Rapid Growth in a pH Auxostat

Population selection is remarkable at high dilution rates because effects of small differences between growth rate and wash-out are magnified. As the cells call for increased pumping of nutrients to hold the concentration constant, there is an accompanying decrease in residence time because the culture volume is constant. As various cells compete, those that do not double during their residence time are washed out with no progeny to contribute to the mixed population. It is a powerful advantage for cells that divide more rapidly to persist in the reactor as slower cells tend to be lost. After several days of competition, very rapidly growing cultures are selected (Bungay, et al., 1981). These rapidly dividing strains did not become contaminated by other organisms. It takes time to acclimate to a culture medium, and random contaminants had insufficient time to become established. Powell (1958) developed the mathematics of competition in continuous culture. The percentage of mature, older cells will depend on the pumping rate with very young cells predominating at high dilution rates. Holding a substrate at elevated concentration offers a novel and potentially valuable approach to high productivity.

A possible alternative explanation of fast dilution rates is that organisms attach to surfaces and persist in the bioreactor. This would give an apparent fast growth rate while, in fact, the organisms were merely extracting nutrients as the media flowed through rapidly. Lin and Bungay (1993) sampled a pH-auxostat and counted the cells by direct microscopic examination of a Petroff-Hauser chamber and have made viable cell counts. One count was made on a fresh sample and the others on samples held for a brief time interval. The difference in numbers confirms fast growth that corresponds to doubling times of ten minutes or less for several independent runs. This happens with either a wild inoculum or a pure culture. Some typical results are shown in Figure 3 in which the times for each line denote when a sample was taken.. There is little chance for mistakes because the samples were simply placed in the chamber, and counts were reported as the ratio of numbers. Viable counts, direct counting under the microscope, and dilution rate in continuous culture all confirm that cells can double in about 10 minutes or less.

Attached cells retard the selection of rapidly-growing suspended cells because of competition for nutrients. It stands to reason that nutrient concentrations are lower when both attached cells and suspended cells are competing. When the attached cells are eliminated or reduced in number, the nutrient concentrations will be greater. Since specific growth rate coefficient is a function of nutrient concentration, the continuous culture will grow faster thus developing more intense competition that will select even faster growing organisms.

Use "BACK" to go with the flow.

R.P.I. archive