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.     

Detection of Ammonia-Oxidizing Archaea in Fish Processing Effluent Treatment Plants

Ammonia oxidation is the rate limiting step in nitrification and thus have an important role in removal of ammonia in natural and engineered systems with participation of both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). However, their relative distribution and activity in fish processing effluent treatment plants (FPETPs) though significant, is hitherto unreported. Presence of AOA in sludge samples obtained from FPETPs was studied by amplification and sequencing of thaumarchaeal ammonia monooxygenase subunit A (AOA-amoA) gene. Different primer sets targeting 16S rRNA and AOA-amoA gene were used for the detection of AOA in FPETPs. Phylogenetic analysis of the gene revealed that the AOA was affiliated with thaumarchaeal group 1.1a lineage (marine cluster). Quantitative real time PCR of amoA gene was used to study the copy number of AOA and AOB in FPETPs. The AOA-amoA and AOB-amoA gene copy numbers of sludge samples ranged from 2.2 × 10⁶ to 4.2 × 10⁸ and 1.1 × 10⁷ to 8.5 × 10⁸ mg⁻¹ sludge respectively. Primer sets Arch-amoAF/Arch-amoAR and 340F/1000R were found to be useful for the sensitive detection of AOA-amoA and Archaeal 16S rRNA genes respectively in FPETPs. Their presence suggests the widespread occurrence and possible usefulness in removing ammonia from FPETPs which is in line with reports from other waste water treatment plants.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0484-6) contains supplementary material, which is available to authorized users.     

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.     

Ammonia-oxidizing archaea versus bacteria in two soil aquifer treatment systems

So far, the contribution of ammonia-oxidizing archaea (AOA) to ammonia oxidation in wastewater treatment processes has not been well understood. In this study, two soil aquifer treatment (SATs) systems were built up to treat synthetic domestic wastewater (column 1) and secondary effluent (column 4), accomplishing an average of 95 % ammonia removal during over 550 days of operation. Except at day 322, archaeal amoA genes always outnumbered bacterial amoA genes in both SATs as determined by using quantitative polymerase chain reaction (q-PCR). The ratios of archaeal amoA to 16S rRNA gene averaged at 0.70?±?0.56 and 0.82?±?0.62 in column 1 and column 4, respectively, indicating that all the archaea could be AOA carrying amoA gene in the SATs. The results of MiSeq-pyrosequencing targeting on archaeal and bacterial 16S rRNA genes with the primer pair of modified 515R/806R indicated that Nitrososphaera cluster affiliated with thaumarchaeal group I.1b was the dominant AOA species, while Nitrosospira cluster was the dominant ammonia-oxidizing bacteria (AOB). The statistical analysis showed significant relationship between AOA abundance (compared to AOB abundance) and inorganic and total nitrogen concentrations. Based on the mathematical model calculation for microbial growth, AOA had much greater capacity of ammonia oxidation as compared to the specific influent ammonia loading for AOA in the SATs, implying that a small fraction of the total AOA would actively work to oxidize ammonia chemoautotrophically whereas most of AOA would exhibit some level of functional redundancy. These results all pointed that AOA involved in microbial ammonia oxidation in the SATs.     

Bacteria dominate the ammonia-oxidizing community in a hydrothermal vent site at the Mid-Atlantic Ridge of the South Atlantic Ocean

Ammonia oxidation is the first and rate-limiting step of nitrification, which is carried out by two groups of microorganisms: ammonia-oxidizing bacteria (AOB) and the recently discovered ammonia-oxidizing archaea (AOA). In this study, diversity and abundance of AOB and AOA were investigated in five rock samples from a deep-sea hydrothermal vent site at the Mid-Atlantic Ridge (MAR) of the South Atlantic Ocean. Both bacterial and archaeal ammonia monooxygenase subunit A (amoA) gene sequences obtained in this study were closely related to the sequences retrieved from deep-sea environments, indicating that AOB and AOA in this hydrothermal vent site showed typical deep ocean features. AOA were more diverse but less abundant than AOB. The ratios of AOA/AOB amoA gene abundance ranged from 1/3893 to 1/242 in all investigate samples, indicating that bacteria may be the major members responding to the aerobic ammonia oxidation in this hydrothermal vent site. Furthermore, diversity and abundance of AOA and AOB were significantly correlated with the contents of total nitrogen and total sulfur in investigated samples, suggesting that these two environmental factors exert strong influences on distribution of ammonia oxidizers in deep-sea hydrothermal vent environment.     

Abundance of amoA genes of ammonia-oxidizing archaea and bacteria in activated sludge of full-scale wastewater treatment plants

In this study, the abundance and sequences of amoA genes of ammonia-oxidizing archaea (AOA) and bacteria (AOB) were determined in seven wastewater treatment plants (WWTPs) whose ammonium concentrations in influent and effluent wastewaters varied considerably (5.6-422.3 mgN l⁻¹ and 0.2-29.2 mgN l⁻¹, respectively). Quantitative real-time PCR showed that the comparative abundance of AOA and AOB amoA genes differed among the WWTPs. In all three industrial WWTPs, where the influent and effluent contained the higher levels of ammonium (36.1-422.3 mgN l⁻¹ and 5.3-29.2 mgN l⁻¹, respectively), more than four orders of magnitude higher numbers of AOB amoA genes than AOA amoA genes arose (with less than the limit of detection of AOA amoA genes). In contrast, significant numbers of AOA amoA genes occurred in all municipal WWTPs (with ammonium levels in the influent and effluent of 5.6-11.0 mgN l⁻¹ and 0.2-3.0 mgN l⁻¹, respectively). Statistical analysis suggested that compared to other plants’ parameters, the ammonium levels in the plants’ effluent showed correlation with the highest p value to the abundance of AOA amoA genes.     

Diversity, abundance, and activity of ammonia-oxidizing bacteria and archaea in Chongming eastern intertidal sediments

Ammonia oxidation plays a pivotal role in the cycling and removal of nitrogen in aquatic sediments. Certain bacterial groups and a novel group of archaea, which is affiliated with the novel phylum Thaumarchaeota, can perform this initial nitrification step. We examined the diversity and abundance of ammonia-oxidizing β-Proteobacteria (β-AOB) and ammonia-oxidizing archaea (AOA) in the sediments of Chongming eastern tidal flat using the ammonia monooxygenase-α subunit (amoA) gene as functional markers. Clone library analysis showed that AOA had a higher diversity of amoA gene than β-AOB. The β-Proteobacterial amoA community composition correlated significantly with water soluble salts in the sediments, whereas the archaeal amoA community composition was correlated more with nitrate concentrations. Quantitative PCR (qPCR) results indicated that the abundance of β-AOB amoA gene (9.11?×?10⁴–6.47?×?10⁵?copies?g^?1 sediment) was always greater than that of AOA amoA gene (7.98?×?10³–3.51?×?10⁵?copies?g^?1 sediment) in all the samples analyzed in this study. The β-Proteobacterial amoA gene abundance was closely related to organic carbon, while no significant correlations were observed between archaeal amoA gene abundance and the environmental factors. Potential nitrification rates were significantly greater in summer than in winter and correlated strongly with the abundance of amoA genes. Additionally, a greater contribution of single amoA gene to potential nitrification occurred in summer (1.03–5.39 pmol?N?copy^?1?day^?1) compared with winter (0.16–0.38 pmol?N?copy^?1?day^?1), suggesting a higher activity of ammonia-oxidizing prokaryotes in warm seasons.     

Abundance and Diversity of Ammonia-Oxidizing Microorganisms in the Sediments of Jinshan Lake

Community structures of ammonia-oxidizing microorganisms were investigated using PCR primers designed to specifically target the ammonia monooxygenase α-subunit (amoA) gene in the sediment of Jinshan Lake. Relationships between the abundance and diversity of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), and physicochemical parameters were also explored. The AOA abundance decreased sharply from west to east; however, the AOB abundance changed slightly with AOB outnumbering AOA in two of the four sediment samples (JS), JS3 and JS4. The AOA abundance was significantly correlated with the NH₄–N, NO₃–N, and TP. No significant correlations were observed between the AOB abundance and environmental variables. AOB had a higher diversity and richness of amoA genes than AOA. Among the 76 archaeal amoA sequences retrieved, 57.89, 38.16, and 3.95 % fell within the Nitrosopumilus, Nitrososphaera, and Nitrososphaera sister clusters, respectively. The 130 bacterial amoA gene sequences obtained in this study were grouped with known AOB sequences in the Nitrosomonas and Nitrosospira genera, which occupied 72.31 % and 27.69 % of the AOB group, respectively. Compared to the other three sample sites, the AOA and AOB community compositions at JS4 showed a large difference. This work could enhance our understanding of the roles of ammonia-oxidizing microorganisms in freshwater lake environment.     

Diversity and abundance of ammonia-oxidizing archaea and bacteria in polluted mangrove sediment

Ammonia oxidation by microorganisms is a critical process in the nitrogen cycle. Recent research results show that ammonia-oxidizing archaea (AOA) are both abundant and diverse in a range of ecosystems. In this study, we examined the abundance and diversity of AOA and ammonia-oxidizing beta-proteobacteria (AOB) in estuarine sediments in Hong Kong for two seasons using the ammonia monooxygenase A subunit gene (amoA) as molecular biomarker. Relationships between diversity and abundance of AOA and AOB and physicochemical parameters were also explored. AOB were more diverse but less abundant than AOA. A few phylogenetically distinct amoA gene clusters were evident for both AOA and AOB from the mangrove sediment. Pearson moment correlation analysis and canonical correspondence analysis (CCA) were used to explore physicochemical parameters potentially important to AOA and AOB. Metal concentrations were proposed to contribute potentially to the distributions of AOA while total phosphorus (TP) was correlated to the distributions of AOB. Quantitative PCR estimates indicated that AOA were more abundant than AOB in all samples, but the ratio of AOA/AOB (from 1.8 to 6.3) was smaller than most other studies by one to two orders. The abundance of AOA or AOB was correlated with pH and temperature while the AOA/AOB ratio was with the concentrations of ammonium. Several physicochemical factors, rather than any single one, affect the distribution patterns suggesting that a combination of factors is involved in shaping the dynamics of AOA and AOB in the mangrove ecosystem.     

Distribution patterns of ammonia-oxidizing archaea and bacteria in sediments of the eastern China marginal seas

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) vary in their contribution to nitrification in different environments. The eastern China marginal seas (ECMS) are featured by complex river runoffs and ocean currents, forming different sediment patches. Here, via quantitative PCR and clone library analysis of the amoA genes, we showed that AOB were more abundant than AOA in ECMS sediments. The abundance, diversity and richness of AOA, but not AOB, were higher in the East China Sea (ECS) than in the Yellow Sea (YS) and Bohai Sea (BS). Nitrosopumilus (AOA) and Nitrosospira (AOB) were predominant lineages, but their abundances varied significantly between ECS, and BS and YS. This was mainly attributed to salinity and dissolved oxygen of the bottom water. The discovery of a high abundance of Nitrosophaera at estuarine sites suggested strong terrigenous influence exerted on the AOA community. In contrast, variations in ocean conditions played more important roles in structuring the AOB community, which was separated by bottom water dissolved oxygen into two groups: the south YS, and the north YS and BS. This study provides a comprehensive insight into the spatial distribution pattern of ammonia-oxidizing prokaryotes in ECMS sediments, laying a foundation for understanding their relative roles in nitrification.     

Abundance and diversity based on amoA genes of ammonia-oxidizing archaea and bacteria in ten wastewater treatment systems

The abundance and diversity of amoA genes of ammonia-oxidizing archaea (AOA) and bacteria (AOB) were investigated in ten wastewater treatment systems (WTSs) by polymerase chain reaction (PCR), cloning, sequencing, and quantitative real-time PCR (qPCR). The ten WTSs included four full-scale municipal WTSs, three full-scale industrial WTSs, and three lab-scale WTSs. AOB were present in all the WTSs, whereas AOA were detected in nine WTSs. QPCR data showed that AOB amoA genes (4.625?×?10⁴–9.99?×?10⁹ copies g^?1 sludge) outnumbered AOA amoA genes (<limit of detection–1.90?×?10⁷ copies g^?1 sludge) in each WTS, indicating that AOB may play an important role than AOA in ammonia oxidization in WTSs. Interestingly, it was found that AOA and AOB coexisted with anaerobic ammonia oxidation (anammox) bacteria in three anammox WTSs with relatively higher abundance. In a full-scale industrial WTS where effluent ammonia was higher than influent ammonia, both AOA and AOB showed higher abundance. The phylogenetic analysis of AOB amoA genes showed that genera Nitrosomonas was the most dominant species in the ten WTSs; Nitrosomonas europaea cluster was the dominant major cluster, followed by Nitrosomonas-like cluster and Nitrosomonas oligotropha cluster; and AOB species showed higher diversity than AOA species. AOA were found to be affiliated with two major clusters: Nitrososphaera cluster and Nitrosopumilus cluster. Nitrososphaera cluster was the most dominant species in different samples and distributed worldwide.     

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.     

High diversity of ammonia‐oxidizing archaea in permanent and seasonal oxygen‐deficient waters of the eastern South Pacific

The community structure of putative aerobic ammonia‐oxidizing archaea (AOA) was explored in two oxygen‐deficient ecosystems of the eastern South Pacific: the oxygen minimum zone off Peru and northern Chile (11°S–20°S), where permanent suboxic and low‐ammonium conditions are found at intermediate depths, and the continental shelf off central Chile (36°S), where seasonal oxygen‐deficient and relatively high‐ammonium conditions develop in the water column, particularly during the upwelling season. The AOA community composition based on the ammonia monooxygenase subunit A (amoA) genes changed according to the oxygen concentration in the water column and the ecosystem studied, showing a higher diversity in the seasonal low‐oxygen waters. The majority of the archaeal amoA genotypes was affiliated to the uncultured clusters A (64%) and B (35%), with Cluster A AOA being mainly associated with higher oxygen and ammonium concentrations and Cluster B AOA with permanent oxygen‐ and ammonium‐poor waters. Q‐PCR assays revealed that AOA are an abundant community (up to 10⁵amoA copies ml^?1), while bacterial amoA genes from β proteobacteria were undetected. Our results thus suggest that a diverse uncultured AOA community, for which, therefore, we do not have any physiological information, to date, is an important component of the nitrifying community in oxygen‐deficient marine ecosystems, and particularly in rich coastal upwelling ones.     

长期集约种植对雷竹林土壤氨氧化古菌群落的影响

应用荧光定量PCR以及PCR-DGGE技术研究了雷竹林长期集约种植过程中土壤氨氧化古菌种群数量及群落结构的演变趋势,并利用典范对应分析(CCA)方法研究影响土壤氨氧化古菌群落的主要环境因子。研究结果表明,水稻田改种雷竹后,土壤氨氧化古菌种群数量显著增加,在4 a时达到最高,但在集约种植后快速下降,9 a雷竹林土壤氨氧化古菌数量最低,随后逐渐稳定。雷竹林土壤氨氧化古菌种群主要为不可培养泉古菌,聚类分析结果表明集约种植前后氨氧化古菌群落结构存在明显差异,长期集约经营雷竹林土壤以适应较低pH值的物种为主要优势种群。CCA分析表明,集约种植时间较长的11 a和15 a林地群落结构较为类似,与7 a和9 a样地明显区分。土壤NO-3-N与氨氧化古菌群落变化的相关性最强,说明氨氧化古菌在雷竹林土壤硝化过程中发挥着重要作用。土壤pH值及速效养分对土氨氧化古菌群落也具有较大影响,它们与NO-3-N合计解释了59.7%的样本总变异,表明土壤酸化以及过量养分的积累对氨氧化古菌群落具有重要影响。     

Diversity and distribution of ammonia-oxidizing Archaea in the seasonally frozen soils in Northeastern China

The genetic diversity and distribution of ammonia-oxidizing Archaea (AOA) in nine seasonally frozen soils sampled around the city of Harbin, China, is analyzed based on archaeal amoA gene. Soil samples are divided into four groups by its properties: fertilized/unfertilized mesic (well-balanced supply of moisture) soils and fertilized/unfertilized hydric (abundant of moisture) soils. Clone libraries based on AOA amoA gene polymerase chain reaction products are constructed, and the phylogenetic analysis at 5 % cutoff level shows that AOA members mainly belong to the soil/sediment lineage which includes four clusters, and very few archaeal amoA gene sequences fall into the marine lineage. The four groups of soils have different archaeal amoA gene assemblage, and the available nitrogen and organic carbon are significantly correlated with diversity indexes. The result shows that long-term artificial amendment such as fertilization and agriculture cultivation has an important impact on AOA community shift in terrestrial environment. Moisture may drive the shape of different AOA communities by changing the aerobic environment into anaerobic. Soil composition is another noticeable factor effect AOA community, which can help the shape of a special AOA community with only two species.     

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.