Biosurfactant Produced by Bacillus subtilis UCP 1533 Isolated from the Brazilian Semiarid Region: Characterization and Antimicrobial Potential

Abstract

The increasing resistance of pathogenic microorganisms to antimicrobials has driven the search for safe and sustainable alternatives. In this context, microbial biosurfactants have gained prominence due to their antimicrobial activity, low toxicity, and high stability under extreme conditions. This study presents the production and characterization of a biosurfactant with antimicrobial potential, obtained from Bacillus subtilis isolated from soil, for application in the control of resistant strains. Bacterial identification was performed using mass spectrometry (MALDI-TOF), confirming it as Bacillus subtilis. The strain B. subtilis UCP 1533 was cultivated using different carbon sources (glucose, soybean oil, residual frying oil, and molasses) and nitrogen sources (ammonium chloride, sodium nitrate, urea, and peptone), with evaluations at 72, 96, and 120 h. The best condition involved a mineral medium supplemented with 2% soybean oil and 0.12% corn steep liquor, resulting in the production of 16 g·L^-1 of biosurfactant, with a critical micelle concentration (CMC) of 0.3 g·L^-1 and a reduction in water surface tension to 25 mN·m^-1. The biosurfactant showed an emulsification index of 100% for used motor oil and ranged from 50% to 100% for different vegetable oils, maintaining stability across a wide range of pH, salinity, and temperature. FT-IR and NMR analyses confirmed its lipopeptide nature and anionic charge. Toxicity tests with Tenebrio molitor larvae showed 100% survival at all the tested concentrations. In phytotoxicity assays, seed germination rates above 90% were recorded for Solanum lycopersicum and Lactuca sativa. Antimicrobial tests revealed inhibitory activity against resistant strains of Escherichia coli and Pseudomonas aeruginosa, as well as against species of the genus Candida (C. glabrata, C. lipolytica, C. bombicola, and C. guilliermondii), highlighting the biosurfactant as a promising alternative in combating antimicrobial resistance (AMR). These results indicate the potential application of this biosurfactant in the development of antimicrobial agents for pharmaceutical formulations and sustainable strategies for phytopathogen control in agriculture.

Keywords: Bacillus subtilis; antibacterial biosurfactant; antimicrobial resistance; caatinga soil.

Figure 1

Flowchart of cultivation and optimization of biosurfactant production by Bacillus subtilis. The microorganism was grown in a mineral medium containing inorganic salts, Tris buffer, and yeast extract. Fermentation was carried out in 250 mL Erlenmeyer flasks with 100 mL of medium at 35 °C and 200 rpm for 120 h. Different carbon sources (glucose, molasses, soybean oil, and used frying oil) and nitrogen sources (corn steep liquor, urea, ammonium chloride, sodium nitrate, and peptones) were evaluated for medium optimization.

Figure 2

Gram staining showing Gram-positive bacilli. (A) Image captured with a 40× objective; (B) Image captured with a 100× objective (oil immersion).

Figure 3

Spore staining using the Wirtz-Conklin method. (A) Overview under 100× objective, showing pink-stained cells. (B) Magnification of the highlighted region, revealing oval spores stained light green inside the cells.

Figure 4

The Catalase test was performed in duplicate. Bubble formation was observed on both slides after the addition of hydrogen peroxide (H₂O₂) solution, indicating the presence of catalase.

Figure 5

Variation in surface tension (mN·m⁻¹) over time (72, 96, and 120 h) in culture media containing different carbon sources.

Figure 6

Variation in surface tension (mN·m⁻¹) over time (72, 96, and 120 h) in culture media containing different nitrogen sources.

Figure 7

Variation of surface tension as a function of the concentration of biosurfactant produced by Bacillus subtilis UCP 1533. The critical micelle concentration (CMC) was determined as the point beyond which increases in concentration no longer resulted in significant reductions in surface tension, indicating the minimum concentration required for micelle formation.

Figure 8

FT-IR spectrum of isolated biosurfactant. The x-axis represents the wavenumber (cm⁻¹), and the y-axis represents the transmittance (%).

Figure 9

¹H NMR spectrum of the isolated biosurfactant.

Figure 10

¹³C NMR spectrum of the isolated biosurfactant.

Figure 11

Images of Tenebrio molitor larvae after exposure to phosphate-buffered saline (PBS) and different biosurfactant concentrations during the toxicity assay. (a) 1/2 CMC; (b) 1 CMC; (c) 2 CMC; (d) Negative control (PBS).