On-line analysis of bacterial metabolism by modern spectroscopic laser techniques

In order to best determine how bioprocesses develop and how to run bioreactors most efficiently, innovative new analytical techniques are required to supplement, or even supersede, conventional methods, many of which are invasive, require sampling and/or do not provide on-line data analysis. Spectroscopic laser techniques are powerful analytical tools that are capable of real-time, non-invasive monitoring of multiple variables simultaneously. Furthermore, spectroscopy offers high selectivity and sensitivity, including the ability to distinguish different isotopomers and isotopologues, which enables isotopic labelling studies to provide greater mechanistic insights into metabolic pathways.

This thesis describes the development of several spectroscopic techniques and their
applications in studying different metabolic modes of Escherichia coli batch cultures. On-line
analysis is achieved in the gas-phase using cavity-enhanced Raman spectroscopy (CERS),
White cell FTIR spectroscopy and photoacoustic detection in a differential Helmholtz resonator (DHR) as well as in the liquid-phase using Raman spectroscopy. The spectral analysis and quantitation of over twenty parameters is discussed, including growth substrates such as glucose and ammonia, metabolites such as acetate, ethanol and formate, headspace gases such as H2, O2 and CO2, and other process variables measured in situ such as the pH and optical density (OD).

The first bacterial study conducted is a revisitation of the classical E. coli experiment
of glucose-lactose diauxie. A new approach for studying mixed sugar metabolism is presented using both the CERS and DHR techniques to distinguish 13CO2 produced from 13C-glucose metabolism from the subsequent production of 12CO2 from unlabelled lactose. Next, gas-phase FTIR and liquid-phase Raman are developed for batch culture analysis and applied to monitoring mixed-acid fermentation. Finally, two further isotopic labelling studies are conducted by using CERS alongside FTIR and liquid-phase Raman analysis. Nitrate and nitrite reduction by E. coli to the major and minor end-products of ammonium and nitrous oxide is studied, respectively. 15N-labelling is used to give mechanistic insights through interpretation of the different 14N/15N-isotopomer products. The final study focuses on the fermentative pathways of E. coli in the absence and presence of O2. Using 13C- and D-labelled formate, evidence is found that the formate hydrogenlyase (FHL) complex can be assembled and functional under micro-aerobic conditions, which could remove some barriers to biotechnological applications such as biohydrogen generation.