Impact of nitrogen loading rate on nitrite accumulation, kinetic analysis and microbial evolution in the partial nitrification process

Yilin Qi , Zhongkuo Guan , Yajun Fan , Lian He , Jun Wu , Miao Zhang

ENG. Environ. ›› 2026, Vol. 20 ›› Issue (7) : 104

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ENG. Environ. ›› 2026, Vol. 20 ›› Issue (7) :104 DOI: 10.1007/s11783-026-2204-y
RESEARCH ARTICLE
Impact of nitrogen loading rate on nitrite accumulation, kinetic analysis and microbial evolution in the partial nitrification process
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Abstract

Partial nitrification (PN) as one of the most efficient and energy-saving processes, has received much attention for process application. However, ensuring its stability remained a significant challenge. This study investigated the effects of reduced nitrogen loading rate (NLR: 0.328–0.057 kg N/(m3·d)) on substrate transformation linked with excitation-emission matrix (EEM) property, nitrification kinetic and functional bacteria. PN was rapidly initiated under higher NLR condition within 23 cycles with nitrite (NO2-N) accumulation ratio (NAR) of 73.35% in the continuous-flow, and the inflection points of DO, pH, and ORP accurately marked the process of ammonium (NH4+-N) oxidation and NO2-N generation in the typical cycle. EEM fluorescence analysis indicated that the main components of soluble microbial products (SMPs) and extracellular polymeric substances (EPS) transformed form tyrosine-like protein, tryptophan--like protein and fulvic acid-like organics to humic acid-like organics, and the fluorescence intensity of SMPs increased significantly under declined NLR. Nitrification kinetic parameters proved that AOB showed higher substrate affinity (KN,AOB < KN,NOB, e.g., 1.65 vs. 17.79 mg/L) and maximum specific oxidation rate (qmax,AOB > qmax,NOB, e.g., 15.15 vs. 11.44 mg N/(gVSS·h)) for efficient PN, revealing the reasons why AOB achieved the dominant position in Phase III. Moreover, Nitrosomonas, which was mainly responsible for NO2-N accumulation, sufficiently enriched (1.12% → 16.94% → 8.25%) and thereby promoted effective retention of functional bacteria. Meanwhile, Nitrospira was eluted (0.56% → 0.31% → 0.49%) and contributed to the stable maintenance of NAR at lower NLR levels. The practical applications and challenges of the PN coupling processes were further summarized for low-strength wastewater.

Graphical abstract

Keywords

Partial nitrification / Nitrogen loading rate / Nitrite accumulation / Nitrification kinetic / EEM fluorescence / Microbial evolution

Highlight

● PN was rapidly activated and maintained as NLR reduced from 0.328 to 0.057 kg N/(m3·d).

● FA and FNA triggered by NLR can alternately inhibit and eliminate NOB.

● Kinetic parameters were analyzed to prove the dominant position of AOB.

Nitrosomonas and Ellin6067 were enriched while Nitrospira was suppressed.

● Low NLR conditions could also provide a possibility for AOB enrichment.

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Yilin Qi, Zhongkuo Guan, Yajun Fan, Lian He, Jun Wu, Miao Zhang. Impact of nitrogen loading rate on nitrite accumulation, kinetic analysis and microbial evolution in the partial nitrification process. ENG. Environ., 2026, 20(7): 104 DOI:10.1007/s11783-026-2204-y

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References

[1]

Abbaszadeh L , Koutra E , Tsigkou K , Gaspari M , Kougias P G , Kornaros M . (2022). Nitrification upon nitrogen starvation and recovery: effect of stress period, substrate concentration and pH on ammonia oxidizers’ performance. Fermentation, 8(8): 387

[2]

APHA AWWA WEF . (2005). Standard Methods for the Examination of Water and Wastewater. 21st ed. New York: American Public Health Association, American Water Works Association, Water Environment Federation

[3]

Aslan S , Miller L , Dahab M . (2009). Ammonium oxidation via nitrite accumulation under limited oxygen concentration in sequencing batch reactors. Bioresource Technology, 100(2): 659–664

[4]

Bu C L , Wang S Y , Yu B H , Pfaender F , Zhu T X , Li Y Y , Liu J Y . (2025). Intelligent FA/FNA alternating strategy for nitrite-oxidizing bacteria inhibition: data-driven prediction and process control. Journal of Environmental Management, 386: 125688

[5]

Chen Y H , Sun Y W , Zhang J H , Li J W , Peng Y Z . (2022). A novel control strategy to strengthen nitrogen removal from domestic wastewater through eliminating nitrite oxidizing bacteria in a plug-flow process. Bioresource Technology, 350: 126856

[6]

Chen Y X , Xu R H , Meng F G . (2025). Biodegradable polylactic acid plastic can aid to achieve partial nitrification/denitrification for low carbon to nitrogen ratio wastewater treatment: performance and microbial mechanism. Bioresource Technology, 427: 132411

[7]

Cui S , Ji S H , Zhao W Z , Wan L G , Li Y Y . (2025). Stoichiometric analysis and control strategy of partial nitrification for treating dewatering liquid from food-waste methane fermentation. Water Research, 276: 123255

[8]

Cui Y C , Gao J F , Zhang D , Li D C , Dai H H , Wang Z Q , Zhao Y F . (2021). Responses of performance, antibiotic resistance genes and bacterial communities of partial nitrification system to polyamide microplastics. Bioresource Technology, 341: 125767

[9]

Duan C X , Zhang Q , Li J L , Feng W Y , Zhang L , Peng Y Z . (2024). Partial nitrification response to dissolved oxygen variation and aerobic starvation: kinetics and microbial community analyses. Chemical Engineering Journal, 481: 148621

[10]

Fan X Y , Niu Y , Li X , Cao S B , Zheng M Y , Zhou S L . (2025). Microbial mechanism of naturally formed stable partial nitrification in sequencing batch biofilm reactors. Journal of Environmental Chemical Engineering, 13(3): 116384

[11]

Fu K M , Bian Y H , Yang F , Xu J , Qiu F G . (2023). Achieving partial nitrification: a strategy for washing NOB out under high DO condition. Journal of Environmental Management, 347: 119186

[12]

Gong F G , Xu A , Gao D W , Gong X F , Wang C C , Huang Z , Liang H . (2025). Insights into magnetic biofilm carriers driving stable partial nitrification by selectively enriching ammonia-oxidizing bacteria. Biochemical Engineering Journal, 221: 109810

[13]

Gong X F , Wang B , Qiao X , Gong Q T , Liu X F , Peng Y Z . (2020). Performance of the anammox process treating low-strength municipal wastewater under low temperatures: effect of undulating seasonal temperature variation. Bioresource Technology, 312: 123590

[14]

Guo J H , Peng Y Z , Wang S Y , Zheng Y N , Huang H J , Wang Z W . (2009). Long-term effect of dissolved oxygen on partial nitrification performance and microbial community structure. Bioresource Technology, 100(11): 2796–2802

[15]

Hou R R , Dong Z C , Yang P , Chen H L . (2025). Insight into the evolution of microbial communities induced by high ammonium loading in a partial nitrification sequencing batch reactor. Process Safety and Environmental Protection, 198: 107182

[16]

Jaramillo F , Orchard M , Muñoz C , Zamorano M , Antileo C . (2018). Advanced strategies to improve nitrification process in sequencing batch reactors - A review. Journal of Environmental Management, 218: 154–164

[17]

Li J , Wang H , Li Z B , Guo J H , Wang Y Y . (2025a). Enhanced nitritation through bubbleless aeration-promoted AOB growth and environmental selective pressures-induced NOB suppression in membrane aerated biofilm reactors. Chemical Engineering Journal, 507: 160519

[18]

Li J W , Peng Y Z , Zhang L , Liu J J , Wang X D , Gao R T , Pang L , Zhou Y X . (2019). Quantify the contribution of anammox for enhanced nitrogen removal through metagenomic analysis and mass balance in an anoxic moving bed biofilm reactor. Water Research, 160: 178–187

[19]

Li S Q , Kang X F , Zuo Z Q , Islam M S , Yang S L , Liu Y C , Huang X . (2024a). Dynamic pH regulation drives Nitrosomonas for high-rate stable acidic partial nitritation. Water Research, 262: 122078

[20]

Li S Y , Wang F , Yang X Z , Wang Y L , Zhang D L , Yang F L , Fu P , Yi W M . (2024b). Effects of aeration coupled with microbial fuel cell on nitrogen removal and electricity production for biogas slurry. Journal of Environmental Chemical Engineering, 12(5): 113761

[21]

Li Y C , Dong W Y , Hou Z L , Zhao Z L , Xie J , Wang H J , Huang X , Peng Y Z . (2024c). Intermittent hydroxylamine dosing to strengthen stability of partial nitrification and nitrogen removal efficiency through continuous-flow anaerobic–aerobic-anoxic reactor treating municipal wastewater. Bioresource Technology, 406: 130947

[22]

Li Z M , Chen Z P , Xie Y , Hu Y B , Xu S L , Yu S , Qiu S , Ge S J . (2025b). Dialysis-assisted control significantly enables stable long-term unaerated partial nitrification in a microalgal-bacterial consortium treating hydrolyzed urine. Bioresource Technology, 438: 133254

[23]

Lin C H , Liu Y X , Li Y Y , Liu J Y . (2023). Difference of high-salinity-induced inhibition of ammonia-oxidising bacteria and nitrite-oxidising bacteria and its applications. Bioresource Technology, 387: 129640

[24]

Liu X G , Kim M , Nakhla G , Andalib M , Fang Y . (2020). Partial nitrification-reactor configurations, and operational conditions: performance analysis. Journal of Environmental Chemical Engineering, 8(4): 103984

[25]

Manser R , Gujer W , Siegrist H . (2005). Consequences of mass transfer effects on the kinetics of nitrifiers. Water Research, 39(19): 4633–4642

[26]

Pan W T , Liu H , Chen Y Z , Tang Z Q , Wang Q , Peng Y Z . (2025). Enhancing nitrogen removal from real oxytetracycline pharmaceutical wastewater via partial nitrification and post-denitrification (PNPDE) process: Insights into performance, mechanism, and application. Journal of Water Process Engineering, 69: 106673

[27]

Park S , Bae W . (2009). Modeling kinetics of ammonium oxidation and nitrite oxidation under simultaneous inhibition by free ammonia and free nitrous acid. Process Biochemistry, 44(6): 631–640

[28]

Shen Y , Huang D M , Chen Y P , Yan P , Gao X . (2020). New insight into filamentous sludge bulking during wastewater treatment: Surface characteristics and thermodynamics. Science of the Total Environment, 712: 135795

[29]

Sun Y Q , Ma Y G , Cao Y W , Liu S Y , Tian X Y , Dai B W . (2025). Nitrogen removal performance and changes in microbial community structure of a single-stage partial nitrification-anammox (PNA) process for treating municipal wastewater with an extremely low carbon-to-nitrogen ratio. Journal of Water Process Engineering, 70: 106931

[30]

Umezawa K , Kojima H , Kato Y , Fukui M . (2020). Disproportionation of inorganic sulfur compounds by a novel autotrophic bacterium belonging to Nitrospirota. Systematic and Applied Microbiology, 43(5): 126110

[31]

Wang J W , Yang H , Zhang F , Su Y , Wang S L . (2020). Activated sludge under free ammonia treatment using gel immobilization technology for long-term partial nitrification with different initial biomass. Process Biochemistry, 99: 282–289

[32]

Wang W , Jiang T , Wang S , Wang L , Li Z Y , Li W J , Wang B . (2024). Low alkalinity, free ammonia, and free nitrous acid cooperatively stabilize partial nitrification under excessive aeration condition. Chemosphere, 352: 141447

[33]

Wang Y Y , Wang J Q , Liu Z P , Huang X H , Fang F , Guo J S , Yan P . (2021a). Effect of EPS and its forms of aerobic granular sludge on sludge aggregation performance during granulation process based on XDLVO theory. Science of the Total Environment, 795: 148682

[34]

Wang Z Y , Zheng M , Hu Z T , Duan H R , De Clippeleir H , Al-Omari A , Hu S H , Yuan Z G . (2021b). Unravelling adaptation of nitrite-oxidizing bacteria in mainstream PN/A process: mechanisms and counter-strategies. Water Research, 200: 117239

[35]

Wu H Y , Bai X L , Li L , Li Z X , Wang M Y , Zhang Z G , Zhu C , Xu Y M , Xiong H Q , Xie X . et al. (2024). Two-stage partial nitrification-denitrification and anammox process for nitrogen removal in vacuum collected toilet wastewater at ambient temperature. Environmental Research, 262: 119917

[36]

Wu J , He C D , van Loosdrecht M C M , Pérez J . (2016). Selection of ammonium oxidizing bacteria (AOB) over nitrite oxidizing bacteria (NOB) based on conversion rates. Chemical Engineering Journal, 304: 953–961

[37]

Wu J , Zhang Y , Zhang M , Li Y H . (2017). Effect of nitrifiers enrichment and diffusion on their oxygen half-saturation value measurements. Biochemical Engineering Journal, 123: 110–116

[38]

Xiao K Q , Tang R , Wei J , Wu J , Shao Y J , Ma Z H , Wang L B , Hu Z H , Zhou Z . (2025). Achieving stable partial nitrification through synergistic inhibition of free ammonia and salinity on nitrite-oxidizing bacteria. Frontiers of Environmental Science & Engineering, 19(4): 42

[39]

Xu J J , Chen X T , Tang R , Feng J W , Yuan S J , Wang W , Hu Z H . (2024). Removal of pathogenic indicator microorganisms during partial nitrification: the role of free nitrous acid. Frontiers of Environmental Science & Engineering, 18(3): 33

[40]

Yang P , Wang Z Z , Zhang C G , Zhu J X , Peng Y Z . (2025a). Engineering application on the combination of simultaneous partial nitrification and denitrification and anammox for advanced nitrogen removal from landfill leachate. Bioresource Technology, 423: 132257

[41]

Yang X F , Zhang L Q , Li S G , Zhang H Y , Zhang S Q , Wan Y X , Yu H R . (2024). Fast start-up of partial nitrification for high-ammonia wastewater treatment using zeolite with in-situ bioregeneration. Journal of Water Process Engineering, 59: 105077

[42]

Yang Z , Lin S X , Wang H , Zhou J , Lin H , Zhou J . (2025b). Simultaneous partial nitrification, endogenous and autotrophic denitrification in a single-stage electrolysis-integrated sequencing batch biofilm reactor (E-SBBR) for stable and enhanced kitchen digested wastewater treatment. Journal of Environmental Management, 375: 124200

[43]

Yuan P K , Wang Y , Lu Y P , Wei Z , Gao P . (2025). Microplastics provide new niches for nitrifiers to maintain nitrification performance in nitrifying bioreactors. Environmental Research, 285(11): 122568

[44]

Zhang D C , Su H , Antwi P , Xiao L W , Liu Z W , Li J Z . (2019a). High-rate partial-nitritation and efficient nitrifying bacteria enrichment/out-selection via pH-DO controls: efficiency, kinetics, and microbial community dynamics. Science of the Total Environment, 692: 741–755

[45]

Zhang F Z , Du Z Y , Qu D , Hao J F , Wang J H , Lu H Y , Zhang Y J , Peng Y Z . (2025a). Unveiling the overlooked impact of idle phase on partial nitrification in sequencing batch reactor: insights from systems with varying operation modes. Water Research, 285: 123984

[46]

Zhang G Y , Liu J M , Han Y , Xia L , Zhang J B , Guo J B , Li H B , Hou Y N , Song Y Y . (2025b). Simultaneously ammonium and perchlorate remove via the partial nitrification-anammox coupled sulfur autotrophic system. Environmental Research, 272: 121195

[47]

Zhang J H , Miao Y Y , Zhang Q , Sun Y W , Wu L , Peng Y Z . (2020). Mechanism of stable sewage nitrogen removal in a partial nitrification-anammox biofilm system at low temperatures: microbial community and EPS analysis. Bioresource Technology, 297: 122459

[48]

Zhang J H , Zhang W K , Bi X J , Gao Z X , Li Y T , Miao Y Y . (2023). Increasing specific biomass nitrogen load facilitated nitrite accumulation in mainstream wastewater treatment. Bioresource Technology, 385: 129337

[49]

Zhang L Y , Zhang Q , Dai J T , Chen Y H , Zhu Z , Li X Y , Peng Y Z . (2021). Rapidly achieving and optimizing simultaneous partial nitrification denitrification and anammox integrated process by hydroxylamine addition for advanced nitrogen removal from domestic wastewater. Bioresource Technology, 342: 125987

[50]

Zhang M , Li B , Guan Z K , Fan Y J , He L , Wu J . (2024). Rapid anammox startup in response to nitrogen loading variations: reactor performance, microbial dynamics and mechanism exploration. Journal of Water Process Engineering, 59: 104963

[51]

Zhang M , Li B , Yang H , Guan Z K , Fan Y J , Wu J . (2025c). Effects of salinity on nitrite accumulation, denitrification kinetic and metagenomic analysis in the partial denitrification (PD) process: metabolic mechanism and feasible application. Journal of Environmental Chemical Engineering, 13(3): 116700

[52]

Zhang M , Tan Y F , Fan Y J , Gao J , Liu Y Z , Lv X F , Ge L Y , Wu J . (2022). Nitrite accumulation, denitrification kinetic and microbial evolution in the partial denitrification process: the combined effects of carbon source and nitrate concentration. Bioresource Technology, 361: 127604

[53]

Zhang M , Wang D B , You Y F , Fan Y J , Cao G H , Wu J , Meng Q G . (2025d). Stable nitrite accumulation, enhanced phosphorus removal and improved sludge aggregation in the partial denitrification (PD) process via calcium addition: The effect of Ca2+/P ratio. Journal of Environmental Chemical Engineering, 13(5): 118255

[54]

Zhang M , Yu M , Wang Y X , He C D , Pang J J , Wu J . (2019b). Operational optimization of a three-stage nitrification moving bed biofilm reactor (NMBBR) by obtaining enriched nitrifying bacteria: nitrifying performance, microbial community, and kinetic parameters. Science of the Total Environment, 697: 134101

[55]

Zhang Y , Fang X H , Ye J M , Huang L , Zhong C M . (2025e). Effect of free ammonia control strategy on partial nitrification-denitrification in MBR reactor under low C/N conditions: performance, SMP and EPS, and microbial community. Journal of Environmental Chemical Engineering, 13(3): 116273

[56]

Zheng M , Li H J , Duan H R , Liu T , Wang Z Y , Zhao J , Hu Z T , Watts S , Meng J , Liu P . et al. (2023). One-year stable pilot-scale operation demonstrates high flexibility of mainstream anammox application. Water Research X, 19: 100166

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