Directing carbohydrates toward ethanol using mesophilic microbial communities

Delft University of Technology

Directing carbohydrates toward ethanol using mesophilic microbial communities

Moscoviz, Roman; Kleerebezem, Robbert; Rombouts, Julius Laurens DOI

10.1016/j.copbio.2021.01.016 Publication date

2021

Document Version Final published version Published in

Current Opinion in Biotechnology

Citation (APA)

Moscoviz, R., Kleerebezem, R., & Rombouts, J. L. (2021). Directing carbohydrates toward ethanol using mesophilic microbial communities. Current Opinion in Biotechnology, 67, 175-183.

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Directing

carbohydrates

toward

ethanol

using

mesophilic

microbial

communities

Roman

Moscoviz

,

Robbert

Kleerebezem

and

Julius

Laurens

Rombouts

Bioethanolproductionisanestablishedbiotechnological

process.Marginsarelowwhichpreventalargerscale

productionofbioethanol.Asalargepartoftheproductioncost

isduetothefeedstock,theuseoflowvalueunsterile

feedstocksfermentedbymicrobialcommunitieswillenablea

morecost-competitivebioethanolproduction.Toselectfor

highyieldethanolproducingcommunities,threeselective

conditionsareproposed:acidwashingofthecellsafter

fermentation,alowpH(<5)duringthefermentationand

microaerobiosisatthestartofthefermentation.Ethanol

producers,suchasZymomonasspeciesandyeasts,compete

forcarbohydrateswithvolatilefattyacidandlacticacid

producingbacteria.Creatingeffectiveconsortiaoflacticacid

bacteriaandhomo-ethanolproducersatlowpHwillleadto

robustandcompetitiveethanolyieldsandtitres.Aconceptual

designofanecology-basedbioethanolproductionprocessis

proposedusingfoodwastetoproducebioethanol,electricity,

digestateandheat.

Addresses

1_{SUEZ,CentreInternationaldeRechercheSurl’Eauetl’Environnement}

(CIRSEE),38rueduPre´sidentWilson,LePecq,France

2_{DelftUniversityofTechnology,vanderMaasweg9,2629HZDelft,The}

Netherlands

Correspondingauthor:

Rombouts,JuliusLaurens(julesrombouts@gmail.com)

CurrentOpinioninBiotechnology2021,67:175–183

ThisreviewcomesfromathemedissueonEnvironmental biotechnology

EditedbyRobbertKleerebezemandDianaMachadodeSousa

https://doi.org/10.1016/j.copbio.2021.01.016

0958-1669/ã2021TheAuthor(s).PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBYlicense(http://creativecommons. org/licenses/by/4.0/).

Introduction

Ethanol production through biotechnological

fermenta-tionprocessesisanestablishedindustry.Theglobalfuel ethanol marketwas estimatedto be110billionlitresin 2018[1].Intheperiodof2012–2019,theethanolpricein

Europe,BrazilandtheUnitedStatesfluctuatedbetween

0.95 and0.28USD perlitre(0.79 0.25sperlitre)and

priceshavedecreasedfrom2012onwards,leadingtoaless

profitablemarketsituation[2].Alargecostcontributorto

productionof bioethanolisthefeedstock.AnEuropean

Commissionreportfrom2002estimatedthat48–60%of

thecostinethanolproductionwasdueto thefeedstock

(in a casestudydescribed for sugar beets),with atotal productioncost of0.42 0.54 sperlitre[3].Margins in thebio-ethanolindustryarelowand,inmanycountries,

subsidies and mandatory blending are main drivers to

sustainethanol production[2].

Using less expensive feedstocks for fermentation can

enable cost-competitive bio-ethanol production. First

generation feedstocks usedarecarbohydrates from

sug-arcane, maize or sugar beets (first generation). Second-generation (i.e.non-foodlignocellulosicfeedstocks)and thirdgeneration(i.e.algaeasfeedstock)ethanol produc-tionarescalingup[1].However,first-generationethanol

remains largelydominantonthemarket andrepresents

morethan97%ofthetotalbioethanolproduction[1].In thelastdecade, agreatdealof researchhasbeen dedi-cated to the use of lignocellulosicmaterials and micro-algaeforbioethanolproduction[4,5].Asthesefeedstocks andtheirpre-treatmenttoreleasefermentablesugarsare relativelyexpensive[3],othersubstratesofinteresthave emerged, suchas theorganicfractionof municipalsolid

waste(OFMSW).Specifically,foodandkitchenwasteare

of interest,as theyare relativelyinexpensive, abundant and rich in readily biodegradable carbohydrates [6,7].

Although food waste collection and sorting remain a

challenge in many countries, such feedstocks have the

potentialtoenableamorecostcompetitivebioprocess,if theethanolyieldsandproductconcentrations(titres)per carbohydrate aresufficient.

It ispredicted that2.5 billiontonnes of food wasteare goingtobegeneratedby2025annuallyworldwide[8].In

Europe,North Americaand Oceania,aonethirdof the

foodwastecorrespondstorestaurantorhouseholdwaste

which maycontainanimalby-products [9].Inthatcase, sanitaryregulationsinplacessuchastheEuropeanUnion limitthescopeoffoodwastevalorisation(e.g.no utiliza-tionasfeedforanimals)[10].Thus,onaglobalscalefood wasteisgenerallylandfilled[8]andiftreated,itismostly valorised bycompostingand/orbiogasproduction[8].

Using food waste as feedstock for conversion towards

higher value products such as bioethanol comes with

some challenges since it isgenerally heterogeneous [7]

[11].Theuse ofpureculturesto fermentfoodwasteto ethanol requires sterilizationof the feedstock andlarge inocula(10%–15%v:v)[12].Sterilizationischallengingas eitherfiltering,heatingorUVsterilizationwilladdtothe costsandinvolvetechnologicalchallenges.Forexample, thermalsterilizationleadstolossofsugarsduetoMaillard reactions[13],whilemicrofiltrationoffoodwasteleadsto alossof atleast25% ofthefermentable sugars[12].In

2007, Kleerebezem and van Loosdrecht proposed to

exclude feedstock sterilization and use mixed-culture

biotechnologyoropenmicrobialcommunitiesto

circum-ventthesechallenges[14].Inlinewiththiswork,

Holt-zappleandGrandaformulatedthecarboxylate platform

asawaytoefficientlytransformbiomassintoalcoholslike

ethanol, usingmixed culturesto produce acetateand a

chemicalreductiontoproduceethanolfromacetateusing

hydrogen [15]. Mixed culture-based processes do not

requiresubstratesterilizationand,duetomicrobial

diver-sity, often display high adaptative capacity regarding

substrate quality and environmental conditions. Van

LoosdrechtandKleerebezemcommentedthatno

selec-tive conditions for bioethanol production were

experi-mentally identified in 2007 [14]. Since then, novel

insightshavebeendescribedthatsuggestspecific opera-tional strategies that allow for effective enrichment of

carbohydrate fermentation to ethanol. The aim of this

article is to explore these ecology-based strategies

enabling bioethanol production from carbohydrates to

pave the way for efficient and competitive biorefinery

approaches. Furthermore, a case study of food waste

valorisationtobioethanolandelectricityintheEuropean

Union is proposed as an example of a biorefinery

effi-cientlyutilising theecology-basedstrategiesfor ethanol production.

A

diversity

of

competing

carbohydrate

fermentation

pathways

One of the main challenges of open mixed culture processes

is product selectivity. Since microbial communities are

diverse,manydifferentmetabolicpathwayscanbecarried outinparallel,leading toavarietyoffermentativeproducts. Underanaerobiosis,ethanolisusuallyproducedfrom

car-bohydratesthroughthreetypesoffermentativepathways

(Figure1).Homo-ethanolproduction(pathway1)leadsto thehighesttheoreticalyieldof0.51gethanolgglucose 1andis

commonly observed to be carried out by yeasts or the

bacteriumZymomonasmobilis[16].Thetwootherpathways

lead to the formation of ethanol together with lactate

(heterofermentation,pathway2)orwithacetateand for-mate or hydrogen(acetate and ethanol production,pathway 3). The latter two catabolic pathways leading to a theoretical yield of 0.26gethanolgglucose 1. Heterofermentation is

describedfor lactic acidbacteria [16] while acetateand

ethanol production has been linked to both lactic acid

bacteriaandEnterobacteriaceaespecies,suchasEscherichia orKlebsiellaspecies[16].Thesethreepathwaysarealsoin directcompetitionforcarbohydrateswithotherpathways

yieldingvolatilefattyacids suchasacetateandbutyrate (pathway4)[17],oftenlinkedtoClostridiumspecies[16],or solelylactateproductionbylacticacidbacteria(pathway5) [16].Ethanolcanalsobeconsumedinanaerobic environ-ments aselectron donorforchainelongationofvolatilefatty acids by species such as Clostridium kluyveri(pathway6)[18], oroxidizedanaerobicallyto acetateandH2through

syn-trophicethanoloxidation(pathway7)[19].Inthepresence

of oxygen ethanol can beconverted to acetate through

incompleteethanoloxidation(pathway8),whichhasbeen linkedtoAcetobacterandGluconobacterspecies[16].

The goal of mixed-culture bioethanol production is to

imposetherightselectiveconditionsthatmaximisethe

carbon conversion from carbohydrates towards ethanol,

whileavoidingethanolconsumption.Ideally,

homo-eth-anolproductionismaximised.However,onlyfewstudies

have focused on mixed-culture biotechnology for

bioethanolproductionand suchselectiveconditionsare

notyetwelldocumentedas reviewed recently [20].In thisworkwewillproposeecologicalstrategiesthatleadto

high ethanol production. These strategies are mainly

based on theabundant literature available ondark

fer-mentation [20], the analysis of spontaneous ethanol

fermentation used in traditional beverage production,

physiological studies of known fermentative organisms

andmicrobialcontaminationsofpureculturebioethanol

productionprocesses.

Acid

wash

during

cell

recycling

and

low

pH

fermentation

enhances

ethanol

production

EarlymodellingworkbyRodriguezetal. [21]predicted that low pH (<5.6) should theoretically favour ethanol productioninsteadoforganicacids, sinceacid transport

outsidecellsbecomesenergeticallyveryexpensivewhen

theextracellular pH is close to the pKa value of these

acids(around4.8).Thistrendseemstobeexperimentally verifiedbytheliteratureondarkfermentation(see

Sup-plementarymaterial).Ameta-analysisof150mesophilic

mixed-culture dark fermentations of carbohydrates

showedthatethanolyieldsweresignificantlyhigherwhen

pHwasbelow5whencomparedtopHbetween5and6

(p-value <0.01) or between 6 and 7 (p-value <0.05). However,ethanolyieldsandtitresremainedlowinthese studies (<0.15gEtOHgglucose-eq. 1 and 5gL 1,

respec-tively).This resultislikelydueto theprimaryfocus of these studies(i.e. H2production optimization) and the

use of inoculum heat treatment (usually 90–100C

between15 30min)inmorethan70%ofthecases.This

heattreatmentiscarriedouttoremovenon-sporeforming

bacteria,suchasthehomo-ethanolproducingZymomonas

species and to promote spore forming bacteria such as

Firmicutesspecieswhichproducehighyieldsofhydrogen.

This heat treatment is also lethal to fungi, including

yeasts[22].Microbialcommunitiesinthesestudieswere likelyhighlydominatedbyheat-shockresistantbacteria, suchasClostridiumspecies[23].Clostridiumspeciesdonot 176 Environmentalbiotechnology

harbour the pyruvate decarboxylase gene to go from

pyruvateto acetaldehyde(Figure 1,searchin GenBank

ofNCBI23rdNov2020)andthereforedonotutilisethe

homo-ethanol pathway.

YeastscanthriveatlowpHandusuallydominateduring

spontaneousethanolproducingfermentationswherepH

istypicallybelow4,suchasgrapejuicefermentationfor

wine production [24], barley fermentation for Belgian

sour ales [25,26] and milk fermentation for kefir [27,28].Peinemannetal.recentlycarriedoutanon-sterile

food waste fermentation inoculated with Saccharomyces

cerevisiae[29].WhenS.cerevisiaecompetedagainstthe

native food waste microbial community, the authors

obtained ethanol yields and titres of 0.33gEtOHg glucose-eq. 1and41gL 1,respectively.WhenS.cerevisiaewas

co-inoculated with a lactic acid bacteria-dominated mixed

culture, they observed that ethanol yields and titres

remained competitive at low pH(uncontrolled, around

4), with values of 0.36gEtOHgglucose-eq. 1and 45gL 1,

respectively. However, when pH was regulated at six,

ethanol yields and titres were reduced twofold

(0.15gEtOHgglucose-eq. 1

and 19gL 1, respectively)

while lactate became the dominantproduct. Yeasts are

alsoshowntoremainviableafterprolongedcyclesofacid treatmentatpH2.0[30]andareshowntoretainasimilar

cellviabilityafter120minofacidtreatment[31].Gibson et al. proposed that one of the selective conditions for

yeasts to dominate a microbial community and cause

ethanol productionis avery lowpH biomass-washstep

during cellrecycling[32].Infact, thispractice is

com-monlyusedinbrewingindustriesandbioethanol

produc-ing facilities to inhibit bacterial growth [32,33]. How-ever,lacticacidbacteriacantolerateverylowpH(below 3).Forinstance,Lactobacillusplantarumshowed50–100% cellsurvivalafter30minatpH2[34].Introducinganacid washstepwhenrecyclingcellsafterthefermentationwill promoteyeastsandtherebyincreasetheethanolyieldin fermentation.

As the meta-analysis points out, low pH fermentation

(pH below 5) seems to be beneficial for ethanol

pro-duction. Clostridiumspecies responsibleforacetateand

butyrate production are less competitive in

environ-ments with lowpHastheproductionofkefir and sour

ales points out [25,26,27,28]. At low pH, ethanol

scavengers are anticipated to be completely inhibited,

as chain elongating organisms and syntrophic ethanol

oxidizers cannot cope with such a low pH [18,35].

Enterobacteriaceae will likely be outcompeted at low

pH since they were notobserved inanaerobic glucose

Figure1

Pentoses Hexoses

Pyruvate

Acetyl-CoA

Acetate Butyrate

Ethanol Lactate Acetaldehyde CO2+ H2 Fd Xyl-5-P CO2 CO2 Formate

Glucose consumption pathways

(1) Homo-ethanol production 1 glucose 2 ethanol + 2 CO2

(2) Heterofermentation 1 glucose 1 ethanol + 1 CO₂+ 1 lactate + 1 H+

(3) Acetate and ethanol production 1 glucose 1 ethanol + 2 CO2+ 2 H2+ 1 acetate + 1 H+

(4) Acetate and butyrate production1 glucose 0.67 acetate + 0.67 butyrate + 2.67 H2 + 2 CO2+ 1.33 H+

(5) Homo-lactate production 1 glucose 2 lactate + 2 H+

Ethanol consumption pathways

(6) Chain elongation 1 ethanol + 0.67 acetate à 0.83 butyrate + 0.5 H2O + 0.33 H2+ 0.17 H+

(7) Syntrophic ethanol oxidation 1 ethanol + 1 H₂O 1 acetate + 2 H₂+ 2 H+

(8) Incomplete ethanol oxidation 1 ethanol + 1 O2 1 acetate + 1 H2O+ 1 H+

Ethanol Acetaldehyde O2 H2O H2O H2 Acetate O2 H2O H2O H2 Butyrate Acetate H2O + H2 1 2 3 4 5 6 7 8 X Pathway number

Current Opinion in Biotechnology

Simplifiedmetabolicpathwaysyieldingdominantfermentativeproductsfromcarbohydratesandsimplifiedmetabolicpathwaysconsuming ethanol.Thehomo-ethanolpathway(1)ishighlightedinpurple.Xyl-5-p=xylulose-5-phosphate,Fd=ferredoxin.Basedon[16].Pentosescanbe xylose,arabinoseandsoon.Hexosescanbeglucose,galactose,fructoseandsoon.

fermentingbatchenvironments[36],andneitherwere

identified as bacterial contaminant in bioethanol

pro-duction plants [37]. Furthermore, Zymomonas mobilis

fermentsactivelydownto pH3.5[38],and thusisable

to co-ferment with yeasts in a low pH environment.

Microbialcommunityanalysesofindustrialbio-ethanol

production contaminants confirmed the presence of

Zymomonasspecies inthefermentation stage[37].

Lac-tic acid bacteria are competitive at pH values below

5usingmainlyheterofermentationasdominantpathway

instead of homolactic acid production[36,39].

Insummary,weproposethatmaintainingalowpH(<5)

during fermentation will prevent ethanol consumption

andfavourethanolproducingpathwaysovervolatilefatty acidproducingroutes.Still,theproductionoflacticacid

as side-product may lower the ethanol yield in the

process.

Initial

microaerobiosis

to

stimulate

yeast

growth

Oxygencan beused to inhibit the growth of microbial

groups responsiblefor low ethanol yields. For instance,

Clostridium species carrying out chain elongation and

syntrophic ethanol oxidizersare obligateanaerobes and

strictlyinhibited in thepresenceof oxygen[16].Lactic acid bacteria can tolerate oxygenand Enterobacteriaceae

are facultative anaerobes [16], thus they will not be

inhibited by introducing oxygen. Incomplete ethanol

oxidationcan occurduring oxygenpresence, but atpH

4or lowerthisactivityislikelynotcompetitive[40].

Conversely, oxygen availability favours the growth of

yeasts. A comparative study showed that only 23% of

thetypespeciesfor75yeastgenerawereinfactableto growanaerobically[41].Ofallthesespecies,S.cerevisiae

wasobservedthemostcompetitivefermenterin

anaero-bicconditions[41],showing amof 0.40h 1inbatch.S. cerevisiaeshowedlittledifferenceingrowthratebetween aerobicananaerobicconditions[41].Early researchhas

shown that S. cerevisiae grows only anaerobically when

ergosteroland unsaturatedfattyacidsaresupplied[42].

Metabolic network models suggest that ergosterol and

oleicacid productionrequireoxygen[43]. Recentwork

has shown anaerobic growth of S. cerevisiae is possible

withoutoxygensupplementation[44],thoughcell viabil-ityunderverylowpH(1.5)andhighethanoltitres(100

gL 1)wasonlyretained whenoxygenwasavailablefor

theyeastcells.

However,oxygenavailabilitywilllikelystimulate

reduc-ing sugar consumption through aerobic respiration and

thus competewith ethanol production.Therefore,

oxy-gen presence must be limited to the initial stage of

fermentationtolimitrespirationandgrowthofunwanted

bacterialspecieswhileprovidingacompetitiveadvantage toyeastsand oxygen-tolerantspecies.

Effective

consortia

of

lactic

acid

bacteria

and

yeasts

Lacticacidbacteriaoftensharethesameenvironmental

nicheasyeastssincetheyarebothtoleranttolowpHand

highethanolconcentrations.Forexample,themaximum

growthrateofLactobacillusplantarumwasreducedbyonly

57%whengrowninpresenceof63gL 1ethanol.

Lacto-bacillus heterohiochii only started to be affected at 100 gL 1 of ethanol [45,46]. These species are regularly foundasbacterialcontaminationinindustrialpureculture yeastfermentations[47].Forinstance,Richetal.

investi-gated the bacterial contamination of five commercial

corn-basedethanolproductionprocessesandfoundthat

Lactobacillus species were systematically the dominant

bacterial contaminant [37]. These contaminants have

been reported to reduce ethanol yields in industrial

processesby up to 30% by directcompetition for

sub-strate, production of inhibitory metabolites (e.g. acetic acid),andalsocauseseveralhazardsofbiofilmandfoam formation[39,47].

Industrialpurecultureprocessesaresusceptibleto

con-taminations as compared to selected ecologically stable

microbial communities. Not all lactic acid bacteria are

detrimentalto ethanol production [39] and somemay

evenhelpprotectingthefermentativecommunityagainst

external contaminants (e.g. by secreting bacteriocins).

For instance, Rich et al. carried out tricultures of S.

cerevisiae,Lactobacillusfermentum(detrimentalforethanol production)andathirdspeciesselectedfromover500

lac-tic acid bacteria isolated from industrial fuel ethanol

fermentations[48].Theauthorsfoundthatover300

iso-lates were able to partially or totally restore ethanol

productionto thelevelsobtained bypure culture ofS.

cerevisiae.Otherbacterialspeciesmayalsohelp prevent-ingbiofilmformationbylacticacidbacteria.Forexample,

several Bacillus spp. werefound to excrete compounds

which did not affect yeast growth but limited biofilm

formation bycommonLactobacillusspeciessuch as

Lac-tobacillusfermentum,L.plantarum andL.brevis[49].

Based on these considerations, we propose that stable

microbialcommunitiesoflacticacidbacteriaandethanol

producers,when grownin a selectiveenvironment that

favoursethanolproduction,willlikelyactsynergistically andyieldhigh ethanoltitres andproductivities.

Designing

novel

bioprocesses:

a

research

outlook

Byusing bothpHstrategiesand initialmicroaerobiosis, we propose that it is possible to effectively enrich and

maintain an ethanol-producing microbial community

(Figure2).Afirstoptionwouldbetocreateaneffective

microbial community by inoculating an efficient pure

strain (e.g. S. cerevisiae) in the unsterile fermentation medium,as carried outbyPeinemannet al.[29].The resultingmixedculturewouldthenbereusedinthenext

fermentations without the need for pure cultivation.

Another option would consist in orienting a complex

mixedcultureintoanefficientethanol-producing

consor-tium.Thissecondstrategyhasonly beendemonstrated

twice so farusing low concentration of glucose as

sub-strate.Tamisetal.(papersubmittedforpublication)have

observedtheenrichmentofamixtureofyeasts(Candida

and Pichia) and lactic acid bacteria (Lactobacillus) in a

sequencingbatchgranularbiomassfermentationprocess

operated at pH 4 and 3.5, where the ethanol yield

increased from 0.28 to 0.31gEtOHgglucose 1 with

Figure2

Acid wash(1)

Low pH fermentation (2)

Microaerobiosis (3) μ Clostridium

Ethanol concentration (%)

Batch time

(h)

Grow

th rate (h

)

Current Opinion in Biotechnology

Thethreeoperationalmeasuresandthehypotheticaleffectonthegrowthrateofdifferentgroupsofcarbohydratefermentingmicroorganismsthat areproposedtofavourcarbohydratefermentationtoethanolusinganecology-baseddesign.Yeasts,lacticacidbacteriaandClostridiumare consideredthemostcompetitivefermentativeorganismsinthisenvironmentandarethereforevisualised.Anewfeedisintroducedattheendof theacidwashphase.Growthrates(_m)areaffectedbyallthreeselectiveconditions,whicharepresentduringthelengthoftheboxofthebatch fermentation. Figure3 Unsterile Fermentation Distillation Anaerobic digestion Food waste Pre-treatment Aerobic acid wash _Ethanol Combined heat and power unit

Electricity Heat

Digestate Heat

Current Opinion in Biotechnology

Aschemeforproducingbioethanolfromfoodwasteinenergyefficientprocessbyintegratingtheexcessheatfromthecombinedheatandpower unittodistillation.Processstagesareblue,mainmaterialsstreamsareingreyboxes,energystreamsinorange.

decreasingpH(maximumtitreof3.1gL 1)[50].Darwin et al. have shown a yield of up to 0.38gEtOHgglucose 1

(titreof15.2gL 1)inacontinuousprocessoperatedata pHofaround3.0[51],showinghomo-ethanol

produc-tioncanoutcompeteheterofermentation.

Mixed culture-based bioethanol production processes would reachlowerethanolyieldsandthustitres,whencomparedto

their pure culture equivalent, with yields of about

0.38gEtOHgglucose-eq.

1

[51] instead of 0.45gEtOHg

glu-cose-eq.-1[52].However,theseprocessescanbedesignedin

aneffectivebiorefineryframeworkwhereethanolisoneof the final products. One of such a process could be designed as follows(Figure3):(i)foodwasteispre-treatedforhydrolysis of particulate substrates such as starch and cellulose;(ii) carbohydratesareisfermentedtoethanolbyamixed micro-bialconsortium;(iii)ethanolisrecoveredfromthe fermenta-tionbrothviadistillation; and(iv) the remaining stillbottomis digestedtobiogasthat(v)isconvertedtoheatandelectricity

usingacombinedheatandpower(CHP)plant.

Pre-treat-mentcan bedesignedin acost-competitive way usinga

fungalmashproducedwithasmallpartofthefoodwaste, containingstarchandcellulosehydrolysingenzymes[12].

Alternatively, a more classic approach involving enzyme

additioncanbeconsidered[53,54].Ahighglucose concen-trationbeforefermentationisthencreated,whichbenefits yeastsandthusethanolproduction[51].Theresidualheat fromtheCHPcanbetransferredbacktothepre-treatment anddistillationtocreateacostandenergyefficientprocess. Anexampleofsuchanintegratedprocesshasbeenproposed

by Bouchez and Richard [55], though no clear ethanol

promotingselectionpressureisproposedforthe fermenta-tionstage.

Usingdatafromtheliterature,asimplifiedmassandheat balanceofsuchprocesswascalculatedandcomparedtoa scenariowhereallfoodwastewasdirectlyusedas

anaer-obicdigestionfeedstock (seeFigure 4 and

Supplemen-tarymaterial).Foranaveragefoodwaste(24.54.5%TS)

[7], depending on carbohydrate content (587%TS),

saccharification (about 0.75gglucose-eqgcarbohydrate 1) and

ethanol production yields (0.28 to 0.38ggglucose-eq 1,

ethanol concentrations between 21 and 59gL 1 were

estimated. These concentrations are largely lower than

what is usually attained in first generation ethanol

fer-mentation (around 120gL 1), leading to a low energy

efficiencyduring thedistillationstep (Figure4b). How-ever,heatproductionfromCHP(e.g.110Cstream)can cover thermal requirementsfor distillation in all cases,

thus showing the potential synergy between ethanol

production and anaerobic digestion. It must also be

mentionedthatthedistillationstepwillalsoensurefood

waste hygienisation, a mandatory step in the EU for

householdorrestaurantwastecontaininganimal

by-pro-ducts[10]Assumingthatall 129million tonneskitchen

waste generated in the EU in 2011 [56] was valorised

throughthisprocess,about4milliontonnesofbioethanol

canbeproducedthroughthis process,which represents

86% of the EU market in 2019 [2]. Yet, more detailed

techno-economicstudieswouldberequiredtooptimize

theprocess configurations.Alternatively,in caseswhere 180 Environmentalbiotechnology Figure4 (a) 0 50 100 150 200 250 300 Anaerobic dig estion

Low Ethanol_{Mean Ethanol}High Ethanol

Biodegradab le COD balance (kg COD .t FW -1) Ethanol Methane Digestate 0 100 200 300 Anaerobic dig estion

Low Ethanol_{Mean Ethanol}High Ethanol

Heat pr oduction or consumption (kWh.t FW -1) Distillation Saccharification CHP Production Consumption (b) Hygienisation

Current Opinion in Biotechnology

Mass(a)andheat(b)balancesofanethanolfoodwastefermentation processcoupledwithanaerobicdigestion.TheAnaerobicdigestion scenarioisprovidedasreferenceandcorrespondstoadirect mesophilicconversionoffoodwasteintomethane,followedbyheat andelectricityproduction.Low,MeanandHighethanolcorrespondto pessimistic,average,andoptimisticscenarios,respectively,for ethanolproductioncoupledtoanaerobicdigestion.Thesethree scenariosfollowtheschemepresentedinFigure3,saccharification beingusedaspre-treatment.Differencesbetweenthethreescenarios lieinthefoodwasteconcentration[7],carbohydratecontent[7], saccharification[53,54]andethanol[51]productionyields.The detailedcalculationisprovidedinSupplementarymaterial.

thedistillationstepistooexpensivedueto lowethanol titres,fermentationbrothcontainingmainlylactate, ace-tate andethanolcouldalsorepresentanidealfeedstock forcaproicacidproductionthroughchainelongation[57].

In conclusion, the main challenge at this stage is to

experimentallydemonstratetheselectivity,productivity,

and robustnessofthemixedculturebioethanol

produc-tionprocess.Twoenrichmentstudieshavedemonstrated

arelativelyhigh yieldbut withlowtitres usingglucose.

Opportunitiesfor futuremixedcultureexperimentation

lieinincreasingtheglucoseconcentration,andthusthe

ethanol titre. Secondly, the effectiveness of all three

describedstrategiesandthesynergyoftheircombination

should beassessed.Thirdly,highyieldethanol

produc-tion needs to be demonstrated using(pre-treated) food

waste. Inadditionto thesekeyexperiments,other

rele-vant ecological parameters deserve to be carefully

explored, suchas fermentationtemperature,theprotein

content or type of fermentablecarbohydrate [51].We

believe that a solid understanding of the ecology of

ethanol production will notonly be usefulfor

develop-ment of mixed culture biotechnology-based processes,

but also strengthens pure culture-based fermentation

processes, by identifying conditions that intrinsically

promote ethanolproduction.

Conflict

of

interest

statement

Nothing declared.

Acknowledgements

J.L.Romboutswishestothanktheearlysupportofthisworkthroughthe SIAMgravitationgrantfromtheNetherlandsOrganisationforScientific Research(NWO)underthenumber024.002.002.Heisalsogratefulforthe discussionswithProf.Dr.G.J.Witkamponthetopicofanintegrated bioethanolproductionprocessesusingmicrobialcommunities.Hefurther wishestothankJ.J.deIonghandB.vandeBergfortheireffortin co-evaluatingthebusinesscaseofbioethanolandelectricityproductionfrom foodwaste(swill)intheRotterdamarea(TheNetherlands).

Appendix

A.

Supplementary

data

Supplementary material related to this article can be

found, in the online version, at doi:https://doi.org/10. 1016/j.copbio.2021.01.016.

References

and

recommended

reading

Papersofparticularinterest,publishedwithintheperiodofreview, havebeenhighlightedas:

 ofspecialinterest ofoutstandinginterest 1.

 Sharmaof2GbioethanolB,Larrocheproduction.C,DussapCG:BioresourComprehensiveTechnol2020,assessment 313:123630

Acomprehensivestudyevaluatingthecurrentstatusoflignocellulosic bioethanolproductionanditschallenges.

2. FlachB,LieberzS,BollaS:EU-28BiofuelsAnnualEUBiofuels Annual2019.2019.

3. Enguı´danos M,SoriaA,ChristidisP,KavalovB:Techno-Economic AnalysisofBio-AlcoholProductionintheEU:AShortSummaryfor DecisionMakers.2002.

4. ZabedHM,AkterS,YunJ,ZhangG,AwadFN,QiX,SahuJN: Recentadvancesinbiologicalpretreatmentofmicroalgaeand lignocellulosicbiomassforbiofuelproduction.RenewSustain EnergyRev2019,105:105-128.

5. TuWC,HallettJP:Recentadvancesinthepretreatmentof lignocellulosicbiomass.CurrOpinGreenSustainChem2019, 20:11-17.

6. HafidHS,RahmanNAA,ShahUKM,BaharuddinAS,AriffAB: Feasibilityofusingkitchenwasteasfuturesubstratefor bioethanolproduction:areview.RenewSustainEnergyRev 2017,74:671-686.

7. Capson-TojoG,RouezM,CrestM,SteyerJP,Delgene`sJP, Escudie´ R:Foodwastevalorizationviaanaerobicprocesses:a review.RevEnvironSciBiotechnol2016,15:499-547.

8. ParthibaKarthikeyanO,TrablyE,MehariyaS,BernetN,WongJW, CarrereH:Pretreatmentoffoodwasteformethaneand hydrogenrecovery:areview.BioresourTechnol2017,249 :1025-1039http://dx.doi.org/10.1016/j.biortech.2017.09.105.

9. GustavssonJ,CederbergdC,SonessonU,vanOtterdijkR, MeybeckA:Globalfoodlossesandfoodwaste.International CongressSAVEFOOD!.FoodandAgriculturalOrganizationofthe UnitedNations;2011:29.

10. EuropeanParliament,CounciloftheEuropeanUnion:Regulation (EC)No1069/2009oftheEuropeanParliamentandoftheCouncil of21October2009LayingDownHealthRulesasRegardsAnimal By-ProductsandDerivedProductsNotIntendedforHuman ConsumptionandRepealing.EuropeanUnion;2009.

11. ChenT,JinY,LiuF,MengX,LiH,NieY:Effectofhydrothermal treatmentonthelevelsofselectedindigenousmicrobesin foodwaste.JEnvironManage2012,106:17-21.

12. MaY,CaiW,LiuY:Anintegratedengineeringsystemfor maximizingbioenergyproductionfromfoodwaste.Appl Energy2017,206:83-89.

13. LiY,JinY,LiJ,LiH,YuZ:Effectsofthermalpretreatmentonthe biomethaneyieldandhydrolysisrateofkitchenwaste.Appl Energy2016,172:47-58.

14. KleerebezemR,vanLoosdrechtMC:Mixedculture

biotechnologyforbioenergyproduction.CurrOpinBiotechnol 2007,18:207-212.

15. HoltzappleMT,GrandaCB:Carboxylateplatform:theMixAlco processpart1:comparisonofthreebiomassconversion platforms.ApplBiochemBiotechnol2009,156.

16. MadiganMT,MartinkoJM:BrockBiologyofMicroorganisms.edn 11.PearsonPrenticeHall;2006.

17. BuckelW,ThauerRK:Energyconservationviaelectron bifurcatingferredoxinreductionandproton/Na+translocating ferredoxinoxidation.BiochimBiophysActa2013,1827:94-113.

18. CavalcanteWdeA,Leita˜oRC,GehringTA,AngenentLT, SantaellaST:Anaerobicfermentationforn-caproicacid production:areview.ProcessBiochem2017,54:106-119.

19. ShenL,ZhaoQ,WuX,LiX,LiQ,WangY:Interspecieselectron transferinsyntrophicmethanogenicconsortia:fromcultures tobioreactors.RenewSustainEnergyRev2016,54:1358-1367.

20.

 Moscovizbiorefinery:R,state-of-the-artTrablyE,BernetonN,Carre`retheproductionH:Theenvironmentalofhydrogen andvalue-addedbiomoleculesinmixed-culturefermentation. GreenChem2018,20:3159-3179

Acomprehensiveandextensivemeta-analysisoffermentative enrich-mentsperformedwithsyntheticorheterogenousfeedstocks(suchas foodwaste).Thismeta-analysiscomprehendsstudiespublishedinthis fieldupto2018.Thereareafewimportantobservationstoaddress.(I) FoodwastecontainsarelativelyhighamountofCODcomparedtoother agri-food residual streams. (II)Afterclustering thedatabase, ethanol productionco-occurswithacetate,butyrateandhydrogenproduction, thusthesepathwaysareincompetitioninthiscluster,withlittlelactate production.

21. RodriguezJ,KleerebezemR,LemaJM,VanLoosdrechtMCM: Modelingproductformationinanaerobicmixedculture fermentations.BiotechnolBioeng2006,93:592-606.

22. MontanariC,TabanelliG,ZamagnaI,BarbieriF,GardiniA, PonzettoM,RedaelliE,GardiniF:Modelingofyeastthermal resistanceandoptimizationofthepasteurizationtreatment appliedtosoftdrinks.IntJFoodMicrobiol2019,301:1-8.

23. CabrolL,MaroneA,Tapia-VenegasE,SteyerJP,Ruiz-FilippiG, TrablyE:Microbialecologyoffermentativehydrogen producingbioprocesses:usefulinsightsfordrivingthe ecosystemfunction.FEMSMicrobiolRev2017,41:158-181.

24. VarelaC,BornemanAR:Yeastsfoundinvineyardsand wineries.Yeast2017,34:111-128http://dx.doi.org/10.1002/ yea.3219.

25. SnauwaertI,RoelsSP,VanNieuwerburghF,VanLandschootA, DeVuystL,VandammeP:Microbialdiversityandmetabolite compositionofBelgianred-brownacidicales.IntJFood Microbiol2016,221:1-11.

26.

 SpitaelsLandschootF,WiemeA,DeVuystAD,JanssensL,VandammeM,AertsP:TheM,microbialDanielH,Vandiversity oftraditionalspontaneouslyfermentedlambicbeer.PLoSOne 2014,9:1-13

Aculture-dependentandindependentstudyofthemicrobialcommunity presentinLambicbeers,wherebothyeastandbacterialcommunitiesare assessed.Arecommendedreadabouttheecologyofthesespontaneous fermentedbeveragesandwhichmicrobialcommunitydetectionmethod yields which information and how to use these different detection methods.

27. MarshAJ,SullivanOO,HillC,RossRP,CotterPD: Sequencing-basedanalysisofthebacterialandfungalcompositionofkefir grainsandmilksfrommultiplesources.PLoSOne2013,8: e69371.

28. GarofaloC,OsimaniA,MilanoviV,AquilantiL,DeFilippisF, StellatoG,DiS,TurchettiB,BuzziniP,ErcoliniDetal.:Bacteria andyeastmicrobiotainmilkkefirgrainsfromdifferentItalian regions.FoodMicrobiol2015,49:123-133.

29.

 PeinemannfermentationJC,ofRheefoodC,wasteShinwithSG,PleissnerindigenousD:Non-sterileconsortiumand yeast–effectsonmicrobialcommunityandproduct spectrum.BioresourTechnol2020,306:123175

Arecentpaperwhichdemonstratesthecompetitivenessof Saccharo-myces cerevisiae infermenting glucoseinunsterilised foodwasteto ethanolathighyieldsandtitres.Amylasewasaddedatthestartofthe fermentationtoacceleratethehydrolysisofstarchtoglucose.

30. BassiAPG,daSilvaJCG,ReisVR,Ceccato-AntoniniSR:Effects ofsingleandcombinedcelltreatmentsbasedonlowpHand highconcentrationsofethanolonthegrowthand

fermentationofDekkerabruxellensisandSaccharomyces cerevisiae.WorldJMicrobiolBiotechnol2013,29:1661-1676.

31. SimpsonWJ,HammondJRM:Theresponseofbrewingyeasts toacidwashing.JInstBrew1989,95:347-354.

32.

 GibsonYeastresponsesBR,LawrencetostressesSJ,LeclaireassociatedJPR,PowellwithCD,industrialSmartKA: breweryhandling.FEMSMicrobiolRev2007,31:535-569

Anoutlineofenvironmentalpressures whichyeaststrainsexperience duringindustrialbioethanolproduction.

33.

 Della-BiancaWhatdoweBE,knowBassoaboutTO,theStambukyeastBU,strainsBassofromLC,theGombertBrazilianAK: fuelethanolindustry? ApplMicrobiolBiotechnol2013, 97:979-991

Areview describingthepersistenceofwildyeaststrainsinindustrial sucarcane-basedbiotethanolfermentationsinBrazil,showingthat com-munitiesinsuchsemiasepcticfermentationsarevariable,butstillleadto competitiveethanolyields.

34. ZottaT,ParenteE,RicciardiA:Viabilitystaininganddetectionof metabolicactivityofsourdoughlacticacidbacteriaunder stressconditions.WorldJMicrobiolBiotechnol2009, 25:1119-1124.

35. YuanH,ZhuN:Progressininhibitionmechanismsandprocess controlofintermediatesandby-productsinsewagesludge anaerobicdigestion.RenewSustainEnergyRev2016, 58:429-438.

36.

 RomboutsKleerebezemJL,R,KranendonkvanLoosdrechtEMM,MCM:RegueiraSelectingA,WeissbrodtforlacticDG,acid producingandutilisingbacteriainanaerobicenrichment

cultures.BiotechnolBioeng2020,117:1281-1293http://dx.doi. org/10.1002/bit.27301

Arecentpaperdemonstratinghowheterofermentativelacticacid bac-teria can be enriched for usingsequential batch reactor cultivation, inoculatingwithanaerobicdigestorsludge.Resourceallocationisused toexplainthecompetitiveadvantageoflacticacidbacteriaasopposedto acetateandbutyrateproducingorganisms,thusrationalizingthedifferent metabolic pathways when enriching with mineral or supplemented medium.

37. RichJO,AndersonAM,LeathersTD,BischoffKM,LiuS,SkoryCD: Microbialcontaminationofcommercialcorn-basedfuel ethanolfermentations.BioresourTechnolReports2020, 11:100433.

38. WangX,HeQ,YangY,WangJ,HaningK,HuY,WuB,HeM, ZhangY,BaoJetal.:Advancesandprospectsinmetabolic engineeringof_{Zymomonasmobilis.}MetabEng2018,50:57-73.

39.

 RichBiofilmJO,formationLeathersTD,andBischoffethanolKM,inhibitionAndersonbybacterialAM,NunnallyMS: contaminantsofbiofuelfermentation.BioresourTechnol2015, 196:347-354

Anextensive ecological studyevaluatingthemicrobialcommunityof corn-basedethanolproductionfacilities.92%ofthe768bacteriaisolated from these facilities were shown to beLactobacillus species. These isolateswereusedtotesttheinfluencewhenco-culturinganisolatewith yeastonthebio-ethanolyield.Acetateproductionappearedtoberelated to the inhibition of bioethanol production, which fits in the general consensusthataceticacidatlowpHinhibitsSaccharomycesspecies.

40. ChenX-H,LouW-Y,ZongM-H,SmithTJ:Optimizationofculture conditionstoproducehighyieldsofactiveAcetobactersp. CCTCCM209061cellsforanti-Prelogreductionofprochiral ketones.BMCBiotechnol2011,11:110.

41. VisserW,ScheffersWA,vanderVegte-BatenburgWH,van DijkenJP:Oxygenrequirementsofyeasts.ApplEnviron Microbiol1990,56:3785-3792.

42. AndreasenAA,StierTJB:Anaerobicnutritionof Saccharomycescerevisiae.II.Unsaturatedfattyand requirementforgrowthinadefinedmedium.JCellComp Physiol1954,43:271-281.

43. O¨ sterlundT,NookaewI,BordelS,NielsenJ:Mapping condition-dependentregulationofmetabolisminyeastthrough genome-scalemodeling.BMCSystBiol2013,7:36.

44. daCostaBLV,RaghavendranV,FrancoLFM,deBrittoChaves FilhoA,YoshinagaMY,MiyamotoS,BassoTO,GombertAK: Foreverpantingandforevergrowing:physiologyof Saccharomycescerevisiaeatextremelylowoxygen availabilityintheabsenceofergosterolandunsaturatedfatty acids.FEMSYeastRes2019,19.

45. vanBokhorst-vandeVeenH,AbeeT,TempelaarsM,BronPA, KleerebezemM,MarcoML:Short-andlong-termadaptationto ethanolstressanditscross-protectiveconsequencesin Lactobacillusplantarum.ApplEnvironMicrobiol2011, 77:5247-5256.

46. IngramLO:Ethanoltoleranceinbacteria.CritRevBiotechnol 1989,9:305-319.

47. Brexo´ RP,AnaASS:Impactandsignificanceofmicrobial contaminationduringfermentationforbioethanolproduction. RenewSustainEnergyRev2017,73:423-434.

48.

 RichResolvingJO,BischoffbacterialKM,contaminationLeathersTD,AndersonoffuelAM,ethanolLiuS,SkoryCD: fermentationswithbeneficialbacteria–analternativeto antibiotictreatment.BioresourTechnol2018,247:357-362

Thisrecentpaperoutlinesanalternativestrategytoantibiotictreatmentto controlbacterialpopulationsinyeastfermentations,throughinoculating beneficialbacteria which support yeastpopulation and high ethanol yieldsandtitres.

49. SaundersLP,BischoffKM,BowmanMJ,LeathersTD:Inhibition of_{Lactobacillus}biofilmgrowthinfuelethanolfermentations by_Bacillus.BioresourTechnol2019,272:156-161.

50. TamisJ,JoosseB,deLeeuwK,KleerebezemR:Flippingthe switch:usingpHtocontroltheproductspectruminanaerobic fermentationbetweenVFAandethanol.BiotechnolBioeng 2021.underreview.

51.

 Darwin,productionCord-RuwischfromsugarR,andCharlesstarchW:wastesEthanolbyandanaerobiclacticacid acidification.EngLifeSci2018,18:635-642

A simpleenrichment study which demonstrates the highest yield of ethanolproductionusingmicrobialcommunitiesknowntotheauthors (&0.7gCODgCOD 1sugar/starch).Greatstartingpointforfutureworkto

explore theniche ofhomoethanol producingcommunitiesand their phylogeny,genomesandproteomes.

52. HuangH,QureshiN,ChenM-H,LiuW,SinghV:Ethanol productionfromfoodwasteathighsolidscontentwith vacuumrecoverytechnology.JAgricFoodChem2015, 63:2760-2766.

53. LiP,ZengY,XieY,LiX,KangY,WangY,XieT,ZhangY:Effectof pretreatmentontheenzymatichydrolysisofkitchenwastefor xanthanproduction.BioresourTechnol2017,223:84-90.

54. LiX,MettuS,MartinGJO,AshokkumarM,LinCSK:Ultrasonic pretreatmentoffoodwastetoaccelerateenzymatic hydrolysisforglucoseproduction.UltrasonSonochem2019, 53:77-82.

55. BouchezT,RichardC:ProcessforProducingEthanolfrom OrganicWasteandIsntallationforCarryingOutSaidMethod.2013.

56. CaldeiraC,DeLaurentiisV,CorradoS,vanHolsteijnF,SalaS: Quantificationoffoodwasteperproductgroupalongthefood supplychainintheEuropeanUnion:amassflowanalysis. ResourConservRecycl2019,149:479-488.

57. DalySE,UsackJG,HarroffLA,BoothJG,KelemanMP, AngenentLT:Systematicanalysisoffactorsthataffect food-wastestorage:towardmaximizinglactateaccumulationfor resourcerecovery.ACSSustainChemEng2020, 8:13934-13944.