Sugarcane straw as a feedstock for xylitol production by Candida guilliermondii FTI 20037

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

Industrial

Microbiology

Sugarcane

straw

as

a

feedstock

for

xylitol

production

by

Candida

guilliermondii

FTI

20037

Andrés

Felipe

Hernández-Pérez

∗

,

Priscila

Vaz

de

Arruda,

Maria

das

Grac¸as

de

Almeida

Felipe

DepartamentodeBiotecnologia,EscoladeEngenhariadeLorena,UniversidadedeSãoPaulo,Lorena,SãoPaulo,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15January2015 Accepted15September2015 Availableonline2March2016 AssociateEditor:JorgeGonzalo FariasAvendano Keywords: Sugarcanestraw Hemicellulosichydrolyzate Xylitol Nutritionalsupplementation Initialoxygenavailability

a

b

s

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t

Sugarcane strawhasbecomeanavailable lignocellulosicbiomasssincetheprogressive introductionofthenon-burningharvestinBrazil.Besideskeepingthisbiomassinthefield, itcanbeusedasafeedstockinthermochemicalorbiochemicalconversionprocesses.This makes feasibleitsincorporationina biorefinery,whoseeconomic profitabilitycouldbe supportedbyintegratedproductionoflow-valuebiofuelsandhigh-valuechemicals,e.g., xylitol,whichhasimportantindustrialandclinicalapplications.Herein,biotechnological productionofxylitolispresentedasapossiblerouteforthevalorizationofsugarcanestraw anditsincorporationinabiorefinery.Nutritionalsupplementationofthesugarcanestraw hemicellulosichydrolyzateasafunctionofinitialoxygenavailabilitywasstudiedinbatch fermentationofCandidaguilliermondiiFTI20037.Thenutritionalsupplementationconditions evaluatedwere:nosupplementation;supplementationwith(NH4)2SO4,andfull supplemen-tationwith(NH4)2SO4,ricebranextractandCaCl2·2H2O.ExperimentswereperformedatpH 5.5,30◦C,200rpm,for48hin125mLErlenmeyerflaskscontainingeither25or50mLof medium inordertovaryinitialoxygenavailability.Without supplementation,complete consumption ofglucose andpartialconsumptionofxylosewereobserved.Inthis con-ditionthemaximumxylitolyield(0.67gg−1)wasobtainedunderreducedinitialoxygen availability.Nutritionalsupplementationincreasedxyloseconsumptionandxylitol produc-tionbyupto200%and240%,respectively.Themaximumxylitolvolumetricproductivity (0.34gL−1h−1)wasreachedatfullsupplementationandincreasedinitialoxygen availabil-ity.Theresultsdemonstratedacombinedeffectofnutritionalsupplementationandinitial oxygenavailabilityonxylitolproductionfromsugarcanestrawhemicellulosichydrolyzate. ©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Brazilisthelargestsugarcaneproducerintheworld, responsi-bleforapproximately38%ofglobalproduction.1_Intheseason

∗ _{Correspondingauthor.}

E-mail:ahernandez@usp.br(A.F.Hernández-Pérez).

2014/2015,theproductionlevelreached634.8millionsmetric tons.2_{Itisestimatedthat28%(onadryweightbasis)ofthe}

harvested sugarcanecorresponds tolignocellulosic byprod-ucts,bagasseandstraw(tops, dryandgreenleaves),which representtwo-thirdsoftheenergeticpotentialofsugarcane.3

http://dx.doi.org/10.1016/j.bjm.2016.01.019

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

Currently, sugarcane bagasse, produced during sugarcane milling,iswidelyusedasafuelinacogenerationprocessfor energyproduction,andasafeedstockinbioprocessesresearch forproductionoflignocellulosicethanolandchemicals.3,4

Also,sugarcanestraw isbecominganavailable lignocel-lulosic biomass,due tothe progressiveintroduction ofthe non-burning harvest (green management), which aims to improvethecropsustainability.3,5_{Accordingtotheseauthors,}

thisharvestsystemleadstodepositionof7–25metrictons/ha of sugarcane straw on the soil, corresponding to a straw-to-stalk ratio of 9–30%. The mulch formed has important agronomicandeconomicimpactsandmaypotentiallycause positiveornegativeeffectsoncropyield,soilfertility,fertilizer management,weedcontrol,soilerosionandorganicmatter dynamics,dependingonthecharacteristicsofeacharea.5

Besidesthe agronomic benefitsofkeeping this biomass in the field, sugarcane straw can be used as a feedstock in thermochemical and biochemical conversion processes, makingfeasibleitsincorporationinafuturesugarcane biore-finery.Infact,ithasbeenproposedthattheincorporationof bothsugarcanebagasseandstrawwouldbeessentialforthe developmentofasugarcanebiorefinerywithcapabilitiesto producesugar,first-andsecond-generationethanol,electrical andthermalenergy,andchemicalproducts,undereconomic, socialandenvironmentalsustainability.6_{Currently,themain}

usesproposed forsugarcanestraware itscomplete utiliza-tionasafuelforenergygenerationanditsdepolymerization forethanolandenergyproduction.3,4,7

It is also known that the profitability of a low-value biofuel-producingbiorefinerycanbesupportedbyintegrated productionofhigh-valuechemicals.8_{Oneofthe high-value}

chemicals as defined by the Department of Energy of the UnitedStates (US-DOE), which can fulfill this approach, is xylitol.9_{Xylitolisanaturalsweetener,whichhasbeenhighly}

valuedforitsimportantapplicationsinfood, odontological, andpharmaceuticalindustries, andasaplatformchemical for the synthesis of other valuable chemicals.10 _Currently,

commercialproductionofxylitolisbasedoncatalytic hydro-genationofxylosepurifiedfromhemicellulosichydrolyzates, aprocessthatisconsideredtobeinefficientandexpensivedue tothecomplexityofthexylosepurificationprocedure.11,12

Biotechnologicalproductionofxylitolfromlignocellulosic biomasshasbeenwidelystudiedasanalternativetothe com-mercialchemicalprocess,sinceneitherpurificationofxylose norhightemperaturesandpressuresarerequiredforthis bio-logicallyselectiveprocess.11–13_{Thisbioprocessisbasedonthe}

abilityofpentose-assimilatingyeaststoreducexyloseto xyli-tolasthefirststepofxylosemetabolism,withparticipation ofNAD(P)H-dependentxylosereductase(XR,E.C.1.1.1.21).14–16

Accordingtotheseauthors,accumulationofxylitoliscaused by a NADH/NAD+ _{imbalance, i.e., low levels of NAD}+ _and

highlevelsofNADH,resultingfromlimitedO2supplyanda

consequentreductionincell respiratoryrate.Thisprevents the oxidation ofxylitol to xylulose by inhibiting NAD(P)+

-dependentxylitoldehydrogenase (XDH,E.C.1.1.1.9), and,as aresult,its integrationinto thecentralcarbon metabolism through the pentoses phosphate pathway (PPP). According toSilvaandcolleagues17_{andYablochkovaandcolleagues,}15

whilesome yeasts can compensate forthe reduced NAD+

regenerationusingNADHasacofactorforXR,thisenzyme

isexclusivelydependentonNADPHinotheryeasts,suchas Candidaguilliermondii,whichmakesthemgoodcandidatesfor xylitolproduction.

Itisknownthat therequirement fornutritional supple-mentationisanimportantfactorfortheuseofhemicellulosic hydrolyzates as suitable fermentationmedia for xylose-to-xylitolbioconversion.18–20 _{Ithasalreadybeendemonstrated}

thatsupplementationrequirementsdependontheraw mate-rial characteristics, due to the fact that the composition ofahemicellulosichydrolyzate variesaccordingtotheraw material, presenting different contents of minerals, trace elements,vitamins,proteins,amongothers,whichare com-poundsthat canbeusedasnutrients formicrobial growth andmetabolism.20,21_{Regardingthefermentativeprocess,itis}

widelyrecognizedthatalimited-oxygen atmosphereisone of the most important factors that must be controlled to increase theprocess efficiency.16,17 _{AccordingtoSomerville}

andProctor,22_{oxygensupplyinbatchculturesatalaboratory}

scaleisafunctionoftheagitationspeedandtheratiobetween thevolumesofaculturemediumandaflask.

Thepresent work aimedto usethe hemicellulosic frac-tion ofsugarcanestraw asa feedstockforxylose-to-xylitol bioconversion by C. guilliermondii FTI 20037, as a possible route for its valorization and incorporation in a biorefin-ery.Tothe best ofour knowledge,thisisthe firstwork on biotechnological productionofxylitolfromsugarcanestraw hemicellulosic hydrolyzate. Therefore, nutritional supple-mentation was studied as a function of initial oxygen availabilityinordertoestablishasuitableformulationofthe fermentationmediumthatwouldfavoroptimalyeast perfor-mance,whichinturnaffectsthecostofthisbioprocess.

Materials

and

methods

Sugarcanestraw

The sugarcane straw was kindly provided by Usina Santa Adélia,Jaboticabal,SãoPaulo,Brazil.Itwascollecteddirectly intheformleftinthefieldaftertheharvest.Thestrawwas drieduntilmoisturecontentwas11.25%.Thebiomassinnature waschemicallycharacterizedforcellulose,hemicelluloseand lignin contents,according tothemethodology proposedby Gouveiaandcolleagues.23

Preparationofhemicellulosichydrolyzate

Thesugarcanestrawwassubjectedtodilute-acidhydrolysis with1.0%(w/v)H2SO4ina35-Lsteelreactorat121◦Cfor20min

at a 1:10 solid/liquidratio. The hemicellulosic hydrolyzate was filtered and vacuum-concentrated at 70◦C to increase theinitialconcentrationofxylosebythreefold.24_Next,itwas

subjected to a detoxificationtreatment consisting ofa pH adjustment to7.0 withCaO (commercial grade) and to2.5 withH3PO4,followedbytheadditionof1.0%(w/v)activated

charcoal (refined powder)and incubation at 60◦C,100rpm for30min.25 _{Alltheprecipitatesformedduringthe process}

were removedbyvacuumfiltration. Thetreated hemicellu-losichydrolyzatewasautoclavedat111◦C,for15mintobe usedasafermentationmedium.

Table1–Structuralchemicalcompositionofsugarcanestrawandcomparisonwithotherlignocellulosicbiomasses.

Rawmaterials Component(%w/v) Reference

Cellulose Hemicellulose Lignin Ash

Sugarcanestraw 31.7 27.0 31.1 1.5 Presentwork

Sugarcanestraw 33.8 27.4 21.3 2.6 Szczerbowskiandcolleagues26

Sugarcanebagasse 43.1 28.6 20.8 2.9 Arruda37

Ricestraw 43.5 22.0 17.2 11.4 MussattoandRoberto21

Wheatstraw 33.8 31.8 20.1 7.0 Canilha38

Oathulls 29.3 28.4 22.2 4.5 Tamaniniandcolleagues39

Microorganismandinoculumpreparation

Forthefermentationexperiments,theyeastC.guilliermondii FTI20037maintainedat4◦Conmalt-extractagarslantswas used.Themediumusedforinoculumpreparationcontained (gL−1):xylose(30.0),ricebranextract(20.0),(NH4)2SO4 (2.0),

andCaCl2·2H2O(0.1). Thericebranwaskindlyprovidedby

thelocalfarmBeneficiadoradeArrozIrmãosLigabo,Canas, SãoPaulo,Brazil.Forricebranextractpreparation,amixture ofthericebran(200g)anddistilledwater(1L)wasautoclaved (111◦C,for15min).Aftercooling,themixturewascentrifuged (2000×g,for20min)underasepticconditionsandthe super-natant,correspondingtothericebranextract,wasaseptically transferredtoasterilizedflask.Aloopfulofcells grownon maltextractagarwastransferredtothemedium(50mL)used forinoculumpreparationinErlenmeyerflasks(125mL)and theculturewasincubatedonarotaryshakerat30◦C,200rpm for24h.Afterwards,thecellswereseparatedby centrifuga-tion(2000×g,for20min),rinsedtwicewithdistilledwater, andthecellpelletwasresuspendedinanappropriatevolume ofdistilledwatertobeusedasaninoculum.

Mediumandfermentationconditions

Thefermentationmediumconsistedofthesugarcanestraw hemicellulosichydrolyzate, withthe followingcomposition (gL−1): xylose (50.60),glucose (9.75),arabinose (9.60),acetic acid(2.87), 5-hydroxymethylfurfural(5-HMF)(0.04).The fol-lowing three nutritional supplementation conditions were evaluated: H1, not supplemented; H2, supplemented with (NH4)2SO4 (2.0gL−1);and H3, fully supplementedwith rice

branextract(20.0gL−1),(NH4)2SO4(2.0gL−1)andCaCl2·2H2O

(0.1gL−1). The initialpH was adjusted to 5.5and was not controlledduringruns.Batchfermentationswerecarriedout in125-mLErlenmeyerflasks,containing 25or50mLofthe fermentationmediuminordertovarytheinitialoxygen avail-ability.Theinitialcellbiomassconcentrationinalltheflasks was1gL−1.Theflaskswereincubatedonarotaryshakerat 30◦C,200rpmfor48h.

Analyticalmethods

The concentrations of xylose, glucose, arabinose, acetic acid, xylitol, glycerol, and ethanol were determined by high-performanceliquidchromatography(HPLC)(Shimadzu LC-10AD,Kyoto,Japan),usingarefractiveindexdetectoranda Bio-Rad(Hercules,CA,USA)AminexHPX-87Hcolumnat45◦C, with0.01NH2SO4asaneluent,ataflowrateof0.6mLmin−1.

Theconcentrations offurfuraland 5-HMFwere also deter-minedbyHPLC,usinganultravioletlightdetector(SPD-10A

UV-VIS,WatersCorp.,Milford,MA,USA)andanRP-18column (Hewlett-Packard,PaloAlto,CA,USA)at25◦C,with acetoni-trile:water(1:8)and10%aceticacidasaneluentandaflow rateof0.8mLmin−1.Cellgrowthwasmonitoredbymeasuring absorbanceat600nm(DU640Bspectrophotometer,Beckman Coulter,Brea,CA,USA)andcalculatedbasedonthe relation-shipbetweenabsorbanceandcelldryweight.

Results

and

discussion

The suitability of the hemicellulosic fraction of sugarcane strawwasstudiedasafeedstockforthebiotechnological pro-ductionofxylitol.Table1showsthestructuralcomposition ofthesugarcanestraw,incomparisonwithother lignocellu-losicbiomasses.Theproportionofthehemicellulosicfraction (27.0%)determined inthis study wassimilar tothatfound by Szczerbowski and colleagues26 _{also in sugarcane straw}

(27.4%), aswell as similar tothe values reportedforother biomassesthatwereevaluatedforxylose-to-xylitol bioconver-sion,thussuggestingthatsugarcanestrawcanbeanadequate feedstockforthisbioprocess.

Thechemicalcompositionofthesugarcanestraw hemi-cellulosichydrolyzateobtainedbydilute-acidhydrolysis(not concentrated and not detoxified) is shown in Table 2 in comparisonwithotherhemicellulosichydrolyzatesobtained under similar hydrolysis conditions. Xylose was the major monosaccharide,representing71%ofthesugarcontent,and itsconcentration(18.6gL−1)wasintherangeofother hemicel-lulosichydrolyzates.Glucoseandarabinoseconstituted14.1 and 14.9%, respectively. For comparison, Szczerbowski and colleagues26_{foundthatxyloserepresented75%ofthe}

hemi-cellulosicfractionofsugarcanestraw.Thehighxylosecontent ofthesugarcanestrawhemicellulosichydrolyzatesupported thesuitabilityofsugarcanestrawasafeedstockfor biotech-nologicalxylitolproduction.

Itisknownthatthepresenceofglucoseinafermentation mediumcan affect,positivelyor negatively, xylitol produc-tion, depending on the glucose/xylose ratio.27,28 _According

toTable2,theglucoseconcentrationinthesugarcanestraw

hemicellulosichydrolyzatewashigherthaninother hemicel-lulosichydrolyzates;nevertheless,theglucose/xyloseratioin thesugarcanestrawhemicellulosichydrolyzatewas approxi-mately1/5,whichhadbeenfoundbySilvaandFelipe28_tobe

theoptimumratioforxylitolproductionbyC.guilliermondiiFTI 20037fromasugarcanebagassehemicellulosichydrolyzate. Regarding toxic compounds, it was confirmed that the

Table2–Compositionofsugarcanestrawhemicellulosichydrolyzateandcomparisonwithotherhemicellulosic hydrolyzates.

Rawmaterials Component(gL−1) Reference

Glucose Xylose Arabinose Aceticacid Furfural 5-HMF

Sugarcanestraw 3.7 18.6 3.9 2.23 0.33 0.57 Presentwork

Sugarcanebagasse 0.84 17.85 1.68 3.15 0.06 0.008 Martonandcolleagues25

Sugarcanebagasse 1.20 18.24 1.71 3.38 0.08 0.003 Rodriguesandcolleagues24

Ricestraw 3.29 18.33 3.4 1.05 0.1 0.17 MussattoandRoberto21

Sorghumstraw 2.1 17.69 1.81 1.87 0.04 1.56 Seneandcolleagues36

concentrations of acetic acid, furfural and 5-HMF in the sugarcane straw hemicellulosic hydrolyzate were in most caseshigherthanthosefoundinthehydrolyzatesofother biomasses,exceptforaceticacidinsugarcanebagasseand 5-HMFinsorghumstraw (Table 2). Nevertheless,the deter-minedvaluesforallthetoxiccompoundswerelowerthanthe reported inhibitory concentrations for xylose consumption andxylitol productionbyC. guilliermondiiinsemi-synthetic media29–31_{andhemicellulosichydrolyzates.}30,32

Theprofilesofxyloseconsumption,cellgrowthand xyli-tolproductionbyC. guilliermondiiFTI20037 growing inthe sugarcanestrawhemicellulosichydrolyzatearepresentedin

Fig.1A–Casfunctionsofnutritionalsupplementationand ini-tialoxygenavailability.Table3summarizesthefermentative parameters for all the experiments. In general,the results obtainedinthepresentworkshowedthatxylose consump-tion, cell growth and xylitol production were improved by nutritional supplementation. Furthermore, it was observed thatxyloseutilizationforcellgrowthorxylitolproductionwas dependentontheinitialoxygenavailability,sincethe exper-iments were performed underconditions ofincreased and reducedinitialoxygenavailability,whichwereestablishedby theuseofdifferentmediumvolumes(25and50mL, respec-tively)intheErlenmeyerflasks(150mL).

0 2 4 6 8 10 12 14 16 18 20 0 10 20 30 40 50 60 50 40 30 20 10 0 Time (h) H1

A

B

0 2 4 6 8 10 12 14 16 18 20 0 10 20 30 40 50 60 50 40 30 20 10 0 Time (h) H2 Cell grow th, Xy litol (gL –1 ) Cell grow th, Xy litol (gL –1 ) Xy lose (gL –1 ) Xy lose (gL –1 )

C

0 2 4 6 8 10 12 14 16 18 20 0 10 20 30 40 50 60 50 40 30 20 10 0 Cell grow th, Xy litol (gL –1 ) Xy lose (gL –1 ) Time (h) H3

Fig.1–Profilesofxylose(square),xylitol(circle)andcellbiomass(triangle)inbatchfermentationwithC.guilliermondiiFTI

20037insugarcanestrawhemicellulosichydrolyzatewithdifferentnutritionalsupplementationandthetwoconditionsof

initialoxygenavailability:(A)withoutsupplementation(H1);(B)supplementationwith(NH4)2SO4(H2);(C)full

supplementationwith(NH4)2SO4,ricebranextractandCaCl2·2H2O(H3).Dashedline(---)andopenstyle:increasedinitial

oxygenavailability(25mLoffermentationmedium).Solidline(—)andsolidstyle:Reducedinitialoxygenavailability(50mL

Table3–ParametersofC.guilliermondiiFTI20037fermentationforeachevaluatedcondition.

Nutritionalsupplementationcondition H1 H2 H3

Volumeoffermentationmedium 25mL 50mL 25mL 50mL 25mL 50mL

Xyloseconsumption(%) 26.12 29.29 60.58 30.95 79.22 57.80

Xyloseuptakerate(gL−1h−1) 0.33b _0.32b _1.08c _0.36b _0.87b _0.53a

Cellbiomass(gL−1) 12.09 6.87 17.30 5.69 19.02 7.16

Cellgrowthrate(gL−1h−1) 0.27b _0.28d _0.41b _0.19e _0.43b _0.27e

Cellbiomassyield(gg−1) 0.57 0.30 0.43 0.23 0.43 0.20

Xylitolproduced(gL−1) 4.78 8.69 4.75 9.61 16.20 14.24

Xylitolyield(gg−1) 0.41 0.67 0.16 0.63 0.47 0.55

Efficiency(%) 45.26 73.32 16.97 68.95 50.91 60.07

Xylitolvolumetricproductivity(gL−1h−1) 0.10 0.18 0.10 0.20 0.34 0.30

H1:withoutsupplementation;H2:supplementationwith(NH4)2SO4;H3:fullsupplementationwith(NH4)2SO4,ricebranextractandCaCl2·2H2O.

25mLoffermentationmedium:increasedinitialoxygenavailability;50mLoffermentationmedium:reducedinitialoxygenavailability.

Xyloseuptakerateandcellgrowthratewerequantifiedduringtheperiodofmaximumxyloseconsumptionandcellgrowth,respectively,asis

denotedbythefollowingsymbols:

a _{Between0hand48h.}

b _{Between8hand48h.}

c _{Between24hand48h.}

d _{Between0hand8h.}

e _{Between0hand24h.}

Cellbiomassyieldwasquantifiedconsideringxyloseandglucoseconsumption.

Itisimportanttopointoutthatintheexperiments with-outnutritionalsupplementation(H1,Fig.1A)the yeastwas ableto completely consumeglucose (datanotshown) and partiallyconsumexylose,resultingincellgrowthandxylitol production(Table3).Thisresultsuggestedthatthesugarcane strawhemicellulosic hydrolyzatecouldsupply abasallevel ofnutrientstosupportthemicrobialmetabolism.Asimilar observationwasmadeforthesameyeastgrownin hemicel-lulosichydrolyzatesofricestraw20_{andbrewer’sspentgrain.}21

Theauthors suggested that, in the case of the rice straw, thisfactwasassociatedtoitshighashcontent(11.4%), indi-catingthattheashcontainedminerals,traceelementsand vitamins20_{;and,inthecaseofthebrewer’sspentgrain,due}

toitselevatedproteincontent(15.25%)andalargevarietyof minerals.21_{Inthepresentwork,theashcontentofthe}

sugar-canestraw(1.5%)waslowerthanthevaluesreportedforthe above-mentionedmaterials(Table1),whiletheproteinand mineralcontentsofthesugarcanestrawwerenotdetermined. Hence,furtherstudiesarenecessarytoestablishthefeasibility ofdevelopingatechnologyforxylitolproductionfrom sug-arcanestrawhemicellulosichydrolyzatewithoutnutritional supplementation.

Withoutsupplementation(H1),xylose consumptionwas 26.12%and29.29%underconditionsofincreasedandreduced initialoxygenavailability,respectively(Table3).Inthe exper-iments with increased initial oxygen availability, xylose consumptionwasincrementedby132%relativetoH1with thesupplementationof(NH4)2SO4(H2).Anadditionalincrease

ofxyloseconsumption by31% relativetoH2was obtained withthefullsupplementationof(NH4)2SO4,ricebrainextract

andCaCl2·2H2O(H3),correspondingtoatotalimprovement

of200%betweenH3andH1(Table3).Ontheotherhand,in theexperimentswithreducedinitialoxygenavailability,the supplementationof(NH4)2SO4 didnotleadtoany

improve-mentinxyloseconsumption;however,a87%increaserelative toH1wasachievedwithfullsupplementation(Table3).Thus,

it can be stated that under the condition of reduced ini-tial oxygenavailability fullsupplementationwas necessary to achieve animprovement inxylose consumption similar tothatreachedwith(NH4)2SO4aloneundertheconditionof

increasedinitialoxygenavailability.

Withthe exception oftheexperimentsconducted with-out nutritional supplementation(H1),inwhich theprofiles of xylose consumption were similar (Fig. 1A, Table 3), the two conditions of initial oxygen availability used in this workpromoteddifferencesintheprofilesandratesofxylose consumption. In the experiments with the reduced initial oxygen availability,xylose begantobeconsumedata con-stant rate sooner than in those with the increasedinitial oxygen availability (Fig. 1BandC). Nonetheless, thexylose consumptionrates(calculatedduringthephaseofmaximum xyloseconsumptionforeachexperiment)werehigherinthe experimentswiththeincreasedinitialoxygenavailability(by 200% withsupplementationof(NH4)2SO4 and by53% with

full supplementation). Consequently, the xylose consump-tion levels were higher than in the experiments with the reducedinitialoxygenavailability(by96%inH2and37%in

H3)(Table3).

The results obtained on xylose consumption suggested thattheyeastcapacitytoassimilatexylosewaslimitedinthe experimentswithreduced initialoxygenavailability, which could be due toa limited supply of NADPH necessary for XR activity.28,33 _{Ithaspreviouslybeen indicatedthatunder}

conditionsofoxygenlimitationthecarbonfluxfromxylose assimilationcouldbeinsufficienttopromoteNADPH regener-ationthroughthePPP,17,29,33_{whichisanimportantfactorthat}

canaffectthexylitolproduction.16,17,27

The cell growth and xylitol production were improved in asimilar wayas xylose consumption. Inregards to cell biomass, in the experiments with increased initial oxy-gen availability the supplementation of(NH4)2SO4 (H2) led

supplementation(H1),andanadditionalincreaseof9%was achievedrelativetoH2withfullsupplementation(H3), cor-respondingtoatotalimprovementof57%betweenH3and

H1(Table3).Intheexperimentswithreducedinitialoxygen

availability,noimprovementincellgrowthwasobservedwith nutritionalsupplementation (Table 3). Itshouldbe empha-sized that supplementation with (NH4)2SO4 alone did not

increasexylitolproductionundereitherconditionofinitial oxygenavailability.Ontheotherhand,fullsupplementation ledto240%and48%improvementsinxylitolproductionunder conditionsofincreasedandreducedinitialoxygenavailability, respectively(Table3).

Consideringtheresultsobtainedonxyloseconsumption, cellgrowth,and xylitolproductionintheexperimentswith reducedinitialoxygenavailability,itcanbestatedthat sim-plesupplementationwithaninorganicnitrogensourcesuch as(NH4)2SO4wasnotsufficienttoimprovexylitolproduction.

Ontheotherhand,withthefullsupplementationofthe sug-arcanestrawhemicellulosichydrolyzatewith(NH4)2SO4,rice

brainextractandCaCl2·2H2O,increasedxyloseconsumption

andxylitolproductionwereachievedunderbothconditions ofinitialoxygenavailability.Thesefactssuggestedthe neces-sityofsupplementationwithacomplexnitrogensourcesuch asthericebranextract,anagro-industrialby-product,which can also supply minerals and vitamins.34 _{Similarly to our}

results,ithasbeendemonstratedthatthexylitolproduction byC.guilliermondiiFTI20037wasimprovedinhemicellulosic hydrolyzatesfromsugarcanebagasse24_{andwheatstraw}19_by

thesupplementationwithricebranextract.

Regardingtheeffectofinitialoxygenavailability,changes wereobservednotonlyinxyloseconsumption,asdiscussed earlier,butalsointhe useofthexylose consumedforcell growthorxylitolproduction.Withthereductionofthe ini-tialoxygenavailability,animprovementinxylose-to-xylitol bioconversion was observed to occur in detriment to cell growth.Intheexperimentswithreducedinitialoxygen avail-ability, the xylitol yield was higher and the cell biomass yieldwas lower compared tothose obtainedunder condi-tionsofincreasedinitialoxygenavailability(Table3).These resultscanbeexplainedbylimitedrespiratory metabolism due tolimited oxygen availability, which notonly reduces theregenerationofNAD+_{,promotingaNADH/NAD}+

imbal-ance that causes a partial inhibition of XDH and xylitol accumulation, but also decreases the energy production and formation of carbon intermediates, affecting the cell growth.16,27,33

Interestingly, a slight reduction in xylitol yield was observedundertheconditionofreducedinitialoxygen avail-ability with the increase of nutritional supplementation, correspondingtoa20%difference betweenthe full supple-mentationand no supplementation(Table 3), and,in fact, thehighestxylitolyield(0.67gg−1)wasobtainedunderthe lattercondition.Thisvaluecanbecomparedwiththe respec-tivevaluesobtainedundersimilarfermentationconditionsin otherstudies,whichusedthesameyeastbutotherfeedstocks, withadifferentcompositionofthefermentationmedia,as wellasdifferentprocessesofconcentrationanddetoxification ofhemicellulosichydrolyzates. Comparedwithasugarcane bagasse hemicellulosic hydrolyzate detoxified with active charcoal,thehighestxylitolyieldachievedinthepresentwork

wassimilartothat(0.66gg−1)obtainedinthestudyofMarton andcolleagues,25_{inwhichxyloseandglucoseconcentrations}

inthefermentationmediumwerehigherandlower, respec-tively,thanthoseinthiswork.Arrudaandcolleagues,35_who

workedwithasugarcanebagassehemicellulosichydrolyzate detoxifiedusingion-exchangeresins,reportedahigher xyli-tolyield(0.81gg−1)thantheoneobtainedinthisstudy.Itis worthnotingthatinthelatterworkthefermentationmedium containeda higherconcentration ofxylose and lower con-centrations ofglucoseand acetic acid than thesefoundin thepresent study.MussattoandRoberto,21 _whousedarice

strawhemicellulosichydrolyzatedetoxifiedwithactive char-coalandwithhigherconcentrationsofxyloseandglucoseand lowerofaceticacidthanthosefoundinthisstudy,obtained ahigherxylitolyield(0.72gg−1)thantheoneachievedinthe present work.Ontheotherhand,inthecaseofasorghum straw hemicellulosichydrolyzatewithlowerconcentrations ofxyloseandglucosethaninthesugarcanestraw hemicellu-losichydrolyzateinthiswork,Seneandcolleagues36_reported

alowerxylitolyield(0.44gg−1)thantheoneobtainedinthe presentstudy.

TheresultssummarizedinTable3showthatthehighest xylitol production (16.20gL−1) and volumetric productiv-ity (0.34gL−1h−1) were obtained in the experiment with increasedinitialoxygenavailabilityandfullsupplementation. This isconsistent with the highestcell concentrationalso found inthis experimentbut notwith thexylitol yield,as discussedpreviously(Table3).Theseresultssuggestedthat, eventhoughthecontrolofoxygenavailabilityisacritical fac-toronxylose-to-xylitolbioconversion,itmaybepossibleto improvethisbioprocess,particularlythevolumetric produc-tivity,byincreasingxyloseconsumptionunderconditionsof oxygen limitation.Anotherpossibilitytoconsiderwouldbe the use ofconditions favoringofcell growth ina previous phase, beforethe xylose-to-xylitol bioconversionbegins. In comparisonwiththeabove-mentionedstudies,whichused otherlignocellulosicbiomassesbutthesameyeastand sim-ilar fermentationconditions, the highestxylitolvolumetric productivityobtainedinthisstudy(0.34gL−1h−1)washigher thantheonereportedbySeneandcolleagues36_(0.19gL−1_h−1₎

and lower than those obtainedbyArrudaand colleagues35

(0.60gL−1h−1),Martonand colleagues25 _(0.50gL−1_h−1_{), and}

MussattoandRoberto21_(0.57gL−1_h−1_).

Conclusion

The hemicellulose content of sugarcane straw, similar to that of other biomasses evaluated for xylitol production, and the high concentrationofxylose inthe hemicellulosic hydrolyzate suggested the suitability of this biomass as a feedstock forxylose-to-xylitol bioconversion. Theabilityof C. guilliermondii FTI 20037 to partially consumexylose and producexylitolinanon-supplementedsugarcanestraw hemi-cellulosichydrolyzatecanbeconsideredastartingpointfor thedevelopmentoftechnologiesforxylitolproductionfrom thissubstratewithoutsupplementationthroughthestudyof fermentationconditions. Atthe sametime, ourstudy con-firmedacombinedeffectofnutritionalsupplementationand initialoxygenavailabilityonxyloseconsumptionandxylitol

production.Itwasfoundthatthesupplementationofan inor-ganicnitrogensource,suchas(NH4)2SO4, wassufficient to

achieveimprovementsinxyloseconsumptionandcellgrowth under a condition of increased initial oxygen availability. However,inordertoimprovethesetwoparametersundera conditionofreducedinitialoxygenavailabilityandtoincrease xylitol production under both oxygen conditions, addition of a complex nitrogen source, as rice bran extract, along with(NH4)2SO4andCaCl2·2H2O,wasnecessary.Althoughthe

reduced initialoxygen availability increasedthe xylose-to-xylitolbioconversionindetrimenttothecellgrowth,italso affected the xylose consumption, which, in turn,hindered improvementsinthexylitolvolumetricproductivity.Whereas thehighestxylitolyieldwasobtainedatreducedoxygen avail-abilityandwithoutnutritionalsupplementation,thehighest volumetric productivity was achieved at increased oxygen availability and fullsupplementation. Consideringthat the conditionsofnutritionalsupplementationandinitialoxygen availabilityrequiredtoachievethemaximumvaluesofxylitol yieldandvolumetricproductivityweredifferent,further stud-iesfocusedontherelationsbetweentheparametersevaluated andotherprocessparameters,suchasthetypeofnutrients, supplementationstrategyandcontroloftheoxygen availabil-ity,arenecessaryforbiotechnologicalproductionofxylitolto becomeacompetitivebiochemicalroutefortheintegrationof sugarcanestrawinabiorefinery.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ThisworkwasfinanciallysupportedbytheBrazilianresearch funding agenciesFAPESP (Fundac¸ãodo amparo àpesquisa do estado de São Paulo, Brazil, process 2013/27142-0) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico,Brazil).

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