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Antimicrobial fatty acids derived from natural sources and renewable feedstocks are promising surface-active substances with a wide range of applications. Their ability to target bacterial membrane in multiple mechanisms offers a promising antimicrobial approach for combating bacterial infections and preventing the development of drug-resistant strains, and it provides a sustainable strategy that aligns with growing environmental awareness compared to their synthetic counterparts. However, the interaction and destabilization of bacterial cell membranes by these amphiphilic compounds are not yet fully understood. Here, we investigated the concentration-dependent and time-dependent membrane interaction between long-chain unsaturated fatty acids-linolenic acid (LNA, C18:3), linoleic (LLA, C18:2), and oleic acid (OA, C18:1)-and the supported lipid bilayers (SLBs) using quartz crystal microbalance-dissipation (QCM-D) and fluorescence microscopy. We first determined the critical micelle concentration (CMC) of each compound using a fluorescence spectrophotometer and monitored the membrane interaction in real time following fatty acid treatment, whereby all micellar fatty acids elicited membrane-active behavior primarily above their respective CMC values. Specifically, LNA and LLA, which have higher degrees of unsaturation and CMC values of 160 µM and 60 µM, respectively, caused significant changes in the membrane with net |Δf| shifts of 23.2 ± 0.8 Hz and 21.4 ± 0.6 Hz and ΔD shifts of 5.2 ± 0.5 × 10-6 and 7.4 ± 0.5 × 10-6. On the other hand, OA, with the lowest unsaturation degree and CMC value of 20 µM, produced relatively less membrane change with a net |Δf| shift of 14.6 ± 2.2 Hz and ΔD shift of 8.8 ± 0.2 × 10-6. Both LNA and LLA required higher concentrations than OA to initiate membrane remodeling as their CMC values increased with the degree of unsaturation. Upon incubating with fluorescence-labeled model membranes, the fatty acids induced tubular morphological changes at concentrations above CMC. Taken together, our findings highlight the critical role of self-aggregation properties and the degree of unsaturated bonds in unsaturated long-chain fatty acids upon modulating membrane destabilization, suggesting potential applications in developing sustainable and effective antimicrobial strategies. Ministry of Education (MOE) Published version This research was supported by the Minister of Education (MOE) in Singapore under grants RG111/20 and RG34/22. This research was also supported by the Procter & Gamble Company (P&G) under grants APG2013/129.

Additional file 1: Table S1. Shear viscosity of sucrose solutions without/with bicelles, and bicelle sizes in sucrose���bicelle mixtures containing various concentrations of sucrose. Figure S1. QCM-D measurements of bicelle adsorption onto silica surfaces in various concentrations of sucrose (A���F, 0���50 wt% sucrose). The baseline of ��f and ��D shifts were recorded in Tris buffer containing 150 mM NaCl, and then measurements were made in the following solutions: (1) sucrose solution in Tris buffer; (2) sucrose���bicelle mixture for SLB formation; (3) sucrose solution in Tris buffer for washing step; (4) Tris buffer for final baseline to check the quality of SLB formation. Figure S2. Air stability of SLBs after 1 month of dehydration. (A) FRAP results and recovery curves for mobility characterization of SLBs made from 20 wt% sucrose and rehydrated after 1 month of dehydration. (B) Corresponding results for SLBs made from 40 wt% sucrose. All scale bars are 20 ��m.