Anaerobic Ammonium-oxidizing Bacteria Research Articles

Harnessing the Potential of Extracellular Polymeric Substances in Enhancing ANAMMOX Processes: Mechanisms, Strategies, and Perspectives

Anaerobic ammonium oxidation (ANAMMOX) has emerged as a promising sustainable nitrogen removal technology that offers significant advantages over conventional nitrification–denitrification processes, such as reduced energy consumption, a 60% reduction in oxygen demand, and a 90% reduction in sludge production. However, the practical application of ANAMMOX is hindered by several challenges, including the slow growth of ANAMMOX bacteria, long start-up periods, and high sensitivity to environmental disturbances. Recent studies have highlighted the crucial role of extracellular polymeric substances (EPSs) in the formation, activity, and stability of ANAMMOX biofilms and granules. An EPS is a complex mixture of high-molecular-weight polymers secreted by microorganisms, mainly composed of polysaccharides, proteins, nucleic acids, and lipids. The diverse physicochemical properties and functional groups of EPSs enable them to serve as a structural scaffold, protective barrier, sorption site, electron shuttle, and nutrient source for ANAMMOX bacteria. This review aims to provide an overview of the latest research progress on harnessing the potential of EPSs to enhance the ANAMMOX process. The characteristics, compositions, and extraction methods of ANAMMOX-derived EPSs are summarized. The mechanisms of how EPSs facilitate the enrichment, immobilization, aggregation, and adaptation of ANAMMOX bacteria are elucidated. The strategies and effects of EPS supplementation on improving the performance and robustness of ANAMMOX reactors under various stresses are critically reviewed. The challenges and future perspectives of the EPS-mediated optimization of the ANAMMOX process are also discussed. This review sheds new light on exploiting EPSs as a renewable bioresource to develop more efficient and stable ANAMMOX applications for sustainable wastewater treatment.

Research Progress,Problems and Future Prospects of A New Combined Anaerobic Ammonia Oxidation and Nitrogen Removal Process

Anaerobic ammonium oxidation (Anammox) is a cost-effective and efficient process for nitrogen removal, which has attracted widespread attention in the field of wastewater treatment due to its significant advantages. However, the slow rate of growth of anaerobic ammonium oxidizing bacteria (AnAOB) and its sensitivity to operational and environmental conditions hinder its wide application. Although various control strategies have been explored and developed, the application of Anammox process still faces many challenges, including unstable nitrogen removal performance and the necessity for additional treatment of effluent nitrate nitrogen. Due to the importance and necessity of Anammox in wastewater denitrification, this paper first introduces the present biological denitrification system based on Anammo. In view of the insufficient source of nitrite nitrogen in nitrogen-containing wastewater, nitrite nitrogen can be provided by partial nitrification (PN) or partial denitrification (PD) , however, the stability of system operation and efficiency of denitrification are still the difficulties in Anammox engineering applications. It is especially crucial to study the combined new processes based on Anammox to achieve stable deep biological nitrogen removal from wastewater. Therefore, this paper focuses on latest research progress in Anammox coupled simultaneous partial nitrification, denitrification and anammox (SPNDA) , constructed wetland (CW) , phosphorus removal/ recovery process, sulfur denitrification removal (SDA) and denitrifying anaerobic methane oxidation (ADAMO) . The nitrogen removal performance, influencing factors, operation advantages, research progress, application prospects and challenges are reviewed. Compared with the single Anammox process, the combined process offers several advantages, including overcoming the issue of nitrate in the effluent (SPNDA, A-DAMO, SDA) , alleviate the inhibition of organic matter on AnAOB (SPNDA, SDA) , recover non-renewable resources (HAP) , and achieve a clean environment with significant economic benefits (A-CW) , providing flexibility for the promotion and application of Anammox process. The study outlines future directionsfor researching, developing, and applying the Anammox combined nitrogen removal process, and at the same time provides as a reference for lowering energy consumption, achieving efficient and stable nitrogen removal processes, and broadensing the scope of engineering application.

The Effect of Electricity Generation on the Performance of Microbial Fuel Cells for Anammox

The Anammox anaerobic fluidized bed microbial fuel cell (Anammox AFB-MFC) exhibits exceptional performance in both nitrogen removal and electricity generation, effectively eliminating ammonia nitrogen (NH4+-N) and nitrite nitrogen (NO2−-N) pollutants. This technology offers the advantages of high efficiency in nitrogen removal and low electricity consumption. By coupling an AFB with an MFC, the Anammox AFB-MFC was developed through the introduction of anaerobic ammonia-oxidizing bacteria (AnAOB) into MFC. Anammox AFB-MFC’s nitrogen removal ability was found to be superior at an influent COD concentration of 200 mg/L, as determined by a study conducted under unchanged conditions. Subsequently, an open and closed-circuit experiment was performed on the Anammox AFB-MFC system while maintaining a COD concentration of 200 mg/L in the influent. Remarkably, the reactor exhibited significantly enhanced nitrogen removal performance when electricity generation occurred. Throughout the entire experimental process, the reactor consistently maintained high nitrogen removal efficiency and electricity production performance. Under optimal experimental conditions, the reactor achieved a remarkable nitrogen removal rate of 91.8% and an impressive output voltage of 439.1 mV. Additionally, the generation of Anammox bioparticles in MFC significantly contributed to efficient pollutant removal. This study elucidates the impact of organic matter on both the nitrogen removal and electricity generation capabilities of Anammox AFB-MFC, as well as highlights the synergistic effect between MFC electricity generation and nitrogen removal in the reactor.

Selective genes expression and metabolites transformation drive a robust nitrite accumulation during nitrate reduction under alternating feast-famine condition

Nitrite production via denitrification has been regarded as a key approach for survival of anaerobic ammonium oxidation (anammox) bacteria. Despite the important carbon substrate, little is known about the role of differential genes expression and extracellular metabolite regulation among diverse microbial communities. In this study, a novel alternating feast-famine strategy was proposed and demonstrated to efficiently accumulate nitrite in a low-nitrogen loading rate (NLR) (0.2∼0.8 kg N/m3/d) denitrification system. Highly selective expression of denitrifying genes was revealed as key regulators. Interestingly, in absence of carbon source (ACS) condition, the expression of narG and narI/V genes responsible for reduction of nitrate to nitrite jumped to 2.5 and 5.1 times higher than that in presence of carbon source (PCS) condition with carbon to nitrate ratio of 3.0. This fortunately facilitated a rapid nitrite accumulation once acetate was added, despite a significantly down-regulated narG and narI/narV and up-regulated nirS/nirK. This strategy selected Thauera as the most dominant denitrifier (50.2 %) with the highest contribution to narG and narI/narV genes, responsible for the high nitrite accumulation. Additionally, extracellular xylose, pyruvate, and glucose jointly promoted carbon-central metabolic pathway of key denitrifiers in ACS stage, playing an important role in the process of self-growth and selective enrichment of functional bacteria. The relatively rapid establishment and robust performance obtained in this study shows an engineering-feasible and economically-favorable solution for the regulation of partial denitrification in practical application.

New insights into functional divergence and adaptive evolution of uncultured bacteria in anammox community by complete genome-centric analysis

Anaerobic ammonium-oxidation (anammox) bacteria play a crucial role in global nitrogen cycling and wastewater nitrogen removal, but they share symbiotic relationships with various other microorganisms. Functional divergence and adaptive evolution of uncultured bacteria in anammox community remain underexplored. Although shotgun metagenomics based on short reads has been widely used in anammox research, metagenome-assembled genomes (MAGs) are often discontinuous and highly contaminated, which limits in-depth analyses of anammox communities. Here, for the first time, we performed Pacific Biosciences high-fidelity (HiFi) long-read sequencing on the anammox granule sludge sample from a lab-scale bioreactor, and obtained 30 accurate and complete metagenome-assembled genomes (cMAGs). These cMAGs were obtained by selecting high-quality circular contigs from initial assemblies of long reads generated by HiFi sequencing, eliminating the need for Illumina short reads, binning, and reassembly. One new anammox species affiliated with Candidatus Jettenia and three species affiliated with novel families were found in this anammox community. cMAG-centric analysis revealed functional divergence in general and nitrogen metabolism among the anammox community members, and they might adopt a cross-feeding strategy in organic matter, cofactors, and vitamins. Furthermore, we identified 63 mobile genetic elements (MGEs) and 50 putative horizontal gene transfer (HGT) events within these cMAGs. The results suggest that HGT events and MGEs related to phage and integration or excision, particularly transposons containing tnpA in anammox bacteria, might play important roles in the adaptive evolution of this anammox community. The cMAGs generated in the present study could be used to establish of a comprehensive database for anammox bacteria and associated microorganisms. These findings highlight the advantages of HiFi sequencing for the studies of complex mixed cultures and advance the understanding of anammox communities.

Self-cultivating anammox granules for enhancing wastewater nitrogen removal in nitrification–denitrification flocculent sludge system

The feasibility of self-cultivating anammox granules for enhancing wastewater nitrogen removal was investigated in a nitrification–denitrification flocculent sludge system. Desirable nitrogen removal efficiency of 84 ± 4 % was obtained for the influent carbon to nitrogen ratio of 1–1.3 (NH4+-N: 150–200 mg N/L) via alternate anaerobic/oxic/anoxic mode. Meanwhile, some red granular sludge was formed in the system. The abundance and activity of anaerobic ammonia oxidation bacteria (AnAOB) increased from ‘not detected’ in seed sludge to 0.57 % and 29.4 ± 0.7 mg N/(g mixed liquor volatile suspended solids·h) in granules, respectively, suggesting successful cultivation of anammox granules. Furthermore, some denitrifying bacteria with capability of partial denitrification were enriched, such as Candidatus Competibacter (2.45 %) and Thauera (5.75 %), which could cooperate with AnAOB, facilitating AnAOB enrichment. Anammox was dominant in nitrogen removal with the contribution to nitrogen removed above 68.8 ± 0.3 %. The strategy of self-cultivating anammox granules could promote the application of anammox.

Microbial community evolution and individual-based model validation of biofilms in single-stage partial nitrification/anammox system

In this study, matrix degradation, microbial community development, and distribution using an individual-based model during biofilm formation on carriers at varying depths within a single-stage partial nitrification/anammox system were simulated. The findings from the application of individual-based model fitting, fluorescence in situ hybridization, and high-throughput sequencing reveal the presence of aerobic bacteria, specifically ammonia-oxidizing bacteria, as discrete particles within the outer layer of the carrier. Facultative anaerobic bacteria exemplified by anaerobic ammonia-oxidizing bacteria, are observed as aggregates within the middle layer. Conversely, anaerobic bacteria, represented by denitrifiers, are enveloped by extracellular polymeric substances within the inner layer. The present study extends the application of individual-based model to the formation of polyurethane-supported biofilms and presents valuable avenues for the design and advancement of pragmatic engineering carriers.

Pangenome-Scale Mathematical Modelling of ANAMMOX Bacteria Metabolism

Removal of fixed nitrogen compounds such as ammonium and nitrite from wastewater is of critical importance for balancing the nitrogen cycle and protecting aquatic environments from eutrophication. ANaerobic AMMonium OXidising (ANAMMOX) bacteria have recently been employed for fixed nitrogen removal purposes in wastewater treatment processes. These specialised bacteria convert ammonium and nitrite into nitrogen gas anaerobically, thereby reducing the amount of energy required for aeration in conventional wastewater treatment processes. However, slow growth rates of ANAMMOX remain a major obstacle towards their widespread use in industrial wastewater treatment processes. Thus, a pangenome-scale, constraint-based metabolic model, iRB399, of ANAMMOX bacteria has been developed to design strategies for accelerating their growth. The main metabolic limitation was identified in the energy metabolism of these bacteria, concerning the production of ATP. The extremely low efficiency of the electron transport chain combined with very high growth-associated maintenance energy is likely to be responsible for the slow growth of ANAMMOX. However, different ANAMMOX species were found to conserve energy using a variety of different redox couples, and the modelling simulations revealed their comparative advantages under different growth conditions. iRB399 also identified dispensable catabolic reactions that have demonstrably beneficial effects on enhancing the growth rates of ANAMMOX bacteria. Thus, the pangenome-scale model will not only help identify and overcome metabolic limitations of ANNAMOX bacteria, but also provide a valuable resource for designing efficient ANNAMOX-based wastewater treatment processes.

The role of the combined nitrogen-sulfur-carbon cycles for efficient performance of anammox-based systems

The combined anammox/mixotrophic denitrification process was conducted in two granular sequencing batch reactors (SBRs) during a 200-day operation. Both reactors were fed with synthetic medium, but SBR2 was enriched with additional sulfate (SO42−) which influenced sulfate reduction ammonium oxidation (SRAO) and heterotrophic reduction of SO42− by sulfate reducing bacteria. It was hypothesized that the addition of SO42− could positively impact the removal rates of N-S-C compounds. A low C/N ratio (0.4–1.6) was maintained to prevent inhibition of anaerobic ammonium oxidizing bacteria (AnAOB), and alternating chemical oxygen demand (COD) on/off conditions were used to regenerate AnAOB during COD-off phases and heterotrophic denitrifiers during COD-on phases. Stoichiometric analysis showed that introducing SO42− in SBR2 enhanced the ammonium utilization rate, which was approximately 10 % higher compared to SBR1 in the final stage of the experiment (25.8 vs. 22.8 mg N/(g VSS·h)). The total nitrogen removal efficiencies ranged from 62 % to 99 % in both reactors, with SBR2 consistently exhibiting approximately 4 % higher efficiency than SBR1. In SBR2, the maximum overall SO42− utilization efficiency reached 27 % under COD-off conditions, while overall COD utilization was almost complete under COD-on conditions. A strong correlation (R2 = 0.98) was observed between SO42− production and COD utilization. The key players responsible for N and S transformations in response to SO42− addition were Candidatus Brocadia and Chloroflexi - Anaerolineae. This study highlights the potential to enhance the overall efficiency of N-S-C removal by implementing an integrated anammox/mixotrophic denitrification process. The combination of cycles emerges as a sustainable approach for treating wastewater rich in N-S-C compounds.

High ammonia loading rate and biofilm reattachment initiated partial nitrification and anammox in a membrane aerated biofilm reactor

Inhibition of nitrite oxidizing bacteria (NOB) is important for efficient wastewater treatment via anammox. Here, a novel strategy was demonstrated, which based on the detachment and reattachment of the biofilm to establish partial nitrification and anammox in a membrane aerated biofilm reactor (MABR). The results showed that using free ammonia as the sole inhibitor to start up partial nitrification took more time, and NOB acclimated to the free ammonia inhibition. Detaching the biofilm made it possible for free ammonia to contact NOB in deeper zones of the biofilm while at the same time washing NOB out of the reactor. Nitrite accumulation efficiency (NAE) increased to 70 % in 50 days when both high ammonia loading and biofilm reattachment were applied. With a short hydraulic retention time and high concentrations of ammonia and nitrite, anammox activity appeared in the reactor and resulted in a nitrogen removal efficiency of 60 %. Microbial community analysis indicated that Nitrosomonas, Nitrospira, and Candidatus Brocadia were the main functional taxa. Anaerobic ammonia oxidizing bacteria (AnAOB) proliferated within the biofilm, inhibiting NOB growth by competing for nitrite. Endogenous denitrifying bacteria also grew, coexisting with AnAOB. Predicted abundance of genes related to fermentation correlated with those involved in denitrification, indicating the possibility of heterotrophic denitrification. These findings help further our understanding of biofilm control-based strategies for NOB management and have implications for establishing autotrophic nitrogen removal using MABRs.

Unraveling potential mechanism of different metal ions effect on anammox through big data analysis, molecular docking and molecular dynamics simulation

Heavy metals (HMs) have been widely reported to pose an adverse effect on anaerobic ammonia oxidation (anammox) bacteria, yet the underlying mechanisms remain unclear. This study provides new insights into the potential mechanisms of interaction between HMs and functional enzymes through big date analysis, molecular docking and molecular dynamics simulation. The statistical analysis indicated that 10 mg/L Cu(II) and Cd(II) reduced nitrogen removal rate (NRR) by 85% and 43%, while 5 mg/L Fe(II) enhanced NRR by 29%. Additionally, the results of molecular simulations provided a microscopic interpretation for these macroscopic data. Molecular docking revealed that Hg(II) formed a distinctive binding site on ferritin, while other HMs resided at iron oxidation sites. Furthermore, HMs exhibited distinct binding sites on hydrazine dehydrogenase. Concurrently, the molecular dynamics simulation results further substantiated their capacity to form complexes. Cu(II) displayed the strongest binding affinity with ferritin for −1576 ± 79 kJ/mol in binding free energy calculation. Moreover, Cd(II) bound to ferritin and HDH for −1052.67 ± 58.49 kJ/mol, −290.02 ± 49.68 kJ/mol, respectively. This research addressed a crucial knowledge gap, shedding light on potential applications for remediating heavy metal-laden industrial wastewater.

Efficient nitrogen removal from real municipal wastewater and mature landfill leachate using partial nitrification-simultaneous anammox and partial denitrification process

Anaerobic ammonia oxidation (anammox) of municipal wastewater is a research focus, especially the combined treatment with mature landfill leachate is a current research hotspot. In this study, municipal wastewater was treated by partial nitrification via sequencing batch reactor (SBR), and its effluent and mature landfill leachate were then mixed into an up-flow anaerobic sludge blanket (UASB) for simultaneous anammox and partial denitrification reaction. Through partial nitrification, a high nitrite accumulation rate (93.0 ± 3.8 %) was achieved by low dissolved oxygen (0.5–1.6 mg/L) and controlled aerobic time (3.5 h) in SBR. The UASB system was responsible for 78.8 ± 2.1 % nitrogen removal of the entire system with a hydraulic reaction time (HRT) of 3.8 h, accompanied by the anammox contribution up to 89.4 ± 6.0 %. The overall partial nitrification-simultaneous anammox and partial denitrification (PN-SAPD) system was controlled at a total COD/TIN of 2.8 ± 0.3 and a total HRT of only 10.2 h, achieving the nitrogen removal efficiency and effluent TIN were 95.2 ± 2.2 % and 3.4 ± 1.5 mg/L, respectively. The qPCR results showed functional genes (hzsA(B), hdh) associated with anaerobic ammonia-oxidizing bacteria (AnAOB), whose high gene copy abundance and transcription expression ensured the removal of major nitrogen from municipal wastewater and mature landfill leachate. 16S amplicon sequencing showed that the Ca. Brocadia (9.72–12.6 %) was further enrichment after sodium acetate was added, and the transcription expression of Thauera (0.5–7.0 %) caused nitrate to nitrite. The high abundance of related enzymes (hao, hzs, hdh, narGHI) involved in anammox and partial denitrification processes were found in the macrogenomic sequencing, and only Ca. Brocadia was involved in multi-pathway nitrogen metabolism in AnAOB. Based on the efficient nitrogen removal by AnAOB and denitrifying bacteria, this modified PN-SAPD process provides a new option for the co-treatment of mature landfill leachate in municipal wastewater treatment plants.

Simultaneous nitrogen and phosphorus removal through an integrated partial-denitrification/anammox process in a single UAFB system

With the aim of obtaining enhanced nitrogen removal and phosphate recovery in mainstream sewage, we examined an integrated partial-denitrification/anaerobic ammonia oxidation (PD/A) process over a period of 189 days to accomplish this goal. An up-flow anaerobic fixed-bed reactor (UAFB) used in the integrated PD/A process was started up with anammox sludge inoculated and the influent composition controlled. Results showed that the system achieved a phosphorus removal efficiency of 82% when the influent concentration reached 12.0 mg/L. Batch tests demonstrated that stable and efficient removal of chemical oxygen demand (COD), nitrogen, and phosphorus was achieved at a COD/NO3−-N ratio of 3.5. Scanning electron microscope (SEM) and X-ray diffraction (XRD) analysis indicated that hydroxyapatite was the main crystal in the biofilm. Furthermore, substrate variation along the axial length of UAFB indicated that partial denitrification and anammox primarily took place near the reactor's bottom. According to a microbiological examination, 0.4% of the PD/A process's microorganisms were anaerobic ammonia oxidizing bacteria (AnAOB). Ca. Brocadia, Ca. Kuenenia, and Ca. Jettenia served as the principal AnAOB generals in the system. Thauera, Candidatus Accumulibacter, Pseudomonas, and Acinetobacter, which together accounted for 27% of the denitrifying and phosphorus-accumulating bacteria, were helpful in advanced nutrient removal. Therefore, the combined PD/A process can be a different option in the future for sewage treatment to achieve contemporaneous nutrient removal.

A microbial-explicit model with comprehensive nitrogen processes to quantify gaseous nitrogen production from agricultural soils

Agricultural soils are a major source of anthropogenic N2O, but their N2O emission estimates are highly uncertain, mainly due to the complexity of nitrogen (N) processes. Most soil N models include only primary N processes such as nitrification and denitrification, which limits their ability to realistically simulate N transformations in soils and accurately estimate N2O emissions from soils. This study introduces a Microbial-Explicit Model incorporating Comprehensive Nitrogen processes (MEMCN) to evaluate and quantify the influences of various N processes on the production of N2O as well as NO and N2, including nitrification, denitrification, anaerobic ammonium oxidation (ANAMMOX), dissimilatory nitrate reduction to ammonium (DNRA), mineralization, and microbial assimilation (i.e., growth) and death. The MEMCN was evaluated in laboratory experiments using agricultural soils with different levels of N additions under anaerobic and aerobic conditions, and reproduced well the dynamics of NH4+, NO3−, NO2−, NO, N2O, and N2. After nitrification and denitrification, ANAMMOX and assimilation were found to be most important in controlling N transformations in agricultural soils. ANAMMOX directly increased N2 emissions by 139% at the beginning of the simulations (i.e., 48 h) under anaerobic conditions, while microbial assimilation indirectly reduced NO, N2O, and N2 emissions by 88%, 54%, and 58%, respectively, at the end of the simulations (i.e., 336 h) under aerobic conditions. Correspondingly, the biomass of ANAMMOX bacteria increased significantly at the beginning of the simulations under anaerobic conditions, while the biomass of nitrite oxidizing bacteria increased substantially under aerobic conditions. In contrast, DNRA, mineralization and microbial death had minor effect on soil N transformations, and the biomass of DNRA bacteria and heterotrophs did not change significantly during the simulations. Our study shows that it is necessary to include ANAMMOX and microbial assimilation in soil N models, while explicit simulation of microbial biomass dynamics may only be necessary if microbial biomass pools change significantly.

Achieving simultaneous removal of carbon and nitrogen by an integrated process of anaerobic membrane bioreactor and flow-through biofilm reactor

In this study, a combined system consisting of an anaerobic membrane bioreactor (AnMBR) and flow-through biofilm reactor/CANON (FTBR/CANON) was developed to simultaneously remove carbon and nitrogen from synthetic livestock wastewater. The average removal efficiencies of total nitrogen (TN) were 64.2 and 76.4% with influent ammonium (NH4+-N) concentrations of approximately 200 and 500 mg/L, respectively. The COD removal efficiencies were higher than 98.0% during the entire operation. Mass balance analysis showed that COD and TN were mainly removed by the AnMBR and FTBR/CANON, respectively. The anammox process was the main nitrogen removal pathway in the combined system, with a contribution of over 80%. High functional bacterial activity was observed in the combined system. Particularly, an increase in the NH4+-N concentration considerably improved the anammox activity of the biofilm in the FTBR/CANON. 16S rRNA high-throughput sequencing revealed that Methanosaeta, Candidatus Methanofastidiosum, and Methanobacterium were the dominant methanogens in the AnMBR granular sludge. In the CANON biofilm, Nitrosomonas and Candidatus Kuenenia were identified as aerobic and anaerobic ammonium-oxidizing bacteria, respectively. In summary, this study proposes a combined AnMBR and FTBR/CANON process targeting COD and nitrogen removal, and provides a potential alternative for treating high-strength wastewater.

Increasing carbon to nitrogen ratio promoted anaerobic ammonia-oxidizing bacterial enrichment and advanced nitrogen removal in mainstream anammox system

The effects of fluctuating organic carbon to nitrogen (C/N) ratios on mainstream simultaneous partial nitrification, anammox, and denitrification (SNAD) process were studied over 376-day period. The nitrogen removal efficiency decreased from 85.0 ± 6.6 % to 75.8 ± 2.8 % as C/N ratio decreased (3.4 → 1.7), but increased to 82.0 ± 1.9 % when C/N ratio raised to 2.9 and to 78.4 ± 3.0 % when C/N ratio decreased again (2.9 → 2.1), indicating that high C/N ratios promoted nitrogen removal. As C/N ratio raised (1.7 → 2.9), anaerobic ammonia-oxidizing bacteria (AnAOB) abundance increased from 1.3 × 109 to 2.0 × 109 copies/L, which explained the improved nitrogen removal. With an elevated C/N ratio, partial nitrification and endogenous partial denitrification reactions were enhanced, providing more nitrite for AnAOB. Additionally, the aerobic_chemoheterotrophy function and particle sizes increased, forming more stable anoxic microenvironment for AnAOB. Overall, increasing C/N ratio promoted the stability of mainstream SNAD.

Insights into the rapid start-up of an inoculated municipal sludge system: A high-height-to-diameter-ratio airlift inner-circulation partition bioreactor based on CFD analysis

The utilization of municipal sludge as a seed sludge for initiating the autotrophic nitrogen removal (ANR) process presents a challenge due to the negligible abundance of anaerobic ammonia-oxidizing bacteria (AnAOB). Here, a computational fluid dynamics model was used to simulate sludge volume fraction and sludge particle velocity. A high-height-to-diameter-ratio airlift inner-circulation partition bioreactor (HHAIPBR) was operated for 175 d to enrich AnAOB from municipal sludge, and the performance of the ANR process was investigated. The start-up period of HHAIPBR inoculated with municipal sludge required approximately 69 d. A high nitrogen removal performance, with a mean total nitrogen removal efficiency of 82.1%, was obtained for 1 month. The simulation results validated the presence of sludge circulation and revealed the distribution characteristics of dissolved oxygen inside the reactor, further supporting the promotion of sludge granulation via the high height-to-diameter ratio. Nitrosomonas (3.31%) of Proteobacteria and Candidatus Brocadia (6.56%) of Planctomycetota were dominant in the HHAIPBR. This study presents a viable approach for the industrial cultivation of anammox sludge and the rapid start-up of the partial nitritation-anammox system.

Migration of microorganisms between nitrification–denitrification flocs, anammox biofilms and blank carriers during mainstream anammox start-up

To solve the shortage of inoculum, the feasibility of establishing simultaneous partial nitrification, anammox, and denitrification (SNAD) reactor through inoculating nitrification–denitrification sludge, anammox biofilm and blank carriers was investigated. Advanced nitrogen removal efficiency of 91.2 ± 3.6 % was achieved. Bacteria related to nitrogen removal and fermentation were enriched in anammox biofilm, blank carriers and flocs, and the abundance of dominant anaerobic ammonia oxidizing bacteria (AnAOB), Candidatus Brocadia, reached 3.4 %, 0.5 % and 0.3 %, respectively. Candidatus Competibacter and Calorithrix became the dominant denitrifying bacteria (DNB) and fermentative bacteria (FB), respectively. The SNAD system was successfully established, and new mature biofilms formed in blank carriers, which could provide inoculum for other anammox processes. Partial nitrification, partial denitrification and aerobic_chemoheterotrophy were existed and facilitated AnAOB enrichment. Microbial correlation networks revealed the cooperation between DNB, FB and AnAOB that promoted nitrogen removal. Overall, the SNAD process was started up through inoculating more accessible inoculum.

Rapid enrichment of anammox bacteria for low-strength wastewater treatment: Role of influent nitrite and nitrate ratios in sequencing batch reactors (SBRs)

Anaerobic ammonium oxidation (Anammox) bacteria (AnAOB) have a long doubling time, which represents one of the key challenges for starting up anammox-based reactors. This study investigated the effect of varied nitrite/nitrate ratios on the anammox reactor startup process using five lab-scale anammox sequencing batch reactors (SBRs) (R1-R5). Nitrate addition significantly accelerated AnAOB enrichment startup, particularly during nitrite accumulation phase. The total start-up time was shortened by 55–71 days, compared to the reactor without nitrate. In R1 (nitrite/nitrate=10:0), anammox contributed the highest nitrogen removal percentage (91%), while R2 (nitrite/nitrate=7:3), with nitrate added, exhibited the highest anammox activity (53.84 mg NH4+-N/g VSS/day) in the steady-state phase. As nitrate proportions increased (R3-R5), anammox activity gradually declined due to limited available COD required for the conversion of NO3- to NO2-. Furthermore, it was found that nitrate-added improved the sludge settleability, with lower SVI30. 16S rRNA gene revealed that Ca. Brocadia was the predominant AnAOB in R1, R2 and R3, where nitrite was the primary NOx--N species (≥50%) in the influent. While in the reactor with low concentration of nitrite (R4: nitrite/nitrate=3:7), Ca. Kuenenia was the dominant AnAOB. This study successfully established a complex and balanced ecosystem with multi-species coexistence, where AnAOB served as the primary functional group on nitrogen removal, supported by denitrification bacteria and dissimilatory nitrate reduction to ammonium (DNRA) bacteria. This study proposes a rapid start-up strategy for the anammox process, which overcomes the bottleneck of long start-up time and helps to promote the application of anammox in wastewater treatment.

Revealing the intrinsic mechanisms for accelerating nitrogen removal efficiency in the Anammox reactor by adding Fe(II) at low temperature

Fe(II) is an essential trace element for anaerobic ammonium oxidation bacteria (AAOB) metabolism, and can improve the nitrogen removal efficiency of anaerobic ammonia oxidation (Anammox). Here we operated two identical expanded granular sludge bed (EGSB) reactors at low temperature (15 ± 3 °C) for 154 days. Reactor 1 (R1) received additional Fe(II) (0.12 mmol/L) during the late startup phase, while reactor 0 (R0) served as the control and did not receive extra Fe(II). Nitrogen removal in R1 became stable at 55 d of operation, ten days earlier than R0. The nitrogen removal rate (NRR) of R1 was 1.64 kg N m−3 d−1 and its TN removal rate was as high as 89%, while R0 only reached 75%. The addition of Fe(II) was further beneficial to aggregation and stability of the granular sludge, and the used sludge of both reactors showed enrichment for AAOB populations compared to the inoculum, for instance, increased abundance of Candidatus-Kuenenia and in particular of Candidatus-Brocadia (from 0.17% to 10.10% in R0 and 7.79% in R1). Diverse microbial species and complex microbial network structure in R1 compared to R0 promoted the coupled denitrogenation by Anammox, dissimilatory nitrate reduction to ammonium (DNRA), nitrate-dependent Fe oxidation (NDFO), and ferric ammonium oxidation (Feammox). In addition, the microbial community in R1 was more resistant to short-term low temperature (2–7 °C) starvation, illustrating a further positive effect of adding Fe(II) during the startup phase of an Anammox reactor.