Metatranscriptomics sheds light on electron transfer in anammox bacteria enhanced by the redox mediator neutral red

Enhancing the activity of key enzymes has been recognized as an effective strategy to improve anammox performance. Neutral red (NR), a potent redox-active electron carrier, has been shown to boost various enzyme activities and microbial reaction rates. However, its potential to enhance anammox performance remains underexplored. This study aimed to investigate the effects of different NR concentrations on anammox nitrogen removal efficiency and gene transcription levels. The results revealed that anammox activity increased with NR doses in the lower concentration range (0.05-0.3 g L^-1). The optimal dosage at 0.1 g L^-1 significantly increased specific anammox activity (SAA) by 16.73 ± 2.68% (p ≤ 0.001), compared to the control without NR addition. Moreover, the total EPS concentration increased by 16.87 ± 1.20% (p ≤ 0.01). Conversely, NR concentrations exceeding the optimal range inhibited anammox activity. Metatranscriptomic analysis showed that appropriate NR supplementation upregulated the expression of cofactor modules related to electron transfer and functional genes (hdh and hzsB) involved in anammox nitrogen removal, thereby enhancing overall performance. Moreover, the mild oxidative stress induced by low NR doses was mitigated through the upregulation of antioxidant genes. In contrast, excessive NR (0.5-1.0 g L^-1) led to an accumulation of reactive oxygen species (ROS) that overwhelmed the antioxidant defense system, resulting in impaired electron transfer and reduced metabolic activity. Specifically, when the NR concentration was increased to 1.0 g L^-1, SAA decreased significantly by 26.45 ± 2.55% (p ≤ 0.001). These findings indicate that appropriately controlled NR supplementation can improve anammox activity, providing a promising strategy for rapid start-up and improved nitrogen removal in practical anammox systems.