Chapter 6: Phenanthrene...

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.


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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


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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


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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.


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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


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


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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


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


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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.


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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).


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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.


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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


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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-


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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 +


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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).


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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.


198

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


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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


200

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)

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.


201

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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


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0 24 48 72 96 120

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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


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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


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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


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Tween 80 Triton X100

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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.


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


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


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.


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

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Wavelength (nm)

24 min


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 -


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.


212

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Petroleum hycrocrabons (0.1%)

0

0.2

0.4

0.6

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1

1.2

1.4

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Ab

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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,


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).


214

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set A Set B Set C set D

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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


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.


216

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