Optimization of Surfactin Production by Bacillus subtilis Is(3)

FromFig.7aandb,itwasfoundthattheoptimumconcentrationsofglucoseandmaltextractforbiosurfactantproductionandgrowthwere2to4g%forglucoseand4g%formaltextract.Inthecaseofmolasses,bothsurfactinproductionandgrowthincreasedsteadilybyincreasingmolassesconcentrationupto16%v/v(Fig.7c).EffectofNitrogenSources

AsshowninFig.8,sodiumnitratewasthebestnitrogensourceforsurfactinproduction.However,othertestednitrogensourcesdecreasedsurfactinproductionwithdifferentdegreesandsuchdecreasewasmorepronouncedincaseofproteinnitrogensources.Itwasobservedthatsurfactinproductivityintermsofsurfactinactivitymayormaynotcoherentlycorrelatewiththatintermsofsurfactinconcentration.Regardingbacterialgrowth,theeffectofnitrogensourceswaseithercomparabletothatofsodiumnitrateorcausedsomedegreesofinhibition.

Fig.4EffectofinitialpHongrowthandsurfactinproduction(intermsofconcentrationandactivity)byB.subtilisisolateBS5inMSM

Biomass (g%)

3.002.752.50

78

2.001.751.501.251.000.75

Crude surfactin conc (g/L)

2.25

6

5

4

0.500.250.003

2

pH

Clear zone diameter by OST (cm) Clear zone diameter (cm)

Biomass (g%)

ApplBiochemBiotechnol(2008)150:305–325Fig.5Effectofincubationtem-peratureongrowthandsurfactinproduction(intermsofconcen-trationandactivity)byB.subtilisisolateBS5inMSM.SurfactinactivitywasmeasuredusingtheclearzonediameterasdeterminedbytheOST

1.81.71.61.5

4.05.04.5

313

Crude Surfactin conc (g/L)

1.41.31.21.11.00.90.8

Biomass (g%)

o

3.53.02.52.01.51.00.5

0.70.60.5Incubation Temperature (C)

Assodiumnitrateprovedtobethebestnitrogensourceforsurfactinproduction,itseffectwasfurtherstudiedatdifferentconcentrations(Fig.9a).Inaddition,thetwoothernitrogensources(ammoniumnitrateandammoniumoxalate),whichshowedcomparablesurfactinproductivityand/orsurfactinconcentration,weresimilarlystudied(Fig.9bandc).TheresultsinFig.9arevealedthatthehighestsurfactinproductivitieswereachievedat0.5–1g%sodiumnitrateconcentration,however,thehighestbiomassvalueswereobtainedat0.25–2.5g%.Figure9bandcalsoshowsthattheoptimumconcentrationsofammoniumnitrateandammoniumoxalatethatproducedthehighestgrowthandsurfactinproductionwere0.25–1g%forammoniumnitrateand1g%forammoniumoxalate.EffectofSomeStructuralAminoAcidsonSurfactinProductioninMSM

TheresultsshowninFig.10revealedthatnoenhancementofsurfactinproductionwasgainedbytheuseofaminoacids,eitherthoseaddedtoMSMat0.1M(D,L-leucine,L

-1.210.80.60.40.20

annoseD-SorbitolGlyGcerlucoolse syrupMolasseMsalt extraSocy betan oilOlive Hoiexlad

ecanPaeraffin oil

ose

ose

cos

to

to

Sucr

ct

uc

La

ac

lu

al

tose

e

se

se

4.54

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

3.532.521.510.50

-g

M

-fr

al

D

(+

) M

D

G

D

Fig.6Effectofdifferentcarbonsourcesongrowthandsurfactinproduction(intermsofconcentrationandactivity)byB.subtilisisolateBS5inMSMafter72hincubation.SurfactinactivitywasmeasuredusingtheclearzonediameterasdeterminedbytheOST

Clear zone diameter (cm)

Clear zone diameter(cm)

314

Fig.7Effectofdifferentcon-centrationsofaglucose,bmaltextract,cmolassesongrowthandsurfactinproduction(intermsofconcentrationandactivity)byB.subtilisisolateBS5inMSMafter72hincubation.SurfactinactivitywasmeasuredusingtheclearzonediameterasdeterminedbytheOST

ApplBiochemBiotechnol(2008)150:305–325

5.0

2.00

4.5

1.751.501.251.000.750.50

4.0

Crude S

urfactin conc (g/L)

3.53.02.52.01.51.00.50.0

0.250.00

Glucose conc (%w/v)

1.5

1.41.31.11.00.90.80.70.60.50.40.30.20.10.01.2

3.0

Biomass (g%)

2.0

1.5

1.0

0.5

0.0

Malt Extract (% w/v)

1.5

1.41.31.2

76

Crude Surfactin conc (g/L)

Biomass (g%)

1.0

0.90.80.70.60.50.40.30.20.10.0

543210

Molasses (% v/v)

asparticacid,L-valine)orthatusedinreplacementofsodiumnitrateinMSM(glutamicacidat0.432g%)althoughthelatterwasrelativelylessinhibitorybetterthanothertestedaminoacids.However,whentheeffectofglutamicacidonsurfactinproductionwasfurtherstudiedatdifferentconcentrations,maximumsurfactinproductionandbacterialgrowthwereobtained

Clear zone diameter (cm)

1.1

Clear zone diameter by OST (cm)

2.5

Crude Surfactin conc (g/L)

Clear zone diameter (cm)

Biomass (g%)

ApplBiochemBiotechnol(2008)150:305–325315Fig.8Effectofdifferentnitrogensourcesongrowthandsurfactinproduction(intermsofconcentrationandactivity)byB.subtilisisolateBS5inMSMafter72hincubation.SurfactinactivitywasmeasuredusingtheclearzonediameterasdeterminedbytheOST

at1g%(Fig.11).Thelevelofsurfactinproduction(intermsofactivity)atthisconcentration(1g%glutamicacid)wascomparabletothatproducedbysodiumnitrateat0.5g%(Fig.9a).EffectofMultivalentCationsAsshowninFig.12,itisclearthatMg++isessentialforgrowthasitsremovalwasaccompaniedbyasubstantialreductioninmicrobialgrowth.However,Ca++wasnotasessentialasMg++becauseitsremovalwasnotaccompaniedbyasignificantchangeinbacterialgrowth.Therefore,amodifiedMSM(glucose–magnesiummedium)thatwasdevoidoftheTESandCa++wasusedforstudyingoftheeffectoftheadditionofdifferentmultivalentcations.

EffectoftheAdditionofMultivalentCationstotheGlucose–MagnesiumMedium

AsshowninFig.13,growthandsurfactinproductionbyB.subtilisisolateBS5weregreatlyenhancedinthepresenceofthefollowingmultivalentcations:zinc,iron(II),iron(III),andmanganese(II)at0.0001Mconcentrations.Regardingsurfactinproduction,iron(III)showedbetterresultsthaniron(II).Othermultivalentcationscausedeithercomparableresultstothatofthecontrolorresultedindifferentdegreesofinhibitionofbothgrowthandsurfactinproduction.Thesecations(Zn,ironIII,Mn)togetherwithmagnesium(whichprovedtobeessentialforbacterialgrowth)werefurtherevaluatedatdifferentconcentrationstofindouttheoptimumconcentrationsofeach(Fig.14a–d).

TheresultsplottedinFig.14a–dshowthattheoptimumconcentrationofzincsulfate,ferricchloride,andmagnesiumsulfateformaximumbiosurfactantproductionis1mM,however,thatofmanganesesulfateis0.1mM.

BiosurfactantProductionbyB.subtilisIsolateBS5inDifferentModifiedMineralSaltsMedia

FromFig.15,itisclearthatthemediumcontainingtheoptimumminerals(M1)increasedsurfactinproductivityoverthatinMSM.Themedium(M2)thatcombinedboth

optimum

316

Fig.9Effectofdifferentcon-centrationsofasodiumnitrate,bammoniumnitrate,

cammoniumoxalateongrowthandsurfactinproduction(intermsofconcentrationandactivity)byB.subtilisisolateBS5inMSMafter72hincubation.SurfactinactivitywasmeasuredusingtheclearzonediameterasdeterminedbytheOST

ApplBiochemBiotechnol(2008)150:305–325

1.00.90.8

5.04.54.0

Crude surfactin conc (g/L)

0.70.60.50.40.3

3.53.02.52.01.51.00.50.0

0.20.10.0

NaNO3 Conc (g%w/v)

0.70.60.50.40.3

1.00.90.8

4.003.753.503.253.002.752.502.252.001.751.501.251.000.750.500.250.00

Crude surfactin conc (g/L)

0.20.10.0

NH4NO3 conc (% w/v)

0.50

6.05.55.0

0.450.40

Crude Surfactin conc (g/L)

Biomass (g%)

0.350.300.250.200.15

4.03.53.02.52.01.51.00.50.0

0.100.050.00

Ammonium oxalate conc (% w/v)

traceelementsandoptimumnitrogensourcetogetherdidnotsignificantlyincreasesurfactinactivityandbacterialgrowth.Itisinterestingtonotethatupontheapplicationofoptimumtraceelements,nitrogensource,andcarbonsource(M3),theireffectstogetherweresodramatic.Anincreaseinsurfactinproduction(intermsofsurfactinconcentrationorsurfactinactivity)ofaboutthreefoldwasreached(ifcomparedwithMSM).ThisoptimumM3medium,whichcontainedoptimumcarbonsource(molasses160ml/l),optimumnitrogensource(NaNO35g/l),andoptimumtraceelements,wasdesignatedasmolassesMSM(MMSM).

Clear zone diameter (cm)

4.5

Clea

r zone diameter (cm)

Biomass (g%)

Clear zone diamete

r (cm)

Biomass (g%)

ApplBiochemBiotechnol(2008)150:305–325Fig.10Effectofdifferentaminoacidsongrowthandsurfactinproduction(intermsofconcen-trationandactivity)byB.subtilisisolateBS5inMSMafter72hincubation.SurfactinactivitywasmeasuredusingtheclearzonediameterasdeterminedbytheOST

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