Strategy for modular tagged high-throughput amplicon sequencing.

The use and validation of a strategy that allows a universal set of bar-coded sequencing primers to be appended to an amplified PCR product is described. The strategy allows a modular approach, in that the same bar code can be used with two or more target-specific primer sets, even simultaneously.

Microbial community differences between propionate-fed microbial fuel cell systems under open and closed circuit conditions.

We report the electrochemical characterization and microbial community analysis of closed circuit microbial fuel cells (CC-MFCs) and open circuit (OC) cells continuously fed with propionate as substrate. Differences in power output between MFCs correlated with their polarization behavior, which is related to the maturation of the anodophilic communities. The microbial communities residing in the biofilm growing on the electrode, biofouled cation-exchange membrane and anodic chamber liquor of OC-and CC-MFCs were characterized by restriction fragment length polymorphism screening of 16S rRNA gene clone libraries. The results show that the CC-MFC anode was enriched in several microorganisms related to known electrochemically active and dissimilatory Fe(III) reducing bacteria, mostly from the Geobacter spp., to the detriment of Bacteroidetes abundant in the OC-MFC anode. The results also evidenced the lack of a specific pelagic community in the liquor sample. The biofilm growing on the cation-exchange membrane of the CC-MFC was found to be composed of a low-diversity community dominated by two microaerophilic species of the Achromobacter and Azovibrio genus.

The introduction of genetically modified microorganisms designed for rhizoremediation induces changes on native bacteria in the rhizosphere but not in the surrounding soil.

A 168-day microcosms experiment was used to assess the possible functional and structural shifts occurring in the bacterial community of a site with a historical record of polychlorinated biphenyl (PCB) contamination, after the introduction of plants whose roots have been inoculated with genetically modified (GM) microorganisms, designed for rhizoremediation. Salix sp. plants were inoculated with two different GM Pseudomonas fluorescens strains or with their parental wild-type strain. Both bulk soil and rhizosphere samples were analyzed. Physiological profiles based on 31 ecologically relevant carbon sources were used to detect differences in bacterial community functions. The community structure of eubacteria, alpha and beta-proteobacteria, actinobacteria and acidobacteria communities were analyzed via a polymerase chain reaction-thermal gradient gel electrophoresis (TGGE) approach. The introduced transgenes had no effect on the function and structure of the bacterial community in bulk soil, although they enhanced biodegradation of PCBs as determined by chemical analysis. However, the transgenes effected the development of functionally and genetically distinct bacterial communities in the rhizosphere. Moreover, structural and functional differences were detected between planted and unplanted soils and between soil and rhizosphere samples. In the case of the different group-specific structures studied, differences were observed between groups because of time-dependant shifts, rhizosphere effect and bacterial strain introduced.

Changes in bacterial populations and in biphenyl dioxygenase gene diversity in a polychlorinated biphenyl-polluted soil after introduction of willow trees for rhizoremediation.

The aim of this study was to analyze the structural and functional changes occurring in a polychlorinated-biphenyl (PCB)-contaminated soil ecosystem after the introduction of a suitable host plant for rhizoremediation (Salix viminalis). We have studied the populations and phylogenetic distribution of key bacterial groups (Alpha- and Betaproteobacteria, Acidobacteria, and Actinobacteria) and the genes encoding iron-sulfur protein alpha (ISPalpha) subunits of the toluene/biphenyl dioxygenases in soil and rhizosphere by screening gene libraries using temperature gradient gel electrophoresis. The results, based on the analysis of 415 clones grouped into 133 operational taxonomic units that were sequence analyzed (>128 kbp), show that the rhizospheric bacterial community which evolved from the native soil community during the development of the root system was distinct from the soil community for all groups studied except for the Actinobacteria. Proteobacteria were enriched in the rhizosphere and dominated both in rhizosphere and soil. There was a higher than expected abundance of Betaproteobacteria in the native and in the planted PCB-polluted soil. The ISPalpha sequences retrieved indicate a high degree of catabolic and phylogenetic diversity. Many sequences clustered with biphenyl dioxygenase sequences from gram-negative bacteria. A distinct cluster that was composed of sequences from this study, some previously described environmental sequences, and a putative ISPalpha from Sphingomonas wittichii RW1 seems to contain greater diversity than the presently recognized toluene/biphenyl dioxygenase subfamily. Moreover, the rhizosphere selected for two ISPalpha sequences that accounted for almost 60% of the gene library and were very similar to sequences harbored by Pseudomonas species.

A molecular fingerprint technique to detect pollution-related changes in river cyanobacterial diversity.

Humans now have a strong influence on almost every major aquatic ecosystem, and our activities have dramatically altered the quality of receiving waters worldwide. Thus, there is a continuous need to develop and apply novel and effective technologies to detect, manage, and correct human-induced degradation of aquatic systems. In the present work, we evaluated the molecular approach using polymerase chain reaction (PCR)-temperature gradient gel electrophoresis (TGGE) to measure changes in cyanobacterial diversity along a pollution gradient in a river and compared it with that of using microscopic observations of field-fixed and cultured samples. The different 16S rDNA genes present in the cyanobacterial community of each sampling point of the river were separated by TGGE, giving a characteristic pattern of bands for each site. This pattern represents a "fingerprint" of the community, allowing direct comparisons of the different samples. The TGGE results revealed that the structure of the cyanobacterial community differed along the pollution gradient of the river. Microscopic and molecular approaches showed that cyanobacterial diversity decreased in a downstream direction. Similar results were obtained by the two methods, as indicated by the high correlation between them. We suggest PCR-TGGE could be a useful and rapidly applied technique for the routine analysis of changes in cyanobacterial diversity in response to pollution, which would allow us to monitor rivers in surveillance networks of watercourse quality.