Optimization of Surfactin Production by Bacillus subtilis Is(5)

biosurfactant

322ApplBiochemBiotechnol(2008)150:305–325productioncanbedividedintothreecategories:carbohydrates,hydrocarbons,andvegetableoils.Inthisstudy,thecarbonsourcestestedwerecarbohydratesources(D-glucose,D-fructose,sucrose,maltose,lactose,galactose,D(+)-mannose,D-sorbitol,glycerol,glucosesyrup,molasses,andmaltextract),vegetableoils(soybeanoilandoliveoil),andfinally,hydrocarbons(hexadecaneandparaffinoil).

Inthisstudy,theresultsrevealedthatmolassesisthebestcarbonsourceforsurfactinproductionandgrowth(Fig.6).However,sucrose,maltose,D-sorbitol,andmaltextractgavelevelsofsurfactinandcellgrowthcomparabletothoseofglucose.Thesugars,lactoseandgalactose,sharplyinhibitedbacterialgrowthandresultedinlowersurfactinproductivitiescomparedtoglucose.Growthandsurfactinproductionincreasedremarkablycomparedtoglucoseandsteadilybyincreasingmolassesconcentrationupto16g%(Fig.7c),however,maltextractat4g%showedbiosurfactantproductivitycomparabletothatofglucose;thisfindingisverypromisingbecausebothmolassesandmaltextractarecheaperandpromisingalternativestoglucose.

Itisworthytonotetheinhibitoryeffectofvegetableoils(soybeanoilandoliveoil)andhydrocarbon(hexadecaneandparaffinoil)onsurfactinproduction(Fig.6),althoughitwaspostulatedthathydrocarbonsenhancetheproductionofbiosurfactantsbybacteria[33].However,manyauthorsdocumentedthat,unlikethemicroorganismsproducingglycolipid,polymeric,orfattyacidtypebiosurfactants,Bacillussp.requiresonlycarbohydratestoproducelipopeptide-typebiosurfactant[34,35,36,37,38].Thefactthatsomebiosurfactantsareproducedevenwhenthecellsarenotgrownonhydrophobicsubstratessuggeststhattheirfunctionisnotsolelyrestrictedtothestimulationofsubstrateavailabilitybuttheyperformotherdefensiveandbiologicalfunctions[39].

Therefore,attheendofthisstudy,thehighestsurfactinproductionbythisBacillusisolateBS5canbeachievedbytheuseofmolassesat160ml/lforB.subtilisisolateBS5.Alternativecarbonsourcescanalsobeeffectivelyusedforhighsurfactinproduction;theseincludemaltextract(40g/l)andglucose(40g/l).

Thenitrogensourceplaysanimportantroleintheproductionofsurface-activecompoundsbymicroorganisms[21].Inthisstudy,thenitrogensourcestestedwereorganic(ammoniumoxalate,urea,yeastextract,peptone,tryptone,andcornsteepliquor)andinorganicsources(sodiumnitrate,potassiumnitrate,ammoniumnitrate,ammoniumchloride,ammoniumbromide,ammoniumcarbonate,andammoniumsulfate).

Inthisstudy,itwasfoundthatsodiumnitratewasthebestnitrogensourceforsurfactinproductionbyBacillusisolate(Fig.8).However,othertestednitrogensourcesdecreasedsurfactinproductionwithdifferentdegreesandsuchdecreasewasmorepronouncedincaseofproteinsources.

Noadvantagewasgainedconcerningsurfactinproductionbytheuseofaminoacids,althoughglutamicacidgavesurfactinproductivitycomparabletothatofsodiumnitrateintermsofactivityonly(Fig.10).Similarresultswerereportedwheredifferentaminoacidsdidnotshowanynotabledifferenceinsurfactinproductionwhentheywereseparatelyaddedtotheculturemedium[40].Althoughtheapproachwasdifferent,similarresultswerealsoreportedbyCooperetal.[6].Theyreportedthatnoimprovementinsurfactinproductionwasobtainedwhenamixtureofalloftheaminoacidsenteringinthestructureofthesurfactinlipopeptidewasaddedtothemedium.Whentheeffectofglutamicacidonsurfactinproductionwasfurtherstudiedatdifferentconcentrations,maximumsurfactinproductionandmaximumbacterialgrowthwereobtainedat1g%(Fig.11).However,thelevelofsurfactinproductionatthisconcentration(1g%glutamicacid)wasstillcomparable(intermsofactivity)tothatproducedbysodiumnitrateatitsoptimumconcentration(0.5g%).Theuseofglutamicacidasthesolenitrogensourcewaspreviouslyreportedbysomeauthors[41].

ApplBiochemBiotechnol(2008)150:305–325323Therefore,attheendofthisstudy,thehighestsurfactinproductionbythisBacillusisolateBS5canbeachievedbytheuseofsodiumnitrateat5g/l.Alternativenitrogensourcescanalsobeeffectivelyusedforhighsurfactinproduction;theseincludeglutamicacid(10g/l),ammoniumoxalate(10g/l),andammoniumnitrate(5g/l).

Thestudyoftheeffectofsomeinorganicsaltsormineralsrevealedthatgrowthandsurfactinproductionweregreatlyenhancedinthepresenceofthefollowingmultivalentcations:zinc,iron(II),iron(III),andmanganese(II)at0.1mMconcentrations(Fig.13)withiron(III)showingbetterresultsthaniron(II)intermsofsurfactinproduction.Theobservedstimulatoryeffectofiron(II)andiron(III)ongrowthandsurfactinproductionbyaBacillusspecieswaspreviouslyreportedbyWeiandChu[42].TheyrecommendedraisingironconcentrationsfromthemicromolartothemillimolarleveltogreatlyenhancethesurfactinproductionfromB.subtilisATCC21332.In2002,thesameauthorsreportedthatagreatenhancementofsurfactinproductivitywasobtainedbyaddingmanganese(II).Itisestablishedthatthismetalisa“key”metalfortheproductionofsecondarymetabolitesbyBacillusspecieswithouthavinganeffectoncellgrowth[6].

Concerningtheinhibitoryeffectofmetalions,Cu2appearedtobethemostpotentinhibitorofbothgrowthandsurfactinproductionbythetestisolate(Fig.13);thisfindingwasinagreementwiththatreportedbyWeiandChu[43].

Consequently,anumberoftraceelementscouldbeusedforoptimumsurfactinproductionbythetestisolate.ThesetraceelementswereZnSO4(1.0mM),FeCl3·6H2O(1.0mM),andMnSO4·H2O(0.1mM).

Basedontheresultsaccumulatedfromthepreviousstudyonnutritionalelementsandmediacomponentsaffectingsurfactinproduction,modifiedmediawereformulated.Thesemediacombinedtheoptimumelementsselectedfromeachnutritionalcategory,i.e.,carbonandnitrogensourcesaswellastraceelements.Amongthedifferentformulatedmedia,MMSM,whichcontainedoptimumcarbonsource(molasses160ml/l),optimumnitrogensource(NaNO35g/l),andoptimumtraceelementsachievedobviousincreaseinsurfactinproductivityofaboutthreefoldcomparedtoMSM.

TimeCourseofSurfactinProductionbyBacillusIsolateBS5inMMSM

ItwasnecessaryattheendoftheoptimizationphasetoredeterminetheoptimumincubationperiodrequiredformaximumgrowthandsurfactinproductioninMMSM.Thisisbecausetheoptimumincubationperiodformaximumsurfactinproductionmayprobablydifferinthemodifiedmediumfromthoseinthebasalmedium(MSM).Theresults(Fig.16)showednearlysimilarprofilestothoseobtainedinMSM(Fig.1)whereproductionwasgrowth-associated,however,ashorterincubationperiodwasrequiredinMMSM.DetectionofPlasmid(s)anditsRelationtoSurfactinProduction

Neitherhighnorlowmolecularweightplasmidswereobservedinthetestisolatebythetechniqueused(Fig.17).Thus,itcanbeconcludedthatthesurfactin-codinggeneislocatedinthechromosomalDNAofthetestedisolateandnotplasmid-associated.Similarly,studiesconductedbyFlecketal.[44]onbiosurfactantproductionbyB.subtilisB1andPseudomonasaeruginosaP1showedthatnoneofthestrainspresentedplasmids,eitherofloworhighmolecularweights.TheyconcludedthatthecodinggenesforbiosurfactantproductionarelocatedinthechromosomalDNA.Thisfindingmaybeadvantageousfromtheindustrialpointofviewbecauseoneofthemajordrawbacksthatpreventaproducingbacteriumfrombeingacandidateindustrialstrainisgeneticinstability.Geneticinstability

324ApplBiochemBiotechnol(2008)150:305–325and,consequently,fluctuationofproductionleveliscommonlyencounteredamongstrainsthathavetheproductiongenescarriedonplasmids.

AcknowledgementsTheauthorswouldliketothankDr.KhaledAbou-Shanab,alecturerofMicrobiologyandImmunology,FacultyofPharmacy,AinShamsUniversity,Cairo,Egypt,forkindlyprovidingthestandardE.coliDH5α/pUC18andgivingadviceonplasmiddetectioninthetestedisolate.

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