Complete Ammonia Oxidation Research Articles

Ammonia-oxidizing archaea and comammox Nitrospira clade B as freeze–thaw resistant nitrifiers in wetland soils

Wetland ecosystems are hotspots for nitrogen cycling, while the community dynamics of nitrification-driving microbes include ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), complete ammonia oxidizer (comammox) Nitrospira, and nitrite-oxidizing bacteria were not well-studied. In this study, we investigated the nitrification process in typical inland and coastal wetlands in northern China and found a high diversity of ammonia oxidizers. To simulate the freezing-thawing state under natural environment, the soil samples were first frozen at −20 °C for two months, and then gradually thawed at 4 °C. Further microcosm incubation combined with 13C-DNA-stable isotope probing analyses showed that after the freeze–thaw cycle, only the activities of AOA and comammox Nitrospira clade B recovered. The results indicated that these two groups of nitrifiers were more freeze–thaw resistant, which is in line with literature showing that AOA and comammox Nitrospira clade B were relatively more abundant than their counterparts in low-temperature environments. We tested, for the first time, the cold-shock resilience of nitrifying microbes in wetland soils and spotted candidates driving nitrification under fluctuating temperatures.

Effects of common afforestation tree species on soil bacterial community and microbial functional guilds in subtropical forests.

To understand the effects of common afforestation tree species on soil microbial community in subtropical forests, seven different tree species were selected as the research object, including Pinus massoniana, Mytilaria laosensis, Liquidambar formosana, Ilex chinensis, Michelia macclurei, Quercus acutissima and Betula luminifera. Based on 16S rRNA high-throughput sequencing and real-time quantitative PCR techniques, we explored the effects of different tree species on soil bacterial community composition, diversity and microbial functional guilds. The results showed that Acidobacteria, Proteobacteria, and Actinobacteria were the dominant bacterial phyla, and that there was no significant difference in bacterial diversity or richness index among different tree species. Results of redundancy analysis suggested that soil bulk density, soil C/N, litter nitrogen content, and litter C/N were the predominant factors determining soil bacterial community composition. The afforestation tree species had significant effects on functional gene abundances of ammonia oxidizing archaea, ammonia oxidizing bacteria and complete ammonia oxidation. Comammox were dominant in abundance. Ammonia oxidizing archaea amoA gene was the only type whose abundance showed significant correlation with soil nitrate content, suggesting that ammonia oxidizing archaea could play a dominant role in the autotrophic nitrification in the acidic subtropical forest soils. The afforestation tree species had significant effects on functional gene abundances of ammonia oxidizing microorganisms. Results of correlation analysis showed that litter nitrogen content was the driving factor for the abundance of ammonia oxidizing microorganisms. Our study provided strong evidence that the responses of soil microbial functional guilds to tree species were more sensitive than bacterial community composition. Future studies should explore the mechanisms of tree plantations on forest ecosystem functioning from the perspective of microbial functional guilds.

Diversity of the Hydroxylamine Oxidoreductase (HAO) Gene and Its Enzyme Active Site in Agricultural Field Soils.

Nitrification is a key process in the biogeochemical nitrogen cycle and a major emission source of the greenhouse gas nitrous oxide (N2O). The periplasmic enzyme hydroxylamine oxidoreductase (HAO) is involved in the oxidation of hydroxylamine to nitric oxide in the second step of nitrification, producing N2O as a byproduct. Its three-dimensional structure demonstrates that slight differences in HAO active site residues have inhibitor effects. Therefore, a more detailed understanding of the diversity of HAO active site residues in soil microorganisms is important for the development of novel nitrification inhibitors using structure-guided drug design. However, this has not yet been examined. In the present study, we investigated hao gene diversity in beta-proteobacterial ammonia-oxidizing bacteria (β-AOB) and complete ammonia-oxidizing (comammox; Nitrospira spp.) bacteria in agricultural fields using a clone library ana-lysis. A total of 1,949 hao gene sequences revealed that hao gene diversity in β-AOB and comammox bacteria was affected by the fertilizer treatment and field type, respectively. Moreover, hao sequences showed the almost complete conservation of the six HAO active site residues in both β-AOB and comammox bacteria. The diversity of nitrifying bacteria showed similarity between hao and amoA genes. The nxrB amplicon sequence revealed the dominance of Nitrospira cluster II in tea field soils. The present study is the first to reveal hao gene diversity in agricultural soils, which will accelerate the efficient screening of HAO inhibitors and evaluations of their suppressive effects on nitrification in agricultural soils.

Effects of dam building on the occurrence and activity of comammox bacteria in river sediments and their contribution to nitrification

The recent discovery of complete ammonia oxidizers (comammox) has fundamentally changed our understanding of nitrification. However, studies on the occurrence and activity of comammox bacteria and their contribution to nitrification remain unclear. Here, we investigated the abundance, activity, and diversity of comammox bacteria and their contribution to nitrification in sediments from dammed rivers in winter and summer. Our results indicated that comammox clade A was ubiquitous in all sediment samples and the community structure in comammox varied between the upper and lower reaches, but not on the time scale (winter and summer). Comammox activity in the dammed river sediments in summer was prominently higher than in winter (summer: 1.08 ± 0.52; winter: 0.197 ± 0.148 mg N kg−1 day−1). Furthermore, the activity of comammox bacteria in summer appeared higher in the vicinity of the dammed river and in the Sanjiang estuary, which is located downstream of the dammed river. The activity of ammonia-oxidizing bacteria (AOB) (0.77 ± 0.478 mg N kg−1 day−1) was higher compared to comammox (0.639 ± 0.588 mg N kg−1 day−1) and ammonia-oxidizing archaea (AOA) (0.026 ± 0.022 mg N kg−1 day−1) in both winter and summer. In terms of contribution to the nitrification process, AOB (winter: 67.13 ± 12.21 %; summer: 50.57 ± 16.14 %) outperformed comammox (winter: 28.59 ± 12.51 %; summer: 48.38 ± 16.62 %) and AOA (winter: <7.39 %; summer: <2.09 %). These findings indicated that the nitrification process in dammed river sediments was mainly dominated by AOB. Additionally, comammox activity was significantly affected by temperature and NH4+, suggesting that these variables were key determinants of the niche partitioning of comammox. Collectively, our findings provide novel perspectives into the widespread distribution and contribution of comammox to nitrification in dammed river ecosystems, thus broadening our understanding of the nitrification processes.

Comammox Nitrospira Clade B is the most abundant complete ammonia oxidizer in a dairy pasture soil and inhibited by dicyandiamide and high ammonium concentrations.

The recent discovery of comammox Nitrospira, a complete ammonia oxidizer, capable of completing the nitrification on their own has presented tremendous challenges to our understanding of the nitrification process. There are two divergent clades of comammox Nitrospira, Clade A and B. However, their population abundance, community structure and role in ammonia and nitrite oxidation are poorly understood. We conducted a 94-day microcosm study using a grazed dairy pasture soil amended with urea fertilizers, synthetic cow urine, and the nitrification inhibitor, dicyandiamide (DCD), to investigate the growth and community structure of comammox Nitrospira spp. We discovered that comammox Nitrospira Clade B was two orders of magnitude more abundant than Clade A in this fertile dairy pasture soil and the most abundant subcluster was a distinctive phylogenetic uncultured subcluster Clade B2. We found that comammox Nitrospira Clade B might not play a major role in nitrite oxidation compared to the role of canonical Nitrospira nitrite-oxidizers, however, comammox Nitrospira Clade B is active in nitrification and the growth of comammox Nitrospira Clade B was inhibited by a high ammonium concentration (700 kg synthetic urine-N ha-1) and the nitrification inhibitor DCD. We concluded that comammox Nitrospira Clade B: (1) was the most abundant comammox in the dairy pasture soil; (2) had a low tolerance to ammonium and can be inhibited by DCD; and (3) was not the dominant nitrite-oxidizer in the soil. This is the first study discovering a new subcluster of comammox Nitrospira Clade B2 from an agricultural soil.

Effects of Dam Building on Niche Differentiation of Comammox Nitrospira in the Main Stream of the Three Gorges Reservoir Area

Complete ammonia oxidizers (comammox) can completely oxidize ammonia to nitrate, and the various habitats of comammox Nitrospira are an important guarantee for their survival. The construction of the Three Gorges Dam, China, made it easier for nitrogen to stay in the reservoir area, which may have caused the niche differentiation of comammox, thereby affecting the natural transformation process of nitrogen in the reservoir area. This study investigated comammox in river sediments in the Three Gorges Reservoir area. Comammox clade A and comammox clade B were detected in all samples, and comammox clade A was dominant. The number of dominant OTUs (Operational Taxonomic Unit) in comammox clade A.1 accounted for 18.69% of the total number of OTUs, followed by comammox clade A.2 (18.58%) and clade B (14.30%). The indicated abundance of comammox Nitrospira clade A and clade B amoA genes in the main stream of the Three Gorges Reservoir increased along the length of the river and reached the maximum in the middle part of the reservoir area. The highest abundance of ammonia-oxidizing archaea (AOA) amoA genes appeared in the upper stream section of the reservoir area. Comammox Nitrospira clade A exhibited the highest abundance (3.00 × 104 ± 8782.37 copies/g), followed by comammox Nitrospira clade B (1.83 × 103 ± 1019.82 copies/g), ammonia-oxidizing bacteria (AOB) (1.28 × 103 ± 574.69 copies/g), and AOA (1.73 × 102 ± 48.05 copies/g). The abundances of both comammox clades A and B were positively correlated with sediment pH, indicating that pH is an important environmental factor affecting the growth of comammox bacteria. Additionally, the relative abundances of both comammox clade A.2 and clade B were significantly correlated with overlying water dissolved oxygen (DO) in the reservoir area. This study thus indicated that there exists a niche differentiation of comammox Nitrospira in the main stream of the Three Gorges Reservoir area. The potential changes in the ammoxidation process and the environmental effects caused by this niche differentiation need further attention.

Benzimidazole fungicide biotransformation by comammox Nitrospira bacteria: Transformation pathways and associated proteomic responses

Benzimidazole fungicides are frequently detected in aquatic environments and pose a serious health risk. Here, we investigated the metabolic capacity of the recently discovered complete ammonia-oxidizing (comammox) Nitrospira inopinata and kreftii to transform a representative set of benzimidazole fungicides (i.e., benzimidazole, albendazole, carbendazim, fuberidazole, and thiabendazole). Ammonia-oxidizing bacteria and archaea, as well as the canonical nitrite-oxidizing Nitrospira exhibited no or minor biotransformation activity towards all the five benzimidazole fungicides. In contrast, the investigated comammox bacteria actively transformed all the five benzimidazole fungicides, except for thiabendazole. The identified transformation products indicated hydroxylation, S-oxidation, and glycosylation as the major biotransformation pathways of benzimidazole fungicides. We speculated that these reactions were catalyzed by comammox-specific ammonia monooxygenase, cytochrome P450 monooxygenases, and glycosylases, respectively. Interestingly, the exposure to albendazole enhanced the expression of the antibiotic resistance gene acrB of Nitrospira inopinata, suggesting that some benzimidazole fungicides could act as environmental stressors that trigger cellular defense mechanisms. Altogether, this study demonstrated the distinct substrate specificity of comammox bacteria towards benzimidazole fungicides and implies their significant roles in the biotransformation of these fungicides in nitrifying environments.

Genomic profiling of Nitrospira species reveals ecological success of comammox Nitrospira

BackgroundThe discovery of microorganisms capable of complete ammonia oxidation to nitrate (comammox) has prompted a paradigm shift in our understanding of nitrification, an essential process in N cycling, hitherto considered to require both ammonia oxidizing and nitrite oxidizing microorganisms. This intriguing metabolism is unique to the genus Nitrospira, a diverse taxon previously known to only contain canonical nitrite oxidizers. Comammox Nitrospira have been detected in diverse environments; however, a global view of the distribution, abundance, and diversity of Nitrospira species is still incomplete.ResultsIn this study, we retrieved 55 metagenome-assembled Nitrospira genomes (MAGs) from newly obtained and publicly available metagenomes. Combined with publicly available MAGs, this constitutes the largest Nitrospira genome database to date with 205 MAGs, representing 132 putative species, most without cultivated representatives. Mapping of metagenomic sequencing reads from various environments against this database enabled an analysis of the distribution and habitat preferences of Nitrospira species. Comammox Nitrospira’s ecological success is evident as they outnumber and present higher species-level richness than canonical Nitrospira in all environments examined, except for marine and wastewaters samples. The type of environment governs Nitrospira species distribution, without large-scale biogeographical signal. We found that closely related Nitrospira species tend to occupy the same habitats, and that this phylogenetic signal in habitat preference is stronger for canonical Nitrospira species. Comammox Nitrospira eco-evolutionary history is more complex, with subclades achieving rapid niche divergence via horizontal transfer of genes, including the gene encoding hydroxylamine oxidoreductase, a key enzyme in nitrification.ConclusionsOur study expands the genomic inventory of the Nitrospira genus, exposes the ecological success of complete ammonia oxidizers within a wide range of habitats, identifies the habitat preferences of (sub)lineages of canonical and comammox Nitrospira species, and proposes that horizontal transfer of genes involved in nitrification is linked to niche separation within a sublineage of comammox Nitrospira.EV8YCFoiwYRfG7zmTEDHpJVideo

Comammox Nitrospira and AOB communities are more sensitive than AOA community to different fertilization strategies in a fluvo-aquic soil

Ammonia oxidizing archaea (AOA), ammonia oxidizing bacteria (AOB) and the newly discovered complete ammonia oxidizers (comammox Nitrospira) are responsible for ammonia oxidation, which is the first and rate-limiting step of nitrification. A better understanding of the complex responses of AOA, AOB and comammox Nitrospira to inorganic and organic fertilization regimes is critical to get the whole picture of soil nitrification. Here, we investigated the abundances and community compositions of AOA, AOB and comammox Nitrospira in a fluvo-aquic soil under different fertilization strategies: nitrogen fertilizer reduction treatments (CK, PK, 50 % NPK and 75 % NPK), conventional fertilizer treatment (100 % NPK), N fertilizer reduction with organic manure substitution treatments (25 % OMNPK, 50 % OMNPK, 100 % OMPK and 200 % OMPK). The results indicated that 100 % NPK treatment significantly increased AOB abundance, while the abundances of AOA and comammox Nitrospira were enhanced significantly by 100 % and 200 % OMPK treatments. Principal coordinate analysis indicated different fertilization strategies had greater effect on the communities of AOB and comammox Nitrospira than those of AOA. Fertilizer application (other than CK) decreased the relative abundance of Nitrosospira cluster 3b of AOB while increased the relative abundance of Nitrosospira cluster 3a.2. Fertilizer, especially high manure application increased the relative abundance of comammox Nitrospira clade A.2 but decreased the relative abundance of clade B. Both redundancy analysis and mantel test showed AOB and comammox Nitrospira communities were regulated by more soil factors than AOA community. These results suggested the communities of AOB and comammox Nitrospira were more sensitive than AOA community to different fertilization strategies in a fluvo-aquic soil of northern China.

Enrichment of comammox bacteria in anammox-dominated low-strength wastewater treatment system within microaerobic conditions: Cooperative effect driving enhanced nitrogen removal

In practical anammox systems, ammonia-oxidizing microbes (AOM) inevitably coexisted with anammox bacteria (AnAOB) due to residue oxygen in influent, but the diversity and impact of AOM in the system remain unclear. In this study, a practical anammox-dominated system with high relative abundance (15.15–19.07 %) of AnAOB (including Ca. Brocadia and Ca. Kuenenia) was studied, which treated low-strength wastewater with dissolved oxygen of 0.24–0.27 mg/L. Results demonstrated that complete ammonia-oxidizing (comammox) bacteria was the most abundant AOM and dominant over other canonical nitrifying bacteria, wherein comammox amoA gene was 8.3-fold more abundant than ammonia-oxidizing bacteria (AOB) amoA gene. Phylogenetic results indicated that the comammox bacteria in this study belonged to comammox Nitrospira clade A. Notably, when treating real nitritation municipal wastewater, cooperative effect of anammox bacteria, comammox bacteria and heterotrophic denitrifiers enhanced nitrogen removal efficiency to 92.9 ± 1.1 %. Hence, these results show that the functional significance of comammox bacteria in engineered ecosystems was previously overlooked. Our findings highlight that comammox bacteria could be an essential functional group in mainstream anammox systems and possess a high potential for energy-efficient nitrogen removal.

Comammox biogeography subject to anthropogenic interferences along a high-altitude river

The recent discovery of comammox Nitrospira performing complete ammonia oxidation to nitrate has overturned the long-held dogma of two-step nitrification on Earth, yet little is known about the effect of urbanization interference on their distribution. Using gene-centric metagenomics, we provided the first blueprints about comammox community, biogeography, and environmental drivers along a high-elevation (> 2000 m) river flowing through the largest city on the vulnerable Qinghai-Tibetan Plateau. Our study confirmed a wide presence and diversity of yet-uncultured comammox clade B across wet and dry seasons, with average 3.0 and 2.0 times as abundant as clade-A amoA genes in water and sediments, respectively. Species identified from freshwater and drinking water treatment plants dominated the comammox guilds (58∼100%), suggesting this plateau river shared a similar comammox assemblage with the above habitat types. Compared with the urban area harboring more abundant canonical Nitrospira identified in wastewater (average 24%), the upstream suburban reach had a smaller human population but larger proportions of comammox in ammonia-oxidizing prokaryotes (24∼72% of abundances) and Nitrospira sublineages I/II. Higher contents of nitrate and nitrite in water, and antibiotics in water and sediments, may restrain comammox niches in nitrifiers over the urban area. Further random forest analysis revealed that lincosamides and quinolones were the most important antibiotic predictors for the niche differentiations between comammox and canonical nitrifiers in water, while macrolides for those in sediments. Finally, by incubation experiments, we demonstrated higher activity contributions of benthic comammox in the suburban area (36.2∼92.8% of potential ammonia-oxidation rates) than in the urban reach, and that the contribution variation had significant negative relations with macrolides and their major components. Overall, this study highlighted that anthropogenic activities hampered the advantage of riverine complete nitrifiers over the canonical two-step ones.

Adding monochloramine to control the growth of nitrite oxidizers and complete ammonia oxidizers in nitrifying moving-bed biofilm reactor

This study investigated a new method of using monochloramine (NH2Cl) to enhance nitrite accumulation in nitrifying moving-bed biofilm bioreactor (MBBR). Firstly, two parallel MBBRs were operated - with 1 mg Cl2 L−1 of NH2Cl in MBBR-1 and without NH2Cl addition in MBBR-2. Both MBBRs oxidized ammonia completely. The NO2− accumulation efficiency increased by twofold in MBBR-1 but remained low in MBBR-2. Subsequently, the carriers from MBBR-1 and 2 were seeded into four sequencing batch reactors (SBRs) for in-situ nitrogen transformation analysis. The results showed that continuous exposure to 1 mg Cl2 L−1 of NH2Cl successfully accumulated 50 mg N L−1 of nitrite in the effluent and suppressed the growth of nitrite-oxidizing bacteria (NOB) and complete ammonia oxidizers (comammox). The finding indicated that adding NH2Cl could be a promising strategy to attain reliable nitrite accumulation for developing two-stage partial nitrification and anaerobic ammonia oxidation process in the future.

Occurrence and Roles of Comammox Bacteria in Water and Wastewater Treatment Systems: A Critical Review

Nitrogen removal is a critical process in water treatment plants (WTPs) and wastewater treatment plants (WWTPs). The recent discovery of a novel bacterial process, complete ammonia oxidation (comammox, CMX), has refuted a century-long perception of the two-step conversion of NH3 to NO3–. Compared with canonical nitrifiers, CMX bacteria offer undeniable advantages, such as a high growth yield propensity and adaptability to nutrient- and growth-limiting conditions, which collectively draw attention to validate the aptness of CMX bacteria to wastewater treatment. As there has been no comprehensive review on the relevance of CMX bacteria for sustainable water and wastewater treatment, this review is intended to discuss the roles and applications of CMX in the removal of nitrogen and pollutants from water and wastewater. We took into account insights into the metabolic versatilities of CMX bacteria at the clade and subclade levels. We focused on the distribution of CMX bacteria in engineered systems, niche differentiation, co-occurrence and interactions with canonical nitrifiers for a better understanding of CMX bacteria in terms of their ecophysiology. Conceptualized details on the reactor adaptability and stress response of CMX bacteria are provided. The potential of CMX bacteria to degrade micropollutants either directly or co-metabolically was evaluated, and these insights would be an indispensable advantage in opening the doors for wider applications of CMX bacteria in WWTPs. Finally, we summarized future directions of research that are imperative in improving the understanding of CMX biology.

Distinct Nitrification Rates and Nitrifiers in Needleleaf and Evergreen Broadleaf Forest Soils.

Research on niche specialization in the microbial communities of ammonia oxidizers is important for assessing the consequences of vegetation shift on nitrogen (N) cycling. In this study, soils were sampled from three tree stands (needleleaf, mixed, and evergreen broadleaf) from the Hannam experimental forest in South Korea in spring (May 2019), summer (August 2019), autumn (November 2019), and winter (January 2020). Quantitative polymerase chain reaction (qPCR) and high-throughput sequencing were used to measure the abundance and community structure of various nitrifiers: ammonia-oxidizing archaea and bacteria (AOA and AOB, respectively) as well as complete ammonia oxidizers (comammox). Nitrification rates and total ammonia oxidizer abundance were significantly higher in needleleaf forest soil than those in other forest stands, and they were lowest in evergreen broadleaf forest soil. Comammox clade B was most abundant in needleleaf and evergreen broadleaf forest soils, while AOA were significantly more abundant in mixed forest soil. The abundances of comammox clade B and AOA were negatively correlated with dissolved organic carbon. Phylogenetic analysis showed that NT-alpha and NS-gamma-2.3.2 were the most abundant AOA lineages in all the samples. The seasonal of AOA, AOB, and comammox varied with the sites, suggesting the need to examine the combinations of environmental factors when considering the effects of seasonal changes in the environment. Overall, the results suggest that potential vegetation shifts in forest ecosystems might affect nitrification activities by regulating the abundance and community structure of ammonia oxidizers.

Niche differentiation and symbiotic association among ammonia/nitrite oxidizers in a full-scale rotating biological contactor

Although the distribution of ammonia/nitrite oxidizers had been profiled in different habitats, current understanding is still limited regarding their niche differentiation in the integrated biofilm reactors, the symbiotic associations of ammonia/nitrite oxidizers, as well as the parasitic interaction between viruses and those functional organisms involved in the nitrogen cycle. Here, the integrated metagenomics and metatranscriptomics are applied to profile the ammonia/nitrite oxidizers communities and transcriptional activities changes along the flowpath of a concatenated full-scale rotating biological contactor (RBC) (frontend Stage-A and backend Stage-B). 19 metagenome-assembled genomes (MAGs) of ammonia/nitrite oxidizers were recovered by using a hybrid assembly approach, including four ammonia-oxidizing bacteria (AOB), two ammonia-oxidizing archaea (AOA), two complete ammonia oxidation bacteria (comammox), eight nitrite-oxidizing bacteria (NOB), and three anaerobic ammonium oxidation bacteria (anammox). Diverse AOB and anammox dominated Stage-A and collectively contributed to nitrogen conversion. With the decline of ammonia concentration along the flowpath, comammox and AOA appeared and increased in relative abundance in Stage-B, accounting for 8.8% of the entire community at the end of this reactor, and their dominating role in nitrogen turnover was indicated by the high transcription activity of their corresponding function genes. Moreover, the variation in the abundance of viruses infecting ammonia and nitrite oxidizers suggests that viruses likely act as a biotic factor mediating ammonia/nitrite oxidizer populations. This study demonstrates that complex factors shaped niche differentiation and symbiotic associations of ammonia/nitrite oxidizers in the RBC and highlights the importance of RBCs as model systems for the investigation of biotic and abiotic factors affecting the composition of microbiomes.

Niche differentiation among comammox (Nitrospira inopinata) and other metabolically distinct nitrifiers.

Due to global change, increasing nutrient input to ecosystems dramatically affects the nitrogen cycle, especially the nitrification process. Nitrifiers including ammonia-oxidizing archaea (AOAs), ammonia-oxidizing bacteria (AOBs), nitrite-oxidizing bacteria (NOBs), and recently discovered complete ammonia oxidizers (comammoxs) perform nitrification individually or in a community. However, much remains to be learned about their niche differentiation, coexistence, and interactions among those metabolically distinct nitrifiers. Here, we used synthetic microbial ecology approaches to construct synthetic nitrifying communities (SNCs) with different combinations of Nitrospira inopinata as comammox, Nitrososphaera gargensis as AOA, Nitrosomonas communis as AOB, and Nitrospira moscoviensis as NOB. Our results showed that niche differentiation and potential interactions among those metabolically distinct nitrifiers were determined by their kinetic characteristics. The dominant species shifted from N. inopinata to N. communis in the N4 community (with all four types of nitrifiers) as ammonium concentrations increased, which could be well explained by the kinetic difference in ammonia affinity, specific growth rate, and substrate tolerance of nitrifiers in the SNCs. In addition, a conceptual model was developed to infer niche differentiation and possible interactions among the four types of nitrifiers. This study advances our understanding of niche differentiation and provides new strategies to further study their interactions among the four types of nitrifiers.

Abundance and Diversity of Nitrifying Microorganisms in Marine Recirculating Aquaculture Systems

Recirculating aquaculture systems (RAS) are important for water quality management in aquaculture facilities, and can help resume water consumption. However, information about the community structure of the micro-ecosystem existing in biofilters, especially the participation of the known nitrifying groups (i.e., AOA, AOB, NOB, and comammox Nitrospira), remains to be fully clarified. In this research, we compared the community structures in three RAS systems operated at different temperatures in a marine aquarium, through both amoA-targeted qPCR assay and 16S rRNA-targeted next-generation sequencing. As result, AOA was the primary nitrifier in the biofilters and was typically abundant and diverse in high-temperature samples (ca. 25 °C). NOB’s relative abundance patterns were numerically similar to that of AOA, suggesting a cooperation relationship between AOA and NOB in the marine RAS system. AOB was at a comparable level with AOA in medium-temperature samples (ca. 19 °C), while their abundance sharply decreased in high-temperature samples. The number of observed OTUs of AOA in high-temperature samples was 1.9 and 1.5 times as much as that detected in low (ca. 10 °C) and medium temperature samples respectively, suggesting a much more diverse and predominant occurrence of AOA at high temperatures. Comammox Nitrospira was only detected at a low level in the biofilter samples, suggesting a negligible contribution to the nitrification process in such ammonia-limited, saline biofilms. Although comammox Nitrospira cannot be detected by 16S rRNA-based analysis, the high diversity and abundance of NOB that were detected in high-temperature samples indicated the prospective possibility of the occurrence of complete ammonia oxidation at high temperatures.

Selective Enrichment of Comammox Nitrospira in a Moving Bed Biofilm Reactor with Sufficient Oxygen Supply.

The recent discovery of comammox (complete ammonia oxidation) Nitrospira has upended the long-held nitrification paradigm. Although comammox Nitrospira have been identified in wastewater treatment systems, the conditions for their dominance over canonical ammonia oxidizers remain unclear. Here, we report the dominance of comammox Nitrospira in a moving bed biofilm reactor (MBBR) fed with synthetic mainstream wastewater. Integrated 16S rRNA gene amplicon sequencing, fluorescence in situ hybridization (FISH), and metagenomic sequencing methods demonstrated the selective enrichment of comammox bacteria when the MBBR was operated at a dissolved oxygen (DO) concentration above 6 mg O2/L. The dominance of comammox Nitrospira over canonical ammonia oxidizers (i.e., Nitrosomonas) was attributed to the low residual ammonium concentration (0.02-0.52 mg N/L) formed in the high-DO MBBR. Two clade A comammox Nitrospira were identified, which are phylogenetically close to Candidatus Nitrospira nitrosa. Interestingly, cryosectioning-FISH showed these two comammox species spatially distributed on the surface of the biofilm. Moreover, the ammonia-oxidizing activity of comammox Nitrospira-dominated biofilms was susceptible to the oxygen supply, which dropped by half with the DO concentration decrease from 6 to 2 mg O2/L. These features collectively suggest a low apparent oxygen affinity for the comammox Nitrospira-dominated biofilms in the high-DO nitrifying MBBR.

Comammox Nitrospira Bacteria Are Dominant Ammonia Oxidizers in Mainstream Nitrification Bioreactors Emended with Sponge Carriers.

Complete ammonia oxidation (i.e., comammox) is a newly discovered microbial process performed by a subset of the Nitrospira genus, and this unique microbial process has been ubiquitously detected in various wastewater treatment units. However, the operational conditions favoring comammox prevalence remain unclear. In this study, the dominance of comammox Nitrospira in four sponge biofilm reactors fed with low-strength ammonium (NH4+ = 23 ± 3 mg N/L) wastewater was proved by coupling 16S rRNA gene amplicon sequencing, quantitative polymerase chain reaction (qPCR), and metagenomic sequencing. The results showed that comammox Nitrospira dominated in the nitrifying guild over canonical ammonia-oxidizing bacteria (AOB) constantly, despite the significant variation in the residual ammonium concentration (0.01-15 mg N/L) under different sets of operating conditions. This result indicates that sponge biofilms greatly favor retaining comammox Nitrospira in wastewater treatment and highlights an essential role of biomass retention in the comammox prevalence. Moreover, analyses of the assembled metagenomic sequences revealed that the retrieved amoA gene sequences affiliated with comammox Nitrospira (53.9-66.0% read counts of total amoA gene reads) were always higher than those (28.4-43.4%) related to β-proteobacterial AOB taxa. The comammox Nitrospira bacteria detected in the present biofilm systems were close to clade A Candidatus Nitrospira nitrosa.

Evaluation of PCR primers for detecting the distribution of nitrifiers in mangrove sediments.

Ammonia-oxidizing archaea and ammonia-oxidizing bacteria (AOA and AOB), complete ammonia oxidizers (Comammox), and nitrite-oxidizing bacteria (NOB) play a crucial role in the nitrification process during the nitrogen cycle. However, their occurrence and diversity in mangrove ecosystems are still not fully understood. Here, a total of 11 pairs of PCR primers were evaluated to study the distribution and abundances of these nitrifiers in rhizosphere and non-rhizosphere sediments of a mangrove ecosystem. The amplification efficiency of these 11 pairs of primers was first evaluated and their performances were found to vary considerably. The CamoA-19F/CamoA-616R primer pair was suitable for the amplification of AOA in mangrove sediments, especially on the surface of rhizosphere sediments. Primer pair amoA1F/amoA2R was better for the characterization of novel AOB in the bacterial community of non-rhizosphere sediments of mangroves. In contrast, primer nxrB169F/nxrB638R showed a low abundance of NOB in mangrove sediments (except for R1). Comammox bacteria were abundant and diverse in mangrove sediments, as indicated by both the amoB gene for Comammox clade A and the amoA gene for Comammox Nitrospira clade B. However, the amoA gene for Comammox Nitrospira clade A was not successful in detecting them in the mangrove sediments. Furthermore, 568 operational taxonomic units (OTUs) were obtained by generating a clone library and a high abundance of OTUs was correlated with ammonium, pH, NO2-, and NO3-. Comammox and Comammox Nitrospira were identified by phylogenetic tree analysis, indicating that mangrove sediments harbor newly discovered nitrifiers. Additionally, many AOA and NOB were mainly distributed in the surface layer of the rhizosphere, whereas AOB and Comammox Nitrospira were in the subsurface of non-rhizosphere, as determined by qPCR analysis. Collectively, our findings highlight the limitations of some primers for the identification of specific nitrifying bacteria. Therefore, primers must be carefully selected to gain accurate insights into the ecological distribution of nitrifiers in mangroves. KEY POINTS: • Several sets of PCR primers perform well for the detection of nitrifiers in mangroves. • Mangroves are an important source of newly discovered nitrifiers. • Ammonium, pH, NO2-, and NO3- are important shapers of nitrifier communities in mangroves.