Metabolic fingerprinting of microbial communities in a Romanian karst cave with bats and low human impact

Natural karst caves are characterized by constant temperature, lack of light, high humidity and low nutrients availability. The diversity and functionality of the microorganisms dwelling the caves micro-habitats are yet underexplored. Therefore, in-depth investigations of these ecosystems aid in enlarging our understanding of the geomicrobial interactions and microbially-driven biogeochemical cycles. Here we aimed at evaluating the diversity, abundance, distribution and organic substrate preferences of microbial communities from Pe?tera cu Apa din Valea Le?ului (Le?u) located in the Apuseni Mountains (North-Western Romania). To achieve this goal, we employed 16S rRNA gene amplicon sequencing and community-level physiological profiling (CLPP) paralelled by the assessment of environmental parameters of cave sediments and water. Proteobacteria was the most prevalent phylum detected across all samples whereas the abundance detected at order level varied among sites and between water and sediment samples. For the majority of the dominant orders the distribution pattern showed a positive correlation with C- sources such as putrescine, ?-amino butyric acid and D-malic acid, while particular cases were positively correlated with polymers (Tween 40, Tween 80 and a-cyclodextrin), carbohydrates (a-D-lactose, i-erythritol, D-mannitol) and most of the carboxylic and ketonic acids. Physicochemical analysis reveals that sediments are geochemically distinct, with increased concentration of Ca, Fe, Al, Mg, Na and K. Water samples presented a low concentration of nitrates and for this pristine cave, this may indicate microbial denitrification processes. Geochemical parameters that may influence the community structure reveal in the PCA plot the clustering of different dominant orders with elements such as Mg, As, P, Fe, Cr. Despite the general similarity at phylum-level in Le?u cave across the sampled area, the results obtained in this study suggest that specific sites drive bacterial community at order-level, perhaps sustaining the enrichment of unique bacterial populations due to microenvironmental conditions. This information serves as starting point for further studies in elucidating the links between taxonomic and functional diversity of subterranean microbial communities.