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Simultaneous production of amylases and proteases by Bacillus subtilis in brewery wastes

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

Industrial

Microbiology

Simultaneous

production

of

amylases

and

proteases

by

Bacillus

subtilis

in

brewery

wastes

Alina

Sánchez

Blanco

a

,

Osmar

Palacios

Durive

a

,

Sulema

Batista

Pérez

a

,

Zoraida

Díaz

Montes

a

,

Nelson

Pérez

Guerra

b,∗

aDepartamentodeFundamentosQuímicosyBiológicos,FacultaddeIngenieríaQuímica,sedeMella,UniversidaddeOriente,Santiagode

Cuba,Cuba

bDepartamentodeQuímicaAnalíticayAlimentaria,FacultaddeCiencias,CampusdeOurense,UniversidaddeVigo,Spain

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received1August2015 Accepted8January2016 Availableonline20April2016 AssociateEditor:AdalbertoPessoa Junior Keywords: Amylases Bacillussubtilis Brewerywastes Proteases

Responsesurfacemethodology

a

b

s

t

r

a

c

t

Thesimultaneousproductionofamylase(AA)andprotease(PA)activitybyBacillussubtilis UO-01inbrewerywasteswasstudiedbycombiningtheresponsesurfacemethodologywith thekineticstudyoftheprocess.Theoptimumconditions(T=36.0◦CandpH=6.8)forhigh biomassproduction(0.92g/L)weresimilartotheconditions(T=36.8◦CandpH=6.6)for highAAsynthesis(9.26EU/mL).However,themaximumPAlevel(9.77EU/mL)wasobtained atpH7.1and37.8◦C.Undertheseconditions,aconsiderablyhighreduction(between69.9

and77.8%)oftheinitialchemicaloxygendemandofthewastewasachieved.Inverification experimentsundertheoptimizedconditionsforproductionofeachenzyme,theAAand PAobtainedafter15hofincubationwere,respectively,9.35and9.87EU/mL.Byusingthe LuedekingandPiretmodel,bothenzymeswereclassifiedasgrowth-associatedmetabolites. Proteaseproductiondelayseemedtoberelated totheconsumptionofnon-proteinand proteinnitrogen.Theseresultsindicatethatthebrewerywastecouldbesuccessfullyused forahighscaleproductionofamylasesandproteasesatalowcost.

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

Introduction

TheamylasesandproteasesproducedbytheBacillusspecies are of world-wide interest for their important industrial applications.1Therefore,productionoftheseenzymesshould

becarriedoutatalowproductioncostbyusingeconomically availableculturemedia(suchasfoodwastesoragro-industrial residues)andoptimizedfermentationconditions.2,3

Correspondingauthor.

E-mail:nelsonpg@uvigo.es(N.PérezGuerra).

Differentstudieshaveshownthatproductionofamylases and proteases is affected by a variety of physicochemical factors, including the type and composition of the sub-strate, incubationtime andtemperature, pH, agitation and the concentration and type of the carbon and nitrogen sources.1,3However,insomecases,theeffectsofthese

vari-ables on enzyme production have been studied by using the “one variable ata time” method1,3,4 rather than using

responsesurfacemethodology(RSM).Thefirstmethodistime

http://dx.doi.org/10.1016/j.bjm.2016.04.019

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

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consuming and couldlead toanincomplete interpretation ofthebehaviorofthesystem,resultinginalackof predic-tiveability,mainlywhenthereareinteractionsbetweenthe independentfactors.5Incontrast,theuseofRSMallowsthe

reductioninthenumberofexperimentsandobtains empiri-calmathematicalmodelsdescribingtheeffect(bothlinearand quadratic)ofeachindependentfactorandtheirinteractions ontheresponsevariables.5

Consideringthesubstantialavailabilityofbrewerywastes (BWs)withahighchemicaloxygendemand(COD)atverylow pricesfromabreweryplantinSantiagodeCuba(Cuba),the useofthiseffluentasafermentationsubstratecouldofferan attractivealternativeforthelowcostproductionofamylases andproteasesbyBacillussubtilisUO-01.However,tothebest ofourknowledge,thereisnoinformationavailableontheuse ofbrewerywasteforproductionofamylasesandproteasesby

Bacillusstrains.

In the present study, we investigated the suitability of thiswastetosupportboththegrowthandenzyme

produc-tionbyB.subtilisUO-01atdifferentinitialvaluesofpHand

incubationtemperatures.Forthispurpose,thetimecourses ofthe synthesisofamylases, proteases,biomassand total sugars consumption were followed for 30h. Subsequently, theconcentrationsofenzymes andbiomassobtainedwere modeledbyusingthecorrespondinglogisticmodeltosmooth theexperimentaldataobtainedandreducetheexperimental error.Thedatapredictedbythelogisticmodelineachcaseat theappropriatefermentationtimewerethenusedfortheRSM analysistoobtaintheoptimumpHandtemperaturevaluesfor highproductionofamylases,proteasesandbiomass.In addi-tion,theenzymeproductionsystemwasstudiedunderthe optimumconditionstoverifytheeffectivenessandthe accu-racyoftheempiricalenzymemodelobtained.Aftertypifying boththeamylaseandproteaseproductionwiththeLuedeking andPiretmodel,6therelationshipbetweenprotease

produc-tion and consumption of total nitrogen and proteins was studiedatdifferentculturepH valuesandatthe optimum temperatureforgrowthofB.subtilisUO-01.

Materials

and

methods

Bacterialcultures

B.subtilisUO-01,theamylase-andprotease-producingstrain,

wasacquiredfromtheBiotechnologyCenteroftheUniversity ofOriente(SantiagodeCuba,Cuba).Stockcultureswere main-tainedat4◦Connutrientagarslants.Theworkingcultures werepreparedmonthlyfromfrozenstockculturesand main-tained at4◦C on nutrient agar (CultimedPanreac Química S.A.).

Culturemediumpreparation,inoculumandfermentation conditions

Brewerywastes(BWs),whichwereusedtopreparethe cul-turemedia,wereobtainedfromalocalbreweryinSantiagode Cuba,Cuba.Thewasteswerecentrifugedat12,000×g/15min toremovethesolidsinsuspension.Thesupernatantobtained fromthe BWs contained(g/L):COD, 3.40;totalsugars,1.98;

Table1–Experimentaldomainandcodificationofthe

independentvariables(T:temperatureandpH)usedin

theexperimentaldesign.

Codedvalues Actualvalues

T(◦C) pH –1.267 28.0 4.0 –1 30.0 4.5 0 37.8 6.2 1 45.5 8.0 1.267 47.6 8.5

reducingsugars,1.46;totalnitrogen,0.095;totalphosphorus,

0.034.

Inoculumwaspreparedbytransferring(withasterile

inoc-ulationloop)somecoloniesofa24-hold slantcultureinto

250mLErlenmeyerflaskscontaining50mLofsterilemedium

composed of(g/L):glucose,20; bacteriologicalpeptone,2.5;

KH2PO4·3H2O,1.5;Na2SO4,1.5;MgSO4·7H2O,0.15,FeSO4·7H2O,

0.03;MnCl2·4H2O,0.1;CaCl2·2H2O,0.45.Afterinoculation,the

mediumwasadjustedtopH6.8andsterilizedat121◦C/15min.

The inoculated culture was then incubated at 36◦C/12h

(200rpm).

The production BWs medium was supplemented with

the same ingredients as the mediumused to prepare the

inoculum,but inthiscase,soluble potatostarch(at a

con-centrationof10g/L)wasusedinsteadofglucose.Themedia

werebufferedatdifferentinitialpHvalueswiththe

appropri-atebuffer(0.1Mpotassiumhydrogenphthalate–HClbufferfor

pHvaluesof4.0,4.5and6.2or0.1Msodiumphosphatebuffer

forpHvaluesof8.0and8.5)accordingtotheexperimental

designdefinedinTable1andthensterilized(121◦C/15min).

Batchcultureswereperformedintriplicatein250mL Erlen-meyerflaskscontaining50mLofthecorrespondingbuffered medium.Eachflaskwasinoculatedwitha2%(v/v)inoculum level(withanabsorbanceof0.5at600nm)ofa12-hinoculum cultureandincubatedinanorbitalshaker(200rpm)for30hat thecorrespondingtemperatureaccordingtotheexperimental matrixdefinedinTable1.

To study the relationshipbetween nitrogenand protein consumptionandproteaseproductionbystrainUO-01at dif-ferent initialpHvalues,theBWsmediawerebufferedwith the same buffers used inthe former experimentto obtain initialpHvaluesof4,5,6,7,and 8.Themediawere incu-bated(200rpm)attheoptimumtemperatureforgrowthofthe enzyme-producingbacteriumfor21h.

Analyticalmethods

In each fermentation, threeflasks were collected each 3h, and triplicatesamples(runs)weretakenfromeach flaskto performanalyticaldeterminations.TheCODvalueswere mea-suredattheendofthecultureperiod(30h).

Growthwasmonitoredbyabsorbanceat600nmand con-vertedtocelldryweight(CDW)fromastandardcurve.Cells were harvested by centrifugation (12,000×g for 15min at 4◦C)ofculturesamplesandwashedtwicewithsaline(0.8% NaCl).Theculturesupernatantswereusedtomeasuretotal sugars (TS), total nitrogen (TN), proteins, enzyme produc-tion and COD. The methods for determining total sugars

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(phenol–sulfuricacidmethod),nitrogen(micro-Kjeldahl, sub-stitutingdistillationfor aspectrophotometric method)and proteins (method of Lowry) were described in a previous work.7

Amylaseactivity(AA)ofthecell-freemediumwas deter-mined at 40◦C by appropriately mixing the diluted crude enzymeextract(80␮L)with400␮Lof0.15Mcitrate–phosphate buffer (pH 5.0) and 800␮L of 4% soluble starch previously maintained at 40◦C/15min. The reaction was stopped by immediatelyadding480␮Lof3,5-dinitrosalicylicacid(DNS). Thereducingsugars(glucoseequivalents)releasedfromthe enzymaticreactionweredetermineafter10minbyusingthe DNSmethod.Oneunitofamylaseactivity (enzymaticunits (EU)/mL) was defined as the amount of enzyme releasing 1mg/mLofreducingsugars/minundertheassayconditions.8

Proteaseactivity (PA)ofthecell-freemediumwas deter-mined as described by Tekin et al.9 The reaction mixture

with1mLof1%(w/v)casein in0.02MNaOH,2mLof0.4M phosphatebufferpH6,and1mLofcell-freemedium (suit-ablydiluted)wasincubatedat30◦C/10min.Thereactionwas stopped with 3mL of 10% trichloroacetic acid, mixed and after5min,centrifugedat12,000×gfor5min,then0.5mLof thesupernatantswereincubatedwith2.5mLof0.1MNaOH in2%(w/v)Na2CO3 for10min.Thereafter,0.25mLofFolin phenolicreagent(commercialsolutiondiluted1:1indistilled water)wasadded,mixedandheldfor30minatroom temper-ature.Theabsorbancemeasuredat750nmwasconvertedto mgoftyrosine/Lusingacalibrationcurve(mgtyrosine/Lvs. absorbance).Thetyrosinesolutions dissolvedin0.01MHCl weretreatedinthe samemannerasthecell-freemedium. Oneunitofprotease(EU/mL)wasdefinedastheamountof enzymethatproducedanabsorbanceat750nmequivalentto 1␮moloftyrosine/minundertheassayconditions.

CODanalyseswerecarriedoutintriplicateintheculture supernatants atthe end ofeach fermentation(30h)using the Standard Methods for the Examination of Water and Wastewater.10

Statisticalanalyses

Afterobtainingtheexperimentalconcentrationsofbiomass (CDW), AA and PA foreach sampling time, the data were smoothedbyusing,incaseofgrowthandPAproduction,the correspondingformofthelogisticmodel6andamodifiedform

ofthelogisticmodel,inthecaseofAA:

CDW(g/L)= CDWmax 1+e(c−b·t), beingc=ln



CDW max CDW0 − 1



(1) AA (EU/mL) = A0·e (−A1·t) 1+e(A2+A3·t+A4·t2)− A0 1+e(A2) (2) PA (EU/mL) = A0 1+e(A2+A3·t+A4·t2)− A0 1+e(A2) (3)

whereCDWmaxandCDW0are,respectively,themaximumand theinitialcelldryweightconcentration(g/L),bisaconstant ofproportionality(h−1),tisthetime(h)andA0,A1,A2,A3and A4areconstantsinmodels(2)and(3).Thelattermodelswere alsoadjustedtodescribeAAproduction.However,thebestfits

wereobtainedwiththeuseofmodel(2)thatincludesaterm (e(−A1·t))thatconsiderstheAAdecayobservedfromthe18h

ofincubationuntilthe endoftheculture.Aftersmoothing thecellgrowthandenzyme(AAandPA)productioncurves, thebestsamplingtimetotakethedataforthecorresponding experimentaldesignwasselectedforstudyingthecombined effectofpHandtemperatureonthegrowthandenzyme syn-thesisbyB.subtilisUO-01intheBWsmedium.

Thecentralcomposite(orthogonal)designwasbasedon twolevelsandtwovariablesandconsistedof13experiments withfour(22)factorialpoints,fouraxialpointstoforma cen-tral composite designwith ˛=1.267 and five center points forreplication (Table1).The corresponding smootheddata obtainedat15hoffermentationwereusedineachcase.The results were analyzed by the Experimental Design Module of the Statistica software package (Statistica 12.0 for Win-dowscomputer programmanual; StatSoftInc., 2013,Tulsa, OK,USA).Thecoefficientsofthemodelswithpvalueslower than0.05wereconsideredstatisticallysignificant.TheFisher’s

F-test foranalysis of variance (ANOVA) was performed on

experimentaldatatoevaluatethe statisticalsignificanceof themodel.

ModelingtheenzymesproductionwiththeLuedekingand Piretmodel

BothAAandPAproductionweremodeledbyusingthe Luedek-ing and Piret model.6 Themodel parameters in each case

wereobtainedbyusingthenon-linearcurve-fittingsoftware ofSigmaPlotforWindowsversion12.0(SystatSoftware,Inc., 2011).Thecoefficientsofthemodelswithpvalueslowerthan 0.05wereconsideredstatisticallysignificant.

Results

and

discussion

Kineticsofgrowth,enzymeproductionandtotalsugars consumptionbyB.subtilisUO-01inBWsmedia

Thekineticsofgrowth(CDW),productionofamylase(AA)and protease(PA)activityandtotalsugars(TS)consumptionbyB.

subtilisUO-01inBWsmediawasfollowedinbatchculturesat

differentinitialpHvaluesandincubationtemperatures(Fig.1) accordingtotheexperimentaldesigndefinedinTable1.

Accordingtothepredictionsofmodel(1),biomass produc-tionincreaseduntil15hofincubationandremainedconstant afterwards inall the culturesalthough,in some fermenta-tions, the total sugars were not totally consumed (Fig. 1). However,theevolutionofenzyme(AAandPA)synthesisand biomassproductionshowedsomedifferences.Thus,AAlevels increaseduntilreachingthemaximumconcentrationat15h ofculture,thentheAAconcentrationwasmaintainedalmost constantuntil18handdecreasedslightlyafterwards(Fig.1), althoughnotalwayssignificantly,becausethevaluesobtained forthecoefficientA1inmodel(2)werenotalwayssignificant. Becausethepresenceofstarchinducestheproductionof ␣-amylases,2duringthefirst15hoffermentation,AAwas

syn-thesizedbyB.subtilisUO-01tohydrolyzethestarchpresent inthemediumandproducemoreeasilyassimilablesugars (glucoseandmaltose)forthegrowingstrain.1

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12 1 5 9 10 6 7 11 8 12 13 2 3 4 0.6 0.4 0.2 0 0.4 0.3 0.2 0.1 0 0.8 0.6 0.2 0.4 0 0.8 0.6 0.2 0.4 0 0.6 0.4 0.2 0 0 10 20

Time (h) Time (h) Time (h)

Time (h) Time (h) Time (h)

Time (h) Time (h) Time (h)

Time (h) Time (h)

CDW: Cell dry weight (g/L),

TS: Total sugars (g/L) AA: Amylase activity (EU/mL), PA: Protease activity (EU/mL),

Time (h) Time (h) TS , AA, P A CD W TS , AA, P A CD W TS , AA, P A CD W TS , AA, P A CD W TS , AA, P A CD W TS , AA, P A CD W TS , AA, P A CD W TS , AA, P A CD W TS , AA, P A CD W TS , AA, P A CD W TS , AA, P A CD W TS , AA, P A CD W CD W TS , AA, P A 30 T=45.5 ºC, pH=8.0 T=47.6 ºC, pH=6.2 T=28.0 ºC, pH=6.2 T=37.8 ºC, pH=8.5 T=37.8 ºC, pH=4.0 T=37.8 ºC, pH=6.2 T=37.8 ºC, pH=6.2 T=37.8 ºC, pH=6.2 T=37.8 ºC, pH=6.2 T=37.8 ºC, pH=6.2 T=45.5 ºC, pH=4.5 T=30.0 ºC, pH=8.0 T=30.0 ºC, pH=4.5 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 0 0.3 4 0.6 8 0.9 12 1.2 0 0.3 0.6 0.9 1.2 0 0.3 0.6 0.9 1.2 0 0.2 0.4 0.6 0.8 0 0.3 0.6 0.9 1.2 0 0.3 0.6 0.9 1.2 16 0 4 8 12 16 0 4 8 12 16 0 4 8 12 16 0 4 8 12 16 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 9 6 3 0 12 0.12 9 0.09 6 0.06 0.03 0 3 0 12 9 6 3 0 12 9 6 3 0 12 9 6 3 0 0.3 0.2 0.1 0 12 9 6 3 0 12 9 6 3 0 12 9 6 3 0

Fig.1–Kineticofproductionofcelldryweight,amylaseandproteaseactivity,andconsumptionoftotalsugarsbyB.subtilis

UO-01attheinitialpHandTvalues,asdefinedinTable1.Eachdatapointrepresentsthemeanofthreeindependent cultures(thestandarderrorswerelessthan5%ofthemeans).

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Table2–Concentrationsofbiomass(CDWing/L),amylase(AAinEU/mL)andprotease(PAinEU/mL)activityobtainedat

15hofincubationandchemicaloxygendemand(COD)reductionpercentages(%)attheendofthecultures(30h).No.:

numberoftheexperiment.

No. Codedvalues Actualvalues Responsevariablevalues

T(◦C) pH T(◦C) pH CDW AA PA CODreduction 1 1 1 45.5 8.0 0.49 3.97 7.16 40.4 2 1 –1 45.5 4.5 0.10 1.73 2.72 8.1 3 –1 1 30.0 8.0 0.68 5.30 7.50 56.3 4 –1 –1 30.0 4.5 0.37 3.20 3.06 30.1 5 1.267 0 47.6 6.2 0.35 3.08 5.42 28.8 6 –1.267 0 28.0 6.2 0.63 4.94 5.91 51.3 7 0 1.267 37.8 8.5 0.68 5.20 8.26 56.7 8 0 –1.267 37.8 4.0 0.25 2.25 2.64 19.9 9 0 0 37.8 6.2 0.91 9.40 8.84 74.4 10 0 0 37.8 6.2 0.89 9.27 8.81 73.1 11 0 0 37.8 6.2 0.93 9.40 9.48 77.8 12 0 0 37.8 6.2 0.81 8.75 9.63 69.9 13 0 0 37.8 6.2 0.87 9.10 9.48 72.1

TheobserveddeclineinAAsynthesisafter18hof

incuba-tioncouldberelatedtoapossibleproteolyticdegradationof

theamylasesbytheactionoftheextracellularproteases

pro-ducedbytheB.subtilisstrain.1,2 However,otherresearchers

attributedthisAAdecreasetoapossibledenaturationand/or decompositionoftheenzymeduetointeractionswithother compoundsinthefermentedmedium.3

Proteaseproductionthat reachedthe maximumPA lev-elsat15hofincubation (Fig.1)wasonlydetectedafter9h offermentationinall cultures, whenthe cells enteredthe post-exponentialphase,asitwasobservedbeforeforother

Bacillus species.11 Thisbehavior hasbeen attributed tothe

needforobtainingnutrientsforpost-exponentialsurvival11

ortoanincreasedneedforturnoverofcell proteinsatthe slowergrowthrate.1

Becauseallthemediawerebufferedatthecorresponding initialpHvalues,thecessationofgrowthfrom15hof fermen-tationcouldberelatedtothefollowingreasons:

1. IntheculturesatinitialpH≥6.2,thecarbonsourcereached levels considerablylower(<3g/L),exceptforthe cultures atinitialpH8.0and T=45.5◦C and atinitialpH6.2and T=47.6◦C(Fig.1).TheselowTSlevelscouldleadtoalow availabilityofthecarbonsource12thatlimitsthegrowthof

B.subtilisUO-01.

2. Theexhaustioninall theculturesofsomenutrient(the sources of nitrogen and/or phosphorus) or micronutri-ent(vitamins,mineralsaminoacidsorcations)essential forthegrowthoftheenzyme-producingstrain,asitwas observedbeforeforotherbacteria.12,13

Theremoval percentagesofinitial CODload in the fer-mented media are shown in Table 2. The higher COD reductions(69.9–77.8%)wereobtainedinthecultures charac-terizedbythehighestbiomassproductioninthecenterpoints oftheexperimentaldesign,suggestingthatB.subtilisUO-01 wasabletoutilizetheorganicmatterpresentinthebrewery wastewaterasasourceofnutrients.Thus,bacterialgrowth andenzymeproductionbyB.subtilisUO-01couldbeusedas analternative forremovingtheinitialCODload present in brewerywastes.

KineticclassificationoftheAAandPAproduction

Totypifytheproductionofthetwoenzymes(E),theclassical LuedekingandPiret(LP)model6wasused:

dE dX=˛·

dX

dt +ˇ·X,ordividingbythebiomass (4)

qE=(˛·+ˇ) (5)

whereXisthebiomassconcentration(gofCDW/L),tisthe time(h),qEisthespecificenzymeproductionrate(EU/mg/h),  isthe specificgrowth rate(h−1), ˛isagrowth-associated constant forenzymeproduction(EU/mg),and ˇisthe non-growth-associatedconstant(EU/mg/h).

TheCDW,AAandPAvalues(symbolsinFig.2)predicted bymodels(1),(2)and(3)(solidlinesinFig.1)wereusedto calculatethevaluesoftheparametersoftheLPmodel(5).

The trajectories for the productiondynamic of the two enzymes(solidlinesinFig.2)werecalculatedbynumerical integrationoftheqEvaluesobtainedwiththeLPmodel(5)for AAandPAsynthesisinthecorrespondingbatchculturesofB.

subtilisUO-01.

Forbothenzymes,significantvalues(p<0.05)for˛and non-significantvalues(p>0.05)forˇwerealwaysobtained(Fig.2). Thus,AAandPAcanbecharacterizedasgrowth-associated metabolites(˛ /= 0,ˇ=0).However,AAproduction(R2

AA

val-uesbetween0.985and0.999)wasmoresatisfactorilydescribed bytheLPmodelthanPAsynthesis(R2

PAvaluesbetween0.876

and0.969),mainlybecauseproductionofthelatterenzyme startedafter9hofincubation,whilethegrowthandAA pro-ductionstartedfromthebeginningoftheculture(Fig.2).

EffectofinitialpHandincubationtemperatureonthe growthandproductionofamylasesandproteases

TheresultsontheeffectofinitialpHandtemperatureon pro-ductionofbiomass(CDW),AAandPAbyB.subtilisUO-01in BWsmediumafter15hofincubationareshowninTable2.

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T=45.5 ºC, pH=8.0

AA, P

A

Time (h) Time (h) Time (h) Time (h)

Time (h) Time (h) Time (h)

Time (h) Time (h)

Time (h) AA: Amylase activity (EU/mL), PA: Protease activity (EU/mL)

Time (h) Time (h) Time (h) AA, P A AA, P A AA, P A AA, P A AA, P A AA, P A AA, P A AA, P A AA, P A AA, P A AA, P A AA, P A T=47.6 ºC, pH=6.2 T=37.8 ºC, pH=6.2 T=37.8 ºC, pH=6.2 T=28.0 ºC, pH=6.2 T=45.5 ºC, pH=4.5 T=30.0 ºC, pH=8.0 T=37.8 ºC, pH=8.5 T=37.8 ºC, pH=6.2 T=37.8 ºC, pH=6.2 T=37.8 ºC, pH=6.2 T=37.8 ºC, pH=4.0 T=30.0 ºC, pH=4.5 9 1 2 6 5 9 10 11 12 13 3 7 4 8 αPA=13.5±0.3 αPA=22.8±0.7 αAA=9.1±0.2 αAA=22.7±1.8 R2 PA=0.946 R2PA=0.876 αPA=11.3±0.8 R2 PA=0.969 αPA=6.8±0.1 R2 PA=0.932 R2 AA=0.999 αPA=14.6±1.0 αPA=8.4±0.5 αAA=9.5±0.8 R2 PA=0.945 R2 PA=0.950 R2 AA=0.997 αAA=8.6±0.7 R2 AA=0.998 αPA=10.5±0.9 R2 PA=0.936 αPA=8.7±0.8 R2 PA=0.943 αAA=8.6±0.7 R2 AA=0.998 αAA=10.9±0.3 R2 AA=0.999 αPA=9.9±0.8 R2 PA=0.932 αAA=11.8±0.9 R2 AA=0.992 αPA=8.6±1.2 R2 PA=0.939 αAA=11.4±1.4 R2 AA=0.992 αPA=9.6±1.2 R2 PA=0.944 αAA=11.9±1.3 R2 AA=0.987 αPA=8.7±1.1 R2 PA=0.939 αAA=11.5±1.2 R2 AA=0.989 αPA=8.3±1.0 R2 PA=0.939 αAA=11.5±0.8 R2 AA=0.993 R2 AA=0.993 αAA=9.4±0.7 R2 AA=0.985 αAA=9.3±0.7 R2 AA=0.992 6 3 0 9 6 3 0 3 2 1 0 6 4 2 0 12 9 6 3 0 12 9 6 3 0 12 9 6 3 0 12 9 6 3 0 12 9 6 3 0 12 9 6 3 0 3.5 2.8 2.1 1.4 0.7 0 10 8 6 4 2 0 5 4 3 2 1 0 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40

Fig.2–ModelingtheproductionofamylaseandproteaseactivitiesbyB.subtilisUO-01inBWsmediaatdifferent temperaturesandpHvalues(experiments1–13,Table1).Thesymbolscorrespondtotheamylaseandproteaselevels obtained,respectively,withmodels(2)and(3).ThesolidlinesdrawnthroughthesymbolswereobtainedaccordingtotheLP model(5).Theparameters˛AAand˛PAarethegrowthassociatedconstants(EU/mg)forAAandPAproductionsintheLP

model,andR2

AAandR2

PAarethecorrespondingcorrelationcoefficients.

The empirical models obtained with their significant coefficientsare:

CDW=0.88−0.11·T+0.17·pH−0.23·T2−0.25·pH2 (6) AA=9.11−0.72·T+1.12·pH−2.88·T23.06·pH2 (7) PA=9.21+2.22·pH−2.07·T2−2.21·pH2 (8) Becausethepvalueswerehigherthan˛(˛=0.050)andthe Fvalueswerelowerthan6.390forthethreemodels,thereis astatisticallysignificant relationshipbetweenthe response andthe independentvariablesatthe 95%confidencelevel. Thehighervaluesoftheadjusteddeterminationcoefficient (adjR2=0.989,0.983and0.989)obtainedforthesemodelsalso

indicatedtheirhighsignificanceandexcellentadequacyfor theexperimentaldata.

Theresponsesurfacesgeneratedwiththeempirical mod-els(6),(7)and(8)areshowninFig.3A–C,respectively.

Thehighestbiomassconcentration(0.92g/L)predictedby model (6) was obtained at T=36.0◦C and pH=6.8. These optimum T and pH values were almost similar to those (T=36.8◦C andpH=6.6) obtainedforhigh amylase produc-tion (9.26EU/mL). The same behavior was observed before

foraB.subtilisstrainisolatedfromsoilsamples3 andthree

mesophilicBacillus(Bacillussp.,B.subtilisandB.

amyloliquefa-ciens)isolates.14

However, comparisons of the optimum amylase level producedbyB.subtilisUO-01withtheoptimumenzyme pro-ductions for other Bacillus strains is very difficult due to the differentmethodologiesusedtodeterminetheenzyme

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CD W (g/L)

A

B

C

AA (EU/mL) pH pH Temp (ºC) Temp (ºC) Temp (ºC) pH P A (EU/mL) 1.0 47.6 37.8 28.0 6.0 6.9 7.7 47.6 37.8 28.0 6.0 6.9 7.7 12 9 6 3 0 0.8 0.6 0.4 0.2 0 1.0 47.6 37.8 28.0 6.0 6.9 7.7 8 6 4 2 0

CDW: Cell dry weight AA: Amylase activity, PA: Protease activity

Fig.3–ResponsesurfacesshowingtheeffectoftemperatureandpHontheproductionofcelldryweight,amylaseand proteaseactivitiesbyB.subtilisUO-01inBWsmedia.

activity,thedifferentformstoexpresstheenzyme concentra-tionandthedifferentformstodefinetheenzymeactivityunit. Forexample,insomecases,theenzymeactivitywas deter-minedbyquantifyingtheamountsofreducingsugarsreleased fromthehydrolysisofstarchbyincubatingthereaction mix-turesattemperaturesof40◦C,150C14or60C15for10min.

Inthesecases,theenzymeactivityunitwasdefinedasthe amountofenzymereleasing1␮molofglucose14,15ormaltose1

equivalentsfromthesubstrateperminundertheassay condi-tions,andthereactionmixtureshaddifferentcompositions.

Withregardtoprotease production,the maximumlevel (9.77EU/mL)predictedbymodel(8)wasobtainedatpH=7.1 and T=37.8◦C. Similar results were obtained forB. subtilis strainRand,whichproducedthemaximumrelativeprotease activity(%)atpH=7andT=37◦C.16

Asindicatedabove,comparisonoftheoptimumprotease levelproduced byB. subtilisstrainUO-01 withtheprotease levelsfoundbyotherresearchersisverydifficultasa con-sequence ofthe different forms ofexpressing the enzyme concentrationsordefiningthe unitofproteaseactivity.For example, protease levels have been expressed as relative productioninU/log10CFU17orasrelativeactivity(%).16In addi-tion,theunitofproteaseactivitywasdefinedastheamount ofenzymerequiredto:(i)produceanincreaseof0.1in opti-caldensityat700nm,1(ii)release1␮goftyrosinepermLper

minute,18or(iii)toobtainanincreaseof0.001absorbanceunit

at450nmperminute.16

The increase in temperature over its optimum value inhibitedproduct(CDW,AAandPA) formationbyB.subtilis strainUO-01,probablybysuppressionofcellviability19 and

enzymaticinactivation.20Incontrast,lowtemperaturevalues

mayslowdownthemetabolismofthemicroorganism20and

consequently,enzymesynthesis,becausethelatterproducts weresynthesizedasgrowth-associated(primary)metabolites, asitwasdemonstratedwiththeLPmodel(Fig.2).

The decrease in growth and enzyme (AA and PA) pro-duction observed at pH levels lower and higher than the optimumvaluecouldberelatedtoareductioninthemetabolic activityofB.subtilisUO-01,3probablycausedbyalimitation

in micronutrients or nutrient transport.13,21 Inactivation or

instabilityoftheenzymescouldbeotherreasonstoexplain thedecreaseobservedinenzymeproduction.22

Verificationofthepredictedresultsintheoptimal conditionsforenzymeproduction

Triplicateincubationexperimentswerecarriedoutunderthe optimizedconditionsforAAandPAproductionstoverifythe resultspredictedbymodels(7)and(8).Theexperimentaldata forgrowthandproductionofAAandPAinbothcaseswere smoothedbyusingthemodels(1),(2)and(3)(Fig.4).Underthe optimalconditionsforAAproduction(T=36.8◦CandpH=6.6), thepredictedvaluesofbiomass,AAandPAafter15hof incu-bationwere,respectively,0.94g/L,9.35EU/mLand9.34EU/mL

(8)

Optimum conditions for AA T=36.8 ºC, pH=6.6

CDW: Cell dry weight (g/L), PA: Protease activity (EU/mL),

AA: Amylase activity (EU/mL), TS: Total sugars (g/L) Time (h) 12 9 6 3 0 1.2 0.9 0.6 0.3 0 0 10 20 30 40 0 10 20 30 40 12 9 6 3 0 1.2 0.9 0.6 0.3 0 Time (h) TS , AA, P A TS , AA, P A CD W CD W

Optimum conditions for PA T=37.8 ºC, pH=7.1

Fig.4–Kineticsoftheproductionofcelldryweight,amylaseandproteaseactivityandconsumptionoftotalsugarsbyB. subtilisUO-01undertheoptimumconditionsforamylaseandproteaseproductions,respectively.Eachdatapointrepresents themeanofthreeindependentassays(thestandarderrorswerelessthan5%ofthemeans).

(leftpartofFig.4).Inaddition,undertheoptimumAA con-ditions,theconcentrations ofbiomassand PApredicted by theempiricalmodels(6)and(8)were0.91g/Land9.51EU/mL, respectively.

Inthesameway,undertheoptimumconditionsforPA pro-duction(T=37.8◦CandpH=7.1),themeanconcentrationsof biomass,AAandPAafter15hofincubationpredictedbythe models(1),(2)and(3)were,respectively,0.91g/L,8.97EU/mL and9.87EU/mL(rightpartofFig.4).UndertheoptimumPA

conditions,theconcentrationsofbiomassandAApredictedby theempiricalmodels(6)and(7)were0.90g/Land8.90EU/mL, respectively.Therefore,themodels(6),(7)and(8)canbe con-sideredtobeaccurateandreliableforpredictingthegrowth andproductionofamylasesandproteasesbyB.subtilisUO-01 asafunctionofinitialpHandincubationtemperatureinBWs media.

Asobservedinthepreviouscultures(Fig.1),protease pro-ductionstartedafter9hofincubationintheculturesatthe

CDW: Cell dry weight (g/L), AA: Amylase activity (EU/mL), PA: Protease activity (EU/mL), TS: Total sugars g/L)

1.2 12 9 6 3 0 12 9 6 3 0 2 0.4 1 0.3 0.2 0 0.9 0.6 0 9 6 3 0 0 5 10 15 Time (h) Time (h) Prot (g/L) AA (EU/mL) CD W (g/L) P A (EU/mL) TN (g/L) TS (g/L) Time (h) 20 25 0 5 10 15 20 25 0 5 10 15 20 25 0.3 pH=4 ( ), pH=5 ( ), pH=6 ( ), pH=7 ( ), pH=8 ( )

Fig.5–Timecourseofcelldryweight,amylaseandproteaseactivityandconsumptionoftotalsugars,proteinsandtotal nitrogenatdifferentinitialpHvaluesandatafixedtemperatureof36◦C.

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optimalconditionsforAAandTAproduction(Fig.4). Inan attempttoexplaintheobserveddelayinproteaseproduction, thekineticsofgrowthandenzymeproductionwerefollowed inparallelwiththeevolutionofthecorrespondinginductor nutrient(totalsugarsandproteins)foreachenzyme.

Becauseboththenutrientconsumptionandproduct for-mation by different bacteria (Lactococcus lactis, L. cremoris,

LactobacillusrhamnosusandLact.casei)dependedontheculture

pH,13,21aseriesofcultureswithstrainUO-01inBWsmedia

adjustedtoinitialpHvaluesof4,5,6,7and8wasstudied. Thecultureswereincubatedat36◦C(theoptimum temper-atureforbiomassproduction)for21h.Consideringthatthe presenceofproteinandnon-proteinnitrogenintheculture mediacanaffectproteinconsumption,7theevolutionoftotal

nitrogenwasalsofollowedinthefermentations.

As observed in Fig. 5, production of biomass and AA increasedinparallelwiththedecreasesinTSandTN.In addi-tion,adirectrelationshipbetweenthetimecoursesofproteins andPAproductionwasobserved.Inthelattercase,proteins decreasedat9hoffermentationwhenproteaseproduction wasdetectedintheculturemedium.However,totalnitrogen wasslightlyconsumedduringthefirst6hofincubation,but afterthistime,theconcentrationoftotalnitrogendecreased rapidly,coincidingwiththestartoftheintenseprotein con-sumption.

IfthetimecoursesofproteinsandTNarejointlyanalyzed, adirectrelationshipbetweenbothvariablesonlyexistsafter 9hofincubation.Accordingtothemanufacturers(Pronadisa, LaboratoriosConda,SA),thebacteriologicalpeptoneusedas thenitrogensourceintheBWsmediahasatotalnitrogenand anaminonitrogencontent of15.5%and 3%(w/w), respec-tively. Thus, B. subtilis UO-01 seemed to consumefirst the non-proteinnitrogenfraction (presumablyeasier to assim-ilate) and then thenitrogen present inthe proteinsofthe culturemedia (Fig. 5), as it was observed before for other bacteria.7,23,24

These observations suggest that the delay in protease productioncould be closely relatedto the consumption of non-proteinandproteinnitrogenbyB.subtilisUO-01(Fig.5). However,theconsumptionofbothtotalnitrogenandproteins dependedon theculturepH (Fig. 5)asobservedbefore for nutrient(sugars,aminoacidsandphosphorus)transportin differentstrainsoflacticacidbacteria.13,21

Conclusions

Theresultsobtainedinthisworkshowedthatbrewerywastes couldbeusedforenzyme(amylasesandproteases)

produc-tionbyB.subtilisUO-01,withtheadditionaladvantageofan

effectiveandcost-effectivereductionoftheinitialCOD.Then, themethodologicalproceduredescribedinthisworkandthe resultsobtained could beused as areference for the pro-ductionofotherimportantmicrobialmetabolitesinbrewery wastesandinothereffluentsfromthefoodindustry.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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5.AgregánR,AlonsoE,TorradoA,GuerraNP.Areviewonsome importantfactorsaffectingbacteriocinproductionby lactococci,lactobacilliandpediococci.CurrBiochemEng. 2014;1:9–24.

6.GuerraNP.Modelingthebatchbacteriocinproduction systembylacticacidbacteriabyusingmodified

three-dimensionalLotka-Volterraequations.BiochemEngJ. 2014;88:115–130.

7.GuerraNP,TorradoA,LópezC,FajardoP,PastranaL. Dynamicmathematicalmodelstodescribethegrowthand nisinproductionbyLactococcuslactissubsp.lactisCECT539in bothbatchandre-alkalizedfed-batchcultures.JFoodEng. 2007;82:103–113.

8.TorradoA,VázquezJA,PrietoMA,etal.Amylaseproduction byAspergillusoryzaeinasolid-statebioreactorwith

fed-batchoperationusingmusselprocessingwastewatersas feedingmedium.JChemTechnolBiotechnol.2013;88:226–236. 9.TekinN,CihanAC¸,Takac¸ZS,TüzünCY,Tunc¸K,C¸ökmüs¸C. AlkalineproteaseproductionofBacilluscohniiAPT5.TurkJ Biol.2012;36:430–440.

10.APHA.StandardMethodsfortheExaminationofWaterand Wastewater.21steditionWashington,DC:AmericanPublic HealthAssociation;2005.

11.AronsonAI,BellC,FulrothB.Plasmid-encodedregulatorof extracellularproteasesinBacillusanthracis.JBacteriol. 2005;187:3133–3138.

12.VanNielEWJ,Hahn-HägerdalB.Nutrientrequirementsof lactococciindefinedgrowthmedia.ApplMicrobiolBiotechnol. 1999;52:617–627.

13.FajardoP,RodríguezI,PastranaL,GuerraNP.Productionofa potentiallyprobioticcultureofLactobacilluscaseisubsp.casei CECT4043inwhey.IntDairyJ.2008;18:1057–1065.

14.NusratA,RahmanSR.Comparativestudiesonthe productionofextracellular␣-amylasebythreemesophilic Bacillusisolates.BanglJMicrobiol.2007;24:129–132.

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