Small-scale biogeography of biofilms and its implications for sequencing-based studies

Biofilm microbial communities are characterized by spatial heterogeneity arising in response to environmental characteristics and the metabolic requirements of their inhabitants, which create biogeographical patterns at both large and small scales. While the impact of small-scale biogeography on several emergent biofilm properties has been characterized by a variety of laboratory studies using simple synthetic communities, the extent of small-scale biogeography in environmental biofilms, as well as its impact on sequencing-based studies, remain poorly characterized. Here, we investigate centimeter-scale biogeography in potable water biofilms developed in environments with distinct levels of heterogeneity. We then estimate microbial diversity at scales relevant for microbial interactions and characterize the impact of sampling strategies on both alpha and beta diversity metrics, as well as on inferred co-occurrences and scaling laws. Our results show that biofilm sections in seemingly homogeneous environments are characterized by similar biogeographic patterns as the ones in a more heterogeneous one, albeit with lower cm-scale variability. We estimated that, despite the overall richness over the entire biofilms, single cells are surrounded by local neighborhoods of limited diversity, strongly limiting their possibility for diverse interactions. Larger sampling areas average such local heterogeneities and distort community structure estimates, leading to an inflated and erroneous estimation of co-occurrences and potential metabolic interactions. Finally, we report how both environmental heterogeneity, as well as sample area size, influence scaling law performances. Our results highlight the ubiquitous nature of biofilm biogeography and call for the adaptation of biofilm sampling strategies to consider this phenomenon relative to specific research questions.