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). 0.10-4- O RESULTS E 008 0 Degradation of chlorophenols in sulfidogenic sediments. The ]> - k -0 If degradation profiles of 2-CP, 3-CP, 4-CP, and 24-DCP in ci 0.06 0 sediment cultures under freshwater and saline conditions are shown in Fig. 1. After an initial lag period of approximately o040.04- 50 to 100 days, the chlorophenols were completely removed K~~~~~~~~ a 0 in 120 to 220 days. After refeeding to the initial concentration 0.02- of 0.1 mM, the monochlorophenols were removed in 45 days or less. After further refeeding, the chlorophenols were n nn removed in <10 days (data not shown). There was little 0 20 40 60 80 100w i: difference observed in the activity of the cultures under time (days) saline or freshwater conditions. No degradation of chlorophenols was observed in the sterile controls. To ensure 3-Chlorophenol that methanogenesis was not taking place, all cultures were 0.12- routinely monitored for CH4, and none was detected. When 24-DCP was degraded, there was a transient appear- 0.10 ance of a metabolite detected by high-performance liquid 0 A chromatography. Based on its retention time and by spiking 2 0.08 with authentic compound, it was tentatively identified as E 6 ~~~~~~0 0----- 4-CP. 4-CP accumulated to 0.03 mM and was then com- ci 0.06 0 pletely removed, with no further metabolites detected (data 04 not shown). This was observed in both saline and freshwater 0.04 cultures. In the cultures fed the monochlorophenols, no metabolites detectable by high-performance liquid chroma- 0.02- tography (UV A280) accumulated. Since no intermediates were observed, it is not clear whether the monochlorophe- _ _ nols undergo dechlorination to phenol. ) 20 40 60 so 100 1; Effect of molybdate on chlorophenol degradation. After time (day3) 2-CP, 3-CP, and 4-CP had been degraded in the initial sediment cultures, the cultures were split into two sets of replicate cultures and refed chlorophenol to the initial 0.1 mM concentration, and the degradation of chlorophenol was monitored. Molybdate (20 mM) was added to one set of replicate cultures to specifically inhibit sulfate reduction. 2 When the chlorophenols had been depleted from the unin- E hibited cultures, they were refed to 0.1 mM. The effect of molybdate on chlorophenol degradation in the freshwater cultures is shown in Fig. 2. As illustrated, 20 mM molybdate 0 completely inhibited degradation of each of the three monochlorophenols. No detectable sulfate loss was observed for these cultures. In the uninhibited cultures, without molybdate 2-CP, 3-CP, and 4-CP were rapidly degraded during repeated feedings. Similar results were observed in the 60 120 cultures under saline conditions (data not shown). time (days) Sulfate depletion during chlorophenol degradation. The active chlorophenol-degrading freshwater and saline cul- FIG. 2. Effect of molybdate on degradation of monochlorophe- tures were repeatedly refed the original substrate seven nols. Symbols: uninhibited (0); 20 mM molybdate added (O). Data times at a concentration of 0.1 to 0.2 mM. When a total of points are the mean of two replicate cultures. approximately 1 mM chlorophenol had been added and metabolized, samples were taken and analyzed for sulfate technique. Degradation of 4-CP resulted in loss of sulfate concentration and compared with samples taken at time with recovery of radioactive sulfide in the Zn acetate traps. zero. Samples were also taken from background cultures, Sulfidogenesis was confirmed by formation of radiolabeled and sulfate reduction due to background organic matter of sulfide in the lactate-fed active controls also. No radioactive the sediment inoculum was subtracted from the results. sulfide was produced in cultures inhibited with molybdate Degradation of 2-CP, 3-CP, and 4-CP resulted in nearly (results not shown). stoichiometric loss of sulfate (Table 1). The measured sulfate Rate of chlorophenol degradation in acclimated cultures. loss corresponded to 86 to 124% of the theoretical values Cultures were repeatedly fed 0.1 to 0.2 mM chlorophenol calculated for complete oxidation of the chlorophenol. No over a period of 6 months. The degradation of monochlo- CH4 was formed in the cultures. rophenols in these acclimated cultures under freshwater Formation of sulfide from sulfate in a 4-CP-degrading conditions is shown in Fig. 3, which shows that degradation culture was determined by using a 35s042- radiotracer of 4-CP was rapid, with 0.17 mmol/liter removed in <6 days. 3258 HAGGBLOM AND YOUNG APPL. ENVIRON. MICROBIOL.

TABLE 1. Sulfate depletion during chlorophenol degradationa TABLE 2. Rate of chlorophenol degradation in acclimated sulfidogenic sediment cultures CP me- Sulfate loss (mM) % of CP fed tabolized Rate (~Lmol (mM) Predicted' Measured' expected Condition Compound liter-' day-')' Freshwater Freshwater 2-CP 10 2-CP 0.86 2.9 2.5 86 3-CP 15 3-CP 0.82 2.8 2.4 86 4-CP 37 4-CP 0.85 2.9 3.6 124 Saline 2-CP 8 3-CP 18 Saline 4-CP 22 2-CP 0.83 2.8 3.2 114 24-DCP 8 3-CP 1.03 3.5 4.2 120 4-CP 0.87 2.9 2.9 100 a The degradation rate was calculated from the linear portion of the degradation curve (the first 6 to 8 days) and given as the mean of duplicate a All values are means from duplicate cultures, except for 2-CP (freshwa- cultures. ter), for which activity in one of the duplicates was lost. b Calculated from the following equation: C6H60CI + 3.375 So42- + 3.5 H20 -+ 3.375 H2S + 6 HC03- + Cl- + 0.25 H+. c Sulfate loss (1.5 mM) in background cultures subtracted. period no CH4 was produced. The bioreactor inoculum was thus able to dechlorinate 24-DCP and 26-DCP reductively under sulfate-reducing conditions, removing the ortho chlo- Degradation of 0.16 mM 3-CP took 12 days, while only 50% rine of 24-DCP and both ortho chlorines of 26-DCP. of 0.18 mM 2-CP was removed in 12 days. The rates of chlorophenol degradation in freshwater and saline cultures DISCUSSION are summarized in Table 2. The results demonstrate that 4-CP was degraded fastest, degradation of 3-CP was some- All three monochlorophenol isomers and 24-DCP are what slower, and degradation of 2-CP and 24-DCP was the degraded in sulfidogenic sediment cultures established under slowest. These differences in rates of degradation were either freshwater or saline conditions. During degradation of consistently observed in all cultures. Each of the monochlo- 2-CP, 3-CP, and 4-CP, there was a concomitant loss of rophenols had been refed to the cultures the same number of sulfate, corresponding to approximately stoichiometric val- times, so that relative comparisons can be made. The ues expected for complete oxidation of the chlorophenol to 24-DCP cultures had been refed only three times to a total CO2. Sulfide production from sulfate during chlorophenol concentration of 0.3 mM, which may partly explain the low degradation was confirmed with a radiotracer technique. No rate of metabolism. methane was formed, verifying that sulfate reduction was Dechlorination of 24-DCP and 26-DCP in bioreactor cul- the main electron sink. Addition of molybdate, a specific tures. We also studied the ability of biomass from an inhibitor of sulfate reduction (25), to the sediment cultures anaerobic bioreactor used in treatment of pulp bleaching completely inhibited degradation of the chlorophenols. effluents to degrade chlorinated phenols under sulfate-reduc- These results indicate that the monochlorophenols are min- ing conditions. The cultures were fed 0.1 mM either 24-DCP eralized under sulfidogenic conditions and that chlorophenol or 26-DCP and 1 mM propionate as an auxiliary carbon oxidation is coupled to sulfate reduction. To our knowledge, source. The results presented in Table 3 show that in 50 days this has not been shown previously. all 24-DCP had been dechlorinated to 4-CP, which persisted. The rate of chlorophenol degradation was greatly en- No phenol or loss of 4-CP was detected. The sterile controls hanced after repeated refeeding of the substrate to the showed a slow abiotic loss of 24-DCP, with no formation of sediment cultures. In acclimated cultures the rate of chlo- dechlorination products. 26-DCP was sequentially dechlori- rophenol degradation varied from 8 to 37 Fmol liter-' day-'. nated first to 2-CP and then to phenol. In sterile controls, Of the three monochlorophenols, 4-CP was degraded the 26-DCP showed a slow abiotic loss. During the incubation fastest and about three times faster than degradation of

TABLE 3. Dechlorination of 24-DCP and 26-DCP in bioreactor cultures' Concn (mM) CP Culture Day 24-DCP 26-DCP 4-CP 2-CP Phenol E 012¢0 24-DCP Active 0 0.066 0 0 50 0 0.064 0 0~~~~ 84 0 0.063 0 Sterile 0 0.097 0 0 0 50 0.054 0 0 0.04- 84 0.04 0 0 26-DCP Active 0 0.105 0 0 0.00 50 0 0.150 0.006 0 2 4 6 8 10 12 84 0 0.037 0.037 time (days) Sterile 0 0.114 0 0 50 0.102 0 0 FIG. 3. Degradation of 2-CP, 3-CP, and 4-CP in acclimated 84 0.066 0 0 sulfidogenic cultures. Cultures (two replicates for each substrate) were set up under freshwater conditions. aActive cultures were set up in duplicate. VOL. 56, 1990 CHLOROPHENOL DEGRADATION AND SULFATE REDUCTION 3259

2-CP. Degradation of 3-CP was somewhat slower than that LITERATURE CITED of 4-CP. These rates of monochlorophenol degradation are 1. Ahlborg, U. G., and T. M. Thunberg. 1980. Chlorinated phe- similar to those found for methanogenic cultures, ranging nols: occurrence, toxicity, metabolism, and environmental im- from 5 to 50 ,umol liter-' day-' (5, 15, 20; M. Haggblom et pact. Crit. Rev. Toxicol. 7:1-35. al., unpublished data). The different relative rates at which 2. Ahling, B., and A. Lindskog. 1982. Emissions of chlorinated under organic substances from combustion, p. 215-225. In 0. Hut- each of the monochlorophenol isomers are degraded zinger, R. W. Frei, E. Merian, and F. Pocchiari (ed.), Chlori- sulfate-reducing conditions are the reverse of those found for nated dioxins and related compounds. Impact on the environ- methanogenic cultures (5, 9, 15, 16; Haggblom et al., unpub- ment. Pergamon Press, Oxford. lished data; 0. O'Connor and L. Y. Young, unpublished 3. Bak, F., and F. Widdel. 1986. Anaerobic degradation of phenol data). These studies showed that 2-CP was the most rapidly and phenol derivatives by Desulfobacterium phenolicum sp. degraded monochlorophenol, while degradation of 4-CP was nov. Arch. Microbiol. 146:177-180. the slowest. The reason for these differences is not clear at 4. Bossert, I. D., and L. Y. Young. 1986. Anaerobic oxidation of p-cresol by a denitrifying bacterium. Appl. Environ. Microbiol. this time. 52:1117-1122. Several pure cultures of sulfate-reducing bacteria which 5. Boyd, S. A., and D. R. Shelton. 1984. Anaerobic biodegradation degrade aromatic compounds have recently been isolated (3, of chlorophenols in fresh and acclimated sludge. Appl. Environ. 29, 30, 34, 36). Most of the strains originated from marine Microbiol. 47:272-277. sediment samples. Compounds degraded by sulfate-reducing 6. Boyd, S. A., D. R. Shelton, D. Berry, and J. M Tiedje. 1983. bacteria include phenol and phenol derivatives, benzoate, Anaerobic biodegradation of phenolic compounds in digested and dihydroxybenzenes. The strains enriched with aromatic sludge. Appl. Environ. Microbiol. 46:50-54. able to use a wide range of different 7. Detrick, R. S. 1977. Pentachlorophenol, possible sources of compounds are often human exposure. For. Prod. J. 27:13-16. aromatic compounds (36). Desulfomonile tiedjei DCB-1, 8. DeWeerd, K. A., L. Mandelco, R. S. Tanner, C. R. Woese, and which dehalogenates 3-chlorobenzoate (31), has been shown J. M. Suflita. 1990. Desulfomonile tiedjei gen. nov. and sp. nov., to be a sulfidogen (8, 32). No sulfate-reducing bacterium able a novel anaerobic dehalogenating, sulfate-reducing bacterium. to degrade chlorinated phenols, however, has yet been Arch. Microbiol. 154:23-30. isolated. 9. Genthner, B. R. S., W. A. Price II, and P. H. Pritchard. 1989. 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Anaerobic important role in certain environments. biodegradation of 2,4-dichlorophenol in freshwater lake sediments at different temperatures. Appl. Environ. Microbiol. 55:348-353. ACKNOWLEDGMENTS 21. Kohring, G.-W., X. Zhang, and J. Wiegel. 1989. Anaerobic dechlorination of 2,4-dichlorophenol in freshwater sediments in We thank Maria Rivera for help in the laboratory. the presence of sulfate. Appl. Environ. Microbiol. 55:2735- This work was supported in part by Environmental Protection 2737. Agency grant CR-814611 and Public Health Service grant ESO 4895 22. Kringstad, K. P., and K. Lindstrom. 1984. Spent liquors from from the National Institute of Environmental Health Sciences. pulp bleaching. Environ. Sci. Technol. 18:236A-248A. 3260 HAGGBLOM AND YOUNG APPL. ENVIRON. MICROBIOL.

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