Environ. Sci. Technol. 2002, 36, 100-103
Sequential Anaerobic-Aerobic Treatment of Soil Contaminated with Weathered Aroclor 1260 EMMA R. MASTER,† VIVIAN W.-M. LAI,‡ BIANCA KUIPERS,‡ WILLIAM R. CULLEN,‡ A N D W I L L I A M W . M O H N * ,† Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Boulevard, Vancouver, British Columbia, V6T 1Z3 Canada, and Environmental Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, V6T 1Z1 Canada
Soil contaminated with weathered Aroclor 1260 was bioremediated by sequential anaerobic and aerobic laboratory-scale treatment. The initial concentration was 59 µg of PCBs/g of soil. Following 4 months of anaerobic treatment with an enrichment culture, all of the major components in Aroclor 1260 were completely or partially transformed to less chlorinated PCB congeners. The major products of reductive dechlorination were 24-24tetrachlorobiphenyl and 24-26-tetrachlorobiphenyl, and the average chlorine substituents per PCB molecule decreased from 6.4 to 5.2. The molar concentration of PCBs did not decrease during the anaerobic treatment. All of the major products formed during the anaerobic treatment were degraded in the subsequent aerobic treatment using Burkholderia sp. strain LB400. After 28 days of the aerobic treatment, the concentration of PCBs was reduced to 20 µg/g of soil. PCBs were not significantly removed in aerobic treatments unless they were bioaugmented with LB400. Also, PCB degradation was not detected in soil bioaugmented with LB400 without prior anaerobic treatment. These results confirm the potential for extensive biological destruction of highly chlorinated, weathered PCB congeners in soil.
Introduction Despite the extreme persistence of highly chlorinated PCBs in the environment, the potential exists for their extensive degradation by naturally occurring microorganisms. Several aerobic bacteria that degrade PCBs have been isolated (1-12). PCB degradation by these organisms proceeds through a series of enzymatic steps that result in the degradation of PCBs to chlorobenzoic acids and chloroaliphatic acids (13-17). Generally, these products are excreted by cells and further transformed by other organisms. The initial enzymes used in aerobic PCB transformation are those of the biphenyl degradative pathway (8, 16), and PCB biodegradation is thought to occur fortuitously during biphenyl metabolism (1, 14, 17). Most of the PCB-degrading bacteria that have been isolated transform PCBs that have up to four * Corresponding author phone: (604)822-4285; fax: (604)822-6041; e-mail:
[email protected]. † Department of Microbiology and Immunology. ‡ Environmental Chemistry Group, Department of Chemistry. 100
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chlorine substituents (18). However, a few bacterial isolates, including Burkholderia sp. strain LB400, can transform certain PCBs with up to six chlorine substituents (5, 18). PCB mixtures used for industrial purposes in North America were mainly sold under the trade name Aroclor. Aroclor mixtures are designated by four digits; the first two indicate that the mixture is composed of chlorinated biphenyls and the last two digits indicate the percent chlorine by weight. Industrial use of Aroclor 1260 has resulted in contamination of many soil and water sites with PCBs containing more than seven chlorine substituents. Such PCB congeners are very recalcitrant to aerobic microbial metabolism. Several researchers have demonstrated anaerobic microbial reductive dehalogenation of PCBs (19-24). Although a bacterium capable of dehalogenating hydroxylated PCBs was recently isolated (25), isolation of bacteria capable of PCB dehalogenation has been unsuccessful to date. Consequently, various anaerobic sediments containing mixed populations of anaerobic microorganisms have been used in experiments investigating PCB dehalogenation. It is currently believed that certain PCB congeners serve as catabolic electron acceptors under anaerobic conditions (26). Interestingly, different dechlorinating microbial consortia appear to have different specificities for PCBs (19). Initial anaerobic treatment of soil contaminated with highly chlorinated PCBs then can potentially transform PCBs resistant to aerobic microbial attack to less chlorinated congeners that are susceptible to aerobic microbial mineralization (1). Sequential anaerobic-aerobic degradation of PCBs has been previously demonstrated. Anid et al. (27) demonstrated sequential anaerobic-aerobic treatment of dehalogenated PCB-contaminated soil spiked with Aroclor 1242. In their experiment, the soil was incubated anaerobically for 76 weeks and then aerobically for 96 days. Moreover, this report showed that anaerobic soil can be aerated with H2O2 and that the aerobic step is enhanced by bioaugmentation. Shannon et al. (28) also reported sequential treatment of soil contaminated with Aroclor 1242 and show profiles of PCBs remaining in a soil matrix after anaerobic and aerobic treatments. However, the details of their experimental method were not revealed. Finally, Evans et al. (29) demonstrated treatment of soil containing weathered PCBs, probably from Aroclor 1248. In their experiment, the anaerobic treatment continued for 19 weeks, followed by a 19-week aerobic treatment that included bioaugmentation with strain LB400. However, this study showed that, when using LB400 as an aerobic inoculum, the initial anaerobic treatment did not contribute to total PCB removal. Here we show for the first time, a detailed quantitative analysis of sequential anaerobic-aerobic treatment of soil contaminated with weathered Aroclor 1260. The soil used was obtained from Saglek, Laborador, Canada. We verified the reproducibility of our method using several replicate experiments. We report on the specificity of both the anaerobic and aerobic treatments and the kinetics of the aerobic step.
Methods Anaerobic Enrichment Culture Preparation. A mixed anaerobic culture (BK81) was enriched from marine sediments having a history of PCB contamination and collected from Esquimalt Harbor, BC, Canada. Sources of contaminated soil and sediment and the procedure of enrichment were previously published (20). The BK81 enrichment culture used in this study was transferred as described (20) four times. 10.1021/es001930l CCC: $22.00
2002 American Chemical Society Published on Web 11/28/2001
Preparation and Sampling of Anaerobic Treatments. Four slurries were prepared in 125-mL serum bottles by mixing 15 g of weathered PCB-contaminated soil from Saglek and 7.5 g of anaerobic pond sediment with 90 mL of mineral medium as previously described (20) with the following modifications. The medium used in this experiment did not contain resazurin or reductant. The medium was not sparged with nitrogen or autoclaved. The medium was added to mixtures of soil and sediment in serum bottles under a headspace of air. Serum bottles were crimp-sealed with butyl rubber stoppers. The bottles were shaken for 2.5 h and then incubated stationary at 21 °C in the dark. After 8 weeks of incubation, oxygen was biologically removed from the bottles and methanogenesis was detected. Methane was measured by using a gas chromatograph equipped with a Haysep DB, 100-120 mesh, 10 in. × 1/8 in. packed column and a thermal conductivity detector. The slurries were then autoclaved and inoculated (10%) with the enriched culture, BK81. 4-Bromobenzoate (200 µg/g of soil plus sediment) was added to the sediment slurries, as this has been shown to stimulate development of PCB dechlorination activity (30). Serum bottles were crimp-sealed with Teflon-faced rubber septa and further stationary incubation was at 21 °C in the dark. Samples of approximately 8 mL were removed from the slurries after inoculation and after 3 and 4 months of incubation. Sampling was done inside an anaerobic chamber (N2-CO2-H2, 85:5:10), and samples were frozen in extraction tubes with Teflon-lined caps until extraction. Prior to extraction, the samples were thawed and centrifuged (2 min at 1500g), and the supernatant (medium) was removed and discarded. Preliminary tests showed that the supernatant did not contain detectable PCBs. Aerobic Culture Preparation. Burkholderia sp. strain LB400 (5) was grown at 21 °C to midexponential phase in mineral medium (18) supplemented with 200 mg/L biphenyl. At midexponential phase, cells were washed and concentrated in mineral medium to 5 × 107 cells/mL. Preparation and Sampling of Aerobic Treatments. In preliminary experiments, anaerobically treated soil was washed to remove potentially inhibitory soluble compounds (e.g., sulfide), aerated, or washed and aerated before bioaugmentation with LB400 grown on biphenyl. The effect of adding biphenyl to the aerobic treatments was also tested. These preliminary tests showed that washing the anaerobically treated soil or adding biphenyl did not improve the aerobic treatment. However, the amount of biphenyl added might not have been significant compared to the amount of biphenyl that was carried over in the inoculum. As a result, for this experiment the anaerobically treated soil and sediment slurries were pooled in a 500 mL Erlenmeyer flask and then aerated on an orbital shaker. Slurry aliquots of 15 g were transferred into six 125-mL serum bottles, and then 10 mL of mineral medium was added to each bottle. Six additional slurries were prepared in 125-mL serum bottles by mixing 15 g of the above soil and 10 mL of mineral medium. Biphenyl (100 µg/g) was added to each treatment. LB400 was added to three of the six anaerobically treated and three of the six nonanaerobically treated slurries, to 2 × 106 cells/ g. Serum bottles were crimp-sealed with Teflon-faced rubber septa and incubated at 21 °C on an orbital shaker at 200 rpm. Following 0, 3, 7, 14, and 28 days of incubation, 2-g samples from each aerobic treatment were transferred to Teflon-lined screw capped glass tubes. Samples were stored at -20 °C before extraction. Each sampling permitted equilibration of culture headspace with air. Extraction of PCBs From Soil. The slurries were extracted by shaking and vortexing soil twice with an equivalent volume of acetone and then twice with an equivalent volume of hexane. Tubes were centrifuged (2 min at 1500g) between extractions. The extracts were pooled and evaporated to
approximately 0.5 mL using nitrogen gas and then passed through a Pasteur pipet packed with hexane washed Florisil topped with sodium sulfate. PCB congeners were eluted from the Florisil column using hexane, and eluates were collected in glass chromatographic vials. Analysis of PCBs. Samples were analyzed with a gas chromatograph fitted with a DB5-ms column (30 m × 0.25 mm × 0.25 µm) and coupled to a mass spectrometer (Varian Saturn model 4D ion trap). The sample volume injected was 2.5 µL, the temperature of the splitless injector was held at 260 °C, and the temperature of the transfer line was 280 °C. The column temperature program used to analyze extracts from the anaerobic treatments was as follows: 104 °C for 3 min, increased at 20 °C/min to 180 °C, increased at 2.5 °C/ min to 272 °C, increased at 30 °C/min to 290 °C, and held at 290 °C for 2 min. The column temperature program used to analyze extracts from the aerobic treatments was as follows: 104 °C for 3 min, increased at 20 °C/min to 160 °C, increased at 2.5 °C/min to 260 °C, increased at 50 °C/min to 290 °C, and held at 290 °C for 3 min. The mass spectrum of each GC peak was used to determine if the peak corresponded to a PCB and, if so, to determine the number of chlorine substituents. Congener assignments were made by comparison of the retention times with those in Aroclor 1221, 1242, 1254, and 1260 (Accu Standard) and comparison of the relative retention times with published results (31, 32). Individual congener standards were used to quantify major product congeners that are present in traces or not present in the Aroclor standards. Linear three-point calibration curves were generated for all congeners using either pure congeners or using the weight percent contributions of the components present in Aroclor standards (31, 32). Amounts of PCBs (µg) per g of dry soil plus sediment were calculated, using as an internal standard, 23456-2345-nonachlorobiphenyl, which is present in the contaminated soil, to correct for variability in sample size and PCB recovery. Importantly, 23456-2345nonachlorobiphenyl is not significantly degraded after 16 weeks of anaerobic incubation (20), and aerobic biodegradation of this PCB congener has not been demonstrated.
Results and Discussion Anaerobic Treatment of PCB-Contaminated Soil. All major components of weathered Aroclor 1260 initially present in Saglek soil were completely or partially removed after 4 months of anaerobic incubation (Figure 1). However, extensive PCB dechlorination was apparent after 3 months. Weathered Aroclor 1260 can have a higher proportion of recalcitrant, highly chlorinated PCB congeners than when initially synthesized (unpublished). Consequently, this demonstration of dechlorination of weathered Aroclor 1260 is encouraging for the application of bioremediation of soil with a history of contamination with PCBs. Dechlorination of PCBs during the anaerobic process was indicated by significant decreases in the amounts of highly chlorinated PCBs and corresponding increases or appearance of less chlorinated PCBs (Figure 1). PCB homologue classes with 5-8 chlorine substituents were removed. The major products of dechlorination were 24-24-tetrachlorobiphenyl and 24-26-tetrachlorobiphenyl. Other less abundant products were 23-2-trichlorobiphenyl or 26-4-trichlorobiphenyl (coeluting) and 235-2-tetrachlorobiphenyl or 24-25-tetrachlorobiphenyl (coeluting). The pattern of dechlorination observed resembled Pattern N as described by Bedard and Quensen (19). As expected, the number of moles of PCBs before and after the anaerobic treatment did not change significantly (Table 1). This result confirms that the anaerobic process results in PCB dechlorination but in little or no degradation of the biphenyl molecule (Table 1). Based on the reduction of chlorine substituents per biphenyl molecule, the weight VOL. 36, NO. 1, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Sequential treatment of PCB contaminated soil. Histograms show the major congeners of weathered Aroclor 1260 (>0.5 µg/g of soil) identified by positions of chlorine substitution. Bars indicate standard error; n ) 3. Percent removal in each treatment phase is indicated for PCBs that were significantly degraded (Student’s t-test, P < 0.1).
TABLE 1. Summary of Treatment of PCB Contaminated Soila treatment
nmol of PCBs/g of soil
µg of PCB/g soil
av Cl/PCB molecule
initial soilb after anaerobic stepc after aerobic stepc
157.4 (15.7) 162.1 (45.4) 55.5 (10.5)
58.7 (5.7) 58.3 (14.4) 19.6 (3.6)
6.4 (0.031) 5.2 (0.068) 5.5 (0.12)
a
Values are means (standard errors).
b
n ) 4. c n ) 3.
of total PCBs should have been reduced by 11%. However, we did not detect significant differences in values for µg of PCBs/g soil before and after the anaerobic step (Table 1). The inhomogeneity of the PCBs appears to have caused large variability in their measurement, which is typical of environmental samples. Further, the anaerobic treatment may have increased the extraction efficiency of PCBs by altering PCB-soil interactions and by converting the PCBs to lesschlorinated, less hydrophobic congeners. Despite this uncertainty about the total PCB concentration, the evidence for their extensive dechlorination is very clear. Aerobic Treatment of PCB-Contaminated Soil. In PCBcontaminated Saglek soil that was not treated anaerobically, 90.2 ( 19.8% of Aroclor 1260 remained after the aerobic treatment. However, the amount of PCBs removed during this treatment was not significant (Students t-test, P < 0.1). This result was not surprising since the majority of PCB congeners present in Saglek soil are substituted with 6-8 chlorine atoms. Consequently, most of these congeners are recalcitrant to biodegradation by LB400 (5, 18). In aerobic treatments bioaugmented with LB400 and previously treated anaerobically, all major PCB congeners generated during the anaerobic stage were completely or partially removed (Figure 1). In treatments bioaugmented with LB400, 67% of total PCBs was removed. Bioaugmentation with LB400 of anaerobically treated soil was necessary to achieve significant aerobic PCB-degradation (Figure 2). Significantly, this result indicates that the products of anaerobic PCB dechlorination are available for aerobic 102
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FIGURE 2. Total PCBs during aerobic treatment of anaerobically treated soil (n ) 3; bars indicate standard error): 9, aerobic treatment without inoculation and [, aerobic treatment with inoculation with Burkholderia sp. strain LB400. microbial degradation and that the anaerobic treatment of the soil did not yield inhibitors of aerobic PCB removal. PCB catabolism is also indicated by a decrease in the total moles of PCBs during the aerobic treatment (Table 1). The selectivity of the aerobic process for catabolism of less chlorinated PCB congeners is indicated by an increase in the average number of chlorine atoms per biphenyl molecule of PCBs (Table 1). Notably, not all congeners known to be degraded by LB400 were removed (5, 18, 33). Also, removal of certain PCB congeners with seven chlorine substituents was detected. The range of PCBs degraded by pure cultures of LB400 might differ from that of LB400 in soil. Also, detectable loss of particular heptachlorobiphenyls may be due to indigenous microbial activity or sampling variation. Certain PCB congeners that were present in the original soil sample were aerobically degraded only after the anaerobic treatment. This result suggests that highly chlorinated PCBs may inhibit PCB biodegradation. Alternatively, the anaerobic treatment might increase the bioavailability of weathered PCBs. Interestingly, the extent of PCB degradation in soil bioaugmented with strain LB400 was similar at 7 days and 28 days (Figure 2), and none of the congeners were completely removed by LB400
(Figure 1). The remaining PCBs may be strongly sorbed to the soil matrix, and poorly available for microbial degradation. Also, metabolites resulting from aerobic PCB biodegradation may have accumulated during the aerobic treatment, which can inhibit further PCB degradation by LB400 (14). Thus, it would be interesting to determine if coinoculation of LB400 and certain chlorobenzoic acid degraders would increase total aerobic PCB removal (34). Finally, we noted that the biphenyl added to each treatment was completely consumed after 7 days. LB400 removes highly chlorinated PCBs less efficiently in the absence of biphenyl, than in the presence of biphenyl (35). It is possible then that adding more biphenyl to the soil treatments during incubation would increase the extent of PCB removal (36). In conclusion, we show that sequential anaerobic-aerobic treatment of soil contaminated with highly chlorinated and weathered PCB congeners results in initial decrease in major components of Aroclor 1260, followed by aerobic degradation of the resulting less chlorinated PCBs. Overall, the concentration of PCBs was decreased by 67% (from 59 µg/g of soil to 20 µg/g of soil), the number of moles PCBs was decreased by 65%, and the average number of chlorine atoms per PCB molecule decreased from 6.4 to 5.5. This successful laboratory-scale demonstration of treatment of weathered PCBs indicates great potential for PCB bioremediation. Our methods are currently being applied to design simple soil slurry bioreactors that can be used on-site. Future experiments will attempt to establish if total aerobic PCB removal is enhanced by coinoculation of additional PCB-degrading bacteria and chlorobenzoic acid degrading bacteria or repeated addition of biphenyl during the aerobic treatment.
Acknowledgments This research was supported by funding provided by the Canadian Department of National Defence, a Canadian Natural Science and Engineering Research Council (NSERC) Strategic Project Grant, and an NSERC graduate scholarship to E.R.M.
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Received for review December 4, 2000. Revised manuscript received October 3, 2001. Accepted October 17, 2001. ES001930L
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