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.     

Low-ammonia niche of ammonia-oxidizing archaea in rotating biological contactors of a municipal wastewater treatment plant

The first step of nitrification is catalysed by both ammonia-oxidizing bacteria (AOB) and archaea (AOA), but physicochemical controls on the relative abundance and function of these two groups are not yet fully understood, especially in freshwater environments. This study investigated ammonia-oxidizing populations in nitrifying rotating biological contactors (RBCs) from a municipal wastewater treatment plant. Individual RBC stages are arranged in series, with nitrification at each stage creating an ammonia gradient along the flowpath. This RBC system provides a valuable experimental system for testing the hypothesis that ammonia concentration determines the relative abundance of AOA and AOB. The results demonstrate that AOA increased as ammonium decreased across the RBC flowpath, as indicated by qPCR for thaumarchaeal amoA and 16S rRNA genes, and core lipid (CL) and intact polar lipid (IPL) crenarchaeol abundances. Overall, there was a negative logarithmic relationship (R(2) =?0.51) between ammonium concentration and the relative abundance of AOA amoA genes. A single AOA population was detected in the RBC biofilms; this phylotype shared low amoA and 16S rRNA gene homology with existing AOA cultures and enrichments. These results provide evidence that ammonia availability influences the relative abundances of AOA and AOB, and that AOA are abundant in some municipal wastewater treatment systems.     

Seasonal changes of freshwater ammonia-oxidizing archaeal assemblages and nitrogen species in oligotrophic alpine lakes

The annual changes in the composition and abundance of ammonia-oxidizing archaea (AOA) were analyzed monthly in surface waters of three high mountain lakes within the Limnological Observatory of the Pyrenees (LOOP; northeast Spain) using both 16S rRNA and functional (ammonia monooxygenase gene, amoA) gene sequencing as well as quantitative PCR amplification. The set of biological data was related to changes in nitrogen species and to other relevant environmental variables. The whole archaeal assemblage was dominated by phylotypes closely related to the crenarchaeal 1.1a group (58% ± 18% of total 16S rRNA gene sequences), and consistent structural changes were detected during the study. Water temperature was the environmental variable that better explained spring, summer, and winter (ice-covered lakes) archaeal assemblage structure. The amoA gene was detected year round, and seasonal changes in amoA gene composition were well correlated with changes in the archaeal 16S rRNA gene pool. In addition, copy numbers of both the specific 1.1a group 16 rRNA and archaeal amoA genes were well correlated, suggesting that most freshwater 1.1a Crenarchaeota had the potential to carry out ammonia oxidation. Seasonal changes in the diversity and abundance of AOA (i.e., amoA) were better explained by temporal changes in ammonium, the substrate for nitrification, and mostly nitrite, the product of ammonia oxidation. Lacustrine amoA gene sequences grouped in coherent freshwater phylogenetic clusters, suggesting that freshwater habitats harbor typical amoA-containing ecotypes, which is different from soils and seas. We observed within the freshwater amoA gene sequence pool a high genetic divergence (translating to up to 32% amino acid divergence) between the spring and the remaining AOA assemblages. This suggests that different AOA ecotypes are adapted to different temporal ecological niches in these lakes.     

Crenarchaeota and their role in the nitrogen cycle in a subsurface radioactive thermal spring in the Austrian Central Alps

Previous results from a 16S rRNA gene library analysis showed high diversity within the prokaryotic community of a subterranean radioactive thermal spring, the "Franz-Josef-Quelle" (FJQ) in Bad Gastein, Austria, as well as evidence for ammonia oxidation by crenarchaeota. This study reports further characterization of the community by denaturing gradient gel electrophoresis (DGGE) analysis, fluorescence in situ hybridization (FISH), and semiquantitative nitrification measurements. DGGE bands from three types of samples (filtered water, biofilms on glass slides, and naturally grown biofilms), including samples collected at two distinct times (January 2005 and July 2006), were analyzed. The archaeal community consisted mainly of Crenarchaeota of the soil-subsurface-freshwater group (group 1.1b) and showed a higher diversity than in the previous 16S rRNA gene library analysis, as was also found for crenarchaeal amoA genes. No bacterial amoA genes were detected. FISH analysis of biofilms indicated the presence of archaeal cells with an abundance of 5.3% (+/-4.5%) in the total 4',6-diamidino-2-phenylindole (DAPI)-stained community. Microcosm experiments of several weeks in duration showed a decline of ammonium that correlated with an increase of nitrite, the presence of crenarchaeal amoA genes, and the absence of bacterial amoA genes. The data suggested that only ammonia-oxidizing archaea (AOA) perform the first step of nitrification in this 45 degrees C environment. The crenarchaeal amoA gene sequences grouped within a novel cluster of amoA sequences from the database, originating from geothermally influenced environments, for which we propose the designation "thermal spring" cluster and which may be older than most AOA from soils on earth.     

Temporal and Spatial Stability of Ammonia-Oxidizing Archaea and Bacteria in Aquarium Biofilters

Nitrifying biofilters are used in aquaria and aquaculture systems to prevent accumulation of ammonia by promoting rapid conversion to nitrate via nitrite. Ammonia-oxidizing archaea (AOA), as opposed to ammonia-oxidizing bacteria (AOB), were recently identified as the dominant ammonia oxidizers in most freshwater aquaria. This study investigated biofilms from fixed-bed aquarium biofilters to assess the temporal and spatial dynamics of AOA and AOB abundance and diversity. Over a period of four months, ammonia-oxidizing microorganisms from six freshwater and one marine aquarium were investigated at 4–5 time points. Nitrogen balances for three freshwater aquaria showed that active nitrification by aquarium biofilters accounted for ≥81–86% of total nitrogen conversion in the aquaria. Quantitative PCR (qPCR) for bacterial and thaumarchaeal ammonia monooxygenase (amoA) genes demonstrated that AOA were numerically dominant over AOB in all six freshwater aquaria tested, and contributed all detectable amoA genes in three aquarium biofilters. In the marine aquarium, however, AOB outnumbered AOA by three to five orders of magnitude based on amoA gene abundances. A comparison of AOA abundance in three carrier materials (fine sponge, rough sponge and sintered glass or ceramic rings) of two three-media freshwater biofilters revealed preferential growth of AOA on fine sponge. Denaturing gel gradient electrophoresis (DGGE) of thaumarchaeal 16S rRNA genes indicated that community composition within a given biofilter was stable across media types. In addition, DGGE of all aquarium biofilters revealed low AOA diversity, with few bands, which were stable over time. Nonmetric multidimensional scaling (NMDS) based on denaturing gradient gel electrophoresis (DGGE) fingerprints of thaumarchaeal 16S rRNA genes placed freshwater and marine aquaria communities in separate clusters. These results indicate that AOA are the dominant ammonia-oxidizing microorganisms in freshwater aquarium biofilters, and that AOA community composition within a given aquarium is stable over time and across biofilter support material types.     

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.     

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。     

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.     

Nitrification of archaeal ammonia oxidizers in acid soils is supported by hydrolysis of urea

The hydrolysis of urea as a source of ammonia has been proposed as a mechanism for the nitrification of ammonia-oxidizing bacteria (AOB) in acidic soil. The growth of Nitrososphaera viennensis on urea suggests that the ureolysis of ammonia-oxidizing archaea (AOA) might occur in natural environments. In this study, ¹⁵N isotope tracing indicates that ammonia oxidation occurred upon the addition of urea at a concentration similar to the in situ ammonium content of tea orchard soil (pH 3.75) and forest soil (pH 5.4) and was inhibited by acetylene. Nitrification activity was significantly stimulated by urea fertilization and coupled well with abundance changes in archaeal amoA genes in acidic soils. Pyrosequencing of 16S rRNA genes at whole microbial community level demonstrates the active growth of AOA in urea-amended soils. Molecular fingerprinting further shows that changes in denaturing gradient gel electrophoresis fingerprint patterns of archaeal amoA genes are paralleled by nitrification activity changes. However, bacterial amoA and 16S rRNA genes of AOB were not detected. The results strongly suggest that archaeal ammonia oxidation is supported by hydrolysis of urea and that AOA, from the marine Group 1.1a-associated lineage, dominate nitrification in two acidic soils tested.     

Ammonia-oxidizing Archaea in the Arctic Ocean and Antarctic coastal waters

We compared abundance, distributions and phylogenetic composition of Crenarchaeota and ammonia-oxidizing Archaea (AOA) in samples collected from coastal waters west of the Antarctic Peninsula during the summers of 2005 and 2006, with samples from the central Arctic Ocean collected during the summer of 1997. Ammonia-oxidizing Archaea and Crenarchaeota abundances were estimated from quantitative PCR measurements of amoA and 16S rRNA gene abundances. Crenarchaeota and AOA were approximately fivefold more abundant at comparable depths in the Antarctic versus the Arctic Ocean. Crenarchaeota and AOA were essentially absent from the Antarctic Summer Surface Water (SSW) water mass (0–45 m depth). The ratio of Crenarchaeota 16S rRNA to archaeal amoA gene abundance in the Winter Water (WW) water mass (45–105 m depth) of the Southern Ocean was much lower (0.15) than expected and in sharp contrast to the ratio (2.0) in the Circumpolar Deep Water (CDW) water mass (105–3500 m depth) immediately below it. We did not observe comparable segregation of this ratio by depth or water mass in Arctic Ocean samples. A ubiquitous, abundant and polar-specific crenarchaeote was the dominant ribotype in the WW and important in the upper halocline of the Arctic Ocean. Our data suggest that this organism does not contain an ammonia monooxygenase gene. In contrast to other studies where Crenarchaeota populations apparently lacking amoA genes are found in bathypelagic waters, this organism appears to dominate in well-defined, ammonium-rich, near-surface water masses in polar oceans.     

Biases in community structures of ammonia/ammonium-oxidizing microorganisms caused by insufficient DNA extractions from Baijiang soil revealed by comparative analysis of coastal wetland sediment and rice paddy soil

Repetitive extraction of DNAs from surface sediments of a coastal wetland in Mai Po Nature Reserve (MP) of Hong Kong and surface Baijiang soils from a rice paddy (RP) in Northeast China was conducted to compare the microbial diversity in this study. Community structures of ammonia/ammonium-oxidizing microorganisms in these samples were analyzed by PCR-DGGE technique. The diversity and abundance of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and anaerobic ammonium-oxidizing (anammox) bacteria were also analyzed based on archaeal and bacterial ammonia monooxygenase subunit A encoding (amoA) and anammox bacterial 16S rRNA genes, respectively. DGGE profiles of archaeal and bacterial amoA and anammox bacterial 16S rRNA genes showed a similar pattern among all five repetitively extracted DNA fractions from both MP and RP, except the anammox bacteria in RP, indicating a more diverse anammox community retrieved in the second to the fifth fractions than the first one. Both soil and marine group AOA were detected while soil and coastal group AOB and Scalindua-anammox bacteria were dominant in MP. Soil group AOA and marine group AOB were dominant in RP, while both Scalindua and Kuenenia species were detected in RP. Pearson correlation analysis showed that the abundance of archaeal and bacterial amoA and anammox bacterial 16S rRNA genes was significantly correlated with the DNA concentrations of the five DNA fractions from MP, but not from RP (except the archaeal amoA gene). Results suggest that anammox bacteria diversity may be biased by insufficient DNA extraction of rice paddy soil samples.     

Shifts in the relative abundance of ammonia-oxidizing bacteria and archaea across physicochemical gradients in a subterranean estuary

Submarine groundwater discharge to coastal waters can be a significant source of both contaminants and biologically limiting nutrients. Nitrogen cycling across steep gradients in salinity, oxygen and dissolved inorganic nitrogen in sandy 'subterranean estuaries' controls both the amount and form of nitrogen discharged to the coastal ocean. We determined the effect of these gradients on betaproteobacterial ammonia-oxidizing bacteria (β-AOB) and ammonia-oxidizing archaea (AOA) in a subterranean estuary using the functional gene encoding ammonia monooxygenase subunit A ( amoA ). The abundance of β-AOB was dramatically lower in the freshwater stations compared with saline stations, while AOA abundance remained nearly constant across the study site. This differing response to salinity altered the ratio of β-AOB to AOA such that bacterial amoA was 30 times more abundant than crenarchaeal amoA at the oxic marine station, but nearly 10 times less abundant at the low-oxygen fresh and brackish stations. As the location of the brackish mixing zone within the aquifer shifted from landward in winter to oceanward in summer, the location of the transition from a β-AOB-dominated to an AOA-dominated community also shifted, demonstrating the intimate link between microbial communities and coastal hydrology. Analysis of ammonia-oxidizing enrichment cultures at a range of salinities revealed that AOA persisted solely in the freshwater enrichments where they actively express amoA . Diversity (as measured by total richness) of crenarchaeal amoA was high at all stations and time points, in sharp contrast to betaproteobacterial amoA for which only two sequence types were found. These results offer new insights into the ecology of AOA and β-AOB by elucidating conditions that may favour the numerical dominance of β-AOB over AOA in coastal sediments.     

太湖竺山湾沉积物中氨氧化原核生物的垂直分布与多样性

原核生物驱动的氨氧化过程对于富营养化湖泊的氮循环具有重要意义。为了解太湖藻型湖区沉积物中氨氧化原核生物的垂直分布和多样性特征,采用分子生态学方法,对竺山湾沉积物剖面中氨单加氧酶基因(amoA)或16S rRNA基因等特征分子标记的变化和序列特征进行了分析。结果表明,氨氧化细菌(ammonia-oxidizing bacteria,AOB)和氨氧化古菌(ammonia-oxidizing archaea,AOA)共存于沉积物各层。AOB的优势种在5cm深度以下发生明显改变,这可能与沉积物氧化还原电位及铵态氮的变化有关;所有细菌amoA序列均属亚硝化单胞菌(Nitrosomonas)。AOA群落结构自表层至7cm深度变化不大,所有古菌amoA序列分属泉古菌CG1.1b和CG1.1a两大类群,这可能与太湖形成历史上的海陆交替过程有关。此外,沉积物各层均未发现典型厌氧氨氧化(anaerobic ammonium oxidation,anammox)细菌16S rRNA基因序列。这些发现丰富了对太湖藻型湖区氨氧化原核生物分布、多样性及环境调控原理的认识,对理解富营养化湖泊氨氧化规律、认识湖泊生态系统氮循环功能具有借鉴意义。     

Abundance and diversity of archaeal ammonia oxidizers in a coastal groundwater system

Nitrification, the microbially catalyzed oxidation of ammonia to nitrate, is a key process in the nitrogen cycle. Archaea have been implicated in the first part of the nitrification pathway (oxidation of ammonia to nitrite), but the ecology and physiology of these organisms remain largely unknown. This work describes two different populations of sediment-associated ammonia-oxidizing archaea (AOA) in a coastal groundwater system in Cape Cod, MA. Sequence analysis of the ammonia monooxygenase subunit A gene (amoA) shows that one population of putative AOA inhabits the upper meter of the sediment, where they may experience frequent ventilation, with tidally driven overtopping and infiltration of bay water supplying dissolved oxygen, ammonium, and perhaps organic carbon. A genetically distinct population occurs deeper in the sediment, in a mixing zone between a nitrate- and oxygen-rich freshwater zone and a reduced, ammonium-bearing saltwater wedge. Both of these AOA populations are coincident with increases in the abundance of group I crenarchaeota 16S rRNA gene copies.     

PCR profiling of ammonia-oxidizer communities in acidic soils subjected to nitrogen and sulphur deposition

Communities of ammonia-oxidizing bacteria (AOB) were characterized in two acidic soil sites experimentally subjected to varying levels of nitrogen and sulphur deposition. The sites were an acidic spruce forest soil in Deepsyke, Southern Scotland, with low background deposition, and a nitrogen-saturated upland grass heath in Pwllpeiran, North Wales. Betaproteobacterial ammonia-oxidizer 16S rRNA and ammonia monooxygenase (amoA) genes were analysed by cloning, sequencing and denaturing gradient gel electrophoresis (DGGE). DGGE profiles of amoA and 16S rRNA gene fragments from Deepsyke soil in 2002 indicated no effect of nitrogen deposition on AOB communities, which contained both Nitrosomonas europaea and Nitrosospira. In 2003, only Nitrosospira could be detected, and no amoA sequences could be retrieved. These results indicate a decrease in the relative abundance of AOB from the year 2002 to 2003 in Deepsyke soil, which may be the result of the exceptionally low rainfall in spring 2003. Nitrosospira-related sequences from Deepsyke soil grouped in all clusters, including cluster 1, which typically contains only sequences from marine environments. In Pwllpeiran soil, 16S rRNA gene libraries were dominated by nonammonia oxidizers and no amoA sequences were detectable. This indicates that autotrophic AOB play only a minor role in these soils even at high nitrogen deposition.     

Quantitative analyses of the abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea of a Chinese upland red soil under long-term fertilization practices

The abundance and composition of soil ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were investigated by using quantitative real-time polymerase chain reaction, cloning and sequencing approaches based on amoA genes. The soil, classified as agri-udic ferrosols with pH (H(2)O) ranging from 3.7 to 6.0, was sampled in summer and winter from long-term field experimental plots which had received 16 years continuous fertilization treatments, including fallow (CK0), control without fertilizers (CK) and those with combinations of fertilizer nitrogen (N), phosphorus (P) and potassium (K): N, NP, NK, PK, NPK and NPK plus organic manure (OM). Population sizes of AOB and AOA changed greatly in response to the different fertilization treatments. The NPK + OM treatment had the highest copy numbers of AOB and AOA amoA genes among the treatments that received mineral fertilizers, whereas the lowest copy numbers were recorded in the N treatment. Ammonia-oxidizing archaea were more abundant than AOB in all the corresponding treatments, with AOA to AOB ratios ranging from 1.02 to 12.36. Significant positive correlations were observed among the population sizes of AOB and AOA, soil pH and potential nitrification rates, indicating that both AOB and AOA played an important role in ammonia oxidation in the soil. Phylogenetic analyses of the amoA gene fragments showed that all AOB sequences from different treatments were affiliated with Nitrosospira or Nitrosospira-like species and grouped into cluster 3, and little difference in AOB community composition was recorded among different treatments. All AOA sequences fell within cluster S (soil origin) and cluster M (marine and sediment origin). Cluster M dominated exclusively in the N, NP, NK and PK treatments, indicating a pronounced difference in the community composition of AOA in response to the long-term fertilization treatments. These findings could be fundamental to improve our understanding of the importance of both AOB and AOA in the cycling of nitrogen and other nutrients in terrestrial ecosystems.     

Growth of ammonia-oxidizing archaea in soil microcosms is inhibited by acetylene

Autotrophic ammonia-oxidizing bacteria were considered to be responsible for the majority of ammonia oxidation in soil until the recent discovery of the autotrophic ammonia-oxidizing archaea. To assess the relative contributions of bacterial and archaeal ammonia oxidizers to soil ammonia oxidation, their growth was analysed during active nitrification in soil microcosms incubated for 30 days at 30 °C, and the effect of an inhibitor of ammonia oxidation (acetylene) on their growth and soil nitrification kinetics was determined. Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial ammonia oxidizer 16S rRNA genes did not detect any change in their community composition during incubation, and quantitative PCR (qPCR) analysis of bacterial amoA genes indicated a small decrease in abundance in control and acetylene-containing microcosms. DGGE fingerprints of archaeal amoA and 16S rRNA genes demonstrated changes in the relative abundance of specific crenarchaeal phylotypes during active nitrification. Growth was also indicated by increases in crenarchaeal amoA gene copy number, determined by qPCR. In microcosms containing acetylene, nitrification and growth of the crenarchaeal phylotypes were suppressed, suggesting that these crenarchaea are ammonia oxidizers. Growth of only archaeal but not bacterial ammonia oxidizers occurred in microcosms with active nitrification, indicating that ammonia oxidation was mostly due to archaea in the conditions of the present study.     

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。