Biodegradation of Emerging

International Journal of
Environmental Research
and Public Health
Article
Biodegradation of Emerging Pharmaceuticals from Domestic
Wastewater by Membrane Bioreactor: The Effect of Solid
Retention Time
Raghad Asad Kadhim ALOBAIDI 1,2, * , Kubra ULUCAN-ALTUNTAS 1, * , Rasha Khalid Sabri MHEMID 3 ,
Neslihan MANAV-DEMIR 1 and Ozer CINAR 1
1 Department of Environmental Engineering, Faculty of Civil Engineering, Yildiz Technical University,

Istanbul 34220, Turkey; nmanav@yildiz.edu.tr (N.M.-D.); ocinar@yildiz.edu.tr (O.C.)
2 Ministry of Health and Environment, Baghdad, Iraq
3 Department of Environmental Technologies, College of Environmental Science and Technologies,
Mosul University, Mosul 41002, Iraq; rashamhemid@uomosul.edu.iq
* Correspondence: env.alobaidi@yahoo.com (R.A.K.A.); kulucan@yildiz.edu.tr (K.U.-A.)
Abstract: Although conventional biological treatment plants can remove basic pollutants, they are
ineffective at removing recalcitrant pollutants. Membrane bioreactors contain promising technology
and have the advantages of better effluent quality and lower sludge production compared to those of
conventional biological treatment processes. In this study, the removal of pharmaceutical compounds
by membrane bioreactors under different solid retention times (SRTs) was investigated. To study
the effect of SRT on the removal of emerging pharmaceuticals, the levels of pharmaceuticals were
Citation: ALOBAIDI, R.A.K.; measured over 96 days for the following retention times: 20, 30, and 40-day SRT. It was found that
ULUCAN-ALTUNTAS, K.; MHEMID, the 40-day SRT had the optimum performance in terms of the pharmaceuticals’ elimination. The
R.K.S.; MANAV-DEMIR, N.; CINAR, removal efficiencies of the chemical oxygen demand (COD) for each selected SRT were higher than
O. Biodegradation of Emerging 96% at steady-state conditions. The highest degradation efficiency was observed for paracetamol.
Pharmaceuticals from Domestic Paracetamol was the most removed compound followed by ranitidine, atenolol, bezafibrate, di-
Wastewater by Membrane Bioreactor: clofenac, and carbamazepine. The microbial community at the phylum level was also analyzed to
The Effect of Solid Retention Time.
understand the biodegradability of pharmaceuticals. It was noticed that the Proteobacteria phylum
Int. J. Environ. Res. Public Health 2021,
increased from 46.8% to 60.0% after 96 days with the pharmaceuticals. The Actinobacteria class,
18, 3395. https://doi.org/10.3390/
which can metabolize paracetamol, carbamazepine, and atenolol, was also increased from 9.1% to
ijerph18073395
17.9% after adding pharmaceuticals. The by-products of diclofenac, bezafibrate, and carbamazepine
Academic Editor: Paul B. Tchounwou
were observed in the effluent samples.
Received: 25 February 2021 Keywords: biodegradation; solid retention time; pharmaceuticals; membrane bioreactor; solid-phase
Accepted: 17 March 2021 extraction; by-products
Published: 25 March 2021
Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in 1. Introduction
published maps and institutional affil- The increase in the use of micropollutants, including pharmaceuticals, has become a
iations. major environmental problem in recent years. While the presence of pharmaceutical com-
pounds is limited in the environment, knowledge of the long-term threats to aquatic species
and human health even with low levels of drugs remains lacking. The concentration and
effects of micropollutants on the environment and, perhaps, human beings have increased
Copyright: © 2021 by the authors. over the past few decades. Many micropollutants are persistent, only partially removed
Licensee MDPI, Basel, Switzerland. during treatment, and when disposed of in the environment over a long period, cause
This article is an open access article substantial pollution. The European directive, Water Framework Directive 2000/60/EC, is
distributed under the terms and a global framework that underlines the importance of micropollutants in superficial water
conditions of the Creative Commons (rivers and lakes), transitional water (estuaries), coastal waters, and groundwater.
Attribution (CC BY) license (https://
Although traditional water and wastewater treatment plants are successful in con-
creativecommons.org/licenses/by/
trolling pollutants such as organic substances and nutrients, they cannot remove the
4.0/).
Int. J. Environ. Res. Public Health 2021, 18, 3395. https://doi.org/10.3390/ijerph18073395 https://www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2021, 18, 3395 2 of 19
micropollutants (MPs). These micropollutants are substances found in low concentrations

(ng/L and µg/L) in water and wastewater and consist of metals, hydrocarbons, surfactants,
hormones, and pharmaceutical products. According to many studies, conventional wastew-
ater treatment systems are insufficient to degrade certain pharmaceutical compounds, as
a result, these compounds can be found in surface and groundwater [1–3]. In particular,
pharmaceutical compounds are biologically active compounds that affect microorganisms
upon their discharge to the aquatic environment. They are recalcitrant compounds that take
a long time to break down in nature, therefore, effective methods of wastewater treatment
that allow for the efficient removal of emerging pollutants at trace levels are increasingly
needed. In wastewater treatment, MBR (membrane bioreactor) systems have been used
more commonly recently because of their high effluent quality performance [4]. MBRs are
effective in the treatment of many organic and inorganic pollutants, offering three basic
advantages: (i) enhanced adsorption capacities through improved biomass characteristics;
(ii) improved high sludge biodegradation, (iii) direct removal of several contaminants
adsorbed on rejected particles through the membrane [5]. These process benefits are com-
monly recognized over the those of the traditional activated sludge process [6]; the most
cited benefit is the decrease of sludge production resulting from operation at high solid
retention times (SRTs). In general, hydrophobic and readily-biodegradable contaminants
are very effectively eliminated by MBRs. However, hydrophilic compounds, in particular
biodegradation-resistant ones, may not be excluded [7]. The substantial difference in MBR
removal efficiency for several pharmaceuticals, from near-complete removal (e.g., paraceta-
mol and bezafibrate) to almost no removal for some others (for example, carbamazepine
and diclofenac) [8–10], has been noted previously.
Removal mechanisms do not follow a general rule as their relative contribution is
dependent on the physicochemical properties of the micropollutant, its origin and composi-
tion, and the wastewater operational parameters [11]. A significant point is to understand
why the removal of micropollutants such as pharmaceuticals by MBRs are affected by
the treatment conditions applied in terms of the solid retention time (SRT). A single com-
pound may vary in removal rate and this may be correlated with the physicochemical
characteristics of the xenobiotics.
Various analytical determination methods of pharmaceutical compounds in wastewa-
ter are already available in the literature, which records specific pharmaceutical existence
at levels from ng/L to g/L [12]. Multi-residual analytical methodologies, including phar-
maceutical products of the multi-class range, are the tools required to provide a reliable
and broad knowledge base on the presence of pharmaceuticals and for monitoring their
removal, partitioning, and final fate. When analyzing several different physicochemical
compounds simultaneously, a high recovery rate for each compound may not be seen.
Therefore, a common experimental condition must be determined. However, the creation
of the multicomponent analysis method is useful due to the reduced number of analyses
that are required. The pre-concentration and isolation of the goal analytes can be per-
formed by solid-phase extraction (SPE). The determination of the overall SPE method is
important because most of these multicomponent analysis methods consist of two or more
different sorbent materials and solvents used for elution, and they involve grouping target
compounds according to their physicochemical properties.
Given these facts, the purpose of this work was the development of a sensitive multi-
component solid-phase extraction method to simultaneously analyze, by LC/MS-MS, six
different therapeutic classified pharmaceuticals in synthetic domestic wastewater and to
investigate the influences of SRT on treatment performance of the MBR in terms of remov-
ing these pharmaceuticals from synthetic domestic wastewater. The change in microbial
species due to the micropollutants was also studied.
viron. Res. Public Health 2021, 18, x FOR PEER REVIEW 3 of 20
2. Materials and Methods

2.1. Design of the2.MBR
Materials
Systemand
andMethods
Its Operation
2.1. Design of the MBR System and Its Operation
The reactor was constructed according to the design and configuration for the size
and dimensions of the Themembrane
reactor wasmodules
constructed according
equipped withtotwo thePES
design and configuration for the size
(polyethersulfone)
membranes with 0.45 µm and 0.20 µm pore sizes (Sterlitech Cooperation,two
and dimensions of the membrane modules equipped with PESWA,
Kent, (polyethersulfone)
USA). membranes with 0.45 µm and 0.20 µm pore sizes (Sterlitech Cooperation, Kent, WA, USA).
The aerobic
The aerobic membrane membrane
bioreactor wasbioreactor
made of awas made of
plexiglass a plexiglass
material with material with a cylindrical
a cylindri-
shape of 17.5 cm diameter corresponding to an 8 L volume
cal shape of 17.5 cm diameter corresponding to an 8 L volume and an active operating and an active operating volume
of 4 L. The peristaltic pumps were used to provide pressure
volume of 4 L. The peristaltic pumps were used to provide pressure for maintaining con- for maintaining constant
stant permeate permeate
flux and forflux and for automatic
automatic inoculationinoculation of the
of the reactor feedreactor feed Coopera-
(Shenchen (Shenchen Cooperation,
tion, Baoding, China). A level sensor was used to balance the water level of thelevel
Baoding, China). A level sensor was used to balance the water of the reactor (Tin
reactor
Muhendislik, Istanbul, Turkey). The reactor’s oxygen
(Tin Muhendislik, Istanbul, Turkey). The reactor’s oxygen demand was provided by am- demand was provided by ambient
air through the air pumps and diffusers. Details of the reactor
bient air through the air pumps and diffusers. Details of the reactor configuration are il- configuration are illustrated
in Figure 1.
lustrated in Figure 1.
Figure 1. Lab-scale set up for submerged membrane bioreactor.

Figure 1. Lab-scale set up for submerged membrane bioreactor.
The aerobic submerged

The aerobic membrane
submerged bioreactor
membrane (AeSMBR) was fed
bioreactor with synthetic
(AeSMBR) was fed do- with synthetic
mestic wastewater prepared
domestic according
wastewater to the following
prepared according recipe: 50 mg·L recipe:
to the following
−1 NH4CI, 50 0.04
mg·L−1 NH4 CI,
mg·L MnCI2·4H
−1 2O,mg
0.04 0.132
·L−mg·L
1 MnCIZnCl
−1 2, 4.5 mg·L K2−
2 ·4H2 O, 0.132 mg·L
−1 HPO 4, 4.2
1 ZnCl mg·L KH−1
2 , 4.5 mg·L
−12PO 4, 0.2 mg·L
K2 HPO
−1
4 , 4.2 mg·L
−1 KH PO ,
2 4
Na2SO3·5H2O, 0.1 0.2 mg·L Na2 SO3 ·5H2 O, 0.1 mg·L CuCl2 ·2H2 O, 2 mg·L FeCl3 ·6H2 O, 0.1 mg·L−1
mg·L −
−1 1CuCl 2 ·2H 2 O, 2 mg·L −1 FeCl − 1
3 ·6H 2 O, 0.1 mg·L −1 NiCl− 2 ·6H
1 2 O, 0.03
mg·L−1 CoCl2·6H 2O, 4.4
NiCl 2 ·6Hmg·L
2 O, 0.03 mg·L−12CoCl
−1 CaCl2·2H O, 12.2 mg·L
2 ·6H
−1 MgSO4−
2 O, 4.4 mg·L ·7H 2O, and
1 CaCl
2 ·2H40
2 O,mg·L
12.2 −1
mg ·L−1 MgSO4 ·7H2 O,
pep-
tone. To obtainanda 750 mg·L −
−1 1concentration of chemical oxygen− 1 demand
40 mg·L peptone. To obtain a 750 mg·L concentration of chemical oxygen demand (COD), glucose
was added. The membrane
(COD), glucose tank
waswas added.equipped with twotank
The membrane polyethersulfones
was equipped with (PES)two sub-
polyethersulfones
merged flat sheet microfiltration
(PES) submerged flat modules, one with a pore
sheet microfiltration size ofone
modules, 0.45withµm aand
pore the
sizeother
of 0.45 µm and the
with a pore sizeother
0.20 µm.
withEach
a poremembrane
size 0.20 module
µm. Each comprised
membrane 12 module
× 12 cm plexiglass,
comprised withan
12 × 12 cm plexiglass,
active surface area
withanof 56.25
activecmsurface area of 56.25 cm2 (7.5 × 7.5 cm).
2 (7.5 × 7.5 cm).
As defined in fluid mechanics,

As defined in fluidfluxmechanics,
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area at aInspecific
this context,
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context, flux can as: be formulated as:
( ) (Volume)
= (1)
( × ) J = ( Time × Area) (1)
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low:
low: Tolow:
low: provide adequate relaxation time for the membrane, the reactor was operated for
low:
low:
4.5 min and stopped for 30 s. In this respect, the flux value needed to be recalculated
××𝐹𝑙𝑢𝑥
𝐹𝑙𝑢𝑥𝐹𝑙𝑢𝑥 𝑅𝑢𝑛𝑛𝑖𝑛𝑔
𝐹𝑙𝑢𝑥
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×× 𝑇𝑖𝑚𝑒𝑇𝑖𝑚𝑒
𝑅𝑢𝑛𝑛𝑖𝑛𝑔
× 𝑅𝑢𝑛𝑛𝑖𝑛𝑔
𝑅𝑢𝑛𝑛𝑖𝑛𝑔 𝑇𝑖𝑚𝑒
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𝑇𝑖𝑚𝑒 11.11
11.1111.11×11.11
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by𝑁𝑒𝑡
considering
𝐹𝑙𝑢𝑥
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𝑁𝑒𝑡 =the
==𝐹𝑙𝑢𝑥
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==relaxation time so that the=net
== = =flux
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≅≅10 10 LMH
≅LMH
≅
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1010LMH
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10 be (2)
LMH
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(2) (2) (2)
(2)
𝑊𝑎𝑖𝑡𝑖𝑛𝑔 𝑡𝑖𝑚𝑒
𝑊𝑎𝑖𝑡𝑖𝑛𝑔
𝑊𝑎𝑖𝑡𝑖𝑛𝑔 ++𝑅𝑢𝑛𝑛𝑖𝑛𝑔
𝑡𝑖𝑚𝑒
+
𝑡𝑖𝑚𝑒 +
++ 𝑇𝑖𝑚𝑒𝑇𝑖𝑚𝑒
𝑅𝑢𝑛𝑛𝑖𝑛𝑔 𝑇𝑖𝑚𝑒(0.5(0.5
(0.5
𝑇𝑖𝑚𝑒
𝑇𝑖𝑚𝑒 + 4.5)
4.5)
(0.5
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below:
2.2.Samples
2.2. Samples Preparation
Preparation
Flux × Running Time 11.11 × 4.5 min ∼
2.2. 2.2.
2.2.Samples
Samples
Net
2.2. Samples
Flux
Samples =Preparation
Preparation
Preparation
Preparation = = 10 LMH (2)
TheThe target
Waiting
compounds were
time +
chosen
Running
because
Time
oftheir
their
(0.5 + 4.5in) the
prevalence
minaquatic envi-
The target
The compounds
The target
target
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compounds
target were
compounds
compounds
compounds chosen
were were
were because
chosen
chosen
were chosen
because
chosen of
because
because
because prevalence
of
of their
of
of their their
their in the
in aquatic
prevalence
prevalence
prevalence
prevalence in
ininthe
the envi-
theaquatic
aquatic
aquatic
the envi-
aquatic envi-
envi-
envi-
ronment
ronment
ronment worldwide,
worldwide,
ronment
ronment
ronment ubiquity,
ubiquity,
worldwide,
worldwide,
worldwide,
worldwide, andand
and
ubiquity,
ubiquity,
ubiquity,
ubiquity, high
high
and human
human
and
high
and high
high
human
high intake.
intake.
human
human
human Target
Target
intake.
intake.
intake. Target
intake. compounds
compounds
Target
Target were
were
compounds
compounds
compounds
Target compounds catego-
catego-
werewere
werecatego-
catego-
catego-
were catego-
2.2.
rized
Samples
as shown
Preparation
in Table 1 according to their therapeutic effect and physicochemical prop-
rizedrized
asrized
shown
rized as
as shown
rized in Table
shown
asas shown
shown in1inTable
in Table
in according
Table
Table to their
11 1according
according
1 according therapeutic
to
tototheir
to their
according their
their effect
therapeutic
therapeutic
therapeutic and
effect
therapeutic physicochemical
effect and
effect
and
effect prop-
andphysicochemical
physicochemical
physicochemical
and physicochemical prop-
prop- prop-
prop-
The
erties.
erties. target compounds were chosen because of their prevalence in the aquatic environ-
erties.
erties.erties.
erties.
ment worldwide, ubiquity, and high human intake. Target compounds were categorized
Table
Table 1.1.
TableasMain
Main
Table
Table 1.characteristics
characteristics
1.
1.shown
Table Main Main
1. Tableofof
characteristics
Main the
1the
incharacteristics
Main characteristics
characteristics selected
ofselected
of the
ofof
according
the the and
and analyzed
toanalyzed
selected and
selected
selected
the their
and
selected and pharmaceuticals
pharmaceuticals
analyzed
analyzed
therapeutic
analyzed
and [13,14].
[13,14].
pharmaceuticals
pharmaceuticals
effect
pharmaceuticals
analyzed and [13,14].
[13,14].
pharmaceuticals [13,14].
physicochemical
properties.
[13,14].
Diclofenac
Diclofenac Diclofenac
DiclofenacDiclofenac
Diclofenac
Table 1. Main Acetaminophen
AcetaminophenAcetaminophen
Acetaminophen
Acetaminophen
characteristics
Acetaminophen of(Sodium
the selected and analyzed pharmaceuticals Ranitidine
Ranitidine Ranitidine
Ranitidine
Ranitidine Hydro-
Hydro-
Ranitidine Hydro-
[13,14]. Hydro-
Hydro-
Hydro-
Carbamazepine
Carbamazepine Carbamazepine
Carbamazepine
Carbamazepine
Carbamazepine (Sodium (Sodium
(Sodium (Sodium
(Sodium Bezafibrate
Bezafibrate Bezafibrate
Bezafibrate chloride
Bezafibrate
Bezafibrate Atenolol
Atenolol Atenolol
Atenolol Atenolol
Atenolol
(Paracetamol)
(Paracetamol)
(Paracetamol)
(Paracetamol)Salt)
(Paracetamol)
(Paracetamol) chloride chloride
chloride chloride
chloride
Acetaminophen Salt) Salt)
Salt)
Salt)Salt)
Diclofenac Ranitidine
Carbamazepine Anti-inflam- Bezafibrate Atenolol
(Paracetamol) Anti-inflam- (SodiumAnti-inflam-
Anti-inflam-
Anti-inflam- Salt)
Anti-inflam- Hydrochloride
Applications
Applications
Applications
Applications
Applications Antiepileptic
Antiepileptic
Applications Antiepileptic Antiepileptic
Antiepileptic
Antiepileptic Therapeutic
Therapeutic Therapeutic
Therapeutic
Therapeutic
Therapeutic Lipid
Lipid Lipid regulator
regulator
Lipid
Lipid regulator
regulator Anti-histamine
Anti-histamine
regulator
Lipid regulator Anti-histamine
Anti-histamine
Anti-histamine
Anti-histamine β-Blockers
β-Blockers β-Blockers
β-Blockers
β-Blockers
β-Blockers
matory
matory matory matory
matory
matory
Anti-
Applications Antiepileptic Therapeutic Lipid regulator Anti-histamine β-Blockers
CAS
CAS CASNumber
Number
CAS
CASCAS
Number Number
Number
Number 298-46-4
298-46-4
298-46-4 298-46-4103-90-2
298-46-4
298-46-4 103-90-2 103-90-2
103-90-2 103-90-2
103-90-2 15307-86-5
15307-86-5
inflammatory
15307-86-5
15307-86-515307-86-5
15307-86-5 41859-67-0
41859-67-0
41859-67-041859-67-066357-59-3
41859-67-0
41859-67-0 66357-59-3 66357-59-3
66357-59-3 66357-59-3
66357-59-3 29122-68-7
29122-68-7 29122-68-7
29122-68-7
29122-68-7
29122-68-7
CAS Number 298-46-4 103-90-2 CC HC
1414 H Cl
10
1014 C
HClNNa
C
C210 NNa
2H
HH
14Cl
15307-86-5
14 14 10 Cl
NNa
21010 22NNa
ClCl 2NNa
NNa 41859-67-0 66357-59-3 29122-68-7
Formula
Formula
FormulaFormula
Formula
Formula CC HC
1515H NH
12
1215 CN
2O
C C2OH
HH
15N
15
12 15 ON
212
12 NN
12 22O
O2OCC8H8HC 98NO
9NO HCC 88H
C
92NOH99NO
2 8H 9NO
2NO 22 2 C C HC
1919 H
2019H
20 ClNO
ClNO
C C
C20
19 HHH
19ClNO
19 20ClNO
420 4CC
420ClNO
ClNO H
4C
13413 H NH
22
42213 N
4O
C
C C4OH
13N
13
22 HS3H
313 ·S22O
422 ·N
HClNHCl
3N
22 ·
S44O
O4O
HClS3··
33S C C
·HCl
SHCl HC
1414
HCl H NH
22
2214 N
2O
C
C C2OH
14N
14
22 H3H
314 ON
222
22 N3N
22 22O
O2O
33 3
Formula C15 H12 N2 O C8 H9 NO2 O O2 2 O2 O
C14 H10 Cl2 NNaO2 O O
22 2 C19 H20 ClNO4 C13 H22 N4 O3 S·HCl C14 H22 N2 O3
LogLog
Log KK Log
owowKLog
Log owKKK 2.45 2.45
owow 2.45 2.452.45 2.45 0.46
0.46 0.460.46 0.46 4.51
0.46 4.51 4.51 4.514.51 4.25 4.25 4.25
4.254.25 4.25 0.27
0.27 0.270.27 0.27 0.16
0.16 0.160.16 0.16
4.514.51 4.25 0.27 0.16
ow
Log Kow 2.45 0.46 0.27 0.16
pKpKa pK
a apK pK
pKa a a 13.9
13.9 13.9 13.913.9
13.9 9.49.4 9.4 9.4
9.4
9.4 4.15
4.15 4.15 4.154.15
4.15 3.61
3.61 3.61 3.61
3.61
3.61 8 8 8 88 8 9.6
9.6 9.6 9.6
9.6 9.6
pKa 13.9 9.4 4.15 3.61 8 9.6
LogLog
Log DDLog D
LogLogDDD 1.89
1.89 1.89
1.891.89 1.89 0.47
0.47 0.47
0.470.47 0.47 1.77
1.77 1.77
1.771.77 1.77 −0.93
−0.93 −0.93 −0.93
−0.93
−0.93 −0.63
−0.63 −0.63 −0.63
−0.63
−0.63 −2.09
−2.09 −2.09 −2.09
−2.09
−2.09
Log D
Henry Con- 1.89 0.47 1.77 − 0.93 − 0.63 − 2.09
Henry Con-
HenryHenry
Henry
Henry Con- Con-Con-
Con-
Henry Constant
stant
stant 1.1
1.1 ×10
1010 −12 −12 −12−126.42 × 10 −13 −13 −13−13 4.7 × 10 −12 −12 −12−12 2.12 × ×1010 −15 −15 −15−15 3.42 × 10 −15 −15 −15−151.37 × 10 −18 −18 −18−18
××1.1 −−12 −126.42 6.42
6.42××10 4.7 × 10 2.12 −15 3.42 3.42 × 10 15 1.37 1.37
× 101.37
−13 −12 −15 −15 −18
stant stant
stant1.1
stant 1.1
×1.1
12 10×× ×10
1.1 1010 6.42
×6.42
6.42
10 − 13×
10 × ×10
1010
−13
4.7×4.7 × 10
4.7 × 10 4.7 × 10
10 −12 −12
4.7 × 10 2.12 2.12
2.12
2.12 ×××10
×2.12
10 ×10
−10
10 15 3.42
×3.42
3.42
3.42 10×××10×10
1010
−−15 ×
1.37
1.3710××××10
1.37 1010
10 −18
−18
(atm-m3 /mole)
(atm-m
(atm-m 3
(atm-m 3/mole)
/mole)
(atm-m
(atm-m
(atm-m 3 3
/mole)
3/mole)
3 /mole)
/mole)
M.W.
M.W. 236.27
236.27 151.17 151.17 318.14
151.17 318.14 361.82 350.86 266.34
M.W. M.W. M.W. 236.27
M.W.
M.W. 236.27 236.27 151.17
236.27
236.27 151.17 151.17 318.14
151.17 318.14 318.14 361.82
318.14
318.14 361.82
361.82 361.82
361.82
361.82 350.86
350.86 350.86 350.86 266.34
350.86
350.86 266.34266.34266.34
266.34
266.34
Molecular
Molecular
Molecular Molecular
Molecular
Molecular
Molecular
Structure
Structure
Structure
Structure
Structure
Structure
Structure
Allpharmaceutical
All pharmaceutical
All All
AllAllpharmaceutical
pharmaceutical
pharmaceutical
pharmaceutical standards
standards
standards used
used
standards
standards
standards usedwere
wereused ofof
used
usedwere high
high
were
were ofpurity
purity
of
of high
were ofhigh
high grade
grade
purity
purity
high purity
grade
purity (>98%).
(>98%).
grade
grade
(>98%).
grade Diclofenac,
Diclofenac,
(>98%).
(>98%).
(>98%). ac-
Diclofenac,ac-ac- ac-
Diclofenac,
Diclofenac,
Diclofenac, ac-
ac-
All
etaminophen,
etaminophen, pharmaceutical
etaminophen,bezafibrate,
bezafibrate,
etaminophen,
etaminophen, bezafibrate, standards
carbamazepine,
carbamazepine,
bezafibrate,
bezafibrate, used
carbamazepine,
carbamazepine,
carbamazepine, were
ranitidine
ranitidine of high
ranitidine
ranitidine
ranitidine
etaminophen, bezafibrate, carbamazepine, ranitidine hydrochloride, and atenolol
purity
hydrochloride,
hydrochloride, grade
and
hydrochloride, and
hydrochloride,
hydrochloride, and (>98%).
atenolol
atenolol
andand
atenolol were
atenololDiclofenac,
were
atenolol
were were
were
were
acetaminophen,
purchased
purchased from
from
purchased
purchased
purchased
purchased from bezafibrate,
Alfa
Alfa
from
fromAesar
Aesar
Alfa
from Alfa
Alfa
Aesar
Alfa (Kandel,
Aesar carbamazepine,
(Kandel,
Aesar
Aesar (Kandel, Germany).
Germany).
(Kandel,
(Kandel,
(Kandel, Germany). ranitidine
The
The
Germany).
Germany).
Germany). Themethod
method
The hydrochloride,
developed
developed
Themethod
method
The method
method consisted
consisted
developed
developed
developed
developed and ofatenolol
ofone
consisted
consistedconsisted
consisted one
of one of were
ofofone
one
one
purchased
extraction
extraction from
step
step
extraction
extraction
extraction
extraction for
for
step Alfa
all
step
step
for
step Aesar
alltarget
target
for
all
for all
fortarget
allall (Kandel,
compounds,
compounds,
target
target Germany).
compounds,
compounds,
compounds,
target compounds, which
which whichwhich
which
which The
greatly
greatly method
simplified
simplified
greatly
greatly greatly developed
sample
sample
simplified
simplified
simplified
greatly simplified samplesample
sample
sample consisted
preparation.
preparation. preparation. of one
preparation.
preparation.
preparation.
Individual
extraction
Individual standard
step
standard
Individual
Individual
Individual
Individual for
standard stock
allstock
stock
standard
standard
standard solutions
target
solutions
stock
stock
solutions
stock ininethanol
compounds,
solutions ethanol
solutions
solutions in
in ethanol were
which
were
ininethanol
ethanol
were
ethanol prepared
greatly
prepared
were
were based
based
prepared
prepared
prepared
were preparedbased on
simplified
on
basedweight
weight
based
on
based on on and
sample
and
weight
weight
on weight
weight andstored
storedpreparation.
and andstored
stored
and stored
stored
atat−2−2
Individual°C.
at°C.
−2at An
atAn
−2
at
−2−2
°C. Anacceptable
acceptable
standard
°C.°C.An
°C. Anacceptabledilution
dilution
stock
acceptable
acceptable
An acceptable of ofindividual
solutions
dilution
dilution individual
dilution
dilution ofin
of stock
stock
ethanol
ofindividual
of individualindividual
individual solutions
solutions
were
stockstock
stock was
was
prepared
solutions
solutions
solutions
stock was
solutions established
established
based
was for
for
on
wasestablished
established
established
was established amixture
afor
weightmixture
foraaand
afor
mixture
for stored
amixture
mixture
mixture
atofall
of −all ◦of
pharmaceuticals.
of2pharmaceuticals.
allC.
of all
of allpharmaceuticals.
An
all pharmaceuticals.
pharmaceuticals.
acceptable dilution of individual stock solutions was established for a mixture
pharmaceuticals.
According
of allAccording totothe
According
According
According
According
pharmaceuticals. the theliterature
to literature
to
totothe
literature
the review
review
theliterature
literature
literature review [15–18],
[15–18],
review
review
review theconcentrations
the
[15–18], concentrations
[15–18],
[15–18], the
[15–18], the ofofthis
theconcentrations
concentrations
concentrations
the concentrations this
of thisstudy
study
of
ofofthis
this
study
this analytes
analytes
study
study
study analytes
analytes analytes
analytes
was
was was chosen
chosen
was as
chosen
was as
waschosen 1212
chosen
chosen µ
asto12µg/L
g/L
as
asasµ12
12 12carbamazepine,
carbamazepine,
g/Lµµg/L g/Lcarbamazepine,
µg/L 20
carbamazepine,
carbamazepine,
carbamazepine, 20 µ µg/L
g/L
20 µ20 diclofenac,
diclofenac,
20µµg/L
g/L
20 µg/L 2020µ
diclofenac,
diclofenac,
diclofenac, µg/L
g/L
20 µ20
g/Lconcentrations
diclofenac, 20 atenolol,
atenolol,
20µµg/L
g/L µg/L
g/L 2020µ20
atenolol,
atenolol,atenolol,
atenolol, µg/L
g/Lµ20 20µµg/L
g/L
20 µg/L
g/L
According the literature review [15–18], the of this study analytes
ranitidine
ranitidine
ranitidine hydrochloride,
hydrochloride,
ranitidine
ranitidine
ranitidine hydrochloride, 1212µ12
hydrochloride,
hydrochloride,
hydrochloride, µg/L
g/Lµ121212
g/Lbezafibrate,
bezafibrate,
µµg/L
µ g/L
g/L andand
and
bezafibrate, 100
bezafibrate,
bezafibrate,
bezafibrate, 100 µ
and µg/L
g/L
and
100
and 100
µ100
100 acetaminophen
acetaminophen
g/Lµ
µ g/L
µ g/Lacetaminophen
acetaminophen
acetaminophen
g/L acetaminophen (paraceta-
(paraceta- (paraceta-
(paraceta-(paraceta-
(paraceta-
was chosen as 12 µg/L carbamazepine, 20 µg/L diclofenac, 20 µg/L atenolol, 20 µg/L
mol).
mol). mol). mol).
mol).
mol).
ranitidine hydrochloride, 12 µg/L bezafibrate, and 100 µg/L acetaminophen (paracetamol).
2.3.Solid-Phase
2.3. Solid-Phase
2.3. 2.3.
2.3. Extraction
Extraction
2.3.Solid-Phase
Solid-Phase
Solid-Phase Method
Method
Extraction
Extraction
Extraction
Solid-Phase Method
Method
Extraction Method
Method
2.3. Solid-Phase Extraction Method
Development,
Development, optimization,
optimization,
Development,
Development,
Development,
Development, andand
and
optimization,
optimization,
optimization,
optimization, validation
validation
and
and ofofa aof
andvalidation
validation
validation
validation method
method
aof
method
of followed
followed
ofaa amethod
method
method bybyliquid
followed liquid
followed
followed
followed by byby
liquid
by chroma-
chroma-
liquid
liquid chroma-
chroma-
liquid chroma-
chroma-
Development,
tography–triple
tography–triple optimization,
quadrupole
quadrupole
tography–triple mass
mass and
spectrometry
spectrometry validation
(LC/MS-MS)
(LC/MS-MS) ofwere a method
were performed
performed followed
for
for the
the by
sim-
sim- liquid
tography–triple quadrupole mass spectrometry (LC/MS-MS) were performed for the
tography–triple
tography–triple quadrupole
quadrupole
quadrupole massmass
massspectrometry
spectrometry
spectrometry (LC/MS-MS)
(LC/MS-MS)
(LC/MS-MS) werewere
were performed
performed
performed for for
for
the thesim-
sim-
the sim-
sim-
chromatography–triple
ultaneous
ultaneous determination
determination
ultaneous
ultaneous
ultaneous quadrupole
of
of
determination
determination
determination six
six
of of
six six
of six mass
multi-class
multi-class spectrometry
pharmaceuticals
pharmaceuticals
multi-class
multi-class
multi-class pharmaceuticals
pharmaceuticals
pharmaceuticals (LC/MS-MS)
using
using offline
offline
using
using using were
solid-phase
solid-phase
offline
offline offline performed
ex-
ex-
solid-phase
solid-phase
solid-phase ex- ex-for
ex-
ultaneous determination of six multi-class pharmaceuticals using offline solid-phase ex-
the simultaneous
traction
traction (SPE).
(SPE).
traction
traction
traction The
The
(SPE).
traction determination
SPE
SPE
(SPE).
(SPE).
The
(SPE). method
method
The
The
SPE
The SPEmethod
SPE was
SPEmethodof
was
method
method six
was multi-class
inspired
inspired
was bybythe
wasinspired
inspired
inspired
was by
inspired pharmaceuticals
theliterature
byliterature
the
bybythe [19].
[19].
theliterature For
literature
literature
the For
[19].
literature using
[19].
[19].
For
[19]. For offline
conditioning
conditioning solid-phase
ap-ap- ap-
ap-
Forconditioning
conditioning
conditioning
For conditioning ap-
ap-
extraction
plications,
plications, (SPE).
acetone
acetone
plications,
plications,
plications, The
acetone
plications, (HPLC
(HPLC
acetone SPE
acetone
(HPLC
acetone method
grade),
grade),
(HPLC
(HPLC
(HPLC was
methanol
methanol
grade),
grade),
grade), inspired
(HPLC
(HPLC
methanol
methanol
methanol
grade), methanol(HPLC by
(HPLC
(HPLC
(HPLC thegrade),
grade),
grade),
grade), literature
andand
and
grade),
grade), and[19].
ultrapure
ultrapure
and For
water
water
ultrapure
ultrapure
ultrapure
and ultrapure conditioning
were
were
water
water water
were
water were
were
were
applications, acetone (HPLC grade), methanol (HPLC grade), and ultrapure water were
used (Merck, Istanbul, Turkey). Table 2 shows the conditions that were applied, including
conditioning, percolation, washing, drying, and elution.
Table 2. Solid-phase extraction (SPE) method determination.
SPE SPE SPE SPE

Applications
Method 1 Method 2 Method 3 Method 4
Oasis HLB Oasis HLB
Types of C18 cartridge, C18 cartridge,
cartridge cartridge
cartridge 500 mg, 6 mL 500 mg, 6 mL
200 mg, 6 mL 200 mg, 6 mL
5 mL acetone, 5 mL acetone,
5 mL MeOH, 5 mL MeOH,
Conditioning 5 mL MeOH, 5 mL MeOH,
5 mL water 5 mL water
5 mL water 5 mL water
Percolation 100 mL 100 mL 100 mL 100 mL
5 mL 5 mL 5 mL 5 mL
Washing
water water water water
Drying 15 min. 15 min. 15 min. 15 min.
Elution 8 mL MeOH 8 mL MeOH 8 mL MeOH 8 mL MeOH
To concentrate the samples, the compounds were extracted via the cartridges given
in Table 2 using the SPE manifold system equipped with a vacuum pump. To optimize
the extraction method, the performances of various SPE cartridge materials (Oasis HLB
(200 mg, 6 mL) from Waters Corporation and C18 (500 mg, 6 mL) from Agilent) were
compared. The detailed procedure is summarized in Table 2; firstly, SPE cartridges for
samples 1 and 3 were conditioned with 5 mL MeOH followed by 5 mL distilled water at
a flow rate of l mL/min. The same procedure was applied for the conditioning of SPE
cartridges for samples 2 and 4 but it was proceeded with a conditioning of 5 mL of acetone.
The water samples were percolated by the cartridges following the conditioning stage. The
cartridge was then rinsed with 5 mL HPLC-grade water and dried for 15 min under a
vacuum to extract the excess water. Elution of 2 × 4 mL of methanol was performed. For
direct testing by LC/MS-MS analysis, the extract was evaporated under a gentle nitrogen
stream and concentrated into 1 mL of sample volume.
2.4. Analytical Methods

The feed wastewater characterization and reactor performance were monitored by
wet analyses. For the suspended solid (TSS) parameter, standard methods (APHA, 1998)
were applied. COD is one of the most important parameters in determining the degree of
organic pollution in wastewaters. COD analyses were carried out using the microdigestion
and titration method specified in the standard methods (SM 5220B) [20].
An HPLC (Shimadzu) equipped with a Diode-Array Detection (DAD) detector and
LC-QTOF-MS/MS (Agilent 6530 Accurate Mass–ESI Interface, Santa Clara, CA, USA) was
used for liquid chromatography analysis. To achieve the chromatographic separation, the
Purospher Star RP-18 column (125 mm × 2.0 mm, particle size 5 µm) was supplied with a
C18 guard column by Merck (Darmstadt, Germany). The analyses were performed in Posi-
tive Ionization mode with eluent A (acetonitrile-methanol (2:1)) and eluent B (ammonium
acetate 5 mM at pH 4.7 (acetic acid)). The flow rate was selected as 0.3 mL/min, and the
injection volume was determined as 10 µL. According to the selected method, the eluent
gradient started from 5% and rose to 95% of eluent A in 5 min (was held for 4 min) and
turned to the initial condition in 5 min.
2.5. Metagenomic Analysis

Biomass samples at the beginning of the steady-state of the 40-day SRT (before adding
pharmaceuticals) and at the end of the 40-day SRT (after adding pharmaceuticals) in the
aerobic membrane bioreactor were collected and stored immediately at −4 ◦ C to evaluate
the percentage of the microbial community. The samples were processed and analyzed as
explained below.
2.6. DNA Isolation

The samples with an amount of 200 mg were transferred to tubes containing 0.1 mm
diameter glass beads and 300 µL of lysis buffer (200 mM Tris-HCl, pH 8.0; 20 mM EDTA;
10% TritonX-100) and were homogenized for 1 min at 6000 rpm. The samples were
transferred to new tubes to isolate the beads and were incubated at the 37 ◦ C for 15 min;
then, 10 µL of the lysozyme solution (200 µg/µL) was added. Next, 250 µL of lysis buffer
(0.5 µg/µL Proteinase K, 5% Tween® 20, 3M Guanidine thiocyanate, 20 mM Tris-HCl,
pH 8.0) was added to each sample, and the samples were incubated for 15 min at 70 ◦ C,
followed by 5 min at 95 ◦ C. After incubation, 250 µL of 2-propanol was appended to each
tube and the samples were loaded onto the silica column. The DNA in the samples were
passed through the silica column by centrifugation at 13,000 rpm for 1 min and kept by
the silica column, then they were washed twice with wash solution (20 mM NaCl, 2 mM
Tris-HCl, pH 8; 80% v/v ethanol). The silica column was dried by centrifugation. DNA
retained in the silica columns was taken from the columns with 50 µL of 100 mM Tris-HCl
prepared with nuclease-free, sterile, deionized water (pH 7) and preserved at −20 ◦ C
until analysis. Spectrophotometric methods measured the quantity and consistency of the
DNA and checked its suitability for the following steps. Other molecular processes were
performed using DNA with an OD260/OD280 ratio of 1.8–2.0, an OD260/OD230 ratio of
2.0–2.2, and at least ten ng/µL (preferably 50–300 ng/µL) concentration.
2.7. Next Generation Sequencing (NGS)

The primary pair was used to create amplicon libraries that covered about a 460 bp
region and the V3-V4 region of the 16S rRNA gene [21]. Connector DNA sequences were
added to the 50 end of the target-specific primer pairs for compatibility with the Illumina
index and sequence adapters of the generated library. The forward primer sequence of
the primer-connector oligos specific for 16Sr RNA was 50 TCGTCGGCAGCGTCAGATGT-
GTATAAGAGACAGCCTACGGGNGGCWGCAG 30 , and the reverse primer sequence
was 50 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCT
AATCC-30 . The first PCR step was performed using “Bio-Speedy® 2X qPCR Mix” and
200 nm from each primer.
The following thermal cycling program was monitored on the Biorad CFX Connect
(Bio-Rad, Hercules, CA, USA): 3 min at 95 ◦ C; 25 cycles of 30 s at 95 ◦ C, 30 s at 55 ◦ C,
and 30 s at 72 ◦ C; 5 min at 72 ◦ C. By carrying out agarose gel electrophoresis of the PCR,
the product size (~550 bp) was confirmed and “Bio-Speedy® PCR Product Cleaning Kit”
(Bioeks, Istanbul, Turkey) was used as an eluent.
By performing the second PCR step, binary index and Illumina sequencing adapters
were included in the first PCR amplicons by using the Nextera XT Index Kit (Illumina, New
York, NY, USA) then, the following program was used for thermal cycling: 95 ◦ C for 3 min;
8 cycles of 30 s at 95 ◦ C, 30 s at 55 ◦ C, and 30 s at 72 ◦ C; 5 min at 72 ◦ C. PCR products were
cleaned up with a “Bio-Speedy® PCR Product Cleaning Kit” (Bioeks, Istanbul, Turkey).
The final library was checked for size (~630 bp) by using the “Bioanalyzer DNA 1000 chip”.
To form a library pool, the final library was diluted to 4 nM using 10 mM Tris pH 8.5 and
5 µL aliquots.
The collected libraries were denatured with NaOH, diluted with hybridization buffer
(HT1), and denatured by temperature for batch forming and sequencing preparation, before
the MiSeq sequencing. The research was performed using Illumina MiSeq v3 reaction kits.
For each reaction, as an internal control, a minimum of 5% PhiX was added.
Mothur version 1.39.1 was used to examine the unprocessed sequence data (forward
and reverse reads merged). The first step was to cut out the index and main sequences and
then classify the particular sequences. The trimmed unique sequences were aligned using
the RDP database sequences and the blastn algorithm. Filtering and error checking were
done to unaligned sequences at both ends of the sequences. Pollution was prevented by
pre-clustering. The UCHIME [22] code was used for the chimera removal. The sequences
were categorized using the Bayesian classifier built into Mothur. With the support of the
and then classify the particular sequences. The trimmed unique sequences were aligned
using the RDP database sequences and the blastn algorithm. Filtering and error checking
were done to unaligned sequences at both ends of the sequences. Pollution was prevented
by pre-clustering. The UCHIME [22] code was used for the chimera removal. The se-
quences were categorized using the Bayesian classifier built into Mothur. With the sup-
portdatabase,
RDP of the RDP database,
reference reference
and taxonomyandfiles
taxonomy files were
were gained. Aftergained. After the taxonomic
the operational operational
taxonomic
unit unit selected
(OTU) was (OTU) wasandselected and determination
taxonomic taxonomic determination
was made was made to
according according
the RDPto
the RDP OTUs
database, database,
were OTUs wereaccording
grouped grouped according to their phylotypes.
to their phylotypes.
3.3.Results
Results
3.1.
3.1.Determination
Determinationofofthe
theSPE
SPEMethod
Method
ToToanalyze
analyzethe
theselected
selected compound
compound by by LC/MS-MS,
LC/MS-MS,the theselected
selectedcompounds
compounds were
were re-
required
quired to be concentrated by the solid-phase extraction method: different solventsand
to be concentrated by the solid-phase extraction method: different solvents and
cartridges
cartridgeswere
werecompared
compared for their recoveries.
for their recoveries. The
Thesummarized
summarizedmethods
methods areare given
given in in
Ta-
Table 2. The concentrations of atenolol, ranitidine, paracetamol, carbamazepine, bezafi-
ble 2. The concentrations of atenolol, ranitidine, paracetamol, carbamazepine, bezafibrate,
brate, and diclofenac were 20, 20, 100, 12, 12, and, 20 µg/L, respectively. The volume of the
and diclofenac were 20, 20, 100, 12, 12, and, 20 µg/L, respectively. The volume of the sam-
samples used to analyze recoveries was 100 mL. The calculated recoveries can be seen in
ples used to analyze recoveries was 100 mL. The calculated recoveries can be seen in Fig-
Figure 2.
ure 2.
100%
80%
Recovery
60%
40%
20%
0%
ATN RND PCT CBZ BZF DCF
SPE Method 1 84% 65% 70% 48% 49% 42%
SPE Method 2 96% 76% 72% 53% 61% 47%
SPE Method 3 45% 0% 29% 59% 74% 72%
SPE Method 4 43% 0% 31% 60% 78% 70%
Pharmaceuticals
Figure 2. Recoveries for the extraction of selected pharmaceuticals in synthetic wastewater by us-
Figure 2. Recoveries for the extraction of selected pharmaceuticals in synthetic wastewater by using
ing four different SPE methods.
four different SPE methods.
Whileranitidine
While ranitidinecan
canbe
beextracted
extractedby byHLB
HLBOASIS,
OASIS,no norecoveries
recoverieswere
wereobserved
observedby by
C18 cartridges. For atenolol, HLB OASIS (higher than 86%) gave better recoveries
C18 cartridges. For atenolol, HLB OASIS (higher than 86%) gave better recoveries than than
didC18
did C18cartridges
cartridges(approximately
(approximately45%).
45%).Similarly,
Similarly,HLB
HLBOASIS
OASISwaswasmore
moreefficient
efficient(70%)
(70%)
thanC18
than C18for
forparacetamol.
paracetamol.Oasis
OasisHLB
HLBprovided
providedbetter
betterresults
resultsfor
foratenolol,
atenolol,paracetamol,
paracetamol,
andranitidine
and ranitidinecompared
comparedtotothose
thosefor
forIsolute
IsoluteC18,
C18,while
whileIsolute
IsoluteC18
C18provided
providedbetter
betterresults
results
for carbamazepine, bezafibrate, and diclofenac compared to those of Oasis
for carbamazepine, bezafibrate, and diclofenac compared to those of Oasis HLB. The HLB. The re-
coveries were 60, 78, and 70% with the SPE method 4 for carbamazepine, bezafibrate,
recoveries were 60, 78, and 70% with the SPE method 4 for carbamazepine, bezafibrate, and
diclofenac,
and respectively.
diclofenac, Accordingly,
respectively. Accordingly, SPESPEmethod
method1 1was was chosen
chosen for the analysis
for the analysisofof
atenolol, ranitidine, and paracetamol, and SPE method 4 was chosen for the analysis of
carbamazepine, bezafibrate, and diclofenac for all the analyses in this study.
Int. J. Environ. Res. Public Health 2021, 18, x FOR PEER REVIEW 8 of 20
atenolol, ranitidine, and paracetamol, and SPE method 4 was chosen for the analysis
Int. J. Environ. Res. Public Health 2021, 18, 3395
of
8 of 19
carbamazepine, bezafibrate, and diclofenac for all the analyses in this study.
3.2. Effect of SRT on COD Removal and MLSS Concentration

3.2. Effect
The of SRT on COD
bioreactor shown Removal and MLSS
in Figure 1 wasConcentration
filled with aerobic activated sludge brought
fromThethebioreactor
Atakoy Wastewater Treatment
shown in Figure 1 wasPlant
filledinwith
Istanbul.
aerobicThe reactor sludge
activated was operated
broughtat
from the Atakoy
hydraulic retention Wastewater
time (HRT)Treatment Plant
(17.7 h), which in Istanbul.
correspondsThe toreactor was operated
a permeate flux of at 10
L/m2·h. retention time (HRT) (17.7 h), which corresponds to a permeate flux of 10 L/m2 ·h.
hydraulic
During
Duringthethetimetimetotoreach
reachthe
thesteady-state,
steady-state,the
thereactor
reactorwas
wasfed
fedwith
withsynthetic
syntheticdomestic
domestic
wastewater
wastewater without MPs. After reaching a steady-state, the addition of MPs wasstarted.
without MPs. After reaching a steady-state, the addition of MPs was started.
All
AllSRT
SRTstudies
studieswerewereevaluated
evaluatedseparately,
separately,and
andfor foreach
eachofofthem,
them,thethereactor
reactorwas
wasprepared
prepared
from
fromthethestarting
startingpoint
pointtotoreach
reachsteady-state
steady-stateconditions.
conditions. To Toevaluate
evaluatethetheeffect
effectofofsludge
sludge
retention
retentiontime
timeon onCOD
CODdegradation
degradationandandbacterial
bacterialgrowth,
growth,thethereactor
reactorwas
wascontrolled
controlledfor for
96
96days
daysafter
afterititreached
reachedsteady-state
steady-stateconditions.
conditions.The Theresults
resultsofofmixed
mixedliquor
liquorsuspended
suspended
solids
solids (MLSS),
(MLSS), mixed liquorvolatile
mixed liquor volatilesuspended
suspendedsolidssolids (MLVSS)
(MLVSS) and,
and, CODCOD areare
seenseen in
in Fig-
Figures 3 and 4, respectively.
ures 3 and 4, respectively.
Figure3.3.The
Figure Theaverage
averageof
ofMLSS
MLSSand
andMLVSS
MLVSSconcentrations
concentrationsinindifferent
differentsolid
solidretention
retentiontimes
times(SRTs).
(SRTs).
During the 20-day SRT, the COD removal efficiency for the 0.45 µm membrane was
betweenDuring
93.1theand20-day
93.5%,SRT,
andthe
forCOD
0.20 removal efficiency
µm membrane for the
it was 0.45 µm membrane
94.2–94.5% (Figure 4). was In
between 93.1 and 93.5%, and for 0.20 µm membrane it was 94.2–94.5%
addition, the MLSS values ranged between 5492 and 5870 mg/L, and the MLVSS varied (Figure 4). In addi-
tion, the 4514
between MLSSand values
4872ranged
mg/L between
(Figure 3).5492 and 5870atmg/L,
Similarly, 30 days andofthe MLVSS
SRT, CODvaried
removal be-
tween 4514 and 4872 mg/L (Figure 3). Similarly, at 30 days of SRT, COD
efficiencies increased slightly (95.6–95.9% for the 0.45 µm membrane and 96.3–96.7% for the removal efficien-
ciesµm
0.2 increased
membrane), slightly (95.6–95.9%
however, for the
MLSS and 0.45 concentrations
MLVSS µm membranewere and 96.3–96.7% for the
1.5 times higher 0.2
than
µmresults
the membrane), however,
of the 20-day SRT. MLSS and MLVSS
Conversely, concentrationsinwere
the improvements COD1.5 times efficiencies
removal higher than
the results
were of the 20-day
much higher and wereSRT. Conversely,
97.3–97.7% andthe improvements
98.0–98.2% in COD
for the 0.45 and removal efficiencies
0.2 µm membranes
were
at much
40-day SRT,higher and were
respectively. 97.3–97.7%
The MLSS and and 98.0–98.2%
MLVSS increased for the 0.45 and 0.2 µm
by approximately mem-
2 times
branes
those of at
the40-day
20-daySRT,
SRT.respectively.
These resultsThe MLSS and
are indeed MLVSS increased
comparable by approximately
with the study of Broeck et al.2
(2012)
times who
thosereported that increasing
of the 20-day SRT results
SRT. These increasesarethe concentration
indeed comparable of sludge
with solids and aof
the study
longer
BroeckSRT boosts
et al. (2012)COD
who removal
reportedtreatment efficiency
that increasing SRT and produces
increases less sludge [23].
the concentration of sludge
Because
solids and a of the high
longer SRTSRT values
boosts COD and complete
removal solid retention
treatment efficiencyinside
and the MBR, the
produces less
biodiversity
sludge [23]. of the microorganisms is promoted and even slowly growing and free-living
bacteria remain in the reactor, which leads to better performance [24].
Because
Int. J. Environ. Res. Public Health 2021, 18, 3395 of the high SRT values and complete solid retention inside the MBR, the bi-
9 of 19
odiversity of the microorganisms is promoted and even slowly growing and free-living
bacteria remain in the reactor, which leads to better performance [24].
a 20 days SRT 30 days SRT 40 days SRT

100%
80%
COD Removal Efficiency
60%
40%
20%
0%
0.2µm membrane 0.45µm membrane
b 0.2µm membrane 0.45µm membrane

100%
80%
COD Removal Efficiency
60%
40%
20%
0%
20 days SRT 30 days SRT 40 days SRT
Figure4.4.COD
Figure CODremoval
removalefficiency
efficiencybased
basedon
onmembrane
membranetype
type(a)
(a)and
anddifferent
differentSRTs
SRTs(b).
(b).
3.3.
3.3.Effect
EffectofofSRT
SRTon
onthe
theRemoval
RemovalofofMPs
MPs
During
Duringthetheacclimatization
acclimatizationphase,
phase,the
thereactor
reactorwas
wasfed
fedwith
withonly
onlysynthetic
syntheticdomestic
domestic
wastewater
wastewater and when the reactor reached steady-state conditions, the mixtureof
and when the reactor reached steady-state conditions, the mixture ofatenolol,
atenolol,
ranitidine,
ranitidine,paracetamol,
paracetamol,carbamazepine,
carbamazepine,bezafibrate,
bezafibrate,and
anddiclofenac
diclofenacwas
waspumped
pumpedwithwithaa
feed
feedpump
pumpshown
shown inin
Figure 1. The
Figure removal
1. The waswas
removal monitored for 1 for
monitored week and the
1 week andsamples were
the samples
collected accordingly for 20, 30, and 40-day SRT. The results are given in Table 3 in detail,
and in Figure 5 the removal efficiencies are shown for different SRTs passed through the
0.2 µm and 0.45 µm membranes-equipped MBR system. The increase in MLSS and MLVSS
µg/L for all conditions. Even the highest concentration within all selected MPs was para-
cetamol (100 µg/L), the highest removal efficiency was obtained for paracetamol for all
SRTs. The lowest removal efficiency was observed for carbamazepine for both membranes
(approximately 28%) regardless of varying SRTs. This low removal efficiency was also
observed
Int. J. Environ. Res. Public Health 2021, 18, 3395 by the authors of [25]. The diclofenac removal efficiency was observed10atof ap- 19
proximately 50%, which was similar to the study conducted by Mousel et al. (2021) [26]
who studied the ultrafiltration membrane bioreactor (UF-MBR) process in the degrada-
tion of several pharmaceuticals from raw hospital wastewater and found that the removal
isofrecognized as one ofdrugs
anti-inflammatory the most important
(ibuprofen and parameters
paracetamol)promoting
was highly theefficient.
biodegradation
Paraceta-
ofmol
pharmaceuticals and personal care products (PPCPs) [25]. According
was the most removed compound followed by ranitidine, atenolol, bezafibrate, to these results,
diclo-
the 40-day
fenac, and SRT was selected
carbamazepine forasboth
the best condition
membranes. for the
These steady-state
results align with conditions. The
many previous
concentrations, standard deviations, LOD, and LOQ of the studied compounds
works [27,28]. It can be concluded that the MBR treatment with several SRT conditions can be
found in Table 3, and the comparison of the removal efficiencies of all compounds
can remove some of the MPs at high removal efficiencies, however, a further treatment is given
inprocess
Figure is
5.required for total removal.
a 20 days SRT 30 days SRT 40 days SRT

100
80
MPs Removal (%)
60
40
20
Int. J. Environ. Res. Public Health 2021, 18, x FOR

0 PEER REVIEW 11 of 20
DCF BZF CBZ ATN RND PCT
b 20 days SRT 30 days SRT 40 days SRT

100
80
MPs Removal (%)
60
40
20
0
DCF BZF CBZ ATN RND PCT
Figure5.5.Micropollutants
Figure Micropollutants(MPs)
(MPs) removal
removal under
under different
different SRTs
SRTs in
in the
the membrane
membrane bioreactor
bioreactor (MBR)
(MBR)
system equipped with (a) 0.2 µm and (b) 0.45 µm membranes (DCF: Diclofenac, BZF: Bezafibrate,
system equipped with (a) 0.2 µm and (b) 0.45 µm membranes (DCF: Diclofenac, BZF: Bezafibrate,
CBZ: Carbamazepine, ATN: Atenolol, RND: Ranitidine, PCT: Paracetamol).
CBZ: Carbamazepine, ATN: Atenolol, RND: Ranitidine, PCT: Paracetamol).
The concentration of all studied compounds was decreased with the increase of SRT.
It can be observed in Figure 3 that MLSS showed an increase in concentration with the
increase of SRT. The increase in MLSS should assist the sorption of selected pharmaceuti-
cals [25,29]. While the increased SRT can generally boost the biological variety of slowly
growing bacteria and longer sludge retention promotes the removal of MPs, some re-
searchers have reported that diclofenac and carbamazepine removal is not affected signif-
icantly by SRT changes in MBR treatment [30].
Table 3. The concentrations of pharmaceuticals before and after biodegradation.

40-Day SRT 30-Day SRT 20-Day SRT
0.2 µm 0.45 µm 0.2 µm 0.45 µm 0.2 µm 0.45 µm

RSD% RSD % RSD % RSD % RSD % RSD %
Comp. R2 LOD LOQ Inf. mem. MBR R.E. % mem. MBR R.E.% Inf. mem. MBR R.E.% mem. MBR R.E% Inf. mem. MBR R.E% mem. MBR R.E%
(n = 3) (n = 3) (n = 3) (n = 3) (n = 3) (n = 3)
Eff. Eff. Eff. Eff. Eff. Eff.
DCF 0.997 1.3 4.39 20.76 ± 0.11 10.173 ± 0.19 1.875 51 10.49 ± 0.38 3.713 49.4 20.48 ± 0.34 11.23 ± 0.17 1.496 45 11.8 ± 0.16 1.4 42.4 20.82 ± 0.23 12.13 ± 0.16 1.281 41.7 12.05 ± 0.134 1.112 42.1
BZF 0.997 0.134 0.445 12.48 ± 0.21 1.72 ± 0.01 0.723 86 1.75 ± 0.02 1.4 86 12.44 ± 0.18 2.093 ± 0.01 0.225 83 2.126 ± 0.038 1.814 82.9 12.31 ± 0.15 2.43 ± 0.01 0.455 80.26 2.34 ± 0.025 0.752 81
CBZ 0.991 1.17 3.9 12.583 ± 0.15 8.96 ± 0.1 1.112 28.7 9.09 ± 0.08 0.933 27.7 12.693 ± 0.15 9.55 ± 0.15 1.539 24.7 9.62 ± 0.237 2.466 24.2 12.428 ± 0.17 9.529 ± 0.09 0.891 23.32 9.731 ± 0.067 0.688 21.7
ATN 0.992 0.64 2.13 20.81 ± 0.12 2.41 ± 0.04 1.79 88 2.51 ± 0.06 2.28 88 20.87 ± 0.04 3.52 ± 0.03 0.877 83 3.5 ± 0.054 1.55 83 20.81 ± 0.26 4.385 ± 0.02 0.522 78.9 4.58 ± 0.037 0.814 78
RND 0.997 0.665 2.22 20.48 ± 0.07 2.02 ± 0.03 1.4 90 2.028 ± 0.05 2.43 90 20.6 ± 0.27 3.28 ± 0.03 0.995 84 3.21 ± 0.088 2.77 84.4 20.52 ± 0.18 3.81 ± 0.01 0.228 81.4 3.73 ± 0.012 0.342 81.8
PCT 0.999 5.3 17.7 103.96 ± 0.81 n.d ≥95 n.d ≥95 104.033 ± 1.95 n.d ≥95 n.d ≥95 103.247 ± 1.26 n.d ≥95 n.d ≥95
DCF: Diclofenac, BZF: Bezafibrate, CBZ: Carbamazepine, ATN: Atenolol, RND: Ranitidine, PCT: Paracetamol, Inf: Influent, Eff. Effluent, LOD: Limit of Detection, LOQ: Limit of Quantification, RSD: Relative
Standard Deviation, R.E.: Removal Efficiency. n.d: not detected.
Paracetamol was detected as under the limit of detection at the 20, 30, and 40-day SRT,
and according to LOD, paracetamol concentrations were obtained lower than 5.3 µg/L
for all conditions. Even the highest concentration within all selected MPs was parac-
etamol (100 µg/L), the highest removal efficiency was obtained for paracetamol for all
SRTs. The lowest removal efficiency was observed for carbamazepine for both membranes
(approximately 28%) regardless of varying SRTs. This low removal efficiency was also
observed by the authors of [25]. The diclofenac removal efficiency was observed at approx-
imately 50%, which was similar to the study conducted by Mousel et al. (2021) [26] who
studied the ultrafiltration membrane bioreactor (UF-MBR) process in the degradation of
several pharmaceuticals from raw hospital wastewater and found that the removal of anti-
inflammatory drugs (ibuprofen and paracetamol) was highly efficient. Paracetamol was
the most removed compound followed by ranitidine, atenolol, bezafibrate, diclofenac, and
carbamazepine for both membranes. These results align with many previous works [27,28].
It can be concluded that the MBR treatment with several SRT conditions can remove some
of the MPs at high removal efficiencies, however, a further treatment process is required
for total removal.
The concentration of all studied compounds was decreased with the increase of SRT. It
can be observed in Figure 3 that MLSS showed an increase in concentration with the increase
of SRT. The increase in MLSS should assist the sorption of selected pharmaceuticals [25,29].
While the increased SRT can generally boost the biological variety of slowly growing
bacteria and longer sludge retention promotes the removal of MPs, some researchers have
reported that diclofenac and carbamazepine removal is not affected significantly by SRT
changes in MBR treatment [30].
3.4. Removal Mechanism and Possible Pathways

The pharmaceutical removal mechanisms in MBR treatment are complex and charac-
terized by four main routes: (i) biotransformation or degradation; (ii) sorption by the sludge;
(iii) volatilization or aeration striping; and, (iv) physical removal through membranes [30].
The constant of Henry’s Law for selected pharmaceuticals is lower than <10−6 , which
shows the removal of these compounds by volatilization is negligible (Table 1). Moreover,
microfiltration (MF) membranes usually have a molecular weight cut off (MWCO) above
several thousand daltons (Da), so that pharmaceutical retention (MWCO from 150 up to
350 Da) is insignificant in the MBR processes because of the exclusion of size. Consequently,
biotransformation and sorption by sludge were presumably the dominating removing
mechanisms.
For non-ionizable compounds, the hydrophobicity is described as log Kow, while it is
described as log D for ionizable compounds; a compound is considered as hydrophobic
when log D > 3.2 or log Kow > 2 [28]. The pKa values of bezafibrate and diclofenac
are 3.61 and 4.15, respectively, (Table 1) so they are ionizable compounds, whereas the
other pharmaceuticals showed no ionization. Accordingly, all the pharmaceuticals used in
this study can be considered hydrophilic except for carbamazepine, which is a moderate
hydrophobic compound.
The increased removal efficiency of hydrophobic pharmaceuticals can be due to
(i) the dominant sorption by the sludge, which results in enhanced biological degradation,
and (ii) having only electron-donating groups (EDGs) that improve oxidation [30]. The
removal of carbamazepine is low in MBR processes because of the lack of an electron-
donating group. Many other studies have shown that carbamazepine is not removed and
recalcitrated in treatment with MBR due to poor degradability [5,8]. On the other hand,
diclofenac was reduced at relatively low efficiency with median removal efficiencies of
49.4%, 42.4%, and 42.1% at SRTs of 40 days, 30 days, and 20 days, respectively. Other
researchers have documented a slow or non-biotransformation rate of diclofenac [28,30].
The lower elimination rates of diclofenac and carbamazepine are due to the existence
of multi-aromatic rings in their structure [11]. Paracetamol is highly degraded because
its structure enables bacteria and enzymes to have unobstructed access to the exposed
molecule that is later amended [31]. The removal of hydrophilic MPs ranged from low
removal (diclofenac) to almost complete removal (paracetamol). The removal of hydrophilic
MPs varies because MPs have different molecular structures and functional groups. Various
removal efficiencies reported for hydrophilic MPs might be because (i) compounds having
only EDGs could accomplish a high degree of removal; (ii) compounds with EDGs and
e-withdrawing (EWGs) like amide and chloride in their molecular structure including
paracetamol, diclofenac, and atenolol have diverse removal efficiencies; (iii) compounds
that only have strong EWGs like carbamazepine showed low removal efficiency [30].
Therefore, the intrinsic biodegradability of these compounds has a great impact on the
dominant removal mechanism of them, as the sludge sorption is less important.
The presence of possible by-products in the samples of the studies with an SRT period
of 40 days and using a 0.2 µm membrane was detected using LC/-MS-MS. Accordingly,
there were no by-products detected for paracetamol, atenolol, and ranitidine. The reason
Int.
Int. J. Environ. Res. Public Health 2021, 18, xxcould be that
FOR PEER the possible by-products could not be concentrated by the method14
REVIEW used
of 20 for
Int. J. Environ. Res. Public Health 2021, 18, x FOR
J. Environ. Res. Public Health 2021, 18, FOR PEER
PEER REVIEW
REVIEW 14
14 of
of 20
20
Int. J. Environ. Res. Public Health 2021, 18, xSPE.
FOR PEER
The REVIEW by-products determined for bezafibrate, carbamazepine, and diclofenac
possible 14 of 20
Int. J. Environ. Res. Public Health 2021, 18, xareFOR PEER in
listed REVIEW
Table 4. 14 of 20
Table
Table 4.
4. The
The possible
possible
4. The by-products
by-products
possible of
of
by-productsdiclofenac,
diclofenac, bezafibrate,
bezafibrate,
of diclofenac, and
and
bezafibrate, carbamazepine
carbamazepine that
that were
were detected
detected [32–37].
[32–37].
Table
Table
Table 4. The
4. The possible
possible by-products
by-products of diclofenac,
of diclofenac, bezafibrate,
bezafibrate, andand
and
carbamazepine
carbamazepine
carbamazepine thatthat
that were
were
were detected
detected
detected
[32–37].
[32–37].
[32–37].
Table 4. The possible by-products of diclofenac, bezafibrate, and carbamazepine that were detected [32–37].
Table
PPCPs
PPCPs 4. The possibleTransformation
by-products of diclofenac,
Transformation bezafibrate, m/z
Products
Products andm/z
carbamazepine that were detected Formula
Structure
Structure [32–37].
Formula
PPCPs
PPCPs Transformation Products
Transformation Products
Products m/z
m/z Structure
Structure Formula
Formula
PPCPs Transformation m/z Structure Formula
PPCPs Transformation Products m/z Structure Formula
PPCPs Transformation
DCF-TP1 Products m/z Structure Formula
DCF-TP1
DCF-TP1
DCF-TP1
Diclofenac
Diclofenac DCF-lactam
DCF-lactam orDCF-TP1
1-(2,6-dichlorophenyl)-
or 1-(2,6-dichlorophenyl)- 280.095
280.095 C 14H10CNOCl
Diclofenac DCF-lactam or 1-(2,6-dichlorophenyl)- 280.095 C 14 H10 NOCl
2
DCF-TP1 14H 10NOCl
2
Diclofenac
Diclofenac DCF-lactam
DCF-lactam or 1-(2,6-dichlorophenyl)- 280.095
orDCF-TP1
1-(2,6-dichlorophenyl)- 280.095 C14
C
14
HH10
10 NOCl
NOCl
2
2
1,3-dihydro-2H-indol-2-one
1,3-dihydro-2H-indol-2-one 2
Diclofenac 1,3-dihydro-2H-indol-2-one
DCF-lactam or 1-(2,6-dichlorophenyl)- 280.095
1,3-dihydro-2H-indol-2-one C14H10NOCl2
Diclofenac DCF-lactam or 1-(2,6-dichlorophenyl)- 280.095
1,3-dihydro-2H-indol-2-one C14H10NOCl2
DCF-TP2
DCF-TP2
DCF-TP2
DCF-TP2 283.036
283.036 C
C 13H9Cl2NO2
DCF-TP2
DCF-benzoic
DCF-benzoic acid acid
283.036
283.036 13H
C13
13 Cl
H999C
Cl NO
1322H9 Cl22 2 NO2
NO
DCF-benzoic
DCF-TP2
DCF-benzoic acid
acid 283.036 C H Cl 2NO2
DCF-TP2acid
DCF-benzoic 283.036 C13H9Cl2NO2
DCF-benzoic acid 283.036 C13H9Cl2NO2
DCF-benzoic acid
Bezafibrate
Bezafibrate 4-Chlorobenzoic acid
4-Chlorobenzoic 113.05 C 7H5ClO2
Bezafibrate
Bezafibrate 4-Chlorobenzoic acid acid
4-Chlorobenzoic acid 113.05
113.05
113.05 C H
C777H ClO
H555ClO
ClO
C7 H225 ClO2
Bezafibrate 4-Chlorobenzoic acid 113.05 C 2
Bezafibrate 4-Chlorobenzoic acid 113.05 C7H5ClO2
Bezafibrate 4-Chlorobenzoic acid 113.05 C7H5ClO2
Carbamazepine
Carbamazepine CBZ-TP1
CBZ-TP1 253.097
253.097 C
C 15H12N2O2
Carbamazepine CBZ-TP1 253.097 15H
C15
15 12N
H12
12 O
N222H
O222 N O
Carbamazepine
Carbamazepine CBZ-TP1
CBZ-TP1 253.097
253.097 C H CN 15 O12 2 2
Carbamazepine CBZ-TP1 253.097 C15H12N2O2
Carbamazepine CBZ-TP1 253.097 C15H12N2O2
CBZ-TP2
CBZ-TP2 210.015
210.015 C
C14 HH11 NO
NO
CBZ-TP2
CBZ-TP2 210.015
210.015 C
C
14H
14 11NO
11
14H11NO
CBZ-TP2
CBZ-TP2 210.015
210.015 C14H11 CNO
14 H11 NO
CBZ-TP2 210.015 C14H11NO
CBZ-TP3
CBZ-TP3 224.125
224.125 C
C14 HH999NO
NO222
CBZ-TP3
CBZ-TP3 224.125
224.125 C
C
14H
14
14H9NO2
NO
CBZ-TP3 224.125 C14H9NO2
CBZ-TP3
CBZ-TP3 224.125
224.125 C14H9C NO
14 H
2 9 NO2
4-Chlorobenzoic
4-Chlorobenzoic acid acid waswas detected,
detected, which
which is is the
the common
common metabolite
metabolite for for bezafibrate
bezafibrate
4-Chlorobenzoic
4-Chlorobenzoic acid
acid was
was detected,
detected, which
which is
is the
the common
common metabolite
metabolite for
for bezafibrate
bezafibrate
[32]. Furthermore,
[32]. Furthermore,
Furthermore,
4-Chlorobenzoic three
three possible
possible
acid by-products
by-products
was detected, were
were
which is thedetermined
determined
common for for
for carbamazepine,
carbamazepine,
metabolite which
which
for bezafibrate
[32].
[32]. 4-Chlorobenzoic
Furthermore, three
three possible
acid
possible by-products
was detected,
by-productswhichwere
were determined
isasthe common
determined carbamazepine,
metabolite
for which
for bezafibrate
carbamazepine, which
are
are
[32].named
named CBZ-TP1,
CBZ-TP1,
4-Chlorobenzoic
Furthermore, threeCBZ-TP2,
CBZ-TP2,
acid
possible wasand
and CBZ-TP3,
CBZ-TP3,
detected,
by-products as
which
were given
given
is in
in
the
determined Table
Table
common
for 4.
4. CBZ-TP1
CBZ-TP1
metabolite
carbamazepine, could
could
for
whichbe
be
bezafi-
are named
[32].
are named CBZ-TP1,
Furthermore,
CBZ-TP1, threeCBZ-TP2,
possible
CBZ-TP2, and CBZ-TP3,
CBZ-TP3,
by-products
and were as determined
as given in
given in Table
Table 4. CBZ-TP1
CBZ-TP1 could
for carbamazepine,
4. could
whichbe
be
formed
formed
are brate
namedby
by oxidation
oxidation
[32]. of
of the
the
Furthermore,
CBZ-TP1, CBZ
CBZ
CBZ-TP2,three molecule
molecule
possible
and (m/z
(m/z =
= 253).
253).
by-products
CBZ-TP3, as Later
Later
given were
inCBZ-TP1
CBZ-TP1
determined
Table 4. could
could for
CBZ-TP1form
form with
with
could the
the
carbamazepine,
be
formed
are named
formed by oxidation
CBZ-TP1,
by oxidation of the
the CBZ
ofCBZ-TP2,
CBZ molecule
and (m/z
CBZ-TP3,
molecule (m/z = 253). Later
as given
= 253). Laterin CBZ-TP1
Table
CBZ-TP1 could
4.could
CBZ-TP1 form with
formcould the
be
withalso
the
lost
lost −CONH
−CONH
which
formed 2 group and
are group
named
by oxidation and then
then
CBZ-TP1,
of the form
form
CBZ CBZ-TP2
CBZ-TP2
CBZ-TP2,
molecule and
and
and
(m/z CBZ-TP3.
CBZ-TP3.
CBZ-TP3,
= CBZ-TP3. as
253). LaterThe The
The diclofenac
diclofenac
given
CBZ-TP1 in Table
could compound
compound
4. CBZ-TP1 also
form withalso could
the
lost
lost −CONH
formed
−CONH 2 group and
2
by by-products
oxidation
2 group and
then
of the
then form
CBZ
form CBZ-TP2
molecule
CBZ-TP2 and
(m/z
and =the
253).
CBZ-TP3.LaterThe diclofenac
CBZ-TP1
diclofenac could compound
form
compound withalso
the
had
lostbe
had some
formed
some
−CONH by-products
2 group andthat
by oxidationthenwere
that of
were
form theobserved
observed
CBZ-TP2 in
CBZ molecule
in
and the activated
(m/z = 253).
activated
CBZ-TP3. Thesludge
Later
sludge treatment
CBZ-TP1
treatment
diclofenac processes.
could
processes.
compound alsoform
had
had some
lost −CONH
someand by-products
2 group andthat
by-products that were
thenwere
form observed
CBZ-TP2 in
observed in
and the activated
CBZ-TP3.
the activated Thesludge treatment
diclofenac
sludge processes.
compound
treatment also
processes.
DCF-TP1
hadwith
DCF-TP1
some the
and DCF-TP2,
lost −CONH
DCF-TP2,
by-products which
2 group
which
that are
areand
were DCF-lactam
DCF-lactam
observed in or
then form or
the 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-
CBZ-TP2
activated andsludge
CBZ-TP3. The diclofenac
1-(2,6-dichlorophenyl)-1,3-dihydro-2H-
treatment processes. com-
DCF-TP1
had someand and DCF-TP2,
by-products which
that wereare DCF-lactam
are observed in oror 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-
the1-(2,6-dichlorophenyl)-1,3-dihydro-2H-
activated sludge
DCF-TP1
indol-2-one
pound
indol-2-one
DCF-TP1 and
also
and
and
DCF-TP2,
had some
which
DCF-benzoic
DCF-benzoic
DCF-TP2, which acid,
by-products
acid,
are
DCF-lactam
were
were observed
that were
observed
DCF-lactam or as given
observed
as given in
in
in Table
the 44treatment
[33,34].
activated
Table [33,34].
1-(2,6-dichlorophenyl)-1,3-dihydro-2H-
processes.
Microorgan-
sludge treatment
Microorgan-
indol-2-one
DCF-TP1 andand DCF-benzoic acid, were observed
observed as given
given in in Table
Table 44 [33,34].
[33,34]. Microorgan-
Microorgan-
indol-2-one
isms
isms played
processes.
played
indol-2-one aaDCF-TP2,
and
and significant
DCF-TP1
significant
which
DCF-benzoic
DCF-benzoic role
and
arethe
acid,
in
DCF-TP2,
role in
acid,
DCF-lactam
were
the
were which
or 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-
are as
transformation
transformation
observed of carbamazepine,
DCF-lactam
of
as given bezafibrate,
or 1-(2,6-dichlorophenyl)-1,3-
carbamazepine,
in Table bezafibrate,
4 [33,34]. and
and
Microorgan-
isms
isms played
indol-2-one
played and
aa significant
DCF-benzoic
significant role
role in
acid,
in the
the transformation
were observed
transformation as of
given
of carbamazepine,
in Table
carbamazepine, 4 bezafibrate,
[33,34]. and
Microorgan-
bezafibrate, and
diclofenac
diclofenac
isms played by
by electrochemical
electrochemical
aelectrochemical reduction
reduction
significant rolereduction and
and oxidative
oxidative
in the transformation transformation
transformation
oftransformation
carbamazepine, [35].
[35].
bezafibrate, and
diclofenac
isms played
diclofenac by
byaelectrochemical
significant rolereduction and oxidative
in the transformation
and oxidativeoftransformation
carbamazepine, [35].
bezafibrate, and
[35].
diclofenac by electrochemical reduction and oxidative transformation [35].
diclofenac
3.5. by electrochemical reduction and oxidative transformation [35].
3.5. Bacterial
3.5. Bacterial Community
Bacterial Community Analysis
Community Analysis
Analysis
3.5. Bacterial Community Analysis
Inorganic
3.5. Bacterial
Inorganic and
Communityorganic
and organic
organic materials
materials are
Analysis are oxidized
oxidized by by microorganisms
microorganisms in in oxidation-reduc-
oxidation-reduc-
Inorganic
3.5. Bacterial
Inorganic and
Community
and materials
Analysis
organic materials are
are oxidized
oxidized by
by microorganisms
microorganisms in
in oxidation-reduc-
oxidation-reduc-
tion
tion reactions to produce growth and maintenance energy. Under aerobic conditions,
reactions to produce growth and maintenance energy. Under aerobic conditions, elec-
elec-
dihydro-2H-indol-2-one and DCF-benzoic acid, were observed as given in Table 4 [33,34].

Microorganisms played a significant role in the transformation of carbamazepine, bezafi-
brate, and diclofenac by electrochemical reduction and oxidative transformation [35].
3.5. Bacterial Community Analysis

Inorganic and organic materials are oxidized by microorganisms in oxidation-reduction
reactions to produce growth and maintenance energy. Under aerobic conditions, electron
exchanges (food substrate for an organism) are often part of oxidation-reduction reactions
and the electron acceptor is oxygen [38,39].
In this study, the 40-day SRT had the optimum performance in pharmaceuticals elimi-
nation and also in COD removal efficiency, so the metagenomic analysis was done for this
period to understand the bacterial community before and after adding the pharmaceuticals.
Figure 6 shows the percentage of the microbial community at the phylum level in-
oculated in the aerobic MBR system of activated sludge, and the microbial community
percentage for the same conditions at the class level is shown in Figure 7 at 0 days during
the steady-state, which is the time before adding the pharmaceuticals, and also 96 days
after adding the pharmaceuticals. As a result of the metagenomic analysis applied to
this sample, a total of 23 phyla and 58 classes were identified. Based on 16S rRNA NGS
data, the percentage of dominant microbial distributions in raw sludge were found to
be 46.8% Proteobacteria, 14.3% Firmicutes, 9.2% Actinobacteria, and 5.4% Bacteroidetes.
Proteobacteria represents the predominate heterotrophic bacterial phylum with Gammapro-
teobacteria, Betaproteobacteria, Deltaproteobacteria, and Alphaproteobacteria classes, the
genus of these bacteria were identified to use paracetamol as the sole carbon and energy
source [40]. It can be noticed that the Proteobacteria phylum was increased to 60.0% three
months after adding the pharmaceuticals (Figure 5) and the Gammaproteobacteria class
increased from 26% to 44% (Figure 6). The Actinobacteria class can metabolize paracetamol,
carbamazepine, and atenolol, and it was also increased from 9.1% to 17.9% after adding
pharmaceuticals [40].
In general, the key mechanism for the removal of pharmaceuticals by activated sludge
is biodegradation. However, the elimination of environmental contaminants is not always
successful due to the lack of degraders in the environment. Biological acclimation and
bioaugmentation will overcome all these limitations [41]. Pure cultures and mixed cul-
tures, which can remove the pollutants, can be dominated by biological acclimation and
bioaugmentation in the biological treatment process. Pure cultures can be used to remove
commonly detected carbamazepine, isolating it from the activated sludge, wastewater,
or sediment. Pure carbamazepine has a stable structure leading to low biodegradability,
however, two pure cultures will degrade carbamazepine in the presence of glucose: uniden-
tified Basidiomycete [42], and Streptomyces MIUG [42,43]. White rot fungus Phanerochaete
Sordida YK-624 can remove diclofenac entirely and in the absence of extra substrate can
eliminate its lethal toxicity to organisms [41–43].
100 Parcubacteria 100 BRC1

Microgenomates
Armatimonadetes
BRC1
90 90 Bacteria_unclassified
Tenericutes
Synergistetes Parcubacteria
80 80
Nitrospirae Microgenomates
Latescibacteria
Tenericutes
70 Cyanobacteria/Chloroplast 70
Nitrospirae
Armatimonadetes
Relative abundance %
Cyanobacteria/Chloroplast
Hydrogenedentes
60 60
Spirochaetes Gemmatimonadetes
Ignavibacteriae Hydrogenedentes
50 50
Gemmatimonadetes
Planctomycetes
Verrucomicrobia
Chloroflexi
40 Candidatus_Saccharibacteria 40
Chloroflexi Verrucomicrobia
Planctomycetes Acidobacteria
30 30
Acidobacteria Candidatus_Saccharibacteria
Bacteria_unclassified
Firmicutes
20 20
Bacteroidetes
Bacteroidetes
Actinobacteria
10 Firmicutes 10 Actinobacteria
Proteobacteria Proteobacteria
0 0
0 Day 96 Day
Figure
Figure 6. Profiles
6. Profiles of bacterial
of bacterial community
community compositionatatthe
composition thephylum
phylumlevel
level of
of activated
activated sludge
sludgebefore
before(at
(at0 0day—left)
day—left)and
and
after adding pharmaceuticals (at 96 day—right) in 40-day SRT.
after adding pharmaceuticals (at 96 day—right) in 40-day SRT.
Chloroplast
Planctomycetes_unclassified
Planctomycetes_unclassified Microgenomates_unclassified
100 Parcubacteria_unclassified
100 Acidobacteria_Gp11
Microgenomates_unclassified Parcubacteria_unclassified
Flavobacteriia Chloroplast
BRC1_unclassified
Acidobacteria_Gp21
Chthonomonadetes
Mollicutes
90 Acidobacteria_Gp22 90 Chthonomonadetes
Acidobacteria_Gp15
Acidobacteria_Gp11 Flavobacteriia
Opitutae Acidobacteria_Gp23
Mollicutes Opitutae
Acidobacteria_Gp12 Bacteria_unclassified
80 Synergistetes
Synergistetes
BRC1_unclassified
Nitrospira
Acidobacteria_Gp22
Acidobacteria_Gp21
Latescibacteria
Nitrospira
Armatimonadetes_gp5
70 Cyanobacteria 70
Armatimonadetes_gp5 Cyanobacteria
Verrucomicrobia_unclassified Acidobacteria_Gp12
Actinobacteria_unclassified
Relative abundance %
Verrucomicrobia_unclassified
Acidobacteria_Gp18
Actinobacteria_unclassified
Acidobacteria_Gp1
60 Candidatus_Hydrogenedens 60 Acidobacteria_Gp1
Acidobacteria_Gp18
Acidobacteria_Gp23
Chloroflexia Chloroflexi_unclassified
Acidobacteria_unclassified Candidatus_Hydrogenedens
Thermomicrobia Firmicutes_unclassified
Acidobacteria_Gp17
50 Bacteroidetes_unclassified
50 Gemmatimonadetes
Spartobacteria
Acidobacteria_Gp4
Chloroflexia
Spirochaetes
Acidobacteria_unclassified
Acidobacteria_Gp10
Ignavibacteriae Thermomicrobia
40 Sphingobacteriia 40 Acidobacteria_Gp17
Firmicutes_unclassified Bacteroidetes_unclassified
Spartobacteria Acidobacteria_Gp7
Acidobacteria_Gp7 Acidobacteria_Gp3
Acidobacteria_Gp16
Acidobacteria_Gp4
Acidobacteria_Gp3
30 Gemmatimonadetes
Proteobacteria_unclassified
Verrucomicrobiae
Chloroflexi_unclassified Acidobacteria_Gp16
Cytophagia Sphingobacteriia
Acidobacteria_Gp6 Acidobacteria_Gp6
20 Verrucomicrobiae 20 Proteobacteria_unclassified
Candidatus_Saccharibacteria_unclassified
Cytophagia
Chloroflexi
Bacilli
Planctomycetes
Candidatus_Saccharibacteria_unclassified
Alphaproteobacteria
Bacteroidia Betaproteobacteria
10 deltaproteobacteria 10 deltaproteobacteria
Bacteria_unclassified Alphaproteobacteria
Actinobacteria Bacteroidia
Betaproteobacteria Actinobacteria
Bacilli
Gammaproteobacteria
0
Gammaproteobacteria
0
0 Day 96 Day
Figure
Figure 7. Profiles
7. Profiles of of bacterialcommunity
bacterial communitycomposition
composition at
at the
the class
class level
levelof
ofactivated
activatedsludge
sludgebefore
before(at(at
0 day—left) andand
0 day—left) after
after
adding pharmaceuticals (at 96 day—right) in 40-day SRT.
adding pharmaceuticals (at 96 day—right) in 40-day SRT.
4. Conclusions
The biodegradation of six pharmaceuticals was investigated in a membrane bioreactor.
After the reactor reached the steady-state condition, the degradation efficiencies of six
targeted pharmaceuticals were determined under different sludge retention times. The
highest degradation efficiencies were observed for paracetamol for each SRT followed
by paracetamol, ranitidine, atenolol, bezafibrate, diclofenac, and carbamazepine. The
bacterial community analysis was evaluated for the sludge in steady-state conditions and
also after adding the pharmaceuticals. It was observed that Proteobacteria in the sludges
increased from 46.8% to 60% while Actinobacteria increased from 9.1% to 17.9%. This
reveals that microorganisms can metabolize the targeted pharmaceuticals. To understand
the degradation mechanism, possible by-products were observed in the effluent samples,
where the first-step degradation by-products of diclofenac, bezafibrate, and carbamazepine
were detected. As a further study, the ratio of sludge adsorption and degradation of
targeted pharmaceuticals can be studied to better understand whether the sludge requires
additional treatment. Since the degradation of diclofenac and carbamazepine was low and
they need to be degraded to reach the non-cancerogenic limits, the development of highly
effective biodegradation of these compounds should also be a future focus.
Author Contributions: Conceptualization, R.A.K.A.; Funding acquisition, R.A.K.A.; Investigation,

R.A.K.A., K.U.-A., R.K.S.M., and N.M.-D.; Methodology, R.A.K.A., K.U.-A., and R.K.S.M.; Project
administration, R.A.K.A. and O.C.; Supervision, O.C.; Writing—Original draft, R.A.K.A., K.U.-A.,
and R.K.S.M.; Writing—Review and editing, R.A.K.A., K.U.-A., N.M.-D., and O.C. All authors have
read and agreed to the published version of the manuscript.
Funding: This study was conducted in 2019 and funded by Yildiz Technical University Scientific
Research Projects Coordination Unit. Project Number: FDK-2018-3276.
Institutional Review Board Statement: Not applicant.
Informed Consent Statement: Not applicant.
Data Availability Statement: All data are included in the article. Data sharing is not applicable to
this article.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Kondor, A.C.; Jakab, G.; Vancsik, A.; Filep, T.; Szeberényi, J.; Szabó, L.; Maász, G.; Ferincz, Á.; Dobosy, P.; Szalai, Z. Occurrence of
pharmaceuticals in the Danube and drinking water wells: Efficiency of riverbank filtration. Environ. Pollut. 2020, 265, 114893.
[CrossRef]
2. Van Driezum, I.H.; Derx, J.; Oudega, T.J.; Zessner, M.; Naus, F.L.; Saracevic, E.; Kirschner, A.K.; Sommer, R.; Farnleitner, A.H.;
Blaschke, A.P. Spatiotemporal resolved sampling for the interpretation of micropollutant removal during riverbank filtration. Sci.
Total Environ. 2019, 649, 212–223. [CrossRef]
3. Kruglova, A.; Kråkström, M.; Riska, M.; Mikola, A.; Rantanen, P.; Vahala, R.; Kronberg, L. Comparative study of emerging
micropollutants removal by aerobic activated sludge of large laboratory-scale membrane bioreactors and sequencing batch
reactors under low-temperature conditions. Bioresour. Technol. 2016, 214, 81–88. [CrossRef] [PubMed]
4. Marszałek, A.; Bohdziewicz, J.; Puszczało, E. Co-Treatment of Municipal Landfill Leachate with Dairy Wastewater in Membrane
Bioreactor. Ecol. Chem. Eng. S 2020, 27, 139–149. [CrossRef]
5. Taheran, M.; Brar, S.K.; Verma, M.; Surampalli, R.; Zhang, T.; Valero, J. Membrane processes for removal of pharmaceutically
active compounds (PhACs) from water and wastewaters. Sci. Total Environ. 2016, 547, 60–77. [CrossRef]
6. Luo, Y.; Jiang, Q.; Ngo, H.H.; Nghiem, L.D.; Hai, F.I.; Price, W.E.; Wang, J.; Guo, W. Evaluation of micropollutant removal and
fouling reduction in a hybrid moving bed biofilm reactor-membrane bioreactor system. Bioresour. Technol. 2015, 191, 355–359.
[CrossRef]
7. Nguyen, L.N.; Hai, F.I.; Kang, J.; Nghiem, L.D.; Price, W.E.; Guo, W.; Ngo, H.H.; Tung, K.-L. Comparison between sequential and
simultaneous application of activated carbon with membrane bioreactor for trace organic contaminant removal. Bioresour. Technol.
2013, 130, 412–417. [CrossRef] [PubMed]
8. Goswami, L.; Kumar, R.V.; Borah, S.N.; Manikandan, N.A.; Pakshirajan, K.; Pugazhenthi, G. Membrane bioreactor and integrated
membrane bioreactor systems for micropollutant removal from wastewater: A review. J. Water Process. Eng. 2018, 26, 314–328.
[CrossRef]
9. Kamaz, M.; Wickramasinghe, S.R.; Eswaranandam, S.; Zhang, W.; Jones, S.M.; Watts, M.J.; Qian, X. Investigation into Micropollu-
tant Removal from Wastewaters by a Membrane Bioreactor. Int. J. Environ. Res. Public Health 2019, 16, 1363. [CrossRef]
10. Kamaz, M.; Jones, S.M.; Qian, X.; Watts, M.J.; Zhang, W.; Wickramasinghe, S.R. Atrazine Removal from Municipal Wastewater
Using a Membrane Bioreactor. Int. J. Environ. Res. Public Health 2020, 17, 2567. [CrossRef]
11. Cirja, M.; Ivashechkin, P.; Schäffer, A.; Corvini, P.F.X. Factors affecting the removal of organic micropollutants from wastewater in
conventional treatment plants (CTP) and membrane bioreactors (MBR). Rev. Environ. Sci. Biotechnol. 2007, 7, 61–78. [CrossRef]
12. Dogan, A.; Płotka-Wasylka, J.; Kempińska-Kupczyk, D.; Namieśnik, J.; Kot-Wasik, A. Detection, identification and determination
of chiral pharmaceutical residues in wastewater: Problems and challenges. TRAC Trends Anal. Chem. 2020, 122, 115710. [CrossRef]
13. Butkovskyi, A.; Leal, L.H.; Zeeman, G.; Rijnaarts, H. Micropollutants in source separated wastewater streams and recovered
resources of source separated sanitation. Environ. Res. 2017, 156, 434–442. [CrossRef] [PubMed]
14. Beijer, K.; Björlenius, B.; Shaik, S.; Lindberg, R.H.; Brunström, B.; Brandt, I. Removal of pharmaceuticals and unspecified
contaminants in sewage treatment effluents by activated carbon filtration and ozonation: Evaluation using biomarker responses
and chemical analysis. Chemosphere 2017, 176, 342–351. [CrossRef] [PubMed]
15. Gallardo-Altamirano, M.; Maza-Márquez, P.; Peña-Herrera, J.; Rodelas, B.; Osorio, F.; Pozo, C. Removal of anti-inflammatory/analgesic
pharmaceuticals from urban wastewater in a pilot-scale A2O system: Linking performance and microbial population dynamics to
operating variables. Sci. Total Environ. 2018, 643, 1481–1492. [CrossRef] [PubMed]
16. Botero-Coy, A.; Martínez-Pachón, D.; Boix, C.; Rincón, R.; Castillo, N.; Arias-Marín, L.; Manrique-Losada, L.; Torres-Palma,
R.; Moncayo-Lasso, A.; Hernández, F. ‘An investigation into the occurrence and removal of pharmaceuticals in Colombian
wastewater’. Sci. Total Environ. 2018, 642, 842–853. [CrossRef] [PubMed]
17. Gros, M.; Petrović, M.; Barceló, D. Development of a multi-residue analytical methodology based on liquid chromatography–
tandem mass spectrometry (LC–MS/MS) for screening and trace level determination of pharmaceuticals in surface and wastewa-
ters. Talanta 2006, 70, 678–690. [CrossRef]
18. Kumar, R.; Sarmah, A.K.; Padhye, L.P. Fate of pharmaceuticals and personal care products in a wastewater treatment plant with
parallel secondary wastewater treatment train. J. Environ. Manag. 2019, 233, 649–659. [CrossRef]
19. Lahti, M. The Fate Aspects of Pharmaceuticals in the Environment: Biotransformation, Sedimentation and Exposure of Fish; University of
Jyväskylä: Jyväskylä, Finland, 2012.
20. APHA; AWWA. Standard Methods for the Examination of Water and Wastewater, 22th ed.; APHA: Washington, DC, USA, 2012.
21. Bozcal, E.; Dagdeviren, M. Bacterial metagenome analysis of Mytilus galloprovincialis collected from Istanbul and Izmir coastal
stations of Turkey. Environ. Monit. Assess. 2020, 192, 1–18. [CrossRef]
22. Edgar, R.C.; Haas, B.J.; Clemente, J.C.; Quince, C.; Knight, R. UCHIME Improves Sensitivity and Speed of Chimera Detection.
Bioinformatics 2011, 27, 2194–2200. [CrossRef] [PubMed]
23. Broeck, R.V.D.; Van Dierdonck, J.; Nijskens, P.; Dotremont, C.; Krzeminski, P.; Van Der Graaf, J.; Van Lier, J.; Van Impe, J.; Smets,
I. The influence of solids retention time on activated sludge bioflocculation and membrane fouling in a membrane bioreactor
(MBR). J. Membr. Sci. 2012, 401–402, 48–55. [CrossRef]
24. Delgado, S.; Villarroel, R.; Gonzalez, E.; Morales, M. Aerobic Membrane Bioreactor for Wastewater Treatment—Performance
Under Substrate-Limited Conditions. Biomass Detect. Prod. Usage 2011. [CrossRef]
25. Chtourou, M.; Mallek, M.; Dalmau, M.; Mamo, J.; Santos-Clotas, E.; Ben Salah, A.; Walha, K.; Salvadó, V.; Monclús, H. Triclosan,
carbamazepine and caffeine removal by activated sludge system focusing on membrane bioreactor. Process. Saf. Environ. Prot.
2018, 118, 1–9. [CrossRef]
26. Mousel, D.; Bastian, D.; Firk, J.; Palmowski, L.; Pinnekamp, J. Removal of pharmaceuticals from wastewater of health care
facilities. Sci. Total Environ. 2021, 751, 141310. [CrossRef] [PubMed]
27. Kim, M.; Guerra, P.; Shah, A.; Parsa, M.; Alaee, M.; Smyth, S.A. Removal of pharmaceuticals and personal care products in a
membrane bioreactor wastewater treatment plant. Water Sci. Technol. 2014, 69, 2221–2229. [CrossRef]
28. Trinh, T.; Akker, B.V.D.; Coleman, H.M.; Stuetz, R.M.; Drewes, J.E.; Le-Clech, P.; Khan, S.J. Seasonal variations in fate and removal
of trace organic chemical contaminants while operating a full-scale membrane bioreactor. Sci. Total Environ. 2016, 550, 176–183.
[CrossRef]
29. Tiwari, B.; Sellamuthu, B.; Piché-Choquette, S.; Drogui, P.; Tyagi, R.D.; Vaudreuil, M.A.; Sauvé, S.; Buelna, G.; Dubé, R.
Acclimatization of microbial community of submerged membrane bioreactor treating hospital wastewater. Bioresour. Technol.
2021, 319, 124223. [CrossRef]
30. Gurung, K.; Ncibi, M.C.; Sillanpää, M. Removal and fate of emerging organic micropollutants (EOMs) in municipal wastewater
by a pilot-scale membrane bioreactor (MBR) treatment under varying solid retention times. Sci. Total Environ. 2019, 667, 671–680.
[CrossRef]
31. Tambosi, J.L.; De Sena, R.F.; Favier, M.; Gebhardt, W.; José, H.J.; Schröder, H.F.; Moreira, R.D.F.P.M. Removal of pharmaceutical
compounds in membrane bioreactors (MBR) applying submerged membranes. Desalination 2010, 261, 148–156. [CrossRef]
32. Quintana, J.B.; Weiss, S.; Reemtsma, T. Pathways and metabolites of microbial degradation of selected acidic pharmaceutical and
their occurrence in municipal wastewater treated by a membrane bioreactor. Water Res. 2005, 39, 2654–2664. [CrossRef]
33. Vieno, N.; Sillanpää, M. Fate of diclofenac in municipal wastewater treatment plant—A review. Environ. Int. 2014, 69, 28–39.
[CrossRef] [PubMed]
34. Vo, H.N.P.; Ngo, H.H.; Guo, W.; Nguyen, T.M.H.; Li, J.; Liang, H.; Deng, L.; Chen, Z. Poly-and perfluoroalkyl substances in water
and wastewater: A comprehensive review from sources to remediation. J. Water Process. Eng. 2020, 36, 101393. [CrossRef]
35. Tahir, K.; Miran, W.; Nawaz, M.; Jang, J.; Shahzad, A.; Moztahida, M.; Kim, B.; Azam, M.; Jeong, S.E.; Jeon, C.O.; et al. Investigating
the role of anodic potential in the biodegradation of carbamazepine in bioelectrochemical systems. Sci. Total Environ. 2019, 688,
56–64. [CrossRef] [PubMed]
36. Wu, G.; Geng, J.; Shi, Y.; Wang, L.; Xu, K.; Ren, H. Comparison of diclofenac transformation in enriched nitrifying sludge and
heterotrophic sludge: Transformation rate, pathway, and role exploration. Water Res. 2020, 184, 116158. [CrossRef] [PubMed]
37. Yao, M.; Duan, L.; Wei, J.; Qian, F.; Hermanowicz, S.W. Carbamazepine removal from wastewater and the degradation mechanism
in a submerged forward osmotic membrane bioreactor. Bioresour. Technol. 2020, 314, 123732. [CrossRef]
38. Mhemid, R.K.S.; Akmirza, I.; Shihab, M.S.; Turker, M.; Alp, K. Ethanethiol gas removal in an anoxic bio-scrubber. J. Environ.
Manag. 2019, 233, 612–625. [CrossRef]
39. Demir, M.N. Determination of microbial community in a pilot scale two-stage step-feed biological nutrient removal process. Glob.
NEST J. 2019, 21, 211–221. [CrossRef]
40. Żur, J.; Piński, A.; Marchlewicz, A.; Hupert-Kocurek, K.; Wojcieszyńska, D.; Guzik, U. Organic micropollutants paracetamol and
ibuprofen—Toxicity, biodegradation, and genetic background of their utilization by bacteria. Environ. Sci. Pollut. Res. 2018, 25,
21498–21524. [CrossRef]
41. Wang, J.; Wang, S. Removal of pharmaceuticals and personal care products (PPCPs) from wastewater: A review. J. Environ.
Manag. 2016, 182, 620–640. [CrossRef]
42. Santos, I.J.S.; Grossman, M.J.; Sartoratto, A.; Ponezi, A.N.; Durrant, L.R. Degradation of the recalcitrant pharmaceuticals
car-bamazepine and 17α-ethinylestradiol by ligninolytic fungi. Chem. Eng. 2012, 27, 169–174. [CrossRef]
43. Popa, C.; Favier, L.; Dinica, R.; Semrany, S.; Djelal, H.; Amrane, A.; Bahrim, G. Potential of newly isolated wild Streptomycesstrains
as agents for the biodegradation of a recalcitrant pharmaceutical, carbamazepine. Environ. Technol. 2014, 35, 3082–3091. [CrossRef]
[PubMed]

Biodegradation of Emerging

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International Journal of

1 Department of Environmental Engineering, Faculty of Civil Engineering, Yildiz Technical University,

Publisher’s Note: MDPI stays neutral

micropollutants (MPs). These micropollutants are substances found in low concentrations

2. Materials and Methods

Figure 1. Lab-scale set up for submerged membrane bioreactor.

The aerobic submerged

As defined in fluid mechanics,

Int. J. Environ. Res. Public Health 2021, 18, 3395 4 of 19

Table 2. Solid-phase extraction (SPE) method determination.

SPE SPE SPE SPE

2.4. Analytical Methods

2.5. Metagenomic Analysis

2.6. DNA Isolation

2.7. Next Generation Sequencing (NGS)

3.2. Effect of SRT on COD Removal and MLSS Concentration

a 20 days SRT 30 days SRT 40 days SRT

b 0.2µm membrane 0.45µm membrane

a 20 days SRT 30 days SRT 40 days SRT

Int. J. Environ. Res. Public Health 2021, 18, x FOR

DCF BZF CBZ ATN RND PCT

b 20 days SRT 30 days SRT 40 days SRT

Table 3. The concentrations of pharmaceuticals before and after biodegradation.

0.2 µm 0.45 µm 0.2 µm 0.45 µm 0.2 µm 0.45 µm

3.4. Removal Mechanism and Possible Pathways

dihydro-2H-indol-2-one and DCF-benzoic acid, were observed as given in Table 4 [33,34].

3.5. Bacterial Community Analysis

100 Parcubacteria 100 BRC1

Author Contributions: Conceptualization, R.A.K.A.; Funding acquisition, R.A.K.A.; Investigation,

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