Erratic calcareous deposits: Biotic formation insights and biomineralising bacterial strain isolation

The present study investigated the contribution of microbial communities in producing "living stones" and the suitability of these clasts as sources of microorganisms with biomineralisation abilities. The calcareous samples were analysed for their microbial community (16S rRNA gene metabarcoding and culturable approach) and in vitro regeneration tests. Scanning electron microscopy and Energy Dispersive Spectroscopy (SEM-EDX) were applied to investigate microbial aggregation structures and footprints in natural and in vitro samples. The metabarcoding unveiled amplicon sequence variants (ASVs) assigned to lineages with biomineralisation abilities (e.g., Proteobacteria and Actinobacteriota). The culturable approach resulted in nineteen calcifying isolates with diverse morphological, metabolic, and mineral precipitation properties. Based on mineralising properties, Stenotrophomonas maltophilia, Lysinbacillus fusiformis, and Microbacterium ginsengiterrae were identified at the molecular level. In vitro regeneration tests and SEM-EDX analyses confirmed the active role of the endogenous microorganisms in forming these "living stones". These findings allow us to hypothesise an essential role of microbial precipitation in forming these "living stones", previously described as of abiotic origin. The current study findings provide a solid scientific foundation for future investigations. The obtained bacterial isolates and their potential applications in bioremediation, construction, and cultural heritage restoration demonstrate the direct applicability of our study in sectors involving biomaterials application. STATEMENT OF SIGNIFICANCE: We studied some "living stones" that can be found worldwide and whose origin is still not completely understood. Geologists have not yet fully explained the origin of these inorganic structures that grow in size over time. The results obtained from our microbiological investigations allowed us to discover that microorganisms play a crucial role in forming these masses. In the investigations of the structures and microbial communities within the stones, we identified specific bacteria that actively contribute to forming minerals and isolated bacteria that can form biominerals. These findings deepen our understanding of natural processes involved in the formation of these structures and show their potential for several applications (e.g., building materials or cultural heritage preservation).