CONTINUOUS CULTURE IN CHEMOSTAT

Cells regulate their rate of growth in response to signals from the external world. As the cell
grows, diverse cellular processes must be coordinated including macromolecular synthesis,
metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of
experimentally controlling cell growth rate, provides a powerful means of systematically studying
how growth rate impacts cellular processe - including gene expression and metabolism - and the
regulatory networks that control the rate of cell growth. When maintained for hundreds of
generations chemostats can be used to study adaptive evolution of microbes in environmental
conditions that limit cell growth.

CHEMOSTAT

A chemostat (from chemical

environment is static) is a bioreactor to which
fresh 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. By
changing the rate with which medium is
added to the bioreactor the specific growth
rate of the microorganism can be easily
controlled within limits.

OPERATION

Steady state
One of the most important features of chemostats is that microorganisms can be grown in a
physiological steady state under constant environmental conditions. In this steady state, growth
occurs at a constant specific growth 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.[4] Microorganisms
growing in chemostats usually reach a steady state because of a negative feedback between growth
rate and nutrient consumption: if a low number of cells are present in the bioreactor, the cells can
grow at growth rates 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 essential for growth, present in the medium at a limiting concentration (all other
nutrients are usually supplied in surplus). However, the higher the number of cells becomes, the
more nutrient is consumed, lowering the concentration of the limiting nutrient. In turn, 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. Due to
the self-regulation, the steady state is stable. This enables the experimenter to control the specific
growth rate of the microorganisms by changing the speed of the pump feeding fresh medium into
the vessel.

Well-mixed
Another important feature of chemostats and other continuous culture systems is that they are
well-mixed so that environmental conditions are homogenous or uniform and microorganisms are
randomly dispersed and encounter each other randomly. Therefore, competition and other
interactions in the chemostat are global, in contrast to biofilms.

Dilution rate
The rate of nutrient exchange is expressed as the dilution rate D. 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 flow of medium per time, F, over the volume V of culture in the bioreactor

D = f/V

Maximal growth rate and critical dilution rate

Specific growth rate μ is inversely related to the time it takes the biomass to double called doubling
time td by:

µ=ln2 / td

therefore, the doubling time td becomes a function of dilution rate D in steady state:

Td = ln2/D
 Each microorganism growing on a particular substrate has a maximal specific growth rate μmax
(the rate of growth observed if growth is limited by internal constraints rather than external
nutrients). If a dilution rate is chosen that is higher than μmax, the cells cannot grow at a rate as
fast as the rate with which they are being removed so the culture will not be able to sustain itself in
the bioreactor, and will wash out.

However, since the concentration of the limiting nutrient in the chemostat cannot exceed the
concentration in the feed, the specific growth rate that the cells can reach in the chemostat is
usually slightly lower than the maximal specific growth rate because specific growth rate usually
increases with nutrient concentration as described by the kinetics of the Monod equation.[citation
needed] The highest specific growth' rates (μmax) cells can attain is equal to the critical dilution
rate (D'c):

D = µmax (S/ Ks + S)
where S is the substrate or nutrient concentration in the chemostat and KS is the half-saturation
constant (this equation assumes Monod kinetics).

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[5][6] and evolutionary biology.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.[11] 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,[14][15] antagonism, predation, and competition among
predators have all been studied in ecology and evolutionary biology using chemostats.[16][17][18]

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]
The chemostat also serves as an experimental model of continuous cell cultures in the
biotechnological industry.
 A chemostat consists of a culture:

1. into which fresh medium is continuously introduced at a constant rate, and

2. the culture volume is kept constant by continuous removal of culture at the same rate, and
3. in which the supply of a single nutrient controls growth rate.

The fermenter is called a chemostat because the growth rate is controlled by the availability of
a single component of the medium (the limiting substrate). Important features are that it is the
continuous introduction of fresh medium that feeds the limiting substrate to the culture and the
dilution rate (that is, the rate of addition of fresh medium) determines the specific growth rate of
the culture

The main features of a chemostat culture are:

1. Volume of the culture remains constant,

2. The time required to mix a small volume of medium with the culture should be small, that is, it
should approach perfect mixing,
3. Environmental conditions can be maintained constant (these include nutrient concentration, pH,
temperature, antibiotic concentration),
4. Specific growth rate can be varied from just above zero to just below µmax,
5. An environmental condition (pH, temperature, oxygen tension, etc.) can be varied whilst
maintaining specific growth rate (µ) constant,
6. Biomass properties such as macromolecular composition and functional characteristics can be
maintained constant,
7. Biomass concentration can be set (by altering the concentration of the limiting substrate in
inflowing medium) and maintained constant, independently of µ,
8. Substrate-limited growth can be maintained indefinitely and this can offer relief of catabolic
repression and induction of secondary metabolism at low substrate (glucose) concentration. When
chemostat cultures are operated for a very long time the organism evolves ; indeed, chemostat
cultures can be used to select mutants with particular physiological characteristics.

As originally conceived, cells are grown in a fixed volume of media that is continually diluted
by addition of new media and simultaneous removal of old media and cells (Figure 1).
The principle of continuous culturing using a chemostat can be realized in a variety of
implementations. In all chemostats it is essential to have
1)methods for maintaining sterility of all components,
2)a well-mixed culture,
3)appropriate aeration of the culture vessel
4)a reliable means5

Chemostats also provide an ideal system for studying microbial evolution. Nutrient limitation is
an ecologically relevant selective pressure and growth rate is a major component of microbial
fitness. The chemostat provides a means of precisely controlling the selective pressure and studying
how molecular networks evolve. Identifying the genetic loci that are targets of selection and
proving their adaptive benefit in the same nutrient-limited environment holds the promise of
understanding the functional basis of adaptive evolution.Chemostats are increasingly being used in
new areas of research including the study of transcriptional dynamics and metabolic oscillations.
Their application in ecology has proved useful in the study of predator-prey dynamics. A renewed
interest in mammalian cell growth regulation, and its impairment in human disease, may motivate a
return to the study of mammalian cells in chemostats using cells that
can be cultured in suspension
TECHNICAL 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,[19] which may be overcome by treating
the glass walls of the vessel with a silane to render them hydrophobic. However, cells will be
selected for attachment to the walls since those that do will not be removed from the system.
Those bacteria that stick firmly to the walls forming a biofilm are difficult to study under chemostat
conditions.
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.