Panreac

Spores were purified as described by [57 (link)] with some modifications. An overnight culture of B. thuringiensis in 4 ml of TSB was grown at 37°C, 180 r.p.m. The overnight culture was diluted 1:100 in 50 ml fresh TSB media containing 0.1 mmol MnSO₄ (PanReac, AppliChem) and grown at 37°C, 180 r.p.m. for 10 days. Cultures were transferred to a 50 ml falcon tube and stored on ice for 20 min. The tubes were spun down at 4°C, 10 000 r.p.m. for 10 min. The pellet was suspended in 20 ml of 50 mM Tris–HCL (pH 7.2; PanReac, AppliChem) with an addition of 50 µg ml⁻¹ lysozyme (from hen egg whites, Fluka). Samples were incubated at 37°C, 180 r.p.m. for 1 h. The sample was spun down at 4°C, 10 000 r.p.m. for 10 min, and the pellet was suspended in cold sterile water. This washing step was repeated twice. The pellet was then suspended in 5 ml 0.05% SDS solution (Sigma, D6750-10G) by vortexing and incubated for 5 min at room temperature. The sample was then spun down at 4°C, 10 000 r.p.m. for 10 min, and the pellet was suspended in cold sterile water. This washing step was repeated five times. After the final washing step, the pellet was suspended in 5 ml of cold, sterile water and stored at 4°C for later use. Spores were counted using a Neubauer-improved chamber (Paul Marienfeld GmbH, Lauda-Konigshofen, Germany) with a chamber depth of 0.1 mm.

The gel forming capacity of the EPS was tested by preparation of cation-mediated gels using divalent cations (FeSO₄·7H₂O, Honeywell Fluka, Seelze, Germany, CuSO₄·5H₂O, AppliChem Panreac, Barcelona, Spain, CaCl₂·2H₂O, AppliChem Panreac, Barcelona, Spain, and MgSO₄·7H₂O, BioChem Chemopharma, Cosne sur Loire, France) and a trivalent cation (FeCl₃·6H₂O, Alfa Aesar, ThermoFisher, Kandel, Germany). The gelation studies were performed according to the procedure described by Shimada et al. [53 (link)], with minor modifications: 5 mL of EPS solution (5 g L⁻¹) was added to the metal salt (10 mg of cation) and agitated until dissolution and gel formation was assessed (standard conditions). Afterwards, 1 mL of NaOH (2 M) was added and the solution was agitated to test gelation in alkaline conditions. Gel formation was assessed by visual inspection, and the gels were categorized according to their strength and homogeneity: (+) for homogeneous gels that maintained their gel structure in a tube-inversion test, (-) for homogeneous gels that did not sustain their structure in the inversion test, and (--) for small non-homogeneous gels.

The compounds 1-amonibenzothiazole (ABT, 97% purity), 2,2′- azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, 98% purity), phenanthrene (PHE, 98% purity), and benz[a]anthracene (BAA, 98% purity) were purchased from Sigma (St. Louis, MO, USA). Copper sulphate and zinc, lead, and silver nitrate metal salts were acquired from Merck (Madrid, Spain). All solvents used were of HPLC grade: acetonitrile (PanReac, AppliChem, Barcelona, Spain), HPLC water (PanReac, AppliChem, Barcelona, Spain), and phosphoric acid (Fisher Chemical, Madrid, Spain). All other chemicals and reagents were of analytical grade or higher purity.
The used fungi were Chaetomium jodhpurense (MH667651.1), Chaetomium maderasense (MH665977.1), Paraconiothyrium variabile (MH667653.1), Emmia lacerata, and Phoma betae (MH667655.1), previously isolated from a salt environment in Tunisia and molecularly identified [19 ]. The fungal genera Chaetomium, Paraconiothyrium, Phoma, belonging to Ascomycota, were known by their large application in bioremediation due to their great ability to produce extracellular enzymes [20 (link),21 (link),22 (link)]. The strain Emmia lacerate is a Basidiomycota belonging to polypore species. As a white rot fungus it can produce a large wide of extracellular enzymes [23 (link)].