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Efficient Microbial Degradation of Bisphenol A in the Presence of Activated Carbon
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 105, No. 2, 157–160. 2008 DOI: 10.1263/jbb.105.157
© 2008, The Society for Biotechnology, Japan
Efficient Microbial Degradation of Bisphenol A in the Presence of Activated Carbon Hayato Yamanaka,1* Kunihiko Moriyoshi,1 Takashi Ohmoto,1 Tatsuhiko Ohe,1 and Kiyofumi Sakai1 Department of Environmental Technology, Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan1 Received 1 August 2007/Accepted 15 November 2007
The biodegradation of bisphenol A (BPA) was carried out with Sphingomonas sp. strain BP-7 and Sphingomonas yanoikuyae BP-11R in the presence of activated carbon (AC). When AC was present, both BPA-degrading bacteria efficiently degraded 300 mg/l BPA without releasing 4-hydroxyacetophenone, the major intermediate produced in BPA degradation, into the medium. The biological regeneration of AC was possible using the BPA-degrading bacteria, suggesting that an efficient system for BPA removal can be constructed by introducing BPA-degrading bacteria into an AC treatment system. [Key words: bisphenol A, biodegradation, activated carbon, Sphingomonas, endocrine-disrupting chemical]
higher BPA-degrading activity (Monri, M., Sakai, K., and Ohe, T., Japan Kokai Tokkyo Koho, 2003-24050, 2003). In this study, BPA degradation was carried out using the abovementioned BPA-degrading bacteria in the presence of activated carbon (AC). AC is known to adsorb hydrophobic compounds including BPA (9) effectively and is extensively used for the removal of contaminants in water or gas treatment systems. However, the major drawback of AC is that regeneration is needed when the adsorption capacity of AC is exhausted, which results in an increase in the total cost of the system. By combining the adsorption property of AC with the microbial degradation of BPA, the lifetime of AC can be extended; thus, the construction of an efficient treatment system is expected. The AC used in this study was Shirasagi C, the commercial product of Takeda Chemical Industries (Osaka), and had the following properties: diameter, 50–100 µm; adsorption capacity for iodine, 980 mg/g; specific surface area, 1003 m2/g; pore volume, 0.579 ml/g; and mean pore diameter, 2.31 nm. BPA was purchased from Nacalai Tesque (Kyoto). 4-Hydroxyacetophenone (4-HAP) was obtained from Kanto Chemical (Tokyo). The beef extract used was the product of Difco (Detroit, MI, USA). Casein peptone and yeast extract were the products of Nihon Pharmaceutical (Tokyo). All other chemicals were also obtained from commercial sources. BPAS-0.1NB medium was composed of (per liter) 0.3 g of BPA, 1 g of peptone, 0.5 g of beef extract, 30 g of NaCl, 1 g of NH4NO3, 0.5 g of KH2PO4, 1 g of K2HPO4, 0.3 g of KCl, 0.5 g of MgSO4 ⋅7H2O, 0.2 g of CaCl2 ⋅2H2O, and 0.01 g of FeCl2 (pH 7.5). BPA-YE medium was composed of (per liter) 0.3 g of BPA, 0.2 g of yeast extract, 0.5 g of NaCl, 2 g of NH4NO3, 1 g of KH2PO4, 1 g of K2HPO4, and 0.5 g of MgSO4 ⋅7H2O (pH 7.0). For the adsorption experiments, 10 mg of AC was added
Bisphenol A (2,2-bis(4-hydroxyphenyl)propane, BPA) is widely used as the starting material for the industrial production of polycarbonates, epoxy resins, and other specialty chemicals. Owing to its mass production and widespread use, the probability of environmental contamination with BPA has increased. Environmental releases are possible via permitted outfalls of industrial wastewater treatment systems or sewage treatment plants that receive BPA. Other possible sources of BPA are found in the environment, such as waste plastics in waste landfills and sewage sludge from wastewater treatment facilities. There is a considerable amount of monitoring data on BPA in Europe, the United States, and Japan, and certain levels of BPA have been detected in many samples (1, 2). BPA was revealed to show acute toxicity within the range of 1–10 mg/l toward algae, invertebrates, and fish (3). The effects of BPA on human health have also been a concern, and BPA was found to show mutagenicity in human RSa cells within the range of 10–7–10–5 M (4). In addition, BPA has been suspected of being an endocrine-disrupting chemical (5). Thus, BPA has become an environmental contaminant of considerable interest (6). There have been many studies on the biodegradation and metabolism of BPA, and several microorganisms capable of degrading BPA have been identified (7). However, little attention has been paid to the application of BPA-degrading microorganisms in BPA-removal systems. We have been interested in the environmental fate of BPA and have isolated several BPA-degrading microorganisms. Sphingomonas sp. strain BP-7, isolated from seawater, was found to degrade BPA in the presence of 3% NaCl (8). Sphingomonas yanoikuyae BP-11R, isolated from river water, showed even * Corresponding author. e-mail: [email protected] phone: +81-(0)6-6963-8065 fax: +81-(0)6-6963-8079 157
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to 10 ml of 80–315 mg/l BPA solutions made with distilled water or 40–350 mg/l 4-HAP solutions made with distilled water, and then incubated at 27°C with shaking. After 24 h, the BPA or 4-HAP in the liquid phase was quantified. Concentrations of BPA and 4-HAP were determined by highperformance liquid chromatography (HPLC) according to a method described previously (10). BPA degradation experiments were repeated at least twice to confirm reproducibility. The BPAS-0.1NB and BPAYE media were used for the degradation experiments with Sphingomonas sp. strain BP-7 and S. yanoikuyae BP-11R, respectively. Prior to the inoculation of the BPA-degrading bacteria, AC (300 mg to the BPAS-0.1NB medium and 200 mg to the BPA-YE medium) was added to 100 ml of the medium in a 500-ml flask and incubated for 15 h at 27°C with shaking to attain equilibrium. The amount of AC was varied because the affinity of BPA for AC in the BPA-YE medium was higher than that in the BPAS-0.1NB medium. Then, each strain was inoculated and cultivated at 27°C. Samples (1 ml each) were withdrawn at various time points to quantify BPA and 4-HAP. The optical density at 660 nm (OD660) was also monitored to estimate cell growth. ACadded media without inoculation were used for blanks. For the continuous degradation of BPA, S. yanoikuyae BP-11R was cultivated in the BPA-YE medium at 27°C, and then 1.3 g of AC was added to 200 ml of 60-h cultures (OD660, 0.3). After incubation for 7 h at 27°C with shaking, the resultant biological activated carbon (BAC) was recovered by filtration and packed into a column (diameter, 7 mm; length, 26 mm). The column temperature was set at 27°C and the solution, containing the same components as the BPAYE medium except that the BPA concentration was changed to 100 mg/l, was continuously fed into the column at a flow rate of 30 ml/h using a peristaltic pump (linear velocity, 0.78 m/h; space velocity, 30 h–1). The samples were intermittently taken from the outlet of the column to quantify BPA. As a control experiment, the same procedure was carried out with an AC column of the same size without BPAdegrading bacteria. Figure 1 shows the adsorption characteristics of BPA and 4-HAP, the major intermediate produced in BPA degradation by Sphingomonas sp. strain BP-7 (8), for AC. In both cases, the adsorption isotherms well fitted the Freundlich equation, which is frequently used for evaluating liquid-phase adsorption (11), q = KC1/n where C and q are the equilibrium concentration in the water phase (mg/l) and the adsorption quantity on AC (mg/g), respectively. AC had a high capacity for adsorbing both BPA and 4-HAP, and BPA was the better adsorbate owing to its more hydrophobic characteristics. The biodegradation of BPA in the presence of AC was carried out. Although several BPA-degrading microorganisms have been isolated, the degradation experiments were usually carried out at a concentration of 100 mg/l or lower. In some cases, higher concentrations of BPA caused the inhibition of the BPA-degrading activity of the microorganisms (12). To confirm the effectiveness of AC, the degradation of 300 mg/l, near the saturated concentration, BPA was
J. BIOSCI. BIOENG.,
FIG. 1. Adsorption isotherms of BPA (closed circles) and 4-HAP (closed triangles) for AC. Freundlich constants are shown in the inset.
attempted. Few studies have been conducted to degrade such a high concentration of BPA, except that strain MV1 was reported to degrade and grow on BPA at a saturated concentration (13). Figure 2 shows the time courses of BPA degradation by Sphingomonas sp. strain BP-7 in the presence and absence of AC. When Sphingomonas sp. strain BP-7 was inoculated into the medium without AC, no BPA degradation or cell growth was observed because of the inhibitory effect of BPA (Fig. 2B). However, when AC was present in the medium, the BPA concentration in the medium decreased to 3.1 mg/l before inoculation as the result of adsorption onto AC, preventing the inhibition caused by BPA (Fig. 2A). Sphingomonas sp. strain BP-7 degraded BPA not adsorbed onto AC, and BPA in the liquid phase became undetectable within 24 h. Thereafter, the cells continued to grow and OD660 reached 0.66 after 48 h of cultivation. When the strain was cultivated in the AC-added medium containing the same components except no BPA, OD660 reached 0.47 at 48 h. This OD difference suggests that the BPA adsorbed onto AC was also degraded by the bacterium. The results of BPA degradation by S. yanoikuyae BP-11R are shown in Fig. 3. S. yanoikuyae BP-11R was found to show BPA-degrading activity at a concentration of 300 mg/l, although a long lag time was required before BPA degradation and cell growth began (Fig. 3B). When AC was present in the medium, BPA concentration decreased to 8.1 mg/l and the BPA remaining in the liquid phase was quickly removed by S. yanoikuyae BP-11R (Fig. 3A). OD660 reached 0.34 after 48 h of cultivation. This value was almost the same as that in the case of the absence of AC and was greater than 0.06, the OD660 at 48 h when the strain was cultivated in the AC-added medium containing the same components except no BPA. This result again indicates that the BPA-degrading bacteria degraded not only BPA in the liquid phase but also the BPA adsorbed onto AC. To quantify the amount of BPA that was degraded, the extraction of BPA adsorbed on AC was attempted. As far as we investigated, however, there was no appropriate solvent for carrying out a quantitative recovery, which should have the properties of both a high solubility for BPA and a high adsorb-
VOL. 105, 2008
FIG. 2. BPA degradation by Sphingomonas sp. strain BP-7 in the presence (A) and absence (B) of AC. The time at which Sphingomonas sp. strain BP-7 was inoculated was set as t = 0. OD660 differences from the blank are shown in the left axis. Symbols: open circles, growth rate; closed circles, BPA concentration; closed triangles, 4-HAP concentration.
ability onto AC. For example, when a known amount of BPA preadsorbed onto AC was extracted with methanol seven times, the recovery varied by about 70–85% and no more BPA was recovered by further extraction. The low recovery and low reproducibility indicate that BPA on some adsorption sites is unextractable and that the ratio of BPA adsorbed onto such sites is variable. In both cases, with Sphingomonas sp. strain BP-7 and with S. yanoikuyae BP-11R, almost no 4-HAP was detected in the medium during BPA degradation in the presence of AC. Sphingomonas sp. strain BP-7 was found to not produce significant amounts of degradation intermediates other than 4-HAP (8). No intermediates other than 4-HAP were detected in the HPLC chromatogram when S. yanoikuyae BP-11R was cultivated in BPA-YE medium in the absence or presence of AC (data not shown). Even if minor intermediates are produced under certain conditions, they will be efficiently adsorbed onto AC and then degraded. Some of the hydrophobic degradation intermediates of BPA have been reported to exhibit estrogenic activity (14, 15). Thus, the application of AC is useful from the viewpoint that the harmful compounds produced during BPA degradation can be trapped without significant leakage into the liquid phase and that BPA concentration in the liquid phase can be immedi-
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159
FIG. 3. BPA degradation by S. yanoikuyae BP-11R in the presence (A) and absence (B) of AC. The time at which S. yanoikuyae BP-11R was inoculated was set as t= 0. OD660 differences from the blank are shown in the left axis. Symbols: open circles, growth rate; closed circles, BPA concentration; closed triangles, 4-HAP concentration.
ately decreased. The surface of the AC samples used in the BPA degradation experiments was observed by scanning electron microscopy (SEM) (JSM 5800 LVC; JEOL, Tokyo). SEM images of the AC samples after the degradation reactions are shown in Fig. 4. The BPA-degrading bacteria were adsorbed onto the surface of AC. Therefore, AC also worked as the support, and the resultant immobilized biomass could work as BAC. Similar to the mechanism of the biological regeneration proposed in the BAC process (16), BPA was considered to be first adsorbed onto the micropores or mesopores of AC that are inaccessible to the bacteria, and then desorbed because of the reverse concentration gradient, owing to the biodegradation of BPA in the liquid phase. The rate of desorption seemed to be slow because the affinity of BPA to AC was high. Desorbed BPA was also biodegraded, and the bacterial cells that increased in number beyond the adsorption capacity of AC were released into the liquid phase. To examine whether BPA adsorbed onto AC can be degraded by BPA-degrading bacteria, BAC with S. yanoikuyae BP-11R was packed into a column and BPA solution was continuously passed through the column. As shown in Fig. 5, the breakthrough time of the BAC column was longer than that of the AC column without BPA-degrading bacteria. Taking into consideration the fact that the BPA loading rate was
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FIG. 4. Scanning electron micrographs of AC after degradation reactions with Sphingomonas sp. strain BP-7 (A) and S. yanoikuyae BP-11R (B).
4.
5. 6.
7. 8. FIG. 5. BPA removal by packed columns of BAC with S. yanoikuyae BP-11R (open circles) and AC without BPA-degrading bacteria (closed circles).
3 mg/h, 23.25 mg of BPA was loaded onto the BAC column in 7.75 h before breakthrough occurred, whereas 13.5 mg of BPA was loaded onto the AC column in 4.5 h before breakthrough occurred. This difference implies that the adsorption property of AC can be regenerated by the action of BPA-degrading bacteria fixed to AC. Moreover, the results also suggest that the lifetime of AC can be extended by introducing BPA-degrading bacteria to an AC system for the removal of BPA. In this experiment, the breakthrough occurred because the BPA loading rate was higher than the rate of degradation by BPA-degrading bacteria fixed to AC. In practice, a longer operation time can be achieved with a proper control of the load. An evaluation of the efficiency of the BAC treatment system using wastewater is in progress. REFERENCES 1. Cousins, I. T., Staples, C. A., Klecka, G. M., and Mackay, D.: A multimedia assessment of the environmental fate of bisphenol A. Hum. Ecol. Risk Assess., 8, 1107–1135 (2002). 2. Yamamoto, T., Yasuhara, A., Shiraishi, H., and Nakasugi, O.: Bisphenol A in hazardous waste landfill leachates. Chemosphere, 42, 415–418 (2001). 3. Alexander, H. C., Dill, L. W., Smith, L. W., Guiney, P. D.,
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and Dorn, P.: Bisphenol A: acute aquatic toxicity. Environ. Toxicol. Chem., 7, 19–26 (1988). Takahashi, S., Chi., X. J., Yamaguchi, Y., Suzuki, H., Sugaya, S., Kita, K., Hiroshima, K., Yamamori, H., Ichinose, M., and Suzuki, N.: Mutagenicity of bisphenol A and its suppression by interferon-alpha in human RSa cells. Mutat. Res., 490, 199–207 (2001). McLachlan, J. A.: Environmental signaling: what embryos and evolution teach us about endocrine disrupting chemicals. Endocr. Rev., 22, 319–341 (2001). Staples, C. A., Dorn, P. B., Klecka, G. M., O’Block, S. T., and Harris, L. R.: A review of the environmental fate, effects, and exposures of bisphenol A. Chemosphere, 36, 2149–2173 (1998). Kang, J. H., Katayama, Y., and Kondo, K.: Biodegradation or metabolism of bisphenol A: from microorganisms to mammals. Toxicology, 217, 81–90 (2006). Sakai, K., Yamanaka, H., Moriyoshi, K., Ohmoto, T., and Ohe, T.: Biodegradation of bisphenol A and related compounds by Sphingomonas sp. strain BP-7 isolated from seawater. Biosci. Biotechnol. Biochem., 71, 51–57 (2007). Bautista-Toledo, I., Ferro-García, M. A., Rivera-Utrilla, J., Moreno-Castilla, C., and Vegas Fernández, F. J.: Bisphenol A removal from water by activated carbon. Effects of carbon characteristics and solution chemistry. Environ. Sci. Technol., 39, 6246–6250 (2005). Yamanaka, H., Moriyoshi, K., Ohmoto, T., Ohe, T., and Sakai, K.: Degradation of bisphenol A by Bacillus pumilus isolated from kimchi, a traditionally fermented food. Appl. Biochem. Biotechnol., 136, 39–52 (2007). Gessner, P. K. and Hasan, M. M.: Freundlich and Langmuir isotherms as models for adsorption of toxicants on activated charcoal. J. Pharm. Sci., 76, 319–327 (1987). Ike, M., Jin, C.-S., and Fujita, M.: Isolation and characterization of a novel bisphenol A-degrading bacterium Pseudomonas paucimobilis strain FJ-4. Jpn. J. Water Treat. Biol., 31, 203– 212 (1995). Lobos, J. H., Leib, T. K., and Su, T.-M.: Biodegradation of bisphenol A and other bisphenols by a gram-negative aerobic bacterium. Appl. Environ. Microbiol., 58, 1823–1831 (1992). Ike, M., Chen, M. Y., Jin, C. S., and Fujita, M.: Acute toxicity, mutagenicity, and estrogenicity of biodegradation products of bisphenol-A. Environ. Toxicol., 17, 457–461 (2002). Ohtani, Y., Shimada, Y., Shiraishi, F., and Kozawa, K.: Variation of estrogenic activities during the bio-degradation of bisphenol A. J. Environ. Chem., 13, 1027–1031 (2003). (in Japanese) Sirotkin, A. S., Koshkina, L. Yu., and Ippolitov, K. G.: The BAC-process for treatment of waste water containing non-ionogenic synthetic surfactants. Water Res., 35, 3265–3271 (2001).
© 2008, The Society for Biotechnology, Japan
Efficient Microbial Degradation of Bisphenol A in the Presence of Activated Carbon Hayato Yamanaka,1* Kunihiko Moriyoshi,1 Takashi Ohmoto,1 Tatsuhiko Ohe,1 and Kiyofumi Sakai1 Department of Environmental Technology, Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan1 Received 1 August 2007/Accepted 15 November 2007
The biodegradation of bisphenol A (BPA) was carried out with Sphingomonas sp. strain BP-7 and Sphingomonas yanoikuyae BP-11R in the presence of activated carbon (AC). When AC was present, both BPA-degrading bacteria efficiently degraded 300 mg/l BPA without releasing 4-hydroxyacetophenone, the major intermediate produced in BPA degradation, into the medium. The biological regeneration of AC was possible using the BPA-degrading bacteria, suggesting that an efficient system for BPA removal can be constructed by introducing BPA-degrading bacteria into an AC treatment system. [Key words: bisphenol A, biodegradation, activated carbon, Sphingomonas, endocrine-disrupting chemical]
higher BPA-degrading activity (Monri, M., Sakai, K., and Ohe, T., Japan Kokai Tokkyo Koho, 2003-24050, 2003). In this study, BPA degradation was carried out using the abovementioned BPA-degrading bacteria in the presence of activated carbon (AC). AC is known to adsorb hydrophobic compounds including BPA (9) effectively and is extensively used for the removal of contaminants in water or gas treatment systems. However, the major drawback of AC is that regeneration is needed when the adsorption capacity of AC is exhausted, which results in an increase in the total cost of the system. By combining the adsorption property of AC with the microbial degradation of BPA, the lifetime of AC can be extended; thus, the construction of an efficient treatment system is expected. The AC used in this study was Shirasagi C, the commercial product of Takeda Chemical Industries (Osaka), and had the following properties: diameter, 50–100 µm; adsorption capacity for iodine, 980 mg/g; specific surface area, 1003 m2/g; pore volume, 0.579 ml/g; and mean pore diameter, 2.31 nm. BPA was purchased from Nacalai Tesque (Kyoto). 4-Hydroxyacetophenone (4-HAP) was obtained from Kanto Chemical (Tokyo). The beef extract used was the product of Difco (Detroit, MI, USA). Casein peptone and yeast extract were the products of Nihon Pharmaceutical (Tokyo). All other chemicals were also obtained from commercial sources. BPAS-0.1NB medium was composed of (per liter) 0.3 g of BPA, 1 g of peptone, 0.5 g of beef extract, 30 g of NaCl, 1 g of NH4NO3, 0.5 g of KH2PO4, 1 g of K2HPO4, 0.3 g of KCl, 0.5 g of MgSO4 ⋅7H2O, 0.2 g of CaCl2 ⋅2H2O, and 0.01 g of FeCl2 (pH 7.5). BPA-YE medium was composed of (per liter) 0.3 g of BPA, 0.2 g of yeast extract, 0.5 g of NaCl, 2 g of NH4NO3, 1 g of KH2PO4, 1 g of K2HPO4, and 0.5 g of MgSO4 ⋅7H2O (pH 7.0). For the adsorption experiments, 10 mg of AC was added
Bisphenol A (2,2-bis(4-hydroxyphenyl)propane, BPA) is widely used as the starting material for the industrial production of polycarbonates, epoxy resins, and other specialty chemicals. Owing to its mass production and widespread use, the probability of environmental contamination with BPA has increased. Environmental releases are possible via permitted outfalls of industrial wastewater treatment systems or sewage treatment plants that receive BPA. Other possible sources of BPA are found in the environment, such as waste plastics in waste landfills and sewage sludge from wastewater treatment facilities. There is a considerable amount of monitoring data on BPA in Europe, the United States, and Japan, and certain levels of BPA have been detected in many samples (1, 2). BPA was revealed to show acute toxicity within the range of 1–10 mg/l toward algae, invertebrates, and fish (3). The effects of BPA on human health have also been a concern, and BPA was found to show mutagenicity in human RSa cells within the range of 10–7–10–5 M (4). In addition, BPA has been suspected of being an endocrine-disrupting chemical (5). Thus, BPA has become an environmental contaminant of considerable interest (6). There have been many studies on the biodegradation and metabolism of BPA, and several microorganisms capable of degrading BPA have been identified (7). However, little attention has been paid to the application of BPA-degrading microorganisms in BPA-removal systems. We have been interested in the environmental fate of BPA and have isolated several BPA-degrading microorganisms. Sphingomonas sp. strain BP-7, isolated from seawater, was found to degrade BPA in the presence of 3% NaCl (8). Sphingomonas yanoikuyae BP-11R, isolated from river water, showed even * Corresponding author. e-mail: [email protected] phone: +81-(0)6-6963-8065 fax: +81-(0)6-6963-8079 157
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to 10 ml of 80–315 mg/l BPA solutions made with distilled water or 40–350 mg/l 4-HAP solutions made with distilled water, and then incubated at 27°C with shaking. After 24 h, the BPA or 4-HAP in the liquid phase was quantified. Concentrations of BPA and 4-HAP were determined by highperformance liquid chromatography (HPLC) according to a method described previously (10). BPA degradation experiments were repeated at least twice to confirm reproducibility. The BPAS-0.1NB and BPAYE media were used for the degradation experiments with Sphingomonas sp. strain BP-7 and S. yanoikuyae BP-11R, respectively. Prior to the inoculation of the BPA-degrading bacteria, AC (300 mg to the BPAS-0.1NB medium and 200 mg to the BPA-YE medium) was added to 100 ml of the medium in a 500-ml flask and incubated for 15 h at 27°C with shaking to attain equilibrium. The amount of AC was varied because the affinity of BPA for AC in the BPA-YE medium was higher than that in the BPAS-0.1NB medium. Then, each strain was inoculated and cultivated at 27°C. Samples (1 ml each) were withdrawn at various time points to quantify BPA and 4-HAP. The optical density at 660 nm (OD660) was also monitored to estimate cell growth. ACadded media without inoculation were used for blanks. For the continuous degradation of BPA, S. yanoikuyae BP-11R was cultivated in the BPA-YE medium at 27°C, and then 1.3 g of AC was added to 200 ml of 60-h cultures (OD660, 0.3). After incubation for 7 h at 27°C with shaking, the resultant biological activated carbon (BAC) was recovered by filtration and packed into a column (diameter, 7 mm; length, 26 mm). The column temperature was set at 27°C and the solution, containing the same components as the BPAYE medium except that the BPA concentration was changed to 100 mg/l, was continuously fed into the column at a flow rate of 30 ml/h using a peristaltic pump (linear velocity, 0.78 m/h; space velocity, 30 h–1). The samples were intermittently taken from the outlet of the column to quantify BPA. As a control experiment, the same procedure was carried out with an AC column of the same size without BPAdegrading bacteria. Figure 1 shows the adsorption characteristics of BPA and 4-HAP, the major intermediate produced in BPA degradation by Sphingomonas sp. strain BP-7 (8), for AC. In both cases, the adsorption isotherms well fitted the Freundlich equation, which is frequently used for evaluating liquid-phase adsorption (11), q = KC1/n where C and q are the equilibrium concentration in the water phase (mg/l) and the adsorption quantity on AC (mg/g), respectively. AC had a high capacity for adsorbing both BPA and 4-HAP, and BPA was the better adsorbate owing to its more hydrophobic characteristics. The biodegradation of BPA in the presence of AC was carried out. Although several BPA-degrading microorganisms have been isolated, the degradation experiments were usually carried out at a concentration of 100 mg/l or lower. In some cases, higher concentrations of BPA caused the inhibition of the BPA-degrading activity of the microorganisms (12). To confirm the effectiveness of AC, the degradation of 300 mg/l, near the saturated concentration, BPA was
J. BIOSCI. BIOENG.,
FIG. 1. Adsorption isotherms of BPA (closed circles) and 4-HAP (closed triangles) for AC. Freundlich constants are shown in the inset.
attempted. Few studies have been conducted to degrade such a high concentration of BPA, except that strain MV1 was reported to degrade and grow on BPA at a saturated concentration (13). Figure 2 shows the time courses of BPA degradation by Sphingomonas sp. strain BP-7 in the presence and absence of AC. When Sphingomonas sp. strain BP-7 was inoculated into the medium without AC, no BPA degradation or cell growth was observed because of the inhibitory effect of BPA (Fig. 2B). However, when AC was present in the medium, the BPA concentration in the medium decreased to 3.1 mg/l before inoculation as the result of adsorption onto AC, preventing the inhibition caused by BPA (Fig. 2A). Sphingomonas sp. strain BP-7 degraded BPA not adsorbed onto AC, and BPA in the liquid phase became undetectable within 24 h. Thereafter, the cells continued to grow and OD660 reached 0.66 after 48 h of cultivation. When the strain was cultivated in the AC-added medium containing the same components except no BPA, OD660 reached 0.47 at 48 h. This OD difference suggests that the BPA adsorbed onto AC was also degraded by the bacterium. The results of BPA degradation by S. yanoikuyae BP-11R are shown in Fig. 3. S. yanoikuyae BP-11R was found to show BPA-degrading activity at a concentration of 300 mg/l, although a long lag time was required before BPA degradation and cell growth began (Fig. 3B). When AC was present in the medium, BPA concentration decreased to 8.1 mg/l and the BPA remaining in the liquid phase was quickly removed by S. yanoikuyae BP-11R (Fig. 3A). OD660 reached 0.34 after 48 h of cultivation. This value was almost the same as that in the case of the absence of AC and was greater than 0.06, the OD660 at 48 h when the strain was cultivated in the AC-added medium containing the same components except no BPA. This result again indicates that the BPA-degrading bacteria degraded not only BPA in the liquid phase but also the BPA adsorbed onto AC. To quantify the amount of BPA that was degraded, the extraction of BPA adsorbed on AC was attempted. As far as we investigated, however, there was no appropriate solvent for carrying out a quantitative recovery, which should have the properties of both a high solubility for BPA and a high adsorb-
VOL. 105, 2008
FIG. 2. BPA degradation by Sphingomonas sp. strain BP-7 in the presence (A) and absence (B) of AC. The time at which Sphingomonas sp. strain BP-7 was inoculated was set as t = 0. OD660 differences from the blank are shown in the left axis. Symbols: open circles, growth rate; closed circles, BPA concentration; closed triangles, 4-HAP concentration.
ability onto AC. For example, when a known amount of BPA preadsorbed onto AC was extracted with methanol seven times, the recovery varied by about 70–85% and no more BPA was recovered by further extraction. The low recovery and low reproducibility indicate that BPA on some adsorption sites is unextractable and that the ratio of BPA adsorbed onto such sites is variable. In both cases, with Sphingomonas sp. strain BP-7 and with S. yanoikuyae BP-11R, almost no 4-HAP was detected in the medium during BPA degradation in the presence of AC. Sphingomonas sp. strain BP-7 was found to not produce significant amounts of degradation intermediates other than 4-HAP (8). No intermediates other than 4-HAP were detected in the HPLC chromatogram when S. yanoikuyae BP-11R was cultivated in BPA-YE medium in the absence or presence of AC (data not shown). Even if minor intermediates are produced under certain conditions, they will be efficiently adsorbed onto AC and then degraded. Some of the hydrophobic degradation intermediates of BPA have been reported to exhibit estrogenic activity (14, 15). Thus, the application of AC is useful from the viewpoint that the harmful compounds produced during BPA degradation can be trapped without significant leakage into the liquid phase and that BPA concentration in the liquid phase can be immedi-
NOTES
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FIG. 3. BPA degradation by S. yanoikuyae BP-11R in the presence (A) and absence (B) of AC. The time at which S. yanoikuyae BP-11R was inoculated was set as t= 0. OD660 differences from the blank are shown in the left axis. Symbols: open circles, growth rate; closed circles, BPA concentration; closed triangles, 4-HAP concentration.
ately decreased. The surface of the AC samples used in the BPA degradation experiments was observed by scanning electron microscopy (SEM) (JSM 5800 LVC; JEOL, Tokyo). SEM images of the AC samples after the degradation reactions are shown in Fig. 4. The BPA-degrading bacteria were adsorbed onto the surface of AC. Therefore, AC also worked as the support, and the resultant immobilized biomass could work as BAC. Similar to the mechanism of the biological regeneration proposed in the BAC process (16), BPA was considered to be first adsorbed onto the micropores or mesopores of AC that are inaccessible to the bacteria, and then desorbed because of the reverse concentration gradient, owing to the biodegradation of BPA in the liquid phase. The rate of desorption seemed to be slow because the affinity of BPA to AC was high. Desorbed BPA was also biodegraded, and the bacterial cells that increased in number beyond the adsorption capacity of AC were released into the liquid phase. To examine whether BPA adsorbed onto AC can be degraded by BPA-degrading bacteria, BAC with S. yanoikuyae BP-11R was packed into a column and BPA solution was continuously passed through the column. As shown in Fig. 5, the breakthrough time of the BAC column was longer than that of the AC column without BPA-degrading bacteria. Taking into consideration the fact that the BPA loading rate was
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FIG. 4. Scanning electron micrographs of AC after degradation reactions with Sphingomonas sp. strain BP-7 (A) and S. yanoikuyae BP-11R (B).
4.
5. 6.
7. 8. FIG. 5. BPA removal by packed columns of BAC with S. yanoikuyae BP-11R (open circles) and AC without BPA-degrading bacteria (closed circles).
3 mg/h, 23.25 mg of BPA was loaded onto the BAC column in 7.75 h before breakthrough occurred, whereas 13.5 mg of BPA was loaded onto the AC column in 4.5 h before breakthrough occurred. This difference implies that the adsorption property of AC can be regenerated by the action of BPA-degrading bacteria fixed to AC. Moreover, the results also suggest that the lifetime of AC can be extended by introducing BPA-degrading bacteria to an AC system for the removal of BPA. In this experiment, the breakthrough occurred because the BPA loading rate was higher than the rate of degradation by BPA-degrading bacteria fixed to AC. In practice, a longer operation time can be achieved with a proper control of the load. An evaluation of the efficiency of the BAC treatment system using wastewater is in progress. REFERENCES 1. Cousins, I. T., Staples, C. A., Klecka, G. M., and Mackay, D.: A multimedia assessment of the environmental fate of bisphenol A. Hum. Ecol. Risk Assess., 8, 1107–1135 (2002). 2. Yamamoto, T., Yasuhara, A., Shiraishi, H., and Nakasugi, O.: Bisphenol A in hazardous waste landfill leachates. Chemosphere, 42, 415–418 (2001). 3. Alexander, H. C., Dill, L. W., Smith, L. W., Guiney, P. D.,
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