chapter 6: phenanthrene...
TRANSCRIPT
Chapter 6: Phenanthrene degradation………
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6.1 Introduction
Pollution by polycyclic aromatic hydrocarbons (PAHs) in soil and sediment has recently
become a matter of great concern due to their toxic, mutagenic and carcinogenic nature
(Fujikawa et al., 1993). PAHs are persistent in environment due to the high
thermodynamic stability of benzene moiety and hydrophobic nature. Most of them tend
to sorb into the soil particulates, rendering them unavailable for biological uptake.
Phenanthrene, a three-ring angular PAH, is a major constituent of coal derivatives and oil
fuels. It is known to be a human skin photosensitizer, an inducer of sister chromatid
exchanger and a potent inhibitor of gap-junctional intercellular communication (Samanta
et al., 2002). Therefore, it is necessary to establish effective methods for removal of
PAH, to protect the environment. Bioremediation is considered as the most significant
and influential for degradation or detoxification of xenobiotic compounds. Many reports
have described phenanthrene degradation by various bacteria such as Alcaligenes,
Acinetobacter, Arthrobacter, Bacillus, Burkholderia, Brevibacterium, Acidovorax,
Micrococcus, Moraxella, Mycobacterium, Pseudomonas, Rhodococcus, Sphingomonas,
Stenotrophomonas, Paenibacillus and others (Juhasz et al., 2000; Zhao et al., 2009; Kim
et al., 2003; Van Hamme et al., 2003; Peng et al., 2008; Keum et al., 2005).
The catabolism of phenanthrene by bacteria has been studied. In general, phenanthrene is
metabolized to 1-hydroxy 2, naphthoic acid (1N2HN) by initial dioxygenation. 1N2HN
was further degraded through two different pathways. In one of the pathway, 1N2HN is
oxidized to 1,2-dihydroxynaphthalene and is later converted to salicylic acid. Salicylic
acid is further degraded via the formation of either catechol or gentisic acid. Both
catechol and gentisic acid undergo ring fission to form TCA-cycle intermediates. In
another pathway, 1N2HN undergoes ring-cleavage leading to formation of o-phthalic
acid, which is further converted to protocatechuic acid which is finally cleaved to form
pyruvic acid and ultimately enters the tricarboxylic acid (TCA) cycle (Peng et al., 2008;
Keum et al., 2005; Habe and Omori, 2003). Although, Pseudoxanthomonas species have
been found to be distributed in a wide variety of contaminated environment; there is no
comprehensive biochemical report on the degradation of PAHs.
Chapter 6: Phenanthrene degradation………
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Another such pollution hotspot area in Gujarat is the region of „The Golden Corridor‟ that
extends from Vapi in the south to Ahmedabad in the north, a stretch of 400Km. This is
called “The Golden Corridor”, from the point of view of the industry (because of good
transport and communication infrastructure, large pool of cheap and unorganized labor
availability). These industrial areas contain thousands of individual industrial units,
including dye factories, textile, rubber, pesticide and paint manufacturers, pulp and paper
producers, pharmaceutical, engineering and chemical companies. Due to the huge
industrialization there is a massive increase in the pollution levels. Amlakhadi, the creek
area, is an earthen storm water drain in Ankleshwar region, carries effluent generated
from these industrial estates located at Ankleshwar, Panoli and Jhagadia, which leads to
the Arabian Sea bearing in all industrial waste.
In this study, we have isolated phenanthrene degrading Pseudoxanthomonas sp. DMVP2
from hydrocarbon contaminated sediment of Gujarat and optimized various
environmental parameters to achieve higher phenanthrene degradation. Phenanthrene
degradation pathway was studied using gas chromatography and mass spectrometry. We
have also examined the effect of presence of other petroleum hydrocarbons on
phenanthrene degradation. Furthermore, we have also determined efficiency of strain
DMVP2 for phenanthrene degradation in stimulated microcosms.
6.2 Materials and methods
6.2.1 Culture medium and chemicals
Bushnell Haas Broth (BHB; Hi-Media, Mumbai, India) and Bushnell Haas Agar (BHA;
Hi-Media, Mumbai, India) supplemented with phenanthrene was used for enrichment and
isolation of phenanthrene degrading bacteria. Phenanthrene, naphthalene, anthracene and
phthalic acid were purchased from Himedia (Mumbai, India). Pyrene and protocatechuic
acid were obtained from Sigma-Aldrich (Steinheim, Germany). All other chemicals used
were of analytical grade.
6.2.2 Enrichment, isolation and screening of PAH degrading strains
Sediment samples were collected from long term polluted Amlakhadi canal, Ankleshwar,
Gujarat, India. Sediment sample (10g) was inoculated in BHB supplemented with 50 ppm
Chapter 6: Phenanthrene degradation………
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of different PAHs such as naphthalene, phenanthrene, pyrene individually and mixture of
these three PAHs and incubated at 37ºC under shaking condition (150 rpm) (Certomate,
B. Braun Biotech International, Goettingen, Germany). After one week, part of this
enriched samples from each enriched flasks was used as inoculum in fresh BHB medium
supplemented with 70 ppm of different PAHs respectively as mentioned above and
incubated at 37ºC under shaking condition (150 rpm). Successively fifteen transfers were
carried out in the same way in fresh BHB medium supplemented with PAHs respectively
and each time PAHs concentration was increased with the increment of 20 ppm. The
appropriate dilutions from each enriched sample were spreaded on BHA containing
respective PAHs and incubated at different temperatures. BHA was spreaded with 25
ppm of phenanthrene or pyrene (PAH was dissolved in acetone) as the sole carbon
source. Total bacterial counts ranging from 1.6x102 to 3.4x10
3 CFU/ml were obtained on
BHA containing different PAHs. Out of that, total 25 bacterial strains were isolated on
the basis their morphological characteristics on BHA and further morphological
characteristics were confirmed on Luria agar. All isolated bacterial strains were examined
for PAHs degradation as well as growth of isolated bacterial strains in presence of
different PAHs (Naphthalene, phenanthrene and pyrene). All isolated bacterial strains
were inoculated in BHB containing 0.1% (w/v) glucose supplemented with 500 ppm of
naphthalene and incubated at 37ºC under shaking condition at 150 rpm. After 48h, flasks
were collected and analyzed for growth as well as remaining naphthalene in the medium.
Similarly, all isolated bacterial strains were inoculated in BHB containing 0.1% (w/v)
glucose supplemented with 300 ppm of phenanthrene and incubated at 37ºC under
shaking condition at 150 rpm. After 48h, flasks were collected and analyzed for growth
as well as residual phenanthrene in the medium. However, we have also determined the
efficiency of all isolated bacterial strain to grow in presence of pyrene. From all isolates,
best phenanthrene degrading strain (DMVP2) was selected for further study.
6.2.3 Identification by 16S rRNA gene sequencing
Genomic DNA was extracted from strain DMVP2 as described by Ausubel et al., (1997).
The 16S rRNA gene was amplified in a 30 µl PCR reaction consisting of 1X buffer (10
mM Tris pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100), 0.33 mM each of
Chapter 6: Phenanthrene degradation………
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dNTPs, 0.66 pmole each of custom synthesized universal primer 8F 5‟-
AGAGTTTGATCCTGGCTCAG-3‟) and 1492R (5‟-GGTTACCTTGTTACGACTT-3‟),
and 1.5U of Taq DNA polymerase. Amplification program was performed with initial
denaturation step at 94ºC for 5 min; followed by 30 cycles of 1 min denaturation step at
94ºC, 1 min annealing step at 55ºC, and 1.2 min elongation step at 72ºC and a final
extension step at 72ºC for 20 min using Biorad icycler version 4,006 (Biorad, CA, USA).
The amplified 16S rRNA gene products were purified using Wizard PCR-Clean-up kit
(Promega, Madison, USA) The purified 1.5 Kb PCR product was sequenced by
automated DNA Analyser 3730 using ABI PRISM®
BigDyeTM
Terminator Cycle
Sequencing 3.1 (Applied Biosystems, Foster City, CA). Nearly full-length bacterial 16S
rRNA gene sequence was analysed using BLAST (n) program at NCBI server (Xia and
Xie, 2001) to identify and downloaded the nearest neighbour sequences from the NCBI
database. All the sequences were aligned using Clustal W version 1.6 program. The
phylogenetic tree was constructed using aligned sequences by the neighbour joining (NJ)
algorithm using Jukes–Cantor evolutionary distances and Kimura 2 parameter with more
than 1000 replicates in MEGA (Molecular Evolutionary Genetic Analysis) version 4.0
software (Tamura et al., 2007).
6.2.4 Preparation of bacterial inoculum
The bacterial strain DMVP2 was inoculated in BHB containing 300 ppm of phenanthrene
and incubated at 37ºC under shaking condition (150 rpm) till the growth reached late
exponential phase (72h). Cells were centrifuged at 5000 x g for 10 min, washed twice
with 0.85% normal saline and finally resuspended in the same buffer to obtain a cell
suspension with an absorbance (A660) of 1.0 and this was then used as the inoculum.
6.2.5 Effect of abiotic factors on phenanthrene degradation
All experiments were carried out in 250 ml Erlenmeyer flasks containing 100 ml of BHB
supplemented with appropriate amount of phenanthrene. The residual phenanthrene was
analyzed using gas chromatography (GC) as described in section 6.2.6.
Chapter 6: Phenanthrene degradation………
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6.2.5.1 Effect of inoculum size
BHB supplemented with 300 ppm of phenanthrene were inoculated with different
inoculum size such as 1, 2, 4 and 6% (v/v), of strain DMVP2 and incubated at 37ºC under
shaking condition (150 rpm). A control, without inoculation of strain DMVP2 was kept
under similar conditions to determine abiotic loss of phenanthrene. Samples were
collected at regular intervals of time (24h) up to 120h and analyzed for residual
phenanthrene.
6.2.5.2 Effect of different carbon and nitrogen source
Pre-grown strain DMVP2 was inoculated in BHB supplemented with 300 ppm
phenanthrene and different carbon and nitrogen sources (0.1%, w/v) (yeast extract,
glucose, peptone, salicylic acid, trypton, sodium succinate, sodium acetate and
ammonium nitrate and phthalic acid). All flasks were incubated at 37ºC under shaking
condition at 150 rpm. A control, without any carbon and nitrogen source, was kept under
similar conditions. After 120h, flasks were collected and analyzed for residual
phenanthrene in medium.
Amongst all carbon and nitrogen source, peptone, yeast extract, sodium succinate and
ammonium nitrate were selected for further study. BHB supplemented with 300 ppm of
phenanthrene along with different carbon and nitrogen source such as peptone, yeast
extract, sodium succinate and ammonium nitrate (0.1%, w/v) were inoculated with strain
DMVP2 (4%, v/v) and incubated at 37ºC under shaking condition (150 rpm). A control,
without any carbon and nitrogen source, was kept under similar conditions. Samples were
collected at regular intervals of time (24h) up to 120h and analyzed for residual
phenanthrene.
6.2.5.3 Effect of temperature
BHB supplemented with 300 ppm of phenanthrene along with peptone (0.1%, w/v) were
inoculated with strain DMVP2 (4%, v/v) and incubated at different temperatures such as
30ºC, 37ºC 40ºC and 45ºC under shaking condition (150 rpm). Controls, without
inoculation of strain DMVP2, were kept under similar conditions. Samples were
Chapter 6: Phenanthrene degradation………
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collected at regular intervals of time (24h) up to 120h and analyzed for residual
phenanthrene.
6.2.5.4 Effect of static as well as different shaking condition
BHB supplemented with 300 ppm of phenanthrene along with peptone (0.1%, w/v) were
inoculated with strain DMVP2 (4%, v/v) and incubated at 37ºC under static as well as
under different shaking condition such as 50, 100 and 150 rpm. Controls, without
inoculation of strain DMVP2, were kept under similar conditions. Samples were
collected at regular intervals of time (24h) up to 120h and analyzed for residual
phenanthrene.
6.2.5.5 Effect of pH
BHB supplemented with 300 ppm of phenanthrene and peptone (0.1%, w/v) was adjusted
to different initial pH such as 6.0, 7.0, 8.0, 9.0 and 10.0 using either HCl or NaOH. The
flasks were inoculated with strain DMVP2 (4%, v/v) and incubated at 37ºC under
shaking condition (150 rpm). Controls, without inoculation of strain DMVP2, were kept
under similar condition with inoculated flasks. Samples were collected at regular
intervals of time (24h) up to 120h and analyzed for residual phenanthrene.
6.2.5.6 Effect of initial concentration of phenanthrene on degradation
BHB supplemented with peptone (0.1%, w/v) along with different concentrations of
phenanthrene such as 300, 500, 750, 1000, 1500, 2000, 4000 and 5000 ppm of
phenanthrene were inoculated with strain DMVP2 (4%, v/v) and incubated at 37ºC under
shaking condition at 150 rpm. Controls, without inoculation of strain DMVP2, were kept
under similar conditions. Samples were collected after 120h to determine residual
phenanthrene.
6.2.5.7 Effect of surfactants
BHB supplemented with 300 ppm of phenanthrene along with 0.02% (w/v)/(v/v) of
different surfactants such as sodium dodecyl sulfate (SDS), Cetyl trimethyl ammonium
bromide (CTAB), Tween 80 and Triton X-100 were inoculated with strain DMVP2
Chapter 6: Phenanthrene degradation………
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(4%, v/v) and incubated at 37ºC under shaking condition at 150 rpm. A control, without
any surfactant, was kept under similar conditions. Samples were collected after 48h and
120h to determine residual phenanthrene.
6.2.6 Phenanthrene extraction, analysis and quantification
The entire content (100 ml) of flask was taken for determination of residual phenanthrene
in all the experiments. For phenanthrene extraction, 20 ml dichloromethane (DCM) was
added in flask and incubated at 37ºC under shaking condition (150 rpm) for 1h. The
content was then centrifuged at 5000 x g for 10 min and the extract was dried using
anhydrous sodium sulfate to remove aqueous phase. Residual phenanthrene in extract
was analyzed using gas chromatography (Clarus 500, Perkin Elmer, USA) equipped with
flame ionization detector (FID) and a Stabliwax column, (30 m length × 0.32 mm inner
diameter, crossbond-PEG) (Restek, USA). The injection volume was 1 μl. Nitrogen gas
was used as the carrier gas at a flow rate of 3 ml/min. The injector temperature and the
detector temperature were kept at 270°C.
6.2.7 Analysis of metabolites during phenanthrene degradation by strain DMVP2
Strain DMVP2 was grown in BHB supplemented with phenanthrene (300 ppm) and
peptone (as nitrogen source). Flasks were incubated under optimized conditions. At
regular interval of time (0 h, 72 h and 144 h), metabolites were extracted from medium as
described by Samanta et al., (1999).
Metabolites were analyzed using GC (Autosystem XL, Perkin Elmer, USA), equipped
with PE-5MS capillary column (30 m long; 0.25 mm internal diameter) and coupled to a
MS (Turbo mass, Perkin Elmer, USA). GC temperature programme was 80°C (1 min),
80–280°C (10°C/min) and 280°C (30 min). The injection volume was 1 μl and helium
was used as carrier gas at the flow rate of 1ml/min. The mass spectrometer was operated
at electron ionization energy of 70 eV. Injector and detector temperature were kept at
250°C.
Chapter 6: Phenanthrene degradation………
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6.2.8 Preparation of cell-free extracts
In addition to this, activity of protocatechuate dioxygenase was determined as described
by Chatterjee and Dutta (2008) with some modification.
Cells grown in BHB containing 0.1% (w/v) peptone supplemented with a phenanthrene
(300 ppm) were harvested at the mid-exponential phase (72h) by centrifugation at 8000 x
g for 10 min at 4°C. The pellet was washed twice with 10-volumes of 50 mM potassium
phosphate buffer pH 7.0 and resuspended in either 1-volume of 50 mM Tris–HCl buffer
(pH 8.0). The cell suspensions were subjected to seven ultrasonic pulses generated with a
sonicator (Sonic Vibra Cell 500W, CT, USA) for 5 min at 4°C till lysis of bacterial cell
culture. The resulting cell homogenates were centrifuged at 20,000 x g for 20 min at 4°C.
The supernatants were used as source of enzymes. Enzyme-catalyzed transformations of
various substrates were monitored by recording changes in UV–Visible spectra of the
compounds using a Cary 100 Bio UV–Visible spectrophotometer (Specod, Analytical
Jena, Jena, Germany). Reactions were scanned in the range of 220–400 nm for 0–24 min.
Data were analyzed by the Varian Cary Win UV Scan application software.
6.2.9 Growth of Pseudoxanthomonas sp. DMVP2 on various other xenobiotic
compounds
BHB containing 0.1% [(w/v)/(v/v)] of different xenobiotic compounds [namely, ketone
(acetone), alkanes (n-hexane), monoaromatic hydrocarbons (benzene, toluene and
xylene), PAH (naphthalene, fluoranthene and pyrene), other petroleum hydrocarbons
(carbazole, phenol, petroleum oil and diesel fuel) and intermediates of phenanthrene
degradation (phthalic acid, protocatechuate, catechol and salicylic acid)] were inoculated
with strain DMVP2 and incubated under optimized conditions. The samples were
withdrawn at regular intervals of time (24h) up to 72h to monitor growth of strain
DMVP2 by measuring absorbance at A660 nm using spectrophotometer (Spectronic
20D+, Milton Roy, USA).
Chapter 6: Phenanthrene degradation………
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6.2.10 Effect of different other petroleum hydrocarbons on phenanthrene degradation
In order to examine the effect of other petroleum hydrocarbons on phenanthrene
degradation, strain DMVP2 was inoculated in BHB supplemented with 300 ppm
phenanthrene along with other petroleum hydrocarbons [0.1% (w/v)/(v/v)] such as
benzene, toluene, xylene, petroleum oil, diesel fuel, carbazole, naphthalene, pyrene and
fluoranthene. All flasks were incubated under optimized conditions. Control flask,
without other petroleum hydrocarbons, was kept under similar conditions. Abiotic
controls, without inoculation of strain DMVP2, were kept under similar conditions.
Samples were collected after 96 h and analyzed for growth of strain DMVP2 as well as
for residual phenanthrene.
6.2.11 Microcosm Studies in soil
The microcosm preparations were done by method of Filonov et al (2006) with certain
modifications. Simulated microcosm was set in duplicates in glass petri dish (90mm
diameter). The soil used was dried and then sieved and 100g soil was taken for each set
and then autoclaved twice for 15min at 121.1°c under 15 lbs pressure.
Four different sets of soil model systems were prepared as : (Set A)-Sterile soil
containing phenanthrene which served as abiotic control, (Set B)-Sterile soil
supplemented with phenanthrene (300ppm) was inoculated by strain DMVP2 to
determine the phenanthrene degradation efficiency by strain DMVP2 in absence of
indigenous organisms, (Set C)- Non sterile Soil with native or indigenous organisms and
phenanthrene, to evaluate the intrinsic ability of soil to degrade phenanthrene, (Set D)-
non sterile soil supplemented with phenanthrene and inoculated with strain DMVP2
culture to check the degradation rate by strain DMVP2 in presence of indigenous flora.
The phenanthrene concentration was 300 ppm and inoculums size was 4ml of pre-grown
culture in each case. The moisture content of soil was maintained 35%. For
determination of residual phenanthrene from medium, dichloromethane (DCM) (20 ml)
was added in flasks and entire content was transferred in stopper flasks. For extraction
of phenanthrene from microcosm after 120h, all flasks were incubated on ultra-sonicator
for 30 min followed by incubation under shaking condition at 150 rpm for 30 min. All
experiment was performed in triplicate.
Chapter 6: Phenanthrene degradation………
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6.3 Results and Discussion
Ankleshwar industrial estate (Gujarat, India) consists of nearly 3,000 manufacturing
units. Majority of the industries manufacture chemicals, dyes, paints, fertilizers,
pharmaceuticals, pesticides and other xenobiotic compounds. The treated and untreated
effluents from various industries of Ankleshwar industrial estate are finally released in
Amlakhadi canal. Amlakhadi canal further ends up in Narmada estuary that enters the
Arabian Sea, Gujarat (Fig. 6.1).
Fig 6.1 Map showing the region of “The Golden Corridor” (Gujarat) (Red line)
Kathuri et al., (2007) found that this canal highly polluted with different toxic metals
such as chromium, cadmium, mercury, zinc and others. Beside this, canal is also
contaminated with various organic compounds such as chlorinated benzene, phenolics,
petroleum hydrocarbons, polychlorinated biphenyl and others. Many of these pollutants
(PAHs) adsorb on to particulate matter due to their hydrophobic nature and settle down as
sediment. Therefore, sediment of this canal has become sink of recalcitrant compounds
and pose a major threat to the ecosystem. Contamination with xenobiotic compounds
exerts selective pressure on microorganisms which results in microbial community shifts
towards bacterial species with enhanced PAH degrading capability. Hence, Amlakhadi
Chapter 6: Phenanthrene degradation………
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canal sediment was selected for isolation of PAH degrading organisms. Recently, Pathak
et al., (2009) had also isolated Pseudomonas sp. HOB1 from the same Amlakhadi canal,
showing ability to tolerate 60,000 ppm of naphthalene.
6.3.1 Isolation and screening of phenanthrene degrading bacterial cultures from
enriched soil sample
The PAH (phenanthrene, naphthalene and pyrene) degrading cultures were enriched
from sediment of polluted Amlakhadi canal, Ankleshwar, Gujarat, India as mentioned in
methods. Plate (Fig.6.2). Twenty PAH degrading cultures were isolated from the
enriched sediment sample essentially based on growth of bacterial culture on solid BHA
plates spread with different PAHs as a sole carbon source in contrast to other strains
which failed to grow on BHA spreaded with PAHs and Luria agar. All isolates had
shown different from their colony characteristics.
Fig. 6.2 Phenanthrene degrading isolates having different morphological characteristics on
Luria agar
Experiment was conducted to determine the ability of all isolated bacterial strain for PAH
degradation (naphthalene at concentration of 500 ppm and phenanthrene at concentration
of 300 ppm) in BHB containing 0.1% (w/v) glucose (Table 6.2). Glucose was added in
medium to enhance the growth of organism. Many organisms do not have ability to
utilize PAHs as sole carbon source but they utilize PAHs through co-metabolism. Co-
Chapter 6: Phenanthrene degradation………
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metabolism as defined by Criddle (1993) consists of the transformation of a non-growth
substrate by growing cells in the presence of a growth substrate.
Table 6.1 PAH degradation by isolated bacterial strains from enriched samples within 48h.
+: Growth of bacterial cultures in BHM containing pyrene as carbon source within 72h
Amongst the twenty five isolates culture, some isolates was able to degrade phenanthrene
and naphthalene both, whereas some isolates capable to degrade only phenanthrene or
only naphthalene. In present, as shown in Table 6.1, Many isolates was capable to
degrade naphthalene efficiently, whereas, it failed to degrade phenanthrene. In
comparison to other isolates, strain DMVP2 was found to be the potential isolates. Strain
DMVP2 was able to degrade 50% phenanthrene within 48h with an abiotic loss of 3%
Culture Naphthalene degradation Phenanthrene degradation Pyrene
O.D at
600nm
Degradation
(%)
O.D at
600nm
Degradation
(%)
Growth
DN1 0.230 33.49 0.317 29.87 +
DN2 0.290 70 0.240 38.79 -
DN3 0.220 56.19 0.098 24.59 +
DN4 0.430 23.32 0.481 7.89 +
DN5 0.350 43.35 0.404 29.97 +
DMVP1 0.298 78.13 0.200 32 --
DN9 0.481 19.23 0.340 2.5 --
DN12 0.240 27.89 0.219 13.45 +
13VP 0.214 12.13 0.589 3.7 --
VMP93 0.210 23.98 0.220 3.98 --
VAL112 0.104 55.78 0.163 3.9 --
VAP92 0.310 39.19 0.240 15.89 --
DMVP2 0.138 17.18 0.404 49.98 +
VMP91 0.090 68,98 0.181 12.10 --
VMP52 0.126 37.19 0.151 10.11 --
6VMP 0.208 70.98 0.240 18.78 --
5VMP 0.101 83.19 0.280 12 +
VMP11 0.130 29.87 0.130 1.67 --
CVP 0.280 80 0.120 29.76 --
Phe-1 0.289 76.19 0.425 11.34 -
Phe-2 0.389 13.87 0.480 39.42 +
Phe-3 0.193 45.98 0.310 45.78 -
VAL-2 0.254 25.67 0.250 34.67 -
3VP 0.327 36.75 0.135 12.34 +
Chapter 6: Phenanthrene degradation………
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and it was also able to grow in presence of higher hydrocarbon such as pyrene. It is an
advantage to exploit this strain for degradation study, because all polluted environment
always contaminated with mixture of hydrocarbons. Therefore, it was selected for further
characterization and degradation studies.
Fig. 6.3 Morphological characteristic of Strain DMVP2 on Luria agar
As shown in Fig. 6.3, Strain DMVP2 was found to be aerobic, gram-negative, non-
endospore forming rod shaped bacterium with smooth colony, entire margin and yellow
pigmentation on Luria agar plate. Further, this phenanthrene degrading strain DMVP2
was identified as Pseudoxanthomonas sp. DMVP2 based on 16S rRNA gene sequence
analysis. The sequence has been deposited in the Gene Bank with an accession number of
JF440626. The phylogenetic cluster of strain DMVP2 along with other closely related
bacterial strains is depicted in Fig. 6.4. Pseudoxanthomonas sp. belongs to the family
Xanthomonadaceae in the order Xanthomonadales and class Gammaproteobacteria.
Strain DMVP2 was 99% similar with Pseudoxanthomonas mexicana (Fig. 6.4).
Chapter 6: Phenanthrene degradation………
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Pseudoxanthomonas sp. LLH-3
Pseudoxanthomonas sp. DMVP2
Pseudoxanthomonas sp. RN402
Pseudoxanthomonas sp. PNK04
Pseudoxanthomonas mexicana strain L2
Pseudoxanthomonas mexicana
Stenotrophomonas sp. TSA3w
Uncultured Pseudoxanthomonas sp. clone ASC90
Uncultured Xanthomonadales bacterium clone PRTAB7892
Uncultured gamma proteobacterium clone MTAB36
Uncultured Pseudoxanthomonas sp. clone W5S36
Stenotrophomonas sp. AGL1
Escherichia coli strain ATCC25922
95
100
96
98
89
99
99
0.02
Fig. 6.4 Phylogenetic tree derived from 16S rRNA gene sequence of Pseudoxanthomonas sp.
DMVP2 and sequences of closest phylogenetic neighbours obtained by NCBI BLAST(n)
analysis. The NJ-tree was constructed using neighbour joining algorithm with Kimura 2
parameter distances in MEGA 4.0 software. E. coli strain ATCC25922 has been taken as an
out-group. Numbers at nodes indicate percent bootstrap values above 50 supported by more
than 1000 replicates. The bar indicates the Jukes–Cantor evolutionary distance.
Klankeo et al., (2009) have reported that Pseudoxanthomonas mexicana carries nidA
gene, situated on megaplasmid, responsible for degradation of pyrene. Recently,
Nopcharoenkul et al., (2012) studied diesel removal by Pseudoxanthomonas sp. There
was little information describing the optimization of various abiotic factors for maximum
phenanthrene degradation and analysis of metabolic pathway for phenanthrene
degradation by Pseudoxanthomonas sp. DMVP2. Therefore, the present work was
initiated to fill a part of this void by optimizing various parameters to achieve better
phenanthrene degradation as well as metabolic mechanism for phenanthrene degradation.
Chapter 6: Phenanthrene degradation………
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0
10
20
30
40
50
60
70
80
90
100
0 24 48 72 96 120
Ph
en
an
thre
ne
de
gra
da
tio
n (
%)
Time (h)
1% 2% 4% 6%
6.3.2 Effect of abiotic factors on phenanthrene degradation
The transformation of PAH compounds in the environment is mainly through microbial
processes, but is also influenced by number of environmental factors (Manilal and
Alexander, 1999). Efforts for degradation of PAHs from environments by bacteria would
require knowledge of the concentration of the toxic compounds and the rate at which the
compound will be degraded. Equally important would be to understand environmental
factors influencing the degradation process. Therefore, in present study effect of pH,
temperature, inoculum size and different incubation conditions was evaluated to obtain
maximum degradation.
6.3.2.1 Effect of inoculum size
Fig. 6.5 shows the effect of inoculum size of strain DMVP2 on phenanthrene
degradation. When inoculum of strain DMVP2 increased up to 4% (v/v), lag period for
growth of strain DMVP2 decreased and subsequently resulted in higher phenanthrene
degradation (88%). As the inoculum of strain DMVP2 was increased further, it resulted
in decreased degradation. This is consistent with earlier report by Abdelhay et al., (2008).
Fig. 6.5 Effect of inoculum size on phenanthrene degradation by strain DMVP2 in BHB
supplemented with 300 ppm of phenanthrene at 37°C under shaking condition at 150 rpm
Chapter 6: Phenanthrene degradation………
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Chen et al, (2008) also made similar observations, which showed inoculum size was the
key factor affecting the speed of phenanthrene biodegradation. Abiotic removal of
phenanthrene was found to be 10.98% within 120h.
6.3.2.2. Effect of carbon and nitrogen sources
The addition of carbon and nitrogen sources has been increased the growth of
microorganisms which resulted in enhanced PAH degradation (Lee et al., 2003). In
present study, various carbon and nitrogen sources such as yeast extract, glucose,
peptone, salicylic acid, trypton, sodium succinate, sodium acetate and ammonium nitrate
and phthalic acid were also studied for phenanthrene degradation.
Table 6.2 Effect of different carbon and nitrogen sources on phenanthrene degradation
by strain DMVP2 within 120h at 37°C under shaking condition at 150 rpm.
As expected, addition of carbon and nitrogen sources in medium increased growth of
organisms, whereas, amongst all carbon and nitrogen sources peptone, yeast extract,
sodium succinate and ammonium nitrate were found to enhance phenanthrene
degradation in comparison to control (without carbon and nitrogen source) (Table 6.2).
Different carbon
and nitrogen source
Phenanthrene
degradation (%)
Control 88
Glucose 60.12
Trypton 65.27
Salicylic acid 40
Sodium acetate 76.17
Yeast extract 92
Peptone 96.28
Ammonium nitrate 90.16
Phthalic acid 65.39
Sodium succinate 88.56
Chapter 6: Phenanthrene degradation………
200
0
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40
50
60
70
80
90
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0 24 48 72 96 120
Ph
en
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de
gra
da
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n (
%)
Time (h)
Yeast extract peptonecontrol Ammonium nitrateSodium succinate
Fig. 6.6 Effect of carbon and nitrogen sources on phenanthrene degradation by strain
DMVP2 in BHB supplemented with 300 ppm of phenanthrene at 37°C under shaking
condition at 150 rpm
To validate, time course study was carried out to determine the effect of selected carbon
and nitrogen sources (peptone, yeast extract, sodium succinate and ammonium nitrate) on
phenanthrene degradation (Fig. 6.6). As observed from Fig. 6.5, 95% phenanthrene was
degraded in presence of peptone within 120 h. Strain DMVP2 was able to degrade 92, 91
and 90% phenanthrene in presence of yeast extract, sodium succinate and ammonium
nitrate, respectively within 120 h. However strain DMVP2 was also able to degrade 89%
of phenanthrene within 120h in control (without carbon and nitrogen source) which
indicated that addition of carbon and nitrogen sources did not significantly improved
phenanthrene degradation by strain DMVP2, but it increased growth of organism
significantly in comparison to control. Therefore, peptone was selected as the nitrogen
source for further studies. Zong et al., (2007) had also reported that supplementation of
different carbon and nitrogen source in the medium enhanced PAH degradation.
Chapter 6: Phenanthrene degradation………
201
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60
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90
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0 24 48 72 96 120
Ph
en
am
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gra
da
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n (
%)
Time (h)
30 37 40 45
6.3.2.3 Effect of Temperature
Temperature variation had also significant effect on phenanthrene degradation. Fig. 6.7
shows phenanthrene degradation at different temperatures (30, 37, 40 and 45ºC), with
maximum degradation (95%) obtained at 37°C within 120 h. Strain DMVP2 was able to
degrade 90% and 78% of phenanthrene within 120 h at 40 and 45°C, whereas it was
unable to grow at 50°C. At 30°C, strain DMVP2 was able to degrade 84% of
phenanthrene within 120 h. This was attributed to the fact that at lower temperature,
enzyme catalyzed reaction slowed down and also the growth of organism was slower and
subsequently phenanthrene degradation decreased. This indicated that isolated strain
DMVP2 is mesophilic in nature.
Fig. 6.7 Effect of temperature on phenanthrene degradation by strain DMVP2 in BHB
containing 0.1% (w/v) peptone and supplemented with 300 ppm of phenanthrene under
shaking condition at 150 rpm
6.3.2.4 Effect of shaking condition
Figure 6.8 shows phenanthrene degradation profile of strain DMVP2 under static as well
as under shaking conditions (50, 100 and 150 rpm). Under all shaking condition, strain
DMVP2 was able to degrade more than 80% of phenanthrene within 120 h, while under
static condition only 49% of phenanthrene was degraded within 120 h at 37ºC. This is an
obvious result, as the shaking condition was increased mass transfer rate was also
Chapter 6: Phenanthrene degradation………
202
0
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60
70
80
90
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0 24 48 72 96 120
Ph
en
an
thre
ne
de
gra
da
tio
n (
%)
Time (h)
Static 50 rpm 100 rpm 150 rpm
increased. Hence availability of phenanthrene toward organisms and dissolution of
phenanthrene in medium was also increased which resulted in increased phenanthrene
degradation. The maximum degradation (97%) was obtained under shaking condition at
150 rpm. The dissolution of phenanthrene and availability of oxygen increased under
shaking condition and thus resulted in increased rate of degradation. The abiotic loss was
found to be 3, 7.6 and 12% under shaking condition of 50, 100 and 150 rpm. However,
under static condition abiotic loss of phenanthrene was negligible.
Fig. 6.8 Effect of shaking condition on phenanthrene degradation by strain DMVP2 in BHB
containing 0.1% (w/v) peptone and supplemented with 300 ppm of phenanthrene at 37°C
6.3.2.5 Effect of pH
pH is a selective environmental factor affecting microbial diversity and activity,
controlling enzyme activity, transport process and nutrient solubility. Consequently, its
effect on phenanthrene degradation was studied as illustrated in Fig. 6.9. Generally, more
than 90% of phenanthrene was degraded in alkaline condition within 120 h. When the
initial pH of medium was 8.0, higher phenanthrene degradation 99.6% was observed.
This can be taken as an advantage in using this isolate for bioremediation of
phenanthrene in coastal environment. Hambrick et al., (1980) have also shown that soil
bacteria which degrade PAH prefer alkaline condition rather than acid condition. During
phenanthrene degradation, pH of the medium decreases from alkaline (pH 8.0-10.0) to
neutral (7.2-8.2), which may be due accumulation of some acidic intermediates during
Chapter 6: Phenanthrene degradation………
203
0
10
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30
40
50
60
70
80
90
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0 24 48 72 96 120 144
Ph
en
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de
gra
da
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n (
%)
Time (h)
pH 6 pH 7 pH 8pH 9 pH 10
phenanthrene degradation by strain DMVP2. Strain DMVP2 was able to degrade and 79
and 89% of phenanthrene within 120h at initial pH of medium pH 6.0 and pH7.0,
respectively. Zhao et al., (2009) also found that 37°C temperature and pH 8.0 were
favorable conditions for phenanthrene degradation by Pseudomonas stutzeri ZP2.
Fig. 6.9 Effect of initial pH of BHB medium on phenanthrene degradation by strain
DMVP2 in BHB containing 0.1% (w/v) peptone and supplemented with 300 ppm of
phenanthrene at 37°C under shaking condition at 150 rpm
6.3.2.6 Effect of Phenanthrene concentration
The phenanthrene consumption at various initial concentrations of phenanthrene is shown
in Fig. 6.10. Increase in phenanthrene consumption (1600 mg/l) was observed till initial
concentration of phenanthrene was increased to 4000 mg/l. When initial phenanthrene
concentration was increased further (5000 mg/l), strain DMVP2 was able to grow.
However, the phenanthrene consumption decreased significantly. The reason for
decreased consumption of phenanthrene at high concentration is attributed to toxicity of
phenanthrene on strain DMVP2 or may be due to saturation of organisms toward
substrate (phenanthrene). Phenanthrene degradation by isolated Pseudoxanthomonas sp.
DMVP2 was higher than Pseudoxanthomonas mexicana, which degraded 100 ppm
Chapter 6: Phenanthrene degradation………
204
0
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20
30
40
50
60
70
80
90
100
300 500 750 1000 1500 2000 4000 5000
Ph
en
an
thre
ne
de
gra
da
tio
n (
%)
Phenanthrene concentration (ppm)
phenanthrene in 8 days (2009). Pseudoxanthomonas sp. DMVP2 was also able to degrade
phenanthrene more efficiently than earlier reported by Zhao et al., (2009) and Kim et al.,
(2005).
Fig. 6.10 Effect of phenanthrene concentration on phenanthrene degradation by strain
DMVP2 in BHB containing 0.1% (w/v) peptone and supplemented with 300 ppm of
phenanthrene at 37°C under shaking condition at 150 rpm
6.3.2.7 Effect of surfactants
The effect of surfactants on phenanthrene degradation by strain DMVP2 is depicted in
Fig. 6.11. Strain DMVP2 was able to degrade 45, 43, 11 and 5.6% phenanthrene (300
ppm) in the presence of Tween 80, Triton X100, SDS and CTAB, respectively within 48
h, whereas, strain DMVP2 was able to degrade 43.45% phenanthrene within 48 h without
any surfactants (control). Strain DMVP2 was able to degrade phenanthrene completely at
300 ppm within 120h in presence of Tween 80 and in control (without any surfactant).
Tween 80, a good dispersing agent, can improve the dispersion and solubility of
phenanthrene. Bautista et al., (2009) had also reported that Tween-80 was the best
amongst non-ionic surfactants in improving the degradation of PAHs. However, addition
Chapter 6: Phenanthrene degradation………
205
0
10
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30
40
50
60
70
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90
100
Tween 80 Triton X100
Control SDS CTAB
Ph
en
an
thre
ne
de
gra
da
tio
n (
%)
Surfactants (0.02%)
48h 120h
of non-ionic surfactants did not show any significant increase in phenanthrene
degradation in comparison to control (without surfactants).
A major limiting factor in hydrocarbon degradation is the lack of bioavailability of the
hydrocarbon due to poor solubility, leading to accumulation and the accompanying toxic
and carcinogenic effects. In nature, a few smart organisms tackle the hydrocarbon
uptake/degradation problem either by direct contact, e.g., attaching themselves to these
compounds, or by producing surface active compounds. Both these mechanisms involve
modulation of cellular physiology, which will lead either to changes in cell surface
properties like hydrophobicity or to secretion of surface-active compounds into the
medium, or a combination of both (Prabhu and Phale, 2002).
Fig. 6.11 Effect of surfactants on phenanthrene degradation by strain DMVP2 in BHB
containing 0.1% peptone and Supplemented with 300 ppm of phenanthrene at 37°C under
shaking condition at 150 rpm
In present study, strain DMVP2 might be producing bio-surfactants, which increased
pseudo-solubilization of phenanthrene in the medium. Nayak et al., (2009) had reported
rhmanolipid production by P. mexicana in presence of PAH which stimulated the
degradation of various xenobiotic compounds.
Chapter 6: Phenanthrene degradation………
206
6.3.3 Analysis of metabolites during phenanthrene degradation
To investigate phenanthrene degradation pathway followed by strain DMVP2, the
intermediates were analyzed using GC-MS at regular time intervals. The GC-MS analysis
of the zero day extract gave one major peak at retention time (Rt) 16.09 min which was
identified as phenanthrene based on the published mass spectra from National Institute of
Standard and Technology (NIST) [Fig. 6.12(a)].
Fig. 6.12(a) GC-MS analysis of metabolites during phenanthrene degradation by strain
DMVP2 at 0 h extract.
After 72 h incubation, peak of phenanthrene decreased and many other new peaks were
observed by GC-MS analysis suggesting the onset of phenanthrene degradation and
formation metabolites were formed [Fig. 6.12(b)]. Amongst all peaks, seven peaks were
identified (Table 6.3). Major peaks found were of phthalate anhydrade (Rt 10.69 min) and
its ester derivatives such as DI-N-octyl phthalate (Rt 23.55 min) which are known
intermediates of phenantherene degradation. Besides that, many long chains alkanes such
as 1-pentadecane (Rt 14.095 min), 1-teradecane (Rt 11.345 min) and ecosanoic acid (Rt
18.349 min) were found during phenanthrene degradation. This corroborates with the
fact, reported by Nayak et al., (2009), that in presence of phenanthrene, P. mexicana
produced rhmanolipid which contain long chain alkanes. Thus strain DMVP2 might also
Chapter 6: Phenanthrene degradation………
207
be producing some surfactant in presence of phenanthrene, leading to formation of long
chain alkanes. In addition to this, peak (Rt 22.69) was identified as phenol 2, 4-bis(1-
phenyl-ethyl) according its mass 406 fragmentation 302 (M+ C22H22O), 287 (M+−OH),
105 (M+−C6H5–CH–CH3) and 77 (M+−C6H5–CH–CH3).The reason for presence of
this metabolite is not known.
Fig. 6.12(b) GC-MS analysis of metabolites during phenanthrene degradation by strain
DMVP2 within 72 h extract.
Table 6.3 GC-MS data for the metabolites of phenanthrene obtained from the extracts of
the BHB medium supplemented with 300 ppm and inoculated by strain DMVP2 within 72h.
Metabolites Retention time
(min)
Molecular ion and
fragmentation pattern
Suggested metabolites
1 10.67 149(M+), 104, 76, 50, 74 Phthalate anhydrade
2 11.34 198(M+), 85 ,71, 57, 43 1-Tetradecane
3 14.09 213(M+),99, 85, 71, 57, 43 1-pentadecane
4 18.34 312(M+), 129,73, 57, 43 Eicosanoic acid
5 22.69 302(M+), 287,105, 77 Phenol 2,4 –Bis(1-
phenyl ethyl)
6 23.07 168(M+), 149, 104, 71, 57 Phthalic Acid, diisoctyl
ester
7 23.55 280(M+), 167, 149, 113, 104 DI-N-octyl phthalate
Chapter 6: Phenanthrene degradation………
208
In present study, we could not find any metabolites of upper metabolic pathway of
phenanthrene degradation. Therefore, further study is required to characterize upper
metabolic pathway of phenanthrene degradation.
Fig. 6.12(c) GC-MS analysis of metabolites during phenanthrene degradation by strain
DMVP2 within 144h extract.
As shown in Fig. 6.12 (C), all these peak completely disappeared within144 h (GC-MS
analysis) indicating that strain DMVP2 completely degraded phenanthrene (300 ppm).
Further investigation of phenanthrene degradation pathway was carried out by
determining procatehuate dioxygenase activity in cell free extracts of strain DMVP2
grown with phenanthrene (Fig. 6.13). Protocatehuate was added as substrate to the crude
cell extract and UV-Visible spectrum was recorded at intervals of 4 min (from 0-24 min).
Characteristic decrease in maxima absorption (250nm and 290nm) indicated the
formation of β-carboxy-cis, cis-muconate, ortho cleavage product of protocatechuic acid
(Chatterjee and Dutta, 2008). Thus it is assumed that protocatechuate is degraded by
ortho cleavage dioxygenase of strain DMVP2. These results indicated that
Pseudoxanthomonas sp. DMVP2 might be degrading phenanthrene by phthalic acid and
protocatechuic acid pathway.
Chapter 6: Phenanthrene degradation………
209
Fig. 6.13 Protocatechuate dioxygenase activity by cell free extract of strain DMVP2 grown
on phenanthrene within 72h.
6.3.4 Growth of Pseudoxanthomonas DMVP2 on other xenobiotic compounds
The efficiency of strain DMVP2 to utilize other xenobiotic compounds as sole source of
carbon and energy is presented in Table 6.4. Besides being able to degrade phenanthrene,
strain DMVP2 was also able to utilize other aromatic hydrocarbons like pyrene,
fluoranthene, benzene, acetone, carbazole and aliphatic hydrocarbons such as petroleum
oil, diesel fuel and hexane as sole carbon source. Comparing the results of our study with
those available in the literature, it is evident that broad catabolic ability is common
among all PAH-degrading strains due to structural similarity of many aromatic
hydrocarbons and broad substrate specificity of dioxygenase enzyme. Moreover,
successive cleavage of fused aromatic rings during biodegradation of multi-ring aromatic
compounds eventually produces mono-aromatic compounds as intermediates; hence one
of the possibilities for the multi-ring degraders is that they utilize mono-aromatic
compound as carbon source (Kim et al., 2005). Recently, Wongwongseea et al., (2013)
also reported that PAH-degrading strains have high versatility to degrade other PAHs.
0 min
16 min
8 min
Ab
sorb
an
ce
Wavelength (nm)
24 min
Chapter 6: Phenanthrene degradation………
210
Churchill et al., (1999) also observed that Mycobacterium sp. strain CH1 could degrade
pyrene and was also capable of using a wide range of branched alkanes and n-alkanes as
sole carbon and energy sources.Strain DMVP2 was not able to utilize toluene, xylene and
naphthalene as sole carbon source.
Table 6.4 Growth of strain DMVP2 on other xenobiotic compounds in BHB containing
0.1% (w/v) peptone and supplemented with 300 ppm of phenanthrene at 37°C under
shaking condition at 150 rpm.
+++ Very fast growth rates, ++ fast growth rates, - no growth
The inability of strain DMVP2 to tolerate mono-aromatic hydrocarbon (xylene and
toluene) is generally believed to be due to disruption of biological membranes. Alteration
of membrane structure can disrupt energy transduction and the activity of membrane-
associated proteins (Heipieper et al., 1994; Sikkema et al., 1995). There are reports
Xenobiotic compounds Growth profile
PAH
Phenanthrene +++
Naphthalene -
Fluoranthene +++
Pyrene +++
Monoaromatic hydrocarbons
Benzene +
Toluene -
Xylene -
Asphaltenes
Acetone ++
Phenol -
Resins
Carbazole ++
Other petroleum hydrocarbons
n-hexane ++
Petroleum oil +++
Diesel fuel +++
Metabolites
Salicylic acid --
Phthalic acid +++
Protocatechuic acid ++
Catechol -
Chapter 6: Phenanthrene degradation………
211
suggesting that bacteria degrading phenanthrene by phthalate pathway cannot grow on
naphthalene as sole source of carbon (Keum et al., 2005; Xia et al., 2005).
Strain DMVP2 was able to utilize intermediates of phenanthrene degradation pathway
such as phthalic acid and protocatechuic acid as sole carbon source but not others such as
catechol, 1-naphthol and salicylic acid. The growth of strain DMVP2 on phthalic acid
and protocatechuic acid again showed that the strain DMVP2 degrades phenanthrene via
phthalic acid – protocatechuic acid pathway.
6.3.5 Effect of presence of other petroleum hydrocarbons on phenanthrene
degradation
Generally bioremediation studies are focused on degradation of single hydrocarbon and
neglect the effect of other co-contaminants which are generally present at the
hydrocarbon contaminated sites. In the open environment, PAHs exist as diverse multi-
component mixtures at contaminated sites. Therefore, studies on biodegradation using
single PAH compounds does not accurately reflects the true complexity of PAH
degradation required in natural environments. In some cases, these interactions may be
positive, resulting in increased degradation of one or more components, whereas in other
cases negative effects have also been observed (Dean-Ross et al., 2005). Keeping this in
view, we determined the effect of presence of other petroleum hydrocarbons on
phenanthrene degradation by strain DMVP2 [Fig. 6.14 (a, b)]. When phenanthrene was
present alone in the medium (control), 97% of the phenanthrene was degraded within
96 h.
The addition of pyrene and fluoranthene to the medium along with phenanthrene did not
affect the pattern of phenanthrene degradation, as 95% of phenanthrene was degraded
within 96 h in presence of pyrene and fluoranthene. Moreover, growth of strain DMVP2
increased when fluoranthene and pyrene were present individually along with
phenanthrene [Fig. 6.14(b)]. Somtrakoon et al., (2008) had also reported that
phenanthrene degradation was not affected in the presence of high molecular weight
PAHs. In contrast, Zong et al., (2007) reported that Sphingomonas sp. Pheb4 took longer
time to degrade phenanthrene in presence of other PAHs. Growth of strain DMVP2 as
well as phenanthrene degradation decreased in presence of naphthalene.
Chapter 6: Phenanthrene degradation………
212
0
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Ph
en
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de
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%)
Petroleum hycrocrabons (0.1%)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Ab
so
rba
nc
e a
t 6
60
nm
Petroleum hydrocarbons (0.1%)
(a)
(b)
Fig. 6.14 Effect of petroleum hydrocarbons on (a) phenanthrene degradation and (b)
growth of strain DMVP2 in BHB containing 0.1% peptone (w/v) and supplemented with
300 ppm of phenanthrene at 37°C under shaking condition at 150 rpm. [Phenanrene depict
as a control: only phenanthrene in medium, whereas other petroleum hydrocarbons:
Petroleum hydrocarbon added individually in medium along with phenanthrene].
Natsuko et al., (2013) also reported that petroleum hydrocarbon mixture influence the rate
of hydrocarbon degradation. The growth of strain DMVP2 was not hindered when diesel
fuel and petroleum oil were present in medium individually along with phenanthrene,
Chapter 6: Phenanthrene degradation………
213
rather it supported the growth of bacteria and enhanced the rate of phenanthrene
degradation. Strain DMVP2 was able to degrade phenanthrene (300 ppm) completely
within 96 h. One of the possible reason, as suggested by Yap et al., (2010), is that lipids
present in the oil weakens the interfacial tension and change the property of interface
between hydrophobic phase and aqueous phase. This promotes the solubilizing of
phenanthrene in the medium and thus increased degradation was observed. Pizzul et al.,
(2006) had also reported that addition of vegetable oil increased the biodegradation of
phenanthrene by Actinomycetes. Therefore, diesel fuel and petroleum oil could be used as
biostimulant agent for bioremediation of phenanthrene contaminated environment.
Interestingly, the presence of carbazole in medium along with phenanthrene significantly
increased the growth of strain DMVP2; however phenanthrene degradation decreased to
56%. Decreased degradation in the presence of multiple substrates can be due to
formation of toxic metabolites and competitive inhibition (Bouchez 1995; Stringfellow
and Aitken, 1995). Strain DMVP2 could degrade 92, 83 and 23% of phenanthrene (300
ppm) in the presence of benzene, toluene and xylene, respectively. Even growth of strain
DMVP2 increased in the presence of benzene whereas decreased in the presence of
toluene and xylene. Therefore, presence of toluene and xylene showed inhibitory effect
on phenanthrene degradation.
6.3.6 Microcosm studies in soil by Pseudoxanthomonas sp. DMVP2
For in-situ bioremediation of contaminated environments, seeding by introduction of
microorganisms has been considered a valuable tool to increase the rate and extent of
biodegradation of the pollutants. Although, an organisms can perform well in optimal
conditions, its efficiency may differ in natural environments when the organism is
subjected to various physico-chemical factors and forced to compete with native
microbial population. Hence, the use of microorganisms depends on the survival and
performance in natural ecosystems, to ensure this, the organisms has to be evaluated for
its survival and degradation efficiency in soil and also in the presence of the indigenous
microbial populations (Kastner et al, 1997).
Chapter 6: Phenanthrene degradation………
214
0
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50
60
70
80
90
100
set A Set B Set C set D
% P
he
na
nth
ren
e d
eg
rad
ati
on
Soil conditions
The microcosm study can provide insight on the soil colonizing ability of the culture by
competition with indigenous microflora along with phenanthrene degradation.
Fig 6.15 phenanthrene degradation by Pseudoxanthomonas sp. DMVP2 during microcosm
studies at 37°C within 120h. (Set A) sterile soil with phenanthrene, (Set B) sterile soil with
phenanthrene inoculated with Pseudoxanthomonas sp. sp. DMVP2, (Set C) non sterile soil
with phenanthrene, (Set D) non sterile soil with phenanthrene inoculated with
Pseudoxanthomonas sp. DMVP2.
The result of the study (Fig. 6.15), with initial phenanthrene concentration of 300 ppm,
showed that in absence of microorganisms, spontaneous phenathrene loss was about 23%
i.e. in the control set (set A), 68.31% phenanthrene degradation was observed by strain
DMVP2 without any indigenous microflora (set B), soil inhabiting microflora was also
able to degrade phenanthrene 39% (set C), in presence of both microflora and strain
DMVP, the phenanthrene degradation 51.49% (set D).
Result indicated that strain DMVP2 was able to degrade maximum phenanthrene
(68.31%) in absence of indigenous flora within 120h. However phenanthrene degradation
rate was decreased in microcosm than BHB. This may be due to the change in soil
characteristics and also may be due to decreased bioavailability of phenanthrene.
However phenanthrene degradation was decreased to 51.49% in presence of indigenous
flora. This may be due to the competition of indigenous micro-organism. Results also
indicated that soil indigenous micro flora has negligible capability to degrade
Chapter 6: Phenanthrene degradation………
215
phenanthrene. Teng et al., (2010) also found that bioaugmentation by Paracoccus sp.
increased phenanthrene degradation.
6.4 Conclusions
To the best of our knowledge this is the first report on optimization as well as analysis of
metabolic profile of phenanthrene degradation by Pseudoxanthomonas genera. Strain
DMVP2 was able to degrade phenanthrene (300 ppm) completely within 120 h under
optimized conditions. This study provides valuable information on optimization of
critical parameters to enhance phenanthrene degradation by Pseudoxanthomonas genera.
Strain DMVP2 was able to utilize many xenobiotic compounds, other than phenanthrene,
as sole carbon source. Moreover, Pseudoxanthomonas sp. DMVP2 can degrade
phenanthrene in presence of other petroleum hydrocarbons and thus it can play an
important role in biodegradation of phenanthrene in sites contaminated with mixture of
pollutants. Moreover, microcosm study revealed that strain DMVP2 could colonize and
able to degrade phenanthrene in presence of ingenious flora. Therefore, bioaugmentation
by strain DMVP2 can be promising bioremediation strategy for PAH-contaminated
sediment.
Chapter 6: Phenanthrene degradation………
216
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Churchill, S.A., Harper, J.P., Churchill, P.F. (1999) Isolation and characterization of a
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Dean-Ross, D., Moody, J., Cerniglia, C. E. (2002) Utilization of mixtures of polycyclic
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Filonov, A.E., Puntus, I.F., Karpov, A.V., Kosheleva, I.A., Akhmetov, L.I., Yonge, D.R.,
Petersen, J.N., Boronin, A.M. (2006) Assessment of naphthalene degradation
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Finkmann, W., Altendorf, K., Stackebrandt, E., Lipski, A. (2000) Characterization of
N2O-producing Xanthomonas-like isolates from biofilters as Stenotrophomonas
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Pseudoxanthomonas broegbernensis gen. nov., sp. nov. Int J Syst Evol Microbiol.
50: 273-282.
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Fujikawa, K., Fort, F.L., Samejima, K., Sakamoto, Y. (1993) Genotoxic potency in
Drosophila melanogaster of selected aromatic amines and polycyclic aromatic
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diverse aerobic bacteria. Biosci Biotechnol Biochem. 67: 225-243.
Hambrick, G.A., De Luane, R.D., Patrick, W.H. (1980) Effect of estuarine sediment pH
and oxidation-reduction potential on microbial hydrocarbon degradation. Appl
Environ Microbiol. 40: 365-36.
Heipieper, H.J., Weber, F. J., Sikkema, J., Keweloh, H., Bont, J.A.M. (1994)
Mechanisms of resistance of whole cells to toxic organic solvents. Trends
Biotechnol. 12: 409-415.
Juhasz, A.L., Stanley, G.A., Britz, M.L. (2000) Microbial degradation and detoxification
of high molecular weight polycyclic aromatic hydrocarbons by Stenotrophomonas
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