Environmental Technology & Innovation 29 (2023) 102983

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Environmental Technology & Innovation

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

Isolation of flurbiprofen-degrading bacteria and evaluating its

degradation characteristics
∗
Tian Liu, Shilin Yang, Zixian Wu, Yufei Cai, Jiawei Zhou, Mingjun Liao
School of Civil Engineering, Architecture and Environment, Hubei University of Technology, Hubei Key Laboratory of Ecological
Remediation for Rivers-Lakes and Algal Utilization, Wuhan 430068, China

article info a b s t r a c t

Article history: We isolated a flurbiprofen-degrading strain LY.1 from the water samples of the Xunsi
Received 28 September 2022 River in Wuhan City, China. The 16S rDNA sequence alignment showed that the LY.1
Received in revised form 8 December 2022 strain was Pseudomonas aeruginosa (PA). Subsequently, we studied the growth and
Accepted 12 December 2022
flurbiprofen-degradation characteristics of LY.1. The results showed that the LY.1 strain
Available online 16 December 2022
grew in the medium containing flurbiprofen as the sole carbon source. Its growth
Keywords: was accompanied by the accumulation of yellow products of aromatic ring-opening
Flurbiprofen reaction. At 400 mg/L initial flurbiprofen concentration, 30 ◦ C, and pH 7, the flurbiprofen
Biodegradation degradation rate of the strain reached 100% at 96 h. GC–MS assay results showed
Pseudomonas aeruginosa that the strain LY.1-mediated flurbiprofen degradation end-product was 3-fluoro-4-
(1-ethylcarboxy) phenylpropionic acid (FCB). The LY.1 strain growing conditions were
25–40 ◦ C, pH 6–8, and substrate concentration 100–800 mg/L. Therefore, our findings
can provide references for the environmental fate of flurbiprofen and its pollution
control.
© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

The presence of pharmaceuticals and personal care products (PPCPs) in natural water bodies has become a hot topic
of concern in recent years due to their potential damages to both the ecological environment and human health (Mei
et al., 2019). Numerous PPCPs have been detected in natural water bodies like rivers, lakes, oceans, and groundwater
(Wang et al., 2014). Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most commonly prescribed drugs for treating
various pain-related disorders (arthritis and other rheumatic diseases) (Amadio and Murphy, 2010) and are also invariably
an important source of PPCPs. Flurbiprofen [2-(2-fluoro-4-biphenyl)propionic acid] is an NSAID, with similar effects as
ibuprofen. Clinically, it is generally used to treat rheumatoid arthritis, degenerative joint disease, osteoarthritis, and low
back pain (Kagkadis et al., 1998). Global sales of flurbiprofen are ∼$100 million annually, with an annual growth rate
of ∼5% (Yanac and Murdoch, 2019). Flurbiprofen is very popular in some European countries, e.g., flurbiprofen ranked
among the top three drugs in Turkey. Recent studies have shown that flurbiprofen could not only be used to treat prostate
and colon cancer (Suthar and Sharma, 2016), but also for inhibiting obesity (Hosoi et al., 2016). Therefore, if the research
results get translated into clinical applications, the use of flurbiprofen is bound to surge in the next few decades.
There are few studies on the status of flurbiprofen presence in the environment. Wolecki et al. (2019). detected
flurbiprofen in the inlet of a Polish municipal sewage plant at a concentration of (51 ± 12) ng/L. Andreozzi et al. detected
flurbiprofen in the effluent of a sewage treatment plant in France and Italy at concentrations of 0.21 µg/L and 0.34 µg/L,

∗ Correspondence to: Hubei Key Laboratory of Ecological Remediation for Rivers-Lakes and Algal Utilization, Wuhan 430068, China.
E-mail address: lmj@hbut.edu.cn (M. Liao).

https://doi.org/10.1016/j.eti.2022.102983
2352-1864/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.
org/licenses/by-nc-nd/4.0/).
T. Liu, S. Yang, Z. Wu et al. Environmental Technology & Innovation 29 (2023) 102983

respectively (Andreozzi et al., 2003). Due to the poor water solubility of flurbiprofen (Know = 4.2), it easily combines
with other surrounding organic substances, thereby providing it high adsorption capacity and bioaccumulation potential
(Indrayanto, 2012). Since drugs usually exist in the environment as complex mixtures, they may have a great toxic effect
on organisms (Backhaus, 2014). Two NSAIDs similar to flurbiprofen, i.e., diclofenac and ibuprofen, have been added to
the EU priority pollutant list due to their ecotoxicological effects (Richardson and Ternes, 2011). These NSAIDs and their
degradation byproducts are toxic to diverse aquatic organisms, like mussels (Gonzalez-Rey and Bebianno, 2011), insects
(Liu et al., 2017), fish (Bickley et al., 2017), etc. Currently, there are few studies on the ecotoxicology of flurbiprofen. Tongur
and Yldz (2021) showed that flurbiprofen had certain physiological toxicity against three commonly used test organisms,
i.e., Lepidium sativum, Daphnia magna, and Aliivibrio fischeri, with the EC50 being 1.9–50 mg/L. Currently, some studies have
shown that the catalytic degradation (Simsek et al., 2018) and chemical oxidative degradation (Barışçı et al., 2015) methods
could remove flurbiprofen residues from medical wastewater at the pilot level. However, these methods are costly and
there is a risk of secondary pollution. Microbial degradation is one of the effective means to treat organic pollutants in
sewage, due to their advantages of high efficiency, good performance, and low cost. There have been extensive studies
on the biodegradation of NSAIDs, like ibuprofen (Li et al., 2015; Murdoch and Hay, 2013; Chopra and Kumar, 2020; Show
et al., 2021)), diclofenac (Żur et al., 2021; Bouju et al., 2016), and naproxen (Górny et al., 2019; Wojcieszyńska et al., 2014),
from the perspectives of isolation and identification of NSAIDs-degrading bacteria, associated degradation pathways, and
the application of strains. However, to the best of our knowledge, no study has been carried out on the screening, isolation,
and characterization of flurbiprofen-degrading bacteria.
In this study, we isolated a strain that could grow with flurbiprofen as the sole carbon source from the water sample of
the Xunsi River in Wuhan City. We also studied its degradation characteristics to provide insights into the ecotoxicology
and biodegradation of flurbiprofen.

2. Materials and methods

2.1. Culture medium and main reagents

Flurbiprofen (98% purity) was purchased from Shanghai Yuanye Biotechnology Co., Ltd, Shanghai, China. The strain
screening water samples were collected from the Xunsi River, Wuhan City, Hubei Province, China. Mineral salt medium
(MSM) (Mccullar et al., 1994) was made of 1 mM MgSO4 , 10 mM K2 HPO4 , 3 mM NaH2 PO4 , 10 mM (NH4 )2 SO4 , 10 µM
Fe(NO3 )3 , and 100 µM Ca(NO3 )2 .

2.2. Enrichment and isolation of flurbiprofen-degrading bacteria

First, 50 mL of raw water collected from the Xunsi River was transferred to a conical flask containing 50 mL of MSM,
with flurbiprofen at a final concentration of 200 mg/L as the carbon source. The bacteria were cultured at 30 ◦ C and 150
rpm for 1 week and then transferred to a fresh flurbiprofen-MSM medium at a 10% inoculation ratio for subculturing for
another week. The process repeated five times. Then, 0.5 mL of enrichment solution was collected for a serial dilution
(10−2 –10−8 ), and 0.1 mL of each dilution was spread on a 400 mg/L flurbiprofen MSM agar plate and incubated at
30 ◦ C. Single colony streaking and culture purification were performed. A single colony after four rounds of streaking
and purification was inoculated on a 400 mg/L flurbiprofen MSM plate, and cultured at 30 ◦ C for further tests.

2.3. Strain identification

Using the bacterial genome as the DNA template, 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R
(5’-GGTTACCTTGTTACGACTT-3’) were used as the upstream and downstream primers for 16S rDNA amplification. PCR
reaction conditions were pre-denaturation at 94 ◦ C for 4 min, denaturation at 94 ◦ C for 25 s, annealing at 72 ◦ C for 30 s,
and extension at 72 ◦ C for 30 s (25 cycles). Last, the final extension was carried out at 72 ◦ C for 5 min. After amplification,
1% agarose gel electrophoresis was used to check the integrity of the PCR products. These were then sent for sequencing to
Sangon Biotech Co., Ltd., Shanghai, China. Blast was used to compare the 16S rRNA gene sequence obtained via sequencing
with the existing 16S rRNA gene sequences in the GenBank database. The MEGA 5.0 software was used to construct a
phylogenetic tree. The 16S rDNA partial sequence of Marinobacterium from the GenBank database was used as outgroup,
and the neighbor-joining method was subsequently used for analysis.

2.4. Determination of the degradation effect of the strain

The fresh bacterial culture was inoculated into the liquid MSM containing a final flurbiprofen concentration of 400
mg/L, according to the inoculation rate of 1% (v/v). The bacterial culture was sampled regularly to measure the OD600 and
the strain growth curve was obtained. The culture of the strain was carried out in an conical flask, specifically, with 1%
(v/v) fresh bacterial culture being inoculated in 100 mL MSM with a final flurbiprofen concentration of 400 mg/L. Then,
the conical flask was placed at 30 ◦ C and 150 rpm, and the bacterial culture solution was sampled every 12 h to determine
the flurbiprofen content by gas chromatography–mass spectrometry (GC–MS) analysis (Yilmaz et al., 2014). Specifically,
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T. Liu, S. Yang, Z. Wu et al. Environmental Technology & Innovation 29 (2023) 102983

1 mL solution was collected and filtered using a 0.22 µm membrane filter, and 0.5 mL of the filtered solution was taken
along with 0.5 mL concentrated phosphoric acid in a test tube, and then it was vortexed for 5 s. Next, 5 mL of the acetic
acid-Ethyl ester-n-hexane (2+3, v/v) solution was added, followed by vortexing for 30 s, and then centrifuged at 3000 g
for 10 min. The organic layer sample was dried with NaCl. After drying, 0.5 mL of the dried sample was further dried
at room temperature by blowing nitrogen. The residue was derivatized with 100 µL of acetonitrile-MSTFA (1+1, v/v).
After derivatization, the sample was transferred to a vial for GC–MS analysis (Agilent 8890-5977B, Agilent Technologies,
CA, USA). The column was an Agilent Technologies HP-5MS UI chromatographic column, with the film thickness being
0.25 µm (30 m×0.25 mm id). Splitless injection was used, the carrier gas was helium, and the flow rate was 1 mL/min.
The temperature of the injector and detector was 250 ◦ C, and the mass spectrometer parameters were: the transmission
line temperature 280 ◦ C, quadrupole, source settings 150 ◦ C and 230 ◦ C, and electron energy 70 eV. The initial temperature
was 150 ◦ C, and the temperature was kept at 150 ◦ C for 1 min. Then, the temperature was increased to 300 ◦ C at a rate
of 10 ◦ C/min, and then the temperature was kept at 300 ◦ C for 1 min. Considering that in the preliminary experimental
finding flurbiprofen was found to be extremely difficult to degrade, and the natural degradation rate within 30 d was
≤5%, we did not include a blank control in this study. The degradation rate of flurbiprofen by the strain was calculated
based on the initial flurbiprofen concentration, and the calculating equation is:

Degradation rate(%) = (C0 − Ct )/C0 ×100% (1)

where, C0 is the initial flurbiprofen concentration, Ct is the concentration at time t. After pretreatment of the culture
solution, GC–MS in full scan mode was used to determine the structure of the degradation products.

2.5. Degradation characteristics of the strain

The bacterial solution cultured to the end of the logarithmic growth phase was inoculated into 100 mL MSM according
to a 1% (v/v) inoculation rate, with flurbiprofen being added to each culture system until a final concentration of 400 mg/L.
At different temperatures (i.e., 20 ◦ C, 25 ◦ C, 30 ◦ C, 35 ◦ C, and 40 ◦ C), the system was cultured at initial pH = 7 and 150
rpm. The MSM without bacterial inoculation was used as the control. Three replicates were used in the experimental
and control groups. During the culture process, the solution was sampled every 24 h to measure the OD600 and OD410
(note: the color of the culture changed during the cultivation process from colorless to yellow, with full-band scanning
showing that it had a maximum absorption peak at 410 nm). Moreover, samples were also collected every 72 h to test
the flurbiprofen concentration change using GC–MS.
The bacterial solution cultured to the end of the logarithmic growth phase was inoculated into 100 mL MSM according
to a 1% (v/v) inoculation rate, and flurbiprofen was added to each culture system at a final concentration of 400 mg/L. At
different pH values (5, 6, 7, 8, and 9), the system was cultured at 30 ◦ C and 150 rpm. The MSM without bacterial inoculation
was used as the control. Three replicates were set for the experimental and control groups. During the culture process,
the solution was sampled every 24 h to measure the OD600 and OD410 . Moreover, samples were also collected every 72 h
to test the flurbiprofen concentration change using GC–MS.
The bacterial solution cultured to the end of the logarithmic growth phase, was inoculated into 100 mL MSM according
to a 1% (v/v) inoculation rate, with flurbiprofen being added to each culture system to obtain different final concentrations
(100, 200, 300, and 400 mg/L). The system was cultured at 30 ◦ C, initial pH = 7, and 150 rpm. The MSM without bacterial
inoculation was used as the control. Three replicates were set for the experimental and control groups. During the culture
process, the solution was sampled every 24 h to measure the OD600 and OD410 . Moreover, samples were also collected
every 72 h to test the flurbiprofen concentration change using GC–MS.
The bacterial solution cultured to the end of the logarithmic growth phase, was inoculated into 100 mL MSM according
to a 1% (v/v) inoculation rate, with different carbon sources being added to the culture systems, including 400 mg/L
flurbiprofen, 400mg/L glycerol, and 400mg/L flurbiprofen plus 400 mg/L glycerol. The system was cultured at 30 ◦ C, initial
pH = 7, and 150 rpm. The MSM without bacterial inoculation was used as the control. Three replicates were set for the
experimental and control groups. During the culture process, the solution was sampled every 24 h to measure the OD600
and OD410 . Moreover, samples were also collected every 72 h to test the flurbiprofen concentration change using GC–MS.
The bacterial solution cultured until the end of the logarithmic growth phase was centrifuged. After the supernatant
was removed, the bacteria were washed with sterile water, and then inoculated into 100 mL MSM. Glycerol was then
added to the culture system at a final concentration of 400 mg/L. The system was cultured for one generation at 30 ◦ C,
initial pH = 7, and 150 rpm. The cultured strains were washed with sterile water and then resuspended in sterile water.
The OD600 was adjusted to 1. The resuspended bacterial cell suspension was inoculated into the MSM according to the
1% (v/v) inoculation rate, and flurbiprofen was sulpplemented at the concentration of 400 mg/L. Additionally, some more
bacterial solution cultured to the end of the logarithmic growth phase was washed and resuspended in the same way, and
then inoculated into the MSM with a final flurbiprofen concentration of 400 mg/L according to the 1% (v/v) inoculation
rate as the control. Three replicates were set for the experimental and control groups. During the culture process, samples
were collected at 2, 6, 12, and 24 h to measure the OD410 .
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Fig. 1. The phylogenetic tree of strain LY.1 derived from neighbor-joining analysis of partial 16S rDNA sequences. Numbers at the nodes refer to
bootstrap approval ratings. The information following the species name in brackets is the GenBank ID of the species. The Marinobacterium from the
GenBank database was chosen as outgroup.

2.6. Testing the application potential of flurbiprofen degrading strains

The bacterial solution cultured to the end of the logarithmic growth period was inoculated into the water sample of
the Xunsi River and MSM at a ratio of 1% (v/v), and flurbiprofen was added to the culture system to a final concentration
of 400 mg/L respectively. The system was incubated at 30 ◦ C and 150 rpm. The water samples of the Xunsi River without
bacterial inoculation was used as the control. Three replicates were set for the experimental and control groups. During
the culture process, the solution was sampled every 24 h to measure the OD600 and OD410 . Moreover, samples were also
collected every 24 h to test the flurbiprofen concentration change using GC–MS.

3. Results and discussion

3.1. Isolation and identification of the strain

After isolation and purification, we obtained a strain that could grow with flurbiprofen as the sole carbon source,
with the strain being named as LY.1. Culturing LY.1 on a beef extract peptone agar plate for 24 h, a round, moist, and
slightly sticky yellowish-green colony with a smooth and prominent surface was formed. The BLASTn analysis results
showed that the LY.1 strain (GenBank accession No. OM865894) shared 100% similarity with multiple Pseudomonas
aeruginosa strains. Using the MEGA 5.0 software, we built a phylogenetic tree via the Neighbor-Joining method (Fig. 1);
we finally named the strain Pseudomonas aeruginosa LY.1. Yanac and Murdoch (2019) obtained an enriched culture from
activated sludge in a municipal sewage treatment plant that could degrade flurbiprofen, which was the first report on the
biodegradation of flurbiprofen. The Pseudomonas aeruginosa LY.1 isolated in this study is the first pure culture with the
ability to degrade flurbiprofen. Pseudomonas aeruginosa has strong degradation ability for a variety of toxic and harmful
organic pollutants, like polychlorinated biphenyls (Cai et al., 2016), aflatoxin (Dong et al., 2021), phenanthrene (Wang
et al., 2015), naphthalene (Wang et al., 2015; Liu et al., 2015), fluoranthene (Lu et al., 2015), and pyrene (Li et al., 2018).
Thus, to better understand the potential applicability in environmental pollution remediation, further tests may be needed
to discover the category of pollutants could be degraded by the LY.1.

3.2. Growth curve and degradation ability of LY.1

During the culture process, the color of the culture changed from colorless to yellow. We performed a full-band
scanning on the yellow culture and found that it had a maximum absorption peak at 410 nm. Then we acidified the yellow
culture and found that the yellow color gradually became lighter with the decreasing pH until it turned colorless. The
yellow color again gradually recovered with increasing pH. This pH-dependent yellow product has a maximum absorbance
of 375–425 nm, and is a typical product of an aromatic ring-opening reaction (Li et al., 2015; Mccullar et al., 1994).
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Fig. 2. Growth curve and degradation ability of LY.1. Fig(a) is the growth of LY.1 on flurbiprofen as measured by increase in LY.1 culture density
via OD600 (White dot), and concomitant yellow color accumulation is measured via OD410 (Black square). Fig(b) is the flurbiprofen concentration
changes during the incubation (Black square), and the flurbiprofen removal ratio is also presented in the figure (White dot).

Table 1
The growth and pollutants-degrading ability of different P. aeruginosa species.
Strains Substrates Degrading Degrading Optimal Optimal
ratio (%) time temperature pH
(◦ C)
P. aeruginosa 10.3 mg/L 40 48 h 28 –
(Gusseme et al., 2011) acetaminophen

P. aeruginosa HJ1012 2200 mg/L 100 75 h 30 7.0

(Hu et al., 2013) paracetamol

P. aeruginosa BAC-6 500 mg/L 46.03 48 h 30 7.0

(Chandrashekar et al., acrylamide
2014)

P. aeruginosa DN1 500 mg/L 90.2 14 d 34–37 5.5–7.5

(Lu et al., 2015) phenanthrene

P. aeruginosa KDQ4 400 mg/L 95.65 24 h 30–37 8–9

(Zhang et al., 2016) quinoline

P. aeruginosa JXJ 1000 mg/L 49.6 7 d 35 7.5

(Cai et al., 2016) PCB77

P. aeruginosa FN 10 mg/L >50 6 h 25 7.0

(Kučić Grgić et al., 2019) diuron

P. aeruginosa M4 10% aflatoxin B1 52.27 56.81 h 36.03 6.79

(Dong et al., 2021)

P. aeruginosa MAPB-2 50 mg/L 27.19 7 d 30 7.0

(Sandhu et al., 2022) polychlorinated
biphenyl

P. aeruginosa LY.1 400 mg/L 100 96 h 36.5 7.6

(this study) flurbiprofen

Its characteristic absorbance OD410 can roughly reflect the degree of flurbiprofen degradation. OD410 is related to the
accumulation of products after ring opening, and when OD410 decreases, it means flurbiprofen is completely degraded.
As shown in Fig. 2a, after culturing in MSM with a final flurbiprofen concentration of 400 mg/L for 30 h, the LY.1 strain
entered the logarithmic growth phase; with the entry into the stationary phase being at 90 h. The growth and pollutants-
degrading ability of different Pseudomonas aeruginosa species are quite different. As shown in Table 1, part of Pseudomonas
aeruginosa strains containing organic pollutant degrading ability are listed. According to the list, Pseudomonas aeruginosa
strains acquired a much lower growth rate and degrading efficiency in persistent organic pollutants than in other organic
pollutants. For example, in the medium with 1 g/L biphenyl or 1 mg/L PCB77 as the sole carbon source, Pseudomonas
aeruginosa JXJ entered the logarithmic growth phase at 85 h (Cai et al., 2016); and in the medium containing 500 mg/L
fluoranthene as the sole carbon source, Pseudomonas aeruginosa DN1 entered the logarithmic growth phase, stationary
phase, and decay phase at days six, eight, and eleven, respectively (Lu et al., 2015). Compared with the above strains,
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Fig. 3. Flurbiprofen metabolism pathway by LY.1. Fig (a) is the spectra of acetonitrile-MSTFA derivatized flurbiprofen identified via GC/MS. Fig(b)
is the putative flurbiprofen metabolite identified via GC/MS, and is suggested to be 3-fluoro-4-(1-ethylcarboxy) phenylpropionic acid. Fig(c) is the
proposed flurbiprofen metabolism pathway by LY.1, and Fig(d) the structure comparison between the flurbiprofen metabolite end product and the
3-fluorocatechol.

the growth rate and degrading efficiency of strain LY.1 are much higher, and which is very close to that of other PPCPs
degrading species in the list.
As shown in Fig. 2a, OD410 peaked at 90 h and decreased to ∼30% of the peak value at 168 h. After 168 h, the pH of
the reaction system was 5.79. The pH of the reaction system was adjusted back to 7, and no rebound of OD410 occurred.
There is a positive correlation between the degrading strain’s growth and its flurbiprofen-degrading ability (Fig. 2b). The
flurbiprofen residue decreased rapidly as the strain entered the logarithmic growth phase, with the degradation rate
increasing significantly (Fig. 2b). After 78 h, the degradation rate of 400 mg/L flurbiprofen was 90%, and it finally reached
100% at 96 h. Yanac and Murdoch (2019) obtained an enriched culture that could degrade flurbiprofen from municipal
sludge at final concentrations of 50 mg/L and 500 mg/L. After monitoring for 80 days, they found that degradation occurred
in the first 34 days, with (32.0 ± 6.3)% and (47.8 ± 2.2)% of the flurbiprofen remaining after 80 days, respectively. The
degradation efficiency of LY.1 is much higher than that of the enriched culture. Considering the above degradation rate of
flurbiprofen, we collected the samples every 72 h in the subsequent test. The culture conditions were 30 ◦ C, initial pH = 7,
and initial flurbiprofen concentration 400 mg/L. We carried out subsequent experiments by changing the environmental
conditions.

3.3. Degradation products of flurbiprofen

We carried out GC–MS analysis on the culture solution of the strain LY.1 and obtained the mass spectrum of flurbipro-
fen and its end product post derivatization (Fig. 3a, b). The results showed that the degradation product was 3-fluoro-4-(1-
ethylcarboxy) phenylpropionic acid, with its mass spectrum, structure, and pathway were shown in Fig. 3b, c, respectively.
Interestingly, the degradation product obtained by Yanac and Murdoch (2019) was also 3-fluoro-4-(1-ethylcarboxy)
phenylpropanoic acid. During the 80-day monitoring process, 3-fluoro -4-(1-ethylcarboxy) phenylpropionic acid was not
degraded, thereby indicating that the biodegradation or conversion of 3-fluoro-4-(1-ethylcarboxy) phenylpropionic acid
is difficult.
Currently, numerous studies exist on the biodegradation of 2-fluorobenzoate and 4-fluorobenzoate. However, few
have reported the biodegradation of 3-fluorobenzoate, possibly due to the generation of toxic intermediates during
its degradation (Engesser et al., 1990). 3-fluorocatechol is a specific cyclodioxygenase inhibitor, which is often used to
inhibit aromatic ring-opening reactions (Engesser et al., 1990, 1988; Dorn and Knackmuss, 1978; Franco et al., 2014;
Schreiber et al., 1980). Through the known degradation pathway of aerobic substituted phenylacetic acid (ipf), the acetic
acid group in 4-(1-carboxyethyl)2-fluorobenzoic acid was removed by 1,2 bis-oxygenation, and we obtained 3-Fluoro-
4-carboxycatechol that has a similar structure to 3-fluorocatechol (Fig. 3d) (Murdoch and Hay, 2005). This compound
may inhibit the meta-ring-opening dioxygenase. Therefore, a dioxygenase-inhibiting intermediate product may have been
produced during the flurbiprofen degradation, which can explain the accumulation of 4-(1-carboxyethyl)2-fluorobenzoic
acid. Therefore, the result suggests that the ecotoxicological effects of flurbiprofen need greater attention.
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Fig. 4. Effect of pH value on the growth and degradation ability of LY.1. Fig(a) is the OD600 represented growth of LY.1 on flurbiprofen under different
pH conditions, and concomitant yellow color accumulation is measured via OD410 (Fig(b)). Fig(c) is the final flurbiprofen concentrations after a 72 h
incubation under different pH conditions (Gray columns), and the flurbiprofen removal ratio is also presented in the figure (White dot). Fig(d) is a
fitted curve of the maximum specific growth rate to the pH value.

3.4. Effect of pH on the growth and degradation ability of the strain

At pH 6–8, the strain LY.1 grew normally (Fig. 4a) and demonstrated flurbiprofen degradation ability (Fig. 4b, c). At pH
= 7, the strain had the highest degradation efficiency (Fig. 4c) (86% degradation of 400 mg/L flurbiprofen within 72 h).
We fitted a curve to the data according to the maximum specific growth rate of each group, with the optimal pH for LY.1
being ∼7.66 (Fig. 4d). The value was close to those of many Pseudomonas aeruginosa strains that could degrade organic
pollutants. For example, the optimum pH of the aflatoxin B1 degrading bacteria Pseudomoas aeruginosa M4 was 6.79 (Dong
et al., 2021), whereas that of the PCB degrading bacteria Pseudomoas aeruginosa JXJ was 7.5 (Cai et al., 2016).

3.5. Effect of temperature on the growth and degradation ability of the LY.1 strain

Although LY.1 grew normally between 25–40 ◦ C (Fig. 5a), there was a long lag period at 25 ◦ C. Moreover, LY.1 showed
extremely strong degradation ability at 30–40 ◦ C (Fig. 5b, c). The highest degradation rate was at 35 ◦ C, where 98% of the
400 mg/L flurbiprofen was degraded within 72 h. We fitted a curve to the data according to the maximum specific growth
rate of each group, with the optimum growth temperature of LY.1 being ∼36.54 ◦ C (Fig. 5d). The value was close to that
of many Pseudomonas aeruginosa strains that could degrade organic pollutants. For instance, the optimum temperature
of fluoranthene-degrading Pseudomoas aeruginosa DN1 was 34–37 ◦ C, and its growth was slow when < 28 ◦ C (Lu et al.,
2015). The optimum temperature of a Pseudomonas aeruginosa strain that could degrade naphthalene and phenanthrene
was 37 ◦ C (Wang et al., 2015). The optimum temperature of the aflatoxin B1 degrading Pseudomoas aeruginosa M4 was
36.03 ◦ C (Dong et al., 2021). It is important to note that it is difficult to maintain the ambient temperature at ∼37 ◦ C,
whether be it natural water bodies or sewage treatment processes. So, if we directly added the LY.1 strain, the expected
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Fig. 5. Effect of temperature on the growth and degradation ability of LY.1. Fig(a) is the OD600 represented growth of LY.1 on flurbiprofen
under different temperature conditions, and concomitant yellow color accumulation is measured via OD410 (Fig(b)). Fig(c) is the final flurbiprofen
concentrations after a 72 h incubation under different temperature conditions (Gray columns), and the flurbiprofen removal ratio is also presented
in the figure (White dot). Fig(d) is a fitted curve of the maximum specific growth rate to the temperature value.

treatment effect may not be achieved. Thus, the strain needs to be acclimated to low temperature for enhancing its
temperature-adaptability, thereby improving its degradation performance in various applications.

3.6. Effect of substrate concentration on the growth and degradation ability of the strain

We can see from Fig. 6a that the LY.1 strain can tolerate high concentrations of flurbiprofen and grow normally at
100–800 mg/L. During the stable phase, there was a significant positive correlation between the biomass and the carbon
source concentration. Thus, the strain enters the stable phase due to the exhaustion of the available carbon source, rather
than the reaction products. When flurbiprofen was the only carbon source, the OD600 of the strain was maintained at
a low level, which may be related to the low flurbiprofen utilization rate of the strain. According to the structure of
the end product of flurbiprofen degradation, the strain could only degrade the non-fluorinated ring of flurbiprofen, and
therefore, cannot fully utilize flurbiprofen. From Fig. 6b and 6c, the 72 h flurbiprofen degradation rate of the strain was
higher than 80% when the concentration was 100–400 mg/L. Furthermore, it still showed degradation ability, even at
a concentration of 800 mg/L. However, it was worth noting that the higher the flurbiprofen concentration; the longer
was the time required for degradation. Compared to the degradation rate obtained by Yanac and Murdoch (2019) (∼50%
degradation of 500 mg/L flurbiprofen in ∼34 days), the LY.1 strain had both a higher substrate tolerance and a higher
degradation rate.
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Fig. 6. Effect of substrate concentration on the growth and degradation ability of LY.1. Fig(a) is the OD600 represented growth of LY.1 on different
concentration of flurbiprofen, and concomitant yellow color accumulation is measured via OD410 (Fig(b)). Fig(c) is the final flurbiprofen concentrations
after a 72 h incubation (Gray columns), and the flurbiprofen removal ratio is also presented in the figure (White dot).

3.7. Effect of mixed carbon sources on the growth and degradation ability of the strain

We can see from Fig. 7a that the LY.1 strain grew rapidly in both the glycerol and the flurbiprofen plus glycerol groups,
with no significant difference in the 72 h biomass (p > 0.05). However, the strain did not degrade flurbiprofen in the
glycerol plus flurbiprofen group (Fig. 7b, c). Thus, the LY.1 strain preferentially utilized glycerol as a carbon source and no
longer degraded flurbiprofen. This may be related to the inhibition of the carbon metabolism of microorganisms, which has
been widely studied in Pseudomonas. The inhibition of carbon metabolism of Pseudomonas aeruginosa is mainly regulated
by CbrAB/Crc (Sonnleitner et al., 2009), with the Crc protein inhibiting the expression of the metabolic genes of inferior
carbon sources, thereby inhibiting the utilization of inferior carbon sources (Moreno and Rojo, 2008), i.e., the metabolic
system of LY.1, considers flurbiprofen an inferior carbon source as compared to glycerol. When flurbiprofen coexists with
other dominant carbon sources, the strain cannot degrade it. Since this can fairly limit the application of the strain, further
studies are needed to solve this.

3.8. Degradation inducibility of the strain

From Fig. 7d, we can see that at 2–12 h, OD410 of the control group (flurbiprofen pre-cultured) was significantly higher
than that of the experimental group (glycerol pre-cultured) (p < 0.05), thus indicating that the flurbiprofen degradation in
experimental group was inhibitated or postponed. At 24 h, OD410 of the experimental group was significantly higher than
that of the control group, thereby indicating that it had begun degrading flurbiprofen. The degradation starting time and
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T. Liu, S. Yang, Z. Wu et al. Environmental Technology & Innovation 29 (2023) 102983

Fig. 7. Effect of carbon sources on the growth and degradation ability of LY.1. Fig(a) is the OD600 represented growth of LY.1 on different carbon
sources, and concomitant yellow color accumulation is measured via OD410 (Fig(b)). Fig(c) is the final flurbiprofen concentrations after a 72 h
incubation on different carbon sources. Fig(d) is the yellow color accumulation measured via OD410 , white square represents a furbiprofen pre-cultured
incubation, white triangle represents a glycerol pre-cultured incubation, and black dot represents a aseptic incubation.

degradation efficiency of the control group were better than those of the experimental group, thereby indicating that the
degradation may be related to the induction of flurbiprofen in the culture system, which can be caused by the key enzyme
in the LY.1. The enzymes in microbial cells can be divided into inducible and constitutive enzymes (Xie et al., 2020), with
the difference lying in the synthesis method and the time of existence (Zhang et al., 2011). The synthesis of constitutive
enzymes is not affected by the external environment, rather controlled only by internal factors, i.e., related genes. For
inducible enzymes, besides related genes, the synthesis is also related to inducers in the environment, i.e., microorganisms
can only produce the enzyme under the action of specific inducers (Zhu et al., 2008; Niu et al., 2011). The key enzyme in
the LY.1-mediated degradation of flurbiprofen is an inducible enzyme whose gene expression is induced by the substrate,
flurbiprofen. The bacterial degrading enzymes are mostly inducible. For instance, for the chlorobenzene-degrading bacteria
Ralstonia pickettii L2, chlorobenzene is the key inducer of 1,2-catechol dioxygenase (Leng et al., 2011). Moreover, as some
degrading enzymes are constitutive enzymes, even though the atrazine-degrading bacteria Shinella granuli BZB-1 was
cultured via continuous transfer in the medium without atrazine for eight generations with no substrate induction, it
showed no significant difference in its atrazine-degradation ability as compared with the atrazine-induced strain (Ying
et al., 2007). Therefore, as compared with the induced enzymes, the constitutive enzymes can be genetically expressed
more stably and do not need to be modified using genetic engineering, and thus have greater future application potential.

3.9. Testing the application potential of flurbiprofen degrading strains

Strain LY.1 grew and degraded flurbiprofen in both MSM and the water sample of the Xunsi River, and entered the
stationary phase earlier in the water sample of the Xunsi River. The OD410 peaked at about 72 h in the water sample
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T. Liu, S. Yang, Z. Wu et al. Environmental Technology & Innovation 29 (2023) 102983

Fig. 8. The application potential test of LY.1. Fig(a) is the OD600 represented growth of microorganisms in river water samples and MSM medium
amended with flurbiprofen and LY.1, and concomitant yellow color accumulation is measured via OD410 . Fig(b) is the flurbiprofen concentration
changes during the incubation (Black square and triangle), and the flurbiprofen removal ratio is also presented in the figure (White square and
triangle).

of the Xunsi River with strain LY.1, which was earlier than that in the MSM, but the peak was lower than that in the
MSM(Fig. 8a). The 72 h degradation rate of flurbiprofen was 94% in the water sample of the Xunsi River, which was 12%
higher than that in the MSM(Fig. 8b). For the isolation and screening of strain LY.1 and later related experiments, MSM
was used, which only ensured the minimum requirements for the growth of the strain, while the nutrients in the water
sample of the Xunsi River were more abundant, which might improve the growth and degradation efficiency of the strain.
The pH of the culture system affects the degradation ability of the strain LY.1. The initial pH of the water sample of the
Xunsi River and MSM were 8.64 and 8.50, and after 72 h, it changed to 6.08 and 6.64. The characteristic absorbance OD410
of the cultures was pH dependent, and the value of OD410 decreased with the decrease of pH. This can explain to some
extent the lower peak OD410 in the water sample of the Xunsi River than in the MSM.

4. Conclusion

In this study, the first pure bacterial strain capable of biodegradation of flurbiprofen was isolated and named as
Pseudomonas aeruginosa LY.1. This bacterial strain could degrade flurbiprofen to the stage of 3-fluoro-4-(1-ethylcarboxy)
phenylpropionic acid. The bacterial strain LY.1 could degrade flurbiprofen at pH 6–8 and temperature 25-40 ◦ C, and
the optimal conditions are when pH = 7.66 and the temperature is at 36.54 ◦ C. The strain was able to tolerate high
concentrations of flurbiprofen (100–800 mg/L) and complete degradation 400 mg/L of flurbiprofen within 96 h at 30 ◦ C
and pH = 7. The degradation enzyme system of the bacterial strain to flurbiprofen fits the category of inducible expression.
There would be no further degradation of flurbiprofen in the presence of glycerol.

CRediT authorship contribution statement

Tian Liu: Designed research, Finished the data analysis, Wrote the manuscript, Revised the manuscript. Shilin Yang:
Designed research. Zixian Wu: Designed research. Yufei Cai: Contributed to experimental work. Jiawei Zhou: Contributed
to experimental work. Mingjun Liao: Revised the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

Acknowledgments

We thank Prof. Anthony Hay for his advises in experiment design. The authors thank EditSprings for language
assistance. This study was supported by the National Natural Science Foundation of China (51579092, 31370148), National
Key Research and Development Program (2016YFC0401702).
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