In vivo phosphorus-31 and carbon-13 nuclear magnetic resonance spectroscopy to study cellular metabolism
Mann, Brenda Kathleen
Shanks, Jacqueline V.
Doctor of Philosophy
Nuclear magnetic resonance spectroscopy has emerged as a valuable tool for engineers for noninvasive, real-time studies of cellular metabolism. Two applications of NMR for studying cellular metabolism were investigated in this work: 31 P NMR for studying yeast in a bioreactor and 13 C NMR for studying bioremediation. One acute challenge in NMR bioreactor design has been supplying enough oxygen for cell respiration in a suspension that contains sufficient cells for NMR signal detection. The use of cytoplasmic pH as an intracellular marker of adequate oxygenation was evaluated from 31 P NMR spectra of two strains of the yeast Saccharomyces cerevisiae at several cell densities, ranging from low (0.9% (v/v)) to very high (45% (v/v)) cell densities, in an airlift bioreactor. 31 P NMR spectra were obtained for derepressed yeast cells prior to, and during, glycolysis under non-growth conditions. During endogenous respiration, pH cyt can be used as an intracellular marker for aeration for both strains and cell densities up to 18% (v/v). During glycolysis, pH cyt values were similar under aerobic and anaerobic conditions for one strain, but were different for the other; hence not all strains behave similarly to environmental changes, and the use of pH cyt during glycolysis as a marker is questionable. While studies in bioremediation are now common, they are generally invasive and may not reflect intracellular conditions accurately. The challenge here was to apply 13 C NMR to a bioremediation problem. 13 C NMR spectra were taken of suspensions of Pseudomonas putida 27G utilizing 13 C-labeled toluene in both in vitro and in vivo studies. Degradation was tentatively determined to follow the TOL pathway through catechol. The degradation conformed to Michaelis-Menten kinetics over a toluene concentration range of 0.1% to 1.0%. Cell phase at harvest had no effect on the degradation. Suspensions bubbled with oxygen produced carbon dioxide faster than those with air. Suspensions bubbled with nitrogen removed toluene at a slower rate than air or oxygen-supplied cells, but no intermediates were found. The airlift reactor used with the yeast was modified and tested with the bacteria. Toluene volatility and foaming were both severe problems that must be overcome before the reactor can be used for bioremediation of an volatile compound.