Compound-Specific Carbon, Nitrogen, and Hydrogen Isotope Analysis to Characterize Aerobic Biodegradation of 2,3-Dichloroaniline by a Mixed Enrichment Culture.

Compound-specific isotope analysis (CSIA) is an established tool to track the in situ transformation of organic chemicals at contaminated sites. In this work, we evaluated the potential of multi-element CSIA to assess biodegradation of 2,3-dichloroaniline (2,3-DCA), which is a major industrial feedstock. Using controlled laboratory experiments, we determined, for the first time, negligible carbon (<0.5‰) and hydrogen (<10‰) isotope fractionation and a significant inverse nitrogen isotope fractionation (>10‰) during aerobic 2,3-DCA biodegradation by a mixed enrichment culture. The tentative identification of a glutamate conjugate of 2,3-DCA as a reaction intermediate indicates that the initial multistep enzymatic reaction may be rate-limiting. The formation of the glutamate adduct would increase the bond energy at the N atom, thus likely explaining the observed inverse N isotope fractionation. The corresponding nitrogen enrichment factor was +6.8 ± 0.6‰. This value was applied to investigate the in situ 2,3-DCA biodegradation at a contaminated site where the carbon and nitrogen isotope signatures from field samples suggested similar aerobic processes by native microorganisms. Under the assumption of the applicability of the Rayleigh model in a pilot wetland treating contaminated groundwater, the extent of biodegradation was estimated to be up to 80-90%. This study proposes multi-element CSIA as a novel application to study 2,3-DCA fate in groundwater and surface water and provides insights into biodegradation pathways.

Identification and widespread environmental distribution of a gene cassette implicated in anaerobic dichloromethane degradation.

Anthropogenic activities and natural processes release dichloromethane (DCM, methylene chloride), a toxic chemical with substantial ozone-depleting capacity. Specialized anaerobic bacteria metabolize DCM; however, the genetic basis for this process has remained elusive. Comparative genomics of the three known anaerobic DCM-degrading bacterial species revealed a homologous gene cluster, designated the methylene chloride catabolism (mec) gene cassette, comprising 8-10 genes encoding proteins with 79.6%-99.7% amino acid identities. Functional annotation identified genes encoding a corrinoid-dependent methyltransferase system, and shotgun proteomics applied to two DCM-catabolizing cultures revealed high expression of proteins encoded on the mec gene cluster during anaerobic growth with DCM. In a DCM-contaminated groundwater plume, the abundance of mec genes strongly correlated with DCM concentrations (R2  = 0.71-0.85) indicating their potential value as process-specific bioremediation biomarkers. mec gene clusters were identified in metagenomes representing peat bogs, the deep subsurface, and marine ecosystems including oxygen minimum zones (OMZs), suggesting a capacity for DCM degradation in diverse habitats. The broad distribution of anaerobic DCM catabolic potential infers a role for DCM as an energy source in various environmental systems, and implies that the global DCM flux (i.e., the rate of formation minus the rate of consumption) might be greater than emission measurements suggest.

Transformation of Chlorofluorocarbons Investigated via Stable Carbon Compound-Specific Isotope Analysis.

Compound-specific isotope analysis (CSIA) is a valuable tool in contaminant remediation studies. Chlorofluorocarbons (CFCs) are ozone-depleting substances previously thought to be persistent in groundwater under most geochemical conditions but more recently have been found to (bio)transform in some laboratory experiments. To date, limited applications of CSIA to CFCs have been undertaken. Here, biotransformation-associated carbon isotope enrichment factors, εC,bulk for CFC-113 (εC,bulk = -8.5 ± 0.4‰) and CFC-11 (εC,bulk = -14.5 ± 1.9‰), were determined. δ13C signatures of pure-phase CFCs and hydrochlorofluorocarbons were measured to establish source signatures. These findings were applied to investigate potential in situ CFC transformation in groundwater at a field site, where carbon isotope fractionation of CFC-11 suggests naturally occurring biotransformation by indigenous microorganisms. The maximum extent of CFC-11 transformation is estimated to be up to 86% by an approximate calculation using the Rayleigh concept. CFC-113 δ13C values in contrast were not resolvably different from pure-phase sources measured to date, demonstrating that CSIA can aid in identifying which compounds may, or may not, be undergoing reactive processes at field sites. Science and public attention remains focused on CFCs, as unexplained source inputs to the atmosphere have been recently reported, and the potential for CFC biotransformation in surface and groundwaters remains unclear. This study proposes δ13C CSIA as a novel application to study the fate of CFCs in groundwater.

Proteogenomics Reveals Novel Reductive Dehalogenases and Methyltransferases Expressed during Anaerobic Dichloromethane Metabolism.

Dichloromethane (DCM) is susceptible to microbial degradation under anoxic conditions and is metabolized via the Wood-Ljungdahl pathway; however, mechanistic understanding of carbon-chlorine bond cleavage is lacking. The microbial consortium RM contains the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, which strictly requires DCM as a growth substrate. Proteomic workflows applied to DCM-grown consortium RM biomass revealed a total of 1,705 nonredundant proteins, 521 of which could be assigned to strain RM. In the presence of DCM, strain RM expressed a complete set of Wood-Ljungdahl pathway enzymes, as well as proteins implicated in chemotaxis, motility, sporulation, and vitamin/cofactor synthesis. Four corrinoid-dependent methyltransferases were among the most abundant proteins. Notably, two of three putative reductive dehalogenases (RDases) encoded within strain RM's genome were also detected in high abundance. Expressed RDase 1 and RDase 2 shared 30% amino acid identity, and RDase 1 was most similar to an RDase of Dehalococcoides mccartyi strain WBC-2 (AOV99960, 52% amino acid identity), while RDase 2 was most similar to an RDase of Dehalobacter sp. strain UNSWDHB (EQB22800, 72% amino acid identity). Although the involvement of RDases in anaerobic DCM metabolism has yet to be experimentally verified, the proteome characterization results implicated the possible participation of one or more reductive dechlorination steps and methyl group transfer reactions, leading to a revised proposal for an anaerobic DCM degradation pathway.IMPORTANCE Naturally produced and anthropogenically released DCM can reside in anoxic environments, yet little is known about the diversity of organisms, enzymes, and mechanisms involved in carbon-chlorine bond cleavage in the absence of oxygen. A proteogenomic approach identified two RDases and four corrinoid-dependent methyltransferases expressed by the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, suggesting that reductive dechlorination and methyl group transfer play roles in anaerobic DCM degradation. These findings suggest that the characterized DCM-degrading bacterium Dehalobacterium formicoaceticum and "Candidatus Dichloromethanomonas elyunquensis" strain RM utilize distinct strategies for carbon-chlorine bond cleavage, indicating that multiple pathways evolved for anaerobic DCM metabolism. The specific proteins (e.g., RDases and methyltransferases) identified in strain RM may have value as biomarkers for monitoring anaerobic DCM degradation in natural and contaminated environments.

Biotic and Abiotic Dehalogenation of 1,1,2-Trichloro-1,2,2-trifluoroethane (CFC-113): Implications for Bacterial Detoxification of Chlorinated Ethenes.

Chlorofluorocarbons including 1,1,2-trichloro-1,2,2-trifluoroethane (CFC-113) often occur in groundwater plumes comingled with chlorinated solvents such as trichloroethene (TCE). We show that CFC-113 inhibits reductive dechlorination by Dehalococcoides mccartyi (Dhc) in a concentration-dependent manner, causing cis-1,2-dichloroethene (cis-DCE) stalls. Following a 17-day exposure of Dhc-containing consortium SDC-9 to 76 µM CFC-113, cis-DCE dechlorination activity did not recover after CFC-113 removal. River sediment microcosms demonstrated that CFC-113 was subject to microbial degradation under anoxic conditions, and chlorotrifluoroethene (CTFE) was observed as a transformation product. No degradation of CFC-113 was observed in killed controls and in incubations with reactive minerals including mackinawite, green rust, magnetite, and manganese dioxide. In vitro experiments with reduced corrinoid (i.e., vitamin B12) mediated reductive dechlorination of CFC-113 to CTFE and trifluoroethene (TFE) followed by reductive defluorination of TFE to cis-1,2-difluoroethene (cis-DFE) as an end product. This biomimetic degradation of CFC-113 to cis-DFE was also demonstrated in vivo using the corrinoid-producing homoacetogen Sporomusa ovata, suggesting the cometabolic microbial reductive dechlorination and reductive defluorination of CFC-113 to cis-DFE is feasible under anoxic in situ conditions. The CFC-113 degradation intermediates CTFE, TFE, and cis-DFE did not inhibit TCE dechlorination by Dhc, indicating that the initial reductive transformation step can overcome cis-DCE stalls.

A Dehalogenimonas Population Respires 1,2,4-Trichlorobenzene and Dichlorobenzenes.

Chlorobenzenes are ubiquitous contaminants in groundwater and soil at many industrial sites. Previously, we demonstrated the natural attenuation of chlorobenzenes and benzene at a contaminated site inferred from a 5 year site investigation and parallel laboratory microcosm studies. To identify the microbes responsible for the observed dechlorination of chlorobenzenes, the microbial community was surveyed using 16S rRNA gene amplicon sequencing. Members of the Dehalobacter and Dehalococcoides are reported to respire chlorobenzenes; however, neither were abundant in our sediment microcosms. Instead, we observed a significant increase in the relative abundance of Dehalogenimonas from <1% to 16-30% during dechlorination of 1,2,4-trichlorobenzene (TCB), 1,2-dichlorobenzene (DCB), and 1,3-DCB over 19 months. Quantitative PCR (qPCR) confirmed that Dehalogenimonas gene copies increased by 2 orders of magnitude with an average yield of 3.6 ± 2.3 g cells per mol Cl- released ( N = 12). In transfer cultures derived from sediment microcosms, dechlorination of 1,4-DCB and monochlorobenzene (MCB) was carried out by Dehalobacter spp. with a growth yield of 3.0 ± 2.1 g cells per mol Cl- released ( N = 5). Here we show that a Dehalogenimonas population respire 1,2,4-TCB and 1,2-/1,3-DCB isomers. This finding emphasizes the need to monitor a broader spectrum of organohalide-respiring bacteria, including Dehalogenimonas, at sites contaminated with halogenated organic compounds.

Identification of Degradation Pathways of Chlorohydrocarbons in Saturated Low-Permeability Sediments Using Compound-Specific Isotope Analysis.

This study aims to investigate whether compound-specific carbon isotope analysis (CSIA) can be used to differentiate the degradation pathways of chlorohydrocarbons in saturated low-permeability sediments. For that purpose, a site was selected, where a complex mixture of chlorohydrocarbons contaminated an aquifer-aquitard system. Almost 50 years after contaminant releases, high-resolution concentration, CSIA, and microbial profiles were determined. The CSIA profiles showed that in the aquitard cis-dichloroethene (cDCE), first considered as a degradation product of trichloroethene (TCE), is produced by the dichloroelimination of 1,1,2,2-tetrachloroethane (TeCA). In contrast, TeCA degrades to TCE via dehydrohalogenation in the aquifer, indicating that the aquifer-aquitard interface separates two different degradation pathways for TeCA. Moreover, the CSIA profiles showed that chloroform (CF) is degraded to dichloromethane (DCM) via hydrogenolysis in the aquitard and, to a minor degree, produced by the degradation of carbon tetrachloride (CT). Several microorganisms capable of degrading chlorohydrocarbons were detected in the aquitard, suggesting that aquitard degradation is microbially mediated. Furthermore, numerical simulations reproduced the aquitard concentration and CSIA profiles well, which allowed the determination of degradation rates for each transformation pathway. This improves the prediction of contaminant fate in the aquitard and potential magnitude of impacts on the adjacent aquifer due to back-diffusion.

Dual Carbon-Chlorine Isotope Analysis Indicates Distinct Anaerobic Dichloromethane Degradation Pathways in Two Members of Peptococcaceae.

Dichloromethane (DCM) is a probable human carcinogen and frequent groundwater contaminant and contributes to stratospheric ozone layer depletion. DCM is degraded by aerobes harboring glutathione-dependent DCM dehalogenases; however, DCM contamination occurs in oxygen-deprived environments, and much less is known about anaerobic DCM metabolism. Some members of the Peptococcaceae family convert DCM to environmentally benign products including acetate, formate, hydrogen (H2), and inorganic chloride under strictly anoxic conditions. The current study applied stable carbon and chlorine isotope fractionation measurements to the axenic culture Dehalobacterium formicoaceticum and to the consortium RM comprising DCM degrader Candidatus Dichloromethanomonas elyunquensis. Degradation-associated carbon and chlorine isotope enrichment factors (εC and εCl) of -42.4 ± 0.7‰ and -5.3 ± 0.1‰, respectively, were measured in D. formicoaceticum cultures. A similar εCl of -5.2 ± 0.1‰, but a substantially lower εC of -18.3 ± 0.2‰, were determined for Ca. Dichloromethanomonas elyunquensis. The εC and εCl values resulted in distinctly different dual element C-Cl isotope correlations (ΛC/Cl = Δδ13C/Δδ37Cl) of 7.89 ± 0.12 and 3.40 ± 0.03 for D. formicoaceticum and Ca. Dichloromethanomonas elyunquensis, respectively. The distinct ΛC/Cl values obtained for the two cultures imply mechanistically distinct C-Cl bond cleavage reactions, suggesting that members of Peptococcaceae employ different pathways to metabolize DCM. These findings emphasize the utility of dual carbon-chlorine isotope analysis to pinpoint DCM degradation mechanisms and to provide an additional line of evidence that detoxification is occurring at DCM-contaminated sites.

Practical application of 1 H benchtop NMR spectroscopy for the characterization of a nonaqueous phase liquid from a contaminated environment.

Nonaqueous phase liquids (NAPLs) located at the surface of the water table and/or below the water table are often a significant source for groundwater contamination near current or former commercial/industrial facilities. Due to the complex and long history of many industrial sites, these NAPLs often contain a complex mixture of contaminants and as such can be difficult to fully characterize using conventional analytical methods. Remediation and risk assessment activities at sites containing NAPLs may, subsequently, be hindered as the contamination profile may not be fully understood. This paper demonstrates the application of bench-scale 1 H nuclear magnetic resonance (NMR) spectroscopy as a practical tool to assist with the characterization of complex NAPLs. Here, a NAPL collected from a contaminated site situated near a former chemical manufacturing facility was analyzed using a combination of one-dimensional (1D) 1 H NMR spectroscopy and two-dimensional (2D) 1 H J-resolved spectroscopy (JRES). It is shown that 1D NMR experiments are useful in the rapid identification of the classes of compounds present, whereas 2D JRES NMR experiments are useful in identifying specific compounds. The use of benchtop NMR spectroscopy as a simple and cost effective tool to assist in the analysis of contaminated sites may help improve the practical characterization of many heavily contaminated sites and facilitate improved risk assessments and remedial strategies.

Mutualistic interaction between dichloromethane- and chloromethane-degrading bacteria in an anaerobic mixed culture.

The microbial mixed culture RM grows with dichloromethane (DCM) as the sole energy source generating acetate, methane, chloride and biomass as products. Chloromethane (CM) was not an intermediate during DCM utilization consistent with the observation that CM could not replace DCM as a growth substrate. Interestingly, cultures that received DCM and CM together degraded both compounds concomitantly. Transient hydrogen (H2 ) formation reaching a maximum concentration of 205 ± 13 ppmv was observed in cultures growing with DCM, and the addition of exogenous H2 at concentrations exceeding 3000 ppmv impeded DCM degradation. In contrast, CM degradation in culture RM had a strict requirement for H2 . Following five consecutive transfers on CM and H2 , Acetobacterium 16S rRNA gene sequences dominated the culture and the DCM-degrader Candidatus Dichloromethanomonas elyunquensis was eliminated, consistent with the observation that the culture lost the ability to degrade DCM. These findings demonstrate that culture RM harbours different populations responsible for anaerobic DCM and CM metabolism, and further imply that the DCM and CM degradation pathways are mechanistically distinct. H2 generated during DCM degradation is consumed by the hydrogenotrophic CM degrader, or may fuel other hydrogenotrophic processes, including organohalide respiration, methanogenesis and H2 /CO2 reductive acetogenesis.

Natural Attenuation of Nonvolatile Contaminants in the Capillary Fringe.

When anoxic polluted groundwater encounters the overlying vadose zone an oxic/anoxic interface is created, often near the capillary fringe. Biodegradation of volatile contaminants in the capillary fringe can prevent vapor migration. In contrast, the biodegradation of nonvolatile contaminants in the vadose zone has received comparatively little attention. Nonvolatile compounds do not cause vapor intrusion, but they still move with the groundwater and are major contaminants. Aniline (AN) and diphenylamine (DPA) are examples of toxic nonvolatile contaminants found often at dye and munitions manufacturing sites. In this study, we tested the hypothesis that bacteria can aerobically biodegrade AN and DPA in the capillary fringe and decrease the contaminant concentrations in the anoxic plume beneath the vadose zone. Laboratory multiport columns that represented the unsaturated zone were used to evaluate degradation of AN or DPA in contaminated water. The biodegradation fluxes of the contaminants were estimated to be 113 ± 26 mg AN·m(-2)·h(-1) and 76 ± 18 mg DPA·m(-2)·h(-1) in the presence of bacteria known to degrade AN and DPA. Oxygen and contaminant profiles along with enumeration of bacterial populations indicated that most of the biodegradation took place within the lower part of the capillary fringe. The results indicate that bacteria capable of contaminant biodegradation in the capillary fringe can create a sink for nonvolatile contaminants.

Sediment Monitored Natural Recovery Evidenced by Compound Specific Isotope Analysis and High-Resolution Pore Water Sampling.

Monitoring natural recovery of contaminated sediments requires the use of techniques that can provide definitive evidence of in situ contaminant degradation. In this study, a passive diffusion sampler, called "peeper", was combined with Compound Specific Isotope Analysis to determine benzene and monochlorobenzene (MCB) stable carbon isotope values at a fine vertical resolution (3 cm) across the sediment water interface at a contaminated site. Results indicated significant decrease in concentrations of MCB from the bottom to the top layers of the sediment over 25 cm, and a 3.5 ‰ enrichment in δ13C values of MCB over that distance. Benzene was always at lower concentrations than MCB, with consistently more depleted δ13C values than MCB. The redox conditions were dominated by iron reduction along most of the sediment profile. These results provide multiple lines of evidence for in situ reductive dechlorination of MCB to benzene. Stable isotope analysis of contaminants in pore water is a valuable method to demonstrate in situ natural recovery of contaminated sediments. This novel high-resolution approach is critical to deciphering the combined effects of parent contaminant (e.g., MCB) degradation and both production and simultaneous degradation of daughter products, especially benzene.

Identification of Anaerobic Aniline-Degrading Bacteria at a Contaminated Industrial Site.

Anaerobic aniline biodegradation was investigated under different electron-accepting conditions using contaminated canal and groundwater aquifer sediments from an industrial site. Aniline loss was observed in nitrate- and sulfate-amended microcosms and in microcosms established to promote methanogenic conditions. Lag times of 37 days (sulfate amended) to more than 100 days (methanogenic) were observed prior to activity. Time-series DNA-stable isotope probing (SIP) was used to identify bacteria that incorporated (13)C-labeled aniline in the microcosms established to promote methanogenic conditions. In microcosms from heavily contaminated aquifer sediments, a phylotype with 92.7% sequence similarity to Ignavibacterium album was identified as a dominant aniline degrader as indicated by incorporation of (13)C-aniline into its DNA. In microcosms from contaminated canal sediments, a bacterial phylotype within the family Anaerolineaceae, but without a match to any known genus, demonstrated the assimilation of (13)C-aniline. Acidovorax spp. were also identified as putative aniline degraders in both of these two treatments, indicating that these species were present and active in both the canal and aquifer sediments. There were multiple bacterial phylotypes associated with anaerobic degradation of aniline at this complex industrial site, which suggests that anaerobic transformation of aniline is an important process at the site. Furthermore, the aniline degrading phylotypes identified in the current study are not related to any known aniline-degrading bacteria. The identification of novel putative aniline degraders expands current knowledge regarding the potential fate of aniline under anaerobic conditions.

Biodegradation of cis-dichloroethene and vinyl chloride in the capillary fringe.

Volatile chlorinated compounds are major pollutants in groundwater, and they pose a risk of vapor intrusion into buildings. Vapor intrusion can be prevented by natural attenuation in the vadose zone if biodegradation mechanisms can be established. In this study, we tested the hypothesis that bacteria can use cis-dichloroethene (cis-DCE) or vinyl chloride (VC) as an electron donor in the vadose zone. Anoxic water containing cis-DCE or VC was pumped continuously beneath laboratory columns that represented the vadose zone. Columns were inoculated with Polaromonas sp. strain JS666, which grows aerobically on cis-DCE, or with Mycobacterium sp. JS60 and Nocardiodes sp. JS614 that grow on VC. Complete biodegradation with fluxes of 84 ± 15 µmol of cis-DCE · m(-2) · hr(-1) and 218 ± 25 µmole VC·m(-2) · h(-1) within the 23 cm column indicated that microbial activities can prevent the migration of cis-DCE and VC vapors. Oxygen and volatile compound profiles along with enumeration of bacterial populations indicated that most of the biodegradation took place in the first 10 cm above the saturated zone within the capillary fringe. The results revealed that cis-DCE and VC can be biodegraded readily at the oxic/anoxic interfaces in the vadose zone if appropriate microbes are present.

Diffusion sampler for compound specific carbon isotope analysis of dissolved hydrocarbon contaminants.

Compound Specific Isotope Analysis (CSIA) is widely utilized to study the fate of organic contaminants in groundwater. To date, however, no method is available to obtain CSIA samples at a fine (cm) spatial scale across the sediment-surface water interface (SWI), a key boundary for discharge of contaminated groundwater to surface water. Dissolved contaminants in such discharged zones undergo rapid temporal and spatial changes due to heterogeneity in redox conditions and microbial populations. The compatibility of a passive sediment pore water sampler ("peeper") to collect 40 mL samples for CSIA of benzene, toluene, monochlorobenzene, and 1,2-dichlorobenzene at field-relevant concentrations (0.1-5 mg L(-1)) was evaluated in laboratory experiments. Results demonstrate that physical diffusion across the polysulfone membrane does not alter the carbon isotope values (±0.5‰). Measured δ(13)C values also remain invariant despite significant adsorption of the compounds on the peeper material, an effect which increased with higher numbers of chlorine atoms and sorption coefficient (Koc) values. In addition, isotope equilibrium between the peeper chamber and the sediment pore water occurred in less than a day, indicating the peeper method can be used to provide samples for CSIA analysis at fine spatial and temporal sampling resolutions in contaminated sediments.

Bioaugmentation with distinct Dehalobacter strains achieves chloroform detoxification in microcosms.

Chloroform (CF) is a widespread groundwater contaminant not susceptible to aerobic degradation. Under anoxic conditions, CF can undergo abiotic and cometabolic transformation but detoxification is generally not achieved. The recent discovery of distinct Dehalobacter strains that respire CF to dichloromethane (DCM) and ferment DCM to nonchlorinated products promises that bioremediation of CF plumes is feasible. To track both strains, 16S rRNA gene-based qPCR assays specific for either Dehalobacter strain were designed and validated. A laboratory treatability study explored the value of bioaugmentation and biostimulation to achieve CF detoxification using anoxic microcosms established with aquifer material from a CF-contaminated site. Microcosms that received 6% (v/v) of the CF-to-DCM-dechlorinating culture Dhb-CF to achieve an initial Dehalobacter cell titer of 1.6 ± 0.9 × 10(4) mL(-1) dechlorinated CF to stoichiometric amounts of DCM. Subsequent augmentation with 3% (v/v) of the DCM-degrading consortium RM to an initial Dehalobacter cell abundance of 1.2 ± 0.2 × 10(2) mL(-1) achieved complete DCM degradation in microcosms amended with 10 mM bicarbonate. Growth of the CF-respiring and the DCM-degrading Dehalobacter populations and detoxification were also observed in microcosms that received both inocula simultaneously. These findings suggest that anaerobic bioremediation (e.g., bioaugmentation) is a possible remedy at CF- and DCM-contaminated sites without CT, which strongly inhibited CF organohalide respiration and DCM organohalide fermentation.

Dichloromethane fermentation by a Dehalobacter sp. in an enrichment culture derived from pristine river sediment.

Dichloromethane (DCM) as the sole substrate supported growth of a Dehalobacter sp. in an enrichment culture derived from noncontaminated river sediment. DCM was not reductively dechlorinated, and acetate was produced, indicating DCM fermentation and further suggesting Dehalobacter growth is not limited to organohalide respiration.

Contribution of coexisting sulfate and iron reducing bacteria to methylmercury production in freshwater river sediments.

We investigated microbial methylmercury (CH(3)Hg) production in sediments from the South River (SR), VA, an ecosystem contaminated with industrial mercury (Hg). Potential Hg methylation rates in samples collected at nine sites were low in late spring and significantly higher in late summer. Demethylation of (14)CH(3)Hg was dominated by (14)CH(4) production in spring, but switched to producing mostly (14)CO(2) in the summer. Fine-grained sediments originating from the erosion of river banks had the highest CH(3)Hg concentrations and were potential hot spots for both methylation and demethylation activities. Sequencing of 16S rRNA genes of cDNA recovered from sediment RNA extracts indicated that at least three groups of sulfate-reducing bacteria (SRB) and one group of iron-reducing bacteria (IRB), potential Hg methylators, were active in SR sediments. SRB were confirmed as a methylating guild by amendment experiments showing significant sulfate stimulation and molybdate inhibition of methylation in SR sediments. The addition of low levels of amorphous iron(III) oxyhydroxide significantly stimulated methylation rates, suggesting a role for IRB in CH(3)Hg synthesis. Overall, our studies suggest that coexisting SRB and IRB populations in river sediments contribute to Hg methylation, possibly by temporally and spatially separated processes.

Compound-specific isotope analysis (CSIA) evaluation of degradation of chlorinated benzenes (CBs) and benzene in a contaminated aquifer.

Compound-specific isotope analysis (CSIA) has become a valuable tool in understanding the fate of organic contaminants at field sites. However, its application to chlorinated benzenes (CBs), a group of toxic and persistent groundwater contaminants, has received less attention. This study employed CSIA to investigate the occurrence of natural degradation of various CBs and benzene in a contaminated aquifer. Despite the complexity of the study area (e.g., installation of a sheet pile barrier and the presence of a complex set of contaminants), the substantial enrichments in δ13C values (i.e., >2‰) for all CBs and benzene across the sampling wells indicate in situ degradation of these compounds. In particular, the 13C enrichments for 1,2,4-trichlorobenzene (1,2,4-TCB) and 1,2-dichlorobenzene (1,2-DCB) display good correlations with decreasing groundwater concentrations, consistent with the effects of in situ biodegradation. Using the Rayleigh model, the extent of degradation (EoD) is estimated to be 47-99% for 1,2-DCB, and 21-73% for 1,2,4-TCB. The enrichments observed for the other CBs (1,4-DCB and chlorobenzene (MCB)) and benzene at the site are also suggestive of in situ biodegradation. Due to simultaneous degradation and production of 1,4-DCB (a major 1,2,4-TCB degradation product), MCB (from DCB degradation), and benzene (from MCB degradation), the estimation of EoD for these intermediate compounds is more complex but a modelling simulation supports in situ biodegradation of these daughter products. In particular, the fact that the δ13C values of MCB and benzene (i.e., daughter products of 1,2,4-TCB) are more enriched than the original δ13C value of their parent 1,2,4-TCB provides definitive evidence for the occurrence of in situ biodegradation of the MCB and benzene.

Impact of multiple drying and rewetting events on biochar amendments for Hg stabilization in floodplain soil from South River, VA.

Frequent drying and rewetting due to flooding/precipitation and drainage events in floodplains induces changes in biogeochemical conditions that may influence the effectiveness of in situ Hg stabilization using biochars as soil amendments. This study evaluated two selected biochars anaerobic digestate (DIG) and sulfurized hardwood (MOAK)) as potential amendment materials in moderately reduced floodplain soil under repeated drying and rewetting events using a modified humidity cell protocol. Enhanced release of filter-passing (0.45-µm) total Hg (THg) and MeHg was observed at early times. Elevated concentrations of 0.45-µm THg were associated with DOC and Mn in sediment control and biochar-amended systems. Elevated concentrations of MeHg were associated with Mn in the MOAK-amended system. Thereafter, decreases in 0.45-µm (up to 57%) and unfiltered THg (up to 93%) were observed. As wetting and drying events continued, decreases in pH and alkalinity as well as increases in SO42- (up to 796 mg L-1) and Ca (up to 215 mg L-1) were observed in the MOAK-amended systems with the microbial community shifted towards sulfur-oxidizing bacteria, indicating microbially-driven oxidation of MOAK. Although results of S K-edge X-ray absorption near edge structure (XANES) analysis suggest polysulfur is the predominant S phase in both MOAK- and DIG-amended systems, microbially-driven oxidation of DIG was not observed. Polysulfur in MOAK from the sulfurization process is more bioavailable to sulfur oxidizing communities than in DIG under the repeated drying and wetting conditions. Results of this study suggest biogeochemical conditions as well as biochar properties should be considered when planning full-scale field applications.