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Continuous Culture in Biotechnology

In this continuous culture in biotechnology post we have briefly explained about continuous cell culture, principle, process, applications, and limitations.

Continuous Cell Culture

Continuous cell culture is a type of open cultivation in which cell development is maintained in a continuous mode of operation. It is an open cultivation system in which fresh nutrient medium is continuously introduced to the culture vessel rather than the nutrients or substrate being recycled and reused.

A continuous cell culture cultivation system has an inlet pump that continually allows the sterile nutrient medium to enter the reservoir. The surplus cells and by-products of the medium are continually eluted by an effluent pump.

The microbial cells grow in a stable exponential phase as a result of this continuous functioning, as there is no depletion of nutrients or accumulation of toxic by-products. Cell density and other parameters such as substrate and product concentrations remain constant in continuous cell culture, but the addition of fresh nutrient medium dilutes the culture medium.

Definition

One of the cultural procedures is continuous cell culture, which involves maintaining a constant cell concentration and volume within the culture vessel or reservoir. It’s a form of open-air farming system. As the name implies, the nutrient medium will be continuously added to and the product will be continuously eluted. This characteristic distinguishes continuous cell culture from batch and fed-batch culture systems.

The growth rate of microbial cells is kept in the exponential phase in continuous cell culture, i.e. the cells do not reach the stationary phase of growth. The steady-state of growth refers to the method of keeping microbial cells in the log or exponential phase.

Keeping the working volume constant in continuous cell culture simplifies culture scale-up based on a constant-power-to-volume technique. The ideal circumstances for maximum and long-term product synthesis can be set up.

Ability to maintain a consistent product quality (the steady-state consists of homogeneous cell culture with a constant biomass and metabolite concentration).

It also leads to increased production per unit volume because time-consuming processes like cleaning and sterilising are no longer required.

Cultures in a steady state can continue for days, weeks, or even months, decreasing downtime and making the process more cost-effective.

Principle

A continuous flow system consists of a reactor into which reactants are continuously pumped and products are emitted.

Their operation is governed by the following factors: how material flows through the reactor (which is determined by its design); the kinetics of the reaction in progress

Continuous cell culture allows growth-limiting nutrients to be kept at steady-state concentrations, allowing microorganisms to grow at submaximal rates.

The cellular growth rate and environmental conditions, such as metabolite concentrations, remain constant in a steady-state.

Furthermore, in continuous cell culture, parameters such as pH, oxygen tension, excretion product concentrations, and population densities can be easily monitored and controlled.

Process

An open system is set up in continuous cell culture, in which one or more feed streams carrying the necessary nutrients are continually fed, while the effluent stream holding the cells, products, and residuals is continuously eliminated. 

Maintaining an identical volumetric flow rate for the feed and effluent streams establishes a steady-state. 

All nutrient concentrations are held at steady-state levels, and the culture volume is kept constant. The exponential growth phase is prolonged during this process, and the generation of by-products is avoided. 

Microbial growth activity or by-product generation are used to monitor continuous fermentation, and these processes are referred to as-

Turbidostat method

To maintain the turbidity inside the vessel constant, a Turbidostat dynamically regulates the flow rate (and thus the dilution rate). 

Sensors that measure light backscatter can be used to track the turbidity created by cells in the growth media.

Maintaining consistent turbidity is a new issue for continuous operation. because cells must be removed on a regular basis without the need to add fresh media during the harvesting process Unlike a chemostat, the cells must be retained in the vessel for the majority of the procedure. 

They must be removed when the culture density becomes too high, while maintaining a steady working volume.

Continuous Culture

Chemostat method

It is the most common approach for regulating population density and cultural growth. Chemostats rely on two factors: the dilution rate and the concentration of the limiting ingredient.

Because fresh nutrients are continuously provided and end products are continuously eliminated in continuous cell culture, end products do not accumulate and nutrients are not entirely depleted, bacteria never enter stationary phase.

The liquid media in a chemostat contain some nutrient at a growth limiting concentration, and the concentration of this nutrient regulates the rate of bacterial growth.

The concentration of the limiting nutrient remains constant in a steady state chemostat because the rate of nutrient supply equals the rate at which it is used by the organism plus flow through the outlet.

The concentration of that vital nutrient in the vessel is examined to see if there is steady cell density or not. When the concentration of a nutrient changes, it means the bacterial density is shifting.

As a result, the flow rate is modified in this situation to maintain a consistent cell density.

Continuous Culture

Perfusion method

The cells are either retained in the bioreactor or recycled back to the bioreactor in this form of continuous bioprocessing mode.

Fresh medium is given here, and the cell-free supernatant is withdrawn at the same time. There are a variety of cell retention techniques available.

Furthermore, based on the chosen medium perfusion rate, also known as dilution rate factor D, the cell density grows continuously in this mode.

Cell development is constrained by food or oxygen limitations, as well as waste product inhibition, resulting in a quasi-steady-state of cells, metabolites, and product concentrations.

Continuous Culture

Plug-flow reactor

The culture solution passes through a tubular reactor without being back mixed in this type of continuous cell culture. Nutrients (reactants) enter the reactor as “plugs” that flow in an axial direction through the reactor in a plug flow reactor. The cells are recycled from the outlet to the inlet while the culture medium flows gradually via a tube.

Continuous Culture in Biotechnology Fig: Plug flow reactor design. Image Source: Jaibiba, P., Vignesh, S. N., & Hariharan, S. (2020). Working principle of typical bioreactors. In Bioreactors. INC. https://doi.org/10.1016/B978-0-12-821264-6.00010-3]

Applications

1. The procedure is economical, because continuous processing is used, the number of phases such as washing, sterilisation, and starter culture preparation are reduced.

2. In continuous cell culture, the cell population can be kept in the exponential phase for a long time at a constant cell concentration.

3. Continuous cell culture also saves time and energy by minimising the number of processes.

4. Because the product is continually eluted, the toxicity of the secondary metabolites has no effect on the growth medium. As a result, continuous culturing prevents hazardous substances from accumulating.

5. Single-cell protein, organic solvents, starter cultures, and other products have all been produced via continuous cell culture fermentation.

6. It’s been used to make beer, fodder yeast, vinegar, and baker’s yeast, among other things.

7. Continuous cell culture has been tested for L-lysine-producing C. glutamicum mutant B-6 in the commercial manufacture of secondary metabolites (such as antibiotics from a Penicillium or a Streptomyces sp.).

Limitations

1. As long as the process isn’t cleaned and sterilised properly, there’s a considerable risk of infection.

2. To carry out the continuous culture method, significant technical skills are required to maintain the steady-state of the bacterial cells.

3. A continuous culture necessitates the use of additional and specialised equipment.

4. It’s difficult to keep track of the manufacturing of some non-growth-related products. As a result, continuous culture frequently necessitates both fed-batch culturing and continuous nutrient input.

5. It may be difficult to keep filamentous organisms alive due to the viscosity and heterogeneity of the mixture.

Further Readings

Reference