- Email: [email protected]
Degradation characteristics of a dibenzofuran-degrader Terrabacter sp. strain DBF63 toward chlorinated dioxins in soil
Chemosphere 48 (2002) 201–207 www.elsevier.com/locate/chemosphere
Degradation characteristics of a dibenzofuran-degrader Terrabacter sp. strain DBF63 toward chlorinated dioxins in soil Hiroshi Habe a, Kazuki Ide b, Mizuyo Yotsumoto b, Hirokazu Tsuji b, Takako Yoshida a, Hideaki Nojiri a, Toshio Omori a,* b
a Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Department of BioEnvironmental Engineering, Obayashi Corporation, 4-640 Shimokiyoto, Kiyose-shi, Tokyo 204-0011, Japan
Received 16 July 2001; received in revised form 3 January 2002; accepted 26 January 2002
Abstract To obtain basic information towards applying a dibenzofuran (DF)-degrader Terrabacter sp. strain DBF63 to bioremediate dioxin-contaminated soil, we investigated the degradative potential of strain DBF63 for either chlorinated or polychlorinated dibenzo-p-dioxins and dibenzofurans (Clx DD/Clx DF) in soil. In the soil slurry system with a soil to water ratio of 1:5 (w/v), the DF-grown DBF63 cells degraded 90% of 1 ppm 2,8-Cl2 DF, whereas only 40% of 1 ppm 2,3Cl2 DD during the 7-day incubation. The degradation rates of 2-ClDF, 2-ClDD, 2,8-Cl2 DF and 2,3-Cl2 DF by strain DBF63 in the soil slurry system (5-day incubation) were approximately 89%, 65%, 78% and 32%, respectively. These results suggest that strain DBF63 was able to degrade mono- to dichlorinated dibenzofurans more effectively than mono- to dichlorinated dibenzo-p-dioxins. Using the same soil slurry system, we performed a preliminary bioremediation experiment using the actual dioxin-contaminated soil at an incineration site. We found that approximately 10% of tetra- to hexa-chlorinated congeners was decreased by a single inoculation with DBF63 cells within a 7-day incubation. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Bioremediation; Bioaugmentation; Polychlorinated dibenzo-p-dioxin; Polychlorinated dibenzofurans; Terrabacter sp.
1. Introduction Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDDs/PCDFs, Clx DDs/Clx DFs, x ¼ 1–8) are mostly produced as unintentional byproducts during combustion of municipal and industrial waste in incinerators, and they bring about serious pollution of the surrounding soil environment because of their extremely high persistence and toxicity. In addition, these pollutants are widely distributed but usually exist at low concentrations. Hence, physicochemical
*
Corresponding author. Tel.: +81-3-5841-3067; fax: +81-35841-8030. E-mail address: [email protected] (T. Omori).
treatment procedures seem not to be useful because vast masses of contaminated soils have to be built up in one place. Recently, biological treatment methods have become popular as an alternative to physicochemical methods because they can be applied in situ at a relatively low cost (Megharaj et al., 1997; Halden et al., 1999; Habe et al., 2001b). There have been no reports on microorganisms capable of utilizing PCDDs/PCDFs for growth, but some PCDDs/PCDFs are subjected to reductive dehalogenations leading to less halogenated congeners that can be attacked by microbial oxidases and dioxygenases (Beurskens et al., 1995; Ballerstedt et al., 1997), and these compounds sometimes can be utilized as carbon and energy sources. Microbial degradation of dioxin-related compounds, i.e., DD, DF, diaryl ethers, and their halogenated derivatives, has been
0045-6535/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 2 ) 0 0 0 6 4 - 4
202
H. Habe et al. / Chemosphere 48 (2002) 201–207
reviewed in its biochemical and biotechnological aspects (Halden and Dwyer, 1997; Wittich, 1998). We also have been studying the degradation of dioxin-related compounds by the DF-mineralizing bacterium, Terrabacter sp. strain DBF63, with biochemical and genetic approaches (Monna et al., 1993; Kasuga et al., 1997, 2001). Studies on the catabolic pathway of DF by strain DBF63 revealed that the initial dioxygenase acted on the angular position adjacent to the ether bridge. The resultant unstable hemiacetal decayed spontaneously to 2,20 ,3-trihydroxybiphenyl (THB), and the dihydroxylated aromatic ring of THB was then metacleaved. By subsequent hydrolysis of the side-chain, salicylic acid was formed, as other groups also reported (Fortnagel et al., 1989; Strubel et al., 1989; Wittich et al., 1992). The genes coding for individual enzymes of these steps have already been cloned and characterized in strain DBF63 (Kasuga et al., 1997, 2001). In addition, DBF63 cells grown on DF or its angular dioxygenase expressed in Escherichia coli cells have been found to transform some mono-to trichlorinated DDs and DFs (10 ppm each) (Habe et al., 2001a). These results have suggested a potential for applying strain DBF63 to bioremediate soil polluted with dioxins. Moreover, because the total concentration of PCDFs was 3–4 times higher than that of PCDDs in the soil samples collected from the polluted incinerator site (Habe et al., 2001b), the development of bioremediation process using the DF-mineralizing bacteria seems to be useful. In this study, as the first step towards bioaugmentation of dioxin-contaminated soil using strain DBF63, we assessed the characteristics of this strain for degradation of chlorinated dioxins in soil.
2. Materials and methods 2.1. Bacterial strains and culture conditions Terrabacter sp. strain DBF63 is a gram positive and DF-utilizing bacterium (Monna et al., 1993, Kasuga et al., 1997). The strain was grown at 30 °C in carbon-free minimal medium (CFMM; containing 2.2 g Na2 HPO4 (15.5 mM), 0.8 g KH2 PO4 (5.88 mM), 3.0 g NH4 NO3 (37.5 mM), 0.2 g MgSO4 7H2 O (0.81 mM), 10 mg FeCl3 6H2 O (36 lM), 10 mg CaCl2 2H2 O (68 lM) and 50 mg yeast extract per liter) supplemented with 1 mg/ml of DF dissolved in dimethylformamide (DMF) (100 mg/ ml). 2.2. Soils The dioxin-contaminated model soil used in this study was granitic soil sieved (2-mm mesh), non-sterilized and stored at room temperature. It consisted of 79%
sand, 10% clay and 11% silt, had a total organic carbon of 0.16%, pH 6.0, cation exchange capacity of 4.81 cmol kg1 and maximum water-holding capacity of 24.5%. Dioxin-contaminated soil was obtained from an incinerator site in Japan. The properties of the soil are the same as those of the model soil described above. The total dioxin concentration (PCDDs and PCDFs) was 725 ng/g dry soil, and the toxicity equivalency quantity (TEQ) was 11 ng/g dry soil (maximum acceptable concentration of dioxins in Japan is 1 ng TEQ/g soil). Since the amounts of mono- to trichlorinated dioxins in the contaminated soil were relatively low in comparison with those of tetra- to octachlorinated dioxins (data not shown) and the authentic 14 C-labeled samples of monoto trichlorinated dioxins for quantification analysis were not available, we analyzed only tetra- and more highly chlorinated DDs and DFs in this study. The quantification results are shown as averages of duplicate determinations. 2.3. 2,8-Cl2 DF degradation study using either the resting cells or the growing cells of strain DBF63 The strain DBF63 was precultivated in 10 ml of CFMM supplemented with DF at a concentration of 1 mg/ml (0.1% (v/v)) at 30 °C for 3 days, and then the culture was transferred to 1 l of the same medium and incubated for another 3 days. For preparation of the resting cells of strain DBF63, the cells were collected by centrifugation, washed twice with CFMM, and resuspended in an appropriate volume of CFMM to be 109 CFU/ml. On the other hand, for preparation of the growing cells of strain DBF63, strain DBF63 cultured for 3 days in CFMM supplemented with DF was directly used (109 CFU/ml). As a control, sterilized DBF63 cells by autoclaving at 120 °C for 20 min were used. One microgram of the 2,8-Cl2 DF dissolved in DMF (0.1 mg/ ml) was added to 5 ml of either the resting, growing, or sterilized cell suspension solution as a model substrate. The reaction mixture was incubated on a reciprocal shaker (300 strokes/min) at 30 °C for 5 days. Samples were then extracted twice with 5 ml of ethyl acetate to determine the concentration of the remaining 2,8-Cl2 DF. 2.4. 2,8-Cl2 DF and 2,3-Cl2 DD degradation study using soil slurry microcosm One-gram aliquots of the granitic soil were introduced into sterile Sakaguchi flasks (100 ml) with aluminum caps. The soil (1 g) was treated with 2,8-Cl2 DF or 2,3-Cl2 DD at a final concentration of 1 ppm (1 lg/gsoil) from 1 mg/ml stock solutions made in DMF, mixed, and then inoculated with 5 ml of CFMM containing the growing cells of strain DBF63 (soil slurry), and incubated on a shaker (120 strokes/min) at 30 °C in
H. Habe et al. / Chemosphere 48 (2002) 201–207
the dark. As a control, each soil with sterilized DBF63 cells was incubated. After incubation for 1, 3, 5 and 7 days, samples were extracted twice with 5 ml of ethyl acetate, and the concentrations of remaining 2,8-Cl2 DF and 2,3-Cl2 DD were determined. 2.5. Some mono- and dichlorinated DDs and DFs degradation study using soil slurry microcosm
203
tection Agency. The decrease of PCDDs and PCDFs concentration was calculated from the amount of the remaining PCDDs and PCDFs, respectively, in soil slurry microcosm. Data are shown as averages of duplicate determinations. PCDDs/PCDFs concentrations as TEQ were calculated based on the toxicity equivalent factors (TEF) quoted from WHO/IPCS (1997). 2.8. Chemicals
In this experiment, 2-ClDF, 2-ClDD, 2,8-Cl2 DF or 2,3-Cl2 DD was singly added to the granitic soil at a final concentration of 1 ppm (1 lg/g-soil). The resultant soils were inoculated with the growing cells of strain DBF63, incubated for 5 days, and the concentrations of each CDD and CDF were determined. As a control, each soil with sterilized DBF63 cells was also analyzed. 2.6. Quantification of CDDs and CDFs The amount of 2-ClDF, 2-ClDD, 2,8-Cl2 DF and 2,3Cl2 DD in each sample was determined with a model JMS-Automass 150 GC-MS system (JEOL, Ltd., Tokyo, Japan) fitted with a fused-silica chemically bonded capillary column (DB-5; 0.25 mm (inside diameter) by 15 m, 0.25 lm film thickness; J&W Scientific Inc., Folsom, CA). As an internal standard, 1 ppm (1 mg/g-soil) of 2-ClDD for quantification of 2-ClDF and 2,8-Cl2 DF, and 1 ppm of 2-ClDF for that of 2-ClDD and 2,3Cl2 DD was added to the samples just before the extraction with ethyl acetate. Each sample was injected into the column at 80 °C in the splitless mode. After 2 min at 80 °C, the column temperature was increased at 16 °C min1 to 280 °C. The head pressure of the helium carrier gas was 65 kPa. We determined the decrease of CDDs and CDFs concentration by comparing the peak area for molecular ion of each compound (m=z ¼ 202 for 2-ClDF, m=z ¼ 218 for 2-ClDD, m=z ¼ 236 for 2,8Cl2 DF and m=z ¼ 252 for 2,3-Cl2 DD) before and after the appropriate incubation.
2-ClDF, 2-ClDD, 2,8-Cl2 DF and 2,3-Cl2 DD were purchased from AccuStandard Inc. (New Haven, CT). All other chemicals were of analytical grade and of the highest purity available.
3. Results and discussion 3.1. Degradation of 2,8-Cl2 DF by either the resting cells or the growing cells of strain DBF63 in soil-free aqueous solution The cell density of strain DBF63 cultured in CFMM supplemented with 1 mg/ml DF, reached the maximum (2 109 CFU/ml) after culturing for 3 days and remained in a stationary state. Hence, in all the following experiments, the 3-day-grown DBF63 cells were used as an inoculum. Liquid CFMM with 1 ppm 2,8-Cl2 DF was inoculated with the growing, resting and sterilized cells of strain DBF63 (109 CFU/ml), and incubated for 5 days. The percentage of 2,8-Cl2 DF transformation by either the resting or growing cells was 75%, while that by sterilized cells (control) was only 19% (Fig. 1). This indicates that the transformation capability of the resting cells of strain DBF63 is almost the same as that of the
2.7. Preliminary bioremediation study using the actual dioxin-contaminated soil Soil slurry microcosms consisting of 20 g dioxincontaminated soil collected from an incinerator site and 100 ml of CFMM containing the growing cells of strain DBF63 at a density of approximately 108 CFU/g-dry soil were prepared in a Sakaguchi flask (500 ml). The same soil slurry inoculated with sterilized DBF63 cells was used as a control. The soil slurry microcosm was incubated on shaker (200 stroke/min) at 30 °C for 7 days in the dark. PCDDs and PCDFs in the soil slurry were extracted by the Soxhlet method, and analyzed at the EAC Corporation (Gumma, Japan) by high resolution mass spectrometry (HRGS/HRMS) according to the protocol recommended by the US Environmental Pro-
Fig. 1. Comparison of degradation rate of 2,8-Cl2 DF (1 ppm) after 5-day treatment with the growing cells, the resting cells, and the sterilized cells (control) of strain DBF63 (109 CFU/ml) cultivated for 3 days in DF-containing medium. For one experiment, the means of three determinations are shown, and the error bars indicate the standard deviations.
204
H. Habe et al. / Chemosphere 48 (2002) 201–207
growing cells of strain DBF63. Therefore, considering the easiness to prepare the cells having the biotransformation ability, we used the growing cells of strain DBF63 in the following studies. 3.2. 2,8-Cl2 DF and 2,3-Cl2 DD degradation in soil slurry microcosm The granitic soil used in this study is one of the major soils in Japan, especially in the Kansai-district. Hence, we examined the degradation characteristics of this strain for dioxin in this soil (soil slurry) to determine whether strain DBF63 is applicable for bioremediation of this major soil. The growing cells of strain DBF63 were inoculated into 2,8-Cl2 DF-contaminated model soil at 1 ppm (soil slurry microcosm), and incubated at 30 °C for various days. As a comparison, 1 ppm of 2,8-Cl2 DF was incubated with DBF63 growing cells in a soil-free aquatic
Fig. 2. Degradation of 2,8-Cl2 DF (a) and 2,3-Cl2 DD (b) by strain DBF63 (growing cells) in soil slurry microcosm (), and in soil-free aqueous solution (). Control (N) shows the degradation rate with sterilized DBF63 cells. For one experiment, the means of three determinations are shown, and the error bars indicate the standard deviations.
solution (Fig. 2a). The degradation rate of 2,8-Cl2 DF in the soil slurry microcosm and the soil-free aqueous solution after a 7-day incubation were 90% and 88%, respectively, but that of 2,8-Cl2 DF in soil slurry inoculated with sterilized DBF63 cells, was only 10%. Although 76–86% of 2,8-Cl2 DF was degraded within 1 day, prolonged incubation did not significantly increase the degradation of 2,8-Cl2 DF. Because similar degradation patterns were observed in these two reaction systems, it was considered that the degradation activity of strain DBF63 towards 2,8-Cl2 DF was not influenced by the presence of this kind of soil under the condition employed in this study. On the other hand, the growing cells of strain DBF63 was also inoculated into the 2,3-Cl2 DD-contaminated model soil slurry system, and incubated at 30 °C. The degradation rate of 2,3-Cl2 DD in the soil slurry microcosm after a 7-day incubation was 40%, while control experiments (inoculated with the sterilized DBF63 cells) showed 22% of degradation (Fig. 2b). This indicated that strain DBF63 degraded 2,8-Cl2 DF more quickly and effectively than 2,3-Cl2 DD in soil (1 ppm each). Recently, we found that the degradation rates of both 2,8-Cl2 DF and 2,3-Cl2 DD (10 ppm each) by the resting cells of strain DBF63 in soil-free aqueous solution were approximately 80–90% (2,8-Cl2 DF) and 70–90% (2,3Cl2 DD), respectively (Habe et al., 2001a), showing that at 10 ppm, these two substrates were degraded similarly by strain DBF63. The transformation capability of strain DBF63 for CDD/CDF congeners at low concentration (1 ppm) may cause the differences in the degradation characteristics of strain DBF63 towards 2,8-Cl2 DF and 2,3-Cl2 DD.
Fig. 3. The degradation characteristics of strain DBF63 toward mono- and dichlorinated DFs and DDs. Black bars represent the rates of degradation of 2-ClDF, 2-ClDD, 2,8-Cl2 DF or 2,3Cl2 DD each at 1 ppm incubated with strain DBF63 for 5 days. White bars represent the degradation rates of respective substrates incubated with sterilized DBF63 cells for 5 days (control). For one experiment, the means of three determinations are shown, and the error bars indicate the standard deviations.
H. Habe et al. / Chemosphere 48 (2002) 201–207
205
trol experiments using sterilized DBF63 cells (Fig. 3, white bars). Similar to degradation of 2,8-Cl2 DF and 2,3-Cl2 DD in the soil slurry system, 2-CDF was degraded much more than 2-CDD, both of which were substituted with a chlorine at the 2-position of DF- and DD-skeleton, respectively. These results suggest that strain DBF63 is able to degrade CDF more efficiently than CDD in soil. Considering that the total concentration of PCDFs was higher than that of PCDDs in the dioxin-contaminated soils used in this study (Table 1), strain DBF63 seems to be applicable for remediation of such kinds of soil. On the other hand, mono-CDD/CDF congeners were degraded much more than di-CDD/CDF congeners
3.3. Degradation of mono- and dichlorinated DDs and DFs in soil slurry microcosm To investigate whether the difference in the degradation rates for 2,8-Cl2 DF and 2,3-Cl2 DD in soil was derived from the differences in the skeletons of DD and DF, we examined the degradation of 2-ClDF, 2-ClDD, 2,8-Cl2 DF and 2,3-Cl2 DD in soil slurry. During 5 days of incubation with strain DBF63 cells, 2-ClDF, 2-ClDD, 2,8-Cl2 DF and 2,3-Cl2 DD singly added to the soil slurry at a concentration of 1 ppm were decomposed by approximately 93%, 79%, 81% and 40%, respectively (Fig. 3, black bars), whereas approximately 38%, 40%, 12% and 12% of respective substrates were degraded in con-
Table 1 Degradation of PCDD and PCDF congeners by strain DBF63 in the contaminated soil collected at an incinerator site Congenersa
Controlb (pg/g-dry soil) Concentration
Single inoculation of DBF63 (pg/g-dry soil) TEQc
Concentration
Degradation amount (pg/g-dry soil)
Degradation rate (%)
TEQc
PCDDs 1,3,6,8-Cl4 DD 1,3,7,9-Cl4 DD 2,3,7,8-Cl4 DD Cl4 DDs
180 103 44 1025
0 0 44 –
155 90 40 870
0 0 40 –
25 13 4 155
13.9 12.6 9.0 15.1
1,2,3,7,8-Cl5 DD Cl5 DDs
765 7000
765 –
705 6300
705 –
60 700
7.8 10.0
1,2,3,4,7,8-Cl6 DD 1,2,3,6,7,8-Cl6 DD 1,2,3,7,8,9-Cl6 DD Cl6 DDs
2150 2800 2650 31 500
215 280 265 –
2000 2450 2300 27 500
200 245 230 –
150 350 350 4000
7.0 12.5 13.2 12.7
1,2,3,4,6,7,8-Cl7 DD Cl7 DDs
28 000 54 500
280 –
28 000 52 500
280 –
0 2000
0.0 3.7
Cl8 DD
45 000
4.5
46 000
4.6
–
PCDFs 1,2,7,8-Cl4 DF 2,3,7,8-Cl4 DF Cl4 DFs
450 265 28 500
0 26.5 –
395 225 25 000
0 22.5 –
55 40 3500
12.2 15.1 12.3
1,2,3,7,8-Cl5 DF 2,3,4,7,8-Cl5 DF Cl5 DFs
1800 4750 71 000
90 2375 –
1550 4350 63 500
78 2175 –
250 400 7500
13.9 8.4 10.6
1,2,3,4,7,8-Cl6 DF 1,2,3,6,7,8-Cl6 DF 2,3,4,6,7,8-Cl6 DF Cl6 DFs
9900 12 000 31 500 176 250
990 1200 3150 –
9150 11 000 29 500 160 000
915 1100 2950 –
750 1000 2000 16 250
7.6 8.3 6.5 9.2
1,2,3,4,7,8,9-Cl7 DF Cl7 DFs
21 500 283 500
215 –
21 500 275 000
215 –
0 8500
0.0 3.0
Cl8 DF
110 000
11
109 500
12
–
a
–
Clx DD and Clx DF represent the congeners other than the listed congeners whose authentic samples were available. b The soil inoculated autoclaved DBF63 cells was used as a control. c TEQ was calculated by multiplying TEF by each concentration and TEF was quoted from WHO/IPCS (1997).
–
206
H. Habe et al. / Chemosphere 48 (2002) 201–207
(Fig. 3), which is commonly observed in the degradation of PCDDs or PCDFs and other chlorinated compounds. In control experiments, the rather soluble mono-CDD/ CDF are much stronger decreased than the di-CDD/ CDF congeners. Because the granitic soil used in this study (both model and actually contaminated soils) were not sterilized prior to use and there were much nutrients like phosphate and nitrogen source in the soil slurry system, there exist the possibility that the indigenous bacteria in the soil might oxidize mono-CDD/CDF substrate, which is easier to be attacked by bacteria than di-CDD/CDF. 3.4. Preliminary examination for bioremediation of the actual dioxin-contaminated soil In the dioxin-contaminated soil at the incinerator site, PCDFs occupy 80% of the total concentration of dioxins, and among the tetra- to heptachlorinated congeners, the higher the concentration of either PCDD or PCDF, the higher the number of chlorine substitution. Among the PCDDs and PCDFs, there is just a small amount of the most toxic compound, 2,3,7,8-Cl4 DDs (Table 1). The soil slurry prepared with dioxin-contaminated soil collected at an incinerator site was incubated with strain DBF63 cells. During a 7-day incubation, the total PCDD and PCDF concentrations decreased from 725 to 715 ng/g-soil and from 11 to 10 ng TEQ/g-soil, respectively. The decrease of each congener is shown in Table 1. The lightly chlorinated congeners among the examined compounds such as Cl4 DDs and Cl4 DFs were decreased at a relatively high rate (12–15%). Dioxins with penta- to hexachlorines were also decreased by DBF63 though only slightly. On the other hand, the levels of Cl7 DD/Cl7 DF and Cl8 DD/Cl8 DF concentrations remained almost unchanged. Considering that Cl7 DD and Cl8 DF are hardly attacked by initial dioxygenases from bacterial strains because no positions of aromatic-rings are free for chlorine substitution, this result seems to be reasonable. In addition, strain DBF63 appears to attack preferably the dioxin congener whose initial concentration was relatively high (Table 1). In this experiment, approximately 10% of tetra- to hexa-CDD/CDF congeners were decreased by the augmentation of strain DBF63. The similar result has been obtained in a preliminary bioremediation experiment of dioxin-contaminated soil using a carbazole-degrader Pseudomonas sp. strain CA10 (Habe et al., 2001b). In order to perform better bioremediation of the actual dioxin-contaminated soils, more improved methods such as the repeated inoculation with dioxin-degrading bacteria, which can maintain a high population density of the cells having biotransformation ability, should be used. In addition, to avoid the absorption of dioxins by soil particles, the use of surfactant may also be useful.
Acknowledgements Part of this work was supported by a Grant-in-Aid for Scientific Research (no. 11794005) to T.O. from the ministry of Education, Science, Sports and Culture of Japan. References Ballerstedt, H., Kraus, A., Lechner, U., 1997. Reductive dehalogenation of 1,2,3,4-tetrachlorodibenzo-p-dioxin and its products by anaerobic mixed cultures from Saale River sediment. Environ. Sci. Technol. 31, 1749–1753. Beurskens, J.E.M., Toussaint, M., de Wolf, J., van der Steen, J.M.D., Slot, P.C., Commandeur, L.C.M., Parsons, J.R., 1995. Dehalogenation of chlorinated dioxins by an anaerobic microbial consortium from sediment. Environ. Toxicol. Chem. 14, 939–943. Fortnagel, P., Harms, H., Wittich, R.-M., Francke, W., Krohn, S., Meyer, H., 1989. Cleavage of dibenzofuran and dibenzodioxin ring systems by a Pseudomonas bacterium. Naturwissenschaften 76, 222–223. Habe, H., Chung, J.-S., Lee, J.-H., Kasuga, K., Yoshida, T., Nojiri, H., Omori, T., 2001a. Degradation of chlorinated dibenzofurans and dibenzo-p-dioxins by two types of bacteria having angular dioxygenases with different features. Appl. Environ. Microbiol. 67, 3610–3617. Habe, H., Ide, K., Yotsumoto, M., Tsuji, H., Hirano, H., Widada, J., Yoshida, T., Nojiri, H., Omori, T., 2001b. Preliminary examinations for applying a carbazoledegrader Pseudomonas sp. strain CA10 to dioxin-contaminated soil remediation. Appl. Microbiol. Biotechnol. 56, 788–795. Halden, R.U., Dwyer, D.F., 1997. Biodegradation of dioxinrelated compounds. Bioremediation J. 1, 11–25. Halden, R.U., Halden, B.G., Dwyer, D.F., 1999. Removal of dibenzofuran, dibenzo-p-dioxin, and 2-chlorodibenzo-pdioxin from soils inoculated with Sphingomonas sp. strain RW1. Appl. Environ. Microbiol. 65, 2246–2249. Kasuga, K., Nojiri, H., Yamane, H., Kodama, T., Omori, T., 1997. Cloning and characterization of the genes involved in the degradation of dibenzofuran by Terrabacter sp. strain DBF63. J. Ferment. Bioeng. 84, 387–399. Kasuga, K., Habe, H., Chung, J.-S., Yoshida, T., Nojiri, H., Yamane, H., Omori, T., 2001. Isolation and characterization of the genes encoding a novel oxygenase component of angular dioxygenase from the gram-positive dibenzofurandegrader Terrabacter sp. strain DBF63. Biochem. Biophys. Res. Commun. 283, 195–204. Megharaj, M., Wittich, R.-M., Blasco, R., Pieper, D.H., Timmis, K.N., 1997. Superior survival and degradation of dibenzo-p-dioxin and dibenzofuran in soil by soil-adapted Sphingomonas sp. strain RW1. Appl. Microbiol. Biotechnol. 48, 109–114. Monna, L., Omori, T., Kodama, T., 1993. Microbial degradation of dibenzofuran, fluorene, and dibenzo-p-dioxin by Staphylococcus auriculans DBF63. Appl. Environ. Microbiol. 59, 285–289. Strubel, V., Rast, H.G., Fietz, W., Knackmuss, H.-J., Engesser, K.H., 1989. Enrichment of dibenzofuran utilizing bacteria
H. Habe et al. / Chemosphere 48 (2002) 201–207 with high co-metabolic potential towards dibenzodioxin and other anellated aromatics. FEMS Microbiol. Lett. 58, 233– 238. Wittich, R.-M., Wilkes, H., Sinnwell, V., Francke, W., Fortnagel, P., 1992. Metabolism of dibenzo-p-dioxin by Sphingo-
207
monas sp. strain RW1. Appl. Environ. Microbiol. 58, 1005– 1010. Wittich, R.-M., 1998. Degradation of dioxin-like compounds by Microorganisms. Appl. Microbiol. Biotechnol. 49, 489– 499.
Degradation characteristics of a dibenzofuran-degrader Terrabacter sp. strain DBF63 toward chlorinated dioxins in soil Hiroshi Habe a, Kazuki Ide b, Mizuyo Yotsumoto b, Hirokazu Tsuji b, Takako Yoshida a, Hideaki Nojiri a, Toshio Omori a,* b
a Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Department of BioEnvironmental Engineering, Obayashi Corporation, 4-640 Shimokiyoto, Kiyose-shi, Tokyo 204-0011, Japan
Received 16 July 2001; received in revised form 3 January 2002; accepted 26 January 2002
Abstract To obtain basic information towards applying a dibenzofuran (DF)-degrader Terrabacter sp. strain DBF63 to bioremediate dioxin-contaminated soil, we investigated the degradative potential of strain DBF63 for either chlorinated or polychlorinated dibenzo-p-dioxins and dibenzofurans (Clx DD/Clx DF) in soil. In the soil slurry system with a soil to water ratio of 1:5 (w/v), the DF-grown DBF63 cells degraded 90% of 1 ppm 2,8-Cl2 DF, whereas only 40% of 1 ppm 2,3Cl2 DD during the 7-day incubation. The degradation rates of 2-ClDF, 2-ClDD, 2,8-Cl2 DF and 2,3-Cl2 DF by strain DBF63 in the soil slurry system (5-day incubation) were approximately 89%, 65%, 78% and 32%, respectively. These results suggest that strain DBF63 was able to degrade mono- to dichlorinated dibenzofurans more effectively than mono- to dichlorinated dibenzo-p-dioxins. Using the same soil slurry system, we performed a preliminary bioremediation experiment using the actual dioxin-contaminated soil at an incineration site. We found that approximately 10% of tetra- to hexa-chlorinated congeners was decreased by a single inoculation with DBF63 cells within a 7-day incubation. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Bioremediation; Bioaugmentation; Polychlorinated dibenzo-p-dioxin; Polychlorinated dibenzofurans; Terrabacter sp.
1. Introduction Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDDs/PCDFs, Clx DDs/Clx DFs, x ¼ 1–8) are mostly produced as unintentional byproducts during combustion of municipal and industrial waste in incinerators, and they bring about serious pollution of the surrounding soil environment because of their extremely high persistence and toxicity. In addition, these pollutants are widely distributed but usually exist at low concentrations. Hence, physicochemical
*
Corresponding author. Tel.: +81-3-5841-3067; fax: +81-35841-8030. E-mail address: [email protected] (T. Omori).
treatment procedures seem not to be useful because vast masses of contaminated soils have to be built up in one place. Recently, biological treatment methods have become popular as an alternative to physicochemical methods because they can be applied in situ at a relatively low cost (Megharaj et al., 1997; Halden et al., 1999; Habe et al., 2001b). There have been no reports on microorganisms capable of utilizing PCDDs/PCDFs for growth, but some PCDDs/PCDFs are subjected to reductive dehalogenations leading to less halogenated congeners that can be attacked by microbial oxidases and dioxygenases (Beurskens et al., 1995; Ballerstedt et al., 1997), and these compounds sometimes can be utilized as carbon and energy sources. Microbial degradation of dioxin-related compounds, i.e., DD, DF, diaryl ethers, and their halogenated derivatives, has been
0045-6535/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 2 ) 0 0 0 6 4 - 4
202
H. Habe et al. / Chemosphere 48 (2002) 201–207
reviewed in its biochemical and biotechnological aspects (Halden and Dwyer, 1997; Wittich, 1998). We also have been studying the degradation of dioxin-related compounds by the DF-mineralizing bacterium, Terrabacter sp. strain DBF63, with biochemical and genetic approaches (Monna et al., 1993; Kasuga et al., 1997, 2001). Studies on the catabolic pathway of DF by strain DBF63 revealed that the initial dioxygenase acted on the angular position adjacent to the ether bridge. The resultant unstable hemiacetal decayed spontaneously to 2,20 ,3-trihydroxybiphenyl (THB), and the dihydroxylated aromatic ring of THB was then metacleaved. By subsequent hydrolysis of the side-chain, salicylic acid was formed, as other groups also reported (Fortnagel et al., 1989; Strubel et al., 1989; Wittich et al., 1992). The genes coding for individual enzymes of these steps have already been cloned and characterized in strain DBF63 (Kasuga et al., 1997, 2001). In addition, DBF63 cells grown on DF or its angular dioxygenase expressed in Escherichia coli cells have been found to transform some mono-to trichlorinated DDs and DFs (10 ppm each) (Habe et al., 2001a). These results have suggested a potential for applying strain DBF63 to bioremediate soil polluted with dioxins. Moreover, because the total concentration of PCDFs was 3–4 times higher than that of PCDDs in the soil samples collected from the polluted incinerator site (Habe et al., 2001b), the development of bioremediation process using the DF-mineralizing bacteria seems to be useful. In this study, as the first step towards bioaugmentation of dioxin-contaminated soil using strain DBF63, we assessed the characteristics of this strain for degradation of chlorinated dioxins in soil.
2. Materials and methods 2.1. Bacterial strains and culture conditions Terrabacter sp. strain DBF63 is a gram positive and DF-utilizing bacterium (Monna et al., 1993, Kasuga et al., 1997). The strain was grown at 30 °C in carbon-free minimal medium (CFMM; containing 2.2 g Na2 HPO4 (15.5 mM), 0.8 g KH2 PO4 (5.88 mM), 3.0 g NH4 NO3 (37.5 mM), 0.2 g MgSO4 7H2 O (0.81 mM), 10 mg FeCl3 6H2 O (36 lM), 10 mg CaCl2 2H2 O (68 lM) and 50 mg yeast extract per liter) supplemented with 1 mg/ml of DF dissolved in dimethylformamide (DMF) (100 mg/ ml). 2.2. Soils The dioxin-contaminated model soil used in this study was granitic soil sieved (2-mm mesh), non-sterilized and stored at room temperature. It consisted of 79%
sand, 10% clay and 11% silt, had a total organic carbon of 0.16%, pH 6.0, cation exchange capacity of 4.81 cmol kg1 and maximum water-holding capacity of 24.5%. Dioxin-contaminated soil was obtained from an incinerator site in Japan. The properties of the soil are the same as those of the model soil described above. The total dioxin concentration (PCDDs and PCDFs) was 725 ng/g dry soil, and the toxicity equivalency quantity (TEQ) was 11 ng/g dry soil (maximum acceptable concentration of dioxins in Japan is 1 ng TEQ/g soil). Since the amounts of mono- to trichlorinated dioxins in the contaminated soil were relatively low in comparison with those of tetra- to octachlorinated dioxins (data not shown) and the authentic 14 C-labeled samples of monoto trichlorinated dioxins for quantification analysis were not available, we analyzed only tetra- and more highly chlorinated DDs and DFs in this study. The quantification results are shown as averages of duplicate determinations. 2.3. 2,8-Cl2 DF degradation study using either the resting cells or the growing cells of strain DBF63 The strain DBF63 was precultivated in 10 ml of CFMM supplemented with DF at a concentration of 1 mg/ml (0.1% (v/v)) at 30 °C for 3 days, and then the culture was transferred to 1 l of the same medium and incubated for another 3 days. For preparation of the resting cells of strain DBF63, the cells were collected by centrifugation, washed twice with CFMM, and resuspended in an appropriate volume of CFMM to be 109 CFU/ml. On the other hand, for preparation of the growing cells of strain DBF63, strain DBF63 cultured for 3 days in CFMM supplemented with DF was directly used (109 CFU/ml). As a control, sterilized DBF63 cells by autoclaving at 120 °C for 20 min were used. One microgram of the 2,8-Cl2 DF dissolved in DMF (0.1 mg/ ml) was added to 5 ml of either the resting, growing, or sterilized cell suspension solution as a model substrate. The reaction mixture was incubated on a reciprocal shaker (300 strokes/min) at 30 °C for 5 days. Samples were then extracted twice with 5 ml of ethyl acetate to determine the concentration of the remaining 2,8-Cl2 DF. 2.4. 2,8-Cl2 DF and 2,3-Cl2 DD degradation study using soil slurry microcosm One-gram aliquots of the granitic soil were introduced into sterile Sakaguchi flasks (100 ml) with aluminum caps. The soil (1 g) was treated with 2,8-Cl2 DF or 2,3-Cl2 DD at a final concentration of 1 ppm (1 lg/gsoil) from 1 mg/ml stock solutions made in DMF, mixed, and then inoculated with 5 ml of CFMM containing the growing cells of strain DBF63 (soil slurry), and incubated on a shaker (120 strokes/min) at 30 °C in
H. Habe et al. / Chemosphere 48 (2002) 201–207
the dark. As a control, each soil with sterilized DBF63 cells was incubated. After incubation for 1, 3, 5 and 7 days, samples were extracted twice with 5 ml of ethyl acetate, and the concentrations of remaining 2,8-Cl2 DF and 2,3-Cl2 DD were determined. 2.5. Some mono- and dichlorinated DDs and DFs degradation study using soil slurry microcosm
203
tection Agency. The decrease of PCDDs and PCDFs concentration was calculated from the amount of the remaining PCDDs and PCDFs, respectively, in soil slurry microcosm. Data are shown as averages of duplicate determinations. PCDDs/PCDFs concentrations as TEQ were calculated based on the toxicity equivalent factors (TEF) quoted from WHO/IPCS (1997). 2.8. Chemicals
In this experiment, 2-ClDF, 2-ClDD, 2,8-Cl2 DF or 2,3-Cl2 DD was singly added to the granitic soil at a final concentration of 1 ppm (1 lg/g-soil). The resultant soils were inoculated with the growing cells of strain DBF63, incubated for 5 days, and the concentrations of each CDD and CDF were determined. As a control, each soil with sterilized DBF63 cells was also analyzed. 2.6. Quantification of CDDs and CDFs The amount of 2-ClDF, 2-ClDD, 2,8-Cl2 DF and 2,3Cl2 DD in each sample was determined with a model JMS-Automass 150 GC-MS system (JEOL, Ltd., Tokyo, Japan) fitted with a fused-silica chemically bonded capillary column (DB-5; 0.25 mm (inside diameter) by 15 m, 0.25 lm film thickness; J&W Scientific Inc., Folsom, CA). As an internal standard, 1 ppm (1 mg/g-soil) of 2-ClDD for quantification of 2-ClDF and 2,8-Cl2 DF, and 1 ppm of 2-ClDF for that of 2-ClDD and 2,3Cl2 DD was added to the samples just before the extraction with ethyl acetate. Each sample was injected into the column at 80 °C in the splitless mode. After 2 min at 80 °C, the column temperature was increased at 16 °C min1 to 280 °C. The head pressure of the helium carrier gas was 65 kPa. We determined the decrease of CDDs and CDFs concentration by comparing the peak area for molecular ion of each compound (m=z ¼ 202 for 2-ClDF, m=z ¼ 218 for 2-ClDD, m=z ¼ 236 for 2,8Cl2 DF and m=z ¼ 252 for 2,3-Cl2 DD) before and after the appropriate incubation.
2-ClDF, 2-ClDD, 2,8-Cl2 DF and 2,3-Cl2 DD were purchased from AccuStandard Inc. (New Haven, CT). All other chemicals were of analytical grade and of the highest purity available.
3. Results and discussion 3.1. Degradation of 2,8-Cl2 DF by either the resting cells or the growing cells of strain DBF63 in soil-free aqueous solution The cell density of strain DBF63 cultured in CFMM supplemented with 1 mg/ml DF, reached the maximum (2 109 CFU/ml) after culturing for 3 days and remained in a stationary state. Hence, in all the following experiments, the 3-day-grown DBF63 cells were used as an inoculum. Liquid CFMM with 1 ppm 2,8-Cl2 DF was inoculated with the growing, resting and sterilized cells of strain DBF63 (109 CFU/ml), and incubated for 5 days. The percentage of 2,8-Cl2 DF transformation by either the resting or growing cells was 75%, while that by sterilized cells (control) was only 19% (Fig. 1). This indicates that the transformation capability of the resting cells of strain DBF63 is almost the same as that of the
2.7. Preliminary bioremediation study using the actual dioxin-contaminated soil Soil slurry microcosms consisting of 20 g dioxincontaminated soil collected from an incinerator site and 100 ml of CFMM containing the growing cells of strain DBF63 at a density of approximately 108 CFU/g-dry soil were prepared in a Sakaguchi flask (500 ml). The same soil slurry inoculated with sterilized DBF63 cells was used as a control. The soil slurry microcosm was incubated on shaker (200 stroke/min) at 30 °C for 7 days in the dark. PCDDs and PCDFs in the soil slurry were extracted by the Soxhlet method, and analyzed at the EAC Corporation (Gumma, Japan) by high resolution mass spectrometry (HRGS/HRMS) according to the protocol recommended by the US Environmental Pro-
Fig. 1. Comparison of degradation rate of 2,8-Cl2 DF (1 ppm) after 5-day treatment with the growing cells, the resting cells, and the sterilized cells (control) of strain DBF63 (109 CFU/ml) cultivated for 3 days in DF-containing medium. For one experiment, the means of three determinations are shown, and the error bars indicate the standard deviations.
204
H. Habe et al. / Chemosphere 48 (2002) 201–207
growing cells of strain DBF63. Therefore, considering the easiness to prepare the cells having the biotransformation ability, we used the growing cells of strain DBF63 in the following studies. 3.2. 2,8-Cl2 DF and 2,3-Cl2 DD degradation in soil slurry microcosm The granitic soil used in this study is one of the major soils in Japan, especially in the Kansai-district. Hence, we examined the degradation characteristics of this strain for dioxin in this soil (soil slurry) to determine whether strain DBF63 is applicable for bioremediation of this major soil. The growing cells of strain DBF63 were inoculated into 2,8-Cl2 DF-contaminated model soil at 1 ppm (soil slurry microcosm), and incubated at 30 °C for various days. As a comparison, 1 ppm of 2,8-Cl2 DF was incubated with DBF63 growing cells in a soil-free aquatic
Fig. 2. Degradation of 2,8-Cl2 DF (a) and 2,3-Cl2 DD (b) by strain DBF63 (growing cells) in soil slurry microcosm (), and in soil-free aqueous solution (). Control (N) shows the degradation rate with sterilized DBF63 cells. For one experiment, the means of three determinations are shown, and the error bars indicate the standard deviations.
solution (Fig. 2a). The degradation rate of 2,8-Cl2 DF in the soil slurry microcosm and the soil-free aqueous solution after a 7-day incubation were 90% and 88%, respectively, but that of 2,8-Cl2 DF in soil slurry inoculated with sterilized DBF63 cells, was only 10%. Although 76–86% of 2,8-Cl2 DF was degraded within 1 day, prolonged incubation did not significantly increase the degradation of 2,8-Cl2 DF. Because similar degradation patterns were observed in these two reaction systems, it was considered that the degradation activity of strain DBF63 towards 2,8-Cl2 DF was not influenced by the presence of this kind of soil under the condition employed in this study. On the other hand, the growing cells of strain DBF63 was also inoculated into the 2,3-Cl2 DD-contaminated model soil slurry system, and incubated at 30 °C. The degradation rate of 2,3-Cl2 DD in the soil slurry microcosm after a 7-day incubation was 40%, while control experiments (inoculated with the sterilized DBF63 cells) showed 22% of degradation (Fig. 2b). This indicated that strain DBF63 degraded 2,8-Cl2 DF more quickly and effectively than 2,3-Cl2 DD in soil (1 ppm each). Recently, we found that the degradation rates of both 2,8-Cl2 DF and 2,3-Cl2 DD (10 ppm each) by the resting cells of strain DBF63 in soil-free aqueous solution were approximately 80–90% (2,8-Cl2 DF) and 70–90% (2,3Cl2 DD), respectively (Habe et al., 2001a), showing that at 10 ppm, these two substrates were degraded similarly by strain DBF63. The transformation capability of strain DBF63 for CDD/CDF congeners at low concentration (1 ppm) may cause the differences in the degradation characteristics of strain DBF63 towards 2,8-Cl2 DF and 2,3-Cl2 DD.
Fig. 3. The degradation characteristics of strain DBF63 toward mono- and dichlorinated DFs and DDs. Black bars represent the rates of degradation of 2-ClDF, 2-ClDD, 2,8-Cl2 DF or 2,3Cl2 DD each at 1 ppm incubated with strain DBF63 for 5 days. White bars represent the degradation rates of respective substrates incubated with sterilized DBF63 cells for 5 days (control). For one experiment, the means of three determinations are shown, and the error bars indicate the standard deviations.
H. Habe et al. / Chemosphere 48 (2002) 201–207
205
trol experiments using sterilized DBF63 cells (Fig. 3, white bars). Similar to degradation of 2,8-Cl2 DF and 2,3-Cl2 DD in the soil slurry system, 2-CDF was degraded much more than 2-CDD, both of which were substituted with a chlorine at the 2-position of DF- and DD-skeleton, respectively. These results suggest that strain DBF63 is able to degrade CDF more efficiently than CDD in soil. Considering that the total concentration of PCDFs was higher than that of PCDDs in the dioxin-contaminated soils used in this study (Table 1), strain DBF63 seems to be applicable for remediation of such kinds of soil. On the other hand, mono-CDD/CDF congeners were degraded much more than di-CDD/CDF congeners
3.3. Degradation of mono- and dichlorinated DDs and DFs in soil slurry microcosm To investigate whether the difference in the degradation rates for 2,8-Cl2 DF and 2,3-Cl2 DD in soil was derived from the differences in the skeletons of DD and DF, we examined the degradation of 2-ClDF, 2-ClDD, 2,8-Cl2 DF and 2,3-Cl2 DD in soil slurry. During 5 days of incubation with strain DBF63 cells, 2-ClDF, 2-ClDD, 2,8-Cl2 DF and 2,3-Cl2 DD singly added to the soil slurry at a concentration of 1 ppm were decomposed by approximately 93%, 79%, 81% and 40%, respectively (Fig. 3, black bars), whereas approximately 38%, 40%, 12% and 12% of respective substrates were degraded in con-
Table 1 Degradation of PCDD and PCDF congeners by strain DBF63 in the contaminated soil collected at an incinerator site Congenersa
Controlb (pg/g-dry soil) Concentration
Single inoculation of DBF63 (pg/g-dry soil) TEQc
Concentration
Degradation amount (pg/g-dry soil)
Degradation rate (%)
TEQc
PCDDs 1,3,6,8-Cl4 DD 1,3,7,9-Cl4 DD 2,3,7,8-Cl4 DD Cl4 DDs
180 103 44 1025
0 0 44 –
155 90 40 870
0 0 40 –
25 13 4 155
13.9 12.6 9.0 15.1
1,2,3,7,8-Cl5 DD Cl5 DDs
765 7000
765 –
705 6300
705 –
60 700
7.8 10.0
1,2,3,4,7,8-Cl6 DD 1,2,3,6,7,8-Cl6 DD 1,2,3,7,8,9-Cl6 DD Cl6 DDs
2150 2800 2650 31 500
215 280 265 –
2000 2450 2300 27 500
200 245 230 –
150 350 350 4000
7.0 12.5 13.2 12.7
1,2,3,4,6,7,8-Cl7 DD Cl7 DDs
28 000 54 500
280 –
28 000 52 500
280 –
0 2000
0.0 3.7
Cl8 DD
45 000
4.5
46 000
4.6
–
PCDFs 1,2,7,8-Cl4 DF 2,3,7,8-Cl4 DF Cl4 DFs
450 265 28 500
0 26.5 –
395 225 25 000
0 22.5 –
55 40 3500
12.2 15.1 12.3
1,2,3,7,8-Cl5 DF 2,3,4,7,8-Cl5 DF Cl5 DFs
1800 4750 71 000
90 2375 –
1550 4350 63 500
78 2175 –
250 400 7500
13.9 8.4 10.6
1,2,3,4,7,8-Cl6 DF 1,2,3,6,7,8-Cl6 DF 2,3,4,6,7,8-Cl6 DF Cl6 DFs
9900 12 000 31 500 176 250
990 1200 3150 –
9150 11 000 29 500 160 000
915 1100 2950 –
750 1000 2000 16 250
7.6 8.3 6.5 9.2
1,2,3,4,7,8,9-Cl7 DF Cl7 DFs
21 500 283 500
215 –
21 500 275 000
215 –
0 8500
0.0 3.0
Cl8 DF
110 000
11
109 500
12
–
a
–
Clx DD and Clx DF represent the congeners other than the listed congeners whose authentic samples were available. b The soil inoculated autoclaved DBF63 cells was used as a control. c TEQ was calculated by multiplying TEF by each concentration and TEF was quoted from WHO/IPCS (1997).
–
206
H. Habe et al. / Chemosphere 48 (2002) 201–207
(Fig. 3), which is commonly observed in the degradation of PCDDs or PCDFs and other chlorinated compounds. In control experiments, the rather soluble mono-CDD/ CDF are much stronger decreased than the di-CDD/ CDF congeners. Because the granitic soil used in this study (both model and actually contaminated soils) were not sterilized prior to use and there were much nutrients like phosphate and nitrogen source in the soil slurry system, there exist the possibility that the indigenous bacteria in the soil might oxidize mono-CDD/CDF substrate, which is easier to be attacked by bacteria than di-CDD/CDF. 3.4. Preliminary examination for bioremediation of the actual dioxin-contaminated soil In the dioxin-contaminated soil at the incinerator site, PCDFs occupy 80% of the total concentration of dioxins, and among the tetra- to heptachlorinated congeners, the higher the concentration of either PCDD or PCDF, the higher the number of chlorine substitution. Among the PCDDs and PCDFs, there is just a small amount of the most toxic compound, 2,3,7,8-Cl4 DDs (Table 1). The soil slurry prepared with dioxin-contaminated soil collected at an incinerator site was incubated with strain DBF63 cells. During a 7-day incubation, the total PCDD and PCDF concentrations decreased from 725 to 715 ng/g-soil and from 11 to 10 ng TEQ/g-soil, respectively. The decrease of each congener is shown in Table 1. The lightly chlorinated congeners among the examined compounds such as Cl4 DDs and Cl4 DFs were decreased at a relatively high rate (12–15%). Dioxins with penta- to hexachlorines were also decreased by DBF63 though only slightly. On the other hand, the levels of Cl7 DD/Cl7 DF and Cl8 DD/Cl8 DF concentrations remained almost unchanged. Considering that Cl7 DD and Cl8 DF are hardly attacked by initial dioxygenases from bacterial strains because no positions of aromatic-rings are free for chlorine substitution, this result seems to be reasonable. In addition, strain DBF63 appears to attack preferably the dioxin congener whose initial concentration was relatively high (Table 1). In this experiment, approximately 10% of tetra- to hexa-CDD/CDF congeners were decreased by the augmentation of strain DBF63. The similar result has been obtained in a preliminary bioremediation experiment of dioxin-contaminated soil using a carbazole-degrader Pseudomonas sp. strain CA10 (Habe et al., 2001b). In order to perform better bioremediation of the actual dioxin-contaminated soils, more improved methods such as the repeated inoculation with dioxin-degrading bacteria, which can maintain a high population density of the cells having biotransformation ability, should be used. In addition, to avoid the absorption of dioxins by soil particles, the use of surfactant may also be useful.
Acknowledgements Part of this work was supported by a Grant-in-Aid for Scientific Research (no. 11794005) to T.O. from the ministry of Education, Science, Sports and Culture of Japan. References Ballerstedt, H., Kraus, A., Lechner, U., 1997. Reductive dehalogenation of 1,2,3,4-tetrachlorodibenzo-p-dioxin and its products by anaerobic mixed cultures from Saale River sediment. Environ. Sci. Technol. 31, 1749–1753. Beurskens, J.E.M., Toussaint, M., de Wolf, J., van der Steen, J.M.D., Slot, P.C., Commandeur, L.C.M., Parsons, J.R., 1995. Dehalogenation of chlorinated dioxins by an anaerobic microbial consortium from sediment. Environ. Toxicol. Chem. 14, 939–943. Fortnagel, P., Harms, H., Wittich, R.-M., Francke, W., Krohn, S., Meyer, H., 1989. Cleavage of dibenzofuran and dibenzodioxin ring systems by a Pseudomonas bacterium. Naturwissenschaften 76, 222–223. Habe, H., Chung, J.-S., Lee, J.-H., Kasuga, K., Yoshida, T., Nojiri, H., Omori, T., 2001a. Degradation of chlorinated dibenzofurans and dibenzo-p-dioxins by two types of bacteria having angular dioxygenases with different features. Appl. Environ. Microbiol. 67, 3610–3617. Habe, H., Ide, K., Yotsumoto, M., Tsuji, H., Hirano, H., Widada, J., Yoshida, T., Nojiri, H., Omori, T., 2001b. Preliminary examinations for applying a carbazoledegrader Pseudomonas sp. strain CA10 to dioxin-contaminated soil remediation. Appl. Microbiol. Biotechnol. 56, 788–795. Halden, R.U., Dwyer, D.F., 1997. Biodegradation of dioxinrelated compounds. Bioremediation J. 1, 11–25. Halden, R.U., Halden, B.G., Dwyer, D.F., 1999. Removal of dibenzofuran, dibenzo-p-dioxin, and 2-chlorodibenzo-pdioxin from soils inoculated with Sphingomonas sp. strain RW1. Appl. Environ. Microbiol. 65, 2246–2249. Kasuga, K., Nojiri, H., Yamane, H., Kodama, T., Omori, T., 1997. Cloning and characterization of the genes involved in the degradation of dibenzofuran by Terrabacter sp. strain DBF63. J. Ferment. Bioeng. 84, 387–399. Kasuga, K., Habe, H., Chung, J.-S., Yoshida, T., Nojiri, H., Yamane, H., Omori, T., 2001. Isolation and characterization of the genes encoding a novel oxygenase component of angular dioxygenase from the gram-positive dibenzofurandegrader Terrabacter sp. strain DBF63. Biochem. Biophys. Res. Commun. 283, 195–204. Megharaj, M., Wittich, R.-M., Blasco, R., Pieper, D.H., Timmis, K.N., 1997. Superior survival and degradation of dibenzo-p-dioxin and dibenzofuran in soil by soil-adapted Sphingomonas sp. strain RW1. Appl. Microbiol. Biotechnol. 48, 109–114. Monna, L., Omori, T., Kodama, T., 1993. Microbial degradation of dibenzofuran, fluorene, and dibenzo-p-dioxin by Staphylococcus auriculans DBF63. Appl. Environ. Microbiol. 59, 285–289. Strubel, V., Rast, H.G., Fietz, W., Knackmuss, H.-J., Engesser, K.H., 1989. Enrichment of dibenzofuran utilizing bacteria
H. Habe et al. / Chemosphere 48 (2002) 201–207 with high co-metabolic potential towards dibenzodioxin and other anellated aromatics. FEMS Microbiol. Lett. 58, 233– 238. Wittich, R.-M., Wilkes, H., Sinnwell, V., Francke, W., Fortnagel, P., 1992. Metabolism of dibenzo-p-dioxin by Sphingo-
207
monas sp. strain RW1. Appl. Environ. Microbiol. 58, 1005– 1010. Wittich, R.-M., 1998. Degradation of dioxin-like compounds by Microorganisms. Appl. Microbiol. Biotechnol. 49, 489– 499.