Temperature impacts community structure and function of phototrophic Chloroflexi and Cyanobacteria in two alkaline hot springs in Yellowstone National Park.

Photosynthetic bacteria are abundant in alkaline, terrestrial hot springs and there is a long history of research on phototrophs in Yellowstone National Park (YNP). Hot springs provide a framework to examine the ecophysiology of phototrophs in situ because they provide natural gradients of geochemistry, pH and temperature. Phototrophs within the Cyanobacteria and Chloroflexi groups are frequently observed in alkaline hot springs. Decades of research has determined that temperature constrains Cyanobacteria in alkaline hot springs, but factors that constrain the distribution of phototrophic Chloroflexi remain unresolved. Using a combination of 16S rRNA gene sequencing and photoassimilation microcosms, we tested the hypothesis that temperature would constrain the activity and composition of phototrophic Cyanobacteria and Chloroflexi. We expected diversity and rates of photoassimilation to decrease with increasing temperature. We report 16S rRNA amplicon sequencing along with carbon isotope signatures and photoassimilation from 45 to 72°C in two alkaline hot springs. We find that Roseiflexus, Chloroflexus (Chloroflexi) and Leptococcus (Cyanobacteria) operational taxonomic units (OTUs) have distinct distributions with temperature. This distribution suggests that, like phototrophic Cyanobacteria, temperature selects for specific phototrophic Chloroflexi taxa. The richness of phototrophic Cyanobacteria decreased with increasing temperature along with a decrease in oxygenic photosynthesis, whereas Chloroflexi richness and rates of anoxygenic photosynthesis did not decrease with increasing temperature, even at temperatures approaching the upper limit of photosynthesis (~72-73°C). Our carbon isotopic data suggest an increasing prevalence of the 3-hydroxypropionate pathway with decreasing temperature coincident with photoautotrophic Chloroflexi. Together these results indicate temperature plays a role in defining the niche space of phototrophic Chloroflexi (as has been observed for Cyanobacteria), but other factors such as morphology, geochemistry, or metabolic diversity of Chloroflexi, in addition to temperature, could determine the niche space of this highly versatile group.

Network-directed efficient isolation of previously uncultivated Chloroflexi and related bacteria in hot spring microbial mats.

The perplexity of the complex multispecies community interactions is one of the many reasons why majority of the microorganisms are still uncultivated. We analyzed the entire co-occurrence networks between the OTUs of Tibet and Yunnan hot spring samples, and found that less abundant OTUs such as genus Tepidimonas (relative abundant <1%) had high-degree centricity (key nodes), while dominant OTUs particularly genus Chloroflexus (relative abundant, 13.9%) formed the peripheral vertexes. A preliminary growth-promotion assay determined that Tepidimonas sp. strain SYSU G00190W enhanced the growth of Chloroflexus sp. SYSU G00190R. Exploiting this result, an ameliorated isolation medium containing 10% spent-culture supernatant of Tepidimonas sp. strain SYSU G00190W was prepared for targeted isolation of Chloroflexi in the Tibet and Yunnan hot spring samples. 16S rRNA gene fingerprinting characterized majority of the colonies isolated from these media as previously uncultivated Chloroflexi, of which 36 are potential novel species (16S rRNA sequence identity <98.5%). Metabolomes studies indicated that the spent-culture supernatant comprises several low-molecular-weight organic substrates that can be utilized as potential nutrients for the growth of these bacteria. These findings suggested that limited knowledge on the interaction of microbes provide threshold to traditional isolation method.

Exploring the operating factors controlling Kouleothrix (type 1851), the dominant filamentous bacterial population, in a full-scale A2O plant.

This study reveals that the abundance of the filament Kouleothrix (Eikelboom type 1851) correlated positively with poor settleability of activated sludge biomass in a Japanese full-scale nutrient removal wastewater treatment plant sampled over a one-year period. 16S rRNA amplicon sequence data confirmed that Kouleothrix was the dominant filament in the plant, with a relative abundance of 3.06% positively correlated with sludge volume index (SVI) (R = 0.691). Moreover, Kouleothrix (type 1851) appeared to form interfloc bridges, typical of bulking sludge, regardless of season. Together with earlier studies that indicated the responsibility of Kouleothrix (type 1851) on bulking events, these data suggest that their high relative abundances alone may be responsible for sludge bulking. 16S rRNA qPCR data for this filament showed changes in its relative abundance correlated with changes in several operational parameters, including mixed liquor temperature, sludge retention time, and suspended solids concentration, and it may be that manipulating these may help control Kouleothrix bulking.

Enhanced reductive dechlorination of trichloroethene with immobilized Clostridium butyricum in silica gel.

Deteriorated environmental conditions during the bioremediation of trichloroethene (TCE)-polluted groundwater cause decreased treatment efficiencies. This study assessed the effect of applying immobilized Clostridium butyricum (a hydrogen-producing bacterium) in silica gel on enhancing the reductive dechlorination efficiency of TCE with the slow polycolloid-releasing substrate (SPRS) supplement in groundwater. The responses of microbial communities with the immobilized system (immobilized Clostridium butyricum and SPRS amendments) were also characterized by the metagenomics assay. A complete TCE removal in microcosms was obtained within 30 days with the application of this immobilized system via reductive dechlorination processes. An increase in the population of Dehalococcoides spp. was observed using the quantitative polymerase chain reaction (qPCR) analysis. Results of metagenomics assay reveal that the microbial communities in the immobilized system were distinct from those in systems with SPRS only. Bacterial communities associated with TCE biodegradation also increased in microcosms treated with the immobilized system. The immobilized system shows a great potential to promote the TCE dechlorination efficiency, and the metagenomics-based approach provides detailed insights into dechlorinating microbial community dynamics. The results would be helpful in designing an in situ immobilized system to enhance the bioremediation efficiency of TCE-contaminated groundwater.

Bacterial, archaeal and micro-eukaryotic communities characterize a disease-suppressive or conducive soil and a cultivar resistant or susceptible to common scab.

Control of common scab disease can be reached by resistant cultivars or suppressive soils. Both mechanisms are likely to translate into particular potato microbiome profiles, but the relative importance of each is not known. Here, microbiomes of bulk and tuberosphere soil and of potato periderm were studied in one resistant and one susceptible cultivar grown in a conducive and a suppressive field. Disease severity was suppressed similarly by both means yet, the copy numbers of txtB gene (coding for a pathogenicity determinant) were similar in both soils but higher in periderms of the susceptible cultivar from conducive soil. Illumina sequencing of 16S rRNA genes for bacteria (completed by 16S rRNA microarray approach) and archaea, and of 18S rRNA genes for micro-eukarytes showed that in bacteria, the more important was the effect of cultivar and diversity decreased from resistant cultivar to bulk soil to susceptible cultivar. The major changes occurred in proportions of Actinobacteria, Chloroflexi, and Proteobacteria. In archaea and micro-eukaryotes, differences were primarily due to the suppressive and conducive soil. The effect of soil suppressiveness × cultivar resistance depended on the microbial community considered, but differed also with respect to soil and plant nutrient contents particularly in N, S and Fe.

Deiodination in the presence of Dehalococcoides mccartyi strain CBDB1: comparison of the native enzyme and co-factor vitamin B12.

Triiodinated benzoic acid derivatives are widely used as contrast media for medical examinations and are found at high concentrations in urban aquatic environments. During bank filtration, deiodination of iodinated contrast media has been observed under anoxic/anaerobic conditions. While several bacterial strains capable of dechlorination and debromination have been isolated and characterized, deiodination has not yet been shown for an isolated strain. Here, we investigate dehalogenation of iodinated contrast media (ICM), triiodobenzoic acids (TIBA), and analogous chlorinated compounds by Dehalococcoides mccartyi strain CBDB1 and its corrinoid co-factor vitamin B12. No cell growth of CBDB1 was observed using iodinated compounds as electron acceptor. Only negligible deiodination occurred for ICM, whereas 2,3,5-TIBA was nearly completely deiodinated by CBDB1 without showing cell growth. Furthermore, TIBA inhibited growth with hexachlorobenzene which is usually a well-suited electron acceptor for strain CBDB1, indicating that TIBA is toxic for CBDB1. The involvement of CBDB1 enzymes in the deiodination of TIBA was verified by the absence of deiodination activity after heat inactivation. Adding iodopropane also inhibited the deiodination of TIBA by CBDB1 cells, indicating the involvement of a corrinoid-enzyme in the reductive TIBA deiodination. The results further suggest that the involved electron transport is decoupled from proton translocation and therefore growth. Graphical abstract.

Colonization and growth of dehalorespiring biofilms on carbonaceous sorptive amendments.

Removal of polychlorinated biphenyls (PCBs) from contaminated sediments is a priority due to accumulation in the food chain. Recent success with reduction of PCB bioavailability due to adsorption onto activated carbon led to the recognition of in situ treatment as a remediation approach. In this study, reduced bioavailability and subsequent break-down of PCBs in dehalorespiring biofilms was investigated using Dehalobium chlorocoercia DF1. DF1 formed a patchy biofilm ranging in thickness from 3.9 to 6.7 µm (average 4.6 ± 0.87 µm), while the biofilm coverage varied from 5.5% (sand) to 20.2% (activated carbon), indicating a preference for sorptive materials. Quantification of DF1 biofilm bacteria showed 1.2-15.3 × 109 bacteria per gram of material. After 22 days, coal activated carbon, bone biochar, polyoxymethylene, and sand microcosms had dechlorinated 73%, 93%, 100%, and 83%, respectively. These results show that a biofilm-based inoculum for bioaugmentation of PCBs in sediment can be an efficient approach.

Estimating bioaugmentation efficacy of TCE dechlorination using long-term field well-to-well tests in a highly recharged and TCE-contaminated aquifer.

This study demonstrates a combined field method accurately assessing the extent of trichloroethylene (TCE) reductive dechlorination activity and the mass fraction of its by-products. A combined method of injecting a known concentration of 1,1,2-trichloro-2-fluoroethene (TCFE) as a TCE bio-surrogate and a data processing technique of forced mass balance (FMB), considering the sorption effect on the mass fraction of chloroethene was evaluated by performing soil column and field bioaugmentation tests. In the soil column test, the FMB resulted in the mass fraction of 6% TCE, 48.3% cis-1,2-dichloroethene, 18.5% vinyl chloride and 27.2% ethylene. In the field bioaugmentation test, TCFE showed equivalent dechlorination pathways of TCE. The mass fractions estimated by FMB were very similar to those observed in the soil column bioaugmentation tests: 4.5% TCFE, 57.1% 1,2-dichloro-1-fluoroethene, 12% 1-chloro-1-fluoroethene and 26.4% fluoroethene (FE). The FMB method gave ∼50% higher mass fraction for more chlorinated ethenes (i.e., TCFE) and ∼10% lower mass fraction of less chlorinated ethenes (i.e., FE) than those considering only the aqueous concentrations of chlorofluoroethenes. A combined method of TCFE and FMB that could accurately estimate both the extent of dechlorination activities and mass distribution of TCE reductive dechlorination would be highly useful.

A short-term stimulation of ethanol enhances the effect of magnetite on anaerobic digestion.

Conductive iron oxides (CIO) have been proved recently to facilitate the anaerobic microbial syntrophy based on the direct interspecies electron transfer (DIET) in batch experiments. However, the effect of CIO was always insignificant in anaerobic digestion (AD) reactor especially when the DIET-based syntrophic partners were absent. In this study, the effect of magnetite on performance of AD system with sucrose as a sole carbon source was investigated, but limited enhancement was achieved during the first 36-day operation. The short-term effect of ethanol addition was further studied in the magnetite-amended AD reactor, and results showed that the AD reactor with 10gFe/L micro-sized magnetite (R3) achieved higher performance of COD removal and methane proportion compared with the other reactors (R1 without magnetite; R2 with 2gFe/L micro-sized magnetite; R4 with 2gFe/L nano-sized magnetite). Meanwhile, the pyridoxine in extracellular polymeric substances (EPS) and conductivity of anaerobic sludge from R3 increased more significantly than those of the others. Analysis of high-throughput sequencing indicated that the abundance of archaea increased in sludge from R3 and Methanosarcina responsible for DIET was dominant (63.64%). Additionally, the abundance of potential electroactive bacteria Chloroflexi in R3 was 7.57-fold, 3.61-fold and 7.37-fold as that of R1, R2 and R4, respectively. These results demonstrated that the electroactive microbes and methanogens could be enriched efficiently in anaerobic sludge via synergetic effect of magnetite addition and ethanol short-term stimulation.

Reductive dechlorination of high concentrations of chloroethenes by a Dehalococcoides mccartyi strain 11G.

Chloroethenes are common groundwater and soil contaminants due to extensive historic utilization and inappropriate discharge. The tendency for chloroethenes to become sequestered as dense non-aqueous phase liquids (DNAPL)-a point source to groundwater contamination and causing high concentrations of chloroethenes in proximal aquifers poses a great challenge for remediation of chloroethene contaminated sites. In this study, we report isolation and characterization of a Dehalococcoides mccartyi strain 11G which couples growth with reductive dechlorination of trichloroethenes (TCE), dichloroethene (DCE) isomers and vinyl chloride (VC) to ethene at a growth yield ranging from 2.47 ± 0.23 × 108 to 5.64 ± 0.43 × 108 cells/µmoles Cl- released and co-metabolically dechlorinates tetrachloroethene (PCE) in the presence of TCE. Compared with previous D. mccartyi strains showing dechlorination of TCE at up to 2.0 mM, strain 11G is distinguished by its capacity to dechlorinate chloroethenes at initial concentrations of DCE isomers as high as 4 mM and TCE as high as 3.5 mM to ethene. Bioaugmentation of a contaminated microcosm with strain 11G resulted in complete detoxification of a mixture of 5 mM chloroethenes (2.5 mM of each TCE and cis-DCE) after 40 days. Strain 11G is a promising candidate for in situ bioremediation of high-concentration-chloroethene contaminated sites.

In situ visualisation of the abundant Chloroflexi populations in full-scale anaerobic digesters and the fate of immigrating species.

Anaerobic digestion is a key process for the conversion of waste organics to biogas for energy and is reliant on the synergistic activities of complex microbial communities. Members of the phylum Chloroflexi are often found to be abundant in these systems, yet little is known of their role, with most members yet to be cultured or identified. The aim of this study was to characterize the Chloroflexi communities present in full-scale anaerobic digesters receiving excess sludge from wastewater treatment plants. The core genus-level-phylotypes were identified from extensive 16S rRNA gene amplicon sequencing surveys of 19 full-scale systems over a 6 year period. The T78 and Leptolinea, and the RB349 and SJA-170, were found to be the most abundant genera of mesophilic and thermophilic digesters, respectively. With the exception of Leptolinea, these phylotypes are known only by their 16S rRNA gene sequence, and their morphology and metabolic potentials are not known. Fluorescence in situ hybridisation (FISH) probes were designed for these phylotypes, with their application revealing a similar thin filamentous morphology, indicating a possible role for these organisms in maintaining floc structure. The new FISH probes provide a useful tool for future efforts to characterize these organisms in situ. FISH also suggests that immigrating Chloroflexi species die off in the anaerobic digester environment and their high abundance in anaerobic digesters, observed with DNA based sequencing surveys, was quite possibly due to the persistence of their DNA after their death. This observation is important for the interpretation of popular DNA-based sequencing methods applied for the characterisation of communities with substantial immigration rates, such as anaerobic digesters.

In situ responses of the sponge microbiome to ocean acidification.

Climate change is causing rapid changes in reef structure, biodiversity, and function, though most sponges are predicted to tolerate conditions projected for 2100. Sponges maintain intimate relationships with microbial symbionts, with previous studies suggesting that microbial flexibility may be pivotal to success under ocean acidification (OA). We performed a reciprocal transplantation of the coral reef sponges Coelocarteria singaporensis and Stylissa cf. flabelliformis between a control reef site and an adjacent CO2 vent site in Papua New Guinea to explore how the sponge microbiome responds to OA. Microbial communities of C. singaporensis, which differed initially between sites, did not shift towards characteristic control or vent microbiomes, even though relative abundances of Chloroflexi and Cyanobacteria increased and that of Thaumarchaeota decreased 7 months after transplantation to the control site. Microbial communities of S. cf. flabelliformis, which were initially stable between sites, did not respond specifically to transplantation but collectively exhibited a significant change over time, with a relative increase in Thaumarchaeota and decrease in Proteobacteria in all treatment groups. The lack of a community shift upon transplantation to the vent site suggests that microbial flexibility, at least in the adult life-history stage, does not necessarily underpin host survival under OA .

Growth of Dehalococcoides mccartyi species in an autotrophic consortium producing limited acetate.

The dechlorinating Dehalococcoides mccartyi species requires acetate as carbon source, but little is known on its growth under acetate limiting conditions. In this study, we observed growth and dechlorination of a D. mccartyi-containing mixed consortium in a fixed-carbon-free medium with trichloroethene in the aqueous phase and H2/CO2 in the headspace. Around 4 mM formate was produced by day 40, while acetate was constantly below 0.05 mM. Microbial community analysis of the consortium revealed dominance by D. mccartyi and Desulfovibrio sp. (57 and 22% 16S rRNA gene copies, respectively). From this consortium, Desulfovibrio sp. strain F1 was isolated and found to produce formate and acetate (1.2 mM and 48 µM, respectively, by day 24) when cultivated alone in the above mentioned medium without trichloroethene. An established co-culture of strain F1 and D. mccartyi strain 195 demonstrated that strain 195 could grow and dechlorinate using acetate produced by strain F1; and that acetate was constantly below 25 µM in the co-culture. To verify that such low level of acetate is utilizable by D. mccartyi, we cultivated strain 195 alone under acetate-limiting conditions and found that strain 195 consumed acetate to below detection (5 µM). Based on the acetate consumption and cell yield of D. mccartyi, we estimated that on average 1.2 × 108 acetate molecules are needed to supply carbon for one D. mccartyi cell. Our study suggests that Desulfovibrio may supply a steady but low amount of fixed carbon to dechlorinating bacteria, exhibiting important implications for natural bio-attenuation when fixed carbon is limited.

Time-dependent bacterial community and electrochemical characterizations of cathodic biofilms in the surfactant-amended sediment-based bioelectrochemical reactor with enhanced 2,3,4,5-tetrachlorobiphenyl dechlorination.

Applying an electric field to stimulate the microbial reductive dechlorination of polychlorinated biphenyls (PCBs) represents a promising approach for bioremediation of PCB-contaminated sites. This study aimed to demonstrate the biocathodic film-facilitated reduction of PCB 61 in a sediment-based bioelectrochemical reactor (BER) and, more importantly, the characterizations of electrode-microbe interaction from microbial and electrochemical perspectives particularly in a time-dependent manner. The application of a cathodic potential (-0.45 V vs. SHE) significantly improved the rate and extent of PCB 61 dechlorination compared to the open-circuit scenario (without electrical stimulation), and the addition of an external surfactant further increased the dechlorination, with Tween 80 exerting more pronounced effects than rhamnolipid. The bacterial composition of the biofilms and the bioelectrochemical kinetics of the BERs were found to be time-dependent and to vary considerably with the incubation time and slightly with the coexistence of an external surfactant. Excellent correlations were observed between the dechlorination rate and the relative abundance of Dehalogenimonas, Dechloromonas, and Geobacter, the dechlorination rate and the cathodic current density recorded from the chronoamperometry tests, and the dechlorination rate and the charge transfer resistance derived from the electrochemical impedance tests, with respect to the 120 day-operation. After day 120, PCB 61 was resistant to further appreciable reduction, but substantial hydrogen production was detected, and the bacterial community and electrochemical parameters observed on day 180 were not distinctly different from those on day 120.

Evolution of the 3-hydroxypropionate bicycle and recent transfer of anoxygenic photosynthesis into the Chloroflexi.

Various lines of evidence from both comparative biology and the geologic record make it clear that the biochemical machinery for anoxygenic photosynthesis was present on early Earth and provided the evolutionary stock from which oxygenic photosynthesis evolved ca. 2.3 billion years ago. However, the taxonomic identity of these early anoxygenic phototrophs is uncertain, including whether or not they remain extant. Several phototrophic bacterial clades are thought to have evolved before oxygenic photosynthesis emerged, including the Chloroflexi, a phylum common across a wide range of modern environments. Although Chloroflexi have traditionally been thought to be an ancient phototrophic lineage, genomics has revealed a much greater metabolic diversity than previously appreciated. Here, using a combination of comparative genomics and molecular clock analyses, we show that phototrophic members of the Chloroflexi phylum are not particularly ancient, having evolved well after the rise of oxygen (ca. 867 million years ago), and thus cannot be progenitors of oxygenic photosynthesis. Similarly, results show that the carbon fixation pathway that defines this clade-the 3-hydroxypropionate bicycle-evolved late in Earth history as a result of a series of horizontal gene transfer events, explaining the lack of geological evidence for this pathway based on the carbon isotope record. These results demonstrate the role of horizontal gene transfer in the recent metabolic innovations expressed within this phylum, including its importance in the development of a novel carbon fixation pathway.

Water table drawdown shapes the depth-dependent variations in prokaryotic diversity and structure in Zoige peatlands.

Microbial communities are important to ecosystem function and sensitive to hydrological dynamics. However, we lack predictable knowledge about how soil microorganisms respond to water table drawdown in different depths. This research used a high-throughput sequencing method to determine the responses of prokaryotic communities to the changes of water table and depth on Zoige peatlands. Our results showed that water table drawdown reduced alpha diversity indices (observed species, Shannon diversity and Chao1 richness) of prokaryotic communities. Intriguingly, the reduction of diversity varied in different depths, and was statistically significant in intermediate layers (20-30 cm and 50-60 cm), but not in the surface (0-10 cm) or deep layer (90-100 cm). In deeper layers there was greater relative abundance of most anaerobic microorganisms (e.g. Chloroflexi, Planctomyctes and NC10), but lesser amounts of most aerobes (e.g. Proteobacteria and Acidobacteria). However, the vertical distribution of prokaryotic microbiota along the depth gradient was altered by water table drawdown, mainly by enriching oligotrophs (e.g. Acidobcteria) over copiotrophs (e.g. Bacteriodetes). In addition, we found that the most important soil parameters influencing community structure were soil pH, total organic carbon and total nitrogen. Our study illuminates that the variations of prokaryotic communities caused by water table drawdown are depth-dependent, and that water table drawdown leads to predictive changes of microbiota in peatlands.

Effects of side-stream ratio on sludge reduction and microbial structures of anaerobic side-stream reactor coupled membrane bioreactors.

An anoxic/oxic membrane bioreactor (AO-MBR) and three anaerobic side-stream reactor (ASSR) coupled MBRs (ASSR-MBR) were operated to investigate effects of side-stream ratio (SR) on sludge reduction and microbial community structure of ASSR-MBRs. The ASSR-MBR achieved efficient COD and ammonium nitrogen removal. SR increased from 0.2 to 1.0 favored nitrogen removal, and increased sludge reduction from 6.0% to 49.7%. The total released COD in the ASSR increased with the rising SR and was inversely proportional to sludge yield of ASSR-MBR. Pyrosequencing analysis showed that phyla Chloroflexi and Armatimonadetes surviving in anaerobic conditions were enriched in the ASSR, while Nitrospirae was dominant in the MBR. Comparison at the genus level revealed that higher SR favored the growth of slow growers, while lower SR enriched hydrolytic and predatory bacteria. The results suggested that SR has a profound effect on nitrogen removal, sludge reduction and microbial community structure in the ASSR-MBR.

Effects of Sulfate Reduction on Trichloroethene Dechlorination by Dehalococcoides-Containing Microbial Communities.

In order to elucidate interactions between sulfate reduction and dechlorination, we systematically evaluated the effects of different concentrations of sulfate and sulfide on reductive dechlorination by isolates, constructed consortia, and enrichments containing Dehalococcoides sp. Sulfate (up to 5 mM) did not inhibit the growth or metabolism of pure cultures of the dechlorinator Dehalococcoides mccartyi 195, the sulfate reducer Desulfovibrio vulgaris Hildenborough, or the syntroph Syntrophomonas wolfei In contrast, sulfide at 5 mM exhibited inhibitory effects on growth of the sulfate reducer and the syntroph, as well as on both dechlorination and growth rates of D. mccartyi Transcriptomic analysis of D. mccartyi 195 revealed that genes encoding ATP synthase, biosynthesis, and Hym hydrogenase were downregulated during sulfide inhibition, whereas genes encoding metal-containing enzymes involved in energy metabolism were upregulated even though the activity of those enzymes (hydrogenases) was inhibited. When the electron acceptor (trichloroethene) was limiting and an electron donor (lactate) was provided in excess to cocultures and enrichments, high sulfate concentrations (5 mM) inhibited reductive dechlorination due to the toxicity of generated sulfide. The initial cell ratio of sulfate reducers to D. mccartyi (1:3, 1:1, or 3:1) did not affect the dechlorination performance in the presence of sulfate (2 and 5 mM). In contrast, under electron donor limitation, dechlorination was not affected by sulfate amendments due to low sulfide production, demonstrating that D. mccartyi can function effectively in anaerobic microbial communities containing moderate sulfate concentrations (5 mM), likely due to its ability to outcompete other hydrogen-consuming bacteria and archaea.IMPORTANCE Sulfate is common in subsurface environments and has been reported as a cocontaminant with chlorinated solvents at various concentrations. Inconsistent results for the effects of sulfate inhibition on the performance of dechlorination enrichment cultures have been reported in the literature. These inconsistent findings make it difficult to understand potential mechanisms of sulfate inhibition and complicate the interpretation of bioremediation field data. In order to elucidate interactions between sulfate reduction and reductive dechlorination, this study systematically evaluated the effects of different concentrations of sulfate and sulfide on reductive dechlorination by isolates, constructed consortia, and enrichments containing Dehalococcoides sp. This study provides a more fundamental understanding of the competition mechanisms between reductive dechlorination by Dehalococcoides mccartyi and sulfate reduction during the bioremediation process. It also provides insights on the significance of sulfate concentrations on reductive dechlorination under electron donor/acceptor-limiting conditions during in situ bioremediation applications. For example, at a trichloroethene-contaminated site with a high sulfate concentration, proper slow-releasing electron donors can be selected to generate an electron donor-limiting environment that favors reductive dechlorination and minimizes the sulfide inhibition effect.

Rapid Enrichment of Dehalococcoides-Like Bacteria by Partial Hydrophobic Separation.

Organohalide-respiring bacteria can be difficult to enrich and isolate, which can limit research on these important organisms. The goal of this research was to develop a method to rapidly (minutes to days) enrich these organisms from a mixed community. The method presented is based on the hypothesis that organohalide-respiring bacteria would be more hydrophobic than other bacteria as they dehalogenate hydrophobic compounds. The method developed tests this hypothesis by separating a portion of putative organohalide-respiring bacteria, those phylogenetically related to Dehalococcoides mccartyi, at the interface between a hydrophobic organic solvent and an aqueous medium. This novel partial separation technique was tested with a polychlorinated biphenyl-enriched sediment-free culture, a tetrachloroethene-enriched digester sludge culture, and uncontaminated lake sediment. Significantly higher fractions, up to 20.4 times higher, of putative organohalide-respiring bacteria were enriched at the interface between the medium and either hexadecane or trichloroethene. The selective partial separation of these putative organohalide-respiring bacteria occurred after 20 min, strongly suggesting that the separation was a result of physical-chemical interactions between the cell surface and hydrophobic solvent. Dechlorination activity postseparation was verified by the production of cis-dichloroethene when amended with tetrachloroethene. A longer incubation time of 6 days prior to separation with trichloroethene increased the total number of putative organohalide-respiring bacteria. This method provides a way to quickly separate some of the putative organohalide-respiring bacteria from other bacteria, thereby improving our ability to study multiple and different bacteria of potential interest and improving knowledge of these bacteria.IMPORTANCE Organohalide-respiring bacteria, bacteria capable of respiring chlorinated contaminants, can be difficult to enrich, which can limit their predictable use for the bioremediation of contaminated sites. This paper describes a method to quickly separate Dehalococcoides-like bacteria, a group of organisms containing organohalide-respiring bacteria, from other bacteria in a mixed community. From this work, Dehalococcoides-like bacteria appear to have a hydrophobic cell surface, facilitating a rapid (20 min) partial separation from a mixed culture at the surface of a hydrophobic liquid. This method was verified in a polychlorinated biphenyl-enriched sediment-free culture, an anaerobic digester sludge, and uncontaminated sediment. The method described can drastically reduce the amount of time required to partially separate Dehalococcoides-like bacteria from a complex mixed culture, improving researchers' ability to study these important bacteria.

Changes in the Bacterial Community Structure of Remediated Anthracene-Contaminated Soils.

Mixing soil or adding earthworms (Eisenia fetida (Savigny, 1826)) accelerated the removal of anthracene, a polycyclic aromatic hydrocarbon, from a pasture and an arable soil, while a non-ionic surfactant (Surfynol® 485) inhibited the removal of the contaminant compared to the untreated soil. It was unclear if the treatments affected the soil bacterial community and consequently the removal of anthracene. Therefore, the bacterial community structure was monitored by means of 454 pyrosequencing of the 16S rRNA gene in the pasture and arable soil mixed weekly, amended with Surfynol® 485, E. fetida or organic material that served as food for the earthworms for 56 days. In both soils, the removal of anthracene was in the order: mixing soil weekly (100%) > earthworms applied (92%) > organic material applied (77%) > untreated soil (57%) > surfactant applied (34%) after 56 days. There was no clear link between removal of anthracene from soil and changes in the bacterial community structure. On the one hand, application of earthworms removed most of the contaminant from the arable soil and had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of the Acidobacteria, Chloroflexi and Gemmatimonadetes, and an increase in that of the Proteobacteria compared to the unamended soil. Mixing the soil weekly removed all anthracene from the arable soil, but had little or no effect on the bacterial community structure. On the other hand, application of the surfactant inhibited the removal of anthracene from the arable soil compared to the untreated soil, but had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of Cytophagia (Bacteroidetes), Chloroflexi, Gemmatimonadetes and Planctomycetes and an increase in that of the Flavobacteria (Bacteroidetes) and Proteobacteria. Additionally, the removal of anthracene was similar in the different treatments of both the arable and pasture soil, but the effect of application of carrot residue, earthworms or the surfactant on the bacterial community structure was more accentuated in the arable soil than in the pasture soil. It was found that removal of anthracene was not linked to changes in the bacterial community structure.