Investigating manganese-oxidizing microbial biofilms in a historic copper mine of Upper Franconia

Redox cycling of iron (Fe) and manganese (Mn) can be an important source of energy for microorganisms in subsurface habitats. While Fe oxidation is well known to support autotrophic growth, the first chemolithoautotrophic Mn-oxidizing organism was only recently enriched from tap water. Truly estimating the distribution and importance of chemolitho(auto)trophic Mn-oxidation remains a major challenge for environmental microbiology. Here, we report on microbial biofilms from a historic mine in Northern Bavaria, which may well represent a natural habitat for autotrophic Mn-oxidizers.

The biofilms are enriched in Mn oxides, with Mn making up to 40% of the biofilms" dry weight. Identification of Mn(II), Mn(IV) and the labile Mn(III) in microbial cells via scanning transmission X-ray microscopy coupled to near-edge x-ray absorption fine structure spectroscopy suggested an ongoing biological Mn oxidation in situ. Characteristic Mn oxide nodules with microbial cells attached were also found in aerobic enrichment cultures set up in minimal media. Lectin staining and confocal laser-scan microscopy showed abundant extracellular polymeric structures coating the mineral particles. Cell numbers and morphologies, as well as taxonomic composition of the microbial communities differed between different types of biofilms and different mining tunnels. From metagenomes of biofilms and mine water, we reconstructed 132 metagenome-assembled genomes (MAGs). Intriguingly, nearly half of the MAGs encoded multi-heme cytochromes indicative of extracellular electron transfer, a necessary capability for electron exchange with a mineral surface. Most of the potential autotrophs MAGs contained multi-heme cytochromes and constituted up to 23% of the community. This further supports our hypothesis that metal-cycling microorganisms belonging to uncultured families of Gammaproteobacteria, Nitrospirota, and Acidobacteriota may be the major primary producers in these biofilms. Based on taxonomic affiliation of the MAGs, the biofilms could be seen as a natural enrichment of Mn-cycling freshwater microorganisms and could help us understand the role of Mn in microbial freshwater ecosystems.