Construction of the laboratory bioreactor
In terms of the construction, the following variants of the laboratory bioreactor can be made:
Glass bioreactor (without the jacket) with an upper stainless steel lid.
Glass bioreactor (with the jacket) with an upper stainless steel lid.
Glass bioreactor (without the jacket) with the upper and lower stainless steel lids.
Two-part bioreactor - glass/stainless steel.
The stainless steel part has a jacket and ports for electrodes installation.
Stainless steel bioreactor with peepholes.
The bioreactor's upper part has:
Ports for electrodes (pH, pO2, T, p).
Ports for the supply of the titration and feeding medium.
Pipes for sampling and chemostate.
Ports for the connection of the outlet air condenser and filter.
Drive sealing (if the mixer's upper drive is used).
Air sparger mounting (if there is no lower lid).
The lid's ports, connections and mountings should ensure the air-tightness and leak- proofness, sterility, convenient removal and installation of the sensors and other elements. The lid is usually connected with the reactor's rest part employing special screws with a circumference diameter (4-8pieces) and a flat or "0" ring silicone rubber sealing.
If the bioreactor has a lower cover, then the following ports and elements should be placed and fastened there:
Mixer's lower drive;
All these connections should secure air-tightness and leak-proofness and should prevent the conditions for infection agents' reproduction ("pockets", unevennesses, etc.).
Practical principle of mixers application
The mixer is mechanically "put on" the mixer's axis. The mixer's diameter is normally 1/3 - 1/2 from the diameter of the reactor vessel. The location of the mixer's axis depends on the fact whether the bioreactor has the upper or lower drive. The upper drive is realisable more easily from the constructive viewpoint. In this case, the construction of the sealing is also easier than in the case of the lower drive. In this case, the servicing of the bioreactor is also simple: the motor is disconnected from the sealing connection places, and then the lid, together with the mixer axis and the mixers, is taken off. In its turn, the lower drive provides a whole range of attractions connected with the operation regimes. In this case, mixing can be ensured in the bioreactor at the removed upper lid. This is important when modelling mixing as well as when cleaning and washing the bioreactor. In this case, it is easier to optimise dispersion, as the mixer can be close to the bioreactor's bottom.
The standard Rushton turbine is most popular
The standard Rushton turbine is the most widespread mixer's construction. Its diameter d is 1/2 - 1/3 (most typically, 1/3) from the vessel's diameter. The Rushton turbine is a typical radial flow generating mixer. For the mixer axis, there are commonly 2-3 standard turbine mixers. The given mixer ensures the highest input power at fixed rotational speed values of the mixer (power index KN = 5-7). This mixing system secures a sufficient course of mixing intensity, bubble dispersion and other mixing operations. This mixing system ensures the highest introduced power by constant rotations speed, comparing with another mixers. Therefore, the most part of bioreactors is equipped with such a mixing system.
The mixing of mechanical sensitive microorganisms
However, in the microorganism cultivation practice, there are fermentations in which the application of the standard turbine mixer does not provide the optimal cultivation result, as irreversible mechanical damages of the microorganisms' cells are caused (this applies mainly to mycelial organisms). For mixing of mechanically sensitive mycelial microorganisms, mixing systems are recommended, which generate dominating axial flows, thereby ensuring a more even mixing throughout the reactor's volume. Ekato Intermig mixing systems are among the most widespread ones. The so-called "Counter-flow mixing system" is known. The approach to the mixing of more sensitive cells (tissue culture, animal cell, etc.) should be different, since, in this case, the mixing regime should have a laminar character.
The mixing by great air flows
Another case when the standard turbine is not effective enough it the dispersion of a great air flow at relatively low rotational speed values of the mixer. With increasing the inlet air amount, it is not dispersed any more, as the "flooding" regime sets in. In this case, to increase the dispersion efficiency, it is recommended to apply SCABA AB 6 SRGT, Chemineer Inc. CD6 or BT6 mixers. The efficiency of these mixers, in comparison with the standard turbine mixer, becomes even more pronounced in the cases of three-phase flows (intensive aeration and the presence of dispersive particles in the medium).
Baffles are vertical radially located plates (their width is about 10% from the bioreactor vessel diameter). There are normally 3-4 such plates in the reactor. Baffles are necessary to prevent the formation of a funnel. As a result of funnel formation, the maximally possible rotational speed of the mixer can be essentially limited. Employing baffles, the consumed electric power of the mixer increases by about 20% at the same rotational speed.
The supply of compressed air is realised through a sparger. The most widespread constructive solution is a loop pipe with small holes in the lower part (d = 0.05 - 0.15 mm). For mycelial culture fermentations, also loop spargers with a conical air outlet channel are employed. This construction prevents the possible overgrowing of cracks, since, in this case, the outlet diameter is greater, as well as pressure, because it is not distributed among the several pipes.
Depending on the construction type, the bioreactor vessel is made of steel, metal or their combination. The relation between the height H and diameter D of the bioreactor is within 1.5-2.5. The bioreactors produced in Europe are commonly more "stately" than those produced in the U.S.A. The reactor filling is about 70%. Thus, it is also the part of the working volume in the reactor vessel. There are high requirements for the reactor vessel materials to prevent the inhibition of the microorganism growth. The same applies also to any other part (sensors, pipes, etc.), which are installed inside the bioreactor vessel. The glass should be 100% borosilicate, e.g Pyrex® and Kimax®. All the metal parts should be made from stainless steel. The most widespread brand of the stainless steel applied in bioreactors is 316L. The letter L indicates that this steel is with a low composition of carbon. The inner surface of the stainless steel bioreactor should be polished to about a mirror surface quality to facilitate the washing and sterilisation process. Welding should be carried out in a fully inert gas medium. The inert gas should be argon, which fully replaces the air. With time, the application of the welding technology not corresponding to the requirements can cause the corrosion of the welds.
Condenser, outlet valve and filter
The task of the condenser is to cool the liquid in the air outlet, resulting in the separation of the foam and other liquid particles.
For this purpose, a pipe is wound inside the condenser (or another solution that ensures, as possible, a longer air pathway); outside, there are two ports for cold water supply. The condenser is usually connected to the port located in the cover's centre. The condenser outlet is connected with the outlet valve. With the outlet valve, it is possible to regulate the necessary overpressure in the bioreactor's vessel. The valve outlet can be connected with a porous microbiological filter.
On the one hand, sampling may seem to be a simple procedure - just open the manual valve in the inlet of the bioreactor vessel, supply as much fermentation broth as required for the sample, and close the tap! In this sampling, we can quite easily guarantee that infection will not be avoided.
The sampling construction should be such so that measures for preventing non-sterility before and after the sampling be avoided. In the sites of the infection origin, sterilisation should be performed promptly with alcohol or steam. Sampling constructions of different companies are known, which are offered for sale:
The essence of sampling is based on one of the following principles:
Simple sampling line. A bladder made of silicone or a similar material is placed into the sampling pipe, and its end is stopped with a clamp. Thereby, it is sterilised together with the bioreactor vessel, and it remains in such a state until the sampling. When sampling, the clamp is removed, and the bladder is also pulled down. With the sample's discharge, the pipe's end is immediately washed with alcohol. Then, in a similar way, the sterilised bladder is put on (doing so, sterility should be certainly observed). This method is applicable if the given fermentation has not very high demands on sterility. Another drawback of this method is a hampered possibility of choosing the sample's amount.
Keofitt sampling device
in 2 and 3 variants, as can be seen from the Figures, all is clear. In principle, variant 2 can be realised by the laboratory staff. Variant 3 is a compact, as if elegant and simple solution, but high accuracy and professionalism are required for its realising. A whole range of industrial sampling valves operates according to this principle.