Surveying and harnessing the genetic, (meta)genomic, and metabolic potential of the deep carbonated biosphere

Abstract

The interaction between microbes and supercritical (sc) carbon dioxide represents an increasingly compelling area of research due to use of scCO₂ in geologic carbon sequestration (GCS) and as a sustainable chemical solvent. To investigate the long-term effects of GCS on the in situ deep subsurface biosphere, I conducted a taxonomic, geochemical and metagenomic survey of the McElmo Dome sCCO₂ reservoir, which serves as a natural analog for GCS environments. Through 16S rRNA amplicon and metagenome sequencing, I identified Sulfurospirillum, Rhizobium, Desulfovibrio and members of the Clostridiales family associated with reservoir fluids. Annotations of complete genomes extracted from metagenomes predict diverse mechanisms for growth and nutrient cycling in deep subsurface sCCO₂ microbial ecosystems at McElmo Dome. Supercritical CO₂ is frequently used as a solvent for compound extraction and in vitro biocatalysis. However, due to its lethal effects, scCO₂ has previously been considered inaccessible for in vivo microbial bioproduct stripping. Utilizing a bioprospecting approach, I isolated strain Bacillus megaterium SR7 through enrichment culture and serial passaging of McElmo Dome scCO₂ reservoir fluids. I then initiated process improvements including media and culturing optimization under 1 atm CO₂ that increased SR7 growth frequency under scCO₂ . After developing a genetic system enabling inducible heterologous enzyme expression, scCO₂ incubations of SR7 transformed with a two-gene isobutanol biosynthesis pathway generated up to 93.5 mg/1 isobutanol. 5.2% of the total isobutanol was directly extracted by the scCO₂ headspace. This finding demonstrates for the first time the feasibility of active bioproduct synthesis and extraction in a single scCO₂-exposed bioreactor.