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Algal Culture Methods
The algal pure strains are kept under standard controlled environment in conditioned
rooms or in especially designed incubators, in which routine work can be performed
under strict hygienic control. The pure strain cultures are usually kept in small glass
containers, such as 10 ml test tubes or 100-ml glass Erlenmeyer flasks - filled with 50 ml
culture medium- and closed by a sterile stopper (screw cap or a folded aluminium foil).
Under routine conditions, strain cultures are usually renewed every week. In the
replication process, an inoculum of 0.1-0.2 ml (from test tubes) or 0.5 to 1 ml (from
Erlenmeyer flasks) is taken from the best old culture which is free of contamination, to
inoculate three - four new vessels of the same size to start a new strain.
The old culture is then either utilized for upscaling (see below), or is discarded. Strain
culture vessels should be stirred at least once a day by hand, paying attention not to stir
bottom debris up.
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Types of algal culture methods
There are two basic methods for culturing algae. One is called a "Batch culture" and the
other is called a "Continuous culture".
1. Batch Culture
The most common culture system is the batch culture, due to its simplicity and low cost.
In this method algal cells are allowed to grow and reproduce in a closed container (i.e.
closed system) in which there is no input or output of materials. The algal population cell
density increases constantly until the exhaustion of some limiting factor, while other
nutrient components of the culture medium decrease over time. When that nutrient is
exhausted, their growth stops and eventually they die. These types of cultures typically
last for about one week. After that, if you wish to continue the culture you must "sub-
culture" by adding some cells from the old culture into a flask containing fresh growth
medium. Any products produced by the cells during growth also increase in
concentration in the culture medium.
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The size of the inoculum
The volume of the algal inoculum is usually 15 to 20% of the new volume. Smaller up
to 2%- or larger inocula could be used to decrease or increase growth rate. In culture up-
scaling, the new vessels have to be inoculated with a sufficiently high microalgae density
in order to ensure a rapid growth and to limit the risk of contamination with different
algae or other micro-organisms (protozoa, nematodes, fungi, etc.).
Small-medium size (0.5 to 10l) cultures of microalgae are usually kept in borosilicate-
glass containers with large necks, such as Erlenmeyer flasks or other flat bottom round
flasks (balloons) and carboys. The flasks are usually closed with a stopper made of
sterilised cotton or of plastic which can stand sterilization in autoclave.
All these cultures are provided with a proper medium to support algal growth (treated and
fertilized seawater), good aeration supplemented with carbon dioxide (CO2) as an
additional carbon source, and a strong light. In this way it is possible to reach quickly the
log-phase growth. Aseptic conditions should be maintained to avoid contamination and
culture crashes.
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Photos of Large volume cultures
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2. Continuous Culture
This method of culturing algae differs from the batch culture method in that fresh
medium is added to the culture at a constant rate and old media (and some of the algae
cells) is removed at the same rate. The culture therefore never runs out of nutrients. As
you can imagine the glassware and tubes for this type of system are much more complex
than the simple batch culture. For most classroom exercises the batch culture system
should do the job, however if you should ever need a constant supply of cells this system
is the best.
Parts of a Continuous Culture System.
The diagram below show the parts of a continuous culture system.
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air air 4
3
clamp
6
8
1 7 Sample
port
outflow
media
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Algal Light
culture
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Setting up the Continuous Culture system
Make up about 6-7 litres of algal growth medium in the large medium vessel. If your
culture flask is 500mL then adding 100 mL of fresh medium each day will give you a
20% dilution rate, and six to seven litters should last about two weeks.
Air is pumped into the culture vessel through a sterile filter. This bubbling air has three
effects: it supplies CO2 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.
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 (ie. growth and cell division where the growth rate
and the total number of cells per milliliter of culture remains constant).
In many continuous culture systems, the nutrient medium is delivered to the culture at a
constant rate by a peristaltic pump. The rate of media flow can be adjusted, and is often
set at approximately 20% of culture volume per day.
A truly continuous culture will have the medium delivered at a constant volume per
unit time. However it has been this researcher's experience that delivery systems such as
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peristaltic pumps or any other device are inherently unreliable. It is difficult to adjust
such systems to deliver equal amounts of medium to several cultures simultaneously. In
order to deliver exactly the same amounts of medium, a semi-continuous approach can
be taken. In a semi-continuous system the fresh medium is delivered to the culture all at
once, by simply opening a valve in the medium delivery line. Fresh medium flows into
the culture vessel, and spent culture flows out into a collecting vessel. Once the required
medium has entered the culture, the valve is closed, and the culture is allowed to grow for
24 hours, when the procedure is repeated.
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