Biosci. Biotechnol. Biochem., 71 (1), 51–57, 2007 Biodegradation of Bisphenol A and Related Compounds by Sphingomonas sp. Strain BP-7 Isolated from Seawater y Kiyofumi SAKAI, Hayato YAMANAKA, Kunihiko MORIYOSHI, Takashi OHMOTO, and Tatsuhiko OHE Department of Biochemistry, Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan Received June 23, 2006; Accepted October 12, 2006; Online Publication, January 7, 2007 [doi:10.1271/bbb.60351] A bacterium capable of assimilating 2,2-bis(4-hydroxy- industry into rivers and seas, and leach out from the phenyl)propane (bisphenol A), strain BP-7, was isolated resins used in plastic wares. Microbial degradation is from offshore seawater samples on a medium containing expected to play a major role in the removal of BPA bisphenol A as sole source of carbon and energy, and from the environment. Rapid and extensive breakdown identified as Sphingomonas sp. strain BP-7. Other of BPA has been demonstrated in a variety of laboratory strains, Pseudomonas sp. strain BP-14, Pseudomonas biodegradation tests.5) sp. strain BP-15, and strain no. 24A, were also isolated Biodegradation of BPA by microorganisms has been from bisphenol A-enrichment culture of the seawater. reported by some workers. Lobos et al.6) have reported These strains did not degrade bisphenol A, but accel- that a BPA-degrading microorganism, strain MV1, was erated the degradation of bisphenol A by Sphingomonas isolated from sludge taken from the wastewater treat- sp. strain BP-7. A mixed culture of Sphingomonas sp. ment plant at a plastics manufacturing facility. Spivack strain BP-7 and Pseudomonas sp. strain BP-14 showed et al.7) have shown that BPA was metabolized by strain complete degradation of 100 ppm bisphenol A within MV1 via a novel pathway involving oxidative skeletal 7 d in SSB-YE medium, while Sphingomonas sp. strain rearrangement. Ike et al.8) have reported that Sphingo- BP-7 alone took about 40 d for complete consumption of monas paucimobilis strain FJ-4 was isolated from an bisphenol A accompanied by accumulation of 4-hydroxy- activated sludge taken from the wastewater treatment acetophenone. On a nutritional supplementary medium, plant at an epoxy resin manufacturing facility. Recent Sphingomonas sp. strain BP-7 completely degraded studies5,9) indicate that BPA is degraded rapidly in bisphenol A and 4-hydroxyacetophenone within 20 h. surface water and sediments taken from a wide variety The strain degraded a variety of bisphenols, such as 1,1- of geographies, suggesting that microorganisms with bis(4-hydroxyphenyl)ethane, 2,2-bis(4-hydroxy-3-meth- the capability to degrade BPA are ubiquitous in the ylphenyl)propane, 2,2-bis(4-hydroxyphenyl)butane, and environment. Environmental pollutants such as endo- 1,1-bis(4-hydroxyphenyl)cyclohexane, and hydroxy aro- crine-disrupting chemicals and chlorinated compounds matic compounds such as 4-hydroxyacetophenone, 4- in soil and river water flow into the sea as the final hydroxybenzoic acid, catechol, protocatechuic acid, and contaminated zone. Hence clarification of the biodegra- hydroquinone. The strain did not degrade bis(4-hydroxy- dation mechanism of BPA in seawater is very important. phenyl)methane, bis(4-hydroxyphenyl)sulfone, or bis- While BPA-degrading microorganisms and the fate of (4-hydroxyphenyl)sulfide. BPA in acclimated activated sludge,5–8) river water,10–14) and soil15,16) have been reported, the biodegradation of Key words: biodegradation; bisphenol A; Sphingomonas BPA in seawater is not well understood. sp. strain BP-7; 4-hydroxyacetophenone; In this paper, we describe the isolation of BPA- symbiosis degrading microorganisms from several offshore sea- water samples, the symbiotic degradation of BPA, and Bisphenol A (BPA), 2,2-bis(4-hydroxyphenyl)pro- the degradability of a variety of bisphenols. pane, is used primarily in the production of polycar- bonate resins and epoxy resins. It is an endocrine- Materials and Methods disrupting chemical that can interfere with mammalian development by mimicking the action of estrogen.1–4) Materials. BPA and 4-hydroxyacetophenone (4-HAP) Trace levels of BPA are discharged by the chemical were obtained from Nacalai Tesque (Kyoto, Japan). y To whom correspondence should be addressed. Fax: +81-6-6963-8079; E-mail: [email protected] Abbreviations: BPA, bisphenol A; 4-HAP, 4-hydroxyacetophenone; 4-HBA, 4-hydroxybenzoic acid 52 K. SAKAI et al. Peptone and yeast extract were obtained from Nihon ng/ml pyrroloquinoline quinone, and 0.1% vitamin Pharmaceutical (Tokyo). Beef extract was obtained mixture solution. The vitamin mixture solution (1,000 from Becton and Dickinson (Franklin Lakes, NJ, ml) contained biotin 1 mg, calcium pantothenate 200 mg, USA). Membrane filters (diameter 47 mm, pore size folic acid 1 mg, inositol 1000 mg, niacin 200 mg, p- 0.45 mm), used for filtration of seawater, were obtained aminobenzoic acid 100 mg, pyridoxine-HCl 200 mg, from Nihon Millipore (Tokyo). Pyrroloquinoline qui- riboflavin 100 mg, thiamine-HCl 200 mg, and choline none was obtained from Kanto Chemical (Tokyo). A 500 mg. Sphingomonas sp. strain BP-7 was cultivated at variety of bisphenols were purchased from Tokyo 27 C for 4 d with reciprocal shaking. Chemical Industry (Tokyo). All other chemicals were of the highest purity commercially available. Biodegradation assay. Biodegradation experiments were done with a OM3001 coulometer (Ohkura, Tokyo), Microorganisms and culture conditions. The organ- described in the modified MITI test,17) which is based on isms used in this study were Sphingomonas sp. strain an electrochemical process to measure the oxygen BP-7 and Pseudomonas sp. strain BP-14 isolated from demand of microorganisms. Biodegradation by seawater seawater (offshore of Matsuyama, Seto Inland Sea, was done in 500-ml flasks containing 300 ml of seawater Japan). They were grown in SSB-YE medium that and 30 mg of BPA at 25 C with stirring. Biodegradation contained 0.02% K2HPO4, 0.1% NH4NO3, 0.001% by activated sludge from the municipal sewage treat- FeCl2.nH2O, 0.02% yeast extract, and 0.01% (0.437 ment plant was done in 300 ml of salt solution, described mM) BPA in the original seawater, pH 7.3. SSB-0.1NB in the modified MITI test, and 30 mg of BPA at 25 C medium contained 0.1% peptone and 0.05% beef extract with stirring, and each flask was inoculated with 1.0 ml instead of the yeast extract in the SSB-YE medium. For of activated sludge. solid media, agar slants and plates, the media contained 0.02% BPA and 1.5% agar. Each strain was maintained Analyses of culture supernatants. BPA, degradation on an SSB-0.1NB slant. Cultures were prepared by intermediates, and various aromatic compounds were transferring cells from a SSB-0.1NB slant to 50 ml of analyzed with a LC-VP high performance liquid chro- SSB-YE medium or SSB-0.1NB medium in 500-ml matography (HPLC) system consisting of an LC-10 shaking flasks. Cultures were incubated at 27 C with ADvp pump and an SPD-M10Avp photodiode array reciprocal shaking. (Shimadzu, Kyoto, Japan) equipped with a Quicksorb column (2:1 Â 100 mm, Chemco, Osaka, Japan). Ana- Enrichment, isolation, and symbiotic cultivation. lytical conditions were as follows: temperature 40 C, Each membrane filter, which filtrated 100 ml of sea- flow rate 0.5 ml/min, and detection 280 nm. The water, was used as a source of microorganisms in the samples were eluted with a linear gradient of 10–30% seawater. Each filter was added to a test tube containing acetonitrile solution containing 0.14% KH2PO4 and 10 ml of SSB-YE medium, and the cultures were 0.34% tetra-n-butylammonium hydrogensulfate. incubated at 27 C with reciprocal shaking. After 3 weeks of cultivation, 0.2 ml of each culture was trans- Identification of degradation intermediate. Sphingo- ferred to 10 ml of the medium, and the cultures were monas sp. strain BP-7 was cultivated in SSB-0.1NB incubated under the same conditions. This procedure medium, and the culture supernatant was obtained by was repeated four times, and then each culture was centrifugation (10;000 Â g, 10 min, 4 C) of the culture. spread on SSB-YE agar plates and BPAS-NB agar The culture supernatant was applied to a LC-VP HPLC plates. BPAS-NB medium contained 0.02% BPA, 3.0% equipped with a Chemcosorb column (4:6 Â 150 mm, NaCl, 0.05% KH2PO4, 0.1% K2HPO4, 0.05% MgSO4. Chemco) using 30% acetonitrile solution. An elution 7H2O, 0.02% CaCl2.4H2O, 0.03% KCl, 0.001% FeCl2. fraction containing a degradation intermediate was 4H2O, 0.1% NH4NO3, 1% peptone, and 0.5% beef collected and extracted with ethyl acetate. extract (pH 7.5). Distinct colonies formed were removed The 1H and 13C nuclear magnetic resonance (NMR) and isolated in pure culture on BPAS-NB agar plates. spectra of the degradation intermediate were obtained Identification of each isolated strains was done by with a JEOL JNM-A600 (Tokyo). Samples for 1H-NMR TechnoSuruga (Shizuoka, Japan). For symbiotic culti- were examined as 1.25% solutions in CD2Cl2/CD3OD vation, 0.2 ml of the cell suspension of each strain, at an (80/20). The spectra were recorded at 25 C at 4.0-s optical density of about 0.5 at 660 nm, from the BPAS- pulse repetition and 256 accumulations. 1H-NMR NB agar plates was inoculated into 10 ml of SSB-YE chemical shifts were referred to internal tetramethylsi- medium. lane, and integrals of samples were calculated as 1.0 of the integral for 1,1,2,2,-tetrachloroethane. The mass Supplementation of nutrients. The nutrients were spectrographs of the intermediate were obtained with a added to SSB-YE medium from which yeast extract Voyager Linear DE-SY apparatus (PE Biosystems, was removed.