Applied and Environmental Microbiology, November 1999, p. 5169-5172, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Department of Civil Engineering, Northwestern University, Evanston, Illinois 60208
Received 17 May 1999/Accepted 31 August 1999
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ABSTRACT |
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Biotransformation of 2-chlorophenol by a methanogenic sediment community resulted in the transient accumulation of phenol and benzoate. 3-Chlorobenzoate was a more persistent product of 2-chlorophenol metabolism. The anaerobic biotransformation of phenol to benzoate presumably occurred via para-carboxylation and dehydroxylation reactions, which may also explain the observed conversion of 2-chlorophenol to 3-chlorobenzoate.
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TEXT |
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Anaerobic biotransformation of a wide variety of aromatic compounds initially involves the formation of a carboxyl group on the aromatic nucleus, e.g., via a carboxylation reaction (6). An anaerobic isolate (21) and a coculture (9) that are able to carboxylate phenol have been characterized. Other aromatics that undergo anaerobic carboxylation include simple polyaromatic hydrocarbons (22), aniline (8), o-cresol (2), and m-cresol (12). However, under anaerobic conditions, biotransformation of most halogenated aromatic compounds is typically initiated by reductive dehalogenation (14). In fact, it appears that removal of the halogen substituent must occur before anaerobic cleavage of the aromatic nucleus is feasible (19). Indeed, in nearly all previous studies, anaerobic transformation of 2-chlorophenol (2-CP) by mixed cultures occurred solely via reductive dehalogenation to phenol (e.g., see references 4 and 15). The pathway by which phenol is biodegraded in mixed cultures under methanogenic conditions has been examined in a number of investigations (e.g., see references 16 and 18). The results of these recent studies indicate that 4-hydroxybenzoate is formed via para-carboxylation of phenol and subsequently dehydroxylated to yield benzoate, which undergoes ring cleavage and ultimately is mineralized.
However, in some cases, more than one pathway may be possible for the biotransformation of aromatic compounds in mixed anaerobic cultures. Which pathway is operative may determine whether or not the parent compound is ultimately mineralized. For example, Londry and Fedorak (12) examined the biotransformation of m-cresol in a methanogenic consortium. Biotransformation of m-cresol initially proceeded via para-carboxylation to yield 4-hydroxy-2-methylbenzoic acid. Metabolism of this intermediate appeared to be the rate-limiting step in the biotransformation of m-cresol. 4-Hydroxy-2-methylbenzoate predominantly underwent demethylation to yield 4-hydroxybenzoic acid, which was ultimately converted to acetate via reductive dehydroxylation to benzoate and subsequent ring cleavage. However, "premature" reductive dehydroxylation of 4-hydroxy-2-methylbenzoate produced a minor metabolite, 2-methylbenzoate, which persisted.
The existence of two m-cresol biotransformation pathways, one leading to mineralization of the parent compound and the other resulting in the production of a persistent substituted benzoate, points to the importance of understanding biotransformation pathways. This information is necessary for predicting the fate of organic pollutants in the environment and for designing bioremediation strategies and wastewater treatment processes. In this paper, we report that in mixed anaerobic cultures, in addition to undergoing the expected pathway of reductive dehalogenation to phenol and ultimate mineralization, transformation of 2-CP may initially involve the formation of a carboxyl group analogous to the anaerobic biotransformation of nonhalogenated aromatic compounds.
Establishment of cultures. Strict anaerobic and aseptic techniques were used throughout the experiments. Experiments were performed with batch reactors consisting of 160-ml or 2-liter glass vessels with serum bottle closures. The glass vessels were sealed with thick black butyl septa and aluminum crimp caps. All of the reactors had the same ratio of headspace volume to slurry-phase volume (3:5, vol/vol). The slurry phase was composed of sediment and anaerobic medium (1:9, vol/vol). The anaerobic sediment inoculum was collected from a depth of 100 m at a site located approximately 16 km east of Fox Point, Wisconsin, in Lake Michigan by using a box corer, transferred to canning jars, and stored at 4°C for approximately three months until used. The techniques used for medium preparation, culture handling, and sampling were based on previously described methods (13). The anaerobic medium used for this study has been previously described (5) and was prepared by combining separate sterile, anoxic solutions of salts, bicarbonate buffer, and reducing agents with the remaining medium components. The sediment inoculum was added to the glass vessels inside an anaerobic glove box (85% N2-10% CO2-5% H2; Coy Laboratories, Ann Arbor, Mich.), and the bicarbonate buffer was equilibrated with 30% CO2-70% N2. 2-CP was added to a final concentration of 200 µM. One 2-liter reactor and two 160-ml viable reactors were prepared in this manner. In the interest of brevity, only the results obtained with the duplicate 160-ml reactors are presented here. Except where noted, the results obtained with the 2-liter reactor were similar. A 2-liter sterile control was prepared in the same manner as the viable reactors, except that the sediment inoculum was autoclaved for 1 h on each of three consecutive days before being combined with sterile medium and 2-CP. The reactors were incubated statically at 30°C.
Analysis of aromatic compounds.
The reactors were continuously
shaken on a platform shaker (100 rpm) during sampling events.
Slurry-phase samples were taken by using deoxygenated and sterile
disposable syringes, filtered (0.45-µm pore size; Gelman Acrodisc),
and either analyzed immediately or frozen (
20°C) prior to analysis.
The aqueous concentrations of 2-CP and its aromatic metabolites were
determined by using a reversed-phase high-performance liquid
chromatography system (Hitachi, Ltd., Tokyo, Japan) equipped with a
computer interface and D-7000 HSM software (Hitachi). Separations were
performed by using a mobile phase of methanol-water-acetic acid
(60:38:2, vol/vol) at a flow rate of 1.0 ml/min and a C18
column (4.7 mm by 235 mm) (Partisil 5 ODS-3; Whatman International
Ltd.). Detection was by UV absorbance with a diode array detector
(Hitachi, model L-4500) operated at 280 nm. Linear calibration factors
were determined from external standards, which were injected along with
each sample set. Identification of aromatic metabolites of 2-CP
biotransformation was based on comparison of their retention times and
UV spectra (250 to 380 nm) with those of authentic compounds.
2-CP fate. Complete removal of 2-CP was observed in the viable reactors within 50 days, as shown for the duplicate 160-ml reactors in Fig. 1A. The concentration of 2-CP in the 2-liter sterile control remained nearly constant during this period, which indicates that transformation of 2-CP in the viable reactors was due to biological activity. Whenever 2-CP was no longer detectable in the sediment slurry reactors, it was replenished.
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ACKNOWLEDGMENTS |
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This research was supported by U.S. Environmental Protection Agency grant R823351.
We thank Gina Berardesco for obtaining the sediment samples and Brian Wrenn for thoughtful comments on the manuscript.
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FOOTNOTES |
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* Corresponding author. Present address: Department of Civil and Environmental Engineering, Lehigh University, 13 E. Packer Ave., Bethlehem, PA 18015-3176. Phone: (610) 758-3543. Fax: (610) 758-6405. E-mail: jgb4{at}lehigh.edu.
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