Candidatus Kuenenia Research Articles

Upflow blanket filter anammox (UBFA) system treating low-nitrogen wastewater: high-efficient nitrogen removal, granules formation, N2O emission, and microbial succession.

This research provides an important approach for low-nitrogen wastewater treatment through anaerobic ammonium oxidation (Anammox), and Anammox granule sludge (AnGS) in the Upflow. Blanket Filter Anammox (UBFA) system through shortening the hydraulic retention time was successfully cultivated. The percentage of medium granules (1.0-2.0mm) with the highest Anammox activity increased from 0 to 28.5%, and the proportion of flocs (0-200μm) reduced from 84.5% to 17.6%. Through the multidimensional analysis of AnGS, the relationship between AnGS and EPS secretion, low SVI, high PN/PS, multiple filamentous bacteria, and AnAOB were explored. Microelectrode tracing tests demonstrated that the main anammox reaction active layer was 0-1500μm, and the highest activity was observed at 200-400μm, whereas denitrification activity and N2O production were mainly distributed in the granules deep layer of 1500-2500μm. The research showed that Candidatus Brocadia and Candidatus Kuenenia were the predominant anammox species in the UBFA system, while the abundance of AnAOB was higher in medium granules.

The start-up of red blood cell-like anammox granular sludge based on substrate volume flux

Enhancing substrate transfer is crucial for promoting the nitrogen removal rate of Anammox granular sludge, as the shape of the granules significantly influences substrate diffusion. In this study, inspired by biomimicry principles, we propose a novel red blood cell-like granular sludge to increase the contact area and improve substrate transfer. The startup and operation of Anammox granular sludge in low-strength ammonia nitrogen wastewater were successfully achieved through an increase in substrate volume flux (SVF). As the SVF increased from 2.38 to 7.43L²/(g VSS·h), specific Anammox activity (SAA) rose from 0.138 to 0.483gN/(g VSS·d). The Anammox granular sludge system achieved a total nitrogen removal efficiency of 85.70% and a nitrogen removal rate (NRR) of 0.792kgN/(m³·d) after 126 days. Notably, red blood cell-like granular sludge was observed by day 116. The rise in SVF induced a granule structure more conducive to substrate transport, improving the granules' efficiency in substrate uptake and thereby enhancing their SAA. High-throughput pyrosequencing revealed that the dominant bacteria using the SVF-enhancing strategy remained anaerobic ammonium oxidizing bacteria (AnAOB) (29.53%-29.89%), with an increased diversity of species, including Candidatus Kuenenia, Candidatus Anammoxoglobus, and Candidatus Brocadia.

Effect of inoculated sludge concentration on start-up of anammox reactor: Nitrogen removal performance and metabolic pathways

The anammox process is efficient for nitrogen removal but faces challenges due to slow bacterial growth and limited inoculated sludge supply. This study examined the effects of different inoculated sludge concentrations (3.5, 7, and 14 g/L) on start-up and nitrogen metabolism in anammox reactors. Three identical reactors were operated under controlled conditions, with comprehensive analysis of nitrogen removal efficiency, sludge characteristics, and microbial community dynamics through metagenomic and transcriptomic approaches. Results demonstrated that higher inoculated sludge concentrations accelerated reactor start-up, with the 14 g/L reactor achieving stable operation in 13 days compared to 44 days for the 3.5 g/L reactor. However, the improvement in nitrogen removal rate showed a boundary effect, not proportional to the increase in sludge concentration. Notably, reactors with higher inoculated sludge concentrations exhibited lower sludge loads but higher sludge yield coefficients. Metagenomic analysis revealed Candidatus Kuenenia as the dominant anammox bacteria, with decreasing hydrazine dehydrogenase (hdh) gene expression levels observed at higher sludge concentrations, suggesting hydrazine synthesis as a potential rate-limiting step. This study provides novel insights into the optimal range of inoculated sludge concentration for anammox reactor start-up and elucidates the underlying metabolic mechanisms, offering valuable guidance for practical engineering applications.

Unraveling the mechanisms of anammox coupled process with thiosulfate-driven denitrification: Community succession and substrate competition

Thiosulfate-driven denitrification coupled with anammox (TDDA) has garnered interest for its efficient and innovative nitrogen removal capabilities. However, the intricate dynamics of the internal microbial community and the specific characteristics of anaerobic ammonium oxidizing bacteria (AnAOB) remain incompletely understood. This study combines experimental methods with density functional theory (DFT) calculations to address these gaps. The TDDA reactor was successfully started-up with an optimal S2O32--S/NO3−-N ratio of 0.6, achieving a nitrogen removal efficiency of 89.6%. Throughout this process, the relative abundance of Candidatus Kuenenia decreased by 10.2%, while the relative abundance of Candidatus Brocadia increased by 9.6%. The elevated concentration of NO₃--N inhibited Candidatus Kuenenia, and simultaneously stimulated the secretion of extracellular polymers, affecting Fe uptake by Candidatus Kuenenia. To further elucidate substrate competition, molecular docking simulations and DFT calculations were employed. The binding energy, compared with the electrostatic potential energy of the protein pocket, clearly demonstrated that Nir in AnAOB has a higher affinity for the substrate (EAnAOB = −163.2 kJ/mol vs. ESOB = -77.7 kJ/mol). By integrating molecular dynamics insights, this study overcomes experimental limitations and deepens the understanding of the mechanisms within the TDDA system.

Analysis of the regulation mechanism for salt-tolerant anammox process: process performance and metabolic insights

ABSTRACT : In this study, the start-up and microbial domestication of a salt-tolerant functional anammox system was investigated by gradually increasing the salinity level in a stabilized anammox system in the laboratory. After 44 days of stable operation, the salt-tolerant system was successfully activated, at which time the salinity of the influent water was 3 g/L, and the maximum removal efficiency of ammonia nitrogen and nitrite nitrogen in the system reached 94.18% and 96.66%, respectively, and then the ammonia nitrogen and nitrite nitrogen removal efficiency were stabilized at 88.17% and 96.48% after the enrichment domestication for 89 days. The system was operated in the salinity of 10 g/L, with the concentration of each nitrogen compound measured at the same time. The ammonia nitrogen removal efficiency decreased to 59.93% at a salinity of 10 g/L, which had a significant impact on the system. High-throughput sequencing revealed that the system was enriched with a large number of Chloroflexi, the relative abundance of which increased from 19.46% to 52.33%, and the genus of AnAOB was transformed from Candidatus Brocadia to Candidatus Kuenenia, Candidatus Kuenenia, with a percentage of 4.78%. The system successfully achieved the simultaneous removal of ammonia nitrogen and nitrite nitrogen under salinity stress, which to a certain extent indicated that AnAOB could achieve the initiation and enrichment domestication under salinity conditions, and could provide a basis for the efficient and low-consumption treatment of high salinity nitrogen-containing wastewater.

Novel ellipsoid-like granules exhibit enhanced anammox performance compared to sphere-like granules

Anammox granular sludge (AnGS) serves as an important platform for cost-effective nitrogen removal from wastewater. Different to the traditionally sphere-like granules, a novel type of AnGS in a unique ellipsoid-like shape was obtained through enhancing shear force. The ellipsoid-like AnGS significantly exhibited a smaller aspect ratio (-25.1 %) and granular size (-11.8 %), compared to traditional sphere-like AnGS (p < 0.01). Comprehensive comparisons showed that ellipsoid-like AnGS possessed a significantly higher extracellular polymeric substances (EPS) content and strength, as well as an enhanced mass transfer and a higher viable bacteria proportion due to the larger substrate permeable zone (p < 0.01). Additionally, the anammox bacterial abundance (Candidatus Kuenenia) was 12.2 % higher in ellipsoid-like AnGS than in sphere-like AnGS. All these characteristics of ellipsoid-like AnGS jointly increased the specific anammox activity by 29.0 % and nitrogen removal capacity by 22.6 %, compared to sphere-like AnGS. Further fluid field simulation suggested the enhanced flow shear on the side surface of AnGS likely drove the formation of ellipsoid-like AnGS. The higher shear force on the side surface led to an increase of EPS content (especially hydrophobic protein) and elastic modulus, thus constraining lateral expansion. This study sheds light on impacts of granular shape, an overlooked morphological factor, on anammox performance. The ellipsoid-like AnGS presented herein also offers a unique and promising aggregate to enhance anammox performance.

Metatranscriptomics provides an in-depth perspective on the resistance and detoxification of anammox bacteria to dissolved oxygen in a pilot CANON process

In the completely autotrophic nitrogen removal over nitrite (CANON) process, the conflicting oxygen requirements of anammox and ammonium-oxidizing bacteria often lead to retardation in anammox activity. However, our study achieved stable nitrogen removal with a maximum capacity of 1096 g-N/m3/d in a 20 m3 CANON reactor under long-term intensive aeration. The anammox bacteria unusually distributed in the outer layer of the biofilm and demonstrated remarkable oxygen tolerance. Their activity only declined by 18.5 % under 2.0 mg/L of dissolved oxygen. When anammox bacteria encountered oxygen exposure, they adopted some strategies. Metatranscriptomics revealed that Candidatus Kuenenia, the dominant anammox species in our system, downregulated its gene expressions involved in carbon metabolism and oxidative phosphorylation. This may reduce electron leakage that combines with O2, thereby minimizing the generation of reactive oxygen species (ROS). By contrast, the secretion of extracellular proteins and conversion of O2·− were upregulated to eliminate ROS promptly. This behavior endowed Ca. Kuenenia with a unique oxygen detoxification pathway: O2·− were initially converted to H2O2 by superoxide dismutase SOD2 and superoxide reductase dfx (major role), followed by reduction to H2O via non-heme chloroperoxidase cpo (a newly recognized mechanism in the oxygen detoxification of anammox) and catalase katE. These results expanded the current knowledge of anammox alleviating oxidative stress.

Differentiation of the Anammox core microbiome: Unraveling the evolutionary impetus of scalable gene flow

Anaerobic ammonium oxidation bacteria (AAOB), distinguished by their unique autotrophic nitrogen metabolism, hold pivotal positions in the global nitrogen cycle and environmental biotechnologies. However, the ecophysiology and evolution of AAOB remain poorly understood, attributed to the absence of monocultures. Hence, a comprehensive elucidation of the AAOB-dominated core microbiome, anammox core, is imperative to further completing the theory of engineered nitrogen removal and ecological roles of anammox. Performing taxonomic and phylogenetic analyses on collected genome repertoires, we show here that Candidatus Brocadia and Candidatus Kuenenia possesses a more compact core than Candidatus Jettenia, which partly explains why the latter has a less common ecological presence. Evidence of gene flow is particularly striking in functions related to biosynthesis and oxygen detoxification, underscoring the evolutionary forces driving lineage and core differentiation. Furthermore, CRISPR spacer traceback of the AAOB metagenome-assembled genomes (MAGs) reveals a series of genetic traces for the concealed phages. By reconceptualizing the functional divergence of AAOB with the historical role of phages, we ultimately propose a coevolutionary framework to understand the evolutionary trajectory of anammox microecology. The discoveries provided in this study offer new insights into understanding the evolution of AAOB and the ecology of anammox.

Enhancing nitrogen removal from digested swine wastewater by anammox with aeration optimization coupling real-time control strategy

The nitrogen removal of anaerobically digested swine wastewater (ADSW) through partial nitritation and anammox is hindered by the challenge of balancing aeration between ammonia oxidizing bacteria (AOB) and anammox bacteria (AnAOB). This study focused on optimizing aeration through a real-time control strategy in an integrated fixed-film activated sludge reactor for treating ADSW. The system implemented a dual aeration mode that included both low dissolved oxygen (DO) (< 0.4 mg/L) and short-term high DO (0.6–1.2 mg/L), with pH, oxidation–reduction potential, and NH4+-N electrode values as real-time control parameters. NH4+-N removal rate increased from 3.37 to 12.82 mgN/(gVSS·h), and total nitrogen (TN) removal rate enhanced from 0.14 to 0.25 kgN/(m3·d). Increasing DO stimulated AOB activity by 31 % and provided sufficient NO2−-N for AnAOB. The r-strategist AnAOB (Candidatus Kuenenia) proliferated well in the biofilm (0.25 % in flocs vs. 1.86 % in biofilm). The enrichment of denitrifiers improved organic matter and TN removal.

Two-stage partial nitrification-denitrification and anammox process for nitrogen removal in vacuum collected toilet wastewater at ambient temperature

Vacuum collected toilet wastewater (VCTW) contains high and fluctuating contents of organics and nitrogen, which exerts technological challenges to biological treatment processes. A partial nitrification–denitrification and anammox (PND-AMX) process was developed in sequencing batch reactor (SBR) and moving bed biofilm reactor (MBBR) to achieve effective nitrogen removal in VCTW at low ambient temperature. Stable PND was achieved, and nitrogen removal efficiency in SBR could be manipulated by adjusting influent COD/N ratios. As temperature ≥18 °C, 91.0% nitrogen was removed in PND-AMX process. In spite of the decreased anammox activity at 13–18 °C, more than 90% nitrogen removal could be obtained by adjusting SBR influent COD/N to 2.43 ± 0.32 with methanol. In MBBR reactor, Candidatus Kuenenia was the dominant anammox bacteria and contributed to more than 90% nitrogen removal capacity. Co-existing anammox and denitrifying bacteria synergistically contributed to the removal of ammonium, nitrite, nitrate, and COD in MBBR.

Response of anammox to organics with different degradation characteristics and exposure time: Performance, sludge characteristics and bacterial community

The effects of typical organic compounds including easily degradable organic matters sodium acetate, yeast and methanol, and refractory organic matter (ROM) humic acid on anaerobic ammonium oxidation (anammox) systems in short-term and medium-term exposure time were studied. During short-term experiments, nitrogen removal activity (NRA) was inhibited at sodium acetate level of 150 mg L−1 total organic carbon (TOC) and methanol level of 30–150 mg L−1 TOC, but humic acid and yeast (≤150 mg L−1 TOC) enhanced nitrogen removal in anammox systems. The greatest NRA of 30.10 mg TN g−1 VSS h−1 was recorded at yeast level of 90 mg L−1 TOC. In medium-term experiments, organics significantly inhibited the nitrogen removal ability. As a ROM, humic acid enhanced sludge aggregation and biological diversity, but decreased the bioactivity and extracellular polymeric substances levels. Due to the endogenous denitrification, the relative abundance of anammox bacteria (AnAOB) was decreased. Candidatus Kuenenia is still dominant in sludge with methanol and humid acid, but AnAOB are not dominant due to the addition of sodium acetate and yeast. This research would be beneficial for the full-scale application of the anammox process in treating real wastewater with organics and ammonia.

Adaptation of the anammox process for high ammonium photovoltaic wastewater treatment

Tunnel Oxide Passivating Contacts (TOPcon) battery in the photovoltaic industry generates high ammonium wastewater during the production process, the adaptability of using the anaerobic ammonia oxidation (Anammox) process for photovoltaic wastewater (PVW) treatment is a research hotspot. Based on the analysis of photovoltaic wastewater quality, the effectiveness of nitrogen removal, sludge characteristics and microbial communities were examined. The results showed that when the influent NH4+-N concentration of PVW was lower than 150 mg·L−1, the nitrogen removal efficiency (NRE) was almost 100 %. In addition, the NRE decreased from 74 % sharply to 20 % when the NH4+-N concentration was increased from 175 mg·L−1 to 200 mg·L−1. The extracellular polymeric substances (EPS) content increased with elevated ammonium concentration in the influent, indicating that microorganisms secreted more EPS to resist elevated nitrogen loading. The main functional populations were Candidatus Kuenenia (0–24 %). The influent ammonium concentration is recommended to be < 200 mg·L−1.

Deciphering retention effect of extracellular polymeric substances to typical heavy metals and their interaction with key inner enzymes of Candidatus Kuenenia

This study employed spectroscopy, metagenomics, and molecular simulation to investigate the inhibitory effects of Cd(II) and Cu(II) on the anammox system, examining both intracellular and extracellular effects. At concentrations of 5 mg/L, Cd(II) and Cu(II) significantly reduced nitrogen removal efficiency by 41.46 % and 62.03 %, respectively. Additionally, elevated metal concentrations were correlated with decreased extracellular polymeric substances (EPS), thereby reducing their capacity to absorb heavy metals, particularly Cu(II), which decreased from 76.47 % to 14.67 %. Spectral analysis revealed alterations in the secondary structures of EPS induced by Cd(II) and Cu(II), decreasing the ratio of extracellular protein α-helix to (β-sheet + random coil), which resulted in looser extracellular protein configurations. The results of the metagenomics study showed that the abundance of Candidatus Kuenenia and its genes encoding nitrogen removal-related enzymes was reduced. The abundance of hzs-γ was reduced by 35.09 % at a concentration of 5 mg/L Cu(II). Conversely, genes associated with metal efflux enzymes, like czcR, increased by 54.86 % at 2 mg/L Cd(II). Molecular docking revealed robust bindings of Cd(II) to HZS-α (−342.299 ± 218.165 kJ/mol) and Cu(II) to HZS-γ (−880.934 ± 55.526 kJ/mol). This study elucidated the inhibitory mechanisms of Cd(II) and Cu(II) on the anammox system, providing insights into the resistance of anammox bacteria to heavy metals.

Metagenomic unveils the promotion of mainstream PD-anammox process at lower nZVI concentration and inhibition at higher dosage

The partial-denitrification-anammox (PdNA) process exhibits great potential in enabling the simultaneous removal of NO3−-N and NH4+-N. This study delved into the impact of exogenous nano zero-valent iron (nZVI) on the PdNA process. Adding 10 mg L−1 of nZVI increased nitrogen removal efficiency up to 83.12 % and maintained higher relative abundances of certain beneficial bacteria. The maximum relative abundance of Candidatus Brocadia (1.6 %), Candidatus Kuenenia (1.5 %), Ignavibacterium (1.3 %), and Azospira (1.2 %) was observed at 10 mg L−1 of nZVI. However, the greatest relative abundance of Thauera (1.3 %) was recorded under 50 mg L−1. Moreover, applying nZVI selectively enhanced the abundance of NO3−-N reductase genes. So, keeping the nZVI concentration at 10 mg L−1 or below is advisable to ensure a stable PdNA process in mainstream conditions. Considering nitrogen removal efficiency, using nZVI in the PD-anammox process could be more cost-effective in enhancing its adoption in industrial and mainstream settings.

Nitrogen removal performance and the biocenosis with microalgae consortium Nitrosifying and anammox bacteria in an upflow reactor

This study introduced an innovative pathway utilizing an algal anaerobic ammonium oxidation (ALGAMMOX) system to treat ammonium wastewater. Lake bottom sludge and anammox sludge were used to cultivate functional microorganisms and microalgae for nitrogen removal in an upflow reactor made of transparent materials. The results showed that the ALGAMMOX system achieved 87.40 % nitrogen removal when the influent NH4+-N concentration was 100 mg-N/L. Further analysis showed that anammox bacteria Candidatus Brocadia (8.87 %) and nitrosobacteria Nitrosomonas (3.74 %) were crucial contributors, playing essential roles in nitrogen removal. The 16S rRNA gene showed that the anammox bacteria in the sludge transitioned from Candidatus Kuenenia to Candidatus Brocadia. The 18S rRNA gene revealed that Chlamydomonas, Bacillariaceae and Pinnularia were the dominant microalgae in the system at a relative abundance of 7.99 %, 3.64 % and 3.14 %, respectively. This novel approach provides a theoretical foundation for ammonium wastewater treatment.

Long-term response of anammox process to hydrazine under different exposure strategies

The enhancing effect of hydrazine (N2H4) on anammox has been extensively studied; however, its long-term inhibitory effect remains controversial. This study aims to evaluate the susceptibility and resistance of the anammox bacteria to N2H4 and to follow the dynamic changes in functional microorganisms under two different N2H4 application strategies (L1–136 days; L2–344 days). In the first strategy (L1), ≥ 88 % of NH4+-N and NO2--N were removed during the N2H4 applications at ≤ 150 mg/L dosages. Above 150 mg/L, nitrogen treatment performance deteriorated. When 150 mg/L of N2H4 was continuously fed to the bioreactor in L2, anammox bacteria performed their activity with ≥ 70 % removal efficiency for both NH4+-N and NO2--N up to 2.74 g/L cumulative N2H4 loading. During these periods in both strategies, Candidatus Kuenenia, a sole anammox genus identified in the bioreactors, was found to adapt to higher N2H4 concentrations than those reported in the literature. Although nitrogen removal capacity deteriorated towards the end of the operation periods, N2H4 was still utilized in the bioreactors. Therefore, this study makes an important contribution to the utilization of anammox bacteria as a potential candidate for the treatment of N2H4-containing wastewaters, in addition to their current widespread applications.

Start-up and Recovery Performance of ANAMMOX Reactors Under Long-term Starvation

The application of ANAMMOX technology is constrained by sluggish growth and difficulty in enriching ANAMMOX bacteria. Long-term starvation of functioning bacteria due to limited substrate supply makes the steady operation of ANAMMOX reactors more difficult. Re-examining the start-up and recovery performance of the ANAMMOX reactor and identifying its resistance mechanism are important from the standpoint of long-term starvation. By inoculating nitrifying and denitrifying sludge under various operating circumstances, the ANAMMOX reactors were successfully started. Under various start-up procedures, the tolerance mechanism and recovery performance were examined. The outcomes demonstrated that the denitrifying sludge-inoculated reactor operated steadily with a high substrate concentration and low flow rate. After 85 days of operation, the removal efficiencies of NH4+-N, NO2--N, and total nitrogen reached 98.7%, 99.3%, and 89.3%, respectively. After 144 days of starvation and 30 days of recovery, the better nitrogen removal performance was achieved at a low substrate concentration and high flow rate, and the removal efficiencies were 99.8% （NH4+-N）, 99.8% （NO2--N）, and 93.6% （total nitrogen）. During the starvation, extracellular polymeric substances wrapped the ANAMMOX bacteria and kept them intact to resist long-term starvation stress. The expression of nirS, hzsA, and hdh genes ensured the synthesis of nitrite/nitric oxide oxidoreductase, hydrazine synthase, and hydrazine dehydrogenase to maintain ANAMMOX activity. There was no significant difference in the relative abundance of ANAMMOX bacteria before and after starvation recovery. Candidatus Kuenenia had better anti-hunger ability, and the relative abundance increased by more than 86% after 30 days of recovery, confirming its tolerance to long-term starvation.

Mechanistic insight into microbial interaction and metabolic pattern of anammox consortia on surface-modified biofilm carrier with extracellular polymeric substances

The extremely slow growth rate of anaerobic ammonia oxidation (anammox) bacteria limits full-scale application of anammox process worldwide. In this study, extracellular polymeric substances (EPS)-coated polypropylene (PP) carriers were prepared for biofilm formation. The biomass adhesion rate of EPS-PP carrier was 12 times that of PP carrier, and EPS-PP achieved significant enrichment of E. coli BY63. The 120-day continuous flow experiment showed that the EPS-PP carrier accelerated the formation of anammox biofilm, and the nitrogen removal efficiency increased by 10.5 %. In addition, the abundance of Candidatus Kuenenia in EPS-PP biofilm was 27.1%. Simultaneously, amino acids with high synthesis cost and the metabolites of glycerophospholipids related to biofilm formation on EPS-PP biofilm were significantly up-regulated. Therefore, EPS-PP carriers facilitated the rapid formation of anammox biofilm and promoted the metabolic activity of functional bacteria, which further contributed to the environmental and economic sustainability of anammox process.

Rapid Start-up of Continuous Autotrophic Nitrogen Removal Reactor by Hybrid-inoculating PN and PN/A Granular Sludges

The rapid cultivation of partial nitritation/ANAMMOX （PN/A） granular sludge in a continuous-flow mode is one of the key technologies for efficient biological nitrogen removal in domestic wastewater treatment. Compared with that in PN/A granular sludge, PN granular sludge demonstrates a shorter incubation period and suitability for batch culture. It is also a good carrier for enriching ANAMMOX （AMX） bacteria. In this study, we established a continuous-flow autotrophic nitrogen removal process in three continuously stirred tank reactors （CSTR） （R1-R3） by hybrid-inoculating PN/A and PN granular sludge at the mass ratios of 3∶1, 1∶1, and 1∶3, respectively. By implementing high ammonium nitrogen loading and short hydraulic retention time, continuous autotrophic nitrogen removal processes were successfully started up in the three CSTRs. The results showed that compared with that of R1 and R2, R3 had a longer start-up time but a similar steady-state nitrogen removal performance. The total nitrogen removal load of R3 could be more than 2.6 kg·（m3·d）-1. Intriguingly, the inoculated PN granular sludge served as a precursor for PN/A granular sludge cultivation. This approach facilitated the enrichment of anaerobic ammonia-oxidizing bacteria （AMX） by introducing abundant ammonium-oxidizing bacteria （AOB） and nitrite nitrogen substrates into the CSTR. According to the results of high-throughput sequencing, the microbial abundance and diversity of the mature granules in R1-R3 were significantly higher than those of the inoculation sludge. AOB （genus Nitrosomonas）, AMX （genera Candidatus Kuenenia and Candidatus Brocadia）, and symbiotic heterotrophs, such as Chloroflexi, Bacteroidetes, and Chlorobi, drove the autotrophic nitrogen removal process and maintained the stability of the granular structure. In summary, a novel start-up strategy of hybrid-inoculating granular sludge was provided for a continuous-flow autotrophic nitrogen removal in engineering application.

High dosage of exogenous glycine in cross-feeding breaks the equilibrium of anammox system

Strategies to break the cross-feeding equilibrium by dosing essential metabolites and thus enriching and isolating anammox bacteria (AnAOB) have been proposed, but amino acids have not been investigated as important cross-feeding pathways in anammox consortia. In this study, long-term experiments were designed to verify the effect of exgenous glycine on anammox consortia and changes in the bacterial community and metabolic activity after the addition of glycine in a highly active anammox system were investigated. The results showed that the impact of glycine on the activity of anammox highly depended on glycine concentrations, which was manifested as a promotive effect at low concentrations (0–150 mg/L) and an inhibitory effect at high concentrations (300–600 mg/L). The addition of low concentrations of glycine not only enhanced the diversity and richness of bacterial communities but also promoted the enrichment of Candidatus Kuenenia. The qPCR results showed that the content of functional genes Hzs, NirS and anammox 16S rRNA increased while the expression of amoA and NrfA genes was suppressed when glycine acted positively on AnAOB; Hzs and amoA were suppressed when glycine acted negatively on AnAOB, while the expression of NirS and NrfA genes increased dramatically. Specific anammox activity (SAA) experimental results show that 50–150 mg/L glycine was the optimal amount for promoting the activity of AnAOB. Analysis of community metabolic functions indicated that the addition of exogenous glycine resulted in a imbalance of glycine cross-feeding and promoted AnAOB enrichment mainly by affecting pathways such as other glycan degradation and Lysosome. Overall, this study provided new insights into understanding the glycine cross-feeding, which is essential for improving anammox-based technologies.