Chlorophenol Degradation Coupled to Sulfate Reduction MAX M

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1990, p. 3255-3260 Vol. 56, No. 11 0099-2240/90/113255-06$02.00/0 Copyright ) 1990, American Society for Microbiology Chlorophenol Degradation Coupled to Sulfate Reduction MAX M. HAGGBLOM' AND L. Y. YOUNG' 2* Departments of Microbiology' and Environmental Medicine,2 New York University Medical Center, 550 First Avenue, New York, New York 10016 Received 13 April 1990/Accepted 10 August 1990 We studied chlorophenol degradation under sulfate-reducing conditions with an estuarine sediment inoculum. These cultures degraded 0.1 mM 2-, 3-, and 4-chlorophenol and 2,4-dichlorophenol within 120 to 220 days, but after refeeding with chlorophenols degradation took place in 40 days or less. Further refeeding greatly enhanced the rate of degradation. Sulfate consumption by the cultures corresponded to the stoichiometric values expected for complete oxidation of the chlorophenol to CO2. Formation of sulfide from sulfate was confirmed with a radiotracer technique. No methane was formed, verifying that sulfate reduction was the electron sink. Addition of molybdate, a specific inhibitor of sulfate reduction, inhibited chlorophenol degradation completely. These results indicate that the chlorophenols were mineralized under sulfidogenic conditions and that substrate oxidation was coupled to sulfate reduction. In acclimated cultures the three monochlorophenol isomers and 2,4-dichlorophenol were degraded at rates of 8 to 37 ,umol liter-' day-'. The relative rates of degradation were 4-chlorophenol > 3-chlorophenol > 2-chlorophenol, 2,4-dichlorophenol. Sulfidogenic cultures initiated with biomass from an anaerobic bioreactor used in treatment of pulp-bleaching effluents dechlorinated 2,4-dichlorophenol to 4-chlorophenol, which persisted, whereas 2,6-dichlorophenol was sequentially dechlorinated first to 2-chlorophenol and then to phenol. Contamination of the environment by chlorinated aro- halogenated compounds (24), suggesting that bacteria capa- matic compounds has been the subject of increased concern ble of (aerobic or anaerobic) dehalogenation could evolve in in the last few years. Chlorinated phenols are common such habitats. Recently, anaerobic degradation of a naturally environmental contaminants; they have been extensively occurring halophenol, 2,4-dibromophenol, was observed in used as biocides, mainly as wood preservatives (26). Chlori- marine sediments (19). nated phenols and other chlorinated phenolic compounds are A number of reports indicate that sulfate appears to inhibit also formed as by-products when chlorine is used for bleach- anaerobic degradation of chlorophenols (11, 33), but there is ing of pulp (22) and for disinfection of drinking water and also evidence of chlorophenol degradation in the presence of wastewater containing phenols (1, 7). They are also formed sulfate (9) or during sulfate reduction (21). Whether degra- during combustion of organic matter (2) and as biological dation of chlorophenols can be linked to sulfate reduction breakdown products of chlorophenoxyacetic acid herbicides has yet to be established. In this paper, we demonstrate that (12, 27). A range of chlorinated organic compounds including the degradation of chlorophenols can be coupled to sulfate chlorophenols can be produced by biologic chlorination as reduction, as observed both with sediment from a polluted well (24). intertidal strait and with inoculum from a bioreactor shown In water, chlorophenols sorb onto particulate material previously to dechlorinate chlorolignin. and, if not degraded, eventually end up in sediments. Chlori- nated phenolics have been found to accumulate in freshwa- MATERIALS AND METHODS ter and marine environments where they may attain concen- Establishment of cultures. Strict anaerobic techniques trations of tens of milligrams per kilogram of dry sediment were followed throughout the study. A sediment sample (28, 38). In anoxic sediments, nitrate, sulfate, or carbonate from an estuarine intertidal strait (East River, New York may serve as a terminal electron acceptor for degradation of City) was used as inoculum. Sediment slurries were added as organic material. Anaerobic degradation of chlorophenols a 10% (vol/vol) inoculum, and a freshwater or saline sulfate has mainly been studied under methanogenic conditions (5, medium was added to a total volume of 50 ml to deoxygen- 6, 10, 11, 15, 16, 20, 23, 33). These studies with freshwater ated 65-ml serum bottles. The freshwater medium was sediments, soil, and sewage sludge as inoculum have shown modified from Widdel (Ph.D. thesis, University of Gottin- that degradation of chlorophenols is initiated by reductive gen, Gottingen, Federal Republic of Germany, 1980) and dechlorination, with complete mineralization to CO2 and contained the following (in grams per liter): NaCl, 1.17; CH4 observed in some cases. MgC12 6H20, 0.41; KCI, 0.3; CaCl2, 0.11; NH4Cl, 0.27; In marine environments, sulfate reduction is the major KH2PO4, 0.2; Na2SO4, 2.84; NaHCO3, 2.52; NaMoO4, 0.018 electron sink during anaerobic degradation of organic mat- mg/liter; Na2S, 1.5 mM. The medium was supplemented ter. In a marine sediment, it accounted for >50% of the with trace elements (37) and vitamins (Widdel, Ph.D. thesis), mineralization of organic matter (35), while in a salt marsh pH 7.2, with resazurin as a redox indicator. The saline environment the sulfate-mediated oxidation of organic mat- sulfidogenic medium contained 23.0 g of NaCl and 1.0 g of ter was 12 times that of 02-mediated oxidation (14). The MgCl2 per liter (otherwise as above), based on the measured marine environment is a rich source of biologically produced salinity of the East River. The headspace gas was N2 (70%)-CO2 (30%). All bottles were sealed with butyl rubber stoppers and aluminum crimp seals. 2-Chlorophenol (2-CP), * Corresponding author. 3-chlorophenol (3-CP), 4-chlorophenol (4-CP), or 2,4-dichlo- 3255 0.12 2-Chlorophenol rophenol (24-DCP) (Aldrich Chemical Co., Milwaukee, Wis.) was added to an initial concentration of 0.1 mM. The 0.10 cultures were established in duplicate with background (no substrate added) and sterile (autoclaved three times on 0.08- A consecutive days) controls. The cultures were incubated E statically at 30°C, in the dark. 0.06-- Another set of experiments was set up with inoculum from C a laboratory-scale anaerobic fluidized-bed bioreactor used 0 U 0.04-\ for treatment of pulp bleaching effluents (M. Haggblom and M. Salkinoja-Salonen, Water Sci. Technol., in press). Bio- 0.02- mass from the bioreactor fluid was collected by centrifuga- tion, washed, and suspended to 1/10 of the original volume in 0.00- IU* ' U- a phosphate buffer; 2 ml was added as inoculum to 50 ml of 0 50 100 150 200 250 sulfate medium (described above). Establishment of cultures time (days) was otherwise as described above. The cultures were fed 1.0 mM propionate as an auxiliary substrate and incubated for 2 0.12, weeks prior to feeding with chlorophenols. 2,6-Dichlorophe- 3-Chlorophenol nol (26-DCP) (Aldrich Chemical Co.) and 24-DCP were fed A U to an initial concentration of 0.1 mM. The cultures were set 0.101 up in duplicate with background and sterile controls and A A incubated as above. 0.08< Analysis. Gas and liquid samples were taken for periodic E A~~~~ analysis with sterile syringes, which had been deoxygenated 0.06! as IA... .A with N2-C02. CH4 in the headspace gas was analyzed c0 described previously (4), with a gas partitioner (model 1200; a 0.04o Fisher Scientific Co., Springfield, N.J.) equipped with a thermal conductivity detector. Sulfate was analyzed by an 0.02 indirect titration as follows (13). Sulfide was first removed by precipitation as ZnS. Sulfate was then precipitated as barium 0.00 sulfate in acid EDTA solution, filtered, and dissolved in an 50 100 150 200 250 excess of EDTA at high pH, and the excess EDTA was time (days) titrated with MgCl2. Chlorophenols were quantified by high-performance liquid chromatography. Prior to analysis, the samples (0.3 to 0.5 ml) were acidified with 10 pAl of 1 N HCl, centrifuged, and filtered (0.45 tLm). Analysis was performed with a Beckman 332 LC chromatograph (Beckman Instruments, Palo Alto, Calif.) equipped with a Spherisorb C-18 column (250 by 4.6 mm; Supelco Inc., Bellefonte, Pa.), with UV detection at 280 E nm, and using a solvent system of methanol (60%, vol/vol)- i 0.06 water (38%, vol/vol)-acetic acid (2%, vol/vol) at a flow rate of 1 ml/min. Uooo0 0.04- Determination of sulfide formation. The reduction of sulfate to sulfide was determined by using a modified radiotracer technique (17, 18). A 4-CP-degrading culture ob- tained through repeated transfers into fresh medium and refeeding with chlorophenol was used. The culture was split into 5-ml subcultures in 10-ml vials, and 5 nCi of [355]NaSO4 (43 Ci/mg, carrier-free, 99% radionuclidic purity; ICN Radi- ochemicals, Irvine, Calif.) was added. Replicate cultures were fed chlorophenol twice to a total of 0.5 mM and incubated for 2 weeks. Lactate was used as a nonselective substrate for sulfate reducers as an active control. Molyb- date-inhibited and unfed cultures served as controls. When the chlorophenol had been degraded, the cultures were 0-% acidified with 25% HCI, which releases sulfide as H2S gas. E H2S was then driven off by flushing the vessel with argon for 30 min and collected in a series of two Zn acetate (2%, C wt/vol) traps, where sulfide was precipitated as ZnS. An 0 U FIG. 1. Degradation of 2-CP, 3-CP, 4-CP, and 24-DCP in sulfidogenic sediment cultures under freshwater and saline conditions. Symbols: saline (U); freshwater (0); sterile control, saline (A); 100 150 sterile control, freshwater (A). Data points are the mean of two time (days) replicate cultures. 3256 VOL. 56, 1990 CHLOROPHENOL DEGRADATION AND SULFATE REDUCTION 3257 aqueous scintillation fluid (ACS; Amersham, Arlington 2-Chlorophenol Heights, Ill.) was added to each trap, and radioactivity was 0.12 -M- measured by scintillation counting (Beckman LS 6000 IC).

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