The cultivation of prokaryotic and eukaryotic cells from laboratory to industrial scale can take place in simple systems represented by flasks or in more complex and accurate systems represented by fermenters or bioreactors, but for both systems the chemical conditions for cell growth are highly variable and dependent on the type of culture. The fermentation parameters to bemonitored throughout the fermentation process are the agitation of the culture soil, its oxygenation (or the absence of O2 in the case of anaerobic processes), pH, temperature, nutrients and foam formation.
Culturing cells in flasks is a simplified method that can be set-up on a rotating platform inside an incubator where control of temperature and oxygenation is feasible, but other parameters like pH and foam formation are difficult to adjust over-time.
Bioreactor on the other hand is a more advanced technology capable of providing an environment suitable for the growth of cells or microorganisms that can be easily adapted from laboratory to industrial scale.. Within bioreactors is possible to maintain sterility while providing the cells with everything they need, with a fine tuning of all growth parameters.
One of the most important parameters is the temperature, which during cell growth tends to increase due to exothermic reactions related to microbial metabolism. An excessive increase would cause a slowdown in cell growth or in extreme cases the death of the culture. For this reason, a thermometer connected to the control unit is immersed in the culture broth to constantly monitor its value while thermic insulation and regulation is guaranteed by a jacket installed on the glass vessel where water flows at controlled temperature.
The agitation is guaranteed by a series of rotating blades connected to a motor (usually placed at the head of the bioreactor). The shape, size, distance, and rotation speed of the blades can be changed according to needs. The blades are essential not only for mixing cells and nutrients (which are supplied sterilized and filtered by an incoming pump) but also for better dissolving the oxygen that is supplied from below in the form of bubbles. Furthermore, a fundamental element inside the glass vessel is represented by the flow-breakers, whose function is precisely to break fixed rotary flows that would create non-homogeneous areas within the culture medium. If the cultured organism is a strict anaerobe, that is, it does not survive in the presence of oxygen, nitrogen is simply blown into the culture medium rather than air.
In the case of culturing cells that are very sensitive to mechanical stress due to blades, such as filamentous fungi, agitation is ensured by the insufflation of a powerful jet of sterile compressed air (called airlift Bioreactors).
The main parameters are monitored by means of pH, temperature, oxygen, and foam sensors connected to the control unit which, if the recorded value deviates from the set-point value, triggers an appropriate response to report the altered conditions to those desired for optimal cell growth. For example, if with the increase of cellular biomass an increase in the CO2 produced and therefore a decrease in pH should occur, due to its solubilization in the culture medium with formation of carbonic acid, the control unit would activate the pump connected to the concentrated basic solution reserve to introduce a small volume of solution into the system to bring the pH back to the optimal set value. A similar mechanism is activated in the event of an increase in pH with the introduction of an acid solution.
Similarly, in the event of foam formation, due to agitation, a special probe, placed at a desired height, detects its presence and activates the pump connected to the anti-foam reserve.
Moreover, through an outlet valve it is possible to collect a sample of the culture medium on which to carry out a series of analyses to measure separately the cell concentration, the nutrients still present in the culture medium and the fermentation products, while maintaining sterility.
Finally, some bioreactors are equipped with gas sensors for the quantification of CO2 or H2 (in the case of methanogenic microorganisms).
In general, bioreactors size ranges from those of 1L in volume, used on the counters of research laboratories, to those of 1 million liters, used for large industrial productions. The shift of production from a smaller to a larger bioreactor is called scale-up.
The cell culture methods in a bioreactor are essentially four, usually classified by the method of administration of the culture medium.
1) Batch cultures (closed system)
The volume of liquid medium is essentially constant. As cells grow, their biomass increases and the amount of nutrients available is reduced, while metabolite concentration increase. The cells thus reach a level (called steady state) that prevents them from further increasing their number.
2) Cultures in fed-batch (closed system fed)
With this technology is possible to prolong the growth time of the cells before reaching the steady state, with continuous add of fresh soil.
3) Cultures in perfusion
This method is characterized by a continuous flow of fresh medium, while the exhausted medium (without cells) and the excreted metabolites are withdrawn from the bioreactor. It is a widely used method in animal cell cultures.
With this technology a certain amount of fresh medium is added to a batch culture in the exponential growth phase and an equivalent amount of medium consumed by cells is subtracted. In this way, by keeping the biomass constant, an almost balanced growth is obtained: even the concentrations of nutrients and metabolites, in fact, remain essentially constant.
4) Cultures on a solid layer
unlike the previous techniques, this culture take place in the absence of free water (in some cases water is present in small quantities). Among the most used solid substrates are legumes, cereals, and other materials of vegetable origin such as straw or sawdust.