The continuous culture is an open cultivation process where cell growth tends to maintain in a continuous mode of operations. Therefore it works like an “Open cultivation system” where the fresh nutrient medium is continuously added into the culture vessel rather than recycling and reuse of the nutrients or substrate.
In a continuous cultivation system, there is an inlet pump which continuously allows the sterile nutrient medium to enter the reservoir. An effluent pump continuously elutes the excess cells and by-products of the medium. By this continuous operation, the microbial cells grow in a steady exponential phase as there is neither depletion of nutrient nor accumulation of toxic by-products.
In continuous culture, cell density and other factors like the concentration of a substrate and product remains constant, whereas the addition of fresh nutrient medium dilutes the culture medium. The increase in the flow rate of the fresh nutrient medium will result in an increased dilution rate and decreased retention time.
Content: Continuous Culture
- Definition of Continuous Culture
- Apparatus for Continuous Culture
Definition of Continuous Culture
Continuous culture can define as the culture process, where the bacterial population grows at a constant cell concentration and volume of the culture vessel or reservoir.
It is a type of open cultivation system. As the name suggests, there is the continuous addition of the nutrient medium and the constant elution of the product simultaneously which is the main distinguishing feature of a continuous culture that differentiates it from the batch and fed-batch culture methods.
In the continuous culture, the growth rate of microbial cells is maintained in the exponential phase, i.e. the cells could not reach the stationary phase of growth. The process of maintaining the microbial cells in the log or exponential phase refers to as “Steady state of growth”.
Apparatus for Continuous Culture
To perform the continuous culture method, one requires some additional equipment to maintain the steady phase of the microbial cells. Chemostat and turbidostat are the two special types of equipment which are most commonly used in the process of continuous culture.
It is an apparatus used for the continuous culture of bacteria where the cell density is kept constant by keeping the dilution rate and flow rate of nutrient medium constant. Chemostat was introduced in the 1950s by three scientists Monod, Novick and Szilard. The sterile nutrient medium is added into the reservoir along with the microbial cells.
The concentration of the nutrient medium is, in turn, controlled by the dilution rate. In a Chemostat, the substrate is continuously added into the reservoir, and the by-products are continuously eluted from the reservoir. Therefore the bacterial cells will grow at the constant rate in the exponential phase because the nutrients are not completely depleted and supplied continuously to the reservoir.
The Chemostat works by two factors like:
- Dilution rate
- The concentration of limiting nutrient
Dilution rate: The dilution rate can define as the flow of the nutrient medium into the reservoir to the culture volume within the vessel. The change in dilution rate will change the growth rate of bacteria, i.e. both cell growth and cell density. Cell density remains constant when the dilution and the flow rate of the medium are kept constant. If the dilution rate alters, cell density and cell growth will also change.
The concentration of limiting nutrient: In Chemostat, an essential nutrient or amino acid is added in limited quantity. By limiting the concentration of an essential nutrient, one can determine the flow rate of the nutrient medium into the reservoir. Therefore, this essential nutrient will also determine the growth rate of the bacteria, i.e. whether the cell density is constant or not. If the concentration of the limiting nutrient alters, the cell density will also change.
Culture of Bacteria by Chemostat
To perform the continuous culture by the chemostat, there are three elements like substrate reservoir, culture vessel and the spent culture bottle. The sterile nutrient medium is continuously added into the culture vessel via flow regulator. The flow regulator allows the constant flow of the fresh substrate into the culture vessel.
In the culture vessel, there is an inoculation port from where the inoculum of bacteria is added. Apart from inoculation port, a culture vessel comprises of magnetic stirrer at the bottom which allows uniform mixing of the cells with the nutrient medium. The remaining dead cells and the by-products from the culture vessel are released out from the sampling outlet into the spent culture bottle.
It also refers as “Biostat”. A turbidostat is a device that maintains the constant cell density by controlling the flow rate of the fresh medium. Turbidostat is an apparatus which was first introduced by the two scientists Bryson and Szybalski in the year 1952.
It is a device which working depends upon the two components like:
- Photoelectric device
- Optical sensing device
Photoelectric device: A photoelectric device like photodiode performs a key role in the examination of the cell density in the culture vessel. Therefore, it determines the alternation in the concentration of the medium.
Optical sensing device: An optical sensing device plays a pivotal role to measure the turbidity or absorbance of the culture in the reservoir. Turbidostat uses a turbidometer which helps to find out the optical density of the medium. The flow rate or the dilution rate is monitored by a turbidostat vigorously to make the turbidity constant.
Culture of Bacteria by Turbidostat
A turbidostat is an apparatus which includes a sterile reservoir, culture vessel, photocell and a light source. In turbidostat also the fresh nutrient medium is automatically regulated which maintains predetermined turbidity. The fresh nutrient medium is added to the culture vessel through a valve which controls the flow of the medium. The fresh nutrient medium is added when the optical density increases.
The cell density is kept constant is the culture vessel by the help of a photoelectric device. The photoelectric device measures the turbidity of the medium by absorbing the light source. The turbidity is monitored throughout the process.
- There is a high risk of contamination, as the process continues without cleaning and proper sterilization.
- To carry out the process of continuous culture, high technical skills are required to maintain the steady state of the bacterial cells.
- To perform a continuous culture, it requires some additional and specific equipment.
- The process is cost-effective.
- As there is continuous processing, it reduces the number of stages like cleaning, sterilization, preparation of starter cultures etc.
- In continuous culture, the cell population can be maintained in the exponential phase at constant cell concentration for a long time.
- By reducing the number of stages, it also saves time and energy.
- As the product is being continuously eluted, so the toxicity of the secondary metabolites do not affect the culture medium. Therefore, continuous culture avoids the accumulation of toxic substance.
- It gives high productivity by utilizing the substrate.
The continuous culture requires two apparatus like Chemostat and Turbidostat to maintain a cell in a “Steady phase of growth”. Both Chemostat and Turbidostat follow some similar criteria like:
- Maintenance of the constant cell density.
- Growth of microbial cells in “Steady-state.”
- The flow rate of the medium and the cells are continuously changing.
Therefore, a continuous culture is preferred over the other types of cell cultivation methods like batch and fed-batch culture. It gives high productivity as the microbial cell exists in their logarithmic phase. As we know, the log phase is a phase of highest metabolic activity where they convert a substrate into a product.