ward 2017 microbial diversity and iron oxidation at okuoku...

Geobiology. 2017;15:817–835. wileyonlinelibrary.com/journal/gbi  | 817© 2017 John Wiley & Sons Ltd

Received:03October2016  |  Accepted:21September2017

DOI: 10.1111/gbi.12266

O R I G I N A L A R T I C L E

Microbial diversity and iron oxidation at Okuoku- hachikurou

Onsen, a Japanese hot spring analog of Precambrian iron

formations

L. M. Ward1 | A. Idei2 | S. Terajima3 | T. Kakegawa3 | W. W. Fischer1 | 

S. E. McGlynn2,4,5

1Division of Geological and Planetary

Sciences,CaliforniaInstituteofTechnology,Pasadena,CA,USA2DepartmentofBiology,TokyoMetropolitanUniversity,Tokyo,Japan3DepartmentofGeosciences,TohokuUniversity,SendaiCity,Japan4Earth-LifeScienceInstitute,TokyoInstituteofTechnology,Tokyo,Japan5BlueMarbleSpaceInstituteofScience,Seattle,WA,USA

Correspondence

L.M.Ward,CaltechGPSDivision,Pasadena,CA,USA.Email:lward@caltech.eduor

S.E.McGlynn,Earth-LifeScienceInstitute,TokyoInstituteofTechnology,Tokyo,Japan.Email:mcglynn@elsi.jp

Funding informationNASANESSF,Grant/AwardNumber:(#NNX16AP39H;NSF,Grant/AwardNumber:#OISE1639454;NSFGROW,Grant/AwardNumber:#DGE1144469;MEXTKAKENHI,Grant/AwardNumber:15K14608;NASAExobiology,Grant/AwardNumber:#NNX16AJ57G;DavidandLucilePackardFoundation;StanfordUniversityBlausteinFellowship

AbstractBanded iron formations (BIFs) are rock deposits common in the Archean andPaleoproterozoic (andregionallyNeoproterozoic)sedimentarysuccessions.Multiplehypothesesfortheirdepositionexist,principallyinvokingtheprecipitationofironviathemetabolicactivitiesofoxygenic,photoferrotrophic,and/oraerobiciron-oxidizingbacteria.SomeisolatedenvironmentssupportchemistryandmineralogyanalogoustoprocessesinvolvedinBIFdeposition,andtheirstudycanaidinuntanglingthefactorsthat lead to ironprecipitation.OnesuchprocessanalogsystemoccursatOkuoku-hachikurou (OHK)Onsen inAkitaPrefecture,Japan.OHK isan iron-andCO

2-rich,

circumneutralhotspringthatproducesarangeofprecipitatedmineraltexturescon-tainingfinelaminaeofaragoniteandironoxidesthatresembleBIFfabrics.Here,wehaveperformed16SrRNAgeneampliconsequencingofmicrobialcommunitiesacrosstherangeofmicroenvironments inOHKtodescribethemicrobialdiversitypresentand to gain insight into the cycling of iron, oxygen, and carbon in this ecosystem.TheseanalysessuggestthatproductivityatOHKisbasedonaerobic iron-oxidizingGallionellaceae.IncontrasttootherBIFanalogsites,Cyanobacteria,anoxygenicpho-totrophs, and iron-reducing micro-organisms are present at only low abundances.Theseobservationssupportahypothesiswherelowgrowthyieldsandthehighstoi-chiometry of iron oxidized per carbon fixed by aerobic iron-oxidizing chemoauto-trophs like Gallionellaceae result in accumulation of iron oxide phases withoutstoichiometricbuildupoforganicmatter.Thissystemsupportslittledissimilatoryironreduction,furthersettingOHKapartfromotherprocessanalogsiteswhereironoxi-dationisprimarilydrivenbyphototrophicorganisms.ThispositionsOHKasastudyareawherethecontrolsonprimaryproductivityiniron-richenvironmentscanbefur-therelucidated.Whencomparedwithgeologicaldata,themetabolismsandmineral-ogyatOHKaremost similar to specificBIFoccurrencesdepositedafter theGreatOxygenationEvent,andgenerallydiscordantwiththosethataccumulatedbeforeit.

818  |     WARD et Al.

1  | BACKGROUND

Banded iron formation (BIF) is a characteristic lithotype in manyPrecambrian basins. These finely laminated, iron-rich (>15% Fe byweight) sedimentary deposits are not only economically criticalsourcesofironore(particularlyafterpost-depositionalweatheringandhydrothermalprocessesoxidize, leach,andupgrade theore,Morris,1980;Beukes,1984),butmayprovidearecordofbiologicalactivityontheearlyEarth(e.g.,Harder,1919;Kappler,Pasquero,Konhauser,&Newman,2005;Konhauseretal.,2002).

Despitetheirprevalenceintheearlyrockrecord,theprocessesbywhichBIFsaredepositedarenotwellunderstood.Itisgenerallyhy-pothesizedthatBIFsformedasaresultoftransportandconcentrationof ferrous iron (as Fe

2+

(aq))inseawaterunderanoxicandsulfur-poorcon-ditions,followedbyoxidationandprecipitationof ironasferric ironphases(Drever,1974;Holland,1973,1984).However,itisimportanttonotethattheprimarymineralogythatmadeuptheprecursorsed-imentstoBIFremainsuncertaininmanycases,andthesignificanceofironoxidationduringBIFdepositionremainsanareaofactivede-bate (Bekker etal., 2014;Kappler&Newman,2004;Kappler etal.,2005; Konhauser etal., 2002; Posth, Konhauser, & Kappler, 2013;Rasmussen,Krapež,Muhling,&Suvorova,2015;Rasmussen,Meier,Krapež,&Muhling,2013;Rasmussen,Muhling,Suvorova,&Krapež,2016;Tosca,Guggenheim,&Pufahl,2016).DifferenthypothesesforBIFdeposition invokearangeof ironoxidationprocesses, includingabioticironoxidationbyUVlight(e.g.,Cairns-Smith,1978;Francois,1986),indirectlybiologicallybyO

2sourcedfromoxygenicphotosyn-

thesisbyCyanobacteria(e.g.,Cloud,1973),ordirectlybiologicallybyaerobiciron-oxidizingbacteria(e.g.,Chan,Emerson,&Luther,2016)or by anaerobic iron-oxidizing phototrophic bacteria (e.g., Kappleretal., 2005;Widdel etal., 1993). BIFs do not form inmarine sedi-mentarybasinstodayduetotheO

2 and sulfate content of seawater

(Canfield, 1998), which prevents the mobilization and concentra-tionofsufficientamountsofdissolvedironinseawaterandshallowpore fluids (Holland, 1973). However, a range of potential analogenvironmentscanbeobservedtodaythatmayrevealkeyprocessesassociatedwith the deposition of BIFs. These include permanentlystratifiedlakes,suchasLakeMatanoinIndonesia(Croweetal.,2008),and iron-rich hot springs, such as Chocolate Pots in YellowstoneNational Park (Pierson, Parenteau, & Griffin, 1999). These systemscontainanoxic, iron-richwaters thatproduce ironoxidesandotherFe-bearingphasesthrougharangeofprocesses.InLakeMatano,ironoxideproductionisthoughttobedrivenlargelybyphotoferrotrophy(Croweetal.,2008),whileatChocolatePots,hotspring ironoxida-tion isdrivenprimarilybyabioticreactionof ironwithO

2produced

byCyanobacteria (Pierson&Parenteau, 2000;Piersonetal., 1999;Trouwborst,Johnston,Koch,Luther,&Pierson,2007).Terrestrialhotsprings can provide unique environments inwhich high concentra-tionsofdissolved ironcanexist at circumneutral conditionsdue torockweathering by anoxic sourcewaters; they also provide accesstonovelmicrobialdiversitywhichisrareorabsentfromtypicalsur-faceenvironments.Whilethesehotspringfluidsarenotperfectcom-positional mimics of Precambrian seawater, the geomicrobiological

processestheysupportandtheresultingfaciesandfabricscanpro-videprocessanalogsforunderstandingdepositionalmechanicsofatleastsomeBIFs.Thisisparticularlyusefulforevaluatingtherelativerolesthatdifferentmicrobiologicalprocesses(e.g.,photoferrotrophy,aerobicironoxidation,andoxygenicphotosynthesis)mayhaveplayedinthedepositionofdifferentBIFfacies.Acrucialbutunderstudiedas-pectofBIFdepositioninmodernanalogenvironmentsistherelativedelivery fluxesof ironoxidesandorganiccarbon tosediments,andtheaverageoxidation stateof the resultant iron formations; trendsin theproportionof ferrous to ferric iron inancientBIFshave longbeenobserved in the rock record (e.g.,Klein,2005)—andthesecanreflecttheredoxstateoftheenvironmentatthetimeofdeposition,aswellastheparticularphysiologiesandmetabolismsresponsibleforironoxidation (Fischer&Knoll,2009) (AppendixS1).Although littleexplored,themetabolicyieldandefficiencyofthemicrobialmetab-olisms driving iron oxidationwill be a factor in themineralogy andredoxchemistryofdiageneticallystabilizedironformationlithologies.Currently, knowledge of the microbial metabolisms and processesthat leadtodifferentmineralassemblages (andferric:ferrous ratios)inmodernenvironmentsremainsmeager.Characterizationofthemi-crobialcommunitiesinthecontextoftheorganiccarboncontentandredoxstateofironinthesolidsaccumulatinginBIFanalogsdepositeddifferentially by photoferrotrophs, Cyanobacteria, and aerobic ironoxidizerswillhelptoconnectobservationsofancientBIFstothegeo-biologicalprocessesresponsibleforironoxidedeposition.

Inthisstudy,weinvestigatedanovelBIFanalogenvironmentatOkuoku-hachikurou Onsen (OHK) in Akita Prefecture, Japan. OHKis an iron- andcarbonate-rich, circumneutralhot springwithanoxicsource waters, which produces extensive iron oxide and aragonitetravertine with mineralogical and textural features resembling BIF(Takashima, Okumura, Nishida, Koike, & Kano, 2011). To study themicrobialdiversityofOHKandevaluatethepotentialrolesofdiffer-entmicrobialmetabolismsinproducingmineraldepositsatOHK,wecollectedmineral precipitates and filtered springwater from pointsalong the outflow of the stream for analysis usingmicroscopy andcharacterizationofthemicrobialcommunityvia16SrRNAgeneam-pliconsequencing.Thesedatarevealthatproductivityandironoxida-tionatOHKisprimarilydrivenbyaerobic,iron-oxidizingtaxarelatedto the genusSideroxydans, and consequently, the environment pro-ducessedimentsthatareorganic-leanandcontainahighproportionof ferric ironphases—anearlydiageneticprecondition to thehighlyoxidizedBIFfaciesdepositedincertainenvironmentsaftertheGreatOxygenationEvent.

2  | MATERIALS AND METHODS

2.1 | Geological context and sedimentology of OHK

OHK is located in Akita Prefecture, Japan, at 40.407925N,140.754744E(Figure1),inanactiveregionoftheTohokuvolcanicarc, generated by the subduction of the Pacific Plate; the localbedrock geology consists of Miocene–Holocene green tuff andfelsicvolcanicrocks (Shimazu,Yamada,Narita,&Igarashi,1965).

     |  819WARD et Al.

OHKconsistsofasinglesubsurfacewatersourcethatoriginatesfromaminingexplorationboreholedrilledinthe1960s.Thisbore-holeemergesintoa2-m-diametersourcepool,withasubmergedshelfaroundtheedgeoftheboreholeat1.2mdepth.Atthetimeof our study inNovember 2015,water emerged from the bore-holeat44.3°C,highinFe(II),verylowinoxygen(Table1),anddis-playedcontinuousandvigorousebullitionofCO

2 (Figure2).The

source pool contains abundant suspended fine flocs of hydrousironoxides.TheearlycrystallineironoxidephasesatOHKconsistof ferrihydrite (Takashimaetal.,2011), thoughthismayage intomore ordered iron oxideminerals such as hematite, goethite, orlepidocrociteintravertines.Fromthesourcepooltotherotenburo(Figure2), the stream flows over a mineralized substrate com-prisedofwell-lithified, finely laminatedaragonite and ironoxidetravertinewith a smooth texture andmottled coloration rangingfrom white aragonite to orange and red iron oxides (Figure2).Surfacesofpools,channels,andcanalsarecoatedinfine,orange-colored ironoxideflocs.Mature ironoxidephaseswithintraver-tine appear dark red. Travertine accumulates around pool edgesas lobate walls ~10cm in width.Where spring water overflowstheedgesofpools travertine terracesdevelopwith a character-istic~5cmstepsizeanduptoaboutahalfmeterintotalheight.Theseterracessuperficiallyresemblethosefoundinbothcircum-neutralandacidic iron-richsystemselsewhere (e.g.,YellowstoneNationalPark,Fouke,2011;andtheTintilloRiver,Spain,España,SantofimiaPastor,&LópezPamo,2007);theoccurrenceofsimilarlarge-scalemorphologiesacrossverygeochemicallydifferenten-vironmentssuggeststhathydrology—asmuchasgeochemistryor

microbial processes—plays a role in the travertinemorphologies(Fouke,2011).

Anoldoutflowchannel emerges from the sourcepool, butwaslargelyinactiveatthetimeofoursampling;flowintothischannelhasbeen blocked and redirected through canals to the rotenburo.Thischannel is ~5cmdeep, partiallymixeswith the source pool due toswashing caused byCO

2 ebullition, but is otherwise stagnant.Thin

platesofaragonite(<1mm)coatthewatersurfaceofthechannel,andinsomeareas,thinmicrobialbiofilmshavedevelopedonthebottomofthechannel.

Wateremergingfromthesourcepoolpredominantlyflowsthroughshallow(5–10cm)canalsintoa~1-m-diameterpoolusedforbathing(rotenburo).Rocksurfacesinthecanalsandrotenburoarecoatedin

F IGURE  1 LocationofOkuoku-hachikurouOnsen(OHK)inAkitaPrefecture,Japan.InsethighlightsthelocalgeologyoftheLakeTowadaregion,modifiedfromGeologicalSurveyofJapan(2012).ThebedrockgeologyatOHKconsistspredominantlyoffelsicvolcanics[Colourfigurecanbeviewedatwileyonlinelibrary.com]

TABLE  1 GeochemicalcharacteristicsofOHKsourcewater

T 44.3°C

pH 6.8

DO <15μm

Fe2+114 μm

NH3/NH

4

+22 μm

Cl− 38 mm

SO4

2−6.5 mm

NO3

− <1.6μm

NO2

− <2.2μm

HPO4

− <1μm

TOC <0.005Cwt%

820  |     WARD et Al.

ironoxide flocs.Water flowsout from the rotenburo throughaddi-tionalshallowcanalsandultimatelydevelopsintoasheetedflowonthehillslope,wherecontinualdegassingofCO

2 leads to formation of

bubbles.Manybubblesbecomeencrustedwitharagoniteandminer-alized(Figure2).

While no substantial accumulation of microbial biomass is visi-blewithinthesourcepool,rotenburo,orcanals,thin(<1mm)patchygreenbiofilmsoccuralongtherimofthesourcepoolandoldstream(Figure2d). These were sampled for the Shallow Source and OldStreamMineralsamples.

Downstream, OHK preserves unique mineralized bubbles. AsCO

2degassesfromthespringwater,thepHrises,dissolvedinorganic

carbon concentrations drop, and the carbonate saturation state in-creases prompting aragonite precipitation. This occurs sufficientlyquickly inOHK that bubbles develop coatings of aragonite and aremineralized in situ. These bubbles encrust and provide a possiblepreservation mechanism for a wide range of organic materials, in-cludingleaves,arthropods,andbiofilmsinthissectionofthespring.Preservationoforganicstructuresbyironmineralshasbeenobservedpreviouslyinacidiciron-richenvironmentsatRioTinto,Spain,wherefossil structures endure formillions of years (Fernández-Remolar &Knoll,2008).TherapidmineralizationofironoxidesandaragoniteatOHKmaythereforealsoserveasamechanismforpreservingkeybio-logicalfeaturesofthisenvironmentovergeologictimescales.

F IGURE  2 ContextphotographsofOHK.(a)PhotographofOHKfacingnorthtowardsourcepool.Sourcepool(1),oldstream(2),canal(3),androtenburo(4)visible.Peopleforscale(~1.8m).(b)ImageofOHKfacingsouthwesttowardrotenburoandbubblepool.Waterflowingfromtherotenburo(4)developsintoasheetedflowwhichspreadsacrossthehillside,fillingthebubblepool(5)wheredegassingCO

2

bubbles become encrusted in aragonite.

Personforscale(~1.8m).(c)Underwaterphotographofsource.WaterandCO

2

bubblesemergefromtheboreholeatright.Scalebaris10cm.(d)Lobatewalloftravertineattherimofthesourcepool,withgreen-pigmentedbiofilms(sampledasShallowSourceMineralsample)visible.Scalebaris10cm.(e)Close-upoftravertineonthewestedgeofthesourcepool.Scalebaris50cm.(f)Close-upphotographofthebubblepool,showingaragonite-mineralizedCO

2bubbles.Scale

bar5cm.(g,h)Close-upsofferrihydriteand aragonite laminations in travertine.

Redlayersareprimarilycomposedofferrihydrite,whilewhitelayersarepredominantlyaragonite.Scalebarsshownare5mm[Colourfigurecanbeviewedatwileyonlinelibrary.com]

(a) (b)

(c) (d)

(e) (f)

(g) (h)

     |  821WARD et Al.

2.2 | Sample collection

Samples for sequencing and geochemical analysis were collectedfromfivesitesatOHK(Figure2):DeepSource(1.2mdeepinthesourcepoolattheborehole),ShallowSource(surfacewaterandathinbiofilmalongtheedgeofthesourcepool),OldStream(waterand thin biofilm along the semi-stagnant blocked outflow), Canal(alongtheflowfromthesourcepooltotherotenburo),andBubblePool (downstream in a 15cm deep pool coated in mineralizedbubbles).

Fromeachsite,botha“Mineral”anda“Water”samplewerecol-lectedfor16Sampliconsequencing,targetingsurface-associatedandpelagicmicrobial communities, respectively. “Mineral” samples con-sistedofscrapingsofthinbiofilms,mineralprecipitates,orwholemin-eralizedbubblesfromsurfacesoftravertinesorcobblesinthebottomofwaterchannels.Mineral sampleswerecollectedusingsterile for-cepsandspatulas(~0.25cm3ofmaterial).“Water”samplesconsistedof cells and sediment filtered from water using sterile syringes and

0.2-μmSansyo (SansyoCo.,Tokyo,JP) filters (~50–200ml ofwaterfiltered,untilfilterbegantoclog).CellswerelysedandDNA-preservedinthefieldusingZymoTerralyzerBashingBeadMatrixandXpeditionLysisBuffer(ZymoResearch,Irvine,CA).Cellsweredisruptedimme-diatelybyattachingtubestothebladeofacordlessreciprocatingsaw(Black&Decker,Towson,MD)andoperatingfor1minute.Samplesforgeochemical analysis consistedofwatercollectedvia sterile syringeandfilteredimmediatelythrougha0.2-μmSansyofilter.

2.3 | Geochemical analysis

Dissolved oxygen (DO), pH, and temperature measurements wereperformedinsituusinganExtechDO7008-in-1PortableDissolvedOxygen Meter (FLIR Commercial Systems, Inc., Nashua, NH). Ironconcentrations were measured using the ferrozine assay (Stookey,1970)followingacidificationwith40mmsulfamicacidtoinhibitironoxidation byO

2 or oxidized nitrogen species (Klueglein & Kappler,

2013).Ammonia/ammoniumconcentrationsweremeasuredusingaTetraTestNH

3/NH

4

+Kit(TetraPond,Blacksburg,VA)followingman-ufacturer’s instructions butwith colorimetry of samples andNH

4Cl

standardsquantifiedwithaThermoScientificNanoDrop2000cspec-trophotometer(ThermoFisherScientific,Waltham,MA)at700nmtoimprovesensitivityandaccuracy.Anionconcentrationsweremeas-ured via ion chromatography on a Shimadzu Ion Chromatograph(ShimadzuCorp.,Kyoto,JP)equippedwithaShodexSI-904Eanioncolumn(ShowaDenko,Tokyo,JP).

Total organic carbon (TOC) contents were assessed for filteredwaterandmineralprecipitatesfromthesourcepool,aswellasmineralprecipitates near the canal. For dissolved organic carbon measure-ments, 3L of springwaterwas filtered using 0.45-μmSansyo glassfiberfilters.Formineralprecipitates,approximately300gofsampleswas collected. Carbonate carbonwas removed via dissolutionwithHCl,andresidueswerefoldedintotincapsulesandanalyzedforcar-bon content via elemental analyzer (Thermo Scientific Flash 2000)withadetectionlimitof0.005%Cbyweight.

SamplesofsedimentfromthesourcepoolwerecharacterizedviaSEM-EDS (SU5500;Hitachi, Tokyo, JP) and μm X-ray diffraction byTEM(JEM-3010;JEOL,Tokyo,JP).

2.4 | 16S rRNA gene amplicon sequencing and analysis

Followingreturntothelaboratory,microbialDNAwasextractedandpurifiedwithaZymoSoil/FecalDNAextractionkit.TheV4-V5regionofthe16SrRNAgenewasamplifiedfromeachextractusingarchaealandbacterialprimers515F(GTGCCAGCMGCCGCGGTAA)and926R(CCGYCAATTYMTTTRAGTTT) (Caporaso etal., 2012). DNA wasquantifiedwithaQubit3.0fluorimeter(LifeTechnologies,Carlsbad,CA)accordingtomanufacturer’s instructionsfollowingDNAextrac-tionandPCRsteps.AllsamplesyieldedPCRampliconswhenviewedonagel after initialpre-barcodingPCR (30cycles).DuplicatePCRswerepooledandreconditionedforfivecycleswithbarcodedprimers.SamplesforsequencingweresubmittedtoLaragen(CulverCity,CA)foranalysisonanIllumniaMiSeqplatform.Sequencedatawerepro-cessed using qiimeversion1.8.0(Caporasoetal.,2010).Rawsequencepairswere joinedandquality-trimmedusing thedefaultparametersin qiime. Sequences were clustered into de novo operational taxo-nomicunits (OTUs)with99%similarityusinguclustopenreferenceclusteringprotocol(Edgar,2010).Then,themostabundantsequencewaschosenasrepresentativeforeachdenovoOTU(Wang,Garrity,Tiedje,&Cole,2007).Taxonomicidentificationforeachrepresenta-tivesequencewasassignedusingtheSilva-115database(Quastetal.,2012)clusteredatseparatelyat99%andat97%similarity.Singletonsandcontaminants(OTUsappearinginthenegativecontroldatasets)were removed; 16S sequences were aligned using mafft (Katoh,Misawa,Kuma,&Miyata, 2002), and a phylogenywas constructedusing fasttree(Price,Dehal,&Arkin,2010).Alphadiversitywasesti-matedusingtheShannonindex(Shannon,1948)andinverseSimpsonmetric(1/D)(Hill,1973;Simpson,1949).Allstatisticswerecalculatedusing scripts inQIIME and are reported at the99%and97%OTUsimilarity levels.Multidimensional scaling (MDS) analyses and plotstoevaluatethesimilaritybetweendifferentsamplesandOHKenvi-ronmentswereproducedinrusingthevegan and ggplot2packages(Oksanenetal.,2016;RCoreTeam2014;Wickham,2009).

3  | RESULTS AND DISCUSSION

3.1 | Geochemistry

GeochemistrymeasurementsofOHKsourcewateraresummarizedinTable1,whilegeochemicalgradientsalongthestreamoutflowaresummarizedinFigure3.Wateremergingfromthesourcewas44.3°C,very lowindissolvedoxygen(<15μm),hadapH6.8,andcontainedsubstantial concentrations of dissolved iron (114 μmFe2+),and22μm

NH3/NH

4

+.Afteremergingfromtheborehole,thespringwaterrapidlyexchangesgaseswiththeairduetoturbulentmixingassociatedwithwaterflowandCO

2ebullition,andDOroseto30μmattheShallow

Source.Dissolvedorganiccarbon in thesourcepoolwasbelowthe

822  |     WARD et Al.

limitofdetection (0.005%Cbyweight).Organiccarboncontentofmineralprecipitatesatthesourcepoolwas0.01%byweight,risingto0.02%nearthecanal.SEM-EDSanalysisofsourcepoolprecipitatesdetectedFeandOasthemajorelements,andselectedareaX-raydif-fractionbyTEMrevealedamorphousFehydroxidesasthesolidphasewithnodetectionofhematiteorothercrystallinephases (Figure4).Aswater flows downstream from the source pool, it cools slightly,degassesCO

2,andcontinuestoabsorbatmosphericO

2.Bythetime

waterreachestheBubblePoolthespringwateris40°C,pH7.5,con-tains 7.7 μmNH

3/NH

4

+,109μm O2,andnodetectabledissolvediron.

Concentrationsofmajoranionswere largelystablealongthespringoutflow, with 1,350mg/L Cl−, 620mg/L SO

4

2−, and no detectableNO

2

−,NO3

−,orHPO4

−(limitofdetection0.1mg/L).

LikemanymodernBIFanaloghotspringsites,undertheenviron-mentalconditionsatOHK,abioticironoxidationisexpectedtopro-ceedspontaneously.FollowingrateequationsforabioticironoxidationfromSingerandStumm(1970),therateofabioticironoxidationpro-ceedsproportionatelytoconcentrationsofdissolvedFe2+,O

2,andthe

activityofOH−,oftheform—d[Fe2+]/dt = k[Fe2+

]PO2[OH−]2,wherek is

arateconstantequaltoabout8×1013L2 mole

−2 atm

−1 min

−1at25°C,andincreasingapproximately10-foldforevery15°Coftemperatureincrease (Stumm&Lee,1961).AttheDeepSource,where[O

2] was

belowthedetectionlimitof0.5mg/L(~15μm),abioticironoxidationratesare thereforeexpected tobe less than~4.35μm/min.Thisex-pectedratewouldbeexpectedto increasebetween~8.7μm/min at

theShallowSourcesiteand~12.7μm/minattheCanalsite,the last

F IGURE  3 Summaryofthegeochemistryandiron-oxidizingcommunitycompositionofOHKordinatedalongtheflowpathsinthesystem.(a)SchematicofOHKflowpaths,keyedtothesamplenames.MineralandWatersampleswerecollectedfromOldStream,DeepSource,ShallowSource,Canal,andBubblePoollocalities.(b)Relativeabundanceofphototrophicandiron-oxidizingtaxainthewater(top,solidbars)andmineral(bottom,crosshatchedbars)samplesalongtheflowpath.DatahereincludetaxalistedinTable3anddiscussedinthetextwithconfidentlyassignedmetabolisms,butnotincludingtaxasuchasAnaerolineaewhosemetabolismscannotbepredictedwithavailabletaxonomicresolution.Aerobicironoxidizersareshowninred,anoxygenicphototrophsinpurple,andoxygenicCyanobacteriaingreen.Aerobicironoxidizersarecommonthroughoutthewatersamples,andintheDeepSourcemineralsample,butdecreaseinrelativeabundancesomewhatdownstream.AnoxygenicphototrophsareconcentratedintheOldStreamandShallowSourceMineralsamples,whileoxygenicCyanobacteriaincreaseinabundancedownstream,particularlyinwatersamples.(c)ConcentrationsofdissolvedO

2andFe2+.Sourcewatersaredepletedin

O2buthighindissolvediron,whiledownstreamFe2+concentrationsdropandO

2increases.Themajorityofironoxidationappearstooccur

undermicrooxicconditionswhereaerobicironoxidizersaremostprevalent,whileanoxygenicphototrophsarelargelyrestrictedtopatchybiofilms,andCyanobacteriaareonlysignificantaftertheironhasalreadybeenremovedfromsolution.Changesintemperature(44.3–40.7°C)andconcentrationsofchloride(~37–39mm)andsulfate(~6.3–6.65mm)alongthehotspringoutflowwereminorandnotexpectedtocontributesignificantlytochangesinmicrobialcommunityormineralprecipitates,andsoarenotdisplayedhere[Colourfigurecanbeviewedatwileyonlinelibrary.com]

(a)

(b)

(c)

     |  823WARD et Al.

samplinglocationatwhichdissolvedironwasdetectable.Thesevaluesaresignificant,andasaresult,abioticironoxidationlikelycontributessubstantially to theproductionof ironoxidesatOHK.Nonetheless,theabundanceofiron-oxidizingbacteriaandmorphologyofironoxideprecipitates demonstrate that there is aviable and active niche for

biologicalironoxideproduction.Thisisfurthersupportedbyestimatesof flowrates throughOHKcorrelatedagainstchanges in ironoxideabundance from theedgeof theShallowSourcePool to theCanal,whereflowratesof~0.33m/sthroughcanalswithcrosssectionsof~10cmby5cmsuggestflowratesof~1.67L/s.Overdistancesof2mfromtheShallowSourcetotheCanal, [Fe2+

] declines from ~114 μm

to ~83 μm, ora changeof31μm inapproximately6seconds; this isequivalenttoabout100-foldfasterthanexpectedforpurelyabioticrates. Even assuming substantial backflow andmixing, this leaves asubstantialroleforaerobiciron-oxidizingbacteriainexplainingthede-clineinironconcentrationsalongthehotspringoutflow.TheseresultsarealsoconsistentwithestimatesfromKasamaandMurakami(2001)that suggest aerobic iron-oxidizingbacteria can increase ironoxida-tionratesbyupto4ordersofmagnitudeabovethoseexpectedfrompurelyabioticreactions.

3.2 | Recovered microbial diversity

In total, we recovered 141,125 sequences from the 10 samples atOHK (Table2). Reads per sample ranged from 2,176 for theCanalMineralsample to27,454reads for theOldStreamMineralsample(median15,247,mean14,112, and standarddeviation8,654).WiththeexceptionoftheOldStreamMineralsample,watersamplescon-sistentlyrecoveredmoresequencereadsthanmineralsamples(meanof5,926versus18,983).AssessmentofsamplingdepthwasestimatedusingGood’sCoverage(Good,1953).Onaverage,90%ofthemicro-bialcommunitywasrecoveredfromOHKsamplesatthe99%OTUlevel(rangingfrom75%coverageintheCanalMineralsampleto96%in theOld StreamMineral sample) and94%at the97%OTU level(83%fortheCanalMineralsampleto98%fortheOldStreamsample).

Mostofthetaxonomicandabundancevariationwasrelatedtolo-cationalong theoutflowand therefore the localenvironmental andredoxconditionsandconsequentmetabolicopportunities (Figures3and5).Thecommunitycompositionofthewatersamplesallappearedrelativelysimilartoeachother,with lowdissimilaritybetweenwatersamplesandmineralsamplesfromthesourcepool.Downstream,min-eralsamplesappeardissimilarbothfromwatersamplesandfromeachother(Figure5).

Relativeabundancesofmicrobialtaxaasrevealedby16Ssurveyscanbeuseful forpredictingmetabolismsdrivinggeochemicalcyclesand producing themineral deposits observed at OHK (Table3 andFigure3).Inparticular,thecontributionsofvariousironoxidizersandphototrophstoprimaryproductivityalongthespringpath (Figure3)can be estimated due to these metabolisms being fairly well con-servedwithinbacterialtaxa(e.g.,Chanetal.,2016;Emerson,Fleming,&McBeth,2010).AnalysisofthemostabundanttaxaatOHKrevealedsignificant roles fororganismsassociatedwithdiverse ironmetabo-lisms, including aerobic and phototrophic iron oxidation, aswell astraceironreducers.Whiletherelativecontributionofabioticironoxi-dationatOHKisnotconstrained,sequencedatasuggestthataerobiciron-oxidizingbacteriaarethedominantbiologicaldriversofironoxi-dationinthehotspring.Alsopresentwerediversephototrophsasso-ciatedwithbothoxygenicandanoxygenicphototrophicclades;these

F IGURE  4 Electronmicroscopyimagingofprecipitatesfromsourcepoolwall.Back-scatteredelectronimagescollectedbySEMofthinsectionsamplesofsolidscollectedfromthetravertinewallofthesourcepool(Figure2d).Thissampleshowsalternatinglaminationsofaragonite-richzonesandironoxide-richzones.(a)Overviewimageillustratesalternationofcarbonate-andironoxide-richlayers.(b)Enlargedimageofironoxide-richzoneof(a),showingaggregationofironoxidenanoparticlesintoadendritictexture.(c)ImageofdendriticironoxideaggregatesfollowingremovalofcarbonatephasesviatreatmentwithHCl,revealingsheath-liketubularandamorphousparticulateironoxidemorphotypes

(a)

(b)

(c)

824  |     WARD et Al.

organismswereobservedatonly traceabundance inmostsamples,butanoxygenicphototrophswereenriched inbiofilmsfromtheOldStream,therimoftheSourcePool,whileCyanobacteriawereabun-dantintheBubblePool.AsmallbutdiverseassortmentofmicrobesassociatedwithnitrogencyclingwaspresentatOHK,despitelowcon-centrationsofnitrogencompoundsinthespringwater(AppendixS1).

3.3 | Aerobic iron- oxidizing bacteria

AmongthemostabundanttaxaatOHKaremembersofthebetapro-teobacterialfamilyGallionellaceae,mostlikelymembersorcloserela-tivesoftheaerobiciron-oxidizinggeneraGallionella or Sideroxydans.

Gallionella and Sideroxydans are neutrophilic iron oxidizers that usemolecularoxygentooxidizedissolvedFe(II)toFe(III)oxideswhilecon-servingenergyforgrowthandautotrophiccarbonfixation(Emerson&Moyer,1997;Emersonetal.,2013;Kucera&Wolfe,1957);theyarecommonlyfoundinterrestrialiron-richsystems(Emersonetal.,2010).Aerobic iron-oxidizing bacteria have characteristically low growthyields (Neubauer, Emerson,&Megonigal, 2002) due to themodestpotentialsofFe(II)/Fe(III)redoxcouplesandresultingrequirementforreverseelectrontransfertoachievethesufficientlylowpotentialelec-tronsneededforcarbonfixation(Bird,Bonnefoy,&Newman,2011).

Gallionellaceaeareabundantinboththemineralandwaterfrac-tionsoftheDeepSource(37%and18%,respectively),butotherwiseappeartobedominantlyassociatedwiththewaterfractionofothersamples(28%averageinwatersamples,8.5%averageinmineralsam-ples) (Table3).Thismay indicateapreferenceof theGallionellaceaeat OHK for a planktonic over surface-attached modes of growth,althoughthis is impossible tostatewithcertainty in theabsenceofcellquantificationbetweenthesesampletypesanddeterminationofdifferencesinabsoluteratherthanrelativeabundance.Basedonthesequencedata,membersoftheGallionellaceaeappeartobethefirstironoxidizersandprimaryproducers toacton theupwelling springwaterasitmixeswithatmosphericO

2,drivingthebulkofearlybio-

logical ironoxidationatOHKandproducingmuchofthe ironoxidesedimentthatistransportedalongthespringoutflow.Gallionellaceaewere fairly diverse, including169OTUs at the97% identity cutoff;however, the twomost abundantOTUswereboth~97% similar toSideroxydans lithotrophicusES-1andrepresentedmorethan92%ofthetotalGallionellaceaesequencesatOHK.TheabundanceofthesetwoOTUsdrivestheoveralltrendinironoxidizerabundance(Figure3).

Also present, albeit at lower abundance (up to ~1.5% relativeabundance) (Table3),aremembersof thezetaproteobacterial familyMariprofundaceae,anothergroupofneutrophilicironoxidizers.Iron-oxidizing Zetaproteobacteria are more commonly found in marinesettings,particularlyindeepoceanbasinsassociatedwithhydrother-malironsources(Emersonetal.,2010).Despiteasimilarphysiology,MariprofundaceaearenotcloselyrelatedtoGallionellaceaeorotheraerobic iron oxidizers, instead forming a distinct class within theProteobacteria(Emersonetal.,2007).Zetaproteobacteriahaveprevi-ouslybeenidentifiedinrelativelysalineterrestrialiron-andCO

2-rich

systems(e.g.,Emerson,Thomas,Alvarez,&Banfield,2016),sometimesco-occurringwithGallionella(Crosseyetal.,2016);thediscoveryoftheT

ABLE 2 DiversitymetricsofOHKsequencing.Diversitymetricscalculatedforboth99%and97%sequenceidentitycutoffsforassigningOTUs

Dee

p So

urce

M

ine

ra

l

Dee

p So

urce

W

ate

r

Shal

low

Sou

rce

Min

era

l

Shal

low

So

urce

Wat

erO

ld S

trea

m

Min

era

l

Old

Str

eam

W

ate

r

Ca

na

l

Min

era

lC

an

al

Wa

te

r

Bubb

le P

ool

Min

era

l

Bubb

le P

ool

Wa

te

r

Re

ad

s6,456

20,067

6,512

25,306

27,454

19,052

2,176

17,893

3,608

12,601

ObservedOTUs(99%)

906

1,928

1,045

2,450

1,775

1,842

74

02,455

85

71,595

Goodcoverage(99%)

0.9035

0.9388

0.8948

0.941

0.9615

0.9423

0.7

46

80.9076

0.8

28

70.911

Shannonindex(99%)

5.9544

6.0339

7.6849

6.4

78

56

.83

02

6.3

40

38

.11

36

.84

82

7.2

74

75.896

InverseSimpson(99%)

9.4959

12.9196

62

.63

37

11

.50

82

37

.01

68

11.4379

10

5.3

15

31

2.7

88

13

2.6

02

58

.64

75

ObservedOTUs(97%)

43

1895

57

21,059

74

7869

490

1,281

55

5910

Goodscoverage(97%)

0.9588

0.9737

0.9459

0.9768

0.9834

0.9738

0.8359

0.9529

0.8911

0.951

Shannonindex(97%)

4.7

60

34.6789

6.4739

5.0

55

45

.57

85

5.0959

7.0

60

35

.47

67

6.1

73

64

.57

11

InverseSimpson(97%)

6.5

62

38.4309

33

.13

31

7.5

28

82

2.6

65

87

.70

54

55

.62

31

8.1

76

31

8.5

50

45

.56

7

     |  825WARD et Al.

co-occurrenceof theseorganisms atOHKprovides further supporttotheoverlappingecologicalnichesoftheseclassically“marine”and“terrestrial” iron-oxidizingbacteria.Thismayberelatedtothe inter-mediatesalinityofOHKwater(~38mmCl−,~6.5mmSO

4

2−),providinganenvironmentconducivetoorganismsadaptedtobothfreshwaterandsaltwater,resultinginthemixedpopulationofGallionellaceaeandMariprofundaceae.

MembersoftheGallionellaceaearetypicallyassociatedwithcoldiron-oxidizingenvironments,andnothotsprings(Hallbeck&Pedersen,2013);whilemembersoftheMariprofundaceaehavebeenobservedtohaveanuppergrowthtemperatureof30°C(Emersonetal.,2010).OHK,withsourcewatertemperatures~44°Cmaythereforesupportuniquethermotolerantstrainsofthesebacteria.

Members of the family Comamonadaceae were also fairlyabundant (~1%–12%) in OHK samples (Table3). This family ofBetaproteobacteria includes members such as Acidovorax ebreus, a

nitrate-reducing anaerobic iron oxidizer (Byrne-Bailey etal., 2010),aswell as iron reducers such asRhodoferax ferrireducens (Finneran,Johnsen,&Lovley,2003)andtheiron-oxidizingbacteriumLeptothrix

(vanVeen,Mulder,&Deinema,1978).However,thetaxonomicaffin-ityoftheComamonadaceaeatOHKisinsufficientlyresolvedtocon-fidently assess the contribution of this group to iron cycling in thisenvironment.

Electronmicroscopyofmineralprecipitatesfromthesourcepoolandcanalrevealedalternatinglaminationsofaragonite-richandironoxide-richmaterial;imagingofironoxidebandsfollowingdissolutionofcarbonateswithHClrevealedthatironoxidesweremadeupofamixture of amorphous and sheath-like tubular structures (Figure4).While iron oxide sheaths are typically associatedwith the betapro-teobacterial iron-oxidizing genus Leptothrix (Emerson etal., 2010),they can alsobeproducedbydiverse ironoxidizers including somestrainsofZetaproteobacteria(Flemingetal.,2013).Althoughweten-tatively regard thesemineralized filaments as biological in origin, it

isunclearwhatorganismsareresponsiblefortheproductionof ironoxide sheaths observed at OHK, particularly as structures appearhighly mineralized, potentially reflecting encrustation of biogeniciron oxides by subsequent abiotic precipitation. Sub-micrometer,amorphous particulate iron oxides are characteristic of iron oxida-tion by Sideroxydans(Emerson&Moyer,1997),andsotheprevalenceof this iron oxide morphology is consistent with this genus of theGallionellaceaebeingmajorcontributorstoironoxidationatOHK.Theabundance of amorphous iron oxide particles also supports the as-signmentofGallionellaceaeOTUsatOHKtoSideroxydansratherthanstalk-formingGallionella(Emersonetal.,2010).However,asdiscussedabove,asignificantportionofironoxidationatOHKlikelyproceedsabioticallyandmaycontributesimilarmorphologiesofamorphousironoxideparticles,sothepresenceoftheseformsisconsistentwith,butnotnecessarilydiagnosticof,theactionofSideroxydans and may in-steadreflectthecombinedactionoftheseorganismswithabioticironoxidation.

3.4 | Cyanobacteria

Cyanobacteria were abundant in the Bubble Pool Water sam-ple,where theymadeup~37%of all sequence reads, butwere ofmuch lower abundance in samples collectedupstream (Table3 andFigure3).AlthoughCyanobacteria are sometimesunderrepresentediniTagdatasetsasaresultofpoorDNAyieldoramplificationbiases(e.g.,Parada,Needham,&Fuhrman,2015;Trembath-Reichertetal.,2016), thepaucityofCyanobacteria inupstreamOHKsampleswasconfirmed by epifluorescencemicroscopy, inwhich cells displayingcyanobacterial autofluorescence were observed abundantly in sam-ples from thedownstreamBubblePool but not in the SourcePool(Fig.S2).

It has been demonstrated that in iron-rich systems whereCyanobacteriaareabundantandproductive,only~1%ofO

2 released

F IGURE  5 MultidimensionalscalinganalysisofOHKsamples.Eachpointrepresentstherecoveredmicrobialcommunityfromagivensample,withsitesidentifiedbycolorandsampletypebyshape.Pointsclosetoeachotherinthistwo-dimensionalspaceshareasimilarcommunitycomposition.Relativeabundance data were transformed by

the4throottodown-weighttheeffectofabundanttaxainthesamples.Watersamplesclustertogether,andwithSourcepoolmineralsamples,whiletheBubblePoolWaterandtheotherMineralsamplesstandoutalongdifferentcurvesinthisspace.Stressvalueis0.0465[Colourfigurecan be viewed at wileyonlinelibrary.com]

Water

Mineral

Bubble Pool

Canal

Deep Source

Old Stream

Shallow Source

826  |     WARD et Al.TABLE 3 RelativeabundanceoftaxatotheFamilylevel.Overall,10mostabundanttaxalisted,aswellasothertaxaofinterestmentionedinthetextandSI,orderedbyrelativeabundance

averagedacrossallsamples.Taxacolorcodedbyputativemetabolism:redforaerobicironoxidizers,purpleforanoxygenicphototrophs,greenforoxygenicphototrophs,bluefornitrifiers,and

yellowfortaxawithmembersperformingadiverserangeofmetabolismsthatcannotberesolvedwithtaxonomicresolutionofavailabledata[Colourtablecanbeviewedatwileyonlinelibrary.com]

Taxo

nD

eep

Sour

ce

Min

era

l (%

)

Dee

p So

urce

W

ate

r (

%)

Shal

low

So

urce

M

ine

ra

l (%

)

Shal

low

So

urce

W

ate

r (

%)

Old

Str

eam

M

ine

ra

l (%

)

Old

Str

eam

W

ate

r (

%)

Ca

na

l

Min

era

l (%

)

Ca

na

l W

ate

r

(%)

Bubb

le P

ool

Min

era

l (%

)

Bubb

le P

ool

Wa

te

r (

%)

Ave

rage

(%)

Bacteria;__

Proteobacteria;__

Betaproteobacteria;__

Nitrosomonadales;__

Gallio

ne

llace

ae

36.94

18

.23

1.09

34

.05

3.2

73

3.5

70

.46

31

.77

0.4

42

3.5

81

8.3

4

Bacteria;__

Proteobacteria;__

Zetaproteobacteria;__

Mariprofundales;__

Mariprofundaceae

0.6

51

.32

0.0

50

.52

1.5

10

.52

1.0

10

.58

0.1

70

.17

0.6

5

Bacteria;__

Cyanobacteria;__

Cyanobacteria;__

SubsectionIII;__FamilyI

0.1

50

.24

2.5

04

.46

0.2

11

.41

0.4

62

.30

0.3

33

7.4

54.95

Bacteria;__

Cyanobacteria;__

Cyanobacteria;__

SubsectionV;__FamilyI

0.0

80

.11

0.09

5.1

60

.01

0.3

10

.00

1.29

0.0

60

.20

0.7

3

Bacteria;__

Proteobacteria;__

Alphaproteobacteria;__

Rhodospirillales;Other

0.09

0.0

56

.68

0.0

42

.57

0.1

70.09

0.1

30.19

0.0

31

.00

Bacteria;__

Proteobacteria;__

Betaproteobacteria;__

Rhodocyclales;__

Rhodocyclaceae

0.94

0.5

50

.15

0.4

22

.61

3.3

10

.00

0.99

0.5

30

.38

0.99

Bacteria;__

Proteobacteria;__

Alphaproteobacteria;__

Rhodobacterales;__

Rhodobacteraceae

0.1

10

.34

1.8

00

.47

2.8

50

.26

0.7

40

.87

0.2

50

.14

0.7

8

(Continues)

     |  827WARD et Al.

Taxo

nD

eep

Sour

ce

Min

era

l (%

)

Dee

p So

urce

W

ate

r (

%)

Shal

low

So

urce

M

ine

ra

l (%

)

Shal

low

So

urce

W

ate

r (

%)

Old

Str

eam

M

ine

ra

l (%

)

Old

Str

eam

W

ate

r (

%)

Ca

na

l

Min

era

l (%

)

Ca

na

l W

ate

r

(%)

Bubb

le P

ool

Min

era

l (%

)

Bubb

le P

ool

Wa

te

r (

%)

Ave

rage

(%)

Bacteria;__

Proteobacteria;__

Alphaproteobacteria;__

Rhodospirillales;__

Rhodospirillaceae

0.0

30

.01

3.1

30

.04

0.0

70

.06

0.09

0.3

00.19

0.29

0.4

2

Bacteria;__Chloroflexi;__

Chloroflexia;__

Chloroflexales;__

Chloroflexaceae

0.0

00

.00

0.0

50

.02

0.0

00

.00

1.19

0.2

70

.83

0.1

20

.25

Bacteria;__Chlorobi;__

Chlorobia;__

Chlorobiales;__OPB56

0.09

0.2

10

.06

0.0

30

.17

0.3

00

.14

0.1

70

.03

0.0

20

.12

Bacteria;__

Proteobacteria;__

Gammaproteobacteria;__

Chromatiales;__

Chromatiaceae

0.4

20

.00

0.0

00

.04

0.0

00

.18

0.0

00

.01

0.0

30

.21

0.09

Bacteria;__

Proteobacteria;__

Gammaproteobacteria;__

Xanthomonadales;__

Xanthomonadaceae

0.2

20

.61

3.0

60

.41

10

.01

0.3

09.33

1.7

024.89

1.39

5.19

Bacteria;__Chloroflexi;__

Anaerolineae;__

Anaerolineales;__

Anaerolineaceae

7.1

31

0.7

51

0.1

03

.52

2.3

20.91

1.4

73

.67

2.2

20

.60

4.2

7

Bacteria;__

Proteobacteria;__

Betaproteobacteria;__

Burkholderiales;__

Comamonadaceae

1.5

01

.88

2.0

32

.35

12

.12

1.5

11

.42

4.1

52

.52

4.3

83.39

Bacteria;__Nitrospirae;__

Nitrospira;__

Nitrospirales;__

Nitrospiraceae

0.39

1.19

0.2

55

.07

4.6

51

2.8

10

.18

3.8

20

.03

2.1

03

.05

Archaea;__

Thaumarchaeota;__

Marine_Group_I;__o;__f

0.99

0.2

30

.00

0.09

0.0

10.98

0.0

00

.44

0.0

00

.04

0.2

8

TABLE 3 (Continued)

(Continues)

828  |     WARD et Al.

Taxo

nD

eep

Sour

ce

Min

era

l (%

)

Dee

p So

urce

W

ate

r (

%)

Shal

low

So

urce

M

ine

ra

l (%

)

Shal

low

So

urce

W

ate

r (

%)

Old

Str

eam

M

ine

ra

l (%

)

Old

Str

eam

W

ate

r (

%)

Ca

na

l

Min

era

l (%

)

Ca

na

l W

ate

r

(%)

Bubb

le P

ool

Min

era

l (%

)

Bubb

le P

ool

Wa

te

r (

%)

Ave

rage

(%)

Bacteria;__Chlorobi;__

Ignavibacteria;__

Ignavibacteriales;Other

6.7

23

4.2

53

.67

5.3

812.95

2.2

30

.37

11

.85

4.49

1.6

68

.36

Bacteria;__

Proteobacteria;__

Deltaproteobacteria;__

Bdellovibrionales;__

Bacteriovoracaceae

2.29

0.97

0.2

14

.31

0.2

11

1.3

80

.14

4.2

10

.08

3.7

52

.76

Bacteria;__Candidate_divi-

sion_OP11;__c;__o;__f

0.1

51

.73

0.1

40

.46

2.09

0.4

26

.34

0.2

51

1.0

30

.06

2.2

7

Bacteria;__

Bacteroidetes;__

Sphingobacteriia;__

Sphingobacteriales;__env.

OPS_17

4.2

03

.28

0.2

57.09

0.1

40

.68

1.2

43

.85

1.1

40

.64

2.2

5

Bacteria;__

Proteobacteria;__

Gammaproteobacteria;__

Enterobacteriales;__

En

tero

bacte

riace

ae

3.4

41

.01

3.49

1.1

60.69

1.4

74

.18

0.39

2.19

0.5

31

.86

Bacteria;__

Proteobacteria;__

Deltaproteobacteria;__

Desulfuromonadales;__

Ge

ob

acte

race

ae

0.0

20.79

0.0

00

.03

0.1

40

.03

0.0

50

.06

0.0

00

.14

0.1

3

Bacteria;__

Cyanobacteria;__

ML635J-21;__o;__f

0.0

30

.06

0.0

20

.06

0.0

00

.34

0.0

00

.02

0.0

00

.23

0.0

8

Bacteria;__

Cyanobacteria;__SHA-

109;__o;__f

0.0

00

.00

0.1

00

.00

0.90

0.0

00

.00

0.0

00

.00

0.0

00

.10

TABLE 3 (Continued)

     |  829WARD et Al.

oxidizesferrousiron,withtheremainderescapingtotheatmosphere(Rantamäki etal., 2016). Thus, given the inefficiency of cyanobac-terial oxygen fluxes for oxidizing dissolved iron, and the scarcity ofCyanobacteriaupstreamatOHKwhereironoxidationistakingplace,Cyanobacteriadonotappeartobemajorcontributorstoironoxida-tionatOHK.

CyanobacteriapresentarepredominantlymembersofSubsectionIII,FamilyI.ThisgroupincludesLeptolyngbya,agenusoffilamentousnon-heterocystousCyanobacteriathathasappearedinclonelibrariesfromOHK(Takashimaetal.,2011)andiscommoninotherhotspringsofsimilartemperatures(e.g.,Bosaketal.,2012;Roeselersetal.,2007).

Members of deeply branching non-phototrophic CyanobacteriacladesareaminorbutnotablecomponentofOHKsamples(upto0.9%abundance).While theCyanobacteriaphylumhas traditionallybeenconsideredtoexclusivelycontainoxygenicphototrophs,severaldeep-branching non-phototrophic clades have recently been describedwithintheCyanobacteriaphylum, includingMelainabacteria,asistergroup tooxygenicCyanobacteria (i.e.,Oxyphotobacteria), aswell asdeeper-branchingclades(DiRienzietal.,2013;Johnsonetal.,2013b;Leyetal.,2005;Soo,Woodcroft,Parks,Tyson,&Hugenholtz,2015;Soo etal., 2014).These deep-branching Cyanobacteria—particularlythecladesSHA-109andML635J-21,whichbranchbasaltoallotherCyanobacteria—are thought to be ancestrally non-phototrophic andcanhelptobetterconstraintheevolutionaryhistoryofCyanobacteriaandthereforeoxygenicphotosynthesis(e.g.,Fischer,Hemp,&Johnson,2016;Shih,Hemp,Ward,Matzke,&Fischer,2017;Soo,Hemp,Parks,Fischer,&Hugenholtz,2017).Thesecladesarefoundathigherabun-danceatOHKthanmostotherenvironments,andOHKcouldprovidea valuable resource for investigatingmembers of this understudiedgroupviametagenomicsequencing,incubations,orisolation.

3.5 | Anoxygenic phototrophs and relatives

Membersofseveraltaxamadeupoforcontaininganoxygenicphoto-trophswerepresentatlowabundanceinOHKsamples(Table3).Theseinclude the Rhodospirillales, Rhodobacteraceae, Rhodocyclaceae,Chloroflexaceae, Chlorobiales, and Chromatiaceae. Some of thesetaxa (e.g.,Chloroflexaceae,Chlorobiales)aremadeupalmostexclu-sivelyofphototrophs,whileothers(e.g.,Rhodobacteraceae)containmemberswithawidediversityofmetabolisms,onlysomeofwhichare phototrophic (Fischer etal., 2016; Overmann & Garcia-Pichel,2013).Shotgunmetagenomicorculture-basedanalysiswillbeneces-sarytoconfirmwhetherthemembersofthesetaxapresentatOHKarephototrophic.Althoughthesetaxaindividuallyrepresentnomorethanafewpercentofthesequencereadsatanygivensite(overallav-erage~0.88%ofreadspertaxon),thispopulationisquitediverseandinsumrepresentsasizablefractionofthetotalmicrobialcommunity(3%–20%)(Figure3).Putativeanoxygenicphototrophsaremostabun-dant intheShallowSourceandOldStreamMineralsamples(13.8%and20.4%oftotalabundance,respectively),droppingto~3%intheDeepSourcesample.EveninthedownstreamBubblePoolsamples,anoxygenicphototrophsmakeup~5%ofthetotalabundanceofse-quences. Sequences associated with anoxygenic phototrophs were

more abundant inmineral samples than inwater samples (~9% vs.~5%),thoughasdiscussedaboveinthecaseofGallionellaceae, it isunclearwhetherabsoluteabundancescaleswithrelativeabundanceandthereforewhethertheseorganismsgrowpreferentiallyattachedtosolidsurfacesratherthanplanktonically.

All samples contained relatively abundant sequence reads be-longingtotheChlorobiphylum(Table3).TheChlorobiareclassicallyknown as the Green Sulfur Bacteria due to the anaerobic sulfur-oxidizing anoxygenic phototrophic lifestyle of its earliest describedmembers (Bryant & Liu, 2013; Davenport, Ussery, & Tümmler,2010). This includes iron-oxidizing anoxygenic phototrophs such asChlorobium ferrooxidans (Heising, Richter, Ludwig, & Schink, 1999),which employ ametabolism thought to be relevant toArcheanBIFdeposition(e.g.,Kappleretal.,2005).Duetoincreasedenvironmentalsequencingandnewisolationefforts,however,theChlorobiphylumis nowknown to also contain aerobic photoheterotrophs (Liu,Klattetal., 2012; Stamps, Corsetti, Spear, & Stevenson, 2014) and non-phototrophs(Podosokorskaya,Kadnikovetal.,2013).ThemajorityofChlorobisequencesfoundinOHKappeartofallwithintheChlorobiorderIgnavibacteria,abasalcladeofChlorobiwhoseknownmembersincludeversatileheterotrophicmetabolismsbutnoknownphototro-phypathways(Iinoetal.,2010;Liu,Frigaardetal.,2012).ItthereforeappearsthatphotoferrotrophybyChlorobi isnotdriving ironoxida-tionatOHK,althoughmetagenomicsequencingandassemblyofOHKChlorobi genomeswill be necessary to confirm that phototrophy isnotpresent in theseorganisms. Ignavibacteria appear tobe a com-moncomponentofhotspringmicrobialcommunities:theseorganismswerefirstisolatedfromaJapanesehotspring(Iinoetal.,2010)andarefoundathighabundanceinChocolatePotshotspringsinYellowstoneNationalPark(Fortneyetal.,2016).

The Ignavibacteria found atOHKhadonly low similarity to de-scribedstrains,withmorethan50%of Ignavibacteriareads (primar-ily from the Deep Source and CanalWater samples) from anOTU~91%similartoMelioribacter roseusP3M,amoderatelythermophilicfacultative anaerobe (Kadnikov etal., 2013). Approximately 20% ofIgnavibacteriareads(primarilyfromtheDeepSourceWaterandOldStreamMineralsamples)were93%similartoIgnavibacteria albumJCM16511.

Members of the bacterial phylum Chloroflexi were remarkablyabundant inOHKsamples (Table3).TheChloroflexiwereclassicallydescribedasthegreennon-sulfurbacteriaduetotheanoxygenicpho-totrophicmetabolismoftheirearliestdescribedmembers(Overmann,2008),butitisnowrecognizedthatthephylumismuchmoregeneti-callyandmetabolicallydiverse(Yamada&Sekiguchi,2009).Metaboliccharacters in the Chloroflexi largely follow class-level taxonomicpatternsbutwithanumberofnotableexceptions,suchas thenon-phototrophicpredatoryHerpetosiphonwithinthepredominantlypho-totrophic Chloroflexia class (Kiss etal., 2011;Ward,Hemp, Pace,&Fischer,2015b).ThemostabundantChloroflexiatOHKbelongtotheclassAnaerolineae,whichwereabundantinallsamples(upto~11%).TheAnaerolineaehavegenerallybeen isolatedasobligatelyanaero-bicheterotrophs(e.g.,Sekiguchietal.,2003;Yamadaetal.,2006),butgenomesequencinghasrevealedthecapacityforaerobicrespiration

830  |     WARD et Al.

indiversemembersofthisclade(e.g.,Hemp,Ward,Pace,&Fischer,2015a, 2015b; Pace, Hemp, Ward, & Fischer, 2015; Ward, Hemp,Pace, & Fischer, 2015a). Furthermore, a genome for an organismcloselyrelatedtotheAnaerolineaewithgenesforphotosynthesishasbeenassembledfromaYellowstoneNationalParkmetagenome(Klattetal.,2011;Tank,Thiel,Ward,&Bryant,2017;Thieletal.,2016).Itisthereforeunclearwhatmetabolismsmaybepresent inAnaerolineaeatOHK,andisolationormetagenomicsequencingoftheseorganismswillbenecessarytodeterminewhatroletheymaybeplayinginthisenvironment.TheAnaerolineaeatOHKwereverydiverse, including480OTUsatthe97%cutoff.ThisincludedadistinctpopulationattheDeepSource (bothWaterandMineralSamples), theShallowSourceMineralsample,andmoredownstreamsamples.Thethreemostabun-dantAnaerolineaeOTUsweremostcloselyrelatedtoThermomarilinea lacunifontana (84%–88% similarity); T. lacunifontana is an anaerobic

heterotroph isolated from a shallow hydrothermal system in Japan(Nunouraetal.,2013).ThemostabundantAnaerolineaeOTUintheShallowSourceWatersample(makingup~7%ofallAnaerolineaeatOHK)was89% identical toOrnatilinea apprima.Themost abundantOTUintheOldStreamMineralsamplewas91%identicaltoLongilinea arvoryzae. Both O. apprima and L. arvoryzae are described as obli-gatelyanaerobicfermenterscapableofdegradingsugarsandproteins(Podosokorskaya, Bonch-Osmolovskaya etal., 2013; Yamada etal.,2007).

3.6 | Low biomass yield of the OHK microbial community

OHKisauniqueecosystemsupportingnovelmicrobialcommunitiesaswellasservingasanintriguingprocessanalogforPrecambrianbandedironformationdeposits.Futureactivitymeasurementsofcommunitymembers,forexample,bymetagenomicsandstableisotopeprobingwillbenecessarytofurtherdefinemicrobialactivitiesinthissystem.Basedonmicroscopyand16Samplicondata,themicrobialcommuni-tiesatOHKappeartobesupportedprimarilybyaerobicironoxida-tionoccurring in andnear the sourcepool.At theOldStream site,mineral-attached anoxygenic phototrophs becomemore significant,whileCyanobacteriabecomeabundantonlyinthemostdownstreamsamples (Figure3).Thispredominanceof lithoautotrophsoverpho-totrophsisrareattheearth’ssurfacetodayandprovidesacontrasttoothermodernBIFanalogsites.Forinstance,inLakeMatano,ironoxidationisthoughttobedrivenlargelybyphotoferrotrophs(Croweetal., 2008). AtChocolate Pots hot spring in YellowstoneNationalPark—perhaps themost geochemically similar system toOHK thathasbeenextensivelystudied—ironoxidationisthoughttobeprimar-ily driven abiotically by O

2 produced in situbycyanobacterialmats

(Trouwborst etal., 2007). Furthermore, relative to Chocolate Pots,OHKsupportsverylittleinthewayofwell-developedmicrobialmats,withonlythin,patchybiofilms.

TheabsenceofsubstantialmicrobialbiomassaccumulationnearthesourcepoolatOHKcanbeconsideredtheresultoftwoseparatephenomena:(i)thepaucityofphotosyntheticCyanobacteriaand(ii)thepoorgrowthyieldsoftheiron-oxidizingbacteriathatdooccur.Neither

oftheseissuesisfullyresolved,buthypothesesfortheircausescanbemadebasedonthegeochemistryandmineralogyofOHKandrelatedsystems,aswellasaspectsofthephysiologiesdrivingironoxidation.

It has been proposed that high iron concentrations are toxic toCyanobacteria and that thismayhaveplayed a role indelaying theoxygenation of the Archean atmosphere (Swanner etal., 2015).In principle, ferrous iron toxicity may help explain the absence ofCyanobacteria in OHK until the most downstream samples, wheremostironhasalreadybeenoxidizedandprecipitated.TheabsenceofCyanobacteriaatOHKischallengingtoexplain,however,asotheriron-richsystems(e.g.,ChocolatePotsHotSpring,Trouwborstetal.,2007)supportproductivecyanobacterialpopulations,andinothersystems,oxygenicphotosynthesisandaerobicironoxidationhavebeenshownto co-occur (Hegler, Lösekann-Behrens, Hanselmann, Behrens, &Kappler,2012;Morietal.,2015).AtFuschnaSpringintheSwissAlps,Gallionella-dominatedcommunitiesoccurintheiron-rich,low-oxygen,high-flowconditionswithintheflowchannel,butCyanobacteria-andiron reducer-richmicrobialmats accumulate along the edgeswhereflowislesspronounced(Hegleretal.,2012).Thissuggeststhatflowregimemayalsoplayarole indeterminingthemicrobialcommunityofiron-richsystems;OHKexperienceshighflowrates,andturbulentmixinginthesourcepool,andthismayplayaroleinlimitingthede-velopmentofphototrophicmicrobialmats.Turbulencemayinhibitthedevelopment of phototrophic microbial mats, while simultaneouslybeingadvantageoustoaerobiciron-oxidizingbacteriabyhelpingthemshed accumulated iron oxides, limiting encrustation by their meta-bolic byproducts.However, futureworkwill be necessary to betterdeterminetheroleoffluidflowregimeandotherfactorsininhibitingCyanobacteriaatOHK.

Areasonablehypothesisforthepoordevelopmentofbiofilmsbyaerobic iron-oxidizing bacteria atOHK could be related to the lowgrowthyieldofaerobic iron-oxidizingmicrobesrelativetophototro-phs. Based on electron balance and assuming an average oxidationstateofzerofororganiccarbon,themaximumpossibleefficiencyofautotrophicironoxidationis1moleofCO

2fixedforevery4molesof

Fe(II)oxidized.However,measuredyieldsofaerobicironoxidizersaretypicallymuch lower,ontheorderof1moleofCO

2 fixedforevery

40molesofFe(II)oxidized (Neubaueretal.,2002).Yieldsforphoto-ferrotrophsappeartobemuchmoreefficient,approximatingtheidealstoichiometryof4Fe:1C (Ehrenreich&Widdel,1994).Whileelec-tron transfer inneutrophilic ironoxidizershasnotbeenextensivelycharacterized,thisdifferenceinyieldappearstofundamentallycomedowntotheredoxpotentialofironoxidationreactions,which(whilequitevariabledependingonenvironmentalpHandmineralogyofironoxides)aretooelectrochemicallypositivetodirectlyreduceNAD(P)+

andthereforebeusefulforcarbonfixation(Birdetal.,2011).Inordertogrow, theseorganismsmustconsumeprotonmotiveforce (PMF)torunelectrons“uphill”tolowerredoxpotentialsinordertogeneratetheNAD(P)HneededtoreduceCO

2(Birdetal.,2011).Inaerobiciron

oxidizers,thisrequireslargefluxesofironoxidationtomaintainsuffi-cientPMFtofixcarbon,whilephototrophicironoxidizerscanrelyoncyclicelectronflowthroughtheirreactioncenterstobuildPMFsuffi-cientlytoallowstoichiometricironoxidationandcarbonfixation.The

     |  831WARD et Al.

relativelypoorgrowthyieldofaerobiciron-oxidizingbacteriaatOHKresults in organic carbon-lean mineral precipitates (<0.02% organiccarbonbyweight),incontrasttophototroph-dominatediron-richsys-tems likeChocolatePotshot springwhereorganic carboncontentscanbeinexcessof1%organiccarbonbyweight(Parenteau&Cady,2010).Ultimately,itislikelythattheoverproductionofferricironrel-ativetocarbonfixationduringaerobicironoxidationsetsthebudgetsforsubsequentcarbonandironcyclinginthisenvironment.

Significantly, 16S sequence reads associated with environ-mentally common iron-reducing microbes (e.g., Shewanellaceae,Geobacteraceae) occur at only very low abundances at OHK,with a maximum abundance of the Deltaproteobacteria familyGeobacteraceaeof0.79%intheDeepSourceWatersample(Table3).Thisisincontrasttootheriron-rich,neutralpHsystemsincludinghotandcoldspringsandgroundwaterseeps(e.g.,Blöthe&Roden,2009;Fortneyetal.,2016;Hegleretal.,2012;Rodenetal.,2012).Thismaypartiallyreflecttherelativefluxesofironoxidationversusorganiccar-bonfixationatOHK;thatis,thereisinsufficientorganiccarbonbeingfixed in this environment to fuel substantial iron oxide respiration.Meanwhile,turbulentmixingensuresthatoxygen-poormicroenviron-mentsdonotdevelop.Asmolecularoxygenisavailableinsubstantialexcessoforganiccarbon,heterotrophyatOHKneverdepletesO

2 suf-

ficientlytomakeironoxidesafavorableelectronacceptor.Incontrast,insystemswithmoresubstantiallydevelopedmicrobialmatsor lessefficientmixing,oxygencanbecomedepleteddeeperinmatfabricsorindiffusion-limitedboundarylayers,drivinglocalanoxiaandtheshifttowardironrespiration.ThelackofsubstantialironreductionatOHKisconsistentwiththepredominanceofferricironmineralsintheOHKdeposits(Takashimaetal.,2011)(Figure3).

4  | CONCLUSIONS

The relative paucity of organic carbon, the dominantly ferric ironcontentofsedimentary laminations,andtheprimaryroleofaerobicironoxidationtogethermakeOHKmostsimilartoProterozoic-typeBIFs,depositedaftertheGOE.BIFcompositionvariesthroughtime,with BIFs of different ages likely forming via different processes.Thisvariabilityislikelydrivenbychangesinprimaryproductivityandremineralizationpathwayscausedbytheevolutionofoxygenicpho-tosynthesisinCyanobacteriaandthesubsequentoxygenationoftheatmosphere at theGOE ca. 2.3Ga.While substantial debate existsabouttheantiquityofCyanobacteria,multiplelinesofevidencesug-gest that oxygenic photosynthesis evolved only shortly before theGOE (Fischer etal., 2016; Johnson etal., 2013a; Shih etal., 2017;Ward,Kirschvink,&Fischer,2016),andtherefore,molecularoxygenderivedfromphotosynthesiswasunlikelytoplayaroleinArcheanBIFformation.ThehypothesisthatthedepositionoftheseBIFsoccurredviaphototrophicironoxidationisconsistentwiththepredominantlyferrous composition of Archean and early Paleoproterozoic ironformations (Fischer&Knoll,2009) (AppendixS1), and isdiscordantwithmechanismsrelyingonaerobicironoxidation;photoferrotrophyresults in stoichiometric amounts of iron oxide and organic carbon

delivered to sediments,whichpromotes an environment conduciveto substantial amounts of iron reduction during burial and diagenesis

(Johnson,Beard,Klein,Beukes,&Roden,2008;Konhauser,Newman,& Kappler, 2005; Li, Konhauser, Kappler, & Hao, 2013). After theGOE,molecularoxygenwassufficientlyabundantintheatmosphereEarthsurfaceenvironmentsthatitcouldbeusedtodriveaerobicironoxidation.Additionally,ithasbeenobservedthattheorganiccarboncontentofBIFtendstobeinverselycorrelatedtotheproportionofresidualferriciron,withthemostironoxide-dominatedBIFscontain-ingontheorderof0.01%orlessorganiccarbonbyweight(Fischeretal., 2014; Klein, 2005). Proterozoic BIFs, such as the syn-glacialiron formations that co-occurwith Snowball Earth episodes late inNeoproterozoic (Cryogenian) time (Hoffman, Kaufman, Halverson,&Schrag,1998;Kirschvink,1992),aredominantlycomprisedoffer-ricironphases.Thisisconsistentwithexpectationsfortheirdeposi-tionviaaerobic ironoxidation (i.e.,excessdepositionof ironoxidesrelative to organic carbon, limiting subsequent iron reduction), andmost closely resembles the mineralogy of the materials currentlybeingdepositedatOHK.BycontrastingOHKwithotherBIFprocessanalogsiteswhereironoxidationpredominantlyoccursbydifferentprocesses (e.g.,phototrophy), itmaybepossible toopenawindowintotheecology,mineralogy,productivity,andotheraspectsofBIFdeposition across theGOE,withOHK representing an endmemberinwhich the ironoxide componentofBIF sedimentwasdepositedprimarilybyaerobicironoxidation.

ACKNOWLEDGMENTS

LMW acknowledges support from NASA NESSF (#NNX16AP39H),NSF (#OISE1639454),andNSFGROW(#DGE1144469).SEMac-knowledges support from MEXT KAKENHI grant-in-aid for chal-lenging exploratory research (grant award number 15K14608).WWF acknowledges the support of NASA Exobiology award#NNX16AJ57G, the David and Lucile Packard Foundation, and aStanford University Blaustein Fellowship. The authors would liketo thank Katsumi Matsuura and the Environmental MicrobiologylaboratoryatTokyoMetropolitanUniversity for laboratorysupport,VictoriaOrphanandStephanieConnonattheCaliforniaInstituteofTechnology for sequencing support, and Nancy Merino and NorioKitadaiattheTokyoInstituteofTechnologyforassistancewith ionchromatographymeasurements.TheauthorswouldalsoliketothankElizabethTrembath-Reichert,JamesHemp,andRolandHatzenpichlerforhelpfuldiscussion,aswellasClaraChanandfouranonymousre-viewersforhelpfulcommentsonthemanuscript.

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How to cite this article:WardLM,IdeiA,TerajimaS,KakegawaT,FischerWW,McGlynnSE.MicrobialdiversityandironoxidationatOkuoku-hachikurouOnsen,aJapanesehotspringanalogofPrecambrianironformations.Geobiology.

2017;15:817–835. https://doi.org/10.1111/gbi.12266