Agriculture receives approximately 25 % of the annual global nitrogen input, 37 % of which subsequently runs off into adjacent low-order streams and surface water, where it may contribute to high nitrification and nitrous oxide (N2O). However, the mechanisms of nitrification and the pathways controlling N2O production in agricultural streams remain unknown. Here, we report that the third microbial ammonia oxidation process, complete ammonia oxidation (comammox), is widespread and contributes to important ammonia oxidation with low ammonia-N2O conversion in both basin- and continental-scale agricultural streams. The contribution of comammox to ammonia oxidation (21.5 ± 2.3 %) was between that of bacterial (68.6 ± 2.7 %) and archaeal (9.9 ± 1.8 %) ammonia oxidation. Interestingly, N2O production by comammox (18.5 ± 2.1 %) was higher than archaeal (10.5 ± 1.9 %) but significantly lower than bacterial (70.2 ± 2.6 %) ammonia oxidation. The first metagenome-assembled genome (MAG) of comammox bacteria from agricultural streams further revealed their potential extensive diverse and specific metabolism. Their wide habitats might be attributed to the diverse metabolism, i.e. harboring the functional gene of nitrate reduction to ammonia, while the lower N2O would be attributed to their lacking biological function to produce N2O. Our results highlight the importance of widespread comammox in agricultural streams, both for the fate of ammonia fertilizer and for climate change. However, it has not yet been routinely included in Earth system models and IPCC global assessments. Synopsis Widespread but overlooked comammox contributes to important ammonia oxidation but low N2O production, which were proved by the first comammox MAG found in agricultural streams.

The waterbodies have been polluted by various natural and anthropogenic activities. The aquatic waste includes ammonia as one of the most toxic pollutants. Several biological treatment systems involving anoxic and semi anoxic bacteria have been proposed for reducing nitrogen loads from wastewater and increasing the efficiency and cost effectiveness. These bacteria play a vital role in the processes involved in the nitrogen cycle in nature. However, the enrichment, sustainability and identification of bacterial communities for wastewater treatment is an important aspect. Most of the chemolithotrophs are unculturable hence their identification and measurement of abundance remains a challenging task. In this study the different bacteria involved in total nitrogen removal from the wastewater are enriched for 700 days under anoxic condition. The synthetic wastewater containing 0.382 g/L of ammonium chloride was used. Molecular identification of the bacteria involved in various steps of the nitrogen cycle was carried out based on amplification of functional genes and 16S rRNA gene Polymerase chain reaction followed by DNA sequencing. Change in the abundance of chemolithotrophs was studied using qPCR. The mutual growth of various nitrifiers along with anaerobic bacteria were identified by molecular characterisation of DNA at various time intervals with the different genes involved in the nitrogen cycle. Nitrosomonas species like Nitrosomonas europaea were identified throughout the batch scale studies possessing the genes associated with ammonia oxidizing bacteria and nitrite oxidizing bacteria which act as a complete ammonia oxidizer. The uncultured species of Nitrospira and anammox bacteria were also observed which predicts the coexistence of the anammox and comammox bacteria in a batch scale study. The coexistence of the semi anoxic and anoxic bacteria helped in the growth of these bacteria for a longer duration of time. The nitrite produced by the comammox during nitrification can be utilized by anammox as an electron carrier. The other species of denitrifiers like Pseudomonas denitrificans and Aminobacter aminovorans were also observed. It is concluded that the enrichment of semi anoxic and anoxic bacteria was faster with the increase in growth of the bacteria involved in nitrification, comammox, anammox and partial denitrification process. The bacterial growth is enhanced and the efficiency is increased which can be further used in the development of small pilot scale bioreactor for total nitrogen removal.

The conventional functional microorganisms for nitrogen and phosphorus removal, such as Nitrosomonas, Nitrobacter, Nitrospira and Candidatus Accumulibacter, were hotspots in past research. However, the role of diverse unconventional functional microorganisms was neglected. In this study, a biofilm system was developed to explore the potential role of unconventional functional microorganisms in nutrients removal. According to the results of microbial community dynamics and metagenomics, complete ammonia oxidizing (comammox) bacteria was 20 times more abundant than ammonia-oxidizing bacteria (AOB) at day 121 and its abundance of amoA gene was almost the same as AOB. Although Nitrospira dominated the nitrite-oxidizing bacteria (NOB), diverse unconventional nxrB-containing microorganisms, particularly Chloroflexi, also significantly contributed to the nitrite oxidation. Binning analysis showed that Myxococcota-affiliated Haliangium had the necessary genes owns by phosphorus-accumulating organisms (PAO) and was likely to be the primary PAO since its abundance (6.38 %) was much higher than other conventional PAO (0.70 %). Comparing metagenome-assembled genomes of comammox bacteria with AOB and ammonia-oxidizing archaea (AOA), it possessed potential metabolic versatility in hydrogen and phosphorus, which may be the primary reason for the positive effect of the alternating anaerobic and aerobic conditions on the enrichment of comammox bacteria. Collectively, our findings broaden the understanding on the microbial mechanism of nitrogen and phosphorus removal in biofilm system.

Simultaneous nitrogen and phosphorus removal (SNPR) biofilm system is an effective wastewater treatment process. However, the understanding on the mechanism of functional microorganisms driving SNPR is still limited, especially the role of complete ammonia oxidation (comammox) Nitrospira and glycogen-accumulating organisms (GAO). In this study, a sequencing batch biofilm reactor (SBBR) performing SNPR was operated for 249 d. Based on the 16S rRNA gene, comammox amoA amplicon sequencing, metagenomics and batch experiment, we found that comammox Nitrospira was the main ammonia-oxidizing microorganisms (AOM) and provided nitrite for anaerobic ammonia oxidation (anammox) bacteria (AnAOB). Besides, GAO was dominated by the bacteria of genus Defluviicoccus and played a primary role in reducing nitrate rather than nitrite. Fluorescent in situ hybridization (FISH) analysis confirmed that Nitrospira was enriched in the inner layer of the biofilm. Thus, we put forward a novel insight into the mechanism of SNPR biofilm system. Comammox Nitrospira was responsible for nitrite and nitrate production in the inner biofilm, and AnAOB consumed the produced nitrite during the anammox process. While GAO reduced nitrate to nitrite and polyphosphate-accumulating organisms (PAO) converted nitrite to dinitrogen via denitrifying phosphorus removal in the outer biofilm. These findings provide a new understanding in SNPR biofilm system.

Exploring nitrifiers in extreme environments is vital to expanding our understanding of nitrogen cycle and microbial diversity. This study presents that complete ammonia oxidation (comammox) Nitrospira, together with acidophilic ammonia-oxidizing archaea (AOA), dominate in the nitrifying guild in sediments of an acid mine lake (AML). The lake water was characterized by acidic pH below 5 with a high ammonium concentration of 175 mg-N/liter, which is rare on the earth. Nitrification was active in sediments with a maximum nitrate production potential of 70.5 μg-N/(g-dry weight [dw] day) for mixed sediments. Quantitative PCR assays determined that in AML sediments, comammox Nitrospira and AOA amoA genes had relative abundances of 52% and 41%, respectively, among the total amoA genes. Further assays with 16S rRNA and amoA gene amplicon sequencing and metagenomics confirmed their dominance and revealed that the comammox Nitrospira found in sediments belonged to comammox Nitrospira clade A.2. Metagenomic binning retrieved a metagenome-assembled genome (MAG) of the comammox Nitrospira from sediments (completeness = 96.76%), and phylogenomic analysis suggested that it was a novel comammox Nitrospira. Comparative genomic investigation revealed that this comammox Nitrospira contained diverse metal resistance genes and an acidophile-affiliated F-type ATPase. Moreover, it had a more diverse genomic characteristic on nitrogen metabolism than the AOA in sediments and canonical AOB did. The results suggest that comammox Nitrospira is a versatile nitrifier that can adapt to acidic environments even with high ammonium concentrations. IMPORTANCE Ammonia-oxidizing archaea (AOA) was previously considered the sole dominant ammonia oxidizer in acidic environments. This study, however, found that complete ammonia oxidation (comammox) Nitrospira was also a dominant ammonia oxidizer in the sediments of an acidic mine lake, which had an acidic pH < 5 and a high ammonium concentration of 175 mg-N/liter. In combination with average nucleotide identity analysis, phylogenomic analysis suggested it is a novel strain of comammox Nitrospira. Moreover, the adaption of comammox Nitrospira to the acidic lake had been comprehensively investigated based on genome-centric metagenomic approaches. The outcomes of this study significantly expand our understanding of the diversity and adaptability of ammonia oxidizers in the acidic environments.

The contribution of agriculture to the sustainable development goals requires climate-smart and profitable farm innovations. Increasing the ammonia fertilizer applications to meet the global food demands results in high agricultural costs, environmental quality deterioration, and global warming, without a significant increase in crop yield. Here, we reported that a third microbial ammonia oxidation process, complete ammonia oxidation (comammox), is contributing to a significant ammonia fertilizer loss (41.9 ± 4.8%) at the rate of 3.53 ± 0.55 mg N kg-1 day-1 in agricultural soils around the world. The contribution of comammox to ammonia fertilizer loss, occurring mainly in surface agricultural soil profiles (0-0.2 m), was equivalent to that of bacterial ammonia oxidation (48.6 ± 4.5%); both processes were significantly more important than archaeal ammonia oxidation (9.5 ± 3.6%). In contrast, comammox produced less N2 O (0.98 ± 0.44 μg N kg-1 day-1 , 11.7 ± 3.1%), comparable to that produced by archaeal ammonia oxidation (16.4 ± 4.4%) but significantly lower than that of bacterial ammonia oxidation (72.0 ± 5.1%). The efficiency of ammonia conversion to N2 O by comammox (0.02 ± 0.01%) was evidently lower than that of bacterial (0.24 ± 0.06%) and archaeal (0.16 ± 0.04%) ammonia oxidation. The comammox rate increased with increasing soil pH values, which is the only physicochemical characteristic that significantly influenced both comammox bacterial abundance and rates. Ammonia fertilizer loss, dominated by comammox and bacterial ammonia oxidation, was more intense in soils with pH >6.5 than in soils with pH <6.5. Our results revealed that comammox plays a vital role in ammonia fertilizer loss and sustainable development in agroecosystems that have been previously overlooked for a long term.

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.

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.

Numerous wastewater treatment processes are designed by engineers to achieve specific treatment goals. However, the impact of these different process designs on bacterial community composition is poorly understood. In this study, 24 different municipal wastewater treatment facilities (37 bioreactors) with various system designs were analyzed by sequencing of PCR-amplified 16S rRNA gene fragments. Although a core microbiome was observed in all of the bioreactors, the overall microbial community composition (analysis of molecular variance; P = 0.001) as well as that of a specific population of Nitrosomonas spp. (P = 0.04) was significantly different between A/O (anaerobic/aerobic) systems and conventional activated sludge (CAS) systems. Community α-diversity (number of observed operational taxonomic units [OTUs] and Shannon diversity index) was also significantly higher in A/O systems than in CAS systems (Wilcoxon; P < 2 × 10-16). In addition, wastewater bioreactors with short mean cell residence time (<2 days) had very low community α-diversity and fewer nitrifying bacteria compared to those of other system designs. Nitrospira spp. (0.71%) and Nitrotoga spp. (0.41%) were the most prominent nitrite-oxidizing bacteria (NOB); because these two genera were rarely prominent at the same time, these populations appeared to be functionally redundant. Weak evidence (AOB:NOB « 2; substantial quantities of Nitrospira sublineage II) was also obtained suggesting that complete ammonia oxidation by a single organism was occurring in system designs known to impose stringent nutrient limitation. This research demonstrates that design decisions made by wastewater treatment engineers significantly affect the microbiome of wastewater treatment bioreactors. IMPORTANCE Municipal wastewater treatment facilities rely on the application of numerous \"activated sludge\" process designs to achieve site-specific treatment goals. A plethora of microbiome studies on municipal wastewater treatment bioreactors have been performed previously; however, the role of process design on the municipal wastewater treatment microbiome is poorly understood. In fact, wastewater treatment engineers have attempted to control the microbiome of wastewater bioreactors for decades without sufficient empirical evidence to support their design paradigms. Our research demonstrates that engineering decisions with respect to system design have a significant impact on the microbiome of wastewater treatment bioreactors.