Insights into the alleviating effects of exogenous α-ketoglutaric acid on free nitrous acid stress in Tribonema minus and ammonium removal in wastewater.

α-Ketoglutaric acid (α-KG) is a key intermediate metabolite in carbon and nitrogen metabolism, playing important roles in regulating microbial stress tolerance. However, its potential to mitigate free nitrous acid (FNA) toxicity, a primary inhibitor of microalgae-nitrifier consortium, has remained unexplored. This study investigated the effects of exogenous α-KG onTribonema minusunder FNA stress and evaluated its application in wastewater treatment. Results showed that 1.0 mmol/L α-KG markedly alleviated FNA stress, but a higher concentration (2.0 mmol/L) was less effective. α-KG mitigated FNA-induced oxidative stress and reduced reactive oxygen species (ROS) and malondialdehyde (MDA) levels by 24.9% and 15.1%, while enhancing superoxide dismutase (SOD) and catalase (CAT) activities by 44.6% and 296.1%, respectively. Additionally, α-KG enhanced nitrogen assimilation enzyme activities and reduced intracellular nitrite content by 31.2-37.2%. Furthermore, the enhanced proton-consuming ammonium assimilation and transcriptional upregulation of transmembrane transporter genes were identified as potential contributors to intracellular pH homeostasis. Under α-KG treatment, photosynthetic carbon fixation efficiency and protein content rose by 181.1%-447.4% and 17.3%, respectively. Transcriptional upregulation of fatty acid degradation-related genes was observed, accompanied by 32.7% reduction in lipid content, likely providing energy for FNA stress tolerance. In microalgae-nitrifier consortium treating ammonium-rich wastewater, α-KG supplementation improved ammonium removal efficiency from 63.8% to 91.0% and increased bio-assimilation contribution from 9.1% to 32.1%. α-KG also enriched bacteria includingNitrospira,Hydrogenophaga, andTrichococcus. This study suggested that α-KG regulated nitrogen and carbon metabolism to enhance microalgal tolerance to FNA stress, offering a promising strategy to stabilize microalgae-based wastewater treatment.