American-Eurasian J. Agric. & Environ. Sci.

, 15 (5): 800-812, 2015

ISSN 1818-6769
© IDOSI Publications, 2015
DOI: 10.5829/idosi.aejaes.2015.15.5.93218

Isolation, Screening and Co-Metabolism of

Polycyclic Aromatic Hydrocarbons by Soil Bacteria
1
Sumera Afzal Khan, 2Muhammad Hamayun, 1Nadia Bibi and 3Sikandar Khan Sherwani,

1
Centre of Biotechnology and Microbiology, University of Peshawar
2
Department of Botany, Abdul Wali Khan university Mardan
3
Department of Microbiology, Federal Urdu University, Karachi, Pakistan

Abstract: Eight bacterial strains were isolated from rhizospheric soil of Morus alba growing at University road
Peshawar and screened for five PAHs namely naphthalene, acenaphthene, anthracene, phenanthrene and
pyrene. Isolated strains were screened on solid nutrient agar and then on mineral media. Strains sprayed with
pyrene produced yellow colour on solid media were due to formation of PAHs degradation metabolites. In order
to confirm the degradation, the strains were inoculated into mineral media containing selected PAHs as sole
carbon source without shaking. A remarkable phenomenon was observed, four strains show best growth on
four- ringed pyrene. The most efficient degrader of pyrene under static condition was further studied for pyrene
and co-metabolically or the mixture. The strain M3 was best in terms of bacterial growth on pyrene individually
and very efficiently degraded PAHs mixture. Naphthalene was lagging behind by anthracene, acenaphthene
and phenanthrene, while pyrene transformation was carried out between 5th to 7th days of incubation.
Acenaphthene was degraded by naphthalene through 1, 8- dicarboxylic acid pathway. The major metabolites
forms were salicylic acid, salicyalaldehyde and catchol. 6, 7-Benzocaumarin was also formed, which is an
indication of the biotransformation of anthracene and phenanthrene via naphthalene pathway. Experiment was
performed in duplicate and data was analysed by 2-way ANOVA and level of significance < 0.002.

Key words: Solid Media Screening Spray Plate Technique Co-metabolism Morus alba

INTRODUCTION such as polycaetes, nematode, bivalves and crustaceans

Polyaromatic hydrocarbons (PAHs) composed of two
or more fused aromatic rings arranged in linear or cluster.
PAHs are carcinogenic, genotoxic and cytotoxic to the
higher organisms and their derivatives have been
documented as the main offenders causing human lung,
bladder, breast cancer, anaemia, asthma, splenomegaly
[1-4]. PAHs are formed as a product of the incomplete
combustion of fossil fuels and are present in a variety of
products such as tar, coal, soot, petroleum, cutting oils
and tobacco smoke. Many PAHs are in the United States
Environmental Protection Agency Priority Pollutant List,
including acenaphthene, naphthalene, anthracene,
phenanthrene, acenaphthylene, fluoranthene, pyrene,
benzo[a]pyrene and benzo[a]anthracene [5]. PAHs impact
the aquatic environment by targeting the bottom feeder
[6, 7], thus generating a disturbance in the aquatic
systems. PAHs may be released into the environment
when the soils or sediments are disturbed. Released PAHs
become accessible to the benthic organisms and other
aquatic life. PAHs are susceptible to bio concentration or
bioaccumulation in food chain [8].
The major concern is about PAHs which act as
ubiquitous pollutant with potential toxic and carcinogenic
effect, persist in the environment for longer period due to
their hydrophobic nature. The genotoxicity of PAHs also
increases with their size, up to at least four or five fused
benzene rings. Release of toxic PAHs by natural and
anthropogenic activities into the ecosystem is the root
cause for air, water and soil pollution which leads to
deleterious effects on plants, animals and human health
[9, 10].

Corresponding Author: Sumera Afzal Khan, Centre of Biotechnology And Microbiology, University of Peshawar, Pakistan.

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 Am-Euras. J. Agric. & Environ. Sci., 15 (5): 800-812, 2015
The fate of hydrocarbons in the environment is
associated with both abiotic and biotic processes which
include volatilization, photo oxidation, chemical oxidation,
bioaccumulation and microbial transformation [11].
Some microorganisms unexpectedly degrade
contaminants while oxidizing or reducing other
compounds for energy and carbon [12]. A great number
of strains capable to metabolize/co-metabolize these
compounds have been isolated. Till twenth century
there were no reports of axenic microbial cultures utilizing
PAHs containing four or more fused rings as the sole
source of carbon and energy. Since then, a number of
pure cultures able to co-metabolize fluoranthene and
pyrene have been reported [13].
Co-metabolic bioremediation is the breakdown of a
contaminant by an enzyme or cofactor that is formed
during microbial metabolism of another compound.
This type of bioremediation is highly targeted because it
ensures that only the microbes that can degrade the
contaminant of concern are stimulated. A significant
advantage of co-metabolic bioremediation is that
biodegradation can be stimulated at low contaminant
concentrations, below concentrations that would make
available a carbon and energy benefit to the
microorganisms if a direct aerobic or anaerobic
bioremediation route was taken. PAHs with two and
three rings, such as naphthalene and phenanthrene,
acenaphthene and acenaphthylene and fluorene are
somewhat easy to degrade [14].
In aerobic Co-metabolic bioremediation, the
contaminant is oxidized by an enzyme or cofactor
produced during microbial metabolism of another
compound with oxygen. In anaerobic co-metabolic
bioremediation, the contaminant is reduced by an enzyme
or cofactor produced during microbial metabolism of
another compound in an environment lacking oxygen.
During co-metabolic biodegradation of the contaminant
does not produce any energy and growth benefit for
the microbe mediating the reaction. Co-metabolic
bioremediation has been used on a variety of
contaminants, including PAHs, explosives, dioxane,
polychlorinated biphenyls, pesticides and halogenated
aliphatic hydrocarbons. This process needs substrate to
stimulate the appropriate reactions. The electron donors
observed in co-metabolic aerobic oxidation of PAHs
include toluene and phenol [14]. The bacteria involved
in co-metabolic degradation do not receive a carbon or
energy benefit from the contaminants. In addition,
intermediate products that act as inhibitors of microbial
801
metabolism can be produced during co-metabolic
bioremediation [15-18]. In present study eight bacterial
strains were isolated and screened on solid media for
utilization of the five selected PAHs individually and
mixture of these by process of co-metabolism.
MATERIALS AND METHODS
Nutrient agar and Mineral salt medium was used for
initial screening. The composition of PNR and PNRG (PNR
+ 5 mM glucose) per liter of distilled water [19, 20], is as;
PN (20x) 50 mL used as 50 mL/L: KH2PO4 13.6 % (w/v),
(NH4)2SO4 2.4 % (w/v), NaOH 2.5 % (w/v) and R salts
used as 7ml/L MgSO4.7H2O 8 % (w/v), FeSO4.7H2O 0.2
% (w/v), HCl 0.4 % (w/v), Agar (2 %) was used as
solidifying agent. All the chemicals naphthalene,
acenaphthene, anthracene, phenanthrene, pyrene,
salicylic acid, salicylaldehyde, dicarboxylic acid and
catechol, purchased from Merck and Fluka of and were
analytical grade.
Collection of Soil Samples: Rhizosphreic soil of Morus
alba growing at Peshawar university campus near a
heavily trafficked road was collected. The samples were
placed into sterile polythene bags and immediately
brought to the laboratory and stored at 4°C.
Isolation of Bacteria from the Soil Samples: Bacteria were
isolated from the soil samples using serial dilution and
spread plate technique on nutrient agar media and
incubation till appearance of growth. Inoculated plates
were purified by repeatedly sub culturing and streak
plate method. Colonies appeared were picked and cultured
in new nutrient plates. Multi steps of purification were
performed until pure strains were obtained. Purification
process involved streak plate method and was done to
ensure that only single strain was obtain for each plating.
Pure cultures obtained were stored in slants of enrichment
medium with 2.0 g/L pure agar and stored at 4°C [20].
Screening on Solid Media for PAHs Degradation:
Screening of the isolates for the ability to use anthracene
as sole source of carbon and energy for biodegradation
was carried out on both solid and liquid mineral media.
A qualitative assay by the spray-plate method was used
to check the degradation potential of the isolated
bacterial strains for growth on PAHs [8, 19, 21-24].
Initially, the strains were screened on nutrient agar
and then on mineral salt medium (PNRG) and (PNR).
 Am-Euras. J. Agric. & Environ. Sci., 15 (5): 800-812, 2015
Initially, the strains were screened on nutrient agar
and then on mineral salt medium (PNRG) and (PNR).
Initial concentration range was 25 ppm-1200 ppm for
anthracene, 100-800 ppm (phenanthrene), 100-1000ppm
(naphthalene and acenaphthene), 10-1000 ppm (pyrene)
and incubated until appearance of growth. PNR plates
were prepared and inoculated with bacteria and sprayed
with 0.1% of all the five selected PAHs which was
appropriately labeled. The plates were incubated at room
temperature (about 28°C) for four days. Appearance of
growth on mineral plates with PAHs spray was an
indication of the utilization of PAH. Those strains with
best growth were selected for further studies. To confirm
the degradation capability, PAHs were added to liquid
mineral media as sole carbon and energy source. Co-
metabolism study was performed according to protocol of
[25] with some modifications.
High Performance Liquid Chromatography (HPLC)
Analysis: Followed by the extraction with ethyl acetate,
the samples were filtered through 0.2mm syringe filter and
analyzed in a high performance liquid chromatography.
HPLC (PerkinElmer) with the C18 reverse phase column
was used to analyze PAHs under isocratic condition
using acetonitrile: water (80:20) (v/v) as mobile phase and
detection wavelength – 254 nm. The flow rate of the
mobile phase (acetonitrile) was maintained at 1.2 mL/min.
The samples volume was 2mL in each vial of HPLC tray.
Metabolites were studied on the basis of retention time
comparison with available authentic standards and
literature values.
RESULT AND DISCUSSION
A number of researches have been carried out on
degradation of low molecular weight and high molecular
weight PAHs by microbes. Up until now, only a few
genera of bacteria have been isolated with the ability to
utilize four-ring PAHs as sole carbon and energy
sources while co metabolism of five-ring compounds
has been described [26, 27]. The biodegradability mainly
depends on the complexity of chemical structures and
physico-chemical properties of PAHs [28]. When mixture
of five PAHs was present in aqueous solution, the
degradation rates were delayed for phenanthrene and
reduced for anthracene and pyrene. In an experiment with
phenanthrene revealed the presence of 2/3-hydroxy-
Phenanthrene, a hydroxy-methoxy-Phenenanthrene, 9,
802
10-Phenanthrenequinone and one unidentified compound
in metabolite form. This indicates that the three PAHs
were reduced to lower molecular compounds. It can be
assumed that the majority of metabolites were not
synthesized in the roots but are a result of microbial
degradation in the rhizosphere [29].
The degradation capabilities of bacterial strains on
solid medium in this study are in line with the previously
reported studies [30, 31]. A total of seventeen strains were
isolated, among which eight were capable of growth on
PNR media supplemented with PAHs. As shown in
(Table 1, 2) three strains showed best growth upto 1200
ppm concentration, two were best at 700 and 900 ppm on
anthracene and naphthalene respectively. While, the
remaining three strains were able to tolerate only 300 ppm
anthracene. The maximum growth conc. range for
acenaphthene, structurally different compound form
naphthalene was 800 ppm and 900 ppm for phenanthrene
(Table 3, 4). When these strains were screened for
pyrene, five of these showed best growth upto 1100 ppm.
Two were best upto 500 ppm and one with capability of
growth upto 800 ppm pyrene (Table 5). It was found that
the degradation rate reduced with an increase in time and
complete degradation was not attained even over a
prolonged time [32- 34]. It is perceived that the percentage
of degradation in studies conducted in solid media is
lower compared to liquid culture studies. These studies
show that complete degradation is not attainable in solid
media [35- 38].
In order to study the utilization potential of these
strains, liquid media screening was carried out as shown
in Fig. 1. Our results of liquid media are comparable with
the previously reported studies. The bacterial growth
(OD 600 nm) values for M2 and M3 were 0.251 and 0.213
for naphthalene and 0.141 and 0.199 for anthracene
respectively. The OD value for M3 on phenanthrene was
0.231 and pyrene it was 0.410. While, for M, M1 and M2
the growth values calculation was 0.215, 0.369 and 0.328
correspondingly. Bacterial growth with pyrene
individually was observed to be increasing rapidly
between 48 to 72 hours and relatively slower during
further 24 hours shown in Fig. 2. While on incubation for
another 24 hours a rapid increase was seen with an OD
value of 0.970. In co-metabolic transformation bacterial
growth was faster with OD of 0.306 than individual four
ringed pyrene with 0.247 in only 24 hours. But, situation
was different after 72 hours with increase bacterial growth
with individual (pyrene 0.590) than mix PAHs (0.481)
 Am-Euras. J. Agric. & Environ. Sci., 15 (5): 800-812, 2015

Table 1: Screening for Anthracene on solid PNRG/PNR for agar media 72 Hours
Anthracene Concentration in ppm
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
S.No. Strain 25 50 100 200 300 400 500 600 700 800 900 1000 1100 1200
1. M +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
2. M2 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
3. M3 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
4. M4 +++ +++ +++ +++ +++ +++ +++ +++ +++ + + + + +
5. M5 +++ +++ +++ +++ +++ ++ ++ + + + + + - -
6. M6 +++ +++ +++ +++ +++ + + + + + + + + -
7. M7 +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ + + + -
8. M8 +++ +++ +++ +++ +++ +++ +++ +++ ++ + + - - -
+++ = Rich growth ++ = Medium growth + =Less growth (-) = No growth

Table 2: Screening for Naphthalene on solid PNRG/PNR agar media for 72 Hours
Naphthalene Concentration in ppm
----------------------------------------------------------------------------------------------------------------------------------------------------------------
S.No. Strain 50 100 200 300 400 500 600 700 800 900 1000 1100
1. M +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ + +
2. M1 +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ + +
3. M2 +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ + +
4. M3 +++ +++ +++ +++ +++ +++ ++ ++ ++ + + +
5. M4 +++ +++ +++ +++ ++ ++ ++ ++ + + + -
6. M5 +++ +++ +++ +++ + + + + + + - -
7. M6 +++ +++ +++ +++ +++ ++ ++ ++ ++ - - -
8. M7 +++ +++ +++ +++ +++ +++ +++ + + + - -
+++ = Rich growth ++ = Medium growth + =Less growth (-) = No growth

Table 3: Screening for Acenaphthene on solid PNRG/PNR agar media for 72 Hours
Acenaphthene Concentration in ppm
----------------------------------------------------------------------------------------------------------------------------------------------------------------
S.No. Strain 50 100 200 300 400 500 600 700 800 900 1000 1100
1. M +++ +++ +++ +++ +++ +++ +++ +++ ++ + + +
2. M1 +++ +++ +++ +++ +++ +++ ++ ++ ++ + + +
3. M2 +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ + +
4. M3 +++ +++ +++ +++ +++ +++ ++ ++ + + + +
5. M4 +++ +++ +++ +++ ++ ++ ++ ++ + + + -
6. M5 +++ +++ +++ +++ + + + + + + - -
7. M6 +++ +++ +++ +++ +++ ++ ++ ++ ++ - - -
8. M7 +++ +++ +++ +++ +++ ++ ++ + + + - -

Table 4: Screening for Phenanthrene on solid PNRG/PNR agar media for 72 Hours
Phenanthrene Concentration in ppm
----------------------------------------------------------------------------------------------------------------------------------------------------------------
S.No. Strain 50 100 200 300 400 500 600 700 800 900 1000 1100
1. M +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ + +
2. M1 +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ + +
3. M2 +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ + +
4. M3 +++ +++ +++ +++ +++ +++ ++ ++ ++ + + +
5. M4 +++ +++ +++ +++ ++ ++ ++ ++ + + + -
6. M5 +++ +++ +++ +++ ++ ++ ++ + + + - -
7. M6 +++ +++ +++ +++ +++ ++ ++ ++ ++ - - -
8. M7 +++ +++ +++ +++ +++ +++ +++ + + + - -
+++ = Rich growth ++ = Medium growth + =Less growth (-) = No growth

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 Am-Euras. J. Agric. & Environ. Sci., 15 (5): 800-812, 2015

Table 5: Screening for Pyrene on solid PNRG/PNR agar media for 72 Hours
Pyrene Concentration in ppm
------------------------------------------------------------------------------------------------------------------------------------------------------------------------
S.No. Strain 10 20 50 100 200 300 400 500 600 700 800 900 1000
1. M +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
2. M1 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
3. M2 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
4. M3 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
5. M4 +++ +++ +++ +++ +++ ++ ++ + + + + + -
6. M5 +++ +++ +++ +++ +++ + + + + + + + +
7. M7 +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ + + +
8. M8 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
+++ = Rich growth ++ = Medium growth + =Less growth (-) = No growth

Fig. 1: Screening for PAHs degradation in liquid media under static conditions

Fig. 2: Bacterial growth on pyrene as sole carbon source

Fig. 3: Growth of bacteria on PAHs mixture by cometabolism

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 Am-Euras. J. Agric. & Environ. Sci., 15 (5): 800-812, 2015
Table 6: Biochemical tests
Microscopy Biochemical tests
----------------------------------------------------------------------------------------------------------------------------------------------------------------------
Rods Gram’s Test
+ive
which also remains same on fourth day of incubation
(Fig. 3). The overall increase in growth was better at single
than mix PAHs but removal efficiency was far better
co-metabolically than individually [39]. The biochemical
characterization of best degrading strain was made as
shown in Table 6 and was selected for co-metabolic
activity of PAHs mixture.
Co-metabolism as mean to enhance the degradation
of high molecular weight PAHs in particular, those with
four or more aromatic rings has been observed. Only few
studies have observed the improved degradation of
recalcitrant PAHs when more liable PAHs are present.
It has recently been reported that stimulation of dibenz
[a, h] anthracene and benzo[a]pyrene degradation by
Burkholderia cepacia was accomplished by the addition
of small quantities of phenanthrene to culture containing
these compounds. In contrast, the degradation of
fluoranthene and pyrene by a Rhodococcus sp. was
inhibited when phenanthrene was added to cultures
containing either fluoranthene or pyrene this effect was
thought to be a result of the blocking of the induction of
PAH-degrading enzymes. In this work, we observed that
phenanthrene could stimulate microbial degradation of
fluoranthene and pyrene when added to the
Sphingomonas strain P2 cultures containing these
compounds [40].
Influence of growth medium on co-metabolic
degradation rate of single and mixed PAHs was studied
on PNRG and PNR media. All the strains show slower
growth on PNRG than PNR which can be attributed to the
fact that the presence of alternate carbon source can
inhibit the PAHs degrading enzymes. On PNR media
assimilated PAHs as sole carbon within 3-4 days showing
a very rich growth. As reported earlier for Sphingomonas
sp. strain PheB4 was able to cometabolize anthracene,
fluorene and fluoranthene using different mineral media
like NB/ PMSM as the growth substrate. On the NB agar
plates coated with anthracene, fluorene, or fluoranthene,
as well as the PMSM agar plates, the colony formed was
surrounded by a clear zone. Growths of the isolate was
directly related to the rate of co-metabolic degradation of
PAH, NB grown culture had higher cell growth and
Catalase Starch hydrolysis Citrate Urease
+ive +ive +ive +ive
co-metabolized single PAH at a more rapid rate compared
to PMSM-grown culture. In these cultures, fluorene and
anthracene were completely removed within 1 and 3 days,
while in PMSM grown culture it took 3 and 7 days for
complete degradation of fluorene and anthracene,
respectively.
Growth was significantly less in PMSM than in NB
that a synthetic mixture of fluorene and fluoranthene is
cometabolized to a larger extent in NB grown culture than
in PMSM-grown culture. In terms of phenanthrene, its
degradation was significantly reduced when other PAH
was present in the medium. When phenanthrene at an
initial concentration of 10 mg l 1 was the sole carbon
source, complete degradation by Sphingomonas sp.
strain PheB4 occurred within 1 day, but it took 3 and 7
days in the presence of fluorene and anthracene,
respectively. If fluoranthene was added, phenanthrene
degradation was incomplete even at day 7 with
phenanthrene remaining in the medium. When this strain
was grown in synthetic mixture of five PAHs, enhanced
degradation of phenanthrene was observe. This strain
was also able to utilize anthracene cometabolically as it
was unable to assimilate it individually [27].
Pseudomonas stutzeri CET 930 was studied for the
first time as bioremediation agent for the degradation of
effluents containing phenanthrene, pyrene and
benzanthracene, both individually and mixed. The
promising results of degradation obtained at flask scale
marks the onset of the operation at bench scale
bioreactor. There is a great adaptability of this strain for
the degradation of structurally different polycyclic
aromatic hydrocarbons; a complete mineralization was
pursued [41].
M3 after twenty hours of incubation a mixture of
salicylic acid, salicylaldehyde and phenanthrene
dicarboxylic acid was observed with 87.01, 1.43 and 3.92%
respectively (Table 7). Our strain has strong analogy with
previously reported study on Sphingomonas VKM has
revealed that it has the capability to transform substances
naphthalene, acenaphthene, phenanthrene and
anthracene separately and PAH mixtures and co-metabolic
oxidization of pyrene. Acenaphthene was degraded by the
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 Am-Euras. J. Agric. & Environ. Sci., 15 (5): 800-812, 2015
Table 7: Cometabolism metabolites by M3
Time in Hours
24
72
120
168
strain via naphthalene-1, 8-dicarboxylic acid and 3-
hydroxyphthalic acid. Transformation of most other
PAHs was restrained to the cleavage of only one aromatic
ring. The major oxidation products of naphthalene,
phenanthrene, anthracene and pyrene were identified as
salicylic acid, 1-hydroxy-2-naphthoic acid, 3-hydroxy-2-
naphthoic acid and dihydroxydihydropyrene respectively.
Quantitative oxidation of phenanthrene and anthracene
to the corresponding hydroxynaphthoic acids occurred
[42].
On 72 hours of incubation the compound
identified were 6, 7-benzocaumarin and 1-methoxy-2-
hydroxyanthracene of 31.34% and 65.55%. After 120
hours 31.07% phthalic acid and 63.05% catechol which
on further degradation of 48 hours transformed to
salicylaldehyde. Phthalic is reported metabolite of
naphthalene degradation via salicylic acid and of
phenanthrene when degraded through naphthalene
pathway [43]. Acenaphthene and fluoranthene were
degraded by the strain via naphthalene-1, 8-dicarboxylic
acid and 3-hydroxyphthalic acid. While according to
reported work on phenanthrene, as a model PAH and
1-hydroxy-2-naphthoic acid (93%), diphenic acid (89%)
and phthalic acid (100%), as putative metabolites, the
extraction and fractionation procedures resulted in
recoveries of and 89%, respectively. The application of
the standardized system to study the biodegradation of
phenanthrene in an agricultural soil with and without
inoculation of the high molecular weight PAH-degrading
strain Mycobacterium sp. AP1, demonstrates its
suitability for determining the environmental fate of PAHs
in polluted soils and for evaluating the effect of
bioremediative treatments [44].
In contrast to earlier work, our strain able to degrade
pyrene as sole carbon and energy source for growth
was examined. It was unable to utilize anthracene and
phenanthrene and shows no growth. To induce its ability
to degrade these compounds, naphthalene, acenaphthene
and phenanthrene was added to anthracene and
pyrene-containing media. Primary oxidation products of
B. thermoleovorans included 1-naphthol, 2-naphthol and
806
HPLC Metabolites
Salicylic acid (87.01%),
Salicylaldehyde (1.43%), Phenanthrene-1,8 dicarboxylic acid (3.92%)
6, 7-benzocaumarin (31.34%)
1-methoxy-2-hydroxyanthracene (65.55%.)
Phthalic acid (32.03%), catechol (63.77%)
Benzocaumarin (93.13%)
2, 3 dihydroxynaphthalene.1-Naphthol and 2-naphthol
have been described as intermediates during the co-
metabolization of naphthalene by other Bacillus strains
[45].
It was reported that bacterial strains can utilize
four-ring PAHs, pyrene and fluoranthene as sole carbon
and energy source for growth after induction this
capability with phenanthrene. In our study the strains
were adopted initially on anthracene proved more efficient
pyrene degraders. Phenanthrene was degraded almost
completely in 72 hour by Sphingomonas sp. strain P2 in
contrast with our results in which phenanthrene was
converted to phenanthrene dicarboxylic acid just in a few
hours of incubation. Anthracene, acenaphthene and
naphthalene and pyrene were also observed to disappear
from the medium in 72 hours. The compound identified
were 6, 7-benzocaumarin and 1-methoxy-2-
hydroxyanthracene. Substantial amount of pyrene was
decreased over 7 days of incubation [8].
Previously reported studies on pyrene and salicylate
medium could support a vast number of bacteria. The
incorporation of salycilate in the mineral medium could
have favoured the induction of PAH degradation,
probably through the salycilate pathway [46]. The
formation of yellow colour product is an indication of
pyrene degradation through catechol pathways as this
colour is produced from meta cleavage of catechol. During
growth on phenanthrene in batch culture, the water-
soluble intermediates causing a change in color of the
medium from clear to yellow was detected. However, the
accumulation of catechol-like compounds and their meta-
ring cleavage products may be attributed to this color
change [47]. However, it cannot be clear whether
genetic induction or degradation by co-metabolism only
occurred among those bacterial populations. Moreover,
pyrene degrading abilities of Ralstonia, would lead to the
assumption that some of the bacterial genera, i.e. could
have developed only by the assimilation of salycilate or
pyrene intermediate metabolites. Our studied strains were
adopted on solid media with selected PAHs individually
and then for co-metabolic degradation [48].
 Am-Euras. J. Agric. & Environ. Sci., 15 (5): 800-812, 2015
Conversion of most other PAHs was limited to the
cleavage of only one aromatic ring. The major oxidation
products of naphthalene, phenanthrene, anthracene,
chrysene and benzo[a]pyrene were identified as salicylic
acid, 1-hydroxy-2-naphthoic acid, 3-hydroxy-2-naphthoic
acid, o-hydroxyphenanthroic acid and o-hydroxypyrenoic
acid, respectively. Fluorene and pyrene were oxidized
mainly to hydroxyfluorenone and
dihydroxydihydropyrene, respectively [49].
Unlikely, soil Pseudomonads which can degrade
naphthalene and anthracene are able to transform the
particular substrate to 1, 2-dihydroxynaphthalene and 2,
3-dihydroxynaphthalene, respectively, degradation
pathway of mesophilic pseudomonads, was not
detected in B. thermoleovorans. Yet, several side chain
degradation products of 2-hydroxybenzal pyruvic acid,
such as coumarin and its cyclization product of 2-
hydroxycinnamic acid, 2-hydroxybenzene-3-propanoic
acid and its cyclization product, 3,4-dihydrocoumarin, as
well as salicylic acid, were identified. Metabolites such
as salicylic acid and coumarin do fit in the previously
published pathways of the bacterial naphthalene
metabolism. 2-Hydroxyphenyl-3 propanoic acid is likely to
result from a hydrogenation reaction of its unsaturated
analogue 2-hydroxycinnamic acid. A further cyclization
reaction would produce 3, 4-dihydrocoumarin. Additional
transformation products of B. thermoleovorans were
identified that have not been reported to be part of the
known naphthalene degradation pathway. 2-
Carboxycinnamic acid, phthalic acid and benzoic acid, as
well as 2, 3-dihydroxynaphthalene, were determined [50].
Salicylic acid could be further oxidized to another
newly identified accumulated intermediate of
acenaphthylene metabolism, gentisic acid. Gentisic acid
was found to be the product of various kinds of PAHs
metabolism, such as the degradation of naphthalene via
salicylic acid and in fluorene degradation. Gentisic acid
could be further oxidized by strain CU-A1 to maleyl
pyruvate and fumaryl pyruvate as reported in Ralstonia
sp. strain U2 [51]. All individual strains were found
capable of co-metabolic degradation of PAH in a range
varying among strains. Inhibition phenomena, sometimes
drastic, were often observed but synergistic interactions
were also detected. Naphthalene was noxious to all strains
not isolated on this compound. Strain associations were
found efficient in relieving inhibition phenomena,
including the toxic effect of naphthalene. Accumulation of
water-soluble metabolites was consistently observed
during PAH degradation [52].
807
Acenaphthene was degraded by the strain via
naphthalene-1, 8-dicarboxylic acid and 3-hydroxyphthalic
acid. Conversion of most other PAHs was restrained to
the cleavage of only one aromatic ring [53]. In contrast, to
the earlier reports on Pseudomonas cepacia F297 and P.
aeruginosa PA01 and PA01 (pRE695) that naphthalene-1,
8-dicarboxylic acid was identified as the dead-end
metabolite of acenaphthene and acenaphthylene. It was
thought that naphthalene-1, 8-dicarboxylic acid could
be decarboxylated at one of the carboxyl groups to
1-naphthoic acid by Ralstonia sp. strain CU-A1can be
compared with the oxidation reaction of phenantherene-4,
5-dicarboxylic acid, an intermediate of pyrene
degradation by Mycobacterium sp. strain PYR-1 [51].
Acenaphthenone was probably further oxygenated to
give the quinone, which underwent hydrolysis to
naphthalene 1, 8-dicarboxylic acid, also detected as its
anhydride [54]. After 72 hours of incubation the
compound identified were 6, 7-benzocaumarin and 1-
methoxy-2-hydroxyanthracene of 31.34% and 65.55%.
After 120 hours 31.07% phthalic acid and 63.05%
catechol which on further degradation of 48 hours
transformed to salicylaldehyde. M3 a few hours after
incubation a mixture of salicylic acid salicylaldehyde and
phenanthrene dicarboxylic acid was observed with 87.01,
1.43 and 3.92% respectively. Data obtained from extract
of pyrene cultures inoculated with Corynebacterium
variabilis sp. Sh42 suggest the proposed pyrene
biodegradation which also involve phenanthrene,
naphthalene and o-phethalate degradation pathways [55].
This pathway was comparable to that suggested
for biodegradation of pyrene by Mycobacterium sp.
Strain KMS. Rendering to the literature review, at least
15 enzymes are involved in the degradation of pyrene and
the o-phethalate degradation from phenanthrene, with
some enzymes being common to the degradation of both
PAHs. Pyrene is first by a dioxygenase to form cis-4, 5-
pyrene-dihydrodiol, cleaved which is rearomatized to form
4, 5-dihydroxy-pyrene by dihrdrodiol dehydrogenase. 4,
5-di-hydroxy-pyrene is subsequently cleaved to produce
phenanthrene-4, 5-dicarboxylic acid by intradiol
dioxygenase, followed by loss of a carboxyl group by
decarboxylase and 4-phenanthroic acid is formed.
Oxidation of 4-phenanthroic acid by ring-hydroxylating
dioxygenase produces 3, 4-phenanthrene dihydrodiol-4-
carboxylic acid, which is further transformed to 3, 4-
dihydroxyphenanthrene by dehydrogenase/
decarboxylase. Once 3, 4-dihydroxyphenanthrene is
formed, it enters the phenanthrene degradation pathway.
 Am-Euras. J. Agric. & Environ. Sci., 15 (5): 800-812, 2015
Where it is metabolized to 1-hydroxy-2-naphthoic acid
and then mineralized through two different pathways, in
one pathway, 1-hydroxy-2-naphthoic acid is oxidized to 1,
2-dihydroxy naphthalene, which is further metabolized via
salicylic acid. In the other pathway, 1-hydroxy-2-
naphthoic acid undergoes ring cleavage and further
metabolize via o-phthalic acid and protocatchuic acid.
Similar pathway was reported for biodegradation of
Phenanthrene by Sphingomonas sp. P2. It has been
demonstrated that a common set of enzymes is
responsible for the conversion of phenanthrene to 1-
hydroxy-2-naphthoic acid as well as that of naphthalene
to salicylic acid [56].
The new metabolite described for pyrene metabolism
and the transformation of the PAHs naphthalene and
phenanthrene via ortho cleavage give new insight into
the understanding of the bio-Metabolites identified in
washed-cell suspensions of Mycobacterium sp. strain
AP1 incubated with PAHs. The results of our study are in
line with that of B. thermoleovorans which produce,
phthalic acid, a second transformation product with
2-carboxy functionality was identified. When anthracene
degradation occurs by means of phthalic acid, may arise
from degradation of 2-carboxycinnamic acid. A
subsequent decarboxylation reaction would provide
benzoic acid. The co-occurrence of 2, 3-
dihydroxynaphthalene, 2-carboxycinnamic acid and
phthalic acid beside cinnamic acid and salicylic acid
strongly suggests that B. thermoleovorans produces
enzymes of different naphthalene degradation pathways
[50].
Phthalic acid (32.03%) and catechol (63.77%) was
obtained after 120 hours. After 168 hour benzocaumarin
(93.13%), the ring-fission product of the oxidative
metabolism of phenanthrene and anthracene by soil
pseudomonads and traces of pyrene metabolites were
detected as previously reported [45, 57, 58, 59, 60, 24].
Sphingomonas VKM B-2434 has the ability to transform
substances naphthalene, acenaphthene, phenanthrene
and anthracene separately and PAH mixtures as a sole
source of carbon and energy. The strain is capable of
carrying co-metabolic oxidization of pyrene. The major
oxidation products of naphthalene, phenanthrene,
anthracene and pyrene were identified as salicylic acid,
1-hydroxy-2-naphthoic acid, 3-hydroxy-2-naphthoic
acid and dihydroxydihydropyrene respectively.
Reckonable oxidation of phenanthrene and anthracene
to the corresponding hydroxynaphthoic acids occurred
[61, 42].
808
Sphingobium strain PNB isolated from municipal
waste-contaminated soil sample, capable of degrading
phenanthrene as sole sources of carbon and energy and
the metabolic pathways involved in the degradation of
phenanthrene was reported earlier [62]. Apart from
phenanthrene, Sphingobium strain was found to be adept
of consuming naphthalene individually as sole sources of
carbon and energy, though complete degradation was
achieved four days. Among the probable metabolic
pathway intermediates, salicylic acid and benzoic acid
sustained growth of this strain, when provided
individually as sole carbon sources. But, growth was not
observed with 3-hydroxy-2-naphthoic acid,
protocatechuic acid, phthalic acid, gentisic acid or
catechol [63].
Apart from dioxygenation at the 3, 4-position, there
are reports of initial dioxygenation at the 1, 2-position of
phenanthrene leading to the formation of 2-hydroxy-1-
naphthoic acid as one of the metabolic intermediates.
Current studies shown that degradation of phenanthrene
was initiated by dioxygenation at both 3, 4- and 1, 2-
positions and was subsequently metabolized to 1-
hydroxy-2-naphthoic acid and 2-hydroxy-1-naphthoic
acid, respectively [64, 65, 66]. These two intermediates
were proposed to be transformed and converged into
naphthalene-1, 2-diol, which was further metabolized
through either salicylic acid or phthalic acid or both
[56, 65-68]. Dioxygenation exclusively at the 1,2-position
of phenanthrene, forming phenanthrene cis-1,2-
dihydrodiol, has also been described, wherever the
dihydrodiol is afterward metabolized to 2-hydroxy-1-
naphthoic acid, which was further degraded by a meta-
cleavage dioxygenase, ultimately leading to TCA cycle
intermediates via salicylic acid and catechol [45, 69].
CONCLUSION
In the present study, the degradation potential of five
PAHs individually and mixed by aerobic bacterial cultures
was performed. In optimal condition, the mixed culture
was capable of complete degradation of all five PAHs.
The results for other PAHs show that as the number of
benzene rings in PAH compounds increases the rate of
degradation decreases. Pyrene and anthracene could
degrade co-metabolically in samples that contain all PAHs
were completely mineralized by the strains which were
unable to degrade these individually. According to
results, the isolated strains cultures could be valuable in
bioremediation of PAH-contaminated environments.
 Am-Euras. J. Agric. & Environ. Sci., 15 (5): 800-812, 2015
A larger variety of new bacteria with degrading abilities
should be isolated and deeper vision into the
biodegradation pathways from the molecular and
genetical point of view is essential. Furthermore, the
exploitation of biodegrading microorganisms worldwide
has a tremendous perspective into speeding up the
bioremediation of polluted areas, hence facilitating the
clean-up of polluted areas and reducing significances on
human and animal health.
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