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Functional diversity of bacteria in a ferruginous hydrothermal sediment

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A microbial community showing diverse respiratory processes was identified within an arsenic-rich, ferruginous shallow marine hydrothermal sediment (20-40 degrees C, pH 6.0-6.3) in Santorini, Greece. Analyses showed that ferric iron reduction with
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  ORIGINAL ARTICLE Functional diversity of bacteria in a ferruginoushydrothermal sediment Kim M Handley 1 , Christopher Boothman 1 , Rachel A Mills 2 , Richard D Pancost 3 and Jonathan R Lloyd 1 1 School of Earth, Atmospheric and Environmental Sciences, The University of Manchester, Manchester, UK; 2 School of Ocean and Earth Science, National Oceanography Centre, University of Southampton,Southampton, UK and   3 Organic Geochemistry Unit, Bristol Biogeochemistry Research Centre, School of Chemistry, University of Bristol, Bristol, UK  A microbial community showing diverse respiratory processes was identified within an arsenic-rich,ferruginous shallow marine hydrothermal sediment (20–40 1 C, pH 6.0–6.3) in Santorini, Greece.Analyses showed that ferric iron reduction with depth was broadly accompanied by manganese andarsenic reduction and FeS accumulation. Clone library analyses indicated the suboxic–anoxictransition zone sediment contained abundant Fe(III)- and sulfate-reducing  Deltaproteobacteria  ,whereas the overlying surface sediment was dominated by clones related to the Fe(II)-oxidizingzetaproteobacterium,  Mariprofundus ferroxydans  . Cultures obtained from the transition zone wereenriched in bacteria that reduced Fe(III), nitrate, sulfate and As(V) using acetate or lactate as electrondonors. In the absence of added organic carbon, bacteria were enriched that oxidized Fe(II)anaerobically or microaerobically, sulfide microaerobically and aerobically and As(III) aerobically.According to 16S rRNA gene analyses, enriched bacteria represented a phylogeneticallywide distribution. Most probable number counts indicated an abundance of nitrate-, As(V)- andFe(III) (s,aq) -reducers, and dissolved sulfide-oxidizers over sulfate-reducers, and FeS-, As(III)-and nitrate-dependent Fe(II)-oxidisers in the transition zone. It is noteworthy that the combinedcommunity and geochemical data imply near-surface microbial iron and arsenic redox cycling weredominant biogeochemical processes. The ISME Journal   advance online publication, 22 April 2010; doi:10.1038/ismej.2010.38 Subject Category:  geomicrobiology and microbial contributions to geochemical cycles Keywords:  microbial community; functional diversity; geochemical cycling; enrichment cultivation;ferruginous; arsenic; redox Introduction Marine hydrothermal sediments are thought to hostan abundance of bacteria and archaea that exploitthe steep geochemical gradients formed at theconfluence of reduced, metal-rich hydrothermalfluids and oxidized seawater ( Jannasch and Mottl,1985; Zierenberg  et al  ., 2000; Luther  et al  ., 2001).However, information on the metabolic diversity of prokaryotes and their effect on redox cyclingremains limited, not the least in iron-rich deposits.Hydrothermal deposits dominated by iron-richmineral phases are found in the vicinity of seafloorspreading centers, intra-plate seamounts and islandarcs. A majority of these have been identified indeep-sea environments (reviewed in Rona, 1988), but shallow marine examples are also known (forexample, Santorini, Aegean Volcanic Arc; AmbitleIsland, Papua New Guinea; Holm, 1987; Alt, 1988;Pichler and Veizer, 1999). Formation proceeds either by the  in situ  precipitation of iron from ascendinghydrothermal fluids or the fall out of iron oxidesformed in hydrothermal plumes (Alt, 1988; Mills et al  ., 1993), with temperatures typically rangingfrom that of ambient seawater to 100 1 C (seereferences in Little  et al  ., 2004).These ferruginous deposits are of particular interestowing to their ability to scavenge and retain traceelements from seawater (German  et al  ., 1991), aswell as the analogies that may be drawn betweentheir development and that of Banded Iron Forma-tions, which formed under oxygen limitation duringthe Precambrian (Krapez  et al  ., 2003). The oxidationof Fe(II) within contemporary iron oxide deposits atcircumneutral pH is increasingly attributed to bacteria that catalyze the transformation of Fe(II) toFe(III) under anaerobic or microaerobic conditions.Currently, much of the evidence for marine micro- bial Fe(II) oxidation is based on the association of stalk- or sheath-like structures within hydrothermal Received 20 November 2009; revised 11 February 2010; accepted1 March 2010Correspondence: Current address: KM Handley, Department of Earth and Planetary Sciences, University of California, Berkeley,307 McCone Hall, Berkeley, CA, USA.E-mail: kim.handley@berkeley.edu The ISME Journal (2010),  1–13 &  2010 International Society for Microbial Ecology All rights reserved 1751-7362/10  $ 32.00 www.nature.com/ismej  iron deposits that are reminiscent of the Fe(II)oxidizers  Gallionella  and  Leptothrix  , respectively(for example, Alt, 1988; Juniper and Fouquet, 1988; Hanert, 2002; Kennedy  et al  ., 2003; Little  et al  .,2004). Both of these taxa are better known fromterrestrial environments. Only a very limited num- ber of marine Fe(II) oxidizers have been isolated todate, namely the zetaproteobacterium  Mariprofun-dus ferrooxydans  and several alphaproteobacteriaand gammaproteobacteria strains related to thegenera  Hyphomonas  and  Marinobacter   (Emersonand Moyer, 2002; Edwards  et al  ., 2003a; Rogers et al  ., 2003; Lysnes  et al  ., 2004; Emerson  et al  ., 2007).The nature of other hypothetical biogeochemicalprocesses in this type of deposit may include themicrobial respiration of Fe(III), SO 42  and NO 3  , andthe biogeochemical cycling of trace elements (forexample, Mn, Cu, U, Cd, As, Ag and Au) typicallyenriched in hydrothermal deposits (cf. Glynn  et al  .,2006; Severmann  et al  ., 2006). Many of these traceelements may be reduced or oxidized by prokaryotesto conserve energy for cell growth or maintenance,or for toxicity resistance (reviewed in Lloyd, 2003).Of these, arsenic is highly enriched in numerousferruginous deposits (Cronan, 1972; Rona, 1988), including those at Santorini and Tutum Bay, PapuaNew Guinea (Varnavas and Cronan, 1988; Price andPichler, 2005). Although the microbial reduction of As(V) and the oxidation of As(III) have been studiedextensively in terrestrial freshwater and geothermal brine environments (Lloyd and Oremland, 2006),little is currently known regarding these processesin marine settings.In this study, we examine the phylogeneticdiversity and geochemical effect of prokaryoticactivity within a temperate shallow marine hydro-thermal sediment at Santorini, Greece that is iron-and arsenic-rich (Varnavas and Cronan, 1988). Thesediment was examined using microbiological,molecular phylogenetic and geochemical techni-ques. In particular, enrichment culturing was usedto analyze the capacity of the indigenous prokar-yotic community to transform a range of geochemi-cally important inorganic species (that is, Fe, Mn,As, S and N). Materials and methods Sampling and analytical methods Samples were collected from a shallow embayment( X 0.3m water depth, 20–40 1 C; Bostrom andWidenfalk, 1984) on the western margin of NeaKameni island, within the Santorini flooded cal-dera. Several cores were extracted in a spatiallyconfined area using hand-push tubes (10cm internaldiameter   50cm long), and sealed with air-tightcaps. One core was selected for geochemicalanalyses. Down-core Eh and pH measurements,initial enrichment inoculations and sedimentsectioning for geochemical analyses (at 5cm intervalsfrom 0–35cm depth) were conducted directly aftercollection under N 2  in a glove bag. Sedimentwas transported immediately to Manchester on ice,where pore-waters were separated by centrifugation(4100rpm, 4min), and filtered (0.45 m m) under N 2 .Pore-water was acidified with HNO 3  for dissolvedelement analyses, or kept anaerobic and chilled forrapid analysis of anions and cations.Total pore-water element concentrations weremeasured by ICP-AES (Perkin-Elmer Optima 5300Dual View, Waltham, MA, USA). Anions weremeasured using a Dionex DX600 ion chromatograph,fitted with a high-capacity ion exchange column(AS9-HC) and an AG9-HC guard column (Sunnyvale,CA, USA), eluted at 1.4ml min  1 with 12m M isocratic Na 2 CO 3  (2600–2800 psi). Dissolved organiccarbon was measured using Shimadzu TOC5050A total carbon analyzer (Milton Keynes, UK).Ammonium concentrations were measured afterreaction with Nessler’s reagent (at 420nm). Ironspecies were quantified by the ferrozine method(Lovley and Phillips, 1986, 1987; Anderson and Lovley, 1999).Solid-phase elements were quantified by an AxiosSequential Wavelength-dispersive X-ray fluores-cence spectrometer (XRF; PANalytical, Almelo,The Netherlands). Total carbon was determined induplicate by flash combustion of freeze-dried finelypowdered samples using a Carlo Erba EA 1108elemental analyzer (CE Elantech, Lakewood, NJ,USA). Total inorganic carbon was determined on aCoulomat 702 C/S analyzer (Stro¨hlein, Karst,Germany). The difference between total carbon andtotal inorganic carbon was taken to be total organiccarbon. Sediment was imaged with a Philips FEG-XL30 environmental scanning electron microscope(Eindhoven, The Netherlands), fitted with a PRISMEDS detector and Spirit software (PGT, Princeton,NJ, USA). Samples were imaged unwashed orwashed (  3) with MilliQ H 2 O, with or withoutfixation by 2.5% glutaraldehyde. Enrichment cultivation Anaerobic, microaerobic and aerobic enrichmentmedia were inoculated in triplicate with 10% (v/v)wet sediment from the suboxic to anoxic transitionzone (5–20cm depth), incubated in the dark at 25 1 Cfor  X 1 month, and subcultured repeatedly beforecommunity composition analysis. An anaerobicmarine minimal medium (MMM) (described inHandley  et al  ., 2009a), supplemented with 10m M acetate or lactate as the electron donor and carbonsource, was used for the reduction of 10m M amorphous Fe(III) oxyhydroxide (FeOOH) (Lovleyand Phillips, 1986), soluble Fe(III)-nitrilotriaceticacid (NTA) or nitrate (KNO 3 ), or 5m M  As(V)(Na 2 HAsO 4 .7H 2 O). Fe(III)-NTA stock (100m M ) wasprepared as described by (Fredrickson  et al  ., 2000),sparged with N 2  and filter sterilized. FeSO 4 .7H 2 O(20m M ) with KNO 3  (10m M ) was used to test Functional diversity of bacteria KM Handley  et al 2 The ISME Journal  anaerobic Fe(II) oxidation. Arsenite oxidizers wereenriched in aerobic MMM using 5m M  Na 3 AsO 2 ,acidified with HCl to pH 6.8–7.0. Marine PostgateMedium B, with 20m M  acetate or 30m M  lactate wasused for sulfate reducer growth (Postgate, 1984).Oxygen gradient tubes using FeS plugs and anagarose slush overlayer of Modified Mineral Wolfe’sMedium in artificial seawater were used to cultivatemicroaerophilic Fe(II) oxidizers following the meth-ods given by Kucera and Wolfe (1957) and modifica-tions by Hanert (1992) and Emerson and Moyer (1997, 2002), with vitamins and minerals as for MMM. Tubes targeting sulfide oxidizers received1% agarose plugs containing 10m M  neutralizedNa 2 S and 25% anoxic artificial seawater ( Jannasch et al  ., 1985). The overlayer consisted of artificialseawater, 1mll  1 vitamins and minerals (MMM),0.001% phenol red ( Jannasch  et al  ., 1985) and 0.1%agarose. A 25 m l stab inoculum was added to over-layers 24h after formation.No organic carbon was added to media usinginorganic electron donors. Most probable number counts (MPN) Enumeration of different microbial functionalgroupswas achieved through the MPN method, using10-fold serial dilutions from 10  1 to 10  8 . Media forcultures was as described for enrichment cultures,with no organic carbon added to media withinorganic electron donors, but acetate and lactateadded in combination to media requiring organicelectron donors. Cell numbers were estimated fromthe three-tube table published in de Man (1983). Isolation of bacteria Sulfate reducers were isolated from enrichmentcultures anaerobically using Postgate Medium Eagar plates with acetate or lactate (Postgate, 1984).Anaerobic Fe(III)-NTA MMM agar plates were usedto isolate from a lactate-dependant FeOOH enrich-ment culture. Fe(III)-reducing bacteria from Fe(III)-NTA and FeOOH/acetate enrichment cultures wereisolated on Luria–Bertani agar plates. As(V)- andAs(III)-oxidizers were isolated on Luria–Bertani and Marinobacter   medium (DSMZ 970) agar plates.Anaerobic nitrate MMM agar plates were used toisolate from the lactate-metabolizing nitrate enrich-ment. Dual-layer plates, based on the gradient tubemethod, but substituting 1.5% agarose in both layers(cf. Wirsen  et al  ., 2002) were used to isolate aerobicsulfide-oxidizers. The metabolism of each isolate wassubsequently tested intheappropriateliquidmedium. Analytical techniques for detection of growth on target substrates The reduction or oxidation of iron in cultureswere determined by the ferrozine method, andwere also evident owing to: the visible formationof the white ferric iron-bearing mineral vivianite[Fe 3 (PO 4 ) 2 .8H 2 O] (confirmed by XRD) from Fe(III)-NTA; the transformation of FeOOH from orange to adark-brown or green precipitate; or the change of Fe(II)/NO 3  medium from green to orange. Ferrousiron oxidation in FeS gradient tubes was indicated by a visible horizon of cell growth stained with anorange ferric precipitate. Similarly, Na 2 S oxidationwas indicated by a horizon of cell growth, and colorchange in phenol red (to pale yellow). Sulfatereduction was evident from the development of FeS. Nitrate reduction was established by anincrease in optical density, and NO 3  , NO 2  andNH 4 þ concentrations. Arsenate and arsenite levelswere measured by the molybdenum blue colori-metric method (see Handley  et al  ., 2009b). DNA extraction and amplification Genomic DNA from sediments, enrichment cultures( X 3rd subculture of the initial 10% v/v dilutions),and sulfate-reducing colonies imbued with FeS wasextracted using the PowerSoil DNA Isolation Kit(MO BIO Laboratories Inc., Carlsbad, CA, USA).DNA from other isolates was amplified directlyfrom colonies. Bacterial 16S rRNA genes wereamplified using the primers 8F and 519R and PCRsettings according to Holmes  et al  ., (2002) with10-min final extensions. Dissimilatory sulfitereductase ( dsr  ) gene primers, DSR1F and DSR4R(Wagner  et al  ., 1998) were also used for analysis of sulfate-reducing communities. Reaction conditionswere: 94 1 C for 1min; 35 cycles of 94 1 C for 30s,54 1 C for 1min, and 72 1 C for 2min; and 72 1 C for10min. PCR products were purified using a QIA-quick purification kit (Qiagen, Crawley, UK), andverified on an ethidium bromide stained 1% agarosetris-borate-EDTA gel. Cloning and restriction fragment length polymorphismanalysis (RFLP) Purified PCR products were cloned using InvitrogenTA Cloning and Top10 kits (Paisley, UK). Colonieswere screened using the vector primers 1F (5 0 -AGTGTGCTGGAATTCGGCTT-3 0 ) and 1R (5 0 -ATATCTGCAGAATTCGGCTT-3 0 ). Between 47 and 57 clones weresampled from enrichment libraries, 57 and 100clones were sampled from surface and transitionzone sediment libraries, respectively, and 6 and 25from  dsr   gene libraries. Sequence diversity wasdetermined by RFLP (Weidner  et al  ., 1996) analysisusing endonucleases EcoRI or Sau3A and MspI(Roche Diagnostics, Lewes, UK), and separationwithin 3% agarose tris-borate-EDTA gels. DNA sequencing and phylogenetic analysis DNA from pure cultures and multiple representa-tives of each RFLP pattern were sequenced using thereverse PCR primers and the ABI Prism BigDye Functional diversity of bacteria KM Handley  et al 3 The ISME Journal  Terminator v1.1 Cycle Sequencing Kit (AppliedBiosystems, Warrington, UK). Electrophoresis wasperformed using an ABI Prism 3100 Genetic Analy-zer. Sequences were verified for chimeras usingChimera Check v. 2.7, RDP-II (Cole  et al  ., 2003). Thephylogenetic affiliations of the nucleotide partialsequences were determined using BLAST analysis(Altschul  et al  ., 1990). Sequences were aligned withphylogenetically related sequences from GenBank,and a neighbor-joining phylogenetic tree was con-structed with 1000 boot-strap replicates, based onevolutionary distances estimated using MEGA v4(http://www.megasoftware.net/index.html) with theMaximum Composite Likelihood method (Tamura et al  ., 2004, 2007). Sequence accession numbers Sequences are deposited in GenBank under thefollowing accession numbers: EU983110–EU983129(surface sediment), EU983130–EU983155 (transitionzone sediment), EU983213–EU983262 (enrichmentcultures) and EU983263–EU983274 (pure cultures). Intergenic spacer profiling  DNA fingerprinting of down-core sediment bacterialcommunities was undertaken by amplifying the 16S(small subunit) and 23S (large subunit) rRNAintergenic spacer region, using the primers SD-Bact-1522-b-s-20 and LD-Bact-132-a-A-18 (Normand et al  ., 1996; Ranjard  et al  ., 2000). These primersyield approximately 20 and 130bp of 16S and 23SrRNA genes (Ranjard  et al  ., 2000), respectively,along with the intergenic spacer region, whichranges from approximately 150–1500bp in prokar-yotes (for example, Fisher and Triplett, 1999;Cardinale  et al  ., 2004). PCR settings were: 94 1 C for3min; 35 cycles of 94 1 C for 1min, 55 1 C for 30s, and72 1 C for 1min; and 72 1 C for 5min. Purified productwasseparatedwithina3%agarosetris-borate-EDTAgel. Results and discussion Sediment geochemistry  The sediment was 25 1 C during sampling (February2006), owing to warm geothermal fluids, andcomprised three distinct geochemical and miner-alogical zones as it graded downwards from suboxicto anoxic. Suboxic surface sediment (zone one,0–5cm depth) was largely characterized by Fe(II)oxidation and Eh values near zero (Figure 1). Thissurface sediment was unconsolidated and rust-colored with a filamentous microtexture (Figure 2)typical of iron oxide deposits (cf. Little  et al  ., 2004and references therein). Deeper anoxic sedimentwas marked by increasingly negative Eh values, andthe reduction of alternative electron acceptors, suchas Fe(III) and Mn(IV). The suboxic to anoxictransition zone (zone two, approximately 5–20cmdepth, Eh   60 to   140mV) graded from poorly towell-consolidated green–brown mud with localized(predominantly millimeter-scale) areas of blackprecipitate, most likely FeS (am) , which continuedthrough the lower anoxic zone (zone three, 20–35cmdepth). From  X 35cm depth the sediment wasgravel-rich.Sediment contained 44–52% Fe, 349–424ppmAs and minor enrichments of Mo (48–56ppm), P(0.2–1.5%) and S (0.3–0.5%) (Supplementary Tables1 and 2). Silicon values were low, although notunusual for similar hydrothermal deposits, andelements typically enriched in hydrothermal sedi-ments, such as Mn, Zn and Cu, were depleted (cf. ElWakeel and Riley, 1961; Bostrom and Widenfalk,1984; Alt, 1988). Inorganic carbon comprised 1.0% of the sediment at 0–5cm depth, 4.0% at 5–20cm,and 3.4% at 20–30cm. Organic carbon was 0.1%,0.5% and 0.3% at the respective sediment depths.Pore-water was significantly enriched indissolved Fe (144.2–316.8mgl  1 ), and containedelevated levels of V (1.07–1.2mgl  1 ), Mn (0.01–0.27mgl  1 ),Si(6.34–17.21mgl  1 )andP(0–0.95mgl  1 )(Supplementary Table 3). Peaks in pore-water Mnand Fe (as ferrous iron) occurred at 5–10cm and10–15cm depth, respectively, while seawater con-centrations of sulfate (2680–3040mgl  1 ) remainedconstant over the 30cm depth analyzed (Figure 1).High levels of ammonium (20–36mgl  1 ) were alsopresent throughout the core (Figure 1, Supplemen-tary Table 4). No nitrate or nitrite were detected.Total dissolved inorganic carbon concentrationswere consistently low (0–1mgl  1 ), whereas dis-solved organic carbon was substantially elevated inthe surface sediment (495mgl  1 ) (Figure 1, Supple-mentary Table 4). In situ  bacterial community and carbon sources Results from ribosomal intergenic spacer analysis(RISA) illustrate a gradational shift in the bacterialcommunity structure with increasing sedimentdepth (Figure 3). An exploration of the surfacesediment (0–5cm depth), based on 16S rRNA geneclone sequencing after RFLP sorting, suggests thecommunity was dominated by bacteria related to themarine Fe(II)-oxidizing zetaproteobacterium,  Mari- profundus ferrooxydans  (33% library abundance;Emerson and Moyer, 2002; Emerson  et al  ., 2007), theFe(III)- and nitrate-reducing deltaproteobacterium, Geothermobacter   sp. (23%; Kashefi  et al  ., 2003) and Chloroflexi   (16%; Figures 3 and 4, SupplementaryTable 5). In the transition zone sediment (5–20cmdepth) bacterial community, the greatest proportionof clone sequences (46%) were most similar to Deltaproteobacteria , with 23% of these sequencessimilar to bacterial species within the  Desulfuromo-nadales , an order characterized by Fe(III)- and S 0 -reducers (Lovley  et al  ., 2004), and 16% similar to the Desulfobulbus  genus, known for sulfate and Fe(III)reduction (for example, Sass  et al  ., 2002; Holmes et al  ., 2004a). These data suggest a broad shift with Functional diversity of bacteria KM Handley  et al 4 The ISME Journal  depth from microbial Fe(II) oxidation (by the M. ferrooxydans -like bacterium) to Fe(III) reduction(for example, by  Desulfuromonodales ).Reactions driven by heterotrophy may have inpart derived organic carbon from photosynthetic bacteria, such as  Chloroflexi  , owing to the shallowwater depth. Other pools of organic carbon srcinatefrom algae, indicated by algal biomarkers (sterols)from this sediment, and allochthonous terrestrialplant matter, indicated by the presence of higherplant biomarkers (for example,  n -alkanols and n -alkanoic acids; Handley, unpublished data). Con-figurations of organic matter indicated a mixture of immature plant and microbial material that isassociated with sedimentation, and mature materialthat has likely been altered by diagenetic and hydro-thermal processes. Current sediment temperaturesdo not account for the maturation, especially giventhe presence of the well-preserved immature com-pound classes. Heterotrophic bacteria might notonly use these organic inputs, but could also usemetabolites arising from their decay. In fact, the verylow dissolved organic matter concentrations in thetransition zone (Figure 1), suggest that the hetero-trophs in this layer are consuming dissolvedsubstrates (that is, metabolites) rather than organicmatter associated with the solid phase. Lithoauto-trophy may be supported by inorganic carbon fromhydrothermal fluids/gases and seawater (that is,CO 2 /HCO 3  ). Cultivated organisms Although bacteria enriched in the first dilution(10% inocula) do not necessarily represent the mostabundant members of their respective functionalgroups in the Nea Kameni sediment (Dunbar  et al  .,1997; Fry, 2003), enrichment experiments clearlyshowed a range of microbial functional processesthat potentially drive key inorganic reactions in thetransition zone and beyond. Prokaryotes performingthese reactions (Figures 4 and 5) were enumerated by MPN techniques (Table 1). No transformationswere detected in uninoculated or heat-killedcontrols. Reactions involving the oxidation of theinorganic electron donors were without addedorganic carbon, and can be attributed to eitherautotrophy supported by bicarbonate buffer in themedia, or heterotrophic consumption of metabolitesproduced by the enrichment community, whileutilizing inorganic electron donors as auxiliaryenergy for cell maintenance. Fe(III) reduction . In experiments with ferric-NTA (aq) , iron was reduced to vivianite. Clone librariesindicated a dominance of bacteria with similar 16SrRNA gene sequences to soluble/insoluble Fe(III)-reducers,  Malonomonas rubra  (97%, 480/491bpidentity) and  Shewanella algae  (100%, 508/508bp) Figure 1  Pore-water profiles with sediment depth, depicting pH (solid symbols) and Eh values (open symbols), and dissolvedconcentrations of total iron (solid symbols), manganese, sulfate, ammonium and organic carbon (DOC). The 0.5 M  HCl soluble(bioavailable) Fe(II) fraction extracted from the sediment is depicted by open symbols. The transition zone is shown in gray. Figure 2  Environmental scanning electron microscope (ESEM)image of the oxidized surface sediment layer, depicting numerousfilamentous (including helical) cells (arrows), associated with theFe(III) oxyhydroxide. Scale bar, 10 m m. Functional diversity of bacteria KM Handley  et al 5 The ISME Journal
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