Environ. Sci. Technol. 1994, 28, 2286-2294
Reductive Dechlorination of Aroclor 1254 by Marine Sediment Cultures Gro D. 0fjord,t Jaakko A. Puhakka,’ and John F. Ferguson” Department of Civil Engineering, University of Washington, Seattle, Washington 98 195
Reductive dechlorination of polychlorinated biphenyls (PCBs) was studied by methanogenic and sulfate-amended laboratory cultures enriched from marine sediments (Puget Sound, WA) in sea-salts media. One sulfate-amended and one methanogenic culture were incubated at a hydraulic retention time of 50 days and fed chitin, sodium benzoate, sodium acetate, and a mixture of four PCB congeners (2,3,3’,5-tetrachlorobiphenyl, 2,2’,4,5,6‘-pentachlorobiphenyl, 2,3’,4,5’,6-pentachlorobiphenyl,and 2,2’,4,4’,5,5‘hexachlorobiphenyl), slowly increasing the concentrations to 6-9 mg/L over 17 months. In these cultures, no dehalogenation of the four congeners was observed. Two other cultures were batch fed Aroclor 1254 at a concentration of 100 mg/L; chitin was used as the PCB carrier and the sole carbon and electron source. Chitin was readily mineralized both under methanogenic and sulfidogenic conditions. Aroclor 1254 was dechlorinated both in the presence and absence of sulfate. The dechlorinations started in both cultures after 4 months of incubation. Under both culturing conditions, 14-15 % meta and 9-10 % of para chlorines were removed over a period of 1 year with the accumulation of mainly tetrachlorobiphenyls; the extent of dechlorination was 7-8%. Introduction Polychlorinated biphenyls (PCBs) are ubiquitous contaminants of the environment with the ocean sediments being the ultimate sink (1). About one-third of the worldwide production of PCBs, estimated to be 10 million t, has been released to the environment (2). High levels of PCBs have been found in, for example, marine samples from the Baltic Sea and the Pacific coastal waters of the United States (3). The open ocean, although low in PCB concentration relative to some lakes and most coastal areas, is the largest environmental PCB reservoir. Due to their hydrophobicity, PCBs sorb to particulate material and sink to the bottom sediments. In harbors, nearshore sediments, and dredge spoils, PCB levels in marine sediments are sometimes present at levels that are of concern because of their lipophilicity and toxicity. PCBs, particularly the higher chlorinated congeners were, until approximately 20 years ago, thought to be resistant to biodegradative processes and hence highly persistent in the environment. However, investigations of PCB spill sites have shown altered profiles of PCB mixtures in sediments as compared to the original source ( 4 , 5). Most attention has been focused on anaerobic dechlorination of PCBs, which produces metabolites that are often less toxic, less likely to bioaccumulate, and more susceptible to further microbial attack by oxidative biodegradative processes. Studies on anaerobic dehalogenation in nonmarine systems both in situ and in laboratory cultures have shown extensive dehalogenation
* Corresponding authors.
t Present address: Aquateam-Norwegian Water Technology Centre AIS, P.O. Box 6326, Etterstad, N-0604 Oslo 6, Norway. 2288
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of PCBs by microbial consortia exhibiting complex patterns of reactivity, mainly loss of non-ortho-substituted chlorines as reviewed in Mohn and Tiedje (6). Sediment decontamination by bioremediation is of significant interest. The emphasis on nonmarine systems in ecological research may have resulted in a restricted view of the potential for reductive dehalogenation. A number of marine organisms, such as macroalgae and invertebrates, produce halogenated aromatic and aliphatic compounds, which may be natural substrates for marine bacteria. The most common of these compounds are polybrominated with some exceptions being polychlorinated (see ref 7 and references cited therein). Further, the ionic strength of seawater ensures that different bacteria are involved, since few if any anaerobic bacteria are tolerant of salt concentrations ranging from freshwater to marine. However sulfate, which is present in marine environments in approximately 27 mM concentrations, has been reported to inhibit the dehalogenation of PCBs (8-10). Reductive dehalogenation of several halogenated phenols has been shown in the presence of sulfate (7, 11, 12). In a few cases, dehalogenation of PCBs has been studied in estuarine and marine systems (8,13-15). Brown and Wagner (13) studied Acushnet Estuary (New Bedford, MA) sediments and found a previously unreported type of PCB dechlorinations which was designated as Process H, This metalpara selective dechlorination of most of the higher PCB congeners was reported to be similar to their unpublished dechlorination patterns at Escambia Bay (FL) and New York Harbor (NY) marine sites, Lake et al. (14) also found environmental PCB dechlorination in New Bedford Harbor sediment cores and, more recently, reported three pathways that involved chlorine removals from congeners with 2,3,4-, 2,4,5-, and 3,4-chlorophenyl groups (15). The most extensively altered PCB distribution was found in the 15-17.5 cm deep core sections. Alder et al. (8) examined reductive dechlorination of Aroclor 1242 by New Bedford Harbor sediments under methanogenic and sulfidogenic culturing conditions. Removal of meta and para chlorines was found in the methanogenic sediment; no dechlorination was observed under sulfatereducing conditions. Chitin is the major structural polymer in invertebrates (marine animals, insects, fungi, and green algae) (16). It is a polysaccharide composed of P-(l-4)-linked monomers of N-acetyl-l-@-D-glucosamine. Billions of tons of chitin are produced annually in the oceans, and substantial amounts become incorporated into anaerobic habitats. Chitin serves as a growth substrate for sediment microorganisms as well as an efficient sorbent of PCBs and, therefore, may affect their bioavailability. In this study, we compared the reductive dechlorination of PCBs by marine sediment cultures in the presence and absence of sulfate in sea-salts media. Chitin was used as the PCB carrier and the main electron donor. The aim of this work was to evaluate the potential of reductive 0013-936X/94/0928-2286$04.50/0
0 1994 American Chemical Society
dechlorination of Aroclor 1254 in semicontinuously electron donor fed, anaerobic marine sediment cultures.
Experimental Section Marine Sediment Samples. The anaerobic cultures used in this study were enriched from Puget Sound sediments. The sediment samples were collected from 10 to 20 cm below the sediment surface near the city of Bremerton and from Elliot Bay, adjacent to Seattle, WA. The salinity and temperature of seawater were 32 ppm and 16 “C a t Sinclair Inlet, Bremerton, and 31 ppm and 12 “C off Pier 63 in Eliot Bay. The average total PCB concentration in Puget Sound sediments varies between 0.5 and 2 mg/kg. Quantitative analysis of PCBs in the sediments was not performed, but blanks and samples before PCB spiking indicate that the sediment inocula had concentrations no greater than 0.5-2 mg/kg. Samples were anaerobically sealed until used. Media. Anaerobic sea-salts medium (ASM) contained a seawater salts solution, nutrient salts, trace minerals, and resazurin as a redox indicator. The concentration of seawater salts was based upon the composition of seawater described by Goldberg (17) and included the major cations and anions except sulfate that are in concentrations greater than 1 mg/L in seawater as follows (g/L): NaCl (22.79), KC1 (0.72), NaHC03 (0.19), NaBr (0.083), H3B03 (0.0271, NaF (0.003),MgC12.6Hz0 (11.18), CaC12.2Hz0 (1.461, and SrCly6Hz0 (0.024). Nutrient salts NazHPOq7HzO and NH&l were added at 0.089 and 0.27 g/L, respectively. The trace mineral mixture was a modification of the formula by Shelton and Tiedje (18) and included the following salts (mg/L): FeC12.4HzO (21, MnCl~4Hz0(1.5) CoC1~6H20(1.5), ZnCl2 (0.15), CuCl2 (0.09),H3B03(0.151, NazMoOq2H20 (0.3), NiCly6HzO (0.3), Na2Se03.5HzO (0.15), and NazWOq2HzO (0.15). The pH of the marine medium was adjusted to 7.6 with NaOH. This medium was used in batch bioassays and in the enrichment and maintenance of methanogenic cultures and supplemented with 2.6 g/L (27 mM) of sodium sulfate for bioassays and enrichment and maintenance of sulfate-amended cultures. The salinity of sulfate-containing media was adjusted to the same level as in the ASM by reducing the NaCl content. PCB Coating. Both Aroclor 1254 and the individual PCB congeners were preadsorbed to chitin (28 mg/g of chitin). The procedure involved preparation of an acetone/ chitin mixture. The PCBs were dissolved in acetone and added to the acetoneichitin suspension. The acetone was evaporated by mixing the suspension on a magnetic stirrer. The dry chitin was then fed to the cultures. Establishment of Anaerobic Cultures. Four different anaerobic sediment cultures were enriched from marine sediments. Sediment samples were added to culture vessels with the ASM to give initial suspended solids concentrations of 10 g/L. One enrichment medium contained ASM with sulfate; the other contained ASM without sulfate. Two of the enrichment vessels consisted of Erlenmeyer flasks with liquid volumes of 5 L and manometers for gas measurement. Continuous mixing was provided by magnetic stirring. These cultures were incubated a t room temperature and fed a mixture of acetate (155 mg/d), benzoate (75 mg/d), and pyruvate (50 mg/d) in ASM with and without sulfate. After 2 months of operation, chitin was added to the organic feed while pyruvate was omitted.
Daily organic feed/sulfate ratio was such that approximately 40% of the reducing equivalents were sufficient for complete sulfate reduction. The organic loading rate was held a t 80 mg of COD L-’ d-1, and the hydraulic retention time (HRT) was held a t 50-100 d i n both systems. A mixture of 2,3,3‘,5-tetrachlorobiphenyl,2,2‘,4,5,6’-pentachlorobiphenyl, 2,3’,4,5’,6-pentachlorobiphenyl, and 2,2r,4,4’,5,5r-hexachlorobiphenyl adsorbed onto chitin was introduced to the feed, and the concentrations of each congener were gradually increased to 6-9 mg/L. Culture contents were periodically sampled and analyzed for PCB congeners. Ferrous chloride was periodically added to the sulfidogenic enrichment to precipitate excess sulfide. Two additional cultures were established after initial testing with the four congeners, one for enrichment of methanogenic and the other of sulfate-reducing cultures. The enrichment cultures were inoculated with media from the cultures described above, together with fresh Puget Sound sediment. The suspended solids concentration was adjusted to 10 g/L. These cultures had a liquid volume of 1.5 L and were operated as batch cultures mixed once a day for approximately 10 s, spiked initially with PCB, and fed weekly with chitin but without removal of sediment or liquid replacement. They were initially fed Aroclor 1254 a t 100 mg/L concentration. The Aroclor 1254 addition was preadsorbed to chitin. Chitin was fed once a week a t a loading rate averaging0.1 g L-l d-l, but without liquid replacement. Sodium sulfate was added once a month (0.1-2.7 g/reactor) so that it was always in excess compared to electron donor in the system. After 18 months of incubation, sulfate concentration in the medium was 1400 mg/L. Samples were taken periodically for 12 months. Anaerobic Batch Bioassays. Biodegradation of chitin and the effect of Aroclor 1254coating on chitin degradation were studied using serum bottle bioassays. Strict anaerobic techniques were followed in the preparation of the ASM for batch bioassays. The medium was boiled under an oxygen-free nitrogen stream and transferred into nitrogen-purged serum bottles (65 mL total volume) with 35 mL of liquid volume. Sodium sulfide was used as the reducing agent (5-10 mg/L). All bottles were sealed with butyl rubber stoppers and aluminum crimp seals. Trace amounts of methanol were added to each bottle to biologically consume any remaining oxygen. The test compounds were then added, and the bottles were monitored for gas and methane production and PCB transformation. The bottles were incubated a t room temperature (20 f 5 “C) in the dark. The gas production was measured as described by Nottingham and Hungate (19).
Reagents. Distilled glass-grade hexane (Baxter Health Care Corp., Mushegon, MI) was used in the PCB extractions. Chitin was a product of Sigma Chemical Co. (St. Louis, MO). Octachloronaphthalene (OCN) (99% minimum purity), chlorobiphenyl congeners (99% ), Aroclor 1242 (99%),Aroclor 1254 (99%),Aroclor 1260 (99%),and individual PCB congeners (2,3,3’,5-CB, 2,2’,4,5,6’-CB, 2,3’,4,5’,6-CB, and 2,2’,4,4’,5,5’-CB, all 99+% 1 were purchased from Accustandard Inc., New Haven, CT. Analytical Procedures. PCB analysis: suspended samples (20 mL) from the enrichment cultures were centrifuged a t 3000 rpm for 3 min. The supernatant was removed by decanting and by using a Pasteur pipet, and decachlorobiphenyl was added as an internal standard Environ. Sci. Technol., Vol. 28, No. 13, 1994
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(1.5 mg/L). The sediments were repeatedly extracted: first twice with acetone (10 mL) and then twice with n-hexane (10 mL). The solvent extracts were combined. Acetone was removed by reverse partitioning into water. The hexane extract was concentrated to 1 mL under a gentle stream of nitrogen and purified in 6-mL Supelclean LCFlorisil SPE tubes (Supelco, Bellefonte, PA). The Florisil column was washed with hexane to give the final solvent volume of 10 mL. Elemental sulfur was removed from the extract using a tetrabutylammonium sulfite reagent (20). After extraction, the remaining solids were dried at 110 “C, the dry weight was measured, and the PCB analysis results were normalized to the sample dry weight. The extraction recoveries were 60 f 10%. PCB analysis was performed on a Varian 3700 gas chromatograph (GC) with a Durabond-5 (J&W Scientific, Folsom, CA) fused silica capillary column (30 m, 0.32 mm i.d., film thickness 0.25 mm) and a 63Nielectron capture detector. Helium was used as the carrier gas (0.75 mL/ min) and argon/methane (95%/5%)as the makeup gas. The injector and detector temperatures were 300 and 350 “C, respectively. The initial oven temperature was 185 “C, ramped a t 1 “C/min to 250 “C,and held a t 250 “C for 5 min. Data were collected on a personal computer using Omega software (Perkin Elmer, Norwalk, CT.). Methane was determined isothermally at 80 “C using a Hach Carle Series A GC with thermal conductivity detector and helium as carrier gas. The GC was equipped with a 6 f t long, W i n . diameter, molecular sieve 5A, mesh 80/100, stainless steel column packed with Hayes Sep Q (Supelco). Sulfide was measured potentiometrically from samples in an alkaline buffer using a Corning pH/ion meter (Corning Ltd., Model 150) with an Orion Research Ag2+/ $32- electrode (Orion Research, Inc., Model 9482000) and a double junction reference electrode (Model 900200). Standards for calibration were prepared from Na2S-9H20 for each measurement. PCB Congener Identification and Quantification. For Aroclor congener identification, Aroclor 1242,Aroclor 1254, and Aroclor 1260were used separately as standards. The congeners were identified by matching the peaks of the three Aroclors with the chromatographic profile of the same Aroclors previously identified by Ballschmitter and Zell(21) and Schulz et al. (22). The single congeners 52, 59, 66, 72, 101, 102, 121, 123, 138, 153, and 190 were also used for direct identification of these congeners. The relative retention times of the single congeners were then used to identify the peaks in the Aroclor chromatograms. Octachloronaphthalene (OCN) was used as the internal standard for determining relative retention times of the congeners, while decachlorobiphenyl was used for the determination of extraction efficiency. RRTs for all 209 congeners were given by Mullin et al. (23). Peak numbers and congener names in this study are given in Table 1. The mole percentage of all PCBs recovered was calculated with the assumption that there was no loss of the biphenyl moiety and that coeluting congeners were present in equal amounts. The extent of dechlorination was calculated from the weighted average number of chlorines (Claverage) in samples before and after incubation. Claverage was calculated according to the following equation: 2288
Environ. Sci. Technoi., Vol. 28, No. 13, 1994
Table 1. Congener Assignment for Each Chromatographic Peak peak no.
IUPAC no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
8+5 18 + 15 16 + 32 31 + 28 33 + 53 52 49 47 + 48 44 59 42 41 + 64 40 74 76 66 + 95 91 56 + 60 84 101 99 83 97 87 + 115 85 136 110 + 77 82 151 144 + 135 124 107 118 + 149 134 146 + 105 153 141 130 137 138 129 128 185 + 167 177 171 156 172 170 + 190
chlorine substitution 2,3-CB + 2,4’-CB 2,2’4-CB + 2,2’,5-CB 2,2’,3-CB + 2,4’,6-CB 2,4,4’-CB + 2,2’,5-CB 2’,3,4-CB + 2,2’,5,6’-CB 2,2’,5,5’-CB 2,2’,4,5’-CB 2,2’,4,5-CB + 2,2’,4,4’-CB 2,2’,3,5’-CB 3,3’,6-CB 2,2’,3,4’-CB 2,2’,3,4-CB+ 2,3,4’,6-CB 2,2’,3,3’-CB 2,4,4’,5-CB 2’,3,4,5-CB 2,3’,4,4’-CB + 2,2’,3,5,6’-CB 2,2’,3,4’,6-CB 2,3,4,4‘-CB + 2,3,3‘,4‘-CB 2,2’,3,3‘,6-CB 2,2’,4,5,5’-CB 2,2’,4,4’,5-CB 2,2’,3,3’,5-CB 2,2’,3’,4,5-CB 2,2’,3,4,5’-CB + 2,3,4,4’,6-CB 2,2’,3,4,4’-CB 2,2‘,3,3‘,6,6‘-CB 2,3,3‘,4‘,6-CB + 3,3‘,4,4‘-CB 2,2’,3,3’,4-CB 2,2’,3,5,5’,6-CB 2,2’,3,4,5’,6-CB + 2,2’,3,3’,5,6’-CB 2’,3,4,5,5’-CB 2,3,3’,4’,5-CB 2,3’,4,4’,5-CB + 2,2’,3,4’,5’,6-CB 2,2’,3,3’,5,6-CB 2,2’,3,4’,5,5’-CB+ 2,3,3’,4,4’-CB 2,2’,4,4’,5,5’-CB 2,2’,3,4,5,5’-CB 2,2’,3,3’,4,5’-CB 2,2’,3,4,4’,5-CB 2,2’,3,4,4’,5’-CB 2,2’,3,3’,4,5-CB 2,2’,3,3’,4,4’-CB 2,3’,4,4’,5,5’-CB+ 2,2’,3,4,5,5’,6-CB 2,2’,3,3’,4’,5,6-CB 2,2’,3,3’,4,4’,6-CB 2,3,3’,4,4’,5-CB 2,2’,3,3’,4,5,5’-CB 2,2’,3,3’,4,4’,5-CB+ 2,3,3’,4,4’,5,6-CB
where cld is the number of chlorines in the peak congener; c d is the molar concentration of the peak congener; Cbt is the total molar concentration; and d is the number of peaks.
Results Startup of Marine Cultures. Daily COD feed to the sediment-inoculated cultures resulted in the onset of methane production after a lag period of 15 weeks in the non-sulfate medium. During the following steady operation period of 2 months, the mean gas production was 135 mL/d (SD=63, 26 measurements) or 340 mL/g of COD added with about 50% methane in the gas. After the establishment of methane production, the PCB congener feed was started. In the sulfate-amended culture, no accumulation of added organics was observed. After 1 month, the PCBcongener/chitin was added to the feed mixture. After 10
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Table 2. Effect of Aroclor Coating on Chitin Degradation
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Figure 1. Cumulativegas and methane production from chitin by marine methanogenic culture (A), marine sulfate-enriched culture in the presence of 27 mM sulfate (B), and marine sulfate-enriched culture in the absence of sulfate (C). Symbols: (B),gas production; (O),methane production.
weeks of operation, the first sulfide measurements showed a 350 mg/L concentration. The daily sulfate amendment was less than that of the electron donor mixture, but sulfate reduction was maintained as observed by sulfide generation. Periodic ferrous chloride addition was started to maintain the sulfide below 300 mg/L. The pH varied between 6.9 and 7.3. These results show slow enrichment of the methanogenic consortium from the Puget Sound sediment samples and faster enrichment of the sulfidogenic culture. This suggests that sulfidogenesiswas the major terminal process in the sediment carbon flow. Effect of PCB Coating on Chitin Degradation, Chitin degradation by the marine sediment cultures was studied in batch bioassays. Figure 1A shows the gas and methane production from chitin by the methanogenic sediment culture in the presence of 50 mg/L Aroclor 1254 (28 mgf g of chitin). After a lag period of 2 weeks, methane production started and resulted in a methane yield of 320
0
mL/g of COD within 10 weeks. With subsequent refeedings of chitin (without Aroclor), methane production started without lag and resulted in a similar methane yield as after the first feeding. The results show efficient conversion of chitin to methane by the marine sediment enrichment. These results indicate enrichment of chitinmineralizing microorganisms during the refeedings. The degradation of chitin by the sulfate-enriched culture was studied both in the presence and absence of sulfate. Aroclor 1254 was preadsorbed onto chitin a t a concentration of 28 mg/g of chitin. The gas and methane production are shown in Figure 1B,C. In the sulfate media, 30 mL of CHJg COD was produced in 24 weeks, while in the absence of sulfate this culture produced about 220 mL of CH4/g of COD. The lag phases prior to the onset of methane production were 6 and 11weeks in the absence and presence of sulfate, respectively. After 24 weeks of incubation in the presence of sulfate (2.6 g/L), sulfide concentrations in the liquid phase were 265,325, and 110 mg/L in bottles fed 50, 100, and 200 mg/L Aroclor 1254 (28 mg/g of chitin), respectively. Sulfate reduction proceeded in the culture with chitin as the sole carbon source. However, sulfate reduction ceased at the highest Aroclor concentration. Methane production in bottles without sulfate showed that this consortium also contained methanogens. Sulfate inhibited methane production by this culture. In the methanogenic enrichment, the methane yield was 220 mL of CH4/g of COD while complete conversion of biodegradable COD would produce approximately 330 mL of CHd/g of COD. Sulfidogenic degradation of chitin was probably also controlled by sulfide accumulation. The effect of Aroclor 1254 coating on chitin degradation was studied further with the methanogenic culture, and the results are shown in Table 2. Aroclor 1254 at 700 mg/g of chitin (2000 mg/L) completely inhibited methane production from chitin. In a glucose medium, 2000 mg/L Aroclor, added in acetone, did not inhibit gas production (results not shown). The inhibition of chitin degradation was likely due to Aroclor coating rather than toxicity. Fate of PCB Congeners. Four PCB congeners (2,3,3’,5CB, 2,2’,4,5,6’-CB, 2,3’,4,5’,6-CB, and 2,2’,4,4’,5,5’-CB) were added in gradually increasing concentrations to both enrichments, reaching the final concentration of 6-9 mg/L for each congener after an incubation of 17 months. The congeners accumulated with no appearance of conversion products. These results show that dechlorination was not induced in Puget Sound sediment cultures by long-term exposure to these PCB congeners a t low concentrations. Aroclor 1254 Dehalogenation. To study the potential of PCB dechlorination, both the methanogenic and sulfateamended cultures were batch fed 100 mg/L Aroclor 1254. To demonstrate the progress of dechlorination with time, Figure 2 shows the decrease of two selected hexachlorobiphenyl congeners [nos. 153 (2,2’,4,4’,5,5’-CB) and 138 (2,2’,3,4,4’,5’-CB)I and the corresponding increase in the Environ. Sci. Technol., Vol. 28, No. 13, 1994
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Figure2. Removal of hexachlorobiphenylcongeners nos. 153 (2,2’,4,4’,5,5‘-CB)and 138(2,2f,3,4,4’,5f-CB)andaccumulationof tetrachlorobiphenyl 48 (2,2’,4,4’-CB/2,2’,4,5-CB) and 49 (2,2’,4,5’-CB) during 12 months of incubation in marine methanogenic (A) and sulfatecongeners 47 reducing (B) culture.
+
following tetrachlorobiphenyl congener concentrations: congeners 47+48 (2,2’,4,4’-CB/2,2’,4,5-CB)and congener 49 (2,2’,4,5’-CB)in both cultures. First changes in Aroclor 1254 chromatograms were observed after 4 months of incubation in both cultures (results not shown). Figure 3 shows the mole percent distribution of the congeners in the two cultures compared to Aroclor 1254 after 12 months incubation. In the methanogenic culture, the main congeners which increased (peak no., mol % change) were as follows: 2,2’,4,5’-CB (7, +6.8%), 2,2’,4,4’CB/2,2’,4,5-CB (8, +8.6%), 2,2’,3,4’-CB (11, +0.5%), and 2,3’,4,4’-CB/2,2’,3,5’,6-CB(16, +6.3%), with simultaneous decrease of 2,2’,4,5,5’-CB (20, -0.7 % 1, 2,3,4,4’,6-CB/ 2,2‘,3,4,5’-CB (24, -2.8%), 2,2’,3,4,4’-CB (25, -1.5%), 2,3,3’,4‘,6-/3,3’,4,4’-CB (27, -5.2 93 ), 2,2‘,3,3’,5,6’-CB (30, -0.7 %), 2,3’,4,4’,5-CB (33, -1.1 %), 2,3,3‘,4,4’-CB (35, -0.1 %), 2,2’,4,4’,5,5’-CB(36, -5.4%), 2,2’,3,4,5,5’-CB (37, -0.7 70 ), 2,2’,3,3’,4,5’-CB (38, -0.4%), 2,2’,3,4,4’,5‘-CB (40, -2.5%), 2,2’,3,3’,4,5-CB (41, -0.5%), 2,2’,3,3’,4,4’-CB (42, -I.()%), 2,3,3’,4,4’,5-CB (46, -0.5%), 2,2’,3,4,5,5’-CB (47, 4.1% ), and 2,Y73,3’,4,4’,5-CB/2,3,3’,4,4’,5,6-CB (48, -0.7 % ). In the sulfidogenic culture, the same congeners increased (peak no., mol % change) as in the methanogenic culture includingthe following: 2,2‘,4,5‘-CB (7, +7.1%), 2,2’,4,4‘,/2,2’,4,5-CB (8, +9.2%), 2,2’,3,4’-CB (11, +0.6%), and 2,3’,4,4’,-/2,2’,3,5’,6-CB(16, +6.7 % ). Simultaneously, the following peaks decreased (peak no., mol 7% change): 2,2’,4,5,5’-CB (20, -0.8 % ), 2,3,4,4’,6-/2,2’,3,4,5’-cB (24, -3.3 %), 2,2’,3,4,4’-CB(25,-1.7% ),2,3,3’74’,6-CB/373’,474’CB (27, -1.9%), 2,2’,3,3’,5,6‘-CB (30, -0.7 %), 2,3‘,4,4’,5CB (33, -1.3%), 2,3,3’,4,4’-CB (35, -0.1%), 2,2’,4,4’,5,5’CB (36, -6.5%), 2,2’,3,4,5,5’-CB (37, -0.9%), 2,2‘,3,3’,4,5‘CB (38, -0.5%), 2,2’,3,4,4’,5’-CB (40, -3.9%), 2,2’,3,3’,4,5CB (41, -0.5%), 2,2’,3,3’,4,4’-CB(42,-1.2%), 2,3,3’,4,4’,52290
Environ. Sci. Technol.,Vol. 28, No. 13, 1994
CB (46, -0.7%), 2,2’,3,4,5,5’-CB (47, -0.1%), and 2,2’,3,3’,4,4’,5-CB/2,3,3’,4,4’,5,6-CB(48, -0.9%). No mono- or dichlorinated biphenyls were detected in either the methanogenic or sulfidogenic culture. The extent of dechlorination was 7 and 8%in the methanogenic and sulfate-reducing culture, respectively. The average numbers of ortho, m e t a , and para chlorines per biphenyl in Aroclor 1254were 1.92,1.82, and 1.15,respectively. After 12 months of incubation, the average numbers of chlorines per biphenyl in the methanogenic system were 1.95 for ortho, 1.55 for m e t a , and 1.03 for para substituents. In the sulfate-reducing system, the respective numbers were 1.89 for ortho, 1.56 for m e t a , and 1.04 for para chlorines per biphenyl. The metabolite accumulation together with changes in average numbers of chlorines per biphenyl show that the dechlorinations were mainly from meta positions, but para dechlorination also occurred. The results show similar dehalogenation patterns in the presence and absence of sulfate. Figure 4 shows the distribution of the different Aroclor 1254 homologs and their abundances in the enrichments after 12 months of incubation. The homolog distribution shows increasing amounts of tri- and tetrachlorobiphenyls and decreases in the penta-, hexa-, and heptachlorobiphenyls. The mol % removals in the sulfidogenic system for highly chlorinated congeners were as follows: 1.1 for heptachlorobiphenyls, 11.4 for hexachlorobiphenyls, and 5.0 for pentachlorobiphenyls. In the methanogenic system, the corresponding mol 96 removals were 0.64, 13.0, and 6.1. These results show partial dechlorination of Aroclor 1254 both in the presence and absence of sulfate. After 12 months, 500 mg/L Aroclor 1254 was added to both enrichments. After 3 months, the dechlorination was
Discussion
B l
C
PEAK NUMBER
Flgure 3. Mol % distribution of PCB congeners in Arwlor 1254 (A) and after 12 months of incubation with marine methanogenic cuiture (E)and sulfate-reducing culture (C).
501 40
leva
penta
hexa
hepta
Figure 4. Homolog distribution of congeners in marine methancgenic ( 0 )and sulfate-reducing0cunure after 12 months of incubation and in Aroclor 1254 (gray box).
about 5% in both enrichments, indicating the enrichment of dechlorinating organisms. After 6 months, the degree of dechlorination was 5 and 7.5% in the methanogenic and sulfate-reducing enrichments, respectively. Figure 5 shows the chromatograms of month 0 and month 6 (after addition of 500 mg/L) in the sulfate-reducing enrichment. The dechlorinations followed the same pattern as in the first incubation period.
Chitin Degradation. In the marine environment, PCBs adsorb to particulate material and sediment particles. Therefore, PCBs were adsorbed onto chitin, the most common polysaccharide in marine environment. Anaerobic degradation of the chitin by methanogenic and sulfate-reducing cultures was efficient as indicated by methane and sulfide production, respectively. Anaerobic cultures are known t o produce carbon dioxide and/or methane from chitin (24,25). Our experiments show that Aroclor 1254 coating at high concentrations decreased the bioavailability of chitin. Aroclor 1254 coating probably decreased chitin degradation by physical hindrance of chitin hydrolyzing exoenzymes. PCB Dechlorination. In Puget Sound sediments, the total PCB concentrations are 2 mg/kg or less. The exposure of two cultures from Puget Sound sediments to selected PCB congeners a t up to 6-9 mg/L for 17 months did not result in dechlorination. 2,2’,4,4’,5,5’-Hexachlorobiphenyl was one of the four selected congeners and is also a constituent of Aroclor 1254. It was almost completely dehalogenated in the Aroclor 1254fed enrichments. With 100mg/LAroclor 1254,dechlorinationstarkdwithin 4 months. Reductive dehalogenation has in some cases been limited by low PCB concentrations (26,27)and not in other cases (28). Our results show the potential for enrichment of Aroclor 1254 dechlorinating microbes from marine sediments exposed only to very low levels of PCBs as previously observed in uncontaminated river sediments (28). These results also suggest that low-level PCB contaminationofmarine sedimentsmay be very persistent. Inorganic electron acceptors in anaerobic environments affect the electron flow and thereby potentially compete with dehalogenation reactions. The first reported aryl dehalogenating organism, Desuljomonile tiedjei, is a sulfate-reducing bacterium (29). It is not known whether PCB dechlorinators are sulfate reducers like D. tiedjei. Sulfate, however, is known to be the major electron sink in marine sediments (30). In our study, Aroclor 1254 dechlorination was similar in the presence and absence of sulfate. In previous studies with cultures from nonmarine sources sulfatehas been reported to inhibit dehalogenation of PCBs (9,10, 31). Reductive dehalogenation of in the presence of sulfate was not detected in the New Bedford Harbor cultures (8). Environmental PCB dehalogenation in New Bedford Harbor sediments was more extensive in deeper (15-17.5 cm) rather than in upper sediment layers ( I @ , which may be related to the prevalence of methanogenesis and sulfate reduction, respectively, in these zones. Sulfate may not inhibit dehalogenation directly but rather the enrichment of dehalogenation activity (for areview, see ref 6). Our result demonstrates the potential of reductive dechlorination of Aroclor 1254 in sulfatecontaining sea-salt environment, which is the largest PCB reservoir as well as the locale of several PCB spills. Dehalogenation of Aroclor 1254 resulted in a decrease in hepta-, bexa-, and pentachlorinated biphenyls, with an accumulation of mainly 2,2‘,4,4‘-CB/2,2‘,4,5-CB,2,2’,4,5’CB, and 2,3’,4,4’-/2,2’,3,5’,6-CB,indicating meta removal of chlorine8 from mainly congeners with 2,3,4-, 2,3,4,5-, and 2,3,4,6-chlorophenyl groups. Table 3 summarizes the suggested meta dechlorination processes in our Puget Sound sediment cultures. This limited dechlorination pattern is very similar to that reported for a sediment core Environ. SCI. Technol.. VOI. 28. No. 13. 1994
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Month 0
t
c I
8
rdm
c. D n
m
$
a a
n
Month 6
m n
9
% .
6
ap'
1
410
cy
a
i m
9
3
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6
n
Flgure 5. Gas chromatogram of Aroclor 1254 after and with incubationtimes of 0 and 6 months after second spiking (500 mg/L) in sulfate-reducing culture. 2292
Envlron. Sci. Technol., Vol. 28, No. 13, 1994
Table 3. Suggested Aroclor 1254 meta-Dechlorination Processes in Methanogenic and Sulfidogenic Marine Sediment Cultures DB-5 peak no. 48 47 46 42 41 40 38 37 36 35 33 30 27 25 24 20
PCB congeners in peak
suggested dechlorination process
---
2,2’,3,3’,4,4’,5 2,2‘,4,4‘ 2,2’,3,3‘,4,4’,5-CB, 2,3,3’,4,4’,5,6-CB 2,2’,4,5’ 2,2‘,3,4,5,5‘ 2,2’,3,4,5,5’-CB 2,3,3’,4,4’,5 2,3‘,4,4‘ 2,3,3’,4,4’,5-CB 2,2’,3,3’,4,4’2,2’,4,4‘ 2,2’,3,3’,4,4’-CB 2,2’,3,3’,4,5 2,2’,3,4’ 2,2‘,3,3’,4,5-CB 2,2’,3,4,4‘,5’2,2‘,4,4’ 2,2’,3,4,4’,5’-CB 2,2’,3,3’,4,5’2,2’,4,5’ 2,2’,3,3‘,4,5‘-CB 2,2’,3,4,5,5’2,2’,4,5’ 2,2’,3,4,5,5‘-CB 2,2’,4,4‘,5,5‘ 2,2‘,4,4’ 2,2’,4,4’,5,5’-CB 2,3,3’,4,4’ 2,3’,4,4’ 2,3,3’,4,4‘-CB 2,3’,4,4’,5 2,3‘,4,4‘ 2,3’,4,4’,5-CB 2,2‘,3,3‘,5,6‘ 2,2‘,3,5‘,6 2,2’,3,3’,5,6’-CB 2,3,3’,4‘,6 2,2‘,5,6 2,3,3’,4’,6-CB, 3,3’,4,4’ 4,4‘ 3,3’,4,4‘-CB 2,2’,3,4,4’- 2,2’,4,4’ 2,2’,3,4,4‘-CB 2,4,4’,6 2,3,4,4’,6-CB,2,2’,3,4,5’-CB 2,3,4,4’6 2,2’,3,4,5’2,2’,4,4’ 2,2’,4,5,5‘ 2,2’,4,5’ 2,2’,4,5,5’-CB
-
(H22) from New Bedford Harbor (15). Lake et al. (15) described conversion of congeners carrying 2,3,4-chlorophenyl groups to those carrying 2,4-chlorophenyls; a close inspection of their chromatograms indicates that 2,3,4,5and 2,3,4,6-~hlorophenylgroups had also been attacked. The decrease in the average number of para chlorines per biphenyl (9-10 % ) indicates also pura dechlorination. Several different dehalogenation patterns were observed in New Bedford Harbor sediments (13-15). Therefore, further sampling and culturing of Puget Sound sediments might also yield a variety of PCB-dechlorinating systems. In addition, the dechlorination in our study was less extensive (7-8%) than has been found in many studies with freshwater sediments (for a review, see ref 6 ) . This research differed from earlier PCB dehalogenation studies in several respects, including the low-contaminated marine sediments used as inoculum, the low solids concentration in the incubation, and the use of marine sea-salts medium. To the best of our knowledge, neither PCB dehalogenation in laboratory cultures enriched from noncontaminated marine sediments has been reported nor has PCB dehalogenation been observed in the presence of sulfate in marine sediments. Conclusions
Anaerobic marine microorganisms have the potential to reductively dechlorinate Aroclor 1254 in the presence and absence of seawater sulfate concentrations and in the presence and absence of sulfate reduction. Reductive dehalogenation of Aroclor 1254 is partial, and chlorines are mainly removed from m e t a and also from para positions. PCB-dehalogenating cultures can be enriched for from marine sediments exposed to background-level PCB contamination by long-term exposure (approximately 4 months) to Aroclor 1254. Chitin is mineralized both by marine methanogenic and sulfidogenic microbial consortia. Aroclor 1254 is available for reductive dechlorination when adsorbed onto chitin substrate. Sorption of Aroclor 1254 onto chitin decreases chitin bioavailability.
Acknowledgments
This work was supported by a grant from the Office of Naval Research (Grant N00014-91-5-1792), by the Valle Scholarship and Scandinavian Exchange Program (G.D.0.), and by the Academy of Finland (J.A.P.). We thank anonymous referees for valuable comments on the manuscript. Author-Supplied Registry Numbers: Aroclor 1242, 53469-21-9;Aroclor 1254,11097-69-1;Aroclor 1260,1109662-5, chitin, 1398-61-4; hexane, 110-54-3; OCN, 2505396-7; tetrabutyl ammonium hydrogen sulfite, 32503-278. Literature Cited (1) Hansen, L. G. In Polychlorinated Biphenyls (PCBs): Mam-
malian and Environmental Toxicology;Safe,S., Hutzinger, O., Eds.; Environmental Toxin Series 1; Springer-Verlag:
Berlin and Heidelberg, 1987; pp 15-48. (2) Eisenreich,S. J. InSources and Fates of Aquatic Pollutants; Hites, R. A., Eisenreich,S. J., Eds.; Advances in Chemistry 216; American Chemical Society: Washington, DC, 1987; pp 394-469. (3) Sissons, D.; Welti, D. J. J. Chromatogr. 1971, 60, 15. (4) Brown, J. F., Jr.; Wagner, R. E.; Feng, H.; Bedard, D. L.;
Brennan,M. J.;Carnahan, J. C.; May, R. J. Enuiron. Toxicol. Chem. 1987,236, 709. ( 5 ) Brown, J. F.; Bedard, D. L.; Brennan, M. J.; Carnahan, J. C.; Feng, H.; Wagner, R. E. Science 1987,236, 709. (6) Mohn, W. W.; Tiedje, J. M. Microbiol. Rev. 1992,56,482. King, G. M. Appl. Enuiron. Microbiol. 1988, 54, 3079. Alder,A. C.; Haggblom, M. M.; Oppenheimer, S. R.; Young, L. Y. Enuiron. Sci. Technol. 1993, 27, 530. May, H. D.; Boyle, A. W.; Price, W. A., 11;Blake, C. K. Appl. Enuiron. Microbiol. 1992, 58, 4051. Suflita,J. M.; Gibson,S. A,; Beeman, R. E. J. Ind. Microbiol. 1988, 3, 179.
Haggblom, M. M.; Young, L. Y. Appl. Enuiron. Microbiol. 1990,56, 3255.
Lee, J. H.; Ferguson, J. F.; Puhakka, J. A.; Herwig, R. P. Abstracts; American Society for Microbiology General Meeting, Atlanta, GA, May 16-20, 1993; Abstract Q369. Brown, J. F., Jr.; Wagner, R. E. Enuiron. Toxicol. Chem. 1990, 9, 1215.
Lake, J. L.; Pruell, R. J.; Osterman, F. A. In Organic Substances and Sediments in Water; Baker, R., Ed.; Lewis Publishers: Chelsea, MI, 1991; pp 173-197. Lake, J. L.; Pruell, R. J.; Osterman, F. A. Mar. Environ. Res. 1992, 33, 31. Gonzhles-Dhvila,M.; Santana-Casiano,J. M.; Millero,F. J. J. Colloid Interface Sci. 1990, 137, 102. Goldberg,E.D.; McGraw-Hill Encyclopedia of Ocean and Atmospheric Sciences; Parker, P. S., Ed.; McGraw-Hill: New York, 1980; pp 415-417. Shelton,D. R.;Tiedje, J. M. Appl. Enuiron. Microbiol. 1984, 47, 850.
Nottingham, P. M.; Hungate, R. E. J. Bacteriol. 1969, 98, 1170.
Jensen, S. L.; Renberg, L.; Reuterghrdh, L. Anal. Chem. 1977, 49, 316.
Ballschmitter, K.; Zell, M. Fresenius 2. Anal. Chem. 1980, 302, 20.
Schultz, D. E.; Petrik, G.; Duimker, J. C. Enuiron. Sci. Technol. 1989, 23, 852. Mullin, D. M.; Pochini, C. M.; McCrindle, S.; Romkes, M.; Safe, S. H.; Safe, L. M.; Enuiron. Sci. Technol. 1984, 18, 468. Boyer, J. N. Appl. Enuiron. Microbiol. 1986, 52, 1415. Boyer, J. N. Appl. Enuiron. Microbiol. 1994, 60, 174. Environ. Sci. Technol., Vol. 28, No. 13, 1094
2293
(26) Quensen, J. F., 111;Tiedje, J. M.; Boyd, S. A. Science 1988, 242, 752. (27) Rhee, G.-Y.;Sokol, R. C.; Bush, B.;Bethoney, C. M. Enuiron. Sci. Technol. 1993, 27, 714. (28) Abramowicz, D. A.; Brennan, M. J.; Van Do% H. Me; Gallagher, E. L.; Environ. Sci. Technol. 1993, 27, 1125. (29) Deweerd, K. A.; Mandelco, L.; Tanner, R. S.; Woese, C. R.; Suflita, J. M. Arch. Microbiol. 1990, 154, 23.
2294
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(30) Oremland, R. S.; Geochim. Cosmochim. Acta 1978,42,209. (31) Morris, P. M.; Mohn, W. W.; Quensen, J. F., 111; Tiedje, J. M.; Boyd, S. A. Appl. Environ. Microbiol. 1992,58, 3088.
Received for review February 2, 1994. Revised manuscript received August 22, 1994. Accepted August 25, 1994.' @
Abstract published in Advance ACS Abstracts, October 1,1994.
