Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Industrial Protein Synthesis Using Recombinant Microorganisms

The production of commercial products using recombinant microorganisms requires collaboration between specialists in two fields: molecular biologists and biotechnologists. The Role of molecular biologists is to identify, characterize, and modify the target genes and to develop efficient expression systems in microbial Cells for the industrial Synthesis of the desired product. The task of biotechnologists, on the other hand, is to optimize the growth conditions of the recombinant microorganism to maximize product yield. In the early days of molecular biotechnology, scientists naively believed that transitioning from laboratory-scale synthesis to industrial production was a simple matter of scale-up—that is, conditions optimal for small volumes would remain optimal for large volumes, so one would only need a larger Reactor and a correspondingly larger volume of culture medium.

This simplistic view does not reflect reality. For example, aerobic microorganisms grow well in a standard 200 mL flask when aerated using a 300 W stirrer. If one were to simply scale up the "flask" to 10,000 liters, a 15 MW stirrer would be required. Its motor would be the size of a house, and the agitation would generate so much heat that the microorganisms would literally cook. While this simple example may not fully convince biotechnologists, they know very well that The Challenge of industrial microbial cultivation is not just a linear scale-up of a laboratory experiment. Of course, increasing the size of the vessel (bioreactor or fermenter) is absolutely necessary, as it makes no sense to use 50,000 individual 200 mL flasks to obtain 10,000 L of Cell suspension. However, to maximize yield in both small (1 to 10 L) and large (>1000 L) bioreactors, numerous parameters must be optimized: Temperature, pH, agitation rate and method, and—for aerobic organisms—oxygen concentration. Furthermore, it must be kept in mind that, as a rule, optimal conditions change with every tenfold increase in bioreactor volume.

There are other highly important considerations. The reactor must maintain strict sterility, and measures must be taken to prevent the escape of genetically modified microorganisms. To allow quick and easy adjustments during Fermentation, the reactor must be equipped with monitoring and control instrumentation to continuously track as many parameters as possible. Since sterilization can alter the COMPOSITION OF THE medium (for example, by destroying Vitamins), it is crucial to ensure that it remains optimal for the growth of the target microorganisms.

As a rule, industrial fermentation and product purification are multi-step processes (Fig. 16.1). Typically, the Procedure begins with the Preparation and Sterilization of the culture medium and equipment. First, a starter culture (5–10 mL) is grown, then incubated in a shake flask (200–1000 mL), transferred to a seed fermenter (10–100 L), and finally introduced into an industrial fermenter (1000–100,000 L). Upon completion of fermentation, cells are harvested from the culture medium by centrifugation or filtration. If the product is intracellular, the cells are lysed, cell debris is removed, and the product is recovered from the clarified lysate. A secreted product is recovered directly from the medium.

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Fig. 16.1. GENERALIZED SCHEME OF the industrial fermentation process. The target product is located either within the cells or in the culture medium, but not in both fractions simultaneously, so subsequent Processing is carried out with only one of these fractions.