Surface response methodology for the optimization of lipase production under submerged fermentation by filamentous fungi

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Industrial

Microbiology

Surface

response

methodology

for

the

optimization

of

lipase

production

under

submerged

fermentation

by

filamentous

fungi

Luciane

Maria

Colla

_,

_Andreiza

_Lazzarotto

_Primaz

_,

_Silvia

_Benedetti

_,

Raquel

Aparecida

Loss

_,

_Marieli

_de

_Lima

_,

_Christian

_Oliveira

_Reinehr

_,

Telma

Elita

Bertolin

_,

_Jorge

_Alberto

_Vieira

_Costa

a_{LaboratoryofFermentations,CourseofFoodEngineering,CollegeofEngineeringandArchitecture,UniversityofPassoFundo,Passo}

Fundo,RS,Brazil

b_{LaboratoryofBiochemicalEngineering,SchollofChemistryandFood,FederalUniversityFoundationofRioGrande,RioGrande,RS,}

Brazil

a

r

t

i

c

l

e

i

n

f

o

Received24March2011 Accepted6January2015 Availableonline2March2016 AssociateEditor:RosaneFreitas Schwan Keywords: Agriculturalresidues Lipase Optimization Submergedbioprocess

a

b

s

t

r

a

c

t

APlackett–BurmanFactorialDesignof16experimentswasconductedtoassesstheinfluence ofninefactorsontheproductionoflipasesbyfilamentousfungi.Thefactorsinvestigated werebrantype(usedasthemaincarbonsource),nitrogensource,nitrogensource concen-tration,inducer,inducerconcentration,fungalstrain(AspergillusnigerorAspergillusflavus

wereselectedasgoodlipaseproducersviasubmergedfermentation),pHandagitation.The concentrationoftheyeastextractandsoybeanoilandthepHhadasignificanteffect(p<0.05) onlipaseproductionandwereconsecutivelystudiedthroughaFullFactorialDesign23_,with

theconcentrationofyeastextractandpHbeingsignificant(p<0.05).Thesevariableswere optimizedusingacentralcompositedesign,obtainingmaximumlipolyticactivitieswiththe useof45g/LofyeastextractandpH7.15.Thestatisticalmodelshoweda94.12%correlation withtheexperimentaldata.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Enzymescurrentlyhavevariousindustrialapplications.1_The

industrial market for enzymes continues to grow due to thedevelopmentofnewproductiontechnologies,theuseof

∗ _{Correspondingauthor.}

E-mail:lmcolla@upf.br(L.M.Colla).

geneticengineeringduringproductionandemergenceofnew fieldsofapplication.Theglobalenzymemarketin2007was 2.3billionUSdollarsandisexpectedtobe2.7billionUS dol-larsin2012.2_{Amongtheseenzymes,lipasesarewidelyused.}

Their applicationsresultfromtheirabilitytocatalyze reac-tions,mainlythehydrolysisandinter-andtransesterification

http://dx.doi.org/10.1016/j.bjm.2016.01.028

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

of lipids,making these enzymes useful in the detergent,3

medical4_{andfoodindustries}5_{aswellasinthepharmaceutical}

industryasdiagnostictools,incosmetics,inteaprocessing,in medicalapplications,asbiosensors,indegreasingofleather andwastetreatment.6_{Otherapplicationsincludethe}

matura-tionofcheeses,7_{thesynthesisofaromas,}8_{theproductionof}

lipidswithhighlevelsofunsaturatedfattyacids9_and

methyl-estersoffattyacids(biodiesel).10

Industrialenzymes areproducedprimarilythrough sub-mergedfermentationinbatchandfed-batchcultures1_using

filamentousfungi.Themost-citedgeneraforlipase produc-tionareAspergillus,Rhizopus,Penicillium,Mucor,Geotrichumand

Fusarium.11,12 _{Submerged processes have some advantages}

oversolid-stateprocesses,suchashigherhomogeneityofthe culturemediumandmorefacilitytocontrolparameterslike temperatureandpH.13

Moreover,Mahadiketal.14_{mentionedthatoneofthe}

disad-vantagesofsolidstateprocessesisthecolorofthefermented media,duetothepresenceoffungalspores,andother com-ponentsremainingintheextractaftertheenzymeextraction process.However,submergedfermentationcanpresent dif-ficultyforthe transferofoxygen inliquid media,which is aggravatedinthecaseoffungiduetothefilamentous mor-phologyofhyphaeinliquidmedia.1_Coradietal.15_mentioned

thatlipaseshavebeenproducedbysubmergedfermentation becausetherecoveryofextracellularenzymesandthe deter-mination ofbiomassare facilitated bybeing performed by simplefiltration orcentrifugation. However,solid-state fer-mentation can improve the use of agricultural waste and demandslesswaterandenergy.

Other factors, such as the types and concentrations of nutrients,pH,agitationandthepresenceandconcentration of inducers can affect the productivity of these biopro-cesses.Researchthatusesisolatedmicroorganismsfromnew environmentsand thatusesagro-industrial residuesinthe compositionofmediaisneededtoobtainhighyieldsatlower costs.

Thestatisticaloptimizationofprocesseshasadvantages comparedtotheclassicalpracticeofchangingonevariable at a time,16,17 _{such as the lower number of experiments}

and the possibility of evaluating the interaction effects amongvariables. Numerous researchershave reported the use of these techniques for the production of lipases by microorganisms.18–20 _{Anefficientandwidelyusedapproach}

istheapplicationofPlackett–Burmandesignsthatallow effi-cientscreeningofkeyvariablesforfurtheroptimizationina rationalway.21,22_{Theaimofthisworkwastoscreensignificant}

variablesforlipaseproductionthroughsubmerged fermenta-tionandtooptimizethesevariablesthroughresponsesurface methodology.

Materials

and

methods

Microorganismsandinoculumpreparation

ThefungiusedinthisstudyweretwospeciesofAspergillus

that were isolated from dairy effluent (isolate E-19) and soilcontaminated with diesel oil (isolated O-8)23 _{and that}

were previously selected as good producers of lipase via

submergedfermentation.24_{Bothisolatesweresubmittedto}

geneticidentificationthroughPhred/PhrapandConsed,using themethodologycitedbySmaniotoetal.,25_{attheCenterof}

NuclearEnergyinAgriculture(Cena),UniversityofSãoPaulo (USP),Brazil.

Sequenceswerecomparedto18SrRNAdataobtainedfrom GenBank(http://www.ncbi.nlm.nih.gov).TheisolateE-19was identifiedasAspergillusnigerstrainDAOM(100%identity, Gen-Bankaccessionnumber:KC545858.1),andtheisolateO-8was identifiedasAspergillusflavusstrainDAOM(99%identity, Gen-Bankaccessionnumber:JN938987.1).

The microorganisms were kept in test tubes with PDA (potato-dextrose-agar)at4◦C.Theinoculumwaspreparedby inoculationofthefungiinPetridishescontaining30mLof solidifiedPDAmediumandincubatedat30◦Cfor5days.

Culturemediumandexperimentalapparatus

Theculturemediumwaspreparedwith10%(m/v)bran(wheat orsoybean),whichwasboiledat100◦Cfor30min.Afterwards, themediumwasfiltered,andthesolubleextractwasaddedto a10%(v/v)salinesolutioncontainingalsothenitrogensource, inducer and distilled water to complete the final volume. Thesaline solutioncontained KH2PO4 (2g/L),MgSO4 (1g/L)

and trace solution (10mL/L). Thecomposition of the trace solution wasFeSO4·7H2O(0.63mg),MnSO4 (0.01mg),ZnSO4

(0.62mg)anddistilledwateruptoavolumeof1L.26_Theliquid

medium was autoclaved, and the pH was adjustedto val-ues pre-definedbytheexperimentaldesignusingsolutions of1.5mol/LHClor1mol/LNaOH.

Theexperimentswerecarriedoutin300-mLErlenmeyer flaskswith100mLofmedium.Theinoculationwas accom-plishedusing10or20mmdiametercircularareascontaining sporesgrowninPetridishes.TheErlenmeyerflasks, contain-ingtheinoculatedculturemedium,wereincubatedat30◦Cfor 10daysinashakerwithalevelofagitationpre-defined accord-ingtotheexperimentaldesign.Aliquots(10mL)wereremoved at24h,48h,72hand96htomeasurelipolyticactivity.

Experimentaldesign

Theoptimizationoflipaseproductionbysubmerged fermen-tation wascarriedout using threesequentialexperimental designs. The first step aimed to assess the influence of ninevariablesonlipaseproductionusingaPlackett–Burman Designwith16trials(1–16).Thevariablesstudiedwerebran typeusedasacarbonsource(wheatbranorsoybeanbran), nitrogen source (sodium nitrate or yeast extract), nitrogen sourceconcentration(10or30g/L),inducer(soybeanorolive oil),inducerconcentration(10or30g/L),culturemediumpH (5 or 7),fungus strainused (A.niger E-19 orA. flavusO-8), inoculumdiameter(fungalsporesgrowthequalto1or2cm diameterinPetridishescontainingPDA)andagitation(120or 160rpm).Next,a23_{FullFactorialDesign(FFD)(Trials17–24)}

wascarriedouttostudytheinfluenceofyeastextract con-centration(YEC),soybeanoilconcentration(SOC)andpHon lipolyticactivity.Subsequently,thepHandtheconcentration ofyeastextract wereoptimized using aCentralComposite RotationalDesign(CCRD),including4factorialpoints,4axial pointsand3centralpointsforevaluatingthepureerrorfora

Table1–RealandcodedlevelsofvariablesusedinthePlackett–BurmanDesignandmaximumresiduallipolytic activitiesobtainedinexperimentscarriedoutinsubmergedfermentation.

Exp. X1(TB) X2(NS) X3(I) X4(NSC) X5(IC) X6(pH) X7(fungus) X8(ID) X9(A) LAMR(U)a

1 −1(WB) −1(SN) −1(OO) −1(10) +1(30) +1(7) +1(E-19) +1(2) +1(160) 1.74±0.16

2 +1(SB) −1(SN) −1(OO) −1(10) −1(10) −1(5) −1(O-8) +1(2) +1(160) 0.31±0.13

3 −1(WB) +1(YE) −1(OO) −1(10) −1(10) +1(7) +1(E-19) −1(1) −1(120) 2.03±0.28

4 +1(SB) +1(YE) −1(OO) −1(10) +1(30) −1(5) −1(O-8) −1(1) −1(120) 1.50±0.12

5 −1(WB) −1(SN) +1(SO) −1(10) +1(30) −1(5) +1(E-19) −1(1) +1(160) 0.19±0.07

6 +1(SB) −1(SN) +1(SO) −1(10) −1(10) +1(7) −1(O-8) −1(1) +1(160) 1.21±0.25

7 −1(WB) +1(YE) +1(SO) −1(10) −1(10) −1(5) +1(E-19) +1(2) −1(120) 0.84±0.44

8 +1(SB) +1(YE) +1(SO) −1(10) +1(30) +1(7) −1(O-8) +1(2) −1(120) 2.93±0.88

9 −1(WB) −1(SN) −1(OO) +1(30) +1(30) +1(7) −1(O-8) +1(2) −1(120) 1.62±0.18

10 +1(SB) −1 (SN) −1 (OO) +1(30) −1 (10) −1 (5) +1(E-19) +1(2) −1(120) 1.36±0.02

11 −1(WB) +1(YE) −1(OO) +1(30) −1(10) +1(7) −1(O-8) −1(1) +1(160) 4.08±0.08

12 +1(SB) +1(YE) −1(OO) +1(30) +1(30) −1(5) +1(E-19) −1(1) +1(160) 0.86±0.01

13 −1(WB) −1(SN) +1(SO) +1(30) +1(30) −1(5) −1(O-8) −1(1) −1(120) 0.37±0.24

14 +1(SB) −1(SN) +1(SO) +1(30) −1(10) +1(7) +1(E-19) −1(1) −1(120) 1.39±0.06

15 −1(WB) +1(YE) +1(SO) +1(30) −1(10) −1(5) −1(O-8) +1(2) +1(160) 3.51±0.54

16 +1(SB) +1(YE) +1(SO) +1(30) +1(30) +1(7) +1(E-19) +1(2) +1(160) 1.68±1.00

Exp.,experiment;TB,typeofbran;NS,nitrogensource;I,inducer;NSC,nitrogensourceconcentration(g/L);IC,inducerconcentration(g/L);ID,

inoculumdiameter(cm);A,agitation(rpm);WB,wheatbran;SB,soybeanbran;SN,sodiumnitrate,YE,yeastextract;OO,oliveoil;SO,soybean

oil;LAMR,maximumresiduallipolyticactivity.

a _{Mean±standarddeviation.}

Table2–Codedandreallevelsofthe23_{FullFactorial}

Designandresultsofmaximumresiduallipolytic

activityobtainedfromsubmergedfermentation.

Experiment X1(YEC,g/L) X2(SOC,g/L) X3(pH) LAMR(U)a

17 −1(10) −1(10) −1(5.0) 0.23±0.13 18 +1(30) −1(10) −1(5.0) 0.89±0.01 19 −1(10) +1(30) −1(5.0) 0.00±0.00 20 +1(30) +1(30) −1(5.0) 0.95±0.14 21 −1(10) −1(10) +1(7.0) 0.84±0.01 22 +1(30) −1(10) +1(7.0) 2.00±0.48 23 −1(10) +1(30) +1(7.0) 0.85±0.08 24 +1(30) +1(30) +1(7.0) 0.95±0.06

YEC,yeastextractconcentration(g/L);SOC,soybeanoil

concentra-tion(g/L);LAMR,maximumresiduallipolyticactivity;fixedvariables,

typeofbran(wheatbran),fungus(O-8),inoculumdiameter(2cm)

andagitation(120rpm).

a _{Mean±standarddeviation.}

totalof11experiments(Trials25–35).Allexperimentswere carriedout in replicates.Lipolytic activity wasused asthe responseinallfactorialdesigns.Tables1–3presentcodedand originalvaluesofthePlackett–BurmanDesign,the23_Factorial

Designandthe22_{CCRD,respectively.}

Lipolyticactivitydeterminations

Thesampleswere filteredwith cottonwool toremovethe hyphae, and the filtrates were used to measure lipolytic activity. Enzymatic activity was determined following the methodologyofBurkertetal.,18_{whichisbasedontheNaOH}

titration offatty acids released bythe action ofthe lipase enzymeintheenzymaticextractontriacylglycerolsofolive oilemulsifiedinarabicgum.

Oneunit oflipolyticactivity wasdefinedastheamount ofenzymethatreleases1␮moloffattyacidperminuteper

Table3–CodedandactualvaluesofpHandyeast extractconcentration(YEC)usedintheCentral

CompositeRotationalDesign(CCRD)fortheoptimization oflipaseproductionbyAspergillusflavus(O-8)via

submergedfermentation.

Experiment X1(pH) X2(YEC,g/L) LAMR(U)a

25 −1(6.0) −1(20) 1.87±0.12 26 +1(8.0) −1(20) 2.20±0.10 27 −1(6.0) +1(40) 2.98±0.05 28 +1(8.0) +1(40) 2.92±0.12 29 −1414(5.5) 0(30) 1.67±0.10 30 +1414(8.5) 0(30) 2.11±0.07 31 0(7.0) −1414(15.9) 1.96±0.02 32 0(7.0) +1414(44.1) 2.98±0.05 33 0(7.0) 0(30) 2.75±0.02 34 0(7.0) 0(30) 2.93±0.10 35 0(7.0) 0(30) 3.04±0.02

Fixedvariables,type of bran (wheat), fungus (O-8),soybeanoil

concentration(1%),inoculumdiameter(2cm),agitation(120rpm);

LAMR,maximumresiduallipolyticactivity.

a _{Mean±standarddeviation.}

mLofenzymeextract (1U=1␮mol/min/mL)underthe test conditions.

Treatmentofdata

Theresultsofthelipolyticactivityovertimeweresubtracted fromthevaluesoflipolyticactivityobtainedatthebeginning offermentation.Theseactivitiescanbeattributedtothe pres-enceoffreefattyacidsintheculturemediumbeforefungal growthandlipaseproduction.Theresultsofmaximum resid-uallipolyticactivitiesobtainedweresubmittedtoanalysisof variance(Anova).Theestimatedeffectsofvariablesandthe regression coefficientsofthegeneratedmodelswere calcu-lated.

3.5 3.0 2.5 2.0 1.5 1.0 0.5 50 40 30 20 10 5.0 6.0 7.0 pH YEC (g/l) 8.0 9.0 0.0 >3.0 <3.0 <2.5 <2.0 <1.5 <1.0 <0.5 LA MR (U ) 2.5 2.0 1.5 1.0 0.5 0.0 35 30 25 20 15 10 5 4.5 5.0 5. 5 _pH 6.0 6.5 7.0 7.5 LA MR (U) YEC (g/l) >2.0 <1.7 <2.0 <1.4 <1.1 <0.8 <0.5

A

B

Fig.1–Responsesurfaceofthemaximumresiduallipolyticactivity(LAMR)asafunctionofpHandyeastextract

concentration(YEC)forlipaseproductionbysubmergedfermentationaccordingto(A)23FFDand(B)CCRD.

Results

and

discussion

Table1shows themaximumlipolyticactivitiesobtainedin thePlackett–Burman(PB)Designexperiments.Themaximum lipolyticactivitiesforallexperimentswerefoundbetweenthe thirdandfourthdaysofcultivation.Inthisway,thevaluesof lipolyticactivityobtainedatthesetimesofcultivationwere usedtoevaluatetheinfluenceofthevariablesstudiedinthe PBdesignonlipaseproduction.Thehighestlipolytic activi-tieswereobtainedintrials3,8,11and15,allofwhichused yeastextractasthenitrogensource.Inrelationtothetypeof branusedasacarbonsource,trials3,11and15werecarried outwithwheatbran,andtrial8wascarriedoutwithsoybean meal.Intrials3and11,oliveoilwasusedastheinducer,and intrials8and15,theinducerwassoybeanoil.Intrials3,8and 11,apHof7.0wasused,andpH5.0wasusedinexperiment 15.TheisolateE-19(A.niger)wasusedintrial3;theisolateO-8 (A.flavus)wasusedintrials4,11and15.Trials3and8were carriedoutwithanagitationrateof120rpm,andtrials11and 15werecarriedoutwithanagitationrateof160rpm.

The results of lipolytic activity obtained in the Plackett–Burman Design trials were evaluated through analysisofvarianceandshowedthatthetypeofbran(TB), inducer (I), inoculum diameter (ID) and agitation had no significant influence(p>0.10) onlipaseproductionthrough submergedfermentation.Thus,thesevariableswerefixedin thenextstagesofoptimization.

Wheatbranwasusedinthissequenceofstatistical opti-mization oflipase production in submerged fermentation, becauseitprovidedthemosthomogeneousculturemedium after boiling. According to Pinto et al.,27 _{agro-industrial}

residuescanberecoveredandusedinfermentativeprocesses. Theadvantageoftherebeingnodifferencebetweenthetype ofbranusedisthat thebrantypecanbechosenin accor-dancewithmarketconditions,suchasprice,transportation andproductavailability.

Soybeanoilwaschosenastheinducerintheoptimization sequencebecauseitischeaperthanoliveoil.Theresultwas similartothatobtainedinthestudybyBurkertetal.,18_who

assessedtheinfluenceofoiltype(oliveoilandsoybeanoil) onlipaseproductionbyGeotrichumsp.andfoundno signifi-cantdifferencebetweentheresultsobtainedusing1%ofthe inducers.

TengandXu19_{studiedtheinfluenceofvariablesonlipase}

production. Agitation and initial inoculum concentration weresignificantvariablesandinfluencedthemorphologyof mycelia.Theformationofgroupsofclumpswasobservedat anagitationrateof200rpm,andthistypeofmorphologywas determinedtobeimportantforlipasesynthesis.Atagitations lower than150rpm,dispersedmyceliawereobtained.High initialconcentrationsofinoculumledtotheformationof dis-persed mycelia;this formationwasafactor thatdecreased lipasesynthesisbyCandidacylindracea.Theseresultswere con-tradictorytothoseobtainedbyHaacketal.,1_{whoreportedthat}

thepresenceofdispersedhyphae,ratherthanclumpsor pel-lets,facilitatesthesynthesisoflipasebystrainsoffilamentous fungibecausetheothermorphologicalformscausediffusion problemsforthesubstrateandproduct.However,thepresence ofdispersedhyphaecausesincreasedviscosityofthemedium, whichreducesoxygentransfer.Inourstudy,thepresenceof pellets wasnotobserved,but therewasan increaseinthe visual viscosity ofthe mediaduringthe fermentation pro-cess,whichcharacterizesthepresenceofdispersedhyphae.

Table4–AnalysisofvarianceofthemaximumresiduallipolyticactivityresultsoftheCCRDexperiments.

Effect SS DF MS Fcalculated Fcritical

Regression 5.137 4 1.284 33.077 2.965

Error 0.660 17 0.039

Total 5.797 21

SS,sumofsquares;DF,degreesoffreedom;MS,meansquare.Theeffectofinteraction,whichwasnotsignificant,wasignoredinthe

imple-mentationofanalysisofvariance.

Theproductionoflipaseswasnotaffectedbyagitationorby theinitialinoculumconcentration,asindicatedbythe anal-ysisofvarianceofthePBdesignexperiments(p>0.10).Thus, insubsequentexperiments,theinoculationwascarriedout usinga20mmdiameteroffungalgrowthafterpreparingthe inoculumandusinganagitationrateof120rpm.

Thenitrogensource(NS)andfungusstrainweresignificant (p<0.001 and p=0.012, respectively), having the estimated effectsof1.15and−0.68,respectively.Thismeansthatlipase activityincreasedapproximately1.15Ubyvaryingthe nitro-gensourcefrom thelowest (sodiumnitrate)tothe highest level(yeastextract)ofthePBdesign.However,aftervarying thelevelofthevariablefungusfromthelowest(A.flavus)to thehighestlevel(A.niger),adecreaseof0.68Uwasobservedin thelipolyticactivity.Thus,yeastextract(nitrogensourceused atthetoplevel)andthefungusO-8(A.flavus,corresponding tothelowestlevel)wereusedthroughouttheexperiments.

Quantitative variables (nitrogen source concentration: NSC,soybean oilconcentration:SOC,and pH)thatshowed asignificantinfluenceonlipaseproductionata10%levelof significancewerestudiedusinga23_{FullFactorialDesign;the} resultsareshowninTable2.

Themaximumlipolyticactivityinthe23_{FFD(2.00U)was}

obtainedintrial22,carriedoutwith30g/Lofyeastextractand 10g/LofsoybeanoilatpH7.Comparingthemaximum lipoly-ticactivitiesobtainedintheupperandlowerconcentrations ofyeastextract,it seemsthatincreasingtheconcentration from10g/Lto30g/Lcausedanincreaseinlipolyticactivities. Increasingthesoybeanoilconcentrationfrom10g/Lto30g/L causedadecreaseinlipolyticactivitywhencomparingtrial 17with19and22with24andcausedanincreasewhen com-paringtrials18and20andtrials21and23.Thisseemingly contradictorybehavior could be explainedby alias effects, which are common in PB designs.However, the statistical analysisoftheresultswillbetterexplainiftheeffectofthis variableonlipaseproductionissignificant.Whenassessing theeffectofpH,thebestresultswereobtainedatpH7.0.

TheYECandthepHweresignificantforlipolyticactivity accordingto theanalysis ofthevariance ofthe data, with levels ofsignificance(p) less than 0.001 forboth variables. Theeffects ofthese variables were positive, 0.71 for yeast extractand0.64forpH.Thisresultindicatesthatthegreatest increasesinlipolyticactivitieswereobtainedusinghigher lev-elsofthesevariables,i.e.,30g/LofyeastextractandpH7.0.The concentrationofsoybeanoilandtheinteractionsbetweenthe variableshadnosignificanteffectonlipolyticactivity(p<0.05). Themathematical model of maximum lipolytic activity presenteda determination coefficient (R2_{) of0.714 and an}

Table5–EstimatedeffectsofvariablesusedintheCCRD andsignificancelevels.

Effect Estimatedeffect t-Test p

Mean 2.922 38.204 <0.001

X1(L) 0.225 2.404 0.035

X1(Q) −0.867 −7.775 <0.001

X2(L) 0.838 8.947 <0.001

X2(Q) −0.278 −3.002 0.029

X1,pH;X2,yeastextractconcentration;L,lineareffect;Q,quadratic effect;p,significancelevel.

R2

adjustedof0.669,asshowninEq.(1)(X1:yeastextract

concen-tration,X3:pH).TheFvalueforregressionwas16.22,andthe

Fcriticalwas3.80,indicatingthatmostofthevariabilityofthe

modelwascausedbyregression,whichstatisticallyvalidates themathematicalmodel.Fig.1(A)showstheresponsesurface ofthemaximumresiduallipolyticactivityasafunctionofpH andyeastextractconcentration;

LAMR=0.839+0.357X1+0.320X3 (1)

Thesignificantvariablesinthe23_{FFD(pHandyeastextract}

concentration)wereoptimizedusingaCCRD,beingthematrix designandtheresultsofmaximumlipolyticactivitypresented inTable3.Astheeffectsofthesevariableswerepositiveinthe 23_{FFD,theirlevelswereincreasedintheCCRD.Thehighest}

lipolyticactivitiesintheCCRDexperimentswereobtainedin 4daysoffermentation,andtheseresultswereusedforthe analysisofvariance.Thelipolyticactivityresultsat4daysof fermentationweresubtractedfromthelipolyticactivityvalues attheinitialtime.

The analysis ofvariance ofmaximum residual lipolytic activity obtained inthe CCRD trials, presented in Table 4, showedthattheFcalculatedvaluefortheregressionwas33.08

whiletheFcriticalvalue(p=0.05,degreesoffreedomfor

regres-sion:4;degreesoffreedomforresidual:17)was2.96,validating thesurfaceresponseobtainedshowninFig.1(B).The mathe-maticalmodelgeneratedisshowninEq.(2),andacoefficient ofdetermination(R2_{)of0.89 andanadjustedcoefficientof}

determination(R2

adjusted)0.86arepresented.

ThepHandyeastextractconcentrationhadsignificant lin-earandquadraticeffects(Table5).Thelineareffectsofboth variableswerepositive,withagreatereffectcausedbythe con-centrationoftheyeastextract(0.838U).Thequadraticeffects ofboth variables were negative,indicatingthe presenceof pointsofmaximumactivityforbothvariables,asshownin

thesurfaceresponsepresentedinFig.1(B):

LAMR=2.922+0.113pH−0.433pH2+0.419YEC−0.139YEC2

(2)

TheoptimumpHlevelsand yeastextractconcentration toobtainmaximumlipolyticactivitywerealsocarriedoutby equalingthefirstderivativeoflipolyticactivityasafunction ofpHandYECtozero.Themaximumlipolyticactivitieswere obtainedatapHlevelof+0.154andaYEClevelof+1.507,which correspondstoapHof7.15andayeastextractconcentration of45g/L.

Several studies have shown the importance of a nitro-gensourceinlipaseproduction.Sharmaetal.5_reviewedthe

conditionsofcultivationtoobtainmaximumlipolyticfungal activitiesandreportedtheuseofmanynitrogensources,such asyeastextract,peptone,soybeanextract,ammonium chlo-ride,andaminoacids,amongothers.

Thesourcesoforganicnitrogenhavebeenusedforthe pro-ductionoflipasesbyfungi,asreportedbyseveral authors. PeptonewasusedbyKaushiketal.17 _{toproducelipasesin}

submergedfermentationusingAspergilluscarneusandbyTeng and Xu19 _{and Wang et al.}20 _{using Rhizopus chinensis. Yeast}

extractandpeptonewere usedincombination byMahadik etal.14_{fortheproductionoflipasebyA.niger.Mirandaetal.}28

evaluatedtheproductionoflipasesusingoilrefinerywaste inthepresenceofnitrogensourcessuchasammonium sul-fate,ureaandammoniumchloride,withthebestresultsbeing obtainedusingammoniumchloride.Forlipaseproductionby

Antrodiacinnamomia,Linetal.29 _{usedorganicandinorganic}

sourcesofnitrogen,includingammoniumandnitratesalts, proteins,peptidesandaminoacids,andhigheractivitieswere obtainedwith sodiumand potassium nitrates,ammonium chlorideand asparagine. In our study,yeastextract gener-atedhigherlipolyticactivitiescomparedtosodiumnitrate,a resultthatcouldbeexplainedbecauseyeastextractcontains othercomponentsbesidesanitrogensourcethatcanactas co-enzymesoftheaerobicmetabolicpathway,forexample, carbonskeletonsandcomplexBvitamins.

SeveralauthorsconsiderpHtobeanimportantvariablein theproductionoflipasebysubmergedfermentation. Muralid-har et al.30 _{used an initial pH of 6.5 in the culture of C.}

cylindracea.TengandXu19_{studiedtheeffectofpHonthe}

pro-ductionoflipasesbyRhyzopuschinensisbetweenpH5and7, andthe bestresultswereachievedatpH5.5.Inthis study, pHprovedtobeanimportantvariablefortheproductionof lipases,withtheoptimalvaluebeingapproximately7.0.This resultisinagreementwithGopinathetal.,31_whostudiedpH

4,7and10intheproductionoflipasebyGeotrichumcandidum.

AmongthefactorsstudiedbyWangetal.20_{fortheproduction}

oflipasesbyR.chinensis,pHwasthevariablethathadalower effectonproduction.EachmicroorganismhasapHforoptimal growth.Inthiscase,pHcouldhaveaffectedlipasesynthesis beyondmicroorganismgrowth.

Byevaluatingthevaluesoflipolyticactivityreached dur-ing the experimental designs, A. flavus showed maximum lipolyticactivitiesof4UinthePlackett–BurmanDesignand 2U inthe23 FullFactorial Design,which canbeattributed

to theloss ofthefungi’sability toproducelipases. Accord-ing to Makhsumkhanov et al.,32 _{this result may berelated}

tothe depletion ofthe maintenanceresourcesduring stor-age. Furthermore,theremaybeanaccumulationofcertain metabolites,decreasingtheculture’sproductivity.Thelossof the fungi’s abilitycan besolved duringperiodsof mainte-nancebyusingreactivationmediacontainingvegetableoilas thesoleassimilablecarbonsource.32_{However,themaximum}

lipolyticactivityobtainedintheCCRDwas3.04U,whilethe valuepredictedbythestatisticalmodelforthehighest lipoly-ticactivityofthefungusO-8was3.24U.Inexperimentscarried outpreviouslyfortheselectionoflipase-producingfungiby submergedfermentation,thefungusA.flavushadalipolytic activityofapproximately2Ufor4daysofcultivation,andthe lipolyticactivityatbaselinewas1U,meaning1Uofenzyme wasproducedduringthistime.Comparedwiththeamount oflipolyticactivityobtainedafteroptimization,therewasan increase,whichmeansthattheoptimizationofvariableswas successfullyperformed.

Conclusion

The optimization of lipase production in submerged fer-mentation was possible through the use of sequential experimentaldesign.Theoptimizedconditionswereobtained using thefungusA. flavus,100g/Lwheatbranasthemain componentoftheculturemedium,45g/Lyeastextractasthe nitrogensource,20g/LsoybeanoilastheinducerandpH7.15. TheoptimizationofpHandyeastextractconcentrationusing acentralcompositedesignledtoamathematicalmodelwith acorrelationcoefficientof94.12%withtheexperimentaldata.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WeacknowledgefundingprovidedbyCNPq,Brazil.Wearealso verygratefultotheresearchersFabioRodrigoDuarteandSiu MuiTsai,fromCena/USP,whocarriedouttheidentificationof thefungiusedinthiswork.

r

e

f

e

r

e

n

c

e

s

1.HaackMB,OlssonL,HansenK,LantzAE.Changeinhyphal morphologyofAspergillusoryzaeduringfed-batch

cultivation.ApplMicrobiolBiotechnol.2006;70:482–487.

2.IyerPV,AnanthanarayanL.Enzymestabilityand stabilization–aqueousandnon-aqueousenvironment.

ProcessBiochem.2008;43:1019–1032.

3.SaisubramanianN,EdwinoliverNG,NandakumarN,Kamini NR,PuvanakrishnanR.EfficacyoflipasefromAspergillus nigerasanadditiveindetergentformulations:astatistical approach.JIndMicrobiolBiotechnol.2006;33:669–676.

4.HasanF,ShahAA,HameedA.Industrialapplicationsof microbiallipases.EnzymeMicrobTechnol.2006;39:235–251.

5.SharmaR,ChistiY,BanerjeeYC.Production,purification, characterization,andapplicationsoflipases.BiotechnolAdv.

2001;19:627–662.

6.SinghAK,MukhopadhyayM.Overviewoffungallipase:a review.ApplBiochemBiotechnol.2012;166:486–520.

7.DupuisC,CorreC,BoyavalP.Lipaseandesteraseactivitiesof

Propionibacteriumfreudenreichiisubsp.freudenreichii.Appl EnvironMicrobiol.1993;59:4004–4009.

8.SalahRB,GhamghuiH,MiledN,MejdoubH,GargouriY. Productionofbutylacetateesterbylipasefromnovelstrain ofRhizopusoryzae.JBiosciBioeng.2007;103:368–372.

9.ReshmaMV,SarithaSS,BalachandranC,ArumughanC. Lipasecatalyzedinteresterificationofpalmstearinandrice branoilblendsforpreparationofzerotransshorteningwith bioactivephytochemicals.BioresourTechnol.

2008;99:5011–5019.

10.ParkEY,SatoM,KojimaS.Fattyacidmethylesterproduction usinglipase-immobilizingsilicaparticleswithdifferent particlesizesanddifferentspecificsurfaceareas.Enzyme MicrobTechnol.2006;39:889–896.

11.D’AnnibaleA,SermanniGG,FedericiF,PetruccioliM. Olive-millwastewaters:apromisingsubstrateformicrobial lipaseproduction.BioresourTechnol.2006;97:1828–1833.

12.HaqI,IdreesS,RajokaMI.ProductionoflipasesbyRhizopus oligosporousbysolid-statefermentation.ProcessBiochem.

2002;37:637–641.

13.PandeyA,SoccolCR,MitchellD.Newdevelopmentsinsolid statefermentation:I.Bioprocessesandproducts.Process Biochem.2000;35:1153–1169.

14.MahadikND,BastawdeKB,PuntambekarUS,KhireJM, GokhaleDV.Productionofacidiclipasebyamutantof

AspergillusnigerNCIM1207insubmergedfermentation.

ProcessBiochem.2004;39:2031–2034.

15.CoradiGV,Visitac¸ãoVL,LimaEA,etal.Comparing submergedandsolid-statefermentationofagro-industrial residuesfortheproductionandcharacterizationoflipaseby

Trichodermaharzianum.AnnMicrobiol.2013;63:533–540.

16.BoxGEP,HunterWG,HunterJS.Statisticsforexperimenters:an introductiontodesign,dataanalysis,andmodelbuilding.New York:JohnWiley&Sons;1978.

17.KaushikR,SaranS,IsarJ,SaxenaRK.Statisticaloptimization ofmediumcomponentsandgrowthconditionsbyresponse surfacemethodologytoenhancelipaseproductionby

Aspergilluscarneus.JMolCatalBEnzym.2006;40: 121–126.

18.BurkertJFM,MaugeriF,RodriguesMI.Optimizationof extracellularlipaseproductionbyGeotrichumsp.using factorialdesign.BioresourTechnol.2004;91:77–84.

19.TengY,XuY.Cultureconditionimprovementforwhole-cell lipaseproductioninsubmergedfermentationbyRhizopus

chinensisusingstatisticalmethod.BioresourTechnol.

2008;99:3900–3907.

20.WangD,XuY,ShanT.Effectsofoilsandoil-related substratesonthesyntheticactivityofmembrane-bound lipasefromRhizopuschinensisandoptimizationofthelipase fermentationmedia.BiochemEngJ.2008;41:30–37.

21.RajendranA,PalanisamyA,ThangaveluV.Evaluationof mediumcomponentsbyPlackett–Burmanstatisticaldesign forlipaseproductionbyCandidarugosaandkinetic modeling.ChinJBiotechnol.2008;24:436–444.

22.RodriguesMI,IemmaAF.Experimentaldesignandprocess optimization.NewYork:CRCPress;2014.

23.CollaLM,RezzadoriK,CâmaraSK,etal.Asolid-state bioprocessforselectinglipase-producingfilamentousfungi.

ZNaturforschCBiosci.2009;64:131–137.

24.CollaLM,PrimazAL,BenedettiS,etal.Selectionof lipase-producingmicroorganismsthroughsubmerged fermentation.ZNaturforschCBiosci.2010;65:483–488.

25.SmaniottoA,SkovronskiA,RigoE,etal.Syntheticlipase’ productionfromanewlyisolatedSporidioboluspararoseus

strainbysubmergedfermentation.BrazJMicrobiol.

2012;43:1490–1498.

26.BertolinTE,CostaJAV,PasqualiGDL.Glucoamylase productioninbatchandfed-batchsolidstatefermentation: effectofmaltoseorstarchaddition.JMicrobiolBiotechnol.

2001;11:13–16.

27.PintoGAS,BritoES,AndradeAMR,FragaSLP,TeixeiraRB. Fermentac¸ãoemestadosólido:umaalternativaparao aproveitamentoevalorizac¸ãoderesíduosagroindustriais tropicais.Embrapa,Fortaleza.ComunicadoTécnico.

2005;102:1–5.

28.MirandaOA,SalgueiroAA,PimentelMCB,LimaFilhoJL,Melo EHM,DuránN.LipaseproductionbyaBrazilianstrainof

Penicilliumcitrinumusinganindustrialresidue.Bioresour Technol.1999;69:145–147.

29.LinES,WangCC,SungSC.Cultivatingconditionsinfluence lipaseproductionbytheedibleBasidiomyceteAntrodia cinnamomeainsubmergedculture.EnzymeMicrobTechnol.

2006;39:98–102.

30.MuralidharRV,ChirumamilaRR,MarchantR,NigamP.A responsesurfaceapproachforthecomparisonoflipase productionbyCandidacylindraceausingtwodifferentcarbon sources.BiochemEngJ.2001;9:17–23.

31.GopinathSCB,HildaA,PriyaTL,AnnaduraiG,AnbuP. PurificationoflipasefromGeotrichumcandidum:conditions optimizedforenzymeproductionusingBox-Behnken design.WorldJMicrobiolBiotechnol.2003;19:681–689.

32.MakhsumkhanovAA,YakubovIT,DavranovK.Conditions forcultivationofthefungusPenicilliummeliniiUzLM-4andits biosynthesisoflipases.ApplBiochemMicrobiol.2003;39:40–43.