Oliva, Feliciana (2026) Sampling, characterization, maintenance and preservation of microbiomes from extreme environments. [Tesi di dottorato]

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Abstract

This thesis, entitled Sampling, characterization, maintenance and preservation of microbiomes from extreme environments, was designed to develop a coherent and reproducible workflow for handling microbiomes from extreme environments under laboratory conditions, with a particular focus on geothermal systems. The overall work was structured as a logical sequence of phases: (i) definition of the conceptual framework and experimental approach, (ii) screening and characterization of a broad set of samples, (iii) selection of representative microbiomes and development of a propagation strategy, and (iv) evaluation of a cryopreservation and post-thaw reactivation workflow. The premise of this work is that microbiomes from extreme environments, although extensively studied and widely exploited in ecology, biotechnology, and molecular biology, are difficult to maintain under laboratory conditions. In particular, the main issue is to propagate and preserve extreme microbiomes ex-situ without introducing shifts in community structure; therefore, to develop a dedicated experimental approach is extremely urgent. This task constitutes a significant experimental challenge due to the physicochemical constraints of extreme habitats and the intrinsic limitations of cultivating highly structured and interdependent microbial communities at laboratory scale. The main objective of the thesis was to define a strategy for the propagation of environmental microbiomes that could preserve, as much as possible, the microbial composition and relative abundance of environmental communities and their capacity to withstand, adapt, and restructure under controlled laboratory conditions. A second objective was to develop and evaluate a cryopreservation approach to enable long-term storage and recovery of the microbiome after freezing, comparing the effect of different preservants (glycerol, DMSO and no_preservant) as well as different preservation temperatures and storage durations. Chapter 1 is a review focused on the Winogradsky column as a culturing model system, and its potential use with complex environmental communities difficult to cultivate using standard approaches. The review presents the Winogradsky column as an experimental system capable of generating gradients and stratified microenvironments, thereby promoting the coexistence of microbial groups with different requirements and supporting trophic and redox interactions. This theoretical basis guided the experimental choices adopted in the subsequent chapters, in particular the use of a “Winogradsky-type” cultivation system both for propagation and for post-freezing reactivation of complex microbial communities from extreme environments. Chapter 2 corresponds to the screening and characterization phase. Fourteen geothermal sites in the Campania region, in Italy, were sampled, and the collected samples were described by integrating environmental data, geochemical features, and sequencing-based microbiological analyses. This phase provided a comparative overview of microbial diversity and associated environmental conditions and, most importantly, enabled the definition of criteria for selecting microbiomes to be used in the subsequent propagation and preservation experiments. Chapter 3 describes the development of a propagation strategy on three microbiomes from geochemically distinct contexts, specifically selected to evaluate the experimental approach on different communities. Propagation was carried out using a cultivation system inspired by the Winogradsky column, designed to promote gradients and stratified microenvironments. Communities were incubated for six months and compared at the end of incubation with the negative control (the sample at time zero) through 16S rRNA gene sequencing, in order to assess the ability of the proposed system to support growth and community reorganization under laboratory conditions. Chapter 4 extends the methodological framework to long-term preservation, focusing on a case study: a microbiome from the geothermal site of Grotta dell’Acqua. A cryopreservation workflow was established by comparing three storage conditions (no_preservant, glycerol and DMSO), two storage temperatures (−20 °C and −80 °C), and three storage times (3, 6 and 12 months). After cryopreservation, samples were reactivated using the same “Winogradsky-type” cultivation approach, in order to evaluate post-thaw recovery and to characterize how the preservants influenced the recovered community. Reactivated communities were analyzed through 16S rRNA gene sequencing, using alpha and beta diversity indices to systematically compare the effects of preservation conditions. Overall, the thesis presents a linear workflow that links a conceptual and methodological framework (Chapter 1) to a screening and selection phase (Chapter 2) and to two complementary applied objectives: propagation (Chapter 3) and cryopreservation with standardized reactivation (Chapter 4). Taken together, this preliminary work demonstrates that the Winogradsky column is a potential cultivation system to maintain and propagate complex environmental microbial communities over time under controlled laboratory conditions, indicating that the preservation of community-level structure is achievable beyond the natural habitat. At the same time, the results highlight that the proposed experimental approach seems to impose selective pressures that favor certain microbial groups over others, leading to partial deviations from the original community composition. Despite these limitations, this study represents a critical proof of concept and provides a valid experimental and conceptual framework paving the way for the systematic optimization of cultivation and preservation strategies aimed at minimizing community shifts while enhancing the long-term stability and ecological relevance of complex microbiomes maintained ex-situ.

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