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Lake Kivu is a meromictic lake in East Africa with enormous amounts of dissolved methane (CH4) and carbon dioxide (CO2) in its deep waters and surprisingly low CH4 in the surface waters. We applied 454 pyrosequencing of 16S rRNA gene fragments to study the bacterial and archaeal community compositions (BCC and ACC, respectively) to provide insight into the ecology of the microbes in Lake Kivu. The vertical distribution of electron donors and acceptors in the chemically stratified water column may be responsible for the stratified distribution of microbial populations, suggesting well-defined functional specialization. The highest microbial richness was detected in the anoxic zone, which hosted high percentages of bacterial sequences related to uncultured and poorly described phyla. This suggests an under-representation of anoxic environments in current databases and the presence of previously undescribed taxa. Microbial diversity is made up of 2 fractions: abundant species (e.g. Galand et al. 2009) and rare species. Abundant species were more stable than rare species over time. The detection of rare candidate divisions (e.g. OP3, WS3, GN02) co-occurring with sulphur-oxidising Epsilonproteobacteria, sulphate- reducing Deltaproteobacteria, hydrogen-oxidising Dehalococcoidetes and methanogens might indicate interactions in the carbon and sulphur cycles in the anoxic waters.

Acetobacter pasteurianus, a Gram-negative bacterium belonging to the α-divison of Proteobacteria, produces acetic acid through ethanol oxidation. A genomic bank of A. pasteurianus 386B DNA was cloned in the low-copy cosmid pRG930Cm vector and the resulting clones were screened for the production of protease using the skimmed-milk agar assay whereby a clearing zone around the inoculated spots indicates casein degradation. Several positive clones were selected and restriction analysis revealed that many contained the same inserts. One clone was further analyzed and the cosmid DNA subjected to in vitro transposon insertion. After electroporation, several clones having lost the capacity to cause casein degradation were isolated and the sequence of the transposon-flanking regions analyzed. The majority of insertions mapped to one gene encoding an NAD(P)(+)-dependent aldehyde dehydrogenase (ALDH) of the PNTB superfamily, whereas one insert was found upstream in a gene encoding an ethanol dehydrogenase. Addition of phenol red to the medium confirmed the ethanol-dependent acidification around the inoculated spots of the clones without transposon insertion, suggesting that casein degradation is due to the production of acetic acid as a result of the combined activities of the alcohol dehydrogenase and ALDH. Quantitative data and pH measurements confirmed a significant acidification, and the presence of acetic acid.

Sewage-contaminated rivers are ecosystems deeply disturbed by human activity due to the release of heavy metals, organic pollutants and pharmaceuticals as well as faecal and pathogenic micro-organisms, which coexist with the autochthonous microbial population. In this study, we compared the percentage of resistance in faecal and heterotrophic bacteria in rivers with different degrees of sewage pollution. As a matter of fact, no correlation was found neither between the degree of sewage pollution and the percentage of antimicrobial resistant heterotrophic bacteria nor between the number of resistant faecal bacteria and that of resistant heterotrophic bacteria. Most of the resistant isolates from the Zenne river downstream Brussels were multi-resistant and the resistance patterns were similar among the strains of each phylogenetic group. The total microbial community in this polluted river (as evaluated through a 16S rRNA gene clone library analysis) appeared to be dominated by the phyla Proteobacteria and Bacteroidetes while the phylum TM7 was the third most represented.

Cocoa bean fermentation is a spontaneous process involving a succession of microbial activities, starting with yeasts, followed by lactic acid bacteria and acetic acid bacteria. So far, all microbiological studies about cocoa bean fermentation were based on culture-dependent (isolation, cultivation, and identification), or, more recently, culture-independent (PCR-DGGE, or polymerase chain reaction denaturing gradient gel electrophoresis) methods. Using a metagenomic approach, total DNA was extracted from heap and box fermentations at different time points and from different locations (Ghana and Brazil, respectively) to generate a 16 S rDNA clone library that was sequenced. The sequencing data revealed a low bacterial diversity in the fermentation samples and were in accordance with the results obtained through culture-dependent and a second, culture-independent analysis (PCR-DGGE), suggesting that almost all bacteria involved in the fermentation process are cultivable. One exception was the identification by 16 S rDNA library sequencing of Gluconacetobacter species of acetic acid bacteria that were not detected by the two other approaches. The presence of Enterobacteriaceae related to Erwinia/Pantoea/Tatumella, as revealed by 16 S rDNA library sequencing, suggests an impact of these bacteria on fermentation.

Aims: The specificity of a method for the enumeration of Escherichia coli (chromocult agar, CC) was tested using freshwater samples from a tropical area (Cuba Island) by isolating colonies and identifying them with API (Appareillage et procede d’identification) strips. Enumerations of E. coli by the most probable number (MPN) microplate method were compared with counts on chromogenic and fluorogenic agar media [CC, rapid E. coli (REC), fluorocult] in tropical and temperate freshwater samples. Methods and Results: A high percentage of specificity (95·7%) for the CC agar enumeration of E. coli was observed. High regression coefficients (log-log linear regressions) were found between E. coli counts on agar media and by the MPN method. In the tropical environment, counts with REC medium were significantly different from those obtained with the other methods. MPN counts were found to be significantly higher than those obtained using the plate counts methods in the temperate environment. Conclusions: Escherichia coli enumeration methods based on glucuronidase activity appear to be suitable for the evaluation of microbiological quality in the tropical environment featured in this study. Significance and Impact of the Study: The methods for the enumeration of E. coli tested in this study should help improve the evaluation of microbiological contamination of Cuban freshwaters.

Le nombre de coliformes fecaux (CF) a ete compare au nombre de Escherichia coli trouves dans des echantillons d'eau douce contamines de facon differentielle. La numeration des CF a ete realisee par comptage sur plaque sur du milieu chlorure triphenilique de 2,3,5-tetrazolium Tergitol. La numeration de E. coli a ete faite par la methode du nombre le plus probable (NPP) sur microplaque, basee sur la detection de l'activite glucuronidase. Les quantites de CF et de E. coli etaient en forte correlation; un ratio E. coli/FC moyen egal a 0,77 a ete trouve, indiquant qu'en moyenne E. coli constituait 77 % des CF. La connaissance du ratio E. coli/FC nous permet de convertir des donnees provenant de l'historique de la qualite microbiologique d'un site, exprimees en nombre de CF, en quantite de E. coli et ainsi les comparer avec les donnees de surveillance actuelles et futures qui sont (ou seront) basees sur la numeration de E. coli.

The microbial community composition in meromictic Lake Kivu, with one of the largest CH4 reservoirs, was studied using 16S rDNA and ribosomal RNA (rRNA) pyrosequencing during the dry and rainy seasons. Highly abundant taxa were shared in a high percentage between bulk (DNA-based) and active (RNA-based) bacterial communities, whereas a high proportion of rare species was detected only in either an active or bulk community, indicating the existence of a potentially active rare biosphere and the possible underestimation of diversity detected when using only one nucleic acid pool. Most taxa identified as generalists were abundant, and those identified as specialists were more likely to be rare in the bulk community. The overall number of environmental parameters that could explain the variation was higher for abundant taxa in comparison to rare taxa. Clustering analysis based on operational taxonomic units (OTUs at 0.03 cutoff) level revealed significant and systematic microbial community composition shifts with depth. In the oxic zone, Actinobacteria were found highly dominant in the bulk community but not in the metabolically active community. In the oxic-anoxic transition zone, highly abundant potentially active Nitrospira and Methylococcales were observed. The co-occurrence of potentially active sulfur-oxidizing and sulfate-reducing bacteria in the anoxic zone may suggest the presence of an active yet cryptic sulfur cycle. ispartof: Microbial Ecology vol:3 issue:3 pages:596-611 ispartof: location:United States status: published

In order to investigate the factors controlling the bacterial community composition (BCC) in reservoirs, we sampled three freshwater reservoirs with contrasted physical and chemical characteristics and trophic status. The BCC was analysed by 16S rRNA gene amplicon 454 pyrosequencing. In parallel, a complete dataset of environmental parameters and phytoplankton community composition was also collected. BCC in the analysed reservoirs resembled that of epilimnetic waters of natural freshwater lakes with presence of Actinobacteria, Alpha- and Betaproteobacteria, Cytophaga-Flavobacteria-Bacteroidetes (CFB) and Verrucomicrobia groups. Our results evidenced that the retrieved BCC in the analysed reservoirs was strongly influenced by pH, alkalinity and organic carbon content, whereas comparatively little change was observed among layers in stratified conditions., SCOPUS: ar.j, info:eu-repo/semantics/published

International audience; In the present study, the antimicrobial resistant (AR) bacteria were quantified and identified in different river samples using in parallel a culture-based approach and a culture-independent one. The objective was to evaluate the importance of the cultivation bias when studying antimicrobial resistance among environmental bacteria. Three different river samples covering a gradient of anthropic influence were tested and three different antimicrobial compounds were used as selective agents: amoxicillin, tetracycline and sulfamethoxazole. From a quantitative point of view, our results highlight the importance of the culture media used, as for the same sample and the same selective agent significant differences were observed in the counts of culturable AR bacteria depending on the culture media used. The identification of AR bacteria through culture or culture-independent methods put on evidence AR bacterial communities that differ dramatically: gamma-proteobacteria and more specifically Aeromonadaceae dominated among the isolates while beta-proteobacteria (Comamonadaceae), dominated among the sequences obtained without culture. Altogether these results highlight the necessity to develop a methodological consensus preferably without culture, to approach this important topic in the coming years. (C) 2013 Elsevier Ltd. All rights reserved.

We review available data on archaea, bacteria and small eukaryotes in an attempt to provide a general picture of microbial diversity, abundances and microbe-driven processes in Lake Kivu surface and intermediate waters (ca. 0–100 m). The various water layers present contrasting physical and chemical properties and harbour very different microbial communities supported by the vertical redox structure. For instance, we found a clear vertical segregation of archaeal and bacterial assemblages between the oxic and the anoxic zone of the surface waters. The presence of specific bacterial (e.g. Green Sulfur Bacteria) and archaeal (e.g. ammonia-oxidising archaea) communities and the prevailing physico-chemical conditions point towards the redoxcline as the most active and metabolically diverse water layer. The archaeal assemblage in the surface and intermediate water column layers was mainly composed by the phylum Crenarchaeota, by the recently defined phylum Thaumarchaeota and by the phylum Euryarchaeota. In turn, the bacterial assemblage comprised mainly ubiquitous members of planktonic assemblages of freshwater environments (Actinobacteria, Bacteroidetes and Betaproteobacteria among others) and other less commonly retrieved phyla (e.g. Chlorobi, Clostridium and Deltaproteobacteria). The community of small eukaryotes (

Approved methods traditionally used for Escherichia coli enumeration in waters are culture-based. However, these methods can underestimate the E. coli abundance in aquatic systems because they do not take into account cells that remain viable but have lost the ability to grow in or on culture media. We investigated, in freshwater samples, the abundance of (i) culturable E. coli, enumerated by the most probable number microplate method and (ii) viable E. coli, estimated using a procedure called DVC–FISH, which couples fluorescent in situ hybridization (FISH) and a viability testing technique (direct viable count (DVC)). The ratio of culturable to viable E. coli was close to 1 in highly contaminated waters (samples with a high concentration of culturable E. coli), but decreased drastically for weakly contaminated samples. This indicates a large fraction of viable but nonculturable (VBNC) E. coli in the latter samples. Microcosm experiments showed that some environmental factors, such as nutrient scarcity and solar irradiation, could lead to the presence of a high proportion of VBNC E. coli.

Aims: The relationships between the β-d-glucuronidase (GLUase) activity, the abundance of culturable Escherichia coli and the number of viable E. coli were investigated in river and wastewater samples. Methods and Results: GLUase activity was measured as the rate of hydrolysis of 4-methylumbelliferyl-β-d-glucuronide. Culturable E. coli were enumerated by the most probale number (MPN) microplate method. Viable E. coli were estimated by fluorescent in situ hybridization (FISH) coupled with a procedure of viability testing (DVC-FISH procedure). Significant correlations were found between the log of GLUase activity and both, the log culturable E. coli and the log of viable E. coli. Conclusions: GLUase activity per viable E. coli gave a broadly constant value from low to highly contaminated waters while GLUase activity per culturable E. coli strongly increased at low contaminated waters because of an underestimation of the number of active E. coli by the culture-based method. Significance and Impact of the Study: GLUase activity is a reliable parameter for the rapid quantification of viable E. coli in waters.

A combination of direct viable count (DVC) and fluorescent in situ hybridization (FISH) procedures was used to enumerate viable Escherichia coli in river waters and wastewaters. A probe specific for the 16S rRNA of E. coli labeled with the CY3 dye was used; enumeration of hybridized cells was performed by epifluorescence microscopy. Data showed that the method was able to accurately enumerate a minimum of 3000 viable E. coli among a large number of non-fecal bacteria. When applied to river water and wastewater samples, the DVC-FISH method gave systematically higher E. coli counts than a reference culture-based method (miniaturized MPN method). The ratio between both counts (DVC-FISH/MPN) increased with decreasing abundance of culturable E. coli indicating that the proportion of viable but non-culturable (VBNC) E. coli (detectable by the DVC-FISH procedure and not by a culture-based method) was higher in low contaminated environments. We hypothesized that the more stressing conditions, i.e. nutritional stress and sunlight effect, met in low contaminated environments were responsible for the larger fraction of VBNC E. coli. A survival experiment, in which sterile mineral water was inoculated with a pure E. coli strain and incubated, confirmed that stressing conditions induced the apparition of non-culturable E. coli detectable by the DVC-FISH procedure. The analysis of the E. coli concentration along a Seine river longitudinal profile downstream a large input of fecal bacteria by a WWTP outfall showed an increasing fraction of VBNC E. coli with increasing residence time of the E. coli in the river after release. These data suggest that the DVC-FISH method is useful tool to analyze the dynamics of fecal bacteria in river water.

The microorganisms inhabiting many petroleum reservoirs are multi-extremophiles capable of surviving in environments with high temperature, pressure and salinity. Their activity influences oil quality and they are an important reservoir of enzymes of industrial interest. To study these microbial assemblages and to assess any modifications that may be caused by industrial practices, the bacterial and archaeal communities in waters from four Algerian oilfields were described and compared. Three different types of samples were analyzed: production waters from flooded wells, production waters from non-flooded wells and injection waters used for flooding (water-bearing formations). Microbial communities of production and injection waters appeared to be significantly different. From a quantitative point of view, injection waters harbored roughly ten times more microbial cells than production waters. Bacteria dominated in injection waters, while Archaea dominated in production waters. Statistical analysis based on the relative abundance and bacterial community composition (BCC) revealed significant differences between production and injection waters at both OTUs0.03 and phylum level. However, no significant difference was found between production waters from flooded and non-flooded wells, suggesting that most of the microorganisms introduced by the injection waters were unable to survive in the production waters. Furthermore, a Venn diagram generated to compare the BCC of production and injection waters of one flooded well revealed only 4% of shared bacterial OTUs. Phylogenetic analysis of bacterial sequences indicated that Alpha-, Beta- and Gammaproteobacteria were the main classes in most of the water samples. Archaeal sequences were only obtained from production wells and each well had a unique archaeal community composition, mainly belonging to Methanobacteria, Methanomicrobia, Thermoprotei and Halobacteria classes. Many of the bacterial genera retrieved had already been reported as degraders of complex organic molecules and pollutants. Nevertheless, a large number of unclassified bacterial and archaeal sequences were found in the analyzed samples, indicating that subsurface waters in oilfields could harbor new and still-non-described microbial species., SCOPUS: ar.j, info:eu-repo/semantics/published