Biochemical Engineering Fundamentals Part 1 - Bailey J., Ollis D. 1989

Kinetics of substrate utilization, metabolite production, and biomass formation in cell cultures
Kinetics of metabolite production
Kinetics of metabolic product formation by filamentous organisms

The kinetics of metabolic product formation and substrate utilization by Molds and other filamentous organisms is usually very complex. A typical example is The Biosynthesis of penicillin in a batch process. As shown in Fig. 7.30, there are three distinct regimes of substrate assimilation. During the first 20 h, rapid sugar utilization takes place, accompanied by active mold growth. In the stationary phase, substrate assimilation slows down, while The rate of penicillin formation reaches its maximum. Afterward, the rate of sugar uptake increases again and remains high until it is depleted.

A similar pattern is observed during the biosynthesis of Other Antibiotics, although each process has its own specific features. In streptomycin production, for instance, the maximum antibiotic synthesis rate is reached later (compared to penicillin), and the final phase of accelerated sugar utilization does not occur at all. The production process of the antibiotic oxytetracycline by the filamentous microorganism Streptomyces rimosus is interesting because the outcome of the entire process also depends on the Morphology of the Organism. Table 7.8 summarizes the main stages and features of this process.

Class="center">Table 7.7. Calculated and experimentally found values of ß-galactosidase activity regulated by the lac promoter-operator system for wild-type E. coli and three mutants (i^-, i^q, i^sq) with various intracellular concentrations of the lac repressor R ([R]₀ = 2∙10^-8 M)^a

^a Reprinted with permission from: Bailey J. E., Hjortso M., Lee S. B., Srienc F., Kinetics of Product Formation and Plasmid Segregation in Recombinant Microbial Populations, Annals N. Y. Acad. Sci., 413, 71 (1983).

^b Relative to the induced wild-type i+ (100%) at an inducer concentration of 10^-3 M.

Listed below are some features of metabolic product formation that are characteristic only of filamentous organisms and have been found experimentally:

1. The maximum yield of a metabolic product is reached in a specific phase of the batch process, provided that an optimal initial Substrate Concentration is used. Increasing the substrate concentration leads to faster growth, but in this case, the substrate is consumed mainly for biomass formation, while the metabolite is synthesized in very low concentrations. Conversely, if the substrate concentration is too low, the resulting minor amount of biomass is unable to synthesize the antibiotic in sufficient concentrations, even during the period of its maximum biosynthesis rate.

Table 7.8. Main phases of the batch oxytetracycline biosynthesis process in submerged culture of Streptomyces rimosus^a

^a Luedeking R., in Biochemical and Biological Engineering, Blakebrough N. (ed.), vol. 1, p. 208, Academic Press, Inc., New York, 1967; see also Doskacil J., Sikyta B., Kasparova J., Poskocilova D., Zajlicek J., J. Gen. Microbiol., 18, 302 (1958).

2. Metabolic product formation is maximized at minimal branching of the actively growing inoculum hyphae. On the other hand, a decrease in the degree of branching increases the duration of the lag phase and, consequently, the length of the entire process. Obviously, there must be some optimal degree of branching for the inoculum hyphae.

3. Since molds and other mycelial microorganisms are aerobes, one might expect that maximum agitation intensity in a submerged culture would promote efficient oxygen transfer to the mold and thus enhance its growth. In the case of penicillin, however, it has been found that maximum antibiotic yields are achieved at an intermediate agitation intensity. Several explanations have been proposed for this fact. Mechanical forces are known to affect mold morphology; it is possible that more vigorous agitation promotes hyphal branching, which is believed to reduce the rate of antibiotic biosynthesis. It is also quite possible that an optimal oxygen supply rate exists, but due to The complexity of the system, verifying this assumption experimentally is extremely difficult. (We will discuss The Effect of agitation in more detail in Chapter 8.)

FIG. 7P2.1. Scheme of morphologically distinct forms of the mold C. acremonium, their interconversions, and the synthesis of cephalosporin C (CPC). [Reprinted with permission from: Matsumura M., Imanaka T., Yoshida T., Taguchi H., Modeling of Cephalosporin C Production and Its Application to Fed-Batch Culture, J. Ferment. Tech. (Japan), 59, 115 (1981).]

Example 7.2. Morphologically structured model of cephalosporin C biosynthesis kinetics [14]. An experimental Study of the biosynthesis of the antibiotic cephalosporin C (CPC) in submerged culture of the mold Cephalosporium acremonium demonstrated that it contains three morphologically distinct forms of microorganisms: hyphae (h), swollen hyphal fragments (s), and arthrospores (a). Depending on the medium composition, these forms can interconvert, as shown schematically in Fig. 7P2.1. The same figure notes that CPC is predominantly produced by the swollen hyphal fragments. The rate of CPC synthesis is directly related to The activity of the corresponding Enzymes, which are induced by intracellular Methionine and repressed by glucose.

This information served as the basis for the structured model of CPC biosynthesis in batch culture presented below:

Mold growth and differentiation:

Where

Note that in this model, the total biomass is divided into three morphologically distinct types observed experimentally. The mass concentrations of glucose and methionine in the medium are denoted by the symbols g and m, respectively.

The substrate utilization model implies that glucose and methionine are assimilated exclusively by the h and s morphological forms.

Glucose and methionine utilization:

where

Equations (7P2.1) – (7P2.8) were used to calculate cell growth and substrate utilization characteristics in a batch process; to achieve a better agreement between the calculated data and experimental results, the model parameters were refined using the simplex optimization method.

Since intracellular methionine plays a special role in regulating the expression of enzymes synthesizing CPC, the chemical kinetic model is structured in such a way that METABOLISM/18.html">The Influence of methionine concentrations m_ih, m_is, and m_ia (expressed in all cases as micromoles per 1 g of Cells) in morphological forms h, s, and a, respectively, can be evaluated separately. The following methionine material balance equations were used for this purpose:

Intracellular methionine

In deriving these equations, it was assumed that the rate of methionine biosynthesis is inversely proportional to its intracellular concentration and that the presence of glucose in the medium increases the rate of methionine utilization in Protein Synthesis. The average intracellular concentration of methionine is equal to

where

Now the model describes all the main aspects of the process: cell growth, substrate utilization, and intracellular methionine concentration. By comparing the found and calculated values of , one can determine the new parameters appearing in equations (7P2.9) – (7P2.11). These parameters are listed In the second Column (Group 2) of Table 7P2.1.

The model assumes that the antibiotic synthesis rate depends on the activity of CPC-synthesizing enzymes e (expressed in mg of CPC per hour per 1 g of cells), which is described by the following equation:

Enzyme synthesis

where

The parameter Q is a measure of glucose-induced catabolite repression. The subscript (t—t_I) indicates The Development of the process during the period t—t_I, reflecting the existence of a lag phase between induction and Gene Expression. Finally, the rate of CPC formation in this model is described by the equation

Antibiotic formation

The parameters for equations (7P2.15) and (7P2.16) are given in the third column (Group 3) of Table 7P2.1.

Table 7P2.1. Parameters of the mathematical model describing the biosynthesis of cephalosporin C (CPC)^a

* Reproduced with permission from: Matsumura M., Imanaka T., Yoshida T., Taguchi H., Modeling of Cephalosporin C Production and Its Application to Fed-Batch Culture, J. Ferment. Tech. (Japan), 59, 115 (1981).

FIG. 7P2.2. Calculated (solid curves) and experimental (dots) data reflecting the course of cephalosporin C (CPC) biosynthesis in batch culture of A. acremonium (the symbol M_i denotes data for intracellular methionine). [Reproduced with permission from: Matsumura M., Imanaka T., Yoshida T., Taguchi H., Modeling of Cephalosporin C Production and Its Application to Fed-Batch Culture, J. Ferment. Tech. (Japan), 59, 115 (1981).]

Fig. 7P2.2 shows experimental data (points) and calculated curves (lines) representing cell growth, substrate utilization, and antibiotic production in a batch process. Note that the concentration of swollen hyphal fragments initially increases and then decreases, while arthrospores are formed predominantly during the later Stages of the process. The model captures the peculiar time-course profile of intracellular methionine concentration remarkably well. At the same time, developing and utilizing such a complex model—whose parameters are extremely laborious to determine—is justified only if it remains applicable to cultivating cultures across a sufficiently wide range of conditions. The described model fully satisfies this requirement. For instance, Fig. 7P2.3 illustrates the model-predicted dependence of The amount of synthesized CPC (per unit volume) on (a) the initial glucose concentration and (b) the timing of methionine addition to the culture. In complete agreement with experimental data, the first curve exhibits a well-defined maximum; the second curve demonstrates the advantage of introducing methionine at the Cytology/cytology/16.html">Early stages of the process, which is likewise corroborated by experiments. Furthermore, this model helped predict that in a batch process, the simultaneous addition of methionine and glucose significantly enhances CPC yield, whereas their separate (sequential) addition is less effective. Subsequent experiments confirmed these predictive findings as well.

FIG. 7P2.3. Simulation of the effect of initial glucose concentration (a) and methionine addition timing (b) on CPC synthesis [Reprinted by permission from: Matsumura M., Imanaka T., Yoshida T., Taguchi H., Modeling of Cephalosporin C Production and Its Application to Fed-Batch Culture, J. Ferment. Technol. (Japan), 59, 115 (1981).]