Exploring the diversity and bioremediation potential of epilithic biofilms living in an acid mine drainage- affected mountain stream

Given the mineralogy of the Alpine bedrock and former mining activities, several Northern Italy areas show arsenic concentrations above the Italian law limit of 20 mg kg-1 of soil. In the Anzasca Valley (Piedmont), an acid mine drainage originated from an abandoned gold mine heavily affects the Rio Rosso stream. Due to the oxidative dissolution of pyrite and arsenopyrite, arsenic and other heavy metals are continuously released into the stream, contaminating the surrounding area. On the whole riverbed, the stream is characterized by the presence of an extensive reddish epilithic biofilm. This study aimed at analyzing the composition and metabolic properties of the Rio Rosso epilithic biofilms to unveil their strategies to cope with extreme metal contamination and evaluate possible metabolic features exploitable for bioremediation purposes. The epilithic biofilms in the vicinity of the mine and at 1.7 km downstream the mine were characterized by 16S amplicon and metagenomic sequencing in relation to the main physicochemical parameters of the biofilm and the surrounding water. In parallel, autotrophic and heterotrophic microorganisms were characterized by enrichment cultivation, isolation and testing for their ability to perform arsenic transformation. Both mine and downstream biofilms were affected by metal contamination, with arsenic accounting for 93.43 and 8.66 g kg-1, respectively. Other heavy metals were accumulated in the downstream biofilm, ranging from 3.59 mg kg-1 to 10 g kg-1. The mine biofilm was further affected by a pH of 2 and dominated by acidophilic iron- and sulfur-oxidizing microorganisms belonging to Acidithrix, Acidiphilium and uncharacterized Planctomycetota. Downstream the mine, a sub-neutral pH (pH 6.7) promoted the establishment of a significantly more diverse community driven by Cyanobacteria. Both metagenomics and cultivation experiments revealed that arsenic resistance was mainly mediated by arsenate reduction to arsenite and consequent extrusion from the cell. Extruded arsenic was likely embedded in an extracellular polysaccharide matrix. To a lesser extent, arsenite oxidation was revealed by the presence of arsenite oxidase genes and the isolation of autotrophic arsenite-oxidizing bacterial strains. These outcomes revealed that the acid mine drainage-affected Rio Rosso epilithic biofilm is a natural sink for arsenic and heavy metals, characterized by the assemblage of bacterial species with a great potential to be exploited for arsenic and heavy metal bioremediation in freshwater environments