Laboratory Study of Treatment of Trichloroethene by Chemical

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Environ. Sci. Technol. 2005, 39, 2888-2897

Laboratory Study of Treatment of Trichloroethene by Chemical Oxidation Followed by Bioremediation L E I L A H R A P O V I C , † B R E N T E . S L E E P , * ,† DAVID J. MAJOR,‡ AND ERIC D. HOOD‡ Department of Civil Engineering, University of Toronto, Toronto, Canada, M5S 1A4, and GeoSyntec Consultants, Guelph, Ontario, Canada, N1G 5G3

Studies were conducted with columns containing soil and emplaced trichloroethene (TCE) to investigate the potential for TCE source zone remediation with chemical oxidation followed by biologically mediated reductive dehalogenation. Following permanganate flushing of four columns, which resulted in rapid but incomplete removal of TCE DNAPL, no biological activity was observed following the addition of distilled water amended with ethanol and acetate, including two of the four columns that were bioaugmented with a TCE-dechlorinating microbial culture. Flushing with unsterilized site groundwater led to consumption of acetate and ethanol, accompanied by manganese reduction and methanogenesis. Reductive dechlorination of TCE to cis-1,2-dichloroethene (cis-DCE) followed the onset of ethanol and acetate biodegradation in bioaugmented columns only. Partial dechlorination of TCE to ethene was observed only in one of the bioaugmented columns after it was inoculated for a third time. At the end of the study (290 days), a trace amount of cis-DCE was observed in one of the two columns which was not bioaugmented. Reduced conditions created by biostimulation were also conducive to reduction of Mn(IV) from MnO2 in both bioaugmented and nonbioaugmented columns resulting in an increased dissolved manganese (Mn2+) concentration in groundwater.

Introduction Chemical oxidation with KMnO4 (potassium permanganate) has been suggested as a suitable technology for in-situ remediation of dense nonaqueous phase liquids (DNAPLs) in the subsurface. This technology has been tested at many field sites (1-6) and under controlled conditions in laboratories (6-11). Batch studies of TCE (trichloroethene) and PCE (tetrachloroethene) oxidation in aqueous solution have characterized reaction kinetics and products (12-15). In TCE, one of the most common groundwater contaminants, oxidation proceeds according to (9, 15)

2KMnO4 + C2HCl3 f

2MnO2 + 2CO2 + 3Cl- + H+ + 2K+

The precipitation of amorphous manganese dioxide (MnO2) * Corresponding author telephone: (416)978-3005; fax: (416)9783674; e-mail: [email protected]. † University of Toronto. ‡ GeoSyntec Consultants. 2888

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may form a rind of low permeability around the TCE DNAPL source (7-10), thereby reducing contact with MnO4- and diminishing the rate of TCE oxidation with time. In addition, the generation of CO2 gas could also lead to local reduction in water-phase permeability, reducing flow of permanganate solution into the source area (6, 9). The extent of these effects would vary with the volume and distribution of the DNAPLs as well as the specific soil hydraulic properties (6, 8-9). It is expected that clogging would be particularly problematic at sites involving DNAPL pools, compared to those with DNAPL sources at residual saturation (8). Channeling of permanganate solution during flushing, because of variations in soil permeabilities, also may lead to mass transfer limited removal of contaminants. As a result of channeling due to soil heterogeneity, and flow and mass transfer reductions due to MnO2 precipitation and carbon dioxide generation, complete removal of contaminants may be very difficult to achieve. Accordingly, oxidation treatment will likely result in some contaminant mass removal from source zones and produce reductions in contaminant concentrations in permeable zones with corresponding reductions in contaminant fluxes from the source zones. With reduced contaminant fluxes from source zones, other remediation technologies such as bioremediation may be better suited to manage the remaining contaminant mass. Bioremediation, either intrinsic or engineered, could be effective in limiting the impacts of postoxidation source zones on groundwater quality. Bioremediation technologies have been applied intensively in recent years for dissolved chlorinated solvent remediation, since the discovery that many dissolved chlorinated solvents could be completely degraded in situ either by indigenous microorganisms or by bioaugmentation with enriched microbial consortia which respire these chlorinated solvents (16-22). However, oxidation technologies have a significant impact on soil geochemistry, which may limit the potential for bioremediation following oxidation treatment. There is little known about the potential impacts of high concentrations of oxidants such as permanganate on indigenous microorganisms in soils. In a study of permanganate flushing combined with soil mixing, no significant change in aerobic or anaerobic bacterial counts were observed before and after permanganate flushing and soil mixing (5). However, this study did not investigate the potential for establishing reductive dechlorination following permanganate flushing. It is possible that for chlorinated solvents, which are typically bioremediated by reductive dechlorination, the high redox levels left after oxidation will not be conducive to initiation of reductive dechlorination, even if bioaugmentation is conducted to replenish dechlorinating populations. There have not been any investigations of the feasibility of establishing a sequential treatment strategy involving permanganate flushing followed by reductive dechlorination to treat chlorinated ethene contaminants remaining after permanganate oxidation. This research investigates the feasibility of using two insitu techniques in sequence to remediate DNAPL TCE: (a) chemical oxidation with potassium permanganate (KMnO4) and (b) biodegradation via reductive dechlorination. The specific objectives of the study were (1) to assess the impact of the permanganate flushing on indigenous microbial activity, (2) to determine whether biostimulation with electron donors of the indigenous microorganisms, after permanganate flushing, is sufficient for bioremediation of TCE, and (3) to determine the impact of bioaugmentation with halorespiring microorganisms. 10.1021/es049017y CCC: $30.25

 2005 American Chemical Society Published on Web 03/12/2005

TABLE 1. Soil Properties and Column Influent Properties soil parameter bulk density

[kg/m3]

hydraulic conductivity [m/s] cation exchange capacity [meq/100 g] total organic carbon [mg/kg dry weight] buffering capacity [mg/ kg dry weight]

site groundwater influent parameter

value (mg/L)

alkalinity, as CaCO3 total dissolved solids total organic carbon chloride sulfate nitrate calcium iron magnesium manganese sodium pH

290 340 5.3 22 13 0.0071 140 2.1 3.7 0.1 5.8 7.3

value 2000 2.2 × 10-6 0.0084 400 120 000

synthetic groundwater influent parameter NH4Cl MgCl2 NaCl CaCl2 Na2HPO4 NaHCO3 KH2PO4

value (mg/L) 26.8 5.1 3.5 88.2 31.9 42.0 13.1

TABLE 2. Treatment Phases of Study phase

activity

day

cumulative pore volumes

I

Baseline Flush with Distilled Water (in all six columns, Qav ) 0.24 L/day, pore volume ) 0.426 L)

-33 to 0

19

II

Oxidation with KMnO4 in Treatment Columns 1-4 flushing with distilled water continued in control columns 5 and 6 to the end of the experiment; average flow rate of 0.19 L/day

0-22

28

III

Post-Oxidation Flush with Distilled Water (Treatment Columns 1-4)

23-33

33

IVa

Biostimulation with Electron Donors (Acetate and Ethanol) in Distilled Water treatment columns 1-4 fed 100 mg/L ethanol and 100 mg/L acetate first bioaugmentation: columns 3 and 4 inoculated with 1 mL fresh KB-1 culture second bioaugmentation: columns 3 and 4 inoculated with 5 mL fresh KB-1 culture

34-112

69

64 75

IVb

Biostimulation with Electron Donors (Acetate and Ethanol) in Groundwater NASA LC-34 groundwater spiked with ethanol and acetate fed to columns 1-4 acetate feeding ceased, 100 mg/L (2.17 mM) ethanol continued

113-180 125-140 151

99

IVc

Biostimulation with Ethanol in Synthetic Groundwater third bioaugmentation: column 3 inoculated with KB-1 culture ethanol feed increased to 200 mg/L in all treatment columns 1-4 dehalogenation of TCE initiated in biostimulated column 1 (trace cis-DCE detected) termination of column tests: control column 6 column 2 (treated with KMnO4 and stimulated with donors) column 3 (treated with KMnO4, stimulated with donors and bioaugmented three times)

180-290 216 218 222

99-148 115 116 118

243 254 260

127 132 135

Materials and Methods Experimental Design. The study was comprised of a set of continuous flow one-dimensional (1D) experiments, employing six columns and soil from Launch Complex 34 (Kennedy Space Center, FL). The columns were constructed of glass (5 cm ID × 60 cm) to allow visual inspection of changes in the soil occurring during the study. Each column had six sampling ports extruded at uniform 10-cm intervals, with the first port located 5 cm above the inlet screen at the bottom of the column. The ports were numbered sequentially with port 1 closest to the inlet at the bottom of the column and port 6 closest to the column outlet at the top of the column. The soil was homogenized by hand, placed into the columns, and compacted lightly in 5-cm lifts (see Table 1 for soil properties). Upward flushing with distilled water and subsequent recycling of the porewater effluent back to the columns followed to achieve full water saturation. Using a syringe pump, liquid TCE was infused at a constant rate into each of the water-saturated soil columns, using a procedure similar to that previously described (9, 23). The amount emplaced was based on a trial infusion of TCE, colored with Sudan red, which resulted in a height of TCE

rise within each column of approximately 10 cm. The direction of the flow was then reversed to displace any mobile TCE phase from the soil. The displaced liquid was collected in a graduated cylinder. An additional two liters of porewater (∼5 pore volumes) were flushed before the columns were switched to upflow operation. Four replicate treatment columns were sequentially flushed with distilled water (Phase I), permanganate solution (Phase II), distilled water (Phase III), distilled water amended with ethanol and acetate (Phase IVa), site groundwater (see Table 1 for composition) amended with ethanol and acetate (Phase IVb), and a mixture of two parts synthetic groundwater (see Table 1 for composition) and one part site groundwater amended with ethanol (Phase IVc). Two of the treatment columns were also bioaugmented during Phase IVa. Two additional columns served as control columns and were only flushed with distilled water for the duration of the study. The phases of the study are summarized in Table 2. Each of the four treatment columns (columns 1-4) was connected through one-eighth-in. Teflon tubing to a 10-L glass feed tank and a peristaltic pump. Distilled water, solutions of KMnO4, and subsequently groundwater, purged VOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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with N2 and spiked with ethanol and acetate (Phase IV), were prepared in the feed tank and continuously distributed to the soil columns during each respective treatment phase. An additional two columns (columns 5 and 6), connected to a separate distilled water tank, were run as controls. For Phase I, the flow rate to each of the columns was approximately 0.24 L/day. For subsequent phases, the flow rate to each of the columns was approximately 0.19 L/day. Permanganate Flushing. Columns 1-4 were flushed with a solution of KMnO4 (2500 mg/L) at a rate of 0.19 L/day for each column, for a total of 22 days. Bioaugmentation Culture. Bioaugmentation of columns 3 and 4 was conducted with the mixed bacterial culture KB-1 (21, 24), enriched on ethanol and TCE in a low sulfate medium. This culture contained two strains of Dehalococcoides ethenogenes (DHC) that are capable of complete dechlorination of TCE under strictly reduced conditions using H2 as sole electron donor. This culture (24) also contained several species of Acetobacterium, a species related to Sulfurospirillum deleyianum (can use acetate as a carbon source with a variety of electron acceptors and can use H2 and sulfide as electron donors), and a species related to Hippea maritima (a thermophilic sulfate reducer). Analytical Methods. Porewater samples with TCE concentration higher than 1 mg/L were extracted in 3 mL of heptane and analyzed on HP series II gas chromatograph (GC) equipped with ECD detector (GC/ECD) and HP7673 autosampler and a fused silica wide bore column (15 m × 0.53 mm ID, 3-µm film, Rtx-200, Restek Corp. U.S.). Samples with volatile organic compounds (VOCs: TCE, cis-DCE, VC, and ethane and methane concentrations less than 1 mg/L) were analyzed on HP 5890 series II GC/FID and HP automated headspace analyzer and a Supel-Q TM Plot fused silica, 30 m × 0.53 mm ID column (Supelco, Bellefonte, U.S.). Detection limits were 20 µg/L for TCE and cis-DCE, 6 µg/L for VC, and 2 µg/L for ethene. Ethanol was separated on a 30 m × 0.32 mm × 0.25 µm film Zebron ZB-FFAP column (Phenomenex) installed in HP 5890 series II GC/FID and HP7673 autosampler. Upon termination of the column tests, soil samples from columns 2, 3, and 6 were analyzed for TCE. To obtain soil samples, the column bottom cap was removed and soil was gradually extruded from the column bottom. Twenty-fivegram samples of soil were immediately taken upon extrusion of approximately a 3-cm-thick layer of soil and placed in 25-mL screw top glass vials containing 10 g of methanol. This sampling procedure was continued for the entire contents of each of the columns, yielding soil samples from the columns at approximately 3-cm intervals along the column lengths. Vials were weighed, capped (with Teflon lined septa), shaken vigorously, and left for 24 h to equilibrate. Fifty to one hundred microliters of methanol extract was subsequently spiked with a gastight syringe into 4 mL deionized water and diluted accordingly. These dilutions were analyzed for TCE using the same GC protocol as for porewater samples. Permanganate (MnO4-) was analyzed using a Spectronic20D+ spectrophotometer set at a wavelength of 525 nm. Soluble manganese ion (Mn2+) was analyzed on a Varian SpectrAA 20 atomic absorption spectrometer at a wavelength of 279.5 nm. Porewater samples were filtered, diluted, and acidified prior to analysis. Soil samples, tested for watersoluble and exchangeable Mn2+, were prepared as outlined in ref 25. Chloride (Cl-) and acetate ions in porewater were separated isocratically on an IonPac AS 11 column (45 mM NaOH in 20% methanol/water eluent) and an IonPac AS 9 column (8 mM Na2CO3 eluent), respectively, using an ion chromatograph equipped with a CD 20 conductivity detector (Dionex Corp.). 2890

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Real Time Quantitative PCR. DNA from column effluent samples was extracted using the Mo Bio Ultraclean water DNA Kit (BIOEnzymTC, Landgraaf, The Netherlands) according to the kit instructions. Total genomic DNA (from Eubacteria and Archaea) was quantified with nucleic acid stain assay (PicoGreen dsDNA Quantification Reagent, Molecular Probes Inc.) by measuring fluorescence on the DNA Engine Opticon 2 system (MJ Research Inc.) within the linear detection range as per manufacturer’s instructions. For the quantitation of DHC, the primers DHC-1F and DHC-264R, (E. I. Du Pont de Nemours & Co., patent pending) that amplify a 264-bp portion of the 16S rRNA gene from base 1 to base 264 of DHC were used. The detection limit for DHC, using 200-mL samples was five copies of 16S rRNA gene/mL

Results Phase I: Baseline Flush with Distilled Water (33 Days). Initial dissolution of TCE from the DNAPL into the soil porewater was not significantly different among the six columns. The maximum concentration of TCE measured was 920 mg/L. As shown in Figure 1(a-c), the concentration of dissolved TCE in port 6 (the top port, 55 cm above the TCE DNAPL) gradually declined after 33 days of upflow flushing with distilled water, prior to chemical oxidation treatment. On the basis of flow rate and dissolved TCE concentration measurements, the average mass of TCE removed during the 33 days of flushing was 4.1 ( 0.8 g (see Table 3). Since the flow rates in each column were identical (Qav ) 0.24 ( 0.016 L/day, total flow of 7.92 L over 33 days), this variation was due to the differences in porewater concentration of TCE observed during this stage of the experiment. Phase II: Chemical Oxidation with Potassium Permanganate (Day 0-22). During permanganate flushing, the course of reaction could be seen through the glass column wall as the purple permanganate front advanced leaving brown manganese dioxide (MnO2) deposit on the sandy soil. The effluent concentration of dissolved TCE decreased, and chloride concentrations increased (see Figure 1a and b and Figure 4e) after 4.5 days of flushing, corresponding to three pore volumes of flushing. In each column, the Cl(chloride) concentration reached a maximum and then diminished with the disappearance of TCE. A maximum Clconcentration of 1120 mg/L was recorded in the effluent of column 4, while maximum chloride in columns 1-3 did not exceed 900 mg/L. Column 3 exhibited the lowest concentrations of TCE and Cl- and the earliest permanganate breakthrough (see Figure 1b). Concentrations of chloride in the control columns (columns 5 and 6) were less than 2 mg/L. When breakthrough of permanganate became evident in each column effluent, permanganate flushing was terminated. Dissolved TCE concentration in the porewater decreased to below the detection limit (20 µg/L). The amounts of TCE oxidized (calculated from the mass of Cl- released) varied from column to column (see Table 3) but were similar to the amounts removed from the columns during the initial 33 days of flushing with distilled water. After cessation of permanganate addition, the TCE concentrations rebounded to about 1 mg/L in the flushed columns, as is evident from TCE concentration profiles in column 3 for day 15 and day 31 (see Figure 4e). Phase III: Postoxidation Flush with Distilled Water (Day 22-33). During Phase III, which lasted 11 days, permanganate was flushed out with distilled water until the concentration dropped below detection (0.1 mg/L). The low chloride concentrations remaining after the treatment were also removed by this postoxidation flushing. The concentration of TCE in the two control columns started dropping markedly after 60 days of continuous flushing with distilled water. The effluent of column 5 (control) had a TCE concentration of 62 mg/L while column

FIGURE 1. Effluent concentrations for (a) columns 1 and 2 (biostimulated), (b) columns 3 and 4 (bioaugmented), and (c) columns 5 and 6 (controls).

TABLE 3. TCE Removal from Columns parameter

col 1

col 2

col 3

col 4

col 5

col 6

column designation

biostimulated

biostimulated

bioaugmented

bioaugmented

control

control

dissolved TCE removed [g]

4.77

Phase I 4.91

4.24

4.31

3.67

5.23

TCE oxidized [g] TCE flushed [g]

4.65 1.48

Phase II & III 3.76 1.54

2.51 1.07

4.82 1.69

1.44

3.63

0.0056 0.0196 10.84

0.09

0.14

5.20

9.01 0.002

TCE biodegraded [g] TCE flushed [g] total TCE removed [g] TCE recovered from soil [g]

0.0345 10.93 0.002

Phase IV 0.0003 0.0208 10.21

6 had a concentration of 199 mg/L, but with a declining trend, as seen in Figure 1c. Phase IVa: Biostimulation with Acetate and Ethanol in Distilled Water (Day 33-112). After an initial 30 days of feeding 100 mg/L each of ethanol and acetate in distilled water (2.17 and 1.67 mM, respectively), no signs of microbial activity were evident. Ethanol and acetate profiles did not show any discernible consumption. The TCE concentrations also stabilized and reached similar levels (about 1 mg/L) as shown in Figures 2 and 3. (The other two treatment columns had similar trends, data not shown.) The measurement of low concentrations (0.4-1.3 mg/L) of dissolved manganese (Mn2+) in column effluents indicated the possibility of reduction taking place in the soil.

0.0057 0.0164 7.84 0.0015

At day 64, columns 3 and 4 were inoculated with the halorespiring KB-1 culture. One milliliter of fresh culture suspension was injected into each of the four bottom ports (ports 1-4) of these two columns. Since the first inoculation did not result in measurable acetate/ethanol consumption or TCE dechlorination after 30 days of electron donor addition, columns 3 and 4 were reinoculated with a larger volume of culture suspension (5 mL) at day 75. Columns 1 and 2, fed with the same mix of ethanol/acetate donors, were not bioaugmented to allow determination of the metabolic capabilities of indigenous microorganisms that may have remained after permanganate flushing. The initial 78 days of biostimulation did not produce any detectable microbial activity in columns 1 and 2. BioaugVOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Concentrations in the effluent of column 2 during biostimulation: IVa, distilled water addition; IVb, NASA groundwater; IVc, synthetic groundwater.

FIGURE 3. Concentrations in the effluent of column 3 during biostimulation and bioaugmentation: IVa, distilled water addition; IVb, NASA groundwater; IVc, synthetic groundwater. mentation of columns 3 and 4 also did not produce any microbial activity. No measurable consumption of donors was detected, and concentrations of dissolved Mn2+ were still very low (0.5-0.8 mg/L). The TCE concentration was steady (about 1 mg/L, see Figures 2 and 3), and neither methane nor products of TCE dechlorination were detected in any of the four treatment columns. During this period of biostimulation, the pH in the column effluents was in the range of 7.3-7.8. Phase IVb: Biostimulation with Acetate and Ethanol in NASA Groundwater (Day 112-180). At day 113, feed to the treatment columns was switched from distilled water to groundwater from LC-34, amended with acetate and ethanol (100 mg/L each). Within 20 days, evidence of a shift to 2892

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increasingly anaerobic conditions was observed in the bioaugmented columns (columns 3 and 4). A small irregular beige (the same color as the original sand color) zone (3.5cm long × 1.7-cm wide) appeared above the sampling port 3 (35 cm from the inlet) of column 3. In addition, the interior of this zone became dark gray indicating that Mn(IV) reduction had started, leading to disappearance of brown MnO2 precipitate. Formation and spreading of the dark grayish zones within the beige ones likely indicated that reduction of sulfates (characteristically precipitated as iron sulfide) was occurring. The concentration of dissolved Mn2+ in column effluent increased over 15 days (from day 125-140, see Figures 2 and 3), thus linking the visual changes to the development of

FIGURE 4. Column 3 concentration variations with distance from column inlet (bottom) during biostimulation and bioaugmentation. reducing conditions favoring Mn reduction. The entire bottom portion of dark brown soil around sampling port 1 of the bioaugmented columns turned beige within 30 h. The same pattern of color change associated with Mn reduction was observed with the soil in columns 1 and 2 (not bioaugmented with dechlorinating bacteria). Complete consumption of ethanol occurred concurrently with manganese reduction, with consumption of ethanol resulting in an increase in acetate concentration. Methanogenesis also started (see Figures 2 and 3). At day 133, cis-DCE was detected in the bioaugmented columns (3 and 4). The TCE concentration in the effluent decreased, coinciding with generation of cis-DCE [see Figure 3b, data for column 4 not shown]. No cis-DCE, VC, or ETH were detected in the biostimulation columns indicating that dechlorination of TCE was not occurring. The depth profiles shown in Figure 4 indicate that all ethanol was fermented to acetate around 142 days since the concentration of acetate almost doubled in the porewater at the bottom of each column at this time (the same was observed for all four treatment columns, data not shown). Concentrations of dissolved Mn2+ increased during groundwater feeding (see Figure 4c). The manganese reduction was the most prominent in column 4 after 162 days when the concentration of Mn2+ reached 1600 mg/L in port 1 but dropped to 100 mg/L in port 2 only 10 cm higher in the column (data not shown, similar trends were persistent during this phase for all four treatment columns). Microbial growth also started in the feed bottle, as indicated by the cloudy appearance of the feed solution, H2S odor and blackening on the inlet tubing walls, and consumption of ethanol and acetate in the inlet lines to the columns. To minimize biodegradation in the feed bottle, feeding of acetate ceased after day 151. The amount of

reducing equivalents available for dechlorination, Mn reduction, and methanogenesis was now reduced from to 39.4 meq/L to 26.1 meq/L with only 100 mg/L of ethanol fed to the columns. Generation of cis-DCE in bioaugmented columns (3 and 4) decreased concurrently with an increase in methane production [see Figure 3b]. A similar increase in methane production (see Figure 2) was observed in the biostimulation columns in which cis-DCE was not detected. Phase IVc: Biostimulation with Ethanol in Synthetic Groundwater (Day 180-290). After 180 days of biostimulation, the composition of the feed was changed and synthetic groundwater was used. The new groundwater contained two parts of distilled water with inorganic salts mixed with one part of site groundwater and 100 mg/L of ethanol (no acetate added). To minimize ethanol degradation in the feeding tank, NASA groundwater was vacuum filtered through a 8-µm pore size cellulose ester membrane (Millipore Corp). This procedure combined with frequent changing of the tubing was useful in controlling the biodegradation of ethanol in the inlet tank and lines. Rapid conversion of ethanol to acetate occurred in both the biostimulation and bioaugmention columns (Figure 4a). At day 218, the ethanol concentration in the feed was increased to 200 mg/L. This did not affect methane concentrations, which were constant after day 180 in both the biostimulation and bioaugmentation columns. In addition, there were no significant increases in Mn2+ concentrations (see Figure 2). At day 216, column 3 was bioaugmented with KB-1 for the third time. Six days after bioaugmentation, both vinyl chloride and ethene were detected [see Figure 3c]. The production of cis-DCE in the effluents of the bioaugmented columns (columns 3 and 4) increased slightly after the ethanol influent concentration was increased, but dechlorination past cis-DCE did not occur in column 4 (data VOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. Concentration of Mn2+ in soil and porewater after termination: (a) column 2 (biostimulated) after 253 days and (b) column 3 (bioaugmented) after 293 days.

TABLE 4. Biomass Quantification Results for Day 148 source

total DNA (ng/mL)

D. Ethenogenes 16S gene copies/mL

raw NASA LC 34 groundwater feed bottle column 1 (biostimulated) column 2 (biostimulated) column 3 (bioaugmented) column 4 (bioaugmented) column 5 (control) column 6 (control)

0.8 23.2 0.5 0.4 0.7 1.1 2.7 2.9

29 nd 147 nd 87 39 44.5 nd

not shown) which was not rebioaugmented on day 216. cisDCE (16 µg/L) was detected for the first time in column 1 (not bioaugmented) at day 222 and persisted at an average concentration of 23 µg/L for 63 days until the end of the study. cis-DCE was not detected in column 2 (not bioaugmented) at any time during the study. Termination of Column Tests. Column 6, which was not treated with permanganate nor stimulated with electron donors, was terminated at day 243 (276 days of flushing with distilled water). The remaining mass of TCE in this column was determined from soil samples taken at 3-cm intervals along the column. The average concentration of TCE from these samples was 1 mg/kg dry soil, and there were no significant trends with distance from the column inlet, indicating that most of the TCE recovered was likely sorbed, rather than TCE DNAPL which would have had much higher concentrations near the column inlet. Flushing of column 2, 2894

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treated with permanganate and subsequently biostimulated with electron donors, was terminated at day 254. Column 3, treated with permanganate and biostimulated with electron donors, was also bioaugmented three times prior to the termination at day 260. In addition to TCE, soil from these two columns was analyzed for soluble and exchangeable manganese. The average concentrations of TCE were 0.9 and 0.7 mg/kg dry soil in columns 2 and 3, respectively. Recovery of manganese from the column soil is shown in Figure 5 a and 5b for columns 2 and 3, respectively. The soil in both columns had significant concentrations of exchangeable manganese (weakly held and easily displaced by other cations). Soluble Mn2+ measured in the soil before placement in the columns was 0.37 mg/kg dry soil. The measured exchangeable background Mn2+ in the soil before placement in the columns was 5.34 mg/kg dry soil. Soluble Mn2+ in the effluents of the control columns 5 and 6 was 0.05 mg/L.

Quantitative PCR Results. Quantitative PCR for total DNA and for DHC was performed on samples of site groundwater, water from the feed bottle, and column effluents at day 148. The results (Table 4) indicate that biomass growth occurred in the feed bottle, as total DNA was higher in the feed bottle than in the site groundwater. The total DNA concentrations were also higher in the control column effluents than in the effluents of columns that were flushed with permanganate. DHC was detected in the site groundwater and in the effluents from one control column, one biostimulation column, and both bioaugmentation columns. The concentration of DHC in the feed bottle, in the second control column, and in the second biostimulated column was below the detection limit of five 16S gene copies/mL.

Discussion Initial TCE Dissolution and Permanganate Flushing. During the initial flushing of the columns with distilled water, effluent dissolved TCE concentrations were in the range of 600-800 mg/L, below the solubility limit of 1100 mg/L (26). Variability in TCE concentrations between columns was likely due to variation in the distribution of the TCE DNAPL between the columns and corresponding variation in DNAPL-water contact areas. The TCE concentrations in the control columns decreased during the first 60 days of flushing to about 1 mg/ L. By the end of the study, dissolved TCE concentrations in the control columns were approximately 0.2 mg/L. This pattern of concentrations is consistent with studies of DNAPL dissolution in which an initial phase of high-dissolved-phase concentrations near solubility limits is followed by a long period of dissolution with low-dissolved-phase concentrations (9). During permanganate flushing of columns 1-4, TCE concentrations were reduced to below detection limits. Chloride concentrations up to 900 mg/L, corresponding to oxidation of approximately 1100 mg/L of TCE, were observed in columns 1-3, while the maximum chloride concentration observed in the effluent of column 4 during permanganate flushing was 1120 mg/L, corresponding to oxidation of 1380 mg/L of TCE. These results indicate slight enhancement of TCE DNAPL dissolution because of oxidation, as amounts of TCE oxidized were greater than the concentrations of aqueous-phase TCE measured in the columns before permanganate flushing (maximum of 920 mg/L). Following cessation of permanganate flushing, the concentration of TCE rebounded to approximately 1 mg/L. Thus, although permanganate flushing removed TCE more rapidly than water flushing, the residual-dissolved TCE concentrations after removal of the bulk of the TCE DNAPL were similar, indicating that a polishing technology such as biostimulation or bioaugmentation is required. Microbial Activity Following Permanganate Flushing. Following permanganate flushing, the biostimulation and bioaugmentation columns were fed ethanol and acetate in distilled water to stimulate microbial activity and establish reducing conditions conducive to reductive dechlorination. In an ongoing study in our laboratory of bioremediation of TCE DNAPL in soil from LC-34, indigenous microorganisms in the soil were capable of degrading ethanol and establishing reducing conditions that resulted in methanogenesis after 30 days. However, in the current study, no microbial activity was detected in either the biostimulated or bioaugmented columns during the addition of acetate and ethanol in distilled water, from day 33 to day 112. After the column influent was switched from distilled water to site groundwater (on day 113), ethanol and acetate degradation commenced within 20 days (day 133). The total DNA results (Table 4) indicate that bacteria were present in the column feed, suggesting that the onset of degradation of ethanol and acetate in the columns may have

been due to the bacteria introduced with the groundwater feed. However, it is also possible that there were insufficient nutrients and salts available in the distilled water and in the column soils to support the growth of indigenous ethanol and acetate utilizing bacteria that either survived permanganate flushing or were present in the KB-1 culture added to the bioaugmented columns. The column effluent total DNA levels were lower for permanganate-flushed columns (columns 1-4) compared to the control columns (columns 5 and 6 fed only distilled water for the entire study). However, the differences are not likely significant, given the low DNA levels measured in all column effluents. In a study involving permanganate flushing combined with soil mixing (5), bacterial enumeration indicated that the natural aerobic and anaerobic microorganisms were not adversely affected by permanganate treatment. However, the site was initially under aerobic conditions, and anaerobic counts before and after treatment were low in both treated and background soil samples. Reductive Dechlorination. Once biodegradation of ethanol and acetate commenced with the feeding of site groundwater to the columns, TCE dechlorination to cis-DCE occurred in the bioaugmented columns. The KB-1 culture, which did not initiate acetate and ethanol degradation between day 33 and day 112, was able to survive and commence TCE dechlorination when reducing conditions were established after switching the column influents from distilled water to groundwater after day 112. This TCE dechlorination was occurring concurrent with the reduction of MnO2 and production of levels of Mn2+ in the range of 400 mg/L. This is in contrast to the findings of ref 16, in which TCE dechlorination under both iron and manganese reducing conditions could not be induced in lab microcosms. Dechlorination to VC and ethene was only observed in column 3 after it was bioaugmented a third time, after ethanol and acetate degradation, Mn reduction, and methanogenesis had commenced. Dechlorination past cis-DCE did not occur in column 4 (data not shown) which was not bioaugmented a third time. Although the KB-1 culture was able to maintain the ability to degrade TCE when inoculated into the column immediately following permanganate flushing, it appears to have lost the ability to degrade cis-DCE. It is also possible that competition for electron donor with methanogens and manganese-reducing organisms inhibited cis-DCE degradation in column 4, but this should have also inhibited cis-DCE degradation after the third bioaugmentation in column 3 if it was the reason for lack of cis-DCE degradation. In contrast, previous bioaugmentation of KB-1 into a reduced subsurface without indigenous terminal halorespirers in a pilot scale field test (21) resulted in successful dechlorination with no stalling at cis-DCE. The quantitative PCR results (Table 4) indicated that detectable levels of D. ethenogenes were present in the site groundwater used for column feed and in column 1 which was not bioaugmented. The onset of transformation of TCE to cis-DCE in column 1 at day 222 may have been associated with these D. ethenogenes. Manganese Reduction. Only trace levels of Mn2+ were detected in the flushing with acetate and ethanol from days 33 to 112 (see Figures 2 and 3). Abiotic reduction and dissolution of MnO2 was not likely to be initiated by ethanol and acetate alone (27). Manganese reduction, mediated by iron- and manganese-reducing bacteria, was however expected to occur given that these bacteria are ubiquitous heterotrophs found in soil and sediments (28-31). The lack of manganese reduction indicates that if manganese-reducing bacteria were initially present in the soil they were inactivated by permanganate flushing or were not able to initiate manganese reduction when the columns were being flushed with distilled water. VOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Reductive dissolution of MnO2, indicated by color changes in the columns, and production of Mn2+ started as soon as ethanol and acetate consumption commenced with addition of unsterilized groundwater indicating the activity of manganese-reducing bacteria that could use acetate as electron donor (30). Levels of Mn2+ were highest at port 1 and decreased significantly between port 1 and port 3 from concentrations above 400 mg/L to concentrations below 100 mg/L (Figure 4c). This decrease in Mn2+ concentrations may have been due to ion exchange of Mn2+ with Ca and Mg (effluent was not analyzed for Ca and Mg) and precipitation as MnCO(3) (31). The maximum exchangeable Mn concentrations were measured in soil from the bottom portions of the columns (approximately from 1 to 20 cm from inlet, Figure 5a, 5b), where the greatest decrease in dissolved Mn2+ concentrations was observed. However, dissolved manganese concentrations at the column outlet still greatly exceeded the U.S. EPA secondary drinking water standard of 50 µg/L. Need for Bioaugmentation. The detection of cis-DCE in column 1 near the end of the study suggests that the first step of TCE reductive dechlorination is possible without bioaugmentation. The significant lag time until detectable dechlorination was likely related to low levels of dechlorinators present in the soil and groundwater and the long time required for a significant dechlorinating population to build up in the column. Bioaugmentation significantly reduced the time for initiation of detectable dechlorination. Bioaugmentation with KB-1 also led to complete conversion of TCE to ethene. It is not known if dechlorination to ethene would eventually have occurred without bioaugmentation. The results for the two bioaugmentation columns suggest that inoculation of dechlorinating cultures into oxidized conditions may impair the ability of the culture to subsequently degrade cis-DCE, even when reducing conditions are reestablished. Therefore, following permanganate flushing, bioaugmentation with a dechlorinating culture should not be implemented until active electron donor fermentation and Mn reduction is established at a site. Implications for Site Remediation. This study has illustrated that a sequential treatment approach consisting of permanganate flushing followed by biostimulation and bioaugmentation is feasible for treatment of TCE contamination. Permanganate flushing is expected to affect the indigenous microbial population, and recolonization of the treatment area from migration of microorganisms from adjacent untreated areas, or through groundwater flushing, may be required before significant microbial activity will be observed in the treatment area. Biostimulation will be necessary to establish reducing conditions conducive to reductive dechlorination. If DHC is not initially present at the site, bioaugmentation with DHC containing cultures, following establishment of reducing conditions through biostimulation, may lead to conversion of chlorinated ethenes to ethene. At sites which contain DHC, bioaugmentation with dechlorinating cultures may significantly reduce the time to onset of significant rates of reductive dechlorination. The potential for biostimulation to release unacceptably high levels of Mn2+ should be considered in design of the site remedy and in the comparison of alternate remediation technologies.

Acknowledgments We express our thanks to Melanie Duhamel for providing KB-1 culture and information she generously shared with us, Dr. Kaiguo Mo for performing DNA analyses, and Prof. Elizabeth Edwards at Department of Chemical Engineering at U of Toronto for use of the analytical equipment and for her valuable advice and help. This research was funded by ESTCP under agreement CU-0116. 2896

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Received for review June 29, 2004. Revised manuscript received January 28, 2005. Accepted February 1, 2005. ES049017Y

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