Two worlds beneath: Distinct microbial strategies of the rock-attached and planktonic subsurface biosphere

Background Microorganisms in groundwater ecosystems exist either as planktonic cells or as attached communities on aquifer rock surfaces. Attached cells outnumber planktonic ones by at least three orders of magnitude, suggesting a critical role in aquifer ecosystem function. However, particularly in consolidated carbonate aquifers, where research has predominantly focused on planktonic microbes, the metabolic potential and ecological roles of attached communities remain poorly understood. Results To investigate the differences between attached and planktonic communities, we sampled the attached microbiome from passive samplers filled with crushed carbonate rock exposed to oxic and anoxic groundwater in the Hainich Critical Zone Exploratory and compared it to a previously published, extensive dataset of planktonic communities. Microbial lifestyle (attached vs. planktonic) emerged as the strongest determinant of community composition, explaining more variance than redox conditions. Metagenomic analysis revealed a striking taxonomic and functional segregation: the 605 metagenome-assembled genomes (MAGs) from attached communities were dominated by Proteobacteria (358 MAGs) and were enriched in genes for biofilm formation, chemolithoautotrophy, and redox cycling (e.g., iron and sulfur metabolism). In contrast, the 891 MAGs from planktonic communities were dominated by Cand. Patescibacteria (464 MAGs) and Nitrospirota (60 MAGs) and showed lower functional versatility. Only 7% of genera were shared, and even closely related MAGs (> 90% ANI) differed in genome size and metabolic traits, demonstrating lifestyle-specific functional adaptation. Analysis of active replication indicated that the active fraction of the attached community was primarily shaped by the most abundant MAGs. Planktonic communities featured more active MAGs, but overall with lower abundances. Conclusions The high abundance, metabolic specialization, and carbon fixation potential of attached microbes suggest that they are key drivers of subsurface biogeochemical processes. Carbonate aquifers may act as much larger inorganic carbon sinks than previously estimated based on CO ₂ fixation rates of the planktonic communities alone. Our findings underscore the need to incorporate attached microbial communities into models of subsurface ecosystem function.