Anammox Research Articles

Novel three-sludge municipal wastewater treatment process coupling denitrifying phosphorus removal with anaerobic ammonium oxidation

The two-sludge anoxic dephosphation (DEPHANOX) process frequently encounters the challenge of elevated effluent ammonia levels in practical applications. In this study, the anaerobic ammonium oxidation (anammox) biofilm was introduced into the DEPHANOX system, transforming it into a three-sludge system, enabling synchronous nitrogen and phosphorus elimination, particularly targeting ammonia. Despite a chemical oxygen demand/total nitrogen ratio of 4.3 ± 0.8 in the actual municipal wastewater and 4.5 h of aeration, the effluent total nitrogen was 13.7 mg/L, lower than the parallel wastewater treatment plant. Additionally, the effluent ammonia reduced to 5.1 ± 2.5 mg/L. Notably, denitrifying phosphorus removal and anammox were coupled in the anoxic zone, yielding 74.5 % nitrogen and 87.8 % phosphorus removal. 16S rRNA gene sequencing identified denitrifying phosphorus-accumulating organisms primarily in floc sludge (Saprospiraceae 7.07 %, Anaerolineaceae 1.95 %, Tetrasphaera 1.57 %), while anammox bacteria inhabited the biofilm (Candidatus Brocadia 4.00 %). This study presents a novel process for efficiently treating municipal wastewater.

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.

Research progress of anaerobic ammonium oxidation (Anammox) process based on integrated fixed-film activated sludge (IFAS).

The integrated fixed-film activated sludge (IFAS) process is considered one of the cutting-edge solutions to the traditional wastewater treatment challenges, allowing suspended sludge and attached biofilm to grow in the same system. In addition, the coupling of IFAS with anaerobic ammonium oxidation (Anammox) can further improve the efficiency of biological denitrification. This paper summarises the research progress of IFAS coupled with the anammox process, including partial nitrification anammox, simultaneous partial nitrification anammox and denitrification, and partial denitrification anammox technologies, and describes the factors that limit the development of related processes. The effects of dissolved oxygen, influent carbon source, sludge retention time, temperature, microbial community, and nitrite-oxidising bacteria inhibition methods on the anammox of IFAS are presented. At the same time, this paper gives an outlook on future research focus and engineering practice direction of the process.

Granulation characteristics of anammox sludge in response to different signal-molecule-stimulants; mediated through programmed cell death

During the anammox process, mitigation of biomass washout to increase sludge retention is an important parameter of process efficiency. Signal molecular stimulants (SMS) initiate the sludge granulations controlled by programmed cell death (PCD) of microorganisms. In this study, the aerobic granular sludge (AGS), cell fragments, extracellular polymeric substances (EPS), and AGS process effluent were tested as SMS to identify their effect on anammox granulation. The results showed that the addition of SMS increased the nitrogen removal efficiency to varying degrees, whereas the addition of AGS process supernatant, as SMS, increased the ammonia removal efficiency up to 96%. The addition of SMS was also found to increase EPS production and contributed to sludge granulation. In this process, the proportion of PCD increased and both Gaiella and Denitratisoma abundance increased from 3.54% to 5.59%, and from 1.8% to 3.42%, respectively. In conclusion, PCD was found important to increase anaerobic ammonia oxidation performance through the granulation mechanism.

Nitrogen loss in coastal sediments driven by anaerobic ammonium oxidation coupled to microbial reduction of Mn(IV)-oxide

Coastal sediments play a central role in regulating the amount of land-derived reactive nitrogen (Nr) entering the ocean, and their importance becomes crucial in vulnerable ecosystems threatened by anthropogenic activities. Sedimentary denitrification has been identified as the main sink of Nr in marine environments, while anaerobic ammonium oxidation with nitrite (anammox) has also been pointed out as a key player in controlling the nitrogen pool in these locations. Collected evidence in the present work indicates that the microbial biota in coastal sediments from Baja California (northwestern Mexico) has the potential to drive anaerobic ammonium oxidation linked to Mn(IV) reduction (manganammox). Unamended sediment showed ammonification, but addition of vernadite (δMnO2 with nano-crystal size ∼15 Å) as terminal electron acceptor fueled simultaneous ammonium oxidation (up to ∼400 μM of ammonium removed) and production of Mn(II) with a ratio ∆[Mn(II)]/∆[NH4+] of 1.8, which is very close to the stoichiometric value of manganammox (1.5). Additional incubations spiked with external ammonium also showed concomitant ammonium oxidation and Mn(II) production, accounting for ∼30 % of the oxidized ammonium. Tracer analysis revealed that the nitrogen loss associated with manganammox was 4.2 ± 0.4 μg 30N2/g-day, which is 17-fold higher than that related to the feammox process (anaerobic ammonium oxidation linked to Fe(III) reduction, 0.24 ± 0.02 μg 30N2/g-day). Taxonomic characterization based on 16S rRNA gene sequencing revealed the existence of several clades belonging to Desulfobacterota as potential microorganisms catalyzing the manganammox process. These findings suggest that manganammox has the potential to be an additional Nr sink in coastal environments, whose contribution to total Nr losses remains to be evaluated.

Denitrification dominates dissimilatory nitrate reduction across global natural ecosystems.

Denitrification, anaerobic ammonium oxidation (anammox), and dissimilatory nitrate reduction to ammonium (DNRA) are three competing processes of microbial nitrate reduction that determine the degree of ecosystem nitrogen (N) loss versus recycling. However, the global patterns and drivers of relative contributions of these N cycling processes to soil or sediment nitrate reduction remain unknown, limiting our understanding of the global N balance and management. Here, we compiled a global dataset of 1570 observations from a wide range of terrestrial and aquatic ecosystems. We found that denitrification contributed up to 66.1% of total nitrate reduction globally, being significantly greater in estuarine and coastal ecosystems. Anammox and DNRA could account for 12.7% and 21.2% of total nitrate reduction, respectively. The contribution of denitrification to nitrate reduction increased with longitude, while the contribution of anammox and DNRA decreased. The local environmental factors controlling the relative contributions of the three N cycling processes to nitrate reduction included the concentrations of soil organic carbon, ammonium, nitrate, and ferrous iron. Our results underline the dominant role of denitrification over anammox and DNRA in ecosystem nitrate transformation, which is crucial to improving the current global soil N cycle model and achieving sustainable N management.

Looking deeper into the effects of scouring and aeration on membrane aerated biofilms: Analysis of nitrogen conversion, oxygen profiles and nitrous oxide emissions

This study investigated the effects of aeration and scouring strategies on the performance of Membrane Aerated Biofilm Reactors (MABRs) and the distribution of oxygen and nitrous oxide in the biofilm. Four flat sheet MABRs were operated with synthetic feed under different conditions: two with intermittent aeration (iMABR) and two with continuous aeration (cMABR). Scouring was induced by bubbling dinitrogen gas through the reactor bulk at low and high frequencies (LF and HF). In the iMABRs, a partial nitritation biofilm initially developed, but the biofilm adapted to the aeration strategy over time and became nitrifying. The cMABRs directly developed a nitrifying biofilm without a significant phase of partial nitritation. Limiting oxygen availability improved the overall performance with regards to total nitrogen (TN) removal by providing a better environment for anaerobic ammonium oxidation (Anammox) while limiting complete nitrification.Oxygen profiles were measured in the iMABR over time at different biofilms depths, showing that intermittent aeration led to various oxygen concentrations and temporal variations in the oxygen availabilities at different depths of the biofilm. Also, N2O emissions from the MABRs differed greatly between the different systems, but still remained lower compared to other reactor configurations for nitrogen removal, making the MABR technology a worthy alternative. The results showed large differences between the operating strategies of the MABRs and can help to gain more insight into the specific properties of MABRs for nitrogen removal.

Deciphering behaviors of 6:2 chlorinated polyfluorinated ether sulfonate (alternative-PFOS) on anammox processes: Nitrogen removal efficiency and microbial adaptability

This study investigates the behaviors and effects of F-53B, an alternative to perfluorooctane sulfonate on anaerobic ammonium oxidation (anammox) processes. Results showed that the nitrogen removal efficiency (NRE) reached 83.8 % at a F-53B concentration of 0.5 mg·L−1, while NRE decreased to 66.9 % with 5 mg·L−1 of F-53B. The defluorination rates of 17.8 % (0.5 mg·L−1) and 9.3 % (5 mg·L−1) were observed, respectively, suggesting the occurrence of F-53B degradation. The relative abundance of Ca. Kuenenia decreased from 26.1 % to 16.2 % with the F-53B concentration increasing from 0.5 mg·L−1 to 5 mg·L−1. Meanwhile, Denitratisoma was selectively enriched with a relative abundance of 40.7 % at an F-53B concentration of 0.5 mg·L−1. Ca. Kuenenia could reduce reactive oxygen species induced by F-53B to maintain the balance of oxidative stress. This study gains insight into the behaviors and metabolic mechanisms of F-53B in anammox consortia, suggesting the feasibility of anammox processes for industrial wastewater.

Nitrogen metabolism pathways and functional microorganisms in typical karst wetlands.

Aha Lake artificial reservoir wetland, Niangniang Mountain karst mountain wetland, and Caohai plateau lake wetland are typical karst wetlands in Guizhou Province with unique topography and geomorphic features. They were selected as research objects in this study to explore microorganisms and functional genes in nitrogen metabolism adopting macro-genome sequencing technology. It was found that Proteobacteria, Actinobacteria, and Acidobacteria were the dominant phyla in nitrogen metabolism in these three wetlands, similar to previous studies. However, at the genus level, there was a significant difference, with the dominant bacteria being Bradyrhizobium, Methylocystis, and Anaeromyxobacter. Six nitrogen metabolism pathways, including nitrogen fixation, nitrification, denitrification, dissimilatory nitrate reduction, assimilatory nitrate reduction, and complete nitrification, comammox, were revealed, but anaerobic ammonia oxidation genes were not detected. Nitrogen metabolism microorganisms and pathways were more affected by SOM, pH, NO3-, and EC in karst wetlands. This study further discussed microorganisms and functions of nitrogen metabolism in karst wetlands, which was of great significance to nitrogen cycles of karst wetland ecosystems.

Aerobic and anoxic utilization of organic matter for flexible nitrite supply in nutrient conversion pathways based on anaerobic ammonium oxidation: Microbial interactive mechanism

This study investigated nutrient conversion pathways and corresponding interactive mechanisms in a mainstream partial-nitritation (PN)/anaerobic ammonium oxidation (anammox)/partial-denitrification-(PD)-enhanced biological phosphorus-removal (EBPR) (PN/A/PD-EBPR) process. A laboratory-scale sequencing batch reactor was operated for 301 days under different operational strategies. Mainstream PN/A/PD-EBPR was successfully operated with aerobic and anoxic utilization of organic matter. Aerobic utilization of organic matter was an effective strategy for conversion to denitrifying polyphosphate-accumulating organism-based phosphorus removal, referring to a biological reaction that outperformed nitrite-oxidizing bacteria. Aerobically adsorbed organic matter could be used as a carbon source for PD, which further enhanced nitrogen removal by PN/A. Ultimately, the interaction between complex nutrient conversion pathways served to achieve stable performance. High-throughput sequencing results elucidated the core microbe functioning in the mainstream PN/A/PD-EBPR process with respect to various nutrients. The outcomes of this study will be beneficial to those attempting to implement mainstream PN/A/PD-EBPR.

Roles of red mud-based biochar carriers in the recovery of anammox activity: characteristics and mechanisms.

Anaerobic ammonium oxidation (anammox) sludge is easily deactivated in the process of treating ammonia-laden wastewater. To investigate an effective recovery method, red mud-based biochar carriers (RMBC) were prepared and added to a deactivated anammox reactor; the operation of this reactor had been interrupted for 6months with starvation and low temperature. The deactivated sludge with added RMBC was recovered rapidly after 31days, with the specific anammox activity rapidly increasing to 0.84g N/(g VSS∙day), and the recovery efficiency of nitrogen removal rate increased by four times compared to the unadded control. The granulation degree and extracellular polymeric substances secretion of the anammox sludge with the added RMBC were significantly higher than that of the control group. In addition, a large number of spherical anammox bacteria were observed moored at the porous channels of RMBC, and the copy numbers of functional genes of anammox bacteria were approximately twice that of the control group. Hence, RMBC is a potential sludge activator, and it can provide a "house" to protect anammox bacteria, enhance the metabolic activity and the agglomerative growth of anammox bacteria, and synergistically achieve rapid recovery of deactivated anammox sludge.

Study on the impact of hydraulic loading rate (HLR) on removal of nitrogen under low C/N condition by modular moving bed constructed wetland (MMB-CW) system

Traditional constructed wetlands often exhibit low nitrogen removal efficiency when treating low C/N wastewater, prompting the need for targeted technological innovation. Modular moving bed constructed wetlands (MMB-CWs) have demonstrated superior performance, achieving over 15% higher TN removal compared to traditional subsurface flow wetlands, even without the addition of carbon, further research is required to fully understand the operational effects of different hydraulic loads and the underlying nitrogen removal mechanisms. To address this gap, the contrast performance of MMB-CWs with three different substrate addition ratios 90% (unit A), 60% (unit B), 30% (unit C) was studied under low C/N conditions and different hydraulic loading rates (HLRs). Results show that there are no significant differences in TN removal among the three systems at 250 mm/d and 500 mm/d. NH4+-N and NO3--N removal rates don't significantly decrease (p>0.05). High HLR leads to significantly lower effluent NO2--N concentrations. HLR minimally affects CODCr removal, with unit C exhibiting better resistance. Functional gene analysis reveals a minor role of nitrification, emphasizing the crucial role of anaerobic ammonium oxidation. The copy number of the Anammox gene is 1.2–4.0 times that of the NIR gene, with a larger ratio in the secondary unit, indicating that denitrification is mainly driven by Anammox. The dominant genera in the first unit are Azospira, Thauera, and Denitratisoma, while Defluviicoccus, Mycobacterium, and Nocardioides dominate the ceramsite and biochar of the second unit. The addition of biochar enhances the composition and abundance of microbial communities in the system, and as biochar addition increases, the removal rates of NH4+-N and TN gradually increase, ensuring effective denitrification under varying loads.

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.

Partial denitrifying phosphorus removal coupling with anammox (PDPRA) enables synergistic removal of C, N, and P nutrients from municipal wastewater: A year-round pilot-scale evaluation

Applying anaerobic ammonium oxidation (anammox) in municipal wastewater treatment plants (MWWTPs) can unlock significant energy and resource savings. However, its practical implementation encounters significant challenges, particularly due to its limited compatibility with carbon and phosphorus removal processes. This study established a pilot-scale plant featuring a modified anaerobic-anoxic-oxic (A2O) process and operated continuously for 385 days, treating municipal wastewater of 50 m3/d. For the first time, we propose a novel concept of partial denitrifying phosphorus removal coupling with anammox (PDPRA), leveraging denitrifying phosphorus-accumulating organisms (DPAOs) as NO2− suppliers for anammox. 15N stable isotope tracing revealed that the PDPRA enabled an anammox reaction rate of 6.14 ± 0.18 μmol-N/(L·h), contributing 57.4 % to total inorganic nitrogen (TIN) removal. Metagenomic sequencing and 16S rRNA amplicon sequencing unveiled the co-existence and co-prosperity of anammox bacteria and DPAOs, with Candidatus Brocadia being highly enriched in the anoxic biofilms at a relative abundance of 2.46 ± 0.52 %. Finally, the PDPRA facilitated the synergistic conversion and removal of carbon, nitrogen, and phosphorus nutrients, achieving remarkable removal efficiencies of chemical oxygen demand (COD, 83.5 ± 5.3 %), NH4+ (99.8 ± 0.7 %), TIN (77.1 ± 3.6 %), and PO43− (99.3 ± 1.6 %), even under challenging operational conditions such as low temperature of 11.7 °C. The PDPRA offers a promising solution for reconciling the mainstream anammox and the carbon and phosphorus removal, shedding fresh light on the paradigm shift of MWWTPs in the near future.

Microbial divergence and evolution. The case of anammox bacteria.

Species differentiation and the appearance of novel diversity on Earth is a major issue to understand the past and future of microbial evolution. Herein, we propose the analysis of a singular evolutive example, the case of microorganisms carrying out the process of anammox (anaerobic ammonium oxidation). Anammox represents a singular physiology active on Earth from ancient times and, at present, this group is still represented by a relatively limited number of species carrying out a specific metabolism within the Phylum Planctomycetota. The key enzyme on the anammox pathway is hydrazine dehydrogenase (HDH) which has been used as a model in this study. HDH and rRNA (16S subunit) phylogenies are in agreement suggesting a monophyletic origin. The diversity of this singular phylogenetic group is represented by a few enriched bacterial consortia awaiting to be cultured as monospecific taxa. The apparent evolution of the HDH genes in these anammox bacteria is highly related to the diversification of the anammox clades and their genomes as pointed by phylogenomics, their GC content and codon usage profile. This study represents a clear case where bacterial evolution presents a paralleled genome, gene and species diversification through time from a common ancestor; a scenario that most times is masked by a web-like phylogeny and the huge complexity within the prokaryotes. Besides, this contribution suggests that microbial evolution of the anammox bacteria has followed an ordered, vertical diversification through Earth history and will present a potentially similar speciation fate in the future.

Insight into microbial adaptability in continuous flow anaerobic ammonium oxidation process for low-strength sewage treatment

Organic matter concentration is a critical factor influencing the adaptability of anaerobic ammonium oxidation (anammox) bacteria to low-strength sewage treatment. To address this challenge and achieve stable anammox activity, a micro-aeration partial nitrification-anammox process was developed for continuous-flow municipal sewage treatment. Under limited ammonium conditions, the effective utilization of organics in denitrification promoted the stable accumulation of nitrite and enhanced anammox activity. This, in turn, led to enhanced nitrogen removal efficiency, reaching approximately 87.7%. During the start-up phase, the protein content of extracellular polymeric substances (EPS) increased. This enhanced EPS intensified the inhibitory effect of denitrifying bacteria (DNB) on nitrite-oxidizing bacteria through competition for nitrite, thereby facilitating the proliferation of anammox bacteria (AnAOB). Additionally, several types of DNB capable of utilizing slowly biodegradable organics contributed to the adaptability of AnAOB. These findings provide valuable insights for ensuring efficient anammox performance and robust nitrogen removal in the treatment of low-strength sewage.

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.

Ammonium-Enhanced Arsenic Mobilization from Aquifer Sediments.

Ammonium-related pathways are important for groundwater arsenic (As) enrichment, especially via microbial Fe(III) reduction coupled with anaerobic ammonium oxidation; however, the key pathways (and microorganisms) underpinning ammonium-induced Fe(III) reduction and their contributions to As mobilization in groundwater are still unknown. To address this gap, aquifer sediments hosting high As groundwater from the western Hetao Basin were incubated with 15N-labeled ammonium and external organic carbon sources (including glucose, lactate, and lactate/acetate). Decreases in ammonium concentrations were positively correlated with increases in the total produced Fe(II) (Fe(II)tot) and released As. The molar ratios of Fe(II)tot to oxidized ammonium ranged from 3.1 to 3.7 for all incubations, and the δ15N values of N2 from the headspace increased in 15N-labeled ammonium-treated series, suggesting N2 as the key end product of ammonium oxidation. The addition of ammonium increased the As release by 16.1% to 49.6%, which was more pronounced when copresented with organic electron donors. Genome-resolved metagenomic analyses (326 good-quality MAGs) suggested that ammonium-induced Fe(III) reduction in this system required syntrophic metabolic interactions between bacterial Fe(III) reduction and archaeal ammonium oxidation. The current results highlight the significance of syntrophic ammonium-stimulated Fe(III) reduction in driving As mobilization, which is underestimated in high As groundwater.

Analyzing the nitrogen removal performance and cold adaptation mechanism of immobilized cold-acclimation ANAMMOX granules at low temperatures.

To improve the treatment performance of anaerobic ammonium oxidation (ANAMMOX) processes at low temperatures, the immobilized cold-acclimation ANAMMOX granules (R3) were prepared and their low-temperature nitrogen removal ability as well as the cold adaptation mechanism were analyzed. The results indicated that the total inorganic nitrogen (TIN) removal efficiency of R3 was significantly higher than that of R2 (cold-acclimation granules without immobilization) and R1 (common granules), especially at 11 ± 2 and 7±2°C (68% and 54%). These were attributed to the remarkable biomass retention capacity of R3, high up to 4.3-4.9mg/gVSS even at 5-18°C. Besides, higher protein (PN) content of tightly bound extracellular polymeric substances (TB-EPS) also facilitated microbial aggregation in R3. Meanwhile, R3 granules retained higher ANAMMOX activity and heme c content at 5-25°C. The original dominant ANAMMOX genus (Candidatus Kuenenia) in R3 kept higher abundance (49%-57%) at 23 ±2 and 16 ±2°C, whereas Candidatus Brocadia became the dominant ANAMMOX genus (25%-32%) in R3 at 11 ±2 and 7±2°C. Notably, different ANAMMOX genera in R3 may adapt to cold environment by regulating the expression of cold-stress proteins (CspA, CspB, PpiD, and UspA). PRACTITIONER POINTS: Immobilized cold-acclimation ANAMMOX granules showed higher nitrogen removal efficiency at 23°C → 5°C. Immobilization method effectively retained biomass (Candidatus Kuenenia and Candidatus Brocadia). Immobilization facilitated TB-EPS release and biological aggregation in cold-acclimation granules. Expression of cold-stress proteins in immobilized cold-acclimation granules was more active.

FAST START-UP MARINE ANAMMOX PROCESS USING INTENSIVE SHRIMP POND SOLID WASTE AS INOCULUM IN FILTER BIOREACTOR

Intensive shrimp culture waste contains high nitrogen, which can reduce water quality and environmental-carrying capacity. Anaerobic ammonium oxidation (anammox) is a potential and economical nitrogen removal process. However, limited information is available on the application of anammox process in aquaculture. The purpose of this study was to start up an anammox process in two filter bioreactors (FtBR) based on different inoculums. Run 1 used intensive shrimp solid waste, and Run 2 used intensive shrimp solid waste and granular of fresh anammox bacteria belonging to Candidatus Brocadia fulgida. Ammonium and nitrite concentrations of 70-100 mg N L-1 each were added to filtered and sterilized seawater as synthetic wastewater and flowed to the reactor with HRT 24-hour. After 120 days, the maximum ammonium conversion efficiency (ACE), nitrogen removal efficiency (NRE), and nitrogen removal rate (NRR) for Run 1 and 2 were 82.48%, 72.58%, 0.12 kg N (m3·d)-1 and 83.06%, 63.59%, 0.10 kg N (m3·d)-1, respectively. Experiment nitrogen stoichiometric ratios of NH4+:NO2-:NO3- were 1:1.40:0.12 and 1:1.13:0.14, which were close to the stoichiometry of the anammox process. Fast start-up of anammox process was achieved using intensive shrimp solid waste as inoculum. Anammox can be a new method for developing intensive shrimp culture wastewater treatment.