Page 257 - Algae

240 Algae: Anatomy, Biochemistry, and Biotechnology

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 CO 2 and
O 2 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 popu-
lation 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.