Washout of cells

Several special terms are used to describe traditional reaction engineering concepts. Examples include yield coefficients for the generally fermentation environment-dependent stoichiometric coefficients, metabolic network for reaction network, substrate for feed, metabolite for secreted bioreaction products, biomass for cells, broth for the fermenter medium, aeration rate for the rate of air addition, vvm for volumetric airflow rate per broth volume, OUR for 02 uptake rate per broth volume, and CER for C02 evolution rate per broth volume. For continuous fermentation, dilution rate stands for feed or effluent rate (equal at steady state), washout for a condition where the feed rate exceeds the cell growth rate, resulting in washout of cells from the reactor. Section 7 discusses a simple model of a CSTR reactor (called a chemostat) using empirical kinetics. 

Steady-state B) is the interesting one, of course, since steady-state (A) corresponds to complete washout of cells. Stability analysis has shown that the two cannot coexist at the same holding time either (A) is stable and (B) is unstable, or (S) is stable and (A) is unstable. Moreover, since (B) is a node if it is stable, we see that Monod s model will not predict oscillations— even damped ones—about a steady state of nonzero cell concentration. Hence, in this sense, there has been no improvement over the Verhulst-Pearl model. 

Without recycle, washout occurs when D is greater than flmax, but recycle permits operation with D far greater than flmax- A family of curves is shown in Fig. 24-24 for concentrations of cell mass and nutrient at different recycle ratios. The distinct differences from Fig. 24-22 with no recycle are obvious. 

There is an interior optimum. For this particular numerical example, it occurs when 40% of the reactor volume is in the initial CSTR and 60% is in the downstream PFR. The model reaction is chemically unrealistic but illustrates behavior that can arise with real reactions. An excellent process for the bulk polymerization of styrene consists of a CSTR followed by a tubular post-reactor. The model reaction also demonstrates a phenomenon known as washout which is important in continuous cell culture. If kt is too small, a steady-state reaction cannot be sustained even with initial spiking of component B. A continuous fermentation process will have a maximum flow rate beyond which the initial inoculum of cells will be washed out of the system. At lower flow rates, the cells reproduce fast enough to achieve and hold a steady state. 

Operating near the washout point maximizes the production rate of cells. A feedback control system is needed to ensure that the limit is not exceeded. The easiest approach is to measure cell mass—e.g., by measuring turbidity— and to use the signal to control the flow rate. Figure 12.5 shows how cell mass varies as a function of t for the system of Examples 12.7 and 12.8. The minimum value for t is 2.05 h. Cell production is maximized at F=2.37h. 

Studies of the efflux of Ca by stimulated rabbit atria have characterized three calcium pools. Phase I may represent extracellular washout of the Ca that binds to the surface of muscle membrane and is characterized by a high rate constant. Phase II may represent loosely bound calcium present in cell membrane and calcium released at the sarcoplasmic reticulum. Calcium in this pool is directly related to contractility.65,84,93 phase III may represent the tightly bound calcium that exchanges very slotrly and does not play a role in maintaining calcium concentrations. Recent study has shown that the storage or release of calcium at the sarcoplasmic reticulum and other loosely bound calcium sites (cell membrane) that are involved in muscle contractility can be directly affected by 2-PAM.21 These results Indicate that 2-PAM increases the rate of release of Phase II calcium. 

A common problem with gap junction measurements is a rundown of gj in these preparations, for example in neonatal rat heart cells Schmilinsky-Fluri et al. [1990] found a decrease in g, of 16.4% in 6 min which could be antagonized by addition of a phospholipase inhibitor, 20 pmol/l bromophenacyl bromide, to 1.8% within 6 min. They suggested that endogenous arachidonic acid is involved in spontaneous uncoupling. Others favored a washout of ATP and cyclic nucleotides as a possible cause and prevented their preparations from spontaneous uncoupling by addition of ATP, GTP or cAMP to the pipette solution [Miiller et al., 1997a, b]. 

In the situation where the left-hand side of Equation 12.24 (i.e., the amount of cells withdrawn from the fermentor per unit time) is greater than the right-hand side (i.e., the cells produced in the fermentor per unit time), continuous operation will become impossible. This is the range where D is greater than /d, as can be seen by dividing both sides of Equation 12.24 by V and such a condition is referred to as a washout. ... 

In the chemostat, the dilution rate is set at a fixed value, and the rate of cell growth then adjusts itself to the set dilution rate. This type of operation is relatively easy to carry out, but becomes unstable in the region near the washout point. 

As in the case of enzymes, whole cells can be immobilized for several advantages over traditional cultivation techniques. By immobilizing the cells, process design can be simplified since cells attached to large particles or on surfaces are easily separated from product stream. This ensures continuous fermenter operation without the danger of cell washout. Immobilization can also provide conditions conducive to cell differentiation and cell-to-cell communication, thereby encouraging production of high yields of secondary metabolites. Immobilization can protect cells and thereby decrease problems related to shear forces. 

In isolated ER vesicles from smooth muscle, the addition of calmodulin induces a submaximal phosphorylation of phospholamban via an endogenous protein kinase (Raeymaekers and Jones, 1986). The addition of calmodulin does not, however, significantly stimulate the Ca2+ uptake in isolated ER vesicles (L. Raeymaekers, unpublished observations) or in skirmed smooth muscle cells (Stout and Silver, 1992). Since this lack of effect could be due to washout of the endogenous kinase, further experiments are needed using purified Ca +/calmodulin-dependent protein kinase. 

At this point, models must be introduced for the fission and washout processes. If we assume that the vessel is perfectly mixed, then washout probability (P(E BC)) is independent of cell age, and moreover... 

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