Journal of Environmental Management 145 (2014) 157e161

Contents lists available at ScienceDirect

Journal of Environmental Management

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

Bacterial degradation of naproxen e Undisclosed pollutant in the

environment

ska*, Dorota Domaradzka, Katarzyna Hupert-Kocurek, Urszula Guzik
Danuta Wojcieszyn
Department of Biochemistry, Faculty of Biology and Environmental Protection, University of Silesia in Katowice, Jagiellonska 28, 40-032 Katowice, Poland

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

Article history: The presence of non-steroidal anti-inflammatory drugs (NSAIDs) in the environment is an emerging
Received 24 March 2014 problem due to their potential influence on human health and biocenosis. This is the first report on the
Received in revised form biotransformation of naproxen, a polycyclic NSAID, by a bacterial strain. Stenotrophomonas maltophilia
17 June 2014
KB2 transformed naproxen within 35 days with about 28% degradation efficiency. Under cometabolic
Accepted 24 June 2014
conditions with glucose or phenol as a carbon source degradation efficiency was 78% and 40%, respec-
Available online
tively. Moreover, in the presence of naproxen phenol monooxygenase, naphthalene dioxygenase,
hydroxyquinol 1,2-dioxygenase and gentisate 1,2-dioxygenase were induced. This suggests that degra-
Keywords:
Naproxen
dation of naproxen occurs by its hydroxylation to 5,7,8-trihydroxynaproxen, an intermediate that can be
Stenotrophomonas maltophilia KB2 cleaved by hydroxyquinol 1,2-dioxygenase. The cleavage product is probably further oxidatively cleaved
Cometabolism by gentisate 1,2-dioxygenase. The obtained results provide the basis for the use of cometabolic systems in
Biodegradation the bioremediation of polycyclic NSAID-contaminated environments.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction Most of non-steroidal anti-inflammatory drugs are not fully

The introduction of non-steroidal anti-inflammatory drugs
(NSAIDs), also known as pain killers, into natural matrices such as
soil, sediments, groundwater and even drinking water is an
emerging problem due to their potential influence on human
health and natural environments (Almeida et al., 2013). These
pollutants enter the environment as a result of pharmaceutical
industry activity, and the improper disposal of unused or expired
drugs, wastes generated in hospitals and stock-raising farms (Wu
et al., 2012). In many countries large amounts of non-steroidal
anti-inflammatory drugs are consumed annually. For example, in
Germany about 836 tonnes of acetylsalicylic acid, 622 tonnes of
paracetamol, 345 tonnes of ibuprofen and 86 tonnes of diclofenac
were consumed in 2001 (Nikolaou et al., 2007). In 2000, 35 tonnes
of naproxen were consumed in England (Nikolaou et al., 2007),
while in Poland the consumption of 58 tonnes of ibuprofen, the
other important drug in this group, was recorded (Sosnowska et al.,
2009). Picquet (2013) announces that Albemarle Company pro-
duces about 500 tonnes of naproxen per year.
* Corresponding author. Tel.: þ48322009454; fax: þ48322009361.

ska),
E-mail addresses: danuta.wojcieszynska@us.edu.pl (D. Wojcieszyn
doroteadomar@gmail.com (D. Domaradzka), katarzyna.hupert-kocurek@us.edu.pl
(K. Hupert-Kocurek), urszula.guzik@us.edu.pl (U. Guzik).
degraded after application, and get into the environment in an
unchanged or slightly modified form. NSAIDs have been detected in
different environments at concentrations ranging from nanograms
to micrograms per liter (Lloret et al., 2010; Wu et al., 2012). Due to
their stability they are not totally eliminated by the sewage treat-
ment plants, and can be unintentionally consumed by humans in
tap water (Gentili, 2007).
Physicochemical processes are frequently used for the removal
of naproxen. However, these processes require harsh reaction
conditions, generate free radicals and/or secondary pollutants, and
incur high operational costs (Zhang et al., 2013). Therefore, biore-
mediation processes are an attractive alternative to these methods.
Bioremediation strategies are cost effective and enable the miner-
alization of NSAIDs into innocuous products (Ahmed et al., 2001).
Quintana et al. (2005) tested the ability of activated sludge to
degrade naproxen and observed its 49% removal with the simul-
taneous appearance of desmethylnaproxen. Carballa et al. (2004)
showed a 40e55% removal of naproxen during biological
treatment.
Although the activated sludge process is used to treat waste-
waters containing non-steroidal anti-inflammatory drugs, it is
not sufficient for the complete removal of these compounds and
thus they are still detected in the effluents (Rodarte-Morales
et al., 2011). Therefore, there is a need to isolate microorgan-
isms with a high capacity for non-steroidal anti-inflammatory

http://dx.doi.org/10.1016/j.jenvman.2014.06.023
0301-4797/© 2014 Elsevier Ltd. All rights reserved.
158
ska et al. / Journal of Environmental Management 145 (2014) 157e161
D. Wojcieszyn
drug degradation, to study their metabolic pathways and en-
zymes, and to characterize optimal conditions for NSAIDs
degradation.
Until now, only a few microorganisms, mainly fungi (Penicillium
sp., Trametes versicolor, Cunninghamella elegans, C. echinulata,
C. blakesleeana, Beauveria bassiana, Phanerochaete chrysosporium,
Ph. sordida, Actinoplanes sp., Bjerkandera sp. R1, Bj. adusta, Irpex
lacteus, Ganoderma lucidum) have been identified to transform or
degrade non-steroidal anti-inflammatory drugs (Lloret et al., 2010;
Marco-Urrea et al., 2010b; Rodarte-Morales et al., 2011, 2012). In the
transformation of naproxen to 2-(6-hydroxynaphthalen-2-yl)pro-
pionic acid and 1-(6-methoxynaphthalen-2-yl)ethanone by fungus
T. versicolor, cytochrome P-450 and laccase were probably engaged
(Marco-Urrea et al., 2010a; Rodriguez-Rodriguez et al., 2010).
Degradation of naproxen by commercial laccase isolated from
Myceliophthora thermophila was also shown by Lloret et al. (2010).
They observed 100% degradation of this pharmaceutical in the
presence of the redox mediator (Lloret et al., 2010).
Much less is known about the degradation of naproxen by
bacteria. Until now, only a few bacterial strains, mainly from genera
Pseudomonas, Sphingomonas, Patulibacter, Nocardia, Rhodococcus
and Stenotrophomonas, able to degrade non-steroidal anti-inflam-
matory drugs, have been isolated (Ahmed et al., 2001; Almeida
et al., 2013; Chen and Rosazza, 1994; Gusseme et al., 2011;
Ivshina et al., 2006; Murdoch and Hay, 2005; Wu et al., 2012;
Zhang et al., 2013). According to our best knowledge the biodeg-
radation of naproxen by pure bacterial strains has not been
described. Therefore, the main aim of our study was to investigate
the treatability of naproxen by the aromatic compound degrader
Stenotrophomonas maltophilia KB2. It was also important to deter-
mine the enzymes engaged in naproxen degradation.
2. Material and methods
2.1. Bacterial strain and growth conditions
S. maltophilia KB2 (VTT E-113197) was routinely cultivated in
BBL nutrient broth at 30  C and 130 rpm for 24 h. Then 6 mg L1
naproxen was added to the culture. After 48 h cells were harvested
by centrifugation (5000g at 4  C for 15 min), washed with fresh
sterile medium and used as inoculum.
Degradation of naproxen in a monosubstrate, as well as com-
etabolic systems, was performed in 500 mL Erlenmeyer flasks
containing 250 mL of the mineral salts medium (Gren
et al., 2010)
inoculated with cells to a final optical density of about 1.5 and 0.1 at
l ¼ 600 nm (OD600) for the monosubstrate and cometabolic sys-
tems, respectively. For degradation experiments two control cul-
tures were prepared. The uninoculated control (I) contained
250 mL of sterile mineral salts medium, while the heat-killed
control (II) consisted of 250 mL of autoclaved culture prepared
under conditions identical to those of the experimental cultures.
Naproxen was added to each flask to obtain a final concentration
of 6 mg L1, and all cultures were incubated with shaking at 30  C
for 35 days.
For studies on the cometabolic transformation of naproxen, as
well as the induction of enzymes, 1 mg L1 glucose or 3 mM
phenol were added respectively. Cultures in 250 mL of sterile
mineral salt medium supplemented with appropriate growth
substrates and 6 mg L1 naproxen were inoculated with cells to a
final optical density of about 0.1 at l ¼ 600 nm (OD600), and
incubated at 30  C with shaking at 130 rpm. If the complete
degradation of the suitable growth substrate was observed, a
successive dose of phenol or glucose was introduced and the cul-
ture was left for incubation until it reached OD600 ¼ 1.0. All cul-
tures were grown in triplicates.
2.2. Determination of substrate concentration
To study the degradation of naproxen, 1 mL samples were taken
periodically (every 7 days) from the culture medium and centri-
fuged (6000g, 15 min). The concentration of aromatic substrate
in the culture supernatant was determined by HPLC (Merck
HITACHI) equipped with a LiChromospher® RP-18 column
(4  250 mm), liChroCART® 250-4 Nucleosil 5 C18 and a DAD
detector (Merck HITACHI). The mobile phase was acetonitrile and
1% acetic acid (50:50 v/v) at a flow rate of 1 mL min1. The
detection wavelength was set at 260 nm. Naproxen in the super-
natant was identified and quantified by comparing the HPLC
retention times and UVevisible spectra with those of the external
standards.
Phenol concentration in the culture supernatant was deter-
mined using a colorimetric method with p-nitroaniline (Lurie and
Rybnikova, 1974). The concentration of glucose in the culture su-
pernatant was determined with a colorimetric method using 3,5-
dinitrosalicylic acid (Miller, 1959).
2.3. Preparation of cell extracts
After 35 days in culture, cells of S. maltophilia strain KB2 were
harvested by centrifugation (4500g for 15 min at 4  C) and the
pellet was washed with 50 mM phosphate buffer, pH 7.0, and
resuspended in the same buffer. Cell-free extracts were prepared by
sonication of the whole cell suspension (6 times for 15 s) and
centrifugation at 9000g for 30 min at 4  C. Clear supernatant was
used as a crude cell extract for enzyme assays.
2.4. Enzyme assays
Monooxygenase activity was determined spectrophotometri-
cally by measuring NADH oxidation (ε340 ¼ 6220 M1 cm1) (Divari
et al., 2003). In order to determine the activity of dioxygenase-
catalyzed dihydroxylation, the formation of cis,cis-dihydrodiol
was measured at 262 nm (ε262 ¼ 8230 M1 cm1) (Cidaria et al.,
1994). The activity of catechol 1,2-dioxygenase was measured
spectrophotometrically by the formation of cis,cis-muconic acid at
260 nm (ε260 ¼ 16,800 M1 cm1). In order to determine catechol
2,3-dioxygenase activity, the formation of 2-hydroxymuconic
semialdehyde was measured at 375 nm
(ε375 ¼ 36,000 M1 cm1) (Wojcieszyn
ska et al., 2011). The activity
of protocatechuate 3,4-dioxygenase was assayed by measuring the
oxygen consumption (Hou et al., 1976). The activity of proto-
catechuate 4,5-dioxygenase was measured spectrophotometrically
by the formation of 2-hydroxy-4-carboxymuconic semialdehyde at
410 nm (ε410 ¼ 9700 M1 cm1) (Wojcieszyn
ska et al., 2011). In
order to determine gentisate 1,2-dioxygenase activity, the forma-
tion of maleylpyruvate was measured at 330 nm
(ε330 ¼ 10,800 M1 cm1) (Feng et al., 1999). The activity of
hydroxyquinol 1,2-dioxygenase was measured spectrophotomet-
rically by the formation of maleylacetate at 243 nm
(ε243 ¼ 44,520 M1 cm1) (Wei et al., 2010).
One unit of enzyme activity was defined as the amount of
enzyme required to generate 1 mmol of product per minute. Protein
concentration in the crude extract was determined by the Bradford
method using bovine serum albumin as a standard (Wojcieszyn
ska
et al., 2011).
2.5. Statistical analysis
All experiments were performed in three replicates. The ob-
tained data were analyzed by one-way ANOVA using STATISTICA
10.0 PL software package.

ska et al. / Journal of Environmental Management 145 (2014) 157e161
D. Wojcieszyn 159

3. Results and discussion

3.1. Biodegradation of naproxen by S. maltophilia KB2

The presence of naproxen in ground and drinking water, even at

low concentrations, is an emerging environmental problem
because of its potential impact on the human health (Gentili, 2007;
Juvancz et al., 2008). This anti-inflammatory drug is known to be
degraded inefficiently by fungi or mixed cultures of activated
sludge (Rodarte-Morales et al., 2011). Degradation of naproxen by a
pure bacterial strain has not yet been reported.
S. maltophilia KB2 is known to metabolize a broad range of ar-
omatic compounds, including phenol, cresols, chloro and nitro-
phenols, and some aromatic compounds of plant origin (Gren
et al.,
2010; Wojcieszyn
ska et al., 2011). The aromatic nature of naproxen,
along with the degradative potential of strain KB2, suggests S.
maltophilia KB2 as a possible candidate for transformation and/or
degradation of this drug.
Determination of naproxen in the uninoculated control (I) as well
as the heat-killed control (II) cultures after 35 days of incubation
revealed no change in the concentration of the compound. This
confirmed the lack of chemical oxidation of naproxen, as well as the
lack of adsorption of this drug to the cells surface. Therefore, the ability
of strain KB2 to grow in the presence of naproxen was determined.
During the preincubation of the strain in the nutrient broth with
6 mg L1 naproxen bacterial growth to an OD600 of about 1.0 was
observed. It indicates that KB2 strain is able to grow in the presence of
naproxen. To determine if S. maltophilia KB2 is able to degrade
6 mg L1 naproxen the bacterial strain was inoculated into a mineral
medium with naproxen as the sole carbon and energy source. As
shown in Fig. 1a, approximately 28% of the naproxen was removed
after 35 days of incubation. However, the degradation of naproxen was
accompanied by a decrease in the number of bacteria. Most likely, this
compound was not a sufficient carbon and energy source for strain
KB2. Degradation of xenobiotics that are resistant to biological
decomposition is frequently observed in the presence of easily
assimilated substrates (Gren
et al., 2010; Rieger et al., 2002;
Sutherland et al., 1981). Therefore, studies on degradation of nap-
roxen under cometabolic conditions were performed. For these
studies glucose was used as a simple source of carbon. The introduc-
tion of glucose into the cultures of S. maltophilia KB2 with naproxen
stimulated biomass production. Under these conditions, after 35 days
incubation, naproxen was degraded at a rate of almost 78% (Fig. 1b).
The degradation of naproxen was accompanied by the emergence of
two unidentified metabolites. The first one appeared on the 7th day of
incubation and disappeared on day 14th with simultaneous appear- Fig. 1. Degradation of 6 mg L1 naproxen by strain KB2 and changes of microbial
biomass monitored as optical density at 600 nm (a e without additional carbon
ance of the second metabolite. After 21 days incubation, neither of source; b e with 1 mg L1 glucose as a simple carbon source; c e with 3 mM phenol as
these metabolites was detected. Intensification of naproxen degra- an aromatic carbon source).
dation by activated sludge under cometabolic conditions was reported
by Quintana et al. (2005). After the introduction of milk powder as an
additional carbon source the authors observed the removal of about in the presence of glucose may result from phenol and naproxen
49% of naproxen after 26 days. Additionally, the increased degradation competition at the active site of enzymes engaged in their degra-
of naproxen by fungi was observed in the presence of peptone or dation (Gren
et al., 2010).
glucose (Rodarte-Morales et al., 2012; Zhong et al., 2003). Even though during our experiment (35 days) only partial
In cometabolic systems, growth substrates with a structure degradation of naproxen was observed, the application of comet-
similar to that of xenobiotics are frequently used. This provides the abolic systems may contribute to faster removal of such pollutants
induction of enzymes required for cometabolite transformation from natural environments. It should be emphasized that bioreme-
(Cornelissen and Sijm, 1996). S. maltophilia KB2 is characterized by diation is a slow process, and the environment undergoes dynamic
intense growth in the presence of different aromatic compounds changes accompanied by a continuous inflow of growth substrates.
(Wojcieszyn
ska et al., 2011). Therefore, in the cometabolic cultures
6 mg L1 naproxen as a cometabolite and 3 mM phenol as the sole 3.2. Induction of enzymes during cometabolic degradation of
carbon source were used. Under this condition almost 40% of naproxen
naproxen was degraded, with a simultaneous increase of bacterial
growth (Fig. 1c). The lower degree of naproxen degradation in the Nowadays, there is little information about the mechanisms of
presence of phenol compared to the degradation of this compound naproxen degradation. Marco-Urrea et al. (2010a) suggested that in
160
ska et al. / Journal of Environmental Management 145 (2014) 157e161
D. Wojcieszyn

degradation of this compound by fungi, laccase and cytochrome P-

450 monooxygenase are involved. Although they identified prod-
ucts of naproxen transformation: 6-O-desmethylnaproxen (2-(6-
hydroxynaphthalen-2-yl)propionoic acid) and 1-(6-
methoxynaphthalen-2-yl)ethanone, subsequent stages of degra-
dation of the aromatic ring of naproxen still remain unknown.
S. maltophilia KB2 is known to synthesize different types of
hydroxylases and dioxygenases depending on the inductor present
in the medium (Gren
et al., 2010; Wojcieszyn
ska et al., 2011). In the
present study, the induction of enzymes which could be involved in
degradation of naproxen was examined. In a monosubstrate system
the significant reduction in the number of cells was observed and
obtained biomass was too low for enzymes isolation. After 35 days
incubation in the presence of naproxen and glucose or phenol as
growth substrates a crude extract of bacterial cells was prepared as
described in Materials and methods. Measurements of enzymes
activity revealed that in the presence of glucose or phenol as a
carbon source strain KB2 produced naphthalene dioxygenase,
phenol monooxygenase and hydroxyquinol 1,2-dioxygenase
(Table 1). Based on these results, and the fact that naproxen is a
derivative of naphthalene, we may assume that dihydroxylation of
this compound, catalyzed by naphthalene dioxygenase (Parales
et al., 2000) is the first step of naproxen transformation by strain
KB2. In the next step, hydroxylation of 7,8-dihydroxynaproxen by
phenol monooxygenase probably occurs and 5,7,8-
trihydroxynaproxen is formed. This molecule can be, in turn, the
substrate for hydroxyquinol 1,2-dioxygenase (Guzik et al., 2013;
Yoshida et al., 2007) (Fig. 2). During the cometabolic degradation
of naproxen using glucose as a carbon source the activity of gen-
tisate 1,2-dioxygenase was revealed. This suggests the participation
of this enzyme in the oxygenolytic cleavage of the product of
gentisate 1,2-dioxygenase activity (Fig. 2). When the cells of KB2
were grown in a medium with phenol as a growth substrate, the
activity of catechol 2,3-dioxygenase was also observed (Table 1).
This enzyme plays a crucial role in phenol degradation by S. mal-
tophilia KB2 (Wojcieszyn
ska et al., 2011). Therefore, its activity was
a result of its induction by this aromatic substrate.

Fig. 2. Proposed degradation pathway of naproxen under cometabolic conditions.

4. Conclusions

This is the first report on the degradation of naproxen by a
bacterial strain. We demonstrated that cometabolic systems can
provide higher efficiency of naproxen transformation with a
simultaneous increase of bacterial growth. The activity of phenol
monooxygenase, naphthalene dioxygenase, hydroxyquinol 1,2-
dioxygenase and gentisate 1,2-dioxygenase under these condi-
tions suggest transformation of naproxen through its hydroxylation
and aromatic ring cleavage. Increase of naproxen degradation ef-
ficiency in the presence of additional carbon sources suggests the
possible application of cometabolic systems for the bioremediation
of degraded areas. The immobilized bacteria able to naproxen
degradation may be used in the future as a biopreparation in waste
water treatment plants.
Acknowledgments
This work was financed by the National Science Centre (Poland),
granted on the basis of decision DEC-2013/09/B/NZ9/00244.
References

Ahmed, S., Javed, M.A., Tanvir, S., Hameed, A., 2001. Isolation and characterization of
Table 1 a Pseudomonas strain that degrades 4-acetamidophenol and 4-aminophenol.
Specific activity of enzymes engaged in naproxen degradation under cometabolic Biodegradation 12, 303e309.
conditions. Almeida, B., Kjeldal, H., Lolas, I., Knudsen, A.D., Carvalho, G., Nielsen, K.L., Barreto
Crespo, M.T., Stensballe, A., Nielsen, J., 2013. Quantitative proteomic analysis of
Enzyme Specific enzyme activity (U mg1 protein) ibuprofen-degrading Patulibacter sp. Strain I11. Biodegradation 24, 615e630.
Naproxen þ glucose Naproxen þ phenol Carballa, M., Omil, F., Lema, J.M., Llompart, M., Garcia-Jares, C., Rodriguez, I.,
Gomez, M., Ternes, T., 2004. Behavior of pharmaceutical, cosmetics and hor-
Phenol monooxygenase 35.2 ± 13.0 19.4 ± 6.6 mones in a sewage treatment plant. Water Res. 38, 2918e2926.
Naphthalene dioxygenase 2.1 ± 1.7 9.7 ± 0.3 Chen, Y., Rosazza, J.P.N., 1994. Microbial transformation of ibuprofen by a Nocardia
Hydroxyquinol 1,2-dioxygenase 179.7 ± 23.5 250.0 ± 14.5 species. Appl. Environ. Microbiol. 60, 1292e1296.
Catechol 1,2-dioxygenase 0.0 ± 0.0 0.0 ± 0.0 Cidaria, D., Deidda, F., Bosetti, A., 1994. A rapid method for naphthalene dioxygenase
Catechol 2,3-dioxygenase 0.0 ± 0.0 60.0 ± 5.9 assay in whole cells of naphthalene cis-dihydrodiol dehydrogenase blocked
Protocatechuate 3,4-dioxygenase 0.0 ± 0.0 0.0 ± 0.0 Pseudomonas fluorescens: screening of potential inducer of dioxygenase activity.
Protocatechuate 4,5-dioxygenase 0.0 ± 0.0 0.0 ± 0.0 Appl. Microbiol. Biotechnol. 41, 689e693.
Cornelissen, G., Sijm, D., 1996. An energy budget model for the biodegradation and
Gentisate 1,2-dioxygenase 440.2 ± 71.3 0.0 ± 0.0
cometabolism of organic substances. Chemosphere 33, 817e830.

ska et al. / Journal of Environmental Management 145 (2014) 157e161
D. Wojcieszyn
Divari, S., Valetti, F., Caposito, P., Pessione, E., Cavaletto, M., Griva, E., Gribaudo, G.,
Gilardi, G., Giunta, C., 2003. The oxygenase component of phenol hydroxylase
from Acinetobacter radioresistens S13. J. Biochem. 270, 2244e2253.
Feng, Y., Khoo, H.E., Poh, Ch.L., 1999. Purification and characterization of gentisate
1,2-dioxygenases from Pseudomonas alcaligenes NCIB 9867 and Pseudomonas
putida NCIB 9869. Appl. Environ. Microbiol. 65, 946e950.
Gentili, A., 2007. Determination of non-steroidal anti-inflammatory drugs in envi-
ronmental samples by chromatographic and electrophoretic techniques. Anal.
Bioanal. Chem. 387, 1185e1202.
Gren
, I., Wojcieszyn
ska, D., Guzik, U., Perkosz, M., Hupert-Kocurek, K., 2010.
Enhanced biotransformation of mononitrophenols by Stenotrophomonas mal-
tophilia KB2 in the presence of aromatic compounds of plant origin. World J.
Microbiol. Biotechnol. 26, 289e295.
Gusseme, B.D., Vanhaecke, L., Verstraete, W., Boon, N., 2011. Degradation of acet-
aminophen by Delftia tsuruhatensis and Pseudomonas aeruginosa in a membrane
bioreactor. Water Res. 45, 1829e1837.
Guzik, U., Hupert-Kocurek, K., Wojcieszyn
ska, D., 2013. Intradiol dioxygenases e the
key enzymes in xenobiotics degradation. In: Chamy, R., Rosenkranz, F. (Eds.),
Biodegradation of Hazardous and Special Products. InTech, Rijeka, Croatia,
pp. 129e153.
Hou, Ch.T., Lillard, M.O., Schwartz, R.D., 1976. Protocatechuate 3,4-dioxygenase from
Acinetobacter calcoaceticus. Biochemistry 15, 582e588.
Ivshina, I.B., Rychkova, M.I., Vikhareva, E.V., Chekryshkina, L.A., Mishenina, I.I., 2006.
Catalysis of the biodegradation of unusable medicines by alkanotrophic Rho-
dococci. Appl. Biochem. Microbiol. 42, 392e395.
Juvancz, Z., Barna, S., Gyarmathy, D., Konoro
t, F., 2008. Study of endocrine dis-
rupting chemicals in environment. Acta Polytech. Hung. 5, 49e58.
Lloret, L., Eibes, G., Lu-Chau, T.A., Moreira, M.T., Feijoo, G., Lema, J.M., 2010. Laccase-
catalyzed degradation of anti-inflammatories and estrogens. Biochem. Eng. J. 51,
124e131.
Lurie, J., Rybnikova, I., 1974. Chemical Analysis of Industrial Sewages. Gaschmizdat,
Moscow (in Russian).
Marco-Urrea, E., Perez-Trujillo, M., Blanquez, P., Vicent, T., Caminal, G., 2010a.
Biodegradation of the analgesic naproxen by Trametes versicolor and identifi-
cation of intermediates using HPLC-DAD-MS and NMR. Bioresour. Technol. 101,
2159e2166.
Marco-Urrea, E., Perez-Trujillo, M., Cruz-Morato, C., Caminal, G., Vicent, T., 2010b.
White-rot fungus-mediated degradation of the analgesic ketoprofen and
identification of intermediates by HPLC-DAD-MS and NMR. Chemosphere 78,
474e481.
Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing
sugar. Anal. Chem. 31, 426e428.
Murdoch, R.W., Hay, A.G., 2005. Formation of catechols via removal of acid side
chains from ibuprofen and related aromatic acid. Appl. Environ. Microbiol. 71,
6121e6125.
161
Nikolaou, A., Meric, S., Fatta, D., 2007. Occurrence patterns of pharmaceuticals in
water and wastewater environments. Anal. Bioanal. Chem. 387, 1225e1234.
Parales, R.E., Lee, K., Resnick, S.M., Jiang, H., Lessner, D.J., Gibson, D.T., 2000. Sub-
strate specificity of naphthalene dioxygenase: effect of specific amino acids at
the active site of the enzyme. J. Bacteriol. 182, 1641e1649.
Picquet, M., 2013. Organometallics as catalysts in the fine chemical industry. Plat-
inum Metals Rev. 75, 272e280.
Quintana, J.B., Weiss, S., Reemtsma, T., 2005. Pathways and metabolites of microbial
degradation of selected acidic pharmaceutical and their occurrence in munic-
ipal wastewater treated by a membrane bioreactor. Water Res. 39, 2654e2664.
Rieger, P.G., Meier, H.M., Gerle, M., Voget, U., Groth, T., Knackmuss, H.J., 2002. Xe-
nobiotics in the environment: present and future strategies to obviate the
problem of biological resistance. J. Biotechnol. 94, 101e123.
Rodarte-Morales, A.I., Feijoo, G., Moreira, M.T., Lema, J.M., 2011. Degradation of
selected pharmaceutical and personal care products (PPCPs) by white-rot fungi.
World J. Microbiol. Biotechnol. 27, 1839e1849.
Rodarte-Morales, A.I., Feijoo, G., Moreira, M.T., Lema, J.M., 2012. Biotransformation
of three pharmaceutical active compounds by the fungus Phanerochaete
chrysosporium in a fed batch stirred reactor under air and oxygen supply.
Biodegradation 23, 145e156.
Rodriguez-Rodriguez, C.E., Marco-Urrea, E., Caminal, G., 2010. Naproxen degrada-
tion test to monitor Trametes versicolor activity in solid-state bioremediation
processes. J. Hazard. Mater. 179, 1152e1155.
Sosnowska, K., Styszko-Grochowiak, K., Goła
s, J., 2009. Leki w
srodowisku e
zro
dła,
_
przemiany, zagrozenia. In: Krakowska Konferencja Młodych Uczonych (in Polish).
Sutherland, J.B., Crawford, D.L., Pometto, A.L., 1981. Catabolism of substituted ben-
zoic acids by Streptomyces. Appl. Environ. Microbiol. 41, 442e448.
Wei, M., Zhang, J.J., Liu, H., Zhou, N.Y., 2010. para-Nitrophenol 4-monooxygenase
and hydroxyquinol 1,2-dioxygenase catalyze sequential transformation of 4-
nitrocatechol in Pseudomonas sp. strain WBC-3. Biodegradation 21, 915e921.
Wojcieszyn
ska, D., Guzik, U., Gren
, I., Perkosz, M., Hupert-Kocurek, K., 2011. In-
duction of aromatic ring e cleavage dioxygenases in Stenotrophomonas malto-
philia strain KB2 in cometabolic systems. World J. Microbiol. Biotechnol. 27,
805e811.
Wu, S., Zhang, L., Chen, J., 2012. Paracetamol in the environment and its degradation
by microorganism. Appl. Microbiol. Biotechnol. 96, 875e884.
Yoshida, M., Oikawa, T., Obata, H., Abe, K., Mihara, H., Esaki, N., 2007. Biochemical
and genetic analysis of the g-resorcylate (2,6-dihydrobenzoate) catabolic
pathway in Rhizobium sp. strain MTP-10005: identification and functional
analysis of its gene cluster. J. Bacteriol. 189, 1573e1581.
Zhang, L., Hu, J., Zhu, R., Zhou, Q., Chen, J., 2013. Degradation of paracetamol by pure
bacterial cultures and their microbial consortium. Appl. Microbiol. Biotechnol.
97, 3687e3698.
Zhong, D.F., Sun, L., Liu, L., Huang, H.H., 2003. Microbial transformation of naproxen
by Cunninghamella species. Acta Pharmacol. Sin. 24, 442e447.