Biofilms in modern CaCO₃-supersaturated freshwater environments reveal viral proxies

Biofilms are mucilaginous-organic layers produced by microbial activity including viruses. Growing biofilms form microbial mats which enhance sediment stability by binding particles with extracellular polymeric substances and promoting growth through nutrient cycling and organic matter accumulation. They preferentially develop at the sediment-water interface of both marine and non-marine environments, and upon the growing surfaces of modern tufa and travertine. In this context, however, little is known about the factors, environmental or anthropogenic, which affect viral communities in freshwater spring settings. To explore this issue, geochemical and metagenomic data were subjected to multidimensional analyses (Principal Component Analysis, Classical Multidimensional Scaling, Partial Least Squares analysis and cluster analysis based on beta-diversity), and these show that viral composition is specific and dependent on environment. Indeed, waters precipitating tufa and travertine do vary in their geochemistry with their viruses showing distinct variability between sites. These differences between virus groups allow the formulation of a viral proxy, based on the Caudoviricetes/Megaviricetes ratio established on the most abundant groups of viruses. This ratio may be potentially used in analysing ancient DNA preserved in carbonate formations as an additional source of information on the microbiological community during sedimentation.