microorganisms
Article
Complete Biodegradation of Diclofenac by New Bacterial
Strains: Postulated Pathways and Degrading Enzymes
Mahmoud S. M. Mohamed 1, * , Ayan A. Asair 1 , Nashwa A. H. Fetyan 2 and Sherif M. Elnagdy 1
Citation: Mohamed, M.S.M.;
Asair, A.A.; Fetyan, N.A.H.;
Elnagdy, S.M. Complete
Biodegradation of Diclofenac by New
Bacterial Strains: Postulated
Pathways and Degrading Enzymes.
Microorganisms 2023, 11, 1445.
https://doi.org/10.3390/
microorganisms11061445
Academic Editor: Edward A. Bayer
Received: 11 May 2023
Revised: 26 May 2023
Accepted: 27 May 2023
Published: 30 May 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Microorganisms 2023, 11, 1445. https://doi.org/10.3390/microorganisms11061445
1 Department of Botany and Microbiology, Faculty of Science, Cairo University, Giza 12613, Egypt
2 Department of Microbiology, Soil, Water and Environment Research Institute, Agriculture Research Center,
Giza 12619, Egypt
* Correspondence: msaleh@sci.cu.edu.eg; Tel.: +20-100-124-3567
Abstract: The accumulation of xenobiotic compounds in different environments interrupts the natural
ecosystem and induces high toxicity in non-target organisms. Diclofenac is one of the commonly
used pharmaceutical drugs that persist in the environment due to its low natural degradation rate
and high toxicity. Therefore, this study aimed to isolate potential diclofenac-degrading bacteria,
detect the intermediate metabolites formed, and determine the enzyme involved in the degradation
process. Four bacterial isolates were selected based on their ability to utilize a high concentration of
diclofenac (40 mg/L) as the sole carbon source. The growth conditions for diclofenac degradation
were optimized, and bacteria were identified as Pseudomonas aeruginosa (S1), Alcaligenes aquatilis (S2),
Achromobacter spanius (S11), and Achromobacter piechaudii (S18). The highest percentage of degradation
was recorded (97.79 ± 0.84) after six days of incubation for A. spanius S11, as analyzed by HPLC. To
detect and identify biodegradation metabolites, the GC-MS technique was conducted for the most
efficient bacterial strains. In all tested isolates, the initial hydroxylation of diclofenac was detected.
The cleavage step of the NH bridge between the aromatic rings and the subsequent cleavage of the
ring adjacent to or in between the two hydroxyl groups of polyhydroxylated derivatives might be
a key step that enables the complete biodegradation of diclofenac by A. piechaudii S18, as well as
P. aeruginosa S1. Additionally, the laccase, peroxidase, and dioxygenase enzyme activities of the
two Achromobacter strains, as well as P. aeruginosa S1, were tested in the presence and absence
of diclofenac. The obtained results from this work are expected to be a useful reference for the
development of effective detoxification bioprocesses utilizing bacterial cells as biocatalysts. The
complete removal of pharmaceuticals from polluted water will stimulate water reuse, meeting the
growing worldwide demand for clean and safe freshwater.
Keywords: Achromobacter; biodegradation; diclofenac; metabolite; Pseudomonas
1. Introduction
The continuous growth of the world population is increasing the demand for water
supply throughout the world. The number of areas on the Earth that are suffering from a
water crisis is growing for many reasons, such as climatic change, war, and the contami-
nation of groundwater. Recently, several micropollutants were detected in water, such as
pharmaceuticals, industrial chemicals, personal care products, and many other xenobiotic
compounds. These pollutants have become a significant problem in the aquatic environ-
ment around the world [1]. In fact, the immoderate usage of pharmaceuticals in human
and veterinary medicine is expanding its occurrence in the environment. In this regard,
many types of pharmaceutical compounds have been found in surface water, groundwater,
and even in drinking water [2].
Diclofenac (DCF), a pharmaceutical compound, is one of the most frequently used
non-steroidal anti-inflammatory drugs. It is used to treat pain, fever, and inflammation
and can be used without a prescription. The fate of DCF after human metabolism and
https://www.mdpi.com/journal/microorganisms
Microorganisms 2023, 11, 1445 2 of 18

excretion in the urine and feces is as metabolites, along with the unaltered parent com-
pounds, which may be subjected to further transformations in wastewater treatment plants
(WWTPs), producing metabolites that may be more toxic than DCF [3]. In several monitor-
ing studies, DCF was detected in wastewater in several countries [4], and the occurrence
of DCF in drinking water was also reported [5,6]. The presence of DCF in the aquatic
environment is anticipated to be a long-term concern due to its potential toxic effects on
non-target organisms [7,8]. In the aquatic environment, a high occurrence percentage of
DCF was reported, and the detected concentrations of DCF were found to be in a range from
24 to 1043 ng/L in surface water [9,10]. DCF was found at a high concentration in the
effluent water of sewage treatment plants (i.e., treated effluent) because the rate of DCF
elimination in WWTPs is low and does not exceed 40%, despite the progress in technologies
for wastewater treatment [11]. Therefore, the European Environmental Agency and the
scientific community consider DCF to be a particular environmental concern (Directive
2013/39/EU).
The degradation of DCF by microorganisms is a cost-efficient, eco-friendly alternative
solution. Only a few studies have reported the ability of bacteria to biodegrade DCF due
to the deleterious effect of DCF on bacterial physiology, such as membrane injury, lipid
peroxidation, and oxidative stress [7,12]. Furthermore, Facey et al. (2018) documented the
aerobic detoxification of DCF by a microbial consortium from forest soil after 10 days in a
low-salt medium into 2,6-dichloroaniline and carboxylated 2-hydroxyphenylacetic acid.
Moreover, the microbial community in activated sludge showed the biodegradation of up
to 21% of DCF in WWTPs through co-metabolic mechanisms and demonstrated the domi-
nance of some bacterial genera, such as Asticcacaulis, Pseudacidovorax, and Nitratireductor
after DCF exposure [13]. However, the first report on the degradation of DCF by a single
bacterial strain revealed the biotransformation of DCF (1.7 µM) by Labrys portucalensis (F11)
after 6 days, with the formation of benzoquinone imine as an intermediate metabolite in
the degradation pathway, but complete degradation was only achieved by co-metabolism
with acetate [14].
However, the specific bacterial mechanism responsible for degradation, the inter-
mediate metabolites, and the enzymes involved in DCF degradation were unknown in
most cases, and there are still gaps in the knowledge associated with the degradation
process. Therefore, in the present study, different samples contaminated with DCF were
used to explore the microbial diversity, isolating a potential local bacterial isolate that
can effectively completely biodegrade high concentrations of DCF (40 mg/L) to less toxic
or nontoxic compounds under optimized conditions. Moreover, the GC-MS technique
was used to postulate the catabolic degradation pathways for DCF based on the detection
of intermediate and residual compounds. In addition, the specific activities of enzymes
involved in biodegradation were estimated. To our knowledge, this may be the first study
to postulate a complete pathway for the biodegradation of high concentrations of DCF by
three different bacterial isolates, with DCF being the sole carbon source.

2. Materials and Methods

2.1. Sample Collection and Isolation of Local Diclofenac-Degrading Bacteria
Soil samples were collected from contaminated Nile River water and soil from Gazira
Park (Giza, Egypt). Other samples were collected from contaminated soil from the upper
(20–30 cm) layer of a vegetable field located at Kaliobia Governorate, Egypt. The enrichment
of DCF-degrading bacteria was performed by inoculating each sample (10 g of soil (wet
weight) or 10 mL of water) into 100 mL of the minimal salt medium (MS) containing the
following (g/L): K2 HPO4 (2.0), KH2 PO4 (1.0), MgSO4 ·7H2 O (0.25), NH4 So4 (0.5), and
trace elements, including FeSO4 ·7H2 O (0.05), MnSO4 ·4H2 O (0.05), and CuSO4 ·5H2 O (0.05),
supplemented with 200 mg/L DCF [15]. The flasks were incubated at 37 ◦ C for 24 h in
a static incubator. In the enrichment culture, all strains used DCF as the sole source of
carbon and energy. One milliliter of enriched bacterial culture was serially diluted up to
a 10−6 dilution. Then, 100 µL was plated onto solid MS medium with DCF (200 µg/mL)
Microorganisms 2023, 11, 1445 3 of 18

and incubated at 37 ◦ C for 48 h. Morphologically different bacterial isolates were selected,

purified, and maintained at 4 ◦ C on nutrient agar slants and glycerol stocks at −20 ◦ C for
further investigation. These isolates were named diclofenac-degrading strains (S1–S23) and
used in the following experiments.

2.2. Optimization of Bacterial Isolate Growth under Different Physicochemical Conditions

Each bacterial isolate was tested for its ability to grow at different concentrations of
DCF (10, 20, 30, and 40 mg/L) as the sole carbon source, and isolates were enumerated by
viable counts using Luria Bertani agar medium (Laboratories Conda SA, Madrid, Spain)
at pH 7.0 after aerobic incubation at 37 ◦ C for 24 h. Bacterial growth was confirmed by
Gram staining; the experiment was repeated three times in the same conditions, and the
viable count was performed in all samples in triplicate. The counted bacterial colonies were
expressed as log10 CFU/mL and plotted against the time of incubation [16].
Based on the preliminary experiment, the initial conditions of the biodegradation
experiment were set as follows: MS medium was supplemented with a DCF concentration
of 10 mg/L, the pH was adjusted to 7, and incubation was carried out with 150 rpm shaking
at 28 ◦ C. The concentration used was 100–400 times higher than the maximum recorded
concentration detected in urban wastewater, i.e., 95 µg/L [1].
MS medium was inoculated with 5 mL of an overnight culture of each bacterium and
then divided into four flasks, and each flask was supplemented with one of the selected
concentrations. Bacterial growth was measured at 600 nm using a spectrophotometer
(Jenway 6305; Bibby Scientific Ltd., Staffordshire, UK) after 1–7 days of incubation. The
experiment was repeated three times, and the average reading was calculated. Similarly,
DCF biodegradation for each bacterial isolate was monitored at different temperatures
(28, 37, and 40 ◦ C) and different pH values (4, 5, 6, 7, 8, and 9) after 1–7 days of incubation.
Oxygen is sometimes essential for cell growth and biodegradation. Finally, to optimize
the oxygen demand of each bacterial isolate, the cultures were incubated under static and
shaking conditions (150 rpm) in MS medium supplemented with 10 mg/L DCF, and the
cell density was evaluated.

2.3. Biochemical Characterization and Molecular Identification of the Selected Bacterial Isolates
A single colony from each bacterial isolate was examined for colony shape, size,
and pigmentation. Gram reactions of all isolates were recorded. All bacterial isolates
were categorized for the different physiological and biochemical tests. The isolates were
first identified to the genus level according to biochemical tests described previously in de-
tail [17], and then 16S rDNA sequence analysis was utilized for identification, as previously
described [18]. The sequence reads obtained from Macrogen (Seoul, Republic of Korea)
were assembled and trimmed, and the contig sequence for each isolate was blasted against
the reference 16S rRNA gene sequences of other bacteria deposited in GenBank. The
phylogenetic tree was generated using the maximum likelihood method via the MEGAX
program with 1000 bootstrap replications [19].

2.4. Quantification of Diclofenac Biodegradation

The percentage of DCF degradation was calculated after measuring the residual DCF
concentration for each bacterial isolate using high-performance liquid chromatography
(HPLC) according to a validated method previously described by Nguyen et al. [13]. In
summary, MS broth medium supplemented with 10 mg/L DCF (500 mL) was used as a
basic medium. The medium was divided into five flasks; each flask was inoculated with
a bacterial isolate (5% overnight grown culture), and one flask was used as a negative
control (without bacteria). Samples were collected after three and six days of incubation
and centrifuged at 8000× g for 10 min, and the cell-free supernatant was filtered using a
0.45 µm membrane filter (Millipore Corp., Burlington, MA, USA). DCF was measured at a
wavelength of 270 nm. The sample volume injected into the HPLC instrument was 100 µL,
Microorganisms 2023, 11, 1445 4 of 18

and the detection limit was 10 µg/L. The percentage of DCF degradation was calculated
using the following equation:

100
Percentage of degradation = (Ci − Cf) ×
Cf
where Ci is the concentration of DCF at the beginning of the experiment, and Cf is the
concentration at the end of the experiment (the cell-free supernatant of the sample).

2.5. Detection of Diclofenac Metabolites Produced by Individual Bacterial Isolates Using Gas
Chromatography–Mass Spectrometry (GC–MS) Analysis
The selected bacterial isolate extracts were analyzed using GC–MS to identify the
metabolic products of DCF during degradation in MS according to the method previously
described by Ivshina et al. [20]. Chromatographic analyses were performed using a Finni-
gan Mat GCQ GC/ITD-MS. Helium was used as a carrier gas with the flow rate adjusted to
constant velocity (40 cm/S). The injector temperature was 250 ◦ C, and 2 µL of the sample
was injected in the splitless injection mode adjusted to a splitless time of 0.8 min on. The
oven temperature was programmed to 100 ◦ C for 1 min following injection, after which the
temperature was increased (30 ◦ C/min) to 150◦ C, maintained for 1 min, then increased to
205 ◦ C (3 ◦ C/min), and finally increased to 260 ◦ C (10 ◦ C/min) for 23 min. The electron
energy for the mass spectra was set at 70 V, and 200 ◦ C was the ion source temperature.
The ITD settings were as follows: mass range of 50–500, 3 microscans, and max ion time of
25 ms. The metabolites were detected and identified by comparing their retention times and
mass spectra with the mass spectral data detailed in the library of WILEY, National Institute
of Standard and Technology (NIST-11), and the mass spectral data of DCF metabolites in
the literature.

2.6. Enzyme Activities

The bacterial isolates were first grown in 50 mL of MS broth supplemented with
10 mg/L DCF or 10 mg/L glucose as a control for 48 h. Laccase, catechol 1,2-dioxygenase,
catechol 2,3-dioxygenase, and peroxidase activities were assayed using cell-free culture
supernatants (extracellular activities). The reaction mixture for the determination of laccase
activity consisted of 2 mL of 10% ABTS in 100 mM acetate buffer (pH 4.9), and the increase
in optical density at 420 nm was recorded [21]. Catechol 1,2-dioxygenase and catechol
2,3-dioxygenase were assayed spectrophotometrically based on the formation of cis, cis-
muconic acid at 260 nm (ε260 = 16,800/M cm) and the formation of 2-hydroxymuconic
semialdehyde at 375 nm (ε375 = 36,000/M cm), respectively [22].
Peroxidase activities were measured using a procedure that determines the rate of
pyrogallol decomposition [23]. All enzyme assays were conducted at 37 (or 30) ◦ C, where
the control was tested in the presence of glucose in the medium instead of DCF, and the
reference blank contained all components but with the boiled enzyme. All enzyme assays
were carried out three times and the average reading was calculated. The unit of enzyme
was defined as a change in absorbance unit/min/mg of protein.

2.7. Statistical Analysis

The data obtained are presented in figures and tables as the mean value ± standard
deviation (SD) of at least three replicates. A one-way ANOVA setting followed by Tukey’s
HSD test (Minitab 18) was used to identify the differences between different treatments,
and differences are represented by letters. Different letters are considered statistically
significant differences at p ≤ 0.05.

3. Results
3.1. Screening for Local Diclofenac-Degrading Bacteria
To isolate bacteria capable of degrading diclofenac, four different polluted soil and
water samples were used. Twenty-three bacterial isolates (S1–S23) were selected and
 3. Results
3.1. Screening for Local Diclofenac-Degrading Bacteria
To isolate bacteria capable of degrading diclofenac, four different polluted soil and
water samples were used. Twenty-three bacterial isolates (S1–S23) were selected and pu-
Microorganisms 2023, 11, 1445
rified based on their ability to utilize diclofenac as a sole carbon source. In order to select
5 of 18

the most active isolates, the ability to degrade diclofenac at a higher concentration (10-
fold) was further evaluated in minimal salt medium supplemented with 2000 µg/mL di-
purified based on their ability to utilize diclofenac as a sole carbon source. In order to
clofenac. The results revealed that only four morphologically different bacterial isolates
select the most active isolates, the ability to degrade diclofenac at a higher concentration
(S1, S2, S11, and S18) were able to grow after 24 h and therefore were selected and further
(10-fold) was further evaluated in minimal salt medium supplemented with 2000 µg/mL
characterized.
diclofenac. The results revealed that only four morphologically different bacterial iso-
lates (S1, S2, S11, and S18) were able to grow after 24 h and therefore were selected and
3.2. Idealcharacterized.
further Growth Parameters for Diclofenac-Degrading Bacteria
Each isolate was tested individually for optimal growth conditions with diclofenac
3.2. Ideal Growth Parameters for Diclofenac-Degrading Bacteria
as the sole carbon source. The results revealed that all bacterial isolates could grow effi-
Each isolate was tested individually for optimal growth conditions with diclofenac as
ciently at all tested concentrations and times. The rise in DCF concentration delayed the
the sole carbon source. The results revealed that all bacterial isolates could grow efficiently
maximum growth reached by all bacteria. The maximum growth was recorded after three
at all tested concentrations and times. The rise in DCF concentration delayed the maximum
days of incubation
growth reached by all forbacteria.
all isolates
The(Figure
maximum 1A–D). At was
growth higher concentrations
recorded after threeof DCF,
days of i.e., 30
and 40 mg/L,
incubation forisolates S1 and
all isolates S2 showed
(Figure 1A–D). aAt delayed
higher lag phase, while
concentrations of S11
DCF,and
i.e.,S18
30 underwent
and
exponential growth after only two days of incubation, and the viable
40 mg/L, isolates S1 and S2 showed a delayed lag phase, while S11 and S18 underwent counts started to
decline aftergrowth
exponential 5 days after
(Figure
only1C,D). Isolate
two days S2 showedand
of incubation, relatively moderate
the viable growth
counts started to during
all incubation times.
decline after 5 days (Figure 1C,D). Isolate S2 showed relatively moderate growth during all
incubation times.

Figure 1. The growth of the four selected bacterial isolates ((A), S1; (B), S2; (C), S11; and (D), S18)
Figure 1. The growth of the four selected bacterial isolates (A, S1; B, S2; C, S11; and D, S18) deter-
determined by the viable count method (log10 CFU/mL) after different time intervals (1–7 days) of
mined by the viable count method (log10 CFU/mL) after different time intervals (1–7 days) of bacte-
bacterial growth in minimal salt media supplemented with different concentrations of diclofenac
rial growth in minimal salt media supplemented with different concentrations of diclofenac (10, 20,
(10, 20, 30, and 40 mg/L) as a single carbon source. The bars represent mean ± SD of three indepen-
30, and 40 mg/L) as a single carbon source. The bars represent mean ± SD of three independent
dent replicates.
replicates.
In order to decrease the effect of different environmental growth conditions on DCF
biodegradation, deviations from the best bacterial growth conditions, including temper-
Microorganisms 2023, 11, 1445 6 of 18

ature, pH, and shaking incubation, were evaluated. The effect of temperature on DCF
degradation demonstrated that 37 ◦ C is the best temperature for the growth of all isolates
relative to other tested growth temperatures, except S1, which had the maximum growth
at 28 ◦ C after 4 days (Supplementary File, Figure S1). The selected isolates were further
tested for biodegradation conditions at different pH values, ranging from 5 to 9. The
results revealed that a neutral or slightly alkaline pH is the optimal pH for the growth and
biodegradation of DCF for all isolates except isolate S2, which grew better at acidic pH
(pH 5 and 6) (Figure S2).
On the other hand, the effect of static and shaking incubation conditions on the
performance of bacterial culture was monitored after different time intervals (1–6 days).
The results showed that bacterial growth was higher under shaking conditions (inferior
under static conditions) for isolates S11 and S18 (Figure S3c,d). The growth of isolates
S1 and S2 increased after 2 days of static incubation, with maximum growth reached at that
time, and then the growth gradually decreased (Figure S3a,b), but with shaking incubation,
the growth began to increase more than under static conditions after 3 days and reached
the maximum for isolate S1 after 5 days and S2 after 3 days.

3.3. Removal Efficiency of Isolated Bacterial Strains

The optimal conditions obtained in previous experiments were used to determine the
efficiency of DCF degradation. The amount of residual DCF remaining in each of the se-
lected bacterial cultures was determined by HPLC analysis (Figure S4). DCF concentrations
were considerably reduced only after 3 days of incubation in all tested isolates with the
initial DCF concentration (Table 1). The highest calculated degradation percentages were
recorded for isolate S11, followed by isolate S18, while isolate S2 had the lowest degradation
percentage of 12.85 among the isolates. A prolonged six-day incubation increased DCF
degradation, and isolate S11 almost completely degraded DCF at 97.79 ± 0.84 percent.
Bacterial isolate S2 had the minimum degradation percentage of 38.99 after six days of
incubation. The bacteria-free control showed almost no change after three and six days of
incubation under the same experimental conditions.

Table 1. The percentages of diclofenac degradation in MS after incubation at 37 ◦ C for three and six days.

Degradation of Diclofenac (%) *

Isolates
3 Days 6 Days
S1 37.76 ± 0.93 c 75.20 ± 0.71 c
S2 12.85 ± 1.58 b 38.99 ± 0.90 b
S11 86.60 ± 1.19 e 97.79 ± 0.84 e
S18 53.15 ±1.55 d 88.72 ± 0.89 d
Control 2. 29 ± 1.32 a 3.51 ± 0.87 a
* The initial diclofenac concentration in MS medium was 40 mg/L. The degradation percentage values are means
of three independent replicates ± standard deviations. Means with different lower-case letters differ significantly
at the probability level of 0.05, as analyzed by Tukey’s test.

3.4. Identification of DCF-Degrading Bacterial Isolates

The DCF-degrading bacterial isolates were initially identified by morphological char-
acters and different biochemical tests. Interestingly, all isolates were motile Gram-negative,
had a short rod shape, and were catalase- and oxidase-positive. Isolate S1 was a lac-
tose fermenter, while isolates S2, S11, and S18 were non-fermentative bacteria and were
first identified to belong to the Alcaligenaceae family. All isolates were identified by sequenc-
ing the 16S rRNA gene to determine the genus and the species. S1 16S rDNA amplicons
exhibited 100% identity with 16S rDNA from P. aeruginosa strains DSM 50071, ATCC 10145,
and NBRC 12689 in the rRNA database of GenBank. The S2 isolate exhibited 98.65 and
98.64% identity with 16S rDNA from Alcaligenes aquatilis LMG 22996 and Alcaligenes faecalis
subsp. parafaecalis strain G, respectively, and therefore, S2 was identified as Alcaligenes
aquatilis. Isolates S11 and S18 were identified as an A. spanius strain and A. piechaudii, respec-
 quencing the 16S rRNA gene to determine the genus and the species. S1 16S rDNA ampli-
cons exhibited 100% identity with 16S rDNA from P. aeruginosa strains DSM 50071, ATCC
10145, and NBRC 12689 in the rRNA database of GenBank. The S2 isolate exhibited 98.65
and 98.64% identity with 16S rDNA from Alcaligenes aquatilis LMG 22996 and Alcaligenes
faecalis subsp. parafaecalis strain G, respectively, and therefore, S2 was identified as Alcali-
Microorganisms 2023, 11, 1445 7 of 18
genes aquatilis. Isolates S11 and S18 were identified as an A. spanius strain and A. piechaudii,
respectively. S11 and S18 were highly associated with A. spanius strain LMG 5911, show-
ing 99.72 and 99.78% identity, respectively, followed by A. kerstersii LMG 3441, A. deleyi
LMG
tively.3458, andS18
S11 and A. piechaudii
were highly NBRC 102461,
associated which
with showedstrain
A. spanius percent
LMG identity ranging 99.72
5911, showing from
99.57 to 99.71. The sequences for isolates S1, S2, S1, and S18 were deposited into the Gen-
and 99.78% identity, respectively, followed by A. kerstersii LMG 3441, A. deleyi LMG 3458,
Bank database under accession numbers OQ504372, OQ504374, OQ504475, and
and A. piechaudii NBRC 102461, which showed percent identity ranging from 99.57 to 99.71.
OQ504476,
The sequences respectively. The
for isolates S1,sequence analysis
S2, S1, and utilizing
S18 were the program
deposited into the MEGAX
GenBankgenerated
database
under accession numbers OQ504372, OQ504374, OQ504475, and OQ504476,
the phylogenetic tree with the related species, with 1000 bootstrap analyses, and revealed respectively.
The sequence
that the tree wasanalysis utilizing
divided the main
into two program MEGAX
groups: the P.generated
aeruginosathe phylogenetic
strain group and tree with
Alacal-
the related
igense group,species,
as wellwith 1000
as an bootstrap analyses,
Achromobacter and revealed
species group (Figure that the tree
2). The latterwas divided
group was
into two main groups: the P. aeruginosa strain group and Alacaligense group,
divided into two different subgroups, one for Alacaligense spp. and the other for Achromo- as well as an
Achromobacter
bacter spp. species group (Figure 2). The latter group was divided into two different
subgroups, one for Alacaligense spp. and the other for Achromobacter spp.

Figure 2. Maximum likelihood phylogenetic tree for 16S rRNA gene sequences of the best diclofenac-
Figure 2. Maximum
degrading likelihood phylogenetic
isolates, Pseudomonas tree
aeruginosa S1, for 16S rRNA
Alacaligense geneS2,sequences
aquatilis of the bestspanius
and Achromobacter diclo-
fenac-degrading isolates, Pseudomonas aeruginosa S1, Alacaligense aquatilis S2, and Achromobacter
strains S11 and S18, with related bacterial species in GenBank. The identified isolates are labeled with
spanius strains
black boxes. S11
The and
tree wasS18, with related
generated using bacterial
MEGAX species
softwareinVersion
GenBank. TheThe
10.1.8. identified isolates
values at are
the nodes
labeled with black boxes. The tree was generated using MEGAX software Version 10.1.8. The values
are the percentage values given by bootstrap sample analysis (1000×).
at the nodes are the percentage values given by bootstrap sample analysis (1000×).
3.5. Detection of DCF Biodegradation Intermediate Compounds Produced by Each Bacterial Strain
3.5. Detection
3.5.1. of DCFofBiodegradation
Identification IntermediateIntermediate
DCF ProductsCompounds Produced
of A. spanius by Each
S11 and Bacterial S18
A. piechaudii
Strain
Based on the previous biodegradation results, the most efficient isolates for degra-
3.5.1.
dationIdentification of Intermediate
were A. spanius S11, A. piechaudii S18, andofP.A.
DCF Products spanius S11
aeruginosa S1,and A. piechaudii
so they S18
were further
Based on the
characterized. To previous biodegradation
detect and results, the metabolites,
identify biodegradation most efficienttheisolates
GC-MS fortechnique
degrada-
tion were
was A. spanius
performed for each A. piechaudii
S11,bacterial S18,
strain. and
The P. aeruginosa
analysis S1, so they
of the GC-MS were further
production char-
profile for
acterized.
A. To detect
spanius S11 andthat
revealed identify
there biodegradation
were nine main metabolites, the GC-MS
metabolites detected technique
among was
the experi-
mental (actual) and predicted degradation metabolites as compared to the parent diclofenac,
with a molecular weight (m/z) of 296 and a retention time (RT) of 12.77 min, which were
the same as those of the authentic standard of diclofenac listed in the library databases and
the control (pure) compound (Tables 2–4). In addition, a monohydroxylated product with a
mass of m/z 312 was detected, which could be attributed to the addition of 16 mass units,
revealing the formation of a singlet OH adduct. In general, the specificity of electrophilic
aromatic substitution is typically driven by the nature of the substitute, which may account
for our report of three different products with the same m/z ratio (312), confirming the
molecular composition of C14 H11 Cl2 NO3 and corresponding to 5-hydroxy diclofenac (2),
4/-hydroxy diclofenac (3), and 3-hydroxy diclofenac (4), indicating the role of cytochrome-
450 (CYP450) monooxygenase as the key enzyme in the biotransformation of diclofenac by
A. spanius S11 (Figure 3). Subsequently, the loss of (-H2 ) from the molecular ion resulted in
the formation of a fragment ion at m/z 309 with the molecular composition C14 H9 Cl2 NO3
(5), which is a quinone imine derivative of 5-hydroxy diclofenac (5-OH-DCF) generated
Microorganisms 2023, 11, 1445 8 of 18

by the action of laccase. The 5-OH-DCF quinone imine derivative (DF-2,5-benzoquinone

imine) was the starting point for further multistep degradation involving decarboxylation,
hydroxylation, and oxidation reactions. These mechanisms help to explain the presence of
the secondary product metabolites at m/z 265 with the molecular formula C13 H9 Cl2 NO (6),
which was converted to metabolite 7 with MW 281, or the molecular formula C13 H9 Cl2 NO2
via the action of peroxidase and a fragment ion at m/z 279 with the molecular composition
C13 H7 Cl2 NO2 (8). Another major metabolite was detected at m/z 298, which corresponds to
the formation of metabolite 9 with the molecular formula C14 H13 Cl2 NO2 with spectral frag-
ments of 4-(2,6-dichlorophenylamino)-1,3-benzenedimethanol. Finally, a fragment at m/z
277 corresponds to the formation of metabolite 10 with the molecular formula C14 H9 Cl2 NO
with spectral fragments of 1-(2,6-dichlorophenyl) indolin-2-one diclofenac amide (Table 2).
Based on these results, a DCF catabolic degradation pathway was postulated (Figure 3).

Table 2. DCF and identified DCF transformation products detected by GC/MS during biodegradation
by Achromobacter spanius S11.

No. Compound Name RT (min) Fragments (m/z) MW Formula

1 Diclofenac 12.77 249, 277, 295 296 C14 H11 Cl2 N2 O2
2 5-Hydroxydiclofenac
3 4/-Hydroxydiclofenac 10.93 214, 231, 249, 312 312 C14 H11 Cl2 NO3
4 3-Hydroxydiclofenac
5 DF-2,5-benzoquinone imine 13.72 242, 279, 309 309 C14 H9 Cl2 NO3
(6E)-6-[(2,5-dichlorophenyl)
6 12.88 111, 222, 265 265 C13 H9 Cl2 NO
imino]-3-oxocyclohexa-1,4-diene-1-carbaldehyde
(4E)-4-[(2,5-dichlorophenyl) imino]-3-(hydroxymethyl)
7 ——– —– 281 C13 H9 Cl2 NO2
cyclohexa-2,5-dien-1-one
(6E)-6-[(2,5-dichlorophenyl)
8 7.66 242, 277, 278 279 C13 H7 Cl2 NO2
amino]-3-oxocyclohexa-1,4-diene-1-carbaldehyde
9 4-(2,6-dichlorophenylamino)-1,3-benzenedimethanol 13.07 179, 214, 242, 279 298 C14 H13 Cl2 NO2
10 1-(2,6-Dichlorophenyl)indolin-2-one diclofenac amide 13.72 151, 214, 242, 277 277 C14 H9 Cl2 NO

Table 3. DCF and identified DCF transformation products detected by GC/MS during biodegradation
by Achromobacter piechaudii S18.

No. Compound Name RT (min) Fragments (m/z) MW Formula

1 Diclofenac 12.77 249, 277, 295 296 C14 H11 Cl2 N2 O2
2 5-Hydroxydiclofenac 6.32 214, 231, 249, 312 312 C14 H11 Cl2 NO3
3 4/ -Hydroxydiclofenac 6.54 214, 231, 249, 312 312 C14 H11 Cl2 NO3
4 (2, 4-Dichloro-3-(2-methylanilino) phenol 7.15 249, 268, 176 268 C13 H11 Cl2 NO
5 3-(3-Chloroanilino)-2-methylphenol 15.73 133, 203, 233, 107 233 C13 H12 ClNO
6 5-(2,4-Dihydroxyanilino)-4-(hydroxymethyl) benzene-1,3-diol 4.6 133, 151, 203, 263 263 C13 H13 No5
7 (2-Aminophenyl) acetic acid 5.04 77, 133, 151 151 C8 H9 NO2
8 Pyruvaldehyde, 1-(diethyl acetal) 47, 101, 144, 146 146 C7 H14 o2
9 2-Ethoxyethyl acetate 4.6 59, 88, 103, 132 132 C6 H12 o3
10 4-Dichlorophenylamino-1,3benzen dimethanol 14.67 214, 242, 277, 298 298 C14 H13 Cl2 NO2
11 4-Ethylbenzoic acid, 6-ethyl-3-octyl ester 55, 70, 135, 290 290 C19 H30 O2
12 4-Methyl-8-hexadecenal, Z 7.17 55, 70, 135, 252 252 C17 H32 O

Table 4. DCF and identified DCF transformation products after degradation by P. aeruginosa S1.

No. Compound Name RT (min) Fragments (m/z) MW Formula

1 Diclofenac 33.66 249, 277, 295 296 C14 H11 Cl2 N2 O2
2 4/ -Hydroxydiclofenac 6.26 214, 231, 249, 312 312 C14 H11 Cl2 NO3
3 5-Hydroxydiclofenac 6.67 214, 231, 249, 312 312 C14 H11 Cl2 NO3
4 2,4-Dichlorophenol ——- ——- 163 C6 H4 Cl2 O
5 (3-Aminophenyl) acetic acid 5 51, 77, 133, 151 151 C8 H9 NO2
6 (3-Hydroxyphenyl) acetic acid 5.21 77, 133, 151 152 C8 H8 O3
4-Amino-3,5-dichlorobenzene
7 5 151, 179, 194 194 C6 H5 Cl2 NO2
-1,2-diol
8 (2,3-Dihydroxyphenyl) acetic acid 7.19 55, 124, 168 168 C8 H8 O4
9 (4,5-Dihydroxy-2-methylphenyl) acetic acid 7.19 151, 179, 182 182 C9 H10 O4
10 (2Z,4E)-2,3,5-Trihydroxypenta-2,4-dienoic acid 9.18 47, 101, 144 146 C5 H6 O5
11 Ethyl ethoxyacetate 4.57 31, 59, 88, 103, 132 132 C6 H12 O3
12 Pyruvaldehyde,1-(diethyl acetal) 9.14 47, 101, 144 146 C7 H14 O3
Microorganisms 2023, 11, x FOR PEER REVIEW 9 of 19

Microorganisms 2023, 11, 1445 9 of 18

Figure 3. The postulated biodegradation pathway of diclofenac by Achromobacter spanius S11.

Figure 3. The postulated biodegradation pathway of diclofenac by Achromobacter spanius S11. (1)
(1) Diclofenac, (2) 5-hydroxydiclofenac, (3) 4-hydroxydiclofenac, (4) 3-hydroxy-diclofenac, (5) DF-
Diclofenac, (2) 5-hydroxydiclofenac, (3) 4-hydroxydiclofenac, (4) 3-hydroxy-diclofenac, (5) DF-2,5-
2,5-benzoquinone imine, (6) (4E)-4-[(2,5-dichlorophenyl)imino]-3-methylcyclohexa-2,5-dien-1-one,
benzoquinone imine, (6) (4E)-4-[(2,5-dichlorophenyl)imino]-3-methylcyclohexa-2,5-dien-1-one, (7)
(7) (4E)-4-[(2,5-dichlorophenyl)imino]-3-(hydroxymethyl)cyclohexa-2,5-dien-1-one,
(4E)-4-[(2,5-dichlorophenyl)imino]-3-(hydroxymethyl)cyclohexa-2,5-dien-1-one, (8)(8)(6E)-6-[(2,5-di-
(6E)-6-[(2,5-
dichlorophenyl)imino]-3-oxocyclohexa-1,4-diene-1-carbaldehyde, (9) 4-(2,6-dichlorophenylamino)-
chlorophenyl)imino]-3-oxocyclohexa-1,4-diene-1-carbaldehyde, (9) 4-(2,6-dichlorophenylamino)-
1,3-benzenedimethanol,
1,3-benzenedimethanol,and and(10)
(10)1-(2,6-dichlorophenyl)indolin-2-one diclofenac
1-(2,6-dichlorophenyl)indolin-2-one diclofenac amide.
amide.
Microorganisms 2023, 11, 1445 10 of 18

On the other hand, the biodegradation of DCF with the A. piechaudii S18 isolate
was different, suggesting divergent degradation pathways. Although the degradation
rates were lower with A. piechaudii S18 than with A. spanius S11, it was more efficient in
breaking down diclofenac into simpler compounds, which are summarized in Table 3.
Figure 4 proposes the biodegradation pathway of A. piechaudii S18 based on the 11 main
metabolites detected by the experimental (actual) and predicted degradation metabolites.
Compared to the parent compound (diclofenac, m/z 296) (1), intermediate products with
the same m/z ratio (312) at two different retention times (6.32 and 6.54) confirmed the
molecular composition of C14 H11 Cl2 NO3 , corresponding to 5-hydroxydiclofenac (2) and
4-hydroxydiclofenac (3), indicating the presence of CYP450 monooxygenase as the key
enzyme in DCF mineralization, and the subsequent hydroxydiclofenacan was dechlorinated
through the loss of CO2 , leading to the formation of the decarboxylated derivative of the
monohydroxyl-DCF metabolite (3) at m/z 268 with the molecular formula C13 H11 NCl2 (4),
corresponding to (2,4-dichloro-3-(2-methylanilino) phenol.
The subsequent dichlorination (-HCl- ) of the molecular ion resulted in the formation
of a fragment ion at m/z 233 with the molecular formula C13 H12 ClNO, corresponding
to the formation of 3-(3-chloroanilino)-2-methylphenol (metabolite 5), followed by the
oxidation of the methyl group, hydroxylation, and the formation of metabolite 5 at m/z
263 with the molecular formula C13 H13 NO5 , which corresponds to 5-(2,4-dihydroxyanilino)-
4-(hydroxymethyl) benzene-1,3-diol. The fragment at m/z 151 corresponds to the cleavage
of the NH bridge between the aromatic rings and the formation of metabolite 6 with
the molecular formula C8 H9 NO2 with spectral fragments of (2-aminophenyl) acetic acid
(metabolite 7). Metabolites 8 and 9 were the result of ring cleavage and the formation
of a fragment at m/z 146 with the molecular formula C7 H14 O3 with spectral fragments
typical of pyruvaldehyde, 1-(diethyl acetal), and a fragment at m/z 132 with the molecular
formula C7 H14 O3 (metabolite 8) and 2-ethoxyethyl acetate (metabolite 9) with the molecular
formula C6 H12 O3 (m/z 132.00).
A major metabolite occurred transiently and was identified by GC-MS as 4-dichloroph
enylamino-1,3 benzendimethanol for a fragment ion at m/z 298 with the molecular formula
C14 H13 Cl2 NO2 (metabolite 10), indicating the role of the laccase enzyme in this transforma-
tion step. However, the presence of metabolite 11 with a fragment ion at m/z 290 and the
molecular formula C19 H30 O2 and metabolite 12 with a fragment ion at m/z 252 and the
molecular formula C17 H32 O, in addition to metabolites 8 and 9, clearly indicated the ability
of A. piechaudii S18 to utilize DCF as the sole carbon source through a strong oxidative
enzyme system (Figure 4). Therefore, the enzyme activities of both Achromobacter strains
were further analyzed to support our suggested transformation map of diclofenac.

3.5.2. Identification of Intermediate DCF Products of P. aeruginosa S1

To understand the mechanism of DCF by P. aeruginosa S1, the GC/MS chromatogram
was analyzed and interpreted, as presented in Figure 5. The chemical structures of these
products were elucidated from the mass library, as well as from their mass fragmentation
patterns (Table 4). The expected initial step for the biodegradation of diclofenac was hy-
droxylation, resulting in 4/-hydroxydiclofenac (2) and 5-hydroxydiclofenac (3), indicating
that CYP450 monooxygenase played the key role in the first step of DCF degradation by
P. aeruginosa S1. As a consequence of hydroxylation, the second step was C-N cleavage between
the aromatic rings, followed by a hydroxylation reaction: the key metabolites formed may be
2,4-dichlorophenol (4), as an expected compound for this step; (3-aminophenyl) acetic acid (5);
(3-hydroxyphenyl) acetic acid (6); and 4-amino-3,5-dichlorobenzene (7). Another two major
metabolites were detected at m/z 168, corresponding to (2,3-dihydroxyphenyl) acetic acid (8),
and m/z 182 with the molecular formula C9 H10 O4 , corresponding to
(4,5-dihydroxy-2-methylphenyl) acetic acid (9), which verifies the assumption of hydroxyl
substitution. Consequently, the results show another intermediate metabolite at m/z 146 with
the molecular formula C5 H6 O5 that corresponds to (2Z,4E)-2,3,5-trihydroxypenta-2,4-dienoic
acid (10); a metabolite at m/z 132 with the molecular formula C9 H10 O4 , corresponding to
Microorganisms 2023, 11, 1445 11 of 18
Microorganisms 2023, 11, x FOR PEER REVIEW 11 of 19

ethyl ethoxyacetate (11); and a metabolite at m/z 146 with the molecular formula C7 H14 O3 ,
composition of C14H11Cl2NO3, corresponding to 5-hydroxydiclofenac (2) and 4-hydroxydi-
corresponding to pyruvaldehyde, 1-(diethyl acetal) (12). Then, the further degradation of ethyl
clofenac (3), indicating the presence of CYP450 monooxygenase as the key enzyme in DCF
ethoxyacetate and pyruvaldehyde, 1-(diethyl acetal) produced CO2 and water. Therefore, DCF
mineralization, and the subsequent hydroxydiclofenacan was dechlorinated through the
by P.ofaeruginosa
biodegradation loss CO2, leadingS1 was catalyzed
to the byofthree
formation major enzymes:
the decarboxylated cytochrome-450
derivative of the monohy-
monooxygenase, droxyl-DCF metabolite (3) at m/z 268 with the molecular formula enzyme
peroxidase, and catechol 1,2-dioxygenase. Accordingly, the C13H11NClactivi-
2 (4), corre-
ties of the three sponding to (2,4-dichloro-3-(2-methylanilino) phenol.
bacterial isolates were further analyzed to support our suggested diclofenac
transformation map of diclofenac.

Figure 4. The proposed biodegradation pathway of diclofenac by Achromobacter piechaudii S18. (1) Di-
Figure 4. The proposed biodegradation pathway of diclofenac by Achromobacter piechaudii S18. (1)
Diclofenac, (2) 5-hydroxydiclofenac,
clofenac, (2) 5-hydroxydiclofenac, (3) 4/-hydroxydiclofenac,
(3) 4/-hydroxydiclofenac, (4) (2, 4-dichloro-3-(2-methylanilino)
(4) (2, 4-dichloro-3-(2-methylanilino) phe-
phenol, (5) 3-(3-chloroanilino)-2-methylphenol, (6) 5-(2-dihydroxyanilino)-4-(hydroxymethyl)
nol, (5) 3-(3-chloroanilino)-2-methylphenol, (6) 5-(2-dihydroxyanilino)-4-(hydroxymethyl) benzene-
1,3-diol, (7) (2-aminophenyl)acetic acid, (8) pyruvaldehyde, 1 (diethyl acetal), (9) 2-ethoxyethyl
acetate, (10) 4-dichlorophenylamino-1,3benzen dimethanol, (11) 4-ethylbenzoic acid, 6-ethyl-3-octyl
ester, and (12) 4-methyl-8-hexadecenal.
 Microorganisms 2023, 11, x FOR PEER REVIEW 13 of 19
Microorganisms 2023, 11, 1445 12 of 18

Figure 5. The proposed biodegradation pathway of diclofenac by P. aeruginosa S1. (1) Diclofenac,
Figure 5. The proposed biodegradation pathway of diclofenac by P. aeruginosa S1. (1) Diclofenac, (2)
(2)4/-hydroxydiclofenac,
4/-hydroxydiclofenac,(3) (3) 5-hydroxydiclofenac,
5-hydroxydiclofenac, (4)(4)2,4-dichlorophenol,
2,4-dichlorophenol,(5)(5)(3-aminophenyl)acetic acid,
(3-aminophenyl)acetic
(6)acid, (6) (3-hydroxyphenyl)acetic acid, (7) 4-amino-3,5-dichlorobenzene-1,2-diol, (8) (2,3-dihydrox-
(3-hydroxyphenyl)acetic acid, (7) 4-amino-3,5-dichlorobenzene-1,2-diol, (8) (2,3-dihydroxyphenyl)acetic
yphenyl)acetic
acid, acid, (9) (4,5-dihydroxy-2-methylphenyl)acetic
(9) (4,5-dihydroxy-2-methylphenyl)acetic acid, (10) (2Z,4E)-2,3,5-trihydroxy-
acid, (10) (2Z,4E)-2,3,5-trihydroxypenta-2,4-dienoic acid,
penta-2,4-dienoic acid, (11) ethyl ethoxyacetate, and (12) pyruvaldehyde,
(11) ethyl ethoxyacetate, and (12) pyruvaldehyde, 1-(diethyl acetal). 1-(diethyl acetal).
Microorganisms 2023, 11, 1445 13 of 18

3.6. Specific Activities of Enzymes Involved in DCF Degradation

To reveal DCF microbial degradation enzymes, the specific activities of selected en-
zymes were determined for A. spanius S11, A. piechaudii S18, and P. aeruginosa S1. Hence,
the specific activities of the selected enzymes (catechol 1,2-dioxygenase, catechol 2,3-
dioxygenase, laccase, and peroxidase) were determined both in the presence of DCF
and in the control medium (glucose instead of DCF). As shown in Table 5, A. piechaudii
S18 was superior in catechol 1,2-dioxygenase activity (2.714 ± 0.147 U/mg), followed by
P. aeruginosa S1 (1.832 ± 0.20 U/mg protein) and A. spanius S11 (0.928 ± 0.026 U/mg).

Table 5. Specific activity of selected enzymes (U/mg protein) measured for Achromobacter spanius S11,
Achromobacter piechaudii S18, and P. aeruginosa (S1) in the presence of DCF or glucose as a control.

Specific Enzyme Activity (mU/mg Protein)

Enzyme A. spanius S11 A. piechaudii S18 P. aeruginosa S1

Control * MS + DCF ** Control MS + DCF Control MS + DCF
Catechol 1,2-dioxygenase 0.777 ± 0.064 0.928 ± 0.026 1.34 ± 0.256 2.714 ± 0.147 1.137 ± 0.293 1.832 ± 0.20
Catechol 2,3-dioxygenase 0.0021 ± 0.008 0.005 ± 0.002 0.334 ± 0.097 0.131 ± 0.083 0.059 ± 0.016 0. 386 ± 0.032
Laccase 0.192 ± 0.037 0.807 ± 0.127 0.390 ± 0.036 0.672 ± 0.096 0.004 ± 0.002 0.002 ± 0.001
Peroxidase 0.0457 ± 0.04 0.107 ± 0.076 0.654 ± 0.041 0.747 ± 0.054 0. 328 ± 0.071 0. 880 ± 0.127
* Control is minimal salt medium supplemented with 10 mg/L glucose. ** MS is a minimal salt medium
supplemented with 10 mg/L diclofenac as the sole carbon source.

Furthermore, the catechol 2,3-dioxygenase assay demonstrated that the highest enzyme-
specific activity was achieved with P. aeruginosa S1 in the presence of DCF (0.386 ± 0.032 U/mg),
followed by A. piechaudii S18, with a specific activity of 0.334 ± 0.097 U/mg, unexpectedly less
than in the control medium (with glucose). The lowest catechol 2,3-dioxygenase activity was
detected with A. spanius S11. The opposite trend was obtained with laccase activity, where the
highest activity was observed with A. spanius S11 and A. piechaudii S18 in the presence of DCF
(0.807 ± 0.127 and 0.672 ± 0.096 U/mg protein, respectively), while no activity was detected
for P. aeruginosa S1. Regarding peroxidase activity, the highest activity was observed with
P. aeruginosa S1 in the presence of DCF (0.880 ± 0.127 U/mg), followed by A. piechaudii
S18 (0.747 ± 0.054 U/mg), while the lowest specific peroxidase activity was achieved with
A. spanius S11 (0.107 ± 0.076 U/mg).

4. Discussion
The global contamination of different water resources with different pharmaceuti-
cal compounds is alarming due to their serious and chronic biological effects, such as
lipophilicity and toxic effects that may occur in living beings [9,24]. DCF is one of the major
pharmaceutical compounds detected in aquatic environments in different countries due to
its high persistence and relatively high concentrations reported in surface and groundwater,
as well as effluent water discharged from WWTPs [9,10]. The natural biodegradation
of DCF in the ecosystem is mainly accomplished by bacteria and other microorganisms.
Therefore, the bacteria-based biodegradation of pharmaceutical compounds is a promising
area of research and a cost-effective strategy, especially for micropollutants. In this study,
four bacterial isolates (S1, S2, S11, and S18) were able to grow in the presence of DCF
as a sole source of carbon and energy (without a co-substrate), which provides evidence
that these strains can use DCF as the sole carbon source, resulting in its biodegradation.
The selected bacterial strains could tolerate exposure to high concentrations of DCF, up
to 40 mg/L, at different magnitudes. It was observed that the selected bacterial strains
required a prolonged lag phase when increasing the DCF concentration, and then the
growth rate became stationary after six days of incubation. This suggests that DCF induces
cellular toxicity, and each bacterial isolate has a different level of resistance. The high
concentration of DCF induces the exacerbation of an oxidative burst inside the bacterial
cells due to the liberation of reactive oxygen species (ROS) [12,25]. In this regard, ROS
intermediate generation after the treatment of cancer cell lines was attributed to the inhi-
bition of the cellular antioxidant enzyme superoxide dismutase [26]. Moreover, ROS can
Microorganisms 2023, 11, 1445 14 of 18

also trigger the bacterial membrane’s unsaturated fatty acids, as well as cellular proteins
and DNA. A high concentration of the lipid peroxidation biomarker (MDA) was formed
during bacterial exposure to DCF [7]. Bacteria can scavenge increased ROS inside the
cells by using different antioxidant enzymes. This response to DCF exposure is specific
to each bacterial strain [25]. Interestingly, the tested DCF concentration is higher than
most of those reported in the previous literature for DCF degradation by a single bacterial
strain, indicating the high efficiency of the scavenging systems of the selected isolates to
cope with the oxidative stress induced by DCF [7,13,14]. However, a microbial consortium
from forest soil samples completely degraded an elevated DCF concentration (100 mg/L)
after only 10 days of aerobic incubation [27]. Remarkably, Klebsiella sp. KSC had the best
degradation rate after only 72 h with an initial concentration of 70 mg/L [28]. The efficiency
of the selected isolates was confirmed by the disappearance of the parent compound, with
the maximum degradation percentage reaching 97.79 ± 0.84 for isolate S11 after only six
days of incubation. These results are a significant improvement compared to 25 days for
Labrys portucalensis F11 [14] and 12 days for the Pseudomonas moorei KB4 strain [25] for the
complete degradation of a comparable concentration of DCF. Fast degradation rates were
reported for Rhodococcus ruber strain IEGM 346 in six days [20] and Bacillus subtilis and
Brevibacillus laterosporus strains after 17 h [29] but with a low initial concentration (50 µg/L)
or with the intermediate product 40 -hydroxy-diclofenac, respectively.
The selected strains were capable of growing in a temperature range of 28–37 ◦ C. This
is in line with the reported literature for mesophilic bacteria that have an optimal growth
temperature range of 35 to 37 ◦ C [10].
pH optimization during exposure to DCF may have an additional advantage for the
degradation process. It was observed that the optimal pH values for the growth of DCF-
degrading bacteria were different among bacterial strains. Three isolates preferred pH
values that were more alkaline (7–8), and only isolate S2 grew better at pH 5. Indeed, it was
reported that for some bacterial strains, the intermediate metabolites of DCF degradation
produced different types of amines that increased the pH value of the culture [7].
The identification of the selected isolates demonstrated that all isolates were Gram-
negative. Three bacterial isolates belonged to the Alcaligenaceae family, and one bacterial
isolate belonged to the Pseudomonadaceae family. In the literature, different genera of both
Gram-positive and Gram-negative bacteria were identified as potential DCF-degrading bac-
teria, such as Klebsiella, Raoultella, Bacillus, and Rhodococcus [20,28,30]. Despite the diversity
of the identified bacterial strains, few bacterial isolates were able to completely degrade
DCF due to the difficulty of degradable intermediates generated during biodegradation [31].
In this study, two strains of the four selected bacteria were new for DCF biodegradation,
namely, A. spanius strain S11 and A. piechaudii S18. Recently, some studies demonstrated the
potentiality of Achromobacter sp. for the degradation of different types of organic pollutants,
such as sulfamethoxazole by Achromobacter sp. JL9 [32] and petroleum bioremediation by
Achromobacter sp. HZ01 [33]. Therefore, their degradation pathways were investigated
alongside the well-known P. aeruginosa S1.
The results obtained from the identification of intermediate metabolites by GC-MS
during DCF biodegradation suggested that the bacterial degradation reactions that oc-
curred were mostly multidirectional [31]. The primary hydroxylation of DCF is considered
a bottleneck step in DCF metabolites, and the detection of 40 -OH-DCF and 50 -OH-DCF in
all investigated isolates indicated that CYP450 monooxygenase is a key enzyme in this
degradation pathway [14,30]. In fact, most of the described microorganisms capable of
DCF biodegradation transformed it into a more toxic hydroxylated form. However, only
a few isolates could further cleave the aromatic structure of this hydroxylated DCF [2,31].
The formation of quinone imine derivatives of 5-hydroxy diclofenac (DF-2,5-QI) in the
A. spanius S11 culture via the action of laccase was previously demonstrated [34]. This
was confirmed by the high laccase activity observed in cells grown with DCF as a sole
carbon source compared to control cells grown in glucose. However, the two-step bio-
transformation to DF-2,5-QI during the human metabolism of DCF demonstrated catalysis
Microorganisms 2023, 11, 1445 15 of 18

by two different CYPs: the highest activity was for the 5-hydroxylation of DF, which was
predominantly catalyzed by CYP3A4, while its subsequent bioactivation to DF-2,5-QI was
catalyzed by CYP2C9 [35]. The further degradation of DF-2,5-QI by A. spanius S11 may
proceed via different pathways (decarboxylation, hydroxylation, and oxidation reactions).
The DCF degradation described for Labrys portucalensis F11 proceeded mainly by hydroxy-
lation reactions, where the formation of benzoquinoneimine species seems to be a central
step in the degradation pathway [14]. The formation of 4-(2,6-dichlorophenylamino)-1,3-
benzenedimethanol (metabolite 8) at m/z 298 via the action of the laccase enzyme was previ-
ously confirmed by the action of purified fungal laccase from the white rot fungus Trametes
versicolor during DCF degradation [36]. The subsequent formation of 1-(2,6-dichlorophenyl)
indolin-2-one diclofenac amide (metabolite 9) by A. spanius S11 could be attributed to
the action of the laccase enzyme. The degradation of DCF with immobilized laccase on
graphene oxide led to the identification of the same compound as a major metabolite in
the degradation of DCF [37]. On the other hand, the complete degradation and distur-
bance of the aromatic structure of hydroxydiclofenac was observed with the isolated strain
A. piechaudii S18. This compound was dechlorinated through CO2 loss, which formed 5-(2,4-
dihydroxyanilino)-4-(hydroxymethyl) benzene-1,3-diol (metabolite 4), where dichlorination
is the central step for degradation and decreases the ecotoxicity of DCF metabolites [38,39].
Ring cleavage occurred subsequent to metabolite 5, producing pyruvaldehyde, 1-(diethyl
acetal) and 2-ethoxyethyl acetate due to subsequent oxidation followed by the cleavage of
the N-C bond and the formation of 5-(2,4-dihydroxyanilino)-4-(hydroxymethyl) benzene-
1,3-diol (metabolite 6). The detection of 4-dichlorophenylamino-1,3 benzen dimethanol
(metabolite 10) produced by the action of laccase was previously reported in a study
performed on DCF using commercial laccase from Trametes versicolor [2]. Many studies
correlated the laccase activity with the microbial degradation of DCF and suggested that
the presence of nitrogen and the negative charge in the DCF ring play important roles
in the degradation of these compounds by the laccase enzyme [40,41]. Furthermore, the
formation of by-products without an aromatic structure (metabolites 8, 9, and 12) allows
their further incorporation into the Krebs cycle [25]. Taken together, these results indicate
that most of the detected metabolites generated by this bacterial strain are less toxic than
the parent compound DCF.
The DCF biodegradation pathway of P. aeruginosa S1 was mostly multidirectional, as
confirmed by the detection of 11 main metabolites [42]. The C-N cleavage between aromatic
rings, followed by a hydroxylation reaction, led to the formation of key metabolites, includ-
ing 2,4-dichlorophenol (4), (3-aminophenyl) acetic acid (5), (3-hydroxyphenyl) acetic acid
(6), and 4-amino-3,5-dichlorobenzene (7). The hydroxyl derivative was exposed, leading to
the subtraction of an electron and H+ from the hydroxyl phenolic group by peroxidases,
generating a phenoxyl radical [43]. Peroxidase could catalyze the first oxidative dechlorina-
tion step in the degradation of several chlorinated phenols. When the electrophilic hydroxyl
radical was added to the aromatic ring, a resonance-stabilized carbon-centered radical was
formed, and the hydrogen radical was eliminated. With further hydroxyl radical oxidation,
multi-dihydroxylation products were formed, including (3,5-dihydroxyphenyl) acetic acid
(8) and (4,5-dihydroxy-2-methyl phenyl) acetic acid (9), which verify the assumption of
hydroxyl substitution, indicating the role of catechol 1,2-dioxygenase, which was indi-
rectly proven by the high enzymatic activity observed in P. aeruginosa S1. The hydroxyl
group enhances the aromatic ring’s electron density; hence, hydroxyl radical electrophilic
adductions occur more quickly [44]. Consequently, another intermediate metabolite at
m/z 132 with the molecular formula C9 H10 O4 , corresponding to ethyl ethoxyacetate (11),
and a metabolite at m/z 146 with the molecular formula C7 H14 O3 , corresponding to pyru-
valdehyde, 1-(diethyl acetal) (12), were generated. Then, the further degradation of ethyl
ethoxyacetate and pyruvaldehyde, 1-(diethyl acetal) produced CO2 and water. Indeed,
recent work demonstrated that P. aeruginosa is capable of mineralizing many persistent
aromatic pollutants, including chlorinated phenols [45,46]. However, DCF degradation
was previously confirmed for Pseudomonas moorei but not for P. aeruginosa [25].
Microorganisms 2023, 11, 1445 16 of 18

From the proposed degradation map, it can be concluded that the first steps of the
oxygenation of DCF occur via phenol hydroxylase enzymes to form monohydroxyl-DCF,
followed by peroxidase, which could catalyze the first oxidative dechlorination step and the
subsequent ring cleavage adjacent to or in between the two hydroxyl groups of polyhydrox-
ylated derivatives. Phenol hydroxylases ranging from simple flavoprotein monooxygenases
to multicomponent hydroxylases, as well as the genes coding for these enzymes, were
described for several aerobic phenol-degrading microorganisms [47].
The postulated degradation pathway of DCF by the three bacterial strains established
the activities of monooxygenase, dioxygenase, peroxidase, and laccase enzymes generated
during degradation by all bacterial species. Although many studies have reported DCF
degradation, reports on the specific enzymatic activities of DCF-degrading bacteria are still
scarce. Therefore, we investigated the enzymatic activities to integrate the biodegradation
pathway with specific enzymatic activities. Catechol 1,2-dioxygenase, peroxidase, and
laccase enzymes were induced in all isolates tested in the presence of DCF compared to
control cells, confirming the involvement of these enzymes in the degradation process.
However, catechol 2,3-dioxygenase activity was high in the glucose-free control culture of
A. piechaudii S18. In this regard, the expression level of catechol 1,2-dioxygenases increased
significantly (145 times) in the Pseudomonas moorei KB4 strain after DCF exposure, but
up-regulation was not detected for catechol 2,3-dioxygenase [25].
The enhanced degradation activities of A. spanius S11 and A. piechaudii S18 are in line
with the ability of different Achromobacter strains to produce monooxygenase and dioxyge-
nase enzymes, such as Achromobacter sp. HZ01, Achromobacter sp. DMS1, and Achromobacter
xylosoxidans DN002 [48]. The presence of several genes encoding the monooxygenase
family, as well as the blue multicopper oxidase family, in the genome of Achromobacter sp.
HZ01 strain confirmed the bioremediation potential of Achromobacter strains [33].

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/microorganisms11061445/s1.
Author Contributions: Manuscript conceptualization, S.M.E., N.A.H.F., and M.S.M.M.; methodology,
A.A.A., M.S.M.M., and N.A.H.F.; data analysis, A.A.A., M.S.M.M., N.A.H.F., and S.M.E.; writing—
original draft preparation, M.S.M.M., A.A.A., and N.A.H.F.; manuscript review and editing, M.S.M.M.,
N.A.H.F., and S.M.E. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: All data are included in the manuscript and the supplementary files.
Conflicts of Interest: The authors declare no conflict of interest.

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