E.coli strain WM04 was determined as a viable option for Bacteria Directed Enzyme Prodrug Therapy. Determination of suitability for animal testing depends on sufficient quantities of bacteria being produced through fermentation. Optimal operating conditions are found for the production of WM04 by observing two important production variables: the quantity of bacteria produced and the capacity for survival of the bacteria.
One limitation to bacterial growth is the amount of oxygen supply. The concentration of dissolved oxygen and oxygen transfer rates are correlated to cell growth. Impellor velocity, ranging from 200-1000 rpm, allowed us to determine the optimal speed at which the greatest oxygen transfer rate was achieved in the bioreactor solution with minimal bacterial shearing.
The dissolved oxygen content of the media in the bioreactor was measured with an oxygen probe at varying impellor speeds. BioCommand Lite software was used to plot the dissolved oxygen concentration against the time of the run and the impellor speed versus time.
[...] Introduction The E.coli strain WM04 has been bioengineered for Bacterial Directed Enzyme Prodrug Therapy for laboratory testing. Preliminary testes for WM04 production were conducted in shake flasks; however we want to use a bioreactor to carry out larger-scale growth. WM04 was altered to produce an antibody on its surface, which recognizes a tumor antigen. This membrane modification increases the sensitivity of the bacterial membrane to shearing at high impellor speeds. We want to determine the optimal operating conditions to minimize negative effects of shearing while still producing sufficient amounts of bacteria for use in animal testing. [...]
[...] A number of standard curves for determining cell concentration were generated by measuring the optical density and counting plated colonies of dilute bacterial concentrations. An initial concentration of 1x108 cells/mL was assumed for the overnight E. coli culture stock5. Figure 3 shows the relationship between the dilution factor of the stock culture and the optical density of the corresponding samples. Figure 3. Relationship between dilution factor of samples and optical density. With very dilute solutions, the absorbance plateaus as the dilution factor increases. [...]
[...] A low initial concentration of E.coli was selected for the fermentation in order to maximize the observed exponential growth curve. Low concentration also enabled us to measure the optical density of the bacteria. The optimal agitation speed must be experimentally established based on conditions specific to the reactor3. When considering the supply of oxygen for fermentation it is necessary to look at the respiration stoichiometry. C6H12O6 + 6O2 6H2O + 6CO2 Equation 2 shows that for every one mole of glucose six moles of oxygen are necessary. [...]
[...] Depending on the allowed fermentation time, a lower OD600nm might be selected for a longer operation time, since bacteria would generate during this time interval. Using the optical density readings obtained during the fermentation run, the cell concentrations in the bioreactor were measured at 10 minute time intervals at each impellor speed. Based on generation time and initial cell concentrations of each run, the expected cell population was determined and graphed with the actual cell population in Figure 8 for comparison. [...]
[...] The bioreactor's capacity for aeration can be described by the volumetric liquid mass transfer coefficient (KLa). The KLa is the product of the mass transfer coefficient and gas/liquid interface area per liquid volume. Aeration capacity is directly proportional to the KLa value since a high KLa indicates low resistance to oxygen transfer from the gas to liquid phases as well as a large transfer interface. The agitation rate and impellor design determine the KLa value of the reactor and have no impact on the dissolved oxygen concentration. [...]
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