Environ. Sci. Technol. 1996, 30, 1352-1357
Extent of Reductive Dechlorination of Chlorobenzoates in Anoxic Sediment Slurries Depends on the Sequence of Chlorine Removal BERNARD J. VAN DER WOUDE,* JAN GERRITSE, RUDOLF A. PRINS, AND JAN C. GOTTSCHAL Department of Microbiology, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands
Distinct capacities for reductive dechlorination from all ring positions of chlorinated benzoates (CBa’s) were present in anoxic slurries from a polluted freshwater marsh sediment. The first detectable reductive dechlorination was obtained after a reproducible time lapse. Model calculations based on measured dechlorination rates suggested that this could be accounted for by CBa-dependent growth of a small starter population of dechlorinating bacteria. Complete reductive dechlorination of CBa’s depended on the specific sequence of chlorine removal. In unacclimated slurries, dechlorination started from the ortho rather than the meta or para position. Thus, 2,3,6-triCBa was completely dechlorinated via 2,5-diCBa and 3-CBa; 2,3,5-triCBa was completely dechlorinated via 3,5-diCBa and 3-CBa. Acclimation to 3-CBa induced meta-dechlorination of 2,3,6- and 2,3,5-triCBa. This in turn prevented complete dechlorination as indicated by the accumulation of 2,6-diCBa and 2-CBa as end products. In contrast, acclimation to 2,5diCBa resulted in a population that first removed chlorine from the ortho position. In this way formation of orthosubstituted “dead-end” products was avoided, resulting in complete dechlorination of di- and triCBa’s with chlorines at ortho and meta positions.
Introduction Chlorinated benzoates (CBa’s) have entered the environment as herbicides and products of the aerobic transformation of PCBs or alkyl benzenes (1-3). They resist mineralization because of the halogen substituent(s). For chlorinated aromatic compounds in general, reductive dechlorination is the most important mechanism for the transformation under anoxic conditions (4). * Corresponding author telephone: 31 (0)50-3632191; fax: 31 (0)50-3632154; e-mail address:
[email protected].
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Predictions have been made about the sequence of reductive removal of chlorine atoms from aromatic rings based on their “electronic properties” or on thermodynamic calculations (5-7). For CBa’s, the thus predicted sequence was meta > para > ortho, and the predicted dechlorination in mixtures of CBa’s was tri > di > mono (5). Indeed, previous studies with methanogenic sediments or sludges from various sources have primarily reported dechlorination of CBa’s from the meta and to a lesser extent the para position (8-11). Nevertheless, in anoxic pond sediment, simultaneous para- and meta-dechlorination of 3,4-diCBa was observed (12), and predominant ortho- over metadechlorination of tri- and diCBa’s was observed in river Rhine and Biesbosch marsh sediment slurries (13). Such exceptions to the predicted sequence may be caused by the history of the sediment. Different bacterial populations could have been selectively enriched on specific chlorinated substrates (5, 14). The combined activities of different dechlorinating bacterial populations may result in complete dechlorination and subsequent mineralization of highly chlorinated contaminants (15). For example, pentachlorophenol was completely dechlorinated by combining populations enriched on different monochlorophenols (16). Nevertheless, some chlorinated aromatic compounds (e.g., 2-CBa, 2,6diCBa, 1,3,5-trichlorobenzene, and ortho-substituted PCBs) often appear to be terminal dechlorination products, probably because the ring positions of the remaining substituents are difficult to attack (5, 7, 9, 17, 18). These observations suggest that the sequence of chlorine removal from different ring positions can significantly affect the extent of dechlorination of polychlorinated compounds. The aim of the present study was to establish complete dechlorination of polychlorinated benzoates by inducing a specific sequence of reductive chlorine removal. Therefore, we compared the sequence of reductive dechlorination for several CBa’s in unacclimated (i.e., with no pre-exposure to CBa in the laboratory) and acclimated anoxic sediment slurries (i.e., exposed to known CBa in the laboratory). In particular, possible factors influencing the occurrence of ortho- or meta-dechlorinating microbial activities were studied.
Materials and Methods Chemicals. All chemicals were of analytical grade (purity >97%), except 2,3,6-trichlorobenzoic acid (technical grade) (Pfaltz & Bauer, Waterbury, CT), which was used as a mixture of trichlorobenzoates (triCBa’s mixture). On the basis of HPLC analysis, it consisted of 2,3,5- (10%), 2,3,6- (39%), 2,4,6- (1%), and a compound tentatively identified as 2,4,5trichlorobenzoic acid (50%). Benzoate (Ba), 2-chlorobenzoate (2-CBa), 3-CBa, 4-CBa, 2,4-diCBa, and 2,6-diCBa were purchased from Merck (Darmstadt, Germany); 2,3-diCBa, 2,5-diCBa, 2,3,5-triCBa, and 2,4,6-triCBa were from Aldrich (Gullingham, England); 3,5-diCBa was from Fluka (Buchs, Switzerland). Source and Preparation of the Inoculum. The sampling site was situated in the De Biesbosch Nature Reserve, a freshwater marsh and sedimentation area of the river Rhine, The Netherlands. Anoxic sediment samples were collected
0013-936X/96/0930-1352$12.00/0
1996 American Chemical Society
in sterile bottles, which were completely filled and stored at 4 °C. Within 2 months, samples were homogenated with a spatula in an anoxic glovebox and used as an inoculum (10 or 20% wet weight/volume) for anoxically prepared sterile medium (see below). The resulting slurries, referred to as unacclimated slurries, were subsequently dispensed into anoxic serum bottles or tubes and sealed with butyl rubber stoppers. Anoxic Medium and Incubation. Batch cultures were routinely incubated at 30 °C and pH ) 7.0 under a N2 gas atmosphere. The low-chloride medium (LCM) was modified from Gerritse and Gottschal (19). The basal medium contained mineral salts, trace elements, resazurin as a redox indicator, and yeast extract (2.0 g/L). For subcultures, Biesbosch sediment (1:10 wet weight/volume) was autoclaved together with the basal medium. After being autoclaved, it was completed by the addition of (concentrations in the LCM): autoclaved KNH4PO4 buffer (25 mM, pH ) 7.0); 1 mL/L of an autoclaved solution of a mixture of volatile organic acids: acetate (120 µM), propionate (40 µM), butyrate (20 µM), 2-methylbutyrate (5 µM), isobutyrate (5 µM), valerate (5 µM), and isovalerate (5 µM); 2 mL/L of a filter-sterilized vitamin solution [0.2 µm; modified from Heijthuijsen and Hansen (20) and DeWeerd and Suflita (21)]. This vitamin solution contained (concentrations in the LCM): p-aminobenzoic acid (0.2 mg), folic acid (0.1 mg), thioctic acid (0.1 mg), riboflavin (0.2 mg), thiamin (0.4 mg), nicotinic acid amide (0.4 mg), pyridoxamine (1.0 mg), pantothenic acid (0.2 mg), cobalamin (0.2 mg), biotin (0.04 mg), pyridoxine (0.05 mg), nicotinamide (0.5 mg), hemin (0.05 mg), 1,4-naphthoquinone (0.2 mg). Growth substrates and chlorinated benzoates (0.5-1 mM) were added from separately autoclaved stock solutions. The medium was prepared anoxically and finally reduced with 5 mL/L of an autoclaved Na2S solution (100 mM). In some cases, further reduction was accomplished by the addition of Ti(III)NTA (1 mM, see text) (22). Chemical Analyses. Identification and quantification of CBa’s in supernatants by capillary gas chromatography was done according to Gerritse and Gottschal (19). After methylation overnight with 2 vol of 50% H2SO4 and with 6 vol of methanol, methylated CBa’s were extracted in chloroform and analyzed. One-point calibration (linear response range from 10 µM to 10 mM) was with standards of known concentrations of CBa’s and 2-bromobenzoate as an internal standard. Detection of benzoates by HPLC was according to Pieper et al. (23), using a Jasco (Tokyo, Japan) UV975 UV/VIS detector and an Alltech (Deerfield, IL) Lichrospher 100RP8 column. Calibration was as above, with a linear response range from 1 µM to 1 mM. Chloride was measured colorimetrically according to the method of Bergman and Sanic (24) with NaCl as a standard, after oxidation of the supernatants (10 min incubation) with H2O2 (3% v/v). Gas chromatographic detection of CH4 was as described by Gerritse et al. (25). Acclimation. The acclimation period preceding detectable dehalogenation of a CBa was defined as the time lapse between the time of addition and the last sampling point before detectable decrease (>10% of initial concentration). For CBa’s produced from parent CBa’s, the acclimation period was defined as the time lapse between the measurement before the first detection and the last measurement before detectable decrease of the produced CBa.
Results Chlorinated Benzoates Tested. In unacclimated Biesbosch sediment slurries, chlorinated benzoates (CBa’s) were tested separately, and reductive dechlorination of most CBa’s was observed within 130 days with concomitant chloride release (Figure 1A). In only one bottle out of two sets of triplicates, 2-CBa disappeared between 150 and 250 days of incubation. Dechlorination of 2,6-diCBa was never observed. No disappearance of CBa’s or production of chloride or methane was observed in autoclaved controls. Position of Chlorine Substituent Removal. The product of 2,5-diCBa dechlorination in unacclimated sediment slurries was 3-CBa, which was dechlorinated subsequently (Figure 1A). Also in the case of other di- or triCBa’s (except 2,6-diCBa), the ortho-chlorines, if present, were removed first (Figure 1A). Dechlorination of the daughter products occurred thereafter, which demonstrated that these slurries also harbored meta- and para-dechlorinating activities (Figure 1A). Transient accumulation of low concentrations (
