CO<sub>3</sub><sup>2−</sup> enhanced electron transfer and enzyme stabilization overcome the ion inhibition paradigm in aerobic denitrification

Jun Zhang; Tian Hu; Zhushuo Zhang; Yihang Ding; Chunling Zheng; Shengtao Zhou; Jun Zhang; Tian Hu; Zhushuo Zhang; Yihang Ding; Chunling Zheng; Shengtao Zhou

doi:10.3934/environsci.2026013

AIMS Environmental Science

2026, Volume 13, Issue 2: 331-347. doi: 10.3934/environsci.2026013

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Research article

CO₃²⁻ enhanced electron transfer and enzyme stabilization overcome the ion inhibition paradigm in aerobic denitrification

1.
College of Water Resources and Modern Agriculture, Nanyang Normal University, Nanyang, 473061, China
2.
Collaborative Innovation Center of Water Security for the Water Source Region of Mid-line of the South-to-North Diversion Project of Henan Province, Nanyang Normal University, Nanyang, 473061, China
3.
College of Life Sciences, Nanyang Normal University, Nanyang, 473061, China

Received: 15 November 2025 Revised: 09 February 2026 Accepted: 06 March 2026 Published: 10 April 2026

Aerobic denitrifying bacteria are often inhibited by inorganic ions during wastewater treatment, and elucidating their regulatory mechanisms is crucial for enhancing nitrogen removal efficiency. In this study, we systematically investigated the effects of varying concentrations of inorganic ions (0–200 mg/L) on the growth, denitrification performance, and metabolic activity of Pseudomonas mosselii H6. Based on enzyme kinetics, energy metabolism indicators, and measurements of electron transport chain activity, CO₃²⁻ was found to significantly enhance denitrification capacity. At 200 mg/L, electron transport system activity (ETSA) increased by 26.42%, ATP synthesis peaked at 1.25 μmol/mg prot, and the NADH/NAD⁺ ratio rose to 22.69. Nitrate reductase (NR) and nitrite reductase (NiR) activities reached 28.37 and 17.76 U/mg prot, respectively. In contrast, Cl⁻ and NO₃⁻ at 200 mg/L markedly suppressed cellular metabolism, reducing NH₄⁺-N removal efficiency to 21.41% and 16.42%, respectively. These findings revealed a concentration-dependent and ion-specific regulatory mechanism. CO₃²⁻ enhances nitrogen removal by optimizing electron transfer and improving enzyme stability. This study provides a theoretical foundation for optimizing the biological treatment of high-salinity wastewater.
- aerobic denitrification,
- Pseudomonas mosselii H6,
- CO₃^2-,
- electron transport,
- enzyme stability
Citation: Jun Zhang, Tian Hu, Zhushuo Zhang, Yihang Ding, Chunling Zheng, Shengtao Zhou. CO₃²⁻ enhanced electron transfer and enzyme stabilization overcome the ion inhibition paradigm in aerobic denitrification[J]. AIMS Environmental Science, 2026, 13(2): 331-347. doi: 10.3934/environsci.2026013

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Abstract

Aerobic denitrifying bacteria are often inhibited by inorganic ions during wastewater treatment, and elucidating their regulatory mechanisms is crucial for enhancing nitrogen removal efficiency. In this study, we systematically investigated the effects of varying concentrations of inorganic ions (0–200 mg/L) on the growth, denitrification performance, and metabolic activity of Pseudomonas mosselii H6. Based on enzyme kinetics, energy metabolism indicators, and measurements of electron transport chain activity, CO₃²⁻ was found to significantly enhance denitrification capacity. At 200 mg/L, electron transport system activity (ETSA) increased by 26.42%, ATP synthesis peaked at 1.25 μmol/mg prot, and the NADH/NAD⁺ ratio rose to 22.69. Nitrate reductase (NR) and nitrite reductase (NiR) activities reached 28.37 and 17.76 U/mg prot, respectively. In contrast, Cl⁻ and NO₃⁻ at 200 mg/L markedly suppressed cellular metabolism, reducing NH₄⁺-N removal efficiency to 21.41% and 16.42%, respectively. These findings revealed a concentration-dependent and ion-specific regulatory mechanism. CO₃²⁻ enhances nitrogen removal by optimizing electron transfer and improving enzyme stability. This study provides a theoretical foundation for optimizing the biological treatment of high-salinity wastewater.

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