Bacillus spp. Isolated from Puba as a Source of Biosurfactants and Antimicrobial Lipopeptides

Abstract

Several products of industrial interest are produced by Bacillus, including enzymes, antibiotics, amino acids, insecticides, biosurfactants and bacteriocins. This study aimed to investigate the potential of two bacterial isolates (P5 and C3) from puba, a regional fermentation product from cassava, to produce multiple substances with antimicrobial and surface active properties. Phylogenetic analyses showed close relation of isolates P5 and C3 with Bacillus amyloliquefaciens and Bacillus thuringiensis, respectively. Notably, Bacillus sp. P5 showed antimicrobial activity against pathogens such as Listeria monocytogenes and Bacillus cereus, in addition to antifungal activity. The presence of genes encoding pre-subtilosin (sboA), malonyl CoA transacylase (ituD), and the putative transcriptional terminator of surfactin (sfp) were detected in Bacillus sp. P5, suggesting the production of the bacteriocin subtilosin A and the lipopeptides iturin A and surfactin by this strain. For Bacillus sp. C3 the presence of sboA and spas (subtilin) genes was observed by the first time in members of B. cereus cluster. Bacillus sp. P5 showed emulsifying capability on mineral oil, soybean biodiesel and toluene, while Bacillus sp. C3 showed emulsifying capability only on mineral oil. The reduction of the surface tension in culture medium was also observed for strain P5, confirming the production of surface-active compounds by this bacterium. Monoprotonated molecular species and adducts of sodium and potassium ions of surfactin, iturin, and fengycin were detected in the P5 culture medium. Comparative MS/MS spectra of the peak m/z 1030 (C14 surfactin A or C15 surfactin B [M+Na]⁺) and peak m/z 1079 (C15 iturin [M+Na]⁺) showed the same fragmentation profile of standards, confirming the molecular identification. In conclusion, Bacillus sp. P5 showed the best potential for the production of antifungal, antibacterial, and biosurfactant substances.

Keywords: Bacillus spp.; MALDI-TOF mass spectrometry; antimicrobial peptides; biosurfactant; cassava; fermented food.

FIGURE 1

Phylogenetic tree based on 16S rDNA gene sequences from Bacillus sp. C3 and Bacillus sp. P5 using the Neighbor-joining method (1000 bootstrap replicates).

FIGURE 2

Antifungal activity of Bacillus strains isolated from puba. Strains were inoculated onto the surface of PDA agar plates containing the filamentous fungi (A) Fusariumoxysporum f. sp. lycopersici and (B) Aspergillus flavus. Plates show three inoculations of Bacillus sp. P5 on the left side and Bacillus sp. C3 on the right side.

FIGURE 3

Cultivation of Bacillus sp. P5 in BHI broth at 42^oC. (A) Cell growth () and antibacterial activity against B. cereus ATCC 14579 () were monitored during cultivation. (B) Emulsification activity (, E₂₄) and antifungal activity against Fusarium oxysporum f. sp. lycopersici () were monitored during growth. Values are the means ± SEM of three independent experiments.

FIGURE 4

Mass spectrum of active fraction from reversed phase HPLC of culture supernatant of Bacillus sp. P5. The spectrum in the m/z range 1030–1110 shown data for the homologous series [M + H]⁺, [M + Na]⁺, [M + K]⁺ for surfactin A C₁₅ (1036.8, 1058.8, 1074) and surfactin A C₁₆ (1050.8, 1072.8, 1088.8). It also shows three additional peaks (1064.8, 1086.8, and 1102.8) that are possibly related to surfactin A C₁₇ molecular species.

FIGURE 5

MS/MS spectra of m/z 1030 peak from Bacillus P5 (A) compared to a commercial surfactin standard (B). MS/MS spectra of m/z 1065 peak obtained from Bacillus P5 (C) compared to a commercial iturin standard (D).