Freshwater Media

Freshwater media are generally selected because they possess characteristics similar to the natural environment or they differentially select for a specific algal component of the habitat. Media of an artificial nature, with known chemical composition, are often employed as additives to natural media with an unknown chemical composition, such as lake water, to enrich them. They are often used to simulate diverse nutritional or physical requirements of a particular species or groups of species, especially when the exact nutritional requirements are unknown.

Media are generally prepared from premixed stock solutions. Aliquots from these stocks are measured and added to a given volume of water. Some, however, must be prepared by weighting or measuring the desired components and adding them directly to a given volume of liquid. Accuracy in measuring liquid aliquots from stock solution or water, and weighing of chemicals is essential. Improper procedures may result in precipitation of one or more of the components of the medium, such as nitrates and phosphates, or a failure of some of the constituents to go into solutions.

Stock solution can be prepared and stored at low temperature in tightly sealed glassware, because evaporation may alter initial concentrations.

Water generally employed for freshwater media should belong to one of the following types: copper-distilled water; single glass-distilled water; double glass-distilled water; membrane filtered water; and deionized water. In most laboratories single or double-distilled water is routinely used, which can be deionized by passing it through a prepacked deionizing column.

As for the marine media, also freshwater media can be “defined” and “undefined.” Defined medium such as Beijerinck or Bold Basal Medium have been proved successful for many algal classes. Most of these defined media can be used for additional algal groups by adding a variety of other components or modifying the amounts of certain reagents. These “undefined” media often have the advantage of supporting growth of large number of different algal species, but, when highly organic, they have also the disadvantage of encouraging more bacterial growth than strictly inorganic media.

Some of the most commonly used freshwater media, defined and undefined, are listed in:

• Table 6.2: BG11 Medium Composition
• Table 6.3: Diatom Medium Composition
• Table 6.4: DY-III Medium Composition
• Table 6.5: Aaronson Medium Composition
• Table 6.6: Cramer and Myers Medium Composition
• Table 6.7: Beijerinck Medium Composition
• Table 6.8: Bold Basal Medium Composition
• Table 6.9: Mes-Volvox Medium Composition

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 incubation, 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 nutrients, 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 atmosphere in an autoclave,CO₂ is driven out of the seawater and the pHis 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 CO₂ 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 ethylenediaminetetraacetic 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 septically 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 4°C (allowing bacteria degradation), by autoclaving (heat may denature inhibitory substances), 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).

The low biomass and continual depletion of many trace elements from the surface waters of the open ocean by biogeochemical processes makes this water much cleaner, and therefore preferable for culturing purposes. Seawater can be stored in polyethylene carboys, and should be stored in cool dark conditions.

Artificial seawater, made by mixing various salts with deionized water, has the advantage of being entirely defined from the chemical point of view, but it is very laborious to prepare, and often does not support satisfactory algal growth. Trace contaminants in the salts used are at rather high concentrations in artificial seawater because so much salt must be added to achieve the salinity of full strength seawater. Commercial preparations are available, which consists of synthetic mixes of the major salts present in natural sea water, such as Tropic Marine Sea Salts produced in Germany for Quality Marine (USA) and Instant Ocean Sea Salts by Aquarium System (USA).

Nutrients, Trace Metals, and Chelators
The term ‘nutrient’ is colloquially applied to a chemical required in relatively large quantities, but can be used for any element or compound necessary for algal growth.

The average concentrations of constituents of potential biological importance found in typical seawater are summarized in Table 6.10. These nutrients can be divided into three groups, I, II, and III; with decreasing concentration:

• Group I. Concentrations of these constituents exhibit essentially no variation in seawater, and high algal biomass cannot deplete them in culture media. These constituents do not, therefore, have to be added to culture media using natural seawater, but do need to be added to deionized water when making artificial seawater media.
• Group II. Also have quite constant concentrations in seawater, or vary by a factor less than 5. Because microalgal biomass cannot deplete their concentrations significantly, they also do not need to be added to natural seawater media. Standard artificial media (and some natural seawater media) add molybdenum (as molybdate), an essential nutrient for algae, selenium (as selenite), which has been demonstrated to be needed by some algae, as well as strontium, bromide, and flouride, all of which occur at relatively high concentrations in seawater, but none of which have been shown to be essential for microalgal growth.
• Group III. All known to be needed by microalgae (silicon is needed only by diatoms and some chrysophytes, and nickel is only known to be needed to form urease when algae are using urea as a nitrogen source). These nutrients are generally present at low concentrations in natural seawater, and because microalgae take up substantial amounts, concentrations vary widely (generally by a factor of 10 to 1000). All of these nutrients (except silicon and nickel in some circumstances) generally need to be added to culture media in order to generate significant microalgal biomass.

Nitrate is the nitrogen source most often used in culture media, but ammonium can also be used, and indeed is the preferential form for many algae because it does not have to be reduced prior to amino acid synthesis, the point of primary intracellular nitrogen assimilation into the organic linkage. Ammonium concentrations greater than 25 µM are, however, often reported to be toxic to phytoplankton, so concentrations should be kept low.

Inorganic (ortho)phosphate, the phosphorus form preferentially used by microalgae, is most often added to culture media, but organic (glycero)phosphate is sometimes used, particularly when precipitation of phosphate is anticipated (when nutrients are autoclaved in the culture media rather than separately, e.g.). Most microalgae are capable of producing cell surface phosphatases, which allow them to utilize this and other forms of organic phosphate as a source of phosphorus. The trace metals that are essential for microalgal growth are incorporated into essential organic molecules, particularly a variety of coenzyme factors that enter into photosynthetic reactions. Of these metals, the concentrations (or more accurately the biologically available concentrations) of Fe, Mn, Zn, Cu and Co (and sometimes Mo and Se) in natural waters may be limiting to algal growth. Little is known about the complex relationships between chemical speciation of metals and biological availability. It is thought that molecules that complex with metals (chelators) influence the availability of these elements. Chelators act as trace metal buffers, maintaining constant concentrations of free ionic metal. It is the free ionic metal, not the chelated metal, which influences microalgae, either as a nutrient or as a toxin. Without proper chelation some metals (such as Cu) are often present at toxic concentrations, and others (such as Fe) tend to precipitate and become unavailable to phytoplankton. In natural seawater, dissolved organic molecules (generally present at concentrations of 1–10 mg l^-1) act as chelators. The most widely used chelator in culture media additions is EDTA, which must be present at high concentrations because most complexes with Ca and Mg, present in large amounts in seawater. EDTA may have an additional benefit of reducing precipitation during autoclaving. High concentrations have, however, occasionally been reported to be toxic to microalgae. As an alternative the organic chelator citrate is sometimes utilized, having the advantage of being less influenced by Ca and Mg. The ratio of chelator:metal in culture medium ranges from 1:1 in f/2 to 10:1 in K medium. High ratios may result in metal deficiencies for coastal phytoplankton (i.e., too much metal is complexed), and many media therefore use intermediate ratios.

In today’s aerobic ocean, iron is present in the oxidized form as various ferric hydroxides and thus is rather insoluble in seawater. While concentrations of nitrogen, phosphorus, zinc, and manganese in deep water are similar to plankton elemental composition, there is proportionally 20 times less iron in deep water than is apparently needed, leading to the suggestion that iron may be the ultimate geochemically limiting nutrient to phytoplankton in the ocean. Very little is known about iron in seawater or phytoplankton uptake mechanisms due to the complex chemistry of the element. Iron availability for microalgal uptake seems to be largely dependent on levels of chelation. It is highly recommended that iron be added as the chemically prepared chelated iron salt of EDTA rather than as iron chloride or other iron salts; the formation of iron chelates is relatively slow, and iron hydoxides will form first in seawater, leading to precipitation of much of the iron in the culture medium.

Apparently, as a result of the extreme scarcity of copper in anaerobic waters, copper did not begin to be utilized by organisms until the earth became aerobic and copper increased in abundance. Consequently copper does not seem to be an obligate requirement, algae either not needing it, or needing so little that free ionic copper concentrations in natural seawater are sufficient to maintain maximum growth rates. Copper may indeed be toxic, particularly to more primitive algae, and hence copper, if added to culture media at all, should be kept at low concentrations. The provision of manganese, zinc, and cobalt in culture medium should not be problematical because even fairly high concentrations are not thought to be toxic to algae.

Vitamins
Roughly all microalgal species tested have been shown to have a requirement for vitamin B12, which appears to be important in transferring methyl groups and methylating toxic elements such as arsenic, mercury, tin, thallium, platinum, gold, and tellurium, around 20% need thiamine, and less than 5% need biotin.

It is recommended that these vitamins are routinely added to seawater media. No other vitamins have ever been demonstrated to be required by any photosynthetic microalgae.

Soil Extract
Soil extract has historically been an important component of culture media. It is prepared by heating, boiling, or autoclaving a 5–30% slurry of soil in fresh water or seawater and subsequently filtering out the soil. The solution provides macronutrients, micronutrients, vitamins, and trace metal chelators in undefined quantities, each batch being different, and hence having unpredictable effects on microalgae. With increasing understanding of the importance of various constituents of culture media, soil extract is less frequently used.