Biodegradation of Bisphenol a and Related Compounds by Sphingomonas Sp. Strain BP-7 Isolated from Seawater

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 (2:1 100 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 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 (4:6 150 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. The nutrients used in the nutritional Tokyo). Identification of an intermediate was confirmed supplementary experiment were 0.1% peptone, 0.1% by comparison of the spectra obtained from an isolated glucose, 0.05% beef extract, 0.05% yeast extract, 100 compound with those of an authentic compound. Biodegradation of Bisphenol A by Sphingomonas sp. Strain BP-7 53 Degradation of various aromatic compounds. Sphin- After prolonged cultivation, the colonies of strain no. 7 gomonas sp. strain BP-7 was cultivated in SSB-0.1NB formed haloes around them, suggesting that the strain medium containing the various aromatic compounds degraded BPA. These isolates were identified on the instead of BPA. Each compound was dissolved in basis of taxonomic characteristics (Table 1) and analysis ethanol at 5% and was aseptically added to the sterile of their 16S rRNA genes, which were submitted to medium. The final concentration of each compound was DDBJ. The 16S rRNA gene (accession no. AB276370) 100 ppm. Cultivation was done at 30 C for 4 d. The of strain no. 7 showed 98.6% homology to that of absorbance at 660 nm of the culture was measured for the type strain of Sphingobium xenophagum (synonym, cell growth, and then the remaining amount of each Sphingomonas xenophaga). The genes (accession compound was analyzed by HPLC. nos. AB276371 and AB276372) of strains nos. 14 and 15 showed 98.2 and 98.5% homology respectively to Results that of the type strain of Pseudomonas pseudoalcali- genes. The gene of strain no. 24A showed less than 90% Biodegradation of bisphenol A in the seawater homology to those of certain type strains, and was samples not identified. A BPA-degrading microorganism, strain Biodegradation experiments were done in the sea- no. 7, was named Sphingomonas sp. strain BP-7. Strains water with an apparatus for measuring biochemical nos. 14 and 15 were named Pseudomonas sp. strain BP- oxygen demand over a 28-d period. The biodegradabil- 14 and Pseudomonas sp. strain BP-15. ity of BPA was 18% for the seawater and 67% for To confirm the roles of isolated strains in BPA activated sludge from the sewage treatment plant. The degradation in the consortium, Sphingomonas sp. strain value of biodegradability in the seawater was change- BP-7 and Pseudomonas sp. strain BP-14 were cultivated able because microbial communities in seawater are in SSB-YE medium. As shown in Fig. 2A, Sphingomo- affected by environmental factors such as nutrients, nas sp. strain BP-7 started to grow at about 20 d of temperature, and water flow. The result indicates that cultivation, accompanied by consumption of BPA. At some microorganisms in the seawater degraded BPA. the same time, 4-hydroxyacetophenone (4-HAP) was detected in the culture medium, and its accumulation Enrichment, isolation, and identification of bisphenol gradually increased. 4-Hydroxybenzoic acid (4-HBA) A-degrading microorganisms was not detected during cultivation. After 40 d, BPA was Enrichment cultivation was done in SSB-YE medium. degraded over 95% in the medium, and the accumu- The cultures that completely degraded BPA were lation of 4-HAP ceased. On the other hand, Pseudomo- transferred to fresh medium. After repeating these nas sp. strain BP-14 did not degrade BPA, and showed procedures several times, a stable microbial consortium, minimum growth using chemicals in the medium no. 21–38, capable of degrading BPA was obtained. The (Fig. 2B). A mixed culture of Sphingomonas sp. strain consortium completely degraded BPA within 22 d BP-7 and Pseudomonas sp. strain BP-14 showed (Fig. 1). Four microorganisms were isolated from the complete degradation of 100 ppm BPA within 7 d culture of the consortium on BPAS-NB agar plates. (Fig. 2C). 4-HAP was slightly detected at 8 d of Three strains, nos. 14, 15, and 24A, of them grew faster cultivation and then was completely degraded. No other than the other strain no. 7. Strain no. 7 formed small degradation intermediates were detected under the colonies on a BPAS-NB agar plate at 5 d of cultivation. analytical conditions of HPLC. Pseudomonas sp. strain BP-15 and strain no. 24A played the same role in the mixed culture with Sphingomonas sp. strain BP-7 as that of Pseudomonas sp. strain BP-14. 0.3 0.6

0.5 ) Effects of nutrients on bisphenol A degradation M ) Table 2 shows the effect of nutrients on BPA

660 0.2 0.4 degradation by Sphingomonas sp. strain BP-7. The 0.3 strain did not degrade BPA on a mineral salt SSB medium. Peptone, among the nutrients tested, was most

Growth (A 0.1 0.2

Concentration (m effective in the stimulation of BPA-degrading activity. 0.1 The strain grew on peptone supplement medium and 0 0 degraded over 98% of BPA within 4 d. In addition, 01020304050 degradation of 4-HAP was also stimulated. The strain Time (d) showed slight growth by assimilating beef extract or yeast extract, but did not degrade BPA efficiently. Fig. 1. Time Course of Bisphenol A Degradation by Microbial Glucose did not support growth. Efficient degradation of Consortium No. 21–38 in SSB-YE Medium. Cultivation conditions are described in ‘‘Materials and Methods.’’ BPA and 4-HAP was achieved in media supplemented , cell growth; , concentration of bisphenol A; , concentration with peptone and beef extract or yeast extract. of 4-hydroxyacetophenone. BPA degradation by Sphingomonas sp. strain BP-7 in 54 K. SAKAI et al. Table 1. Taxonomic Characteristics of Microorganisms Isolated from Bisphenol A-Degrading Microbial Consortium No. 21–38

Characteristic Strain No. 7 Strain No. 14 Strain No. 15 Strain No. 24A Shape Rod Rod Rod Rod Gram stain a Spore Motility + + + + Color of colony Yellow Translucent Translucent Yellowish Catalase + + + + Oxidase ++ + OF test Nitrate reductase + + Indole test Acid production from glucose Arginine dihydrolase ++ Urease Esculin hydrolysis + + Gelatin hydrolysis -Galactosidase + + Utilization of: Glucose + + + + L-Arabinose + ++ D-Mannose ++ + D-Mannitol ++ + N-Acetyl-D-glucosamine + Maltose + Gluconate ++ Caproate ++ Adipate DL-Malate ++ + Citrate ++ Phenyl acetate

a+, positive; , week; , negative.

Table 2. Effect of Nutrients on Bisphenol A Degradation by Sphingomonas sp. Strain BP-7 Cultivation conditions are described in the Materials and Methods.

Bisphenol A Concentration Growth 4-Hydroxyacetophenone Nutrient remaining (%) (A ) (mM) 660 (%) None — 0.073 100 0 Peptone 0.1 0.301 1.77 0.109 Glucose 0.1 0.058 96.7 0.004 Beef extract 0.05 0.117 98.1 0.011 Yeast extract 0.05 0.108 86.8 0.044 Vitamins — 0.072 98.9 0 Pyrroloquinoline quinone 100 ng/ml 0.064 99.1 0

Peptone, Beef extract 0.1, 0.05 0.393 0.812 0.013 Peptone, Yeast extract 0.1, 0.05 0.474 0.466 0 Peptone, Vitamins 0.1, — 0.322 0.339 0.106 Peptone, Pyrroloquinoline quinone 0.1, 100 ng/ml 0.314 0 0.158

Glucose, Beef extract 0.1, 0.05 0.142 94.2 0.016 Glucose, Yeast extract 0.1, 0.05 0.152 75.5 0.077 Glucose, Vitamins 0.1, — 0.058 98.9 0 Glucose, Pyrroloquinoline quinone 0.1, 100 ng/ml 0.076 96.2 0.005

SSB-0.1NB medium, a nutritional supplementary me- strain BP-14 grew faster than Sphingomonas sp. strain dium, was examined. Sphingomonas sp. strain BP-7 BP-7, but did not degrade BPA. started to grow after 1 d of cultivation, and at the same time BPA degradation began. BPA in the medium was Degradability of Bisphenol derivatives and various completely degraded in 4 d (Fig. 3). 4-HAP accumulated aromatic compounds temporarily and then was degraded. Pseudomonas sp. The degradability of various compounds by Sphingo- Biodegradation of Bisphenol A by Sphingomonas sp. Strain BP-7 55

0.3 0.6 A 0.5 0.6 0.5 0.5 )

0.4 M

0.2 0.4 ) 0.4 660 0.3 0.3 0.3 0.1 0.2 0.2

0.1 Growth (A 0.2 0.1 Concentration (m 0 0 0.1

0.3 0.6 B ) 0 0 M 02468

) 0.5 Time (d) 660 0.2 0.4

0.3 Fig. 3. Time Courses of Bisphenol A Degradation by Sphingomonas sp. Strain BP-7 in SSB-0.1NB Medium. 0.1 0.2 Cultivation conditions are described in ‘‘Materials and Methods.’’ Growth (A 0.1 , cell growth; , concentration of bisphenol A; , concentration Concentration (m of 4-hydroxyacetophenone. 0 0

0.3 0.6 C 0.5 5–8) 10–14) 0.2 0.4 environment, as in activated sludge, river water, and soil,15,16) and that some BPA-degrading micro- 0.3 organisms are isolated from such places, but not from 0.1 0.2 seawater. Kang and Kondo18) suggested that BPA in 0.1 seawater may be caused more by chemical degradation 0 0 than by biological degradation, while organisms such as 01020304050 bacteria and flagellates can have an important effect on Time (d) the chemical degradation of BPA. We succeeded in isolating a BPA-degrading bacterium, Sphingomonas sp. Fig. 2. Time Courses of Bisphenol A Degradation by Sphingomonas strain BP-7, from offshore seawater samples. sp. Strain BP-7 and/or Pseudomonas sp. Strain BP-14 in SSB-YE Medium. Sphingomonas sp. strain BP-7 in a microbial con- Cultivation conditions are described in ‘‘Materials and Methods.’’ sortium no. 21–38 capable of degrading BPA always A, Sphingomonas sp. strain BP-7; B, Pseudomonas sp. strain BP-14; grew as secondary colonies appearing on primary C, Sphingomonas sp. strain BP-7 and Pseudomonas sp. strain BP-14. colonies growing faster on SSB-YE medium, which , cell growth; , concentration of bisphenol A; , concentration basically consisted of seawater supplemented with of 4-hydroxyacetophenone. various salts and a small amount of yeast extract. On nutritious BPAS-NB medium, Sphingomonas sp. strain monas sp. strain BP-7 was studied in SSB-0.1NB BP-7 formed small colonies independent from the other medium. The strain degraded bisphenols such as 1,1- microorganisms at 5 d of cultivation. Three other strains bis(4-hydroxyphenyl)ethane, 2,2-bis(4-hydroxy-3-meth- isolated from the consortium, Pseudomonas sp. strain ylphenyl)propane, 2,2-bis(4-hydroxyphenyl)butane, and BP-14, Pseudomonas sp. strain BP-15, and strain 1,1-bis(4-hydroxyphenyl)cyclohexane the same as BPA no. 24A, supported the degradation of BPA by Sphin- (Table 3). Some metabolites were detected during the gomonas sp. strain BP-7, and the accumulation of 4- biodegradation of these compounds and were not HAP ceased, while these strains did not degrade 4-HAP. identified. Bis(4-hydroxyphenyl)sulfone was not degrad- By the addition of nutrients such as peptone, beef ed, and bis(4-hydroxyphenyl)methane and bis(4-hydroxy- extract, and yeast extract to mineral salt basal medium, phenyl)sulfide inhibited the growth of the strain. The Sphingomonas sp. strain BP-7 was able to degrade BPA. strain degraded the degradation intermediates6) of BPA, These results suggest that Sphingomonas sp. strain BP-7 such as 4-HAP and 4-HBA, and various aromatic requires certain nutrients for effective growth and that compounds such as 4--cumylphenol, 4-nonylphenol, the other microorganisms supply certain compounds catechol, protocatechuic acid, hydroquinone, and 4- instead of these nutrients. It is probable that BPA is hydroxybenzaldehyde. However, 4-HAP of the signifi- degraded by a synergistic action of bacteria in seawater, cant accumulation compound was degraded at a slow which is a nutrient-poor environment. rate. Pseudomonas sp. strain BP-14 did not degrade 4- 4-HAP was detected as a degradation intermediate of HAP. BPA, while 4-HBA was not detected. Degradation of 4- HAP by Sphingomonas sp. strain BP-7 was slower than Discussion that of 4-HBA. These results suggest that the degrada- tion pathway of 4-HAP of the strain is a rate-limiting Many studies have found that BPA is degraded in the step in the degradation of BPA, because of low activities 56 K. SAKAI et al. Table 3. Degradation of Bisphenol Derivatives and Various Aromatic Compounds by Sphingomonas sp. Strain BP-7 Cultivation conditions are described in the Materials and Methods.

Growth Residual Degradation Compound Structure a (A660) (%) intermediates None 0.398 — — Bisphenol A 0.527 0 nd H3C CH3

HO OH

Bis(4-hydroxyphenyl)methane H H 0.035 102 nd

HO OH 1,1-Bis(4-hydroxyphenyl)ethane 0.443 9.34 d H CH3

HO OH 2,2-Bis(4-hydroxy-3-methylphenyl)propane 0.313 5.94 d H3C CH3 H3C CH3

HO OH 2,2-Bis(4-hydroxyphenyl)butane 0.256 0 d H3C CH3

HO OH 1,1-Bis(4-hydroxyphenyl)cyclohexane 0.488 0 d

HO OH

Bis(4-hydroxyphenyl)sulfone O 0.356 97.2 nd

S

O HO OH

Bis(4-hydroxyphenyl)sulfide S 0.041 98.3 nd

HO OH 4--Cumylphenol 0.347 0 d H3C CH3

OH 4-tert-Butylphenol 0.491 99.5 nd 4-Nonylphenol 0.431 0 nd Catechol 0.653 47.2 nd Protocatechuic acid 0.492 0 nd Hydroquinone 0.592 0 nd 4-Hydroxyacetophenone 0.277 78.9 nd 4-Hydroxybenzaldehyde 0.534 0 nd 4-Hydroxybenzoic acid 0.529 0 nd

ad, detected; nd, not detected. of initial enzymes in the pathway or an inhibition effect yl)butane, and 1,1-bis(4-hydroxyphenyl)cyclohexane as of 4-HAP on the growth of the strain. The degradation of well as BPA. However, the strain did not degrade bis(4- BPA by the strain proceeded via 4-HAP, as previously hydroxyphenyl)methane, bis(4-hydroxyphenyl)sulfone, reported by Lobos et al.6) Sphingomonas sp. strain BP-7 or bis(4-hydroxyphenyl)sulfide. Bis(4-hydroxyphenyl)- showed features different from strain MV-1,6) including methane and bis(4-hydroxyphenyl)sulfide inhibited the the following: no accumulation of 4-HAB, acceleration growth of the strain. These results indicate that the of degradation of BPA and 4-HAP by the addition of presence of at least one methyl or methylene group nutrients, and degradation of a various bisphenols by bonded at the carbon atom between two phenol groups living cells. in the BPA molecule is necessary for biodegradation of Sphingomonas sp. strain BP-7 degraded bisphenols the bisphenols by the strain. Spivack et al.7) reported that such as 1,1-bis(4-hydroxyphenyl)ethane, 2,2-bis(4-hy- the degradation of BPA by strain MV-1 occurs via a droxy-3-methylphenyl)propane, 2,2-bis(4-hydroxyphen- pathway involving oxidative skeletal rearrangement of Biodegradation of Bisphenol A by Sphingomonas sp. Strain BP-7 57 an unactivated aliphatic methyl group in the BPA of bisphenol A and other bisphenols by a gram-negative molecule, judging from the metabolic intermediates of aerobic bacterium. Appl. Environ. 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