Beyond surface scraping to understand fluvial biofilms: an electron microscopy analysis of the rock–biofilm interface in Antarctic meltwater streams

Fluvial biofilms develop through the establishment of microbial communities on submerged mineral surfaces. However, they are routinely studied after detachment from their colonized substrate by scraping or brushing, a standard procedure that homogenizes biofilm architecture and disrupts the biofilm–rock interface. Using an in situ microscopy approach that examines biofilms without separating them from their colonized substrate, we studied undisturbed biofilm-covered rocks collected from Antarctic meltwater streams. Our observations revealed: (i) a highly organized three-dimensional architecture characterized by spatially structured microbial assemblages embedded in a matrix of extracellular polymeric substances; (ii) a close association among microbial cells, the colonized rock surface and fine-grained sedimentary components; (iii) the development of an endolithic biofilm fraction through the colonization of internal rock fissures and cavities; and (iv) a frequent laminated structure with alternating layers rich in microbial cells and layers composed mainly of sediment particles, which may preserve a physical signatures of hydrological variability and sediment deposition. Together, these observations challenge the widespread view of fluvial biofilms as uniform microbial layers developing on inert mineral surfaces. Integrating in situ microscopy approaches with conventional destructive approaches offers new opportunities to link microbial diversity with its spatial organization, providing a more complete understanding of fluvial biofilm ecology and microbial–mineral interactions.