Microbial Consortia Development and Microcosm and Column Experiments for Enhanced Bioremediation of Chlorinated Volatile Organic Compounds, West Branch Canal Creek Wetland Area, Aberdeen Proving Ground, Maryland

By Michelle M. Lorah¹, Emily H. Majcher², Elizabeth J. Jones³, and Mary A. Voytek³

¹ U.S. Geological Survey, Baltimore, MD
² Formerly of U.S. Geological Survey, Baltimore, MD
³ U.S. Geological Survey, Reston, VA

Prepared in cooperation with
U.S. Army Garrison, Aberdeen Proving Ground
Environmental Conservation and Restoration Division
Aberdeen Proving Ground, Maryland

Abstract

Chlorinated solvents, including 1,1,2,2-tetrachloroethane, tetrachloroethene, trichloroethene, carbon tetrachloride, and chloroform, are reaching land surface in localized areas of focused ground-water discharge (seeps) in a wetland and tidal creek in the West Branch Canal Creek area, Aberdeen Proving Ground, Maryland. In cooperation with the U.S. Army Garrison, Aberdeen Proving Ground, Maryland, the U.S. Geological Survey is developing enhanced bioremediation methods that simulate the natural anaerobic degradation that occurs without intervention in non-seep areas of the wetland. A combination of natural attenuation and enhanced bioremediation could provide a remedy for the discharging ground-water plumes that would minimize disturbance to the sensitive wetland ecosystem. Biostimulation (addition of organic substrate or nutrients) and bioaugmentation (addition of microbial consortium), applied either by direct injection at depth in the wetland sediments or by construction of a permeable reactive mat at the seep surface, were tested as possible methods to enhance anaerobic degradation in the seep areas. For the first phase of developing enhanced bioremediation methods for the contaminant mixtures in the seeps, laboratory studies were conducted to develop a microbial consortium to degrade 1,1,2,2-tetrachloroethane and its chlorinated daughter products under anaerobic conditions, and to test biostimulation and bioaugmentation of wetland sediment and reactive mat matrices in microcosms. The individual components required for the direct injection and reactive mat methods were then combined in column experiments to test them under groundwater- flow rates and contaminant concentrations observed in the field. Results showed that both direct injection and the reactive mat are promising remediation methods, although the success of direct injection likely would depend on adequately distributing and maintaining organic substrate throughout the wetland sediment in the seep area.

For bioaugmentation, two mixed anaerobic cultures, named the "West Branch Consortia" (WBC-1 and WBC-2), were developed by enrichment of wetland sediment collected from two contaminated sites in the study area where rapid and complete reductive dechlorination naturally occurs. WBC are capable of degrading 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane, 1,2-dichloroethane, tetrachloroethene, trichloroethene, cis- and trans-1,2-dichloroethene, and vinyl chloride to the non-chlorinated end-products ethene and ethane. In addition, the column experiments showed that the consortia could completely degrade carbon tetrachloride and chloroform, although they were not grown on these contaminants. No other cultures are known that can degrade the broad mixture of chlorinated alkanes, alkenes, and methanes as shown for WBC. WBC-2 (suspended in the culture media) is capable of complete dechlorination of 50 micromolar 1,1,2,2-tetrachloroethane to ethene in 1 to 2 days with little transient accumulation of chlorinated daughter products. Only about 5 percent of the clones sequenced from WBC-1 and WBC-2 were related to dechlorinating bacteria that have been studied previously in culture, indicating the presence of unknown dechlorinators. Dehalococcoides spp. comprised about 1 percent of WBC-1 and WBC-2, which is minor compared to the population size of about 30 percent in other dechlorinating consortia for chlorinated alkenes. Although both WBC-1 and WBC-2 showed efficient degradation in laboratory tests in this study, long-term cultivation of WBC-1, which was developed using hydrogen as the organic substrate, was determined to be infeasible. Thus, WBC-2, cultivated with lactate as the organic substrate, would be used in future tests.

Nutrient (ammonia and phosphate mixture) addition to anaerobic microcosms constructed with wetland sediment and ground water collected from the study area showed some enhancement in the degradation rate of 1,1,2,2-tetrachloroethane, but degradation of 1,1,2,2-tetrachloroethane's anaerobic daughter compounds (1,1,2-trichloroethane, 1,2-dichloroethane, trichloroethene, 1,2-dichloroethene, and vinyl chloride) was not enhanced. Similarly, nutrient amendment did not enhance degradation of 1,1,2,2-tetrachloroethane or its anaerobic daughter products in microcosms constructed with mixtures of BionSoil (a dairy-derived compost) and commercial peat that were evaluated as potential reactive mat materials. Biostimulation by addition of organic donor (lactate) also did not enhance 1,1,2,2-tetrachloroethane degradation in microcosms with wetland sediment and BionSoil/peat mixtures compared to unamended microcosms. In contrast, microcosms constructed with wetland sediment or the compost/peat mixture and augmented with the developed anaerobic mixed culture in media showed 1,1,2,2-tetrachloroethane biodegradation rates that were two to three times higher than those in controls amended with culture media alone. In addition, bioaugmentation substantially enhanced degradation of the chlorinated daughter compounds, with typically a maximum of about 20 to 30 percent of the initial 1,1,2,2-tetrachloroethane added in the bioaugmented microcosms recovered as daughter products compared to 50 to 100 percent in the unamended microcosms during a 42-day incubation. Bioaugmentation with WBC-2 was equally effective in enhancing 1,1,2,2-tetrachloroethane degradation in the mixture of BionSoil compost and peat as observed in wetland sediment from the study area. Wetland sediment from the study area and bioaugmented BionSoil/peat also was able to rapidly degrade carbon tetrachloride and chloroform to non-chlorinated endproducts. Out of five additional composts tested in microcosms with WBC-2, only two others provided a suitable environment for the culture, indicating the need to test individual composts before use in a bioremediation effort.

Column tests were used to (1) combine biostimulation and bioaugmentation to simulate direct injection in seep sediment, and to combine the matrix zones conceptualized for the permeable reactive mat (zero-valent iron (ZVI) and organic layer with an overlying bioaugmented organic layer), (2) better simulate the bioremediation methods under field conditions, including continuous ground-water flow at measured rates and increased contaminant concentrations, and (3) evaluate possible adverse water-quality effects that could result from the proposed near-surface bioremediation methods in the wetland. The enriched, mixed microbial culture did not appear to stimulate enhanced biodegradation of site contaminants in the seep sediment columns without the adequate addition of continuous, soluble electron donor. With the addition of ethanol, methanogenic conditions were rapidly established and efficient degradation of 1,1,2,2-tetrachloroethane, as well as tetrachloroethene and trichloroethene, was observed in the columns. Degradation efficiency was highly sensitive to changes in the ethanol dosing and indicated that success of direct injection may be largely dependent on the ability to adequately distribute electron donor.

The bioaugmented microbial culture stimulated enhanced degradation of 1,1,2,2-tetrachloroethane in both matrices for the reactive mat (organic mix and zero-valent iron/organic mix) without lactate and then, with improved efficiency, with lactate addition. A zero-valent iron layer underlying the bioaugmented organic mix layer was tested primarily because of the high carbon tetrachloride and chloroform concentrations in some seeps— these compounds typically are toxic to many microorganisms but can be degraded abiotically in zero-valent iron. In both matrices, 1,1,2,2-tetrachloroethane and all associated daughter compounds were typically removed within 46 centimeters (cm) along the column. In the presence of chloroethenes and chloromethanes, rapid 1,1,2,2-tetrachloroethane degradation was maintained, and all daughter compounds were removed within the 76-centimeter column length in both sediment matrices. Thus, the microbial community established within each column matrix after bioaugmentation with WBC was able to facilitate complete degradation of the chloroethene (tetrachloroethene and trichloroethene) and chloromethane (carbon tetrachloride and chloroform) co-contaminants and associated daughter compounds without any substantial toxicity effects. The overall comparable degradation efficiencies in the mix and iron-organic mix columns indicate that zero-valent iron would not be a necessary component of an organic-based, bioaugmented reactive mat, even in seep areas with high chloromethane concentrations (up to 25 micromolar was tested). Neither organic-based matrix is likely to cause any adverse effects on water quality. The results of this laboratory study show the broad dechlorinating capabilities of the developed WBC and support the feasibility of applying the consortia in the field for enhanced bioremediation of groundwater discharge seep areas, either by direct injection at depth in the wetland sediment or by construction of a reactive, flowthrough mat on the seep surface.