Nitrogen Removal Pathway Research Articles

Mainstream deammonification at ambient temperature treating real sewage by a plug-flow fixed-bed reactor based on zeolite/tourmaline-modified polyurethane carriers

A plug-flow fixed-bed reactor (PFBR) with zeolite/tourmaline-modified polyurethane (ZTP) carriers (PFBRZTP) was constructed to realize mainstream deammonification for real domestic sewage treatment. The PFBRZTP and PFBR were operated for 111 days treating aerobically pretreated sewage in parallel. A higher nitrogen removal rate of 0.12 kg N·(m3·d)−1 was achieved in PFBRZTP despite lowering the temperature (16.8–19.7 °C) and fluctuating water quality. Meanwhile, it was indicated that anaerobic ammonium oxidation dominated (64.0 ± 13.2%) in PFBRZTP, by nitrogen removal pathway analysis and high anaerobic ammonium-oxidizing bacteria (AnAOB) activity (2.89 mg N·(g VSS·h)−1). And, the lower protein/polysaccharides (PS) ratio further indicated a better biofilm structure in PFBRZTP owing to a higher abundance of microorganisms relevant to PS and cryoprotective EPS secretion. Furthermore, partial denitrification was an important nitrite supply process in PFBRZTP based on low AOB activity/AnAOB activity ratio, higher Thauera abundance and a remarkably positive correlation between Thauera abundance and AnAOB activity.

Improvement of nitrate-dependent anaerobic ferrous oxidation by EDTA-2Na addition: An effective way for dealing with iron crusts

The nitrate-dependent anaerobic ferrous oxidation (NAFO) is a newly discovered biological denitrification process. However, the formation of iron crusts on the surface of microorganisms by residual ferric iron poses a significant challenge to the maintenance of NAFO performance. To solve the aforementioned issue, this study set up two continuous NAFO systems with (R2) and without EDTA-2Na (R1), and investigated the impact of EDTA-2Na on nitrate removal and microbial community. After 91 days of operation, EDTA-2Na demonstrated the ability to improve the ferrous utilization rate in NAFO process, and the average effluent ferrous concentration was 15.71 mg/L in R2, which was superior to the 50.03 mg/L of R1. Compared with the conventional system, R2 increased nitrate removal by 17 % and stabilized the growth of microorganisms by mitigating iron crusts in the long-term operation. Batch experiments indicated that the optimal nitrate removal efficiency was achieved at an initial Fe/N of 3.24 and a pH of 6.42. High-throughput analysis implied that the reactor with EDTA-2Na contained numerous nitrogen removal pathways. This study provided a practical method for the long-term stable operation of a NAFO system.

Revealing the response of community structure and metabolic pathway to varying organic matter stress in a dissolved oxygen-differentiated airlift internal circulation partial nitritation-anammox system

In practice, the influent organic matter is often pre-treated to reduce the impact on partial nitritation-anammox (PNA) process. However, the influent organics may also drive the denitrification process and improve total nitrogen removal efficiency of the PNA process. Thus, we designed and operated a novel dissolved oxygen-differentiated airlift internal circulation PNA (PNA-DOAIC) system in this study at various influent C/N ratios of 0–4.0. Nitrogen removal performance, microbial activity and community, and metabolic pathways in response to varying organic matter stress were investigated via the continuous experiment combined with batch test. The results showed that the optimum influent C/N ratio was 2.0 in this system, and the efficient and stable operation was still maintained at the C/N ratios of 0–3. At this time, the TN removal efficiency and removal rate could reach 95.1 % and 0.93 kg-N/m3/d, respectively, while COD efficiency remained at 95.4 %. Efficient removal performance was achieved via the PNA coupled with denitrification. However, the anammox bacteria (AnAOB) activity and abundance declined persistently as the influent C/N ratio was further raised, and heterotrophic bacteria gradually replaced AnAOB as dominate genus. Meanwhile, metabolic functions involving the material exchange and organic degradation were significantly enhanced. Nitrogen removal pathways changed from PNA to the nitrification-denitrification process. This study provides deep insights into effects of organic matter on the PNA process and can expand the application scope of this novel PNA-DOAIC bioreactor.

Deciphering the effect of temperature reduction on the biofilm in partial nitritation-anammox system: Insights from microbial community to metabolic pathways

The concept of partial nitritation-anammox (PN/A) process has been proposed for many years, however seasonal temperatures and low ammonia concentrations under mainstream conditions always challenge the application in practical engineering. Thus, we operated a PN/A moving bed biofilm reactor (PN/A-MBBR) for treating 50 mg-NH4/L wastewater at various temperatures of 35 °C, 25 °C, and 15 °C. The effect of temperature reduction on biofilm system was investigated from process performance, microbial community and activity, to metabolic pathways. The results showed that total nitrogen removal efficiency declined from 76.6 % to 48.6 % as the temperature was reduced from 35 °C to 15 °C, while nitrogen removal rate decreased to 0.28 kg-N/m3/d. Low temperature stimulated the secretion of polysaccharide in loosely bound-EPS and protein in tightly bound-EPS. Meanwhile, specific activity of anammox declined from 1.53 to 0.56 g-N/g-VSS/d, and the relative abundance of Candidatus Brocadia dropped from 20.1 % to 9.4 %. Temperature reduction had significant effect on the expression of functional genes (hzsA and hdh) for anammox reaction. Nitrification-denitrification process was enhanced during low temperatures and recovery phases. Biofilm system might respond to temperature reduction by the decline of substance transport and energy consumption, as well as the enhancement of biofilm formation and diversity of nitrogen removal pathways. The findings of this study provide a deep insight into the effect of temperature reduction on PN/A process, also promote the practical applications of mainstream PN/A in the future.

Characterization and removal mechanism of a novel enrofloxacin-degrading microorganism, Microbacterium proteolyticum GJEE142 capable of simultaneous removal of enrofloxacin, nitrogen and phosphorus

In the study, a novel ENR-degrading microorganism, Microbacterium proteolyticum GJEE142 was isolated from aquaculture wastewater for the first time. The ENR removal of strain GJEE142 was reliant upon the provision of limited additional carbon source, and was adaptative to low temperature (13 ℃) and high salinity (50‰). The ENR removal process, to which intracellular enzymes made more contributions, was implemented in three proposed pathways. During the removal process, oxidative stress response of strain GJEE142 was activated and the bacterial toxicity of ENR was decreased. Strain GJEE142 could also achieve the synchronous removal of ammonium, nitrite, nitrate and phosphorus with the nitrogen removal pathways of nitrate → nitrite → ammonium → glutamine → glutamate → glutamate metabolism and nitrate → nitrite → gaseous nitrogen. The phosphorus removal was implemented under complete aerobic conditions with the assistance of polyphosphate kinase and exopolyphosphatase. Genomic analysis provided corresponding genetic insights for deciphering removal mechanisms of ENR, nitrogen and phosphorus. ENR, nitrogen and phosphorus in both actual aquaculture wastewater and domestic wastewater could be desirably removed. Desirable adaptation, excellent performance and wide distribution will make strain GJEE142 the hopeful strain in wastewater treatment.

Removal efficacy of fly ash composite filler on tailwater nitrogen and phosphorus and its application in constructed wetlands.

Constructed wetlands (CWs) have been widely used in tailwater treatment. However, it is difficult to achieve considerable removal efficiency of nitrogen and phosphorus in tailwater solely by CWs-an efficient green wetland filler is also important. This study investigated 160 domestic sewage treatment facilities (DSTFs) in rural areas from two urban areas in Jiaxing for TP and NH3-N and found that TP and NH3-N concentrations in rural domestic sewage (RDS) in this plain river network are still high. Therefore, we selected a new synthetic filler (FA-SFe) to enhance nitrogen and phosphorus reduction, and we discuss the importance of filler in constructed wetlands. Experiments revealed the adsorption capacity of the new filler: the maximum adsorption amounts of TP and NH3-N reached 0.47g m-2 d-1 and 0.91gm-2 d-1, respectively. The application potential of FA-SFe was verified in actual wastewater treatment, with the removal rates of ammonia nitrogen and TP reaching 71.3% and 62.7%, respectively. This study provides a promising pathway for nitrogen and phosphorus removal from rural tailwaters.

Insight into the roles of microalgae on simultaneous nitrification and denitrification in microalgal-bacterial sequencing batch reactors: Nitrogen removal, extracellular polymeric substances, and microbial communities

This study explored the influence and mechanism of microalgae on simultaneous nitrification and denitrification (SND) in microalgal-bacterial sequencing batch reactors (MB-SBR). It particularly focused on nitrogen transformation in extracellular polymeric substances (EPS) and functional groups associated with nitrogen removal. The results showed that MB-SBR achieved more optimal performance than control, with an SND efficiency of 68.01% and total nitrogen removal efficiency of 66.74%. Further analyses revealed that microalgae changed compositions and properties of EPS by increasing EPS contents and improving transfer, conversion, and storage capacity of nitrogen in EPS. Microbial community analysis demonstrated that microalgae promoted the enrichment of functional groups and genes related to SND and introduced diverse nitrogen removal pathways. Moreover, co-occurrence network analysis elucidated the interactions between communities of bacteria and microalgae and the promotion of SND by microalgae as keystone connectors in the MB-SBR. This study provides insights into the roles of microalgae for enhanced SND.

Efficient nitrogen removal of a novel Pseudomonas chengduensis strain BF6 mainly through assimilation in the recirculating aquaculture systems

Biological nitrogen removal has received increasing attention in wastewater treatment. A bacterium with excellent nitrogen removal performance was isolated from biofilters of recirculating aquaculture systems (RAS) and identified as Pseudomonas chengduensis BF6. It was indicated that inorganic nitrogen is transformed into gaseous and biological nitrogen by the metabolic pathways of denitrification, anammox, and assimilation, which is the main nitrogen removal pathway of strain BF6. The strain BF6 could effectively remove nitrogen within 24 h under the conditions of ammonia, nitrate, nitrite, and mixed nitrogen sources with maximum total nitrogen removal efficiencies reaching 97.00 %, 61.40 %, 79.10 %, and 84.98 %, respectively. The strain BF6 exhibited total nitrogen removal efficiency of 91.14 %, altered the microbial diversity and enhanced the relative abundance of Pseudomonas in the RAS biofilter. These findings demonstrate that Pseudomonas sp. BF6 is a highly efficient nitrogen-removing bacterium with great potential for application in aquaculture wastewater remediation.

Performance of simultaneous carbon and nitrogen removal of high-salinity wastewater in heterotrophic nitrification-aerobic denitrification mode

In this work, heterotrophic nitrification-aerobic denitrification (HN-AD) mode was successfully established after a 105-day acclimation in a sequencing batch reactor (SBR) system using mustard tuber wastewater (MTWW) as substrate. The performance of simultaneous carbon and nitrogen removal (SCNR) was investigated using manual wastewater within the salinity from 1% to 6%. Results indicate that high salinity caused little effect on carbon removal, while it brought a significant influence on nitrogen removal. At the salinity of 1% and 3%, the heterotrophic nitrification rates were 2.83 and 3.96-fold of those of the autotrophic nitrification, respectively. As the salinity was up to 6%, the autotrophic nitrification was significantly inhibited, while the anoxic denitrification was slightly affected and the aerobic denitrification became dominant. The analysis on microbial community structure of the acclimated wastewater sludge reveals that the genera with autotrophic nitrification were gradually eliminated with an increasing salinity during the acclimation. Paracoccus, Pseudomonas, Alcaligenes and Bacillus were the dominant genera related to HN-AD process. The optimal operation parameters for SCNR were investigated, including C/N ratio (25), DO concentration (6 mg/L) and temperature (35 °C). This work provides an insight into the migration of nitrogen removal pathway and the succession of HN-AD microbial community in the process of high-salt wastewater biotreatment.

Metagenomic analysis revealed the mechanism of extracellular polymeric substances on enhanced nitrogen removal in coupled MABR systems with low C/N ratio containing salinity

A combined membrane-aerated biofilm reactor (MABR) was conducted to treat various C/N ratios with saline wastewater. The influence of C/N on nitrogen removal property, extracellular polymeric substances (EPS) features, bacterial community and relevant functional genes were investigated. Fourier-transform infrared spectrum (FTIR), three-dimensional exaction and emission matrix (3D-EEM) fluorescence spectra and metagenomics analysis were conducted to study the mechanism of EPS for enhanced nitrogen removal at low C/N ratio. The results showed that TN removal rate of the whole MABR system were 73.21%, 69.51% and 67.04%, respectively, with the C/N ratio of 6, 4.5 and 3. MABR-2 biofilms secreted more EPS under low C/N and high-salinity conditions to maintain intracellular and extracellular osmotic pressure balance and to participate in cellular metabolic processes. Meanwhile, under the stimulation of low C/N, MABR-2 system with low influent loading was in a “starvation state”, thus EPS could be utilized by the biofilm of MABR-2 system as carbon source to enhance nitrogen removal. Besides, metagenomics analysis showed that aerobic denitrification and dissimilatory nitrite reduction to ammonia (DNRA) were dominant nitrogen removal pathways in MABR-2 system at low C/N ratio. EPS might participate in related metabolic processes in the electron donors and energy production processes as well as the electron transfer pathways in denitrification system. This work provided a feasible practical application scheme for the enhanced TN removal of low C/N ratio containing salinity.

Simultaneous removal of typical antibiotics and nitrogen by SWIS assisted by iron carbon micro-electrolysis

In this study, a combination of subsurface wastewater infiltration system (SWIS) with iron carbon micro-electrolysis (namely ICMESWIS) was used to enhance the simultaneous removal of nitrogen and typical antibiotics (SMX, OTC and HCl-Lev was selected). The results indicated that assisted by 10 % dopant amount of ICME, SMX, OTC, Lev-HCl, NH4+-N, NO3--N and NO2--N removal rates increased by 26.6 %, 19.7 %, 10.2 %, 33.7 %, 28.5 % and 19.3 %, respectively via ICMESWIS than SWIS. The higher TFe release rate, organic matter content and richer microbial populations encouraged ICMESWIS to resist the impact of antibiotics. High-throughput sequencing revealed that electrochemical effect enhanced the microbial community structure, which was the main pathway for nitrogen and antibiotics removal. The total proportions of nitrogen and antibiotics removal relating bacteria were 18.38 % and 46.67 %, 5.49 % and 4.72 % higher than those of SWIS, respectively. Anammox became an additional N removal way in ICMESWIS.

Reactive and microbial inhibitory mechanisms depicting the panoramic view of pH stress effect on common biological nitrification

pH is a crucial factor of microbial nitrification, which often combines with high-strength ammonium to influence nitrogen removal pathway in wastewater treatment. However, the detailed inhibitory mechanisms of pH stress are not sufficiently disclosed yet. In this study, the pH stress effect on nitrification was comprehensively studied by a set of experiments which identified the reactivity of nitrification processes and activity of nitrifiers, the time dependence of inhibition effect and the hybrid pH stress effect with ammonium. The results revealed two distinct inhibitory mechanisms dominating in alkaline and acid ranges. In alkaline range (pH > 8), pH stress causes physiological damages on microorganisms which is named as microbial inhibition. It has the features of less recoverability of nitrifiers, time-dependent inhibition effect and low pH-tolerance of nitrite oxidation bacteria. Free ammonia enhanced microbial inhibition and greatly promoted nitrite accumulation. A novel reactive inhibition mechanism dominated in acid range (pH < 7) was disclosed. It only impedes ammonia oxidation process (AOP) but not impair microbial activity obviously and the effect is time-independent. The mechanism was clarified from H+ transport because AOP involved H+ production. The H+ transport was impeded under acid stress owing to the decrease of pH gradient across cell membrane. The two mechanisms formed a panoramic view of pH stress effect on nitrification advancing the understanding of nitrifier adaptability and nitritation regulation in wastewater treatment processes.

Quantification of enhanced nitrogen removal pathways of pyrite interaction with anammox sludge system

Fe and S could improve the anammox performance by coupling with the N cycle. However, the coexistence of Fe and S complicates the metabolic process, troubling the quantification of nitrogen removal pathways. Herein, the nitrogen removal pathways were quantificationally analyzed by 15N stable isotope tracing and elaborate batch tests, and the enhancing mechanism was illuminated by physiological and metatranscriptome analyses in pyrite (FeS2)-amended anammox system. We found that anammox, denitrification and Feammox (NH4+ oxidation coupled with Fe(III) reduction) contributed to 80.22 %, 19.53 % and 0.25 % nitrogen removal in this process. With pyrite addition, Fe, S release and the extracellular polymeric substances secretion were responsible for the enhancement of nitrite and nitrate removal via chemical, autotrophic and heterotrophic denitrification, improving nitrogen removal by 14 % and 19 %. The network analyses further confirmed that the multiple nitrogen removal pathways (i.e., anammox, Feammox and denitrification) contribute to pyrite-induced nitrogen removal enhancement in the anammox system. This work enables the in-depth understanding, future design and optimization of the pyrite-amended anammox system, providing a fundamental basis for developing Fe- and S-based anammox enhancement technologies.

Biofilm stratification and autotrophic-heterotrophic interactions in a structured bed reactor (SBRIA) for carbon and nitrogen removal

The structured-bed reactor with intermittent aeration (SBRIA) is a promising technology for simultaneous carbon and nitrogen removal from wastewater. An in depth understanding of the microbiological in the reactor is crucial for its optimization. In this research, biofilm samples from the aerobic and anoxic zones of an SBRIA were analyzed through 16S rRNA sequencing to evaluate the bacterial community shift with variations in the airflow and aeration time. The control of the airflow and aeration time were essential to guarantee reactor performances to nitrogen removal close to 80%, as it interfered in nitrifying and denitrifying communities. The aeration time of 1.75 h led to establishment of different nitrogen removal pathways by syntrophic relationships between nitrifier, denitrifier and anammox species. Additionally, the predominance of these different species in the internal and external parts of the biofilm varied according to the airflow.

Metagenomics reveals the microbial community and functional metabolism variation in the partial nitritation-anammox process: From collapse to recovery

Mainstream partial nitritation-anammox (PNA) process easily suffers from performance instability and even reactor collapse in application. Thus, it is of great significance to unveil the characteristic of performance recovery, understand the intrinsic mechanism and then propose operational strategy. In this study, we combined long-term reactor operation, batch tests, and metagenomics to reveal the succession of microbial community and functional metabolism variation from system collapse to recovery. Proper aeration control (0.10-0.25 mg O2/L) was critical for performance recovery. It was also found that Candidatus Brocadia became the dominant flora and its abundance increased from 3.5% to 11.0%. Significant enhancements in carbon metabolism and phospholipid biosynthesis were observed during system recovery, and the genes abundance related to signal transduction was dramatically increased. The up-regulation of sdh and suc genes showed the processes of succinate dehydrogenation and succinyl-CoA synthesis might stimulate the production of amino acids and the synthesis of proteins, thereby possibly improving the activity and abundance of AnAOB, which was conducive to the performance recovery. Moreover, the increase in abundance of hzs and hdh genes suggested the enhancement of the anammox process. Changes in the abundance of key genes involved in nitrogen metabolism indicated that nitrogen removal pathway was more diverse after system recovery. The achievement of performance recovery was driven by anammox, nitrification and denitrification coupled with dissimilatory nitrate reduction to ammonium. These results provide deeper insights into the recovery mechanism of PNA system and also provide a potential regulation strategy for the stable operation of the mainstream PNA process.

Isolation and characteristics of two heterotrophic nitrifying and aerobic denitrifying bacteria, Achromobacter sp. strain HNDS-1 and Enterobacter sp. strain HNDS-6

In order to solve nitrogen pollution in environmental water, two heterotrophic nitrifying and aerobic denitrifying strains isolated from acid paddy soil were identified as Achromobacter sp. strain HNDS-1 and Enterobacter sp. strain HNDS-6 respectively. Strain HNDS-1 and strain HNDS-6 exhibited amazing ability to nitrogen removal. When (NH4)2SO4, KNO3, NaNO2 were used as nitrogen resource respectively, the NH4+-N, NO3−-N, NO2−-N removal efficiencies of strain HNDS-1 were 93.31%, 89.47%, and 100% respectively, while those of strain HNDS-6 were 82.39%, 96.92%, and 100%. And both of them could remove mixed nitrogen effectively in low C/N (C/N = 5). Strain HNDS-1 could remove 76.86% NH4+-N and 75.13% NO3−-N. And strain HNDS-6 can remove 65.07% NH4+-N and 78.21% NO3−-N. A putative ammonia monooxygenase, nitrite reductase, nitrate reductase, assimilatory nitrate reductase, nitrate/nitrite transport protein and nitric oxide reductase of strain HNDS-1, while hydroxylamine reductase, nitrite reductase, nitrate reductase, assimilatory nitrate reductase, nitrate/nitrite transport protein, and nitric oxide reductase of strain HNDS-6 were identified by genomic analysis. DNA-SIP analysis showed that genes Nxr, narG, nirK, norB, nosZ were involved in nitrogen removal pathway, which indicates that the denitrification pathway of strain HNDS-1 and strain HNDS-6 was NO3−→NO2−→NO→N2O→N2 during NH4+-N removal process. And the nitrification pathway of strain HNDS-1 and strain HNDS-6 was NO2−→NO3−, but the nitrification pathway of NH4+→ NO2− needs further studies.

Start-up phase optimization of pyrite-intensified hybrid sequencing batch biofilm reactor (PIHSBBR): Mixotrophic denitrification performance and mechanism

Pyrite-based autotrophic denitrification (PAD) is an emerging biological process to diminish nitrate pollution, but the relatively low NO3−-N removal rate limits its practical application. In this research, a pyrite-intensified hybrid sequencing batch biofilm reactor (PIHSBBR) was designed to treat low C/N ratio domestic wastewater. The results showed that PIHSBBR could achieve optimal removal of COD, NH4+-N, and TN under the aeration rate of 1.0 L/L∙min and the hydraulic retention time (HRT) of 8 h, with removal rates of 69.67 ± 4.37%, 77.04 ± 4.84%, and 63.92 ± 6.66%, respectively. The PAD efficiency in PIHSBBR during the stable operation was not high (13.05–31.01%), and the main nitrogen removal pathway in PIHSBBR, especially in the aerobic zone, was simultaneous nitrification and denitrification (SND). High-throughput sequencing analysis unraveled that Planctomycetota (3.65%) had a high abundance in the anoxic zone of PIHSBBR, implying that anaerobic ammonium oxidation (anammox) might have occurred in the anoxic zone. In addition, the nitrogen cycle function gene with the highest abundance was nirBD, indicating the possible presence of dissimilatory nitrate reduction to ammonium (DNRA) within the system (aerobic and anoxic zones). Our research can provide useful information for the improvement and future application of PIHSBBR.

Impact of aeration frequency on performance of mixotrophic sequencing batch biofilm reactor (SBBR) treating real domestic wastewater: Removal efficiency, pathways, and mechanisms

Real domestic wastewater usually contains multiple pollutants including nitrogen, phosphorus and organics, which are not easy to be polished by a single process due to the lack of electron donors for denitrification, especially for low carbon to nitrogen (C/N) ratio wastewater. Autotrophic denitrification with inorganic substances as electron donors is a potent pathway for nitrogen removal and can be coupled with the heterotrophic process to achieve high performance. In this study, three double-layered mixotrophic sequencing batch biofilm reactors (SBBRs) involving pyrite-based autotrophic denitrification (PAD) were constructed to treat real domestic wastewater under different aeration frequencies. By adjusting the frequency of intermittent aeration to 16 times per operating cycle, the mean percent removals of ammonium-nitrogen (NH4+-N), total nitrogen (TN), and orthophosphate (PO43--P) reached 83.74 ± 6.95%, 54.41 ± 4.42%, and 90.45 ± 9.70%, respectively. Meanwhile, the increase of aeration frequency enhanced the removal of NH4+-N and PO43--P from the system, but high aeration frequency had an inhibitory effect on both heterotrophic denitrification and the PAD process. The 16S rRNA sequencing analysis revealed that the dominant taxa appeared in the system were Microscillaceae_unclassified, Nitrospira, Thiobacillus, and Candidatus_Competibacter, etc., which were contributable to the multiple nitrogen metabolism. The relative abundance of genes involved in complete nitrification, denitrification, assimilatory nitrate reduction to ammonium (ANRA), and dissimilatory nitrate reduction to ammonium (DNRA) was predicted based on PICRUSt2. In addition, two sulfur metabolic pathways, i.e. the sulfur oxidation (Sox) and sulfide:quinone oxidoreductase (SQR) pathways were found in the system. These findings might provide new insight into the mechanism of nutrient removal in PAD-based mixotrophic denitrification systems treating real domestic wastewater in an intermittent aeration mode.

Evaluation of the stability of shortcut nitrification-denitrification process based on online specific oxygen uptake rate monitoring

Shortcut nitrification-denitrification (SCND) is widely concerned because of its low energy consumption and high nitrogen removal efficiency. However, the current difficulty lies in the stable maintenance of SCND performance, which leads to the challenge of large-scale application of this new denitrification technology. In this study, the nitrogen removal pathway from complete nitrification-denitrification (CND) to SCND was rapidly realized under high free ammonia (FA), high pH and low dissolved oxygen (DO) conditions. The variations of specific oxygen uptake rate (SOUR) of activated sludge in both processes were investigated by an online SOUR monitoring device. Different curves of SOUR from CND to SCND process were observed, and the ammonia peak obtained based on SOUR monitoring could be used to control aeration time accurately in SCND process. Accordingly, the SOUR ratio of ammonia oxidizing bacteria (AOB) to nitrite oxidizing bacteria (NOB) (SOURAOB/SOURNOB) was increased from 1.40 to 2.93. 16S rRNA Miseq high throughput sequencing revealed the dynamics of AOB and NOB, and the ratio of relative abundance (AOB/NOB) was increased from 1.03 to 3.12. Besides, SOURAOB/SOURNOB displayed significant correlations to ammonia removal rate (P < 0.05), ammonia oxidation rate / nitrite oxidation rate (P < 0.05), nitrite accumulation rate (P < 0.05) and the relative abundance of AOB/NOB (P < 0.05). Thus, a strategy for evaluation the SCND process stability based on online SOUR monitoring is proposed, which provides a theoretical basis for optimizing the SCND performance.

Overlooked pathways of endogenous simultaneous nitrification and denitrification in anaerobic/aerobic/anoxic sequencing batch reactors with organic supplementation

The anaerobic/aerobic/anoxic (A/O/A) process is a promising biotechnology to intensify denitrification in low carbon/nitrogen (C/N) wastewater treatment, but the neglected typical rate-limiting step—nitrification—would hinder its wider application. Heterotrophic nitrification driven by intracellular carbon (PHAs) could enhance nitrification and achieve endogenous simultaneous nitrification and denitrification (ESND) in the A/O/A process, but its feasibility remains unexamined. Here we established four A/O/A-SBRs at different C/N ratios (3, 7.5, 12, and 16.5) to address the above-mentioned knowledge gaps. The results showed that organic supplementation promoted both nitrification and denitrification (performance and relevant enzymatic activities) until organic overdose (C/N = 16.5) exacerbated niche competitions from other non-functional heterotrophs. qPCR and batch tests indicated that high C/N ratios inhibited autotrophic nitrifiers, and heterotrophic nitrifiers (HNB) dominated in the enhanced nitrification. Given the high HNB contribution (43.7%) and low COD variation (< 10 mg L−1) in the SND (76.4%) of CN12, we proposed a potential SND pathway based on heterotrophic nitrification and denitrification driven by PHAs and verified it with batch tests. Microbial and functional analyses suggested that CN12 favored the intracellular carbon transformation and harbored the minimum autotrophic nitrifiers, supporting the dominance of ESND in the enhanced SND. Our findings expand the understanding of the relationships between intracellular carbon transformation and SND and provide a novel nitrogen removal pathway for the practical application of the A/O/A process.