Presentation Information
[P02-229]Biodegradation and assimilation of high concentrations of Bisphenol S by a black yeast Exophiala xenobiotica strain BPS1
○Masahiro Takeo1, Taiju Fukushima1, Emina Okano1, Yuki Chinzei2, Hidehiro Ishizawa1, Seiji Negoro1 (1. Graduate School of Engineering, University of Hyogo (Japan), 2. Faculty of Engineering, University of Hyogo (Japan))
Keywords:
Bisphenol S,endocrine disrupting compound,biodegradation,black yeast,degradation pathway
[Purpose]
Bisphenol S (4,4'-dihydroxydiphenylsulfone, BPS) has been used in large quantity for the synthesis of super engineering plastics, and as color developers for thermal papers and liner materials for cans. Through such applications, BPS has widely polluted soil and aquatic environments. However, BPS has endocrine disrupting effects to higher organisms and is known to be persistent in the environment. Therefore, to understand the biodegradation mechanisms is the need of the hour. Recently, we isolated a black yeast Exophiala xenobiotica strain BPS1 as a BPS-assimilating microorganism. In this study, we introduce its high degradation and assimilation potential for BPS and show clues leading to the elucidation of the BPS degradation pathway.
[Method]
Strain BPS1 was routinely cultivated in a mineral salts medium including yeast extract at 25 ℃ and at 150 rpm on a rotary shaker. BPS or its structural analog, 2,4'-dihydroxydiphenylsulfone (24'BS) was supplemented to the medium at various concentrations as a major carbon and sulfur source. The growth of BPS1 was measured as OD600 using a spectrophotometer. For the rapid and enhanced biodegradation, the immobilized cells of BPS1 was prepared by mixing 4 mL of the cell suspension (OD600=4) and 20 mL of 3.0%(w/v) sodium alginate solution and further dropping it to 1.5%(w/v) CaCl2 solution using a syringe. The beads (3mm diameter) of the immobilized cells were added to 50 mL of 1/2-diluted LB medium in a 300-mL flask. The flask was shaken under the above-mentioned condition. The concentrations of BPS, 24'BS, and their metabolites in the culture supernatant were analyzed by HPLC (Prominent series, Shimadzu) equipped with a Mightysil RP-18 GP Aqua column (150-4.6 mm, 5 µm, Kanto Kagaku) and by GC/MS (GCMS-QP-2020 NX, Shimadzu) equipped with a RXi-5SilMS column (30 m x 0.25 mm ID, 0.25 µm, GL Science).
[Results]
Strain BPS1 was able to completely degrade and assimilate up to 5 mM (1.25g/L) BPS during one-month cultivation and showed good growth. When 6 mM BPS was supplied to the medium, the apparent growth was observed but it reduced only 2 mM BPS for the same period. Since the degradation proceeded slowly, it was difficult to detect the metabolites from BPS in the culture. Therefore, we employed an enhanced degradation test using the immobilized cells and a BPS analog 24'BS. As expected, the immobilized cells could degrade 0.8 mM 24'BS completely in three days and the culture supernatant was analyzed by HPLC or GC/MS. As a result, hydroquinone and o-phenolsulfonic acid were identified as the metabolites from 24'BS. In addition, a mono-nucleus metabolite, whose substituted group has a C-S bond, was also detected.
[Consideration]
This is the first report on the biodegradation and assimilation of such a high concentration of BPS (5 mM). In the enhanced 24'BS degradation test, the detection of hydroquinone and o-phenolsulfonic acid indicates the cleavage of the C-S bond of 24'BS. In addition, the detection of the mono-nucleus metabolite with a C-S bond suggests the cleavage of one of the two aromatic-rings of 24'BS.
[Conclusion]
In this study, we showed that Exophiala xenobiotica strain BPS1 could degrade and assimilate high concentrations of BPS. In addition, by the enhanced 24'BS degradation test, we suggest two possible BPS degradation pathways: the cleavage of the C-S bond of BPS and the cleavage of one of the two aromatic-rings of BPS.
Bisphenol S (4,4'-dihydroxydiphenylsulfone, BPS) has been used in large quantity for the synthesis of super engineering plastics, and as color developers for thermal papers and liner materials for cans. Through such applications, BPS has widely polluted soil and aquatic environments. However, BPS has endocrine disrupting effects to higher organisms and is known to be persistent in the environment. Therefore, to understand the biodegradation mechanisms is the need of the hour. Recently, we isolated a black yeast Exophiala xenobiotica strain BPS1 as a BPS-assimilating microorganism. In this study, we introduce its high degradation and assimilation potential for BPS and show clues leading to the elucidation of the BPS degradation pathway.
[Method]
Strain BPS1 was routinely cultivated in a mineral salts medium including yeast extract at 25 ℃ and at 150 rpm on a rotary shaker. BPS or its structural analog, 2,4'-dihydroxydiphenylsulfone (24'BS) was supplemented to the medium at various concentrations as a major carbon and sulfur source. The growth of BPS1 was measured as OD600 using a spectrophotometer. For the rapid and enhanced biodegradation, the immobilized cells of BPS1 was prepared by mixing 4 mL of the cell suspension (OD600=4) and 20 mL of 3.0%(w/v) sodium alginate solution and further dropping it to 1.5%(w/v) CaCl2 solution using a syringe. The beads (3mm diameter) of the immobilized cells were added to 50 mL of 1/2-diluted LB medium in a 300-mL flask. The flask was shaken under the above-mentioned condition. The concentrations of BPS, 24'BS, and their metabolites in the culture supernatant were analyzed by HPLC (Prominent series, Shimadzu) equipped with a Mightysil RP-18 GP Aqua column (150-4.6 mm, 5 µm, Kanto Kagaku) and by GC/MS (GCMS-QP-2020 NX, Shimadzu) equipped with a RXi-5SilMS column (30 m x 0.25 mm ID, 0.25 µm, GL Science).
[Results]
Strain BPS1 was able to completely degrade and assimilate up to 5 mM (1.25g/L) BPS during one-month cultivation and showed good growth. When 6 mM BPS was supplied to the medium, the apparent growth was observed but it reduced only 2 mM BPS for the same period. Since the degradation proceeded slowly, it was difficult to detect the metabolites from BPS in the culture. Therefore, we employed an enhanced degradation test using the immobilized cells and a BPS analog 24'BS. As expected, the immobilized cells could degrade 0.8 mM 24'BS completely in three days and the culture supernatant was analyzed by HPLC or GC/MS. As a result, hydroquinone and o-phenolsulfonic acid were identified as the metabolites from 24'BS. In addition, a mono-nucleus metabolite, whose substituted group has a C-S bond, was also detected.
[Consideration]
This is the first report on the biodegradation and assimilation of such a high concentration of BPS (5 mM). In the enhanced 24'BS degradation test, the detection of hydroquinone and o-phenolsulfonic acid indicates the cleavage of the C-S bond of 24'BS. In addition, the detection of the mono-nucleus metabolite with a C-S bond suggests the cleavage of one of the two aromatic-rings of 24'BS.
[Conclusion]
In this study, we showed that Exophiala xenobiotica strain BPS1 could degrade and assimilate high concentrations of BPS. In addition, by the enhanced 24'BS degradation test, we suggest two possible BPS degradation pathways: the cleavage of the C-S bond of BPS and the cleavage of one of the two aromatic-rings of BPS.
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