Vibrational Spectroscopic Applications of Fourier Transform Infrared and Raman Spectroscopy in Biochemistry and Microbiology

Vibrational spectroscopy has a number of applications due to its high sensitivity and selectivity, including the distinction of isotopomers, making it a powerful analytical technique. Unlike typical chromatographic methods that require sampling, vibrational spectroscopic methods give in situ data acquisition in a closed system, with high time resolution. These advantages make vibrational spectroscopy suitable for monitoring rapidly developing biological processes. A number of applications of both infrared and Raman spectroscopies are presented in this thesis, including the monitoring of metabolites produced and consumed by microbiological cultures and volatiles released during fruit ripening. A home-built 2.0 m pathlength multiple-pass absorption White cell was constructed, characterised and employed for long-path Fourier Transform Infrared (FTIR) Spectroscopy. CO2, acetaldehyde and ethanol production were monitored by FTIR spectroscopy during both Escherichia coli growth and banana ripening. Other volatiles, such as ethyl acetate and the ripening hormone ethylene, were also monitored during fruit ripening. Cavity Enhanced Raman Spectroscopy (CERS) was used to monitor the consumption of IR inactive O2 by bacterial cultures and bananas transitioning from aerobic respiration to fermentation. H2 production was also monitored using CERS for anaerobic cultures of E. coli. Alongside FTIR and CERS, TMAO reduction by E. coli cultures was monitored using in situ optical density measurements and liquid phase Raman spectroscopy to monitor TMAO, glucose, acetate, formate and monobasic and dibasic phosphate. Phosphate anion concentration could be used to calculate the pH of the growth medium using a modified Henderson Hasselbalch equation. Deuterated formate (formate-d) could be distinguished from unlabelled formate allowing the monitoring of exogenous formate metabolism and the link to H2 production in the presence of TMAO. FTIR and CERS are proven to be cost-effective, highly specific analytical methods in biochemistry and bioscience, complementing and in some cases superseding existing conventional techniques. They also offer new possibilities for mechanistic insights in biochemistry and display a significant promise for measuring isotopically labelled bioassays.