Efficient production of pullulan by Aureobasidium pullulans grown on mixtures of potato starch hydrolysate and sucrose

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

Biotechnology

and

Industrial

Microbiology

Efficient

production

of

pullulan

by

Aureobasidium

pullulans

grown

on

mixtures

of

potato

starch

hydrolysate

and

sucrose

Chao

An

1

_,

_Sai-jian

_Ma

1

_,

_Fan

_Chang,

_Wen-jiao

_Xue

∗

MicrobiologyInstituteofShaanxi,Xi’an,PRChina

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received1June2015 Accepted16August2016

Availableonline19November2016 AssociateEditor:GiseleMonteirode Souza

Keywords:

Mixedsugar

Aureobasidiumpullulans

Pullulan

Potatostarchhydrolysate Batchkinetics

a

b

s

t

r

a

c

t

Pullulanisanaturalexopolysaccharidewithmanyusefulcharacteristics.However, pullu-lanismorecostlythanotherexopolysaccharides,whichlimitsitseffectiveapplication.The purposeofthisstudywastoadoptanovelmixed-sugarstrategyformaximizingpullulan production,mainlyusingpotatostarchhydrolysateasalow-costsubstrateforliquid-state fermentationbyAureobasidiumpullulans.Basedonfermentationkineticsevaluationof pullu-lanproductionbyA.pullulans201253,thepullulanproductionrateofA.pullulanswith mix-turesofpotatostarchhydrolysateandsucrose(potatostarchhydrolysate:sucrose=80:20) was0.212h−1,whichwassignificantlyhigherthanthoseofpotatostarchhydrolysatealone (0.146h−1)andmixturesofpotatostarchhydrolysate,glucose,andfructose(potatostarch hydrolysate:glucose:fructose=80:10:10,0.166h−1_{)with 100gL}−1 _{totalcarbon source.The}

resultssuggest thatmixtures ofpotato starchhydrolysate and sucrosecould promote pullulansynthesisandpossiblythatasmallamountofsucrosestimulated theenzyme responsibleforpullulansynthesisandpromotedeffectivepotatostarchhydrolysate conver-sioneffectively.Thus,mixedsugarsinpotatostarchhydrolysateandsucrosefermentation mightbeapromisingalternativefortheeconomicalproductionofpullulan.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Pullulanisanextracellularwater-soluble homo-polysaccha-ridecomposedoflinearmaltotriose-unitsinterconnectedvia ␣-1,6-glycosidiclinkages.Theselinkagesendowpullulanwith structuralflexibilityandhighaqueoussolubility.1,2_Pullulanis

∗ _{Correspondingauthorat:ShaanxiInstituteofMicrobiology,No.76XiyingRd,Xi’an,ShaanxiProvince710043,PRChina.}

E-mail:x-wenjiao@163.com(W.Xue).

1 _{ChaoAnandSai-jianMacontributedequallytothiswork.}

usuallybiosynthesizedbystrainsoftheyeast-like polymor-phicfungusAureobasidiumpullulans.3–5_{Becauseofitsstrictly}

linearstructure,pullulanhasbeenusedinapplicationssuch asbloodplasmasubstitutesandasanadditiveforfood, adhe-sives,andcosmetics.1,6_{Despitetheseapplications,pullulanis}

morecostly($25kg−1)thanotherexopolysaccharides,which isamajorlimitingfactorforitseffectiveapplication.7

http://dx.doi.org/10.1016/j.bjm.2016.11.001

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

Aprimarystrategytosolvetheproblemofhighproduction costsistosearchforcheapcarbonandnitrogensources,which arenutritionallyrichenoughtosupportgrowthofthe microor-ganismandtheproductionofpullulan.8_{Leathersconcluded}

thatanagriculturalwasteproduct,maizeresidue,canbeused asacarbon sourceforpullulanproductionbyA.pullulans.1

Otherwaste productsfromthe agricultureandfood indus-tries, such as deproteinized whey, cassava starch residue, beetmolasses,sugarcanejuice,andsweetpotatoandpeat hydrolysates,havealsobeenconsideredaseconomical sub-stratesforpullulanproduction.8,9

Potato, acheap and availableagricultural product, con-tainsalargeamountofstarch,whichisasuitablefeedstock forindustrial fermentation. Potato isalso a major crop in ShaanxiProvinceofChina.However,lowproductivityof pul-lulan fermentationusing potatostarch as asubstrate is a drawback that has not yet been solved.10 _{Available data}

showthatboththeamount ofexopolysaccharideproduced andtherateofcarbohydratesourceconsumptionareclearly influenced by the particular carbohydrate used, and Duan proposed thathigh pullulan yieldis relatedto high activi-tiesof␣-phosphoglucosemutase,UDPG-pyrophosphorylase, and glucosyltransferase in A. pullulans Y68 grown on dif-ferentsugars.11_{Basedontheseconsiderations,mixed-sugar}

fermentation might be an efficient fermentation strategy thatleadstohigh productivitybyprovidingmorethan one substrate.

In our previous research, sucrose was demonstrated to besuperiortoothersugars,basedon pullulanyield,and a mixtureofsucroseandpotatostarchhydrolysate(PSH)was foundtosignificantlyenhancethepullulanformationrateof

A. pullulans201253 whenusing acarbon source inaflask.

Finally,basedonfermentationkineticsevaluationfor pullu-lanformationbyA.pullulans201253,themodelparameters associatedwithpolysaccharideformation(m,n,and(dP/dt)/X) andsubstrateconsumption˛,ˇand(dS/dt)/Xwereassayedto elaboratethefermentationkineticprocessesusingamixture ofpotatostarchhydrolysateandsucrose.

Material

and

methods

Microorganismandculture

A.pullulans201253wasobtainedfromtheAmericanType

Cul-tureCollection(Rockville,MD,USA)andwasmaintainedat 4◦Con potatodextroseagar (PDA)slants andsub-cultured every 2weeks. For long-term storage,cultures were main-tained at −80◦_{C in a 20% glycerol solution. For inoculum}

preparation, A. pullulans was grown at 28◦C for 48h in a medium containing 50g glucose, 2.5g yeast extract, 0.6g (NH4)2SO4,1gNaCl,5gK2HPO4,and0.2gMgSO4·7H2Operliter

ofdeionized water,ataninitialpHof6.5withagitation at 230rpm.

Basic fermentation medium consisted of 50g of car-bon source, 2.5g yeast extract, 0.6g (NH4)2SO4, 1g NaCl,

5gK2HPO4, and 0.2g MgSO4·7H2O per liter of deionized

water,12_{ataninitialpHof6.5withagitationat230rpm.To}

evaluatetheeffectsofmixedcarbonsourcesonpullulan pro-duction,thecarbonsourcewasvariedinthisstudy.

Preparationofpotatostarchhydrolysate

Potatostarch waspurchased from alocalagricultural mar-ket.ThepHofaslurryofpotatostarchataconcentrationof 40%(w/v)wasadjustedtopH6.5andinstantlyheatedto70◦C within3mininthepresenceof0.1%␣-amylase.After lique-faction,thetemperaturewasquicklyreducedto55◦Cwithin 1min,andthepHwasadjustedto5.0.Amyloglucosidasewas thenaddedataconcentrationof0.3%(w/w)andincubatedfor prolongedhydrolysis.Afterhydrolysis,thehydrolysateswere filtered.8

Analyticalmethods

Theculturewascentrifugedat10000×gfor10mintoremove themicroorganism.Thebiomass(myceliaandyeast-likecells) dryweight(BDW)wasdeterminedbywashingthesediment with distilledwaterand dryingat105◦Covernight.Fifteen millilitersofthesupernatantwastransferredintoatesttube, andthen30mLofcoldethanolwasaddedtothetesttubeand mixedthoroughly.Thesolutionwasthenheldat4◦Cfor12h toprecipitatetheexopolysaccharide,whichwasseparatedby centrifugationat8000×gfor10min.Theprecipitated mate-rial wasdriedat80◦C,and the finalweightdetermined as thatofexopolysaccharide.Thetotalconcentrationof reduc-ing sugarwasdeterminedbythedinitrosalicylicacid (DNS) method.13_{FTIRFouriertransforminfraredspectroscopy(FTIR)}

spectra ofpullulan samples forcomposition analysiswere directlyacquiredusingtheSmart-iTRsetupwithoutfurther preparation.14

Batchfermentationofpullulanina10-Lbioreactor

Batch fermentation was carried out in a 10-L bioreactor (YanggeBioengineeringEquipmentCo.,Ltd,Shanghai,China) containing7.0Lfermentationmediumat28◦Cwithagitation of 500rpm. The inoculum volumewas 5.0%(v/v). Samples weretakenperiodicallytodeterminetheconcentrationof pul-lulan,biomass,andreducingsugar.

Determinationofparametersduringthefermentation process

Biomassformation

Thelogisticequation wasusedtofitthebiomasscurves.A commonautonomousrateequationisthedifferentialformof thelogisticequation:

dX dt =maxX



1− X Xmax



, (1)

wheremaxandXmaxaretheinitialspecificgrowthrateand

themaximumbiomassconcentration,respectively.The inte-gratedformusingX0=X(t=o)givesasigmoidalvariationX(t)

thatmayempiricallyrepresentbothanexponentialanda sta-tionaryphase:

X(t)= X0emaxt

1−(X0/Xmax)(1−emaxt).

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Sp ecific growt h rate, µ (h -1) ,

Specific production rate,dP/dt/X,(h)

-1

Specific substrate consumption rate,dS/dt/X,(h)

-1

Specific growth rate Specific production rate

Specific substrate consumption rate

A B C D

Fig.1–Effectofcarbonsourceongrowthrate.(A)Sucrose; (B)PSH;(C)PSH:sucrose=80:20;(D)

PSH:glucose:fructose=80:10:10.

Productformation

Productformationisdescribed byLuedeking–Piretkinetics. Theproductformationratehasbeenshowntodependupon boththe instantaneous biomassconcentration, X, and the growthrate,dX/dt,inalinearmanner:

dP

dt =nX+m dX

dt, (3)

wheremandnareempiricalconstants(forgrowthand non-growth associated polysaccharide formation, respectively) thatmay varywith formationconditions.Integrationof(3)

using(2)givesanequationwithtwoinitialconcentrations(X0,

P0),afinalcondition(Xmax),andthreeparameters(,n,and

m): P(t)=P0+mX0



emaxt 1−(X0/Xmax)(1−emaxt)− 1



+nXmax max ln[1−(X0/Xmax)(1−emaxt)]. (4) Substrateconsumption

Thesubstratebalanceforpolysaccharideformationmaybe writtenasfollows(modificationofLuedekingandPiret):

dS dt =− 1 Yx/s dX dt − 1 YP/s dP dt −keX (5)

whereYx/sandYp/saretheyieldcoefficientsforthebiomass

andproduct,respectively,andkeisthespecificmaintenance rate(i.e.,thesubstrateusedtosupportcellactivityeveninthe absenceofgrowth).From(3)and(5):

dS dt =−˛

dX

dt −ˇX (6)

where˛ and ˇare constants forsubstrate consumptionat growthandnon-growthphases,respectively.Substituting(2) into(6)andintegrating,

S(t)=S0−˛



X0Xmaxemaxt Xmax−X0(1−emaxt)−X0



−ˇXmax max ln



1− X0 Xmax (1−emaxt₎



_{. (7)}

The 1stOpt (First Optimization, 7D-Soft High Technol-ogyInc.)softwarewasappliedtosolvingthe parametersof biomass formation,pullulan formation, and substrate con-sumption.OriginPro8softwarewasappliedtobuildthekinetic curves.

Results

and

discussion

Kineticsofbiomassformation

WecultivatedA.pullulansina10-Lstirred-tankbioreactoron sucrose, PSH, PSH:sucrose=80:20, and PSH:glucose:fructose =80:10:10.Samplesweretakenatdifferenttimeintervalsand analyzed.

Thelogistic equation wasused todescribethe biomass formation,and thecalculatedmodelparametersassociated withbiomass(Xmax,,andX0)aresummarizedinTable1and

representedinFig.1.

Table1–Kineticparametersforthefermentation.

Parameter Carbonsource

A B C D

Specificgrowthratem(h−1) 0.046 0.043 0.042 0.044

MaximumattainablebiomassXmax(gL−1) 13.12 15.72 14.5 14.73

CalculatedinitialbiomassX0(gL−1) 1.38 1.72 1.64 1.29

Growthassociatedconstantm(gproductg−1_{biomass) 6.37 3.405 5.054 3.779}

Non-growthassociatedconstantn(gproductg−1_{biomassh) −0.154 −0.478 −0.291 −0.232}

CalculatedinitialproductP0(gL−1) 0.809 1.473 0.857 1.816

Growthassociatedconstantforsubstrateconsumption˛(gsubstrateg−1biomass) 6.539 4.359 6.308 5.152 Non-growthassociatedconstantforsubstrateconsumptionˇ(gsubstrateg−1biomassh) 0.474 0.263 0.294 0.206

CalculatedinitialsubstrateS0(gL−1) 95.06 91.09 97.77 98.6

14

A

a

b

c

B

C

D

A

B

C

D

A

B

C

D

12 10 8 6 4 2 0 14 12 10 8 6 4 2 0 0 20 40 60 80 100 Time (h) Biomass (g.L –1) Pullulan (g.L –1) Residual sugar (g.L –1) 120 0 20 40 60 80 100 120 0 0 100 80 60 40 20 0 100 80 60 40 20 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 20 40 60 80 100 Time (h) 120 0 20 40 60 80 100 120 0 20 40 60 80 100 Time (h) 120 0 20 40 60 80 100 120

Fig.2–GrowthofA.pullulans201253withdifferentcarbon sources.(a)Comparisonofexperimentaldata()with simulationsusingthelogisticequation().(b)Comparison ofthesimulationusingtheLuedeking–Piretmodel()with theexperimentalpolysaccharideclonal().(c)Comparison

The value of Xmax showed a peak of 15.72gL−1 with

100gL−1PSH,whilethespecificgrowthrate,,showedapeak of0.046h−1with100gL−1sucrose.ThevalueofXmaxvaried

from 13.12gL−1 to15.72gL−1, andthe specificgrowth rate, , variedfrom 0.042h−1 to 0.046h−1 with different carbon sources(allataconcentrationof100gL−1)inthisstudy.Thus, weconcludedthatthetypeofcarbon sourcedoesnotlead toasignificantdifferenceinthebiomass.Theresultsmight beattributedtothepresenceofthesameamountsof nitro-gen sourceavailableforassimilationinthemediumduring differentcultivations.15 _{Accordingtotheresultsofprevious}

studies,nitrogensourcelevelscouldshiftthepatternof car-boneffluxduringthefermentationofpullulanbyA.pullulans,

andpullulanproductiondecreasedbutbiomassaccumulation increasedasthelevelofnitrogensourceincreased.16,17

Kineticsofpullulanformation

Product formation is described byLuedeking–Piret kinetics andthecalculatedmodelparametersassociatedwith polysac-charideformation(m, n, P0, and(dP/dt)/X) arepresentedin

Table1andFig.2(a).

Variationsinthespecificproductionrate(dP/dt)/Xshowed a peak of 0.243h−1 with 100gL−1 sucrose as the carbon source.Theresultsalsoindicated thatthe specific produc-tion rate (dP/dt/X, h−1) on mixtures of PSH and sucrose (PSH:sucrose=80:20) was 0.212h−1, which was significantly higherthanthoseofPSH(0.146h−1)andmixturesofPSH, glu-cose,andfructose(PSH:glucose:fructose=80:10:10,0.166h−1) with100gL−1totalcarbonsource.

Thevaluesofmandn(Table1)indicatethatpolysaccharide formationbyanon-growth associatedmechanism ismuch lessimportantcomparedtothatbyagrowth-associated mech-anism,regardlessoftheinitialcarbonsource.Similarresults werereportedbyKlimek,Boa,andMulchandani.18–20

Fig. 2(b) also indicates that a mixed substrate of PSH andsucrosecanincreasetherateofpullulanformationand shortenthefermentationperiod.Thehighestyieldofpullulan was110hwhenPSHwasusedasasolecarbonsource; how-ever,toachievethesameyield,sucrose,PSH:sucrose=80:20, andPSH:glucose:frutcose=80:10:10required50,60,and80h, respectively.

Accordingtopreviousstudies,co-feedingfermentationis anefficientfermentationstrategythatenableshigh produc-tivity with lowcostby providingmorethan onesubstrate. Xu et al. adopted aco-feeding strategy toproduce l-lactic acidbasedoncanemolasses/glucosecarbonsources,withthe aimofremovingsubstancesfromcanemolassesthatinhibit fermentation.21 _{Li et al.appliedamixed-sugar strategyfor}

highlyefficientandeconomicproductionofdocosahexaenoic

acid byAurantiochytrium limacinum SR21.22 _{Our resultsalso}

indicated thata mixtureofPSH (alowcost substrate) and sucrose,whichwasdemonstratedtobesuperiortoother sug-arswithrespecttothetiterofpullulanproducedinourstudy

oftheexperimentaldata()oftheconsumptionbyA.

pullulans201253andthesimulationusingthemodified

Luedeking–Piretmodel().(A)Sucrose;(B)PSH;(C) PSH:sucrose=80:2;(D)PSH:glucose:fructose=80:10:10.

4000 3500 3000 2500 2000 1500 1000 500 10 20 30 40 50 60 70 80 90 100 Wavenumber (cm-1 ) Tr a n smitta n ce, %

Fig.3–FITRforpullulanproducedfromthemixtureofsucroseandPSHasacarbonsource.

andpreviousreports,15,23,24_{cansignificantlyenhancethe}

pul-lulanformationrateofA.pullulans201253atalowcost.

Kineticsofsubstrateconsumption

Substrate consumption is described by modification of LuedekingandPiretkinetics,andthecalculatedmodel param-eters associated with substrate consumption are given in

Table1.

Variations in the specific substrate consumption rate (dS/dt)/Xshowedapeak of0.675h−1 with100gL−1 sucrose asthecarbonsource.Thespecificsubstrateconsumptionrate (dS/dt)/XonmixturesofPSHandsucrose(PSH:sucrose=80:20) was 0.559h−1, which was significantly higher than those of PSH (0.45h−1) and mixtures of PSH, glucose, and fruc-tose(PSH:glucose:fructose=80:10:10,0.52h−1)with 100gL−1 oftotalcarbonsources.AsFig.2(c)shows,after96hof fermen-tation,thecarbonsourceinthefermentationbrothwasalmost entirelyconsumedwhensucroseonlywasusedasthe sub-strate,andonly1.15gL−1sugarwasnotconverted.Residual sugar(17.40gL−1,6.32gL−1,and12.24gL−1)remainedwhen PSH,PSH:sucrose=80:20,andPSH:glucose:fructose=80:10:10, respectively.

These resultsshow that sucrosewas the most efficient carbonsourceforsubstrateconsumption,whichwasin agree-ment with a previous study.15 _{Additionally, these results}

indicatethatmixturesofPSHandsucrosecansignificantly enhance the utilization rate ofA. pullulans for the carbon source. The reason may be that A. pullulans could simul-taneously consumedifferent carbonsources andeliminate the carbon source repression effect. Similar results have been widely reported for the fermentation of a variety of products.25,26

IRcharacteristics

Fig.3showstheFT-IRspectraforpullulanobtainedfroma mix-tureofsucroseandPSH.Thepeaksappearingat3400cm−1and 2930cm−1wereattributedtoOHstretchingandCHstretching, respectively.Furthercharacteristicsignalsobtainedat1368, 1155,and1080cm−1wereattributedtoC–OHbending,C–O–C stretching,andC–Ostretching,respectively.27_{Otherfeatures}

ofthebiopolymerwerealsofoundfromthespectra, includ-ingO–C–Ostretch(1656cm−1),andC–O–Cstretch(1155cm−1), as reported previously.28 _{The absorption that appeared at}

755cm−1and931cm−1wasattributedto␣(1,4)and␣(1,6) link-ages between glucose units withinpullulan, respectively.29

These resultssuggest thatthe exopolysaccharideproduced fromthemixtureofPSHandsucroseispullulan.

Conclusions

Inourresearch,sucrosewasthebestcarbonsourcefor pul-lulan productionbyA.pullulans201253,whilethepotatois arelativelycheaprawmaterialandamajorcropinShaanxi Province.Hence,anovelmixed-sugarstrategywasdeveloped foreconomicalpullulanproductionbyA.pullulans. Eventu-ally, theyieldofpullulanincreasedto54.57gL−1 whenthe ratioofPSHtosucrosewas20:80,whichisslightlybelowthe yieldofpullulanusedbysucrose,buthigherthanthatwith PSHandasugarmixture(PSH:glucose:fructose=80:10:10).The results suggest that sucrose canpromote pullulan synthe-sis,becauseasmallamountofsucrosemightstimulatethe enzymeresponsibleforpullulansynthesisandpromote effec-tivePSH conversioneffectively.Overall, thisstudy provides anefficientandeconomicaloptionfortheutilizationofraw materialsinmicrobialproduction.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

This project was funded bythe Scientificand Technologic Research Program of Shaanxi Academy ofSciences, China (No.2013k-06,2014k-01),theWesternChineseAcademyof Sci-ences (2013DF01), the Natural ScienceBasic Research Plan in Shaanxi Province ofChina (Program No. 2014JM2-2015), Shaanxi Science& Technology Co-ordination & Innovation

Project(2014SZS07-K03),andtheScienceandTechnology Pro-gramofXi’an(NC1505(3)).

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