Anammox Activity Research Articles

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

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

Sulfide addition accelerates anammox sludge granulation and promotes microbial cooperation

The granular anaerobic ammonium oxidation (anammox) system has attractive advantages in tolerance to environmental-stress and enhancement of nitrogen removal capacity. Sulfide addition can improve nitrogen removals in anammox systems via inducing sulfur denitrification, yet its function in the improvement of the property of anammox granular sludge remains unclear. Herein, we investigated the variations in the morphological and microbial properties of the anammox sludge response to different sulfide concentrations (Na2S: 10–100 mg/L) through a long-term experiment. By comparing the sludge diameter and heme c content, it comes that a relatively low sulfide (S/N [nitrate] molar ratio of 0.18–0.50) significantly promoted the average diameter and heme c concentration of sludge by 25–175 % and 75–95 %, respectively, compared to that of both without sulfide addition and a high sulfide addition (S/N > 0.85). This enhancement is primarily because a low amount of sulfide had stimulated the secretion of extracellular polymeric substance, induced slight biogenic sulfur accumulation as microbial nuclei, and facilitated the appropriate amount of filamentous bacteria proliferation. Microbial metabolism functions analyses revealed a robust granular anammox coupled with sulfur denitrification in the sulfide-mediated anammox reactor, and the assembled granules exhibited exceptional tolerance to environmental stress. Significantly, the anammox bacteria (Candidatus_Brocadia) dominating the granules displayed satisfactory anammox activity (21.8 ± 2.1 mg N/g VSS h), and their produced nitrate was efficiently removed by the sulfur-oxidizing bacteria (Thiobacillus) that predominantly occurred in the flocs. This collaboration ensured an efficient sulfide-mediated anammox granules system, achieving nitrogen removal efficiency exceeding 95 %. These results highlight the function of sulfide in improving the morphological property of anammox sludge as well as the creation of a favorable ecological niche for the functional microorganism, which is important to maintain the efficiency and robustness of the anammox process in treating wastewater.

Comparation on the responses and resilience of single-anammox system and synergistic partial-denitrification/anammox system to long-term nutrient starvation: Performance and metagenomic insights

Starvation disturbance was a common problem in biological sewage treatment processes. However, understanding about the responses and resilience of different active anammox biomass in autotrophic and heterotrophic systems to long-term nutrient starvation remains limited. This study compared responses and potential recovery mechanisms of autotrophic single-Anammox and heterotrophic synergistic partial-denitrification/anammox (PD/anammox) systems to prolonged starvation (31–40 days). After starvation, total inorganic nitrogen (TIN) removal efficiency of single-Anammox and synergistic PD/anammox systems decreased to 62.16 % and 78.52 %, respectively, of their original level. After the nutrient resupply, the performance of both systems gradually recovered to a similar-to-pre-starvation level at the rate of 1.26 %/day and 1.89 %/day, respectively. Compared with single-Anammox system, complex synergistic relationship of microorganisms and effective quorum sensing (QS) regulation strategies might mitigate the negative influences were caused by starvation and ensure the performance quickly return of synergistic PD/anammox system. This study would contribute to promote the application of Anammox technology.

Optimizing nitrogen removal in PD/A reactors: Effects of influent composition and temperature on system stability and microbial dynamics

This study investigates the performance and microbial community dynamics in two partial denitrification/anammox (PD/A) reactors with different influent wastewater compositions (differ in the presence/absence of NO2−) subjected to a controlled temperature gradient reduction from mesophilic (30 °C) to room temperature (20.92 °C) over 76 days. Two lab-scale PD/A reactors (R1 and R2), both operated with a total inorganic nitrogen (TIN) concentrations of 70 mg N/L. R1 maintained a NH4+/NO2−/NO3− ratio of 3:3:1 and a COD/NO3− ratio of 2.0, while R2 had an NH4+/NO3− ratio of 3:4, and COD/NO3− ratios of 2.0 and 2.5. Our findings reveal distinct responses to the temperature transitions: the optimization of the NH4+/NO2−/NO3− ratio at 3:3:1 facilitated more stable nitrogen removal as temperatures decreased. This stability can be attributed to the enhanced synchronization between anammox bacteria and denitrifiers, promoting a balanced bioconversion process that is less susceptible to temperature-induced disruptions. Notably, the specific anammox activity (SAA) in both reactors declined linearly with the decrease in temperature, but the relative abundance of anammox bacteria (Ca. Brocadia) in R1 increased from 2.1 % to 9.7 %. Furthermore, the percentage of anammox-related key genes was higher in R1 than in R2, suggesting a microbial mechanism underlying the stable performance of R1. These results underscore the significant impact of influent nitrogen composition on PD/A performance amid temperature gradients and highlight the critical role of optimizing influent ratios for maintaining efficient nitrogen removal. This study offers valuable insights into enhancing the stability of PD/A systems under varying thermal conditions.

Enhancing the nitrogen removal of anammox sludge by setting up novel redox mediators-mediated anammox process

Anaerobic ammonium oxidizing (anammox) bacteria have been proven weak-electroactive. However, the impact of exogenous anthraquinone-2,6-disulfonate (AQDS) on the anammox activity, although it usually plays essential roles in the life activities of many other electroactive microorganisms, is still unknown. Therefore, this study further explored the influences of AQDS on the anammox activity and the interaction mechanism with anammox bacteria, as well as the behaviors of NH4+, NO2−, and NO3−. The results showed that exogenous AQDS increased the ammonium and total nitrogen removal rates by 12.8% and 10.7%, respectively. Interestingly, the conversion from NO2− to NO3− was significantly reduced after adding AQDS, resulting in a 40.1% reduction in NO3− production of anammox process. In this study, we found for the first time that anammox bacteria could not only carry out the conventional anammox process but also perform a weak redox mediator-mediated anammox process, which could achieve the 1:1 consumption of NH4+ and NO2−. The redox mediator-mediated anammox process was related to an endogenous redox mediator (ERM) synthesized and secreted by anammox bacteria, whose redox midpoint potential was around −0.26 V (vs. Ag/AgCl). After adding AQDS, not only the ERM-mediated anammox process was enhanced, but also two novel redox mediator-mediated anammox processes were introduced, including the AQDS-mediated anammox process and ERM-AQDS-mediated anammox process. These three redox mediator-mediated anammox processes significantly improved the nitrogen removal performance of anammox bacteria and reduced energy consumption. These findings will help reduce the dependence of anammox technology on NO2−, reduce the cost of subsequent treatment of NO3−, and provide new visions for optimizing and applying anammox technology.

Polystyrene nanoparticles regulate microbial stress response and cold adaptation in mainstream anammox process at low temperature

Nanoplastic pollution has become one of the most pressing environmental issues, and its bioaccumulation in aquatic environment also causes a great difficulty in treatment. Therefore, this work investigated the microbial dynamics of mainstream anaerobic ammonia oxidizing (anammox) process to treat the wastewater containing typical nanoplastics, as well as the fate and regulation mechanism of polystyrene nanoparticles (PS-NPs) with different concentrations. The results showed that 0.1–0.5 mg L-1 of PS-NPs had no significant effect on the nitrogen removal efficiency (NRE). When the concentration of PS-NPs increased from 0.5 mg L-1 to 2 mg L-1, the NRE of R1 with PS-NPs decreased from 94.9 ± 2.3 % to 77.0 ± 1.6 %, while the control reactor R0 maintained a stable NRE. Notably, the relative abundance of Ca. Kuenenia decreased from 17.4 % to 14.8 %, and that of Ca. Brocadia slightly decreased from 5.9 % to 5.0 % in R1. In addition, PS-NPs induced oxidative stress in anammox consortia, leading to the significant increase in reactive oxygen species (ROS) and lactate dehydrogenase (LDH) as well as cell membrane damage. PS-NPs also downregulated the content of heme c and further inhibited anammox activity. Based on the molecular docking simulation and western blotting, cold shock proteins (CSPs) could bind to PS-NPs and reduce the performance of anammox processes at low temperatures.

Strategy to mitigate substrate inhibition in wastewater treatment systems

Global urbanization requires more stable and sustainable wastewater treatment to reduce the burden on the water environment. To address the problem of substrate inhibition of microorganisms during wastewater treatment, which leads to unstable wastewater discharge, this study proposes an approach to enhance the tolerance of bacterial community by artificially setting up a non-lethal high substrate environment. And the feasibility of this approach was explored by taking the inhibition of anammox process by nitrite as an example. It was shown that the non-lethal high substrate environment could enhance the nitrite tolerance of anammox bacterial community, as the specific anammox activity increasing up to 24.71 times at high nitrite concentrations. Moreover, the system composed of anammox bacterial community with high nitrite tolerance also showed greater resistance (two-fold) in response to nitrite shock. The antifragility of the system was enhanced without affecting the operation of the main reactor, and the non-lethal high nitrite environment changed the dominant anammox genera to Candidatus Jettenia. This approach to enhance tolerance of bacterial community in a non-lethal high substrate environment not only allows the anammox system to operate stably, but also promises to be a potential strategy for achieving stable biological wastewater treatment processes to comply with standards.

Synergetic effects of chlorinated paraffins and microplastics on microbial communities and nitrogen cycling in deep-sea cold seep sediments

Chlorinated paraffins (CPs) and microplastics (MPs) are commonly found in deep-sea cold seep sediments, where nitrogen cycling processes frequently occur. However, little is known about their combined effects on sedimentary microbial communities and nitrogen cycling in these environments. This study aimed to investigate the synergistic impacts of CPs and MPs on microbial communities and nitrogen cycling in deep-sea cold seep sediments through microcosm experiments. Our results demonstrated that the presence of CPs and MPs induced significant alterations in microbial community composition, promoting the growth of Halomonas. Furthermore, CPs and MPs were found to enhance nitrification, denitrification and anammox processes, which was evidenced by the higher abundance of genes associated with nitrification and denitrification, as well as increased activity of denitrification and anammox in the CPs and MPs-treatment groups compared to the control group. Additionally, the enhanced influence of CPs and MPs on denitrification was expected to promote nitrate-dependent and sulfate-dependent anaerobic oxidation of methane, thereby resulting in less methane released into the environment. These findings shed light on the potential consequences of simultaneous exposure to CPs and MPs on biogeochemical nitrogen cycling in deep-sea cold seep sediments.

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

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

Enhancing collaboration of anammox with heterotrophic microbes mediated selectively by iron of different valences: Activities balance, metabolic mechanism, and functional genes regulation

The partial denitrification/anammox (PD/A) process is receiving increasing attention due to its cost-effectiveness advantages. However, effective strategies to alleviate organic matter inhibition and promote anammox activity have been proven to be a big challenge. This study investigated the effects of three types of iron (nano zero-valent iron (nZVI), Fe(II), and Fe(III)) on the PD/A process. It is worth noting that nZVI of 5–50 mg/L and Fe(III) of 5–120 mg/L promoted both PD and anammox activity. Long-term intermittent addition of nZVI (50 mg/L) resulted in a nitrogen removal efficiency of 98.2% in the mixotrophic PD/A system driven by iron and organic matter. The contribution of anammox for nitrogen removal reached as high as 93.8%. The organic carbon demand decreased due to the external electron donor provided by nZVI for PD. Multiple Fe–N metabolic pathways, primarily involving ammonia oxidation by Fe(III) and nitrate reduction by nZVI, play a crucial role in facilitating nitrogen transformation. Conversely, the direct addition of 30–120 mg/L Fe (II) resulted in a significant decrease in pH to below 5.0 and severe inhibition of PD and anammox activity. Following prolonged operation in the presence of nZVI, it was demonstrated that there is an enhancing effect on robust nitrite production for anammox. This was accompanied by a remarkable up-regulation of genes encoding nitrate reductase and iron-transporting proteins dominated by Thauera. Overall, this study has provided an efficient approach for advanced nitrogen removal through organic- and iron-driven anammox processes.

Integration of batch assays and microbial community analysis for partial nitritation and anammox process monitoring

This study investigates the feasibility of using batch assays combined with microbial community analysis to control the partial nitritation/anammox (PN/A) process. A Moving Bed Sequencing Batch Biofilm Reactor (MBSBBR) was operated over 130 days with varying ammonia influent concentrations. The highest anammox activity (22.4 mg N/gVSS∙h) was observed at an influent concentration of 200 mg N-NH4/L, correlating with high total inorganic nitrogen (TIN) removal efficiency. However, increasing the ammonia influent concentration to 340 mg N/L led to decreased anammox and nitrifying bacteria activities and reduced TIN removal efficiency. Sequencing revealed Candidatus Brocadia as the predominant genus, with its abundance correlating with TIN removal performance. High correlation coefficients (above 0.76) among batch tests highlighted the interdependence of key bacterial activities in the PN/A, emphasizing the need for a balanced microbial community for effective nitrogen removal. Batch assays provided immediate data on metabolic activities, while sequencing offered comprehensive insights into microbial community structure. Integrating these methods allowed for a holistic understanding of the microbial dynamics and their impact on process stability. This approach enhances the accuracy of wastewater treatment monitoring, aiding in the development of effective management strategies for optimizing PN/A processes.

Rapid start-up of mainstream partial denitrification /anammox and enhanced nitrogen removal through inoculation of precultured biofilm for treating low-strength municipal sewage

This study investigated the rapid start-up of mainstream partial denitrification coupled with anammox (PD/A) and nitrogen removal performance by inoculating precultured PD/A biofilm. The results showed mainstream PD/A in the anaerobic-anoxic-aerobic (A2O) process was rapidly established within 30 days. Nitrogen removal efficiency (NRE) improved by 23.8 % contrasted to the traditional A2O process. The mass balance showed that anammox contribution to total nitrogen (TN) removal were maintained at 37.9 %∼55.7 %, and reducing hydraulic retention time (HRT) strengthened simultaneously denitrification and anammox activity. The microbial community showed that the dominant bacteria such as denitrifying bacteria (DNBs) and glycogen accumulating organisms (GAOs) both in biofilm and flocculent sludge (floc), integrating with anammox bacteria (AnAOB) in biofilm might lead to enhanced nitrogen removal. Overall, this study offered a fast start-up strategy of mainstream PD/A with enhanced nitrogen removal, which are valuable for upgradation and renovation of existed municipal wastewater treatment plants (WWTPs).

Increased anaerobic conditions promote the denitrifying nitrogen removal potential and limit anammox substrate acquisition within paddy irrigation and drainage units

Microbial nitrogen (N) removal is crucial for purifying surface water quality in paddy irrigation and drainage units (IDUs). However, the spatiotemporal microbial N removal potential characteristics within these IDUs and the effects of changing anaerobic conditions on this potential remain insufficiently studied. In this study, we investigated the microbial N removal potential of conventional rice-wheat rotation and anaerobically enhanced rice-crayfish rotation IDUs using field measurements, isotope tracing techniques, and quantitative PCR. Our findings reveal that paddy fields were identified as hotspots for anammox activity, contributing to 76.0 %–97.4 % of the total anammox N removal potential in the IDU, while denitrification processes in ditches accounted for 43.5 %–77.4 % of the IDU's denitrification potential. During the rice transplanting period, the anammox N removal potential peaked, representing 35.8 % and 71.8 % of the total anammox N removal potential of the paddy fields in rice-wheat and rice-crayfish IDUs, respectively. An increase in anaerobic conditions diminished the anammox N removal potential while amplifying denitrification capabilities. The N removal potential in paddy fields decreased with increasing depth, contrasting with the relative stability in ditches. Spatiotemporal fluctuations in N removal potentials within these units are influenced by Fe2+ concentration, carbon and N content, WFPS, and pH levels. This study provides a scientific basis for improving nitrogen removal and water quality treatment in IDUs.

Start-up strategy of single-stage partial nitrification-anammox process for anaerobic digestion effluent

The objective of this study was to improve the nitrogen removal efficiency and reduce the start-up period of a single-stage partial nitritation-anammox (SPNA) system using iron particle-integrated anammox granules (IP-IAGs). Anammox granules were enriched in sequencing batch and expanded granular sludge bed (EGSB) reactors. The EGSB reactor produced larger and more uniform granules with higher specific anammox activity. IP-IAGs were then inoculated into a two-stage partial nitritation-anammox reactor treating anaerobic digestion (AD) effluent, followed by an internal recirculation strategy to acclimate the granules to oxygen exposure for SPNA. Finally, the SPNA process operated to treat real AD effluent under optimal conditions of 0.05 L/min aeration intensity (0.01 vvm) and 24 h of hydraulic retention time, achieving TNRE of 86.01 ± 2.64 % and nitrogen removal rate of 0.74 ± 0.04 kg-N/m3·d for 101 d.

Effect of long-term dosage of hydrazine on mainstream anammox process: Biofilm characteristics and microbial community

The impact of the long-term trace hydrazine (N2H4) exogenous supplementation on activity of the anaerobic ammonium oxidation (anammox) biofilm was investigated in a moving bed biofilm reactor (MBBR) for mainstream wastewater treatment. The results of this study demonstrated that the addition of 2–5 mg/L N2H4 enhanced anammox biofilm activity, as evidenced by the augmented nitrogen removal rate (NRR), which increased from 113.4 g/(m3·d) to 126.7 g/(m3·d) with the introduction of 2 mg/L N2H4. However, a higher concentration of N2H4 (10 mg/L) suppressed anammox activity, leading to a reduced NRR of 91.5 g/(m3·d). Bioindicators revealed that the long-term addition of 2 mg/L N2H4 fostered the accumulation of anammox bacteria (AnAOB) biomass, elevating the volatile suspended solids (VSS) content by 12%. Moreover, the structural composition of extracellular polymeric substances (EPS) within the biofilm was altered, resulting in enhanced biofilm strength within the reactor. The protective mechanism of the biofilm was activated, and EPS secretion was stimulated by the continuous N2H4 supplementation. The introduction of an excess dosage of N2H4 led to alterations in the microbial communities, ultimately resulting in a decline in the performance of the reactor. These findings collectively illustrate that N2H4, as an intermediate product, can effectively enhance anammox activity within the MBBR for mainstream wastewater treatment. This study contributes to the understanding of the optimization strategies for anammox processes in wastewater treatment systems.

Low-cost, reliable, and highly efficient removal of COD and total nitrogen from sewage using a sponge-filled trickling filter.

Development of low-cost and reliable reactors demanding minimal supervision is a need-of-the-hour for sewage treatment in rural areas. This study explores the performance of a multi-stage sponge-filled trickling filter (SPTF) for sewage treatment, employing polyethylene (PE) and polyurethane (PU) media. Chemical oxygen demand (COD) and nitrogen transformation were evaluated at hydraulic loading rates (HLRs) ranging from 2 to 6 m/d using synthetic sewage as influent. At influent COD of ∼350 mg/L, PU-SPTF and PE-SPTF achieved a COD removal of 97% across all HLRs with most of the removal occurring in the first segments. Operation of PE-SPTF at an HLR of 6 m/d caused substantial wash-out of biomass, while PU-SPTF retained biomass and achieved effluent COD < 10 mg/L even at HLR of 8-10 m/d. The maximum Total Nitrogen removal by PE-SPTF and PU-SPTF reactors was 93.56 ± 1.36 and 92.24 ± 0.66%, respectively, at an HLR of 6 m/d. Simultaneous removal of ammonia and nitrate was observed at all the HLRs in the first segment of both SPTFs indicating ANAMMOX activity. COD removal data, media depth, and HLRs were fitted (R2 > 0.99) to a first-order kinetic relationship. For a comparable COD removal, CO2 emission by PU-SPTF was 3.5% of that of an activated sludge system.

Deciphering the anammox microbial community succession with humic acid exposure to optimize large anammox granules for robust nitrogen removal

The robustness of the anaerobic ammonia oxidation (anammox) process in treating wastewater with high concentrations of humic acids (HAs), including landfill leachate and sludge anaerobic digestion liquid, has been paid great attention. This study revealed that the anammox sludge granule size of 1.0–2.0 mm could be robust under the HA exposure with high concentrations. The total nitrogen removal efficiency (NRE) was 96.2% at the HA concentration of 20–100 mg/L, while the NRE was 88.5% at the HA concentration of 500 mg/L, with reduced by 7.7%. The increased extracellular polymeric substances (EPS) content which was stimulated by the HA exposure favored the formation of large granules (1.0–2.0 mm) by enveloping medium and micro granules (0.2–1.0 mm). The abundance of anammox bacteria Candidatus Brocadia was found to be higher (14.2%) in large anammox granules sized 1.0–2.0 mm, suggesting a potentially high anammox activity. However, the abundance of denitrifiers Denitratisoma increased by 4.3% in ultra-large anammox granules sized >2.0 mm, which could be attributed to the high EPS content for heterotrophic denitrifiers metabolism as organic matter. The feedback mechanism of the anammox community for maintaining the ecological function under the HA exposure resulted in a closely related microbial community, with positive and negative correlations in the ecological network increased by 64.3%. This study revealed that the HA exposure of the anammox system resulted in the anammox granules of 1.0–2.0 mm size being the dominant granules with robust nitrogen removal, providing significant guidance for the optimization of anammox granules for an efficient treatment of HA-containing wastewater in anammox applications.

Evidence of Potential Anammox Activities from Rice Paddy Soils in Microaerobic and Anaerobic Conditions.

Anammox, a reaction in which microorganisms oxidize ammonia under anaerobic conditions, is used in the industry to remove ammonium from wastewater in an environmentally friendly manner. This process does not produce intermediate products such as nitrite or nitrate, which can act as secondary pollutants in soil and water environments. For industrial applications, anammox bacteria should be obtained from the environment and cultivated. Anammox bacteria generally exhibit a slow growth rate and may not produce a large number of cells due to their anaerobic metabolism. Additionally, their habitats appear to be limited to specific environments, such as oxidation-reduction transition zones. Consequently, most of the anammox bacteria that are used or studied originate from marine environments. In this study, anammox bacterial evidence was found in rice paddy soil and cultured under various conditions of aerobic, microaerobic, and anaerobic batch incubations to determine whether enrichment was possible. The anammox-specific gene (hzsA) and microbial community analyses were performed on the incubated soils. Although it was not easy to enrich anammox bacteria due to co-occurrence of denitrification and nitrification based on the chemistry data, potential existence of anammox bacteria was assumed in the terrestrial paddy soil environment. For potential industrial uses, anammox bacteria could be searched for in rice paddy soils by applying optimal enrichment conditions.

The effects of graphene on low-temperature anammox process: The insights from short-term tests and long-term operation

Effluent quality deterioration caused by seasonal low temperature is a great challenge to the application of anammox technology. Here, the effects of different graphene materials on anammox process were investigated under both optimal temperature and low-temperature. The batch tests showed that at 30 °C, 300 mg/L of reduced graphene oxide‑sodium alginate gel (RGOSA) had the most significant promoting effect, reaching nitrogen removal efficiency (NRE) and nitrogen removal rate (NRR) of 95 % and 8.88 mgN/L/d, respectively. The changes of EPS secretion patterns and increasing of key enzymes activity might contribute to the enhanced anammox activity. During the long-term operation of anammox reactor, the NRE and NRR of the reactor decreased when the temperature dropped to 15 °C, showing an NRE of 50 %–57 % with the addition of 200 mg/L of reduced graphene oxide (RGO) and 40 %–45 % with the addition of 20 mg/L of RGO. Furthermore, specific anammox activity (SAA) of the RGO200 reactor at 15 °C increased by 57.1 % compared to the UASB reactor without graphene addition. Additionally, 16S rRNA and metagenomic analysis results revealed anammox bacteria Ca. Kuenenia was the dominant bacteria. Moreover, the RGO can significantly increase the relative abundance of N-converting functional genes. This study demonstrates the graphene materials can help anammox process adapting to low temperatures, providing a possible solution for the application of anammox technology.

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

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