Mechanisms driving anammox bacteria enrichment in constructed wetlands for self-purification of high-nitrogen polluted wastewater: Environmental gradients and microbial interactions

Anammox bacteria in constructed wetlands (CWs) play pivotal role in sustainable nitrogen transformation, yet existing studies lack comprehensive analysis of environmental gradients and microbial interactions, both key factors in anammox bacteria enrichment. This study investigated the mechanisms driving anammox bacteria enrichment in lab-scale simulated CWs treating high-nitrogen wastewater, focusing on bacterial community responses across wetland layers with various strategies, including continuous up-flow influent, nitrogen loading increase, effluent recirculation, intermittent influent, and anammox bacteria inoculation. Results showed that total relative and absolute abundances of anammox bacteria ranged from 0.77 % to 12.50 % and from 0.13 to 6.46 × 10⁷ copies/g, respectively. Dissolved oxygen and pH had significant positive correlations with the absolute abundance of anammox bacteria, while organic matter and nitrate negatively impacted their relative abundance. Permutational multivariate analysis of variance indicated that spatial heterogeneity explained more variation in anammox bacteria abundance (43.44 %) compared to operational strategies (8.58 %). In terms of microbial interactions, 60 dominant species exhibited potential correlations with anammox bacteria, comprising 170 interactions (105 positive and 65 negative), which suggested that anammox bacteria generally foster cooperative relationships with dominant bacteria. Notably, significant interspecies interactions were observed between Candidatus Kuenenia (dominant anammox bacteria in CWs) and species within the genera Chitinivibrionia and Anaerolineaceae, suggesting that microbial interactions primarily manifest as indirect facilitative effects rather than direct mutualistic relationships. Given that the Normalized Stochasticity Ratio in CWs were <50 %, this study inferred that environmental gradients have greater influence on anammox bacteria than microbial interactions.