Enhancement of Activated Sludge Biofilm Formation and Microbial Community Modulation by Exogenous Amino Acid Supplementation Under Different Environmental Conditions.

Stable biofilm formation is essential for improving the efficiency of biofilm-based biological processes, including wastewater treatment systems and membrane bioreactors. In recent decades, biofilms have attracted considerable research interest because of their efficiency, ease of use, and minimal environmental impacts. However, uncontrolled biofilm development and insufficient microbial attachment under variable environmental conditions remain major limitations in practical applications. Although external nutrient addition has been widely investigated for improving microbial growth, the effects of specific amino acids on biofilm formation and microbial community organization are still poorly understood. In this study, exogenous amino acids (arginine, ornithine, and lysine) were applied to activated sludge systems to evaluate their potential for enhancing biofilm formation and influencing microbial community structure under different environmental conditions. The effects of amino acid type, concentration (0.5-10 mM), incubation time, temperature, light/dark, carrier surface properties, light/dark, and hydrodynamic stress conditions on biofilm development were investigated. The results demonstrated that amino acid supplementation significantly promoted biofilm formation compared with control. The highest biofilm biomass was observed at 1 mM arginine and 3 mM ornithine and lysine concentrations under the tested conditions. Among the tested amino acids, arginine exhibited the strongest biofilm-promoting capability, followed by ornithine and lysine. Evaluation of environmental conditions revealed that moderate temperatures (24-32◦C), suitable rotational speeds (100-150 rpm), porous and rigid carriers, and appropriate maturation time (21-28 days) favored biofilm development. Furthermore, high-throughput 16S rRNA sequencing indicated that selected amino acid supplementation was associated with changes in microbial diversity and biofilm-associated community structure. Overall, this study demonstrates that exogenous amino acid supplementation can influence biofilm formation and microbial community structure in activated sludge under different environmental conditions. The outcomes of this study enhance our understanding of the influence of different environmental conditions on biofilm development and provide possible strategies for promoting biofilm formation under different environmental conditions.