science

PergamonPII:S0043-135~97)00144-9Wat.Res.Vol.31,No.I1,pp.2771-2774,1997©1997ElsevierScienceLtd.AllrightsreservedPrintedinGreatBritain0043-1354/97$17.00+0.00EFFECTOFFREEPHOSPHINEONANAEROBICDIGESTIONF.EISMANN~*,D.GLINDEMANN~,A.BERGMANN~andP.KUSCHK2tUniversityofLeipzig,InstituteforAnimalHygieneandVeterinaryPublicHealth,Semmelweisstrasse4,D-04103Leipzig,Germanyand2CentreforEnvironmentalResearchLeipzig-HalleLtd,Permoserstrasse15,D-04318Leipzig,Germany(ReceivedNovember1996;acceptedinrevisedformApril1997)Abstract---Gaseousphosphinewasfoundtoinhibitbiogasformationduringtheanaerobicfermentationofswinemanure.Alogarithmicdose-responsecorrelationwasobtainedwith50%inhibitionataphosphineconcentrationofapproximately150ppm.Anaerobicfermentationofacetateandyeastextractmeasuredbybiogasformationwerenotaffectedatconcentrationsupto1000ppm.Despitetherelativetoleranceofanaerobicbacteria,phosphinemayobviouslyinhibitthedegradationofmanureconstituents.©1997ElsevierScienceLtdKeywords--phosphine,toxicity,manure,anaerobicdigestionINTRODUCTIONPhosphine(phosphate,PH3)iscommerciallyusedingrainfumigationandmicroelectronics.Matrix-boundphosphinewasreportedtobepresentinriverandseasediments(Gassmann,1994),aswellasinthefaecesofhumans,ruminantsandswine(GassmannandGlindemann,1993).Consequently,matrix-boundphosphineendsupinbiotechnologicalsystemsfortheremovaloffaecalresidues.Freephosphinegashasbeenfoundinsewagetreatmentplants,manurestoragetanksandfermenters,aswellasintheanaerobicdigestionofexcesssludgefrommunicipalwastewatertreatment(Devaietal.,1988;GlindemannandBergmann,1995;Glindemannetal.,1996).Theoriginofphosphineinanaerobictechnologieshasyettobeestablished.Thehypothesisthatmicrobialphosphatereductionisthepredomi-nantmechanismresponsibleforphosphineformationissubjecttocontroversialdiscussion(Tsubota,1959;Iverson,1968;Williams,1978)andnomicroorganismcapableofbiochemicallyproducingphosphinehasbeendescribedtodate.Thus,othersourcesmightalsobepossible.Nevertheless,whateverthesourcesofphosphineare,itstoxicityremainsundisputed.Aconcentrationof538ppmwasfoundbyDevaietaL(1988)inbiogasfromananaerobicdigestiontankcouldcauseacutedeathifinhaled.Acutetoxicityandmutageniceffectshavebeenobservedinplants,insects,rodentsandhumans(Bakheitet'al.,1985;Nakakita,1987;Dumaetal.,1977;Garryetal.,1989).Suppressionofbothfungalgrowthandthe*Authortowhomallcorrespondenceshouldbeaddressed[Fax;[phone]].productionofmycotoxinsprovidesevidenceofthetoxicactionofphosphineonmicroorganisms(Leitaoetal.,1987).Thepurposeofthisworkwastoexaminetheimplicationsofhighphosphineconcentrationsinanaerobicsewagetechnologiesforthemetabolicactivityofthemicrobialcommunity.Threeanaerobicculturesweretestedfortheirshort-termresponsetogaseousphosphineasmeasuredbybiogasformationkinetics.Anenrichmentonacetateaddressedtheactivityofacetoclasticmethanogenicbacteria,anenrichmentonyeastextractcomprisedacidogenicbacteriaandasyntrophicconsortiumofhydrogen-producingacetogenicbacteriaandhydrogen-consumingmethanogens,andthecomplexnatureofananaerobiccommunityinmanurefermentationwasmodelledbymanurefromswine.MATERIALSANDMETHODSEnrichmentculturesAnaerobicsludgesamplesfromriversediment(WeisseElster,Leipzig)wereusedtoenrichbacterialculturesonacetateandyeastextractassubstrates.Thesamplesweredilutedataratioofl:10withmineralsaltmediumcontaining1.0gNH4C1,0.9gNaCl,0.1gCaC12o2H20,0.2gMgCl2.6H20,1.0gK2HPO4,0.5gNaHCO3,0.128gnitrilotriaceticacid,13.5mgFeCl~.6H20,lmgMnCI2.4H20,0.24mgCoCl2.6H20,lmgZnCl2,0.25mgCuCl:.2H20,0.1mgH3BO3,0.24mgNa2MoO4.2H20,1.2mgNiCl2.6H20and0.26mgNa2SeO3.5H20perlitreofwater.Sodiumacetate(1.6g1-~)andyeastextract(2gl-~)wereaddedascarbonsources,respectively.Thecultureswereincubatedin!.21bottlesundernitrogenat37°CandpH7.0withthefrequentsubstitutionofculturesamplesbyfreshnutrientsolutionsresultinginanaverageliquidretentiontimeof25days.27712772F.Eismannetal.Formanurefermentation,a11sampleoffreshswinemanurewasusedforthetoxicitytest.Toguaranteepropermicrobialactivity,thesamplewasincubatedat37'~Cfor1weekpriortothetest.Usually,thebiogasproductionfrommanurelastslongerthan1month;thus,theorganicsubstratesdonotbecomelimitingwithin1weekoffermentationandthesubsequenttestperiod.PhosphinetoxicitytestPortionsoftheenrichmentculturesonacetateandyeastextractdilutedwithabuffersolution(1g1-~KEHPO4,pH7.0)weretransferredinto150mlserumbottlesresultinginafinaldryweightof0.4g1-~.Sodiumacetate(0.8g1-~)andyeastextract(1gl-~)wereadded,respectively.Formanurefermentation20mlportionsofthepre-incubatedmanuresamplewereaddedto30mlofthebuffersolution.ThebuffercapacityofthemediumwasappropriatetoguaranteeanearlystableneutralpHvalueintheseveralculturesduringthetoxicitytest.Thebottles,withafinalliquidvolumeof50ml,wereflushedwithnitrogentodisplaceairandsealedwithphosphine-inertstoppers.Avolumeof20mlheadspacegaswasremovedbyasyringeandreplacedby20mlofpreparedphosphinestandards(phosphineinnitrogen),resultinginfinalconcentrationsof0,10,50,100,500and1000ppm.Thecultureswereincubatedat37°Cinthreereplicatesforeachconcentration.Biogasproductionwasm