ILLUMINATING MICROBIAL DARK MATTER IN MEROMICTIC SAKINAW LAKE

Advances in genomic sequencing started to uncover the metabolic potential of many uncultivated candidate divisions, aka Microbial Dark Matter (MDM), however, fundamental questions regarding their population structure, interactions, and biogeochemical roles in the environment remain. Multiple molecular surveys have indicated that anoxic, sulfidic, and/or methane-rich ecosystems tend to be naturally enriched in MDM. Here we use a cultivation independent approach to illuminate diversity, abundance, and co-occurrence patterns of MDM in the permanently stratified waters of meromictic Sakinaw Lake, Canada. The Sakinaw Lake water column partitions into oxic, fresh surface waters referred to as the mixolimnion, a redox transition zone (RTZ), and anoxic bottom waters rich in hydrogen sulfide and methane referred to as the monimolimnion. Samples were taken from 12 depth intervals spanning the water column, and DNA extracts were processed for small subunit ribosomal RNA gene amplification and pyrosequencing using universal primers targeting domains Bacteria, Archaea and Eukarya. The microbial population structure revealed pronounced trends. Eukaryotic sequences vanish almost completely below the mixolimnion, followed by a rapid and sustained increase in unassigned Archaeal sequences and Bacterial candidate divisions within and below the RTZ, comprising up to 40% of the microbial community. Co-occurrence network analysis revealed highly correlated and depth-dependent assemblages between abundant OTUs affiliated with bacterial candidate divisions (WWE1, JS-1/OP9, OP8, and OD1), unassigned Archaea, Chloroflexi, and methanogenic Archaea affiliated with Methanomicrobiales and Methanosarcinales. Moreover, metabolic reconstruction focused on the Wood-Ljungdahl pathway using published Sakinaw Lake OP8 and OP9/JS-1 single-cell amplified genome sequences revealed complete pathway coverage in both candidate divisions. It was proposed that the Wood-Ljungdal cycle is run in reverse during syntrophic acetate oxidation (SAO), leading to the release of two CO₂ molecules and the transfer of four H₂ molecules to methanogens. Given that OP8 and OP9/JS1 have been recovered from various aquatic environments, SAO might contribute significantly to global greenhouse gas production and emission.