!!!!! Tall!Grass!Biomass!for!Biogas:!Investigating!the!Use!of!Phragmites+ australis!(cav.)!trin.!ex.!steud.!(common!reed)!as!an!energy!

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1 TallGrassBiomassforBiogas:InvestigatingtheUseofPhragmites+ australis(cav.)trin.ex.steud.(commonreed)asanenergy FeedstockinOntario,Canada by KurtisBaute AThesis presentedto TheUniversityofGuelph Inpartialfulfilmentofrequirements forthedegreeof MasterofScience in EnvironmentalScience Guelph,Ontario,Canada KurtisA.Baute,August,2015

2 ABSTRACT TALLGRASSBIOMASSFORBIOGAS:INVESTIGATINGTHEUSEOFPHRAGMITES+ AUSTRALISTRIN.EXSTEUD.(COMMONREED)ASANENERGYFEEDSTOCKIN ONTARIO,CANADA KurtisAlexanderBaute Advisors: UniversityofGuelph,2015 ProfessorB.H.Gilroyed ProfessorL.L.VanEerd We#evaluated#the#use#of#wild#Phragmites+australis#as#a#biogas#feedstock#along#with#two# perennial#grass#crops:#miscanthus+x+giganteus+and#panicum+virgatum.#july#biomass#yields#for#p.+ australis+were#not#statistically#different#than#those#of#m"xgiganteus+and#were#greater#than# those#of#p.+virgatum,+at#1.85#±0.16,#1.28#±0.15,#and#0.49#±0.06#kg#dry#matter#(dm)#m K2,# respectively.#the#methane#production#potential#of#julykharvested#p.+australis+was#lower#than#m" xgiganteus,#at#172.4#±15.3#and#229.8#±15.2#normalized#liters#ch 4 #kg K1 #volatile#solids,# respectively.#findings#from#a#secondary#study#showed#that#grass#seeds#were#unable#to#survive# long#durations#of#anaerobic#digestion.#seed#viability#of#p.+australis,+p.+virgatum,+phalaris+ arundinacea,+and+solanum+lycopersicum+was#reduced#by#95%##(lt 95 )#after#29,#52,#98,#and#105# hours#of#commercialkscale#anaerobic#digestion,#respectively,#whereas#typical#anaerobic#digesters# have#operating#times#ranging#from#240#to#1,480#hours.## # #

3 Acknowledgements Science:it sateameffort.firstly,thankyoutorobbuchanan,grahamhoogterp, DonNott,andthelateDeanTiessen,foryourgenerousdonationsofbiomass,seeds,and practicaladvice.supportfromthisprojectalsocamefromtheagriculturaladaptation Council(AAC),theNaturalSciencesandEngineeringResearchCouncilofCanada(NSERC), andtheuniversityofguelphridgetowncampuscentreforagriculturalrenewableenergy andsustainability(cares);thankyouformakingmyresearchpossible.iwouldalsoliketo thankthegermanacademicexchangeservice(daad),thecanadianassociation ofresearchlibraries(carl),andeuraxess,forgrantingmeopportunitiestotravelthe worldinthenameofscience.theseadventureshavehelpedmedevelopasastudent,asa researcher,andasaperson,andiamgratefulforthat. Thankyou,KimberlyVanOverloopforkeepingthatgiantCARESbiogasreactor happywhilesomehowmanagingtosparetimetohelpmewithmyresearch.toresearch assistantsandcotopstudentsmichaeledson,paulinehalder,stevenharvey,colinlittle, EmiliePhilippe,IanPrins,andLauraVanVliet,thankyouforbearingendlesshoursof microscopeworkandmosquitos,andfordoingitwithasmile.iamsimplyluckytohave hadsuchadeterminedandpositivecrewasyou. ToDaveHooker,myexternalexaminer,thankyouforyourthoughtfulfeedbackon howtoimprovethisthesis.todarrenrobinsonandpetersikkema,thankyouforbeinga partofmysupervisorycommittee,andforallofyourwordsofwisdom.thankyoutomy advisors,brandongilroyedandlauravaneerdforthisopportunity,foryourexpert guidancebothinresearchandinlife. Mostimportantly,I dliketothankallofmyfriendsandfamily.thankyouforyour endlesssupportandinspiration.youareawesome. iii

4 TableofContents 0 leofcontents ABSTRACT...ii Acknowledgements...iv TableofContents...v ListofTables...vii ListofFigures...xi ListofAbbreviations...xii 1 LiteratureReview:theUseofPerennialGrassBiomassasaBiogas Feedstock Introduction GrowthoftheBiogasIndustryandFeedstockDemand AnOverviewofAnaerobicDigestion VariationsinAnaerobicDigesterParametersandDesigns PerennialGrassesForBiomass BiomassFeedstocks DesiredTraitsforBiogasSpecies Phragmites"australis:TheCommonReed PerennialGrassCrops BiogasfromGrassBiomass DifferencesbetweenSpeciesandCultivar HarvestTimingandBiomassQuality EffectsofMultipleHarvestsonBiogasYield StorageandPreTtreatmentOptionsforBiomass SeedSurvivalDuringAnaerobicDigestion PotentialFutureResearch ObjectivesandHypothesis TablesandFigures Chapter MethanePotentialofPhragmites+australis,+Miscanthusx giganteus+andpanicum+virgatumgrowninsouthernontario,canada Abstract: Introduction: MaterialsandMethods: SiteHistory&FieldSampling: BiologicalMethanePotentialAssay: StatisticalMethods: Results: BiomassYieldandChemicalComposition...35 iv

5 2.4.2 BiologicalMethanePotentialAssay Discussion: HarvestandBiomassYields BiologicalMethanePotential ResearchSignificance LimitationsandFutureDirections Conclusions Tables Figures Chapter SurvivalofPerennialGrassSeedsDuringCommercialbScale AnaerobicDigestion Abstract: Introduction: MaterialsandMethods: SeedSelectionandPreparation AnaerobicDigestionConditions SeedViability PreliminaryTrial SeedSurvivalExperiment StatisticalAnalysis: Results: Discussion: Conclusions: Tables Figures Chapter Discussion Context Significance RecommendationsforFutureResearch...88 Bibliography...90 v

6 ListofTables Table Comparisonofbiogaspotentialforseveralgrassspecieswith42to95dayretention timesatmesophilictemperatures(35to38 C)inlabTscalereactors(workingvolume 0.25to30L). Table ReporteddrybiomassyieldsforseveralperennialgrassspeciesintheUnitedStates andeurope.adaptedfromlewandowskietal.,2003. Table ChemicalcharacteristicsoffourperennialgrassescomparedtoannualZea"mays,as reportedinthescientificliterature. Table ListofperennialgrassspeciesresearchedforbiogaspotentialinNorthAmericaand Europe. Table Characterizationofthedigestateusedasinoculumineachbiologicalmethanepotential runpriortouseinthebiologicalmethanepotentialassay. Table Mean(±standarderror)compositionof2013samplesofPhragmites"australis,"Panicum" virgatum,and"miscanthus"x"giganteus(n=3);andzea"mayssilage(n=1).meansfollowed bythesameletterineachcolumnarenotsignificantlydifferentaccordingtotukey s rangetest(p>0.05).zea"mays"wasnotanalyzedduetolackofreplication.dm:dry Matter. Table Mean(±standarderror)plantcharacteristicsforyearbyspeciesinteractionsfor Phragmites"australis,"Panicum"virgatum,"and"Miscanthus"x"giganteus Nagara,atthree sitesinsouthwesternontario,canada,harvestedin2013and2014(n=6).means followedbythesameletterineachcolumnarenotsignificantlydifferentaccordingto Tukey srangetest(p>0.05).otherresultsofthreetwayanovashownontable2.4. vi

7 Table Mean(±standarderror)plantcharacteristicsforPhragmites"australis,"Panicum" virgatum,"and"miscanthus"x"giganteus Nagara,atthreesitesinSouthwesternOntario, Canada,harvestedovertwoseasonsin2013and2014(n=6).ResultsofthreeTway ANOVAalsoshownonTable2.3.Meansfollowedbythesameletterineachcolumn werenotsignificantlydifferentaccordingtotukey srangetest(p>0.05). Table Mean(±standarderror)nutrientconcentrationsinPhragmites"australis,"Panicum" virgatum,"and"miscanthus"x"giganteus"(n=3)andzea"mays"silage"(n=1)duringjulyand Octoberharvestsfor2013samples.Meansfollowedbythesameletterineachcoloumn werenotsignificantlydifferentaccordingtotukey srangetest(p>0.05).zea"mayswas notanalyzedduetolackofreplication. Table MeannutrientconcentrationsinPhragmites"australis,"Panicum"virgatum,"and" Miscanthus"X"giganteus"(n=3)andZea"mays"silage(n=1)duringsummerandfall harvestsfor2013samples.zea"mayswasnotanalyzedduetolackofreplication. Table MeanmethaneyieldsfrommesophilicbenchTtopreactorsforMiscanthus"xgiganteus" Nagara,"Panicum"virgatum,and"Phragmites"australiswhenharvestedinJulyor October(n=3).Meansforcornsilage(Zea"mays)displayed(n=9),butnotincludedin thetwotwayanova.meansfollowedbythesameletterineachcolumnwerenot significantlydifferentaccordingtotukey srangetest(p>0.05).dm:drymatter;fm: FreshMatter;NL:NormalizedLiters;VS:VolatileSolids. Table Regressionmodelsfitforchemicalcomponentsofperennialgrasses(gkg T1 ; independentvariable)inrelationtomethaneproduction(nlch4kg T1 VS;dependent variable)asdeterminedbylaboratorytestsofanaerobicdigestionofsamplesfrom 2013(n=6). Table AveragedailyconditionsandfeedinputofthecommercialTscaleanaerobicdigesterat theuniversityofguelphridgetowncampusduringseedviabilitystudies. vii

8 Table Proceduresusedforseedstainingwith2,3,5Ttriphenyltetrazoliumchloride(TZ)totest forviability,incubatedfor18t24hat25 C,basedontheguidelinesintheTetrazolium TestingHandbook. Table InitialseedviabilityoffourspeciesdeterminedbypetriTdishgerminationfollowedby tetrazoliumstaininglotsof100seeds(n=3),priortoexposuretocommercialtscale anaerobicdigestion.lettersdenotesignificantresultsfromatukey srangetest (p>0.05). Table ResultsofANOVAondatafrom2014experiment(Figure3.3)modifiedsothateach specieshadaninitialgerminationvalueof100%.seedsurvivalduringaftervarious durationsofanaerobicdigestion(0,2,6,12,24,48,72,and168hours)forfourspecies (Panicum"virgatum,"Phalaris"arundinacea,"Phragmites"australis,and"Solanum" lycopersicum)(n=3). Table NonTlinearGompertzmodelparametersofseedviabilityoffourspecieswithtwo methods:petritdishgerminationwithtetrazoliumstaining,andgerminationinsoilina greenhousewithcoldtmoiststratification,basedonexperimentaldatafrom preliminarytrial(figure3.2). Table Thelengthoftimeinananaerobicdigesterthatittookfourspeciestoreduceseed viabilityby50%,80%,and95%,referredtoaslethaltime(lt)values,basedon GompertznonTlinearmodelsfrom2014data(Figure3.3). Table ResultsofANOVAcomparingtwotypesofviabilitytests:twoTweeksofgerminationin petritdishesfollowedbytetrazoliumstaining,andgreenhousegerminationfollowedby coldtmoiststratification.resultsrepresentdatafromapreliminarystudyonseed survivalduringincreasedtimeexposuretoanaerobicdigestion(figure3.2),withthree pseudotreplicates viii

9 Table Estimatedmarginalmeans(±standarderror)andpartialresultsofmultipleANOVAsfor comparingtwomethodsofviabilitytesting:twotweeksofgerminationinpetritdishes followedbytetrazoliumstaining(petri),andgreenhousegerminationfollowedbycoldt moiststratification(greenhouse).resultsrepresentdatafromapreliminarystudyon seedsurvivalduringincreasedtimeexposuretoanaerobicdigestion(figure3.2),with threepseudotreplicates. Table ResultsofANOVAondatafrom2014experimentmodifiedsothateachspecieshadan initialgerminationvalueof100%.seedsurvivalduringaftervariousdurationsof temperaturetreatmentof38 C(0,2,6,12,24,48,72,and168hours)forfourspecies (Panicum"virgatum"Phalaris"arundinacea,"Phragmites"australis,and"Solanum" lycopersicum)(n=3). Table PartialresultsofANOVAregardingtheeffectofeightdurationsofexposuretoa temperaturetreatmentof38 C(0,2,6,12,24,48,72,and168hours)forseedsoffour differentspecies(n=3),includingestimatedmarginalmeansforoverallseedviability. Table HypotheticalscenariosofseedsurvivalforPhalaris"arundinacea"biomassharvestedfor useinanaerobicdigestionandtheresultingdigestatespreadontolandaftervarious durationsofexposure. ix

10 ListofFigures Figure LocationofcultivatedMiscanthus"xgiganteus"(purplemarkers)andPanicum"virgatum" (greenmarkers)biomass,aswellaswildstandsofphragmites"australis"(redmarkers) usedinthisexperimentconductedinsouthwesternontario.stripedmarkersdenote locationsofthesamplesusedinthebiologicalmethanepotentialanalysis. Figure Organizationalschematicforpreliminaryseedsurvivabilitystudy,usingmeshTbagsto exposeseedstoanaerobicdigestioninacommercialtscaledigester.seedswere separatedby:exposuretimetpoints(a),species(b),andviabilitymethod(c).bags werepseudotreplicated. Figure Resultsofpreliminarytrialforseedviabilityandgerminationduringincreasing durationsofexposureinacommercialtscaledigester,comparingfourspecies:phralaris" arundinacea(topleft),phragmites"australis"(topright),"panicum"virgatum"(bottomleft), andsolanum"lycopersicum"(bottomright).solidlinesdepictnontlineargompertz model(parametersintable8),andmarkersareobservedvalues,fortwomethods: petritdishgerminationwithtetrazoliumstaining(lightlineandcircles),and germinationinsoilinagreenhousewithcoldtmoiststratification(darklineand squares).eightdurationsofexposure(0,2,6,12,24,48,72,and168hours)were assessedinpseudottriplicate(n=100). Figure Seedviability(asmeasuredbypetriTdishgerminationfollowedbytetrazolium staining)duringincreasinghoursofexposuretoacommercialtscaledigesterforfour species:phralaris"arundinacea(topleft),phragmites"australis"(topright),"panicum" virgatum"(bottomleft),andsolanum"lycopersicum"(bottomright).solidlinesdepict nontlineargompertzmodelanddotsdepictobservedvaluesofeightdurationsof exposure(0,2,6,12,24,48,72,and168hours)wereassessedintriplicate(n=3).exp: Exponent. x

11 ListofAbbreviations AD ANOVA BMP DM FM LT NL TS VS AnaerobicDigestion AnalysisofVariance BiologicalMethanePotential DryMatter FreshMatter LethalTime NormalizedLiters TotalSolids VolatileSolids xi

12 1 LiteratureReview:theUseofPerennialGrassBiomass asabiogasfeedstock" 1.1 Introduction Withconcernsoverclimatechangeontheriseglobally,thereductionofgreenhouse gasemissionsanddevelopmentofrenewableenergytechnologiesarebecomingof increasingimportance[1].anaerobicdigestion(ad)utilizesmicrobialactivitytodegrade organicagriculturalandindustrialbytproductsinordertoyieldmethanetrichbiogasthat canbeusedasarenewablefuelsource.biogasisamixtureofgasesmicrobiologically producedduringthebreakdownoforganicmaterials,anditisoneofthethreemajor bioenergyproducts(alongwithethanolandbiodiesel)producedatcommerciallevels globally[1 3].Renewableenergyproductionincludingbiofuels,biomass,geothermal, hydropower,solar,andwind,stillonlyaccountsforanestimated10%ofglobalenergy consumption[4]andsubstantialdevelopmentinthecarbontneutralenergysectorisstill requiredinordertohelpmitigatetheeffectsofclimatechange[5] GrowthoftheBiogasIndustryandFeedstockDemand Thebiogasindustryhasdevelopedsubstantiallyoverthepast25years,withmany countriesshowinggrowthinthenumberofanaerobicdigestersinoperation.thereare currentlyapproximately33biogasplantsinoperationinontario,canada[6],mostofwhich werecommissionedinthelast10years.intheunitedstates,thenumberofanaerobic digestersoncommerciallivestockfarmsincreasedfrom150to264between2012and 2015respectively[7,8].ThenumberofagriculturalbiogasplantsinPolandincreasedfrom 25in2000toaround150in2010[9],withplanstoreach2000plantsbytheyear2020 [10].AlthoughmoresubstantialthanNorthAmericanbiogasindustrygrowth,Poland s developmenthasstillbeenmuchlesssizablethanothereuropeancountriessuchas 1

13 Denmark,Austria,Sweden,orGermany[9].InGermany,agriculturalbiogasfacilitieshave increasedfromapproximately100to4000to7900in1990,2008,and2014,respectively [1,11,12].In2010,biogasoperationsintheEuropeanUnionreachedenergyproduction capacityequivalentto10.9milliontonnesofoil[13].anaerobicdigestersineuropehave extensivelyincludedz."mays"silageasabiogasfeedstock,andgermanyalonegrows0.5 millionhectaresz."maysforbiogas,muchofwhichisdevotedtovarietiesspecifically tailoredforbiogasproduction[14]. Feedstockdemandhasbeenrisingalongsidetheincreaseinthenumberofoperational anaerobicdigesters.accordingly,feedstockacquisitionisbecomingmoredifficultin locationsthatdohavegovernmentincentivisedbiomassprograms[15].forexample,ad facilitiesinontariowith ontfarm designation(usingfeedstockssuchasmanureandzea" mays"silage)receivehigherfeedtintariffrates(afeeabovetheretailrateofelectricity)than facilitiesnotonfarms,andarerequiredtohavenomorethan50%oftheirfeedstockscome fromofftfarmsources(suchasindustrialwastes)[16].however,somefacilitieshave difficultysecuringasufficientsupplyofofftfarmfeedstocks[17]duetoissuessuchas competitionfromotherindustriesandthelackofestablisheddistributionnetworks.these aforementionedfactorsaredrivingontariobiogasproducerstolookforcropswithhigh biogasyieldsthatcanbeproducedeconomicallyandinclosephysicalproximityto anaerobicdigesters.specifically,therehasbeenasurgeofresearchonthepotentialuseof perennialgrassspeciesasbiogasfeedstocks.morethantwicethenumberofprimary researcharticlesonbiogasfromperennialgrassbiomasswerepublishedfrom2010to 2014(22)thanintheprevioustenyearsfrom2000to2010(9). 2

14 1.1.2 AnOverviewofAnaerobicDigestion Anysourceoforganicmatterhasthepotentialforuseasabiogasfeedstock.Desired feedstocksarelowcostandyieldhighamountsofmethaneperunitoffeedstock.typical feedstocksincludemanures,agriculturalplantresidues,energycrops,andindustrialbyt products.dependingontheoriginandcharacteristicsofthefeedstock,additional processingsuchasparticlesizereduction,pasteurization,orprettreatmentstoenhance conversiontobiogas(seesection3.4)maybenecessarybeforeitcanbefedintoan anaerobicdigester[1].onceinsideananaerobicdigester,feedstocksaredegradedthrough ananaerobicbiologicalprocesstoproducebiogasandresidualdigestate.afterdigestion, digestateisstoreduntilitcanbeappliedtoagriculturallandsasafertilizer[18]. ThebiologicalprocessofADhasbeencategorizedintofourphases.Intheinitial hydrolysisphase,complexpolymersarebrokendownintosimplermonomersincluding sugars,aminoacids,andlongchainfattyacids[1].duringtheacidogenesisphase,these simplemonomersarebrokendownfurthertocarbondioxide,ammonia,hydrogen,and carbonicacids[19].intheacetogenesisphasevolatilefattyacidsareconvertedintoacetic acid,carbondioxide,andhydrogenviaobligatehydrogentproducingacetogenicbacteria. Duringmethanogenesis,thefinalphase,methanogenicArchaeaconvertacetateinto methaneandcarbondioxide,whileconsuminghydrogen[19].methanogensinclude acetotrophs(obligateanaerobes"whichconvertacetateintomethaneandcarbondioxide) andhydrogenotrophs(whichconverthydrogengas andcarbondioxideintomethane)[20]. Biogasistypicallycomposedofmethane(40to75%),carbondioxide(20to45%), nitrogen(upto17%),oxygen(lessthan1%),andafewothercompoundsusuallyfoundin tracequantities,suchashydrogensulphide(32to169mgkg T1 )andwatervapour[9,21,22]. Biogasistypicallydesulfurizedtopreventdamagetogasutilizationequipment[1],andis 3

15 eitherusedasafuelsource(requiringstorageand/ortransportation)orisburnedina generatortocreateelectricity[21] VariationsinAnaerobicDigesterParametersandDesigns Therearemanypossibleanaerobicdigesterdesigns,andresearchhasshownthat factorssuchasorganicloadingrate[23],hydraulicretentiontime[10],andreactortype [24]allplayrolesinoptimizingefficiencyofconvertingbiomasstobiogas. Anaerobicdigesterscanoperateatvarioustemperatures,rangingfrom psychrophilic(15to20 C),tomesophilic(30to37 C),tothermophilic(55to65 C)[25], withmesophilicconditionsbeingthemostcommon.fluctuationsintemperaturecan adverselyaffectmicrobialpopulationsbecausedifferentspeciesthriveatdifferent temperatures.mesophilicbacteriacantoleratechangesof±3 Cwithoutsignificanteffectsto methaneproduction[1]. TheoptimalpHrangeformethaneformationisbetween7.0and8.0,withsevere inhibitionofadoccurringbelow6.0andabove8.5[1].thefirsttwobiochemicalphasesof digestionmustbeinequilibrium:iftheinitialdegradationphasehappenstoorapidlythen thephwilldropduetoacidaccumulation;ifthesecondphasehappenstooquickly,the methaneproductionwillbelimitedbythehydrolyticstage[1].thefactorthatismost limitingtherateofaddependsonthecompositionofthesubstratebeingusedforbiogas production[1].thephvaluerisesduringproteindegradationduetoammonia accumulation,andfallswhenvolatilefattyacidsaccumulate[1].volatilefattyacid concentrationisanimportantperformanceindicator,becauseofitscloserelationtoph, alkalinity,andmethanogenactivity[19]. 4

16 Anaerobicmicroorganismsrequiremacronutrients(carbon,nitrogen,phosphorus, andsulphur)inlargequantities,andmicronutrients(suchasnickel,iron,cobalt,selenium, tungsten,andmolybdenum)intracequantities.theidealratioofmacronutrientsforadis 600:15:5:1(C:N:P:S),andmicronutrientsneedtobeaddedincaseswhereenergycropsare theonlybiogassubstrate[1]. DigesterTypes: Inadditiontooperatingatdifferenttemperatures,thereareseveralothervariations ofanaerobicdigesterdesign.hydraulicretentiontimedescribestheaveragedurationfor whichfeedstocksremaininareactor.insingletstagereactors,hydraulicretentiontime rangesfrom10to60days.themajorityofthebiogasproducedfrombiomassistypically formedinunder20days,thoughreactorstypicallyoperateoverlongerperiodsoftime[26]. Theorganicloadingrate(ie.theratethatfeedstocksarefedintoananaerobicdigester) variesbetweensystems,withcontinuouswetreactorsrangingbetween2to4kgorganic drymatter(dm)perm 3 perday[1].themostcommonanaerobicdigesterdesignis: mesophilic,onetstage,continuous,vertical,andwet[1].insuchreactorsallbiochemical phasesarealwayshappeningsimultaneously.anaerobicdigestersvarygreatlyinscale, rangingfromsmallbenchttopreactorsof<1linsizetocommercialscaleanaerobic digesterswithmultitmillionlcapacity.muchoftheresearchonusingbiomassforbiogas hasusedlabtscalemesophilicwetbatchreactors. 1.2 PerennialGrassesForBiomass BiomassFeedstocks Whereasfirstgenerationbioenergywasfocussedonliquidbiofuels(i.e.usingstarch orsugartomakebioethanolorbiodiesel),secondgenerationfeedstocksbasedonplant 5

17 biomasshavebecomethefocusformuchofthebioenergyindustryandresearchsectors. Manureshavehighbufferingcapacityandprovideawiderangeofnutrients,withdairy manurec:nratiosofaround8:1[27].cotdigestionofmanurewithbiomasscropscreatesa carbontonitrogenratiocloserto40:1[1]forhighermethaneyields[28].totalbiogas productionfromcattlemanurerangesfromapproximately140to266normalizedlitres (NL)kg T1 volatilesolids(vs),whichislowrelativetoenergycropsonavsbasis.cattle manureperformsevenworsecomparedtoenergycropsonafreshtmatterbasisduetoits lowsolidscontent[29].feedstocksupplementationwithpurposetgrownbiomassis economicallybeneficialbecauseitincreasesbiogasproduction,allowsforgreaterfeedstock continuityandreliability,canbestoredassilagefortimesofincreaseddemandsorenergy prices,improvesthebiodegradationofotherfeedstocks,dilutescompoundsthatmay inhibitbiogasproduction,andenhancesthenutrientqualityofthedigestatefertilizer[15]. Forsystemsthatuseenergycropsasfeedstocks,twoTstagedesignshavebeenshownto havehigherbiogasyieldsthansingletstagesystems[1]. ThemajorityofcurrentagriculturalbiogasfacilitiesusepurposeTgrownZ."mays silageandbreedingeffortsarebeingmadetofurtherimprovethedigestibilityofbiogasz." mays[14].whilez."mays"hasachievedhigherbiogaspotentialcomparedwiththeperennial grassesdiscussedhere(table1.1),z."maysisgenerallygrownonthebestarablelandsand thuscompetesforfoodproduction[30].incomparisontoalternativecropssuchasarundo" donax"(giantreed)"andpanicum"virgatum"(switchgrass),z."maysrequireslandofhigher quality[13],andincreasedproductioncostsduetorequirementssuchasfertilizersand annualplanting. Thereisacontinualdemandforreducingthecostsofbioenergyfeedstockswhile increasingtheirenergyyield.assuch,demandforlowtinput,hightyieldingfeedstockshas 6

18 increasedinterestintheuseofperennialspecies.perennialgrassspecieshavemany advantagesovertraditionalrowcrops[31].unlikemanyannualcropssuchasz."mays, perennialsrequireonlyoneplanting,persistformanyyearsthereafter,canbegrownwith fewerfertilizerandpesticideinputs,onmarginallands,andcanbeharvestedmultipletimes inasingleseasondependingonthespecies(section3.2)[32].perennialfeedstockshave otherbenefits,including:anegativecarbonbalanceduetocarbonsequestrationin undergroundbiomass,reductionofsoilerosion,waterqualityimprovement,protectionof ecologicaldiversity,andcancomplementfoodproductionratherthancompetewithit [3,24,32]. Inbioenergycropdevelopment,therehasbeenafocusontallperennialgrass species,suchasp."virgatum,miscanthus"spp."(elephantgrass),phalaris"arundinacea"l."(reed canarygrass),anda."donax(foracomprehensivelist,seetable1.2).unlikenatural grasslands,whicharegeographicallyvariableintheirspeciescomposition,monocultural standsofbiomasscropsallowformorecomparisonstobemadeacrossdifferentpartsof thescientificliterature.additionally,thebiomassofperennialgrassspeciessuchasdactylis" glomerata"(l.),festuca"arundinaceae"schreb.,"miscanthus"spp.,"panicum"virgatum,"p." arundinacea,"andphleum"pratense"(l.)speciesproducebiogasyieldsthatarecomparableto Z."mays(seeTable1.1)[29,33 38],makingthemworthyofconsiderationforpotential biogascrops.thefocusofthisliteraturereviewwillbeontheproductionofbiogasfrom variouslignocellulosicperennialgrassspeciesthatproducehighquantitiesofbiomassper unitarea,withspecificattentiontophragmites"australis DesiredTraitsforBiogasSpecies Desiredbiomasscharacteristicsforbiogasfeedstockspeciesinclude:lowcost,low inputs(fertilizer,pesticides,fuel,andlabour),highbiomassandbiogasyields,highbiogasto 7

19 biomassratio(cheapertransport),andlowconcentrationsoflignin(lessneedforpret treatment).weiland[1]suggeststhatthemostimportantparametertoconsiderisnet energyyieldperhectare,buttherearemanyfactorsthatcontributetothatmetric,suchas theefficiencyofthemachineryused,transportationcosts,andenergyconversion calculations,anditisnotreportedintheliteratureasoftenasbiogasyieldperhectare. Thesemanyaforementionedfactorscontributingtofeedstockpotentialhasledtothe investigationofalargenumberofspeciesascandidatebiomasssources Phragmites+australis:TheCommonReed Phragmites"australis"Trin.Ex.Steud.(commonreed)hasnotattractedmuchattention asabioenergyfeedstock[39,40],thoughitsbasicbiologyandecologyhasbeenwelltstudied [41].Phragmites"australisformslargemonoculturalstandsandisaprolificbiomass producer[42],withyieldscomparabletothoseofcultivatedp."arundinacea"(seetable1.2). WhileP."australis"isahighlyinvasivespecieswithinNorthAmerica[43],itmaybepossible thatwildstandsofp."australis"couldbecontrolledwithregularharvestforuseasabiomass feedstock[44].manyofthedesiredbiomasstraitsdiscussedpreviously(section2.2) overlapwithtraitsresponsibleforinvasiveness[45].usingnontcultivatedplantsfromnont croplandassourcesforbioenergyhasnotbeenresearchedextensively,thoughthereis potentialtoremoveinvasiveandunwantedgrassspeciesforuseasbioenergyfeedstocks [42]. Phragmites"australisisaperennialgrassintheArudinoideaesubfamilythatexhibits C3TC4intermediatephotosynthesis[46].Thespecieshasanearlycosmopolitan distribution,andhasahaplotype(referredtoashaplotypem)thatsuggestp."australis"was likelyintroducedtonorthamericafromeurasiaintheearly19 th century[47]andhassince spreadrapidly[48].unlikeitsnativenorthamericancounterpart,haplotypemis 8

20 recognizedasinvasiveinnorthamericawhereithasreplacedmillionsofhectaresofnative wetlandplants[48].theecologicalimpactsofitsspreadarenumerous,includingreducing thediversityofregionalandlocalplants,changingthecompositionoffaunaassociatedwith thoserespectiveplants,andalteringecosystemfunction[44]. NumerouseradicationmethodsofP."australis"havebeenattempted,including burning,spraying,grazing,andcutting,butallhaveshownlimitedsuccessandexpensive [44].ThemostsuccessfulandcommonlyusedmethodistosprayP."australiswitha herbicide,mechanicallyrollthedeadbiomass,andburnit[44],atanapproximatecostof $US175ha T1 year T1 untilitissuccessfullyeradicated(frankletourneau,doveragritserve; personalcommunication).evenafterp."australishasbeensuccessfullyremoved,thereis nothingtopreventretestablishmentinthesamelocation. InadditiontopotentialendTusebenefits,suchastheproductionofbiogasenergy andgreenmanure,theharvestandremovalofp."australis"fromwetlandecosystemscould haveseveralecologicaladvantages.asmentioned,harvestingforbiomassmayprovidea methodoflongttermsustainablecontrolforp."australis. InseveralEuropeanandAfricancountries,P.""australis"biomassisharvestedforuse asbuildingmaterials,includingroofthatching,walls,andweavingmats,paperpulp,forage feed,litter,andheat[49,50].averageyieldshavebeenshowntorangebetween0.6to1.0kg DMm T2 insweden[51],anditcomparesfavourablytomanyotherperennialspecies(table 1.2).MethaneyieldsforP."australis"havebeencalculatedat180Lkg T1 VS[51],and measuredat220lch 4kg T1 VSinathermophilic(52 C)anaerobicdigester[52].Thisbiogas yieldislessencouragingthanthebiomassyield,asitisatthelowendofthespectrum comparedwithperennialgrassspeciesmeasuredforbiogaspotentialinmesophilic(35to 9

21 38 C)experimentswhichrangedfrom187to347NLCH 4kg T1 VS[34,36]withtheexception ofmiscanthus"spp.,"whichhadyieldsaslowas84nlch 4kg T1 VS[34](Table1.1).However, biologicalmethanepotentialforp."australis"hasnotyetbeenexperimentallymeasuredat mesophilictemperatures. Thegrossmethaneproductionvalueof1tDMofP."australis"wasestimatedat approximately$us90(sweden,2003)[51].however,whenexpensessuchasharvest, chopping,transport,storage,treatment,andspreadingwereincludedinthecalculation, therewasatotalnetlossof$us120pertdmharvested[51].inswedenitwasconcluded thatharvestingp."australisforbiogasproductioniseconomicallyreasonableonlyincertain situationswherereedgrowthisaproblemorwhereinsomesortofphytoremediationgoal issought[51].assuminga6to20tdmyieldperha,usingp."australis"asabiogasfeedstock wouldresultinanetlossof$us720to2400perhaharvested[51].however,thisstudy onlyaccountedforinputcostsandtheprofitofbiogassales[51].benefitsofremovingthe invasiveplantintermsofbiodiversityandphytoremediationshouldalsobeconsidered. Furthereconomicanalysiswouldberequiredtoproperlyassesstheseadditionalfactors andtheoveralleconomicviabilityofp."australisasafeedstock. AnotherstudythatevaluatedtheenergyinputsandoutputsofaP."australisbiogas systemreportedamorepositiveoutcome[52].intermsofthenetrenewableenergyvalue, 1tfreshmatterofreedroughlycorrespondedto38Lofpetrol.Risénet"al."determinedthat thedominantenergyinputrequirementsinsuchasystemwereheating,electricity,andfuel forharvesting,whereasprocessingstepsincludingensiling,transport,prettreatment,and handling;eachonlyaccountedforlessthan10%oftheenergyinputcosts[52].further researchonp."australis"biomassandbiogasyieldsisrequiredtohelpdeterminewhetheror notp."australis"isaviablefeedstock. 10

22 1.2.4 PerennialGrassCrops TheUSDepartmentofEnergychosethegrassspeciesP."virgatumasamodel organismforlignocellulosicbioenergycropsinthe1990s,andithassincebeenresearched extensively[53].panicum"virgatumiswelladaptedtonorthamerica,withlow requirementsforfertilizerinputs,andgooddiseaseandpestresistance[54]."breeding effortsarebeingmadetofurtherdevelopmanyperennialbioenergycropssuchasp." virgatum,"miscanthus,"andp."arundinacea"[55]. NumerouseffortshavebeenmadetoestimateyieldsofbiomasscropsbothintheUS andineurope,ascompiledinareviewlewandowskiet"al.[56].table1.2showsbiomass yieldestimatesforseveralperennialgrassspeciesfortheusandeurope,whichincludesa numberofhightyieldingspecies,suchasa."donax,miscanthus"spp.,"p."virgatum,pennisetum" purpureum"schum(napiergrass),andp."australis.however,thechemicalconstituentsof thesespeciesvarygreatly(table1.3).incomparingtable1.2andtable1.3,itbecomes clearthatspeciesdifferintheirbiomassquantityandquality,bothofwhichareimportant factorsforuseasbiogasfeedstocks.forinstance,comparedwithz."mays,botha."donaxand P."virgatumhave>2xmoreligninandcellulose(undesirabletraits);6xlessstarch,and>3x lesslipids(desirabletraits). Intermsofagronomy,idealbiogascropswillestablishrapidly,easilyandproduce highqualitybiomasswithminimaltimeandresourceinputs.suchidealcropsshouldhave thepotentialforbeinggrownonmarginallands,butshouldalsobegrowninwaysthat minimizetheriskofinvasiveness. 11

23 SpeciesSelection: WhileZ."maysremainsthepredominantlygrownbiomasscrop,Miscanthusspp.and P."virgatumaretwoofthemostcultivatedperennialgrassesforbiomass.Thereare approximately1300,400,325,and50haofmiscanthusspp.growninfrance,austria, Germany,andItaly,respectively[34].Thoroughreviewsoftheliteraturehavebeen conductedforp."virgatum"[32,53]andmiscanthus"[57]. Whilemanyhaveproposedthatidealbioenergycropsshouldbesuitableforgrowth onpoorgradesoils,muchoftheresearchhasassessedtheseplantswithtrialsonhigh qualitylands."panicum"virgatumhasbeenshowntobestronglyaffectedbyvariationsinsoil parameterssuchasn,p,moistureandph[58]. Invasivenessisanotherimportantfactorincropselection.Forexample,P."virgatum" isanunlikelybiomasscropinnewzealandduetothecountry sspeciesintroduction qualificationrequirements[30].thedecisiontoadoptp."virgatumormiscanthus"spp.by particulargrowerspresentsaninterestingcasestudyinthedebateoverbiomasshaving potentialtobeinvasiveorweedy[45].whilemiscanthusspp.hasbeenshowntohave higheryields,manyearlybiomassgrowersraisedconcernsaboutit spotentialtobecome invasive.incontrast,p."virgatumwasrecognizedasanativespeciesandwasmorewidely accepteddespiteit sloweryieldpotential.concernsoverthepotentialspreadofinvasives hasresultedinlimitedresearchbeingconductedontheuseofinvasivesforbiomass. BiomassYields: Theyieldsofperennialgrassesvarydrasticallybetweenclimatesandcontinents (Table1.2),andbiomasscropspeciesoptionswillvaryaccordingly.Tocompareyieldsfrom 12

24 perennialgrassesgrownineuropeandnorthamerica,seetable1.2.thespeciesthathad thehighestyield(over20tdmha T1 )area."donax,p."purpureum,p."virgatum,miscanthus" spp.,and"saccharum"spp.(energycane)[56].inthemidwestusa,miscanthusspp.yieldan averageof38.2tdmha T1 year T1,whichisthreetimesgreaterthanP."virgatum(12.5tDMha T 1year T1 )[5].However,whilebiomassyieldisimportantanddiffersgreatlybetweenspecies andregion,thestructuralandchemicalqualityofthebiomassalsovaries(table1.3). Thereispotentialforcertainperennialspeciestobeharvestedmultipletimes duringasinglegrowingseason,dependingonstandmaturity,environmentalconditions, andmachineryrequirements,[59].dueinparttotherhizomatousrootmassesofperennial grasses,manyhavetheabilitytobecuttwiceinthegrowingseason.speciessuchasp." arundinaceahavebeenshowntohavethehighestannualyieldsunderatwotcutstrategy [60].Arundo"donax"alsorespondsverywelltomultipleTcutstrategies,evenwhengrownin marginalland[61].panicum"virgatumhasonlyminorbiomassyieldimprovementsundera twotcutstrategy.economicefficiencyofmultipleharvestregimesrequirefurther investigation[35]sincemultipleharvestingalsointroducesanadditionalcostinthecrop thatmustbeconsidered[30].othercropssuchasmiscanthusspp.arebestusedundera singletharvestregime,asitrespondspoorlytomultipletcuts,evenonhighlyfertilesoil[61]. FormanyspecieswithinOntario,suchasP."australis,itisstillunclearifmultipleTcutsare possible. UsingbiomassforbiogasproductionhassomebenefitsoverotherendTuses.Biogas feedstocksdonotrequiredrying,asdomanyotheralternativeendtusesofbiomass(e.g. pelletizingforthermalenergy,theproductionofmanybiomaterials).theabilitytouse greentharvestedbiomassissimplerthanrequiringadryingprocess,andcanmeanhigher biomassyieldsthanbiomassharvestedafterintfieldoverwinteringwhereinbiomassislost 13

25 intheformofleafmatter.thesefactorsmakebiogasproductionanattractiveendtusefor perennialgrassbiomass. 1.3 BiogasfromGrassBiomass Researchersareworkingtobetterunderstandtherelationshipbetweenbiomass characteristics,suchaschemicalcomposition,anddigestibilitywithafocusonbiogas production.inherbaceousplants,thecellwallaccountsfor40to80%ofthedmbiomass, dependingonspecies(table1.3),cultivar,andplantmaturity[31].plantcellwallsare matricesofcellulose,hemicellulose,andpectin(polysaccharides),withembeddedlignin(a phenolicpolymer)thatprovidesstrengthandstructureaswellasprotectionagainst pathogensandherbivory[31].lignocellulosicfeedstocksaresonamedafterprominent traitsintheircellwalls.whilecellwallcomponentscontributetooverallbiomass,their recalcitranceprotectsalargeproportionofbiomassagainstmicrobialdigestion,lowering biogasyields." Someplantcharacteristicshavebeenshowntocorrelatewithbiogasproduction, thoughstandardizedbiologicalmethanepotentialassaysarestillthebestwaytoevaluate methanepotential.theconversionefficiencyofcelluloseandhemicellulosetobiogas dependsonthespeciesbeingstudied,andcorrelatewiththeratioofpolysaccharidesto ligninconcentration[10].furthermore,increasesindmcontentandlignificationhavebeen showntonegativelycorrelatedbiogasquantityandquality[62,63] SpeciesVariationinBiomassQuality Biogasproductionpotentialvariesdependingonspeciesandcultivarused(Table 1.4).Generally,withoutpreTtreatment(seeSection3.4),specieswithhigherligninandDM contentarelessdigestible,andtheytendtoproducelessbiogasperunitfeedstock.for 14

26 instance,miscanthusspp.isahighbiomassyielding,highlytlignifiedgrass,withlow potentialasabiogascropwhenusedwithoutprettreatment[61].however,steampret treatmentsofmiscanthusspp.canallowforhighbiogaspotentialtobereached(374nlch 4 kg T1 VS)[34],withbioagasyieldssimilartothatofZ."mays"(289T390NLCH 4kg T1 VS)[29,38]. Speciesandcultivarsofmanyperennialgrassspecieshavebeencomparedtoone another,includingseveralmiscanthus"species(miscanthus"x"giganteus"and"miscanthus" sacchariflorus"[10]).furthermore,wildp."arundinacea"biomasshasbeencomparedto commercialcultivarsofp."arundinacea"[62]."inthesesidetbytsidecomparisons,miscanthus" sacchariflorusproducedbiogasatnearlydoubletheratethanmiscanthus"x"giganteus"[10], andthep."arundinaceacultivarproduced>3xthemethaneasthewildtype[62].these differencesinbiogaspotentialinconsiderationwiththelimitedamountofbreedingthat hassofarbeenconductedonthesespeciessuggestthatthereispotentialforcreating cultivarsoflignocellulosicgrasseswithgreaterdigestibilitythanwhatiscurrentlyavailable HarvestTimingandBiomassQuality Variationsinthechemicalcompositionofbiomassoccuracrossdifferentharvest times,andtherefore,propertimingofharvestplaysacriticalroleontheproductionof methane.harvesttimeplaysanimportantroleonthebiomassquality,impactingfactors thatareconsideredimportanttobiodegradabilityandbiogasproduction.forz."mays, optimalharvesttimeforbiogasproductionhasbeenwellestablishedtobeatthevegetative stageof waxripeness "[38].However,suchtimingisnotaswellunderstoodfor lignocellulosicbiomasscrops.changesovertimeareexpectedintheshoottoleafratio,the carbontonitrogenratio,theamountofnontstructuralcarbohydrates,andcellwall components[59].biogasproductionisnegativelyaffectedbyharvestingperennialcrops suchasa."donaxatlaterstagesofmaturity[59]. 15

27 GreaterleafinessinZ."mayshasbeenlinkedtogreaterin"vitrodigestibility[64].The chemicalcompositionofperennialgrassfeedstocksbecomesprogressivelylessfavourable forbiogasproductionoverthecourseofseasonalmaturationascropmoisturecontent decreases.aswitnessedwithp."arundinacea,throughouttheseasoncropsbecomeless leafy,andintermsofpercentdm,nitrogenandashcontentdecreases,whilelignin, cellulose,andhemicellulosecontentincrease[37].forexample,lignincontentcanvary seasonallyfrom133to173gkg T1 DMinP."virgatum,andfrom109to148gkg T1 DMinP." arundinacea,forearlyandlateharvestsrespectively[65].inp."virgatum,celluloseand hemicelluloseconcentrationshaveagetdependentrangesfrom273to322gkg T1 DM,and from235to279gkg T1 DM,respectively[19].Theoptimalharvesttimeforreaching maximumadpotentialisthereforedependentonmanyvariables,butitisanessentialstep indeterminingthefeasibilityofusingperennialgrassesasbiogasfeedstocks EffectsofMultipleHarvestsonBiogasYield CertainspeciessuchasP."arundinaceacanbeharvesteduptofourtimesayear. Digestibilityhasbeendemonstratedtoincreasewithcuttingfrequency[37,60].However, thevaluethatmattersmostisnetenergyproductionperhectare.thebiogasandbiomass yieldsmustbeassessedwithconsiderationofenergyinputs. Methaneyieldcanbemuchhigherforplantsthatareharvestedearlierinthe season,orthosethatareharvestedforasecondtimeinasingleseason,duetomaturity factorssuchaslignification.forp."arundinaceaandp."virgatum,methaneyieldsdecreased significantlywithgreatercropmaturity,andanoptimalmanagementstrategymayrequire twoharvestspergrowingseason(oneinsummerandasecondinautumn)[35,37].atwot cutharvestingregimecouldresultinapproximately25%greaterbiogasyieldperhectare forp."virgatum,thoughnetenergyandeconomicefficiencieshavenotyetbeenevaluated 16

28 [35].ArundodonaxalsoshowspromiseforbiogasproductionunderdoubleTcuttingregimes [61] StorageandPrebtreatmentOptionsforBiomass FeedstockStorage Harvestedfeedstocksneedtobestockpiledforusethroughouttheyear(oftenuntil thenextharvest),whichrequiresastorageandpreservationmethodologytobeemployed. Therearetwogeneralmethodsofpreservationforenergycrops:ensilingfreshmaterialand harvestingdriedbiomass.foruseinbiogasproduction,ensilingisthemorelikelystrategy. Ensilingisabiologicalprocessthatrequiresbiomasstobeharvestedat25to35% moisture,choppedtoaparticlesizeof10to20mm[1],compactedtodensityrangingfrom approximately150to250kgm T3 [66],andsealedfromaircirculation.Assoluble carbohydratesareconvertedintolacticacidandvolatilefattyacid,phofthebiomass decreasestobetween3and4.thelowphpreventsspoilageofthebiomass,thuspreserving thefeedstockassilage[1].ensilinghasbeenshowntobeagoodmethodofstoringad feedstocksforlateruseforseveralperennialgrassspecies[28],buthasprovenmore difficultforothercropssuchasmiscanthusspp.[61]. Ensilingpartlydegradesthestructuralpolysaccharidesofthebiomass,thusacting asakindofprettreatment.however,undesirableaerobicdegradationofsubstratecanalso occurduringensiling,resultingin8to20%energylosses[1].itisthereforecriticaltoensile materialproperlyandminimizespoilageduetooxygenexposure. 17

29 PreTtreatments PreTtreatmenttechnologieshavebeenresearchedextensively,withseveralliterature reviewsdevotedtothetopic[67 70].Thereare26categoriesofpreTtreatment technologies,asdiscussedandcomparedinacomprehensivereviewbytaherzadehand Karimi[68].Thesemethodsareaimedateitherremovinglignin,solubilizinghemicellulose, reducingcellulosecrystallization,orincreasingsurfaceareaoffeedstocks[71].pret treatments"areeitherphysical(e.g.,milling,irradiation,thermal),chemical(e.g.,acid, explosion,oxidization),orbiological(e.g.,addingenzymes,ensiling)[68,71].furthermore, foreachoftheprospectiveprettreatments,therearenumerouscombinationsofvariablesto consider.forexample,thereisnogeneralconsensusonhowvariablessuchastiming, temperature,andcatalystsshouldbecombinedinhydrothermalprettreatment,andthere arecorrespondinglyvariableresultsinhydrothermalprettreatmentefficacyinthe literature[71]. Plantcellwallsarematricesofcrystallinecelluloseembeddedinthepolymerslignin andhemicellulose[67].inlignocellulosiccrops,thisstructureisveryresistantto destruction[68].dependingonspecies,prettreatmentshavebeenshowntovaryinthe effectthattheyhaveonincreasingmethaneyield"[71],butincertaincasesprettreatments cantriplethemethaneyieldofcellulosicbiomass.inoneexample,steamttreatedmiscanthus spp.produced374nlch 4kg T1 VSincomparisonto84NLCH 4kg T1 VSfromuntreated biomass[34].forcertainhighlylignifiedspeciesthatproducesubstantialbiomasssuchas Miscanthus"spp.,preTtreatmentisnotonlyoptimal,butisrequired[34].LessTlignified specieswithlowerbiomassyieldssuchasd."glomerata,"f."arundinaceae,"and"p."pretense," havepromisingbiogasyieldswithoutprettreatment[33].thisrepresentsatradetoff betweenbiomassquantityandqualitythathasbeensuggestedbyotherauthors[37,72].if 18

30 thistradetoffcanbeovercomeeitherbymanagementstrategiesasmultipleharvesting(see Section3.3),preTtreatments,orbreedingimprovements,thenitmaybepossibletoreach highquantityandqualityofbiomasssimultaneously. TheusefulnessofpreTtreatmenttechnologiescanbeevaluatedbythefollowing criteria:energycostscomparedwithenergygained,costofcapitalinvestment,associated hazardsandenvironmentalimpact[34],andscalability.aneffectiveandeconomicalpret treatmentshouldmakecellulosicfibreavailableformicrobialdigestion,avoidformationof chemicalsthatinhibithydrolyticenzymesandfermentingmicroorganisms,andavoidthe useofexpensivechemicals[68].prettreatmentssuchassteamexplosion[34],lime,liquid hotwater,andammoniahavebeenidentifiedashavingahighoverallpotential[69]. EfficacyofpreTtreatmentsisdependentonthelignocellulosesource,soaneffectivemethod foronetypeoffeedstockmaybeineffectiveforanother[68].moreresearchisrequiredto determinewhichpretreatmentsaremosteconomicallyefficientwithwhichlignocellulosic species. 1.4 SeedSurvivalDuringAnaerobicDigestion IfperennialgrassbiomassspeciesaretobeusedasbiogasfeedstocksinAD,then therisksassociatedwithspreadingviableseedsindigestatemustbeevaluated.as discussedpreviouslyinsection2.3,manyofthesespeciesarerecognizedasinvasivein NorthAmericaorhaveinvasivetraits,anddesiretoplantbiomassfromseedhasresultedin apreferenceforfertilespeciesratherthaninfertileones[45].therefore,ifseedsofgrass biomassreadilysurvivetheprocessofcommercialtscalead,thenthiscouldpresentarisk ofintroducingweedstofieldswheredigestatewasapplied. 19

31 StudiesonseedsurvivalduringADhavesofarfocussedonweedspeciesthatare presentinsmallquantitiesinharvestedbiomasssuchasz."mays"[73].thesestudieshave shownthatseedviabilityinadisspeciestspecific,withupto10%ofchenopodium"album"l." (lamb squarters)seedssurviving20daysofdigestion[74].however,mostspeciesstudied havesurvivedonlyafewdaysofdigestion[74].reactortypehasalsobeenshowntohave animpactonseedsurvivalrates.moststudieshaveusedbenchttopreactorstoassessseed survival,butcommercialtscaleanaerobicdigestershavebeenshowntobelesseffectiveat reducingseedsurvivability[74,75].thusthereisaneedtoconductcommercialtscale assessmentsofseedviability. Whileapproximately35specieshavebeenassessedforseedviabilityafterAD,no biomassspeciesandonlyfivegrassspecieshavebeeninvestigated[18,74 77].Considering thevariabilityinseedsurvivalratesbetweenspeciesthusfarstudied(withsurvivalranging from0to10%after20daysofad),andthatthegreatestriskisforthosespeciesthatare recognizedasquarantinedorinvasive[74],ariskassessmentofseedsurvivalforbiomass specieswouldproveusefultogrowers,biogasoperators,andindustryregulators. 1.5 PotentialFutureResearch ThebiomasspotentialofwildP."australis"hasnotbeenreportedintheliteraturefor SouthwesternOntario,whereitisamajorinvasiveproblem[78,79].Therehasbeenlimited researchontheuseofp."australis"asabiogasfeedstock[51,52],andnoreportedevaluation ofwhenisthebesttimetoharvestitforuseinad.furthermore,itisunclearhowan invasivep."australis"standwouldcomparetoothercultivatedbiomasscropsintermsof biomassorbiogasyield.lastly,thereisconcernformanyoftheseperennialbiomass speciesthattheirpropagationandusecouldleadtoproblemswithinvasivenessofftsite [45].Iftheseperennialspeciesaretobeusedforbiogasproduction,researchisrequiredto 20

32 evaluatethetheriskofseedssurvivingtheprocessofdigestionandbecomingweedsin otherhabitatsorfarmfields.beforep."australis"maybeconsideredforad,theseconcerns willneedtobeaddressed. 1.6 ObjectivesandHypothesis Thehypothesesofthisresearchare: 1. ThatthebiomassandbiogaspotentialofwildPhragmites"australisstandswillbe lessthanthoseofcultivatedperennialbiomassgrasscrops(m"xgiganteusandp."" virgatum). 2. ThatbiomassyieldwillincreaseandbiogasyieldwilldecreaseforOctober harvesteddatevs.julyforallgrassesstudied. 3. ThatamultipleTcutregime(JulyandOctober)willyieldthegreatestquantityof biomassforp."australisandp."virgatum,butnotform"xgiganteus. 4. Thatseedsofperennialgrassspecies(P."australis,"P."virgatum,"andPhalaris" arundinaceae)willdiewithinaweekofexposuretocommercialtscalead. Theobjectivesofthisresearchare: 1. Toevaluatebiomassyieldsofthreespecies(P."australis,"M"xgiganteusandP." virgatum)attwoharvesttimes(julyandoctober)insouthwesternontario. 2. ToassessregrowthofgrassinOctoberforareaspreviouslyharvestedinJuly. 3. Todeterminethemethanepotentialofeachspeciesandharvesttimescomparedto ensiledzea"mays,"usingalabscaleanaerobicdigester. 4. Todetermineiftherearerelationshipsbetweenplantcharacteristicsandmethane yield. 5. Toassessthelevelofriskofusingperennialgrassspecies(P."australis,"P." 21

33 arundinacea,"andp."virgatum)asasourceofbiogasfeedstockintermsofabilityfor seedstosurvivead,comparedtosolanum"lycopersicum"(tomato). 22

34 1.7 TablesandFigures Table1.1.Comparisonofbiogaspotentialforseveralgrassspecieswith42to95day retentiontimesatmesophilictemperatures(35to38 C)inlabTscalereactors(working volume0.25to30l). LatinName Prebtreatments Biogaspotential(NLCH 4kg b1 VS) Dactylis"glomerata"(L.)" Chopped 342[33] Festuca"arundinaceae"Schreb." Chopped 336[33] Miscanthus"spp." Chopped,steamed 84T347[34] Panicum"virgatum" Ensiled 191T309[35] Phalaris"arundinacea" Ensiled 187T325[36,37] Phleum"pratense"(L.)" Chopped 335[33] Zea"mays" Ensiled 289T390[29,38] anl:normalizedlitres;andvs:volatilesolids. 23

35 Table&1.2.ReporteddrybiomassyieldsforseveralperennialgrassspeciesintheUnitedStatesandEurope.AdaptedfromLewandowski etal.,2003[56]. English&Name& Latin&name& United& States& Europe& Citation& (t&dm&ha?1 &yr?1 )& Crestedwheatgrass Agropyron(desertorum(FischexLink)Schult.( 16.3 NA [80] Bigbluestem Andropogon(gerardii(Vitman( 6.8R11.9 8R15 [56,80] Giantreed Arundo(donaxL.( 35 36R55 [81,82] Tufteddigitgrass Digitaria(eriantha(Stued( 5.5 NA [83] Weepinglovegrass Eragrostis(curvula(Schrad.)Nees( 8.2 NA [83] Tallfescue Festuca(arundinacea(Schreb.( R6 [33,84] Elephantgrass Miscanthus(spp.( 29.6R38.2 7R44 [5,85] Kleingrass Panicum(coloratumL.( 5.0 NA [83] Switchgrass Panicum(virgatum(L.( 10.4R23.5 3R20 [5,58,81] Napiergrass Pennisetum(purpureum(Schum( [56,86] Reedcanarygrass Phalaris(arundinaceaL.( R13.7 [33,84] Timothy Phleum(pratense(L.( R3.8 [33,84] Commonreed Phragmites(australis(Trin.Ex.Steud.)( NA 5.9R21.8 [87] Kentuckybluegrass Poa(pratensisL( 6.1 NA [84] Energycane Saccharum(spp.( [56] Giantcordgrass Spartina(cynosuroides(L.( NA 9 [88] Prairiecordgrass Spartina(pectinata(Bosc.( NA 4R18 [88] ana:notavailable. & & 24

36 Table&1.3ChemicalcharacteristicsoffourperennialgrassescomparedtoannualZea(mays,asreportedinthescientificliterature. Constituent& Arundo'donax' Panicum' virgatum' Phalaris' arundinacea' Miscanthus'spp.' Zea'mays' %Moisture 48.8R63.2[89] 15[56] 10R23[56] 16R62[56] 58.7R63.1[90] Ash(%ofDM) 4.8R7.8[56] 4.5R10.5[56] 1.9R11.5[56] 1.6R4.0[56] 2.77R5.37[90] N(%ofDM) 0.2R0.4[56] 0.71R1.37[56] 0.45R1.54[56] 0.19R0.67[56] K(%ofDM) 0.65[91] 1.28[91] 0.06R0.81[56] 0.31R1.28[56] 1.06R1.3[90] Ca(%ofDM) 0.33[91] 0.28R0.73[56] 0.08R0.32[56] 0.08R0.14[56] 0.26R0.4[90] Cl(%ofDM) 0.22[91] 0.15[91] 0.01R0.30[56] 0.10R0.50[56] 0.30R0.31[90] S(%ofDM) 0.22[91] 0.12[56] 0.06R0.11[56] 0.04R0.19[56] 0.12R0.15[90] Si(%ofDM) 1.39[91] 1.50[91] 0.56R3.70[56] [56] Proteins(mgg R1 VS) 51.2[13] 23.8[13] 64.7[13] Lipids(mgg R1 VS) 9.5[13] 9.6[13] 33.6[13] Solublesugars(mgg R1 VS) 24[13] 39.9[13] 63.7[13] Starch(mgg R1 VS) 39[13] 54[13] 319[13] Cellulose(mgg R1 VS) 315[13] 283[13] 130[13] Hemicellulose(mgg R1 VS) 237[13] 235[13] 123[13] AcidInsolubleLignin(mgg R1 VS) 193[13] 177[13] 77[13] 25

37 Table&1.4.ListofperennialgrassspeciesresearchedforbiogaspotentialinNorthAmericaandEurope. Common&Name& Species&Name& Country& Meadowfoxtail Alopecurus(pratensis( Germany[92] Giantreed Arundo(donax( Italy[71] Cocksfoot Dactylis(glomerata((L.)( Finland[33],Germany[92] Tallfescue Festuca(arundinaceae(Schreb.( Finland[33] Perennialryegrass Lolium(perenne( Germany[92] Miscanthus Miscanthus(spp.( Denmark[93],Norway[34],Poland[10] Switchgrass Panicum(virgatum( Canada[94],USA[63] Napiergrass Pennisetum(purpureum(( USA[21] Reedcanarygrass Phalaris(arundinacea( Canada[36],Denmark[37],Finland[33], Latvia[95],Poland[62] Timothy Phleum(pratense((L.)( Finland[33] Commonreed Phragmites(australis( Finland[96] Sorghumsilk Sorghum(x(almum(Parodi)( Italy[13] 26

38 2 Chapter( Methane(Potential(of(Phragmites+australis,+ Miscanthus(x(giganteus+and(Panicum+virgatum(Grown(in( southern(ontario,(canada( 2.1 Abstract:( Theproductionofbioenergyfromplantbiomasshasthepotentialtoreducefossil fueluse.thenumberofanaerobicdigestionfacilitiesaroundtheworldproducinguseable CH 4hasrisendramatically,leadingtodemandforfeedstocks.Phragmites+australis,an invasiveperennialgrassspeciesinsouthwesternontario,canada,thatformslarge monoculturalstands,wasevaluatedasabiogasfeedstockcomparedwithtwobiomass crops:miscanthus+x+giganteus+andpanicum+virgatum.biomassyieldsforp.+australis+ averagedapproximately1.82±0.9kgdrymatter(dm)m N2,thiswascomparabletothatofM. xgiganteus,whichrangedfrom1.28±0.15to2.43±0.32kgdmm N2 injulyandoctober, respectively.phragmites+australis+ch 4yieldwasgreaterthanP.+virgatum+CH 4yield,which rangedfrom0.49±0.06to0.69±0.07kgdmm N2 injulyandoctober,respectively.+in mesophilicbenchntopdigesterexperiments(n=3),methaneyieldsweregreaterforjulyn thanforoctobernharvestedperennialgrasses,rangingfrom172.4±15.3to229.8±15.2 normalizedliters(nl)ch 4kg N1 volatilesolidsforallperennialgrasses.theseyieldswere lowerthaninthecontrolsamplesofzea+mayssilage,whichproduced334.9±4.3nlch 4kg N1 volatilesolids.biogasperhectareyieldswerehighestforoctobernharvestedm.xgiganteus, followedbyjulynharvestedm.xgiganteus+andp.+australis,whilebothp.+virgatumharvest timesyieldedlowerthanotherspecies.panicum+virgatumshowedlesspotentialthanother speciesintermsofbothitsbiomassyieldanditsbiogasyieldperhectare.theseresults indicatethatp.+australis+generallyisnotaneconomicallyviablebiogasfeedstockoption. 27

39 + Keywords:(Anaerobicdigestion,commonreed,methane,perennialgrass,biomass Abbreviations:BMP,biologicalmethanepotential;DM,drymatter;FM,freshmatter;NL, normalizedliters;ts,totalsolids;vs,volatilesolids. 2.2 Introduction:( ThenumberofbiogasfacilitiesinCanadaandaroundtheworldhasincreasedwith mostofthe33biogasplantsinoperationinontariohavingbeencommissionedinthelast 10years[6,94].Astheindustrycontinuestogrowsodoesthedemandforsustainable, reliable,andaffordablefeedstockswithhighmethaneyields.energycropsarecurrently usedasbiogasconsubstrates,with0.5millionhectaresofbiogasmaizegrowningermany alone[14].energycropsarerecognizedashavingthemostpotentialforuseasbiogascon substratesintheeuropeanunion[1].theuseofenergycropsinanaerobicdigestioncan improvebiogasyields,provideareliableandconsistentfeedstocksupply,andovercome someofthelimitationsofmonondigestion,namelybyimprovingthec:nratioandthe bufferingcapacity[97]. Therehasbeenconsiderablescientificresearchontheuseofperennialgrassspecies asfeedstocksforuseinanaerobicdigestion,includingthepublicationofover30peern reviewedarticlessince2000.perennialgrassspeciescanbeharvestedformanyyearsafter plantingduetotheirrootmasses,whichstorenutrientsfromyearntonyear.althoughdozens ofgrassspeciesarebeingscreenedforsuitabilityindifferentclimatesaroundtheworld, threeperennialgrassesarethefocusofresearchinnorthamerica[98],includingpanicum+ virgatum(switchgrass)[32,53],miscanthus+spp.(miscanthus)[57]andphalaris+ arundinacea(reedcanarygrass)[84]. 28

40 TheUSDepartmentofEnergychoseP.+virgatumasamodelplantspeciesfor lignocellulosicbioenergycropsinthe1990s.panicum+virgatumiswelladaptedtonorth America,withlowfertilizerrequirementsandgoodresistancetodiseaseandinsectpests [54].+Researchershaveinvestigatedvariousharvesttimings[7],evaluatedmultipleharvests inasinglegrowingseason[35],comparedthebiogaspotentialofdifferentcultivars[10], andassessedtheeffectsofvariouspretreatmenttechnologiesonenhancingbiogasyield [70]. Typically,positivebiomasscropcharacteristicsincludinghighyield,lowproduction cost,andabilitytogrowonmarginallands,arealsocharacteristicsofinvasivespecies. Consideringinvasivespeciesasasourceofbiomassmaymonetizeecologicalcontrolwith simultaneousbioenergyproduction,providingincentiveformanagementthatotherwise maynotexist.thereisaneedtostudybiogaspotentialofsuchinvasivespecies. ThisstudyinvestigatedthebiogaspotentialofPhragmites+australis(Cav.)Trin.ex. Steud.(CommonReed),aninvasiveperennialgrassspeciesinNorthAmerica.Phragmites+ australisisfrequentlyfoundinthegreatlakesregion,includingmorethan24,600haof wildmonoculturalstandsmappedalongtheamericansideofthelakesalone[99].this specieshasbeenharvestedforuseinthatchedroofsandbiomaterialsineasterneurope andafrica[100],andhasanestimatedmarketvalueofapproximately$us45,000ha N1 [50] inbotswana. ThesametraitsthatmakeP.+australisinvasivemakeitattractiveasabiomass species.thesetraitsincludeperennialregrowthfromrhizomes,abilitytospreadfrom seeds,growthonmarginallands,abilitytooutcompetemanyotherplantspecies,and havingfewpests[100].reportedbiomassyieldsforp.+australisvaryconsiderablybasedon location,rangingfrom0.3kgdmm N2 fromasiteinsweden,0.7kgdmm N2 inthebritishisles 29

41 [101],1.5kgDMm N2 inthenetherlands[102]to4.2kgdmm N2 inromania[103].inone experimentinscotland,yieldsvariedconsiderablybetweenthreesitesinthesameregion, rangingfrom0.7to4.0kgdmm N2 [104].However,researchonboththeuseofP.+australis forbiogasandonitsabovengroundbiomassyieldsinnorthamericaislimited.considering thevariabilityinp.+australis+biomass,thereisaneedtodeterminetheproductivityofp.+ australis+innorthamerica. BenchtopreactorexperimentssuggestthatP.+australis+maypotentiallybeasuitable consubstrate,producing220lch 4kg N1 VSfeedstock[52].ResultsfromaSwedishstudy whichcalculatedefficiencycostsincludingharvest,transport,storage,andtreatment, concludedthattherewasanenergyoutputof4.36mjkg N1 DMusinganaerobicdigestion [105].Phragmites+australishasalsobeenchosenforresearchasacoNsubstrateforitsability toimprovethec:nratioduringdigestion[97].aseparateswedishstudyevaluatingthe economicsofusingwildp.+australis+asabiogasfeedstockcalculatedanetlossof$us720to 2400perhaharvested[51].Theseauthors[51]didnotincludepotentialsocialand environmentalbenefits(suchascarbonsequestration,andwaterremediation[103,106])in theircalculations. ManybiomasscropyieldestimateshavebeenmadeintheUSandinEurope,as compiledinareviewbylewandowskiet+al.[56].ofparticularinterestisthepossibilityof cuttingperennialgrassesmultipletimeswithinasingleyear,allowingforregrowthtobe harvestedinordertoincreaseannualyields.thispossibilityofmultiplenharvestsfor perennialgrasseshasnotyetbeenextensivelyexaminedinnorthamerica,andtheoptimal harvesttimeofmanyperennialgrassspeciesinnorthamericastillneedstobedetermined [69].Whileperennialgrasscropbiomassyieldsincreasethroughoutthegrowingseason, thebiogasyieldistypicallyadverselyaffectedbyincreasingconcentrationoflignocellulose 30

42 inthebiomass[36].iftheoptimalharvesttimingofperennialgrassesforuseasbiogas feedstockistobedetermined,thenbothbiomassandbiogasyieldmeasurementsneedtobe considered. Theobjectivesofourstudywere:toquantifytheamountofP.+australisbiomass fromwildstandsattwoharvesttimesinsouthwesternontarioincomparisontotwo biomasscrops(m.xgiganteus+and+p.+virgatum);tomeasurethebiogasproducedfromthe threeperennialgrassescomparedtozea+mays+l.silage;andtocalculatethebiogasyieldper unitarea.wehypothesizedthateachspecieswouldbedifferentintermsofbiomassyield, biogasproduction,andbiogasproductionperunitareaharvested.wealsohypothesized thatbiomassyieldswouldincreasefromsummercomparedtofallharvestdates.ourfinal hypothesiswasthatanincreaseinlignificationineachgrasswouldbecorrelatedwith decreasedbiogasyields. 2.3 Materials(and(Methods:( Site(History(&(Field(Sampling:( Toassessthebiomassandbiogaspotentialof+P.+australisinSouthwesternOntario, threewildstandswereselectedandcomparedtothreecultivatedfieldsofthebiomass cropsmiscanthus+x+giganteusgreef&deuterexhodkinson&renvoizecultivar Nagara (miscanthus),andp.+virgatum+l.cultivar CaveNinNRock (switchgrass).allcultivatedfields (Figure1)hadreachedstandmaturity,withestablishmentoccurringatleastthreeyears priortothebeginningofthisstudy(personalcommunication:deantiessenandrob Buchanan).Afterestablishment,P.+virgatumandM.xgiganteus+cropsreceivednofertilizer orpesticideinputs.allm.xgiganteussiteswerelefttoovernwinterinthefieldandwere harvestedannuallyinthespring,whereasp.+virgatum+siteswereeitherfallharvestedornot harvestedaspermanentprairie.phragmites+australissiteswereselectedfromwildinvasive standsthatwerenotmaintainedorharvested,thoughnaturalburningeventshadoccurred 31

43 previouslyatbothsitesnearlakestclair(figure2.1).selectedp.+australissiteswere absentofthenativep.+australis+phenotype,typicallynonnflooded,andpreferencewasgiven tolargersiteswithlownlikelihoodoferadicationduringthetwonyearstudy.preparedz.+ mays+silagewascollectedfromuniversityofguelphridgetowncampus,andbiomassyield assumedatavalueof1.85kgdmm N2 [107]. In2013and2014allsitesweresampledinearlyJulyandmidNOctober.These samplingtimeswerechoseninordertoevaluatebiomassyieldpotentialofp.+australis harvestedtwiceperyear[41].in2013,noregrowthwasobservedafterthejulyharvest, andmultipleharveststrategieswerenotinvestigatedin2014. Ateachsiteandtimepoint,plantsampleswereharvestedusinga1m 2 quadratplaced randomlyatdistances>3mfromothersampleareasandfromthefieldedge(n=3).the meanofthreequadratsateachharvesttimewasusedasthesamplevalueforeachsite. Quadratswereassembledonthesoilsurfaceandpruningshearswereusedtomanuallycut thebiomassapproximately10cmabovegroundlevel.ateachharvest,freshbiomasswas weighed(activescale,ohaus1n10;canada),stemscountedandtheirlengthsmeasured.a subsamplewascutto<25cmsegments,frozen,andstoredatn20 CfortheBiological MethanePotential(BMP)experiments,andasecondsubsamplewasdriedfor48hoursat 60 CinacustomNbuiltwalkNindryer.Subsampledryweightwasrecorded,andleaveswere separatedfromstemsattheliguleandweighedseparately.driedsamplesfromjulyand Octoberof2013werefinelygroundtoamaximumparticlesizeof2mm(MillModel4, ThomasScientific,USA)andsenttoSGSAgrifoodLaboratoriesInc.(Guelph,Canada)forwet chemistryconstituentanalysis. 32

44 2.3.2 Biological(Methane(Potential(Assay:( DigestateusedforBMPassaywastakenfromthe250kWcommercialanaerobic digester(ad)attheuniversityofguelphridgetowncampus.characteristicsofthedigester dietvariedineachrun(table2.1),whichistypicalofcommercialnscalead.digestatewas storedanaerobicallyat38 Candagitateddaily(foradurationof~1minute)forthree weekspriortoinitiatingeachtrialinordertominimizebackgroundgasproductionfrom inoculumduringtheexperiment. Aloadingrateof1:2volatilesolids(VS)ratioofsubstrate(grassbiomass)to inoculum(digestate)wasused[59].totalsolids(ts)andvsweredeterminedby combustionusingamufflefurnace(lingbergbluem,thermoscientific,usa)accordingto standardmethods[108]. Frozengrasseswerefinelychoppedtoatargetparticlesizeof1cmwithscissors [33],andweremixedwithdigestatetoafinalvolumeof400mLin500mLglassbottles (650mLeffectivevolumeincludingheadspace[109])(BioProcess,Lund,Sweden).ThepH ofthecontentsofeachbottlewasrecordedbeforeandaftertheexperimentusingaseven ExcellencepHmeter(MettlerToledo,Switzerland). AfterpreliminaryanalysisonDMbiomassyieldrevealednosignificantdifferences betweensites(p=0.17),locationschosenforbmpanalysiswereselectedonthebasisof quantity(asweight)offrozensubsamples.acompletelyrandomizedblockdesignwas utilizedwiththreeruns;eachrunincludedmaterialfromallplantspecies,atbothharvest timenpointsfromthe2013samplingyear,fromonesamplelocation(figure2.1)(n=6). Samplesfrom2014werenotassessedforBMPduetotimeconstraintsandagenerallackof yeareffectorinteractionsonbiomassfmordmyield(table2.3).inaddition,threebottles ofz.+mayssilagewereincludedineachruntoassessintranrunvariability,andadigestaten 33

45 onlycontrolbottlewasincludedtomeasureresidualgasproductionfrominoculum, resultingintenbottlesperrun. Bottleswerekeptat38 CinaBioProcessAMPTSII(Lund,Sweden)waterNbathand weremechanicallystirredfor60seconds(mixerspeedat80%)followedby60seconds withoutstirring,forthedurationoftheexperiment.individualbottleheadspaceswere flushedusing250mlofnitrogengasafterbeingsealedinordertopromoteanaerobic conditions.biogasproducedwaschannelledthroughco 2Nfixationbottlescontaining0.5% phindicatingsolution(0.4%thymolphthaleininethanolindistilledwater)and95.5%3m NaOHsolution[109].Methaneproductionwasmeasuredcontinuallyvialiquid displacementandbuoyancyusinganamptsiibioprocessflowcellarrayanddata acquisitionunit.onaweeklybasis,1mlgassamplesweretakenfromtheheadspaceof eachbottleandimmediatelyassessedforbiogascompositioninasri8610cgas chromatographequippedwitha1.8m,1.83mx3.18mms.s.molecularsieve(13xpacked column)(sriinstruments,usa),andathermalconductivitydetectorsetat150 C.Oven temperaturewaskeptat40 Cduring11minassessment,withheliumasacarriergasata flownrateof20mlmin N1. Eachexperimentranfor21days,atwhichpoint>90%ofgasproductionwaslikelyto haveoccurred[93].afterdigestion,subsampleswerefrozenandsenttosgsagrifood Laboratories(Guelph,Canada)forconstituentanalysis,andTSandVScontentdetermined. DigestionofgrassbiomasswasdeterminedbycalculatingthedifferencebetweenpreNand postndigestionvaluesfortsandvs Statistical(Methods:( DatawereanalyzedusingSPSSStatistics(Version21),withanerrorratesetas0.05. Forthefieldstudy,theindependentvariableswere:species(fixed),harvesttime(fixed), 34

46 year(fixed),fieldsite(random),andinteractionsthereof.dependentvariableswere:fresh matter(fm)yield,drymatter(dm)yield,shootheight,stemsperm N2,percentleafmatter, andchemicalcomponents.dataweretestedfornormality,independenterror,homogeneity, andoutlierswithgrubbs test.datathatdidnotmeettheassumptionsweretransformed usingeithersquarerootorbasen10logarithmicandbackntransformedforpresentationof results.fournwayanovaswereusedtoassesseachdependentvariableagainstsite,year, speciesandharvesttime.tukey srangetestswereconductedtoassesstheeffectofharvest timing. FortheBMPexperiment,theindependentvariablesspeciesandharvesttimewere treatedasfixedeffects,runwastreatedasarandomeffect,andthedependentvariables weremethaneyield,percentmethane,andvsreduction.interactionsforharvesttimeby species,harvesttimebyrun,andrunbyspecieswerealsotested.dataweretestedfor normality,heterogeneityoferror,andindependenterror.aonenwayanovawasconducted onthetriplicatevaluesofz.+mays+withnlch4kg N1 VSasthedependentvariable,andrunas theindependent.atwonwayanovawasconductedtoevaluateperennialgrassspecies methaneyieldsvs.harvesttime.tukey srangetestswerecalculatedforalltreatments. Principalchemicalcomponents(cellulose,hemicellulose,lignin,andprotein;Table2.8)and methaneproduction(nlch 4kg N1 VS)wereregressedforlinear,quadraticandcubicmodels. LackNofNfittestswereconducted. 2.4 Results:( Biomass(Yield(and(Chemical(Composition( AthreeNwayANOVAonthefielddatashowednoyeareffectforFMorDMbiomass yieldorothercharacteristics(table2.2).drymatteryieldsform.xgiganteus+(1.85±0.24kg m N1 )andp.+australis+(1.82+±0.09kgm N1 )werenotstatisticallydifferent,whereasp.+virgatum+ (0.59+±0.05kgm N1 )hadloweryields(table2.2).drymatteryieldform.xgiganteus+ 35

47 increasedwhentheharvestwasdelayedfromjulytooctoberbutnotforp.+virgatum+or+p.+ australis+(table2.2).+freshmatteryieldsweredifferentforeachspecies,wherem.x giganteus+(4.85±0.32fmkgm N2 )hadthehighestyield,followedbyp.+australis+(3.80±0.25 FMkgm N2 )andp.+virgatum+(1.56±0.12fmkgm N2 )(Table2.2).+ Therewasaninteractionbetweenspeciesandyearforstemcount,plantheight,and percentleafmatter(table2.3).noothereffectsorinteractionswerefoundforyear. Phragmites+australis+stemcountwas33%higherin2013comparedto2014,whilethere wasnodifferenceamongyearsforp.+virgatum+orm.xgiganteus.therewasnodifferencein Panicum+virgatum,P.+australis+andM.xgiganteus+inplantheightinpercentleafmatterin 2013and2014(Table2.3).Phragmites+australis+hadthelowestpercentageofleafmatter, andallspecieshadlowerpercentagesofleafmatterinoctobercomparedwithjuly(table 2.2).StemsofP.+australis+thathadoverwinteredfromthepreviousyearaccountedfor14.5 ±4.8%ofthefreshmatterbiomass(datanotshown).Panicum+virgatum+hadtheshortest averageplantheight,followedbym.xgiganteus+andp.+australis,at103(±8.3),192(±10), and274(±5.9)cm,respectively.correspondingly,p.+virgatum+hadthehigheststemcount, followedbym.xgiganteus+andp.+australis,at283,93and75stemsperm N2,respectively (Table2.4). Thereweredifferencesbetweenspeciesandharvesttimesforcertainchemical constituents(table2.2andtable2.5).specifically,p.+australis+biomasshadlower concentrationsofhemicelluloseandhigherconcentrationsofsodiumthanbothother perennialgrasses,percentdmwashighestinm.xgiganteus+andnotdifferentthanp.+ virgatum,andproteinwaslowestforp.+virgatum(table2.2andtable2.5).manganese concentrationwashigherinp.+australis+thaninp.+virgatum,thoughm.xgiganteuswasnot differentthaneither(table2.5).intermsofmaineffects,therewasanincreaseof16.2%in theoveralldmcontentbetweenjulyandoctoberharvests(table2.2).therewasa 36

48 significantreductioninoverallproteincontent,from90.8(±11.2)gkg N1 DMinJulyto50.5 (±5.5)gkg N1 DMinOctober(Table2.2),aswellasreductionsintheconcentrationofcopper, nitrogen,andpotassiumbetweenjulyandoctoberharvestdates(table2.5).nitrogen concentrationvariedbyspeciesandharvesttime,withm.xgiganteus+droppingfrom17.9 (±0.9)to7.4(±0.9)gkg N1 fromjulytooct,respectively,butnochangeinotherspecies (Table2.5).Severalchemicaltraits,includingcalcium,cellulose,iron,ligninandzincwere notinfluencedbyplantspeciesorharvesttime(table2.5andtable2.6). Thoughitcouldnotbeincludedinthestatisticalanalysisduetolackofreplication, Z.+mays+silagedatatendedtobedifferentthanthoseoftheperennialgrasses.Specifically, comparedtoothertreatmentsinourstudy,z.+mayssilagehad:loweramountsofcellulose, hemicellulose,iron,lignin,anddm;andcontainedthehigherconcentrationsofcalcium, copper,andphosphorus Biological(Methane(Potential(Assay( MeanpHofdigestateNgrasssolutionsdidnotvarybetweenexperimentalgroups (p=0.641),butdidchangeoverthedurationoftheexperiment(p<0.001):phwasinitially 7.9±0.04,andwasreducedto7.5(±0.02)bytheendofthestudy.ThedigestateNonlycontrol maintainedaconstantphof8.1±0.1(0.965).meanelectricalconductivityformixtureswith perennialgrasssampleswas17.12±0.32mmhoscm N1 anddidnotvarybetween experimentalgroups(p=0.638)orduringthedurationoftheexperiment(p=0.163).after 21daysofeachrunthemethaneproductionratesrangedfrom1.1to1.5NmLCH 4g N1 VS day N1. Cornsilagebiogasyieldswere337.0±7.2,347.4±6.0,and324.6±1.7NLCH 4kg N1 VS forbmprunsone,two,andthree,respectively,thoughnodifferenceswerefoundamong runs(p=0.089).methaneyieldvariedamongperennialgrassspeciesonadmbasis (p=0.049),withp.+australis+havingloweryieldsthanm.xgiganteus+andp.+virgatum(table 37

49 2.7).Harvesttimealsoaffectedmethaneyields,withtheJulyNharvestsamplesyielding greaterquantitiesofmethanethantheoctobernharvestsamples,withdifferencesof26,23, and6%basedonvs,dm,andfm,respectively(table2.7).therewasnodifferencebetween harvesttimeforbiogasyieldsperm 2,percentmethane,orVSreduction.Therewasa significantinteractionbetweenspeciesandharvesttimeformethaneyieldperarea harvested(table2.7).themethaneyieldperareaofp.+virgatumshowednodifference betweenjulyandoctoberharvesttimes,whereasp.+australis+yieldsdecreased,andm+x giganteus+yieldsincreased(correspondingtoabiomassyieldincrease,table2.4). Biomassyieldhadagreaterimpactonmethaneyieldperareathanwasmethane yieldperkgvsoffeedstock.whileoverallp.+virgatum+hadthehighestbiogasyieldonadm basis(114.5±11.3(nlch4kg N1 DM),ithadthelowestperNareayield(116.7±12.4NLCH 4m N 2)atlessthanhalfofthenextclosestspecies(P.+australis,+249.9±33.3NLCH 4m N2 )(Table 2.7).FallNharvestedM+xgiganteus+hadthehighestpotentialNLCH 4yieldm N2 (371.4±19.8) comparedtotheotherperennialspecies(table2.7).however,thepernareayieldsofall perennialspeciesweremuchlessthanthoseofz.+mays,whichhadpotentialfor581.2±7.5 NLCH 4yieldm N2.ThesameistrueforZ.+mays+yieldsintermsofVS,DM,orFM(Table2.7). Lignin,cellulose,hemicellulose,andproteinconcentrationoftheperennialgrasses wereregressedwithmethaneproduction(nlch 4kg N1 VS)(n=6).However,onlyligninhada significantrelationship(p=0.013),whereinincreasesinlignincontentwerenegatively correlatedwithmethaneproduction,asexpressedinthequadraticmodelintable Discussion:( Harvest(and(Biomass(Yields( WedidnotobserveanyregrowthbetweentheJulyandOctoberharvesttimesfor anyofthespeciestested.thismayhavebeenduetothelowcuttingheight,leavingonly10 cmofstubble.othershaverecommendedastubbleheightof20cmforseasonalregrowthto 38

50 occurinp.+virgatum+[32],or25n30cminp.+australis+[110].+possiblythelowcuttingheight forharvestusedinthisexperimentmayhaveinfluencedtheamountofregrowth.thelack ofregrowthmayhavealsobeenduetotheharvesttimenpointsthatweselected,ortothe shortergrowingseasoncomparedtostudiesintheusa,ortolimitedsoilnitrogen[56]. RegrowthhasbeenreportedforP.+australis[110]andP.+virgatum+[36],whichresultedina 25%increaseinmethaneyieldperunitareainthecaseofP.+virgatum.ForlongNtermstand health,miscanthusisbestmanagedwithdelayednharvests,typicallyinthewinterorspring afterthegrowingseason,allowingtimebetweentheendofthegrowingseasonandharvest [111].Therefore,whilemoreresearchisrequiredtodeterminetheprecisevariablesthat preventedregrowthinourstudy,suchlackofregrowthmaybeseenasapossible advantageforusingandcontrollingwildp.+australis+stands. OurM+xgiganteusbiomassyieldsof1.28±0.15to2.43±0.32kgDMm N2 inthisstudy weresimilartothosereportedinillinois(1.4to4.4kgdmm N2 )[112].Panicum+virgatum yieldsinourstudyof0.49to0.69kgdmm N2 weresimilartothosefoundinotherstudiesin easterncanada,rangingfrom0.40to1.26kgdmm N2 [35,36].Phragmites+australishasbeen showntohavehighlyvariableyieldsdependingonlocation,rangingfrom0.7to4.0kgdm m N2 atthreescottishsites[104].phragmites+australisyieldsinourstudyfellwithinthis range(mean1.83kgdmm N2 ).ResultsforstudiesonP.+australisinScotland[104]and Sweden[113]hadmaximumDMyieldsinlatesummer(AugustandSeptember),though therewasnoincreaseinyieldbetweenthejulyandoctoberharvestdatesinourstudy.the stemsthatremainedstandingfromthepreviousyearincreasedtheinitialbiomass measuredinourstudy,possiblyaccountingforwhyinitialbiomassyieldwascomparableto thelaterharvestdate.phragmites+australis+stemswhichvisuallyappearedtohave overwintered+accountedfor14.5±4.8%ofthefmbiomassinthesamplesofourstudy, whichpossiblytranslatestoamuchgreaterpercentageonadmbasis,sincethesestems 39

51 appearedtobeextremelydry.however,separatedmmeasurementswerenottakenfor seasonalandoverwinteredstems,duetotheconstraintsofourexperimentaldesign. TheoptimalDMcontentformaterialtobeensiledisbetween28and35%[114].At thetimeofjulyharvests,m+xgiganteusandp.+virgatumwereatthelowendofthisrange (27.2±3.2and28.2±2.1%DM,respectively),andP.+australiswasdrierat42.7±1.7%(Table 2.2).ThusthehighermoisturecontentofallspeciesinourstudyintheJulyharvestrelative tooctoberharvestsuggestthattheoptimaltimeforharvestingthesespeciesforsilage purposesinontariomaybeatsomepointinearlyormidsummer(e.g.june). Panicum+virgatum+haddifferentcharacteristicsthantheothertwoperennial grasses,having:theshorteststems,higheststanddensity,andtheleafiestbiomass(table 2.4).+Panicum+virgatumhadthelowestproteinconcentrationoftheplantsinourstudy (Table2.2).ThedatafortheaforementionedvariableswemeasuredinP.+virgatumwere consistentwiththeliterature[32],suggestingthatp.+virgatum+maybeapoorchoiceasa modelorganismforperennialgrassbiomassspecies,asitisnotnecessarilyrepresentative ofothertallperennialgrasses. BothharvesttimesofP.+australis+hadlowerconcentrationsofhemicellulose,ranging from238.4±2.9to252±3.0gkg N1 ofdm,thantheotherspecies(table2.2).thesevalues werelowerthantheonlyotherreportedhemicellulosevalueforp.+australis(320gkg N1 of DM)[96].Thisdifferenceinhemiceullulosecontentcouldbeduetodifferencesinharvest timing,biogeographicandnaturalvariation,asthepreviousstudywasfromfinland (harvesttimingnotreported).also,p.+australis+potassiumandproteinconcentrations (Table2.2andTable2.5)tendedtobehigherinsamplestakeninthesummerthaninthe fall,atrendthatislikelyduetosheddingleavesandtranslocationofnutrientsintorhizomes duringsenescence[115]. 40

52 TheproteinconcentrationsoftheperennialgrassesinourstudywerelowerinJuly comparedtotheoctoberharvesttime,at90.8(±11.2)and50.5(±5.5)gkg N1 DM, respectively(table2.2).proteinconcentrationsalsovariedbyspecies,withp.+virgatum+ havingtheleast,at43.8(±4.9)gkg N1 DM,andM+xgiganteus+and+P.+australis+havingnot differentconcentrations,at79.2(±15.2)and88.9(±11.7)gkg N1 DM,respectively.Alinkhas beenshownbetweenhigherproteinandhighermethaneyields[116],suggestingthatthe JulyharvesttimecouldbeamoreproductiveoptionthantheOctoberharvesttimeinterms ofbiogasproduction.however,therewasnorelationshipbetweenproteinconcentrations andmethaneyieldinthisstudy,whichwaspossiblyduetothelimitedscopeofthechemical analysis. Giventhedesignofthestudy,specificallythelimitedscopeofchemicalanalysis (withdataonlyfrom2013samples)andthelackofgrowingspeciessidenbynside,the conclusionsthatcouldbedrawnabouttherelationshipsbetweenfielddataandchemical constituentsofbiomassneedtobecarefullyconsidered.however,whencombiningall speciestogether,therewasapositivelinearrelationshipbetweenplantheightandlignin content(lignin=0.28(height)+31.37;r 2 =0.36,p=0.008)(Table2.4andTable2.8).This correlationmakesbiologicalsense,giventhattheprimaryfunctionofligninisstructural support[69],howeveramoreinndepthinvestigationoftherelationshipsbetweenplant height,lignin,andbmpshouldbeundertaken,asplantheightisaveryeasilynmeasured characteristic.phragmites+australis+wastallerthantheotherspeciesinourstudy,and regularlycontainedyearnoldstemsatharvest(morethananyotherspeciesinthisstudy); bothofthesefactorslikelycontributedtop.+australishavingthehighestlignincontentofthe specieswestudied. 41

53 ThebiologyandagronomyofZ.+mays+areconsiderablydifferentthantheperennial grassesinvestigatedinthisstudy,andthiswasreflectedinitschemicalcomposition,asit wasregularlyatextremeendsoftherangeswemeasured.inadditiontherewerelarge differencesbetweenz.+maysandtheperennialgrassesintermsofbiogaspotential Biological(Methane(Potential(( Itisoftendifficulttocomparebiogasyieldsamongstudies,duetodifferencesin plantspecies,cultivarsgeography,standtype,anddigestionparameters(including retentiontime,reactorsize,reactorstyleandmicrobialenvironment).however,previous studieshavereportedsimilarbiogasyieldsforcertainperennialgrassspecies.for untreated,latenharvestedmiscanthus+spp.,biogasyieldsbetween80to200nlch 4kg N1 VS havebeenreported[34,117],andourdatavariedseasonallybetween167.5±8.9and229.8 ±15.2NLCH 4kg N1 VS.BasedonthedecreaseinBMPbetweenharvesttimesweobservedin thisstudy,thelowerbmpreportedbyothers[34,117]mayhavebeenduetothetimeat whichmiscanthus+spp.washarvestedinotherstudies(december).panicum+virgatumyields havebeenreportedtorangefrom190to310nlch 4kg N1 VS[35,36],whichwasslightly higherthanthe160.1±25.2to186.5±9.4nlch 4kg N1 VSbiogasyieldsfoundinthisstudy. ThehigherP.+virgatumyieldsinthepreviousstudiescomparedtoourswerelikelydueto ensilingofgrasssamplespriortodigestion,andlongerdigestionperiodsof60days comparedwith21days[35,36].ensiledz.+mays+methaneyieldsinourstudy,334.9(±4.3) NLCH 4kg N1 VS,weresimilartovaluesfromstudieswithsimilarmethodology,rangingfrom 289N390NLCH 4kg N1 VS[29,38].MethaneyieldsreportedforP.+australisrangewidelyfrom 34to220NLCH 4kg N1 VS[52,96],withourresultsfromthisstudyatthehigherendofthe rangeat107.6±3.9to172.4±15.3nlch 4kg N1 VS. Zea+mayssilageisthemostwidelyusedpurposeNgrownbiogasfeedstockduetoits highbiogaspotential,typicallyyieldingfrom289to390nlch 4kg N1 VS[29,38]or589.7to 42

54 877.8NLCH 4m N2 [29,118].Zea+mayssilageyieldsinthisstudywere334.9±4.3NLCH 4kg N1 VS,fallingclosetothecenterofthisrange.Digestateinoculumusedintheaforementioned studieswithhigherbmppossiblyhadmicrobialecosystemsthatwerebetteracclimatedto digestingz.+mays+silage,asthedigestatecamefromcommercialreactorsutilizingcorn silageasaprimaryfeedstock,whiletheinoculumusedinourstudywasnotacclimatedto largeinputsofz.+mays.consideringthesimilarityofz.+mays+silagebmpresultsinthisstudy tothoseinotherstudies,thebmpassayusedinthisstudycanbesaidtohavetypical methaneyieldperformance. Thereisashortageofdataformethaneyieldsperunitareaofharvestedbiomass. Masséet+al.[35]reportedsummermethaneyieldsof260±33andfallyieldsof176±26NL CH 4m N2 forp.+virgatum+grownineasterncanada,whichwasconsiderablyhigherthanthe summeryieldsof139.2±7.0andfallyieldsof94.2±14.8nlch 4m N2 inthisstudy.this aforementioneddiscrepancybetweentheresultsfromthisstudyandthoseofmasséet+al.+ [35]islikelyduetodifferencesintheexperimentaldesignofdigestion.Specifically,these authorsusedalargerdigestionscale(30l),adifferentdiet(withtotalmethanefrom inoculumreachingamaximumofonly17.1%oftotalbiogasproduced,comparedwiththis study>95%),andrecirculatedmixingasopposedtomechanicalstirring.methanebyarea comparisonsform.+xgiganteus+andp.+australiscouldnotbemadeduetolackofavailable data. Similartoourstudy,otherstudiesshowedthatgreaterbiomassyieldsresultedin greaterbiogasperareayields,despitedeclinesinqualityanddigestibilityofbiomass[1]. Withineachofthespecieswestudied,thisaforementionedruleofbiomassyieldhavinga greaterimpactonmethaneperareafeedstockharvestedthanbiomassqualitywastrue. However,thisruledidnotremainvalidwhencomparingbetweenspecies.Bothsummer 43

55 andfallnharvestedp.+australisyieldedgreateramountsofdmperareathansummern harvestedm+xgiganteusbutthequalityofthematerialharvestedwassuchthatoverall biogasyieldsperareawerehighestforfallnharvestedm+xgiganteus.thusbasedonthe harvesttimesevaluatedinthisstudygreatestmethaneyieldperareaharvestedofp.+ australis,p.+virgatum,andm+xgiganteus+canbeachievedattimeofmaximumbiomassyield. Methaneconcentrationsmeasuredinourstudywereslightlyhigherthanthetypical rangeof50n75%ch 4[119],atupto77.4±9.3.ThereductionofVSoverthedurationofour experiments,whichrangedfrom5.4±4.0to18.1±7.2forallgrasses(table2.7),waslower thantherangeof38n70%reportedinotherstudies[7].asitisdifficulttoexplainthebiogas yieldsinourexperimentasaresultofsuchlowreductioninvs,itispossiblethattherewas someformofexperimentalerror(eg.digestatesamplingmethods,orconditionsofmuffle furnace).however,therewasasignificantpositivelinearrelationshipbetweenhigher reductioninvsandincreasesinmethaneproduction(mlch 4=7.91(%VSreduction) ;p=0.014,R 2 =0.278).Therefore,whilethereasonforthelowvolatilesolidsreduction wemeasuredintheseexperimentsisunknown,thevsreductionswemeasuredwerelinked asexpectedwithincreasedmethaneproduction. Asotherauthorshaveobserved[111],thebiomassyieldsofgrasses(M+xgiganteus+ andp.+virgatum)increasedovertime,thoughmethaneyieldperunitfeedstock(intermsof eithervsordmcontents)decreasedasgrassmatured.thistradenoffbetweenbiomass yieldandbiogasyieldisimportanttoconsiderwhenscreeningotherspeciesforbiomass applications.iftheprimarygoalistohavethehighestpossiblemethaneyieldperareaof perennialgrassbiomassharvestedthenfeedstockshouldbeharvestedatthetimeof maximumbiomassyield. 44

56 2.5.3 Research(Significance( TheyieldswemeasuredinwildinvasivestandsofP.+australisinSouthwestern Ontario,Canada,werehigherthanthoseofcultivatedP.+virgatum+cropsandcomparableto thoseofm+xgiganteus+crops.thisfindingmaybeusefultomanymembersofthebiomass industry,asp.+australisbiomassmayhavepotentialendnuseswedidnotinvestigate,suchas combustion,pyrolysis,andvariousbiomaterialuses.theinabilityofthethreeperennial grassspecieswestudiedtoregrowbetweentheharvestdatesexaminedinourstudymay beindicativethatthesespeciesarenotwellnsuitedformultipleharvestregimesin SouthwesternOntario,thoughvariationsharvestNtimingandcuttingNheight[32]needtobe furtherevaluated.ourstudyhasalsohelpedtodefinetherangeinbiomassandbiogas yieldsforthethreespecieswestudied,withallthreespeciesrangingfrom0.49(±0.06)to 2.43(±0.32)kgDMm N2 andfrom107.6(±3.9)to229.8(±15.2)nlch 4kg N1 VS.Theinverse relationshipbetweenligninconcentrationsandbiogasyields,andthegeneraltrendof lowerbiogasyieldsperfeedstockformaterialharvestedlaterintheseasonwere reconfirmed.thebiomassandbiogasyieldsrecordedinourstudyprovideabaselinefor thesegrassesinthisregion,whichmaybeausefulbenchmarkforfuturestudies Limitations(and(Future(Directions( Asitwasnotofenvironmentalinteresttogrowinvasive+P.+australisinabiomass plot,butrathertoevaluatewildstands,ourfieldstudydidnothavesidenbynsidegrowthof allthreespecies,butarandomsamplingofvariouslocalbiomassstands.whilethe environmentsdiffered,thisstudyisusefulfordeterminingaveragepopulationtrendsin SouthwesternOntario,andspecificallyfordeterminingthepotentialuseofP.+australisfrom thelocationsthatithasinvaded. Thedesignofthisexperimentwasoptimizedforcomparinghowdifferentperennial grassspeciesyieldintermsofbiogaspotentialataspecificmomentintime,ratherthanfor 45

57 creatingamodelregardingtime;weonlymeasuredbmpforsamplesfrom2013,not samplesfrom2014.whilethisisusefulformakingcomparisonsamongp.+australis+and otherbiomassoptions,itisnotnecessarilybestforcomparingmaximumbiomassorbiogas yieldpotentialsoroptimizedharvestregimesforthesedifferentspecies,sinceeachhave peakyieldsatdifferenttimesoftheyear.ourpreliminarylookatthistopicfromanontario perspectivesuggeststhatfutureresearchbefocussedonp.+australisandm+xgiganteus, whichhadahigheryieldthanp.+virgatum. Phragmites+australistypicallygrowsinwetlandsandmarginallands,someofwhich arehardtoaccessoroperatemachineryon.whilespecializedmachineryforharvestingp.+ australis+exists,andalthoughtheplantdoessometimesgrowsvigorouslyonperiodicallyn floodedmarginallands,largernscaletrialswillundoubtedlybefacedwithtechnical difficulties.examplesofsuchdifficultiesmayincludewetandnonnuniformconditionsfor machineryandtransportationtopointnofnuse.futureresearchisneededtodeterminethe differencesbetweensmallandlargenscaletrials,andtocalculatecostcomparisonsbetween thesespecies.(( ( WerecommendthatfutureresearchshouldevaluatebiogasfromP.+australis biomassofnewgrowthratherthanpreviousnyearsgrowth.sinceweobservedthatdead shootscanremainstandingformultipleyears,andthatoldershootsdidnotproducebiogas asreadily(foroctobervs.julymaterial),thereforeshootsremainingfromthepreviousyear shouldberemovedinthespringpriortoregrowthinordertomaximizebiomassquality. Whilenotinvestigatedinourstudy,thepossibilityofimprovingbiogasyields throughthepretreatmentoffeedstocksisofmuchinterestinthescientificcommunity[67 69].Viablepretreatmenttechnologies(thosewhichyieldanetenergygain)couldhave substantialimplicationsonthebiogaspotentialofthespeciesstudiedintheseexperiments. 46

58 Theeffectthatensilingbiomasshasonbiogasyieldshouldalsobeevaluatedforspecies suchasp.+australis.however,thebiogasyieldsofz.+mays+(intermsofbothareaharvested andfeedstockinput)wereroughly150to700%ofanyoftheperennialgrassesatanyofthe harvesttimesinvestigated.therefore,substantialbiogasnyieldincreasesforthechopped perennialgrassbiomassinthisstudywouldberequiredforthesespeciestobecome competitivefeedstockoptionscomparedwithz.+mays.+ AnotherstudyonusingP.+australisforbiogascalculatednetcosts,associatedwith harvest,chopping,transport,storage,treatment,incomefromgas,andspreading,as$us 120pertDMharvested(estimatedas 90in2004)[51].Thisaforementionedestimatewas recognizedasaneteconomicloss,whichmeansthatuseofp.+australisforbiogas productionwouldonlybeeconomicallyreasonableinsituationswhereinremovalisalready deemedrequired,orwhereinthereisagoaltophytoremediatetheareaitoccupies[51].a socioneconomicandecologicalcomparisonwouldberequiredtoproperlyaddressthese factorsanddeterminewhetherornotp.+australis+couldbeaviablebiogasfeedstockoption undervariousscenarios. 2.6 Conclusions( Basedontheresultsfromthisstudythebesttimetoharvestthesespeciesinterms ofreachingmaximumbiogaspotentialperunitfeedstockwouldbeinearlysummer. Specifically,thisislikelytobeatsomepointbeforethebeginningofJuly,whenbiomasshas highermoisturecontentandproteinconcentrations,makingitbetterforensilingand digesting,respectively.however,inordertoachievemaximumbiogasyieldperarea harvested,biomassoftheseperennialgrassesshouldbeharvestedattimeofpeakbiomass yield(e.g.anoctoberharvesttime).determiningwhichscenarioisoptimaldependsonthe processinvolved(ie.ifthematerialneedstobeensiledandstored),andonlocalfeedstock availability. 47

59 WildstandsofinvasiveP.+australishavebiomassyieldssimilarwiththoseoftwo biomassgrassescurrentlygrowninsouthwesternontario:p.+virgatumandm+xgiganteus. However,usingP.+australis+forbiogasproductionisunlikelytobeaneconomicallyviable biogasfeedstockoptionexceptpotentiallyinextenuatingcircumstanceswhereitsremoval isalreadyrequired.lastly,z.+mays+silageyieldedsubstantiallygreateramountsofmethane thancultivatedp.+virgatumandm+xgiganteusperunitfeedstock,makingitthemost attractivefeedstockoptionweexaminedinthisstudy. 48

60 2.7 Tables( ( ( Table(2.1.Characterizationofthedigestateusedasinoculumineachbiologicalmethane potentialrunpriortouseinthebiologicalmethanepotentialassay. ( Variable( Run(1( Run(2( Run(3( DateSampled 02NJulN NJulN NOctN2014 Temperature( C) RetentionTime(day) DietComponents(kgday N1 ): DairyManure 20,161 22,097 31,167 CornSilage Fats,Oils,andGrease Total 21,171 22,987 32,215 TotalSolids(%) VolatileSolids(%) ph NA* ElectricalConductivity(mmhoscm N1 ) ChemicalComposition: N(%) P(%) K(%) NH 3(mgkg N1 DM) 1,780 1,650 1,550 *NA:notavailablebecausemeasurementnottaken 49

61 Table&2.2.Mean(±standarderror)compositionof2013samplesofPhragmites+australis,+Panicum+virgatum,and+Miscanthus+X+giganteus (n=3);andzea+mayssilage(n=1).meansfollowedbythesameletterineachcolumnarenotsignificantlydifferentaccordingtotukey s rangetest(p>0.05).zea+mays+wasnotanalyzedduetolackofreplication.dm:drymatter. Dry&Matter& Cellulose& Hemicellulose& Lignin&& Protein&& IIIIII%IIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIgkg I1 DMIIIIIIIIIIIIIIIIIIIIIIIIIII Species& +++M+xgiganteus+ 46.5(±2.9)b 360(±7.7) 267.9(±3.9)b 74.8(±20.7) 79.2(±15.2)b +++P.+australis+ 37.2(±4.1)a 374.5(±9.6) 245.2(±3.6)a 108.0(±5.9) 88.9(±11.7)b P.#virgatum 35.1(±5.4)a 360.9(±4.9) 274.6(±3.3)b 67.4(±4.7) 43.8(±4.9)a Z.+mays Harvest&Time& July 31.5(±2.5)m 367.4(±8.6) 262.7(±4) 77.8(±14.7) 90.8(±11.2)m October 47.7(±2.5)n 362.9(±2.9) 262.4(±6.3) 89(±7.6) 50.5(±5.5)n Species&*&Harvest&Time& M+xgiganteus*July 40.3(±0.5) 363.7(±16.4) 261.2(±1.7)xy 76.3(±46.3) 112.2(±5.5)x M+xgiganteus*October 52.8(±1.6) 356.3(±3.7) 274.7(±5.2)x 73.3(±2.9) 46.3(±5.8)z P.+australis*July 30.8(±1.8) 380.9(±20.3) 252(±3.0)yz 98.5(±7.2) 108.6(±16)xy P.+australis*October 43.6(±6.2) 368.1(±3.5) 238.4(±2.9)z 117.6(±5.6) 69.2(±6.4)yz P.+virgatum*July 23.6(±0.6) 357.6(±8.4) 274.8(±5.5)x 58.8(±0.3) 51.7(±6.8)z P.+virgatum*October 46.6(±3.3) 364.3(±6.1) 274.3(±2.2)x 76.1(±0.8) 36(±2.7)z Effect& IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIP+valueIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Species < <0.001 HarvestTime < <0.001 HarvestTime*Species

62 & Table&2.3.&Mean(±standarderror)plantcharacteristicsforyearbyspeciesinteractionsforPhragmites+australis,+Panicum+virgatum,+and+ Miscanthus+x+giganteus Nagara,atthreesitesinSouthwesternOntario,Canada,harvestedin2013and2014(n=6).Meansfollowedbythe sameletterineachcolumnarenotsignificantlydifferentaccordingtotukey srangetest(p>0.05).otherresultsofthreeiwayanova shownontable2.4. Fresh&Matter& Yield& Dry&Matter& Yield&& Stems*& Height& Leaf& IIIIIIIIIIIIIkgm I2 IIIIIIIIIIIII IINumberm I2 II IIIIIIcmIIIIII %oftotaldm Species&*&Year& M+xgiganteus* (±0.48) 1.71(±0.25) 107b 180(±10)b 40(±1)c M+xgiganteus* (±0.44) 1.99(±0.43) 86b 203(±17)b 33(±4)bc P.+australis* (±0.43) 1.74(±0.14) 62a 268(±5)c 25(±2)ab P.+australis* (±0.3) 1.9(±0.12) 97b 280(±11)c 17(±1)a P.+virgatum* (±0.16) 0.6(±0.08) 276c 109(±14)a 32(±4)bc P.+virgatum* (±0.19) 0.58(±0.08) 300c 97(±9)a 36(±4)bc Effect& PvalueIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Year Species*Year Harvest*Year *Datawastransformedusingalogarithmicbase10transformation. & 51

63 Table&2.4.Mean(±standarderror)plantcharacteristicsforPhragmites+australis,+Panicum+virgatum,+and+Miscanthus+x+giganteus Nagara, atthreesitesinsouthwesternontario,canada,harvestedovertwoseasonsin2013and2014(n=6).resultsofthreeiwayanovaalso shownontable2.3.meansfollowdbythesameletterineachcolumnwerenotsignificantlydifferentaccordingtotukey srangetest (p>0.05). Fresh&Matter& Yield& Dry&Matter& Yield&& Stems*& Height& Leaf& IIIIIIIIIIIIIkgm I2 IIIIIIIIIIIII IINumberm I2 II IIIIIIcmIIIIII %oftotaldm Species& +++M+xgiganteus+ 4.85(±0.32)c 1.85(±0.24)b 93b 192(±10)b 37(±2.4)b +++P.+australis+ 3.80(±0.25)b 1.82(±0.09)b 75a 274(±5.9)c 21(±1.7)a +++P.+virgatum+ 1.56(±0.12)a 0.59(±0.05)a 282c 103(±8.3)a 34(±2.6)b Harvest&Time& July 3.62(±0.85) 1.20(±0.28)m (±19.8)m 34(±8.0)n October 3.19(±0.75) 1.64(±0.39)n (±15.4)n 27(±6.3)m Species&*&Harvest&Time& M+xgiganteus*July 4.79(±0.40) 1.28(±0.15)xy (±7.9)x 38(±2.8) M+xgiganteus*October 4.91(±0.54) 2.43(±0.32)w (±10.7)w 35(±4.0) P.+australis*July 4.34(±0.38) 1.85(±0.16)wx (±8.1)v 22(±2.2) P.+australis*October 3.27(±0.12) 1.80(±0.10)wx (±9.5)v 19(±2.5) P.+virgatum*July 1.75(±0.15) 0.49(±0.06)z (±6.8)z 41(±2.8) P.+virgatum*October 1.38(±0.15) 0.69(±0.07)yz (±6.4)y 27(±1.7) Effect& PvalueIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Species <0.001 <0.001 <0.001 <0.001 <0.001 HarvestTime < Species*HarvestTime < Species*Harvest*Year *Datawastransformedusingalogarithmicbase10transformation. & 52

64 Table&2.5.Mean(±standarderror)nutrientconcentrationsinPhragmites+australis,+Panicum+virgatum,+and+Miscanthus+X+giganteus+(n=3) andzea+mays+silage+(n=1)duringjulyandoctoberharvestsfor2013samples.meansfollowedbythesameletterineachcoloumnwere notsignificantlydifferentaccordingtotukey srangetest(p>0.05).zea+mayswasnotanalyzedduetolackofreplication. Magnesium*& Nitrogen& Phosphorus**& Potassium& Sodium& Copper& Manganese** Species& IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIgkg I1 DMIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIImgkg I1 DMIIIIII +++M+xgiganteus+ 2.7ab 12.7(±2.4)b 1.5b 12.5(±2.1) 0.2(±0)a 3.9(±1.0)b 43.5ab +++P.+australis+ 4.4b 14.2(±1.9)b 0.7a 9.5(±2.2) 0.4(±0)b 1.2(±0.7)a 123.7b P.+virgatum 1.6a 7.0(±0.8)a 1.5b 10.2(±1.6) 0.1(±0)a 1.8(±0.7)a 17.9a Z.+mays Harvest&Time& July (±1.8)m (±0.9)m 0.2(±0) 4.0(±0.6)m 46.7 October (±0.9)n (±0.7)n 0.3(±0.1) 0.6(±0.4)n 45 Species&*&Harvest&Time& M+xgiganteus*July (±0.9)x (±2.2) 0.2(±0.0) 5.86(±0.8) 50.9 M+xgiganteus*October (±0.9)z (±0.9) 0.2(±0.0) 1.8(±0.4) 19.8 P.+australis*July (±2.6)xy (±1.4) 0.3(±0.0) 2.7(±0.5) 99.5 P.+australis*October (±1)yz (±1) 0.4(±0.1) I0.3(±0.3) P.+virgatum*July (±1.2)z (±0.2) 0.2(±0.0) 3.3(±0.6) 20.1 P.+virgatum*October (±0.2)z (±0.3) 0.1(±0.0) 0.3(±0.2) 15.9 Effect& IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIP+valueIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Species < <0.001 < HarvestTime < < < HarvestTime*Species *SquarerootbackItransformed;**BaseI10logarithmbackItransformed. & 53

65 Table&2.6.MeannutrientconcentrationsinPhragmites+australis,+Panicum+virgatum,+and+Miscanthus+X+giganteus+(n=3)andZea+mays+silage (n=1)duringsummerandfallharvestsfor2013samples.zea+mayswasnotanalyzedduetolackofreplication. Calcium& Iron& Zinc& Species& IIIgkg I1 III IIIIIIIIImgkg I1 IIIIIIIII +++M+xgiganteus+ 2.8(±0.3) 55.5(±2.6) 21.4(±4.4) +++P.+australis+ 3.2(±0.3) 53.5(±4) 27.8(±3.8) P.+virgatum 3.2(±0.6) 54.9(±5.9) 14.5(±1.1) Z.+mays Harvest&Time& July 2.7(±0.3) 51.4(±3.1) 23.1(±3.2) October 3.4(±0.3) 58.2(±3.6) 19.4(±3.3) Species&*&Harvest&Time& M+xgiganteus*July 2.7(±0.5) 56.9(±4.7) 25.3(±5.4) M+xgiganteus*October 2.8(±0.2) 53.4(±2.0) 17.5(±7.3) P.+australis*July 3.1(±0.6) 48.2(±6.6) 29.1(±6) P.+australis*October 3.3(±0.2) 58.8(±3.2) 26.4(±6) P.+virgatum*July 2.3(±0.3) 49.0(±4.9) 15.0(±2.0) P.+virgatum*October 4.2(±0.8) 60.8(±10.6) 14.1(±1.4) Effect& Species HarvestTime HarvestTime*Species

66 Table&2.7.MeanmethaneyieldsfrommesophilicbenchItopreactorsforMiscanthus+xgiganteus+ Nagara,+Panicum+virgatum,and+ Phragmites+australiswhenharvestedinJulyorOctober(n=3).Meansforcornsilage(Zea+mays)displayed(n=9),butnotincludedinthe twoiwayanova.meansfollowedbythesameletterineachcolumnwerenotsignificantlydifferentaccordingtotukey srangetest (p>0.05).dm:drymatter;fm:freshmatter;nl:normalizedliters;vs:volatilesolids. NL&CH4&kg M1 &VS& NL&CH4&kg M1 &DM& NL&CH4&kg M1 &FM& NL&CH4&m M2 & Methane&(%)& Reduction&in&VS&(%)& Species& ++++M+xgiganteus (±16) 175.8(±12.3)b 69.2(±5) 339.0(±27.3)c 72.3(±7.1) 13.1(±4.1) +++P.+australis (±16.1) 114.5(±11.3)a 67.8(±6.4) 249.9(±33.3)b 72.8(±1.3) 3.8(±2.4) +++P.+virgatum (±13.4) 159.2(±12.4)b 72.4(±5.4) 116.7(±12.4)a 66.5(±2.3) 9.6(±2.9) +++Z.+mays (±4.3) 314.1(±4.1) 123.5(±1.6) 497.5(±6.4) 75.6(±3.9) 15.7(±0.4) Harvest&Time& July 196.2(±2.8)y 169.1(±2.8)y 72.1(±1.1)y 247.5(±4.4) 69.7(±4.3) 9.0(±0.4) October 145.1(±12.2)z 130.6(±11.9)z 67.5(±4.8)z 222.9(±4.5) 71.3(±2.8) 9.3(±3.5) Species&*&Harvest&Time& +++M+xgiganteus++*July (±15.2) 198.6(±13.2) 54.0(±3.6) 253.2(±16.8)x 77.4(±9.3) 11.3(±7.2) ++++M+xgiganteus++*October (±8.9) 153.0(±8.2) 75.5(±4.0) 371.4(±19.8)w 68.7(±5.8) 18.1(±7.2) +++P.+australis+*July (±15.3) 136.4(±12.1) 58.2(±5.2) 251.7(±22.3)x 70.6(±5.0) 6.2(±3.5) +++P.+australis+*October (±3.9) 92.6(±3.4) 50.8(±1.9) 166.2(±6.1)y 75.9(±5.2) 5.4(±4.0) +++P.+virgatum*July (±9.4) 172.2(±8.7) 48.5(±2.5) 83.6(±4.2)z 63.0(±3.4) 14.5(±1.2) +++P.+virgatum*October (±25.2) 146.2(±23.0) 75.3(±11.9) 101.2(±15.9)z 71.2(±4.6) 5.4(±4.6) Interactions& P+valueIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Species HarvestTime Run NA* HarvestTime*Run Species*Run Species*HarvestTime *NA:Notavailable,measurementsforfirstrunnottaken. 55

67 Table&2.8.Regressionmodelsfitforchemicalcomponentsofperennialgrasses(gkg I1 ;independentvariable)inrelationtomethane production(nlch4kg I1 VS;dependentvariable)asdeterminedbylaboratorytestsofanaerobicdigestionofsamplesfrom2013(n=6). Independent& Variable& Regression& Model& R&Square& F&value& Pr&>&F& Lignin* Linear =266.9I1.342x Quadratic =304.6I2.67x+0.009x Cellulose* Linear =I x Quadratic =I xI0.008x Hemicellulose* Linear =I x Quadratic =I xI0.251x Protein Linear = x Quadratic =203.7I2.5x+0.02x Cubic =I xI0.20x x *ThecubicmodelcouldnotbefittedduetonearIcollinearityamongmodelterms. 56

68 2.8 Figures+ Figure+2.1.LocationofcultivatedMiscanthus*xgiganteus*(purplemarkers)andPanicum* virgatum*(greenmarkers)biomass,aswellaswildstandsofphragmites*australis*(red markers)usedinthisexperimentconductedinsouthwesternontario.stripedmarkers denotelocationsofthesamplesusedinthebiologicalmethanepotentialanalysis.* 57