Resonance Raman spectroscopy of NADH, NAD+ , ferredoxin and cytochrome c in Sporomusa ovata and Clostridium carboxidivorans for microbial electrosynthesis applications

Microbial electrosynthesis (MES) offers a sustainable alternative for the production of platform chemicals from CO 2 . Label-free Raman spectroscopy can provide direct insights into biomolecular changes in MES, reflecting metabolic activity and production. In this study defined co-cultures of electro-active bacteria, i.e. Sporomusa ovata and Clostridium carboxidivorans, were investigated by confocal resonance Raman micro-spectroscopy tuned to 532 nm to gain insights into the microbial processes of biomarkers involved in the Wood-Ljundahl pathway. The results were correlated to high-performance liquid chromatography (HPCL) and optical density measurements regarding the production rate of acetate, butyrate, ethanol and butanol. Pre-processed difference Raman spectra of co-cultures from S. ovata and C. carboxidivorans at ratios 1:10, 10:1, and 1:1 were compared to monocultures on day 1 and 2, revealing substantial variations in Raman intensity and thus the relative metabolic activity of NADH, NAD + , ferredoxin and cytochrome c. Such information may point to high metabolic activity at the start of acetate, butyrate, ethanol, and butanol production followed by a steady state at day four. This was verified by Raman difference spectra between fresh cultures and > 4 days old cultures, indicating a similar degree of metabolic activity after a certain time during ongoing production. Interestingly, the Raman spectra did not reveal any differences in metabolism depending on feedstocks, i.e. CO 2 and H 2 versus betaine or different ratios of co-cultures that were significantly more productive. This qualitative study demonstrates that resonance Raman spectroscopy is a viable tool for metabolic investigations of microbial electrosynthesis systems with potential for in situ investigations.