Lipid-based biofuel production from wastewater

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Lipid-based biofuel production from wastewater

^§

Emilie EL Muller, Abdul R Sheik and Paul Wilmes

Increasingworldpopulation,urbanizationandindustrialization aredrivingglobalincreasesinwastewaterproduction.

Wastewatercomprisessignificantamountsofchemicalenergy primarilyintheformoforganicmolecules(inparticularlipids), whicharecurrentlynotbeingrecoveredcomprehensively.

Withinbiologicalwastewatertreatment(BWWT)systems, specializedmicroorganismsassimilateandstorelipids anaerobically.Theseintracellularstoresrepresentinteresting feedstocksforbiofuelsynthesis.Here,wereviewourcurrent understandingofthegeneticandfunctionalbasisforbacterial lipidaccumulationandprocessing,andrelatethistolipid accumulatingbacterialpopulationswhichoccurnaturallyin BWWTplants.Agrandchallengeformicrobialecologistsand engineersnowliesintranslatingthisknowledgeintothedesign ofnewBWWTprocessesforthecomprehensiverecoveryof lipidsfromwastewaterstreamsandtheirsubsequent conversionintobiofuel.

Addresses

Eco-SystemsBiologyGroup,LuxembourgCentreforSystems Biomedicine,UniversityofLuxembourg,CampusBelval,7avenuedes Hauts-Fourneaux,Esch-sur-AlzetteL-4362,Luxembourg

Correspondingauthor:Wilmes,Paul(paul.wilmes@uni.lu, paul.wilmes@icloud.com)

CurrentOpinioninBiotechnology2014,30:9–16

ThisreviewcomesfromathemedissueonChemicalbiotechnology EditedbyCurtRFischerandSteffenSchaffer

AvailableonlineXXX

0958-1669/$–seefrontmatter,#2014TheAuthors.Publishedby ElsevierLtd.Allrightsreserved.

http://dx.doi.org/10.1016/j.copbio.2014.03.007

Introduction

Microorganismspredominate Earth’s biota. Theyencode themajorityofgeneticdiversityontheplanetandunderpin nearlyallbiogeochemicalprocesses.Advancesinmolecular methodsarenowallowingustouncovertheextensivegene poolcontainedwithmicrobialecosystems,andthisresource willultimatelydrive andsustainoursocietalandenviron- mental needs. Among the renewable commodities of immediate interest which can be produced by microbes in significant quantities are biofuels. It is projected that

microbially produced biofuels will eventually replace petroleum-basedfuelsas wellasfirst-generationandsec- ond-generationbiofuels[1].First-generationbiofuelssyn- thesizedfromedibleplantmaterialandsecond-generation biofuelsderivedfromnon-foodvegetablefeedstocks(e.g.

lignocellulosicmaterial)areoftenconsideredunsustainable due totheircompetitionwitharable landandtheirinsig- nificantimpactintermsofreducinganthropogenicgreen- house gas emissions[1]. Nonetheless, second-generation biofuels may have asignificantimpact in specific spatio- temporalcontextsinwhichenhancedfeedstockavailabil- itymayforexampleturnthetideintheirfavor[2].Third- generationbiofuelsthatarebasedonoleaginousmaterial derivedfrommicroorganisms(microalgae,yeasts,bacteria) capableof growing(photo-)heterotrophicallyonorganic wasteorphototrophicallyoninorganiccarbon,donotshare theselimitations[1].Althoughresearchanddevelopment has mainly focused on algae-derived and yeast-derived lipids (for recentreviewssee[3–5]),certainwastewater- borne microorganisms exhibit exciting phenotypictraits whichmaybeharnessedforbiofuelproduction,especially sinceincreasingworldpopulation,urbanizationandindustrialization are leading to an increasing production of wastewater globally. In particular, oleaginous biomass from biological wastewater treatment (BWWT) plants representsapotentiallyimportantfeedstockfor thepro- ductionofthird-generationliquidbiofuelssuchasbiodie- sel(fattyacidalkylester)aswellasotherhigh-valueorganic andinorganicresources[6,7].

Current BWWT systems are based on the activated sludge processandthisprimarilyreliesonthemicrobial oxidation of organic molecules to CO2. Only a small proportion of the wastewaterorganic fraction is assimi- lated into the sludge biomass. Subsequent anaerobic digestion of primary (mainly floating solids and greases collected from primary clarifier tanks) and secondary (activated sludge) sludge to biogas only allows limited chemicalenergyrecovery.

Inmunicipalwastewater,lipidscanrepresentinexcessof 40%ofthetotalorganicfraction[8],withthevastmajority consistingoftriacylglycerols(TAGs)andaminorpartof free long-chain fatty acids [9]. In order to potentially facilitate significant chemical energy recovery from wastewater, for example through direct biodiesel syn- thesisfromlipid-richbiomass,specializedbacteriawhich occur within BWWT plants and accumulate copious amountsof intracellularlipidsmaybeexploited.These oleaginous microorganisms either assimilate lipids from the wastewater or synthesize them de novo from other carbon sources,andstorethemintracellularlyas neutral

§Thisisanopen-access articledistributedunderthetermsofthe CreativeCommonsAttribution-NonCommercial-NoDerivativeWorks License, whichpermits non-commercialuse,distribution,andrepro- duction inanymedium,providedtheoriginalauthorandsource are credited.

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lipids,forexample,TAGs,waxesters (WEs)or polyhydroxyalkanoates(PHAs)[10].

Preliminary studies have demonstrated that lipid-rich bacterial biomass from biological wastewater treatment plants can be directly used for biofuel synthesis. The predominantfattyacidsinwastewaterareintherangeof C14–C18whichareidealchainlengthsfortheproduction of biodiesel [11,12]. Mondala et al. [11] were able to producefattyacidmethylesters(FAMEs)—abiodiesel form—fromprimaryandsecondarysludgebychemical transesterification. On the basis of their method, they estimatedayieldof10%FAMEsperdryweightofsludge resultingin an estimated production cost of $ 3.23 per gallon (approximately 0.20 s per liter), which is lower thancurrentconsumerpricesforpetroleum-baseddiesel and alternative biodiesels [11]. Furthermore, they esti- matedthat theintegrationof dedicated lipid extraction and transesterification processes in 50% of all existing municipal BWWT plants in the United States could produceanequivalentof0.5%oftheUSyearlypetroleum diesel demand [12]. However, the future utilization of oleaginous biomass feedstocks could dramatically improvetheyieldand cost-effectivenessofwastewater- derived biodiesel production. Therefore, together with projectedimprovementsinengineefficiency,wastewater biodieselcould becomeanimportant partofany future renewableenergy portfolio.

Knowing and understanding the function of the genes involvedinmicrobiallipidassimilation,accumulationand processingarecriticalconsiderationsfortheoptimization andlarge-scaleproductionofbiofuelsfromwastewateras well as from other complex lipid-rich feedstocks. The presentreview discussesour currentknowledgeofbac- teriallipidmetabolisminrelationtorepresentativelipid accumulating organisms in BWWT plants, that is the long-chainfattyacidaccumulatingorganism‘Candidatus Microthrixparvicella’ (henceforthreferredtoas M.parvicella)andthePHAaccumulatingorganism‘Candidatus Accumulibacterphosphatis’(henceforthreferredtoasA.

phosphatis), as well as in relation to metagenomic sequencedataobtainedfromBWWTplants.Inaddition, lipidic wastewater-derived biofuels such as bio-oils and hydrocarbonsproducedusingtheFischer-Tropschprocess arealsosuccinctlypresented.Finally,a‘wastewaterbiorefinerycolumn’conceptforfuturerecoveryofenergy-rich lipidsfromwastewaterisalsobrieflydiscussed.

Bacteriallipid accumulation

Triacylglycerolsandwaxesters

Inbacteria,thelaststepinthebiosynthesisofTAGsand WEsiscatalyzedbythesameenzyme,waxestersynthase/

acyl-CoA:diacylgycerolacyltransferase(WS/DGAT),which assuresthetransferofanacyl-CoAontoafattyalcoholto produceaWEorontoadiacylglycerol(DAG)toyieldaTAG [13] (Figure 1). Recent studies have demonstrated the

general broad substrate specificity of the WS/DGAT [14,15,16]andhavehighlightedspecificaminoacidresi- dueswhich determine itssubstrate spectrum [17,18,19].

Theanalysisofarecentlypublishedactivatedsludgemeta- genome [20] and the genome sequence of M. parvicella strainBio17-1[21]highlights the importance of the WS/

DGATgeneinactivatedsludgebiomass.Interestingly,M.

parvicellastrainBio17-1 [21]encodes 4 homologsof WS/

DGAT (Figure 1) suggestingthat thisenzymatic redun- dancyplaysaroleinthisorganism’sabilitytoaccumulate largeamountsoflipidsintracellularly.

Ourunderstandingofthestructureanddynamicsoflipid bodiesformedduringTAGorWEaccumulationhasbeen greatly improved by recent work. The current model posits that WS/DGAT docks to the cytoplasmic mem- brane,whereitcatalyzesthesynthesisoflipidmicrodro- plets. These microdroplets are then released into the cytoplasm after conglomeration and surrounded by a phospholipid monolayer decorated with proteins [22].

Therecentisolationoflipidbodieshasledtotheidenti- ficationof many associated proteins, including ahighly abundant protein referred to as TadA, MLDS or LPD06283[23,24,25].Thisproteinisinvolvedin con- trollingthesizeof lipiddroplets [23,24,25]and therefore potentially represents a key gene which regulates overall lipid accumulation phenotypes. The search for Rhodococcus’ TadA (HM625859.1) or LPD06283 (4218150) homologs in M. parvicella did not result in any significant hits suggesting that the discovery of additionalsequences of TadAhomologsexperimentally validatedinphylogeneticallyunrelatedorganismswillbe necessaryto unravelthedistributionof thisgeneinthe bacterialdomain.Additionally,themechanismof action ofthisproteinneedstobeclarifiedinordertoexploitits biotechnologicalapplication.

Ourlatestunderstanding ofbacteriallipidaccumulation isalreadybeingusedtobioengineerlaboratorystrainsto accumulategreaterquantitiesofTAGsorWEs(forrecent reviews see [26,27]). Strikingly, the complete list of bacterial genes involved in WE and TAG biosynthesis wasnotknownuntilquiterecently.Thesegenesinclude thefatty aldehyde reductasenecessaryfor fatty alcohol synthesisbeforeWEsynthesis[28–30]andthebacterial phosphatidicacidphosphatase(PAP)[31]whichcatalyzes the biosynthesis of DAG, a first dedicated reaction for thesynthesisofTAGsorWEsratherthanphospholipids in bacteria (Figure 1). Moreover, other studies have suggested the existence of an alternative TAG biosyn- thesispathwayinvolvingadifferentenzymetoWS/DGAT [32,33]butdetailssofarremainelusive.

Bacterial populations known to accumulate TAGs and WEs(mainly actinobacterialgenera such as Mycobacter- ium, Rhodococcus, Nocardia or Microthrix, but also from othertaxasuchasAcinetobacter)arepresentinthebiomass

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of activated sludge [34,35]. These organisms typically assimilate long-chain fatty acids under anaerobic con- ditions, can become very abundant [36] and are often discernibleasfoamonthesurfaceofanoxicBWWTtanks

[34].Therefore,wesuggestthattherecuperationofthis lipid-richbiomassbysimplyskimmingthesurfacecould providethefeedstockforsubsequenthigh-yieldbiodiesel production.

Figure1

Acyl-ACP or Acyl-CoA

Lyso-phosphatidic

acid Phosphatidic acid DAG

Fatty alcohol Fatty aldehyde

(Fatty) acyl-CoA

Acetoacetyl-CoA

Enzyme

# Enzyme name

glycerol-3-phosphate acyltransferase (GPAT)

lyso-phosphatidic acid acyltransferase (LPAT)

phosphatidic acid phosphatase (PAP)

fatty acid-CoA synthase fatty acyl-CoA reductase (FAR) fatty aldehyde reductase (FALDR)

acetoacetyl CoA reductase (AAR) PHB polymerase β-ketothiolase (BKD)

wax ester synthase/

acyl-CoA:diacylgycerol acyltransferase (WS/DGAT)

Metagenomic EBPR Aalborg

Candidatus Microthrix parvicella

Candidatus Accumulibacter

phosphatis 2

2

2 3 4

6 1 1

1 1 1 9

25

13

4 28 16

0^b 0^b

nd^a

nd^a 19^c 143

135 18

5

6 0^e 0^e

0^b 0^b

Acetyl-CoA Acetyl-

CoA CoA

3-hydroxy-

butyryl-CoA PHB

TAG

WE Phospholipid

FA

G3P

1 2 3 4

7 4

10 6

6

8 9

1

2 3

4

5 6 7 8 9 10

5 ACP or CoA

Acyl- ACP

Acyl- CoA

ACP Pi CoA

CoA

CoA NADP⁺ NADPH

NADP⁺ NADPH

CoA + ATP

PPi +AMP

NAD(P)⁺ NAD(P)H

H₂O

PHB_n-1

Current Opinion in Biotechnology

Themainbacterialmetabolicpathwaysinvolvedinlipidaccumulation.Thefrequenciesofspecifichomologousgenesarereportedinthe correspondingtablefor(i)ametagenomicdatasetgeneratedfromthebiomassofaBWWTplantoperatedforenhancedbiologicalphosphorus removal(EBPR)[20],(ii)thegenomeofthelipidaccumulatingfilamentousactinobacteriumM.parvicellaBio17-1[21]and(iii)thepolyphosphate accumulatingbetaproteobacteriumA.phosphatisUW-1[63],basedontheirannotation(fordetailsonannotations,seeSupplementaryTables).

Abbreviations:ACP:acylcarrierprotein;DAG:diacylglycerol;FA:fattyacid;G3P:glycerol-3-phosphate;PHB:polyhydroxybutyrate;Pi:inorganic phosphate;PPi:pyrophosphate;TAG:triacylglycerol;WE:waxester.^anotdetermined;^bnotfoundbyBLASTsearchofnucleotidesequencesofthe3 characterizedPAPsequences(StreptomycescoelicolorA3(2)genesSCO1102andSCO1753[31]aswellasgeneEF535727.1fromGeobacillustoebii strainT85[64])orFALDRofMarinobacteraquaeoleiVT8(accessionnumberyp_959486[30]);^cannotatedasdiacylglycerolacyltransferaseonly.

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In activated sludge-based BWWT processes, specific TAG accumulating organisms excrete extracellular lipases, catalyzing the lipid hydrolysis present in the surrounding wastewater before its assimilation (Figure 1) [37]. M. parvicella possesses 8 lipases and due to the complexity of lipid mixtures in wastewater and extensiveinter-organismalcompetition, these must havebroadsubstratespecificityandhighenzymaticeffi- ciency.Theseenzymecharacteristics shouldprovevery usefulfortransesterificationreactionsinvolvedinbiodie- selproductionusingarangeofdifferentlipidfeedstocks (seealsosection‘Fromoleaginousbiomasstobiofuelsvia biocatalysis’).

Polyhydroxyalkanoates

Themostcommonbacteriallipidinclusionsareso-called carbonosomesconsisting ofPHAs. PHAshave typically been promoted as replacement for petroleum-derived plastics [38]. However, an alternative use of PHAs includestheproduction byesterification ofhydroxyalk- anoatemethylesters (HAMEs),whichcanserveasbio- fuelsorasfueladditives [39,40](seealsoBox1).

Polyhydroxybutyrate(PHB) typicallyis themost abundant PHA. The genes encoding the key enzymes involved in the synthesis of the monomeric precursor ofPHBas wellas itspolymerizationanddepolymeriza- tion are well established [10] (Figure 1). Our current understanding based on work in model organisms suggeststhatPHBgranule formation followsa‘scaffold model’inwhichPHBsynthases attachfirstto ascaffold molecule to produce the initiation complex for PHA biosynthesis.These scaffold molecules likelyare DNA andPHAgranule-associateproteinPhaM,whichalsoaids in the dimerizationof PHAsynthases [41,42]. Other proteinscoatingPHAgranuleshavealsobeenidentified

andrecentlyreviewed[43].Duetotheindustrialinterest inPHAfortheproductionofbioplastics,extensivework hasbeen carried out to enhance the productionof this polymer through for example translating it into heter- ologouscontexts[44].

InthebacterialcommunitiesunderpinningBWWT,PHA accumulationcanbeveryrapidandpronounced(e.g.up to77%ofcelldryweightinfivehours[45]).Twomajor categories of organisms,namely phosphorusaccumulat- ing organisms and glycogen accumulating organisms accumulatePHAwhenexposedtoanaerobicconditions.

Theseorganisms encodehomologs of the genes impli- catedinPHAsynthesis(Figure1).

Fromoleaginous biomasstobiofuels via biocatalysis

ApartfromtheWEsofethanolormethanol,inordertobe usedin common internalcombustion engines,the con- tent of lipid granules has to be converted into a less viscousform.Toachievethis,transesterificationofTAGs ismostcommonlycarriedouttoproducefattyacidalkyl esters(FAAEs),aswellastheesterificationofPHAsinto HAMEs.

TAGandPHA(trans-)esterificationreactionsrequirean alcohol(methanolforFAMEsandHAMEs,orethanolfor fattyacidethylesters—FAEE)andacatalyst.TheTAG

Box2 Alternativestrategiesforusingwastewaterand microorganismstoproduceenergy.

Alternativestrategiesforexploitingwastewaterorwastesludgehave involvedeukaryoticphotoheterotrophic(e.g.micro-algae[67])or heterotrophicorganisms(e.g.oleaginousyeast[68]).

Additionally,anaerobicdigestionofsludgetobiogasisanestab- lishedbioenergyrecoverystrategyfromwastewater,butfewnotable limitationsexistsuchasthelimitedrecoveryofchemicalenergy, qualityofgasgenerated,storageproblemsandoverallcapital investment[69].

Apartfromlipid-basedbiofuels,electrochemicalenergyproduction representsanotherinterestingavenueforenergyrecoveryfrom wastewater.Whenmicroorganismsoxidizesubstrates,electronsare transferredtoanelectronacceptor.Microbialfuelcells(MFCs) containanodesandcathodestogenerateelectricitybyharnessing thisflowofelectrons.Generally,microorganismsinvolvedinthe oxidationofsubstratesarelocatedattheanodeandelectron acceptingmicroorganismsatthecathodeofMFCs.Theensuing differenceinpotentialbetweentheanodeandacathodeleadstothe generationofanelectricalcurrent.Thepowerproducedisakey factorforevaluatingtheperformanceofMFCs.Atpresent,theyields ofMFCsfedwithwastewaterarefarbelowwhatwouldberequired tomakethemeconomicallysustainable.However,intenseresearch inthepastfiveyearssuggeststhatenergyoutputofMFCsisfarfrom reachingitsfullpotential(overallwehavewitnesseda10-fold increaseinpowergenerationfromMFCscomparedtopreliminary outputs)and,consequently,thistechnologyshowsgreatpotentialas afuturemeansofrecoveringchemicalenergyfromwastewater streams[70].

Box1 Fuelblending.

Crudeoilisacomplexmixtureofhydrocarbonscomposedof branched-chainalkanes,alkenesandaromaticsrangingfrom4to23 carbonsinlength.Therefore,thehydrocarboncompositionofcrude oilhastobemodifiedbydistillationandrefiningprocessesto achievedistinctcetanenumbers,whichreflectignitionqualityin dieselengines.Althoughbiodieselingeneralsatisfiesstandardfuel specifications,variationsinoxidativestability,cold-flowproperties andexhaustemissionscancausequalityconcerns[65].However, theuseofadditivesandcetaneenhancersaswellastheblendingof variousfuelscanminimizetheselimitations,therebyimproving overallfuelpropertiesandensuringconsistentquality[66].Giventhe chemicaldifferencesofbiodieselwhencomparedtopetroleum- baseddieselandshort-chainalcohols,blendingallowsfine-tuningof fuelcharacteristics.Preliminarystudiessuggestthatblendsof ethanol–dieselandofethanol–biodiesel–dieselexhibitincreasedfuel energyrecoveryduetoincreasedfueloxygenationaswellas reducedexhaustemissions.Mixedfeedstockbiodieselproduction hasrecentlybeenusedtoimprovethephysicalpropertiesoffuels.

Suchblendingstrategiescanalsobeusedinthecontextof wastewaterbiodieselproduction.

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transesterification catalystcanbe alipaseor achemical suchasanacidorabase(e.g.ofcatalyst-freemethods,see [46,47]).Thecurrentindustrialproductionofbiodieselby chemical transesterification of TAGs hasseveral disad- vantages including high energy consumption, theneed forsaltandwaterremovaltoavoidsaponificationoffree fatty acidsandtherequirementfor downstreamproces- sing,forexample,glycerolremoval.Recently,enzymatic transesterification(recentlyreviewedin[48]),catalyzed by intracellular or extracellular lipases, has been suggestedasafuturealternative. Theinvivoenzymatic method appearsto bethemostconvenientpathto pro- ducebiodiesel,butinmanycasesrequiresbioengineering of strainsand such strainsmaynot beable to compete withotherorganismsinopenbioreactorsystemssuchas BWWTplants.Ontheotherhand,invitroprocessesmay beusedinvolvingforexampleenzymeimmobilizationor encapsulation[48].Interestingly,glycerol,thebyproduct of biodiesel production via transesterification of TAGs, canalsobereusedtosynthesizeothercommodities[49], notablybiodiesel[50]andbioethanol[51](forshort-chain alcoholicbiofuelproductionusingwastebiomass,seethe next section).

Alternative routes ofliquidbiofuel production from wastewater

The production of bio-oils (also called pyrolysis oils), short-chain alcoholic biofuels or complex hydrocarbons by the Fischer-Tropsch process represent alternative methodsfortheproductionofliquidbiofuelsfromwaste- water biomass. Other strategies to produce bioenergy (biogas,electricity)fromwastewaterarealsohighlighted in Box2.

Theproductionofbio-oilsfromwastesludgebypyrolysis hasgainedsignificantattentioninthepastfewyears[52].

Biomassfeedstockqualitymajorlyaffectspyrolysisyield, but recentadvancesinaccuratelydefiningreactioncon- ditions according to sludge composition have demonstrated the feasibility of cost-effective and commercial production of bio-oilsfrom wastewaterbiomass[53,54].

For the sustainable full-scale production of sludge- derivedbio-oils,thecompositionandcombustionquality of produced fuel should provide the required perform- ancewithoutcausingengineandinfrastructuredamages (drop-infuels)[55](Box1).Thecalorificvalueofpyrol- ysis-derived sludge bio-oil (36MJkg ¹) is comparable

Figure2

Substrates

Extracellular lipases Lipids

Lipid accumulation

TAG accumulators

WE accumulators

PHB accumulators

PHB accumulation WE accumulation TAG accumulation

PHB granules

HAME FAME FAEE

Lipid microdroplets Lipid microdroplets

Conglomeration and release WS/DGAT

docking

WS/DGAT docking

PhaM, DNA and PHB pol

scaffold Influent

wastewater General Mechanism

de novo synthesis

Current Opinion in Biotechnology

Conceptualschemeofa‘biorefinerycolumn’forbiofuelproductionfromwastewaterunderanaerobicconditionsusingspecificallyenrichedlipid accumulatingbacterialpopulations.Abbreviations:FAEE:fattyacidethylester;FAME:fattyacidmethylester;HAME:hydroxyalkanoatemethylester;

PHB:polyhydroxybutyrate;PHBpol:PHBpolymerase;TAG:triacylglycerol;WE:waxester.

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with that of commercial diesel (45MJkg ¹) [56].

Furthermore, the bio-oil from BWWT sludge contains various hydrocarbons ranging from C6 to C20 [54], including isoprenoid lipids such as farnesene [57] with substantial calorific values. By monitoring the hydro- carbon composition of sludge-based bio-oils and their further targeted refinement, the conversion of sludge- derivedbio-oilsintoavarietyofspecificproductsopens upexcitingcommercial prospects.

A widerange of residues from wastewatercan beused to produce short-chain alcoholic biofuels but these are typically confined to small-scale plants [58]. Although poorly studied, preliminary studies have highlighted thefermentationcapabilitiesofmicroorganismsproducing industrialrelevant organic acids within BWWTbiomass [59]. Additional research on identifying and cultivating microorganismsthatarecapableof producingsignificant quantities of short-chain alcohols from wastewater may improve large-scale short-chain alcoholic biofuel pro- ductioninthefuture.

Another alternative process to produce liquid biofuels such as biodiesel and short-chain alcoholsusing wastewater-derived gasified biomass involves the Fischer- Tropsch process or microbial syngas fermentation [60,61]. Both processes involve the conversion of CO andH2intohydrocarbons.Althoughstillintheirinfancy when applied to wastewater biomass, large-scale pro- ductioniscurrentlybeingpiloted(e.g.SYNPOLproject, URL:http://www.synpol.org/).

Towards abiorefinery column forbiofuel productionfromwastewater

In the context of increasing global population growth coupled to environmental deterioration due to human activity,futuresustainabledevelopmentscenariosshould notonlyincludetherecoveryofenergy(biofuelorelec- tricity)from wastewaterto reduceouroverallfossilfuel footprint,butalsoincludeprovisionstomeetothercom- modityneeds.Inthiscontext,wehaverecentlyproposed theconceptof a‘wastewaterbiorefinerycolumn’which would leveragethe existing and futurewealth of infor- mationconcerningthegeneticreservoirofmicroorganisms andtheirfunctionalcapacityforsustainableproductionof bioenergy (Figure 2), in addition to other commodities suchasbioplasticsandfertilizers[6].However,inorderto makethisconceptcometofruition,itisessentialthatwe first obtain detailed descriptions of the niches of the individual community members, using global in situ monitoring methods (‘meta-omics’ [62]). Once detailed knowledge has been obtained, BWWT processes may be (re-)engineered using bottom-up design principles whichtakeintoaccountforexampletheecologicalniches oftheindividualorganismalgroups.Theoptimizationof processes may theninvolve adiscovery-driven planning approach[62],ratherthetop-downstrategiespursuedso

far.Westillhavealongwayto gotobringthisvisionto fruitionbutitmayrepresentagrandchallengeformicrobial ecologistsandengineersaliketotackleatthecentenaryof Ardern & Lockett’s discovery of the activated sludge wastewatertreatmentprocess.

Acknowledgements

ThisworkwasfundedbyaLuxembourgNationalResearchFund(FNR) ATTRACTprogramgrant(A09/03)andaEuropeanUnionJointProgramin NeurodegenerativeDiseasesgrant(INTER/JPND/12/01)toPWaswellas postdoctoralgrantsAidea` laFormationRecherche(AFR)toEELM(PDR- 2011-1/SR)andARS(PDR-2013-1/5748561).WethanktheLuxembourg CentreforSystemsBiomedicineandtheUniversityofLuxembourgfor supportofEELM.

AppendixA. Supplementary data

Supplementarymaterialrelatedtothisarticlecanbefound,intheonline version,athttp://dx.doi.org/10.1016/j.copbio.2014.03.007.

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