GENOMIC CHARACTERIZATION OF EXTREMELY ACIDOPHILIC BACTERIA IN ACIDIC CAVE WALL BIOFILMS

Caves form by sulfuric acid speleogenesis when anoxic fluids bearing hydrogen sulfide (H₂S) encounter air-filled pores or oxygenated groundwater. Substantial cave enlargement occurs above the water table where hydrogen sulfide degasses into the cave atmosphere and is oxidized to sulfuric acid by sulfur-oxidizing bacteria and archaea. In order to study the acidophilic microorganisms responsible for sulfide oxidation, we enriched and isolated Acidithiobacillus spp. from highly acidic wall biofilms from the Villa Luz and Frasassi cave systems. Physiological experiments showed that both isolates grew at pH values as low as 0.2 and as high as 6, with the highest growth rates observed at pH 2-3. While both isolates were able to grow chemoautotrophically elemental sulfur, only the Frasassi isolate was able to grow on thiosulfate. We used complete genome sequencing to characterize biochemical pathways for inorganic sulfur oxidation and compare other metabolic differences among the isolates. Then, in order to further explore other acidophiles from the Cueva de Villa Luz community, we combined short- and long-read sequencing (Illumina and Pacific Biosciences) to generate a metagenomic dataset from extremely acidic wall biofilms collected from this cave in 2013. Analysis of these communities reveals an acidophilic community comprised primarily of bacteria, with minor amounts of archaea and fungi. The dominant bacterial genera in this dataset are Acidithiobacillus, Metallibacterium, and Acidimicrobium. Acidithiobacillus spp. from this cave have pathways for inorganic sulfur compound oxidation that include a partial SOX system, sulfur dioxygenase, sulfur oxygenase reductase, and multiple sulfide:quinone oxidoreductases. Metagenome-assembled genomes showed that the Metallibacterium also have multiple pathways for sulfur oxidation, while the Acidimicrobium appear to be organo-heterotrophs. Further analysis into the functional genes of each of these three groups revealed their metabolic capability, thereby elucidating their role in snottite biogeochemistry. This has important implications for sulfur cycling in cave environments, as well as sulfur metabolisms being discussed in both astrobiological and biotechnical applications.