International Biodeterioration & Biodegradation xxx (2016) 1e6

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Bisphenol A degradation by Ralstonia eutropha in the absence and

presence of phenol
Hamed Heidari a, Mahsa Sedighi b, Seyed Morteza Zamir a, *, Seyed Abbas Shojaosadati a
a
Biotechnology Group, Chemical Engineering Department, Tarbiat Modares University, Tehran, Iran
b
Bioenergy and Bioconversion group, Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran, Iran

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

Article history: In the present study, a pure culture of Ralstonia eutropha was used to degrade bisphenol A (BPA). About
Received 1 July 2016 15e56% of BPA with the initial concentrations ranging from 1 to 20 mg l
1, was degraded. Since the
Received in revised form bacterial culture was not able to degrade BPA completely, the phenol-adapted cells were used for the
30 October 2016
investigation of the co-metabolic degradation of BPA at concentration of 20 mg l
1 by using of phenol as
Accepted 30 October 2016
Available online xxx
the growth substrate with the concentration of 200 mg l
1. Three different approaches was used for BPA
biodegradation: (i) by using it as the sole carbon source, (ii) by phenol-adapted cells (resting cells), and
(iii) by growing cells in the simultaneous presence of both phenol and BPA. The removal efciency (RE) of
Keywords:
Bisphenol A
BPA was about 10% when it was used as the sole carbon source. RE increased to 36% by using resting cells
Phenol and to 50% in the presence of phenol, simultaneously. This improvement was due to the advantages of
Ralstonia eutropha co-metabolism due to the presence of enzymes involved in phenol degradation, which affects BPA as a
Co-metabolism co-substrate in a co-metabolic degradation process. Kinetic data for BPA biodegradation by phenol-
Kinetic modeling adapted cells of R. eutropha best tted to the Monod model. The kinetic parameters were Vmax 7.4
(mg BPA g
1 biomass h
1) and Ks 10.8 (mg l
1).
2016 Elsevier Ltd. All rights reserved.

1. Introduction concentrations in nature, is a recalcitrant compound for

Bisphenol A (BPA) is a raw material for manufacturing some
polycarbonate plastics and epoxy resins, with two phenol rings,
connected to two methyl branches ((CH3)2C(C6H4OH)2) (Li et al.,
2012; Kamaraj et al., 2014). BPA is widely used in CDs/DVDs, bot-
tles, toys, medical instruments, etc. (Ferro Orozco et al., 2013). In
2011, global production of BPA reached over 2.6 million tons per
year with annual growth of 7% (Zhou et al., 2011). BPA is a com-
pound disrupting the endocrine system and has become a threat to
the environment and human being after some discoveries about its
estrogenic and hazardous activities (Ferro Orozco et al., 2016; Zhao
et al., 2008). It is suspected to raise the risk of prostate and breast
cancer (Kabiersch et al., 2011). BPA is found in natural and synthetic
environments at low concentrations (Michalowicz, 2014). It was
reported that even at picogram levels, the BPA can affect physio-
logical functions of brain (Sun et al., 2016). BPA, despite of its low
* Corresponding author.
E-mail addresses: hamed.h1382@gmail.com (H. Heidari), m_sedighi@aut.ac.ir
(M. Sedighi), zamir@modares.ac.ir (S.M. Zamir), shoja_sa@modares.ac.ir
(S.A. Shojaosadati).
biodegradation.
In some petrochemical wastewater treatment plants, phenol is
found along with BPA. Phenol is a common petrochemical sub-
stance which is more biodegradable than BPA. There are many
studies on the biodegradation of phenol and its derivatives (Wang
et al., 2010). To our best knowledge, there are no reports regarding
any possible interactions between these two components during
biodegradation.
BPA can be degraded by Pseudomonas sp. and Pseudomonas
putida (Kang and Kondo, 2002), Bacillus sp.(GZB) (Li et al., 2012),
Cunninghamella elegans ATCC36112 (Keum et al., 2010) and Heliscus
lugdunensis (Omoike et al., 2013). While some of these microor-
ganisms are able to degrade phenol, too. Ralstonia eutropha is
among the well-known bacterial species, capable of degrading
phenol. R. eutropha was used extensively in co-metabolic degra-
dation of recalcitrant compounds in the presence of phenol as the
growth substrate, such as PNP (para-nitrophenol) (Jamshidian
et al., 2013), ethyl mercaptan (Sedighi et al., 2016), and TCE
(trichloroethylene) (Chen et al., 2008). Many pollutants such as BPA
cannot be used efciently as the growth substrates by the micro-
organisms. Biodegradation of such molecules by co-metabolism has
been reported (Velasco et al., 2017). In the co-metabolism, the

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2 H. Heidari et al. / International Biodeterioration & Biodegradation xxx (2016) 1e6
microorganism is able to biodegrade a pollutant without using it as
a growth substrate (non-growth substrate), while it grows by
assimilating a different substrate (growth substrate). By growing
the microorganism on the growth substrate, specic mono-
oxygenase enzymes and electrons are produced for degrading the
target pollutant (Nzila, 2013). Bio-stimulation of BPA degradation
by co-cultivation with phenol by Cupriavidus basilensis JF1 was re-
ported by Fischer et al. (2010). The detected degradation metabo-
lites (4-isopropenylphenol, p-hydrochinone, 4-
hydroxyacetophenone, 4-(2-propanol)-phenol, 4-hydroxybenzoic
acid and 4-hydroxy-benaldehyde) revealed the presence of
mono-oxygenases introducing the oxygen into the ring system of
BPA. The mineralization pathway of BPA was similar to that pro-
posed for Sphingomonas sp. by Kolvenbach et al. (2007), where the
cleavage of the ring system was catalyzed by a mono-oxygenase in
the presence of NADPH.
The aim of this study is to evaluate the potential ability of
R. eutropha to degrade BPA as the sole carbon/energy source and co-
metabolically with phenol as the growth substrate. Two different
processes, resting cells and simultaneous biodegradation, were
studied for the co-metabolic removal of both BPA and phenol. The
kinetics of degradation of each compound was also studied.
2. Materials and methods
2.1. Chemicals and reagents
BPA was purchased from Incheon Chemical Co. (South Korea).
All other chemicals were purchased from Merck with analytical
grade. 4-AAP (4-aminoantipyrine) and potassium ferry cyanide
solutions were kept in dark and cool place (4  C) and renewed every
month in order to maintain their quality.
2.2. Microorganism and culture conditions
The bacterium R. eutropha (PTCC 1615) was obtained from Per-
sian Type Culture Collection (Iranian Research Organization for
Science and Technology IROST) in lyophilized form, and was
maintained at 4  C, on slants containing nutrient agar. Subcultures
were routinely prepared every month. The nutrient medium con-
tained the following ingredients (in g l
1): glucose, 3; yeast extract,
2; peptone, 2; KH2PO4, 1; K2HPO4, 1; (NH4)2SO4, 1; MgSO4$7H2O,
0.05; 15 g l
1. Agar was added for solid growth media. The pH of the
medium was adjusted to 7, and it was sterilized by autoclaving at
121  C for 20 min. The mineral salts medium (MSM) used for
adapting the bacterial culture to phenol and BPA contained (g l
1):
KH2PO4, 1; K2HPO4, 1; (NH4)2SO4, 1; and MgSO4$7H2O, 0.05. For BPA
biodegradation using resting cells and simultaneously with phenol,
the same medium was also used.
2.3. Experiments
Batch experiments were conducted using 70 ml of MSM with
phenol and/or BPA as the sole carbon/energy source in a conical
ask, in a rotary shaker (shaker speed 150 rpm and
temperature 30  C). The pH of the medium was xed at 7 and all
experiments were carried out in triplicates. Before the experiments,
inoculum was prepared by suspending the bacterial cells in the
distilled water. The optical density of the cell suspension was
adjusted to 1.0 at 590 nm 30 ml of the cell suspension was inocu-
lated into 70 ml MSM.
To determine the possibility of chemical degradation of BPA or
adsorption by biomass, parallel control tests were also conducted,
without the introduction of biomass or with autoclaved bacterial
cells. Results showed negligible change in BPA concentration under
Please cite this article in press as: Heidari, H., et al., Bisphenol A degradation by Ralstonia eutropha in the absence and presence of phenol,
International Biodeterioration & Biodegradation (2016), http://dx.doi.org/10.1016/j.ibiod.2016.10.052
the operating conditions.
Adaptation of R. eutropha to phenol and BPA was performed
separately using the MSM by gradually and sequentially increasing
the concentration of phenol or BPA as the growth substrate ac-
cording to Table 1.
To study the degradation capacity of R. eutropha, different initial
concentrations of BPA ranging from 1 to 20 mg l
1 were introduced
to the bacterial culture. BPA biodegradation by the use of resting
cells was carried out by adding BPA to batch cultures of cells that
previously degraded constant phenol concentration (200 mg l
1).
As the phenol was exhausted, BPA was added to the medium at a
constant concentration of 20 mg l
1. For simultaneous biodegra-
dation of BPA and phenol, pre-determined concentrations of
phenol and BPA (200 and 20 mg l
1, respectively) were added
simultaneously into the growing medium at the beginning of each
experiment. Samplings from the liquid phase were carried out
during the experimental period and the samples were analyzed for
phenol and/or BPA concentrations.
For evaluating the BPA mineralization during the biodegrada-
tion process, separate experiments were conducted using 20 ml of
MSM with BPA at initial concentrations of 0.5, 1 and 2 mg l
1 in
120 ml sterile serum bottles, sealed with Teon-coated silicone
septa and aluminum crimp caps. Samplings from the gas phase
were carried out by a gas-tight syringe (Hamilton) at the end of the
degradation process (10 days).
2.4. Analytical methods
Phenol concentration was measured by a spectrophotometer
(CARY-100) at 765 nm using the Folin-Ciocalteau reagent as the
standard procedure (Box, 1983). BPA concentration was also
measured by the spectrophotometer using 4-AAP reagent as the
standard procedure at 506 nm (Modaressi et al., 2005). Biomass
concentration was determined by the measurement of the optical
density (OD) of the solution at 590 nm, by using the spectropho-
tometer. The CO2 concentration in the gas phase was analyzed by
using a gas chromatograph (GC) (Teif Gostar TG-2552, Iran),
equipped with a thermal conductivity detector (TCD), and a packed
column (Porapak Q). The temperature of injector, oven, and de-
tector were xed at 40, 40, and 100  C, respectively. At given times,
1 ml of the gas phase was extracted from each of the serum-bottles,
by a 5 ml gas-tight syringe.
3. Results and discussions
3.1. Phenol and BPA adaptation of the bacteria cells
R. eutropha was adapted to BPA up to 20 mg l
1 via gradual
increasing of BPA concentration (Fig. 1a). Although R. eutropha
could partially utilize BPA solely, complete removal of BPA was not
achieved at any initial concentrations (the RE of BPA was 15e56%)
and the biomass growth was also negligible. Since BPA and phenol
are similar in the chemical structure, it is likely that the key en-
zymes involved in phenol degradation affect BPA as a co-substrate
in a co-metabolic process. The co-substrate competes with the
primary substrate to occupy the active sites and irreversibly in-
activates enzymes with its toxicity (Wang et al., 2003; Arp et al.,
2001). In order to decrease the possibility of competitive inhibi-
tion owing to the presence of BPA, R. eutropha was adapted to
phenol up to 800 mg l
1 (Fig. 1b). As it is shown, free cells of bac-
terium remained active at the end of adaptation. This is comparable
to the results of other studies about phenol degradation using
R. eutropha (Nickzad et al., 2012; Dursun and Tepe, 2005). Although
the bacterial cells were adapted to phenol, up to 800 mg l
1, the
maximum degradation rate of phenol was achieved at the
 H. Heidari et al. / International Biodeterioration & Biodegradation xxx (2016) 1e6 3

Table 1
Increasing trend of phenol and BPA concentrations during adaptation process.

No. Phenol concentration BPA concentration

(mg l
1)/days of incubation (mg l
1)/days of incubation

1 20/1 1/10
2 50/1 1.5/10
3 100/2 2/10
4 200/2 2.5/10
5 300/3 3/10
6 400/3 5/10
7 500/4 8/10
8 600/4 12/10
9 700/5 14/10
10 800/5 20/10

concentration of 200 mg l
1 (i.e. 548 mg phenol g
1 biomass h
1).

Therefore, BPA degradation using phenol-adapted cells of
R. eutropha was investigated at the constant concentration of
200 mg l
1 either by using resting cells or simultaneously.

3.2. BPA as the sole carbon source

To study the effect of initial concentration on the bacterial

degradation capacity, BPA at different concentrations ranged from 1
to 20 mg l
1, was introduced into the BPA-adapted bacterial culture.
BPA concentration was monitored during the incubation. Fig. 2
shows the degradation trends of BPA during the batch experi-
ments. Complete BPA removal was not achieved at any initial
concentrations and the bacterial culture could not degrade BPA Fig. 2. Variations of BPA concentrations during the biodegradation as sole carbon/
efciently. The RE of BPA was 15e56% and did not follow any pro- energy source a) concentrations<10 mg l
1 b) concentrations>10 mg l
1.
portional pattern to the initial concentration. This could be due to
the more complicated structure of BPA, comparing with phenol, or
biodegradation efciencies were decreased from 92% to 66% with
its inhibition effect on the cell activity. Li et al. (2012), investigated
increasing the BPA concentrations from 5 to 30 mg l
1. Comparing
the aerobic degradation of BPA by a facultative anaerobic bacterial
the results, investigating the degradation capacity of R. eutropha in
strain, Bacillus sp. GZB. They showed that after 96 h incubation, the
using BPA as the sole carbon source can be concerned due to its rst
use as a pure environmentally safe culture for the biological
oxidation of BPA. The results are also in agreement with the earlier
studies showing that under environmental conditions and with
strains cultivated in mineral medium, BPA was found not to be
easily degradable (Badiefar et al., 2015).

3.3. Kinetic model for BPA or phenol degradation as the sole carbon
source

Changes in the concentration of BPA were monitored during the

biodegradation to obtain a specic degradation rate (SDR) for BPA.
SDR was veried for each initial concentration by dividing the
degradation rate to the initial biomass.
Fig. 3a shows the SDRs for the different concentrations of BPA.
Regarding the SDR trend, the Monod model was tted to the
experimental data, which is represented by Equation (1):

Vmax S
SDR (1)
Ks S

where S, is the concentration of substrate (mg l
1), Vmax is the

Fig. 1. Adaptation process for R. eutropha to BPA and phenol separately.
maximum specic degradation rate (mg g
1 h
1), and Ks is the half-
velocity constant (mg l
1). Application of Monod equation to the
experimental data gave a good t, with R2 0.95. The model pa-
rameters were evaluated by using GraphPad Prism 5, obtained as
follows:

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4 H. Heidari et al. / International Biodeterioration & Biodegradation xxx (2016) 1e6

Fig. 3. Kinetic model parameters estimations a) BPA as sole carbon source b) Phenol as sole carbon source.

  produced a ten-fold increase in the rst-order degradation rate

Vmax 7:4 mg g
1 h
1 constant. This could be related to the simpler chemical structure of
phenol, comparing with BPA, which resulted in efcient biodeg-
  radation of phenol and incomplete removal of BPA even at low
Ks 10:8 mg l
1 initial concentrations.

Finally, the Monod equation for the biodegradation of BPA by

3.4. Co-metabolic degradation of BPA using phenol as the growth
R. eutropha can be represented as follows (Equation (2)):
substrate
ds Vmax SX 7:37SX

SDR:X
(2) As mentioned before, complete removal of BPA was not ach-
dt Ks S 10:75 S
ieved at any initial concentrations and the biomass growth was also
In Equation (2), X represents the biomass concentration (g l
1). negligible. Therefore, its co-metabolic degradation using phenol
Li et al. (2012), showed that the aerobic biodegradation of BPA by was investigated. Although the bacterial cells were adapted to
Bacillus sp. GZB, follows the pseudo-rst-order kinetic model phenol up to 800 mg l
1, the maximum SDR of phenol was achieved
within the concentration range from 5 to 30 mg l
1, and the cor- at concentration of 200 mg l
1. The maximum degradation rate of
responding degradation rate constants are decreased from 0.030 to
0.013 h
1. Although such models have a strong ability to predict the
kinetic behavior of biodegradation, they are not general, and can
only be applied in distinct cases, which limits their capability of
comparing their results with other kinetic data. Application of the
general kinetic models such as Monod or Haldane, provides an
opportunity to compare the different biodegradation kinetic be-
haviors (Sedighi et al., 2013).
By monitoring the changes in the initial concentration of phenol
during biodegradation, SDR for phenol was also obtained and
incorporated into a model (Fig. 3b). As it is shown, the SDR curve
can be divided into two parts. At low phenol concentrations, SDR
increased (0e548 mg phenol g
1 biomass h
1) as the phenol con-
centration increased (0e200 mg l
1). However, SDR decreased after
the phenol concentration exceeded the threshold value of
200 mg l
1. Therefore, phenol had a substrate-inhibition effect
above a certain concentration. The Haldane model is the best
general inhibitory kinetic model to describe the phenol removal
rate (Maleki et al., 2015), as shown in Equation (3):

Vmax S
SDR 2
(3)
Ks S SKi

where Ki, is the inhibition constant (mg l
1). The application of the
Haldane equation to the experimental data gave a good t
(R2 0.95) and the model parameters evaluated as Vmax 7973 mg
phenol g
1 biomass h
1, Ks 1174 mg l
1, and Ki 19.7 mg l
1. The
ratio of VKmax s
is representative of the rst-order degradation rate
constant at low substrate concentrations. This value was about 0.7
and 6.8 for BPA and phenol, respectively. By comparing the values Fig. 4. Co-metabolic degradation of BPA a) By using phenol-adapted cells (resting
for VKmax
s
, it can be observed that using phenol as the carbon source cells) b) In the presence of phenol (Simultaneous).

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 H. Heidari et al. / International Biodeterioration & Biodegradation xxx (2016) 1e6 5

Table 2
Degradation of BPA by use of different microbial pure cultures.

Microorganism Operating conditions Co-substrate BPA concentration Removal Degradation References

efciency (%) time (day)

Ralstonia eutropha Sequential addition Phenol (200 ppm) 20 ppm 36 10 This study
Simultaneous addition Phenol (200 ppm) 20 ppm 50 7
Cupriavidus basilinsis JF1 Sequential addition Phenol (1.7 mM) 0.17 mM (39 ppm) 90 225 Fischer et al., 2010
Simultaneous addition Phenol (1.5 mM) 0.21 mM (48 ppm) 33 150
Pseudomonas sp. strain KU1, Simultaneous addition Sodium 1000 ppm 81e85 12 Kamaraj et al., 2014
Pseudomonas sp. glutamate (0.5%)
strain KU2, Bacillus sp.
strain KU3
Bacillus amyloliquefaciens e e 60 ppm 77 2 Zuhlke et al., 2016
Bacillus sp. GZB e e 5 ppm 51 4 Li et al., 2012
Heliscus lugdunensis e e 10 ppm 70 12 Omoike et al., 2013

BPA was also achieved at 20 mg l
1. Therefore, BPA degradation
using phenol-adapted cells of R. eutropha was investigated at a
constant concentration of 20 mg l
1 of BPA and 200 mg l
1 of
phenol either by using resting cells or during simultaneous
biodegradation. Fig. 4a shows the RE of BPA at concentration of
20 mg l
1 by using of resting cells. As it is shown, after 10 days of
incubation, only 36% of BPA was degraded. Although complete
removal of BPA was not achieved by resting cells, RE improved in
comparison with using BPA as the sole carbon source, which was
only 10% at the same initial concentration of BPA. This improve-
ment could be due to the presence of enzymes involved in phenol
degradation, which affect BPA as a co-substrate in a co-metabolic
degradation process. However, the toxicity of BPA as a co-
substrate on the cell growth and the inhibition and/or inactiva-
tion of enzymes involved in the co-metabolic process could result
in incomplete degradation. Similar results were also observed by
Sedighi et al. (2016) for the co-metabolic degradation of ethyl
mercaptan (EM) by phenol-utilizing cells of R. eutropha. The ex-
periments at EM concentrations ranging from 1.2 to 14.4 mg l
1,
showed almost complete removal of EM at concentrations below
10.1 mg l
1, which was much higher than the maximum biode-
gradable EM concentration (2.5 mg l
1) obtained in the absence of
phenol as the primary substrate (Sedighi et al., 2016).
In another approach, co-metabolic degradation of BPA
(20 mg l
1) in the presence of phenol (200 mg l
1) was also
investigated. Fig. 4b shows the simultaneous biodegradation of
phenol and BPA. As it is shown, the RE of BPA increased to 50% in
the presence of phenol probably due to the many co-metabolic
enzymes and co-factors induced by the primary substrate and
also the supportive effect of phenol on cell-growth. Complete
removal of phenol shows that BPA had no inhibitory effect on
phenol biodegradation.
Biodegradation of BPA by different pure strains, as the sole
carbon source or co-metabolically, has been considered in different
studies. Table 2 reviews the results of some researches in com-
parison with this study. Although the degradation capability of
R. eutropha is relatively low compared to some previous strains,
according to the initial degradable concentration of BPA and the
degradation time, the degradation capacity of R. eutropha can be
considerable for the biological oxidation of BPA. It is probable that
by optimizing its environmental conditions and/or modifying the
degradation pathways using genetic engineering techniques, the
potential of this strain for BPA degradation in wastewater con-
taining BPA will be increased.
was observed compared to the initial value. It can be concluded that
complete mineralization was not occurred during biodegradation
and that BPA has been transformed to other metabolites. Produc-
tion of different metabolites and their presence in the medium
could have negative impact on the cell activity and inhibition or
inactivation of involved enzymes. This could be also the reason of
incomplete biodegradation of BPA by bacterial cells of R. eutropha.
3.6. Practical implications
BPA is frequently found in wastewater, sewage sludge, water and
sediments and its concentration depends on the production source.
Some publications suggested that BPA could affect aquatic organ-
isms at concentrations below 1 mg l
1 (Weltin et al., 2002). Bio-
stimulation by primary substrates is a very promising strategy for
the bioremediation of petrochemical wastewater containing BPA. In
this study, the increase of RE of BPA in the presence of phenol to
50% from 10% in the absence of phenol shows that co-metabolic
degradation of pollutants can be effectively used in industrial
scales. Both phenol and BPA can be found in petrochemical
wastewater. It is hypothesized that the addition of a growth sub-
strate such as glucose can increase or stimulate biodegradation of
recalcitrant compounds like BPA. However, it will be economically
applicable when another pollutant acts as the growth substrate to
synergistically facilitate the degradation of both compounds. In this
case, enrichment of activated sludge in wastewater treatment
plants by those microorganisms capable of handling co-metabolic
degradation can be an advantage for the treatment process. How-
ever, it is necessary to characterize the BPA biodegradation
pathway in the absence and presence of phenol by indicating the
produced metabolites and measuring their estrogenic activities.

3.5. CO2 production and mineralization of BPA

The results of CO2 production are shown in Fig. 5. The CO2 Fig. 5. Normalized production of CO2 during the biodegradation of BPA as the sole
concentration did not increase obviously at the examined BPA carbon source. C0: CO2 concentration in the blank sample with no BPA, C: CO2 con-
concentrations and just a 1.3-fold or 2.3-fold increase (mass ratio) centration after 10 days.

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6 H. Heidari et al. / International Biodeterioration & Biodegradation xxx (2016) 1e6
4. Conclusion
The role of bacteria in the degradation of emerging contami-
nants such as BPA has been characterized in the different studies. To
the best of our knowledge, the present study is the rst demon-
stration that R. eutropha can partially degrade BPA as the sole car-
bon source to about 10%. R. eutropha degraded almost 36% and 50%
of BPA in the presence of phenol as a growth substrate in resting
cells and simultaneous co-metabolic degradation processes,
respectively. The Monod model successfully predicted kinetic data
obtained from batch experiments for BPA biodegradation, while
Haldane model can be used for phenol biodegradation by
R. eutropha. Further work is needed to identify and characterize the
metabolites of R. eutropha degradation and transformation of BPA
and also to evaluate the estrogenic activity of these compounds
during BPA biodegradation.
Acknowledgement
The authors gratefully acknowledge Dr. Mehrdad Azin for his
practical comments.
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