Degradation of Chlorinated Biphenyl, Dibenzofuran, and Dibenzo-P-Dioxin by Marine Bacteria That Degrade Biphenyl, Carbazole, Or Dibenzofuran

Biosci. Biotechnol. Biochem., 67 (5), 1121–1125, 2003

Note Degradation of Chlorinated Biphenyl, Dibenzofuran, and Dibenzo-p-dioxin by Marine Bacteria That Degrade Biphenyl, Carbazole, or Dibenzofuran

Hiroyuki FUSE,† Osamu TAKIMURA,KatsujiMURAKAMI, Hiroyuki INOUE, andYukihoYAMAOKA

Institute for Marine Resources and Environment, National Institute of Advanced Industrial Science and Technology, 2-2-2 Hirosuehiro, Kure, Hiroshima 737-0197, Japan

Received August 19, 2002; Accepted December 20, 2002

Marine bacterial strains (BP-PH, CAR-SF, and DBF- tetrachlorodibenzo-p-dioxin.7) Most of these bacteria MAK) were isolated using biphenyl, carbazole (CAR), have been isolated from soil and freshwater. By con- or dibenzofuran (DF) respectively as substrates for trast there have been few reports of marine bacteria growth. Their 16S ribosomal DNA sequences showed that can mineralize BP, CAR, or DF, or of the degra- that the species closest to strain BP-PH, strain CAR-SF, dation of chlorinated BPs, DFs, or DDs by marine and strain DBF-MAK are Alteromonas macleodii bacteria, though only some marine Cycloclasticus (96.3% identity), Neptunomonas naphthovorans spp. are able to grow on BP.8,9) We have therefore set (93.1% identity), and Cycloclasticus pugetii (97.3% out to isolate bacteria from marine samples that can identity), respectively. The metabolites produced use BP, CAR, or DF as a carbon source and have suggested that strain CAR-SF degrades CAR via dioxy- investigated the degradation of chlorinated BPs, genation in the angular position and by the meta- DFs, and DDs by the isolates we obtained. cleavage pathway, and that strain DBF-MAK degrades The basal medium for isolating and culturing

DF via both lateral and angular dioxygenation. Poly- microorganisms contained 100 mg NH4 NO3,10mg chlorinated biphenyl (KC-300) and 2,3-dichlorodiben- KH2PO4,2.5mgFeEDTA,5mgyeastextract,and zo-p-dioxin were partially degraded by strain BP-PH 0.4–2 mg growth substrate in 1 l ˆltered sea water and strain DBF-MAK, while 2,7-dichlorodibenzo-p- (pH 8.1). Microorganisms were grown at 21–249C. dioxin and 2,4,8-trichlorodibenzofuran remained vir- Three isolates capable of assimilating BP, CAR, or tually unchanged. DF were obtained from seawater samples taken near the shore. They were named BP-PH, CAR-SF, and Key words: carbazole; dibenzofuran; biphenyl; diben- DBF-MAK respectively. Their 16S rDNA sequences zo-p-dioxin; marine bacteria (1529, 1537, and 1536 bases, respectively) were analyzed by NCIMB Japan (Shimizu, Japan) and Polychlorinated dibenzo-p-dioxins, dibenzofu- analyzed phylogenetically using CLUSTAL W rans, and biphenyls are widespread toxic pollutants. (DDBJ, Mishima, Japan) (Fig. 1). The 16S rDNA They are also found in marine sediments and marine sequence data of strain BP-PH, strain CAR-SF, and organisms.1,2) Some of these chlorinated compounds strain DBF-MAK have been deposited in the DDBJ are degraded by biphenyl (BP), carbazole (CAR), nucleotide sequence databases with the accession or dibenzofuran (DF)-mineralizing bacteria. BP- number AB086226, AB086227, and AB086228, assimilating Beijerinkia sp. (later assigned to the respectively. Based on a BLAST search of DDBJ, the genus Sphingomonas)andBurkholderia sp. JB1 species closest to strain BP-PH, strain CAR-SF, and oxidized several mono- to trichlorinated dibenzo-p- strain DBF-MAK are Alteromonas macleodii (96.3z dioxins (DDs) and DFs.3,4) The DF- and DD-miner- identity), Neptunomonas naphthovorans (93.1z alizing bacterium Sphingomonas wittichii RW1 oxi- identity) , and Cycloclasticus pugetii (97.3z identi- dized several mono- and dichlorinated DDs and ty), respectively. All three of these species are marine DFs.5) DF-utilizing Terrabacter sp. DBF63 and CAR- bacteria. N. naphthovorans is able to grow on utilizing Pseudomonas resinovorans CA10 also naphthalene but not on biphenyl, and C. pugetii is degraded several mono- to trichlorinated DDs and able to use biphenyl, naphthalene, anthracene, DFs.6) DF-utilizing Sphingomonas sp. HH69 de- phenanthrene, and toluene as carbon sources,8,10) pleted 2,3,7,8-tetrachlorodibenzofuran and 2,3,7,8- though A. macleodii is not able to grow on benzo-

† To whom correspondence should be addressed. Fax: ＋81-823-73-3284; Tel: ＋81-823-72-1936; E-mail: h-fuse＠aist.go.jp Abbreviations: BP, biphenyl; CAR, carbazol; DF, dibenzofuran; GC-MS, gas chromatograph-mass spectrometry; 2,3-DCDD, 2,3- dichlorodibenzo-p-dioxin; 2,7-DCDD, 2,7-dichlorodibenzo-p-dioxin; 2,4,8-TCDF, 2,4,8-trichlorodibenzofuran 1122 H. FUSE et al.

Fig. 1. Phylogenetic Relationship of Strain BP-PH, CAR-SF, and DBF-MAK Based on 16S rDNA Sequences. The scale bar corresponds to an approximately 0.01 change per nucleotide position. Bootstrap values from 1000 replicates are also shown near the clades.

ate.11) Strain BP-PH used only BP and strain CAR- degrades CAR via dioxygenation in the angular SF only CAR, whereas strain DBF-MAK used BP position and the meta-cleavage pathway, as do P. and DF in the three substrates. resinovorans CA10, Pseudomonas sp. LD2, and Each isolate was cultured for ˆve days in two Pseudomonas stutzeri OM1.13–15) though detection of 500-ml ‰asks containing 100 ml of basal media with meta-cleavage enzyme activity for 2,3-dihydrox- 0.2z of the substrate used for its isolation. Super- ybiphenyl or further identiˆcation of metabolites is natants were extracted with ethyl acetate at pH 2 and required for drawing this conclusion. Salicylic acid concentrated after drying with anhydrous sodium produced by strain DBF-MAK when grown on DF sulfate. Samples were derivatized by methylation was also identiˆed by comparison with an authentic with diazomethane in a microapparatus (GL Science, sample. One methylated metabolite has a mass spec- Tokyo, Japan) or by silylation with TMSI-H (hex- trum with fragments at m Wz (z) 220(6), 147(100), amethyldisilazone:trimethylchlorosilane:pyridine 121(15), 120(71), and 92(54), which is virtually identi- ＝2:1:10) (GL Science, Tokyo, Japan), before analy- cal to that of (chroman-4-on-2-yl)-acetic acid methyl sis by gas chromatography-mass spectrometry (GC- ester16) and another has a mass spectrum with frag- MS). A QP-5000 (Shimadzu, Kyoto, Japan) with a ments at m Wz (z) 260 (6), 202 (13), 201(100), 30 m DB-5 capillary column (J&W Scientiˆc, CA, 186(43), 158(19), 145(8), 102(14), and 76(18), which USA) was used for GC-MS. Benzoic acid was identi- is virtually identical to that of 4-(3?-methoxy- 2?-ben- ˆed by comparison with an authentic sample in cul- zofuranyl)-2-oxo-3-butenoic acid methyl ester.17,18) tures of strain BP-PH grown on BP. The metabolite (Chroman-4-on-2-yl)-acetic acid would be produced and the yellow color of the culture at the ˆrst stage of by decarboxylation of 3-(chroman-4-on-2-yl)-pyruvic growth on BP indicates that strain BP-PH degrade acid, which would be made by inter molecular BP via the same oxidative route as described in many cyclization of 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)- other bacteria.12) A methylated metabolite produced hexa-2,4-dienoic acid.16) These facts suggest that by strain CAR-SF when grown on CAR has a mass strain DBF-MAK is able to degrade DF via lateral spectrum with fragments at m Wz (z) 261(4), 147(9), dioxygenation and also via angular dioxygenation. 146(100), 91(6), and 77(7). This is virtually identical DF-utilizing Terrabacter sp. DBF63, Sphingomonas to that of the methylated derivative of 2-hydroxy-6- sp. HH69, and S. wittichii RW1, as well as CAR- oxo-6-(2?-aminophenyl)hexa-2,4-dienoic acid tenta- utilizing P. resinovorans CA10 and biphenyl-utilizing tively identiˆed by Ouchiyama et al.13) This result and Burkholderia JB1, degrade DF via angular dioxygen- also the yellow color of the culture at the ˆrst stage of ation.4,6,16) On the other hand, biphenyl-utilizing growth on CAR indicates that strain CAR-SF Ralstonia sp. SBUG 290, naphthalene-utilizing Pseu- Degradation of Dioxin-like Compounds by Marine Bacteria 1123

Table 1. Degradation of 2,3-DCDD, 2,7-DCDD, and 2,4,8- Table 2. Degradation of PCB Congeners by Isolated Strains TCDF by Isolated Strains Degradation (z) Degradation (z) PCB congener Substrate 2,2? 67 2 6 BP-PH CAR-SF DBF-MAK 2,3? 100 2 71 2,4? 100 3 59 2,3-DCDD 90 0 52 2,2?,6 0 1 1 2,7-DCDD 1 0 4 2,4,6 0 0 1 2,4,8-TCDF 6 2 3 2,2?,5 0 2 1 2,2?,4 12 2 2 Data values are averages of two experiments. All strains were pre-grown with the substrates used for their isolation. Resting cells were incubated for 2,3?,6 97 2 3 26 hours with one ppm of chlorinated compounds. 3,4; 3,4; 2,3,6 25 3 6 2,2?,3 82 2 3 4,4? 98 2 69 2,4?,6 16 2 0 domonas spp., and ‰uorene-utilizing Burkholderia 2,3?,5; 2,4,5 77 2 5 cepacia F297 degrade DF to 4-(3?-methoxy-2?-ben- 1,2?,4; 2,2?,5,6? 49 2 5 2,4?,5 48 1 1 zofuranyl)-2-oxo-3-butenoic acid via lateral dioxy- 2,3,3?;2,4,4?;2,2?,3,6; 2,2?,4,6? 98 1 13 genation, and the product turns the medium bright 2,3,4; 2?,3,4 97 2 11 orange.17–19) When strain DBF-MAK was grown on 2,2?,3,6? 80 0 DF, the medium also turned orange and the color 2,2?,5,5? 99 2 24 never faded, suggesting that the responsible product 3,3?,5 0 1 0 3,3?,5; 2,2?,4,5? 19 2 0 was terminal. There have been few reports on wild- 2,2?,4,5 0 1 0 type bacteria which are able to oxidize an aromatic 3,4?,5; 2,2?,3,5?;2,2?,4,4?; 2,3,5,6 0 1 0 compound both by lateral and angular dioxygena- 2,3,4,6 0 1 0 tion. The DF-WDD-degrading bacterium Rhodococ- 2,3,3?,6; 2,4,4?,6; 2,2?,3,6,6? 53 5 cus opacus SAO101 also oxidizes DD by lateral and 3,4,5; 2,2?,3,4? 50 0 2,2?,3,4 5 1 0 angular dioxygenation, though no ring cleavage 3,3?,4; 2,2?,3,3?;2,3?,4?,6 0 2 0 product was detected except for the one supposed to 2,3,4?,6 16 1 0 come from the angular dioxygenation.20) 3,4,4? 11 0 We next investigated the degradation of 2,3- 2,3,3?,5; 2,2?,3,5?,6; 2,2?,4,4?,6 19 2 18 dichlorodibenzo-p-dioxin (2,3-DCDD), 2,7- 2,2?,3,5,6 2 1 0 2,3?4,5; 2,2?,3?,4,6 0 0 0 dichlorodibenzo- -dioxin (2,7-DCDD), 2,4,8- p 2,3,4?,5; 2,2?,3,4,6 33 1 1 trichlorodibenzofuran (2,4,8-TCDF), and PCB 2,3,4,5; 2,2?,3,4?,6 53 1 0 (KC-300) by resting cells of the isolated strains. 2,4,8- 2,3?,4?,5; 2,4,4?,5; 2?,3,4,5; 2,2?,4,4?,6 2 0 0 TCDF was purchased from Aldrich (WI, USA) and 2,3?,4,4? 22 0 the other chlorinated compounds were from GL 2,3,3?,4; 2,2?,3,4,6? 71 0 2,3,3?,4?;2,3?,4,5?,6 28 3 2 Science (Tokyo, Japan). Each strain was cultivated in 2,3,4,4? 78 2 1 a 500-ml ‰ask containing 100 ml of the basal medi- 2,2?,3,5,5?;2,2?,4,4?,6,6? 42 2 um, with 0.2z of the substrate used for its isolation. 2,2?,3,4?,5; 2,2?,4,5,5?; 2,3,3?,5?,6; 01 1 StrainBP-PHandstrainCAR-SFwereculturedfor 2,2?,3,4?,6,6?;2,2?,3,5,6? ˆve days and strain DBF-MAK for eight days. Cells 2,2?,3,3?,5; 2,2?,4,4?,5; 2,2?,3,3?,6,6? 01 0 2,2?,3,4,5; 2,2?,3?,4,5; 2,3,3?,4,6; 01 0 were harvested by centrifugation, washed twice with 2,3?,4,4?,6; 2?,3,4,5,6? ˆltered seawater, and suspended in 14 ml of ˆltered 2,2?,3,4,5? 01 0 seawater. Ten ml of acetone containing 0.2z KC-300 2,2?,3,4,4?; 2,3,4,5,6; 2,3,4?,5,6 0 2 0 or 0.01z of the other chlorinated compounds was 2,3,3?4?,6; 2,3,4,4?,6 0 2 0 added to one ml of each cell suspension and the cells 2,2?,3,3?,4 0 1 1 3,3?,4,4?; 2,3,3?;2,2?,3,3?,5,6?; 02 0 incubated for 26 h at 24 C on a shaker. Control cells 9 2,2?,3,5,5?,6; 2,2?,4,4?,5,6? were heat-inactivated (1109C, 5 min). The extraction 2,2?,3,4?,5,6; 2,2?,3,4?,5?,6 0 1 0 procedures used were those of Bedard et al.21) PCB 2,3,3?,4,4?;2,2?,3,4?5,5? 02 0 congeners were separated on a 100-m (0.25-mm 2,2?,4,4?,5,5?;2,3?,4,4?,5?,6 0 1 1 2,2?,3,4,4?,5? 00 1 inside diameter) Chrompack CP-Sil 5WC18 CB for 63 PCB capillary column using a Ni electron capture Data values are averages of two experiments. All strains were pre-grown detector, and identiˆed by their relative retention with the substrates used for their isolation. Resting cells were incubated for times as indicated by the Varian-Chrompack, using 26 hours with 20 ppm of KC-300. 12 congeners as standards. The other chlorinated compounds were also separated on that column. The percentage transformation of the chlorinated com- pounds was calculated by comparing the area of each peak with the control. Strain CAR-SF hardly degrad- 1124 H. FUSE et al. ed any of the chlorinated compounds (Tables 1, 2). 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