Bioremediation of Chlorinated Ethenes in Fractured Bedrock and

Article pubs.acs.org/est

Bioremediation of Chlorinated Ethenes in Fractured Bedrock and Associated Changes in Dechlorinating and Nondechlorinating Microbial Populations Alfredo Pérez-de-Mora,*,†,‡,# Anna Zila,† Michaye L. McMaster,§ and Elizabeth A. Edwards*,† †

Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario M5S 3E5N, Canada ‡ Research Unit Analytical Biogeochemistry, Department of Environmental Sciences, Helmholtz Zentrum München, Ingolstädterlandstrasse 1, 85764 Neuherberg, Germany § Geosyntec Consultants, 2-130 Research Lane, Guelph, Ontario N1G 5G3, Canada S Supporting Information *

ABSTRACT: The use of enhanced in situ anaerobic bioremediation (EISB) and bioaugmentation in fractured bedrock is limited compared to its use in granular media. We evaluated EISB for the treatment of trichloroethene (TCE)-impacted groundwater in fractured carbonate rock at a site in Southern Ontario, Canada, with cool average groundwater temperature (∼ 13 °C). Borehole-connectivity, contaminant concentrations, and groundwater properties were investigated. Changes in dechlorinating and nondechlorinating populations (fermenters, acetogens, methanogens, and sulfate reducers) were assessed via quantitative PCR (qPCR). During biostimulation with ethanol, concentrations of TCE daughter products cisdichloroethene (cDCE) and vinyl chloride (VC) decreased in association with an enrichment of vcrA (VC reductive dehalogenase)-carrying Dehalococcoides, whereas ethene production was only moderate. Following bioaugmentation with the mixed dechlorinating culture KB-1, greater concentrations of chloridea product of dechlorination was observed in most wells; in addition, ethene production increased significantly in monitoring well locations that had strong hydraulic connectivity to the groundwater recirculation system, while Dehalococcoides and vcrA concentrations did not appreciably vary. Interestingly, increases of 3−4 orders of magnitude of an ethanol-fermenting Bacteroidetes population also present in KB-1 were correlated to improved conversion to ethene, an observation which suggests there could be a causal relationshipfor example, better syntrophy and/or synergy among bacterial populations.

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INTRODUCTION Bioremediation has become a viable remedial alternative for treatment of groundwater contaminated with chlorinated ethenes such as tetrachloroethene (PCE), TCE, isomers of DCE, and VC.1 The primary biotransformation mechanism for higher chlorinated ethenes (PCE, TCE) is reductive dechlorination under anaerobic conditions, which involves the sequential replacement of Cl atoms with H atoms leading to nontoxic ethene.2−4 Although anaerobic reductive dechlorination might occur naturally, at many sites incomplete or slow transformation results in the accumulation of cDCE and VC.5 To overcome this, a fermentable organic substrate (e.g., lactate, ethanol, or vegetable oil) can be added for the purpose of stimulating the native dechlorinating populations (biostimulation).6−9 In cases where dechlorinating microorganisms are not detected, inoculation of groundwater with mixed Dehalococcoides-containing consortia can be beneficial (bioaugmentation).10,11 Dehalococcoides is the only organism shown so far that is capable of dechlorinating beyond cDCE to VC and ethene.12,13 © 2014 American Chemical Society

Biostimulation and bioaugmentation approaches have been successfully used at a variety of sites for treatment of dissolved chlorinated solvent plumes and even source areas.14−17 While most bioremediation demonstrations have taken place in granular porous media, there is scant information on the use of bioremediation for in situ treatment of chlorinated ethenes in fractured bedrock.18 Fractured bedrock presents a significant remediation challenge due to complex fracture networks and unpredictable contamination patterns resulting from the penetration and distribution of contaminants within the fractures. This coupled with a slow dissolution rate means these dense nonaqueous phase liquids (DNAPLs) can provide a persistent source of contamination for decades.19,20 Detecting the presence or quantifying the abundance of Dehalococcoides organisms by molecular biology tools such as Received: Revised: Accepted: Published: 5770

September 17, 2013 April 9, 2014 April 11, 2014 April 11, 2014 dx.doi.org/10.1021/es404122y | Environ. Sci. Technol. 2014, 48, 5770−5779

Environmental Science & Technology

Article

Figure 1. Sketch of the ISSO field site. Groundwater was extracted in the vicinity of the northern property boundary (EW wells) and transferred through buried piping to building 1 where it was combined into a central manifold (composite) and directed through a filter system to remove particulates. Following filtration, the groundwater was amended (continuously or periodically depending on system operation) with chlorine dioxide (ClO2) to control biofouling. Then the water was conducted through a central force main to building 2 where the groundwater was amended with electron donor (ethanol) and distributed to individual recharge wells through a manifold to stimulate the indigenous microorganisms.

regular or quantitative PCR has become common practice at sites contaminated with chlorinated ethenes where anaerobic bioremediation is the primary method of treatment.21−23 Because Dehalococcoides strains differ in their dechlorinating capabilities, the development of molecular probes targeting genes coding for reductive dehalogenase enzymes responsible for the dechlorination reaction such as tceA, bvcA, or vcrA, has further refined our ability to evaluate the need for enhancing reductive dechlorination in the field.22−24 Whereas only Dehalococcoides is known to perform the critical final reductive dechlorinating steps, Dehalococcoides grows best in mixed consortia, relying on nondechlorinating members to provide essential nutrients and maintain anaerobic conditions.25 Various laboratory studies have assessed the diversity and relative abundance of nondechlorinating organisms in mixed microbial enrichments.26−30 In field trials, qualitative assessment of microbial communities has gained relevance, but so far there are no quantitative studies of Dehalococcoides-accompanying nondechlorinating microorganisms.31,32 Thus, the population dynamics of such accompanying organisms and their potential to enhance the overall reductive dechlorinating activity at field sites require further investigation. The main aim of this study was to evaluate the potential of enhanced in situ bioremediation (EISB) for treatment of chlorinated ethenes in groundwater in fractured bedrock and relatively cool average groundwater temperatures (∼ 13 °C); the study was part of a remedial project. To underpin the potential biogeochemical interactions responsible for successful or limited dechlorination of cVOCs at particular wells and

stages of the active treatment process we used a combined approach including the following: (a) a conservative tracer test to assess formation connectivity; (b) analysis of chlorinated volatile organic contaminants (cVOCs) and ethene concentrations; (c) analysis of groundwater geochemical data; and (d) quantitative real-time PCR analysis targeting Dehalococcoides and the vcrA gene as well as nondechlorinating microorganisms (fermenters, acetogens, methanogens) characteristic of the mixed microbial consortium KB-1. Because of the high concentration of sulfate in the groundwater, concentrations of sulfate and sulfide were measured and sulfate-reducing microorganisms, which can compete with Dehalococcoides for H2, were assessed by means of the dsrAB gene (dissimilatory sulfite reductase).

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MATERIALS AND METHODS

Field Site Overview and Recirculation System. The field demonstration took place at an active industrial site in southern Ontario (ISSO), Canada, where TCE was used as a degreasing agent. ISSO geology consists of 3−6 m of overburden materials (glaciolacustrine tills and clays) overlying a series of fractured carbonate (limestone and dolostone) formations.33,34 The bedrock formations, which subcrop in the general area, are (from youngest to oldest) (i) the Lower Devonian Bois Blanc Formation (cherty limestone with shale), (ii) the Upper Silurian Bertie (dolostone with thin beds of shale); and (iii) the Salina Formations (shale and dolostone with evaporites (i.e., gypsum)).35,36 5771

dx.doi.org/10.1021/es404122y | Environ. Sci. Technol. 2014, 48, 5770−5779


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Figure 2. Concentrations of cVOC and ethene, and microbiological data of composite (a and b), well-2 intermediate (c and d), well-1 deep (e and f), and well-1 shallow (g and h). Composite is a central pipeline where water from all three extraction wells is collected. The complete data set for all locations is provided in SI Figures S4, S5, and S6. 5772



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archaeal Operational Taxonomic Units (OTUs) in KB-1, as well as for the quantification of the functional genes vcrA and dsrAB via real-time quantitative PCR. The following details are provided in the SI: (a) the OTU composition of the KB1 culture (Figure S3), (b) primers and annealing temperatures (Table S3), (c) amplification efficiencies and standard curves (Table S4), (d) method detection limits (Table S5), (e) quality check of qPCR data between field duplicates (Table S6), (f) taxonomic information on the OTU consensus sequences utilized for primer design (Table S7), and (g) concentrations of all genes assessed (Table S10). In addition, a supplementary Excel file provides information on the BLAST hits of the primers against the NCBI nucleotide nr/nt database.

The water table is located within the overburden at a depth of 1−2 m below the ground surface. Groundwater typically flows to the northeast. Groundwater flow in the bedrock aquifer occurs primarily through subparallel bedding plane fractures, and also via secondary porosity features resulting from dissolution of calcite and gypsum. TCE was released to the subsurface through historical facility operations contaminating the underlying fractured carbonate bedrock, where concentrations of TCE above 1000 μM (140 mg L−1) were detected. The presence of TCE daughter products, mainly cDCE, but also VC, suggests natural dechlorination is occurring at ISSO. In May 2008, an EISB system was installed at ISSO to enhance biodegradation of chlorinated ethenes. The system consisted of a recirculation loop with injection and extraction wells operating as indicated in Figure 1. The EISB system groundwater flow was approximately 300−350 L min−1 or 430000 L day−1. Table S1 in the Supporting Information (SI) provides additional information on the amount of groundwater extracted from each extraction well. Details on monitoring dates and other important events are provided in SI Figure S1. Electron donor (ethanol) addition began in July 2008 (month 3). Ethanol was selected based on prior laboratory treatability studies, its high water solubility, ease of use, and low cost. The daily dose of ethanol ranged from 2 to 6 kg day−1 and targeted an average groundwater concentration of 7−14 mg L−1. At the end of October 2009 (month 18), ISSO was bioaugmented with 100 L of the KB-1 culture (SiREM Laboratories, Guelph, Canada). KB-1 was directly injected into the injection lines of the recharge wells. The recirculation system remained off for 3 days to allow for subsurface colonization of KB-1, and was restarted thereafter. A conservative tracer test with bromide was conducted in October 2009, just prior to bioaugmentation with KB-1 (SI Text S1 and Figure S2). This test was not intended to characterize the complex fracture network but to improve the knowledge of formation connectivity as it related to the EISB recirculation system. Collection of Groundwater Samples and Field Site Analytical Methods. Groundwater from various depths was sampled from multilevel monitoring wells, instrumented with low density polyethylene (LDPE) sample tubing and equipped with mini-inertial pumps (Waterra Pumps, Ltd., Mississauga, ON) by standard low flow sampling protocols (i.e., the well was purged at low purging rate, ∼0.45 L min−1). Stabilization of field groundwater parameters (pH, temperature, dissolved oxygen, redox potential, and turbidity) was achieved prior to collection of analytical samples using a multiparameter sensor (Horiba, Japan), which was previously calibrated according to the manufacturer’s instructions. For determination of chlorinated ethenes, ethene, and general geochemical parameters in groundwater, samples were express-shipped, on ice, to ALS Laboratories (Waterloo, ON). SI Table S2 provides the various analytes that were sampled for and the corresponding method. The locations sampled (see Figure 1) included the extraction wells (EW1, EW2, and EW3), the composite pipeline (composite) into which EW wells merged, monitoring wells within the treatment target area (well-1, well-2, and well-3), and monitoring wells outside the treatment target area (well-4, well5, and well-6). The depths investigated are also indicated in Figure 1. DNA Extraction and Molecular Analysis. Groundwater intended for genomic DNA (gDNA) analysis was collected and processed as detailed in SI text S2 and S3. Extracted DNA was used for quantification of the most representative bacterial and

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RESULTS AND DISCUSSION Connectivity of Wells. Conservative Tracer Test Study. Time analysis of the breakthrough curves of the conservative tracer sodium bromide led to grouping of well locations into the following three categories: (a) strong hydraulic connectivity, (b) moderate hydraulic connectivity, and (c) weak hydraulic connectivity (SI Figure S2). Therefore, the groundwater flow pattern within the recirculation zone is variable, ranging from good hydraulic connection to poor connection. Generally, the recirculation system seemed to largely constrain groundwater flow within the treatment area. Monitoring wells located outside of the treatment area (wells-4, -5, and -6) were sampled to assess changes in these areas. However, these wells were sampled less frequently so the data are less definitive. Biotransformation of cVOCs to Ethene. Despite reducing conditions (∼ −300 mV) and the presence of dechlorinators, the lack of labile C was likely limiting dechlorination and allowing the VC plume to expand at ISSO. Because molecular hydrogen is used by Dehalococcoides as the primary electron donor in the reductive dechlorination of cVOCs, a fermentable organic substrate (either a soluble or slowly soluble viscous substrate) to produce hydrogen is generally added.8,37 At ISSO, ethanol was added via the recharge wells (RW; Figure 1) on a daily basis to serve as the primary electron donor. Time trends of cVOC concentrations during biostimulation (months 3−18) differ greatly between those locations showing strong connectivity to the recirculation system (EW wells, composite, and well-1, well-2, and well-3 intermediate) and those showing moderate or weak connectivity (Figures 2 and SI S4−S6). In wells showing strong connectivity, concentrations of cDCE decreased steadily from 10 to 15 μM to 2 μM (Figures 2 and SI S4−S5). Concentrations of VC initially increased, reaching a maximum between months 5 and 7; following that peak, concentrations of VC decreased to approximately 5 μM at the end of the biostimulation phase (Figures 2 and SI S4−S5). These results suggest that biostimulation enhanced reductive dechlorination in the intermediate bedrock. During the same period, in the shallow and deep bedrock (moderate and weak connectivity) concentrations of cDCE generally did not decrease over time (Figures 2e and g and SI S5a and c). On occasion, concentrations of cDCE increased as time progressed (well-1 deep, Figure 2e) or sudden spikes in the concentrations of cDCE (well-2 shallow and deep, SI Figure S5a and c, respectively) and TCE (well-1 deep; Figure 2e) were observed. These data indicate that the groundwater extracted through the EW wells mainly flows through the intermediate bedrock as suggested by the tracer test experiment. It is possible that the 5773



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Table 1. Statistical Comparison of Ethene and Chloride Concentrations before and after Bioaugmentation with KB-1a compound/ location ethene/EW1 ethene/EW2 ethene/EW3 ethene/ composite ethene/W-1 shallow ethene/W-1 int. ethene/W-1 deep ethene/W-2 shallow ethene/W-2 int. ethene/W-2 deep chloride/all wellsb

mean biostim. (μmol L−1) (over 15 months)

mean bioaug. (μmol L−1) (over 18 months)

fold difference (bioaug./ biostim.)

N

P

± ± ± ±

3.8 2.2 3.3 2.9

2.3 1.5 2.3 2.0

5.6 3.1 5.4 4.8

12 12 12 12

0.000 0.000 0.000 0.000

0.2 ± 0.05

0.7 ± 0.7

3.5

0.5

9

0.043

2.2 ± 0.9

4.8 ± 1.8

2.2

2.6

9

0.013

2.1 ± 0.8

4.4 ± 1.9

2.1

2.3

9

0.023

4.3 ± 1.7

8.1 ± 3.1

1.9

3.8

9

0.013

5.1 ± 1.3

9.8 ± 3.3

1.9

4.7

9

0.013

1.1 ± 0.4

1.0 ± 0.3

0.9

−0.1

9

0.432

3100 ± 820

4900 ± 2300

1.6

29

0.000

4.3 6.0 4.3 4.7

± ± ± ±

1.1 1.2 1.3 2.1

9.9 9.1 9.7 9.5

average difference (μmol L−1) (bioaug. − biostim.)

1800

a

N = Number of samples considered for each period (e.g. 9 for biostimulation and 9 for bioaugmentation). The P values are based on a 2-tailed test. bioaug. = bioaugmentation; biostim. = biostimulation; int. = intermediate; w = well. The paired t test was conducted with SPSS 16.0. bNote that for chloride, data from different wells were pooled together because there were fewer data points for each individual well.

bioaugmentation was accompanied by a significant increase in chloride concentrations (Tables 1 and SI S8 and SI Figure S8a and b) and a decrease in cDCE and VC to a final concentration of below 0.5 μmol L−1 (Figures 2 and SI S4−S5). These trends also were supported by positive (ethene−chloride) and negative (ethene−cDCE and VC) bivariate correlations (SI Table S11). During the last months of the trial (months 30− 36), concentrations of ethene decreased steadily to 4−6 μmol L−1 (Figures 2 and SI S4−S5). This was attributed to a lower overall VOC load. The average cVOC concentration at the extraction wells over the 36 months had decreased to less than 0.5 μmol L−1. Mundle et al. found no isotopic evidence of microbial degradation of ethene in groundwater samples collected from selected monitoring wells at ISSO;39 therefore the decrease in ethene toward the end of the experiment is likely due to low ethene production (cDCE and VC depleted) and partial dilution through mixing with fresh noncontaminated groundwater. Concentrations of cVOC and ethene at two locations outside the treatment area were measured on months 18 and 30 (wells 4 and 5). In all cases concentrations of cVOC and ethene were below the method detection limits (∼ 0.5 μg L−1 or 0.004 μmol L−1 for TCE), except for well-5 on month 30, in which a VC concentration of 4 μmol L−1 was recorded. Although this location might be connected in some capacity to the recirculation area, it seems that the recirculating system effectively constrained contaminants within the treatment area. The locations upstream of the target treatment area (well 6 intermediate and shallow) showed the highest concentrations of TCE and cDCE at ISSO (max. of 1300 μmol L−1 of TCE and 200 μmol L−1 of DCE; SI Figure S6). Concentrations of cVOC throughout the study remained constant in well-6 with VC and ethene below detection limits. Concentrations of TCE above 100 mg L−1 (760 μmol L−1) are indicative of residual DNAPL being present in the proximity of this monitoring location.37 Groundwater Physical and Geochemical Properties. General groundwater properties affecting anaerobic microbial activity such as pH (∼ 7), redox potential (∼ −300 mV),

alteration in the natural groundwater circulation imposed by the EISB system caused dissolution and/or resuspension of TCE and cDCE from the bedrock matrix or fractures situated in the shallow and deep bedrock. This may have led to greater concentrations of these compounds in the groundwater, although on occasion this phenomenon was reverted as in well-2 at the shallow depth (SI Figure S5a). Similar phenomena have been previously reported during another bioremediation trial to treat a DNAPL source zone at a site where a recirculation system was employed.17 It should be noted that fractured media with a high porosity matrix (e.g., dolostone and limestone) might initially attenuate DNAPL constituents that partition into groundwater; however, back diffusion can sustain dissolved-phase concentrations in groundwater flowing in the fractures even after the source zone is apparently depleted.38 Despite the decrease of cDCE and VC concentrations at the end of the biostimulation phase, there was no net increase in ethene production from months 8 to 18 of the biostimulation period, even in locations with strong hydraulic connectivity to the EISB system (Figures 2 and SI S4−S5). Because treatability studies showed that reductive dechlorination could be improved with the addition of the mixed dechlorinating culture KB-1 and the cost of bioaugmenting is small compared to that of prolonged system operation, 100 L of KB-1 was inoculated into the subsurface through RW1 and RW2 on month 19 (Figure 1). Shortly after inoculation, ethene concentrations in locations with strong connectivity to the EISB started to increase, peaking approximately on month 27; here, ethene reached concentrations similar to those of cDCE prior to biostimulation treatment (Figures 2 and SI S4−S5). The increase in ethene concentrations following bioaugmentation was significant in all strongly connected wells (except well-3 intermediate), as well as in the moderately connected wells (Table 1). In the latter, ethene concentrations were still substantially lower than those of cDCE (Figures 2 and SI S5). This could be attributed to either limited reductive dechlorination or limited transfer of ethene produced in strongly connected wells (e.g., well-1 and well-2 intermediate) through recirculation. The increase in ethene production during 5774



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temperature (∼ 13 ± 4 °C, which varied seasonally), conductivity (1.5 mS cm−1), and dissolved oxygen (< 0.5 mg L−1) differed little among sampling locations (SI Figure S7). Although the relatively low groundwater temperatures might have slowed microbial activity compared to other sites with warmer temperatures (e.g., 16−22 °C), the small temperature variation across the various wells suggests that temperature did not have a major impact on the differences in biodegradation performance among wells. In addition to cVOC and ethene, chloride concentrations were monitored as a product of dechlorination. SI Table S8 shows changes in chloride concentrations between different time periods for both biostimulation and bioaugmentation. Despite periods of no net increase in chloride, all locations within the treatment area, regardless of their connectivity, showed a net increase in chloride concentrations at the end of the study indicating an underestimation of cVOC transformations based on ethene production alone (Table 1). The net change in chloride concentrations within the same well was significantly greater during bioaugmentation than during biostimulation and for strongly connected wells compared to moderately or weakly connected wells. These results indicate that differences in biodegradation of cVOC between strongly and moderately or weakly connected wells were likely due to poor delivery of electron donor to less connected wells. Concentrations of total organic C and total volatile organic acids were measured as indicators of donor availability. Total organic C measurements did not show any consistent patterns between wells with different connectivity (SI Figure S8c and d). In general, total organic C concentrations ranged from 2 to 20 mg C L−1 with few instances above 20 mg L−1. For volatile organic acids, however, concentrations above 40 mg L−1 during biostimulation and above 20 mg L−1 during bioaugmentation were common among strongly connected locations in contrast to moderately or weakly connected locations (SI Figure S8e and f). It should be noted that increases in VOAs can result from donor fermentation but also from acetogenesis. It is unclear as to whether the differences in cVOC biodegradation performance between strongly and moderately or weakly connected wells were solely due to electron donor delivery. For instance, ethene production was moderate during biostimulation in the strongly connected wells despite donor availability and significant abundance of native Dehalococcoides. The detection of substantial concentrations of sulfate, sulfide, and methane in the groundwater suggested that the occurrence of other electron-consuming processes such as sulfate reduction and methanogenesis could interfere with reductive dechlorination. Whereas high concentrations of sulfate (> 500 mg L−1) might be problematic in terms of donor competititon (SI Figure S9a and b), sulfide, the product of sulfate reduction, might be more inhibitory for dechlorination due to its toxicity.8,40 High levels of sulfide were observed during biostimulation in both strongly and moderately connected wells when ethene production was only moderate (SI Figure S9c and d). Still the higher concentrations of sulfide in strongly connected wells suggest enhanced sulfate reduction due to donor availability. This hypothesis is supported by the negative correlations between sulfide and sulfate and the positive correlations between sulfide and TOC and VOAs (SI Table S11). Methanogenesis can be of concern in terms of efficient donor utilization and aquifer biofouling.41 For most wells, methane concentrations increased between months 0 and 15 (biostimulation) and months 21 and 36 (bioaugmentation),

while they were close to pretreatment values between months 16 and 20. This trend was observed in most wells regardless of their connectivity, except for well-1 at the shallow depth and well-6 at the intermediate and shallow depths (SI Figure S9e and f). Here, methane concentrations were notably higher (above 5000 μg L−1 and up to 25 000 μg L−1) compared to the other wells (∼ 2500 μg L−1). It is important to note that these wells shared the highest concentrations of chlorinated ethenes and no evidence of biodegradation, although high cVOC concentrations are known to inhibit methanogenesis.17,41 It is also possible that these locations are partially isolated from the primary groundwater flow paths allowing for methane accumulation over time. Another possibility for such high methane concentrations could be abiogenic methane. This could also explain the copresence of ethane at concentrations above those of ethene in groundwater at ISSO (data not shown). Overall geochemical data indicate that various electron-accepting processes (methanogenesis, acetogenesis, sulfate reduction) were co-occurring at ISSO in addition to dechlorination. We made an estimation of the relative contribution of these processes in the “composite pipeline” during the biostimulation and the bioaugmentation periods (SI Table S9). Our estimations suggest that sulfate reduction was the dominant electron consuming process during biostimulation (59%), whereas acetate production via fermentation− acetogenesis dominated the bioaugmentation phase (58%). Electron equivalent consumption by dechlorination significantly increased after bioaugmentation (from 9 to 19%). It is possible that a shift from sulfate reduction to acetogenesis benefited reductive dechlorination during bioaugmentation as Dehalococcoides can solely use acetate as C source. Methanogenesis played a minor role (only 2−4% of total electron equivalents), possibly due to partial inhibition by sulfate reduction.42 Dynamics of Dehalococcoides and Nondechlorinating Populations. The dechlorinating and nondechlorinating members of the bioaugmentation culture KB-1 were monitored in the study. A total of eight OTUs were screened via real-time PCR, in addition to general bacteria and general archaea (Figures 2 and SI S4−S6 and Table S10). The choice of OTUs was based on their relative abundance in a clone library of KB-1 consisting of 216 bacterial and 57 archaeal clones (SI Figure S3). Despite the fact that clone libraries provide qualitative rather than quantitative information, previous work with various KB-1 subcultures showed that OTUs appearing only once or twice in clone libraries did not account for more than 2% of the total 16S rRNA gene copies measured in a DNA sample.42 Over the course of the field trial the absolute abundance of various OTUs significantly represented in KB-1, including putative acetogens and fermenters, was either low (e.g., Acetobacterium, 103−104 gene copies L−1), or below the detection limits of the methods employed (Spirochaetes, Pelobacter and Synergistetes; SI Table S10). At ISSO, changes above an order of magnitude were reported for Dehalococcoides, Bacteroidetes, Methanomethylovorans, and Geobacter over the course of the experiment in addition to general bacteria and archaea (SI Table S10). Prebiostimulation (i.e., background) molecular data exist for the three EW wells (SI Figure S4e). The average of these wells was taken as a baseline for OTU abundance at ISSO (predonor data in all graphs). In addition, a well transgradient and southeast of the treatment area (well-4) with cVOC concentrations below the method detection limits was sampled 5775



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was so abundant at ISSO. In subcultures of KB-1 maintained on other electron donors such as methanol or H2, but not ethanol, Methanomethylovorans, while present, was not the dominating methanogen.43,44 The most significant change after bioaugmentation with KB1 was the increase in Bacteroidetes abundance in all locations reaching titers similar to or even greater than those of other OTUs. In the moderately and weakly connected wells, Bacteroidetes numbers went from below the detection limit before bioaugmention to 106−107 gene copies L−1 (Figures 2 and SI S4−S6). Changes in Bacteroidetes concentrations were positively and better correlated to those of ethene than concentrations of Dehalococcoides or Methanomethylovorans (SI Table S11). The recent completion of the genome of Bacteroidales strain CF from a 1,1,1-trichloroethane (TCA)and chloroform-dechlorinating culture shows that this organism is predicted to ferment L-lactacte and ethanol.45 Bacteroidales strain CF shares 91% nucleotide sequence identity at the 16S rRNA level with the Bacteroidetes OTU present in KB-1. Interestingly, the Bacteriodetes forward and backward primers used in this study only have one mismatch each with the 16S rRNA of Bacteroidales strain CF. This primer set was tested with DNA extracted from the 111-TCA- and chloroformdechlorinating culture and verified to yield specific PCR amplicons of the same size and melting temperature as those obtained in KB-1 (unpublished data). Members of the Bacteroidetes phylum have been reported as important constituents of other mixed dechlorinating cultures including the ANAS and DONNA2 cultures.25 It is possible that the KB1-like Bacteroidetes population, in addition to fermenting ethanol syntrophically to H2, also provides other key (and limiting) nutrients to Dehalococcoides and thus plays a significant role in providing conditions conducive to better ethene production by Dehalococcoides. Not only do Dehalococcoides organisms require H2 as a direct electron donor, they also require acetate as a carbon source, and derivatives of vitamin B12 as a cofactor. Similarly Dehalococcoides may have stimulated growth of Bacteroidetes by detoxification of contaminants or by other nutrient exchanges.46The enhancement of syntrophic and synergistic interactions between Dehalococcoides and the Bacteroidetes population may thus have led to increased ethene production during the bioaugmentation phase. Given the low initial abundance of most of the monitored OTUs in the EW wells and at the various depths of well-4 (SI Figure S4e and f), it is evident that in situ biostimulation and/ or bioaugmentation increased the abundance of key organisms involved directly (dechlorinators) or indirectly (nondechlorinators) in reductive dechlorination. The low numbers of most OTUs in well-5 (downstream) and well-6 (upstream) during the bioaugmentation phase further highlight the dynamics observed within the recirculation area, that is, the late arrival/ growth of microorganisms in moderately and weakly connected wells (Figures 2 and SI S4−S5). The relatively low numbers of Geobacter compared to Dehalococcoides and other OTUs (by at least 1 order of magnitude) might be due to the low concentrations of TCE in most wells compared to cDCE and VC. Geobacter is known to dechlorinate PCE and TCE to cDCE, but does not further dechlorinate cDCE.46 This provides an advantage for Dehalococcoides over Geobacter at ISSO. The fact that Geobacter can also grow using other electron acceptors (e.g., Fe3+) may have still allowed limited growth of Geobacter at ISSO.47 Although we did not measure

at various depths; this location could serve as a background reference point for the area (SI Figure S4e). Initially (prior to donor addition), the only specific OTU found above the method detection limits at ISSO was Dehalococcoides (∼ 106 copies L−1; SI Figure S4e). By contrast, the vcrA gene was below the MDL (6.2 × 103 gene copies L−1). These results might explain why cDCE and VC were found in groundwater prior to remediation in the absence of significant ethene concentrations. In well-4, Dehalococcoides titers ranged from 8 × 102 (MDL) to 105 gene copies L−1 in the various depths; vcrA was below the MDL (6.2 × 103 gene copies L−1). These data underpin two important points for this study: (a) Dehalococcoides organisms were present in the contaminated subsurface at ISSO prior to any intervention and (b) the dominant Dehalococcoides population present originally did not carry the vcrA gene, and thus suboptimal conditions were initially present for achieving complete reductive dechlorination. Biostimulation had a significant impact on the abundance of the different OTUs at ISSO, particularly in strongly connected locations. This allowed populations previously below detection limits such as Methanomethylovorans, Bacteroidetes, Geobacter, and sulfate reducers to thrive (Figure 2b and d). The increase in Bacteroidetes and Geobacter lagged compared to that of Methanomethylovorans and the sulfate reducers, appearing only in the late biostimulation phase. Whereas Dehalococcoides titers did not change drastically following biostimulation (106−107 gene copies L−1), there was a shift in the genetic makeup of the Dehalococcoides population as shown by the increase in vcrA abundance, with matching levels of Dehalococcoides and vcrA (Figures 2 and SI S4−S5). This suggests that Dehalococcoides populations containing vcrA became dominant during biostimulation. In the moderately connected wells (well-1 deep and well-2 shallow) similar trends were observed, except that Bacteroidetes and Geobacter remained below the method detection limits during biostimulation (Figures 2f and SI S5b). In the weakly connected wells (well-1 shallow, Figure 2h and well-2 deep, SI Figure S5d), the increase in Methanomethylovorans and dsrAB lagged compared to wells with greater connectivity; the abundance of other OTUs was lower, with Dehalococcoides and vcrA titers close to 105−5 × 105 gene copies L−1 and Bacteriodetes and Geobacter below the method detection limits (103−105 gene copies L−1). The change in OTU abundance was also reflected in general bacteria and archaea titers, which increased between 1 and 3 orders of magnitude following biostimulation (SI Table S10). In general, Dehalococcoides titers represented around 1% of all bacteria, whereas titers of sulfate reducers as assessed by the quantification of the dsrAB gene represented about 10%. In a small proportion of samples (SI Table S10), the quantification of Dehalococcoides and dsrAB abundance exceeded that achieved with primers targeting “all” bacteria. This could be due to preferential binding of universal primers to other OTUs less abundant. Similar discrepancies were sometimes observed for specific versus universal detection of archaea. However, for archaea, Methanomethylovorans and general archaea titers were generally similar suggesting that Methanomethylovorans was an important methanogen at ISSO during the biostimulation and bioaugmentation phases. Methanomethylovorans is an H2−CO2dependent methanogen and was the only methanogen found in the archaeal clone libraries of KB-1 amended with both methanol and ethanol as donor sources and was very abundant (SI Figure S3). It is unclear as to why Methanomethylovorans 5776



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Fe3+ concentrations, total Fe concentrations in groundwater were generally below the method detection limits (0.05 mg L−1; data not shown). The primers targeting KB-1 OTUs were designed to be generally highly specific (OTU level) and not intended to cover other taxonomically related organisms at the genus or order level, with the exception of Dehalococcoides (due to the high similarity of the 16S rRNA sequences within the Dehalococcoides genus). Thus, it is possible that other OTUs within the Bacteroidetes, Spirochaetes, and other phyla not targeted by our primers might have flourished at ISSO. To provide context to the microbial community at ISSO a small set of samples was subjected to pyrotagsequencing analysis (SI Figure S10). An indepth analysis of the pyrotagdata is beyond the scope of this work and will be discussed elsewhere. The pyrotagsequencing data confirmed the presence of the monitored KB-1 OTUs which were found above the method detection limits of the qPCR analysis as well as other OTUs representing putative functional groups such as fermenters, acetogens, methanogens, and sulfate reducers also relevant to the overall effectiveness of enhanced reductive dechlorination. Dehalococcoides and Methanomethylovorans represented a high percentage of the total Chloroflexi- and archaea-related sequences, respectively. Various groups of sulfate reducers within the δ-proteobacteria (Desulfobacterales, Desulfovibriales, and Syntrophobacterales) and within the Nitrospirae (Thermodesulfovibrio) taxa were identified. Sequences from putative fermenters within the Firmicutes taxon (Chlostridium and Bacteroidetes) and putative acetogens within the Spirochaetes were also retrieved. Proteobacteria were highly represented in all samples including Geobacter. Other OTUs with significant representation included anaerobic autotrophic Chlorobi and candidate phyla most affine to Chloroflexi (whose functional role in groundwater systems is yet to be unraveled) such as ABHY1_OD1, TM7, and OP3, but also others (e.g., OP11).48,49 Implications and Perspectives for using in Situ Bioremediation in Fracture Bedrock. This study shows that fractured rock settings are not only complex in terms of their hydrology, but also in their biogeochemistry. Monitoring of cVOCs and ethene concentrations as well as Dehalococcoides and the vcrA gene may therefore not suffice in uncovering why reductive dechlorination is successful or fails in the field. In addition, monitoring of ethene production may underestimate cVOC removal compared to production of chloride. The main question at this site remains: Why did ethenogenesis take so long despite abundant Dehalococcoides and a ratio of Dehalococcoides/vcrA gene that remained close to one once biostimulation was established? The growth of an-ethanolfermenting Bacteroidetes population to high concentrations (107 gene copies L−1) during the late biostimulation and the bioaugmentation phases suggests a causal link between enhanced syntrophy and/or synergy and ethene/chloride production during the bioaugmentation phase. Our data also suggest that a shift in the dominating competing electronconsuming process at this site from sulfate reduction to acetate production via fermentation−acetogenesis following bioaugmentation may also have enhanced reductive dechlorination, by providing a C source to dechlorinators and decreasing production of sulfide, which is toxic. Another possibility is that bioaugmentation may have introduced Dehalococcoides expressing a more efficient vcrA protein. A combination of all these factors cannot be ruled out. Given sufficient time, complete cDCE and VC dechlorination could have been

achieved in strongly and moderately connected wells without bioaugmenting; however, the low-cost of bioaugmentation and the clear potential to reduce remediation time frames and prolonged system maintenance were compelling arguments in favor of this approach. It is generally accepted that complete treatment of cVOCs where residual sources remain may take decades. Our study shows that bioremediation can be used to treat cVOCs in fractured bedrock, even at relatively low groundwater temperatures. In wells with poor hydraulic connectivity to the engineered system a lag in certain key parameters (including electron donor and microorganisms) underpins limitations not only for bioremediation, but any technology requiring the introduction of reagents. However, two advantages of bioremediation include (i) extended treatment beyond the active treatment period is common and (ii) less contaminant rebound is generally observed compared to chemical treatments.50 Given sufficient time, microorganisms may be able to colonize areas with poor connectivity, providing some continued activity at low levels on limited donor provided by the rock matrix. Such strategy, while not cleaning up ISSO completely, may help at least to protect downgradient receptors in fractured settings.

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ASSOCIATED CONTENT

S Supporting Information *

Timeline (Figure S1); conservative tracer test (Text S1 and Figure S2); volume of groundwater extracted (Table S1); standard methods (Table S2); additional details on groundwater filtering and DNA extraction (Text S2 and S3); composition of the KB-1 culture (Figure S3) and molecular data analysis including: primers and annealing temperatures (Table S3), amplification efficiencies and standard curves (Table S4), method detection limits (Table S5), quality check of qPCR data between field duplicates (Table S6), and taxonomic information on the OTU consensus sequences utilized for primer design (Table S7); concentrations of cVOC, ethene and microbiological OTUs (Figures S4−S6 and Table S10); changes in chloride concentrations (Table S8); electron equivalent consuming processes (Table S9); groundwater geochemical properties (Figures S7−S9); Pearson’s correlations (Text S4 and Table S11); pyrotagsequencing data (Text S5 and Figure S10); Excel file containing BLAST results for the primers employed. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (A.P.-d.-M); elizabeth. [email protected] (E.A.E.). Present Address

# Institute of Groundwater Ecology, Dept. of Environmental Sciences, Helmholtz Zentrum München, Neuherberg, Germany.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A. P.-d.-M. thanks the European Commission for financial support through an IOF Marie Curie Fellowship within the project AnDeMic (Contract 23974/7th Framework). We thank the Natural Sciences and Engineering Council of Canada 5777



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(NSERC), the U.S. Department of Defense Strategic Environmental Research and Development Program (SERDP), and Sustainable Development Technology Canada (SDTC) for providing funding. We also acknowledge R&D in kind contributions by Geosyntec and SiREM to the project study. We thank Adria Bells, Jeff Roberts, and Carey Austrins for field assistance, as well as the three anonymous reviewers who greatly improved the quality of the manuscript.

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Bioremediation of Chlorinated Ethenes in Fractured Bedrock and

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