A Comparison of the Effect of Bisphenol A on Activated Sludge
Microbial Communities at Different Exposure Levels
Jeff Tee Jia Jun
School of Chemical and Biomedical Engineering
Abstract – Bisphenol A (BPA) is an endocrine
disruptor that constantly leaks into the environment
through wastewater, putting us and aquatic systems at
risk. Previous work on BPA degradation was focused
on isolate-based studies, instead of community-based
studies that translate better into natural and engineered
systems. Thus, this study was conducted to investigate
the fate of BPA and characterize the associated
microbial behavior when microbial communities are
exposed to BPA. Triplicates of bioreactors using
activated sludge were operated and exposed to 50 μg/L
and 500 μg/L of BPA. Their performance was
monitored through chemical oxygen demand (COD),
volatile suspended solids (VSS), and BPA removal. For
both sets of bioreactors, COD removal stabilized
between < 90% and < 93% by the end of the study
while VSS values remained relatively stable. For the
reactors exposed to 50 μg/L of BPA, effluent
concentrations stabilized at undetectable levels while
for reactors exposed to 500 μg/L of BPA, effluent
concentrations stabilized at the order of 10 μg/L. The
acclimated communities showed no resistance even
after 91 days. These results suggest that BPA is readily
removed in the activated sludge and the microbial
communities fully acclimated without toxic effect.
Keywords – BPA, activated sludge, microbial
communities, toxic effects of BPA on bacteria.
1 INTRODUCTION
Bisphenol A (BPA; 4,4’-isopropylidenediphenol) is
mainly used as an intermediate in the synthesis of
epoxy resins (nearly 20% of total BPA consumption)
and polycarbonate plastics (74% of total BPA used).
Products containing BPA include but are not limited to:
plastic food packaging, walls of cans that isolate food
from metal, healthcare and dental equipment, toys,
spectacles, and contact lenses. In 2008, the estimated
total world production of BPA is around 5.2 million
tons, with lead producers being the United States
(22.9% of global BPA production), Taiwan (13.1% of
global BPA production) and Japan (13% of BPA
production) [1]. In 2015, the total global consumption
of BPA is estimated to be about 7.7 million tons, and is
projected to reach 10.6 million tons by 2022 [2].
Due to its widespread use and mass production, BPA
is constantly released and escapes into the environment
through a multitude of diffuse and point sources, one of
Assistant Professor Oh Seungdae
Dr. Choi Donggeon
School of Civil and Environmental Engineering
the main point sources being municipal wastewater
treatment plant (WWTP) effluents. A study published in
July 2015 set out to assess the global occurrence and
bioaccumulation of BPA by examining over 500 peer
reviewed studies. According to the study, most of the
water occurrence studies were focused on Europe, Asia
or North America and there is a lack of studies
identified from Russia, India, South America (barring
Brazil), Central America, and Africa (barring Tunisa).
They reported that levels of BPA in effluents of
WWTPs ranged from nondetectable to 370 μg/L, with
most cases being lower than 5 μg/L. For surface water,
BPA levels ranged from nondetectable to 56 μg/L, with
most samples representing river sites upstream of a
WWTP. The concentrations of BPA in the sludge of
WWTPs ranged from 10 to 10,000 μg/kg DW, with
higher levels (>100 000 μg/kg) being found in WWTPs
receiving elevated industrial effluents. In soil, the
concentration of BPA varied across several orders of
magnitude (<0.01-1000 μg/kg), while for soils
specifically treated with sewage sludge, BPA
concentration generally ranged from 1 to 150 μg/kg. For
sediments, the concentration of BPA also varied across
several orders of magnitude, depending on the upstream
loading. Concentrations from 100 to 1000 μg/kg were
most commonly reported downstream of WWTPs,
industrial discharges and heavily populated urban areas
[3]. In 2010, a separate study investigated the
atmospheric aerosol concentrations. In urban areas of
India, China, Japan, New Zealand, and the United
States, BPA levels ranged from 0.004 to 17 ng/m3,
while in rural areas of China and Germany, the
concentration of BPA ranged from 0.005 to 0.2 ng/m 3.
In marine areas of the Pacific, Atlantic, and Indian
Oceans, BPA atmospheric aerosol concentrations was
detected between 0.001 and 0.03 ng/m3. BPA was even
detected in samples collected from polar regions, with
concentrations between 0.001 and 0.017 ng/m3 [4].
“How much and at what rate atmospheric BPA is
differentially deposited to the world oceans remains to
be determined” [3]. On the topic of bioaccumulation,
there has been relatively less work done. Among the 63
papers that were reviewed in the study, 62% of them
reported data directly from the field, while the rest is
derived from laboratory settings. A trend that was found
is that laboratory bioconcentration factors (BCFs) are
typically much lower than reported bioaccumulation
factors (BAFs). The paper did not take a conclusive
stance on the ability of BPA to accumulate and advised
Proceedings of the URECA@NTU 2016-17
that more data (especially in vitro metabolism
experiments) is required to better understand the
potential of BPA to accumulate through food chains. In
the context of BPA bioaccumulation in humans, the
paper pointed out that the scientific community heavily
relies on a study concluding that BPA was rapidly
eliminated and has a half-life of 5.3 hours, though there
is conflicting evidence, for example rapid clearance of
BPA was not observed in fasting adults (food was
thought to be the main source of BPA intake for
humans) and there are concerns such as detection limits
raised [3]. Still, we must keep in mind that being
degradable does not mean posing no harm to an
organism, as concluded by another review on the
metabolic fate of BPA from microorganisms to
mammals [7].
The health risks of BPA on humans is a long-debated
topic. While being heralded as an endocrine disruptor
involved in the pathogenesis of a plethora of disorders
such as infertility, precocious puberty, breast cancer,
prostate cancer and polycystic ovary syndrome [5],
most of the concerns of BPA on humans were derived
from animal studies and there is a general lack of
conclusive proof concerning the effects of BPA on
humans. In 2009, a review of endocrine disrupting
compounds (EDCs) stated that “there is no endocrine
system safe from EDCs” and that “even infinitesimally
low levels of exposure—indeed, any level of exposure
at all—may cause endocrine or reproductive
abnormalities, particularly if exposure occurs during a
critical developmental window” [6]. However, the
paper is aimed towards EDCs in general, and how well
the generalized conclusions apply to BPA remains to be
seen. The non-traditional dose-response dynamics,
perceived low potential to bioaccumulate, low
concentrations in the environment, high
biodegradability of BPA and the lack of conclusive
proof that BPA is indeed harmful to us makes for a
compelling case for those arguing against the potential
health risks on humans. To deal with these
uncertainties, safety factors/uncertainty factors are often
employed, for example, the reference dose for BPA in
2015 as set by the U.S. Environmental Protection
Agency (EPA) is 50 μg/kg per day, based on the Lowest
Observed Adverse Effect Level (LOAEL) reported in a
National Toxicology Program chronic rat oral study
published in 1982 and a safety factor of 1000. Recently,
the European Safety Authority (EFSA) lowered the
recommended daily intake from 50 μg/kg per day to 4
μg/kg per day [3,5], based on the observations reported
in a 2-generation toxicity study in mice, using an
uncertainty factor of 150. The EFSA also estimated that
the highest aggregate exposure is 1.449 μg/kg per day
for adolescents, concluding that there is no health
concern for any age group for current exposure levels to
BPA [3].
Regardless, it would still be beneficial to develop
strategies to lower BPA levels in the environment
because: (1) The environmental risks of BPA are quite
significant, and can be attested by the animal studies
that have been carried out to date, for example a study
in Japan showed that BPA was able to induce the
production of female specific proteins (FSP) in male
Oryzias latipes at concentrations similar to those found
in Japan’s fresh waters [8]. Another example other than
the 2 studies on rats mentioned in the previous
paragraph would be a study in 2016 conducted on C.
elegans. The study predicts that BPA could cause
adverse chronic effects at exposure levels as low as
0.228 μg/L [9]. (2) While current exposure levels are
believed to be harmless, it would still be in our best
interests to prevent it from rising to potentially
dangerous levels.
Biodegradation of BPA (by bacteria in particular) has
been seen as a desirable pathway because the
degradation of BPA by bacteria leads to no toxic and
estrogenic effects of BPA [7]. In 1994, the metabolic
routes of BPA by bacteria was found using a gram-
negative bacterial strain MV1. A major and a minor
pathway was identified in the study, both having 2
primary metabolites. The major pathway produces 4-
hydroxyacetephenone and 4-hydroxybenzoic acid while
the minor pathway produces 2,2-bis(4-hydrozyphenyl)-
1-propanal and 2,3-bis(4-hydroxyphenyl)-1,2-
propanediol. The study also suggested that in the
breakdown of BPA, 60% of the carbon was mineralized
to form CO2 while 20% is associated with the bacterial
cells and the remaining 20% is concerted to soluble
organic compounds, based on total carbon analysis [7].
On the other hand, another pathway has been found
recently through the isolation and studying of an
aerobic strain Bacillus sp. GZB with seven metabolites
identified as: 4-(29Hydroxypropan-2-yl)phenol, 4-
(prop-1-en-2-yl)phenol, 1-(4-hydroxyphenl)ethenone,
4-hydroxybenzaldehyde, benzoic acid, 2-
Hydroxypropanoic acid and 2-methylbutanoic acid [10].
In WWTPs, the use of microbial communities is
preferred over that of individual populations because
microbial communities are more resilient to changes in
their working environment. This is supported by the
results of a study in 2002, which “indicate that
functionally stable wastewater treatment bioreactors
have stable microbial community structures under
normal operating conditions but are able to adapt in
response to perturbations to sustain high effluent
quality.” [11]. However, to the best of our knowledge,
there is a lack of work done studying potential effects of
high BPA concentrations on bacteria in a community
setting, which is an important consideration when
designing a microbial community (engineered system)
to deal with BPA under the working conditions of a
WWTP. Although studies have been done to investigate
the toxicity of BPA to microbial populations in the past
using 2 freshwater Pseudomonas species, it is not
reasonable to assume that the results (Fabig reported an
18-h EC50 of >320,000 μg/L while Stone and
Watkinson reported an IC50 of 54,500 μg/L, according
to a review done in 1998 [12]) automatically translate
Proceedings of the URECA@NTU 2016-17
well into a community made up of many populations
combined.
Hence, this study sets out to investigate the potential
toxic effects on microbial communities through (1)
studying the fate of BPA (2) characterizing and
comparing the associated microbial behavior when
triplicates of bioreactors containing activated sludge
(microbial communities) are exposed to 50 μg/L
(mimics environmentally relevant concentrations) and
500 μg/L (simulates 10 times environmentally relevant
concentrations) of BPA.
2 METHODS AND MATERIALS
2.1 BIOREACTOR OPERATION
The sludge used in this study was obtained from
Jurong Water Reclamation Plant which and was
acclimated in the laboratory for 2 months. The
operating volume (volume of bioreactor just after
feeding) was maintained at 600 mL. Six bioreactors
were operated in this study, three of which were
exposed to 50 µg/L of BPA (introduced through mixing
with feed solution) while the other three were exposed
to 500 µg/L of BPA. The feed solution used was
prepared as follows: 3 mL of glucose (1830 mg/L),
CaCl2 (53 mg/L), KH2PO4 (340 mg/L), K2HPO4 (600
mg/L), MgSO4·7H2O (270 mg/L), NH4Cl (30 mg/L),
trace mineral (200x), along with 0.3 mL of
FeSO4·7H2O (9.8 mg/L) was diluted in Milli-Q water
until the volume of the feed reaches 600 mL. Starting
from the 4th cycle, the appropriate concentrations of
BPA was mixed within the feed solution. The
bioreactors were aerated using an air pump and a simple
tube connected to an air stone diffuser. In this study, the
operation period was 26 cycles (3.5 days each), or 91
days after exposure to BPA.
2.2 SAMPLING
At the end of every cycle, 200 mL of each bioreactor
is withdrawn and replaced by 200 mL of feed solution.
The withdrawn samples of each bioreactor were then
used in chemical oxygen demand (COD) removal tests,
mixed liquor volatile suspended solids (MLVSS)
measurements, high-performance liquid
chromatography (HPLC) tests, and minimum inhibitory
concentration (MIC) tests as described in the following
sections. MLVSS measurements were carried out on
every cycle, while COD tests were carried out on cycles
1, 2, 4, 8, 12, 20, 26. HPLC tests were carried out
before feeding of the following cycles (and after
feeding for the previous cycles): 1, 4, 6, 8, 12, 16, 18,
20, 26
2.3 COD REMOVAL AND MLVSS
Aluminum dishes were first weighed (Mempty dish) and
labelled. A 15 mL sample was then extracted from each
bioreactor after mixing and placed into separate dishes.
The dishes were then placed at 105 °C overnight. After
cooling, the dishes were weighed again (Mdried sample).
Next, the dishes were placed in a furnace at 550 °C for
15 minutes and weighed (Mevaporated dish) after cooling.
For COD measurements before feeding, a 4 mL sample
was extracted from all bioreactors and centrifuged at
8000 rpm for 4 mins. 2 mL of the supernatant is added
into 20-1500 mg/L range COD vials. The mixture is
heated in a COD reactor for 2 hours at 120 °C. The
COD concentration was then found using 435 method
(620 nm) (high range COD). For effluent COD
measurements, a 6 mL sample is withdrawn from the
bioreactors and centrifuged at 8000 rpm for 4 minutes.
2 mL of the supernatant was added into low range COD
vials and heated up in a COD reactor for 2 hours at 120
°C. The COD concentration was then found using 435
method (420 nm) (low range COD).
2.4 HPLC MEASUREMENT
To determine BPA removal rate, HPLC tests were
conducted to measure BPA concentrations before and
after feeding. The mobile phase used was a 40:60%
(v/v) mixture of solution A and solution B. Solution A
is 20 mM sodium dihydrogen phosphate prepared by
dissolving 1.38 g of sodium dihydrogen phosphate in 1
L of Milli-Q water and then adding 0.68 mL phosphoric
acid so that pH reaches 2.5. Solution B is acetonitrile.
The mobile phase flow rate used was 1.8 mL/min.
2.5 RELATIVE GROWTH
MEASUREMENT
Relative growth was measured to determine the
resistant effect of BPA in activated microbial
communities. The activated microbial communities
exposed to BPA for 91 days were used as an inoculum.
Muller Hinton (MH) broth medium was used for their
growth. Briefly, 5 mL of MH broth medium was added
into 15 mL of autoclaved test tube. BPA from 40 g/L of
stock solution was diluted at the range of concentration
as 2, 4, 8, 16, 32, 64, and 128 mg/L in each test tube.
The same amount of inoculum volumes (1%) was used
as the initial cell numbers using activated sludge from
the bioreactors exposed to 50 and 500 g/L of BPA
respectively. The relative growth corresponding to BPA
concentrations from 2 to 128 mg/L was measured by
optical density at 600 nm using a spectrophotometer.
The control group which was not acclimated by BPA
but fed by a synthetic wastewater containing glucose as
a carbon source was used to compare the resistance of
BPA. The p-values of relative growth between control
and BPA-exposed bioreactors (both with a
concentration of 50 µg/L and 500 µg/L) were calculated
using Mann-Whitney U rank sum test.
Proceedings of the URECA@NTU 2016-17

3 RESULTS AND DISCUSSIONS

3.1 VSS AND COD CURVES

Figure 2. Effluent concentration of BPA from 500

g/L added bioreactors at cycle 1, 4, 8, 12, 16, 18, 20,
26.

After 4 cycles, the effluent concentration of BPA in

Figure 1. Bioreactor performance with VSS (g/L) and
COD removal rate (%) in each cycle (3.5 days). Red
line indicates that BPA was added at cycle 4. (a)
bioreactor performance exposed to 50 μg/L of BPA, (b)
bioreactor performance exposed to 500 μg/L of BPA.
For both figures, the top curve shows COD trends while
the bottom curve shows VSS trends.
reactors exposed to 50 μg/L were consistently at
undetectable levels. For reactors exposed to 500 μg/L of
BPA, the effluent concentration was constantly detected
at the order of 10 μg/L. While the removal rate of the
reactors exposed to 50 μg/L of BPA cannot be
computed since the effluent concentration was
undetectable, the removal rate of BPA in reactors
exposed to 500 μg/L of BPA was found to stabilize at
roughly 95%.
From Figure 2, we can see a big drop in the effluent
concentration of BPA after cycle 4, this can be
interpreted as a sharp rise in removal rate (after
acclimatization) and is characteristic to biodegradation.
This is a strong indicator that biodegradation was the
main driving force behind the breakdown of BPA in our
study, as a sharp increase in degradation rate is not
expected to happen for photolysis, volatilization or
hydrolysis.

The VSS curves of the reactors exposed to 50 μg/L

and 500 μg/L of BPA show similar trends after adding
BPA. The VSS values of both reactors remained
relatively stable except for a sharp drop on cycle 8. The
fact that the sharp drop was observed on the same cycle
suggests that what caused it is likely a time-dependent
factor as opposed to BPA concentration.
4 cycles (14 days) after exposing the reactors to BPA,
the COD removal rate reached and proceeded to
stabilize in the range of <90% to <93%. This suggests
that the microbial communities have fully acclimated
by 14 days upon exposure to BPA.

3.2 BPA REMOVAL RATE

Proceedings of the URECA@NTU 2016-17
3.3 RELATIVE GROWTH OF CELLS
Figure 3. Relative growth (%) linked to BPA
concentration on both 50 and 500 g/L of BPA exposed
bioreactors at cycle 26 (91 days).
Relative growth was measured and compared with the
control bioreactor after 91-day operation with BPA. The
growth of microbial communities exposed to both BPA
concentrations was not significantly changed (Fig. 3).
The p-values of Mann-Whitney U rank sum test were
shown as 0.902 (P>0.05) of both two bioreactors at
cycle 26.
4 CONCLUSION
Our results agree with previous studies that show that
BPA is ‘highly biodegradable’. The fact that MLVSS,
COD removal rate and BPA removal rate were so stable
after 14 days and that there was no resistance based on
MIC tests even after 91 days suggest that the microbial
communities fully acclimated without toxic effect. This
study has shown that BPA’s effects on microbial
communities are largely similar under exposure levels
of 50 μg/L and 500 μg/L which suggests that bacteria
acclimated to BPA under environmentally relevant
concentrations will be able to handle at least 10 times
increase in BPA concentration if used to treat WWTP.
This further validates the viability of microbial
communities to deal with BPA in MP form because the
BPA levels (non-detectable to 370 μg/L) reported in
WWTP effluents are likely to be within tolerance levels
of acclimated communities.
ACKNOWLEDGMENT
We wish to acknowledge the funding support for this
project from Nanyang Technological University under
the Undergraduate Research Experience on CAmpus
(URECA) programme.
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