Evaluationoftheextracellularproteinsinfullscaleact

Evaluationoftheextracellularproteinsinfull-scaleactivatedsludgesChulParka,*,JohnT.Novakb,RichardF.Helmc,Young-OckAhnd,AsimEsendaDepartmentofCivilandEnvironmentalEngineering,UniversityofMassachusetts,Amherst,MA01003,USAbDepartmentofCivilandEnvironmentalEngineering,VirginiaTech,Blacksburg,VA24061,USAcDepartmentofBiochemistry,VirginiaTech,Blacksburg,VA24061,USAdDepartmentofBiologicalSciences,VirginiaTech,Blacksburg,VA24061,USAarticleinfoArticlehistory:Received3February2008Receivedinrevisedform28May2008Accepted30May2008Availableonline21June2008Keywords:ActivatedsludgeflocsCationsEPSHydrophobicityProteomicsSDS-PAGEabstractTheproteinspresentintheextracellularpolymericsubstances(EPS)ofactivatedsludgeflocswereinvestigatedusingthreecation-associatedextractionmethods.Thesubpro-teomesgeneratedfromfourfull-scaleactivatedsludgesweresubsequentlyfractionatedbyammoniumsulfateprecipitationandanalyzedbysodiumdodecylsulfatepolyacryl-amidegelelectrophoresis(SDS-PAGE).TheresultsshowedthateachextractionmethodledtouniqueSDS-PAGEproteinprofiles,whichprovidedstrongevidencethattheextractedproteinsareuniquelyassociatedwithspecificcationsinactivatedsludgeflocs.Thecom-parisonofproteinprofilesacrosssludgesfromdifferenttreatmentplantsrevealedthatex-tractsobtainedusingacation-exchangeresinexhibitedsimilarproteinbandingpatternswhilesulfide-andbase-extractedEPSledtomorevariableproteinprofiles.Analysisofsev-eralSDS-PAGEbandsbyliquidchromatography–tandemmassspectrometryoftrypticdi-gestsledtotheidentificationofseveralbacterialproteinsaswellassewage-derivedpolypeptides(humanelastaseIIIAandkeratins).Theirputativerolesinactivatedsludgesandtheirassociationwithtargetedcationsareproposed.ª2008ElsevierLtd.Allrightsreserved.1.IntroductionTheactivatedsludgeprocessisthemostwidelyusedbiologi-calprocessfortreatingbothdomesticandindustrialwastewa-ter.Degradationoftheorganicmatterpresentintheinfluentsewagebymicroorganismsleadstocellgrowthinaflocculentmode.Thelarge,denseflocsformedduringtheprocesscanbeseparatedfromtheaqueoussuspensionbygravity,withtheeffectivenessofthisseparationdeterminingeffluentquality(Gradyetal.,1999).Althoughtheprocesshasbeenusedfornearlyacentury,anin-depthunderstandingofthebiofloccu-lationprocessiscurrentlylimited.Theinconsistentbehaviorofwastewatertreatmentplants(WWTPs)isgenerallyconsid-eredtobeduetovariationsinbioflocculationprocesses.AsitisnotunusualtofindWWTPsthatdonotmeetregulatoryeffluentrequirements,understandingthebioflocculationpro-cessisessentialfordesigningandoperatingconsistentlyper-formingfacilities.Itisbelievedthattheextracellularpolymericsubstances(EPS)producedduringmicrobialgrowthareresponsibleforlinkingmicrobialcoloniesandotherparticulatematerialstogether,leadingtotheflocculentcharacteristicofactivatedsludges(Wingenderetal.,1999).ActivatedsludgeEPSarethoughttoconsistofvariousbiopolymerssecretedbyacti-vatedsludgemicroorganisms,intracellularmaterialsandcel-lulardebrisderivedfromcelllysis,andorganicmatteradsorbedfrominfluentsewagebeingfedtothesystem(Hig-ginsandNovak,1997;Dignacetal.,1998).Recentstudiesusing*Correspondingauthor.Tel.:þ14135459456;fax:þ14135452202.E-mailaddress:[e-mail](C.Park).Availableat–seefrontmatterª2008ElsevierLtd.Allrightsreserved.doi:10.1016/j.watres.2008.05.014waterresearch42(2008)3879–3889visualandchemicalexamination(Frølundetal.,1996;LiuandFang,2002;Matiasetal.,2003)havedemonstratedthatEPSaremajororganiccomponentsinactivatedsludge,indicatingtheircriticalroleinbioflocculationandflocstability.Charac-terizationoftheEPSinactivatedsludgeisthereforeofcriticalimportance,however,thechemicalcompositionofthissludgecomponentmakesthisaratherformidablechallenge.Thecomplexityofactivatedsludgemicrobiota,thevariablenatureofinfluentwastewater,andthelackofstandardizedEPSex-tractionmethodsalsomakemolecularcharacterizationdiffi-cult.Consequently,thetrueimpactofEPSonsludgeproperties,especiallyregardingsludgesettlinganddewater-ing,remainscontroversial(ParkandNovak,2007).WhileactivatedsludgeEPSiscomprisedofawidevarietyoforganicandinorganicmolecularspecies,proteinshavebeenreportedtobeapredominantorganiccomponentoftheextracellularmatrix(Urbainetal.,1993;Frølundetal.,1996).Proteinscaninfluencebothsurfaceandbulkpropertiesofactivatedsludgeflocs,therebymodulatingthehydropho-bicityofactivatedsludge,andhenceimpactingflocculation,dewaterability,anddigestibility.Abetterunderstandingofthefunctionandfateofextracellularproteinsintheactivatedsludgeisthereforecriticalforimprovingourunderstandingofbioflocculationaswellasunitdesignandoperations.SDS-PAGEisafundamentalbiochemicaltoolforthesize-basedseparationandanalysisofproteinsorproteomesinbiologicalsystems.Duncanetal.(2000)reportedthatSDS-PAGEoffull-scaleactivatedsludgewaschallengingasbandingpatternswerediffuseandsmeared,althoughthepresenceofselectedproteinswaspossiblebyWesternblottingproce-dures.EhlersandCloete(1999)usedSDS-PAGEforthepurposeofcomparingthecompositionofmicrobialcommunitiesinvariousfieldactivatedsludges.Whileprot