Various methods of establishing a persistent mixed culture were examined in continuous culture. A well-defined system consisting of Esherichia coli and Saccharomyces cerevisiae was used. The primary interaction was competition for glucose, the rate limiting nutrient. When this was the only interaction no coexistence is possible in a well mixed fermentor at constant conditions (i.e., a chemostat).
The two microorganisms while competing for glucose were maintained in a stable cycle of coexistence by alternating the growth advantage between the two organisms by oscillating the pH in a chemostat. Pure culture experiments found S. cerevisiae to be insensitve to pH between 5 and 4.3 with a maximum specific growth rate (Umax) of 0.4/hr; while Umax of E. coli decreased from 0.6/hr at pH 5 to 0.1/hr at pH 4.3. Steady state and crossinoculation chemostat runs at a dilution rate of 0.17/hr confirmed the expectation that the mixed culture system is unstable at constant pH with E. coli dominating at pH 5 and S. cerevisiae dominating at pH 4.3. Three pH oscillation experiments were performed at D=0.17/hr with 1 gm/1 glucose feed. The 16 hr/16 hr cycle was stable for six periods with a stable alternating cycle of E. coli and S. cerevisiae being quickly established. A 18 hr pH5./14 hr pH4.3 cycle was found to be stable with smaller yeast concentrations. A 6hr/6hr cycle was found unstable with yeast washout. Simulation results were compared with these runs and were used to predict the onset of instability. Oscillations of pH can force stable persistence of a competing mixed culture that is otherwise unstable. Thus time varying conditions are experimentally demonstrated to be one explanation for competitive coexistence.
A mixed culture of Saccharomyces cerevisiae and Esherichia coli was established in a stable coexistence steady state in a chemostat under constant operating conditions at higher feed concentrations. The species competed for glucose, the growth limiting resource, and produced acetate and ethanol. The acetic acid was shown to be very inhibitory to E. coli in pure culture at pH 5 while ethanol inhibition was only marginal. No significant inhibition of S. cerevisiae growth was observed by either acetate or ethanol. Pure culture paramenters were measured and used in the analysis. Linearized stabilty analysis for the case when both organisms produce the inhibitor showed that a transition through three stable outcomes was possible as the feed concentration is lowered. Experimental studies verified these predictions and successive transitions from a yeast growth steady state, to a coexistence steady state, and to a E. coli growth steady state were obtained by lowering the glucose concentration in the feed from 10 to 5 to 2.5 g/l. This dynamic behavior is distinctly different form other competition-inhibition combinations and demostrates for the first time that coexistence is possible due to substrate competition and product inhibition.
A bioreactor with simultaneous fermentation and cell recycle was investigated. The reactor consisted of a typical fermentor and an attached inclined side-arm that allowed enhanced sedimentation. Due to the enhanced sedimentation in the side-arm settler the cells precipitate quickly and flow back into the reactor. A virtually cell free broth can be withdrawn through the side-arm while maintaining both a high flowrate and a high cell density. Continuous fermentations with S. cerevisiae demonstrated these features and the possibility of high cell densities at flowrates that ordinarially would lead to washout. Ethanol productivities and yields were high. Increased resistance to contamination was feasible and tested using E. coli as the model contaminant.
This new reactor with size-selective properties was found to allow a coexistent mixed culture of Esherichia coli and Saccharomyces cerevisiae. The larger yeast population was retained and recycled at high efficiencies, while the smaller yet faster growing bacteria were removed preferentially through the side-arm. Stability analysis indicated that the coexistence of this system could be stable only if the yeast removal rate as a function of biomass was concave up. This would occur if growth continued in the side-arm. Another experimental system was devised to measure this removal rate function. A negative removal rate (i.e., a net addition of yeast to the fermentor) was observed at low biomass indicating growth in the settler and explaining the stability of the coexistence steady state.
A mixed culture, that was unstable during pure competition under constant well-mixed conditions as expressed in the Competitive Exclusion Principle, was made to persist indefinitely by the use of time-varying conditions (such as pH oscillations), or the addition of other interactions (such as inhibition), or the design of spatially nonuniform reactors (such as the side-arm settler).
