(119c) Engineering an Environmentally-Isolated Strain of Bacillus megaterium for Biofuel Production and Recovery Under Supercritical CO2

Using a targeted bioprospecting approach by sampling fluid from a deep subsurface supercritical carbon dioxide (scCO₂) well, a strain of Bacillus megaterium was isolated that is rarely able to demonstrate consistent, robust growth in the presence of scCO₂. Due to the broad microbial lethality and solvent chemistry of scCO₂, we hypothesize that a dual-phase reactor of growth media and scCO₂ will simultaneously provide a sterile growth environment and the capacity to continuously strip off strain-produced biofuels (short chain alcohols) to alleviate product toxicity. Additionally, scCO₂ is a sustainable, labile solvent that can be separated from desired products through depressurization, leaving these products at high concentrations and providing advantages over other co-solvent extraction systems. We have developed a transformation protocol for our strain of B. megaterium and evaluated a xylose-inducible promoter under scCO₂, which was found to have similar expression compared to anaerobic cultures. We engineered the scCO₂ tolerant strain to produce isobutanol by introducing a two-enzyme (2-ketoisovalerate decarboxylase (KivD) and alcohol dehydrogenase (Adh)) pathway. Combining our recombinant biofuel strain with scCO₂ culturing, isobutanol production was observed for cultures under scCO2 as well as was extracted into the scCO₂ phase. Currently we are analyzing the transcriptome for our B. megaterium strain, which we expect will provide hypotheses for scCO₂ tolerance as well as aid in further metabolic engineering of this unique host. Additionally, we have developed an integrated process model for culturing and product extraction to analyze the energetics of producing and separating isobutanol using scCO₂. We have found process conditions that result in comparable if not better energetics than existing in situ extraction techniques such as gas stripping. Lastly, we are working on building a genome scale model of our strain of Bacillus to incorporate our transcriptome data, creating metabolic pathways to make fuels directly from common carbon substrates, and developing genomic integration/knockout protocols to enhance the metabolic engineering of this unique host.