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Standardized protocol for determination of biohydrogen potential
Julian Carrillo-Reyes, German Buitron, Iván Moreno-Andrade, Aida Cecilia Tapia-Rodríguez, Rodolfo Palomo-Briones, Elías Razo-Flores, Oscar Aguilar-Juárez, Jorge Arreola-Vargas, Nicolas Bernet, Adriana Ferreira Maluf
Braga, et al.
To cite this version:
Julian Carrillo-Reyes, German Buitron, Iván Moreno-Andrade, Aida Cecilia Tapia-Rodríguez, Rodolfo
Palomo-Briones, et al.. Standardized protocol for determination of biohydrogen potential. MethodsX,
Elsevier, 2020, 7, pp.100754. �10.1016/j.mex.2019.11.027�. �hal-02921485�
Protocol Article
Standardized protocol for determination of biohydrogen potential
Julián Carrillo-Reyes
a,*, Germán Buitrón
a,
Iván Moreno-Andrade
a, Aida Cecilia Tapia-Rodríguez
b, Rodolfo Palomo-Briones
b, Elías Razo-Flores
b,
Oscar Aguilar-Juárez
c, Jorge Arreola-Vargas
d, Nicolas Bernet
e, Adriana Ferreira Maluf Braga
f, Lucia Braga
g, Elena Castelló
g, Lucile Chatellard
e, Claudia Etchebehere
h, Laura Fuentes
h, Elizabeth León-Becerril
c, Hugo Oscar Méndez-Acosta
i, Gonzalo Ruiz-Filippi
j, Estela Tapia-Venegas
j, Eric Trably
e, Jorge Wenzel
h, Marcelo Zaiat
faLaboratoryforResearchonAdvancedProcessesforWaterTreatment,InstitutodeIngeniería,Unidad AcadémicaJuriquilla,UniversidadNacionalAutónomadeMéxico,Blvd.Juriquilla3001,Queretaro,76230, Mexico
bDivisióndeCienciasAmbientales,InstitutoPotosinodeInvestigaciónCientíficayTecnológicaA.C.,Caminoa laPresaSanJoséNo.2055,Col.Lomas4aSección,C.P.78216,SanLuisPotosí,SLP,Mexico
cDepartmentofEnvironmentalTechnology,CentrodeInvestigaciónyAsistenciaenTecnologíayDiseñodel EstadodeJalisco,A.C.,Av.Normalistas800,Col.ColinasdelaNormal,C.P.44270,Guadalajara,Jalisco,Mexico
dDivisióndeProcesosIndustriales,UniversidadTecnológicadeJalisco,LuisJ.JiménezNo.577,1odeMayo,C.
P.44979,Guadalajara,Jalisco,Mexico
eINRAE,Univ.Montpellier,LBE,Narbonne,France
fBiologicalProcessLaboratory,SãoCarlosSchoolofEngineering,UniversityofSãoPaulo(LPB/EESC/USP),Av.
JoãoDagnone1100,SãoCarlos,SãoPaulo,13563-120,Brazil
gLaboratorioBioProA,FacultaddeIngeniería,UniversidaddelaRepúblicadeUruguay,Av.JulioHerreray Reissig565,Montevideo,Uruguay
hLaboratoriodeEcologíaMicrobiana,DepartamentodeBioquímicayGenómicaMicrobiana,Institutode InvestigacionesBiológicasClementeEstable,Av.Italia3318,Montevideo,Uruguay
iDepartamentodeIngenieríaQuímica,CUCEI-UniversidaddeGuadalajara,Blvd.M.GarcíaBarragan1451,C.P.
44430,Guadalajara,Jalisco,Mexico
jEscueladeIngenieríaBioquímica,FacultaddeIngeniería,PontificiaUniversidadCatólicadeValparaíso,Av.
Brasil2085,Valparaíso,Chile
DOIoforiginalarticle:http://dx.doi.org/10.1016/j.ijhydene.2019.08.124
*Correspondingauthor.
E-mailaddress:JCarrilloR@ii.unam.mx(J.Carrillo-Reyes).
http://dx.doi.org/10.1016/j.mex.2019.11.027
2215-0161/©2019TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://
creativecommons.org/licenses/by/4.0/).
ContentslistsavailableatScienceDirect
MethodsX
journal homepage:www.elsevier.com/locate/mex
ABSTRACT
Biohydrogenproductionpotential(BHP)dependsonseveralfactorslikeinoculumsource,substrate,pH,among manyothers.Batchassaysarethemostcommonstrategytoevaluatesuchparameters,wherethecomparisonisa challengingtaskduetothedifferentproceduresused.Thepresentmethodintroducesthefirstinternationally validatedprotocol,evaluatedby8independentlaboratoriesfrom5differentcountries,toassessthebiohydrogen potential.Asqualitycriteria,acoefficientofvariationofthecumulativehydrogenproduction(Hmax)wasdefined
tobe<15%.TwooptionstorunBHPbatchtestswereproposed;amanualprotocolwithperiodicmeasurementsof
biogasproduction,needingconventionallaboratorymaterialsandanalyticalequipmentforbiogascharacteriza- tion;andanautomaticprotocol,whichisruninadevicedevelopedforonlinemeasurementsoflowbiogas production.Thedetailedproceduresforbothprotocoloptionsarepresented,aswellasdatavalidatingthem.The validation showed acceptable repeatability and reproducibility, measured as intra- and inter-laboratory coefficientofvariation,whichcanbereducedupto9%.
©2019TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://
creativecommons.org/licenses/by/4.0/).
ARTICLE INFO
Protocolname:Biohydrogenpotentialprotocol
Keywords:Automaticprotocol,Darkfermentation,Heat-treatedinoculum,Manualprotocol Articlehistory:Received25October2019;Accepted29November2019;Availableonline4December2019
SpecificationTable
SubjectArea: EnvironmentalScience Morespecificsubject
area:
Biofuelsproduction
Protocolname: Biohydrogenpotentialprotocol Reagents/tools: Requiredreagents
NH4Cl
MES(2-(N-Morpholino)ethanesulfonicacid,4-Morpholineethanesulfonicacid) MgCl26H2O
FeSO47H2O CoCl26H2O MnCl24H2O KI NiCl26H2O ZnCl2
Anhydrousglucose HCl(37%) NaOH
Thymolphthalein(onlyfortheautomaticversion) Distilledwater
Requiredmaterialsandequipment
10-mLgraduatedpipettesoranairdisplacementpipette(110mL)withtips Spatula
pHmeter Pasteurpipettes
Digitalanalyticalbalance(readabilitydownto0.1mg)
Industrialblender,electriccoffeegrinderorporcelainmortarandpestle Standardmesh#20(sievesize850mm)
Laboratoryovenat1051C
Gaschromatographtoanalyzebiogascomposition(H2andCO2) Specificmaterialsforthemanualprotocol
120-mLserumbottles(Fig.1a)
Rubberstoppersforserumbottles(foranaerobiccultures)(Fig.1b)(Note:Becarefultonotuse thinstoppersthatcanleadtoleakageofthegasafterthefirstpunctureoftheneedle)
Aluminumrings(Fig.1b)
18-Gx11/2-inhypodermicneedlesandtubing(Fig.1c) Medicaldisposablethree-waystopcockvalve(Fig.1d) 50-mLplasticsyringe
100-mLgraduatedcylinder 500-mLglassbeaker
Crimpertosealaluminumrings Incubatorwithorbitalagitation Glassthermometer
Specificmaterialsfortheautomaticprotocol
AutomaticMethanePotentialTestSystem(AMPTSII,BioprocessControlAB;LundSweden) (Fig.2)
500-mLSchottglassbottles(Fig.2d) 50-mLgraduatedcylinder
500-mLgraduatedcylinder 50-mLglassbeakers
Experimentaldesign: TheproposedmethodpresentstwooptionstorunBHPbatchtests;amanualprotocolwithperiodic measurementsofbiogasproduction;andanautomaticprotocol,whichisruninadevicedevelopedfor onlinemeasurementsoflowbiogasproduction.
Trialregistration: n/a
Ethics: n/a
ValueoftheProtocol
Thismethodstandsforthefirststandardizedprotocolvalidatedinternationallytoestimatethehydrogenpotential.
Twoversionsoftheprotocolareincluded,amanualonewithlowequipment’srequirementsandanautomaticone.
Theaverageintra-laboratorycoefficientofvariationcanbereducedupto9%.
Descriptionofprotocol
Background
Hydrogenproductionbydarkfermentationisanemergingtechnologyofincreasinginterestdueto itsrenewablefeature.TheassessmentoftheBHPisacommonproceduretoevaluatenewpotential substrates, and other parameters [1–3]. This method stands for the first standardized protocol validated internationally to estimate the BHP using batch tests. A complete discussion of the developmentofthisprotocolhasbeenpublished[4].
Fig.1.Serumbottles,rubberstoppers,andaluminumrings.
Stocksolutions
SolutionA:nutrientsandbuffersolution,modifiedfrom[5]
Dissolveinavolumetricflasktomake1Lsolutionwithdistilledwater:41.6gNH4Cl,19.52gMES, 2gMgCl2
6H2O,1.6gFeSO47H2O,40mgCoCl26H2O,40mgMnCl24H2O,40mgKI,8mgNiCl26H2O,8mgZnCl2.
SolutionB:substrate,50gL1glucose
Dry28gofanhydrousglucose(105C,2h)andallowtocoolinadesiccator.Weigh25gofdry glucoseanddissolvewithdistilledwatertomakea500-mLsolutioninavolumetricflask.Thesource ofcarbohydratescanvaryaccordingtotheaimofeachdetermination,e.g.,usinganagroindustrial effluent. The recommendation to get the maximum hydrogen potential is to keep the initial carbohydrateconcentrationat5gL1duringtheassay.
SolutionC:pHadjustment,5NHCl
Add25mLofdistilledwatertoa50-mLvolumetricflask.Add20mLofHCl(37%)anddilutetothe graduationmarkingwithdistilledwater.
SolutionD:pHadjustment,5NNaOH
Add10gofNaOHto30mLofdistilledwater,dissolvewithmagneticstirringandallowtocool.
Dilutethesolutionwithdistilledwatertocomplete50mLinavolumetricflask.
SolutionE:pHindicator,0.4%(w/v)thymolphthalein
Dissolve40mgofthymolphthaleinin9mLofethanol(99.5%);dilutethesolutionwithdistilled watertocomplete10mLinavolumetricflask.
SolutionF:CO2absorptionsolution,3NNaOH
Add144g ofNaOHto1Lofdistilledwater,dissolve withagitation;diluteitto1.2L.Add6mLofsolutionE.
Note:SolutionsEandFareonlyrequiredfortheautomaticprotocol.
Inoculumpreparation
Use sludge froman anaerobic wastewater treatmentreactoras inoculum. Perform a thermal pretreatment drying the sludge at 105C during 24h, for the selection of hydrogen-producing microorganisms[6]. Letthesludge coolandhomogenizeit bygrinding(with theblender,coffee grinderorthemortar)andselecttheparticlesizelesserthan850
m
mbysieving.Determinevolatilesolids(VS)contentaccordingtostandardmethods[7].
Manualprotocol Preparation
1 Labelallbottles;triplicatesofeachevaluatedconditionmustbeincluded,aswellasendogenous control(withoutsubstrate)intriplicate.
Fig.2. Sample incubation unit (a), CO2absorption unit (b), gas volume measuring device (c), and Schott bottles with agitation system (d).
2CalculatethenecessaryamountofinoculumtoaddtoeachserumbottletomaintainanS/Xratio (substrate/inoculum,gsubstrategVS1)of2.7,consideringaworkingvolumeof80mLandan initialcarbohydrateconcentrationof5gL1.Weightheexactamountofinoculumdirectlyinthe serumbottleusingtheanalyticalbalance.
3Add5mLofsolutionAineachbottleusingagraduatedpipette(oranairdisplacementpipette).
4Add4mLofsolutionB(withtheexceptionoftheendogenouscontrol)usingagraduatedpipette(or anairdisplacementpipette);theinitialglucoseconcentrationwillbe5gL1.
5Addtheneededamountofdistilledwaterusingagraduatedcylinder,71mLor75mL,forbottles withcarbohydrates(withsolutionB)orendogenouscontrols,respectively;tocomplete80mLof workingvolumeintheserumbottles.Theheadspacewillbeof40mL.
6AdjusttheinitialpHto7.5bydrippingsolutionCorDasnecessaryusingaPasteurpipette.
7Closethebottleswiththerubberstopperandsealthemwiththealuminumrings(usethecrimper).
8Fortheheadspaceexchange,insertaneedleconnectedtoahosetoflushwithN2for30sinthe serumbottlesandwiththehelpofanotherneedletoletthegasout(Fig.3).After30s,getoutthe needlessimultaneously.
IncubationandmonitoringofH2production
1Consideringpreviousexperimentalexperiencestovalidatethisprotocol,starttheincubationclose tonoon(12:00h).
2Incubatethebottlesat37Cwithanorbitalshakingof150rpm.Afterthisstep,preparethewater- filledinvertedgraduatedcylinderaccordingtothefollowingsection.
3Afteronehourofincubation,dischargetheexcesspressureofeachserumbottlesintheinverted graduatedcylinder.
4Performgasproductionmeasurementseverythreeorfourhours.Recordthevolumeproduced,as wellasthetemperatureoftheacidwaterinthewater-filledinvertedgraduatedcylinder.
5Totakeagassample,purgethehosebeforetakingthesample.Analyzethecompositionofthe biogas(H2andCO2)atleastonceadaybygaschromatography.
6Todeterminetheendofthetest,applythestoptimecriterion(ST),whichisobtainedwhentheH2
productioncurve becomesasymptotic(FT) (Fig.4)[8].Additionally,you canusethecoefficient ofvariation betweenthelastthreerecordsofcumulativehydrogenasstopcriterionwhenitislowerthan5%.
Fig.3. Headspaceexchangeontheserumbottles.
7 Tostopthetest,removetheserumbottlesfromtheincubator.Seethesamplingandanalysissection forfurtherinstructions.
NOTE. Todetermine theendof thetestproperly, youwillhavetoplotimmediatelyeveryH2
productionreading.
Water-filled-invertedgraduatedcylindertomeasurebiogas
1 Acidify500mLofdistilledwaterwith10mLofsolutionC(pH<2)topreventCO2dissolutioninthe liquid.
2 Placea100mLinvertedcylinderasshowninFig.5usingahosetothebottomofthecylinder,a syringe,andathree-waystopcocktofillthecylinderwiththeacidwater(Fig.5c).
3 Tomeasurethebiogasproduction,connectaneedletoonesideofthethree-waystopcockandkeep itclosed,theninserttheneedlethroughtherubberstopperoftheserumbottlesandopenthe stopcocktoallowthebiogasdisplacetheliquidinthecylinder,registerthevolume(Fig.5d).
Fig.4.Kineticovertime.LT:latencytime;FT:finaltime;SP:timetostopthekinetic.
Fig.5.Requiredmaterialforthebiogasmeasuring(a),fillingtheinvertedcylinderwithasyringe(b),water-filled-inverted graduatedcylinder(c),andbiogasproductionmeasuring(d).
4The biogas samples can be taken from the displaced volume in the cylinder, or using a chromatographysyringedirectlyfromtheserumbottle.
Automaticprotocol
Fortheautomaticdeterminationofthebiohydrogenpotential,theprotocolwasadaptedtothe manufacturer recommendations of the Automatic Methane Potential Test System II (Bioprocess ControlAB;Lund,Sweden),intheoperationandmaintenancemanual[https://www.bioprocesscon- trol.com/media/1511/bioprocess-control-manual-ampts-ii-ampts-ii-light.pdf].
Preparation
1For theCO2 absorption unit preparation, add 80mLof F solution to each bottle of the CO2
absorptionunit(Fig.3b),placeaplasticstopperwiththetwotubingports.
2ConnectthebiogashosesofeachbottleoftheCO2absorptionunittothecorrespondentbottlein theincubationunit(Fig.3a)accordingtotheAMPTSIIinstructions.
3Label all Schott bottles;triplicates of each evaluatedcondition must beincluded, as wellas endogenouscontrol(orblank)intriplicate.
4CalculatethenecessaryamountofinoculumtoaddtoeachSchottbottletomaintainanS/Xratio (substrate/inoculum,gsubstrategVS1)of2.7,consideringaworkingvolumeof360mLandan initialcarbohydrateconcentrationof5gL1.Weightheexactamountofinoculumforeachbottle inglassbeakersusingtheanalyticalbalance.
5Add22.6mLofsolutionAineachglassbottleusingagraduatedpipette(oranair-displacement pipette).
6Add36mLofsolutionBineachbottle(excepttoendogenouscontrol)usingagraduatedpipette(or anairdisplacementpipette);theinitialglucoseconcentrationwillbe5gL1.
7Addtheweighedinoculumtoeachglassbottle,suspendtheremainingsludgethatstaysintheglass beakerusingthewaterpointedoutinthefollowingstep,andaddedtothecorrespondingbottle.
8Tocomplete360mLofworkingvolumeintheserumbottles,addtheneededamountofdistilled waterusingagraduatedcylinderandpipette,301.4and337.4mL,tothebottleswithcarbohydrates (withsolutionB)orforendogenouscontrols,respectively.Theheadspacewillbeof240mL.A workingvolume/headspaceratioof1.5willbemaintainedaccordingtotherecommendationsof theAMPTSIImanual.
9AdjusttheinitialpHto7.5bydrippingsolutionCorDasnecessaryusingaPasteurpipette.
10Putathinlayerofstopcockgreaseinthebottlethreadandtheoutsidepartofthebottleopening,to avoidgasleaks.
Fig.6. HeadspaceexchangeonAMPTSIIbottlesusingagassamplingbagwithN2.
11 Place thebottlestopperswithtwo tubingportsandrotatingshaft for mixingintheopening ofthe bottles.
12AttachthemultifunctionbrushlessDCmotor.
13PuttheTygon1hoseintheportsfromthestopper.Connectoneofthehosestothecorresponding bottleintheCO2absorptionunitandsealtheotherhosewithapipeplug.
Headspaceexchange
1 Fortheheadspaceexchange,temporallydisconnecttheTygon1hosefromtheCO2absorptionunit andintroducethehoseinabeakerwithwatertodischargetheexcesspressure.Removethepipe plugandconnectittoaN2flow,flushtheheadspaceduring30s(Fig.6).
2 ClosetheTygon1hosesagainwiththepipeplugandreconnecttheotherhosetothecorresponding bottleintheCO2absorptionunit.
AMPTSIIsystemincubationandmonitoring
1 Filltheincubationunitwithdistilledwaterandsettoatemperatureof37C.
2 Connecttheagitationsystemsofeachbottleseriallywiththemotorcables,andthefirstmotorto theMotorController.
3 Connectthe12VpoweradaptertotheGasVolumeMeasuringDevice,andtoa100240V50/
60Hzstandardpowersocket.Afterthis,connectthe24VpoweradaptertotheMotorController andtoa100240V50/60Hzstandardpowersocket.
4 SettheSystemswitchontheMotorControllertoON,andsettheON/OFFswitchesoneachmotor unittoON.
5 Emptyeachflowcellinthegasvolumemeasuringdevice.
6 Adjustthemotorspeedto60%(approx.120rpm),withintermittentmixingat60sONand180s OFF.ActivatethemotorsettingthecontroltoONandapplythesettings.
7 StartthedataloggingprogramaccordingtotheAMPTSIIsoftwaremanual.
8 Analyzethegascompositiontodeterminethehydrogenfractioninthebiogassamples,foreach bottleatleastonceduringthekinetics.DisconnecttheTygon1hosefromtheabsorberunitand sealitwithapipeplugandtakethebiogassamplewithasyringefromtheotherhose.
9 Checkdailythatthehardtubingpiecesthatconnectthebentstirrodtothemotorareundamaged andthemixingworksproperly.Otherwise,changethetubingpieces.
10Checkdailythatthewaterlevelinthethermostaticwaterbathreachestheplasticlidandfillitwith additionaldistilled water ifnecessary. Makesurethat theTygon1 hoseisnot bentabruptly, interferingwiththegasflowandnotbeingdamagedbytherotatingpartsoftheagitator/motor.
11 Verifythatthewaterlevelinthegasvolumemeasurementdeviceiswithintherecommended range.Fillwithadditionaldeionizedordistilledwatertotherecommendedwaterlevelwhen necessary.
12ThethymolphthaleinpHindicatorinsolutionFwillchangefrombluetocolorlesswhentheCO2
absorption capacity of the NaOH solution drops below the optimum. At this point, it is recommendedtoreplacethebottlewithnewsolutionFtopreventtheCO2gasfrompassingtothe gasvolumemeasuringdevice.Approximately2.9LofCO2canbecaptured(at22C)ineachbottle beforeitneedstobechanged.
13TochangethesolutionF,placepipeplugsonthehosesoftheincubationunitandgasmeasuring device.Replacetheoldsolutionwithanewone,reconnectthehosesandremovethepipeplugs.
NOTE.BeforeusingtheAMPTSIIsystem,readtheoperationandmaintenancemanualcarefully, detailsofsettinguptheequipment,connection,andsoftwaremanagementareincluded.
Endofthetest
1TheAMPTSIIsoftwareautomaticallyupgradesthecumulativehydrogenproductioncurve;review thecurvedailytodeterminetheendoftheessayfollowingthestopcriteria.Thestoptimecriterion (ST) is obtained when theH2 production curvebecomes asymptotic (FT) (Fig. 4) [8]. Asan
additionalstopcriterion,youcanusethecoefficientofvariationbetweenthelastthreerecordsof cumulativehydrogenwhenitbecomeslowerthan5%.
2 IntheAMPTSIIsoftware,generateareportintheDownloadreportmenu.Downloadthereportand openittomakesurethatthereporthasbeengeneratedcorrectlyandthatnoerrorshaveoccurred duringthedownloadofthefile.
3 Stoptheregistrationbypressingthepausebuttonandthenthestopbutton.Note:Bypressingthe stopbutton,theexperimentassociatedwiththatparticularcellcannolongercontinue.
4 Turnoffthethermostaticwaterbath.
5 Settheon/offswitchofeachmotorunittoOFF.
6 SetthesystemswitchonthemotorcontrollertoOFF.
7 Unplugthepoweradapters(forthemotorcontrollerandthegasvolumemeasuringdevice)from thepowersupply.
8 DisconnectthehosesbetweenthereactorsandtheNaOHbottles.
9 DisconnectthehosesbetweentheNaOHbottlesandthegasvolumemeasuringdevice.
10Emptythewaterbathusingthemanualwaterpumpincludedwiththeequipment.
Samplingandanalysis
1Oncethekineticshavebeencompletedaccordingtothepreviouslymentionedcriteria,openthe bottlesandrecordthepHvalueofitsliquidcontent.
2Keep the bottles in constant agitation with the help of a magnetic stirrer and grill. Take a homogeneoussampleofatleast20mLfromeachbottleanddeterminetheconcentrationoftotal andvolatilesuspendedsolidsbyduplicate(5mLpersample)accordingtostandardmethods[7].
3Take a sample of at least20mL fromeach bottle todeterminethechemical oxygen demand (COD),fermentationproducts,andresidualcarbohydrates.Acidifythesampleswithconcentrated H2SO4(pH<2).
4Centrifugethe20-mLsamples(3600rpmfor10min,oratahigherspeed)andfilterthesupernatant with0.45
m
mnitrocellulosefilters.Thesamplecanbekeptrefrigerated(4C)fornomorethan 7daysuntilanalysis.Ifsamplescontainmanysuspendedsolids,beforethenitrocellulosefilteritis advisabletousea1.5m
mglassfiberfilter.5FromthecentrifugedsampleanalyzetheCODaccordingtostandardmethods[7],thefermentation productsbygasorliquidchromatography,aswellastheresidualcarbohydratesbythephenol- sulfuricacidmethod[9].
Fig.7. Cumulativehydrogenproduction(Hmax)specifictotheworkingvolume(LH2L1),forthemanualandautomatic protocols,anditscorrespondingintra-andinter-laboratoryvariation.Forthemanualprotocolevaluationdifferentinoculum weretestedbydifferentlaboratories,meanwhilefortheautomaticprotocolthreedifferentsourceofinoculumwereevaluated indifferentlaboratories.Thedifferentinoculumsourceswereheat-treatedanaerobicsludgefromathermophilicdigester (HTT);heat-treatedanaerobicsludgefromamesophilicdigester(HTM);biomassfromanauto-fermentedeffluentrichin sucrose(AF);aerobicsludgepretreatedbycellwash-out(WO);compostfromkitchenwastes(C);andheat-treatedanaerobic sludgefromadigestertreatingsolidwastes(HTS).L1–L8standsforresultsfromdifferentlaboratories.
Validationofanalyticalmethods
It is highly recommended to validate the analytical methods to determine COD, sugars and fermentationproductsasindicatedbelow:
1 Weigh1gofanhydrousglucose(driedat105Cfor2handstoredinadesiccator)anddiluteina1L volumetricflaskwithdistilledwater.
2 Analyzethe CODand carbohydrates of thesolutionwith themethodologyof each laboratory.
ConsiderthatglucosehasatheoreticalCODof1.067gCODgglucose1.
3 IftheresultsofCODorcarbohydratesanalysisarenotasexpected,youshouldreviewyouranalysis procedureandcalibrationcurves.
4 Tovalidatethemethodsofmeasuringfermentationproductsitissuggestedtousecertifiedreference materials(e.g.VolatileFreeAcidMixCRM46975-Supelco,andLacticacidPHR1215-Supelco;Merck KGaA,Germany).
Calculations
H2cumulativevolume
FromthecumulativehydrogenvolumegraphadjustthecurvetothemodifiedGompertzmodel [10],recordthesoftwareusedandthecorrelationcoefficient,maketheadjustmentforeachreplicate:
HðtÞ¼Hmaxexp exp 2:71828Rmax
Hmax ð
l
tÞþ1WhereH(t)(mL)isthecumulativehydrogenproductionattimet(h);Hmax(mL)isthemaximumgas produced;Rmaxisthemaximumproductionrate(mLh1);and
l
isthelag-phasetimebeforetheexponentialhydrogenproduction(h).Forcumulativehydrogenproductionresultsusingthemanual version,adjusttherecordedvolumetostandardconditions(0Cand1atm).Theautomaticdevice AMPTSIIreportsthegasproductionatstandardconditions,accordingtointernaltemperatureand pressuresensors.
CODbalanceoffermentationproducts
It is highlyrecommended toquantifythenon-identified(N.I.CODeq)products followinga COD balance[11]:
N:I:CODeq¼CODfGlucoseCODeqX
MetabolitesCODeq
WhereCODf(gCODL1)istheanalyticalsolubleCODconcentrationdeterminedattheendofthetest;
GlucoseCODeq (gCODeqL1)istheresidualglucoseconcentration;and P
MetabolitesCODeq isthetotal solubleproductsconcentration(gCODeqL1)determinedbychromatography.TheCODequivalenceof the different fermentation products and carbohydrates, can be calculated with the following equations,basedontheaverageoxidationreactionsoftheanalyzedcompoundsandthewater-oxygen aselectronacceptor.
aÞ mg CODace
L ¼ X mgace L
1 mmol 60 mgace
8 meq e 1 mmolace
8 mg OD 1 meq e
bÞ mg CODpro
L ¼ X mgpro L
1 mmol 74 mgpro
14 meq e 1 mmolpro
8 mg OD 1 meq e
cÞ mg CODbut
L ¼ X mgbut L
1 mmol 88 mgbut
20 meq e 1 mmolbut
8 mg OD 1 meq e
dÞ mg CODval
L ¼ X mgval
L
1 mmol 102 mgval
12 meq e 1 mmolval
8 mg OD 1 meq e
eÞ mg CODcap
L ¼ X mgcap L
1 mmol 116 mgcap
24 meq e 1 mmolcap
8 mg OD 1 meq e
fÞ mg CODeth
L ¼ X mgeth L
1 mmol 46 mgeth
12 meq e 1 mmoleth
8 mg OD 1 meq e
gÞ mg CODlac
L ¼ X mglac L
1 mmol 90 mglac
12 meq e 1 mmollac
8 mg OD 1 meq e
hÞ mg CODfor
L ¼ X mgfor L
1 mmol 46 mgfor
2 meq e 1 mmolfor
8 mg OD 1 meq e
iÞ mg CODsuc
L ¼ X mgsuc
L
1 mmol 118 mgsuc
14 meq e 1 mmolsuc
8 mg OD 1 meq e
jÞ mg CODglu
L ¼ X mgglu L
1 mmol 180 mgglu
24 meq e 1 mmolglu
8 mg OD 1 meq e
kÞ mg CODH2¼X mL H2; STP 1 mmol 22:4 mLH2
2 meq e 1 mmolH2
8 mg OD 1 meq e
WhereXmgcompoundL ¼producedmetabolitesconcentration mgL
;ace,aceticacid;pro,propionicacid;but, butyricandisobutyricacids;val,valericandisovalericacids;cap,caproicandisocaproicacids;eth, ethanol;lac,lacticacid;for,formicacid;suc,succinicacid;glu,glucose;H2,hydrogen;XmLH2,STP, hydrogen volume at standard temperature (0C) and pressure (1atm) conditions. The COD equivalenceisalsousefultodeterminethestoichiometryofthereactionsandmetabolicpathways followedbytheinoculumused.Anexampleofsuchcalculationisdetailedelsewhere[12].
Qualitycriteriafortheresults
Inordertovalidatethehydrogenproductiontests,thecoefficientofvariationofHmaxmustbe<15
%,evenafterapplyingastatisticalanalysistoeliminateoutliers[4].UsetheDixontesttoeliminatea uniqueoutlieroftriplicates,consideringapvalue=0.05.
Methodvalidation
The present method was validated in 8 independent laboratories from 5 different countries (Brazil, Chile, France,MexicoandUruguay);adetaileddiscussionaboutitsdevelopment,validationandconsiderationof workloadtimewerealreadypublished[4].Averagevaluesofcumulativehydrogenproduction(Hmax)and thecorrespondingstandarddeviationswereusedtocalculatetheintra-laboratoryandinter-laboratory coefficientofvariation(CV)asrepeatabilityandreproducibilityindicators,respectively.
Applyingthepresentprotocol,theaverageintra-laboratorycoefficientofvariationrangedfrom13
%to9%usingthemanualandtheautomaticprotocol,respectively.Butusingtheautomaticprotocol, theinterlaboratoryvariationcouldbereducedupto15%withtheHTTasinoculum;whichisan acceptableresultaccordingtostudiesanalyzingthebiochemicalmethanepotential(Fig.7)[13].
Conclusions
Thismethodfeaturesanewandvalidatedprotocolforbiohydrogenpotential,providingatoolfor inter-laboratory studies and reliable comparisons of results. Indeed, following the protocol, the variationofresultscanbereducedtoacceptablelevels,usingeitherthemanualorautomaticversion.
DeclarationofCompetingInterest
The authors declare that they have no known competing financial interests or personal relationshipsthatcouldhaveappearedtoinfluencetheworkreportedinthispaper.
Acknowledgements
ThisworkwasperformedintheframeoftheBiohydrogenLatinAmericanetwork.Theauthors thanksthefundingofthefollowingprojects:FondodeSustentabilidadEnergéticaSENERCONACYT, ClústerBiocombustiblesGaseosos,247006andCONACYT240087;Fondecyt3160219;ProgramECOS- CONICYTprojectN C12E06, GRAIL613667 (FP7-KBBE-2013);ANII-FSE102488 (Uruguay); FAPESP (2015/06246-7)and CNPq processes 406751/2013-7,150641/2015-0,150475/2016-0 (Brazil); and programEUWaste2bioHyproject(FP7-MC-IEF-326974).ThecofundingofBioprocessControlAB (Lund,Sweden)fortheopenaccesspublicationofthismethodisalsoacknowledged.Theauthors thankthe technicalassistance of Carolina MejíaSaucedo, Gloria MorenoRodríguez,Jaime Pérez Trevilla,ÁngelA.HernándezHuerta,GuillermoVidriales-EscobarandRobertoE.BolañosRosales.
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