Advances in Chemical Engi neering and Science , 2011, 1, 90-95
doi:10.4236/aces.2011.13015 Published Online July 2011 (http://www.SciRP.org/journal/aces)
Copyright © 2011 SciRes. ACES
Bioremediation of Bisphenol A by Glycosylation with
Immobilized Marine Microalga Amphidinium crassum
Bioremediation of Bisphenol A by Immobilized Cells
Kei Shimoda1*, Ryohei Yamamoto2, Hiroki Hamada2*
1Department of Chemistry, Faculty of Medicine, Oita University, Oita, Japan
2Department of Life Science, Faculty of Science , Okayama Uni vers i t y of Sc ience, Okayama, Japan
E-mail: *shimoda@med.oita-u.ac.jp, *hamada@dls.ous.ac.jp
Received April 14, 2011; revised May 17, 2011; accepted May 26, 2011
Abstract
Glycosylation of bisphenol A, which is an endocrine disrupting chemical, was investigated using immobi-
lized marine microalga and plant cells from the viewpoint of bioremediation of bisphenol A. Immobilized
marine microalga of Amphidinium crassum glucosylated bisphenol A to the corresponding glucoside. On the
other hand, bisphenol A was glycosylated to its glucoside, diglycoside, gentiobioside, and gentiobiosylgluco-
side, which was a new compound, by immobilized plant cells of Catharanthus roseus.
Keywords: Glycosylation, Biosphenol A, Amphidinium crassum, Catharanthus roseus, Immobilized Cells
1. Introduction
Bisphenol A is widely used as the starting material for the
production of polyacrylates, ether resins, phenol resins,
photostabilizers, insecticides, fragrance ingredients, ag-
ricultural chemicals, pharmaceuticals, and coatings, and
are released as pollutants and toxic compounds into rivers
and seas [1]. Recently, bisphenol A has attracted consid-
erable attention as it exhibited estrogenic activity in bio-
assays [2] and has been listed among “chemicals sus-
pected of having endocrine disrupting effects” by the
World Wildlife Fund, the National Institute of Environ-
mental Health Sciences in the USA and the Japanese En-
vironment Agency. From the viewpoint of pollution con-
trol, many studies on the biological metabolites of aro-
matic compounds have been reported, e.g., the benzene
rings of aromatic compounds are degraded through the
gentisic acid intermediate by some soil bacteria [3-8].
However, little attention has been paid to the biological
degradation of endocrine disrupting chemicals. On the
other hand, the metabolic pathway of aromatic compounds
in plant cells is quite different from that in microorganisms;
plant cells glycosylate phenols and accumulate them as
glycosides in the cells [9-15].
Recently, the biotransformation of exogenous sub-
strates by cultured marine microalga and plant cells has
been reported [16,17]. These cells have the abilities of
hydroxylation, glycosylation, oxido-reduction, hydrogena-
tion, and hydrolysis for various organic compounds. Par-
ticularly, glycosylation seems to be an efficient procedure
for the bioremediation of environmental pollution, be-
cause the estrogenicity of endocrine disrupting compound,
i.e., bisphenol A, was eliminated by formation of its
glycosides [18]. This paper describes the glycosylation of
bisphenol A by the immobilized marine microalga of Am-
phidinium crassum and immobilized plant cells of Ca-
tharanthus roseus.
2. Experimental
2.1. General
Bisphenol A was purchased from Aldrich Chemical Co.
The 1H and 13C NMR, H-H COSY, C-H COSY, and
HMBC spectra were recorded in CD3OD using a Varian
XL-400 spectrometer (Varian Inc.). The chemical shifts
were expressed in δ (ppm) referring to tetramethylsilane.
The FABMS spectra were measured using a JEOL
MStation JMS-700 spectrometer (JEOL Ltd.). HPLC
was carried out on a YMC-Pack R&D ODS column (150
× 30 mm) at 25˚C [solvent: methanol-water (9:11, v/v);
detection: UV (280 nm); flow rate: 1.0 ml/min].
2.2. Cell Line and Culture Conditions
A. crassum, a gift from Ehime Prefectural Fisheries Ex-
K. SHIMODA ET AL.
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91
perimental Station, Japan, cells were cultivated in a syn-
thetic seawater (500 ml) for 2 weeks at 20˚C with con-
stant aeration by air (1 l/min) in 1 l flasks under illumi-
nation (1000 lx). The synthetic seawater contained 20.747
g NaCl, 0.8 g MnCl2·4H2O, 9.474 g MgCl2·6H2O, 1.326
g CaCl2·6H2O, 3.505 g Na2SO4, 597 mg KCl, 171 mg
NaHCO3, 85mg KBr, 34mg Na2B4O7·10H2O, 12 mg SrCl2,
3 mg NaF, 1 mg LiCl, 0.07 mg KI, 0.2g CoCl2·6H2O, 8
g AlCl3·6H2O, 5 g FeCl3·6H2O, 0.2 g Na2WO4·2H2O,
0.02 mg (NH4)6Mo7O24, 0.0045% Na2SiO 3 and 1.07 ml of
NM solution per 1 l of distilled water. The NM solution
(1 l) is a kind of vitamin solutions and composed of
NaNO3 (150 g), Na2HO4 (10 g), EDTA-2Na (0.9 g), Vi-
tamin B12 (1.5 mg), thiamine·HCl (75 mg), biotin (1 mg),
EDTA-Fe (2.5 g), and H2NC(CH3OH)3 (5 g) in distilled
water.
The cultured plant cells of C. roseus have been culti-
vated over 20 years in our laboratory and subcultured in
300 ml conical flasks containing Schenk and Hildebrand
(SH) medium (100 ml, pH 5.7) on a rotary shaker (120
rpm) at 25˚C in the dark for every 3 - 5 weeks. Part of
the callus tissues (fresh weight 30 g) was transplanted to
freshly prepared SH medium (100 ml in a 500 ml conical
flask, pH 5.7) containing 3% sucrose and was incubated
for 3 weeks prior to use for this work.
2.3. Glycosylation of Bisphenol A by A. crassum
and C. roseus
Cultured A. crassum cells were harvested by centrifuga-
tion at 3000 rpm for 15 min and washed twice by adding
100 ml of synthetic seawater followed by centrifugation
(3000 rpm for 15 min). To the 500 ml flask containing 9
g of cultured A. crassum cells and 300 ml of a synthetic
seawater was added 0.2 mmol of bisphenol A. The cul-
tures were incubated at 20˚C on a rotary shaker (120
rpm) for five days under illumination (1000 lx). After the
incubation period, the cells and synthetic seawater were
separated by centrifugation at 1000 g for 15 min. The syn-
thetic seawater was extracted with ethylacetate and then
n-butanol. The cells were extracted (three times) by ho-
mogenization with methanol, and the methanol fraction
was concentrated and partitioned between water and
ethylacetate. The ethylacetate fractions were analyzed by
HPLC, combined, and concentrated. The water and n-
butanol fractions were analyzed by HPLC, combined,
evaporated, and re-dissolved in water. This water fraction
was applied to a Diaion HP-20 column and the column
was washed with water followed by elution with metha-
nol. The methanol eluate was subjected to preparative
HPLC [column: CAPCELLPAK R&D C18 column (250
× 30 mm); solvent: MeOH: H2O (9:11, v/v); detection:
UV (340 nm); flow rate: 1.0 ml/min] to give glycosyla-
tion products.
Bisphenol A (0.2 mmol) was administered to the 500
ml flask containing 300 ml of SH medium and 70 g of
the suspension cultured cells of C. roseus, and the cul-
tures were incubated at 25˚C for five days on a rotary
shaker (120 rpm) under illumination (1000 lx). After the
incubation, the cells and medium were separated by fil-
tration with suction. The filtered medium was extracted
with EtOAc. The medium was further extracted with n-
BuOH. The cells were extracted (x3) by homogenization
with MeOH. The MeOH fraction was concentrated and
partitioned between H2O and EtOAc. The EtOAc fractions
were combined and concentrated. The H2O fraction was
applied to a Dianion HP-20 column and the column was
washed with H2O followed by elution with MeOH. The
MeOH eluate was subjected to HPLC to give products.
Spectral data of a new compound, 2-(4--gentiobiosy-
loxphenyl)-2-(4--D-glucopyranosyloxyphenyl)propane
(5): FAB MS: m/z 737 [M + Na]+; 1H NMR (400 MHz,
CD3OD, in ppm): 1.60 (s, 6H, H-1, 3), 3.25-3.90 (m,
18H, H-2', 3', 4', 5', 6', 2'', 3'', 4'', 5'', 6'', 2''', 3''', 4''', 5''',
6'''), 4.50 (d, 1H, J=7.6 Hz, H-1'''), 4.86 (d, 1H, J=8.0 Hz,
H-1'), 4.87 (d, 2H, J = 8.0 Hz, H-1''), 6.95 (d, 2H, J = 8.5
Hz, H-12, 14), 7.00 (d, 2H, J = 8.5 Hz, H-6, 8), 7.12 (d,
2H, J = 8.5 Hz, H-11, 15), 7.15 (d, 2H, J = 8.5 Hz, H-5,
9); 13C NMR (CD3OD): 31.5 (C-1, C-3), 42.9 (C-2),
62.3, 62.5 (C-6', C-6'''), 68.8 (C-6''), 71.2, 71.5 (C-4', C-4'',
C-4'''), 75.0, 75.1 (C-2', C-2'', C-2'''), 77.7, 78.0, 78.1
(C-3', C-3'', C-3'''), 78.2, 78.3, 78.8 (C-5', C-5'', C-5'''),
102.5 (C-1', C-1''), 105.1 (C-1'''), 117.0 (C-12, C-14),
117.2 (C-6, C-8), 128.1 (C-11, C-15), 128.7 (C-5, C-9),
146.1 (C-10), 146.8 (C-4), 157.0 (C-13), 157.6 (C-7).
2.4. Preparation of Immobilized A. crassum and
C. roseus in Sodium Alginate Gel
Sodium alginate (2%) was suspended in water (500 ml),
which was autoclaved at 120˚C for 30 min. The cultured
cells in the stationary growth phase have been used for
experiments. Cultured cells of A. crassum (9 g) and C.
roseus (70 g) were individually added to this solution
and the mixture was stirred for 2 h until it became ho-
mogeneous. The suspension was added dropwise from a
dropping funnel with a glass tube into a 5% CaCl2 solu-
tion (1 l) with stirring to form pieces of spherical sodium
alginate gel with 5 mm diameter immediately. Washing
with water gave each immobilized cells of A. crassum
and C. roseus which were used for biotransformation of
bisphenol A.
2.5. Time Course Experiments
Time course experiments to examine the biotransforma-
K. SHIMODA ET AL.
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92
tion of bisphenol A by A. crassum were carried out using
eight flasks containing cultured cells (9 g) or immobi-
lized cells, which included 9 g cells. In the case of the
biotransformation by the cultured and immobilized C.
roseus cells, cultured cells (70 g) or immobilized cells,
which included 70 g cells, were partitionated to each
flask. Substrate (0.2 mmol) was administered to each of
flasks and the mixtures were incubated on a rotary shaker
at 25˚C. At a day interval, one of the flasks was taken out
from the rotary shaker, and the cells (or immobilized
cells) and medium were separated by filtration. The ex-
traction and analysis procedures were same as described
above. The yield of the products was determined on the
basis of the peak area from HPLC and expressed as a
relative percentage to the total amount of the whole reac-
tion products extracted.
3. Results and Discussions
The biotransformation product was isolated from A.
crassum cell cultures, which had been incubated with
bisphenol A (1) for five days, by a combination of Diaion
HP-20 column chromatography and preparative HPLC in
4% yield. The glycosylation product 2 was detected by
HPLC. No additional conversion products were observed
in spite of careful analyses by HPLC. Incubation of the
substrate in medium without cells gave no transformation
products. The structure of the product 2 was determined
as 2-(4--D-glucopyranosyloxyphenyl)-2-hydroxypheny-
lpropane (bisphenol A glucoside) by FABMS, 1H and 13C
NMR analyses (Figure 1). To investigate the biotrans-
formation pathway, the time course in the conversion of
1 was followed. Figure 2 showed that the amount of pro-
duct 2 increased with time during the reaction with cul-
tured A. crassum cells.
Next, A. crassum cells were immobilized with sodium
alginate at concentrations of 2%. The immobilized A.
crassum cells were incubated with bisphenol A (1) for
five days. The product 2 was obtained in 6% yield. The
time course of the conversion of bisphenol A (1) with
immobilized A. crassum cells was investigated. As shown
in Figure 3, the glycosylation activity for bisphenol A (1)
was increased and the compound 2 was produced in
higher yield in comparison with the case of the biotrans-
formation using normal cells.
On the other hand, four biotransformation products
2-5 were isolated by a combination of Diaion HP-20
column chromatography and preparative HPLC after five
days incubation of cultured plant cells of C. roseus with
bisphenol A (1). The yields of 2-5 were 5, 12, 8, and 2%.
The structures of the products 3-6 were identifined as
2,2-bis(4--D-glucopyranosyloxyphenyl)propane (di-
glucoside, 3), 2-(4--genbiobiosyloxyphenyl)-2-(4-
hydroxyphenyl) propane (gentiobioside, 4), and 2-(4--
gentiobiosylophenyl)-2-(4--D-glucopyranosyloxyph-
enyl) propane (gentiobiosylglucoside, 5) (Figure 4). The
CHO OH
CH3
CH3
COOH
CH3
CH3
O
OH
HO
HO OH
Bisphenol A (1)2
1
2
3
4
56
7
89
10
11 12
13
1415 A. crassum
Figure 1. Glycosylation of bisphenol A (1) by cultured cells of A. crassum.
50
100
48
Time/day
Figure 2. Time course of the glycosylation of bisphenol A (1) by the cultured cells of A. crassum. Yields of 1 () and 2 () are
plotted.
K. SHIMODA ET AL.
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93
50
100
48
Time/day
Figure 3. Time course of the glycosylation of bisphenol A (1) by the immobilized cells of A. crassum. Yields of 1 () and 2 ()
are plotted.
product 5 was a new compound. To investigate the bio-
transformation pathway, the time course in the conver-
sion of 1 by cultured C. roseus cells was examined.
Products 2, 3, and 4 were produced at an early stage of
incubation. On the other hand, 5 was accumulated after 3
days of incubation (Figure 5). These findings indicated
that 1 was first converted to 2-4 and further glycosylation
gave 5 as shown in Figure 4.
Immobilized C. roseusm cells were tested for their
ability to convert bisphenol A (1). The substrate, bisphe-
nol A (1), was converted into products 2-5 in 7, 17, 11,
and 4% yields by five days incubation. The time course
of the conversion of bisphenol A (1) with immobilized A.
crassum cells was investigated. As shown in Figure 6,
the products 2-5 were obtained in higher yields in com-
parison with the case of the biotransformation using nor-
CHO OH
CH3
CH3
COOH
CH3
CH3
O
OH
HO
HO OH
COO
CH3
CH3
O
OH
HO
HO OH
COOH
CH3
CH3
O
OH
HO
HO
O
O
HO
OH
OH
HO
O
OH
HO
HO OH
COO
CH3
CH3
O
OH
HO
HO
O
O
HO
OH
OH
HO
O
OH
HO
HO OH
1
2
C. roseus
3
4
5
Figure 4. Glycosylation of bisphenol A (1) by cultured cells of C. roseus.
50
100
48
Time/day
Figure 5. Time course of the glycosylation of bisphenol A (1) by the cultured cells of C. roseus. Yields of 1 (), 2 (), 3 (), 4
(), and 5 () are plotted.
K. SHIMODA ET AL.
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94
50
100
48
Time/day
Figure 6. Time course of the glycosylation of bisphenol A (1) by the immobilized cells of C. rose us. Yields of 1 (), 2 (), 3 (),
4 (), and 5 () are plotted.
mal cells.
The results of this experiment demonstrate that cul-
tured marine microalga of A. crassum converted bisphe-
nol A into its glucoside and that cultured plant cells of C.
roseus glycosylate bisphenol A to its glucoside, digluco-
side, gentiobioside, gentiobiosylglucoside.The use of
immobilized cells of both A. crassum and C. roseus in
sodium alginate gel much improved the yield of the
products.
Recently, it has been reported that freshwater micro-
alga of Pseudokirchneriella subcapitata, Scenedes-
musacutus, and Coelastrum reticulatum converted
bisphenol A into its glucoside [19]. Also, bisphenol A
was shown to be transformed to its glucoside, digluco-
side, gentiobioside, and trisaccharide, i.e., O--D-
glucopysyl-(14)-[-D-glucopyranosyl-(16)]-D-
glucopyranoside, by cultured plant cells of Nicotiana
tabacum [20]. On the other hand, recent paper revealed
that estrogenicity of bisphenol A was eliminated by for-
mation of the diglucoside and that reduced activity re-
mained in the glucoside [18]. These studies demonstrate
that metabolism of bisphenol A by freshwater microalga
and plants offers the possibility of bioremediation of con-
taminated water. The present study showed that immobi-
lized marine microalga of A. crassum and plant cells of C.
roseus are useful bioreactors for bioremediation of bis-
phenol A, which is an environmental pollutant released
into seas and rivers. Studies of the physiological activi-
ties of bisphenol A glycosides, such as gentiobioside and
gentiobiosylglucoside, are now in progress.
4. Acknowledgements
This work was supported by grant from The Salt Science
Research Foundation, No. 1105.
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