Ethanol production in Brazil: a bridge between science and industry

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

Biotechnology

and

Industry

Microbiology

Ethanol

production

in

Brazil:

a

bridge

between

science

and

industry

Mario

Lucio

Lopes

,

Silene

Cristina

de

Lima

Paulillo,

Alexandre

Godoy,

Rudimar

Antonio

Cherubin,

Marcel

Salmeron

Lorenzi,

Fernando

Henrique

Carvalho

Giometti,

Claudemir

Domingos

Bernardino,

Henrique

Berbert

de

Amorim

Neto,

Henrique

Vianna

de

Amorim

Fermentec,Piracicaba,SãoPaulo,SP,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received8September2016 Accepted5October2016 Availableonline25October2016 AssociateEditor:AdalbertoPessoaJr

Keywords: Ethanol Yeast Fermentation Distillery

a

b

s

t

r

a

c

t

Inthelast40years,severalscientificandtechnologicaladvancesinmicrobiologyofthe fermentationhavegreatlycontributedtoevolutionoftheethanolindustryinBrazil.These contributionshaveincreasedourviewandcomprehensionaboutfermentationsinthefirst and,morerecently,second-generationethanol.Nowadays,newtechnologiesareavailable toproduceethanolfromsugarcane,cornandotherfeedstocks,reducing theoff-season period.Bettercontroloffermentationconditionscanreducethestressconditionsforyeast cellsandcontaminationbybacteriaandwildyeasts.Therearegreatresearch opportu-nitiesinproductionprocessesofthefirst-generationethanolregardinghigh-valueadded products,costreductionandselectionofnewindustrialyeaststrainsthataremorerobust andcustomizedforeachdistillery.Newtechnologieshavealsofocusedonthereduction ofvinassevolumesbyincreasingtheethanolconcentrationsinwineduringfermentation. Moreover,conversionofsugarcanebiomassintofermentablesugarsforsecond-generation ethanolproductionisapromisingalternativetomeetfuturedemandsofbiofuelproduction inthecountry.However,buildingabridgebetweenscienceandindustryrequires invest-mentsinresearch,developmentandtransferofnewtechnologiestotheindustryaswellas specializedpersonneltodealwithnewtechnologicalchallenges.

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

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

Introduction

Theautomobilewasoneofthemostimpressiveinventions thatchangedthelifestyleofhumanity.Thefirstcarswere designedtorunonethanol.However,duetothediscoveryof

∗ _{Correspondingauthorat:Av.AntoniaPazinattoSturion,1155Piracicaba,Brazil.}

E-mail:[email protected](M.L.Lopes).

newoilfields,itsabundanceandlowpricesatthebeginning ofcentury20,engineswerebeingmodified,givingpreference to petroleum based-fuels. Then, gasoline became the pri-maryfueloptionforcarsworldwide.Nevertheless,because of 1970’s oilcrisis, the Braziliangovernment launched the “Proalcool” program in 1975 with the aim to reduce the

http://dx.doi.org/10.1016/j.bjm.2016.10.003

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

country’sdependenceon oilimports.1 _{Inthe first phaseof}

theprogram,ethanolwasaddedtogasoline.Afterthesecond oilcrisisin1979,theautomobileindustrystartedtoproduce thefirstcartorunonethanolonlyfortheBrazilianmarket.2

In1980,productionoflightvehiclesthatranonethanol reached 95% of all fleet produced in Brazil. It increased ethanolconsumptionand reducedsignificantlyoil depend-ence.However,acombination offactors involvingoilprice drops,reductionofsubsidiestoproducersandriseofsugar pricescontributedtofuelshortagethatledtoamajor down-turninthedemandforethanol-runcars.2

Atthebeginningof21stcentury,theuseofethanolasfuel wasresumedchieflymotivatedbyhighoilpricesinthe inter-nationalmarketandthedevelopmentofflex-fueltechnology.1

Flex-fuel cars can run either on 100% hydrous ethanol or ondifferentblendsofethanolandgasoline.Electronic sen-sors detect the fuelblending and automatically adjustthe enginecombustion.Furthermore,theuse ofethanolblends replacedtoxicadditivesingasoline,suchastetraethylleadand oil-derivedbenzene.Leadedgasolinewasbannedinseveral countriesduetoseriouscontaminationproblemstothe envi-ronmentandhumans.3_{Moreover,theuseofethanolblends}

hasimprovedairqualityinlargeurbancenters,reducing emis-sionsofcarbonmonoxidefrom50g/kmdriventolessthan 5.8g/kmdriven.4_{Nowadays,gasolinecontainsablendingof}

27%ofanhydrousethanol.

The flex-fuel car technology allowed the consumer to chooseforthemostconvenientandcheaperfuel.The produc-tionandsellingofflexfuelcarsandlightcommercialvehicles represented85.45%ofthetotalin2015.5_{Currently,theuseof}

ethanolasbiofuelinBrazilhasbeenthemostsuccessful pro-gramtoreplacefossilfuelsworldwide.Severalcountrieshave startedtheirownprogramsforproductionanduseofethanol asfueltoreduceoildependenceandGHG(greenhousegases) emissions.6–8

Ethanol canbeproducedeither bychemical or microbi-ologicalprocesses.Thechemicalrouteisbasedonethylene hydrationwhilethemicrobiologicalprocessischieflycarried outbyyeastSaccharomycescerevisiae,althoughother microor-ganismsmayalsoproduceethanol.9–11

Currently, the main industrial route used for ethanol production worldwide is the microbiological process, also referred asalcoholicor ethanolicfermentation.12–15 _During

thisprocess,sugarsareconvertedintoethanol,energy,cellular biomass,CO2andotherbyproductsbyyeastcells.These sug-arsmaycomefromdifferentfeedstocksandcropwastes.16,17

InBrazil,themainfeedstockissugarcanewhiletheUnited StatesofAmerica(USA)producesethanolfromcorn.16,18

Sug-arcane,cornandsorghumareC4plantswithhighefficiencyto convertatmosphericCO2andwaterintosugarsandpolymers suchasstarch,celluloseandhemicellulosethrough photo-synthesis. Thisprocess usessunlight energyto fixcarbon andreleaseoxygenintotheair.19_Then,allCO

2resultingfrom ethanolburningisrecycledthroughphotosynthesis(Fig.1).

Ethanol produced from sugarcane has reduction rates between40and62%inGHGemissionscomparedtogasoline.20

Readily fermentable sugars such as sucrose, glucose and fructosearedirectlyconvertedintoethanolduringalcoholic fermentationbytheyeastS.cerevisiaewhilestarch,cellulose andhemicelluloseneedtobehydrolyzedtosimplesugarsto befermented.21,22 _{Inbothcases,yeastcellsconvert asolid}

substrateintoaliquidfuel.Moreover,toconvertsugarsinto ethanol, yeasts charge only7%ofthe energy containedin sugar molecules. The end balance shows that 93% of the energypresentinsugarsisconservedinethanol.23_Inaddition,

ethanolisamoleculethatcanbeeasilyseparatedfromwater throughdistillationprocessbecauseitsboilingpoint(78◦C)is verydifferentfromwater(100◦C).Moreover,ethanol produc-tionfromsugarcaneinBraziliandistilleriespresentsahighly positiveenergybalance.Intermsofequivalence,foreachunit

Sun light Photosynthesis Sugarcane Sucrose glucose fructose

Crushing Fermentation Distillation Distillery

Fuel station

Flex fuel cars Ethanol

O

CO

CO

O

H

O

H

O

Fig.1–Throughphotosynthesis,sugarcaneconvertscarbondioxide(CO2),water(H2O)andenergyfromlightintosugars

andpolysaccharidesreleasingoxygen(O2)intoatmosphere.Afterharvestingandcrushing,sugarsarefermentedbyyeasts

andconvertedintoethanol.Itisseparatedbythedistillationandusedasbiofuelbycars.Theburningofethanolgenerates CO2thatreturnstotheatmosphereclosingthecycle.

offossil energy consumed during its production, 9.3units ofrenewableenergywereproducedin2005,butwith poten-tialtoreach11.6 in2020.6 _{Moreover,forsecond-generation}

ethanol processes, energy balance for ethanol production fromcellulosicmaterialsisexpectedtobebetterthanthat ofsugarcaneorcorn.24 _{Allthese characteristicsturnedthe}

Braziliandistilleriesarenewableandenvironmentalfriendly biofuelindustry.25

Severalscientificstudiesinthemicrobiologicalfieldhave beencrucialforevolutionofthe ethanolindustryinBrazil, namelytheimprovementanddevelopmentofnew fermen-tationprocesses,selectionofrobustindustrialyeaststrains, better control of bacterial contaminants, improvement of chemicalandmicrobiologicalcontrolatdistilleries.26–29

More-over,investmentsinresearchonsecond-generationethanol havestimulatedtheparticipationofsomecompaniesto pro-duce ethanol from sugarcane bagasse and vegetal trash. Nowadays,newscientificandtechnologicalfrontiersarefaced by the industry to improve the fermentation process for ethanolproductioninBrazil.

The

main

alcoholic

fermentation

processes

in

the

world

Nowadays,theUSAandBrazilaretheworld’slargestethanol producers.Together, both countriesaccount formorethan 94 billion liters of ethanol produced per year, accounting foraround 85% ofworldwide production.30 _{However, there}

arehugedifferencesinthefermentationprocesses(Table1). Besidesfeedstock,aprimarydifferenceistherecyclingofyeast cells.Braziliandistilleriesuseanimprovedfermentation pro-cesspatentedin1937byFirminoBoinotfromtheMelleregion, France.31 _{Afterthe endofeach fermentation,therawwine}

iscentrifuged toseparatetheyeastcells inaconcentrated creamwhilethewinegoestodistillation.Afteratreatment withwater-diluted sulfuricacid(pH2.0–2.5for1–2h),these yeastcellsreturntolarge-volumetanks(250–3000L)foranew fermentationcycle.21,26,27_{DistilleriesintheUSAdonotrecycle}

yeastcells due tohigh concentration ofsolids and all fer-mentedmedia(includingyeastcells)isdistilled.32_Then,yeast

cellsarenotrecycledandmoresugarisdeviatedfor multipli-cationofcellsratherthantoethanolproduction.Fermentation yieldislowerinprocesseswithoutrecyclingofcells.33

Table1–Maincharacteristicsofethanolproductionin theUSAandBrazil.

Characteristics USA Brazil Mainfeedstock Corn Sugarcane Fermentationprocess Withoutyeast

recycling Withyeast recycling Solidsinsuspension >30% <1% Fermentationyield 85–90% 90–92% Fermentationtime 45–60h 6–12h Yeastconcentration 3–4% 8–12% Ethanolconcentration 12–18%(v/v) 7–12%(v/v) Daysofplantoperation 345 200–240 Main by-product/residue DDGSforanimal feed Vinassefor ferti-irrigation

Once therecyclingprocesses startwith highercell

con-centrations (8–12%, v/v), fermentation times of Brazilian

distilleriesarefaster(6–12h)whencomparedtofermentations

inUSA(54–72h)withoutyeastcellrecycling.However,corn

fermentationsreachhigherethanolconcentrations(12–18%,

v/v)incomparisontoBrazilianprocesses(7–12%,v/v).Once

corngrainscanbestoredforseveralweeks,fermentationscan

becarriedoutduring345daysayearwhileBrazilian

distiller-iesruntheirprocessesfor200–240days.Differentlyfromcorn

grains,sugarcaneneedstobecrushedsoonafterharvesting

toavoidlosscausedbymicrobialcontamination.Inaddition,

Braziliandistilleriesaresubjecttointerruptionsofsugarcane

harvesting,crushingandfermentationduetorains.1,21

The mainco-product from corn fermentationsis DDGS (distillersdriedgrainswithsolubles)usedforanimalfeedand hasahighmarketvalueduetonutritionalcharacteristics.34

On the other hand, Brazilian distilleries generate huge volumesofpotassium-richvinasse,whichisusedfor sugar-caneferti-irrigation,reducing costsonimportsofchemical fertilizers.35

Braziliandistilleriespresentseveraldifferencesfromoneto anotherintermsofthefermentationprocessandsugarcane mustcomposition.Distilleriesattachedtosugarfactories usu-allyfermentsugarcanemustsofmolassesdilutedwithwater oramixofjuiceandmolasseswhileautonomousdistilleries fermentonlyjuice.36

Concerningfermentation,fed-batchprocessesaccountfor around 83% of the distilleries while continuous fermenta-tionsare17%.37_{Despitehigherinvestmentsforinstallation,}

fed-batchprocesseshavepresentedbetterresultsthan con-tinuous fermentation (Table 2). A study carried out during eightconsecutiveyearswith62distilleries(51fed-batchand 11continuousfermentation)showedthatfed-batchprocesses reachedthehighestfermentationyields.Inaddition,itwas observedalowerbacterialcontaminationinwineandlower consumptionofchemicalssuchassulfuricacidandantibiotics incomparisontocontinuousprocesses.37_However,a

miscon-ductedfed-batchprocesswillbeworsethanawell-managed continuousfermentation.Sometimes,thebenefitsofa con-tinuous fermentationare masked byimproperengineering conceptionandlowcostadaptations.38

Infed-batchfermentations,each tankisfilled,managed andcleanedseparatelyfromtheother,whileincontinuous processesalltanksfermentsimultaneouslyinlineand can-notbecleanedwiththesamefrequency.Aftercentrifugation, theyeastcreamreceivesanacidictreatmentandreturnsto fermentationtanks(Figs.2and3).

Fermentation:

a

living

process

AlthoughEduardBuchnershowedthatalcoholicfermentation couldbecarriedoutwithextractofdeadyeast,39_the

Melle-Boinot fermentation depends on living cells.31 _{These cells}

mustbealiveattheendoffermentationandaftertheacidic treatmentforreuseinthenextcycle.TypicalBrazilian distill-eriesrun400fermentationcyclesduringthesugarcane har-vestingseason.Becausetheyarerecycledseveraltimes(two cyclesaday),yeastcellsaresubjecttostressfulconditionsof industrialfermentations(Fig.4).Theseconditionsmaychange

Table2–Comparisonofcontinuousandfed-batchfermentationswithcellrecyclingforethanolproductioninBrazil.37

Characteristics Unit Continuous Fed-batch Fermentationyield % 87.0–89.5 88.9–90.5 Bacterialcontaminationofwinea _{bacterialrods/mL 3.8–9.9×10}7 _{2.0–3.9×10}7

Consumptionofsulfuricacid g/Lethanol 8–14 6–7

Consumptionofantibiotics mg/Lethanol 5.7–13.0 3.8–8.0

Consumptionofantifoaming mg/Lethanol 0.47–0.75 0.45–0.70

a _{Livingbacterialrodsarecountedbyopticalmicroscopy.}

fromonedistillerytoanother,aswellas,inthesamedistillery duringthesugarcaneharvestingseason.Moreover,yeastcells aresubjecttoeffectsoftwoormorestressingconditions.40,41 However,yeastcellshavemechanismsofstressresponseto industrialconditionsoffermentationandacidtreatment.42,43

Trehaloseandglycogenare reservecarbohydratesfor addi-tionalenergywhencellsarestarvedforsugars.44,45_Moreover,

trehaloseisaprotectingcompoundtomembranethathelps cellstotoleratedehydrationandhighethanolconcentrations aswellasotherindustrialstressingfactors.46,47_Succinicacid

in combination with ethanol has an antibacterial effect,48

whileglycerolisaregulatorforosmoticshocks.49,50

Accordingtothe GayLussac equation,theoretically, fer-mentationproduces511gofethanoland489gofCO2 from 1000gofglucose. However,inindustrialprocessesthis fer-mentation yield is not achieved because yeast cells drive sugarstotheproductionofcellularbiomassandsecondary compoundssuchasglycerol,succinic,malicandaceticacids,

fuseloiland other minor by-products. Forthis reason, the maximum fermentation yield achieved is 92–93%.28

More-over, fermentation yield is highly dependent on the yeast strain.Afermentationcarriedoutwithcellrecyclesinbench scaleshowedthatbaker’syeasthadafermentationyield3.9% lower thanthatofindustrialstrains.26 _{Itseemslittle,but it}

means8.5millionlitersofethanolduring200workingdays foranautonomousdistillerythatcrushes14,000sugarcane tonsdaily.

Monitoring

and

selection

of

industrial

and

tailored-yeast

strains

Before1990,identificationandmonitoringofindustrialstrains belongingtoS.cerevisiaewereoftenambiguousanduncertain whenperformedbycellandcolonymorphology, physiolog-ical and biochemical methodologies.51 _{Morphology of cells}

Juice Molasses Water

Heat exchanger Must

Tank 1 Tank 2 Tank 3 Tank 4

Raw wine Water Heat exchanger Recycled y e ast Yeast treatment Yeast cream Centrifugation Distillation Tank of wine Sulfuric acid

Heat exchanger Juice Molasses Must Water + Acid Recycled y east Yeast cream Centrifugation Tank of wine Distillation Raw wine Fermentation tanks Water

Fig.3–Simplifieddrawingofafed-batchfermentationprocesswiththerecyclingofyeastcellscurrentlyadoptedby Braziliandistilleries. Acetic acid Lactic acid Sulfuric acid Nutrient starvation Aluminum High ethanol concentrations High concentration of salts Osmotic shocks

Trehalose

glycogen

succinic acid

glycerol

Fig.4–Stressingfactorsthataffectyeastcellsinindustrial fermentationsforethanolproductionandcommon mechanismsofcelldefensebasedontrehalose,glycogen, succinicacidandglycerol.

and coloniesisquitesimpletodistinguishstrains and dif-ferentstrainscanfrequentlypresentthesamemorphology. Ontheotherhand,thesamestraincanchangeits celland colonymorphologyinresponsetostressconditionsor nutri-entstarvationandoverexpressionofgenesrelatedtocellular dimorphism likePHD1.50,52 _{Several strains ofSaccharomyces}

canalternatetheirmultiplicationpatternfromsinglecell bud-ding to pseudohyphae,when the buds remain attached to mothercells.53_{Physiologicalandbiochemicaltraitsarebased}

ontheabilitytoassimilateand/orfermentdifferentkindsof sugars,the useofnitrogensources,tolerancetoinhibitors, hydrolysis ofcomplex molecules, growth atdifferent tem-peraturesandotherconditionsthatmayallowtodistinguish differentstrainsfromothers.However,allthese methodolo-gies have shown limitations toidentify and monitoryeast populationsinpractice.

During several years,the dynamic ofyeast populations in industrial processes of alcoholic fermentation in Brazil remained unknown and unexplored due to difficulties for strain identification by traditionalmethodologies based on morphologicalandphysiologicaltraits.Whilesome distiller-iesclaimedthatthebaker’syeasts,M300AandIZ1904,usedas starterstrainswereverygoodfermenters,othershadan oppo-siteopinionduetolowfermentationyields.54_Duetolackof

propermethodologiestoidentifyandmonitorstarterandwild strains, itwasnotpossibletofollowchangesinpopulation

duringsuccessiverecycles.Forthisreason,whentheinitial yeastpopulationwasreplacedbywildSaccharomyceswithgood characteristics,the people believed that thestrain used as starterwassuperior.Onthe otherhand,whenwildstrains withpoorfermentationabilities contaminatedthe process, starter yeasts were appointed as responsible for problems atthe distillery.Theadventofmoleculartechniquesbased onDNA analyses allowedtouse differences in genome as molecularmarkersforstrainidentification,monitoringand selection.

After1990,severallaboratorieshaveintroducedmolecular techniques to identify new yeast strains from indus-trial fermentations worldwide. One methodology was the electrophoretic karyotyping based on chromosome-length polymorphismofyeastcells.55–57_{Afterapulsed-fieldgel}

elec-trophoresisto separatelarge DNA molecules, yeaststrains show differencesin chromosomeprofile regardingnumber and size that make each karyotype unique and strain-specific.58–60 _{This techniquehad beenused toidentifyand}

monitor yeast strains in grape fermentations with excel-lent results.56 _{In 1990, after a visit to INRA, Montpelier,}

France,ProfessorLuizCarlosBasso(ESALQ-USP)introduced theelectrophoretickaryotypingtoidentify,monitorandselect industrialyeaststrainsbasedonchromosomepolymorphism. Karyotypingshowedthatthebaker’syeast,IZ1904,and labo-ratorystrainsdonotsurvivesuccessivefermentationcycles.56

Afterthreeorfourweeks,thesestrainsarereplacedbywild

Saccharomycesthatcontaminatetheindustrialprocessof alco-holicfermentation.26_{However,severalyearswerenecessary}

toconvincepeoplefromdistilleriesthatbaker’syeast,IZ1904 andlaboratory strainsdonotsurviveinindustrial fermen-tationswithcellrecyclingandare quicklyreplacedbywild

Saccharomyces.61_{Inaddition,karyotypinghasshownthatPE2}

andCAT1,usedasstarterstrains,wereabletoreplacebaker’s yeastevenatproportionsaslowas0.5kgfor12tonsofpressed baker’syeast.26_{Nowadays,thereareonlysixindustrialstrains}

ofmajor importancefor ethanolproduction inBrazil: PE2, CAT1,FT858LandFermel®_{(selectedbyFermentec),BG1and} SA1(selectedbyCTC).Thesestrainshavebeenusedby Brazil-iandistilleriesthataccountfor70%ofallethanolproduction inthecountry.

Industrial yeast strains have more complexes and het-erogeneouschromosomesthanlaboratorystrainsdo.62 _The

highpolymorphismisuniqueforeachstrain.However,these chromosomesaresubjecttomitoticandmeiotic recombina-tionaswellassegregationerrorsduringsuccessivemitoses understressconditionsoraftersporulationandconjugation ofspores.58,59,63 _{Theintroduction ofasimplified}

methodol-ogyforthemitochondrialDNAanalysisallowedtodistinguish variantsfromstartersandcontaminatingstrains.64_The

com-binationofkaryotypingandfragmentlengthpolymorphism ofmitochondrial DNA hasprovento bea powerfultool to monitoryeastpopulationdiversityinindustrialfermentations as well as to select newstrains and variants from starter yeaststhatbecomemoreadaptedtoconditionsofeach dis-tilleryinparticular.61 _{VariabilityofmitochondrialDNAdoes}

notfollowtheMendelianlawsofsegregationand indepen-dentassortmentofchromosomes.65_{Themaindifferencesare}

related to occasionalmutations that are incorporated into DNA moleculesofmitochondria. Thus,the combination of

karyotypingandmitochondrialDNAanalyseshasallowedto distinguishstrainsderivedfromstarteryeastsand contam-inants aswell as toselect robustyeaststrains forethanol production.54

The genome complexity of industrial yeast strains rep-resents a new opportunity to select variants with infinite combinations ofgenesindifferent fermentationprocesses. Twoveryimportantcontributionsongenomesequencingof twoindustrialyeaststrainstraditionallyusedbyBrazilian dis-tilleriesarePE266_andCAT1.67,68_{TheyeaststrainPE2showeda}

highgenomeplasticitymainlyintelomericandsubtelomeric regions ofchromosomes.Thisplasticitycould explainwhy thisstrainadaptstodifferentfermentationprocesses.66_CAT1,

selectedfromacontinuousfermentationprocesspresented large genomic regions ofheterozygosity losswhile 58% of the6652predictedprotein-codinggenesweredifferentalleles whencomparedtoreferencegenomeofstrainS288c.68_Both

strains,PE2andCAT1,haveanincreasedcopynumberofgenes forbiosynthesisofthiamin(B1)andpyridoxine(B6)in compar-isontothebaker’syeastandlaboratorystrains.67_Anincreased

copy number can confer better fitness to these strains in vitamin-freefermentationmediumwithhighconcentration ofsugars.CAT1presentedashortlagphaseincomparisonto S288c,alaboratorystrain,whenbothyeastsweresubjected tohighsugar concentrationinamediumwithoutvitamins B1 and B6.67 _{These results are directly related to factors}

thataffecttheyeastdominanceandpersistenceinindustrial fermentation processes of ethanol production and explain whyindustrialstrainsaremorerobustandtolerantthanthe baker’syeastandlaboratorystrains.Thismeansthat monitor-ing yeastpopulationsinindustrialfermentationsregarding theirdominanceandpersistencecharacteristicsisthebasis toselecttailoredandhigh-performancestrainsaccordingto thepeculiarcharacteristicsofeachdistillery.Currently,18 dis-tilleriesuseTailoredYeastStrains®_{intheirprocess.Someof} themhavetwo,threeorevenfourcustomizedstrains(Fig.5). ThisapproachwasdesignedasProcess-DrivenSelection®_.54

However,yeastselectionandbiodiversityofwild strains stillremainascientificchallengetobeexplored.22,69_Moreover,

0 2008 2 2 22 33 3 3 6 7 7 10 10 16 14 21 18 26 2009 2010 2011 2012 2013 2014 2015 2016 5 10 15 20 25 30

Fig.5–NumberofdistilleriesthatuseTailoredYeast Strains®_{asstarterforethanolproductioninBrazil(gray}

bars)andtotalnumberofstrains(darkbars)selectedsince 2008bykaryotypingandthemitochondrialDNAanalysis.

ithasbeenproposedthatbiodiversityofyeastsisolatedfrom differentenvironmentscanrevealnewstrainswithdesired characteristics to industrial applications such as ethanol production.70

Contaminating

yeast

and

bacteria

Becauseoflargevolumes,industrialfermentationsaresubject tobacterialcontamination, suchasSaccharomycesand

non-Saccharomyceswildspecies.26,71,72_{Severalwildyeastscompete}

withstarterstrainscausingseriousoperationaldifficultiesto industrialprocessesofethanolproduction.26,54,73

Aresearchcarriedoutduringfiveyearsdemonstratedthat all73wildyeastsisolatedfromtwodistillerieswereclassified asS.cerevisiae.74_{However,contaminantstrainscanpresent}

differentcharacteristicsinrelationtostarterstrains.Afterto evaluate340contaminatingyeastsisolatedfromfermentation tanksof50 Braziliandistilleriesitwasshownthat80%had undesirablecharacteristicsforanoptimumfermentationsuch as foaming, flocculation and high concentrations of resid-ualsugars inthe wine.26Moreover,somestrainspresented acombinationofthesetraitsandmostofthembelongedto genusSaccharomyces.Themaincontaminantyeasts,classified asnon-Saccharomyces, wereDekkera,Schizosaccharomycesand

Candida.Dekkeraisacommon genusofcontaminantyeasts causingsignificantreductionoffermentationyieldin distill-eriesthatdoesnotwarmthesugarcanejuiceand worksat verylowethanolconcentrationsinthewine.75,76

ContaminatingwildSaccharomycesshowahighfrequency of undesirable traits such as flocculation, pseudohyphae development and foam excess. Although flocculation and chainformationofcellsarecharacteristicswelldocumented, foamproductionbydifferentstrainsisnotwellknown. How-ever,ithasbeenassociatedtohydrophobicityoftheyeastcell wallinethanolandsakeproduction.77,78

Althoughcontaminantscanentertheprocessthrough dif-ferentways,sugarcaneisthemajorsourceandnewstrategies basedonnon-culturaltechniqueshavebeenappliedtoaccess microbial communitiesthat canaffect industrialprocesses ofbioethanol production.79,80 _{Besides wild yeasts, bacteria}

competeforsugarsand nutrientswithstarteryeaststrains causingmanyproblems,suchasinhibitionoffermentation, productionoforganicacids,reductionofindustrialyieldsand flocculation.81–83

AcommonprocedureadoptedbyBraziliandistilleriesthat recycle yeast cells is the use of diluted sulfuric acid (pH 2.0–2.5for1–2h)tokillbacteria.26_{Withoutacidictreatment,}

industrialfermentationsaresubjecttosugarlossesdueto bac-terialcontamination.Microbiologicalanalysescarriedoutin adistillerydemonstratedthatsulfuricacidtreatmentofthe yeastcreamreducedbacterialpopulationby44.55%.84

Con-trolofbacterialcontaminationinindustrialfermentationsfor ethanolproductionhasbeenoneofthemostimportant fac-torsthat contributed to increasethe fermentation yieldin Braziliandistilleries.37

Thedevelopmentofamethodologytocountliving bacte-riabymicroscopyin15min,evolutionoffastteststoidentify the best antibiotics in only 6h and the use of antimicro-bialproductsreducedsignificantlybacterialcontaminationin

theindustry.29,71_{From2007onward,newantimicrobialswere}

introducedtocontrolbacterialcontamination.85_Thischange

wasnecessaryduetopressuretoreducetheuseofantibiotics, mainlybydistilleriesthatproduceinactivedryyeastfor ani-malfeed.Chlorinedioxideandhopacids derivatives(alpha and beta fraction)areamongthe newantimicrobialsused. Theseproductsarealternativestocontrolbacterial contami-nantswithoutuseofantibiotics.Microscopiccountingsaslow as105 _{bacterialrodspermLofwinehavebeenobtainedin} severaldistilleries.Thismeansathousandandtenthousand timeslessbacteriainfermentationthan40yearsago.37

More-over,severalcompoundscanaffectnotonlybacteriabutalso theyeastcellsandshouldbeevaluatedbeforetobeusedby theindustry.86

Regarding bacterial biodiversity in industrial fermenta-tions,334strainswereisolatedfromBraziliandistilleriesand classified using a matrix of similarity.84 _{The main}

bacte-rialcommunitieswererepresentedbyGram-positive(98.52%), rods(87.76%)andnot-sporulated(73.95%).Amongthemain bacterial groups, thelargest were Lactobacillus (59.75%) and

Bacillus (26.58%) whileother representatives were atminor proportion (14.67%). For the Lactobacillus group, the main speciesobservedwasL.fermentum(15.04%).84Despite bacte-rialcontaminationiscausedchieflybyGram-positivebacteria, itwasreportedacasewithdrasticreductionoffermentation yieldandethanolproductionduetoGram-negativebacteria identifiedasAcetobacterpasteurianusbythesequencingof16S rDNAgene.83

Lacticacidbacteriaarethemaincontaminantsofalcoholic fermentations.Thesebacteriaareclassifiedintotwo biochem-icalgroups,according tometabolismofglucose:homo and heterofermentative. Thefirst group ischaracterizedby the productionoflactatefromglucose,whiletheseconddrives themetabolismfortheproductionoflactate,acetate,ethanol andCO2.87

Since lacticacid bacteria are the maincontaminantsof alcoholicfermentations,lacticacidisanimportantindicator tomonitorbacterialcontamination.However,somebacterial speciescanproducedifferentisomersoflacticacidoramix ofboth.Aresearchcarriedoutwith27strainsofLactobacillus

isolatedfromindustrialfermentationsofethanolproduction demonstrated that70% ofthem produced dl-lactatewhile the other 30% produced onlyoneisomer (dor l)-lactate.88 Furthermore,heterofermentativebacteriaalsoproduce man-nitol which has been used as an indicator of sugarcane deteriorationbymicroorganismsaswellasofcontamination of alcoholic fermentation processes by heterofermentative bacteria.89,90_{Homoandheterofermentativelacticacid}

bacte-riacanaffectthefermentationofdifferentways.91_Because

oftheirimportance,somedistillerieshavemonitored manni-tol,lactate,acetateandresidualsugarsinwinethroughhigh performanceliquidchromatographyimprovingtheirprocess control.29

Another very important issue is the effect of bacterial contamination onthe yeast.Avery common effectis floc-culationofyeastcells.Thiskindofflocculationdependson physicalcontactbetweenbacteriaandyeastcells.Among lac-ticacidbacteria,L.fermentum,L.fructosus,L.buchneriandL. plantarumcancauseyeastflocculation.92_Whentheratio

afermentationassaycarriedoutwiththreestrains(baker’s yeast,PE-2andM-26),contaminatedbyL.fermentum, demon-strated that baker’s yeast was the mostsensible strain to bacterial contamination. Fermentations with baker’s yeast presentedthe lowestrates ofcellviability and the highest ratesofbacterialmultiplication.Theresultsshowedthatdead cellsreleasenutrientssuchasaminoacids,vitaminsand min-eralsthat stimulatebacterialmetabolism.Inaddition, PE-2 and M-26 showed the highestcell viability rates and low-estbacterialcontaminationbyLactobacillus.94_{Later,thehigh}

toleranceofindustrialandtailoredyeaststrainstobacterial contaminationbyL.fermentumwhilethe baker’syeastwas moresensible.54

Oneofthemostimportantbacterialcontaminationsources ofsugarcane juice is the soil. There is onebillion of bac-terialcells foreach 1gofsoil.95 _{Another importantsource}

ofbacterialcontaminationissugarcaneinfestationbyborer. Inaddition,sugarcanestalksperforatedbyboreraccumulate organicacidsandphenoliccompoundsthatcaninhibitthe fermentation.96

Bacterialcontaminationcanaffecttheethanolproduction differentlyaswellastheindirectdeterminationof fermen-tation yield based on by-products such as glycerol, yeast biomass,residualsugarsandacidity.Someyearsago,a dis-tillerypresentedafermentationprocesswithcontamination above107_{bacterialrods/mLandawineaciditythatwaslower} than acidity of sugarcane must. It was not expected for anindustrialfermentation. Afterisolation andbiochemical testswereidentifiedbacterialspeciesbelongingto malolac-ticgroup.97_{Thesebacterialspecieshavebeenisolatedfrom}

grapewinefermentationsbutnotfromethanolproduction. Malolactic bacteriaare ableto convertmalic acid into lac-tic acid for ATP production.98 _{Malic acid is a dicarboxylic}

acidpresentinsugarcanejuice,butitcanalsobeproduced by yeasts during the fermentation.49,99 _{Once lactic acid is}

monocarboxylic,thesebacteriareducetheoverall acidityof fermentedmedium.Becausetheaciditybalanceattheendof fermentationwasnegative,itoverestimatedthefermentation yieldbyindirectmethodologybasedonby-products.In2013, anothercaseobservedwasthecontaminationbyAcetobacter indonesiensis.ItisaGram-negativebacteriaassociatedtosoil androotsofsugarcane.Thiscontaminationwasassociatedto adrasticglycerolreductioninwine,overestimatingthereal fermentationyieldbasedonby-products.

Allofthese casesshowthe diversityand complexity of yeastandbacterialcommunitiesthatcontaminateindustrial process ofethanol production and the different waysthat thesemicroorganismscanaffectalcoholicfermentationand compromisethecurrentethanolproduction.Thebiodiversity ofcontaminantsmay affectnotonlyethanolproductionof firstgenerationbutalsonewtechnologies.

New

technologies

for

production

of

first

and

second-generation

ethanol

Despite reaching alcoholic fermentation yields as high as 92%,thereareseveralscientificandtechnologicalchallenges tobeovercomeforfirstandsecond-generationethanol.22,100

Incomparisonto cornfermentations inthe USA, Brazilian

distilleries runtheirprocessesforeightmonths(orless)in a year because sugarcane cannot be harvested during the rainy seasonand cannot be stored as corn can. However, new Braziliandistilleries were built to ferment cornstarch, reducing the off-season without ethanol production.101 _It

has opened a newopportunity to increase the production of first-generation ethanol, reducing industrial fixed costs, employing specialized labor force and improving competi-tiveness of distilleries. Moreover, these processes have an enormousadvantagebecausetheydonotgeneratevinasses. Themainby-productisdistillersdriedgrainswithsolubles (DDGS).Ithasacommercialvalueashighasthatofethanol, itisricherinproteinthancorngrainandcanbeuseddirectly foranimalfeeding.102

Morerecently,anewBrazilianprocessofcornfermentation hasbeendevelopedbasedonthereuseofsurplusyeastcells fromsugarcanefermentationthatwouldbediscardedbythe plant.ThisBrazilianprocess integratesthe fermentationof sugarcanejuice(ormolasses)andstarchyfeedstocks, provid-inganumberofadvantagesoverconventionaldistilleries.103

Thisprocesshasafasterfermentation(34–36h)incomparison withthetraditionalprocess(45–60h)adoptedbydistilleries in the USA and less sugar is deviated for yeast multipli-cationandproductionofcellularbiomass.Inaddition, this technologyallowstouse thesamedistillationsystemused forsugarcaneand extendthe periodofethanolproduction to 345 days a year, reducing initial investments and fixed costs.Eachcorntonallowstoproduce415Lofethanoland 250kgofDDGS.103_{Moreover,integrationofethanolproduction}

from sugarcane and corn has the potential tooffer signif-icant economic advantages in comparison to stand-alone units, since importantoperations, yeast, feedstock, labora-toriesandtechnicalpersonnelmaybesharedbetweenboth processes.

Flexfuelplantsofcornandsugarcanehaveanenergy bal-anceandreductionofGHGemissionsthatdonotcompromise thefermentationperformance.Theyareeconomicallyviable inregionswithcornsupplyatlowpricesandhighdemand ofDDGS foranimal feed.Itisanopportunity forBrazilian distilleriesincornproducingregionsthatarefarfromports.104 Besidesethanol productionfrom starchyfeedstocks, the last frontier of biofuels has been the use of lignocellu-losicbiomass-derivedsugars.105_{Celluloseandhemicellulose}

accountformorethan60%ofdryweightofsugarcanebagasse andcanbeconvertedintofermentablesugarseitherbyacidor enzymatichydrolysis.106_{Toachievethisgoal,severalresearch}

groupsandcompaniesworldwidehaveexpendedeffortsand resourcestoproducenotonlyethanol,butalsootherbiofuel moleculessuchasbutanolandisobutanol.107–109

Researchonsyntheticbiologyandmetabolicengineering hasimprovedproductivityandexpectationsofhighyieldfor differentbiofuels.Moreover,selectionofmicroorganismsto beusedasbiotechnologicalplatformformetabolic engineer-ingapproacheshasbeenconsideredasuccesskeyfactor.110

These strains need to be improved regarding velocity of sugarassimilation,co-fermentationofpentosesandhexoses, tolerancetoinhibitorsproducedfromhydrolysates,tolerance toethanolconcentrationsandrecyclingprocesses.22,110

Selec-tion ofindustrial yeaststrains hasbeen a viableapproach foranewgenerationofstrainsthatcanbeusedasplatform

toproducesecond-generationethanolwhileotherinitiatives havefocusedonbacteria.111,112

Nowadays,inBrazil,therearetwoindustrialplantsin oper-ation forthe productionofsecond-generationethanol and onethatuseasemi-industrialprocess.Theseprocesseswere originatedfrominvestmentprogramsofBraziliangovernment in second-generation ethanol launched in 2011. Neverthe-less,productioncostsofsecond-generationethanolarestill high,mainlyconcerningequipmenthandlingatbagasse pre-treatmentandtheuseofenzymes.113_{Severalinitiativeshave}

focusedonselectionofmicrobialstrainsandoptimizationof conditionsforproductionofenzymesrequiredforhydrolysis andfermentationinindustrialprocesses.114–118

Lignocellulosicpre-treatments,suchassteamexplosion, alkalinehydrogen peroxide, dilutedacid, ammonia, use of solvents,among others,are carried out priortoenzymatic hydrolysis in order to make cellulose more accessible to attackbyenzymes.119_{However,thepre-treatmentof}

sugar-canebagassestillhastechnologicalchallengestobeovercame inindustrialscale.Newtechnologiestodisassemblythe sug-arcanefiberaswellastheuseofenzymeswillimprovethe efficiencyofbagassehydrolysisaswellastheproductionof ethanol2G.120,121

Reduction

of

vinasse

volumes

Vinasse is the resulting residue after distillation of wine. ForeachliterofethanolproducedinBrazil,onaverage,are generatedother 12Lofvinasse.122 _{Consideringtheethanol}

productionin2015of30billionliters,itmeansaproduction of360billionlitersofvinasseperyear.Vinasseis potassium-richandithasbeenusedinferti-irrigationofsugarcanefields, reducingcostswithchemicalfertilizers.35_{Inaddition,vinasse}

increasesthesoilpHsomedaysafteritsapplicationdueto microbialactivitystimulatedbyorganiccompoundsandother nutrients.123

However,vinassetransportandapplicationinthefieldare costlyandrequirecarefulprocedurestoavoidcontamination oftablewatersandsoilsaturationwithcations.124_A

reduc-tion ofvinassevolumeincreases the economicdistanceof transportandapplicationinsugarcanecrops.125–127_More

con-centrated,vinassecanbetransportedtodistantareasfrom distilleryreducingcostsofapplicationinthefield.

The vinasse volume is directly related to ethanol con-centration in wine (Table 3). Despite the overall yield of fermentation process increased from 75–80% to 90–92% in thelast30years,ethanolconcentrationsinwine(8.0–8.5)has remainedalmostthesamewithexceptionofsomedistilleries thathaveruntheirprocesseswithalcoholiccontentsabove 10%(v/v).22,128,129

A key factor to increase the ethanol concentrations in wine and reduce vinasse volumes has been the selection anduse ofrobustyeaststrains. Testscarriedout atapilot scalerevealed thatsomeindustrialstrainsaremorerobust and viablefor fermentationswithhigh ethanol concentra-tions in winethan other traditionalyeasts.130 _{These yeast}

strainsopenanewperspectivetoBraziliandistilleriesincrease theconcentrationofethanolinwineforreductionofvinasse volumes.

Table3–Increaseofethanolconcentrationinwineand reductionofvinassevolumesforadistillerywithan ethanolproductionof5200m3_/week.

Ethanol concentrationin wine(v/v)

Lvinasse/Lethanol Vinassevolume (m3_/week) 8 11.9 61,880 9 10.6 55,120 10 9.4 48,880 11 8.5 44,200 12 7.7 40,040 13 7.0 36,400 14 6.4 33,280 15 5.9 30,680 16 5.4 28,080 Source:Fermentec.

Final

remarks

Nowadays,severalBraziliandistilleriesarelookingfor

tech-nological innovations to improve their performance and

ensurecompetitiveness ofethanol production.Evolutionof

first and second-generation ethanol depends ofnew

feed-stocksandacontinuousimprovementofthemicrobiological

processes.22,131,132

Althoughproductionoffirst-generationethanolhasbeen considered amaturetechnology,therearehuge opportuni-ties of research, development and innovation forBrazilian distilleries.133 _{It includes reduction of sugars losses, new}

strategiestocontrolbacterialcontaminants,selectionofnew yeaststrains,technologiesforreductionofvinassevolumes and alternative uses bythe industry, energysaving, better useofwater,developmentofnewfermentationprocessesfor alternativefeedstocksandbiorrefineriesforhigh-valueadded products.22

Moreover,it isnecessarytodevelopstrategiesfor trans-ferofnewtechnologiestotheindustry.134_{Thismovementof}

technologiesisfundamentaltoincreaseefficiencyandreduce costs. Astudy conductedin2016showed thatforevery R$ 1.00investedinresearchanddevelopment,thereispotential toreturnR$ 17.11onlyintermsofreductionofproduction costsinBraziliandistilleries.135_{Finally,investmentsin}

scien-tificandtechnologicaldevelopment,formationofresearchers andspecializedprofessionalswillbuildsolidbridgesbetween scienceandindustryforsustainablefutureofethanol produc-tioninBrazil.136

Acknowledgment

ToMartaMariaMoraesLeitãofordrawingsandillustrations.

r

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