Occurrence of ammonia-oxidizing archaea in wastewater treatment plant bioreactors

We report molecular evidence that ammonia-oxidizing archaea (AOA) occur in activated sludge bioreactors used to remove ammonia from wastewater. Using PCR primers targeting archaeal ammonia monooxygenase subunit A (amoA) genes, we retrieved and compared 75 sequences from five wastewater treatment plants operating with low dissolved oxygen levels and long retention times. All of these sequences showed similarity to sequences previously found in soil and sediments, and they were distributed primarily in four major phylogenetic clusters. One of these clusters contained virtually identical amoA sequences obtained from all five activated sludge samples (from Oregon, Wisconsin, Pennsylvania, and New Jersey) and accounted for 67% of all the sequences, suggesting that this AOA phylotype may be widespread in nitrifying bioreactors.     

Relative abundance and diversity of ammonia-oxidizing archaea and bacteria in the San Francisco Bay estuary

Ammonia oxidation in marine and estuarine sediments plays a pivotal role in the cycling and removal of nitrogen. Recent reports have shown that the newly discovered ammonia-oxidizing archaea can be both abundant and diverse in aquatic and terrestrial ecosystems. In this study, we examined the abundance and diversity of ammonia-oxidizing archaea (AOA) and betaproteobacteria (beta-AOB) across physicochemical gradients in San Francisco Bay--the largest estuary on the west coast of the USA. In contrast to reports that AOA are far more abundant than beta-AOB in both terrestrial and marine systems, our quantitative PCR estimates indicated that beta-AOB amoA (encoding ammonia monooxygenase subunit A) copy numbers were greater than AOA amoA in most of the estuary. Ammonia-oxidizing archaea were only more pervasive than beta-AOB in the low-salinity region of the estuary. Both AOA and beta-AOB communities exhibited distinct spatial structure within the estuary. AOA amoA sequences from the north part of the estuary formed a large and distinct low-salinity phylogenetic group. The majority of the beta-AOB sequences were closely related to other marine/estuarine Nitrosomonas-like and Nitrosospira-like sequences. Both ammonia-oxidizer community composition and abundance were strongly correlated with salinity. Ammonia-oxidizing enrichment cultures contained AOA and beta-AOB amoA sequences with high similarity to environmental sequences. Overall, this study significantly enhances our understanding of estuarine ammonia-oxidizing microbial communities and highlights the environmental conditions and niches under which different AOA and beta-AOB phylotypes may thrive.     

Abundance and composition of epiphytic bacterial and archaeal ammonia oxidizers of marine red and brown macroalgae

Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are important for nitrogen cycling in marine ecosystems. Little is known about the diversity and abundance of these organisms on the surface of marine macroalgae, despite the algae's potential importance to create surfaces and local oxygen-rich environments supporting ammonia oxidation at depths with low dissolved oxygen levels. We determined the abundance and composition of the epiphytic bacterial and archaeal ammonia-oxidizing communities on three species of macroalgae, Osmundaria volubilis, Phyllophora crispa, and Laminaria rodriguezii, from the Balearic Islands (western Mediterranean Sea). Quantitative PCR of bacterial and archaeal 16S rRNA and amoA genes was performed. In contrast to what has been shown for most other marine environments, the macroalgae's surfaces were dominated by bacterial amoA genes rather than those from the archaeal counterpart. On the basis of the sequences retrieved from AOB and AOA amoA gene clone libraries from each algal species, the bacterial ammonia-oxidizing communities were related to Nitrosospira spp. and to Nitrosomonas europaea and only 6 out of 15 operational taxonomic units (OTUs) were specific for the host species. Conversely, the AOA diversity was higher (43 OTUs) and algal species specific, with 17 OTUs specific for L. rodriguezii, 3 for O. volubilis, and 9 for P. crispa. Altogether, the results suggest that marine macroalgae may exert an ecological niche for AOB in marine environments, potentially through specific microbe-host interactions.     

Aquarium nitrification revisited: Thaumarchaeota are the dominant ammonia oxidizers in freshwater aquarium biofilters

Ammonia-oxidizing archaea (AOA) outnumber ammonia-oxidizing bacteria (AOB) in many terrestrial and aquatic environments. Although nitrification is the primary function of aquarium biofilters, very few studies have investigated the microorganisms responsible for this process in aquaria. This study used quantitative real-time PCR (qPCR) to quantify the ammonia monooxygenase (amoA) and 16S rRNA genes of Bacteria and Thaumarchaeota in freshwater aquarium biofilters, in addition to assessing the diversity of AOA amoA genes by denaturing gradient gel electrophoresis (DGGE) and clone libraries. AOA were numerically dominant in 23 of 27 freshwater biofilters, and in 12 of these biofilters AOA contributed all detectable amoA genes. Eight saltwater aquaria and two commercial aquarium nitrifier supplements were included for comparison. Both thaumarchaeal and bacterial amoA genes were detected in all saltwater samples, with AOA genes outnumbering AOB genes in five of eight biofilters. Bacterial amoA genes were abundant in both supplements, but thaumarchaeal amoA and 16S rRNA genes could not be detected. For freshwater aquaria, the proportion of amoA genes from AOA relative to AOB was inversely correlated with ammonium concentration. DGGE of AOA amoA genes revealed variable diversity across samples, with nonmetric multidimensional scaling (NMDS) indicating separation of freshwater and saltwater fingerprints. Composite clone libraries of AOA amoA genes revealed distinct freshwater and saltwater clusters, as well as mixed clusters containing both freshwater and saltwater amoA gene sequences. These results reveal insight into commonplace residential biofilters and suggest that aquarium biofilters may represent valuable biofilm microcosms for future studies of AOA ecology.     

辉腾锡勒草原干涸湖泊中氨氧化微生物群落结构分析

【目的】以内蒙古辉腾锡勒草原九十九泉湿地为对象,研究湖泊干涸过程中氨氧化微生物的群落结构及其变化。【方法】通过MPN-PCR定量测定氨氧化古菌(AOA)和氨氧化细菌(AOB)的数量;构建amoA基因克隆文库,进行系统发育分析;结合土壤环境因子,探讨湿地退化过程中影响氨氧化微生物的潜在因素。【结果】依湖泊湿地退水梯度的不同样点中,有75%的样点AOB的数量高于AOA,AOB与AOA的数量比率为0.3-18.1。从湖心到湖岸草原带,AOA和AOB的数量有明显增加,但生物多样性呈降低趋势,二者没有呈现正相关。研究发现,AOB的数量与土壤中NH 4+-N的变化存在良好响应。系统发育分析显示,退化湖泊湿地AOA克隆序列均来自于泉古菌门(Crenarchaeota);AOB的amoA基因的克隆序列大部分与亚硝化单胞菌属(Nitrosomonas)有一定同源性,较少部分与亚硝化螺菌属(Nitrosospira)有一定同源性。【结论】湖泊退水过程增加了湿地土壤氨氧化微生物的数量,而氨氧化微生物的种群丰度有所降低。AOA和AOB群落对湖泊湿地的退化过程做出了响应,其中AOB的响应较为明显,氧化条件和土壤铵浓度的改变可能是促成这种响应的重要原因。     

南美白对虾养殖底泥中氨氧化细菌与氨氧化古菌多态性分析

【目的】本研究皆在了解虾养殖底泥中氨氧化细菌与氨氧化古菌群落多态性。【方法】以功能基因为基础,构建氨氧化细菌(AOB)与氨氧化古菌(AOA)的氨单加氧酶α亚基基因(amoA)克隆文库。利用限制性片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)技术将克隆文库阳性克隆子进行归类分析分成若干个可操作分类单元(Operational Taxa Units,OTUs)。【结果】通过序列多态性分析,表明AOB amoA基因克隆文库中所有序列都属于变形杆菌门β亚纲(β-Proteobacteria)中的亚硝化单细胞菌属(Nitrosomonas)及Nitrosomonas-like,未发现亚硝化螺旋菌属(Nitrosospira)。AOA amoA基因克隆文库中只有一个OTU序列属于未分类的古菌(Unclassified-Archaea),其余序列都属于泉古菌门(Crenarchaeote)。AOA群落结构单一且存在一个绝对优势类群OTU3,其克隆子数目占克隆文库的57.45%。AOB和AOA amoA基因克隆文库分别包括13个OTUs和9个OTUs,其文库覆盖率分别为73.47%和90.43%。AOB amoA基因克隆文库的Shannon-Wiener指数、Evenness指数、Simpson指数、Richness指数均高于AOA。【结论】虾养殖塘底泥中存在氨氧化古菌的amoA基因,且多态性低于氨氧化细菌,表明氨氧化古菌在虾养殖塘底泥的氮循环中可能具有重要的作用。     

RETRACTED ARTICLE: Archaeal <Emphasis Type="Italic">amoA</Emphasis> Genes Outnumber Bacterial <Emphasis Type="Italic">amoA</Emphasis> Genes in Municipal Wastewater Treatment Plants in Bangkok

The contribution of ammonia-oxidizing archaea (AOA) to nitrogen removal in wastewater treatment plants (WWTPs) remains unknown. This study investigated the abundance of archaeal (AOA) and bacterial (ammonia-oxidizing bacteria (AOB)) amoA genes in eight of Bangkok’s municipal WWTPs. AOA amoA genes (3.28 × 10⁷ ± 1.74 × 10⁷–2.23 × 10¹¹ ± 1.92 × 10¹¹ copies l⁻¹ sludge) outnumbered AOB amoA genes in most of the WWTPs even though the plants’ treatment processes, influent and effluent characteristics, removal efficiencies, and operation varied. An estimation of the ammonia-oxidizing activity of AOA and AOB suggests that AOA involved in autotrophic ammonia oxidation in the WWTPs. Statistical analysis shows that the numbers of AOA amoA genes correlated negatively to the ammonium levels in effluent wastewater, while no correlation was found between the AOA amoA gene numbers and the oxygen concentrations in aeration tanks. An analysis of the AOB sequences shows that AOB found in the WWTPs limited to only two AOB clusters which exhibit high or moderate affinity to ammonia. In contrast to AOB, AOA sequences of various clusters were retrieved, and they were previously recovered from a variety of environments, such as thermal and marine environments.     

Ammonia‐oxidizing archaea: important players in paddy rhizosphere soil?

The diversity (richness and community composition) of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in paddy soil with different nitrogen (N) fertilizer amendments for 5 weeks were investigated using quantitative real-time polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE) jand clone library analysis based on the ammonia monooxygenase α-subunit ( amoA ) gene. Ammonia-oxidizing archaea predominated among ammonia-oxidizing prokaryotes in the paddy soil, and the AOA:AOB DNA-targeted amoA gene ratios ranged from 1.2 to 69.3. Ammonia-oxidizing archaea were more abundant in the rhizosphere than in bulk soil. Rice cultivation led to greater abundance of AOA than AOB amoA gene copies and to differences in AOA and AOB community composition. These results show that AOA is dominant in the rhizosphere paddy soil in this study, and we assume that AOA were influenced more by exudation from rice root (e.g. oxygen, carbon dioxide) than AOB.     

Diversity of ammonia-oxidizing archaea and bacteria in the sediments of a hypernutrified subtropical estuary: Bahía del Tóbari, Mexico

Nitrification within estuarine sediments plays an important role in the nitrogen cycle, both at the global scale and in individual estuaries. Although bacteria were once thought to be solely responsible for catalyzing the first and rate-limiting step of this process, several recent studies have suggested that mesophilic Crenarchaeota are capable of performing ammonia oxidation. Here we examine the diversity (richness and community composition) of ammonia-oxidizing archaea (AOA) and bacteria (AOB) within sediments of Bahía del Tóbari, a hypernutrified estuary receiving substantial amounts of ammonium in agricultural runoff. Using PCR primers designed to specifically target the archaeal ammonia monooxygenase alpha-subunit (amoA) gene, we found AOA to be present at five sampling sites within this estuary and at two sampling time points (January and October 2004). In contrast, the bacterial amoA gene was PCR amplifiable from only 40% of samples. Bacterial amoA libraries were dominated by a few widely distributed Nitrosomonas-like sequence types, whereas AOA diversity showed significant variation in both richness and community composition. AOA communities nevertheless exhibited consistent spatial structuring, with two distinct end member assemblages recovered from the interior and the mouths of the estuary and a mixed assemblage from an intermediate site. These findings represent the first detailed examination of archaeal amoA diversity in estuarine sediments and demonstrate that diverse communities of Crenarchaeota capable of ammonia oxidation are present within estuaries, where they may be actively involved in nitrification.     

Diversity and spatial distribution of amoA-encoding archaea in the deep-sea sediments of the tropical West Pacific Continental Margin

Aims:  The ecological characteristics of the deep-sea amoA -encoding archaea (AEA) are largely unsolved. Our aim was to study the diversity, structure and distribution of the AEA community in the sediments of the tropical West Pacific Continental Margin, to develop a general view of the AEA biogeography in the deep-sea extreme environment.
Methods and Results:  Archaeal amoA clone libraries were constructed. Diverse and novel amoA sequences were identified, with the Bohol Sea, Bashi Strait and Sibuyan Sea harbouring the highest and the Bicol Shelf the lowest AEA diversity. Phylogenetic and statistical analyses illustrate a heterogeneous distribution of the AEA community, probably caused by the differential distribution of the terrestrial or estuarine AEA in the various sampling sites.
Conclusions:  The deep-sea sedimentary environments potentially harbour diverse and novel AEA in the tropical West Pacific Continental Margin. The stations in the Philippine inland seas (including station 3043) may represent AEA assemblages with various terrestrial influences and the stations connected directly to the open Philippine Sea may represent marine environment-dominant AEA assemblages.
Significance and Impact of Study:  Our study indicates the potential importance of geological and climatic events in the transport of terrestrial micro-organisms to the deep-sea sedimentary environments, almost totally neglected previously.     

Viral composition and context in metagenomes from biofilm and suspended growth municipal wastewater treatment plants

Wastewater treatment plants (WWTPs) contain high density and diversity of viruses which can significantly impact microbial communities in aquatic systems. While previous studies have investigated viruses in WWTP samples that have been specifically concentrated for viruses and filtered to exclude bacteria, little is known about viral communities associated with bacterial communities throughout wastewater treatment systems. Additionally, differences in viral composition between attached and suspended growth wastewater treatment bioprocesses are not well characterized. Here, shotgun metagenomics was used to analyse wastewater and biomass from transects through two full-scale WWTPs for viral composition and associations with bacterial hosts. One WWTP used a suspended growth activated sludge bioreactor and the other used a biofilm reactor (trickling filter). Myoviridae, Podoviridae and Siphoviridae were the dominant viral families throughout both WWTPs, which are all from the order Caudovirales. Beta diversity analysis of viral sequences showed that samples clustered significantly both by plant and by specific sampling location. For each WWTP, the overall bacterial community structure was significantly different than community structure of bacterial taxa associated with viral sequences. These findings highlight viral community composition in transects through different WWTPs and provide context for dsDNA viral sequences in bacterial communities from these systems.     

The demonstration of a novel sulfur cycle-based wastewater treatment process: Sulfate reduction, autotrophic denitrification, and nitrification integrated (SANI®) biological nitrogen removal process

Saline water supply has been successfully practiced for toilet flushing in Hong Kong since 1950s, which saves 22% of freshwater in Hong Kong. In order to extend the benefits of saline water supply into saline sewage management, we have recently developed a novel biological organics and nitrogen removal process: the Sulfate reduction, Autotrophic denitrification, and Nitrification Integrated (SANI®) process. The key features of this novel process include elimination of oxygen demand in organic matter removal and production of minimal sludge. Following the success of a 500‐day lab‐scale trial, this study reports a pilot scale evaluation of this novel process treating 10 m³/day of 6‐mm screened saline sewage in Hong Kong. The SANI® pilot plant consisted of a sulfate reduction up‐flow sludge bed (SRUSB) reactor, an anoxic bioreactor for autotrophic denitrification and an aerobic bioreactor for nitrification. The plant was operated at a steady state for 225 days, during which the average removal efficiencies of both chemical oxygen demand (COD) and total suspended solids (TSS) at 87% and no excess sludge was purposefully withdrawn. Furthermore, a tracer test revealed 5% short circuit flow and a 34.6% dead zone in the SRUSB, indicating a good possibility to further optimize the treatment capacity of the process for full‐scale application. Compared with conventional biological nitrogen removal processes, the SANI® process reduces 90% of waste sludge, which saves 35% of the energy and reduces 36% of fossil CO₂ emission. The SANI® process not only eliminates the major odor sources originating from primary treatment and subsequent sludge treatment and disposal during secondary saline sewage treatment, but also promotes saline water supply as an economic and sustainable solution for water scarcity and sewage treatment in water‐scarce coastal areas. Biotechnol. Bioeng. 2012; 109: 2778–2789. © 2012 Wiley Periodicals, Inc.     

喹啉与吲哚驯化的反硝化反应器的微生物群落结构分析比较

[目的]本研究旨在比较分析分别以喹啉和吲哚为底物,在相同条件下驯化的两个反硝化生物反应器的微生物群落结构.[方法]采用相同的种子污泥和相同的驯化条件,经过大约6周的驯化后,两个反应器均达到稳定而高效的污染物去除能力,通过16S rDNA克隆文库技术对两个反应器的微生物群落结构进行研究.[结果]研究发现,微生物群落结构表现出很大的差异.喹啉驯化的群落中所有的OTU都属于Betaproteobacteria,而吲哚驯化的群落中Betaproteobacteria占56.3%,吲哚驯化的群落具有更高的多样性.两个群落的优势OTU也不同,喹啉驯化群落中Thauera及其它Rhodocyclaceae科的微生物占整个群落的73%,而吲哚驯化群落中优势OTU为Comamonadaceae科、Alcaligenaceae科和Rhodocyclaceae科等类型的微生物,其中Comamonadaceae科的一个OTU占整个群落的28.7%.[结论]不同的驯化底物对微生物群落的组成具有较强的选择作用.首次报道并比较了可高效降解喹啉和吲哚的反硝化生物反应器的微生物群落结构.     

Abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea communities of an alkaline sandy loam

The abundance and composition of soil ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) communities under different long-term (17 years) fertilization practices were investigated using real-time polymerase chain reaction and denaturing gradient gel electrophoresis (DGGE). A sandy loam with pH (H(2)O) ranging from 8.3 to 8.7 was sampled in years 2006 and 2007, including seven fertilization treatments of control without fertilizers (CK), those with combinations of fertilizer nitrogen (N), phosphorus (P) and potassium (K): NP, NK, PK and NPK, half chemical fertilizers NPK plus half organic manure (1/2OMN) and organic manure (OM). The highest bacterial amoA gene copy numbers were found in those treatments receiving N fertilizer. The archaeal amoA gene copy numbers ranging from 1.54 x 10(7) to 4.25 x 10(7) per gram of dry soil were significantly higher than those of bacterial amoA genes, ranging from 1.24 x 10(5) to 2.79 x 10(6) per gram of dry soil, which indicated a potential role of AOA in nitrification. Ammonia-oxidizing bacteria abundance had significant correlations with soil pH and potential nitrification rates. Denaturing gradient gel electrophoresis patterns revealed that the fertilization resulted in an obvious change of the AOB community, while no significant change of the AOA community was observed among different treatments. Phylogenetic analysis showed a dominance of Nitrosospira-like sequences, while three bands were affiliated with the Nitrosomonas genus. All AOA sequences fell within cluster S (soil origin) and cluster M (marine and sediment origin). These results suggest that long-term fertilization had a significant impact on AOB abundance and composition, while minimal on AOA in the alkaline soil.     

ERIC-PCR-based strain-specific detection of phenol-degrading bacteria in activated sludge of wastewater treatment systems

Aims:  To evaluate the use of Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR)-derived probes and primers to specifically detect bacterial strains in an activated sludge microbial community.
Methods and Results:  ERIC-PCR was performed on two phenol-degrading bacterial strains, Arthrobacter nicotianae P1-7 and Klebsiella sp. P8-14. Their amplicons were DIG labelled for use as probes and then hybridized with ERIC-PCR fingerprints. The results showed the distinct band patterns for both bacterial strains. Strain-specific PCR primers were designed based on the sequences of ERIC-PCR bands. The DNA of each of these strains was successfully detected from its mixture with activated sludge DNA, either by using their respective ERIC-PCR-based probes for hybridization or by using species-specific primers for amplification, with higher sensitivity by latter method.
Conclusions:  Two phenol-degrading bacterial strains were identified from a mixture of activated sludge by using ERIC-PCR-based methods.
Significance and Impact of the Study:  The study demonstrated that the bacteria, which have important functions in complex wastewater treatment microbial communities, could be specifically detected by using ERIC-PCR fingerprint-based hybridization or amplification.     

Comparative analysis of archaeal 16S rRNA and <Emphasis Type="Italic">amoA</Emphasis> genes to estimate the abundance and diversity of ammonia-oxidizing archaea in marine sediments

Considering their abundance and broad distribution, non-extremophilic Crenarchaeota are likely to play important roles in global organic and inorganic matter cycles. The diversity and abundance of archaeal 16S rRNA and putative ammonia monooxygenase alpha-subunit (amoA) genes were comparatively analyzed to study genetic potential for nitrification of ammonia-oxidizing archaea (AOA) in the surface layers (0-1 cm) of four marine sediments of the East Sea, Korea. After analysis of a 16S rRNA gene clone library, we found various archaeal groups that include the crenarchaeotal group (CG) I.1a (54.8%) and CG I.1b (5.8%), both of which are known to harbor ammonia oxidizers. Notably, the 16S rRNA gene of CG I.1b has only previously been observed in terrestrial environments. The 16S rRNA gene sequence data revealed a distinct difference in archaeal community among sites of marine sediments. Most of the obtained amoA sequences were not closely related to those of the clones retrieved from estuarine sediments and marine water columns. Furthermore, clades of unique amoA sequences were likely to cluster according to sampling sites. Using real-time PCR, quantitative analysis of amoA copy numbers showed that the copy numbers of archaeal amoA ranged from 1.1 x 10(7) to 4.9 x 10(7) per gram of sediment and were more numerous than those of bacterial amoA, with ratios ranging from 11 to 28. In conclusion, diverse CG I.1a and CG I.1b AOA inhabit surface layers of marine sediments and AOA, and especially, CG I.1a are more numerous than other ammonia-oxidizing bacteria.     

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。     

西藏米拉山土壤古菌16S rRNA及amoA基因多样性?分析

摘要：【目的】硝化作用在全球土壤氮循环中具有重要的作用,虽然细菌一度被认为单独负责催化这个过程的限速步骤,但是最近一些研究结果表明泉古菌具有氨氧化的能力。本文通过构建古菌16S rRNA 基因克隆文库和氨氧化古菌amoA基因文库,分析西藏米拉山高寒草甸土壤中古菌及氨氧化古菌群落结构组成情况,为揭示青藏高原高寒草甸土壤古菌的多样性提供理论基础。【方法】采用未培养技术直接从土壤中提取微生物总DNA,分别利用通用引物构建古菌16S rRNA 基因和氨氧化古菌amoA基因克隆文库。【结果】通过构建系统发育树,表明古菌16S rRNA 基因克隆文库包括泉古菌门和未分类的古菌两大类,并且所有泉古菌均属于热变形菌纲。氨氧化古菌amoA基因克隆文库中序列均为泉古菌。通过DOTUR软件分析,古菌16S rRNA基因和古菌amoA基因克隆文库分别包括64个OTUs和 75个OTUs。【结论】西藏米拉山高寒草甸土壤中古菌多样性比较丰富,表明古菌在高寒草甸土壤的氮循环中可能具有重要的作用。所获得的一些序列与已知环境中土壤、淡水及海洋沉积物中获得的一些序列具有很高的相似性,其古菌及氨氧化古菌来自不同环境的可能性比较大,可能与青藏高原的地质历史变迁过程有关。米拉山古菌及氨氧化古菌与陆地设施土壤中相似性最高,说明与西藏米拉山高寒草甸土壤的退化有关。