Access to this full-text is provided by Wiley.
Content available from ISRN Biotechnology
This content is subject to copyright. Terms and conditions apply.
Hindawi Publishing Corporation
ISRN Biotechnology
Volume , Article ID , pages
http://dx.doi.org/.//
Research Article
Biofilm-Mediated Enhanced Crude Oil Degradation by
Newly Isolated Pseudomonas Species
Debdeep Dasgupta, Ritabrata Ghosh, and Tapas K. Sengupta
Department of Biological Sciences, Indian Institute of Science Education & Research-Kolkata, Mohanpur Campus,
Nadia 741252, India
Correspondence should be addressed to Tapas K. Sengupta; senguptk@iiserkol.ac.in
Received December ; Accepted January
Academic Editors: W. J. Ernst, W. A. Kues, O. Pontes, S. Sanyal, and J. Sereikaite
Copyright © Debdeep Dasgupta et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
e bioavailability of organic contaminants to the degrading bacteria is a major limitation to ecient bioremediation of sites
contaminated wit h hydrophobic pollutants. Such limitation of bioavailability can be overcome by ste ady-state biolm-base d reactor.
e aim of this study was to examine the eect of suchmulticellular aggregation by naturallyexisting oil-degrading bacteria on crude
oil degradation. Microorganisms, capable of utilizing crude oil as sole carbon source, were isolated f rom river, estuary and sea-water
samples. Biochemical and S rDNA analysis of the best degraders of the three sources was found to belong to the Pseudomonas
species. Interestingly, one of the isolates was found to be close to Pseudomonas otitidis family which is not reported yet as a degrader
of crude oil. Biodegradation of crude oil was estimated by gas chromatography, and biolm formation near oil-water interface was
quantied by confocal laser scanning microscopy. Biolm supported batches of the isolated Pseudomonas specieswereableto
degrade crude oil much readily and extensively than the planktonic counterparts. Volumetric and topographic analysis revealed
that biolms formed in presence of crude oil accumulate higher biomass with greater thickness compared to the biolms produced
in presence of glucose as sole carbon source.
1. Introduction
World marine ecosystem has been studied extensively since
the second half of the last century. Oil spillage and oil
pollution in marine environment have been a major threat
to the ecosystem including the ocean life as well as to the
human being through the transfer of toxic organic mate-
rials including polycyclic aromatic hydrocarbons (PAHs)
into the food chain [–]. Presence of polycyclic aromatic
hydrocarbons (PAHs) in soil and water is major problem
as environmental contaminants and most of these PAHs are
recalcitrant in nature. PAHs mean a potential risk to the
marineanimalsaswellastothehumanhealthasmanyof
them are carcinogenic []. Physical and chemical methods
like volatilization, photooxidation, chemical oxidation, and
bioaccumulation [] are rarely successful in rapid removal
and in cleaning up PAHs [], and also these methods are
not safe and cost eective when compared to microbial
bioremediation. Bacteria have long been considered as one
of the predominant hydrocarbon degrading agents found
in the environment, which are free living and ubiquitous.
Over twenty genera of bacteria of marine origin have been
documented to be hydrocarbon degrading [–]. Bacteria
belonging to subphyla 𝛼-, 𝛽-, and 𝛿-proteobacteria [–]are
wellestablishedtobeofsuchnature.
One of the major factors that impedes the process biore-
mediation is bioavailability of hydrophobic contaminants to
the hydrocarbon utilising microorganisms. Although numer-
ousstudiesfocusedonbiolmreactorintheeldofbiore-
mediation [], pollution of marine bodies by oil and other
hydrocarbons solely during the oil spillage needs further
attention in the context of bioavailability of microorganisms.
It has been investigated earlier that this major limitation
can be improved by exploiting chemotactic bacteria [–
]. Microbial chemotaxis plays important role in surface
colonization and biolm formation [,]. Microbes have
a natural tendency to form multi-cellular aggregates being
gluedtoformbiolm[,].Biolmcanbeformedby
ISRN Biotechnology
single bacterial species or even by a group of bacteria, fungi,
algae, and protozoa. e potential of microbial aggregates in
the biolm communities for bioremediation is always a safer
and more adept method than planktonic microorganism as
the biolm matrix protects them during stress, and therefore
organism gets a better chance of adaptation []. Interest-
ingly, Klein et al. [] reported that hexadecane assimilation
by Marinobacter hydrocarbonoclasticus SP occurs through
the formation of a biolm at the alkane-water interface and
how the cell behavior changes with the presence of utilizable
or nonutilizable alkanes at the interface. e biolm-based
reactors furnish high microbial biomass accessible for better
microbialactivitythanplanktoniccellsforotherbiological
activities like biomineralization []. Recently the eciency
of biolm-associated cells in degradation of naphthalene
over planktonic had been elucidated for strain Pseudomonas
stutzeri T, and the survival of cells in petroleum contam-
inated soil is well documented []. Chandran and Das []
demonstrated % degradation of diesel oil over a period of
days by yeast biolm on gravel particle. Faster and intense
depletion of linear and brunched hydrocarbon was observed
in biolm microbial community of Alcanivorax borkumensis
[]. Microbial consortia on gravel particle were found as
conducive tools for self-cleaning of oily gulf coast throughout
all the sites and season []. Biolm community is diverse
and relatively stable for longer period of time []. e lm
consortia were isolated from petroleum contaminated urban
subway drainage system where they were capable of degra-
dation at een-degree centigrade []. e phenomenon
of chemotaxis by the organisms towards the pollutants and
the simultaneous attachment-detachment process maintains
a constant load of biomass to the aected site in the water
bodies.
Oil spillage has taken place in India for more than one
instance. Notably, in early , a Japanese tanker collided
with a small Indian vessel km west of the Nicobar and
Andaman Archipelago, spilling over , tons of oil into
the Indian Ocean. More recently, a ship carrying iron ore is
reported to be spilling oil in the sea near Paradip Port (Orissa,
India) since it has gone down under sea in September .
Kolkata Port and nearby areas like Haldia port, part of Bay
of Bengal close to the ports and Haldia Renery are major
concerns for possible oil spillage due to everyday transport
of fuel oil and other means since it is a major shipping
corridor for eastern region of India. In spite of possible
threat of contamination of water sources by spilled oil in
these areas, little work has been done so far on presence and
characterization of oil-degrading microorganisms, naturally
existing in water sources near Kolkata port and nearby areas.
Our present work was to emphasize the multicellular
aggregation of biolm formation by naturally occurring
hydrocarbon degrading strains from this region and to inves-
tigate the applicability of biolm amendment on enhance-
ment of biodegradation of crude oil. Briey, microorganisms,
capabletoutilizecrudeoilassolecarbonsource,wereisolated
from the water samples of the previously mentioned sources
through serial enrichment culture technique. Based on better
crude oil utilization ability, three of the isolated strains from
the three mentioned sources were screened. e organisms
werecharacterizedandidentiedbybiochemicaltestandS
rDNA sequencing and further tested for utilization of various
fuel oils and their ability to form biolm. e volumetric and
topological properties of biolm near oil water interface were
estimated by confocal laser scanning microscopy (CLSM).
Gas chromatography-mass spectroscopic analysis was carried
out to measure the eect of biolm amendment on crude oil
degradation in comparison to planktonic culture alone.
2. Materials and Methods
2.1. Source of Microorganisms. Water samples were collected
from Kolkata port of Hooghly River (∘N, .E),
River Haldi at Haldia port (∘N, ∘E) and Bay of
Bengal at Digha (∘N
∘E) for isolation of crude
oil degrading microorganism. Water sample from Gomukh
glacier (∘N, ∘E), the source of Ganges river at the
altitudeofm,wasusedascontrolnonpollutedwaterand
tested for a presence of crude oil degrading bacteria.
2.2. Culture Enrichment Isolation and Characterization of
Strain. e enrichment of crude oil degrading bacteria was
carried out under aerobic condition with crude oil as sole
source of carbon. Crude oil was obtained from Indian
Oil Corporation Limited (IOCL, Haldia, West Bengal). e
mineral salt media (MSM) [] were amended with % crude
oil (v/v), and enrichment of culture was carried out in
three consecutive batches each having a span of days and
enriched by using previous growth as inoculums for the next.
Bacterial growth was measured by using spectrophotometer
(Chemito Instruments UV ) at nm and compared
with control without inoculation. Selective solid inorganic
media (SSIM) [] were inoculated by spreading 𝜇Lof
broth from last batch of enriched culture incubated at ∘C
for days. Representative pure colonies were isolated and
further conrmed for oil degradation by growing in MSM
media provided with % crude oil (lter sterilized using
. 𝜇m syringe lter).
Selection of microorganisms was based on better ability
to grow in presence of crude oil as sole source of carbon
in growth media. e isolated microorganisms were tested
for Gram staining and biochemical properties as described
previously [–].Motilitytestsweredonebystabbingcells
in semisolid nutrient agar (.% agar) [].
2.3. Growth Characteristics in Dierent Oil and Biodegrada-
tion Analysis. Studies on growth characteristics of the iso-
lated microorganisms were carried in Bushnell-Hass (Difco)
media using crude oil, diesel, kerosene, unused engine oil
(Bharat petroleum), and used engine oil (obtained from local
service station). ese oil samples were lter-sterilized using
. 𝜇msyringelterandaddedintomLofBHmedia
(composition (gm/lit) MgSO4(.), CaCl2(.), KH2PO4
(.), (NH4)2HPO4(.), KNO3(.), FeCl3(.) and % of
oilsample),pH.eBHmediawereinoculatedwithisolated
bacteria and incubated at ∘C under static condition for
days. Aliquots of bacterial cultures were collected, serially
diluted, and plated on nutrient agar plates. e numbers of
ISRN Biotechnology
colonies were counted to determine bacterial growth in terms
of colony forming units (CFU/mL).
For biodegradation studies gas chromatography-mass
spectroscopic (GC-MS) analysis of crude oil was carried
out. Isolated microorganisms were grown in mL of BH
medium (pH 7 ± .02)at
∘C for days in presence of
% crude oil as sole carbon source. Aer days of growth,
the residual crude oil components were extracted with equal
volume of organic solvent dichloromethane (DCM). e
aqueous phase and the organic phase were separated in
separating funnel. e residual water from the organic phase
was absorbed by anhydrous sodium sulphate ( gm/ mL).
microliter samples of the DCM extracts were then analyzed
by GC-MS (Agilent N GC-MS-N) with a column
of (m) ×. (𝜇m) at a ow rate of . mL/min
[].esampleswereheldat
∘C for minutes initially
and increased at the rate of ∘C/min to reach the nal
temperature of ∘C. e nal temperature was held for
minutes. mL of BH medium with % crude oil was also
kept at ∘C for days as control and crude oil components
were extracted with DCM and analyzed by GC-MS as stated
before.
2.4. 16S rDNA Sequencing and Phylogenetic Analysis. A
colony of each isolate was grown overnight in LB medium
incubated at ∘C. mL sample of each culture was cen-
trifuged at g for min. e bacterial DNA was iso-
lated using bacterial genomic DNA isolation kit (Chromous
Biotech), and S rDNAs were amplied by using PCR master
mix (Fermentas). Bacterial universal primers Forward-f
(-AGAGTTTGATCATGGCTCAG-)andReverse-r
(TAC GGYTACC T TGTTAC G ACTT-)wereusedfor
amplication []. e PCR products were puried with
QIAquick Gel extraction kit (Qiagen). Nucleotide sequences
were determined from the puried product by automated
sequencer with an ABI PRISM II Dye Terminator Cycle
Sequencing kit (Chromous-Biotech) with the same primers.
e identity of S rDNA sequences of isolates was deter-
minedbyusingtheBLASTdatabasesearch[]. Eighteen
sequences (rst hits for each isolate) of the cultivable
organisms were procured from NCBI-Blast search, and the
alignment was done by using CLUSTALX . soware. e
alignment was thoroughly checked in soware SEAVIEW
for any gaps and edited accordingly. A phylogenetic tree
was constructed by neighbour joining method (Kimura -
parameter) using MEGA v-., and the tree was subsequently
bootstrapped (random speed , replicates).
2.5. Oil Biolm Development and Quantication. ree iso-
lated strains were rst tested for biolm formation on mm
glass cover slips being immersed in mL BH media with
% crude oil in mL sterile falcon tubes. e organism was
inoculated and incubated at ∘Cfordays.ecoverslips
were recovered from the culture tubes, washed thoroughly
in % saline solution aseptically, air-dried and Gram-stained.
Formation of biolm was viewed under X oil immersion
objective using Nikon’s DN microscope. e formation of
biolm on thin glass cover slips was also studied for hourly
development of lm by the strain KPW.-S and stained at
dierent time intervals of growth at th, th, th, th, th
and th hours.
Biolm load in presence of crude oil was estimated by the
confocal laser scanning microscopic (CLSM) image stacks
[]. Briey, the isolated strains were grown on glass surface
in presence of % crude oil (glass slide: 25 × 75 mm) and %
glucose (v/v) (cover glass: 12 × 12mm), respectively, in BH
media. e surface of the substratum was washed with PBS
(X) thrice and stained with .% acridine orange (w/v) for
minutes in dark and washed twice. e slides were observed
under Carl Zeiss CLSM- (Axio observer microscope
version Z.) using nm excitation argon laser with MBS
(main beam splitter) and emission wavelength detected from
to nm. e image acquisition was done under X
oil immersion lens (NA: .). Series of measurement was
taken at random vertically across the oil water interface (in
caseofcrudeoil)andairwaterinterfaceincaseofglucose.
All samples were viewed from the clean side of the cover
slip under oil, and the height was measured from these
transects vertically from the base of cover slip to the top of the
biolm (frame size 512 × 512,-bitimage,𝑍stacks interval
.𝜇m). Volumetric and topological parameters (thickness,
biovolume, biomass, surface area, skewness, and kurtosis)
of biolm were calculated using the soware provided (Zen
) along with the microscope.
2.6. Eect of Biolm Amendment on Biodegradation. Oil
degradation was also compared for biolm amended and
unamended planktonic cultures (with equal starter inocu-
lums) with KPW.-S and strain with biolm defect DSW.-S.
Glass slide of dimension mm × mm was immersed in
mL falcon tube containing mL BH media with % crude
oil and incubated for days at ∘C. For biodegradation
analysis other sets of batch cultures with and without biolm
carrying the residual crude oil were extracted with equal
volume of organic solvent dichloromethane. e aqueous
phaseandoilwereseparated.eresidualwaterwasabsorbed
by anhydrous sodium sulphate ( gm/ mL). e extract was
analyzed by gas chromatography-mass Spectroscopy (Agilent
N GC-MS-N) as described in the previous section.
Percentage of degradation for ten detectable peaks (with
respect to control) was calculated by the method described
earlier [].
2.7. Statistical Analysis. Graph plotting and multiple compar-
isons of optical densities were assessed by Origin followed
by SigmaPlot soware version , San Jose, California, USA.
e data were expressed as mean ±standard error.
3. Results
3.1. Enrichment and Screening of Organism. During incuba-
tion of water samples (collected from Kolkata Port, Haldia
Port, and Bay of Bengal areas) in MSM media (containing
crude oil as sole carbon source) no visual change in turbidity
duetobacterialgrowthwasobservedtillthethirddayof
incubation. e optical density (for bacterial growth) kept
ISRN Biotechnology
T : Growth characteristics of isolated organisms by utilization of various oil/hydrocarbon as sole carbon source.
Oil/hydrocarbon
Colony forming unit/mL
Strain
K P W. - S H R W. - S D S W. - S
Crude oil (1.5 ± 0.17)×(6.8 ± 0.11)×(1.4 ± 0.09)×
Diesel (6.7 ± 0.072)×(6.1 ± 0.08)×(6.5 ± 0.12)×
Kerosene (7.7 ± 0.06)×(1.6 ± 0.04)×(1.7 ± 0.07)×
Hexadecane (2.94 ± 0.13)×(4.4 ± 0.02)×(2.4 ± 0.07)×
Engine oil (6.1 ± 0.07)×(6.4 ± 0.11)×(3.6 ± 0.07)×
T : Preliminary biochemical tests for isolated bacterial strains.
Characteristics KPW.-S HRW.-S DSW.-S
Gram nature Negative Negative Negative
Shape Rod Coccobacillary Coccobacillary
Diusible pigments ++ −
Motility ++ +
Citrate utilization ++ ++ ++
Lysine utilization +++
Ornithine utilization ++ +
Urease test −− −
Phenylalanine test NC NC NC
Nitrate reduction NC NC NC
HS production +++ ++ +
Production of acid by
utilization of sugar∗
(i) Glucose −− −
(ii) Adonitol −− −
(iii) Lactose −− −
(iv) Arabinose −− −
(v) Sorbitol −− −
NC: not conclusive.
∗e test attributed the change of the color of pH indicator by production of
acid and gas when grown in presence of various sugars.
increasing slowly for the next – days. e second enrich-
ment using inoculums from the rst enrichment showed
a little early response with a shorter lag period of growth,
and the culture reached stationary phase of growth within
– days. Finally, in the third serial enrichment, culture
inoculated from the second batch showed an early response,
and signicant growth was observed in the second day
onwards and reached stationary phase of growth within
days (Figures (a)–(c)). Interestingly, water sample, collected
from Gomukh glacier, showed no growth even aer weeks
of incubation in MSM medium, containing crude oil as sole
carbon source (data not shown).
From the third batch of enriched cultures, pure colonies
were isolated on SSIM crude oil agar plates. Total of ,
, and pure colonies were thus obtained from three
water samples collected from Kolkata port, Haldi River
water, and Bay of Bengal near Digha site, respectively. ese
colonies (in duplicate) were restreaked on nutrient agar (NA),
and they were dierentiated by colony characteristics based
on morphology and pigmentation. e distinct colonies
with unique characteristics were isolated as pure culture
in NA media. All isolated colonies were tested for further
conrmation of their ability of oil degradation, and three
isolates from three dierent sources were selected based on
their rapid growth in presence of crude oil as sole carbon
source (data not shown). e organisms were named as
KPW.-S (isolated from Kolkata Port water, MTCC ),
HRW.-S (isolated from Haldi River water, MTCC ),
and DSW.-S (isolated from Digha sight of the shore of Bay
of Bengal, MTCC ) and currently deposited at Microbial
Type Culture Collection and Gene Bank (MTCC), India.
3.2. Growth Characteristics in Dierent Oil and Biodegrada-
tion Analysis. e selected bacteria (KPW.-S, HRW.-S,
and DSW.-S) were subjected to grow in presence of various
other oils as carbon source. e bacterial growth was found
to be uneven depending on the bacterial species and oil
type (Table ). Signicant dierence was observed between
the growth of the three strains in case of crude oil and
hexadecane (𝑃 < 0.05). e strain KPW.-S showed best
growth in presence of hexadecane as carbon source and the
DSW.-S in presence of crude oil and higher hydrocarbons
presentinengineoil.ekeroseneshowedtobetheleast
supportive as a carbon source, and it had no signicant
eect on dierence of growth among the three strains (𝑃>
0.05). e strain HPW.-S showed average growth rate and
crude oil degradation (Figure (c),Table). ese results
clearly indicate that dierent oils were degraded and utilized
by all the strains in various proportions, depending on the
complexity and aliphatic and aromatic nature of the sample
oil dependent on bacterial species as well.
Organisms KPW.-S, HRW.-S, and DSW.-S were
growninBushnell-Hass(BH)mediainpresenceofcrudeoil
as only carbon source at ∘C for days. e hydrocarbon
prole in the growth media aer days of growth was ana-
lyzed by GC-MS and compared with the hydrocarbon prole
from a control ask where the BH medium and crude oil
were kept together for days under identical conditions. e
hydrocarbon prole obtained by GC-MS analysis showed the
relative abundance of various hydrocarbons in the complex
mixture (Figure ). e control sample (Figure (a))shows
the presence of various hydrocarbons in the unresolved
complexmixture.Alltheisolatedbacteriawereabletoreduce
at least % of relative abundance of various hydrocarbons
ISRN Biotechnology
02468101214161820
22
0
0.5
1
1.5
2
2.5
3
Incubation time (days)
O.D at 600 nm
Enrichment-I
Enrichment-II
Enrichment-III
(a)
02 4 6 8 10 12 14 16 18 20 22
0
0.5
1
1.5
2
2.5
3
Incubation time (days)
O.D at 600 nm
Enrichment-I
Enrichment-II
Enrichment-III
(b)
Enrichment-I
Enrichment-II
Enrichment-III
0246810121416182022
Incubation time (days)
0
0.5
1
1.5
2
2.5
3
O.D at 600 nm
(c)
F : Enrichment of crude oil-degrading bacteria. Bacterial populations in water samples, collected from (a) Kolkata Port, (b) Haldi River
and (c) Digha site at Bay of Bengal were subjected to three-step enrichment process in MSM in presence of crude oil as sole carbon source.
Inallthecasesthestandarderrorvaluesrangedfrom.%to%.
present in crude oil compared to control experiment (Fig-
ures (b)–(d)) within days of incubation. Interestingly,
bacterial strains isolated from Kolkata port (KPW.-S) and
Digha area of Bay of Bengal (DSW.-S) showed minimum
and maximum overall hydrocarbon degradation abilities,
respectively.
3.3. Taxonomic Identication of Bacterial Strains. Isolated
three organisms (KPW.-S, HRW.-S, and DSW.-S) were
tested for a series of biochemical and morphological tests
(Table ). All three organisms, KPW.-S, HRW.-S, and
DSW.-S, were found to be Gram-negative, citrate positive,
rod-shaped (similar to coccobacillus shape), and motile.
When crude oil was used as carbon source, all the isolated
organisms showed similar growth characteristic at dierent
temperatures ranging from ∘Cto
∘Cwithanoptimum
temperature at ∘C.FurtheranalysisofSrDNAgene
sequences was done for taxonomic identication. Ampli-
cation and sequencing of S rDNA followed by phyloge-
netic analysis (together with biochemical and morphological
analyses as described in Table )revealedthattheisolated
strains (KPW.-S (FJ), HPW.-S (FJ), and
DSW.-S (FJ)) belong to phylum Proteobacteria, class
Betaproteobacteria [], and genus Pseudomonas (Figure ).
ISRN Biotechnology
90
45
5
Abundance
Time (min)
58 11 13.5
×105
(a)
45
25
5
Abundance
×105
Time (min)
58 11 13.5
(b)
20
12
1
Abundance
Time (min)
58 11 13.5
×105
(c)
24
12
2
Abund ance
×105
Time (min)
58 11 13.5
(d)
F : Biodegradation of crude oil (at ∘C, -day incubation) analyzed by GC-MS. (a) Without microorganism (control) and with
organisms (b) KPW.-S, (c) HRW.-S, (d) and DSW.-S.
e strains KPW.-S and HPW.-S are the closest neigh-
bour of Pseudomonas aeruginosa strain Tsaydam--ASA
(KC.), whereas DSW-S is nearest to the candidate
of Pseudomonas otitidis strain f (AB.).
3.4. Oil Biolm Development. Preliminary studies demon-
strated that the isolated bacterial strains possess the ability
to form biolm on glass surface when grown in BH medium
in presence of crude oil as sole carbon source (Figures
(a)–(c)). Interestingly, a thick biomass was observed to
be aggregated near the oil water interface on the glass
bioreactor. Transitional episode of oil biolm development
was observed vividly using light microscopy. e initial event
of bacterial attachment was found in the rst sixth hour
of growth (Figure (a)). In the next hours, the cells start
forming nascent cell cluster being cemented on the glass
substratum (Figure (b)). At around hours of growth
cell clusters become more mature and initiate aggregation
(Figure (c)). is was followed by evacuation and release of
cells from the matrix with further incubation (Figures (d)–
(f)). Interestingly, KPW.-S cells were again able to form
well-dened biolm on the same glass surface at around
hours of incubation (Figure (f)).
3.5. Quantication of Biolm by Confocal Laser Scan-
ning Microscopy. Volumetric and topologic quantication of
biolm formations by the isolated Pseudomonas species at oil-
water interface was carried out by confocal laser scanning
microscopy (CLSM). All of the three Pseudomonas strains
tested showed ability to form biolm near air-water interface
when grown in presence of glucose as sole carbon source
(Figures (a)–(c)). e average thickness of KPW.-S and
HRW.-S was found to be and 𝜇minpresenceof
glucose whereas it was found to be .𝜇mincaseofthethird
strain DSW.-S (Table ). Interestingly, enhanced biolm
production was observed when the Pseudomonas cells were
growninpresenceofcrudeoilassolesourceofcarbon
(Figures (d)–(f)). e average thicknesses of KPW.-S,
HRW.-S, and DSW.-S were found to be , , and
. 𝜇m, respectively, in presence of crude oil representing at
least -fold increase of biolm thickness for all the strains
(Table ). ese results clearly showed that presence of crude
ISRN Biotechnology
Pseudomonas aeruginosa strain 6A (bc4) (JX661716.2)
Pseudomonas aeruginosa strain R8-769-1 (JQ659982.1)
Bacterium H1C (JX149543.1)
Endophytic bacterium 202P-2 (JF901362.1)
Pseudomonas aeruginosa strain IFS (JQ041638.1)
Pseudomonas aeruginosa strain N002 (JX035794.1)
Pseudomonas aeruginosa strain Tsaydam-5-ASA (KC137277.1)
Pseudomonas aeruginosa strain JYR-Pk-2011 (JQ792038.1)
Pseudomonas aeruginosa strain R7-734 (Q659920.1)
Pseudomonas aeruginosa strain RI-1 (JQ773431.1)
Pseudomonas sp. CEBP1 (JQ894531.1)
Pseudomonas sp. a-1-8 (JX416374.1)
Bacterium P2A (JX149546.1)
Pseudomonas aeruginosa strain DSM 50071T (HE978271.1)
Pseudomonas sp. KPW. 1-S1 (FJ897721.1)
Pseudomonas sp. HRW. 1-S3 (FJ897723.1)
Pseudomonas sp. DSW. 1-S4 (FJ897724.1)
Pseudomonas otitidis strain 81f (AB698739.1)
Pseudomonas sp. GD6(2010) (GU566307.1)
Pseudomonas sp. 8.2 (EF426444.1)
Pseudomonas sp. 7.5 (EF426443.1)
Pseudomonas sp. AHL 2 (AY379974.1)
Pseudomonas otitidis strain R6-410 (JQ659846.1)
Pseudomonas sp. TSH17 (AB508848.1)
0.001
60
8
62
100
60
F : Phylogenic tree of all sequenced 𝛽-Proteobacteria (∙) KPW.-S, (◼)HRW.-S,(⧫) DSW.-S. e bootstrap values are indicated
for the major nodes.
oil in growth medium enhanced biolm production by all
of the three strains, although the DSW.-S strain has lesser
potential to form biolm under the experimental conditions.
us, the spatial biomass accumulated near the oil water
interface by KPW.-S and HRW.-S was observed to be –
units per unit area, and for the low biolm former DSW.-
S, spatial biomass accumulation was - units per unit area.
In presence of crude oil, the maximum thickness of biolm
obtained by KPW.-S and HRW.-S was and 𝜇m
(Figures (d)–(f))whereasthestrainpossessingbiolm
defect could develop up to . 𝜇m of maximum thickness.
To further substantiate our nding, topological parame-
ters of biolm load were investigated through the measure-
ment of CLSM. Here, the mean thickness and biomass were
found inversely proportional to skewness (𝑆ku ). Since higher
skewness indicates lack of porosity [], it is likely that lesser
skewness and hence greater porosity enable biolm to access
higher nutrient and other essential factors which attributes
in greater thickness of biolm. e statement is validated
in six cases comprising three strains and two conditions
(Table ). Strikingly, no signicant dierence was observed
in other topological parameters, kurtosis (𝑆sk) which implies
that adherence of organism with substratum was more or less
similarinallthesixcasesirrespectiveofstrainwithprofound
biolm forming ability (KPW.-S and HRW.-S) or strain
DSW.-S with lesser potential to make thick biolm.
3.6. Comparative Degradation Study. e aggregation of cells
near oil-water interface observed through CLSM prompted
to examine the relation between biolm formation at the
oil-water interface and utilization/degradation of crude oil
components by the Pseudomonas isolates. A comparative
account of GC-MS proles of hydrocarbons present in crude
oil was analyzed in presence and absence of substratum
support. Biolm forming strain KPW.-S and biolm defec-
tive DSW.-S were taken into account for oil degradation
analysis. e GC-MS result shows that the relative abundance
of the peak reduced considerably for both of the strains
depending on potential of oil degradation (Figure ). Oil
degradation in terms of reduction of total numbers of hydro-
carbon peaks and depletion of total area of chromatogram
of various components of crude oil was observed with the
introduction of biolm amendment. 20 ± 10%to40 ±
10% increment of degradation was achieved in presence of
matrix enclosed biolm in comparison to planktonic cells
alone for both of the strains. Short chain hydrocarbon peaks
were out of detection limit. From the percentage of degra-
dation measured using the method described earlier, it is
evident that increased degradation of individual hydrocarbon
is mediated or induced by the biolm near oil air-water
interface (Figure ). Additionally for the strain KPW.-S
the biolm assisted culture could target the short chain low
molecular weight hydrocarbons (Table ,Figure), whereas
the strain DSW.-S could successfully degrade both short
and long chain hydrocarbons present in crude oil eciently.
4. Discussion
Ability to form biolm on various surfaces is always ad-
vantageous for the microorganisms in terms of survival,
ISRN Biotechnology
T : Percentage degradation of individual hydrocarbon present in crude oil.
Peak no. Retention time (minute) Percentage degradation
𝑃/KPW.-S 𝐵𝐿/KPW.-S 𝑃/DSW.-S 𝐵𝐿/DSW.-S
. . . . .
. . . . .
. . . . .
. . . . .
. . . . .
. . . . .
. . . . .
. . . . .
. . . . .
. . . . .
Overall∗.–. . . . .
𝑃represents degradation of hydrocarbon without amendment of biolm (only planktonic).
𝐵𝐿represents degradation of hydrocarbon with amendment of biolm (planktonic + biolm).
∗Represents overall degradation of all the hydrocarbon considering the total area of control and samples under dierent conditions.
T : Mean thickness, biomass, substratum coverage, kurtosis (𝑆ku), and skewness (𝑆sk)ofbiolmofPseudomonas sp. KPW.-S, HRW.-S,
and DSW.-S.
Carbon source Strain Avg. thickness (𝜇m) Total biomass (𝜇m/𝜇m)Kurtosis(𝑆ku )Skewness(𝑆sk) Max. thickness (𝜇m)
KPW.-S 22.36 ± 0.98 14.17 ± 1.71 3.5 ± 0.43 −0.68 ± 0.31
Crude oil HWR.-S 26.51 ± 1.2 18.45 ± 1.36 4.19 ± 1.2 −1.01 ± 0.33
DSW.-S 4.55 ± 1.6 1.6 ± 0.55 4.06 ± 0.2 0.86 ± 0.16
KPW.-S 12.5 ± 0.89 9.33 ± 0.9 3.91 ± 0.5 −0.49 ± 0.34
Glucose HWR.-S 10.32 ± 0.37 6.7 ± 0.49 2.72 ± 0.3 −0.46 ± 0.15 .
DSW.-S 1.51 ± 0.08 0.9 ± 0.08 3.66 ± 0.2 0.48 ± 0.15 .
Values are means of data from image stacks. e standard error is calculated as the square root of the mean of the variances of each of the four groups (image
stacks from two glass slides in two independent experiment rounds).
metabolism, adaptation, and propagation [,]. One of the
major limitations faced in the process of bioremediation is the
bioavailability of organic compounds on site []. Early stud-
ies that indicate biolm forming bacteria can be employed to
overcome this limitation although the application of steady-
state biolm in bioremediation is not well established. Studies
indicate that biolm-mediated bioremediation is a procient
approach and safer option since cells in biolm have better
chance of survival and adaptability especially during the
stressed conditions [,].Establishmentofbiolmon
gravel particles and glass slides was reported previously
[] where the articially glued microorganisms showed
excellent attenuation of crude oil in liquid waste in batch
culture. Vaysse et al. [] showed altered prole of expressed
proteins, specically type VI secretion system in biolm
forming Marinobacter hydrocarbonoclasticus SP17 at alkane-
water interface.
Crude oil degrading bacteria were unrued from three
dierent locations which are the prominent risk zones of oil
contamination. ree-step enrichment process was employed
to enrich and isolate microorganisms with greater degrees of
oil-degrading capabilities. ese bacteria were then preferred
for additional characterization and identication. Out of the
selected organisms, KPW.-S, HRW.-S, and DSW.-S were
isolated from water of Kolkata Port, Haldi River, and Digha
at Bay of Bengal, respectively. All three bacteria were Gram-
negative, motile, oxidase positive coccobacilli in nature, and
were preliminarily identied as of class Betaproteobacteria.
Ability of the isolated bacteria for utilization of crude oil
as carbon source was investigated by GC-MS analysis. e
data revealed that the isolated strains could be able to reduce
dierent hydrocarbons present in crude oil samples up to
% within days of growth under laboratory conditions
(Figure ). e isolated organisms also showed their ability
to use other complex oils like petrol, diesel, and kerosene
ascarbonsourcesaswell(Table). It is interesting to note
that S rDNA sequence and phylogenetic analysis revealed
that KPW.-S and HRW.-S are closest to Pseudomonas
aeruginosa and DSW.-S to Pseudomonas otitidis (Figure ).
Previous studies on naturally existing oil-degrading bacteria
in these regions were elucidated although the molecular
phylogenetic analysis based on S rDNA sequencing was
not initiated [].usDSW.-SisanovelPseudomonas sp.
in terms of its ability to degrade crude oil since there is no
report so far for crude oil degradation ability of Pseudomonas
otitidis or any closely related species of Pseudomonas otitidis,
although, recently, Venketaswar Reddy et al. []reported
anewlyisolatedPseudomonas otitidis as a potential biocat-
alyst for polyhydroxyalkanoates (PHA) synthesis. Moreover,
results of the present study strongly suggest the existence of
ISRN Biotechnology
(a) (b) (c)
F : (a) Oil biolm formation on glass surface by the three isolates (a) KPW.-S, (b) HRW.-S, and (c) DSW.-S. Bars, 𝜇m.
(a) (b) (c)
(d) (e) (f)
F : Hourly development of biolm on thin glass cover slip at dierent time interval by KPW.-S at (a) th, (b) th, (c) th, (d) th,
(e) th, and (f ) th hours. Bars, 𝜇m.
hydrocarbon degrading consortia in the river water sample
although the degradation of crude oil by the strains was
limitedto%basedonthepeakheights(Figure). It was
presumptuous to assume that the major factor that governs
the low rate of degradation was bioavailability of hydrocarbon
to the microbial biomass. To address this limitation, we
developed a laboratory scale steady-state biolm reactor
which circumvents the limitation to an appreciable extent
as biolm formation near oil-water interface accumulates
substantially higher amount of biomass.
e isolated Pseudomonas strains in this study showed
their ability to form biolm on glass surface in BH medium
in presence of crude oil as the only carbon source (Figures
(a)–(c)). In addition to this, the chemotaxis elucidated by
the bacteria adhering on biolm could also support the fact of
bacterial motility towards the crude oil and other pollutants
present in it (data not shown). Moreover, there is a wealth
of evidence supporting the fact that biolm development
and maturation follow a cycle of attachment and release.
e time lapse image of oil biolm development indicated
that the multicellular aggregation near oil-water interface
cycles at regular interval. ese data further supports the
fact that the biolm amendment presents the hydrocarbon
degrading consortia to the oil and toxic compounds oating
on the aqueous surface for a prolonged duration. It has been
documented earlier that the mass-transfer limitations that
impede the bioremediation process can be overcome by cells
displaying chemotaxis that can sense chemicals such as those
adsorbed to soil particles in a particular niche and swim
towards them [].uswetestedtheeectofoilbiolm
(static) amendment on the overall degradation of crude oil
by GC-MS analysis and characterized the biolm formation
at oil water interface by confocal laser scanning microscopy
(CLSM).
First the biolm forming ability of the three isolates was
estimatedby-wellmicrotitreassaymethodinpresence
of various carbon source including glucose, glycerol, and
hexadecane(datanotshown).Biolmloadinpresenceof
ISRN Biotechnology
20 𝜇m
(a)
20 𝜇m
(b)
20 𝜇m
(c)
20 𝜇m
(d)
20 𝜇m
(e)
20 𝜇m
(f)
F : Confocal laser scanning microscopy image of biolm hive near air water interface by three isolates (a) KPW.-S, (b) HRW.-S,
and (c) DSW.-S growing in presence of glucose as carbon source. Similar image was taken in % crude oil by the three isolates (d) KPW.-S,
(e) HRW.S, and (f) DSW.-S to compare the anity of biolm formation in presence of oil. Biolms with maximum thicknesses achieved
by the three isolates in presence of crude oil are represented in the gure. Bars, 𝜇m.
Abunda nce
Time (min)
510 15 20 25 30 35
4.2𝑒+07
4𝑒+07
3.8𝑒+07
3.6𝑒+07
3.4𝑒+07
3.2𝑒+07
3𝑒+07
2.8𝑒+07
2.6𝑒+07
2.4𝑒+07
2.2𝑒+07
2𝑒+07
1.8𝑒+07
1.6𝑒+07
1.4𝑒+07
1.2𝑒+07
1𝑒+07
8000000
6000000
4000000
2000000
F : GC-MS analysis of oil degradation record of strain DSW.-
S for biolm amended and unamended conditions. Chromatogram
showing decrease in peak height and corresponding area. Control
(black), Planktonic only (blue), and biolm + planktonic cells (Red).
crude oil was not measured by this procedure as oil interfered
with the assay process. Based on the characteristic pattern
of biolm development of the isolated strains, KPW.-S and
biolm defective DSW.-S were selected for oil degradation
analysis. Our hypothesis got more impetus with GC-MS
prole of the oil degradation in presence and absence of
biolm amendment. e calculation of percentage of degra-
dation also suggests that the biolm amended culture could
successfully degrade the individual hydrocarbon with much
greater eciency (Table ). Notably, the overall degradation
was enhanced by %–% for both of the strains when
amended with matrix enclosed biolm cells in comparison
to planktonic cells alone (Figure ). e comparative study of
biolm formation showed that the oil biolm can accumulate
a large number of hydrocarbon degrading organisms near
the oil water interface. is biolm hive in oil had unusually
higher biomass compared to the lm produced in glucose
as carbon source. e degradation of individual hydrocar-
bons was also tested in representative ten peaks where the
similar reduction of peak area was observed. Although the
enhanced degradation by biolm defective strain DSW.-S
was counterintuitive, the comparative GC-MS data suggests
that the strain is better degrader when compared with others,
and therefore the oil degradation was further enhanced by
accumulated cells of DSW.-S enclosed in biolm near oil
water interface. Additionally biolm formation follows a
dynamic cycle of attachment and release from its substratum
during its meal on oil observed by Gram staining at dierent
time points (Figures (a)–(f)). e phenomenon is also
reected in its mean thickness and total biomass formation
observed by confocal imaging of biolm (Table ). e three
isolated strains varied in thier total thicknesses depending on
the phase in which they were stained and photographed in
independent experiments. e maximum thickness achieved
by all the three strains was found higher than the mean
due to the cycle of attachment and release. ese ndings
prompted us to examine the overall degradation prole in
a span of een days by the biolm amended and unamended
conditions. e GS-MS data strongly supports the fact that
the greater degradation is due to higher population density
in the biolm amended condition.
ISRN Biotechnology
e topological parameters of the biolm architecture
also highlight the intrinsic properties of these lms. e mean
thickness and average biomass calculated from confocal
imaging soware correlated with our biolm assay result.
e thickness and biomass were highest for KPW.-S and
HRW.-S whereas they were least for DSW.-S. e test was
repeated for biolm image obtained using glucose as carbon
source. From the result it was evident that the multicellular
aggregation was signicantly higher in presence of crude oil.
e greater skewness value of the poor biolm forming strain
DSW.-S makes the lm more porous. e higher standard
deviation relates the higher heterogeneity of the biolm for
theallthethreestrains.ekurtosisvalueforallthethree
strains was roughly the same making the adhesion potential
nearly similar.
Hence, from the previous nding it was conclusive that
in water bodies natural oil degrading strains capable of
formation of biolm degrade oil much faster and eciently
when compared to planktonic cells alone. Recently Al-Bader
et al. [] depict the role of phototrophic, diazotrophic,
and hydrocarbon-utilizing bacterial biolm consortia leav-
ing the promise of bioremediation of aquatic hydrocarbon
pollutants. Secondly, the enhancement of degradation is
profound in all oil degrading naturally exiting strains with
various degrees of biolm forming capacity. ese results
tempt us to speculate that natural oil degrading strains can
accumulate near oil water interface during oil spillage and
eciently circumvent the limitation of bioavailability of in
situ bioremediation to a considerably greater extent.
5. Conclusion
e results in the present study consolidate our nding
that potent hydrocarbon degrading bacterial consortia exist
naturally in the water body near Kolkata port, Haldia
Renery, and nearby areas in the eastern regions of India
in contrary to the water sources from Himalayan Glacier
where pollution through various sources is negligible. is
signies that the hydrocarbon degrading bacteria exist or
evolved to exist with the ever increasing intensity of marine
pollution. Pseudomonas strains, isolated from water sources
near Kolkata port, Haldi River, and Digha Sea shore, showed
that their ability to degrade various complex hydrocarbons
andbiolmsformedbytheisolatedbacteriacouldenhance
degradation ability. erefore, these isolated Pseudomonas
strains could be considered for future use for bioremediation
of contaminated spilled oil in water sources. However, further
studies are needed to evaluate the potential of the isolated
strains to degrade hydrocarbons in situ, in natural environ-
mental conditions. us, the oil degradation capability, ability
to form biolm, greater survival in the nutrient stressed con-
dition, cycle of attachment and release of biolm-associated
cells, and cooperative nature of these natural isolates could
be exploited as a better option for bioremediation technology.
is could be equally applicable for any Pseudomonas or other
bacterial strains ubiquitously available in nature having the
previously mentioned criteria, and the technology could be
further developed for targeting of any pollutants present on
earth creating enormous environmental and health hazards.
Acknowledgments
e authors thank TCG Life Sciences and National Test
House—Kolkata for GC-MS analysis. e authors also thank
Drs. Balaram Mukhopadhyay and Punyasloke Bhaduri of
IISER-Kolkata for their valuable suggestions and Mr. Sud-
hangsu Maity and Mr. Mrinmoy Bose for their technical assis-
tance. e authors also acknowledge the confocal imaging
facility of IISER Kolkata. D. Dasgupta is recipient of Institute
Fellowship from Government of India.
References
[] M.Blumer,H.L.Sanders,J.F.Grassle,andG.R.Hampson,“A
small oil spill,” Environment,vol.,no.,pp.–,.
[] M.B.Fernandes,M.A.Sicre,A.Boireau,andJ.Tronczynski,
“Polyaromatic hydrocarbon (PAH) distributions in the Seine
River and its estuary,” Marine Pollution Bulletin, vol. , no. ,
pp. –, .
[] A.SeiandB.Z.Fathepure,“BiodegradationofBTEXathigh
salinity by an enrichment culture from hypersaline sediments of
Rozel Point at Great Salt Lake,” Journal of Applied Microbiology,
vol. , no. , pp. –, .
[]E.Deziel,G.Paquette,R.Villemur,F.Lepine,andJ.G.
Bisaillon, “Biosurfactant production by a soil Pseudomonas
strain growing on polycyclic aromatic hydrocarbons,” Applied
and Environmental Microbiology,vol.,no.,pp.–,
.
[]H.P.Zhao,L.Wang,J.R.Ren,Z.Li,M.Li,andH.W.Gao,
“Isolation and characterization of phenanthrene-degrading
strains Sphingomonas sp. ZP and Tistrella sp. ZP,” Journal of
Hazardous Materials, vol. , no. , pp. –, .
[] R. C. Prince, “Bioremediation of marine oil spills,” Tre nds in
Biotechnology,vol.,no.,pp.–,.
[] A. Bruns and L. Berthe-Corti, “Fundibacter jadensis gen.
nov., sp. nov., a new slightly halophilic bacterium, isolated
from intertidal sediment,” International Journal of Systematic
Bacteriology,vol.,no.,pp.–,.
[] M. M. Yakimov, P. N. Golyshin, S. Lang et al., “Alcanivorax
borkumensis gen. nov., sp. nov., a new, hydrocarbon-degrading
and surfactant-producing marine bacterium,” Inter national
Journal of Systematic Bacteriology, vol. , no. , pp. –,
.
[] M. J. Gauthier, B. Lafay, R. Christen et al., “Marinobacter
hydrocarbonoclasticus gen. nov., sp. nov., a new, extremely
halotolerant, hydrocarbon-degrading marine bacterium,” Inter-
national Journal of Systematic Bacteriology,vol.,no.,pp.
–, .
[] M. A. Engelhardt, K. Daly, R. P. J. Swannell, and I. M.
Head, “Isolation and characterization of a novel hydrocarbon-
degrading, Gram-positive bacterium, isolated from intertidal
beach sediment, and description of Planococcus alkanoclasticus
sp. nov.,” Journal of Applied Microbiology,vol.,no.,pp.–
, .
[] I. M. Head and R. P. J. Swannell, “Bioremediation of petroleum
hydrocarbon contaminants in marine habitats,” Current Opin-
ion in Biotechnology,vol.,no.,pp.–,.
[] G. D. Floodgate, “Some environmental aspects of marine
hydrocarbon bacteriology,” Aquatic Microbial Ecology,vol.,
no. , pp. –, .
ISRN Biotechnology
[]A.D.Geiselbrecht,R.P.Herwig,J.W.Deming,andJ.T.
Staley, “Enumeration and phylogenetic analysis of polycyclic
aromatic hydrocarbon-degrading marine bacteria from Puget
Sound sediments,” AppliedandEnvironmentalMicrobiology,
vol. , no. , pp. –, .
[] R. Singh, D. Paul, and R. K. Jain, “Biolms: implications in
bioremediation,” Trends i n Mic rob i olo g y ,vol.,no.,pp.–
, .
[] D. Paul, G. Pandey, J. Pandey, and R. K. Jain, “Accessing micro-
bial diversity for bioremediation and environmental restora-
tion,” Tre n ds in Bi ote chn olog y ,vol.,no.,pp.–,.
[] G. Pandey and R. K. Jain, “Bacterial chemotaxis toward envi-
ronmental pollutants: role in bioremediation,” Applied and
Environmental Microbiology,vol.,no.,pp.–,
.
[]P.L.Stelmack,M.R.Gray,andM.A.Pickard,“Bacterial
adhesion to soil contaminants in the presence of surfactants,”
AppliedandEnvironmentalMicrobiology,vol.,no.,pp.–
, .
[] R. Morgan, S. Kohn, S. H. Hwang, D. J. Hassett, and K. Sauer,
“BdlA, a chemotaxis regulator essential for biolm dispersion in
Pseudomonas aeruginosa,” Journal of Bacteriolog y,vol.,no.
, pp. –, .
[] J. Schmidt, M. Musken, T. Becker et al., “e Pseudomonas
aeruginosa chemotaxis methyltransferase CheR impacts on
bacterial surface sampling,” PLoS ONE,vol.,no.,ArticleID
e.
[] J. W. Costerton, P. S. Stewart, and E. P. Greenberg, “Bacterial
biolms: a common cause of persistent infections,” Science,vol.
, no. , pp. –, .
[] J. Wimpenny, W. Manz, and U. Szewzyk, “Heterogeneity in
biolms,” FEMS Microbiology Reviews,vol.,no.,pp.–
, .
[] A. W. Decho, “Microbial biolms in intertidal systems: an
overview,” Continental Shelf Research, vol. , no. -,pp. –
, .
[] B. Klein, P. Bouriat, P. Goulas, and R. Grimaud, “Behavior of
Marinobacter hydrocarbonoclasticus SP cells during initiation
of biolm formation at the alkane-water interface,” Biotechnol-
ogy and Bioengineering,vol.,no.,pp.–,.
[] T. Barkay and J. Schaefer, “Metal and radionuclide bioremedia-
tion: issues, considerations and potentials,” Current Opinion in
Microbiology,vol.,no.,pp.–,.
[] K. Shimada, Y. Itoh, K. Washio, and M. Morikawa, “Ecacy
of forming biolms by naphthalene degrading Pseudomonas
stutzeri T towa rd bioremediation technolo gy and its molecu-
lar mechanisms,” Chemosphere,vol.,no.,pp.–,.
[] P. Chandran and N. Das, “Degradation of diesel oil by immo-
bilized Candida tropicalis andbiolmformedongravels,”
Biodegradation,vol.,no.,pp.–,.
[] C. Gertler, G. Gerdts, K. N. Timmis, M. M. Yakimov, and P.
N. Golyshin, “Populations of heavy fuel oil-degrading marine
microbial community in presence of oil sorbent materials,”
Journal of Applied Microbiology,vol.,no.,pp.–,
.
[] H. Mahmoud, R. Al-Hasan, M. Khanafer, and S. Radwan,
“A microbiological study of the self-cleaning potential of oily
Arabian Gulf coasts,” Environmental Science and Pollution
Research,vol.,no.,pp.–,.
[] G.Bødtker,T.orstenson,B.L.P.Lillebøetal.,“eeectof
long-term nitrate treatment on SRB activity, corrosion rate and
bacterial community composition in oshore water injection
systems,” Journal of Industrial Microbiology and Biotechnology,
vol. , no. , pp. –, .
[]K.S.Cho,O.K.Choi,Y.H.Joo,K.M.Lee,T.H.Lee,and
H. W. Ryu, “Characterization of biolms occurred in seepage
groundwater contaminated with petroleum within an urban
subway tunnel,” Journal of Environmental Science and Health
Part A,vol.,no.,pp.–,.
[] Z. Liu, A. M. Jacobson, and R. G. Luthy, “Biodegradation of
naphthalene in aqueous nonionic surfactant systems,” Applied
and Environmental Microbiology,vol.,no.,pp.–,.
[] N. A. Sorkhoh, M. A. Ghannoum, A. S. Ibrahim, R. J. Stretton,
and S. S. Radwan, “Crude oil and hydrocarbon-degrading
strains of Rhodococcus rhodochrous isolated from soil and
marine environments in Kuwait,” Environmental Pollution,vol.
, no. , pp. –, .
[]J.G.Holt,N.R.Krieg,P.H.A.Sneat,J.T.Staley,andS.
T. Williams, Bergey’s Manual of Determinative Bacteriology,
Williams & Willkins,, Baltimore, Md, USA, .
[] S.T.Williams,M.E.Sharpe,andJ.G.Holt,Bergey’s Manual of
Systematic Bacteriology, Vol. IV, Williams & Wilkins, Baltimore,
Md,USA,.
[] J. T. Staley, M. P. Bryant, N. Pfenning, and J. G. Holt, Bergey’s
Manual of Systematic Bacteriology, Vol. III, Williams & Wilkins,
Baltimore, Md, USA, .
[] M. Hasanuzzaman, A. Ueno, H. Ito et al., “Degradation of long-
chain n-alkanes (C and C) by Pseudomonas aeruginosa
strain WatG,” International Biodeterioration and Biodegrada-
tion,vol.,no.,pp.–,.
[] V. Kisand, R. Cuadros, and J. Wikner, “Phylogeny of culturable
estuarine bacteria catabolizing riverine organic matter in the
northern Baltic Sea,” AppliedandEnvironmentalMicrobiology,
vol.,no.,pp.–,.
[] S.F.Altschul,W.Gish,W.Miller,E.W.Myers,andD.J.Lipman,
“Basic local alignment search tool,” Journal of Molecular Biology,
vol. , no. , pp. –, .
[] V. J. M. Allan, M. E. Callow, L. E. Macaskie, and M. Paterson-
Beedle, “Eect of nutrient limitation on biolm formation and
phosphatase activity of a Citrobacter sp.,” Microbiology,vol.,
no. , pp. –, .
[] A. Rajasekar, T. G. Babu, S. Maruthamuthu, S. T. K. Pandian, S.
Mohanan, and N. Palaniswamy, “Biodegradation and corrosion
behaviour of Serratia marcescens ACE isolated from an Indian
diesel-transporting pipeline,” World Jo u r n a l o f Microb i o l o g y a n d
Biotechnology,vol.,no.,pp.–,.
[] H.P.Zhao,L.Wang,J.R.Ren,Z.Li,M.Li,andH.W.Gao,
“Isolation and characterization of phenanthrene-degrading
strains Sphingomonas sp. ZP and Tistrella sp. ZP,” Journal of
Hazardous Materials, vol. , no. , pp. –, .
[] M. Raulio, M. J¨
arn, J. Ahola et al., “Microbe repelling coated
stainless steel analysed by eld emission scanning electron
microscopy and physicochemical methods,” Journal of Indus-
trial Microbiology and Biotechnology,vol.,no.,pp.–,
.
[] A. D. Peacock, Y. J. Chang, J. D. Istok et al., “Utilization of
microbial biolms as monitors of bioremediation,” Microbial
Ecology,vol.,no.,pp.–,.
[] P. J. Vaysse, L. Prat, S. Mangenot, S. Cruveiller, P. Goulas,
and R. Grimaud, “Proteomic analysis of Marinobacter hydro-
carbonoclasticus SP biolm formation at the alkane-water
interface reveals novel proteins and cellular processes involved
ISRN Biotechnology
in hexadecane assimilation,” Research in Microbiology,vol.,
no. , pp. –, .
[] R. Singh, D. Paul, and R. K. Jain, “Biolms: implications in
bioremediation,” Trends i n Mic rob i olo g ,vol.,no.,pp.–
, .
[] H.Al-Awadhi,R.H.Al-Hasan,N.A.Sorkhoh,S.Salamah,and
S. S. Radwan, “Establishing oil-degrading biolms on gravel
particlesandglassplates,”International Biodeterioration and
Biodegradation,vol.,no.,pp.–,.
[] R. Kumar, R. Subarna, H. Dipak, B. Debabrata, and B. Dipa,
“Survey of petroleum-degrading bacteria in coastal waters of
Sunderban Biosphere Reserve,” Wor l d J ournal of Mi c r o biolog y
and Biotechnology,vol.,no.,pp.–,.
[] M.VenkateswarReddy,G.N.Nikhil,S.VenkataMohan,Y.V.
Swamy, and P. N. Sarma, “Pseudomonas otitidis as a potential
biocatalyst for polyhydroxyalkanoates (PHA) synthesis using
synthetic wastewater and acidogenic euents,” Bioresource
Techn o l o g y,vol.,pp.–,.
[]D.Al-Bader,M.K.Kansour,R.Rayan,andS.S.Radwan,
“Biolm comprising phototrophic,diazotrophic, and hydrocar-
bon-utilizing bacteria: a promising consortium in the biore-
mediation of aquatic hydrocarbon pollutants,” Environmental
Science and Pollution Research International,.
Available via license: CC BY
Content may be subject to copyright.
Content uploaded by Tapas K Sengupta
Author content
All content in this area was uploaded by Tapas K Sengupta on Jun 11, 2015
Content may be subject to copyright.
