![]() It has been the principle organism used for the study of polyhydroxybutyrate (PHB) polymer biosynthesis. Ralstonia eutropha (also known as Cupriavidus necator) is a Gram-negative, facultatively chemolithoautotrophic bacteria. This 105 page technical report summarizing the three years of research includes 72 figures and 11 tables of findings. This final technical report presents the findings of both the biological engineering work at MIT that extended the native branched-chain amino acid pathway of the wild type Ralstonia eutropha H16 to perform this biosynthesis, as well as the unique design, modeling, and construction of more » a bioreactor for incompatible gasses at Michigan State that enabled the operational testing of the complete system. The goal of the project is to produce Isobutanol (IBT), a branched-chain alcohol that can serve as a drop-in transportation fuel, through the engineered microbial biosynthesis of Carbon Dioxide, Hydrogen, and Oxygen using a novel bioreactor. ![]() This research project is a collaboration between the Sinskey laboratory at MIT and the Worden laboratory at Michigan State University. ![]() 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 scCO 2 extraction to be potentially competitive, if not superior. After establishing induced-expression under scCO 2, isobutanol production from 2-ketoisovalerate is observed with greater than 40% yield more » with co-produced isopentanol. Towards this goal, we report the domestication and engineering of a scCO 2-tolerant strain of Bacillus megaterium, previously isolated from formation waters from the McElmo Dome CO 2 field, to produce branched alcohols that have potential use as biofuels. 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 (scCO 2), which is also known for its broad microbial lethality. megaterium (Aims 1 & 2) 3) conducting de novo pathway engineering for biosynthesis of longer chain fuels (Aim 2) and 4) developing and modeling a two-phase stripping chemostat for continuous production of biofuels with in situ scCO2 extraction (Aim = ,Ĭulture contamination, end-product toxicity, and energy efficient product recovery are long-standing bioprocess challenges. megaterium during growth under scCO2 (Aim 1) 2) engineering heterologous butanol production pathways for anaerobic and aerobic expression in B. ![]() To this end we pursued a series of Aims and Sub-Aims with the ultimate goals of 1) determining the systems physiology of B. We proposed an interdisciplinary research and development effort involving bioprocess engineering, systems biology, and chemical separation engineering, centered around a recently isolated microorganism that is tolerant to scCO2. We aimed to develop a system that utilizes supercritical CO2 (scCO2) to address challenges associated with microbial contamination, product toxicity, and product recovery that currently limit the potential of advanced biofuel and bio-based chemical production. The goal of our project was to develop Bacillus megaterium as a host for continuous biofuel production coupled with in situ product extraction by supercritical (sc) CO2 stripping.
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