Read this article to learn about the features and operations of a conventional bioreactor.
Features of a Conventional Bioreactor:
The different types and designs of bioreactors are described. The most common features of a typical bioreactor are diagrammatically represented in Fig. 19.5, and briefly described hereunder.
Conventional bioreactors are cylindrical vessels with domed top and bottom. The reaction vessel, surrounded by a jacket, is provided with a sparger at the bottom through which air (or other gases such as CO2 and NH3 for pH maintenance) can be introduced.
The agitator shaft is connected to a motor at the bottom. The reaction vessel has side ports for pH, temperature and dissolved O2 sensors. Above the liquid level of the reaction vessel, connections for acid, alkali, antifoam chemicals and inoculum are located.
The bioreactor is usually designed to work at higher temperature (150-180°C), higher pressure (377-412 kPa). The reaction vessel is also designed to withstand vacuum, or else it may collapse while cooling. The materials used for the construction of bioreactor must be non-toxic and must withstand the repeated sterilization with high pressure steam.
The bioreactor vessel is usually made up of stainless steel. It should be free from crevices and stagnant areas so that no solids/liquids accumulate. Easy to clean channels and welded joints (instead of couplings) are preferred. Transparent material should be used wherever possible, since it is advantageous to inspect medium and culture frequently.
Operation of a Conventional Bioreactor:
The operation of a bioreactor basically involves the following steps:
1. Sterilization
2. Inoculation and sampling
3. Aeration
4. Control systems
5. Cleaning.
Sterilization:
Aseptic conditions are the basic requirements for successful fermentation. That is the bioreactor and its accessories, the growth medium and the air supplied during fermentation must be sterile.
In situ sterilization:
The bioreactor filled with the required medium is injected with pressurized steam into the jacket or coil surrounding the reaction vessel. The whole system is heated to about 120°C and held at this temperature for about 20 minutes. In situ sterilization has certain limitations. It is not energy- efficient (i.e., energy is wasted) since the bioreactor has to be heated for a long period to rise the temperature of the whole system to 120°C. Prolonged heating may destroy vitamins, besides precipitating the medium components.
Continuous heat sterilization:
In this technique, empty bioreactor is first sterilized by injecting pressurised steam. The medium is rapidly heated to 140°C for a short period, by injecting the pressurised steam. Alternately, the medium can be sterilized by passing through a heat exchanger heated by pressurised steam. Subjecting the medium to high temperature for a short period does not precipitate medium components. Further, there is no energy wastage in continuous heat sterilization method.
Inoculation and Sampling:
The bioreactor with the growth medium under aseptic conditions is ready for inoculation with the production organism. The size of the inoculum is generally 1-10% of the total volume of the medium. A high yielding production strain of the organism taken from a stock culture (lyophilized and stored in a deep freezer or in liquid nitrogen) is used.
During the course of fermentation, samples are regularly drawn from the bioreactor. This is required to check the contamination (if any) and measurement of the product formed.
Aeration:
Aeration of the fermentation medium is required to supply O2 to the production organisms and remove CO2 from the bioreactor. The aeration system is designed for good exchange of gases. Oxygen (stored in tanks in a compressed form) is introduced at the bottom of the bioreactor through a sparger.
The small bubbles of the air pass through the medium and rise to the surface. The bioreactor usually has about 20% of its volume as vacant space on the upper part which is referred to as head space. The bioreactor has about 80% working volume. The gases released during fermentation accumulate in the headspace which pass out through an air outlet.
Air-lift system of aeration:
In this type of aeration, sparging of air is done at the bottom of the fermenter. This allows an upward flow of air bubbles. The more is the aeration capacity of the fermenter, the more is the dissolved O2 in the medium. Further, the aeration capacity of the air-lift system is directly proportional to the airflow rate and the internal pressure.
Oxygen demand refers to the rate at which the growing culture requires O2. For all the aerobic organisms, the aeration capacity should be more than the oxygen demand or else the growth of the organisms will be inhibited due to oxygen depletion (starvation).
Stirred system of aeration:
The aeration capacity of the medium can be enhanced by stirring. This can be done by using impellers driven by a motor. The aeration capacity of the stirred fermenter is proportional to the stirring speed, rate of air flow and the internal pressure. Stirred fermenters are better suited than air-lift fermenters to produce better aeration capacities.
Control Systems:
It is essential to maintain optimal growth environment in the reaction vessel for maximum product formation. Maximal efficiency of the fermentation can be achieved by continuously monitoring the variables such as the pH, temperature, dissolved oxygen, adequate mixing, nutrient concentration and foam formation. Improved sensors are now available for continuous and automated monitoring of these variables (i.e., on line measurement of pH).
Most of the microorganisms employed in fermentation grow optimally between pH 5.5 and 8.5. In the bioreactor, as the microorganisms grow, they release metabolites into the medium which change pH. Therefore, the pH of the medium should be continuously monitored and maintained at the optimal level.
This can be done by the addition of acid or alkali base (as needed) and a thorough mixing of the fermentation contents. Sometimes, an acid or alkaline medium component can be used to correct pH, besides providing nutrients to the growing microorganisms.
Temperature:
Temperature control is absolutely essential for a good fermentation process. Lower temperature causes reduced product formation while higher temperature adversely affects the growth of microorganisms. The bioreactors are normally equipped with heating and cooling systems that can be used as per the requirement, to maintain the reaction vessel at optimal temperature.
Dissolved oxygen:
Oxygen is sparingly soluble in water (0.0084 g/1 at 25°C). Continuous supply of oxygen in the form of sterilized air is done to the culture medium. This is carried out by introducing air into the bioreactor in the form of bubbles. Continuous monitoring of dissolved oxygen concentration is done in the bioreactor for optimal product formation.
Adequate mixing:
Continuous and adequate mixing of the microbial culture ensures optimal supply of nutrients and O2, besides preventing the accumulation of toxic metabolic byproducts (if any). Good mixing (by agitation) also creates favourable environment for optimal and homogeneous growth environment, and good product formation. However, excessive agitation may damage microbial cells and increase the temperature of the medium, besides increased foam formation.
Nutrient concentration:
The nutrient concentration in a bioreactor is limited so that its wastage is prevented. In addition, limiting concentrations of nutrients may be advantageous for optimal product formation, since high nutrient concentrations are often associated with inhibitory effect on microbial growth. It is now possible to do on-line monitoring of the nutrient concentration, and suitably modify as per the requirements.
Foam formation:
The media used in industrial fermentation is generally rich in proteins. When agitated during aeration, it invariably results in froth or foam formation that builds in head space of the bioreactor. Antifoam chemicals are used to lower surface tension of the medium, besides causing foam bubbles to collapse. Mineral oils based on silicone or vegetable oils are commonly used as antifoam agents.
Mechanical foam control devices, referred to as mechanical foam breakers, can also be used. Such devices, fitted at the top of the bioreactor break the foam bubbles and the throw back into the fermentation medium.
Cleaning:
As the fermentation is complete, the bioreactor is harvested i.e. the contents are removed for processing. The bioreactor is then prepared for the next round of fermentation after cleaning (technically called turn round). The time taken for turn round referred to as down time should be as short as possible (since it is non-productive). Due to large size of the bioreactors, it is not possible to clean manually. The cleaning of the bioreactors is carried out by using high-pressure water jets from the nozzles fitted into the reaction vessel.