Salinity responsive mechanisms of sulfur-based mixotrophic denitrification and ectoine induced tolerance enhancement

Increasing discharge of saline wastewater poses a challenge for heterotrophic and sulfur autotrophic denitrification (HSAD) in nitrogen removal. This study explored the response mechanism of mixotrophic denitrifying microorganisms to high-salt stress and feasible mitigation strategies. Results showed that denitrification efficiency was promoted at 2 % salinity but significantly decreased at 6 %, with NO₃^–-N removal rate decreased from 95.77 % to 38.01 %. The contribution of sulfur autotrophic denitrification (SAD) remained higher than heterotrophic denitrification (HD). High salinity stimulation led to an increase in reactive oxygen species (ROS) content, while the nicotinamide adenine dinucleotide (NADH) content, adenosine triphosphate (ATP) level, and electron transport system activity (ETSA) decreased by 10.74 %, 46.6 % and 56.28 % at 6 % salinity, respectively. Additionally, high salt inhibited the activity of nitrate reductase (NAR) and nitrite reductase (NIR), reducing the abundance of denitrifying bacteria. Notably, the addition of 250 mg/L of ectoine at 6 % salinity alleviated stress, promoted the secretion of extracellular polymeric substances (EPS) to protect the microorganisms, reduced microbial oxidative stress, and maintained metabolic and denitrification enzyme activities. Ectoine acted as an osmotic pressure regulator and protective agent, maintaining intracellular enzymes and macromolecular structure, thus enhancing denitrification efficiency. Microbial community analysis demonstrated a 3.99 % increase in HD functional bacteria abundance with ectoine addition, highlighting its crucial role in regulation community succession and stability. Structural equations modeling analysis identified the regulatory relationship paths involved in the response and mitigation of HSAD to high salinity stress. This study deepens understanding of the inhibition mechanism of HSAD caused by high-salt wastewater and provides a feasible technical solution for salt stress alleviation in sulfur-based mixotrophic denitrification.