Environ. Sci. Technol. 1997, 31, 1749-1753
Reductive Dechlorination of 1,2,3,4-Tetrachlorodibenzo-p-dioxin and Its Products by Anaerobic Mixed Cultures from Saale River Sediment H E N D R I K B A L L E R S T E D T , * ,† ANGELIKA KRAUS,‡ AND UTE LECHNER† Institute of Microbiology and Institute of Analytics and Environmental Chemistry, Martin-Luther-University Halle, D-06099 Halle/Saale, Germany
The capability of anaerobic bacterial consortia from different environmental sources including soils, sewage sludges, and sediment of the river Saale (Germany) to dehalogenate chlorinated dioxins was compared using 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) as the model compound. The inocula were amended with mineral medium and organic acids and spiked with a high concentration (50 µM) of 1,2,3,4-TCDD to stimulate microbial dehalogenating activity. Reductive dechlorination was observed to 1,3dichlorodibenzo-p-dioxin (1,3-DCDD) as the main product and to minor amounts of the 1,2,4- and 1,2,3-trichlorodibenzop-dioxins (TrCDD) using incubations with Saale River sediment. No reaction was observed in the controls and in incubations with soils or sewage sludges. The dechlorination of 1,2,4- and 1,2,3-TrCDD was analyzed in separate subcultures. Reductive dechlorination of 1,2,4-TrCDD was a relative fast process (about 6 µM converted within 58 days) and yielded only one product (1,3-DCDD). 1,2,3-TrCDD was slowly dechlorinated to equal amounts of 1,3- and 2,3DCDD. These observations suggest that the main dechlorination route of 1,2,3,4-TCDD to 1,3-DCDD proceeds primarily via the removal of a lateral chlorine atom with 1,2,4TrCDD as the intermediate.
Introduction Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/ Fs) are ubiquitous environmental contaminants. PCDD/Fs are a group of chlorinated tricyclic, essentially planar aromatic compounds characterized by extremely low water solubilities, low vapor pressures, and strong absorption to particles and surfaces (high Kow) (1). A number of the 75 PCDD congeners are extremely toxic to several organisms, particularly those chlorinated in lateral (2, 3, 7, and 8) positions and with four to six chlorine atoms (2). They are emitted into the environment as unwanted byproducts of anthropogenic processes. The main sources of PCDDs are the production of herbicides and pentachlorophenol (PCP), bleach processes in the pulp and paper industry, metal smelting, and the incineration of municipal and industrial waste (3-6). Important nonindustrial diffuse sources are household wastewater (7), automobile exhausts (8), home heating, and combustion of landfill gas. Accidents, e.g., forest fires and fires in buildings, * Corresponding author phone: (49) 345-5526374; fax: (49) 3455527010; E-mail:
[email protected]. † Institute of Microbiology. ‡ Institute of Analytics and Environmental Chemistry.
S0013-936X(96)00737-7 CCC: $14.00
1997 American Chemical Society
lead to the formation of PCDDs. Environmental sinks for the persistent lipophilic PCDDs include compost, sewage sludge, and sediments (8, 9). Reuse of sewage sludge or compost in agriculture may contribute to further dispersal and, caused by repeated treatments, accumulation of the persistent compounds in soils and consecutively in human and animal food. Therefore, it is important not only to know the fate of the PCDD/Fs in different environments but also to elucidate possible mechanisms of their destruction. Aerobic biodegradation was restricted to dioxins with a low degree of chlorination (e.g., refs 10 and 11). The most promising approach to biodegradation of highly chlorinated dioxins represents the reductive dehalogenation using anaerobic bacteria. A wide variety of highly oxidized haloaromatic compounds has been reported to be the subject of reductive dehalogenation (12). The products containing fewer halogen substituents are more susceptible to mineralization by anaerobic or aerobic bacteria and, in most cases, possess a lower toxicity than the parent compounds. Recently, laboratory studies have demonstrated that PCDDs can be reductively dechlorinated by sediment and aquifer microorganisms in the anaerobic environment (13-16). Analyses of the dechlorination products showed for highly chlorinated PCDD/Fs (five to seven chlorines per molecule) that the chlorines were removed in both the peri and the lateral positions (16). The authors concluded that the 2,3,7,8-residue patterns in historically contaminated sediments are likely affected by microbial activity. Beurskens et al. (13) reported on dominant dechlorination of 1,2,3,4-TCDD to 1,3-DCDD and with 2,3DCDD as a minor product of peri-dechlorination (product ratio was 5:2) by anaerobic cell suspensions previously adapted to hexachlorobenzene as the electron acceptor. In this study, we examined the ability of anaerobic consortia from different environmental samples to reductively dehalogenate 1,2,3,4-TCDD. The extent of dechlorination and the order of chlorine removal from 1,2,3,4-TCDD, 1,2,4-, and 1,2,3-TrCDD, respectively, by enrichment cultures from Saale River sediment were investigated.
Materials and Methods Cultures and Medium. An inorganic culture medium (medium 1) as proposed by Holliger et al. (17) was used for cultivation. This medium was supplemented with the following organic components to stimulate bacterial growth and dehalogenating activity: 0.002% (w/v) fermented yeast extract (18) and 10% (v/v) filter sterilized culture medium of an active 2,4,6-trichlorophenol dechlorinating enrichment (containing 300 µM 4-chlorophenol). This medium was mixed with appropriate amounts of inocula. Pyruvate, acetate, benzoate, and fumarate were added to the medium to a final concentration of 5 mM each, and nonchlorinated dibenzo-p-dioxin was added to a final concentration of 100 µM. The latter compound as well as the spent medium from the chlorophenol dechlorinating culture were applied in the primary enrichment culture but were omitted in all further incubations because no stimulating effect was observed in a separate experiment (unpublished data). Enrichments were carried out in 125-mL serum bottles with a culture volume of 80 mL. The dechlorination in these enrichment cultures was followed by sampling 2 g after manually shaking. The kinetics of dechlorination of the TrCDDs was measured by using replicate cultures (3-5 mL in 20-mL Hungate tubes), which were sacrificed in triplicate at the times indicated and stored at -20 °C until analysis. Chemicals. 1,2,3,4-TCDD, 1,2,3-TrCDD, 1,2,4-TrCDD, 2,3DCDD, and 1- and 2-monochlorodibenzo-p-dioxin (MCDD)
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were synthesized by AccuStandard Inc. (New Haven, CT) and supplied in Germany by Restek GmbH (Sulzbach/ Taunus). The 1,3-DCDD was a kind gift from John R. Parsons, Department of Environmental and Toxicological Chemistry, University of Amsterdam. 2,4,8-Trichlorodibenzofuran (2,4,8-TrCDF), 5,6-dibromoacenaphthene (DBAN), silver nitrate, and 2,2,4,4,6,8,8-heptamethylnonane (HMN) were obtained from Aldrich GmbH; the silica gel (63-200 µm, 60 Å) was supplied by ICN Biomedicals GmbH (Eschwege). All other chemicals were of the highest purity commercially available. Inocula. Soils, sewage sludges, and river sediment were used to prepare the inocula. The soil samples originated from the following: (A) Bitterfeld, industrial area (PCDD/F, up to 50 000 ng of I-TE/kg dry weight); (B) Bitterfeld-Greppin, garden soil (PCDD/F, up to 5590 ng of TE/kg dry weight) (19); (C) Lu ¨ tzgendorf, mineral oil factory, aquifer borehole depth, 5.60-5.70 m (mineral oil products); and (D) herbicide storage (herbicides). Sewage sludges were sampled in municipal sewage works from digesters (E) Schu ¨ ttorf (PCDD/F, 200 ng of TE/kg dry weight) and (F) Wittmund (PCDD/F, 9 ng of TE/kg dry weight) and from clarifyers (G) Halle/Saale and (H) Eisleben (PCDD/F, 124 ng of I-TE/kg dry weight). The sediment sample (I) originated from a meander of the river Saale (Wilde Saale) in Halle (Germany) and was collected from the top to 30 cm depth of the sediment in February 1995 and kept in serum bottles sealed with butyl rubber stoppers (PCDD/F, 0.89-57.6 ng of I-TE/kg dry weight at different sampling sites in the area of Halle) (19). The sediment was characterized by 30% dry weight and a content of organic carbon of 11 mg/g dry weight. Soil Samples. The soil samples were stored under nitrogen at 4 °C. Soil samples (0.125 g/mL) were mixed with prereduced organic medium and glass beads (5 mm in diameter) and were shaken at 500 rpm on a rotary shaker for about 48 h at 4 °C. The soil particles were allowed to sediment (15 min), and 16 mL of the supernatant was transferred as inoculum (20% of the total volume) to 64 mL of sterilized organic mineral salt medium, containing 1.6 g of air-dried sieved non-contaminated municipal sewage sludge as complex organic supplement (20). Samples from Sediment and Sewage Sludges. Prior to use as inoculum, the samples were mixed with 1 µL of ethanol/ mL as substrate for aerobic bacteria to consume the residual oxygen and incubated at 8 °C. When methane production appeared, the sediment was transferred to a glovebox (Coy Box, Toeppfer, Go¨ppingen), passed through a sieve (2-mm bores) to remove small branches and leaves, and inoculated into the organic mineral medium (50% (v/v)). Incubations with 1,2,3,4-TCDD. Volumes of 6.35 mL (serum bottle) or 0.4 mL (controls in Hungate tubes) of a stock solution of 1,2,3,4-TCDD in acetone (0.2 g/L) were added with sterile glass syringes to empty autoclaved vessels to yield a final concentration of 50 µM. The acetone was vaporized under a gentle stream of a filter-sterilized CO2/N2 (20/80) gas mixture, and after that the sterilized medium was added in appropriate volumes to bottles and Hungate tubes. The inocula from soils, sewage sludges, and sediment were prepared as described above and transferred (20, 50, and 50% [v/v], respectively); the vessels were sealed with butyl rubber stoppers and aluminum screw caps. During this procedure, the headspace was flushed with sterile N2/CO2. The cultures were incubated in the dark at 20 °C except the cultures inoculated with sewage sludge, which were incubated at 37 °C. Culture vessels were gentle shaken on rotary shakers to improve the dispersal of the low water-soluble chlorinated compounds within the heterogeneous sample material. In addition, biological controls supplemented with autoclaved sample material (three times 1 h at 121 °C, on three consecutive days) and chemical controls, which were not inoculated, were established in Hungate tubes, and they were
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completely extracted and analyzed closely with the living cultures. Incubations with 1,2,3- and 1,2,4-TrCDD. A volume of 500 µL of an acetone stock solution (17.2 µg/mL) of the corresponding TrCDD (final concentration 10 µM) was added to autoclaved empty Hungate tubes. The acetone was vaporized. The medium was prepared by mixing organic mineral medium with 50% (v/v) of γ-sterilized Saale sediment (35 kGy dose) as a complex organic supplement. This medium (2.7 mL) was filled into Hungate tubes and mixed with the inoculum (0.3 mL of the primary incubation). Hungate tubes containing γ-sterilized sediment with organic mineral medium served as biological controls. Chemical controls consisted of organic mineral medium only. Analytical Procedures. The frozen samples were freezedried for about 24 h. Teflon-sealed sample vials were used as extraction vessels. After the addition of 2,4,8-TrCDF as an internal (recovery) standard, each sample was vigorously shaken with 10 mL of n-hexane for 10 min on a Vortex shaker. Subsequently, the tubes were heated in a water bath at 80 °C and sonicated for 30 min. The organic phase was saved, and the extraction vessel was washed two times with 2 mL of n-hexane. The combined hexane extracts were concentrated on ice under a stream of nitrogen to a volume of 2 mL. To remove sulfur ions and other electronegative ions and molecules, a silver nitrate-silica column was used (ca. 0.5 g in 10-mL glass syringes with glass wool stoppers on the top and the bottom). The silver nitrate-covered silica was prepared as described elsewhere (21). The PCDDs were eluted with 40 mL of n-hexane. The surrogate standard for GC-ECD analysis DBAN was added in 50 µL of HMN. Prior to analysis by GCECD, the n-hexane was vaporized under a gentle stream of nitrogen and the residual HMN phase (keeper) was directly injected or, if necessary, diluted with n-hexane containing the same concentration of the surrogate standard. Chemical analyses were performed on a Shimadzu 14A gas chromatograph equipped with an AOC 14 autosampler and a C-R4 AX Chromatopac integrator, using a split/splitless injector (split: 1:50), a DB-5 capillary column (J&W Scientific, 30 m × 0.25 mm i.d., film thickness 0.25 µm), and a 63Ni electron-capture detector. Helium was used as the carrier gas (head pressure, 35 psi; linear velocity, 0.298 m/s), with nitrogen as the makeup gas. The injector and detector temperatures were 270 and 340 °C, respectively. The oven temperature was held at 190 °C for 5 min, ramped at 2 °C/ min to 198 °C, held for 2 min at 198 °C, ramped at 2 °C/min to 222 °C, and ramped at 40 °C/min to 320 °C, and finally held for 5 min. Compounds were identified by comparing the retention times with external standards and by mass spectra obtained with the aid of a Varian GC 3400 coupled to an ion trap detector ITD 800 (Finnigan MAT). A DB-5 ms capillary column (J&W Scientific, 60 m × 0.25 mm i.d., film thickness 0.25 µm) was used with helium as carrier gas at 30 psi, and injections of 0.6 µL were made via a split injector at 260 °C. The GC oven temperature was held at 220 °C for 0.5 min, ramped at 50 °C/min to 260 °C, held for 9 min at 260 °C, ramped at 20 °C/min to 280 °C, and finally held for 10 min. The transfer line was held at 230 °C. Electron impact spectra (70 eV) were obtained in full-scan mode (50-320 u). The identification of the 1- and 2-MCDD was realized using mass spectra combined with the relative retention values referring to 1,3- and 2,3-DCDD and a GC start temperature of 160 °C held for 20 min, followed by the above-mentioned program. Fatty acids and alcohols were analyzed in the culture fluid after centrifugation (3500 rpm for 30 min) and acidification with sulfuric acid (50% v/v) to pH 2.0 by gas chromatography on a polar HP-Innowax capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm). Gas samples of the headspace were taken with a gas-tight syringe. The headspace gas was
monitored for methane and hydrogen using a gas chromatograph (GCHF 18.3, Chromatron, Berlin) equipped with a thermal conductivity detector. For hydrogen determination, a stainless steel column packed with activated charcoal (3 m × 3 mm i.d.; carrier gas, nitrogen; 60 kPa head pressure) was used, and for methane determination, a Porapak Q packed steel column (2 m × 3 mm i.d.; carrier gas, hydrogen; 50 kPa head pressure) was used. Fumarate, benzoate, and pyruvate were determined on a high-pressure liquid chromatography (HPLC) system equipped with the pump system 325, UV/VIS detector 332 (Kontron Instruments, Neufahrn) and a LiChrospher 100-RP 18 column (244 × 4 mm, 10 µm, 100 Å; Merck, Darmstadt). The eluent for separation was methanol-water in a ratio of 50:50 (v/v) acidified with 1 g/L H3PO4 and a flow rate of 1 mL/min. Quantification. The dioxin congeners were quantified using DBAN as an internal standard and taking into account the recovery efficiency of 2,4,8-TrCDF (70-90%) during sample cleanup. The recovery of 1,2,3,4-TCDD, 1,2,4- and 1,2,3-TrCDD, and 2,3-DCDD was examined and was in the same range. The calibration mixture contained 1,2,3,4-TCDD, 1,2,3-TrCDD, 1,2,4-TrCDD, 2,3-DCDD, 1,3-DCDD, 1-MCDD, 2-MCDD, and 2,4,8-TrCDF. Each congener was quantified by use of a calibration curve (CR4AX Chromatopac software, Version 3.7; 1989) generated from reference standards at eight calibration levels ranging from 0.3 to 180 µM.
FIGURE 1. Relative molar distribution of 1,2,3,4-TCDD (50 µM) and its dechlorination product 1,3-DCDD as a function of time. The data shown are from two parallel cultures of Saale river sediment. The intermediate TrCDDs accounted for less than 1 mol %.
Results and Discussion Dechlorination of 1,2,3,4-TCDD. To search for 1,2,3,4TCDD-dehalogenating microbial activity, several samples from soils, sewage sludges, and sediment of the river Saale with different historically present concentrations of PCDD/ Fs and other chlorinated and non-chlorinated pollutants were used as inocula for anaerobic culture setups. The conditions of culture establishment followed the suggestions of Tiedje et al. (22) and Quensen et al. (23) for enhanced reductive dechlorination of PCBs, which are similar to PCDD/Fs in their environmental persistence and some physicochemical properties. We used (i) a high concentration of 1,2,3,4-TCDD (50 µM) as compared with environmental PCDD/F contaminations with the aim to enhance the bioavailable fraction. For low PCB concentrations, it was shown that dechlorination often was very slow or non-measurable, which was explained by sorption into the organic phase of soils and sediments (22). We added (ii) a high content of complex organic material to the culture setups using an inoculum volume of 50% (v/v) or a supplement of air-dried sewage sludge (23, 20). The fate of the spiked 1,2,3,4-TCDD in the primary enrichment cultures was investigated over a period of 13 months with sampling intervals of 1 month. Increasing concentrations of lower chlorinated dioxins appeared in the two cultures inoculated with sediments from the river Saale. In contrast, this was not seen in any of the biological and chemical controls as well as in the enrichments from soils and sewage sludges. This fact suggests the presence of dechlorinating bacteria only in the river sediment. Recently Nowak et al. (24) reported on a methanogenic consortium enriched from the sediment of a quite different part of the Saale River that is able to transform a wide spectrum of chlorobenzenes via monochlorobenzene to unsubstituted benzene. Interestingly, the known PCDD-dechlorinating cultures (13, 14) were also derived from freshwater sediments and aquifer material. Concerning the PCB dechlorination, most studies refer to river sediments (e.g., refs 25-31), with few exceptions, where the dechlorination by soil populations is described (20, 22). In the sediment cultures, the trichlorinated isomers 1,2,3TrCDD and 1,2,4-TrCDD were both detected at low concentrations during the course of this study and identified by GC/ MS, using the selective ion mode. The main product of dechlorination was 1,3-DCDD, which accumulated in the
FIGURE 2. Comparison of the mass spectra of the main dechlorination product 1,3-DCDD from 1,2,3,4-TCDD in the primary culture (A) with an authentic standard (B). cultures. Inhomogeneous dispersion of the low water-soluble dioxins [water solubilities at 20 °C of 1,2,3,4-TCDD, 1,2,4TrCDD, and 2,3-DCDD are 2, 30, and 59 nM, respectively (1)] resulted in different total amounts of the sum of congeners (10-57 µM) recovered from the 2-g samples. Therefore Figure 1 gives the relative molar distribution of 1,2,3,4-TCDD and 1,3-DCDD at different sampling times. After 388 days, 37 mol % of 1,2,3,4-TCDD was converted to 1,3-DCDD. The mass spectra of the authentic 1,3-DCDD and the dechlorination product showed high similarity (Figure 2). 2,3-DCDD and lower chlorinated dioxins were not detected (detection limit was 0.5 µM). During the period of the highest dechlorinating activity (50-250 d), a transformation rate of 0.06 µM/d (r 2 ) 0.97) was calculated by linear regression analysis, based upon the known amount of 1,2,3,4-TCDD added and the increase of the relative mol % of 1,3-DCDD evolved. This value is in the same order of magnitude as that described for the dechlorination of 1,2,3,4-TCDD by cell suspensions acclimatized to hexachlorobenzene (13). However, in our experiment, the dechlorination did not stop after some time
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but continued with a slightly reduced rate over 1 year, probably due to the use of primary, non-adapted inocula and to the application of a higher concentration of 1,2,3,4-TCDD. After 256 days, the primary enrichment cultures were examined for the presence of methane, hydrogen, acetate, propionate, butyrate, and short-chain alcohols (C2-C5). The high concentrations of methane (1-13 µmole/mL culture fluid) and traces of hydrogen formed in all living cultures during the last sampling interval (ca. 1 month) indicated the establishment of anaerobic conditions. Ethanol was detected at low concentrations (10-30 µM) in all cultures. Four samples inoculated with soil eluates (A and D) and sludges (E and F), respectively, contained considerable amounts of at least one of the compounds acetate, propionate, and butyrate (A: 1.7 mM, 1.8 mM, 0.3 mM; D: 14.5 mM, 0.5 mM, 0.25 mM; E: 3.1 mM, 5.8 mM, 0.25 mM; F: 1.6 mM, 0.4 mM, 0 mM, respectively). The analyses of the cultures B, C (soil samples), and G (sewage sludge) revealed lower amounts of acetate and propionate (B: 0.3 mM, traces; C: 0.2 mM, traces; G: 0.15 mM, 0.35 mM, respectively) and no butyrate. The cultures H (sludge) and I (sediment) exhibited the lowest contents of organic compounds in the medium (H: 0.01 mM ethanol, 0.08 mM acetate, 0.03 mM propionate; I:
