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

## TL;DR

Rock-attached microbes in groundwater ecosystems are more abundant and functionally distinct from planktonic microbes, playing a key role in subsurface biogeochemical processes.

## Contribution

This study reveals the distinct taxonomic and functional profiles of attached versus planktonic microbial communities in carbonate aquifers.

## Key findings

- Attached microbial communities are dominated by Proteobacteria and enriched in genes for biofilm formation and chemolithoautotrophy.
- Planktonic communities are dominated by Cand. Patescibacteria and Nitrospirota with lower functional versatility.
- Attached microbes likely contribute significantly to inorganic carbon sequestration in carbonate aquifers.

## Abstract

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.

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 from the same aquifer ecosystem. Microbial lifestyle (attached vs. planktonic) explained more variance in community composition than redox conditions, prompting us to further investigate its role in shaping functional and activity profiles. 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 a few genera were shared, and even closely related MAGs (> 90% average nucleotide identity) differed in assembly size and metabolic traits, demonstrating lifestyle-specific functional adaptation. Analysis of active replication indicated that the active fraction of the attached community was primarily represented by the most abundant MAGs. Planktonic communities featured a higher fraction of active MAGs compared to attached communities, but overall with lower relative abundances.

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 CO2 fixation rates of the planktonic communities alone. Our findings underscore the need to incorporate attached microbial communities into models of subsurface ecosystem function.

Video Abstract

Video Abstract

The online version contains supplementary material available at 10.1186/s40168-025-02325-1.