Bacterial degradation of pyrene in minimal salt medium mediated

by catechol dioxygenases: Enzyme purication and molecular size

determination
S.N. Singh
a,
, Babita Kumari
a
, Santosh Kumar Upadhyay
b
, Shweta Mishra
a
, Dileep Kumar
c
a
Environmental Science Division, CSIR-National Botanical Research Institute, Lucknow 226001, UP, India
b
Plant Molecular Biology and Genetic Engineering Division, CSIR-National Botanical Research Institute, Lucknow 226001, UP, India
c
Microbial Biotechnology, CSIR-North-East Institute of Science and Technology, Jorhat, India
h i g h l i g h t s
" Microbial degradation of pyrene in MSM.
" Involvement of catechol 1,2 dioxygenase and catechol 2,3 dioxygenase in pyrene degradation.
" Purication and molecular size determination of catechol 1,2 dioxygensae.
a r t i c l e i n f o
Article history:
Received 12 November 2012
Received in revised form 11 January 2013
Accepted 12 January 2013
Available online 4 February 2013
Keywords:
Pyrene degradation
Bacterial strains
Catechol dioxygenases
Enzyme purication
Molecular size determination
a b s t r a c t
In vitro degradation of pyrene was studied in MSM by three bacterial strains individually, designated as
BP10, NJ2 and P2. Among these strains, NJ2 was the highest degrader (60%) of pyrene, followed by BP10
(44%) and the least was P2 (42%) in MSM with pyrene (50 lg ml
1
) in 8 days. During pyrene degradation,
catechol 1,2 dioxygenase (C12O) activity was induced by 13 folds in BP10 and 17 folds in P2 as compared
to catechol 2,3 dioxygenase (C23O). However, in NJ2, C23O activity was augmented 1.3 times more than
C12O. This clearly indicated that C12O played a major role in pyrene degradation by BP10and P2, while in
NJ2, C23O contributed more to degradation process than C12O. Molecular weight of highly inducible
C12O was determined as 64 kDa by size exclusion chromatography and as 32 kDa on denaturing
SDS PAGE in BP10 which indicated dimeric nature of the enzyme.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous con-
taminants in the environment, mainly originating from anthropo-
genic activities like burning of fossil fuels, coal liquefaction and
gasication process, oil seepage and accidental spillage of hydro-
carbons and petroleum industries (Guerin and Jones, 1988; Juhasz
and Naidu, 2000). PAHs are a group of compounds mainly com-
posed of two or more fused aromatic rings in linear, angular and
clustered arrangements. They are classied as low molecular
weight (LMW) as well as high molecular weight (HMW) PAHs
depending upon the number of aromatic rings. LMW PAHs are
characterized by higher volatility, solubility and greater ease of
degradation than HMW PAHs which are thermodynamically stable
and hydrophobic in nature. Hence, HMW PAHs strongly bind to the
soil particles and therefore, become recalcitrant to microbial deg-
radation. Besides, due to their high toxicity and/or mutagenic, tet-
ratogenic and carcinogenic properties (Abd-Elsalam et al., 2009;
Abou-Arab et al., 2010), PAHs pose a serious health risk to human
beings on persistent exposure.
Possible fates of PAHs in the environment are volatilization,
photooxidation, chemical oxidation, bioaccumulation and adsorp-
tion to soil particles. But, microbial transformation and degrada-
tion are projected as an environmentally benign and public
acceptable potential strategy to address PAH contamination in
the soils (Gibson et al., 1975). However, a major constraint for bio-
degradation of PAHs is their low availability to bacteria due to their
high hydrophobicity and strong adsorption to soil (Thomas et al.,
1986; Volkering et al., 1998). A lot of reports are available on bac-
terial degradation of PAHs (Cerniglia, 1992; Wilson and Jones,
1993), but in most of the cases, the organisms have been isolated
from the oil sludge contaminated soil or crude oil spills with the
likelihood that they could have developed the catabolic capability
0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.01.068

Corresponding author. Tel.: +91 522 2297823; fax: +91 522 2205839.
E-mail address: drsn06@gmail.com (S.N. Singh).
Bioresource Technology 133 (2013) 293300
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to degrade PAHs as a result of selective pressure of the contami-
nated site on the organisms as compared to uncontaminated sites.
However, there are reports also available for occurrence of PAH
degrading bacteria from uncontaminated sites (Juhasz and Naidu,
2000; Kanaly et al., 2000). Several species of bacterial genera Pseu-
domonas, Alcaligenes, Mycobacterium, Rhodococcus, Sphingomonas
have been found highly capable of degrading PAHs (Cerniglia,
1992; Mueller et al., 1997). Lease et al. (2011) compared the PAH
mineralization ability of Mycobacterium isolates from both con-
taminated and uncontaminated sites.
Among the HMW PAHs, pyrene is an important compound
which is recalcitrant for microbial degradation due to their high
hydrophobicity which restricts its cellular uptake by microbes
(Seo et al., 2009). The rst report of the pyrene degradation as a
sole source of carbon and energy was demonstrated by Rhodococ-
cus sp. strain UWI (Walter et al., 1991). Liang et al. (2006) studied
the catabolic enzymes involved and metabolites formed during
pyrene degradation by Mycobacterium sp. strain KMS. Interestingly,
Khan et al. (2009) reported more pyrene degradation by the rhizo-
spheric bacteria than non-rhizospheric bacteria and identied the
catabolic genes nahAc and pdo 1 responsible for PAH degradation.
Krivobok et al. (2003) studied involvement of two ring hydroxylat-
ing dioxygenases and also identied pyrene induced proteins in
Mycobacterium sp. Besides, an enteric bacterium Leclercia adecarb-
oxylate PS4040 was reported by Sarma et al. (2010) for pyrene deg-
radation. Recently, Ceyhan (2012) identied a novel strain Proteus
vulgaris 4Bi which degraded pyrene more effectively with the for-
mation of non-toxic and non-persistant metabolites in nature.
In this study, pyrene was chosen as a model compound of HMW
PAHs for degradation in isolation by three bacterial strains (BP10,
P2 and NJ2) isolated from the oil-contaminated soil to examine
their differential degradation ability and to investigate the involve-
ment of catechol 1,2 dioxygenase and catechol 2,3 dioxygenase in
pyrene degradation. Molecular size of highly expressed catechol
1,2 dioxygenase in BP10 was also determined after enzyme puri-
cation and the co-factor with oxidation state was identied.
2. Methods
2.1. Materials
Catechol, folin and pyrene were purchased from SigmaAldrich
while Na
2
HPO
4
, NaH
2
PO
4
, EDTA, NaOH, ammonium potassium tar-
trate, CuSO
4
5H
2
O, hexane, benzene etc. were procured from Fisher
Scientic and culture media like MSM without dextrose, nutrient
agar and nutrient broth were all procured from Himedia.
2.2. Isolation and screening of bacterial strains
2 g of crude oil, collected from Barauni Oil Renery, Barauni (Bi-
har), India was added to 100 ml sterilized minimal salt medium
(MSM) (composition: 7 g dipotassiumphosphate, 2 g monopotassi-
um phosphate, 0.5 g sodium citrate, 1 g ammonium sulfate, 0.1 g
magnesium sulfate in 1 l medium pH 7.0 0.2) and then incubated
in an orbital shaker set at 37 C and 150 rpm for the enrichment of
petroleumhydrocarbon degrading bacterial strains. After 1 week of
incubation, 1 ml of active inoculum was transferred to the asks
having fresh sterile MSM supplemented with petroleum oil
(2% w/v). After 3 transfers in MSM (100 ml) with 2% (w/v) of petro-
leum oil, the active inoculum was used for the isolation of TPH (to-
tal petroleum hydrocarbon) degrading bacteria by serial dilution
method and spreading over the nutrient agar (NA, composition:
10 g peptic digest of animal tissue, 5 g beef extract, 5 g sodium
chloride and 15 g agar in 1 l medium pH 7.4 + 0.2) plates. Separate
colonies were picked up for the purication of bacteria by streak-
ing method on MSM agar plates supplemented with 50 mg l
1
pyrene.
Bacterial strains isolated from petroleum oil and NJ2 (supplied
by NIEST, Jorhat, India) were rst screened on the basis of the uti-
lization of pyrene (50 mg l
1
) in MSM agar. Three strains out of 10
bacterial isolates designated as BP10, P2 and NJ2 showed faster
growth on MSM agar plates compared to other strains. Therefore,
these strains were selected for further study of pyrene degradation.
2.3. Pyrene degradation
Three selected bacterial strains (BP10, P2 and NJ2) were en-
riched separately in 100 ml of nutrient broth (NB) (composition:
5 g of peptic digest of animal tissue, 5 g of sodium chloride, 1.5 g
of beef extract and 1.5 g of yeast extract in 1 l of media) in
250 ml asks in an orbit shaker set at 150 rpm and 35 C till
OD
600
was equivalent to 1. Bacterial cells were harvested by centri-
fugation at 5000g at 4 C for 5 min. Harvested cells were resus-
pended in MSM. Degradation of pyrene by individual strains was
monitored in 100 ml ask containing 20 ml MSM with 50 mg l
1
of pyrene as a source of carbon and energy. For this, sterilized
MSM with 50 mg l
1
of pyrene was inoculated separately with
1 ml of active inoculum of each bacterial strain. Uninoculated ster-
ilized medium was treated as control. Each treatment was pre-
pared in 6 sets, of which 3 sets were used for the extraction of
remaining amount of pyrene after degradation and another 3 sets
for analysis of other parameters like pH, protein, enzyme etc. to
prevent any loss of pyrene during cell harvesting. All asks were
incubated in an orbital shaker set at 150 rpm and 37 C. Samples
were harvested every day after 24 h intervals from both uninocu-
lated and inoculated asks for 8 days.
2.4. Extraction and analysis of pyrene
Residual amount of pyrene after degradation was extracted
from MSM by liquidliquid extraction (1:1 v/v MSM: Benzene).
Samples were extracted three times and pooled together. Extrac-
tion efciency was 98%. Extract was evaporated under a gentle
nitrogen hood and residue was dissolved in 2 ml acetone. Samples
were analyzed by the gas chromatograph (Agilent GC model
7890A) with FID using capillary BP-5 column (5% phenyl methyl
polysiloxane column, 30 m 0.32 mm 0.25 lm). Both injection
and detector temperatures were maintained at 280 C. The initial
oven temperature was kept 80 C for 2 min and then increased to
300 C with 10 C increase per min. The injection volume (5 ll)
of sample was taken for GC analysis.
2.5. Bacterial growth
Bacterial growth was determined by CFU counting. CFU ml
1
of
bacteria was determined by counting colonies on NA plates incu-
bated at 37 C for 24 h through serial dilution method.
2.6. Protein extraction and analysis
Ten ml of sample was centrifuged at 5000g for 10 min and the
cell pellet was washed with 0.01 M phosphate buffer (pH 7.0).
Again the cell pellet was resuspended in the same buffer, sonicated
(for 5 min at 0 C) and then centrifuged at 20,000g for 25 min at
4 C. Supernatant was used for the protein estimation and enzyme
assays. Protein was determined following the standard method of
Lowry et al. (1951).
294 S.N. Singh et al. / Bioresource Technology 133 (2013) 293300
2.7. Enzyme assays
2.7.1. Catechol 1,2 dioxygenase
Catechol 1,2 dioxygenase (C12O, EC 1.13.11.1) was assayed by
the method of Hegeman (1966) using the colorimetric method to
detect the product ciscis muconate (pH 7.0, k = 260 nm;
e = 25,600 mol
1
cm
1
). Reaction mixture contained 1 lmol of
EDTA, 0.1 lM of catechol, 8.7 lM of sodium phosphate buffer
(pH 7.0) and cell extract (0.020.06 mg of protein) in a nal volume
of 1 ml. The increase in OD
260
, as a result of accumulation of cis, cis
muconic acid, was measured by UVVIS spectrophotometer (Per-
kin Elmer Lambda 35).
2.7.2. Catechol 2,3 dioxygenase
Catechol 2,3 dioxygenase (C23O, EC 1.13.11.2) was determined
with an increase in OD
375
concomitant with the formation of
2-hydroxymuconic semialdehyde (pH 7.5, k = 375 nm;
e = 33,400 mol
1
cm
1
) using UVVIS Spectrophotometer. Reaction
mixture contained 48 lM of sodium phosphate buffer (pH 7.5),
0.1 lM catechol and cell extract (0.020.06 mg of protein) in a nal
volume of 1 ml. Cell extract was heated for 10 min at 60 C before
the enzyme activity was assayed following the method of Klecka
and Gibson (1981).
2.8. Expression and purication of catechol 1,2 dioxygenase
MSM with 50 mg l
1
pyrene (as a sole source of carbon) was
used for the growth of bacterial strains for high expression of
C12O. The expression and purication were performed as de-
scribed by Kumar et al. (2011) with some modications. A loop
of single isolated colony was inoculated in 5 ml LB (Luria Bertani
Broth) and grown as primary culture for 24 h at 37 C in orbital
shaker set at 150 rpm. 1 ml primary culture was inoculated in
500 ml MSM with 50 mg l
1
pyrene and grown in an orbital shaker
for 96 h at 37 C with 150 rpm. Cells were sonicated and then har-
vested by centrifugation (6,000g at 4 C for 10 min) and medium
containing extracellular catechol 1,2 dioxygenase enzyme was
used for further purication. Ammonium sulfate (80%) was added
to the medium and left over night at 4 C on stirrer for the precip-
itation of the proteins present in medium. Medium was further
centrifuged at 12,000g at 4 C for 15 min. Precipitated protein
was dissolved in 20 mM Tris HCl buffer (pH 8.0) by 2 h incubation
at room temperature with gentle shaking. Solubilized protein was
further centrifuged at 12,000g at 4 C for 15 min. Supernatant was
dialyzed against 20 mM Tris HCl buffer (pH 8.0) and ltered by
0.22 lm syringe lter (Milipore, USA). Supernatant was loaded
on mono-Q anion exchange column pre-equilibrated with 20 mM
Tris HCl buffer (pH 8.0) at the ow rate of 0.5 ml min
1
. Column
was washed many times till OD
280
became stable and bound pro-
teins were subsequently eluted by 01 M linear gradient solutions
of NaCl. All the fractions were used for the detection of C12O activ-
ity. Fractions containing the higher enzyme activity were analyzed
on SDSPAGE and further puried by size exclusion chromatogra-
phy (superdex 200 column; GE healthcare, USA) in PBS (pH 7.5).
Eluted proteins were analyzed for enzyme activity and purication.
All the steps of purications were performed at 4 C and analyzed
on 12% SDSPAGE.
2.9. Molecular size estimation
The molecular size of the C12O was determined by size exclu-
sion chromatography (on Superdex 200 column) in PBS (pH 7.5)
using standard proteins as marker. First, the column was calibrated
with aldolase (158 kDa), albumin (67 kDa) and ovalbumin (43 kDa)
and then puried C12O protein was used for the determination of
molecular size.
2.10. Detection of enzyme co-factor and its oxidation state
The co factor associated with C12O isolated from BP10 was
determined by atomic absorption spectrophotometer (GBC
P
AAS). To identify the oxidation state of cofactor in C12O, an en-
zyme fraction puried by size exclusion chromatography (Super-
dex 200 column; GE healthcare, USA) in PBS (pH 7.5) with
highest activity of C12O was reacted with the 100 ll of 1 M sodium
thiocyanate to know the oxidation state.
2.11. Identication of bacteria
Bacterial strains were identied by M/S Chromous Biotech, Ben-
galuru (India) on the basis of 16S rDNA technology using ABI 3130
Genetic Analyzer and their homology (>99%) of DNA sequence with
NCBI databases of bacteria. They used 16s forward primer (5
0
-AGA-
GTRTGATCMTYGCTWAC-3
0
) and 16s reverse primer (5
0
-CGY-
TAMCTTWTTACGRCT-3
0
). The sequencing reaction (10 ll)
contained 4 ll Big Dye Terminator Ready reaction mixture, 1 ll
of 100 gg ll
1
, 2 ll of 10 pmol ll
1
primer and 3 ll Milli Q water
and PCR conditions (25 cycles) were: Initial Denaturation: 96 C for
1 min, Denaturation: 96 C for 10 s, Hybridization: 50 C for 5 s and
Elongation: 60 C for 4 min.
3. Result and discussion
3.1. Degradation of pyrene
The pattern of pyrene degradation in MSM by individual bacte-
rial strains clearly indicates that the pyrene degradation was en-
hanced continuously with incubation period till 7 days and then
tended to stabilize. Among the bacterial strains, a maximumdegra-
dation of 60% in pyrene was achieved by NJ2, followed by BP10
(44%) and the least (42%) was recorded in P2 during 8 days of incu-
bation (Fig. 1). However, the level of pyrene degradation also in-
cluded 11% degradation by abiotic factor as observed in control
with no bacterial inoculum. After deduction of pyrene degradation
by abiotic factor, 49% pyrene degradation was in fact contributed
by NJ2, 33% by BP10 and 31% by P2 during this period. Difference
in pyrene degradation ability of bacterial strains was clearly re-
ected even after 1 day incubation which was further magnied
with incubation period. As compared to rst day incubation, the le-
vel of pyrene degradation was enhanced by more than ve folds in
BP10 and more than 6 folds in NJ2 and P2 in 8 days incubation.
Thus, pyrene was degraded by all three bacterial strains, but they
differed widely in their inherent ability. Although there was no
marked difference in the ability of BP10 and P2, but NJ2 degraded
pyrene 1.3 times faster than other two strains.
Biodegradation ability of Sphingomonas and Mycobacterium
strains for PAH degradation has been reported by various workers
(Guo et al., 2010; Kim et al., 2007; Molina et al., 1999). Besides, an
enteric bacterium Leclercia adecarboxylata PS4040 also metabo-
lized pyrene as reported by Sarma et al. (2010). Okparanma et al.
(2009) studied the effectiveness of Bacillus subtilis and P. aeruginosa
in degradation of uoranthene, pyrene, chrysene and
benzo(a)anthracene. Their metabolic differences may be linked to
difference in PAH degrading enzymes. Surprisingly, Kalzadeh
et al. (2012) isolated bacterial strains i.e. Mycobacterium sp., Corny-
bacterium sp., Nocardia sp., Pseudomonas sp., Rhodococcus sp. and
Micrococcus sp. from landlls in Shiraz (Iran) and found them
highly capable of degrading pyrene up to 89.1%, 79.4%, 75.3%,
68.2%, 62.3% and 56.8%, respectively, after 10 days of incubation
period. Recently, Moscoso et al. (2012) isolated microbial consor-
tium of Staphylococcus warneri and Bacillus pumilus from soil pol-
luted with PAHs and heavy metals. This consortium could
S.N. Singh et al. / Bioresource Technology 133 (2013) 293300 295
degrade the phenanthrene, pyrene and benzo (a) anthracene, sep-
arately more than 85% in ask and 90% in bioreactor in 3 days of
incubation. Wu et al. (2009) reported 19% degradation of pyrene
by Ochrobactrum sp. in 30 days which was only half of the degrada-
tion ability of Ochrobactrum intermedium observed in our case.
Pyrene degradation was initially slow due to hydrophobic nat-
ure (non-polar) of the PAH compound which restricts its adherence
to the microbial cells. However, after initial degradation, the polar-
ity was introduced into pyrene which hastened its degradation
process. A complete and integrated pyrene degradation pathway
in Mycobacterium vanbaalenii PYR1 has been elucidated by Kim
et al. (2007) based on systems biology. They used metabolic, geno-
mic, and proteomic approaches to construct complete and inte-
grated pyrene degradation pathway. Earlier, Heitkamp et al.
(1988) had proposed pyrene degradation pathway and also identi-
ed ring oxidation and ring ssion products. M. vanbaalenii can
oxidize pyrene via initial dioxygenation at C-1 and C-2 positions
to form o-methylated derivatives of pyrene 1,2-diol. However,
the predominant pathway is dioxygenation which is initiated at
C-4 and C-5 positions (K-region) to produce cis-4,5-dihydroxy-
4,5-dihydropyrene (Pyrene cis-4,5-dihydrodiol). Rearomatization
of dihydrodiol and subsequent ring cleavage dioxygenation lead
to the formation of 4,5-dicarboxyphenanthrene to be carboxylated
to 4-phenanthroate. The similar pathway of pyrene degradation
was also observed by Ceyhan (2012) in a new bacterial strain P.
vulgaris 4Bi. Interestingly, metabolites formed in this degradation
pathway were found non-carcinogenic (Enke et al., 2007) as phen-
anthrene derivatives are low molecular PAHs and their half life
time is very short as compared to pyrene. Subsequently, cis 3,4-
dihydroxyphenanthrene-4-carboxylate is produced by second
dioxygenation reaction. Rearomatization then forms 3, 4-
dihydroxyphenanthrene which is further metabolized to 1-hydro-
xy-2-naphthoate. The next enzymatic reactions including intradiol
ring cleavage dioxygenation result in the production of o-phthalate
which is further transformed via the keto-adipate pathway to tri-
carboxylic acid (TCA) cycle intermediates.
3.2. Bacterial growth and protein
During 7 days incubation with pyrene, all the three bacterial
strains continued to grow as reected by increasing CFU value
(Fig. 2). This indicates that pyrene was used as a sole carbon and
energy source. The maximum growth was recorded in NJ2
(1.7 10
11
CFU ml
1
), followed by P2 (5.6 10
10
CFU ml
1
) and
the least was observed in BP10 (3.3 10
10
CFU ml
1
) after 7 days
of incubation and then a decline in growth was observed in all
the strains. The growth pattern of different strains largely corre-
sponded to level of pyrene degradation except in BP10. In BP10,
Incubation period (days)
P
y
r
e
n
e

d
e
g
r
a
d
a
t
i
o
n

(
%
)
BP10 NJ2 P2 Control
Fig. 1. Pyrene degradation (50 mg l
1
in MSM) by three bacterial strains; NJ2, BP10 and P2, in isolation during 8 days of incubation.
Fig. 2. Bacterial growth and cell protein during 8 days of incubation in MSM supplemented with pyrene.
296 S.N. Singh et al. / Bioresource Technology 133 (2013) 293300
the growth in terms of CFU was lower than P2 strain, but the level
of pyrene degradation was found to be more in BP10 than P2 strain
showing inherent ability to degrade pyrene faster due to more
induction of catechol 1,2 dioxygenase than P2.
In case of all the bacterial strains, the growth was reduced be-
yond 7 days incubation. Klankeo et al. (2009) also observed an in-
crease in CFU value from 6.32 to 7.55 log CFU ml
1
in
Diaphorobacter sp. and from6.52 to 8.05 log
10
CFU ml
1
in Pseudox-
anthomonas sp. during 99% degradation of pyrene (100 ppm).
Protein content of the bacteria also corresponded to the bacte-
rial growth. It continued to increase with the incubation period and
reached to peak at 7 days incubation in all the bacterial strains and
then declined (Fig. 2). The maximum protein (368 lg ml
1
) was
observed in NJ2, followed by BP10 (342 lg ml
1
) and the least
was found in P2 (298 lg ml
1
). Sarma et al. (2004) also reported
a concomitant increase in cell protein with increasing growth of
L. adecarboxylata strain PS4040 during pyrene degradation.
Although all bacterial strains used pyrene in MSM as a sole
source of carbon and energy, but higher degradation of pyrene by
BP10 compared to P2 despite of its lower CFU value ensures its
inherent ability to degrade pyrene faster.
3.3. Degradative enzymes
3.3.1. Catechol 1,2 dioxygenase (C12O) and catechol 2,3 dioxygenase
(C23O)
When the activity of C12O was examined during pyrene degra-
dation, it was found that C12O activity continued to increase with
incubation period, attaining maximum value after 6 days of incu-
bation. This trend continued up to 7 days of incubation and then
sharply declined at 8 days of incubation. It is evident from
Fig. 3A that after 7 days incubation, the maximum activity of
C12O was recorded as high as 9140 gmol mg
1
protein in BP10,
followed by P2 (6829 gmol mg
1
of protein) and the least was ob-
served in NJ2 (3964 gmol mg
1
of protein). Thus, the activity of
C12O in BP10 was observed 3 times more than NJ2 and 1.5 times
more than P2.
Like C12O, C23O activity also continued to increase with the
incubation period attaining the peak at 7 days incubation and then
declined (Fig. 3B). However, maximum activity of C23O was ob-
served in NJ2 strain instead of BP10 as in case of C12O. In compar-
ison, the C23O activity was found 9 times more in NJ2 than BP10
and 13 times more than P2. Except in NJ2, C23O activity was
invariably lower in BP10 and P2 than C12O. Higher activity of
C12O and C23O in the bacterial strains when incubated with pyr-
ene in MSM, indicates their active involvement in the pyrene deg-
radation. However, C12O activity in BP10 was found more twofold
higher than C23O in NJ2. This shows that cleavage between 1 and 2
positions of benzene rings was of high signicance in the break-
down of pyrene compound mediated by BP10 and P2, but not in
NJ2, as there was no signicant difference in the activity of C12O
and C23O in bacterial strain NJ2.
Catechol is an important intermediate formed in the metabolism
of PAHs by bacteria. They are degraded by two pathways, the ortho
cleavage pathway and the meta cleavage pathway, in which ring
cleavage is the rst step mediated by C12O and C23O, respectively
(Smith, 1994). Differentiated induction of these enzymes in differ-
ent bacterial strains indicates clearly that they followed different
pathways of ring cleavage of pyrene. Dhote et al. (2010) reported
that meta cleavage by C23O was a predominant process of uo-
ranthene degradation in contrast to chrysene degradation. This
observation was further conrmed by Kumar et al. (2011) in uo-
ranthene degradation by uncultured Acinetobacter sp. Involvement
of two ring hydroxylating dioxygenase was supported by Krivobok
et al. (2003) in pyrene degradation. However, Sarma et al. (2010) in-
ferred that in pyrene degradation by an enteric bacterium L. ade-
carboxylata PS4040, the initial attack on pyrene was made by
mono-oxygenation of C-1 position, contradicting the earlier reports
which suggested formation of a dihydrodiol by dioxygenase cata-
lyzed reaction (Kim et al., 2007; Liang et al., 2006). Besides, other
key enzymes, such as dihydrodiol dehydrogenase, oxidoreductase
and epoxide hydrolase are also signicantly induced by the pres-
ence of pyrene as reported by Liang et al. (2006).
3.4. Media pH
A slight decline in medium pH was observed during the degra-
dation of pyrene as reected in Fig. 4. The maximum drop in med-
ium pH was observed in NJ2 (pH 7.196.82), followed by BP10 (pH
7.26.9) and P2 (pH 7.26.96) and the least change was noted in
Fig. 3. Activities of catechol 1,2 dioxygenase (C12O) and catechol 2,3 dioxygenase (C23O) during pyrene degradation.
S.N. Singh et al. / Bioresource Technology 133 (2013) 293300 297
control (pH 7.217.09). A change in medium pH indicated forma-
tion of acidic intermediates during degradation of pyrene by bacte-
ria. pH 7.0 was reported as optimum condition for the degradation
of pyrene by Meng and Jin (2011).
3.5. Expression, purication and molecular size determination of C12O
Since activity of C12O was found highest in BP10 during pyrene
degradation, this enzyme was considered for purication and
molecular size determination. Sample was precipitated by (NH
4
)
2-
SO
4
to minimize the non-protein fraction. A total of 120 mg protein
was extracted in MSM medium from 500 ml culture after 96 h
incubation. Out of which, 102 mg protein was precipitated by
(NH
4
)
2
SO
4
, dialyzed against 20 mM Tris HCl buffer (pH 8.0) and
then fractionated on mono-Q Anion exchange column. Although
several fractions showed the C12O activity, but a fraction showing
highest C12O activity (4802.44 gmole mg
1
min
1
) was only ana-
lyzed on SDSPAGE and then puried on size exclusion column.
Purity of the desired protein eluted from mono-Q column was en-
hanced from 50% to 80% after size exclusion chromatography (Ta-
ble 1). Fold enrichments after several steps of purications were
1.14, 6.23 and 8.09. Then after, puried C12O protein was used
for molecular size determination. The elution volume was com-
pressed with standard proteins such as ovalbumin, albumin and
aldolase. The puried protein eluted from one peak from the size
exclusion column corresponded to 64 kDa. However, the molecu-
lar mass on SDSPAGE was 32 kDa which conrmed the dimeric
nature of puried C12O (Fig. 5).
3.6. Identication of co-factor of C12O and its oxidation state
The presence of iron as a cofactor was conrmed by atomic
absorption Spectrophotometer. Subsequently, when a puried
fraction of C12O was reacted with sodium thiocyanate, the activity
was completely inhibited. This indicated that sodium thiocyanate
made a complex (Fe(SCN)
2+
) with the iron in the Fe
3+
state which
is responsible for inhibition of enzyme activity. This conrms that
iron was present in the Fe
3+
state as a co-factor in C12O.
3.7. Identication of bacterial strains
Three bacterial strains BP10, P2 and NJ2 used for degradation
study of pyrene in in vitro conditions were identied by M/s Chro-
mous Biotech, Bengaluru (India) based on 16S rDNA technology.
The 16S rDNA sequences of BP10, P2 and NJ2 showed 100%, 99%
and 92% similarity to Pseudomonas YL8 (NCBI No. EU363698),
Fig. 4. Changes in pH of MSM during pyrene degradation.
Table 1
Enrichment of catechol 1,2-dioxygenase after different steps of purication.
Step Total
protein
(mg)
Specic
activity
Total
activity
Fold
enrichment
Medium after 96 h of expression 120 769.82 92378.4 0
Ammonium sulphate
precipitation
102 884.92 90261.84 1.14
Anion exchange chromatography 6.85 4802.44 32896.71 6.23
Size exclusion chromatography 2.40 6234.86 14963.66 8.09
Fig. 5. SDS page analysis of catechol 1,2 dioxygenase purication steps; M:
molecular weight marker, (1) total protein in medium, (2) puried on mono-Q
column, (3) puried on superdex size exclusion column.
298 S.N. Singh et al. / Bioresource Technology 133 (2013) 293300
Ochrobactrum intermedium; ADV1 (NCBI No. AF526509) and Rhodo-
coccus pyridinvorans (T); PDB9 (NCBI No. AF173005), respectively.
However, the 16s rDNA sequence of BP10 has been only reected
in Fig. 6 as C12O was puried from this strain for molecular size
determination.
4. Conclusion
Among the three bacterial strains, isolated from petroleum oil,
R. pyridinvorans NJ2 was found to be highest degrader of pyrene
in MSM, followed by Pseudomonas BP10 and O. intermedium P2 in
decreasing order. It was also conrmed that both C12O and C23O
were actively involved in pyrene degradation, but their activities
were differentially induced expressed in the different bacteria.
The molecular size of C12O isolated from Pseudomonas BP10 was
determined as 64 kDa showing dimeric nature. Presence of Fe
3+
as a cofactor in C12O was also conrmed.
Acknowledgements
Authors are thankful to Director, National Botanical Research
Institute, Lucknow for his active support and CSIR for nancial sup-
port to the Project NWP019. Help rendered by lab trainees Ms.
Priyanka Gaur and Ms. Sucheta Rajput are also duly acknowledged.
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