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Anaerobic Degradation of 1,1,2,2-Tetrachloroethane and Association with Microbial Communities in a Freshwater Tidal Wetland, Aberdeen Proving Ground, Maryland: Laboratory Experiments and Comparisons to Field Data

By Michelle M. Lorah, Mary A. Voytek, Julie D. Kirshtein, and Elizabeth J. (Phillips) Jones

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Abstract

Defining biodegradation rates and processes is a critical part of assessing the feasibility of monitored natural attenuation as a remediation method for Groundwater containing organic contaminants. During 1998-2001, the U.S. Geological Survey conducted a microbial study at a freshwater tidal wetland along the West Branch Canal Creek, Aberdeen Proving Ground, Maryland, as part of an investigation of natural attenuation of chlorinated volatile organic compounds (VOCs) in the wetland sediments. Geochemical analyses and molecular biology techniques were used to investigate factors controlling anaerobic degradation of 1,1,2,2-tetrachloroethane (TeCA), and to characterize the microbial communities that potentially are important in its degradation. Rapid TeCA and daughter product degradation observed in laboratory experiments and estimated with field data confirm that natural attenuation is a feasible remediation method at this site. The diverse microbial community that seems to be involved in TeCA degradation in the wetland sediments varies with changing spatial and seasonal conditions, allowing continued effective natural attenuation throughout the year.

Rates of TeCA degradation in anaerobic microcosm experiments conducted with wetland sediment collected from two different sites (WB23 and WB30) and during three different seasons (March-April 1999, July-August 1999, and October-November 2000) showed little spatial variability but high seasonal variability. Initial first-order degradation rate constants for TeCA ranged from 0.10±0.01 to 0.16±0.05 per day (half-lives of 4.3 to 6.9 days) for March-April 1999 and October-November 2000 microcosms incubated at 19 degrees Celsius, whereas lower rate constants of 0 ± 0.03 and 0.06 ± 0.03 per day were obtained in July-August 1999 microcosms incubated at 19 degrees Celsius. Microbial community profiles showed that low microbial biomass and microbial diversity in the summer, possibly due to competition for nutrients by the wetland vegetation, could account for these unexpectedly low degradation rates. In microcosms incubated at 5 degrees Celsius, about 50 percent of the initial TeCA in solution was converted to daughter products within a 35-day incubation period, indicating that biodegradation in the wetland sediments can continue during cold winter temperatures.

Initial pathways of TeCA degradation were the same in the wetland sediment microcosms regardless of the season or sediment collection site, the reduction-oxidation conditions, and the previous exposure of the sediment to contamination. Immediate and simultaneous dichloroelimination and hydrogenolysis, producing 1,2-dichloro-ethene (12DCE) and 1,1,2-trichloroethane (112TCA), respectively, were the initial TeCA degradation pathways in all live microcosm experiments. The production and degradation of vinyl chloride (VC), which is the most toxic of the TeCA daughter compounds, was affected by spatial and seasonal variability, reduction-oxidation condition, and pre-exposure of the wetland sediment. TeCA-amended microcosms constructed with WB30 sediment showed approximately twice as much VC production as those constructed with WB23 sediment. Results of 112TCA-amended microcosms indicated that the greater production of VC in the WB30 sediment resulted from a greater predominance of the 112TCA dichloro-elimination pathway in these sediments. VC degradation also was substantially higher in microcosms constructed with WB30 sediment than those constructed with WB23 sediment, resulting in lower VC concentrations at the end of WB30 microcosms. Enrichment experiments in which microcosm slurry was amended with high initial VC concentrations showed that the spatial difference in VC degradation was negligible after prolonged incubation under methanogenic conditions. Inhibition of methanogenic activity in microcosms by addition of sulfate or of 2-bromoethanesulfonic acid inhibited production and degradation of VC. Inhibition of methanogenesis by addition of ferric iron or of 2-bromoethanesulfonic acid also completely inhibited VC degradation in VC-amended enrichment experiments. Pre-exposure to VC substantially increased degradation in VC-amended enrichment experiments.

A microbial consortium, rather than one microbial species or group, likely is involved in the degradation of TeCA, as indicated by the occurrence of multiple degradation pathways and the variability in VC production and degradation. A bacterial peak at 90 base pair (bp) fragment length in terminal-restriction fragment length polymorphism (TRFLP) profiles was associated with TeCA hydrogenolysis to 112TCA, and bacterial species represented by 198 and 170 bp fragment lengths were associated with TeCA dichloroelimination to 12DCE. Dichloroelimination of 112TCA to VC was associated with increasing dominance of the 198 bp bacterial peak in March-April 1999 and October-November 2000 microcosms, whereas an 86 bp or the 170 bp bacterial peak was associated with 112TCA dichloroelimination in the summer experiment. Hydrogenolysis of 12DCE to VC was associated with a carbon dioxide-utilizing methanogen at 307 bp in the March-April 1999 and October-November 2000 microcosm experiments, whereas production of VC occurred despite low methanogen biomass and methane production in the July-August 1999 experiments. Production of VC in the absence of methane production also occurred in 12DCE-amended enrichment cultures. The exponential production of VC in the 12DCE-amended enrichment cultures after an initial lag indicated growth of a microbial species or group, possibly one of the known dehalorespiring bacteria. Molecular analyses using specific primers targeting dehalorespiring bacteria of the Dehalococcoides group (Dehalococcoides ethenogenes and Dehalococcoides sp. strain FL2) and of the acetate-oxidizing Desulfuromonas group (Desulfuromonas sp. strain BB1 and Desulfuromonas chloroethenica) showed the presence of these bacteria in microcosm slurry from site WB30 but not from site WB23. Addition of hydrogen, which is the favored substrate of Dehalococcoides, tripled VC production in 12DCE-amended enrichment cultures. VC degradation showed a marked association with an increase in the relative proportion of Methanosarcinaceae, a family of methanogens that includes all those capable of utilizing acetate as a substrate, in the total methanogen community.

Half-lives for TeCA and TCE estimated from field data were in the range of 60 to 100 days, which agrees well with laboratory estimates of degradation rates considering the inherent differences in the laboratory and field systems. Both laboratory microcosm experiments and field data showed that 12DCE and VC are the predominant, persistent daughter compounds from TeCA degradation. In addition, porewater chemistry showed higher accumulation of VC in the wetland sediment at site WB30 than at site WB23, as was observed in the microcosm experiments. Molecular analyses of grab samples of surficial wetland sediment showed that all the microbial species or groups linked to TeCA degradation in the microcosm experiments were present in all sediment samples. Microbial biomass and diversity were lowest in an area of the wetland (transect C-C ¢) where porewater VOC concentrations are highest, indicating that the higher VOC concentrations could result from lower degradation rates. The lower microbial biomass and diversity in this area could be caused by toxic effects of the contaminants, or possibly from differences in frequency and duration of tidal inundation.


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