Anammox Granular Sludge Research Articles

Potential role of N-acyl homoserine lactone-mediated quorum sensing in the adaptation of anammox granular sludge system to salinity stress

Anammox granular sludge (AnGS) systems efficiently remove nitrogen from saline wastewater, but their adaptation mechanisms to salt stress are unclear. This study explores the adaptability of the AnGS system when exposed to salinity (0–30 g NaCl/L), focusing on the role of N-acyl homoserine lactone-mediated quorum sensing (AHL-QS) in microbial responses and community symbiosis under stress. Based on Hill model assessments, AnGS tolerates salt stress up to 15.73 g/L. Within this range, AnGS maintains cellular stability by enhancing extracellular polymeric substances (EPS) release, regulating oxidative stress; and drives nitrogen metabolism by increasing cytochrome c-activity to maintain electron transfer. With the mantel test and validation experiments, salt stimulates QS, leading to increased AHL (C6-HSL and C8-HSL) secretion associated with EPS release, extracellular electron transfer, and oxidative stress. Stabilization of AHL-QS genera supports AHL secretion and microbial symbiosis, promoting AnGS adaptation to salt stress. These insights facilitate optimizing AnGS for saline wastewater treatment.

Effect of polyethylene microplastics on anammox sludge characteristics and microbial communities

Microplastics (MPs), a class of emerging pollutants, pose significant potential risks to microbial metabolism and ecology. By adding different concentration gradients of polyethylene (PE), the effects of PE on the nitrogen removal efficiency and bacterial colony structure of anammox granular sludge (AnGS) were examined. The results of the 40 d experiment with various concentrations (0.05~1.0g/L) of PE MPs on AnGS showed that the elimination of ammonia and nitrite nitrogen was less affected than in the control group. Analysis of extracellular polymer (EPS) content revealed a decreasing trend in EPS content under high stress concentrations (0.2~1.0g/L) compared to the control, and protein/polysaccharide (PN/PS) showed a similar situation, suggesting that the stability of AnGS was compromised by elevated PE concentrations. Specific anammox activity (SAA) indicated a declining trend with increasing polyethylene microplastic (PE MP) concentrations between 0.2 and 1.0g/L, and the nitrogen removal performance of AnGS decreased. From the micro-morphological point of view, the AnGS subjected to 0.5g/L stress formed blocky large particle aggregates. PE MPs were adsorbed on the surface of AnGS and combined with it to form dense particles, and both particle size and mechanical strength of the granular sludge increased proportionally the concentration of PE MPs. High-throughput sequencing data indicated that exposure to 0.5g/L PE MPs stress enhanced the complexity of the microbial community structure in AnGS, while reduced the relative abundance of the anammox bacteria Candidatus Brocadia and Planctomycetes by 2.2% and 4.2%, respectively.

Formate-facilitated recovery of wastewater anammox granular sludge from oxygen inhibition: Effects and mechanisms

Oxygen leakage due to poor reactor sealing or accidental aeration can have detrimental impacts on the performance of wastewater anammox processes. Strategies of restoring functionality of anammox consortia are scarce, and providing appropriate chemical reducing reagents may help reduce oxidative damage. This study characterized the responses of anammox granular sludge (AnGS) to oxygen inhibition, and identified formate as an effective promoter capable of facilitating activity recovery, while other organics (acetate, glucose, methanol) were less effective. Upon 12 h of air exposure (DO: 1.2 ± 0.1 mg/L), the total nitrogen removal efficiency of a lab-scale AnGS reactor decreased from 85.15 % to 45.46 %. Compared with natural recovery (without chemical addition), potassium formate at 10 mg COD/L for 3 days significantly increased the total nitrogen removal efficiency, both in the short-term (66.68 % vs 57.09 %, 3 days post inhibition) and long-term (82.34 % vs 72.88 %, 30 days post inhibition). By examining sludge morphology, metagenome and transcriptome, the following potential mechanisms were proposed: 1) rapidly decreasing intracellular ROS; 2) enhancing nitrite turnover; 3) maintaining granular sludge integrity; 4) facilitating interspecies metabolic interactions. The ability of formate to facilitate sludge recovery from oxygen inhibition also implies its potential in mitigating other oxidative stresses in wastewater anammox reactors.

Aerobic granular sludge enhances start-up and granulation in single-stage partial nitritation anammox granular sludge systems: Performance, mechanism, and shifts in bacterial communities

The rapid start-up and granulation of a single-stage partial nitritation anammox granular sludge (PN/AnGS) system under limited seed sludge conditions is crucial for its practical application. This study proposed an aerobic granular sludge (AGS) − based strategy, enhanced the enrichment of anammox bacteria (AnAOB), and shortened the start-up time of PN/AnGS system by 20.5%. In addition, the inoculation of AGS can ensure the stable operation of the system during the selective sludge discharge to washout the flocs. Microbial community structure, particle size distribution, morphology results showed that niche shift was the key to promote the enrichment of AnAOB and AGS played a decisive role in the particle characteristics of PN/AnGS. Since AGS can be directly obtained from full-scale AGS wastewater treatment plants, integrating PN/AnGS with AGS processes can transition wastewater treatment from a “linear economy” to a “circular economy”, enhancing nitrogen removal efficiency and delivering significant economic and environmental benefits.

Novel ellipsoid-like granules exhibit enhanced anammox performance compared to sphere-like granules

Anammox granular sludge (AnGS) serves as an important platform for cost-effective nitrogen removal from wastewater. Different to the traditionally sphere-like granules, a novel type of AnGS in a unique ellipsoid-like shape was obtained through enhancing shear force. The ellipsoid-like AnGS significantly exhibited a smaller aspect ratio (-25.1 %) and granular size (-11.8 %), compared to traditional sphere-like AnGS (p < 0.01). Comprehensive comparisons showed that ellipsoid-like AnGS possessed a significantly higher extracellular polymeric substances (EPS) content and strength, as well as an enhanced mass transfer and a higher viable bacteria proportion due to the larger substrate permeable zone (p < 0.01). Additionally, the anammox bacterial abundance (Candidatus Kuenenia) was 12.2 % higher in ellipsoid-like AnGS than in sphere-like AnGS. All these characteristics of ellipsoid-like AnGS jointly increased the specific anammox activity by 29.0 % and nitrogen removal capacity by 22.6 %, compared to sphere-like AnGS. Further fluid field simulation suggested the enhanced flow shear on the side surface of AnGS likely drove the formation of ellipsoid-like AnGS. The higher shear force on the side surface led to an increase of EPS content (especially hydrophobic protein) and elastic modulus, thus constraining lateral expansion. This study sheds light on impacts of granular shape, an overlooked morphological factor, on anammox performance. The ellipsoid-like AnGS presented herein also offers a unique and promising aggregate to enhance anammox performance.

Surface-Adhered Microbubbles Enhance the Resistance of ANAMMOX Granular Sludge to Sulfamethoxazole Stress.

The granule-based anammox process has been reported to be more resistant to the stress of antibiotics; however, the underlying resistance mechanism is still not fully understood. In this study, we found that more microbubbles stably adhered to the surface layer of anammox granular sludge (AnGS, Gs) operating under long-term sulfamethoxazole (SMZ) stress of 2 mg/L, compared to that in the control reactor (Gc). The difference in covering content can be up to over three times (1.0 ± 0.1% vs 0.3 ± 0.0%). Batch tests showed that the coverage ratio of microbubbles on Gs reached approximately 32.5%, which significantly reduced SMZ transfer into AnGS due to the adsorption of SMZ by bubbles, thus alleviating the inhibition of anammox bacterial activity by 36.5%. The adhesion force between the microbubbles and the surface layer of Gs was found to be largely enhanced by 75.0% compared to that of Gc. The increased hydrophobicity of surface layer due to the increased extracellular polymer substance (EPS, mainly proteins) content, and the larger capillary force of surface layer, owing to the unique micronano structure, were identified as key factors responsible for the stable adhesion of microbubbles on the Gs. Consequently, this study demonstrated the vital roles of the surface-adhered microbubbles in resisting the stress of SMZ and shed light on the regulation and development of robust granule-based anammox processes.

Insight into the responses of the anammox granular sludge system to tetramethylammonium hydroxide (TMAH) during chip wastewater treatment

Tetramethylammonium hydroxide (TMAH), an extensively utilized photoresist developer, is frequently present in ammonium-rich wastewater from semiconductor manufacturing, and its substantial ecotoxicity should not be underestimated. This study systematically investigated the effects of TMAH on the anammox granular sludge (AnGS) system and elucidated its inhibitory mechanisms. The results demonstrated that the median inhibitory concentration of TMAH for anammox was 84.85 mg/L. The nitrogen removal performance of the system was significantly decreased after long-term exposure to TMAH (0–200 mg/L) for 30 days (p < 0.05), but it showed adaptability to certain concentrations (≤50 mg/L). Concurrently, the stability of the granules decreased dramatically, resulting in the breakdown of AnGS. Further investigations indicated that TMAH exposure increased the secretion of extracellular polymeric substances but weakened their defense function. The increase in reactive oxygen species resulted in damage to the cell membrane. Reduced activity of anammox bacteria, impeded electron transfer, and changes in enzyme activity suggested that TMAH affected the metabolic activity. Microbiological analysis revealed that TMAH caused a decrease in the abundance of anammox bacteria and a weakening of symbiotic interactions within the microbial community. These results provide valuable guidance for the AnGS system application in chip wastewater treatment.

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

Anaerobic ammonium oxidation (Anammox) process is an economical and energy-efficient method of wastewater nitrogen removal. However, they are highly susceptible to starvation stress caused by sudden environmental changes. Rapid reactivation of starved anammox sludge is a crucial method to address seed sludge shortages and expand practical applications. This study investigated the impact of gravel balls on the reactivation of long-term starved anammox granular sludge (628 days). The results showed that gravel balls enhanced the recovery of nitrogen removal performance in starved anammox sludge, with nitrogen removal efficiency being 19.88% higher than the control group at the end of the recovery phase. The gravel balls also increased extracellular polymeric substance (EPS) secretion, contributing to the stability of the anammox system. Furthermore, the gravel balls promoted the proliferation of anammox bacteria, with the relative abundance of anammox bacteria reaching 38.25% on the 80th day. The analyses of microbial functions indicated that gravel balls facilitated cross-feeding and co-metabolism among microbes, while enhancing quorum sensing associated with anammox bacteria, forming a multifunctional community network centered on anammox bacteria. This indicates that gravel balls can effectively accelerate the reactivation process of long-term starved anammox sludge, aiding the reutilization of long-term starved anammox sludge.

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.

Oxygen-induced evolution of anammox granular sludge explains its unique responses during preservation

Anammox granular sludge (AnGS) preservation is indispensable for the application of anammox technology. Oxygen is a common and crucial factor for anammox, yet its long-term effects on AnGS during preservation remain incomplete clarification. This study investigated the effect of oxygen on AnGS in two simulated preservation systems with open and sealed conditions, and the mechanism was discussed. The results showed that the open system was in an oxidized state with an average dissolved oxygen (DO) concentration and oxidation-reduction potential (ORP) of (3.10 ± 1.36) mg·L-1 and (112.58 ± 46.78) mV, while a reduced state for the sealed system with no detected DO and a lower average ORP of (-153.96 ± 64.32) mV. Both systems showed declines in AnGS activity, while with different responses of AnGS demonstrated by the evolution in terms of granular morphology and structure, bacterial communities, bacteria survival, and bacteria antioxidation. In the open system, reactive oxygen species were generated and destroyed the unsaturated fatty acids in the cell membrane, further leading to the destructed cell structure and declined activity. However, in the sealed system, AnAOB tended to enter a dormant state after long-term preservation, contributing to better conditions in granular morphology and structure, higher AnAOB abundance, and higher live cell ratio. The findings of this study are expected to offer vital information and guidelines for the preservation technologies of AnGS.

Characteristics and nontargeted metabolomics analysis of anammox granular sludge under short-term exposure to polypropylene and polylactic acid microplastics

A significant quantity of microplastics (MPs) is concealed within biological sludge. Current research predominantly examines the effects of non-biodegradable MPs on traditional sludge. However, the influence of biodegradable MPs on the functional microbial activity of anaerobic ammonium oxidation granular sludge (AnGS) remains inadequately explored. This study specifically investigated the effects of non-biodegradable polypropylene (PP) MPs and biodegradable polylactic acid (PLA) MPs on AnGS under short-term stress. The study found that PP MPs inhibited nitrogen removal performance, reducing nitrogen removal efficiency by 1.14% (100 mesh) and 5.77% (1000 mesh) respectively, along with decreased hydrazine dehydrogenase (HDH) activity. Conversely, PLA promoted denitrification performance, increasing efficiency by 8.21% (100 mesh) and 6.54% (1000 mesh). In response to MPs-induced environmental stress, the secretion of extracellular polymeric substances (EPS) increased in all experimental groups. Additionally, non-targeted metabolomic analysis revealed a decrease in the abundance of KEGG pathways related to carbon and amino acid metabolism in the experimental groups, while the enrichment of terephthalate and benzamide in the PLA groups significantly impacted its process. These findings offered valuable insights into the impact of MPs on anaerobic ammonium oxidation (anammox) and could potentially enhance its application.

Efficient alleviation granular sludge floatation in a high-rate anammox reactor by dosing folate

Although granular floatation has been recognized as a significant issue hindering the application of high-rate anammox biotechnology, limited knowledge is available about its causes and control strategies. This study proposed a novel control strategy by adding folate, and demonstrated its role in the granular floatation alleviation through long-term operation and granular characterizations. It was found that the floatation of anammox granular sludge was obviously relieved with the decreased sludge floatation potential by 67.1% after dosing with folate (8 mg/L) at a high nitrogen loading rate of 12.3 kg-N/(m3·d). Physiochemical analyses showed that the decrease of extracellular polymeric substances (EPS) content (mainly protein), the alleviation of granular surface pore plugging in conjunction with the smooth discharge of generated nitrogen gas were collectively responsible for efficient floatation control. Moreover, metagenomic analysis suggested that the synergistic interactions between anammox bacteria and their symbionts were attenuated after dosing exogenous folate. Anammox bacteria would reduce their synergistic dependence on the symbionts, and decline the supply of metabolites (e.g., amino acids and carbohydrates in EPS) to symbiotic bacteria. The declined EPS excretion contributed to the alleviation of granular floatation by dredging pores blockage, thus leading to a stable system performance. The findings not only offer insights into the role of microbial interaction in granular sludge floatation, but also provide a feasible approach for controlling the floatation issue in anammox granular-based processes.

A novel coupling process to replace the traditional multi-stage anammox process-sulfur autotrophic denitrification coupled anammox system.

A novel coupling process to replace the traditional multi-stage anammox process-sulfur autotrophic denitrification (SAD) coupled anaerobic ammonium oxidation (anammox) system was designed, which solved problems of nitrate produced in anammox process and low nitrate conversion rate caused by nitrite accumulation in SAD process. Different filter structures (SAD filter and anammox granular sludge) were investigated to further explore the excellent performance of the novel integrated reactor. The results of sequential batch experiments indicated that nitrite accumulation occurred during SAD, which inhibited the conversion of nitrate to dinitrogen gas. When SAD filter and anammox granular sludge were added to packed bed reactor simultaneously, the nitrate removal rate increased by 37.21% and effluent nitrite concentration decreased by 100% compared to that achieved using SAD. The stratified filter structure solved groove flow. Different proportion influence of SAD filter and anammox granular sludge on the stratified filter structure was evaluated. More suitable ratio of SAD filter to anammox granular sludge was 2:1. Proteobacteria (57.26%), Bacteroidetes (20.12%) and Chloroflexi (9.95%) were the main phyla. The dominant genera of denitrification functional bacteria were Thiobacillus (39.80%), Chlorobaculum (3.99%), norank_f_PHOs-HE36 (2.90%) and Ignavibacterium (2.64%). The dominant genus of anammox bacterium was Candidatus_Kuenenia (3.05%).

Metagenomic insights into microbial metabolic mechanisms of a combined solid-phase denitrification and anammox process for nitrogen removal in mainstream wastewater treatment

To overcome the significant challenges associated with nitrite supply and nitrate residues in mainstream anaerobic ammonium oxidation (anammox)-based processes, this study developed a combined solid-phase denitrification (SPD) and anammox process for low-strength nitrogen removal without the addition of nitrite. The SPD step was performed in a packed-bed reactor containing poly-3-hydroxybutyrate-co-3-hyroxyvelate (PHBV) prior to employing the anammox granular sludge reactor in the continuous-flow mode. The removal efficiency of total inorganic nitrogen reached 95.7 ± 1.2% under a nitrogen loading rate of 0.18 ± 0.01 kg N·m3·d−1, and it required 1.02 mol of nitrate to remove 1 mol of ammonium nitrogen. The PHBV particles not only served as biofilm carriers for the symbiosis of hydrolytic bacteria (HB) and denitrifying bacteria (DB), but also carbon sources that facilitated the coupling of partial denitrification and anammox in the granules. Metagenomic sequencing analysis indicated that Burkholderiales was the most abundant HB genus in SPD. The metabolic correlations between DB (Betaproteobacteria, Rhodocyclaceae, and Anaerolineae) and anammox bacteria (Candidatus Brocadiac and Kuenenia) in the granules were confirmed through microbial co-occurrence networks analysis and functional gene annotations. Additionally, the genes encoding nitrate reductase (Nap) and nitrite reductase (Nir) in DB primarily facilitated nitrate reduction, thereby supplying nitric oxide to anammox bacteria for subsequent nitrogen removal with hydrazine synthase (Hzs) and hydrazine dehydrogenase (Hdh). The findings provide insights into microbial metabolism within combined SPD and anammox processes, thus advancing the development of mainstream anammox-based processes in engineering applications.

Mechanisms of anammox granular sludge reactor effluent as biostimulant: Shaping microenvironment for anammox metabolism

Effluent from anammox granular sludge (AnGS) bioreactor contains microbes and microbial products. This study explored mechanisms of utilizing AnGS-effluent as biostimulant for anammox process enhancement. Compared with no AnGS-effluent supplemented control reactor, 5.0 and 1.3 times higher ammonium nitrogen and total inorganic nitrogen removal rates, respectively were obtained with continuous AnGS-effluent supplementation after 98 days’ operation. Anammox bacteria from Candidatus Brocadia accounted for 0.1 % (DNA level) and 1.3 %–1.5 % (RNA level) in control reactor, and 2.9 % (DNA level) and 54.5 %–55.4 % (RNA level) in the AnGS-effluent-fed reactor. Influent microbial immigration evaluation showed that bacterial immigration via AnGS-effluent supplementation was not the main contributor to active anammox community development. Amino acids biosynthesis, B-vitamins and coenzymes metabolism related pathways were facilitated by AnGS-effluent supplementation. AnGS-effluent supplementation aided anammox metabolic activity by shaping microenvironment and microbial interactions. This study provides insights into enhancing anammox bacterial metabolism with AnGS-effluent microbial products as biostimulant.

Diversity and dynamic response of anaerobic ammonia oxidation granular sludge

Red anaerobic ammonia oxidation (Anammox) granular sludge (AnGS) has been reported in successfully operating Anammox systems, and its color is associated with sludge activity. However, in long-term operating systems, AnGS exhibits different sensory colors, physical structures, community structures, and denitrification performance, but the relationship between them has not yet been elucidated. The AnGS of the Anammox system, which has been in operation for more than a decade, can be divided into two main categories: red and white. The specific Anammox activity (SAA) in conventional red AnGS increased continuously as the particle size increased from <0.51 mm to 6.02 ± 0.84 mm. The SAA of white AnGS were slightly lower than those of red AnGS with similarly-size granules but significantly higher than AnGS with smaller red granules. Compared with red AnGS, the extracellular polymeric substances of white AnGS were significantly reduced, mainly due to the higher intracellular iron content, resulting in lower heme c concentration. Thus, heme c may prove not to be an evaluative tool for measuring Anammox activity. Red and white AnGS, whether through self-aggregation or adsorption by hydroxyl apatite and other carriers, will face the fate of internal voids during particle size growth. White AnGS exhibited a more complex microbial community than red AnGS. Candidatus Brocadia was abundant in red AnGS and the abundance increased with increasing granule size. Candidatus Kuenenia and Candidatus Jettenia made significant contributions to denitrification in white AnGS. This study provides a new perspective on particle selection for anammox engineering applications.

Rapid reactivation of long-term frozen preservation anammox granular sludge by biochar: Performance, mechanism and artificial neural network optimization

Anaerobic ammonia oxidation (Anammox) is an efficient nitrogen removal technology for wastewater, and has been successfully applied in practical wastewater treatment. The preservation and rapid reactivation of anammox bacteria (AnAOB) play an important role in the anammox application. Biochar has been widely used in the water treatment industry due to its stable structure, economic and environmental protection characteristics. Hence, this study used biochar to accelerate anammox granular sludge (AnGS) performance recovery after 60 d preservation under low-temperature frozen (−20 °C). The time of AnGS recovery in the group with biochar addition was shortened by 15 d than the natural recovery group. Meanwhile, biochar addition improved the shock loading resistance of the anammox system. EPS secretion, and the types and quantities of functional groups in EPS increased under biochar stimulation, especially tighter PN. The functional groups in biochar can act as electron shuttles during anammox process, accelerating the electron transfer rate. Biochar addition enhanced the relative abundance of AnAOB, especially Candidatus Brocadia. PICRUSt analysis indicated that biochar promoted key gene related to anammox process and accelerated the recovery of frozen sludge. In addition, the changes in effluent nitrogen removal efficiency were successfully predicted by artificial neural network modeling with R = 0.9895 and MSE = 0.0048. This result may provide a new technological idea for the rapid recovery of AnGS under long time frozen conditions.

Novel insights into intrinsic mechanisms of magnetic field on long-term performance of anaerobic ammonium oxidation process

The performance of an anaerobic ammonium oxidation (anammox) reactor with the magnetic field of 40 mT was systematically investigated. The total nitrogen removal rate was enhanced by 16% compared with that of the control group. The enhancing mechanism was elucidated from the improved mass transfer efficiency, the complicated symbiotic interspecific relationship and the improved levels of functional genes. The magnetic field promoted formation of the loose anammox granular sludge and the homogeneous and well-connected porous structure to enhance the mass transfer. Consequently, Candidatus Brocadia predominated in the sludge with an increase in abundance of 13%. Network analysis showed that the positive interactions between Candidatus Brocadia and heterotrophic bacteria were strengthened, which established a more complicated stable microbial community. Moreover, the magnetic field increased the levels of hdh by 26% and hzs by 35% to promote the nitrogen metabolic process. These results provided novel insights into the magnetic field-enhanced anammox process.

Enhancing nitrogen removal efficiency for anammox granular sludge at low temperature by fulvic acids from different sources

The stable and efficient operation of the anaerobic ammonia oxidation (anammox) process in mainstream wastewater treatment at low temperatures still faces significant challenges. In this study, the effect of mineral fulvic acid (MFA) and biochemical fulvic acid (BFA) on the nitrogen removal efficiency of the anammox reactor at low temperature (20 °C) was investigated. The results demonstrated that both MFA and BFA could improve the nitrogen removal performance of the anammox reactor, with MFA exhibiting a more pronounced promotional effect than BFA. At concentrations of 30 mg total organic carbon (TOC)/L for both MFA and BFA, the nitrogen removal rate (NRR) increased by 26.68 % and 21.93 %, respectively. MFA and BFA stimulated the secretion of extracellular polymeric substances (EPS), preventing the disintegration of granular sludge at low temperatures, with a notable increase in the particle size of granular sludge, especially at high concentrations of MFA. Moreover, serving as electron shuttles, MFA demonstrated superior electron transfer ability compared to BFA, significantly enhancing the metabolic activity of anammox bacteria (AnAOB) under low temperatures. In addition, compared with BFA, MFA exhibited the potential to promote the growth of Candidatus_Kuenenia, offering a beneficial contribution to improving the nitrogen removal performance of the anammox reactor at low temperatures.

Insights into the roles of EDTA in enhancing the bioavailability of Fe(Ⅲ) and stabilizing the anammox granular sludge system

The adsorption and utilization of Fe(Ⅲ) can be impeded by precipitation in anaerobic reactors. In this study, we investigated whether the bioavailability of Fe(Ⅲ) can be enhanced by EDTA-Fe(Ⅲ), thereby improving the stability of anammox system. Fe(Ⅲ) and EDTA-Fe(Ⅲ) were added to two parallel up-flow anaerobic sludge blanket (UASB) reactors, respectively. After 186 days of operation, when the nitrogen loading rate (NLR) was 0.8 g-N/L/d, the nitrogen removal rate (NRR) of R2 was stable at 0.675 g-N/L/d, which was higher than that of R1 at 0.557 g-N/L/d. The anammox activity of R2 reached its peak in phase Ⅲ (158.55 ± 4.53 mg-N/g-VSS/d), while the anammox activity of R1 was only 28.37 ± 5.96 mg-N/g-VSS/d. Moreover, in phase Ⅱ-Ⅴ, the bioavailability of Fe(Ⅲ) in R2 was increased by 15.63%, 38.37%, 65.90%, and 39.63%, respectively, compared with R1. The results showed that EDTA-Fe(Ⅲ) can prevent precipitation encasing anammox granular sludge, which can enhance substrate transport and Fe(Ⅲ) adsorption utilization. High throughput sequencing suggested that EDTA-Fe(Ⅲ) could better increase the abundance of anammox bacteria (AnAOB). In conclusion, this research proved that EDTA could increase the bioavailability of Fe(Ⅲ) and thereby ensuring the stability of the reactors.