# Exploring microbial diversity using cell-size fractionated enrichment incubations from subsurface aquifers at Äspö, Sweden

## TL;DR

This study shows that subsurface groundwater in Äspö, Sweden, hosts diverse small-celled microbes that form strong co-occurrence networks to survive in low-energy conditions.

## Contribution

The study introduces cell-size fractionated enrichment incubations to reveal previously underexplored microbial diversity in subsurface aquifers.

## Key findings

- Fractionated incubations enriched for small-genome microbes like Patescibacteria, Nanobdellota, and Omnitrophota.
- High microbial diversity was observed in fractionated incubations, but community structure remained stable over four months.
- Network analysis showed strong co-occurrences between Patescibacteria and Desulfobacterota populations in groundwater.

## Abstract

The continental subsurface hosts energy-constrained groundwaters with a high diversity of ecologically elusive microorganisms adapted to the prevailing low-energy conditions. This study explored potential interactions among microbes using anaerobic enrichment incubations with three types of groundwater of contrasting hydrochemistry from the Äspö Hard Rock Laboratory, Sweden. Removing cells larger than 0.45 µm from the inoculum resulted in incubations enriched in populations characterized by very small genomes, including Patescibacteria, Nanobdellota, and Omnitrophota. These incubations had a higher diversity than non-fractionated incubations. However, cell numbers and community structure of the fractionated incubations did not change over an incubation period up to four months, despite high microbial diversity and experimental amendments with either simple (acetate) or more complex (cell lysate) carbon sources. In addition, network analysis on the groundwaters revealed multiple co-occurrences between populations affiliated with the Patescibacteria and the Desulfobacterota. Overall, these findings support that a considerable part of microbial diversity has a small cell size in these low energy groundwaters and strong co-occurrences among populations as an important survival strategy.

Enrichment incubations of subsurface groundwaters (Äspö, Sweden) reveal a highly diverse microbial community dominated by small cells, with strong co-occurrences among populations that may support survival in this low-energy environment.