Addition of Heterotrophic Nitrification and Aerobic Denitrification Bacterial Agents to Enhance Bio-Nests Treating Low Carbon-to-Nitrogen Ratio Municipal Wastewater

Municipal wastewater with a low carbon-to-nitrogen (C/N) ratio presents challenges for conventional nitrogen removal processes, often requiring costly external carbon sources. This study investigated the enhancement of nitrogen removal in a simultaneous nitrification and denitrification (SND) system by incorporating heterotrophic nitrification and aerobic denitrification (HN-AD) bacterial agents (Klebsiella variicola L3, Acinetobacter beijerinckii W4, and Acinetobacter sp. Z1) with modified basalt fiber carriers. Three reactors were compared: mixed HN-AD strains (M), mixed strains with activated sludge (A+M), and activated sludge alone (A). Results demonstrated that the A+M reactor achieved superior performance, with median removal efficiencies of 82.2% for NH4+-N, 52.9% for total nitrogen (TN), and 51.6% for COD, outperforming the M reactor (75.2%, 43.6%, and 51.6%) and the A reactor (63.2%, 29.3%, and 44.8%). The A+M reactor also exhibited a 40% reduction in COD consumption per unit TN removed (2.55 ± 1.75) compared to the control reactor A (4.25 ± 3.99). Microbial analysis revealed Acinetobacter sp. Z1 (6.1%) and K. variicola L3 (1.1%) as dominant species, with the A+M reactor showing higher microbial diversity (56.4% Proteobacteria, 10.2% Bacteroidota) and biological viability (VSS/SS ratio of 0.70 ± 0.01). Extracellular polymeric substance (EPS) content in A+M reached 242.26 ± 15.52 mg/g-VSS, with a protein-to-polysaccharide ratio of 2.77 ± 0.00, indicating robust biofilm activity. These findings highlight the potential of HN-AD bacterial agents to enhance nitrogen removal in low C/N wastewater treatment, offering a cost-effective and sustainable alternative to traditional methods by reducing reliance on external carbon sources and improving system efficiency.