Determinants of the microbial community structure of eutrophic, hyporheic river sediments polluted with chlorinated aliphatic hydrocarbons.

Chlorinated aliphatic hydrocarbons (CAHs) often discharge into rivers as contaminated groundwater baseflow. As biotransformation of CAHs in the impacted river sediments might be an effective remediation strategy, we investigated the determinants of the microbial community structure of eutrophic, CAH-polluted sediments of the Zenne River. Based on PCR-DGGE analysis, a high diversity of Bacteria, sulfate-reducing bacteria, Geobacteraceae, methanogenic archaea, and CAH-respiring Dehalococcoides was found. Depth in the riverbed, organic carbon content, CAH content and texture of the sediment, pore water temperature and conductivity, and concentrations of toluene and methane significantly contributed to the variance in the microbial community structure. On a meter scale, CAH concentrations alone explained only 6% of the variance in the Dehalococcoides and sulfate-reducing communities. On a cm-scale, however, CAHs explained 14.5-35% of the variation in DGGE profiles of Geobacteraceae, methanogens, sulfate-reducing bacteria, and Bacteria, while organic carbon content explained 2-14%. Neither the presence of the CAH reductive dehalogenase genes tceA, bvcA, and vcrA, nor the community structure of the targeted groups significantly differed between riverbed locations showing either no attenuation or reductive dechlorination, indicating that the microbial community composition was not a limiting factor for biotransformation in the Zenne sediments.

Evolution of bacterial community during bioremediation of PAHs in a coal tar contaminated soil.

The monitoring of a windrow treatment applied to soil contaminated by mostly 2-, 3- and 4-ring PAHs produced by coal tar distillation was performed by following the evolution of both PAH concentration and the bacterial community. Total and PAH-degrading bacterial community structures were followed by 16S rRNA PCR-DGGE in parallel with quantification by bacterial counts and 16 PAH measurements. Six months of biological treatment led to a strong decrease in 2-, 3- and 4-ring PAH concentrations (98, 97 and 82% respectively). This result was associated with the activity of bacterial PAH-degraders belonging mainly to the Gamma-proteobacteria, in particular, the Enterobacteria and Pseudomonas genera, which were detected over the course of the treatment. This group was considered to be a good bioindicator to determine the potential PAH biodegradation of contaminated soil. Conversely, other species, like the Beta-proteobacteria, were detected after 3months, when 2-, 3- and 4-ring PAHs were almost completely degraded. Thus, presence of the Beta-proteobacteria group could be considered a good candidate indicator to estimate the endpoint of biotreatment of this type of PAH-contaminated soil.

Characterization of cultures enriched from acidic polycyclic aromatic hydrocarbon-contaminated soil for growth on pyrene at low pH.

Two polycyclic aromatic hydrocarbon (PAH)-contaminated soils of pH 2 were successfully used as inoculum to enrich cultures growing on phenanthrene and pyrene at different pHs, including pH 3. Selected pyrene-utilizing cultures obtained at pH 3, pH 5, and pH 7 were further characterized. All showed rapid [14C]pyrene mineralization at pH 3 and pH 5 and grew on pyrene at pH values ranging from 2 to 6. Eubacterial and mycobacterial 16S rRNA gene denaturing gradient gel electrophoresis fingerprinting and sequencing indicated that the cultures were dominated by a single bacterium closely related to Mycobacterium montefiorense, belonging to the slow-growing Mycobacterium sp. In contrast, a culture enriched on pyrene at pH 7 from a slightly alkaline soil sampled at the same site was dominated by Pseudomonas putida and a fast-growing Mycobacterium sp. The M. montefiorense-related species dominating the pyrene-utilizing cultures enriched from the acidic soils was also the dominant Mycobacterium species in the acidic soils. Our data indicate that a slow-growing Mycobacterium species is involved in PAH degradation in that culture and show that bacteria able to degrade high-molecular-weight PAHs at low pH are present in acidic PAH-contaminated soil.

Distribution of the Mycobacterium community and polycyclic aromatic hydrocarbons (PAHs) among different size fractions of a long-term PAH-contaminated soil.

Summary Mycobacterium is often isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soil as degraders of PAHs. In model systems, Mycobacterium shows attachment to the PAH substrate source, which is considered to be a particular adaptation to low bioavailability as it results into increased substrate flux to the degraders. To examine whether PAH-degrading Mycobacterium in real PAH-contaminated soils, in analogy with model systems, are preferentially associated with PAH-enriched soil particles, the distribution of PAHs, of the PAH-mineralizing capacity and of Mycobacterium over different fractions of a soil with an aged PAH contamination was investigated. The clay fraction contained the majority of the PAHs and showed immediate pyrene- and phenanthrene-mineralizing activity upon addition of (14)C-labelled pyrene or phenanthrene. In contrast, the sand and silt fractions showed a lag time of 15-26 h for phenanthrene and 3-6 days for pyrene mineralization. The maximum pyrene and phenanthrene mineralization rates of the clay fraction expressed per gram fraction were three to six times higher than those of the sand and silt fractions. Most-probable-number (MPN)-polymerase chain reaction demonstrated that Mycobacterium represented about 10% of the eubacteria in the clay fraction, while this was only about 0.1% in the sand and silt fractions, indicating accumulation of Mycobacterium in the PAH-enriched clay fraction. The Mycobacterium community composition in the clay fraction represented all dominant Mycobacterium populations of the bulk soil and included especially species related to Mycobacterium pyrenivorans, which was also recovered as one of the dominant species in the eubacterial communities of the bulk soil and the clay fraction. Moreover, Mycobacterium could be identified among the major culturable PAH-degrading populations in both the bulk soil and the clay fraction. The results demonstrate that PAH-degrading mycobacteria are mainly associated with the PAH-enriched clay fraction of the examined PAH-contaminated soil and hence, that also in the environmental setting of a PAH-contaminated soil, Mycobacterium might experience advantages connected to substrate source attachment.

Effect of bioaugmentation and supplementary carbon sources on degradation of polycyclic aromatic hydrocarbons by a soil-derived culture.

The degradation of polycyclic aromatic hydrocarbons (PAHs) by an undefined culture obtained from a PAH-polluted soil and the same culture bioaugmented with three PAH-degrading strains was studied in carbon-limited chemostat cultures. The PAHs were degraded efficiently by the soil culture and bioaugmentation did not significantly improve the PAH degrading performance. The presence of PAHs did, however, influence the bacterial composition of the bioaugmented and non-bioaugmented soil cultures, resulting in the increase in cell concentration of sphingomonad strains. the initial enhancement of the degradation of the PAHs by biostimulation gradually disappeared and only the presence of salicylate in the additional carbon sources had a lasting slightly stimulating effect on the degradation of phenanthrene. The results suggest that bioaugmentation and biostimulation have limited potential to enhance PAH bioremediation by culture already proficient in the degradation of such contaminants.

Occurrence and community composition of fast-growing Mycobacterium in soils contaminated with polycyclic aromatic hydrocarbons.

Fast-growing mycobacteria are considered essential members of the polycyclic aromatic hydrocarbons (PAH) degrading bacterial community in PAH-contaminated soils. To study the natural role and diversity of the Mycobacterium community in contaminated soils, a culture-independent fingerprinting method based on PCR combined with denaturing gradient gel electrophoresis (DGGE) was developed. New PCR primers were selected which specifically targeted the 16S rRNA genes of fast-growing mycobacteria, and single-band DGGE profiles of amplicons were obtained for most Mycobacterium strains tested. Strains belonging to the same species revealed identical DGGE fingerprints, and in most cases, but not all, these fingerprints were typical for one species, allowing partial differentiation between species in a Mycobacterium community. Mycobacterium strains inoculated in soil were detected with a detection limit of 10(6) CFU g(-1) of soil using the new primer set as such, or approximately 10(2) CFU g(-1) in a nested PCR approach combining eubacterial and the Mycobacterium specific primers. Using the PCR-DGGE method, different species could be individually recognized in a mixed Mycobacterium community. This approach was used to rapidly assess the Mycobacterium community structure of several PAH-contaminated soils of diverse origin with different overall contamination profiles, pollution concentrations and chemical-physical soil characteristics. In the non-contaminated soil, most of the recovered 16SrRNA gene sequence did not match with previous described PAH-degrading Mycobacterium strains. In most PAH-contaminated soils, mycobacteria were detected which were closely related to fast-growing species such as Mycobacterium frederiksbergense and Mycobacterium austroafricanum, species that are known to include strains with PAH-degrading capacities. Interestingly, 16S rRNA genes related to M. tusciae sequences, a Mycobacterium species so far not reported in relation to biodegradation of PAHs, were detected in all contaminated soils.

Streptomycin as a selective agent to facilitate recovery and isolation of introduced and indigenous Sphingomonas from environmental samples.

Sphingomonas is an organism of major interest for the degradation of organic contaminants in soils and other environments. A medium based on the aminoglycoside antibiotic streptomycin (Sm) was developed, which, together with the yellow pigmentation of Sphingomonas, facilitated the detection, recovery and quantification of culturable Sphingomonas from soils. All 29 previously described bacterial strains belonging to 17 different Sphingomonas species were able to grow on mineral media containing 200 microg ml(-1) streptomycin, showing that the capacity to resist high concentrations of Sm is a common characteristic within Sphingomonas. Incorporation of Sm into the mineral medium led to a significant reduction in the background microbial population and a concomitant 100 times more sensitive detection of Sphingomonas inoculated in non-sterile soil matrices. The Sm-containing medium was used to examine a variety of hydrocarbon-contaminated soils for the presence and biodiversity of Sphingomonas. Incorporation of Sm in the medium led to a significant increase in the number of yellow-pigmented colonies. Comparison of contaminated and non-contaminated soils derived from the same site revealed colonization by culturable yellow-pigmented Sm-resistant bacteria of the polluted location solely. Both yellow and non-yellow-pigmented colonies were purified from plates containing glucose and Sm, and BOX-polymerase chain reaction (PCR) was used to sort out clonally related strains. Representative strains from the major BOX-PCR clusters were identified using FAME and partial 16S rRNA gene sequencing. Forty-eight of 58 Sm-resistant isolates were identified as Sphingomonas sp. Streptomycin-resistant Sphingomonas isolates generated BOX-PCR diversity patterns that were site dependent and represented different species mainly belonging to Sphingomonas subgroups containing species formerly designated as Sphingopyxis and Sphingobium. The ability to degrade phenanthrene was only found in a minority of the Sphingomonas isolates, which all originated from soils containing high phenanthrene concentrations.

Occurrence and phylogenetic diversity of Sphingomonas strains in soils contaminated with polycyclic aromatic hydrocarbons.

Bacterial strains of the genus Sphingomonas are often isolated from contaminated soils for their ability to use polycyclic aromatic hydrocarbons (PAH) as the sole source of carbon and energy. The direct detection of Sphingomonas strains in contaminated soils, either indigenous or inoculated, is, as such, of interest for bioremediation purposes. In this study, a culture-independent PCR-based detection method using specific primers targeting the Sphingomonas 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assess Sphingomonas diversity in PAH-contaminated soils. PCR using the new primer pair on a set of template DNAs of different bacterial genera showed that the method was selective for bacteria belonging to the family Sphingomonadaceae.Single-band DGGE profiles were obtained for most Sphingomonas strains tested. Strains belonging to the same species had identical DGGE fingerprints, and in most cases, these fingerprints were typical for one species. Inoculated strains could be detected at a cell concentration of 10(4) CFU g of soil(-1). The analysis of Sphingomonas population structures of several PAH-contaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowest Sphingomonas diversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA gene Sphingomonas sequences significantly different from sequences from known cultivated isolates (i.e., sequences from environmental clones grouped phylogenetically with other environmental clone sequences available on the web and that possibly originated from several potential new species). In conclusion, the newly designed Sphingomonas-specific PCR-DGGE detection technique successfully analyzed the Sphingomonas communities from polluted soils at the species level and revealed different Sphingomonas members not previously detected by culture-dependent detection techniques.

Acinetobacter diversity in environmental samples assessed by 16S rRNA gene PCR-DGGE fingerprinting.

A primer pair was designed to selectively amplify a fragment of the Acinetobacter 16S rRNA gene from environmental samples by PCR. 16S rRNA gene products were only obtained in PCRs with DNA from members of the genus Acinetobacter and not with DNA from other bacterial species. Denaturing gradient gel electrophoresis (DGGE) of the Acinetobacter 16S rRNA gene amplicons enabled discrimination between different Acinetobacter species. PCR using the Acinetobacter primer pair allowed detection of Acinetobacter in soil with a detection limit of 10(4) cells g(-1) soil, but attachment of the GC-clamp to the forward primer resulted in a 100-fold decrease in sensitivity. Using a nested PCR approach, the detection limit could be lowered to at least 10 cells g(-1) of soil. The method was applied to assess Acinetobacter diversity in soil samples originating from different historically hydrocarbon-contaminated sites. In addition, for one oil-contaminated soil, the dynamics of the Acinetobacter community in response to different treatments was monitored over time in a laboratory biostimulation experimental set-up. In all cases, bands in the DGGE fingerprints were cloned and sequenced. Environmental samples taken from a mineral oil-contaminated site and from a kerosene-contaminated site demonstrated relatively simple Acinetobacter 16S rRNA gene fingerprints with A. lwoffii and A. johnsonii as dominant members. In contrast, soils derived from MTBE- and BTEX-contaminated sites did not harbor detectable Acinetobacter populations. Although Acinetobacter was detected in the soil employed for the biostimulation experiment prior to treatment, substantial changes in its populations were observed depending on the treatment.

Community shifts in a seeded 3-chlorobenzoate degrading membrane biofilm reactor: indications for involvement of in situ horizontal transfer of the clc-element from inoculum to contaminant bacteria.

Pseudomonas putida BN210, carrying the self- transferable clc-element encoding degradation of 3-chlorobenzoate on the chromosome, was used as inoculum in different membrane biofilm reactors treating 3-chlorobenzoate-contaminated model wastewater. Analysis of the bacterial population in the effluent and in the biofilm showed the loss of BN210 beyond detection from the reactors and the appearance of several novel 3-chlorobenzoate mineralizing bacteria mainly belonging to the beta-proteobacteria. In contrast, in non-inoculated reactors, no 3-chlorobenzoate degradation was observed and no 3-chlorobenzoate degraders could be recovered. Southern blots hybridization of genomic DNA using clc-element-specific probes and FIGE analysis indicated the presence of the complete clc-element in one or more copies in the isolates. Moreover, the isolates could transfer the clc genes to Ralstonia metallidurans recipients. Two representative reactor isolates, Ralstonia sp. strains KP3 and KP9 demonstrated a higher growth rate on 3-chlorobenzoate than strain BN210 in batch cultures. When BN210, KP3 and KP9 were simultaneously inoculated in a membrane reactor supplied with 3-chlorobenzoate, strain KP3 outcompeted the two other strains and remained the major 3-chlorobenzoate degrading population in the reactor. Our data suggest that in situ horizontal transfer of the clc-element from the inoculum to contaminant bacteria in the reactors was involved in the establishment of novel 3-chlorobenzoate degrading populations that were more competitive under the defined reactor conditions than the inoculum strain.