Synergistic coupling of anammox and mixotrophic sulfide-driven denitrification for extensive nitrogen removal

Abstract

The integration of anaerobic ammonium oxidation (anammox) with sulfide-dependent autotrophic denitrification (S-SADN) offers a promising low-carbon route for biological nitrogen removal from large volumes of wastewater generated worldwide. However, its application is hindered by sulfide toxicity, nitrite competition, and excessive sulfate production. Here, we developed a stable mixotrophic model system (KAS1–AutoDN2) by integrating an anammox-enriched culture (KAS1) with a sulfide-oxidizing denitrifier, Thauera sp. AutoDN2. At a low carbon-to-nitrogen ratio (C/N) of 0.8, with acetate and sulfide serving as co-electron donors, the KAS1–AutoDN2 system achieved 98.1% ± 2.1% ammonium removal and 99.4% ± 2.2% total nitrogen (TN) removal. Long-term mixotrophic fed-batch operation demonstrated that anammox accounted for 71.2%–77.1% of TN removal, while mixotrophic S-SADN provided a complementary pathway with markedly reduced sulfate yields (63%–68% lower than those of conventional S-SADN systems). Stable transcription of hzsA/hzsB (anammox) and narG/napA (denitrification) confirmed the coexistence and metabolic synergy between the two functional microbial guilds. Notably, no detectable nitrous oxide (N₂O) emission was observed, likely due to the high nitrite affinity of anammox bacteria. This study demonstrates that strategic mixotrophy mitigates sulfide inhibition, balances denitrification stoichiometry, suppresses sulfate overproduction, and stabilizes integrated C-N-S cycling. The anammox-mixotrophic S-SADN platform therefore represents a scalable, energy-efficient biotechnology for treating carbon-limited, sulfide-rich wastewaters in both industrial and municipal sectors.

Graphical abstract

Keywords

Anammox / Sulfide-dependent autotrophic denitrification / Synergistic nitrogen removal

Highlight

	● A stable anammox-mixotrophic S-SADN system was developed using Thauera sp. AutoDN2.
	● Mixotrophic metabolism mitigated sulfide inhibition and sustained anammox activity.
	● Near-complete nitrogen removal (99.4% TN) was achieved at a low C/N ratio (0.8).
	● Anammox accounted for 71%–77% of TN removal during long-term operation.
	● No N2O emission occurred, indicating an environmentally robust C-N-S pathway.

References

[1] Arora A S , Nawaz A , Qyyum M A , Ismail S , Aslam M , Tawfik A , Yun C M , Lee M . (2021). Energy saving anammox technology-based nitrogen removal and bioenergy recovery from wastewater: inhibition mechanisms, state-of-the-art control strategies, and prospects. Renewable and Sustainable Energy Reviews, 135: 110126

[2] Cai J , Zheng P , Qaisar M , Zhang J Q . (2017). Elemental sulfur recovery of biological sulfide removal process from wastewater: a review. Critical Reviews in Environmental Science and Technology, 47(21): 2079–2099

[3] Cao S B , Du R , Zhou Y . (2021). Coupling anammox with heterotrophic denitrification for enhanced nitrogen removal: a review. Critical Reviews in Environmental Science and Technology, 51(19): 2260–2293

[4] Costerton J W , Cheng K J , Geesey G G , Ladd T I , Nickel J C , Dasgupta M , Marrie T J . (1987). Bacterial biofilms in nature and disease. Annual Review of Microbiology, 41: 435–464

[5] Dalsgaard T , De Brabandere L , Hall P O J . (2013). Denitrification in the water column of the central Baltic Sea. Geochimica et Cosmochimica Acta, 106: 247–260

[6] 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

[7] Fajardo C , Mora M , Fernández I , Mosquera-Corral A , Campos J L , Méndez R . (2014). Cross effect of temperature, pH and free ammonia on autotrophic denitrification process with sulphide as electron donor. Chemosphere, 97: 10–15

[8] 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

[9] 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

[10] Jin R C , Yang G F , Zhang Q Q , Ma C , Yu J J , Xing B S . (2013). The effect of sulfide inhibition on the ANAMMOX process. Water Research, 47(3): 1459–1469

[11] Jing C , Ping Z , Mahmood Q . (2010). Influence of various nitrogenous electron acceptors on the anaerobic sulfide oxidation. Bioresource Technology, 101(9): 2931–2937

[12] Joss A , Derlon N , Cyprien C , Burger S , Szivak I , Traber J , Siegrist H , Morgenroth E . (2011). Combined nitritation–anammox: advances in understanding process stability. Environmental Science & Technology, 45(22): 9735–9742

[13] Kong Z , Li L , Feng C P , Dong S S , Chen N . (2016). Comparative investigation on integrated vertical-flow biofilters applying sulfur-based and pyrite-based autotrophic denitrification for domestic wastewater treatment. Bioresource Technology, 211: 125–135

[14] Li X , Shi M , Zhang M , Li W , Xu P L , Wang Y Y , Yuan Y , Huang Y . (2022). Progresses and challenges in sulfur autotrophic denitrification-enhanced Anammox for low carbon and efficient nitrogen removal. Critical Reviews in Environmental Science and Technology, 52(24): 4379–4394

[15] Liu Z H , Lin W M , Luo Q J , Chen Y C , Hu Y Y . (2021). Effects of an organic carbon source on the coupling of sulfur (thiosulfate)-driven denitration with Anammox process. Bioresource Technology, 335: 125280

[16] Lotti T , Kleerebezem R , Abelleira-Pereira J M , Abbas B , Van Loosdrecht M C M . (2015). Faster through training: the anammox case. Water Research, 81: 261–268

[17] Lotti T , Kleerebezem R , Lubello C , Van Loosdrecht M C M . (2014). Physiological and kinetic characterization of a suspended cell anammox culture. Water Research, 60: 1–14

[18] Manonmani U , Joseph K . (2018). Granulation of anammox microorganisms for autotrophic nitrogen removal from waste-water. Environmental Chemistry Letters, 16(3): 881–901

[19] Matsuto T , Zhang X , Matsuo T , Yamada S . (2015). Onsite survey on the mechanism of passive aeration and air flow path in a semi-aerobic landfill. Waste Management, 36: 204–212

[20] Mora M , Guisasola A , Gamisans X , Gabriel D . (2014). Examining thiosulfate-driven autotrophic denitrification through respiro-metry. Chemosphere, 113: 1–8

[21] 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

[22] Qin Y J , Wu C L , Chen B Q , Ren J Y , Chen L Y . (2019). Short term performance and microbial community of a sulfide-based denitrification and Anammox coupling system at different N/S ratios. Bioresource Technology, 294: 122130

[23] Qiu Y Y , Zhang L , Mu X T , Li G B , Guan X Q , Hong J Y , Jiang F . (2020). Overlooked pathways of denitrification in a sulfur-based denitrification system with organic supplementation. Water Research, 169: 115084

[24] Russ L , Speth D R , Jetten M S M , Op Den Camp H J M , Kartal B . (2014). Interactions between anaerobic ammonium and sulfur-oxidizing bacteria in a laboratory scale model system. Environmental Microbiology, 16(11): 3487–3498

[25] Saxena G, Chandra R, Bharagava R N (2017). Environmental pollution, toxicity profile and treatment approaches for tannery wastewater and its chemical pollutants. In: De Voogt P, ed. Reviews of Environmental Contamination and Toxicology Volume 240. Cham: Springer, 31–69

[26] Strous M , Kuenen J G , Jetten M S M . (1999). Key physiology of anaerobic ammonium oxidation. Applied and Environmental Microbiology, 65(7): 3248–3250

[27] Su J J , Liu B Y , Chang Y C . (2003). Emission of greenhouse gas from livestock waste and wastewater treatment in Taiwan. Agriculture, Ecosystems & Environment, 95(1): 253–263

[28] Sun C Y , Mao S Y , Zhao W Y , Chen Y S , Cao X , Tian T , Ma X Y , Li B , Qiu Y . (2025). Multi-objective comparison of conventional and emerging wastewater treatment processes based on simulation to reduce greenhouse gas emissions. Frontiers of Environmental Science & Engineering, 19(3): 29

[29] Vaiopoulou E , Melidis P , Aivasidis A . (2005). Sulfide removal in wastewater from petrochemical industries by autotrophic denitrification. Water Research, 39(17): 4101–4109

[30] Verania L R B , Satoh H , Sotelo T J . (2026). Sulfide removal from wastewater via self-purification by porous media: effect of flow intermittency and various feed sulfide loadings. Biochemical Engineering Journal, 226: 109975

[31] Wang J J , Huang B C , Li J , Jin R C . (2020). Advances and challenges of sulfur-driven autotrophic denitrification (SDAD) for nitrogen removal. Chinese Chemical Letters, 31(10): 2567–2574

[32] Wu C L , Qin Y J , Yang L , Liu Z J , Chen B Q , Chen L Y . (2020). Effects of loading rates and N/S ratios in the sulfide-dependent autotrophic denitrification (SDAD) and Anammox coupling system. Bioresource Technology, 316: 123969

[33] Xie H T, Ji D D, Zang L H (2017) Effects of inhibition conditions on anammox process. IOP Conference Series: Earth and Environmental Science, 100: 012149

[34] Xu G H , Yin F J , Chen S H , Xu Y J , Yu H Q . (2016). Mathematical modeling of autotrophic denitrification (AD) process with sulphide as electron donor. Water Research, 91: 225–234

[35] 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

[36] Yan J , Wen H J , Li Q Q , Wang S J , Xie J H , Chen M P , Li J Y , Chang X Y , Zhang H G , Hong Y G . (2020). Enhanced elemental sulfur recovery and nitrogen removal through coupling of sulfide-dependent denitrification and anammox processes during ammonium-and sulfide-laden waste stream treatment. International Biodeterioration & Biodegradation, 155: 105086

[37] Yuan Z L , Chen Y Z , Zhang M , Qin Y R , Zhang M N , Mao P Y , Yan Y . (2022). Efficient nitrite accumulation and elemental sulfur recovery in partial sulfide autotrophic denitrification system: insights of seeding sludge, S/N ratio and flocculation strategy. Chemosphere, 288: 132388

[38] Zhang J T , Fan C Z , Zhao M , Wang Z Q , Jiang S F , Jin Z , Bei K , Zheng X Y , Wu S Q , Lin P . et al. (2023). A comprehensive review on mixotrophic denitrification processes for biological nitrogen removal. Chemosphere, 313: 137474

[39] Zhang L , Cui Y F , Dou Q H , Peng Y Z , Yang J C . (2024). Sulfur-carbon loop enhanced efficient nitrogen removal mechanism from iron sulfide-mediated mixotrophic partial denitrification/ anammox systems. Bioresource Technology, 403: 130882

[40] Zhang L , Qiu Y Y , Sharma K R , Shi T , Song Y R , Sun J L , Liang Z S , Yuan Z G , Jiang F . (2023). Hydrogen sulfide control in sewer systems: a critical review of recent progress. Water Research, 240: 120046

[41] Zhang Y F , Wang Q K , Rogers M J , He J Z . (2025). Autotrophic denitrification under anoxic conditions by newly discovered mixotrophic sulfide-oxidizing bacterium. Bioresource Technology, 430: 132553

[42] 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

[43] Zheng R , Zhang K , Kong L R , Liu S T . (2024). Research progress and prospect of low-carbon biological technology for nitrate removal in wastewater treatment. Frontiers of Environmental Science & Engineering, 18(7): 80

[44] Zhou Y F , Zhu Y Y , Zhu J Y , Li C R , Chen G . (2023). A comprehensive review on wastewater nitrogen removal and its recovery processes. International Journal of Environmental Research and Public Health, 20(4): 3429

[45] Zhu W Q , Zhang P Y , Dong H Y , Li J . (2017). Effect of carbon source on nitrogen removal in anaerobic ammonium oxidation (anammox) process. Journal of Bioscience and Bioengineering, 123(4): 497–504

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