Bioprocess development with Clostridium ljungdahlii based on metabolic modelling

Abstract

Bacterial synthesis gas (syngas) fermentation offers a promising solution for the reduction of greenhouse gas emissions - the greatest challenge of today’s society. The substrate gas, which mainly consists of CO2, CO, and H2, represents an inexpensive feedstock originating from agricultural, industrial, and municipal wastes. It can be metabolized to a multitude of valuable commodity chemicals and biofuels using different autotrophic bacteria. With syngas fermentation, fossil-based resources are replaced with the simultaneous diminution of the greenhouse gas CO2 and usage of the waste gas CO. In this regard, Clostridium ljungdahlii (C. ljungdahlii) is a good representative of gas-fermenting acetogens, as it is natively endowed to convert syngas components into acetic acid, ethanol, 2,3-butanediol (2,3-BD) and lactate. In addition, C. ljungdahlii is genetically accessible and, therefore, a promising platform for the recombinant formation of high-value products like isobutanol. The autotrophic central metabolism of C. ljungdahlii refers to the Wood-Ljungdahl pathway (WLP), an ancient and energy-limited reductive pathway that relies on a proton gradient for ATP conservation. The conversion of reducing equivalents within this pathway is essential for the establishing of the proton gradient needed for ATP formation, and also for product formation based on several reductive steps starting from CO2. The provision of crucial reducing equivalents depends on the oxidation of the electron source in the substrate gas - CO via carbon monoxide dehydrogenase (CODH) or H2 by a bifurcating hydrogenase (HYD). Hence, for the optimized formation of natural and non-natural reduced products, it is decisive to thoroughly understand the cellular link between energy management, growth, by-product formation, and the electron availability in the substrate gas. In the framework of this thesis, controlled bioreactor batch cultivations with continuous gas supply in 2 L scale were performed to study the growth and product formation of C. ljungdahlii in dependence on varying substrate compositions. In addition, a stoichiometric metabolic model was manually reconstructed for subsequent analysis of intracellular carbon fluxes, redox and energy metabolism using flux balance analysis. Subsequently, the heterologous syngas-based isobutanol production of C. ljungdahlii was investigated. Finally, with regard to the scale-up of syngas fermentations to commercial scales, possible performance losses during CO-based cultivation of C. ljungdhalii in a 125 m3 bubble column reactor were analysed using a kinetic correlation model.