Identification and characterization of indigenous actinobacteria biofilms under optimal growth conditions

Abstract

Actinobacteria play an important ecological role in bioremediation processes in the natural world, in which the formation of biofilms is an important characteristic for bacterial communities since it favors adaptation to adverse environmental conditions. Therefore, the proposed objectives were: 1-Evaluate actinobacteria strains in their biofilm production capacity, 2- Study and characterize optimal conditions for biofilm formation. Different Streptomyces sp. (M7, A5, MC1, A12, A14) producers of biofilms (Urrutia et al., 2021), and then from a suspension that was previously grown in a culture medium of casein starch agar (CAA), was inoculated in Tryptone Soy Agar (TSB) medium, incubated at 30 °C by shaking for 72 h. For such growth, the influence of incubation time at 48, 72, and 96 h, amount of inoculum using TSB medium at DO 540nm (0.5; 1.0 and 1.5) was analyzed; different pH (5, 7, and 9), production at different osmolarity with D-sorbitol and NaCl (0.03; 0.06; 0.3 and 0.6 M) and biofilm formation capacity on different surfaces, on Polysorp (hydrophobic surface) and Maxisorp (hydrophilic surface) microplates. The biofilms quantification was determined by the crystal violet staining method at DO 590 nm. Preliminary results determinated that Streptomyces sp., A14, A5, MC1, M7, and A12 produced biofilm and of them, particularly the A14, A5, and MC1 strains showed the highest production under the pre-established conditions. Likewise, the higher production incubation time was determined at 96 h. In respect of the inoculum density, it was evidenced that whith an OD of 1 the highest biofilm formation was reached since no significant differences were observed at an OD of 1.5. It was determined that an alkaline pH allowed a greater biofilm formation capacity that was significantly higher than those obtained at pH 7 and 5 (p ≤ 0.05). The presence of 0.6 M D-sorbitol significantly favored the biofilm synthesis in A5, A14, M7, and in the presence of 0.03 NaCl, a positive but not significant effect was observed. Regarding the surface used for its formation, it was different and greater in hydrophilic surface than in hydrophobic surface in A5, A14, M7, and MC1 where the values obtained were 3.9; 3.4; 3.2, and 3.1 respectively. While in A12 with an OD of 1.2, no increase in biofilm formation was observed; this would be explained by the fact that the hydrophilic surfaces have high surface energy and a greater attraction force to keep bacteria attached. From the results, we can conclude that in the optimization of the parameters they determined that the best biofilms producers were Streptomyces sp A14, A5, and MC1.