urease gene-containing archaea dominate autotrophic ammonia oxidation in...

Urease gene-containing Archaea dominate autotrophicammonia oxidation in two acid soils

Lu Lu1,2 and Zhongjun Jia1*1State Key Laboratory of Soil and SustainableAgriculture, Institute of Soil Science, Chinese Academyof Sciences, Nanjing, 210008, Jiangsu Province, China2University of Chinese Academy of Sciences, Beijing,100049, China.

Summary

The metabolic traits of ammonia-oxidizing archaea(AOA) and bacteria (AOB) interacting with their envi-ronment determine the nitrogen cycle at the globalscale. Ureolytic metabolism has long been proposedas a mechanism for AOB to cope with substratepaucity in acid soil, but it remains unclear whetherurea hydrolysis could afford AOA greater ecologicaladvantages. By combining DNA-based stable isotopeprobing (SIP) and high-throughput pyrosequencing,here we show that autotrophic ammonia oxidation intwo acid soils was predominately driven by AOA thatcontain ureC genes encoding the alpha subunit of aputative archaeal urease. In urea-amended SIP micro-cosms of forest soil (pH 5.40) and tea orchard soil(pH 3.75), nitrification activity was stimulated signifi-cantly by urea fertilization when compared with water-amended soils in which nitrification resulted solelyfrom the oxidation of ammonia generated throughmineralization of soil organic nitrogen. The stimu-lated activity was paralleled by changes in abundanceand composition of archaeal amoA genes. Time-course incubations indicated that archaeal amoAgenes were increasingly labelled by 13CO2 in bothmicrocosms amended with water and urea. Pyrose-quencing revealed that archaeal populations werelabelled to a much greater extent in soils amendedwith urea than water. Furthermore, archaeal ureCgenes were successfully amplified in the 13C-DNA,and acetylene inhibition suggests that autotrophicgrowth of urease-containing AOA depended onenergy generation through ammonia oxidation. Thesequences of AOB were not detected, and activeAOA were affiliated with the marine Group 1.1a-

associated lineage. The results suggest that ureolyticN metabolism could afford AOA greater advantagesfor autotrophic ammonia oxidation in acid soil, butthe mechanism of how urea activates AOA cellsremains unclear.

Introduction

The ubiquity of ammonia-oxidizing archaea (AOA)across a wide variety of environments implies that meta-bolically distinct AOA may be adapted to life under physi-cochemically contrasting niches on Earth (Erguder et al.,2009). The availability of ammonia substrate might beone of the most important factors that led to the meta-bolic divergence of ammonia oxidizers. This is clearlyevidenced by the dependence of ammonia-oxidizing bac-teria (AOB) on a ureolytic function under acidic condi-tions (Burton and Prosser, 2001). A recent study indeeddemonstrated that the first soil AOA isolate could growon urea as an alternative energy source (Tourna et al.,2011); however, it remains unclear whether ureoltyticmetabolism may also afford AOA greater ecologicaladvantage for nitrification in acid soil. The concentrationof ammonia substrate declines exponentially withdecreasing pH due to the ionization of ammonia toammonium. In acid soil with pH < 5.5, ammonia concen-tration is generally three orders of magnitude lower thanthe threshold value of 1.9–4.2 mM required for the growthof AOB (Koops and Pommerening-Röser, 2001). Ureoly-sis has long been proposed as a mechanism for nitrifi-cation in acid soil by AOB capable of using urea as asource of ammonia (De Boer et al., 1989). For instance,AOB isolated from acid soil are generally ureolytic, and aureolytic physiological function enables AOB to grow atlower pH (De Boer and Kowalchuk, 2001). Environmen-tal metagenomics has revealed urea utilization genes inassociation with AOA in marine habitats (Hallam et al.,2006; Konstantinidis et al., 2009; Yakimov et al., 2011;Tully et al., 2012), but it remains unknown whether mostsoil AOA have the genetic ability for ureolytic N metabo-lism to enhance archaeal nitrification in soil (Lu et al.,2012).

The growth of Nitrososphaera viennensis on urea inbatch culture suggests that ureolytic metabolism of AOAmight occur in natural environments (Tourna et al., 2011).The application of pig manure stimulated the growth of

Received 21 July, 2012; revised 3 December, 2012; accepted 5December, 2012. *For correspondence. E-mail [email protected]; Tel.(+86) 25 8688 1311; Fax (+86) 25 8688 1000.

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Environmental Microbiology (2012) doi:10.1111/1462-2920.12071

© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd

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AOA in an agricultural soil, providing the first hint thatarchaeal ammonia oxidation is supported by hydrolysis ofurea (Schauss et al., 2009). The addition of urea to anacidic forest soil stimulated nitrification activity, which wasparalleled by the increase in abundance of archaealamoA genes (Levicnik-Höfferle et al., 2012). Moreover, itis intriguing that nitrification activity in acid soils is notaffected by inorganic ammonium amendment (Levicnik-Höfferle et al., 2012) and soil pH elevation (De Boer et al.,1989). This implies a mechanism for sequential intracel-lular generation and oxidation of ammonia through ureahydrolysis that is independent of extracellular pH (DeBoer et al., 1989; Burton and Prosser, 2001). It is note-worthy that very recently urea-fuelled nitrification of che-moautotrophic AOA was observed in polar seas (Alonso-Sáez et al., 2012), although Nitrosopumilus maritimusand Nitrosoarchaeum limnia showed no genetic potentialto grow on urea (Walker et al., 2010; Blainey et al., 2011).Our previous results demonstrated that urea-based nitri-fication likely occurred under in situ field conditions, andwas most likely ascribed to AOA in two acid soils (Lu et al.,2012). The role of AOB in these soils and the question ofwhether the active AOA in soil contain genes encoding theurease enzyme remained to be shown. DNA-based stableisotope probing (SIP) is a powerful technique to linkfunctional activity with taxonomic identity of active micro-organisms in complex environment (Radajewski et al.,2000). Recent studies demonstrate that high-throughputsequencing provides a new strategy for interpretation of13C-labelled microorganisms with unprecedented levels ofcoverage (Xia et al., 2011; Nelson and Carlson, 2012).Therefore, in this study a microcosm-based SIP experi-ment was employed to link nitrification with the ammoniaoxidizers in the two acid soils described previously (Luet al., 2012), and to assess whether there is geneticpotential of active ammonia oxidizers for ureolytic Nmetabolism.

Results

Soil nitrification activity

In the absence of 100 Pa acetylene (C2H2), a suicidesubstrate for ammonia monooxygenase and widely usedinhibitor for soil nitrification, stepwise production of nitratewas observed in microcosms of forest (Fig. 1a) and teaorchard soils (Fig. 1b). We observed no significant differ-ence in soil nitrate production between 13CO2 labelled and12CO2 control microcosms (Fig. 1a and b) as reportedpreviously (Jia and Conrad, 2009). The mean value of soilnitrification activities from triplicate 13CO2 and 12CO2

microcosms thus was used (Table S1). As for water-amended treatment, nitrification activity resulted solelyfrom the oxidation of ammonia released through minerali-zation of soil organic nitrogen, and was estimated to be

0.95 and 1.21 mg NO3--N g-1 d.w.s. day-1 in forest and tea

orchard soils after incubation for 56 days respectively(Table S1). The production rate of soil nitrate from 0 to 28days was similar to that between 28 to 56 days in bothacid soils. As for urea-amended microcosms, 100 mgurea-N g-1 d.w.s. instead of water was added once a weekto soil microcosms, and urea-amended microcosmreceived a total of 800 mg urea-N g-1 d.w.s. over the incu-bation course of 56 days (Fig. 1a and b). Nitrificationactivity was significantly stimulated by urea fertilizationwith 4.13 and 2.01 mg NO3

--N g-1 d.w.s. day-1 in forest andtea orchard soils respectively (Table S1). In stark contrastto water-amended soils, nitrification activity exhibitedclear biphasic kinetics in urea-amended microcosms. Forinstance, nitrate was formed at a rate of 2.33 mg NO3

--N g-1 d.w.s. day-1 in forest soil from 0 and 28 days, whilea significantly higher rate of 5.93 mg NO3

--N g-1 d.w.s.day-1 was observed between 28 and 56 days (Table S1).Acetylene completely abolished the production of nitratein both soils.

Soil mineralization activity

Mineralization activity was evaluated as the increase inammonium generated solely from soil organic nitrogen inmicrocosms where ammonia oxidation by nitrifying com-munities was eliminated by acetylene addition. Ammo-nium concentrations showed an increasing trend inmicrocosms of forest (Fig. 1c) and tea orchard (Fig. 1d)soils following the addition of water or urea. For instance,soil NH4

+-N concentrations increased from 3.01 mg N g-1

d.w.s. at day 0, to 25.9 and 48.4 mg N g-1 d.w.s. at days 28and 56 respectively in water-amended microcosms offorest soil (Fig. 1c). Mineralization activities were esti-mated to be 0.83 and 1.70 mg NH4

+-N g-1 d.w.s. day-1 inforest and tea orchard soils respectively (Table S1).Repeated fertilization of forest soil with urea resulted insignificant accumulation of soil NH4

+-N concentrations upto 471 and 1484 mg N g-1 d.w.s. at day 28 and 56 respec-tively (Fig. 1c). Similar results were obtained for teaorchard soil after incubation with urea for 28 days (298 mgNH4

+-N g-1 d.w.s.) and 56 days (1464 mg NH4+-N g-1

d.w.s.) (Fig. 1d). The release of ammonium from urea wasexcluded for the calculation of mineralization activities,and the mineralization rate of ammonium from soil organicnitrogen showed clear biphasic patterns in urea-amendedmicrocosms. Mineralization activities increased dramati-cally from 2.47 mg NH4

+-N g-1 d.w.s. day-1 between 0 and28 days, to 21.9 mg NH4

+-N g-1 d.w.s. day-1 between 28and 56 days in forest soil (Table S1). As for tea orchardsoil, mineralization activity was significantly higherbetween 28 and 56 days (16.6 mg NH4

+-N g-1 d.w.s.day-1), when compared with that between 0 and 28 days(6.42 mg NH4

+-N g-1 d.w.s. day-1).

2 L. Lu and Z. Jia

© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

Soil AOA communities

The abundance of AOA was evaluated by quantitativePCR of amoA genes in soil microcosms incubated for0, 28 and 56 days following the addition of water or urea.The copy number of archaeal amoA genes increasedsignificantly from 1.35 ¥ 108 at day 0, to 4.63 ¥ 108

and 5.50 ¥ 108 in water-amended microcosms of forestsoil after incubation for 28 and 56 days respectively(Fig. 2a). Urea fertilization led to significantly higherabundances of archaeal amoA genes at days 28 and 56,representing 15- and 23-fold increases respectively.Similar results were observed for tea orchard soil(Fig. 2b). The amoA gene copies showed a 10-foldincrease from 2.65 ¥ 107 at day 0 to 2.59 ¥ 108 at day 56in water-amended microcosms, while a 53-fold increaseof amoA gene copies was observed in urea-amendedmicrocosms at day 56.

The composition of AOA was analysed by denaturinggradient gel electrophoresis (DGGE) fingerprinting ofamoA genes in soil microcosms. DGGE fingerprintinganalysis demonstrated highly reproducible patterns ofamoA genes among triplicate microcosms of each treat-ment (Fig. 3). There was no discernable difference inDGGE banding patterns of amoA genes between 13CO2-labelled and 12CO2-control microcosms, suggesting thatthe assimilation of 13CO2 did not result in apparent biasagainst AOA communities involved in soil nitrification. Sig-nificant changes in AOA communities were observed insoil microcosms after incubation for 56 days following theaddition of either water or urea. As for forest soil, theintensities of DGGE band-1 and band-2 were significantlyenhanced in the absence of acetylene (Fig. 3a) andsignificantly reduced in acetylene-treated microcosms,in agreement with previous findings (Lehtovirta-Morleyet al., 2011). A similar result was observed for tea orchard

Fig. 1. Change in the concentrations of nitrate (a, b) and ammonium (c, d) in the soil microcosms incubated with 12CO2, or 13CO2 or13CO2 + C2H2 for 56 days. FS and TS represent forest soil and tea orchard soil respectively. Day-56 indicates the soil microcosms incubatedfor 56 days. Urea and H2O denote the soil microcosms that received 100 mg urea-N g-1 d.w.s. or an equal volume of H2O on a weekly basisrespectively. 13CO2 + C2H2 represents the soil microcosms incubated with 13CO2 in the presence of a nitrification inhibitor, acetylene (C2H2). Alltreatments were conducted in triplicate microcosms. The error bars represent the standard errors of the mean of the triplicate microcosms.Different letters above the columns for each treatment indicate a significant difference (P < 0.001) using ANOVA.

Urea-linked archaeal ammonia oxidation in acid soil 3


soil (Fig. 3b). The intensity of DGGE band-6 was stimu-lated in soil microcosms that received water or urea andwas inhibited when nitrification activity was blocked byacetylene.

13C-labelled active AOA in soils

Real-time quantitative PCR indicated the increasing label-ling of archaeal amoA genes (Fig. 4) during active soilnitrification (Fig. 1). In acetylene-treated microcosms asSIP controls (13CO2 + C2H2-Day-56) of forest soil, thehighest copy number of archaeal amoA genes wasobserved in the ‘light’ fractions typical for the unlabelledDNA with buoyant density of 1.715 g ml-1. However, in the13CO2-labelled microcosms incubated without acetylenefor 28 and 56 days, archaeal amoA genes peaked in the‘heavy’ DNA fractions with buoyant densities of 1.725 and1.737 g ml-1 respectively (Fig. 4a). Similar results wereobtained for tea orchard soil (Fig. 4b), suggesting thegenomes of AOA cells were gradually labelled over thecourse of incubation for 56 days. As for urea-amendedmicrocosms, the results remained unchanged for bothacid soils (Fig. 4c and d), and archaeal amoA genespeaked in the ‘heavy’ DNA fractions only when nitrificationwas not inhibited by C2H2.

Pyrosequencing analysis of 16S rRNA genes at thewhole microbial community level was further performed inDNA gradient fractions from the labelled microcosms(13CO2-Day-28 and 56) and the control microcosms(13CO2 + C2H2-Day-56 and 12CO2-Day-56) of both soils(Fig. 5). About 303 741 and 451 475 high-qualitysequence reads were obtained from SIP microcosms offorest soil (Table S2) and tea orchard soil (Table S3)respectively. Across the SIP gradients from DNA fractions4–9, the highly enriched archaeal 16S rRNA gene

Fig. 2. Change in the copy number of archaeal amoA genes in themicrocosms of forest soil (a) and tea orchard soil (b) incubated with12CO2, or 13CO2 or 13CO2 + C2H2 for 56 days. Day-56-13CO2 + C2H2

denotes the soil microcosm incubated for 56 days with 13CO2 in thepresence of a nitrification inhibitor, acetylene (C2H2). The error barsrepresent the standard errors of the means of the microcosmtriplicates. All other designations are the same as thosein Fig. 1.

Fig. 3. DGGE fingerprints of the archaeal amoA genes in microcosms of forest soil (a) and tea orchard soil (b) incubated with 12CO2, or 13CO2

or 13CO2 + C2H2 for 56 days. Day-56-Urea and Day-56-H2O indicate the incubations for 56 days of soil microcosms that received 100 mgurea-N g-1 d.w.s. or an equal volume of H2O on a weekly basis respectively. The numbers 1, 2 and 3 represent the microcosm triplicates ofeach treatment. All other designations are the same as those in Figs 1 and 2.

4 L. Lu and Z. Jia


sequences were observed in the ‘heavy’ DNA fractions(i.e. fractions 4–7) only from the labelled microcosm andnot from control microcosms (Fig. 5). For instance, 21.7%of the total 16S rRNA genes in DNA fraction-4 wereassigned to archaeal communities in the labelled micro-cosms, whereas archaeal 16S rRNA genes accountedonly for 0.6% of the total 16S rRNA genes in controlmicrocosms of forest soil incubated with acetylene(Fig. 5a). In urea-amended microcosms, the proportionalincrease of archaeal 16S rRNA genes was stimulated upto 73.5% in the ‘heavy’ DNA fraction-5 from the labelledmicrocosms (Fig. 5c), while low frequency of archaeal16S rRNA genes were observed in control microcosms of13CO2 + C2H2-Day-56 (0.1%) and of 12CO2-Day-56 (0.8%).Similar results were obtained for tea orchard soil. In thelabelled microcosms amended with water, the relativefrequency of archaeal 16S rRNA genes was up to 9.1% ofthe total 16S rRNA sequence reads in the ‘heavy’ DNA

fraction (Fig. 5b). Urea fertilization led to significant stimu-lation of archaeal proportions up to 36.7% in the ‘heavy’DNA fraction-5 from the labelled microcosms after incu-bation for 56 days (Fig. 5d).

Phylogenetic analysis of active AOA in soils

Phylogenetic analysis revealed that DGGE band-2 andthe cloned amoA genes in the 13C-DNA from forest soilwere most closely affiliated with N. devanaterra, while13C-labelled amoA genes and DGGE band-6 in teaorchard soil formed an unique cluster which is distantlyrelated to N. devanaterra within the Group 1.1a-associated lineage (Fig. 6). Clone library construction ofarchaeal 16S rRNA genes further demonstrated thatN. devanaterra-like sequences dominated archaeal popu-lations in the 13C-DNA from forest soil microcosms, whileall cloned archaeal 16S rRNA genes in the 13C-DNA were

Fig. 4. Quantitative distribution of the archaeal amoA genes in the fractionated DNA across the entire density range of the SIP gradients fromsoil microcosms amended with water (a, b) or urea (c, d) over an incubation course of 56 days. The normalized data are the ratios of thegene copy number in each DNA gradient to the maximum quantities from each treatment. The standard error of the triplicate samples isshown, with some error bars smaller than the symbol size. All other designations are the same as those in Figs 1 and 2.



N. devanaterra-related in tea orchard soil (Fig. 7). Similarresults were observed for archaeal 16S rRNA genes frompyrosequencing reads in the 13C-DNA (Fig. 7). As forforest soil, 99% of archaeal 16S rRNA gene reads showedhigh similarity with N. devanaterra in urea-amendedmicrocosms (Table S4), while N. devanaterra-likesequences comprised 96% of the total archaeal 16SrRNA genes in water-amended microcosms. As for teaorchard soil, N. devanaterra-related sequences domi-nated archaeal 16S rRNA gene reads in the 13C-labelledDNA, whereas ~ 4% of the 13C-labelled 16S rRNAsequences in water-amended microcosms were related toCrenarchaeota Group 1.1c (Fig. 7). None of the 755 216sequence reads were affiliated with AOB, consistent withour previous findings (Lu et al., 2012).

The ureC genes encoding the alpha subunit of a puta-tive archaeal urease were successfully amplified in the13C-DNA fractions from the labelled microcosms of forestand tea orchard soils, but not in the ‘heavy’ DNA fractionsfrom the control microcosms of 13CO2 plus acetylene or12CO2 treatments (Fig. S1). Clone libraries of putativearchaeal ureC genes from 13C-DNA were constructed.Phylogenetic analysis demonstrated that the ureC genesin this study formed two distinct clusters that containedsequences solely from either tea orchard soil or forest soil(Fig. 8). The closest relative of archaeal ureC genes wasN. viennensis, the only soil AOA isolate containing ureasegenes determined so far (Tourna et al., 2011). ArchaealureC genes in soils were phylogenetically distantly asso-ciated with the ureC of Cenarchaeum symbiosum (Hallam

Fig. 5. Relative frequency of the archaeal 16S rRNA gene reads in the fractionated DNA from microcosms amended with water (a, b) or urea(c, d) over an incubation course of 56 days. The relative frequency is expressed as the percentage of the archaeal 16S rRNA genes to thetotal 16S rRNA gene reads of the total microbial communities in each DNA fraction. The numbers 4–9 represent the fractionated DNA withbuoyant density ranging from heavy to light (the higher the number, the lower the buoyant density), following the total DNA ultracentrifugationfrom the labelled or control microcosms. All other designations are the same as those in Fig. 4.

6 L. Lu and Z. Jia


et al., 2006) and putative archaeal urease gene fragmentsfrom marine habitats (Konstantinidis et al., 2009; Yakimovet al., 2011).

Discussion

Metabolic diversity can afford AOA selective advantagesfor colonization across a wide variety of environmentson Earth from Yellowstone hot springs (de la Torre et al.,2008) to frozen Mount Everest soil (Zhang et al.,2009). The results of this study demonstrate that AOAhave the genetic potential for ureolytic N metabolism inacid soil in which ammonia concentration is remarkablylow. The stimulation of nitrification activity by urea ferti-

lization coupled well with changes of AOA communitiesin two acid soils. DNA-SIP indicated that nitrificationwas predominantly carried out by AOA affiliated withN. devanaterra within the marine Group 1.1a lineage.The actively growing cells of AOA contain ureC genesencoding the alpha subunits of a putative archaealurease enzyme, and acetylene inhibition suggests thatthe replication of urease-containing AOA cells dependedon energy generation from ammonia oxidation, consist-ent with recent findings of N. viennensis in liquid batchculture (Tourna et al., 2011). AOB sequences were notdetected in the acid soils tested and the metabolicmechanism whereby urea activates AOA cells remainsunclear.

Fig. 6. Phylogenetic tree showing the relationship of the archaeal amoA genes in the 13C-DNA from the forest soil (�) and tea orchard soil(�) microcosms to those in the GenBank. Clone library was constructed from the 13C-labelled ‘heavy’ DNA fractions (HF) of the labelledmicrocosms incubated with urea for 56 days. The FS-HF-AOA-Clone-1 designation represents that the archaeal amoA gene sequence ofclone-1 was retrieved from the ‘heavy’ DNA fractions of the labelled forest soil microcosms. The heavy DNA fractions 4 or 5 were used forclone library construction. The archaeal amoA genes of the DGGE bands 2 and 6 in Fig. 3 are also included for the phylogenetic treeconstruction. Bootstrap values higher than 85% are indicated at the branch nodes. The scale bar represents two changes per 100 nucleotidepositions.



DNA-SIP is a powerful technique to link nitrificationactivity with the taxonomic identity of ammonia oxidizersthat usually comprise only a tiny fraction of the total micro-bial communities in soil. High-throughput pyrosequencingcould greatly facilitate the interpretation of isotopicallyenriched DNA from less abundant AOB and AOA in soil(Xia et al., 2011). Without prior knowledge of which SIPgradient fractions are highly enriched with the labelledDNA of active microorganisms following isopycnic densitygradient ultracentrifugation (Lueders et al., 2004), pyrose-quencing of the total 16S rRNA genes revealed a remark-

able enrichment of archaeal communities in the ‘heavy’DNA fractions from the labelled microcosms and not fromcontrol microcosms. For instance, archaeal 16S rRNAgenes accounted for 21.7% of the total sequence reads inthe labelled treatment, while only 0.6% was observed inthe unlabelled control. Urea fertilization further facilitatedthe enrichment of archaeal communities up to 73.5% inforest soil. Acetylene inhibition completely eliminated theenrichment of archaeal communities in the ‘heavy’ DNAfraction, despite the presence of 13CO2 substrate (Fig. 5).These results are consistent with the distribution patterns

Fig. 7. Phylogenetic tree showing the relationship of archaeal 16S rRNA genes in the 13C-DNA from forest soil (�) and tea orchard soil (�)microcosms to those in the GenBank. The archaeal 16S rRNA pyrosequencing reads in the 13C-labelled DNA are included from theurea-amended microcosms, indicated in red, and water-amended microcosms, indicated in green. The 454-FS-Urea-HF-OTU-1-4176-(55%)designation indicates that the OTU-1 contains 4176 reads, with sequence similarities of > 97%, and accounts for 55% of the total archaeal16S rRNA reads in the 13C-DNA fractions isolated from forest soil microcosms after incubation with urea for 56 days. One representativesequence from each OTU was extracted using the mothur software package for tree construction. Clone library was constructed from the13C-DNA of the labelled microcosms incubated with urea for 56 days. The cloned archaeal 16S rRNA genes, with an expected size of 920 bp,are also included. The FS-HF-Archaea-Clone-1 (9) designation represents that the OTU-1 containing nine sequences, with sequencesimilarities of > 97%, was retrieved from the ‘heavy’ DNA fractions of the labelled forest soil microcosms amended with urea. Bootstrap valueshigher than 70% are indicated at branch nodes. The scale bar represents two changes per 100 nucleotide positions.

8 L. Lu and Z. Jia


of archaeal amoA gene abundances in DNA templateacross the entire density of SIP gradients (Fig. 4), sug-gesting that autotrophic growth of AOA depended onenergy that was generated from ammonia oxidation. Fur-thermore, the universal primer pair of 515F and 907R isgenerally thought to favour the amplification of bacterialbut not archaeal 16S rRNA genes (Lane, 1991; Stubner,2002). The exceptionally high proportions of archaeal 16SrRNA genes in the ‘heavy’ DNA fractions thus providestrong evidences for 13CO2 assimilation by archaeal com-munities that catalysed ammonia oxidation in the acidsoils tested (Fig. 4).

The increasing line of evidence has suggested thatAOA play an important role in autotrophic ammoniaoxidation in acid soil. Statistical analyses indicate therewas a significantly positive correlation between archaealamoA gene copies and soil nitrate production in this study(Fig. S2), and the estimated cell-specific rate of ammoniaoxidation by AOA fell well within those reported previously(Table S5), implying archaeal predominance of ammoniaoxidation in the two acid soils tested. This could be wellexplained by the unusually high substrate affinity of AOAbecause ammonia concentration is generally too low tosupport bacterial nitrification in acid soil. Ammonia ratherthan the ammonium ion acts as the substrate for ammoniaoxidizers because the Michaelis constant calculated fromammonia plus ammonium exponentially declines withdecreasing pH value but remains almost unchangedwhen calculated as a function of ammonia concentration(Suzuki et al., 1974; Frijlink et al., 1992). A recent studyhas shown that the acidophilic N. devanaterra has the

greatest substrate affinity determined so far for anyknown ammonia oxidizer (Lehtovirta-Morley et al., 2011).Ammonia concentrations were estimated to be 7.0 nM intea orchard soil and 221 nM in forest soil, which is suffi-ciently high for the growth of N. devanaterra but not ofAOB in culture. Phylogenetic analyses suggest thatN. devanaterra-like AOA dominated the nitrification activi-ties in forest soil, whereas N. devanaterra-related AOAwere active ammonia oxidizers in tea orchard soil on thebasis of the labelled amoA (Fig. 6) and 16S rRNA genes(Fig. 7), in agreement with our previous findings (Lu et al.,2012). These results indicate that DGGE band-2 andband-6 could largely reflect the changes of AOA commu-nities upon urea fertilization in forest soil (Fig. 3a) and teaorchard soil (Fig. 3b) respectively, and the DGGE bandsin the upper gel likely represented the artefacts becausethe excised bands could not be amplified for sequencing.It appears that archaeal predominance in soil nitrificationis frequently associated with N. devanaterra-relatedorganisms within the marine Group 1.1a (Zhang et al.,2010; 2012; Lehtovirta-Morley et al., 2011). However, itremains to be shown whether N. devanaterra in culturepossesses a ureolytic function to support nitrification, inaddition to having a high affinity for ammonia (Lehtovirta-Morley et al., 2011).

The results of this study demonstrate that autotrophicammonia oxidation of AOA was significantly stimulated inacid soils by urea fertilization. Urea hydrolysis has longbeen proposed as a mechanism to enable nitrification inacid soil. Batch study experiments support a mechanismfor intracellular urea hydrolysis and subsequent oxidation

Fig. 8. Phylogenetic tree showing therelationship of putative archaeal ureC genesin the 13C-DNA from forest soil (FS) and teaorchard soil (TS) microcosms to those in theGenBank. The ‘heavy’ DNA fractions 4 and 5were used for construction of the archaealureC gene clone library. Bootstrap valueshigher than 90% are indicated at branchnodes. The scale bar represents two changesper 100 nucleotide positions.



of the released ammonia for autotrophic growth of AOB atlowered pH (Burton and Prosser, 2001). 15N isotopetracing indicated that urea-based nitrification of archaealammonia oxidizers could likely occur under in situ condi-tions (Lu et al., 2012). Our results demonstrate that thenet nitrification activity was stimulated up to fourfold inmicrocosms of acid soils upon urea fertilization (Table S1).The presence of ureC genes in active AOA in acid soildetected in this study indicates that intracellular ureahydrolysis could be an important mechanism behind theobserved urea stimulation of AOA. Nevertheless, inacetylene-treated microcosms the remarkable accumula-tion of soil inorganic ammonium (Fig. 1) has suggested arapid hydrolysis of urea which is most likely ascribed toextracellular urease activity because ureolytic metabolismis a common feature in soil microbes (Mobley et al.,1995). It remains unknown to what extent intracellularurea hydrolysis could contribute to archaeal nitrification inacid soils tested. An inhibition assay that specifically pre-vents the entry of urea into the cells or inhibits intracellularurea hydrolysis would provide further evidence for theecological significance of this ureolytic N metabolism insoil AOA. Furthermore, there was no significant phyloge-netic divergence between active AOA in microcosmstreated with water and urea (Figs 6 and 7). This impliesthat the active AOA in this study were capable of growingwithout urea even though they contain urease genes thatcould potentially facilitate nitrification in acid soil.

The stimulation of nitrification activity by urea fertiliza-tion was accompanied by the enhanced mineralization ofsoil organic nitrogen. It has been shown that nitrificationoccurred only when net mineralization started, despite thepresence of an excess of ammonium ion (De Boer et al.,1988; Stopnisek et al., 2010; Levicnik-Höfferle et al.,2012). Mineralization activity showed clear biphasic pat-terns that coupled well with nitrification kinetics in soilmicrocosms amended with urea (Table S1). For instance,mineralization activities increased from 0.77 mg NH4

+-N g-1 d.w.s. day-1 between 0 and 28 days, to 14.9 mgNH4

+-N g-1 d.w.s. day-1 between 28 and 56 days in forestsoil, while nitrification activity was stimulated from 2.33 to5.93 mg NO3

--N g-1 d.w.s day-1. It appears that nitrificationin acid soils was fuelled by the oxidation of ammoniagenerated from soil mineralization. This observation lendsstrong support for a mechanism that nitrification in acidsoil was activated by direct diffusion of ammonia from anadjacent ammonifying cell into the cells of nitrifying organ-isms within soil aggregates (De Boer and Kowalchuk,2001). In fact, all other DNA-SIP studies available so fardemonstrate that archaeal predominance in soil nitrifica-tion resulted from oxidation of the ammonia releasedthrough mineralization of soil organic nitrogen (Zhanget al., 2009; 2012; Lehtovirta-Morley et al., 2011). Itseems plausible that intimately interacting associations

might occur among distinctly different processes of soilnitrogen transformation, but the underlying mechanismsremains largely unknown in particular for soil AOA.

Taken together, the results of this study demonstratethat nitrification activity was predominantly catalysed byAOA that contain ureC genes encoding the alpha subunitof an archaeal urease in two acid soils tested. Archaealammonia oxidation was supported by hydrolysis of urea,but ureolytic N metabolism of AOA remains elusive in acidsoil. Acetylene inhibition demonstrated that autotrophicgrowth of AOA depended on the energy generation fromammonia oxidation. The results of this study suggest thatAOA may play more important roles for nitrogen cyclingthan previously recognized in urea-rich environment.

Experimental procedures

Site description and soil sampling

Tea orchard soil (pH 3.75) was collected from the long-termexperimental field at the Tea Research Institute of theChinese Academy of Agricultural Sciences, Hangzhou City(120°09′E, 30°14′N) in Eastern China, where the famousChinese premium tea, Westlake Longjin, is produced. Thefield plot received approximately 600 kg urea-N·ha-1·a-1, withthree to four split dressing application, and was also appliedwith 1125 kg·ha-1 organic fertilizer (mainly rapeseed cake)over the last decades (Han et al., 2007). In addition, forestsoil (pH 5.45) was also sampled from an adjacent area fromwhich the tea orchard was converted in 1974. The forest sitewas about 80 m away from the tea orchard fields. Both soilswere developed on quaternary red earth, and classified asUltisols. The soil was loamy clay with Anshan quartz-freeporphyry parent material, and the dominant clay mineralsconsist of kaolinite, chlorite, as well as Fe and Al oxides. Theannual mean temperature is 17°C, ranging from 1.7°C inJanuary to 33.0°C in July. The annual mean precipitation is1533 mm, with 74% of the total rainfall occurring during thetea growing season from March to September. Soil samplingwas carried out in triplicate fields using a hand trowel. Threesubsamples from each field were randomly obtained at adepth of 15 cm. Plant residues and other materials, such asstones and obvious macrofauna, were removed before thesubsamples from each field were homogenized through a2 mm mesh. The soil samples were kept at -4°C until use.Moreover, the soil characteristics were described previously(Lu et al., 2012).

DNA–SIP microcosms

For both acid soils, three treatments were performed includ-ing 13CO2-labelled microcosms, 12CO2-control microcosms,and 13CO2 + C2H2 control microcosms. Pairwise comparisonbetween the 13CO2 and 12CO2 treatments was used to assesswhether AOA assimilated CO2 for autotrophic growth,whereas the 13CO2 + C2H2 treatment was employed to assesswhether the 13CO2 assimilation by AOA depends on theenergy generation from ammonia oxidation (Xia et al., 2011)that could be completely abolished by acetylene (Berg et al.,

10 L. Lu and Z. Jia


1982). SIP microcosm incubations were performed asdescribed previously (Lu et al., 2012), except for the use of alabelled substrate and/or acetylene nitrification inhibitor in thecurrent study.

The tea orchard and forest soil DNA-SIP microcosms wereconstructed in triplicate, as described previously (Jia andConrad, 2009), with slight modification. Each microcosm con-tained ~ 6.0 g of fresh soil (equivalent to 5.0 g dry weightgram soil, i.e. d.w.s.) in a 120 ml serum bottles capped withblack butyl stoppers for incubation at 28°C in darkness for 8weeks. Urea fertilization using 100 mg urea-N g-1 d.w.s. wasperformed once a week through dropwise addition of freshlymade urea solution. Thus, the soil microcosms received atotal of 800 mg urea-N g-1 d.w.s. over an incubation period of8 weeks. The control treatment was carried out by addingsterilized water instead of urea to the soil microcosms. Theheadspace of the bottle was flushed weekly with synthetic air(20% O2, 80% N2) for 45 s to maintain oxic conditions. Waterloss was replaced immediately after flushing, and the treat-ments, such as the addition of urea-N, sterilized water, 5%CO2, and/or 100 Pa C2H2, were renewed if stated. The head-space CO2 concentration was measured using a gas chro-matograph equipped with a thermal conductivity detector(Shimadzu GC-14B, Japan). A destructive sampling wascarried out after incubation for 0, 28, and 56 days. About 2.0 gof fresh soil was removed from each triplicate microcosmsand immediately frozen at -20°C for molecular analysis. Therest of the soil was homogenized with 10 ml of 2 M KCl byshaking at 200 r.p.m. for 60 min, and then passed through afilter paper for the determination of NH4

+-N, NO2--N, and

NO3--N using a Skalar SAN Plus segmented flow analyser

(Skalar, Breda, the Netherlands). The nitrite concentrationwas below the detection limit. Nitrification activity wasassessed by the changes in soil NO3

--N content, whereasNH4

+-N formation was used for the soil mineralization activityevaluation based on the oven-dried soil weight.

The total DNA from 0.5 g (fresh weight) of each soil wasextracted using a FastDNA spin kit for soil (Qbiogene, Irvine,CA) according to the instructions of the manufacturer. Celllysis was performed through vigorous shaking in a beadbeater at a rate of 6.0 for 40 s. DNA pellets were washedusing 5.5 M guanidine thiocyanate solution to remove humicsubstance contamination. The purified DNA was eluted with100 ml of TE buffer. DNA quality and quantity were checkedusing a NanoDrop spectrophotometer (NanoDrop Technolo-gies, Wilmington, DE). The soil DNA was stored at -20°C.

Real-time quantitative PCR and DGGE fingerprinting

AOA abundance was assessed by quantifying the amoAgene copies on a CFX96 Optical Real-Time DetectionSystem (Bio-Rad Laboratories, Hercules, CA) using the Arch-amoAF/Arch-amoAR primer pairs (Francis et al., 2005). Thereal-time PCR standard was generated using a plasmid DNAfrom one representative clone containing archaeal amoAgenes. A standard template dilution series from 4.37 ¥ 101 to4.37 ¥ 107 copies per assay were used. In addition, a seriesdilution of soil DNA extract was also used to assess whethera PCR inhibition by humic substance co-extraction occurredin the acid soils. The amoA gene copies decreased propor-tionally with the diluted soil DNA template concentrations. A

10-fold diluted soil DNA was used for subsequent analysis.Quantitative PCR was performed in a reaction mixture con-taining 1¥ SYBR® Premix Ex Taq™ (TaKaRa Biotech, Dalian,China), 0.5 mM of each primer, and approximately 1.0–10 ngDNA template. The amplification was started by denaturing at95°C for 3 min, followed by 40 cycles of 30 s at 95°C, 30 s at55°C, 30 s at 74°C, and plate read at 85°C (Table S6). Blankswere run with water as template instead of soil DNA extract.Real-time PCR was performed in triplicate. Amplification effi-ciencies of 96–103% were obtained with R2 values of 0.996–0.999. The specific amplification was verified using themelting curve analysis. The amplification always resulted in asingle peak. Bacterial amoA genes were not detected despitethe use of a range of degenerate primers and PCR conditions(Stephen et al., 1999; Nicolaisen and Ramsing, 2002), aspreviously reported (Stopnisek et al., 2010; Lu et al., 2012).

AOA composition was analysed by a DGGE of the amoAgenes using the D-Code system (Bio-Rad Laboratories,Hercules, CA), as described previously (Lu et al., 2012).Archaeal amoA genes were amplified using theCrenamoA23f and CrenamoA616r primer pairs (Nicol et al.,2008; Lehtovirta-Morley et al., 2011). The PCR reactionmixture consisted of 0.2 mM of each deoxynucleoside tri-phosphate, 1¥ PCR buffer (Mg2+ Plus), 0.1 mM of each primer,1.25 U of TaKaRa Taq HS polymerase (TaKaRa Biotech,Dalian, China), and 2 ml of soil DNA template in a final volumeof 50 ml. PCR reaction was carried out in a thermal cycler(Bio-Rad Laboratories, Hercules, CA) using the following pro-gramme: 5 min of incubation at 94°C, followed by 35 cycles of94°C for 30 s, 56°C for 45 s, and 72°C for 45 s, with a 7 minextension at 72°C (Table S6). The PCR products werechecked using 1.2% agarose gel to ascertain the specificity ofarchaeal amoA gene amplification with a size of ~ 629 bp.The 150 to 200 ng PCR amplicons were separated using 6%(wt/vol) polyacrylamide [acrylamide–bisacrylamide (37.5:1)]gels with a denaturing gradient of 30–50% (100% denaturantcontains 7 M urea and 40% formamide). A 1 mm thick gelwith 20 wells was poured from bottom to top using a gradientformer and peristaltic pump at a speed of 4.5 ml min-1. A5.0 ml stacking gel containing no denaturants was subse-quently added on top before polymerization to insert a combto make wells. An electrophoresis separation of archaealamoA gene amplicons was carried out using a 0.5¥ Tris-acetate-EDTA buffer at 80 V for 17 h at 60°C. The gels werestained with 1:10000 (v/v) SYBR Green I for 30 min, and thenscanned with a Molecular Imager FX using Quantity Onesoftware package (Bio-Rad Laboratories, Hercules, CA,USA). The bacterial amoA genes were not analysed becauseof the difficulties in obtaining the PCR amplicons, as men-tioned above.

The DGGE fingerprinting patterns of the amoA genes wereconsistent with our previous results. The representativebands were excised for sequencing as reported previously(Lu et al., 2012). The re-amplified PCR product of the DGGEbands was cloned using pEASY-T3 vector (TransGenBiotech, Beijing, China) according to the instructions of themanufacturer. The clones containing the exact archaealamoA gene insert from each DGGE band were sequenced(Invitrogen, Shanghai, China). DNASTAR software was usedto check the clone sequences manually for subsequentanalyses.



Isopycnic centrifugation and gradient fractionation

A density gradient centrifugation was performed for eachtreatment to separate the 13C-labelled DNA from the 12C-DNAin triplicate SIP microcosms. About 3.0 mg of soil DNA extractwas mixed with the CsCl stock solution. The DNA mixturebuoyant density was adjusted to 1.725 g ml-1 using the gra-dient buffer (pH 8.0; 100 mM Tris HCl; 100 mM KCl; 1.0 mMEDTA). The DNA was spun in 5.1 ml Beckman polyallomerultracentrifuge tubes in a Vti65.2 vertical rotor (BeckmanCoulter, Palo Alto, CA, USA) at 177 000 g for 44 h at 20°C(Neufeld et al., 2007). The centrifuged gradient was fraction-ated from bottom to top by displacing the gradient mediumwith sterile water using an NE-1000 single syringe pump(New Era Pump Systems, Farmingdale, NY, USA) (Jia andConrad, 2009). Fourteen to 15 DNA gradient fractions ofequal volumes of about 340 ml were generated, and a 6 mlaliquot of each fraction was used for refractive index meas-urement using an AR200 digital hand-held refractometer(Reichert, Buffalo, NY, USA). The buoyant density of eachDNA gradient fraction was determined as specified previously(Lueders et al., 2004). The ‘light’ DNA fractions contaminatedwith water were discarded (Freitag et al., 2006). Nucleicacids were separated from the CsCl solution by precipitationusing 2 vols of polyethylene glycol 6000 at 37°C for 1 h,followed by centrifugation at 13 000 g for 30 min. The frac-tionated DNA was further purified with 70% ethanol. Thefractionated DNA was then dissolved in 30 ml TE buffer.

Real-time quantitative PCR and pyrosequencingof 13C-DNA

A real-time quantitative PCR of amoA genes was performedin triplicate to verify the efficacy of 13C incorporation into thegenomic DNA of the AOA communities by analysing thearchaeal amoA gene distribution patterns over the entiredensity range of the SIP gradient (Jia and Conrad, 2009).This analysis was performed using a CFX96 Optical Real-Time Detection System (Bio-Rad Laboratories, Hercules,USA). The PCR conditions and primers are described above(Table S6). Amplification efficiencies of 96–101% wereobtained with R2 values of 0.996–0.999.

A bidirectional pyrosequencing was carried out using aRoche 454 GS FLX Titanium sequencer by analysing the V4regions of the 16S rRNA genes at the whole microbial com-munity level in the DNA gradient fractions 4–9 from thelabelled and control microcosms, as reported previously (Xiaet al., 2011). A slight modification was made by PCR-amplifying the 16S rRNA genes, with the universal primers515F and 907R extended as amplicon fusion primers usingthe respective primer A or B adapters, key sequence, and taqsequence. The triplicate amplicons were pooled, purified, andvisualized using 1.8% agarose gels. The concentration ofpurified PCR amplicon was determined, and then the purifiedPCR amplicons were combined in equimolar ratios into asingle tube for the pyrosequencing analysis.

The Ribosomal Database Project (RDP) online pyrose-quencing analysis resulted in 303 741 and 451 475 high-quality sequence reads for the forest soil (Table S2) and teaorchard soil (Table S3) SIP microcosms respectively. Thetaxonomy of the high-quality sequence reads was furtherassigned using the RDP classifier, with a minimum support

threshold of 60%, and the RDP taxonomic nomenclature(Cole et al., 2009). None of the 16S rRNA gene sequencesreads were related to the bacterial ammonia oxidizers. The16S rRNA genes affiliated with the putative nitrifying commu-nities were extracted for further analysis. The targetedsequence reads from the 13C-labelled ‘heavy’ DNA fractionswere clustered into an operational taxonomic unit (OTU) at97% cut-off using mothur software (Schloss et al., 2009); theOTUs containing less than 10 reads were removed. A repre-sentative sequence was then used from each OTU for phy-logenetic analysis.

Cloning, sequencing, and phylogenetic analysis

The archaeal 16S rRNA, amoA, and putative ureC genes inthe 13C-DNA were also amplified for clone library constructionfrom the forest soil and tea orchard soil SIP microcosm incu-bated with urea for 56 days (Table S6). The PCR primer pairswere Arch21f/Arch958R for the 16S rRNA genes (DeLong,1992), Arch-amoAF/Arch-amoAR for the amoA genes(Francis et al., 2005), and Crur-F155/CRUR-R1420 forthe ureC genes (Yakimov et al., 2011). The triplicate PCRamplicons were pooled and purified before cloning intothe pEASY-T3 vector (TransGen Biotech, Beijing, China)according to the instructions of the manufacturer. Escherichiacoli JM109 competent cells were used for transformation.About 10 clones containing the correct insert weresequenced for each clone library (Invitrogen, Shanghai,China). DNASTAR software package was used to check theclone sequences manually for analysis (Lu et al., 2012).

Phylogenetic analysis was performed using the MolecularEvolutionary Genetics Analysis (MEGA 4.0) softwarepackage (Tamura et al., 2007). The basic tree of sequencesfrom known AOA cultures and fosmid clones of the archaealureC genes was constructed through a neighbour-joiningalgorithm. The closest relatives to the 13C-labelled ureCgenes in this study were selected to reconstruct a phyloge-netic tree based on the Jukes–Cantor correction, and testedby bootstrap calculation (1000 replications) and a rand-omized input order of sequences. The tree topology waschecked using the neighbour-joining algorithm and theminimum evolution method. Similarly, the phylogenetic tree ofarchaeal 16S rRNA genes was constructed by including thesequences of > 1.0 kb from known AOA and the clonedsequences in this study. Moreover, the pyrosequencing 16SrRNA reads in the 13C-DNA was included for the phylogeneticanalysis. The phylogenetic analysis of the archaeal amoAand 16S rRNA genes was performed as described previously(Lu et al., 2012).

Statistical analysis

Soil nitrate concentration, ammonium concentration andamoA gene copy numbers were compared though multiple-sample comparisons using one-way ANOVA analysis followedby Student–Newman–Keulstest to check for quantitativevariance between different treatments. The two-way ANOVA

analysis was performed to examine the effect of urea fertili-zation and of soil type on nitrification activity. Pairwisecomparisons between treatments were determined with two-tailed Student’s t-tests. Pearson’s correlation analyses were

12 L. Lu and Z. Jia


performed to assess the relationships between soil nitrateproduction and amoA gene copy numbers. All analyses wereconducted using SPSS version 17.0 (IBM, Armonk, NY, USA).

Accession numbers of nucleotide sequences

The nucleotide sequences were deposited at the GenBank/DNA Data Bank of Japan (DDBJ) with Accession NumbersJX239722–JX239736 and JX239696–JX239706 for the 13C-labelled archaeal 16S rRNA genes and archaeal amoA genesin this study respectively. The 13C-labelled archaeal ureCgenes were deposited with Accession Numbers JX239707–JX239721. The pyrosequencing reads were deposited at theGenBank DNA Data Bank of Japan (DDBJ) with AccessionNumber DRA000587.

Acknowledgements

This work was financially supported by the National ScienceFoundation of China (40971153 and 41090281), the Knowl-edge Innovation Programs of the Chinese Academy of Sci-ences (KSCX2-EW-G-16), and the Distinguished YoungScholar Programme of Jiangsu Province (BK2012048). Weare grateful to Dr Marc G. Dumont and the two anonymousreviewers for comments that improved the manuscriptgreatly. We thank Dr Wenyan Han for the access to thelong-term experimental trail of the Chinese Academy ofAgricultural Sciences, Dr Yucheng Wu for soil collection, DrBenli Chai at Michigan State University for bioinformaticsassistance.

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Supporting information

Additional Supporting Information may be found in the onlineversion of this article:

Fig. S1. Agarose gel electrophoresis of putative archaealureC genes (with an expected size of ~ 1260 bp) amplifiedfrom ultracentrifuged DNA over the entire density range ofthe SIP gradient from the microcosms of forest soil (A) andtea orchard soil (B) after incubation for 56 days. The ultra-centrifuged DNA gradient diagram shows buoyant densityfractions (from light to heavy, from top to bottom respec-tively). 13CO2 and 13CO2 + C2H2 indicate that the soil micro-cosms were incubated with 13CO2 as labelled treatment and13CO2 + C2H2 as control treatment. Day-56-H2O and Day-56-Urea indicate that the soil microcosms were amended withwater and urea, respectively, over an incubation course of56 days. The marker denotes the DNA ladder. PCR ampli-cons, indicated by the blue-box, were excised from thelabelled treatment for the construction of the target ureCgene clone library (indicated by arrows) and for sequencinganalysis.Fig. S2. Relationship between archaeal amoA gene copiesand soil NO3

--N concentrations in soil microcosms amendedwith urea and water for incubation. The sampling volume forlinear regression was 60 from triplicate microcosms of forestand tea orchard soils. Significant correlation was observed atP < 0.001.Table S1. The rates of net nitrification and mineralizationin SIP microcosms incubated with urea or water for 56days.

14 L. Lu and Z. Jia


Table S2. Pyrosequencing results of the total 16S rRNAgenes in the fractionated DNA isolated from SIP microcosmsof forest soil.Table S3. Pyrosequencing results of the total 16S rRNAgenes in the fractionated DNA isolated from SIP microcosmsof tea orchard soil.

Table S4. Summary of the dominant OTUs of archaeal 16SrRNA genes in 13C-DNA isolated from forest soil and teaorchard soil.Table S5. The estimated cell number of AOA and cell-specific rate of soil archaeal nitrification.Table S6. Primers and conditions used in this study.



urease gene-containing archaea dominate autotrophic ammonia oxidation in...

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