ARTIGO_2-Phenylethanol and 2-phenylethylacetate production by nonconventional yeasts using tequila vinasses as a substrate

2-Phenylethanol

and

2-phenylethylacetate

production

by

nonconventional

yeasts

using

tequila

vinasses

as

a

substrate

José

de

Jesús

Rodríguez-Romero

a

,

César

Arturo

Aceves-Lara

b,c

,

Cristina

Ferreira

Silva

d

,

Anne

Gschaedler

a

,

Lorena

Amaya-Delgado

a

,

Javier

Arrizon

a,

*

a_{IndustrialBiotechnologyDepartment,CentrodeInvestigaciónyAsistenciaenTecnologíayDiseñodelEstadodeJalisco,A.C.,Jalisco,Mexico} b

TBI,UniversitédeToulouse,CNRS,INRA,INSA,Toulouse,France c

TBI(ex.LISBP)-INSA,Toulouse135AvenuedeRangueil,31077,Toulouse,France d

DepartmentofBiology,FederalUniversityofLavras,PostalCode3037,37200-000,Lavras,MG,Brazil

ARTICLE INFO Articlehistory: Received30May2019

Receivedinrevisedform4January2020 Accepted4January2020 Keywords: 2phenylethanol 2phenylethylacetate Nonconventionalyeasts Tequilavinasses Metabolicmodeling ABSTRACT

Vinassesfromthetequilaindustryarewastewaterswithhighlyelevatedorganicloads.Therefore,to obtainvalue-addedproductsbyyeastfermentations,suchas2-phenylethanol(2-PE)and 2-phenyl-ethylacetate(2-PEA),couldbeinterestingforindustrialapplicationsfromtequilavinasses.Inthisstudy, four yeasts species (Wickerhamomyces anomalus, Candida glabrata, Candida utilis, and Candida parapsilosis) were evaluated with two different chemically defined media and tequila vinasses. Differencesinthearomacompoundsproductionwereobserveddependingonthemediumandyeast speciesused.Intequilavinasses,thehighestconcentration(65mg/L)of2-PEAwasreachedbyC.glabrata, theinhibitorycompoundsdecreasedbiomassproductionandsynthesisof2-PEA,andbiochemicaland chemicaloxygendemandswerereducedbymorethan50%.Tequilavinassesweresuitableforthe productionof2-phenylethylacetatebytheshikimatepathway.Ametabolicnetworkwasdevelopedto obtainaguidelinetoimprove2-PEand2-PEAproductionusingfluxbalanceanalysis(FBA).

©2020PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

1.Introduction

TequilaisthemostrecognizedMexican distillatearoundthe

world.Productionandconsumptionhavegrownsignificantlyover

thelast_fiveyears,reaching309millionlitersin2018[1].According

tothe Tequila RegulatoryCouncil (CRT), this industrial activity

generatessignificantamountsofsolidandliquidwastes,suchas

bagasse and vinasses,which both constitute an environmental

problem,butparticularlyvinasses.

Foreachliter oftequilaproduced, 7–10litersofvinasses are

generated,producingapproximately 2500–3000millionlitersof

vinasses each year. In addition, since there is not an updated

regulationfordisposingoftheseresidues,theyarepouredintowater bodies,whichrepresentsasevereenvironmentalissue[2].Vinasses

havecharacteristicsthatmakesthemhighlycontaminantresidues;

suchasahighbiochemicalandchemicaloxygendemand(BODand

CODrespectively),lowpH,hightemperatureandturbidity[2–5].

Non-Saccharomyces yeasts, also known as nonconventional

yeasts, have emerged as novel microbial sources for the

development of newbioprocesses,and someof them exhibita

highercapacity foraromametaboliteproductionthan

Saccharo-mycescerevisiae[6,7].Therefore,thesemicroorganismscouldoffer

anexcellentoptionforthedevelopmentofbioprocessestoobtain

high-valueproducts.Furthermore,theuseoftequilavinassesina

biorefinerymodelisemergingasanalternativefortheintegraluse

ofbyproductsofthetequilaindustry.

Several studies have beenperformed to obtain added-value

products and toreduce theenvironmentalimpact oftequila vinasses,

includingphysicochemicaltreatments[8]fermentativeprocesses

[9,10], biohydrogen production [11], the xylitol process [12], feedstockproteinproduction[13],andfertilizerproduction[14].

Thefirststudyofaromaproductionfromtequilavinasseshas

recently been addressed by DosReis et al. [9], who evaluated

Saccharomyces and non-Saccharomyces yeasts in vinasses from

cachaçaandtequila.Nevertheless,thisstudyfocusedto

character-ize a single kind of tequila vinasses and to compare them to

cachaçavinasses.Theydidnotcarryoutacorrelationbetweenthe

vinasses composition and the aroma compound that was

produced,ortheeffectofthepresenceofinhibitorycompounds

inthefermentation.

Aromacompoundsareimportantforthefood,pharmaceutical,

tobacco,andcosmeticindustries[15–18].Intheparticularcaseof

* Correspondingauthor.

E-mailaddress:jparrizon@ciatej.mx(J.Arrizon). https://doi.org/10.1016/j.btre.2020.e00420

2215-017X/©2020PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

Biotechnology

Reports

2-phenylethanol (2-PE) and its ester, 2-phenylethylacetate

(2- PEA), they have industrial applications by providing the

characteristicaromaofhoneyandroses.Aftervanillin,2-PEand

2-PEAarethesecondmostusedaromacompoundsintheindustry

(10,000tonsperyear).2-PEand2-PEAcanbeobtainedbychemical

synthesis,however,benzeneanditsderivatives,whenusedasraw

material,arehazardousregulatedcompounds.Alternatively,the

extraction of essential oils from plants presents low yield and

generates industrial wastes [19]. Most of the studies of 2-PE

synthesis by fermentation, have been carried out from the

catabolicbreakdownofphenylalanine,whichisa moreefficient

pathway than that of anabolic synthesis [16,20_–24]. This is

probably due to a more direct set of steps: the first pathway

involvesadeaminationofaminoacidstoproducetheα-ketoacid

(precursor of higher alcohols); the second pathway involves

several steps from sugars and nitrogen sources through the

shikimatepathwaytosynthesisoftheα-ketoacid[24].

For the production of 2-PE by fermentation, research has

focusedmainlyin themicroorganismsS.cerevisiaeand

Kluyver-omycesmarxianus[16,17,25,18,24].Ithasalsobeenobservedthat

thenatureandconcentrationofcarbonandnitrogensourceshave

aneffectontheproductionofthesearomaticcompounds.Fabre

etal.[26]reportedthatdifferentcarbonsourceshaveaneffecton

2-PEproductioninfermentationswithyeastK.marxianus.Martin

etal.[27]observedthatnitrogensourceotherthanaminoacids

significantlyreducedhigheralcoholsproductionwith

Hansenias-poravinaeyeasts.Hence,cultureconditionsandyeastspeciesare

importantfactorsinthearomacompoundsproduction.

For theimprovementofbiotechnological productionof2-PE,

metabolicmodelsareusefultoolsinthestudyofbioprocessesto

predictoptimalfluxesduringgrowthandmetaboliteproduction.

Tothebestofourknowledge,thisisthefirsttimethatmetabolic

modelingtoolswereusedinaromacompoundproductionfrom

tequilavinassesasasubstratebyusingnonconventionalyeasts.

A flux balance analysis (FBA) was applied in this study to

understandthemetabolicinteractionbetweentheprecursorsand

products 2-PE and 2-PEA, which considered the carbon and

nitrogenfluxes,andcharacterizedthesteady-statesolutionspace

within the stoichiometric network [28]. The construction of

metabolicnetworksisachievedbyanextensiveliteraturereview

todefinetheequationsof thereactionsfor thedesiredsystem.

Nevertheless, the biomass formation equations of a specific

microorganismrequireconcreteassumptions for thesystem,or

carbontrackingexperiments(whichis adifficultandexpensive

tasktoaccomplish).Therefore,severalstudieswithnon-common

microorganismshaveinferredthesebiomassformationequations

fromothermodelmicroorganisms,suchasS.cerevisiaeorE.coli.

Eventhoughdifferencesmaybeobserved intheresultsof this

parameter,theirvalidityisaccepted[29–33]

Thesetoolshavebeenusedtooptimizebioprocesses,suchas

methane production [34], lipids accumulation and citric acid

production [31], clavulanic acid production [35], and alcohols

[29,32,33].

Theaimsofthepresentworkwere:(i)toevaluatethemetabolic

capacity of four nonconventional yeasts (Wickerhamomyces

anomalus,Candidaglabrata,Candidautilis,andCandidaparapsilosis) intwodifferentmetabolicconditions,(ii)toevaluatetheeffectof

tequila vinasse variability as a substrate for the production of

aromacompounds,suchas2-PEand2-PEA;(iii)toformadeeper

characterization of aroma compounds precursors in tequila

vinasses, such as amino acids; (iv) to determine the effect of

inhibitorycompoundspresentintequilavinassesfermentations,

and (v) to implement metabolic modeling in the carbon and

nitrogenfluxesandevaluatethefeasibilityofdifferentmetabolic

scenariosfortheproductionof2-PEand2-PEA.Theproductionof

biomass, 2-PE and its ester (2-PEA) were quantified during

fermentation, and the reduction in BOD and COD in tequila

vinasseswasalsoevaluated.

2.Materialsandmethods

2.1.Microorganisms

The strainsthat wereevaluatedin this studybelongtotwo

different culture collections, yeasts Candida glabrata (119) and

Candida parapsilosis (448), which were isolated from coffee

fermentation and belong to the Culture Collection of the

Agricultural Microbiology Department (CCMA), the Federal

UniversityofLavras,(Brazil);Candidautilis(CUT)and

Wickerha-momycesanomalus(DB)wereisolatedfrommezcalfermentation

andbelongtotheCultureCollectionoftheIndustrial

Biotechnolo-gyDepartment(CCIB),CIATEJ(Mexico).

2.2.Evaluationofcatabolicanddenovosynthesisof2-phenylethanol

and2-phenylethylacetate

The production capacity of 2-PE and 2-PEA by yeasts was

evaluatedintwodifferentmetabolicconditions,eitherthroughthe

Ehrlichcatabolicpathway(ECP)orthedenovopathway(DNP).The

yeastswereactivated(overnightculture)inYPDmediumat30C

and 250rpm. The initial inoculum used was at a cellular

concentration of 1106 _{cells/mL in 100mL of medium in}

250mL Erlenmeyer flasks. ECP evaluation was carried out as

reportedby Yin et al.[24] byusing a specific culturemedium

composed of 40g/L sucrose, 0.5g/L Na2HPO4, 1.8g/L yeast

nitrogen base without amino acids (BD DifcoTM_{, NJ, USA) and}

7g/L L-phenylalanine as a nitrogen source (pH 5.0). DNP was

evaluatedaccordingto[17,18],withaculturemediumcomposedof

20g/Lglucose,6.7g/Lyeastnitrogenbasewithoutaminoacids(BD

DifcoTM_{, NJ, USA) and 0.77g/L complete supplement mixture}

withouturacil(Adenine10mg/L,L-isoleucine50mg/L,L-leucine

100mg/L, L-methionine, L-phenylalanine 50mg/L, L-threonine

100mg/L, L-tryptophan 50mg/L, L-lysine 50mg/L, L-tyrosine

50mg/L,L-valine 140mg/L). Fermentations werecarried out at

30C and 250rpm agitation for 96h for both culture media.

Samplesweretakenat6,12,24,48,72and96htomonitorgrowth

bytheopticaldensitywithabsorbancerecordedat600nm;2-PE

and 2-PEA analyses were performed by gas chromatography.

Biomassproductionwasquantifiedusingthedryconstantweight

method (60C). Substrate consumption was measured using

spectrophotometric methods, dinitrosalicylic acid reagent for

reducingsugars[36]andanthronefortotalsugars.

2.3.Gaschromatography

2-PE and 2-PEA quantifications were carried out under the

methodologybyArellanoetal.[37].Sampleswereanalyzedusinga

GCAgilent7890Bsystem(AgilentTechnologies,CA,USA),a7890A

HeadspaceSampler,andanFIDdetector,usinganHP-INNOWax

(60m x250

m

m_0.25

m

m) column. Then, 2mLsamples were

placed in 20mL vials, to take them to the 7890A Headspace

Sampler withthefollowing conditions:vialtemperature: 90C,

loop temperature: 110C, transfer line: 115C, vialequilibrium

time:5min,pressurizationtime:2min,loopfilling:0.2min,loop

equilibriumtime:0.5min,injectiontime:1min,injectionvolume:

10

m

L.GCAgilent7890Bsystemconditionswereasfollows:the

initialsetupwasprogrammedat45Cfor8min,withincreased

stepsof2Cuntil80C,followedbya5Cincreaseuntil160C,and

finally, an increased step of 25C up to 220C for 4min. The

detectorand injectorweresetat250C,ina split-lessinjection

mode. The analysis time was 55min, including the headspace

by using external standards of 2-PE and 2-PEA (Millipore

Corporation,MA,USA).

2.4.Physicalandchemicalcharacterizationoftequilavinasses

Vinasseswerecollectedfromtendifferenttequilafactoriesin

twotequilaproducingregionsintheJaliscostate,Mexico.Region

“DelValle”correspondstothetownsof“Tequila”,Amatitán”and

“ElArenal”,whiletheregion“LosAltosSur”includesthetownsof

“Arandas”,“TepatitlándeMorelos”and“SanIgnacioCerroGordo”.

Freshvinasseswerecollectedin plastic containersimmediately

afterdistillationandwerestoredat 20_{Cinafreezingchamber}

prioruse.AminoacidswerequantifiedbyHPLC(AOAC982.30-A,

modifiedfromWATERSACCQTAGultra(WatersCorporation,MA,

USA))(2007),biochemicaloxygendemand(BOD)wasdetermined

byaBODTrakIIrespirometricapparatus(Hachcompany,CO,USA)

and chemical oxygen demand (COD) was determined by the

Reactor Digestion Method, using a DRB 200 Reactor (Hach

company,CO, USA). Both parameters werecalculated from the

amount of oxygen that is needed to oxidize the organic and

inorganiccomponents,respectively.Totalnitrogenand

ammoni-um-nitrogenwereestimatedbytheKjeldahlmethod,accordingto

the guidelines of the American Public Health Association [38],

photocolorimetric methods were used in the determination of

total sugars using the anthrone method at 620nm; the Miller

method(DNS)wasusedat540nmforreducingsugars,and the

total solids werecalculated by the dry weight method(60C).

Mineralquantificationsofcalcium,copper,nickel,magnesium,and

ironweredeterminedbyatomicabsorptionspectrometrybytheU.

S.EnvironmentalProtectionAgencymethod6010B(EPA,1996).

2.5.Evaluationoftheinhibitorycompoundsintequilavinasses

Inhibitorycompoundswere quantified bya 1220Infinity LC

high-performanceliquid chromatographysystem (Agilent

Tech-nologies, CA, USA) equipped with a Zorbax Eclipse Plus C18

(4.6mmx250mm, 5

m

m) (Agilent Technologies, CA, USA). The

mobilephaseswereA:(methanol100%)andB:formicacid-water

(2.5%v/v);theoperatingconditionswereasfollows:flowrateof

0.8ml/minat30C,withaflowgradientduring55minfrom0%to

48%ofphaseB,apressureof1200100psi,anddetectionbya

diodearraydetector(DAD)atwavelengthscreeningsat262,275,

295and342nm.

2.6.Nonconventionalyeastsfermentationsontequilavinassesfor

aromacompoundsproduction

Tequila vinasses were pretreated as follows: centrifugation

wasperformed (13,000rpm, 20min, 4C) to removeinsoluble

solids. The centrifuged vinasses were added into 250mL

Erlenmeyer_flasks(100mL),withthefollowingformulation:0.1

%yeastextractw/v,0.05%K2HPO4w/v,0.2%glucosew/v,0.5%

peptone w/v, and fresh vinasses without dilution. Prior to

inoculation,thesemediasolutions weresterilizedat121_{C for}

15min.Cultureswereincubatedforeachstrainwith1106_cells/

mLat30Cand150rpmagitationfor120h.Samplesweretaken

every24h.Thefactorsthatwereevaluatedwerethefourdifferent

strains and the 10 sampling locations. The response variables

were the 2-PE and 2-PEA titers, biomass production, pH

measurements,andsubstrateconsumption.

2.7.Evaluationofthechemicalandbiochemicaloxygendemand

reductionbyyeastsduringtequilavinassefermentations

The reductionin CODandBODin tequilavinassesafter yeast

fermentationwasevaluatedasstatedinthesectionofPhysicaland

chemicalcharacterizationoftequilavinasses,asacomplementtothe

productionofthearomacompounds.Aselectionofanalyzedvinasses

wasinaccordwiththeresultsofcharacterization,aswellasyeasts

speciesfromtheresultsoftequilavinassefermentations.Analyses

were performed intriplicate from a singlebatch of the selected

vinasses,whichcorrespondstotheinitialvalueoftheCODandBOD

parameters.1106_{cells/mLwereinoculatedintheselectedtequila}

vinasses, and fermentationwas performedin 250ml Erlenmeyer

flasks at30_{Cand150rpmfor120h.Thecellswereremovedby}

centrifugationat4000rpmfor15minat4Cpriortoanalysis.

2.8.Metabolicnetworkmodeling

Cellularmetabolicpathwaysfromametabolicnetworkcould

bedescribedbythesetofelementarymodes(EMs).TheEMsarea

setofnondecomposablepathwaysconsistingofaminimalsetof

reactions thatfunctionin steady-state[32,33].In thiswork,the

constructednetworkwas basedonthemetabolicconversion of

carbonandnitrogensourcesassubstratestobiomass,2-PEand

2-PEAasproductsforyeastsinanaerobicconditions.Itconsistsof48

reactions, with58 internalmetabolitesand5 mainmetabolites

(glucose,biomass,2-PE,2-PEA,maintenance),whichincludesthe

glycolysispathway,pyruvatemetabolism,thepentosephosphate

pathway, the Krebs cycle,the shikimateand Ehrlichpathways,

glutamate and glutamine metabolism, and biomass formation

[31–33,39].Theaminoacidmetabolismofphenylalanineisrelated

tothenitrogenuptakethatislinkedthroughglutamate

metabo-lismintheconstructedmetabolicnetwork[40].Onlythecytosol

and mitochondria were considered in order to simplify the

transportreactions.Thecomputationofelementarymodesfrom

the metabolic network was performed through the CellNet

Analyzer toolbox [41] in the MATLAB1 software (Mathworks

Inc.,MA,USA).Acompletesetofreactionsandabbreviationscanbe

foundassupplementarymaterialinappendixA.

2.9.Statisticalanalysis

Experiments wereperformed as a triplicate of independent

samples.Dataareexpressedasthemeanstandarddeviation.The

resultswerestatisticallyanalyzedusingone-wayANOVA(p<0.05)

andcomparisonamonggroupswasperformedusingTukey’stest

toobservethedifferencesamongthedifferentculturemediaand

strains. Analyses were performed using the Minitab1 17.1.0

software(MinitabInc.,PA,USA)

3.Resultsanddiscussion

3.1.Evaluationof2-phenylethanoland2-phenylethylacetate

productionbyfermentationundercatabolicanddenovosynthesis

conditions

Productionof2-PEand2-PEAintheECPandDNPinductor

media are shown in Table 1. Significant differences in the

percentage of the consumed sugar can beobserved depending

on the medium and the yeast strains. Complete depletion of

glucosewasobservedintheDNPmediumwithallyeasts,whileC.

glabrata and W. anomalus were not able to achieve total

consumption ofsucrosein theECPmedium.However,biomass

washigherincomparisonwithC.parapsilosisandC.utilis.Glucose

consumptionwas similartothose studiesinwhich mostofthe

carbonsourcewascompletelydepletedafter24 28h,eitherby

yeasts(S.cerevisiaeandK.marxianus)[16,23]orbacteria

(Enter-obacter sp.) [42]. Previous studies in the same DNP medium

reporteda biomassproductionfor K.marxianusintherange of

2–3g/L[17,18],whichissimilartotheonethatwasobtainedwith

2-PEwasproducedonlybyC.utilisintheECPmedium,whilein

theDNPmediumitwasobservedasignificantdecreaseof98%.For

2-PEA, the highest concentration was produced by yeasts C.

glabrataandW.anomalusintheECPmedium,asshowninTable1,

with 665 and 689mg/L, respectively. The same behavior was

observedforthesetwoyeastsintheDNPmedium,withadecrease

of95%.Therearereportsthatmentionthatthenitrogensourceis

ofgreatimportanceinthesynthesisofphenylpropanoids.Martin

etal.(2016)observedthattheadditionofammoniumsalts(75mg/

Lofyeastassimilablenitrogen)totheculturemediumnegatively

affectedtheproductionofthesearomaticcompounds(reduction

from13to2

m

g/Lapproximately)withH.vineaeyeast.Thisresultis

consistentwiththeresultsobtainedinthiswork.

Yinetal.,[24]producedamaximumconcentrationof800mg/L

of2-PEwiththewild-typeS.cerevisiaefromtheErhlichpathway

withaphenylalanine(7g/L)basedmediumandsucroseascarbon

source(40g/L),which was differentfromtheresultsthat were

obtainedinthisstudy,sincemetaboliteproductionwasobserved

tobedirectedtowards2-PEAaccumulation,probablybecausethe

biosynthesisof2-PEdependsonthecapabilityofeachoneofthe

microorganismsintoleratingthiscompound,aswellastheactivity

ofalcoholacetyltransferasethatisneededfor2-PEAproduction

[43,17,18]reportedaconcentrationof200mg/Lof2-PEina

wild-typestrainofK.marxianusfromglucose(20g/L)andammonium

sulfate(5g/L)asnitrogensource,withamaximumamountof1g/L

inarecombinantstrain.Hence,thecapacityforsynthesisofthese

compoundsdependsontheculturemediacompositionandthe

inherentmetaboliccapabilityofthemicroorganismusingdifferent

pathwaysfortheproductionof thesemetabolites.Forexample,

Jimenez-Martiand del Olmo, [44] observed that for the same

microorganism(S.cerevisiae)inalcoholfermentationconditions,

the use of ammonia increased the expression of ARO8 gene

(involvedin aromatic amino acids synthesis), while the useof

aminoacidsrepressedtheSfp1pprotein(transcriptionalfactorof

ribosomalproteinsynthesis).Thus,thenatureofnitrogensourceis

themostimportantfactortoinducedifferentmetabolicpathways

fortheproductionofaromaticcompounds.Thiscouldbeinferred

fromthedifferencesobservedbetweenECPandDNPconditionsfor

thefournonconventionalyeasts(W.anomalus,C.glabrata,C.utilis, andC.parapsilosis).

It also shouldbe highlightedthat in themajorityof yeasts,

accumulationof2-PEcausesintracellulartoxicity,asreportedby

severalauthors[16,45,46].Thiscouldleadtothetransformationof

thishigheralcoholtotheesterform,affectingthelevelsof2-PE

accumulation in the culture medium. Even though there is no

preciseevidenceforthebiologicalfunctionofthesecompounds,it

has been suggested that the alcohol acetyltransferase (Atf2p)

reactionisrelatedtoadetoxi_ficationprocess[47].Itisinteresting

to point out, however, that this detoxification process may be

revertedovertime.Wittmannetal.[23]observedinK.marxianus

that 2-PEA production is significantly lower than 2-PE, and

determinedthataccumulationof thishigher alcoholin thelate

stagesoffermentationisdueto2-PEAcleavage.

3.2.Physicalandchemicalcharacterizationoftequilavinasses

Resultsofthephysicochemicalcharacterizationofvinassesare

presentedinTable2.Threedifferentagavecookingprocessesand

two distillation systems were identified in the elaboration

process of tequila from thetwo regions thatwere sampled.A

lowlevelofreducingsugarswasobserved(1.79_–4.5g/L),which

was similar to the findings reported in previous vinasses

characterization studies [2,5,9,13]. There were differences in

thetotalnitrogenconcentration,vinasseAhadthelowestamount

at50mg/L,whiletheotherevaluatedvinassespresentedarange

between177 482mg/L.

Table2

Physicalandchemicalcharacterizationofthedifferentvinasses.

Region “DelValle” “LosAltos”

Vinasse A B C D E F G H I J Parameter ReducingSugars(g/L) 4.250.5 3.621.2 3.41.0 1.790.1 3.310.6 3.890.1 2.551.6 4.340.1 2.721.4 3.060.6 Totalsugars(g/L) 7.782.9 9.314.5 7.171.5 5.820.2 7.541.5 7.190.7 7.221.6 17.156.7 7.290.8 8.680.3 Totalnitrogen(mg/L) 50.420.1 482.457.2 232.6110.0 226.5124.0 439.646.3 341.846.5 388.5138.7 312.425.0 177.8121.9 315.9205.7 NH4+-nitrogen(mg/L) 8.83.9 14.90.98 8.13.23 11.475 16.680.58 11.421.87 27.039.9 7.641.47 8.772.65 9.096.71 Organicnitrogen(mg/L) 41.6316.12 467.4756.18 224.48106.8 215117.99 34663.1 330.3944.64 361.445128 304.7526.48 169.0115.66 306.79199.02 Phenylalanine(_mg/L) <0.01* <0.01* <0.01* 0.026 <0.01* 0.011 0.026 <0.01* 0.013 0.019 Valine(mg/L) 0.039 <0.01* 0.022 0.041 <0.01* <0.01* 0.016 <0.01* <0.01* <0.01* Leucine(mg/L) 0.02 <0.01* <0.01* 0.036 <0.01* 0.011 0.017 0.01 <0.01* <0.01* Isoleucine(mg/L) <0.01* <0.01* <0.01* 0.01 <0.01* <0.01* <0.01* <0.01* <0.01* <0.01* C/Nratio 154.0 19.0 31.0 26.0 17.0 21.0 19.0 55.0 41.0 27.0 COD(g/L) 68.715.6 55.734.8 58.80.4 53.513.6 50.026.7 50.632.8 59.817.4 66.22.0 46.917.2 69.12.0 BOD(g/L) 23.10.8 22.72.1 20.63.4 18.51.3 22.82.1 20.71.7 28.08.6 28.612.9 22.56.0 29.812.9 TotalSolids(mg/L) 33.523.4 23.719.0 44.313.9 17.30.1 26.51.4 20.710.7 38.86.7 66.712.8 20.712.3 56.416.7

Resultsareshownasanaveragewithastandarddeviationoftworeplicatesfromvinassescollectedintwodifferentregionsatdifferentproductionperiods.Region“DelValle” (A,B,C,D,E),andtheregion“LosAltos”(F,G,H,I,J).*0.01Minimumdetectionlimit.

Table1

Evaluationofconsumedsugar,biomass,2-phenylethanol(2-PE)and2-phenylethylacetate(2-PEA)productionbyyeastsindifferentmedia.

DNP ECP Yeast Yx/s (gbiomass/gglucose) Consumed sugar(%) 2-PE[mg/L] 2-PEA[mg/L] Yx/s (gbiomass/gsucrose)

Consumedsugar(%) 2-PE[mg/L] 2-PEA[mg/L]

C.glabrata 0.230.06aA 99.420.05aA ND 35.080.54aA 0.240.01aA 61.291.09aB ND 665.3325.55aB C.parapsilossis 0.140.05aA _99.110.13aA _{ND 28.564.91}bA _0.150.04aA _95.512.58aB _{ND 357.4619.32}aB W.anomalus 0.260.06bA 99.510.01aA 2.690.57aA 36.930.11aA 0.530.09bA 44.621.23bB ND 689.1666.63aB C.utilis 0.310.08bA 99.630.04aA 4.471.87bA 29.501.97bA 0.180.01aA 96.472.31aB 242.6526.9bA 6.670.49bB

DNP=denovopathwaymedium,ECP=Ehrlichcatabolicpathwaymedium.Resultsareshownasanaverageofduplicateswithstandarddeviation;lowercaselettersshow comparisonsbetweenstrainsanduppercaselettersshowcomparisonsbetweenmedia.Statisticalsignificancegivenatp<0.05.ND-Notdetected.

Totalnitrogen is given by thesum of organicand inorganic

nitrogen.Inwastewaters,thelatterisconstitutedbythesumof

ammonium,nitrites,andnitrates[48].Previousstudiesoftequila

vinasses indicated that the main portion of available nitrogen

comesfromorganicnitrogenbythepresenceofaminoacidsand

proteinsfromthefermentationpreviousdistillationofthemust

[49]. The quantity of total amino acids that were found

corresponds approximately totheorganic nitrogenavailable in

vinasses(datanotshown).

Other reports for tequila vinasses found a low content of

nitrogen(20 50mg/L) [2].However,mezcal vinassesshowed a

higher concentration of this parameter, with oscillations from

660 5650mg/L[5], which is more consistentwithresults that

werefoundbyDosReisetal.[9](818mg/L).Therefore,valuesthat

areobservedinTable2couldbeassociatedwithdifferencesinthe

tequilaelaborationprocess.AstheC/Nratiowithalownitrogen

contenthasnegativeeffectsonthemetabolismofmicroorganisms

[50], the C/N ratio that was found for the vinasses samples

(12 67g/g)indicatesthatthereisnosurplusthatmayinterferein

thefermentationprocess,exceptforvinasseA,whichobtaineda

lownitrogenconcentrationandahighC/Nratio(154g/g).

ThemaximumresultsofCODandBODwerehigher(6.7g/Land

3.8g/L,respectively)thanthevaluesthatwerepreviouslyreportedfor mezcalvinasses(6.0g/Land2.2g/L)[5],Braziliancachaçaproduction vinasses(5.9g/Land1.9g/L)foundbySilvaetal.[13],andtequila vinasses (4.2g/L and1.8g/L) [9]. This could indicate thattequila

vinasses are more recalcitrant wastewaters than other similar

beverages, and the amount depends on the origin ofthe corresponding

vinasses.CODandBODvaluesinvinassescanbeaffectedbytheraw

material origin, the preparation of the agavemust, the alcoholic

fermentation system, the types of yeast, the distillation and the

separation processes [2–4,51,52]. Nevertheless, the results observed in

Table 2did not show correlation between the origin ofthe vinasses and

theprocessfromwhichtheywereobtained.

Incontrast,differences in theprocesshad aneffectontotal

solids.Totalsolidsandreducingsugarsweresignificantlylowerin

thevinassesobtainedfromadiffusorprocesscomparedtovinasses

fromthetraditionalprocess.Thus,theextractionofsugarsisakey

parameterthathasaninfluenceinthefinalcompositionoftequila

vinasses,andthereforecouldaffectthefermentationperformance

ofyeastsusingthesewastewatersasasubstrate.

AromaticaminoacidquantificationisalsopresentedinTable2.

Itshowedthattheamountofaromaticaminoacidsislow

(0.01-0.026

m

g/L), which is consistent with the results that were

obtainedbyDíaz-Montañoetal.[53],whodeducedthatthelow

concentrationsofhigher alcoholsandotherbyproducts maybe

linkedtotheverypooraminoacidconcentrationinagavejuiceand

consequently,intequilavinasses.

Inhibitory compounds that are present in tequila vinasses

normallycomefromthehydrolysisofagavefructansduringthe

cookingprocess intheelaborationof tequila, suchasthefuran

hydroxymethylfurfural (HMF)orterpeniccompoundsthatcome

from theagaveplant, suchas vanillin[54]. Eventhough these

compoundsarepresumednottobemetabolizedbyyeasts,ithas

beenfoundthattheyaffectthefermentationyields[49].However,

some species are able to tolerate these compounds at a

concentrationcloseto2.5g/L expressedas totalinhibitors[55].

Theresultsofthemeasuredinhibitorycompoundsaredisplayedin

Table 3. Vinasse H presented the highest measure of these

compounds with a total of 455mg/L, while vinasses B and D

obtainedtheleastamountwithatotal of85mg/Land 99mg/L,

respectively.Eventhoughthereisnoexhaustiveinformationabout

thesecompoundsintheliterature,theresultsbyGarcíaetal.[56]

in vinassesfrom sugarcane ethanol production showa similar

inhibitorycompoundsconcentration,presentedastotal phenols

(469mg/L).

3.3. Nonconventional yeast fermentations on tequila vinasses for

2-phenylethanoland2-phenylethylacetateproduction

Fig.1showsthefermentationperformanceofevaluatedyeasts

withdifferenttequilavinasses.Therewerenodifferencesinthe

sugarconsumptionpercentageamongstrainsinalmostalltequila

vinasses,exceptinvinassesGandH,whereiscanbeobservedthat

W.anomaluswastheyeastthatconsumedlesssubstrate(Fig.1a).

Thebiomassforthefouryeastsoftheevaluatedvinassesareshown

in Fig.1b.There was asimilar behaviorfor allyeasts,withthe

exception of G and H vinasses, wherein the lowest yields of

biomass were observed. These two vinasses showed a high

inhibitory compounds concentration (in particular vinasse H),

where C. parapsilosis was able to produce high biomass,

which indicatesthat itpossessesbettertoleranceand abilityto

surviveunderstressconditions,aswasobservedbyDosReisetal.

[9],withabiomassproductionof5g/Linbothcachaçaandtequila

vinasses fermentations. In contrast, W. anomalus showed the

lowest biomass in vinasses G and H. Nevertheless, this yeast

produced4 7g/Lofbiomassintequilavinasses(datanotshown),

whichwassimilartothebiomassquantitythatwasproducedfor

thissameyeaststrainincachaçavinasses(4 8g/L)accordingto

Silvaetal.[13].

Vinassescontainaninitialconcentrationofaromacompounds

(ranging from 10mg/Lto 25mg/L) which wereconsidered and

subtractedbeforeperformingthecalculationsofthefinalyieldsof

2-PEA(C.glabrata:32 60mg/L;W.anomalus:3 47mg/L;C.utilis:

4 40mg/L; C. parapsilosis: 1 11mg/L). The presence of aroma

compoundsintheinitialcompositionofthemediumisduetothe

Table3

Inhibitorycompoundspresentintequilavinasses.

Vinasse A B C D E F G H I J Compound(mg/L) Hydroquinone 4.292.64g 12.660.08f 4.880.18g 37.420.04d 42.061.12c 43.600.01c 49.071.46b 96.430.30a 1.820.05g 22.740.12e Hydroxymethylfurfural 127.943.53c _18.180.32f _140.250.51b _22.050.02f _126.380.06c _21.790.03f _115.630.24d _227.131.09a _85.910.37e _137.470.85b Furfural 8.170.37b _5.870.11d _4.470.01e _0.180.01h _10.610.05a _7.390.01c _2.710.15f _8.700.12b _2.000.02g _4.950.01e 2-Furoicacid 24.957.05b 20.491.00b 17.070.07b 2.030.01e 11.490.01b 10.430.01b 19.890.25b 38.590.37a 9.930.04b 18.750.17b Hydroxybenzoicacid 5.093.14b _5.060.11b _7.020.25b _3.100.05c _3.700.18c _5.201.19b _5.320.30b _4.710.07b _9.050.09a _8.870.07b Hydroxybenzaldehyde 0.540.76g _0.920.04g _22.170.10d _23.570.01d _19.400.32e _25.070.14c _2.600.78f _49.500.19a _22.690.06d _31.600.23b Vanillicacid 37.781.27a 16.460.26c 4.250.02d 4.270.04d 3.840.02d 4.210.001d 3.560.19d 2.910.12d 26.810.19b 37.380.27a Vanillin 1.570.63d 1.0250.02d 4.230.05c 5.860.01c 7.220.01c 6.210.03c 22.630.74a 24.511.10a 1.840.47d 10.570.20b Acetovanillone 0.440.62a _1.450.86a _{ND ND 0.680.01}a _0.890.01a _0.860.08a _0.810.11a _{ND ND} Acetosyringone 1.510.03a 1.180.04c 1.180.02c 1.190.01c 1.360.01b 1.190.002c ND 1.410.03a ND 1.340.06b Coniferylaldehyde 5.260.65a 2.280.01b ND ND ND ND ND ND ND ND

Resultsareshownasanaverageofduplicatevinassesampleswithstandarddeviation.Comparisonsweremadebetweenvinassesforeachcompound;differentlower-case lettersshowStatisticalsignificancegivenatp<0.05.ND-notdetected.

previousfermentation and distillation of the agave must. This

findinghasbeenpreviouslyreportedinanotherkindofbeverages,

such as wine and beer [7,57]. Highest 2-PEA production was

accomplishedbyyeastC.glabratawithaconcentrationof60mg/L,

followedbyW.anomaluswith47mg/L,whichwassimilartothe

amountsreportedbyDosReisetal.[9] (19mg/Lto68mg/L).C.

parapsilosisshowedthelowestamountof2-PEAaccumulation,as

itcanbeseeninFig.1c.Asit wasstatedabove,theaminoacid

contentintequilavinassesisnotenoughforcatabolicbreakdown

pathwayproduction,andtherefore,thearomaticcompoundswere

producedthroughtheDenovopathwayfromglucose.

Several studies have approached the production of these

aromaticcompound fromglucose. For example, overexpression

oftheARO10geneina S.cerevisiaestrainimplementedbyShen

etal.[22]reportedconcentrationsof2-PEoflessthan10mg/Lfor

thewild-type strain,and a maximumconcentrationof 90mg/L

witha transformantstrain.Another approachwas evaluatedby

Rolleroetal.[58]totestthecombinedeffectofnutrientsinaroma

compounds production in wine fermentations, wherein they

observedbyasurfaceresponsemethodologythat,2-PEAand

2-PE,whichcanassimilatenitrogen,hadanegativequadraticeffect

inthesynthesisandthus,establishedanadequateconcentrationof

200mg/Lofnitrogenforaproductionof16mg/Lof2-PE,whichis

similartothenitrogenamountinmostvinasses.Anotherexample

isthestudyofEtschmannetal.[20],whoobtainedconcentrations

of 0.31g/L for 2-PEA from an optimized molasses medium

supplementedwithphenylalaninebyusingaK.marxianusyeast

before applying in situ product removal strategies (ISPR), and

obtainingfinalconcentrationsof1.27g/Lof2-PEA.

Regarding 2-PE and 2-PEA production, similar results were

found in the previous section. 2-PE is not accumulated in the

mediumandislikelydirectlytransformedto2-PEA;thisbehavior

hasbeenreportedinfermentationswithagavejuicetocompare

theperformanceofnonconventionalyeastsKloeckeraafricanaand

KloeckeraapiculatawiththatofS.cerevisiaestrains,wherethere

wasasignificantlyhigher2-PEAaccumulationthanthe2-PE,with

similarresults(51 60mg/L)[53].

Ithasbeenfoundthataromacompoundsynthesisisassociated

withgrowth[20],whichwasalsoobservedforbiomassandaroma

compoundsproductionsintequilavinassesfermentations.These

twoparametersdecreasedastheconcentrationoftheinhibitory

compoundincreased.Thus,thelevelsof2-PEAweremoreaffected

bytheconcentrationoftheinhibitorspresentintequilavinasses

thanbythenutrientconcentration(C/Nratio).

3.4.Chemicalandbiochemicaloxygendemandreduction

VinasseJwasselectedduetothehighestCODandBODlevels

(Table2).Thisvinassewasusedinafermentationprocessusing

theyeastsC. glabrata andW. anomalus. Itwas foundthat after

120h,bothmicroorganismsreducedapproximatelyover50%of

theCODandBOD.Previousstudieshavereportedsimilarfindings

forwastewatertreatments;SeluyandIsla[10]foundareductionof

60 % in beer breweries effluents; Pires et al. [59] obtained a

removalofCOD(39%–76%)andBOD(42%–56%)fromcachaça

vinasses(50 %v/vdilution), bymixing inoculum of yeastsand

bacteria;andDosReisetal.[9]reducedover80%forbothCODand

BODintequilavinasses(70%v/vdilution).Itmustbepointedout

thatin thepresent work,thetequilavinasseswerenotdiluted,

whichcouldexplainthedifferencesintheobtainedCODandBOD

reductions.

3.5.Metabolicpathwaysandelementarymodesanalysis

Fig. 2 displays the proposedreduced metabolic network of

nonconventional yeasts for 2-PE and 2-PEA production. 48

reactions were taken in to account in this network, with 58

internalmetabolitesand5mainmetabolites(glucose,biomass,

2-PE,2-PEA,maintenance),whichincludestheglycolysispathway,

pyruvatemetabolism,thepentosephosphatepathway,theKrebs

cycle, the shikimate and Ehrlich pathways, glutamate and

glutamine metabolism, and biomass formation [31–33,39]. The

aminoacidmetabolismofphenylalaninerelatedtothenitrogen

uptake, which is linked through glutamate metabolism in the

constructedmetabolicnetwork[40].

The computational analysis of the network using CellNet

Analyzerledto142elementarymodes(EMs),whichwerelower

thanthemodelsconstructedbyRobles-Rodriguezetal.[31]with

1944elementarymodes,andtheoneevaluatedby[32,33]with

369computedEMs.Thesedifferencesaredue tothenumberof

metabolites,and reactionsinvolvedintheevaluatedprocess,as

wellasthenumberofinputsorsubstrateintakes.Therefore,each

system must be delimited by every modeler_’s judgment and

objectives.

Fig.3showsthedistributionofthefoundEMsforthebiomass,

2-PE,2-PEAandethanolproduction,wherethediagonaldepicts

thehistogramsthatrelatethenumberofEMsversusmetabolite

yields. From142 EMs,136 included biomass and 139 included

ethanolproduction,while72and62EMswereinvolvedin2-PEA

and2-PEproductionrespectively.Therefore,biomassandethanol

productionsarecoupledwith2-PEAand2-PEsynthesis.Thelower

diagonalmatrixinFig.3showstheyieldplotsofthemetabolites

(biomass,ethanol, 2-PE,2-PEA) in theaxeswithrespecttothe

consumed carbonsource(glucose). It canbe observedthat the

yields found in the convex space hull, biomass and ethanol

Fig.1.Fermentationperformancesindifferenttequilavinassesforpercentageof sugarconsumed(a),Biomass(b)and2-Phenylethylacetateproduction(c)ofC. glabrata(&),C.parapsilosis(&),W.anomalus(&)andC.utilis(&)yeasts.

Fig.3.Distributionofelementarymodesofthereducedmetabolicnetwork.Histogramsofelementarymodesdistribution(diagonal)showthenumberofsolutionsfoundfor eachproduct.Yieldplotsdepictthegeometryofthefoundsolutionsforpredictedyieldsofthemetabolites(biomass,ethanol,2-PE,2-PEA)intheaxeswithrespecttothe consumedcarbonsource(lowerdiagonal).Unitsofyieldsaregivenin(mmol/mmolGLUC)exceptforbiomass(gBIOM/mmolGLUC).

production are favored in comparison with the ones obtained

for2-PEand2-PEA.

Thisinformationpermitstounderstand(fromastoichiometric

point of view), theimportance of each set of reactions in the

metabolicnetwork.Therefore,forthesynthesisof2-PEand2-PEA,

theuseoflowethanol-producingyeastspeciesmayincreasethe

carbon flux to these metabolites. Thus, these nonconventional

yeastsaresuitablefortheproductionbyfermentationinanaerobic

conditions of2-PE and 2-PEA,as they arenot considered high

ethanol-producingyeasts[60].

TheconvexhullspaceshowninFig.4wasusedtovalidatethe

experimentaldataoftheyields(Y2PEA/YGLUC)obtainedinECPand

DNPmediaforthefournonconventionalyeasts.Theexperimental

dataof 2-PEAwereselected asit was the metabolitewiththe

highestproduction (Table1).It canbeobserved thatall ofthe

evaluateddataarewithinthesolutionspace,whichcorroborates

thattheconstructedmetabolicnetworkpredictsthemetabolism

ofthestudiedyeasts.

Thedifferencesobservedamongtheseyeastsshowedthatthe

best2-PEAproducerswereW.anomalousand C.glabrata.These

strainsproducehighbiomassconcentrations,whichiscorrelated

withthebehaviorobservedinFig.3.Themetabolicnetworkalso

showedthatcouldbetheoreticallypossibletoincreasethe2-PEA

yieldfortheyeastsevaluatedinECPand DNP,especiallyinthe

secondone(lowphenylalanineconcentration).Thus,otherfactors

wouldbeinvestigatedtoimprovetheyieldofthismetabolite;such

asC/N ratio,type of carbon and nitrogensource,2-PE toxicity,

amongothers.

AnalysisoftheEMsobtainedfromthismetabolicnetworkin

theshikimatepathway(denovobiosynthesis)andphenylalanine

catabolicpathway,allowedtoobservethat theydidnot

coexis-tenceatthesametime(Datanotshown).Thishasbeenstatedin

other studies [44], where the gene expression of different

metabolicconditionswasstudied.Theyreportedthatyeastscells

are capable of selectively use different nitrogen sources as a

regulatorymechanism,showingadifferentialreprogrammingof

thegeneexpressiondependingonthenitrogensourceadded.They

alsofoundthatammoniaadditionresultedinahigherexpression

ofgenesinvolvedinaminoacidsbiosynthesiswhileaminoacid

additionpreparesthecellsforproteinbiosynthesis.

Inthecaseoftequilavinasses,theircompositionshowedthat

thearomaticaminoacids(Table2),includingphenylalanine,are

very low. Thus, the production of 2-PEA in tequila vinasses is

carriedoutbytheshikimatepathway.AnanalysisofEMs(35)was

performedtakingonlyintoaccounttheshikimatepathway,where

itwasfoundthat23ofthemwerenotbiologicallyfeasibledueto

an interruption in the citric acid cycle and a lack of biomass

production(evenwhenoneofthesescenarioswastheEMwiththe

highestyieldfor2-PEproduction,thusitwasdiscarded),resulting

in12biologicallypossiblescenariosforthemetabolitesofinterest (Table4).Themaximumyieldvaluesattainedfor2-PEAand2-PE

were0.13and0.21mmol/mmolGLUCrespectively,whichmustbe

consideredasvaluesofreferencefortheirproductionfromtequila

Fig.4. Yieldanalysisfor2-PEAproduction.RedsymbolsrepresenttheDenovopathway(DNP)experimentaldata,whilebluesymbolsrepresenttheEhrlichcatabolicpathway (EPC)for(*)C.glabrata;(o)C.parapsilosis;(x)W.anomalus;(^)C.utilisyeasts.Unitsofyieldsaregivenin(mmol/mmolGLUC)(Forinterpretationofthereferencestocolourin thisfigurelegend,thereaderisreferredtothewebversionofthisarticle).

Table4

Analysisofelementarymodes(EMs)inthemetabolismofnonconventionalyeasts fortheshikimatepathway.

EM YBIOM/GLUC Y2-PE/GLUC Y2-PEA/GLUC YN/GLUC YETH/GLUC

18 0.003 0.0091 0 0.0221 0.7589 20 0.0028 0 0.0069 0.0208 0.7597 26 0.0268 0.0819 0 0.1985 0.8935 27 0.0264 0 0.0646 0.1958 0.84 53 0.0015 0 0.0232 0.0107 0.7464 55 0.012 0 0.192 0.089 0.7204 80 0.0032 0.0005 0 0.0236 0.7291 82 0.0032 0 0.0005 0.0235 0.7296 122 0.0021 0.0295 0 0.0157 0.7448 124 0.0021 0 0.0149 0.0158 0.7532 128 0.0155 0.2155 0 0.1146 0.7123 129 0.0189 0 0.1314 0.1398 0.7779

EMsareexpressedasyieldswithrespecttoglucose.Unitsofyieldsaregivenin (mmol/mmolGLUC)exceptforbiomass,givenin(gBIOM/mmolGLUC).

vinasses.Toimprove2-PEand2-PEAproduction,yieldanalysiswill

beconsideredasafirstguidelinetooptimizetheprocess.

4.Conclusions

Theresultsindicatethatvinassescouldbeofinterestforthe

productionofindustrialmetabolites,suchas2-phenylethylacetate,

throughtheShikimatepathway.TheyeastsW. anomalus andC.

glabrataobtainedthebestperformancesforcellgrowthandaroma

compoundproductionandaccomplishedover50%CODandBOD

reductionfromtequilavinasses.However,thecompositionofthe

differentvinassesplaya majorroleintheusageofthisresidue,

sincethepresenceofinhibitorycompoundsnegativelyaffectedcell

growthand2-PEAproduction;thus,thesecompoundsshouldbe

monitoredpriortovinassefermentation.Elementarymodesand

yieldanalysisobtainedfromtheFBAshowedthedistributionand

the theoretical fluxes for aroma compound production, which

offersaguidelineandstartingpointforimprovementusingtequila

vinassesassubstrate.

CRediTauthorshipcontributionstatement

José de Jesús Rodríguez-Romero: Writing - original draft.

CésarArturoAceves-Lara:Datacuration,Formalanalysis.Cristina

FerreiraSilva:Conceptualization.AnneGschaedler:Investigation,

Methodology.LorenaAmaya-Delgado:Fundingacquisition.Javier

Arrizon:Projectadministration,Supervision,Writing-review&

editing.

DeclarationofCompetingInterest

The authors declare noconflict of interests involved in the

study.

Acknowledgments

ThisworkwassupportedbyaCONACYTgrant,announcement

291137;FSE-CONACYT-SENER248090and245750projects,aswell

asthe“PlandeMovilidadAGARED2017”program.The authors

thanktheTequilafactoriesinvolvedinthestudyforprovidingthe

vinassesusedinthetequilavinassesfermentations.

AppendixA

Reactionsonthereducedmetabolicnetworkofnonconventional

yeastsholdingdifferentmetabolicpathways.Irreversiblereactions

aredescribedby‘=>’,whereasreversiblereactionsaredenotedby‘=’.

Biomassequationwasmodifiedfromtheoneproposedby

Robles-Rodriguezetal.[31],SongandRamkrishna[32]

Glycolysis R1 GLCx=>GLC R2 GLC+ATP=>G6P+ADP R3 G6P=F6P

R4 F6P+ATP=DHAP+GAP+ADP R5 DHAP=GAP

R6 DHAP+NADH=GOL+NAD R7 GOL=GOLx

R8 GAP+NAD+ADP=PG3+NADH+ATP R9 PG3=PEP

R10 PEP+ADP=PYR+ATP Pyruvatemetabolism R11 PYR=>ACD+CO2 R12 ACD+NADH=>ETH+NAD R13 ACD+NADHm=>ETH+NADm R14 ACD+NADP=>ACT+NADPH R15 ACT=>ACTx

R16 ACT+CoA+2ATP=>AcCoA+2ADP R17 PYR+ATP+CO2=>OAA+ADP

Pentosephosphatepathway

R18 G6P+2NADP=>Ru5P+CO2+2NADPH R19 Ru5P=X5P R20 Ru5P=R5P R21 R5P+X5P=S7P+GAP R22 S7P+GAP=E4P+F6P R23 E4P+X5P=F6P+GAP Krebscycle

R24 PYR+NADm+CoAm=>AcCoAm+CO2+NADHm R25 OAA+NADm+NADH=OAAm+NADHm+NAD R26 OAAm+AcCoAm=>ICT+CoAm

R27 ICT+NADm=>AKG+CO2+NADHm R28 ICT+NADPm=>AKG+CO2+NADPHm R29 AKG+ADP+NADm=>SUC+ATP+CO2+NADHm R30 SUC+0.5NADm=MAL+0.5NADHm

R31 MAL+NADm=OAAm+NADHm Shikimate-Ehrlichpathway R32 E4P+PEP=>DHA7P R33 DHA7P+NADH=SHKT+NAD R34 SHKT+PEP+ATP=CHO+ADP R35 CHO=>PHP+CO2 R36 PHE+AKG=GLUT+PHP R37 PHP=>PHAC+CO2 R38 PHAC+NADH=>2_PE+NAD R39 2_PE+AcCoA=>2_PEA+CoA Biomassformation

R40 1.04AKG+0.57E4P+0.11GOL+2.39G6P+1.07OAA+0.99PEP+0.57 PG3+1.15PYR+0.74R5P+2.36+AcCoA+0.31AcCoAm+2.68NAD+0.53 NADm+11.55NADPH+1.51NADPHm+30.48ATP=>1gBIOM+2.36CoA +0.31CoAm+2.68NADH+0.53NADHm11.55NADP+1.51NADPm+ 30.48ADP

Glutamine,glutamatemetabolism R46 NH4=>NH3

R47 NADPH+AKG+NH3=>NADP+GLUT R48 ATP+GLUT+NH3=>ADP+GLUM Others

R41 ATP=>ADP+MAINT R42 NADH=>NAD R43 2_PE=>2_PEx R44 2_PEA=>2_PEAx R45 PHEx=>PHE

Abreviations

AcCoAAcetylCoenzymeA(cytosol)

AcCoAmAcetylCoenzymeA(mitochondria)

ACDAcetaldehyde

ACTAcetate

ACTxAcetate(extracellular)

ADPAdenosineBisphosphate

AKGα-ketoglutarate

ATPAdenosineTriphosphate

BIOMCatalyticBiomass

CHOChorismate

CoACoenzymeA

CoAmCoenzymeA(mitochondria)

CO2Carbondioxide

DHAPDihydroxyacetonephosphate

DHA7P3-Deoxy-D-arabino-heptulosonicacid7-phosphate

E4PErythrose4phosphate

ETHEthanol

F6PFructose6-phosphate

G6PGlucose6-phosphate

GAPGlucose3-phosphate

GLCGlucose

GLCxGlucose(extracellular)

GLUMGlutamine

GLUTGlutamate

GOLGlycerol3-phosphate

GOLxGlycerol3-phosphate(extracellular)

ICTIsocitrate

MAINTMaintenance

NADNicotinamideadeninedinucleotideoxidized(cytosol)

NADHNicotinamideadeninedinucleotidereduced(cytosol)

NADHm Nicotinamide adenine dinucleotide reduced

(mito-chondria)

NADmNicotinamideadeninedinucleotideoxidized

(mitochon-dria)

NADPNicotinamideadeninedinucleotidephosphateoxidized

(cytosol)

NADPHNicotinamideadeninedinucleotidephosphatereduced

(cytosol)

NADPHm Nicotinamide adenine dinucleotide phosphate

re-duced(mitochondria)

NADPmNicotinamideadeninedinucleotidephosphateoxidized

(mitochondria)

NH3Ammonia

NH4Ammonium

OAAOxaloacetate

OAAmOxaloacetate(mitochondria)

PEPPhospho-enolpyruvate

PG3Glyceraldehyde3-phosphate

PHACPhenylacetaldehyde

PHEPhenylalanine

PHExPhenylalanine(extracellular)

PHPPhenylpyruvate

PYRPyruvate

R5PRibose5-phosphate

Ru5PRibulose5-phosphate

S7PSedoheptulose7-phosphate SHKShikimate SUCSuccinate X5PXylose5-phosphate 2PE2-phenylethanol 2PEA2-phenylethylacetate

BOD5Biochemicaloxygendemand

CODChemicaloxygendemand

EMsElementarymodes

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