Preparation of Spore Suspensions

Clostridium beijerinckii NCIMB 8052 and Bacillus subtilis DBM 3006 were stored in the form of spore suspensions in sterile distilled water at 4°C. Spores of C. beijerinckii NCIMB 8052 were obtained after 48 h incubation of the culture in 250 mL Erlenmeyer flasks containing 100 mL of TYA medium in an anaerobic chamber (Concept 400, Ruskinn Technology, United Kingdom) at 37°C. TYA medium contained in g/L: glucose 40, yeast extract (Merck) 2, tryptone (Sigma-Aldrich) 6, potassium dihydrogenphosphate 0.5, ammonium acetate 3, magnesium sulfate heptahydrate, 0.3, ferrous sulfate heptahydrate 0.01; the pH of the medium was adjusted prior to sterilization in the autoclave (20 min, 121°C, 0.1 MPa) to 6.8. Spores of B. subtilis DBM 3006 were obtained after 48 h incubation of the culture in 250 mL Erlenmeyer flasks containing 50 mL of MP broth shaken on a rotary shaker (New Brunswick Scientific) at 300 rpm and 30°C. MP broth contained in g/L: meat extract (Roth) 3, peptone (Roth) 5; the pH of the medium was adjusted prior to sterilization in the autoclave (20 min, 121°C) to 7.0. Salts, HCl and NaOH for medium preparation and pH adjustment were purchased from Penta, Czech Republic. After cultivation, spores of both bacterial cultures were harvested by centrifugation (Hettich MIKRO 220R) for 5 min, 5,000 rpm, at 4°C. Spores were washed with sterile water, centrifuged under the same conditions and re-suspended in 20 mL of sterile water. Finally, the spore suspension was pipetted in 1 mL portions into Eppendorf tubes, which were stored at 4°C. All spore handling was performed under aseptic conditions using sterile tools and materials. Spore concentrations were estimated by flow cytometry (Branska et al., 2018) to be 2^∗10⁸ spores/mL and 8^∗10⁸ spores/mL for C. beijerinckii and B. subtilis, respectively. Prior to inoculation of medium for germination assays, spore suspensions were heat shocked (80°C for 30 s followed by cooling on ice for 2 min) to accelerate germination.