Partial Denitrification-anammox Research Articles

Robust PD/A biofilms sustain advanced real municipal wastewater treatment: Reinforcement of interspecific communication and electron transfer

In this study, a robust partial denitrification/anammox (PD/A) biofilm system was efficiently established within merely 83 days using solely conventional activated sludge as the inoculum, employing a multivariate regulation strategy involving the addition of biofillers, optimization of municipal wastewater feeding ratios, prolongation of hydraulic retention time, and introduction of hydrazine. The performance variability of PD/A system within a long-term, intricate mainstream substrate environment was further investigated by incrementally elevating municipal wastewater proportion (50 % → 75 % → 100 %). The results demonstrated that PD/A system ultimately achieved an impressive total nitrogen removal efficiency of 93.6 % and an effluent total nitrogen concentration of 8.6 mg/L, accompanied by anammox contributing over 85 % to nitrogen removal. The biofilm’s layered spatial structure nurtured Candidatus Brocadia, enabling it to reach an abundance of 2.5 % under high real municipal wastewater loading. Metabolic function analyses revealed that biofilm enhanced the abundance of genes associated with interspecies electron transfer and quorum sensing communication in PD/A consortia after prolonged exposure to municipal wastewater. These findings underscore the significances of interspecific interactions, bacterial communications, and synergistic metabolic traits within PD/A biofilm systems in the stabilization of real municipal wastewater treatment.

Enhanced nitrogen removal from secondary effluent of municipal wastewater using denitrification filter: Feasibility of refractory organics as a carbon source

Conventional advanced nitrogen removal in municipal wastewater is hindered by the limited availability of carbon sources in the secondary effluent. However, refractory organics present in it had the potential to serve as intrinsic carbon sources after hydrolysis for nitrogen removal via simultaneous denitrification and partial-denitrification anammox (PDA) processes. To assess this potential, a denitrification filter was set up in this study to evaluate its feasibility of concurrent processes. Results showed that increasing influent ammonium (NH4+-N) from 1.0 to 7.0 mg/L increased total nitrogen (TN) removal from 52.4 % to 89.9 %. Simultaneous occurrence of PDA and denitrification process were confirmed by the actual chemical oxygen demand (COD) consumption (0.8–1.2 mg/mg TN removal) from non-fluorescent organics. The presence of the anammox, hydrolytic and denitrifying bacteria further supported the achievement of nitrogen removal through PDA and denitrification processes by utilizing hydrolytic products biodegraded from refractory organics.

Unique ecology of biofilms and flocs: Bacterial composition, assembly, interaction, and nitrogen metabolism within deteriorated bioreactor inoculated with mature partial nitrification-anammox sludge

This work unraveled discrepant ecological patterns between biofilms and flocs in a deteriorated bioreactor inoculated with mature partial nitrification-anammox (PN/A) sludge. Based on 16S rRNA analysis, a comprehensive evaluation of neutral and null models, along with niche width, delineated that the bacterial community assembly in biofilms and flocs was dominantly driven by the stochastic process, and dispersal limitation critically shaped the community assembly. Co-occurrence network analysis revealed that environmental stress caused decentralized and fragmented bacterial colonies, and anammox bacteria were mainly peripheral in biofilms network and less involved in interspecific interactions. Simultaneous PN/A and partial denitrification-anammox (PD/A) processes were identified, whereas PN and PD process primarily occurred in the biofilms and flocs, respectively, as evidenced by metagenomics. Collectively, these outcomes are expected to deepen the basic understanding of complex microbial community and nitrogen metabolism under environmental disturbance, thereby better characterizing and serving the artificial ecosystems.

Increasing biomass concentration facilitates simultaneous nitrogen removal and sludge reduction under low C/N conditions

To overcome the issues of limited carbon source and high sludge production in partial denitrification/anammox (PD/A) process, the effects of mixed liquor suspended solids (MLSS) and carbon/nitrogen ratio (C/N) on PD/A were investigated through parallel experiments. Nitrogen removal efficiencies decreased significantly when C/N was reduced (1.5 → 0.75). When MLSS was doubled, the nitrogen removal efficiencies in the two parallel reactors increased from 75.3 %, 72.9 % to 86.9 %, 89.7 %, respectively, and sludge yields decreased obviously. Combining with in-situ test, it was speculated when MLSS increased, fermentation was enhanced, providing substrate for partial denitrification. Thauera, involved in partial denitrification, decreased obviously with reduced C/N, but increased from 9.93 % to 38.16 % when MLSS doubled, which could promote the PD/A process. Terrimonas and Ignavibacterium (fermentative bacteria) increased from 1.26 %, 5.22 % to 6.62 %, 6.30 %, respectively. These results proved that increasing MLSS under low C/N ratios promoted fermentation in PD/A system, facilitating efficient nitrogen removal and sludge reduction.

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.

Efficient partial denitrification-anammox process enabled by a novel denitrifier with truncated nitrite reduction pathway

Partial denitrification coupled with anaerobic ammonium oxidation (PD-anammox) is a promising technology for cost-effective nitrogen removal from wastewater. Nitrite availability is crucial to anammox performance but often limited by the slow partial denitrification process. Here we report an efficient PD-anammox system driven by the novel denitrifier Bacillus velezensis C1–3 with truncated nitrite reduction pathway. Whole-genome sequencing analysis revealed that the lack of nitrite reductase genes nirS/nirK and norBC in strain C1–3 enabled nitrite accumulation without the need for precise control of carbon dosage. By coupling it with anammox sludge, over 79 % total nitrogen (TN) removal was stably achieved, under a TN loading rate of 660 mg/L/d and a carbon/nitrogen ratio below 1.0. Mechanism explorations indicate that the niche differentiation of C1–3 and anammox bacteria facilitated their mutualism while avoiding nitrite competition. This study demonstrates a novel strategy for establishing efficient PD-anammox process by harnessing the unique metabolic deficiency of denitrifiers, shedding light on the development of stable and sustainable biological nitrogen removal technologies with minimal carbon footprint.

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.

Mechanistic insight into assimilation capture: Implication for high-efficiency nitrogen removal from mature landfill leachate

The AO (Anoxic/oxic) process for treating high-strength ammonia in landfill leachate requires external carbon source and alkalinity. Alternatively, the processes of partial nitrification-Anammox (PNA) and partial denitrification-Anammox (PDA) with less or no carbon source have been developed, but enabled modulation difficulty due to nitrogen valence transformation. Ammonia assimilation for nitrogen removal immobilize ammonia (NH4+-N) as organic nitrogen, without nitrogen valence conversion and reducing greenhouse gases emission. This study offers a novel approach for nitrogen removal from low chemical oxygen demand (COD) to nitrogen (C/N) ratio leachate. The nitrogen removal response of biomass to ammonia assimilation in landfill leachate was established. Results demonstrated that the nitrogen removal contribution of ammonia assimilation and organics utilization by microorganisms significantly increased with biomass. When the initial mixed liquor suspended solid (MLSS) was 11200 mg/L, the removal efficiency of total nitrogen (TN) and COD in the landfill leachate achieved 98.42 ± 0.12 % and 80.60 ± 0.35 % after 30 days of operation, respectively. The corresponding assimilation contributions for TN and COD removals were 82.00 ± 0.96 % and 52.24 ± 0.82 %, respectively. Cell protein analysis showed that higher biomass significantly increased the production of intracellular and extracellular proteins or amino acids. The enrichment of bacterial genera such as Defluviicoccus, Aequorivita, Truepera, and Proteiniphilum played a crucial role in organics removal and NH4+-N assimilation. Functional gene analysis further demonstrated that most NH4+-N and organics were removed through bacterial assimilation. This research offered a green, efficient, and economical solution for the treatment of low C/N ratio landfill leachate.

Starvation resilience in anammox-based bioreactors: A stable nitrogen removal route on partial denitrification/anammox (PD/A)

This study investigates the performance, resilience and microbial community dynamics of two anaerobic processes, i.e. pure anammox (R1) and partial denitrification/anammox (PD/A) (R2), following a 30-day starvation period. The tolerance to starvation was assessed by comparing nitrogen removal efficiency and microbial activity across both reactors. Results show that the PD/A process recovery to pre-starvation performance levels within just one day, as compared to the pure anammox process. Notably, although the activity of anammox bacteria decreased in both processes during starvation, the decay rate in R1 was 69.59 % higher than in R2, potentially explaining the quicker recovery of R2. Furthermore, enhanced secretion of extracellular polymeric substance (EPS) during starvation served as a protective mechanism. The potential functions and genes in microorganisms, as well as the pathway of nitrogen cycling, were demonstrated through analyses using the KEGG database. This research reveals essential mechanistic insights and strategic guidance for the effective implementation of anammox-based biological nitrogen removal processes.

Carbon-efficient Nutrients removal from real municipal wastewater under conditions of highly variable influent quality and low temperature

In order to maximize carbon efficient nutrient removal of real primary effluent under the conditions of highly variable influent quality and low temperature, this study integrated four advanced bioprocesses, namely partial nitrification, endogenous denitrification, partial denitrification-anammox, enhanced biological phosphorus removal, into one advanced pilot design. The pilot system has been on-site operated for 212 days and was able to handle highly variable conditions with the aid of automatic feedback and feedforward controllers nested in secondary and tertiary treatment. Comparing to the case when full nitrification and full dentification technologies had to be used to meet the effluent requirement of total inorganic nitrogen (TIN) ≤ 3 mg/L and orthophosphate phosphorus ≤ 0.1 mg/L, the pilot results showed that 100 wt% carbon and 55.5 wt% oxygen could be saved in the secondary process and another 45.2 wt% carbon could be saved in the tertiary process. 44.5 wt% influent TIN was removed through endogenous denitrification in the secondary treatment with another 39.5 wt% TIN polished by partial denitrification-anammox in tertiary treatment. Because post-anoxic endogenous denitrification has removed majority of the NOX-N, it was concluded that the energy intensive mixed liquor recirculation that has been popular in traditional design for pre-anoxic zone denitrification can be eliminated.

Simultaneous hydroxyapatite-based phosphorus recovery and partial denitrification-anammox-based nitrogen removal during sludge leachate treatment

Anammox has been widely applied for treating high ammonia‑nitrogen leachate, but challenges remain due to the phosphorus in the wastewater and by-products nitrate (NO3−). Therefore, we combined partial denitrification-anammox and hydroxyapatite crystallization within a single UASB reactor. This method achieved an effluent with a 4.27 ± 0.45 mg TN/L and a maximum PO43− removal efficiency of 68.31 ± 5.42 %. The addition of excess calcium led to an increase in phenylalanine-like substances, the humification index (HIX) of extracellular polymeric substances (EPS), and the MLVSS/MLSS ratio. These changes indicated that hydroxyapatite coated the initial granular sludge, causing it to crack and re-granulate, but the MLVSS didn't decrease significantly. Therefore, calcium addition didn't significantly impact the function of biomass. Metagenomic analysis revealed that by-products NO3− was removed through partial denitrification using in-situ refractory dissolved organic matters in SL. The formation of hydroxyapatite crystals is the main pathway for PO43− removal, and biological process also contributed to some PO43− removal. Overall, our study provided a viable solution for the simultaneous removal of nitrogen and recovery of phosphorus from high ammonia‑nitrogen wastewater.

Metagenomic unveils the promotion of mainstream PD-anammox process at lower nZVI concentration and inhibition at higher dosage

The partial-denitrification-anammox (PdNA) process exhibits great potential in enabling the simultaneous removal of NO3−-N and NH4+-N. This study delved into the impact of exogenous nano zero-valent iron (nZVI) on the PdNA process. Adding 10 mg L−1 of nZVI increased nitrogen removal efficiency up to 83.12 % and maintained higher relative abundances of certain beneficial bacteria. The maximum relative abundance of Candidatus Brocadia (1.6 %), Candidatus Kuenenia (1.5 %), Ignavibacterium (1.3 %), and Azospira (1.2 %) was observed at 10 mg L−1 of nZVI. However, the greatest relative abundance of Thauera (1.3 %) was recorded under 50 mg L−1. Moreover, applying nZVI selectively enhanced the abundance of NO3−-N reductase genes. So, keeping the nZVI concentration at 10 mg L−1 or below is advisable to ensure a stable PdNA process in mainstream conditions. Considering nitrogen removal efficiency, using nZVI in the PD-anammox process could be more cost-effective in enhancing its adoption in industrial and mainstream settings.

Treatment of low-C/N nitrate wastewater using a partial denitrification-anammox granule system: Granule reconstruction, stability, and microbial structure analyses

Industrial wastewater discharged into sewer systems is often characterized by high nitrate contents and low C/N ratios, resulting in high treatment costs when using conventional activated sludge methods. This study introduces a partial denitrification-anammox (PD/A) granular process to address this challenge. The PD/A granular process achieved an effluent TN level of 3.7 mg/L at a low C/N ratio of 2.3. Analysis of a typical cycle showed that the partial denitrification peaked within 15 min and achieved a nitrate-to-nitrite transformation ratio of 86.9%. Anammox, which was activated from 15 to 120 min, contributed 86.2% of the TN removal. The system exhibited rapid recovery from post-organic shock, which was attributed to significant increases in protein content within TB-EPS. Microbial dispersion and reassembly were observed after coexistence of the granules, with Thauera (39.12%) and Candidatus Brocadia (1.25%) identified as key functional microorganisms. This study underscores the efficacy of PD/A granular sludge technology for treating low-C/N nitrate wastewater.

Unraveling the role of formate in improving nitrogen removal via coupled partial denitrification-anammox

Unraveling the role of formate in improving nitrogen removal via coupled partial denitrification-anammox

Response and self-regulation of PD/A granular sludge to oxytetracycline stress

This paper investigated the operational characteristics and self-regulation mechanism of the partial denitrification/anammox (PD/A) granular system under the stress of oxytetracycline (OTC), an emerging pollutant that accumulates in municipal wastewater treatment plants through various pathways, posing significant challenges for its future promotion in engineering applications. The results indicated that OTC concentrations below 100 mg/L intensified its short-term inhibition on the PD/A granular sludge system, decreasing functional bacterial activity, while between 150 and 300 mg/L, PD's NO3−-N to NO2−-N conversion ability diminished, and Anammox activity was significantly suppressed. Under long-term high OTC stress (20–30 mg/L), nitrogen removal suffered, and batch tests revealed significant inhibition of PD's NO3−-N to NO2−-N conversion, dropping from 73.77 % to 50.17 %. Anammox bacteria activity sharply declined from 1.81 to 0.39 mg N/gVSS/h under OTC stress. Extracellular polymeric substances (EPS) content rose from 185.39 to 210.86 mg/gVSS, indicating PD/A sludge's self-protection mechanism. However, EPS content fell due to cell lysis at high OTC (30 mg/L). The decreasing relative abundance of Candidatus_Brocadia (2.32 % to 0.93 %) and Thaure (12.63 % to 7.82 %) was a key factor in the gradual deterioration of denitrification performance. This study was expected to provide guidance for the PD/A process to cope with the interference of antibiotics and other emerging pollutants (short-term shock and long-term stress).

Influence of temperature on performance and mechanism of advanced synergistic nitrogen removal in lab-scale denitrifying filter with biogenic manganese oxides

Temperature is a significant operational parameter of denitrifying filter (DF), which affects the microbial activity and the pollutants removal efficiency. This study investigated the influence of temperature on performance of advanced synergistic nitrogen removal (ASNR) of partial-denitrification anammox (PDA) and denitrification, consuming the hydrolytic and oxidation products of refractory organics in the actual secondary effluent (SE) as carbon source. When the test water temperature (TWT) was around 25, 20, 15 and 10 °C, the filtered effluent total nitrogen (TN) was 1.47, 1.70, 2.79 and 5.52 mg/L with the removal rate of 93.38%, 92.25%, 87.33% and 74.87%, and the effluent CODcr was 8.12, 8.45, 10.86 and 12.29 mg/L with the removal rate of 72.41%, 66.17%, 57.35% and 51.87%, respectively. The contribution rate of PDA to TN removal was 60.44%∼66.48%, and 0.77–0.96 mg chemical oxygen demand (CODcr) was actually consumed to remove 1 mg TN. The identified functional bacteria, such as anammox bacteria, manganese oxidizing bacteria (MnOB), hydrolytic bacteria and denitrifying bacteria, demonstrated that TN was removed by the ASNR, and the variation of the functional bacteria along the DF layer revealed the mechanism of the TWT affecting the efficiency of the ASNR. This technique presented a strong adaptability to the variation of the TWT, therefore, it has broad application prospect and superlative application value in advanced nitrogen removal of municipal wastewater.

Influence of hydraulic retention time on advanced synergistic nitrogen removal using internal carbon source

Hydraulic retention time (HRT) is a significant operational eter of denitrifying filter (DF). The influence of HRT on the performance and mechanism of advanced synergistic nitrogen removal (ASNR) of partial-denitrification anammox (PDA) and denitrification was investigated, consuming the hydrolytic and oxidation products of refractory organics in the secondary effluent (SE) as carbon source. When the HRT was 8, 6, 4 and 2 h, the filtered effluent total nitrogen (TN) was 1.47, 3.47, 4.17 and 4.49 mg/L, and the effluent CODcr was 8.12, 9.95, 9.40 and 9.87 mg/L, respectively. Removing 1 mg of TN actually consumed 0.87–0.96 mg CODcr, and the contribution rate of PDA to TN removal was 62.10–70.02 %. A variety of manganese oxidizing (MnOB), hydrolytic, anammox and denitrifying bacteria were identified, and their abundance was obviously affected by the HRT. The lack of carbon source was the main factor limiting the ASNR efficiency, rather than the HRT.

Rapid start-up and operational characteristics of partial denitrification coupled with anammox driven by innovative strategies

The Partial Denitrification-Anammox (PD/A) process established a low-consumption, efficient and sustainable pathway for complete nitrogen removal, which is of great interest to the industry. Rapid initiation and stable operation of the PD/A systems were the main issues limiting its engineering application in wastewater nitrogen removal. A PD/A system was initiated in a continuous stirred-tank reactors (CSTRs) in the presence of low concentration of organic matter, and the effects of organic matter types and COD/NO3−-N ratios on the performance of the PD/A system, and microbial community characteristics were explored. The results showed that low concentrations of organic matter could promote the rapid initiation of the Anammox process and then the strategy of gradually replacing NO2−-N with NO3−-N could successfully initiate the PD/A system at 70 days. The type of organic matter had a significant effect on the initiation of the Anammox and the establishment of the PD/A system. Compared to glucose, sodium acetate was more favorable for rapid start-up and the synergy among microorganisms, and organic matter was lower, with an optimal COD/NO3−-N ratio of 3.0. Microorganisms differed in their sensitivity to environmental factors. The relative abundance of Planctomycetota and Proteobacteria in R2 was 51 %, with the presence of three typical anammox bacteria, Candidatus_Brocadia, Candidatus_Kuenenia, and Candidatus_Jettenia in the system. This study provides a new strategy for the rapid initiation and stable operation of the PD/A process.

Rapid establishment of municipal sewage partial denitrification-anammox for nitrogen removal through inoculation with side-stream anammox biofilm without domestication

Mainstream partial denitrification anammox was achieved through inoculation of side-stream mature partial nitritation anammox biofilm without domestication. The contribution of anammox to nitrogen removal was 29.4 %. Moreover, prolonging anoxic hydraulic retention time and introducing side-stream nitrite under different carbon/nitrogen ratios enriched anammox bacteria. The abundance of anammox bacteria increased by ∼ 10 times ((2.19 ± 0.17) × 1012 copies gene / g dry sludge) with a total relative abundance of 18.51 %. During 258 days of operation, the contribution of anammox to nitrogen removal gradually increased to 68.8 %. The total nitrogen in the effluent decreased to 8.84 mg/L with a total nitrogen removal efficiency of 76.4 % under a carbon/nitrogen ratio of 3. This paper proposes a novel way to rapidly achieve mainstream partial denitrification anammox via inoculation with side-stream mature partial nitritation anammox biofilm. This method achieves advanced nitrogen removal from municipal wastewater, even under low carbon/nitrogen ratios.

Interactions and coupling mechanisms between Anammox, partial denitrification (PD) and hydroxyapatite (HAP) biomineralization in a PD/A-HAP system

In this study, hydroxyapatite (HAP) crystallization was introduced into a partial denitrification/Anammox (PD/A) process to develop PD/A-HAP granules and achieve simultaneous removal of nitrogen and phosphorus from wastewater. During the formation of PD/A-HAP granules, an unbalanced relationship between the biomass growth and phosphorus crystallization was observed, which resulted in a mass transfer inhibition inside the system. PD and Anammox activities were both impacted by the impeded nutrient contact, and PD process was revealed to be more susceptible to inhibition. Through the regulation of COD and NO3–-N, a dynamic balance between microbial proliferation and phosphorus crystallization was achieved. As a result, high nitrogen removal efficiency (NRE) was attained at 97.8±1.2 % in the final stage of operation, accompanied by a phosphorus removal efficiency (PRE) of 59.4±2.9 %. Furthermore, the recovered phosphorus was transformed into HAP crystals and encapsulated by the PD/A biomass to form granules, which greatly promoted the settling performance of the sludge. This study underlines the importance of reconciling biomass and HAP growth for a strengthened PD/A granular sludge and an enhanced nitrogen removal system. The constructed PD/A-HAP process will expand the application of PD/A with its ability to remove phosphorus while efficiently removing nitrogen.