Community Structure of Subsurface Biofilms in the Thermal Sulfidic Caves of Acquasanta Terme, Italy

D. S. Jones
D. J. Tobler
I. Schaperdoth

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We performed a microbial community analysis of biofilms inhabiting thermal (35 to 50°C) waters more than 60 m below the ground surface near Acquasanta Terme, Italy. The groundwater hosting the biofilms has 400 to 830 μM sulfide, <10>μM O2, pH of 6.3 to 6.7, and specific conductivity of 8,500 to 10,500 μS/cm. Based on the results of 16S rRNA gene cloning and fluorescent in situ hybridization (FISH), the biofilms have low species richness, and lithoautotrophic (or possibly mixotrophic) Gamma- and Epsilonproteobacteria are the principle biofilm architects. Deltaproteobacteria sequences retrieved from the biofilms have <90% 16S rRNA similarity to their closest relatives in public databases and may represent novel sulfate-reducing bacteria. The Acquasanta biofilms share few species in common with Frasassi cave biofilms (13°C, 80 km distant) but have a similar community structure, with representatives in the same major clades. The ecological success of Sulfurovumales-group Epsilonproteobacteria in the Acquasanta biofilms is consistent with previous observations of their dominance in sulfidic cave waters with turbulent water flow and high dissolved sulfide/oxygen ratios. Despite rapid progress in the past decade, the deep subsurface remains one of the least explored microbial habitats on earth. Recent studies illustrate the presence of significant spatial heterogeneity (13, 53) and the strong influence of mineralogy and fluid flow on subsurface microbial biodiversity (9, 16, 31, 61). Data obtained by drilling are complemented by an increasing number of studies that exploit subsurface passages navigable by humans (17). These subsurface passages include caves (14, 46) and mines (22, 32, 47, 52, 57). Approximately 10% of known caves (49) and perhaps more (33) are formed where reduced, sulfidic groundwaters interact with oxidized water descending from surface environments. Limestone dissolution in these groundwater mixing zones results in deep caves that receive few organic inputs from the surface. Due to the presence of b