Running Head: Anaerobic Ammonia Oxidation 1

AStudyinAnaerobicAmmoniaOxidation(Anammox)

ThesisBy

JonasReeder

NatureofthePhenomena

Introduction

Throughout its history no single organism has acquired the capacity to influence or alter theEarth’sdelicatelybalancedbiospheresquitelikethespeciesHomosapiens insuchashorttimeperiod.Humankind’sabilitytoaffectenvironmentalchangehasbeenbroughtaboutoutofnecessity,greed,inthe name of progress and through a general, though often innocent, lack of comprehensiveunderstanding. This ability to shape our world has led to many extraordinary discoveries andtechnologicaladvancesbutitshouldalsoinferasolemnresponsibilitytothesustainedpreservationofeach and every environmental ecosystem and a sincere accountability to the overall health of ourplanet.Natureitselfhasproventobeaforcemostthoroughinourplanet’srecyclingandbioremedialmaintenance processes. Therefore it will likely be from methodical study and faithful emulation ofnature that true understanding of our world can be realized and the capacity to accommodatecontinuousavailabilityof resources (resource sustainability) forevery inhabitantof theglobe. It is tothisend that the topicofnutrient removalandmore intentionally, themeticulous removaloforganicnitrogen from source waters, was chosen for investigation in the development of this thesis. Theimportance of this explorationwill be obvious and continue to grow in urgency aswe take notice ofincreased reports of validated indicator events describing this category of environmental pollution,indicatoreventssuchastoxicalgalblooms,fishandotherbiologicalkills;thisallinthepresenceofalessthaneffectualmeansatourcurrentdisposaltomanagetheseissues.

Whilereadingthroughanumberofreferencematerialsaimedattheisolation,oridentificationrather, of novel nitrifying bacteria and specifically themore recent discovery of the aptly designatedannamox microbes and the unique processes that they show evidence of, the thought provokingquestions or conceptual arguments which never strayed far from focus were; can the anammoxtreatment process replace the classic model for nitrification/denitrification commonly controlled bymeans of an activated sludge system design? If so what are the advantages and are there anylimitations?Arethereanycostsavingsbenefits?Wouldtherebeasignificant increase inbio-remedialproductivity,or justanotherpromisingyet impracticalprocess?Inshort, isanammoxaneffectivenewnutrient treatment process or one that simply declared cannot deliver on any real promise tosignificantly improve on our ability, as remediators, to clean up and responsibly manage theenvironment? These are all relevant questions that deserve further investigation, because theimplications for any new treatment process that could, in theory, provide a cheaper,more efficient,cleaner and/or safer, alternative to current treatment processes could have lasting influence on thefutureofallnutrientremovalinwastewatertreatment.

Theanswerstothesequestionsarenotnecessarilystraightforwardanddependultimatelyonhowpersistentorpatientrather,apotentialimplementermaybe,which,afterfurtherthesis

Anaerobic Ammonia Oxidation 2

developmentshouldbeapparent.Atpresent,thisreallyraisesmorequestionsthancanbeansweredwithoutlookingalittledeeperintothebiological,chemical,andphysicalprocessesthatareinvolvedinboththeanammoxandtheclassictreatmentmodels.Nitrogen,thoughcalculatedatroughly80%(3.9X10¹⁵metrictons)ofthevolumeoftheearth’satmosphereand0.002%(7.7X10¹⁴metrictons)intheearth’scrustorlithosphere,islargelyunavailabletoalmostallknownorganisms.However,initsreducedoroxidizedformitisundeniablyoneoftheprimarynutrientscriticaltothesurvivalofalllifeonearthandalternately,oneoftheprimarypollutantintheeutrophicationofaquaticecosystems.Again,sincethemajorityofatmosphericnitrogenisintheformofdinitrogen(N₂)andthenitrogenouscompoundsthatarefoundinthelithospherearenottypicallyinbio-availableform,thesereservoirsfornitrogenareprincipallyunusabletoallbutafeworganismsdirectly.Dinitrogengasneedstothereforeberenderedusefulthroughaprocessknownasnitrogenfixation.Thisnaturalprocessismadelargelypossiblebyagroupofmicroorganisms,whichforthemostpartwillnotbediscussedhereandissubsequentlyatopicforanothertime,andalsotoasignificantlylesserextentthechemicalreactionsassociatedwithlightningstrikes.AnothermethodforthefixationofnitrogenistheindustrialchemicalmanufactureofammoniafromnitrogenandhydrogenthroughTheHaberProcess.Thisprocesswasdevelopedintheearly1900’sbytheGermanphysicalchemistFritzHaber.Thisprocesscombinesnitrogenfromtheairwithhydrogenderivedmainlyfromnaturalgasormethaneintoammoniaasseeninthefollowingequation,

andvisualizedusingthefollowingschematic:

Figure1:“TheHaberProcess,”byJ.Clark,2002,(modifiedApril2013).

Fascinatingthoughthisman-madeprocessmaybeittoowillnotbefurtherdevelopedinthisthesis.

Theimportantthingtonotehereisthatafternitrogenfixationthenitrogenisnowinformsthatcanbeutilizedby livingorganismsforsimplenutritionandfortheconstructionofcomplexmolecules,suchasproteins,DNA,andChlorophyll,tonameafew.Theinorganicnitrogenatthisstagewillbefoundaseitherdissolvedammoniagas (NH₃),or theammonium ion (NH₄⁺), andmore likely the latter since

Anaerobic Ammonia Oxidation 3

fixed nitrogen and molecular compounds, in general, are reasonably dependent on pH and typicalenvironmentalpHvaluestendtodrivethenitrogenfixationreactiontoyieldmoreammoniumions incontrasttotheproductionofammoniagasmolecules.Rememberthatdependingontheconcentrationsof the various forms of nitrogen containingmolecules present, they could be considered significantlydetrimentalenvironmentalpollutants.Thereareothertransformationsthatbothorganicandinorganicnitrogencananddoesundergowithinthenitrogencycle;andeachwillhaveitsownsignificance,butintheinterestoftimeandthepurposeofthisprojectwewilllimitourfocusofthetraditionallyvalidatednitrogen cycle to the specific processes of nitrification and denitrification and even further limit thereview of these processes with the exceptions of how they relate to, or as a comparison for, the“anammoxreaction.”

ReviewofLiterature

ClassicDenitrificationSystems

Thetraditionalorconventionalconceptofactivatedsludgenitrificationanddenitrification,theprocessmainlyreliedupontosomeextentoranotherbythemajorityoftreatmentfacilities,seemstobethemostcomprehensivelyunderstoodmechanismfortheremovalofnitrogenouscompoundsfromwastewater.Assuch,therearenumerousexcellentsourcesofinformationouttherethatindetailmakeclearthisprocessbutsinceanammoxitselfisthefocusofthisresearchandnotthetraditionalprocessnecessarily, going intodetailaboutawelldocumentedandreasonably reliable treatmentstrategybywayofan indepthreviewofpublishedsources isnotentirelynecessary.Agoodgeneric referenceoronline “white paper” on the subject was published online by TheWater Planet Company and titled“Nitrification&Denitrification.”Thereisnospecificauthorcreditedwiththepublishedarticlebutsincethetraditionalmodelcanbeeasily researchedfromcountlessothersourcesandthisparticulararticlecontainsinformationthatisrelevantasacomparativetoolbetweenthecurrenttreatmentprocessandanynewer technologies, it’s thisarticle thatwascitedwhiledevelopingabackgroundwith regards toactivatedsludgenitrificationanddenitrification.

Theconventionalprocesstechnicallyhappensinthreesteps,firstammonificationtakesplaceasorganicnitrogenisconvertedtoammonia.Secondly,andmoreimportantwithregardstothisresearch,ammoniumnitrogen(NH₄-N)isconvertedtonitratenitrogen(NO₃-N),initself,atwostepprocesscallednitrification (“Nitrification & Denitrification,” 2015). It has been long known that Nitrosomonas andparticularlyNitrosomonasEurophaisthemicroorganismmostcommonlyassociatedwiththeconversionof ammonia tonitriteor rathernitrosofication, the first step in theprocessof nitrification. There areotherspeciesofcourse,namelyNitrosospira,Nitrosococcus,andevenammonia-oxidizingArchaeahavebeen grown in pure cultures (“Nitrification & Denitrification,” 2015). After these organisms haveconvertedsomeoftheNH₄-Ntonitritenitrogen(NO₂-N)asecondgroupofmicroorganismscanfinishtheconversionofNO₂-NtoNO₃-Nandthegroupofbacteriacommonlyassociatedwiththenitrificationprocess is called Nitrobacter. Other members of the nitrite-oxidizing genera include Nitrospira,Nitrococcus,andNitrospina(“Nitrification&Denitrification,”2015).Bothgroupshavebeenextensivelystudied in all manner of natural environments and play important roles in wastewater treatmentfacilities by removing harmful levels of ammonia helping to prevent pollution to receiving waters.Together these bacteria are the “nitrifiers” and are all strict aerobes,meaning that theirmetabolismfunctionsinconditionswherethereisoptimallyatleast1.0mg/Loffreedissolvedoxygen(“Nitrification& Denitrification,” 2015). Also, keep in mind that nitrification itself does not remove nitrogen fromwastewateritsimplyremoveoxygendemandbyoxidizingNH₄-NtoNO₃-Nitselfalessdamagingvariantofnitrogen.

Anaerobic Ammonia Oxidation 4

The reason for choosing the current articlewill now be apparent by looking at the followingequations as detailedby thedocuments author regarding thenitrificationprocess. The carbonaceousBOD at this point is reduced to a low value (<15mg/L). This does not affect nitrifiers since they arechemolithotrophic (chemoautotrophic)organisms. TheseorganismsuseCO2as their carbon source togrow.

Alkalinitybufferingequation

H₂O↔H₂CO₃↔HCO₃+H⁺↔CO₃+2H+

Nitrificationequations

NH₄⁺+1.5O₂→2H⁺+2H₂O+NO₂⁻

NO₂⁻+0.5O₂→NO₃

NH₄⁺+1.83O₂+1.98HCO₃⁻→0.021C₅H₇O₂N+0.98NO₃⁻+1.041H₂O+1.88H₂CO₃⁻

NH₄⁺+1.9O₂+2HCO₃⁻→1.9CO₂+2.9H₂O+0.1CH₂

Thoughnotentirelycorrectlybalancedinthisdocument,theauthorsuggestsusingtheseequationstocalculate the amount of resources required. In a typical treatment facility these resources are notnecessarilyavailabletooxidizeammoniatonitrate.UsingtheInternationalSystemofUnits(SI)itiseasyto convert these calculations to themetric system for simplicity and consistency. These resources inmostcasesrepresentacostandtheyconsistof:

4.18poundsor1.90Kilograms(Kg)ofoxygen

7.14poundsor3.24KgofalkalinityforpHbufferingascalciumcarbonate(asCaCO₃)

12poundsor5.44KgofalkalinityforpHbufferingassodiumbicarbonate(asNaHCO₃)

Theaboveprocesscombinedwiththethirdandfinalstepinthetraditionalnitrificationand denitrification model, that being the actual denitrification step, allows for some interestingconclusionstobedrawn.Thedenitrificationprocessoccursunderanoxicconditionswherethedissolvedoxygenislessthan0.2mg/Lideally,meaningthatnitratebecomestheprimaryelectronacceptorfortherelevantfacultativeheterotrophicmicroorganisms(“Nitrification&Denitrification,”2015).Thisresultsinreduction of nitrates to nitrite and subsequently to nitrogen gas and nitrous oxide. The nitrogen gasthenescapesback into theatmospherewhere ifa startingpointweresubjectivelychosen it couldbestated that the process begins all over. It’s worth pointing out that because these afore mentionedmicroorganisms areheterotrophic, theywill require a sourcesof carbonandenergy for their survival(“Nitrification&Denitrification,” 2015).Asmentionedabove,most conventional treatmentplants arelimitedintheirabilitytoeffectivelyutilizethisprocessasthereistypicallynotenoughorganiccarbon,resultinginlowBODatthispoint,toremoveallreactivenitrogenbydenitrificationandassuchacarbonsource would need to be added for denitrification to occur completely. The formula describing thisreactionisthus:

6NO₃⁻+5(CH₃OH)→3N₂+5CO₂+7H₂O+6OH.

Anaerobic Ammonia Oxidation 5

Figure2:“ActivatedSludgeTreatmentProcess,”byM.Sondossi,2015.

In conclusion, for complete nitrogen removal to occur in the traditionalmodel, the ammoniaload requires the addition of aeration and alkalinity, throughmultiple carbonate sources, to achievenitrificationandtheadditionofareadilybiodegradableorganicsubstrate(mostcommonlytheadditionof methanol) to carry out denitrification. These additions obviously add to the overall cost of atreatment plants operation; either through power consumption, from blowers aerating the wastestream, or the addition of massive quantities of costly chemicals. Additionally, the more traditionalprocessfornitrogenremovalalsorequirestheuseoflongerretentiontimes.Thisistoensuretheslowergrowing nitrifying bacteria are not washed out of the system but consequently this generates aconsiderable quantity of bio-solids that will require some method of disposal. Now how does thiscomparetotheanammoxreaction?

AnammoxDenitrificationSystems

The definition or explanation rather of the anammox process is as follows. The acronymanammoxstandsfor“anaerobicammonia-oxidation”andisspecificallyassociatedwithaspecialgroupofmicroorganisms(bacteria)thatoxidizeammoniawithnitriteasanelectronacceptor,twoofthemostpotentially dangerous formsof inorganic nitrogen, under anoxic conditions toproducedinitrogen gas(van Nifrik et al.,2012). There evidence to show that, under NH4 concentration limitation, alternateelectrondonorsandacceptorsotherthanNH4

+andNO2-maybeused.Anammoxbacteriaunderthese

conditionscanuseacetateorpropionateaselectrondonorsandsomespeciescanevenusemanganeseoxidesandmetals,likeiron,insteadofnitriteaselectronacceptors(vanNifriketal.,2012).Inonestudyexploringtheinfluenceofmetalionadditionsonthespecificanammoxactivity(SAA)itwasconcludedthat thecontrolledadditionofFe³⁺produced“amaximumSAA increaseof533.2%”,which lead to“a50% increase in thenitrogen removal capacityobservedatambient temperature” (Chanetal.,2014).Theseadaptationswill notbe focusedon for this research, but it canbe cautiously stated that thesemicroorganismsarereasonablyadaptabletoavarietyofvery inhospitableenvironmentsandaretrulyadmirableintheirabilitytoutilizewhateverresourcesmaybeavailabletothemintheirsimplepursuitofsurvival.

Anammoxbacteriaarecoccoidshapedwithanaveragediameterrangingbetween800and1100nmandtheyareanaerobicchemolithoautophicmicroorganismsthatusenitriteasanelectronacceptorcombined with ammonia to produce the already mentioned dinitrogen gas as its final product.

Anaerobic Ammonia Oxidation 6

Hydrazine(H₂N₄),or“rocketfuel”,andnitricoxide(NO)arealsoproduced,butasintermediatestothisprocess (van Nifrik et al.,2012). There have been five thus far described belonging to the orderBrocadiales,fromthephylumPlanctomycetes.Inbacterialnomenclature,Candidatusisacomponentofthetaxonomicnameforabacteriumthatcannotbemaintainedinlaboratoryculture.Thefiveare,innoparticular order, Candidatus Brocadia, Candidatus Jettenia, Candidatus Kuenenia, CandidatusAnammoxoglobus,andCandidatusScalindua.Thefirstfourofwhichhavebeenenrichedfromactivatedsludge,whileC.Scalinduaseemstobethepredominantspeciesfoundinnaturalenvironments,suchasmarinesediments(vanNifriketal.,2012).

Studiesconductedasearlyasthe1940’sindicatedthatamicroorganismwas“missing”,inotherwordsasyetunidentified, fromnaturethatcouldanaerobicallyoxidizeammonium.Thiswasbased inpart,onobservationsthattherewassignificantlylessammoniumthataccumulatedinanoxicconditionsthanmathematicalcalculationsshouldhavepredicted(vanNifriketal.,2012).Itwasnotuntiltheearly1990’s, however, that the first indications of this process could be experimentally obtained and thisoccurred at Gist-Brocades, “when ammonium was found to be converted to dinitrogen gas at theexpense of nitrate in an anoxic fluidized-bed bioreactor”, a Netherlands yeast factory (van Nifrik etal.,2012).Thisdevelopmentshowedthattherewaswithinthenitrogencycleatrulyidentifiablemissinglink but no one could have predicted that these microorganisms would defy many conventionalmicrobiological concepts. Itappears that far fromconforming to the typical characteristicsofbacteriatheseanammoxbacteriahavefeaturesincommonwithallthreedomainsoflife,Bacteria,Archaea,andEukarya,and their “unusualmetabolism”makes themofextreme interest in the fieldsofwastewaterapplication andmicrobial ecology. It has beenproposed that they lack both anoutermembrane andpeptidoglycan.Further, theyhavebeenreferred toasproteinaceousandseemto, for themajorityofthe currently identified species, have cell envelopes that resemble neither Gram-positive nor Gram-negativebacteria.TheonlyexceptionbeingthatofC.KueneniawhichdoeshavecharacteristicsthatcansuggestthepresenceofaGram-negative-likecellwall,includingtheperiplasmicspaceencapsulatedbyan outer membrane. It turns out that anammox bacteria grow slowly (van Nifrik et al.,2012).Comparably, traditional chemolithotrophic nitrifying organisms tend to also have slow growth rates.Under optimal conditions measured in laboratories the anammox microbes double on average onlyaround11daysandas resultof this lengthygeneration time thesemicrobeshaveyet tobegrown inpureculture.Todatetheanammoxbacteriadefystandardmicrobialcultivationmethods(vanNifriketal.,2012). It goes without saying that pure cultures could optimize the application of anammox inwastewatertreatmentplants.

So how do these unusual microorganisms make the anammox process work specifically?Thoughtheprocess isstillnotclearlyunderstood,theredoesseemenoughevidenceexiststosupportthe general hypothesis that the anammox process takes place in a membrane bound compartmentcalledtheanammoxosome,oneoftheanammoxbacteria’smaincellularcharacteristics.Thisstructurehasbeentheorizedtobesimilarinfunctiontothemitochondriaineukaryotes,inotherwordsacellularcompartmentdedicatedtoenergymetabolism.Anotherfeaturethatcanbefoundinanammoxbacteriaandonethatisthusfaruniqueinnatureiscalledtheladderanemembranelipids.Speculationregardingtheladderanemembranelipidshasmaintainedtheyarehighlyimpermeable,whichwouldpreventionsand metabolites from excessive loss (van Nifrik et al.,2012). It could also provide integrity to thestructureoftheanammoxbacteria’scell.Itisfurtherspeculatedthatthisuniquemembraneadaptationisone ifnot themainreasonthesemicroorganismsarecapableof theanammoxreaction.Thetightlyfoldedstructurewouldpreventthetoxic intermediatephasesofnitrogen,beingthatof thehydrazineandnitric oxide fromescaping the anammoxosomeanddoingdamage toother structureswithin thebacterium’scell.In2007itwasproposedthatthefirststepintheanammoxprocessisthereductionof

Anaerobic Ammonia Oxidation 7

nitritebyanitritereducingenzymetohydroxylamine(Brandesetal.,2007).Thisprocess,theprocessofcatalysis is completed by several cytochrome c proteins and other hydrazine (HAO) oxidizing-likeproteins to convert any hydroxylamine into nitric oxide, an essential intermediate in anammoxmetabolism(vanNifriketal.,2012).Togetherwithammonium,thenitricoxidethathasbeenconvertedfromhydroxylamineandthefreenitritearecombinedtoformthehighlyvolatileandreactivehydrazinebytheenzymehydrazinehydrolase.Thenhydrazineoxidizingenzymesoxidizehydrazinetodinitrogengas (N₂) and indoing so liberateprotons. It shouldbepointedout thathydrazine synthaseandnitricoxide reductase found in denitrifying microorganisms are the only enzymes known to be able tofacilitatethebondingoftwonitrogenatoms.ThismechanismestablishesaprotongradientwhichcanbeusedtoproduceATP(vanNifriketal.,2012).

DenitrificationStrategies

Obviouslythishasbothsimilarities,aswellasdifferences,fromthetraditionalnitrificationanddenitrificationmodelat least inall thespecificmicroorganismmetabolicactivitiesor thoseassociatedwith each relevant process, though the final resultsmay not necessarily be noticeably any different.However,twonoteworthypointsforconsiderationarethattheanammoxreactiontookplaceinanoxicconditionsoranaerobicprocesses,meaningnomanuallysupplementedsourceofoxygenwasrequired.Secondly, the addition of costly biodegradable organic substrateswas unnecessary for the anammoxmodel.What thismeans iswhile the complete process of nitrogen removal (carrying the reaction orammonia rather, to complete denitrification by forming dinitrogen gas) can occur through all thedomainsof life.Bacteria,archaea,andeveneukaryotescandenitrifynitratenitrogentosomedegree;howeverecosystemswithasurplusoforganicelectrondonorswill favor thetraditionaldenitrificationprocess.Nitrateassimilationisachievedthroughitsbiologicalreductiontoammonium.Thesubsequentutilizationofammoniumtoformaminoandamido-Ncompoundsprovidesthelinkamongtheplethoraofpathwaysoforganicnitrogenmetabolismand thepathwaysof inorganicnitrogenassimilation.Thecapability toassimilatenitrate ispossessedbycertainbacteria, some fungi,andvirtuallyallalgaeandhigherplants. It isabsentfromtheanimalkingdom.Nitrateassimilationrepresentssubstantialenergyexpenditurebythecellwhencomparedwithammoniumutilizationbecauseeightreducingequivalentsare consumed in the reduction of nitrate to ammonium. Consequently, cells that assimilate nitrate,regulate this pathway to avoid wasteful use of reducing power when the product, ammonium, isavailable.Therefore,ecosystemswithlowoxygenandorganiccarbon,withpresenceofanamplesupplyof ammonium will likely favor the anammox process. Looking at a diagram borrowed from NatureEducationbelow,abetterunderstandingofhowthetwoprocessesultimatelydiffercanbeobtained.

Anaerobic Ammonia Oxidation 8

Figure3:“MajorTransformationsinthenitrogencycle,”byNatureEducation.2010.

Fromtheabovediagramit’sapparentthattheanammoxmicrobesliterallyskipmultiplestepsutilizedbythecombinedeffortsofthemicroorganismsassociatedwiththetraditionalconceptofnitrification.Infactwhatistypicallyathreestepprocess,forconventionalnitrifyinganddenitrifyingbacteria,canliterallybeaccomplishedbytheanammoxmicroorganismsinonestepunderoptimalconditions.

Thequestionnowshouldbehowtoutilizethesemicrobesandtheanammoxprocessspecifically?Asithasalreadybeenstated,theanammoxreactiontakesplaceinanoxiczones;infactthepresenceofoxygeneveninsmallconcentrationsisinhibitorytotheanammoxmicroorganisms.Anoxiczonesinnatureandtreatmentplantsofferthesemicrobestheirbestchanceforsurvivalbutdomakethemtrickytostudy.Innature,deepwithinoceansedimentsisoneplacetheanammoxprocesshasbeenisolated(Kotayetal.,2013).Anotherlocationwheretheprocesshasbeenobservediswastewatertreatmentplantswhereoxygendemandisparticularlyhigh.Thisisespeciallytrueforsludgeandmixedliquors,commonlyreferredtoascentrateorfiltrate,andisextremelyrichinammonianitrogenaswellasothernutrients.Innormaltreatmentplantoperationsthefiltratewouldberecycledbacktotheplantheadworkswheretheextraammonianitrogenposessignificanttreatmentchallenges.Thiscanbeovercomeintheorybytreatingthefiltrateusingsidestreamreactors(Kotayetal.,2013).Further,conventionalnitrificationanddenitrificationofferenoughchallengestomaketheanammoxprocessseemvaluableasanalternativetreatmentoptionandultimatelythemostpracticaloptionforsidestreamtreatmentofthenutrientrichblackliquor.Thisprocesswasfirstmanuallyattemptedin2002atawastewatertreatmentplantinRotterdam,intheNetherlands.Sincethattimetherehavebeenadditionaltreatmentplantsadoptingthisprocesswithavarietyofvariationsonthesametheme,thatbeingtheutilizationofanammoxtechnologytoimprovetheirtreatmentcapabilities(Kotayetal.,2013).Sincethetechnologyseemstoworkeffectivelyundertherightconditionsitishighlylikelythatfutureplanningregardingnewtreatmentfacilitiesorexpansionsandupgradestoexistingplantswillprobablyconsideroperatingsomeversionofanammoxtechnologytoatleastaideintheirtreatmentprocess.

Anaerobic Ammonia Oxidation 9

AnammoxReactorConstruction

SowhatdoesanexampleofanammoxreactororFluidizedBedReactor(FBR)looklikeandhowdoesitoperate?Fromthediagrambelow,thereactoritselfappearstobeafairlysimpleapparatus.Itconsistsofasealedcylinderthatcontainsheatingcoilstocontrolthetemperature,apHprobeattachedtoapHmetertomonitorand, ifneedsbe,adjustthepH,andacarriergassupplylineusedtopipeininertgasessuchasnitrogenorargontocontroloxygenlevels inthecylinder. It is fedfromaseparatecontainerwhere it appears the influent stockwouldget theadjustments indicatedby themonitoringequipment attached to the actual anammox reactor. It is also plumbed to an effluent carboywhereadditionalanalysiswouldpresumablybeperformedtofurtherevaluatetheeffectivenessoftheongoingprocess(Kotayetal.,2013).Thereactorinthisdiagramwasoperatedaccordingtothefollowingstartupprotocols; the anammox reactor was semi-continuously fed and maintained to achieve anaerobicammoniaoxidation.Itwasinoculatedwithabiomass,presumablyofanammoxmicroorganismsfromapublicsource,andwasfeedanaerobicdigesterfiltrate(blackliquid)fromalocalwastewatertreatmentplant. During the startup procedure the filtrate was fed a diluted supplement of NO₂-N by adding apreparedsolutionofsodiumnitrite.Thiswasnecessaryforreasonstobeshortlydiscussed.Thestartupprocess continued for nearly 40 days during which time nitrogen removal efficiency was constantlyanalyzed and recorded. Ultimately the reactor was deemed to be at a ready state. The reactor wasmaintainedatapHof7.8±0.3andthedissolvedoxygen(DO)wascontrolledbypurgingthesystemwithroughly95%argonanda5%CO₂gasmixture intermittentlydependingonthemeasuredpH.Also thereactorwaskeptat32°C(Kotayetal.,2013).

Anaerobic Ammonia Oxidation 10

Figure4:“Anammox(FBR)ReactorDiagram,”byM.Kotay,2013.

Asimilar strategywasusedwhereotheranammoxreactorswere inoculated frommixturesofsludge’sobtainedfrommunicipalwastewatertreatmentplantswhichwereoperatedunderoxic/anoxicconditions (Dapena-Mora et al., 2004). In this example the author’s reactorwasmaintained at 35 °CwithastringentpHof7.8to8.Constantmixingwasprovidedtothemediuminthereactorat50rpmand the reactorwas continually flushedwith the typical 95% argon to a 5% CO₂mixture in order tomaintain the desired anaerobic conditions (Dapena-Mora et al., 2004). From this and other similarlypublished experimentation, interesting inferences may be drawn. Whether using a commerciallyobtainedbiomassorcultivatingyourownanammoxbiomassenrichmentculturesbothofthediscussedmethodshavesome limitationsthatneedseriousattentiontobepaidduringthestartupofeitherofthespecificseededanammoxreactors.Namely,astheanammoxprocessisanaerobictheexclusionofoxygenisessentialatthiscriticalstage.Alsosincetheanammoxbacteriahaveveryslowgrowthratesitis important toutilize a system thatminimizes thebiomasswashout.Additionally, anammoxbacteriahave been shown to be inhibited in nitrite concentrations between 0.5mg/L and 10.0 mg/L.Furthermore, it has also been demonstrated to cause 50 % microbial activity inhibition at aconcentration of 0.4g/L. This means that during startup the addition of NO₂-N must be closelymonitored and in most cases carefully adjusted (Dapena-Mora et al., 2004). That being said theanammoxreactionconsumesapproximately1:1uptoa1.7:1ratioofNO₂-NtoNH₄-Nandsoitbecomeschallenging to treat high strength, ammonia rich filtrate because there is little or noNO₂-N available(Kotayetal.,2013).

Anaerobic Ammonia Oxidation 11

Thishurdlecanbeovercomebytheadditionofanotherprocessorparallelcomponenttotheanammoxmethod.EarlieritwasmentionedthatduringthestartupoftheanammoxreactorthefiltratewassupplementwithNO₂-Nassodiumnitrite.Thiswasnecessarybecause,asithasalreadybeenstatedthereislittletonoavailableNO₂-Ninthemixedliquororblackliquidfiltratefromtheanaerobicdigestertreatmentprocess.ThoughthisliquidhasampleNH₄-Ntosustaintheanammoxmicroorganismsitlacksthe necessary electron acceptors needed to truly begin the process (Kotay et al.,2013). This can beaddressed by avoiding the oxidation of all the NH₄-N in a waste water flow to NO₃-N and thendenitrifying this product to nitrogen gas by controlling the reaction. If only half of the NH₄-N wereoxidized to NO₂-N then the remaining NH₄-N and the newly formed nitrite could be converted tonitrogengasbyanammoxbacteriainananaerobic(absenceofoxygen)environment.Thewaythiscouldbeaccomplishedinapracticalmannerresultingintheneedforlessthanhalfasmuchoxygenandlessexcess sludge produced for disposal, in comparisonwith the traditional nitrification to denitrificationprocessesfortheremovalofnitrogenfromwastewater(Kotayetal.,2013).Akeyfactorthatfacilitatescontrolling thisprocess is the fact thatbacteria thatoxidizenitrite tonitrate (nitrobacter) growmoreslowly than both the bacteria that oxidize ammonia to nitrite (nitrosomonas) and the heterotrophicbacteria that oxidize carbon and hydrogen containing organic matter. Thus, maintaining the solidsretention time low (keeping microorganisms in the system for a short time) allows the oxidation oforganic carbon as Biochemical Oxygen Demand (BOD) and the oxidation of ammonia to nitrite butdoesn’t allow thepopulationofnitrobacter tobuildup to levels that allow theoxidationofnitrite tonitrate(Kotayetal.,2013).

Ideally thisprocessormethodof controlling themicroorganismpopulationwouldbe throughtheuseofwhatcanbecalledapartialnitritationreactor.Thispre-steptotheanammoxreaction,anddependingon the reference sited, hasbeen called a sequencingbatch reactor (SBR)or single reactorsystem for high ammonia removal over nitrite (SHARON). Other processes and technologies include:Oxygen limited autotrophic nitrification-denitrification (OLAND); completely autotrophic nitrogenremoval over nitrite (CANON). Regardless of the designation chosen to reference this process thereaction is ultimately the same as all have a high potential for reactive nitrogen removal (Pardes etal,2007).Thediagrambelowillustrateshowapartialnitritationreactormightlook.Clearlythistypeofreactorisnomorecomplicatedthananactualanammoxreactorandisessentiallycomposedofsimilarcomponents.Thedifferenceboilsdowntohowtheunitisoperated.Thepartialnitritationreactorwouldbe seeded with a biomass appropriate to the desired process results, which would likely includeenriched cultures of nitrosomonas. Itwould then be operated on 12 hour cycleswith two cycles perbatch(Kotayetal.,2013).Thisiscalledthehydraulicretentiontime(HRT).Atthebeginningofeachcyclerawfiltratewouldbeaddedandthenthereactionfollowedforthe12hourHRT.Thiswasfollowedby30minutesofoperator inactivity toallow forsettlingso thesupernatantcouldbedecanted.ThereactortemperatureandpHwerekeptessentially thesameas theanammoxreactororat32 °CandapHofroughly 8.0 ± 0.5 respectively (Kotay et al.,2013). Themajor difference between this reactor and theanammoxreactorwasthatthenitritationreactorwasaeratedatapproximately2.5L/minbypumpstomaintain a dissolved oxygen concentration of 1.5 ± 0.5mg/L (Kotay et al.,2013). It is the addition ofoxygentothereactorthatallowsforanenvironmentsuitableforthenitrosomonasmicroorganismstoestablishthemselvesandtoconverttheavailableNH₄-Nthroughammoniaoxidationtothedesired50%NO₂-N.Additionally,thehydraulicretentiontimewaskeptrelativelyshort,beingonly12hourcycles,sothenitrobactermicrobesneverreallyhadachancetoestablishapopulationsuitabletotheconversionof NO₃-N from the available NO₂-N. Finally, alkaline pH and controlled dissolved oxygen levels alsocontributed to limiting the oxidation of ammonia to nitrite without oxidation to nitrate (Kotay etal.,2013). This process was initiated around the sixtieth day in the start-up cycle of the anammox

Anaerobic Ammonia Oxidation 12

reactor. When it consistently yielded a ratio of 1.2:1 for NO₂-N to NH₄-N at around its 45th day ofoperation,itwasplacedinlinewiththeanammoxreactor(Kotayetal.,2013).

Figure5:“Partialnitritation(SBR)ReactorDiagram,”byM.Kotay,2013.

Usingthisstrategytheinitialreactiontookapproximately40daystoreachroughly80%removalof the total inorganicnitrogen suppliedby the filtrate feed (Kotayetal.,2013).Keep inmind thiswaswhentheNO₂-Nwasbeingsupplementedmanually.Aftertheintroductionofthenitritationreactortothe operational system the removal efficiency jumped up another 10% and ultimately stabilized atroughly 90 ± 4% of removal efficiency for the total inorganic nitrogen, at least after the nitritationreactor stabilized with an average nitrite to ammonia ratio of about 1.18:1 respectively (Kotay etal.,2013).Thiseffectivelydemonstratesthatifthenitritationtreatedflowgoestoananaerobictank,oranammox reactor, seededor culturedwithapopulationof anammoxbacteria theanammox reactionwill takeplace andbegin converting the incomingNO₂-Nwith the incomingNH₄-N tonitrogengas atmarkedlyhigherremovalrates.Thisandotherexperimentallyobtainedexamplesprovethatwhilethereare challenges in operating an anammox reactor successfully, it is possible to use this technology totreatnitrogenouscompoundsinwastewatertreatmentplantsandmoreexperiencewiththeoutlinedprocess will only help to refine the process and redefine the waste water industry by making theanammoxprocessthatmuchmoreviableasanefficienttreatmentoption.

Anaerobic Ammonia Oxidation 13

Methodology

MethodsDevelopment

Using what is currently understood about the previous processes we can develop throughexperimentaldesignafunctionalhybridizedpartialnitrationtoanammoxreactorinanefforttofurtherthecurrenttechnology.Thefollowingsetuporvariationthereofwillbeusedforthepurposeofnitrogenreductioninallfutureinvestigationthroughoutthisthesisdevelopment:

Figure6.“ProposedPartialDenitrification/AnammoxReactorDiagram,”byJ.Reeder,2015.

The system itself will be operated initially by the following recommendations. First theanammox (FBR) reactor will be seeded with an anammox biomass culture and the partial nitritationreactor (SBR) will be inoculated with a mixture of sludge’s both obtained from a local wastewatertreatmentplant.Thesystemwillbemaintainedat35°CwithapHrangecontrolledbetween7.8and8.0.Bothreactorswillbesubjecttoconstantmixingatabout50rpmwithmagneticstirbarsandallowedtoequilibrateaminimumofaweekpriortobeginningofanalysis.ItwillnotbenecessaryduringtheinitialstartuptoinstalltheanammoxfeedcylinderinlineasitcouldtakeseveralweeksfortheSBRtoachievethedesired50%conversionofammoniumtonitrite.ItmayalsobenecessarytoaddanartificialsourceofnitritenitrogentotheFBRduringthisphaseasareadysourceofelectronacceptorsfortheanammoxbiomass’s specific biological activity. The SBR will be purged with compressed air in an effort tomaintainadissolvedoxygen(DO)concentrationofatleast1.0mg/LwhiletheFBRwillbepurgedwithagas mixture designed to force DO out of the reactor and at a concentration < 0.5mg/L. After theprescribedequilibrationtimehasbeenmet,sampleanalysismaybeginasdetailedinAppendix.Priorto

HeatedStirPlateFeed

HeatedStirPlate HeatedStirPlateCompressedAir Nitrogen

Argon/CO₂Vent

GasAnalysis

SamplePort

PartialNitritationReactor(SBR)

AnammoxFeedCylinder

AnammoxReactor(FBR)

SamplePort

Anaerobic Ammonia Oxidation 14

bringingtheanammoxfeedcylinder in line itwillbenecessarytovalidatethroughanalyticalmethodsthattheSBR’sratioofnitritetoammoniumisstoichiometricallyquantifiedtobetween1.1and1.2(Ruietal.,2015).

Once this ratio is achieved the full system can be considered operational and therefore beplacedsequentiallyinoperation,asdetailabove.Initiallytheinstrumentationwillbeoperatedfollowingdocumentedproceduresregardingretentionandcycletimes.Thisreferssimplytotheamountoftimethat thebiomass isallowed to reside inanyoneparticular reactorandwhat rateanydesiredvolumepassesfromonevesseltothenext.Thisisanareainthisinvestigationwhereexperimentationwillbewarrantedinevitablyinanefforttofullyoptimizethepartialdenitrificationtoanammoxprocess.ItmayalsobebeneficialtoadjustpHandtemperaturesincombinationwith,stirringandsettlingtimes,astheprocessmaturestofurthervalidateinstrumentaloptimization.Theoreticallywenowhaveafunctioninganammoxreactorwithpartialdenitrificationandthisiswherewecanstarttoanswerthequestionsthatoriginallypresentedwiththeintroductionofthisinvestigation.

Findings

UsingeitheroftheIC(IonChromatography)methodsfromthesisappendixwillgenerateusabledata in under 10 minutes per aliquot. Though significantly more cost prohibitive initially, a two ICinstrumentation simultaneous setup,where each unit is optimized for specific analyte quantification,will save aminimumof 35minutes per sample andwhile not specifically addressed, amuch smallervolumeof sample is required for ionchromatographyanalysis. It shouldbenoted thatwhile IC is thequickermethod and spectroscopy is the least costlymethod, eithermethodwill generate equivalentdataeasilywithin5%uncertainty.

Finally, sincedenitrificationdoesoccurwithorwithout thepresenceofanammoxbiomass, itwould be interesting to use the concept of radioactive tagging and head space gas analysis todifferentiate,throughoutthisinvestigation,therelativeratiobetweeneachofthebiologicalprocesses.This couldbeused topredict the likelihood that theanammox reactionwillbe thedominant force indenitrification or as means to encourage this process above conventional nitrification/denitrificationmodels. For example, using ¹⁵NO2

--N (¹⁵N a naturally occurring stable nitrogen isotope) to essentiallyspike the anammox reaction it should theoretically be possible to differentiate anammox fromnitrification/denitrification.Assuming¹⁴N2(¹⁴N≡¹⁴N)isthetypicalproductofnitrification/denitrification,where ¹⁴NH3-N is origin. Itwould be possible to calculate themass abundance of each isotope andthereforequantifytheprocessmostresponsibleforthereductionofnitrogen in¹⁵NO2

—Nspikedstudy.Asmentionedaboveinnitrification/denitrificationthe¹⁴N2ismadeupof¹⁴N≡¹⁴N,whereinanammoxN2producedwillbemadeupof¹⁴N≡15N(seebelowequations).Withtheseassumptionsaheadspacegassamplewouldbecollectedfromthegassampleportdescribedaboveinfigure6onpage12,andthenanalyzedmostlikelythroughGasChromatography/MassSpectroscopy(GC-MS).Theratioof¹⁵Nrelativeto¹⁴Ncannowbeusedto,hypothetically,describewhichofthetwoprocessesarefavoredintheaboveprocess design. Using the ratio of ¹⁵N to ¹⁴N in N2 it should be possible to begin to improve processdesigninanefforttofurtherpromoteNH3

-Nreductioninfavorofanammoxratherthanconventionalnitrification/denitrification treatment strategies. This in turnwill yieldmore definitive answers to thequestionsinitiallyposed.

Anammox:¹⁴NH3-N+¹⁵NO2—N→→¹⁴N≡15N+2H2O

Nitrification:¹⁴NH3-N→¹4NO2—N→Denitrification:¹4NO2

—N→→¹⁴N≡¹⁴N

Anaerobic Ammonia Oxidation 15

Recommendations

Conclusions

Insummary,thepartialnitrificationtoanammoxreactionthough,dueinparttotheslowgrowthoftheanammoxbacteriaandtheavailabilityofnitritenitrogenneededtosatisfytheirmetabolism,isaviablealternativetoconventionalnitrificationanddenitrificationtreatment.Generallyspeakingtheanammoxreactioncanbeachievedwithin50daysifthestartupreactors,inotherwordsthepartialnitrificationandanammoxreactors,andtheirenrichmentculturesareoperatedandmaintainedwithastrategybasedonthefollowingprinciples.First,starttheanammoxreactorbysupplementingNO₂-Nataconcentrationofnotmorethan0.5–10.0mg/Linthefeedbutkeepingthenitritetoammoniaratioaround1.2:1.Next,flushthefeedwithanargon/carbondioxideoranitrogen/carbondioxidegasmixtureat95/5%forde-oxygenationoftheanammoxreactoranduseanairtightfeedtankwithacovertopreventlightpenetration.Finally,aftertheSBRhasequilibratedusingsimilarmechanismsforcontroltemperatureat35°C,pHat7.8,andgasfeedfordesireddissolvedoxygenconcentrationsandthereactorisyieldinga1.2:1ratioforNO₂-NtoNH₄-N,thenitcanbeplacedinline.Thenitritationreactorcanthenbecomethefeedsourcefortheanammoxreactor,thusreplacingtheneedfortheadditionofnitritetoactastheelectronacceptorsessentialfortheanammoxreaction.Tosavetime,asthisreactorsstart-upcycletimeisalsoaround50days,itwouldbeprudenttostartthenitritationreactorsimultaneouslywiththeanammoxreactor.Initially,itwouldbenecessarytokeepthereactorsindependentasthenitritationreactorwouldnotbeoptimizedandwouldneedthecycleperiodtogeneratethenitritenitrogenlevelsrequiredbytheanammoxreactor.

Thisprocess,oncetotaloptimizationiscompleted,isexpectedtoconvertammoniawithnitritetonitrogengasatroughly90%efficiencyorbetter.Sothisprocesscouldbe,withmoredevelopment,anexcellentoptionforthetreatmentofinorganicnitrogeninwastewater.ThestateofUtahdoesnothavelimitsonnutrientremovalinmostcircumstances;buttheyarecoming.Sotreatmentfacilitiesthatalreadyhaveissueswithhighammoniaresidualsintheireffluents,whichcanobviouslybecomeenvironmentallyproblematicinandofitself,canalsocombinewithchlorine.Manyplantsstilltreattheirfinaleffluentswiththisdatedbutcheapdisinfectionchemicalandstrugglewiththeformationofchloramines.Monochloramines,dichloramines,andtrichloraminesthatareproducedhavetheirowntreatmentdrawbacks,sotreatmentfacilitiesneedtobethinkingaheadofhowtobesttreatfornutrientsremovalinordertosatisfyupandcomingregulatorylimitsandresponsiblyspendtaxpayerdollars.

Thatbeingsaid,evenwiththeadditionoftheSBRtypereactorintothesystem,itisestimatedthattreatmentfacilitieswouldstilluse60%lessoxygenovertheconventionaltreatmentmodelswhichinturncouldyieldsubstantialsavingsinutilitycosts.Inaddition,thereisnoneedforanexternalsourceofcarbon,usuallyintheformofmethanol,sinceanammoxmicroorganismsarenotdependantonorganiccarbonsourcesfortheirmetabolism.Thiswouldsavetreatmentplantsthatarealreadyregulatedonnitratesandthosethatsoonwillbethecostofhavingtoaddcostlychemicalstotheirtreatmentprocessfornitrogenremoval.Ithasbeenspeculatedthatintotal,theanammoxprocesscouldreduceoperationalcostbyasmuchas90%whencomparedwithtraditionalnitrificationanddenitrificationmethodologies.Ifthisstatementistruethenobviouslytherewouldbeasignificantfinancialadvantagetoadoptingtheanammoxprocess.

Anaerobic Ammonia Oxidation 16

Inconventionaltreatmentthepercentremovalofnitratesforaproperlyoperatednitrificationanddenitrificationsystemdoesitselfyieldremovalratesessentiallyequaltothatoftheproperlysetupanammoxprocesswithin±5%,soadramaticincreaseinproductivityisunlikely.Therealsellingpointforanammoxwouldthenhavetobethecostsavings.Thatbeingsaidtheonlyrealdisadvantagetoananammoxsetupwouldbetheslowerstart-uptimewhichwillalwaysbelongerthanthatofcurrenttreatmentstrategies.Todatetheanammoxbacteriahaveyettobegrowninpureculturebutwhencommercialcultivationbecomespossiblethatwillbearealgamechangerinanammoxtechnology.Theabilitytohaveaccesstoreadilyavailablepurecultureofananammoxbiomasscansignificantlyreduceinitialstart-upcycles.Hopefullythisclarifieswhyitshouldbeimportanttocontinuethepursuitofgettingtheseremarkablemicroorganismsinpureculture.Theanammoxbacteriapromiseanexcitingfuturefornutrientremovalbecausetimeandtimeagainithasbeenproventhatnoengineeredmechanicalmethodofremediationalonewilleverbeasefficientasnaturalsystemsutilizedforthebenefitofremediationandgeneralmaintenanceofournaturalenvironment.

Anaerobic Ammonia Oxidation 17

REFERENCES

ASTM International, Inorganic Constituents in Water Subcommittee D19.05. (2013). MethodD6919-03StandardTestMethod forDeterminationofDissolvedAlkali andAlkalineEarthCations andAmmoniuminWaterandWastewaterbyIonChromatography,AnnualBookofASTMStandards,11.01.

Brandes, J.A., Devol, A.H., andDeutsch, C. (2007). Newdevelopments in themarine nitrogencycle.ChemicalReviews,107,577-589.

Paredes,D.,Kuschk,P.,Mbwette,T.S.A.,Stange,F.,Müller,R.A.,Köser,H.(2007).Newaspectsofmicrobialnitrogentransformationsinthecontextofwastewatertreatment–Areview.Eng.LifeSci,7(1),13-25.

Dapena-Mora,A.,VanHulle,S.W.H.,LuisCampos,J.,Mendez,R.,Vanrolleghem,P.A.,Jetten,M. (2004). Enrichment of anammox biomass from municipal activated sludge: Experimental andmodeling results. Journal of Chemical Technology and Biotechnology, 79, 1421-1428. DOI:10.1002/jctb.1148.

EncyclopædiaBritannica,Inc.(2015).HaberBoschProcess.Retrievedfromhttp://www.britannica.com/technology/Haber-Bosch-process.html

ChenH.,Yu, J. J., Jia,X.Y., Jin,R.C. (2014).EnhancementofanammoxperformancebyCu(II),Ni(II),andFe(III)supplementation.Chemosphere,117,610-616.

Schmidt,I.,KatimkavandePas-Schoonen,C.H.,Strouse,M.,opdenCamp,H.J.,Kuenen,J.G.,Jetten,M.S.M.(2002).Anaerobicammoniaoxidationinthepresenceofnitrogenoxides(NOχ)bytwodifferentlithotrophs.AppliedandEnvironmentalMicrobiology,5351-5357.

Kotay,S.M.,Mansell,B.L.,Hogsett,M.,Pei,H.,Goel,R.(2013).Anaerobicammoniaoxidation(ANAMMOX™) for side-stream treatment of anaerobic digester filtrate process performance andmicrobiology.BiotechnologyandBioengineering,110(4).

Strous,M.,Heijnen,J.J.,Kuenen,J.G.,Jetten,M.S.M.,Huang,Y.(1998).Thesequencingbatchreactorasapowerfultoolforthestudyofslowlygrowinganaerobicammoniaoxidizingmicroorganisms.ApplMicrobiolBiotechnol,50,589-596.

Ruscalleda,M.,Seredynska-Sobecka,B.,Ni,B.J.,Arvin,E.,Balaguer,M.D.,Colprim,J.,Smets,B.F. (2014). Spectrometric characterization or the effluent dissolved organicmatter from an anammoxreactorshowscorrelationbetweentheEEMsignatureandanammoxgrowth.Chemosphere,117,271-277.

Ali,M.,Oshiki,M.,Okabe,S.(2014).Simple,rapidandeffectivepreservationandreactivationofanaerobicammoniumoxidizingbacterium“CandidatusBrocadiasinica”.Sciencedirect,57,215-222.

The Water Planet Company. (2015). Nitrification & Denitrification. Retrieved fromhttp://www.yumpu.com/en/document/view/11509528/nitrification-amp-denitrification-the-water-planet-company.html

Du,R.,Peng,Y.,Cao,S.,Wang,S.,Wu,C.(2015).Advancednitrogenremovalfromwastewaterbycombininganammoxwithpartialdenitrification.BioresourceTechnology,179,497-504.

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Suneethi, S., Joseph, K. (2011). Batch culture enrichment of ANAMMOX populations fromanaerobicandaerobicseedcultures.BioresourceTechnology,102,585-591.

Greenberg,A.E.,Clesceri,L.S.,Eaton,A.D.(1992).4500-NH₃-D.Nitrogen(Ammonia)PhenateMethod.StandardMethodsfortheExaminationofWaterandWastewater18thEdition,4:81–4:84.

Greenberg,A.E.,Clesceri,L.S.,Eaton,A.D.(1992).4500-NO₂-B.Nitrogen(Nitrite)ColorimetricMethod.StandardMethodsfortheExaminationofWaterandWastewater18thEdition,4:85–4:87.

Greenberg, A. E., Clesceri, L. S., Eaton, A. D. (1992). 4500-NO₃-E.Nitrogen (Nitrate) CadmiumReductionMethod.StandardMethodsfortheExaminationofWaterandWastewater18thEdition,4:89–4:92.

Clark,J.(2015).TheHaberProcess.Retrievedfromhttp://www.chemguide.co.uk/physical/equilibria/haber.html

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Van Niftrik, L., Jetten, M. S. M. (2012). Anaerobic ammonium-oxidizing bacteria: Uniquemicroorganisms with exceptional properties. Microbiol. Mol. Biol. Rev. 76 (3) 585. DOI:10.1128/MMBR.05025-11.

Anaerobic Ammonia Oxidation 19

Appendix

AnalyticalMethods

Adiscussionregardingtheoreticalmethodsforanalysiswillfollowhereas itwillbeimpossibletomeasurethereactorsefficacywithoutaccurateandappropriateanalytical techniques.Thenutrientanalysis itself can be done using a variety of certified technicalmethods. However, only twowill bediscussed here. They consist of colorimetric or UV/VIS spectroscopy and ion chromatography. Bothhave been proven to be robust and highly accuratemethods andwill be invaluable in the followingstudytoprecisely followthenitrogenouscompoundstransitionsthroughouttheanammoxandpartialdenitrificationprocesses.Ofthetwotechnologies,spectroscopyisthemethodmostcommonlyutilizedfortheanalysisofthenutrientsofinterestinthisstudy.Thisisdueinparttomethodologiesthatofferfewifanyanalytical interferents, lowerinitialcostsandoperationalsimplicity. Onedrawbacktousingcolorimetricspectroscopyastheanalyticalmethodgoto,isthateachanalytemustbemeasuredusingitsownspecificstandardoperatingprocedureandassuchmustberunseparatelytotheotheranalytesinagivenaliquot. Againtherearespectrophotometrictechniquesthatcan,throughtheuseofphotodiode array detectors, like those found in Inductively Coupled Plasma Optical Emission Spectroscopy(ICP-OES), which can analyze multiple analytical components at once but this would be consideredexcessive for the three analyteswe are investigatinghere. The following is a generalizationof howaspectrophotometerworks.A lampprovides thesourceof light.Thisbeamof light strikesadiffractiongrating,whichworks likeaprismandseparatesthe light into itscomponentwavelengths.Thegratingcan typicallybe rotatedso thatonlya specificwavelengthof light reaches theexit slit.Then the lightinteractswiththesample.Fromthispoint,thedetectormeasuresthetransmittanceandabsorbanceofthesample.Transmittancereferstotheamountoflightthatpassescompletelythroughthesampleandstrikesthedetector.Absorbanceisameasurementoflightthatisabsorbedbythesample.Thedetectorsenses the light being transmitted through the sample and converts this information into a digitaldisplay.Forcolorimetricspectroscopywewillneedtodiscussthreeindividualmethods.ThefirsttobedetailedwillbeammoniumandtheanalyticalmethodusedwhenammoniumionsreactatapHof12.6withhypochloriteionsandsalicylateionsinthepresenceofsodiumnitroprusside,thecatalyst,toformindophenolbluecompound,whichcanthenbequantifiedbyaproperlycalibratedspectrophotometerwithawavelengthof630nmandalightpathofroughly1cm.

UsingaHachDR2800SpectrophotometerfirstcarefullyremovethefoilfromtheDosiCapZipandunscrewcaponthesamplevial.Pipette0.2mLofsampleintothevial.ImmediatelyscrewtheDosiCapZipbackon.Shakefirmly2-3times.Wait15minutesforthereactiontocomplete.Invert2-3times.ThoroughlycleantheoutsideofthevialwithaKimWipe,andinsertintothespectrophotometer.Afterthevialisinsertedcollectthedisplayedconcentrationdata.Thenumberisreportedasmg/LNH4-N.Thedatacanthenbeenteredintoabenchsheet,wherestatisticalcalculationscanbemadeinanexcelspreadsheet.Thecalculatedresultscanbeevaluatedfortrendsrelevanttotheanammoxinstrumentationoperation.

Anaerobic Ammonia Oxidation 20

Nexttobedetailedarenitritesandtheanalyticalmethodusedwhennitritesreactwithprimaryaromaticaminesinacidicsolutiontoformdiazoniumsalts.Thesecombinewitharomaticcompoundsthatcontainanaminegrouporahydroxylgrouptoformintensivelycoloredreddishpurpleazodyes,whichcanthenbequantifiedbyaproperlycalibratedspectrophotometerwithawavelengthof543nmandalightpathofroughly1cm.Thecolorsystem,upto180μg/L,obeysBeer’sLaworthelinearrelationshipbetweenconcentrationandabsorbance.

UsingaHachDR2800Spectrophotometerfirstremovefoilcaponsamplevialandunscrewcap.Pipette0.2mLofsampleintothevial.FlipovertheDosicapandscrewbackonthevial.Shakefirmly2-3times.Wait10minutes.Thoroughlycleantheoutsideofthevialandinsertitintothespectrophotometer.Afterthevialisinsertedcollectthedisplayedconcentrationdata.Thenumberisreportedasmg/LNO2-N.Thedatacanthenbeenteredintoabenchsheet,wherestatisticalcalculationscanbemadeinanexcelspreadsheet.Thecalculatedresultscanbeevaluatedfortrendsrelevanttotheanammoxinstrumentationoperation.

Lastlytobedetailedarenitratesandtheanalyticalmethodusedwhennitrateionsinsolutioncontainingsulfuricandphosphoricacidsreactwith2,6-dimethylphenoltoform4-nitro-2,6-dimethylphenol,whichcanthenbequantifiedbyaproperlycalibratedspectrophotometerwithawavelengthof543nmandalightpathofroughly1cm.ToremoveanyNO₂¯asreportedabovetheadditionofareductionstepisrequiredforthismethod.Thisstepisnothingmorethanapreparedcolumnwhichconsistsofroughly18.5cmofcommercialCu-Cdgranulesthroughthesamplemustthenpass.

AgainusingaHachDR2800Spectrophotometerfirstslowlypipettes1.0mLofsampleintothesamplevial.Slowlypipette0.2mLofsolutionAintothevial.Capandinvert2-3timesuntilnomorestreakscanbeseen.Wait15minutes.Thoroughlycleantheoutsideofthevialandinsertitintothespectrophotometer.Afterthevialisinsertedcollectthedisplayedconcentrationdata.Theresultsarereportedasmg/LNO3-N.Thedatacanthenbeenteredintoabenchsheet,wherestatisticalcalculationscanbemadeinanexcelspreadsheet.Thecalculatedresultscanbeevaluatedfortrendsrelevanttotheanammoxinstrumentationoperation.Fromthedetailsaboveitisclearthateachsamplecollectedmustberunatleast3separatetimes

usingthe3differentcolorimetricmethodsandtotalingaminimumof40minutespersample.HoweverthesamplescanalsobeanalyzedfollowingthespecificationsbelowwhicharereferencedfromEPAMethod300.0section2.0Summaryofmethodandequipmentmanufacturer’srecommendations.Typically,IonExchangeChromatographyreliesoncharge-chargeinteractionsbetweenproteinsinasampleandthechargesimmobilizedonthecolumnbedresin.Ionexchangechromatographycanbe

Anaerobic Ammonia Oxidation 21

subdividedintocationexchangechromatography,inwhichpositivelychargedionsbindtoanegativelychargedresin;andanionexchangechromatography,inwhichthebindingionsarenegative,andtheimmobilizedfunctionalgroupispositive.Oncethesolutesarebound,thecolumniswashedtoequilibrateitinanappropriatestartingbuffer,whichshouldbeoflowionicstrength.Thentheboundmoleculesareelutedoffusingagradient,orasecondbuffer,whichsteadilyincreasestheionicstrengthoftheeluentsolution.Alternatively,thepHoftheeluentbuffercanbemodifiedastogivethesamplematrixachargeatwhichtheywillnotinteractandmoleculesofinterestelutesfromtheresin.IfsampleisnegativelychargedattheapplicablepH,useananionexchanger;ifitispositive,useacationexchanger.Inmanycasesitmaybemoreadvantageoustoactuallyselectconditionsatwhichsampleanalyteswillflowthroughwhileanycontaminantswillbind.Thismodeofbindingisoftenreferredtoas"flowthroughmode".Thisisaparticularlygoodmodetouseinthecaseofanionexchange.Hereonecouldusethistypeofmodetobinduphighlynegativelychargedsubstanceswhilemaintainingrelativelysimpleflowingsamplealiquotsthroughtheanalyticalmatrix.Detailedbelowcanbefoundtheactualsamplepreparation,instrumentoperation,anddataprocessingmethodsforbothanionandcationexchangechromatographymethods,beginningwithanions.

SamplePreparation:Priortoanalysisallsamplesarefilteredatorbelowthemanufacturersrecommendedmaximumfilterporediameterof0.45microns.Pretreatmentofsamplesmaybedonepriortofiltration,suchas;homogenizationorcentrifugation,butfiltrationisstillstronglyadvised.Samplesarethenbypipetteintocleanandappropriatelylabeled5mLtubesata1to5dilutionconcentration.Thedilutedsampleshelpsalleviatecolumnoverloadwhichmayinterferewithdataquantificationandfurtherpreventplugging.Samplesarethencappedwithfiltercapsandloadedaccordingtobatchsequencefile(setupinChromeleonsoftware)intheautomatedsampler5mLcassettes.Thesecannowbeloadedontheionchromatographyautosampler.PresstheHold/Runbuttononfrontoftheautosamplertocompletetheloadingprocedureandpreparetheautosamplertoreceiveinstructionsfromthecontrollingsoftware.

InstrumentPreparation:MakesuretheChromeleonserverisworking.Itislocatedinthelowerrightcornerof

theWindowstaskbar.OnlyICsoftwaresystemadministratorscanturnonorofftheserver,sobesurethatitisrunningbeforeproceedingfarther.TheIChardwarewillnotoperateiftheserverisnotconnected.

OpentheChromeleonsoftwareonthecomputersdesktoporinitsprogramfiles.Thesoftwareautomaticallyopenswiththelastworkspacefileused.Moreadvanceduserswillbeabletocreateormodifythelayoutofmostsoftwarefeatures,includingtheworkspacefile,butthisismorecomplexthancanbecoveredhere.Notethattherearetwowindowsopenwiththecurrentworkspacefile.Oneisthebrowserandislabeledassuch.Theotheristhecontrolpanel.Withthecontrolpanelontoplocatethesystemsstartupbuttonandpressit.

TheIChardwareisnowwarmingup.Thestartupbuttonturnsonthehardwarepumps,suppressors,trapcolumns,heaters,andanyotherhardwaretheuserhasprogrammedittoo.TheIChardwareshouldbeallowedtostabilizeforaboutonehouroruntilthebackgroundconductivityisstabilizedatorbelow2.0µS.ThismeasurementcanbefoundontheupperrightcornerofthefrontpaneloftheIChardwareorthelowercornersofthesoftwarecontrolpanel.

Anaerobic Ammonia Oxidation 22

Whilethehardwarestabilizesthebatchsequencefile,programfile,methodfile,etc.,canbesetupormodified.Theeasiestwaytodothiswouldbetoopenupapriorbatchsequencelocatedinthebrowserwindowunderdataandsaveasthenewbatchname.Besuretoopenamorerecentlycompletedrunasitwillhavethemostcurrentversionoftheappropriatefiles.Thiswillcontaintheprogramfiles,whichisthesoftwareinstructionstotheIChardwareandautosampler.Itwillalsocontainthemethodfilewhichrequiresacurrentcalibrationanddefinestheparametersbywhichallanalyticaldataforabatchisquantified.

InadditiontheQCandsamplesequenceisalreadyproperlyarrangedandordered.ThiscanbedonefromscratchthroughthesoftwarebyselectFileintheupperleftcornerofthewindow,followedbynewandtheselectingthedesiredfiletocreate.Thistakesconsiderablylongerandrequiresmorefamiliaritywiththesoftware,butisanotheroption.Also,inthebrowserunderDIONEX_localabackupofallcurrentandnecessaryfileshasbeencreatedundertheirrespectivefiletypenames.

Withthesamplespreparedandloadedontheautosampler,thebatchsequence,program,andmethodfilessetup,andtheIChardwarestabilizedthebatchisreadytobestarted.

Analysis:Tostarttheanalyticalbatch,checkthatthebrowserwindowisthecurrentlyviewed

window(thebrowserwindowisontopofthecontrolpanelwindow)intheopenworkspace.

LocatetheBatchcommandatthetopofthewindownexttotheToolscommandandbyleftclickingthemouseadropdownmenuwillappear.Selectthestartfunction.Thiswillopenthebatchfiledirectorycontainingallsequencefilesthatrequirecompletion.Atthispointtheonlyfilelistedshouldbethecurrentsequencefile.Ifthecurrentsequencefileisnotlistedorifotherfilesarependinginthedirectorytheycanbeaddedordeletedusingtheappropriatebuttonsontherightsideofthedirectory.Note:Previoussequencefilesinthedirectoryshouldbeinspectedtodeterminewhytheydidnotcompletebeforetheyaredeletedoutoftherundirectory.

SelecttheReadycheckfunctionatthebottomofthedirectorytoperformacheckonthestatusofthesequence.Anyunsavedchangestothebatchfilesormissingbatchinformationwillresultinawarningmessagetoappear.Ifawarningmessagedoesappearmaketheappropriatecorrectionsandcontinue.Aftermakingtherequiredcorrectionsoriftherearenorequiredcorrectionthesoftwarewilladviseonhowmuchdiskspaceontheharddriveandhowmucheluent(water)willberequiredtocompletethesequence.Makesurethatthereisadequateofeachtosafelycompletethesequence.ClickOK

Withthebatchfiledirectorystillupselecttherunfunctionatthebottomofthedirectoryandthebatchwillbegin.Note:TheIChardwarewillnotautomaticallyshutdownaftersequencecompletionandsoitwasnecessarytocreateashutdownprogramfile.Besurethisprogramlabeledastheshutdownsampleisontheendofanysequencewhichwillcompleteafterattheendofthebatchsequence.Alsobesurethisfileiscurrentwiththecurrentbatchsequencefilesoinadvertenttemperaturechangesoreluentconcentrations,etc.,doesnotoccur.

Withthebatchrunning,dataquantificationcanbeginassamplescomplete.Todothisamethodfileneedstobegeneratedbasedonavalidcalibration.

CalibrationsforICanalysisaresaidtobestableforupwardsofsixmonthsoruntilQCdataisoutofcontroloriftherehavebeensignificantchangestotheIChardware.Select

Anaerobic Ammonia Oxidation 23

Newfromthefilemenuintheupperleftcornerofthemainwindowandfromthatselectmethod.

Savethemethodfilebyanappropriatenameandbeginupdating.Onthegeneraltab(middleleft)underdimensionofamountchoosetheappropriateunitofmeasurement(mg/L).Thedetectiontabcontainsallthetoolsforhowthesoftwarequantifiespeaks,suchasbutnotlimitedto,peakheadingandtailing,baselineresolution,etc.Thepeaktabletabiswherepeaksfromthecalibrationforallanalytesofinterestarecreatedwiththeirrespectiveretentiontimes,curvefittings,etc.

Theamounttabiswheretheconcentrationfortheanalytesofthestandardsattheappropriatedilutionisstated.ThecorrectquantificationofpeakdataneedstobedonebyorunderthesupervisionofananalystexperiencedwiththeICsoftwareuntiladequateproficiencycanbedemonstratedbyanewICuser.Thequickestwaytoquantifydailybatchrunsistogenerateonemethodfilepercalibrationandsaveittoeverybatchusingthesamecalibration.Thisisdonebypullingupthepriorday’sbatchandlocatingthe.qntfileatthetopandcenterofthebrowsertab.Thenrightclickandholdwiththemouseanddragthendropthefileintothecurrentbatch.Thesoftwarewillaskwhethertocopyormovefile,besuretocopyonly.IfthecalibrationisstillvalidandnosignificantchangeshaveoccurredtosamplematrixesorIChardwarethennooronlyslightmodificationswillberequiredtoprocessthebatchdata.

AlltheQCsamplesneedstobecopiedbyhighlightingthesample,rightclickingthemouse,andselectingthecopycommand.ThefilecanthenbepastedontheICQCcalculatorbatchcoversheetlocatedinDocumentLocatorunderreferencebenchsheets.ThissheetcanbeprintedoutandallQCerrors,ifany,flagged.

Withthebrowsertabopenselecttheappropriatefileandbesuretohighlightit.Thiswillbenoticeableasabluesquaresurroundingthebatchname.Atthetopofthewindowselectthefilecommandandtheselectbatchreport.Thisbringsupthereportdefinitionwindow.Selectandprintthecurrentprintreportdefinitionfollowedbythecurrentexportreportdefinition.Ifthebatchnamewasnotproperlyhighlightedthebatchwillnotbeprintedorexported.Oncethebatchhasbeenprinted,allQCcanbereviewedandapprovedorflaggedasnecessary.OncetheQChasbeenapprovedorflaggedandthebatchhasbeenexportedandsavedlocallythebatchanalyticaldatacanbeevaluatedfortrendsrelevanttotheanammoxinstrumentationoperation.

IfionchromatographyisusedtoevaluatetheanionsNO₂¯andNO₃¯thenitwillbenecessaryto

optimizetheICinstrumentationtoruncationsastheabovemethodwillnotmeasurethese.ThesimplestwaytoaccomplishthiswouldbetohaveasecondICunitoptimizedforcationsandrunsimultaneously,throughtheuseofsamplesplittervalves,withanionanalysis.Otherwiseitwillbenecessarybetweenrunstoswitchanalyticalandtrapcolumns,suppressors,andeluentcartridges.EitherwaythecationswillbeanalyzedbyfollowingthespecificationsbelowwhicharereferencedfromASTMD6919-03summaryofmethodandequipmentmanufacturersrecommendations.

Asamplealiquotispreparedbyfilteringwithasyringefilterandthendilutingpriortoanalysis.Additionalpreparationsuchashomogenizationorcentrifugationmaybeusedpriortofiltration.

Thesamplealiquotisinjectedintoanionchromatographwheretheanalytesofinterestareseparatedandthenmeasuredusingaconductivitydetector.Theresultsarequantitated

Anaerobic Ammonia Oxidation 24

againstastandardcurve.

VerifythattheChromeleonserverisworking.ItislocatedinthelowerrightcorneroftheWindowstaskbar.Note:OnlyICsoftwaresystemadministratorscanturnonorofftheserver,sobesurethatitisrunningbeforeproceedingfurther.TheIChardwarewillnotoperateiftheserverisnotconnected.Tofullyoperatethesoftware,setupormodifyreports,workspaces,etc.,itisadvisabletoobtainappropriatelevelsofpermissionfromLabsupervisorandNDSDITdepartment.OpentheChromeleonsoftwareonthecomputer’sdesktoporinitsprogramfiles.Thesoftwareautomaticallyopenswiththelastworkspacefileused.MoreadvancedChromeleonuserswillbeabletocreateormodifythelayoutofmostsoftwarefeatures,includingtheworkspacefile.Seethesoftwaremanualsforfurtherinstruction.Notethattherearetwowindowsopenwiththecurrentworkspacefile.Oneisthebrowserandislabeledassuch.Theotheristhecontrolpanel.Withthecontrolpanelontoplocatethesystemsstartupbuttonandpressit.Thiswillstartthehardwarewarmupcycle,whichincludesthepumps,suppressors,trapcolumns,heaters,andanyotherhardwaretheuserhasprogrammedittostart.AllowtheIChardwaretostabilizeforaboutonehouroruntilthebackgroundconductivityisstabilizedatorbelow2.0µS.ThismeasurementcanbefoundontheupperrightcornerofthefrontpaneloftheIChardwareorthelowercornersofthesoftwarecontrolpanel.Whilethehardwarestabilizesthebatchsequencefile,programfile,methodfile,etc.,canbesetupormodified.

Theeasiestwaytodothisistoopenupapriorbatchsequencelocatedinthebrowserwindowunderdataandsavethesequenceasthenewbatchname.Besuretoopenamorerecentlycompletedrunasitwillhavethemostcurrentversionoftheappropriatefiles.Thesequencefilecontainstheprogramfiles,whichisthesoftwareinstructionstotheIChardwareandautosamplerandthemethodfile,whichrequiresacurrentcalibrationanddefinestheparametersbywhichallanalyticaldataforabatchisquantified.Inaddition,theQCandsamplesequenceisalreadyproperlyarrangedandordered.Thesequencefilecanalsobepreparedfromscratchthroughthesoftware.Tocreatethefileusingthisoption,select“File”intheupperleftcornerofthewindow,thenselect“New”andthenselectthedesiredfiletocreate.Thistakesconsiderablylongerandrequiresmorefamiliaritywiththesoftware,butisanotheroption.

Anaerobic Ammonia Oxidation 25

Thethirdoptionistouseabackupfile.BackupfilescanbefoundinthebrowserunderDIONEX_local.Abackupofallcurrentandnecessaryfileshasbeencreatedundertheirrespectivefiletypenames.

SamplePreparation

Priortoanalysis,filterallsamplesatorbelowthemanufacturersrecommendedmaximumfilterporediameterof0.45microns.Pretreatmentofsamplesmaybedonepriortofiltration,suchas;homogenizationorcentrifugation,butfiltrationisstillstronglyadvised.Pipettethefilteredsamplesarethenpipettedintocleanandappropriatelylabeled5-mLtubesata1to5dilutionconcentration.Thedilutionofsampleshelpsalleviatecolumnoverloadwhichmayinterferewithdataquantificationandfurtherpreventplugging.Thetypicalpreparationis1.0mLofsampleand4.0mLofreagentwater.Capthesamplevialswithfiltercapsandloadaccordingtobatchsequencefile(setupinChromeleonsoftware,seedescriptioninprevioussection,“InstrumentPreparation”)intheautomatedsampler5-mLcassettes.Thesecannowbeloadedontheionchromatographyautosampler.PresstheHold/Runbuttononfrontoftheautosamplertocompletetheloadingprocedureandpreparetheautosamplertoreceiveinstructionsfromthecontrollingsoftware.Withthesamplespreparedandloadedontheautosampler,thebatchsequence,program,andmethodfilessetup,andtheIChardwarestabilizedthebatchisreadytobestarted.

SampleAnalysis

Tostarttheanalyticalbatch,checkthatthebrowserwindowisthecurrentlyviewedwindow(thebrowserwindowisontopofthecontrolpanelwindow)intheopenworkspace.LocatetheBatchcommandatthetopofthewindownexttotheToolscommand.Leftclickandadropdownmenuwillappear.Selectthestartfunction.Thiswillopenthebatchfiledirectorycontainingallsequencefilesthatrequirecompletion.Atthispointtheonlyfilelistedshouldbethecurrentsequencefile.Ifthecurrentsequencefileisnotlistedorifotherfilesarependinginthedirectorytheycanbeaddedordeletedusingtheappropriatebuttonsontherightsideofthedirectory.Note:Previoussequencefilesfoundinthedirectoryshouldbeinspectedtodeterminewhytheydidnotcompletebeforetheyaredeletedoutoftherun

Anaerobic Ammonia Oxidation 26

directory.Selectthe“Ready”checkfunctionatthebottomofthedirectorytoperformacheckonthestatusofthesequence.

Anyunsavedchangestothebatchfilesormissingbatchinformationwillresultinawarningmessageappearing.Ifawarningmessagedoesappear,maketheappropriatecorrectionsandcontinue.Aftermakingtherequiredcorrectionsoriftherearenorequiredcorrectionthesoftwarewilladviseonhowmuchdiskspaceontheharddriveandhowmucheluent(water)willberequiredtocompletethesequence.Ensurethatthereisanadequateamountofeachtosafelycompletethesequence.ClickOK.

Withthebatchfiledirectorystillup,selectthe“Run”functionatthebottomofthedirectoryandthebatchwillbegin.Note:TheIChardwarewillnotautomaticallyshutdownaftersequencecompletionandsoitwasnecessarytocreateashutdownprogramfile.Besurethisprogramlabeledastheshutdownsampleisontheendofanysequencewhichwillcompleteafterworkhours.AlsobesurethisfileiscurrentwiththecurrentBatchsequencefilesoinadvertenttemperaturechangesoreluentconcentrations,etc.,donotoccur.Withthebatchrunning,dataquantificationcanbeginassamplescomplete.Todothisamethodfileneedstobegeneratedbasedonavalidcalibration.ICcalibrationsshouldtypicallyberunmonthly.SelectNewfromthefilemenuintheupperleftcornerofthemainwindowandfromthatselectmethod.Savethemethodfiletoanappropriatenameandbeginupdating.Typically,thefilenameisbasedonthemonthforwhichthecalibrationisperformed.

Onthegeneraltab(middleleft)underdimensionofamountchosetheappropriateunitofmeasurement(mg/L).Thedetectiontabcontainsallthetoolsusedbythesoftwaretoquantifypeaks,suchasbutnotlimitedto,peakheadingandtailing,baselineresolution,etc.Thepeaktabletabiswherepeaksfromthecalibrationforallanalytesofinterestarecreatedwiththeirrespectiveretentiontimes,curvefittings,etc.Theamounttabiswheretheconcentrationfortheanalytesofthe

Anaerobic Ammonia Oxidation 27

standardsattheappropriatedilutionisstated.ThecorrectquantificationofpeakdataneedstobedonebyorunderthesupervisionofananalystexperiencedwiththeICsoftwareuntiladequateproficiencycanbedemonstratedbyanewICuser.Thequickestwaytoquantifydailybatchrunsistogenerateonemethodfilepercalibrationandsaveittoeverybatchusingthesamecalibration.

Thisisdonebypullingupthepriorday’sbatchandlocatingthe.qntfileatthetopandcenterofthebrowsertab.Then,rightclickandholdwiththemouseanddragthendropthefileintothecurrentbatch.Thesoftwarewillaskwhethertocopyormovefile,besuretocopyonly.IfthecalibrationisstillvalidandnosignificantchangeshaveoccurredtosamplematricesorIChardware,thennooronlyslightmodificationswillberequiredtoprocessthebatchdata.

CopyQCDatatoBenchSheet

AlltheQCsamplesneedtobecopiedbyhighlightingthesample,rightclickingthemouse,andselectingthecopycommand.ThefilecanthenbepastedontheICQCcalculatorbatchcoversheetlocatedinDocumentLocatorunderreferencebenchsheets.ThissheetcanbeprintedoutandallQCerrors,ifany,flagged.Withthebrowsertab,openselecttheappropriatefileandbesuretohighlightit.Thiswillbenoticeableasabluesquaresurroundingthebatchname.

Atthetopofthewindowselectthefilecommandandtheselectbatchreport.Thisbringsupthereportdefinitionwindow.Selectandprintthecurrentprintreportdefinitionfollowedbythecurrentexportreportdefinition.Ifthebatchnamewasnotproperlyhighlightedthebatchwillnotbeprintedorexported.Oncethebatchhasbeenprinted,allQCcanbereviewedandapprovedorflaggedasnecessary.