General Microbiology

CHEMOSTAT (A CONTINOUS CULTURE SYSTEM)

Written by MicroDok

A chemostat is a bioreactor to which fresh growth medium is continuously added, while culture liquid containing left over nutrients, metabolic end products and microorganisms are continuously removed at the same rate to keep the culture volume constant. It can be likened to a fermentation vessel or fermentor. A chemostat is a growth vessel into which fresh growth medium is delivered at a constant rate and cells and spent medium overflow at that same rate.

A practical advantage of the chemostat is that a microbial cell population may be maintained in the exponential growth phase for long periods, days, and even weeks. And because exponential (log) phase cells (e.g., microbial cells) are usually most desirable for physiological experiments, with a chemostat such cells (i.e., exponential phase cells) can be readily available at any time.  The chemostat was originally introduced in the 1950s as a method to culture a bacterial population at a reduced growth rate for an indefinite period and is the most widely used approach to establish steady-state culture for various microbial and/or industrial applications.

The chemostat provides a powerful means of systematically studying how growth rate impacts cellular processes – including gene expression and metabolism – and the regulatory networks that control the rate of cell growth. The chemostat is also a method of experimentally controlling cell growth rate. When maintained for hundreds of generations, chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. The chemostat is a typical example of a continuous cultivation or culture system of microbial growth maintenance in the laboratory or industry.

BRIEF OVERVIEW OF BATCH AND CONTINUOUS CULTIVATION

In batch cultivation, the bacteria are inoculated into the bioreactor (always stirred tank bioreactor). Then, under certain environmental conditions (temperature, pH, aeration) the bacteria go through all the growth phases (lag, exponential, stationary). At last, the fermentation is stopped and the product is collected. Then, after cleaning and sterilization of the fermenter, the fermenter is ready for another batch.

In continuous cultivation, the fresh medium flows into the fermentor continuously, and part of the medium in the reactor is withdrawn from the fermenter at the same flow rate of the inlet flow.

The most common type of continuous culture device is the chemostat. In the chemostat, both the growth rate and the population density of the culture can be controlled independently and simultaneously. Two factors are important in such control:

  1. The Addition Rate: This is the rate at which fresh medium is pumped in and spent medium is removed;
  2. The concentration of a limiting nutrient, such as a carbon or nitrogen source, present in the sterile medium entering the chemostat vessel.

In a batch culture as explained above, the nutrient concentration can affect both growth rate and growth yield. At very low concentrations of a given nutrient, the growth rate is submaximal because the nutrient cannot be transported into the cell fast enough to satisfy metabolic demand. At moderate or higher nutrient levels, however, the growth rate plateaus, but the final cell yield may continue to increase in proportion to the concentration of nutrients in the medium up to some fixed limit. In a chemostat, by contrast, growth rate and growth yield are controlled independently: The growth rate by the dilution rate and the growth yield (cell number/milliliter) by the limiting nutrient. Independent control of these two growth parameters is impossible in a batch culture because it is a closed system where growth conditions are constantly changing with time.

A chemostat is a technique used to grow microorganisms or cells continually in a specific phase of growth. In this technique, a bioreactor is provided with fresh medium which is continuously added. Culture liquid containing left over nutrients and metabolic end products are simultaneously removed. And microorganisms are continuously removed at the same rate in order to keep the culture volume constant.
The growth rate of the microorganisms in bioreactor can be changed by changing the rate with which medium is added hence growth can be easily controlled within limits.
In continuous culture the bacterial cultures can be stabled at state of exponential growth phase over long periods of time. The process involved in the chemostat growth technique is designed in such a way to relieve the conditions that stop exponential growth in batch cultures.

The chemostat is a steady state technique of microbial growth or cultivation. Some of the main features or benefits of this steady state include:

  • The main feature of this technique is that microbial growth in a continuous culture takes place under steady state conditions i.e., growth occurs at a constant rate and in a constant environment.
  • All culture parameters like dissolved oxygen concentration, culture volume, nutrient and cell density, product concentrations, pH, etc. remain constant in the process.
  • The flow of media (i.e., growth medium or nutrient) in the chemostat vessel is related to the volume of vessel in the form of dilution factor. This dilution factor is exemplified in the formular below:

D = F/V

Where:
F= flow rate
V= volume of vessel
D= dilution factor

  • The environmental conditions in the chemostat can be controlled by the experimenter.
  • Microorganisms cultured in the chemostat bioreactor usually grow at a steady state because of a negative feedback between growth rate and nutrient consumption: if the number of cells present in the bioreactor is low, the cells growth rates will be higher than the dilution rate as they consume little nutrient so growth is less limited by the addition of limiting nutrient with the inflowing fresh medium. The limiting nutrient is a nutrient component required for growth, present in the medium at a limiting concentration. However, if the number of cells becomes higher, the more nutrient is consumed, lowering the concentration of the limiting nutrient. Hence this will reduce the specific growth rate of the cells which will lead to a decline in the number of cells as they keep being removed from the system with the outflow. This results in a steady state.
  • As the process is regulated by the experimenter, there is more stability of the steady state. This helps the experimenter to control the specific growth rate of the microorganisms which can be done by changing the speed of the pump feeding fresh medium into the vessel.

Maintenance of purity of culture: In the chemostat bioreactor there is chances of contamination. The contamination of continuous culture with the foreign organisms and mutation of parent organism is sufficient to alter the basic characteristics of the culture. To find out the occurrences the sample taken directly from the chemostat bioreactor must be repeatedly examined, microscopically, by plating out and examination of colonies, by regular subculturing and examination by biochemical tests of typical colonies.

EXPERIMENTAL USES OF THE CHEMOSTAT

  • Chemostats are used in enzyme studies to study enzyme activities.
  • They are used in microbial ecology studies.
  • Chemostats are used in microbial physiology studies.
  • They are used for the enrichment and isolation of bacteria, especially from natural samples in the environment.

APPLICATIONS OF CHEMOSTAT

  • Chemostats are used in Biomedical Research: A continuous culture is used for investigations in cell biology, as they form the source of large volumes of uniform cells or protein. In order to generate a mathematical model relating to its metabolic processes, the chemostat is often used to gather steady state data about an organism. Continuous cultures are also used as microcosms in ecology and evolutionary biology. In the one case, mutation is a problem, in the other case; it is the desired process under study. Chemostats can also be used to enrich for specific types of bacterial mutants in culture such as auxotrophs or those that are resistant to antibiotics or bacteriophages for further scientific study. Variations in the dilution rate permit the study of the metabolic strategies pursued by the organisms at different growth rates. Competition for single and multiple resources, the evolution of resource acquisition and utilization pathways, cross-feeding/symbiosis, antagonism, predation, and competition among predators have all been studied in ecology and evolutionary biology using chemostats.
  • Chemostats are used in the Industry (e.g., Biotechnology): Chemostats are frequently used in the industrial manufacturing of ethanol. In this case, several chemostats are used in series, each maintained at decreasing sugar concentrations. The chemostat also serves as an experimental model of continuous cell cultures in the biotechnological industry.

ADVANTAGES OF CONTINUOUS CULTURES

  1. Bacterial growth rate is higher due to the continuous addition of nutrients to the fermentation tank.
  2. Efficiency of continuous cultures is high as the fermentor operates continuously.
  3. The chemostat or continuous culture system is very useful for the production of primary metabolites.

DISADVANTAGES OF CONTINUOUS CULTURES

  1. The fermentor set up is highly difficult.
  2. The maintenance of required growing conditions is also difficult to achieve.
  3. There is more chances of contamination in the use of the chemostat; and if contamination occurs, large volumes of product may be lost.

 

References

https://orbitbiotech.com/chemostat-a-continous-culture-culture-microbial-growth-chemostat/

Hall MN, Raff MC, Thomas G, editors. Cell Growth: Control of Cell Size. CSHL Press; 2004.

Monod J. La technique de culture continue, theorie et applications. Ann. Inst. Pasteur. 1950;79:390–410.

Novick A, Szilard L. Description of the chemostat. Science. 1950;112:715–716.

Madigan, Michael (2015). Brock Biology of Microorganisms. Pearson. pp. 152–153.

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