Environ. Sci. Technol. 2006, 40, 3623-3633
Biological Enhancement of Tetrachloroethene Dissolution and Associated Microbial Community Changes BRENT E. SLEEP,‡ DAVID J. SEEPERSAD,† KAIGUO MO,† CHRISTINA M. HEIDORN,† LEILA HRAPOVIC,‡ PENNY L. MORRILL,§ MICHAYE L. MCMASTER,| ERIC D. HOOD,| CARMEN LEBRON,⊥ BARBARA SHERWOOD LOLLAR,§ DAVID W. MAJOR,| AND E L I Z A B E T H A . E D W A R D S * ,† Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada, M5S 3E5, Department of Civil Engineering, University of Toronto, Toronto, Ontario, Canada, M5S 1A4, Department of Geology, University of Toronto, Toronto, Ontario, Canada, M5S 3B1, GeoSyntec Consultants, Guelph, Ontario, Canada, N1G 5G3, and Naval Facilities Engineering Service Center, Port Hueneme, California, 93043-4370
A bench-scale study was performed to evaluate the enhancement of tetrachloroethene (PCE) dissolution from a dense nonaqueous phase liquid (DNAPL) source zone due to reductive dechlorination. The study was conducted in a pair of two-dimensional bench-scale aquifer systems using soil and groundwater from Dover Air Force Base, DE. After establishment of PCE source zones in each aquifer system, one was biostimulated (addition of electron donor) while the other was biostimulated and then bioaugmented with the KB1 dechlorinating culture. Biostimulation resulted in the growth of iron-reducing bacteria (Geobacter) in both systems as a result of the high iron content of the Dover soil. After prolonged electron donor addition methanogenesis dominated, but no dechlorination was observed. Following bioaugmentation of one system, dechlorination to ethene was achieved, coincident with growth of introduced Dehalococcoides and other microbes in the vicinity and downgradient of the PCE DNAPL (detected using DGGE and qPCR). Dechlorination was not detected in the nonbioaugmented system over the course of the study, indicating that the native microbial community, although containing a member of the Dehalococcoides group, was not able to dechlorinate PCE. Over 890 days, 65% of the initial emplaced PCE was removed in the bioaugmented, dechlorinating system, in comparison to 39% removal by dissolution from the nondechlorinating system. The maximum total ethenes concentration (3 mM) in the bioaugmented system occurred approximately 100 days after bioaugmentation, indicating * Corresponding author phone: (416) 946-3506; fax: (416) 9788605; e-mail:
[email protected]. † Department of Chemical Engineering and Applied Chemistry, University of Toronto. ‡ Department of Civil Engineering, University of Toronto. § Department of Geology, University of Toronto. | GeoSyntec Consultants. ⊥ Naval Facilities Engineering Service Center. 10.1021/es051493g CCC: $33.50 Published on Web 04/19/2006
2006 American Chemical Society
that there was at least a 3-fold enhancement of PCE dissolution at this time. Removal rates decreased substantially beyond this time, particularly during the last 200 days of the study, when the maximum concentrations of total ethenes were only about 0.5 mM. However, PCE removal rates in the dechlorinating system remained more than twice the removal rates of the nondechlorinating system. The reductions in removal rates over time are attributed to both a shrinking DNAPL source area, and reduced flow through the DNAPL source area due to bioclogging and pore blockage from methane gas generation.
Introduction Chlorinated ethenes such as tetrachloroethene (PCE) are very common groundwater contaminants often present as dense nonaqueous phase liquids (DNAPLs) that form long-term source zones and present formidable challenges for remediation. Since Freedman and Gossett (1) first demonstrated that PCE could be biotransformed under anaerobic conditions through reductive dehalogenation to nontoxic ethene, there has been growing interest in using in situ bioremediation to treat subsurface contamination from chlorinated ethenes. Many microbial cultures have now been described that can metabolically reduce PCE and trichloroethene (TCE) to cis-1,2-dichloroethene (cDCE) (2-6). However, organisms in the Dehalococcoides phylogenetic group are the only known organisms capable of complete dechlorination of cDCE and VC to ethene (7-14). All mixed cultures and most field sites shown to dechlorinate PCE and TCE completely to ethene contain Dehalococcoides (8-10, 15). It therefore is important and informative to track and quantify Dehalococcoides and associated microbial communities at contaminated sites. The potential of intrinsic or enhanced reductive dechlorination for control of dissolved PCE plumes emanating from PCE DNAPL source zones is now fairly well accepted as a result of a large number of lab- and field-scale studies. In contrast, the potential of using reductive dechlorination for treatment of source zones is much more uncertain (16). Initially it was thought that toxicity associated with saturation levels of PCE typical of DNAPL zones would be problematic (17). However, a number of studies (18-20) demonstrated that dechlorination of aqueous phase PCE at concentrations near or at saturation levels was possible. This raised the possibility that dechlorination could occur in source zones in close proximity to DNAPL-water interfaces, leading to increased pore-scale PCE concentration gradients and enhanced dissolution of PCE (21). The biological enhancement of PCE dissolution has been studied under a variety of conditions (see Table S1 in the Supporting Information). Carr et al. (22) found that reductive dechlorination of PCE to cDCE produced a factor of 14 increase in PCE removal compared to dissolution alone for a NAPL consisting of PCE (0.13 mole fraction) and tridecane in a continuous flow stirred tank reactor. Cope and Hughes (23) observed a biological enhancement of 5-6.5 for removal of PCE from a PCE (0.13 mole fraction) and tridecane DNAPL emplaced in glass bead columns, with vinyl chloride (VC) as the terminal dechlorination product. Adamson et al. (24), investigating bioaugmentation in a three-dimensional pilotscale tank with several small PCE DNAPL source zones, were not able to establish that biological activity enhanced PCE dissolution rates, as they primarily monitored effluent concentrations; cDCE was the predominant ethene compound produced after 225 days. VOL. 40, NO. 11, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Schematic diagram of experimental setup showing column and row labels used to identify sampling ports (b), and approximate location of sintered stainless steel diffuser used during PCE DNAPL injection and injection ports used for inoculation of Box 2 with KB1 culture. Yang and McCarty (20) observed a 5-fold increase in PCE removal with reductive dechlorination (primarily to cDCE) compared to dissolution alone in a column containing PCE DNAPL. In a follow-up study, they investigated the effects of electron donor type and similarly observed that reductive dechlorination of PCE DNAPL in columns led to an enhancement in PCE removal by a factor of 2.1 to 3.0, compared to dissolution alone, and also observed VC and ethene production, again with cDCE as the major product (25). Columns supplemented with oleate and pentanol exhibited high levels of methanogenesis, raising concerns about the efficiency of electron donor usage for dechlorination, and about clogging due to biomass growth and gas generation (25). The model simulations of Chu et al. (26) indicated that biological growth could substantially alter groundwater flow profiles due to bioclogging, often leading to reduced flow through the source zone and difficulties in electron donor delivery to the DNAPL. In all studies of PCE source remediation to date, the composition and distribution of the microbial community have not been characterized or quantified in the vicinity of the DNAPL zone over the duration of bioremediation. In this investigation, we evaluated the potential for reductive dechlorination to enhance the dissolution of PCE from a DNAPL source zone through long-term (890 days) multidimensional bench experiments. PCE DNAPL source zones were established in two rectangular boxes after they were packed with site soil and groundwater from Dover Air Force Base (AFB). Both boxes were biostimulated with electron donors, and one box was bioaugmented with the KB1 dechlorinating culture to evaluate the potential for bioaugmentation to accelerate reductive dechlorination and PCE removal (dissolution, biological transformation, and flushing from boxes). We evaluated the biological enhancement of PCE dissolution, inhibition due to the presence of PCE DNAPL, potential difficulties of electron donor delivery, and the impact of methanogenesis. Additional compound-specific stable carbon isotope analyses are reported separately (27). This is the first comprehensive study of the spatial and temporal trends in microbial composition and chlorinated ethene transformation during biological enhancement of dissolution from a PCE DNAPL source zone in a realistic porous medium system.
Materials and Methods Bench-Scale System Setup. Two stainless steel boxes (inner dimensions of 76 cm × 38 cm × 2.54 cm) approximating a two-dimensional system were constructed using 0.32 cm thick 316 stainless steel (Figure 1). Each box had a 2.54 cm long clear well at each end separated from the soil compartment by a perforated stainless steel wall lined with stainless 3624
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steel mesh, and was equipped with 25 liquid sampling ports on the front (1/8 in. M-NPT Mininert valves) and 36 soil sampling ports on the back (sealed with 3/8 in. brass plugs), offset from the liquid sampling ports. A PCE injection port was installed on the back of each box and consisted of a PTFE valve connected by 1/8 in. PTFE tubing to a 10 cm long piece of sintered stainless steel tubing (i.d.. 9.5 mm, 5 micron pore size, internal volume of 7 mL) on the inside of each box (Figure 1). The total volume of each box (excluding clear wells) was 7.3 L and soil pore volume was 2.0 L. Both boxes were packed with soil from Dover AFB (field properties given in Table S2 in the Supporting Information), which was first mixed in an anaerobic chamber to improve soil homogeneity and to remove large pebbles. Soil was placed into the boxes in 2-3 cm lifts while maintaining a freeboard of groundwater and a continuous stream of argon gas. A 1 cm layer of dry bentonite was added to the top of each box before sealing to prevent channeling along the box tops. The boxes were sealed with an iron plate isolated and sealed from the box by a Viton rubber gasket. Soil and groundwater VOCs were below detection. Molecular analyses were also conducted to determine the initial Dehalococcoides concentrations (see Methods in the Supporting Information). Site groundwater was pumped from the feed reservoirs (2 × 20 L glass bottles) through each box at a rate of 0.21 mL/min (6.5 day residence time) using a peristaltic pump. The headspace of each feed reservoir was continually purged with argon to maintain anaerobic conditions. Site groundwater was collected from two locations at Dover AFB, with a switch made between locations at Day 130. There were significant differences in the concentrations of sulfate and chloride between the two locations (Table S2, Supporting Information). Groundwater, collected in 20 L plastic carboys and shipped to the University of Toronto, was kept frozen until use. System Startup and DNAPL Injection. Following packing and sealing of the boxes, groundwater was flushed through the boxes for 100 days to ensure complete water saturation. Subsequently, 10 mL of neat PCE was injected into each box through the PCE injection port using a Hamilton gastight syringe and a syringe pump at a rate of 2 mL/hour. The injected PCE was followed by injection of water to flush PCE from the sintered steel tubing. After PCE injection, groundwater flow (0.21 mL/min) without electron donor was maintained through each box for 112 days. Over the course of the study liquid samples (0.2-1 mL, depending on concentration) were collected from liquid sampling ports and effluent lines using 0.5 or 1 mL glass gastight syringes for analysis (see Methods in the Supporting Information) for chlorinated ethenes, ethene, methane, methanol, ethanol, and major anions (acetate, chloride, nitrite, bromide, nitrate,
TABLE 1. Details of Timeline and Treatments days 0b-112 113-164 165-212 213-275 276-380 381-450 451-480 481-500 501-580 581-821 822-880 881-890
donor type
delivery method
location
ratio of Box 2:Box 1 PCE removal ratesa
PHASE I: Groundwater Flushing none
n/a
n/a
0.43
MeOH acetate EtOH
PHASE II: Biostimulation of both systems (Days 113-275) continuous injection at a target concentration of 180 mg/L weekly injection of 0.9 mL of 90 mM sodium acetate per port weekly injection of 1.8 mL of 3% EtOH solution per port
inlet line ports A1-A5 ports A1- A5
1.0 1.2 1.2
PHASE III: Bioaugmentation of Box 2, continued biostimulation of both systems (Days 276-890) EtOH weekly injection of 55 µL neat EtOH per port ports A1-A5 EtOH weekly injection of 55 µL neat EtOH per port ports A1-A5 EtOH weekly injection of 69 µL neat EtOH per port ports A2-A5 1:1 MeOH/EtOH weekly injection of 83 µL of neat MeOH and EtOH ports A2-A5 mixturec per port 1:1 MeOH/EtOH weekly injection of 110 µL of neat MeOH and EtOH ports A3-A5 mixturec per port 1:1 MeOH/EtOH weekly injection of 110 µL of neat MeOH and EtOH ports A3-A5 mixturec per port 1:1 MeOH/EtOH weekly injection of 110 µL of neat MeOH and EtOH ports B3-B5 mixturec per port 1:1 MeOH/EtOH weekly injection of 110 µL of neat MeOH and EtOH ports B3-B5 mixturec per port
1.9 2.5 2.5 2.5 2.5 3.8 2.3 10.7
a Removal rate calculated from outlet concentrations and groundwater flow rate, includes estimate of ethenes removed during sampling (
