Optimising the Wider Energy Metabolome of Microbes Using Targeted UHPLC-MS/MS Present in Bio-Electrochemical Systems for Carbon Dioxide Conversion to Platform Chemicals

This thesis addresses a major analytical gap in microbial biotechnology by developing and validating a rapid, sensitive UHPLC-MS/MS platform for simultaneous quantification of the key intracellular energy metabolites that govern redox balance, energy charge, and carbon flux in industrial microorganisms. The method was designed to improve and optimise quenching, extraction, and analysis of yeast and bacterial cultures, enabling robust measurement of NAD+, NADH, NADP+, NADPH, FAD, AMP, ADP, and ATP in a single analytical run using Zwitterionic Hydrophilic Interaction Chromatography (Z-HILIC) coupled to tandem mass spectrometry. The resulting workflow offers high sensitivity, strong reproducibility, and low limits of detection and quantification, making it suitable for the analysis of low-biomass microbial samples and dynamic physiological transitions.<br/><br/>The importance of this method was demonstrated through its application to Saccharomyces cerevisiae and Escherichia coli, where it was used to investigate how environmental conditions and targeted metabolite supplementation influence intracellular redox state and platform chemical production. Application of the method to S. cerevisiae and E. coli showed that timed environmental and nutritional interventions can significantly reshape redox balance and energy status, leading to improved ethanol and lactate production without genetic modification. <br/><br/>In S. cerevisiae, aerobic and anaerobic growth conditions, oxygen-shift experiments, and AMP supplementation were used to examine their effects on ethanol production, showing that timed AMP addition at the onset of stationary phase substantially increased ethanol output without genetic modification. In E. coli, aerobic and anaerobic conditions, oxygen-shift experiments, and dextrose spiking were used to assess lactate formation, demonstrating that timed dextrose addition during oxygen transition increased lactic acid production.<br/><br/>Overall, this work shows that quantitative energy metabolomics can do more than describe microbial physiology, it can identify actionable process interventions that improve product formation in a non-genetic manner. The thesis therefore provides both a validated analytical platform and new biological insight into how energy and redox metabolism can be manipulated to enhance industrial bioprocesses. These findings are relevant to biorefinery development, where improving carbon conversion efficiency, reducing by-product formation, and maximising platform chemical yields are central goals. The approach also has broader potential in microbial biotechnology, biofuel production, and bio-electrochemical systems where intracellular cofactor dynamics influence product formation.

Name dc:type.qualificationname

Doctoral Thesis

Level dc:type.qualificationlevel

Student thesis

Year dc:date.issued

2026

Author dc:creator

• Oliver, Jordan

Advisors dc:contributor.advisor

• Dinsdale, Richard
• Davies, Antony

Language dc:language

eng

Identifier

oai:pure.atira.dk:studenttheses/77786460-92a5-41bb-8c21-d54efcd6ed6c

OAI identifier oai:identifier

oai:pure.atira.dk:studenttheses/77786460-92a5-41bb-8c21-d54efcd6ed6c