An <Emphasis Type="Italic">Azospira oryzae</Emphasis> (syn <Emphasis Type="Italic">Dechlorosoma suillum</Emphasis>) Strain That Reduces Selenate and Selenite to Elemental Red Selenium

A bacterium that reduces the soluble selenium oxyanions, selenate and selenite, to insoluble elemental red selenium (Se⁰) was isolated from a laboratory reactor developed to remove selenate from groundwater. Gene sequence alignment of the 16S rRNA allowed identification of the isolate as Azospira oryzae. Biochemical and morphologic characterization confirm the identification. The isolate reduces selenate and selenite to Se⁰ under microaerophilic and denitrifying conditions but not under aerobic conditions. It does not use selenate or selenite as terminal eˉ donors. Se oxyanion reduction causes the formation of Se nanospheres that are 0.25 ± 0.04 μm in diameter. Nanospheres may be associated with the cells or free in the medium. The enzymatic activity associated with the reduction of selenate has a molecular mass of approximately 500 kD, and the enzymatic activity associated with the reduction of selenite has a mass of approximately 55 kD. Selenite reduction was inhibited by tungsten. The molecular masses of these activities were different from those associated with the reduction of dimethylsulfoxide, sulfate, and nitrite. This bacterium, or perhaps its enzymes or DNA, might be useful for the remediation of waters contaminated with Se oxyanions.     

Microbial genetic diversity and ciliate community structure along an environmental gradient in coastal soil

We investigated the microbial genetic diversity and ciliate community in coastal soil from five sites with an environmental gradient using denaturing gradient gel electrophoresis (DGGE), gene sequencing and the Ludox–QPS method. The analyses of both the 16S ribosomal RNA (rRNA) gene and 18S rRNA gene DGGE resulted in equal or even a higher number of bands found in the samples taken from the high-salinity sites IV and V than in those taken from the low-to-moderate-salinity sites I–III. Cluster analysis of both DGGE profiles classified the five sites into three main groups (sites I, II and III and IV and V), which corresponded well to the analysis of environmental factors. There were 13 species observed at site I, three species at site II and nine species at site III, while no active ciliates were observed at the high-salinity sites IV and V. By contrast, the ciliate-specific DGGE revealed a higher number of bands in the samples taken from the high-salinity soil. Furthermore, gene sequencing suggested that the ciliates in the high-salinity soil comprised forms originating not only from soil but also from marine environments. The data indicate that saline soil may maintain a high diversity of ciliates and soil salinity might be the most influential factor regulating the community structure of ciliates.     

Physiological and molecular characterization of a microbial community established in unsaturated, petroleum-contaminated soil

The microbial communities established in soil samples from an unsaturated, petroleum-contaminated zone and from an adjacent uncontaminated site were characterized by physiological and molecular approaches. Possible electron acceptors such as sulfate and nitrate had been completely depleted in these soil samples. Slurries of these soil samples were incubated in bottles in the presence of hydrocarbon indicators (benzene, toluene, xylene and decane), and the degradation of these compounds was examined. Supplementation with electron acceptors stimulated hydrocarbon degradation, although the stimulatory effect was small in the contaminated soil. The initial degradation rates in the contaminated soil under fermentative/methanogenic conditions were comparable to those under aerobic conditions. The microbial populations in the original soil samples were analysed by cloning and sequencing of polymerase chain reaction (PCR)-amplified bacterial and archaeal 16S rRNA gene fragments, showing that the sequences retrieved from these soils were substantially different. For instance, Epsilonproteobacteria, Gammaproteobacteria, Crenarchaeota and Methanosarcinales could only be detected at significant levels in the contaminated soil. Denaturing gradient gel electrophoresis (DGGE) analyses of 16S rRNA gene fragments amplified by PCR from the incubated soil-slurry samples showed that supplementation of the electron acceptors resulted in a shift in the major populations, while the DGGE profiles after incubating the contaminated soil under the fermentative/methanogenic conditions were not substantially changed. These results suggest that petroleum contamination of the unsaturated zone resulted in the establishment of a fermentative/methanogenic community with substantial hydrocarbon-degrading potential.     

Bacterial diversity based on 16S rRNA and gyrB genes at Yinshan mine, China

The diversity of bacterial communities at three sites impacted by acid mine drainage (AMD) from the Yinshan Mine in China was studied using comparative sequence analysis of two molecular markers, the 16S rRNA and gyrB genes. The phylogenetic analyses retrieved sequences from six classes of bacteria, Nitrospira, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Acidobacteria, and Actinobacteria, as well as sequences related to the plastid of the cyanobacterium Cyanidium acidocaldarium and also some unknown bacteria. The results of phylogenetic analyses based on gyrB and 16S rRNA were compared. This confirmed that gyrB gene analysis may be a useful tool, in addition to the comparative sequence analysis of the 16S rRNA gene, for the analysis of microbial community compositions. Moreover, the Mantel test showed that the geochemical characteristics, especially the pH value and the concentration of iron, strongly influenced the composition of the microbial communities.     

Bacterial community structure associated with the Antarctic soft coral, Alcyonium antarcticum

The structure and composition of microbial communities inhabiting the soft coral Alcyonium antarcticum were investigated across three differentially contaminated sites within McMurdo Sound, Antarctica. Diverse microbial communities were revealed at all sites using culture-based analysis, denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene clone-library analysis, and FISH. Phylogenetic analysis of isolates and retrieved sequences demonstrated close affiliation with known psychrophiles from the Antarctic environment and high similarity to Gammaproteobacteria clades of sponge-associated microorganisms. The majority of bacteria detected with all techniques reside within the Gammaproteobacteria, although other phylogenetic groups including Alpha- and Betaproteobacteria, Bacteroidetes, Firmicutes, Actinomycetales, Planctomycetes, and Chlorobi and bacteria from the functional group of sulfate-reducing bacteria were also present. Multivariate (nMDS) analysis of DGGE banding patterns and principal component analysis of quantitative FISH data revealed no distinct differences in community composition between differentially contaminated sites. Rather, conserved coral-associated bacterial groups were observed within and between sites, providing evidence to support specific coral-microbial interactions. This is the first investigation of microbial communities associated with Antarctic soft corals, and the results suggest that spatially stable microbial associations exist across an environmental impact gradient.     

Isolation and characterization of selenite- and selenate-tolerant microorganisms from selenium-contaminated sites

Eight bacterial strains designated ARI 1-8 were isolated from soil and water samples collected from selenium-contaminated sites in India using the enrichment culture technique. They exhibited very high MIC values ranging from 300 to 750 mM for different forms of selenium (selenite and selenate). On the basis of various biochemical tests, fatty acid methyl ester profile and 16S rRNA gene sequencing, these isolates were identified belonging to the classes β-Proteobacteria and Bacilli. These microorganisms could be further used for bioremediation of contaminated sites.     

Characterization by denaturing gradient gel electrophoresis of bacterial communities in deep groundwater at the Kamaishi Mine,Japan

Bacterial communities in groundwater collected from five different sites at the Kamaishi Mine were investigated by using denaturing gradient gel electrophoresis (DGGE). The bacterial cells in groundwater were collected on Millipore filters, and their nucleic acid was extracted by freeze-thaw cycles. A partial 16S rRNA gene was amplified by using a universal primer set by PCR. The PCR products were analyzed by DGGE. The band pattern of DGGE was essentially identical between two samples obtained from different depths in the same borehole (KH-1). Samples from the other sites differed from one another. The partial sequences of 16S rRNA genes (about 350 base pairs) isolated from bands were determined and analyzed for phylogenetic position. Almost half the sequences from two samples of the KH-1 belonged to the cluster of spore-forming, gram-positive sulfate reducer, Desulfotomaculum. The other bands also were related to those of obligate anaerobes. This suggests that the environment in both sites of KH-1 was highly anaerobic. Although only a few sequences were retrieved from the other sites, they were phylogenetically distanced from known isolates.     

昆明盐矿古老岩盐沉积中的原核生物多样性

应用PCR-DGGE和rRNA分析法研究了昆明盐矿古老岩盐沉积中的原核生物多样性。样品的细菌DGGE分析得到27条带,古菌得到18条带。样品与纯培养得到的19个属菌株的DGGE图谱对比分析发现,细菌18个属菌株,只有1个属菌株与样品中的1条带迁移位置都不一致;古菌1个属的菌株不与样品中任何条带迁移位置一致。表明纯培养所得菌株并非该环境中的优势类群。同时,建立了样品细菌和古菌的16S rDNA克隆文库,从中分别挑取36个细菌克隆和20个古菌克隆进行ARDRA分析。细菌可分为10个OTUs,其中3个OTUs是优势类群,分别占38.9%,25.0%,16.7%,其余7个OTUs各含有1个克隆。古菌分为8个OTUs,没有明显的优势类群。每个OTU的代表克隆16S rDNA序列分析表明,细菌分属3大类群:α-Proteobacteria,γ-Proteobacteria和Actinobacteria,以Pseudomonas属菌为优势,含有其它岩盐沉积中没有发现的Actinobacteria。古菌主要是Halorubrum属、Haloterrigena属菌和未培养古菌。本研究表明,昆明盐矿古老岩盐沉积具有较丰富的原核生物多样性,含有大量未知的、未培养或不可培养的原核生物,但在原核生物物种组成和丰度上,免培养与此前的纯培养研究结果存在一定差异。因此,结合使用两类方法才能较全面地认识高盐极端环境微生物的多样性。     

Change of bacterial communities in sediments along Songhua River in Northeastern China after a nitrobenzene pollution event

More than 100 tons of nitrobenzene and related compounds were released into Songhua River due to the explosion of an aniline production factory in November, 2005. Sediment samples were taken from the heavily polluted drainage canal, one upstream and three downstream river sites. The change of bacterial community structures along the river was studied by denaturing gradient gel electrophoresis (DGGE) and cloning and sequencing of 16S rRNA genes with five clone libraries constructed and 101 sequences acquired representing 172 clones. Both DGGE profiles and sequences of 16S rRNA genes from clone libraries demonstrated that the contaminated drainage canal and three downstream river sites were similar in that all had Betaproteobacteria , mainly grouped into Comamonadaceae , as the dominant group of bacteria, and all had Firmicutes , primarily as Clostridium spp. These results suggest that these latter two groups of bacteria may play potential roles in degradation and detoxification of nitrobenzene in the present contaminated river environments.     

Identification and Characterization of Bacteria in a Selenium-Contaminated Hypersaline Evaporation Pond

Solar evaporation ponds are commonly used to reduce the volume of seleniferous agricultural drainage water in the San Joaquin Valley, Calif. These hypersaline ponds pose an environmental health hazard because they are heavily contaminated with selenium (Se), mainly in the form of selenate. Se in the ponds may be removed by microbial Se volatilization, a bioremediation process whereby toxic, bioavailable selenate is converted to relatively nontoxic dimethylselenide gas. In order to identify microbes that may be used for Se bioremediation, a 16S ribosomal DNA phylogenetic analysis of an aerobic hypersaline pond in the San Joaquin Valley showed that a previously unaffiliated group of uncultured bacteria (belonging to the order Cytophagales) was dominant, followed by a group of cultured γ-Proteobacteria which was closely related to Halomonas species. Se K-edge X-ray absorption spectroscopy of selenate-treated bacterial isolates showed that they accumulated a mixture of predominantly selenate and a selenomethionine-like species, consistent with the idea that selenate was assimilated via the S assimilation pathway. One of these bacterial isolates (Halomonas-like strain MPD-51) was the best candidate for the bioremediation of hypersaline evaporation ponds contaminated with high Se concentrations because it tolerated 2 M selenate and 32.5% NaCl, grew rapidly in media containing selenate, and accumulated and volatilized Se at high rates (1.65 μg of Se g of protein⁻¹ h⁻¹), compared to other cultured bacterial isolates.     

Impacts on microbial communities and cultivable isolates from groundwater contaminated with high levels of nitric acid-uranium waste

Microbial communities were characterized at contaminated sites that had elevated levels of nitrate, nickel, aluminum, and uranium (up to 690 mM, 310 microM, 42 mM, and 30 microM, respectively). The bacterial community structure based upon clonal libraries of the SSU rRNA genes (screened clones = 876) was diverse at the background site, but the three acidic samples had decreased diversity and the majority of clones were closely related to Azoarcus and Pseudomonas species. Arthrobacter and Novosphingobium sequences were recovered from the background samples but not the acidic sites, and similar pseudomonad populations were present at the background and acidic sites albeit at different relative abundances. Heterologous sequence coverage analyses indicated the microbial communities at the contaminated sites were very similar (p = 0.001) but different from the background site. Bacterial isolates (n = 67) classified as beta-or gamma-Proteobacteria, high G+C Gram-positive or low G+C Gram-positive were obtained from the background and one contaminated sample, and some of the isolates had less than 95% sequence identity with previously observed microorganisms. Despite variations in nitrate and heavy metal levels and different proximities to the source ponds, the three acidic samples had similar microbial populations. However, the least contaminated site (lowest nitrate and aluminum) had increased diversity compared to the other acidic samples. The results suggested that the combined contamination has decreased the microbial diversity, and Azoarcus populations were observed at a drastically increased frequency compared to the background site that had a more even distribution of multiple taxa.     

Rapid intrinsic biodegradation of benzene, toluene, and xylenes at the boundary of a gasoline-contaminated plume under natural attenuation

A groundwater plume contaminated with gasoline constituents [mainly benzene, toluene, and xylenes (BTX)] had been treated by pumping and aeration for approximately 10 years, and the treatment strategy was recently changed to monitored natural attenuation (MNA). To gain information on the feasibility of using MNA to control the spread of BTX, chemical and microbiological parameters in groundwater samples obtained inside and outside the contaminated plume were measured over the course of 73 weeks. The depletion of electron acceptors (i.e., dissolved oxygen, nitrate, and sulfate) and increase of soluble iron were observed in the contaminated zone. Laboratory incubation tests revealed that groundwater obtained immediately outside the contaminated zone (the boundary zone) exhibited much higher potential for BTX degradation than those in the contaminated zone and in uncontaminated background zones. The boundary zone was a former contaminated area where BTX were no longer detected. Denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified bacterial 16S rRNA gene fragments revealed that DGGE profiles for groundwater samples obtained from the contaminated zone were clustered together and distinct from those from uncontaminated zones. In addition, unique bacterial rRNA types were observed in the boundary zone. These results indicate that the boundary zone in the contaminant plumes served as a natural barrier for preventing the BTX contamination from spreading out.     

Screening and Isolation of PHB-Producing Bacteria in a Polluted Marine Microbial Mat

The characteristics of microbial mats within the waste stream from a seafood cannery were compared to a microbial community at a pristine site near a sandy beach at Puerto San Carlos, Baja California Sur, Mexico. Isolation of poly-beta-hydroxybutyrate (PHB)-producing bacteria, recognition of brightly refractile cytoplasmatic inclusions, lipophilic stains with Sudan Black and Nile Red, and chemical extraction of PHB were used as a culture-dependent strategy for the detection of PHB-producing bacteria. The culture-independent approach included denaturing gradient gel electrophoresis of phylotypes of 16S rRNA of microbial communities from environmental samples. Significant differences in community structure were found among the polluted and pristine sites. These differences were correlated with the physicochemical characteristics of the seawater column. At the polluted site, the seawater was rich in nutrients (ammonia, phosphates, and organic matter), compared to the pristine location. Partial sequencing of 16S rDNA of cultures of bacteria producing PHB included Bacillus and Staphylococcus at both sites; Paracoccus and Micrococcus were found only at the polluted site and Rhodococcus and Methylobacterium were found only at the pristine site. Bands of the sequences of 16S rDNA from both field samples in the denaturing gradient gel electrophoresis (DGGE) analyses affiliated closely only with bacterial sequences of cultures of Bacillus and Staphylococcus. High concentrations of organic and inorganic nutrients at the polluted site had a clear effect on the composition and diversity of the microbial community compared to the unpolluted site.     

Site-specific variation in Antarctic marine biofilms established on artificial surfaces

The community structure and composition of marine microbial biofilms established on glass surfaces was investigated across three differentially contaminated Antarctic sites within McMurdo Sound. Diverse microbial communities were revealed at all sites using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) techniques. Sequencing of excised DGGE bands demonstrated close affiliation with known psychrophiles or undescribed bacteria also recovered from the Antarctic environment. The majority of bacterial sequences were affiliated to the Gammaproteobacteria, Cytophaga/Flavobacteria of Bacteroidetes (CFB), Verrucomicrobia and Planctomycetales. Principal components analysis of quantitative FISH data revealed distinct differences in community composition between sites. Each of the sites were dominated by different bacterial groups: Alphaproteobacteria, Gammaproteobacteria and CFB at the least impacted site, Cape Armitage; green sulfur and sulfate reducing bacteria near the semi-impacted Scott Base and Planctomycetales and sulfate reducing bacteria near the highly impacted McMurdo Station. The highest abundance of archaea was detected near Scott Base (2.5% of total bacteria). Multivariate analyses (non-metric multidimensional scaling and analysis of similarities) of DGGE patterns revealed greater variability in community composition between sites than within sites. This is the first investigation of Antarctic biofilm structure and FISH results suggest that anthropogenic impacts may influence the complex composition of microbial communities.     

Recovery of novel bacterial diversity from a forested wetland impacted by reject coal

Sulphide mineral mining together with improperly contained sulphur-rich coal represents a significant environmental problem caused by leaching of toxic material. The Savannah River Site's D-area harbours a 22-year-old exposed reject coal pile (RCP) from which acidic, metal rich, saline runoff has impacted an adjacent forested wetland. In order to assess the bacterial community composition of this region, composite sediment samples were collected at three points along a contamination gradient (high, middle and no contamination) and processed for generation of bacterial and archaeal 16S rDNA clone libraries. Little sequence overlap occurred between the contaminated (RCP samples) and unimpacted sites, indicating that the majority of 16S rDNAs retrieved from the former represent organisms selected by the acidic runoff. Archaeal diversity within the RCP samples consisted mainly of sequences related to the genus Thermoplasma and to sequences of a novel type. Bacterial RCP libraries contained 16S rRNA genes related to isolates (Acidiphilium sp., Acidobacterium capsulatum, Ferromicrobium acidophilium and Leptospirillum ferrooxidans) and environmental clones previously retrieved from acidic habitats, including ones phylogenetically associated with organisms capable of sulphur and iron metabolism. These libraries also exhibited particularly novel 16S rDNA types not retrieved from other acid mine drainage habitats, indicating that significant diversity remains to be detected in acid mine drainage-type systems.     

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.     

Effects of site and plant species on rhizosphere community structure as revealed by molecular analysis of microbial guilds

The bacterial and fungal rhizosphere communities of strawberry (Fragaria ananassa Duch.) and oilseed rape (Brassica napus L.) were analysed using molecular fingerprints. We aimed to determine to what extent the structure of different microbial groups in the rhizosphere is influenced by plant species and sampling site. Total community DNA was extracted from bulk and rhizosphere soil taken from three sites in Germany in two consecutive years. Bacterial, fungal and group-specific (Alphaproteobacteria, Betaproteobacteria and Actinobacteria) primers were used to PCR-amplify 16S rRNA and 18S rRNA gene fragments from community DNA prior to denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial fingerprints of soil DNA revealed a high number of equally abundant faint bands, while rhizosphere fingerprints displayed a higher proportion of dominant bands and reduced richness, suggesting selection of bacterial populations in this environment. Plant specificity was detected in the rhizosphere by bacterial and group-specific DGGE profiles. Different bulk soil community fingerprints were revealed for each sampling site. The plant species was a determinant factor in shaping similar actinobacterial communities in the strawberry rhizosphere from different sites in both years. Higher heterogeneity of DGGE profiles within soil and rhizosphere replicates was observed for the fungi. Plant-specific composition of fungal communities in the rhizosphere could also be detected, but not in all cases. Cloning and sequencing of 16S rRNA gene fragments obtained from dominant DGGE bands detected in the bacterial profiles of the Rostock site revealed that Streptomyces sp. and Rhizobium sp. were among the dominant ribotypes in the strawberry rhizosphere, while sequences from Arthrobacter sp. corresponded to dominant bands from oilseed rape bacterial fingerprints.     

DGGE fingerprinting of bacteria in soils from eight ecologically different sites around Casey Station,Antarctica

Bacterial community structures in soils collected from eight sites around Casey Station, Antarctica, were investigated using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rRNA gene fragments. Higher bacterial diversity was found in soils from protected or relatively low human-impacted sites in comparison to highly impacted sites. However, the highest diversity was detected in samples from Wilkes Tip, a former waste disposal site that has been undisturbed for the last 50 years. Comparison of community structure based on non-metric multidimensional scaling plots revealed that all sites, except the hydrocarbon-contaminated (oil spill) site, were clustered with a 45% similarity. A total of 23 partial 16S rRNA gene sequences were obtained from the excised DGGE bands, with the majority of the sequences closely related to those of the Cytophaga–Flexibacter–Bacteroides group. No significant correlation was established between environmental variables, including soil pH, electrical conductivity, carbon, nitrogen, water content and heavy metals, with bacterial diversity across the eight study sites.     

Molecular analysis of prokaryotic diversity in the deep subsurface of the former Homestake gold mine,South Dakota,USA

A culture-independent molecular phylogenetic analysis was carried out to study the prokaryotic diversity in two soil samples collected from the subsurface (1.34 km depth) of the former Homestake gold mine, Lead, South Dakota, USA at two sites, the Ross shaft and number 6 Winze. Microbial community analyses were performed by cloning and sequencing of 16S rRNA genes retrieved directly from soil samples. Geochemical characterization of soils revealed high amount of toxic metals such as As, Cd, Co, Cr, Cu, Ni, Pb, Zn, and U at both the sites. Phylogenetic analyses showed that soil samples were predominantly composed of phylotypes related to phylum Proteobacteria. Other phyla detected in libraries were Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Chlorobi, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, Verrucomicrobia, and candidate divisions OP10 and TM7. The majority (>95%) of the phylotypes retrieved in the libraries were most closely related to environmental sequences from yet-uncultured bacteria representing a hitherto unidentified diversity. The archaeal communities at both the sites exhibited lower diversity and were most closely affiliated to uncultivated species within the Crenarchaeota. Results showed the existence of diverse microbial populations in deep subsurface environment of the Homestake gold mine. Statistical analyses demonstrated that each site harbored phylogenetically distinct microbial populations that were more diverse at Ross site compare to winze site.