Micro-mechanism of rhamnolipid promoting acid production during anaerobic digestion: protein structures, metagenomics...

The addition of rhamnolipid (RL) is a promising strategy to enhance volatile fatty acids (VFAs) production in anaerobic digestion (AD) systems. However, the microscopic mechanisms underlying this enhancement remain poorly understood. This study investigates the micro-mechanisms by which RL promotes VFAs production, integrating protein structural analysis, metagenomics, and molecular dynamics simulations. Experimental results revealed that adding RL at 0.08 g/g TS significantly increased VFAs production to 11,441.8 mg COD/L. Protein structural analysis revealed disruption of amide I-related C = O groups and amide II-related CN and NH bonds, indicating the release or structural alteration of sludge proteins. Metagenomic analysis indicated an increase in the abundance of microbial communities and related genes, suggesting that RL enhanced the activity of acid-producing microorganisms and related metabolic pathways. Furthermore, molecular docking and molecular dynamics simulations indicated that RL spontaneously aggregated and absorbed acetate kinase (AK), altering its conformation and reducing structural compactness, which made acetyl phosphate (AcP) more accessible to the binding site of AK. RL reduced the energy barrier associated with the polar solvation interactions, increasing the contributions of key residues (LYS176 and GLU234) to the binding free energy, which enhanced the binding affinity of AK-AcP complex. This study provides a comprehensive molecular basis for how RL promotes VFAs production in AD, offering a promising strategy for optimizing acid production.