Structure of a Natural Microbial Community in a Nitroaromatic Contaminated Groundwater Is Altered during Biodegradation of Extrinsic,but Not Intrinsic Substrates

A BSTRACTThis study demonstrates microbial community changes over time in a nitroaromatic-contaminated groundwater upon amendment with hydrocarbons previously unknown to the microbial community (extrinsic) and hydrocarbons previously known to the microbial community (intrinsic). Sealed flasks, shaken and incubated at 25 degrees C, containing contaminated groundwater and salts were amended twice with extrinsic hydrocarbons including phenol, benzoic acid, and naphthalene, and intrinsic hydrocarbons including 2,4-dinitrotoluene (2,4-DNT) and para-nitrotoluene ( p-NT). Microbial growth, biodegradation, and community structure changes measured by random amplified polymorphic DNA (RAPD) and quantitative PCR (qPCR) targeting catechol-2,3-dioxygenase (C23O) genes were monitored over time. All amended substrates were biodegraded after both substrate amendments except for 2,4-DNT, which was only partially degraded after the second amendment. Unique microbial communities were developed in flasks amended with phenol, benzoic acid, and naphthalene. However, in the flasks amended with intrinsic hydrocarbons the microbial community remained similar to the unamended control flasks. The relative amount of C23O genes detected by qPCR correlated with the biodegradation of phenol and naphthalene but not with 2,4-DNT. The results showed that a selection for microorganisms capable of catabolizing extrinsic hydrocarbons naturally and initially present in the nitroaromatic-contaminated groundwater occurred. However, growth-linked biodegradation of added intrinsic hydrocarbons was not selective.     

Microbial analysis of soil and groundwater from a gasworks site and comparison with a sequenced biological reactive barrier remediation process

AIMS: To investigate the distribution of a polymicrobial community of biodegradative bacteria in (i) soil and groundwater at a former manufactured gas plant (FMGP) site and (ii) in a novel SEquential REactive BARrier (SEREBAR) bioremediation process designed to bioremediate the contaminated groundwater. METHODS AND RESULTS: Culture-dependent and culture-independent analyses using denaturing gradient gel electrophoresis (DGGE) and polymerase chain reaction (PCR) for the detection of 16S ribosomal RNA gene and naphthalene dioxygenase (NDO) genes of free-living (planktonic groundwater) and attached (soil biofilm) samples from across the site and from the SEREBAR process was applied. Naphthalene arising from groundwater was effectively degraded early in the process and the microbiological analysis indicated a dominant role for Pseudomonas and Comamonas in its degradation. The microbial communities appeared highly complex and diverse across both the sites and in the SEREBAR process. An increased population of naphthalene degraders was associated with naphthalene removal. CONCLUSION: The distribution of micro-organisms in general and naphthalene degraders across the site was highly heterogeneous. Comparisons made between areas contaminated with polycyclic aromatic hydrocarbons (PAH) and those not contaminated, revealed differences in the microbial community profile. The likelihood of noncultured bacteria being dominant in mediating naphthalene removal was evident. SIGNIFICANCE AND IMPACT OF THE STUDY: This work further emphasizes the importance of both traditional and molecular-based tools in determining the microbial ecology of contaminated sites and highlights the role of noncultured bacteria in the process.     

Anaerobic degradation of naphthalene by a pure culture of a novel type of marine sulphate-reducing bacterium

Incubation of marine sediment in anoxic, sulphate-rich medium in the presence of naphthalene resulted in the enrichment of sulphate-reducing bacteria. Pure cultures with short, oval cells (1.3 by 1.3–1.9 μm) were isolated that grew with naphthalene as the only organic carbon source and electron donor for sulphate reduction to sulphide. One strain, NaphS2, was characterized. It affiliated with completely oxidizing sulphate-reducing bacteria of the δ-subclass of the Proteobacteria, as revealed by 16S rRNA sequence analysis. 2-Naphthoate, benzoate, pyruvate and acetate were used in addition to naphthalene. Quantification of substrate consumption, sulphide formation and formed cell mass revealed that naphthalene was completely oxidized with sulphate as the electron acceptor.     

Determination of viability within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii

Concern has been raised about the percentage of viable cells within soil rhizobia populations measured by the immunofluorescence direct count method. The purpose of this study was to evaluate a direct viable count technique which is based on the fact that viable bacteria in natural populations undergo cell elongation when they are exposed to a combination of substrate and the inhibitor of DNA gyrase, nalidixic acid. A soil extraction procedure was developed to recover a high proportion of soil bacteria (ca. 10(9)/g of soil) in suspensions with an optical clarity suitable for accurate microscopic enumeration. After incubation for 16 to 20 h at 27 degrees C in the presence of yeast extract (200 mg/liter) and nalidixic acid (10 mg/liter), between 65 and 74% of the bacteria in soil suspension became significantly elongated (greater than or equal to 4.2 microns). In contrast, less than or equal to 0.5% of the same population could be cultured, regardless of the medium composition, nutrient concentration, or incubation conditions. The direct viable count method was combined with immunofluorescence to compare the percent viability and kinetics of appearance of elongated cells within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii. Although the majority of these organisms were viable, as observed by immunofluorescence, we obtained evidence that subpopulations within the soil rhizobia community were in different states of competence to respond to substrate. A consistently low percentage (less than or equal to 30%) of the population of serotype 23 was elongated even after 24 h of incubation and regardless of when the soil was sampled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of viability within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii.

Concern has been raised about the percentage of viable cells within soil rhizobia populations measured by the immunofluorescence direct count method. The purpose of this study was to evaluate a direct viable count technique which is based on the fact that viable bacteria in natural populations undergo cell elongation when they are exposed to a combination of substrate and the inhibitor of DNA gyrase, nalidixic acid. A soil extraction procedure was developed to recover a high proportion of soil bacteria (ca. 10(9)/g of soil) in suspensions with an optical clarity suitable for accurate microscopic enumeration. After incubation for 16 to 20 h at 27 degrees C in the presence of yeast extract (200 mg/liter) and nalidixic acid (10 mg/liter), between 65 and 74% of the bacteria in soil suspension became significantly elongated (greater than or equal to 4.2 microns). In contrast, less than or equal to 0.5% of the same population could be cultured, regardless of the medium composition, nutrient concentration, or incubation conditions. The direct viable count method was combined with immunofluorescence to compare the percent viability and kinetics of appearance of elongated cells within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii. Although the majority of these organisms were viable, as observed by immunofluorescence, we obtained evidence that subpopulations within the soil rhizobia community were in different states of competence to respond to substrate. A consistently low percentage (less than or equal to 30%) of the population of serotype 23 was elongated even after 24 h of incubation and regardless of when the soil was sampled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of the MIDI-FAME technique to characterize groundwater communities

Fatty acid methyl ester (FAME) profiles were identified directly from groundwater microbial communities concentrated on and extracted with polycarbonate filters. The sensitivity of this direct extraction method was determined using pure cultures of Acinetobacter junii, Pseudomonas putida and Stenotrophomonas maltophilia. A minimum concentration of 107 cells filter-1 was required to identify the predominant fatty acids from each culture. However, at least 3.7 x 109 cells filter-1 were required to obtain fatty acid profiles that matched the signature profiles for pure cultures in a commercial database. While several saturated fatty acids (i.e. 14 : 0, 16 : 0, 18 : 0) were extracted from the polycarbonate filters, they were readily subtracted from microbial fatty acid profiles and did not interfere with the characterization of pure cultures or environmental samples. For the environmental samples, 3 l of groundwater from the Savannah River Site, Aiken, SC, (USA) contained sufficient biomass for direct extraction. A comparative analysis of FAME groundwater profiles demonstrated a qualitative difference among communities sampled from spatially discrete locations, while a groundwater well that was sampled at two time points showed strong similarities over time. Concentration of microbial biomass on polycarbonate filters coupled with the MIDI-FAME extraction of both biomass and filter was a useful technique to characterize microbial communities from groundwater.     

Analysis of BIOLOG GN Substrate Utilization Patterns by Microbial Communities

BIOLOG GN plates are increasingly used to characterize microbial communities by determining the ability of the communities to oxidize various carbon sources. Studies were done to determine whether the BIOLOG GN plate assay accurately reflects the catabolic potential of the inoculum used. To gain insight into which populations of microbial communities contribute to the BIOLOG patterns, denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis (TGGE) were used to assess the diversity of ribotypes in the inocula and individual wells of BIOLOG plates following incubation. These studies were done with microbial communities from the rhizosphere of potatoes and an activated sludge reactor fed with glucose and peptone. TGGE analyses of BIOLOG wells inoculated with cell suspensions from the potato rhizosphere revealed that, compared with the inoculum, there was a decrease in the number of 16S rRNA gene fragments obtained from various wells, as well as a concomitant loss of populations that had been numerically dominant in the inoculum. The dominant fragments in TGGE gels could be assigned to the gamma subclass of the class Proteobacteria, suggesting that fast-growing bacteria adapted to high substrate concentrations were numerically dominant in the wells and may have been primarily responsible for the patterns of substrate use that were observed. Similarly, the community structure changed in wells inoculated with cells from activated sludge; one or more populations were enriched, but all dominant populations of the inoculum could be detected in at least one well. This study showed that carbon source utilization profiles obtained with BIOLOG GN plates do not necessarily reflect the functional potential of the numerically dominant members of the microbial community used as the inoculum.     

Stable carbon isotope analysis of nucleic acids to trace sources of dissolved substrates used by estuarine bacteria.

The natural abundance of stable carbon isotopes measured in bacterial nucleic acids extracted from estuarine bacterial concentrates was used to trace sources of organic matter for bacteria in aquatic environments. The stable carbon isotope ratios of Pseudomonas aeruginosa and nucleic acids extracted from cultures resembled those of the carbon source on which bacteria were grown. The carbon isotope discrimination between the substrate and total cell carbon from bacterial cultures averaged 2.3% +/- 0.6% (n = 13). Furthermore, the isotope discrimination between the substrate and nucleic acids extracted from bacterial cultures was 2.4% +/- 0.4% (n = 10), not significantly different from the discrimination between bacteria and the substrate. Estuarine water samples were prefiltered through 1-micron-pore-size cartridge filters. Bacterium-sized particles in the filtrates were concentrated with tangential-flow filtration and centrifugation, and nucleic acids were then extracted from these concentrates. Hybridization with 16S rRNA probes showed that approximately 90% of the nucleic acids extracted on two sample dates were of eubacterial origin. Bacteria and nucleic acids from incubation experiments using estuarine water samples enriched with dissolved organic matter from Spartina alterniflora and Cyclotella caspia had stable carbon isotope values similar to those of the substrate sources. In a survey that compared diverse estuarine environments, stable carbon isotopes of bacteria grown in incubation experiments ranged from -31.9 to -20.5%. The range in isotope values of nucleic acids extracted from indigenous bacteria from the same waters was similar, -27.9 to -20.2%. Generally, the lack of isotope discrimination between bacteria and nucleic acids that was noted in the laboratory was observed in the field.(ABSTRACT TRUNCATED AT 250 WORDS)     

A modified method for the detection of microbial proteases on agar plates using tannic acid

In routine assay for the screening of microbes producing proteases, 10% trichloroaceticacid (TCA) is flooded on the milk agar plates after inoculation and required incubation to precipitate the protein. However, the clarity of the hydrolyzed zone is not very sharp and distinct. We herein present an improved assay for detecting the presence of extracellular protease from microorganisms on agar plates. In this method 10% tannic acid is flooded on the milk agar plate (in place of, TCA) to observe the zone of hydrolysis. Tannic acid sharply increases the colour intensity of the plate, as it favours the precipitation of the unhydrolyzed protein in the plate, thereby improving the contrast between the intact zones and the enzymatic lyses zones of the substrate. Our results indicate that this method is useful to detect extracellular proteases produced by both fungi as well as bacteria. The method used in the present study is sensitive, and can be easily performed for screening of large number of microbial cultures. This is the first report on the use of tannic acid for the detection of microbial proteases.     

A modified method for the detection of microbial proteases on agar plates using tannic acid

In routine assay for the screening of microbes producing proteases, 10% trichloroaceticacid (TCA) is flooded on the milk agar plates after inoculation and required incubation to precipitate the protein. However, the clarity of the hydrolyzed zone is not very sharp and distinct. We herein present an improved assay for detecting the presence of extracellular protease from microorganisms on agar plates. In this method 10% tannic acid is flooded on the milk agar plate (in place of, TCA) to observe the zone of hydrolysis. Tannic acid sharply increases the colour intensity of the plate, as it favours the precipitation of the unhydrolyzed protein in the plate, thereby improving the contrast between the intact zones and the enzymatic lyses zones of the substrate. Our results indicate that this method is useful to detect extracellular proteases produced by both fungi as well as bacteria. The method used in the present study is sensitive, and can be easily performed for screening of large number of microbial cultures. This is the first report on the use of tannic acid for the detection of microbial proteases.     

Distribution of culturable microorganisms in Fennoscandian Shield groundwater

Microbial populations in 16 groundwater samples from six Fennoscandian Shield sites in Finland and Sweden were investigated. The average total cell number was 3.7×10⁵ cells ml⁻¹, and there was no change in the mean of the total cell numbers to a depth of 1390 m. Culture media were designed based on the chemical composition of each groundwater sample and used successfully to culture anaerobic microorganisms from all samples between 65 and 1350 m depth. Between 0.0084 and 14.8% of total cells were cultured from groundwater samples. Sulfate-reducing bacteria, iron-reducing bacteria and heterotrophic acetogenic bacteria were cultured from groundwater sampled at 65–686 m depth in geographically distant sites. Different microbial populations were cultured from deeper, older and more saline groundwater from 863 to 1350 m depth. Principal component analysis of groundwater chemistry data showed that sulfate- and iron-reducing bacteria were not detected in the most saline groundwater. Iron-reducing bacteria and acetogens were cultured from deep groundwater that contained 0.35–3.5 mM sulfate, while methanogens and acetogens were cultured from deep sulfate-depleted groundwater. In one borehole from which autotrophic methanogens were cultured, dissolved inorganic carbon was enriched in ¹³C compared to other Fennoscandian Shield groundwater samples, suggesting that autotrophs were active. It can be concluded that a diverse microbial community is present from the surface to over 1300 m depth in the Fennoscandian Shield.     

The potential of a polyphasic PCR-dGGE approach in evaluating microbial diversity of natural whey cultures for water-buffalo Mozzarella cheese production: bias of culture-dependent and culture-independent analyses

A polyphasic PCR-DGGE approach was used to describe the microbial population occurring in natural whey cultures (NWCs) for water-buffalo Mozzarella cheese production. Total microbial community was assessed without cultivation by analyzing DNA directly extracted from the original samples of NWC. In addition, DNA extracted from bulks of cells formed by harvesting colonies from the serial dilution agar plates of a variety of culture media was used to profile the "cultivable" community. The 16S rDNA V3 region was amplified using DNA from NWC as well as DNA from bulks as templates and the amplicons were separated by DGGE. The microbial entities occurring in NWCs were identified by partial 16S rDNA sequencing of DGGE bands: four lactic acid bacteria (LAB) closest relative of Streptococcus thermophilus, Lactococcus lactis, Lactobacillus delbrueckii and Lactobacillus crispatus were revealed by the analysis of DNA directly extracted from NWC while two other LAB, Lactobacillus fermentum and Enterococcus faecalis, were identified by analyzing DNA from the cultivable community. The developed PCR-DGGE analysis of the "cultivable" community showed good potential in evaluating microbial diversity of a dairy environment: it usefully highlighted the bias introduced by selective amplification when compared to the analysis of the total community from NWC and allowed suitability of media and growth conditions to be evaluated. Moreover, it could be used to complete the culture independent study of microbial diversity to give information on concentration ratios among species occurring in a particular environment and can be proposed for rapid identification of dominant microorganisms in alternative to traditional tools.     

Evidence for microbial iron reduction in a landfill leachate-polluted aquifer (Vejen, Denmark).

Aquifer sediment samples obtained from the anaerobic part of a landfill leachate plume in Vejen, Denmark, were suspended in groundwater or in an artificial medium and incubated. The strictly anaerobic suspensions were tested for reduction of ferric iron [Fe(III)] oxides, which was measured as an increase in the concentration of dissolved Fe(II). Iron reduction did not occur when the medium was inoculated with inactive sediment and when the organisms in the inoculated medium were killed by formaldehyde, by chloroform, or by pasteurization, whereas the level of iron reduction was significant when living bacteria were present. Mixed cultures were obtained from the sediment samples, and differences in apparent iron reduction rates among the different cultures were maintained during several transfers. In addition, iron reduction was observed in unamended incubation mixtures containing whole sediment and groundwater. Synthetic amorphous Fe(III) oxides, as well as naturally occurring sediment-bound Fe(III) oxides, could be reduced by the cultures. Together, our results provide evidence that iron-reducing bacteria are present and microbial iron reduction occurs in the polluted aquifer sediments which we studied.     

Soil and plant effects on microbial community structure

We investigated the effects of two different plant species (corn and soybean) and three different soil types on microbial community structure in the rhizosphere. Our working hypothesis was that the rhizosphere effect would be strongest on fast-growing aerobic heterotrophs, while there would be little or no rhizosphere effect on oligotrophic and other slow-growing microorganisms. Culturable bacteria and fungi had larger population densities in the rhizosphere than in bulk soil. Communities were characterized by soil fatty acid analysis and by substrate utilization assays for bacteria and fungi. Fatty acid analysis revealed a very strong soil effect but little plant effect on the microbial community, indicating that the overall microbial community structure was not affected by the rhizosphere. There was a strong rhizosphere effect detected by the substrate utilization assay for fast-growing aerobic heterotrophic bacterial community structure, with soil controls and rhizosphere samples clearly distinguished from each other. There was a much weaker rhizosphere effect on fungal communities than on bacterial communities as measured by the substrate utilization assays. At this coarse level of community analysis, the rhizosphere microbial community was impacted most by soil effects, and the rhizosphere only affected a small portion of the total bacteria.     

The use of a solid adsorber resin for enrichment of bacteria with toxic substrates and to identify metabolites: degradation of naphthalene, O-, and m-xylene by sulfate-reducing bacteria

Anaerobic sulfate-reducing bacteria were enriched from contaminated aquifer samples with naphthalene, o-, and m-xylene as sole carbon and energy source in the presence of Amberlite-XAD7, a solid adsorber resin. XAD7 served as a substrate reservoir maintaining a constantly low substrate concentration in the culture medium. In equilibration experiments with XAD7, the aromatic hydrocarbons needed up to 5 days to achieve equilibrium between the water and the XAD7 phase. The equilibrium concentration was directly correlated with the amount of added substrate and XAD7. In the enrichments presented here, XAD7 and aromatic hydrocarbons were adjusted to maintain substrate concentrations of 100 microM m-, or o-xylene, or 50 microM naphthalene. After five subsequent transfers, the three cultures were able to grow with higher substrate concentrations in the absence of XAD7 although they grew best with lower hydrocarbon concentrations. Two new xylene-degrading cultures were obtained that could not utilise toluene as carbon source. O-xylene was degraded anaerobically by a culture, which could also oxidise m-xylene but not p-xylene. Eighty-three percent of the electrons from o-xylene oxidation were recovered in the produced sulfide, indicating a complete oxidation to CO2. Another sulfate-reducing enrichment culture oxidised m-xylene completely to CO2 but not o-, or p-xylene. A naphthalene-degrading sulfate-reducing enrichment culture oxidised naphthalene completely to CO2. Metabolites of naphthalene degradation were recovered from the XAD7 phase and subjected to GC/MS analysis. Besides the metabolites 2-naphthoic acid and decahydro-2-naphthoic acid which were identified by the mass spectrum and coelution with chemically synthesised reference compounds, the reduced 2-naphthoic acid derivatives 5,6,7,8-tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. Cultivation of bacterial cultures in the presence of XAD7 and subsequent derivatisation and extraction of metabolites directly from the solid XAD7 resin provides a new method for the isolation of sensitive bacteria and identification of metabolites.     

Epimerization of the four 3,7-dihydroxy bile acid epimers by human fecal microorganisms in anaerobic mixed cultures and in feces

The conversion of 3,7-dihydroxy bile acids by anaerobic mixed cultures of intestinal microorganisms was studied in fecal samples from eight healthy adult males. Incubations using substrate chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) were performed simultaneously in separate microbial suspensions from the same fecal samples. A time course study was done on four samples, chosen randomly from the eight. In the incubation of CDCA, substrate CDCA always decreased rapidly in amount; UDCA increased in amount, as did 3 beta, 7 beta-dihydroxy-5 beta-cholanoic acid (3 beta, 7 beta) and 3 beta, 7 alpha-dihydroxy-5 beta-cholanoic acid (3 beta, 7 alpha). In the incubation of UDCA, UDCA gradually decreased in amount; (3 beta, 7 beta), CDCA, and (3 beta, 7 alpha) increased gradually in amount. All reactions involved four epimers. After 48-72 hr UDCA was predominant and the reactions appeared to have reached equilibrium. In cultures from all eight samples, after 72-96 hr, a predominance of beta-hydroxy configurations at 7-position and alpha-hydroxy configurations at 3-position was observed. To compare these bile acid compositions to those in feces, an in vivo study using nine subjects was carried out. Concurrent with the collection of feces, transit time of food through the gut was measured. In samples from five subjects, in which amounts of lithocholic acid (LCA) was small, four 3,7-dihydroxy epimers were found. In samples from the other four, however, CDCA, the predominant epimer in bile, had apparently been converted to LCA by 7-dehydroxylation, and four epimers were not always found. In contrast to the incubation study, UDCA was not always the predominant 3,7-dihydroxy epimer in the fecal study. This may have been due to the transit times, which averaged 26.4 +/- 8.9 SD hr, being much shorter than the time it took for the incubation reactions to reach equilibrium.     

Monitoring of accelerated naphthalene-biodegradation in a bioaugmented soil slurry

The effect of microbial inoculation on the mineralization of naphthalene in a bioslurry treatment was evaluated in soil slurry microcosms. Inoculation by Pseudomonas putida G7 carrying the naphthalene dioxygenase (nahA) gene resulted in rapid mineralization of naphthalene, whereas indigenous microorganisms in the PAH-contaminated soil required a 28 h adaptation period before significant mineralization occurred. The number of nahA-like gene copies increased in both the inoculated and non-inoculated soil as mineralization proceeded, indicating selection towards naphthalene dioxygenase producing bacteria in the microbial community. In addition, 16S rRNA analysis by denaturing gradient gel electrophoresis (DGGE) analysis showed that significant selection occurred in the microbial community as a result of biodegradation. However, the indigenous soil bacteria were not able to compete with the P. putida G7 inoculum adapted to naphthalene biodegradation, even though the soil microbial community slightly suppressed naphthalene mineralization by P. putida G7.     

Identification of bacteria in biofilm and bulk water samples from a nonchlorinated model drinking water distribution system: detection of a large nitrite-oxidizing population associated with Nitrospira spp

In a model drinking water distribution system characterized by a low assimilable organic carbon content (<10 microg/liter) and no disinfection, the bacterial community was identified by a phylogenetic analysis of rRNA genes amplified from directly extracted DNA and colonies formed on R2A plates. Biofilms of defined periods of age (14 days to 3 years) and bulk water samples were investigated. Culturable bacteria were associated with Proteobacteria and Bacteriodetes, whereas independently of cultivation, bacteria from 12 phyla were detected in this system. These included Acidobacteria, Nitrospirae, Planctomycetes, and Verrucomicrobia, some of which have never been identified in drinking water previously. A cluster analysis of the population profiles from the individual samples divided biofilms and bulk water samples into separate clusters (P = 0.027). Bacteria associated with Nitrospira moscoviensis were found in all samples and encompassed 39% of the sequenced clones in the bulk water and 25% of the biofilm community. The close association with Nitrospira suggested that a large part of the population had an autotrophic metabolism using nitrite as an electron donor. To test this hypothesis, nitrite was added to biofilm and bulk water samples, and the utilization was monitored during 15 days. A first-order decrease in nitrite concentration was observed for all samples with a rate corresponding to 0.5 x 10(5) to 2 x 10(5) nitrifying cells/ml in the bulk water and 3 x 10(5) cells/cm(2) on the pipe surface. The finding of an abundant nitrite-oxidizing microbial population suggests that nitrite is an important substrate in this system, potentially as a result of the low assimilable organic carbon concentration. This finding implies that microbial communities in water distribution systems may control against elevated nitrite concentrations but also contain large indigenous populations that are capable of assisting the depletion of disinfection agents like chloramines.     

Biosurfactant Production by a Soil Pseudomonas Strain Growing on Polycyclic Aromatic Hydrocarbons

The capacity of polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria to produce biosurfactants was investigated. Twenty-three bacteria isolated from a soil contaminated with petroleum wastes were able to form clearing zones on mineral salt agar plates sprayed with solutions of PAHs. Naphthalene and phenanthrene were utilized as sole substrates. Biosurfactant production was detected by surface tension lowering and emulsifying activities from 10 of these strains grown in an iron-limited salt medium supplemented with high concentrations of dextrose or mannitol, as well as with naphthalene or phenanthrene. Glycolipid determinations showed that in cultures of Pseudomonas aeruginosa 19SJ on naphthalene, the maximal productivity of biosurfactants was delayed compared with that in cultures grown on mannitol. However, when small amounts of biosurfactants and naphthalene degradation intermediates were present at the onset of the cultivation, the delay was markedly shortened. Production of biosurfactants was accompanied by an increase in the aqueous concentration of naphthalene, indicating that the microorganism was promoting the solubility of its substrate. Detectable amounts of glycolipids were also produced on phenanthrene. This is the first report of biosurfactant production resulting from PAH metabolism.