Microbial Dechlorination of 2,3,5,6-Tetrachlorobiphenyl under Anaerobic Conditions in the Absence of Soil or Sediment

Bacterial enrichment cultures developed with Baltimore Harbor (BH) sediments were found to reductively dechlorinate 2,3,5,6-tetrachlorobiphenyl (2,3,5,6-CB) when incubated in a minimal estuarine medium containing short-chain fatty acids under anaerobic conditions with and without the addition of sediment. Primary enrichment cultures formed both meta and ortho dechlorination products from 2,3,5,6-CB. The lag time preceding dechlorination decreased from 30 to less than 20 days as the cultures were sequentially transferred into estuarine medium containing dried, sterile BH sediment. In addition, only ortho dechlorination was observed following transfer of the cultures. Sequential transfer into medium without added sediment also resulted in the development of a strict ortho-dechlorinating culture following a lag of more than 100 days. Upon further transfer into the minimal medium without sediment, the lag time decreased to less than 50 days. At this stage all cultures, regardless of the presence of sediment, would produce 2,3,5-CB and 3,5-CB from 2,3,5,6-CB. The strict ortho-dechlorinating activity in the sediment-free cultures has remained stable for more than 1 year through several transfers. These results reveal that the classical microbial enrichment technique using a minimal medium with a single polychlorinated biphenyl (PCB) congener selected for ortho dechlorination of 2,3,5,6-CB. Furthermore, this is the first report of sustained anaerobic PCB dechlorination in the complete absence of soil or sediment.Anaerobic dechlorination of polychlorinated biphenyls (PCBs) has been demonstrated in situ and with laboratory microcosms containing sediment (reviewed in reference 1a). However, sustained PCB dechlorination has never been shown to occur in the absence of soil or sediments. Morris et al. (6) demonstrated a sediment requirement for the stimulation of PCB dechlorination within freshwater sediment slurries. Wu and Wiegel have recently described PCB-dechlorinating enrichments which required soil for the successful transfer of PCB-dechlorinating activity (9). In addition, no anaerobic microorganisms that dechlorinate PCBs have been isolated or characterized, and this may be due in part to the soil or sediment requirement. The inability to isolate dechlorinating organisms or maintain dechlorination without sediment has limited biogeochemical and physiological investigations into the mechanisms of PCB dechlorination.Dechlorination (ortho, meta, and para) of single PCB congeners has been observed following anaerobic incubation of Baltimore Harbor (BH) sediment under estuarine or marine conditions (2). While sediments from several sites within BH are contaminated with PCBs (1, 5), background contamination of sediment is not necessarily a prerequisite for the development of PCB dechlorination in laboratory microcosms. Wu et al. (8) recently demonstrated meta and ortho dechlorination of Aroclor 1260 when it was added to the same BH sediments. These results showed that more than one dechlorinating activity could be developed with these sediments. It has been proposed that discrete microbial populations are responsible for specific PCB dechlorinations (1a). Consistent with this idea, the ortho dechlorination observed with BH sediments may be catalyzed by discrete microbial populations. In addition, these organisms may be able to couple PCB dechlorination with growth. Therefore we have attempted to select for ortho PCB-dechlorinating organisms by enrichment under minimal conditions with high levels of 2,3,5,6-tetrachlorobiphenyl. We also speculated that given the proper conditions, a PCB-dechlorinating population could be maintained in an actively dechlorinating state in the absence of sediment. Here we report that a distinct PCB-dechlorinating activity, namely, ortho dechlorination, was selected for through sequential transfer initiated with sediments from BH and sustained in the absence of soil or sediment. This is the first report of sustained anaerobic PCB-dechlorinating activity in the total absence of sediment.     

The Synergistic Effects of High Nitrate Concentrations on Sediment Bioreduction

Groundwaters at nuclear sites can be characterized by low pH and high nitrate concentrations (10–100 mM). These conditions are challenging for bioremediation, often inhibiting microbial Fe(III)-reduction which can limit radionuclide migration. Here, sediment microcosms representative of the UK Sellafield site were used to study the influence of variable pH and nitrate concentrations on microbially-mediated TEAP (terminal electron accepting processes) progression. The rate of reduction through the terminal electron accepting cascade NO^? ₃ > NO^? ₂ > Mn(IV)/Fe(III) > SO^2? ₄ at low pH (～5.5) was slower than that in bicarbonate buffered systems (pH ～ 7.0), but in the low pH systems, denitrification and associated pH buffering resulted in conditioning of the sediments for subsequent Fe(III) and sulfate-reduction. Under very high nitrate conditions (100 mM), bicarbonate buffering (pH ～ 7.0) was necessary for TEAP progression beyond denitrification and the reduction of 100 mM nitrate created alkaline conditions (pH 9.5). 16S rRNA gene analysis showed that close relatives of known nitrate reducers Bacillus niacini and Ochrobactrum grignonense dominated the microbial communities in this reduced sediment. In Fe(III)-reducing enrichment cultures from the 100 mM nitrate system, close relatives of the Fe(III)-reducing species Alkaliphilus crotonatoxidans and Serratia liquifaciens were observed. These results highlight that under certain conditions and contrary to expectations, denitrification may support bioreduction via pH conditioning for optimal metal reduction and radionuclide immobilization.     

The occurrence and ecology of microbial chain elongation of carboxylates in soils

Chain elongation is a growth-dependent anaerobic metabolism that combines acetate and ethanol into butyrate, hexanoate, and octanoate. While the model microorganism for chain elongation, Clostridium kluyveri, was isolated from a saturated soil sample in the 1940s, chain elongation has remained unexplored in soil environments. During soil fermentative events, simple carboxylates and alcohols can transiently accumulate up to low mM concentrations, suggesting in situ possibility of microbial chain elongation. Here, we examined the occurrence and microbial ecology of chain elongation in four soil types in microcosms and enrichments amended with chain elongation substrates. All soils showed evidence of chain elongation activity with several days of incubation at high (100 mM) and environmentally relevant (2.5 mM) concentrations of acetate and ethanol. Three soils showed substantial activity in soil microcosms with high substrate concentrations, converting 58% or more of the added carbon as acetate and ethanol to butyrate, butanol, and hexanoate. Semi-batch enrichment yielded hexanoate and octanoate as the most elongated products and microbial communities predominated by C. kluyveri and other Firmicutes genera not known to undergo chain elongation. Collectively, these results strongly suggest a niche for chain elongation in anaerobic soils that should not be overlooked in soil microbial ecology studies.Subject terms: Soil microbiology, Microbial ecology     

Characterization of a Defined 2,3,5,6-Tetrachlorobiphenyl-ortho-Dechlorinating Microbial Community by Comparative Sequence Analysis of Genes Coding for 16S rRNA

Defined microbial communities were developed by combining selective enrichment with molecular monitoring of total community genes coding for 16S rRNAs (16S rDNAs) to identify potential polychlorinated biphenyl (PCB)-dechlorinating anaerobes that ortho dechlorinate 2,3,5,6-tetrachlorobiphenyl. In enrichment cultures that contained a defined estuarine medium, three fatty acids, and sterile sediment, a Clostridium sp. was predominant in the absence of added PCB, but undescribed species in the δ subgroup of the class Proteobacteria, the low-G+C gram-positive subgroup, the Thermotogales subgroup, and a single species with sequence similarity to the deeply branching species Dehalococcoides ethenogenes were more predominant during active dechlorination of the PCB. Species with high sequence similarities to Methanomicrobiales and Methanosarcinales archaeal subgroups were predominant in both dechlorinating and nondechlorinating enrichment cultures. Deletion of sediment from PCB-dechlorinating enrichment cultures reduced the rate of dechlorination and the diversity of the community. Substitution of sodium acetate for the mixture of three fatty acids increased the rate of dechlorination, further reduced the community diversity, and caused a shift in the predominant species that included restriction fragment length polymorphism patterns not previously detected. Although PCB-dechlorinating cultures were methanogenic, inhibition of methanogenesis and elimination of the archaeal community by addition of bromoethanesulfonic acid only slightly inhibited dechlorination, indicating that the archaea were not required for ortho dechlorination of the congener. Deletion of Clostridium spp. from the community profile by addition of vancomycin only slightly reduced dechlorination. However, addition of sodium molybdate, an inhibitor of sulfate reduction, inhibited dechlorination and deleted selected species from the community profiles of the class Bacteria. With the exception of one 16S rDNA sequence that had the highest sequence similarity to the obligate perchloroethylene-dechlorinating Dehalococcoides, the 16S rDNA sequences associated with PCB ortho dechlorination had high sequence similarities to the δ, low-G+C gram-positive, and Thermotogales subgroups, which all include sulfur-, sulfate-, and/or iron(III)-respiring bacterial species.The extensive industrial use of polychlorinated biphenyls (PCBs) during the 20th century has resulted in the release of an estimated several million pounds of PCBs into the environment (2). Due to the hydrophobicity and chemical stability of these compounds, PCBs ultimately accumulate in subsurface anaerobic sediments, where reductive dechlorination by anaerobic microorganisms is proposed to be an essential step in PCB degradation and detoxification (6). Although anaerobic reductive dechlorination has been documented in the environment and in the laboratory, attempts to identify and isolate anaerobic PCB-dechlorinating microbes by classical enrichment and isolation techniques have been unsuccessful (for a review, see reference 2). Isolation of anaerobic PCB-dechlorinating microbes has been hindered in part by the inability to maintain and sequentially transfer dechlorinating consortia in defined medium. May et al. (24) were the first to demonstrate that single colonies could be obtained by plating highly enriched PCB-dechlorinating enrichment cultures on agar-solidified media. Although two of the colonies exhibited para dechlorination activity when transferred back to liquid enrichment medium, the colonies contained a mixed community of microorganisms and dechlorination required the addition of sediment to the medium. More recently, highly enriched PCB-ortho-dechlorinating enrichment cultures were developed from Baltimore Harbor sediments in minimal media that contained sediments and a single congener (3) or Aroclor 1260 (37). These were the first confirmed reports of sustained ortho dechlorination of PCBs throughout sequential transfers in medium with estuarine sediments. Finally, Cutter et al. demonstrated that a consortium of PCB-ortho-dechlorinating anaerobes from Baltimore Harbor could be sequentially transferred and maintained in minimal medium without the addition of sterile sediment (9). With the ability to maintain PCB dechlorination in a completely defined medium, highly enriched PCB-dechlorinating consortia could be developed by sequential transfers in medium that contained the minimal growth requirements for dechlorinating species.The current study identifies putative PCB-dechlorinating anaerobes in ortho-dechlorinating enrichment cultures by a comprehensive approach that combines traditional selective enrichment techniques with molecular monitoring (SEMM). Microbial consortia enriched for PCB ortho dechlorination in minimal medium were analyzed by comparative sequence analysis of genes coding for 16S rRNA (16S rDNA) amplified from total community DNAs. Protocols were developed for chromosomal DNA extraction from sediment, 16S rDNA amplification by PCR, cloning of partial 16S rDNA PCR fragments, screening by restriction fragment length polymorphism (RFLP) analysis, and DNA sequencing for comparative sequence analysis. By utilizing these techniques, shifts in the microbial community were monitored as the cultures were further enriched for PCB-dechlorinating anaerobes by elimination of undefined medium components (i.e., sediment), changes in carbon source, and addition of selective physiological inhibitors. The results presented herein demonstrate the applicability of the SEMM approach for the selection and monitoring of highly defined PCB-dechlorinating microbial consortia.     

Introduction of anaerobic dechlorinating bacteria into soil slurry microcosms and nested-PCR monitoring.

Desulfomonile tiedjei and Desulfitobacterium dehalogenans were chosen as model bacteria to demonstrate the introduction of an anaerobic microbia reductive dechlorination activity into nonsterile soil slurry microcosms by inoculation. De novo 3-chlorobenzoate dechlorination activity was established with the bacterium D. tiedjei in microcosms normally devoid of this dechlorination capacity. The addition of D. tiedjei to microcosms supplemented with 20 mM pyruvate as the cosubstrate resulted in total biotransformation of 1.5 mM 3-chlorobenzoate within 7 days. The introduction of the bacterium Desulfitobacterium dehalogenans into nonsterile microcosms resulted in a shortening of the period required for dechlorination activity to be established. In microcosms inoculated with Desulfitobacterium dehalogenans, total degradation of 6 mM 3-chloro-4-hydroxy phenoxyacetic acid (3-Cl-4-OHPA) was observed after 4 days in contrast to the result in noninoculated microcosms, where the total degradation of 3-Cl-4-OHPA by indigenous microorganisms was observed after 11 days. Both externally introduced bacterial strains were detected in soil slurry microcosms by a nested-PCR methodology.     

Effect of Nitrate and Sulfate on Dechlorination by a Mixed Hydrogen-Fed Culture

A novel hollow-fiber membrane remediation technology developed in our laboratory for hydrogen delivery to the subsurface was shown to support the dechlorination of perchloroethene (PCE) to cis-dichloroethene. In previous research, the presence of nitrate or sulfate has been observed to inhibit biological reductive dechlorination. In this study hollow-fiber membranes were used to supply hydrogen to a mixed culture to investigate whether adequate hydrogen could be added to support dechlorination in the presence of alternative electron acceptors. By continuously supplying hydrogen through the membrane, the hydrogen concentrations within the reactor were maintained well above the hydrogen thresholds reported to sustain reductive dechlorination. It was hypothesized that by preventing nitrate and sulfate reducers from decreasing hydrogen concentrations to below the dehalorespirer threshold, the inhibition of PCE dechlorination by nitrate and sulfate might be avoided and dechlorination could be stimulated more effectively. Enough membrane-fed hydrogen was supplied to completely degrade the alternative electron acceptors present and initiate dechlorination. Nevertheless, nitrate and sulfate inhibited dechlorinating activity even when hydrogen was not limiting. This suggests that competition for hydrogen was not responsible for the observed inhibition. Subsequent microcosm experiments demonstrated that the denitrification intermediate nitrous oxide was inhibitory at 13 µM.     

Bar-coded pyrosequencing reveals the responses of PBDE-degrading microbial communities to electron donor amendments

Polybrominated diphenyl ethers (PBDEs) can be reductively degraded by microorganisms under anaerobic conditions. However, little is known about the effect of electron donors on microbial communities involved in PBDEs degradation. Here we employed 454 Titanium pyrosequencing to examine the phylogenetic diversity, composition, structure and dynamics of microbial communities from microcosms under the conditions of different electron donor amendments. The community structures in each of the five alternate electron donor enrichments were significantly shifted in comparison with those of the control microcosm. Commonly existing OTUs between the treatment and control consortia increased from 5 to 17 and more than 50% of OTUs increased around 13.7 to 186 times at least in one of the microcosms after 90-days enrichment. Although the microbial communities at different taxonomic levels were significantly changed by different environmental variable groups in redundancy analysis, significant correlations were observed between the microbial communities and PBDE congener profiles. The lesser-brominated PBDE congeners, tri-BDE congener (BDE-32) and hexa-BDE, were identified as the key factors shaping the microbial community structures at OTU level. Some rare populations, including the known dechlorinating bacterium, Dehalobacter, showed significant positive-correlation with the amounts of PBDE congeners in the consortia. The same results were also observed on some unclassified bacteria. These results suggest that PBDEs-degrading microbial communities can be successfully enriched, and their structures and compositions can be manipulated through adjusting the environmental parameters.     

Two anaerobic polychlorinated biphenyl-dehalogenating enrichments that exhibit different para-dechlorination specificities.

Two anaerobic polychlorinated biphenyl (PCB)-dechlorinating enrichments with distinct substrate specificities were obtained: a 2,3,4,6-tetrachlorobiphenyl (2346-CB) para-dechlorinating enrichment derived from Aroclor 1260-contaminated Woods Pond (Lenox, Mass.) sediment and a 2,4,6-trichlorobiphenyl (246-CB) unflanked para-dechlorinating enrichment derived from PCB-free Sandy Creek Nature Center (Athens, Ga.) sediment. The enrichments have been successfully transferred to autoclaved soil slurries over 20 times by using 300 to 350 microM 2346-CB or 246-CB. Both enrichments required soil for successful transfer of dechlorination activity. The 2346-CB enrichment para dehalogenated, in the absence or presence of 2346-CB, only 4 of 25 tested para halogen-containing congeners: 234-CB, 2345-CB, 2346-CB, and 2,4,6-tribromobiphenyl (246-BrB). In the presence of 246-CB, the 246-CB enrichment para dehalogenated 23 of the 25 tested congeners. However, only three congeners (34-CB, 2346-CB, and 246-BrB) were dehalogenated in the absence of 246-CB, indicating that these specific congeners initiate dehalogenation in this enrichment culture. The addition of the 2346-CB (para)-dechlorinating enrichment did not further stimulate the 2346-CB-primed dechlorination of the Aroclor 1260 residue in Woods Pond sediment samples. Compared to the addition of the primer 246-CB or the 246-CB unflanked para-dechlorinating enrichment alone, the addition of both 246-CB (300 microM) and the 246-CB enrichment stimulated the unflanked para dechlorination of the Aroclor 1260 residue in Woods Pond sediments. These results indicate that the two enrichments contain different PCB-dechlorinating organisms, each with high substrate specificities. Furthermore, bioaugmentation with the enrichment alone did not stimulate the desired dechlorination in PCB-contaminated Woods Pond sediment.     

Dechlorination of three tetrachlorobenzene isomers by contaminated harbor sludge-derived enrichment cultures follows thermodynamically favorable reactions

Dechlorination patterns of three tetrachlorobenzene isomers, 1,2,3,4-, 1,2,3,5-, and 1,2,4,5-TeCB, were studied in anoxic microcosms derived from contaminated harbor sludge. The removal of doubly, singly, and un-flanked chlorine atoms was noted in 1,2,3,4- and 1,2,3,5-TeCB fed microcosms, whereas only singly flanked chlorine was removed in 1,2,4,5-TeCB microcosms. The thermodynamically more favorable reactions were selectively followed by the enriched cultures with di- and/or mono-chlorobenzene as the main end products of the reductive dechlorination of all three isomers. Based on quantitative PCR analysis targeting 16S rRNA genes of known organohalide-respiring bacteria, the growth of Dehalococcoides was found to be associated with the reductive dechlorination of all three isomers, while growth of Dehalobacter, another known TeCB dechlorinator, was only observed in one 1,2,3,5-TeCB enriched microcosm among biological triplicates. Numbers of Desulfitobacterium and Geobacter as facultative dechlorinators were rather stable suggesting that they were not (directly) involved in the observed TeCB dechlorination. Bacterial community profiling suggested bacteria belonging to the phylum Bacteroidetes and the order Clostridiales as well as sulfate-reducing members of the class Deltaproteobacteria as putative stimulating guilds that provide electron donor and/or organic cofactors to fastidious dechlorinators. Our results provide a better understanding of thermodynamically preferred TeCB dechlorinating pathways in harbor environments and microbial guilds enriched and active in anoxic TeCB dechlorinating microcosms.

     

Microcosm evaluation of bioaugmentation after field-scale thermal treatment of a TCE-contaminated aquifer

This paper investigates effects of combining thermal and biological remediation, based on laboratory studies of trichloroethene (TCE) degradation. Aquifer material was collected 6 months after terminating a full-scale Electrical Resistance Heating (ERH), when the site had cooled from approximately 100°C to 40°C. The aquifer material was used to construct bioaugmented microcosms amended with the mixed anaerobic dechlorinating culture, KB-1^TM, and an electron donor (5 mM lactate). Microcosms were bioaugmented during cooling at 40, 30, 20, and 10°C, as temperatures continually decreased during laboratory incubation. Redox conditions were generally methanogenic, and electron donors were present to support dechlorination. For microcosms bioaugmented at 10°C and 20°C, dechlorination stalled at cis-dichloroethene (cDCE) and vinyl chloride (VC) 150 days after bioaugmentation. However, within 300 days of incubation ethene was produced in the majority of these microcosms. In contrast, dechlorination was rapid and complete in microcosms bioaugmented at 30°C. Microcosms bioaugmented at 40°C also showed rapid dechlorination, but stalled at cDCE with partial VC and ethene production, even after 150 days of incubation when the temperature had decreased to 10°C. These results suggest that sequential bioremediation of TCE is possible in field-scale thermal treatments after donor addition and bioaugmentation and that the optimal bioaugmentation temperature is approximately 30°C. When biological and thermal remediations are to be applied at the same location, three bioremediation approaches could be considered: (a) treating TCE in perimeter areas outside the source zone at temperatures of approximately 30°C; (b) polishing TCE concentrations in the original source zone during cooling from approximately 30°C to ambient groundwater temperatures; and (c) using bioremediation in downgradient areas taking advantages of the higher temperature and potential release of organic matter.     

滨海湿地生态系统微生物驱动的氮循环研究进展

滨海湿地生态系统介于陆地生态系统和海洋生态系统之间,其类型多种多样,环境差异极大,微生物种类丰富。近年来,随着人为氮源的大量输入,造成滨海湿地生态系统富营养化污染问题日趋严重。本文主要总结了滨海湿地生态系统微生物驱动的固氮、硝化、反硝化、厌氧氨氧化、NO_3~-还原成铵等主要氮循环过程,并综述了通过功能基因(如nifH、amoA、hzo、nirS、nirK、nrfA)检测微生物群落多样性及其环境影响因素的相关研究,旨在更好理解微生物驱动氮循环过程以去除氮,以期为减轻富营养化和危害性藻类爆发提供科学依据。     

Contribution of quinone-reducing microorganisms to the anaerobic biodegradation of organic compounds under different redox conditions

The capacity of two anaerobic consortia to oxidize different organic compounds, including acetate, propionate, lactate, phenol and p-cresol, in the presence of nitrate, sulfate and the humic model compound, anthraquinone-2,6-disulfonate (AQDS) as terminal electron acceptors, was evaluated. Denitrification showed the highest respiratory rates in both consortia studied and occurred exclusively during the first hours of incubation for most organic substrates degraded. Reduction of AQDS and sulfate generally started after complete denitrification, or even occurred at the same time during the biodegradation of p-cresol, in anaerobic sludge incubations; whereas methanogenesis did not significantly occur during the reduction of nitrate, sulfate, and AQDS. AQDS reduction was the preferred respiratory pathway over sulfate reduction and methanogenesis during the anaerobic oxidation of most organic substrates by the anaerobic sludge studied. In contrast, sulfate reduction out-competed AQDS reduction during incubations performed with anaerobic wetland sediment, which did not achieve any methanogenic activity. Propionate was a poor electron donor to achieve AQDS reduction; however, denitrifying and sulfate-reducing activities carried out by both consortia promoted the reduction of AQDS via acetate accumulated from propionate oxidation. Our results suggest that microbial reduction of humic substances (HS) may play an important role during the anaerobic oxidation of organic pollutants in anaerobic environments despite the presence of alternative electron acceptors, such as sulfate and nitrate. Methane inhibition, imposed by the inclusion of AQDS as terminal electron acceptor, suggests that microbial reduction of HS may also have important implications on the global climate preservation, considering the green-house effects of methane.     

Responses of the Anaerobic Bacterial Community to Addition of Organic C in Chromium(VI)- and Iron(III)-Amended Microcosms

Chromium (VI) is toxic to microorganisms and can inhibit the biodegradation of organic pollutants in contaminated soils. We used microcosms amended with either glucose or protein (to drive bacterial community change) and Fe(III) (to stimulate iron-reducing bacteria) to study the effect of various concentrations of Cr(VI) on anaerobic bacterial communities. Microcosms were destructively sampled based on microbial activity (measured as evolution of CO₂) and analyzed for the following: (i) dominant bacterial community by PCR-denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA gene; (ii) culturable Cr-resistant bacteria; and (iii) enrichment of iron-reducing bacteria of the Geobacteraceae family by real-time PCR. The addition of organic C stimulated the activities of anaerobic communities. Cr(VI) amendment resulted in lower rates of CO₂ production in glucose microcosms and a slow mineralization phase in protein-amended microcosms. Glucose and protein amendments selected for different bacterial communities. This selection was modified by the addition of Cr(VI), since some DGGE bands were intensified and new bands appeared in Cr(VI)-amended microcosms. A second dose of Cr(VI), added after the onset of activity, had a strong inhibitory effect when higher levels of Cr were added, indicating that the developing Cr-resistant communities had a relatively low tolerance threshold. Most of the isolated Cr-resistant bacteria were closely related to previously studied Cr-resistant anaerobes, such as Pantoea, Pseudomonas, and Enterobacter species. Geobacteraceae were not enriched during the incubation. The studied Cr(VI)-contaminated soil contained a viable anaerobic bacterial community; however, Cr(VI) altered its composition, which could affect the soil biodegradation potential.     

Metagenomic analysis reveals distinct patterns of denitrification gene abundance across soil moisture,nitrate gradients

This study coupled a landscape-scale metagenomic survey of denitrification gene abundance in soils with in situ denitrification measurements to show how environmental factors shape distinct denitrification communities that exhibit varying denitrification activity. Across a hydrologic gradient, the distribution of total denitrification genes (nap/nar + nirK/nirS + cNor/qNor + nosZ) inferred from metagenomic read abundance exhibited no consistent patterns. However, when genes were considered independently, nirS, cNor and nosZ read abundance was positively associated with areas of higher soil moisture, higher nitrate and higher annual denitrification rates, whereas nirK and qNor read abundance was negatively associated with these factors. These results suggest that environmental conditions, in particular soil moisture and nitrate, select for distinct denitrification communities that are characterized by differential abundance of genes encoding apparently functionally redundant proteins. In contrast, taxonomic analysis did not identify notable variability in denitrifying community composition across sites. While the capacity to denitrify was ubiquitous across sites, denitrification genes with higher energetic costs, such as nirS and cNor, appear to confer a selective advantage in microbial communities experiencing more frequent soil saturation and greater nitrate inputs. This study suggests metagenomics can help identify denitrification hotspots that could be protected or enhanced to treat non-point source nitrogen pollution.     

Short-term influence of recolonisation by the polycheate worm [2pt] Nereis succinea on oxygen and nitrogen fluxes and [2pt] denitrification: a microcosm simulation

The short-term effects of sediment recolonisation by Nereis succinea on sediment-water column fluxes of oxygen and dissolved inorganic nitrogen, and rates of denitrification, were studied in microcosms of homogenised, sieved sediments. The added worms enhanced oxygen uptake by the sediments, due to the increased surface area provided by the burrow walls and the degree of stimulation was stable with time. Similarly, ammonium fluxes to the water column were stimulated by N. succinea, but declined over the 3 day incubation in all microcosms including the controls. Nitrate fluxes were generally greater in the faunated microcosms, but highly variable with time. Denitrification rates were positively stimulated by N. succinea populations, denitrification of water column nitrate was stimulated 10-fold in comparison to denitrification coupled to nitrification in the sediments. Rates of denitrification of water column nitrate were not significantly different from rates in undisturbed sediment cores with similar densities of N. succinea, whereas rates of coupled nitrification–denitrification were 3-fold lower in the experimental set-up. These results may reflect the relative growth rates of nitrifying and denitrifying bacteria, which allow more rapid colonisation of new burrow surfaces by denitrifier compared to nitrifier populations. The data indicate that recolonisation by burrowing macrofauna of the highly reduced sediments of the Sacca di Goro, Lagoon, Italy, following the annual dystrophic crisis, may play a significant role in the reoxidation and detoxification of the sediments. The increased rates of denitrification associated with the worm burrows, may promote nitrogen losses, but due to the low capacity of nitrifying bacteria to colonise the new burrow structures, these losses would be highly dependent upon water column nitrate concentrations.     

Reductive dechlorination of hexachlorocyclohexane (HCH) isomers in soil under anaerobic conditions

The biological anaerobic reductive dechlorination of beta-hexachlorocyclohexane under methanogenic conditions was tested in a number of contaminated soil samples from two locations in the Netherlands. Soils from a heavily polluted location showed rapid dechlorination of beta-hexachlorocyclohexane to benzene and chlorobenzene with lactate as electron donor. Soils from an adjacent slightly polluted location did not show substantial dechlorination of beta-hexachlorocyclohexane within 4 months. A heavily polluted sample was selected to optimise the dechlorination. All tested hexachlorocyclohexane isomers (alpha-, beta-, gamma-, and delta-), either added separately or simultaneously, were dechlorinated in this soil sample. The most rapid dechlorination was observed at a temperature of 30 degrees C. Dechlorination of beta-hexachlorocyclohexane was observed with acetate, propionate, lactate, methanol, H2, yeast extract and landfill leachate as electron donors. In a soil percolation column, packed with a selected heavily polluted soil sample, the presence of 10 mM sulphate in the influent led to simultaneous dechlorination of beta-hexachlorocyclohexane and sulphate reduction. When the column was fed with 10 mM nitrate instead of sulphate, dechlorination ceased immediately. After omitting nitrate from the influent, dechlorination activity recovered in about 1 month. Also in a separate column, the addition of nitrate from the start of the experiment did not result in dechlorination of beta-HCH. The significance of these experiments for in situ bioremediation of polluted soils is discussed.     

Development and characterization of DehaloR^2, a novel anaerobic microbial consortium performing rapid dechlorination of TCE to ethene

A novel anaerobic consortium, named DehaloR^2, that performs rapid and complete reductive dechlorination of trichloroethene (TCE) to ethene is described. DehaloR^2 was developed from estuarine sediment from the Back River of the Chesapeake Bay and has been stably maintained in the laboratory for over 2 years. Initial sediment microcosms showed incomplete reduction of TCE to DCE with a ratio of trans- to cis- isomers of 1.67. However, complete reduction to ethene was achieved within 10 days after transfer of the consortium to sediment-free media and was accompanied by a shift to cis-DCE as the prevailing intermediate metabolite. The microbial community shifted from dominance of the Proteobacterial phylum in the sediment to Firmicutes and Chloroflexi in DehaloR^2, containing the genera Acetobacterium, Clostridium, and the dechlorinators Dehalococcoides. Also present were Spirochaetes, possible acetogens, and Geobacter which encompass previously described dechlorinators. Rates of TCE to ethene reductive dechlorination reached 2.83 mM Cl⁻ d⁻¹ in batch bottles with a Dehalococcoides sp. density of 1.54E+11 gene copies per liter, comparing favorably to other enrichment cultures described in the literature and identifying DehaloR^2 as a promising consortium for use in bioremediation of chlorinated ethene-impacted environments.     

Biodegradation of Trichloroethylene and Dichloromethane in Contaminated Soil and Groundwater

Soil column and serum bottle microcosm experiments were conducted to investigate the potential for in situ anaerobic bioremediation of trichloroethy lene (TCE) and dichloromethane (DCM) at the Pinellas site near Largo, Florida. Soil columns with continuous groundwater recycle were used to evaluate treatment with complex nutrients (casamino acids, methanol, lactate, sulfate); benzoate and sulfate; and methanol. The complex nutrients drove microbial dechlorination of TCE to ethene, whereas the benzoate/sulfate and methanol supported microbial dechlorination of TCE only to cis-1 ,2-dichloroethylene (cDCE). Microbial sulfate depletion in the benzoate/sulfate column allowed further dechlorination of cDCE to vinyl chloride. Serum bottle microcosms were used to investigate TCE dechlorination and DCM biodegradation in Pinellas soil slurries bioaugmented with liquid from the soil columns possessing TCE-dechlorinating activity and DCM biodegradation by indigenous microorganisms. Bioaugmented soil microcosms showed immediate TCE dechlorination in the microcosms with methanol or complex nutrients, but no dechlorination in the benzoate/sulfate microcosm. DCM biodegradation by indigenous microorganisms occurred in soil microcosms amended with either benzoate/sulfate or methanol, but not with complex nutrients. Bioaugmentation stimulated DCM biodegradation in both complex nutrient and methanol-amended microcosms, but appeared to inhibit DCM biodegradation in benzoate/sulfate-amended microcosms. TCE dechlorination occurred before DCM biodegradation in bioaugmented microcosms when both compounds were present.     

Metabolic versatility of haloalkaliphilic bacteria from soda lakes belonging to the Alkalispirillum–Alkalilimnicola group

Four new isolates were obtained from denitrifying enrichments with various electron donors using sediment samples from hypersaline soda lakes. Based on 16S rRNA gene analysis and DNA-DNA hybridization results, they were all identified as members of the Gammaproteobacteria closely associated with the Alkalispirillum–Alkalilimnicola group. Two isolates were obtained from samples enriched with nitrate as electron acceptor and H₂ or polysulfide as electron donors, and another two strains were obtained with N₂O as the electron acceptor and sulfide or acetate as electron donors. All four new isolates, together with the type strains of the genera Alkalispirillum and Alkalilimnicola originally described as obligate aerobes, were capable of anaerobic growth with acetate using either nitrate or N₂O as electron acceptors. Their denitrification pathway, however, was disrupted at the level of nitrite. RuBisCO form I gene was detected and sequenced in the new isolates and in Alkalilimnicola halodurans but not in Alkalispirillum mobile. These data, together with the evidence of Oremland et al. (Appl Environ Microbiol 68:4795–4802, 2002) on the potential of Alkalilimnicola sp. MLHE-1 for autotrophic growth with arsenite as electron donor and nitrate as electron acceptor, demonstrate much higher metabolic diversity of this specific group of haloalkaliphilic Gammaproteobacteria than was originally anticipated.     

Frequent concomitant presence of Desulfitobacterium spp. and "Dehalococcoides" spp. in chloroethene-dechlorinating microbial communities

The presence of chloroethene dechlorination activity as well as several bacterial genera containing mainly organohalide-respiring members was investigated in 34 environmental samples from 18 different sites. Cultures inoculated with these environmental samples on tetrachloroethene and amended weekly with a seven organic electron donor mixture resulted in 11 enrichments with cis-DCE, ten with VC, and 11 with ethene as dechlorination end product, and only two where no dechlorination was observed. “Dehalococcoides” spp. and Desulfitobacterium spp. were detected in the majority of the environmental samples independently of the dechlorination end product formed. The concomitant presence of Dehalococcoides spp. and Desulfitobacterium spp. in the majority of the enrichments suggested that chloroethene dechlorination was probably the result of catalysis by at least two organohalide-respiring genera either in parallel or by stepwise catalysis. A more detailed study of one enrichment on cis-DCE suggested that in this culture Desulfitobacterium spp. as well as Dehalococcoides spp. dechlorinated cis-DCE whereas dechlorination of VC was only catalyzed by the latter.