Research Article Phylogenetic Framework and Biosurfactant Gene …downloads.hindawi.com/archive/2014/860491.pdf · 2019-07-31 · Research Article Phylogenetic Framework and Biosurfactant

Research ArticlePhylogenetic Framework and BiosurfactantGene Expression Analysis of Marine Bacillus spp ofEastern Coastal Plain of Tamil Nadu

Sreethar Swaathy1 Varadharajan Kavitha1 Arockiasamy Sahaya Pravin1

Ganesan Sekaran2 Asit Baran Mandal1 and Arumugam Gnanamani1

1 Microbiology Division CSIR-Central Leather Research Institute Adyar Chennai Tamil Nadu 600 020 India2 Environmental Technology Division CSIR-Central Leather Research Institute Adyar Chennai Tamil Nadu 600 020 India

Correspondence should be addressed to Arumugam Gnanamani gnanamani3gmailcom

Received 15 October 2013 Accepted 17 December 2013 Published 12 February 2014

Academic Editor Rodrigo E Mendes

Copyright copy 2014 Sreethar Swaathy et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The present study emphasizes the diversity assessment ofmarineBacillus species with special reference to biosurfactant productionrespective gene expression and discrimination among Bacillus licheniformis and Bacillus subtilis Among the 200 individual speciesof eastern coastal plain of Tamil Nadu screened five biosurfactant producing potential bacterial species with entirely differentmorphology were selected Biochemical and 16S rRNA gene sequence analysis suggested that all the said five species belong toBacillus genera but differ in species levels Biosurfactant of all the five species fluctuates in greater levels with respect to activity aswell as to constituents but showed partial similarity to the commercially available surfactinThe expression of srf gene was realizedin all of the five species However the sfp gene expression was observed only in three species In conclusion both B licheniformisand B subtilis demonstrate srf gene nevertheless sfp gene was expressed only by Bacillus subtilis

1 Introduction

Biodiversity is given by a variety of species living on earthresulting from billions of years of evolution Molecular-phylogenetic studies have revealed that the main diversity oflife is microbial and it is distributed among three domainsprokaryotes eukaryotes and archaea The functioning the ofwhole biosphere depends absolutely on the activities of themicrobial world Due to versatility microbes are the majornatural providers of ecological services as well as playingmajor role in semiartificial systems such as sewage treatmentplants landfills and in toxic waste bioremediation Howeverbecause of the continuous exposure to man-made syntheticmolecules a large changereduction in microbial diversityhas been realized Several publications document the effectof chemical pollutants on microbial community structurebased on the available sequencing technologies [1] Metage-nomics metatranscriptomics metaproteomics and single-cell sequencing are the approaches providing a view not only

of the community structure (species phylogeny richness anddistribution) but also of the functional (metabolic) potentialof a community

Among the various sources microbes of marinewaterssludges received good attention because of the widepotential biological products with immense applications[2] Most of the products are used to recover oil [3ndash5] Inaddition the interest in marine bacteria arises from theirbiotechnological potential in bioremediation [6] and inoceanic biogeochemical cycles [7] and as a source of novelmetabolites different from those isolated from terrestrialbacteria such as antibiotics [8] and products for industrialuse like exopolysaccharides enzymes [9] biosurfactants andcompatible solutes [10] Most of the available investigationsfocused on the two groups of bacteria the members of thegenera Bacillus or Pseudomonas due to the resourcefulnessand applications [5 6 11ndash15] With regard to Bacillus generait consists of more than 222 recognized species (httpwwwbacteriocictfr) distributed widely across many terrestrial

Hindawi Publishing CorporationInternational Journal of BacteriologyVolume 2014 Article ID 860491 10 pageshttpdxdoiorg1011552014860491

2 International Journal of Bacteriology

and aquatic habitats [15 16] including marine sediments [17]Members of the genusBacillus comprise Grampositive sporeforming rod-shaped and aerobic bacteria Bacillus speciesare phenotypically and genotypically heterogenous [18]Few studies have focused on the phylogeny and biodiversityof marine Bacilli [15ndash17 19] These reports were based ona small number of isolates or a unique sampling site andshowed that Bacillus subtilis Bacillus licheniformis Bacilluscereus Bacillus amyloliquefaciens and Bacillus pumilusare the common inhabitants of marine environments andconsequently often isolated However a more extensiveanalysis to assess the real biodiversity and phylogeneticrelationship between Bacilli from marine origin is becomingnecessary Recent studies on marine bacilli showed thatstrains of B marinus B subtilis B pumilus B licheniformisB cereus and B mycoides are common inhabitants of themarine habitat [15] They constitute closely related taxa andtheir differentiation has become difficult and laborious asthey cannot be distinguished from each other by phenotypictests

Furthermore with regard to biosurfactant productioncertain strains of the closely related species B subtilis Bmojavensis B sonorensis and B licheniformis produce thecyclic lipopeptide biosurfactants surfactin and lichenysin[20ndash23] B subtilis produces a known lipopeptide biosurfac-tant called surfactin which is coded by four open readingframes (ORFs) named as SrfA SrfB SrfC and SrfD [24]Similarly lichenysin is a lipopeptide biosurfactant producedby B licheniformis coded by lichenysin operon (LchA) andconsists of three peptide synthetase genes LicAA LicABLicAC and LicAD [21] B cereus produces a lipopeptidebiosurfactant called plipastatin [25]

The speciesmost closely related toB subtilis are unusuallysimilar at the phenotypic level For example fatty acidcomposition is the only known phenotypic character thatdistinguishes Bacillus mojavensis and Bacillus vallismortisfrom one another or from B subtilis [26 27] and Bacil-lus atrophaeus is distinguishable from B subtilis only bydifferences in pigmentation [28] A heterogeneous groupof moderately halophilic bacteria which comprises Bacillussalexigens and three species of the new genus HalobacillusH halophilus H litoralis and H trueperi [29ndash31] may bedifferentiated by their ability to grow at 10 to 20 of total saltsand the possession of an unusual type of murein Molecularmethods have proven to be more effective in distinguishingclose relatives of B subtilis

From the above-summarized information it has beenunderstood that discrimination of the Bacillus species basedon the morphology phylogenetic framework and geneexpression of molecule of interest is possible neverthelessthe closely related species with similarity in the byproductsneed intensive explorations

Thus exploring the phylogenetic markers such as 16SrRNA genes to reveal microbial diversity and further explo-ration of gene expression to reveal the functional powerof the microbes is essential Hence in the present studyphylogenetic framework of marine Bacillus spp and expres-sion of gene responsible for biosurfactant production has

been explored in detail which provided interesting infor-mation and highly relevant to identification of novel strainswith desirable functional characteristics and biotechnologicalapplications

2 Materials and Methods

21 Isolation and Maintenance of Organism Marine samplesof water sediments mussels shells sea weeds and sandwere collected from Kalpakkam Ennore port Besant NagarBeach Marina beach Mahabalipuram beach MandapamVedaranyam Tuticorin Cuddalore in Tamil Nadu India(Figure 1) Zobell marine brothagar (for bacteria) was themedia used for the isolation of microbial species accordingto the standard procedure employed for the isolation andmaintenance of marine organisms Morphologically distinctorganisms were selected and stored in 30 glycerol stock atminus20∘C

22 Screening of Biosurfactant Producing Microbes Biosur-factant producers were screened based on the surfactantactivity exhibited by the culture media after 24ndash72 hours ofincubation at 37∘C in the presence of the bacterial isolatesIn brief Zobell marine broth (Hi Media) (100mL in 1000mLcapacity conical flask) was sterilized and inoculated with theisolated cultures and was incubated at 37∘C for 24ndash72 hoursunder shaking condition Followed by incubation the sam-ples weremade cell-free by centrifugation at 10 000timesg at 4∘CBiosurfactants activity of the cell free medium was assessedaccording to the procedure summarized in the followingparagraphs and the isolates exhibiting appreciable surfactantactivity were selected and subjected to identification andfurther production

23 Morphological Physiological and Biochemical Character-istics ofMarine Isolates Thestrains isolated frommarine sed-iments were identified by conventional biochemical tests inaccordance with Bergeyrsquos Manual of Systematic Bacteriology[32]

24 DNA Extraction Genomic DNA was isolated andpurified by ldquoDneasy Tissue Kitrdquo (Qiagen GmgH HildenGermany) according to the manufacturerrsquos directions Thequality of the extracted DNA was checked by agarose gelelectrophoresis 2 (wv)

25 PCR Amplification 16S rRNA Gene Sequencing Toconfirm the identities of the isolates PCR amplificationand sequencing of the 16S rRNA gene were performedThe 16S rRNA genes (1500 bp) were purified using thegel elution kit (Sigma-Aldrich USA) as per the manu-facturerrsquos protocol using the bacterial universal primer setof 8F 51015840-AGAGTTTGATCCTGGCTCAG-31015840 and 1492R 51015840-GGCTACCTTGTTACGACTT-31015840 as described by Turner etal [33] Amplification was carried out with the eppendorfthermal cycler (Eppendorf North America Inc) in a totalvolume of 25 120583L containing about 50 ng of genomic DNA5U of Taq DNA polymerase 20 pmol of each primer 200 120583M

International Journal of Bacteriology 3

India

80∘E 81

∘E79∘E78

∘E77∘E76

∘E

80∘E 81

∘E79∘E78

∘E77∘E76

∘E

13∘N

12∘N

11∘N

10∘N

9∘N

8∘N

13∘N

12∘N

11∘N

10∘N

9∘N

8∘N

A

BChennai

C

D

E

N

Tamil Nadu

Figure 1 Location of the five sampling sites Site coordinates limits (marine samples of water sediments mussels shells sea weeds and sand)A is Ennore Chennai Tamilnadu B is Mahabalipuram Tamilnadu C is Kalpakkam township Tamilnadu D is Cuddalore Tamilnadu E isMandapam Ramanathapuram Tamilnadu

dNTPs and 1X Taq buffer with 15mM MgCl2according

to the following program 93∘C for 2min 35 cycles of93∘C for 1min 52∘C for 2min 72∘C for 2min and finalextension at 72∘C for 5min PCR products were analyzed byelectrophoresis in 1 agarose

26 Phylogenetic Analysis For phylogenetic identificationthe acquired sequences were used for a gene homology searchwith the 16S rRNA gene sequences available in the publicdatabases obtained fromGenBank using the BLAST program(httpblastncbinlmnihgov) [34] and ribosomal databaseproject (httprdpcmemsueduseqmatchseqmatch introjsp) [35] Sequences retrieved from the GenBankdatabase were aligned using the CLUSTAL omega[36] and tree was performed using three tree-makingalgorithms neighbor-joining using Tamura-Nei distanceparameters in the Geneious R6 program (available fromhttpwwwgeneiouscom)

27 Experimental Setup and Extraction of Surface ActiveAgents All the selected isolates were cultured individuallyin 1000mL Erlenmeyer flask containing 100mL of Zobellmarine broth and incubated at 37∘C for 24ndash48 hours undershaking condition Followed by incubation the cell-freesupernatant was subjected to (equal amount of ice cold)ethanol precipitation according to Vater et al [37] Theresidual pellet was obtained upon centrifugation dissolvedin water subjected to surface activity measurements anddesignated as ldquomicrosurf rdquo

28 Surface Activity Measurements

281 Drop Collapse Test It is a rapid and crude method toassess the surfactant activity according to Jain et al [38]In brief about 10mL of cell free broth was added in thecenter of an oil drop (20120583L of any oil) taken in a clean glassslide The collapse of oil drop has been visualized and theless time taken indicates the higher activity of a surfactantActivity of microbial surfactant was compared with watersynthetic (Tween 80 SDS CTAB) and commercially availablebiosurfactant (Surfactin)

282 Oil Displacement Method This method is so sensitiveand only a small amount of sample was required to measurethe surfactant activity About 10mL of cell-free broth wasplaced in the center of oil (50120583L of any oil) which wasformed on the surface of water in a large size petridish (15 cmdiameter) The size of the resultant oil-displaced circle areareflects the activity of a surfactant The larger the size thehigher the activity of a surfactant [39] A 50 120583L distilled waterwas used for negative control and 50120583L of 01 Triton X 100was used for positive control experiments

283 Tensiometer Measurements Surface tension of bio-surfactant was measured by plate method using GBX-3Stensiometer (DM) at room temperature at different dilutionsranging from 1- to 10-fold [40] A 10mL of the cell free brothwas taken in a clean glass beaker (20mL) and was placedon tensiometer platform A sterile plate was submerged into


a solution and then slowly pulled through the liquid-airinterface Stabilization was allowed to occur until a standarddeviation of 10 successive measurements was lt04mNmEach result was the average of 10 determinations after stabi-lization

29 Assessment of Emulsifying Activity Emulsification Indexand Stability of Emulsion of ldquoMicrosurf (1 to 5)rdquo Followed bythe measurement of biosurfactant activity bioemulsificationactivity was determined using heptadecane (hydrocarbon) assubstrate In brief we mixed 50 120583L of the sample (Microsurf(1 to 5)) with 10mL of sodium acetate buffer (pH 30)then added 2120583L of heptadecane and vortex After 2-3minabsorbance was measured at 540 nm using UV-visible spec-trophotometer (Shimadzu Japan) One unit of emulsifyingactivity was defined as the amount of biosurfactant thataffected an emulsion with an absorbance at 540 nm of 10 Toassess the emulsification index 10ml of cell free broth wasmixed with 10ml of various hydrocarbons (oils of sesamepeanut sunflower soybean rice bran and crude) benzenetoluene and kerosene individually vortex for 30min and keptat room temperature Emulsification index (E24) was calcu-lated after 24 hours bymeasuring the height of emulsion layerwith respect to original volume

For the assessment of stability of emulsion emulsionsobtained from above experiments were kept at room temper-ature for the period of 0ndash100 days Visual observations weremade for any phase separation such as flocculation creamyand coalescence Samples exhibiting nil phase separationwere considered as stable emulsion

210Hemolytic Activity onHumanErythrocytes To assess thehemolytic activity of ldquomicrosurf (1 to 5)rdquo obtained from theabove experiments human RBC cells were used as substrates[41] RBC suspension was incubated in the presence of iso-tonic solution of biosurfactants solution at room temperaturefor 10min in the dark The amount of hemoglobin releasedupon centrifugation (3000timesg) wasmeasured at 540 nm RBClysis with water was taken as 100 and sodium dodecylsulfate (SDS) as reference compound

211 Antimicrobial Activity Antimicrobial activity of theldquomicrosurf (1 to 5)rdquo was made according to the CLSI [42]Standards for Antimicrobial Susceptibility Testing usingGram positive and Gram negative species obtained fromMicrobial Type Culture Collection (MTCC) ChandigarhIn detail Mueller-Hinton agar (MHA) was prepared froma commercially available dehydrated base according to themanufacturerrsquos instructions Followed by sterilization themedia was poured in a glass petridishes The agar mediumwas then allowed to cool to room temperature For antimicro-bial activity of ldquomicrosurf (1 to 5)rdquo 50 120583L of (05McfarlandODof MTCC strains) MTCC strains spread over the MHA plateand 05mmwell was made on the plate and different dilutionof microsurf (1 to 5) was poured in the well and incubated at37∘C

212 Surfactant Gene (srfsfp) Expression Analysis Followedby identification and ldquomicrosurf rdquo production the geneof interest was designed from earlier reports [43] andstudied according to the standard procedures The primerpair srfA3licA3F CAAAAKCGCAKCATATGAG andsrfA3licA3R AGCGGCAYATATTGATGCGGYTC wasdesigned to amplify a 268 bp portion of the srfA3 or thehomologous licA3 gene present in surfactinlichenysin-producing strains of the B subtilisB licheniformis Theseprimers were tested against five Bacillus strains ThesrfA3licA3 PCR reaction mix in a volume of 25120583L reactionmixture consists of 25U of Taq DNA polymerase 100 ngof genomic DNA 20 pmol of each primer 200120583M dNTPsand 1X Taq buffer with 2mMMgCl

2 The thermal cycler was

programmed for 2min at 93∘C for an initial denaturationcycle 38 cycles of 35 s denaturation at 93∘C 35 s annealingat 48∘C 45 s extension at 72∘C and followed by a 5min finalextension at 72∘C

With reference to sfp gene (the second gene essential tothe production of surfactin) [44ndash46] PCR amplification wasstudied for all the five isolates with primer pairs specific tothe sfp gene The sfp 675 bp fragment corresponding to theB subtilis sfp gene (GenBank accession number X63158) atpositions 167ndash841 was amplified from the genomic DNA byPCR using two oligonucleotide primers sfp F (51015840-ATG-AAGATTTACGGAATTTA-31015840) and sfp-R (51015840-TTATAA-AAGCTCTTCGTACG-31015840) Amplification was carried outwith eppendorf thermal cycler (Eppendorf North AmericaInc) in a total volume of 25120583L containing about 200120583MdNTPs 1XTaq buffer 50 ng of genomicDNA 20 pmol of eachprimer and 3 U Taq DNA polymerase The thermal cyclerwas programmed for 1min at 94∘C for an initial denaturationcycle 25 cycles of 1min denaturation at 94∘C 30 s annealingat 46∘C 1min extension at 72∘C and followed by a 10minfinal extension at 72∘C

PCR products were analyzed by electrophoresis on 1agarose The expected DNA bands of srfsfp 268675 bpwere excised from gel and purified using the gel elution kit(Sigma-Aldrich USA) as per the manufacturerrsquos protocolThe sequencing reactions were carried out for both the geneusing a Big Dye Terminator Cycle Sequencing Kit (AppliedBiosystems Foster City CA USA) The sequences thusobtained were assembled and edited using Geneious R6

213 DNA Sequence Accession Numbers Accession numbersof sequences determined in this study were deposited inGenbank as isolate 1 HM145910 isolate 2 HM194725 isolate3 HM222944 isolates 4 and 5 sequences submitted to NCBI

3 Results and Discussion

31 The Marine Samples of Eastern Coastal Plain of TamilNadu The marine samples collected from eastern coastalplain of Tamil Nadu were subjected to the microbial profilewith respect to biosurfactant production which implied thatonly 90 of the bacterial have population shown biosur-factant production However the remaining populationsdisplayed secondary metabolites other than biosurfactants It


Isolate 1 Isolate 2

Isolate 3 Isolate 4 Isolate 5

Figure 2 Plate morphology of the five different marine isolates (isolate-1 isolate-2 isolate-3 isolate-4 isolate-5)

could be reasoned to the less oil pollution but with greaterpharmaceutical waste contaminations Even the samples ofEnnore port and Tuticorin port also showed less biosurfac-tant producers It can be argued that the sampling seasonsmay play the role in the specific microbial populationsHowever to avoid this argument sampling was done at threedifferent seasons and in all the scheduled time periods onlyless number of biosurfactant producers were encounteredBut still the sampling studies need much more explorations

32 Phenotypic and Biochemical Characteristics Screeningand identifications studies revealed that the selected isolateswere confirmed as the members of the genus Bacillus phe-notypically and genotypically With respect to phenotypiccharacteristics most of the colonies were smooth white incolour and having regular margin and some of the microbeshave rough dry irregular margin and some have slimywith smooth colonies and one isolate exhibited filamentousgrowth with pigment production on agar plate (Figure 2)Though most of the reports on Bacillus species infer nilpigmentation however the pigmented colonies of one ofthe isolates observed in the present study correlate well withthe report of Palmisano et al [47] With reference to Gramstaining the isolates were Gram positive rod-shaped anddominated in the size range of 05ndash12 120583m All the isolateswere motile and spore producers

Biochemical analysis revealed that all five isolates werepositive for catalase and one isolate showed positive for

oxidase All five isolates showed hydrolysis of casein gelatinstarch and lipid Few isolates were not responsive to theIMViC test Table 1 describes the results on the biochemicalanalysis of the biosurfactant producers

33 Molecular Characterization Results on phenotypic char-acteristics and 16S rRNA gene sequence homology suggestedthat all the five isolates have shown close relativeness togenus Bacillus (Table 2) Isolates 1 and 2 share an almostidentical 16S rRNA gene sequence with the published speciesB licheniformis ATCC 14580 and B licheniformis DSM13 =ATCC 14580 (94 and 99 similarity) whereas isolates 34 and 5 share an almost identical 16S rRNA gene sequencewith the published species B subtilis RO-NN-1 chromosomeBacillus spp JS chromosome and B subtilis BEST7613 DNAwith sequence homology (100 similarity)The phylogeneticframework was constructed based on the sequence analysisconstructed by neighbor-joining method based on 16S rRNAgene sequence (Figure 3) It has been understood that allfive isolates are closely related to B subtilis with meagervariations Results of Roberts et al [26 27] and Nakamura[28] also observed the similar relativeness in B subtilis withother Bacillus spp

With respect to biosurfactant activity of the isolatesthe cell-free broth was subjected to drops collapse test oildisplacement test and tensiometer measurements The bio-surfactant activity was comparedwith commercially availablebiosurfactant synthetic surfactant and water All the isolates


Table 1 Phenotypic and biochemical characteristic of marine isolates

Characteristics Isolate 1 Isolate 2 Isolate 3 Isolate 4 Isolate 5

Morphology Pink dry andfilamentous

White smooth andirregular colonies

White smoothcircular and undulate

margin

White circular andsmooth margin

White smooth slimyand entire margin

Gram stain + + + + +Shape Rod Rod Rod Rod RodMotility Motile Motile Motile Motile MotileIndole minus minus minus minus minus

Methyl red minus minus minus minus minus

Voges Proskauer + + + + +Citrate + + + + +Urease minus minus minus minus +Nitrate + + minus minus +Oxidase + minus minus minus minus

Catalase + + + + +Starch hydrolysis + + + + +Casein hydrolysis + + + + +Lipid hydrolysis + + + + +Gelatin hydrolysis + + + + +Growth at 55∘C + + minus minus minus

Growth in NaCl5 + + + + +75 + + + + +

(+) positive (minus) negative

Table 2 16S rRNA sequence homology Identification and compar-ison of the bacterial isolates from marine environment

Isolates The best GenBank matchClassification

(accession number)

homology

coverage

Isolate 1 B licheniformis ATCC14580 (NC0062703) 94 99

Isolate 2 B licheniformis DSM13 =ATCC14580 (NC0062701) 99 100

Isolate 3 B subtilis RO-NN-1chromosome (NC0199481) 100 100

Isolate 4 Bacillus spp JS chromosome(NC0177431) 100 100

Isolate 5 B subtilis BEST7613 DNA(NC0199481) 100 99

collapse the oil but differ at the time taken for collapsing theoil with respect to organism Table 3 describes results on thedrops collapse test and oil displacement test and tensiometermeasurements suggested that all the five marine isolatesexhibit appreciable biosurfactants activity which was on parwith synthetic surface active agents and Table 4 illustratesthe chemical composition of biosurfactant obtained frommarine isolates Followed by the production recovery ofbiosurfactant by precipitation processes was carried outEthanol precipitated biosurfactant (designated as ldquomicrosurf

Table 3 Surfactant activity of the 5 marine isolates

Isolate DCT ODT TM (mNm)Isolate 1 +++ +++ 28 plusmn 3Isolate 2 +++ +++ 31 plusmn 2Isolate 3 ++ + 29 plusmn 4Isolate 4 +++ +++ 30 plusmn 2Isolate 5 +++ +++ 26 plusmn 4(+++) excellent (++) good (+) average [DCT drop collapse test ODT oildisplacement test TM tensiometer measurement]

(1 to 5) respective to isolate 1 to isolate 5rdquo) was creamy whitein colour and soluble in water and dimethyl sulfoxide

Examination of emulsification activity emulsificationindex and stability of emulsion reveal emulsification indexof 85ndash95was observed with vegetable oils and crude oil andlt75with keroseneWith regard to the stability of emulsionsstable emulsion was observed with vegetable oils (more than90 days) more than emulsion formed with kerosene (onlyup to 30 days) and crude oil Followed by the emulsion for-mation we observed a complete transformation of emulsionto thread like structures in crude oil and reasons to lessstability With reference to hemolytic activity no haemolysiswas exhibited by the ldquomicrosurf (1 to 5)rdquo even at highconcentration The nonhemolytic behaviour of ldquomicrosurf (1to 5)rdquo in comparison with standard surfactin observed inthe present study may be due to the nonionic nature of the


Table 4 Chemical composition of biosurfactants obtained from marine isolates

Marine isolates Lipid content(120583gmL)

Protein content(120583gmL)

Carbohydratecontent (120583gmL) Haemolytic activity Antimicrobial

activity Ionic character

Microsurf-1 175 1033 861 Nonhaemolytic No activity NonionicMicrosurf-2 759 8028 265 Nonhaemolytic No activity NonionicMicrosurf-3 691 1673 072 Nonhaemolytic No activity NonionicMicrosurf-4 1778 2055 719 Nonhaemolytic No activity NonionicMicrosurf-5 97 539 0235 Nonhaemolytic No activity NonionicStandard(surfactin) mdash mdash mdash Haemolytic Broad spectrum Anionic

00060

Bacillus amyloliquefaciens DSM7 complete genomeBacillus amyloliquefaciens TA208 chromosome complete genome

Bacillus amyloliquefaciens XH7chromosome complete genome

Bacillus amyloliquefaciens LL3 chromosome complete genome

Bacillus amyloliquefaciens subsp Plantarum AS433 complete genome

Bacillus amyloliquefaciens subsp Plantarum CAU B946 complete genome

Bacillus amyloliquefaciens subsp Plantarum YAU B9601-Y2 complete genome

Bacillus spp JS chromosome complete genome

Bacillus amyloliquefaciens Y2 chromosome complete genome

Bacillus atrophaeus 1942 chromosome complete genome

Bacillus licheniformismdashisolate 1


Bacillus licheniformis ATCC 14580 chromosome complete genome

Bacillus subtilismdashisolate 4



Bacillus subtilis subsp spizizenii str W23 chromosome complete genome

Bacillus subtilis subsp spizizenii

Bacillus subtilis BSn5 chromosome complete genome

Bacillus subtilis BEST7003 DNA complete genome

Bacillus subtilis subsp subtilis str 168 chromosome complete genome

Bacillus subtilis subsp subtilis str RO-NN-1 chromosome complete genome

Bacillus subtilis QB928 chromosome complete genome

Bacillus subtilis BEST7613DNA complete genome

Bacillus subtilis subsp subtilis str BSP1 complete genome

TU-B-10 chromosome complete genome

Bacillus licheniformis DSM 13 = ATCC 14580 chromosome complete genome

Figure 3 Phylogenetic tree of 16S rRNA gene sequences of the marine isolates Bar 0006 substitutions per nucleotide position

ldquomicrosurfrdquo The reason for the nonionic nature has not beenunderstood yet However according to the literatures thepresence of aminoacid determines the ionic characteristic ofbiosurfactant And moreover the presence of alanine mayalso contribute to the nonhemolytic behaviour [48] Further

the nonantimicrobial activity of ldquomicrosurf rdquo has also beenreasoned to the nonionic nature

34 Identification of Gene of Interest and Sequence Deter-mination of PCR Amplified Product Figure 4(a) illustrates


(bp) (bp)

300

200

268

M 1 2 3 4 5

(a)

(bp) (bp)

700

600675

M 1 2 3 4 5

(b)

Figure 4 PCR amplification of srf (a) and sfp (b) gene in the chosen marine isolates [M-molecular size marker (100 bp ladder) Lane 1 is Blicheniformis (isolate 1) Lane 2 is B licheniformis (isolate 2) Lane 3 is B subtilis (isolate 3) Lane 4 is B subtilis (isolate 4) and Lane 5 is Bsubtilis (isolate 5)]

the PCR amplification of srf genes in all the five isolatesIt has been observed that all of the five isolates expresssrf gene amplification at 268 bp However the expressionof sfp gene amplified at 675 bp was realized only for threeisolates (Figure 4(b)) The sfp gene is an essential componentof peptide synthesis systems and also plays a role in theregulation of surfactin biosynthesis gene expression [42]Thesequences srf sfp thus obtained were verified in the NCBIdatabases for the genespecies confirmation thus validatingthe presence of the genes in the selected strains of Bacillus

According to the existing literatures discrimination ofBacillus species was assessed based on the (i) morphology(ii) biochemical (iii) genes responsible for the particularmetabolite productionor molecular profile and finally (iv)the structural features of the metabolitesenzymes Nev-ertheless still the variation at subspecies levels has beenrealized by a number of researchers In the present studydiscrimination in Bacillus species was made based on thebiosurfactant production Since there was no clear report onthe actual steps involved in the synthesis of surface activeagents by Bacillus species and numbers of hypotheses havebeen explored by the researchers The entire said hypothesisas a whole suggested that biosurfactant produced has beencategorized as surfactin though there has been the differencein the structural features The major difference was observedin lipid moiety and the peptide sequence Furthermore theresearchers have also realized the difference in the ionicnature hemolytic behavior and antimicrobial propertiesalthough the organisms displayed the gene responsible forsurfactin It has been observed that surfactin of one Bacillusspecies is entirely different from the surfactin of otherBacillusspecies But only few reports illustrate gt99 matching withthe commercially available surfactin

In the present study though the surface active agent of Blicheniformis and B subtilis showed similarity among themthe gene of expression clearly demonstrated the differencesrf expression was observed in both B licheniformis and Bsubtilis whereas sfp expression was realized only with Bsubtilis It has been suggested that in B licheniformis theintegration between srf and sfp genes may not occur which

limits the expression of sfp gene whereas the integrationbetween said genes in B subtilis expresses the sfp gene It mayalso be argued that the selection of primers could be one ofthe reasons or the directed de nova synthesis may hinder theexpression of sfp

4 Conclusion

The present study deals with the discrimination in surfactin-like biosurfactant extracted from marine Bacillus speciesof Tamil coastal plain that was studied in detail and theexpression of genes responsible for surface active agentdemonstrated wide variations which added an additionalinformation for the Gram positive bacterial species of marineorigin

On comparing these observations with the reportedliteratures it has been concluded that Bacillus spp of TamilNadu coastal plains have the capacity to produce biosur-factant having similar or more or less equal surfactantactivity compared to other Bacillus spp but displayed non-ionic and nonhemolytic behaviour This property of thebiosurfactant is interesting and needs explorations Amongthe B licheniformis and B subtilis species the gene specificto biosurfactant production (sfp) has been expressed in Bsubtilis and not in B licheniformis

Conflict of Interests

The authors declare that there is no conflict of interests re-garding the publication of this paper

Acknowledgment

The authors greatly acknowledge DBT New Delhi CSIRNetwork Project (CSC0127) for financial assistance in theform of projects


References

[1] E Baath M Dıaz-Ravina A Frostegard and C D CampbellldquoEffect of metal-rich sludge amendments on the soil microbialcommunityrdquo Applied and Environmental Microbiology vol 64no 1 pp 238ndash245 1998

[2] G M Konig S Kehraus S F Seibert A Abdel-Lateff andD Muller ldquoNatural products from marine organisms and theirassociated microbesrdquo ChemBioChem vol 7 no 2 pp 229ndash2382006

[3] G Georgiou S-C Lin and M M Sharma ldquoSurface-activecompounds from microorganismsrdquo Nature Biotechnology vol10 no 1 pp 60ndash65 1992

[4] S H Kim E J Lim S O Lee J D Lee and T H Lee ldquoPurifi-cation and characterization of biosurfactants fromNocardia spL-417rdquo Biotechnology and Applied Biochemistry vol 31 no 3 pp249ndash253 2000

[5] N Youssef M S Elshahed and M J McInerney ldquoChapter6 microbial processes in oil fields culprits problems andopportunitiesrdquo Advances in Applied Microbiology vol 66 pp141ndash251 2009

[6] W-Q Zhuang J-H Tay A M Maszenan and S T L TayldquoBacillus naphthovorans sp nov from oil-contaminated tropicalmarine sediments and its role in naphthalene biodegradationrdquoApplied Microbiology and Biotechnology vol 58 no 4 pp 547ndash553 2002

[7] G J Dick Y E Lee and B M Tebo ldquoManganese(II)-oxidizingBacillus spores in Guaymas basin hydrothermal sediments andplumesrdquoApplied and Environmental Microbiology vol 72 no 5pp 3184ndash3190 2006

[8] N Fusetani D Ejima S Matsunaga et al ldquo3-Amino-3-deoxy-D-glucose an antibiotic produced by a deep-sea bacteriumrdquoExperientia vol 43 no 4 pp 464ndash465 1987

[9] Y Ohta Y Nogi M Miyazaki et al ldquoEnzymatic propertiesand nucleotide and amino acid sequences of a thermostable 120573-agarase from the novel marine isolate JAMB-A94rdquo BioscienceBiotechnology and Biochemistry vol 68 no 5 pp 1073ndash10812004

[10] C R Harwood ldquoBacillusrdquo in Biotechnology Handbooks TAtkinson and R F Sherwood Eds Plenum New York NYUSA 1989

[11] S Lang and D Wullbrandt ldquoRhamnose lipidsmdashbiosynthesismicrobial production and application potentialrdquoAppliedMicro-biology and Biotechnology vol 51 no 1 pp 22ndash32 1999

[12] I M Banat R S Makkar and S S Cameotra ldquoPotentialcommercial applications of microbial surfactantsrdquo AppliedMicrobiology and Biotechnology vol 53 no 5 pp 495ndash5082000

[13] T T Nguyen NH YoussefM JMcInerney andDA SabatinildquoRhamnolipid biosurfactant mixtures for environmental reme-diationrdquoWater Research vol 42 no 6-7 pp 1735ndash1743 2008

[14] A S Nerurkar ldquoStructural and molecular characteristics oflichenysin and its relationship with surface activityrdquo in Biosur-factants vol 672 of Advances in Experimental Medicine andBiology pp 304ndash315 Springer New York NY USA 2010

[15] E P Ivanova M V Vysotskii V I Svetashev et al ldquoChar-acterization of Bacillus strains of marine originrdquo InternationalMicrobiology vol 2 no 4 pp 267ndash271 1999

[16] J L Siefert M Larios-Sanz L K Nakamura et al ldquoPhylogenyof marine Bacillus isolates from the Gulf of Mexicordquo CurrentMicrobiology vol 41 no 2 pp 84ndash88 2000

[17] C A C Miranda O B Martins and M M ClementinoldquoSpecies-level identification of Bacillus strains isolates frommarine sediments by conventional biochemical 16S rRNA genesequencing and inter-tRNA gene sequence lengths analysisrdquoAntonie van Leeuwenhoek vol 93 no 3 pp 297ndash304 2008

[18] F G Priest ldquoSystematics and ecology of Bacillusrdquo in BacillusSubtilis and Other Gram-Positive Bacteria Biochemistry Physi-ology andMolecular Genetics A L Sonenshein J A Hoch andR Losick Eds pp 3ndash16 American Society for MicrobiologyWashington DC USA 1993

[19] E AGontangW Fenical and P R Jensen ldquoPhylogenetic diver-sity of gram-positive bacteria cultured frommarine sedimentsrdquoApplied and Environmental Microbiology vol 73 no 10 pp3272ndash3282 2007

[20] P Cosmina F Rodriguez F de Ferra et al ldquoSequence andanalysis of the genetic locus responsible for surfactin synthesisinBacillus subtilisrdquoMolecularMicrobiology vol 8 no 5 pp 821ndash831 1993

[21] D Konz S Doekel and M A Marahiel ldquoMolecular andbiochemical characterization of the protein template control-ling biosynthesis of the lipopeptide lichenysinrdquo Journal ofBacteriology vol 181 no 1 pp 133ndash140 1999

[22] N H Youssef K E Duncan andM J McInerney ldquoImportanceof 3-hydroxy fatty acid composition of lipopeptides for biosur-factant activityrdquo Applied and Environmental Microbiology vol71 no 12 pp 7690ndash7695 2005

[23] R Sen ldquoSurfactin biosynthesis genetics and potential applica-tionsrdquo in Biosurfactants vol 672 of Advances in ExperimentalMedicine and Biology pp 316ndash323 Springer New York NYUSA 2010

[24] F Peypoux J M Bonmatin and J Wallach ldquoRecent trendsin the biochemistry of surfactinrdquo Applied Microbiology andBiotechnology vol 51 no 5 pp 553ndash563 1999

[25] T Nishikiori H Naganawa Y Muraoka T Aoyagi and HUmezawa ldquoPlipastatins new inhibitors of phospholipase A2produced by Bacillus cereus BMG302-fF67 II Structure of fattyacid residue and amino acid sequencerdquo Journal of Antibioticsvol 39 no 6 pp 745ndash745 1986

[26] M S Roberts L K Nakamura and F M Cohan ldquoBacillusmojavensis sp nov distinguishable from Bacillus subtilis bysexual isolation divergence in DNA sequence and differencesin fatty acid compositionrdquo International Journal of SystematicBacteriology vol 44 no 2 pp 256ndash264 1994

[27] M S Roberts L K Nakamura and F M Cohan ldquoBacillusvallismortis sp nov a close relative of Bacillus subtilis isolatedfrom soil in Death Valley Californiardquo International Journal ofSystematic Bacteriology vol 46 no 2 pp 470ndash475 1996

[28] L K Nakamura ldquoTaxonomic relationship of black-pigmentedBacillus subtilis strains and a proposal forBacillus atrophaeus spnovrdquo International Journal of Systematic Bacteriology vol 39 no3 pp 295ndash300 1989

[29] A Ventosa M T Garcia M Kamekura H Onishi and FRuiz-Berraquero ldquoBacillus halophilus sp nov a moderatelyhalophilic Bacillus speciesrdquo Systematic and Applied Microbiol-ogy vol 12 no 2 pp 162ndash166 1989

[30] S Spring W Ludwig M C Marquez A Ventosa and K-H Schleifer ldquoHalobacillus gen nov with descriptions ofHalobacillus litoralis sp nov and Halobacillus trueperi sp novand transfer of Sporosarcina halophila toHalobacillus halophiluscomb novrdquo International Journal of Systematic Bacteriology vol46 no 2 pp 492ndash496 1996


[31] M J Garabito D R Arahal E Mellado M C Marquez andA Ventosa ldquoBacillus salexigens sp nov a new moderatelyhalophilic Bacillus speciesrdquo International Journal of SystematicBacteriology vol 47 no 3 pp 735ndash741 1997

[32] P H A Sneath ldquoEndospore-forming gram-positive rods andcoccirdquo in Bergeyrsquos Manual of Systematic Bacteriology R G EMurray D J Brenner andM P Bryant Eds pp 1104ndash1207TheWilliams ampWilkins Baltimore Md USA 1986

[33] S Turner K M Pryer V P W Miao and J D Palmer ldquoInves-tigating deep phylogenetic relationships among cyanobacteriaand plastids by small subunit rRNA sequence analysisrdquo TheJournal of Eukaryotic Microbiology vol 46 no 4 pp 327ndash3381999

[34] 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

[35] J R Cole QWang E Cardenas et al ldquoThe ribosomal databaseproject improved alignments and new tools for rRNA analysisrdquoNucleic Acids Research vol 37 supplement 1 pp D141ndashD1452009

[36] F Sievers A Wilm D Dineen et al ldquoFast scalable generationof high-quality protein multiple sequence alignments usingClustal OmegardquoMolecular Systems Biology vol 7 p 539 2011

[37] J Vater B Kablitz C Wilde P Franke N Mehta and S SCameotra ldquoMatrix-assisted laser desorption ionization-time offlight mass spectrometry of lipopeptide biosurfactants in wholecells and culture filtrates of Bacillus subtilis C-1 isolated frompetroleum sludgerdquo Applied and Environmental Microbiologyvol 68 no 12 pp 6210ndash6219 2002

[38] D K Jain D L CThompson H Lee and J T Trevors ldquoA drop-collapsing test for screening surfactant-producing microorgan-ismsrdquo Journal of Microbiological Methods vol 13 no 4 pp 271ndash279 1991

[39] M Morikawa H Daido T Takao S Murata Y Shimonishiand T Imanaka ldquoA new lipopeptide biosurfactant produced byArthrobacter sp strain MIS38rdquo Journal of Bacteriology vol 175no 20 pp 6459ndash6466 1993

[40] A A Bodour and R M Miller-Maier ldquoApplication of a mod-ified drop-collapse technique for surfactant quantitation andscreening of biosurfactant-producingmicroorganismsrdquo Journalof Microbiological Methods vol 32 no 3 pp 273ndash280 1998

[41] M Rosenberg E A Bayer J Delarea and E Rosenberg ldquoRoleof thin fimbriae in adherence and growth of Acinetobacter cal-coaceticus RAG-1 on hexadecanerdquo Applied and EnvironmentalMicrobiology vol 44 no 4 pp 929ndash937 1982

[42] CLSI Performance Standards for Antimicrobial SusceptibilityTesting Seventeenth Informational Supplement Wayne PaUSA 2007

[43] D R Simpson N R Natraj M J McInerney and K E Dun-can ldquoBiosurfactant-producing Bacillus are present in producedbrines from Oklahoma oil reservoirs with a wide range ofsalinitiesrdquo Applied Microbiology and Biotechnology vol 91 no4 pp 1083ndash1093 2011

[44] M M Nakano N Corbell J Besson and P Zuber ldquoIsolationand characterization of sfp a gene that functions in the pro-duction of the lipopeptide biosurfactant surfactin in BacillussubtilisrdquoMolecular and General Genetics vol 232 no 2 pp 313ndash321 1992

[45] F-C Hsieh M-C Li T-C Lin and S-S Kao ldquoRapid detectionand characterization of surfactin-producing Bacillus subtilis

and closely related species based on PCRrdquo Current Microbiol-ogy vol 49 no 3 pp 186ndash191 2004

[46] G Seydlova and J Svobodova ldquoReview of surfactin chemicalproperties and the potential biomedical applicationsrdquo CentralEuropean Journal of Medicine vol 3 no 2 pp 123ndash133 2008

[47] M M Palmisano L K Nakamura K E Duncan C A Istockand F M Cohan ldquoBacillus sonorensis sp nov a close relative ofBacillus licheniformis isolated from soil in the Sonoran DesertArizonardquo International Journal of Systematic and EvolutionaryMicrobiology vol 51 no 5 pp 1671ndash1679 2001

[48] M R Infante L Perez A Pinazo P Clapes and M CMoran ldquoAmino acid-based surfactantsrdquo in Novel SurfactantsPreparation Application and Biodegradability K HolmbergEd Surfactant Science Series pp 193ndash216 Marcel Dekker NewYork NY USA 114th edition 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


and aquatic habitats [15 16] including marine sediments [17]Members of the genusBacillus comprise Grampositive sporeforming rod-shaped and aerobic bacteria Bacillus speciesare phenotypically and genotypically heterogenous [18]Few studies have focused on the phylogeny and biodiversityof marine Bacilli [15ndash17 19] These reports were based ona small number of isolates or a unique sampling site andshowed that Bacillus subtilis Bacillus licheniformis Bacilluscereus Bacillus amyloliquefaciens and Bacillus pumilusare the common inhabitants of marine environments andconsequently often isolated However a more extensiveanalysis to assess the real biodiversity and phylogeneticrelationship between Bacilli from marine origin is becomingnecessary Recent studies on marine bacilli showed thatstrains of B marinus B subtilis B pumilus B licheniformisB cereus and B mycoides are common inhabitants of themarine habitat [15] They constitute closely related taxa andtheir differentiation has become difficult and laborious asthey cannot be distinguished from each other by phenotypictests

Furthermore with regard to biosurfactant productioncertain strains of the closely related species B subtilis Bmojavensis B sonorensis and B licheniformis produce thecyclic lipopeptide biosurfactants surfactin and lichenysin[20ndash23] B subtilis produces a known lipopeptide biosurfac-tant called surfactin which is coded by four open readingframes (ORFs) named as SrfA SrfB SrfC and SrfD [24]Similarly lichenysin is a lipopeptide biosurfactant producedby B licheniformis coded by lichenysin operon (LchA) andconsists of three peptide synthetase genes LicAA LicABLicAC and LicAD [21] B cereus produces a lipopeptidebiosurfactant called plipastatin [25]

The speciesmost closely related toB subtilis are unusuallysimilar at the phenotypic level For example fatty acidcomposition is the only known phenotypic character thatdistinguishes Bacillus mojavensis and Bacillus vallismortisfrom one another or from B subtilis [26 27] and Bacil-lus atrophaeus is distinguishable from B subtilis only bydifferences in pigmentation [28] A heterogeneous groupof moderately halophilic bacteria which comprises Bacillussalexigens and three species of the new genus HalobacillusH halophilus H litoralis and H trueperi [29ndash31] may bedifferentiated by their ability to grow at 10 to 20 of total saltsand the possession of an unusual type of murein Molecularmethods have proven to be more effective in distinguishingclose relatives of B subtilis

From the above-summarized information it has beenunderstood that discrimination of the Bacillus species basedon the morphology phylogenetic framework and geneexpression of molecule of interest is possible neverthelessthe closely related species with similarity in the byproductsneed intensive explorations

Thus exploring the phylogenetic markers such as 16SrRNA genes to reveal microbial diversity and further explo-ration of gene expression to reveal the functional powerof the microbes is essential Hence in the present studyphylogenetic framework of marine Bacillus spp and expres-sion of gene responsible for biosurfactant production has

been explored in detail which provided interesting infor-mation and highly relevant to identification of novel strainswith desirable functional characteristics and biotechnologicalapplications

2 Materials and Methods

21 Isolation and Maintenance of Organism Marine samplesof water sediments mussels shells sea weeds and sandwere collected from Kalpakkam Ennore port Besant NagarBeach Marina beach Mahabalipuram beach MandapamVedaranyam Tuticorin Cuddalore in Tamil Nadu India(Figure 1) Zobell marine brothagar (for bacteria) was themedia used for the isolation of microbial species accordingto the standard procedure employed for the isolation andmaintenance of marine organisms Morphologically distinctorganisms were selected and stored in 30 glycerol stock atminus20∘C

22 Screening of Biosurfactant Producing Microbes Biosur-factant producers were screened based on the surfactantactivity exhibited by the culture media after 24ndash72 hours ofincubation at 37∘C in the presence of the bacterial isolatesIn brief Zobell marine broth (Hi Media) (100mL in 1000mLcapacity conical flask) was sterilized and inoculated with theisolated cultures and was incubated at 37∘C for 24ndash72 hoursunder shaking condition Followed by incubation the sam-ples weremade cell-free by centrifugation at 10 000timesg at 4∘CBiosurfactants activity of the cell free medium was assessedaccording to the procedure summarized in the followingparagraphs and the isolates exhibiting appreciable surfactantactivity were selected and subjected to identification andfurther production

23 Morphological Physiological and Biochemical Character-istics ofMarine Isolates Thestrains isolated frommarine sed-iments were identified by conventional biochemical tests inaccordance with Bergeyrsquos Manual of Systematic Bacteriology[32]

24 DNA Extraction Genomic DNA was isolated andpurified by ldquoDneasy Tissue Kitrdquo (Qiagen GmgH HildenGermany) according to the manufacturerrsquos directions Thequality of the extracted DNA was checked by agarose gelelectrophoresis 2 (wv)

25 PCR Amplification 16S rRNA Gene Sequencing Toconfirm the identities of the isolates PCR amplificationand sequencing of the 16S rRNA gene were performedThe 16S rRNA genes (1500 bp) were purified using thegel elution kit (Sigma-Aldrich USA) as per the manu-facturerrsquos protocol using the bacterial universal primer setof 8F 51015840-AGAGTTTGATCCTGGCTCAG-31015840 and 1492R 51015840-GGCTACCTTGTTACGACTT-31015840 as described by Turner etal [33] Amplification was carried out with the eppendorfthermal cycler (Eppendorf North America Inc) in a totalvolume of 25 120583L containing about 50 ng of genomic DNA5U of Taq DNA polymerase 20 pmol of each primer 200 120583M


India

80∘E 81

∘E79∘E78

∘E77∘E76

∘E

80∘E 81

∘E79∘E78

∘E77∘E76

∘E

13∘N

12∘N

11∘N

10∘N

9∘N

8∘N

13∘N

12∘N

11∘N

10∘N

9∘N

8∘N

A

BChennai

C

D

E

N

Tamil Nadu

























Isolate 1 Isolate 2















margin







Growth in NaCl5 + + + + +75 + + + + +




(accession number)

homology

coverage


















00060
































(bp) (bp)

300

200

268

M 1 2 3 4 5

(a)

(bp) (bp)

700

600675

M 1 2 3 4 5

(b)






4 Conclusion





Acknowledgment



References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


India

80∘E 81

∘E79∘E78

∘E77∘E76

∘E

80∘E 81

∘E79∘E78

∘E77∘E76

∘E

13∘N

12∘N

11∘N

10∘N

9∘N

8∘N

13∘N

12∘N

11∘N

10∘N

9∘N

8∘N

A

BChennai

C

D

E

N

Tamil Nadu

























Isolate 1 Isolate 2















margin







Growth in NaCl5 + + + + +75 + + + + +




(accession number)

homology

coverage


















00060
































(bp) (bp)

300

200

268

M 1 2 3 4 5

(a)

(bp) (bp)

700

600675

M 1 2 3 4 5

(b)






4 Conclusion





Acknowledgment



References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















Isolate 1 Isolate 2















margin







Growth in NaCl5 + + + + +75 + + + + +




(accession number)

homology

coverage


















00060
































(bp) (bp)

300

200

268

M 1 2 3 4 5

(a)

(bp) (bp)

700

600675

M 1 2 3 4 5

(b)






4 Conclusion





Acknowledgment



References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Isolate 1 Isolate 2















margin







Growth in NaCl5 + + + + +75 + + + + +




(accession number)

homology

coverage


















00060
































(bp) (bp)

300

200

268

M 1 2 3 4 5

(a)

(bp) (bp)

700

600675

M 1 2 3 4 5

(b)






4 Conclusion





Acknowledgment



References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







margin







Growth in NaCl5 + + + + +75 + + + + +




(accession number)

homology

coverage


















00060
































(bp) (bp)

300

200

268

M 1 2 3 4 5

(a)

(bp) (bp)

700

600675

M 1 2 3 4 5

(b)






4 Conclusion





Acknowledgment



References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








00060
































(bp) (bp)

300

200

268

M 1 2 3 4 5

(a)

(bp) (bp)

700

600675

M 1 2 3 4 5

(b)






4 Conclusion





Acknowledgment



References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(bp) (bp)

300

200

268

M 1 2 3 4 5

(a)

(bp) (bp)

700

600675

M 1 2 3 4 5

(b)






4 Conclusion





Acknowledgment



References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Phylogenetic Framework and Biosurfactant Gene …downloads.hindawi.com/archive/2014/860491.pdf · 2019-07-31 · Research Article Phylogenetic Framework and Biosurfactant