Ecotoxicology and Environmental Safety 167 (2019) 505–512

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Ecotoxicology and Environmental Safety

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

Naproxen ecotoxicity and biodegradation by Bacillus thuringiensis B1(2015b) T

strain
Dorota Górny, Urszula Guzik, Katarzyna Hupert-Kocurek, Danuta Wojcieszyńska
⁎

Department of Biochemistry, Faculty of Biology and Environmental Protection, University of Silesia in Katowice, Jagiellońska 28, 40-032 Katowice, Poland

ARTICLE INFO ABSTRACT

Keywords: High level of naproxen consumption leads to the appearance of this drug in the environment but its possible
Naproxen effects on non-target organisms together with its biodegradation are not well studied.
Biodegradation The aim of this work was to evaluate naproxen ecotoxicity by using the Microbial Assay for Risk Assessment.
Toxicity Moreover, Bacillus thuringiensis B1(2015b) was tested for both ecotoxicity and the ability of this strain to degrade
Bacteria
naproxen in cometabolic conditions. The results indicate that the mean value of microbial toxic concentration
estimated by MARA test amounts to 1.66 g/L whereas EC50 of naproxen for B1(2015b) strain was 4.69 g/L. At
toxic concentration, Bacillus thuringiensis B1(2015b) showed 16:0 iso 3OH fatty acid presence and an increase in
the ratio of total saturated to unsaturated fatty acids. High resistance of the examined strain to naproxen cor-
related with its ability to degrade this drug in cometabolic conditions.
The results of bacterial reverse mutation assay (Ames test) revealed that naproxen at concentrations above
1 g/L showed genotoxic effect but the response was not dose-dependent.
Maximal specific naproxen removal rate was observed at pH 6.5 and 30 °C, and in the presence of 0.5 g/L
glucose as a growth substrate. Kinetic analysis allowed estimation of the half saturation constant (Ks) and the
maximum specific naproxen removal rate (qmax) as 6.86 mg/L and 1.26 mg/L day, respectively. These results
indicate that Bacillus thuringiensis B1(2015b) has a high ability to degrade naproxen and is a potential tool for
bioremediation.

1. Introduction
Naproxen is one of the most popular non-steroidal anti-in-
flammatory drugs (NSAID) with antipyretic and analgesic properties. It
shows wide working spectrum in mild to moderate pain treatment.
Naproxen inhibits two types of cyclooxygenases (I and II) influencing
on prostaglandins and thromboxanes level. High consumption of this
drug leads to its appearance in the environment. Naproxen enters
aquatic environments mainly through the effluents of wastewater
treatment plants. It is classified as a high priority pharmaceutical
(Ginebreda et al., 2012; Grenni et al., 2013, 2018). Because of its bio-
logical activity naproxen may influence living organisms and reduce
biodiversity of the natural environmental communities. Brozinski et al.
(2013) observed seasonal variation of naproxen concentration in the
water of Haapajarvi Lake, Finland ranging from 40 ng/L (November) to
210 ng/L (February). At the same time, concentration of naproxen de-
tected in the bile of bream and roach caught from this lake was almost
1000-fold higher and ranged from 6 to 32 ng/mL and from 11 to
103 ng/mL, respectively. It is known that naproxen may affect mRNA
expression and has negative influence on gastrointestinal tract and
kidneys of zebrafish (Ding et al., 2017). According to Li et al. (2016),
Lethal Dose 50% (LC50) values of naproxen for embryos and larvae of
zebrafish were 115.2 mg/L and 147.6 mg/L, respectively. Because na-
proxen causes pericardial edema and histopathological liver damage it
may be considered as a potential threat to aquatic organisms (Li et al.,
2016). It is also suggested that naproxen may induce genotoxicity
(Ahmad et al., 2018).
The main method of naproxen elimination from the environment is
its phototransformation. Additionally, for purification of wastewaters
loaded with naproxen, the advanced oxidation processes are used.
However, during these reactions 1-(6-methoxy-2-napthyl)ethanol, 1-(6-
methoxynaphthalen-2-yl)ethanone, 2-ethyl-6-methoxynaphthalene, 1-
(6-methoxynaphthalen-2-yl)ethylhydroxyperoxide can be formed
(Jallouli et al., 2016). It was noted that both acute and chronic median
effective concentration (EC50) or LC50 for these compounds were up to
17-fold lower than the corresponding EC50 or LC50 values for the

⁎
Corresponding author.
E-mail addresses: ddomaradzka@us.edu.pl (D. Górny), urszula.guzik@us.edu.pl (U. Guzik), katarzyna.hupert-kocurek@us.edu.pl (K. Hupert-Kocurek),
danuta.wojcieszynska@us.edu.pl (D. Wojcieszyńska).

https://doi.org/10.1016/j.ecoenv.2018.10.067
Received 30 January 2018; Received in revised form 15 October 2018; Accepted 17 October 2018
0147-6513/ © 2018 Elsevier Inc. All rights reserved.
D. Górny et al.
naproxen. Therefore, it should be emphasized that this method of na-
proxen removal, although effective, may lead to its increased toxicity in
the environment (Straub and Stewart, 2007). For that reason it seems to
be very important to develop a new method of naproxen utilization.
The answer to this problem may be the use of biological systems with
microorganisms being able to degrade non-steroidal anti-inflammatory
drugs. However, until now only few bacterial and fungal strains char-
acterized with this ability have been described. These include Bjer-
kandera sp. R1, Bjerkandera adusta, Phanerochaete chrysosporium, Tra-
metes versicolor, Cunninghamella bakeslesna AS3.153, Cunninghamella
echinulate AS3.2004, Cunninghamella elegans AS3.156 from fungi and
only Stenotrophomonas maltophilia KB2, Bacillus thuringiensis B1(2015b)
and Planococcus sp. S5 from bacteria (Zhong et al., 2003; Marco-Urrea
et al., 2010; Rodarte-Morales et al., 2011; Wojcieszyńska et al., 2014;
Domaradzka et al., 2015; Li et al., 2015; Marchlewicz et al., 2016).
Because there is no information on naproxen toxicity to micro-
organisms, the aim of this work was to examine the influence of this
drug on Bacillus thuringiensis B1(2015b) and the model microorganisms.
Since changes in the cellular fatty acids composition have been reported
for bacteria exposed to various xenobiotics (Nowak et al., 2016; Zdarta
et al., 2017), influence of naproxen on the fatty acids profile of Bacillus
thuringiensis B1(2015b) was also examined.
Biological processes depend on the enzyme stability. Therefore, it is
important to determine the optimal conditions for naproxen degrada-
tion such as pH, temperature or additional carbon source. Due to the
mentioned above, the aim of our work was to evaluate the ability of
Bacillus thuringiensis B1(2015b) to degrade naproxen in cometabolic
conditions. We also studied the impact of the environmental factors on
naproxen removal rate and estimated the kinetic parameters of de-
gradation process.
2. Materials and methods
2.1. Bacterial strains and growth conditions
Bacillus thuringiensis B1(2015b) (Marchlewicz et al., 2016) was
routinely cultivated in the nutrient broth (BBL® Nutrient broth, Becton
Dickinson, USA) at 30 °C and 130 ×g for 24 h. After this, cells were
harvested by centrifugation (5000 ×g at 4 °C for 15 min), washed with
a fresh sterile mineral salts medium (Greń et al., 2010) and used to start
a new culture.
Salmonella typhimurium TA98 and Salmonella typhimurium TA100
were used as model organisms in bacterial reverse mutation assay
(Ames test). These strains were cultivated in Growth Medium with
ampicillin (Xenometrix) according to manufacturer's instruction.
In MARA (Microbial Assay for Risk Assessment) test, lyophilized
model microorganisms: Microbacterium sp., Brevundimonas diminuta,
Citrobacter freudii, Comamonas testosteroni, Enterococcus casseliflavus,
Delftia acidovorans, Kurthia gibsonii, Staphylococcus warneri,
Pseudomonas aurantiaca, Serratia rubidaea, Pichia anomala were used.
These microorganisms were cultivated on 96-well plates in the presence
of gradient concentration of naproxen at 30 °C during 18 h.
2.2. Ecotoxicity bioassays
To assess ecotoxicity of naproxen, the MARA test was used. This test
was conducted in 96-well microtitre plates according to Marchlewicz
et al. (2017a). The microbial growth after exposure to the concentration
gradient of naproxen (0–5.5 g/L) was determined by a reduction of
tetrazolium salt. The MTC (Microbial Toxic Concentration) value was
calculated according to the following formula:
Ptot
1 one-way ANOVA. The statistical significance (p < 0.05) of differences
MTC = cmin × d Po
where: cmin - the lowest concentration in the gradient; Po -pellet size in
the control; d - dilution factor; Ptot - sum of all the pellets sizes across
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Ecotoxicology and Environmental Safety 167 (2019) 505–512
the concentration gradient (Marchlewicz et al., 2017a).
Genotoxicity of naproxen was evaluated with Ames MPF test
(Xenometrix). It was performed with Salmonella typhimurium TA98 and
Salmonella typhimurium TA100 according to the manufacturer's re-
commendations. 10 mL of the growth medium was inoculated with
10 µL of refrozen bacterial strains and incubated for 16 h at 37 °C with
shaking at 250 ×g in the presence of 50 µg/mL ampicillin until culture
density reached > 2.0 (OD600). Next, the cultures were diluted 10-fold
in the exposure medium and 240 µL of the mixture was introduced into
every well of the 24-well plate. Simultaneously, an appropriate volume
of 4 g/L naproxen stock solution was introduced into the wells to obtain
the final concentrations of 0.125, 0.25, 0.5, 1.0, 2.0, and 4.0 g/L. Ames
test was performed according to Domaradzka et al. (2016).
2.3. Bacterial growth inhibition test
To determine the inhibitory effect of naproxen on bacterial growth,
a pure culture of Bacillus thuringiensis B1(2015b) was grown in the
nutrient broth supplemented with naproxen in the concentration range
0–5.6 g/L. The initial optical density of each culture was 0.05 (OD600).
After 24 h incubation with shaking at 30 °C, the optical density of the
cultures was measured. The EC50 value was estimated using five para-
meter logistic regression with SigmaPlot 12.0 software. The equation is
formulated below:
(max min)
y = min + ;
Hillslope s
x
1+
xb
where:
1 log 2 s (1) 1
xb = EC50 × 10 Hillslope
;
min – bottom of the curve; max – top of the curve; Hillslope – char-
acterizes the slope of the curve at its midpoint; s – asymmetry para-
meter; x – naproxen concentration; y – optical density of the bacterial
culture
2.4. Fatty acids extraction and analysis
The fatty acids composition of Bacillus thuringiensis B1(2015b) was
determined after 24 h of cultivation of the strain on (1) a nutrient broth
(control sample); (2) a nutrient broth containing 4.8 g/L naproxen; (3) a
nutrient broth containing 5.2 g/L naproxen. Bacteria were harvested by
centrifugation (8000 ×g) at 4 °C for 20 min. The cell pellets were wa-
shed twice with 0.85% NaCl to remove the residue of the culture
medium. The fatty acid isolation and identification was conducted by
the MIDI-MIS (MIDI Sherlock® Microbial Identification System) method
(Sasser, 1990). Analysis of the fatty acid methyl esters (FAMEs) was
performed using an HP 5890 gas chromatograph (Hewlett Packard,
Rolling Meadows, IL, US) equipped with a HP 25 m x 0.2 mm cross-
linked methyl-silicone capillary column. The initial oven temperature
was 170 °C. Then it was increased to 260 °C at 5 °C/min, and then to
320 °C at 40 °C/min, and was held constant for 1.5 min. Hydrogen was
used as the carrier gas. FAMEs were identified using Sherlock software
(TSBA library, version 3.9, Microbial ID, Newark, NJ, USA) based on
the actual calibration retention times run prior to sample analysis.
Results were evaluated by the analysis of variance. Three replicates
of data obtained from each treatment were analysed statistically by
was assessed by a post hoc comparison of the means using the least
significant differences (LSD) test. All analyses were performed using the
Statistica 13.1 PL software package.
D. Górny et al.
2.5. Naproxen degradation experiments
Cometabolic degradation of naproxen was performed in 250 mL
Erlenmeyer flasks containing 130 mL of the mineral salts medium (Greń
et al., 2010) inoculated with bacterial cells to a final optical density of
about 0.1 at λ = 600 nm (OD600). Naproxen was added to a final con-
centration of 6 mg/L. As growth substrates 500 mg/L or 1000 mg/L
glucose, 414 mg/L 4-hydroxybenzoic acid, 432 mg/L sodium benzoate,
462 mg/L 3,4-dihydroxybenzoic acid or 282 mg/L phenol were used.
After complete degradation of suitable growth substrates, successive
doses of the growth substrates were introduced into the culture.
Determination of the optimal temperature and pH for naproxen
degradation was conducted in the range of 4–40 °C and 4–7.3 pH, re-
spectively. All cultures were grown in triplicate and incubated with
shaking at 30 °C. Chromatographic analyses of the culture supernatant
and measurements of the culture growth were carried out. All statistical
analyses were performed using the Statistica 13.1 PL software package.
All curves were fitted with the non-linear least squares regressions
method according to the matrix:
y = f (X , ) +
where: y is an n-by-1 vector of responses, f is a function of β and X, β is a
m-by-1 vector of coefficients, X is the n-by-m design matrix for the
model, ε is an n-by-1 vector of errors.
2.6. Determination of kinetic parameters of naproxen degradation
For studying the degradation kinetics in cometabolic systems, pure
cultures of the Bacillus thuringiensis B1(2015b) strain were separately
inoculated in a series of 500 mL Erlenmeyer flasks containing 250 mL of
mineral salts medium (Greń et al., 2010) supplemented with 500 mg/L
glucose and naproxen at initial concentrations of 1, 3, 6, 9, 12, 15 or
18 mg/L to a final optical density of 0.1 (OD600). Flasks were incubated
with shaking at 30 °C. 1 mL samples were taken every 24 h during 35
days from the cometabolic cultures. Growth of strain was monitored by
the optical density measurements at 600 nm and the degradation ki-
netics was studied by determination of the residual concentration of
naproxen in the medium. The concentration of glucose was measured
by anthrone method (Gerhardt et al., 1994).
For studying microbial growth in the cometabolic cultures, the
Andrew model was used, which is presented by the following equation:
=( + S + (S 2/ Ki )]
max · S )/[K s
where: µ is the specific growth rate (mg/L day), µmax is the maximum
specific growth rate (mg/L day), Ksµ is substrate concentration in which
µ = 1/2µmax (mg/L), Ki is inhibition constant (mg/L); S is naproxen
concentration (mg/L) (Gąszczak et al., 2010). Kinetic constants were
estimated by the Rosenbrock and quasi-Newton method using Statistica
13.1 software (Tomas et al., 2004).
The Monod equation, by which the biodegradation of naproxen in
cometabolic cultures was evaluated, is formulated below:
q = (qmax ·S )/(Ks + S )
where: q is the specific naproxen removal rate (mg/L day), qmax is the
maximum specific naproxen removal rate (mg /L·day), Ks is the half
saturation constant (mg/L), S is the naproxen concentration (mg/L)
(Marchlewicz et al., 2017a). Kinetic constants were estimated using
SigmaPlot 12.0 software.
2.7. HPLC analysis
The concentration of naproxen, hydroxybenzoic acid, sodium
benzoate, 3,4-dihydroxybenzoic acid and phenol was determined with
the HPLC technique using Merck Hitachi HPLC reversed-phase chro-
matograph equipped with an Ascentis Express ® C18 HPLC Column
(100 × 4.6 mm), an Opti-Solw ® EXP pre-column, and a UV/VIS DAD
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Ecotoxicology and Environmental Safety 167 (2019) 505–512
detector. The analysis of aromatic compounds concentration was per-
formed using acetonitrile and 1% acetic acid (50:50 v/v) as the mobile
phase at a flow rate of 1 mL/min was. The detection wavelength was set
at 260 nm for naproxen, 4-hydroxybenzoic acid, and 3,4-dihydrox-
ybenzoic acid and at 272 nm for phenol and sodium benzoate.
Naproxen, hydroxybenzoic acid, sodium benzoate, 3,4-dihydrox-
ybenzoic acid and phenol in supernatant were identified by comparing
the HPLC retention times and UV–visible spectra with that of the ex-
ternal standards.
3. Results and discussion
3.1. Toxicity of naproxen
Naproxen is a member of non-steroidal anti-inflammatory drugs,
which are inhibitors of cyclooxygenases officially known as pros-
taglandin-endoperoxide synthases, belonging to the peroxidase-cy-
clooxygenase superfamily. It is known that enzymes of this superfamily
are found in organisms belonging to various domains of life (Zamocky
et al., 2008, 2015; Zamocky and Obinger, 2010). Phylogenetic analysis
shows close relationship between bacterial and mammalian peroxidase-
cyclooxygenases. The physiological role of these enzymes in mammals
is well known and concerns both cyclooxygenase and peroxidase ac-
tivity, whereas role of related enzymes from prokaryotic organisms is
completely unknown (Zamocky et al., 2008, 2015). However, the
structural similarity among peroxidase-cyclooxygenases may suggest
sensitivity of bacterial enzymes to inhibitors of the mammalian en-
zymes. This phenomenon was observed e.g. for flavin-containing
monooxygenases from Bacillus sphaericus JS905 and human liver mi-
crosomes inhibited by methimazole (Guo et al., 1997; Kadiyala and
Spain, 1998). As a consequence of the inhibitory effect of methimazole,
inhibition of para-nitrophenol degradation by JS905 strain has been
observed (Kadiyala and Spain, 1998). Although the effect of naproxen
on microbial community was studied, there is no information about the
influence of naproxen on pure bacterial strains. Grenni et al., (2013,
2014) showed that after 3 h exposure of River Tiber bacterial commu-
nity to naproxen, live bacterial abundance significantly increased re-
latively to the control. In the presence of naproxen changes in microbial
community structure as well as the shift in the dominance of Alpha- and
Gamma-Proteobacteria groups in comparison to non-treated microcosms
was observed. At the end of the experiment a collapse in live bacterial
abundance was noticed, which was probably caused by the toxic effect
of transformation products (Grenni et al., 2013, 2014). In other studies,
Jiang et al. (2017) observed high removal efficiency (85.64%) of na-
proxen in sequencing batch reactor and on the base of bacterial ribo-
somal small subunit gene sequencing indicated changes in microbial
community. Presence of naproxen resulted in the increase strength of
Actinobacteria and Bacteroidetes, whereas contribution of Micropruina
and Nakamurella decreased.
Due to the lack of information about the influence of naproxen on
bacterial cells it was interesting to evaluate the effect and potential
toxicity of this drug to them. In our study the toxicity of naproxen to
model microorganisms was examined with the use of MARA test. The
most sensitive microorganism was Gram-negative Delftia acidovorans
(Fig. 1a). The MTC for this strain was 0.34 ± 0.00 g/L. Microbacterium
sp., Brevundimonas diminuta and Staphylococcus warneri were also sus-
ceptible to naproxen. As earlier studies have shown (Domaradzka et al.,
2016; Marchlewicz et al., 2017a), these strains were also sensitive to
the presence of other non-steroidal anti-inflammatory drugs: diclofenac
and ibuprofen. The most tolerant to naproxen (MTC above 3.4 g/L)
were Pseudomonas aurantiaca and Serratia rubidaea. Earlier studies have
also revealed resistance of these two strains to NSAIDs (Domaradzka
et al., 2016; Marchlewicz et al., 2017a). The mean value of microbial
toxic concentration MTCavg (equivalent of EC50) for naproxen was
1.66 ± 0.03 g/L and it was higher than MTCavg obtained for diclofenac
and ibuprofen. It indicates that naproxen is less toxic to
D. Górny et al. Ecotoxicology and Environmental Safety 167 (2019) 505–512

Fig. 1. a - Mean Microbial Toxic Concentration (MTC) values in the Microbial Assay for Risk Assessment (MARA) test, with naproxen exposure ranging between
0.48 g/L and 3.67 g/L. Microorganisms used in the MARA test: 1 - Microbacterium sp. G(+); 2 - Brevundimonas diminuta G(-); 3 - Citrobacter freudii G(-); 4 - Comamonas
testosteroni G(-); 5 - Enterococcus casseliflavus G(+); 6 - Delftia acidovorans G(-); 7 - Kurthia gibsonii G(+); 8 - Staphylococcus warneri G(+); 9 - Pseudomonas aurantiaca
G(-); 10 - Serratia rubidaea G(-); 11 - Pichia anomala (yeast); Bars showing the mean values of three independent trials ± standard deviation. Different letters indicate
significant differences (P < 0.05, LSD test) related to the effects of naproxen on bacterial strain. b- Inhibition of Bacillus thuringiensis B1(2015b) growth in the
presence of naproxen at various concentrations. Shown are means ± standard deviation and the fitted 5-parameter logistic regressions in dependence of naproxen
concentrations.

Table 1
Mutagenic activity expressed as the mean and standard deviations of the number of revertants/plate of bacterial strains TA98 and TA100 treated with naproxen, at
various doses, without metabolic activation system or with metabolic activation system (S9). Data represent the average of three independent trials ± standard
deviation.
Dose level (g/L) Without metabolic activation system

TA98 TA100

Number of Fold increase (over t-test p-value Number of Fold increase (over t-test p-value
revertants/plate baseline 1.84) revertants/plate baseline 17.89)

0 1.00 ± 0.84 – – 14.83 ± 3.06 – –

0.125 1.00 ± 1.00 0.75 0.22 12.33 ± 0.58 0.69 0.11
0.25 1.67 ± 0.58 1.25 0.03 12.33 ± 2.52 0.69 0.13
0.50 0.67 ± 0.58 0.50 0.38 18.33 ± 4.16 1.02 0.10
1.00 4.00 ± 1.00 2.99 0.00 15.33 ± 1.15 0.86 0.40
2.00 1.33 ± 0.58 1.00 0.09 15.67 ± 1.53 0.88 0.34
4.00 3.00 ± 1.00 2.24 0.00 10.33 ± 2.08 0.58 0.30

Dose level (g/L) With metabolic activation system

TA98 TA100

Number of Fold increase (over t-test p-value Number of Fold increase (over t-test p-value
revertants/plate baseline 1.92) revertants/plate baseline 17.08)

0 1.17 ± 0.75 – – 12.67 ± 4.41 – –

0.125 0.67 ± 1.15 0.35 0.23 13.33 ± 2.52 0.78 0.41
0.25 1.67 ± 1.15 0.87 0.23 14.33 ± 4.04 0.84 0.30
0.50 1.67 ± 1.53 0.87 0.26 19.33 ± 3.06 1.13 0.03
1.00 1.33 ± 1.53 0.69 0.41 25.00 ± 3.00 1.46 0.00
2.00 3.33 ± 1.53 1.74 0.01 26.33 ± 3.21 1.54 0.00
4.00 3.33 ± 0.58 1.74 0.00 11.33 ± 2.25 0.66 0.32

microorganisms. This may be connected with the mechanism of na-
proxen transport into the bacteria cells or the synthesis of inner mem-
brane proteins and/or enzymes (Martins et al., 2013). It is known that
hydrocarbons with condensed aromatic ring may be transported by
three different mechanisms: passive diffusion, passive facilitated dif-
fusion or active uptake (Hua and Wang, 2014). Presence of negatively
charged carboxylic group in naproxen unable its passive transport and
facilitated diffusion across a membrane. Therefore, low toxicity of na-
proxen to the examined strains may suggest lack of the proper carriers
of this drug in their cells. Moreover, resistance to naproxen may be
connected with synthesis of the enzymes engaged in its transformation
into less toxic products or lack of the enzymes sensitive to naproxen
(peroxidase-cyclooxygenases or other ones with the similar mechanism
reaction).
Genotoxicity of naproxen was evaluated using Ames microplate
assays. The obtained results are ambiguous (Table 1). In our study,
slightly above 2-fold increase in mutant frequency was observed in the
presence of naproxen at concentration above 1 g/L, when Salmonella
typhimurium TA98 was used as a model organism. It indicates that na-
proxen at higher concentration may show genotoxic effect but the re-
sponse is not dose-dependent. Similar results were obtained by
Philipose et al. (1997). In the presence of metabolic activation system,
genotoxic impact was not observed. We assume that enzymatic acti-
vation of naproxen did not lead to mutagenic intermediates. Our results

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D. Górny et al. Ecotoxicology and Environmental Safety 167 (2019) 505–512

Table 2 3.2. Naproxen degradation by Bacillus thuringiensis B1(2015b)

Percentage of total fatty acids of Bacillus thuringiensis B1 grown on nutrient
broth (1), nutrient broth supplemented with 4.8 g/L naproxen (2), nutrient Potential negative impact of naproxen on living organisms enforces
broth supplemented with 5.2 g/L naproxen (3). Data represent the average of searching methods for its degradation. Due to the safety and low costs,
three independent trials ± standard deviation. ND - implies fatty acid con-
biological methods are good alternatives to the physical ones.
centrations below detection limit < 10 ng/L. Different letters indicate sig-
Our previous studies showed the ability of Bacillus thuringiensis
nificant differences (P < 0.05, LSD test) related to the effects of naproxen
B1(2015b) to degrade non-steroidal anti-inflammatory drugs
concentration.
(Marchlewicz et al., 2016, 2017b). However, in monosubstrate culture
Fatty acids % of total fatty acids with naproxen as a sole carbon and energy source the degradation ef-
ficiency was very low. Simultaneously we observed that glucose as an
1 2 3
additional carbon source significantly increased decomposition of na-
Saturated proxen (Marchlewicz et al., 2016). It is generally known that micro-
12:0 0.11 ± 0.00a NDb NDb organisms may benefit from the presence of additional growth substrate
14:0 1.17 ± 0.15a 0.42 ± 0.01b 0.40 ± 0.04b
in the form of cell growth support or/and induction of the catabolic
16:0 8.12 ± 0.24a 5.28 ± 0.32b 5.95 ± 0.47b
16:0 2OH 0.33 ± 0.03a 0.16 ± 0.00b 0.13 ± 0.01c enzymes in the case of structural analogue of degraded compound
16:0 iso 3OH NDa 25.62 ± 5.89b 33.84 ± 2.04c (Domaradzka et al., 2015; Nowak et al., 2016). For that reason the
17:0 0.39 ± 0.00a 0.31 ± 0.04b 0.33 ± 0.04b cometabolic conditions are frequently used for degradation of persistent
18:0 2.14 ± 0.19a 1.23 ± 0.20b 1.67 ± 0.27c pollutants including non-steroidal anti-inflammatory drugs.
18:0 2OH 0.49 ± 0.06a 0.38 ± 0.06ab 0.34 ± 0.01b
18:0 anteiso 46.13 ± 1.64a 45.25 ± 1.17ab 37.57 ± 1.83b
Domaradzka et al. (2015) showed that in the presence of glucose or
Unsaturated phenol degradation of naproxen by Planococcus sp. S5 increased by 45%
16:1 ω7c 0.54 ± 0.08a 0.19 ± 0.03b 0.19 ± 0.03b and 56%, respectively. In this study, in the presence of 0.5 and 1.0 g/L
16:1 ω9c NDa NDa 0.12 ± 0.00b glucose, Bacillus thuringiensis B1(2015b) degraded 6 mg/L of naproxen.
17:1 ω8c 0.64 ± 0.04a 0.55 ± 0.06a 0.39 ± 0.03b
However, there was no increase in degradation efficiency with the in-
18:1 ω9c 40.44 ± 0.91a 20.68 ± 4.39b 19.20 ± 2.22b
19:1 ω11c NDa 0.10 ± 0.00b NDa crease of glucose concentration from 0.5 g/L to 1 g/L. Maximal de-
Sat./unsat. ratio 1.41 3.65 4.03 gradation rate in glucose was reached at 0.5 g/L (Fig. 2a, b). This was
significant improvement in the degradation efficiency in comparison to
ω – methyl end of fatty acid. monosubstrate condition where only 25% of naproxen was degraded
c – cis configuration of the double bound. after 35 days (Marchlewicz et al., 2016). Non-steroidal anti-in-
-OH indicates the position of the hydroxyl group from the acid end.
flammatory drugs include in their structures aromatic ring which may
iso, anteiso - branched fatty acids.
induce enzymes engaged in the decomposition of this ring. This group
of enzymes are commonly characterized by broad range enzyme spe-
cificity (Guzik et al., 2014). It can therefore be supposed that the usage
of easily degradable aromatic compound may accelerate drug de-
confirm earlier reports which revealed that naproxen had not generated gradation not only through increase of biomass but also by induction of
genotoxicity in the model Salmonella typhimurium strains. The genotoxic proper enzymes. In our preliminary studies, phenol, sodium benzoate,
effect has been previously reported only in in vivo studies on bone 4-hydroxybenzoic acid and 3,4-dihydroxybenzoic acid as an aromatic
marrow cells of mice and in Hyalella azteca cells (Philipose et al., 1997; growth substrate for Bacillus thuringiensis B1(2015b) were used (data
Garcia-Medina et al., 2015). It is suggested that the genotoxic effect is not presented). Many microorganisms are able to degrade phenol be-
connected with the oxidative stress induced by naproxen (Garcia- cause aromatic structure of this compound is hydroxylated. The pre-
Medina et al., 2015; Ahmad et al., 2018). sence of hydroxyl group ensures the omission of aromatic ring hydro-
Our earlier study showed that Bacillus thuringiensis B1(2015b) is able xylation by monooxygenases, the first stage of degradation. For that
to degrade naproxen (Marchlewicz et al., 2016). It would be interesting reason this compound is very often used as a growth substrate in con-
to know if its ability to degrade naproxen is correlated with the higher centration range 100–300 mg/L during degradation of persistent xe-
resistance of the strain to this drug. The EC50 value of naproxen exposed nobiotics (Liu, 2009; Li et al., 2014; Nowak et al., 2016). In the pre-
to the Bacillus thuringiensis B1(2015b) was 4.69 ± 0.09 g/L (Fig. 1b). sence of phenol at 282 mg/L, B1(2015b) strain degraded naproxen
This value is almost three-fold higher than the average MTC estimated during 28 days. In addition, bacterial growth was 33% lower than in the
for the model microorganisms. High resistance to naproxen together presence of glucose with naproxen (Fig. 2c). Despite the induction of
with the resistance to ibuprofen makes Bacillus thuringiensis B1(2015b) similar enzymes engaged in aromatic ring degradation, lower bacterial
an ideal tool in bioremediation of sites contaminated with non-steroidal growth may result due to toxic effect of phenol. Secondary plant me-
anti-inflammatory drugs (Marchlewicz et al., 2017a). In response to the tabolites are an important source of nutrients for environmental bac-
toxic compounds, changes in bacterial cellular fatty acids composition teria (Greń et al., 2010; Fraraccio et al., 2017). Typical aromatic
are frequently observed (Nowak et al., 2016). After incubation of Ba- compounds synthesized by plants are benzoic acids (Widhlam and
cillus thuringiensis B1(2015b) in the presence of naproxen at con- Dudareva, 2015). However, presence of benzoate which is less toxic
centration: 4.8 and 5.2 g/L significant changes in the ratio of saturated than phenol did not improve degradation of naproxen (Fig. 2d). Similar
and unsaturated total fatty acids were observed. In the presence of effects were observed after addition of 4-hydroxybenzoic acid and 3,4-
naproxen there was significant decrease in the quantity of four satu- dihydroxybenzoic acid as growth substrates (Fig. 2e, f). This may in-
rated fatty acids: 12:0, 14:0, 16:0, 17:0 and two unsaturated fatty acids: dicate that aromatic carboxylic acids and naproxen are utilised by Ba-
16:1 ω7c and 18:1 ω9c. Moreover, at these conditions appearance of cillus thuringiensis B1(2015b) through different pathways. The other
16:0 iso 3OH fatty acid was correlated with the reduction of un- possibility is that aromatic carboxylic acids compete with naproxen for
saturated fatty acids contribution (Table 2). the active site of enzyme because of the presence of carboxylic group
Increased ratio of saturated to unsaturated total fatty acids results in both in aromatic plant compounds and naproxen (Wojcieszyńska et al.,
the decrease of membrane fluidity as a response to the presence of toxic 2011). To date, there are no reports related to the effect of additional
factor. Additional hydroxyl group may stabilize structure of membrane carbon source on naproxen degradation, and therefore it is hard to refer
by intensification of the interaction with membrane proteins and it is to results obtained by other authors. However, obtained results clearly
known as an adaptive mechanism against stress conditions (Zdarta showed that glucose at concentration 0.5 g/L constitutes optimal
et al., 2017). carbon source for cometabolic degradation of naproxen by Bacillus

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D. Górny et al. Ecotoxicology and Environmental Safety 167 (2019) 505–512

Fig. 2. Cometabolic naproxen degradation and changes in the optical density of cultures in the presence of glucose 0.5 g/L (a), glucose 1.0 g/L (b), phenol (c), sodium
benzoate (d), 4-hydroxybenzoic acid (e) and 3,4-dihydroxybenzoic acid (f). Shown are means ± standard deviation and the fitted the least squares regressions.
Different small and capital letters indicate significant differences in naproxen degradation and optical density of cultures, respectively, amongst the tested come-
tabolic systems (ANOVA, p < 0.05, LSD-test).

thuringiensis B1(2015b). more amenable to nucleophilic attack (Dolgopyatova et al., 2011).

Biodegradation efficiency depends on the physicochemical proper-
ties of the environment. Our earlier study on ibuprofen degradation
showed that specific removal rate of this compound depended on pH
and temperature. Optimal temperature for utilization of ibuprofen and
naproxen was 30 °C. Optimal ibuprofen degradation was observed at pH
7.2 (Marchlewicz et al., 2017b) while optimal pH for naproxen de-
gradation was lower by 0.7 unit (Fig. 3a, b). It may be connected with
different ionization degree of naproxen and ibuprofen at the same pH.
At pH 6.5, naproxen percent ionization is 100% while complete ioni-
zation of ibuprofen is observed at pH 7.0. Dependence of compound
degradation rate on its ionization was described by Dolgopyatova et al.
(2011). These authors suggested that completely ionized compound is
To the best of our knowledge, there is no information about the
kinetics of biological degradation of naproxen. There is only some in-
formation on kinetics of physico-chemical decomposition of naproxen
(Gao et al., 2017). Therefore, kinetics of naproxen degradation by Ba-
cillus thuringiensis B1(2015b) was analysed in cometabolic systems with
0.5 g/L glucose as a growth substrate (Fig. 4a). The half saturation
constant (Ks) and the maximum specific naproxen removal rate (qmax)
were 6.86 ± 0.80 mg/L and 1.26 ± 0.06 mg/L per day, respectively.
Definitely higher Ks value for naproxen compared with ibuprofen in-
dicates much lower bacterial affinity for this drug. It was also reflected
in the lower value of maximum specific naproxen removal rate in
comparison to maximum specific ibuprofen removal rate (Marchlewicz

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D. Górny et al. Ecotoxicology and Environmental Safety 167 (2019) 505–512

Fig. 3. Influence of pH (a) and temperature (b) on the specific naproxen removal rate. Shown are means ± standard deviation and the fitted the least squares
regressions. Different letters indicate significance of pH (a) and temperature (b) on specific naproxen removal rate; tested according to the Student's t-test (p < 0.05).

Fig. 4. Kinetic models of naproxen degradation (a) and bacteria survival (b) in the cometabolic system with 0.5 g/L glucose as a growth substrate. Shown are
means ± standard deviation and the fitted the non-linear estimation with usage Monod equation (5a) and the Rosenbrock and quasi-Newton method (5b). Plot in
Fig. 4a presents inhibition by substrate and was fitted with the least squares regressions. Different letters indicate significance of naproxen concentration on specific
naproxen removal rate (a) and specific growth rate (b); tested according to the Student's t-test (p < 0.05).

et al., 2017a). At naproxen concentration above 12 mg/L, inhibition of Acknowledgments

degradation process by the substrate was observed (Fig. 4a). A similar
effect was observed during ibuprofen biodegradation (Marchlewicz
et al., 2017a). However, the concentration of naproxen at which de-
gradation of this compound was inhibited was significantly higher than
the environmentally relevant concentrations for this compound. For
that reason this effect should not have an impact on applicable im-
portance of Bacillus thuringiensis B1(2015b).
The maximum specific growth rate of strain B1(2015b) in the pre-
sence of glucose as an additional carbon source was 0.55 ± 0.03 mg/L
day, Ksµ was 0.19 ± 0.07 mg/L, Ki was 36.69 ± 6.88 mg/L.
Significant decrease in specific growth rate was observed above 12 mg/
L naproxen, corresponding to the concentration of naproxen which
inhibited the degradation process (Fig. 4a, b).
In conclusion, in the light of the foregoing results naproxen does not
show high toxicity at the environmentally relevant concentrations.
However, because of its biological activity it may influence the activity
of prokaryotic and eucaryotic cyclooxygenases or related enzymes and
in this way affect regulation and functioning of organisms in ecosys-
tems. For that reason it is very important to select microorganisms with
the extended ability to decompose naproxen. Bacillus thuringiensis
B1(2015b) seems to fulfil this expectation. This strain in cometabolic
conditions is able to degrade naproxen at concentrations up to 12 mg/L
with maximal specific removal rate. This, together with high resistance
of this strain to naproxen (EC50 = 4.69 g/L), makes B1(2015b) strain a
good potential tool in bioremediation processes.
This work was financed by the National Science Centre (Poland),
granted on the basis of decision DEC-2013/09/B/NZ9/00244.
Conflict of interest
The authors declare that they have no conflicts of interest.
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