Anaerobic Ammonium-oxidizing Bacteria Research Articles

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.

Hydrogeochemical differences drive distinct microbial community assembly and arsenic biotransformation in unconfined and confined groundwater of the geothermal system

High‑arsenic (As) groundwater in geothermal aquifers poses a serious threat to public health. Assembly processes governing groundwater microbial community related to As biotransformation are still unexplored in geothermal groundwater across different aquifers. To fill this gap, groundwater microorganisms, community assembly processes, and microbially metabolic coupling of carbon (C), nitrogen (N), phosphorus (P), sulfur (S), and arsenic (As) were investigated in unconfined and confined groundwater in the thermal reservoirs of the Guide Basin. The difference in groundwater hydrogeochemicals led to the heterogeneity of the microbial community and microbially mediated C, N, P, S, and As cycling between unconfined and confined groundwater. Higher temperature and As concentrations, low nutrient supply, and reduced conditions in confined groundwater supported stronger interspecific coexistence and environmental selection, thus promoting the proliferation of As-resistant microorganisms (ARMs) and simplifying the community assemblage. Abundant available nutrient supply and oxidizing conditions supported an increased species diversity and metabolic functionality in unconfined groundwater. S oxidizers, C fixation, and C degradation bacteria potentially contributed to the decreased As concentrations in unconfined groundwater. However, ARMs, ammonification, and anaerobic ammonia-oxidizing bacteria potentially caused As mobilization in confined groundwater. Overall, our results give a comprehensive insight into the interaction between As and microorganisms in geothermal groundwater.

Advanced N removal from low C/N sewage via a plug-flow anaerobic/oxic/anoxic (AOA) process: Intensification through partial nitrification, endogenous denitrification, partial denitrification, and anammox (PNEnD/A)

Achieving low-cost advanced nitrogen (N) removal from municipal wastewater treatment plants (WWTPs) remains a challenge. A plug-flow anaerobic/oxic/anoxic (AOA) system with a mixtures bypass (MBP) integrating partial nitrification (PN), endogenous carbon denitrification (EnD), partial denitrification (PD), and anaerobic ammonium oxidation (Anammox), was constructed to treat actual sewage with a low C/N ratio. The effluent concentrations and removal efficiency of total inorganic nitrogen (TIN) during stable operation were 2.9 ± 0.9 mg/L and 93.1 ± 2.0 %, respectively. EnD was enhanced by the MBP through the efficient utilization of polyhydroxyalkanoates generated in the anaerobic zone. PD was promoted by the addition of carries and sodium acetate to the anoxic tank and the subsequent implantation of the Anammox biofilm successfully coupled PD/A. Stable PN was obtained with a satisfactory nitrite accumulation ratio of 92.6 %, facilitated by carriers and the introduction of hydroxylamine in the oxic zone. Mass balance analysis revealed that EnD and Anammox contributed 40.8 % and 48.2 % of TIN removal, respectively. The enrichment and synergistic effects of ammonia-oxidizing bacteria, denitrifying bacteria, glycogen-accumulating organisms, and anaerobic ammonia-oxidizing bacteria formed a diverses bacterial basis for the establishment of PN, EnD, PD, and Anammox (PNEnD/A) in the AOA system. The successful integration of PNEnD/A into the AOA process provides an innovative approach for low-cost advanced N removal in WWTPs.

Insights into the individual and combined effects of Cu(Ⅱ) and Ni(Ⅱ) on anammox: Nitrogen removal performance, enzyme activity and microbial community

Anaerobic ammonium oxidation (anammox) is an efficient and economical nitrogen removal process for treating ammonium-rich industrial wastewaters. However, Cu(Ⅱ) and Ni(Ⅱ) present in industrial wastewaters are toxic to anaerobic ammonium-oxidizing bacteria (AnAOB). Unfortunately, the effects of Cu(Ⅱ) and Ni(Ⅱ) on anammox have not been thoroughly investigated, especially when Cu(Ⅱ) and Ni(Ⅱ) coexist. This work comprehensively investigated the individual and combined effects of Cu(Ⅱ) and Ni(Ⅱ) on anammox and revealed the inhibitory mechanisms. With the influent NH4+-N and NO2−-N concentration of 230 and 250 mg L−1, the inhibition thresholds on anammox are 2.00 mg L−1 Cu(Ⅱ), 1.00 mg L−1 Ni(Ⅱ) and 1.00 mg L−1 Cu(Ⅱ) + 1.00 mg L−1 Ni(Ⅱ), and higher Cu(Ⅱ) or Ni(Ⅱ) concentrations resulted in sharp deteriorations of nitrogen removal performance. The inhibition of Ni(Ⅱ) on anammox was mainly attributed to the adverse effect on NiR activity, while the inhibition mechanism of Cu(Ⅱ) seemed to be unrelated to the four functional enzymes, but associated with disruption of cellular and organellar membranes. The behavior of extracellular polymeric substances (EPS) contributed to the antagonistic effect between Cu(Ⅱ) and Ni(Ⅱ) on anammox. In addition, the niche of Candidatus Brocadia and Candidatus Jettenia shifted under the Cu(II) and Ni(II) stress, and Candidatus Jettenia displayed greater tolerance to Cu(II) and Ni(II) stress. In conclusion, this research clarified the combined effect and the inhibitory mechanism of multiple heavy metals on anammox, and provide the guidances for anammox process application in treating high-ammonium industrial wastewaters containing heavy metals.

Effect of zero-valent iron (ZVI) on nitrogen and Fe(II) metabolism in anammox-hydroxyapatite (HAP) system under low-temperature stress

Low temperatures could inhibit the activity of anaerobic ammonium oxidizing bacteria (AnAOB). This study investigated the effect of exogenous zero-valent iron (ZVI) on Anammox-hydroxyapatite (HAP) system at 17 ± 1 °C. The results showed that long-term addition of ZVI increased the nitrogen removal efficiency by 8.59 %, and alleviated the inhibition of AnAOB under the condition of low temperature. After addition of ZVI, the activity of AnAOB and the heme c content increased by 58.20 % and 4.83 %, respectively. Moreover, the functional enzyme activities of NIR, HZS, and HZO increased by 94.12 %, 36.36 %, and 16.67 %, respectively. The concentrations of quorum sensing molecules, i.e., C6-HSL and C8-HSL, increased by 191.17 % and 41.54 %, respectively, after ZVI addition. Metagenomic analysis revealed that the addition of 5 g/L ZVI stimulated the relative abundance of Fe(II) metabolism-related genes in AnAOB at 17 ± 1 °C, thereby contributing to the uptake, transport, utilization, and storage of Fe(II). In summary, the addition of ZVI could improve the nitrogen removal performance of the anammox-HAP system at the low temperature.

Synergy between Nitrogen Removal and Fermentation Bacteria Ensured Efficient Nitrogen Removal of a Mainstream Anammox System at Low Temperatures.

Simultaneous partial nitrification, anammox, denitrification, and fermentation (SNADF) is a novel process achieving simultaneous advanced sludge reduction and nitrogen removal. The influence of low temperatures on the SNADF reactor was explored to facilitate the application of mainstream anammox. When temperature decreased from 32 to 16 °C, efficient nitrogen removal was achieved, with a nitrogen removal efficiency of 81.9-94.9%. Microbial community structure analysis indicated that the abundance of Candidatus Brocadia (dominant anaerobic ammonia oxidizing bacteria (AnAOB) in the system) increased from 0.03% to 0.18%. The abundances of Nitrospira and Nitrosomonas increased from 1.6% and 0.16% to 2.5% and 1.63%, respectively, resulting in an increase in the ammonia-oxidizing bacteria (AOB) to nitrite-oxidizing bacteria (NOB) abundance ratio from 0.1 to 0.64. This ensured sufficient nitrite for AnAOB, promoting nitrogen removal. In addition, Candidatus Competibacter, which plays a role in partial denitrification, was the dominant denitrification bacteria (DNB) and provided more nitrite for AnAOB, facilitating AnAOB enrichment. Based on the findings from microbial correlation network analysis, Nitrosomonas (AOB), Thauera, and Haliangium (DNB), and A4b and Saprospiraceae (fermentation bacteria), were center nodes in the networks and therefore essential for the stability of the SNADF system. Moreover, fermentation bacteria, DNB, and AOB had close connections in substrate cooperation and resistance to adverse environments; therefore, they also played important roles in maintaining stable nitrogen removal at low temperatures. This study provided new suggestions for mainstream anammox application.

Scavenging of reactive oxygen species in Candidatus Brocadia fulgida through nanocompartments

The antioxidant defense mechanisms for anaerobic ammonia oxidation (anammox) bacteria are still unclear. In this study, the potential antioxidant ability of nanocompartments in Candidatus Brocadia fulgida to typical reactive oxygen species (ROS) was investigated. The results showed that the copies of genes involved in anammox central metabolism were inhibited with hydrogen peroxide (H2O2), while the genes encoded putative anti-oxidative protein (nanocompartments and cargo HAO) up-regulated. The genetically engineered bacteria grew better and maintained the lower ROS levels (65.60 %-78.07 %) and higher electron transport activities (∼5–21 times) than the wild bacteria under H2O2 stimulus. Molecular docking confirmed that nanocompartment proteins could provide diverse sites to bind with H2O2 based on heme as the redox center. Additionally, the nanocompartments induced up-regulation of multiple protective pathways for coping with oxidative stress from H2O2, including antioxidant enzymes and other non-enzymatic pathways. Thus, the heme-containing nanocompartments presented great potential in preventing and relieving oxidative stress.

Exploring the promoting behavior of weak electric mediation on indole and pyridine biodegradation under anaerobic condition

Indole and pyridine, which are highly produced refractory compounds in the industrial wastewater, exhibit poor degradation capabilities in natural environments. In this study, we developed an anaerobic digestion system coupled with weak electric mediation (ED), and investigated the promoting effect of weak electricity on indole and pyridine biodegradation. The degradation characteristics were systematically explored, and the results showed that the degradation rate and mineralization of indole and pyridine were significantly enhanced, the production of CH4 was increased 1.4-fold, and the optimal voltages were 1.0 V and 0.8 V in the ED, respectively. Moreover, simultaneous removal of carbon and nitrogen was achieved. Gas chromatography–mass spectrometry analysis verified the transformation products, and possible pathways were proposed. Several byproducts of indole and pyridine were identified, with oxindole and glutaric dialdehyde being the main metabolites, respectively. Additionally, density functional theory (DFT) analysis was performed to investigated the radical indices and stabilities of the molecules to further confirm the degradation pathway. Microbial structure analysis demonstrated that the electrically mediated enhanced metabolism and activity of functional microbes, led to the promotion of indole and pyridine mineralization. Moreover, such species as degrading bacteria (Alicycliphilus, Shinella) and electroactive bacteria (Achromobacter), anaerobic ammonia-oxidizing bacteria (SM1A02), and denitrifying bacteria (Thiobacillus) coexisted. This study demonstrates that weak electric mediation is a promising methodology for enhancing the removal of indole and pyridine from wastewater under anaerobic conditions.

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.

Magnetite addition reduces nitrite requirement for efficient anaerobic ammonium oxidation by facilitating mutualism of ANAMMOX and FEAMMOX bacteria

Partial nitrification (PN) is crucial for anaerobic ammonium oxidation (ANAMMOX), but faces challenges such as high energy demands and process control. Recent research has highlighted additives like magnetite as potential alternatives to conventional electron acceptors (O₂ and NO₂−) for enhancing ammonium (NH4+) oxidation with lower energy consumption. This study investigated the effect of adding 50 mg/L of magnetite to ANAMMOX reactors, resulting in improved nitrogen (N) removal efficiency. The magnetite-added ANAMMOX (M-ANA) reactor yielded N removal efficiencies of 71 %, 66 %, and 57 % for NH4+:NO2− molar ratios of 1:1.3, 1:0.8, and 1:0.5, respectively. The M-ANA reactor operated under a 0.5 mol lower NO2− concentration achieved similar performance to the control ANAMMOX (C-ANA) reactor operated with a theoretical amount of NO2−. Moreover, the M-ANA reactor showed the potential to remove NH4+ by 56 % without any NO2− supplementation. Metagenomic analysis showed that the addition of magnetite significantly improved the relative abundance of microorganisms involved in the FEAMMOX reaction, such as Fimbriimonas ginsengisoli and Pseudomonas stutzeri. It also facilitated positive mutualism between ANAMMOX and FEAMMOX reactions. In addition, M-ANA granules exhibited a dense and compact structure compared with C-ANA, and the presence of magnetite facilitated the formation of resilient granules. Notably, the useful protein (Heme C) concentration and specific microbial activity in the M-ANA reactor were 1.3 and 2.2 times higher than those in the C-ANA reactor. Overall, the results demonstrate that an appropriate amount of magnetite can enhance the N removal efficiency while reducing the energy input requirements and associated carbon emissions. These findings can guide the future development of carbon- and energy-neutral N removal processes.

Coupling Nitrate-Dependent Anaerobic Ethane Degradation with Anaerobic Ammonium Oxidation.

The microbial oxidation of short-chain gaseous alkanes (SCGAs, consisting of ethane, propane, and butane) serves as an efficient sink to mitigate these gases' emission to the atmosphere, thus reducing their negative impacts on air quality and climate. "Candidatus Alkanivorans nitratireducens" are recently found to mediate nitrate-dependent anaerobic ethane oxidation (n-DAEO). In natural ecosystems, anaerobic ammonium-oxidizing (anammox) bacteria may consume nitrite generated from nitrate reduction by "Ca. A. nitratireducens", thereby alleviating the inhibition caused by nitrite accumulation on the metabolism of "Ca. A. nitratireducens". Here, we demonstrate the coupling of n-DAEO with anammox in a laboratory-scale model system to prevent nitrite accumulation. Our results suggest that a high concentration of ethane (6.9-7.9%) has acute inhibition on anammox activities, thus making the coupling process a significant challenge. By maintaining ethane concentrations within the range of 1.7-5.5%, stable ethane and ammonium oxidation, nitrate reduction, and dinitrogen gas generation without nitrite accumulation were finally achieved. After the accomplished coupling of n-DAEO with anammox, nitrate reduction rates increased by 8.1 times compared to the rate observed with n-DAEO alone. Microbial community profiling via 16S rRNA gene amplicon sequencing showed "Ca. A. nitratireducens" (6.6-12.9%) and anammox bacteria "Candidatus Kuenenia" (3.4-5.6%) were both dominant in the system, indicating they potentially form a syntrophic partnership to jointly contribute to nitrogen removal. Our findings offer insights into the cross-feeding interaction between "Ca. A. nitratireducens" and anammox bacteria in anoxic environments.

Dating Ammonia-Oxidizing Bacteria with Abundant Eukaryotic Fossils.

Evolution of a complete nitrogen (N) cycle relies on the onset of ammonia oxidation, which aerobically converts ammonia to nitrogen oxides. However, accurate estimation of the antiquity of ammonia-oxidizing bacteria (AOB) remains challenging because AOB-specific fossils are absent and bacterial fossils amenable to calibrate molecular clocks are rare. Leveraging the ancient endosymbiosis of mitochondria and plastid, as well as using state-of-the-art Bayesian sequential dating approach, we obtained a timeline of AOB evolution calibrated largely by eukaryotic fossils. We show that the first AOB evolved in marine Gammaproteobacteria (Gamma-AOB) and emerged between 2.1 and 1.9 billion years ago (Ga), thus postdating the Great Oxidation Event (GOE; 2.4 to 2.32 Ga). To reconcile the sedimentary N isotopic signatures of ammonia oxidation occurring near the GOE, we propose that ammonia oxidation likely occurred at the common ancestor of Gamma-AOB and Gammaproteobacterial methanotrophs, or the actinobacterial/verrucomicrobial methanotrophs which are known to have ammonia oxidation activities. It is also likely that nitrite was transported from the terrestrial habitats where ammonia oxidation by archaea took place. Further, we show that the Gamma-AOB predated the anaerobic ammonia-oxidizing (anammox) bacteria, implying that the emergence of anammox was constrained by the availability of dedicated ammonia oxidizers which produce nitrite to fuel anammox. Our work supports a new hypothesis that N redox cycle involving nitrogen oxides evolved rather late in the ocean.

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.