Licata, Marzia (2026) Cryopreservation of Photosynthetic Microorganisms: Integrated Assessment of Post-Cryogenic Recovery across Phylogenetically Distinct Lineages. [Tesi di dottorato]

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Abstract

The long-term preservation of photosynthetic microorganisms represents a strategic undertaking for biodiversity conservation and for the maintenance of culture collections, as well as for ensuring the reproducibility of physiological and evolutionary studies and supporting biotechnological applications. Among the available strategies, cryopreservation has become an effective tool for the long-term storage of biological material, reducing the risks associated with continuous subculturing, such as contamination, genetic drift, and the loss of functional traits. Nevertheless, the assessment of cryopreservation success still presents significant conceptual limitations. Chapter 1 provides a critical review of the literature on photosynthetic microorganisms cryopreservation, examining its fundamental principles, operational variables, and current evaluation strategies. A critical appraisal of the literature highlights that the success of microalgae and cyanobacteria cryopreservation has been primarily evaluated through post-thaw viability measurements, implicitly assuming that survival constitutes a reliable indicator of functional integrity. Alongside this parameter, several studies have considered individual physiological indicators, such as photosynthetic efficiency or pigment content. However, these evaluations are often fragmented, limited to a restricted number of parameters and short time scales, and lack an integrated assessment of the different functional levels involved in post-cryogenic recovery. Moreover, the application of multiparametric and omics-based approaches remains limited, as do comparative studies involving microalgae and cyanobacteria from phylogenetically distinct divisions. From this analysis emerges the need to consider post-cryogenic recovery as a complex and multidimensional process, for which the integration of physiological and kinetic parameters is essential, including molecular parameters where applicable. In light of these limitations, the present thesis investigates the cryopreservation of photosynthetic microorganisms from a biological and comparative perspective, in which the interpretation of post-thaw physiological and molecular responses plays a central role, integrated with the evaluation of the technical aspects of preservation protocols. Cryopreservation is thus employed as a controlled experimental stress, capable of revealing lineage-specific differences in recovery strategies, functional priorities, and biological timing through which phylogenetically distinct photosynthetic microorganisms cope with and overcome a common cryogenic perturbation. Chapter 2 focuses on a green microalga belonging to the genus Stichococcus, commonly cryopreserved. This chapter provides a methodological baseline for both cryopreservation and assessment of post-cryogenic recovery through the comparison of different freezing and thawing strategies, and the introduction of a kinetic approach combined with physiological analyses. Chapter 3 extends this framework to an extremophilic red microalga of the genus Galdieria, for which cryopreservation studies are extremely limited. This chapter enables the investigation of how organisms adapted to extreme environmental conditions respond to a cryogenic perturbation occurring outside their natural ecological range, offering the opportunity to characterize more complex response and recovery programs through the integration of physiological and molecular analyses. Chapter 4 further expands the comparative dimension by including a cyanobacterium, allowing the comparison of prokaryotic and eukaryotic organisms characterized by different photosynthetic architectures. The contrast between systems based on membrane-integrated antenna complexes and those relying on peripheral, modular light-harvesting structures provides an ideal framework to examine how photosynthetic organization and phylogenetic context shape post-cryogenic recovery strategies. Across all systems analyzed, the ability to successfully cryopreserve biological material constitutes a fundamental prerequisite of this study. Within this perspective, the analysis extends to the characterization of the quality and dynamics of post-cryogenic recovery, going beyond the mere demonstration of preservation feasibility. Understanding recovery mechanisms and functional differences among phylogenetically distant microalgae and cyanobacteria not only contributes to the improvement of long-term conservation strategies, but also provides a privileged perspective for investigating the resilience and functional diversity of photosynthetic systems.

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