Black Carbon Impacts on Paraburkholderia xenovorans Strain LB400 Cell Enrichment and Activity: Implications toward Lower-Chlorinated Polychlorinated Biphenyls Biodegradation Potential.

Volatilization of lower-chlorinated polychlorinated biphenyls (LC-PCBs) from sediment poses health threats to nearby communities and ecosystems. Biodegradation combined with black carbon (BC) materials is an emerging bioaugmentation approach to remove PCBs from sediment, but development of aerobic biofilms on BC for long-term, sustained LC-PCBs remediation is poorly understood. This work aimed to characterize the cell enrichment and activity of biphenyl- and benzoate-grown Paraburkholderia xenovorans strain LB400 on various BCs. Biphenyl dioxygenase gene (bphA) abundance on four BC types demonstrated corn kernel biochar hosted at least 4 orders of magnitude more attached cells per gram than other feedstocks, and microscopic imaging revealed the attached live cell fraction was >1.5× more on corn kernel biochar than GAC. BC characteristics (i.e., sorption potential, pore size, pH) appear to contribute to cell attachment differences. Reverse transcription qPCR indicated that BC feedstocks significantly influenced bphA expression in attached cells. The bphA transcript-per-gene ratio of attached cells was >10-fold more than suspended cells, confirmed by transcriptomics. RNA-seq also demonstrated significant upregulation of biphenyl and benzoate degradation pathways on attached cells, as well as revealing biofilm formation potential/cell-cell communication pathways. These novel findings demonstrate aerobic PCB-degrading cell abundance and activity could be tuned by adjusting BC feedstocks/attributes to improve LC-PCBs biodegradation potential.

Genome-Resolved Metagenomics and Metatranscriptomics Reveal Insights into the Ecology and Metabolism of Anaerobic Microbial Communities in PCB-Contaminated Sediments.

Growth of organohalide-respiring bacteria such as Dehalococcoides mccartyi on halogenated organics (e.g., polychlorinated biphenyls (PCBs)) at contaminated sites or in enrichment culture requires interaction and support from other microbial community members. To evaluate naturally occurring interactions between Dehalococcoides and key supporting microorganisms (e.g., production of H2, acetate, and corrinoids) in PCB-contaminated sediments, metagenomic and metatranscriptomic sequencing was conducted on DNA and RNA extracted from sediment microcosms, showing evidence of both Dehalococcoides growth and PCB dechlorination. Using a genome-resolved approach, 160 metagenome-assembled genomes (MAGs), including three Dehalococcoides MAGs, were recovered. A novel reductive dehalogenase gene, distantly related to the chlorophenol dehalogenase gene cprA (pairwise amino acid identity: 23.75%), was significantly expressed. Using MAG gene expression data, 112 MAGs were assigned functional roles (e.g., corrinoid producers, acetate/H2 producers, etc.). A network coexpression analysis of all 160 MAGs revealed correlations between 39 MAGs and the Dehalococcoides MAGs. The network analysis also showed that MAGs assigned with functional roles that support Dehalococcoides growth (e.g., corrinoid assembly, and production of intermediates required for corrinoid synthesis) displayed significant coexpression correlations with Dehalococcoides MAGs. This work demonstrates the power of genome-resolved metagenomic and metatranscriptomic analyses, which unify taxonomy and function, in investigating the ecology of dehalogenating microbial communities.

Reconnaissance of Oxygenic Denitrifiers in Agriculturally Impacted Soils.

Row crop production in the agricultural Midwest pollutes waterways with nitrate, and exacerbates climate change through increased emissions of nitrous oxide and methane. Oxygenic denitrification processes in agricultural soils mitigate nitrate and nitrous oxide pollution by short-circuiting the canonical pathway to avoid nitrous oxide formation. Furthermore, many oxygenic denitrifiers employ a nitric oxide dismutase (nod) to create molecular oxygen that is used by methane monooxygenase to oxidize methane in otherwise anoxic soils. The direct investigation of nod genes that could facilitate oxygenic denitrification processes in agricultural sites is limited, with no prior studies investigating nod genes at tile drainage sites. Thus, we performed a reconnaissance of nod genes at variably saturated surface sites, and within a variably to fully saturated soil core in Iowa to expand the known distribution of oxygenic denitrifiers. We identified new nod gene sequences from agricultural soil and freshwater sediments in addition to identifying nitric oxide reductase (qNor) related sequences. Surface and variably saturated core samples displayed a nod to 16S rRNA gene relative abundance of 0.004% to 0.1% and fully saturated core samples had relative nod gene abundance of 1.2%. The relative abundance of the phylum Methylomirabilota increased from 0.6% and 1% in the variably saturated core samples to 3.8% and 5.3% in the fully saturated core samples. The more than 10-fold increase in relative nod abundance and almost 9-fold increase in relative Methylomirabilota abundance in fully saturated soils suggests that potential oxygenic denitrifiers play a greater nitrogen cycling role under these conditions. IMPORTANCE The direct investigation of nod genes in agricultural sites is limited, with no prior studies investigating nod genes at tile drains. An improved understanding of nod gene diversity and distribution is significant to the field of bioremediation and ecosystem services. The expansion of the nod gene database will advance oxygenic denitrification as a potential strategy for sustainable nitrate and nitrous oxide mitigation, specifically for agricultural sites.

Lab-scale biodegradation assay using passive samplers to determine microorganisms' ability to reduce polychlorinated biphenyl (PCB) volatilization from contaminated sediment.

Many PCB-degrading aerobes have been identified which may serve as bioaugmentation strains for aerobic, in situ bioremediation or in combination with dredging operations. The present work describes a lab-scale PCB biodegradation assay which can be used to screen potential bioaugmentation strains or consortia for their ability to decrease PCB mass flux from contaminated sediment to air through biodegradation of freely dissolved PCBs that have desorbed from sediment particles. The assay uses two types of passive samplers to simultaneously measure PCB mass that is freely dissolved in aqueous solution and PCB mass that has volatilized to the headspace of the bioreactor. Using this approach, relative comparisons of PCB mass accumulated in passive samplers between bioaugmented treatments and controls allow for practical assessment of a microbial strain's ability to reduce both freely dissolved and vapor phase PCB concentrations. The method is designed to be conducted using aliquots of homogenized, well-characterized, PCB-contaminated sediment gathered from a field site. This work details the experimental design methodology, required materials, bioreactor set-up, passive sampling, PCB-extraction, sample cleanup, and quantification protocols such that the biodegradation assay can be conducted or replicated. A step-by-step protocol is also included and annotated with photos, tips, and tricks from experienced analysts.•Relative comparisons of PCB mass accumulated in passive samplers between experimental treatments and controls allow for practical assessment of bioaugmentation strain's ability to reduce both freely dissolved and vapor phase PCB concentrations•Passive sampler preparation, deployment, PCB-extraction, cleanup procedures, and quantification are detailed step-by-step and annotated by experienced analysts.

Aerobic Bioaugmentation to Decrease Polychlorinated Biphenyl (PCB) Emissions from Contaminated Sediments to Air.

We conducted experiments to determine whether bioaugmentation with aerobic, polychlorinated biphenyl (PCB)-degrading microorganisms can mitigate polychlorinated biphenyl (PCB) emissions from contaminated sediment to air. Paraburkholderia xenovorans strain LB400 was added to bioreactors containing PCB-contaminated site sediment. PCB mass in both the headspace and aqueous bioreactor compartments was measured using passive samplers over 35 days. Time-series measurements of all 209 PCB congeners revealed a 57% decrease in total PCB mass accumulated in the vapor phase of bioaugmented treatments relative to non-bioaugmented controls, on average. A comparative congener-specific analysis revealed preferential biodegradation of lower-chlorinated PCBs (LC-PCBs) by LB400. Release of the most abundant congener (PCB 4 [2,2'-dichlorobiphenyl]) decreased by over 90%. Simulations with a PCB reactive transport model closely aligned with experimental observations. We also evaluated the effect of the phytogenic biosurfactant, saponin, on PCB bioavailability and biodegradation by LB400. Time-series qPCR measurements of biphenyl dioxygenase (bphA) genes showed that saponin better maintained bphA abundance, compared to the saponin-free treatment. These findings indicate that an active population of bioaugmented, aerobic PCB-degrading microorganisms can effectively lower PCB emissions and may therefore contribute to minimizing PCB inhalation exposure in communities surrounding PCB-contaminated sites.

Contrasting regulatory effects of organic acids on aerobic vinyl chloride biodegradation in etheneotrophs.

Vinyl chloride (VC) is a common groundwater pollutant generated during anaerobic biodegradation of chlorinated solvents (e.g., trichloroethene (TCE) or tetrachloroethene (PCE)). Aerobic VC biodegradation by etheneotrophs can support anaerobic PCE and TCE bioremediation to achieve complete removal in situ. However, anaerobic bioremediation strategies necessitate biostimulation with electron donors that are fermented in situ, generating organic acids that could influence aerobic VC biodegradation processes. We examined the effect of organic acids (lactate, acetate, propionate, and butyrate) on aerobic VC biodegradation by VC-assimilating etheneotrophs Mycobacterium strain JS60 and Nocardioides strain JS614. Strain JS60 grew on all organic acids tested, while strain JS614 did not respond to lactate. VC-grown strain JS60 fed VC and one or more organic acids showed carbon catabolite repression (CCR) behavior where VC biodegradation occurred only after organic acids were depleted. In contrast, CCR was not evident in VC-grown strain JS614, which degraded VC and organic acids simultaneously. Acetate-grown JS60 showed similar CCR behavior when fed VC and a single organic acid, except that extended lag periods (5-12 days) occurred before VC oxidation ensued. Acetate-grown JS614 fed VC and either acetate or butyrate displayed 5-8 day lag periods before simultaneous VC and organic acid biodegradation. In contrast, acetate-grown JS614 degraded VC and propionate without a significant lag, suggesting a regulatory link between propionate and VC oxidation in JS614. Different global regulatory mechanisms controlling VC biodegradation in the presence of organic acids in etheneotrophs have implications for developing combined anaerobic-aerobic bioremediation strategies at chlorinated ethene-contaminated sites. KEY POINTS: • With organic acids present, VC utilization was repressed in JS60, but not in JS614 • Strain JS60 grew readily on lactate, while strain JS614 did not • Propionate alleviated lag periods for VC utilization in acetate-grown JS614.

Combined read- and assembly-based metagenomics to reconstruct a Dehalococcoides mccartyi genome from PCB-contaminated sediments and evaluate functional differences among organohalide-respiring consortia in the presence of different halogenated contaminants.

Microbial communities that support respiration of halogenated organic contaminants by Dehalococcoides sp. facilitate full-scale bioremediation of chlorinated ethenes and demonstrate the potential to aid in bioremediation of halogenated aromatics like polychlorinated biphenyls (PCBs). However, it remains unclear if Dehalococcoides-containing microbial community dynamics observed in sediment-free systems quantitatively resemble that of sediment environments. To evaluate that possibility we assembled, annotated, and analyzed a Dehalococcoides sp. metagenome-assembled genome (MAG) from PCB-contaminated sediments. Phylogenetic analysis of reductive dehalogenase gene (rdhA) sequences within the MAG revealed that pcbA1 and pcbA4/5-like rdhA were absent, while several candidate PCB dehalogenase genes and potentially novel rdhA sequences were identified. Using a compositional comparative metagenomics approach, we quantified Dehalococcoides-containing microbial community structure shifts in response to halogenated organics and the presence of sediments. Functional level analysis revealed significantly greater abundances of genes associated with cobamide remodeling and horizontal gene transfer in tetrachloroethene-fed cultures as compared to halogenated aromatic-exposed consortia with or without sediments, despite little evidence of statistically significant differences in microbial community taxonomic structure. Our findings support the use of a generalizable comparative metagenomics workflow to evaluate Dehalococcoides-containing consortia in sediments and sediment-free environments to eludicate functions and microbial interactions that facilitate bioremediation of halogenated organic contaminants.

Metagenomes, Metagenome-Assembled Genomes, and Metatranscriptomes from Polychlorinated Biphenyl-Contaminated Sediment Microcosms.

We present a comprehensive data set that describes an anaerobic microbial consortium native to polychlorinated biphenyl (PCB)-contaminated sediments. Obtained from sediment microcosms incubated for 200 days, the data set includes 4 metagenomes, 4 metatranscriptomes (in duplicate), and 62 metagenome-assembled genomes and captures microbial community interactions, structure, and function relevant to anaerobic PCB biodegradation.

Natural Biodegradation of Vinyl Chloride and cis-Dichloroethene in Aerobic and Suboxic Conditions.

Chlorinated ethene (CE) groundwater contamination is commonly treated through anaerobic biodegradation (i.e., reductive dechlorination) either as part of an engineered system or through natural attenuation. Aerobic biodegradation has also been recognized as a potentially significant pathway for the removal of the lower CEs cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC). However, the role of aerobic biodegradation under low oxygen conditions typical of contaminated groundwater is unclear. Bacteria capable of aerobic VC biodegradation appear to be common in the environment, while aerobic biodegradation of cDCE is less common and little is known regarding the organisms responsible. In this study, we investigate the role of aerobic cDCE and VC biodegradation in a mixed contaminant plume (including CEs, BTEX, and ketones) at Naval Air Station North Island, Installation Restoration Site 9. Sediment and groundwater collected from the plume source area, mid-plume, and shoreline were used to prepare microcosms under fully aerobic (8 mg/L dissolved oxygen (DO)) and suboxic (< 1 mg/L DO) conditions. In the shoreline microcosms, VC and cDCE were rapidly degraded under suboxic conditions (100% and 77% removal in < 62 days). In the suboxic VC microcosms, biodegradation was associated with a > 5 order of magnitude increase in the abundance of functional gene etnE, part of the aerobic VC utilization pathway. VC and cDCE were degraded more slowly under fully aerobic conditions (74% and 30% removal) in 110 days. High-throughput 16S rRNA and etnE sequencing suggest the presence of novel VC- and cDCE-degrading bacteria. These results suggest that natural aerobic biodegradation of cDCE and VC is occurring at the site and provide new evidence that low (< 1 mg/L) DO levels play a significant role in natural attenuation of cDCE and VC.

Two Metatranscriptomic Profiles through Low-Dissolved-Oxygen Waters (DO, 0 to 33 µM) in the Eastern Tropical North Pacific Ocean.

We present 16 seawater metatranscriptomes collected from a marine oxygen-deficient zone (ODZ) in the eastern tropical North Pacific (ETNP). This data set will be useful for identifying shifts in microbial community structure and function through oxic/anoxic transition zones, where overlapping aerobic and anaerobic microbial processes impact marine biogeochemical cycling.

Microbial communities in polychlorinated biphenyl (PCB)-contaminated wastewater lagoon sediments: PCB congener, quantitative PCR, and 16S rRNA gene amplicon sequencing datasets.

The potential for aerobic and anaerobic microbial natural attenuation of PCBs in freshwater sediments is described by PCB congener, quantitative PCR, and 16S rRNA gene amplicon sequencing datasets generated, in duplicate, from 27 sediment samples collected from a PCB-contaminated freshwater lagoon (54 samples total). Sediment samples were subjected to a hexane PCB extraction protocol and the concentrations of 209 PCB congeners were determined in hexane extracts by gas chromatography with a tandem mass spectrometry detection. DNA was extracted from sediments sediment samples and used for qPCR and 16S rRNA amplicon sequencing. The abundance of 16S rRNA genes (i.e., Dehalococcoides and putative dechlorinating Chloroflexi) and functional genes (i.e., reductive dehalogenase (rdhA) and biphenyl dioxygenase (bphA)) associated with aerobic and anaerobic PCB biodegradation, along with the total 16S rRNA genes abundance, was determined by SYBR green qPCR. The microbial community composition and structure in all sediment samples was obtained by 16S rRNA gene amplicon sequencing. Primers targeting the 16S rRNA gene V4 region were used to produce 16S rRNA gene amplicons that were sequencing with the high-throughput Illumina MiSeq platform and sequencing chemistry. The 16S rRNA gene sequencing dataset along with PCB congener and qPCR datasets included as metadata, could be reused in meta-analyses that aim to determine microbial community interactions in contaminated environments, and uncover relationships between microbial community structure and environmental variable (e.g., PCB congener concentrations).

Dataset describing biodegradation of individual polychlorinated biphenyl congeners (PCBs) by Paraburkholderia xenovorans LB400 in presence and absence of sediment slurry.

This dataset describes the biodegradation of polychlorinated biphenyl (PCB) congeners by Paraburkholderia xenovorans LB400 in absence and presence of PCB-contaminated sediment slurry, over time [1]. In absence of sediment, PCBs were extracted from aqueous bioreactors by liquid-liquid extraction (LLE) with hexane. In presence of sediment, the extraction method used was a modification of U.S. EPA Method 3545 [3]. Sediment slurry samples were extracted from bioreactors using pressurized fluid extraction (Accelerated Solvent Extractor; Dionex ASE-200) with equal parts acetone and hexane. GC-MS/MS triple quadrapole technology in multiple reaction monitoring mode (MRM) was used for identification and quantification of 209 PCBs as 174 chromatographic peaks. Samples were processed in batches of five along with one method blank per batch. All materials used in sample extraction had either been triple rinsed with solvent (methanol, acetone, and hexane) or combusted overnight at 450 °C to prevent background PCB contamination. Results from the method blanks were used to determine the limit of quantification (LOQ) as the upper limit of the 95% confidence interval (average mass plus two times the standard deviation). PCB congener masses were corrected for surrogate recoveries less than 100%. The PCB concentration dataset was dichotomized at the threshold of the congener specific LOQ. Concentrations of congeners below the LOQ were treated as zero. During analysis, PCB concentration data was filtered to include only congeners belonging to the commercial PCB mixture, Aroclor 1248. LOQ corrected data can inform future experimental design and be reused by other researchers for further analysis and / or interpretive insights.

Biodegradation of PCB congeners by Paraburkholderia xenovorans LB400 in presence and absence of sediment during lab bioreactor experiments.

Experiments were conducted to measure biodegradation of polychlorinated biphenyl (PCB) congeners contained in mixture Aroclor 1248 and congeners present in wastewater lagoon sediment contaminated decades earlier at Altavista, Virginia. A well-characterized strain of aerobic PCB-degrading bacteria, Paraburkholderia xenovorans LB400 was incubated in laboratory bioreactors with PCB-contaminated sediment collected at the site. The experiments evaluated strain LB400's ability to degrade PCBs in absence of sediment and in PCB-contaminated sediment slurry. In absence of sediment, LB400 transformed 76% of Aroclor 1248 within seven days, spanning all homolog groups present in the mixture. In sediment slurry, only mono- and di-chlorinated PCB congeners were transformed. These results show that LB400 is capable of rapidly biodegrading most PCB congeners when they are freely dissolved in liquid but cannot degrade PCB congeners having three or more chlorine substituents in sediment slurry. Finally, using GC/MS-MS triple quadrupole spectrometry, this work distinguishes between physical (sorption to cells) and biological removal mechanisms, illuminates the process by which microorganisms with LB400-type congener specificity can selectively transform lower-chlorinated congeners over time, and makes direct comparisons to other studies where individual congener data is reported.

Detection of an alkene monooxygenase in vinyl chloride-oxidizing bacteria with GeneFISH.

Fluorescence in situ hybridization (FISH) can provide information on the morphology, spatial arrangement, and local environment of individual cells enabling the investigation of intact microbial communities. GeneFISH uses polynucleotide probes and enzymatic signal amplification to detect genes that are present in low copy numbers. Previously, this technique has only been applied in a small number of closely related organisms. However, many important functional genes, such as those involved in xenobiotic degradation or pathogenesis, are present in diverse microbial strains. Here, we present a geneFISH method for the detection of the functional gene etnC, which encodes the alpha subunit of an alkene monooxygenase used by aerobic ethene and vinyl chloride oxidizing bacteria (etheneotrophs). The probe concentration was optimized and found to be 100 pg/µl, similar to previous geneFISH reports. Permeabilization was necessary for successful geneFISH labeling of Mycobacteria; sequential treatment with lysozyme and achromopeptidase was the most effective treatment. This method was able to detect etnC in several organisms including Mycobacteria and Nocardioides, demonstrating for the first time that a single geneFISH probe can detect a variety of alleles (>80% sequence similarity) across multiple species. Detection of etnC with geneFISH has practical applications for bioremediation. This method can be readily adapted for other functional genes and has broad applications for investigating microbial communities in natural and engineered systems.

Metabolic flexibility of SUP05 under low DO growth conditions.

Chemoautotrophic bacteria from the SUP05 clade often dominate anoxic waters within marine oxygen minimum zones (OMZs) where they use energy gained from the oxidation of reduced sulfur to fuel carbon fixation. Some of these SUP05 bacteria are facultative aerobes that can use either nitrate or oxygen as a terminal electron acceptor making them ideally suited to thrive at the boundaries of OMZs where they experience fluctuations in dissolved oxygen (DO). SUP05 metabolism in these regions, and therefore the biogeochemical function of SUP05, depends largely on their sensitivity to oxygen. We evaluated growth and quantified differences in gene expression in Ca. T. autotrophicus strain EF1 from the SUP05 clade under high DO (22 µM), anoxic, and low DO (3.8 µM) concentrations. We show that strain EF1 cells respire oxygen and nitrate and that cells have higher growth rates, express more genes, and fix more carbon when oxygen becomes available for aerobic respiration. Evidence that facultatively aerobic SUP05 are more active and respire nitrate when oxygen becomes available at low concentrations suggests that they are an important source of nitrite across marine OMZ boundary layers.

Impacts of ammonia loading and biofilm age on the prevalence of nitrogen-cycling microorganisms in a full-scale submerged attached-growth reactor.

This study reports the impacts of seasonal ammonia load changes and biofilm age on the quantity of biomass and on the prevalence of ammonia- and nitrite-metabolizing organisms within a submerged attached-growth reactor (SAGR™) following lagoon treatment. Ammonia (NH3 ) loadings (0.12-3.17 kg/d) in the primary SAGR were measured over 223 days from May to December in 2017. Adjustment of the wastewater flow path on September 1 successfully increased NH3 loading to the primary SAGR, which subsequently caused reactor biomass to increase. The NH3 removal rate in October (0.5 kg/d) was greater than rates in June and July (0.3 and 0.2 kg/d) despite a water temperature decrease from >24 to 15.6°C. This elevated removal rate in October, and the sustained removal rate in December (0.4 kg/d, 5.3°C) were associated with a measured increase in microbial biomass. The relative abundance of the anammox organism C. Brocadia was 5 times greater in the mature biofilm after 686 days of growth, and the genus Pseudomonas increased sevenfold. The presence of Pseudomonas, which contains denitrifying species, and anammox suggests a high potential for removal of total nitrogen in SAGRs. PRACTITIONER POINTS: Pseudomonas prevalence and the presence of anammox suggest a high potential for total nitrogen removal in mature SAGR biofilms. The abundance of the anammox microorganism C. Brocadia was greater after 686 days of biofilm growth compared with 33 days. Simple operational changes can increase biomass in the SAGR to maintain, or even increase, NH3 transformation rates during cold weather.

Growth of Dehalococcoides spp. and increased abundance of reductive dehalogenase genes in anaerobic PCB-contaminated sediment microcosms.

Polychlorinated biphenyls (PCBs) contaminate 19% of US Superfund sites and represent a serious risk to human and environmental health. One promising strategy to remediate PCB-contaminated sediments utilizes organohalide-respiring bacteria (OHRB) that dechlorinate PCBs.However, functional genes that act as biomarkers for PCB dechlorination processes (i.e., reductive dehalogenase genes) are poorly understood. Here, we developed anaerobic sediment microcosms that harbor an OHRB community dominated by the genus Dehalococcoides. During the 430-day microcosm incubation, Dehalococcoides 16S rRNA sequences increased two orders of magnitude to 107 copies/g of sediment, and at the same time, PCB118 decreased by as much as 70%. In addition, the OHRB community dechlorinated a range of penta- and tetra-chlorinated PCB congeners including PCBs 66, 70 + 74 + 76, 95, 90 + 101, and PCB110 without exogenous electron donor. We quantified candidate reductive dehalogenase (RDase) genes over a 430-day incubation period and found rd14, a reductive dehalogenase that belongs to Dehalococcoides mccartyi strain CG5, was enriched to 107 copies/g of sediment. At the same time, pcbA5 was enriched to only 105 copies/g of sediment. A survey for additional RDase genes revealed sequences similar to strain CG5's rd4 and rd8. In addition to demonstrating the PCB dechlorination potential of native microbial communities in contaminated freshwater sediments, our results suggest candidate functional genes with previously unexplored potential could serve as biomarkers of PCB dechlorination processes.

Cryogenic soil coring reveals coexistence of aerobic and anaerobic vinyl chloride degrading bacteria in a chlorinated ethene contaminated aquifer.

Vinyl chloride (VC) is a common groundwater contaminant and known human carcinogen. Three major bacterial guilds are known to participate in VC biodegradation: aerobic etheneotrophs and methanotrophs, and anaerobic organohalide-respiring VC-dechlorinators. We investigated the spatial relationships between functional genes representing these three groups of bacteria (as determined by qPCR) with chlorinated ethene concentrations in a surficial aquifer at a contaminated site. We used cryogenic soil coring to collect high-resolution aquifer sediment samples and to preserve sample geochemistry and nucleic acids under field conditions. All samples appeared to be anaerobic (i.e., contained little to no dissolved oxygen). VC biodegradation associated functional genes from etheneotrophs (etnC and/or etnE), methanotrophs (mmoX and/or pmoA), and anaerobic VC-dechlorinators (bvcA and/or vcrA) coexisted in 48% of the samples. Transcripts of etnC/etnE and bvcA/vcrA were quantified in contemporaneous groundwater samples, indicating co-located gene expression. Functional genes from etheneotrophs and anaerobic VC-dechlorinators were correlated to VC concentrations in the lower surficial aquifer (p < 0.05). Methanotroph functional genes were not correlated to VC concentrations. Cryogenic soil coring proved to be a powerful tool for capturing high-spatial resolution trends in geochemical and nucleic acid data in aquifer sediments. We conclude that both aerobic etheneotrophs and anaerobic VC-dechlorinators may play a significant role in VC biodegradation in aquifers that have little dissolved oxygen.

Integrated methodological approach reveals microbial diversity and functions in aerobic groundwater microcosms adapted to vinyl chloride.

Vinyl chloride (VC), a known human carcinogen, is often formed in groundwater (GW) by incomplete reductive dechlorination of chlorinated ethenes. An integrated microbial ecology approach involving bacterial enrichments and isolations, carbon stable-isotope probing (SIP) and metagenome and genome sequencing was applied to ethene-fed GW microcosms that rapidly transitioned to aerobic growth on VC. Actinobacteria, Proteobacteria and Bacteroidetes dominated the microbial communities in ethene- and VC-grown cultures. SIP with 13C2-VC demonstrated that Nocardioides spp. significantly participated in carbon uptake from VC (52.1%-75.7% enriched in heavy fractions). Sediminibacterium, Pedobacter and Pseudomonas spp. also incorporated 13C from VC into genomic DNA. Ethene- and VC-assimilating Nocardioides sp. strain XL1 was isolated. Sequencing revealed a large (∼300 kbp) plasmid harboring genes encoding alkene monooxygenase and epoxyalkane: coenzyme M transferase, enzymes known to participate in aerobic VC and ethene biodegradation. The plasmid was 100% identical to pNOCA01 found in VC-assimilating Nocardioides sp. strain JS614. Metagenomic analysis of enrichment cultures indicated other bacteria implicated in carbon uptake from VC possessed the genetic potential to detoxify epoxides via epoxide hydrolase or glutathione S-transferase (Pseudomonas) and/or metabolize VC epoxide breakdown products and downstream VC metabolites. This study provides new functional insights into aerobic VC metabolism within a GW microbial community.

PCB dechlorination hotspots and reductive dehalogenase genes in sediments from a contaminated wastewater lagoon.

Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that are distributed worldwide. Although industrial PCB production has stopped, legacy contamination can be traced to several different commercial mixtures (e.g., Aroclors in the USA). Despite their persistence, PCBs are subject to naturally occurring biodegradation processes, although the microbes and enzymes involved are poorly understood. The biodegradation potential of PCB-contaminated sediments in a wastewater lagoon located in Virginia (USA) was studied. Total PCB concentrations in sediments ranged from 6.34 to 12,700 mg/kg. PCB congener profiles in sediment sample were similar to Aroclor 1248; however, PCB congener profiles at several locations showed evidence of dechlorination. The sediment microbial community structure varied among samples but was dominated by Proteobacteria and Firmicutes. The relative abundance of putative dechlorinating Chloroflexi (including Dehalococcoides sp.) was 0.01-0.19% among the sediment samples, with Dehalococcoides sp. representing 0.6-14.8% of this group. Other possible PCB dechlorinators present included the Clostridia and the Geobacteraceae. A PCR survey for potential PCB reductive dehalogenase genes (RDases) yielded 11 sequences related to RDase genes in PCB-respiring Dehalococcoides mccartyi strain CG5 and PCB-dechlorinating D. mccartyi strain CBDB1. This is the first study to retrieve potential PCB RDase genes from unenriched PCB-contaminated sediments.