Environ. Sci. Technol. 1997, 31, 3138-3147
Metabolic Adaptation and in Situ Attenuation of Chlorinated Ethenes by Naturally Occurring Microorganisms in a Fractured Dolomite Aquifer near Niagara Falls, New York RICHARD M. YAGER,† SHARON E. BILOTTA,‡ CYNTHIA L. MANN,‡ AND E U G E N E L . M A D S E N * ,‡ U.S. Geological Survey, Ithaca, New York 14850, and Section of Microbiology, Division of Biological Sciences, Cornell University, Ithaca, New York 14853
A combination of hydrogeological, geochemical, and microbiological methods was used to document the biotransformation of trichloroethene (TCE) to ethene, a completely dechlorinated and environmentally benign compound, by naturally occurring microorganisms within a fractured dolomite aquifer. Analyses of groundwater samples showed that three microbially produced TCE breakdown products (cis1,2-dichloroethene, vinyl chloride, and ethene) were present in the contaminant plume. Hydrogen (H2) concentrations in groundwater indicated that iron reduction was the predominant terminal electron-accepting process in the most contaminated geologic zone of the site. Laboratory microcosms prepared with groundwater demonstrated complete sequential dechlorination of TCE to ethene. Microcosm assays also revealed that reductive dechlorination activity was present in waters from the center but not from the periphery of the contaminant plume. This dechlorination activity indicated that naturally occurring microorganisms have adapted to utilize chlorinated ethenes and suggested that dehalorespiring rather than cometabolic, microbial processes were the cause of the dechlorination. The addition of pulverized dolomite to microcosms enhanced the rate of reductive dechlorination, suggesting that hydrocarbons in the dolomite aquifer may serve as electron donors to drive microbially mediated reductive dechlorination reactions. Biodegradation of the chlorinated ethenes appears to contribute significantly to decontamination of the site.
Introduction Knowledge of microbially mediated anaerobic transformations of chlorinated ethenes has advanced dramatically during the last decade (1-3). A variety of laboratory studies using mixed microbial cultures have demonstrated sequential reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE) to lesser chlorinated ethenes such as cis-1,2-dichloroethene (DCE), vinyl chloride (VC), and ethene * Corresponding author phone: 607-255-3086; fax: 607-255-3904; e-mail: elm
[email protected]. † U.S. Geological Survey. ‡ Cornell University.
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under methanogenic (e.g., refs 4 and 5) and sulfate-reducing (e.g., ref 6) conditions. More recently, both pure cultures of individual bacteria (7-11) and bacterial transition metal coenzymes (12) have been shown to catalyze the replacement of chlorines on the ethene molecule by hydrogen atoms. The physiological principles that emerge from such studies suggest that the widely distributed chlorinated contaminants PCE and TCE may be transformed anaerobically through (i) cometabolic reductive dechlorination in anaerobic bacteria, especially methanogens, that fortuitously produce reactive transition metal coenzymes or (ii) dehalorespiration by anaerobic bacteria that carry out growth-coupled metabolism by utilizing chlorinated ethenes as physiological terminal electron acceptors and hydrogen as the electron donor (3, 10, 13, 14). The evolutionary origin of dehalorespiration is unknown because chlorinated ethenes have no known naturally occurring analogs. In addition, dehalorespiration is considered a far more effective decontamination mechanism than cometabolism (2, 13, 15). The above-mentioned laboratory experiments provide a foundation for predicting and interpreting the fate of chlorinated ethenes in field sites. Field observations in contaminated sites have revealed the presence of the reductive dehalogenation daughter products, DCE and VC (16-19). These compounds, not present in the original contaminant spill, provide evidence for in situ microbial dechlorination activity. The fate of VC in field sites is of particular concern because the compound is a human carcinogen. Several field studies have documented the transformation of VC to ethene, an environmentally benign compound, under anaerobic conditions in groundwater contaminated by chlorinated ethenes. Major et al. (18) concluded that PCE was undergoing complete dechlorination in a sand and silt aquifer based on field detection of ethene and the limited spatial extent of its precursor, VC. This conclusion was supported by microcosm studies in which PCE was consumed and ethene produced (18). Beeman et al. (16) implemented a pilot-scale treatment system in a sand aquifer that resulted in the conversion of PCE to ethene under sulfate-reducing conditions. Semprini et al. (19) observed a correlation between high concentrations of ethene (up to 7 mg/L) and methane (up to 12 mg/L) in a sand aquifer contaminated with TCE and concluded that VC was reduced in situ under methanogenic conditions. Ethene has also been observed in fractured carbonate aquifers contaminated with TCE and PCE (17, 20). The fate of VC and its persistence in groundwater is determined by the geochemical setting. Anaerobic conditions, such as those at the site discussed here, provide only two known means of microbial degradation of VC: (i) reductive dechlorination to ethene and (ii) oxidation to carbon dioxide by iron-reducing microorganisms (21). Identification of the terminal electron-accepting processes (TEAPs) that dominate in contaminated environments is important because chlorinated ethenes may compete as electron acceptors with naturally occurring chemical species in microbially mediated redox processes. Chapelle et al. (22) have discussed the identification of TEAPs through measurements of electron acceptors (e.g., ferric iron, sulfate, and bicarbonate); final redox products (e.g., ferrous iron, sulfide, and methane); and hydrogen, an intermediary metabolite of anaerobic microbial processes. The purpose of this study was to examine the biogeochemical reactions influencing TCE, DCE, and VC in a fractured dolomite aquifer. This paper describes (i) the hydrogeology of a contaminated study site near Niagara Falls, NY; (ii) sampling and analytical methods used in the study; (iii) the distribution of the chlorinated ethenes at the site; (iv)
S0013-936X(97)00105-3 CCC: $14.00
1997 American Chemical Society
FIGURE 1. Well monitoring network and the distribution of aqueous phase liquid (APL) contaminants at a manufacturing facility near Niagara Falls, NY. the geochemistry of groundwater at the site; and (v) the results of laboratory microcosm studies conducted to establish whether microorganisms in groundwater from the site were adapted to completely dechlorinate the contaminants. Site Hydrogeology. The study area is adjacent to a manufacturing facility 8 km east of Niagara Falls in western New York (Figure 1). Contaminants from the facility have entered the upper part of the Lockport Group, a 52 m thick dolomite of the Niagaran Series (Middle Silurian) that strikes east-west and dips gently to the south at about 4.6 m/km (23). The Lockport Group is a petroliferous dolomite that contains both gypsum and metal sulfides (24). The Guelph Formation, the uppermost part of the Lockport Group beneath the facility, is a fine-grained, thin-bedded dolomite with black, bituminous bedding partings associated with gypsum coatings (25). The underlying Eramosa Formation is a saccharoidal textured, thinly to thickly-bedded bituminous dolomite that emits a petroliferous odor. The hydraulic properties of the Lockport Group are related primarily to secondary permeability (fractures and and vugs). The principal water-bearing zones in the Lockport Group are
the weathered bedrock surface and horizontal-fracture zones near stratigraphic contacts (26). These horizontal-fracture zones are connected by high-angle fractures and by subcrop areas where the fracture zones intersect the bedrock surface. Groundwater flows southward and westward through the Lockport Group from recharge areas near the Niagara Escarpment, 8 km north of the facility, toward discharge areas in low-lying areas near the Niagara River 5 km to the south (27). The manufacturing facility that was the source of TCE overlies 5-12 m of till and lacustrine silt and clay (28). These unconsolidated deposits are underlain by the upper part of the Lockport Group that has been classified into four zones by previous investigators (Figure 2). Zone 1, which corresponds to the Guelph Formation, is 3-6 m thick and comprises the weathered bedrock layer that contains many open fractures, some of which have been widened by dissolution of dolomite and gypsum (28). Zone 1 also contains a water-bearing, horizontal-fracture zone near its lower contact with zone 2, the upper part of the Eramosa Formation. Zone 2 is a massive, 2.4 m thick dolomite that appears
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FIGURE 2. Generalized geologic section through chlorinated ethene plume. The A-A′ cross section is shown in Figure 1. relatively unfractured, but the presence of chlorinated ethenes in a limited area within the underlying zone 3 indicates that some high-angle fractures probably penetrate zone 2 and form pathways for downward migration from zone 1. The TCE remains primarily within zone 1, the primary focus of this paper (Figure 2). The zone 1 water levels in August 1990 indicated that groundwater flowed southward and westward in the vicinity of the manufacturing facility under the natural hydraulic gradient (Figure 3A). The transmissivity of zone 1 was estimated to be about 6 m2/d from a cross-hole pumping test conducted by Golder Associates (28). The pumping test showed hydraulic connection throughout zone 1, and no vertical connection was apparent between zones and 1 and 3. Groundwater velocity was computed to range from 0.2 to 0.9 m/d, from an assumed effective porosity of 3%. A system of five offsite recovery wells began pumping groundwater from zone 1 in 1993 at a combined rate of 265 m3/d to limit the migration of the aqueous phase contaminants. The gradient induced by pumping has altered the natural direction of groundwater flow south of the facility (Figure 3B) and induced an upward vertical gradient from zone 3 to zone 1 (29).
Methods Sampling. Groundwater samples were collected from nine monitoring wells in January 1995 after the removal of three well volumes. Samples analyzed for ethene were collected with dedicated stainless steel bailers, while all other samples were collected with a peristaltic pump and dedicated highdensity polyethylene tubing. Duplicate samples for analysis of ethene and methane were collected in 40-mL Ichem vials (New Castle, DE) prefilled
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with 3 mL of acidic mercuric chloride fixative (0.25 M HgCl2 in 5% HCl). Approximately 25 mL of well water was added to each vial; the precise volume added was determined later by weight in the laboratory. The vials were tightly capped and sealed with Teflon-lined septa, inverted, and kept on ice or stored at 4 °C until analyzed. Samples analyzed for hydrogen were collected with a gasstripping procedure described by Chapelle et al. (22), in which a glass gas-sampling bulb is used to create a bubble of gases that equilibrate with the stream of water produced from the well. The gas sample is then obtained through a septum with a needle for analysis by gas chromatography (GC). Groundwater used in laboratory microcosms was collected by filling precleaned, sterile 160-mL serum bottles to the brim. A new sterile Teflon-lined butyl rubber septum was then attached with an aluminum crimp seal in a manner that prevented air entrapment. The bottles were then placed on ice or stored at 4 °C until use in microcosm preparation. Samples of rock core for the microcosm studies were collected from zone 1 during drilling before the installation of monitoring well 94-02(1) in October 1994 (see Figure 1). The borehole was cored using (8.8 cm, NW) wireline coring with air and a diamond bit. The rock core was submerged in water pumped from the borehole and kept on ice or stored at 4 °C until being dried, pulverized, and autoclaved for use in microcosm preparation. Laboratory Microcosms. Microcosm experiments were prepared using established procedures (10, 18, 30). Sterile serum bottles (120 or 36 mL) were transferred at least 24 h before microcosm preparation to an anaerobic hood containing an atmosphere with a nitrogen to hydrogen ratio of 95:5 and a trace of CO2. Groundwater was transferred to the serum bottles and mixed (1:1) with a sterile anaerobic nutrient
FIGURE 3. Hydraulic head distribution in zone 1 in the vicinity of the manufacturing facility: (A) August 1990 and (B) January 1995. solution of mineral salts (NaHCO3 to 0.1%, NH4Cl [0.5 g/L], K2HPO4 [0.4 g/L], MgCl2 [0.1 g/L], CaCl2 [0.05 g/L], and rezasurin [1 mg/L]) (30). Each of the 120-mL bottles received 52 mL of the water/nutrient mixture; the 36-mL bottles each received 28 mL of the water/nutrient mixture. Yeast extract (to 200 ppm) was then added to each bottle. Before the bottles were sealed, amendments were anaerobically prepared and added to promote five distinctive TEAPs: 2 mM Na2S (reducing agent) for methanogenesis, 2 mM Na2S and 200 mM Na2SO4 for sulfate reduction, 4.6 mg/mL sterile iron(III) oxide (31) for iron reduction, 1 mg/mL sterile manganese(IV) oxide (31) for manganese reduction, and 200 mM NaNO3 for nitrate reduction. To assess the influence of potential electron donors in the aquifer solids, 60 mg/mL sterile pulverized dolomite was added to the methanogenic treatment. Each bottle was sealed with a Teflon-faced butyl rubber stopper after all additions had been made. The bottles were then amended with TCE (approximately 10 µmol/bottle or ≈0.2 mM; equivalent to the maximum site concentration) with a glass syringe. Microcosm treatments were prepared either as single bottles or in triplicate. A singlebottle abiotic control was also prepared for each treatment by adding 2.0 mL of an acidic (5% HCl) mercuric chloride solution (0.2 M) to bottles prepared identically to the live treatments. The bottles were incubated in the dark at room temperature (22 ( 2 °C) in an inverted position on a rotary shaker operating at 50 rpm. Samples of headspace gases were obtained with a 250-µL sampling syringe (fitted with a gas-tight mininert valve) flushed with O2-free nitrogen gas and analyzed as described below. Analytical Techniques. Stable inorganic constituents in groundwater samples were measured by the U.S. Geological Survey National Water Quality Laboratory using standard water-quality techniques (32). Unfiltered samples were analyzed in the field for dissolved oxygen using a colorimetric
procedure and for ferrous iron and sulfide using spectroscopic procedures (33). Concentrations of gaseous hydrogen were measured using a GC equipped with a reduction gas detector. Concentrations of aqueous hydrogen were then calculated from hydrogen solubility data as described by Chapelle et al. (22). Field measurements of chlorinated ethenes were conducted by a commercial laboratory using the U.S. Environmental Protection Agency’s Method 8260 for volatile organic compounds (29). Ethene and methane in headspaces of mercury-fixed water samples and microcosms were analyzed using a GC (Perkin Elmer, Model 8500) equipped with a flame ionization detector and a stainless steel (1.8 m × 0.3 cm o.d.) column containing 80/100 Chromosorb 102 packing. The oven temperature was held constant at 50 °C, and the carrier gas (helium) flowed at 16 mL/min. The retention times of ethene and methane were 4.7 and 1.4 min, respectively. The presence of ethene in the headspace of groundwater samples was confirmed by injecting 50 µL into a GC (HewlettPackard, Model 5890) equipped with a PoraPLOT-Q (H-P) capillary column connected to quadrupole mass selective detector (H-P, Model 5971A) operated at an electron energy of 70 eV and a detector voltage of 2000 V. A splitless injection was used with a 1-min delay before septum purge and helium as the carrier gas at a linear velocity of 46 cm/s. The injector and detector temperatures were 120 and 300 °C, respectively, and the oven temperature was isothermal at 70 °C. The ion source pressure was maintained at 1.0 × 10-5 Torr. Ethene in groundwater samples and in prepared standards eluted at 3.0 min. The mass spectrometer was operated in single ion monitoring mode for all 10 key ions to allow spectral comparison between analytes in field samples and ethene standards.
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TABLE 1. Water Quality Constituents in Groundwater Sampled from Monitoring Wells near Manufacturing Facility monitoring wellsa
water constituent pHb
87-02(1) -c
calcium magnesium sodium potassium alkalinity, as calcium carbonateb chloride sulfate dissolved solids dissolved oxygenb dissolved organic carbon ferrous iron sulfideb hydrogen (nmol/L)b methaned ethened trichloroethenee cis-1,2-dichloroethenee vinyl chlorided
140 710
