Chemostat

Stirred bioreactor operated as a chemostat, with a continuous inflow (the feed) and outflow (the effluent). The rate of medium flow is controlled to keep the culture volume constant.

A chemostat (from Chemical environment is static) is a bioreactor to which fresh medium is continuously added, while culture liquid is continuously removed to keep the culture volume constant.^[1][2] By changing the rate with which medium is added to the bioreactor the growth rate of the microorganism can be easily controlled.

Operation

Steady State

One of the most important features of chemostats is that micro-organisms can be grown in a physiological steady state. In steady state, growth occurs at a constant rate and all culture parameters remain constant (culture volume, dissolved oxygen concentration, nutrient and product concentrations, pH, cell density, etc.).In addition environmental conditions can be controlled by the experimenter.^[3] Micro-organisms grown in chemostats naturally strive to steady state: if a low amount of cells are present in the bioreactor, the cells can grow at growth rates higher than the dilution rate, as growth isn't limited by the addition of the limiting nutrient. The limiting nutrient is a nutrient essential for growth, present in the media at a limiting concentration (all other nutrients are usually supplied in surplus). However, if the cell concentration becomes too high, the amount of cells that are removed from the reactor cannot be replenished by growth as the addition of the limiting nutrient is insufficient. This results in an equilibrium situation (steady state), where the rate of cell growth is equal to the rate of cell removal.

Because obtaining a steady state requires at least 5 volume changes, chemostats require large nutrient and waste reservoirs.

Dilution Rate

At steady state the specific growth rate (μ) of the micro-organism is equal to the dilution rate (D). The dilution rate is defined as the rate of flow of medium over the volume of culture in the bioreactor:

Maximal growth rate

Each microorganism growing on a particular substrate has a maximum specific growth rate (μ_max) (the rate of growth observed if none of the nutrients are limiting). If a dilution rate is chosen that is higher than μ_max, the culture will not be able to sustain itself in the bioreactor, and will wash out.

Applications

Research

Chemostats in research are used for investigations in cell biology, as a source for large volumes of uniform cells or protein. The chemostat is often used to gather steady state data about an organism in order to generate a mathematical model relating to its metabolic processes. Chemostats are also used as microcosms in ecology^[4][5] and evolutionary biology^[6][7][8][9]. In the one case, mutation/selection is a nuisance, 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.^[10]

Competition for single and multiple resources, the evolution of resource acquisition and utilization pathways, cross-feeding/symbiosis^[11][12], antagonism, predation, and competition among predators have all been studied in ecology and evolutionary biology using chemostats.^[13][14][15]

Industry

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.^{[citation needed]}

Concerns

• Foaming results in overflow with the volume of liquid not exactly constant.
• Some very fragile cells are ruptured during agitation and aeration.
• Cells may grow on the walls or adhere to other surfaces^[16], which is easily overcome by treating the glass walls of the vessel with a silane to render them hydrophobic.
• Mixing may not truly be uniform, upsetting the "static" property of the chemostat.
• Dripping the media into the chamber actually results in small pulses of nutrients and thus oscillations in concentrations, again upsetting the "static" property of the chemostat.
• Bacteria travel upstream quite easily. They will reach the reservoir of sterile medium quickly unless the liquid path is interrupted by an air break in which the medium falls in drops through air.

Continuous efforts to remedy each defect lead to variations on the basic chemostat quite regularly. Examples in the literature are numerous.

• Antifoaming agents are used to suppress foaming.
• Agitation and aeration can be done gently.
• Many approaches have been taken to reduce wall growth^[17][18]
• Various applications use paddles, bubbling, or other mechanisms for mixing^[19]
• Dripping can be made less drastic with smaller droplets and larger vessel volumes
• Many improvements target the threat of contamination

Variations

Fermentation setups closely related to the chemostats are the turbidostat, the auxostat and the retentostat. In retentostats culture liquid is also removed from the bioreactor, but a filter retains the biomass. In this case, the biomass concentration increases until the nutrient requirement for biomass maintenance has become equal to the amount of limiting nutrient that can be consumed.

See also

• Fed-batch
• Biochemical engineering
• Continuous stirred-tank reactor (CSTR)
• Bacterial growth
• E. coli long-term evolution experiment

References

External links

1. http://www.midgard.liu.se/~b00perst/chemostat.pdf
2. http://www.rpi.edu/dept/chem-eng/Biotech-Environ/Contin/chemosta.htm
3. A final thesis including mathematical models of the chemostat and other bioreactors
4. A page about one laboratory chemostat design