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We describe a method by which the degree of bubble saturation can be determined by measuring the velocity of single bubbles at different heights from the bubble source in pure water containing increasing concentrations of surfactants. The highest rising velocities were measured in pure water. Addition of surfactants caused a concentration-dependent and height-dependent decrease in bubble velocity; thus, bubbles are covered with surfactants as they rise, and the distance traveled until saturation is reached decreases with increased concentration of surfactant. Pluronic F68 is a potent effector of bubble saturation, 500 times more active than serum. At Pluronic F68 concentrations of 0.1% (w/v), bubbles are saturated essentially at their source. The effect of bubble saturation on the interactions between animal cells and gas bubbles was investigated by using light microscopy and a micromanipulator. In the absence of surfactants, bubbles had a killing effect on cells; hybridoma cells and Chinese hamster ovary (CHO) cells were ruptured when coming into contact with a bubble. Bubbles only partially covered by surfactants adsorbed the cells. The adsorbed cells were not damaged and they also could survive subsequent detachment. Saturated bubbles, on the other hand, did not show any interactions with cells. It is concluded that the protective effect of serum and Pluronic F68 in sparged cultivation systems is based on covering the medium-bubble interface with surfaceactive components and that cell death occurs either after contact of cells with an uncovered bubble or by adsorption of cells through partially saturated bubbles and subsequent transport of cells into the foam region. (c) 1994 John Wiley & Sons, Inc.

A general methodology is proposed to estimate the actual specific growth and death rate of mammalian cells in continuous perfusion reactors from the monitoring of the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) in the culture medium. The procedure is illustrated on a perfusion culture of human tumor kidney cells growing on microcarriers and producing prourokinase (PUK). The intracellular LDH content of living attached cells is checked to be constant during the culture. However, cells detached from the microcarriers, and counted dead because of the uptake of trypan blue, have only released part of their intracellular LDH. In the culture medium, LDH is relatively stable as the loss of activity does not exceed 5% per day. The time variation of the LDH concentration in the medium is used to calculate the total amount of lysed and actually produced cells in the reactors, hence, the actual specific rates of cell growth and death. It is thus found that the stationary phase observed after 400 h of perfusion culture is the result of equal growth and death rates, with a daily renewal of living cells on the microcarriers near 10%. Moreover, for the cell line tested, the production of PUK is associated with cellular growth.

SummaryThe kinetics of C. tropicalis growth were investigated with pure n-hexadecane as dispersed phase substrate. Two distinct growth phases were found: in the first phase, exponential growth waa independent of stirrer speed. The onset of the second phase, one of linear growth, was determined by stirrer speed. By the use of two different fermenter types, it was shown that the drop size of the dispersed phase was not primarily responsible for the observed kinetics. It was considered that the formation of biological flocs determined the observed growth pattern. This was substantiated by the results of continuous cultures in the different fermenter types, with various substrate concentrations.

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