The “Infernaccio” Gorges: Microbial Diversity of Black Deposits and Isolation of Manganese-Solubilizing Bacteria

Abstract

The present study explored the microbial diversity of black deposits found in the “Infernaccio” gorge. X-ray Powdered Diffraction (XRPD) was used to investigate the crystallinity of the samples and to identify the minerals. Scanning electron microscope and energy-dispersive X-ray spectroscopy (SEM-EDS) were used to detect the bacterial imprints, analyze microbe–mineral interactions, and highlight the chemical element distribution in the black deposits. 16S rRNA gene metabarcoding allowed the study of Archaea and Bacteria communities. Mn-oxide-solubilizing isolates were also obtained and characterized by culturable and molecular approaches. The multidisciplinary approach showed the occurrence of deposits composed of birnessite, diopside, halloysite, and leucite. Numerous bacterial imprints confirmed the role of microorganisms in forming these deposits. The Bacteria and Archaea communities associated with these deposits and runoff waters are dynamic and shaped by seasonal changes. The uncultured and unknown taxa are the most common and abundant. These amplicon sequence variants (ASVs) were mainly assigned to Proteobacteria and Bacteroidetes phyla. Six isolates showed interesting Mn solubilization abilities under microaerophilic conditions. Molecular characterization associated isolates to Brevibacterium, Bacillus, Neobacillus, and Rhodococcus genera. The findings enriched our knowledge of geomicrobiological aspects of one of the Earth’s hidden habitats. The study also unveiled the potential of this environment as an isolation source of biotechnologically relevant bacteria.

Preliminary indication of the role of AHL-dependent quorum sensing systems in calcium carbonate precipitation in Gram-negative bacteria

“…The results of the morphological characterization of the calcifying strains collected during this study showed the prevalence (67%) of Gram-positive bacteria; only strains B4, B6 and B15 were Gram-negative. This finding is in line with the results of previous studies [41] , [42] . Among all studied bacteria, 7 strains (A7, A11, A14, LMG 11351, B4, B6 and B15), all GN bacteria, were selected for further analysis based on their ability to precipitate CaCO 3 in vitro .…”

“…Inoculated B-4 plates were incubated at 27 °C for 7 days. All colony types were then described based on pigmentation, form, elevation, margin, opacity and surface area [42] . Colonies were classified according to morphology and chromatogenesis and characterized by cell shape and Gram type determination using the Gram 2 Kit (bioMérieux, Marcy-l'Etoile, France).…”

Exploring structure, microbiota, and metagenome functions of epigean and hypogean black deposits by microscopic, molecular and bioinformatic approaches

“…Microbial contribution is usually described by the mineralization/lithification of microbial cells and their aggregation structures, such as biofilms 12 , which can occur internally and externally. Many authors have described the presence of these biosignatures for several speleothems 15 – 18 . Our previous investigations described already the presence of these imprints within “Grotta Grande dei Cervi” black deposits 12 , 19 .…”

“…Microbial contribution is usually described by the mineralization/ lithification of microbial cells and their aggregation structures, such as biofilms 12 , which can occur internally and externally. Many authors have described the presence of these biosignatures for several speleothems [15][16][17][18] .…”

“…In our previous studies we investigated and described black deposits of gorge and cave environments, finding similar biological and structural traits 12 , 15 . For this reason, we hypothesized that black deposits of gorge and cave environments could present similar Bacteria and Archaea communities and common metabolic processes, especially concerning manganese metabolism.…”