Matrix-mediated reversal of gram-dependent removal in antimicrobial flocculation

Microbial contamination continues to threaten safe drinking water and sanitation. Antimicrobial flocculation is being increasingly explored as an upstream strategy to reduce disinfection demand and associated risks, yet the impact of environmental matrices on its performance has been overlooked in the past. Here, we report a previously unrecognized, matrix-induced reversal of Gram-dependent bacterial removal during antimicrobial flocculation using cationic flocculants. In matrix-free suspensions, Gram-positive S. aureus is removed more efficiently than Gram-negative E. coli. However, in the presence of representative matrices, involving inorganic suspended particles (ISPs) and natural organic matter (NOM), both bacteria are removed more effectively, but the enhancement is substantially stronger for E. coli, reversing the relative removal ranking. Biomolecular signatures, microscopy and flow cytometry reveal that environmental matrices induce Gram-dependent cell envelope damage, with stronger vulnerability in Gram-negative bacteria: ISPs cause shear-enabled physical abrasion with strong hydrodynamic dependence, whereas NOM induces chemically mediated envelope injury and permeability enhancement through cell-envelope binding. These findings demonstrate that antibacterial flocculation performance is not determined by flocculant properties alone, but by complex interactions among flocculants, bacteria, and co-existing matrices, highlighting the importance of representative water-matrix conditions to microbial control strategies.