Environ. Sci. Technol. 1996, 30, 1667-1674
Microbial Reduction of Cr(VI) during Anaerobic Degradation of Benzoate HAI SHEN,† P. HAP PRITCHARD,‡ AND G U Y W . S E W E L L * ,† U.S. EPA, National Risk Management Research Laboratory, Subsurface Protection and Remediation Division, Ada, Oklahoma 74820, and U.S. EPA, National Health and Environment Exposure Research Laboratory, Gulf Ecology Division, Gulf Breeze, Florida 32561
A series of microcosms and enrichments were conducted to evaluate the potential for microbially mediated Cr(VI) reduction linked to benzoate catabolism. Bacterial degradation of benzoate with the transport of electrons to Cr(VI) was achieved using nitrate or molecular oxygen as an initial stimulator. After depletion of nitrate or oxygen, microcosms and enrichments still retained the capacity for benzoate degradation linked to Cr(VI) reduction. Experiments demonstrated that benzoate degradation occurred concurrently with the reduction of Cr(VI) and was strongly dependent on the presence of Cr(VI). Benzoate degradation proceeded as long as Cr(VI) was present, ceased when Cr(VI) was completely removed, and continued once Cr(VI) was added. Furthermore, the observed benzoate removal, linearly correlated with Cr(VI) consumed, and the stoichiometric ratio were in good agreement to the theoretical ratio for the complete oxidation of benzoate to carbon dioxide coupled to the reduction of Cr(VI) to Cr(III). The addition of nitrate up to a concentration of 5.0 mM did not inhibit Cr(VI) reduction but enhanced benzoate degradation in the enrichments. This process may provide the opportunity for in-situ biotreatment of Cr(VI) in subsurface environments.
Introduction Microorganisms are capable of using metals, including Fe(III), Mn(IV), Se(VI), U(VI), Hg(II), As(V), and Cr(VI), as terminal electron acceptors in anaerobic metabolism (15). The microbially mediated metal transformation usually alters the toxicity and solubility of the metals and thus may provide the potential for in-situ bioremediation of subsurface contamination (5-7). Microbial reduction of Cr(VI), U(VI), As(V), and Se(VI) to their insoluble reduced forms may offer a cost-effective mechanism for controlling the * Corresponding author telephone: (405) 436-8566; fax: (405) 4368703. † National Risk Management Research Laboratory. ‡ National Health and Environment Exposure Research Laboratory.
This article not subject to U.S. Copyright. Published 1996 by the American Chemical Society.
mobility of these contaminates in the subsurface and thus eliminate or mitigate their impacts on human health and subsurface ecosystems. Microbial reduction of Fe(III) and Mn(IV), similar to the reduction of nitrate and sulfate, may be coupled to the anaerobic oxidation of aromatic compounds and serve as an important mechanism for the removal of these contaminants when oxygen becomes limited in the subsurface environment (1). Microbial reduction of Cr(VI) and its potential application have been extensively studied and reviewed (6, 7). The microbial transformation of Cr(VI) to the more immobile and less toxic Cr(III) may be developed as useful alternatives for the remediation of contaminated groundwater since conventional pump-and-treat technologies have limitations in terms of time, cost, and effectiveness (8). Although anaerobic reduction of Cr(VI) by bacteria has been demonstrated in several studies, the quantitative analysis of the relationship between Cr(VI) and oxidizable substrates was only recently determined by Lovley and Phillips, using H2 as an electron donor (9). No evidence has been presented that Cr(VI) can be used as an electron acceptor for microbial degradation of aromatic compounds under anaerobic conditions. The use of aromatic compounds as an electron donor for microbial reduction of Cr(VI) represents the potential for simultaneous detoxification of organic and inorganic contaminants. In the real world, polluted sites often contain a mixture of metals and organic compounds since industrial processes, including leather tanning, metal finishing operations, and petroleum refining, may discharge Cr(VI) and aromatic compounds simultaneously (10). For example, a commonly used woodpreserving pesticide consists of chromate, copper, arsenate, and aromatic compounds (11). Therefore, it is not surprising that Cr(VI) and aromatic compounds have been concurrently detected at contaminated sites (12, 13). Recently, efforts have been devoted to investigating the simultaneous detoxification of Cr(VI) and phenolic compounds using either a consortia of aerobic bacteria or abiotic redox reactions under acidic conditions (14, 15). However, the in-situ bioremediation of contaminated subsurface soils and groundwater may require an anaerobic process occurring at a near-neutral pH range to minimize impacts of remediation processes on impacted ecosystems. In this study, evidence is provided that biological processes may facilitate simultaneous reduction of Cr(VI) and degradation of benzoate.
Experimental Section Aquifer Material. Subsurface core material contaminated with chromium (e12 mg/kg of soil) was obtained from Elizabeth City, NC. The sample was stored at 4 °C in a glass container sealed with an air-tight snap lid before use. Medium. A mineral salt medium was prepared by dissolving the following chemicals in 1.0 L of distilled water: 800 mg of (NH4)2HPO4, 200 mg of KH2PO4, 100 mg of NaCl, 200 mg of MgSO4‚7H2O, 100 mg of CaCl‚2H2O, 5 mg of FeCl3‚6H2O, and 1 mg of Na2MoO4‚2H2O. Following autoclaving at 121 °C for 25 min, the medium, while still warm, was moved into a vacuum/gas interchange of an anaerobic chamber (Coy) where it was flushed twice with N2 before being equilibrated with the incubation gas mix
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(92.7% N2 and 7.5% H2). The medium was allowed to sit overnight or longer in the anaerobic glovebox to remove any traces of oxygen before use. The chamber atmosphere was continuously circulated by two fan boxes through palladium catalysts, which in the presence of hydrogen quickly removed any trace of oxygen. A Coy oxygenhydrogen analyzer (Laboratory Products Inc. Model 10, Grass Lake, MI) in the chamber indicated that no detectable oxygen (e1 ppm) was present during constructing or sampling of the experimental systems in the anaerobic chamber. After sitting overnight, 1 mL of sterilized trace metal solution (2850 mg of H3BO3, 1360 mg of FeSO4‚7H2O, 1800 mg of MnCl2‚4H2O, 1770 mg of sodium tartrate, 20.8 mg of ZnCl2, 26.9 mg of CuCl2‚2H2O, and 40.4 mg of CoCl2‚ 6H2O/L of distilled water) and 10 mL of filter-sterilized (0.45 µm) vitamin stock solution (2 mg of biotin, 2 mg of folic acid, 10 mg of pyridoxine hydrochloride, 5 mg of riboflavin, 5 mg of thiamine, 5 mg of nicotinic acid, 5 mg of pantothenic acid, 5 mg of p-aminobenzoate, 0.1 mg of cyanocobalamin, and 5 mg of thioctic acid/L of distilled water) were added. Both the trace metal and vitamin solutions were maintained under anaerobic conditions following the same procedure prepared for the medium. The final solution was designated as NMS medium and had a pH of approximately 7. Solid media were prepared using purified agar (Difco) with the NMS medium at 2% (wt/vol). Following autoclaving at 121 °C for 25 min, the medium, while still hot, was moved into the glovebox, and acetate, lactate, benzoate, Cr(VI), and nitrate were added from stock solutions before the liquified media were poured into sterile Petri plates. The chromium-agar contained 100 mg/L of benzoate, lactate, or acetate with 10, 25, or 50 mg/L of Cr(VI). The nitrate supplemented chromium-agar included the same concentration as above of the individual carbon sources with 10 mg/L of Cr(VI) and 620 mg/L of nitrate. Microcosms. Microcosms were prepared aseptically using 1-L or 250-mL screw-cap bottles and were incubated at room temperature (22 °C) in the dark in the anaerobic chamber. All glasswares, caps, and preparation supplies were autoclaved at 121 °C for 25 min and were stored in the glovebox 1 day before use to remove any traces of oxygen. All operations and samplings were conducted at chamber atmospheric oxygen concentrations of less than 1 ppm (v/v) as determined by a Coy oxygen-hydrogen analyzer. Nitrate-Induced Experiments. In the initial test, 250 g of core materials was added into a 1-L bottle. The bottle was filled with 1.0 L of NMS medium, and then Cr(VI) and benzoate were added from stock solutions of K2CrO4 (20 g/L Cr) and sodium benzoate (14 g/L benzoate) to yield initial concentrations of 10 mg/L Cr(VI) and 15 mg/L benzoate. Following anaerobic incubation for 7 days, KNO3 was added into one bottle to achieve a final concentration of 1.0 mM, while no nitrate was added to others. The combined concentration of nitrate and nitrite was determined to be below 0.2 mg/L (N) after incubation for 20 days. Nitrate was not added again. When Cr(VI) or benzoate were depleted in the bottle, however, they were re-added. Following repeated respiking, the enrichment was used as a seed for subsequent experiments. For the subsequent tests, batch experiments with or without inoculations from the above enrichment were used to evaluate the potential of bacteria to reduce Cr(VI) with benzoate as the electron donor. A 50-g sample of the same aquifer material was placed into 250-mL bottles, and then
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200 mL of NMS medium was added. Two of the bottles were inoculated with 20 mL of liquid of the above enrichment while the others were not. Then Cr(VI) and benzoate were added into the bottle to achieve the desired concentrations. Abiotic controls were prepared according to the same procedure, except that the containers and aquifer material were autoclaved for 1 h at 121 °C on two successive days before Cr(VI) and benzoate were added. Oxygen-Induced Experiments. Enrichments induced with oxygen were made by mixing 50 g of the same aquifer solids with 200 mL of fresh NMS medium in 250-mL bottles. Instead of adding nitrate as a stimulating step, the bottles were first incubated aerobically in a stationary mode for 3 weeks after Cr(VI) and benzoate were added. The aerobic systems were maintained in an incubator with the screw caps loosened to allow for gas exchange with the laboratory atmosphere. The bottles were then moved into the anaerobic chamber at day 21 for incubation after being purged with nitrogen. Measurements with a DO probe (YSI Model 75, Yellow Spring, OH) indicated that oxygen levels in the liquid system were below detection limit (
