exposed to chlorine, relative to unchlorinated controls. Effluent turbidity and total suspended solids from settled mixed liquor increased significantly in both short-term batch and sequencing batch reactor experiments when chlorine mass load increased above 6 milligrams of chlorine per gram mixed liquor volatile suspended solids (mg Cl2/g MLVSS) in a single dose, which correlated with a localized chlorine concentration at the dose point of 10 mg/L as Cl2 or greater. The results support the hypothesis that the glutathione-gated potassium efflux (GGKE) bacterial stress response may contribute to increased effluent turbidity associated with high doses of mixed-liquor chlorination. It is suggested that potassium is a useful parameter to monitor at full-scale facilities when determining chlorine mass doses that should be used to control filaments and minimize increases in effluent turbidity.

Use of chlorate as a selective inhibitor to distinguish membrane-bound nitrate reductase (Nar) and periplasmic nitrate reductase (Nap) of dissimilative nitrate reducing bacteria in sediment

The use of chlorate as a selective inhibitor of dissimilative nitrate reduction was studied using pure cultures of Comamonas testosteroni (a denitrifier) and Klebsiella pneumoniae (a nitrate-ammonifier) isolated from estuarine sediment, and in sediment slurry. Pure culture experiments demonstrated that chlorate selectively inhibited membrane-bound nitrate reductase (Nar) activity, probably by blocking nitrate transporters (NarK). Sediment slurry experiments showed that chlorate inhibited nitrate reduction and N 2O formation, but did not inhibit nitrite reduction and its N 2O formation, indicating that chlorate selectively inhibited only the first step of nitrate reduction. Chlorite chemically oxidized nitrite to nitrate and could not be used as a selective inhibitor of nitrite metabolism, although chlorite apparently selectively inhibited formation of N 2O from nitrite. Chlorate can be used as a specific inhibitor to distinguish between nitrate reduction by Nap or Nar in natural communities of microorganisms.

Linking autotrophic activity in environmental samples with specific bacterial taxa by detection of 13C‐labelled fatty acids

A method for the detection of physiologically active autotrophic bacteria in complex microbial communities was developed based on labelling with the stable isotope 13C. Labelling of autotrophic nitrifying, sulphur-oxidizing and iron-oxidizing populations was performed in situ by incubation with NaH[13C]O3. Incorporated label into fatty acid methyl esters (FAMEs) was detected and quantified using gas chromatography-mass spectrometry in single ion monitoring mode. Before the analyses of different environmental samples, the protocol was evaluated in pure culture experiments. In different environmental samples a selective labelling of fatty acids demonstrated which microbial taxa were responsible for the respective chemolithoautotrophic activity. The most strongly labelled fatty acids of a sample from a sulphide treating biofilter from an animal rendering plant were cis-7-hexadecenoic acid (16:1 cis7) and 11-methyl hexadecanoic acid (16:0 11methyl), which are as-yet not known for any sulphide-oxidizing autotroph. The fatty acid labelling pattern of an experimental biotrickling filter sample supplied with dimethyl disulphide clearly indicated the presence and activity of sulphide-oxidizing bacteria of the genus Thiobacillus. For a third environmental sample from an acid mining lake sediment, the assignment of autotrophic activity to bacteria of the genus Leptospirillum but not to Acidithiobacillus could be made by this method, as the fatty acid patterns of these bacteria show clear differences.

The Role of Enhanced Heterotrophic Bacterial Growth on Iron Oxidation by Acidithiobacillus ferrooxidans

Addition of organic carbon substrate (glucose) profoundly affected the growth of cultures of acidophilic bacteria typical of acid mine drainage (AMD) sites: the iron-oxidizing autotrophic bacteria, Acidithiobacillus ferrooxidans , and a common heterotrophic strain, Acidiphilium acidophilum . Growth of A. ferrooxidans on soluble ferrous iron media was significantly inhibited in the presence of 1,000 mg/L glucose, regardless of the initial cell density, and in spite of favorable pH and aeration conditions. Interestingly, inhibition of A. ferrooxidans was reduced with addition of the heterotroph, apparently due to the consumption of glucose because the onset of iron oxidation coincided with reduction in glucose concentration in the medium. Another mechanism, local production of CO 2 by A. acidophilum provided inorganic carbon required by A. ferrooxidans cells, was investigated. Although no direct proof of interspecies CO 2 exchange was identified, iron oxidation was enhanced and glucose inhibition reduced with incubation of A. ferrooxidans cultures with 5% CO 2 in air. When oxygen was limited and glucose was added to the acidic coculture, the ultimate amount of iron oxidized was significantly lower and the ferric iron produced was subsequently reduced by the heterotrophs as conditions became anoxic. Attribution of ferric iron reduction to A. acidophilum was confirmed in pure culture experiments where a zero-order iron-reduction rate of 458 - 26.9 w mol Fe/L-day (25.6 - 1.5 mg Fe/L-day) was observed. Bacterial iron reduction also led to an increase in pH from 2.5 to 4.0. Thus, the addition of glucose or some other organic electron donor could provide an in situ or ex situ bioremediation strategy to raise pH at AMD sites resulting in a lower amount of metal leaching into drainage water by promoting reducing conditions favorable to iron reduction.

Microbial fossilization in carbonate sediments: a result of the bacterial surface involvement in dolomite precipitation

ABSTRACT Recent dolomitic sediment samples from Lagoa Vermelha, Brazil, were examined microscopically to study the process of bacterial fossilization in carbonate sediments. Bacteria‐like bodies were intimately associated with carbonate mineral surfaces, and coatings on the former demonstrate the calcification of single bacterial cells. The bacterial fossilization process in Lagoa Vermelha sediments was simulated in the laboratory by cultivation of mixed and pure cultures of sulphate‐reducing bacteria, which were isolated from the Lagoa Vermelha sediments. These cultures produced carbonate minerals that were studied to provide insight into the initiation of the fossilization process. In mixed culture experiments, bacterial colonies became calcified, whereas in pure culture experiments, single bacterial cells were associated with dolomite surfaces. Dolomite nucleated exclusively in bacterial colonies, intimately associated with extracellular organic matter and bacterial cells. Electrophoretic mobility measurements of the bacterial cells in electrolyte solutions demonstrated the specific adsorption of Ca2+ and Mg2+ onto the cell surfaces, indicating the role of the bacterial surface in carbonate nucleation and bacterial fossilization. The affinity of the cells for Mg2+ was related to the capability of the strains to mediate dolomite formation. Combined with sulphate uptake, which dissociates the [MgSO4]0 ion pair and increases the Mg2+ availability, the concentration of Mg2+ ions in the microenvironment around the cells, where the conditions are favourable for dolomite precipitation, may be the key to overcome the kinetic barrier to dolomite formation. These results demonstrate that bacterial fossilization is a consequence of the cell surface involvement in carbonate precipitation, implying that fossilized bacterial bodies can be used as a tool to recognize microbially mediated carbonates.

The effect of temperature and pH on the growth of lactic acid bacteria: a pH-auxostat study

The growth of Streptococcus salivarius subsp. thermophilus, Lactococcus lactis subsp. lactis, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus and Lactobacillus paracasei was studied in a pH-auxostat. The computer-controlled smooth change of pH or temperature was used to study the effect of pH and temperature on the culture characteristics (specific growth rate μ, growth yield Y ATP and specific lactate production rate Q ATP). The behaviour of μ and Q ATP with an increase of temperature suggested that ATP production capacity was an important factor in determining the maximum growth rate of lactic acid bacteria (LAB). The decrease of Y ATP with increase of temperature resulted in the decrease of specific growth rate while Q ATP remained constant or even increased. With the decrease of pH, decrease of both Y ATP and Q ATP was observed. S. thermophilus St20 having the highest maximum specific growth rate of 2.2 h −1 at 44 °C was the most acid sensitive strain. The L. paracasei E1H3 having the lowest μ max was the most tolerant strain to pH change. The behaviour of mixed culture of L. bulgaricus and S. thermophilus in milk in a pH-auxostat with pH-decrease was in agreement with the pure culture experiments of the same species. The study showed that pH-auxostat with smooth controlled change of pH and temperature is an effective method for determination of technological characteristics and comparative physiological study of LAB.

Competitive growth of Gordonia and Acinetobacter in continuous flow aerobic and anaerobic/aerobic reactors

Gordonia amarae and Acinetobacter calcoaceticus pure culture experiments were conducted in continuous flow chemostats fed with an acetate/mineral salts medium under aerobic and anaerobic-aerobic conditions. The growth parameters such as volatile suspended solids (VSS) suggest that Acinetobacter can successfully out-compete Gordonia under both aerobic and anaerobicaerobic conditions when both are present in a dual culture. Gordonia was reduced to near detection limits (<10 mg VSS/1), while Acinetobacter successfully grew to about 300 mg VSS/1 when both were grown as a dual culture in a single-stage aerobic chemostat with an initial concentration of 200 mg VSS/1 each. In a two-stage anaerobic-aerobic chemostat with recycling, Gordonia was completely washed out in about 24 h, whereas, Acinetobacter successfully grew to over 170% of its initial concentration. In a single-stage aerobic chemostat, a mathematical model describing the competitive growth of the two species based on single culture kinetic parameters predicted Gordonia washout in <30, and the same was observed from experiments. The model predicted an Acinetobacter concentration of 200% of its initial concentration in about 30 h, while experimentally this value was about 150%. Under anaerobic-aerobic conditions, the two-stage model predicted Gordonia washout and an Acinetobacter concentration of about 170% of the initial concentration. The experimental results agreed with the prediction, but the decrease in the Gordonia and increase in the Acinetobacter concentrations were much more rapid than predicted by the model.

ACTIVATED SLUDGE DEFLOCCULATION IN RESPONSE TO CHLORINE ADDITION: THE POTASSIUM CONNECTION

Chlorination is often used to control filamentous bulking in activated sludge systems. Pure culture and mixed-liquor experiments showed that soluble potassium (K+) concentrations increased by 2.4 mg/L (80%) and 1.5 to 3.6 mg/L (11 to 30%) in the bulk liquid phase of pure and activated sludge cultures that were exposed to chlorine, relative to unchlorinated controls. Effluent turbidity and total suspended solids from settled mixed liquor increased significantly in both short-term batch and sequencing batch reactor experiments when chlorine mass load increased above 6 milligrams of chlorine per gram mixed liquor volatile suspended solids (mg Cl2/g MLVSS) in a single dose, which correlated with a localized chlorine concentration at the dose point of 10 mg/L as Cl2 or greater. The results support the hypothesis that the glutathione-gated potassium efflux (GGKE) bacterial stress response may contribute to increased effluent turbidity associated with high doses of mixed-liquor chlorination. It is suggested that potassium is a useful parameter to monitor at full-scale facilities when determining chlorine mass doses that should be used to control filaments and minimize increases in effluent turbidity.

Decolorization of reactive dyes by the white rot fungus Trametes versicolor in sequencing batch reactors.

The white rot fungus Trametes versicolor was shown to be capable of decolorizing three reactive dyes in a sequencing batch process, using glucose as the carbon and energy source over an extended period without supplementation of new mycelium. Decolorization activity was related to the expression of extracellular peroxidases and could be continuously reactivated by sheering the suspended pellets. Pure culture experiments were carried out simultaneously in agitated Erlenmeyer flasks and in completely stirred tank reactors with two azo dyes, C.I. Reactive Black 5 and C.I. Reactive Red 198 as well as the anthraquinone dye C.I. Reactive Blue 19 (Brilliant Blue R). Results show high and stable degrees of decolorization of 91%-99% in both systems, which could be repeated without decrease in activity over time. Under nonsterile conditions only five cycles of decolorization could be achieved. An increasing bacterial population suppressed fungal growth and the formation of peroxidases.

Use of isotopic and molecular techniques to link toluene degradation in denitrifying aquifer microcosms to specific microbial populations.

Microcosms were inoculated with sediments from both a petroleum-hydrocarbon (PHC)-contaminated aquifer and from a nearby pristine aquifer and incubated under anoxic denitrifying conditions with [methyl-13C]toluene. These microcosms served as a laboratory model system to evaluate the combination of isotope (13C-labeling of polar-lipid-derived fatty acids) and molecular techniques (16S rRNA-targeting gene probes) to identify the toluene-metabolizing population. After total depletion of toluene, the following bacterial phospholipid fatty acids (PLFA) were 13C-enriched: 16:1omega7c, 16:1omega7t, 16:0, cy17:0, and 18:1omega7c. Pure culture experiments demonstrated that these compounds were also found in PLFA profiles of PHC-degrading Azoarcus spp. (beta-Proteobacteria) and related species. The origin of the CO2 evolved in the microcosms was determined by measurements of stable carbon isotope ratios. Toluene represented 11% of the total pool of mineralized substrates in the contaminated sediment and 54% in the pristine sediment. The microbial community in the microcosm incubations was characterized by using DAPI staining and whole-cell hybridization with specific fluorescently labeled 16S rRNA-targeted oligonucleotide probes. Results revealed that 6% of the DAPI-stained cells in the contaminated sediment and 32% in the pristine sediment were PHC-degrading Azoarcus spp. In biotic control microcosms (incubated under denitrifying conditions, no toluene added), Azoarcus spp. cells remained at less than 1% of the DAPI-stained cells. The results show that isotope analysis in combination with whole-cell hybridization is a promising approach to identify and to quantify denitrifying toluene degraders within microbial communities.

Reclamation of an activated-sludge microbial consortium by selective biostimulation.

Our previous study showed that an activated-sludge process broke down at the phenol-loading rate of 1.5 g l(-1) day(-1), when non-flocculating bacteria (called R6T and R10) overgrew the sludge, resulting in a sludge washout. In this study, we attempted to circumvent this breakdown problem by reclaiming the consortium structure. Activated sludge was fed phenol, and the phenol-loading rate was increased stepwise from 0.5 g l(-1) day(-1) to 1.0 g l(-1) day(-1) and then to 1.5 g l(-1) day(-1). Either galactose or glucose (at 0.5 g l(-1) day(-1)) was also supplied to the activated sludge from the phenol-loading rate of 1.0 g l(-1) day(-1). Pure culture experiments have suggested galactose to be a preferential substrate for a floc-forming bacterium (R6F) that predominantly degrades phenol under low phenol-loading conditions. Supplying galactose allowed sustainment of the R6F population and suppression of the overgrowth of R6T and R10 at the phenol-loading rate of 1.5 g l(-1) day(-1). This measure allowed the activated-sludge process to treat phenol at a phenol-loading rate up to 1.5 g l(-1) day(-1), although it broke down at 2.0 g l(-1) day(-1). In contrast, supplying glucose reduced the R6F population and allowed the activated-sludge process to break down at the phenol-loading rate of 1.0 g l(-1) day(-1). This study demonstrated that reclamation of the activated-sludge consortium by selective biostimulation of the floc-forming population improved the phenol-treating ability of the process.

Use of Hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine as a Nitrogen Source in Biological Treatment of Munitions Wastes

The purpose of this study was to develop a biological treatment process for the degradation of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX), a nitramine explosive present in munitions manufacturing and processing wastes. A real waste mixture containing RDX, nitrate and nitrite (NO x), and several solvents (primarily cyclohexanone and acetone) was used as basic feed for bench‐scale experiments. Pure culture experiments indicated that bacteria can use RDX as a nitrogen source and organic solvents as carbon sources under aerobic conditions. In addition, it has been found that inorganic nitrogen compounds might interfere with the use of RDX as a nitrogen source. Because NOx are problematic constituents that should be removed anyway, anaerobic microbial denitrification was applied as a pretreatment stage to simultaneously remove NOx and organics and to form a nitrogen‐deficient environment for further aerobic degradation of RDX and residual organics. This conceptual approach was performed using two reactor systems: a two‐stage anaerobic–aerobic system and a single reactor with anaerobic and aerobic stages in its operation cycle. All laboratory reactors were operated in the sequencing batch reactor mode. Both systems achieved complete NOx removal in the denitrification stage with acetone serving as the carbon source and complete aerobic biodegradation of RDX with cyclohexanone serving as the carbon source.

Development of a Protocol to Retrieve Microorganisms from Ancient Salt Crystals

Previous research has indicated that halophilic microorganisms are associated with salt crystals from ancient formations. However,this research has generally failed to convincingly demonstrate that these organisms were originally trapped inside the crystals during their deposition and were not recent surface contaminants. This paper presents techniques for crystal selection; verifiable, noninvasive, surface sterilization of salt crystals; and sterile extraction of biological material from the crystals. Immersing salt crystals in 10 M NaOH for 5 min; rinsing with sterile, saturated brine before immersing for 5 min in 10 M HCl; and rinsing with saturated brine is an effective method for surface sterilization. No growth has resulted from contamination tests of 216 faces from 36 natural salt crystals exposed to these treatments. Pure culture experiments using the halotolerant eubacteria, Halomonas elongata and Bacillus sp. (2-9-3), and the archeon, Halogeometricium borinquense, showed that exposure to either 10 M HCl or NaOH reduced the population of these organisms by a factor of 107 to 108 colony-forming units/ml. The fluid is extracted from inclusions by drilling into surface-sterilized salt crystals with a variable-speed drill using sterilized 0.5-mm-diameter carbide drill bits and extracting the fluid with a sterilized microliter syringe. Drilling and extraction are performed in a Class II biosafety cabinet. All procedures are carried out in a biosafety level 3 facility. All personnel involved wear cleanroom coveralls, shoe covers, hair caps, and gloves. The above protocol has resulted in the isolation of a Bacillus sp. from a Permian-age salt crystal.

Electricity generation in microbial fuel cells using neutral red as an electronophore.

Neutral red (NR) was utilized as an electron mediator in microbial fuel cells consuming glucose to study both its efficiency during electricity generation and its role in altering anaerobic growth and metabolism of Escherichia coli and Actinobacillus succinogenes. A study of chemical fuel cells in which NADH, NR, and ferricyanide were the electron donor, the electronophore, and the electron acceptor, respectively, showed that electrical current produced from NADH was proportional to the concentration of NADH. Fourfold more current was produced from NADH in chemical fuel cells when NR was the electron mediator than when thionin was the electron mediator. In microbial fuel cells in which E. coli resting cells were used the amount of current produced from glucose when NR was the electron mediator (3.5 mA) was 10-fold more than the amount produced when thionin was the electron mediator (0.4 mA). The amount of electrical energy generated (expressed in joules per mole of substrate) and the amount of current produced from glucose (expressed in milliamperes) in NR-mediated microbial fuel cells containing either E. coli or A. succinogenes were about 10- and 2-fold greater, respectively, when resting cells were used than when growing cells were used. Cell growth was inhibited substantially when these microbial fuel cells were making current, and more oxidized end products were formed under these conditions. When sewage sludge (i.e., a mixed culture of anaerobic bacteria) was used in the fuel cell, stable (for 120 h) and equivalent levels of current were obtained with glucose, as observed in the pure-culture experiments. These results suggest that NR is better than other electron mediators used in microbial fuel cells and that sludge production can be decreased while electricity is produced in fuel cells. Our results are discussed in relation to factors that may improve the relatively low electrical efficiencies (1.2 kJ/mol) obtained with microbial fuel cells.

Modelling the interactions between Lactobacillus curvatus and Enterobacter cloacae: II. Mixed cultures and shelf life predictions

The modelling approach presented in this study can be used to predict when interactions between microorganisms in homogenous systems occur. It was tested for the interaction between Lactobacillus curvatus and Enterobacter cloacae. In this binary system, L. curvatus produces lactic acid which decreases the pH in the system. The pH decrease was found to be the main limiting factor of growth of both E. cloacae and L. curvatus. This resulted in E. cloacae reaching its final concentration earlier when compared to its growth in pure culture. The models consisted of a set of first order ordinary differential equations describing the growth, consumption and production rates of both microorganisms. The parameters for these equations were obtained from pure culture studies and from literature. These equations were solved using a combination of analytical and numerical methods. The prediction of growth in mixed culture using parameters from pure culture experiments and literature were close to the experimental data. Both model predictions and experimental validation indicated that interaction occurs when the concentration of L. curvatus reaches 10 8 cfu/ml. At that moment in time, the pH had decreased to inhibiting levels. These concentrations of microorganisms (10 8 cfu/ml ) do occur in fermented products where interactions obviously are important. In nonfermented foods however, this level of microorganisms indicate that spoilage has occurred or is about to start. Microbial interactions can therefore be neglected when predicting shelf life or safety of food products in most cases.

Enumeration and detection of anaerobic ferrous iron-oxidizing, nitrate-reducing bacteria from diverse European sediments.

Anaerobic, nitrate-dependent microbial oxidation of ferrous iron was recently recognized as a new type of metabolism. In order to study the occurrence of three novel groups of ferrous iron-oxidizing, nitrate-reducing bacteria (represented by strains BrG1, BrG2, and BrG3), 16S rRNA-targeted oligonucleotide probes were developed. In pure-culture experiments, these probes were shown to be suitable for fluorescent in situ hybridization, as well as for hybridization analysis of denaturing gradient gel electrophoresis (DGGE) patterns. However, neither enumeration by in situ hybridization nor detection by the DGGE-hybridization approach was feasible with sediment samples. Therefore, the DGGE-hybridization approach was combined with microbiological methods. Freshwater sediment samples from different European locations were used for enrichment cultures and most-probable-number (MPN) determinations. Bacteria with the ability to oxidize ferrous iron under nitrate-reducing conditions were detected in all of the sediment samples investigated. At least one of the previously described types of bacteria was detected in each enrichment culture. MPN studies showed that sediments contained from 1 x 10(5) to 5 x 10(8) ferrous iron-oxidizing, nitrate-reducing bacteria per g (dry weight) of sediment, which accounted for at most 0.8% of the nitrate-reducing bacteria growing with acetate. Type BrG1, BrG2, and BrG3 bacteria accounted for an even smaller fraction (0.2% or less) of the ferrous iron-oxidizing, nitrate-reducing community. The DGGE patterns of MPN cultures suggested that more organisms than those isolated thus far are able to oxidize ferrous iron with nitrate. A comparison showed that among the anoxygenic phototrophic bacteria, organisms that have the ability to oxidize ferrous iron also account for only a minor fraction of the population.

Microbiological safety of kinema, a fermented soya bean food

Kinema is a fermented soya bean food of Nepal and the hilly regions of North-eastern States of India. Generally, the fermentation is dominated by Bacillus spp. that often cause alkalinity and desirable stickiness in the product. The present study was undertaken in a limited number of commercial (market) kinema samples to test for the presence of foodborne pathogens and their properties. Bacillus cereus was present in numbers exceeding 10 4 cfu/g product in five of the tested 15 market samples. Enterobacteriaceae and coliform bacteria exceeded 10 5 cfu/g in 10 of the 15 samples. Escherichia coli exceeding 10 5 cfu/g was found in two samples. Staphylococcus aureus was not detected in any of the tested samples. Of 31 isolated typical and atypical strains of B. cereus, 18 representative strains were tested qualitatively for the ability to produce diarrhoeal type enterotoxin (BCET) using an Oxoid BCET-RPLA test kit. Overall, BCET was formed by 12 strains in BHIG (brain heart infusion broth +1% glucose), by seven strains on sterilized cooked rice, and by five strains on sterilized cooked soya beans. Semi-quantitative tests on BCET revealed that levels exceeding 256 ng/g soya beans, produced by single pure culture inoculation with the isolated B. cereus strains, were reduced to ≤ 8 ng/g by frying kinema in oil, a common procedure when making kinema curry. It was also shown in a mixed pure culture experiment that a kinema strain B. Subtilis DK-W1, is able to suppress growth and BCET formation by a selected toxin producing strain (BC7-5) of B. cereus. It is concluded that the traditional way of making kinema and its culinary use in curries is safe. However, for novel applications of kinema, safety precautions are advisable.

Evidence for differential effects of 2,4,6‐trinitrotoluene and other munitions compounds on specific subpopulations of soil microbial communities

AbstractThe effects of 2,4,6‐trinitrotoluene (TNT) and other munitions compounds on indigenous microbial communities in several soils were examined. Culturable heterotrophs, concentrations of phospholipid fatty acid (PLFA), and basal respiration rates exhibited slight negative correlations with high TNT and 1,3,5‐trinitrobenzene (TNB) levels. Heat‐shock‐resistant culturable heterotrophs, percentage of gram‐positive soil isolates, mole percent of branched PLFA, and 10Me18:0 (tuberculostearic acid) were observed to be significantly lower in highly contaminated soils. Total soil nitrogen levels were positively correlated with high TNT and TNB concentrations, whereas total soil carbon exhibited no significant correlation with either compound. Multivariate analysis of PLFA data resulted in distinct separation of soils with respect to their degree of contamination, with specific signature PLFAs for gram‐positive bacteria, fungi, and protozoa being negatively associated with high contaminant levels. Apparent concentrations of TNT resulting in 50% reductions in indicators of gram‐positive populations were much higher than values from pure culture experiments, possibly as a result of low bioavailability due to sorption onto clay and soil organic matter. Few effects of other munitions compounds were observed. Closer examination of a highly contaminated soil revealed that the number of culturable heterotrophs growing on 0.3% molasses plates decreased by 50% when 67 μg TNT/ml was added to the medium; a 99% decrease was observed for soil contaminated with less than 20 μg TNT/g. Highly contaminated soil harbored a greater number of organisms that were able to grow on plates amended with greater than 10 μg TNT/ml. Gram‐positive isolates from both soils demonstrated marked growth inhibition when greater than 8‐16 μg TNT/ml was present in the culture medium. These results indicate that chronic exposure to munitions compounds can dramatically alter soil microbial communities.

Expression of a nitrogen regulated lux gene fusion in Pseudomonas fluorescens DF57 studied in pure culture and in soil

The biological availability of assimilable nitrogen in soil was assessed by introducing a Pseudomonas fluorescens DF57 reporter strain, carrying a Tn 5:: luxAB insertion in a nitrogen-starvation-inducible gene, displaying no homology with any known genes. Initial pure culture experiments demonstrated that bioluminescence was induced in response to limitation for ammonium or other nitrogen sources that could be easily assimilated by the P. fluorescens strain. Conditions conducive to the expression of bioluminescence existed at ammonium concentrations below 10–90 μM. Upon introduction into sterile or natural soil microcosms a nitrogen-starvation signal was not observed, demonstrating that the cells were not encountering a limited supply of nitrogen sources. However, amendment of the soil with carbon+phosphorus sources led to conditions where a specific nitrogen-starvation signal was observed. Attempts to repress the reporter system by subsequent addition of ammonium were unsuccessful in the soil, but not in pure culture experiments. A possible explanation for this result is that the luciferase enzyme is stable in cells inhabiting the soil environment, imposing limitations to the use of luxAB as a real-time monitoring system.

Expression of a nitrogen regulated lux gene fusion in Pseudomonas fluorescens DF57 studied in pure culture and in soil

The biological availability of assimilable nitrogen in soil was assessed by introducing a Pseudomonas fluorescens DF57 reporter strain, carrying a Tn5::luxAB insertion in a nitrogen-starvation-inducible gene, displaying no homology with any known genes. Initial pure culture experiments demonstrated that bioluminescence was induced in response to limitation for ammonium or other nitrogen sources that could be easily assimilated by the P. fluorescens strain. Conditions conducive to the expression of bioluminescence existed at ammonium concentrations below 10–90 μM. Upon introduction into sterile or natural soil microcosms a nitrogen-starvation signal was not observed, demonstrating that the cells were not encountering a limited supply of nitrogen sources. However, amendment of the soil with carbon+phosphorus sources led to conditions where a specific nitrogen-starvation signal was observed. Attempts to repress the reporter system by subsequent addition of ammonium were unsuccessful in the soil, but not in pure culture experiments. A possible explanation for this result is that the luciferase enzyme is stable in cells inhabiting the soil environment, imposing limitations to the use of luxAB as a real-time monitoring system.