Microbially Driven Fenton Reaction for Transformation of

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Environ. Sci. Technol. 1999, 33, 1886-1891

Microbially Driven Fenton Reaction for Transformation of Pentachlorophenol ADONIA M. MCKINZI AND THOMAS J. DICHRISTINA* School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332

Fe(II) + H2O2 w •OH + Fe(III) + OH-

(1)

Under acidic conditions, the Fenton reaction is autocatalytic since Fe(III) catalytically decomposes H2O2 to regenerate Fe(II) (eqs 2 and 3) provided there is a continuous supply of H2O2 and HO2• remains protonated (pKa) 4.8) (4):

Fe(III) + H2O2 + H+ S {Fe-O2H}2+ S Fe(II) + HO2• (2) Fe(III) + HO2• w Fe(II) + H+ + O2

(3)

•OH

A microbially driven transformation system was developed for the oxidative degradation of pentachlorophenol (PCP). The system was based on a free radical-generating Fenton reaction between bacterially produced Fe(II) and H2O2. The Fe(III)-reducing, facultative anaerobe Shewanella putrefaciens strain 200 was used as a catalyst for both Fe(III) reduction and H2O2 production by alternating between anaerobic and aerobic conditions in liquid batch cultures supplemented with Fe(III). The highest observed PCP degradation rate was approximately 0.31 µM h-1. Tetrachlorohydroquinone (TCHQ) and tetrachlorocatechol (TCC) were formed as the principal PCP transformation products, indicating that PCP oxidation proceeded via hydroxyl radical (•OH) attack on the ortho and para positions of the aromatic ring. PCP was degraded, and TCHQ and TCC were produced in a chemically driven (biomimetic) system where H2O2 and Fe(II) were supplied at concentrations comparable to those detected in the microbially driven system. PCP was not degraded (and PCP transformation products were not produced) in a set of control experiments that included (i) the presence of Fe(II)-chelating agents or radical scavenging compounds, (ii) strict aerobic or anaerobic conditions, (iii) the substitution of NO3- for Fe(III) as anaerobic electron acceptor, and (iv) the omission of S. putrefaciens. The microbially driven Fenton reaction system operated at neutral pH and required neither addition of exogenous H2O2 nor UV irradiation to regenerate Fe(II). The newly developed system may provide the basis for novel Fenton-type bioremediation strategies.

Introduction Free radical-based oxidation technologies provide an attractive alternative to conventional treatment strategies for elimination of hazardous wastes (e.g., adsorption to activated carbon or reverse osmosis) (1). These treatment technologies harness the oxidizing potential of hydroxyl radicals (•OH) to degrade a wide variety of hazardous organic compounds, including the widely applied pesticide and wood preservative pentachlorophenol (PCP). •OH radicals are among the most reactive oxidants found in aqueous environments and can readily oxidize a variety of naturally occurring and contaminating organic compounds (2). •OH initiates a cascade of oxidation reactions that can lead to total mineralization of organic pollutants. •OH is produced by the reaction between Fenton reagents Fe(II) and H2O2 (eq 1) (3): * Corresponding author phone: (404)894-8419; fax: (404)894-0519; e-mail: [email protected]. 1886

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subsequently reacts nonselectively with most organic compounds at diffusion-controlled rates (k ) 106-1010) via extremely fast H-abstraction and electron addition to C-C unsaturated bonds (5). Although •OH is generally regarded as the primary reactant in Fenton reaction-based oxidations, other Fe(II)- and H2O2-dependent intermediates have also been implicated as active oxidants, including the peroxo (Fe(OOH)+) and ferryl (FeO2+) radicals (6). Fenton reaction-generated •OH has been used to treat a wide variety of hazardous organic compounds, including landfill leachates (7), groundwater contaminated with chlorinated aliphatics and aromatics (8, 9), drycleaning solvents (10, 11), PCB congeners (12), nitroaromatic compounds (13, 14), azo dyes (15), and PCP (16). Although Fenton reactionbased processes can effectively degrade a wide variety of hazardous organic compounds, continuous addition of Fe(II) and H2O2 is required. In •OH technologies operating at pH > 5, the continuous addition of Fe(II) also results in the generation of large quantities of particulate Fe(III), which contributes to sludge disposal problems (17). Radical oxidizing systems that utilize UV irradiation to induce photolytic radical production include O3-, H2O2-, TiO2-, and photo-Fenton-based systems. Such systems can be limited, however, by problems associated with UV light penetration (17-20). The photo-Fenton system has been used to overcome problems associated with the accumulation of Fe(III) (20). The photo-Fenton reaction (eq 4) is initiated by photoreduction of dissolved Fe(III) complexes and continuously regenerates Fe(II) in acidic solutions:

Fe(OH)2+ + hv w Fe(II) + •OH

(4)

Radical oxidizing systems based on the photo-Fenton reaction have been used to degrade a variety of hazardous organic wastes, including nitroaromatic compounds (21), polychlorinated phenols (22), chlorophenoxy herbicides 2,4-D and 2,4,5-T (23), compounds found in landfill leachates (17), and PCP (19). Although the photo-Fenton systems require orders of magnitude less Fe(II) than the conventional Fenton reaction-based oxidation systems, H2O2 must still be continuously supplied to the system (24), and acidic conditions must be maintained. The purpose of the present study was to design a microbially driven, Fenton reaction-based radical-generating system that operates under neutral pH conditions and requires neither the addition of H2O2 nor the photolysis of Fe(III) to catalyze the oxidative degradation of PCP. The system utilized the Fe(III)-reducing facultative anaerobe Shewanella putrefaciens strain 200 as a catalyst to produce H2O2 and reduce Fe(III) under alternating aerobic/anaerobic conditions. The expression of an Fe(III)- and O2-reductiondependent agar-hydrolyzing (AGR) phenotype by strain 200 provided the impetus for the present study (25). After several days of aerobic growth on iron(III) citrate-supplemented 10.1021/es980810z CCC: $18.00

 1999 American Chemical Society Published on Web 04/28/1999

nutrient agar, wild-type colonies hydrolyzed the agar support and sank to the bottom of the Petri dish (AGR-positive phenotype). It was hypothesized that the AGR-positive phenotype resulted from a classical Fenton reaction between H2O2 and Fe(II) (3, 26), the metabolic byproducts of aerobic respiration and Fe(III) reduction (which occurred simultaneously in the colony interior). The purpose of the present study was to determine if S. putrefaciens was capable of generating •OH to drive the oxidative transformation of PCP by simultaneously producing Fenton reagents (H2O2 and Fe(II)) as the metabolic byproducts of aerobic respiration and Fe(III) reduction.

Materials and Methods Culture Medium and Chemical Reagents. Unless otherwise noted, chemical reagents and growth media were obtained from Fisher Scientific. S. putrefaciens strains were grown aerobically on LB medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) (27). PCP-degradation experiments were conducted in a lactate (15 mM)-supplemented salt solution (LS; pH, 7.0) (0.5 g/L K2HPO4, 2.0 g/L Na2SO4, 1.0 g/L NH4Cl, 0.15 g/L MgSO4‚7H2O, and 0.5 g/L yeast extract) (28). Iron(III) citrate was prepared by previously described methods (25) and added at 10 mM final concentration. PCP was obtained from Aldrich. Tetrachlorohydroquinone (TCHQ), tetrachlorocatechol (TCC), diethylenetriaminepentacetic acid (DETAPAC), N,N-diethyl-1,4-phenylenediammonium sulfate (DPD), mannitol, sodium benzoate, and thiourea were obtained from Sigma. Peroxidase (POD) reagent was obtained from Boehringer, Mannheim. Development of a PCP-Acclimated Strain of S. putrefaciens Strain 200. Wild-type S. putrefaciens strain 200 was originally isolated from a crude oil pipeline (28) and has been alternately referred to in the literature as Pseudomonas sp. 200 (29), Pseudomonas ferrireductans (30), Alteromonas putrefaciens 200 (31), and more recently S. putrefaciens 200 (32). Aerobic growth of the wild-type strain 200 was inhibited by PCP at concentrations >1 mg/L (data not shown). A PCPacclimated strain was developed for use in the present study by exposing wild-type strain 200 to LB growth medium supplemented with increasing concentrations of PCP (1100 mg mL-1) in a continuous flow bioreactor over a 2-month incubation period. The continuous flow bioreactor consisted of a Biostat B bioreactor (B. Braun Biotech, Allentown, PA) that provided digital feedback control of temperature, pH, and dissolved oxygen concentration. The Biostat B was equipped with dosing pumps that allowed continuous input of PCP-containing LB from sterile stock solutions. Aliquots were withdrawn on a weekly basis, and purified colonies of the PCP-acclimated culture were tested for aerobic growth on PCP-supplemented nutrient agar (Difco) containing PCP at levels equal to that of the continuous culture. After the 2-month exposure period, a purified PCP-acclimated colony was isolated from the bioreactor (designated strain 200P) and was shown to grow aerobically at near wild-type rates in LB supplemented with 100 mg/L PCP (data not shown). Inhibition of AGR Phenotype of S. putrefaciens Strain 200P. S. putrefaciens strain 200P was grown aerobically for 5-7 days (30 °C) on nutrient agar supplemented with 50 mM iron(III) citrate (FNA medium). The resulting colonies were visually scored for an AGR phenotype by noting colony sinking over time. Previous studies had demonstrated that the AGR phenotype was expressed only by those colonies grown aerobically (and not anaerobically) on FNA (25). To determine if the AGR phenotype depended on the presence of Fe(II) and •OH, strain 200P was grown on FNA supplemented with the Fe(II) chelating agent, DETAPAC (7 mM), and the radical scavenging compounds (RSCs) mannitol (50 mM) and sodium benzoate (5 mM). S. putrefaciens anaerobic

respiratory mutant strain T121 (33) was included in this set of experiments as an Fe(III) reduction-deficient control strain. PCP Transformation by Microbially Driven Fenton Reaction. S. putrefaciens strain 200P was grown aerobically in LB on a rotary shaker (100 rpm, 30 °C) to early stationary phase (A600, 1.5), harvested by centrifugation at 6000g (4 °C), and resuspended in LS solution to a final cell density of 2 × 109 cells/mL. The cell suspension was immediately transferred to the Biostat B bioreactor, and iron(III) citrate and PCP were added to final concentrations of 10 mM and 3.5 µM, respectively. The culture was allowed to reduce the pool of Fe(III) for approximately 30 min (or until Fe(II) levels increased to approximately 75% of maximum) by continuous sparging with compressed nitrogen (gas flow rate, 1 L/min; stirrer speed, 300 rpm). Reactor temperature (30 °C) and pH (7.0) were held constant in all experiments. Aerobic conditions were initiated by sparging the culture with compressed air for approximately 60 min (or until Fe(II) levels decreased to 25% of maximum). Dissolved oxygen concentrations were monitored with an Ingold O2 sensor inserted through the reactor headplate. During the initial 8 h of incubation, six complete anaerobic/aerobic cycles were carried out by changing the inlet gas stream from compressed nitrogen to compressed air every 1.5 h. During the next 16-h incubation period, the PCP-containing culture was held under strict anaerobic conditions by continuous sparging with compressed nitrogen (and Fe(II) levels remained constant). During the final 8-h incubation period, the PCP-containing culture was exposed to two additional complete anaerobic/ aerobic cycles. PCP Transformation by Chemically Driven (Biomimetic) Fenton Reaction. A biomimetic (abiotic) PCP transformation system was set up to mimic the chemical conditions of the microbially driven system. The bioreactor configuration and environmental conditions were identical to those described above, except for the omission of S. putrefaciens strain 200P. LS solution was supplemented with 1.5 mM Fe(II)SO4, 1.5 mM iron(III) citrate, 1.5 mM citrate, and 6.8 µM PCP and held under constant anaerobic conditions for 22 h. H2O2 (30 µM) was added every 15 min during the first 7 h and final 9 h of the reaction. No H2O2 was added during the intervening 13-h period. Analytical Techniques. Fe(II) concentrations were determined along the time course of experiments using a previously described ferrozine-based, colorimetric assay (34). Absorbance measurements were carried out on a UV160U double-beam spectrophotometer (Shimadzu). Cl- concentrations were determined using a Dionex 100 chromatograph coupled to an ED40 electrochemical detector (Ionpac AS14 anion column with isocratic elution at 1.2 mL min-1, 3.5 mM Na2CO3/1.0 mM NaHCO3 mobile phase). H2O2 spectrophotometric measurements were not possible in iron-containing LS due to interference by iron(III) citrate. H2O2 concentrations were determined in iron-free aerobic cultures using a previously described spectrophotometric assay based on the peroxidase-catalyzed oxidation of N,N-diethyl-p-phenylenediamine (DPD) (35). Inhibition of PCP Degradation. A series of biotic and abiotic control experiments were carried out to confirm that the PCP degradation process was catalyzed by •OH generated via exposure of S. putrefaciens 200P to 10 mM iron(III) citrate and alternating anaerobic/aerobic conditions. All control experiments were carried out (in the dark) using bacterial growth and bioreactor conditions identical to those described above for the microbially driven PCP-degradation system. In the first set of control experiments, PCP degradation was monitored (under alternating anaerobic/aerobic conditions) in the presence of either the Fe(II) complexing compound DETAPAC (20 mM) or the RSCs mannitol (120 mM), thiourea (40 mM), and benzoate (8 mM). Previous control experiments VOL. 33, NO. 11, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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demonstrated that aerobic growth of strain 200P was not inhibited by either DETAPAC or the RSCs at these concentrations (data not shown). In the second set of control experiments, PCP degradation was monitored in the presence of iron(III) citrate under either strict aerobic or anaerobic conditions. In the third set of control experiments, PCP degradation was monitored in the presence of nitrate (15 mM KNO3) under alternating aerobic/anaerobic conditions. In the fourth set of (abiotic) control experiments, PCP degradation was monitored in the presence of Fe(II) (10 mM FeSO4) under strict anaerobic conditions with strain 200P omitted from the bioreactor. Sampling and Analyses of PCP and Dechlorinated Transformation Products. Triplicate samples (2.0 mL) were withdrawn at preselected time intervals, and PCP (and intermediate transformation products) was extracted using a modified version of a previously described liquid extraction technique (36). Each sample was spiked with an internal standard of tribromophenol (TBP; 1.25 mg/L final) and centrifuged at 12000g (2 min, 4 °C). The supernatant was diluted (2×) into a second vial, and the pH raised to >10 by the addition of 50 µL of 7.6 g/L K2CO3. Each sample was subsequently derivitized for 30 min with 50 µL of acetic anhydride (Sigma). The resulting pentachlorophenyl acetate and derivitized transformation products were extracted into 5.0 mL of hexane (Sigma), dried with anhydrous Na2SO4, and analyzed by gas chromatography. PCP concentrations were determined via GC analysis (HP5890, Hewlett-Packard) on a 30 m × 0.32 mm HP-5 column with a stationary phase film thickness of 0.25 µm coupled to a Ni63 electron capture detector (ECD). The carrier gas (He) was introduced at a constant flow rate of 1.0 mL/ min. Samples (0.5 µL) were injected in split mode (50:1 ratio). The injector and detector temperatures were 200 and 220 °C, respectively. The temperature program ramped from 55 °C for 2 min to 210 °C at a rate of 4 °C/min. Peaks corresponding to pentachlorophenyl acetate (and derivitized transformation products) were identified by coelution with known standards. The detector response was calibrated via the relative response of the analyte to the internal standard (TBP). The intermediate transformation products (TCHQ) and tetrachlorocatechol (TCC) were confirmed by GC-MS analysis. Samples were analyzed on a HP-5MS 5% phenyl methyl siloxane column (30 m × 250 µm diameter x 0.25 µm film thickness) using a HP6890 GC (Hewlett-Packard) coupled to a mass spectrometer. Inlet temperature was 250 °C, and the oven temperature program consisted of 5 min at 70 °C, ramped to 100 °C at a rate of 5 °C/min, and finally ramped at 15 °C intervals to 280 °C over a 5-min period. The inlet gas flow rate was held constant at 1.2 mL/min.

Results and Discussion Inhibition of AGR Phenotype of S. putrefaciens Strain 200P. Previous studies showed that S. putrefaciens strain 200P displayed an AGR-positive phenotype during aerobic growth on FNA, yet displayed an AGR-negative phenotype during anaerobic growth on the same medium (25). The results of this study showed that S. putrefaciens strain 200P also displayed an AGR-negative phenotype during aerobic growth on FNA supplemented with the Fe(II) complexing agent DETAPAC and the •OH scavenging compounds mannitol and benzoate (data not shown). This finding suggested that •OH may be the key reactant in hydrolysis of the alternating β-1,4 and β-1,3 agar linkages. Fe(III) reduction-deficient mutant T121 also displayed an AGR-negative phenotype during aerobic growth on FNA. These experiments demonstrated that expression of the AGR phenotype required microbially catalyzed Fe(III) reduction, aerobic incubation conditions, and the presence of •OH. 1888

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FIGURE 1. H2O2 production by cell suspensions of S. putrefaciens strain 200P exposed to aerobic conditions for 8 h in iron-free LS. Values shown are means of three independently processed samples with error bars corresponding to the standard deviation. H2O2 Formation. Under aerobic conditions, S. putrefaciens strain 200P was capable of producing extracellular H2O2 at 30 µM levels in 30 min (Figure 1). This level was well below the concentration (1 mM) considered to be toxic to microbial cells (37). H2O2 is generally produced by aerobically respiring bacteria via one of several O2 activation pathways, including amino acid oxidase-catalyzed reactions that result in the twoelectron reduction of O2 to H2O2 (eqs 5 and 6):

O2 + e- w O2•-

(5)

O2 + O2•- w H2O2

(6)

Although the physiological mechanism by which S. putrefaciens strain 200P transfers electrons to O2 is unknown, previous studies have shown that S. putrefaciens strain 200P is capable of oxidizing a wide range of amino acids as sole carbon/energy source during aerobic growth (28) and contains a large amount of flavin-containing proteins (putative amino acid oxidases) (30). H2O2 may also be produced under aerobic conditions via (purely chemical) Fe(II) autoxidation pathways (eqs 7 and 8) (2):

Fe(II) + O2 w Fe(III) + O2•-

(7)

Fe(II) + O2•- + 2H+ w H2O2 + Fe(III)

(8)

Therefore, exposure of Fe(II)-containing liquid cultures of S. putrefaciens 200P to aerobic conditions may result in H2O2 production via a combination of microbially catalyzed O2 reduction and Fe(II) autoxidation. PCP Transformation by Microbially Driven Fenton Reaction and Identification of Dechlorinated Transformation Products. During the first 8-h incubation period, the concentration of Fe(II) was rapidly oscillated (six anaerobic/ aerobic cycles; Figure 2C), and PCP was degraded at a linear rate (0.31 µM h-1) (Figure 2A). The linear decrease in PCP was reflected in a concomitant linear increase in two transformation products that eluted at 36.7 and 37.3 min, respectively (Figure 2B). The transformation products coeluted with standard derivitized extracts of tetrachlorohydroquinone (TCHQ; 36.7 min) and tetrachlorocatechol (TCC; 37.3 min). The identity of the transformation products was confirmed via GC-MS analysis. No other chlorinated transformation products were detected. A comparison of the PCP degradation rate with the sum of the TCHQ (0.24 µM h-1)

FIGURE 2. Degradation of PCP (A), production of TCHQ and TCC (B), and oscillation of Fe(II) concentrations (C) in the microbially driven Fenton reaction system. Cell suspensions of S. putrefaciens strain 200P were supplemented with 3.5 µM PCP, 10 mM iron(III) citrate and exposed to alternating anaerobic/aerobic conditions during the first and last 8 h of the reaction. Strict anaerobic conditions were maintained during the intervening time period. Values shown in panels A and B are means of three independently processed samples with error bars corresponding to the standard deviation. and TCC (0.03 µM h-1) production rates indicated that approximately 87% of PCP degradation could be accounted for in TCHQ/TCC produced. The inability to fully account for PCP degradation as the sum of TCHQ and TCC production may be due to the possibility that TCHQ and TCC reacted with •OH to form further transformation products (coupling products or ring-opened intermediates) (38). Previous studies of Fenton reaction-catalyzed PCP degradation demonstrated that •OH attack is more rapid on dechlorinated transformation

products than on PCP due to lowered oxidation states and increased water solubilities of the hydroxylated products (9, 39). Strict anaerobic conditions were maintained during the next 16-h incubation period (iron therefore remained in its Fe(II) form; Figure 2C), and PCP levels remained constant (Figure 2A). PCP degradation resumed only after oxygen was reintroduced into the reactor during the final 8-h incubation period (two additional anaerobic/aerobic cycles; Figure 2C). This finding corroborated with the previous observation that S. putrefaciens strain 200P colonies grown anaerobically on FNA displayed an AGR-negative phenotype, while colonies grown aerobically on FNA displayed an AGR-positive phenotype (presumably via •OH generation during concomitant Fe(III) reduction and aerobic respiration) (25). The detection of TCHQ and TCC (Figure 2B) provided further evidence that the microbially driven Fenton system generated •OH as the PCP-oxidizing agent. Previous studies (40, 41) have demonstrated that catechol is formed from •OH attack at the ortho position of phenol. The conversion of phenol to its hydroxylated products catechol and hydroquinone has also been used as a quantitative assay for •OH formation (42). Furthermore, in a previous study on the chemical oxidation of PCP using low concentrations of Fenton’s reagent (100 µM H2O2, 0.329 mM Fe(II)), Lee and co-workers (43) concluded that hydrolytic dechlorination of PCP yielded stoichiometric amounts of TCHQ and TCC. In the microbially driven system of the present study, comparable H2O2 concentrations (30 µM) were detected 30 min after exposing liquid cultures of S. putrefaciens strain 200P to aerobic conditions in iron-free LS (Figure 1). A mass balance on Cl- could not be carried out due to the presence of high background levels of Cl- (18 mM) in LS. Efforts were made to substitute Cl--free buffers (44) for LS in the microbially driven system. However, the metabolic activity of S. putrefaciens strain 200P decreased markedly when minimal salt solutions containing less than 150 µM NaCl were used in the place of LS (data not shown). The high background Cl- levels in LS therefore masked Cl- production (17.9 µM if PCP were fully dechlorinated) and prevented Clmass balance calculations from being carried out. PCP Transformation by Chemically Driven (Biomimetic) Fenton Reaction. A chemically driven (biomimetic) Fenton reaction experiment was carried out to confirm the results of the microbially driven Fenton reaction experiment. The biomimetic process was designed to mimic the concentrations of Fe(II) and H2O2 determined in the microbially driven process. The reactor vessel was held under anaerobic conditions throughout the time course of the biomimetic experiment. Equimolar concentrations of Fe(II) and Fe(III) were added at time zero followed by continual additions of 30 µM H2O2 every 15 min. PCP was degraded (0.13 µM h-1) (Figure 3A), and TCHQ (0.06 µM h-1) and TCC (0.03 µM h-1) (Figure 3B) were produced at linear rates during the first 8-h time interval. As in the microbially driven system, no other dechlorination products were detected in the biomimetic system. A comparison of the PCP degradation rate and the sum of the TCHQ and TCC production rates indicated that nearly 70% of PCP degradation was accounted for by TCHQ and TCC production. PCP was degraded only when H2O2 was added to the reactor vessel (first and last 8 h of incubation) (Figure 3A). PCP was not degraded, and TCHQ and TCC were not produced during the intervening 13-h anaerobic period when H2O2 was not added. Fe(II) was oxidized only when H2O2 was added (Figure 3C), indicating that the transformation of PCP to TCHQ and TCC depended on Fe(II) oxidation by H2O2. The results of the chemically driven (biomimetic) system (Figure 3) are similar to those of the microbially driven system VOL. 33, NO. 11, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Degradation of PCP (A), production of TCHQ and TCC (B), and oxidation of Fe(II) (C) in the chemically driven (biomimetic) Fenton reaction system. LS solution was supplemented with 6.8 µM PCP, 1.5 mM Fe(II)SO4, 1.5 mM iron(III) citrate, and 1.5 mM citrate and held under anaerobic conditions for 22 h with the addition of 30 µM H2O2 every 15 min during the first 7 h and final 9 h of the reaction. Values shown are means of three independently processed samples with error bars corresponding to the standard deviation.

(Figure 2), suggesting that PCP was transformed to TCHQ and TCC via similar mechanisms in both systems. 1890

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Inhibition of PCP Transformation and Absence of Dechlorinated Transformation Products. A series of control experiments were carried out to confirm that the PCPdegradation process depended on the presence of •OH generated via exposure of S. putrefaciens 200P to Fe(III) and alternating anaerobic/aerobic conditions. PCP was not degraded (and transformation products were not detected) in cell suspensions supplemented with either the Fe(II) chelating agent DETAPAC or the •OH radical scavenging compounds (RSCs) mannitol, thiourea, and sodium benzoate. Fe(II) levels in the DETAPAC-supplemented culture increased to maximum levels under anaerobic conditions and remained constant throughout the incubation period, despite alternating anaerobic/aerobic conditions (data not shown). This finding suggested that DETAPAC inhibited •OH formation by effectively complexing bacterially produced Fe(II) and preventing the interaction of Fe(II) and H2O2. In a similar manner, DETAPAC and the RSCs inhibited expression of the AGR-positive phenotype displayed by strain 200P during aerobic growth on FNA (data not shown). In the RSCsupplemented control experiments, Fe(II) levels oscillated with the alternating aerobic/anaerobic conditions. Although Fe(II)/H2O2 interaction was possible, PCP degradation was not observed, and transformation products were not detected. This finding suggested that PCP degradation required the presence of •OH, which was otherwise scavenged by the RSCs. Although previous studies demonstrated that a Pseudomonas strain could enzymatically transform PCP to TCHQ and TCC under aerobic conditions (45), PCP transformation was not observed (and transformation products were not detected) in control experiments with S. putrefaciens strain 200P exposed to continuous aerobic conditions. This suggests that the aerobically expressed enzymes of S. putrefaciens strain 200P are not responsible for PCP transformation. Another set of control experiments examined the importance of iron in the PCP degradation process. PCP transformation was not observed (and transformation products were not detected) in control experiments where Fe(III) was replaced by NO3- in an otherwise identical reaction setup (including two cycles of alternating aerobic/anaerobic conditions). This finding demonstrated that PCP degradation required Fe(III) and not NO3- as a catalyst for PCP degradation. This finding also indicated that enzymes expressed under NO3--reducing anaerobic conditions were not responsible for PCP degradation. PCP degradation was not observed (and transformation products were not detected) in an abiotic control experiment where Fe(II) was maintained at constant levels under strict anaerobic conditions during an 8-h incubation period. PCP degradation was not observed (and transformation products were not detected) in cell suspensions maintained under strict (Fe(III)-reducing) anaerobic conditions where Fe(II) was the predominant form of iron, nor was PCP degradation observed under strict aerobic conditions where Fe(III) was the predominant form of iron. These findings indicated that PCP degradation was not due to Fe(II)-catalyzed (chemical reduction) or Fe(III)-catalyzed (chemical oxidation) pathways. These findings also indicated that enzymes produced under Fe(II)-oxidizing or Fe(III)reducing conditions were not responsible for PCP degradation and that a redox transition from Fe(II)- to Fe(III)-dominated conditions (i.e., via alternating anaerobic/aerobic conditions) was required for PCP degradation. Summary. In contrast to traditional Fenton- or photoFenton-based oxidation systems, the microbially driven Fenton reaction system operates at neutral pH and requires neither addition of exogenous H2O2 nor UV irradiation to regenerate Fe(II). The anaerobic Fe(III) reduction system of S. putrefaciens functionally replaces UV light as the Fe(II) regenerator, while the aerobic electron-transport system produces H2O2 as a metabolic byproduct of aerobic respira-

tion. Current work focuses on enhancement of microbially driven Fe(II) and H2O2 production and optimization of PCP degradation rates.

Acknowledgments Financial support for this study was provided in part by the National Science Foundation Graduate Research Traineeship Program and the Technical Association of the Pulp and Paper Industry (TAPPI). We thank Jay Day, Julie Turner, and SheauYun Chiang for technical assistance. We thank Dr. Michael Saunders for access to the GC-ECD in the School of Civil and Environmental Engineering at Georgia Tech and Dr. Matt Tarr of the University of New Orleans Chemistry Department for ion chromatographic analysis.

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Received for review August 6, 1998. Revised manuscript received March 24, 1999. Accepted March 24, 1999. ES980810Z

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