Optimization of Surfactin Production by Bacillus subtilis Is(4)

317

Biomass (g%); Crude surfactin conc (g/L)

TimeCourseofSurfactinProductionbyB.subtilisIsolateBS5inMMSM

TheresultsinFig.16showedthatsurfactinproductionstartedearlyintheexponentialphaseandtheproductionkineticsparalleledthebiomasskineticsupto2daysofincubation.Onthebasisofthesefacts,itcouldbeconcludedthatsurfactinproductionisgrowth-associated,thesamefindingwasobservedwhenusingthebasalmedium(MSM).Itwasfoundthatthemaximumlevelofcellbiomasswasobtainedafter48hofincubation;however,maximumsurfactinconcentrationwasobtained12hlater,i.e.,after60hofincubation.Afterthoseperiods,asharpreductionineitherbiomassorsurfactinproductionlevelswasobserved.

DetectionofPlasmid(s)inB.subtilisIsolateBS5

TheplasmidextractionprocesswasconductedonculturegrowninbothLuria–Bertani(LB)brothandinMSMmedium.Plasmidswerenotdetected(Fig.17).Thus,itcanbeconcludedthatneitherhighnorlowmolecularweightplasmidsdoexistinthetestedisolate.

Discussion

Thechemicalcompositionoftheculturemediumandenvironmentalfactorsinfluencecellgrowthandbiosurfactantproduction[17].Abetterunderstandingofthemedium

Fig.11Effectofdifferentcon-centrationsofglutamicacidongrowthandsurfactinproduction(intermsofconcentrationandactivity)byB.subtilisisolateBS5inMSMafter72hincubation.Surfactinactivitywasmeasuredusingtheclearzonediameterasdeterminedbythe

OST

1.25

1.00

0.750.500.250.002.50

5.0

2.25

4.5

2.00

4.0

Crude Surfactin conc (g/L)

1.751.50

Biomass (g%)

3.53.02.52.01.51.00.50.0

Glutamic acid (% w/v)

Clear zone diameter (cm)Clear zone diameter (cm)

318

Fig.12GrowthofB.subtilis

isolateBS5inMSMwithand

withoutdifferentmultivalent

cationsafter48hincubation

Biomass (g%)0.080.070.060.050.040.03

0.02

0.01

0MSM devoid of

TES, Mg & CaApplBiochemBiotechnol(2008)150:305–325MSM devoid ofTES & MgMSM devoid ofTES & CaMSM devoid ofTES only

componentsandenvironmentalfactorsandtheiroptimalcontrolcan,therefore,beusedtoimprovethebiosurfactantproduction.Abasalsyntheticmedium(MSMsupplementedwith2%glucose)wasappliedforstudyingdifferentfactorsaffectingbiosurfactantproductionby

B.subtilisisolateBS5.

TimeCourseExperimentsinMSM

Theresults(Fig.1)showedthatsurfactinproductionstartedearlyintheexponentialphaseandtheproductionkineticsparalleledthebiomasskineticsthroughthelogarithmicphase.Accordingly,surfactinproductionbythisisolateisgrowth-associated.Growth-associatedproductionofbiosurfactanthasbeenreportedforB.subtilis[6].Insubsequentexperiments,resultsweretakenafter72hofincubationasproductionwasincreasingataslowerrate

thereafter.

Fig.13Effectofadditionofdifferentmultivalentcationsongrowthandsurfactinproduction(intermsofconcentrationandactivity)byB.subtilisisolateBS5inmodifiedMSMafter72hincubation.SurfactinactivitywasmeasuredusingtheclearzonediameterasdeterminedbytheOST

ApplBiochemBiotechnol(2008)150:305–325

319

3.503.253.002.75

3.753.503.253.002.502.252.001.751.501.251.000.750.500.250.002.75

Clear zone diamet

er (cm)

Crude surfactin conc (g/L)

Biomass (g%)

2.001.751.501.251.000.750.50

Biomass (g%)

2.250.250.00

ZnSO4 conc. (M)

MgSO4 conc. (M)

3.753.503.253.002.502.252.001.751.501.251.000.750.500.250.002.75

Biomass (g%)

FeCl3 conc. (M)

3.503.253.002.752.502.252.001.751.501.251.000.750.500.25

Biomass (g%)

0.00

MnSO4 conc. (M)

Fig.14Effectofdifferentconcentrationsofazincsulfate,bferricchloride,cmanganesesulfate,dmagnesiumsulfateongrowthandsurfactinproduction(intermsofconcentrationandactivity)byB.subtilisisolateBS5inmodifiedMSMafter72hincubation.SurfactinactivitywasmeasuredusingtheclearzonediameterasdeterminedbytheOST

EffectofDifferentEnvironmentalFermentationConditionsonSurfactinProductionTheenvironmentalfactors:pH,temperature,andaerationsignificantlyinfluencethebacterialcellgrowthandbiosurfactantproduction[17].Inoculumsizeandinoculumconditionalsohaveparamounteffectsonbacterialgrowthandproductivity[29].

Thestudyoftheeffectofaerationonsurfactinproduction(Fig.2)revealedthatalinearrelationshipbetweenaerationandproductionexistedandasharpdeclineinsurfactin

Clear zone diameter (c

m)

Crude surfactin conc (g/L)

Clear zone diameter (cm

)

Crude surfactin conc (g/L)

Clear zone diameter (cm)

Crude surfactin conc (g/L)

2.50

320ApplBiochemBiotechnol(2008)150:305–325

Fig.15Growthandsurfactinproduction(intermsofconcentrationandactivity)byB.subtilisisolateBS5inMSMandindifferentmodifiedculturemedia(M1,M2,andM3)after72hincubation.SurfactinactivitywasmeasuredinCFSusingtheclearzonediameterasdeterminedbytheOSTandthepercentageofthereductionofST

productionoccurredupondecreaseofaeration.Thisisinaccordancewiththeoxygenrequirementsforsuchanaerobicmicroorganism.

Theeffectofdifferentinoculumsizesongrowthandsurfactinproductionbythetestedisolatewasinvestigated.Therewasagradualdecreaseingrowthaccompaniedbyagradualslowincreaseinsurfactinproductionuponincreasingtheinoculumsizeupto6%v/v(Fig.3).Astherewasnodramaticincreaseinsurfactinproductionuponincreasingtheinoculumsize,2%v/vinoculumsize,whichgavereasonablyhighproductivity,wasappliedinsubsequentexperiments.

ThepHofthemediumisoneoftheenvironmentalfactorsthatmayaffectsurfactinproductionandbacterialgrowth.Fromtheresults(Fig.4),itappearedthatthetestBacillusisolategrewatpH6to9.0.HighestcellgrowthandsurfactinproductionwasrecordedatpH6.8.

Fig.16Timecourseofgrowthandsurfactinproduction(intermsofconcentrationandactivity)byB.subtilisisolateBS5inMMSM.SurfactinactivitywasdeterminedbymeasuringtheclearzonediameterasdeterminedbytheOSTandmeasuringthesurfacetensionoftheCFSstalagmomet-rically

5.04.5

4.0

3.0

60555045403530

0.5

2520

4.0

70

3.5

65

Clear zone diameter (cm)

Biomass (g%)

3.02.52.01.5

2.5

2.0

1.5

1.00.50.0

1.0

0.0

Time (days)

Surface Tension (mN/m)

3.5

Surfactin conc. (g/L)

ApplBiochemBiotechnol(2008)150:305–325

Fig.17Agarosegel(0.7%)elec-

trophoresisoftheplasmidprepa-

rationsofB.subtilisisolateBS5

andE.coliDH5α/pUC18.Lanes

1and2plasmidpreparationfrom

E.coliDH5α/pUC18grownin

LBmedium(asapositivecon-

trol);lanes3and4plasmid

preparationfromBacillusisolate

BS5growninLBmediumand

MSM,respectively;M.W.1Kb

DNAladder321

Thereisalwaysanoptimumtemperatureatwhichthegrowthrateofamicroorganismisfastest[30].Inthisstudy,theresults(Fig.5)showedthatB.subtilisisolateBS5couldgrowatalltestedtemperatureswithrelativelyhighcellgrowthandbiosurfactantproductionbeingobtainedatanincubationtemperatureof30°C.ThistemperaturewassimilartotheincubationtemperaturecommonlyusedforsurfactinproductionbyB.subtilis[7,6,31].Accordingly,bytheendofthestudyoftheeffectofdifferentenvironmentalfermentationconditions,itcanbeconcludedthattheoptimumfermentationconditionsforhighsurfactinproductionbyB.subtilisisolateBS5inshakeflasksare:incubationperiod,72h;volumetricoxygenpercentage,90%;inoculumsize,2%v/v;initialpH,6.5–

6.8;andincubationtemperature,30°C.

EffectofDifferentMediumComponentsonSurfactinProduction

Thebasicnutritionalrequirementsofmicroorganismsareanenergyorcarbonsource,anavailablenitrogensource,inorganicelements,andforsomecelltypes,specificgrowthfactors[32].

Duringthecourseofstudyingtheeffectofnutritionalfactorsonsurfactinproduction,aprotocolofthreephaseswasfollowed:first,screeningofthedifferentnutritionalsourcesbelongingtoeachnutritionalcategory(carbonsources,nitrogensources,andminerals)thatgivemaximumsurfactinproduction,thenselectingthepromisingsourcesforproduction;second,studyingtheeffectoftheselectedsourcesonbiosurfactantproductionatdifferentconcentrations.Finally,collectivecombinationofthedifferentnutritionalsourcesattheiroptimumconcentrationsinanewlyformulatedmediumandstudyingtheircollectiveeffectonsurfactinproduction.

Thecarbonsourceusedinbacterialculturehasaverypronouncedeffectonbiosurfactantproduction[19,20].Thecarbonsourcesgenerallyusedin

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