Microbial Engineering and Process Modeling Toward the Development of Electromicrobial Production Systems

Biochemical engineering has long held potential for large-scale production of fuels, plastics, commodity chemicals, and other products. Biochemical processes hold numerous advantages over traditional petrochemical processes, including their chemical selectivity for complex molecules, low operating temperatures and pressures, ability to self-regenerate biocatalysts, and in particular, their theoretical carbon neutrality. Previous biochemical processes have faced difficulties for widespread industrial chemical production, however, due to their reliance on sugar-based substrates produced through the agricultural sector as well as overall economic considerations. Electromicrobial production (EMP) processes are next-generation biotechnologies that use electricity or electrochemically generated molecules as energy sources in the place of sugars for microbial product formation. In particular, mediator molecules such as hydrogen gas or formic acid, both of which can be produced electrochemically, can be metabolized by Knallgas bacteria and formatotrophs respectively and can provide the energy required to biochemically convert carbon dioxide to value-added products. Following a description of electromicrobial production and a summary of important previous work on these systems in Chapter 1, in this dissertation I describe work in process modeling and analysis as well as microbial engineering to evaluate and advance electromicrobial technologies. I describe my analytical work first (Chapters 2 and 3), wherein I use life cycle analysis and techno-economic assessment to evaluate EMP on a systems-level basis. In Chapter 2, I introduce a three-part framework, relying on first principles-based bioreactor modeling, process modeling, and life cycle assessment to examine the potential environmental impacts (namely global warming potential and land use) of three proposed EMP schemes. This framework allows me to compare these proposed EMP systems to each other as well as to a traditional glucose-based bioprocess. This analysis identified environmental hotspots of these EMP processes that should be addressed prior to large-scale deployment and established targets for various metrics such as product yield. In Chapter 3, I take a similar approach to develop a techno-economic model of a hypothetical EMP system that converts air-captured CO2 to the biofuel n-butanol. I use this model to identify specific economic bottlenecks that currently prevent viability of this specific process and demonstrate what conditions must be met in the path to the marketability of this system. In the experimental portion of this dissertation (Chapters 4 and 5), I describe efforts in engineering microbial strains with specific functions that can be used in electromicrobial processes, demonstrating the utility of two major strain development techniques: rational genetic engineering and adaptive laboratory evolution. The model Knallgas bacteria Cupriavidus necator, one of the primary strains studied in the field of EMP, is the major microbial chassis used in these chapters. In Chapter 4, I address the issue of bioseparations in EMP, focusing on the cell lysis step required to recover intracellular biomolecules. I developed a method to confer susceptibility to osmolysis, or cell lysis in distilled water, to bacteria used for intracellular biomolecule production. This method combines adaptive laboratory evolution to improve a strain’s halotolerance and rational knockout of mechanosensitive channel genes to confer this desired phenotype. Variations of this approach led to engineered strains of C. necator and Escherichia coli that undergo significant cell lysis in distilled water, demonstrating the method’s broad applicability. In Chapter 5, I engineer a strain of C. necator capable of n-butanol production and use adaptive laboratory evolution to improve the tolerance of C. necator to n-butanol. This work establishes a foundation for further engineering, enabling the development of a C. necator strain capable of producing the biofuel n-butanol from electrochemically produced substrates with high titers and yields. I then conclude this dissertation in Chapter 6 with a discussion of further academic research that should be pursued in the field of EMP and potential pathways for the real-world use of these systems in industrial settings. This dissertation combines process modeling and microbial engineering toward the development of electromicrobial production systems, with these two parallel efforts informing each other. This dissertation provides insight on key problems of EMP that must be solved prior to their practical deployment and details examples of how microbial engineering strategies can be used to address those problems. The content of this dissertation can guide research and development of electromicrobial technologies, from laboratory research to industrial adoption.

Advisor: Clark, Douglas S.