Journal of Hazardous Materials 178 (2010) 777–785

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Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Bisphenol A removal by the Dracaena plant and the role of plant-associating

bacteria
S. Saiyood a , A.S. Vangnai b,c , P. Thiravetyan d , D. Inthorn a,∗
a
Department of Environmental Health Sciences, Center of Environmental health, Toxicology and Management of Chemicals (ETM), Mahidol University, Bangkok 10400, Thailand
b
Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
c
National Center of Excellence for Environmental and Hazardous Waste Management (NCE-EHWM), Chulalongkorn University, Bangkok 10330, Thailand
d
School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkok 10150, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Dracaena sanderiana and Dracaena fragrans plants, as representatives of native, tropical, evergreen plants
Received 16 November 2009 with fibrous root systems, were evaluated for bisphenol A (BPA) tolerance and uptake capability. D. sande-
Received in revised form 2 February 2010 riana demonstrated significantly higher BPA removal capability than D. fragrans. Therefore, it was chosen
Accepted 2 February 2010
for further study. D. sanderiana tolerated BPA toxicity levels up to 80 ␮M, while higher BPA concentra-
Available online 12 February 2010
tions damaged the plant. In the sterile hydroponic system with an initial BPA concentration of 20 ␮M, the
plant could uptake approximately 50% of the BPA. The plant’s ability to translocate BPA was confirmed
Keywords:
by the detection of BPA that accumulated at the roots and stems, but not at the leaves of the plant. Upon
Bisphenol A
Dracaena plant
BPA exposure, the D. sanderiana secreted extracellular plant mucilage as a protective barrier to the toxic
Plant-associating bacteria compound. In the non-sterile treatment, the BPA dissipation was contributed not only by the D. sande-
Plant-associating Enterobacter sp. riana plant, but also by the co-existing microbes. The BPA reached 85% of the initial concentration at
Plant-associating Bacillus sp 20 ␮M. Among the six plant-associating bacterial isolates, Bacillus cereus strain BPW4 and Enterobacter
sp. strain BPW5 colonized the D. sanderiana root surface and facilitated BPA dissipation in the hydro-
ponic treatment system. In addition, the success of the BPA treatment in the hazardous waste landfill
leachate demonstrated the potential application of D. sanderiana plant in the phytoremediation of BPA
contaminated wastewater or industrial leachate.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction the biodegradation ability of microorganisms (bioremediation) or

Bisphenol A [BPA, 2,2-bis(4-hydroxyphenyl)propane] has been
used widely as an intermediate for the production of epoxy-
phenolic resins, polycarbocates, polyacrylates, plastics, food-drink
packaging coating and other specialty chemicals [1]. It has also been
used as an inert ingredient in pesticides, antioxidant, flame retar-
dant, and rubber chemicals [2]. Due to its mass production and
widespread use, the environmental release and contamination of
BPA have been found through permitted discharges of industrial
wastewater treatment systems, sewage sludge, and leachate from
waste plastic in landfills. BPA has become one of the major toxic
environmental pollutants of concern due to BPA’s acute toxicity
towards algae, invertebrates, fish within the range of 0.04–0.4 ␮M
[3], as well as its mutagenic and estrogenic effects on humans
within the range of 0.1–10 ␮M [4].
The biological treatment of environmental toxic pollutants is
an alternative remediation technique which depends on either
∗ Corresponding author. Tel.: +66 2 35 48525; fax: +66 2 35 48525.
E-mail address: phdit@mahidol.ac.th (D. Inthorn).
several mechanisms by intact higher plants (phytoremediation).
Previous reports have shown that BPA can be biodegraded by
microorganisms distributed in the environment, or metabolized
by plant and animal enzymes [5], or absorbed, biotransformed
and bioaccumulated by higher plants. For instance, a Polygonaceae
dicot plant such as a Rumex crispus could completely remove BPA
(1.7 ␮M) from the liquid medium after 15 days. It showed higher
BPA removal capability than rice under the same test conditions [6].
Moreover, the use of a plant such as Ipomoea aquatica to remove BPA
indicated a partial metabolization of BPA. Possibly, over 50% of the
initial BPA content was polymerized or tightly bound to the plant
residues [7]. Although there have been reports of BPA removal by
several plants, the success of phytoremediation depends mainly on
the suitability of the plant candidate to the contaminated environ-
ment, i.e., soil/water type and climate. Thus, the use of native plant
species towards the treatment of toxic pollutant targets has been
completed with encouragingly.
In this study, native, tropical, evergreen Dracaena plants were
tested for their ability to remove BPA. Dracaena plants are in the
Agavaceae family. They are commonly classified as tropical, ever-
green and foliage plants which can be grown indoors and outdoors.

0304-3894/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jhazmat.2010.02.008
778 S. Saiyood et al. / Journal of Hazardous Materials 178 (2010) 777–785
Propagation of the plants occurs by means of seeding or herba-
ceous stem cutting [8]. Dracaena plants are generally recognized
as sources for chemical constituents with bioactivities [9], such as
an antifungal activity of Dracaena cochinchinensis [10] in medici-
nal application. In addition, the application of Dracaena plant was
previously carried out for environmental pollutant removal. For
example, Dracaena warneckii was used to remove benzene, an
indoor volatile pollutant [11].
In the present work, the ability of two Dracaena plant species,
Dracaena sanderiana (ribbon plant) and Dracaena fragrans (corn
plant), to remove BPA was evaluated in a hydroponic system. D.
sanderiana showed higher BPA removal ability and was selected
for further study. The uptake, translocation, and accumulation of
BPA in the selected Dracaena species were illustrated. In addition,
the role of plant-associated bacteria that facilitate the remediation
of BPA was determined. Finally, a feasibility study of D. sanderiana
on the remediation of BPA from hazardous waste landfill leachate
was demonstrated.
2. Materials and methods
2.1. Chemicals and reagents
BPA (C15 H16 O2 ) (99.9% chemical purity) was purchased from
Kishada Chemical Co., Ltd. (Osaka, Japan). HPLC solvents (HPLC
grade) and other chemicals (analytical grade) were purchased from
Merck, Germany and Scharlau Chemie, Spain.
2.2. Plant species and plant cultivation conditions
Two Dracaena plant species, D. sanderiana (ribbon plant) and D.
fragrans (corn plant), were used in this study. Plants that had stems
with an approximate length of 40 cm and 10–15 leaves were pur-
chased from a plant nursery at Jatujak market in Bangkok, Thailand.
The stems were washed by tap water to remove dirt, rinsed with
distilled water and the brown leaves were removed. The washed
stems were cultivated in a hydroponic system using a 400 ml half-
strength Hoagland’s nutrient solution [12] in a 2-l glass jar. The
plant was placed 30 cm under natural luminescence using four
cool-white fluorescent lamps (25 ␮mol m−2 s−1 PAR). The plant cul-
tivation was a daily 12 h/12 h light–dark cycle at room temperature
(ca. 31 ◦ C) for 4 weeks until the roots grew. The nutrient solution
was removed and replaced with a new one once a week. The plant
was allowed to grow to 2 months in age and then the roots were
washed with distilled water several times. The initial fresh weights
were determined before the experiment was set up. At 2 months
old, each stem of D. sanderiana and D. fragrans generally had stem
diameters of approximately 0.5 and 3 cm, respectively and initial
fresh weights of approximately 20 and 200 g, respectively.
2.3. The BPA treatment using the selected plant species
The selected plant species that were 2 months in age were used
for BPA removal in a hydroponic treatment system. Prior to the
treatment, the roots and stems were washed several times with dis-
tilled water. The fresh weight of plants that weighed approximately
80 g (approximately 16 g dry weight) was transferred to a 0.8-l glass
jar that contained 400 ml of half-strength Hoagland’s nutrient solu-
tion supplemented with BPA at various concentrations, i.e., 20, 40,
60, 80, 100, 150, and 200 ␮M. The jar was covered with a sterile
cloth and aluminum foil. The plant was cultivated under the stated
conditions stated for 20 days. Every 4 days, samples were collected
for analyses including the remaining BPA, bacterial cell growth, pH
change and the formation of extracellular polysaccharide mucilage
(EPM), as described below. The plant with sterile roots and stems
immersed in a sterile BPA-containing hydroponic system was used
as a control. A sterile half-strength Hoagland’s solution with 20 ␮M
BPA without the plant was used as a natural degradation control.
BPA removal ability and BPA uptake capacity were calculated by
the following equation:
C0 − Ct
BPA removal ability (%) = × 100 (1)
C0
(C0 − Ct ) × V
BPA uptake capacity (q; mM/g dry weight) = (2)
W
where C0 was the initial BPA concentration (␮M); Ct was the BPA
concentration at the time indicated (␮M); V was the volume of BPA
solution (l), and W was the dry weight of the plant biomass (g).
To carry out the sterile treatment control, the plant roots and
stems were surface sterile according to Weyens et al. [13]. Briefly,
the roots and stems were washed several times with sterile dis-
tilled water. Then, they were soaked with 1.5% (v/v) active chloride
solution supplemented with 0.04% (v/v) Tween 80 for 5 min. They
were subsequently rinsed at least three times in sterile distilled
water. The final rinsing solution was plated on a Luria Bertani (LB)
agar plate and incubated at 30 ◦ C overnight to check for growth or
no growth as indication of failure or success of surface sterilization,
respectively.
2.4. Extraction and determination of BPA accumulated in the
plant parts
After 20 days of BPA treatment, the entire plant was collected.
Roots, stems and leaves were cut, separated, and dried in a hot air
oven at 80 ◦ C for 48 h until a constant dry weight was reached. The
dried sample was cooled down to room temperature (ca. 30 ◦ C) in a
desiccator before grinding to a fine powder. BPA was extracted from
each plant part by mixing it with ethanol (10 ml for every 1.5 g plant
dry weight) for 24 h and analyzed using a high performance liquid
chromatography (HPLC).
2.5. BPA analysis using HPLC
The BPA concentration was analyzed by a reverse phase HPLC
using a C18 column (Alltima, 250 mm × 46 mm, Mandel Scien-
tific, OH, USA) equipped with a UV detector. The HPLC mobile
phase was a solution containing acetonitrile–potassium dihydro-
gen phosphate (pH 2.5) (1:1, v/v). Under this analytical method
and at a flow rate of 1 ml min−1 , the retention time of BPA was
8.53 ± 0.005 min. It was monitored at the wavelength of 221 nm.
2.6. Isolation and identification of plant-associating bacteria
The plant roots and spent medium were used as sources for iso-
lation of plant-associating bacteria. The plant roots were washed
several times with sterile water before placing it on a LB agar plate
containing BPA (20 ␮M) for bacterial isolation and potato dextrose
agar containing BPA (20 ␮M) for fungal isolation, while the spent
medium was directly spread on to the media. Isolates grown on
these media were picked and re-inoculated onto a new agar plate
containing BPA until pure cultures were obtained. The chromoso-
mal DNA of each isolate was isolated using a standard protocol
[14] and identified by a 16S rRNA gene sequence analysis following
PCR amplification using primer 63f and 1387r [15]. The PCR mix-
ture and the thermocycling conditions were carried out according
to Kongpol and Vangnai [16]. The PCR products were cloned into
the pGEM-T Easy vector (Promega, Madison, USA) and sequenced
(Macrogen, Seoul, Korea). The partial 16S rRNA gene sequence of
the selected isolates was analyzed using the BLAST search program
[17].
 S. Saiyood et al. / Journal of Hazardous Materials 178 (2010) 777–785
2.7. Analysis of EPM using the phenol–sulfuric acid method
The formation of EPM secreted by the plants was analyzed for
total sugar content using the phenol–sulfuric acid method and glu-
cose as a standard [18,19]. A 2 ml sample from the solution was
mixed with 0.05 ml of 80% phenol solution and 5 ml of concen-
trated sulfuric acid. Then, the absorbance was measured at 490 nm
by using a spectrophotometer (DU800, Beckman Coulter, USA).
2.8. BPA dissipation by D. sanderiana plant and the
plant-associating bacteria
BPA dissipation was completed with a half-strength Hoagland’s
nutrient solution containing 20 ␮M BPA for 20 days under the fol-
lowing conditions: (1) D. sanderiana plants with their sterile roots
and stems soaked in the nutrient solution, (2) same as (1) but
under the non-sterile condition, (3) same as (1) but bioaugmented
with each of the plant-associating bacteria and (4) same as (1) but
bioaugmented with a mixture of the plant-associating bacteria. The
concentration of BPA was analyzed by HPLC under the conditions
stated above and bacterial cell growth was expressed with the opti-
cal density (OD) at 560 nm by using a spectrophotometer (DU800,
Beckman Coulter, USA).
2.9. Electron microscopy
The plant root was cut to the dimensions of
3 mm × 5 mm × 3 mm. Then, the bacterial cells associated with
the plant roots were fixed with 2.5% glutaraldehyde in 0.1 M
phosphate buffer, at pH 7.2 for 2 h at 4 ◦ C. They were washed
twice with the phosphate buffer and then by sterile distilled
water (10 min each). Afterward, the sample was dehydrated in
an ascending concentration series of ethanol, starting with 30%
and ending with 90% ethanol (10 min each). Next, the sample
was dehydrated three times with absolute ethanol. Subsequently,
the sample was subjected to critical-point drying with dry ice
(Balzer CPD020, Balzers AG, Liechtenstein), mounted on metal
stubs coated with gold-palladium (Ion sputter, Balzer CPD040,
Balzers AG, Liechtenstein), and examined under JEOL SEM, model
JSM-5410LV (JEOL, Tokyo, Japan) at 15 kV.
2.10. Feasibility test for BPA removal from the hazardous waste
landfill leachate using the selected plant
The physical properties, chemical properties and other char-
acteristics, i.e., odor, color, pH, chemical oxygen demand (COD),
as well as biological oxygen demand (BOD), total solid (TS), total
dissolved solid (TDS), total suspended solid (TSS), total Kjeldahl
nitrogen (TKN), cadmium (Cd), mercury (Hg), and lead (Pb) were
determined according to standard methods [20]. The concentration
of BPA was analyzed by HPLC under the conditions stated above.
The leachate used for the feasibility test was filtered through
filter paper GF/C and No. 5 and prepared by two means. First, the
leachate was diluted with deionized water to the final concentra-
tion of 20, 40, 60, 80, and 100% (v/v) of its initial concentration.
Second, the leachate was precipitated by 25% (w/v) alum and the
supernatant was used for the treatment experiment. Then, the
Table 1
The capability of two Dracaena plant species for BPA removal and their growth rate.
Plant species Plant growth rate (g dry wt)
In the control unit In BPA treatment unit
D. sanderiana 0.0219 ± 0.0020 0.0159 ± 0.0012
D. fragrans 0.0024 ± 0.0030 0.0011 ± 0.0057
a
The data are presented as the mean ± SD of three individual experiments.
779
two types of the leachate samples were used to grow the selected
plant for 20 days under the indicated experimental conditions. The
remaining BPA was analyzed using HPLC.
3. Results and discussion
3.1. BPA removal ability by D. sanderiana and D. fragrans
Plant selection is one of the most important factors for phy-
toremediation [21]. Dracaena plants were considered to be good
candidates for phytoremediation because of their characteristics
which meet the initial criteria of being non-edible, tropical, and
evergreen plants with a fibrous root system. They have a com-
paratively high growth rate and can grow in a wide range of
environmental conditions, from full sunlight to shaded conditions.
They also require low growth maintenance. Therefore, their abili-
ties to uptake and tolerate toxicity of the xenobiotic of interest, i.e.,
BPA, should be evaluated. In this study, D. sanderiana and D. fragrans
at 2 months old were chosen as the representatives of Dracaena
plants to evaluate for BPA tolerance and uptake capability. Both
plant species were exposed to an initial BPA concentration of 20 ␮M
in a glass jar containing 400 ml half-strength Hoagland’s nutrient
solution for 8 days. Exposure to BPA at the initial concentration
of 20 ␮M slightly caused an adverse growth effect for D. sanderiana
and D. fragrans in the hydroponic system. Under these conditions, D.
sanderiana and D. fragrans showed 30 and 45% slower growth rates,
respectively, compared to that of the non-exposed plant control
unit (Table 1). After 8 days of BPA exposure, the leaves of D. fragrans
were wilted and scorched indicating that the plant could not toler-
ate BPA exposure and was damaged. By comparison, the leaves and
stems of D. sanderiana appeared healthy after exposure. Although
the removal percentage of BPA by both plant species was within a
similar range (Table 1), their BPA uptake capacities were distinc-
tively different. D. sanderiana demonstrated a 17 times higher BPA
removal capability than D. fragrans. Therefore, D. sanderiana was
selected for further study.
3.2. Effect of BPA concentrations on BPA removal capability
The most important characteristics of plant candidates for phy-
toremediation are the removal capability and the tolerance of the
plant towards various concentrations of the organic pollutant tar-
get. In this study, D. sanderiana was used to treat BPA at various
concentrations. During the treatments with low initial BPA con-
centrations (20–60 ␮M), D. sanderiana plants appeared healthy,
although a few black spots could be observed at the roots after
4 days of the treatment. On the contrary, when D. sanderiana plants
were exposed to higher BPA concentrations ranging from 80 to
200 ␮M, some toxic symptoms due to BPA toxicity were observed.
These symptoms included brownish spots on the leaves and several
black spots on the roots. These results indicated that D. sanderiana
plants were able to tolerate BPA toxicity up to a certain level.
D. sanderiana plants demonstrated BPA uptake capability to
approximately 50% of the initial BPA concentration at 20 ␮M
within the first 4 days of the treatment in a sterile hydroponic
system (Fig. 1). In the sterile hydroponic system, the BPA dissi-
pation solely resulted from the plant uptake. In the non-sterile
BPA removal (%) BPA uptake capacitya (q; ␮M g/dry wt)
82.7 ± 1.3 0.370 ± 0.030
73.9 ± 1.4 0.021 ± 0.010
780 S. Saiyood et al. / Journal of Hazardous Materials 178 (2010) 777–785
Fig. 1. Dissipation of BPA at various concentrations in a hydroponic system containing the D. sanderiana plant at different treatment periods. The data are presented as the
mean ± SD of three individual experiments.
treatment, the dissipation resulted not only from plant uptake,
but also from the contribution of the microbes associated with the
plants. After 20 days of the treatment, the total BPA uptake by D.
sanderiana plants in the sterile hydroponic system reached approxi-
mately 60% of the initial BPA concentration. At the same time,
the BPA dissipation contributed by D. sanderiana plant and the co-
existing microbes progressed to 85% of the initial concentration at
20 ␮M.
Relatively, high BPA removal was achieved through the coop-
eration of D. sanderiana plant uptake and BPA degradation by its
associating microbes. Therefore, a similar system was used to inves-
tigate the effect of BPA concentration on the BPA removal capability
of the system. At the low initial BPA concentration, BPA was dissi-
pated in the hydroponic treatment system at a relatively higher
rate (Fig. 1). The BPA removal rate and the total BPA removal per-
centage were gradually decreased with an increase in the initial
BPA concentrations. When the treatment was completed with the
initial BPA concentrations of 20 and 200 ␮M, BPA was removed at
50 and 25% of the initial amounts within the first 4 days, respec-
tively. After 20 days of treatment, the removal rose to 85 and 40%,
respectively. The decrease of BPA removal was likely caused by the
increase in BPA toxicity at higher concentrations for both the plant
and the microbes. During the treatment, the level of BPA dissipation
by natural hydrolysis was negligible (Fig. 1).
Previous reports showed that several plants were able to uptake
and tolerate BPA in a hydroponic system. BPA at various initial con-
centrations was efficiently taken up by some edible plants, e.g., rice
(88 ␮M) [6], Ipomoea aquatica (22 ␮M) [7], and non-edible plants,
e.g., Rumex cripus (176 ␮M) [6], and Portulaca oleracea (250 ␮M)
[22]. Direct comparison of BPA removal efficiency for each plant
type is difficult due to variation in the experimental parameters,
including the BPA initial concentrations, the liquid media used,
and the BPA concentration to plant mass ratios. In this study, D.
sanderiana removes 85% of 20 ␮M BPA within 20 days. D. sanderiana
showed a comparatively high tolerance and good uptake capability
towards BPA, demonstrating its potential use in a phytoremedia-
tion. Its tolerance was particularly high when it co-existed with an
associating microbial consortium.
3.3. BPA uptake and accumulation in the D. sanderiana plant
The use of plants to cleanup toxic organic chemicals contami-
nated in soil and water depends upon the natural abilities of the
plants to absorb and uptake the toxic chemicals through their root
systems, as well as to stabilize, transform or accumulate the chem-
ical in their plant parts [23]. In order to confirm that BPA was
uptaken by D. sanderiana and translocated to those plant parts, the
levels of BPA in the roots, stems and leaves of the D. sanderiana
plant were determined in this study. A control using an auto-
claved whole plant was employed by putting the plant in a 400 ml
hydroponic solution containing 1826 ␮g BPA (20 ␮M). Then, the
BPA sorption by the roots and stems was determined. The result
showed that less than 5% of BPA was abiotically absorbed into the
plant roots and stems. Therefore, abiotic sorption was determined
to be a minor cause of BPA dissipation in this system. Uptake and
translocation by D. sanderiana indicated that the plant could uptake,
translocate and accumulate about 81% of BPA. In the roots, BPA
equaled about 519.63 ± 78.97 ␮g g−1 total plant dry weight and in
the stems, it equaled about 790.87 ± 105.51 ␮g g−1 total plant dry
weight. However, BPA did not equal comparable concentration lev-
els in the leaves (Fig. 2). The BPA remained in the solution at levels of
approximately 19% (338.72 ± 0.14 ␮g l−1 BPA). These results indi-
cated that the plant did not completely metabolize or transform
BPA. However, the D. sanderiana plant could uptake, translocate,
and accumulate BPA, demonstrating that this plant has potential
use in phytoremediation.
3.4. The plant-associating bacteria and the formation of EPM by
D. sanderiana
During the BPA treatment by the D. sanderiana plant, the
increase of the solution turbidity was observed after 4 days of treat-
ment for both the sterile and the non-sterile hydroponic systems.
The turbidity detected under the sterile hydroponic condition was
caused by the plant secretion of EPM under the non-sterile hydro-
ponic condition turbidity was caused by a combination of EPM
secretion and microbial growth.
The turbid solution with microbial growth was used as a source
for microbial isolation on half-strength Hoagland’s nutrient agar
supplemented with 20 ␮M of BPA. Six bacterial isolates, namely
BPR1, BPR7, BPW4, BPW5, BPW6, and BPW10, were obtained under
the isolation conditions indicated, but no fungal isolate was found.
The ability of each bacterial isolate to contribute to BPA biodegrada-
tion was individually examined in a half-strength Hoagland’s liquid
medium. All bacterial isolates to contribute to able to grow in a
half-strength Hoagland’s medium BPW4 had the highest growth
while BPR7 had the least growth (Fig. 3a). Among the six isolates,
only three of them, i.e., BPR1, BPW4, and BPW5, exhibited high lev-
els of biodegradability. After 20 days of incubation, the total BPA
degradation equaled 93 ± 2, 60 ± 2, 96 ± 2%, respectively (Fig. 3b).
 S. Saiyood et al. / Journal of Hazardous Materials 178 (2010) 777–785
Fig. 2. The translocation and accumulation of BPA in each D. sanderiana plant at dif-
ferent treatment periods. The data are presented as the mean ± SD of three individual
experiments.
The 16S rRNA sequence analysis of BPR1 and BPW5 revealed that
they are Gram-negative Enterobacter sp., while BPW4 was a Gram-
positive Bacillus cereus. The growth curve of each strain grown
in a half-strength Hoagland’s medium with BPA has a relatively
short lag phase and a short stationary phase. Generally, a short
lag phase of bacterial growth resulted from the acclimatization
before cell inoculation [24]. In this study, Enterobacter sp. strain
BPR1, strain BPW5 and B. cereus strain BPW4 demonstrated a quick
response to BPA exposure as a short lag phase of growth without cell
acclimatization. The short stationary phase of bacteria growth dur-
ing BPA biodegradation was previously reported in BPA-degrading
bacteria, i.e., Achromobacter xylosoxidans strain B-16 [24] and
Sphingomonas sp. strain BP-7 [25] This short phase was the result
Fig. 3. Growth and BPA biodegradation of the plant-associating bacterial isolates in a
sterile half-strength Hoagland’s nutrient-hydroponic treatment system with 20 ␮M
BPA. (a) Bacterial growth and (b) the remaining BPA (%). The data are presented as
the mean ± SD of three individual experiments.
781
of accumulation of toxic intermediates during BPA biodegradation.
This result was unlikely in this case because no intermediate accu-
mulation was detected during BPA biodegradation by the strains
BPR1, BPW4, or BPW5. Nevertheless, the strains BPR1, BPW4, and
BPW5 were grown in a half-strength Hoagland’s nutrient solu-
tion without any supplementary nutrients in order to imitate the
hydroponic system in this study. Therefore, the short stationary
phase likely occurred as a result of a carbon source limitation. The
bacterial growth declined after 10 days of incubation after which
the remaining BPA was lower than 20% of the initial BPA concen-
tration (BPR1 and BPW5) or when BPA was no longer degraded
(BPW4) (Fig. 3b). In addition, many BPA-degrading bacteria have
been reported such as Pseudomonas paucimobilis strain FJ-4, B-16,
Sphingomonas sp. strain AO1, Achromobactor xylosoxidans strain
B-16 [26,27,24]. Moreover, unidentified Gram-negative bacteria,
including MV1 and WH1 strains [28,29,30] utilize BPA as their sole
carbon and energy source.
As stated before, the turbidity of the treated solution found with
the non-sterile hydroponic condition was caused by a combination
of EPM secreted from the plant and microbial growth. EPM is one
of the diverse classes of biological hydrocolloid macromolecules
commonly found in many higher plants [31,32]. Although the
physiological function of the mucilages is not clear, there are
reports suggesting that they may play a role in water transport
[33], as carbohydrate reserves [34] or in response to physiological
stresses. Plant host–pathogen interactions [35] and drought stress
[36] are examples of these roles. In this study, the formation of
EPM was determined as the total sugar equivalent value by the
phenol–sulfuric acid method using glucose as a standard [18,37].
Although previous reports stated that plant mucilage composi-
tion consists of several types of sugar constituents, e.g., rhamnose,
glucose, galactose, arabinose, and glucose [36,38], the main sugar
component was glucose [36]. Therefore, glucose was used for com-
parison. The formation of EPM was observed when the D. sanderiana
plant was exposed to BPA (Fig. 4a). The amount of EPM that formed
was significantly higher when the plant was grown in the presence
of the bacteria (Fig. 4a). Perhaps, the D. sanderiana plant secreted the
polysaccharide mucilages as a protective barrier to prevent harm-
ful effects from environmental stresses such as the toxic chemicals,
i.e., BPA and the high density of plant-associating bacteria enriched
from the presence of BPA.
3.5. Dissipation of BPA by D. sanderiana plant and the
plant-associating bacteria
The bioaugmentation of each plant-associating bacterium to the
hydroponic system affected the growth of the D. sanderiana plant
differently. BPR7, BPW6, and BPW10 (at 1%, v/v inoculation, or
approximately 106 CFU), caused unhealthy conditions for the D.
sanderiana plant. The leaves were wilted, scorched and became
blackish-yellow. The roots had several black spots within 2 days.
Therefore, these three bacterial strains were no longer examined
for their contribution to BPA dissipation. The strain BPR1 illus-
trated high levels of growth (Fig. 3a) and BPA biodegradation ability
when it was individually tested in the half-strength Hoagland’s liq-
uid medium without the plant (Fig. 3b). However, those properties
were relatively poor when they were grown in the hydroponic sys-
tem containing the D. sanderiana plant (Fig. 4a and b). Poor growth
of the strain BPR1 leading to a decrease of BPA biodegradation
was likely due to the pH drop caused by EPM secretion (Fig. 4b).
Moreover, the presence of a relatively higher cell bioaugmented
density of strain BPR1 in the hydroponic system than that found
associated with the plant adversely affected the plant health. Gen-
erally, the survival of the introduced bacteria is one of the essential
prerequisites for the success of the coupling process for bioaugmen-
tation and phytoremediation [39]. This study demonstrated that
782 S. Saiyood et al. / Journal of Hazardous Materials 178 (2010) 777–785

Fig. 4. BPA dissipation in half-strength Hoagland’s nutrient-hydroponic system containing the D. sanderiana plant with 20 ␮M BPA and its associating bacteria (BPR1, BPW4
and BPW5). (a) Bacterial growth and EPM secreted from the D. sanderiana plant, and (b) the remaining BPA (%) and pH of the system. The data are presented as the mean ± SD
of three individual experiments.

the imbalanced cell density of the bioaugmented bacteria into the
plant system could damage the plant itself as well as reduce the
phytoremediation efficiency. A similar study was performed using
the mixture of plant-associating Enterobacter sp. strain BPW5 and
B. cereus strain BPW4. When both strains were used in the treat-
ment system no harmful effects to the plant were detected. The
growth of Enterobacter sp. strain BPW5 was enhanced whereas
that of B. cereus strain BPW4 was moderately reduced (Fig. 4a).
When tested individually the BPA biodegradation with each strain
in the system was significantly reduced compared to samples with
the mixed culture. This result may suggest the role of the plant-
associating Enterobacter sp. strain BPW5 and B. cereus strain BPW4
in facilitating the coupling process of bacterial bioaugmentation
and phytoremediation for BPA dissipation.
Another important characteristic of bacteria in augmenting
phytoremediation is the colonization ability on the root surface
[40]. The colonization of each plant and the mixture of the plant-
associating Enterobacter sp. strain BPW5 and B. cereus strain BPW4
on the surface of D. sanderiana plant roots after 10 days of the
incubation treatment was determined with an electron microscope
(Fig. 5). The results showed that Enterobacter sp. strain BPW5 or
B. cereus strain BPW4 with the mixed culture could colonize on
the plant roots and contribute to the enhancement of the BPA dis-
sipation in the plant hydroponic system. This result agrees with
previous reports indicating that bioaugmentation by bacteria is
associated with the plant-enhanced phytoremediation of toxic
environmental contaminants [39–42].
3.6. Removal feasibility test of BPA in the hazardous waste
landfill leachate using the D. sanderiana plant
Leachates emitted from hazardous waste landfill sites are of
primary concern due to their toxicity impact. Coupling leachate
irrigation with a phytoremediation system has been considered
as one of the potential remediation treatments to detoxify the
toxic compound target in the leachate [43]. Therefore, we investi-
gated the ability of the D. sanderiana plant to dissipate BPA from
the hazardous waste landfill leachate. The characteristics of the
hazardous waste landfill leachate were odor (foul odor), color
(dark brown), pH (7.72), COD (92,398 mg l−1 ), BOD (26,400 mg/l),
TS (48,024 mg l−1 ), TDS (47,635 mg l−1 ), TSS (380 mg l−1 ), TKN
(2889 mg l−1 ), Cd (<0.10 mg l−1 ), Hg (non-detectable), and Pb
(0.28 mg l−1 ) with an initial BPA concentration of 169 ± 10 ␮M. Dur-
ing the 20 days of the treatment, the results showed that the D.
 S. Saiyood et al. / Journal of Hazardous Materials 178 (2010) 777–785 783

Fig. 5. Scanning electron micrographs showing the colonization of Bacillus cereus strain BPW4 (a), Enterobacter sp. Strain BPW5 (b), and the mixed culture (c) on the root
surface of the D. sanderiana plant grown for 10 days in a haft-strength Hoagland’s nutrient solution supplemented with 20 ␮M BPA. Bar = 5 ␮m (a and b) and 1 ␮m (c).

sanderiana plant could survive. There was no sign of plant dam- days of the treatment, D. sanderiana plants could survive with no
age in the diluted hazardous waste landfill leachate which had a sign of plant damage. The plants could tolerate the leachate tox-
final concentration ranging from 20 to 40% (v/v) and correspond- icity and were capable of removing BPA up to 60% of the initial
ing BPA concentrations of 34–68 ␮M, respectively. During the 20 BPA (34 ␮M) in the leachate (Fig. 6a). This result indicated that the

Fig. 6. Dissipation of BPA presented in the hazardous waste landfill leachate by the D. sanderiana plant. (a) Remaining BPA (%) in the various leachate concentrations. (b) The
remaining BPA (%) in the leachate after precipitation by 25% (w/v) alum. The data are presented as the mean ± SD of three individual experiments.
784 S. Saiyood et al. / Journal of Hazardous Materials 178 (2010) 777–785
high organic compounds and other toxic substances present in the
leachate might have adverse influences to the plants. These influ-
ences could lead to the decrease in the BPA uptake rate. After 10
days of the treatment period, the leachate and the plant roots were
used as sources for bacterial isolation. The results indicated that
no bacterial isolate was obtained and that the leachate was highly
toxic. The dissipation of BPA in the leachate treatment was mainly
caused by the uptake of the D. sanderiana plant.
In another case, the leachate was pretreated by precipitation
with alum and the selected plant was grown for 20 days. Results
indicated that BPA could reduce to 82.30 ␮M after precipitation by
alum. After treatment by the D. sanderiana plant for 20 days, this
plant could reduce BPA to 52.51 ␮M (36%). In this case, the plant still
survived (Fig. 6b). It was possible to use the plant as a secondary
treatment to avoid high toxicity of the leachate. In addition, the
efficiency of BPA uptake by plants could be increased by increasing
the number of plants in the treatment system.
4. Conclusions
This present study demonstrated the success of phytoremedia-
tion as one of the most cost-effective and efficient techniques for
remediation of toxic environmental pollutants. We initially started
by selecting an appropriate plant candidate with a high tolerance
towards the toxic compound target. D. sanderiana, a native, tropical,
low-maintenance plant, not only demonstrated its ability to toler-
ate BPA toxicity, but also to uptake, translocate, and accumulate
BPA. The D. sanderiana plant secreted polysaccharide mucilages,
possibly, as a protective barrier to prevent harmful effects from
BPA toxicity. In addition, the D. sanderiana plant was found to be
associated with bacteria including Gram-negative Enterobacter sp.
strains BPR1 and BPW5 as well as Gram-positive B. cereus. These
bacteria, which adhere at the root surface played a significant role
for BPA biodegradation and could enhance the BPA dissipation in
the plant hydroponic system. In addition, the successful treatment
of the BPA present in the hazardous waste landfill leachate demon-
strates the potential application of the D. sanderiana plant in the
phytoremediation of BPA contamination in wastewater or indus-
trial leachate.
Acknowledgements
The research was financially supported by the Thailand Research
Fund (TRF) and the Commission on Higher Education (CHED).
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