Abstract
The crustal subseafloor is the least explored and largest biome on Earth. Interrogating crustal life is difficult due to habitat inaccessibility, low-biomass and contamination challenges. Subseafloor observatories have facilitated the study of planktonic life in crustal aquifers, however, studies of life in crust-attached biofilms are rare. Here, we investigate biofilms grown on various minerals at different temperatures over 1-6 years at subseafloor observatories in the Eastern Pacific. To mitigate potential sequence contamination, we developed a new bioinformatics tool - TaxonSluice. We explore ecological factors driving community structure and potential function of biofilms by comparing our sequence data to previous amplicon and metagenomic surveys of this habitat. We reveal that biofilm community structure is driven by temperature rather than minerology, and that rare planktonic lineages colonize the crustal biofilms. Based on 16S rRNA gene overlap, we partition metagenome assembled genomes into planktonic and biofilm fractions and suggest that there are functional differences between these community types, emphasizing the need to separately examine each to accurately describe subseafloor microbe-rock-fluid processes. Lastly, we report that some rare lineages present in our warm and anoxic study site are also found in cold and oxic crustal fluids in the Mid-Atlantic Ridge, suggesting global crustal biogeography patterns.