Optimization of Surfactin Production by Bacillus subtilis Is

Optimization of Surfactin Production by Bacillus subtilis Isolate BS5

ApplBiochemBiotechnol(2008)150:305–325

DOI10.1007/s12010-008-8155-x

OptimizationofSurfactinProductionbyBacillussubtilisIsolateBS5

A.MohammadAbdel-Mawgoud&

M.MabroukAboulwafa&

NadiaAbdel-HaleemHassouna

Received:17October2007/Accepted:15January2008/

Publishedonline:29February2008#HumanaPress2008

AbstractBacillussubtilisBS5isasoilisolatethatproducespromisingyieldofsurfactinbiosurfactantinmineralsaltsmedium(MSM).ItwasfoundthatcellulargrowthandsurfactinproductioninMSMweregreatlyaffectedbytheenvironmentalfermentationconditionsandthemediumcomponents(carbonandnitrogensourcesandminerals).OptimumenvironmentalconditionsforhighsurfactinproductionontheshakeflasklevelwerefoundtobeaslightlyacidicinitialpH(6.5–6.8),anincubationtemperatureof30°C,a90%volumetricaerationpercentage,andaninoculumsizeof2%v/v.Formediacomponents,itwasfoundthattheoptimumcarbonsourcewasmolasses(160ml/l),whereastheoptimumnitrogensourcewasNaNO3(5g/l)andtheoptimumtraceelementswereZnSO4·7H2O(0.16g/l),FeCl3·6H2O(0.27g/l),andMnSO4·H2O(0.017g/l).AmodifiedMSM(molassesMSM),combiningtheoptimummediumcomponents,wasformulatedandresultedinthreefoldincreaseinsurfactinproductivitythatreached1.12g/l.Noplasmidcouldbedetectedinthetestedisolate,revealingthatbiosurfactantproductionbyB.subtilisisolateBS5ischromosomallymediatedbutnotplasmid-mediated.KeywordsSurfactin.Bacillus.Production.Optimization

Introduction

Naturallyoccurringsurface-activecompoundsderivedfrommicroorganismshavegainedattentioninthepastfewdecadesbecauseoftheirbiodegradability,lowtoxicity,ecologicacceptability,andabilitytobeproducedfromrenewableandcheapersubstrates[1,2].Biosurfactantsfindapplicationsinthecosmetic,pharmaceutical,andfoodindustriesasemulsifiers,humectants,dispersants,anddetergents[3,4].Moreover,theyaresuitedfor

A.M.Abdel-MawgoudM.M.Aboulwafa(*):N.A.-H.Hassouna

DepartmentofMicrobiologyandImmunology,FacultyofPharmacy,AinShamsUniversity,

Cairo,Egypt

e-mail:maboulwafa@yahoo.com

306ApplBiochemBiotechnol(2008)150:305–325environmentalapplicationssuchasbioremediation,dispersionofoilspills,andwastetreatment[3].

Amongthemanyclassesofbiosurfactants,lipopeptidesareofgreatinterestbecauseoftheirhighsurfaceactivitiesandtherapeuticpotential[5].SurfactinisoneofthemostefficientbiosurfactantssofarknownwhichbelongstothelipopeptidefamilyexcretedbyBacillussubtilisspp.[6].Theincreasinginterestinsurfactinisbecauseofitsamphiphiliccharacter,whichisresponsibleforitsexcellentsurface-activepropertiesasitreducesthesurfacetensionofwaterfrom72to27mN/mataconcentrationaslowas0.005%[7].Inaddition,surfactinsexhibitdiversebiologicalactivitiessuchasantiviralandantimycoplasma[8,9],antitumoral

[10],inhibitionoffibrinclot,andantibacterialproperties[7].

Althoughpromising,biosurfactants,ingeneral,competewithdifficultyagainstthechemicallysynthesizedcompoundsonthesurfactantmarketbecauseoftheirhighproductioncosts(atleast50timesmoreexpensive,dependingonthebiosurfactantanditspurity)[11,12].Productioncostisconsideredthebottleneckofmanybiotechnolog-icalprocesses[5].Thesuccessofbiosurfactantproductiondepends,inoneofitsstrategies,onthedevelopmentofcheaperprocessesandtheuseoflow-costrawmaterials,whichaccountfor10–30%oftheoverallcost[5].Molasses[13],peathydrolysate[14],andpotatoprocesseffluents[15]areexamplesofalternativesubstratesthathavebeensuggestedforbiosurfactantproductionbyB.subtilis.Otherstrategiesincludeoptimizingthedifferentenvironmentalandmediacomponentswhichaffectproduction[16].Manyauthorsreportedtheeffectofenvironmentalfactors,pH,temperature,andaerationonthebacterialcellgrowthandbiosurfactantproduction

[17].Theinfluenceofmetalionsonbiosurfactantproductionhasbeenreportedbysomeotherauthors[18,6].Othersreportedthepronouncedeffectofcarbonsourceusedinbacterialcultureonbiosurfactantproduction[19,20].Someauthorsalsoreportedtheeffectandtheroleofnitrogensourcesontheproductionofsurface-activecompoundsbymicroorganisms[21].Therefore,inthisstudy,theeffectofthesefactorsonsurfactinproductionbyB.subtilisisolateBS5wasstudied.ThisisolatewasrecoveredfromEgyptiansoilthroughanextensivescreeningprogramandshowedpotentialsurfactinproductivity[22].Thestudyaimedatimprovingsurfactinproduction,takingintoaccounttheeconomicconsiderationsoftheproductionprocess;thiswasaccomplishedthroughoptimizingtheenvironmentalandnutritionalproductionconditions.PlasmidextractionprocedurewasalsoperformedintheB.subtilisisolateBS5toknowwhetherproductionischromosomallyorplasmid-mediated.

MaterialsandMethods

Microorganisms

B.subtilisisolateBS5isapromisingsurfactinproducerobtainedthroughanextensivescreeningprogram[22].EscherichiacoliDH5α/pUC18(Hanahan1983;VieiraandMessing1982)waskindlyprovidedbyDr.KhaledAbou-Shanab(FacultyofPharmacy,AinShamsUniversity,Cairo,Egypt).

CultureMedia

Thebasalmineralsaltsmedium(MSM)andthemodifiedMSM(M1,M2,andM3)wereusedinthisstudyandtheircompositionsarelistedinTable1.

ApplBiochemBiotechnol(2008)150:305–325

Table1CompositionoftheculturemediausedforsurfactinproductionbyB.subtilisisolateBS5.Natureofingredients

BasalmediumMSM

C-source(amount/l)N-source(amount/l)Minerals(amount/l)

Glucose(20g)NaNO3(2.5g)ZnSO4·7H2O(0.0015g)FeSO4·7H2O(0.0005g)MnSO4·H2O(0.0015g)H3BO3

(0.0003g)CuSO4·5H2O(0.00015g)Na2MoO4·2H2O(0.0001g)CaCl2·2H2O(0.05g)MgSO4·7H2ONaClKCl

H3PO4(85%)dH2O

ModifiedmediaM1Glucose(20g)NaNO3(2.5g)

ZnSO4·7H2O(0.16g)FeCl3·6H2O(0.27g)MnSO4·H2O(0.017g)

M2Glucose(20g)NaNO3(5g)

M3a

307

Molassesb(160ml)NaNO3(5g)

Othercommoningredients(amount/l)

KOHpellets(forpHadjustment)

0.4g1g1g10ml

To1,000mlabout16g

(togivepH7.2)

M3istheoptimizedmedium.

Stockmolassessolutionwaspreparedbysuspendinganamountoflocalgrademolasses(SugarsandComplementaryIndustries,Hawamdeya,Egypt)inanequalamountofdH2Oandthenexposedtothesteamofboilingwaterbathfor1hinameasuringcylinder.Thesupernatantwasthencollectedandusedformediumpreparation.

FermentativeProductionofSurfactin

Theseedculturewaspreparedbytransferringaloopfulfromafreshculturegrownontonutrientagarinto25mlMSMcontainedin250mlErlenmeyerflask.Theflaskwasincubatedat250rpmand30°Cfor36h.

Theproductionprocesswascarriedout,unlessotherwisementioned,inErlenmeyerflasks(250ml)containing50mlaliquotsofthefermentationmediumundertest.Theflaskswereinoculatedwiththeseedcultureat2%v/vandincubatedinashakingincubator(250rpm)at30°Cfor7days(fortimecourseexperiments)and3days(otherexperiments).Atdifferenttimeintervals(timecourseexperiments)orattheendtheincubationperiod(otherexperiments),thefermentationbrothwassampledfordeterminationofbiomass,biosurfactantconcentration,andbiosurfactantactivity.

308ApplBiochemBiotechnol(2008)150:305–325AnalyticalMethods

BiomassDetermination

Cellulargrowthwasexpressedintermsofdrycellweightwhichwascalculatedfromtheequationofacalibrationcurveconstructedbetweenopticaldensity(OD578nm)anddrycellweightofthetestedisolateB.subtilisBS5[23].

Dryweighteg%T?OD578nm=65:648

SurfactinConcentration

ThegravimetricmethoddescribedbyNitschkeandPastore[5]wasusedforthedeterminationofsurfactinconcentrationwithminormodification.Theculturebrothwascentrifugedat10,000×gfor10mintopreparethecellfreesupernatant(CFS).AnaliquotoftheCFSwasacidifiedtopH2.0using1NHClinpreweighedplastictubes,leftovernightat4°C,andthencentrifugedat10,000×gfor20min.Theresultingsupernatantwasdiscardedandtheremainingpelletwasdriedinanincubatorat37°Cfor24–48h(tillreachingaconstantweight).Thenetweightofthecrudeprecipitatewasdetermined,andthecrudesurfactinconcentration(ing/l)wascalculated.

SurfactinActivity

Thiswascarriedoutusingthefollowingmethods:

OilspreadingtestTheoilspreadingtest(OST)wasconductedasdevelopedbyMorikawaetal.[24]andrecommendedbyYoussefetal.[25].Thediameterofthevisuallydetectableclearhalowasmeasured.

MeasuringsurfacetensionstalagmometricallyThebiosurfactantactivitywasmonitoredbymeasuringsurfacetensionusingTraube’sstalagmometer(dropweightmethod)at25°C.Thesurfacetensionofthetestsamplewasmeasuredusingthefollowingequation: