Enhancing nitrogen removal in constructed wetlands: The role of influent substrate concentrations in integrated vertical-flow systems

Recent advancements in constructed wetlands (CWs) have highlighted the imperative of enhancing nitrogen (N) removal efficiency. However, the variability in influent substrate concentrations presents a challenge in optimizing N removal strategies due to its impact on removal efficiency and mechanisms. Here we show the interplay between influent substrate concentration and N removal processes within integrated vertical-flow constructed wetlands (IVFCWs), using wastewaters enriched with NO₃^--N and NH₄⁺-N at varying carbon to nitrogen (C/N) ratios (1, 3, and 6). In the NO₃^--N enriched systems, a positive correlation was observed between the C/N ratio and total nitrogen (TN) removal efficiency, which markedly increased from 13.46 ± 2.23% to 87.00 ± 2.37% as the C/N ratio escalated from 1 to 6. Conversely, in NH₄⁺-N enriched systems, TN removal efficiencies in the A-6 setup (33.69 ± 4.83%) were marginally 1.25 to 1.29 times higher than those in A-3 and A-1 systems, attributed to constraints in dissolved oxygen (DO) levels and alkalinity. Microbial community analysis and metabolic pathway assessment revealed that anaerobic denitrification, microbial N assimilation, and dissimilatory nitrate reduction to ammonium (DNRA) predominated in NO₃^--N systems with higher C/N ratios (C/N ≥ 3). In contrast, aerobic denitrification and microbial N assimilation were the primary pathways in NH₄⁺-N systems and low C/N NO₃^--N systems. A mass balance approach indicated denitrification and microbial N assimilation contributed 4.12-47.12% and 8.51-38.96% in NO₃^--N systems, respectively, and 0.55-17.35% and 7.83-33.55% in NH₄⁺-N systems to TN removal. To enhance N removal, strategies for NO₃^--N dominated systems should address carbon source limitations and electron competition between denitrification and DNRA processes, while NH₄⁺-N dominated systems require optimization of carbon utilization pathways, and ensuring adequate DO and alkalinity supply.