Continuous Cultures

In continuous cultures, resources are potentially infinite: cultures are maintained at a chosen point on the growth curve by the regulated addition of fresh culture medium. In practice, a volume of fresh culture medium is added automatically at a rate proportional to the growth rate of the alga, while an equal volume of culture is removed. This method of culturing algae permits the maintenance of cultures very close to the maximum growth rate, because the culture never runs out of nutrients.

Fresh growth medium is stored in the large vessel. Air is pumped into the airspace in this medium vessel. This air pressure will push the medium through a tube which is connected to the culture vessel. By opening and closing the clamp on this medium line one can add medium to the culture vessel. Air is also pumped into the culture vessel. This air passes down a long glass tube to the bottom of the culture and bubbles up.

This serves to keep the culture well suspended as well as high in oxygen and CO₂. The air flowing into the culture vessel flows out through an outflow tube. As fresh medium is added to the culture vessel the level of the liquid in the culture vessel rises; when that level reaches the bottom of the outflow tube, old medium and cells flow out of the culture vessel into a waste flask. There is another glass tube in the culture vessel, the sample port. When a sample of cells from the culture vessel is needed the clamp on the sample port can be opened up and medium and cells flow out.

Continuous culture systems have been widely used to culture microbes for industrial and research purposes. The early development of a continuous culture system can be traced back to the 1950s, when the first chemostat, also called bactogen, was developed.

Batch and continuous culture systems differ in that in a continuous culture system, nutrients are supplied to the cell culture at a constant rate, and in order to maintain a constant volume, an equal volume of cell culture is removed. This allows the cell population to reach a “steady state” (i.e., growth and cell division where the growth rate and the total number of cells per milliliter of culture remains constant).

Two categories of continuous cultures can be distinguished sensu Fogg and Thake (1987):

• Turbidostat culture, in which fresh medium is delivered only when the cell density of the culture reaches some predetermined point, as measured by the extinction of light passing through the culture. At this point, fresh medium is added to the culture and an equal volume of culture is removed. The diluted culture increases in cell density until the process is repeated.
• Chemostat culture, in which a flow of fresh medium is introduced into the culture at a steady, predetermined rate. The latter adds a limiting vital nutrient (e.g., nitrate) at a fixed rate and in this way the growth rate and not the cell density is kept constant. In a chemostat, the medium addition ultimately determines growth rate and cell density.

In many chemostat continuous culture systems, the nutrient medium is delivered to the culture at a constant rate by a peristaltic pump or solenoid gate system. The rate of media flow can be adjusted, and is often set at approximately 20% of culture volume per day. Air is pumped into the culture vessel through a sterile filter. This bubbling air has three effects: it supplies CO₂ and O₂ to the culture, aids in circulation and agitation of the cultures, and pressurizes the head space of the culture vessel so as to provide the force to “remove” an amount of media (and cells) equal to the volume of inflowing media. The culture may be aseptically sampled by opening the clamp on a sample port. The magnetic stirrer and aeration help to prevent the cells from collecting in the bottom of the culture vessel. A truly continuous culture will have the medium delivered at a constant volume per unit time. However, delivery systems such as peristaltic pumps or solenoid gates are inherently unreliable. In order to deliver exactly the same amounts of medium to several cultures growing at once, a “semicontinuous” approach can be taken.

The rate of flow of medium into a continuous culture system is known as the “dilution rate.” When the number of cells in the culture vessel remains constant over time, the dilution rate is said to equal the rate of cell division in the culture, as the cells being removed by the outflow of medium are being replaced by an equal number through cell division in the culture. The principal advantage of continuous culture is that the rate of dilution controls the rate of microbial growth via the concentration of the growth-limiting nutrient in the medium. As long as the dilution rate is lower than the maximum growth rate attainable by the algal species, the cell density will increase to a point at which the cell division rate (“birth rate”) exactly balances the cell washout rate (“death rate”). This steady-state cell density is also characterized by a constancy of all metabolic and growth parameters. On the other hand, if the dilution rate exceeds the maximum cell division rate, then cells are removed faster than they are produced and total washout of the entire cell population eventually occurs.

The disadvantages of the continuous system are its relatively high cost and complexity. The requirements for constant illumination and temperature mostly restrict continuous systems to indoors and this is only feasible for relatively small production scales. Continuous cultures have the advantages of producing algae of more predictable quality. Furthermore, they are amenable to technological control and automation, which in turn increases the reliability of the system and reduces the need for labor.