Anammox Bacteria Research Articles

Ultra-stable mixotrophic denitrification coupled with anammox under organic stress for mainstream municipal wastewater treatment

Sulfur-based autotrophic denitrification (SAD) coupled with anammox is a promising process for autotrophic nitrogen removal in view of the stable nitrite accumulation during SAD. In this study, a mixotrophic nitrogen removal system integrating SAD, anammox and heterotrophic denitrification was established in a single-stage reactor. The long-term nitrogen removal performance was investigated under the intervention of organic carbon sources in real municipal wastewater. With the shortening of hydraulic retention time, the nitrogen removal rate of the mixotrophic system dominated by the autotrophic subsystem reached 0.46 Kg N/m³/d at an organic loading rate of 0.57 Kg COD/m³/d, with COD and total nitrogen removal efficiencies of 82.5 % and 94 %, respectively, realizing an ideal combination of autotrophic and heterotrophic systems. The 15NO3−-N isotope labeling experiments indicated that thiosulfate-driven autotrophic denitrification was the main pathway for nitrite supply accounting for 80.6 %, while anammox exhibited strong competitiveness for nitrite under the dual electron supply of sulfur and organic carbon sources and contributed to 65.1 % of nitrogen removal. Sludge granulation created differential functional distributions in different forms of sludge, with SAD showing faster reaction rate as well as higher nitrite accumulation rate in floc sludge, while anammox was more active in granular sludge. Real-time quantitative PCR, RT-PCR and high-throughput sequencing results revealed a dynamically changing community composition at the gene and transcription levels. The decrease in heterotrophic denitrification bacteria abundance indicated the effectiveness of the operational strategy for introduction of thiosulfate and maintaining the dominance of SAD in denitrification process in suppressing the excessive growth of heterotrophic bacteria in the mixotrophic system. The high transcriptional expression of sulfur-oxidizing bacteria (SOB) (Thiobacillus and Sulfurimonas) and anammox bacteria (Candaditus_Brocadia and Candidatus_Kuenenia) played a crucial role in the stable nitrogen removal.

Single-stage partial nitrification/anammox process performance restoration by bio-augmenting nitrifiers with or without nitrite oxidizing bacteria suppression

The use of low dissolved oxygen (DO) is common to suppress the growth of nitrite oxidizing bacteria (NOB) in the partial nitrification/anammox (PN/A) process. However, this approach can negatively impact the activity of ammonium oxidizing bacteria (AOB) and reduce the oxygen consumption rate, disrupting the essential low DO environment required for anammox bacteria (AMX) and leading to a decline in PN/A performance. We innovatively harnessed the strategy of common nitrifiers (AOB + NOB) bioaugmentation in an impaired single-stage PN/A reactor after operation for a period of time. The results demonstrated an immediate improvement, with the total nitrogen removal efficiency increasing from approximately 60 % to 80 %. This enhancement was attributed to decreased NOB activity and increased AMX activity, which occurred at reduced DO level (0.24 to 0.12 mg O2/L) following nitrifiers bioaugmentation. Partial nitrification (PN) and PN/A batch experiments with nitrifiers bioaugmentation further supported these findings. This strategy pioneered a fresh perspective that the diversion of nitrite from NOB to AMX was the pivotal underlying mechanism. Mathematical simulations complemented these results by demonstrating that nitrifiers bioaugmentation broadened the optimal oxygen supply window.

Advance nitrogen removal from anaerobic sludge digestion liquor using partial nitrification and denitrification coupled with simultaneous partial nitrification, anammox, and denitrification process

A novel two-stage continuous-flow partial nitrification and denitrification coupled with simultaneous partial nitrification, anammox, and denitrification (PND-SNAD) process was developed to treat anaerobic sludge digestion liquor. During the stable phase, the total nitrogen and chemical oxygen demand (COD) removal efficiencies were 93 ± 3 % and 59 ± 7 %, respectively. Free ammonia suppression (26.2 ± 12.7 mg/L) and low dissolved oxygen (DO, 0.12 ± 0.07 mg/L) were key factors in the operation of the PND process, while the SNAD process was restricted successfully by limited oxygen (DO < 0.1 mg/L) and short solids retention time (9.7 d). The PND process was an important pretreatment process that could remove biodegradable dissolved COD by denitrification and supply ammonium-oxidizing bacteria (AOB) to the SNAD process. Nitrosomonas and Ca. Brocadia were the dominant AOB and anammox bacteria, respectively. Overall, this research presents a distinctive SNAD combined process for anaerobic sludge digestion liquor treatment.

Enhanced resuscitation of anammox granular sludge by adding folate: A perspective from the growth factor

Quick resuscitation of preserved anammox granular sludge (AnGS) is crucial for its engineering applications. In this study, the stimulation effect and its mechanism of folate addition on resuscitation of AnGS after the long-term cryopreservation were investigated. The folate addition was found to enhance the recovery of AnGS activity to 76.4% of the original level on the 8th day, which was 28.1% higher than the folate-free group (CK). The folate addition could also promote the community recovery of AnGS, in which the live cells and abundance of anammox bacteria (AnAOB) were 10.2% and 14.0% higher than those in the CK in the early phase of resuscitation. Besides, the folate addition was revealed to stimulate the AnAOB recovery of AnGS, with higher activity and cell number by 58.7% and 40.9% than those in the CK. Furthermore, the mechanism of folate stimulation was proposed on the resuscitation of preserved AnGS. The exogenous folate addition could enhance the CO2 fixation capacity of AnAOB, which further accelerated both activity and cell number recovery of AnAOB, leading to the strong stimulation effect on the resuscitation of AnGS. These findings could offer valuable insights for optimizing and innovating the preservation technology of AnGS.

Partial denitrification-anammox granular sludge domesticated from high-strength anammox granules and the high-efficiency performance in treating low-nitrogen wastewater

Partial denitrification (PD) is an effective approach to supply nitrite for the anaerobic ammonium oxidation (anammox) reaction, and the PD-anammox process can achieve nearly 100 % nitrogen removal when denitratation, denitritation and anammox processes are well balanced. However, most of the previous studies initiated the PD-anammox process by a mixed inoculation of denitrification and anammox sludges, leading to low anammox bacteria abundance and a risk of unstable operation in the long-term. To address this, this study used high-strength anammox granules as seed sludge for rapid PD-anammox process start-up. During the continuous operation, nitrogen removal efficiencies of 97.6 ± 1.6 % and 91.0 ± 2.0 % were attained under influent conditions of 159.4 ± 3.4 mg N/L and 78.1 ± 3.5 mg N/L, respectively. In addition, the nitrite conversion rate of the sludge reached 5.34 ± 0.37 g N/g VSS/d during the operation, and the anammox activity was stable at 0.27–0.41 g N/g VSS/d, indicating a synergistic PD-anammox relationship. Furthermore, PD bacteria were efficiently enriched in the reactor within a short period of time, with Thauera accounting for 7.6 % of the relative abundance. Notably, the abundances of dominating anammox bacteria, Ca. Brocadia and Ca. Kuenenia were 16.6 % and 4.9 %, respectively, which were among the highest of the reported PD-anammox processes. High anammox microbial abundance is conducive to ensuring the dominance and stability of the anammox reaction, enabling efficient nitrite conversion and nitrogen removal throughout the continuous operation. Finally, nitrogen removal pathways based on mass balance were proposed to clarify the fate of different nitrogen compounds in the PD-anammox system.

A pilot-scale SNAD-MBBR process for treating anaerobic digester liquor of swine wastewater: performance and microbial community.

In this pilot-scale study, simultaneous partial nitrification, anammox, and denitrification (SNAD) process was achieved successfully in a moving bed biofilm reactor (MBBR) for treating anaerobic digester liquor of swine wastewater. After 95days of operation, when the total nitrogen loading rate of SNAD-MBBR process was 1.09kg TN/m3/day, the total nitrogen removal rate could reach 0.87kg TN/m3/day, and the removal efficiencies of ammonium and total nitrogen were 92.0% and 79.7%, respectively. The optimum pH and temperature for SNAD-MBBR process were 8.5 and 35°C, respectively, and the optimum dissolved oxygen for SNAD1 and SNAD2 were 0.30 and 0.07mg/L, respectively. The 16S rRNA sequencing suggested that Candidatus Kuenenia, Candidatus Brocadia, Nitrosomonas, and Denitratisoma were the dominant nitrogen removal bacteria. Some of the co-existing bacteria (Truepera, Limnobacter, and Anaerolineaceae uncultured) promoted ammonium oxidation and guaranteed the growth of the anammox bacteria under adverse environmental conditions. Overall, this study demonstrated that the SNAD-MBBR process would be an energy-saving and cost-effective method for the removal of nitrogen from swine wastewater and provided important process parameters for stable operation of the full-scale SNAD process.

Successful start-up of a novel integrated denitrifying phosphorus removal and partial denitrification coupled with anammox process for simultaneous nitrogen and phosphorus removal with fully ordinary suspended sludge

Partial Denitrification coupled with Anammox (PDA) process was used as for nitrogen removal from wastewater. However, the phosphate removal still relies on chemical methods in wastewater treatment plants. Denitrifying Phosphorus Removal (DPR) process allows phosphorus uptake under anoxic conditions using nitrate (NO3−) as electron acceptor. In this study, the PDA and DPR processes were successfully coupled into a novel integration (DPR-PDA) for co-treatment of simulated domestic wastewater and NO3− wastewater to achieve simultaneous nitrogen and phosphorus removal. The effluent TN and TP were 0.9 and 0.2 mg/L during phase Ⅵ (day450-513) under influent COD/TN of 1.8, respectively. Anammox bacteria was self-enriched in this fully suspended sludge system, with abundances increasing from undetected at day19 to 2.0 × 1010 copies/g dry sludge at day468. Anammox bacteria was effectively retained and was responsible for the main nitrogen removal, accompanied by increasing activity up to 3.1 mg N/g VSS/h. Batch tests demonstrated that denitrifying polyphosphate accumulating organisms exhibited DPR capacity immediately after the introduction of NO3−, but not after the introduction of nitrite (NO2−) only. Candidatus_Competibacter with endogenous denitrification ability was enriched from 13.30 % to 35.42 %, which coexisted with anammox bacteria stably. The novel DPR-PDA process is a promising technology for simultaneous removal of nitrogen and phosphorus with low carbon demand and without aeration.

Rapid enrichment of anammox bacteria for low-strength wastewater treatment: Role of influent nitrite and nitrate ratios in sequencing batch reactors (SBRs)

Anaerobic ammonium oxidation (Anammox) bacteria (AnAOB) have a long doubling time, which represents one of the key challenges for starting up anammox-based reactors. This study investigated the effect of varied nitrite/nitrate ratios on the anammox reactor startup process using five lab-scale anammox sequencing batch reactors (SBRs) (R1-R5). Nitrate addition significantly accelerated AnAOB enrichment startup, particularly during nitrite accumulation phase. The total start-up time was shortened by 55–71 days, compared to the reactor without nitrate. In R1 (nitrite/nitrate=10:0), anammox contributed the highest nitrogen removal percentage (91%), while R2 (nitrite/nitrate=7:3), with nitrate added, exhibited the highest anammox activity (53.84 mg NH4+-N/g VSS/day) in the steady-state phase. As nitrate proportions increased (R3-R5), anammox activity gradually declined due to limited available COD required for the conversion of NO3- to NO2-. Furthermore, it was found that nitrate-added improved the sludge settleability, with lower SVI30. 16S rRNA gene revealed that Ca. Brocadia was the predominant AnAOB in R1, R2 and R3, where nitrite was the primary NOx--N species (≥50%) in the influent. While in the reactor with low concentration of nitrite (R4: nitrite/nitrate=3:7), Ca. Kuenenia was the dominant AnAOB. This study successfully established a complex and balanced ecosystem with multi-species coexistence, where AnAOB served as the primary functional group on nitrogen removal, supported by denitrification bacteria and dissimilatory nitrate reduction to ammonium (DNRA) bacteria. This study proposes a rapid start-up strategy for the anammox process, which overcomes the bottleneck of long start-up time and helps to promote the application of anammox in wastewater treatment.

Advanced nitrogen removal from municipal wastewater through partial nitrification-denitrification coupled with anammox in step-feed continuous system

Combined partial nitrification-denitrification/anammox (PN-PD/A) processes have attracted great attention from researchers in recent years to achieve high nitrogen removal from low carbon /nitrogen (C/N) municipal wastewater. In this context, a step-feed anoxic/oxic (A/O) process was conducted in this study through the combination of the partial nitrification-anammox (PN/A) and partial denitrification-anammox (PD/A) to remove N from municipal wastewater with low C/N. The enhancement of the PN-PD/A process resulted in N removal efficiency of 85.6% at C/N of 2.8. The contributions of the anammox reached 36.4 and 8.8% in the anoxic and oxic chambers, respectively. The biocarriers added to the anoxic and oxic chambers increased the relative abundance of the anammox bacteria in biofilms from 0.61% to 1.51% and 1.02%, respectively. This study demonstrated that employing the step-feed A/O process can create optimal conditions for the anammox bacteria growth, thereby ensuring advanced N removal from low C/N municipal wastewater.

Environmental pollution of fluoroquinolones and its relationship with nitrogen cycling mediated by microorganisms.

Fluoroquinolone antibiotics (FQs) are one of the most widely used antibiotics, which are new pollutants with 'pseudo persistence' in the environment, causing great ecological risks. FQs could change the structure and function of microbial communities and affect nitrogen cycling mediated by microorganisms. Consequently, FQs would change the composition of various types of nitrogen in the environment and exert a significant impact on the global nitrogen cycling. We encapsulated the distribution of FQs in the environment and its impacts on nitrogen cycling mediated by microorganisms, explained the role of FQs in each key process of nitrogen cycling, aiming to provide an important reference for revealing the ecological effects of FQs. Generally, FQs could be detected in various environmental media, with significant differences in the concentration and types of FQs in different environments. Ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin are the four types of FQs with the highest detection frequency and concentration. The effect of FQs on nitrogen cycling deeply depends on typical characteristics of concentration and species. FQs mainly inhibit nitrification by reducing the abundance of amoA gene related to ammoxidation process and the abundance and composition of ammoxidation bacteria. FQs inhibits nitrification by reducing the abundance and composition of microbial communities. The denitrification process is mainly inhibited due to the reduction of the activity of related enzymes and the abundance of genes such as narG, nirS, norB, and nosZ genes, as well as the abundance and composition of denitrifying functional microorganisms. The process of anammox is restricted due to the reduction of the abundance, composition and hzo gene abundance of anaerobic anammox bacteria. FQs lead to the reduction of active nitrogen removal and the increase of N2O release in the environment, with further environmental problems such as water eutrophication and greenhouse effect. In the future, we should pay attention to the effects of low concentration FQs and complex antibiotics on the nitrogen cycling, and focus on the effects of FQs on the changes of nitrogen cycle-related microbial monomers and communities.

Quantitative effect of adding percentages of anammox granules on the start-up process and microbial community analysis

The anaerobic ammonium oxidation (anammox) process is challenging due to its long start-up duration and high demand for mature anammox seed sludge. However, adding a small amount of anammox sludge to the inoculum can be a reasonable solution. This study investigated the effect of adding percentage of anammox granules (0, 1, 2, 4, and 8%) in the seed sludge on the anammox start-up process. The anammox process was achieved in all five reactors after 55, 6, 5, 3 and 0 days. Increasing the adding percentage effectively shortened the duration of lag phase and cell lysis, but had little effect on the final nitrogen removal performance, except for 4% adding percentage. Families of Brocadiaceae, Burkholderiaceae, Ignavibacteriaceae, SJA-28, and Rhodocyclaceae were dominant, with a core microbiota of eight operational taxonomic unites (OTUs), and Candidatus Brocadia fulgida became the dominant anammox species. Seven synergistic members with anammox bacteria were identified by correlation network analysis. Major potential functional groups involved in C and N cycle were also observed by FAPROTAX. Together with the qPCR and sequencing results, it was suggested that more than 2% of adding percentages would result in a short lag phase, rapid growth rate in elevation stage, high final performances, and anammox bacteria abundance comparable to that in the anammox seed sludge. This crucial finding indicated the feasibility of economical and rapid start-up of the anammox process with a minimum amount of anammox seed sludge.

Microbial stratification protects denitrifying anaerobic methane oxidation archaea and bacteria from external oxygen shock in membrane biofilm reactor

Different gradients of dissolved oxygen (DO) regulate the microbial community and nitrogen removal pathways of denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (Anammox) coupled process in a batch biofilm reactor. Under completely anaerobic condition, approximately 72 mg NO3–-N/L was removed at a daily rate of 6.55 mg N/L, whereas a peak accumulation of 95 mg NO3–-N/L was observed during DO reached 0.5 mg/L. There is a decrease in the abundance of Candidatus Methylomirabilis (24.1%), Candidatus Methanoperedens (23.3%), and Candidatus Kuenenia (22.6%) to below 5% when DO levels reached 0.2 mg/L. Moreover, key genes associated with the reverse methanogenesis (mcrA) and anaerobic ammonium oxidase (hzo) decreased. These findings indicate that during oxygen shock, methanotrophs and denitrifiers replace Anammox bacteria on the outer sphere of the biofilm, whereas DAMO bacteria and archaea are protected from external oxygen shock due to the microbial stratification of biofilm.

Efficient nitrogen removal and substrate usage in integrated fixed-film activated sludge-anammox system under seasonal temperature variation

To elucidate how integrated fixed-film activated sludge (IFAS) system favors nitrogen removal performance under seasonal temperature variations, two push-flow reactors were operated with and without carriers under the same operating conditions. The results show that the IFAS system had significant advantages in shock response and low temperature adaptation, with a nitrogen removal rate of 0.37–0.53 kg-N(m3·d)-1 at the temperature of 8–12 °C. Anammox bacteria on carriers were almost unaffected by temperature variation, and its nitrogen removal contribution rate stabilized at 55 % in the IFAS system. The Haldane model reveals that the specific anammox activity in the IFAS system was 28 % to 49 % higher than that in the control system at 13 °C. Candidatus_Jettenia, with the highest abundance of 45 %, was the dominant species in the IFAS system and preferred to attach to the carriers. This study provides a feasible scheme for the application of anammox process.

Microbial electrolysis drives ammonium removal from saline wastewater through marine anammox bacteria

Currently, ammonium removal from saline wastewater still poses a challenge to conventional biological nitrogen removal process. Herein, the conversion of NH4+-N to N2 was successfully driven by marine anammox bacteria (MAB) in a single chamber microbial electrolytic cell (MEC) using NH4+-N as the sole substrate. Compared with no ammonium removal in the abiotic reactor with applied voltage (0–1.5 V), the maximum ammonium and total nitrogen removal rate (ARR and TNRR) of 55 and 50 g/m3•d were obtained at the voltage of 1.1 V, which were 78 % and 76 % higher than that of 0.5 V, respectively. At the voltage ≥ 1.1 V, the accumulation of NO2–-N and NO3–-N was observed in the MEC, and the nitrifying bacteria Nitrosmonas and Nitrospira were detected on the anode surface with the relative abundances of 0.84 % and 0.21 %, respectively. It was proved that the production of NO2–-N and NO3–-N was an electrochemical biological process by electroactive nitrifying bacteria. Candidatus Scalindua was the main functional genus for ammonium removal at anode biofilm with the relative abundance of 19.27 %. Therefore, ammonium was successfully removed by integrating anodic anammox and anodic nitrification. Applied voltage increased the microbial diversities at anode surface and promoted the formation of electrode biofilm. These findings indicated a promising saline wastewater treatment by employing salt-tolerant MAB for ammonium removal.

Enhancing anammox bacteria enrichment in integrated fixed-film activated sludge partial nitritation/anammox process via floc retention control

A promising technology for partial nitritation/anammox (PN/A) processes to treat ammonium wastewater is integrated fixed-film activated sludge (IFAS). For practical applications, achieving efficient enrichment of anammox bacteria (AnAOB) remains a challenge. In this study, membranes were temporarily used to separate solid and liquid components to induce changes in the mixed liquor suspended solids of the flocs. With membrane separation, AnAOB proliferated rapidly with a seven-fold increase in the maximum specific growth rate (μ) (from 0.009 to 0.072 d−1) and a three-fold increase in the nitrogen removal rate (from 0.91 to 3.20 kg N/(m3·d)). Moreover, microbial community analysis showed significant changes in bacterial species richness and diversity with and without membrane separation. Overall, the regulation of flocs significantly influenced the microbial community structure of both flocs and biofilms leading to improved nitrogen removal efficiency in the IFAS-PN/A system.

Potential of nitrite-absent anaerobic ammonium oxidation by mixed culture ANAMMOX granules in a single chamber bio-electrochemical system

Nitrogen (N) removal from wastewater is essential, but it a process that demands a substantial amount of energy. Therefore, there is an urgent need to develop treatment processes that can conserve and use energy effectively. This study investigated the potential of a single chamber bio-electrochemical system (BES) for ammonium (NH4+) removal. Various NH4+:NO2− ratios (1:1, 1:0.5, and 1:0) were tested at an applied potential of 0.4 V vs. Ag/AgCl. Potential in the reactors (R-1, R-2, and R-3) significantly improved NH4+ removal efficiencies. Specifically, R-1, R-2, and R-3 exhibited removal efficiencies of 68.12%, 64.22%, and 57.86%, respectively. NH4+ oxidation in R-3 involved using a carbon brush electrode as an electron acceptor. Significant electric charge generation was observed in all reactors (R-1, R-2, and R-3) during NH4+ removal. Particularly, the use of a carbon brush as an electron acceptor in R-3 resulted in higher electric charge generation compared to those in R-1 and R-2, where NO2− served as an electron acceptor. Upon NH4+ removal and concurrent electric charge generation, nitrate (NO3−) accumulation was observed in reactors with applied potential (R-1, R-2, and R-3), demonstrating greater accumulation compared to reactors without potential (R-7, R-8, and R-9). The mechanism involves ammonium oxidizing bacteria (AOB) oxidizing NH4+ to NO2−, which is then further oxidized by nitrite-oxidizing bacteria (NOB) to NO3−. ANAMMOX bacteria could directly produce N2 from NH4+ and NO2− or NH4+ could be oxidized to N2 through extracellular electron transfer (EET). A carbon brush electron acceptor reduces NO2− requirement by 1.65 g while enhancing NH4+ oxidation efficiency. This study demonstrates the potential of mixed culture ANAMMOX granules for efficient NO2-free NH4+ removal.

Challenges of suppressing nitrite-oxidizing bacteria in membrane aerated biofilm reactors by low dissolved oxygen control

Membrane aerated biofilm reactor (MABR) and shortcut nitrogen removal are two types of solutions to reduce energy consumption in wastewater treatment, with the former improving the aeration efficiency and the latter reducing the oxygen demand. However, integrating these two solutions, i.e., achieving shortcut nitrogen removal in MABR, is challenging due to the difficulty in suppressing nitrite-oxidizing bacteria (NOB). In this study, four MABRs were established to demonstrate the feasibility of initiating, maintaining, and restoring NOB suppression using low dissolved oxygen (DO) control, in the presence and absence of anammox bacteria, respectively. Long-term results revealed that the strict low DO (< 0.1 mg/L) in MABR could initiate and maintain stable NOB suppression for more than five months with nitrite accumulation ratio above 90 %, but it was unable to re-suppress NOB once they prevailed. Moreover, the presence of anammox bacteria increased the threshold of DO level to maintain NOB suppression in MABRs, but it was still incapable to restore the deteriorated NOB suppression in conjunction with low DO control. Mathematical modelling confirmed the experimental results and further explored the differences of NOB suppression in conventional biofilms and MABR biofilms. Simulation results showed that it is more challenging to maintain stable NOB suppression in MABRs compared to conventional biofilms, regardless of biofilm thickness or influent nitrogen concentration. Kinetic mechanisms for NOB suppression in different types of biofilms were proposed, suggesting that it is difficult to wash out NOB developed in the innermost layer of MABR biofilms because of the high oxygen level and low sludge wasting rate. In summary, this study systematically demonstrated the challenges of NOB suppression in MABRs through both experiments and mathematical modelling. These findings provide valuable insights into the applications of MABRs and call for more studies in developing effective strategies to achieve stable shortcut nitrogen removal in this energy-efficient configuration.

Study on rapid start-up and stable nitrogen removal efficiency of carrier enhanced continuous flow PD/A granular sludge system

Anaerobic ammonia oxidation (anammox) is a vital process for nitrogen removal, but challenges like slow start-up and unstable operation persist. To tackle these issues and assess carrier efficiency in improving the partial denitrification/anammox (PD/A) process’s start-up and stability under low nitrogen loading rates (NLR) (0.11–0.19 kg/(m3·d)), two continuous flow PD/A systems were set up: one with self-immobilized granular sludge (R1) and the other with carrier-based granular sludge (R2). The results showed that R2 achieved over 70% total nitrogen (TN) removal within 45 days, 16 days shorter than R1. Scanning electron microscope (SEM) characterization revealed that the ecological maturity of biofilm granular sludge was approximately 14 days earlier in R2 than in R1. Comparative analysis of protein/polysaccharide (PN/PS) ratios within extracellular polymeric substances (EPS) and volatile suspended solids/suspended solids (VSS/SS) demonstrated that carrier-based granular sludge exhibited superior settling characteristics and structural stability compared to self-immobilized granular sludge. Moreover, nitrogen removal stability within the R2 reactor system was superior to R1 under diverse operating conditions, highlighting carrier reinforcement’s efficacy in enhancing the PD/A process’s operational stability. The increased abundance of anammox bacteria Candidatus_Kuenenia in R2 ensured complete nitrogen removal in the PD/A coupling process, elucidating the microscopic mechanism of carrier-enhanced nitrogen removal. The hydrophilic porous biological carrier not only expedited the PD/A system’s start-up in the upflow sludge bed (USB) reactor but also enhanced the enrichment and collaboration of effective functional microorganisms. This study provides technical reference and theoretical evidence for rapid start-up and long-term stable operation of PD/A process.

Performance optimization and nitrogen removal mechanism of up-flow partial denitrification/anammox process

This study aimed to remediate the problems of sludge floating and uneven mass transfer in up-flow partial denitrification/anammox (PDA) reactors and dissect the nitrogen removal mechanism. Two up-flow PDA reactors were operated, whereby in R1 combined biological carriers were added, while in R2 mechanical stirring was applied, the reactors were inoculated with PD sludge and anammox sludge. Results showed the TN removal rates at the end of the operation were 89% (R1) and 92% (R2). The addition of both strategies suppressed the occurrence of sludge upwelling and deterioration of settling performance, even when the granule diameter of the granular zone in R1 and R2 reached 1.921 and 2.006 mm, respectively. 16SrRNA sequencing revealed R1 had a higher abundance of anammox bacteria (AAOB, 14.53%-R1, 9.06%-R2, respectively), and R2 had a higher quantity of denitrifying bacteria (61.92%-R1, 67.11%-R2, respectively). And the nitrogen removal was contributed by anammox and denitrification in combination, with contributions of 82.17%, 17.83% (R1), and 85.07%, 14.93% (R2), respectively. In summary, both strategies prevented sludge flotation and uneven nitrogen mass transfer. However, mechanical agitation had a more substantial positive effect on the performance of PDA than the addition of biocarriers because it achieved a more adequate mass transfer.

Extracellular polymeric substances and microbial community shift during the start-up of a single-stage partial nitritation/anammox process.

A sequencing batch reactor (SBR) was operated to investigate variations of extracellular polymeric substances (EPS) and microbial community during the start-up of the single-stage partial nitritation/anammox (SPN/A) process at intermittent aeration mode. The SPN/A system was successfully started on day 34, and the nitrogen removal efficiency and total nitrogen loading rate were 82.29% and 0.31 kg N/(m3 ·d), respectively. Furthermore, the relationship between the protein secondary structures and microbial aggregation was strongly related. The α-helix/ (β-sheet + random coil) ratios increased obviously from 0.20 ± 0.03 to 0.23 ± 0.01, with the sludge aggregation mean size increased from 56 to 107 μm during the start-up of SPN/A. During the start-up of SPN/A, Candidatus Kuenenia was the primary anammox bacteria, whereas Nitrospira was the main functional bacteria of nitrite-oxidizing bacteria. Correlation between the microbial community and EPS components was performed. The EPS and microbial community played important roles in keeping stable nitrogen removal and the formation of sludge granules.