Functional genomic analysis reveals bacterial response of thiosulfate-driven denitrification to artificial sweetener “aspartame”

Artificial sweeteners such as aspartame (ASP) have raised increasing concerns due to their environmental persistence and biological toxicity, yet their impacts on sulfur-driven denitrification systems remain poorly understood. This study systematically investigated the microbial responses and functional gene dynamics of a thiosulfate-driven anaerobic denitrification system under gradient ASP exposure (0–25 mg/L). Results showed that low concentrations of ASP caused transient inhibition of denitrification, with nitrate accumulation, nitrite buildup, and a drop in nitrogen removal efficiency from 97 % to a minimum of 78 %. However, the system rapidly recovered and maintained stable performance even under higher ASP loads. This resilience was attributed to the enrichment of the core microbial module dominated by Thiobacillus and Rhodocyclaceae , along with upregulated expression of key sulfur and nitrogen cycling genes (soxB, nar, nir, nos). This module functionally resembles an immune-like regulatory network, coupling sulfur oxidation with denitrification and enabling metabolic stability under stress. Correlation and random forest analyses further identified nasC, aprB, soxB, and nosZ as key stress-responsive genes, highlighting their role in adaptive reprogramming and ecosystem robustness. • Microbial resilience was driven by a sulfur–nitrogen coupled core module. • Random forest identified predictive genes of ASP-induced functional shifts. • Key genes (soxB, nar, nir, nos) formed a functional immune-like module.