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

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

Evaluation of methanotrophic bacterial communities capable of biodegrading trichloroethene (TCE) in acidic aquifers.

While bioremediation technologies for trichloroethene (TCE), a suspected carcinogen, have been successfully demonstrated in neutral pH aquifers, these technologies are often ineffective for remediating TCE contamination in acidic aquifers (i.e., pH < 5.5). Acidophilic methanotrophs have been detected in several low pH environments, but their presence and potential role in TCE degradation in acidic aquifers is unknown. This study applied a stable isotope probing-based technique to identify active methanotrophs that are capable of degrading TCE in microcosms prepared from two low pH aquifers. A total of thirty-five clones of methanotrophs were derived from low pH microcosms in which methane and TCE degradation had been observed, with 29 clustered in γ-Proteobacteria and 6 clustered in α-Proteobacteria. None of the clones has a high similarity to known acidophilic methanotrophs from other environments. The presence and diversity of particulate MMO and soluble MMO were also investigated. The pmoA gene was detected predominantly at one site, and the presence of a specific form of mmoX in numerous samples suggested that Methylocella spp. may be common in acidic aquifers. Finally, a methane-grown culture at pH 4 was enriched from an acidic aquifer and its ability to biodegrade various chlorinated ethenes was tested. Interestingly, the mixed culture rapidly degraded TCE and vinyl chloride, but not cis-dichloroethene after growth on methane. The data suggest that aerobic biodegradation of TCE and other chlorinated solvents in low pH groundwater may be facilitated by methanotrophic bacteria, and that there are potentially a wide variety of different strains that inhabit acidic aquifers.

In situ bioremediation of 1,2-dibromoethane (EDB) in groundwater to part-per-trillion concentrations using cometabolism.

1,2-Dibromoethane (ethylene dibromide; EDB) is a probable human carcinogen that was historically added to leaded gasoline as a scavenger to prevent the build-up of lead oxide deposits in engines. Studies indicate that EDB is present at thousands of past fuel spill sites above its stringent EPA Maximum Contaminant Level (MCL) of 0.05 µg/L. There are currently no proven in situ options to enhance EDB degradation in groundwater to meet this requirement. Based on successful laboratory studies showing that ethane can be used as a primary substrate to stimulate the aerobic, cometabolic biodegradation of EDB to <0.015 µg/L (Hatzinger et al., 2015), a groundwater recirculation system was installed at the FS-12 EDB plume on Joint Base Cape Cod (JBCC), MA to facilitate in situ treatment. Groundwater was taken from an existing extraction well, amended with ethane, oxygen, and inorganic nutrients and then recharged into the aquifer upgradient of the extraction well creating an in situ reactive zone. The concentrations of EDB, ethane, oxygen, and anions in groundwater were measured with time in a series of nested monitoring wells installed between the extraction and injection well. EDB concentrations in the six monitoring wells that were hydraulically well-connected to the pumping system declined from ~ 0.3 µg/L (the average concentration in the recirculation cell after 3 months of operation without amendment addition) to <0.02 µg/L during the 4-month amendment period, meeting both the federal MCL and the more stringent Massachusetts MCL (0.02 µg/L). The data indicate that cometabolic treatment is a promising in situ technology for EDB, and that low regulatory levels can be achieved with this biological approach.

Biological treatment of N-nitrosodimethylamine (NDMA) and N-nitrodimethylamine (NTDMA) in a field-scale fluidized bed bioreactor.

The ex situ treatment of N-nitrosodimethylamine (NDMA) and N-nitrodimethylamine (NTDMA) in groundwater was evaluated in a field-scale fluidized bed bioreactor (FBR). Both of these compounds, which originally entered groundwater at the test site from the use of liquid rocket propellant, are suspected human carcinogens. The objective of this research was to examine the application of a novel field-scale propane-fed fluidized bed bioreactor as an alternative to ultraviolet irradiation (UV) for treating NDMA and NTDMA to low part-per-trillion (ng/L) concentrations. Previous laboratory studies have shown that the bacterium Rhodococcus ruber ENV425 can biodegrade NDMA and NTDMA during growth on propane as a primary substrate and that the strain can effectively reduce NDMA concentrations in propane-fed bench-scale bioreactors of different design. R. ruber ENV425 was used as a seed culture for the FBR, which operated at a fluidization flow of ∼19 L-per-min (LPM) and received propane, oxygen, and inorganic nutrients in the feed. The reactor effectively treated ∼1 µg/L of influent NDMA to effluent concentrations of less than 10 ng/L at a hydraulic residence time (HRT) of only 10 min. At a 20 min HRT, the FBR reduced NDMA to <4.2 ng/L in the effluent, which was the discharge limit at the test site where the study was conducted. Similarly, NTDMA was consistently treated in the FBR from ∼0.5 µg/L to <10 ng/L at an HRT of 10 min or longer. Based on these removal rates, the average NDMA and NTDMA elimination capacities achieved were 2.1 mg NDMA treated/m3 of expanded bed/hr of operation and 1.1 mg NTDMA treated/m3 of expanded bed/hr of operation, respectively. The FBR system was highly resilient to upsets including power outages. Treatment of NDMA, but not NTDMA, was marginally affected when trace co-contaminants including trichloroethene (TCE) and trichlorofluoromethane (Freon 11) were initially added to feed groundwater, but performance recovered over a few weeks in the continued presence of these compounds. Strain ENV425 appeared to be replaced by native propanotrophs over time based on qPCR analysis, but contaminant treatment was not diminished. The results suggest that a FBR can be a viable alternative to UV treatment for removing NDMA from groundwater.

Enhancing aerobic biodegradation of 1,2-dibromoethane in groundwater using ethane or propane and inorganic nutrients.

1,2-Dibromoethane (ethylene dibromide; EDB) is a probable human carcinogen that was previously used as both a soil fumigant and a scavenger in leaded gasoline. EDB has been observed to persist in soils and groundwater, particularly under oxic conditions. The objective of this study was to evaluate options to enhance the aerobic degradation of EDB in groundwater, with a particular focus on possible in situ remediation strategies. Propane gas and ethane gas were observed to significantly stimulate the biodegradation of EDB in microcosms constructed with aquifer solids and groundwater from the FS-12 EDB plume at Joint Base Cape Cod (Cape Cod, MA), but only after inorganic nutrients were added. Ethene gas was also effective, but rates were appreciably slower than for ethane and propane. EDB was reduced to <0.02 µg/L, the Massachusetts state Maximum Contaminant Level (MCL), in microcosms that received ethane gas and inorganic nutrients. An enrichment culture (BE-3R) that grew on ethane or propane gas but not EDB was obtained from the site materials. The degradation of EDB by this culture was inhibited by acetylene gas, suggesting that degradation is catalyzed by a monooxygenase enzyme. The BE-3R culture was also observed to biodegrade 1,2-dichloroethane (DCA), a compound commonly used in conjunction with EDB as a lead scavenger in gasoline. The data suggest that addition of ethane or propane gas with inorganic nutrients may be a viable option to enhance degradation of EDB in groundwater aquifers to below current state or federal MCL values.

Ex situ treatment of N-nitrosodimethylamine (NDMA) in groundwater using a fluidized bed reactor.

N-nitrosodimethylamine (NDMA) is a suspected human carcinogen that has traditionally been treated in water using ultraviolet irradiation (UV). The objective of this research was to examine the application of a laboratory-scale fluidized bed reactor (FBR) as an alternative technology for treating NDMA to part-per-trillion (ng/L) concentrations in groundwater. Previous studies have shown that the bacterium Rhodococcus ruber ENV425 is capable of cometabolizing NDMA during growth on propane as a primary substrate in batch culture (Fournier et al., 2009) and in a bench-scale membrane bioreactor (Hatzinger et al., 2011) to low ng/L concentrations. R. ruber ENV425 was inoculated into the FBR during this study. With a hydraulic residence time (HRT) of 20 min, the FBR was found to be an effective means to treat 10-20 µg/L of NDMA to effluent concentrations less than 100 ng/L. When the HRT was increased to 30 min and oxygen and propane addition rates were optimized, the FBR system demonstrated treatment of the NDMA to effluent concentrations of less than 10 ng/L. Short-term shutdowns and the presence of trichloroethene (TCE) at 6 µg/L as a co-contaminant had minimal effect on the treatment of NDMA in the FBR. The data suggest that the FBR technology can be a viable alternative to UV for removing NDMA from groundwater.

Aerobic treatment of N-nitrosodimethylamine in a propane-fed membrane bioreactor.

N-Nitrosodimethylamine (NDMA) is a suspected human carcinogen that has recently been detected in wastewater, groundwater and drinking water. Treatment of this compound to low part-per-trillion (ng/L) concentrations is required to mitigate cancer risk. Current treatment generally entails UV irradiation, which while effective, is also expensive. The objective of this research was to explore potential bioremediation strategies as alternatives for treating NDMA to ng/L concentrations. Batch studies revealed that the propanotroph Rhodococcus ruber ENV425 was capable of metabolizing NDMA from 8 µg/L to <2 ng/L after growth on propane, and that the strain produced metabolites that do not pose a significant risk at the concentrations generated (Fournier et al., 2009). A laboratory-scale membrane bioreactor (MBR) was subsequently constructed to evaluate the potential for long-term ex situ treatment of NDMA. The MBR was seeded with ENV425 and received propane as the primary growth substrate and oxygen as an electron acceptor. At an average influent NDMA concentration of 7.4 µg/L and a 28.5 h hydraulic residence time, the reactor effluent concentration was 3.0 ± 2.3 ng/L (>99.95% removal) over more than 70 days of operation. The addition of trichloroethene (TCE) to the reactor resulted in a significant increase in effluent NDMA concentrations, most likely due to cell toxicity from TCE-epoxide produced during its cometabolic oxidation by ENV425. The data suggest that an MBR system can be a viable treatment option for NDMA in groundwater provided that high concentrations of TCE are not present.

Biotransformation of N-nitrosodimethylamine by Pseudomonas mendocina KR1.

N-Nitrosodimethylamine (NDMA) is a potent carcinogen and an emerging contaminant in groundwater and drinking water. The metabolism of NDMA in mammalian cells has been widely studied, but little information is available concerning the microbial transformation of this compound. The objective of this study was to elucidate the pathway(s) of NDMA biotransformation by Pseudomonas mendocina KR1, a strain that possesses toluene-4-monooxygenase (T4MO). P. mendocina KR1 was observed to initially oxidize NDMA to N-nitrodimethylamine (NTDMA), a novel metabolite. The use of 18O2 and H(2)18O revealed that the oxygen added to NDMA to produce NTDMA was derived from atmospheric O2. Experiments performed with a pseudomonad expressing cloned T4MO confirmed that T4MO catalyzes this initial reaction. The NTDMA produced by P. mendocina KR1 did not accumulate, but rather it was metabolized further to produce N-nitromethylamine (88 to 94% recovery) and a trace amount of formaldehyde (HCHO). Small quantities of methanol (CH3OH) were also detected when the strain was incubated with NDMA but not during incubation with either NTDMA or HCHO. The formation of methanol is hypothesized to occur via a second, minor pathway mediated by an initial alpha-hydroxylation of the nitrosamine. Strain KR1 did not grow on NDMA or mineralize significant quantities of the compound to carbon dioxide, suggesting that the degradation process is cometabolic.

Enhancing transport of hydrogenophaga flava ENV735 for bioaugmentation of aquifers contaminated with methyl tert-butyl ether.

The gasoline oxygenate methyl tert-butyl ether (MTBE) has become a widespread contaminant in groundwater throughout the United States. Bioaugmentation of aquifers with MTBE-degrading cultures may be necessary to enhance degradation of the oxygenate in some locations. However, poor cell transport has sometimes limited bioaugmentation efforts in the past. The objective of this study was to evaluate the transport characteristics of Hydrogenophaga flava ENV735, a pure culture capable of growth on MTBE, and to improve movement of the strain through aquifer solids. The wild-type culture moved only a few centimeters in columns of aquifer sediment. An adhesion-deficient variant (H. flava ENV735:24) of the wild-type strain that moved more readily through sediments was obtained by sequential passage of cells through columns of sterile sediment. Hydrophobic and electrostatic interaction chromatography revealed that the wild-type strain is much more hydrophobic than the adhesion-deficient variant. Electrophoretic mobility assays and transmission electron microscopy showed that the wild-type bacterium contains two distinct subpopulations, whereas the adhesion-deficient strain has only a single, homogeneous population. Both the wild-type strain and adhesion-deficient variant degraded MTBE, and both were identified by 16S rRNA analysis as pure cultures of H. flava. The effectiveness of surfactants for enhancing transport of the wild-type strain was also evaluated. Many of the surfactants tested were toxic to ENV735; however, one nonionic surfactant, Tween 20, enhanced cell transport in sand columns. Improving microbial transport may lead to a more effective bioaugmentation strategy for MTBE-contaminated sites where indigenous oxygenate degraders are absent.