Chemostat

A type of bioreactor

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 is 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.

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. 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 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.

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.

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 unit of time, F, over the volume V of culture in the bioreactor

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 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. 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.

The steady state concentration of the limiting substrate in the chemostat is independent of the influx concentration. The influx concentration will affect the cell concentration and thus the steady state OD. Even though the limiting substrate concentration in the chemostat is usually very low, and is maintained by discrete highly concentrated influx pulses, in practice the temporal variation in the concentration within the chemostat is small (a few percent or less) and can thus be viewed as quasi steady state. The time it takes for the cell density (OD) to converge to a steady state value (overshoot/undershoot) will often be long (multiple chemostat turnovers), especially when the initial inoculum is large. But, the time can be minimized with proper parameter choice.

A chemostat might appear to be in steady state, but mutant strain takeovers can occur continuously, even though they are not detectable by monitoring macro scale parameters like OD or product concentrations. The limiting substrate is usually at such low concentrations that it is undetectable. As a result, the concentration of the limiting substrate can vary greatly over time (percentage wise) as different strains takeover the population, even if resulting changes in OD are too small to detect. A “pulsed” chemostat (with very large influx pulses) has a substantially lower selective capacity than a standard quasi continuous chemostat, for a mutant strain with increased fitness in limiting conditions. By abruptly lowering the influx limiting substrate concentration it is possible to temporarily subject the cells to relatively harsher conditions, until the chemostat stabilizes back to the steady state (on the time order of the dilution rate D).

When the conditions are right (a large enough population, and multiple targets in the genome for simple advantageous mutations) multiple strains are expected to successively takeover the population, and to do so in a relatively timed and paced manner. The timing depends on the type of mutations. In a takeover succession, even if the selective improvement of each of the strains stays constant (e. g. each new strain is better than the previous strain by a constant factor) – the takeover rate does not stay constant, but rather diminishes from strain to strain. There are cases where successive takeovers occur so rapidly that it is very difficult to differentiate between strains, even when examining allele frequency. Thus, a lineage of multiple takeovers of consecutive strains might appear as the takeover of a single strain with a cohort of mutations.

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.