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Removal of phenolic endocrine disruptors by Portulaca oleracea
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 103, No. 5, 420–426. 2007 DOI: 10.1263/jbb.103.420
© 2007, The Society for Biotechnology, Japan
Removal of Phenolic Endocrine Disruptors by Portulaca oleracea Sofue Imai,1 Atsuhiko Shiraishi,1 Kazuaki Gamo,1 Ippei Watanabe,1 Hiroshi Okuhata,2 Hitoshi Miyasaka,2 Kazunori Ikeda,3 Takeshi Bamba,1 and Kazumasa Hirata1* Department of Applied Environmental Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan,1 The Kansai Electric Power Co., Inc., Environmental Research Center, 1-7 Hikaridai, Seikacho, Sourakugun, Kyoto 619-0237, Japan,2 and The General Environmental Technos Co., Ltd., 1-3-5 Azuchi-cho, Chuo-ku, Osaka 541-0052, Japan3 Received 12 December 2006/Accepted 7 February 2007
Portulaca oleracea, a garden plant prevalent from spring to autumn in Japan, showed the ability to efficiently remove from water bisphenol A (BPA), which is well known as an endocrine disrupting compound (EDC) having estrogenic properties. In water culture, 50 µM BPA was almost completely removed within 24 h when the ratio of whole plant weight to the water volume was set up at 1 g to 25 ml. The estrogenic activity of the water decreased in parallel with the elimination of BPA. This plant also rapidly removed other EDCs having a phenol group including octylphenol (OP), nonylphenol (NP), 2,4-dichlorophenol (2,4-DCP) and 17β-estradiol and, thereby, removed the endocrine disrupting activities. In addition, the ability of P. oleracea to remove BPA was not affected by BPA concentration (up to 250 µM), by cultivation in the dark, by temperatures ranging from 15°C to 30°C, or by pH ranging from 4 to 7. Moreover, the ability of P. oleracea to individually remove BPA, NP, and OP was the same as when they were all present. These results suggest that P. oleracea is a promising material for practical phytoremediation of landfill leachates and industrial wastewater contaminated with the tested EDCs. [Key words: phytoremediation, endocrine disrupting compounds, bisphenol A, Portulaca oleracea, estrogenic activity]
have described the degradation of BPA by microorganisms, and several strains have shown a propensity for high BPA degradability. For example, Streptomyces sp. isolated from river water degrades 90% of 1 ppm BPA within 10 d (3). However, the practical application of such microorganisms to polluted sites might be difficult because it is necessary to add external nutrients such as carbon sources. Further, it is necessary to control the environmental conditions including maintaining the biological correlations to native organisms in order for microorganisms to retain their high degradation abilities. Phytoremediation, the use of higher plants in bioremediation, has been recently proposed as an innovative technology for removal of pollutants from soil and water. Unlike microorganisms, addition of external nutrients and maintaining control of environmental conditions are not essential to achieve removal of pollutants by plants. This is because plants can grow photoautotrophically and maintain their metabolic activities even under poor nutrient conditions and in the presence of native organisms. In addition, this technique is expected to be carried out at a lower cost and to be more publicly acceptable than physicochemical methods or bioremediation using microorganisms (4, 5). In general, however, the removal rates of pollutants by plants are much lower than these conventional methods, and it often takes several years or more to complete remediation. This is the
Recently, various toxic chemical compounds have been discharged into the environment along with our industrial activities. Those having hormonal activities are called endocrine disrupting compounds (EDCs). Bisphenol A (BPA, Fig. 1) is a well-known EDC having estrogenic properties. BPA is widely used as a monomeric material for the production of polycarbonate and epoxy-phenolic resins and as a stabilizer or an antioxidant for many types of plastics such as polyvinyl chloride. Annual world production of BPA has been estimated at more than 500,000 t (1). High levels of this compound were found in leachates of waste landfills and wastewaters from plastic industries (2). It is suspected that contamination of food is caused when BPA diffuses into the environment from those wastes and by its direct migration from food and drink packaging. Therefore, establishment of an efficient BPA removal system for landfill leachates and industrial wastewaters is being demanded to avoid its threat to human health. Because BPA is difficult to remediate by conventional physicochemical methods once it diffuses into the leachates and wastewaters, bioremediation using organisms with high BPA degradability is alternatively available. Many reports * Corresponding author. e-mail: [email protected] phone: +81-(0)6-6879-8236 fax: +81-(0)6-6879-8239 420
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FIG. 1. Chemical structure of BPA and other EDCs with a phenol group.
rate-limiting step that renders phytoremediation unsuitable for treating environmental pollution. Moreover, although metabolisms of BPA by plants and plant cultured cells have been well-studied, a plant suitable for remediation of environmental pollution has never been found. From more than 100 different garden plants tested, we found that P. oleracea was the best at removing BPA very rapidly from a polluted water model. Therefore, our study characterized the ability of this plant to remove BPA from water cultures and evaluated its applicability for practical phytoremediation. MATERIALS AND METHODS Selection of plant able to efficiently remove BPA from water About 100 different garden plants were purchased from a local market in Osaka, Japan. Their roots were washed with distilled water to remove the soil completely. The plants were incubated at 25°C under continuous illumination (50 µmol s–1 m–2 per µA) while the roots were immersed in distilled water. After 24 h, they were transferred to water culture bottles containing 50 µM BPA (Nacalai Tesque, Kyoto). This solution was prepared by adding a 10 mM stock of BPA (dissolved in ethanol) into pure water, and it was named the polluted water model. The ratio of whole plant weight to water volume was approximately 1 g to 25 ml. Water models were incubated under the same conditions for 168 h. After incubation, 40 µl of the water was sampled and analyzed by HPLC (C-18 column 250 ×4.6 [Nacalai Tesque]; 40% aqueous methanol was used as the eluent at a flow rate of 1 ml/min; absorption at 280 nm was monitored). The ability of each plant to remove BPA was evaluated by determining the amount of BPA remaining in the water after 168 h. Culture of sterile P. oleracea and evaluation of its ability to remove endocrine disruptors Sterile P. oleracea was obtained by successive transfer and cultivation of shoots cut from whole plants on Murashige–Skoog medium (including vitamins) containing 1% sucrose and 0.2% Gellan-gum in a culture bottle at 25°C. The plants were incubated under the same conditions for 2 months before use. Their roots were washed with sterile distilled water, and the plants were transferred to polluted water models containing EDCs, as described above. Then, they were incubated under
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the same conditions for 96 h. Two grams of plant material were incubated in 50 ml of water containing EDCs, even when roots and shoots were separately tested. Heat treatment of roots was carried out by autoclave treatment (at 121°C, 2 atoms for 15 min). As a control, water was incubated for the indicated period in the absence of a plant. Two milliliters of the water was sampled after 0, 1, 3, 6, 12, 24, 48, 72, and 96 h of exposure to EDCs. For BPA, 2,4-dichlorophenol (2,4-DCP; Nacalai Tesque), and diethylphtharate (Nacalai Tesque), 40 µl of the water were analyzed by HPLC as described above. For octylphenol (OP; Nacalai Tesque) and nonylphenol (NP; Nacalai Tesque) 400 µl was analyzed by HPLC using a C-18 column with 75% aqueous acetonitrile as an eluent. For 17β-estradiol (Nacalai Tesque), 400 µl was analyzed by HPLC using the same column, but with 80% aqueous methanol as an eluent. Dibuthylphtharate and diethylhexylphtharate were analyzed by gas chromatography (GC-18; Shimadzu, Kyoto). The column was INERT CAP 5 (df = 0.25 µm, 0.32 mm I.D. × 30 m; GL Science, Tokyo). The injection temperature was 250°C, and the column temperature was a gradient from 180°C to 300°C at the rate of 10°C/min. These compounds were detected using flame ionization detector (FID). Estrogenic activity assay Estrogenic activity of the polluted water models was assayed by a modified yeast two-hybrid assay (6). Water models containing 250 µM BPA were treated with P. oleracea for 96 h at 25°C. This concentration was chosen because it was the lowest amount that could be used to detect estrogenic activity using this method. Saccharomyces cerevisiae cells were inoculated on SD medium lacking tryptophan and leucine and incubated at 30°C for 40 h with shaking. Then 50 µl of this culture was added to a mixture containing 200 µl of fresh medium and 250 µl of test water. After incubation for 4 h at 30°C, β-galactosidase activities of the yeast were determined by the manufacturer’s protocol (Clontech Laboratories, Mountain View, CA, USA), with a slight modification as follows. The incubated yeasts were collected by centrifugation and resuspended in 200 µl of Z buffer (0.1 M sodium phosphate [pH 7.0], 10 mM KCl, 1 mM MgSO4). The suspension was incubated for 15 min at 37°C, and the enzymatic reaction was started by adding 40 µl of 4 mg/ml 2-nitrophenyl β-D-galactoside (ONPG). After incubation for 30 min at 30°C, 100 µl of 1 M Na2CO3 were added to stop the reaction. β-Galactosidase activity was calculated with the following equation: U = 1000 × ([OD415] − [1.753OD570]/([t] × [v] × [OD595]) where t is time of reaction (min), v is volume of culture used in assay (ml), OD595 is cell density at the start of the assay, OD415 is absorbance by o-nitrophenol at the end of the reaction, and OD570 is light scattering at the end of the reaction. Analysis of BPA in P. oleracea Extraction of BPA from P. oleracea was carried out by the following method. After incubation with 50 µM BPA for the indicated period, plants were washed with large amounts of distilled water and divided into roots, stems and leaves. They were individually freeze-dried and extracted with methanol (7). After evaporation to dryness, the extracts were dissolved in ethanol and analyzed by HPLC as described above.
RESULTS Removal of BPA by P. oleracea To select plants able to efficiently remove BPA from water, about 100 different garden plants were cultivated in polluted water models containing 50 µM BPA at 25°C under continuous illumination of light (50 µmol s–1 m–2 per µA). This concentration approximately corresponds to the emission standard in water for phenols, and BPA around this concentration is frequently
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FIG. 2. Photograph of culture bottle containing sterile P. oleracea.
detected in landfill leachates. Several plants could remove more than 90% BPA within 24 h. Among them, we employed P. oleracea as the material for further investigation because it is an easily cultivable garden plant capable of growing rapidly even under strong sunlight and at high temperatures. Easy maintenance of materials under outdoor conditions is very important for practical phytoremediation to achieve good results. To characterize its ability to remove BPA, sterile P. oleracea plants were used (Fig. 2). As shown in Fig. 3A, about 90%, more than 95%, and almost all of the BPA added was removed from the water after 12, 24, and 48 h, respectively. Removal of BPA did not occur in the absence of the plant. Therefore, P. oleracea seems to remove BPA very rapidly without any cooperation from co-existing bacteria. To investigate the change in endocrine-disrupting activity of BPA resulting from its removal by P. oleracea, estrogenic activity of the water was measured by a yeast-
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FIG. 4. Distribution of BPA in water and plant during its removal by P. oleracea. The water containing 50 µM BPA was incubated with plants. BPA concentrations in the water (black bars) and plant (white bars) were individually determined. Data are represented as the mean± SD (n= 3).
two-hybrid assay. As show in Fig. 3B, the activity rapidly decreased in parallel with removal of BPA. These results strongly indicate that P. oleracea alone can efficiently remove BPA and, thus, the endocrine-disrupting activity. Metabolism of BPA by P. oleracea To confirm the removal mechanism of BPA by P. oleracea, BPA concentrations in the polluted water model and in the plant itself were determined after 3 and 24 h. As shown in Fig. 4, BPA was never detected in the plant after 3 h, although 35% of BPA was eliminated from the water. After 24 h, BPA was scarcely detected both in the water and the plant. This result indicates that P. oleracea has no ability to absorb BPA on its surface or to accumulate it inside. Alternatively, it may have the ability to metabolize BPA into other compounds that have no endocrine-disrupting activity. When shoots and roots were individually examined, roots removed higher levels of BPA than shoots (Fig. 5). Moreover, heat-treated roots did
FIG. 3. Removal of BPA (A) and its estrogenic activity (B) from water by P. oleracea. Water containing 50 µM BPA was incubated with (closed circles) or without (open circles) plants. Data are represented as the mean ± SD (n= 3).
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FIG. 5. Removal of BPA by roots and shoots of P. oleracea. Whole plants were divided into roots and shoots. Then, the abilities of roots (circles), heat-treated roots (triangles), and shoots (squares) to remove 50 µM BPA were individually tested. Data are represented as the mean±SD (n= 3).
not remove BPA. These results suggest that this plant might metabolize BPA using an enzyme(s) mainly located in the root. Removal of other endocrine disruptors We investigated the ability of P. oleracea to remove several other EDCs from water. Phenol compounds OP, NP, and 2,4-DCP are suspected EDCs. Among them, OP and NP were rapidly removed by P. oleracea and mostly disappeared within 24 h in a manner similar to BPA (Fig. 6A, B). 2,4-DCP also could be rapidly removed, and 60% was eliminated after 24 h (data not shown). 17β-Estradiol is a typical female hormone, but its contamination in wastewater has attracted considerable attention because it shows strong endocrine-disrupting activity to fish and birds (8, 9). As shown in Fig. 6C, P. oleracea could also remove this EDC, although the removal rate was considerably slower than for BPA, OP, and NP. For all four of these EDCs, estrogenic activity disappeared along with their removal from the water in a similar manner to that of BPA (data not shown). We also examined the ability
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of P. oleracea to remove phthalates such as diethylphtharate, dibuthylphtharate, and diethylhexylphtharate from water. However, significant decreases in their concentrations were not observed (data not shown). Based on the above results, P. oleracea is a promising material for the removal of EDCs having phenol groups in their structure. Further investigation of plants that can remove other typical EDCs such as dioxins and PCBs is now in progress. Effects of environmental conditions on removal ability When we consider the practical application of P. oleracea for phytoremediation, it is necessary to evaluate not only the ability of the plant to remove EDCs, but also the effects of various environmental conditions at polluted sites on the plant’s ability to target EDCs. The concentrations of EDCs differ from one another depending upon the conditions of polluted sites. Therefore, the ability of P. oleracea to remove various concentrations of BPA was first evaluated. As shown in Fig. 7, BPA concentrations lower than 250 µM were almost completely removed within 24 h. Even at the highest concentration (500 µM), more than 95% of BPA could be removed after 24 h. Since more than 7.5 µmol BPA per one gram plant was eliminated after only 6 h, the average removal rate is about 1.25 µmol per one gram plant per hour. Because the highest concentration of BPA detected in landfill leachates is 74 µM (2), the ability of P. oleracea to remove EDCs is thought to be high enough to complete remediation sufficiently. In general, metabolic activity in plants depends on photosynthesis. If the removal ability markedly decreases at night, it is very disadvantageous for practical application in comparison with bioremediation using microorganisms which are expected to work day and night. Therefore, the effect of a dark period on removal ability was investigated. As shown in Fig. 8, almost the same ability to remove BPA was obtained under three different conditions: continuous light, continuous dark, and an 8 h light/16 h dark cycle. This result indicates that the removal of BPA by P. oleracea occurs independently of photosynthesis. Thus, phytoremediation using this plant can be carried out the whole day and does not requires light illumination in the night to maintain its ability. Because temperature and pH are very important factors
FIG. 6. Removal of octylphenol (OP) (A) nonylphenol (NP) (B), and 17β-estradiol (C) by P. oleracea. Water containing 50 µM OP, 40 µM NP, or 25 µM 17β-estradiol was incubated with (closed circles) or without (open circles) plants. Data are represented as the mean ±SD (n= 3).
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FIG. 7. Effect of initial concentration of BPA on its removal by P. oleracea. Water containing 50 µM (open circles), 100 µM (closed circles), 250 µM (open triangles), or 500 µM (closed triangles) BPA was incubated with plants. Data are represented as the mean ± SD (n= 3).
FIG. 8. Effect of light condition on removal of BPA by P. oleracea. Water containing 50 µM BPA was incubated with plants under continuous illumination of light (closed circles), continuous dark (open circles), or an 8 h light and 16 h dark cycle (closed triangles). Data are represented as the mean ± SD (n= 3).
affecting the ability of plant materials to remove pollutants at practical polluted sites, the effects of them were investigated. When P. oleracea was cultivated at different temperatures, 15°C, 25°C, and 30°C, the rate of BPA removal at 15°C decreased to about half of that at 25°C. On the contrary, the rate increased slightly at 30°C (Fig. 9). After 24 h, however, BPA was almost completely eliminated from the water at all three temperatures. With regard to pH as shown in Fig. 10, the removal abilities at pHs 5 and 6 were about two times higher than that at pH 7. At pH 4, the ability was still higher than pH 7, but it markedly decreased at pHs higher than 7. These results indicate that the ability of P. oleracea to remove BPA from water was not affected by
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FIG. 9. Effect of temperature on removal of BPA by P. oleracea. Water containing 50 µM BPA was incubated with plants at 15°C (closed circles), 25°C (open circles), or 35°C (closed triangles). Data are represented as the mean ± SD (n= 3).
FIG. 10. Effect of pH on removal of BPA by P. oleracea. Water containing 50 µM BPA was incubated with plants for 12 h in the presence of 20 mM citrate buffer at pH from 4 to 6 (circles), 20 mM MES buffer at pH 6 to 7 (triangles), or 20 mM HEPES buffer at pH from 7 to 9 (squares).
temperatures ranging from 15°C to 30°C. On the other hand, the optimal pH for BPA removal was 6. Further, the acidic side was more preferable for removal of BPA than the alkaline side. At practical polluted sites, it is suspected that pollution with plural EDCs frequently occurs. Therefore, the effect of EDC co-existence on the ability of P. oleracea to remove each EDC was also investigated. Even in the presence of 50 µM BPA, 25 µM NP, and 25 µM OP, all EDCs were almost completely eliminated within 24 h (Fig. 11). This indicates that the ability of P. oleracea to remove each EDC was not affected by the simultaneous co-existence of other EDCs. Based on these results, P. oleracea has a high potential for practical application to landfill leachates and industrial
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FIG. 11. Removal of co-existing BPA, octylphenol (OP), and nonylphenol (NP) by P. oleracea. Water containing 50 µM BPA (closed circles), 25 µM OP (open circles) or 25 µM NP (closed triangles) was incubated with plants.
wastewaters including relatively higher concentrations of BPA. To extend the applicability to whole seasons and more varied wastewaters, it is necessary to evaluate the effect of wider temperature ranges and to solve the problem of its reduced ability at high pHs. DISCUSSION We found that P. oleracea, a common garden plant, can efficiently remove BPA from water after examining about 100 different plants. From water containing 50 µM BPA, most was removed within 24 h. Further, the endocrine-disrupting activity disappeared simultaneously (Fig. 3A, B). Such high removal ability has never been found in any plant materials previously reported, although direct comparison is quite difficult because experimental conditions in those reports were different from those employed in this study (7, 10–15). Moreover, OP, NP, 2,4-DCP, and 17β-estradiol
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were also rapidly eliminated in parallel with endocrine-disrupting activities (Fig. 6A–C). These five EDCs commonly contain a phenol group in their structure. Because no evidence of absorption or accumulation of BPA in the plant was observed during the removal period (Fig. 4), BPA may be metabolized into compounds having no endocrine-disrupting activity. Enzymes mainly located in the root may catalyze this metabolism since heat-treated root could not remove BPA. On the other hand, this plant scarcely removes phthalates, which are also well known as EDCs but contain no phenol group. These results suggest that P. oleracea may possess the enzyme(s) that specifically metabolizes EDCs containing a phenol group. In the polluted water model containing BPA, several compounds were detected by HPLC analysis during treatment with P. oleracea. The concentration of one of these compounds increased along with decreasing BPA until after 6 h and then decreased and almost disappeared after 48 h. This observation suggests that this unknown compound, named product A, may be the first metabolite of BPA that is further metabolized into other compounds. By the EI-MS analysis of product A, the molecular ion peak at m/z 244 was detected. Therefore, product A is estimated as a hydroxylation product of BPA, probably 4[1-(4-hydroxyphenyl)-1-methylethyl]-benzen-1,2-diol (Fig. 12). In previous reports, the common BPA metabolic pathway in plant materials consists of complex reactions of hydroxylation at 2 and 2′ and glycosylation of original (1 and 1′) or newly formed (2 and 2′) hydroxyl groups (13). On the other hand, Yoshida et al. detected monoquinone and bisquinone derivatives were detected when BPA was treated with the crude enzyme fraction of potato (16, 17). Because these quinone derivatives were not found in this study, the first metabolizing reaction of BPA is thought to be hydroxylation at the 2 site and this intermediate may be further hydroxylated and/or glucosylated as described above. To evaluate the applicability of P. oleracea for phytoremediation of polluted wastewater, the effects of various environmental conditions on the removal ability were investigated. This plant could remove BPA almost completely within 24 h up to a concentration of 250 µM (Fig. 7). Re-
FIG. 12. EI-MS spectrum of product A and pathway for hydroxylation of BPA by plant materials reported in previous studies (13, 15).
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moval was unaffected by light cycles (Fig. 8) or temperature fluctuations ranging from 15°C to 30°C (Fig. 9). Optimal pHs ranged from 4 to 7 (Fig. 10). Decrease in removal rate of BPA at alkaline side may be caused by suppression of enzyme activity for BPA metabolism, because BPA was completely soluble at alkaline side and similar decrease in the rate was also observed when BPA was treated with the crude enzyme fraction of P. oleracea roots. Finally, the presence of other phenolic EDCs did not affect EDC removal from water (Fig. 11). The maximum removal rate obtained in this study was about 1.25 µmol BPA per one gram plant per hour (Fig. 7). Therefore, P. oleracea is a promising material for efficient phytoremediation of BPA and other EDCs containing a phenol group from landfill leachates and industrial wastewaters. Moreover, this plant might be applicable to common water sources and soil where the contamination with those EDCs is suspected but are in concentrations much lower than their emission standards, for example, passive remediation such as a biotope system for wastewater or cultivation on polluted soil by using flowering plants ACKNOWLEDGMENTS We express our thanks to Assoc. Prof. J. Nishikawa, Osaka University, for advice on yeast-two-hybrid assay. A part of this study was carried out as the Regional New Consortium Project on “Development of advanced wastewater treatment system using recycled filter elements and plants” which was entrusted by the Ministry of Economy, Trade and Industry, Japan (METI), the Kansai Bureau Economy, Trade and Industry (METIKANSAI), and NPO Kinki Bio-industry Development Organization (KBDO).
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Removal of Phenolic Endocrine Disruptors by Portulaca oleracea Sofue Imai,1 Atsuhiko Shiraishi,1 Kazuaki Gamo,1 Ippei Watanabe,1 Hiroshi Okuhata,2 Hitoshi Miyasaka,2 Kazunori Ikeda,3 Takeshi Bamba,1 and Kazumasa Hirata1* Department of Applied Environmental Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan,1 The Kansai Electric Power Co., Inc., Environmental Research Center, 1-7 Hikaridai, Seikacho, Sourakugun, Kyoto 619-0237, Japan,2 and The General Environmental Technos Co., Ltd., 1-3-5 Azuchi-cho, Chuo-ku, Osaka 541-0052, Japan3 Received 12 December 2006/Accepted 7 February 2007
Portulaca oleracea, a garden plant prevalent from spring to autumn in Japan, showed the ability to efficiently remove from water bisphenol A (BPA), which is well known as an endocrine disrupting compound (EDC) having estrogenic properties. In water culture, 50 µM BPA was almost completely removed within 24 h when the ratio of whole plant weight to the water volume was set up at 1 g to 25 ml. The estrogenic activity of the water decreased in parallel with the elimination of BPA. This plant also rapidly removed other EDCs having a phenol group including octylphenol (OP), nonylphenol (NP), 2,4-dichlorophenol (2,4-DCP) and 17β-estradiol and, thereby, removed the endocrine disrupting activities. In addition, the ability of P. oleracea to remove BPA was not affected by BPA concentration (up to 250 µM), by cultivation in the dark, by temperatures ranging from 15°C to 30°C, or by pH ranging from 4 to 7. Moreover, the ability of P. oleracea to individually remove BPA, NP, and OP was the same as when they were all present. These results suggest that P. oleracea is a promising material for practical phytoremediation of landfill leachates and industrial wastewater contaminated with the tested EDCs. [Key words: phytoremediation, endocrine disrupting compounds, bisphenol A, Portulaca oleracea, estrogenic activity]
have described the degradation of BPA by microorganisms, and several strains have shown a propensity for high BPA degradability. For example, Streptomyces sp. isolated from river water degrades 90% of 1 ppm BPA within 10 d (3). However, the practical application of such microorganisms to polluted sites might be difficult because it is necessary to add external nutrients such as carbon sources. Further, it is necessary to control the environmental conditions including maintaining the biological correlations to native organisms in order for microorganisms to retain their high degradation abilities. Phytoremediation, the use of higher plants in bioremediation, has been recently proposed as an innovative technology for removal of pollutants from soil and water. Unlike microorganisms, addition of external nutrients and maintaining control of environmental conditions are not essential to achieve removal of pollutants by plants. This is because plants can grow photoautotrophically and maintain their metabolic activities even under poor nutrient conditions and in the presence of native organisms. In addition, this technique is expected to be carried out at a lower cost and to be more publicly acceptable than physicochemical methods or bioremediation using microorganisms (4, 5). In general, however, the removal rates of pollutants by plants are much lower than these conventional methods, and it often takes several years or more to complete remediation. This is the
Recently, various toxic chemical compounds have been discharged into the environment along with our industrial activities. Those having hormonal activities are called endocrine disrupting compounds (EDCs). Bisphenol A (BPA, Fig. 1) is a well-known EDC having estrogenic properties. BPA is widely used as a monomeric material for the production of polycarbonate and epoxy-phenolic resins and as a stabilizer or an antioxidant for many types of plastics such as polyvinyl chloride. Annual world production of BPA has been estimated at more than 500,000 t (1). High levels of this compound were found in leachates of waste landfills and wastewaters from plastic industries (2). It is suspected that contamination of food is caused when BPA diffuses into the environment from those wastes and by its direct migration from food and drink packaging. Therefore, establishment of an efficient BPA removal system for landfill leachates and industrial wastewaters is being demanded to avoid its threat to human health. Because BPA is difficult to remediate by conventional physicochemical methods once it diffuses into the leachates and wastewaters, bioremediation using organisms with high BPA degradability is alternatively available. Many reports * Corresponding author. e-mail: [email protected] phone: +81-(0)6-6879-8236 fax: +81-(0)6-6879-8239 420
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FIG. 1. Chemical structure of BPA and other EDCs with a phenol group.
rate-limiting step that renders phytoremediation unsuitable for treating environmental pollution. Moreover, although metabolisms of BPA by plants and plant cultured cells have been well-studied, a plant suitable for remediation of environmental pollution has never been found. From more than 100 different garden plants tested, we found that P. oleracea was the best at removing BPA very rapidly from a polluted water model. Therefore, our study characterized the ability of this plant to remove BPA from water cultures and evaluated its applicability for practical phytoremediation. MATERIALS AND METHODS Selection of plant able to efficiently remove BPA from water About 100 different garden plants were purchased from a local market in Osaka, Japan. Their roots were washed with distilled water to remove the soil completely. The plants were incubated at 25°C under continuous illumination (50 µmol s–1 m–2 per µA) while the roots were immersed in distilled water. After 24 h, they were transferred to water culture bottles containing 50 µM BPA (Nacalai Tesque, Kyoto). This solution was prepared by adding a 10 mM stock of BPA (dissolved in ethanol) into pure water, and it was named the polluted water model. The ratio of whole plant weight to water volume was approximately 1 g to 25 ml. Water models were incubated under the same conditions for 168 h. After incubation, 40 µl of the water was sampled and analyzed by HPLC (C-18 column 250 ×4.6 [Nacalai Tesque]; 40% aqueous methanol was used as the eluent at a flow rate of 1 ml/min; absorption at 280 nm was monitored). The ability of each plant to remove BPA was evaluated by determining the amount of BPA remaining in the water after 168 h. Culture of sterile P. oleracea and evaluation of its ability to remove endocrine disruptors Sterile P. oleracea was obtained by successive transfer and cultivation of shoots cut from whole plants on Murashige–Skoog medium (including vitamins) containing 1% sucrose and 0.2% Gellan-gum in a culture bottle at 25°C. The plants were incubated under the same conditions for 2 months before use. Their roots were washed with sterile distilled water, and the plants were transferred to polluted water models containing EDCs, as described above. Then, they were incubated under
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the same conditions for 96 h. Two grams of plant material were incubated in 50 ml of water containing EDCs, even when roots and shoots were separately tested. Heat treatment of roots was carried out by autoclave treatment (at 121°C, 2 atoms for 15 min). As a control, water was incubated for the indicated period in the absence of a plant. Two milliliters of the water was sampled after 0, 1, 3, 6, 12, 24, 48, 72, and 96 h of exposure to EDCs. For BPA, 2,4-dichlorophenol (2,4-DCP; Nacalai Tesque), and diethylphtharate (Nacalai Tesque), 40 µl of the water were analyzed by HPLC as described above. For octylphenol (OP; Nacalai Tesque) and nonylphenol (NP; Nacalai Tesque) 400 µl was analyzed by HPLC using a C-18 column with 75% aqueous acetonitrile as an eluent. For 17β-estradiol (Nacalai Tesque), 400 µl was analyzed by HPLC using the same column, but with 80% aqueous methanol as an eluent. Dibuthylphtharate and diethylhexylphtharate were analyzed by gas chromatography (GC-18; Shimadzu, Kyoto). The column was INERT CAP 5 (df = 0.25 µm, 0.32 mm I.D. × 30 m; GL Science, Tokyo). The injection temperature was 250°C, and the column temperature was a gradient from 180°C to 300°C at the rate of 10°C/min. These compounds were detected using flame ionization detector (FID). Estrogenic activity assay Estrogenic activity of the polluted water models was assayed by a modified yeast two-hybrid assay (6). Water models containing 250 µM BPA were treated with P. oleracea for 96 h at 25°C. This concentration was chosen because it was the lowest amount that could be used to detect estrogenic activity using this method. Saccharomyces cerevisiae cells were inoculated on SD medium lacking tryptophan and leucine and incubated at 30°C for 40 h with shaking. Then 50 µl of this culture was added to a mixture containing 200 µl of fresh medium and 250 µl of test water. After incubation for 4 h at 30°C, β-galactosidase activities of the yeast were determined by the manufacturer’s protocol (Clontech Laboratories, Mountain View, CA, USA), with a slight modification as follows. The incubated yeasts were collected by centrifugation and resuspended in 200 µl of Z buffer (0.1 M sodium phosphate [pH 7.0], 10 mM KCl, 1 mM MgSO4). The suspension was incubated for 15 min at 37°C, and the enzymatic reaction was started by adding 40 µl of 4 mg/ml 2-nitrophenyl β-D-galactoside (ONPG). After incubation for 30 min at 30°C, 100 µl of 1 M Na2CO3 were added to stop the reaction. β-Galactosidase activity was calculated with the following equation: U = 1000 × ([OD415] − [1.753OD570]/([t] × [v] × [OD595]) where t is time of reaction (min), v is volume of culture used in assay (ml), OD595 is cell density at the start of the assay, OD415 is absorbance by o-nitrophenol at the end of the reaction, and OD570 is light scattering at the end of the reaction. Analysis of BPA in P. oleracea Extraction of BPA from P. oleracea was carried out by the following method. After incubation with 50 µM BPA for the indicated period, plants were washed with large amounts of distilled water and divided into roots, stems and leaves. They were individually freeze-dried and extracted with methanol (7). After evaporation to dryness, the extracts were dissolved in ethanol and analyzed by HPLC as described above.
RESULTS Removal of BPA by P. oleracea To select plants able to efficiently remove BPA from water, about 100 different garden plants were cultivated in polluted water models containing 50 µM BPA at 25°C under continuous illumination of light (50 µmol s–1 m–2 per µA). This concentration approximately corresponds to the emission standard in water for phenols, and BPA around this concentration is frequently
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FIG. 2. Photograph of culture bottle containing sterile P. oleracea.
detected in landfill leachates. Several plants could remove more than 90% BPA within 24 h. Among them, we employed P. oleracea as the material for further investigation because it is an easily cultivable garden plant capable of growing rapidly even under strong sunlight and at high temperatures. Easy maintenance of materials under outdoor conditions is very important for practical phytoremediation to achieve good results. To characterize its ability to remove BPA, sterile P. oleracea plants were used (Fig. 2). As shown in Fig. 3A, about 90%, more than 95%, and almost all of the BPA added was removed from the water after 12, 24, and 48 h, respectively. Removal of BPA did not occur in the absence of the plant. Therefore, P. oleracea seems to remove BPA very rapidly without any cooperation from co-existing bacteria. To investigate the change in endocrine-disrupting activity of BPA resulting from its removal by P. oleracea, estrogenic activity of the water was measured by a yeast-
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FIG. 4. Distribution of BPA in water and plant during its removal by P. oleracea. The water containing 50 µM BPA was incubated with plants. BPA concentrations in the water (black bars) and plant (white bars) were individually determined. Data are represented as the mean± SD (n= 3).
two-hybrid assay. As show in Fig. 3B, the activity rapidly decreased in parallel with removal of BPA. These results strongly indicate that P. oleracea alone can efficiently remove BPA and, thus, the endocrine-disrupting activity. Metabolism of BPA by P. oleracea To confirm the removal mechanism of BPA by P. oleracea, BPA concentrations in the polluted water model and in the plant itself were determined after 3 and 24 h. As shown in Fig. 4, BPA was never detected in the plant after 3 h, although 35% of BPA was eliminated from the water. After 24 h, BPA was scarcely detected both in the water and the plant. This result indicates that P. oleracea has no ability to absorb BPA on its surface or to accumulate it inside. Alternatively, it may have the ability to metabolize BPA into other compounds that have no endocrine-disrupting activity. When shoots and roots were individually examined, roots removed higher levels of BPA than shoots (Fig. 5). Moreover, heat-treated roots did
FIG. 3. Removal of BPA (A) and its estrogenic activity (B) from water by P. oleracea. Water containing 50 µM BPA was incubated with (closed circles) or without (open circles) plants. Data are represented as the mean ± SD (n= 3).
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FIG. 5. Removal of BPA by roots and shoots of P. oleracea. Whole plants were divided into roots and shoots. Then, the abilities of roots (circles), heat-treated roots (triangles), and shoots (squares) to remove 50 µM BPA were individually tested. Data are represented as the mean±SD (n= 3).
not remove BPA. These results suggest that this plant might metabolize BPA using an enzyme(s) mainly located in the root. Removal of other endocrine disruptors We investigated the ability of P. oleracea to remove several other EDCs from water. Phenol compounds OP, NP, and 2,4-DCP are suspected EDCs. Among them, OP and NP were rapidly removed by P. oleracea and mostly disappeared within 24 h in a manner similar to BPA (Fig. 6A, B). 2,4-DCP also could be rapidly removed, and 60% was eliminated after 24 h (data not shown). 17β-Estradiol is a typical female hormone, but its contamination in wastewater has attracted considerable attention because it shows strong endocrine-disrupting activity to fish and birds (8, 9). As shown in Fig. 6C, P. oleracea could also remove this EDC, although the removal rate was considerably slower than for BPA, OP, and NP. For all four of these EDCs, estrogenic activity disappeared along with their removal from the water in a similar manner to that of BPA (data not shown). We also examined the ability
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of P. oleracea to remove phthalates such as diethylphtharate, dibuthylphtharate, and diethylhexylphtharate from water. However, significant decreases in their concentrations were not observed (data not shown). Based on the above results, P. oleracea is a promising material for the removal of EDCs having phenol groups in their structure. Further investigation of plants that can remove other typical EDCs such as dioxins and PCBs is now in progress. Effects of environmental conditions on removal ability When we consider the practical application of P. oleracea for phytoremediation, it is necessary to evaluate not only the ability of the plant to remove EDCs, but also the effects of various environmental conditions at polluted sites on the plant’s ability to target EDCs. The concentrations of EDCs differ from one another depending upon the conditions of polluted sites. Therefore, the ability of P. oleracea to remove various concentrations of BPA was first evaluated. As shown in Fig. 7, BPA concentrations lower than 250 µM were almost completely removed within 24 h. Even at the highest concentration (500 µM), more than 95% of BPA could be removed after 24 h. Since more than 7.5 µmol BPA per one gram plant was eliminated after only 6 h, the average removal rate is about 1.25 µmol per one gram plant per hour. Because the highest concentration of BPA detected in landfill leachates is 74 µM (2), the ability of P. oleracea to remove EDCs is thought to be high enough to complete remediation sufficiently. In general, metabolic activity in plants depends on photosynthesis. If the removal ability markedly decreases at night, it is very disadvantageous for practical application in comparison with bioremediation using microorganisms which are expected to work day and night. Therefore, the effect of a dark period on removal ability was investigated. As shown in Fig. 8, almost the same ability to remove BPA was obtained under three different conditions: continuous light, continuous dark, and an 8 h light/16 h dark cycle. This result indicates that the removal of BPA by P. oleracea occurs independently of photosynthesis. Thus, phytoremediation using this plant can be carried out the whole day and does not requires light illumination in the night to maintain its ability. Because temperature and pH are very important factors
FIG. 6. Removal of octylphenol (OP) (A) nonylphenol (NP) (B), and 17β-estradiol (C) by P. oleracea. Water containing 50 µM OP, 40 µM NP, or 25 µM 17β-estradiol was incubated with (closed circles) or without (open circles) plants. Data are represented as the mean ±SD (n= 3).
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FIG. 7. Effect of initial concentration of BPA on its removal by P. oleracea. Water containing 50 µM (open circles), 100 µM (closed circles), 250 µM (open triangles), or 500 µM (closed triangles) BPA was incubated with plants. Data are represented as the mean ± SD (n= 3).
FIG. 8. Effect of light condition on removal of BPA by P. oleracea. Water containing 50 µM BPA was incubated with plants under continuous illumination of light (closed circles), continuous dark (open circles), or an 8 h light and 16 h dark cycle (closed triangles). Data are represented as the mean ± SD (n= 3).
affecting the ability of plant materials to remove pollutants at practical polluted sites, the effects of them were investigated. When P. oleracea was cultivated at different temperatures, 15°C, 25°C, and 30°C, the rate of BPA removal at 15°C decreased to about half of that at 25°C. On the contrary, the rate increased slightly at 30°C (Fig. 9). After 24 h, however, BPA was almost completely eliminated from the water at all three temperatures. With regard to pH as shown in Fig. 10, the removal abilities at pHs 5 and 6 were about two times higher than that at pH 7. At pH 4, the ability was still higher than pH 7, but it markedly decreased at pHs higher than 7. These results indicate that the ability of P. oleracea to remove BPA from water was not affected by
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FIG. 9. Effect of temperature on removal of BPA by P. oleracea. Water containing 50 µM BPA was incubated with plants at 15°C (closed circles), 25°C (open circles), or 35°C (closed triangles). Data are represented as the mean ± SD (n= 3).
FIG. 10. Effect of pH on removal of BPA by P. oleracea. Water containing 50 µM BPA was incubated with plants for 12 h in the presence of 20 mM citrate buffer at pH from 4 to 6 (circles), 20 mM MES buffer at pH 6 to 7 (triangles), or 20 mM HEPES buffer at pH from 7 to 9 (squares).
temperatures ranging from 15°C to 30°C. On the other hand, the optimal pH for BPA removal was 6. Further, the acidic side was more preferable for removal of BPA than the alkaline side. At practical polluted sites, it is suspected that pollution with plural EDCs frequently occurs. Therefore, the effect of EDC co-existence on the ability of P. oleracea to remove each EDC was also investigated. Even in the presence of 50 µM BPA, 25 µM NP, and 25 µM OP, all EDCs were almost completely eliminated within 24 h (Fig. 11). This indicates that the ability of P. oleracea to remove each EDC was not affected by the simultaneous co-existence of other EDCs. Based on these results, P. oleracea has a high potential for practical application to landfill leachates and industrial
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FIG. 11. Removal of co-existing BPA, octylphenol (OP), and nonylphenol (NP) by P. oleracea. Water containing 50 µM BPA (closed circles), 25 µM OP (open circles) or 25 µM NP (closed triangles) was incubated with plants.
wastewaters including relatively higher concentrations of BPA. To extend the applicability to whole seasons and more varied wastewaters, it is necessary to evaluate the effect of wider temperature ranges and to solve the problem of its reduced ability at high pHs. DISCUSSION We found that P. oleracea, a common garden plant, can efficiently remove BPA from water after examining about 100 different plants. From water containing 50 µM BPA, most was removed within 24 h. Further, the endocrine-disrupting activity disappeared simultaneously (Fig. 3A, B). Such high removal ability has never been found in any plant materials previously reported, although direct comparison is quite difficult because experimental conditions in those reports were different from those employed in this study (7, 10–15). Moreover, OP, NP, 2,4-DCP, and 17β-estradiol
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were also rapidly eliminated in parallel with endocrine-disrupting activities (Fig. 6A–C). These five EDCs commonly contain a phenol group in their structure. Because no evidence of absorption or accumulation of BPA in the plant was observed during the removal period (Fig. 4), BPA may be metabolized into compounds having no endocrine-disrupting activity. Enzymes mainly located in the root may catalyze this metabolism since heat-treated root could not remove BPA. On the other hand, this plant scarcely removes phthalates, which are also well known as EDCs but contain no phenol group. These results suggest that P. oleracea may possess the enzyme(s) that specifically metabolizes EDCs containing a phenol group. In the polluted water model containing BPA, several compounds were detected by HPLC analysis during treatment with P. oleracea. The concentration of one of these compounds increased along with decreasing BPA until after 6 h and then decreased and almost disappeared after 48 h. This observation suggests that this unknown compound, named product A, may be the first metabolite of BPA that is further metabolized into other compounds. By the EI-MS analysis of product A, the molecular ion peak at m/z 244 was detected. Therefore, product A is estimated as a hydroxylation product of BPA, probably 4[1-(4-hydroxyphenyl)-1-methylethyl]-benzen-1,2-diol (Fig. 12). In previous reports, the common BPA metabolic pathway in plant materials consists of complex reactions of hydroxylation at 2 and 2′ and glycosylation of original (1 and 1′) or newly formed (2 and 2′) hydroxyl groups (13). On the other hand, Yoshida et al. detected monoquinone and bisquinone derivatives were detected when BPA was treated with the crude enzyme fraction of potato (16, 17). Because these quinone derivatives were not found in this study, the first metabolizing reaction of BPA is thought to be hydroxylation at the 2 site and this intermediate may be further hydroxylated and/or glucosylated as described above. To evaluate the applicability of P. oleracea for phytoremediation of polluted wastewater, the effects of various environmental conditions on the removal ability were investigated. This plant could remove BPA almost completely within 24 h up to a concentration of 250 µM (Fig. 7). Re-
FIG. 12. EI-MS spectrum of product A and pathway for hydroxylation of BPA by plant materials reported in previous studies (13, 15).
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moval was unaffected by light cycles (Fig. 8) or temperature fluctuations ranging from 15°C to 30°C (Fig. 9). Optimal pHs ranged from 4 to 7 (Fig. 10). Decrease in removal rate of BPA at alkaline side may be caused by suppression of enzyme activity for BPA metabolism, because BPA was completely soluble at alkaline side and similar decrease in the rate was also observed when BPA was treated with the crude enzyme fraction of P. oleracea roots. Finally, the presence of other phenolic EDCs did not affect EDC removal from water (Fig. 11). The maximum removal rate obtained in this study was about 1.25 µmol BPA per one gram plant per hour (Fig. 7). Therefore, P. oleracea is a promising material for efficient phytoremediation of BPA and other EDCs containing a phenol group from landfill leachates and industrial wastewaters. Moreover, this plant might be applicable to common water sources and soil where the contamination with those EDCs is suspected but are in concentrations much lower than their emission standards, for example, passive remediation such as a biotope system for wastewater or cultivation on polluted soil by using flowering plants ACKNOWLEDGMENTS We express our thanks to Assoc. Prof. J. Nishikawa, Osaka University, for advice on yeast-two-hybrid assay. A part of this study was carried out as the Regional New Consortium Project on “Development of advanced wastewater treatment system using recycled filter elements and plants” which was entrusted by the Ministry of Economy, Trade and Industry, Japan (METI), the Kansai Bureau Economy, Trade and Industry (METIKANSAI), and NPO Kinki Bio-industry Development Organization (KBDO).
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