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This work was carried out in collaboration between all authors. Author MM designed the study and
wrote the protocol. Author VNM wrote the first draft of the manuscript. Authors VNM and MM
managed the analyses of the study. Author VNYM reviewed the experimental design and all drafts of
the manuscript. All authors read and approved the final manuscript.
Article Information
DOI: 10.9734/MRJI/2017/30484
Editor(s):
(1) Grzegorz Cieslar, Department and Clinic of Internal Diseases, Angiology and Physical Medicine, Medical University of
Silesia, Poland.
Reviewers:
(1) S. R. Khandelwal, HPT Arts and RYK Science College, Nasik, Maharashtra, India.
(2) Yousif Abd El-Aziz Elhassaneen, Minoufiya University, Shebin El-Kom, Egypt.
(3) P. N. Palanisamy, Kongu Engineering College, India.
Complete Peer review History: http://www.sciencedomain.org/review-history/17290
ABSTRACT
Aim: Polycyclic aromatic hydrocarbons (PAHs) are categorised as potentially harmful chemicals of
environmental and health apprehension. Microbial degradation is the principle practice for effective
elimination and abolition of PAHs from polluted environment. Phenanthrene, a PAH compound,
recalcitrant xenobiotic causes severe damage to kidney, liver, fat tissues and causes cancer.
Removal of this from environmental pollutants is expensive by existing methods. To overcome this,
an attempt has been made for the degradation of phenanthrene by using microorganisms.
Materials and Methods: Seven organisms were isolated from thirty six soil samples using M9
media with phenanthrene. One organism that shows highest degradation was identified by
morphological and molecular (16S rRNA sequencing) analysis. Physical parameters such as
incubation period, concentration, pH and temperature and chemical parameters like carbon,
_____________________________________________________________________________________________________
*Corresponding author: E-mail: drmahesh.azyme@gmail.com;
nitrogen and metal ion sources were used for efficient degradation of phenanthrene.
Results: Results revealed that, among the seven organisms, one organism was significantly
effective in degradation and was identified as Pseudomonas aeruginosa by 16S rRNA sequence
and strain kar5, accession No KT225506 was registered in National Centre for Biotechnology
Information (NCBI). Maximum degradation was noticed using 100 ppm concentration at 37C with
pH 7 and 96 hr. Starch showed slightly high degradation compared to other carbon sources such as
sucrose, lactose, maltose and cellulose. In nitrogen sources, tryptone revealed more degradation
than casein, gelatin, egg albumin and bovine serum albumin. Metal ions like MgCl2 and ZnCl2
recoded significant degradation measured by spectrophotometer at 340 nm and confirmed by
HPLC. Maximum growth of microorganism measured by spectrophotometer at 600 nm and reached
to OD 0.851 0.7.
Conclusion: Study confirms that, physico-chemical parameters play a significant role in degrading
soil with high PAHs concentration.
Keywords: HPLC; microbial degradation; spectrophotometer; polycyclic aromatic hydrocarbon;
16S rRNA.
formed
as
products
during
incomplete
combustion of organic matter and their extensive
occurrence in the environment is of great
concern, while many of them are toxic,
mutagenic and carcinogenic [8-9].
1. INTRODUCTION
Polycyclic aromatic hydrocarbons (PAHs) are an
abundant class of organic compounds existing in
the atmosphere. PAHs are ubiquitous compound
originate from natural and anthropogenic
pyrolysis of organic material like forest fire,
automobiles exhaust, coal refining practices and
oil industry [1]. These compounds assumed as
the maximum abundant organic amalgams on
the earth pose a threat to the environment and
mankind [2]. Significant levels are detected in air,
water, soils and sediments [3]. Microbial
degradation of PAHs by bacteria and fungi is
considered as a safe and environment friendly
technique to eradicate contaminated impurities.
Biodegradation of PAHs in soil by microbial
action is of great importance because soil is a
sink for the accumulation of hydrophobic
pollutants including PAHs. Potentially, the rate of
biodegradation could be changed by the addition
of some nutrients [4]. Phenanthrene found as
pollutant in soil, estuarine waters, sediments and
other terrestrial and aquatic sites. When
phenanthrene is ingested, it is lipid soluble and
easily absorbed by the human body. During
metabolism, phenanthrene breaks down in to
epoxy compounds that are highly mutagenic and
carcinogenic affecting lungs, liver and skin [5].
PAHs are commonly found due to atmospheric
activities which include incomplete combustion of
organic matter. Since PAHs are known to
encompass bioaccumulation possesses serial
threat to aquatic and terrestrial habitats [6]. PAHs
are the most important pollutants have been
recognized as potentially toxic with high durability
that causes accumulation of these compounds
in terrestrial organisms. They reduce the
biodiversity rate and reproduction as well as
destroying terrestrial regions [7]. PAHs can be
(953FACAAGCGGTGGAGCATGTGGT) and 10
picomoles
of
reverse
primer
(1507RaACCTTGTTACGACTTCACCCCAG)was
obtained from Sigma-Aldrich and makeup the
volume to 50 micro litre. The amplified condition
was 95C for 5 min and subjected to
denaturation at 94C for 1 min, annealing at
55C for 1 min, primer extension at 72C for 1
min and for 30 cycles further final extension was
at 72C for 5 min. Amplified sample was run on
agarose gel with 1 kb plus ladder (Invitrogen
Tracklt) to confirm the amplified fragment length.
Sequencing data obtained from Eurofins lab,
Bengaluru was used for identification of organism
compare with 16S rRNA database from NCBI for
similar sequence.
2.2 Chemicals
Phenanthrene (Sigma-Aldrich, purity 96%),
Muller-Hilton media and Luria Bertani (HiMedia), Acetonitrile (Merck) and all
chemicals used for extraction, preparation of
minimal media and bacteria culture were of
analytical grade and supplied by SD fine, India.
Spectrophotometer (Chemito 2000 India) and
HPLC, (Waters 510 Isochratic USA).
nm respectively by UV Spectrophotometer at
regular intervals of 12 hrs.
2.6.2 Concentration
Phenanthrene aliquots with concentration 50
ppm, 100 ppm, 150 ppm, 200 ppm and 250 ppm
were added to 100 ml of M9 media separately in
250 ml conical flasks. 1 ml of seed culture was
inoculated and incubated at 37C in shaker
incubator at 150 rpm for 96 hrs. Degradation and
growth of organism was measured at 340 nm
and
600
nm
respectively
by
UV
Spectrophotometer.
2.6.3 pH
of
Phenanthrene
2.6.4 Temperature
Temperature used for the degradation of
phenanthrene ranges at 20, 25, 30, 37, 40, 45
and 50C. Inoculate 1 ml of enriched culture and
incubated at above mentioned temperatures in
shaker incubator at 150 rpm for 96 hrs.
Degradation and growth of organism was
measured at 340 nm and 600 nm respectively by
UV Spectrophotometer.
3. RESULTS
Six organisms were isolated from thirty six soil
samples collected from different industrial
areas in Bengaluru. The organism showing
maximum degradation was confirmed by
spectrophotometer at 340 nm and growth was
measured at 600 nm to found CFU/ml. The
organism was identified using 16S rRNA gene
sequencing and biochemical tests. Universal
primers (27 F and 1492 R) for the amplification
of 16S rRNA were able to amplify the region
giving 1.5 kb size fragment in isolated strain.
Amplicon envisaged on 1% agarose gel with 1X
Tris-acetate EDTA buffer at constant voltage of
80 V. Based on 16S rRNA sequence data,
BLAST search showed 99% resemblance with
Pseudomonas aeruginosa (Fig. 1) and
biochemical tests also shows same characters.
Fig. 1. Phylogenic tree of Pseudomonas aeruginosa based on 16S rRNA gene sequences and
numbers at the nodes show bootstrap support stages
(25 mcg), Cefotaxime (30 mcg), Piperacillin (100
mcg), Chloramphenicol (30 mcg), Ciprofloxacin
(5 mcg), Ceftizoxime (30 mcg), Tetracycline (30
mcg), Ofloxacin (5 mcg), Gentamicin (10 mcg),
Amikacin (30 mcg) and Gatifloxacin (10 mcg),
Co-Trimoxazole recorded least zone of inhibition
and it is indicating less resistance to this
antibiotic. On the other hand, Ciprofloxacin
shows highest zone of inhibition indicating more
sensitivity (Fig. 2).
4. DISCUSSION
Bengaluru is well known for rapid increase in
urbanisation, industrialisation and globalisation.
Soil samples collected areas are highly polluted
with hydrocarbons, heavy metals, pesticides etc.
Polluted soils with hydrocarbons are good
sources for the segregation of PAHs degrading
bacteria that can be used for the exclusion of
such compounds from the location [23-24].
Number of bacterial species is known to degrade
PAHs and most of them are isolated from
contaminated
soil
or
sediments
[25].
Pseudomonas aeruginosa [26], Pseudomonas
fluoresens, Mycobacterium sp., Haemophilus sp.,
Rhodococcus sp. are some of the commonly
studied PAHs degrading bacteria [27-28].
Supplementation of contaminated soils with
compost
materials
can
also
enhance
biodegradation without long-term accumulation of
extractable polar. A DNA fragment commonly
used for taxonomic determinations of bacteria is
the 16S rRNA gene [29]. 16S rRNA gene
5. CONCLUSION
Pseudomonas aeruginosa isolated from soil with
hydrocarbon residues has the potential in
degrading
phenanthrene
under
optimum
conditions in bioremediation process of the
contaminated environment. In congruence with
8
8.
9.
COMPETING INTERESTS
Authors have
interests exist.
declared
that
no
10.
competing
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