Antimicrobial activity enhancement of cationic lignin by phenolation

In this work, we developed a new class of highly cationic antimicrobials (QPALs) from alkali lignin (AL). Unlike conventional cationization of AL, lignin was first phenolated here to increase its phenolic hydroxyl content, followed by quaternization with glycidyltrimethylammonium chloride (GTMAC). By varying phenolation conditions, a library of phenolated lignins (PALs) was synthesized, from which 3 representative samples with low, intermediate, and high phenolic contents were down-selected (PAL1, PAL2, PAL3) and converted to QPAL (QPAL1, QPAL2, QPAL3) with enhanced ion exchange capacities: 2.1-2.7 mmol g^-1 vs. 1.2 mmol g^-1 for non-phenolated cationic lignin (QAL). QPAL exhibited markedly improved antimicrobial activity against wild-type, ampicillin-resistant, and kanamycin-resistant E. coli. With a short 5 min treatment, 40 μg mL^-1QPAL1, QPAL2, and QPAL3 achieved 75%, 90%, and >98% colony forming unit (CFU) reduction, respectively, outperforming PALs (PAL1: <15%, PAL2: <16%, PAL3: <16%), AL (<11%), and QAL (59%). Complete bacterial inactivation (100% CFU reduction) was achieved within 1 h for all QPAL samples (40 μg mL^-1), corroborating with predominantly dead cells seen in live/dead assays upon QPAL treatment. In contrast, QAL required 150-300 µg mL^-1 to reach comparable % CFU reduction efficacy. Due to higher cationic charge density and more branched architecture (achieved by phenolation before cationization), QPAL was able to cause severe membrane disruption, ROS generation, morphological deformation, and intracellular leakage upon QPAL treatment. Importantly, QPAL maintained low cytotoxicity toward human embryonic kidney cells. These findings demonstrate phenolation-enabled enhanced cationization as an effective strategy for developing high-performance lignin-based antimicrobials.