Optimization of Surfactin Production by Bacillus subtilis Is(2)

γtest?γ0=m0?mtest

whereγ0isthesurfacetensionofdistilledwater,m0istheweightofdistilledwaterperonedrop,andmtestistheweightofthesample(CFS)peronedrop[26].Thepercentageofsurfacetension(ST)reductionwasdeterminedfromthefollowingequation:

%STreduction??eSTinitialàSTfinalT=STinitial???100:

DifferentFactorsAffectingSurfactinProduction

ThetimecourseofsurfactinproductioninMSMwasstudiedtodeterminethetimerequiredformaximumsurfactinproduction.Theeffectofdifferentenvironmentalandmediacomponentsonsurfactinproductionwasstudied.Inallcases,themonitoredfermentationparametersincludedbiomass,biosurfactantconcentration,andbiosurfactantactivity.

ApplBiochemBiotechnol(2008)150:305–325309EffectofDifferentEnvironmentalFermentationConditions

Thefactorsstudiedincluded:effectofaeration(studiedbyvaryingthevolumeofheadspacein250mlconicalflaskscreatedbyvariationofthevolumeofthemediumintheflaskwhichwillbereflectedinvariationofthelevelofaeration),initialpH,incubationtemperature,inoculumsize.

EffectofDifferentMediaComponentsonSurfactinProduction

Theeffectofdifferentcarbonsources,nitrogensources,andmineralswasstudied.Forstudyingtheeffectoftheadditionofdifferentcarbonsourcesonsurfactinproductionbythetestedisolate,thebasalC-source(glucose)presentinMSMwasreplacedwithothercarbonsources.Thetestedcarbonsourcewasaddedataconcentrationequivalentinitscarboncontenttothatofglucose(20g/l)exceptforoilsandhydrocarbonswheretheywereusedat2%v/v.Thetestedcarbonsourceswerecarbohydratesources(D-glucose,D-fructose,sucrose,maltose,lactose,galactose,D(+)-mannose,D-sorbitol,glycerol,glucosesyrup,molasses,andmaltextract),vegetableoils(soybeanoilandoliveoil),andhydrocarbons(hexadecaneandparaffinoil).

Theeffectoftheadditionofdifferentnitrogensourcesonsurfactinproductionwasstudiedsimilarly.Thestudiednitrogensourceswereclassifiedintoorganic(ammoniumoxalate,urea,yeastextract,peptone,tryptone,andcornsteepliquor)andinorganicsources(sodiumnitrate,potassiumnitrate,ammoniumnitrate,ammoniumchloride,ammoniumbromide,ammoniumcarbonate,andammoniumsulfate).

Theaminoacids(leucine,asparticacid,valine,andglutamicacid)thatconstitutethepeptidemoietyofsurfactinwereincorporatedintheculturemediumforstudyingtheireffectonsurfactinproduction.TheaminoacidsD,L-leucine,L-asparticacid,L-valinewereaddedtoMSMat0.1mM,whereasglutamicacidwasaddedinreplacementofsodiumnitrateinMSM(ataconcentrationequivalentinnitrogencontent).

Theeffectoftheadditionofdifferentmultivalentcationsonsurfactinproductionwasstudiedbyaddingthemtotheglucose–magnesiummediumataconcentrationof0.1mM.Theresultswerecomparedtothecontrolflaskwhichreceivednoadditionalmultivalentcations(glucose–magnesiummedium),i.e.,itwasdevoidoftraceelementsandcalcium.Glucose–magnesiummediumwasderivedfrombasalMSMbystepwiseremovalofthemultivalentcationsandthestudyoftheeffectofremovalongrowth.Thedivalentcationsalreadypresentinthebasalmediumwerecalciumchloride,magnesiumsulfate,andthosepresentinthetraceelementssolution(FeSO4·7H2O,ZnSO4·7H2O,MnSO4·H2O,K3BO3,CuSO4·5H2O,andNa2MoO4·2H2O).Growthunderfourdifferentconditionswasstudiedasfollows:growthinMSMthatwasdevoidofdivalentcationsandtraceelementssolution(TES),growthinMSMdevoidofTESandMg++butcontainingCa++,growthinMSMdevoidofTESandCa++butcontainingMg++,andfinally,MSMdevoidofTESonlybutcontainingbothMg++andCa++.

BiosurfactantProductionbyB.subtilisIsolateBS5inDifferentModifiedMineralSaltsMedia

Basedontheresultsaccumulatedfromthestudyonmediacomponentsaffectingsurfactinproduction,threemodifiedmedia(M1,M2,andM3)wereformulated.Thesemediacombinedtheoptimumelementsselectedfromeachnutritivecategory,i.e.,fromcarbonandnitrogensourcesaswellastraceelements(Table1;“MaterialsandMethods”section).

310ApplBiochemBiotechnol(2008)150:305–325

Bothgrowthandsurfactinproductionweretestedinsuchmedia.Theresultsofthemodifiedmedia(M1,M2,andM3)werecomparedwiththoseofMSM.IsolationandDetectionofPlasmidsinB.subtilisIsolateBS5PlasmidExtraction

PlasmidextractionwasperformedusingthealkalinelysismethodofBirnboimandDoly[27].E.coliDH5α/pUC18wasusedasastandardE.colistrainbearingthepUC18plasmidtoactasapositivecontrol.AgarosegelelectrophoresiswascarriedoutessentiallyasdescribedbySambroukandRussell[28]atanagaroseconcentrationof0.7%.

Results

DifferentFactorsAffectingSurfactinProduction

Beforestudyingtheeffectofenvironmentalfactorsandthedifferentmediumcomponentsaffectingbiosurfactantproduction,theincubationperiodatwhichmaximumbiosurfactantproductionoccurswasdetermined.

TimeCourseofSurfactinProductioninMSM

Theresults(Fig.1)showedthatsurfactinproductionstartedearlyintheexponentialphaseandtheproductionkineticsparalleledthebiomasskineticstoalargeextentduringlogarithmicgrowth.Theproductionprofilewasbiphasic,thefirstphaseextendedupto72hduringwhichproductionwasincreasingatahighrate,whereasinthesecondphase,productionwasincreasingataslowerrateandaslightincreaseinsurfactinproductionwasobtained.Forgrowth,maximumbiomasswasobtainedafter72hofincubationfollowedbyaplateaufor2days,thenadeclinetilltheendoftheincubation(Fig.1).Therefore,theresultsofsubsequentexperimentsweretakenafter72hofincubation.

Crude surfactin conc. (g/L)

Biomass (g%)

1.751.501.25

76543210

1.00

0.75

0.500.250.00

Time (days)

Clear zone diameter (cm)

Fig.1TimecourseofgrowthandsurfactinproductionbyB.subtilisisolateBS5inMSM.SurfactinactivitywasmeasuredusingtheclearzonediameterasdeterminedbytheOST

2.502.252.00

1098

ApplBiochemBiotechnol(2008)150:305–325311

EffectofDifferentEnvironmentalFermentationConditionsEffectofAeration

TheresultsinFig.2showedthatthehighestbacterialgrowthwasattainedatanaerationpercentageof80%(volumemediumis20%).However,maximumsurfactinproductionwasobtainedatanaerationpercentageof90%(volumeofmediumis10%)andtherewasasharpdeclineinsurfactinproductionupondecreaseofaeration.EffectofInoculumSize

Therewasagradualandslowincreaseinsurfactinproductionuponincreasingtheinoculumsizeupto6%v/v.However,bacterialgrowthdecreaseduponincreasinginoculumsize(Fig.3).EffectofMediumInitialpH

FromtheresultspresentedinFig.4,itappearsthatB.subtilisisolateBS5grewatpH6to9.0withthehighestlevelsofcellgrowthrecordedatpHvaluesrangingfrom6.5to9.0.However,thehighestlevelsofsurfactinproductionwereobtainedatpH6.8.OtherpHvalueswereaccompaniedbydecreasedsurfactinproduction.EffectofIncubationTemperature

TheresultsdisplayedinFig.5showthatB.subtilisisolateBS5couldgrowatalltestedtemperatures.However,relativelyhighcellgrowthandsurfactinproductionwasobtainedatincubationtemperaturesof25and30°C.

EffectofDifferentMediaComponentsonSurfactinProductionEffectofCarbonSources

TheresultsinFig.6showedthatthebestcarbonsourceforsurfactinproductionandgrowthismolasses.Noneoftheothercarbonsourceswasfoundtobesuperiortoglucose;

Fig.2Effectofaeration(differentvolumesofMSMin250mlflasks)ongrowthandsurfactinproduction(intermsofconcentrationandactivity)byB.subtilisisolateBS5inMSM.SurfactinactivitywasmeasuredusingtheclearzonediameterasdeterminedbytheOST

1.2

1.11.00.90.80.7

0.60.50.44.55.0

Biomass (g%)

4.0

3.5

3.0

2.5

Volumetric percentage of medium per flask volume (%v/v)

Clear zone diameter by OST (cm)

Crude surfactin conc (g/L)

312

Fig.3Effectofinoculumsizeongrowthandsurfactinproduction(intermsofconcentrationandactivity)byB.subtilisisolateBS5inMSM.SurfactinactivitywasmeasuredusingtheclearzonediameterasdeterminedbytheOST

ApplBiochemBiotechnol(2008)150:305–325

10.09.59.08.58.07.57.06.56.05.55.04.54.03.53.02.52.01.51.00.50.0

1.6

1.51.4

Crude surfactin conc (g

/L)

1.3

1.21.11.00.90.8

0.70.60.5

Inoculum size (% v/v)

however,sucrose,maltose,D-sorbitol,andmaltextractachievedlevelsofsurfactinproductionandcellgrowththatwerecomparabletothoseofglucose.Thesugars,lactoseandgalactose,aswellasthetestedoilsandhydrocarbon(HC)sharplyinhibitedbacterialgrowthandresultedinlowersurfactinproductivitiescomparedtoglucose.Glucose,maltextract,andmolasseswerefurtherstudiedatdifferentconcentrations(Fig.7a–c).