Characterization of Incubation Experiments and Development of an Enrichment Culture Capable of Ammonium Oxidation Under Iron Reducing Conditions S

Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | bac- ) to serve − 2 oxidizing bacteria, re- + 4 Acidimicrobiaceae , and ferric iron [Fe(III)] is − 2 Acidimicrobium ferrooxidans family, whose closest cultivated rela- 12296 12295 oxidation process coupled to iron reduction was first (with 92 % identity) and + 4 [email protected]) ff Acidimicrobiaceae é (ja ff é ff ) oxidation because they lack dissolved oxygen or nitrite (NO + 4 is the electron donor, which is oxidized to NO + 4 Ferrimicrobium acidiphilum This discussion paper is/has beenPlease under refer review to for the the corresponding journal final Biogeosciences paper (BG). in BG if available. the electron acceptor, which is reduced to ferrous iron [Fe(II)]. The stoichiometry and The conventional removal of nitrogenganic nitrogen), from followed soil by environments nitrificationSaturated is and anoxic mineralization then soils (for denitrification are or- (Canfield notmonium et considered (NH al., suitable 2010). for eitheras aerobic electron or acceptors anaerobic by am- aerobicspectively. A or novel anaerobic anaerobic NH (anammox) NH noted in a forestedaction, riparian NH wetland in New Jersey (Clement et al., 2005). In this re- ferric iron as an electron acceptorwas while oxidized oxidizing to ammonium nitrite, to nitrite. nitrogen After loss ammonium proceeded via denitrification and/or anammox. 1 Introduction or goethite and ammoniumwere chloride. observed Significant after changesbrane in 180 days reactor, the of using microbial incubation community 454-pyrosequencing, 16S and as rRNA real-time well quantitative genethe PCR as PCR-denaturing dominant analysis. in gradient microbial We believe species a gel thatterium in electrophoresis, continuous one our A6), of system flow belonging (an to mem- tive uncultured is the (with 90 % identity), might play a key role in this anaerobic biological process that uses Incubation experiments were conductedwetland using where soil we samplespled have from to a previously iron forested reduction. observedpeatedly riparian Production during anaerobic of incubations ammonium both when nitrite oxidation the and soil cou- ferrous slurry iron was were supplied measured with re- either ferrihydrite Abstract Characterization of incubation experiments and development of an enrichment culture capable of ammonium oxidation under iron reducing conditions S. Huang and P. R. Ja Department of Civil and Environmental08544, Engineering, USA Princeton University, Princeton, NJ Received: 14 July 2014 – Accepted: 21Correspondence July to: 2014 P. R. – Ja Published: 14 August 2014 Published by Copernicus Publications on behalf of the European Geosciences Union. Biogeosciences Discuss., 11, 12295–12321, 2014 www.biogeosciences-discuss.net/11/12295/2014/ doi:10.5194/bgd-11-12295-2014 © Author(s) 2014. CC Attribution 3.0 License. 5 20 25 10 15 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 2 + 4 − 2 + 4 O) 30 2 :N ) as = NH NH 2 2 Fe(III) n 15 + H to NO 2 1 0.5H − + 4 · 3 O 2 addition ( + 4 , which conclusively NH ) in these sediments 2 2 + N ) (Sawayama, 2006), or 15 − 3 addition; either only ferri- + 4 − 2 NH in anaerobic environments has + NO − 2 cult. Here we focus on a series of + ffi O 2 to NO + 4 4 to 4.5. The vials were sealed tightly with erent Fe(III) sources to determine which 8.5H ff ∼ concentration, and acetylene gas (C + 12298 12297 + 4 + 2 6Fe . Following the aeration treatment, the soil was divided → 1 addition; ferric citrate − + 4 + 4 NH NH + ) (Eq. (1), Supplementary Information 1.1) + 1 + − 4.0 in the ferric chloride/citrate augmented samples. Soil-slurry 0.20 mg g ∼ 10H ± + addition; and autoclaved soil with ferrihydrite O + 4 2 (Yang et al., 2012), using Fe(III) as electron acceptor. 2 10 g (air-dry equivalent) subsamples, and added into 50 mL serum vials, oxidation, or a very similar one, was also observed in a biological reactor 145.08kJ mol 0.5H × · + 4 3 ≤ − O r 2 G Our understanding of the Feammox process is still incomplete, particularly informa- ∆ and to between 3.5 samples, which were prepared to have an initial concentration of 12.0 mmol L 2.2 Batch incubation experiments Soil samples weresource first could incubated be with usedor goethite di by [FeO(OH)] the [prepared Feammox accordingaddition; to process: ferric Cornell 6-line and chloride ferrihydrite Schwertmann,hydrite (Fe 2003] or NH per treatment). pH was adjusted to 4.5 in the ferrihydrite/goethite augmented samples, into 400 with 30 mL deionized water.(80 The : 20) soil mixture, slurries resulting wererubber in purged a stoppers thoroughly final and pH with of temperature a were to CO stored allow for in stabilization an before starting anaerobic the glove incubations. box for 30 days at ambient Ten soil coresumn were collected containers from (8 cmwithin 10 cm diameter 2 h. below and The thetected. soil 30 cm The pH surface long) detailed was with physicochemical between anddescribed polyethylene characteristic 3.5 transported elsewhere of col- and (Clement these to 4.5, wetland etslurry and the soils al., from no laboratory have 2005). Manganese the been Prior oxide fieldbon. to was site After de- all was a incubation aerated 30was day experiments, stable for at aeration soil a 2.06 treatment, month the to dissolved degrade organic the carbon labile (DOC) organic content car- 2.1 Sample collection and processing Soils for allforested the riparian experiments wetland described at in the this Assunpink study Wildlife Management were Area, taken New from Jersey. a temperate 2 Methods terial community change during incubations. community responsible forShrestha the et process al., described 2009).for Soil previously laboratory samples (Clement incubation were et collected experimentsFeammox in from al., as a the continuous well 2005; flow same as membrane location reactor.sources, to Various and incubation inorganic set conditions used carbon [Fe(III) up content, ana NH selected enrichment inhibitor] system were for methods, used such to as study denaturing the gradienting, Feammox gel and mechanism. electrophoresis real-time Molecular (DGGE), quantitative 454 PCR biology pyrosequenc- (qPCR) analysis were used to investigate the bac- directly to N tion about the microorganism(s)into responsible for the it mechanism is of lacking.incubations This the and makes Feammox establishing further process a study di Feammox enrichment culture to identify the microbial been described in the literature beforein this process a was reported. microcosm Usingshowed labeled experiment, that resulted ammonium-N inunder was the iron converted production to reducing of for nitrogen conditions NH gas (Shrestha (N (Sawayama, et 2006) and al., a tropical 2009).mox rainforest (Sawayama, soil Either 2006). (Yang et These this al., pathways(Clement 2012), have same et and been pathway coined al., reported Feam- 2005; to oxidize Shrestha NH et al., 2009), to nitrate (NO 3Fe ( No proven pathway for the oxidation of NH change in free energy when ferrihydrite is the Fe(III) source is: 5 5 20 25 10 15 25 20 10 15 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 1 5 + 4 Cl − 4 ∼ NH PO4, 1 2 NH − Cl, and C), and concen- 1 ◦ 4 − amended 2 , detection sodium cit- H 3 1 2 1 − − was added on O analysis, and 3 2 Cl were added to 4 in solution was ex- NaHCO , 9.00 mg KH 3 concentrations in the + 4 1 − . One mmol L + 4 5, and dissolve oxygen 1.0 mL min NaHCO and 1.00 mmol L 1 of NH ∼ − 1 1 = 1 − − − 0.016 ppm). For the total or- were then incubated anaero- . For this incubation, samples Ion Chromatograph (LC3000) 3 = 2 C for molecular biology analysis. ™ H ◦ 2 (80 : 20) mixture. Triplicate samples sodium citrate was feed to the re- 20 O. After autoclaving, 1 mL trace el- 2 1 2 − − , 71.0 mg KHCO 3 :N 2H 2 of NaHCO plus 2 mmol L · 1 2 3 − 12300 12299 , detection limit Cl additions after the NH 1 4 − were extracted with DI water for 1 h anaerobically, erent inorganic carbon contents on Feammox. Either − 2 of NaHCO ff ects the Feammox bacteria, 1.00 mmol L 1 . Soil slurries were first incubated for 90 days in eighty ff − 2 was added on days 24, 60, and 90. After this incubation, , 19.8 mg NaHCO H 3 4 2 , were incubated in a series of 50 mL vials with an oxygen- 1.2 mL min + 4 and NO = SO ect of di was analyzed using a Dionex additions were inoculated into a continuous flow membrane re- 2 ff . Samples with and without C − 3 ) NH + 4 1 3 O, and 60.0 mg CaCl 4 − 2 1 was then added every 10 days, which increased the soil pH to NaHCO − gas were added to 40 vials, which resulted in a finial C trough the reactor’s headspace at a room temperature (25 3 1 2 7H 2 . Samples form the outflow were collected every two days, and sludge − · 1 H or 0.20 mmol L − 2 4 NaHCO 1 − 1 − 0.1 mg L 0.012 ppm). NO < = Cl, 77.9 mg (NH 4 The enrichment medium contained the following components per liter: 177 mg In the third experiment, inorganic nitrogen species were quantified through incuba- The second incubation was conducted to extend the anoxic incubation with ferri- guard column (flow rate ganic carbon (TOC) and total nitrogen (TN) analyses a Shimadzu TOC-5000(A) was pled in a gloveately box under using oxygen-free a conditionsroom pH and temperature the electrode. to pH An determine wassoils. extraction acid-extractable measured Fe(II) Fe(II) with immedi- and was 0.5N NH analyzedet HCl using al., was the 2007), conducted ferrozine and assay for NH with method 24 a h (Stookey, CS-16 1970; at Colum Komlos limit and a CS-16and guard measured column via (flow Ion rate Chromatography, using an AS-22 Colum along with an AG-22 was samples from reactor were collected and kept at 2.4 Chemical analyses For each sample collection during the incubations, a set of vials was destructively sam- with a 48 h hydraulic retention time. NH 100 mg MgSO ement solution (Van deferrihydrite Graaf were et added al., once 1996) everytaining two was anaerobic weeks added directly conditions, to into 0.10–0.20 theactor mmol the L about medium. reactor. twice To 50.0 mmol aid per L in month. main- pH was controlled at around 4 Soil samples collected on1.2 mmol day L 180 fromactor the (Abbassi incubation et with al.,stantly ferrihydrite, 2014), purging NH which N was operated under anaerobic conditions by con- 2.3 Continuous flow membrane Feammox reactor tions in the presence50 of mL C vials, with anand initial 0.20 mmol Fe(III) L concentration of5 mL 25 of mmol L pure C tration of 100 µmol L bically for 20 days. Thesoil headspace samples gas were was analyzed sampled every every two 24 h days for for Fe N and N species. bations were used tohow enrich organic the carbon Feammox content bacteriarate a in was a also membrane suppliedsamples. reactor. on To study day 125 to four of the 1.20 mmol L hausted. The initial concentration of Fe(III)was was added 25.0 on mmol L days 4,day 24, 50 and 60, and furthermore, day 0.20two 90 mmol sets L of to the study the1.20 incubation. mmol e L On day 125,each incubation set. vials NaHCO were dividedin into the samples amended withwere 1.20 collected mmol L every four days. Finally, soil samples collected on day 180 of the incu- and/or 2.00 mmol L free headspace, created by purging withwere a CO collected destructively every two days to analyze iron andhydrite nitrogen to species. 180 days, with repeated NH 5 5 25 20 10 15 25 20 10 15 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ® C. ◦ Aci- -related Acidimi- bacteria 10 ng DNA PrimeScript ∼ ® ) and ammonia nirS C, and 30 s at 70 ◦ pro Soil Direct Kit. The ® Acidimicrobiaceae Acidimicrobiaceae gene numbers was initiated erent time points during the Real-Time PCR System (Life ff SYBR Green I and visualized ™ and 1 − amoA C, 30 s at 57 ◦ and nirS spin kit for soil (MP Biomedicals, USA) as C as annealing temperature respectively, with 12302 12301 ) were quantified with primer sets NirS3/NirS5 ® ◦ Acidobacteriaceae amoA C, and 58 ◦ and acm342f (5’-GCA ATG GGG GAA ACC CTG AC -3’) , C, 55 ◦ II (Takara, Japan), 0.8 µL 10 µM of each primer, and bacteria ® C, followed by 40 cycles of 5 s at 94 ◦ bacteria (Supplementary Information 1.3). For the detection of denitrifiers O concentrations were determined on a gas chromatograph Shimadzu 2014 2 the same program.those For described RNA for measuring quantification, gene the numbers, with cycling the exception conditions that were an initial identical incuba- to Premix Ex Taq template. RNA quantification wastranscription-PCR conducted (RT-qPCR) through analysis, a by real-timeRT-PCR using Kit quantitative (Takara, the reverse Japan) One accordingmal to Step cycling the SYBR conditions manufacturer’s for recommendations. total Ther- for 16S 30 s rDNA, at 94 16S rDNA numbers ofwere anammox, performed at 56 crobiaceae and ammonia-oxidizing bacteria,monooxygenase denitrifying structural gene functional ( (Braker genes et ( al., 1998)For DNA and quantification, amoA-1F/amoA-2R, each respectively qPCR (Rotthauwe mixture (20 et µL) al., was composed 1997). of 10 µL of SYBR 2.7 Quantitative PCR (qPCR) assay qPCR experiments were carried usingTechnologies, USA), a represented StepOnePlus by 16Sfor rRNA genes, total using bacteria primer(Schmid sets et (Harms 1055f/1392r al., et 2000,432r 2003), al., (5’-GAC acd320f AGG 2003), (5’-CGG GTTdobacteriaceae Amx368f/Amx820r TCC TTA CAG AGA for TCC CTC– anammox GAA CTA 439r CGG GA bacteria (5’-ACC GA -3’) GTC -3’) which AAT – TTC we GTC developed CCT for GC -3’) which we developed for samples collected from the incubationreactor on after days 150 0, days 30, ofV5 90, reactor region 160 operation. of and Domain-specific the from primers, 16S thePinto targeting rDNA membrane et the of al. V3- bacteria (2012) were amplified following methods suggested by firm the changes in the bacterial community, 454 pyrosequencing was performed with (Supplementary Information 1.2). Sequences obtainedGenBank in this database study under are accession available in numbers the KC581755–KC581779. To further con- with an UV transilluminator.templates All for visible bands re-amplification, were usingand excised followed the from by primer the cloning.cloned gel set PCR and into V3-1/V3-2 products used (Jensen a were as clones et purified pGEM-T for al., via vector each agarose 1998) (Promega). bandto gel A avoid were extraction erroneous identified total and interpretations. by DNA ofInc. sequencing colony Bacteria 10 was PCR, then were to and conducted classifiedsequences by 30 were was Genewiz, and sent positive constructed the for recombinant using phylogenetic sequencing the tree Bayesian of inference (Huelsenbeck et al., 2001) Bacterial universal 16S rRNAused gene for primer PCR amplification. sets DGGE V3-2/V3-3 wastaining (Jensen performed a with et gradient an from al., 8 % 40 1998)system % polyacrylamide were to gel (C.B.S. con- 80 SCIENTIFIC, % denaturant USA).15 using h. The the After gradient electrophoresis gel that was electrophoresis the carried gel out was at stained 60 V with for 0.1 µL mL any laboratory incubation, fromincubation the experiments samples and taken at fromor di the sludge reactor. samples DNA usingdescribed by was the the extracted FastDNA manufacturer, and fromconcentrations RNA were 500 using mg measured the soil using FastRNA Scientific, a USA). Nano-drop 2000 spectrophotometer (Thermo 2.6 PCR-DGGE and 454 pyrosequencing analysis equipped with an electron capture detector. 2.5 DNA and RNA isolation DNA and RNA samples were extracted from soils collected at the wetland prior to used. N 5 5 25 20 10 15 20 25 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 3 in Cl, 1 sp., oxi- 4 − . The + 4 1 − Geobacter 0.17 mmol L 3.13 mg L (Fig. S3b). During Acidimicrobiaceae ± ± + 4 appeared within a few Cl, the microbial com- 4 − 2 erent Fe(III) sources for (Fig. S3b). ff 1 − added. The increased NaHCO Cl resulted in the fastest NH + 4 4 value than goethite (Clement et al., production showed a similar pattern 50 mg L G − 3 NH Cl was supplied repeatedly, especially ∼ additions were increased from 0.20 to ∆ 4 family. This family contains mainly den- erent operational conditions 1 3 − ff family are represented by band A8. Some removal was observed in ferrihydrite than in 12304 12303 + 4 did not accumulate in the system and was imme- C to facilitate reverse transcriptase activity. Each − 2 ◦ , with a maximum concentration 0.44 Rhodocyclaceae + 4 Acidobacteriaceae oxidation (Figs. 1 and S1). In samples incubated with ferric citrate and removal, between inflow and outflow was achieved in the membrane oxidation cycle. NO oxidation rate increased as NH + 4 + 4 in addition to the 2.00 mmol L sp., which showed a strong presence in the samples at 30 days of in- + 4 + 4 (Fig. S3a), and TN loss similar to the decrease in NH 1 − 2 − concentration remained fairly constant (Fig. 1). No detectable Fe(II) reduction oxidation was found the sterilized soils amended with ferrihydrite and NH + 4 + 4 Cl, Fe(III) reduction was much faster than in those supplied with Fe(III) oxides, but 4 A 64.5 % NH Since samples incubated with ferrihydrite and NH Huang et al., 2011). cubation and was attenuatedA6, after A8 longer and A9 incubation became times. morethere In significant were as contrast, three the DGGE incubation groups bands timebation. of increased, Band showing bacteria that A6, dominating representsfamily. in Bacteria a the from group system the of afterspecies bacteria 160 in days belonging this of family to incu- have(Kishimoto the been et described al., 1991; as Rowe ironA9 et reducers represents al., and 2007; bacteria obligate Coupland of heterotrophs and the itrifying Johnson, bacteria, 2008). DGGE which band exhibit very versatile metabolic capabilities (Smith et al., 2005; lane 1–4). Some DGGE bandsband disappeared A7. gradually Band with time, A5,which such represents as existed a band in dissimilatory A5 this and the iron-reducing Fe(III)-rich initial bacteria, anaerobic wetland incubation. soil BandNitrosomonas A7, and represents reappeared an ammonia-oxidizing for bacterium, a short time during 3.2 Phylogenetic analysis of the microbial communityAll based visible on bands 16S rRNA observed gene inFig. the 3) DGGE were analysis excised (significant12 bands from samples were the resulted marked, gel in see groups and 721 of sequences sequenced bacteria of were after classifieding partial cloning. via this 16S Clone a 180 rRNA day libraries phylogenetic gene anaerobic analysismunities from fragments, (Tables incubation shifted 1 and with and dramatically six ferrihydrite S1). and and Dur- the NH microbial diversity decreased with time (Fig. 3, 180 days of incubation, the systemDOC experienced a content loss fluctuated of TN slightlyconcentration of in 57.2 was the relatively stable early at stage around of 45 incubation, butreactor overall, after the 150 DOC days of operation. to that of NO as the Fe(III) source results in2005). a The larger NH negative after 125 days of incubation when1.20 mmol the L NaHCO dosing also increased the generation ofdays after Fe(II) (Fig. the 2a, addition b). ofthe NO NH second NH diately consumed after generation (Fig. S3a). NO or NH (Fig. S2). Faster irongoethite-amended reduction sediments and (Fig. NH S1). dation, the anaerobic incubation with ferrihydrite was extended to 180 days. Ferrihydrite 3 Results 3.1 Change in Fe and NAfter species under incubating di the30 pre-treated days, only soil samples slurry tomeasurable which NH with either the ferrihydrite or fourNH goethite di had been added showed the NH assay contained a standard using agenes, serial independent dilution triplicate of templates plasmids for containing eachcontrols specific soil (NTC). target sample, and triplicate no template tion was conducted for 5 min at 42 5 5 25 15 20 10 20 25 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | , 3 + 4 ) had 3 gener- , and 6 − 2 bacteria, NaHCO bacterium Acidobac- 10 1 , × − Acidimicrobi- added. In the NaHCO liated, was de- bacterium was 3 amendment 1 ffi − 3 , 0.13 7 bacteria (DGGE band 10 × Actinobacteria Acidimicrobiaceae , had a decreased diversity in Acidimicrobiaceae dw over 50 days of incubation + 4 cell number increased the most ). From day 125 to day 180 of 1 1 − − Acidimicrobiaceae , the rRNA copy number increased 3 gene, representing the abundance of gene), increased over the 180 day incu- bacteria (DGGE band A8), and that of bacteria, represented by band A6, were Acidimicrobiaceae copies g 6 nirS NaHCO gene were 0.37 amoA 12306 12305 10 1 Actinobacteria − × vs. 1.20 mmol L bacteria (DGGE band A6), increased during the nirS 1 ered between samples incubated with various Fe(III) − 0.09 ff and have shown Feammox activity (Fig. 3). ± 3 groups (which band A6, A8 and A9 belong to) was consis- Acidobacteriaceae Acidimicrobiaceae bacterium, represented by band A6, which belongs to the phylum, with which anammox bacteria are a (Fig. 6). The rRNA copies of the -related sequences is shown using a phylogenetic tree (Fig. 5). erences in response to the amounts of NaHCO to 0.19 3 ff dw, respectively, while the amoA gene was not detected. 6 1 were also the dominant species in the Feammox enrichment reac- sodium citrate) and inorganic carbon (1.20 mmol L 10 − 1 − × Acidimicrobiaceae phylum, was the dominant species in the incubation experiments after NaHCO Cl, and NaHCO 1 added (0.20 mmol L 4 0.06 − -Proteobacteria 3 copies g ± β 6 Planctomycetes bacteria were higher in the soils with the higher inorganic carbon content. Acidimicrobiaceae 10 Acidimicrobiaceae reactor and Actinobacteria × In the Feammox reactor, the copy number of Changes in the microbial community after 180 days of incubation were also con- The Microbial communities also di from 0.04 of NaHCO the incubation, bothaceae 16S rRNA geneThe and 16S rRNA fragment rRNAwere numbers gene four of times copies0.20 higher mmol of L than samples thoseshowed augmented in even larger with samples di that 1.20 mmolsamples L had augmented been with augmented 1.20 mmol L with only 0.92 3.4 Changes of bacterial abundance andAbundance activities and with activity NaHCO of compared between samples incubated under the same conditions except the amounts denitrifiers (represented by the numberbation of (Fig. S4a, b),A6). although Increase less in than the the ated denitrifier via activity Feammox. was Theammonia-oxidizing most bacteria, number likely decreased of stimulated sharply the by withter 90 time the days and of NO was incubation hardly (Fig. detected S4d). af- A6, Anammox bacteria and incubation, particularly after 90 days.first The 3 rRNA months numbers and(Fig. increased 6). slowly doubled rRNA during between as the a dayas biomarker 130 mRNA, for changes is and of a day2010). protein good level, 140 The even indicator of though abundance for not the of as bacterial incubation specific activity period (Poulsen et al., 1993; Park et al., ment copies of The total bacterial abundancecreased determined during via the the 180 day 16S incubation rRNA (Fig. gene 6). Both, copy 16S number, rRNA de- gene and rRNA frag- other Unlike the bacteria represented bythat band A8 did and A9, not whichonly show were detected also Feammox in found transformations, incubations in (or samples this rihydrite, reactor) NH that were augmented simultaneously with fer- 3.3 Changes of bacterial abundance and activity during incubations and in the tor based on the results of the 16S rDNAActinobacteria library obtained via pyrosequencing (Fig.180 4). days of incubation (14.8reactor % after in 150 terms days of of cell operation numbers) (40.2 % as well in as terms in of the cell membrane numbers). Its similarity to a higher bacterial diversity (DGGE Fig. 3, lane 8). firmed via 454-pyrosequencing, andteria the obvious growth of tent with the DGGE results,(Fig. where 4). the tected in the first 90 days160. of incubation, but disappeared or was below detection on day sources, and between samples withples or supplied without with the either additionand ferric of chloride samples inorganic or supplied carbon. ferric with Sam- citratetheir just bacterial as ferrihydrite communities the and (Fig. Fe(III) 3, no sourcebon lane NH (1.00 plus 5–7). mmol L NH Samples supplied with both organic car- 5 5 25 20 10 15 25 20 10 15 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | , 3 2 5 + 4 2 H dw H 10 2 2 2 O to 1 , and 2 H × − 2 2 H removal 2 NaHCO + 4 0.10 1 Acidimicro- Acidimicro- − ± reduction, ac- copies g 5 − 2 10 , which belongs to × were conducted. Af- in sample incubated 2 1 − H bacterium was only de- 2 as an electron donor, and 0.01) , which is postulated to be + 4 ± − 2 F. acidiphilum gene at a time of increasing NH bacterium was enriched from an O, a product of NO 0.67 mmol L 2 ected by the presence of C ± ff under aerobic conditions, and the re- , which inhibits the reduction of N amoA 2 − 2 H (Fig. S5a). NO Acidimicrobiaceae 2 2 sp. has been detected in mine water, but so H sp. (Table 1). 12308 12307 to NO 2 (Fig. S5c). N + 4 bacterium became the dominant species during was oxidized in the samples amended with C 2 0.72 and 5.71 bacterium, also has a 90 % identity with H dw, indicating a substantial increase in its activity + 4 ± 2 Acidimicrobiaceae 1 dw during the same 50 days incubation (Fig. 6). − oxidation, accumulated slowly in the samples incubated 1 a facultative autotroph in the same family, which can re- − reached a higher concentration in samples without C + 4 , − 3 strain T23 is the only pure strain with a comprehensive char- Ferrimicrobium sp. is an acidophilic heterotrophic ferrous iron oxidizing bac- copies g as well as the anammox pathway under anaerobic conditions family, was first isolated from mine environments (Johnson et al., bacterium may play an important role in the Feammox reactions , respectively. was supplied as a carbon source, NH as compared to the samples amended only with ferrihydrite, in- 2 2 copies g 3 6 H + 4 (Table 1) 5 2 10 Acidimicrobiaceae 10 × Acidimicrobiaceae × O to N 2 Ferrimicrobium acidiphilum F. acidiphilum (Fig. S5b). NO 0.07) 0.05 2 bacterium increased along with the Feammox activity during the incubations. ± H Ferrimicrobium ± gene, decreased after 30 days of incubation (Fig. S4d). Oxygen deficiency was 2 can inhibit the oxidation of NH oxidation in the later incubation times. 2 Acidimicrobiaceae Acidimicrobiaceae (Fig. S5d). Fe(II) production was not much a + 4 H Ammonia-oxidizing bacteria represented by DGGE band A7 (Fig. 3), as well as the An uncultured 2 2 duce Fe(III) in anaerobic environments while oxidizing sulfide to sulfur and exists widely identity with the 2009), and acterization. Uncultured far not in wetland soils (Gonzalez-Toril2011). et al., 2003; Johnson etterium, al., 2009; which Bruneel can et also al., The reduce uncultured Fe(III) under anoxicbium conditions ferrooxidans (Johnson et al., 2009). (Fig. 6). Inand the Fe(III) continuous reduction flowinitial membrane rate, 14.8 reactor, % this which tothis had 40.2 % a after high 150 daysdescribed NH in operation this study. (Fig. According to 4). a These phylogenetic analysis, results this bacterium indicate has that a 92 % the 180 day anaerobicof incubation cell period, numbers to increasing 14.8tected % from when on NaHCO 0.92 day % 160. on This ferrihydrite as day electron acceptor 0 (Fig. in 3).biaceae The terms abundance and activity ofDuring this the incubation periodto its rRNA (0.28 changed from (0.22 amoA the most likely reason fortime the (Laanbroek decline et in al., ammonia-oxidizing 1994). bacteria Thisoxidation in decrease indicates this in that system aerobic over ammonia-oxidizingNH bacteria were not the drivers of the dicating that most offrom the dissimilatory Fe(III) Fe(III) reduction reduction (Fig. came 1a from vs. the Fig. S2a). Feammox reaction, and not 4 Discussion DGGE band A5 representsa short dissimilatory time iron-reducing atthese bacteria, the heterotrophic beginning which bacteria of appeared the decreasedriod, for anaerobic rapidly more incubation. (Fig. than For 3). three longer Over times incubationferrihydrite the a times and mass 25 of NH day Fe(II) incubation was pe- produced in samples amended with than in samples incubated withcumulated C in the samples incubatedN with C after 20 days incubation was 4.36 with and without C C duction of N (Yoshinari et al., 1977; Jensen etinto the al., nitrogen 2007; removal Kartal process observed, etter incubations al., with 20 2011). C days To of gain incubation, furthercompared less insights to NH those incubated withoutthe C direct product of thewith NH C (day 130 to daywere 180). added However, on in day the 125,to samples the 0.19 rRNA to number which gradually only deceased from 0.2 mmol 0.29 L 3.5 Nitrogen species changes in samples incubated in the presence of C 5 5 25 20 10 15 20 25 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | , + 4 2 H was pro- 2 gene + 4 − 2 Acidimi- oxidation oxidation oxidizers, nirS has never + 4 , and AMO + 4 + 4 2 2 was used to H H 2 2 2 can also inhibit H ) plus NO 2 2 1 − H Acidimicrobiaceae Acidimicrobiaceae 2 oxidation was reduced oxidation after 125 days + 4 + 4 . When C as the oxidant (Kartal et al., − 2 via denitrification makes the oxidation by Fe(III) reducers − 2 is a major product of the NH 0.23 mmol L − 2 + 4 − 2 ± consumed, showing that NH bacterium A6, which became the erent from common NH amendments (Figs. 2 and 6), which ff + 4 3 erent Feammox studies (Sawayama, ff O (0.72 2 coupled to Fe(III) reduction), as well as its + 4 oxidation in the presence of C 12310 12309 coupled to iron reduction under anaerobic con- + 4 bacterium is an autotroph. Growth of + 4 bacterium A6 was more active and the Feammox removal, and denitrification became more dominant , generated through the Feammox process are ther- , the total N Acidimicrobiaceae − 2 2 2 did not accumulate in the system. oxidation pathway that is not inhibited by C ) was equal to the NH dw of anammox rRNA gene were found, which decreased − 2 1 1 + 4 in our samples, and that NO − − and N oxidized to Fe(III) reduced increased gradually from 1 : 1.9 to 2 O to N and NO 2 − 2 + 4 + 4 is an inhibitor of ammonia monooxygenase (AMO), and can re- oxidation through Feammox was NO on day 130 (Fig. S4c). We postulate that anammox was responsi- 2 oxidizers from using oxygen by binding covalently to AMO (Hynes Acidimicrobiaceae , NO 5 + 4 H + 4 copies g 2 − 3 Acidimicrobiaceae 10 6 0.07 mmol L × 10 ± bacterium A6 and the Feammox process studied here. × that was being produced during the anaerobic NH 5 µM, Lam et al., 2007), NH 0.06 − 2 < removal later during the incubation period (Figs. 5 and S4b, c). -activation step of anammox cells, which use NO ± + 4 0.05 − 2 by denitrifiers, and NO ± 2 The results and analyses described here have shown that a Feammox enrichment The role of anammox during the incubations was also evaluated. During the incu- The Feammox reaction studied here proceeded only when iron oxides (ferrihydrite crobiaceae reactor has the capacity ofditions, oxidize NH and that andominant species uncultured over timeaccess might be to responsible samples for from(Sawayama, this 2006; other Feammox Yang reaction. reported et Without the biological al., processes NH 2012), reported it by isIsolating these the not investigators pure possible are bacterial to the strain know same will if as allow those the to identified organisms establish here. for a direct link between ratio, especially at earlier incubation0.17 times. In the samples takento before 0.09 the incubation, ble for some initial NH for NO by using an alternative NH might not play a role in Feammox. bations the ratio of NH 1 : 5.3, which isthe close Feammox to reaction the became more stoichiometry dominant ofof in 1 terms incubation : of due 6, NH to shown a inallel Eq. relative pathway (1). increase to This in Feammox, indicates the such that activity as of the anammox, Feammox could bacteria. explain A the par- lower stoichiometric been reported. C strain aerobic NH and Knowles, 1982; Hyman andthe Wood, NH 1985; Gilch et al.,2011). 2009). Therefore these C Feammox bacteria might di (oxygen not oxidized directly to N oxidation via Feammox. Although nitrification might happen in suboxic environments Fe(II) buildup due toFeammox sorption, reaction, and as rapid shown in removal Eq.products, of (1), i.e. NO energetically NO favorable. Various NH modynamically feasible, and were reported2006; in Shrestha et di al., 2009;the Yang et product al., 2012). of In NH ourstop incubations amended the with reduction C of N duced (0.13 or goethite) were suppliedric as chloride electron or acceptor, ferric whereas citrate(Figs. samples as incubated 1 the with and Fe(III) fer- source S1).Feammox showed Since reaction, no iron measurable the NH oxides concentrationslimit adsorb of through dissolved the Fe(II) Fe(II) incubation. in The that solution acidic is were conditions generated of below via the the incubations, the detection lack of dissolved family that mightdescribed be here. either responsiblepathway or was faster play in samples a with higherindicates key NaHCO that role this inindicated the that Feammox denitrification process pathwayshere. NO were also activeto in N the incubations described According to a phylogeneticthe comparison GenBank with (Fig. 5), similarulated and clones taking by from into inorganic studies account carbon, reportedactivity its oxidizing special in increase NH growth with characteristics increasedbacterium (stim- Feammox A6 activity, is this uncultured probably a previously unreported species in the in soil environments (Clark and Norris, 1996; Bond et al., 2000; Hartmann et al., 2009). 5 5 25 20 10 15 25 20 10 15 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | gen. nov., 10.5194/bg-8-3041- er, C., Van Dorsselaer, A., ff gen. nov., sp. nov.: mixed-culture Ferrithrix thermotolerans é, P. R.: Ammonium oxidation cou- ff ect of specific inhibition on anammox and ff . ) to detect denitrifying bacteria in environmental é, P. R.: Laboratory study of nitrification, denitri- ff 12312 12311 nirS species, Microbiology, 142, 785–790, 1996. -encoding bacterial community related to environmental and nirS gen. nov., sp. nov., and nirK ect of acetylene on autotrophic and heterotrophic nitrification, ff Acidimicrobium ferrooxidans Sulfobacillus This research was supported by Project X from Princeton University. We , 2011. Ferrimicrobium acidiphilum of sp. nov.: heterotrophic iron-oxidizing, extremelyMicr., acidophilic 59, actinobacteria, 1082–1089, Int. 2009. J. Syst. Evol. agricultural soil revealed by DGGE separationMicrobiol. of Ecol., PCR 26, amplified 17–26, 16 1998. s rDNA fragments, FEMS by acetylene, Biochem. J., 227, 719–725, 1985. Can. J. Microbiol., 28, 334–340, 1982. denitrification in marine sediments, Appl. Environ. Microb., 73, 3151–3158, 2007. phylogeny and its impact on evolutionary biology, Science, 294, 2310–2314, 2001. pled to dissimilatory reductionBiochem., of 37, iron 2323–2328, under 2005. anaerobic conditions in wetland soils,currences, Soil and Biol. Uses, John Wiley and Sons Ltd, 2003. tional genes and diversity ofcharacteristics the of river sediments,2011 Biogeosciences, 8, 3041–3051, doi: ferrous iron oxidation with community structures throughout soilstands, Environ. horizons Microbiol., of 11, 3045–3062, harvested 2009. and naturally disturbed forest tion of nitrite reductase genessamples, ( Appl. Environ. Microb., 64, 3769–3775, 1998. Morin, G., Brown Jr, G.Bertin, E., P. Personné, N., C. Elbaz-Poulichet, J., F., and Leterial Paslier, Arsène-Ploetze, community D., F.: Schae Characterization involved of in theMicrob. natural Ecol., active attenuation 61, bac- processes 793–810, in 2011. arsenic-rich creek sediments, cycle, Science, 330, 192–196, 2010. somonas europaea, Biol. 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Bioeng., 101, 70–72, Park, H., Rosenthal, A., Ramalingam, K., Fillos, J., and Chandran, K.: Linking community pro- Poulsen, L. K., Ballard, G., and Stahl, D. A.: Use of rRNA fluorescence in situ hybridization Lam, P., Jensen, M. M., Lavik, G., McGinnis, D. F., Müller, B., Schubert, C. J., Amann, R., Laanbroek, H. J., Bodelier, P. L. E., and Gerards, S.: Oxygen consumption kinetics of Komlos, J., Kukkadapu, R. K., Zachara, J. M., and Ja Kishimoto, N., Kosako, Y., and Tano, T.: Kartal, B., Maalcke, W. J., de Almeida, N. M., Cirpus, I., Gloerich, J., Geerts, W., Op den 5 5 25 20 15 30 10 20 25 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

erent Fe(III) sources: ﬀ 35 35 The The values represent

three three different Fe(III) . ) 30 30 ▲ (

with with

25 25 ). The values represent the mean and N incubation

20 20 in

+ 4

, and ferric citrate 27 ) ■ NH (

15 Time (days) Time in incubation with three di Time (days) Time 12316 12315 and + 4

10 10 Fe(II) 16S ribosomal RNA gene (JX505108) ribosomal RNA gene (JQ259419) Acidimicrobium ferrooxidans strain TH3 16Sribosomal RNA gene (EF621760) 90 3OL11 16S ribosomal RNA gene(GQ342349) Geothrix sp. culture clone AP-FeEnrich1ribosomal 16S RNA gene (JX828409) 94 , ferric chloride ), and ferric citrate ( 5 ) 5 error (n=3). error ○ (

b a 0 0 A6 Uncultured Ferrimicrobium sp. clone D.an-41 95 A9 Comamonas sp. “ARUP UnID 223” 16S 97 D6 ribosomal RNA gene (AF251436) 9 6 3 0 1 0 3 2 18 15 12

Concentration Concentration of A6, B1, Ferrimicrobium acidiphilum strain T23 16S 92 A9, B9, Uncultured Rhodocyclus sp. clone W4S68 97

3.5 2.5 1.5 0.5

A8, D11 Uncultured Acidobacteria bacterium clone 97

C4, D14 16S ribosomal RNA gene (AY691423)

) L (mmol NH ) L (mmol Fe(II)

+ + -1 -1 ferrihydrite . 1 : 3). = n Figure sources standard and mean the

), ferric chloride (

Concentration of Fe(II) and NH 535 536 537 538 539 532 533 534 Sequence analysis of bands excised from DGGE gels of soil samples with Feammox Actinobacteria Acidobacteria BetaProteobacteria Phylogenetic group Band Related sequence Identity (%) standard error ( Figure 1. ferrihydrite ( Table 1. Activity. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 3 1 1 + − − 3

Cl (lane NaHCO 4 Cl Cl + 4 NaHCO + NH + Cl + 4 25 Cl were added Cl 4 . 4 mmol

2 NH

+ 4, 24, and +

+ s 1 of NH - with with ferrihydrite + 200 200 1

− 3). ferrihydrite ferrihydrite + NH mmol L 180 180 =

; n 180 day incubation 1.2 7); 7); 120 days incubation with added added on day

160 160 . the the incubation 2 mmol L with with only ferrihydrite (lane 5); was 90

lane + + 4 1 140 140 (band (band 8) day − during during Cl Cl ( NH

4 + 1 - 4 120 120 NH NH 50 and

during the 180 day incubation. 25 mmol L

)

b + 4 ( s 100 100 organic carbon (lane 9). Samples from 6 and mmol mmol L 28

day 29

was added on days 4, 24, and 60. 0.2 mmol L + NH The The values represent the mean and standard error and + 4 Time (days) Time Time (days) Time

80 80 12318 12317 Cl 4 (b) NH days of incubation

Fe(II) 1 , 90 and 160 days of + organic carbon 60

60 NH − ) 3 a ferric ferric citrate + ( +

day day 125. was added on ;

3 40 40 6)

4); 4); 160 - 1 Fe(II) and

20 20 lane . Samples from 6 and 120 days of incubation without any NaHCO added added on

1 - was added on day 0. 1.0 b a (a) ere Cl Cl ( lane 0 0 4 ( w

Cl Cl + NaHCO

4 3 2 1 0 6 0

3 Concentration Concentration of

24 18 12

Cl 4 lane 9)

) L (mmol Fe(II) ) L (mmol NH Fe(III)

Cl (lane 7); 120 days incubation with ferrihydrite mmol L

-1 -1 + + . 1 4 -

2 Samples Samples from 0, 30 10 and 11) were use as control 11) were 10 and NH

0.2

NH of Comparison Comparison of DGGE analysis profiles of soil communities during anaerobic

- + lane mmol mmol L 60. L (n=3). Figure . 3

Cl Cl + NaHCO 4 542 543 544 545 546 547 548 549 550 540 541 Concentration of Comparison of DGGE analysis proﬁles of soil communities during anaerobic incuba- was added on day 50 and day 90. 1.2 mmol L 3

Figure incubations. NH ferric chloride + NH ferrihydrite + NH organic carbon ( ( addition

556 557 558 559 560 561 554 555 552 553 551 120 days of incubation without any addition (lane 10 and 11) were use as controls. Figure 3. tions. Samples from 0, 30, 90 and 1606); days of ferric incubation citrate withorganic ferrihydrite carbon (band 8); ferrihydrite (lane 1–4); 160 days of incubation with only ferrihydrite (lane 5); ferric chloride Figure 2. NaHCO on day 125. The values represent the mean and standard error ( Fe(III) was added on day 0. 1.0 mmol L Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

was

sequences sequences The The tree

Nitrospira Nitrospira Bacteroidetes β-Protebacteria Armatimonadetes Armatimonadetes Fusobacteria Planctomycetes Acidobacteria Firmicutes Actinobacteria Verrucomicrobia Verrucomicrobia Chlamydiae -Protebacteria δ γ-Protebacteria -Protebacteria α Unclassilfied bacteria bacteria Unclassilfied Chloroflexi Chloroflexi

during during anaerobic family family from other studies.

16S rRNA gene

Reactor Reactor related related sequences. - 160 . Bootstrap values were based on 1000 -related sequences. The tree was constructed

Acidimicrobiaceae 31 30 12320 12319 90 family from other studies. Sequences determined in

Acidimicrobiaceae ian ian inference (BI) method with 30 bacteria bacteria from the Acidimicrobiaceae and and

A6 0 Acidimicrobiaceae sequence divergence. sequence Phylogenetic Phylogenetic tree of 50 % bootstrap support. The scale bar represents 10 % sequence divergence.

. Relative abundance of bacterial phyla for each soil samples > 4 0% 5.

20% 60% 40% 80% ations (days 0, 30, 90, 160) and enrichment culture from the reactor. from the culture enrichment (days 0, 30, 90, 160) and ations

100%

DGGE band percentages of total sequences total of percentages Relative abundance of bacterial phyla for each soil samples during anaerobic incu- Phylogenetic tree of

Figure Figure incub

Figure constructed using the Bayes from Sequences determined in this study are in bold replicates each and are shown at the nodes with >50 % bootstrap support. The scale bar 10% represents

562 563 564 569 570 571 572 573 574 567 568 565 566 Figure 5. using the Bayesian inference (BI) methodand with 16S bacteria rRNA from gene sequences the this from DGGE study band are A6 in bold.the Bootstrap nodes with values were based on 1000 replicates each and are shown at Figure 4. bations (days 0, 30, 90, 160) and enrichment culture from the reactor. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

. )

. ) 8 -1

Total bacterial abundce (×10 g dw)

8 -1 . )

Total bacterial abundce (×10 g dw)

0 1.2 0.8 0.4 . ) 0.8 0.4 0 1.2 8 -1

Total bacterial abundce (×10 g dw) 8 -1

Total bacterial abundce (×10 g dw) 0 1.2 0.8 0.4 1.2 0.8 0.4 0 180 180 bacterium bacterium A6 in

) and RNA ( bacterium bacterium A6 in

) and RNA (

☐

. . ) (

180 ☐

. (

bacterium bacterium A6 in

. 180 160

8 -1 )

) and RNA (

Total bacterial abundce (×10 g dw) ) copy numbers of bacterium bacterium bacterium A6 in

) and RNA (

160

8 -1 ☐

0 1.2 0.8 0.4

addition .

Total bacterial abundce (×10 g dw) (

3 ☐

. ( bacterium A6 in soil samples with 0 1.2 0.8 0.4

addition 160 140

80 days of anaerobic incubation 3 Acidimicrobiaceae 160 16S rRNA gene 180

NaHCO .

addition 140 1

- 80 days of anaerobic incubation bacterium bacterium A6 in 3

) and RNA ( ) and RNA ( 180 L addition

ays) Acidimicrobiaceae

during during 1 130 3

bacterium bacterium A6 in 16S rRNA gene ☐

)

) and RNA (

. NaHCO ( 140 32 .

80 days of anaerobic incubation

addition 1 ◆

- 140 ☐

3 Acidimicrobiaceae 80 days of anaerobic incubation . ime (d ime ( L 160

16S rRNA gene

T ays) Acidimicrobiaceae

12321 during during 1 NaHCO 130 . 90

16S rRNA gene 1 160 )

Acidimicrobiaceae ) during 180 days of anaerobic incubation. 16S rRNA

NaHCO - .

) copy numbers of 32 addition 1 L addition

NaHCO -

◆ ays)

n during during 1 3 130 1 ( 3 L -

addition ime (d ime ays) ) during during 1

130

3 32

T 60 140 ) addition

80 days of anaerobic incubation ◆ 90

32 3 addition. addition Acidimicrobiaceae ime (d ime ◆ 140

3 80 days of anaerobic incubation mmol 16S rRNA gene ) copy numbers of 3

NaHCO ime (d ime . 0 NaHCO

Acidimicrobiaceae 90

n 1 ) and RNA nce nce of total bacteria ( T - 16S rRNA gene 1 ( 30 a -

NaHCO

90 n . L

bacterium A6 0.20 mmol with bacterium ) copy numbers of (

L ays) 1

during during 1

130 addition. 16S rRNA gene ( - NaHCO 60

) copy numbers of NaHCO ) copy numbers of

n 3 L )

1 (

ays) NaHCO - 1 during during 1 32

130 Abund

n − addition

1 L ( ◆

- ) 0

3 60 32 mmol L ime (d ime 6.

addition

◆

60 0 T 3 NaHCO 90 ime (d ime ) and RNA nce nce of total bacteria (

30 a

mmol

/10 10 × ( A6 bacterium dw ) dw g T

− 0.2 0.6 0.4

-1 -1 6 7 n

bacterium A6 0.20 mmol with bacterium 90 ) copy numbers of

(

soil soil samples with 1.2 of numbers copy Figure 16S rRNA gene

0 mmol Acidimicrobiaceae of copies rRNA 16S Abundance of total bacteria (

NaHCO ) and RNA nce nce of total bacteria ( ) and RNA (

n 0 1 ) copy numbers of ( 30 a

) and RNA nce nce of total bacteria ( n NaHCO bacterium A6 0.20 mmol with bacterium L (

30 Abund a n

589 590 591 592 593 594 595 596 575 576 577 578 579 580 581 582 583 584 585 586 587 588 1 ( n

bacterium A6 0.20 mmol with bacterium - 60

( 0

60 6. Abund Figure 6. gene ( 1.20 mmol L A6 with 0.20 mmol L

mmol Abund 0

0 0

mmol

) and RNA nce nce of total bacteria ( 10 × ( A6 bacterium /10 dw ) dw g

0.2 0.4 0.6 7 -1 -1

6 30 a

16S rRNA gene soil samples with 1.2 of numbers copy Figure

n bacterium A6 0.20 mmol with bacterium 16S rRNA copies of of copies rRNA 16S Acidimicrobiaceae

) and RNA nce nce of total bacteria ( (

0 30 a

10 × ( A6 bacterium /10 dw ) dw g

bacterium A6 0.20 mmol with bacterium

0.2 0.4 0.6

7 -1 -1 6

(

10 × ( A6 bacterium /10 dw ) dw g 16S rRNA gene soil samples with 1.2 of numbers copy Figure

16S rRNA copies of of copies rRNA 16S Acidimicrobiaceae 0.2 0.4 0.6 7 -1 -1

6 Abund

16S rRNA gene soil samples with 1.2 of numbers copy Figure

595 596 593 594 590 591 592 587 588 589 584 585 586 582 583 579 580 581 576 577 578 575 16S rRNA copies of of copies rRNA 16S Acidimicrobiaceae 0

Abund

595 596 593 594 590 591 592 587 588 589 584 585 586 582 583 579 580 581 576 577 578 575

10 × ( A6 bacterium /10 dw ) dw g

0.2 0.4 0.6

6 7 -1 -1

16S rRNA gene soil samples with 1.2 of numbers copy

Figure

16S rRNA copies of of copies rRNA 16S Acidimicrobiaceae

10 × ( A6 bacterium /10 dw ) dw g

0.2

0.4 0.6

7 6 -1 -1

16S rRNA gene soil samples with 1.2 of numbers copy

Figure

16S rRNA copies of of copies rRNA 16S Acidimicrobiaceae

595 596

593 594

590 591 592

587 588 589

584 585 586

582 583

579 580 581

576 577 578 575 595 596 593 594 590 591 592 587 588 589 584 585 586 582 583 579 580 581 576 577 578 575