Soilbacterialcommunitiesinconstructedwetlandstreat

SoilbacterialcommunitiesinconstructedwetlandstreatedwithswinewastewaterusingPCR-DGGEtechniqueXiuliDong,GudigopuramB.Reddy*DepartmentofNaturalResourcesandEnvironmentalDesign,NorthCarolinaAgriculturalandTechnicalStateUniversity,Greensboro,NC27411,USAarticleinfoArticlehistory:Received9March2009Receivedinrevisedform14August2009Accepted18September2009Availableonline12October2009Keywords:WetlandNutrientremovalDenaturinggradientgelelectrophoresisCulture-dependenttechniqueCulture-independenttechniqueabstractMarsh-pond-marsh(MPM)constructedwetlandsweredesignedforthetreatmentofswinewastewater.Thegoalofthisstudywastocharacterizebacterialcommunitiesinthesewetlandsanddeterminethenutrientremovalfrominfluenttoeffluent.Surfacesoilsampleswerecollectedandanalyzedbycul-ture-dependentandculture-independenttechniques.Theresultsshowedthatthebacterialcolonyform-ingunits(CFU)andtheaverageconcentrationsoftotalnitrogen,NHþ4,totalphosphorous(TP)andPO34fromtheinfluenttotheeffluentdecreased.TheNHþ4andthePO34concentrationsshowedthemostdra-maticchanges,withdecreasesof39.97%and16.92%,respectively.Dataofculture-independentsamplesproducedbyusingPCR-denaturinggradientgelelectrophoresis(DGGE)techniqueshowedthattheShan-nondiversityindexandrichnessdecreasedsignificantly(P0.05)frominfluenttoeffluent.BacteriumspeciesdistributionsstronglycorrelatedwiththeconcentrationsofTP,NHþ4andthePO34.Sequencingofpartial16SrRNAgenesfragmentsrevealedthatthetotalbacterialcommunitycompositionwasdom-inatedbyPseudomonassp.,Arthrobactersp.,Bacillussp.andothersoilbacteria.Anammox(anaerobicammoniumoxidation)stainsweredetected.Phylogeneticanalysisdemonstratedthatsomeofthepartial16SrRNAgenesequenceshadcloserelationshipswithunculturabledenitrificationbacteria.Theactivitiesofthesebacteriamightcontributetothenutrientremovalinthewetlands.2009ElsevierLtd.Allrightsreserved.1.IntroductionSwinewastewatercontainshighconcentrationofpollutants,includingsuspendedsolids,organicsandnutrientsthatcangreatlydeterioratethequalityofaquaticenvironmentsintowhichtheyaredischarged(Leeetal.,2004).Traditionally,swinehousesflushthewasteintoananaerobiclagoonwhichisthensprayedonagricul-turalfields.However,continuoussprayingoflagoonwastewateronfieldscanleadtohighNandPaccumulationsinsoilormaycon-taminatesurfaceorgroundwater.Tolessensuchenvironmentalimpact,atreatmentmustbeperformedpriortothesprayingappli-cation.Onepossiblesolutionistouseconstructedwetlandsinasso-ciationwithstabilizationponds.Thepreviousstudiesconductedonmarsh-pond-marsh(MPM)constructedwetlandsshowedpromis-ingresultsinremovingnutrients(Reddyetal.,2001;Poachetal.,2004).Also,thesewetlandsshowedhighefficiencyindenitrifica-tionbutlimitationinnitrification(Huntetal.,2006).However,theextentofwastewatertreatmentinnaturalorconstructedwet-landsdependsuponthewetlanddesign,microbialcommunityandtypesofplantsinvolved(Ibekweetal.,2003).Microbialcommunitiesinfunctionalwetlandsplayanimpor-tantroleinpollutantremoval.Nitrogenremovalinconstructedwetlandstreatedwithswinewastewaterdependslargelyonmicrobialactivitiesassociatedwithnitrification,followedbyan-oxicdenitrification.Onamassbasis34–55%ofNwasremovedinMPMwetlandswhenswinewastewaterwasappliedbetween2and50kgNha1day1(Poachetal.,2004).AthigherNloadingrates,gaseouslosseswerealsothemajorNremovalpathwayinMPMwetlands(Poachetal.,2003,2004).Denitrifyingbacteriaplayakeyroleinnitrogenremovalandthusconstituteanimportantgroupinwetlandmicrobialcommunity.Duringthedenitrificationprocess,theinorganicnitrate-N(NO3)andnitrite-N(NO2)arere-ducedinastepwisemannertonitricoxide(NO),nitrousoxide(N2O)andnitrogengas(N2)(Enwalletal.,2005;Zumft,1992).Anammox(anaerobicammoniumoxidation)bacteriaconstituteanotherbacterialgroup,thePlanctomycetes,thatcontributestotheremovalofnitrogenbyoxidizingammoniatonitrogengasusingnitriteasanelectronaccepterunderanoxicconditions(Yamamotoetal.,2008).Determiningthestructureofwetlandmicrobialcommunitiesisimportantforunderstandingthebiologicalprocessesthatoccurinsoil–water–plantwetlandsystem.Studiesinthelasttwodecadeshaverevealedthatmorethan99%ofthebacteriapresentinmanyenvironmentalsamplescannotbecultivatedinthelaboratory(Sharmaetal.,2005).Someofthepossiblereasonsareasfollows:arequirednutrientisnotpresentintheculturemedium;thecul-turemediumitselfistoxic;orotherbacteriainthesampleproduce0960-8524/$-seefrontmatter2009ElsevierLtd.Allrightsreserved.doi:10.1016/j.biortech.2009.09.071*Correspondingauthor.Tel.:[phone];fax:[phone].E-mailaddress:[e-mail](G.B.Reddy).BioresourceTechnology101(2010)1175–1182ContentslistsavailableatScienceDirectBioresourceTechnologyjournalhomepage:(Wade,2002).Regard-lessofthereason,bacterialpopulationsbasedoncurrentcultiva-tionmethodslikelymisrepresentthetruediversityofmicrobialpopulationsintheenvironment(LeungandTopp,2001).Theadventofmolecularbiologyhasmadestudyingbacterialcommunitiesintheirentiretywithouttheneedtoculturethecom-munities.Denaturinggradientgelelectrophoresis(DGGE)ofPCR-amplified16SrRNAge