Research ArticleColiform Bacteria for Bioremediation of Waste Hydrocarbons

Majida Khanafer Husain Al-Awadhi and Samir Radwan

Microbiology Program Department of Biological Sciences Faculty of Science Kuwait University PO Box 5969 13060 Safat Kuwait

Correspondence should be addressed to Samir Radwan samirradwankuedukw

Received 30 May 2017 Revised 26 July 2017 Accepted 6 August 2017 Published 10 September 2017

Academic Editor Sardar Khan

Copyright copy 2017 Majida Khanafer 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

Raw domestic sewage of Kuwait City contained about 106mlminus1 colony forming units of Enterobacter hormaechei subsp oharae(566)Klebsiella spp (36) andEscherichia coli (74) as characterized by their 16S rRNA-gene sequencesThe isolated coliformsgrew successfully on a mineral medium with crude oil vapor as a sole source of carbon and energy Those strains also grew albeitto different degrees on individual 119899-alkanes with carbon chains between C

9and C

36and on the individual aromatic hydrocarbons

toluene naphthalene phenanthrene and biphenyl as sole sources of carbon and energy These results imply that coliforms likeother hydrocarbonoclastic microorganisms oxidize hydrocarbons to the corresponding alcohols and then to aldehydes and fattyacids which are biodegraded by 120573-oxidation to acetyl CoAThe latter is a well-known key intermediate in cell material and energyproduction E coli cells grown in the presence of 119899-hexadecane (but not in its absence) exhibited typical intracellular hydrocarboninclusions as revealed by transmission electron microscopy Raw sewage samples amended with crude oil 119899-hexadecane orphenanthrene lost these hydrocarbons gradually with time Meanwhile the numbers of total and individual coliforms particularlyEnterobacter increased It was concluded that coliform bacteria in domestic sewage probably in other environmental materials tooare effective hydrocarbon-biodegrading microorganisms

1 Introduction

Environmental pollution with spilled oil and waste hydrocar-bons has become a problem of major concern on the globalscale Those compounds contaminate water and terrestrialand atmospheric ecosystems Early reports estimated thatabout 008 to 04 of the internationally produced crude oilis spilled into the environment [1] Probably this value hascurrently increased because of the ever increasing activitiesrelated to oil extraction from reservoirs transportation pro-cessing use as an energy source and accidents and militaryconflicts Spilled oil may be removed using physiochemicalor microbiological means The former approach comprisesthe use of sorbents [2 3] solidifiers [4 5] dispersants [6 7]and gelators [8 9] However the use of these products is notalways environmentally safe and may even cause additionalpollution [1 10] Bioremediation is the alternative approachcommonly accepted as the most cost-effective and eco-friendly biotechnology available Two practices are suggestedfor bioremediation bioaugmentation and biostimulation [11]

In the former approach oil-contaminated areas are inoc-ulated with cocktails of hydrocarbonoclastic microorgan-isms [12ndash14] Biostimulation on the other hand dependson enhancing already existing microorganisms by addingnutrients [11] surfactants [12 15] and other materials Inthis context the characteristic feature of hydrocarbonoclasticmicroorganisms lies in their ability to utilize hydrocarbonsas sole sources of carbon and energy through the well-established metabolic pathways They possess hydroxylases(also called oxygenases) which introduce oxygen atoms frommolecular oxygen into the hydrocarbon substrate producingfatty alcoholsThe latter are further oxidized via the aldehydes(or ketones) to the corresponding fatty acids The latter arebiodegraded into the key intermediatemetabolite acetyl CoA

In bioaugmentation an important question regardingthe choice of hydrocarbonoclastic microorganisms to beinoculated should be considered Essential prerequisites arethat those organisms should easily succeed in colonizingthe new environment and in standing competition with thealready existing inhabitants [16 17] Therefore the term

HindawiBioMed Research InternationalVolume 2017 Article ID 1838072 8 pageshttpsdoiorg10115520171838072

2 BioMed Research International

ldquoautochthonous bioaugmentation (ABA)rdquo has been sug-gested recently for such a practice [18ndash20]

In the course of our work on bioremediation of desertand aquatic ecosystems that had been heavily polluted withoil during the greatest man-made oil spill at the end ofthe 1991 Gulf-war we used to isolate hydrocarbonoclasticbacteria that according to their 16S rRNA-gene sequenceswere coliforms Such bacteria belonged predominantly to thegenera Enterobacter Klebsiella Escherichia and others Theliterature includes several reports by other investigators onhydrocarbon-degradation by such coliforms [21ndash23] Eventhe hazardous simple and polyaromatic hydrocarbons werereported to be degraded by coliforms such as Klebsiella andEnterobacter [24ndash26] Also diesel oil [27] and constituenthydrocarbons of crude oil [28ndash31] were found to be degradedby various coliforms However in many of those studies thehydrocarbonoclastic potential was based solely on the abilityof the strains to grow in mineral media with hydrocarbonsas sole sources of carbon and energy In some of thosereports the coliform taxa were just listed in tables includingthe identified hydrocarbonoclastic isolates without furtherelaboration on criteria for considering them as hydrocarbondegraders

Coliform bacteria belong to the family Enterobacteri-aceae which comprises facultative anaerobic bacteria nat-urally inhabiting digestive tracts of warm-blooded animalsincluding man Therefore such microorganisms are contin-uously discharged with feces and E coli for example makesup about 10 of the domestic sewage microflora worldwide

The major objective of this study was to investigate thehydrocarbonoclastic potential of coliforms from local domes-tic sewage samples For this microbiological biochemicaland cytological techniques of analysis were integrated toobtain a more comprehensive evidence for the hydrocarbon-oclastic potential of the obtained isolates Should coliformsprove to be effective oil-biodegraders domestic sewagewouldbe a cost-effective accessible source of microorganisms to beused in oil-bioremediation via bioaugmentation Within thiscontext domestic sewage frequently receives waste hydrocar-bons as constituents of disposed plant and animal wastes

2 Materials and Methods

21 Counting and Characterization of Coliforms in RawSewage Three local raw domestic sewage samples werecollected from Al-Jahra station and used to count and isolatetheir indigenous coliformsThe conventional dilution-platingmethod was adopted and the coliform-selective mediumEosin Methylene Blue Agar (EMB) [32 33] was used Thecoliform colonies were counted subcultured and purified onthe above selective medium Individual coliforms were alsocounted after they had been differentiated according to theircharacteristic morphologies on this medium Enterobactercolonies were pink with dark centers Klebsiella coloniesmucoid pink and Escherichia coli colonies black with greenmetallic sheenThe isolateswere further characterized accord-ing to their potential for acid and gas production from lactosefermentation at 37∘C and 44∘C following established routineprocedures Representative strains were isolated purified

and characterized by comparing the sequences of their 16SrRNA-genes with sequences of type strains in the GenBankdatabase The method was essentially as follows The totalgenomic DNA was extracted with PrepMan Ultra SamplePreparation Reagent (Applied Biosystems USA) followingthe manufacturerrsquos description The 16S rRNA-genes wereamplified by polymerase chain reaction (PCR) using a VeritiThermal Cycler (Applied Biosystems USA) and a standardprotocol with initial denaturation at 95∘C for 5min and32 cycles of denaturation at 94∘C for 1min annealing at55∘C for 1min and primer extension at 72∘C for 1minThe mixture PCR contained puReTaq Ready-To-Go PCRBeads (Amersham Biosciences UK) 25 ng of DNA tem-plate 1 120583L of each of the universal primer combinationsGM5F (51015840-CCTACGGGAGGCAGCAG-31015840) and 907R (51015840-CCGTCAATTCMTTTGAGTTT-31015840) [34] The reaction vol-ume was completed to 25 120583L with molecular water (SigmaUK) Partial sequencing of the 16S rRNA-genes was doneusing a BigDye version Terminator Kit (Applied BiosystemsUSA)Themixture contained 20 ng of the DNA template 2120583lof the BigDye version 31 terminator 2 120583l of the 5x sequencingbuffer l 120583L of either 907R or GM5F and the final volumewas brought up to 10 120583L with molecular water Labeling wascompleted in the Veriti Thermal Cycler (Applied BiosystemsUSA) using one cycle of 96∘C for lmin then 25 cycles of lminat 96∘C 5 s at 50∘C and 4min at 60∘C The pure templateDNA samples were processed in a 3130xl genetic analyzer(Applied Biosystems USA) Sequencing analysis version 52software (Applied Biosystems USA) was used to analyze theresults Sequences were subjected to basic local alignmentsearch tool analysis with the National Center for Biotech-nology Information (NCBI Bethesda MD USA) GenBankdatabase [35] A phylogenetic tree was constructed usingneighbor joining including bootstrap analysis using PAUPlowastv4 [36] Bootstrap proportions were used on 2000 replicates

22 Hydrocarbon Utilization Potential of Coliforms All thecoliform strains were tested for their ability to grow on amin-eralmedium [37] with oil vapor as a sole source of carbon andenergy The mineral medium had the following composition(glminus1) 50 NaNO

3 056 KH

2PO4 086Na

2HPO4 017 K

2SO4

037 MgSO4sdot7H2O 0007 CaCl

2sdotH2O and 25ml lminus1 of a trace

element solution consisting of (g lminus1) 232 CuSO4sdot5H2O 039

Na2MoO4sdot2H2O 066 KI 10 EDTA 04 FeSO

4sdot7H2O and

0004 NiCl2sdot6H2OThe pH was adjusted to 7 Strains capable

of growth on that medium were considered ldquopresumptiverdquohydrocarbonoclastic

To study the range of pure hydrocarbons that couldbe utilized by the individual strains a common inoculum(loopful in 5ml sterile water) was prepared for each strainand one-loopful aliquots were streaked on the solid mineralmedium containing 05 of individual 119899-alkanes rangingin chain lengths between C

9ndashC20 C23ndashC26 C28 C30 and

C36 The aromatics toluene naphthalene phenanthrene and

biphenyl were also investigated Cultures were incubated at30∘C for 15 d and examined for growth

23 Hydrocarbon Consumption by Coliforms The crude oiland pure-hydrocarbon consumption by individual isolates

BioMed Research International 3

was measured quantitatively by gas liquid chromatography(GLC) Aliquots 100ml of the mineral medium [37] in250ml flasks were supplied with 03 (wv) crude oil orpure hydrocarbons Each flask was inoculated with 1ml cellsuspension (a loopful of 48 h biomass in 5ml sterile water)The cultures were sealed to avoid hydrocarbon losses byvolatilization and incubated at 30∘C on an electrical shaker200 rpm for 2 weeks Three parallel replicates were preparedthroughout The residual hydrocarbons were recovered withthree successive 10ml portions of pentane The combinedextracts were completed to 35ml using pentane and 10 120583lwas analyzed byGLCThepercent loss of the peak areas basedon the peak areas of the control samples (similarly preparedbut using previously autoclaved bacteria) was calculated as aquantitativemeasure of the oil-removal For GLC we used anAgilent 7890A GC (USA) system equipped with FID a DB-5capillary column (Agilent Technologies USA) and He as acarrier gas The oven temperature started at 50∘C for 3minthen rising at 3∘Cmin to 80∘C then rising at 8∘Cmin to256∘C then rising at 30∘Cmin to 330∘C and holding at thistemperature for 11min

24 Hydrocarbon-Uptake by Coliforms In this experimenttwo strains of E coli one from domestic sewage and theother from wheat straw [38] were investigated Using trans-mission electron microscopy (TEM) we examined sectionsof cells previously grown in conventional nutrient broth andof cells grown in the mineral medium containing 03119899-hexadecane [37] for intracellular hydrocarbon inclusionsBiomass samples were fixed with 25 glutaraldehyde insodium cacodylate buffer (01M pH 74) for 2 h [39]The cellswere washed in the same buffer postfixed with 10 OsO

4

(at 4∘C for 2 h) and washed three times in the same bufferCells were centrifuged at 5000timesg for 10min and the pelletwas mixed with an equal volume of 20 noble agar Theagar was spread on a slide left to solidify and cut into smallcubes The cubes were dehydrated in an ascending series (3050 70 and 100) of acetone in water and rinsed twice in100 propylene oxide Finally the samples were embeddedin Epon resin Ultrathin sections [40] were obtained using anultramicrotome Leica (Leica Microsystems Germany) Thesections were mounted on carbon-coated grids stained withsaturated uranyl acetate and Reynolds lead citrate [41] andviewed in an electron microscope JEM 1200-EX II (JEOLUSA)

25 Oil-Removal in Sewage Batches Aliquots 100ml offreshly harvested raw sewage (not previously sterilized) in250ml flasks were provided separately with 10 and 50 gportions of crude oil 10 g portions of 119899-hexadecane and10 g portions of phenanthrene The flasks were sealed toavoid hydrocarbon volatilization and incubated at 30∘C onan electrical shaker 200 rpm for 20 d At time zero andin 5 d intervals cultures were harvested for quantitative oiland coliform analysis as already described Three replicatesamples were analyzed throughout

A similar parallel experiment was run but using previ-ously autoclaved raw sewage aliquots as controls

26 Statistical Analysis Three determinations for each anal-ysis were done and the mean values plusmn standard deviationvalues were calculated using Microsoft Excel 2007 AlsoStatistical Package for Social Sciences version 12 was usedto assess the degree of significance where the analysis ofvariance (ANOVA) was used to differentiate between themeans of the tested parameters

3 Results and Discussion

31 Local Coliform Bacterial Strains The three raw sewagesamples studied were found to contain 95 times 105 98 times105 and 96 times 105mlminus1 colony forming units (CFU) oftotal coliform bacteria as counted on the selective coliformmedium Based on their 16S rRNA-gene sequences thosecoliforms consisted ofEnterobacter hormaechei subsp oharae56ndash58 Klebsiella variicola 23ndash25 Klebsiella pneumonia10-11 and Escherichia coli 7ndash10 The results in Table 1show that the sequence similarities between the isolatesand the standard strains in the GenBank database werehigh between 99 and 100 The phylogenetic relationshipsamong the isolates are shown in the phylogenetic tree inFigure 1 The results of the conventional test for acid and gasproduction from lactose by the 4 coliform isolates (Table 2and Figure S1 in the Supplementary Material available onlineat httpsdoiorg10115520171838072) were those expectedaccording to their generic identities

32 Oil and Hydrocarbon Utilization by the Coliform IsolatesA total of 313 pooled coliform colonies (on the selective EMBmedium)were subcultured and purified All the pure culturesgrew on a solid mineral medium [37] with crude oil vaporas a sole source of carbon and energy This result indicatesthat all the isolated coliforms presumably have potential forhydrocarbon utilization

Five haphazardly selected strains for each coliformspecies were found to grow albeit with different intensitieson the solid mineral medium [37] supplied with the indi-vidual 119899-alkanes C

9ndashC20 C23ndashC26 C28 C30 and C

36 and

the individual aromatic hydrocarbons toluene naphthalenephenanthrene and biphenyl The growth on such a widerange of aliphatic and aromatic hydrocarbons reflects a highpotential for hydrocarbon utilization Earlier investigatorsalso found that the same coliform species isolated elsewherecould grow on oil and hydrocarbons as sole sources ofcarbon and energy [21ndash23] Successful growth of all tested col-iforms on aliphatic and aromatic hydrocarbons suggests thatthey like all hydrocarbonoclastic microorganisms producemonooxygenases and dioxygenases (hydroxylases) respec-tively [42ndash44] Moreover the further metabolic pathwaysin such coliforms are apparently similar to those in otherhydrocarbonoclastic microorganisms The fatty alcohols areoxidized to aldehydes (ketones) and ultimately to fatty acidswhich in turn are degraded by 120573-oxidation to acetyl CoAunitsThe latter could then be used for synthesis of cell mate-rial and for the production of ATP through the tricarboxylicacid cycle

The results in Table 3 show that representatives of thelocal coliform isolates removed quantitatively significant

BioMed Research International 3

was measured quantitatively by gas liquid chromatography(GLC) Aliquots 100ml of the mineral medium [37] in250ml flasks were supplied with 03 (wv) crude oil orpure hydrocarbons Each flask was inoculated with 1ml cellsuspension (a loopful of 48 h biomass in 5ml sterile water)The cultures were sealed to avoid hydrocarbon losses byvolatilization and incubated at 30∘C on an electrical shaker200 rpm for 2 weeks Three parallel replicates were preparedthroughout The residual hydrocarbons were recovered withthree successive 10ml portions of pentane The combinedextracts were completed to 35ml using pentane and 10 120583lwas analyzed byGLCThepercent loss of the peak areas basedon the peak areas of the control samples (similarly preparedbut using previously autoclaved bacteria) was calculated as aquantitativemeasure of the oil-removal For GLC we used anAgilent 7890A GC (USA) system equipped with FID a DB-5capillary column (Agilent Technologies USA) and He as acarrier gas The oven temperature started at 50∘C for 3minthen rising at 3∘Cmin to 80∘C then rising at 8∘Cmin to256∘C then rising at 30∘Cmin to 330∘C and holding at thistemperature for 11min

24 Hydrocarbon-Uptake by Coliforms In this experimenttwo strains of E coli one from domestic sewage and theother from wheat straw [38] were investigated Using trans-mission electron microscopy (TEM) we examined sectionsof cells previously grown in conventional nutrient broth andof cells grown in the mineral medium containing 03119899-hexadecane [37] for intracellular hydrocarbon inclusionsBiomass samples were fixed with 25 glutaraldehyde insodium cacodylate buffer (01M pH 74) for 2 h [39]The cellswere washed in the same buffer postfixed with 10 OsO

4

(at 4∘C for 2 h) and washed three times in the same bufferCells were centrifuged at 5000timesg for 10min and the pelletwas mixed with an equal volume of 20 noble agar Theagar was spread on a slide left to solidify and cut into smallcubes The cubes were dehydrated in an ascending series (3050 70 and 100) of acetone in water and rinsed twice in100 propylene oxide Finally the samples were embeddedin Epon resin Ultrathin sections [40] were obtained using anultramicrotome Leica (Leica Microsystems Germany) Thesections were mounted on carbon-coated grids stained withsaturated uranyl acetate and Reynolds lead citrate [41] andviewed in an electron microscope JEM 1200-EX II (JEOLUSA)

25 Oil-Removal in Sewage Batches Aliquots 100ml offreshly harvested raw sewage (not previously sterilized) in250ml flasks were provided separately with 10 and 50 gportions of crude oil 10 g portions of 119899-hexadecane and10 g portions of phenanthrene The flasks were sealed toavoid hydrocarbon volatilization and incubated at 30∘C onan electrical shaker 200 rpm for 20 d At time zero andin 5 d intervals cultures were harvested for quantitative oiland coliform analysis as already described Three replicatesamples were analyzed throughout

A similar parallel experiment was run but using previ-ously autoclaved raw sewage aliquots as controls

26 Statistical Analysis Three determinations for each anal-ysis were done and the mean values plusmn standard deviationvalues were calculated using Microsoft Excel 2007 AlsoStatistical Package for Social Sciences version 12 was usedto assess the degree of significance where the analysis ofvariance (ANOVA) was used to differentiate between themeans of the tested parameters

3 Results and Discussion

31 Local Coliform Bacterial Strains The three raw sewagesamples studied were found to contain 95 times 105 98 times105 and 96 times 105mlminus1 colony forming units (CFU) oftotal coliform bacteria as counted on the selective coliformmedium Based on their 16S rRNA-gene sequences thosecoliforms consisted ofEnterobacter hormaechei subsp oharae56ndash58 Klebsiella variicola 23ndash25 Klebsiella pneumonia10-11 and Escherichia coli 7ndash10 The results in Table 1show that the sequence similarities between the isolatesand the standard strains in the GenBank database werehigh between 99 and 100 The phylogenetic relationshipsamong the isolates are shown in the phylogenetic tree inFigure 1 The results of the conventional test for acid and gasproduction from lactose by the 4 coliform isolates (Table 2and Figure S1 in the Supplementary Material available onlineat httpsdoiorg10115520171838072) were those expectedaccording to their generic identities

32 Oil and Hydrocarbon Utilization by the Coliform IsolatesA total of 313 pooled coliform colonies (on the selective EMBmedium)were subcultured and purified All the pure culturesgrew on a solid mineral medium [37] with crude oil vaporas a sole source of carbon and energy This result indicatesthat all the isolated coliforms presumably have potential forhydrocarbon utilization

Five haphazardly selected strains for each coliformspecies were found to grow albeit with different intensitieson the solid mineral medium [37] supplied with the indi-vidual 119899-alkanes C

9ndashC20 C23ndashC26 C28 C30 and C

36 and

the individual aromatic hydrocarbons toluene naphthalenephenanthrene and biphenyl The growth on such a widerange of aliphatic and aromatic hydrocarbons reflects a highpotential for hydrocarbon utilization Earlier investigatorsalso found that the same coliform species isolated elsewherecould grow on oil and hydrocarbons as sole sources ofcarbon and energy [21ndash23] Successful growth of all tested col-iforms on aliphatic and aromatic hydrocarbons suggests thatthey like all hydrocarbonoclastic microorganisms producemonooxygenases and dioxygenases (hydroxylases) respec-tively [42ndash44] Moreover the further metabolic pathwaysin such coliforms are apparently similar to those in otherhydrocarbonoclastic microorganisms The fatty alcohols areoxidized to aldehydes (ketones) and ultimately to fatty acidswhich in turn are degraded by 120573-oxidation to acetyl CoAunitsThe latter could then be used for synthesis of cell mate-rial and for the production of ATP through the tricarboxylicacid cycle

The results in Table 3 show that representatives of thelocal coliform isolates removed quantitatively significant

4 BioMed Research International

Bacillus subtilis

Escherichia coli NR_112558

Escherichia fergusonii NR_027549

Escherichia fergusonii NR_114079

Escherichia coli LC069032

Escherichia coli NR_114042

Escherichia coli KX279366

520

1000

Enterobacter hormaechei subsp oharae AJ853889

Enterobacter hormaechei subsp oharae KX279369

Enterobacter hormaechei NR_042154

Enterobacter tabaci NR_146667

Enterobacter tabaci KP990658

Enterobacter asburiae KY285194762

962

849

905

Klebsiella quasipneumoniae subsp quasipneumoniae NR_134062

Klebsiella quasipneumoniae subsp similipneumoniae NR_134063

Klebsiella pneumonia KX279368

Klebsiella pneumoniae KX274129791

866

646

Klebsiella pneumoniae NR_119278

Klebsiella variicola KX279367

Klebsiella variicola KP783170

Klebsiella variicola NR_025635

840

Klebsiella pneumoniae NR_112009

Klebsiella pneumoniae NR_117686

580

746

998

Figure 1 Phylogenetic tree showing relationships among coliforms isolated from domestic sewage

Table 1 Information about the 16S rDNA sequencing of the coliforms isolated from domestic sewage

Nearest GenBank match Total bases Similarity () Compared bases Accession numberEscherichia coli 513 100 513513 KX279366Klebsiella variicola 482 99 484485 KX279367Klebsiella pneumoniae 507 100 507507 KX279368Enterobacter hormaechei subsp oharae 506 99 511514 KX279369

Table 2 Acid and gas production from lactose by coliform isolates

Isolates Acid production Gas production37∘C 44∘C 37∘C 44∘C

Escherichia coli (wheat straw) + + + +Escherichia coli (domestic sewage) + + + +Klebsiella variicola + + + minus

Klebsiella pneumoniae + + + minus

Enterobacter hormaechei subsp oharae + + + minus

BioMed Research International 5

Table 3 Crude oil and pure-hydrocarbon consumption by coliform isolates

Isolates hydrocarbon consumptionCrude oil C16 Phenanthrene

Escherichia coli (wheat straw) 183 plusmn 11 171 plusmn 10 163 plusmn 09Escherichia coli (domestic sewage) 222 plusmn 32 190 plusmn 22 181 plusmn 30Klebsiella variicola 166 plusmn 10 123 plusmn 08 110 plusmn 05Klebsiella pneumoniae 142 plusmn 12 106 plusmn 08 109 plusmn 10Enterobacter hormaechei subsp oharae 303 plusmn 15 359 plusmn 20 185 plusmn 08Pseudomonas stutzeri 156 plusmn 09 184 plusmn 08 141 plusmn 07Pseudomonas stutzeri from our private culture collection was analyzed as a classic hydrocarbon-utilizer for the purpose of comparison

(a) (b)

(c) (d)

Figure 2 Transmission electronmicrographs of ultrathin sections of E coli cells from domestic sewage grown in 119899-hexadecane-free medium(a) and in 119899-hexadecane-containing medium (b) and of E coli cells from wheat straw grown in 119899-hexadecane-free medium (c) and in 119899-hexadecane-containing medium (d) Magnification (a) and (b) times60000 (c) and (d) times40000

proportions of the available crude oil as well as of the purealiphatic (C

16) and aromatic (phenanthrene) hydrocarbons in

15 days Those hydrocarbons were provided in the mineralmedium [37] as sole sources of carbon and energy Thesequantitative determinations provide a clear-cut evidence forthe hydrocarbonoclastic potential of the studied coliforms

33 Cytological Evidence for 119899-Hexadecane (11986216) Uptake by Ecoli For this experiment two strains of the typical coliformE coli one from sewage and the other from wheat straw[38] were used The TEM images in Figure 2 show thatthe cell cytoplasm of hydrocarbon-incubated cells in con-trast to that of cells harvested from nutrient broth (withouthydrocarbons) was enriched with less electron-dense areas

(hydrocarbon inclusions) of varying dimensions Our mea-surements showed that those inclusions occupied roughly50 of the total cell volumes Earlier researchers alsodescribed similar intracytoplasmic hydrocarbon inclusionsin classical hydrocarbonoclastic bacteria incubated in mediacontaining 119899-hexadecane [37] Cells incubated in the pres-ence of C

16also showed clear morphological deformations

34 Self-Cleaning ofOily Sewage by IndigenousColiforms Theresults in Figure 3 show that crude oil C18 and phenan-threne added to freshly harvested raw sewage (not previouslysterilized) were steadily removed during the 20 d incubationperiod At the end the remaining proportions were only 70of 3 crude oil 45 of 1 crude oil 66 of C18 and 55

6 BioMed Research International

0 5 10 15 20

3 crude oil

Phenanthrene

0 5 10 15 20

1 crude oil

0

20

40

60

80

100

120

cr

ude o

il co

ncen

trat

ion

0

20

40

60

80

100

120

cr

ude o

il co

ncen

trat

ion

0

5

10

15

20

25

30

(Col

ony

form

ing

units

times103

mlminus

1)

0

10

20

30

40

50

60

(Col

ony

form

ing

units

times103

mlminus

1)

0

2

4

6

8

10

12

14

16

18

(Col

ony

form

ing

units

times103

mlminus

1)

0

20

40

60

80

100

120

p

hena

nthr

ene c

once

ntra

tion

0

2

4

6

8

10

12

14

16

18

20(C

olon

y fo

rmin

g un

itstimes103

mlminus

1)

18

0

20

40

60

80

100

120

18

conc

entr

atio

n

5 10 15 200Incubation period (days)

5 10 15 200Incubation period (days)

Total CFU mＦminus1

CFU of Enterobacter

CFU of Klebsiella

CFU of Escherichiaspp mＦ

minus1

spp mＦminus1

spp mＦminus1 spp mＦ

minus1

spp mＦminus1

spp mＦminus1phenanthrene percent

Total CFU mＦminus1

CFU of Enterobacter

CFU of Klebsiella

CFU of Escherichiaphenanthrene percent

Crude oil 18 or Crude oil 18 or

Figure 3 Consumption of crude oil added to freshly harvested sewage and parallel increase of total coliform members with time

of phenanthrene based on the amount added at time zeroMeanwhile there was a parallel increase of the total numbersof the total coliforms from 146 times 106 to 265 times 106 CFUmlminus1in 3 crude oil to 527 times 106 CFUmlminus1 in 1 crude oil to189 times 106 CFUmlminus1 in C18 and to 158 times 106 CFUmlminus1 inphenanthrene Figure 3 shows further that Enterobacter wasthe coliform that exhibited the highest increase in numberwhich may reflect a main role of this coliform in oil con-sumption Other hydrocarbonoclastic noncoliform bacteriaindigenous to sewage are also probably involved in the mea-sured crude oil removal However the significant increase(119901 lt 005) in numbers of coliforms parallel to the signi-ficant decrease (119901 lt 005) in the concentration of the remain-ing oil provides an experimental evidence for a major con-tribution of indigenous coliforms to self-cleaning of oil in

sewage This finding implies that the indigenous domesticsewage microflora is capable of controlling potential oil spillsin this waste product Furthermore raw sewage is an acces-sible cost-effective source of hydrocarbonoclastic micro-bial cocktails that can be used for bioaugmenting oil-con-taminated areas

4 Conclusions

The results of this paper provide experimental evidence forthe role of coliforms in biodegradation of oil spilled in theenvironment This group should be particularly effective incleaning oil spills in sewage where those organisms areautochthonous (indigenous) Conditions prevailing duringconventional sewage treatment probably bioenhance the

BioMed Research International 7

coliforms potential for oil-bioremediation On one handsewage treatment involves the routine practice of extensiveaeration to reduce the sewage BOD values (ie the organicmatter content) In view of the fact that the initial step ofmicrobial attack on hydrocarbons is catalyzed by oxygenases(also called hydroxylases) aeration should bioenhance thespilled-oil bioremediation Oil-bioremediation is also bioen-hanced by nitrogen-fertilizers During sewage treatmentnitrates and other nitrogenous compounds are continuouslyproduced and in addition several coliforms for exampleKlebsiella spp and Enterobacter spp are diazotrophic Inother words nitrogen fertilization is also guaranteed insewage for the hydrocarbonoclastic coliform communities

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

Thiswork has been supported by Kuwait University ResearchGrant SL0616 Thanks are due to SAF Analysis Facilityfor providing Genetic Analysis through Project GS0102 toNUERS for providing GLC analysis through Project SRUL0113 and to the Nanoscopy Science Center for the technicalassistance with transmission electron microscopy

References

[1] National Research Council (NRC) Using Oil Spill Dispersantson the Sea National Academy Press Washington DC WashUSA 1989

[2] A A Al-Majed A R Adebayo and M E Hossain ldquoA sustain-able approach to controlling oil spillsrdquo Journal of EnvironmentalManagement vol 113 pp 213ndash227 2012

[3] A Bayat S F Aghamiri A Moheb and G R Vakili-NezhaadldquoOil spill cleanup from seawater by sorbentmaterialsrdquoChemicalEngineering and Technology vol 28 no 12 pp 1525ndash1528 2005

[4] D Sundaravadivelu M T Suidan A D Venosa and P IRosales ldquoCharacterization of solidifiers used for oil spill reme-diationrdquo Chemosphere vol 144 pp 1490ndash1497 2016

[5] W Dahl R R Lessard E Cardello et al ldquoSolidifiers for oil spillresponserdquo in Proceedings of the SPE Health Safety and Environ-ment in Oil and Gas Exploration and Production Conference pp803ndash810 New Orleans LA USA 1996

[6] L Graham C Hale EMaung-Douglass et al ldquoOil spill scienceChemical dispersants and their role in oil spill responserdquoMASGP-15-015 2016

[7] A Zolfaghari-Baghbaderani M Emtyazjoo P Poursafa et alldquoEffects of three types of oil dispersants on biodegradationof dispersed crude oil in water surrounding two Persian Gulfprovincesrdquo Journal of Environmental and Public Health vol2012 Article ID 981365 8 pages 2012

[8] C Ren G H B Ng H Wu et al ldquoInstant room-temperaturegelation of crude oil by chiral organogelatorsrdquo Chemistry ofMaterials vol 28 no 11 pp 4001ndash4008 2016

[9] J Bachl S Oehm J Mayr C Cativiela J J Marrero-Telladoand D D Dıaz ldquoSupramolecular phase-selective gelation bypeptides bearing side-chain azobenzenes effect of ultrasoundand potential for dye removal and oil spill remediationrdquo

International Journal of Molecular Sciences vol 16 no 5 pp11766ndash11784 2015

[10] E E Cordes D O Jones T A Schlacher et al ldquoEnvironmentalimpacts of the deep-water oil and gas industry a review to guidemanagement strategiesrdquo Frontiers in Environmental Science vol4 2016

[11] R M Atlas and R Bartha Microbial Ecology Fundamentalsand Applications vol 70 BenjaminCummings PublishingCompany Inc 4th edition 1998

[12] A Szulc D Ambrozewicz M Sydow et al ldquoThe influence ofbioaugmentation and biosurfactant addition on bioremediationefficiency of diesel-oil contaminated soil feasibility during fieldstudiesrdquo Journal of Environmental Management vol 132 pp121ndash128 2014

[13] I Kuiper E L Lagendijk G V Bloemberg and B J J Lugten-berg ldquoRhizoremediation a beneficial plant-microbe interac-tionrdquoMolecular Plant-Microbe Interactions vol 17 no 1 pp 6ndash15 2004

[14] H Van Limbergen E M Top and W Verstraete ldquoBioaug-mentation in activated sludge current features add futureperspectivesrdquo Applied Microbiology and Biotechnology vol 50no 1 pp 16ndash23 1998

[15] Ł Ławniczak R Marecik and Ł Chrzanowski ldquoContributionsof biosurfactants to natural or induced bioremediationrdquoAppliedMicrobiology and Biotechnology vol 97 no 6 pp 2327ndash23392013

[16] S S Radwan N A Sorkhoh I M El-Nemr and A F El-Desouky ldquoA feasibility study on seeding as a bioremediationpractice for the oilyKuwaiti desertrdquo Journal of AppliedMicrobio-logy vol 83 no 3 pp 353ndash358 1997

[17] S S Radwan ldquoGulf oil spillrdquoNature vol 350 no 6318 pp 456ndash460 1991

[18] S Di Gregorio M R Castglione A Gentini and R LorenzildquoBiostimulation of the autochthonous bacterial community andbioaugmentation of selected bacterial strains for the depletionof polycyclic aromatic hydrocarbons in a historically con-taminated soilrdquo Geophysical Research Abstracts vol 17 2015EGU2015-14690

[19] M Nikolopoulou N Pasadakis and N Kalogerakis ldquoEvalu-ation of autochthonous bioaugmentation and biostimulationduring microcosm-simulated oil spillsrdquo Marine Pollution Bul-letin vol 72 no 1 pp 165ndash173 2013

[20] A Ueno Y Ito I Yumoto and H Okuyama ldquoIsolation andcharacterization of bacteria from soil contaminated with dieseloil and the possible use of these in autochthonous bioaugmenta-tionrdquoWorld Journal of Microbiology and Biotechnology vol 23no 12 pp 1739ndash1745 2007

[21] S K Shekhar J Godheja and D R Modi ldquoHydrocarbon bio-remediation efficiency by five indigenous bacterial strains iso-lated from contaminated soilsrdquo International Journal of CurrentMicrobiology and Applied Sciences vol 4 no 3 pp 892ndash9052015

[22] J K Nduka L N Umeh I O Okerulu et al ldquoUtilization ofdifferent microbes in bioremediation of hydrocarbon contam-inated soils stimulated with inorganic and organic fertilizersrdquoJournal of Petroleum amp Environmental Biotechnology vol 3 no2 p 116 2012

[23] E Dıaz A Ferrandez M A Prieto and J L Garcıa ldquoBiodegra-dation of aromatic compounds by Escherichia colirdquo Microbiol-ogy and Molecular Biology Reviews vol 65 no 4 pp 523ndash5692001

8 BioMed Research International

[24] G Zafra A E Absalon M D C Cuevas and D V Cortes-Espinosa ldquoIsolation and selection of a highly tolerant microbialconsortium with potential for PAH biodegradation from heavycrude oil-contaminated soilsrdquoWater Air and Soil Pollution vol225 no 2 article 1826 2014

[25] S E Hassan S E Desouky A Fouda M El-Gamal and AAlemam ldquoBiodegradation of phenanthrene by klebsiella sp iso-lated fromorganic contaminated sedimentrdquo Journal of Advancesin Biology amp Biotechnology vol 4 no 4 pp 1ndash12 2015

[26] J Godheja S K Shekhar and D R Modi ldquoBiodegradationof one ring hydrocarbons (benzene and toluene) and two ringhydrocarbons (acenapthene and napthalene) by bacterial iso-lates of hydrocarbon contaminated sites located in chhattisgarha preliminary studyrdquo Journal of Petroleum amp EnvironmentalBiotechnology vol 6 no 1 2015

[27] S Ramasamy A Arumugam and P Chandran ldquoOptimizationof Enterobacter cloacae (KU923381) for diesel oil degradationusing response surface methodology (RSM)rdquo Journal of Micro-biology vol 55 no 2 pp 104ndash111 2017

[28] R Akpe A O Ekundayo S P Aigere et al ldquoBacterial degra-dation of petroleum hydrocarbons in crude oil polluted soilamended with cassava peelsrdquo American Journal of ResearchCommunication vol 3 pp 99ndash118 2015

[29] L U Obi H I Atagana and R A Adeleke ldquoIsolation and cha-racterisation of crude oil sludge degrading bacteriardquo Springer-Plus vol 5 no 1 article 1946 2016

[30] F A Ikuesan B E Boboye and F C Adetuyi ldquoComparativebioremediation of crude oil - contaminated soil samples usingactivated soil and activated cow dungrdquo Sky Journal of Microbi-ology Research vol 4 pp 021ndash030 2016

[31] G Ghoreishi A Alemzadeh M Mojarrad and M DjavaherildquoBioremediation capability and characterization of bacteriaisolated frompetroleumcontaminated soils in Iranrdquo SustainableEnvironment Research vol 27 no 4 pp 195ndash202 2017

[32] D J Leininger J R Roberson and F Elvinger ldquoUse of eosinmethylene blue agar to differentiate Escherichia coli from othergram-negative mastitis pathogensrdquo Journal of Veterinary Diag-nostic Investigation vol 13 no 3 pp 273ndash275 2001

[33] M Levine ldquoDifferentiation of E Coli and A Aerogens on a sim-plified eosin-methylene blue agarrdquo Journal of InfectiousDiseasesvol 23 no 1 pp 43ndash47 1918

[34] C M Santegoeds T G Ferdelman G Muyzer and D DeBeer ldquoStructural and functional dynamics of sulfate-reducingpopulations in bacterial biofilmsrdquo Applied and EnvironmentalMicrobiology vol 64 no 10 pp 3731ndash3739 1998

[35] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[36] D L Swafford PAUP Phylogenetic Analysis Using Parasimanyand Other Method Version 4 USA Sinauer Association Sun-derland MA USA 1998

[37] 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

[38] N Dashti N Ali M Khanafer and S S Radwan ldquoOil uptake byplant-based sorbents and its biodegradation by their naturallyassociated microorganismsrdquo Environmental Pollution vol 227pp 468ndash475 2017

[39] I Esteve E Montesinos J G Mitchell and R Guerrero ldquoAquantitative ultrastructural study of Chromatium minus in thebacterial layer of Lake Ciso (Spain)rdquo Archives of Microbiologyvol 153 no 4 pp 316ndash323 1990

[40] E Diestra A Sole MMartı T Garcia De Oteyza J O Grimaltand I Esteve ldquoCharacterization of an oil-degrading Micro-coleus consortium by means of confocal scanning microscopyscanning electron microscopy and transmission electronmicroscopyrdquo Scanning vol 27 no 4 pp 176ndash180 2005

[41] E S Reynolds ldquoThe use of lead citrate at high pH as anelectron-opaque stain in electron microscopyrdquo The Journal ofCell Biology vol 17 pp 208ndash212 1963

[42] H J Rehm and I Reiff ldquoMechanisms and occurrence of micro-bial oxidation of long-chain alkanesrdquo Advances in BiochemicalEngineering vol 19 pp 175ndash215 1981

[43] Ratledge ldquoDegradation of aliphatic hydrocarbonsrdquo in Develop-ments in Biodeterioration of Hydrocarbons I Watkinson Edvol 1 pp 1ndash44 Applied Science Essex 1978

[44] S S Radwan and N A Sorkhoh ldquoLipids of n-alkane-utilizingmicroorganisms and their application potentialrdquo Advances inApplied Microbiology vol 39 pp 29ndash90 1993

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

Table 3 Crude oil and pure-hydrocarbon consumption by coliform isolates

Isolates hydrocarbon consumptionCrude oil C16 Phenanthrene

Escherichia coli (wheat straw) 183 plusmn 11 171 plusmn 10 163 plusmn 09Escherichia coli (domestic sewage) 222 plusmn 32 190 plusmn 22 181 plusmn 30Klebsiella variicola 166 plusmn 10 123 plusmn 08 110 plusmn 05Klebsiella pneumoniae 142 plusmn 12 106 plusmn 08 109 plusmn 10Enterobacter hormaechei subsp oharae 303 plusmn 15 359 plusmn 20 185 plusmn 08Pseudomonas stutzeri 156 plusmn 09 184 plusmn 08 141 plusmn 07Pseudomonas stutzeri from our private culture collection was analyzed as a classic hydrocarbon-utilizer for the purpose of comparison

(a) (b)

(c) (d)

Figure 2 Transmission electronmicrographs of ultrathin sections of E coli cells from domestic sewage grown in 119899-hexadecane-free medium(a) and in 119899-hexadecane-containing medium (b) and of E coli cells from wheat straw grown in 119899-hexadecane-free medium (c) and in 119899-hexadecane-containing medium (d) Magnification (a) and (b) times60000 (c) and (d) times40000

proportions of the available crude oil as well as of the purealiphatic (C

16) and aromatic (phenanthrene) hydrocarbons in

15 days Those hydrocarbons were provided in the mineralmedium [37] as sole sources of carbon and energy Thesequantitative determinations provide a clear-cut evidence forthe hydrocarbonoclastic potential of the studied coliforms

33 Cytological Evidence for 119899-Hexadecane (11986216) Uptake by Ecoli For this experiment two strains of the typical coliformE coli one from sewage and the other from wheat straw[38] were used The TEM images in Figure 2 show thatthe cell cytoplasm of hydrocarbon-incubated cells in con-trast to that of cells harvested from nutrient broth (withouthydrocarbons) was enriched with less electron-dense areas

(hydrocarbon inclusions) of varying dimensions Our mea-surements showed that those inclusions occupied roughly50 of the total cell volumes Earlier researchers alsodescribed similar intracytoplasmic hydrocarbon inclusionsin classical hydrocarbonoclastic bacteria incubated in mediacontaining 119899-hexadecane [37] Cells incubated in the pres-ence of C

16also showed clear morphological deformations

34 Self-Cleaning ofOily Sewage by IndigenousColiforms Theresults in Figure 3 show that crude oil C18 and phenan-threne added to freshly harvested raw sewage (not previouslysterilized) were steadily removed during the 20 d incubationperiod At the end the remaining proportions were only 70of 3 crude oil 45 of 1 crude oil 66 of C18 and 55

6 BioMed Research International

0 5 10 15 20

3 crude oil

Phenanthrene

0 5 10 15 20

1 crude oil

0

20

40

60

80

100

120

cr

ude o

il co

ncen

trat

ion

0

20

40

60

80

100

120

cr

ude o

il co

ncen

trat

ion

0

5

10

15

20

25

30

(Col

ony

form

ing

units

times103

mlminus

1)

0

10

20

30

40

50

60

(Col

ony

form

ing

units

times103

mlminus

1)

0

2

4

6

8

10

12

14

16

18

(Col

ony

form

ing

units

times103

mlminus

1)

0

20

40

60

80

100

120

p

hena

nthr

ene c

once

ntra

tion

0

2

4

6

8

10

12

14

16

18

20(C

olon

y fo

rmin

g un

itstimes103

mlminus

1)

18

0

20

40

60

80

100

120

18

conc

entr

atio

n

5 10 15 200Incubation period (days)

5 10 15 200Incubation period (days)

Total CFU mＦminus1

CFU of Enterobacter

CFU of Klebsiella

CFU of Escherichiaspp mＦ

minus1

spp mＦminus1

spp mＦminus1 spp mＦ

minus1

spp mＦminus1

spp mＦminus1phenanthrene percent

Total CFU mＦminus1

CFU of Enterobacter

CFU of Klebsiella

CFU of Escherichiaphenanthrene percent

Crude oil 18 or Crude oil 18 or

Figure 3 Consumption of crude oil added to freshly harvested sewage and parallel increase of total coliform members with time

of phenanthrene based on the amount added at time zeroMeanwhile there was a parallel increase of the total numbersof the total coliforms from 146 times 106 to 265 times 106 CFUmlminus1in 3 crude oil to 527 times 106 CFUmlminus1 in 1 crude oil to189 times 106 CFUmlminus1 in C18 and to 158 times 106 CFUmlminus1 inphenanthrene Figure 3 shows further that Enterobacter wasthe coliform that exhibited the highest increase in numberwhich may reflect a main role of this coliform in oil con-sumption Other hydrocarbonoclastic noncoliform bacteriaindigenous to sewage are also probably involved in the mea-sured crude oil removal However the significant increase(119901 lt 005) in numbers of coliforms parallel to the signi-ficant decrease (119901 lt 005) in the concentration of the remain-ing oil provides an experimental evidence for a major con-tribution of indigenous coliforms to self-cleaning of oil in

sewage This finding implies that the indigenous domesticsewage microflora is capable of controlling potential oil spillsin this waste product Furthermore raw sewage is an acces-sible cost-effective source of hydrocarbonoclastic micro-bial cocktails that can be used for bioaugmenting oil-con-taminated areas

4 Conclusions

The results of this paper provide experimental evidence forthe role of coliforms in biodegradation of oil spilled in theenvironment This group should be particularly effective incleaning oil spills in sewage where those organisms areautochthonous (indigenous) Conditions prevailing duringconventional sewage treatment probably bioenhance the

BioMed Research International 7

coliforms potential for oil-bioremediation On one handsewage treatment involves the routine practice of extensiveaeration to reduce the sewage BOD values (ie the organicmatter content) In view of the fact that the initial step ofmicrobial attack on hydrocarbons is catalyzed by oxygenases(also called hydroxylases) aeration should bioenhance thespilled-oil bioremediation Oil-bioremediation is also bioen-hanced by nitrogen-fertilizers During sewage treatmentnitrates and other nitrogenous compounds are continuouslyproduced and in addition several coliforms for exampleKlebsiella spp and Enterobacter spp are diazotrophic Inother words nitrogen fertilization is also guaranteed insewage for the hydrocarbonoclastic coliform communities

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

Thiswork has been supported by Kuwait University ResearchGrant SL0616 Thanks are due to SAF Analysis Facilityfor providing Genetic Analysis through Project GS0102 toNUERS for providing GLC analysis through Project SRUL0113 and to the Nanoscopy Science Center for the technicalassistance with transmission electron microscopy

References

[1] National Research Council (NRC) Using Oil Spill Dispersantson the Sea National Academy Press Washington DC WashUSA 1989

[2] A A Al-Majed A R Adebayo and M E Hossain ldquoA sustain-able approach to controlling oil spillsrdquo Journal of EnvironmentalManagement vol 113 pp 213ndash227 2012

[3] A Bayat S F Aghamiri A Moheb and G R Vakili-NezhaadldquoOil spill cleanup from seawater by sorbentmaterialsrdquoChemicalEngineering and Technology vol 28 no 12 pp 1525ndash1528 2005

[4] D Sundaravadivelu M T Suidan A D Venosa and P IRosales ldquoCharacterization of solidifiers used for oil spill reme-diationrdquo Chemosphere vol 144 pp 1490ndash1497 2016

[5] W Dahl R R Lessard E Cardello et al ldquoSolidifiers for oil spillresponserdquo in Proceedings of the SPE Health Safety and Environ-ment in Oil and Gas Exploration and Production Conference pp803ndash810 New Orleans LA USA 1996

[6] L Graham C Hale EMaung-Douglass et al ldquoOil spill scienceChemical dispersants and their role in oil spill responserdquoMASGP-15-015 2016

[7] A Zolfaghari-Baghbaderani M Emtyazjoo P Poursafa et alldquoEffects of three types of oil dispersants on biodegradationof dispersed crude oil in water surrounding two Persian Gulfprovincesrdquo Journal of Environmental and Public Health vol2012 Article ID 981365 8 pages 2012

[8] C Ren G H B Ng H Wu et al ldquoInstant room-temperaturegelation of crude oil by chiral organogelatorsrdquo Chemistry ofMaterials vol 28 no 11 pp 4001ndash4008 2016

[9] J Bachl S Oehm J Mayr C Cativiela J J Marrero-Telladoand D D Dıaz ldquoSupramolecular phase-selective gelation bypeptides bearing side-chain azobenzenes effect of ultrasoundand potential for dye removal and oil spill remediationrdquo

International Journal of Molecular Sciences vol 16 no 5 pp11766ndash11784 2015

[10] E E Cordes D O Jones T A Schlacher et al ldquoEnvironmentalimpacts of the deep-water oil and gas industry a review to guidemanagement strategiesrdquo Frontiers in Environmental Science vol4 2016

[11] R M Atlas and R Bartha Microbial Ecology Fundamentalsand Applications vol 70 BenjaminCummings PublishingCompany Inc 4th edition 1998

[12] A Szulc D Ambrozewicz M Sydow et al ldquoThe influence ofbioaugmentation and biosurfactant addition on bioremediationefficiency of diesel-oil contaminated soil feasibility during fieldstudiesrdquo Journal of Environmental Management vol 132 pp121ndash128 2014

[13] I Kuiper E L Lagendijk G V Bloemberg and B J J Lugten-berg ldquoRhizoremediation a beneficial plant-microbe interac-tionrdquoMolecular Plant-Microbe Interactions vol 17 no 1 pp 6ndash15 2004

[14] H Van Limbergen E M Top and W Verstraete ldquoBioaug-mentation in activated sludge current features add futureperspectivesrdquo Applied Microbiology and Biotechnology vol 50no 1 pp 16ndash23 1998

[15] Ł Ławniczak R Marecik and Ł Chrzanowski ldquoContributionsof biosurfactants to natural or induced bioremediationrdquoAppliedMicrobiology and Biotechnology vol 97 no 6 pp 2327ndash23392013

[16] S S Radwan N A Sorkhoh I M El-Nemr and A F El-Desouky ldquoA feasibility study on seeding as a bioremediationpractice for the oilyKuwaiti desertrdquo Journal of AppliedMicrobio-logy vol 83 no 3 pp 353ndash358 1997

[17] S S Radwan ldquoGulf oil spillrdquoNature vol 350 no 6318 pp 456ndash460 1991

[18] S Di Gregorio M R Castglione A Gentini and R LorenzildquoBiostimulation of the autochthonous bacterial community andbioaugmentation of selected bacterial strains for the depletionof polycyclic aromatic hydrocarbons in a historically con-taminated soilrdquo Geophysical Research Abstracts vol 17 2015EGU2015-14690

[19] M Nikolopoulou N Pasadakis and N Kalogerakis ldquoEvalu-ation of autochthonous bioaugmentation and biostimulationduring microcosm-simulated oil spillsrdquo Marine Pollution Bul-letin vol 72 no 1 pp 165ndash173 2013

[20] A Ueno Y Ito I Yumoto and H Okuyama ldquoIsolation andcharacterization of bacteria from soil contaminated with dieseloil and the possible use of these in autochthonous bioaugmenta-tionrdquoWorld Journal of Microbiology and Biotechnology vol 23no 12 pp 1739ndash1745 2007

[21] S K Shekhar J Godheja and D R Modi ldquoHydrocarbon bio-remediation efficiency by five indigenous bacterial strains iso-lated from contaminated soilsrdquo International Journal of CurrentMicrobiology and Applied Sciences vol 4 no 3 pp 892ndash9052015

[22] J K Nduka L N Umeh I O Okerulu et al ldquoUtilization ofdifferent microbes in bioremediation of hydrocarbon contam-inated soils stimulated with inorganic and organic fertilizersrdquoJournal of Petroleum amp Environmental Biotechnology vol 3 no2 p 116 2012

[23] E Dıaz A Ferrandez M A Prieto and J L Garcıa ldquoBiodegra-dation of aromatic compounds by Escherichia colirdquo Microbiol-ogy and Molecular Biology Reviews vol 65 no 4 pp 523ndash5692001

8 BioMed Research International

[24] G Zafra A E Absalon M D C Cuevas and D V Cortes-Espinosa ldquoIsolation and selection of a highly tolerant microbialconsortium with potential for PAH biodegradation from heavycrude oil-contaminated soilsrdquoWater Air and Soil Pollution vol225 no 2 article 1826 2014

[25] S E Hassan S E Desouky A Fouda M El-Gamal and AAlemam ldquoBiodegradation of phenanthrene by klebsiella sp iso-lated fromorganic contaminated sedimentrdquo Journal of Advancesin Biology amp Biotechnology vol 4 no 4 pp 1ndash12 2015

[26] J Godheja S K Shekhar and D R Modi ldquoBiodegradationof one ring hydrocarbons (benzene and toluene) and two ringhydrocarbons (acenapthene and napthalene) by bacterial iso-lates of hydrocarbon contaminated sites located in chhattisgarha preliminary studyrdquo Journal of Petroleum amp EnvironmentalBiotechnology vol 6 no 1 2015

[27] S Ramasamy A Arumugam and P Chandran ldquoOptimizationof Enterobacter cloacae (KU923381) for diesel oil degradationusing response surface methodology (RSM)rdquo Journal of Micro-biology vol 55 no 2 pp 104ndash111 2017

[28] R Akpe A O Ekundayo S P Aigere et al ldquoBacterial degra-dation of petroleum hydrocarbons in crude oil polluted soilamended with cassava peelsrdquo American Journal of ResearchCommunication vol 3 pp 99ndash118 2015

[29] L U Obi H I Atagana and R A Adeleke ldquoIsolation and cha-racterisation of crude oil sludge degrading bacteriardquo Springer-Plus vol 5 no 1 article 1946 2016

[30] F A Ikuesan B E Boboye and F C Adetuyi ldquoComparativebioremediation of crude oil - contaminated soil samples usingactivated soil and activated cow dungrdquo Sky Journal of Microbi-ology Research vol 4 pp 021ndash030 2016

[31] G Ghoreishi A Alemzadeh M Mojarrad and M DjavaherildquoBioremediation capability and characterization of bacteriaisolated frompetroleumcontaminated soils in Iranrdquo SustainableEnvironment Research vol 27 no 4 pp 195ndash202 2017

[32] D J Leininger J R Roberson and F Elvinger ldquoUse of eosinmethylene blue agar to differentiate Escherichia coli from othergram-negative mastitis pathogensrdquo Journal of Veterinary Diag-nostic Investigation vol 13 no 3 pp 273ndash275 2001

[33] M Levine ldquoDifferentiation of E Coli and A Aerogens on a sim-plified eosin-methylene blue agarrdquo Journal of InfectiousDiseasesvol 23 no 1 pp 43ndash47 1918

[34] C M Santegoeds T G Ferdelman G Muyzer and D DeBeer ldquoStructural and functional dynamics of sulfate-reducingpopulations in bacterial biofilmsrdquo Applied and EnvironmentalMicrobiology vol 64 no 10 pp 3731ndash3739 1998

[35] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[36] D L Swafford PAUP Phylogenetic Analysis Using Parasimanyand Other Method Version 4 USA Sinauer Association Sun-derland MA USA 1998

[37] 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

[38] N Dashti N Ali M Khanafer and S S Radwan ldquoOil uptake byplant-based sorbents and its biodegradation by their naturallyassociated microorganismsrdquo Environmental Pollution vol 227pp 468ndash475 2017

[39] I Esteve E Montesinos J G Mitchell and R Guerrero ldquoAquantitative ultrastructural study of Chromatium minus in thebacterial layer of Lake Ciso (Spain)rdquo Archives of Microbiologyvol 153 no 4 pp 316ndash323 1990

[40] E Diestra A Sole MMartı T Garcia De Oteyza J O Grimaltand I Esteve ldquoCharacterization of an oil-degrading Micro-coleus consortium by means of confocal scanning microscopyscanning electron microscopy and transmission electronmicroscopyrdquo Scanning vol 27 no 4 pp 176ndash180 2005

[41] E S Reynolds ldquoThe use of lead citrate at high pH as anelectron-opaque stain in electron microscopyrdquo The Journal ofCell Biology vol 17 pp 208ndash212 1963

[42] H J Rehm and I Reiff ldquoMechanisms and occurrence of micro-bial oxidation of long-chain alkanesrdquo Advances in BiochemicalEngineering vol 19 pp 175ndash215 1981

[43] Ratledge ldquoDegradation of aliphatic hydrocarbonsrdquo in Develop-ments in Biodeterioration of Hydrocarbons I Watkinson Edvol 1 pp 1ndash44 Applied Science Essex 1978

[44] S S Radwan and N A Sorkhoh ldquoLipids of n-alkane-utilizingmicroorganisms and their application potentialrdquo Advances inApplied Microbiology vol 39 pp 29ndash90 1993

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

0 5 10 15 20

3 crude oil

Phenanthrene

0 5 10 15 20

1 crude oil

0

20

40

60

80

100

120

cr

ude o

il co

ncen

trat

ion

0

20

40

60

80

100

120

cr

ude o

il co

ncen

trat

ion

0

5

10

15

20

25

30

(Col

ony

form

ing

units

times103

mlminus

1)

0

10

20

30

40

50

60

(Col

ony

form

ing

units

times103

mlminus

1)

0

2

4

6

8

10

12

14

16

18

(Col

ony

form

ing

units

times103

mlminus

1)

0

20

40

60

80

100

120

p

hena

nthr

ene c

once

ntra

tion

0

2

4

6

8

10

12

14

16

18

20(C

olon

y fo

rmin

g un

itstimes103

mlminus

1)

18

0

20

40

60

80

100

120

18

conc

entr

atio

n

5 10 15 200Incubation period (days)

5 10 15 200Incubation period (days)

Total CFU mＦminus1

CFU of Enterobacter

CFU of Klebsiella

CFU of Escherichiaspp mＦ

minus1

spp mＦminus1

spp mＦminus1 spp mＦ

minus1

spp mＦminus1

spp mＦminus1phenanthrene percent

Total CFU mＦminus1

CFU of Enterobacter

CFU of Klebsiella

CFU of Escherichiaphenanthrene percent

Crude oil 18 or Crude oil 18 or

Figure 3 Consumption of crude oil added to freshly harvested sewage and parallel increase of total coliform members with time

of phenanthrene based on the amount added at time zeroMeanwhile there was a parallel increase of the total numbersof the total coliforms from 146 times 106 to 265 times 106 CFUmlminus1in 3 crude oil to 527 times 106 CFUmlminus1 in 1 crude oil to189 times 106 CFUmlminus1 in C18 and to 158 times 106 CFUmlminus1 inphenanthrene Figure 3 shows further that Enterobacter wasthe coliform that exhibited the highest increase in numberwhich may reflect a main role of this coliform in oil con-sumption Other hydrocarbonoclastic noncoliform bacteriaindigenous to sewage are also probably involved in the mea-sured crude oil removal However the significant increase(119901 lt 005) in numbers of coliforms parallel to the signi-ficant decrease (119901 lt 005) in the concentration of the remain-ing oil provides an experimental evidence for a major con-tribution of indigenous coliforms to self-cleaning of oil in

sewage This finding implies that the indigenous domesticsewage microflora is capable of controlling potential oil spillsin this waste product Furthermore raw sewage is an acces-sible cost-effective source of hydrocarbonoclastic micro-bial cocktails that can be used for bioaugmenting oil-con-taminated areas

4 Conclusions

The results of this paper provide experimental evidence forthe role of coliforms in biodegradation of oil spilled in theenvironment This group should be particularly effective incleaning oil spills in sewage where those organisms areautochthonous (indigenous) Conditions prevailing duringconventional sewage treatment probably bioenhance the

BioMed Research International 7

coliforms potential for oil-bioremediation On one handsewage treatment involves the routine practice of extensiveaeration to reduce the sewage BOD values (ie the organicmatter content) In view of the fact that the initial step ofmicrobial attack on hydrocarbons is catalyzed by oxygenases(also called hydroxylases) aeration should bioenhance thespilled-oil bioremediation Oil-bioremediation is also bioen-hanced by nitrogen-fertilizers During sewage treatmentnitrates and other nitrogenous compounds are continuouslyproduced and in addition several coliforms for exampleKlebsiella spp and Enterobacter spp are diazotrophic Inother words nitrogen fertilization is also guaranteed insewage for the hydrocarbonoclastic coliform communities

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

Thiswork has been supported by Kuwait University ResearchGrant SL0616 Thanks are due to SAF Analysis Facilityfor providing Genetic Analysis through Project GS0102 toNUERS for providing GLC analysis through Project SRUL0113 and to the Nanoscopy Science Center for the technicalassistance with transmission electron microscopy

References

[1] National Research Council (NRC) Using Oil Spill Dispersantson the Sea National Academy Press Washington DC WashUSA 1989

[2] A A Al-Majed A R Adebayo and M E Hossain ldquoA sustain-able approach to controlling oil spillsrdquo Journal of EnvironmentalManagement vol 113 pp 213ndash227 2012

[3] A Bayat S F Aghamiri A Moheb and G R Vakili-NezhaadldquoOil spill cleanup from seawater by sorbentmaterialsrdquoChemicalEngineering and Technology vol 28 no 12 pp 1525ndash1528 2005

[4] D Sundaravadivelu M T Suidan A D Venosa and P IRosales ldquoCharacterization of solidifiers used for oil spill reme-diationrdquo Chemosphere vol 144 pp 1490ndash1497 2016

[5] W Dahl R R Lessard E Cardello et al ldquoSolidifiers for oil spillresponserdquo in Proceedings of the SPE Health Safety and Environ-ment in Oil and Gas Exploration and Production Conference pp803ndash810 New Orleans LA USA 1996

[6] L Graham C Hale EMaung-Douglass et al ldquoOil spill scienceChemical dispersants and their role in oil spill responserdquoMASGP-15-015 2016

[7] A Zolfaghari-Baghbaderani M Emtyazjoo P Poursafa et alldquoEffects of three types of oil dispersants on biodegradationof dispersed crude oil in water surrounding two Persian Gulfprovincesrdquo Journal of Environmental and Public Health vol2012 Article ID 981365 8 pages 2012

[8] C Ren G H B Ng H Wu et al ldquoInstant room-temperaturegelation of crude oil by chiral organogelatorsrdquo Chemistry ofMaterials vol 28 no 11 pp 4001ndash4008 2016

[9] J Bachl S Oehm J Mayr C Cativiela J J Marrero-Telladoand D D Dıaz ldquoSupramolecular phase-selective gelation bypeptides bearing side-chain azobenzenes effect of ultrasoundand potential for dye removal and oil spill remediationrdquo

International Journal of Molecular Sciences vol 16 no 5 pp11766ndash11784 2015

[10] E E Cordes D O Jones T A Schlacher et al ldquoEnvironmentalimpacts of the deep-water oil and gas industry a review to guidemanagement strategiesrdquo Frontiers in Environmental Science vol4 2016

[11] R M Atlas and R Bartha Microbial Ecology Fundamentalsand Applications vol 70 BenjaminCummings PublishingCompany Inc 4th edition 1998

[12] A Szulc D Ambrozewicz M Sydow et al ldquoThe influence ofbioaugmentation and biosurfactant addition on bioremediationefficiency of diesel-oil contaminated soil feasibility during fieldstudiesrdquo Journal of Environmental Management vol 132 pp121ndash128 2014

[13] I Kuiper E L Lagendijk G V Bloemberg and B J J Lugten-berg ldquoRhizoremediation a beneficial plant-microbe interac-tionrdquoMolecular Plant-Microbe Interactions vol 17 no 1 pp 6ndash15 2004

[14] H Van Limbergen E M Top and W Verstraete ldquoBioaug-mentation in activated sludge current features add futureperspectivesrdquo Applied Microbiology and Biotechnology vol 50no 1 pp 16ndash23 1998

[15] Ł Ławniczak R Marecik and Ł Chrzanowski ldquoContributionsof biosurfactants to natural or induced bioremediationrdquoAppliedMicrobiology and Biotechnology vol 97 no 6 pp 2327ndash23392013

[16] S S Radwan N A Sorkhoh I M El-Nemr and A F El-Desouky ldquoA feasibility study on seeding as a bioremediationpractice for the oilyKuwaiti desertrdquo Journal of AppliedMicrobio-logy vol 83 no 3 pp 353ndash358 1997

[17] S S Radwan ldquoGulf oil spillrdquoNature vol 350 no 6318 pp 456ndash460 1991

[18] S Di Gregorio M R Castglione A Gentini and R LorenzildquoBiostimulation of the autochthonous bacterial community andbioaugmentation of selected bacterial strains for the depletionof polycyclic aromatic hydrocarbons in a historically con-taminated soilrdquo Geophysical Research Abstracts vol 17 2015EGU2015-14690

[19] M Nikolopoulou N Pasadakis and N Kalogerakis ldquoEvalu-ation of autochthonous bioaugmentation and biostimulationduring microcosm-simulated oil spillsrdquo Marine Pollution Bul-letin vol 72 no 1 pp 165ndash173 2013

[20] A Ueno Y Ito I Yumoto and H Okuyama ldquoIsolation andcharacterization of bacteria from soil contaminated with dieseloil and the possible use of these in autochthonous bioaugmenta-tionrdquoWorld Journal of Microbiology and Biotechnology vol 23no 12 pp 1739ndash1745 2007

[21] S K Shekhar J Godheja and D R Modi ldquoHydrocarbon bio-remediation efficiency by five indigenous bacterial strains iso-lated from contaminated soilsrdquo International Journal of CurrentMicrobiology and Applied Sciences vol 4 no 3 pp 892ndash9052015

[22] J K Nduka L N Umeh I O Okerulu et al ldquoUtilization ofdifferent microbes in bioremediation of hydrocarbon contam-inated soils stimulated with inorganic and organic fertilizersrdquoJournal of Petroleum amp Environmental Biotechnology vol 3 no2 p 116 2012

[23] E Dıaz A Ferrandez M A Prieto and J L Garcıa ldquoBiodegra-dation of aromatic compounds by Escherichia colirdquo Microbiol-ogy and Molecular Biology Reviews vol 65 no 4 pp 523ndash5692001

8 BioMed Research International

[24] G Zafra A E Absalon M D C Cuevas and D V Cortes-Espinosa ldquoIsolation and selection of a highly tolerant microbialconsortium with potential for PAH biodegradation from heavycrude oil-contaminated soilsrdquoWater Air and Soil Pollution vol225 no 2 article 1826 2014

[25] S E Hassan S E Desouky A Fouda M El-Gamal and AAlemam ldquoBiodegradation of phenanthrene by klebsiella sp iso-lated fromorganic contaminated sedimentrdquo Journal of Advancesin Biology amp Biotechnology vol 4 no 4 pp 1ndash12 2015

[26] J Godheja S K Shekhar and D R Modi ldquoBiodegradationof one ring hydrocarbons (benzene and toluene) and two ringhydrocarbons (acenapthene and napthalene) by bacterial iso-lates of hydrocarbon contaminated sites located in chhattisgarha preliminary studyrdquo Journal of Petroleum amp EnvironmentalBiotechnology vol 6 no 1 2015

[27] S Ramasamy A Arumugam and P Chandran ldquoOptimizationof Enterobacter cloacae (KU923381) for diesel oil degradationusing response surface methodology (RSM)rdquo Journal of Micro-biology vol 55 no 2 pp 104ndash111 2017

[28] R Akpe A O Ekundayo S P Aigere et al ldquoBacterial degra-dation of petroleum hydrocarbons in crude oil polluted soilamended with cassava peelsrdquo American Journal of ResearchCommunication vol 3 pp 99ndash118 2015

[29] L U Obi H I Atagana and R A Adeleke ldquoIsolation and cha-racterisation of crude oil sludge degrading bacteriardquo Springer-Plus vol 5 no 1 article 1946 2016

[30] F A Ikuesan B E Boboye and F C Adetuyi ldquoComparativebioremediation of crude oil - contaminated soil samples usingactivated soil and activated cow dungrdquo Sky Journal of Microbi-ology Research vol 4 pp 021ndash030 2016

[31] G Ghoreishi A Alemzadeh M Mojarrad and M DjavaherildquoBioremediation capability and characterization of bacteriaisolated frompetroleumcontaminated soils in Iranrdquo SustainableEnvironment Research vol 27 no 4 pp 195ndash202 2017

[32] D J Leininger J R Roberson and F Elvinger ldquoUse of eosinmethylene blue agar to differentiate Escherichia coli from othergram-negative mastitis pathogensrdquo Journal of Veterinary Diag-nostic Investigation vol 13 no 3 pp 273ndash275 2001

[33] M Levine ldquoDifferentiation of E Coli and A Aerogens on a sim-plified eosin-methylene blue agarrdquo Journal of InfectiousDiseasesvol 23 no 1 pp 43ndash47 1918

[34] C M Santegoeds T G Ferdelman G Muyzer and D DeBeer ldquoStructural and functional dynamics of sulfate-reducingpopulations in bacterial biofilmsrdquo Applied and EnvironmentalMicrobiology vol 64 no 10 pp 3731ndash3739 1998

[35] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[36] D L Swafford PAUP Phylogenetic Analysis Using Parasimanyand Other Method Version 4 USA Sinauer Association Sun-derland MA USA 1998

[37] 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

[38] N Dashti N Ali M Khanafer and S S Radwan ldquoOil uptake byplant-based sorbents and its biodegradation by their naturallyassociated microorganismsrdquo Environmental Pollution vol 227pp 468ndash475 2017

[39] I Esteve E Montesinos J G Mitchell and R Guerrero ldquoAquantitative ultrastructural study of Chromatium minus in thebacterial layer of Lake Ciso (Spain)rdquo Archives of Microbiologyvol 153 no 4 pp 316ndash323 1990

[40] E Diestra A Sole MMartı T Garcia De Oteyza J O Grimaltand I Esteve ldquoCharacterization of an oil-degrading Micro-coleus consortium by means of confocal scanning microscopyscanning electron microscopy and transmission electronmicroscopyrdquo Scanning vol 27 no 4 pp 176ndash180 2005

[41] E S Reynolds ldquoThe use of lead citrate at high pH as anelectron-opaque stain in electron microscopyrdquo The Journal ofCell Biology vol 17 pp 208ndash212 1963

[42] H J Rehm and I Reiff ldquoMechanisms and occurrence of micro-bial oxidation of long-chain alkanesrdquo Advances in BiochemicalEngineering vol 19 pp 175ndash215 1981

[43] Ratledge ldquoDegradation of aliphatic hydrocarbonsrdquo in Develop-ments in Biodeterioration of Hydrocarbons I Watkinson Edvol 1 pp 1ndash44 Applied Science Essex 1978

[44] S S Radwan and N A Sorkhoh ldquoLipids of n-alkane-utilizingmicroorganisms and their application potentialrdquo Advances inApplied Microbiology vol 39 pp 29ndash90 1993

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

coliforms potential for oil-bioremediation On one handsewage treatment involves the routine practice of extensiveaeration to reduce the sewage BOD values (ie the organicmatter content) In view of the fact that the initial step ofmicrobial attack on hydrocarbons is catalyzed by oxygenases(also called hydroxylases) aeration should bioenhance thespilled-oil bioremediation Oil-bioremediation is also bioen-hanced by nitrogen-fertilizers During sewage treatmentnitrates and other nitrogenous compounds are continuouslyproduced and in addition several coliforms for exampleKlebsiella spp and Enterobacter spp are diazotrophic Inother words nitrogen fertilization is also guaranteed insewage for the hydrocarbonoclastic coliform communities

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

Thiswork has been supported by Kuwait University ResearchGrant SL0616 Thanks are due to SAF Analysis Facilityfor providing Genetic Analysis through Project GS0102 toNUERS for providing GLC analysis through Project SRUL0113 and to the Nanoscopy Science Center for the technicalassistance with transmission electron microscopy

References

[1] National Research Council (NRC) Using Oil Spill Dispersantson the Sea National Academy Press Washington DC WashUSA 1989

[2] A A Al-Majed A R Adebayo and M E Hossain ldquoA sustain-able approach to controlling oil spillsrdquo Journal of EnvironmentalManagement vol 113 pp 213ndash227 2012

[3] A Bayat S F Aghamiri A Moheb and G R Vakili-NezhaadldquoOil spill cleanup from seawater by sorbentmaterialsrdquoChemicalEngineering and Technology vol 28 no 12 pp 1525ndash1528 2005

[4] D Sundaravadivelu M T Suidan A D Venosa and P IRosales ldquoCharacterization of solidifiers used for oil spill reme-diationrdquo Chemosphere vol 144 pp 1490ndash1497 2016

[5] W Dahl R R Lessard E Cardello et al ldquoSolidifiers for oil spillresponserdquo in Proceedings of the SPE Health Safety and Environ-ment in Oil and Gas Exploration and Production Conference pp803ndash810 New Orleans LA USA 1996

[6] L Graham C Hale EMaung-Douglass et al ldquoOil spill scienceChemical dispersants and their role in oil spill responserdquoMASGP-15-015 2016

[7] A Zolfaghari-Baghbaderani M Emtyazjoo P Poursafa et alldquoEffects of three types of oil dispersants on biodegradationof dispersed crude oil in water surrounding two Persian Gulfprovincesrdquo Journal of Environmental and Public Health vol2012 Article ID 981365 8 pages 2012

[8] C Ren G H B Ng H Wu et al ldquoInstant room-temperaturegelation of crude oil by chiral organogelatorsrdquo Chemistry ofMaterials vol 28 no 11 pp 4001ndash4008 2016

[9] J Bachl S Oehm J Mayr C Cativiela J J Marrero-Telladoand D D Dıaz ldquoSupramolecular phase-selective gelation bypeptides bearing side-chain azobenzenes effect of ultrasoundand potential for dye removal and oil spill remediationrdquo

International Journal of Molecular Sciences vol 16 no 5 pp11766ndash11784 2015

[10] E E Cordes D O Jones T A Schlacher et al ldquoEnvironmentalimpacts of the deep-water oil and gas industry a review to guidemanagement strategiesrdquo Frontiers in Environmental Science vol4 2016

[11] R M Atlas and R Bartha Microbial Ecology Fundamentalsand Applications vol 70 BenjaminCummings PublishingCompany Inc 4th edition 1998

[12] A Szulc D Ambrozewicz M Sydow et al ldquoThe influence ofbioaugmentation and biosurfactant addition on bioremediationefficiency of diesel-oil contaminated soil feasibility during fieldstudiesrdquo Journal of Environmental Management vol 132 pp121ndash128 2014

[13] I Kuiper E L Lagendijk G V Bloemberg and B J J Lugten-berg ldquoRhizoremediation a beneficial plant-microbe interac-tionrdquoMolecular Plant-Microbe Interactions vol 17 no 1 pp 6ndash15 2004

[14] H Van Limbergen E M Top and W Verstraete ldquoBioaug-mentation in activated sludge current features add futureperspectivesrdquo Applied Microbiology and Biotechnology vol 50no 1 pp 16ndash23 1998

[15] Ł Ławniczak R Marecik and Ł Chrzanowski ldquoContributionsof biosurfactants to natural or induced bioremediationrdquoAppliedMicrobiology and Biotechnology vol 97 no 6 pp 2327ndash23392013

[16] S S Radwan N A Sorkhoh I M El-Nemr and A F El-Desouky ldquoA feasibility study on seeding as a bioremediationpractice for the oilyKuwaiti desertrdquo Journal of AppliedMicrobio-logy vol 83 no 3 pp 353ndash358 1997

[17] S S Radwan ldquoGulf oil spillrdquoNature vol 350 no 6318 pp 456ndash460 1991

[18] S Di Gregorio M R Castglione A Gentini and R LorenzildquoBiostimulation of the autochthonous bacterial community andbioaugmentation of selected bacterial strains for the depletionof polycyclic aromatic hydrocarbons in a historically con-taminated soilrdquo Geophysical Research Abstracts vol 17 2015EGU2015-14690

[19] M Nikolopoulou N Pasadakis and N Kalogerakis ldquoEvalu-ation of autochthonous bioaugmentation and biostimulationduring microcosm-simulated oil spillsrdquo Marine Pollution Bul-letin vol 72 no 1 pp 165ndash173 2013

[20] A Ueno Y Ito I Yumoto and H Okuyama ldquoIsolation andcharacterization of bacteria from soil contaminated with dieseloil and the possible use of these in autochthonous bioaugmenta-tionrdquoWorld Journal of Microbiology and Biotechnology vol 23no 12 pp 1739ndash1745 2007

[21] S K Shekhar J Godheja and D R Modi ldquoHydrocarbon bio-remediation efficiency by five indigenous bacterial strains iso-lated from contaminated soilsrdquo International Journal of CurrentMicrobiology and Applied Sciences vol 4 no 3 pp 892ndash9052015

[22] J K Nduka L N Umeh I O Okerulu et al ldquoUtilization ofdifferent microbes in bioremediation of hydrocarbon contam-inated soils stimulated with inorganic and organic fertilizersrdquoJournal of Petroleum amp Environmental Biotechnology vol 3 no2 p 116 2012

[23] E Dıaz A Ferrandez M A Prieto and J L Garcıa ldquoBiodegra-dation of aromatic compounds by Escherichia colirdquo Microbiol-ogy and Molecular Biology Reviews vol 65 no 4 pp 523ndash5692001

8 BioMed Research International

[24] G Zafra A E Absalon M D C Cuevas and D V Cortes-Espinosa ldquoIsolation and selection of a highly tolerant microbialconsortium with potential for PAH biodegradation from heavycrude oil-contaminated soilsrdquoWater Air and Soil Pollution vol225 no 2 article 1826 2014

[25] S E Hassan S E Desouky A Fouda M El-Gamal and AAlemam ldquoBiodegradation of phenanthrene by klebsiella sp iso-lated fromorganic contaminated sedimentrdquo Journal of Advancesin Biology amp Biotechnology vol 4 no 4 pp 1ndash12 2015

[26] J Godheja S K Shekhar and D R Modi ldquoBiodegradationof one ring hydrocarbons (benzene and toluene) and two ringhydrocarbons (acenapthene and napthalene) by bacterial iso-lates of hydrocarbon contaminated sites located in chhattisgarha preliminary studyrdquo Journal of Petroleum amp EnvironmentalBiotechnology vol 6 no 1 2015

[27] S Ramasamy A Arumugam and P Chandran ldquoOptimizationof Enterobacter cloacae (KU923381) for diesel oil degradationusing response surface methodology (RSM)rdquo Journal of Micro-biology vol 55 no 2 pp 104ndash111 2017

[28] R Akpe A O Ekundayo S P Aigere et al ldquoBacterial degra-dation of petroleum hydrocarbons in crude oil polluted soilamended with cassava peelsrdquo American Journal of ResearchCommunication vol 3 pp 99ndash118 2015

[29] L U Obi H I Atagana and R A Adeleke ldquoIsolation and cha-racterisation of crude oil sludge degrading bacteriardquo Springer-Plus vol 5 no 1 article 1946 2016

[30] F A Ikuesan B E Boboye and F C Adetuyi ldquoComparativebioremediation of crude oil - contaminated soil samples usingactivated soil and activated cow dungrdquo Sky Journal of Microbi-ology Research vol 4 pp 021ndash030 2016

[31] G Ghoreishi A Alemzadeh M Mojarrad and M DjavaherildquoBioremediation capability and characterization of bacteriaisolated frompetroleumcontaminated soils in Iranrdquo SustainableEnvironment Research vol 27 no 4 pp 195ndash202 2017

[32] D J Leininger J R Roberson and F Elvinger ldquoUse of eosinmethylene blue agar to differentiate Escherichia coli from othergram-negative mastitis pathogensrdquo Journal of Veterinary Diag-nostic Investigation vol 13 no 3 pp 273ndash275 2001

[33] M Levine ldquoDifferentiation of E Coli and A Aerogens on a sim-plified eosin-methylene blue agarrdquo Journal of InfectiousDiseasesvol 23 no 1 pp 43ndash47 1918

[34] C M Santegoeds T G Ferdelman G Muyzer and D DeBeer ldquoStructural and functional dynamics of sulfate-reducingpopulations in bacterial biofilmsrdquo Applied and EnvironmentalMicrobiology vol 64 no 10 pp 3731ndash3739 1998

[35] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[36] D L Swafford PAUP Phylogenetic Analysis Using Parasimanyand Other Method Version 4 USA Sinauer Association Sun-derland MA USA 1998

[37] 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

[38] N Dashti N Ali M Khanafer and S S Radwan ldquoOil uptake byplant-based sorbents and its biodegradation by their naturallyassociated microorganismsrdquo Environmental Pollution vol 227pp 468ndash475 2017

[39] I Esteve E Montesinos J G Mitchell and R Guerrero ldquoAquantitative ultrastructural study of Chromatium minus in thebacterial layer of Lake Ciso (Spain)rdquo Archives of Microbiologyvol 153 no 4 pp 316ndash323 1990

[40] E Diestra A Sole MMartı T Garcia De Oteyza J O Grimaltand I Esteve ldquoCharacterization of an oil-degrading Micro-coleus consortium by means of confocal scanning microscopyscanning electron microscopy and transmission electronmicroscopyrdquo Scanning vol 27 no 4 pp 176ndash180 2005

[41] E S Reynolds ldquoThe use of lead citrate at high pH as anelectron-opaque stain in electron microscopyrdquo The Journal ofCell Biology vol 17 pp 208ndash212 1963

[42] H J Rehm and I Reiff ldquoMechanisms and occurrence of micro-bial oxidation of long-chain alkanesrdquo Advances in BiochemicalEngineering vol 19 pp 175ndash215 1981

[43] Ratledge ldquoDegradation of aliphatic hydrocarbonsrdquo in Develop-ments in Biodeterioration of Hydrocarbons I Watkinson Edvol 1 pp 1ndash44 Applied Science Essex 1978

[44] S S Radwan and N A Sorkhoh ldquoLipids of n-alkane-utilizingmicroorganisms and their application potentialrdquo Advances inApplied Microbiology vol 39 pp 29ndash90 1993

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 BioMed Research International

[24] G Zafra A E Absalon M D C Cuevas and D V Cortes-Espinosa ldquoIsolation and selection of a highly tolerant microbialconsortium with potential for PAH biodegradation from heavycrude oil-contaminated soilsrdquoWater Air and Soil Pollution vol225 no 2 article 1826 2014

[25] S E Hassan S E Desouky A Fouda M El-Gamal and AAlemam ldquoBiodegradation of phenanthrene by klebsiella sp iso-lated fromorganic contaminated sedimentrdquo Journal of Advancesin Biology amp Biotechnology vol 4 no 4 pp 1ndash12 2015

[26] J Godheja S K Shekhar and D R Modi ldquoBiodegradationof one ring hydrocarbons (benzene and toluene) and two ringhydrocarbons (acenapthene and napthalene) by bacterial iso-lates of hydrocarbon contaminated sites located in chhattisgarha preliminary studyrdquo Journal of Petroleum amp EnvironmentalBiotechnology vol 6 no 1 2015

[27] S Ramasamy A Arumugam and P Chandran ldquoOptimizationof Enterobacter cloacae (KU923381) for diesel oil degradationusing response surface methodology (RSM)rdquo Journal of Micro-biology vol 55 no 2 pp 104ndash111 2017

[28] R Akpe A O Ekundayo S P Aigere et al ldquoBacterial degra-dation of petroleum hydrocarbons in crude oil polluted soilamended with cassava peelsrdquo American Journal of ResearchCommunication vol 3 pp 99ndash118 2015

[29] L U Obi H I Atagana and R A Adeleke ldquoIsolation and cha-racterisation of crude oil sludge degrading bacteriardquo Springer-Plus vol 5 no 1 article 1946 2016

[30] F A Ikuesan B E Boboye and F C Adetuyi ldquoComparativebioremediation of crude oil - contaminated soil samples usingactivated soil and activated cow dungrdquo Sky Journal of Microbi-ology Research vol 4 pp 021ndash030 2016

[31] G Ghoreishi A Alemzadeh M Mojarrad and M DjavaherildquoBioremediation capability and characterization of bacteriaisolated frompetroleumcontaminated soils in Iranrdquo SustainableEnvironment Research vol 27 no 4 pp 195ndash202 2017

[32] D J Leininger J R Roberson and F Elvinger ldquoUse of eosinmethylene blue agar to differentiate Escherichia coli from othergram-negative mastitis pathogensrdquo Journal of Veterinary Diag-nostic Investigation vol 13 no 3 pp 273ndash275 2001

[33] M Levine ldquoDifferentiation of E Coli and A Aerogens on a sim-plified eosin-methylene blue agarrdquo Journal of InfectiousDiseasesvol 23 no 1 pp 43ndash47 1918

[34] C M Santegoeds T G Ferdelman G Muyzer and D DeBeer ldquoStructural and functional dynamics of sulfate-reducingpopulations in bacterial biofilmsrdquo Applied and EnvironmentalMicrobiology vol 64 no 10 pp 3731ndash3739 1998

[35] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[36] D L Swafford PAUP Phylogenetic Analysis Using Parasimanyand Other Method Version 4 USA Sinauer Association Sun-derland MA USA 1998

[37] 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

[38] N Dashti N Ali M Khanafer and S S Radwan ldquoOil uptake byplant-based sorbents and its biodegradation by their naturallyassociated microorganismsrdquo Environmental Pollution vol 227pp 468ndash475 2017

[39] I Esteve E Montesinos J G Mitchell and R Guerrero ldquoAquantitative ultrastructural study of Chromatium minus in thebacterial layer of Lake Ciso (Spain)rdquo Archives of Microbiologyvol 153 no 4 pp 316ndash323 1990

[40] E Diestra A Sole MMartı T Garcia De Oteyza J O Grimaltand I Esteve ldquoCharacterization of an oil-degrading Micro-coleus consortium by means of confocal scanning microscopyscanning electron microscopy and transmission electronmicroscopyrdquo Scanning vol 27 no 4 pp 176ndash180 2005

[41] E S Reynolds ldquoThe use of lead citrate at high pH as anelectron-opaque stain in electron microscopyrdquo The Journal ofCell Biology vol 17 pp 208ndash212 1963

[42] H J Rehm and I Reiff ldquoMechanisms and occurrence of micro-bial oxidation of long-chain alkanesrdquo Advances in BiochemicalEngineering vol 19 pp 175ndash215 1981

[43] Ratledge ldquoDegradation of aliphatic hydrocarbonsrdquo in Develop-ments in Biodeterioration of Hydrocarbons I Watkinson Edvol 1 pp 1ndash44 Applied Science Essex 1978

[44] S S Radwan and N A Sorkhoh ldquoLipids of n-alkane-utilizingmicroorganisms and their application potentialrdquo Advances inApplied Microbiology vol 39 pp 29ndash90 1993

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology