Lipopeptide surfactin, a biosurfactant, is synthesized by numerous strains of Bacillus subtilis [1]. It comprises a heptapeptide head group, characterized by the sequence (L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu), connected to a β-hydroxy fatty acid enclosed by a lactone ring [2]. The range of carbon atoms within the β-hydroxy fatty acid moiety was reported to be between 11 to 17 [3], [4], [5]. The molecules display amphiphilic properties, characterized by a hydrophobic segment comprising a lengthy fatty acid chain and several lipophilic amino acids (Leu2, Leu3, Val4, Leu6, Leu7). Additionally, they possess a hydrophilic component consisting of the cyclic backbone and two anionic residues (Glu1 and Asp5) [6]. Surfactin, known for its potent biodegradability, demonstrates remarkable surfactant properties by significantly lowering the water surface tension at concentrations around 20 μM. It can effectively reduce the water surface tension from 72 to 27 mN m−1 [7], [8]. At extremely low concentrations, it aggregates into sizable micelles. The critical micellar concentration of the various analogs is approximately 10−5 M [8]. Thorough investigations have revealed that surfactin displays diverse biological effects, such as antiviral [9], [10], antibacterial [10], [11], [12], antitumor [13], antifungal [14], hemolytic [15], and membrane activities [16]. Additionally, surfactin exhibits noticeable physicochemical properties, making it highly effective for enhanced oil recovery [17], and the bioremediation of the environmental pollutants [18].

Based on 1H-NMR study, Bonmatin proposed the first three- dimensional structure of surfactin in DMSO. They introduced two models, each illustrating the peptidic fragment assuming a “horse-saddle” arrangement. In both models, two hydrophilic residues create a possible binding “claw” on one side, whereas the fatty acid chain’s five hydrophobic residues are oriented towards the opposite side. The primary distinction between the two conformers lies in their intramolecular hydrogen bonds, specifically [NH(5)–CO(2)] for one conformer and [NH(7)–CO(5), NH(4)–CO(2), and NH(6)–C1O] for the other conformer [19].

In a separate NMR study conducted in SDS micellar aqueous solution, Lancelin and colleagues revealed that the surfactin adopts a three-dimensional structure that belongs to a unique low-energy family. However, the backbone maintains a horse-saddle conformation. On one side of the molecule, all hydrophobic residues are exposed, except for Val4. Meanwhile, both acidic residues are positioned together on the opposite side of the molecule [20].

Surfactin, a powerful biosurfactant, has been extensively studied for its unique surface properties under various conditions. Research by Maget-Dana and Ptak explored how pH, temperature, and electrolytes influence these properties, suggesting multiple possible orientations at interfaces [21]. Further Bo-Zhong Mu et al. showed that increasing temperature has little impact on micelle size and shape but causes the peptide ring backbone to shift from a horse-saddle to a flat conformation [22]. Besides, surfactin’s interfacial concentration influences its orientation, backbone flexibility, and monolayer organization, with higher concentrations enhancing the packing and stabilizing the β-turn [23]. Ishigami et al. revealed that surfactin adopts a β-sheet secondary structure at the air/water interface [8]. Gallet et al. employed molecular modeling and proposed that surfactin’s peptide ring aligns parallel to the interface, with acidic residues extending into the water and the β-hydroxy fatty acid interacting with a leucine side chain [6]. Simulations by Nicolas highlighted the high flexibility of surfactin’s peptide backbone at the hexane/water interface, influenced by concentration gradients [24]. Additionally, neutron reflectometry studies demonstrated surfactin’s hydrophobic, ball-like structure at the air/water interface, showing that its structure and hydrophilicity are influenced by charge and the presence of cations [25], [26].

Alkyl chain length plays a critical role in influencing surfactant’s hydrophobic–hydrophilic balance, which governs key physicochemical properties such as surface tension reduction, critical micelle concentration, and adsorption at interfaces [27]. Even though the impact of chain length on surfactant activity is known, the molecular-level mechanisms by which it affects self-assembly and interfacial organization remain little understood for the surfactin. A detailed atomistic perspective is essential to bridge this knowledge gap and to guide rational design or modification of surfactin analogs for specific applications. In this study, we have explored the aggregation behavior and interfacial properties of surfactin molecules by varying the chain length of the hydrocarbon moiety using atomistic molecular dynamics (MD) simulations. We investigated the self-assembly behavior of surfactin in aqueous solutions, its interactions with water, and conducted a hydrogen bond analysis of surfactin aggregates. We have also simulated the liquid–vapor interface to understand the surface activity of surfactin. These insights enhance our understanding of how structural variations in surfactin influence its self-assembly and interfacial behavior, which is critical for optimizing its applications in areas such as emulsification, drug delivery, and environmental remediation.