Effects of operational parameters and quorum sensing signal molecules on the performance of marine anaerobic ammonium oxidation-based microbial electrolysis cells treating mariculture wastewater

Abstract

The application of anaerobic ammonium oxidation (anammox) is often constrained by the limited availability of nitrite (NO₂^–-N). Microbial electrochemical technology offers a promising strategy to circumvent this limitation. Therefore, we constructed microbial electrolysis cells (MECs) incorporating marine-anammox electroactive biofilm and conducted batch experiments to investigate the effects of operational parameters and quorum sensing (QS) signal molecules on their performance. Under NO₂^–-N-deficient conditions, the anodic biofilm utilized the graphite felt electrode as an extracellular electron acceptor to drive anammox process, achieving ammonia (NH₃-N) removal while generating a sustained electrical current. Notably, no nitrate was produced, and NO was not a key intermediate in electrode-dependent anammox. NH₃-N removal followed first-order kinetics, with a substantially lower rate constant for the electrode as electron acceptor than for NO₂^–-N. Higher initial NH₃-N concentrations (25–100 mg/L) and temperatures (10–35 °C) significantly enhanced nitrogen removal and current generation. NO₂^–-N below 5 mg/L had a negligible impact, whereas higher levels (5–75 mg/L) slightly improved nitrogen removal but markedly suppressed current generation. Dissolved oxygen below 1.0 mg/L exerted minimal influence. Externally-added C₆-HSL, C₁₂-HSL, and 3-oxo-C₆-HSL (5–80 µmol/L) promoted both nitrogen removal and current generation. Conversely, 3-oxo-C₁₂-HSL (5–80 µmol/L) and acylase I (10–80 mg/L) inhibited these processes. The promoting or inhibitory effects exhibited concentration- and time-dependence, with lower concentrations requiring longer exposure to achieve stable effects. These findings demonstrate the potential of marine anammox-based MECs for treating NO₂^–-N-deficient mariculture wastewater and highlight the role of QS in regulating their performance.

Graphical abstract

Keywords

Mariculture wastewater / Microbial electrolysis cell / Marine anammox / Quorum sensing / N-Acyl homoserine lactones

Highlight

	● Marine-anammox MECs removed NH3-N from mariculture water in the absence of NO2–-N.
	● NO2–-N (> 5 mg/L) was the preferred electron acceptor over the electrode in MECs.
	● High initial NH3-N, elevated temperature, and low DO favored the MEC performance.
	● Adding different AHLs and acylase I elicited distinct effects on MEC performance.
	● The promoting/inhibitory effects of AHLs were concentration- and time-dependent.

References

[1] Ahmad A , Al Senaidi A S , Mubarak M S . (2024). Microbial approach towards anode biofilm engineering enhances extracellular electron transfer for bioenergy production. Journal of Environmental Management, 370: 122696

[2] Bao R L , Peng X L , Ma C , Xue L Q , Wu C Y , Zhou X K . (2025). A review of electron transfer mechanisms and their strategies for process enhancement in anaerobic ammonia oxidizing bacteria: from intracellular to extracellular. Process Safety and Environmental Protection, 201: 107515

[3] Berger C , Rosenbaum M A . (2017). Spontaneous quorum sensing mutation modulates electroactivity of Pseudomonas aeruginosa PA14. Bioelectrochemistry, 117: 1–8

[4] Chen S S , Jing X Y , Tang J H , Fang Y L , Zhou S G . (2017). Quorum sensing signals enhance the electrochemical activity and energy recovery of mixed-culture electroactive biofilms. Biosensors and Bioelectronics, 97: 369–376

[5] Chen Y C , Dong K , Zhang Y M , Zheng J J , Jiang M M , Wang D Q , Zhang X H , Huang X W , Zhou L J , Li H X . (2024). Enhancing biofilm formation in the hydrogen-based membrane biofilm reactor through bacterial Acyl-homoserine lactones. Frontiers of Environmental Science & Engineering, 18(11): 142

[6] Chien I C , Chu C Y , Huang C Y . (2022). Isolation of bacteria capable of degrading various AHLs for biofouling control in membrane bioreactors. Water, 14(11): 1712

[7] Du R , Peng Y Z , Ji J T , Shi L L , Gao R T , Li X C . (2019). Partial denitrification providing nitrite: opportunities of extending application for anammox. Environment International, 131: 105001

[8] Efremenko E , Senko O , Stepanov N , Aslanli A , Maslova O , Lyagin I . (2023). Quorum sensing as a trigger that improves characteristics of microbial biocatalysts. Microorganisms, 11(6): 1395

[9] Elsayed A , Lee T , Kim Y . (2025). Maximizing the efficiency of single-stage partial nitrification/Anammox granule processes and balancing microbial competition using insights of a numerical model study. Water Environment Research, 97(3): e70059

[10] Fang Y L , Deng C S , Chen J , Lü J , Chen S S , Zhou S G . (2018). Accelerating the start-up of the cathodic biofilm by adding acyl-homoserine lactone signaling molecules. Bioresource Technology, 266: 548–554

[11] Fu J W , He Y F , Zhao H X , Yang H , Li Q , Chen R , Li Y Y . (2025). Biochar mediated microbial synergy in Partial nitrification-anammox systems: enhancing nitrogen removal efficiency and stability. Frontiers of Environmental Science & Engineering, 19(7): 95

[12] Gao B , Zhang X N , Zhao R , Zhu X R , Ji L M , Wu P . (2025). Advances in the application of carbon materials for anammox enhancement: a comprehensive review. Journal of Environmental Management, 393: 127255

[13] Gao D W , Wang X L , Liang H , Wei Q H , Dou Y , Li L W . (2018). Anaerobic ammonia oxidizing bacteria: ecological distribution, metabolism, and microbial interactions. Frontiers of Environmental Science & Engineering, 12(3): 10

[14] Guo H H , Li Y W , Ge H , Sha H , Luo X Z , Zou G W , Liang H W . (2025). Competitive bio-accumulation between ammonia and nitrite results in their antagonistic toxicity to Hypophthalmichthys molitrix: antioxidant and immune responses and metabolic detoxification evidence. Antioxidants, 14(4): 453

[15] Guo M W , Wang C , Qiao S . (2023). Light-driven ammonium oxidation to dinitrogen gas by self-photosensitized biohybrid anammox systems. iScience, 26(5): 106725

[16] He S Y , Yu P , Shao Y , Gao X T , Sakamaki T , Li X N . (2025). Enhanced activity of mixed-culture electroactive biofilms and sulfamethoxazole removal efficiency by adding N-acyl-homoserine lactones in bio-electrochemical system. Environmental Technology, 46(12): 2254–2267

[17] Hu P F , van Loosdrecht M , Gu J D , Yang Y C . (2025). The core anammox redox reaction system of 12 anammox bacterial genera and their evolution and application implications. Water Research, 281: 123551

[18] Huang J H , Shi Y H , Zeng G M , Gu Y L , Chen G Q , Shi L X , Hu Y , Tang B , Zhou J X . (2016). Acyl-homoserine lactone-based quorum sensing and quorum quenching hold promise to determine the performance of biological wastewater treatments: an overview. Chemosphere, 157: 137–151

[19] Ismail I N , Taufik M , Umor N A , Norulhuda M R , Zulkarnaini Z , Ismail S . (2022). Anammox process for aquaculture wastewater treatment: operational condition, mechanism, and future prospective. Water Science & Technology, 86(12): 3093–3112

[20] Jing X Y , Liu X , Deng C S , Chen S S , Zhou S G . (2019). Chemical signals stimulate Geobacter soli biofilm formation and electroactivity. Biosensors and Bioelectronics, 127: 1–9

[21] Kallistova A Y , Dorofeev A G , Nikolaev Y A , Kozlov M N , Kevbrina M V , Pimenov N V . (2016). Role of anammox bacteria in removal of nitrogen compounds from wastewater. Microbiology, 85(2): 140–156

[22] Khanthong K , Jang H , Kadam R , Jo S , Lee J , Park J . (2023). Bioelectrochemical system for nitrogen removal: fundamentals, current status, trends, and challenges. Chemosphere, 339: 139776

[23] Kristensen R , Andersen J B , Rybtke M , Jansen C U , Fritz B G , Kiilerich R O , Uhd J , Bjarnsholt T , Qvortrup K , Tolker-Nielsen T . et al. (2024). Inhibition of Pseudomonas aeruginosa quorum sensing by chemical induction of the MexEF-oprN efflux pump. Antimicrobial Agents and Chemotherapy, 68(2): e0138723

[24] Kumwimba M N , Lotti T , Şenel E , Li X Y , Suanon F . (2020). Anammox-based processes: how far have we come and what work remains? A review by bibliometric analysis. Chemosphere, 238: 124627

[25] Li J , Qi P Q , Qiang Z M , Dong H Y , Gao D W , Wang D . (2018). Is anammox a promising treatment process for nitrogen removal from nitrogen-rich saline wastewater?. Bioresource Technology, 270: 722–731

[26] Li J L , Li Z F , Xie J , Xia Y , Gong W B , Tian J J , Zhang K , Yu E M , Wang G J . (2023). Quorum-quenching potential of recombinant PvdQ-engineered bacteria for biofilm formation. International Microbiology, 26(3): 639–650

[27] Li Q Q , Mao S , Wang H , Ye X Y . (2022). The molecular architecture of Pseudomonas aeruginosa quorum-sensing inhibitors. Marine Drugs, 20(8): 488

[28] Liang Y Q , Gao P K , Jiao Y H , Zhang Z Z , Xin Y F , Xia W J , Gu J D . (2025). Dinitrogen production modes of diverse anammox bacteria and the contribution in nitrogen cycle: a review. International Biodeterioration & Biodegradation, 203: 106113

[29] Liu L Y , Xie G J , Xing D F , Liu B F , Ding J , Cao G L , Ren N Q . (2021). Sulfate dependent ammonium oxidation: a microbial process linked nitrogen with sulfur cycle and potential application. Environmental Research, 192: 110282

[30] Lu S M , Liu X G , Liu C , Cheng G F , Zhou R F , Li Y Y . (2021). A review of ammonia-oxidizing archaea and anaerobic ammonia-oxidizing bacteria in the aquaculture pond environment in China. Frontiers in Microbiology, 12: 775794

[31] Ma M J , An T Y , Zou X Y , Adams M , Chen C J . (2023). Influence and mechanisms of humic substances on distinct anammox-based systems: a critical review. Journal of Water Process Engineering, 56: 104543

[32] Mulder A , van de Graaf A A , Robertson L A , Kuenen J G . (1995). Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor. FEMS Microbiology Ecology, 16(3): 177–183

[33] Okabe S , Nukada K , Horiguchi K , Oshiki M . (2026). Oxygen scavenging enables microoxic survival of the marine anammox bacterium Scalindua sp. The ISME Journal, 20(1): wrag075

[34] Oshiki M , Satoh H , Okabe S . (2016). Ecology and physiology of anaerobic ammonium oxidizing bacteria. Environmental Microbiology, 18(9): 2784–2796

[35] Pan M , Jiang T , Wang Z H , Huang X Z , Liu S J , Huang X M . (2025). Enhanced nitrogen removal via simultaneous nitrification-denitrification (SND) and Feammox in a magnetic zeolite modified intermittently aerated sequential biological reactor. Frontiers of Environmental Science & Engineering, 19(9): 115

[36] Parde D , Behera M , Dash R R , Bhunia P . (2024). A review on anammox processes: strategies for enhancing bacterial growth and performance in wastewater treatment. International Biodeterioration & Biodegradation, 191: 105812

[37] Rahimi S , Modin O , Mijakovic I . (2020). Technologies for biological removal and recovery of nitrogen from wastewater. Biotechnology Advances, 43: 107570

[38] Ren Z Q , Wang H , Zhang L G , Du X N , Huang B C , Jin R C . (2022). A review of anammox-based nitrogen removal technology: from microbial diversity to engineering applications. Bioresource Technology, 363: 127896

[39] Rios-Del Toro E E , Cervantes F J . (2019). Anaerobic ammonium oxidation in marine environments: contribution to biogeo-chemical cycles and biotechnological developments for wastewater treatment. Reviews in Environmental Science and Bio/Technology, 18(1): 11–27

[40] Shaw D R , Ali M , Katuri K P , Gralnick J A , Reimann J , Mesman R , van Niftrik L , Jetten M S M , Saikaly P E . (2020). Extracellular electron transfer-dependent anaerobic oxidation of ammonium by anammox bacteria. Nature Communications, 11(1): 2058

[41] Shaw D R , Gonzalez J T , Guadarrama C B , Saikaly P E . (2024). Emerging biotechnological applications of anaerobic ammonium oxidation. Trends in Biotechnology, 42(9): 1128–1143

[42] Tang J, Li Y F, Wu J B, Wang Z B, Lv L, Li Q Q (2025). Impact of salinity on anammox process operation: mechanisms and mitigation strategies. Environmental Research, 286(Pt 2): 122898

[43] Thakur K , Kuthiala T , Singh G , Arya S K , Iwai C B , Ravindran B , Khoo K S , Chang S W , Awasthi M K . (2023). An alternative approach towards nitrification and bioremediation of wastewater from aquaponics using biofilm-based bioreactors: a review. Chemosphere, 316: 137849

[44] Tom A P , Jayakumar J S , Biju M , Somarajan J , Ibrahim M A . (2021). Aquaculture wastewater treatment technologies and their sustainability: a review. Energy Nexus, 4: 100022

[45] Wang J L , Pan Y B , Mou W B , Meng H , Wen X , Zeng G M . (2026). Review of salinity adaptation and application of functional microorganisms in biological ammonia removal from high-salinity wastewater. Desalination and Water Treatment, 325: 101625

[46] Wang K C , Li J , Gu X , Wang H , Li X , Peng Y Z , Wang Y Y . (2023a). How to provide nitrite robustly for anaerobic ammonium oxidation in mainstream nitrogen removal. Environ-mental Science & Technology, 57(51): 21503–21526

[47] Wang N , Gao J , Liu Y , Wang Q Y , Zhuang X L , Zhuang G Q . (2021). Realizing the role of N-acyl-homoserine lactone-mediated quorum sensing in nitrification and denitrification: a review. Chemosphere, 274: 129970

[48] Wang S, Zhang K M, Miao Y, Wang Z (2024). The enhancement of anammox by graphene-based and iron-based nanomaterials in performance and mechanisms. npj Clean Water, 7(1): 100

[49] Wang S , Zhuang X L , Dong W L , Xin F X , Jia H H , Wu X Y . (2023b). Advances in the application of quorum sensing to regulate electrode biofilms in bioelectrochemical systems. Fermentation, 9(7): 625

[50] Wang T , Chen M , Zhu J X , Li N , Wang X . (2023c). Anodic ammonium oxidation in microbial electrolysis cell: towards nitrogen removal in low C/N environment. Water Research, 242: 120276

[51] Wei Q Y , Kawagoshi Y , Huang X W , Hong N , van Duc L , Yamashita Y , Hama T . (2016). Nitrogen removal properties in a continuous marine anammox bacteria reactor under rapid and extensive salinity changes. Chemosphere, 148: 444–451

[52] Xia Y G , Zhou J H , Yang X L , Shi F F , Fan R , Xu J Y . (2026). Anodic potential and conductive carrier synergistically drive nitrite-free extracellular electron transfer-dependent anaerobic ammonium oxidation (Anammox) in mixed microbial communities. Bioresource Technology, 446: 134180

[53] Yang Y , Zhou H H , Mei X X , Liu B F , Xing D F . (2018). Dual-edged character of quorum sensing signaling molecules in microbial extracellular electron transfer. Frontiers in Microbiology, 9: 1924

[54] Zhang L , Okabe S . (2020). Ecological niche differentiation among anammox bacteria. Water Research, 171: 115468

[55] Zhang Q , Fan N S , Fu J J , Huang B C , Jin R C . (2021). Role and application of quorum sensing in anaerobic ammonium oxidation (anammox) process: a review. Critical Reviews in Environmental Science and Technology, 51(6): 626–648

[56] Zhao Q , Peng Y Z , Li J W , Jia T P , Zhang Q , Zhang L . (2024). Pilot-scale implementation of mainstream anammox for municipal wastewater treatment against cold temperature. Nature Communications, 15(1): 10314

[57] Zhao Z C , Xie G J , Liu B F , Xing D F , Ding J , Han H J , Ren N Q . (2021). A review of quorum sensing improving partial nitritation-anammox process: functions, mechanisms and prospects. Science of the Total Environment, 765: 142703

[58] Zhen J Y , Zheng M , Wei W , Ni S Q , Ni B J . (2024). Extracellular electron transfer (EET) enhanced anammox process for progressive nitrogen removal: a review. Chemical Engineering Journal, 482: 148886

[59] Zheng D C , Li D P , Wang J T , Zhang Y F . (2024). Recent advances in anodic anaerobic ammonia oxidation in microbial electrolysis cells. Current Opinion in Electrochemistry, 45: 101470

[60] Zhu J C , Zou X Y , Zheng Y , Li M M , Ma M J , Ahmed M S , Chen C J . (2023a). Typical community structure and functional genes of anammox and changes in the effects of saline wastewaters: a critical review. Journal of Environmental Chemical Engineering, 11(6): 111481

[61] Zhu Q , Zheng Y Y , Zhou X W , Wang D J , Yuan M J , Qian D K , Liang S , Yu W B , Yang J K , Hou H J . et al. (2024). c-di-GMP and AHL signals-triggered chemical communication under electrical signaling disruption restores Geobacter sulfurreducens biofilm formation. ISME Communications, 4(1): ycae096

[62] Zhu Y J , Li D , Qi S Y , Yang S , Zhang J , Wang W Q , Li S . (2023b). Anticipating on potential electron acceptors for Anammox. Chemical Engineering Journal, 467: 143458

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