Hydrocarbon degraders establish at the costs of microbial richness, abundance and keystone taxa after crude oil contamination in permafrost environments.

Oil spills from pipeline ruptures are a major source of terrestrial petroleum pollution in cold regions. However, our knowledge of the bacterial response to crude oil contamination in cold regions remains to be further expanded, especially in terms of community shifts and potential development of hydrocarbon degraders. In this study we investigated changes of microbial diversity, population size and keystone taxa in permafrost soils at four different sites along the China-Russia crude oil pipeline prior to and after perturbation with crude oil. We found that crude oil caused a decrease of cell numbers together with a reduction of the species richness and shifts in the dominant phylotypes, while bacterial community diversity was highly site-specific after exposure to crude oil, reflecting different environmental conditions. Keystone taxa that strongly co-occurred were found to form networks based on trophic interactions, that is co-metabolism regarding degradation of hydrocarbons (in contaminated samples) or syntrophic carbon cycling (in uncontaminated samples). With this study we demonstrate that after severe crude oil contamination a rapid establishment of endemic hydrocarbon degrading communities takes place under favorable temperature conditions. Therefore, both endemism and trophic correlations of bacterial degraders need to be considered in order to develop effective cleanup strategies.

An assessment of the diversity of culturable bacteria from main root of sugar beet.

The partial sequences of the 16S rRNA genes of 531 bacteria isolated from the main root of the sugar beet (Beta vulgaris L.) were determined and subsequently grouped into 155 operational taxonomic units by clustering analysis (≥99% identity). The most abundant phylum was Proteobacteria (72.5-77.2%), followed by Actinobacteria (9.8-16.6%) and Bacteroidetes (4.3-15.4%). Alphaproteobacteria (46.7-64.8%) was the most dominant class within Proteobacteria. Four strains belonging to Verrucomicrobia were also isolated. Phylogenetic analysis revealed that the Verrucomicrobia bacterial strains were closely related to Haloferula or Verrucomicrobium.

Performance assessment PCR-based assays targeting bacteroidales genetic markers of bovine fecal pollution.

There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from Bacteroidales. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require a priori characterization at each watershed of interest.

Phylogenetic analysis of Bacteroidales 16S rRNA gene sequences from human and animal effluents and assessment of ruminant faecal pollution by real-time PCR.

AIMS: The aims of this study were to evaluate the host-specific distribution of Bacteroidales 16S rRNA gene sequences from human- and animal-related effluents and faeces, and to define a ruminant-specific marker. METHODS AND RESULTS: Bacteroidales 16S rRNA gene clone libraries were constructed from samples of effluent (sewage, bovine manure and pig slurry) and faeces (human, bovine, pig and wild bird), using PCR primers targeting order Bacteroidales. The phylogenetic analysis revealed six main distinct human-, bovine-, pig- and wild bird-specific clusters. From the bovine-specific cluster II, we designed a ruminant-specific marker, Rum-2-Bac, and this showed 97% sensitivity (n=30) and 100% specificity (n=40) when tested by TaqMan real-time PCR. Average concentrations of this marker in bovine and sheep faeces and in bovine manure were 8.2+/-0.5, 8.4+/-1.3 and 7+/-0.5 log10 copies per gram, respectively. It was also quantified in samples of runoff water impacted by bovine manure, with average concentrations of 5.1+/-0.3 log10 copies per millilitre water. CONCLUSIONS: Our results confirmed that some members of Bacteroidales isolated from effluents and faeces had host-specific distributions. Identification of a bovine-specific cluster made it possible to design a reliable ruminant-specific marker. SIGNIFICANCE AND IMPACT OF THE STUDY: The host-specific distribution of Bacteroidales sequences from effluents mirrored the host-specific distribution of sequences observed in individual faeces. This efficient new ruminant-specific Bacteroidales 16S rRNA marker represents a useful addition to the microbial source tracking toolbox.

Seasonality in bacterial diversity in north-west Mediterranean coastal waters: assessment through clone libraries, fingerprinting and FISH.

We combined denaturing gradient gel electrophoresis (DGGE), catalysed reporter deposition-FISH (CARD-FISH) and clone libraries to investigate the seasonality of the bacterial assemblage composition in north-west Mediterranean coastal waters. DGGE analysis indicated that bacterial diversity changed gradually throughout the year, although with a clear distinction of the summer period. Alphaproteobacteria were the dominant group on an annual basis [29% of the DAPI (4',6-diamidino-2-phenylindole) counts by CARD-FISH, and 70% of the bacterial clones]. The SAR11 clade was most abundant during spring and summer (>20% of DAPI counts), while the Roseobacter clade was abundant primarily in winter and spring (up to 7% of DAPI counts). The phylum Bacteroidetes constituted the second most important group and was quantitatively uniform throughout the year (average 11% of the DAPI counts). Gammaproteobacteria showed a peak during summer (8% of DAPI counts), when most of them belonged to the NOR5 cluster. Clone libraries and CARD-FISH showed reasonable agreement in the quantitative proportions of Bacteroidetes and Gammaproteobacteria, but Alphaproteobacteria were overrepresented in clone libraries. Sequencing of the most predominant DGGE bands failed to detect the SAR11 group despite their high abundance. The combination of the three molecular approaches allowed a comprehensive assessment of seasonal changes in bacterial diversity.