Integrated Advanced Molecular Tools Predict In Situ cVOC Degradation Rates: Field Demonstration.

Chlorinated volatile organic compound (cVOC) degradation rate constants are crucial information for site management. Conventional approaches generate rate estimates from the monitoring and modeling of cVOC concentrations. This requires time series data collected along the flow path of the plume. The estimates of rate constants are often plagued by confounding issues, making predictions cumbersome and unreliable. Laboratory data suggest that targeted quantitative analysis of Dehalococcoides mccartyi (Dhc) biomarker genes (qPCR) and proteins (qProt) can be directly correlated with reductive dechlorination activity. To assess the potential of qPCR and qProt measurements to predict rates, we collected data from cVOC-contaminated aquifers. At the benchmark study site, the rate constant for degradation of cis-dichloroethene (cDCE) extracted from monitoring data was 11.0 ± 3.4 yr-1, and the rate constant predicted from the abundance of TceA peptides was 6.9 yr-1. The rate constant for degradation of vinyl chloride (VC) from monitoring data was 8.4 ± 5.7 yr-1, and the rate constant predicted from the abundance of TceA peptides was 5.2 yr-1. At the other study sites, the rate constants for cDCE degradation predicted from qPCR and qProt measurements agreed within a factor of 4. Under the right circumstances, qPCR and qProt measurements can be useful to rapidly predict rates of cDCE and VC biodegradation, providing a major advance in effective site management.

Sustained detoxification of 1,2-dichloroethane to ethylene by a symbiotic consortium containing Dehalococcoides species.

1,2-Dichloroethane (1,2-DCA) is a ubiquitous volatile halogenated organic pollutant in groundwater and soil, which poses a serious threat to the ecosystem and human health. Microbial reductive dechlorination has been recognized as an environmentally-friendly strategy for the remediation of sites contaminated with 1,2-DCA. In this study, we obtained an anaerobic microbiota derived from 1,2-DCA contaminated groundwater, which was able to sustainably convert 1,2-DCA into non-toxic ethylene with an average dechlorination rate of 30.70 ± 11.06 µM d-1 (N = 6). The microbial community profile demonstrated that the relative abundance of Dehalococcoides species increased from 0.53 ± 0.08% to 44.68 ± 3.61% in parallel with the dechlorination of 1,2-DCA. Quantitative PCR results showed that the Dehalococcoides species 16S rRNA gene increased from 2.40 ± 1.71 × 108 copies∙mL-1 culture to 4.07 ± 2.45 × 108 copies∙mL-1 culture after dechlorinating 110.69 ± 30.61 µmol of 1,2-DCA with a growth yield of 1.55 ± 0.93 × 108 cells per µmol Cl- released (N = 6), suggesting that Dehalococcoides species used 1,2-DCA for organohalide respiration to maintain cell growth. Notably, the relative abundances of Methanobacterium sp. (p = 0.0618) and Desulfovibrio sp. (p = 0.0001995) also increased significantly during the dechlorination of 1,2-DCA and were clustered in the same module with Dehalococcoides species in the co-occurrence network. These results hinted that Dehalococcoides species, the obligate organohalide-respiring bacterium, exhibited potential symbiotic relationships with Methanobacterium and Desulfovibrio species. This study illustrates the importance of microbial interactions within functional microbiota and provides a promising microbial resource for in situ bioremediation in sites contaminated with 1,2-DCA.

Mechanistic insight into co-metabolic dechlorination of hexachloro-1,3-butadiene in Dehalococcoides.

Hexachloro-1,3-butadiene (HCBD) as one of emerging persistent organic pollutants (POPs) poses potential risk to human health and ecosystems. Organohalide-respiring bacteria (OHRB)-mediated reductive dehalogenation represents a promising strategy to remediate HCBD-contaminated sites. Nonetheless, information on the HCBD-dechlorinating OHRB and their dechlorination pathways remain unknown. In this study, both in vivo and in vitro experiments, as well as quantum chemical calculation, were employed to successfully identify and characterize the reductive dechlorination of HCBD by Dehalococcoides. Results showed that some Dehalococcoides extensively dechlorinated HCBD to (E)-1,2,3-tri-CBD via (E)-1,1,2,3,4-penta-CBD and (Z,E)-1,2,3,4-tetra-CBD in a co-metabolic way. Both qPCR and 16S rRNA gene amplicon sequencing analyses suggested that the HCBD-dechlorinating Dehalococcoides coupled their cell growth with dechlorination of perchloroethene (PCE), rather than HCBD. The in vivo and in vitro ATPase assays indicated ≥78.89% decrease in ATPase activity upon HCBD addition, which suggested HCBD inhibition on ATPase-mediated energy harvest and provided rationality on the Dehalococcoides-mediated co-metabolic dechlorination of HCBD. Interestingly, dehalogenation screening of organohalides with the HCBD-dechlorinating enrichment cultures showed that debromination of bromodichloromethane (BDCM) was active in the in vitro RDase assays but non-active in the in vivo experiments. Further in vitro assays of hydrogenase activity suggested that significant inhibition of BDCM on the hydrogenase activity could block electron derivation from H2 for consequent reduction of organohalides in the in vivo experiments. Therefore, our results provided unprecedented insight into metabolic, co-metabolic and RDase-active-only dehalogenation of varied organohalides by specific OHRB, which could guide future screening of OHRB for remediation of sites contaminated by HCBD and other POPs.

Effects of ferrous iron supplementation on reductive dechlorination of tetrachloroethene and on methanogenic microbial community.

Chloroethenes are common soil and groundwater pollutants. Their dechlorination is impacted by environmental factors, such as the presence of metal ions. We here investigated the effect of ferrous iron on bacterial reductive dechlorination of chloroethenes and on methanogen community. Reductive dechlorination of tetrachloroethene was assayed with a groundwater sample originally containing 6.3 × 103 copies mL-1 of Dehalococcoides 16S rRNA gene and 2 mg L-1 of iron. Supplementation with 28 mg L-1 of ferrous iron enhanced the reductive dechlorination of cis-dichloroethene (cis-DCE) and vinyl chloride in the presence of methanogens. The supplementation shortened the time required for complete dechlorination of 1 mg L-1 of tetrachloroethene to ethene and ethane from 84 to 49 d. Methanogens, such as Candidatus 'Methanogranum', Methanomethylovorans and Methanocorpusculum, were significantly more abundant in iron-supplemented cultures than in non-supplemented cultures (P < 0.01). Upon methanogen growth inhibition by 2-bromoethanesulfonate and in the absence of iron supplementation, cis-DCE was not dechlorinated. Further, iron supplementation induced 71.3% dechlorination of cis-DCE accompanied by an increase in Dehalococcoides 16S rRNA and dehalogenase vcrA gene copies but not dehalogenase tceA gene copies. These observations highlight the cooperative effect of iron and methanogens on the reductive dechlorination of chloroethenes by Dehalococcoides spp.

'Candidatus Oscillochloris kuznetsovii' a novel mesophilic filamentous anoxygenic phototrophic Chloroflexales bacterium from Arctic coastal environments.

Chloroflexales bacteria are mostly known as filamentous anoxygenic phototrophs that thrive as members of the microbial communities of hot spring cyanobacterial mats. Recently, we described many new Chloroflexales species from non-thermal environments and showed that mesophilic Chloroflexales are more diverse than previously expected. Most of these species were isolated from aquatic environments of mid-latitudes. Here, we present the comprehensive characterization of a new filamentous multicellular anoxygenic phototrophic Chloroflexales bacterium from an Arctic coastal environment (Kandalaksha Gulf, the White Sea). Phylogenomic analysis and 16S rRNA phylogeny indicated that this bacterium belongs to the Oscillochloridaceae family as a new species. We propose that this species be named 'Candidatus Oscillochloris kuznetsovii'. The genomes of this species possessed genes encoding sulfide:quinone reductase, the nitrogenase complex and the Calvin cycle, which indicate potential for photoautotrophic metabolism. We observed only mesophilic anaerobic anoxygenic phototrophic growth of this novel bacterium. Electron microphotography showed the presence of chlorosomes, polyhydroxyalkanoate-like granules and polyphosphate-like granules in the cells. High-performance liquid chromatography also revealed the presence of bacteriochlorophylls a, c and d as well as carotenoids. In addition, we found that this bacterium is present in benthic microbial communities of various coastal environments of the Kandalaksha Gulf.

Preparation and characterization of site-specific dechlorinating microbial inocula capable of complete dechlorination enriched in anaerobic microcosms amended with clay mineral.

Short-chain halogenated aliphatic hydrocarbons (e.g. perchloroethene, trichloroethene) are among the most toxic environmental pollutants. Perchloroethene and trichloroethene can be dechlorinated to non-toxic ethene through reductive dechlorination by Dehalococcoides sp. Bioaugmentation, applying cultures containing organohalide-respiring microorganisms, is a possible technique to remediate sites contaminated with chlorinated ethenes. Application of site specific inocula is an efficient alternative solution. Our aim was to develop site specific dechlorinating microbial inocula by enriching microbial consortia from groundwater contaminated with trichloroethene using microcosm experiments containing clay mineral as solid phase. Our main goal was to develop fast and reliable method to produce large amount (100 L) of bioactive agent with anaerobic fermentation technology. Polyphasic approach has been applied to monitor the effectiveness of dechlorination during the transfer process from bench-scale (500 mL) to industrial-scale (100 L). Gas chromatography measurement and T-RFLP (Terminal Restriction Fragment Length Polymorphism) revealed that the serial subculture of the enrichments shortened the time-course of the complete dechlorination of trichloroethene to ethene and altered the composition of bacterial communities. Complete dechlorination was observed in enrichments with significant abundance of Dehalococcoides sp. cultivated at 8 °C. Consortia incubated in fermenters at 18 °C accelerated the conversion of TCE to ethene by 7-14 days. Members of the enrichments belong to the phyla Bacteroidetes, Chloroflexi, Proteobacteria and Firmicutes. According to the operational taxonomic units, main differences between the composition of the enrichment incubated at 8 °C and 18 °C occurred with relative abundance of acetogenic and fermentative species. In addition to the temperature, the site-specific origin of the microbial communities and the solid phase applied during the fermentation technique contributed to the development of a unique microbial composition.

Spatial distribution and diversity of organohalide-respiring bacteria and their relationships with polybrominated diphenyl ether concentration in Taihu Lake sediments.

It is acknowledged that organohalide-respiring bacteria (OHRB) can degrade polybrominated diphenyl ethers (PBDEs); however, very little is known about the distribution of OHRB or their response to PBDE contamination in natural sediments. We collected sediments from 28 sampling sites in Taihu Lake, China, and investigated the spatial distribution and diversity of OHRB, and the relationships between the PBDE contamination levels and the PBDE removal potential. The abundances of five typical OHRB genera, namely Dehalobacter, Dehalococcoides, Dehalogenimonas, Desulfitobacterium, and Geobacter, ranged from 0.34 × 104 to 19.4 × 107 gene copies g-1 dry sediment, and varied significantly among different areas of Taihu Lake. OHRB were more abundant in sediments from Meiliang and Zhushan Bay, where the PBDE concentrations were higher, and the phylotype diversity of the OHRB belonging to the family Dehalococcoidaceae was lower, than reported for other areas. While the sulfate concentrations explained much of the spatial distribution of OHRB, PBDE concentrations were also a strong influence on the abundance and diversity of OHRB in the sediments. For Dehalococcoides, Dehalogenimonas and Geobacter, the abundance of each genus was positively related to its own potential to remove PBDEs. The dominant OHRB genus, Dehalogenimonas, may contribute most to in situ bioremediation of PBDEs in Taihu Lake.

Grape pomace compost harbors organohalide-respiring Dehalogenimonas species with novel reductive dehalogenase genes.

Organohalide-respiring bacteria have key roles in the natural chlorine cycle; however, most of the current knowledge is based on cultures from contaminated environments. We demonstrate that grape pomace compost without prior exposure to chlorinated solvents harbors a Dehalogenimonas (Dhgm) species capable of using chlorinated ethenes, including the human carcinogen and common groundwater pollutant vinyl chloride (VC) as electron acceptors. Grape pomace microcosms and derived solid-free enrichment cultures were able to dechlorinate trichloroethene (TCE) to less chlorinated daughter products including ethene. 16S rRNA gene amplicon and qPCR analyses revealed a predominance of Dhgm sequences, but Dehalococcoides mccartyi (Dhc) biomarker genes were not detected. The enumeration of Dhgm 16S rRNA genes demonstrated VC-dependent growth, and 6.55±0.64 × 108 cells were measured per µmole of chloride released. Metagenome sequencing enabled the assembly of a Dhgm draft genome, and 52 putative reductive dehalogenase (RDase) genes were identified. Proteomic workflows identified a putative VC RDase with 49 and 56.1% amino acid similarity to the known VC RDases VcrA and BvcA, respectively. A survey of 1,173 groundwater samples collected from 111 chlorinated solvent-contaminated sites in the United States and Australia revealed that Dhgm 16S rRNA genes were frequently detected and outnumbered Dhc in 65% of the samples. Dhgm are likely greater contributors to reductive dechlorination of chlorinated solvents in contaminated aquifers than is currently recognized, and non-polluted environments represent sources of organohalide-respiring bacteria with novel RDase genes.

Hypersaline sapropels act as hotspots for microbial dark matter.

Present-day terrestrial analogue sites are crucial ground truth proxies for studying life in geochemical conditions close to those assumed to be present on early Earth or inferred to exist on other celestial bodies (e.g. Mars, Europa). Although hypersaline sapropels are border-of-life habitats with moderate occurrence, their microbiological and physicochemical characterization lags behind. Here, we study the diversity of life under low water activity by describing the prokaryotic communities from two disparate hypersaline sapropels (Transylvanian Basin, Romania) in relation to geochemical milieu and pore water chemistry, while inferring their role in carbon cycling by matching taxa to known taxon-specific biogeochemical functions. The polyphasic approach combined deep coverage SSU rRNA gene amplicon sequencing and bioinformatics with RT-qPCR and physicochemical investigations. We found that sapropels developed an analogous elemental milieu and harbored prokaryotes affiliated with fifty-nine phyla, among which the most abundant were Proteobacteria, Bacteroidetes and Chloroflexi. Containing thirty-two candidate divisions and possibly undocumented prokaryotic lineages, the hypersaline sapropels were found to accommodate one of the most diverse and novel ecosystems reported to date and may contribute to completing the phylogenetic branching of the tree of life.

Soil bacterial quantification approaches coupling with relative abundances reflecting the changes of taxa.

Understanding the abundance change of certain bacterial taxa is quite important for the study of soil microbiology. However, the observed differences of relative abundances by high-throughput techniques may not accurately reflect those of the actual taxon abundances. This study investigated whether soil microbial abundances coupling with microbial quantities can be more informative in describing the microbial population distribution under different locations. We analyzed relative abundances of the major species in soil microbial communities from Beijing and Tibet grasslands by using 16 S rRNA high-throughput sequencing technique, and quantified the absolute bacterial cell numbers directly or indirectly by multiple culture-independent measurements, including adenosine tri-phosphate (ATP), flow cytometry (FCM), quantitative real-time PCR (qPCR), phospholipid fatty acids (PLFA) and microbial biomass Carbon (MBC). By comparison of the relative abundance and the estimated absolute abundances (EAA) of the major components in soil microbial communities, several dominant phyla, including Actinobacteria, Bacteroidetes, Verrucomicrobia, Chloroflexi, Gemmatimonates and Planctomycetes, showed significantly different trends. These results indicated that the change in EAA might be more informative in describing the dynamics of a population in a community. Further studies of soil microbes should combine the quantification and relative abundances of the microbial communities for the comparisons among various locations.

Dehalogenimonas formicexedens sp. nov., a chlorinated alkane-respiring bacterium isolated from contaminated groundwater.

A strictly anaerobic, Gram-stain-negative, non-spore-forming bacterium designated NSZ-14T, isolated from contaminated groundwater in Louisiana (USA), was characterized using a polyphasic approach. Strain NSZ-14T reductively dehalogenated a variety of polychlorinated aliphatic alkanes, producing ethene from 1,2-dichloroethane, propene from 1,2-dichloropropane, a mixture of cis- and trans-1,2-dichloroethene from 1,1,2,2-tetrachloroethane, vinyl chloride from 1,1,2-trichloroethane and allyl chloride (3-chloro-1-propene) from 1,2,3-trichloropropane. Formate or hydrogen could both serve as electron donors. Dechlorination occurred between pH 5.5 and 7.5 and over a temperature range of 20-37 °C. Major cellular fatty acids included C18 : 1ω9c, C14 : 0 and C16 : 0. 16S rRNA gene sequence-based phylogenetic analysis indicated that the strain clusters within the class Dehalococcoidia of the phylum Chloroflexi, most closely related to but distinct from type strains of the species Dehalogenimonas alkenigignens (97.63 % similarity) and Dehalogenimonas lykanthroporepellens (95.05 %). A complete genome sequence determined for strain NSZ-14T revealed a DNA G+C content of 53.96 mol%, which was corroborated by HPLC (54.1±0.2 mol% G+C). Genome-wide comparisons based on average nucleotide identity by orthology and estimated DNA-DNA hybridization values combined with phenotypic and chemotaxonomic traits and phylogenetic analysis indicate that strain NSZ-14T represents a novel species within the genus Dehalogenimonas, for which the name Dehalogenimonas formicexedens sp. nov. is proposed. The type strain is NSZ-14T (=HAMBI 3672T=JCM 19277T=VKM B-3058T). An emended description of Dehalogenimonas alkenigignens is also provided.

The Relative Abundance and Transcriptional Activity of Marine Sponge-Associated Microorganisms Emphasizing Groups Involved in Sulfur Cycle.

During the last decades, our knowledge about the activity of sponge-associated microorganisms and their contribution to biogeochemical cycling has gradually increased. Functional groups involved in carbon and nitrogen metabolism are well documented, whereas knowledge about microorganisms involved in the sulfur cycle is still limited. Both sulfate reduction and sulfide oxidation has been detected in the cold water sponge Geodia barretti from Korsfjord in Norway, and with specimens from this site, the present study aims to identify extant versus active sponge-associated microbiota with focus on sulfur metabolism. Comparative analysis of small subunit ribosomal RNA (16S rRNA) gene (DNA) and transcript (complementary DNA (cDNA)) libraries revealed profound differences. The transcript library was predominated by Chloroflexi despite their low abundance in the gene library. An opposite result was found for Acidobacteria. Proteobacteria were detected in both libraries with representatives of the Alpha- and Gammaproteobacteria related to clades with presumably thiotrophic bacteria from sponges and other marine invertebrates. Sequences that clustered with sponge-associated Deltaproteobacteria were remotely related to cultivated sulfate-reducing bacteria. The microbes involved in sulfur cycling were identified by the functional gene aprA (adenosine-5'-phosphosulfate reductase) and its transcript. Of the aprA sequences (DNA and cDNA), 87 % affiliated with sulfur-oxidizing bacteria. They clustered with Alphaproteobacteria and with clades of deep-branching Gammaproteobacteria. The remaining sequences clustered with sulfate-reducing Archaea of the phylum Euryarchaeota. These results indicate an active role of yet uncharacterized Bacteria and Archaea in the sponge's sulfur cycle.

High-throughput sequencing of microbial diversity in implant-associated infection.

Few molecular studies have shown that the number of bacterial species in implant-associated infection may have been underestimated. To determine the actual microbial diversity in implant-associated infection, a high-throughput sequencing method was adopted to sequence the DNAs extracted from the tissues of infected and uninfected patients. Principal component analysis (PCA) and ß diversity showed an obvious divergence of infected and uninfected groups, and that the overgrowth of Proteobacteria (80.87%), Firmicutes (13.41%) in the positive deep infection group (P.d, via biopsy) and Proteobacteria (91.68%) in the positive surface infection group (P.s, via swabs) might be the causative factors in implant-associated infection. Moreover, Venn results indicated that a mean of 330 common operational taxonomic units (OTUs) was obtained in all groups, of which 113, 109, 45, 20, 13 and 12 OTUs belonging to Proteobacteria, Actinobacteria, Acidobacteria, Planctomycetes, Gemmatimonadetes and Chloroflexi were identified. In conclusion, many traditional "pathogenic bacteria" were identified as the common bacteria in operation sites, and the disruption of their complex interaction caused infection; therefore, further work is need to illustrate the aetiology of implant-associated infection using in-depth systems-level analyses.

The corrinoid cofactor of reductive dehalogenases affects dechlorination rates and extents in organohalide-respiring Dehalococcoides mccartyi.

Corrinoid auxotrophic organohalide-respiring Dehalococcoides mccartyi (Dhc) strains are keystone bacteria for reductive dechlorination of toxic and carcinogenic chloroorganic contaminants. We demonstrate that the lower base attached to the essential corrinoid cofactor of reductive dehalogenase (RDase) enzyme systems modulates dechlorination activity and affects the vinyl chloride (VC) RDases BvcA and VcrA differently. Amendment of 5,6-dimethylbenzimidazolyl-cobamide (DMB-Cba) to Dhc strain BAV1 and strain GT cultures supported cis-1,2-dichloroethene-to-ethene reductive dechlorination at rates of 107.0 (±12.0) µM and 67.4 (±1.4) µM Cl(-) released per day, respectively. Strain BAV1, expressing the BvcA RDase, reductively dechlorinated VC to ethene, although at up to fivefold lower rates in cultures amended with cobamides carrying 5-methylbenzimidazole (5-MeBza), 5-methoxybenzimidazole (5-OMeBza) or benzimidazole (Bza) as the lower base. In contrast, strain GT harboring the VcrA RDase failed to grow and dechlorinate VC to ethene in medium amended with 5-OMeBza-Cba or Bza-Cba. The amendment with DMB to inactive strain GT cultures restored the VC-to-ethene-dechlorinating phenotype and intracellular DMB-Cba was produced, demonstrating cobamide uptake and remodeling. The distinct responses of Dhc strains with BvcA versus VcrA RDases to different cobamides implicate that the lower base exerts control over Dhc reductive dechlorination rates and extents (that is, detoxification), and therefore the dynamics of Dhc strains with discrete reductive dechlorination capabilities. These findings emphasize that the role of the corrinoid/lower base synthesizing community must be understood to predict strain-specific Dhc activity and achieve efficacious contaminated site cleanup.

Reductive Dehalogenases Come of Age in Biological Destruction of Organohalides.

Halogenated organic compounds (organohalides) are globally prevalent, recalcitrant toxic, and carcinogenic environmental pollutants. Select microorganisms encode enzymes known as reductive dehalogenases (EC 1.97.1.8) that catalyze reductive dehalogenation reactions resulting in the generation of lesser-halogenated compounds that may be less toxic and more biodegradable. Recent breakthroughs in enzyme structure determination, elucidation of the mechanisms of reductive dehalogenation, and in heterologous expression of functional reductive dehalogenase enzymes have substantially increased our understanding of this fascinating class of enzymes. This knowledge has created opportunities for more versatile (in situ and ex situ) biologically-mediated organohalide destruction strategies.

Contributions of Abiotic and Biotic Dechlorination Following Carboxymethyl Cellulose Stabilized Nanoscale Zero Valent Iron Injection.

A pilot scale injection of nanoscale zerovalent iron (nZVI) stabilized with carboxymethyl cellulose (CMC) was performed at an active field site contaminated with a range of chlorinated volatile organic compounds (cVOC). The cVOC concentrations and microbial populations were monitored at the site before and after nZVI injection. The remedial injection successfully reduced parent compound concentrations on site. A period of abiotic degradation was followed by a period of enhanced biotic degradation. Results suggest that the nZVI/CMC injection created conditions that stimulated the native populations of organohalide-respiring microorganisms. The abundance of Dehalococcoides spp. immediately following the nZVI/CMC injection increased by 1 order of magnitude throughout the nZVI/CMC affected area relative to preinjection abundance. Distinctly higher cVOC degradation occurred as a result of the nZVI/CMC injection over a 3 week evaluation period when compared to control wells. This suggests that both abiotic and biotic degradation occurred following injection.

Manganese and iron as structuring parameters of microbial communities in Arctic marine sediments from the Baffin Bay.

The Arctic Baffin Bay between Canada and Greenland is sea ice-covered during the majority of the year, restricting primary production to the summer months. Sediments receive low amounts of mostly terrestrial- and less marine-derived organic matter. To study microbial communities constrained by physicochemical conditions changing with distance from land and ocean depth, we applied high-throughput 16S rRNA gene sequencing and compared sequence diversity with biogeochemical parameters in 40 different sediment samples. Samples originated from seven cores down to 470 cm below seafloor along a shelf-to-basin transect. Bacterial diversity decreased faster with depth in basin than in shelf sediments, suggesting higher organic matter content sustained diversity into greater depths. All samples were dominated by Betaproteobacteria (mostly order Burkholderiales), which were especially abundant in basin sediments with low organic carbon and high Mn and Fe pore water concentrations. Strong statistical correlations between concentrations of reduced Mn and/or Fe and the relative abundances of Betaproteobacteria suggest that this group is involved in metal reduction in Baffin Bay sediments. Dehalococcoidia (phylum Chloroflexi) were abundant in all samples, especially in shelf sediments with high organic content. This study indicates that Mn and/or Fe play important roles structuring microbial communities in Arctic sediments poor in organic matter.

Polyphasic characterization of an anaerobic hexachlorobenzene-dechlorinating microbial consortium with a wide dechlorination spectrum for chlorobenzenes.

An anaerobic consortium that was capable of reductively dechlorinating hexachlorobenzene (HCB) to benzene was enriched from contaminated sediment. The consortium was capable of dechlorinating all chlorobenzene isomers except 1,4-dichlorobenzene. Singly and doubly flanked chlorines, as well as unflanked meta-substituted chlorines, were dechlorinated, although doubly flanked chlorines were preferred. Formate, acetate and lactate (but not ethanol) could be utilized as optimum electron donors for reductive dechlorination. Alternative electron acceptors, including nitrate and sulfate, completely inhibited HCB degradation, whereas amorphous iron oxide (FeOOH) did not suppress dechlorination activity. No degradation was found in chloramphenicol-treated consortium; however, vancomycin, molybdate, and 2-bromoethanesulfonate did not inhibit HCB dechlorination. The results of inhibitory treatments suggested that the dechlorinators were non-sulfate-reducing gram-negative or vancomycin resistant gram-positive bacteria. In addition to physiological characterization, analyses of 16S rRNA gene library of the consortium and quantitative PCR of 16S rRNA genes suggested that Dehalococcoides sp. was involved in the reductive dechlorination of HCB, and Geobacter sp. may serve as a dechlorinating candidate.

Biochemical and EPR-spectroscopic investigation into heterologously expressed vinyl chloride reductive dehalogenase (VcrA) from Dehalococcoides mccartyi strain VS.

Reductive dehalogenases play a critical role in the microbial detoxification of aquifers contaminated with chloroethenes and chlorethanes by catalyzing the reductive elimination of a halogen. We report here the first heterologous production of vinyl chloride reductase VcrA from Dehalococcoides mccartyi strain VS. Heterologously expressed VcrA was reconstituted to its active form by addition of hydroxocobalamin/adenosylcobalamin, Fe(3+), and sulfide in the presence of mercaptoethanol. The kinetic properties of reconstituted VcrA catalyzing vinyl chloride reduction with Ti(III)-citrate as reductant and methyl viologen as mediator were similar to those obtained previously for VcrA as isolated from D. mccartyi strain VS. VcrA was also found to catalyze a novel reaction, the environmentally important dihaloelimination of 1,2-dichloroethane to ethene. Electron paramagnetic resonance (EPR) spectroscopic studies with reconstituted VcrA in the presence of mercaptoethanol revealed the presence of Cob(II)alamin. Addition of Ti(III)-citrate resulted in the appearance of a new signal characteristic of a reduced [4Fe-4S] cluster and the disappearance of the Cob(II)alamin signal. UV-vis absorption spectroscopy of Ti(III)citrate-treated samples revealed the formation of two new absorption maxima characteristic of Cob(I)alamin. No evidence for the presence of a [3Fe-4S] cluster was found. We postulate that during the reaction cycle of VcrA, a reduced [4Fe-4S] cluster reduces Co(II) to Co(I) of the enzyme-bound cobalamin. Vinyl chloride reduction to ethene would be initiated when Cob(I)alamin transfers an electron to the substrate, generating a vinyl radical as a potential reaction intermediate.

Molecular diversity of fungal and bacterial communities in the marine sponge Dragmacidon reticulatum.

The present work aimed to investigate the diversity of bacteria and filamentous fungi of southern Atlantic Ocean marine sponge Dragmacidon reticulatum using cultivation-independent approaches. Fungal ITS rDNA and 18S gene analyses (DGGE and direct sequencing approaches) showed the presence of representatives of three order (Polyporales, Malasseziales, and Agaricales) from the phylum Basidiomycota and seven orders belonging to the phylum Ascomycota (Arthoniales, Capnodiales, Dothideales, Eurotiales, Hypocreales, Pleosporales, and Saccharomycetales). On the other hand, bacterial 16S rDNA gene analyses by direct sequencing approach revealed the presence of representatives of seven bacterial phyla (Cyanobacteria, Proteobacteria, Actinobacteria, Bacteroidetes, Lentisphaerae, Chloroflexi, and Planctomycetes). Results from statistical analyses (rarefaction curves) suggested that the sampled clones covered the fungal diversity in the sponge samples studied, while for the bacterial community additional sampling would be necessary for saturation. This is the first report related to the molecular analyses of fungal and bacterial communities by cultivation-independent approaches in the marine sponges D. reticulatum. Additionally, the present work broadening the knowledge of microbial diversity associated to marine sponges and reports innovative data on the presence of some fungal genera in marine samples.