Nitrification Inhibition to Nitrifying Bacteria and Activated Sludge by Silver Nanoparticles.docx

Nitrification Inhibition to Nitrifying Bacteria and Activated Sludge by Silver Nanoparticles.docx

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NitrificationInhibitiontoNitrifyingBacteriaandActivatedSludgebySilverNanoparticles

NitrificationInhibitiontoNitrifyingBacteriaandActivatedSludgebySilverNanoparticles

Keywords:

nitrificationinhibition;silvernanoparticles;Nitrosomonas;Nitrobacter;SBR;activatedsludge

ABSTRACT

Thisstudyfocusedonthenitrificationinhibitiononnitrifiersandactivatedsludgetreatmentprocessbysilvernanoparticles（AgNPs）.AgNPswiththediametersof9,13,15and23nmandconcentrationsrangefrom0.12to4.82mg/LwereusedtotesttheinhibitioneffectofNitrosomonasandNitrobacter.ItwasfoundthattheNH4+-NremovalefficiencyofNitrosomonaswasdecreasedbynearly70%bytheadditionofnanoparticleswithsizeof23nmwhenthesilverconcentrationreached4.82mg/L,whileNO2--Nremovalefficiencywasmerelyaffected.Comparisonshowedthatparticleswiththesizeof9nmhadthemostseveretoxicitytoNitrosomonas.Nitrificationinhibitionbysilvernanoparticlesonactivatedsludgeprocesswasstudiedwithbenchscalesequencingbatchreactor（SBR）withfourdifferentsizesandfourdifferentconcentrationsofAgNPs.Theinhibitioneffectwas30%ofreductionofnitrogenremovalwasmonitoredinSBRprocess.

INTRODUCTION

Withtheincreasingutilityofnanotechnologyandnano-materials,theenvironmentaleffectsfromnanoparticleshaveattractedmoreandmoreattention.Withremarkablydifferentphysiochemicalcharacteristicssuchasincreasedoptical,electromagneticandcatalyticpropertiesfromthebulkcounterpart,silvernanoparticle,thenano-sizeparticleofsilverelement,becomesoneofthemostcommonlyusedmaterialinconsumer’sproducts（Wenseleers,etal,2002;Kelly,etal,2003）.Asof2007,theProjectonEmergingNanotechnologiesattheWoodrowWilsonInternationalCenterforScholarshadcompiledalistofmorethan500consumerproductsthatclaimtoincludesomeformofengineerednanoparticle（WoodrowWilsonInternationalCenterforScholars,2007）.Oftheseproducts,about20%containsilvernanoparticles.Socks,paint,bandage,foodcontainer,washingmachine,feedingbottle,cosmetic,andsoapincorporatesilvernanoparticlestoexploititsantimicrobialproperties.

Likeasmanyothernanoparticles,silvernanoparticleswouldalsoenterwastewatertreatmentplants（WWTPs）（MuellerandNowack,2008）.Itwasreportedthatsockscontainingsilvernanopartilceswouldlikelytoreleasesilverafterseveraltimeswashing（BennandWesterhoff,2008）,andasmuchas1.3mgAg/L（650μgsilverin500mLdistilledwater）wouldbereleasedinidealcondition.SilvernanoparticlesdisposedinthesewerpipemayaggregatedependingonpH,redoxpotential,andionicstrengthintapwater（YakutikandShevchenko,2004;Zhang,etal,2008）.Itwasreportedthattheseparticleswouldlikelybeoxidizedtosilverionswhichwerecapabilitytocomplexwithanionsinwatereasily（Lok,etal,2007）.TherewereconsiderableamountofsilvernanoparticlesenteringWWTPslastfewyearsandtheamountofsilverisincreasinggradually,however,notmuchabouttheiradverseeffecttowastewatertreatmentwasknown.

Asitwasknown,nitrificationwasakeyprocessinbiologicalnitrogenremoval.BecauseofitssensitivitytoexternalconditionssuchaspH,dissolvedoxygenconcentration,andtoxicchemicals（BlumandSpeece,1991;Hu,etal,2003）,nitrificationwasknownasthecontrollingstepinwastewatertreatmentprocess.Itwasreportedthatnitrificationprocess,especiallytheammoniaoxidationstep,wassensitivetosilvernanoparticles（ChoiandHu1,2008;Choi,etal,2008;ChoiandHu2,2008;ChoiandHu,2009）.Insynthesiswastewater,nitrificationwouldbeinhibitedbynearly90%whensilverconcentrationachieved1mg/Linbatchextantrespirometricassay.Therefore,nitrificationprocessandnitrifyingbacteriawerechosentoevaluatethetoxicityofsilvernanoparticlesinthisresearch.Thisstudyfocusedontheinhibitioneffectbysilvernanoparticlestonitrifyingbacteria,bothtotheNitrosomonasandNitrobacter,andactivatedsludgeinmunicipalwastewatertreatment.

MATERIALSANDMETHODS

NitrifyingBacteria

Nitrifyingbacteriausedinthisresearchwereisolatedfromtheactivatedsludgefromlocalwastewatertreatmentplant.ActivatedsludgefromWWTPwasputintoculturesofNitrosomonas（ammonia-nitrogenoxidationbacteria,shortforAOB）andNitrobacter（nitrite-nitrogenoxidationbacteria,shortforNOB）,respectively.ThecompositionsofbothculturesareshownasTable2.1.

Table2.1CompositionofTwoCultures

AOBCulture

NOBCulture

Compound

Weight（g）

Compound

Weight（g）

（NH4）2SO4

2.0

NaNO2

1.0

NaH2PO4

0.25

Na2CO3

1.0

MnSO4•4H2O

0.01

NaH2PO4

0.25

K2HPO4

0.75

CaCO3

1.0

MgSO4•7H2O

0.03

K2HPO4

0.75

CaCO3

5.0

MnSO4

0.01

MgSO4•4H2O

0.03

Dilutedto1000mLwithdistillingwater,adjustedpHto7.2with1.0MHClorNaOH,thensterilizedunder121°Cfor30min.

Cultureshadbeensterilizedunder121°C,0.1MPabeforeuse.Allcultureswerethenplacedinshakingtable（180rpm）under35°C.ThebacteriasolutionweretestedbyGriessagentanddiphenylamineeveryday,respectively.AftertheAOBculturesappearedredaftertheadditionofGriessagent,andtheNOBculturesblueaftertheadditionofconcentratedsulfuricacidanddiphenylamine,NitrosomonasandNitrobacterwereenriched.Afterenrichment,allbacteriasolutionswerethenkeptunder4°C.

ActivatedSludgeandBenchReactor

ActivatedsludgewasfromthelocalWWTPandthencultivatedbymunicipalwastewaterfromlocalwastewatersysteminareactorwiththetotalvolumeof20L.CharacteristicsofwastewaterwereshownasTable2.2.

Table2.2CharacteristicsofLocalMunicipalWastewater

Influent

mg/L

COD

TotalNitrogen

AmmoniaNitrogen

TotalPhosphorus

180~225

32~40

21~28

3~5

Thebenchscalereactorwasdividedinto5zonesandeachzonehadthevolumeof4L,respectively.Aliquotsofdriedsludgewereaddedinto5zonesandthencultivatedby4Lmunicipalwastewater（Figure2.1）.Thereactorwasoperatedasthestyleofsequencebatchreactor（SBR）,withsolidretentiontime（SRT）of10dandhydraulicretentiontime（HRT）of8h,contained0.5hourfillingtime,6hoursofreactiontime,1hourofsettlingtime,0.5hourdrawandidletime.Thequalityofinfluentandeffluentwastestedevery2days,includingtotalnitrogen,ammonianitrogen,CODandMLSS.MLSSofeachzonewascontrolledtobearound2,500mg/Lbydischargingexceedingsludge.ActivatedsludgewascultivatedtoachievesteadystatewithstableammonianitrogenremovalrateandsteadyMLSSvalue.

SRT=10d,HRT=8h

Influent（20Ltotallyand4Leach）

Figure2.1SchematicoftheBenchReactor

SilverNanoparticles

Agnanoparticleswerepreparedbythesodiumborohydride（NaBH4）reductionprocess（ChoiandHu1,2008）,inwhichsilvernitratewasreducedwithsodiumborohydrideandaddingpolyvinylalcohol（PVA）asthecappingagenttocontrolthegrowthofnanocrystalsandagglomerationofnanoparticles.TodissolvePVA,asolutioncontaining0.06%（wt）PVAwasheatedto100°C,then1mLsilvernitratewiththeconcentrationof14mMwasaddedinto20mLPVAsolutionandthenthemixturewasboiled.Silverparticleswerepreparedbyrapidlyinjecting0.1mLof10mMNaBH4intotheboiledPVAsolution.WiththeadditionofNaBH4,thesolutionbecameyellowimmediately,indicatingtheproductionofsilvernanoparticles.After5minofstirring,thereactionmixturewasstoredat4°Cbeforeuse.

Itwasreportedthataddingdifferentconcentrationofsodiumborohydride,madethemolarratiosofBH4-/Ag+become0.1,0.2,0.38,0.6,1.2inthemixture,couldsynthesizesilvernanoparticleswiththeaveragesizesfrom9to21nm（ChoiandHu2,2008）.Inthisstudy,4BH4-/Ag+ratioswereadapted,including0.1,0.3,0.7,1.0,tosynthesizesuspensionwithdifferentconcentrationsanddifferentsizesparticles.

InhibitionExperiments

InhibitionExperimenttoNitrifier

ItwasreportedthattheinhibitioneffectofNitrosomonasandNitrobacterbytoxicsubstancescanbeobservedinpureculturesandtheexperimentcanbecarriedinshakingtableusingtubes（Svenson,etal,1999;GrunditzmandDalhammar,2001）.Inthisstudy,theenrichedNitrosomonasandNitrobacterwereexposedtosilvernanoparticlesinwastewatertreatmentandtheexperimentswerecarriedoutinmixedculturescontainingmunicipalwastewaterandnitrifiersolution.Alltubes（aftersterilizing）weredividedintotwogroups,oneforNitrosomonasandtheotherforNitrobacter.Then,aliquotsof10mLmunicipalwastewaterwereaddedintoeachgroupoftubes,respectively.Inthisresearch,agradientof4concentrationswaschosen,andsowasagradientof4differentsizes.Everyconcentrationorsizehad3parallelinthisexperiment.

Aliquotsof1mLNitrosomonasandNitrobactersolutionswereinoculatedintotwogroupsoftubes,respectively.Ineachgroup,threetubesofmixedliquidweresetasidetotesttheinitialNH4+-Nvalueofthemixturewithbacteriasolutionandmunicipalwastewater.Afterdosing,bothgroupsoftubeswerepackedandplacedintoshakingtable,themixtureswouldhavereactionunder35

1°Catanoscillationspeedof200rpminshakingtable.

NitrosomonasandNitrobacterwouldhaveareactiontimeof48hoursand120hours,respectively.AsthegroupoftubesforNitrosomonaswasstoppedreactionafter48hoursinmixture,whiletheoneforNitrobacterwasstoppedafter120hours,blanksamplesofbothgroupsshouldbetestedtogettheinitialNH4+-NvaluesandNO2--NvaluesforNitrosomonasandNitrobacter.Aftersampling,bothofthesetwogroupsoftubeswerethendividedinto8groups（A11~A14,A21~A24,A31~A34,A41~