Long-Term Cryopreservation of Pseudomonas spp. for Biobanking and Biotechnology Applications

The genus Pseudomonas encompasses one of the most metabolically versatile and ecologically diverse groups among Gram-negative bacteria, comprising over 300 validly described species distributed across virtually all ecosystems. Members of this genus are of considerable relevance in medicine, environmental science, and industrial biotechnology, owing to their capacity to produce a wide array of bioactive compounds, as well as their involvement in biodegradation of xenobiotics and plant growth promotion. Among the most extensively studied representatives are P. aeruginosa and P. putida, which serve as contrasting model strains in pathogenicity research and environmental biotechnology, respectively. Improper storage conditions may compromise key physiological traits of these strains, including pigment production and biosurfactant-synthesizing capacity, thereby limiting their utility in downstream applications. The long-term preservation of Pseudomonas strains in biobanks is therefore essential to safeguard biological material of scientific, biotechnological, and biomedical value. In this chapter, we describe a standardized glycerol-based cryopreservation protocol at -80°C for the long-term preservation of Pseudomonas strains, along with the evaluation of their functional stability following reactivation. The method relies on glycerol as a penetrating cryoprotectant, which minimizes cellular damage associated with ice crystal formation and osmotic stress while preserving cell viability, genetic integrity, and metabolic functionality. Post-reactivation validation includes the assessment of fluorescence emission and the capacity to produce biosurfactants. This protocol provides a reliable and reproducible tool for the preservation of biotechnologically relevant microbial genetic resources.