Lyophilization Protocol for Long-Term Preservation of Ureolytic Sporosarcina Strains with Biotechnological Applications.

The long-term preservation of biotechnologically relevant bacterial genetic resources is essential, particularly for Sporosarcina spp. strains capable of microbially induced carbonate precipitation (MICP) for toxic metal immobilization. Standard glycerol-based cryopreservation requires an ultra-low-temperature cold chain (-80°C), thereby increasing operational costs and limiting field-scale inoculum transport. To address this bottleneck, lyophilization offers a robust, cost-effective alternative compatible with standard freezing temperatures (-4°C to -20°C). Here, we standardize and validate an optimized lyophilization protocol for Sporosarcina spp. using 20% skim milk as a cryoprotectant. Following a 2-year storage period at -20°C, the lyophilized cells demonstrated high viability and fully retained the urease enzymatic activity required for MICP. These findings demonstrate that this protocol preserves the phenotypic stability and bioremediation potential of Sporosarcina strains without the need for specialized ultra-cold equipment. Consequently, this method provides a highly scalable and viable framework for the formulation of bacterial inocula tailored for in situ field applications.