Types of bioreactors and their applications
Once in the stationary growth phase, the action is taken on two levels i.e., the type of bioreactor and the composition/condition of the culture medium. The first parameter allows us to calibrate the culture towards an adequate production of active principles. Depending on the type of bioreactor and the growth conditions it imposes, the following are distinguished:
Closed cycle bioreactors
The cells of the inoculum are grown until the stationary phase of growth is reached; the soil always remains the same, and the system remains closed. In this way, the cells begin to produce secondary metabolites on their own since they perceive the lack of one of their nutrients as a sufficient element of stress. The cells are kept in a closed system to produce secondary metabolites for a certain time, which varies from a few days up to an entire week. After this time, the system is opened, and the secondary metabolites extracted from the soil and cells (some metabolites are trapped in the vacuole while others are released into the environment).
Semi-continuous cycle bioreactors
Cycle adapted to those types of crops that need additional elements of stress, in addition to the lack of nutritional factors and soil growth. It is said to be semi-continuous because, already halfway through exponential growth, the cells begin to produce secondary metabolites. This is probably due to a change in the culture medium due to the massive extrusion of a secondary waste catabolite, toxic to cells, and an element of stress. At this point, 50% of the cell mass is taken from the bioreactor, with 50% of the culture medium; the various active principles are then extracted from this sample.
To the bioreactor in which the remaining percentage of cells and culture medium is contained, an equivalent aliquot of a new medium is added, to allow the cells to resume an exponential growth phase from zero time. In this case, the system is opened when the cells are still in the exponential phase. The bioreactor is defined as "semi-continuous" because small but sufficient quantities of active principles are extracted from it at regular intervals.
The closed or semi-continuous growth system is chosen based on the production capacity of the cell type; some cells produce more metabolites with a given growth system rather than another.
Continuous cycle bioreactors
They are the most used, the most modern, and engineered. They allow the crop to reach the stationary growth phase; at this point, very small aliquots of cells and soil are taken in continuum at regular and close intervals of time. The small sampling allows the callus to regenerate the same number of cells that have been removed, while the collected soil is replaced with a new one. In this way, the cells are kept in balance on the thread of the production of active principles, a thread that represents the optimization in terms of quality and quantity of the production of active principles. This constant and continuous withdrawal, as well as the addition of new soil, is monitored automatically by chemostats and turbo stats. The chemostat is the equipment for monitoring crop conditions, such as the pH and the nutritional elements that make up the soil; when these are not sufficient, the equipment intervenes by entering the corrective means. Turbostats, on the other hand, measure the optical density of the culture, which is directly proportional to the number of cells. When this reaches a maximum threshold value, a small portion is taken, subsequently subjected to the extraction of the active principles.
Immobilized cell bioreactors
modality similar to the closed cycle typology, it differs when the ability of the cell culture to produce secondary metabolites is ascertained, gelatinizing compounds or solid supports are introduced into the bioreactor, which remains closed. These supports allow the suspended culture to become solid culture always inside a bioreactor, where the cells are in the form of macroaggregates equally in contact with the culture medium, therefore equally sensitive to soil stimuli.
For certain cell cultures belonging to certain plant species, solid support represents a mechanical stimulus capable of inducing a sensitive morphological and functional differentiation. In other words, the cells begin, albeit slowly, to differentiate into organized tissues; a morpho / physiological macro or microscopic differentiation most often corresponds to a metabolic differentiation.
Bioreactors and the synthesis of active principles
The bio fermenters or bioreactors containing the liquid medium can have different volumes from 100 ml to several liters. These containers are adapted to an in vitro culture and connected to mechanical systems, which guarantee the adequate growth and aeration of the cells. The possible mechanical stirring modes are different from vane stirring to air blowing. The structural diversification of the bioreactors is justified by the ultimate aim of the process, the best yield in the production of active principles; in fact, in nature, plants synthesize secondary metabolites concerning conditions of environmental stress of any kind.
The secondary metabolites represent the relationship mechanism that the plant establishes with the environment that surrounds it. Similarly to what happens in nature, in the laboratory, we try to recreate the optimal stress conditions, which are often significantly different from those found in nature, since there are different tools available. In any case, the condition recreated in the suspension culture is aimed not only at the production of active principles but also at the determination of those stressors that induce cells to produce secondary metabolites.
The cultures inside the bioreactor must be adequately subjected to various stresses, which mimic elements that stimulate the production of secondary metabolites. The bioreactors are structured in such a way as to determine different types of the cell cycle. As it happens in solid soil, before inducing cells to produce secondary metabolites, it is necessary to stimulate their multiplication; this allows them to obtain a large and adequate number for the synthesis of the active principles.
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