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.     

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.     

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.     

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.     

Low Temperature Decreases the Phylogenetic Diversity of Ammonia-Oxidizing Archaea and Bacteria in Aquarium Biofiltration Systems

The phylogenetic diversity and species richness of ammonia-oxidizing archaea (AOA) and bacteria (AOB) were examined with aquarium biofiltration systems. Species richness, deduced from rarefaction analysis, and diversity indices indicated that the phylogenetic diversity and species richness of AOA are greater than those of AOB; the diversity of AOA and of AOB is minimized in cold-water aquaria. This finding implies that temperature is a key factor influencing the population structure and diversity of AOA and AOB in aquarium biofiltration systems.     

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.     

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。     

Changes in N-transforming archaea and bacteria in soil during the establishment of bioenergy crops

Widespread adaptation of biomass production for bioenergy may influence important biogeochemical functions in the landscape, which are mainly carried out by soil microbes. Here we explore the impact of four potential bioenergy feedstock crops (maize, switchgrass, Miscanthus X giganteus, and mixed tallgrass prairie) on nitrogen cycling microorganisms in the soil by monitoring the changes in the quantity (real-time PCR) and diversity (barcoded pyrosequencing) of key functional genes (nifH, bacterial/archaeal amoA and nosZ) and 16S rRNA genes over two years after bioenergy crop establishment. The quantities of these N-cycling genes were relatively stable in all four crops, except maize (the only fertilized crop), in which the population size of AOB doubled in less than 3 months. The nitrification rate was significantly correlated with the quantity of ammonia-oxidizing archaea (AOA) not bacteria (AOB), indicating that archaea were the major ammonia oxidizers. Deep sequencing revealed high diversity of nifH, archaeal amoA, bacterial amoA, nosZ and 16S rRNA genes, with 229, 309, 330, 331 and 8989 OTUs observed, respectively. Rarefaction analysis revealed the diversity of archaeal amoA in maize markedly decreased in the second year. Ordination analysis of T-RFLP and pyrosequencing results showed that the N-transforming microbial community structures in the soil under these crops gradually differentiated. Thus far, our two-year study has shown that specific N-transforming microbial communities develop in the soil in response to planting different bioenergy crops, and each functional group responded in a different way. Our results also suggest that cultivation of maize with N-fertilization increases the abundance of AOB and denitrifiers, reduces the diversity of AOA, and results in significant changes in the structure of denitrification community.     

Relationship of temporal and spatial variabilities of ammonia-oxidizing bacteria to nitrification rates in Monterey Bay, California

Temporal and spatial dynamics of ammonia-oxidizing bacteria (AOB) were examined using genes encoding 16S rRNA and ammonia monooxygenase subunit A (AmoA) in Monterey Bay, Calif. Samples were collected from three depths in the water column on four dates at one mid-bay station. Diversity estimators for the two genes showed a strong positive correlation, indicating that overlapping bacterial populations had been sampled by both sets of clone libraries. Some samples that were separated by only 15 m in depth had less genetic similarity than samples that were collected from the same depth months apart. Clone libraries from the Monterey Bay AOB community were dominated by Nitrosospira-like sequences and clearly differentiated from the adjacent AOB community in Elkhorn Slough. Many Monterey Bay clones clustered with previously identified 16S rRNA and amoA groups composed entirely of marine sequences, supporting the hypothesis that these groups are specific to the marine environment and are dominant marine AOB. In addition, novel, phylogenetically distinct groups of AOB sequences were identified and compared to sequences in the database. Only one cluster of gammaproteobacterial AOB was detected using 16S rRNA genes. Although significant genetic variation was detected in AOB populations from both vertical and temporal samples, no significant correlation was detected between diversity and environmental variables or the rate of nitrification.     

Nitrospira-like bacteria associated with nitrite oxidation in freshwater aquaria

Oxidation of nitrite to nitrate in aquaria is typically attributed to bacteria belonging to the genus Nitrobacter which are members of the alpha subdivision of the class Proteobacteria. In order to identify bacteria responsible for nitrite oxidation in aquaria, clone libraries of rRNA genes were developed from biofilms of several freshwater aquaria. Analysis of the rDNA libraries, along with results from denaturing gradient gel electrophoresis (DGGE) on frequently sampled biofilms, indicated the presence of putative nitrite-oxidizing bacteria closely related to other members of the genus Nitrospira. Nucleic acid hybridization experiments with rRNA from biofilms of freshwater aquaria demonstrated that Nitrospira-like rRNA comprised nearly 5% of the rRNA extracted from the biofilms during the establishment of nitrification. Nitrite-oxidizing bacteria belonging to the alpha subdivision of the class Proteobacteria (e.g., Nitrobacter spp.) were not detected in these samples. Aquaria which received a commercial preparation containing Nitrobacter species did not show evidence of Nitrobacter growth and development but did develop substantial populations of Nitrospira-like species. Time series analysis of rDNA phylotypes on aquaria biofilms by DGGE, combined with nitrite and nitrate analysis, showed a correspondence between the appearance of Nitrospira-like bacterial ribosomal DNA and the initiation of nitrite oxidation. In total, the data suggest that Nitrobacter winogradskyi and close relatives were not the dominant nitrite-oxidizing bacteria in freshwater aquaria. Instead, nitrite oxidation in freshwater aquaria appeared to be mediated by bacteria closely related to Nitrospira moscoviensis and Nitrospira marina.     

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.     

The phylogenetic affiliation of an uncultured population of ammonia-oxidizing bacteria harboring environmental sequences of amoA cluster-3

We investigated the phylogenetic diversity of ammonia-oxidizing bacteria (AOB) in Yellow Sea continental shelf sediment by the cloning and sequencing of PCR-amplified amoA and 16S rRNA genes. Phylogenetic analysis of the amoA-related clones revealed that the diversity of AOB was extremely low at the study site. The majority (92.7%) of amoA clones obtained belonged to a single cluster, environmental amoA cluster-3, the taxonomic position of which was previously unknown. Phylogenetic analysis on AOB-specific 16S rRNA gene sequences also demonstrated a very low diversity. All of the cloned 16S rRNA gene sequences comprised a single phylotype that belonged to the members of uncultured Nitrosospira cluster-1, suggesting that AOB belonging to the uncultured Nitrosospira cluster- 1 could carry amoA sequences of environmental amoA cluster-3.     

Comparative analysis of ammonia monooxygenase (amoA) genes in the water column and sediment-water interface of two lakes and the Baltic Sea

The functional gene amoA was used to compare the diversity of ammonia-oxidizing bacteria (AOB) in the water column and sediment-water interface of the two freshwater lakes Plusssee and Sch?hsee and the Baltic Sea. Nested amplifications were used to increase the sensitivity of amoA detection, and to amplify a 789-bp fragment from which clone libraries were prepared. The larger part of the sequences was only distantly related to any of the cultured AOB and is considered to represent new clusters of AOB within the Nitrosomonas/Nitrosospira group. Almost all sequences from the water column of the Baltic Sea and from 1-m depth of Sch?hsee were related to different Nitrosospira clusters 0 and 2, respectively. The majority of sequences from Plusssee and Sch?hsee were associated with sequences from Chesapeake Bay, from a previous study of Plusssee and from rice roots in Nitrosospira-like cluster A, which lacks sequences from Baltic Sea. Two groups of sequences from Baltic Sea sediment were related to clonal sequences from other brackish/marine habitats in the purely environmental Nitrosospira-like cluster B and the Nitrosomonas-like cluster. This confirms previous results from 16S rRNA gene libraries that indicated the existence of hitherto uncultivated AOB in lake and Baltic Sea samples, and showed a differential distribution of AOB along the water column and sediment of these environments.     

Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys

The current perception of evolutionary relationships and the natural diversity of ammonia-oxidizing bacteria (AOB) is mainly based on comparative sequence analyses of their genes encoding the 16S rRNA and the active site polypeptide of the ammonia monooxygenase (AmoA). However, only partial 16S rRNA sequences are available for many AOB species and most AOB have not yet been analyzed on the amoA level. In this study, the 16S rDNA sequence data of 10 Nitrosomonas species and Nitrosococcus mobilis were completed. Furthermore, previously unavailable 16S rRNA sequences were determined for three Nitrosomonas sp. isolates and for the gamma-subclass proteobacterium Nitrosococcus halophilus. These data were used to revaluate the specificities of published oligonucleotide primers and probes for AOB. In addition, partial amoA sequences of 17 AOB, including the above-mentioned 15 AOB, were obtained. Comparative phylogenetic analyses suggested similar but not identical evolutionary relationships of AOB by using 16S rRNA and AmoA as marker molecules, respectively. The presented 16S rRNA and amoA and AmoA sequence data from all recognized AOB species significantly extend the currently used molecular classification schemes for AOB and now provide a more robust phylogenetic framework for molecular diversity inventories of AOB. For 16S rRNA-independent evaluation of AOB species-level diversity in environmental samples, amoA and AmoA sequence similarity threshold values were determined which can be used to tentatively identify novel species based on cloned amoA sequences. Subsequently, 122 amoA sequences were obtained from 11 nitrifying wastewater treatment plants. Phylogenetic analyses of the molecular isolates showed that in all but two plants only nitrosomonads could be detected. Although several of the obtained amoA sequences were only relatively distantly related to known AOB, none of these sequences unequivocally suggested the existence of previously unrecognized species in the wastewater treatment environments examined.     

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。     

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。     

Diversity and quantity of ammonia-oxidizing Archaea and Bacteria in sediment of the Pearl River Estuary, China

The diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in the sediment of the Pearl River Estuary were investigated by cloning and quantitative real-time polymerase chain reaction (qPCR). From one sediment sample S16, 36 AOA OTUs (3% cutoff) were obtained from three clone libraries constructed using three primer sets for amoA gene. Among the 36 OTUs, six were shared by all three clone libraries, two appeared in two clone libraries, and the other 28 were only recovered in one of the libraries. For AOB, only seven OTUs (based on 16S rRNA gene) and eight OTUs (based on amoA gene) were obtained, showing lower diversity than AOA. The qPCR results revealed that AOA amoA gene copy numbers ranged from 9.6 × 10⁶ to 5.1 × 10⁷ copies per gram of sediment and AOB amoA gene ranged from 9.5 × 10⁴ to 6.2 × 10⁵ copies per gram of sediment, indicating that the dominant ammonia-oxidizing microorganisms in the sediment of the Pearl River Estuary were AOA. The terminal restriction fragment length polymorphism results showed that the relative abundance of AOB species in the sediment samples of different salinity were significantly different, indicating that salinity might be a key factor shaping the AOB community composition.     

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.     

Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria

Denaturing gradient gel electrophoresis (DGGE) of PCR amplicons of the ammonia monooxygenase gene (amoA) was developed and employed to investigate the diversity of ammonia-oxidizing bacteria (AOB) in four different habitats. The results were compared to DGGE of PCR-amplified partial 16S rDNA sequences made with primers specific for ammonia-oxidizing bacteria. Potential problems, such as primer degeneracy and multiple gene copies of the amoA gene, were investigated to evaluate and minimize their possible impact on the outcome of a DGGE analysis. amoA and 16S rDNA amplicons were cloned, and a number of clones screened by DGGE to determine the abundance of different motility types in the clone library. The abundance of clones was compared to the relative intensity of bands emerging in the band pattern produced by direct amplification of the genes from the environmental sample. Selected clones were sequenced to evaluate the specificity of the respective primers. The 16S rDNA primer pair, reported to be specific for ammonia-oxidizing bacteria (AOB), generated several sequences that were not related to the known Nitrosospira-Nitrosomonas group and, thus, not likely to be ammonia oxidizers. However, no false positives were found among the sequences retrieved with the modified amoA primers. Some phylogenetic information could be deduced from the position of amoA bands in DGGE gels. The Nitrosomonas-like sequences were found within a denaturant range from 30% to 46%, whereas the Nitrosospira-like sequences migrated to 50% to 60% denaturant. The majority of retrieved sequences from all four habitats with high ammonia loads were Nitrosomonas-like and only few Nitrosospira-like sequences were detected.