This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg)Nanyang Technological University, Singapore.

Optimization of micronutrient supplement forenhancing biogas production from food waste intwo‑phase thermophilic anaerobic digestion

Menon, Ajay; Wang, Jing‑Yuan; Giannis, Apostolos

2016

Menon, A., Wang, J.‑Y., & Giannis, A. (2017). Optimization of micronutrient supplement forenhancing biogas production from food waste in two‑phase thermophilic anaerobicdigestion. Waste Management, 59, 465‑475.

https://hdl.handle.net/10356/80484

https://doi.org/10.1016/j.wasman.2016.10.017

© 2016 Elsevier Ltd. This is the author created version of a work that has been peerreviewed and accepted for publication by Waste Management, Elsevier Ltd. It incorporatesreferee’s comments but changes resulting from the publishing process, such ascopyediting, structural formatting, may not be reflected in this document. The publishedversion is available at: [http://dx.doi.org/10.1016/j.wasman.2016.10.017].

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1IntroductionFoodwaste (FW) isoneof the largest componentsofmunicipal solidwaste (MSW) inmostdevelopedcountries.Approximately36.4and89million tonnesofFWaregeneratedannually inUSAandEU,

respectively(USEPA,2012;EU,2010).IndevelopingcountriessuchasChina,FWformsupto50%ofthetotalMSWanditisexpectedtoincreasedrasticallyinthefuture(Daietal.,2013).Ineffectivemanagementof

FW can causemany environmental issues such as hygiene and odour problems, greenhouse gas emissions, pollution, and loss of resources, etc. (Zhang et al., 2014). Food waste composition is highly variable

dependingonthesourcewithcharacteristichighmoisturecontentof74–90%,VS/TSratioof80–97%,andC/Nratioof14.7–36.4(MillerandClesceri,2003).ThismakesFWeasilybiodegradableandgoodsourcefor

renewableenergyproductionthroughbioconversion.Consideringenergysecurity,worldhungerandenvironmentalimpacts,itwouldbemoreaccuratetocallFWamisplacedresourceratherthanwaste.

Anaerobic digestion (AD) is a promising approach for the bioconversion of FW into biogaswith relatively low energy consumption, space requirement and cost (Speece, 1983). The twomain groups of

microorganisms(acidogensandmethanogens)involvedinADdifferwidelyintheirnutritionalneeds,growthkineticsandenvironmentalrequirements.Anyimbalanceamongthemcouldcauseprocessfailure(Demirel

andYenigün,2002).Inparticular,ADsufferswitheasilybiodegradablesubstratelikeFWduetoenhancementofacidogenicstage(Wardetal.,2008).Thescarcityofessentialmicronutrients,thepresenceofinhibitory

agentssuchaslongchainfattyacids(LCFA)andtheexcessofammoniaaresomeotherfactorsthatcouldfurtherlimitADprocess(Chenetal.,2008).

PhaseseparationcanimprovetheADprocessthroughoptimizationoftheacidogenicand/orthemethanogenicstages(Jiangetal.,2012).Thetwo-phaseADprocessmitigatesimbalanceandoverloading,while

higherorganic loadingrates(OLRs)orbiogasproductivitycouldbeachieved(Shenetal.,2013).ThermophilicAD(carriedoutat50–60°C) isanotheroperationmode to increasebiogasproductivity.Thehigher

temperaturesimprovehydrolysisandphysicaldegradationofthesubstrate,increasedestructionoforganicmatterandenhancemethaneyields(Kim,2004).

Recently, there has been a spotlight on the role of certain trace metals or micronutrients in AD process. The micronutrients play significant role in several metabolic pathways in AD, however, their

concentrationisusuallyinadequatetoaffectthem(Schattaueretal.,2011;Zhangetal.,2007).Micronutrientsofparticularinterestarecobaltnickel,iron,magnesiumandcalcium.Thesemicronutrientsareessential

Optimizationofmicronutrientsupplementforenhancingbiogasproductionfromfoodwasteintwo-phasethermophilicanaerobicdigestion

AjayMenon

Jing-Yuan Wang

ApostolosGiannis⁎

agiannis@ntu.edu.sg

ResiduesandResourceReclamationCentre(R3C),NanyangEnvironmentandWaterResearchInstitute,NanyangTechnologicalUniversity,1CleantechLoop,CleanTechOne,Singapore637141,Singapore

⁎Correspondingauthor.

Abstract

Theaimofthisstudywastoenhancethebiogasproductivityoftwo-phasethermophilicanaerobicdigestion(AD)usingfoodwaste(FW)astheprimarysubstrate.Theinfluenceofaddingfourtrace

metals(Ca,Mg,Co,andNi)asmicronutrientsupplementinthemethanogenicphaseofthethermophilicsystemwasinvestigated.Initially,ResponseSurfaceMethodology(RSM)wasappliedtodeterminethe

optimalconcentrationofmicronutrientsinbatchexperiments.Theresultsshowedthatoptimalconcentrationsof303,777,7and3mg/LofCa,Mg,CoandNi,respectively,increasedthebiogasproductivity

asmuchas50%andsignificantlyreducedtheprocessingtime.Theformulatedsupplementwastestedincontinuoustwo-phasethermophilicADsystemwithregardtoprocessstabilityandproductivity.Itwas

foundthatadestabilizedthermophilicADprocessencounteringhighVFAaccumulationrecoveredinlessthantwoweeks,whilethebiogasproductionwasimprovedby40%yielding0.46LCH4/gVSadded/day.

TherewasalsoamajorincreaseinsolubleCODutilizationupontheadditionofmicronutrientsupplement.TheresultsofthisstudyindicatethatamicronutrientsupplementcontainingCa,Mg,CoandNi

couldprobablyremedyanytypeofthermophilicADprocess.

Keywords:Micronutrients;ThermophilicAD;Responsesurfacemethodology;Biogas

foravarietyofchemical,biochemicalandmicrobiologicalreactionsrelatedtoVFAutilization,methanegenerationandcelllysis.Cobaltandnickelformapartofcarbonmonoxidedehydrogenase(CODH),whichplays

anessentialroleinmethanogenesisfromacetate(Madiganetal.,20 15;Zandvoortetal.,2006).NickelisalsofoundinthecofactorcoenzymeF430(TakashimaandSpeece,1990),whichplaysanimportantrolein

autotrophicmethanogenesis. Iron is part of cytochromes and ferredoxin inmethylotrophicmethanogens, and as iron-sulphur proteins in some enzymes (Takashima and Speece, 1990).Magnesium is known to

stimulatetheproductionofsinglecells(SchmidtandAhring,1993),whicharerequiredforavoidingthelossofaceticlasticactivityinanaerobicreactors.CalciumcanaddresstheproblemofoveracidificationinAD

duetolongchainfattyacidinhibitionbyforminginsolublesaltswithLCFAs(Kleybockeretal.,2012).

Themicronutrientsoperateatthemetaboliclevel,andtheirrequirementisindependentofsourceofthesubstrate.Assuch,micronutrientsupplementationhasbeenshowntobeimportantforstabilizingand

optimizingbiogasproductionfromfoodwaste(ZhangandJahng,2012;Weietal.,2014).WhilesomeresearchhasbeenconductedtoformulateamicronutrientsupplementforuseinAD,littlehasbeendonetowards

tailoringamicronutrientsupplementforenhancingthermophilicADoffoodwaste.

Usually,themicronutrientsupplementisexternallyaddedinADsystem(Facchinetal.,2013).However,itisimperativetodeterminetheoptimallevelsasoverdosecanleadtoexcesscostandeco-toxicity.

Responsesurfacemethodology(RSM),acollectionofmathematicalandstatisticaltechniquesthatcanbeusedforanalysingtheeffectsofsuchindependentvariablesandoptimizetheireffectonasingleresponse

(BoxandDraper,1987).RSMcanbeappliedinDesignofExperimentstoimproveprocessdesignandoptimizetheresponseoftwoormorevariablewithoutknowingtherelationshipbetweenthefactors.Theobjective

ofusingRSMistodeterminetheoptimaloperatingconditionsforthesystemtoachievearequiredoutput.Hence,RSMhasbeenusedtooptimizevariousfactorsinanaerobicdigestionsuchassubstratemixingratio,

feedingrate,C/Nratioandsubstratepretreatment(Kim,2004;Wangetal.,2012;Menonetal.,2015).

Inthisstudy,amicronutrientsupplementcontainingCa,Mg,Co,andNiwasformulatedtoimprovemethanegenerationintwo-phasethermophilicADsystemusingfoodwasteassubstrate.RSMwasinitially

appliedtooptimizetheconcentrationofCa,Mg,Co,andNiinbatchexperiments.TheefficiencyoftheformulatedsupplementwastestedincontinuousmodethermophilicADsystemconsideringprocessstabilityand

biogasyield.

2Materialsandmethods2.1Substrateandinoculum

Theprimary substratewasFWcollected fromacanteen inNanyangTechnologicalUniversity,Singapore. Itmostlyconsistedof leftoverscontaining rice,noodles, fruitpeels,boiledvegetables, cookedmeatandoil.Non-

biodegradablematerialssuchasbonesorplasticsweremanuallyseparated.Afterwards,theFWwasgrinded,andfilteredthroughafinewiresieve,creatingapastewithparticlessmallerthan1mmindiameterwhichwasthendiluted

withwatertotheconcentrationof3gVS/L.ThiswasusedasfeedstockintheacidogenicphaseofthermophilicADprocess.TheeffluentfromtheacidogenicphasewasusedassubstrateinthemethanogenicphaseofADwithoutany

pre-treatmentordilution.Table1presentsthemaincharacteristicsofFWandacidogeniceffluent.

Table1CharacteristicsoffoodwasteoriginalfeedstockandacidogenicADeffluent.

Parameter FW(3gVS/L) Acidogeniceffluent

pH 5.1 4.2

ORP(mV) −59 −388

TS(%) 3.2 2.1

VS/TS(%) 93.51 74.54

Acetate(mM) 1.22 40.17

Propionate(mM) 0.15 1.86

Butyrate(mM) 0.05 15.52

TVFAs(mM) 1.98 59.69

CODt(g/L) 33.55 28.26

03

CODs(g/L) 8.95 16.57

Ca(mg/L) 13.3 36.2

Co(mg/L) 2.27 0.84

Fe(mg/L) 51.79 16.25

Mg(mg/L) 15.8 5.18

Ni(mg/L) 14.18 n.d

n.d.–notdetected.

TheoriginalseedinoculumwasmesophilicADsludgethatobtainedfromUluPandanWaterReclamationPlant,Singapore.ItsconversiontothermophilicsludgewasdoneusingamethoddescribedbydelaRubiaetal.(2005)

withsomechanges.Briefly,a20LCSTRbioreactorwasoperatedwithorganicloadingrate(OLR)of0.5gVS/L/dayFWassubstrateandhydraulicretentiontime(HRT)of30daysforaperiodof2weeksatmesophilicconditions.The

temperaturewasthendirectlyraisedtothermophilicconditions(55°C)usingheating jacketandafteraperiodofstarvation for2days, thereactorwas fedwith0.5gglucose/L/day for2weeks,whileother conditions remained

unchanged.ThesubstratewasthenchangedtoFWwithanOLRof0.5gVS/L/day.Oncebiogasproductionandsoliddestructionwerestabilized,atwo-phasethermophilicADsystemwasadoptedwithanOLRof3gVS/L/day(more

detailsinSection2.3).Theeffluentfromthemethanogenicphasewascollectedandusedasinoculuminthebatchexperiments.

2.2MicronutrientsupplementTheconcentrationofmicronutrients(Ca,Mg,Co,andNi)wasmeasuredbythequantityofindividualcomponenttypicallyfoundinfoodwasteandtheactualrequirementinADprocess.Themaximumconcentrationswere

chosenbasedonextensiveliteraturereviewandpreliminaryexperiments.Assuch,themaximumconcentrationsusedwere500mg/LforCa,1000mg/LforMg(Loetal.,2010),10mg/LforCo(Pobeheimetal.,2010)and5mg/LforNi

(TakashimaandSpeece,1990).Alltracemetalswereaddedasdissolvedsalts(CaCl2·4H2O,MnCl2·4H2O,CoCl2·6H2O,NiCl2·6H2O)inthefeedforthemethanogenicphase.Inaddition,VisualMinteq,achemicalequilibriummodelwas

appliedtocalculatethemetalspeciationandsolubilityequilibria.

2.3ExperimentalsetupsTheoptimalconcentrationofmicronutrientsupplementwasdeterminedusingbiochemicalmethanepotential(BMP)tests.Thesubstratetoinoculumratio(SIR)wasmaintainedat0.3(basedonVS)(Angelidakietal.,2009).

TheBMPtestswereconductedintheAutomatedMethanePotentialTestingSystem(AMPTSII)(BioprocessControl,Sweden).AMPTSconsistedof15parallelreactorsof500mLcapacity(workingvolumeof400mL),andthesame

numberofgasflowmeterswasconnectedtotheacquisitionsystem.Thereactorsweresealedwithrubberstoppersandthenconnectedtomechanicalagitatorwhichstirredthesludgefor1minonandoffat80rpm.Thereactorswere

spargedwithnitrogengasbeforesealedtocreateanaerobicconditionsandincubatedat55°C.Thegasproducedwasmonitoredusingautomateddataloggingsystem.Threeseparaterunswerecarriedout–Run1:Ca-Mgmixture

(highconcentrationmetals);Run2:Co-Nimixture(lowconcentrationmetals);andRun3:optimizedmicronutrientsupplement.Run1and2wereconductedinduplicate,whileRun3intriplicate.Allexperimentslastedfor20days.

Theeffectoftheformulatedmicronutrientsupplementonmethaneproductionwasfurtherstudiedincontinuoustwo-phasethermophilicADsystemconsistingofa10Lreactor forhydrolysisandacidogenesis,anda20 L

reactor formethanogenisis (Winpact,USA).Thereactorsweremonitored forpH,ORPandbiogasproduction.Bothreactorsweremaintainedat55°Cusingheating jackets,while theHRTwas5and15days for acidogenic and

methanogenic phase, respectively. The feedstock in the acidogenic phase was FWwith OLR 3 g VS/L/day and pH 4.5± 0.3. The effluent from the acidogenic phase was the feedstock in the methanogenic phase. Before the

micronutrientsupplementation,thetwo-phaseADsystemwasatstableoperationfor6months,butatlaterstagethemethanogenicphaseexperiencedsomeVFAaccumulationandthemethaneproductiondropped.

2.4ResponseSurfaceMethodology(RSM)Acentralcompositedesignwithtwofactorswasusedtodesigntheexperimentsinthebatchstudies.MethaneproductionwaschosenastheresponsevariableandtheconcentrationsofCa,Mg,CoandNiwerechosenasthe

independentvariables.Thisdesignrequiredtheuseofthreelevelsoftheindividualvariables-alowerlimit(-(−1),anupperlimit(1)andthecentrepoint(0).Thelowerlimitwaschosen0mg/Lforeveryelementandtheupperlimit

waschosenbasedonliteraturereviewandpreliminaryexperimentsasexplainedinSection2.2.Hence,thethreeconcentrationsusedforeachmicronutrientwere:0,250and500mg/LforCa2+;0,500and1000mg/LforMg2+;0,5

and10mg/LforCo2+;and0,2.5and5mg/LforNi2+.Themicronutrientsweredividedintotwogroupsforevaluationonthebasisoftheirsimilarityandsynergisticeffect:Ca-Mg(similarchemicalcharacteristics,requiredinlarger

concentrations),andNi-Co(bothinvolvedinmethanogenesisandsubstrateutilization,requiredinlowerconcentrations).ThestatisticalanalysissoftwareMINITAB17(SBTI,SanMarcos,Texas)wasusedforthispurpose.

2.5AnalyticaltechniquesThe pH-readings in the batch experiments were conducted with a compact titrator (Mettler Toledo) equipped with a pH probe (Mettler Toledo DGi 115-SC). Total (CODt) and soluble (CODs) chemical oxygen demand

measurementsweremadeusingCODdigestionvials(Hach)andaspectrophotometerDR2800(Hach,USA)asperstandardmethods(APHA,1995).AmmoniawasmeasuredcolorimetricallyusingaDR2800spectrophotometer(Hach,

USA). The micronutrient content in the AD effluent was analysed using Inductively Coupled Plasma-Mass Spectrometer (ICP-MS NexION 300D, Perkin Elmer). Volatile fatty acids (VFAs) content was measured using a gas

chromatograph (Agilent Technologies 7890A,USA), equippedwith a flame ionization detector (FID) (Agilent Technologies,USA). A selective capillary column (DB-FFAP)was applied for good separation of nine VFA compounds

includingacetic,propionic,iso-butyric,n-butyric,iso-valeric,n-valeric,iso-caproic,n-caproicandheptanoicacids.Agaschromatograph(AgilentTechnologies7890A,USA)equippedwithathermalconductivitydetector(TCD)was

usedtomeasurethemethane,hydrogenandcarbondioxidecontentinbiogas.TheC/NratioofFWwasdeterminedbymeasuringthetotalcarbonandnitrogenusinganelementalanalyzer(VarioELcubeCHNOS,Germany).

3Resultsanddiscussion3.1Micronutrientavailability

ThebioavailabilityofmicronutrientelementswasdeterminedusingtheVisualMinteqsoftware.TableS1showstheconcentrationofthespeciesusedtomodelthemetalspeciationanalysis.Normalmethanogenicconditionsof

pH=7,ORP=−500mVandammonia,carbonateandphosphatespecieswereused.Forthedeterminationofbioavailabilitythemaximumconcentrationoftracemetalswasused(Ca:500mg/L,Mg:1000mg/L,Co:10mg/L,Ni:

5mg/L).TableS2showstheresultsofthespeciationanalysisfortracemetalions.94%and91.6%ofaddedCaandMgwerepredictedtoremaininsolutionasCa2+andMg2+ions,respectively,while92%ofCowaspredictedtoremain

intheformofbioavailableCo2+ions.83.3%ofNi(addedinthesmallestamount)waspredictedtobeasNi2+ions,whileapproximately15%ofNiwaspredictedtoremaininsolubleammoniumandphosphatecomplexes.Therefore,all

tracemetalswouldbemostlyinbioavailableforms,andtheADsystemwouldnotsufferwithaccumulationofprecipitants(insolublecrystals)thatinhibitbiogasproductionandcausetoxiceffects.

3.2Optimizationofmicronutrientmixtures3.2.1Run1:Ca-Mgmixture

Fig.1presentstheeffectofCa-Mgmixtureonmethaneproduction.Itseemsthattheadditionofmicronutrientshadapositiveeffectinmethaneproduction.Morespecifically,theadditionofmicronutrientsnotonlyincreases

theproduction,butitacceleratedtheproductionratesaswell.ThissuggeststhatthetraceelementsCa-MgwereatsuboptimalconcentrationsintheADsystemandtheiradditionwasnecessaryforbetterprocessperformance.The

productionofmethanewasincreasedby30–60%atvariousdosagesofthemicronutrients,withthecombinationof250mg/LCa2+and500mg/LMg2+showingthehighestyield.

TheRSManalysisofCa-MgconcentrationsforoptimizedmethaneproductionisshowninFig.2intheformofthreedimensionalresponsesurfaceofthepredictedmethaneproduction.Theregressionequationusedforthe

modelwas:

Fig.S1showsthe2DsurfacegraphsforoptimizingCa-Mgconcentrationsformaximummethaneproduction.ThepredictedoptimalconcentrationsforCaandMgwere303and777mg/L,respectively,whichcouldtheoretically

produce2.3Lofmethaneina20daysbatchexperiment,leadingtoa75%increaseinproduction.

Table2showstheANOVAresultsfortheresponsesurfaceanalysisoftheadditionofCa-Mgmixtureforoptimizingmethaneproduction.Basedonthesignificantstrongrelationshipbetweenthemodelandtheresponsevariable

(p-value<0.005),thehighR-squaredandR-squared(predicted)valuesof93.31%and88.53%,respectively,andthesignifyingacceptablegoodness-of-fitstatistics,themodelcouldreliablybeusedtopredicttheeffectofadditionofCa-

Mgmixtureonmethaneproduction.Intheregressionmodel,thep-valuesofthebothCa-Mglinearcoefficientswerehighlysignificant(<0.005),signifyingthatbothhadalargeeffectonmethaneproduction.Thesquaredvalueof

Ca∗Cawashighlysignificant(p-value<0.005)andthesquaredvalueofMg∗Mgwassignificant(<0.05),whichsuggeststhatbothelementswereimportantformethaneproduction,butcalciumwasmoreimportantamongthetwo.On

theotherhand,theCa-Mghadahighp-value(>0.05),whichsuggeststhatthesynergisticeffectofCa-Mgonmethaneproductionwasnotthatprominent.Thiswasexpectedasbothmicronutrientsaffectdifferentmetabolicpathways.

CaandMgaresimilarlyimportantasactivatorsofvariousenzymeswhichareimportantforthegrowthandfunctioningofbacterialcommunities,buttheprimaryroleofCaistoinhibitthetoxicityoflongchainfattyacids(Kleybocker

etal.,2012).Longchainfattyacids(LCFAs)arethemajorinhibitorsforbiogasproduction(Kuangetal.,2006;Luostarinenetal.,2009),whenoveracidificationhappens.TheroleofMgistostimulatetheproductionofsinglecells

(Harris,1987; SchmidtandAhring,1993).Thehighsensitivityofsinglecells(whicharenecessaryforcatabolizingaceticacid)tolysisisanimportantfactorinthelossofaceticlasticactivityinanaerobicreactors.This

problemisheightenedinthermophilicreactorsascelllysisisaccelerated.

Table2ANOVAresultsforresponsesurfaceanalysisofadditionofCaandMgmixture.

Fig.1EffectofCa-Mgmixturesonmethaneproduction.

(1)

Fig.2Three-dimensionalresponsesurfaceoftheeffectsofCa-Mgsupplementsonmethaneproduction.

Xunetal.,1988;

Source DF AdjSS AdjMS F-value P-value

Model 5 1,299,958 259,992 19.53 0.001

Linear 2 558,910 279,455 20.99 0.001

Ca 1 260,625 260,625 19.57 0.003

Mg 1 298,285 298,285 22.4 0.002

Square 2 694,758 347,379 26.09 0.001

Ca∗Ca 1 366,375 366,375 27.52 0.001

Mg∗Mg 1 89,551 89,551 6.73 0.036

2-Wayinteraction 1 46,290 46,290 3.48 0.105

Ca∗Mg 1 46,290 46,290 3.48 0.105

Error 7 93,201 13,314

Lack-of-fit 3 87,812 29,271 21.73 0.006

Pureerror 4 5389 1347

Total 12 1,393,159

R-sq R-sq(adj) R-sq(pred)

93.31% 88.53% 37.66%

ItisdifficulttoevaluatethederiveddatawithotherstudiesasveryfewwereconductedtodeterminetheoptimalconcentrationsofthesemicronutrientsforthermophilicADoffoodwaste.Theresultsagreedinsomeextent

withsimilarstudiesastheoptimalconcentrationsdeterminedherefellsquarelywithinthesuggestedconcentrationsforCaandMg(TakashimaandSpeece,1990).

3.2.2Run2:Co-NimixtureFig.3showsthemethaneproductionafteraddingmixturesofCo(0,5and10mg/L)andNi (0,2.5and5mg/L).Similar toRun1, theadditionofmicronutrientsgreatly increasedmethaneproductionof thermophilicAD

process.Theenhancementwasbetween35%and40%overcontrol.Fig.4showstheRSMbasedonoptimizationofCo-Niconcentrationsformethaneproduction.Thequadraticequationusedforthismodelwas:

(2)

Fig.S2showsthe2DplotsofCo-Niconcentrationsoptimizedformaximizingmethaneproduction.Theoptimizedcombinationwas7mg/LCo2+and3mg/LNi2+,whichcouldtheoreticallyincreasebiogasyieldby45%.

Table3showsthedetailedANOVAdataoftheadditionofCo-Niforoptimizingmethaneproduction.Therelationshipbetweenthemodelandtheresponsevariable(methanegeneration)washighlysignificant(p-value<0.005),

Fig.3EffectofCo-Nimixturesonmethaneproduction.

Fig.4Three-dimensionalresponsesurfaceoftheeffectsofCo-Nisupplementsonmethaneproduction.

whilest thehighR-squared value (>93%) suggests that themodel had considerablegoodness-of-fit statistics.Hence, thismodel couldbe safely used to predict the relationshipbetween the concentrations ofCo-Ni andmethane

production.Theeffectofadditionofbothmicronutrientswashighlysignificantformethanegeneration(p-value<0.005).ThesquaretermofNiwashighlysignificant(p-value<0.005),whileCowasonlysignificantat lowp-value

(p<0.1).ThisimpliesthatNiadditionwasextremelyimportantforincreasingbiogasproduction,whileCowasstillimportantbutnotthatcritical.Moreover,theadditionofbothmicronutrientswassignificant(p<0.05),suggesting

thatCo-Nihadanimportantsynergisticeffect.Thisisbecausebothelementsaredirectlyinvolvedinmetabolicprocessesrelevanttomethanogenesis.CoisakeyelementofvitaminB12,whichisinvolvedintheactivityofmethyl

transferaserequiredforthetransferofmethylgroupsfromcompounds includingmethanol inmethanogenesis (Madiganetal.,2003).Co isalso found incarbonmonoxidedehydrogenase(CODH),whichplaysanessentialrole in

methanogenesisfromacetate(Madiganetal.,2003,Zandvoortetal.,2006).CODHalsorequiresNi,whichisfoundincofactorF430inmethyl-coenzymeMreductase(MCR)(TakashimaandSpeece,1990).F430isaporphinoid(Shima

etal.,2002)thatplaysaroleinautotrophicmethanogenesis,makingNiespeciallyimportantwhenH2andCO2arethesolesourcesofenergy.Niisatthecatalyticcentreofmosthydrogenases(Shimaetal.,2002)andmaybeinvolved

inmembranestability(JarrellandSprott,1982).

Table3ANOVAresultsforresponsesurfaceanalysisofadditionofCoandNimixture.

Source DF AdjSS AdjMS F-value P-value

Model 5 176,639 35327.8 19.16 0.001

Linear 2 88,849 44424.7 24.1 0.001

Co 1 31,284 31284.3 16.97 0.004

Ni 1 57,565 57565.2 31.23 0.001

Square 2 60,688 30,344 16.46 0.002

Co∗Co 1 7814 7814.1 4.24 0.079

Ni∗Ni 1 32,016 32015.6 17.37 0.004

2-Wayinteraction 1 27,101 27101.4 14.7 0.006

Co∗Ni 1 27,101 27101.4 14.7 0.006

Error 7 12,904 1843.5

Lack-of-fit 3 12,644 4214.5 64.66 0.001

Pureerror 4 261 65.2

Total 12 189,543

R-sq R-sq(adj) R-sq(pred)

93.19% 88.33% 32.60%

Itshouldbementionedthattherearenosimilarstudies,butthefindingsherearecomparablewiththosereportedbyPobeheimetal.(2010).ThesuggestedconcentrationofCoagreeswiththerangementionedinthecurrent

study,whileNiishigherbyanorderofmagnitude.Thisisduetothemesophilicconditionsanddifferentsubstratesthatwereapplied.

3.3FormulationofmicronutrientsupplementAmicronutrientsupplementwas formulatedwithCa,Mg,CoandNicontaining303,777,7,and3mg/L, respectively,basedon theprevious findings.Researchdataon theeffectof formulatedmicronutrientsupplement,

optimizedCa-Mgmixture,andoptimizedCo-Nimixture inmethaneproductionarepresented inFig.5. It seemsthat thesynergisticeffectofmicronutrientsupplementgreatlyenhanced the thermophilicADprocesscompared to

individualmixtures.Theadditionofmicronutrientsupplementwashighlysignificant(p<0.005)inimprovingmethaneproduction.Thetotalmethaneproductionwas1722mL,anincreaseof2.7timesovercontrol.Therewasalso

significant improvement (p<0.05) in theproduction in comparison to theadditionof individualmixtures (Ca-MgandCo-Ni, respectively)withat least20% increase in totalmethaneproduction.Thebatcheswith the individual

mixtures(Ca-Mg,Co-Ni)hadsimilarproductionimplyingthatthebenefitsofaddingbothgroupofelementswereequivalent.Thegasproductionprofiles,however,presentedsomedifferences.ThebatchfedwiththeCa-Mgmixture

appeared a lag phase likely resulted from the improvements in cellular stability and inhibition as described earlier in Section 3.2. The batch fedwithCo-Ni had a shorter lag phase,which could be attributed to themetabolic

enhancementsinmethanegenerationpathways.Thecombinedbenefitsofthetwogroupsasmicronutrientsupplementwasnotonlythehigherproduction,butalsoamajorreductioninprocessingtime.Ittooklessthan5daysfor85%

ofthetotalmethaneproduction.

Table4 presents the effluent characteristics (VFAs, TS removal, total and solubleCOD, and pH) at the end of the batch experiments. The experimentwith the formulatedmicronutrient supplement showed a significant

reduction ineffluentVFAsandCODandshowedaminor increase inTSdestructioncomparedtoexperimentswith individualmixturesandcontrol.Morespecifically, theresidualVFAs in thebatchwithmicronutrientsupplement

showed28–30%and45%reductioncomparedtoCa-MgandCo-Nimixturesandcontrol,respectively.TheadditionofformulatedmicronutrientsupplementgreatlyenhancedVFAutilization,whichcontributedtoimprovedmethane

productionanddecreasedacidification.ThelatterwasapparentinthenoticeablyhigherpHofthebatchwithformulatedmicronutrientsupplement(7.8comparedto7.1incontrol).Therewasanalmost3-timesreductioninCODson

additionofthemicronutrients,againpointingtoimprovedsubstrateutilization.TherewasaminorincreaseinTSdestruction(76.8%inmicronutrientsupplementvs.72.5incontrol),whilesimilarsoliddestructionpresentedtheother

batches.Regardingtracemetalsresidualconcentration,itwasfoundthatmorethan95%ofelementsinmicronutrientsupplementwereutilizedbythemicrobialactivity(datanotshown).Thissuggeststhatminoraccumulationof

precipitateswaspossible,whichcouldbetoxicinlongrun,anditcouldalsoaffecttheeconomicviabilityandsustainabilityofmicronutrientsaddition.

Table4Effluentcharacteristicsaftermicronutrientsaddition.

VFA(mM)

TSreduction(%)

CODs(g/L)

CODt(g/L)

pH

Micronutrientsupplement 1.8 76.8±0.03 3.03±0.04 18.14±0.14 7.8

Ca-Mgmixture 2.3 74.7±0.12 7.80±0.06 22.58±0.16 7.4

Co-Nimixture 2.5 76.8±0.04 6.97±0.02 24.36±0.04 7.5

Control 3.1 72.5±0.14 8.36±0.09 26.56±0.15 7.1

3.4Effectofmicronutrientsupplementoncontinuoustwo-phasethermophilicAD

Fig.5Methaneproductionusingtheformulatedmicronutrientsupplement,andmixturesofCa-Mg,andCo-Ni.

Theformulatedmicronutrientsupplementwastestedinthemethanogenicphaseoftwo-phasethermophilicADsystem.Fig.6aillustratesthemethaneyieldinthemethanogenicreactor.Thetwo-phasethermophilicADprocess

wascarriedoutwithoutanymicronutrientsupplementation for150days.Themethaneyieldaveraged0.3–0.35LCH4/gVSadded/day tillday80,when therewassomesludgewash-out.Thiscaused temporary reduction inmethane

productivityandafterwardsitfluctuatedduetounstableconditions.Atday150,themethaneyieldreachedthelowestvalue0.2LCH4/gVSadded/day.Atthispoint,themicronutrientsupplementwasaddedtothereactor.Thiswasdone

bypreparinga20×stocksolutionindistilledwaterandaddingittothefeed(effluentfromtheacidogenicphase).Theconcentrationofthevarioustracemetalsinthemethanogenicphasewasadjustedtotheformulatedmicronutrient

supplement.

Themethaneproductivitywasenhancedcontinuously forseveralweeksuntil thetraceelementsdepleted.Themicronutrientsupplementwasaddedagainonday170and190tomaintaintheoptimalconcentrationof the

micronutrientsinthemethanogenicreactor.Themethaneproductionreachedtheultimateyield0.46LCH4/gVSadded/day,whichwasnearlytwicetheproductionbeforetheadditionofthesupplement.Themethaneproductionstabilized

ataround0.43LCH4/gVSadded/day,whichwas40%increaseoverthestablemethaneproductionintheearlydaysoftheADsystem.Thisindicatesthatthemicronutrientsupplementnotonlyincreasedthemethaneproductivity,butalso

stabilizedthefailingthermophilicADprocess.

ThemethanogenicreactorwasalsomonitoredforpHandORPvariations(Fig.6b).Similartomethaneyield,pHandORPshowedfairlystableprofilestillday80,afterwhichsomedestabilizationappeared.ThepHdropped

below6afterday110,whichwasatsuboptimallevelformethanogenesis.ThepHdecreasesignifiedpossibleVFAaccumulation.ORPpresentedsimilartrend(atsuboptimallevel),withalotofdestabilizationafterday110.However,

upontheadditionofthemicronutrientsupplementonday150,bothpHandORPquicklyrecovered.Theyreachedoptimalandstablelevelsagainwithintwoweeksandremainedatthoselevelsforthedurationoftheexperiment.

Asmentionedpreviously,theeffectofformulatedmicronutrientsupplementcanbecomparedtootherstudieswithsimilarsubstrates.ClimenhagaandBanks(2008)usedamicronutrientrecipedescribedbyGonzales-Giletal.

(2000),whichconsistedof2000mg/LofCoCl2·6H2Oand142mg/LofNiCl2·6H2OamongstothertracemetalssuchasFe,Al,Zn,Cu,Mn,etc.tostabilizemethaneproductionfromsinglestagemesophilicADofcateringwastesinsemi-

CSTR.Inthatstudy,theADprocesspresentedpartlyfailureandstoppedproducingbiogasafter60daysofoperationat25dayHRT,whilethemicronutrientsupplementationallowedforstablemethaneyieldat0.4–0.5L/gVSadded/day.

Inthecurrentstudy,themethaneyieldwasstabilizedatsimilarlevels,butlessmicronutrients(4opposedto10)wasusedandatmuchlowerconcentrations.Theoptimalconcentrationscouldalsohelptominimizetheadditionalcost

and reducepotential toxicity.Furthermore,Zhangand Jahng (2012)used tracemetal supplementation to stabilizemethaneproduction from foodwaste inCSTR reactor.Amixture ofCo,Fe,MoandNi (2, 100, 10 and5mg/L,

Fig.6MonitoringthemethanogenicphaseofcontinuousthermophilicADprocess,(a)methaneyield;(b)pHandORPvariations.

respectively)wasshowntostabilizemethaneyieldat0.3–4.3L/gVSadded/day,consistentwiththeresultsfoundinthisstudy.Althoughallstudieshaddifferentsubstratesandreactorsetups,animprovementinsubstrateutilizationand

methaneproductionwasnoticedontheadditionoftracemetals.

Fig.7ashows the influentandeffluentVFAsconcentrationof themethanogenic reactor.Although theconcentrationofVFAs in the influentwasnearlyconstant, therewasamajorbuild-upofVFAs in thereactor (during

destabilization stage).By the day 150, the amount ofVFAswas nearly doubled compared to initial stage of theADprocess. This causedpHdrop (as seen inFig.6) and hence reducedmethane production. Aftermicronutrient

supplementadditiononday150,therewasadrasticincreaseofVFAsutilizationandmethaneproductionindicatingthedirectimpactofmicronutrientavailability.TheVFAsconcentrationwasfurtherreducedafterthesecondaddition

ofthesupplementandstabilizedbythetimeofthethirdaddition.TheVFAsconcentrationpresented50%reductioncomparedtodestabilizationstage.TheseresultscouldbeparticularlyattributedtotheadditionofCaandCo.Calcium

iswellknownttoplayanimportantroleincellpermeability(Chenetal.,2008)aswellasenhancetheactionofothermetals(OleszkiewiczandSharma,1990).CobaltadditionisparticularlyimportantinimprovingVFAutilization

(Zandvoortetal.,2006;GallertandWinter,2008)andforactingsynergisticallywithNiforVFAuptakeandenhancingmethanogenicactivity(MurrayandvandenBerg,1981).

Fig.7Methanogenicreactormonitoring,(a)influent/effluentVFAs;(b)influent/effluentsolubleCOD;(c)solidsdestruction.

Fig.7bshowstheinfluentandeffluentCODsvariationsofthemethanogenicreactor.WhiletheeffluentCODswasfairlyconstanttillday150,varyingbetween6and8g/L,adrasticreductionwasseenaftertheadditionofthe

micronutrientsupplement.Afterthefirstaddition,theCODslevelsdroppedrapidlyto3g/Landremainedaround2g/Lafterthesecondandthirdaddition.ThisiscomparablewitheffluentCODslevelsseeninotherstudies(Zhanget

al.,2011;ZhangandJahng,2012).SuchdrasticreductioninCODscouldexplainthesignificantimprovementinmethaneproductivity.Theinfluenceofeachmicronutrientwasexplainedatearlierparts.

TheVSandTSdestructionefficiencyprofilesarepresentedinFig.7c.VSdestructionwasaround70%beforeprocessdestabilization.Afterday70,theVSdestructionwasreducedto35%suggestinglowsubstrateutilization

efficiencyand increasedwash-outofuntreatedwaste.Although therewasa slow recoveryafterday120,micronutrient supplementationon150 lead toan immediate improvement inVSdestruction.Byday190,VSdestruction

efficiencywashigher than80%.RegardingTSdestruction efficiency, it showedaminor improvement aftermicronutrient supplementation. This improvement couldbe attributed to a healthier biomasswith enhanced substrates

utilization rates as statedabove.Studieshave shown similar improvement inVS removal, butnot to this level (Yirongetal.,2014).Coadditionhasbeen identified to improve thehealthof thebiomass (Agleretal.,2008). The

supplementationwithMgisvital,sinceitisknowntospurthegrowthofcellsdirectlyinvolvedinsubstrateutilizationandpreventbiomasswashout.Thisisespeciallysignificantastheroleofmagnesiumismostlyneglectedinmost

studiesrelatedtomicronutrientsupplementation.

ItshouldbenotedthatthebeneficialeffectsofCa,Mg,CoandNionsubstrateutilizationandmethanogenicpathwayscouldonlybeinferredbasedonpreviousstudies.Moreresearchisrequiredtoidentifythedirecteffectsof

these micronutrients. This can be achieved through investigations on gene products and bacterial community dynamics which could pinpoint the changes caused by the addition of micronutrients to the composition of the

methanogeniccommunity.Whilecommunitydynamicsstudiescouldshedsomelightontheeffectofsuchadditions(Karlssonetal.,2012),metagenomicstudieswouldbeofparticularinterest,consideringmostmicrobialcommunities

presentinADprocessareuncultured(StreitandSchmitz,2004).

4ConclusionsItwasfoundthatamicronutrientsupplementconsistedofCa,Mg,CoandNi(303,777,7and3mg/L,respectively)couldenhancemethaneproductionandstabilizethemethanogenicphaseofanaerobic

digestionprocess.Thebiogasyieldwas0.46LCH4/gVSadded/day,whiletherewasincreasedutilizationofsolubleCOD.Inmostcases,theseelementsarenotpresentinoptimalconcentrations,thus,theADprocess

usuallyoperatesatsuboptimalratesorevenfails.Thesupplementcouldbesimplymixedwiththesubstrate(oracidifiedeffluent)andaddedtothereactor.Specialemphasisandattentionshouldbegiventometal

accumulationincasethesludgeisutilizedassoilconditioner.

5Uncitedreferences

AcknowledgmentsThisstudywassupportedbytheNationalResearchFoundation,Singapore,programnumberNRF-CRP5-2009-02,fortheSchoolofCivilandEnvironmentalEngineering/ResiduesandResourceReclamation

Centre,NanyangTechnologicalUniversity,Singapore.

AppendixA.SupplementarymaterialSupplementarydataassociatedwiththisarticlecanbefound,intheonlineversion,athttp://dx.doi.org/10.1016/j.wasman.2016.10.017.

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AppendixA.SupplementarymaterialMultimediaComponent1

Supplementarydata1

Highlights

• CH4yieldoftwophasethermophilicADwasenhancedbymicronutrientaddition.

• ThesupplementconsistedofCa,Mg,CoandNi.

• Optimalconcentrationofmicronutrientsidentifiedusingresponsesurfaceanalysis.

QueriesandAnswersQuery:Yourarticleisregisteredasaregularitemandisbeingprocessedforinclusioninaregularissueofthejournal.IfthisisNOTcorrectandyourarticlebelongstoaSpecialIssue/Collectionpleasecontacte.j@elsevier.comimmediatelypriortoreturningyourcorrections.Answer:Correct.Thearticleshouldbeincludedinaregularissue.

Query:Theauthornameshavebeentaggedasgivennamesandsurnames(surnamesarehighlightedintealcolor).Pleaseconfirmiftheyhavebeenidentifiedcorrectly.Answer:Allnamesarecorrect.

Query:References‘Madiganetal.(2003),Zandvoortetal.(2006),Loetal.(2010),Kuangetal.(2006),Luostarinenetal.(2009),Harris(1987),Xunetal.(1988),Shimaetal.(2002),JarrellandSprott(1982),Gonzales-Giletal.(2000)andGallertandWinter(2006)’arecitedinthetextbutnotprovidedinthereferencelist.Pleaseprovidetheminthereferencelistordeletethesecitationsfromthetext.Answer:Referencesaddedinthelist.Reference:Xunetal.(1988)isdeleted.

Query:Pleasecheckthehierarchyofthesectionheadings.Answer:Weagreewithhierarchygiven.

Query:Thecitations‘Chenetal.(2007),GallertandWinter(2006)andYirongetal.(2015)’havebeenchangedtomatchtheauthordateinthereferencelist.Pleasecheckhereandinsubsequentoccurrences,andcorrectifnecessary.Answer:Weagree.Thanksforthecorrection.

Query:Thissectioncomprisesreferencesthatoccurinthereferencelistbutnotinthebodyofthetext.Pleasepositioneachreferenceinthetextor,alternatively,deleteit.Anyreferencenotdealtwithwillberetainedinthissection.Answer:Deleleallreferences.

Query:Oneormoresponsornamesmayhavebeeneditedtoastandardformatthatenablesbettersearchingandidentificationofyourarticle.Pleasecheckandcorrectifnecessary.Answer:Agree.

Query:ThecountrynamesoftheGrantSponsorsareprovidedbelow.Pleasecheckandcorrectifnecessary.‘NationalResearchFoundation’-‘SouthAfrica’.Answer:Agree.

Query:Pleasecheckthepagerangeinreference‘BoxandDraper(1987)’.Answer:Revised.

• VSdestructionefficiencywasmorethan80%.