2.7.1. Bacteria Encapsulation in Alginate Beads

B. subtilis was cultured in LB broth and incubated at 25 °C, with rotation of 120 rpm for 24 h. Bacteria were recovered by centrifugation and resuspended in 0.9% NaCl until final OD600 of 1.8. Alginate beads were obtained by preparing a 1.25% sodium alginate solution, constantly stirred at 120 °C until a homogeneous solution was obtained. The composition also included glycerin (as an osmoprotector) and sucrose to better dissolve the alginate, and as an additional source of nutrition. The sodium alginate mixture was cooled to 50 °C, and the bacteria in suspension were added and mixed slowly. With the aid of a positive pressure pump, the alginate solution mixture containing the bacterial cells was added dropwise to a 2% CaCl₂ solution while maintaining constant agitation for 45 min until stabilization of the beads. Beads were then removed from the CaCl₂ solution and washed in 0.9% sterile NaCl. Beads were immediately used for bacterial counting and plant growth promotion tests [46].