Next-generation sequencing for microbial characterization of biovermiculations from a sulfuric acid cave in Apulia (Italy)

Sulfuric acid cave systems host abundant microbial communities that can colonize several environments displaying a variety of morphologies, i.e. white filamentous mats floating on the water surface, white creamy moonmilk deposits on the walls, and biovermiculations. Up to date, only few reports have described the microbiological aspects behind biovermiculation geomicrobiology of Italian sulfuric acid caves despite their overall abundance. Here, we present the first characterization of biovermiculation microbial populations from the Santa Cesarea Terme (Apulia, Italy) using next-generation sequencing and field emission scanning electron microscopy (FESEM) approaches. We focused our analysis on biovermiculations from Fetida Cave located along the Adriatic Sea coastline. This cave is at sea level, and moving from the entrance to its inner part, it is possible to observe a decrease of marine influence accompanied by a corresponding increase in the acidic effect of the upwelling waters. Biovermiculations are copiously observed covering the ceiling and walls mainly in the inner and middle environments of the cave, while they are absent near the entrance. Biovermiculations have a widespread range of colors going from grey to dark brown with an overall slimy appearance; dendritic morphologies alternate to very dense wall-covering sheets. Total DNA was extracted from each sample and 16S rDNA sequences were analyzed through Ilumina MiSeq platform. The main lineages found in all the samples included Gammaproteobacteria, Betaproteobacteria, Alphaproteobacteria, Bacteroidetes, Planctomycetes, Actinobacteria and Acidobacteria. In particular, the samples from the inner part of the cave had the highest abundance of Acidobacteria and showed the presence of Epsilonproteobacteria that can be related to bacterial sulfuroxidizing activity. FESEM images revealed microbial mats composed of filamentous cells including Actinobacteria and reticulated filaments of unknown origin, as well as prosthecate bacteria. This study shows that the biovermiculation microbial communities from Fetida cave is in accordance with previous data reported in acidic caves from Italy and support the role of Epsilonproteobacteria typically occurring in sulfidic environments.