Lipopeptide production by Bacillus subtilis R1 and its possible applications

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h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Industrial

Microbiology

Lipopeptide

production

by

Bacillus

subtilis

R1

and

its

possible

applications

Sujata

S. Jha

a,b

_,

_Sanket

_J.

_Joshi

a,c,∗

_,

_Geetha

_S.J.

a,c

a_M._S._University_of_Baroda,_Department_of_Microbiology_and_{Biotechnology}_Centre,_Vadodara,_Gujarat,_India

b_Northwestern_University,_Feinberg_School_of_Medicine,_Department_of_Microbiology_and_Immunology,_Chicago,_IL,_United_States

c_Sultan_Qaboos_University,_Department_of_Biology,_College_of_Science,_Oman

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received20June2015 Accepted7April2016 Availableonline26July2016 AssociateEditor:AdalbertoPessoa Junior

Keywords:

Bacillussubtilis

Biosurfactant

Ethylmethanesulfonate Microbialenhancedoilrecovery Antifungalactivity

a

b

s

t

r

a

c

t

Thepossibleapplicationofabacterialstrain–BacillussubtilisR1,isolatedfromanoil contam-inateddesertsiteinIndia,asbiocontrolagentanditsbiosurfactantinmicrobialenhanced oilrecoveryarediscussed.Thebiosurfactantproductioninminimalmediumwascarried outatdifferenttemperaturesandsaltconcentrations,whereitproducedanefficient bio-surfactantat30–45◦Candinpresenceofupto7%salt.Itsignificantlyreducedthesurface tensionfrom66±1.25mN/mto29±0.85mN/mwithin24h.Inordertoenhancethe bio-surfactantproduction,randommutagenesisofB.subtilisR1wasperformedusingchemical mutagen–ethylmethanesulfonate.Majorityoftheisolated42mutantsshowed biosurfac-tantproduction,butthedifferencewasstatisticallyinsignificantascomparedwithparent strainR1.Thereforenoneofthemutantswereselectedforfurtherstudy,andonlyparent strainR1wasstudied.Thebiosurfactantwasquitestableunderharshconditionsforupto 10days.Thebiosurfactantwasextractedandcharacterizedassimilartothelipopeptide group–surfactinsandfengycin.Thecrudeoildisplacementexperimentsusing biosurfac-tantbrothinsandpackglasscolumnsshowed33±1.25%additionaloilrecovery.Thestrain alsoshowedinhibitionofvariousplantpathogenicfungionpotatodextroseagarmedium. ©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction

Chemical compounds are extensively used in petroleum industries fordifferent operations, mainly inenhanced oil recovery(EOR),likesurfactantsandpolymers,whichare gen-erallytoxicandrecalcitrantleadingtodisposalproblemsfor

∗ _{Corresponding}_author_at:_College_of_Science,_Department_of_Biology,_Sultan_Qaboos_University,_PO_Box_36,_123,_Oman.

E-mail:[email protected](S.J.Joshi).

oilﬁeld producedwater, ground watercontamination, and obvious healthrisks to humans. Biosurfactantsare oneof theenvironmentalfriendlyalternativestothecurrentlyused chemical surfactants. Biosurfactants are amphiphilic com-pounds produced by microorganisms, similar to chemical counterparts, but with better environmental compatibility, activity, stability, lower toxicity, and versatile applications

http://dx.doi.org/10.1016/j.bjm.2016.07.006

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in health, food and agriculture. Even though biosurfac-tants have many advantages over chemical surfactants, widespreadapplicationsareweigheddownbythehighcosts of production. Researchers have reported several ways to reducethe costsofbiosurfactant productionlike, selection ofcheaper rawmaterialor agro-industrial waste products, applicationofstatisticalmethodstooptimizetheproduction, and mutationof the strains toenhance the production.1–5

Nevertheless the biosurfactant market is growing rapidly, and accordingtoarecent report,global biosurfactant mar-ket was worth USD 1.7 billion in 2011, which is expected to reach USD 2.2 billion in 2018, growing at an aver-age annual growth rate of3.5% from 2011to 2018 (http:// www.prweb.com/releases/2012/11/prweb10158903.htm – as accessed on 12th April, 2015). Amongst different types of biosurfactants,smallermoleculeslikelipopeptidesproduced

byBacilli sp.are reportedtobethe mostactiveinreducing

thesurfacetensionandinterfacialtensionbetweenoiland water,leadingtoenhancedoilrecovery.Thoselipopeptides arealsoreportedtoinhibitmicrobialgrowth,and thusalso have potential applications in plantprotection and health applications.Severaltypesofbiosurfactantsarereportedto have applications in environmentalbioremediation and in microbialenhancedoilrecovery(MEOR).MEORisoneofthe tertiaryrecoverytechniquesforenhancingorimprovingthe recoveryofcrudeoilfromdeclining oilreservoirs, whichis a well-anticipated technology, to be widely applied in the oil ﬁelds.6–9 We have isolated Bacillus subtilis R1 from oil contaminatedsite,identiﬁedandscreenedforbiosurfactant productioninglucosebasedminimalsaltmedia,atdifferent saltconcentrationandtemperatures.Theisolatewastreated withchemicalalkylatingmutagen–ethylmethanesulfonate (EMS),forrandommutagenesistoenhancebiosurfactant pro-duction.Theisolateproducedapotentbiosurfactantwhich wasextracted,structurallycharacterized,andstudiedfurther forcriticalmicelleconcentration(CMC),stabilityunderharsh conditions(hightemperature,pHandsalinity),MEOR stud-iesbyglasssand-packmodels,andantifungalactivityonagar plates.

Materials

and

methods

Isolationandidentiﬁcationofmicroorganism

Severalmicroorganismswereisolatedasdescribedpreviously, from Kutch desert (Gujarat, India) and screened on blood agarplate.2 _One_of_the_selected_isolate_R1 _was_used_for_all

experiments.Theisolatewasmaintainedaerobicallyon Luria-Bartani(LB) agar plates and was regularlytransferred into fresh LB mediumfor short-term storage. Stockcultures of pureisolatewerepreparedin40%glycerolandstoredbelow −80◦_C,_for_long-term_{preservation.} _The_isolate_was _studied morphologically, different biochemical tests and 16S rRNA sequencing.

ColonyPCRand16SrDNAampliﬁcation

ThegenomicDNAwasextracteddirectlyfromcoloniesgrown onLBagar andused asthetemplateforpolymerasechain

reaction(PCR)aspreviouslydescribed.10_Isolated_pure_colonies

(2–3 colonies) were suspended in 20–30␮L sterile distilled water,heatedat99◦Cfor25minandcentrifugedat10,000rpm for3min.ThesupernatantwasusedasatemplateDNAinthe PCRsystem.The16SrRNAgenefragmentwasamplifiedusing universaleubacterialprimers27F(5-GAGAGTTTGATCCTG GCTCAG-3)and1107R(5-GCTCGTTGCGGGACTTAA CC-3).TheamountofDNAtakenforamplificationwas∼10ng. ThePCRcomponentsandconditions(for50␮Lsystem)used foramplification were asfollows (␮L):TargetDNA (100ng), 15.0;Taqpolymerasebuffer(10×),5.0;dNTPs(total10mMwith 2.5mMeach),2.0;forwardprimer(27F)20pmoles,1.0;reverse primer(1107R)20pmoles,1.0;Taqpolymerase3units/␮L,0.5; PCRwater,25.5.PCRprogram(ThermalCycler,Applied Biosys-tems)wasasfollows:Lidtemperature,105◦C;Step1:Initial Denaturation,94◦C–60s;Step2:Denaturation, 94◦C–45s; Step3:Primerannealing,56◦C–30s;Step4:Primer exten-sion,72◦C–1.5min;Steps2,3,4repeatedfor30cycles;Step5: Finalextension,72◦C–10min.Thepurityandsizeofeach PCR productwas examined bygel electrophoresis on0.8% agarose gel in0.5× TBE buffer. The100bpladder (molecu-lar weightmarker) wasused forcomparisonofsizeofthe PCR products.Thegel was visualized inUV transillumina-tor and photographed subsequently. All the PCR reagents, primers,Taqpolymeraseandothermolecularbiologyreagents wereobtainedfromBangaloreGeneiPvt.Ltd,Indiaandused aspermanufacturer’sinstructions.Theamplified16SrDNA genewassequencedatBangaloreGeneiPvt.Ltd,India,and the nucleotide sequence was deposited in NCBI GenBank database.

Biosurfactantproductionstudies

For preparation ofinoculum LB brothwas used as a seed medium,wheretheloopfulofbacteriaweretransferredand incubated at30◦C for8–10h (OD600 –0.8–0.9). For produc-tionofbiosurfactant,2%(v/v)inoculumwastransferredinto 50mLsterileglucosebasedminimalmediain250-mL Erlen-meyer ﬂasks.Themediumcomposition was (g/L):Glucose, 20;NaNO3,4;MgSO4,0.4;KCl,0.2;CaCl2,0.1;H3PO4,0.5mL, trace elements (mg/L): H3BO4, 1.53; CuSO4, 0.284; MnSO4, 1.71;Na2MoO4,0.7;ZnSO4, 2.9;FeSO4,4.3;CoCl2,0.1;EDTA, 200. The minimal medium pH was adjusted to 7.2 using 1.0NNaOH. Theﬂaskswereincubated atdifferent temper-atures(30◦C,40◦C,45◦C,50◦Cand 80◦C)and tostudy the effectofsalinity,differentconcentrationsofNaCl(0%,5%,7%) wereaddedintotheminimalmedia.Sampleswerecollected at every 24h forgrowth (OD600) and biosurfactant produc-tion (Surfacetension)analysistill96h.Surfacetension(ST) wasmeasuredbyDu-Nouytensiometerbyringdetachment methodusingdistilledwater(72±0.50mN/m)and uninocu-latedmedia(66±1.25mN/m)asabioticnegativecontrols.All measurementswere performedintriplicatesusingcellfree broth.

Randommutagenesisforenhancedbiosurfactant production

Alkalyting agent–ethyl methanesulfonate (EMS, Sigma–Aldrich, USA) was used for random mutagenesis

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of B. subtilis R1. The culture was grown in LB broth till OD600 reached0.8–0.9,then centrifugedand the pelletwas suspended in sterile normal-saline (0.85% NaCl) and EMS (15␮L/mL)wasaddedandincubatedincoldcondition.After incubation,cellswerewashedtwicebysaline,centrifugedto removeEMSandsuspendedinsterilenormal-saline. Appro-priatedilutionswerespreadonLBagarplatesandincubated at30◦C for 24h. The well isolated mutantcolonies on LB agarplateswerefurthertransferredon10%bloodagarplates for screening putative mutants. Culture showing zone of hemolysiswerefurtherusedforproductionstudieswherein theyweregrownin2%glucosebasedminimalmediaandST wasmeasuredafter72h.

Stabilitystudies

Stability studies were carried out using cell free broth, obtainedbycentrifugingtheculturesat11,292×gfor20min, as reported previously.2,11 _For _temperature _stability _study,

tightly closed bottles with 50mL broth were incubated at 30–80◦Cfor10daysandcooledatroomtemperaturebeforeST measurement.ThepHstabilitywasstudiedbyadjustingthe pHtodifferentvalues(2.0,4.0,6.0,8.0,10.0and12.0)with1N NaOHor1NHCl,andsaltstabilitywasdeterminedbyadding differentconcentrationsofsalt(NaCl–5%,7%,10%,15%,and 20%(w/v))tothecellfreebrothfollowedbyincubationfor10 daysat30◦C.TheSTwasmeasuredfromallthesamplesat differentintervalstill10thday.

Biosurfactantextraction

Themethodbasedonacidprecipitationwasusedfor extrac-tion andpartialpuriﬁcation ofbiosurfactant.12 _The_culture

broth was centrifuged at 11,292×g for 20min to get cell freebroth. Biosurfactant was precipitatedbyadjusting the pHofthecellfreebrothto2.0using6NHClandkeepingit at4◦C overnight. Afterdecanting off the supernatant, the precipitate was collected and suspended in distilled water (pH8.0)andlyophilized.Thelyophilizedproductwasweighed toﬁnd out the yieldofbiosurfactant and usedfor further characterization.

Criticalmicelleconcentration(CMC)

Critical micelleconcentration (CMC)isdeﬁned asthe con-centration of the surfactant necessary to initiate micelle formation.UponreachingtheCMC,theSTdoesnotcontinue todecreaseevenafterincreasingthesurfactant concentra-tion.TheCMCwasdeterminedbyplottingthesurfacetension asafunctionofthebiosurfactantconcentration.The concen-trationsrangingfrom0.6to10,000mg/Lwerepreparedwith distilledwaterandSTwasdeterminedusingDu-Nouy’sring Tensiometeratroomtemperature(30±2◦C).

Analyticaltechniques

Thinlayerchromatography(TLC)

TLCanalysisofpartiallypuriﬁedbiosurfactantwasperformed usingSilicagel60 (Merck)TLCplates,where20␮Lsamples were spotted on TLC plates and developed in a chamber

saturatedwithsolventsystem–chloroform:methanol:water (65:25:4).Theseparatedcomponentsweredetectedwiththree differentmethods–saturationofiodinevapors;sprayingwith methanol:sulphuricacid(95:5)reagent,followedbyheatingat 100◦Cfor30min;andsprayingtheplateswithbloodreagent.

Fouriertransforminfraredspectroscopy(FTIR)

FTIR(PerkinElmer,SpectrumGX,FT-IRsystem)spectrumof partially puriﬁed biosurfactant was performed at Sophisti-catedInstrumentationCentreforAppliedResearchand Test-ing(SICART),VallabhVidyanagar,Gujarat,India.Sampleswere analyzedinKBrcell.Datawascollectedbetween400and4000 wavenumbers/cm,andprocessedwithIRanalyticalsoftware.

Matrixassistedlaserdesorptionionizationtime-of-ﬂight

massspectroscopy(MALDI-TOFMS)

Thebiosurfactant wascharacterizedbyULTRFLEX-TOF/TOF (Bruker Daltonics) in reﬂector mode using a matrix of 2, 5–Dihydroxybenzoic acid (DHB). The reﬂector voltage was 25kVA and nitrogen laser was used with 150 laser shots (337nm,50Hz).

Antifungalactivity

A bacterial culture grown in LB to a concentration of 108_cfu/mL _was_spotted _on_the_Petri_dish _containing_potato dextroseagar(PDA).Simultaneouslya1cm2_(diameter)_agar plug containing 48h grown fungal mycelium in PDA was placedinthecenteroftheplate.Theinhibitioneffecton fun-galgrowthwasevaluatedafter4–5daysincubationat28◦C. Allinvitroantagonismassayswereperformedintriplicates, asdescribedpreviously.13

Oildisplacementstudiesusingglasssandpackcolumns TheapplicationofthebiosurfactantinMEORwasevaluated usingtheglasssandpackcolumnsasdescribedpreviously.14 Wherebriefly,glass columns(45.7cm×2.5cm)werepacked with 100gofacid washedsand (100mesh size),and satu-ratedwithbrine(5.0%NaCl).Eachcolumnwasthenvertically floodedupwardswithlightcrudeoiltoirreduciblewater satu-ration,theamountofoilinplacewascalculatedandthenthe columnwasfloodedwithbrineuntilnofurtheroilappeared intheeffluent.Theamountofoilrecovered wasmeasured and thencellfreeBrothwaspumpedupwardsthroughthe sandpack column.Therateofmovement ofthebrothwas about5in.perhour.Theamountofoilreleasedwith biosur-factantfloodingwasmeasured,andtheadditionaloilrecovery percentagewascalculated.

Statisticalanalysis

Alloftheexperimentaldataareexpressedintermsof arith-meticaveragesofatleastthreeindependentreplicates,with standarddeviation(±),andthemutantswereanalyzedforany signiﬁcantincreaseinefﬁciencyusingSigmaPlot12.5(Systat Software,Inc.,USA).

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Table1–Morphologicalandbiochemicalcharacteristics ofB.subtilisR1.

Tests Results

Gramstaining Gram+ Shapeofcell Shortrods Sporeformation + Motility + Anaerobicgrowth − Catalase + VPtests + NO3reduction + Utilizationofcitrate + ProductionofIndole + Hemolysis +

Carbohydratefermentation(acidproduction)

Glucose + Arabinose − Xylose − Mannose + Enzymeproduction Proteinase + Gelatinase + Amylase + Chitinase −

‘+’,positivereaction;‘−’,negativereaction.

Results

and

discussion

Differentisolateswerescreenedforbiosurfactantproduction on blood agar plates and one isolate R1 was selected for further screening. It was studied for morphological and biochemicaltests,andwasfoundtobeGrampositive,motile short rods, with spores. The details are as presented in

Table 1. It was identiﬁed as B. subtilis by 16S rRNA gene

sequencing and the nucleotidesequence was deposited in NCBIGenBank(GenBankaccessionno:DQ922949).Previously we’ve reported biosurfactantproductionbyB. subtilisR1 in molasses media.2,14 _Molasses _is _a _cheaper _{agro-industrial}

byproduct. The production of molasses is related to the productionofsugarcanecrop.Itwasreportedthatthecane molasses productionhasﬂuctuatedsigniﬁcantlyinthe last onedecadeinIndia(India’sSugarIndustry:Analysing Domes-ticDemandandRecentTrends,accessedon13thSeptember 2015).Molassescontainssubstantialamountofsugar,thatis notextractableandiswidelyutilizedforalcoholproduction byfermentation.Ithasbeenestimatedthatalmostallofthe molasses(∼95%)producedinIndiaisusedfortheproduction of Alcohol, and it is the raw material forpotable alcohol, industrialalcoholandbioethanol.Thusalmostallavailable molasses is expended by ethanol industry and is tightly regulated inthe Indian market.Most of the states donot allowfreeinterstatemovementsandsellingofanymolasses requires a permitNOC from the state excise departments (http://www.indiansugar.com/NewsDetails.aspx?nid=1774;

andIndia’sSugarIndustry:AnalysingDomesticDemandand RecentTrends,accessedon13thSeptember2015).Duetoall those uncertainties inavailability and ﬂuctuating pricesof canemolasses,wetestedglucosebasedminimalmediumfor biosurfactantproduction.

Effectofdifferentconditionsongrowthandbiosurfactant production

Inordertostudyeffectofvariousenvironmentalfactorson growthandbiosurfactantproductionB.subtilisR1wasgrown atdifferenttemperatures(30,40,45,50and80◦C)anddifferent concentrationsofNaCl(0%,5%,7%),inminimalmediumwith 2%(w/v)glucoseasacarbonsource.Thegrowthwas mon-itoredbymeasuringOD600andSTwasmeasuredafterevery

12

A

C

D

B

10 8 6 4 2 0 0 6 12 24 30 36 48 Time (h) OD 600 54 60 72 78 84 96 12 10 8 6 4 2 0 0 6 12 24 30 36 48 Time (h) OD 600 54 60 72 78 84 96 80 70 60 50 40 30 20 10 0 0 24 48 Time (h) Surf ace tension (mN/m) 72 96 80 0% 30ºC 40ºC 45ºC 50ºC 80ºC 30ºC 40ºC 45ºC 50ºC 80ºC 5% 7% 0% 5% 7% 70 60 50 40 30 20 10 0 0 24 48 Time (h) Surf ace tension (mN/m) 72 96

Fig.1–ThegrowthandbiosurfactantproductionproﬁleofBacillussubtilisR1atdifferenttemperatures(A,B),andin presenceofdifferentsaltconcentrations(C,D).

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Fig.2–Thescreeningofmutants(A)andparentstrainR1(B)onBloodAgarplates.Thezoneofhemolysiscanbeseen surroundingbiosurfactantproducingcolonies.

24htill96h.B.subtilisR1showedgrowthattemperaturerange

of30–50◦C,butnotat80◦C(Fig.1A).Wherehighestgrowth wasobservedat30◦C,followedby40–50◦C.B.subtilisR1also producedbiosurfactantfrom30to45◦C,whichsubstantially reducedtheSToftheculturemediumfrom66±1.25mN/m to29±0.85mN/mwithin24h(Fig.1B).Productionof biosur-factantatthermophilic conditionhasapparent advantages whilelargescaleproductionorscale-up,suchasiforganism cangrowathightemperatureitwouldreducecostofcooling andalsotherearelesschancesofcontamination.B.subtilis

R1alsoshowedgrowthinpresenceofupto7%salt,wherein absenceofsaltbettergrowthwasobserved(Fig.1C).B.subtilis

R1producedbiosurfactantthusreducingtheSTinpresenceof upto5%salt,whereSTwasreducedfrom66±1.25mN/mto 29±0.85mN/mwithin24h(Fig.1D).Inpresenceof7%saltthe STwasreducedto∼41mN/m.Elzzazyetal.15_reported_similar

observationforthehalophilicstrainV.salarius(KSA-T)where highestbiosurfactantwasproducedinpresenceof4%(w/v) salt(NaCl),however,inpresenceofhighersalt (10%w/v)it lost25%ofitsactivity.

Randommutagenesisusingchemicalmutagenfor enhancedproductionofbiosurfactant

Production economy is the limiting factor of any biotech-nologicalprocess,evenincaseofbiosurfactantproduction. Thesuccessofbiosurfactantproductiondependson devel-opmentofprocess whichreducesthe overallcosteitherby use of cheaper raw material or by enhancing the biosur-factant productioncapacity of the bacteria byaltering the geneticmakeupbyeitherrandomorsite-directed mutagen-esis.Wereporteduseofcheaperrawmaterialslikemolasses and cheese whey for biosurfactant production by B.

sub-tilis R1,whereit produced biosurfactant thus reducing the

ST to29–34mN/m.2 _In _present _paper_we _describe_the

ran-dom mutagenesis of B. subtilis R1 using alkylating agent EMS to enhance the biosurfactant production. EMS is an organicmutageniccompound(C3H8SO3),whichinduces ran-dommutationsingeneticmaterialbynucleotidesubstitution; particularly by guanine alkylation.16 _Random _mutagenesis

by using physical (U.V. light) or chemical mutagens (like

45 40 35 30 25 20 15 10 5 0 BS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Mutants Surface tension CMD1 CMD2 CZ/CS CZ/CS ST (mN/m), CMD 1, CMD 2 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Fig.3–Thescreeningofmutantsforbiosurfactantproductiononbloodagarplates(Cz/Cs)andsurfacetensionreductionat differentdilutions(undiluted–ST;10×diluted–CMD−1;and100×diluted–CMD−2).

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EMS,NTGetc.)are reportedtobetimeconsumingbut suc-cessful in ﬁnding out improved mutants. Tahzibi et al.17

reportedbiosurfactantproductionbyPseudomonasaeruginosa

mutantsderivedbyrandommutagenesiswithN-methyl-N -nitro-N-nitrosoguanidine(MNNG).ThemutantdesignatedP.

aeruginosaPTCC1637producedrhamnolipidatconcentration

10 times higher than parent strain. Lin et al.18 _reported _a

B.licheniformismutantderivedbyrandommutagenesiswith

MNNG, thus producing 12 times more biosurfactant than theparentstrain.Inpresentstudytotal42mutantcolonies obtainedaftermutagenesiswerescreenedforenhanced bio-surfactant productionusingBA plates (Fig. 2).Theisolated mutants(Fig.2A)andparentstrainR1(Fig.2B)werecompared forzoneofhemolysisonBAplates.Thehemolysiszone sur-roundingtheparentstrainB.subtilisR1andmutantcolonies onBAplate(Cz),andthesizeofcolonies(Cs)weremeasured (mm)andtheefﬁciencywascalculatedasaratioofCz/Cs.Total 11mutantsshowed biggerhemolysiszone(1.7–4mm)than parentstrainR1(1.4±0.2mm),and9mutantsshowednozone ofhemolysis(Fig.3).Allthemutantswerescreenedfor biosur-factantproductioninminimalmedia,andSTwasmeasured (undiluted,10×diluted–CMD−1,and100×diluted–CMD−2), where7mutantsshowednogrowthinminimalmedium.The mutantnumber10,13,25,26,28,and35showedsimilaror lower ST than parent strain R1 (28–29mN/m), but showed

0 2 4 6 pH % activity 8 10 12 20 40 60 80 100

A

B

0 0% 5%

0th day 9th day 10th day 7% 10% % salt 15% 20% % activity 20 40 60 80 100

Fig.4–ThestabilitystudiesofbiosurfactantatdifferentpH (A)andinpresenceofdifferentsaltconcentrations(B),for durationof10days. 0 10 20 30 40 50 60 100 000 1000 10 0.1 Surface tension (mN/m) Biosurfactant concentration (mg/l)

Fig.5–TheCMC(mg/L)ofBacillussubtilisR1biosurfactant.

similar CMD−1 andCMD−2 valuesafter10× and100× dilu-tions(Fig.3).WhenthedataofCz/Cs,ST,CMD−1andCMD−2 ofall42mutantsandparentstrainR1werestatistically ana-lyzedusing‘One-SampleSignedRankTest’,itwasobserved tobestatisticalsigniﬁcantwithpvalue<0.001.Hence,thesix selectedmutantsandparentstrainR1werefurtheranalyzed by‘Kruskal–WallisOneWayAnalysisofVariance(ANOVA)on Ranks’,wherethedifferenceinthemedianvaluesamongthe treatmentwere statisticalinsigniﬁcant(p=0.423forST and

p=1.000forCMD−1andCMD−2).Henceasobserved,basedon zoneofhemolysis,mutantsshowedbetterbiosurfactant pro-duction,butwhenthedataforST,CMD−1andCMD−2 were analyzed,allthemutantswereeithersimilarorpoor biosur-factantproducers,ascomparedtoparentstrainB.subtilisR1 (Fig.3).Thereforenoneofthemutantswereselectedandonly parentstrainR1wasusedforallfurtherexperiments. Stabilitystudies

Theenhancedstabilityunderharshreservoirconditionsisone ofthe importantcriterionsforselectingany compoundfor

Fig.6–TheTLCofbiosurfactantdevelopedusing methanol:sulphuricacid(A),iodinevapor(B)andblood reagent(C).

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EORapplications.Thuswestudiedtheproducedbiosurfactant underdifferenttemperatures,saltconcentrationsandpH val-ues.Itwascompletelystablefor10daysat30–80◦C,andno changewasobservedinST.Italsoretained64.28–80.96% sur-faceactivitytill10thdayatpH4.0–12.0(Fig.4A).Thesurface activitywasreducedto40%atpH2.0,anditstarted precipi-tating.Whereas,itretained≥64%surfaceactivityevenat10% salt(NaCl),after10days(Fig.4B),andat15–20%salt,the bio-surfactantactivitywasreducedto64–52%,after5thdayitself (Fig.4B).ThusthebiosurfactantproducedbyB.subtilisR1was foundtobestableupto80◦C,pHrangeof4.0–12.0,and10%salt till10days.Itissimilartootherrepotsforstabilityof biosur-factantproducedbydifferentmicroorganisms.4,11,15,19–22_The

useofsuchstablebiosurfactantisadvantageousinanyfuture commercialapplicationinoilindustryforcleaningoil-sludge instoragetanks,oilimmobilizationandMEOR.

BiosurfactantyieldandCMCdetermination

Thebiosurfactantwasextractedandpartiallypuriﬁedusing acidprecipitationmethodfollowedbylyophilizationtocollect crudeoff-white biosurfactantpowder.Theobserved biosur-factantyieldwas0.7±0.23g/L.TheCMCvaluecharacterizes the efﬁciencyofbiosurfactant,wherelower theCMC – bet-terthebiosurfactant.FormeasurementofCMC,biosurfactant obtainedafterlyophilizationwasdissolvedindistilledwater andSTwasmeasured.WeobservedmaximumreductioninST (∼30to29mN/m)at19–20mg/Lbiosurfactant,beyondwhich concentration STremained unchanged (Fig. 5), thus it was considered as CMC for current biosurfactant.Previously in molassesmediaweobservedbiosurfactantproductionbyB.

subtilisR1 withCMCvalueof39.5±0.66mg/L,14 _whereas_in

currentglucosebasedminimalmediatheCMCobservedwas 100.0

A

B

98 96 94 92 90 88 86 84 82 80 78 76 74 72 70 68 66 64 62 4000.0 3600 3200 2800 2924.23 3070.02 3316.52 _2924.36 2862.74 1731.46 1650.34 1544.05 1468.53 2952.38 2851.54 3315.82 1712.21 1658.41 1462.19 1377.63 722.15 2400 2000 1800 1600 cm-1 %T %T 1400 1200 1000 800 600 400.0 60.0 52.0 50 45 40 35 30 25 20 15 10 5 0.0 4000.0 3600 3200 2800 2400 2000 1800 1600 cm-1 1400 1200 1000 800 600 400.0

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20±0.56mg/L(Fig.5).Thusweobservedbiosurfactant with betterCMCvalueinglucosecontainingminimalmedium,as comparedtopreviouslyreportedmolassesmedium.

Biosurfactantcharacterization

Preliminary characterization of Biosurfactant was done by TLC.Theseparatedbiosurfactantcomponentsweredetected bythree different methods– saturating plates withIodine vapor,sprayingwithmethanol:sulphuricacid(95:5)reagent followedbycharringplatesat100◦Cfor30min,andalsoby sprayingtheplateswithbloodreagent.Browncoloredspots were detectedafter saturation with Iodine vapors and the spots were charredand observed asdark blackspots with methanol–sulfuricacidreagent (Fig. 6),whichcanbe corre-lated to lipidic-organic compounds. When the plates were sprayed with 20% blood reagent, hemolysis was observed surroundingtheseparatedbiosurfactantafter20min,which showed the presence of hemolytic biosurfactant fractions. TheFTIRspectrumofpartiallypuriﬁedbiosurfactant(Fig.7A) showedbandscharacteristicofpeptidesat3315cm−1 (N H stretchingmode) andat1658cm−1 (stretchingmodeofthe CO Nbond).Thebandsat2952–2924cm−1,2851cm−1andat 1462cm−1,1377cm−1reﬂectaliphaticchains( CH3, CH2 ). TheFTIRspectrumofbiosurfactantshowedsimilaritytocyclic lipopeptideslikesurfactin(Fig.7B)andlichenysinproducedby bacilli,asreportedbyotherresearchers.8,13_The_{biosurfactant}

wasfurtheranalyzedbyMALDI-TOF-MS,inthepositivemode, whereitprimarilyshowedthesodiumadductsforthe com-pounds.MALDIshowedthreemainions, beingthesodium adductsatm/z1030,1044and1058(Fig.8).TheMALDI analy-sisshowedthatthesamplealsocontainedasmallamountof otherlipopeptidesatm/z1485,1499,1531,1559,1661and1639 (Fig.8).Howeverthoselipopeptidesarepresentinminor quan-tities,andmaybesimilartolipopeptide–fengycin.23,24 _From

theanalysisitwasobservedthatbiosurfactantcontainedlipid andpeptidemoiety,andmajorfractionwassimilartoa mix-tureofC13–15fattyacid-surfactinhomologues.25–27

Antifungalactivity

BacteriaofthegenusBacillusareknownproducersofanumber of lipopeptides with antimicrobial activity like iturin, sur-factinand fengycin.Thefengycin isreportedtoinhibitthe growth of ﬁlamentous fungi, and surfactin is reported for inhibiting the growth of bacteria and viruses, but has lit-tleornoinhibitioneffectsonﬁlamentousfungi.28–30 _Asaka

and Shoda,31_reported_a_B._subtilis_strain_producing_iturin_A

andsurfactin,tobeeffectiveforthecontrolofdamping-off causedbyR.solaniintomatoplants.Joshietal.13_also_reported

thebiosurfactantproductionandinhibitionofvariousplant pathogenicfungilikeChrysosporiumindicum,Alternariaburnsii,

Fusariumoxysporium,andRhizoctoniabataticolabythegrowth

ofB.subtilis20B.Incurrentpaperalsowechecked

antifun-galactivityoftheproducedbiosurfactantagainstsomefungal plantpathogensonPDAplates.B.subtilisR1showed produc-tion ofantifungal compound on PDA plates, co-inoculated withvariousfungi.Amongthefungitested,growthof

Rhizocto-niabataticolaandFusariumoxysporium(Fig.9)wasinhibitedby

B.subtilisR1,whereas,thegrowthofAspergillusnigerand

Peni-cilliumchrysogeniumwerenotinhibited.B.subtilisR1showedno

chitinaseproductionbutantifungalactivityagainstmycelial growthofplantpathogenicfungi,soitcanbehypothesized thatitproducesantifungalcompoundsimilartofengycin,as alsoevidentbyMALDI-TOFresults.Kimetal.23_reported

co-productionofbiosurfactantlipopeptides–IturinA,Fengycin, and Surfactin A, from B. subtilis CMB32, and its inhibitory effectonfungalpathogenColletotrichumgloeosporioides.They observedthatamongthethreelipopeptidesproduced,iturin Aandfengycinshowedantifungalactivity,whereassurfactin

×105 1.50 1.25 1.00 0.75 0.50 0.25 0.00 1000 1016.584 1030.591 1134.564 1118.564 1090.623 1074.599 1044.613 1058.632 1485.692 1499.703 _1531.727 1559.754 1661.747 1693.770 1102.589 1100 1200 1300 1400 1500 1600 1700 m/z Intens . [a.u.]

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B.subtilis R1

Rhizoctonia bataticola

Fusarium oxysporium

B. subtilis R1

Isolate k51

Isolate new

Isolate HS3

Fig.9–AntifungalactivityofB.subtilisR1againstplantpathogenicfungi–RhizoctoniabataticolaandFusariumoxysporium.

Measuring cylinder Column holder Sand pack column Brine tank

Crude oil tank

Peristaltic pump Flow

of Brine / Oil

Fig.10–SchematicrepresentationoftheSandPackColumnsystemusedforMEORstudies.

Aonlyactedasasynergisticfactorfortheantifungaleffect ofiturin.Theco-productionofsurfactantsandfungicideisan interestingcharacteristicwithpotentialpracticalapplications inpetroleum,environmentalandagriculturalindustries.The observedresultsshowedthattheco-productionof biosurfac-tantandantifungalcompoundshavepotentialapplicationsto reducetheusageofsyntheticsurfactantsandchemical fungi-cides,thuscontributingtotheenvironmentalprotection.

Oildisplacementstudiesusingsandpackcolumn

Previouslyweobserved32.18%additionaloilrecovery(AOR) usingbiosurfactantproducedbydifferentisolatesincluding

B. subtilis R1, using molasses as a carbon source.14 _In _the

present paper wedescribe the production ofbiosurfactant using glucosebased medium and its application inMEOR. Insand pack experiments(Fig. 10) residual oilin columns was mobilized during passage of the biosurfactant broth and nearly 33±1.25% of residual oil was recovered using biosurfactantbroth.Theresultsindicatedthatbiosurfactant mobilizedoilin the sand columnmodeland aided in bio-surfactantbasedMEOR.ItissimilartootherreportsofAOR

usingbiosurfactants,wheretheyhaveobserved20–37%AOR insand-packcolumnsorcore-ﬂoodexperiments.11,22,32,33

Conclusion

It wasconcluded thatisolate B. subtilisR1 could grow and producebiosurfactantatupto45◦Candinpresenceof7% salt,inglucosebasedminimalmedium.Itwassubjectedto randommutagenesisusingEMS,where42mutantswere iso-latedandscreenedforbiosurfactantproduction.Noneofthe mutantsshowedstatisticallysigniﬁcantdifferencein biosur-factant production as compared toparent strain R1. Thus parentstrainR1wasselectedforfurtherexperiments.It pro-duced0.7±0.23g/LbiosurfactantwithCMCof20±0.56mg/L. Itwasalsoquitestableathightemperature,salinityandpH valuesfor10days.Itwasstructurallysimilartolipopeptides– surfactinandfengycin.Sandpackcolumnstudiesshowedthat biosurfactantcanrecover33±1.25%additionalcrudeoilandit alsoinhibitedsomeoftheplantpathogenicfungi.ThestrainB.

subtilisR1canbepotentiallyexploredforbiosurfactantbased

MEORapplicationsand asabiocontrolagentinagriculture ﬁeld.

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Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

AllauthorskindlyacknowledgetheguidanceprovidedbyProf. AnjanaJ.Desai,andDepartmentofMicrobiologyand Biotech-nology Centre, TheM.S.University ofBarodafor providing theresearchfacilityandconsumablesneededfortheresearch project.

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