Abstract
The “Nitrite Bottleneck” in nitrite-based nitrogen removal processes undermines the efficiency of partial nitrification-anammox (PN/A). This perspective proposes leveraging bacteriophages for precise microbial community engineering in nitrogen removal: selectively lysing nitrite-oxidizing bacteria (NOB) via targeted lysis, enhancing ammonia-oxidizing and anammox bacteria through auxiliary metabolic genes, and facilitating nutrient redistribution via the viral shunt. We explore the feasibility, technical challenges, and potential biosafety risks, offering a roadmap for phage-based advancements in wastewater treatment systems.
Data availability
No datasets were generated or analyzed during the current study.
References
Tian, X. et al. Will reaching the maximum achievable yield potential meet future global food demand?. J. Clean. Prod. 294, 126285 (2021).
Rockstrom, J. et al. A safe operating space for humanity. Nature 461, 472–475 (2009).
Voets, J., Vanstaen, H. & Verstraete, W. Removal of Nitrogen From Highly Nitrogenous Wastewaters. J. Water Pollut. Control Fed. 47, 394–398 (1975).
Kartal, B., Kuenen, J. G. & van Loosdrecht, M. C. M. Sewage treatment with Anammox. Science 328, 702–703 (2010).
Oshiki, M., Shimokawa, M., Fujii, N., Satoh, H. & Okabe, S. Physiological characteristics of the anaerobic ammonium-oxidizing bacterium ‘Candidatus Brocadia sinica’. Microbiology 157, 1706–1713 (2011).
Wang, Z. et al. Unravelling adaptation of nitrite-oxidizing bacteria in mainstream PN/A process: Mechanisms and counter-strategies. Water Res. 200, 117239 (2021).
Feng, L. et al. Intracellular electron competition in response to the oxygen pressure of the aerobic denitrification process in an O2-based membrane biofilm reactor (MBfR) for nitrate removal. Sci. Total Environ. 875, 162592 (2023).
Huang, R., Meng, T., Liu, G., Gao, S. & Tian, J. Simultaneous nitrification and denitrification in membrane bioreactor: Effect of dissolved oxygen. J. Environ. Manag. 323, 116183 (2022).
Zhang, X. et al. Enhancement of nitrite production via addition of hydroxylamine to partial denitrification (PD) biomass: Functional genes dynamics and enzymatic activities. Bioresour. Technol. 318, 124274 (2020).
Zhao, J. et al. Selective Enrichment of Comammox Nitrospira in a Moving Bed Biofilm Reactor with Sufficient Oxygen Supply. Environ. Sci. Technol. 56, 13338–13346 (2022).
Zheng, M. et al. Predominance of comammox bacteria among ammonia oxidizers under low dissolved oxygen condition. Chemosphere 308, 136436 (2022).
Teklemariam, A. D. et al. The Battle between Bacteria and Bacteriophages: A Conundrum to Their Immune System. Antibiotics 12, 381 (2023).
Saw, P. E. & Song, E.-W. Phage display screening of therapeutic peptide for cancer targeting and therapy. Protein Cell 10, 787–807 (2019).
Karimi, M. et al. Bacteriophages and phage-inspired nanocarriers for targeted delivery of therapeutic cargos. Adv. Drug Deliv. Rev. 106, 45–62 (2016).
Penades, J. R., Chen, J., Quiles-Puchalt, N., Carpena, N. & Novick, R. P. Bacteriophage-mediated spread of bacterial virulence genes. Curr. Opin. Microbiol. 23, 171–178 (2015).
Touchon, M., Moura de Sousa, J. A. & Rocha, E. P. Embracing the enemy: the diversification of microbial gene repertoires by phage-mediated horizontal gene transfer. Curr. Opin. Microbiol. 38, 66–73 (2017).
Mathieu, J., Yu, P., Zuo, P., Da Silva, M. L. B. & Alvarez, P. J. J. Going viral: emerging opportunities for phage-based bacterial control in water treatment and reuse. Acc. Chem. Res. 52, 849–857 (2019).
Sohail, H. A. et al. Bacteriophages: emerging applications in medicine, food, and biotechnology. Phage 1, 75–82 (2020).
Wang, D. et al. Distributions, interactions, and dynamics of prokaryotes and phages in a hybrid biological wastewater treatment system. Microbiome 12, 134 (2024).
Qi, H. et al. Symbiotic bacteriophages exhibit multiple adaptive strategies in activated sludge flocs and contribute to floc stability. Chem. Eng. J. 492, 152448 (2024).
Daims, H. et al. Complete nitrification by Nitrospira bacteria. Nature 528, 504 (2015).
Wang, Y. et al. Heterogeneity of soil bacterial and bacteriophage communities in three rice agroecosystems and potential impacts of bacteriophage on nutrient cycling. Environ. Microbiome 17, 17 (2022).
Jin, M., Cai, L., Lu, L., Yu, M. & Zhang, R. Combined metabolomic and genomic analyses reveal phage-specific and infection stage-specific alterations to marine Roseobacter metabolism. ISME Commun. 5, ycaf047 (2025).
Duan, H., Ye, L., Lu, X. & Yuan, Z. Overcoming nitrite oxidizing bacteria adaptation through alternating sludge treatment with free nitrous acid and free ammonia. Environ. Sci. Technol. 53, 1937–1946 (2019).
Yu, L. et al. The differential proliferation of AOB and NOB during natural nitrifier cultivation and acclimation with raw sewage as seed sludge. RSC Adv. 10, 28277–28286 (2020).
Gruber-Dorninger, C. et al. Functionally relevant diversity of closely related Nitrospira in activated sludge. ISME J. 9, 643–655 (2015).
Ghimire-Kafle, S., Weaver, M. E. & Bollmann, A. Ecophysiological and genomic characterization of the freshwater complete ammonia oxidizer Nitrospira sp. Strain BO4. Appl. Environ. Microbiol. 89, (2023).
Wei, Y., Chen, Y., Xia, W., Ye, M. & Li, Y.-Y. Dynamic pulse approach to enhancing mainstream Anammox process stability: Integrating sidestream support and tackling nitrite-oxidizing bacteria challenges. Bioresour. Technol. 395, 130327 (2024).
Duan, H. et al. Nitrite oxidizing bacteria (NOB) contained in influent deteriorate mainstream NOB suppression by sidestream inactivation. Water Res. 162, 331–338 (2019).
Ali, M. et al. Physiological characterization of anaerobic ammonium oxidizing bacterium ‘Candidatus Jettenia caeni’. Environ. Microbiol. 17, 2172–2189 (2015).
Cui, B. et al. The effect of dissolved oxygen concentration on long-term stability of partial nitrification process. J. Environ. Sci. 90, 343–351 (2020).
Al-Hazmi, H. E., Yin, Z., Grubba, D., Majtacz, J. B. & Makinia, J. Comparison of the Efficiency of Deammonification under Different DO Concentrations in a Laboratory-Scale Sequencing Batch Reactor. Water 14, 368 (2022).
Yuan, Y., Xie, Y., Xu, P. & Li, X. Verification of inhibition effects of anoxic/aerobic alternation on NOB in nitrosation system under mainstream conditions. J. Water Process Eng. 45, 102479 (2022).
Qiu, J., Li, X., Peng, Y. & Jiang, H. Advanced nitrogen removal from landfill leachate via a two-stage combined process of partial nitrification-Anammox (PNA) and partial denitrification-Anammox (PDA). Sci. Total Environ. 810, 151186 (2022).
Hubaux, N., Wells, G. & Morgenroth, E. Impact of coexistence of flocs and biofilm on performance of combined nitritation-anammox granular sludge reactors. Water Res. 68, 127–139 (2015).
Dong, W., Lu, G., Yan, L., Zhang, Z. & Zhang, Y. Characteristics of pellets with immobilized activated sludge and its performance in increasing nitrification in sequencing batch reactors at low temperatures. J. Environ. Sci. 42, 202–209 (2016).
Seuntjens, D. et al. Mainstream partial nitritation/anammox with integrated fixed-film activated sludge: Combined aeration and floc retention time control strategies limit nitrate production. Bioresour. Technol. 314, 123711 (2020).
Li, J. et al. A critical review of one-stage anammox processes for treating industrial wastewater: Optimization strategies based on key functional microorganisms. Bioresour. Technol. 265, 498–505 (2018).
Wang, Z. et al. Nitrite accumulation in comammox-dominated nitrification-denitrification reactors: Effects of DO concentration and hydroxylamine addition. J. Hazard. Mater. 384, 121375 (2020).
Wu, W. et al. Contrasting the relative importance of species sorting and dispersal limitation in shaping marine bacterial versus protist communities. ISME J. 12, 485–494 (2018).
Winkler, M.-K. H. et al. An integrative review of granular sludge for the biological removal of nutrients and recalcitrant organic matter from wastewater. Chem. Eng. J. 336, 489–502 (2018).
Chamblee, J. et al. Endolysin Regulation in Phage Mu Lysis. mBio 13, (2022).
Nair, G. & Jain, V. An intramolecular cross-talk in D29 mycobacteriophage endolysin governs the lytic cycle and phage-host population dynamics. Sci. Adv. 10, eadh9812 (2024).
Voigt, E., Rall, B. C., Chatzinotas, A., Brose, U. & Rosenbaum, B. Phage strategies facilitate bacterial coexistence under environmental variability. PeerJ 9, e12194 (2021).
Lee, S., Hazard, C. & Nicol, G. W. Activity of novel virus families infecting soil nitrifiers is concomitant with host niche differentiation. ISME J. 18, wrae205 (2024).
Quiros, P. et al. Identification of a virulent phage infecting species of Nitrosomonas. ISME J. 17, 645–648 (2023).
Kotay, S. M., Datta, T., Choi, J. & Goel, R. Biocontrol of biomass bulking caused by Haliscomenobacter hydrossis using a newly isolated lytic bacteriophage. Water Res. 45, 694–704 (2011).
Petrovski, S., Seviour, R. J. & Tillett, D. Prevention of Gordonia and Nocardia Stabilized Foam Formation by Using Bacteriophage GTE7. Appl. Environ. Microbiol. 77, 7864–7867 (2011).
Guo, J. et al. VirSorter2: a multi-classifier, expert-guided approach to detect diverse DNA and RNA viruses. Microbiome 9, 37 (2021).
Paez-Espino, D. et al. IMG/VR v.2.0: an integrated data management and analysis system for cultivated and environmental viral genomes. Nucleic Acids Res. 47, D678–D686 (2019).
Baquero, D. P. et al. Stable coexistence between an archaeal virus and the dominant methanogen of the human gut. Nat. Commun. 15, 7702 (2024).
Rodriguez-Brito, B. et al. Viral and microbial community dynamics in four aquatic environments. ISME J. 4, 739–751 (2010).
Knowles, B. et al. Lytic to temperate switching of viral communities. Nature 531, 466–470 (2016).
Silveira, C. B. & Rohwer, F. L. Piggyback-the-Winner in host-associated microbial communities. npj Biofilms Microbiomes 2, 16010 (2016).
Silveira, C. B. & Rohwer, F. L. Piggyback-the-Winner in host-associated microbial communities. npj Biofilms Microbomes 2, 16010 (2016).
Mara, P. et al. Viral elements and their potential influence on microbial processes along the permanently stratified Cariaco Basin redoxcline. ISME J. 14, 3079–3092 (2020).
Hurwitz, B. L. & U’Ren, J. M. Viral metabolic reprogramming in marine ecosystems. Curr. Opin. Microbiol. 31, 161–168 (2016).
Sun, M., Yuan, S., Xia, R., Ye, M. & Balcázar, J. L. Underexplored viral auxiliary metabolic genes in soil: Diversity and eco-evolutionary significance. Environ. Microbiol. 25, 800–810 (2023).
Yuan, L. & Ju, F. Potential auxiliary metabolic capabilities and activities reveal biochemical impacts of viruses in municipal wastewater treatment plants. Environ. Sci. Technol. 57, 5485–5498 (2023).
Wang, D. et al. Niche differentiation and symbiotic association among ammonia/nitrite oxidizers in a full-scale rotating biological contactor. Water Res. 225, 119137 (2022).
Fuhrman, J. A. Marine viruses and their biogeochemical and ecological effects. Nature 399, 541–548 (1999).
Weitz, J. S. & Wilhelm, S. W. Ocean viruses and their effects on microbial communities and biogeochemical cycles. F1000 Biol. Rep. 4, 17 (2012).
Weitz, J. S. et al. A multitrophic model to quantify the effects of marine viruses on microbial food webs and ecosystem processes. ISME J. 9, 1352–1364 (2015).
Shelford, E. J., Middelboe, M., Møller, E. F. & Suttle, C. A. Virus-driven nitrogen cycling enhances phytoplankton growth. Aquat. Microb. Ecol. 66, 41–46 (2012).
Pourtois, J., Tarnita, C. E. & Bonachela, J. A. Impact of Lytic Phages on Phosphorus- vs. Nitrogen-Limited Marine Microbes. Front. Microbiol. 11, (2020).
Suttle, C. A. Viruses in the sea. Nature 437, 356–361 (2005).
Bolsan, A. C. et al. Bacteriophages in wastewater treatment: can they be an approach to optimize biological treatment processes?. Environ. Sci. Pollut. Res. Int 29, 89889–89898 (2022).
Lood, C. et al. Digital phagograms: predicting phage infectivity through a multilayer machine learning approach. Curr. Opin. Virol. 52, 174–181 (2022).
Doudna, J. A. & Charpentier, E. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).
Shalem, O., Sanjana, N. E. & Zhang, F. High-throughput functional genomics using CRISPR-Cas9. Nat. Rev. Genet. 16, 299–311 (2015).
Carlson, H. K. et al. Geochemical constraints on bacteriophage infectivity in terrestrial environments. ISME Commun. 3, 78 (2023).
Bacteriophage application in inhibiting corrosion- producing bacteria | BMC Microbiology | Full Text. https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-025-03952-2.
Zhang, G. et al. Bacteriophage application in inhibiting corrosion-producing bacteria. BMC Microbiol. 25, 241 (2025).
Huang, D. et al. Adaptive strategies and ecological roles of phages in habitats under physicochemical stress. Trends Microbiol. 32, 902–916 (2024).
Letten, A. D. & Ludington, W. B. Pulsed, continuous or somewhere in between? Resource dynamics matter in the optimisation of microbial communities. ISME J. 17, 641–644 (2023).
Chen, L., Zhao, X., Wongso, S., Lin, Z. & Wang, S. Trade-offs between receptor modification and fitness drive host-bacteriophage co-evolution leading to phage extinction or co-existence. ISME J. 18, wrae214 (2024).
Borin, J. M., Avrani, S., Barrick, J. E., Petrie, K. L. & Meyer, J. R. Coevolutionary phage training leads to greater bacterial suppression and delays the evolution of phage resistance. Proc. Natl. Acad. Sci. 118, e2104592118 (2021).
Costa, P., Pereira, C., Romalde, J. L. & Almeida, A. A game of resistance: War between bacteria and phages and how phage cocktails can be the solution. Virology 599, 110209 (2024).
Reisch, C. R. & Prather, K. L. J. The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli. Sci. Rep. 5, 15096 (2015).
Yehl, K. et al. Engineering Phage Host-Range and Suppressing Bacterial Resistance through Phage Tail Fiber Mutagenesis. Cell 179, 459 (2019).
Meile, S., Du, J., Dunne, M., Kilcher, S. & Loessner, M. J. Engineering therapeutic phages for enhanced antibacterial efficacy. Curr. Opin. Virol. 52, 182–191 (2022).
Peng, H., Chen, I. A. & Qimron, U. Engineering phages to fight multidrug-resistant bacteria. Chem. Rev. 125, 933–971 (2024).
Chaudhary, N. et al. CRISPR-Cas-assisted phage engineering for personalized antibacterial treatments. Indian J. Med. Microbiol. 53, 100771 (2025).
Steiner, L. X. et al. Phage-induced disturbance of a marine sponge microbiome. Environ. Microbiome 19, 97 (2024).
Balleste, E. et al. Bacteriophages in sewage: abundance, roles, and applications. FEMS Microbes 3, xtac009 (2022).
Pfeifer, E., Bonnin, R. A. & Rocha, E. P. C. Phage-plasmids spread antibiotic resistance genes through infection and lysogenic conversion. mBio 13, (2022).
Shi, L.-D. et al. A mixed blessing of viruses in wastewater treatment plants. Water Res. 215, 118237 (2022).
Jiang, S. et al. Generic Diagramming Platform (GDP): a comprehensive database of high-quality biomedical graphics. Nucleic Acids Res. 53, D1670–D1676 (2024).
Acknowledgements
The authors would like to thank the contributors for their valuable support. We would like to express our gratitude to Dr. Linchen He from Peking University for his valuable contributions in revising and enhancing the language throughout the manuscript. This work was supported by the National Natural Science Foundation of China (Grant Nos. 42407161, 42230411, and 42177099) and the Jing-Jin-Ji Regional Integrated Environmental Improvement-National Science and Technology Major Project (Grant No. 2025ZD1204700).
Author information
Authors and Affiliations
Contributions
J.P. conceived and designed the study, led the writing of the original draft, and was responsible for visualization. C.J. and X.L. defined the key information and provided funding support. X.W. contributed to the development of the content structure and assisted in reviewing and editing. S.X., A.S., and X.Z. supervised the study. All authors have read and agreed to the published version of the manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Reprints and permissions
About this article
Cite this article
Peng, J., Jiang, C., Wang, X. et al. The nitrite bottleneck revisited: exploring the potential of phage-mediated microbial regulation for advancing novel nitrogen removal strategies.
npj Clean Water (2025). https://doi.org/10.1038/s41545-025-00543-6
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41545-025-00543-6
Source: Resources - nature.com
