Altitudinal Distribution of Ammonia-Oxidizing Archaea and Bacteria in Alpine Grassland Soils Along the South-Facing Slope of Nyqentangula Mountains,Central Tibetan Plateau

Nitrogen is a major limiting nutrient for the net primary production of terrestrial ecosystems, especially on sentinel alpine ecosystem. Ammonia oxidation is the first and rate-limiting step on nitrification process and is thus crucial to nitrogen cycle. To decipher climatic influence on ammonia oxidizers, their communities were characterized by qPCR and clone sequencing by targeting amoA genes (encoding the alpha subunit of ammonia mono-oxygenase) in soils from 7 sites over an 800 m elevation transect (4400–5200 m a.s.l.), based on “space-to-time substitution” strategy, on a steppe-meadow ecosystem located on the central Tibetan Plateau (TP). Archaeal amoA abundance outnumbered bacterial amoA abundance at lower altitude (<4800 m a.s.l.), but bacterial amoA abundance was greater in surface soils at higher altitude (≥4800 m a.s.l.). Archaeal amoA abundance decreased with altitude in surface soil, while its abundance stayed relatively stable and was mostly greater than bacterial amoA abundance in subsurface soils. Conversely, bacterial amoA abundance gradually increased with altitude at all three soil depths. Statistical analysis indicated that altitude-dependent factors, in particular pH and precipitation, had a profound effect on the abundance and community of ammonia-oxidizing bacteria, but only on the community composition of ammonia-oxidizing archaea along the altitudinal gradient. These findings imply that the shifts in the relative abundance and/or community structure of ammonia-oxidizing bacteria and archaea may result from the precipitation variation along the altitudinal gradient. Thus, we speculate that altitude-related factors (mainly precipitation variation combing changed pH), would play a vital role in affecting nitrification process on this alpine grassland ecosystem located at semi-arid area on TP.     

Community structures of ammonia‐oxidising archaea and bacteria in high‐altitude lakes on the Tibetan Plateau

1. Community structures of planktonic ammonia‐oxidising archaea (AOA) and bacteria (AOB) were investigated for five high‐altitude Tibetan lakes, which could be classified as freshwater, oligosaline or mesosaline, to develop a general view of the AOA and AOB in lakes on the Tibetan Plateau. 2. Based on PCR screening of the ammonia monooxygenase α‐subunit (amoA) gene, AOA were present in 14 out of 17 samples, whereas AOB were detected in only four samples. Phylogenetic analyses indicated that the AOB communities were dominated by a unique monophylogenetic lineage within Nitrosomonas, which may represent a novel cluster of AOB. AOA, on the other hand, were distinct among lakes with different salinities. 3. Multivariate statistical analyses indicated a heterogeneous distribution of the AOA communities among lakes largely caused by lake salinity, whereas the uniform chemical properties within lakes and their geographical isolation may favour relatively homogeneous AOA communities within lakes. 4. Our results suggest a wide occurrence of AOA in Tibetan lakes and provide the first evidence of salinity‐related differentiation of AOA community composition as well as potential geographical isolation of AOA in inland aquatic environments.     

Dynamics of ammonia-oxidizing archaea and bacteria populations and contributions to soil nitrification potentials

It is well known that the ratio of ammonia-oxidizing archaea (AOA) and bacteria (AOB) ranges widely in soils, but no data exist on what might influence this ratio, its dynamism, or how changes in relative abundance influences the potential contributions of AOA and AOB to soil nitrification. By sampling intensively from cropped-to-fallowed and fallowed-to-cropped phases of a 2-year wheat/fallow cycle, and adjacent uncultivated long-term fallowed land over a 15-month period in 2010 and 2011, evidence was obtained for seasonal and cropping phase effects on the soil nitrification potential (NP), and on the relative contributions of AOA and AOB to the NP that recovers after acetylene inactivation in the presence and absence of bacterial protein synthesis inhibitors. AOB community composition changed significantly (P⩽0.0001) in response to cropping phase, and there were both seasonal and cropping phase effects on the amoA gene copy numbers of AOA and AOB. Our study showed that the AOA:AOB shifts were generated by a combination of different phenomena: an increase in AOA amoA abundance in unfertilized treatments, compared with their AOA counterparts in the N-fertilized treatment; a larger population of AOB under the N-fertilized treatment compared with the AOB community under unfertilized treatments; and better overall persistence of AOA than AOB in the unfertilized treatments. These data illustrate the complexity of the factors that likely influence the relative contributions of AOA and AOB to nitrification under the various combinations of soil conditions and NH₄⁺-availability that exist in the field.     

亚高山/高山森林土壤有机层氨氧化细菌和氨氧化古菌丰度特征

为了解季节性冻融作用对川西亚高山/高山地区土壤氨氧化微生物群落的影响,采用qPCR技术,以氨单加氧酶基因的α亚基(amoA)为标记,在生长阶段、冻结阶段、融化阶段中的9个关键时期调查了该地区不同森林群落：岷江冷杉(Abies faxoniana)原始林(PF)、岷江冷杉(A. faxoniana)和红桦(Betula albosinensis)混交林(MF)、岷江冷杉次生林(SF)土壤有机层的氨氧化细菌(ammonia-oxidizing bacteria, AOB)和氨氧化古菌(ammonia-oxidizing archaea, AOA)丰度的特征。结果表明,三个森林群落土壤有机层中都具有相当数量的氨氧化细菌和古菌,均表现出从生长阶段至冻结阶段显著降低,在冻结阶段最低,但冻结阶段后显著增加,在融化阶段为全年最高的趋势。土壤氨氧化微生物类群结构(AOA/AOB)受负积温影响明显。冻结后期三个森林群落土壤负积温最大时,AOA数量明显高于AOB,但其他关键时期土壤氨氧化微生物类群结构与群落类型密切相关。高海拔的PF群落土壤有机层表现为AOA>AOB(冻结初期除外),低海拔的SF群落中表现为AOB>AOA(冻结后期除外),而MF群落则仅在融冻期和生长季节末期表现为AOB>AOA。这些结果为认识亚高山/高山森林及其相似区域的生态过程提供了一定的科学依据。     

基于amoA基因扩增子高通量测序的氨氧化古菌多样性分析方法

【背景】对于环境样品中氨氧化古菌(Ammonia-oxidizing archaea,AOA)多样性的研究,利用amoA功能基因作为分子标记会比16SrRNA基因有更强的特异性和更高的分辨率,能更准确地反映环境样品中氨氧化古菌的种群结构和分布特征。然而,目前对amoA基因扩增子高通量测序的分析存在两大限制因素:一是缺乏相应的amoA基因参考数据库;二是AOA amoA基因在种水平上的相似性阈值未知,分析过程中没有明确的划分种水平操作分类单元(Operational taxonomic unit,OTU)的阈值。【目的】构建基于amoA功能基因序列分析氨氧化古菌多样性的方法,为基于高通量测序的功能微生物多样性分析提供参考。【方法】基于目前已通过分离纯化或富集培养获得的34株氨氧化古菌及功能基因数据库中收录的环境样品amoA基因序列,构建氨氧化古菌amoA基因参考数据库。通过菌株间两两比对获得的amoA基因相似度与16SrRNA基因相似度的相关性分析,确定amoA基因在种水平上的相似性阈值。基于MOTHUR软件平台,利用建立的参考数据库和确定的阈值对南海一个垂直水体剖面样品的amoA基因序列进行多样性分析。【结果】构建了含有26 091条序列信息的古菌amoA基因参考数据库,确定了89%作为分析过程中古菌amoA基因划分种水平OTU的阈值,对南海水体样品氨氧化古菌的多样性分析结果很好地显示了南海不同深度水层水体中氨氧化古菌的种群结构和系统发育关系,有效揭示了南海氨氧化古菌的垂直分布差异。【结论】建立了基于amoA基因高通量测序的氨氧化古菌多样性分析方法,此方法可以有效分析环境样品中氨氧化古菌的多样性。     

Ammonia-oxidizing archaea have more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils

Increasing evidence demonstrated the involvement of ammonia-oxidizing archaea (AOA) in the global nitrogen cycle, but the relative contributions of AOA and ammonia-oxidizing bacteria (AOB) to ammonia oxidation are still in debate. Previous studies suggest that AOA would be more adapted to ammonia-limited oligotrophic conditions, which seems to be favored by protonation of ammonia, turning into ammonium in low-pH environments. Here, we investigated the autotrophic nitrification activity of AOA and AOB in five strongly acidic soils (pH<4.50) during microcosm incubation for 30 days. Significantly positive correlations between nitrate concentration and amoA gene abundance of AOA, but not of AOB, were observed during the active nitrification. ¹³CO₂-DNA-stable isotope probing results showed significant assimilation of ¹³C-labeled carbon source into the amoA gene of AOA, but not of AOB, in one of the selected soil samples. High levels of thaumarchaeal amoA gene abundance were observed during the active nitrification, coupled with increasing intensity of two denaturing gradient gel electrophoresis bands for specific thaumarchaeal community. Addition of the nitrification inhibitor dicyandiamide (DCD) completely inhibited the nitrification activity and CO₂ fixation by AOA, accompanied by decreasing thaumarchaeal amoA gene abundance. Bacterial amoA gene abundance decreased in all microcosms irrespective of DCD addition, and mostly showed no correlation with nitrate concentrations. Phylogenetic analysis of thaumarchaeal amoA gene and 16S rRNA gene revealed active ¹³CO₂-labeled AOA belonged to groups 1.1a-associated and 1.1b. Taken together, these results provided strong evidence that AOA have a more important role than AOB in autotrophic ammonia oxidation in strongly acidic soils.     

Spatial distribution of ammonia-oxidizing bacteria and archaea across a 44-hectare farm related to ecosystem functioning

Characterization of spatial patterns of functional microbial communities could facilitate the understanding of the relationships between the ecology of microbial communities, the biogeochemical processes they perform and the corresponding ecosystem functions. Because of the important role the ammonia-oxidizing bacteria (AOB) and archaea (AOA) have in nitrogen cycling and nitrate leaching, we explored the spatial distribution of their activity, abundance and community composition across a 44-ha large farm divided into an organic and an integrated farming system. The spatial patterns were mapped by geostatistical modeling and correlations to soil properties and ecosystem functioning in terms of nitrate leaching were determined. All measured community components for both AOB and AOA exhibited spatial patterns at the hectare scale. The patchy patterns of community structures did not reflect the farming systems, but the AOB community was weakly related to differences in soil pH and moisture, whereas the AOA community to differences in soil pH and clay content. Soil properties related differently to the size of the communities, with soil organic carbon and total nitrogen correlating positively to AOB abundance, while clay content and pH showed a negative correlation to AOA abundance. Contrasting spatial patterns were observed for the abundance distributions of the two groups indicating that the AOB and AOA may occupy different niches in agro-ecosystems. In addition, the two communities correlated differently to community and ecosystem functions. Our results suggest that the AOA, not the AOB, were contributing to nitrate leaching at the site by providing substrate for the nitrite oxidizers.     

白洋淀湖滨湿地岸边带氨氧化古菌与氨氧化细菌的分布特性

摘要：本研究通过分子生物学分析方法,以amoA基因为标记,考察了氨氧化古菌（Ammonia-Oxidizing Archaea, AOA）和氨氧化细菌（Ammonia-Oxidizing Bacteria,AOB）在华北平原的白洋淀这一典型湖泊的湖滨湿地岸边带系统中的生物多样性和丰度分布。在前人的研究中,氨氧化古菌在海洋、原生态土壤和人为干扰土壤等环境中主导氨氧化过程的完成。但本研究发现,在湿地岸边带系统中氨氧化过程并不是完全由氨氧化古菌主导完成,即氨氧化古菌和氨氧化细菌在不同区域分别占据主导地位。根据主导微生物的不同,可以将湿地岸边带区域划分为陆相区、中间区和湖相区。在湿地岸边带陆相区,氨氧化古菌主导氨氧化过程,氨氧化古菌的amoA基因丰度是氨氧化细菌的526倍（AOA：1.23?108每克干土;AOB：2.34?105每克干土）;在岸边带湖相区,氨氧化细菌主导氨氧化过程,氨氧化古菌的amoA基因丰度只有氨氧化细菌的1/50倍（AOA：3.17?106每克干土;AOB：1.39?108每克干土）;在岸边带中间区,两种微生物对氨氧化过程的贡献相当,二者的amoA基因丰度也相当 (AOA：9.83?106, AOB：4.08?106)。研究还发现,湿地中间区的微生物生物多样性高于陆相区和湖相区。在湿地中间区,氨氧化古菌和氨氧化细菌的生物多样性都最高,分别有5和7个操作分类单元（OTUs）;相比之下,岸边带陆相区和湖相区的多样性依次降低,陆相区的氨氧化古菌和氨氧化细菌分别有3和6个操作分类单元,湖相区的氨氧化古菌和氨氧化细菌分别有2和6个分类单元。本研究的两个结论进一步反映了湿地岸边带极强的空间异质性。     

Ammonia-oxidizing bacteria dominates over ammonia-oxidizing archaea in a saline nitrification reactor under low DO and high nitrogen loading

A continuous nitrification reactor treating saline wastewater was operated for almost 1 year under low dissolved oxygen (DO) levels (0.15-0.5 mg/L) and high nitrogen loadings (0.26-0.52 kg-N/(m(3) day)) in four phases. The diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) were analyzed by cloning, terminal restriction fragment length polymorphism (T-RFLP) and quantitative polymerase chain reaction (qPCR). The results showed that there were only one dominant AOA species and one dominant AOB species in the reactor in all of the four experimental phases. The amoA gene of the dominant AOA only had a similarity of 89.3% with the cultured AOA species Nitrosopumilus maritimus SCM1. All of the AOB species detected in the reactor belong to Nitrosomonas genus and it was found that the AOB populations changed with the ammonium loadings and DO levels. The abundance of AOB in the reactor was ～40 times larger than that of AOA, and the ratio of AOB to AOA increased significantly up to ～2,000 to ～4,000 with the increase of ammonium loading, indicating that AOB are much more competitive than AOA in high ammonium environments and probably AOA play a less important role than AOB in the nitrification reactors.     

围栏对青藏高原不同类型草地土壤原核微生物多样性的影响

草地退化是草地植被的倒退演替,导致生物多样性丧失和生态系统功能退化,围栏是恢复退化草地生态系统功能的有效管理措施。微生物是土壤中的重要组成部分,在维持草地生态系统稳定性和功能方面发挥着重要作用。然而,目前尚不清楚围栏如何影响不同类型草地土壤微生物群落。以青藏高原草甸、草原和荒漠草地三种草地类型的退化草地为研究对象,设置围栏和放牧两种处理,采用Illumina HiSeq高通量测序技术研究了围栏对土壤原核微生物群落多样性和群落结构的影响。结果表明：围栏未显著影响草甸土壤原核微生物的丰富度、Shannon多样性和均匀度,但显著增加了草原土壤的原核微生物的丰富度、Shannon多样性和均匀度（P<0.05）,稍降低了荒漠草地土壤原核微生物的丰富度、Shannon多样性和均匀度（P=0.086、0.072和0.099）。在围栏处理的草地中,土壤原核微生物丰富度、Shannon多样性和均匀度与年均温、干旱度和pH显著负相关（P<0.01）,与年平均降水量、溶解性有机碳、地上生物量和植物多样性显著正相关（P<0.01）。在放牧处理的草地中,土壤原核微生物丰富度、Shannon多样性和均匀度与年均温和干旱度显著负相关（P<0.05）,但原核微生物丰富度和Shannon多样性与所有土壤理化和植被因素均无显著相关性。冗余分析（RDA）表明,不同类型草地土壤原核微生物群落结构发生了显著的变化,并沿草甸、草原和荒漠草地的过渡逐渐转变（P<0.001）。方差分解分析（VPA）进一步表明,原核微生物群落结构变化主要受年均温、年平均降水量、干旱度和pH的驱动。围栏显著改变了不同类型草地中部分样点土壤原核微生物群落结构。三种草地类型的主要原核微生物优势门均为放线菌门（Actinobacteria）、变形菌门（Proteobacteria）和酸杆菌门（Acidobacteria）。放线菌门（Actinobacteria）的相对丰度在荒漠草地土壤中最高,而变形菌门（Proteobacteria）和酸杆菌门（Acidobacteria）的相对丰度在草甸土壤中最高。此外,不同类型围栏和放牧草地土壤原核微生物类群的相对丰度均无显著差异。研究表明不同类型草地土壤原核微生物群落对围栏的响应不同,这为因地制宜制定草地管理措施提供了数据支持,为草地退化的防治提供了理论支持。     

Responses of community structure of amoA‐encoding archaea and ammonia‐oxidizing bacteria in ammonia biofilter with rockwool mixtures to the gradual increases in ammonium and nitrate

Aims

To investigate community shifts of amoA‐encoding archaea (AEA) and ammonia‐oxidizing bacteria (AOB) in biofilter under nitrogen accumulation process.

Methods and Results

A laboratory‐scale rockwool biofilter with an irrigated water circulation system was operated for 436 days with ammonia loading rates of 49–63 NH₃ g m^?3 day^?1. The AEA and AOB communities were investigated by denaturing gradient gel electrophoresis, sequencing and real‐time PCR analysis based on amoA genes. The results indicated that changes in abundance and community compositions occurred in a different manner between archaeal and bacterial amoA during the operation. However, both microbial community structures mainly varied when free ammonia (FA) concentrations in circulation water were increasing, which caused a temporal decline in reactor performance. Dominant amoA sequences after this transition were related to Thaumarchaeotal Group I.1b, Nitrosomonas europaea lineages and one subcluster within Nitrosospira sp. cluster 3, for archaea and bacteria, respectively.

Conclusions

The specific FA in circulation water seems to be the important factor, which relates to the AOB and AEA community shifts in the biofilter besides ammonium and pH.

Significance and Impact of the Study

One of the key factors for regulating AEA and AOB communities was proposed that is useful for optimizing biofiltration technology.     

高含氮稻田深层土壤的氨氧化古菌和厌氧氨氧化菌共存及对氮循环影响

随着海洋生态系统中的厌氧氨氧化反应和氨氧化古菌的发现,自然生态系统的氮循环过程被重新认识,但是目前尚无在陆地深层的相关报道。结合同位素示踪与分子生物学技术探索了稻田深层土壤中anammox与AOA的存在及特性。结果表明,在沼渣处理废水浇灌的高含氮稻田深层土壤中,anammox与AOA共存。通过构建克隆文库发现,此土壤中厌氧氨氧化菌的生物多样性相对较低,35个克隆序列只分为4个独立操作单元(OTU),代表序列与Genebank数据库中已探明的厌氧氨氧化菌Candidatus 'Kuenenia stuttgartiensis’的同源性超过95%;对氨氧化古菌的分析发现,20个克隆子共得到5个OTU,其与基因库中土壤/沉积物进化分支关系最近,序列的同源性部分超过98%。同位素示踪的初步结果表明,anammox产生的氮气占此土壤总氮气生成量的24.1%-29.8%。AOA与anammox的共存为anammox反应的广泛存在与发生提供了新思路。     

Review of ammonia-oxidizing bacteria and archaea in freshwater ponds

Aquaculture ponds are simple and unique ecosystems, which are affected intensively by human activities. In this mini-review, we focus our attention on the distribution and community diversity of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in pond water and sediments, as well as the possible ecological mechanisms involved. Moreover, we discuss the possibility of increasing the activity of ammonia-oxidizing organisms in order to improve the water quality in aquaculture ponds. Compared with eutrophic lakes, the significantly higher ammonia concentration in pond water does not lead to significantly higher AOB levels, and the abundance of AOA is too low to quantify accurately. Similar to eutrophic lakes, high abundances of AOA and AOB are present in the surface sediments at the same time, where the oxidation of ammonia is performed mainly by AOB. AOB and AOA exhibit significant seasonal variations in aquaculture ponds, which are affected by the temperature, pH, and dissolved oxygen. The dominant AOB species are Nitrosomonas and the Nitrosospira lineage in pond environments. Nitrososphaera or members of the Nitrososphaera-like cluster dominate the AOA species in surface sediments, whereas the Nitrosopumilus cluster dominates the deeper sediments. AOB and AOA can be enriched on artificial substrates suspended in the pond water, thereby potentially improving the water quality.

     

长期不同施肥制度对水稻土氨氧化微生物数量和群落结构的影响

由氨氧化微生物驱动的氨氧化过程是硝化作用的限速步骤,在土壤氮素循环过程中扮演着重要角色.以湖南省宁乡县长达30 a定位试验水稻土壤为研究对象,采用荧光定量PCR和Illumina MiSeq高通量测序分析方法,以amoA基因为靶标,研究了4种施肥制度[不施肥(CK)、化肥(CF)、70%化肥+30%有机肥(CFM₁)和40%化肥+60%有机肥(CFM₂)]水稻土壤氨氧化微生物的数量和群落结构变化.结果表明: 不同施肥处理氨氧化古菌(AOA)和氨氧化细菌(AOB) amoA基因拷贝数分别为3.09×10⁷～8.37×10⁷和1.04×10⁷～7.03×10⁷ copies·g^-1干土.施肥显著提高了AOA和AOB数量,但处理CFM₂中AOB数量与CK差异不显著.有机肥配施比例对AOB群落α多样性指数的影响强于AOA,处理CFM₁中AOA群落的多样性指数(Shannon)和AOB群落的丰富度指数(ACE和Chao1)均显著高于CK.奇古菌门和泉古菌门是AOA群落的优势门类群,占AOA amoA基因总序列的83.4%;亚硝化螺菌属、environmental_samples_norank、Bacteria_unclassified和Nitrosomonadales_unclassified是AOB群落的优势属类群,占AOB amoA基因总序列的97.8%.维恩分析结果显示,有机肥配施比例对AOB群落操作分类单元(OTU)数量的影响强于AOA,但对各处理共有AOA和AOB amoA基因序列条数的影响均较小.冗余分析结果显示,不同施肥处理AOB群落结构差异强于AOA,且所有土壤理化性质均与AOA和AOB群落结构存在显著相关关系.综上可知:有机肥配施比例显著改变了AOA和AOB数量、多样性和群落结构,配施30%有机肥时,AOA群落的Shannon指数最高,AOB群落数量、ACE和Chao1指数均最高.研究结果可为进一步探讨农业系统中氨氧化微生物对不同施肥制度的响应机制及其在氮素转化中的作用提供科学依据.     

Evidence for different contributions of archaea and bacteria to the ammonia-oxidizing potential of diverse Oregon soils

A method was developed to determine the contributions of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) to the nitrification potentials (NPs) of soils taken from forest, pasture, cropped, and fallowed (19 years) lands. Soil slurries were exposed to acetylene to irreversibly inactivate ammonia monooxygenase, and upon the removal of acetylene, the recovery of nitrification potential (RNP) was monitored in the presence and absence of bacterial or eukaryotic protein synthesis inhibitors. For unknown reasons, and despite measureable NPs, RNP did not occur consistently in forest soil samples; however, pasture, cropped, and fallowed soil RNPs commenced after lags that ranged from 12 to 30 h after acetylene removal. Cropped soil RNP was completely prevented by the bacterial protein synthesis inhibitor kanamycin (800 μg/ml), whereas a combination of kanamycin plus gentamicin (800 μg/ml each) only partially prevented the RNP (60%) of fallowed soils. Pasture soil RNP was completely insensitive to either kanamycin, gentamicin, or a combination of the two. Unlike cropped soil, pasture and fallowed soil RNPs occurred at both 30°C and 40°C and without supplemental NH(4)(+) (≤ 10 μM NH(4)(+) in solution), and pasture soil RNP demonstrated ～ 50% insensitivity to 100 μM allyl thiourea (ATU). In addition, fallowed and pasture soil RNPs were insensitive to the fungal inhibitors nystatin and azoxystrobin. This combination of properties suggests that neither fungi nor AOB contributed to pasture soil RNP and that AOA were responsible for the RNP of the pasture soils. Both AOA and AOB may contribute to RNP in fallowed soil, while RNP in cropped soils was dominated by AOB.     

Isotopic signatures of N2O produced by ammonia-oxidizing archaea from soils

N₂O gas is involved in global warming and ozone depletion. The major sources of N₂O are soil microbial processes. Anthropogenic inputs into the nitrogen cycle have exacerbated these microbial processes, including nitrification. Ammonia-oxidizing archaea (AOA) are major members of the pool of soil ammonia-oxidizing microorganisms. This study investigated the isotopic signatures of N₂O produced by soil AOA and associated N₂O production processes. All five AOA strains (I.1a, I.1a-associated and I.1b clades of Thaumarchaeota) from soil produced N₂O and their yields were comparable to those of ammonia-oxidizing bacteria (AOB). The levels of site preference (SP), δ¹⁵N^bulk and δ¹⁸O -N₂O of soil AOA strains were 13–30%, −13 to −35% and 22–36%, respectively, and strains MY1–3 and other soil AOA strains had distinct isotopic signatures. A ¹⁵N-NH₄⁺-labeling experiment indicated that N₂O originated from two different production pathways (that is, ammonia oxidation and nitrifier denitrification), which suggests that the isotopic signatures of N₂O from AOA may be attributable to the relative contributions of these two processes. The highest N₂O production yield and lowest site preference of acidophilic strain CS may be related to enhanced nitrifier denitrification for detoxifying nitrite. Previously, it was not possible to detect N₂O from soil AOA because of similarities between its isotopic signatures and those from AOB. Given the predominance of AOA over AOB in most soils, a significant proportion of the total N₂O emissions from soil nitrification may be attributable to AOA.