research article biofilm-mediated enhanced crude oil ...glass cover slips being immersed in ml bh...

Hindawi Publishing CorporationISRN BiotechnologyVolume 2013 Article ID 250749 13 pageshttpdxdoiorg1054022013250749

Research ArticleBiofilm-Mediated Enhanced Crude Oil Degradation byNewly Isolated Pseudomonas Species

Debdeep Dasgupta Ritabrata Ghosh and Tapas K Sengupta

Department of Biological Sciences Indian Institute of Science Education amp Research-Kolkata Mohanpur CampusNadia 741252 India

Correspondence should be addressed to Tapas K Sengupta senguptkiiserkolacin

Received 31 December 2012 Accepted 25 January 2013

Academic Editors W J Ernst W A Kues O Pontes S Sanyal and J Sereikaite

Copyright copy 2013 Debdeep Dasgupta et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The bioavailability of organic contaminants to the degrading bacteria is a major limitation to efficient bioremediation of sitescontaminatedwith hydrophobic pollutants Such limitation of bioavailability can be overcome by steady-state biofilm-based reactorThe aimof this studywas to examine the effect of suchmulticellular aggregation bynaturally existing oil-degrading bacteria on crudeoil degradationMicroorganisms capable of utilizing crude oil as sole carbon source were isolated from river estuary and sea-watersamples Biochemical and 16S rDNA analysis of the best degraders of the three sources was found to belong to the Pseudomonasspecies Interestingly one of the isolates was found to be close to Pseudomonas otitidis family which is not reported yet as a degraderof crude oil Biodegradation of crude oil was estimated by gas chromatography and biofilm formation near oil-water interface wasquantified by confocal laser scanning microscopy Biofilm supported batches of the isolated Pseudomonas species were able todegrade crude oil much readily and extensively than the planktonic counterparts Volumetric and topographic analysis revealedthat biofilms formed in presence of crude oil accumulate higher biomass with greater thickness compared to the biofilms producedin presence of glucose as sole carbon source

1 Introduction

World marine ecosystem has been studied extensively sincethe second half of the last century Oil spillage and oilpollution in marine environment have been a major threatto the ecosystem including the ocean life as well as to thehuman being through the transfer of toxic organic mate-rials including polycyclic aromatic hydrocarbons (PAHs)into the food chain [1ndash3] Presence of polycyclic aromatichydrocarbons (PAHs) in soil and water is major problemas environmental contaminants and most of these PAHs arerecalcitrant in nature PAHs mean a potential risk to themarine animals as well as to the human health as many ofthem are carcinogenic [4] Physical and chemical methodslike volatilization photooxidation chemical oxidation andbioaccumulation [5] are rarely successful in rapid removaland in cleaning up PAHs [6] and also these methods arenot safe and cost effective when compared to microbialbioremediation Bacteria have long been considered as one

of the predominant hydrocarbon degrading agents foundin the environment which are free living and ubiquitousOver twenty genera of bacteria of marine origin have beendocumented to be hydrocarbon degrading [7ndash9] Bacteriabelonging to subphyla 120572- 120573- and 120575-proteobacteria [10ndash13] arewell established to be of such nature

One of the major factors that impedes the process biore-mediation is bioavailability of hydrophobic contaminants tothe hydrocarbon utilisingmicroorganisms Although numer-ous studies focused on biofilm reactor in the field of biore-mediation [14] pollution of marine bodies by oil and otherhydrocarbons solely during the oil spillage needs furtherattention in the context of bioavailability of microorganismsIt has been investigated earlier that this major limitationcan be improved by exploiting chemotactic bacteria [15ndash17] Microbial chemotaxis plays important role in surfacecolonization and biofilm formation [18 19] Microbes havea natural tendency to form multi-cellular aggregates beingglued to form biofilm [20 21] Biofilm can be formed by

2 ISRN Biotechnology

single bacterial species or even by a group of bacteria fungialgae and protozoa The potential of microbial aggregates inthe biofilm communities for bioremediation is always a saferand more adept method than planktonic microorganism asthe biofilm matrix protects them during stress and thereforeorganism gets a better chance of adaptation [22] Interest-ingly Klein et al [23] reported that hexadecane assimilationby Marinobacter hydrocarbonoclasticus SP17 occurs throughthe formation of a biofilm at the alkane-water interface andhow the cell behavior changes with the presence of utilizableor nonutilizable alkanes at the interface The biofilm-basedreactors furnish high microbial biomass accessible for bettermicrobial activity than planktonic cells for other biologicalactivities like biomineralization [24] Recently the efficiencyof biofilm-associated cells in degradation of naphthaleneover planktonic had been elucidated for strain Pseudomonasstutzeri T102 and the survival of cells in petroleum contam-inated soil is well documented [25] Chandran and Das [26]demonstrated 97 degradation of diesel oil over a period of10 days by yeast biofilm on gravel particle Faster and intensedepletion of linear and brunched hydrocarbon was observedin biofilm microbial community of Alcanivorax borkumensis[27] Microbial consortia on gravel particle were found asconducive tools for self-cleaning of oily gulf coast throughoutall the sites and season [28] Biofilm community is diverseand relatively stable for longer period of time [29] The filmconsortia were isolated from petroleum contaminated urbansubway drainage system where they were capable of degra-dation at fifteen-degree centigrade [30] The phenomenonof chemotaxis by the organisms towards the pollutants andthe simultaneous attachment-detachment process maintainsa constant load of biomass to the affected site in the waterbodies

Oil spillage has taken place in India for more than oneinstance Notably in early 2006 a Japanese tanker collidedwith a small Indian vessel 470 km west of the Nicobar andAndaman Archipelago spilling over 4500 tons of oil intothe Indian Ocean More recently a ship carrying iron ore isreported to be spilling oil in the sea near Paradip Port (OrissaIndia) since it has gone down under sea in September 2009Kolkata Port and nearby areas like Haldia port part of Bayof Bengal close to the ports and Haldia Refinery are majorconcerns for possible oil spillage due to everyday transportof fuel oil and other means since it is a major shippingcorridor for eastern region of India In spite of possiblethreat of contamination of water sources by spilled oil inthese areas little work has been done so far on presence andcharacterization of oil-degrading microorganisms naturallyexisting in water sources near Kolkata port and nearby areas

Our present work was to emphasize the multicellularaggregation of biofilm formation by naturally occurringhydrocarbon degrading strains from this region and to inves-tigate the applicability of biofilm amendment on enhance-ment of biodegradation of crude oil Briefly microorganismscapable to utilize crude oil as sole carbon source were isolatedfrom the water samples of the previously mentioned sourcesthrough serial enrichment culture technique Based on bettercrude oil utilization ability three of the isolated strains fromthe three mentioned sources were screened The organisms

were characterized and identified by biochemical test and 16SrDNA sequencing and further tested for utilization of variousfuel oils and their ability to form biofilmThe volumetric andtopological properties of biofilm near oil water interface wereestimated by confocal laser scanning microscopy (CLSM)Gas chromatography-mass spectroscopic analysiswas carriedout to measure the effect of biofilm amendment on crude oildegradation in comparison to planktonic culture alone

2 Materials and Methods

21 Source of Microorganisms Water samples were collectedfrom Kolkata port of Hooghly River (22∘321015840N 88241015840E)River Haldi at Haldia port (22∘051015840N 88∘031015840E) and Bay ofBengal at Digha (21∘371015840N 87∘251015840E) for isolation of crudeoil degrading microorganism Water sample from Gomukhglacier (30∘581015840N 78∘551015840E) the source of Ganges river at thealtitude of 3819m was used as control nonpolluted water andtested for a presence of crude oil degrading bacteria

22 Culture Enrichment Isolation and Characterization ofStrain The enrichment of crude oil degrading bacteria wascarried out under aerobic condition with crude oil as solesource of carbon Crude oil was obtained from IndianOil Corporation Limited (IOCL Haldia West Bengal) Themineral salt media (MSM) [31] were amended with 1 crudeoil (vv) and enrichment of culture was carried out inthree consecutive batches each having a span of 15 days andenriched by using previous growth as inoculums for the nextBacterial growth was measured by using spectrophotometer(Chemito Instruments UV 2600) at 600 nm and comparedwith control without inoculation Selective solid inorganicmedia (SSIM) [32] were inoculated by spreading 100 120583L ofbroth from last batch of enriched culture incubated at 30∘Cfor 10 days Representative pure colonies were isolated andfurther confirmed for oil degradation by growing in MSMmedia provided with 1 crude oil (filter sterilized using02 120583m syringe filter)

Selection of microorganisms was based on better abilityto grow in presence of crude oil as sole source of carbonin growth media The isolated microorganisms were testedfor Gram staining and biochemical properties as describedpreviously [33ndash35] Motility tests were done by stabbing cellsin semisolid nutrient agar (07 agar) [33]

23 Growth Characteristics in Different 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 localservice station) These oil samples were filter-sterilized using02 120583m syringe filter and added into 50mL of BH media(composition (gmlit) MgSO

4(02) CaCl

2(002) KH

2PO4

(10) (NH4)2HPO4(10) KNO

3(10) FeCl

3(005) and 1 of

oil sample) pH7TheBHmediawere inoculatedwith isolatedbacteria and incubated at 30∘C under static condition for 5days Aliquots of bacterial cultures were collected seriallydiluted and plated on nutrient agar plates The numbers of

ISRN Biotechnology 3

colonies were counted to determine bacterial growth in termsof colony forming units (CFUmL)

For biodegradation studies gas chromatography-massspectroscopic (GC-MS) analysis of crude oil was carriedout Isolated microorganisms were grown in 50mL of BHmedium (pH 7 plusmn 02) at 30∘C for 15 days in presence of1 crude oil as sole carbon source After 15 days of growththe residual crude oil components were extracted with equalvolume of organic solvent dichloromethane (DCM) Theaqueous phase and the organic phase were separated inseparating funnel The residual water from the organic phasewas absorbed by anhydrous sodium sulphate (1 gm10mL) 10microliter samples of the DCM extracts were then analyzedby GC-MS (Agilent 6890N GC-MS-5973N) with a columnof 30 (m) times 025 (120583m) at a flow rate of 100mLmin[36] The samples were held at 60∘C for 2 minutes initiallyand increased at the rate of 20∘Cmin to reach the finaltemperature of 325∘C The final temperature was held for 5minutes 50mL of BH medium with 1 crude oil was alsokept at 30∘C for 15 days as control and crude oil componentswere extracted with DCM and analyzed by GC-MS as statedbefore

24 16S rDNA Sequencing and Phylogenetic Analysis Acolony of each isolate was grown overnight in LB mediumincubated at 30∘C 1mL sample of each culture was cen-trifuged at 2300 g for 10min The bacterial DNA was iso-lated using bacterial genomic DNA isolation kit (ChromousBiotech) and 16S rDNAswere amplified by using PCRmastermix (Fermentas) Bacterial universal primers Forward-27f(51015840-AGAGTTTGATCATGGCTCAG-31015840) and Reverse-1492r(51015840 TACGGYTACCTTGTTACGACTT-31015840) were used foramplification [37] The PCR products were purified withQIAquick Gel extraction kit (Qiagen) Nucleotide sequenceswere determined from the purified product by automatedsequencer with an ABI PRISM II Dye Terminator CycleSequencing kit (Chromous-Biotech) with the same primers

The identity of 16S rDNA sequences of isolates was deter-mined by using the BLAST database search [38] Eighteensequences (first 6 hits for each isolate) of the cultivableorganisms were procured from NCBI-Blast search and thealignment was done by using CLUSTALX 182 software Thealignment was thoroughly checked in software SEAVIEWfor any gaps and edited accordingly A phylogenetic treewas constructed by neighbour joining method (Kimura 2-parameter) usingMEGAv-40 and the tree was subsequentlybootstrapped (random speed 64328 1000 replicates)

25 Oil Biofilm Development and Quantification Three iso-lated strains were first tested for biofilm formation on 18mmglass cover slips being immersed in 15mL BH media with1 crude oil in 50mL sterile falcon tubes The organism wasinoculated and incubated at 30∘C for 4 days The cover slipswere recovered from the culture tubes washed thoroughlyin 1 saline solution aseptically air-dried and Gram-stainedFormation of biofilm was viewed under 100X oil immersionobjective using Nikonrsquos DN100 microscopeThe formation ofbiofilm on thin glass cover slips was also studied for hourly

development of film by the strain KPW1-S1 and stained at 6different time intervals of growth at 6th 12th 18th 24th 36thand 48th hours

Biofilm load in presence of crude oil was estimated by theconfocal laser scanning microscopic (CLSM) image stacks[39] Briefly the isolated strains were grown on glass surfacein presence of 1 crude oil (glass slide 25 times 75mm) and 2glucose (vv) (cover glass 12 times 12mm) respectively in BHmedia The surface of the substratum was washed with PBS(1X) thrice and stained with 0005 acridine orange (wv) for5minutes in dark andwashed twiceThe slides were observedunder Carl Zeiss CLSM-710 (Axio observer microscopeversion Z1) using 488 nm excitation argon laser with MBS(main beam splitter) and emission wavelength detected from493 to 586 nm The image acquisition was done under X100oil immersion lens (NA 14) Series of measurement wastaken at random vertically across the oil water interface (incase of crude oil) and air water interface in case of glucoseAll samples were viewed from the clean side of the coverslip under oil and the height was measured from thesetransects vertically from the base of cover slip to the top of thebiofilm (frame size 512 times 512 8-bit image 119885 stacks interval037120583m) Volumetric and topological parameters (thicknessbiovolume biomass surface area skewness and kurtosis)of biofilm were calculated using the software provided (Zen2010) along with the microscope

26 Effect of Biofilm Amendment on Biodegradation Oildegradation was also compared for biofilm amended andunamended planktonic cultures (with equal starter inocu-lums)withKPW1-S1 and strainwith biofilmdefectDSW1-S4Glass slide of dimension 25mm times 75mm was immersed in50mL falcon tube containing 20mL BHmedia with 1 crudeoil and incubated for 15 days at 30∘C For biodegradationanalysis other sets of batch cultures with and without biofilmcarrying the residual crude oil were extracted with equalvolume of organic solvent dichloromethane The aqueousphase andoil were separatedThe residualwaterwas absorbedby anhydrous sodium sulphate (1 gm10mL)The extract wasanalyzed by gas chromatography-mass Spectroscopy (Agilent6890N GC-MS-5973N) as described in the previous sectionPercentage of degradation for ten detectable peaks (withrespect to control) was calculated by the method describedearlier [40]

27 Statistical Analysis Graph plotting andmultiple compar-isons of optical densities were assessed by Origin 7 followedby SigmaPlot software version 11 San Jose California USAThe data were expressed as mean plusmn standard error

3 Results

31 Enrichment and Screening of Organism During incuba-tion of water samples (collected from Kolkata Port HaldiaPort and Bay of Bengal areas) in MSM media (containingcrude oil as sole carbon source) no visual change in turbiditydue to bacterial growth was observed till the third day ofincubation The optical density (for bacterial growth) kept


Table 1 Growth characteristics of isolated organisms by utilization of various oilhydrocarbon as sole carbon source

OilhydrocarbonColony forming unitmL

StrainKPW1-S1 HRW1-S3 DSW1-S4

Crude oil (15 plusmn 017) times 108 (68 plusmn 011) times 108 (14 plusmn 009) times 109

Diesel (67 plusmn 0072) times 108 (61 plusmn 008) times 108 (65 plusmn 012) times 108

Kerosene (77 plusmn 006) times 107 (16 plusmn 004) times 107 (17 plusmn 007) times 108

Hexadecane (294 plusmn 013) times 109 (44 plusmn 002) times 108 (24 plusmn 007) times 108

Engine oil (61 plusmn 007) times 108 (64 plusmn 011) times 108 (36 plusmn 007) times 108

Table 2 Preliminary biochemical tests for isolated bacterial strains

Characteristics KPW1-S1 HRW1-S3 DSW1-S4Gram nature Negative Negative NegativeShape Rod Coccobacillary CoccobacillaryDiffusible pigments + + minus

Motility + + +Citrate utilization ++ ++ ++Lysine utilization + + +Ornithine utilization + + +Urease test minus minus minus

Phenylalanine test NC NC NCNitrate reduction NC NC NCH2S production +++ ++ +Production of acid byutilization of sugarlowast

(i) Glucose minus minus minus

(ii) Adonitol minus minus minus

(iii) Lactose minus minus minus

(iv) Arabinose minus minus minus

(v) Sorbitol minus minus minus

NC not conclusivelowastThe test attributed the change of the color of pH indicator by production ofacid and gas when grown in presence of various sugars

increasing slowly for the next 16ndash18 days The second enrich-ment using inoculums from the first enrichment showeda little early response with a shorter lag period of growthand the culture reached stationary phase of growth within14ndash16 days Finally in the third serial enrichment cultureinoculated from the second batch showed an early responseand significant growth was observed in the second dayonwards and reached stationary phase of growth within 7days (Figures 1(a)ndash1(c)) Interestingly water sample collectedfrom Gomukh glacier showed no growth even after 6 weeksof incubation in MSM medium containing crude oil as solecarbon source (data not shown)

From the third batch of enriched cultures pure colonieswere isolated on SSIM crude oil agar plates Total of 3630 and 16 pure colonies were thus obtained from threewater samples collected from Kolkata port Haldi Riverwater and Bay of Bengal near Digha site respectively Thesecolonies (in duplicate) were restreaked on nutrient agar (NA)

and they were differentiated by colony characteristics basedon morphology and pigmentation The distinct 13 colonieswith unique characteristics were isolated as pure culturein NA media All isolated colonies were tested for furtherconfirmation of their ability of oil degradation and threeisolates from three different sources were selected based ontheir rapid growth in presence of crude oil as sole carbonsource (data not shown) The organisms were named asKPW1-S1 (isolated from Kolkata Port water MTCC 10087)HRW1-S3 (isolated from Haldi River water MTCC 10088)and DSW1-S4 (isolated from Digha sight of the shore of Bayof BengalMTCC 10089) and currently deposited atMicrobialType Culture Collection and Gene Bank (MTCC) India

32 Growth Characteristics in Different Oil and Biodegrada-tion Analysis The selected bacteria (KPW1-S1 HRW1-S3and DSW1-S4) were subjected to grow in presence of variousother oils as carbon source The bacterial growth was foundto be uneven depending on the bacterial species and oiltype (Table 1) Significant difference was observed betweenthe growth of the three strains in case of crude oil andhexadecane (119875 lt 005) The strain KPW1-S1 showed bestgrowth in presence of hexadecane as carbon source and theDSW1-S4 in presence of crude oil and higher hydrocarbonspresent in engine oil The kerosene showed to be the leastsupportive as a carbon source and it had no significanteffect on difference of growth among the three strains (119875 gt005) The strain HPW1-S3 showed average growth rate andcrude oil degradation (Figure 2(c) Table 1) These resultsclearly indicate that different oils were degraded and utilizedby all the strains in various proportions depending on thecomplexity and aliphatic and aromatic nature of the sampleoil dependent on bacterial species as well

Organisms KPW1-S1 HRW1-S3 and DSW1-S4 weregrown in Bushnell-Hass (BH) media in presence of crude oilas only carbon source at 30∘C for 15 days The hydrocarbonprofile in the growth media after 15 days of growth was ana-lyzed by GC-MS and compared with the hydrocarbon profilefrom a control flask where the BH medium and crude oilwere kept together for 15 days under identical conditionsThehydrocarbon profile obtained by GC-MS analysis showed therelative abundance of various hydrocarbons in the complexmixture (Figure 2) The control sample (Figure 2(a)) showsthe presence of various hydrocarbons in the unresolvedcomplexmixture All the isolated bacteria were able to reduceat least 50 of relative abundance of various hydrocarbons


0 2 4 6 8 10 12 14 16 18 20 22

0

05

1

15

2

25

3

Incubation time (days)

OD

at 6

00 nm

Enrichment-IEnrichment-IIEnrichment-III

(a)

0 2 4 6 8 10 12 14 16 18 20 22

0

05

1

15

2

25

3


OD

at 6

00 nm


(b)


0 2 4 6 8 10 12 14 16 18 20 22Incubation time (days)

0

05

1

15

2

25

3

OD

at 6

00 nm

(c)

Figure 1 Enrichment of crude oil-degrading bacteria Bacterial populations in water samples collected from (a) Kolkata Port (b) Haldi Riverand (c) Digha site at Bay of Bengal were subjected to three-step enrichment process in MSM in presence of crude oil as sole carbon sourceIn all the cases the standard error values ranged from 01 to 1

present in crude oil compared to control experiment (Fig-ures 2(b)ndash2(d)) within 15 days of incubation Interestinglybacterial strains isolated from Kolkata port (KPW1-S1) andDigha area of Bay of Bengal (DSW1-S4) showed minimumand maximum overall hydrocarbon degradation abilitiesrespectively

33 Taxonomic Identification of Bacterial Strains Isolatedthree organisms (KPW1-S1 HRW1-S3 and DSW1-S4) weretested for a series of biochemical and morphological tests(Table 2) All three organisms KPW1-S1 HRW1-S3 andDSW1-S4 were found to be Gram-negative citrate positive

rod-shaped (similar to coccobacillus shape) and motileWhen crude oil was used as carbon source all the isolatedorganisms showed similar growth characteristic at differenttemperatures ranging from 20∘C to 37∘C with an optimumtemperature at 30∘C Further analysis of 16S rDNA genesequences was done for taxonomic identification Amplifi-cation and sequencing of 16S rDNA followed by phyloge-netic analysis (together with biochemical and morphologicalanalyses as described in Table 2) revealed that the isolatedstrains (KPW1-S1 (FJ897721) HPW1-S3 (FJ897723) andDSW1-S4 (FJ897724)) belong to phylumProteobacteria classBetaproteobacteria [41] and genus Pseudomonas (Figure 3)


90

45

5

Abun

danc

e

Time (min)5 8 11 135

times105

(a)

45

25

5

Abun

danc

e

times105

Time (min)5 8 11 135

(b)

20

12

1

Abun

danc

e

Time (min)5 8 11 135

times105

(c)

24

12

2

Abun

danc

e

times105

Time (min)5 8 11 135

(d)

Figure 2 Biodegradation of crude oil (at 30∘C 15-day incubation) analyzed by GC-MS (a) Without microorganism (control) and withorganisms (b) KPW1-S1 (c) HRW1-S3 (d) and DSW1-S4

The strains KPW1-S1 and HPW1-S3 are the closest neigh-bour of Pseudomonas aeruginosa strain Tsaydam-5-ASA(KC1372771) whereas DSW1-S4 is nearest to the candidateof Pseudomonas otitidis strain 81f (AB6987391)

34 Oil Biofilm Development Preliminary studies demon-strated that the isolated bacterial strains possess the abilityto form biofilm on glass surface when grown in BH mediumin presence of crude oil as sole carbon source (Figures4(a)ndash4(c)) Interestingly a thick biomass was observed tobe aggregated near the oil water interface on the glassbioreactor Transitional episode of oil biofilm developmentwas observed vividly using light microscopyThe initial eventof bacterial attachment was found in the first sixth hourof growth (Figure 5(a)) In the next 6 hours the cells startforming nascent cell cluster being cemented on the glasssubstratum (Figure 5(b)) At around 18 hours of growthcell clusters become more mature and initiate aggregation(Figure 5(c)) This was followed by evacuation and release ofcells from the matrix with further incubation (Figures 5(d)ndash5(f)) Interestingly KPW1-S1 cells were again able to form

well-defined biofilm on the same glass surface at around 48hours of incubation (Figure 5(f))

35 Quantification of Biofilm by Confocal Laser Scan-ning Microscopy Volumetric and topologic quantification ofbiofilm formations by the isolatedPseudomonas species at oil-water interface was carried out by confocal laser scanningmicroscopy (CLSM) All of the three Pseudomonas strainstested showed ability to form biofilm near air-water interfacewhen grown in presence of glucose as sole carbon source(Figures 6(a)ndash6(c)) The average thickness of KPW1-S1 andHRW1-S3 was found to be 10 and 12 120583m in presence ofglucose whereas it was found to be 15120583m in case of the thirdstrain DSW1-S4 (Table 4) Interestingly enhanced biofilmproduction was observed when the Pseudomonas cells weregrown in presence of crude oil as sole source of carbon(Figures 6(d)ndash6(f)) The average thicknesses of KPW1-S1HRW1-S3 and DSW1-S4 were found to be 22 26 and45 120583m respectively in presence of crude oil representing atleast 2-fold increase of biofilm thickness for all the strains(Table 4) These results clearly showed that presence of crude


Pseudomonas aeruginosa strain 6A (bc4) (JX6617162)Pseudomonas aeruginosa strain R8-769-1 (JQ6599821)Bacterium H1C (JX1495431)Endophytic bacterium 202P-2 (JF9013621)Pseudomonas aeruginosa strain IFS (JQ0416381)Pseudomonas aeruginosa strain N002 (JX0357941)Pseudomonas aeruginosa strain Tsaydam-5-ASA (KC1372771)Pseudomonas aeruginosa strain JYR-Pk-2011 (JQ7920381)Pseudomonas aeruginosa strain R7-734 (Q6599201)Pseudomonas aeruginosa strain RI-1 (JQ7734311)Pseudomonas sp CEBP1 (JQ8945311)Pseudomonas sp a-1-8 (JX4163741)Bacterium P2A (JX1495461)Pseudomonas aeruginosa strain DSM 50071T (HE9782711)Pseudomonas sp KPW 1-S1 (FJ8977211)Pseudomonas sp HRW 1-S3 (FJ8977231)Pseudomonas sp DSW 1-S4 (FJ8977241)

Pseudomonas otitidis strain 81f (AB6987391)Pseudomonas sp GD6(2010) (GU5663071)Pseudomonas sp 82 (EF4264441)Pseudomonas sp 75 (EF4264431)Pseudomonas sp AHL 2 (AY3799741)Pseudomonas otitidis strain R6-410 (JQ6598461)

Pseudomonas sp TSH17 (AB5088481)

0001

60

8

62

100

60

Figure 3 Phylogenic tree of all sequenced 120573-Proteobacteria (∙) KPW1-S1 (◼) HRW1-S3 (⧫) DSW1-S4 The bootstrap values are indicatedfor the major nodes

oil in growth medium enhanced biofilm production by allof the three strains although the DSW1-S4 strain has lesserpotential to form biofilm under the experimental conditionsThus the spatial biomass accumulated near the oil waterinterface by KPW1-S1 and HRW1-S3 was observed to be 14ndash18 units per unit area and for the low biofilm former DSW1-S4 spatial biomass accumulation was 1-2 units per unit areaIn presence of crude oil the maximum thickness of biofilmobtained by KPW1-S1 and HRW1-S3 was 35 and 73120583m(Figures 6(d)ndash6(f)) whereas the strain possessing biofilmdefect could develop up to 45 120583m of maximum thickness

To further substantiate our finding topological parame-ters of biofilm load were investigated through the measure-ment of CLSM Here the mean thickness and biomass werefound inversely proportional to skewness (119878ku) Since higherskewness indicates lack of porosity [42] it is likely that lesserskewness and hence greater porosity enable biofilm to accesshigher nutrient and other essential factors which attributesin greater thickness of biofilm The statement is validatedin six cases comprising three strains and two conditions(Table 4) Strikingly no significant difference was observedin other topological parameters kurtosis (119878sk) which impliesthat adherence of organismwith substratumwasmore or lesssimilar in all the six cases irrespective of strain with profoundbiofilm forming ability (KPW1-S1 and HRW1-S3) or strainDSW1-S4 with lesser potential to make thick biofilm

36 Comparative Degradation Study The aggregation of cellsnear oil-water interface observed through CLSM promptedto examine the relation between biofilm formation at the

oil-water interface and utilizationdegradation of crude oilcomponents by the Pseudomonas isolates A comparativeaccount of GC-MS profiles of hydrocarbons present in crudeoil was analyzed in presence and absence of substratumsupport Biofilm forming strain KPW1-S1 and biofilm defec-tive DSW1-S4 were taken into account for oil degradationanalysisTheGC-MS result shows that the relative abundanceof the peak reduced considerably for both of the strainsdepending on potential of oil degradation (Figure 7) Oildegradation in terms of reduction of total numbers of hydro-carbon peaks and depletion of total area of chromatogramof various components of crude oil was observed with theintroduction of biofilm amendment 20 plusmn 10 to 40 plusmn10 increment of degradation was achieved in presence ofmatrix enclosed biofilm in comparison to planktonic cellsalone for both of the strains Short chain hydrocarbon peakswere out of detection limit From the percentage of degra-dation measured using the method described earlier it isevident that increased degradation of individual hydrocarbonis mediated or induced by the biofilm near oil air-waterinterface (Figure 7) Additionally for the strain KPW1-S1the biofilm assisted culture could target the short chain lowmolecular weight hydrocarbons (Table 3 Figure 7) whereasthe strain DSW1-S4 could successfully degrade both shortand long chain hydrocarbons present in crude oil efficiently

4 Discussion

Ability to form biofilm on various surfaces is always ad-vantageous for the microorganisms in terms of survival


Table 3 Percentage degradation of individual hydrocarbon present in crude oil

Peak no Retention time (minute) Percentage degradation119875KPW1-S1 119861

119871KPW1-S1 119875DSW1-S4 119861119871DSW1-S4

1 12635 4208 6987 4870 70802 14066 8169 8912 7657 87453 14953 9076 9288 9384 95764 1655 981 3235 8045 87225 17808 6999 8187 7374 85626 1875 5572 7328 5725 74047 1977 5670 7041 6969 81958 2252 6532 6984 5929 70609 2336 4011 6775 5854 685210 27077 5719 5877 4130 6598Overalllowast 1097ndash2948 4293 6635 4793 7545119875 represents degradation of hydrocarbon without amendment of biofilm (only planktonic)119861

119871 represents degradation of hydrocarbon with amendment of biofilm (planktonic + biofilm)lowastRepresents overall degradation of all the hydrocarbon considering the total area of control and samples under different conditions

Table 4Mean thickness biomass substratum coverage kurtosis (119878ku) and skewness (119878sk) of biofilmofPseudomonas sp KPW1-S1 HRW1-S3and DSW1-S4

Carbon source Strain Avg thickness (120583m) Total biomass (120583m3120583m2) Kurtosis (119878ku) Skewness (119878sk) Max thickness (120583m)KPW1-S1 2236 plusmn 098 1417 plusmn 171 35 plusmn 043 minus068 plusmn 031 35

Crude oil HWR1-S3 2651 plusmn 12 1845 plusmn 136 419 plusmn 12 minus101 plusmn 033 73DSW1-S4 455 plusmn 16 16 plusmn 055 406 plusmn 02 086 plusmn 016 24

KPW1-S1 125 plusmn 089 933 plusmn 09 391 plusmn 05 minus049 plusmn 034 23Glucose HWR1-S3 1032 plusmn 037 67 plusmn 049 272 plusmn 03 minus046 plusmn 015 126

DSW1-S4 151 plusmn 008 09 plusmn 008 366 plusmn 02 048 plusmn 015 32Values are means of data from 8 image stacksThe standard error is calculated as the square root of the mean of the variances of each of the four groups (imagestacks from two glass slides in two independent experiment rounds)

metabolism adaptation and propagation [43 44] One of themajor limitations faced in the process of bioremediation is thebioavailability of organic compounds on site [45] Early stud-ies that indicate biofilm forming bacteria can be employed toovercome this limitation although the application of steady-state biofilm in bioremediation is not well established Studiesindicate that biofilm-mediated bioremediation is a proficientapproach and safer option since cells in biofilm have betterchance of survival and adaptability especially during thestressed conditions [21 46] Establishment of biofilm ongravel particles and glass slides was reported previously[46] where the artificially glued microorganisms showedexcellent attenuation of crude oil in liquid waste in batchculture Vaysse et al [44] showed altered profile of expressedproteins specifically type VI secretion system in biofilmforming Marinobacter hydrocarbonoclasticus SP17 at alkane-water interface

Crude oil degrading bacteria were unruffled from threedifferent locations which are the prominent risk zones of oilcontaminationThree-step enrichment processwas employedto enrich and isolate microorganisms with greater degrees ofoil-degrading capabilitiesThese bacteria were then preferredfor additional characterization and identification Out of theselected organisms KPW1-S1 HRW1-S3 and DSW1-S4 wereisolated 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 andwere preliminarily identified as of class Betaproteobacteria

Ability of the isolated bacteria for utilization of crude oilas carbon source was investigated by GC-MS analysis Thedata revealed that the isolated strains could be able to reducedifferent hydrocarbons present in crude oil samples up to50 within 15 days of growth under laboratory conditions(Figure 2) The isolated organisms also showed their abilityto use other complex oils like petrol diesel and keroseneas carbon sources as well (Table 2) It is interesting to notethat 16S rDNA sequence and phylogenetic analysis revealedthat KPW1-S1 and HRW1-S3 are closest to Pseudomonasaeruginosa and DSW1-S4 to Pseudomonas otitidis (Figure 3)Previous studies on naturally existing oil-degrading bacteriain these regions were elucidated although the molecularphylogenetic analysis based on 16S rDNA sequencing wasnot initiated [47] Thus DSW1-S4 is a novel Pseudomonas spin terms of its ability to degrade crude oil since there is noreport so far for crude oil degradation ability of Pseudomonasotitidis or any closely related species of Pseudomonas otitidisalthough recently Venketaswar Reddy et al [48] reporteda newly isolated Pseudomonas otitidis as a potential biocat-alyst for polyhydroxyalkanoates (PHA) synthesis Moreoverresults of the present study strongly suggest the existence of


(a) (b) (c)

Figure 4 (a) Oil biofilm formation on glass surface by the three isolates (a) KPW1-S1 (b) HRW1-S3 and (c) DSW1-S4 Bars 23120583m

(a) (b) (c)

(d) (e) (f)

Figure 5 Hourly development of biofilm on thin glass cover slip at different time interval by KPW1-S1 at (a) 6th (b) 12th (c) 18th (d) 24th(e) 36th and (f) 48th hours Bars 23 120583m

hydrocarbon degrading consortia in the river water samplealthough the degradation of crude oil by the strains waslimited to 50 based on the peak heights (Figure 2) It waspresumptuous to assume that the major factor that governsthe low rate of degradationwas bioavailability of hydrocarbonto the microbial biomass To address this limitation wedeveloped a laboratory scale steady-state biofilm reactorwhich circumvents the limitation to an appreciable extentas biofilm formation near oil-water interface accumulatessubstantially higher amount of biomass

The isolated Pseudomonas strains in this study showedtheir ability to form biofilm on glass surface in BH mediumin presence of crude oil as the only carbon source (Figures4(a)ndash4(c)) In addition to this the chemotaxis elucidated bythe bacteria adhering on biofilm could also support the fact ofbacterial motility towards the crude oil and other pollutantspresent in it (data not shown) Moreover there is a wealthof evidence supporting the fact that biofilm developmentand maturation follow a cycle of attachment and release

The time lapse image of oil biofilm development indicatedthat the multicellular aggregation near oil-water interfacecycles at regular interval These data further supports thefact that the biofilm amendment presents the hydrocarbondegrading consortia to the oil and toxic compounds floatingon the aqueous surface for a prolonged duration It has beendocumented earlier that the mass-transfer limitations thatimpede the bioremediation process can be overcome by cellsdisplaying chemotaxis that can sense chemicals such as thoseadsorbed to soil particles in a particular niche and swimtowards them [46] Thus we tested the effect of oil biofilm(static) amendment on the overall degradation of crude oilby GC-MS analysis and characterized the biofilm formationat oil water interface by confocal laser scanning microscopy(CLSM)

First the biofilm forming ability of the three isolates wasestimated by 96-well microtitre assay method in presenceof various carbon source including glucose glycerol andhexadecane (data not shown) Biofilm load in presence of


20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)

20 120583m

(f)

Figure 6 Confocal laser scanning microscopy image of biofilm hive near air water interface by three isolates (a) KPW1-S1 (b) HRW1-S3and (c) DSW1-S4 growing in presence of glucose as carbon source Similar image was taken in 1 crude oil by the three isolates (d) KPW1-S1(e) HRW1S3 and (f) DSW1-S4 to compare the affinity of biofilm formation in presence of oil Biofilms with maximum thicknesses achievedby the three isolates in presence of crude oil are represented in the figure Bars 20 120583m

Abun

danc

e

Time (min)

5 10 15 20 25 30 35

42119890+074119890+07

38119890+0736119890+0734119890+0732119890+07

3119890+0728119890+0726119890+0724119890+0722119890+07

2119890+0718119890+0716119890+0714119890+0712119890+07

1119890+078000000600000040000002000000

Figure 7 GC-MS analysis of oil degradation record of strainDSW1-S4 for biofilm amended and unamended conditions Chromatogramshowing decrease in peak height and corresponding area Control(black) Planktonic only (blue) and biofilm+ planktonic cells (Red)

crude oil was notmeasured by this procedure as oil interferedwith the assay process Based on the characteristic patternof biofilm development of the isolated strains KPW1-S1 andbiofilm defective DSW1-S4 were selected for oil degradationanalysis Our hypothesis got more impetus with GC-MSprofile of the oil degradation in presence and absence ofbiofilm amendment The calculation of percentage of degra-dation also suggests that the biofilm amended culture couldsuccessfully degrade the individual hydrocarbon with muchgreater efficiency (Table 3) Notably the overall degradation

was enhanced by 20ndash40 for both of the strains whenamended with matrix enclosed biofilm cells in comparisonto planktonic cells alone (Figure 7)The comparative study ofbiofilm formation showed that the oil biofilm can accumulatea large number of hydrocarbon degrading organisms nearthe oil water interface This biofilm hive in oil had unusuallyhigher biomass compared to the film produced in glucoseas carbon source The degradation of individual hydrocar-bons was also tested in representative ten peaks where thesimilar reduction of peak area was observed Although theenhanced degradation by biofilm defective strain DSW1-S4was counterintuitive the comparative GC-MS data suggeststhat the strain is better degrader when compared with othersand therefore the oil degradation was further enhanced byaccumulated cells of DSW1-S4 enclosed in biofilm near oilwater interface Additionally biofilm formation follows adynamic cycle of attachment and release from its substratumduring its meal on oil observed by Gram staining at differenttime points (Figures 5(a)ndash5(f)) The phenomenon is alsoreflected in its mean thickness and total biomass formationobserved by confocal imaging of biofilm (Table 4) The threeisolated strains varied in thier total thicknesses depending onthe phase in which they were stained and photographed inindependent experimentsThemaximum thickness achievedby all the three strains was found higher than the meandue to the cycle of attachment and release These findingsprompted us to examine the overall degradation profile ina span of fifteen days by the biofilm amended and unamendedconditions The GS-MS data strongly supports the fact thatthe greater degradation is due to higher population densityin the biofilm amended condition


The topological parameters of the biofilm architecturealso highlight the intrinsic properties of these filmsThemeanthickness and average biomass calculated from confocalimaging software correlated with our biofilm assay resultThe thickness and biomass were highest for KPW1-S1 andHRW1-S3 whereas they were least for DSW1-S4The test wasrepeated for biofilm image obtained using glucose as carbonsource From the result it was evident that the multicellularaggregation was significantly higher in presence of crude oilThe greater skewness value of the poor biofilm forming strainDSW1-S4 makes the film more porous The higher standarddeviation relates the higher heterogeneity of the biofilm forthe all the three strains The kurtosis value for all the threestrains was roughly the same making the adhesion potentialnearly similar

Hence from the previous finding it was conclusive thatin water bodies natural oil degrading strains capable offormation of biofilm degrade oil much faster and efficientlywhen compared to planktonic cells alone Recently Al-Baderet al [49] depict the role of phototrophic diazotrophicand hydrocarbon-utilizing bacterial biofilm consortia leav-ing the promise of bioremediation of aquatic hydrocarbonpollutants Secondly the enhancement of degradation isprofound in all oil degrading naturally exiting strains withvarious degrees of biofilm forming capacity These resultstempt us to speculate that natural oil degrading strains canaccumulate near oil water interface during oil spillage andefficiently circumvent the limitation of bioavailability of insitu bioremediation to a considerably greater extent

5 Conclusion

The results in the present study consolidate our findingthat potent hydrocarbon degrading bacterial consortia existnaturally in the water body near Kolkata port HaldiaRefinery and nearby areas in the eastern regions of Indiain contrary to the water sources from Himalayan Glacierwhere pollution through various sources is negligible Thissignifies that the hydrocarbon degrading bacteria exist orevolved to exist with the ever increasing intensity of marinepollution Pseudomonas strains isolated from water sourcesnear Kolkata port Haldi River and Digha Sea shore showedthat their ability to degrade various complex hydrocarbonsand biofilms formed by the isolated bacteria could enhancedegradation ability Therefore these isolated Pseudomonasstrains could be considered for future use for bioremediationof contaminated spilled oil in water sources However furtherstudies are needed to evaluate the potential of the isolatedstrains to degrade hydrocarbons in situ in natural environ-mental conditionsThus the oil degradation capability abilityto form biofilm greater survival in the nutrient stressed con-dition cycle of attachment and release of biofilm-associatedcells and cooperative nature of these natural isolates couldbe exploited as a better option for bioremediation technologyThis could be equally applicable for anyPseudomonas or otherbacterial strains ubiquitously available in nature having thepreviously mentioned criteria and the technology could befurther developed for targeting of any pollutants present onearth creating enormous environmental and health hazards

Acknowledgments

The authors thank TCG Life Sciences and National TestHousemdashKolkata for GC-MS analysis The authors also thankDrs Balaram Mukhopadhyay and Punyasloke Bhaduri ofIISER-Kolkata for their valuable suggestions and Mr Sud-hangsuMaity andMrMrinmoyBose for their technical assis-tance The authors also acknowledge the confocal imagingfacility of IISER Kolkata D Dasgupta is recipient of InstituteFellowship from Government of India

References

[1] M Blumer H L Sanders J F Grassle and G R Hampson ldquoAsmall oil spillrdquo Environment vol 13 no 2 pp 1ndash12 1971

[2] M B Fernandes M A Sicre A Boireau and J TronczynskildquoPolyaromatic hydrocarbon (PAH) distributions in the SeineRiver and its estuaryrdquo Marine Pollution Bulletin vol 34 no 11pp 857ndash867 1997

[3] A Sei and B Z Fathepure ldquoBiodegradation of BTEX at highsalinity by an enrichment culture fromhypersaline sediments ofRozel Point at Great Salt Lakerdquo Journal of Applied Microbiologyvol 107 no 6 pp 2001ndash2008 2009

[4] E Deziel G Paquette R Villemur F Lepine and J GBisaillon ldquoBiosurfactant production by a soil Pseudomonasstrain growing on polycyclic aromatic hydrocarbonsrdquo Appliedand Environmental Microbiology vol 62 no 6 pp 1908ndash19121996

[5] H P Zhao L Wang J R Ren Z Li M Li and H W GaoldquoIsolation and characterization of phenanthrene-degradingstrains Sphingomonas sp ZP1 and Tistrella sp ZP5rdquo Journal ofHazardous Materials vol 152 no 3 pp 1293ndash1300 2008

[6] R C Prince ldquoBioremediation of marine oil spillsrdquo Trends inBiotechnology vol 15 no 5 pp 158ndash160 1997

[7] A Bruns and L Berthe-Corti ldquoFundibacter jadensis gennov sp nov a new slightly halophilic bacterium isolatedfrom intertidal sedimentrdquo International Journal of SystematicBacteriology vol 49 no 2 pp 441ndash448 1999

[8] M M Yakimov P N Golyshin S Lang et al ldquoAlcanivoraxborkumensis gen nov sp nov a new hydrocarbon-degradingand surfactant-producing marine bacteriumrdquo InternationalJournal of Systematic Bacteriology vol 48 no 2 pp 339ndash3481998

[9] M J Gauthier B Lafay R Christen et al ldquoMarinobacterhydrocarbonoclasticus gen nov sp nov a new extremelyhalotolerant hydrocarbon-degradingmarine bacteriumrdquo Inter-national Journal of Systematic Bacteriology vol 42 no 4 pp568ndash576 1992

[10] M A Engelhardt K Daly R P J Swannell and I MHead ldquoIsolation and characterization of a novel hydrocarbon-degrading Gram-positive bacterium isolated from intertidalbeach sediment and description of Planococcus alkanoclasticussp novrdquo Journal of Applied Microbiology vol 90 no 2 pp 237ndash247 2001

[11] I M Head and R P J Swannell ldquoBioremediation of petroleumhydrocarbon contaminants in marine habitatsrdquo Current Opin-ion in Biotechnology vol 10 no 3 pp 234ndash239 1999

[12] G D Floodgate ldquoSome environmental aspects of marinehydrocarbon bacteriologyrdquo Aquatic Microbial Ecology vol 9no 1 pp 3ndash11 1995


[13] A D Geiselbrecht R P Herwig J W Deming and J TStaley ldquoEnumeration and phylogenetic analysis of polycyclicaromatic hydrocarbon-degrading marine bacteria from PugetSound sedimentsrdquo Applied and Environmental Microbiologyvol 62 no 9 pp 3344ndash3349 1996

[14] R Singh D Paul and R K Jain ldquoBiofilms implications inbioremediationrdquo Trends in Microbiology vol 14 no 9 pp 389ndash397 2006

[15] D Paul G Pandey J Pandey and R K Jain ldquoAccessing micro-bial diversity for bioremediation and environmental restora-tionrdquo Trends in Biotechnology vol 23 no 3 pp 135ndash142 2005

[16] G Pandey and R K Jain ldquoBacterial chemotaxis toward envi-ronmental pollutants role in bioremediationrdquo Applied andEnvironmental Microbiology vol 68 no 12 pp 5789ndash57952002

[17] P L Stelmack M R Gray and M A Pickard ldquoBacterialadhesion to soil contaminants in the presence of surfactantsrdquoApplied and Environmental Microbiology vol 65 no 1 pp 163ndash168 1999

[18] R Morgan S Kohn S H Hwang D J Hassett and K SauerldquoBdlA a chemotaxis regulator essential for biofilmdispersion inPseudomonas aeruginosardquo Journal of Bacteriology vol 188 no21 pp 7335ndash7343 2006

[19] J Schmidt M Musken T Becker et al ldquoThe Pseudomonasaeruginosa chemotaxis methyltransferase CheR1 impacts onbacterial surface samplingrdquo PLoS ONE vol 6 no 3 Article IDe18184

[20] J W Costerton P S Stewart and E P Greenberg ldquoBacterialbiofilms a common cause of persistent infectionsrdquo Science vol284 no 5418 pp 1318ndash1322 1999

[21] J Wimpenny W Manz and U Szewzyk ldquoHeterogeneity inbiofilmsrdquo FEMS Microbiology Reviews vol 24 no 5 pp 661ndash671 2000

[22] A W Decho ldquoMicrobial biofilms in intertidal systems anoverviewrdquoContinental Shelf Research vol 20 no 10-11 pp 1257ndash1273 2000

[23] B Klein P Bouriat P Goulas and R Grimaud ldquoBehavior ofMarinobacter hydrocarbonoclasticus SP17 cells during initiationof biofilm formation at the alkane-water interfacerdquo Biotechnol-ogy and Bioengineering vol 105 no 3 pp 461ndash468 2010

[24] T Barkay and J Schaefer ldquoMetal and radionuclide bioremedia-tion issues considerations and potentialsrdquo Current Opinion inMicrobiology vol 4 no 3 pp 318ndash323 2001

[25] K Shimada Y Itoh K Washio and M Morikawa ldquoEfficacyof forming biofilms by naphthalene degrading PseudomonasstutzeriT102 toward bioremediation technology and itsmolecu-lar mechanismsrdquo Chemosphere vol 87 no 3 pp 226ndash233 2012

[26] P Chandran and N Das ldquoDegradation of diesel oil by immo-bilized Candida tropicalis and biofilm formed on gravelsrdquoBiodegradation vol 22 no 6 pp 1181ndash1189 2011

[27] C Gertler G Gerdts K N Timmis M M Yakimov and PN Golyshin ldquoPopulations of heavy fuel oil-degrading marinemicrobial community in presence of oil sorbent materialsrdquoJournal of Applied Microbiology vol 107 no 2 pp 590ndash6052009

[28] H Mahmoud R Al-Hasan M Khanafer and S RadwanldquoA microbiological study of the self-cleaning potential of oilyArabian Gulf coastsrdquo Environmental Science and PollutionResearch vol 17 no 2 pp 383ndash391 2010

[29] G Boslashdtker T Thorstenson B L P Lilleboslash et al ldquoThe effect oflong-term nitrate treatment on SRB activity corrosion rate and

bacterial community composition in offshore water injectionsystemsrdquo Journal of Industrial Microbiology and Biotechnologyvol 35 no 12 pp 1625ndash1636 2008

[30] K S Cho O K Choi Y H Joo K M Lee T H Lee andH W Ryu ldquoCharacterization of biofilms occurred in seepagegroundwater contaminated with petroleum within an urbansubway tunnelrdquo Journal of Environmental Science and HealthPart A vol 39 no 3 pp 639ndash650 2004

[31] Z Liu A M Jacobson and R G Luthy ldquoBiodegradation ofnaphthalene in aqueous nonionic surfactant systemsrdquo Appliedand Environmental Microbiology vol 61 no 1 pp 145ndash151 1995

[32] N A Sorkhoh M A Ghannoum A S Ibrahim R J Strettonand S S Radwan ldquoCrude oil and hydrocarbon-degradingstrains of Rhodococcus rhodochrous isolated from soil andmarine environments in Kuwaitrdquo Environmental Pollution vol65 no 1 pp 1ndash17 1990

[33] J G Holt N R Krieg P H A Sneat J T Staley and ST Williams Bergeyrsquos Manual of Determinative BacteriologyWilliams ampWillkins Baltimore Md USA 1994

[34] S T Williams M E Sharpe and J G Holt Bergeyrsquos Manual ofSystematic Bacteriology Vol IVWilliams ampWilkins BaltimoreMd USA 1989

[35] J T Staley M P Bryant N Pfenning and J G Holt BergeyrsquosManual of Systematic Bacteriology Vol III Williams ampWilkinsBaltimore Md USA 1989

[36] M Hasanuzzaman A Ueno H Ito et al ldquoDegradation of long-chain n-alkanes (C36 and C40) by Pseudomonas aeruginosastrain WatGrdquo International Biodeterioration and Biodegrada-tion vol 59 no 1 pp 40ndash43 2007

[37] V Kisand R Cuadros and J Wikner ldquoPhylogeny of culturableestuarine bacteria catabolizing riverine organic matter in thenorthern Baltic Seardquo Applied and Environmental Microbiologyvol 68 no 1 pp 379ndash388 2002

[38] S F AltschulW GishWMiller EWMyers and D J LipmanldquoBasic local alignment search toolrdquo Journal ofMolecular Biologyvol 215 no 3 pp 403ndash410 1990

[39] V J M Allan M E Callow L E Macaskie and M Paterson-Beedle ldquoEffect of nutrient limitation on biofilm formation andphosphatase activity of a Citrobacter sprdquoMicrobiology vol 148no 1 pp 277ndash288 2002

[40] A Rajasekar T G Babu S Maruthamuthu S T K Pandian SMohanan and N Palaniswamy ldquoBiodegradation and corrosionbehaviour of Serratia marcescensACE2 isolated from an Indiandiesel-transporting pipelinerdquoWorld Journal ofMicrobiology andBiotechnology vol 23 no 8 pp 1065ndash1074 2007


[42] M Raulio M Jarn J Ahola et al ldquoMicrobe repelling coatedstainless steel analysed by field emission scanning electronmicroscopy and physicochemical methodsrdquo Journal of Indus-trial Microbiology and Biotechnology vol 35 no 7 pp 751ndash7602008

[43] A D Peacock Y J Chang J D Istok et al ldquoUtilization ofmicrobial biofilms as monitors of bioremediationrdquo MicrobialEcology vol 47 no 3 pp 284ndash292 2004

[44] P J Vaysse L Prat S Mangenot S Cruveiller P Goulasand R Grimaud ldquoProteomic analysis of Marinobacter hydro-carbonoclasticus SP17 biofilm formation at the alkane-waterinterface reveals novel proteins and cellular processes involved


in hexadecane assimilationrdquo Research in Microbiology vol 160no 10 pp 829ndash837 2009

[45] R Singh D Paul and R K Jain ldquoBiofilms implications inbioremediationrdquo Trends in Microbiolog vol 14 no 9 pp 389ndash397 2006

[46] H Al-Awadhi R H Al-Hasan N A Sorkhoh S Salamah andS S Radwan ldquoEstablishing oil-degrading biofilms on gravelparticles and glass platesrdquo International Biodeterioration andBiodegradation vol 51 no 3 pp 181ndash185 2003

[47] R Kumar R Subarna H Dipak B Debabrata and B DipaldquoSurvey of petroleum-degrading bacteria in coastal waters ofSunderban Biosphere Reserverdquo World Journal of Microbiologyand Biotechnology vol 18 no 6 pp 575ndash581 2002

[48] M Venkateswar Reddy G N Nikhil S Venkata Mohan Y VSwamy and P N Sarma ldquoPseudomonas otitidis as a potentialbiocatalyst for polyhydroxyalkanoates (PHA) synthesis usingsynthetic wastewater and acidogenic effluentsrdquo BioresourceTechnology vol 123 pp 471ndash479 2012

[49] D Al-Bader M K Kansour R Rayan and S S RadwanldquoBiofilm comprising phototrophicdiazotrophic and hydrocar-bon-utilizing bacteria a promising consortium in the biore-mediation of aquatic hydrocarbon pollutantsrdquo EnvironmentalScience and Pollution Research International 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


single bacterial species or even by a group of bacteria fungialgae and protozoa The potential of microbial aggregates inthe biofilm communities for bioremediation is always a saferand more adept method than planktonic microorganism asthe biofilm matrix protects them during stress and thereforeorganism gets a better chance of adaptation [22] Interest-ingly Klein et al [23] reported that hexadecane assimilationby Marinobacter hydrocarbonoclasticus SP17 occurs throughthe formation of a biofilm at the alkane-water interface andhow the cell behavior changes with the presence of utilizableor nonutilizable alkanes at the interface The biofilm-basedreactors furnish high microbial biomass accessible for bettermicrobial activity than planktonic cells for other biologicalactivities like biomineralization [24] Recently the efficiencyof biofilm-associated cells in degradation of naphthaleneover planktonic had been elucidated for strain Pseudomonasstutzeri T102 and the survival of cells in petroleum contam-inated soil is well documented [25] Chandran and Das [26]demonstrated 97 degradation of diesel oil over a period of10 days by yeast biofilm on gravel particle Faster and intensedepletion of linear and brunched hydrocarbon was observedin biofilm microbial community of Alcanivorax borkumensis[27] Microbial consortia on gravel particle were found asconducive tools for self-cleaning of oily gulf coast throughoutall the sites and season [28] Biofilm community is diverseand relatively stable for longer period of time [29] The filmconsortia were isolated from petroleum contaminated urbansubway drainage system where they were capable of degra-dation at fifteen-degree centigrade [30] The phenomenonof chemotaxis by the organisms towards the pollutants andthe simultaneous attachment-detachment process maintainsa constant load of biomass to the affected site in the waterbodies

Oil spillage has taken place in India for more than oneinstance Notably in early 2006 a Japanese tanker collidedwith a small Indian vessel 470 km west of the Nicobar andAndaman Archipelago spilling over 4500 tons of oil intothe Indian Ocean More recently a ship carrying iron ore isreported to be spilling oil in the sea near Paradip Port (OrissaIndia) since it has gone down under sea in September 2009Kolkata Port and nearby areas like Haldia port part of Bayof Bengal close to the ports and Haldia Refinery are majorconcerns for possible oil spillage due to everyday transportof fuel oil and other means since it is a major shippingcorridor for eastern region of India In spite of possiblethreat of contamination of water sources by spilled oil inthese areas little work has been done so far on presence andcharacterization of oil-degrading microorganisms naturallyexisting in water sources near Kolkata port and nearby areas

Our present work was to emphasize the multicellularaggregation of biofilm formation by naturally occurringhydrocarbon degrading strains from this region and to inves-tigate the applicability of biofilm amendment on enhance-ment of biodegradation of crude oil Briefly microorganismscapable to utilize crude oil as sole carbon source were isolatedfrom the water samples of the previously mentioned sourcesthrough serial enrichment culture technique Based on bettercrude oil utilization ability three of the isolated strains fromthe three mentioned sources were screened The organisms

were characterized and identified by biochemical test and 16SrDNA sequencing and further tested for utilization of variousfuel oils and their ability to form biofilmThe volumetric andtopological properties of biofilm near oil water interface wereestimated by confocal laser scanning microscopy (CLSM)Gas chromatography-mass spectroscopic analysiswas carriedout to measure the effect of biofilm amendment on crude oildegradation in comparison to planktonic culture alone

2 Materials and Methods

21 Source of Microorganisms Water samples were collectedfrom Kolkata port of Hooghly River (22∘321015840N 88241015840E)River Haldi at Haldia port (22∘051015840N 88∘031015840E) and Bay ofBengal at Digha (21∘371015840N 87∘251015840E) for isolation of crudeoil degrading microorganism Water sample from Gomukhglacier (30∘581015840N 78∘551015840E) the source of Ganges river at thealtitude of 3819m was used as control nonpolluted water andtested for a presence of crude oil degrading bacteria

22 Culture Enrichment Isolation and Characterization ofStrain The enrichment of crude oil degrading bacteria wascarried out under aerobic condition with crude oil as solesource of carbon Crude oil was obtained from IndianOil Corporation Limited (IOCL Haldia West Bengal) Themineral salt media (MSM) [31] were amended with 1 crudeoil (vv) and enrichment of culture was carried out inthree consecutive batches each having a span of 15 days andenriched by using previous growth as inoculums for the nextBacterial growth was measured by using spectrophotometer(Chemito Instruments UV 2600) at 600 nm and comparedwith control without inoculation Selective solid inorganicmedia (SSIM) [32] were inoculated by spreading 100 120583L ofbroth from last batch of enriched culture incubated at 30∘Cfor 10 days Representative pure colonies were isolated andfurther confirmed for oil degradation by growing in MSMmedia provided with 1 crude oil (filter sterilized using02 120583m syringe filter)

Selection of microorganisms was based on better abilityto grow in presence of crude oil as sole source of carbonin growth media The isolated microorganisms were testedfor Gram staining and biochemical properties as describedpreviously [33ndash35] Motility tests were done by stabbing cellsin semisolid nutrient agar (07 agar) [33]

23 Growth Characteristics in Different 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 localservice station) These oil samples were filter-sterilized using02 120583m syringe filter and added into 50mL of BH media(composition (gmlit) MgSO

4(02) CaCl

2(002) KH

2PO4

(10) (NH4)2HPO4(10) KNO

3(10) FeCl

3(005) and 1 of

oil sample) pH7TheBHmediawere inoculatedwith isolatedbacteria and incubated at 30∘C under static condition for 5days Aliquots of bacterial cultures were collected seriallydiluted and plated on nutrient agar plates The numbers of











3 Results



























0 2 4 6 8 10 12 14 16 18 20 22

0

05

1

15

2

25

3


OD

at 6

00 nm


(a)

0 2 4 6 8 10 12 14 16 18 20 22

0

05

1

15

2

25

3


OD

at 6

00 nm


(b)



0

05

1

15

2

25

3

OD

at 6

00 nm

(c)






90

45

5

Abun

danc

e

Time (min)5 8 11 135

times105

(a)

45

25

5

Abun

danc

e

times105

Time (min)5 8 11 135

(b)

20

12

1

Abun

danc

e

Time (min)5 8 11 135

times105

(c)

24

12

2

Abun

danc

e

times105

Time (min)5 8 11 135

(d)










0001

60

8

62

100

60






4 Discussion





119871KPW1-S1 119875DSW1-S4 119861119871DSW1-S4













(a) (b) (c)


(a) (b) (c)

(d) (e) (f)







20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)

20 120583m

(f)


Abun

danc

e

Time (min)

5 10 15 20 25 30 35

42119890+074119890+07

38119890+0736119890+0734119890+0732119890+07

3119890+0728119890+0726119890+0724119890+0722119890+07

2119890+0718119890+0716119890+0714119890+0712119890+07

1119890+078000000600000040000002000000







5 Conclusion


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











3 Results



























0 2 4 6 8 10 12 14 16 18 20 22

0

05

1

15

2

25

3


OD

at 6

00 nm


(a)

0 2 4 6 8 10 12 14 16 18 20 22

0

05

1

15

2

25

3


OD

at 6

00 nm


(b)



0

05

1

15

2

25

3

OD

at 6

00 nm

(c)






90

45

5

Abun

danc

e

Time (min)5 8 11 135

times105

(a)

45

25

5

Abun

danc

e

times105

Time (min)5 8 11 135

(b)

20

12

1

Abun

danc

e

Time (min)5 8 11 135

times105

(c)

24

12

2

Abun

danc

e

times105

Time (min)5 8 11 135

(d)










0001

60

8

62

100

60






4 Discussion





119871KPW1-S1 119875DSW1-S4 119861119871DSW1-S4













(a) (b) (c)


(a) (b) (c)

(d) (e) (f)







20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)

20 120583m

(f)


Abun

danc

e

Time (min)

5 10 15 20 25 30 35

42119890+074119890+07

38119890+0736119890+0734119890+0732119890+07

3119890+0728119890+0726119890+0724119890+0722119890+07

2119890+0718119890+0716119890+0714119890+0712119890+07

1119890+078000000600000040000002000000







5 Conclusion


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


























0 2 4 6 8 10 12 14 16 18 20 22

0

05

1

15

2

25

3


OD

at 6

00 nm


(a)

0 2 4 6 8 10 12 14 16 18 20 22

0

05

1

15

2

25

3


OD

at 6

00 nm


(b)



0

05

1

15

2

25

3

OD

at 6

00 nm

(c)






90

45

5

Abun

danc

e

Time (min)5 8 11 135

times105

(a)

45

25

5

Abun

danc

e

times105

Time (min)5 8 11 135

(b)

20

12

1

Abun

danc

e

Time (min)5 8 11 135

times105

(c)

24

12

2

Abun

danc

e

times105

Time (min)5 8 11 135

(d)










0001

60

8

62

100

60






4 Discussion





119871KPW1-S1 119875DSW1-S4 119861119871DSW1-S4













(a) (b) (c)


(a) (b) (c)

(d) (e) (f)







20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)

20 120583m

(f)


Abun

danc

e

Time (min)

5 10 15 20 25 30 35

42119890+074119890+07

38119890+0736119890+0734119890+0732119890+07

3119890+0728119890+0726119890+0724119890+0722119890+07

2119890+0718119890+0716119890+0714119890+0712119890+07

1119890+078000000600000040000002000000







5 Conclusion


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


90

45

5

Abun

danc

e

Time (min)5 8 11 135

times105

(a)

45

25

5

Abun

danc

e

times105

Time (min)5 8 11 135

(b)

20

12

1

Abun

danc

e

Time (min)5 8 11 135

times105

(c)

24

12

2

Abun

danc

e

times105

Time (min)5 8 11 135

(d)










0001

60

8

62

100

60






4 Discussion





119871KPW1-S1 119875DSW1-S4 119861119871DSW1-S4













(a) (b) (c)


(a) (b) (c)

(d) (e) (f)







20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)

20 120583m

(f)


Abun

danc

e

Time (min)

5 10 15 20 25 30 35

42119890+074119890+07

38119890+0736119890+0734119890+0732119890+07

3119890+0728119890+0726119890+0724119890+0722119890+07

2119890+0718119890+0716119890+0714119890+0712119890+07

1119890+078000000600000040000002000000







5 Conclusion


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





0001

60

8

62

100

60






4 Discussion





119871KPW1-S1 119875DSW1-S4 119861119871DSW1-S4













(a) (b) (c)


(a) (b) (c)

(d) (e) (f)







20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)

20 120583m

(f)


Abun

danc

e

Time (min)

5 10 15 20 25 30 35

42119890+074119890+07

38119890+0736119890+0734119890+0732119890+07

3119890+0728119890+0726119890+0724119890+0722119890+07

2119890+0718119890+0716119890+0714119890+0712119890+07

1119890+078000000600000040000002000000







5 Conclusion


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




119871KPW1-S1 119875DSW1-S4 119861119871DSW1-S4













(a) (b) (c)


(a) (b) (c)

(d) (e) (f)







20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)

20 120583m

(f)


Abun

danc

e

Time (min)

5 10 15 20 25 30 35

42119890+074119890+07

38119890+0736119890+0734119890+0732119890+07

3119890+0728119890+0726119890+0724119890+0722119890+07

2119890+0718119890+0716119890+0714119890+0712119890+07

1119890+078000000600000040000002000000







5 Conclusion


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)


(a) (b) (c)

(d) (e) (f)







20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)

20 120583m

(f)


Abun

danc

e

Time (min)

5 10 15 20 25 30 35

42119890+074119890+07

38119890+0736119890+0734119890+0732119890+07

3119890+0728119890+0726119890+0724119890+0722119890+07

2119890+0718119890+0716119890+0714119890+0712119890+07

1119890+078000000600000040000002000000







5 Conclusion


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)

20 120583m

(f)


Abun

danc

e

Time (min)

5 10 15 20 25 30 35

42119890+074119890+07

38119890+0736119890+0734119890+0732119890+07

3119890+0728119890+0726119890+0724119890+0722119890+07

2119890+0718119890+0716119890+0714119890+0712119890+07

1119890+078000000600000040000002000000







5 Conclusion


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




5 Conclusion


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article biofilm-mediated enhanced crude oil ...glass cover slips being immersed in ml bh...

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