Linking nitrite accumulation to shift in carbon utilization of denitrification: from single to composite electron donor

Abstract

The role of organic carbon source as electron donor in incomplete denitrification, particularly in nitrite (NO₂⁻) accumulation, remains crucial yet poorly understood. A detailed understanding of carbon and nitrogen metabolic interactions is essential for advancing technologies that integrate partial denitrification (PD) with anammox. In this study, the carbon transformation and gradient utilization of various volatile fatty acids (VFAs) were explored to elucidate their impacts on nitrate (NO₃⁻) and NO₂⁻ reduction during PD. Long-term experiments revealed that composite VFAs (a mixture of acetate, propionate and butyrate) achieved the highest nitrate-to-nitrite transformation ratio (NTR) of 79.1%, outperforming single VFA (69.5% with acetate and 69.4% with propionate). The NO₂⁻ accumulation during PD was strongly influenced by the utilization of exogenous, endogenous and extracellular carbon, which varied significantly with VFAs type and dosage. Polyhydroxybutyrate (PHB) served as the primary endogenous electron donor in acetate-driven PD, promoting modest NO₂⁻ accumulation, while polyhydroxyvalerate (PHV) along with glycogen (Gly) was the key contributor in propionate-driven PD, supporting complete NO₃⁻ reduction. In contrast to single VFA-driven PD, the lower levels and delayed utilization of PHB and PHV in composite VFAs-driven PD enabled more stable and efficient NO₂⁻ accumulation. Furthermore, metagenomic analysis illuminated that the transition from single VFA to composite VFAs strengthened the potential for both electron production and their transport to NO₃⁻ reductase. Thauera was always the core denitrifier demonstrating strong adaptability to various VFAs. This study provides mechanistic insights into organic carbon-regulated NO₂⁻ accumulation, filling the gap regarding dynamic changes in carbon utilization during PD.

Graphical abstract

Keywords

Partial denitrification / Single VFA / Composite VFAs / Gradient carbon utilization / Nitrite accumulation and reduction

Highlight

	● Robust NO2− production with NTR of 79.1% was achieved using composite VFAs.
	● Delayed PHA utilization improved PD resilience and efficiency with composite VFAs.
	● Composite VFAs induced a more resilient collective to function.
	● Composite VFAs enhanced electron production and their transport to NO3− reductase.

References

[1] APHA (2012). Standard Methods for the Examination of Water and Wastewater. Washington, DC: American Public Health Association

[2] Chen G J , Lin L , Wang Y , Zhang Z K , Cao W Z , Zhang Y L . (2023). Unveiling the interaction mechanisms of key functional microorganisms in the partial denitrification-anammox process induced by COD. Frontiers of Environmental Science & Engineering, 17(8): 103

[3] Coyte K Z , Schluter J , Foster K R . (2015). The ecology of the microbiome: networks, competition, and stability. Science, 350(6261): 663–666

[4] Du R , Cao S B , Li B K , Niu M , Wang S Y , Peng Y Z . (2017). Performance and microbial community analysis of a novel DEAMOX based on partial-denitrification and anammox treating ammonia and nitrate wastewaters. Water Research, 108: 46–56

[5] Du R , Cao S B , Zhang H Y , Li X C , Peng Y Z . (2020). Flexible nitrite supply alternative for mainstream anammox: advances in enhancing process stability. Environmental Science & Technology, 54(10): 6353–6364

[6] Elefsiniotis P , Li D . (2006). The effect of temperature and carbon source on denitrification using volatile fatty acids. Biochemical Engineering Journal, 28(2): 148–155

[7] Elefsiniotis P , Wareham D G , Smith M O . (2004). Use of volatile fatty acids from an acid-phase digester for denitrification. Journal of Biotechnology, 114(3): 289–297

[8] Feng F , Qu C Y , Tang J , Wu X , Tang X , Yao F B , Chai L Y , Xiao R Y , Tang C J . (2023). Quantification of enhanced nitrogen removal pathways of pyrite interaction with anammox sludge system. Chemical Engineering Journal, 459: 141519

[9] Feng J , Zhao J , Xiang H , You Z P , Shi L L , Yu Z D , Qiu Y L , Yu D S , Wang X X . (2025). Establishment of continuous flow partial denitrification biofilm module with short hydraulic retention time. Water Research, 268: 122743

[10] Fu J W , Hou Z Y , Zhao H X , Li Q , Chen R , Li Y Y . (2024). Enhanced nitrogen removal from low strength anaerobic membrane bioreactor (AnMBR) permeate using complete nitrification and partial denitrification-anammox processes. Frontiers of Environmental Science & Engineering, 18(12): 155

[11] Goldford J E , Lu N X , Bajić D , Estrela S , Tikhonov M , Sanchez-Gorostiaga A , Segrè D , Mehta P , Sanchez A . (2018). Emergent simplicity in microbial community assembly. Science, 361(6401): 469–474

[12] Huang H , Ma R , Ren H Q . (2024). Scientific and technological innovations of wastewater treatment in China. Frontiers of Environmental Science & Engineering, 18(6): 72

[13] Jeon Y , Li L , Calvillo J , Ryu H , Santo Domingo J W , Choi O , Brown J , Seo Y . (2020). Impact of algal organic matter on the performance, cyanotoxin removal, and biofilms of biologically-active filtration systems. Water Research, 184: 116120

[14] Ji Z Y , Chen Y G . (2010). Using sludge fermentation liquid to improve wastewater short-cut nitrification-denitrification and denitrifying phosphorus removal via nitrite. Environmental Science & Technology, 44(23): 8957–8963

[15] Kartal B , Kuenen J G , Van Loosdrecht M C M . (2010). Sewage treatment with anammox. Science, 328(5979): 702–703

[16] Kondrotaite Z , Valk L C , Petriglieri F , Singleton C , Nierychlo M , Dueholm M K D , Nielsen P H . (2022). Diversity and ecophysiology of the genus OLB8 and other abundant uncultured Saprospiraceae genera in global wastewater treatment systems. Frontiers in Microbiology, 13: 917553

[17] Lei Z , Yang S M , Wang L X , Huang X Y , Wang X C , Li Y Y , Li Q , Zhao Y , Chen R . (2020). Achieving successive methanation and low-carbon denitrogenation by a novel three-stage process for energy-efficient wastewater treatment. Journal of Cleaner Production, 276: 124245

[18] Li C , Cao J S , Ren H Q , Li Y , Tang S Y . (2015). Comparison on kinetics and microbial community among denitrification process fed by different kinds of volatile fatty acids. Process Biochemistry, 50(3): 447–455

[19] Li X , Li X Z , Li C N , Li N , Zou P , Gao X L , Cao Q . (2023). Nitrogen removal performances and metabolic mechanisms of denitrification systems using different volatile fatty acids as external carbon sources. Chemical Engineering Journal, 474: 145998

[20] Li X C , Peng Y Z , Zhang J W , Du R . (2022). Multiple roles of complex organics in polishing THP-AD filtrate with double-line anammox: inhibitory relief and bacterial selection. Water Research, 216: 118373

[21] Liu Q T , Li C , Fan J R , Peng Y Z , Du R . (2023). Evaluation of sludge anaerobic fermentation driving partial denitrification capability: in view of kinetics and metagenomic mechanisms. Science of the Total Environment, 884: 163581

[22] Luo J Y , Feng L Y , Chen Y G , Sun H , Shen Q T , Li X , Chen H . (2015). Alkyl polyglucose enhancing propionic acid enriched short-chain fatty acids production during anaerobic treatment of waste activated sludge and mechanisms. Water Research, 73: 332–341

[23] Miao L , Wang S Y , Li B K , Cao T H , Zhang F Z , Wang Z , Peng Y Z . (2016). Effect of carbon source type on intracellular stored polymers during endogenous denitritation (ED) treating landfill leachate. Water Research, 100: 405–412

[24] Owusu-Agyeman I , Bedaso B , Laumeyer C , Pan C , Malovanyy A , Baresel C , Plaza E , Cetecioglu Z . (2023). Volatile fatty acids production from municipal waste streams and use as a carbon source for denitrification: the journey towards full-scale application and revealing key microbial players. Renewable and Sustainable Energy Reviews, 175: 113163

[25] Pan Y , Sun R Z , Wang Y , Chen G L , Fu Y Y , Yu H Q . (2023). Carbon source shaped microbial ecology, metabolism and performance in denitrification systems. Water Research, 243: 120330

[26] Qu J H , Chen J P . (2024). Pathways toward a pollution-free planet and challenges. Frontiers of Environmental Science & Engineering, 18(6): 67

[27] Torresi E , Gülay A , Polesel F , Jensen M M , Christensson M , Smets B F , Plósz B G . (2018). Reactor staging influences microbial community composition and diversity of denitrifying MBBRs- Implications on pharmaceutical removal. Water Research, 138: 333–345

[28] Wang H B , Zhang L , Dan Q P , Zhang Y X , Wang S Y , Zhang Q , Li X Y , Wang C X , Peng Y Z . (2023). Ultra-high nitrogen removal from real municipal wastewater using selective enhancement of glycogen accumulating organisms (GAOs) in a partial nitrification-anammox (PNA) system. Water Research, 230: 119594

[29] Xu L , Su J F , Li K , Hu R Z , Yan H , Liang E L , Zhou Z N , Shi J . (2022). Performance of hydrogel immobilized bioreactors combined with different iron ore wastes for denitrification and removal of copper and lead: optimization and possible mechanism. Water Research, 225: 119196

[30] Yang Z C , Zhou Q , Sun H M , Jia L X , Zhao L , Wu W Z . (2021). Metagenomic analyses of microbial structure and metabolic pathway in solid-phase denitrification systems for advanced nitrogen removal of wastewater treatment plant effluent: a pilot-scale study. Water Research, 196: 117067

[31] Zhang J W , Peng Y Z , Li X C , Du R . (2022). Feasibility of partial-denitrification/ anammox for pharmaceutical wastewater treatment in a hybrid biofilm reactor. Water Research, 208: 117856

[32] Zhang W L , Zhang Q , Li M , Wang H Y , Li Y , Peng H J , Feng J P . (2021). Microbial community and function evaluation in the start-up period of bioaugmented SBR fed with aniline wastewater. Bioresource Technology, 319: 124148

[33] Zhang Z Z , Zhang Y , Chen Y G . (2020). Comparative metagenomic and metatranscriptomic analyses reveal the functional species and metabolic characteristics of an enriched denitratation community. Environmental Science & Technology, 54(22): 14312–14321

[34] Zhao Q , Li J W , Deng L Y , Jia T P , Zhao Y , Li X Y , Peng Y Z . (2023). From hybrid process to pure biofilm anammox process: suspended sludge biomass management contributing to high-level anammox enrichment in biofilms. Water Research, 236: 119959

[35] Zhu W L , Xiao R , Xu M , Chai W B , Liu W L , Jin Z Y , Ikumi D , Lu H J . (2024). Unraveling the role of formate in improving nitrogen removal via coupled partial denitrification-anammox. Frontiers of Environmental Science & Engineering, 18(9): 112

RIGHTS & PERMISSIONS

Higher Education Press 2026