Microbial Biosurfactants: Antimicrobial Agents Against Pathogens

Abstract

Microbial biosurfactants (mBSs) are bioactive molecules with diverse applications, notably as antimicrobial agents against antibiotic-resistant pathogens. Produced by bacteria and yeasts, mBSs are classified as glycolipids, lipopeptides, polymeric, and particulate types. The global rise in multidrug-resistant organisms, such as Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Pseudomonas aeruginosa, and Acinetobacter baumannii, underscores the urgent need for new antimicrobial strategies. mBSs disrupt microbial growth by interacting with the lipid components of pathogens, offering promising alternatives to conventional antibiotics. This review highlights the sources, chemical structures, and properties of mBSs, their antimicrobial activities, synergistic effects with antibiotics, and structure–activity relationships. Special emphasis is placed on surfactant modification, where targeted changes—such as valine substitution in surfactin—significantly lower critical micelle concentrations (CMC) and enhance antimicrobial potency. Such rational engineering demonstrates how biosurfactants can be tailored for improved biomedical performance while minimizing cytotoxicity. In parallel, artificial intelligence (AI) algorithms, including artificial neural networks and genetic algorithms, optimize yields, predict substrate suitability from agricultural residues, and guide microbial strain engineering. AI models can predict interfacial behavior and synchronize fermentation with purification. Advancing the understanding of mBS interactions with microbial membranes, combined with modification strategies and AI-guided optimization, is essential for developing targeted therapies against resistant infections. Future research should integrate these approaches to engineer novel derivatives, reduce costs, and validate clinical potential through comprehensive in vivo studies.

1. Introduction

The emergence of several multidrug-resistant pathogens is mainly attributed to the abuse of traditional antibiotics [1]. The 2024 WHO BPPL (Bacterial Priority Pathogen List) covers 24 pathogens, including Mycobacterium tuberculosis, a Gram-negative bacterium resistant to last-resort antibiotics, and other high-burden resistant pathogens such as Salmonella, Shigella, Neisseria gonorrhoeae, Pseudomonas aeruginosa, and Staphylococcus aureus. Wound infections are commonly polymicrobial, with both Gram-positive (+) and Gram-negative (−) pathogens, as listed in

Table 1. Antibiotics and causative pathogens.

Antibiotics	Resistant Microorganism(s)
Carbapenem	(−): Acinetobacter, Enterobacteriaceae
β-lactamase	(−): Enterobacteriaceae
Methicillin	(+): Staphylococcus aureus
Vancomycin	(+): Enterococci
Erythromycin	(+): Group A Streptococcus or S. pyogenes
Clindamycin	(+): Group B Streptococcus or S. agalactiae
Multidrug	(+): Clostridioides difficile, Streptococcus pneumoniae (−): Neisseria gonorrhoeae, Campylobacter, P. aeruginosa, Shigella, nontyphoidal Salmonella Yeast: Candida, Candida auris

[2].

The US Centers for Disease Control and Prevention has recommended the combined use of antibiotics with novel compounds with antimicrobial properties [2]. In this scenario, biosurfactants (BSs) might be a new source of antimicrobial agents [3]. Synthetic surfactants and BSs are organic compounds composed of a head group with an affinity for polar phases and a tail group that is attracted to nonpolar phases. The molecule’s head group can be anionic, cationic, non-ionic, zwitterionic, and anionic surfactants (synthetic or bio-based), with a negative charge being the most commonly available surfactant [4]. Due to their distinctive structural properties, surfactants are extensively employed to lower surface and interfacial tension between two or more phases. In solution, they assemble into micelles—self-organized structures that form at a specific concentration known as the critical micelle concentration (CMC), with diameters ranging from nanometers to microns. For over a century, synthetic or chemically derived surfactants originating from petroleum have been integral to a wide array of applications, including industrial products, detergents and cosmetics, fabric softeners, pharmaceuticals, and drug delivery systems.

Besides their stability at high temperatures, pH, and salinity [5], microbial biosurfactants (mBSs) are biodegradable and expected to be non-toxic [6], compared with their synthetic counterparts. Considerable research has been dedicated to the production of mBSs by various microorganisms, as well as to elucidating the genetic and biochemical mechanisms underlying their synthesis. Among their distinguishing properties, mBSs demonstrate exceptional emulsifying capabilities, characterized by notably low CMC, resulting in significant reductions in air/water surface tension. The ability of several microorganisms—including bacteria, yeasts, and fungi—to grow on hydrophobic substrates, particularly P. aeruginosa, has been extensively documented [7]. Bioremediation is one of the potential applications of bacteria to remove C₁₀–C₂₆ n-alkanes and polycyclic aromatic hydrocarbons polluted by heavy oil spills. The glycolipid BS rhamnolipid (RL) was first isolated from Pseudomonas in 1949 [8], followed by the related sophorolipid (SL) isolated from Candida apicola in 1961 [9]. Intensive purification is required to purify a specific BS with the highest purity for biomedical applications because microorganisms often produce a mixture of different BS compounds.

This review examines the potential application of mBSs as antimicrobial agents, offering a comprehensive overview of their classification, origins, and characteristics. It highlights that human-associated bacteria can produce mBSs with antimicrobial properties effective against a range of pathogens. Nevertheless, the subject of microbial mBSs is complex with an extensive body of literature, and this review does not address plant-derived mBSs.

2. Classification of Biosurfactants (BSs)

Unlike synthetic surfactants, microbial biosurfactants (mBSs) are categorized based on their polymeric composition rather than ionic charge. Low-molecular-weight mBSs (<500 Da), such as glycolipids, lipopeptides, fatty acids, and phospholipids, effectively lower surface tension at the air-water interface and interfacial tension at the oil-water interface. High-molecular-weight-mBSs (>1000 Da) are generally polymeric or particulate, as shown in

Table 2. Classification of Biosurfactants based on their molecular weights.

Low molecular weight (<500 Da)

- Glycolipids: Their mono, di, tri, and tetrasaccharide types are linked to one or two fatty acid chains [11] with rhamnose and sophorose, with two major types. - Rhamnolipids: one or two fatty acid chains (8 to 16 carbons) are linked to one or two rhamnose [12]. - Sophorolipids (lactonic or acidic): Their hydrophilic disaccharide sophorose comprises two monomers connected by β-1,2 bonds [13]. - Mannosylerythritol lipids (MELs) have mannoses, linked to fatty acids. Their subdivision is based on the hydrophobic fatty acid chain length, the degree of saturation and/or acetylation [14]. - Lipopeptides: They consist of surfactin, iturin, fengycin, lichenysin, and viscosin. Over 30 types of surfactin are known [15]. - Fatty acids and phospholipids: Both bacteria and yeasts grown on n-alkanes produce phospholipids. Phospholipids have an amphipathic structure: a hydrophilic phosphate “head” attached to a glycerol backbone, which also links to two hydrophobic fatty acid “tails”

High molecular weight (>1000 Da)

- Polymeric: Emulsan (~1000 kDa) is a polyanionic amphipathic heteropolysaccharide. - Liposan (10-20 kDa, 83% carbohydrates and 17% proteins) is an emulsifier with surfactant capacity. - Alasan (~1000 kDa) [16], an anionic polysaccharide and proteins - Particulate: Macromolecules of proteins, phospholipids, polysaccharides [17], and phosphatidyl ethanolamine-rich vesicles [5]. Whole cells are represented by Cyanobacteria.

, and exhibit pronounced ability to stabilize oil-in-water emulsions [10].

3. Microbial Sources for the Synthesis of Biosurfactants

Various representative mBSs are synthesized by bacteria, yeast, and fungi, as outlined in

Table 3. Representative Biosurfactants Produced from Microorganisms.

Biosurfactants vs. Microbial Sources
Glycolipids Rhamnolipds: Pseudomonas aeruginosa [19,20,21], P. cepacia [22], Pseudomonas ssp. [23], Lysinibacillus sphaericus [24], Serratia rubidaea [25] Trehalolipids: Nocardia farcinica [26], Rhodococcus sp. [27], C. bombicola [28] Sophorolipids: C. sphaerica, [29] Starmerella bombicola [30,31], Cutaneotrichosporon mucoides [32]
Lipopeptides Surfactin: Bacillus subtilis, B. nealsonii [33,34,35,36,37] Lichenysin: B. licheniformis [38]
Phospholipids Thiobacillus thiooxidans [39], K. pneumoniae [35]
Polymeric Surfactants Liposan: C. lipolytica [39] Rufisan; Candida species [40] Emulsan: Acinetobacter lwoffii [41] Alasan: A. radioresistens [42]

; however, other microorganisms, not listed in this table, also produce various BSs [18]. These surfactants remain insufficiently characterized, and their respective microbial origins are therefore not cited here. Typically, many microorganisms produce a complex mixture of mBSs as secondary metabolites during stationary growth on water-immiscible substrates. The general structure of a surfactant comprises a long-chain fatty acid, hydroxyl fatty acid, or α-alkyl-β-hydroxy fatty acid, together with a hydrophilic moiety such as a carbohydrate, amino acid, alcohol, cyclic peptide, carboxylic acid, or phosphate.

As summarized in Table 3, biosurfactants encompass structurally diverse amphiphiles whose chemical architecture governs their physicochemical behavior and microbial distribution.

-

Glycolipids such as rhamnolipids, sophorolipids, trehalolipids, and MELs share a common motif of carbohydrate headgroups linked to one or two fatty acid chains, resulting in low-molecular-weight molecules with strong surface activity and low CMC values.

-

Lipopeptides, including surfactin, iturin, fengycin, lichenysin, and viscosin, combine peptide rings with β-hydroxy fatty acids, conferring exceptional interfacial tension reduction, high thermal stability, and robust foaming properties.

-

Fatty acids and phospholipids exhibit classical amphipathic glycerol–phosphate structures that readily form bilayers and vesicles.

-

High-molecular-weight polymeric and particulate biosurfactants, such as emulsan, liposan, alasan, and vesicle-rich complexes, provide strong emulsification and steric stabilization due to their polysaccharide–protein matrices. These structural features align with the microbial producers listed in Table 3, as each taxonomic group preferentially synthesizes specific biosurfactant classes according to its metabolic pathways and cell envelope composition.

Numerous microbially derived biosurfactant (mBS)-producing bacteria, notably within the family of the Lactobacilli genus, have been isolated from various food sources. For instance, L. paracasei A20 was obtained from Portuguese dairy facilities [43], whereas strains such as L. acidophilus, L. pentosus, and L. fermentum have been isolated from assorted fruits and dairy products. Additionally, Pediococcus dextrinicus SHU1593, now reclassified as Lactobacillus [44], synthesizes a cell-bound lipoprotein biosurfactant [45]. Other bacterial species associated with human microbiota are also recognized for their capacity to produce biosurfactants. The intestinal environment is of interest due to its population of lactic acid bacteria, such as those from the Lactobacillus genus, which generate biosurfactants primarily constituted of proteins, polysaccharides, and phosphates in varying proportions [46]; these are mainly classified as glycolipids or glycolipoproteins [47,48]. A further notable inhabitant of the human body, P. aeruginosa, is distinguished by its robust mBS production. Cell-free rhamnolipids secreted by P. aeruginosa ATCC 10145 show antimicrobial and antifungal activity [49]. Most antimicrobial biosurfactants originating from bacteria related to human health are categorized as lipopeptides, glycolipids, glycopeptides, and glycolipoproteins [50,51,52,53]. P. aeruginosa ATCC 10145 exhibits both antibacterial and antifungal effects [49].

Regarding Lactobacilli, both cell-free and cell-associated antimicrobial biosurfactant production has been observed [54]. Among the cell-free forms, small glycolipids released by L. acidophilus NCIM 2903 [55] and lipopeptides produced by a Lactobacillus strain derived from homemade curd [53] are particularly noteworthy. However, the processes of isolation, purification, and characterization remain labor-intensive and complex. Consequently, further research is necessary to elucidate the full range of properties and potential applications of these biosurfactants.

4. Key Chemical Compositions and Properties of Biosurfactants

The two most extensively studied low-molecular-weight (LMW) glycolipids are rhamnolipids and sophorolipids. Rhamnolipids comprise one or two rhamnose sugar moieties (Figure 1A,B), which are linked via an O-glycosidic bond to a hydrophobic tail containing one or two fatty acid chains ranging from C8 to C16/C18 [56,57]. P. aeruginosa is known to produce more than 30 distinct rhamnolipid congeners [58]. Sophorolipids have a hydrophobic fatty acid tail containing 16 or 18 carbon atoms and a head group, sophorose. A glucose-derived disaccharide with a β-1,2 bond is acetylated in the 6′ and/or 6′′ positions (Figure 1). Detailed information on the structure of sophorolipids is well described by Davila et al. [59].

Surfactins, iturins, and fengycin are the most prominent lipopeptides. Surfactins are characterized by a cyclic peptide comprising seven different amino acids attached to a β-hydroxyl fatty acid with variable chain lengths ranging from 13 to 15 carbon atoms. This cyclic peptide is conjugated to an acylated fatty acid molecule (Figure 2A). The key structural difference for three of the main types involves the amino acid at position 7 (involved in lactone ring formation): surfactin A has L-leucine; surfactin B has L-valine; surfactin C has L-isoleucine. Surfactin D also contains eight depsipeptides with variations in the number of carbon atoms in the fatty acid chain, typically between 13 and 16, and possibly differences in methylation.

The presence of glutamic and aspartic acids imparts hydrophilic properties and negative charges to the ring structure, while the valine residue, oriented toward the fatty acid chain, establishes a significant hydrophobic domain. The overall range for various surfactin preparations is broad, but specific values for pure homologues fall within a more defined range. A frequently cited value for natural surfactin (often a mixture) is around 20 µM (or 10–20 mg/L). The CMC value of surfactin decreases as its fatty acid chain becomes longer [60]. The length of the fatty acid chain also affects properties; for example, C14-rich surfactin exhibits exceptional emulsifying activity with hexadecane, which C15-rich surfactin does not.

Iturin exhibits a cyclic peptide configuration [61], as illustrated in Figure 2B. Iturins have been widely studied for their antifungal activities; they comprise C14 to C17 β-amino fatty acids and heptapeptides. The iturin family encompasses several notable members, including iturin A, C, D, and E. The amino acid sequence of the heptapeptide of iturin A is Asn-Tyr-Asn-Gln-Pro-Asn-Ser. Iturins D and E differ from iturin A by the presence of a free carboxyl group in iturin D and a carboxymethyl group in iturin E. Iturin A is less potent than surfactin and forms vesicles at higher concentrations. The iturinic lipopeptides also contain lipopeptides. The iturinic lipopeptides also contain bacillomycin D, bacillomycin F, bacillomycin L, mycosubtilin, and mojavensin, which are different in the amino acid sequences of the heptapeptides.

To illustrate the structural diversity of biosurfactants, Figure 3 presents representative examples from two distinct classes: lipopeptides and glycolipids. Fengycin (Plipastatin A1) is a cyclic lipopeptide produced by Bacillus subtilis, characterized by a peptide ring linked to a long fatty acid tail. Its amphiphilic architecture underlies its potent antifungal activity against filamentous fungi while sparing bacteria and yeasts. In contrast, mannosylerythritol lipids (MELs) are glycolipids composed of a mannosylerythritol sugar backbone esterified to fatty acid chains, with structural variants (A–D) differing in acetylation and chain branching. These differences modulate emulsification, antimicrobial properties, and potential industrial applications. Together, fengycin and MELs highlight how biosurfactant classes employ distinct chemical strategies to achieve amphiphilicity and biological function. Surfactin and rhamnolipids are the main focus in research because of their notable antimicrobial and industrial significance. Iturin and fengycin are gaining traction in antifungal and biocontrol contexts. Sophorolipids and MELs are newer but increasingly studied for sustainable applications in food, cosmetics, and medicine.

Rhamnolipids decrease the water surface tension from 72 to 25.19 mN/m [62], compared with 30–35 mN/m [63] of acidic and lactonic sophorolipids, 27 mN/m of surfactin [64], and 27 mN/m of iturin [61]. The intermolecular forces between two immiscible liquids produce an interfacial tension, e.g., 40 mN/m of a water–hexadecane mixture. A surfactant is evaluated by its ability to decrease surface and interfacial tensions with minimal CMC. In general, BSs outperform synthetic surfactants to attain lower surface tension with lower concentrations.

In brief, there is a correlation between the CMC value and the molecular weight. LMW biosurfactants including glycolipids and lipopeptides are highly effective at reducing surface tension and generally have CMC values ranging from 1 to 200 mg/L. HMW biosurfactants including polymeric substances such as bioemulsifiers (e.g., emulsan, alasan) function better as emulsion stabilizers and can have CMCs up to 1000 times lower than LMW variants.

Within a specific biosurfactant family, an increase in the molecular weight of the hydrophobic segment (e.g., a longer carbon chain) typically results in a lower CMC. For example, in surfactin homologs, the CMC decreases from 0.35 mM for a C12 chain to 0.08 mM for a C15 chain. Surfactin A (mixed C14–C15) with a CMC of 10–20 mg/L, is often used as the standard reference for natural mixtures [65].

There is also a hydrophilic/hydrophobic balance, as the CMC is sensitive to the ratio of the hydrophilic head to the hydrophobic tail. Increasing the molecular weight of the hydrophilic block (e.g., adding more sugar units) increases the CMC by increasing the overall solubility of the molecule.

For high-molecular-weight polymeric biosurfactants, the CMC drops exponentially as the size of the hydrophobic block increases, which enhances the thermodynamic stability of the resulting micelles.

The CMC values of some mBSs and their surface tension at the CMC are summarized in

Table 4. CMC and surface tension at CMC (γCMC) of some commonly used biosurfactants.

Biosurfactants	CMC (mg/L)	γCMC (mN/m)
Rhamnolipids from P. aeruginosa
- Mono [66]	50	25.9
- Di [66]	15	33.5
- Mono [67]	25	25.9
- Di [67]	15	31.7
Sophorolipids from S. bombicola [68]-diacetylated
- L-C18:0	29.2	35.7
- L-C18:1	31.2	36.3
- L-C18:2	35.0	38.5
- L-C18:3	39.1	38.8
Surfactins from B. subtilis grown on sucrose, peptone, yeast extract, and other mineral salts [69]	250.0	27.9
Surfactins [70] B. subtilis grown on a mineral salt solution with:
- Glucose	325.1	29.2
- Glycerol	154.1	29.7
- Lactose	65.3	30.7
Iturin [61] B. subtilis isolated from crude oil samples	40	29

. Experimental CMC values can vary significantly, depending on the purity of the sample and environmental factors like pH and solvent.

5. Antimicrobial Activities of Biosurfactants

5.1. Minimum Inhibitory Concentration (MIC) and Zone of Inhibition (ZOI)

The outer leaflet of the Gram-negative bacterial membrane contains lipopolysaccharides (LPS). In contrast, Gram-positive bacteria possess teichoic acids—anionic polymers—covalently attached to the peptidoglycan layer and integrated into the lipid membrane. Amphiphilic microbial biosurfactants (mBSs), characterized by distinct hydrophilic and hydrophobic regions, can interact with bacterial membranes through hydrophobic, dipole–dipole, and hydrogen bonding interactions. This amphiphilic nature facilitates the incorporation of rhamnolipids into bacterial membranes at subcritical micelle concentrations (sub-CMC), subsequently altering membrane structure and extracting lipopolysaccharides [71].

Both microdiffusion and microdilution have been extensively performed to quantify the antimicrobial effects of various biosurfactants against diverse pathogens. Rhamnolipids bind to bacterial membranes via electrostatic interactions [72], specifically involving the polar groups of positively charged surfactants and the negatively charged biomolecules present in bacterial membranes, such as LPS in Gram-negative bacteria and lipoteichoic acid in Gram-positive bacteria. Additionally, hydrophobic interactions between the alkyl chains of surfactants and the lipid bilayer may disrupt membrane integrity, leading to cytoplasmic leakage and eventual cell death [73]. Rhamnolipids may incorporate their acyl chains into bacterial lipid membranes, leading to disruption and increased membrane permeability [12], ultimately resulting in cell lysis. The antimicrobial efficacy of rhamnolipids is influenced by pH and NaCl concentration, which correlate with the type and size of molecular aggregates formed [74,75]. Planktonic cells are effectively eliminated at pH 7.0 in the presence of 5% NaCl [76]. A notable distinction is that biosurfactants tend to integrate into membranes rather than dissolve them, differing from the mechanism of chemical surfactants. Specifically, rhamnolipids can extract LPS below their critical micelle concentration (CMC) [71].

Sophorolipids, like rhamnolipids, interact with bacterial surfaces to alter their hydrophobic characteristics, resulting in compromised membrane integrity and subsequent cell death [77]. Iturin can penetrate the cell wall and disrupt the plasma membrane, leading to the formation of small vesicles. The aggregation of these intramembranous particles adversely affects the structural and morphological stability of microbial membranes [78]. In general, microbial biosurfactants (mBSs) demonstrate greater efficacy against Gram-positive bacteria than against Gram-negative bacteria, as the latter possess a protective lipopolysaccharide barrier [79]. Glycolipids, especially rhamnolipids and sophorolipids, have undergone extensive evaluation for their antimicrobial activity against a broad spectrum of pathogenic organisms. Among the lipopeptides, surfactins, fengycin, and iturin also exhibit antimicrobial activities (

Table 5. Antimicrobial activities of microbial biosurfactants and target pathogens.

mBS and Microbial Sources	Target Microorganism(s) and MIC (μg/mL)
Rhamnolipids
P. aeruginosa BM02 [80]	S. aureus and E. faecium (50)
P. aeruginosa PAO1 [81]	Cutibacterium acnes (15.62); MBC (31.25)
P. aeruginosa B5 [82]	P. capsici (10); C. cucumerinum, C. orbiculare (25); C. destructans
P. aeruginosa 47T2 [68]	Gram-negative: K. pneumoniae (0.5), E. aerogenes (4), S. marcescens (8), A. faecalis (64), E. coli (64), B. bronchiseptica (128), S. typhimurium (128), P. aeruginosa (256) Gram-positive: B. subtilis (16), S. aureus (32), S. epidermidis (32) M. luteus (64), A. oxidans (128), M. phlei (128), C. perfringens (128).
P. aeruginosa AT10 [83]	Gram-negative: S. marcescens (16), A. faecalis (32), E. coli (32), B. bronchiseptica (128), S. typhimurium (128) Gram-positive: S. epidermidis (8), A. oxidans (16), M. phlei (16), M. luteus (32), B. subtilis (64), S. faecalis (64), S. aureus (128)
P. aeruginosa MR01 Diffusion test [84]	Zone of inhibition (ZOI) diameter (mm), based on 0.3 mg of biosurfactant E. coli and P. aeruginosa (no affected), B. cereus (30 mm), S. epidermidis (15 mm), S. aureus (14 mm)
P. aeruginosa DR1 Diffusion test [85]	Mycelial growth inhibition: M. phaseolina (60.46%, 9 μg), F. oxysporium (55%, 12 μg), P. nicotianae (63.63%, 13.5 μg)
Soropholipids Candida sp. AH62 [86] S. bombicola [87] C. tropicalis RA1 [88] R. babjevae YS3 [89]	S. aureus (1), B. subtilis (2), E. coli, and P. aeruginosa (4) S. aureus (31.25), L. monocytogenes (62.50). S. aureus (250), L. monocytogenes (500), E. coli (1000) T. mentgrophytes (1 mg/mL, 62% of inhibition); (4 mg/mL, 100% of inhibition)
C. bombicola ATCC 22214 [90] Microdilution	% Cell survival at the biosurfactant concentration (μg/mL) B. subtilis: 91.04% (0.6), 57.41% (0.8), 5.25% (1.0) P. aeruginosa: 8.77% (1), 2.19% (3), 0.31% (5) S. aureus: 9.62% (6), 1.03% (8), 0.34% (10) E. coli: 58.01% (10), 34.09% (20), 2.05% (30) C. albicans: 10.34% (25), 10.34% (50), 6.89% (75)
mBS and Microbial Sources	Target Microorganism(s)
Glycolipids S. saprophyticus SBPS 15 Diffusion test [91]	ZOI (mm) at a given surfactant concentration. Several bacteria were affected, using 0.2–3.2 μg/mL or 1.6–2.4 μg. The zone of inhibition diameter ranges from 15 to 23 mm.
Surfactins B. circulans [92]	Diffusion test: ZOI diameter (mm) ranges from 10.66 to 17 mm using 1000 μg/mL of surfactins against various bacteria Microdilution, MIC (μg/mL): M. flavus (200), B. pumilis (30), M. smegmatis (50), E. coli (40), S. marcescens (30), P. vulgaris (10), A. faecalis (10), and K. aerogenes (80)
B. velezensis H3 [93]	Diffusion test, zones of inhibition diameter (mm) at 1000 μg/mL of biosurfactant: K. peneumoniae (10 mm), S. aureus (11 mm), C. albicans (14 mm), and P. aeruginosa (14 mm)
B. subtilis [94]	Diffusion test, percentage of growth inhibition of A. flavus (%) at a given concentration of surfactins (mg/L): 36% (20), 54% (40), 84% (80), 100% (160)
Fengycin B. thuringiensis [95]	Microdilution, MIC (μg/mL) C. albicans, A. niger (15.62); S. epidermidis, E. coli (1000)
Iturins B. subtilis K1 [96]	Microdilution, MIC(μg/mL) A. niger and A. brunsii (2.5)
Lipopeptide B. cereus [97]	- Diffusion test: Zones of inhibition (mm in diameter) range from 11.4 to 20.2 mm with 30 mg/mL of biosurfactant against A. flavus, C. albicans, K. pneumoniae, P. aeruginosa, S. aureus, and E. coli, - Microdilution, MIC (mg/mL) ranges from 0.5 to 7.6 against S. aureus, E. coli, P. aeruginosa, K. pneumoniae, C. albicans, and A. flavus.
Surfactins and Fengycin B. subtilis fmbj [98]	MIC (μg/mL): B. cereus (156.25).
Mannosylerythritol lipids (MELs) [99]	MELs produced by P. aphidis show antimicrobial properties at an MIC = 1.25 mg/mL against B. cereus, and the antibacterial effect is correlated with the MEL concentration.
mBSs produced by human-associated bacteria	- Biosurfactants from Lactobacilli have an antimicrobial effect against Neisseria gonorrhoeae [100], E. coli, S. saprophyticus, E. aerogenes, and K. pneumoniae, and antifungal activity against C. albicans [48]. - Cell-free BS of L. paracasei ssp. paracasei A20 exhibits antimicrobial and antiadhesive activities against several bacteria and fungi [43] - L. acidophilus, L. pentosus, and L. fermentum produce cell-free BSs with an antimicrobial activity [50,51]. - Pediococcus dextrinicus SHU1593 (re-classified as Lactobacillus) [44] produces cell-bound lipoprotein against B. cereus, E. aerogenes, and S. typhimurium [45]. - Cell-associated BSs produced by L. casei LBI and L. casei ATCC 393 prevent oral diseases with antimicrobial and antibiofilm activities against Staphylococcus aureus [101]. - P. aeruginosa ATCC 10145 produces a cell-free rhamnolipid BS with antimicrobial and antifungal activities [49].

).

5.2. Chemical Structures of Biosurfactants and Their Antimicrobial Properties

Microbial biosurfactants (BSs) are frequently produced as mixtures exhibiting variations in size and hydrophobic region saturation. Across biosurfactant families, the hydrophobic domain—chain length, branching, and degree of unsaturation—plays a central role in membrane insertion, aggregation behavior, and ultimately antimicrobial potency. As expected, biosurfactants significantly reduce surface and interfacial tension, enabling their interaction with cell surfaces to emulsify lipid components, destabilizing cellular structures. Above the CMC, all biosurfactants can completely solubilize membrane lipids into micelles, causing total cell lysis

Specifically, rhamnolipids (RL) and sophorolipids (SL) differ in their sugar residues and levels of acetylation, resulting in diverse surface-active properties. Consequently, the chemical structures of various microbial BSs influence their antibacterial, antifungal, and antiviral activities. For example, when oleic acid (C18:1) serves as the primary carbon source, sophorolipids can be generated in both acidic and lactonic forms. The alkyl chains of these sophorolipids vary in length (C16 or C18) and in the number of double bonds (0–2), while SL molecules may undergo acetylation at up to two positions on the sugar ring [102]. Similarly, RLs are synthesized with one or two rhamnose units, and their fatty acid moieties differ in alkyl chain length (C10 or C12) and saturation (zero or one double bond) [102]. Distinct RL variants exhibit unique biophysical properties, such as critical micelle concentration (CMC), surface tension, acyl chain fluidity, aggregate structure, and membrane insertion capability. Increasing the number of rhamnose groups enhances acyl chain fluidity and promotes loose packing, which is likely attributable to the greater steric bulk of the saccharide groups [103]. In contrast, incorporation of an additional acyl chain expands the hydrophobic region.

Research has explored associations between the chemical structure of microbial BSs and their antimicrobial efficacy, highlighting the following key points:

-

Positively charged biosurfactants have double killing effects. First, they attract the negatively charged bacterial membrane and disrupt the membranes. The second antimicrobial activity is attributed to their insertion into the bacterial membrane, resulting in physical disruption, like a needle bursting a balloon [104]. However, excessively long chains (above C16–C18) may decrease in activity due to solubility limits.

-

Mono-rhamnolipids show a greater inhibition diameter of five different microbial strains than di-rhamnolipids [105], but lower antifungal activity [67].

-

The lactonic variants of SL exhibit stronger antimicrobial properties [97] and cytotoxic effects than the acidic SLs [68].

-

The chain unsaturation in diacetylated lactonic sophorolipids is also attributed to the antimicrobial activity against S. aureus, as C18:0 and C18:1 show a MIC (50 μg/mL), significantly lower than the values of C18:2 and C18:3 (200 μg/mL). Therefore, the presence of one or two double bonds in lactonic sophorolipids plays an important role in antimicrobial activity [106].

-

Antimicrobial properties of sophorolipids depend on the degree of acetylation. The MIC against B. cereus of diacetylated sophorolipids is 12 μM, which is significantly lower than 25 μM for sophorolipids [106].

-

Among 20 MEL molecules with C6, C8, C10, C12, and C14 alkyl chain lengths [107], MELs with C10 are more effective against M. luteus, whereas very short or very long chains (C6 and C14, respectively) exhibit weaker antimicrobial activities [107].

-

Some biosurfactants, like surfactin, can also chelate essential mono- or divalent cations, which further destabilizes the membrane and compromises its integrity [108].

Notably, if MIC < CMC, the antimicrobial activity is driven by individual surfactant monomers (unimers). If MIC > CMC, the micelles formed create a larger “effective solubilization area” that increases interaction with the bacterial cell wall. In this context, inhibitory effects only become significant at concentrations close to or higher than the CMC. Biosurfactants are more efficient than their synthetic counterparts because they can achieve both a lower surface tension and a lower CMC, often resulting in effective antimicrobial activity at lower total concentrations. However, biosurfactant activity is complex, but generally, the MIC values for biosurfactants tend to be higher than their CMC values. The MIC often varies widely depending on the specific biosurfactant type, the producing microorganism, and the target pathogen, as well as experimental conditions like purity and medium. As an example, the MIC (μg/mL) of surfactin shown in Table 5 varies significantly depending on the pathogen: M. flavus (200), M. smegmatis (50), E. coli (40), S. marcescens (30), B. pumilis (30), P. vulgaris (10), A. faecalis (10), and K. aerogenes (80), whereas its CMC value ranges from 10 to 20 mg/L (μg/mL) [65]. Thus, the MIC cannot be used to predict the outcome of a specific antimicrobial biosurfactant.

6. Cytotoxicity

Biosurfactants exhibiting antimicrobial activity represent an emerging category of agents with potential pharmaceutical applications. As such, it is essential that these compounds undergo rigorous evaluation procedures, including comprehensive toxicity assessments. Microbial biosurfactants are composed of lipids, sugars, and proteins, which generally confer lower toxicity and enhanced biodegradability than their synthetic counterparts. Although their antimicrobial effects are well documented, the amphiphilic nature of microbial biosurfactants indicates a possible risk of cytotoxicity to human cells. Among rhamnolipids, one fraction of mBSs produced by Sphingobacterium detergents reduces cell proliferation and induces apoptosis in 3T3 fibroblasts and HaCaT keratinocytes. The IC₅₀ value is 699.9 ± 120.6 µg/mL (for 3T3 fibroblasts) and 1041.6 ± 118.9 µg/mL (for HaCaT keratinocytes), compared with 53.5 ± 3.3 µg/mL of dodecyl sodium sulfate [109]. A rhamnolipid with both mono and di-rhamnolipid congeners exhibits no toxicity to the mouse L292 fibroblastic cell line [110]. The level of toxicity can be related to the percentage of viable cells at a given concentration. The zero value indicates 90% of cell viability, whereas 10% indicates the maximal scale of toxicity (scale = 5) [111]. The toxicity of different mBSs obtained from some bacteria is summarized in

Table 6. Cytotoxicity of some selected biosurfactants.

Biosurfactants and Microbial Sources	Conc. (g/L)	Viability (%)-Toxicity Scale (0–5)
L pentosus (BS5), amphoteric, lipopeptides [112]	1	91-Zero
L pentosus (BS5), amphoteric, lipopeptides [112]	1	80-2 (Moderate)
L. pentosus (BS5), amphoteric, a mixture of lipopeptides and glycolipids [112]	1	96 (Zero)
L. pentosus (BS5), non-ionic, lipopeptides [112]	1	97-Zero
L. pentosus (BS5), non-ionic, cell-bound biosurfactant, glycoprotein or glycolipopeptides [112]	1	113
Lipopeptide from Bacillus cereus [97]	10	63-2 (Moderate)
Lipopeptide from B. stratosphericus [113]	1	96-Zero
Glycolipid from Enterococcus faecium [114] is cell-compatible with mouse fibroblast cells	6.25	90-Zero
Glycolipid from Cyberlindnera saturnus [115]	1	70-2 (Moderate)
Cell-associated biosurfactant of L. pentosus NCIM 2912 [116]	0.5	- Human embryonic kidney (HEK 293): 90.3 ± 0.1% - Mouse fibroblast ATCC L929: 99.2 ± 0.43% - Human epithelial type (HEP-2): 94.3 ± 0.2%
Biosurfactants produced by Marinobacter strain MCTG107b and Pseudomonas strain MCTG214(3b1) [117]	O.25-3	HaCaT cells and THLE cells - Negligible cytotoxicity up to 0.25 g/L - Viability is lower than 50% at 1 g/L

.

As shown in Table 5, most of the tested mBSs are not cytotoxic at a concentration of 1 g/L, except for one glycolipid from Cyberlindnera saturnus, which is 70% viable. However, the MIC of several mBSs to target different pathogens is in μg/mL or mg/L, which is well below 1 g/L. Of interest is the promotion of fibroblast growth up to 113% by the L. pentosus biosurfactant [112].

A discussion of surfactin cytotoxicity is warranted, given their demonstrated anticancer [118] and antiviral, including anti-COVID-19 properties [119]. However, surfactin purified from B. subtilis 573 also exhibits cytotoxic effects on human normal MCT-3T3-E1 fibroblast cells at concentrations and exposure times comparable to those inhibiting human T47D and MDA-MB-231 breast cancer cell lines [120,121]. Additionally, surfactin exhibits hemolytic activity when concentrations exceed 0.05 g/L [122]. To mitigate this limitation, linear derivatives of surfactin have been engineered, showing no significant hemolysis up to 1 mM [123]. Furthermore, polymeric nanocapsules loaded with 1′,4′′-Sophorolactone 6′,6′′-diacetate demonstrated efficacy against human cervical cancer cells [124,125], while exhibiting non-significant toxicity toward normal cell lines (CCD-841). A combination of acidic and lactonic sophorolipids (SLs) with varying lipophilic chain lengths and saturation (monounsaturated versus saturated), derived from Starmerella bombicola and tested up to 0.1% w/v, does not induce cytotoxicity in fibroblasts, which are essential for wound healing [126]. In a related publication [19], the same research group investigated the viability of human keratinocyte (HaCaT) cells following exposure to various microbial biosurfactants (mBSs), yielding more complex results. Acidic sophorolipids at concentrations up to 0.50 mg/mL do not affect HaCaT cell viability. In contrast, di-rhamnolipids administered more than 70 μg/mL reduced cell viability to below 30%, whereas lactonic sophorolipids also demonstrated effects at this concentration [127].

7. Synergistic Use of Biosurfactants with Conventional Antibiotics

Biosurfactants—such as rhamnolipids, surfactin, sophorolipids, and lipopeptides—have attracted increasing interest as adjuvants to conventional antibiotics owing to their ability to disrupt microbial membranes, enhance antibiotic penetration, and destabilize biofilms. When used in combination with antibiotics, biosurfactants often reduce the minimum inhibitory concentration (MIC) of the drug, restore susceptibility in resistant strains, and markedly improve biofilm eradication. Mechanistically, biosurfactants interact with bacterial cell envelopes by altering surface tension and membrane fluidity, which increases permeability and facilitates intracellular antibiotic uptake. In biofilm settings, biosurfactants can solubilize extracellular polymeric substances (EPS), exposing embedded bacteria to antibiotics that would otherwise be ineffective. Importantly, this synergy allows for lower antibiotic dosages, potentially reducing toxicity and slowing the emergence of resistance.

The adjunct use of biosurfactants represents a promising strategy to rejuvenate existing antibiotics, particularly against multidrug-resistant pathogens and biofilm-associated infections (

Table 7. Representative studies on biosurfactant–antibiotic synergy.

Biosurfactant	Antibiotic	Target Microorganism	Key Outcome	Ref.
Rhamnolipids	Ciprofloxacin	P. aeruginosa (biofilm)	MIC of ciprofloxacin reduced 4–8×; >90% biofilm disruption	[128]
Rhamnolipids	Tetracycline	S. aureus (MRSA)	Synergistic killing; enhanced membrane permeabilization	[129]
Surfactin	Ampicillin	E. coli	MIC reduced ~4×; membrane destabilization confirmed by SEM	[130]
Surfactin	Vancomycin	S. epidermidis (biofilm)	Biofilm biomass reduced >80% compared to antibiotic alone	[131]
Sophorolipids	Gentamicin	Candida albicans (mixed biofilm)	Significant reduction in CFU and biofilm thickness	[132]
R5Lipopeptide (Bacillus sp.)	Rifampicin	M. smegmatis	Enhanced intracellular drug uptake; lowered MIC	[133]

).

8. Other Biomedical Applications of Biosurfactants

Biosurfactants—most prominently glycolipids (e.g., rhamnolipids, sophorolipids, mannosylerythritol lipids) and lipopeptides (e.g., surfactin and related Bacillus lipopeptides)—are amphiphilic molecules produced by microorganisms that combine surface activity with bioactivity. Compared with many petrochemical surfactants, biosurfactants are often highlighted for their biodegradability, formulation versatility, and relatively favorable biocompatibility profiles, making them attractive in medical and pharmaceutical contexts. Their applications span antimicrobial/antibiofilm strategies, drug delivery, oncology, wound care/regenerative medicine, immunomodulation/antiviral support, and selected diagnostic uses [134,135].

8.1. Antimicrobial and Antibiofilm Agents

A key biomedical value of biosurfactants is their ability to interfere with microbial membranes and adhesion, which are central to both acute and chronic biofilm-associated infections. At the cellular level, many biosurfactants insert into lipid bilayers or disturb membrane organization, causing permeabilization, leakage of intracellular components, and cell death. At the community level, they can reduce initial adhesion, disrupt extracellular polymeric substances (EPS), and help penetrate established biofilms—structures that typically show elevated tolerance to antibiotics and host defenses [136].

Medical-device protection (anti-adhesion/antifouling). Biosurfactants have been explored as surface-adsorbed coatings (or covalently attached layers) on biomaterials to reduce colonization of catheters, silicone, and implant surfaces. As an example, rhamnolipid-coated medical-grade silicone has shown antibiofilm activity against clinically relevant pathogens [137]. Covalent immobilization strategies (e.g., rhamnolipids bonded onto PDMS) have also been reported to improve the stability of the antimicrobial/antifouling effect under clinically relevant exposure conditions [138]. As discussed above, beyond direct activity, biosurfactants may act as antibiotic adjuvants, enhancing antibiotic diffusion into biofilms and increasing intracellular drug access by weakening membrane and matrix barriers. This concept is supported by studies showing improved outcomes when Bacillus lipopeptides are paired with antibiotics against mature biofilms [139].

8.2. Drug Delivery Systems

Biosurfactants can function as formulation excipients that improve drug solubility and delivery to form micelles, microemulsions, and nanoemulsions. These systems can enhance the apparent solubility of hydrophobic drugs, protect labile compounds, and improve biodistribution depending on the route of administration.

-

Enhanced solubility and bioavailability: Reviews highlight biosurfactant-enabled microemulsion/nanoemulsion platforms (notably with rhamnolipids and sophorolipids) as strategies to increase the solubilization of poorly water-soluble actives and broaden the formulation design space [140].

-

Targeted/functional delivery: Biosurfactant-containing assemblies can be co-formulated with nanoparticles or engineered with targeting ligands (conceptually similar to other surfactant-based delivery systems) to improve tissue selectivity and reduce off-target toxicity [141].

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Transdermal and mucosal delivery: Surfactant-based systems are widely studied as permeation enhancers; biosurfactants are increasingly discussed within this context for topical/transdermal approaches, including microemulsion-based gels and advanced topical delivery frameworks [142].

8.3. Oncology (Anticancer Activity)

Several biosurfactants show selective cytotoxicity in cancer models, often linked to membrane interactions, mitochondrial dysfunction, oxidative stress pathways, and apoptosis-related signaling. Recent reviews synthesize evidence that sophorolipids can inhibit proliferation and induce apoptosis in multiple cancer cell models, while highlighting remaining challenges (structure–activity relationships, delivery, and translational safety) [143].

Apoptosis induction and growth inhibition: Sophorolipid candidates have demonstrated cytotoxic efficacy in breast cancer models in vitro (including advanced 3D culture approaches in some studies) [144].

Glycolipid MELs and melanoma: Mannosylerythritol lipids (MELs) have long been reported to induce apoptosis-related phenotypes and growth inhibition in melanoma models, supporting the broader theme that glycolipid biosurfactants can directly affect tumor cell viability [145].

8.4. Wound Healing and Regenerative Medicine

Biosurfactants are being investigated in wound care as antimicrobials (biofilm control on wound surfaces) and bioactive agents that may support repair processes.

-

Tissue repair effects. Bacillus-derived lipopeptides have been reported in experimental models to support wound closure outcomes and antioxidant-linked benefits, with histological improvements in some animal studies [146].

-

Formulation opportunities. Because chronic wounds frequently involve polymicrobial biofilms and high protease burden, biosurfactants are also attractive as components of hydrogels/films/coatings that combine antibiofilm action with a moist healing environment (an area still actively developing) [147].

8.5. Immunomodulation and Antiviral Support

Beyond surface activity, biosurfactants can influence host–pathogen interactions through immune modulation and virucidal actions against enveloped viruses.

Immunomodulation: A literature review indicates that microbial surfactants (including surfactin) can modulate inflammatory signaling in immune cells (e.g., changes in cytokine production in stimulated macrophage models), suggesting potential value in inflammation control or as components in immunologically active formulations [148].

Antiviral activity (enveloped viruses): Rhamnolipid mixtures have been reported to inactivate enveloped viruses, including coronaviruses, in laboratory studies; notably, antiviral activity against SARS-CoV-2 has been reported for rhamnolipid mixtures under experimental conditions [149]. Sophorolipids have also been discussed as candidates for antiviral strategies against enveloped viruses, primarily from a mechanistic and translational review perspective [150].

8.6. Specialized Medical and Diagnostic Uses

Some clinically established “surfactant medicines” are not microbial biosurfactants per se, but they illustrate the broader medical importance of surfactant chemistry and formulation. Pulmonary surfactant replacement therapy: Exogenous surfactant therapy is a standard intervention in neonates with respiratory distress syndrome (RDS), with clinical guidelines supporting early rescue strategies and product selection based on evidence and local practice [151]. Diagnostics and imaging (notably ultrasound microbubbles): Surfactants can stabilize microbubbles used as ultrasound contrast agents; biosurfactant-based approaches for microbubble preparation have been reviewed, supporting feasibility for diagnostic/theranostic formats even though this remains more niche than antimicrobial and drug-delivery applications [152].

9. Perspectives and Conclusions

Among the various microorganisms capable of synthesizing microbial biosurfactants with diverse chemical structures, the leading bacterial genera include Candida, Pseudomonas, Bacillus, Mycobacterium, and Acinetobacter. These organisms are characterized by their ability to reduce surface tension and emulsify lipophilic substances, thereby facilitating growth on hydrophobic substrates such as hydrocarbons and aromatic hydrocarbons. To date, microbial biosurfactants have demonstrated significant value in a range of biomedical applications, particularly in antimicrobial and antifungal interventions. While their anticancer [153] and antiviral activities [154] are noteworthy, these topics fall outside the scope of this review.

Microbial biosurfactants (mBSs) constitute a promising category of antimicrobial agents with notable efficacy against antibiotic-resistant pathogens. Their attributes, including high biocompatibility, minimal toxicity, and biodegradability, have attracted considerable research attention. In Gram-positive bacteria, mBSs compromise and disrupt bacterial membranes, leading to cell lysis. Empirical evidence also supports their activity against Gram-negative bacteria and fungi. However, biosurfactants are typically synthesized as heterogeneous mixtures that vary in chemical structure and molecular size, which can affect their antibacterial efficacy. Consequently, future work should focus on reassessing the antimicrobial activity of purified and well-characterized mBSs to clarify their underlying mechanisms. A comprehensive understanding of the biological pathways governing biosurfactant production is essential to control their chemical composition. Moreover, standardizing production processes and improving cost efficiency are critical steps for the large-scale deployment of these agents.

There are several reports on the synergistic effect between mBSs and antibiotics. Rhamnolipids have been proven to enhance the efficacy of ampicillin, chloramphenicol, erythromycin, kanamycin, and tetracycline against E. coli and B. megaterium [155]. Similarly, sophorolipids enhanced the antimicrobial efficacy of tetracycline [3]. It was reasoned that sophorolipids interacted with the microbial lipid bilayer to increase permeability and facilitate the penetration of the antibiotic. Another example of the combined treatment using a glycolipid synthesized by Shewanella algae and ciprofloxacin or gentamicin against biofilm formation caused by MRSA and A. baumanii [156]. The glycolipid alone effectively inhibited biofilm formation by about 84–93%. However, in conjunction with gentamicin or ciprofloxacin, the effectiveness approached 99% as glycolipid-induced cell membrane disruption, leading to cytoplasm leakage and subsequent cell death.

A further illustration is the synergistic antimicrobial effect observed with sophorolipid-hexyl esters in conjunction with piscidins [157], which resulted in a two-fold enhancement of antimicrobial activity against B. cereus. It has been proposed that sophorolipid-picaridin complexes form and accumulate on microbial membranes, interfering with essential microbial processes and compromising the integrity of the lipid bilayer, ultimately leading to cell lysis [157]. In this regard, future research should prioritize the combined application of diverse sophorolipids, glycolipids, and other microbial biosurfactants. Hydrogenation of glycolipid biosurfactants enables the production of fully saturated lipid analogs. Notably, an engineered strain of S. bombicola has been used to produce 24 novel derivatives, including sophoroside amines with varying alkyl chain lengths (ranging from ethyl to octadecyl) and their corresponding quaternary ammonium salts [158]. Lactonic sophorolipids, sophoroside amines, and quaternary ammonium salts, with some derivatives, exhibit MIC values as low as 0.0137 mg/mL [158] against B. subtilis, S. aureus, L.monocytogenes, E. coli, P. aeruginosa, and C. albicans. Given the diverse structures of mBSs, variations in hydrophobic region saturation, and other unique attributes, as well as their potential combinations with antibiotics or alternative antimicrobial agents, there is considerable scope for fundamental research into the molecular mechanisms responsible for their antimicrobial activity. Despite these promising characteristics, current medical applications remain limited. To address this gap, novel strategies are being developed to improve multifunctionality and extend applications, including those with antiviral potential.

Although the exact antimicrobial mechanisms of microbial biosurfactants necessitate further empirical clarification, these compounds interact with bacterial cell membranes via several pathways. For example, rhamnolipids adhere to bacterial membranes through electrostatic interactions between their cationic polar moieties and the anionic components of bacterial membranes—specifically, lipopolysaccharides in Gram-negative bacteria and lipoteichoic acid in Gram-positive strains. Additionally, the alkyl chains present in surfactants interact with microbial lipid bilayers through hydrophobic mechanisms. Experimental evidence demonstrates that mBSs exhibit antimicrobial effects by associating with microbial membranes, resulting in altered surface energy, increased membrane permeability, and subsequent cell lysis. The interactions between mBSs and model lipid membranes have been extensively investigated [159,160,161] and summarized in recent reviews [162].

Microorganisms strategically modify their lipid composition to regulate various functions; notably, membrane structure and properties are linked to alterations in cell size, morphology, and adaptive responses to external stressors, including drug resistance [163]. A comprehensive understanding of the dynamic variations in bacterial lipid diversity and membrane characteristics under stress conditions may facilitate novel strategies for selective antibacterial drug development.

Microbial biosurfactants (mBSs) also require thorough purification and characterization to assess their efficacy, cytotoxicity, and hemolytic properties. The in vitro hemolysis assay, also known as the hemolytic potential assay, provides an initial evaluation of the toxic effects of chemicals, drugs, and biomaterials before subsequent in vivo testing. Each biosurfactant must also conform to established specifications for purity, potency, and quality. Furthermore, standardized production protocols for mBSs with tailored properties to target specific pathogens remain underdeveloped. Future research is warranted to fully elucidate their scope and potential applications within the medical sector. Notably, rhamnolipids and sophorolipids have already been incorporated into various cosmetic and personal care formulations [164].

Several biosurfactants have demonstrated the ability to kill or significantly inhibit the growth of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) in laboratory settings. They are considered promising alternatives to conventional antibiotics because of their unique mechanisms of action that make bacterial resistance less likely.

Notably, rhamnolipids produced primarily by Pseudomonas aeruginosa, have shown broad-spectrum activity and can kill all ESKAPE bacteria, with high killing percentages (>90% for some, >86% for K. pneumoniae). Surfactin and other lipopeptides produced by Bacillus subtilis and related species. Lipopeptides like surfactin, fengycin, and iturin exhibit strong antibacterial and antibiofilm properties. A lipopeptide from Bacillus amyloliquefaciens could inhibit biofilm formation by up to 99% [165]. Sophorolipids produced by non-pathogenic yeasts like Starmerella bombicola, are effective against both Gram-positive and Gram-negative bacterial biofilms [77]. Biosurfactants can prevent initial bacterial attachment to surfaces and break down the extracellular matrix of mature biofilms, dispersing the protective layer and exposing the bacteria. They can interfere with the chemical signaling pathways (quorum sensing) that bacteria use to coordinate biofilm formation and express virulence factors. In general, Gram-positive bacteria are more sensitive to selected biosurfactants than Gram-negative bacteria. Sophoro- and rhamnolipids prevented the growth of Gram-positive bacteria but P. aeruginosa was not inhibited by those surfactants, as these glycolipids were hydrolyzed by bacterial enzymatic reactions [166].

While promising, most research on biosurfactants for ESKAPE pathogens has been conducted in a laboratory setting (in vitro and in some animal models). More studies, particularly human clinical trials, are needed to translate these findings into widespread clinical practice, though ongoing research in nanotechnology and combination therapies (e.g., biosurfactants combined with antibiotics) shows potential to overcome current limitations.

Further investigation into the synergistic effects of microbial biosurfactants and conventional antibiotics could promote the development of novel combination therapies targeting multidrug-resistant infections. This targeted approach, which prioritizes purified components and collaborative mechanisms, is crucial for realizing the full therapeutic potential of microbial biosurfactants for treating infectious diseases.

Modification of biosurfactants deserves a brief discussion, as both chemical modification and genetic engineering have emerged as effective strategies to improve their physicochemical and biological properties. Surfactin, a cyclic lipopeptide produced by Bacillus subtilis, provides a representative example in which targeted amino-acid substitutions can markedly alter surface activity and antimicrobial potential. Replacement of valine residues within the peptide ring by other hydrophobic amino acids (e.g., leucine or isoleucine) has been shown to significantly reduce the CMC, thereby lowering the amount of biosurfactant required to achieve micelle formation and functional activity [167]. Such modifications enhance efficiency in surface tension reduction and emulsion stabilization, rendering the modified surfactin both more potent and cost-effective.

In parallel, variation in the length or branching of the fatty-acid tail directly modulates lipopeptide hydrophobicity. Extension of the alkyl chain generally decreases CMC values, whereas controlled branching can fine-tune aqueous solubility and biological activity [167]. Natural biosurfactants typically occur as complex mixtures of isoforms; therefore, selective enrichment or engineering of specific congeners has been exploited to maximize antimicrobial efficacy against resistant pathogens while minimizing cytotoxicity [168]. Beyond conventional amino-acid substitutions, rational genetic engineering of Bacillus strains enables the incorporation of non-canonical amino acids into surfactin, offering additional flexibility for tailoring biosurfactants to specialized biomedical applications, including antibiofilm coatings and drug delivery systems [167,168].

Notably, artificial intelligence (AI) is emerging as a transformative tool in biosurfactant research and commercialization, particularly in addressing the long-standing challenge of high production costs, which are currently estimated at approximately USD 5–20/kg. Artificial neural networks (ANNs) and genetic algorithms (GAs) have already demonstrated strong predictive capability for optimizing fermentation yields and media composition [169]. In addition, AI-enabled “soft sensors” provide real-time estimates of key process variables, such as biomass concentration and product formation, which are otherwise difficult to monitor experimentally [170]. AI-driven metabolic models further support the rational design of microbial strains by predicting the effects of genetic modifications on biosynthetic pathways [170].

In parallel, AI-based tools facilitate the evaluation of complex agricultural residues (e.g., palm oil mill effluent, rice bran, and orange peels) as low-cost feedstocks, supporting circular bioeconomy strategies. Because downstream processing accounts for approximately 70–80% of total production costs, AI-based surrogate and hybrid models are increasingly applied to optimize purification steps, predict interfacial behavior, and integrate fermentation with extraction processes to reduce energy consumption and waste generation [170]. Collectively, these advances suggest a realistic pathway toward reducing biosurfactant production costs to below USD 2.50/kg, thereby unlocking their full biomedical and industrial potential [171,172].

In this context, AI serves as a critical bridge between laboratory-scale biosurfactant innovation and industrial-scale implementation by enabling predictive design, cost-efficient bioprocess optimization, and scalable manufacturing strategies. By integrating molecular engineering with data-driven process control, AI not only accelerates translation but also reduces the economic barriers that have historically limited widespread adoption of biosurfactant-based biomaterials.