Spatial distribution of sponge-associated bacteria in the Mediterranean sponge Tethya aurantium

The local distribution of the bacterial community associated with the marine sponge Tethya aurantium Pallas 1766 was studied. Distinct bacterial communities were found to inhabit the endosome and cortex. Clear differences in the associated bacterial populations were demonstrated by denaturing gradient gel electrophoresis (DGGE) and analysis of 16S rRNA gene clone libraries. Specifically associated phylotypes were identified for both regions: a new phylotype of Flexibacteria was recovered only from the sponge cortex, while Synechococcus species were present mainly in the sponge endosome. Light conduction via radiate spicule bundles conceivably facilitates the unusual association of Cyanobacteria with the sponge endosome. Furthermore, a new monophyletic cluster of sponge-derived 16S rRNA gene sequences related to the Betaproteobacteria was identified using analysis of 16S rRNA gene clone libraries. Members of this cluster were specifically associated with both cortex and endosome of T. aurantium.     

Thauera and Azoarcus as functionally important genera in a denitrifying quinoline-removal bioreactor as revealed by microbial community structure comparison

Structural shifts associated with functional dynamics in a bacterial community may provide clues for identifying the most valuable members in an ecosystem. A laboratory-scale denitrifying reactor was adapted from use of non-efficient seeding sludge and was utilized to degrade quinoline and remove the chemical oxygen demand. Stable removal efficiencies were achieved after an adaptation period of six weeks. Both denaturing gradient gel electrophoresis profiling of the 16S rRNA gene V3 region and comparison of the 16S rRNA gene sequence clone libraries (LIBSHUFF analysis) demonstrated that microbial communities in the denitrifying reactor and seeding sludge were significantly distinct. The percentage of the clones affiliated with the genera Thauera and Azoarcus was 74% in the denitrifying reactor and 4% in the seeding sludge. Real-time quantitative PCR also indicated that species of the genera Thauera and Azoarcus increased in abundance by about one order of magnitude during the period of adaptation. The greater abundance of Thauera and Azoarcus in association with higher efficiency after adaptation suggested that these phylotypes might play an important role for quinoline and chemical oxygen demand removal under denitrifying conditions.     

<Emphasis Type="Italic">Methanobrevibacter</Emphasis> Phylotypes are the Dominant Methanogens in Sheep from Venezuela

Rumen methanogens in sheep from Venezuela were examined using 16S rRNA gene libraries and denaturing gradient gel electrophoresis (DGGE) profiles prepared from pooled and individual PCR products from the rumen contents from 10 animals. A total of 104 clones were examined, revealing 14 different 16S rRNA gene sequences or phylotypes. Of the 14 phylotypes, 13 (99 of 104 clones) belonged to the genus Methanobrevibacter, indicating that the genus Methanobrevibacter is the most dominant component of methanogen populations in sheep in Venezuela. The largest group of clones (41 clones) was 97.9-98.5% similar to Methanobrevibacter gottschalkii. Two sequences were identified as possible new species, one belonging to the genus Methanobrevibacter and the other belonging to the genus Methanobacterium. DGGE analysis of the rumen contents from individual animals also revealed 14 different bands with a range of 4-9 bands per animal.     

Phylogenetic diversity, composition and distribution of bacterioplankton community in the Dongjiang River, China

Bacterioplankton community compositions in the Dongjiang River were characterized using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library construction. Water samples in nine different sites were taken along the mainstem and three tributaries. In total, 24 bands from DGGE gels and 406 clones from the libraries were selected and sequenced, subsequently analyzed for the bacterial diversity and composition of those microbial communities. Bacterial 16S rRNA gene sequences from freshwater bacteria exhibited board phylogenetic diversity, including sequences representing the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteriodetes, Verrucomicrobia, and candidate division TM7. Members of Betaproteobacteria group were the most dominant in all sampling sites, followed by Gammaproteobacteria, Alphaproteobacteria, and Actinobacteria. DGGE profiles and the ∫-LIBSHUFF analysis revealed similar patterns of bacterial diversity among most sampling sites, while spatial distribution variances existed in all sites along the river basin. Statistical analysis showed that bacterial species distribution strongly correlated with environmental variables, such as nitrate and ammonia, suggesting that nitrogen nutrients may shape the microbial community structure and composition in the Dongjiang River. This study had important implications for the comparison with other rivers elsewhere and contributed to the growing data set on the factors that structure bacterial communities in freshwater ecosystems.     

Replicability of dominant bacterial populations after long-term surfactant-enrichment in lab-scale activated sludge

Bacterial communities were examined in replicate lab-scale activated sludge reactors after a period of several months of enrichment with non-ionic nonylphenol ethoxylate (NPE) surfactants. Four sequential batch reactors were fed with synthetic sewage, two of which received additionally NPE. Small subunit rDNA-derived denaturing gel gradient electrophoresis (DGGE) profiles and 16S rDNA clone libraries were dominated by clones of Gammaproteobacteria class. Sequences of the other codominant rDNA phylotypes observed only in DGGE from NPE-amended reactors were, respectively, associated with the Group III of the Acidobacteria phylum. Intriguingly, 16S rRNA content from abundant Gammaproteobacteria cells was unexpectedly low. In addition to Acidobacteria, rRNA-derived DGGE profiles were dominated by members of the order Burkholderiales (of the Betaproteobacteria) and of the genus Sphingomonas (a member of the Alphaproteobacteria). Specific oligonucleotide probes for the selected ribotypes were designed and applied for quantitative real time polymerase chain reaction and fluorescence in situ hybridization, confirming their dominance in treated reactors. The parallel abundance of unique phylotypes in replicate reactors implies a distinctive selection of dominant organisms, which are better adapted to specialized niches in the highly selective environment.     

Bacterial community structure, compartmentalization and activity in a microbial fuel cell

AIMS: To characterize bacterial populations and their activities within a microbial fuel cell (MFC), using cultivation-independent and cultivation approaches. METHODS AND RESULTS: Electron microscopic observations showed that the fuel cell electrode had a microbial biofilm attached to its surface with loosely associated microbial clumps. Bacterial 16S rRNA gene libraries were constructed and analysed from each of four compartments within the fuel cell: the planktonic community; the membrane biofilm; bacterial clumps (BC) and the anode biofilm. Results showed that the bacterial community structure varied significantly between these compartments. It was observed that Gammaproteobacteria phylotypes were present at higher numbers within libraries from the BC and electrode biofilm compared with other parts of the fuel cell. Community structure of the MFC determined by analyses of bacterial 16S rRNA gene libraries and anaerobic cultivation showed excellent agreement with community profiles from denaturing gradient gel electrophoresis (DGGE) analysis. CONCLUSIONS: Members of the family Enterobacteriaceae, such as Klebsiella sp. and Enterobacter sp. and other Gammaproteobacteria with Fe(III)-reducing and electrochemical activity had a significant potential for energy generation in this system. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that electrochemically active bacteria can be enriched using an electrochemical fuel cell.     

Archaeal Microbial Diversity of Hypersaline Lake Acıgöl,Denizli, Turkey

Archaeal diversity in Lake Ac?göl, a closed-basin, alkaline, hypersaline lake located at the northern edge of western Tourides in southwest Anatolia, was investigated using culture-independent methods. Microbial mat samples were collected from six different points. Archaeal 16S rRNA gene libraries were generated using domain specific oligonucleotide primers, and 16S rRNA gene sequences of clone libraries were analyzed phylogenetically. Denaturing gradient gel electrophoresis of 16S rRNA genes showed a variance in diversity with spatial differences. Archaeal diversity of Ac?göl is dominated by the members of family Halobacteriaceae which requires both high salt concentration and high pH for growth. Sequence analysis of archaeal 16s rRNA genes indicates the presence of the phylotypes affiliated with the genera Halorubrum, Halosimplex, Halorhabdus, Haloterrigena and Natronococcus in the analyzed samples.     

Molecular diversity studies of bacterial communities of oil polluted microbial mats from the Etang de Berre (France)

The biodiversity of microbial mats inhabiting the oil-contaminated lagoon Etang de Berre was determined by molecular approaches. The fingerprint of denaturing gradient gel electrophoresis (DGGE) and automatic ribosomal intergenic spacer analysis (ARISA) of mats exposed to different pollution levels showed specific microbial communities for each site but similar diversity richness. Species composition of the mats were compared by constructing 16S rRNA libraries. Amplified rDNA restriction analysis (ARDRA) of clone libraries confirmed their similar level of diversity richness. Phylogenetic analysis of the 16S rRNA sequences showed that the classes gamma and alpha of Proteobacteria were abundantly present in both sites whereas phylotypes related to the delta-Proteobacteria and to the uncultured WS3 group were mainly found in the site with the highest pollution. Identification of the species involved in oil degradation by combining culture-based approaches and DGGE, showed that enrichment cultures were constituted by members of the Rhodobacterales and species related to Rhodococcus, Sphingomonas, Xanthomonas and Microbacterium, all of them known for their ability to degrade hydrocarbons. Our findings suggest that oil pollution has not affected the biodiversity richness of the mats. However, the populations involved in hydrocarbon degradation represent a minor fraction of the mat communities in the Etang de Berre.     

Changes in Bacterial Communities of the Marine Sponge Mycale laxissima on Transfer into Aquaculture

The changes in bacterial communities associated with the marine sponge Mycale laxissima on transfer to aquaculture were studied using culture-based and molecular techniques. M. laxissima was maintained alive in flowthrough and closed recirculating aquaculture systems for 2 years and 1 year, respectively. The bacterial communities associated with wild and aquacultured sponges, as well as the surrounding water, were assessed using 16S rRNA gene clone library analysis and denaturing gradient gel electrophoresis (DGGE). Bacterial richness and diversity were measured using DOTUR computer software, and clone libraries were compared using S-LIBSHUFF. DGGE analysis revealed that the diversity of the bacterial community of M. laxissima increased when sponges were maintained in aquaculture and that bacterial communities associated with wild and aquacultured M. laxissima were markedly different than those of the corresponding surrounding water. Clone libraries of bacterial 16S rRNA from sponges confirmed that the bacterial communities changed during aquaculture. These communities were significantly different than those of seawater and aquarium water. The diversity of bacterial communities associated with M. laxissima increased significantly in aquaculture. Our work shows that it is important to monitor changes in bacterial communities when examining the feasibility of growing sponges in aquaculture systems because these communities may change. This could have implications for the health of sponges or for the production of bioactive compounds by sponges in cases where these compounds are produced by symbiotic bacteria rather than by the sponges themselves.     

Effect of environmental variables on eukaryotic microbial community structure of land‐fast Arctic sea ice

Sea ice microbial community structure affects carbon and nutrient cycling in polar seas, but its susceptibility to changing environmental conditions is not well understood. We studied the eukaryotic microbial community in sea ice cores recovered near Point Barrow, AK in May 2006 by documenting the composition of the community in relation to vertical depth within the cores, as well as light availability (mainly as variable snow cover) and nutrient concentrations. We applied a combination of epifluorescence microscopy, denaturing gradient gel electrophoresis and clone libraries of a section of the 18S rRNA gene in order to compare the community structure of the major eukaryotic microbial phylotypes in the ice. We find that the community composition of the sea ice is more affected by the depth horizon in the ice than by light availability, although there are significant differences in the abundance of some groups between light regimes. Epifluorescence microscopy shows a shift from predominantly heterotrophic life styles in the upper ice to autotrophy prevailing in the bottom ice. This is supported by the statistical analysis of the similarity between the samples based on the denaturing gradient gel electrophoresis banding patterns, which shows a clear difference between upper and lower ice sections with respect to phylotypes and their proportional abundance. Clone libraries constructed using diatom‐specific primers confirm the high diversity of diatoms in the sea ice, and support the microscopic counts. Evidence of protistan grazing upon diatoms was also found in lower sections of the core, with implications for carbon and nutrient recycling in the ice.     

Recruitment of members from the rare biosphere of marine bacterioplankton communities after an environmental disturbance

A bacterial community may be resistant to environmental disturbances if some of its species show metabolic flexibility and physiological tolerance to the changing conditions. Alternatively, disturbances can change the composition of the community and thereby potentially affect ecosystem processes. The impact of disturbance on the composition of bacterioplankton communities was examined in continuous seawater cultures. Bacterial assemblages from geographically closely connected areas, the Baltic Sea (salinity 7 and high dissolved organic carbon [DOC]) and Skagerrak (salinity 28 and low DOC), were exposed to gradual opposing changes in salinity and DOC over a 3-week period such that the Baltic community was exposed to Skagerrak salinity and DOC and vice versa. Denaturing gradient gel electrophoresis and clone libraries of PCR-amplified 16S rRNA genes showed that the composition of the transplanted communities differed significantly from those held at constant salinity. Despite this, the growth yields (number of cells ml(-1)) were similar, which suggests similar levels of substrate utilization. Deep 454 pyrosequencing of 16S rRNA genes showed that the composition of the disturbed communities had changed due to the recruitment of phylotypes present in the rare biosphere of the original community. The study shows that members of the rare biosphere can become abundant in a bacterioplankton community after disturbance and that those bacteria can have important roles in maintaining ecosystem processes.     

Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis.

The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1, 632 bp by using CE and laser-induced fluorescence detection. Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs. Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected. We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR. Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared. Distinct clusters were identifiable for different sampling sites. Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities.     

Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis

The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1,632 bp by using CE and laser-induced fluorescence detection. Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs. Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected. We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR. Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared. Distinct clusters were identifiable for different sampling sites. Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities.     

Postinoculation protozoan establishment and association patterns of methanogenic archaea in the ovine rumen

Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.     

Temporal and spatial changes in the microbial bioaerosol communities in green-waste composting

In this study, the microbial community within compost, emitted into the airstream, downwind and upwind from a composting facility was characterized and compared through phospholipid fatty acid analysis and 16S rRNA gene analysis using denaturing gradient gel electrophoresis and bar-coded pyrosequencing techniques. All methods used suggested that green-waste composting had a significant impact upon bioaerosol community composition. Daily variations of the on-site airborne community showed how specific site parameters such as compost process activity and meteorological conditions affect bioaerosol communities, although more data are required to qualify and quantify the causes for these variations. A notable feature was the dominance of Pseudomonas in downwind samples, suggesting that this genus can disperse downwind in elevated abundances. Thirty-nine phylotypes were homologous to plant or human phylotypes containing pathogens and were found within compost, on-site and downwind microbial communities. Although the significance of this finding in terms of potential health impact was beyond the scope of this study, it clearly illustrated the potential of molecular techniques to improve our understanding of the impact that green-waste composting emissions may have on the human health.     

Archaeal diversity in naturally occurring and impacted environments from a tropical region

AIMS: To evaluate archaeal diversity in natural and impacted habitats from Rio de Janeiro state, Brazil, a tropical region of South America. METHODS AND RESULTS: 16S rRNA gene was amplified directly by polymerase chain reaction (PCR) from genomic DNA, extracted from Guanabara Bay (GB) water, halomarine sediment (HS), municipal landfill leachate, agricultural soil and wastewater treatment (WT) system. Five archaeal 16S rDNA clone libraries were constructed. A total of 123 clones, within the five libraries analysed, were clustered into 29 operational taxonomic units, related to cultivated (24%) and uncultivated (76%) organisms. Rarefaction analysis showed that the libraries contained different levels of diversity. PCR-denaturing gradient gel electrophoresis (DGGE) of 16S-23S intergenic spacer regions confirmed the presence of a dominant phylotype, revealed by the WT system clone library. CONCLUSIONS: Archaeal communities of impacted environments seem to be confined to specific ecosystems with similar physicochemical properties, while communities from natural environments appear to be widely distributed. The presence of a high number of phylotypes related to uncultivated organisms suggests new archaeal lineages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports, for the first time, the analysis of archaeal diversity in tropical environments from Brazil, and adds sequences from this region to the developing database of 16S rRNA clone libraries from environmental samples.     

Molecular characterization of mesophilic and thermophilic sulfate reducing microbial communities in expanded granular sludge bed (EGSB) reactors

The microbial communities established in mesophilic and thermophilic expanded granular sludge bed reactors operated with sulfate as the electron acceptor were analyzed using 16S rRNA targeted molecular methods, including denaturing gradient gel electrophoresis, cloning, and phylogenetic analysis. Bacterial and archaeal communities were examined over 450 days of operation treating ethanol (thermophilic reactor) or ethanol and later a simulated semiconductor manufacturing wastewater containing citrate, isopropanol, and polyethylene glycol 300 (mesophilic reactor), with and without the addition of copper(II). Analysis, of PCR-amplified 16S rRNA gene fragments using denaturing gradient gel electrophoresis revealed a defined shift in microbial diversity in both reactors following a change in substrate composition (mesophilic reactor) and in temperature of operation from 30°C to 55°C (thermophilic reactor). The addition of copper(II) to the influent of both reactors did not noticeably affect the composition of the bacterial or archaeal communities, which is in agreement with the very low soluble copper concentrations (3–310 μg l⁻¹) present in the reactor contents as a consequence of extensive precipitation of copper with biogenic sulfides. Furthermore, clone library analysis confirmed the phylogenetic diversity of sulfate-reducing consortia in mesophilic and thermophilic sulfidogenic reactors operated with simple substrates.     

Who Is in There? Exploration of Endophytic Bacteria within the Siphonous Green Seaweed Bryopsis (Bryopsidales,Chlorophyta)

Associations between marine seaweeds and bacteria are widespread, with endobiotic bacterial-algal interactions being described for over 40 years. Also within the siphonous marine green alga Bryopsis, intracellular bacteria have been visualized by electron microscopy in the early '70s, but were up to now never molecularly analyzed. To study this partnership, we examined the presence and phylogenetic diversity of microbial communities within the cytoplasm of two Bryopsis species by combining fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Sequencing results revealed the presence of Arcobacter, Bacteroidetes, Flavobacteriaceae, Mycoplasma, Labrenzia, Phyllobacteriaceae and Xanthomonadaceae species. Although the total diversity of the endobiotic communities was unique to each Bryopsis culture, Bacteroidetes, Mycoplasma, Phyllobacteriaceae, and in particular Flavobacteriaceae bacteria, were detected in several Bryopsis samples collected hundreds of kilometres apart. This suggests that Bryopsis closely associates with well-defined endophytic bacterial communities of which some members possibly maintain an endosymbiotic relationship with the algal host.