Dechlorination of Polychlorinated Biphenyl Congeners by Anaerobic Microorganisms From River Sediment

Abstract

The microbial dechlorination of seven kinds of polychlorinated biphenyls (PCBs) by anaerobic microorganisms from river sediment was investigated. Dechlorination rates were found to be affected by the chlorine level of PCB congeners; dechlorination rates decreased as chlorine levels increased. Dechlorination rates were fastest under methanogenic conditions and slowest under nitrate-reducing conditions. The addition of individual electron donors (acetate, pyruvate, and lactate) enhanced the dechlorination of PCB congeners under methanogenic and sulfate-reducing conditions but delayed the dechlorination of PCB congeners under nitrate-reducing conditions. PCB congener dechlorination also was delayed by the addition of various polycyclic aromatic hydrocarbons (PAHs) under three reducing conditions and by surfactants, such as brij30, triton SN70, and triton N101. The results suggest that methanogen, sulfate-reducing bacteria, and nitrate-reducing bacteria all are involved in the dechlorination of PCB congeners. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT:

The microbial dechlorination of seven kinds of polychlorinated biphenyls (PCBs) by anaerobic microorganisms from river sediment was investigated. Dechlorination rates were found to be affected by the chlorine level of PCB congeners; dechlorination rates decreased as chlorine levels increased. Dechlorination rates were fastest under methanogenic conditions and slowest under nitrate-reducing conditions. The addition of individual electron donors (acetate, pyruvate, and lactate) enhanced the dechlorination of PCB congeners under methanogenic and sulfate-reducing conditions but delayed the dechlorination of PCB congeners under nitrate-reducing conditions. PCB congener dechlorination also was delayed by the addition of various polycyclic aromatic hydrocarbons (PAHs) under three reducing conditions and by surfactants, such as brij30, triton SN70, and triton N101. The results suggest that methanogen, sulfate-reducing bacteria, and nitrate-reducing bacteria all are involved in the dechlorination of PCB congeners. Water Environ. Res., 78, 764 (2006).

KEYWORDS: PCB, dechlorination, river sediment.

doi:10.2175/106143006X107380

Introduction

The hydrophobic quality of polychlorinated biphenyls (PCBs) and other anthropogenic pollutants enhances their ability to adsorb to sediments. As sediments settle, PCBs accumulate in the lower anoxic layers of the sediment column. The reductive PCB dechlorination power of anaerobic microorganisms in sediment has been demonstrated in the laboratory and in situ (Abramowicz, 1994; Bedard and May, 1996; Brown et al., 1987). Furthermore, the turnover of naturally formed halogenated compounds in marine coastal regions suggests that such environments hold significant dechlorination potential (King, 1988). The environmental transformation of PCBs in sediments has been documented (Brown and Wagner, 1990; Lake et al., 1992). Precise measurements of biodegradation rates are considered important for accurately forecasting the fates of potential pollutants and assessing risk.

The Taiwan Environmental Protection Administration (1997) found high concentrations of such PCB congeners as 2,2,5-CB; 2,4',5-CB; 2,2',5,5'-CB; 2,2',3,5-CB; 2,2',4,5,5'-CB; 2,2',4,4', 5,5'-CB; and 2,2',3,4,4',5,5'-CB in the Tanshui River. In the current study, the authors assessed the dechlorination potential for these seven PCB congeners and the effect of such factors as adding electron donors, polycyclic aromatic hydrocarbons (PAHs), nonionic surfactants, or microbial inhibitors on the dechlorination of PCB congeners in sediment samples collected from the Keelung River, a branch of the Tanshui River that also contains heavily contaminated sites. The microbial inhibitors included bromoethanesulfonic acid (BESA), molybdate, and vancomycin, which have been identified as selective inhibitors of methanogen (Lovely and Philips, 1988), sulfate-reducing bacteria (Distefano et al., 1992), and eubacteria (Distefano et al., 1992), respectively.

Materials and Methods

Chemicals. Polychlorinated biphenyls with 99.0% analytical standards were obtained from Chem Service (West Chester, Pennsylvania). Polycyclic aromatic compounds (phenanthrene, acenaphthene, anthracene, fluorene, and pyrene) with 99.0% analytical standards were purchased from Aldrich Chemical Co. (Milwaukee, Wisconsin). All other chemicals were purchased from Sigma (St. Louis, Missouri). Solvents were purchased from Mallinckrodt, Inc. (Paris, Kentucky).

Sampling. The locations of the sampling sites are presented in Figure 1. Samples were collected between January and August 1999. Sediment samples were collected from the top 10-cm layer with an Ekman grab sampler and stored at 4°C until used. There were five sample points at each site. All bottles or grabs for one site were mixed manually until homogeneous by visual estimate and then sampled for analysis. The collection sites were along the Keelung River-one of the most heavily contaminated rivers in northern Taiwan. Samples were taken from sites K6, K8, K10, K11, and K12, as described in Yuan et al. (2001). Samples from K6 were collected in the middle portion, and samples from K8, K10, K11, and K12 were collected downriver. For the K6, K8, K10, K11, and K12 samples, biochemical oxygen demand (BOD) values were 14, 52, 34, 17, and 27 mg/L; NH^sub 3^-N values were 2.1, 5.8, 5.3, 4.8, and 4.9 mg/L; pH values were 6.8,6.5, 6.8,7.0, and 6.8; and anaerobic microbial populations were measured as 2.2 × 10^sup 5^, 3.9 × 10^sup 6^, 2.8 × 10^sup 6^, 4.7 × 10^sup 5^, and 1.6 × 10^sup 6^ cells/g of sediment, respectively (Chang et al., 2001; Yuan et al., 2001). The samples contained various amounts of 11 out of 28 PCB congeners listed by the Taiwan Environmental Protection Administration (1997) as being of special concern. Concentrations ranged from below measurable levels to 15.9 ng/g dry weight (average 6.7 ng/g).

Polychlorinated Biphenyl Dechlorination in River Sediment

The following factors were adjusted to study their effects on the dechlorinating effectiveness of the mixed culture used in this research:

* Sampling site (K6, K8, K10, K11, or K12);

* PCB congeners (2,2',5-CB; 2,4',5-CB; 2,2',3,5-CB; 2,2',5, 5'-CB; 2,2',4,5,5'-CB; 2,2',4,4',5,5'-CB; and 2,2',3,4,4',5, 5'-CB, all at a concentration of 1 µg/g), either alone or in combination;

* Treatment with one of three electron donors (20 mM sodium acetate, 20 mM sodium pyruvate, or 20 mM sodium lactate);

* Methanogenic conditions (20 mM sodium hydrogen carbonate), sulfate-reducing conditions (20 mM sodium sulfate), or nitrate-reducing conditions (20 mM sodium nitrate);

* Inoculated control (absence of sodium sulfate, sodium nitrate, and sodium hydrogen carbonate);

* Various PCBs and PAHs (1 µg/g mixtures of phenanthrene, pyrene, anthracene, fluorene, and acenapthene), either alone or in combination under methanogenic, sulfate-reducing, or nitrate-reducing conditions;

* Non-ionic surfactants (Brij 30, Triton SN70, or Triton N101 at concentrations of 0.01, 0.1, and 1.0 % [vol/vol]), respectively;

* Treatment with a microbial inhibitor (50 mM bromoethanesulfonic acid (BESA), 50 mM vancomycin, or 20 mM sodium molybdate-2-hydrate).

All experiments were performed using 125-mL serum bottles containing 45 mL medium, 5 g river sediment, and 1 µg/g each of 2,2',5-CB; 2,4',5-CB; 2,2',3,5-CB; 2,2',5,5'-CB; 2,2',4,5,5'-CB; 2,2',4,4',5,5'-CB; and 2,2',3,4,4',5,5'-CB in combination. Bottles were capped with butyl rubber stoppers and wrapped in aluminum foil to prevent photolysis, then incubated without shaking at 30°C in darkness. Our experimental medium consisted of (all concentrations in g/L): NH^sub 4^Cl, 2.7; MgCl^sub 2^ . 6H^sub 2^O, 0.1; CaCl^sub 2^ . 2H^sub 2^O, 0.1; FeCl^sub 2^ . 4H^sub 2^O, 0.02; K^sub 2^HPO^sub 4^, 0.27; KH^sub 2^PO^sub 4^, 0.35; yeast extract, 2.0; and resazurin, 0.001. Medium pH was adjusted to 7.0 following autoclaving; 0.9 mM titanium citrate was added as a reducing reagent. All experiments were conducted in an anaerobic glove box (Forma Scientific, model 1025 S/N, California) filled with N^sub 2^ (85%), H^sub 2^ (10%), and CO2 (5%) gases. Bottle contents were periodically sampled to measure residual concentrations of PCB congeners, oxidation-reduction potential (ORP) values, pH values, and methane production. All experiments were performed in duplicate.

Procedure. The extraction and analysis of PCB congeners were performed as described in a previous report (Chang et al., 2001). Supernatant ORP and pH values were measured with a pH/ ORP meter (HI 9017, Hanna, Italy). Methane levels were measured using a gas chromatograph (Sigma 3B Perkin-Elmer, Wellesley, Massachusetts) equipped with a packed column (200 × 0.5 cm, 80/100 porapak Q; Supecol, Bellefonte, Pennsylvania) and flameionization detector. Column, injector, and detector temperatures were maintained at 55°C, 90°C, and 90°C, respectively. Nitrogen was used as a carrier gas at a flow rate of 20 mL/min. Degradation rates (microgram PCB per gram of sediment (dry weight) per day) were determined according to PCB congeners reduction time courses, using 2 to 4 points in the linear portion of graphs plotting the concentration of PCB congeners against time. To calculate remaining percentages, the concentrations of PCB congeners residue were divided by the original concentrations of PCB congeners. Statistically significant differences were calculated using an F-test.

Results and Discussion

PCB congener dechlorination rates among the five sediment samples are presented in Table 1. For each PCB congener, the high-to-low order of dechlorination rates by sample site was K8 > K10 > K12 > K11 > K6. We also found that the order for BOD values was K8 > K10 > K12 > K11 > K6. The order of sample sites indicates a correlation between dechlorination rates and BOD values. The order of dechlorination rates by sample site was similar to that in our previous report about the biodegradation of phenanthrene in river sediment (Yuan et al., 2001). The results suggest that microorganisms are capable of adapting to site-specific conditions to achieve optimum dechlorination efficiency. Because dechlorination rates for all seven congeners were highest in the K8 sediment samples, discussion is restricted to those specific results.

The dechlorination rates for seven additional PCB congeners (mixed or individual) in K8 sediment samples are shown in Table 2. As shown, the high-to-low order of observed dechlorination rates was 2,2',5-CB > 2,4',5-CB > 2,2',3,5'-CB > 2,2',5,5'-CB > 2,2',4,5,5'-CB > 2,2',4,4',5,5'-CB > 2,2',3,4,4',5,5'-CB. These results show that chlorine removal depends on the chlorine content of individual PCB congeners. Because of increased toxicity levels, dechlorination rates were lower for the simultaneous presence of all compounds than for their individual presence. These results support our previous finding of delayed dechlorination in the simultaneous presence of 2,3,5,6-CB; 2,3,4,5-CB; and 2,3,4,5,6-CB (Chang et al., 2001).

Data on dechlorination rates, pH values, methane production, and ORP values for 2,4',5-CB; 2,2',5,5'-CB; and 2,2'4,5,5'-CB are presented in Figure 2. The results show that the three congeners were dechlorinated within 41, 84, and 110 days of incubation, respectively, and that pH values ranged between 6.6 and 7.5 during the 110-day incubation period. These results are similar to those reported in two previous studies (Chang et al., 1999, 2001). The data show no significant differences in maximum methane production during the 110-day incubation period (950 mg/L for 2,4', 5-CB; 910 mg/L for 2,2',5,5'-CB; and 900 mg/L for 2,2'4,5, 5'-CB). Initial ORP values were measured at -160 to -250 mV for the three congeners, but these values decreased to -400 mV before eventually increasing to approximately -300 mV within 110 days. No significant differences were noted for pH values, ORP values, or methane production for any of these three PCB congeners. In other words, no relationship was noted between PCB congener dechlorination and ORP value. It may be that the observed changes in ORP values were a result of growth in the anaerobic consortium. These results match those reported in our previous study (Chang et al., 1998).

As shown in Table 3, the dechlorination of 2,4',5-CB; 2,2',5, 5'-CB; and 2,2',4,5,5'-CB was enhanced under various reducing conditions. We found that, compared with our inoculated control, dechlorination rates for the three congeners were enhanced under methanogenic and sulfate-reducing conditions but delayed under nitrate-reducing conditions. The high-to-low dechlorination order was methanogenic conditions > sulfate-reducing conditions > nitrate-reducing conditions. This suggests that methanogens play a significant role in the dechlorination potential of the experimental consortium. Treatment with carbonate can also lead to increased dechlorination via its stimulative effect on methanogen growth; the addition of nitrate as an electron acceptor has the opposite effect (Madsen and Aamand, 1991; Haggblom et al., 1993). The data in Table 3 also show that the dechlorination for the three congeners was delayed following the addition of the five PAHs under the three reducing conditions. Furthermore, the degradation of anaerobic PAHs was delayed when PCB congeners were present under the three reducing conditions (data not shown). It may be that the microorganisms involved prefer using PAHs as carbon sources, which would reduce their dechlorination activity.

The data in Table 4 show the effects on the dechlorination of 2,2',5,5'-CB of adding various electron donors to the K8 sediment samples under the three reducing conditions. Under nitrate-reducing conditions, the dechlorination of 2,2',5,5'-CB was delayed (compared to the inoculated control) following the addition of pyruvate, lactate, or acetate. Under methanogenic and sulfate-reducing conditions, the dechlorination of 2,2',5,5'-CB was enhanced by the addition of pyruvate, lactate, or acetate. It may be that lactate, pyruvate, and acetate promote growth, resulting in faster dechlorination (Oremland, 1988; Chang et al., 1999). The fastest dechlorination rate was observed following treatment with lactate. Lactate or pyruvate metabolism by acidogens may produce hydrogen or hydrogen carbonate, both of which enhance methanogenic activity (Oremland, 1988). It is also possible that the action of sulfate-reducing bacteria on lactate produces pyruvate plus two electrons, and that pyruvate produces acetate plus two electrons (Legall and Fauque, 1988)-both processes resulting in faster dechlorination rates. Similar results were found for the dechlorination of 2,4',5-CB and 2,2',4,5,5'-CB.

Figure 3 presents data on the effects of surfactants on the dechlorination of the three PCB congeners in the sediment sample. The individual addition of Brij 30, Triton SN70, or Triton N101 at a concentration of 0.1% inhibited dechlorination in all cases. We also found that methane production was inhibited by the addition of any of the three surfactants at concentrations of 0.01, 0.1, and 1.0 % (Figure 4). Treatment with any of the three surfactants inhibited methane production, thus delaying PCB congener dechlorination. The use of surfactants is a common practice to improve the solubility of a poorly soluble component (Martel et al., 1998). We also added Brij 30, Triton SN70, or Triton N101 to the sediment sample and found that methane production was inhibited in each case. Thus, as also suggested by Tartakovsky et al. (2000), the results indicate a toxic effect of surfactants on anaerobic bacteria.

To investigate the significant dechlorination population in river sediment, we added various microbial inhibitors to the sediment. As shown in Figure 5, the addition of molybdate, BESA, or vancomycin delayed PCB congener dechlorination in the order of combined vancomycin/molybdate/BESA [arrow right] BESA [arrow right] molybdate [arrow right] vancomycin. Methane production also was inhibited by the addition of BESA or molybdate. The addition of molybdate delayed dechlorination under methanogenic conditions, and the addition of BESA delayed dechlorination under sulfate-reducing conditions (data not shown). These results suggest that sulfate-reducing bacteria, methanogen, and eubacteria are involved in dechlorination processes in general, and that sulfate-reducing bacteria and methanogen play significant roles in PCB congener dechlorination. We isolated six strains of PCB-dechlorinating organisms from our experimental mixed culture after 2 weeks of incubation. However, the dechlorination potential of these strains was weak, suggesting that the organisms predominantly responsible for dechlorination were nonculturable or that close synthrophic relationships exist among the culture's various organisms.

Conclusions

The five sediments used in these experiments were capable of dechlorinating the seven PCB congeners. In the simultaneous presence of the seven PCB congeners, dechlorination rates decreased. The addition of electron donors, PAHs, and surfactants all affected the rate of dechlorination of PCB congeners. We will use similar conditions to further define operating parameters for river sediment bioremediation and use the same anaerobic consortium to further define operating parameters for soil or sludge bioremediation.

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