微生物培养及在岩石或混凝土上沉积.docx

微生物培养及在岩石或混凝土上沉积.docx

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微生物培养及在岩石或混凝土上沉积

TheoryandApplicationofcement-basedmaterialsremediationbyCarbonatePrecipitation（MCP）inducedbymicroorganism

QianChunxiang

（1）

（2）,ChengLiang

（1）,WangRuixing

（1）andWangJianyun

（1）

（1）SchoolofMaterialsScience&Engineering,SoutheastUniversity

（2）JiangsuKeyLabinCivilEngineeringMaterials,Nanjing,211189China

AbstractBacterialinducedcarbonatemineralizationhasbeendemonstratedasanewpotentialmethodforrestorationoflimestonesinhistoricbuildingsandmonuments.Weclaimheretheformationofcalciumcarbonatewascontrolledbyextracellularpolymericsubstances（EPS）isolatedfromBacilluspasteurii.TheprocessofcrystallizationnucleationwasacceleratedinthepresenceofcellsandinhibitedinthepresenceofEPS.TheCaCO3filmdepositedoncementpastesurfacewasabout100μmafter7daystreatment.Theresultsofvariousrestoringmethodsshowedthathigherdecreaseofwaterabsorptionofcementpastewasgainedinbrushingapplicationinthepresenceofagar,whichcouldmaintainureasewithhighactivityinlongtermcomparedtosprayingmethod.Thecoefficientofcapillarysuctionofcementpastetreatedwithbrushingmethodwasreducedby90%.Mixedmediaconsistedofsands,urea,Ca2+andconcentratedbiomass,wasinjectedintoartificialcracksofcementpastefollowedbycontinualnutrientsupplement,andCaCO3particleswereprecipitatedgraduallybetweensandsparticleswhichwerecombinedwithcementmatrix.Theresultsshowedthatthecompressivestrengthofrecoveredspecimenswasrestoredto84%,whichdemonstratedthatthiskindofbio-restorationmethodiseffectiveinrepairingsurfacedefectsofcement-basedmaterials.

KeywordsBacteria;deposited-film;cementpaste;calciumcarbonate;coefficientofcapillarysuction;CaCO3

1.INTRODUCTION

Bacteriallyinducedmineralizationhasrecentlyemergedasamethodforprotectingandconsolidatingdecayedconstructmaterials.Calcite（CaCO3）isoneofthemostcommonandwidespreadmineralsonEarth,constituting4%byweightoftheEarth’scrust.CaCO3precipitationisacommonphenomenon,formsnaturalrockandexistsinenvironmentssuchasmarinewater,freshwater,andsoils[1].Microbialcarbonateprecipitation（MCP）hasgainedinterestinthelast20years.MCPcanbeinducedbyorganismsinvolvedinthenitrogencycle,viathehydrolysisofureabyenzymeurease,whichresultsintheproductionofcarbonateionsinthepresenceofammonium.Calciumcarbonateisreadilyprecipitatedundertheseconditions,inthepresenceofcalcium.

Thebasicreactionofthecalcocarbonicsystemcanbeaddressedasfollows.Duringmicrobialureaseactivity,1moleofureaishydrolyzedintracellularlyto1moleofammoniaand1moleofcarbonate（Eq.1）,whichspontaneouslyhydrolyzestoformadditional1moleofammoniaandcarbonicacid（Eq.2）[2].

（1）

（2）

Theseproductssubsequentlyequilibrateinwatertoformbicarbonateand1moleofammoniumandhydroxideionswhichgiverisetoapHincrease.

（3）

（4）

（KSO=3.8×10-9）（5）

Where,KSOisthesolubilityproduct.ThedrivingforceforprecipitationofCaCO3isthesupersaturationlevelS,definedbytheratiooftheionicproductasshowninEq.6.

（6）

Ureahydrolysisisthemosteasilycontrolledofthecarbonategeneratingreactions,withthepotentialtoproducehighconcentrationsofcarbonatewithinashorttime.AnumberofapplicationsinvolvingMCPhavebeenpreviouslyattempted.Todate,mostofthepublishedworkhasfocusedonMCPforpurposesofstrengthdevelopmentandmaterialsremediation（Table1）.

Table1reactionconditionsreportedintheliteraturefortheproductionofCaCO3viamicrobialproductionofcarbonatefromureahydrolysis

Aim

Urea

（mM）

Ca2+

（mM）

Ureaseactivity

（mMurea.min-1）

Reference

Stoneremediation

333

12-50

0,19

[3]

Stoneremediation

66

25

0.041

[4]

Portlandcementremediation

333

50

n/s

[5]

Pluggingofrockpros

333

0.025

n/s

[6]

Physical,biologicalandchemicalfactorsmaycausetheweatheringofmonumentalstones.Consequently,alossofcohesionofstonematerialandprogressivemineralmatrixdissolutionwillbeenhanced.Inthecaseofcalcareousstones,theporositywillincreaseduetoCaCO3leachingandweakeningofthesuperficialstructureofthestone[7].Theattemptwhichwasdoneby[7]instoppingorslowingdownthedeteriorationofmonumentalstatuarybyureolyticbacteriawasunsuccessful.Recently,microbialsealantatwhichanewadditionallayeronthesurfaceofanoldconcretelayerwasachieved.ItwasconcludedthatcracksremediationbySporosarcinapasteuriimayenhancethestrengthandthedurabilityofthestructure.[8]foundthatBacilluscereuswasabletointroduceextracellularprecipitationofcalciumcarbonateondecayedlimestone.[9]testedtheabilityofMyxococcusxanthustoinducecalciumcarbonateprecipitationonsterilizedporouslimestone,findingthat（i）acoherentcarbonatecementof10–50lmcoatedthetreatedstones;（ii）thenewcementwascompatiblewiththesubstrate;and（iii）thiscementwasrooteddowntoadepthof1mmwhileatthesametimestoneporosityremainedcompletelyunaltered.Thenewlyformedbacterialcementwasmoreresistanttomechanicalstress,i.e.moreconsolidated,thantheoriginalcarbonate.

Inbiologicallycontrolledmineralization（BCM）,theorganismexertsagreatdegreeofcrystallochemicalcontroloverthenucleationandgrowthofthemineralparticles.Forthemostpart,themineralsaredirectlysynthesizedataspecificlocationwithinoronthecellandonlyundercertainconditions.Inmostcases,BCMhappensintracellularly,whereasthelipids,proteins,polysaccharides,etc.makeastablematrixforthecationstocondense,andforthemineraltogrowinaconstrainedspace.Bacterialcellshavethemselvesbeenshowntobeexcellentnucleationsitesforgrowingmineralsduringtheformationofrock[10],withmanystudiesconfirmingtheprecipitationofcalciteonthebacterialcellsurface.

ThisstudyinvestigatesthepotentialrelationshipsbetweencellsandextracelluarsecretionandcalciumcarbonatecrystalsprecipitatedbyBacilluspasteuriibacterialculturesandabioticallymediatedcalciumcarbonatesynthesis.ThisisdoneusingextracellularsecretionisolatedfromBacilluspasteuriiinabatchsystem.Meanwhile,theeffectofdifferenttreatmentssuchasimmersing,sprayingandbrushingoncementpastesamplessurfaceremediationandstrengthrecoverywasstudied.

2.MATERIALSANDMETHODS

2.1Microorganismandgrowthconditions

B.pasteuriiwasusedthroughoutthestudy.Liquidculturemediaconsistedof3g/Lnutrientbroth,5g/Lpeptoneand2.4g/Lurea.ThepHofthemediawasadjustedto7.0with10NNaOHpriortoautoclaving.Liquidmediaweresterilizedbyautoclavingfor25min.at121°.Brothcultureswerecultivatedat30℃onashakerat170rpmfor24h.

2.2DifferentcircumstancesofCaCO3precipitation

ThreedifferentmediumsolutionswereusedtoallowCaCO3crystalformation.

（1）DifferentofconcentrationofovernightB.pasteuriicultures,whichODvalueswere0.4,0.8,1.6and3.2respectivelyat550nm,werepreparedforCaCO3precipitation.

（2）Cellssolution:

CellsharvestedfromovernightculturewithOD1.6at550nmbycentrifuging（5000r/min,5min）werere-suspendedindeionizedwaterwithfinalconcentrationof3×108cellsmL-1.（3）Extracellularpolymericsubstances（EPS）solution:

Extractedfromovernightculture（OD5501.6）.byremovingcells.

EquimolarCaCl2andNa2CO3wereintroducedintoeachsamplementionedabovewithfinalconcentrationof6.67mM.After4hourscrystalswerecollectedbycentrifuging（5000r/min,5min）forSEMandIRexamination.Allexperimentswerecarriedoutatroomtemperature（20℃）.

2.3CEMENTPASTEREMEDIATIONEXPERIMENTS

2.3.1Cementpasteproportionsandspecimensdimensions

CementpastesamplesweremadewithnormalPortlandcement（P.O32.5）and0.4water-cementratios,whichwascastinto30×30×30mmprismsandcuredatroomtemperaturefor7days.

2.3.2Surfacecoatingbyimmersing

Preparedcementpastesamplesweresubmergedbybacterialcultureconsistedof0.2MequimolarCa（NO3）2andureaand5×108cell/mLbacterialcells.Liquidlevelwas10cmhigherthantopsurfaceofcementpaste.Depositedfilmwasobservedafter7dayscuring..

2.3.2SurfacecoatingbySpraying

Cellsharvestedfrom100mLculture（5×108cell/mL）re-suspendedin2.5mlfreshmediaandwassprayedontotopsurfaceofcementpastefollowedby4mLcementationliquidconsistedof0.5MequimolarureaandCaCl2.Duringnext3days,4mLcementationliquidwasintroducedtotreatedplaneat6hoursintervals（Fig.1）.

Figure1:

Theprocedureofbio-precipitatingCaCO3filmwithsprayingmethod

2.3.3Brushingsurfacecoatingbyagar

Bacteriawereintroducedintofreshmedia,whichconsistedof3g/Lnutrientbroth,5g/Lpeptone,2g/Lagarand5mol/LequimolarureaandCa（NO3）2,withfinalconcentrationof2×1010cells/mL.Mixedmediawasbrushedonthesurfaceofspecimenfollowedbyconcentratedbacteriasolution（2×1010cells/mL）after6hours.Thespecimenwascuredatroomtemperature,andthefilmcoveringwasobservedafter7days（Fig.2）.

Figure2:

Theprocedureofbio-precipitatingCaCO3filmwithbrushingmethodimmobilizedbyagar

2.3.4Artificialcracksofcementremediation

Cellspremixedwithsand,urea,agarandCa（NO3）2wasinjectedintoartificialcracks（50（L）×3（W）×20（D）（mm））ofcementplaster.Theproportionofingredientofinjectionwasshownintable2.Every2days2mlnutrientsolution（Table3）wasinjectedintocracks.Allexperimentswereconductedatroomtemperature.

Table2:

Bio-restorationmaterialsforcracksinspecimens

Group

Sand（g