Michaels et al. (1996) showed that these phenomena did not cause significant cell
damage. Finally, Yang and Wang (1992) studied the reduction in growth of the alga
Ochromonas malhamensis at different agitation and sparging rates. The first-order death-
rate constant was proportional to the overall oxygen mass-transfer coefficient, which was
used as a measure for the specific surface area. They concluded that the cell damage in a
sparged and agitated bioreactor was higher than the damage at sparging or agitation
alone. However, it is not clear whether this is due to the occurrence of damaging bubble
coalescence and break-up in the bulk or due to the fact that bubbles are broken up
resulting in more small bubbles causing more cell death at the surface.
In conclusion, there is no clear proof that cell death due to bubble coalescence and
break-up is significant.
Protective AdditivesProtective additives prevent cell death by a combination of mechanisms depending on the
type of additive. All additives adsorb to the cell membrane, making the cell less fragile.
In addition, Pluronic moderates the bursting event. Finally, Pluronics, PVA and Methocel
decrease attachment of cells to the air bubbles and cause rapid draining of cells and
medium out of bubble films, which expels the cells from the danger zone. PVP and PEG
cause an increase in cell concentration near the bursting bubbles due to increased cell-
bubble attachment. In addition, cells and medium drain relatively slowly from the film.
Despite this, these compounds effectively protect the cells against shear. This indicates
that strengthening of the cells and reduction of hydrodynamic forces are the main
protective mechanisms, although the effect of these additives on the burst process is not
known. Also for Pluronic the drainage of cells out of the danger zone seems to not be
required for offering protection. Finally, serum decreases cell attachment to bubbles and
gives medium drainage rates of cells and medium from the bubble film. Furthermore,
serum has also been shown to moderate the bursting event. Yet it only provides moderate
protection compared to the other additives. Notably, in the absence of additives, medium
drains faster than the cells, which may be partly responsible for the concentration factor
of cells in the ejected liquid drops and in foams. With the use of Pluronic, which is the
main additive used in commercial media, the problem of bubble-associated cell death is
largely solved. However, one should consider that these additives might affect cell
physiology. For instance, Al-Rubeai et al. (1992) showed that addition of Pluronic
(0.025–0.1%) led to a decrease in cell concentration and increase in productivity in batch
and steady-state continuous cultures. In addition, Pluronic may decrease the oxygen
transfer coefficient (Murhammer and Pfalzgraf, 1992), thus leading to higher required
gassing rates. Finally, upon using additives foaming may occur, which would result in
physical loss of cells and operating problems. If possible, antifoam can be added.
However, antifoam may make the cells more shear sensitive (van der Pol et al., 1993) and
often the use of antifoam is not allowed because of GMP regulations.
REACTOR DESIGN
Lethal effects of bubbles in animal-cell culture 483