Page 242 - Algae Anatomy, Biochemistry, and Biotechnology

Algal Culturing 225

MARINE MEDIA
Seawater is an ideal medium for growth of marine species, but it is an intrinsically complex
medium, containing over 50 known elements in addition to a large but variable number of
organic compounds. Usually it is necessary to enrich seawater with nutrients such as nitrogen,
phosphorus, and iron. Synthetic formulations have been designed primarily to provide simplified,
defined media. Marine species generally have fairly wide tolerances, and difficulties attributed to
media can frequently be related to problems of isolation, conditions of manipulations and incu-
bation, and physiological state of the organism. A single medium will generally serve most
needs of an investigator. Many media are only major variations of some widely applicable, and
often equally effective media. Whatever the choice, a medium should be as simple as possible in
composition and preparation.
Media for the culture of marine phytoplankton consist of a seawater base (natural or artificial)
which may be supplemented by various substances essential for microalgal growth, including nutri-
ents, trace metals and chelators, vitamins, soil extract, and buffer compounds.
The salinity of the seawater base should first be checked (30–35‰ for marine phytoplankton),
and any necessary adjustments (addition of fresh water/evaporation) made before addition of
enrichments.
Seawater, stock solutions of enrichments, and the final media must be sterile in order to prevent
(or more realistically minimize) biological contamination of unialgal cultures. Autoclaving is a
process which has many effects on seawater and its constituents, potentially altering or destroying
inhibitory organic compounds, as well as beneficial organic molecules. Because of the steam atmos-
phere in an autoclave, CO 2 is driven out of the seawater and the pH is raised to about 10, a level which
can cause precipitation of the iron and phosphate added in the medium. Some of this precipitate may
disappear upon re-equilibriation of CO 2 on cooling, but both the reduced iron and phosphate levels
and the direct physical effect of the precipitate may limit algal growth. The presence of ethylene-
diaminetetraacetic acid (EDTA) and the use of organic phosphate may reduce precipitation
effects. Addition of 5% or more of distilled water may also help to reduce precipitation (but may
affect final salinity). The best solution, however, if media are autoclaved, is to sterilize iron and
phosphate (or even all media additions) separately and add them aseptically afterwards.
Some marine microalgae grow well on solid substrate. A 3% high grade agar can be used for the
solid substrate. The agar and culture medium should not be autoclaved together, because toxic
breakdown products can be generated. The best procedure is to autoclave 30% agar in deionized
water in one container and nine times as much seawater base in another. After removing from
the autoclave, sterile nutrients are added aseptically to the water, which is then mixed with the
molten agar. After mixing, the warm fluid is poured into sterile Petri dishes, where it solidifies
when it cools. The plate is inoculated by placing a drop of water containing the algae on the
surface of the agar, and streaking with a sterile implement. The plates are then maintained under
standard culture conditions.

Seawater Base

The quality of water used in media preparation is very important. Natural seawater can be collected
near shore, but its salinity and quality is often quite variable and unpredictable, particularly in
temperate and polar regions (due to anthropogenic pollution, toxic metabolites released by algal
blooms in coastal waters). The quality of coastal water may be improved by ageing for a few
months at 48C (allowing bacteria degradation), by autoclaving (heat may denature inhibitory sub-
stances), or by filtering through acid-washed charcoal (which absorbs toxic organic compounds).
Most coastal waters contain significant quantities of inorganic and organic particulate matter,
and therefore must be filtered before use (e.g., Whatman no. 1 filter paper).