4.2. Isolation and Screening of Isolates for BSs Production

A 0.5 g seaweed sample was homogenized (Stomacher Lab Blender 80, Seward Medical Limited, London, UK) with 4.5 mL of a sterile diluent. A series of 10-fold dilutions was prepared in an isotonic saline solution. From the appropriate dilutions, 0.1 mL aliquots were spread onto brain heart infusion agar (BHI agar pH 7; HiMedia) and the plates were incubated at 27 °C for 48 h under aerobic conditions. Using the Gram and Wirtz-Conklin staining methods, preparations were made from the pure colonies, and the microscopic images—shape, color, size, arrangement and the presence of spores—were observed and evaluated. Gram-positive, spore-forming, rod-shaped isolates were tested for BSs production. BHI broth (HiMedia) was used as a seed medium, which was inoculated with a loop-full of the previously obtained isolates on BHI agar and incubated at 27 °C for 18 h. Subsequently, a liquid McKeen medium [74] (20 g/L glucose, 5 g/L glutamic acid, 1 g/L K₂HPO₄, 1.02 g/L MgSO₄, 0.5 g/L KCl, pH 7.0) supplemented with 1 mL of mineral solution (0.5 g/L MnSO₄·7H₂O, 0.16 g/L CuSO₄·5H₂O, 0.015 g/L FeSO₄·7H₂O) was inoculated with 2% (v/v) seed media. After inoculation, the flasks were incubated on a water bath shaker (JULABO SW 2C, Labor Technic GMBH Selbach, Germany) at 27 °C and 120 rpm for 72 h. The cell-free supernatants (CFS) obtained by centrifugation (4754× g/45 min/4 °C) were screened for BSs production by an oil spreading test. This test was performed according to Morikawa et al. [75] with the following modification: 20 µL of crude oil (Slovnaft, Vlčie hrdlo, Slovakia) was added to the surface of 10 mL of distilled water in a 60 mm diameter Petri dish to form a thin oil layer. Then, 100 µL of CFS was gently applied to the centre of the oil layer. In a positive case, the oil was displaced, and a clearing zone was formed. The diameter of this clearing zone on the oil surface correlated with the surfactant activity (oil displacement). The reference surfactin-producing strain B. subtilis subsp. subtilis DSM 3257 was used as a positive control, and McKeen medium served as a negative control. The 3/22 isolate that produced the largest clearing zone was selected for further study.