Characterization and optimization of 1-hexanol production in CO-utilizing clostridia

Kottenhahn, Patrick; Blank, Lars M. (Thesis advisor); Büchs, Jochen (Thesis advisor)

Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022

Abstract

The goal of this thesis is the optimization of hexanol production from syngas. Hexanol can be used i.e.for the production of plastics, fuels and additives. Two potential biocatalysts, C. carboxidivorans and C. ljungdahlii, were characterized and evaluated for this application.These are the most important results obtained in this study: Bioenergetic calculations revealed that hexanol could be produced as the sole fermentation product from syngas. Hexanol toxicity was identified as a crucial limiting factor in hexanol production and could be circumvented by product extraction, increasing yields ~2.5 fold to 24 mM hexanol. The overall toxicity of hexanol towards both tested species was similar with acute inhibition at ≤ 20 mM. The hexanol tolerance of C. carboxidivorans was higher at 30°C (IC50 17.5 mM) than at 37°C (IC50 11.8 mM) and extraction led to detoxification only at the lower temperature. During scale-up from bottles to CSTR, gas delivery, measured as dissolved CO tension, and different nutrient requirements were identified as important bottlenecks. The highest hexanol titer produced in the CSTR was 37 mM. This is ~3 times the currently highest titer reported in other studies. To improve heterologous hexanol production with C.ljungdahlii, different enzymes were analyzed in transgenic C. ljungdahlii strains but the produced hexanol titers still remained lower than the titers obtained with C. carboxidivorans. In conjugation experiments with C. carboxidivorans, colonies could only be obtained either after heat shock of the recipient or after use of a donor strain expressing the C. carboxidivorans methyl transferases. These colonies displayed resistance towards the selection markers, but no verifiable liquid cultures could beobtained. The methylation pattern of C. carboxidivorans was investigated and the discovered motifs were attributed to respective methyl transferases. Converter gas (CG) was compared to synthetic CG with the same composition to investigate toxic effects of minor gas components. Without dilution of the gas and low cell densities, no growth was observed. High cell densities could partially circumvent inhibition and final product- and growth yields were similar with real and synthetic CG. If the gas phase was renewed, growth ceased even at high cell densities. No adaptation towards CG was observed. Compared to synthetic CG, hexanol production and growth yields were reduced with only H2+CO2 as substrate. Screening of different media compositions revealed improvement through addition of 2 g L-1 carbonate and a lower pH. In conclusion, C. carboxidivorans is a promising candidate for hexanol production from syngas. The hexanol titers obtained in this study of 37 mM (3.8 g L-1) is higher than titers reported for transgenic biocatalysts as well as co-cultivations. The major drawback is the lack of reliable methods for genetic manipulation. By addressing the crucial points identified in this work, hexanol production from syngas could be developed into a commercial process that can decrease net carbon emissions while simultaneously reducing dependence on fossil carbon sources. This is a step towards a greener andmore sustainable economy.

Institutions

• Department of Biology [160000]
• Chair of Applied Microbiology [161710]