Engineered microbial biofuel production and recovery under supercritical carbon dioxide.

Culture contamination, end-product toxicity, and energy efficient product recovery are long-standing bioprocess challenges. To solve these problems, we propose a high-pressure fermentation strategy, coupled with in situ extraction using the abundant and renewable solvent supercritical carbon dioxide (scCO2), which is also known for its broad microbial lethality. Towards this goal, we report the domestication and engineering of a scCO2-tolerant strain of Bacillus megaterium, previously isolated from formation waters from the McElmo Dome CO2 field, to produce branched alcohols that have potential use as biofuels. After establishing induced-expression under scCO2, isobutanol production from 2-ketoisovalerate is observed with greater than 40% yield with co-produced isopentanol. Finally, we present a process model to compare the energy required for our process to other in situ extraction methods, such as gas stripping, finding scCO2 extraction to be potentially competitive, if not superior.

Isolation, Development, and Genomic Analysis of Bacillus megaterium SR7 for Growth and Metabolite Production Under Supercritical Carbon Dioxide.

Supercritical carbon dioxide (scCO2) is an attractive substitute for conventional organic solvents due to its unique transport and thermodynamic properties, its renewability and labile nature, and its high solubility for compounds such as alcohols, ketones, and aldehydes. However, biological systems that use scCO2 are mainly limited to in vitro processes due to its strong inhibition of cell viability and growth. To solve this problem, we used a bioprospecting approach to isolate a microbial strain with the natural ability to grow while exposed to scCO2. Enrichment culture and serial passaging of deep subsurface fluids from the McElmo Dome scCO2 reservoir in aqueous media under scCO2 headspace enabled the isolation of spore-forming strain Bacillus megaterium SR7. Sequencing and analysis of the complete 5.51 Mbp genome and physiological characterization revealed the capacity for facultative anaerobic metabolism, including fermentative growth on a diverse range of organic substrates. Supplementation of growth medium with L-alanine for chemical induction of spore germination significantly improved growth frequencies and biomass accumulation under scCO2 headspace. Detection of endogenous fermentative compounds in cultures grown under scCO2 represents the first observation of bioproduct generation and accumulation under this condition. Culturing development and metabolic characterization of B. megaterium SR7 represent initial advancements in the effort toward enabling exploitation of scCO2 as a sustainable solvent for in vivo bioprocessing.

Microbial potential for carbon and nutrient cycling in a geogenic supercritical carbon dioxide reservoir.

Microorganisms catalyze carbon cycling and biogeochemical reactions in the deep subsurface and thus may be expected to influence the fate of injected supercritical (sc) CO2 following geological carbon sequestration (GCS). We hypothesized that natural subsurface scCO2 reservoirs, which serve as analogs for the long-term fate of sequestered scCO2 , harbor a 'deep carbonated biosphere' with carbon cycling potential. We sampled subsurface fluids from scCO2 -water separators at a natural scCO2 reservoir at McElmo Dome, Colorado for analysis of 16S rRNA gene diversity and metagenome content. Sequence annotations indicated dominance of Sulfurospirillum, Rhizobium, Desulfovibrio and four members of the Clostridiales family. Genomes extracted from metagenomes using homology and compositional approaches revealed diverse mechanisms for growth and nutrient cycling, including pathways for CO2 and N2 fixation, anaerobic respiration, sulfur oxidation, fermentation and potential for metabolic syntrophy. Differences in biogeochemical potential between two production well communities were consistent with differences in fluid chemical profiles, suggesting a potential link between microbial activity and geochemistry. The existence of a microbial ecosystem associated with the McElmo Dome scCO2 reservoir indicates that potential impacts of the deep biosphere on CO2 fate and transport should be taken into consideration as a component of GCS planning and modelling.

Microbial growth under supercritical CO2.

Growth of microorganisms in environments containing CO2 above its critical point is unexpected due to a combination of deleterious effects, including cytoplasmic acidification and membrane destabilization. Thus, supercritical CO2 (scCO2) is generally regarded as a sterilizing agent. We report isolation of bacteria from three sites targeted for geologic carbon dioxide sequestration (GCS) that are capable of growth in pressurized bioreactors containing scCO2. Analysis of 16S rRNA genes from scCO2 enrichment cultures revealed microbial assemblages of varied complexity, including representatives of the genus Bacillus. Propagation of enrichment cultures under scCO2 headspace led to isolation of six strains corresponding to Bacillus cereus, Bacillus subterraneus, Bacillus amyloliquefaciens, Bacillus safensis, and Bacillus megaterium. Isolates are spore-forming, facultative anaerobes and capable of germination and growth under an scCO2 headspace. In addition to these isolates, several Bacillus type strains grew under scCO2, suggesting that this may be a shared feature of spore-forming Bacillus spp. Our results provide direct evidence of microbial activity at the interface between scCO2 and an aqueous phase. Since microbial activity can influence the key mechanisms for permanent storage of sequestered CO2 (i.e., structural, residual, solubility, and mineral trapping), our work suggests that during GCS microorganisms may grow and catalyze biological reactions that influence the fate and transport of CO2 in the deep subsurface.