Convergent enrichment of core functional guilds involved in Fe-N metabolism in anammox and activated sludge: Insights into genome-resolved metagenomics.

Effects of the C/N ratio and bacterial populations on nitrogen removal in the simultaneous anammox and heterotrophic denitrification process: Mathematic modeling and batch experiments

Because of its economic benefits, the combined partial nitritation-anaerobic ammonium oxidation (PN-Anammox) process is increasingly adopted/recommended for efficient nitrogen removal, despite its application limitations such as the concomitant nitrate production. The discovery of denitrifying anaerobic methane oxidation (DAMO) processes offers a potential solution to overcome the limitations of the PN-Anammox process and enables complete/high-level nitrogen removal through coupling PN-Anammox-DAMO, which could be favourably supported in membrane biofilm reactor (MBfR) systems. However, this novel integrated process involves complex microbial interactions between functional microorganisms including ammonium-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), Anammox bacteria, DAMO archaea, and DAMO bacteria, which determine the treatment performance. Although mathematical modeling is widely applied to generate multifaceted analysis of emerging technologies, limited work has been dedicated to investigating the integrated PN-Anammox-DAMO process. Hence, this thesis aims to develop mathematical models to understand/evaluate the novel MBfR-based complete/high-level nitrogen removal technology with focus on the system performance and microbial interactions under different conditions of operations and reactor configurations. To this end, a mathematical model was firstly developed to describe the coupled Anammox-DAMO process in a lab-scale MBfR. The MBfR with an Anammox-DAMO dominated biofilm was fed with methane through gas-permeable membranes, while nitrate and ammonium fed in the bulk liquid outside the biofilm. The key stoichiometric and kinetic parameters of DAMO microorganisms were calibrated using the long-term dynamic experimental data, and the model was successfully validated using two independent batch tests at different operational stages of the MBfR. The developed model was then extended with nitrite inhibition terms and further verified by another sets of batch experimental data obtained from the MBfR with different practically applied feeding compositions (i.e., ammonium and nitrite/nitrate). The verified model was applied to assess the feasibility of achieving complete nitrogen removal by a partial nitritation reactor followed by an MBfR performing Anammox and DAMO. The optimum NO2-/NH4+ ratio produced from the preceding partial nitritation for the Anammox-DAMO MBfR was found to be 1.0 in order to achieve the maximum total nitrogen (TN) removal of over 99.0%, irrespective of the TN surface loading applied, while the corresponding optimal methane supply increased with the increasing TN surface loading, accompanied by the decreasing methane utilization efficiency. Through coupling the verified model with the well-established stoichiometry and kinetics of AOB and NOB, the feasibility of integrating PN-Anammox-DAMO into a one-stage MBfR for high-level nitrogen removal was tested through controlling the bulk liquid dissolved oxygen (DO) concentration in the system. The maximum TN removal was found to be achieved at a low bulk DO concentration (depending on process parameters), with AOB, Anammox bacteria, and DAMO microorganisms coexisting in the biofilm. The coupling PN-Anammox-DAMO process could potentially treat anaerobic digestion liquor through utilizing the dissolved methane remaining, avoiding the dissolved methane stripping and thus reducing the carbon footprint of wastewater treatment. A single-stage MBfR was therefore proposed for simultaneous ammonium and dissolved methane removal from side-stream anaerobic digestion liquor through integrating PN-Anammox-DAMO. In such an MBfR, ammonium and dissolved methane are provided in the bulk liquid, while oxygen via gas-permeable membranes. The previously verified model was applied to assess the MBfR system under different operational conditions. The simulation results demonstrated that both influent and oxygen surface loadings significantly influenced the TN and dissolved methane removal. The maximum simultaneous removal efficiencies of TN and dissolved methane could reach up to 96% and 98%, respectively, by adjusting the influent and oxygen surface loadings whilst maintaining a sufficient and suitable biofilm thickness (e.g., 750 mm). The counter-diffusional supply via the biofilm and the concentration gradients of substrates caused microbial stratification in the biofilm, where AOB attached close to the membrane surface where oxygen and ammonium were available, while Anammox and DAMO microorganisms jointly grew in the biofilm layer close to the bulk liquid where methane, ammonium, and nitrite were available with the latter produced by AOB. In addition to ammonium and dissolved methane, anaerobic digestion liquor might also contain sulfide which needs to be managed properly. A mathematical model was therefore developed through incorporating sulfide related metabolisms into the previously verified model and applied to evaluate the system performance and the associated microbial community structure of the single-stage MBfRs, which integrate desired microbial consortia to treat main-stream and side-stream anaerobic digestion liquors containing ammonium, dissolved methane, and sulfide simultaneously. The simulation results showed that the dissolved methane and sulfide remaining could be utilized as electron donors by DAMO bacteria and sulfide oxidizing bacteria (SOB), respectively, to further enhance the overall nitrogen removal. The high-level (g97.0%) simultaneous removal of ammonium, dissolved methane, and sulfide could be achieved by adjusting the influent and oxygen surface loadings. AOB, DAMO bacteria, aerobic methane-oxidizing bacteria (MOB), and SOB dominated the biofilm of the main-stream MBfR, while AOB, Anammox bacteria, DAMO bacteria, and SOB coexisted in the side-stream MBfR and cooperated to remove ammonium, dissolved methane, and sulfide simultaneously.

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The eutrophication of a body of water is a globally serious water-polluting problem caused by the excess discharge of nutrients. Nitrogen and phosphorus removal from wastewater is extremely necessary. The biological method is the major technology used for nitrogen removal. With the increasingly strict discharging standard worldwide, the problems of insufficient efficiency, considerable energy consumption, and secondary pollution involved in the traditional nitrogen removal process have become increasingly apparent, resulting in an urgent need for an economic, efficient, and sustainable technology. The widely investigated anaerobic ammonium oxidation (Anammox) process discovered in the 1990s is regarded as the most energy-efficient technology for nitrogen removal without aeration and organic carbon demand. However, it has not been widely adopted because for a long time now, it has faced bottlenecks in stable substrate nitrite (NO2−) acquisition. Recent studies have demonstrated that the NO2−-produced partial denitrification (PD) process provides a completely novel concept for solving this problem. China takes the lead in the investigation and practice of the discovery, development, and microbial mechanism of this process. A novel technology combining PD and Anammox is opening a new window for the low-carbon and high-efficiency treatment of municipal wastewater, which has been regarded as a hot and cutting-edge topic in this field. Compared with the conventional nitrification/denitrification process, it could save aeration energy by 100% and organic carbon source by 80% and reduce the sludge production and greenhouse gas (CO2, N2O) emission in the simultaneous treatment of municipal wastewater and nitrate (NO3−) contained in wastewater. In comparison with the present Anammox process, it is little affected by the substrate concentrate, temperature, and other environmental factors, could break the top limit of nitrogen removal efficiency by Anammox (89%), and reach up to 100% of the total nitrogen removal efficiency, thereby exhibiting great significance and applicable value. In the future, the separation, purification, and molecular metabolic mechanism of the PD and Anammox bacteria should be focused on. Furthermore, the engineering application of the novel technology should also be promoted.

Autotrophic ammonium removal by sulfate-dependent anaerobic ammonium oxidation (S-Anammox) process was studied in an upflow anaerobic sludge bed reactor inoculated with Anammox sludge. Over an operation period of 371 days, the reactor with a hydraulic retention time of 16 h was fed with influent in which NH4+ concentration was fixed at 70 mg N L-1, and the molar ratio of NO2-:NO3-:SO42- was 1:0.2:0.2, 0.5:0.1:0.3 and 0:0:0.5 in stages I, II and III, respectively. As the NO2- in influent was entirely replaced by SO42-, the NH4+ removal rate was 31.02 mg N L-1 d-1, and the conversion rate of SO42- was 8.18 mg S L-1 d-1. On grounds of the high NH4+:SO42- removal ratio (8.67:1), the S2- accumulation and pH drop in effluent, as well as the analysis results of microbial community structure, the S-Anammox process was speculated to play a dominant role in stage III. The NH4+ over-transformation was presumably as a consequence of the cyclic regeneration of SO42-. Concerning the microbial characteristics in the system, the Anammox bacteria (Candidatus Brocadia), sulfate-reducing bacteria (SRB) (Desulfatiglans and Desulfurivibrio) and sulfur-oxidizing bacteria (SOB) (Thiobacillus) in biomass was enriched in the case of without addition of NO2- in influent. Sulfate reduction driven ammonium anaerobic oxidation was probably attributed to the coordinated metabolism of nitrogen- and sulfur-utilizing bacteria consortium, in which Anammox bacteria dominates the nitrogen removal, and the SRB and SOB participates in the sulfur cycle as well as accepts required electrons from Anammox bacteria through a direct inter-species electron transfer (DIET) pathway.

The effects of adding organic carbon on the performance of different partial nitrification-anammox (PNA) process (the activated sludge process and biofilm process) were studied, especially nitrogen removal, functional microbial activity, and microbial community structure. The potential influences of quorum sensing (QS) on the nitrogen metabolism were also analyzed. The results showed that the addition of organic carbon in biofilm systems could reduce total nitrogen (TN) removal percentages, while in activated systems it could increase TN removal percentages. The TN removal percentages in SBBR-CN (the biofilm system with addition of organic carbon) and SBR-CN (the activated sludge system with addition of organic carbon) were 15% and 45%, respectively, and those in SBBR-N (the biofilm system without addition of organic carbon) and SBR-N (the activated sludge system without addition of organic carbon) were 75% and 21%, respectively. Batch experiments have proved that organic carbon inhibited the activities of nitrite-oxidizing bacteria (NOB) and anaerobic ammonia oxidation (anammox) bacteria, and organic carbon could promote the activity of denitrifying bacteria in activated sludge systems. Compared with activated sludge systems, biofilm systems could protect the activity of anammox bacteria. The relative abundances of ammonia oxidizing bacteria (AOB) and anammox bacteria were decreased, while the relative abundances of denitrifying bacteria (Thauera) were increased with the addition of organic carbon. The biofilm systems were more conducive to the growth of anammox bacteria. Metagenomics revealed that the same bacteria might be involved in different nitrogen metabolism, and nitrogen metabolism was achieved through the complex cooperation among functional bacteria. Besides, functional bacteria involving in the nitrogen metabolism had genes related to QS, indicating that QS might affect the nitrogen metabolism by regulating the functional bacteria activity. PRACTITIONER POINTS: PNA was achieved through SBBR and complete nitrification was achieved through SBR under the low ammonia nitrogen concentration condition. The effect of organic carbon on biofilm and activated sludge PNA process was different under the low ammonia nitrogen concentration condition. QS and QQ may affect the nitrogen removal performance by regulating the expression of nitrogen metabolism-related genes.

Start-up characteristics and microbial nitrogen removal mechanisms in ANAMMOX systems with different inoculations under prolonged starvation

BACKGROUND: This study was conducted to investigate the feasibility and performance of nitrogen removal through the complete autotrophic nitrogen removal over nitrite (CANON) process for saline wastewater in a continuous reactor, and to characterize microorganisms in the sludge from the reactor using DNA‐based techniques.RESULTS: The nitrogen removal experiment in the reactor was operated over five phases for 286 days treating a synthetic sewage of 1.2% salinity at 21–25 °C. At dissolved oxygen (DO) concentrations of 0.5–1.0 mg L−1 and in the presence of glucose, NO2− was accumulated, indicating the activity of ammonia‐oxidizing bacteria (AOB). At DO concentration of 0.5 mg L−1 without organic substrate, the anaerobic ammonium oxidation (Anammox) process was the major pathway responsible for nitrogen removal, with a total nitrogen removal of 70% and an ammonium conversion efficiency of 96%. A maximum ammonium removal rate of 0.57 kg‐N m−3 d−1 was achieved during the experimental period. The concentrations of AOB and Anammox bacteria were monitored over the operation of reactor using quantitative real‐time polymerase chain reaction (qRT‐PCR).CONCLUSION: In this study, autotrophic nitrogen removal process was achieved under salinity condition in a one‐reactor system. An over 100 fold increase of AOB was found due to the increased supply of ammonium at the beginning, then AOB concentration decreased temporarily in correspondence with the decreased DO, and the AOB resumed their concentration at the last phase. The Anammox bacteria abundance was about 150 fold higher than that at the beginning, indicating the successful enrichment of Anammox bacteria in the reactor. Copyright © 2010 Society of Chemical Industry

Community assembly and succession of the functional membrane biofilm in the anammox dynamic membrane bioreactor: Deterministic assembly of anammox bacteria.

Application of an enrichment culture of the marine anammox bacterium “Ca. Scalindua sp. AMX11” for nitrogen removal under moderate salinity and in the presence of organic carbon

Acidic partial nitritation (PN) has emerged to be a promisingly stable process in wastewater treatment, which can simultaneously achieve nitrite accumulation and about half of ammonium reduction. However, directly applying anaerobic ammonium oxidation (anammox) process to treat the acidic PN effluent (pH 4-5) is susceptible to the inhibition of anammox bacteria. Here, this study demonstrated the adaptation of anammox process to acidic pH in a moving bed biofilm reactor (MBBR). By feeding the laboratory-scale MBBR with acidic PN effluent (pH=4.6±0.2), the pH of an anammox reactor was self-sustained in the range of pH 5-6. Yet, a high total nitrogen removal efficiency of over 80% at a practical loading rate of up to 149.7±3.9mg N/L/d was achieved. Comprehensive microbial assessment, including amplicon sequencing, metagenomics, cryosection-FISH, and qPCR, identified that Candidatus Brocadia, close to known neutrophilic members, was the dominant anammox bacteria. Anammox bacteria were found present in the inner layer of thick biofilms but barely present in the surface layer of thick biofilms and in thin biofilms. Results from batch tests also showed that the activity of anammox biofilms could be maintained when subjected to pH 5 at a nitrite concentration of 10mg N/L, whereas the activity was completely inhibited after disturbing the biofilm structure. These results collectively indicate that the anammox bacteria enriched in the present acidic MBBR could not be inherently acid-tolerant. Instead, the achieved stable anammox performance under the acidic condition is likely due to biofilm stratification and protection. This result highlights the biofilm configuration as a useful solution to address nitrogen removal from acidic PN effluent, and also suggests that biofilm may play a critical role in protecting anammox bacteria found in many acidic nature environments.

Enhancement of the reactivation process of long-term starved anammox granular sludge with gravel balls: Microbial succession and metabolic impact

A two-stage partial nitritation (PN)-ANAMMOX process was successfully carried out for low-strength NH4+-N (50 mg·L-1) wastewater treatment at ambient/low temperatures. The results show that an average total nitrogen removal rate and removal efficiency above 0.6 kg·(m3·d)-1and 80% could be maintained, respectively, at temperatures between 20℃ and 14℃. The two-stage PN-ANAMMOX process was successful both under NO2--N-limited and NH4+-N-limited conditions. When the two-stage PN-ANAMMOX process was operated under NH4+-N-limited conditions, the "limit of technology" for nitrogen removal (TN<3 mg·L-1) could be easily achieved by following anoxic denitrification. Lowering the temperature to 12℃ resulted in the reduction of the total nitrogen removal rate to~0.5 kg·(m3·d)-1. Due to the low temperature, the anammox reaction became the rate-limiting step for nitrogen removal, while the PN reaction was not impacted. In the temperature range of 10-20℃, the activity-temperature coefficients (θ) of the PN granules and ANAMMOX sludge were 1.056 and 1.172, respectively, suggesting that the anammox bacteria have a higher temperature sensitivity than the ammonium oxidizing bacteria (AOB). Overall, the results clearly indicate that the nitrogen removal loading rate of the two-stage PN-ANAMMOX process is mainly controlled by the activity and quantity of anammox biomass. In contrast, the process nitrogen removal efficiency mainly depends on the performance of the first-stage PN (i.e., effluent NO2--N/NH4+-N ratio and NO3--N concentration) under a constant nitrogen removal loading rate (no overload). Based on these results, a hierarchically separate control strategy was proposed to obtain a high-rate nitrogen removal during the two-stage mainstream PN-ANAMMOX process.

Anaerobic ammonium oxidation (Anammox) process is a new type of biological nitrogen removal technology that is highly efficient, consumes low energy, and is cost-effective. However, from a practical perspective, there are operational problems involved with the technology, due to its special low temperature environmental conditions. As such, the technology is currently a key research direction in the field of sewage control engineering. This study investigated the effect of salinity on the performance of the anammox process at the stress of a low temperature (15℃) and the role salinity has on extracellular polymeric substance (EPS) secretion and by extension, anammox activated sludge. The study tested the technology used to adjust and control salinity at a low temperature. The study found that at a low temperature of 15℃, low salinity can promote the nitrogen removal efficiency of anammox bacteria. Low salinity can also activate anammox bacteria activity. However, in contrast with low salt concentrations, high salt concentrations can inhibit anammox activity. When the temperature was 15℃ and the salinity was 4 g/L, the nitrogen removal efficiency of the reactor was 1.79 times higher than in the environment with unadjusted salinity at 15℃. At a low temperature, as salinity increased, the water binding capacity and flocculation capacity of sludge also increased. Salinity can promote the secretion of EPS and changes its composition. Under low temperature stress, the concentration of salt was less than 12 g/L, and the anammox activity improved. However, a high salinity level significantly inhibited anammox activity.

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.