Autotrophic Ammonia Oxidation Research Articles

15N-DNA stable isotope probing reveals niche differentiation of ammonia oxidizers in paddy soils

Chemoautotrophic canonical ammonia oxidizers (ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB)) and complete ammonia oxidizers (comammox Nitrospira) are accountable for ammonia oxidation, which is a fundamental process of nitrification in terrestrial ecosystems. However, the relationship between autotrophic nitrification and the active nitrifying populations during 15N-urea incubation has not been totally clarified. The 15N-labeled DNA stable isotope probing (DNA-SIP) technique was utilized in order to study the response from the soil nitrification process and the active nitrifying populations, in both acidic and neutral paddy soils, to the application of urea. The presence of C2H2 almost completely inhibited NO3−-N production, indicating that autotrophic ammonia oxidation was dominant in both paddy soils. 15N-DNA-SIP technology could effectively distinguish active nitrifying populations in both soils. The active ammonia oxidation groups in both soils were significantly different, AOA (NS (Nitrososphaerales)-Alpha, NS-Gamma, NS-Beta, NS-Delta, NS-Zeta and NT (Ca. Nitrosotaleales)-Alpha), and AOB (Nitrosospira) were functionally active in the acidic paddy soil, whereas comammox Nitrospira clade A and Nitrosospira AOB were functionally active in the neutral paddy soil. This study highlights the effective discriminative effect of 15N-DNA-SIP and niche differentiation of nitrifying populations in these paddy soils.Key points• 15N-DNA-SIP technology could effectively distinguish active ammonia oxidizers.• Comammox Nitrospira clade A plays a lesser role than canonical ammonia oxidizers.• The active groups in the acidic and neutral paddy soils were significantly different.Graphical

Applications of autotrophic ammonia oxidizers in bio-geochemical cycles

Nitrogen is essential for life and to produce food. Still, nitrogen loss of the unused nitrogen in wastewater, air, freshwater, and oceans has caused nitrogen pollution, which impacts the environment and leads to eutrophication, climate change, biodiversity loss, and ozone depletion. It also causing cardio respiratory issues in humans. Anthropogenic activities such as food processing, fertilizer production, mining, and other impacting the global biogeochemical nitrogen cycle. Autotrophic ammonia-oxidizing microbial moieties such as ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and anaerobic ammonia-oxidizing bacteria (AnAOB) are being extensively used to remove nitrogen (present in the ammonia and ammonium forms). The conventional nitrogen removal process, “nitrification and denitrification,” has been highly used in wastewater treatment plants by employing a various bacterial communities. Since it required high organic compounds and an energy-intensive process, new strategies were being developed to reduce carbon footprint. “Anammox,” “Feammox,” and “Comammox” are a few processes that utilize specific bacterial moieties and do not require organic carbon. Among these, Anammox has become the most potential nitrogen removal system. In various studies, the combined Anammox and partial nitrification (PN/A) have shown the maximum nitrogen removal rate (NRR). This review discusses the applications of autotrophic ammonia oxidizers in agriculture, wastewater treatment plants, and engineered ecosystems. We have also addressed various stress impacts on ammonia oxidizers.

Removal of tetracycline in nitrification membrane bioreactors with different ammonia loading rates: Performance, metabolic pathway, and key contributors

Membrane bioreactors (MBRs) have been widely applied for the treatment of wastewater that contains high concentrations of both ammonium and antibiotics. Nonetheless, information about tetracycline (TC) removal in nitrification MBRs with high ammonium loading rates (ALRs) is still very limited. Herein, the fate of TC at four different concentrations of 1, 5, 20, and 50 mg/L in three parallel lab-scale nitrification MBRs with different ALRs (named AN50, AN500, and AN1000) were investigated in this study. Excellent nitrification performance and high TC removal efficiency (90.46%) were achieved in AN1000 at influent TC concentration of 50 mg/L. Higher ALRs promoted the removal of TC at lower influent TC concentration (≤5 mg/L), while no significant difference was observed in TC removal efficiencies among different ALRs MBRs at higher influent TC concentration (≥20 mg/L), implying that the heterotrophic degradation could be strengthened after long-term exposure to high concentration of TC. Batch tests demonstrated that adsorption and biodegradation were the primary TC removal routes by nitrification sludge, of which both autotrophic ammonia oxidizers and heterotrophic microorganisms played an important role in the biodegradation of TC. FT-IR spectroscopy confirmed that amide groups on the sludge biomass contributed to the adsorption of TC. Mass balance analyses indicated that biodegradation (63.4–88.6% for AN50, 74.5–88.4% for AN500 and 74.4–91.4% for AN1000) was the major mechanism responsible for the removal of TC in nitrification MBRs, and its contribution increased with influent TC concentration, while only 1.1%–15.0% of TC removal was due to biosorption. TC was progressively degraded to small molecules and the presence of TC had no notable effect on membrane permeability. These jointly confirmed TC could be effectively removed via initial adsorption and subsequent biodegradation, while biodegradation was the primary mechanism in this study.

Responses of various carbon to nitrogen ratios to microbial communities, kinetics, and nitrogen metabolic pathways in aerobic granular sludge reactor

The role of different ammonia concentrations (mg N/L) (of 100 (carbon to nitrogen ratio (C/N) = 12; Stage I), 200 (C/N = 6; Stage II), 400 (C/N = 3; Stage III) and 200 (C/N = 6; Stage IV)) in nitrogen metabolic pathways, microbial community, and specific microbial activity were investigated in an aerobic granular sludge reactor. Heterotrophic ammonia oxidizing bacteria (HAOB) showed higher ammonia oxidation rates (AORs) than autotrophic ammonia oxidizing bacteria (AAOB) at higher C/N conditions (Stages I and II). Paracoccus was the dominant HAOB. AAOB, with only 0.2–0.3 % in relative abundance, showed 2.7-fold higher AORs than HAOB at elevated ammonia and free ammonia (FA) concentrations with C/N at 3. Nitrosomonas and a genus in Nitrosomondaceae family were the major AAOB. This study proposed that FA inhibition on heterotrophic bacteria might be the mechanism that contributes to the development of the autotrophic ammonia oxidation pathway and enrichment of AAOB.

Ammonia oxidizing bacteria dominate soil nitrification under different fertilization regimes in black soils of northeast China

Although autotrophic ammonia oxidizing archaea (AOA) and bacteria (AOB) and comammox coexist in soils, their abundances and contributions to nitrification are not well addressed, especially in arable black soils. In this study, using the nitrification inhibitors acetylene and octyne, the contributions to nitrification potential provided by different ammonia oxidizers and heterotrophic nitrifiers in four long-term fertilization treatments, i.e., no fertilization (NoF), manure fertilization (M), chemical fertilization (CF), and chemical fertilization plus manure (CFM), were investigated in samples collected from the northern, middle and southern black soil regions of northeast China. The amoA gene abundances of AOA, AOB, comammox Clade A and comammox Clade B in these soils were quantified by qPCR. The results showed that the amoA gene abundances of the three ammonia oxidizers tended to increase from the northern to southern sites, while the opposite trend was observed in the ratios of AOA/AOB and comammox Clade B/Clade A; the different fertilization treatments significantly influenced the amoA abundances, and the effects varied with the ammonia oxidizers and across the sites. Autotrophic ammonia oxidizers accounted for 62%–100% of the gross soil nitrification potential across all soils. Among them, AOB dominated soil autotrophic nitrification in the fertilized soils, while no fertilization may have increased the nitrification potential provided by AOA plus comammox. Pairwise analysis showed that the amoA gene abundances of AOA and AOB were significantly and positively correlated with their respective nitrification-driven potentials. Correlation heatmaps indicated that soil pH and inorganic nitrogen content were the main soil factors related to nitrification activities and the abundances of the ammonia oxidizers. Our findings highlight that AOB are the dominant ammonia oxidizers contributing to nitrification in arable fertilized black soils, although AOA and comammox are highly abundant.

Compartmentation of microbial communities in structure and function for methane oxidation coupled to nitrification–denitrification

A hollow-fiber membrane biofilm reactor was designed and constructed to achieve simultaneous nitrification–denitrification coupled to methane oxidation in low O2/CH4 ratio and high nitrogen removal rate. Three O2/CH4 ratio stages were operated. Ammonia removal rates reached 77.5 and 95 mg/(L·d) at the O2/CH4 ratio of 1.47 and 2.1, respectively. Microbial community analysis revealed that aeration through physical partition and O2/CH4 ratio stages achieved compartmentation of microbial community in structure and function. Combined functional genes analysis using qPCR, the aeration through gas distributer was proved to promote the enrichment of autotrophic ammonia oxidizers in the suspended liquid/mixed filler samples, and the aeration through hollow-fiber membrane favored the growth of methanotrophs and heterotrophic nitrification-aerobic denitrification bacteria. This study helps to develop effective regulatory strategies for high nitrogen removal based on the understanding of the community assembly process and the key driving factors.

Cyanate is a low abundance but actively cycled nitrogen compound in soil

Cyanate can serve as a nitrogen and/or carbon source for different microorganisms and as an energy source for autotrophic ammonia oxidizers. However, the extent of cyanate availability and utilisation in terrestrial ecosystems and its role in biogeochemical cycles is poorly known. Here we analyse cyanate concentrations in soils across a range of soil types, land management practices and climates. Soil cyanate concentrations were three orders of magnitude lower than ammonium or nitrate. We determined cyanate consumption in a grassland and rice paddy soil using stable isotope tracer experiments. We find that cyanate turnover was rapid and dominated by biotic processes. We estimated that in-situ cyanate production rates were similar to those associated with urea fertilizer decomposition, a major source of cyanate in the environment. We provide evidence that cyanate is actively turned over in soils and represents a small but continuous nitrogen/energy source for soil microbes.

Evaluating CO2 emissions from continuous flow and batch growth systems under autotrophic mode: Implications for GHG accounting of biological nutrient removal

The oxidation of ammonia by autotrophic bacteria is a central part of the nitrogen cycle and a fundamental aspect of biological nutrient removal (BNR) during wastewater treatment. Autotrophic ammonia oxidation produces protons and results in net-CO2 production due to the neutralizing effect of bicarbonate alkalinity. Attention must be paid to the propensity for this produced CO2 to be transferred to the atmosphere where it can act as a greenhouse gas (GHG). In the context of BNR systems, bicarbonate-derived CO2 emissions should be considered distinct from the biogenic CO2 that arises from cellular respiration, though this distinction is not made in current GHG accounting practices. The aim of this study was to evaluate the performance of two experimental systems operated under autotrophic mode and buffered with bicarbonate, to investigate the relationship between ammonia removal and gaseous CO2 emissions. The first system consisted of continuously aerated lab-scale batch reactors, which were effective in demonstrating the important link between ammonia oxidizer activity, pH, and gaseous CO2 production. Depletion of the buffer system always led to a rapid decline in system pH and cessation of CO2 emissions when the pH fell below 7.0. The second system was a tubular continuous-flow biofilm reactor which permitted comparison of ammonia removal and CO2 emission rates. A linear relationship between ammonia removal and CO2 emissions was demonstrated and the quantified CO2 production was relatively close to that which was predicted based on the stoichiometry of nitrification, with this CO2 being detected in the gas phase. It was apparent that this system offered minimal resistance to the mass transfer of CO2 from the liquid to gas, which is an important factor that determines how much of the bicarbonate-derived CO2 may contribute to greenhouse gas emissions in engineered systems such as those used for BNR.

Effects of Different Land Use Types on Active Autotrophic Ammonia and Nitrite Oxidizers in Cinnamon Soils.

Land use types with different disturbance gradients show many variations in soil properties, but the effects of different land use types on soil nitrifying communities and their ecological implications remain poorly understood. Using 13CO2-DNA-based stable isotope probing (DNA-SIP), we examined the relative importance and active community composition of ammonia-oxidizing archaea (AOA) and bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in soils under three land use types, forest, cropland, and greenhouse vegetable soil, representing three interference gradients. Soil net nitrification rate was in the order forest soil > cropland soil > greenhouse vegetable soil. DNA-SIP showed that active AOA outcompeted AOB in the forest soil, whereas AOB outperformed AOA in the cropland and greenhouse vegetable soils. Cropland soil was richer in NOB than in AOA and AOB. Phylogenetic analysis revealed that ammonia oxidation in the forest soil was predominantly catalyzed by the AOA Nitrosocosmicus franklandus cluster within the group 1.1b lineage. The 13C-labeled AOB were overwhelmingly dominated by Nitrosospira cluster 3 in the cropland soil. The active AOB Nitrosococcus watsonii lineage was observed in the greenhouse vegetable soil, and it played an important role in nitrification. Active NOB communities were closely affiliated with Nitrospira in the forest and cropland soils, and with Nitrolancea and Nitrococcus in the greenhouse vegetable soil. Canonical correlation analysis showed that soil pH and organic matter content significantly affected the active nitrifier community composition. These results suggest that land use types with different disturbance gradients alter the distribution of active nitrifier communities by affecting soil physicochemical properties. IMPORTANCE Nitrification plays an important role in the soil N cycle, and land use management has a profound effect on soil nitrifiers. It is unclear how different gradients of land use affect active ammonia-oxidizing archaea and bacteria and nitrite-oxidizing bacteria. Our research is significant because we determined the response of nitrifiers to human disturbance, which will greatly improve our understanding of the niche of nitrifiers and the differences in their physiology.

Investigations of soil autotrophic ammonia oxidizers in farmlands through genetics and big data analysis

Soil ammonia oxidation is a key and rate-limiting process of nitrogen cycle in agriculture. Diverse autotrophic ammonia oxidizers, including ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), anaerobic ammonium oxidation (anammox), complete ammonia oxidation (comammox), and anaerobic ammonium oxidation coupled to iron reduction (feammox), were found to drive the process. However, limited studies have investigated the co-occurrence, abundance and relative contributions of these microbes in agricultural soils. Here, we report the diversity, abundance, environmental factors influencing these microbes and their contributions to soil ammonia oxidation. Results suggest that AOA, AOB, comammox, anammox, and feammox commonly co-existed in soil microbial community regardless of land uses (rice, soybean, orchard, nursery stock, and weed); and had a higher abundance in the topsoil (0–15 cm) than the subsoil (15–30 cm). Both AOA and AOB were important drivers of autotrophic ammonia oxidation in rice soils, with predicted ammonia oxidation rates of 0.48 ± 0.56 and 0.93 ± 1.32 μmol N·g dry soil−1·d−1, respectively. Interestingly, feammox, Acidimicrobiaceae sp. A6, showed an equivalent or even greater predicted ammonia oxidation rate (1.23 ± 0.98 μmol N·g dry soil−1·d−1) than AOA or AOB in rice soils, suggesting the significant contribution of feammox to soil ammonia oxidation. Multiple factors, including nutrients, pH, moisture content, total organic carbon, and reactive heavy metals were confirmed to directly or indirectly affect the autotrophic ammonia oxidizers. Notably, the increase of heavy metal availability enriched the autotrophic ammonia oxidizers, which implied the negative effect of heavy metals on soil nitrogen management. These results suggest that multi-engineering measures, e.g., slow-release fertilizer, soil acidification mitigation, and heavy metal immobilization should be taken together to slow the ammonia oxidation for improving nitrogen-use efficiency in agriculture.

Comammox Nitrospira play an active role in nitrification of agricultural soils amended with nitrogen fertilizers

The recent discovery of complete ammonia oxidizers (comammox Nitrospira) challenged the paradigm of the two-step nitrification mediated by two distinct groups of nitrifiers, and raised fundamental questions regarding their niche specialization and relative contribution to nitrification in agricultural soils. Previous studies suggest that comammox Nitrospira have an oligotrophic lifestyle and would outcompete canonical ammonia oxidizers (ammonia-oxidizing bacteria and ammonia-oxidizing archaea) under ammonia-limited conditions. Here, we demonstrated that comammox Nitrospira clade A were significantly more abundant than canonical ammonia oxidizers and 13CO2-DNA-stable isotope probing revealed that comammox Nitrospira clade A incorporated 13CO2 into their genomes in fertilized agricultural soils during the microcosm incubation. Phylogenetic analysis of the amoA gene revealed that 13CO2-labelled comammox Nitrospira clade A belonged to the Nitrospira inopinata-related cluster and a new cluster that was distinct from the known comammox isolates. These results demonstrated the potential important role of comammox Nitrospira in autotrophic ammonia oxidation in agricultural soils amended with nitrogen fertilizers and their lifestyle may be not strictly restricted to oligotrophic habitats. There is a potential contribution of comammox Nitrospira to soil nitrification, which calls re-evaluation of the microbial nitrogen cycling processes and the subsequent impacts on agriculture and the environment.

The dominance of non-halophilic archaea in autotrophic ammonia oxidation of activated sludge under salt stress: A DNA-based stable isotope probing study

Dynamics of nitrification activity, ammonia-oxidizing archaea (AOA) and bacteria (AOB) abundance and active ammonia oxidizers of activated sludge were explored under different salinities. Results showed that specific ammonium oxidation rates were significantly negative with increasing salinity. The responses of AOA and AOB populations to salt stress were distinct. AOA abundance decreased at moderate salinities (2.5, 5 and 7 g L−1) and increased at high salinities (10, 15, 20 and 30 g L−1), while AOB abundance showed opposite tendency. DNA-based stable isotope probing assays indicated AOA exclusively dominated active ammonia oxidation of test samples under different salinities. The active AOA communities retrieved were all non-halophilic and regulated by salinities. Candidatus Nitrosocosmicus exaquare and Ca. Nitrosocosmicus franklandus were the predominantly active AOA in both salt-free and salt-containing microcosms, while 13C-labeled Nitrososphaera viennensis and Ca. Nitrososphaera gargensis were only retrieved from the microcosms amended with 0 and 30 g L−1 salinity, respectively.

Long-term fertilization regimes change soil nitrification potential by impacting active autotrophic ammonia oxidizers and nitrite oxidizers as assessed by DNA stable isotope probing.

Chemoautotrophic ammonia-oxidizers and nitrite-oxidizers are responsible for a significant amount of soil nitrate production. The identity and composition of these active nitrifiers in soils under different long-term fertilization regimes remain largely under-investigated. Based on that soil nitrification potential significantly decreased in soils with chemical fertilization (CF) and increased in soils with organic fertilization (OF), a microcosm experiment with DNA stable isotope probing was further conducted to clarify the active nitrifiers. Both ammonia-oxidizing archaea (AOA) and bacteria (AOB) were found to actively respond to urea addition in soils with OF and no fertilizer (CK), whereas only AOB were detected in soils with CF. Around 98% of active AOB were Nitrosospira cluster 3a.1 in all tested soils, and more than 90% of active AOA were Nitrososphaera subcluster 1.1 in unfertilized and organically fertilized soils. Nitrite oxidation was performed only by Nitrospira-like bacteria in all soils. The relative abundances of Nitrospira lineage I and VI were 32% and 61%, respectively, in unfertilized soils, and that of Nitrospira lineage II was 97% in fertilized soils, indicating long-term fertilization shifted the composition of active Nitrospira-like bacteria in response to urea. This finding indicates that different fertilizer regimes impact the composition of active nitrifiers, thus, impacting soil nitrification potential.

Long-term effects of environmentally relevant concentrations of silver nanoparticles on major soil bacterial phyla of a loamy soil

BackgroundThe growing production and use of engineered AgNP in industry and private households make increasing concentrations of AgNP in the environment unavoidable. Although we already know the harmful effects of AgNP on pivotal bacterial driven soil functions, information about the impact of silver nanoparticles (AgNP) on the soil bacterial community structure is rare. Hence, the aim of this study was to reveal the long-term effects of AgNP on major soil bacterial phyla in a loamy soil. The study was conducted as a laboratory incubation experiment over a period of 1 year using a loamy soil and AgNP concentrations ranging from 0.01 to 1 mg AgNP/kg soil. Effects were quantified using the taxon-specific 16S rRNA qPCR.ResultsThe short-term exposure of AgNP at environmentally relevant concentration of 0.01 mg AgNP/kg caused significant positive effects on Acidobacteria (44.0%), Actinobacteria (21.1%) and Bacteroidetes (14.6%), whereas beta-Proteobacteria population was minimized by 14.2% relative to the control (p ≤ 0.05). After 1 year of exposure to 0.01 mg AgNP/kg diminished Acidobacteria (p = 0.007), Bacteroidetes (p = 0.005) and beta-Proteobacteria (p = 0.000) by 14.5, 10.1 and 13.9%, respectively. Actino- and alpha-Proteobacteria were statistically unaffected by AgNP treatments after 1-year exposure. Furthermore, a statistically significant regression and correlation analysis between silver toxicity and exposure time confirmed loamy soils as a sink for silver nanoparticles and their concomitant silver ions.ConclusionsEven very low concentrations of AgNP may cause disadvantages for the autotrophic ammonia oxidation (nitrification), the organic carbon transformation and the chitin degradation in soils by exerting harmful effects on the liable bacterial phyla.

Neutrophilic bacteria are responsible for autotrophic ammonia oxidation in an acidic forest soil

Increasing evidence suggests that autotrophic ammonia oxidation in acidic soils is largely performed by archaea. However, ammonia oxidizing bacteria (AOB) are also found in acidic soils, and mechanisms have been demonstrated that enable the growth of AOB in low pH soils. This work used DNA stable isotope probing to examine the response of autotrophic ammonia oxidizers in an acidic forest soil (pH 5.0) following urea amendment. After application, increases in nitrate concentrations correlated only with increases in AOB and not ammonia oxidizing archaeal (AOA) amoA gene abundance, indicating that AOB may be responsible for urea-derived ammonia oxidation. To identify growing autotrophic populations associated with ammonia oxidation, qPCR and high throughput sequencing analysis of 16S rRNA gene amplicons demonstrated that 13CO2 was assimilated by Nitrosospira AOB rather than archaeal ammonia oxidizing populations, and Nitrospira-associated nitrite-oxidizing bacteria (NOB). Phylogenetic analysis of amoA genes demonstrated that the active ammonia oxidizers were closely related to neutrophilic Nitrosospira multiformis within Nitrosospira cluster 3a.2. These results demonstrate that AOB can dominate ammonia oxidation in acidic soils under specific conditions.

Effects of the nitrification inhibitor acetylene on nitrous oxide emissions and ammonia-oxidizing microorganisms of different agricultural soils under laboratory incubation conditions

Acetylene (C2H2) is an effective nitrification inhibitor targeting autotrophic ammonia oxidizers, and has shown promise for improving nitrogen use efficiency by mitigating greenhouse gas nitrous oxide (N2O) emissions and reducing nitrate leaching. Its efficacy, however, varies considerably with edaphic and environmental conditions and remains largely less studied in dryland agricultural soils. Here we conducted two laboratory microcosm incubations to explore the efficacy of C2H2 across various agricultural soils and under different conditions. The first incubation was with four agricultural soils at 25°C and 60% water-filled pore space (WFPS), and the second incubation included one cropping soil under a range of conditions (15°C, 25°C, 35°C and 50%, 70% WFPS). Our results showed that incubation of soil with 1% v/v C2H2 resulted in complete or partial inhibition of nitrification, N2O emission, and AOA or AOB growth under the experimental conditions. Acetylene can totally inhibit nitrification in acidic cropping and dairy pasture soils through retarding both AOA and AOB growth, while C2H2 partly inhibited nitrification and N2O emission in the alkaline vegetable soil through impeding only AOB growth. The highest inhibition effect of C2H2 was achieved at 25°C and 50% WFPS, while there was no inhibitory effect of C2H2 when soil was incubated at 15°C and 50% WFPS suggesting soil temperature may have a significant influence on C2H2 effectiveness. The inhibition of C2H2 on cumulative N2O emission increased with increasing temperature at 50% WFPS. In contrast, at 70% WFPS, the inhibition of C2H2 on cumulative N2O emission decreased with increasing temperature. Since the effect of C2H2 varied with soils and environmental conditions, this highlights the assumption that N2O production and nitrification can be affected by low concentrations of C2H2 may be not appropriate in some occasions.

The response patterns of community traits of N2O emission-related functional guilds to temperature across different arable soils under inorganic fertilization

Temperature is an important factor governing the community traits of N2O-emission related functional guilds (mainly autotrophic ammonia oxidizers and heterotrophic denitrifiers) and their activities. However, there have been few attempts to explore the broad response patterns of these guilds to temperature changes across arable soils. For this, a temperature-controlled (15, 25 and 35 °C) microcosm experiment was conducted using three arable soils (Fujian, Gansu, and Jiangsu) in China under two different fertilizations (no fertilization control (CK) and inorganic fertilization (NPK)). In conjunction with the measurement of N2O emission, the community structure and abundance of ammonia oxidizing archaea (AOA) and bacteria (AOB), as well as nirS- and nirK-denitrifiers were assessed using T-RFLP and quantitative PCR, respectively. The analysis of community traits indicated a consistent response pattern of AOAs to temperature in terms of guild abundance, and a consistent effect of inorganic fertilization on the abundance of AOBs, but soil-dependent response patterns to fertilization and temperature were found for nirS- and nirK-denitrifiers in terms of abundance and community structure. The correlation analysis suggested that AOAs possibly assumed a role in N2O emission in all the tested soils, and nirS-denitrifiers probably participated in N2O emission in both the Fujian and Gansu soil, while a considerable amount of N2O emission in the Jiangsu soil might have been derived from heterotrophic nitrification.

Retraction Note to: Archaeal amoA Genes Outnumber Bacterial amoA 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.

Characterisation of terrestrial acidophilic archaeal ammonia oxidisers and their inhibition and stimulation by organic compounds.

Autotrophic ammonia oxidation is performed by two distinct groups of microorganisms: ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB). AOA outnumber their bacterial counterparts in many soils, at times by several orders of magnitude, but relatively little is known of their physiology due to the lack of cultivated isolates. Although a number of AOA have been cultivated from soil, Nitrososphaera viennensis was the sole terrestrial AOA in pure culture and requires pyruvate for growth in the laboratory. Here, we describe isolation in pure culture and characterisation of two acidophilic terrestrial AOA representing the Candidatus genus Nitrosotalea and their responses to organic acids. Interestingly, despite their close phylogenetic relatedness, the two Nitrosotalea strains exhibited differences in physiological features, including specific growth rate, temperature preference and to an extent, response to organic compounds. In contrast to N. viennensis, both Nitrosotalea isolates were inhibited by pyruvate but their growth yield increased in the presence of oxaloacetate. This study demonstrates physiological diversity within AOA species and between different AOA genera. Different preferences for organic compounds potentially influence the favoured localisation of ammonia oxidisers within the soil and the structure of ammonia-oxidising communities in terrestrial ecosystems.

Mutagenesis of the hydrocarbon monooxygenase indicates a metal centre in subunit-C, and not subunit-B, is essential for copper-containing membrane monooxygenase activity

The hydrocarbon monooxygenase (HMO) of Mycobacterium NBB4 is a member of the copper-containing membrane monooxygenase (CuMMO) superfamily, which also contains particulate methane monooxygenases (pMMOs) and ammonia monooxygenases (AMOs). CuMMOs have broad applications due to their ability to catalyse the oxidation of difficult substrates of environmental and industrial relevance. Most of our understanding of CuMMO biochemistry is based on pMMOs and AMOs as models. All three available structures are from pMMOs. These share two metal sites: a dicopper centre coordinated by histidine residues in subunit-B and a 'variable-metal' site coordinated by carboxylate and histidine residues from subunit-C. The exact nature and role of these sites is strongly debated. Significant barriers to progress have been the physiologically specialized nature of methanotrophs and autotrophic ammonia-oxidizers, lack of a recombinant expression system for either enzyme and difficulty in purification of active protein. In this study we use the newly developed HMO model system to perform site-directed mutagenesis on the predicted metal-binding residues in the HmoB and HmoC of NBB4 HMO. All mutations of predicted HmoC metal centre ligands abolished enzyme activity. Mutation of a predicted copper-binding residue of HmoB (B-H155V) reduced activity by 81 %. Mutation of a site that shows conservation within physiologically defined subgroups of CuMMOs was shown to reduce relative HMO activity towards larger alkanes. The study demonstrates that the modelled dicopper site of subunit-B is not sufficient for HMO activity and that a metal centre predicted to be coordinated by residues in subunit-C is essential for activity.