Introduction:

The Eger Rift in Western Bohemia (CZ) stands out as a unique subsurface ecosystem due to frequent tectonic activity, high CO₂ fluxes, and periodic seismic release of H₂. This environment offers a scientifically relevant opportunity to study microbial subsurface processes, particularly carbon cycling. Genomic analyses of drill core and formation water samples reveal a surprisingly diverse microbial community, including methanogenic archaea, acetogens, sulfate reducers, and cyanobacteria, suggesting Eger Rift microorganisms to employ a range of metabolic approaches to utilize CO₂.

Goal:

Our study aims to investigate native Eger Rift organisms by employing diverse culturing methods to enrich, isolate, and characterize potential CO₂ utilizers. The overarching objective is to identify and describe native microbial carboxylation reactions.

Methods:

We conducted anaerobic enrichments over six months under a high H₂/CO₂ atmosphere to enrich methanogenic archaea, and six-week aerobic enrichments focused on the growth of cyanobacteria under atmospheric conditions. Characterization involved amplicon and metagenomic ONT sequencing. Metagenome assembled genomes (MAGs) were reconstructed and functionally annotated. Ongoing isolation efforts using agar shakes (anaerobic) and streak plates (aerobic) are in progress.

Results:

Anaerobic enrichments yielded a circular Methanobacterium genome and a MAG of a novel species of hydrogenotrophic Methanosphaerula from 60 m deep Eger sediments. Cyanobacteria only grew in cultures inoculated with 118 m and 194 m deep sediments, while other aerobic microorganisms, including Arthrobacter, Polaromonas, and Rhizobium, could be enriched from different depth. Genomic and functional characterization revealed the metabolic potential of anaerobic (Wood Ljungdahl) and aerobic (CBB) CO₂ fixation, as well as H2 utilization pathways.

Summary:

Our ongoing cultivation work supports previous findings, suggesting that, in addition to methanogenic archaea, several aerobic microorganisms, including cyanobacteria, likely inhabit the Eger Rift subsurface and utilize naturally occurring CO₂.