Geomicrobiology Journal

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Geomicrobiology Journal Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713722957 Iron Sulfide and Pyrite as Potential Electron Donors for Microbial Nitrate Reduction in Freshwater Wetlands

Online Publication Date: 01 July 2007 To cite this Article: Haaijer, Suzanne C. M., Lamers, Leon P. M., Smolders, Alfons J. P., Jetten, Mike S. M. and Camp, Huub J. M. Op den (2007) 'Iron Sulfide and Pyrite as Potential Electron Donors for Microbial Nitrate Reduction in Freshwater Wetlands', Geomicrobiology Journal, 24:5, 391 - 401 To link to this article: DOI: 10.1080/01490450701436489 URL: http://dx.doi.org/10.1080/01490450701436489

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Op den] At: 08:00 14 August 2007 irt-eedn rnsld islto nfehae ecosystems. freshwater in dissolution sulfide iron nitrate-dependent sulfide is iron and mineral anoxic oxidation, sulfide of mineral occurrence iron the of for type determinant important the an that suggest results these gether and L62 D h ehrad.Emi:[email protected] E-mail: Netherlands. The ED, NL-6525 1, Toernooiveld Nijmegen, University Radboud IWWR, Microbiology, discussion. and advice assistance, practical for eateto irbooy WR abu nvriyNjee,62 DNjee,TeNetherlands The Nijmegen, Camp ED 6525 den Nijmegen, Op University Radboud M. IWWR, J. Microbiology, of Huub Department and Jetten M. S. Mike Nijmegen, Netherlands University The Radboud Nijmegen, IWWR, ED Biology, Environmental 6525 and Smolders Ecology P. Aquatic J. of Alfons Department and Lamers M. P. Netherlands The Leon Nijmegen, ED 6525 Nijmegen, University Radboud IWWR, Microbiology, of Department Haaijer M. C. Suzanne Wetlands Freshwater for in Donors Reduction Electron Nitrate Potential Microbial as Pyrite and Sulfide Iron Copyright Journal Geomicrobiology et(.. oekr19;Cba n gaids20;McGuire 2001; Ignatiadis and ex- Cabral some 1997; to Bosecker studied (e.g., min- been tent sulfide has metal microorganisms by aerobic oxidation anoxic decades, eral 6 and last oxic the Over under conditions. environments freshwater and marine-, INTRODUCTION Keywords otie eune of A biomass sequences nitrite. reactors or contained the from nitrate generated of gene) rRNA expense (16S the library at clone sulfate, to biore- the sulfide as amended of calculations, sulfide oxidation stoichiometric upon iron based the identified, was in actor process dominant active The iron an of sulfide. contrast, addition In upon donor. developed Crystalline electron community microbial an conditions. denitrifying as freshwater function not anoxic, did under pyrite bioreactor investigated a was reduction in function nitrate to microbial sulfide) for iron donors and electron (pyrite as minerals sulfide iron poten- two the of study, present tial inter- the as In toxicity. such metal processes, or adverse eutrophication to nal lead ultimately sulfide- could iron soils, in rich influxes Nitrate ecosystems. wetland freshwater consequences for important has reduction nitrate microbial for donors O:10.1080/01490450701436489 DOI: online 1521-0529 / print 0149-0451 ISSN: irba rnsld iea xdto cusi both in occurs oxidation mineral sulfide iron Microbial h oeta fio ufiemnrl ofnto selectron as function to minerals sulfide iron of potential The drs orsodnet .J .O e ap eatetof Department Camp, den Op M. J. H. to correspondence Kartal Address Boran and Pas-Schoonen de van Katinka 2007. thank February authors 13 The accepted 2006; October 2 Received Acidovorax c alr&FacsGop LLC Group, Francis & Taylor rba irt reduction, nitrate crobial mi- oxidation, mineral sulfide iron ecosystem, Freshwater lk atra hc oee 0 falcoe.To- clones. all of 90% covered which bacteria, -like 43141 2007 24:391–401, , Thiobacillus Thiobacillus -like, seismyb e lyr in players key be may -species Thiobacilli Geothrix- ie OP10-like, like, 391 02 oil ta.20) nmrn n rcihsystems, pyrite brackish of and especially marine oxidation, (FeS mineral In al. sulfide 2002). et metal Lucassen al. microbial 2002; et Fontbot Morillo and Dold 2002; 2000; al. waters natural et to threat (Edwards serious a sul- that pose as metals, is toxic such and oxidation, aspect acid furic mineral Another sulfide (Agate 2003). metal anoxic al. ores of et from products Olson metals 2001; valuable microbial Suzuki of of 1996; recovery importance the economic rea- in the One is leaching 2003). al. attention et this Rohwerder for 2003; son al. et Okibe 2001; al. et ieas.Oc h olo remtlin sdpee,sulfide depleted, is ions metal free of pool sulfide iron the of Once (reformation ions minerals). metal binding other or by iron immobilized ferrous is with Sulfide sulfide. com- produce reducing will When sulfate munity availibility. the sulfate available, becomes by sulfate limited additional therefore, is, bacte- activity Sulfate-reducing rial 2000). Jørgensen marine and to (Kasten comparison in systems low are wetlands freshwater in concentrations sulfate Normally, production. sulfate to ox- lead Furthermore, will process. idation the of course the in released trace are these metals as metal-toxicity, to lead therefore could availability, Luther and 2001). Morse al. et 1997; Yu al. 2000; et Ledin 1999; (White poten- metals for, sink trace a toxic, as tially function minerals sulfide iron quality. example, ecosystem For freshwater al. of mediators preservation et microbial for relevance Buss its of and is 1993; process Golterman this and Understanding 2005). Garcia-Gil freshwater 1992; Engesgaard in 1991; Kipp occur al. and et to Postma suggested 1991; Golterman the been (e.g., at systems has oxidation nitrate mineral of sulfide on expense not Iron but mediators. process, microbial the of the effects environmental mainly the has on systems focused anoxic freshwater in on oxidation Research mineral 2004). sulfide al. sulfur iron et and Simmons 2003; iron al. 2002; the et 2001, Edwards in Jørgensen and minerals (Schippers sediments sulfide these of iron cycles of role central the rnsld iea xdto,cue yicesdnitrate increased by caused oxidation, mineral sulfide Iron 2 n rnsld FS,hsas ensuidbcueof because studied been also has (FeS), sulfide iron and ) Downloaded By: [Camp, Huub J. M. Op den] At: 08:00 14 August 2007 bet xdz e,btntFeS not but are FeS, bacteria oxidize that to reported able Schip- previously environment. (2002) freshwater Jørgensen and a pers in oxidation iron nitrate-dependent mineral is of sulfide mineral occurrence sulfide the iron in factor of determinant type a the whether envi- out natural find the and resembles ronment closely more that environment an in sulfides iron of oxidation nitrate-dependent investigate to for wished therefore We responsible environment. natural necessarily microorganism the in the not process sought-after of the do identification therefore the and to organ- 1998) lead fast-growing al. favor et to known (Grosskopf are isms (2006) used microbial al. as et to such Haaijer subject efforts in were enrichment batch sulfides deter- Furthermore, iron not oxidation. of was types it minerals, which sulfide mined iron of oxidation evidence dependent indirect, betaproteobacterial albeit of involvement strong, the for provided study oxidation. this mineral sulfide Although iron nitrate-dependent in players key genus that betaproteobacterial suggested the 2006) of un- al. members fully et yet (Haaijer findings not Previous are derstood. environments freshwater in oxidation eral 2006). al. et Welle der (van animals and effect toxic plants direct on 1991; a has Roelofs sulfide Additionally, 1989; 2001). al. al. et Lamers et (Caraco leads eutrophication phosphate (internal) released to The phosphate. releasing and sulfides iron forming complexes, iron-phosphate from iron ferric reduces 392 aeocre ntentr eev e wr ae hc is (51 which Netherlands Water the Zwart of south Het the reserve in nature situated the in occurred have Source Soil METHODS AND MATERIALS ecosystems. freshwater in oxidation mineral sulfide terial betaproteobac- that 2006, al. et Haaijer in the formulated supported hypothesis, composition community microbial reactors the analyses of stoichiomet- molecular on from based findings Furthermore, identified the calculations. is ric at nitrite, or oxidation nitrate sulfide of expense bioreactor, in process sulfide-amended dominant iron- is The the reduction experiments. nitrite activity and function with nitrate to evaluated microbial sulfide for iron donors and electron freshwater pyrite as under of nitrite potential in and The investigated nitrate conditions. further with is amended minerals bioreactor sulfide a iron previ- micro- on The work presented. activity the are bial 2006), to al. parallel et (Haaijer in published performed ously was which study, tor environment. marine a e el 07.Asi ape(. et)fo h horizon the from depth) m (1.5 sample (van soil weight A dry the 2007). of Welle 2% der was samples organic soil The the De of reserve. lake content nature matter freshwater this in the situated around pre- is (2006), which described Venkoelen, al. as et collected, Haaijer were in site samples sampling viously a Soil as studies. relevant our highly it for making 2002), al. et cassen h irba rcse novdi nxcio ufiemin- sulfide iron anoxic in involved processes microbial The nteps,polm ihaiicto n eutrophication and acidification with problems past, the In bioreac- a from results the therefore, article present the In Thiobacilli a ekypaesi irt-eedn iron nitrate-dependent in players key be may 2 ihntaea h xdn in oxidant the as nitrate with , Thiobacilli Thiobacillus ◦ 24 N:6 ◦ 11E nnitrate- in .C .HAJRE AL. ET HAAIJER M. C. S. a be may (Lu- ) otiigtehget(0no g nmol (60 highest the containing hog uewt imtro m h eco a kept was reactor The mm. 17 4 and of 15 diameter between a with proceeded analysis tube for a sampling through Liquid sulfide. iron and with experiments pyrite between adjusted was gas-flow the properties, of settling therefore and a donors. reactor ensured electron the reactor the of the top of the top at the turbulence combi- at low in separator reactor phase the a of with of shape nation mixing conical provided The and contents. anoxic reactor system the the kept flow gas (95/5) ciiy htrsl rmtefloigeuto:C equation: following no the (assuming from values result expected that activity) the with (mea- concentrations actual continuous the sured) of in comparison nitrite by and performed nitrate were experiments sulfate, of production and tion consump- on Calculations below). described (as performed were ciiyExperiments Activity the A and time reactor. the the t in rate, concentration dilution initial the D medium, inflowing the m,gslf eco.A h otmo h eco nArgon/CO an reactor the of bottom the At reactor. gas-lift were ume, shaped donors conically electron a was the end-result which The retained. in min- designed the was to reactor tubes a attached erals, of possibly clogging microorganisms avoid of To wash-out design. and reactor- special a for called System Reactor additions. consecutive with or reactor single the in syringes to tight supplied oxy- gas were with donors reaction electron chemical Mineral avoid closed gen. to a gas in nitrogen stored under was de- suspension bottle were The ions suspension. for iron the or checked in sulfide tected was free sulfate, suspension No resulting ions. contaminant The (1999). described Hanert as by prepared chemically was A reactor. sulfide the iron to of addition suspension and before dis- dried products was with oxidation pyrite rinsing The possible sulfur. remove by free to followed order acetone in with water once tilled with and times HCl 3 washed M were size grains 1 Pyrite in mm. varying 3 to grains, up mm small 0.5 into from ground was pyrite The electron donor. an as used was Nijmegen) University Radboud Heijnen, Donors Electron Mineral most the iron-sulfide-oxidizing as of 2006) enrichments al. bacteria. start et to Haaijer sample in suitable detail se- was in content (described content) mineral lected sulfide iron reflect to assumed le ih8go rsaln yie hratr continuous a Thereafter, pyrite. crystalline (D medium of of g flow 8 with plied wit reactor inoculated The was performed. system con- was following experiment The medium-flow tinuous assessed. reduction.was nitrate microbial e − Dt eas yieadio ufiepsesdfeetsettling different possess sulfide iron and pyrite Because is,tesiaiiyo yie(FeS pyrite of suitability the First, h oi aueo h lcrndnr sdi hsstudy this in used donors electron the of nature solid The F.K.W. Dr. of (courtesy pyrite crystalline highly a Initially, ) + A · e − Dt nwihC which in , ◦ = .Bt ac n otnosexperiments continuous and batch Both C. .0 h 0.005 h8gofthe 0 − 1 ersnstecnetainof concentration the represents m irt)wssatdand started was nitrate) 5mM , − 1 cdvltl ufr(AVS, sulfur volatile acid ) 2 sa lcrndnrfor donor electron an as ) eetdsi n sup- and soil selected ,2lw rigvol- orking = C 0 · (1 − 2 Downloaded By: [Camp, Huub J. M. Op den] At: 08:00 14 August 2007 hkricbtra 5 rpm. 150 at incubator shaker a topeeo Argon/CO of atmosphere gas iron (50 and for suspension each) later mM sulfide (see (0.5 medium nitrite mineral nitrate, of containing ml composition) 20 sul- experiment iron this of reaction In chemical fide. possible assess to performed was later). situ described in (as Fluorescence analysis for of (FISH) fixed rpm. Hybridization extraction and 4,000 DNA for at weight used molecular min were high 30 samples for precip- concentrated the centrifugation of Volumes 6x, by concentrated 30x was fraction fraction itate the vf generate The to precipitates fraction. the pfp suspend to used remain- was The volume volume. reactor ing vf total The the of population. enable 5/6 of bacterial to consisted fraction the (vf), of fraction into analysis liquid molecular divided a detailed and was (pfp) content fraction reactor precipitate the a operation), of months 18 halted. when production stopped sulfate was and experiment consumption of The nitrate concentration present. was residual nitrite a iron mM and of 0.5 ml added, 5 was experiment, the suspension of sulfide start of mM the 1 At containing supplied. ni- medium was of of nitrate addition flow single continuous a a by then mM and 1 the trate to of adjusted first course was reactor the the in concentration in nitrate The increased experiments. had iron-sulfide-amended denitrification au- of to contribution the totrophs whether medium evaluate to continuous performed a was and flow, nitrate, final sulfide, a iron con- Therefore, with experiments. nitrite experiment first and/or the soil, in nitrate the especially to sumption, from contributed organics have of could Het- however, traces reduction. on nitrite and denitrification nitrate erotrophic to coupled oxidation 84. sulfide and 72 49, 22, 0, days ml on (5 added suspension was sulfide Iron aliquots) reactor. the to and supplied nitrite mM was 0.5 nitrate containing and medium previously of flow described continuous as a then flushed first was reactor eval- the this facilitate uation, To additions. sulfide iron sequential of by evaluated means was 0 activity nitrite-reducing day and/or nitrate- on mmoles). crobial (1.4 added 23 was day and Nitrite mmoles) 0. (1.7 15 day day on mmoles), (0.84 performed was suspension ml) sulfide iron (5 of addition single a and medium stopped the was Subsequently, flow experiments. previous sulfate the and from nitrite nitrate, ions residual any remove to nitrite wit or nitrate flushed first was occurred. reactor oxidation The sulfide iron nitrite-driven whether assess to 25. and 16 9, 0, days on added suspension ml sulfide 5 iron and of sulfide, portions iron ad- finer-grained the gas-flow of the settling supplied, allow to was justed, sediment of selected inoculum the additional of the an grams experiment from 6 the removed of not start was the iron At pyrite reactor. on The activity nitrate. assess and to (FeS) used sulfide was setup reactor same the ment, anandfrapro f35mnh.AtrteFeS the After months. 3.5 of period a for maintained nadto oteratreprmns ac experiment batch a experiments, reactor the to addition In (after performed been had experiments activity the all After iron autotrophic favor to was study our of intention The mi- and availability sulfide iron between relation the Third, operation batch to switched was system reactor the Second, μ )wsicbtdfr6dy ne a under days 6 for incubated was l) 2 9/)a omtmeauei a in temperature room at (95/5) iea eimwithout medium mineral l 6 h RNSLIEMNRL N IRBA IRT REDUCTION NITRATE MICROBIAL AND MINERALS SULFIDE IRON 2 experi- fteseiemnrlmdu upidws( l (g was supplied medium mineral composition sterile The the of reduction. nitrite and nitrate autotrophic favor Medium Mineral xrce sn tnadpoeue.HtsatPRreactions was PCR DNA Hot-start weight procedures. molecular frac- standard High vf using and preceding). extracted pfp the the (see on separately tions performed was cloning and Analysis Gene rRNA 16S 4053 Cambridge, LKB Ltd., England). 7.3). a Biochrom pH (LKB with M spectrophotometer performed (0.15 Kinetics buffer were phosphate measurements potassium Extinction with ml filled ortho- 100 and of ethanol to absolute g of 0.54 ml of 10 in consisted dissolved reagent phtaldialdehyde OPA mea- The extinctions nm). the (420 and sured RT), min, (20 incubated 40 reagent, OPA of volume al. et A Taylor 1974). 1971; with (Roth determined reagent (OPA) were ortho-phtaldialdehyde concentrations Ammonium (Haaijer 2006). previously al. described et as sul- performed turbidometry-based were and analyses analyses fate used Colorimetry- nitrite was determinations. and supernatant nitrate ammonia resulting based and The nitrite rpm. nitrate, 13,000 for at min 5 for n rcpttdio,lqi ape eesplmne with supplemented were samples aNa liquid iron, precipitated and rpm) 13,000 min; de- (5 samples. was centrifuged iron from soluble ferrous supernatants per- free in samples, of was termined of species oxidation analysis direct iron chemical by exposure water-insoluble formed limit limit of therefore to Determination and reactor iron. air the the from min extinction to samples 15 the within of performed measuring removal were before upon determinations Iron water supplemented nm. distilled 562 and of at 7) ml pH 1 HEPES-buffer, with mM 50 200 20 in with treatment ferrozine this mixed After was min. tem- ple room 15 at for incubated (RT) and determination) perature iron with (total or HCl M in determination) 1 hydroxylammonium-dihydrochloride iron of either; (ferrous solution were saturated HCl a Samples 1M modifications. with 1:1 minor mixed with (1970) and al. ammonia Stookey et to of experi- according performed per presence were twice determinations determined Iron pH, ment. were The iron soluble week. free reac- a of the presence once from least taken at samples liquid tor, in determined were nitrite Analysis Chemical stock sterile from salts in sodium medium their mineral solutions. as the or amounts reactor required the the to added Nitrate 1996). were al. nitrite et Graaf and de (van solution element trace a of ml/l KH iea eimlcigogncsbtae a upidto supplied was substrates organic lacking medium mineral A ihmlclrwih N xrcinfloe yPCR by followed extraction DNA weight molecular High osprt irt,ntieadamnu osfo free from ions ammonium and nitrite nitrate, separate To and nitrate, sulfate, of concentrations reactivity, assess To 2 PO 2 CO 4 3 .6 CaCl 0.06, ouin(tabe l 96,mxd n centrifuged and mixed, 1996), al. et (Straub solution 2 (H 2 μ O) fsml a ie ih760 with mixed was sample of l μ 2 erzn egn 00 w/w % (0.05 reagent ferrozine l .2 MgCl 0.12, 2 (H 2 − O) 1 KHCO ) 2 .8ad0.5 and 0.08 μ fsam- of l 3 1.25, 393 μ l Downloaded By: [Camp, Huub J. M. Op den] At: 08:00 14 August 2007 e nTbe1 he eaaePRratos ifrn ntean- (50 the temperature in differing nealing reactions, PCR separate Three 1. Table in fied G¨ a in performed 394 tool their searches BLASTN with < by database compared GenBank were the in sequences relatives sequences. closest gene gene rRNA rRNA 16S 16S (Infor- whole Cloned package assemble software to used 7.0 was Suite Max) NTI Vector the of ContigXpress program The 1. Table in specified gene, rRNA univer- the 16S of bacterial the sites conserved with targeting obtained primers, sequencing were bacterial sal Almost sequences gene nucleotides. rRNA 200–990 16S se- full between Partial varied 1). (Table of lengths M13R sequencing quence primer Partial with kit. performed the was with clones according supplied Biotech instructions Pharmacia the Amersham to of with Kit performed FlexiPrep was the isolation DNA Plasmid manufacturer. by the supplied instructions the to according Technologies) Life (Invitro-gen cells competent with TOP10F Kit and vector Cloning pCR2.1-TOPO TOPO-TA the the each with for separately performed sample was pooled Cloning sample procedure. cloning each the for to pooled prior were the temperatures with obtained annealing (nt), different nucleotides 1500 prod- approximately PCR of gene rRNA ucts, 16S bacterial resulting The sample. each raie ngop ae pnteecasfiainrsls Fur- results. classification these upon based groups in organized pro- release hierarchy Bacteriology, taxonomical Systematic 6.0 the of Manual to Bergey’s clones in posed assign to used was 32 eunigAGGCGTGGTC19–46(ae1991) (Lane 2000) Horikoshi and (Takai 1994) al. multiple 1998) et Tebbe flanking (Greisen and Region (Schwieger 1993) al. et (Muyzer 1996) al. et multiple (Ferris flanking 1998) Region al. et 1998) 1392–1406 AC (Juretschko al. 1991) ATG et (Lane GCT (Juretschko ACA GAA CAG 517–531 Sequencing 1991) al. et (Weisburg G M13R CCA CGG 1177–1196 CGA AAA GTA TAC GTG 510–526 GGT Sequencing GGC CC ACG CTT CAC CCC 1053–1072 CAT 341–357 GGC CGT GCT Sequencing TGA M13F GCT GCG 1529–1545 ACC CG GCT Sequencing 1392R TCA TCG CTG CC Sequencing TGG CAG CAG GCA 1177R TGC CCG ACT AG AGC 8–27 GGC Sequencing 1522–1541 8–27 517R GGA ACG CC GAT Sequencing CCT GGT GGA AG CA AG 1072F AAA CTC CCG CTC CAK TGG CAG TGG ATC TCM Sequencing TYM GTG TGA TGA GAG GTT AAG GTT 526F AGA AGA (5 Sequence Sequencing 357F Sequencing PCR 1522R PCR 27F 630R 616F use Primer Primer tign emn) eal ftePRpiesue r speci- are used primers PCR the of Details Germany). ottingen, http://www.ncbi.nih.nlm.edu/BLAST # coli *E. rmrue nti eeec ssmlrt h rmrue nti study. this in used primer the to similar is reference this in used primer < < http://bergeysoutline.com http://rdp.cme.msu.edu/classifier/ ubrn 6 RAgene. rRNA 16S numbering T rdetPRaprts(hta Biometra, (Whatman apparatus PCR gradient ◦ ,54 C, ◦ Cor60 > h ln irr aawas data library clone The . ◦ > ) eepromdfor performed were C), > h D classifier RDP The . Cl ta.2005), al. et (Cole , -3 oiin References Position* ) .C .HAJRE AL. ET HAAIJER M. C. S. rmrSpecifications Primer AL 1 TABLE eso irognss(AI,Bcei bceilprobemix) betaproteobacterial num- (bacterial Total and Bacteria 3.1). (DAPI), microorganisms (version of package bers software standard used the was Germany) with Jena, (Zeiss, microscope epifluorescence 2 at performed were 1000 inspections a Microscopic well on label. as FLUOS label labeling Cy-3 a a as of with used choice was (Betthio1001) the probe specific all betaproteobacterial of the almost outcome, effect hybridize hybridization an to the used exclude was To EUB338, III Bacteria. probes EUB338 and Cy-5-labeled II of of EUB338 probemix concentration bacterial formamide A a 30%. at performed Hy- were Germany). (Ulm, bridizations and Thermohybaid Cy-5 from Cy-3, derivatives as labeled purchased (FLUOS) pre- were ester are Probes 5(6)-carboxyfluoresein-N-hydroxysuccinimide study 2. this Table DNA. in in all used stain sented probes and of signal details fluorescent and the Specifications enhance to used (4,6-diamidino-2-phenylindole) was DAPI with medium mounting CA) Burlingame, inc., Laboratories, (Vector Vectashield (1990). (FISH) Hybridization situ in Fluorescence DQ975214-DQ975219. are GenBank, to submitted study, Numbers Accession 2004). al. et (Kumar program 3.1 MEGA the with formed per- were analyses evolutionary molecular and phylogenetic ther vector 2.1 pCR site cloning vector 2.1 pCR site cloning IHaaye eepromda ecie yAane al. et Amann by described as performed were analyses FISH this from sequences gene rRNA 16S of numbers Accession × anfiain o mg custo es Axioplan Zeiss a acquisition image For magnification. Thiobacilli Ivtoe,Goign the Groningen, (Invitrogen, kit cloning TA TOPO Manual the Groningen, (Invitrogen, kit cloning TA TOPO Manual Netherlands) Netherlands) poeBto01 were Betio1001) (probe # # Thiobacilli # # - Downloaded By: [Camp, Huub J. M. Op den] At: 08:00 14 August 2007 diino rnsld cluae vrtetm eidfo o7days); 7 to 0 days). from 30 period to 28 time from riod the over (calculated ( sulfide iron of addition nrae ntecus fteeprmn.Tersetv rates respective The nitrate experiment. of the rate of consumption course the the and in increased nitrite, and production sulfate The 1). of Figure rates in A the see (for period: week this 1 in of mM rates period calculated 1.1 a during to respectively) mM 0 0.05 to (from the 0 and increased and concentrations mM) 2 nitrite to and 4.7 sulfate (from decreased concentration nitrate the Nitrate for Donor Electron an Reduction as Sulfide Iron of Potential occurred. indicat- oxidation medium-feed, the pyrite the no in of that sulfate that ing as and same nitrite ac- the electron remained nitrate, the reactor of as concentrations nitrate and the donor ceptor, electron the as pyrite with Nitrate for Donor Electron Reduction an as Pyrite of Potential RESULTS sample. per each bacteria for (50–200 images image) 14 of analysis through determined I.1 rdcinadcnupinrtso e n NO and FeS on rates consumption and Production 1. FIG. eimfe (D feed medium consumption. B U 3 C C C G G G 6 RA 3–5 atra(am ta.1999) al. et (Daims 1999) al. 1999) et al. (Daims et (Daims Verrucomicobiales 2006) al. et Planctomycetales (Haaijer Bacteria Betaproteobacterial 338–355 rRNA, 16S 338–355 1001–1021 rRNA, 338–355 rRNA, 16S rRNA, 23S 16S ACC GGG TTT TCA ACG TGT AGC AGG CTT CGT ACC TGT GCC AGG GCT CGT AGT ACC AGG Betthio1001 GCC CGT GCA TCC GCC III GCT 338 EUB II 338 EUB 338 EUB aeSqec (5 Sequence Name aeatrtefut diino rnsld cluae vrtetm pe- time the over (calculated sulfide iron of addition fourth the after rate ) eyqikyatrtefis rnsld diino a 0; day on addition sulfide iron first the after quickly Very experiment the of period month 3.5 entire the Throughout * .coli E. numbering. = .0 h 0.005 uft production; sulfate , − 1 otiig5m irt.( nitrate. mM 5 containing ) RNSLIEMNRL N IRBA IRT REDUCTION NITRATE MICROBIAL AND MINERALS SULFIDE IRON -3 fpoeadpsto pcfiiyReference Specificity * position and probe of ) irt production; nitrite , A aeatrtefirst the after rate ) 3 − Continuous . rb Specifications Probe nitrate , AL 2 TABLE agtmolecule Target rosidct irt additions. nitrite indicate arrows h ocnrtoso h noigmdu eg,Fgr :day 3: Figure (e.g., towards medium concentrations inflowing sulfate the and of concentrations nitrate the the of monitored returning be could a sulfide as iron of Depletion sulfide. iron addi- of upon tion 49–56) day 3: and Figure decrease (e.g., increased not even interme- sometimes did an occasionally as concentrations (nitrite nitrite reduction diate), nitrate the and the inflow both of medium result a is concentration sul- concentration nitrite sulfate iron the Because and of increased. addition decreased each concentration Upon nitrate 3. fide, Figure in shown activity tor Sequential Additions Nitrite: Sulfide and Iron Nitrate Sulfide, Iron on Activity simultaneous 2. the Figure from in derived visible production be sulfate can and consumption as nitrite culture this in place Culture Batch in Nitrite and Sulfide Iron on Activity 1) Figure rates. in these (B in sulfide increase iron 2-fold of a addition indicate fourth the in and (A 1) first the Figure after consumption nitrate and production sulfate of I.2 ciiyo e n NO and FeS on Activity 2. FIG. h eeiiefeigwt rnsld eutdi h reac- the in resulted sulfide iron with feeding repetitive The took clearly nitrite of expense the at oxidation sulfide Iron Thiobacilli 2 − ac culture. Batch . nitrite; , uft.The sulfate. , 395 Downloaded By: [Camp, Huub J. M. Op den] At: 08:00 14 August 2007 in.Cniuu eimfe (D feed medium Continuous tions. hmcloiaino rnsld i o cu nti time this in occur not did sul- significant sulfide or period. iron that nitrite of indicating nitrate, oxidation observed, in chemical were changes no concentration nitrite; fate and nitrate with Sulfide Iron of Oxidation Chemical the after observed sulfide. iron not of was depletion production sulfate or nitrite was consumption, consumption nitrate pyrite residual reactor, Although the in de- depletion. available day to still sulfide start After iron reduction stable. nitrate indicating and relatively crease, rate 5–14, remain production days sulfate and Between the lower 15, operation. of are days rates 4 these first the in were consumption recorded nitrate and production sulfate of rates highest Denitrification to Autotrophs of Contribution Increased the of slopes the nitrate-decrease. of the steepness and which increasing sulfate-increase as experiment 3 the Figure of in visible course is the produc- during sulfate increased and rates consumption tion nitrate sulfide The iron activity. additional restored with reactor the Supplementing 16–22). NO FeS, on Activity 3. FIG. 396 . Mntie h rosidct rnsld additions. sulfide iron indicate arrows The nitrite. mM 0.5 uigte6dy fteseieicbto fio sulfide iron of incubation sterile the of days 6 the During The 4. Figure in shown are experiment this of results The 3 − n NO and = .0 h 0.005 2 − h fet frpttv e addi- FeS repetitive of effects the ; − 1 otiig05m irt and nitrate mM 0.5 containing ) .C .HAJRE AL. ET HAAIJER M. C. S. I.4 ciiyo e n NO and FeS on Activity 4. FIG. 7 otefl-eghcoe f1ado f3 Fgr ) The 5). identities (Figure pfp37 genus sequence and/or this pfp1 exhibited clones to clones full-length the directly all to 97% assigned of be 30% could 3), (Table clones genus total betaproteobacterial of the 20% affiliated of sequences members of consisted with sequences of group abundant and (9%), clones), total of (62% contained and clones) total of (71% teobacteria the in clones similar of 5 3). Figure abundance (Table in library the included of was shown consideration pfp8 partial is Clone upon 97% of sequence. than number clone higher each the identity for addition, sequence vf28) In a and with (pfp8). 23 sequences vf clone vf5, partial pfp37, 1 positions (pfp1, and clones phylogenetic sequenced the fully shows 5 5 of Figure fraction. and liquid fraction the precipitate the from sequences clone of phylogenetic distribution the between differences are data significant sequence data identify the not library did of examination clone Detailed (vf). the 3. Table of fraction in organization presented liquid the the from from results 16 The the and from (pfp) originating fraction 29 clones, precipitate 45 on performed was screening Sequencing Gene rRNA iron, 16S ferric mM (7 fraction pfp iron). ferrous the mM in 0.2 detected were species remained pH The time. (7 any stable at detected free was nor microorganisms iron, ammonia, water-soluble of Neither possible. association was as precipitates these cleaned with not -carbonates). were and walls fer- and The -hydroxides, (ferric -oxides, precipitation iron iron-phosphates, of rous result the likely precipitates most These reactor. were the of walls the on accumulated itates otiig1m nitrate. mM 1 containing vrl,tecoelbaydt a oiae yBetapro- by dominated was data library clone the Overall, preliminary a enable to genes rRNA 16S of sequencing Partial precip- orange and green experiments, sulfide iron the During ± Acidovorax .) ihcnetain f ae-noul,iron water-insoluble, of, concentrations High 0.2). Geothrix lk 4)sqecs(al ) h most The 3). (Table sequences (4%) -like nitrite; , 3 − otnosmdu ed(D feed medium Continuous lk 1%,O1-iesequences OP10-like (13%), -like sulfate; , • nitrate. , Thiobacillus Thiobacillus = .0 h 0.005 -like − ≥ 1 ; ) Downloaded By: [Camp, Huub J. M. Op den] At: 08:00 14 August 2007 oiae yBcei.Tebceilcnetaino h pre- the of and (1.2 concentration fraction active cipitates bacterial highly The was indicates Bacteria. reactor by which the dominated analyzed, of DAPI images community and microbial all probemix the in bacterial identical the were for signal counts (data The differences shown). significant not no showed betaproteobacterial probe Cy3-labeled the and of Comparison 2006). Thiobacilli al. et (Haaijer bac- the with betaproteobacterial hybridized of respectively) the portion 44%, major and a (53% community cases, terial both in that showed fraction FISH study this in obtained sequences clone 5). length (Figure full near level to genus ( tity) a resemblance strong to any classified have not con- be did library, not and the could in that sequences sequences of clone sisted the of 10% remaining con- with The 3), affiliated (Table sequences clones all of of sisted 18% group, abundant most second ntelqi rcin(6.8 fraction liquid the in oe hnta ftev rcin h oa otiuino the of contribution total times The 5 fraction. was vf content the reactor of the that to than lower fraction pfp the of tribution eu hoails72 1 7 clone bacterium 0 Uncultured 1 Thiobacillus Genus 0 1 Acidovorax Genus 1 2 Rhodocyclaceae GC55 bacterium Uncultured 5 1 6 Burkholderiales 2 bacterium sludge Uncultured 4 Betaproteobacteria 1 2 1 Betaproteobacteria 2 Proteobacteria 2 4 OP10 Genus Geothrix Genus Bacteria soito lnscoe ers relative Nearest clones clones * association hlgntc Pfp Phylogenetic* A eemndwt h lsie olo h iooa aaaePoet(oee l 05,(http://rdp.cme.msu.edu.) 2005), al. et (Cole Project Database A Ribosomal the of Tool Classifier # the with determined *As IHaayi ftefie ape rmteppadv reactor vf and pfp the from samples fixed the of analysis FISH yesriso utrdrpeettvsaepeetdweee hs xiie eunesimilarity sequence a exhibited these whenever presented are representatives cultured or strains Type rcptt rcinreactor, fraction precipitate ubr bandwt h LO-aee probe FLUOS-labeled the with obtained numbers ± Thiobacilli 0.6 ± 2.3 · A 10 B iudfato reactor fraction liquid 7 · 10 ml RNSLIEMNRL N IRBA IRT REDUCTION NITRATE MICROBIAL AND MINERALS SULFIDE IRON Vf 5 seicpoeBetthio1001 probe -specific − hoailsplumbophilus Thiobacillus ml 1 B a 7tmshge than higher times 17 was ) − 1 .Tevlmti con- volumetric The ). < hoailsthioparus Thiobacillus defluvii Acidovorax Dechloromonas plumbophilus Thiobacillus thioparus Thiobacillus plumbophilus Thiobacillus fermentans Geothrix 7 euneiden- sequence 97% lsicto 6 RAgn sequences gene rRNA 16S Classification ZC (AJ224618) 5Z-C1 (AF288772) (AJ316618) (AJ224618) 5Z-C1 (AJ316618) (U41563) 7(AJ412674) 87 (AJ271048) (AF234701) A22 p FL8 sp. AL 3 TABLE Y81)Atvtdsug,nitrate sludge, Activated (Y18616) . # strain strain irt-euigatvt.Teasneo moi throughout ammonia of absence The ni- activity. sulfide-oxidizing, nitrate-reducing and iron production increasing indicating in sulfate consumption, resulted trate of 3) rates (Figure sulfide increasing iron simultaneously (Figure of observed additions were Repeated 1–4). consumption iron nitrite to and reaction nitrite accumulation consumption, In nitrate production, reduction. sulfate nitrite addition, sulfide and nitrate donor microbial electron accessible for an provided hand, other Iron the 4). on Figure sulfide, 3, (Figure depleted was sulfide iron when nitrite stopped completely production, consumption nitrate sulfate and production, observed: or consumption were oxidation indications no donor pyrite again, system), experimentsfor electron the sulfide from an removed iron not the as was In (pyrite function reduction. nitrate not microbial did for pyrite crystalline that Oxidation Sulfide Sulfide: Iron on Activity DISCUSSION donors. addition electron in mineral biomass the retained to the reactor vf in the situated the indicating were of fraction, (77%) pfp contribution reactor the the in than bacteria Most higher fraction. reactor times the 3.4 in present therefore bacteria of was number total the to fraction pfp h otnoseprmn ihprt n irt showed nitrate and pyrite with experiment continuous The prclrt euto 95 reduced oxidation Aerobic reduction (per)chlorate sulfide, of oxidation Aerobic 95–97 reduced oxidation Aerobic sulfide, of oxidation Aerobic reduction iron Ferric eirfigratrtreating 94–96 reactor Denitrifying 90–98 sludge Activated sludge Activated ufrcompounds sulfur reduction hydrogen and galena compounds sulfur hydrogen and galena adl leachate landfill ers eaiecoe onearest to clones relative Nearest soitdwt eaie(%) relative with associated > 0 oteclones. the to 90% euneidentity Sequence 96–100 95–99 92–98 95 98 99 397 Downloaded By: [Camp, Huub J. M. Op den] At: 08:00 14 August 2007 r niae ewe rces ihteecpino h P0gop nyrfrnesqecsfo utrdseiswr sd ob elsie,s reclassified, be to * used. were species cultured from sequences reference only group, OP10 ( the affiliated of strongly exception sequences the With partial brackets. of between number indicated The are substitutions/site. indicates bar scale The (arrow). reference irt/uft ai ste h irt osmto iie by divided production. consumption sulfate expected nitrate expected The the the later). then (see is respectively ratio 2 nitrate/sulfate production and sulfate 1 expected pro- equations the nitrite using calculate and to consumption used were nitrate oxidized duction was measured sulfide The and sulfate. gas, to dinitrogen a and to nitrite reduced was of nitrate mixture that ex- assumption nitrate-amended the on for based periments calculated were ratio) (nitrate/sulfate stoichiometries. observed and expected of comparison the in process important an not was 1997) reactor. al. al. et et (Eisenmann Moura bacteria several 1995; for reduction described nitrate as ammonia, dissimilatory to that suggested experiments the seque nt). The (1,385 shown. sequences are full-length 50% th the than with of higher generated alignment replicates) tree after (1000 consensus tree values bootstrap the Bootstrap Neighbor-joining (1969). to clones. Cantor manually rRNA and added 16S Jukes was sequenced of nt) fully method (524 the distance pfp8 of the clone positions and the 2004) showing al. tree et Phylogenetic (Kumar program 5. FIG. 398 eeorpsta xdz eiulognccro rmtesoil nitrate. the of expense from denitrifying the carbon at of organic activity residual by oxidize for explained that 1.7 heterotrophs be A, can de- for deviation 1) (1.6 This Figure values B). expected 3.5, the and from (2.7 sto- considerably B observed viate and the A the nitrate situations and In for sulfide ichiometries production. iron sulfate with measured experiment first the by divided sumption xetdrto fntaecnupint uft production sulfate to consumption nitrate by of evaluated ratios was Expected sulfide iron on reactor the of activity The h bevdntaeslaertoi h esrdntaecon- nitrate measured the is ratio nitrate/sulfate observed The 3H 4NO + 3 − + + 5HS HS − − + → 8NO SO 4 2 3 − − → + 4NO 5SO 4 2 2 − −+ + H 4N + 2 + 4H .C .HAJRE AL. ET HAAIJER M. C. S. 2 [1] O [2] ope ordcino irt odntoe gas. dinitrogen to nitrite sulfate of to reduction sulfide of to oxidation expectedcoupled for the 3, nitrite to equation identical from of derived is ratio ratio, (2.7) and observed produced consumed sulfate The were to produced. consumed nitrite sulfate of of mmoles mmoles 3.8 experiment 1.4 of sulfide total iron a 2) nitrite-amended (Figure the In oxidation. fide the in increased have experiments. to iron-sulfide-amended considered the of is course nitrate and on reactor sulfide the in iron denitrification the to autotrophs the of Therefore, contribution negligible. experiment, became first denitrification the to heterotrophic the contrast in to that, ratio indicates nitrate/sulfate value expected observed the of The explained approximation intermediates. be close denitrification can of 4) accumulation Figure 3.5–17.5, transient day by from from (2.7 1.3 periods dif- and 2 the 0–4 these day and in ratios rate nitrate/sulfate reduced con- the a nitrate in with fast ferences period initial a The by 1.7. of followed is sumption ratio ratio nitrate/sulfate expected the observed while con- an 1.8, sul- were in of nitrate resulting mmoles 2.0 of produced, and mmoles fate produced nitrite 3.6 of total; mmoles 0.27 very In sumed, other. are each stoichiometries to observed close and expected the however, mne xeiet dnie ufieoiaina the at nitrate- oxidation last sulfide the identifies and experiment experiment, this amended of stoichiometry The irt losre sa lcrnacpo o irba sul- microbial for acceptor electron an as served also Nitrite 4), (Figure nitrate and sulfide iron with experiment last the In 8NO 2 − + 3HS qie pyrophilus Aquifex − > + 7 euneiett)wt h ln sequences clone the with identity) sequence 97% 5H + → 3SO M34)wsue sa outgroup an as used was (M83548) 4 2 − + 4H 2 O + eGenBank. ee N 2 MEGA e c of nce [3] Downloaded By: [Camp, Huub J. M. Op den] At: 08:00 14 August 2007 eco eit ohntaedpnetfrosio oxidation iron oxidation. ferrous sulfide and nitrate-dependent both mediate reactor open possibility the betaproteobacterial leaves the which that 1996), al. nitrate-reducing et (Straub iron-oxidizing, capacity possess to however, described previously reported, any bacteria. of oxidizing iron presence ferrous the library distinct clone indicate The not 1). did (eq. situation data the oxidation for sulfide 1.7 for of ratio accounting expected of only the to ratio close nitrate/sulfate still observed is which overall 2, an in result would This irt codn oeuto ol euti osmto of consumption of in expense nitrate. result the mmoles would 0.4 at 4 equation mmoles to 2 in- according these the nitrate from of available Oxidation were FeS. iron troduced ferrous released, of were sulfate mmoles of 2 mmoles 2 implying total in sulfide, iron and trate the on influence little ratio. only exert nitrate/sulfate would observed nitrate oxidation of iron expense the microbial at simultaneous result oxidation, to shown sulfide was reduction from nitrate conditions. of part oxygen major (Hanert low the acceptor under Although 2004) electron Weiss the and as Emerson oxygen 1999; Buchholz- with and or Straub 1996; 1998) as al. Cleven nitrate et with (Straub acceptor oxidation electron anoxic microor- the through by proceed iron activity can ferrous reactor of ganisms the Oxidation which in recorded. period generally of time was frame the time was a which within days, activity 6 exhibit not did furthermore trite negligible. experiment, is this nitrite in by oxidation least iron at ferrous that, abiotic the indicating than and higher observed, sulfide been now have iron one would on ratio nitrite experiment nitrite/sulfate to the batch extent nitrite, reactor large the a in to contributed reduction have would of oxidation sulfide abiotic iron If compounds. sev- iron of ferrous oxidation of solid-phase extent eral microbial and rate nitrate-dependent the Weber increases that although greatly reported activity study, already our (2001) in al. oxidation et iron con- ferrous theory to in could tribute process as This 1991). 1977; proposed Thorling Buresh oxide and and nitrous Sørensen (Moraghan with reduction nitrite 1996) from al. product et the Straub 1994; (Hansen al. previously et reported been has nitrite with ferrous oxidation iron, Abiotic iron ferric oxidation. iron mainly ferrous contained predominant wall, suggesting reactor precip- the The study. on this formed in itates obtained results the from apparent not Iron of Fate The Sulfide: Iron the on Activity in process dominant the as nitrite bioreactor. and nitrate of expense o xml,i h otnoseprmn ih1m ni- mM 1 with experiment continuous the in example, For ni- and nitrate with sulfide iron of incubation abiotic The was sulfide iron of portion iron ferrous the of fate exact The 0FeCO 10 3 + + N 2NO 2 + 3 − 0HCO 10 Thiobacillus + 4H2O 24 RNSLIEMNRL N IRBA IRT REDUCTION NITRATE MICROBIAL AND MINERALS SULFIDE IRON 3 − + .denitrificans T. → 8H ebr on nthe in found members + 0Fe(OH) 10 3 a been has [4] ntecoelbaycnitdof consisted library clone the mem- in After oxidation. genus the sulfide of in bers involved likely suggest most the library in reactor, group the physiological dominant in a represent sequences sequences clone these genus these the of to dominance assigned the be could that quences possessing bacteria be- ( the sulfur-oxidizing to belonging chemolithoautotrophic bacteria of consisted genus taproteobacterial reactor the in tion Community Microbial 6 RAgn eune(55n)wsasge otegenus the to assigned was nt) (1515 sequence reason- vf5’s gene a Clone rRNA phylum. as 16S this 2002) of members al. for role et physiological (Stein able previously suggested been metal-cycling although has unknown, is physiology the and phylum 2001). al. et OP10- (Dalevi uncultured, GC55 the clone sludge of previously activated sequence the like, gene to se- rRNA identity gene 16S sequence rRNA 95% published 16S had The nt) OP10-like library. (1505 the clone quence for the representative in sequences considered clone was 23 vf the Clone on and 95%, assumptions the only allow of was to physiology 1999) insufficient al. as et regarded Coates therefore 1996; al. et (Lonergan genus cultured, the the of of sequence representative representa- gene iron-reducing, rRNA a 16S the as to functioned identity quence nt) (524 the 8 for tive pfp gene rRNA clone 16S of partial The sequence reactor. the in detected bacteria like the of physiology the on sumptions to 94% only of of sequence identity gene sequence rRNA a 16S had the nt) not (1535 vf28 does clone apparently of bacterium genus this the that to belong 5) (Figure reclassification, of shows view and in determined (accession sequence was gene AJ316618) rRNA number 16S The 1992). al. et (Drobner hydrogen-oxidizing aerobic, and new, sulfide- a as galena-, hybridization DNA-DNA and physiology on sequences. with (1%) affiliated genus the to longing the of with sequence 98% gene and 99% was rRNA pfp37 16S clone the of identity with sequence The 95%. both were denitrificans T. the to pfp1 clone of sequence identities 96% shared Sequence identity. and genus (1531 this to pfp37 assigned and be both nt), could (1491 nt) pfp1 clones of sequences rRNA 16S full-length nearly The capacity. nitrate-reducing 2006) Madigan ( ing hoailsdenitrificans Thiobacillus h hsooyo h utrdrltvso h ln se- clone the of relatives cultured the of physiology The popula- dominant most the showed data library clone The hr r opr utrsaalbefrteO1 candidate OP10 the for available cultures pure no are There as- informed any make to low too as regarded was value This hoailsthioparus Thiobacillus hoailsplumbophilus Thiobacillus Geothrix .denitrificans T. 6 RAgn eune hw nFgr 5, Figure in shown sequences gene rRNA 16S Thiobacillus Geothrix Thiobacillus .thioparus T. lk eune ntecoelbay t se- Its library. clone the in sequences -like Thiobacillus Thiobacillus el n od20)adlack- and 2000) Wood and Kelly , lk atradtce ntereactor. the in detected bacteria -like tbe ta.19;Stlyand Sattley 1998; al. et Stubner , vrl;alcoesqecsbe- sequences clone all Overall; . h eodms bnatgroup abundant most second the , hoailsplumbophilus Thiobacillus h 6 RAgn sequence gene rRNA 16S The . hoailsplumbophilus Thiobacillus hnwith than eeol lgtystronger slightly only were hoailsplumbophilus Thiobacillus hsgnsencompasses genus This . a rtdsrbdbased described first was etrxfermentans Geothrix Thiobacillus .denitrificans T. .thioparus T. Thiobacillus .thioparus T. species strains, . and , -like 399 and . - Downloaded By: [Camp, Huub J. M. Op den] At: 08:00 14 August 2007 REFERENCES in dentirification microbial environments. soil to and/or sedimentary importance of are likely compounds, pyrite-like seems crystalline therefore than It rather pyrite. minerals, for iron-sulfide-like that oxidant an (2002) not Jørgensen find- is and nitrate Our Schippers that of not. conclusion was the for support pyrite donor ings whereas electron reduction an as nitrate function microbial to able was sulfide iron freshwater study, sulfide our important In iron environments. an microbial freshwater in is anoxic oxidation available mineral of mineral occurence the sulfide for iron determinant of suggest type study this the from that results freshwa- the under Furthermore, oxidation conditions. mineral ter sulfide iron anoxic in genus players betaproteobacterial the of members process. relevant ecologically an therefore as is regarded and metals) toxic of is release eutrophication, stimulation (internal quality ecosystem This freshwater on oxidation. effects adverse have sulfide expected to expected iron are anoxic deposition, stimulate nitrogen-rich to of agricultural infiltration from run-off and e.g., land nitrate, of influxes Water, Zwart resolved. be to remains detected bacteria) OP10-like and like nteratr h ucinlt fteohrpyoeei groups phylogenetic ( other the of functionality The reactor. the oxidation in sulfide nitrate-driven genus performing the group of functional the members it the analysis that FISH probable the seems in demonstrated community bacterial total h ag otiuino betaproteobacterial of contribution large the the data, physiological of the dominance by suggested nitrate of expense the at Implications depletion soil. after the reactor from matter the organic in of compounds biomass organic of decaying amounts from trace resultant on sur- possible bacteria and these system of reactor vival our in retention biomass explained the de- be by can system growth reactor chemolithoautotrophic our favor in to bacteria signed al. these et of (Gentile presence The bioreactors 2006). denitrifying of constituents major as genus the of Members genus; the of sentative Acidovorax 400 usS,Rvt O ognP emn D 05 sn cec ocreate to science Using 2005. 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