Step-feeding ratios affect nitrogen removal and related microbial communities in multi-stage vertical flow...

Step-feeding (SF) strategies have been adopted in several types of constructed wetlands (CWs) to enhance nitrogen (N) removal. However, it is unclear how SF affects the N-transforming bacterial communities in CWs. Herein, four multi-stage vertical flow constructed wetlands (MS-VFCWs), each including three vertical flow stages (stage 1-3), were operated under different SF ratios (0%, 10%, 20% and 30%) in the stage 2. The physicochemical influent and effluent parameters, i.e., redox potential (ORP), pH value, chemical oxygen demand (COD), total nitrogen (TN), ammonia (NH₄⁺-N), nitrate (NO₃^--N), and nitrite (NO₂^--N), free-ammonia (FA) concentration, COD/TN ratio, as well as the abundance, structure, and activity of N-transforming bacteria were investigated. Results showed that N removal in a multi-stage vertical flow constructed wetland in the absence of SF was 45.0 ± 7.74%. Alternatively, a combined SF ratio of 20% increased N removal to 61.7% ± 4.50%, accounting for a 37.1% increase compared to the SF ratio of 0%. In the microbial community, FA was determined to be the primary physicochemical parameter governing nitrification processes in MS-VFCWs. Further, partial nitrification processes played an important role in ammonium removal during stage 1, while ammonia-oxidizing archaea were major contributors to ammonium removal in stage 3. Furthermore, abundance of nitrite reductase genes (nirS, nirK) and relative abundance of denitrifying bacteria increased with increasing SF ratio; while the nirS/nirK ratio and the alpha diversity of nirK denitrifiers were significantly affected by SF ratios, and the influent NO₃^--N concentration was related to a shift in denitrifier composition toward strains containing the nirS gene. Autotrophic (e.g., Thiobacillus, Sulfurimonas, Arenimonas, Gallionella and Methyloparacoccus) and facultative chemolithoautotrophic (e.g., Pseudomonas and Denitratisoma) denitrifying bacteria were enriched in stage 2. Hence, the synergy between heterotrophic and autotrophic denitrifying bacteria promoted excellent N removal efficiency with a low COD/TN ratio.