Spatial and functional differentiation of microbial biofilms in a traditional cheese ripening environment.

Biofilms in historic buildings represent stable microbial ecosystems shaped by long-term environmental filtering. We investigated bacterial and fungal communities forming biofilms on walls and ceilings in a 19th-century cheese ripening cellar in Poland, characterized by low temperature, high humidity, and minimal light - conditions resembling natural subterranean habitats. Using high-throughput 16 S rRNA and ITS sequencing, we revealed distinct taxonomic and predicted functional profiles associated with surface type (wall vs. ceiling) and material (brick vs. stone). The wall biofilms exhibited greater taxonomic and functional diversity, with enrichment in heterotrophic, fermentative, and polymer-degrading taxa and pathways, whereas ceiling biofilms showed predicted enrichment in aerobic, stress-tolerant, and potentially methanogenic lineages. The co-occurrence network analysis revealed more complex and tightly connected associations in wall biofilms, dominated by Actinobacteriota (21-97%) and Ascomycota (60-97%), suggesting stable ecological organization despite the limited sample size. Environmental factors, such as pH, redox potential, and electrolytical conductivity, explained a substantial proportion of the variance in the microbial diversity and predicted functional traits. Overall, this study highlights traditional ripening cellars as semi-natural built ecosystems that sustain specialized, spatially structured microbiomes. The results provide new insights into microbial adaptation, functional potential, and ecological resilience in heritage food environments.