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Primary and secondary metabolites
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Primary and secondary metabolites
There are two basic types of microbial metabolites: primary and secondary. A primary metabolite is one that is formed during the active growth phase of the MO. Ethanol is a product of anaerobic metabolism of yeast and it is formed as part of energy metabolism. Because growth can occur only if energy production occurs, ethanol formation takes place in parallel with growth. It is a typical primary metabolite (fig. 3a).
Fig. 3. Primary (a) and secondary (b) metabolites.
In contrast to ethanol production by yeast in some processes, a secondary metabolite is not made during the active growth phase, but only in a stationary phase (fig. 3b). Many important products such as antibiotics are optimally formed during the stationary phase of the growth cycle in batch cultivation.
Fermentation control and monitoring
During microbial fermentation, it is necessary not only to measure growth and product formation, but also to control the process by moving environmental parameters.
Environmental factors that are frequently controlled include:
ü Temperature
ü Oxygen concentration
ü pH
ü cell mass
ü levels of key nutrients
ü product concentration
Not all cells grow at the same temperatures. In addition, the needs of oxygen for growing, depend on if they are aerobic or anaerobic MO. Yeast grow at acidic pH and bacteria for instance, at neutral pH. Therefore, it is important to keep the optimal conditions of growth to achieved adequate results in product formation.
During growth and product formation in large-scale fermentation, it is essential to obtain data in real time. For instance, it may be desirable to alter one or more of the environmental parameters as the fermentation progresses or to feed a nutrient at a rate that exactly balances growth.
Characteristics of large-scale fermentation. The bioreactor
In its simplest form, the bioprocess is just the mixing of MO with a nutrient broth and allowing the components to react, e. g. yeast cells with a sugar solution to give alcohol. All biotechnological processes are essentially performed within containment systems or bioreactors. Large numbers of cells are invariably involved in these processes and the bioreactor ensures their close involvement with the correct medium and conditions for growth and product formation. It also should restrict the release of the cells into the environment. A main function of a bioreactor is to minimize the cost of producing a product or service.
Bioreactors range from simple stirred or non-stirred open containers to complex aseptic integrated systems involving varying levels of advanced computer control. They can operate in batch, fed-batch and continuous manner as we saw when studied the microbial growth.
Bioreactors can vary in size from the small 5 to 10 L laboratory scale to the enormous 500 000 L industrial scale. The size of bioreactor depends on the process and how it is operated. They can be divided in two classes, those for anaerobic processes and those for aerobic processes.
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Anaerobic bioreactors require little special equipment, except for removal of heat generated during the fermentation.
Aerobic bioreactors, however require much more elaborate equipment to ensure that mixing and adequate aeration are achieved. They are constructed of stainless steel. Because sterilization of the culture medium and removal of heat are vital for successful operation, the bioreactor is fitted with an external cooling jacket through which steam (for sterilization) or cooling water (for cooling) can be run.
One of the most used bioreactors is the centrally stirred tank reactor consisting of a cylindrical vessel with a motor-driven central shaft that supports one or several agitators with the shaft entering either through the top or through the bottom of the vessels (Fig. 4).
Fig. 4. Centrally stirred tank bioreactor.
A critical part of the bioreactor is the aeration system. Oxygen is poorly soluble in water and in a bioreactor with a high microbial density-population, there is tremendous oxygen demand by the culture.
The primary function of agitation is to suspend the cells and nutrient evenly throughout the medium, to ensure that the nutrients, including oxygen, are available to the cells and to allow heat transfer. Most industrial organisms are aerobic and, in most fermentations, the organisms will exhibit a high oxygen demand. Since oxygen is sparingly soluble in aqueous solutions (solubility of CO2 in water is about 30 times higher than that of O2), aerobic fermentations can only be supported by vigorous and constant aeration of the medium.
The second main approach to aerobic bioreactor design uses air distribution (with low power consumption) to create forced and controlled liquid fl ow in a recycle or loop bioreactor. In this way, the contents are subjected to a controlled recycle fl ow, either within the bioreactor or involving an external recycle loop. Thus, stirring has been replaced by pumping, which may be mechanical or pneumatic, as in the case of the airlift bioreactor.
In a small bioreactor, the use of sparger alone may be sufficient to ensure adequate aeration. However, in industrial–size bioreactors, stirring with an impeller is essential. The speed of the impellers is related to the degree of fragility of the cells. Mammalian cells are extremely fragile when compared to most MO.
For successful commercial operation of these bioprocesses, quantitative description of the cellular processes is an essential prerequisite: the two most relevant aspects, yield and productivity, are quantitative measures that will indicate how the cells convert the substrate into the product. The yield represents the amount of product obtained from the substrate while the productivity specifi es the rate of product formation.
In almost all fermentation processes performed in a bioreactor there is generally a need to measure specifi c growth-related and environmental parameters, record them and then use the information to improve and optimize the process. Bioreactor control measurements are made in either an on-line or an off-line manner. With an on-line measurement, the sensor is placed directly within the process stream whereas for off-line measurement a sample is removed aseptically from the process stream and analyzed. On-line measurement is readily available for temperature, pH, dissolved oxygen and carbon dioxide analyses.
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The media design is also very important in fermentation processes. It is important to consider the main nutrients that require the MO to grow optimally. They include water and source of carbon, as the main components, nitrogen, and others depending on the process that will occur.
Media preparation may seem to be a relatively uninteresting part of the overall bioprocess but it is in fact the cornerstone of the whole operation. Poor media design will lead to low effi ciency of growth and concomitant poor product formation.
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