Algae Culture

Microalgae are a diverse group of unicellular organisms comprising eukaryotic protists,

prokaryotic cyanobacteria, and blue-green algae, their numerous characteristics have been used
in different industrial biotechnological processes. There are several advantages offered
by microalgae over higher plants as a source of biodiesel or bioethanol, for example:
(1) The yield of storage compounds per unit of area for microalgae can greatly exceed that of
traditional crops;
(2) Microalgae can be cultivated in areas unsuitable for conventional agriculture;
(3) They can be cultured in controlled aquaculture;
(4) Microalgae grow either in seawater or in brackish water which is unusable for normal
agriculture;
(5) Microalgae allow the recovery of P and N from wastewater, and the utilization of waste
CO2 (e.g. from flue gases).
The need to culture microalgae arises because of the many benefits that can be used for human
life, so there are efforts to support optimal growth of the high density of maintained microalgae.
The basic methods of algae culture have changed little over the years and various steps in the
process leading to laboratory scale production culture are introduced in Figure 1.

Figure 1 Steps in the production of algae.

(1) Stock cultures (250 ml or less) remain in isolation under light and climate control (low
temperature) and are only used to inoculate starter cultures when necessary.
(2) Starter cultures (250 ml to 4 l in volume) are grown quickly for 7 to 14 days at higher
temperatures and light intensity with a supply of carbon dioxide enriched air.Small
portion of the volume is used to start a new starter culture and the main portion to
begin an intermediate-scale culture.
(3) Intermediatescale cultures (usually of between 4 l and 20 l in volume) may be used as
food for larvae or to start a large-scale culture.
(4) Large-scale cultures are generally of a minimum of 50 l and are frequently much greater
in volume.
After inoculation these cells grow and divide faster because they adjust to the culture conditions.
This acclimatization period, which lasts for 2 to 3 days, is called the lag phase. After adjusting for
conditions, the rate of cell division accelerates and increases the number of cells in the culture is
logarithmic. This period lasts 4 to 6 days and is called the exponential growth phase. The level of
cell division then slows down because the penetration of light through culture and / or nutrition
is limited. Culture then enters the stationary phase, which can last for days. Culture remains in
this phase by recycling nutrients from dead and decaying cells, but in the case of diatoms, which
can produce their own inhibiting metabolites, which attract bacterial growth, the culture
collapses.

Small-scale Microalgae Cultures Protocols

1. Clean all the inner surfaces of the inoculation chamber with 75% ethanol.
2. Place everything that will be needed at the laminar flow cabinet; that is, all the flaks to be
transferred from (flaks transfer) and the flaks contains sterilized media to be transferred
to (new flaks).Turn off the lights. Turn on the small stove.
3. Remove the cover from one transfer and one new flaks. Turn on the neck of each flaks by
turning the neck slowly through the flame.
4. Use a 1000 ml flaks. Tilt the flaks neck transfer to the new flaks. Put 100 ml of BG II media
- modified and 1 ml of trance metal solution into a new flaks. Add sterile distilled water
to reach 900ml then transfer about 100 ml of microalgae (This volume might change
based on density of original culture). Avoid touching the neck of both flasks. Never touch
the stopper that is inserted into the flaks.
5. Replace the cover above the new flaks neck. By using a waterproof marker, label the new
flaks with algae species inoculated and the transfer date.
6. Repeated procedures for all flakss in the laminar flow cabinet. When finished, turn off the
stove and open the laminar flow cabinet.
(Source: Bourne, Hodgson and Whyte, 1989)

BG II (Blue-Green Medium)- modified Recipe

Freshwater algae

Composition g L-1
NaNO3 3
K2HPO4 0.08
MgSO47H2O 0.15
CaCl2 2H2O 0.072
Citric Acid 0.012
Ammonium Citrate 0.012
Na2CO3 0.002
EDTA 0.004
Table 1. The composition of BG-II medium
Composition g L-1
FeCl3 6H2O 3.15
MnCl2 4H2O 0.18
ZnSO4 7H2O 0.022
COCl2 6H2O 0.01
CuSO4 5H2O 0.01
Na2MoO4 2 H2O 0.006
Table 2. Composition of trace metal solution

Make up to 1 liter with distilled water, then autoclave at 15 psi for 15 minutes.
(source: Stanier RY, Kunisawa R, Mandel M & Cohen-Bazire G, 1971)