Article pubs.acs.org/est
Biodegradation of cis-Dichloroethene and Vinyl Chloride in the Capillary Fringe Zohre Kurt,† E. Erin Mack,‡ and Jim C. Spain*,† †
School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0512, United States DuPont, Corporate Remediation Group, P.O. Box 6101, Glasgow 300, Newark, Delaware 19714-6101, United States
‡
S Supporting Information *
ABSTRACT: Volatile chlorinated compounds are major pollutants in groundwater, and they pose a risk of vapor intrusion into buildings. Vapor intrusion can be prevented by natural attenuation in the vadose zone if biodegradation mechanisms can be established. In this study, we tested the hypothesis that bacteria can use cis-dichloroethene (cis-DCE) or vinyl chloride (VC) as an electron donor in the vadose zone. Anoxic water containing cis-DCE or VC was pumped continuously beneath laboratory columns that represented the vadose zone. Columns were inoculated with Polaromonas sp. strain JS666, which grows aerobically on cis-DCE, or with Mycobacterium sp. JS60 and Nocardiodes sp. JS614 that grow on VC. Complete biodegradation with fluxes of 84 ± 15 μmol of cis-DCE·m−2·hr−1 and 218 ± 25 μmole VC·m−2·h−1 within the 23 cm column indicated that microbial activities can prevent the migration of cis-DCE and VC vapors. Oxygen and volatile compound profiles along with enumeration of bacterial populations indicated that most of the biodegradation took place in the first 10 cm above the saturated zone within the capillary fringe. The results revealed that cis-DCE and VC can be biodegraded readily at the oxic/anoxic interfaces in the vadose zone if appropriate microbes are present.
■
INTRODUCTION Chlorinated compounds are major pollutants in groundwater due to their wide use as solvents and in synthetic chemical production.1 Polluted groundwater may encounter an oxic/ anoxic interface at or near the water table. If there is no biodegradation, volatile chlorinated compounds in the subsurface can migrate through the vadose zone and impact a variety of receptors.2 Most reports of vapor intrusion focus on trichloroethene (TCE) and perchloroethene (PCE), which resist biodegradation under aerobic conditions in the vadose zone.3 Incomplete reductive dechlorination of TCE and PCE in groundwater leads to cis-DCE and VC accumulation.1 Dehalococcoides strains catalyze the anaerobic transformation of cis-DCE to ethene via VC.4−7 In the absence of Dehalococcoides sp. or in the absence of sufficient electron donors cis-DCE and VC can accumulate.8 However, cis-DCE and VC are biodegradable under oxic conditions and potentially subject to natural attenuation in the vadose zone. Field observations suggest the potential for cis-DCE biodegradation in oxic groundwater,8,9 but Polaromonas sp. JS666 is the only isolated bacterium reported to biodegrade cisDCE aerobically as a sole carbon source.10 The isolate has been studied extensively, its genome has been sequenced,11,12 and the degradation pathway has been established.13 The potential for bioaugmentation with Polaromonas sp. JS666 in contami© 2014 American Chemical Society
nated subsurface sediments and groundwater was studied by Giddings et al.14 Polaromonas sp. JS666 was capable of biodegrading cis-DCE even when municipal primary effluent was added as an additional carbon or organism source. Further site-specific studies suggested that Polaromonas sp. JS666 could be used to enhance biodegradation in groundwater.15 Aerobic biodegradation of VC, on the other hand, is widespread in contaminated sites.16 Several strains were isolated for their ability to degrade VC under oxic conditions, and the VC degradation pathway was established rigorously.17−19 Field studies have demonstrated rapid aerobic biodegradation of VC in groundwater suggesting that VC detoxification can be achieved as long as oxygen is present.9 Biodegradation of hydrocarbons in the vadose zone is well established, and several models describing the process are available.20−23 Biodegradation of chlorinated compounds in the vadose zone, however, has not been studied extensively.24 Biodegradation of chlorobenzenes in the vadose zone was established recently using laboratory columns packed with either sand or site materials.25 Similarly, VC biodegradation in the vadose zone was previously reported.26 Very recently, Received: Revised: Accepted: Published: 13350
June 25, 2014 October 14, 2014 October 20, 2014 October 20, 2014 dx.doi.org/10.1021/es503071m | Environ. Sci. Technol. 2014, 48, 13350−13357
Environmental Science & Technology
Article
CA) at a flow rate of 1 mL/min for the column experiments. Porosity of the sand was determined as described by Marion et al.41 Growth Conditions. Polaromonas sp. JS666 was routinely grown with cis-DCE (25 μmol/bottle) as the sole carbon and energy source in 165 mL serum bottles containing 50 mL of 1/ 4 strength minimal salt medium (MSB).42 Mycobacterium sp. JS60 and Nocardioides sp. JS614 were grown under the same conditions except that full strength MSB was used with VC vapor (170 μmol/bottle). cis-DCE and VC disappearance were monitored by GC analysis of the headspace and estimated oxygen stoichiometry was 2 for cis-DCE and 2.5 for VC. Onequarter strength trypticase soy agar (1/4-TSA) was used to check the purity of the cultures based on colony morphology. Microcosms. Microcosms were constructed in 35 mL serum bottles sealed with Teflon lined stoppers where cis-DCE was monitored via headspace sampling. The bottles contained 3.3 mL of 1/4-strength MSB with cis-DCE (120 μmol/bottle) and Polaromonas sp. JS666 at a final OD600 of 0.1. The microcosms intended to simulate the upper layer of the vadose zone received an additional 30 g of sand. The moisture content in the microcosms was based on a previous study describing naphthalene biodegradation in the vadose zone (0.11 g of water/g of soil).43 Controls were prepared without Polaromonas sp. JS666 or without MSB. The microcosms were incubated at room temperature with no stirring. The initial biodegradation rates were calculated using the slope of the concentrations between the second and fourth data points in Figure 1.
several lines of evidence were used to rigorously establish VC degradation in the vadose zone at a field site.27 To our knowledge, cis-DCE biodegradation in the vadose zone has not been studied. There is a growing awareness of the importance of redox discontinuities in biodegradation processes in the field25,28 because they provide steep gradients where electron donors and electron acceptors support high microbial activity.29−31 Such redox gradients play key roles in the fringes of contaminant plumes,32,33 at oxic/anoxic interfaces in sediments,28 and the interface of the water table with the overlying vadose zone.23,25,34 Nothing is known, however, about the spatial relationships among steep redox gradients, distribution of microbes and the resulting aerobic biodegradation of cisDCE and VC. The goal of the present study was to test the hypothesis that Polaromonas sp. JS666 can biodegrade cis-DCE vapor in unsaturated systems and to determine the location of the active zones for cis-DCE and VC degradation with respect to the capillary fringe and the oxic/anoxic interface above the saturated zone. Laboratory columns representing the vadose zone were inoculated with Polaromonas sp. JS666 or with Mycobacterium sp. JS6018 and Nocardioides sp. JS61435 and used to determine whether aerobic cis-DCE or VC biodegradation could be sufficient to prevent migration in the vadose zone. The results reveal a surprisingly active biodegradation zone within the capillary fringe.
■
MATERIALS AND METHODS Chemicals. cis-DCE (97%) and VC (>99.5%) were from Sigma-Aldrich, and cis-DCE epoxide was synthesized as described previously.36,37 All other chemicals were reagent grade. Analytical Methods. cis-DCE (97%) and VC in gas phase samples (100 μL) were analyzed on an HP6890 GC equipped with an electron capture detector (ECD) as described previously13 or with an Agilent 6890N GC equipped with a flame ionization detector (FID) and a Supelco 1% SP-1000 on 60/80 Carbopack B column using the method described by Coleman et al.10 with a detection limit of 0.1 μM. Oxygen was quantified with an Agilent Technologies 6850 Network GC equipped with a thermal conductivity detector (TCD) as described previously.38 All of the gas samples were analyzed in duplicate except the first port above the saturated layer of the multiport columns where there was only enough volume for single samples. Liquid concentrations of cis-DCE in the multiport columns were measured by high performance liquid chromatography (HPLC) on a Merck Chromolith RP18e column (4.6 × 100 mm) with a mobile phase that consisted of 98% part A (% 0.1 trifluoroacetic acid (TFA) in water) and 2% part B (% 0.05 TFA in acetonitrile) at a flow rate of 1 mL/min. The mobile phase was kept constant for 5 min and changed to 50% part A to 50% part B over a 1 min period while cis-DCE was monitored at 210 nm with a detection limit of 5 μM. Aqueous concentrations of VC were measured by injecting 5 μL samples on the GC. Protein was measured with a Pierce bicinchoninic acid protein assay kit (Rockford, IL) after bacterial cells were dislodged from the sand by bead beating,25 lysed with 0.1 M sodium hydroxide and boiled for 10 min. cisDCE epoxide was measured with a colorimetric method described by Fox et al.39 Chloride was measured by the method of Bergmann and Sanik40 or with an ion chromatograph (IC) containing an IonPac AS14A anion-exchange column (Dionex,
Figure 1. Degradation of cis-DCE in microcosms. cis-DCE in 1/4strength MSB (blue triangle), or cis-DCE in 1/4-strength MSB and sand (red triangle). Controls containing cis-DCE prepared with 1/4strength MSB (blue circle), air (green circle), or unsaturated sand (red circle). cis-DCE concentrations were measured in the gas phase, and cis-DCE was respiked twice at 30 and 48 h (results are the means of duplicate microcosms).
Column Designs. Ottawa sand (0.6 to 0.42 mm sieve size) used for the columns was baked at 550 °C. The single-port glass column, 21 cm tall and 2.2 cm inside diameter, was packed with 125 g of sand by the procedure described by Oliveira et al.44 Teflon tubing (0.125 in. OD and 0.02 in. wall) was used for all connections to prevent sorption of cis-DCE. Glass beads (10.2 g of 1 mm diameter and 13.3 g of 3 mm dia.) were added to the top and bottom to create uniform inflow and outflow (Figure S1-A, Supporting Information). The column was held at 23 °C and humidified air mixed with cis-DCE vapor was pumped into the bottom of the column at flow rates between 8 13351
dx.doi.org/10.1021/es503071m | Environ. Sci. Technol. 2014, 48, 13350−13357
Environmental Science & Technology
Article
and 32 mL/h. The cis-DCE concentrations (4 μmoles or 0.4 mg of cis-DCE·L−1 of air) were based on groundwater contamination values reported to be between 0.2 and 4.5 μM cis-DCE (0.02 and 0.5 mg of cis-DCE·L−1 of water).45,46 cisDCE concentrations in the gas phase were measured at the inlet and outlet of the column. The column was inoculated by injecting 20 mL of Polaromonas sp. JS666 cells (suspended with 1/4-strength MSB to an OD600 of 0.09) into the bottom of the column until the liquid reached the top of the sand. The liquid was immediately allowed to drain from the bottom of the column. All the liquids introduced to the column were drained by gravity and analyzed for chloride and protein. To provide enough liquid for microbial growth, the water-filled porosity in the column (0.16 v/v) was kept higher than the air filled porosity (0.13 v/v). The moisture content in the column was measured at the end of the experiment and was determined to be equivalent to 12.8 mL of water in the column. Rain was simulated by adding 23 mL of 1/100-strength MSB to the top of the column. The volume was chosen to be equivalent to the volume of the water-filled porosity, and the concentration was chosen to provide a small amount of buffering and nutrients and to avoid the possibility of lysing the cells with deionized water. The salinity of the liquid in the column (measured with an ATC salinity meter) was 0.8 ppt at the end of the experiment; therefore, the salting-out effect47 was neglected. A 30 cm tall multiport column with inner diameter of 2.2 cm, packed with sand (196 g) as described previously25 (Figure S1A, Supporting Information), was used to evaluate cis-DCE or VC biodegradation in the capillary fringe in the vadose zone. The feed containing water with either cis-DCE or VC was prepared under nitrogen. The water entered the bottom of the column and exited at 1 cm to simulate a water table. The feed and air flows were maintained at 1 mL·h−1. Abiotic columns were uninoculated columns filled with autoclaved sand. The cisDCE column was inoculated by adding 25 mL of Polaromonas sp. JS666 cells suspended in 1/4-strength MSB to an OD600 of 0.2 to the top of the column. The VC column was inoculated by adding 12.5 mL of Mycobacterium sp. JS60 and 12.5 mL of Nocardioides sp. JS614 cells both suspended in MSB to an OD600 of 0.2. The liquid was drained immediately from the lowest ports of the column by gravity. Prior to inoculation, columns were operated until equilibrium was established (8 days) to determine oxygen and contaminant gradients along with mass transfer under abiotic conditions. The visually observable capillary fringe thickness in both the columns was 13 cm above the saturated zone, which was consistent with the calculated capillary rise (12 to 17 cm) based on the mesh size of the sand.22 At the end of the study, the moisture content in each section of the column was measured by weighing samples before and after heating at 105 °C for 12 h. Quantitative Polymerase Chain Reaction (qPCR). DNA used for the experiments was extracted from 2 cm segments of the column packing by using a PowerSoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA). An established qPCR protocol48 was used with 2× Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA) and an ABI 7500 Fast Real-Time PCR System equipped with SDS v. 2.0.3 software using the default SYBR Green cycling parameters. BacF/BacR primers were used to amplify total bacterial 16S rRNA genes,49 AceA 276F/AceA 414R were used for quantification of the isocitrate lyase gene of Polaromonas sp. JS666,14 and RTC_F/ RTC_R and RTE_F/RTE_R were used to quantify ethene monooxygenase (etnC) and epoxyalkane transferase (etnE)
genes in VC degraders.50 Calibrations were performed in triplicate with serial 10-fold dilutions of the 16S rRNA gene and the isocitrate lyase gene of Polaromonas sp. JS666, and etnC and etnE genes from Mycobacterium sp. JS60 cloned using a pGEMT Easy Vector System. Standards were diluted from approximately 3 × 108 copies down to approximately three copies per reaction, calculated based on the method described by Whelan et al.51 Standard curves gave a linear slope of 3.2− 3.4 and an R2 value higher than 0.90 where 33 copies per reaction was the minimum detection limit. Polaromonas sp. JS666 contains only one isocitrate lyase gene14 and one copy of the 16S rRNA gene per cell. Similarly, for Mycobacterium sp. JS60 and Nocardioides sp. JS614 there was one copy of etnC and etnE genes52 and two copies of the 16S rRNA gene per cell.
■
RESULTS AND DISCUSSION Microcosms. Because previous studies analyzing cis-DCE concentrations and stable isotope fractionation revealed no cisDCE biodegradation in the vadose zone,53 we tested whether physical constraints prevent Polaromonas sp. JS666 biodegradation of cis-DCE in the upper layer of the vadose zone by constructing microcosms with sand containing 11% moisture which was below field capacity (13% w/w). The results indicate that bacteria in the unsaturated sand could biodegrade cis-DCE as rapidly as in liquid cultures (Figure 1). The initial cis-DCE biodegradation rate was 30 ± 4 μmol·h −1 per 10 g of sand, which indicated that biodegradation of the cis-DCE vapor can be robust. Both the abiotic liquid and air controls as well as the release of chloride in all microcosms (with a chloride to cisDCE ratio between 2.4 and 1.7) support the conclusion (Figure 1). Single Port Column Study. The cis-DCE disappearance, stoichiometric chloride release, and protein production (13.6 mg) observed during the single port column experiment all clearly indicated that Polaromonas sp. JS666 biodegraded cisDCE in the system designed to simulate the vadose zone above the capillary fringe (Figures S2 and S3, Supporting Information). Biodegradation fluxes in the column (63−252 μmol/m2/h within a column height of 15 cm) were calculated by eq 1 F=
(Ceff − C inf ) × f A
(1)
where F is the flux, f is the flow rate of the feed, Ceff and Cinf are the effluent and the influent concentrations, respectively, and A is the cross-sectional column area. When the flow rate was increased to 32 mL/h between 2100 and 2800 h substantial breakthrough (Figure S1) suggested that the flux exceeded the capacity of the biomass due to insufficient contact time. Trace amounts of an unknown volatile compound were detected in the effluent by ECD but not by FID as in previous studies describing the biodegradation pathway of cis-DCE.13 The mass balance determined by measuring chloride release indicated 92% mineralization of the cis-DCE. Thus, the unknown metabolite amounted to less than 8% of the degraded cisDCE; further investigations about the properties of the metabolite were not conducted. At intervals the column was washed with 1/100-strength MSB for two reasons. First, Polaromonas sp. JS666 is a nonmotile organism54 that is not known to produce extensive biofilms,55 which suggests that rainfall could cause it to wash out. The simulated rain seemed to wash some of the bacteria 13352
dx.doi.org/10.1021/es503071m | Environ. Sci. Technol. 2014, 48, 13350−13357
Environmental Science & Technology
Article
Figure 2. cis-DCE concentration profiles in the gas phase of the multiport column. Uninoculated control column after 72 h (A), active column 196 h after inoculation with JS666 (B), and anoxic column 62 h after the air in the feed was replaced with nitrogen (C).
Figure 3. VC concentration profiles in the gas phase of the multiport column. Sterile column after 52 h (A), active column 85 h after the column was inoculated (B), and active column 56 h after nitrogen was substituted for oxygen in the headspace (C).
feed contained 41 ± 7 μM cis-DCE the aqueous effluent concentration was 38 ± 4 μM cis-DCE. After inoculation with Polaromonas sp. JS666, biodegradation was evaluated by monitoring the changes in cis-DCE and oxygen profiles. The cis-DCE and oxygen profiles stabilized within 196 h and the system remained stable for an additional 120 h. cis-DCE was not detectable 15 cm above the saturated zone (Figure 2B). The influent and effluent cis-DCE concentrations for the active column were 42 ± 9 μM and 10 ± 3 μM, respectively, and cisDCE was not detectable in the gas effluent. The effect of oxygen limitation on biodegradation was evaluated by switching from air to nitrogen in the top of the column. Biodegradation in the column stopped within 62 h (Figure 2C), and the system remained stable for 86 h. The activity in the column recovered within 82 h after air was reintroduced. When similar experiments were performed with VC (145 ± 18 μM), the VC and oxygen profiles in the sterile column stabilized after 52 h (Figure 3A) and the aqueous effluent VC concentration was 132 ± 11 μM. The profile remained stable for an additional 160 h before the column was inoculated. The VC and oxygen profiles in the active column stabilized within 85 h (Figure 3B) and remained constant for an additional 201 h. Measured influent and effluent VC concentrations were 148 ± 13 μM and 65 ± 8 μM, respectively. Activity in the column operated with nitrogen ceased within 56 h (Figure 3C) and remained undetectable for an additional 92 h. The activity in
from the column, but the biodegradation in the column resumed within a few hours after each wash (Figure S2, Supporting Information). Additionally, at high concentrations cis-DCE degradation causes hydrochloric acid formation which decreases pH and limits biodegradation. The optimum pH for Polaromonas sp. JS666 is 7.2.10 Therefore, the column was washed with 1/100-strength MSB to prevent inhibition due to potential pH fluctuations (Figure S2, Supporting Information). The biodegradation of cis-DCE in the column increased slightly after each wash, which suggested that buffering capacity in the field might be important if high concentrations of contaminant are present. The above results clearly indicated that Polaromonas sp. JS666 could biodegrade cis-DCE in the systems designed to simulate the vadose zone above the capillary fringe. However, with only one sampling port and constant moisture content, the single port column design failed to simulate the oxic/anoxic interface and the gradient of moisture within the capillary fringe associated with contaminated groundwater plumes. Therefore, a multiport column experiment was performed. Multiport Column Studies. The multiport columns were designed to simulate biodegradation in a configuration more representative of field conditions at contaminated sites. The abiotic column containing cis-DCE reached equilibrium within 72 h, and the concentrations in the sampling ports remained stable for an additional 120 h (Figure 2A). When the anoxic 13353
dx.doi.org/10.1021/es503071m | Environ. Sci. Technol. 2014, 48, 13350−13357
Environmental Science & Technology
Article
Figure 4. Estimated microbial biomass in the cis-DCE (A) and VC (C) multiport columns at the end of the experiments. Dashed lines represent the average initial bacterial inoculum estimated by quantifying 16S rRNA genes in the inoculum divided by the mass of sand in the column.
number of cis-DCE or VC degraders (Figure 4). The majority of the active microbes in the multiport columns were within 10 cm above the saturated zone (Figure 4), which was within the capillary fringe. There was a clear peak in the 7−9 cm section. The results were consistent with the recently published study describing accumulation of bacteria in the capillary fringe.60 The results presented here reveal sustained cis-DCE biodegradation in systems designed to simulate the vadose zone. Additionally, microbial estimates and contaminant concentration profiles throughout the vadose zone clearly demonstrated that biodegradation of cis-DCE and VC takes place within the capillary fringe. Several factors could influence the microbial activity and growth during the above experiments. Moisture levels, contaminant concentrations, pH, and oxygen concentrations are the most obvious. The fact that biodegradation in the liquid cultures was similar to that in the unsaturated cultures in the microcosms containing Polaromonas sp. JS666 indicated that moisture content is not a limiting factor in the capillary fringe for cis-DCE degradation. Protein measurements in the single port column studies demonstrated that even low concentrations of cis-DCE will support bacterial growth. Similar results would be expected with VC because VC degraders are active and widely distributed in the vadose zone.27 In the multiport column study a minimal medium was provided with the initial inoculations, therefore the added nitrogen and buffering capacity could have enhanced the degradation. Because only 5.7 ± 0.4 μmol of cis-DCE and 16 ± 2 μmol of VC were degraded during the multiport column studies, the buffering capacity seemed to be sufficient but would need to be considered in the field. Previous studies indicated that microbial activities are enhanced at oxic/anoxic interfaces.25,28,61,62 The multiport column results confirm these findings and revealed that chlorinated ethenes also could be readily degraded at the oxic/anoxic interface. The increase in microbial biomass where the oxygen and contaminants intersect demonstrates that the biological activity is concentrated in a narrow zone consistent with previous predictions.33 The above results indicate that aerobic biodegradation of cisDCE and VC depends on oxygen and substrate availability and occurs in a narrow zone. The profiles in the uninoculated columns were consistent with Fick’s first law of diffusion
the column recovered within 48 h after air was reintroduced. The above results indicate clearly that cis-DCE and VC were biodegraded within the lower part of the capillary fringe and that the biodegradation was oxygen dependent. In previous work to determine the contaminant fluxes in the vadose zone, the measured aqueous or gas concentrations of the contaminants in the field (1−260 nmole·L−1 for cis-DCE56 and 0.9−126 nmole·L−1 for VC56) were multiplied by the average vapor fluxes in the field (0.01−0.1 L·m−2·min−157). The vapor fluxes for cis-DCE and VC were estimated as 1−1560 nmole·m−2·h−1 and 1−756 nmol·m−2·h−1, respectively. The flux under consideration here is the contaminant flux from the saturated zone to the capillary fringe and vadose zone. The horizontal plane is the intersection between the top of the saturated zone and the base of the capillary fringe. The column design allows measurement of the change in concentration between the aqueous influent and effluent, which is the flux to the overlying regions of the column. The fluxes (loss to the atmosphere) of the contaminants in the multiport columns were calculated by using eq 1 where Cinf and Ceff are the influent and effluent concentrations in the anoxic feed, respectively. The fluxes in the sterile multiport columns were 8 ± 1 μmol·m−2· h−1 for cis-DCE and 35 ± 9 μmol·m−2·h−1 for VC and the biodegradation flux in the multiport columns was 84 ± 15 μmol·m−2·h−1 for cis-DCE and 218 ± 25 μmol·m−2·h−1 for VC. The results revealed enhancement of the flux (removal from the aqueous phase) caused by biodegradation. There was no evidence in the multiport column experiments of volatile intermediates or metabolites resulting from biodegradation. Because the columns were packed with sand instead of field materials the void volume and therefore the contaminant fluxes are likely to be higher than in the field. Fluxes estimated here indicate that biodegradation of both cis-DCE and VC in the vadose zone could be sufficient to prevent vapor migration. Microbial biomass in the vadose zone increases with increasing biodegradation fluxes.58,59 To determine the location of the bacteria that degrade cis-DCE and VC in the vadose zone multiport columns, the active microbial biomass was measured by qPCR throughout the column at the end of the experiments. Although no attempt was made to maintain pure cultures in the columns most of the bacteria detected at the end of the experiments were derived from the inocula as indicated by the similarity between the numbers of total bacteria and the 13354
dx.doi.org/10.1021/es503071m | Environ. Sci. Technol. 2014, 48, 13350−13357
Environmental Science & Technology assuming that the top of the saturated layer is a sink for oxygen. Attempts to model inoculated columns using the “BioVapor” modeling tool63 and the instantaneous model described by Davis et al.64 did not provide adequate descriptions of the results. The currently available models for vadose zone biodegradation emphasize vapor phase diffusion and do not have a term for biomass. In the system described here most of the biodegradation takes place in the saturated portion of the capillary fringe, biomass is highly concentrated at the interface, the source is dissolved in the underlying groundwater, and aqueous phase diffusion controls the transport of the contaminant, whereas gas-phase diffusion controls the transport of oxygen. A model more focused on the capillary fringe and including the nonlinear moisture content, limiting factors such as biomass, substrate and oxygen concentrations, and biodegradation will be required to more accurately predict the aerobic biodegradation of xenobiotics in the field. Hydrocarbon biodegradation in the vadose zone has been extensively studied, and higher aerobic biodegradation rates have been measured in the capillary fringe.65 Because petroleum hydrocarbons are often found as light nonaqueous phase liquids they can be concentrated at the capillary fringe and enter the vadose zone by evaporation from the free product. In contrast, chlorinated aliphatic compounds are found as dense nonaqueous phase liquids and mostly enter the vadose zone by volatilization from aqueous solution. Recent studies by Patterson et al.27 provided an excellent description of VC biodegradation in the vadose zone with emphasis on the area above the capillary fringe and concluded that temperature was the main parameter affecting microbial activity.27 They also measured VC and oxygen concentrations near the saturated layer and provided a good mass balance based on carbon dioxide measurements. Their results revealed the most if not all of the biodegradation took place in a zone from 1 m below to 1 m above the water table. In field studies it is difficult to investigate processes at finer scales. Lower effluent concentrations in the active columns compared to abiotic columns indicates that biodegradation enhanced the diffusion of the contaminants from the saturated layer by steepening the gradients. The finding can be used to evaluate anoxic contaminated groundwater plumes and suggests that biodegradation in the capillary fringe might substantially reduce the contamination in the anoxic plume beneath it. The overall results of this study establish that as long as the cis-DCE and VC degrading bacteria are present, natural attenuation in the vadose zone can eliminate cis-DCE and VC in a thin layer at the capillary fringe. In contrast to trichloroethylene and perchloroethylenes,3 cis-DCE and VC in the vadose zone would pose a lower risk for vapor intrusion.
■
ACKNOWLEDGMENTS
■
REFERENCES
Funding was provided by DuPont Corporate Remediation Group.
(1) McCarty, P. L. Groundwater contamination by chlorinated solvents: History, remediation technologies and strategies. In In situ remediation of chlorinated solvent plumes; Stroo, H. F., Ward, C. H., Eds.; Springer: New York, 2010; pp 1−28. (2) Little, J. C.; Daisey, J. M.; Nazaroff, W. W. Transport of subsurface contaminants into buildings. Environ. Sci. Technol. 1992, 26 (11), 2058−2066. (3) Office of Underground Storage Tanks. Petroleum hydrocarbons and chlorinated hydrocarbons differ in their potential for vapor intrusion; U.S. Environmental Protection Agency: Washington, DC, 2012; pp 1− 13. (4) He, J. Z.; Ritalahti, K. M.; Aiello, M. R.; Loffler, F. E. Complete detoxification of vinyl chloride by an anaerobic enrichment culture and identification of the reductively dechlorinating population as a Dehalococcoides species. Appl. Environ. Microbiol. 2003, 69 (2), 996− 1003. (5) He, J. Z.; Ritalahti, K. M.; Yang, K. L.; Koenigsberg, S. S.; Loffler, F. E. Detoxification of vinyl chloride to ethene coupled to growth of an anaerobic bacterium. Nature 2003, 424 (6944), 62−65. (6) MaymoGatell, X.; Chien, Y. T.; Gossett, J. M.; Zinder, S. H. Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethene. Science 1997, 276 (5318), 1568−1571. (7) Hendrickson, E. R.; Payne, J. A.; Young, R. M.; Starr, M. G.; Perry, M. P.; Fahnestock, S.; Ellis, D. E.; Ebersole, R. C. Molecular analysis of Dehalococcoides 16S ribosomal DNA from chloroethenecontaminated sites throughout North America and Europe. Appl. Environ. Microbiol. 2002, 68 (2), 485−495. (8) Bradley, P. M.; Chapelle, F. H. Microbial mineralization of dichloroethene and vinyl chloride under hypoxic conditions. Groundwater Monit. Rem. 2011, 31 (4), 39−49. (9) Davis, G. B.; Patterson, B. M.; Johnston, C. D. Aerobic bioremediation of 1,2 dichloroethane and vinyl chloride at field scale. J. Contam. Hydrol. 2009, 107 (1−2), 91−100. (10) Coleman, N. V.; Mattes, T. E.; Gossett, J. M.; Spain, J. C. Biodegradation of cis-dichloroethene as the sole carbon source by a βproteobacterium. Appl. Environ. Microbiol. 2002, 68, 2726−2730. (11) Jennings, L. K.; Chartrand, M. M. G.; Lacrampe-Couloume, G.; Lollar, B. S.; Spain, J. C.; Gossett, J. M. Proteomic and transcriptomic analyses reveal genes upregulated by cis-dichloroethene in Polaromonas sp. strain JS666. Appl. Environ. Microbiol. 2009, 75, 3733−3744. (12) Mattes, T. E.; Alexander, A. K.; Richardson, P. M.; Munk, A. C.; Han, C. S.; Stothard, P.; Coleman, N. V. The genome of Polaromonas sp. strain JS666: insights into the evolution of a hydrocarbon- and xenobiotic-degrading bacterium, and features of relevance to biotechnology. Appl. Environ. Microbiol. 2008, 74 (20), 6405−6416. (13) Nishino, S. F.; Shin, K. A.; Gossett, J. M.; Spain, J. C. Cytochrome P450 initiates degradation of cis-dichloroethene by Polaromonas sp strain JS666. Appl. Environ. Microbiol. 2013, 79 (7), 2263−2272. (14) Giddings, C. G. S.; Jennings, L. K.; Gossett, J. M. Microcosm assessment of a DNA probe applied to aerobic degradation of cis-1,2dichloroethene by Polaromonas sp. strain JS666. Groundwater Monit. Rem. 2010, 30 (2), 97−105. (15) Jennings, L. K.; Giddings, C. G. S.; Gossett, J. M.; Spain, J. C., Bioaugmentation for aerobic degradation of cis-1,2-dichloroethene. In Bioaugmentation for aerobic degradation; Stroo, H. F., Leeson, A., Ward, C. H., Eds.; Springer: New York, 2013; Vol. 5. (16) Chartrand, M. M. G.; Waller, A.; Mattes, T. E.; Elsner, M.; Lacrampe-Couloume, G.; Gossett, J. M.; Edwards, E. A.; Lollar, B. S. Carbon isotopic fractionation during aerobic vinyl chloride degradation. Environ. Sci. Technol. 2005, 39 (4), 1064−1070. (17) Coleman, N. V.; Mattes, T. E.; Gossett, J. M.; Spain, J. C. Phylogenetic and kinetic diversity of aerobic vinyl chloride-assimilating
ASSOCIATED CONTENT
S Supporting Information *
Figures S1−S3 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.
■
■
Article
AUTHOR INFORMATION
Corresponding Author
*Phone: (404) 894-0628. Fax: (404) 894-8266. E-mail:
[email protected]. Notes
The authors declare no competing financial interest. 13355
dx.doi.org/10.1021/es503071m | Environ. Sci. Technol. 2014, 48, 13350−13357
Environmental Science & Technology
Article
bacteria from contaminated sites. Appl. Environ. Microbiol. 2002, 68, 6162−6171. (18) Coleman, N. V.; Spain, J. C. Epoxyalkane:coenzyme M transferase in the ethene and vinyl chloride biodegradation pathways of Mycobacterium strain JS60. J. Bacteriol. 2003, 185, 5536−5545. (19) Jin, Y. O.; Cheung, S.; Coleman, N. V.; Mattes, T. E. Association of missense mutations in epoxyalkane coenzyme M transferase with adaptation of Mycobacterium sp. strain JS623 to growth on vinyl chloride. Appl. Environ. Microbiol. 2010, 76 (11), 3413−3419. (20) Lundegard, P. D.; Johnson, P. C.; Dahlen, P. Oxygen transport from the atmosphere to soil gas beneath a slab-on-grade foundation overlying petroleum-impacted soil. Environ. Sci. Technol. 2008, 42 (15), 5534−5540. (21) Abreu, L. D. V.; Johnson, P. C. Simulating the effect of aerobic biodegradation on soil vapor intrusion into buildings: Influence of degradation rate, source concentration, and depth. Environ. Sci. Technol. 2006, 40 (7), 2304−2315. (22) Kristensen, A. H.; Hosoi, C.; Henriksen, K.; Loll, P.; Moldrup, P. Vadose zone biodegradation of benzene vapors in repacked and undisturbed soil cores. Vadose Zone J. 2012, 11, 1. (23) Davis, G. B.; Patterson, B. M.; Trefry, M. G. Evidence for instantaneous oxygen limited biodegradation of petroleum hydrocarbon vapors in the subsurface. Ground Water Monit. Remediat. 2009, 29 (1), 126−137. (24) Bekele, D. N.; Naidu, R.; Bowman, M.; Chadalavada, S. Vapor intrusion models for petroleum and chlorinated volatile organic compounds: Opportunities for future improvements. Vadose Zone J. 2013, 12, 2. (25) Kurt, Z.; Spain, J. C. Biodegradation of chlorobenzene, 1,2dichlorobenzene, and 1,4-dichlorobenzene in the vadose zone. Environ. Sci. Technol. 2013, 47 (13), 6846−6854. (26) Graves, D.; Pohlmann, D.; Ingram, S., Intrinsic biodegradation of vinyl chloride in the vadose zone. In Natural attenuation considerations and case studies: remediation of chlorinated and recalcitrant compounds, Wickramanayake, G. B., Gavaskar, A. R., Kelley, M. E., Eds.; Battelle Press: Columbus, 2000; pp 121−127. (27) Patterson, B. M.; Aravena, R.; Davis, G. B.; Furness, A. J.; Bastow, T. P.; Bouchard, D. Multiple lines of evidence to demonstrate vinyl chloride aerobic biodegradation in the vadose zone, and factors controlling rates. J. Contam. Hydrol. 2013, 153 (1), 69−77. (28) Kurt, Z.; Shin, K.; Spain, J. C. Biodegradation of chlorobenzene and nitrobenzene at interfaces between sediment and water. Environ. Sci. Technol. 2012, 46 (21), 11829−11835. (29) Larentis, M.; Hoermann, K.; Lueders, T. Fine-scale degrader community profiling over an aerobic/anaerobic redox gradient in a toluene-contaminated aquifer. Environ. Microbiol. Rep. 2013, 5 (2), 225−234. (30) Winderl, C.; Anneser, B.; Griebler, C.; Meckenstock, R. U.; Lueders, T. Depth-resolved quantification of anaerobic toluene degraders and aquifer microbial community patterns in distinct redox zones of a tar oil contaminant plume. Appl. Environ. Microbiol. 2008, 74 (3), 792−801. (31) Bauer, R. D.; Maloszewski, P.; Zhang, Y.; Meckenstock, R. U.; Griebler, C. Mixing-controlled biodegradation in a toluene plume Results from two-dimensional laboratory experiments. J. Contam. Hydrol. 2008, 96 (1−4), 150−168. (32) Vieth, A.; Kastner, M.; Schirmer, M.; Weiss, H.; Godeke, S.; Meckenstock, R. U.; Richnow, H. H. Monitoring in situ biodegradation of benzene and toluene by stable carbon isotope fractionation. Environ. Toxicol. Chem. 2005, 24 (1), 51−60. (33) Chu, M.; Kitanidis, P. K.; McCarty, P. L. Modeling microbial reactions at the plume fringe subject to transverse mixing in porous media: When can the rates of microbial reaction be assumed to be instantaneous? Water Resour. Res. 2005, 41, 6. (34) Ma, J.; Luo, H.; DeVaull, G. E.; Rixey, W. G.; Alvarez, P. J. J. Numerical model investigation for potential methane explosion and benzene vapor intrusion associated with high-ethanol blend releases. Environ. Sci. Technol. 2014, 48 (1), 474−481.
(35) Mattes, T. E.; Coleman, N. V.; Spain, J. C.; Gossett, J. M. Physiological and molecular genetic analyses of vinyl chloride and ethene biodegradation in Nocardioides sp. strain JS614. Arch. Microbiol. 2005, 183 (2), 95−106. (36) Janssen, D. B.; Grobben, G.; Hoekstra, R.; Oldenhuis, R.; Witholt, B. Degradation of trans-l,2-dichloroethene by mixed and pure cultures of methanotrophic bacteria. Appl. Microbiol. Biotechnol. 1988, 29 (4), 392−399. (37) Verce, M. F.; Gunsch, C. K.; Danko, A. S.; Freedman, D. L. Cometabolism of cis-1,2-dichloroethene by aerobic cultures grown on vinyl chloride as the primary substrate. Environ. Sci. Technol. 2002, 36 (10), 2171−2177. (38) Pumphrey, G. M.; Hanson, B. T.; Chandra, S.; Madsen, E. L. Dynamic secondary ion mass spectrometry imaging of microbial populations utilizing 13C-labelled substrates in pure culture and in soil. Environ. Microbiol. 2009, 11 (1), 220−229. (39) Fox, B. G.; Borneman, J. G.; Wackett, L. P.; Lipscomb, J. D. Haloalkene Oxidation by the Soluble Methane Monooxygenase from Methylosinus trichosporium OB3b: Mechanistic and Environmental Implications. Biochemistry 1990, 29 (27), 6419−6427. (40) Bergmann, J. G.; Sanik, J. Determination of trace amounts of chlorine in naphtha. Anal. Chem. 1957, 29 (2), 241−243. (41) Marion, D.; Nur, A.; Yin, H.; Han, D. Compressional velocity and porosity in sand-clay mixtures. Geophysics 1992, 57 (4), 554−563. (42) Stanier, R. Y.; Palleroni, N. J.; Doudoroff, M. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 1966, 43, 159− 271. (43) Andersen, R. G.; Booth, E. C.; Marr, L. C.; Widdowson, M. A.; Novak, J. T. Volatilization and biodegradation of naphthalene in the vadose zone impacted by phytoremediation. Environ. Sci. Technol. 2008, 42 (7), 2575−2581. (44) Oliveira, I. B.; Demond, A. H.; Salehzadeh, A. Packing of sands for the production of homogeneous porous media. Soil Sci. Soc. Am. J. 1996, 60 (1), 49−53. (45) Bradley, P. M.; Chapelle, F. H. Effect of contaminant concentration on aerobic microbial mineralization of DCE and VC in stream-bed sediments. Environ. Sci. Technol. 1998, 32 (5), 553−557. (46) Wise, D. L.; Trantolo, D. J.; Cichon, E. J.; Inyang, H. I.; Stottmeister, U. Bioremediation of contaminated soils; Marcel Dekker: New York, 2000. (47) Zhou, X. L.; Mopper, K. Apparent partition coefficients of 15 carbonyl compounds between air and seawater and between air and freshwater; implications for air-sea exchange. Environ. Sci. Technol. 1990, 24 (12), 1864−1869. (48) Baldwin, B. R.; Nakatsu, C. H.; Nies, L. Detection and enumeration of aromatic oxygenase genes by multiplex and real-time PCR. Appl. Environ. Microbiol. 2003, 69 (6), 3350−3358. (49) Chung, J.; Krajmalnik-Brown, R.; Rittmann, B. E. Bioreduction of trichloroethene using a hydrogen-based membrane biofilm reactor. Environ. Sci. Technol. 2008, 42 (2), 477−483. (50) Jin, Y. O.; Mattes, T. E. A quantitative PCR assay for aerobic, vinyl chloride- and ethene-assimilating microorganisms in groundwater. Environ. Sci. Technol. 2010, 44 (23), 9036−9041. (51) Whelan, J. A.; Russell, N. B.; Whelan, M. A. A method for the absolute quantification of cDNA using real-time PCR. J. Immunol. Methods 2003, 278 (1−2), 261−269. (52) Mattes, T. E.; Alexander, A. K.; Coleman, N. V. Aerobic biodegradation of the chloroethenes: pathways, enzymes, ecology, and evolution. FEMS Microbiol. Rev. 2010, 34, 445−475. (53) Hunkeler, D.; Aravena, R.; Shouakar-Stash, O.; Weisbrod, N.; Nasser, A.; Netzer, L.; Ronen, D. Carbon and chlorine isotope ratios of chlorinated ethenes migrating through a thick unsaturated zone of a sandy aquifer. Environ. Sci. Technol. 2011, 45 (19), 8247−8253. (54) Margesin, R.; Spröer, C.; Zhang, D. C.; Busse, H. J. Polaromonas glacialis sp. nov. and Polaromonas cryoconiti sp. nov., two novel bacteria from alpine glacier cryoconite. Int. J. Syst. Evol. Microbiol. 2011, 1466− 5026. (55) Major, D. W. Enhancing natural attenuation through bioaugmentation with aerobic bacteria that degrade cis-1,2-dichloroethene; 13356
dx.doi.org/10.1021/es503071m | Environ. Sci. Technol. 2014, 48, 13350−13357
Environmental Science & Technology
Article
Environmental Security Technology Certification Program; Environmental ProtectionAgency: Washington, DC, 2008. (56) Environmental Protection Agency, U. S. Vapor intrusion database. http://www.epa.gov/oswer/vaporintrusion/vi_data.html (accessed Dec 9, 2013),. (57) Devaull, G. E. Indoor vapor intrusion with oxygen-limited biodegradation for a subsurface gasoline source. Environ. Sci. Technol. 2007, 41 (9), 3241−3248. (58) Fuller, M. E.; Mu, D. Y.; Scow, K. M. Biodegradation of trichloroethylene and toluene by indigenous microbial populations in vadose sediments. Microb. Ecol. 1995, 29 (3), 311−325. (59) Ma, J.; Rixey, W. G.; Alvarez, P. J. J. Microbial processes influencing the transport, fate and groundwater impacts of fuel ethanol releases. Curr. Opin. Biotech. 2012, 24, 1−10. (60) Jost, D.; Winter, J.; Gallert, C. Noninvasive quantification of green fluorescent protein labeled Escherichia coli in a dynamic capillary fringe by fluorescence intensity. Vadose Zone J. 2014, 13 (10), 1−9. (61) Lee, C. Controls on organic carbon preservation: The use of stratified water bodies to compare intrinsic rates of decomposition in oxic and anoxic systems. Geochim. Cosmochim. Acta 1992, 56 (8), 3323−3335. (62) Lin, X.; Kennedy, D.; Fredrickson, J.; Bjornstad, B.; Konopka, A. Vertical stratification of subsurface microbial community composition across geological formations at the Hanford Site. Environ. Microbiol. 2012, 14 (2), 414−425. (63) API BioVapor. A 1-D vapor intrusion model with oxygen-limited aerobic biodegradation. http://www.api.org (accessed Jun 23). (64) Davis, G. B.; Patterson, B. M.; Trefry, M. G. Evidence for instantaneous oxygen-limited biodegradation of petroleum hydrocarbon vapors in the subsurface. Ground Water Monit. Remed 2009, 29 (1), 126−137. (65) Lahvis, M. A.; Baehr, A. L.; Baker, R. J. Quantification of aerobic biodegradation and volatilization rates of gasoline hydrocarbons near the water table under natural attenuation conditions. Water Resour. Res. 1999, 35 (3), 753−765.
13357
dx.doi.org/10.1021/es503071m | Environ. Sci. Technol. 2014, 48, 13350−13357
