Comammox Nitrospira cooperate with anammox bacteria in a partial nitritation-anammox membrane bioreactor treating low-strength ammonium wastewater at high loadings.

Research has revealed that comammox Nitrospira and anammox bacteria engage in dynamic interactions in partial nitritation-anammox reactors, where they compete for ammonium and nitrite or comammox Nitrospria supply nitrite to anammox bacteria. However, two gaps in the literature are present: the know-how to manipulate the interactions to foster a stable and symbiotic relationship and the assessment of how effective this partnership is for treating low-strength ammonium wastewater at high hydraulic loads. In this study, we employed a membrane bioreactor designed to treat synthetic ammonium wastewater at a concentration of 60 mg N/L, reaching a peak loading of 0.36 g N/L/day by gradually reducing the hydraulic retention time to 4 hr. Throughout the experiment, the reactor achieved an approximately 80 % nitrogen removal rate through strategically adjusting intermittent aeration at every stage. Notably, the genera Ca. Kuenena, Nitrosomonas, and Nitrospira collectively constituted approximately 40 % of the microbial community. Under superior intermittent aeration conditions, the expression of comammox amoA was consistently higher than that of Nitrospira nxrB and AOB amoA in the biofilm, despite the higher abundance of Nitrosomonas than comammox Nitrospira, implying that the biofilm environment is favorable for fostering cooperation between comammox and anammox bacteria. We then assessed the in situ activity of comammox Nitrospira in the reactor by selectively suppressing Nitrosomonas using 1-octyne, thereby confirming that comammox Nitrospira played the primary role in facilitating the nitritation (33.1 % of input ammonium) rather than complete nitrification (7.3 % of input ammonium). Kinetic analysis revealed a specific ammonia-oxidizing rate 5.3 times higher than the nitrite-oxidizing rate in the genus Nitrospira, underscoring their critical role in supplying nitrite. These findings provide novel insights into the cooperative interplay between comammox Nitrospira and anammox bacteria, potentially reshaping the management of nitrogen cycling in engineered environments, and aiding the development of microbial ecology-driven wastewater treatment technologies.

Genome-centered metagenomics illuminates adaptations of core members to a partial Nitritation-Anammox bioreactor under periodic microaeration.

The partial nitritation-anaerobic ammonium oxidation (anammox; PN-A) process has been considered a sustainable method for wastewater ammonium removal, with recent attempts to treat low-strength wastewater. However, how microbes adapt to the alternate microaerobic-anoxic operation of the process when treating low ammonium concentrations remains poorly understood. In this study, we applied a metagenomic approach to determine the genomic contents of core members in a PN-A reactor treating inorganic ammonium wastewater at loading as low as 0.0192 kg-N/m3/day. The metabolic traits of metagenome-assembled genomes from 18 core species were analyzed. Taxonomically diverse ammonia oxidizers, including two Nitrosomonas species, a comammox Nitrospira species, a novel Chloroflexota-related species, and two anammox bacteria, Ca. Brocadia and Ca. Jettenia, accounted for the PN-A reactions. The characteristics of a series of genes encoding class II ribonucleotide reductase, high-affinity bd-type terminal oxidase, and diverse antioxidant enzymes revealed that comammox Nitrospira has a superior adaptation ability over the competitors, which may confer the privileged partnership with anammox bacteria in the PN-A reactor. This finding is supported by the long-term monitoring experiment, showing the predominance of the comammox Nitrospira in the ammonia-oxidizing community. Metagenomic analysis of seven heterotrophs suggested that nitrate reduction is a common capability in potentially using endogenous carbohydrates and peptides to enhance nitrogen removals. The prevalence of class II ribonucleotide reductase and antioxidant enzymes genes may grant the adaptation to cyclically microaerobic/anoxic environments. The predominant heterotroph is affiliated with Chloroflexota; its genome encodes complete pathways for synthesizing vitamin B6 and methionine. By contrast, other than the two growth factors, Nitrospira and anammox bacteria are complementary to produce various vitamins and amino acids. Besides, the novel Chloroflexota-related ammonia oxidizer lacks corresponding genes for detoxifying the reactive oxygen species and thus requires the aid of co-existing members to alleviate oxidative stress. The analysis results forecast the exchanges of substrates and nutrients as well as the collective alleviation of oxidative stress among the core populations. The new findings of the genomic features and predicted microbial interplay shed light on microbial adaptation to intermittent microaeration specific to the PN-A reactor, which may aid in improving its application to low-strength ammonium wastewater.

Glutamate Enhances Osmoadaptation of Anammox Bacteria under High Salinity: Genomic Analysis and Experimental Evidence.

An osmoprotectant that alleviates the bacterial osmotic stress can improve the bioreactor treatment of saline wastewater. However, proposed candidates are expensive, and osmoprotectants of anammox bacteria and their ecophysiological roles are not fully understood. In this study, a comparative analysis of 34 high-quality public metagenome-assembled genomes from anammox bacteria revealed two distinct groups of osmoadaptation. Candidatus Scalindua and Kuenenia share a close phylogenomic relation and osmoadaptation gene profile and have pathways for glutamate transport and metabolisms for enhanced osmoadaptation. The batch assay results demonstrated that the reduced Ca. Kuenenia activity in saline conditions was substantially alleviated with the addition and subsequent synergistic effects of potassium and glutamate. The operational test of two reactors demonstrated that the reduced anammox performance under brine conditions rapidly recovered by 35.7-43.1% as a result of glutamate treatment. The Ca. Kuenenia 16S rRNA and hydrazine gene expressions were upregulated significantly (p < 0.05), and the abundance increased by approximately 19.9%, with a decrease in dominant heterotrophs. These data demonstrated the effectiveness of glutamate in alleviating the osmotic stress of Ca. Kuenenia. This study provides genomic insight into group-specific osmoadaptation of anammox bacteria and can facilitate the precision management of anammox reactors under high salinity.

Comammox Nitrospira Species Dominate in an Efficient Partial Nitrification-Anammox Bioreactor for Treating Ammonium at Low Loadings.

Bacteria capable of complete ammonia oxidation (comammox) are widespread and contribute to nitrification in wastewater treatment facilities. However, their roles in partial nitrification-anaerobic ammonium oxidation (anammox) systems remain unclear. In this study, a bench-scale bioreactor with continuous stirring was operated for more than 1000 days with limited oxygen supply to achieve efficient nitrogen removal (70.1 ± 2.7%) at a low ammonium loading of 35.2 mg-N/L/day. High-throughput amplicon sequencing analysis of the comammox ammonia monooxygenase subunit A (amoA) gene revealed seven sequence types from two clusters in clade A of comammox Nitrospira. Quantitative polymerase chain reaction analyses suggested that the comammox species dominated the ammonia-oxidizing community, with an abundance as high as 89.2 ± 7.9% in total prokaryotic amoA copies. Multiple linear regression further revealed the substantial contribution of the comammox Nitrospira to ammonia oxidation in the bioreactor. The investigation with bioreactor and batch experiments consistently showed that activities of comammox Nitrospira were inhibited by free ammonia far more severely than other ammonia-oxidizing microbes. Overall, this study provided new insight into the ecology of comammox Nitrospira under hypoxic conditions and suggested comammox-associated partial nitrification-anammox as a potential method for treating low-strength ammonium-containing wastewater.

System Performance Corresponding to Bacterial Community Succession after a Disturbance in an Autotrophic Nitrogen Removal Bioreactor.

Performance of a bioreactor is affected by complex microbial consortia that regulate system functional processes. Studies so far, however, have mainly emphasized the selective pressures imposed by operational conditions (i.e., deterministic external physicochemical variables) on the microbial community as well as system performance, but have overlooked direct effects of the microbial community on system functioning. Here, using a bioreactor with ammonium as the sole substrate under controlled operational settings as a model system, we investigated succession of the bacterial community after a disturbance and its impact on nitrification and anammox (anaerobic ammonium oxidation) processes with fine-resolution time series data. System performance was quantified as the ratio of the fed ammonium converted to anammox-derived nitrogen gas (N2) versus nitrification-derived nitrate (npNO3 -). After the disturbance, the N2/npNO3 - ratio first decreased, then recovered, and finally stabilized until the end. Importantly, the dynamics of N2/npNO3 - could not be fully explained by physicochemical variables of the system. In comparison, the proportion of variation that could be explained substantially increased (tripled) when the changes in bacterial composition were taken into account. Specifically, distinct bacterial taxa tended to dominate at different successional stages, and their relative abundances could explain up to 46% of the variation in nitrogen removal efficiency. These findings add baseline knowledge of microbial succession and emphasize the importance of monitoring the dynamics of microbial consortia for understanding the variability of system performance.IMPORTANCE Dynamics of microbial communities are believed to be associated with system functional processes in bioreactors. However, few studies have provided quantitative evidence. The difficulty of evaluating direct microbe-system relationships arises from the fact that system performance is affected by convolved effects of microbiota and bioreactor operational parameters (i.e., deterministic external physicochemical forcing). Here, using fine-resolution time series data (daily sampling for 2 months) under controlled operational settings, we performed an in-depth analysis of system performance as a function of the microbial community in the context of bioreactor physicochemical conditions. We obtained statistically evaluated results supporting the idea that monitoring microbial community dynamics could improve the ability to predict system functioning, beyond what could be explained by operational physicochemical variables. Moreover, our results suggested that considering the succession of multiple bacterial taxa would account for more system variation than focusing on any particular taxon, highlighting the need to integrate microbial community ecology for understanding system functioning.