GROWTH DYNAMICS AND CARBON-SUBSTRATE OXIDATION AND INCORPORATION PATTERNS OF METHYLOMONAS L3 (METHYLOTROPHS, CARBON METABOLISMS, DYNAMIC BEHAVIOR, GROWTH MODEL, MIXED-SUBSTRATE CULTURES)
Doctor of Philosophy
The Ribulose Monophosphate-type (RuMP), obligate methanol-utilizing bacterium Methylomonas L3 has been used in the study of methylotrophic metabolism and fermentation dynamics. The dynamic behavior of M. L3 in continuous cultures was studied in order to understand the regulatory mechanisms of cell growth under various transient situations. Transients generated by dilution-rate shifts and methanol-pulse additions were employed. The methanol-uptake rate (MUR) profiles of most experiments displayed the unusual phenomenon of showing negative MUR values for a time period following the methanol pulse. This phenomenon suggested the existence of a methanol active transport system. In single-substrate cultures, the specific growth rate decreased immediately after the methanol addition. In mixed-substrate cultures (with formaldehyde as methanol co-substrate), the specific growth rate increased after the methanol addition. The biomass yield decreased after the methanol addition in all experiments; however, the drop was more severe in the single-substrate experiments. In dilution-rate shift experiments, overshoot and damped oscillatory behavior of the specific growth rate was observed. Radioactive labels were employed to elucidate the carbon substrate oxidation and incorporation patterns of strain L3. A thorough mathematical derivation of the proposed procedure was presented. ('14)C-labeled methanol, formate and 1-('14)C-glucose were used as tracers in a series of experiments with batch-grown, exponential-phase cells with different initial levels of substrate(s). From the distribution of the radioactivities in biomass and carbon dioxide, the rates of the carbon substrate oxidation reactions, the rates of the general decarboxylation and carboxylation reactions, as well as the rate of the direct biomass incorporation were obtained. The results showed that the two known oxidation pathways were operating in vivo, and that the extent of utilization of each pathway depended on the initial substrate(s) concentrations as well as on the state of the inoculum. An unstructured growth model, incorporating the basic Monod formulation with substrate-inhibition and the maintenance energy concept, was constructed based on steady-state data. The ability of this model to predict the transient cell behavior was tested by comparison to the transient data obtained. The model performed better in predicting transient behavior due to dilution-rate shifts. For methanol-pulse transients, the model performed better at higher dilution rates.