Environ. Sci. Technol. 1998, 32, 1222-1229
Anaerobic Benzene Oxidation in the Fe(III) Reduction Zone of Petroleum-Contaminated Aquifers ROBERT T. ANDERSON Department of Civil and Environmental Engineering, University of Massachusetts, Amherst, Massachusetts 01003 JULIETTE N. ROONEY-VARGA, CATHERINE V. GAW, AND DEREK R. LOVLEY* Department of Microbiology, Morrill Science Center IV North, University of Massachusetts, Amherst, Massachusetts 01003
The potential for anaerobic benzene oxidation in the Fe(III)reduction zone of petroleum-contaminated aquifers was evaluated. Sediments were incubated under strict anaerobic conditions without any amendments in order to simulate in situ conditions. [14C]Benzene was not oxidized to 14CO2 at most sites examined, which is consistent with previous studies that have found that benzene tends to persist in unamended, anaerobic aquifer materials and/or long periods of time are required in order to adapt the microbial population for benzene degradation. However, at one site located in Bemidji, MN, [14C]benzene was oxidized to 14CO2 in unamended sediments without an apparent lag, suggesting that benzene was anaerobically degraded in situ. Benzene was not significantly oxidized in sediments from nearby Fe(III)-reducing sites nor in sediments collected from an uncontaminated background site in the same aquifer. Culturing and 16S rRNA-based molecular studies of the Bemidji aquifer demonstrated that while all sites contained similar numbers of Fe(III)-reducing microorganisms closely related to Geothrix fermentens, the site at which anaerobic benzene degradation was observed was greatly enriched with microorganisms in the family Geobacteraceae. This study provides the first data consistent with in situ anaerobic oxidation of benzene to carbon dioxide in the Fe(III)reducing zone of a petroleum-contaminated aquifer and suggests that comparative studies on the size of the Geobacteraceae community in petroleum-contaminated aquifers might aid in the location of zones in which benzene degradation coupled to Fe(III) reduction is taking place.
Introduction The potential for benzene to be oxidized with the reduction of Fe(III) in petroleum-contaminated aquifers is of interest because petroleum-contaminated aquifers often contain extensive anaerobic zones and Fe(III) is generally the most abundant electron acceptor for organic matter oxidation in these systems (1-3). Thus, benzene oxidation coupled to Fe(III) reduction could potentially remove significant quantities of benzene from contaminated aquifers. Previous studies with samples from a petroleum-contaminated aquifer located in Hanahan, SC, demonstrated * Corresponding author e-mail:
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that benzene can be oxidized with the reduction of Fe(III) in laboratory incubations of aquifer sediments, but only under artificial conditions not typically found in aquifers. For example, benzene was oxidized to carbon dioxide with the reduction of Fe(III) in aquifer sediments that were amended with synthetic Fe(III) chelators (4). The chelators stimulated Fe(III) reduction by solubilizing Fe(III) from insoluble Fe(III) oxides (5) and making the Fe(III) more accessible for microbial reduction. Addition of humic substances also resulted in anaerobic benzene oxidation under Fe(III)-reducing conditions (6), presumably because humics can also alleviate the need for Fe(III) reducers to directly access insoluble Fe(III) oxides by acting as an electron shuttle between Fe(III) reducers and insoluble Fe(III) oxides (7). However, when the sediments were incubated without amendments of chelators or humics in order to simulate in situ conditions, there was no benzene degradation (4, 6). Thus, these studies suggested that although benzene oxidation coupled to Fe(III) reduction was possible in aquifer sediments, this process did not take place in situ in the aquifer studied. Studies in another petroleum-contaminated aquifer, located in Bemidji, MN, offered preliminary evidence that benzene might be degraded under Fe(III)-reducing conditions. There was a loss of benzene relative to a conservative tracer along the groundwater flow path within the anaerobic zone (8), and it was suggested that benzene was degraded under Fe(III)-reducing conditions in laboratory incubations of some of these sediments (9). However, close inspection of the data in that study reveals that the evidence for anaerobic benzene degradation in the laboratory incubations is not convincing. At the end of the 61 day anaerobic incubation study, the biologically active sediments still contained 74% of the initial benzene. Benzene concentrations in killed controls had decreased to 83% of the initial level. No statistics were presented to determine whether this slight difference between the two treatments was statistically significant. Furthermore, the fate of the lost benzene was not determined. Although it was reported that there was a “trace” of phenol in the biologically active sediments and it was suggested that this phenol was a product of benzene degradation, there was no direct evidence that the phenol was derived from benzene oxidation. To further evaluate the potential for anaerobic benzene oxidation in the Fe(III)-reducing zone of petroleumcontaminated aquifers, oxidation of [14C]benzene in relatively undisturbed sediments from the Fe(III) reduction zones of several petroleum-contaminated aquifers was monitored. The results demonstrate that anaerobic benzene oxidation is rarely detected, and when it is, it may be restricted to narrowly confined portions of the Fe(III) reduction zone. However, the results also suggest that assaying for specific Fe(III)-reducing populations may aid in predicting the zones in which anaerobic benzene oxidation is taking place.
Materials and Methods Study Sites and Sample Collection. Sediments from three petroleum-contaminated aquifers were evaluated for the capacity to anaerobically degrade benzene under Fe(III)reducing conditions. One aquifer was the same site at which anaerobic benzene oxidation coupled to Fe(III) reduction was first documented (4). This aquifer is located under a tank farm facility in Hanahan, SC, that has had several petroleum releases over the last 50 years (10, 11). Sediments for the study reported here were collected in 1995. Sediments were also collected in 1995 from an underground storage tank site in Rocky Point, NC (12). At the time of sediment S0013-936X(97)00494-X CCC: $15.00
1998 American Chemical Society Published on Web 03/18/1998
FIGURE 1. Map of the Bemidji site showing the positions of the major sampling well clusters and core sample locations relative to the source area. This map is modified from those available through the USGS (71). The background site (not shown) is approximately 100 m upgradient from the source area. collection, this site had been contaminated for approximately 9 years. Previous geochemical studies had indicated that aromatic hydrocarbon components were being removed from this site, and the accumulation of Fe(II) in the groundwater suggested that microbial Fe(III) reduction might be an important process (12). The third site was the USGS Groundwater Toxics Site in Bemidji, MN. This site has been contaminated with crude oil for 18 years as a result of a break in an oil pipeline (8, 13-15). As discussed in the Introduction, previous studies had suggested that benzene might be degraded anaerobically at this site and that Fe(III) reduction was taking place in the anaerobic zone of the aquifer. Sediments were collected from Bemidji in 1995 and 1997 at the sites shown in Figure 1. Sediment samples were obtained either from drilling cores (16) if a drill rig was available or by split spoon sampler (hand auger) and transported immediately to the laboratory. Sediments were homogenized and transferred to sample vessels in a N2-filled glovebag. For MPN analyses, sediment samples (1-2 g) were added to pressure tubes for later dilution as described below. For molecular analyses, 20 g aliquots were placed into sterile centrifuge tubes and stored at -80 °C until used. For sediment incubations, 30 g was transferred to 50 mL serum bottles and the headspace of each serum bottle was then flushed with a stream of N2:CO2 (95:5) which had been passed through a furnace containing hot copper filings to remove residual oxygen. No liquid amendments other than the solutions of radiolabeled substrates were added to the sediment bottles in order to preserve the in situ conditions of the sediment (moisture, ionic strength, etc.) as much as possible. Total liquid amendments rarely exceeded 1 mL/30 g of sediment. Sediment Incubations. All sediments and associated pore waters had a pH of 6.16 ( 0.30 units (mean ( standard deviation). All the sediments were anaerobic and nitrate depleted. Total HCl-extractable iron was greater than HClextractable Fe(II) at all sites, indicating that they contained microbially reducible Fe(III) (17). In order for Fe(III) reduction to be considered the terminal electron-accepting process (TEAP) in the sediments the following previously described criteria (18) had to be met: (1) sediments must be
depleted of nitrate; (2) sediments, must contain Fe(II); (3) [2-14C] acetate must be converted to 14CO2 with no 14CH4 production in anaerobic sediment incubations; and (4) molybdate, a specific inhibitor of sulfate reduction, must not inhibit anaerobic oxidation of [2-14C]acetate to 14CO2. [U-14C]Benzene (58.2 mCi/mmol), [U-14C]toluene (60 mCi/mmol), and [2-14C]acetate (44.5 mCi/mmol) were obtained from Sigma Chemical Co., St. Louis, MO. The chemical purity of all compounds was greater than 98%. Aqueous stock solutions of the hydrocarbons were prepared by aseptic, anaerobic transfer of the radiolabeled compounds to sterile, anaerobic, deionized water to a concentration of 1-2.5 µCi/mL using an anaerobic adaptation of the manufacturer’s recommended transfer method. Sediment incubations were amended with 1 µCi of radiolabeled benzene or toluene. Acetate-amended samples contained 0.25-0.5 µCi of [2-14C]acetate. Production of 14CO2 and 14CH4 in the headspace was monitored over time using gas chromatography coupled with a gas proportional counter detector as previously described (18). Total 14CO2 was determined from 14CO in the headspace and the partitioning of H14CO - added 2 3 to sediments as previously described (19). Cultivation-Based Evaluation of Microbial Populations. The Fe(III)-reducing population in all sediments was estimated with a 3-tube most probable number analysis (MPN) using an adaptation of a previously described Fe(III) reducer media (20). Briefly, this media contains a poorly crystalline Fe(III) oxide (30 mM) as an electron acceptor, 4 mM nitrilotriacetic acid (NTA) as an Fe(III) chelator, 2 mM acetate as the electron donor, and 1.3 mM Fe(II) chloride as a reductant. Each initial dilution tube (10-1) also contained 0.1% pyrophosphate to release cells bound to the sediment (21, 22). Fe(III) reduction was initially monitored by noting the production of magnetite and later confirmed by measuring Fe(II) content with ferrozine (23). To determine the phylogeny of the dominant acetateoxidizing Fe(III) reducers recovered in the MPN analysis from the Bemidji site, partial 16S rRNA sequences of the organisms in the highest positive dilutions were determined. A total of 0.5 mL of each culture was added to 1 mL of filter-sterilized 300 mM oxalate in order to chelate traces of Fe(III) from the sample which can inhibit the PCR (24). The cells were pelleted by centrifugation, the supernatant discarded, and the pellet resuspended in 250 µL of 10 mM Tris buffer (pH 8.0). Nucleic acids were released from cell suspensions by subjecting them to three freeze-thaw cycles at -70 and 65 °C, and the resulting lysate was extracted with phenol and chloroform-isoamyl alcohol (25). A total of 5 µL of the resulting aqueous phase was used as template for the PCRs. A portion of the 16S rDNA was amplified with primer 8F-GC (26) containing a 40 base pair GC clamp (27) (5′-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGAACACATGCAAGTCGAACG-3′) and a reverse primer (5′- GTATTACCGCGGCTGCTGG-3′) derived from primer 519R (28). PCR conditions were essentially as previously described (29), except that each 100 µL reaction contained 1.5 mM MgCl2 and 2.5 units of AmpliTaq (Perkin-Elmer Cetus, Norwalk, CT). To minimize production of nonspecific PCR products, touchdown primer annealing (30) from 65 to 55 °C (decreasing 0.5 °C per cycle) was used followed by 10 cycles at 55 °C. The PCR products were separated on denaturing gradient (5570%) acrylamide gels (27) and stained with ethidium bromide (25). Isolated bands were excised and reamplified as described above, except that no GC clamp was incorporated into primer 8F. These PCR products were sequenced as previously described (31). The sequences were compared to sequences in GenBank and the Ribosomal Database Project (RDP) using BLASTN (32) and SIMILARITY-RANK (33), respectively. The sequences were manually aligned to 16S rDNA sequences of bacteria obtained from the RDP (33). VOL. 32, NO. 9, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Potential Electron Acceptor Availability in a Variety of Petroleum-Contaminated Aquifer Sediments, Methane Production, the Effect of Molybdate on Acetate Mineralization, and MPN Values for Acetate-Oxidizing Fe(III) Reducers [2-14C]acetate
North Carolina Hanahan MW20 Hanahan W1 Hanahan E10 Bemidji 95-1 Bemidji 95-2 Bemidji 97-1 Bemidji 97-2 Bemidji 97-3 a
nitrate (µM)
sulfate (µM)
Fe(II) (mmol/kg)
Fe(III) (mmol/kg)
14CH 4 produced?
14CO production 2 inhibited by molybdate?
acetate-oxidizing Fe(III) reducers MPN (g sed)
