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Biotransformation of dibenzothiophene to dibenzothiophene sulfone by Pseudomonas putida

MELANIE R. MORMILE AND RONALD M. ATLAS' Department of Biology, University of Louisville, Louisville, KY 40292, U.S.A.

Received September 18, 1988 Accepted December 9, 1988

MORMILE, M. R., and ATLAS, R. M. 1989. Biotransformation of dibenzothiophene to dibenzothiophene sulfone by Pseudo- monas putida. Can. J . Microbiol. 35: 603-605.

A strain of Pseudomonas putida was isolated that transforms dibenzothiophene (DBT) to DBT sulfone (DBT-5-dioxide) via DBT-5-oxide. It also degrades DBT to 3-hydroxy-2-formyl benzothiophene via an alternate and previously described path- way, and to an unidentified red crystalline compound. Neither DBT sulfone nor 3-hydroxy-2-formyl benzothiophene are further degraded by this organism.

Key words: dibenzothiophene, microbial degradation, Pseudomonas, dibenzothiophene sulfone, sulfur metabolism.

MORMILE, M. R., et ATLAS, R. M. 1989. Biotransformation of dibenzothiophene to dibenzothiophene sulfone by Pseudo- monas putida. Can. J . Microbiol. 35 : 603 -605.

Nous avons is016 une souche de Pseudomoms putida qui transforme le dibenzothiophene (DBT) en DBT sulfone (DBT-5-dioxyde) via DBT-5-oxyde. Cette souche dCgrade aussi le DBT en 3-hydroxyl-2-formyl benzothiophene par un sentier mktabolique alterne dkcrit antkrieurement et un compost5 crystallin rouge non-identifik. Cet organisme ne dt5grade ni le DBT sulfone ni le 3-hydroxy-2-formyl benzothiophene en d'autres composCs.

Mots clis : dibenzothioph&ne, dkgradation microbieme, Pseudomonas, dibenzothiophkne sulfone, mktabolisme du soufre. [Traduit par la revue]

Dibenzothiophene (DBT) has been shown to be biodegraded aerobically to 3-hydroxy-2-formyl benzothiophene by Pseudo- monas (Hou and Laskins 1976; Kodama 1977a, 1977b; Kodama et al. 1970, 1973; Monticello et al. 1985; Sagardia et al. 1975), Acinetobacter (K. A. Malik and D. Laus. 1976. Fifth International Fermentation Symposium, June 28 - July 3, 1976, Berlin. Verlag Versuchs- und Lehtranstalt fiir Spiritufabrikation und Fermentationtechnologie, Berlin. Abstr. 23.03, p. 421), and Beijerinckia (Laborde and Gibson 1977). Kodama et al. (1973) also reported that Pseudomonas convert DBT to DBT-5-oxide by an alternate pathway. Recently, DBT degradation via the DBT-5-oxide pathway to o,ol-biphenol (D. Dutt, P. Saadi, S. Ciatti, and S. Krawiec. 1988. Annu. Meet. Am. Soc. Microbiol. Abstr. K112, p. 225) has been reported.

In this study we examined metabolism of DBT by a strain of Pseudomoms putida isolated from Ohio River sediment at Louisville, KY. The organism was identified according to the criteria outlined by Stolp and Godkari (1981). The isolated strain of P. putida contains a single 48.5 kilobase (kb) plas- mid, loss of which results in the inability to attack DBT (A. Bej , personal communication). Pseudomoms putida was inoculated into a medium consisting of 4 g NaH2P04, 4 g K2HP04, 2 g (NH4)$04, 0.2 g MgS04, 1 mg CaC12 . 2H20, 1 mg FeSO, . 7H20, and 0.25 g yeast extract per litre of dis- tilled water (pH 7.2). DBT was added as a 10% solution dis- solved in dirnethylformamide, after the medium had been sterilized, to achieve a final DBT concentration of 0.05%. Approximately lo6 cells of P. putida were used to inoculate

'Author to whom correspondence should be addressed.

cultures. The cultures were incubated at 28°C with continuous shaking at 100 rpm for 1-16 days. Disappearance of DBT was measured by extracting the cultures with methylene chlo- ride and quantitating the residual DBT by gas -liquid chroma- tography using a 25 m ultra-2 fused silica column (Hewlett Packard) and a Hewlett Packard 5840 gas chromatograph with flame ionization detector. Operational parameters were as follows: injector, 240°C; detector, 320°C; column, 100°C for 5 min, then 5"CImin to 250°C; helium as carrier and makeup gas.

Metabolic products were identified by using TLC, light absorbance spectral characterization (UV - vis absorbance), energy dispersive x-ray analysis (EDAX), GLC, and gas- liquid chromatography - mass spectrometry (GC -MS). For these analyses, the medium in replicate flasks was acidified with concentrated HC1 and extracted with diethyl ether. A portion of the ether extract was evaporated to dryness and dis- solved in acetone. The compounds in acetone were purified by silica gel column chromatography by eluting with chloro- form - acetone (1 : 1) and acetone - water (1 : 1) (Monticello et al. 1985). Fractions collected from the silica gel column were analyzed by UV spectroscopy and by TLC. Portions of the original ether extracts were also concentrated and applied to glass TLC plates precoated with cellulose (0.25 mrn thick- ness). The cellulose TLC plates were developed by using ethanol - water -NH40H (20: 15: 1, by volume). Products were detected visually and by viewing with short wavelength UV light. Products were scraped from TLC plates and dis- solved in butyl acetone. These extracts were subjected to GLC and GC-MS analyses. The original ether extracts were also subjected to GLC and GC -MS analyses. Besides extracting

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604 CAN. J . MICROBIOL. VOL. 35. 1989

dibenzothiophene (DBT)

trans-4-[2-(3-hydroxy)-benzothiophene]-2-0~0-3-butenoic ocid dibanzothiophans-5-oxide

3-hydroxy-2-formyl benzothiophene dibenzothiophene sulfone

FIG. 1. The two pathways of DBT degradation of Pseudomonas putida showing the identified biodegradation products that accumulate in the culture medium.

the media, cells and products were pelleted by centrifugation at 5000 x g for 30 min. The pellet was extracted with acetone and this extract was further analyzed by TLC, UV -vis absor- bance, EDAX, GLC, and GC-MS.

Pseudomonas putida was found to produce products pre- viously shown to be formed by the biodegradation of DBT, including 3-hydroxy-2-formyl benzothiophene, trans- 4-[2-(3-hydroxy)-benzothiophene]-2-oxo-3-butenoic acid, the hemiacetal form of 4-[2-(3-hydroxy)-benzothiophenel-2-0x0- 3-butenoic, and 3-0x0-[3 '-hydroxybenzothiophene-(2)-methy- lene-dihydrothionaphthene, which were identified by their Rf values after TLC and their absorbance characteristics. The identity of 3-hydroxy-2-formyl benzothiophene was confirmed by GC-MS analyses. In addition to these DBT biodegrada- tion products, all of which were previously reported by Kodama et al. (1973), GC - MS analyses showed that the ether extract contained DBT-5-oxide and appreciable amounts of DBT sulfone (DBT-5-dioxide). These products, which had identical retention times and mass spectral patterns as known standards, were not found in extracts from sterile controls incubated for identical time periods that were extracted and treated in an identical manner. Although dibenzothiophene-5- oxide has previously been reported as a microbial DBT bio- transformation product (Kodama et al. 1970), DBT sulfone has not been previously identified as a product of microbial DBT metabolism, but has been found as an oxidation product of DBT in rat liver microsomes (Vignier et al. 1985). When P. putida was grown in media containing yeast extract and DBT-5-oxide (Aldrich) (absorbance maximum, 262 nm), UV -vis absorbance measurement scans from 220 to 500 nm showed the production of DBT sulfone (absorbance maxi- mum, 271 nm) and no other products. Thus, DBT sulfone appears to be the end product of this DBT transformation path-

way by P. putida. Based upon the identification of these products, it appears that this strain of P. putida has two differ- ent pathways for the metabolism of DBT, one that produces 3-hydroxy-2-formyl benzothiophene as the end product and the other that produces the end product DBT sulfone (Fig. 1).

With regard to the rate of DBT metabolism and the sequence of product formation, we found that DBT was rapidly degraded during the first 2 days and that disappearance of DBT ceased for the next 4 days after which DBT disappear- ance was again observed (Fig. 2). During the initial 2-day period of metabolism only 3-hydroxy-2-formyl benzothio- phene and intermediates in this pathway could be detected. UV-vis absorbance scans from 220 to 500 nm showed that both trans4-[2-(3-hydroxy)-benzothiophene]-2-0~0-3-butenoic acid (absorbance maximum, 480 nm) and 3-hydroxy-2-formyl benzothiophene (absorbance maximum, 390 nm) accumulated during this time and that no additional accumulation of these products occurred after 2 days. DBT sulfone was not detected by GLC analyses until after 6 days. Based upon these observa- tions it appears that P. putida first metabolizes DBT via the 3-hydroxy-2-formyl benzothiophene pathway and then later transforms DBT via the alternate DBT sulfone pathway.

Besides the products shown in these pathways, we observed a red crystalline product when the cells and products were pelleted by centrifugation. Pseudomonas putida is a cream- colored organism and the red-colored product was observed only when this bacterium was grown in a medium containing yeast extract and DBT. EDAX analysis of the crystals only showed a major sulfur peak. Even after repeated purification by TLC, using a solvent system (ethanol - water -NH40H, 20:15:1) in which the red product migrates well (Rf = 0.3) and DBT does not migrate (Rf = 0.0), GC-MS analysis identified DBT as the product. Clearly the red color and the

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NOTES 605

Time (days)

FIG. 2. Time course for the biodegradation of dibenzothiophene by cultures of P. putida as determined by gas liquid chromatography. 3-Hydroxy-2-formyl benzothiophene was produced between 0 and 2 days. Dibenzothiophene sulfone was produced between 6 and 12 days.

TLC migration characteristics preclude the possibility that this product is DBT but its identity remains unknown. It appears that this red product is thermally unstable and reverts to DBT during the mass spectral analysis. Dibenzothiophene with a charged methyl sulfonium ion might be thermally unstable and red (H. Hurst, personal communication). Further analyses are being performed to try to identify this compound. It is not clear whether this product is an intermediate in either of the two DBT degradation pathways of P. putida or whether it is formed by yet another pathway.

Acknowledgement Mass spectral analyses were kindly performed by Dr.

Harrell Hurst.

Hou, C. T., and LASKIN, A. I. 1976. Microbial conversion of dibenzothiophene. Dev. Ind. Microbiol. 17: 35 1 - 361.

KODAMA, K. 1977a. Induction of dibenzothiophene oxidation by Pseudomonas jianii. Agric. Biol. Chem. 41: 1193 - 1196.

1977b. Co-metabolism of dibenzothiophene by Pseudo- monas jianii. Agric. Biol. Chem. 41: 1305 - 1306.

KODAMA, K., NAKATANI, S., UMEHARA, K., SHIMIZU, K., MINODA, Y., and YAMADA, K. 1970. Microbial conversion of petro-sulfur compounds. III. Isolation and identification of products from dibenzothiophene. Agric. Biol. Chem. 34: 1320 - 1324.

KODAMA, K., UMEHARA, K., SHIMIZU, K., NAKATINI, S., MINODA, Y., and YAMADA, K. 1973. Identification of products from dibenzo- thiophene and its proposed oxidation pathway. Agric. Biol. Chem. 37: 45-50.

LABORDE, A. L., and GIBSON, D. T. 1977. Metabolism of dibenzo- thiophene by a Beijerinckia species. Appl. Environ. Microbiol. 34: 783 -790.

MONTICELLO, D. J., BAKKER, D., and FINNERTY, W. R. 1985. Plasmid-mediated degradation of dibenzothiophene by Pseudo- moms species. Appl. Environ. Microbiol. 49: 756-760.

SAGARDIA, F., RIGAU, J. J . , MARTINEZ-LAHOZ, A., FUENTES, F., LOPEZ, C., and FWRES, W. 1975. Degradation of benzothiophene and related compounds by a soil Pseudomoms in an oil-aqueous environment. Appl. Microbiol. 29: 722-725.

STOLP, H., and GODKARI, D. 1981. Nonpathogenic members of the genus Pseudomonas. In The prokaryotes. Edited by M. P. Starr, H. Stolp, H. G. Truper, A. Ballows, and H. G. Schlegel. Springer-Verlag, Berlin. pp. 719 - 743.

VIGNIER, V., BERTHOU, F., DREANO, Y., and FLOCH, H. H. 1985. Dibenzothiophene sulphoxidation: a new and fast high-perform- ance liquid chromatographic assay of mixed-function oxidation. Xenobiotica, 15: 991 -999.

Erratum: Why can't a cell grow infinitely fast?ly2

ARTHUR L. KOCH Biology Department, Indiana University, Bloomington, IN 47405, U. S. A.

(Ref. Can. J. Microbiol. 34: 421 -426. 1988.)

There is an inconsistency between eq. 1 and eq. 4 on p. 422. I should have chosen the proportionality constant in eq. 1 as Ilk instead of k. Then k would have had the correct units for a first order rate constant for protein synthesis per unit amount of ribosome. This usage would be consistent with my earlier publications. Then eq. 1 would read R/P = plk and eq. 4 would be correct.

'Received at NRC February 15, 1989. 2Key words: cell growth, ribosomal synthesis, protein synthesis.

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