TEMPERATURE
The temperature at which cultures are maintained should ideally be as close as possible to the temperature at which the organisms were collected; polar organisms (<10°C); temperate (10–25°C); tropical (>20°C). Most commonly cultured species of microalgae tolerate temperatures between 16 and 27°C, although this may vary with the composition of the culture medium, the species, and strain cultured. An intermediate value of 18–20°C is most often employed. Temperaturecontrolled incubators usually use constant temperature (transfers to different temperatures should be conducted in steps of 2°C per week), although some models permit temperature cycling. Temperatures lower than 16°C will slow down growth, whereas those higher than 35°C are lethal for a number of species.

LIGHT

As for plants, light is the source of energy which drives photosynthetic reactions in algae and in this regard intensity, spectral quality, and photoperiod need to be considered. Light intensity plays an important role, but the requirements greatly vary with the culture depth and the density of the algal culture: at higher depths and cell concentrations the light intensity must be increased to penetrate through the culture. Too high light intensity (e.g., direct sunlight, small container close to artificial light) may result in photoinhibition. Most often employed light intensities range between 100 and 200 µE sec-1 m-2, which corresponds to about 5–10% of full daylight (2000 µE sec-1 m-2). Moreover, overheating due to both natural and artificial illumination should be avoided. Light may be natural or supplied by fluorescent tubes emitting either in the blue or the red light spectrum, as these are the most active portions of the light spectrum for photosynthesis. Light intensity and quality can be manipulated with filters. Many microalgal species do not grow well under constant illumination, although cultivated phytoplankton develop normally under constant illumination, and hence a light/dark (LD) cycle is used (maximum 16:8 LD, usually 14:10 or 12:12).

pH
The pH range for most cultured algal species is between 7 and 9, with the optimum range being 8.2–8.7, though there are species that dwell in more acid/basic environments. Complete culture collapse due to the disruption of many cellular processes can result from a failure to maintain an acceptable pH. The latter is accomplished by aerating the culture. In the case of high-density algal culture, the addition of carbon dioxide allows to correct for increased pH, which may reach limiting values of up to pH 9 during algal growth.

SALINITY
Marine algae are extremely tolerant to changes in salinity. Most species grow best at a salinity that is slightly lower than that of their native habitat, which is obtained by diluting sea water with tap water. Salinities of 20–24 g l21 are found to be optimal.

MIXING
Mixing is necessary to prevent sedimentation of the algae, to ensure that all cells of the population are equally exposed to the light and nutrients, to avoid thermal stratification (e.g., in outdoor cultures), and to improve gas exchange between the culture medium and the air. The latter is of primary importance as the air contains the carbon source for photosynthesis in the form of carbon dioxide. For very dense cultures, the CO₂ originating from the air (containing 0.03% CO₂) bubbled through the culture is limiting the algal growth and pure carbon dioxide may be supplemented to the air supply (e.g., at a rate of 1% of the volume of air). CO₂ addition furthermore buffers the water against pH changes as a result of the CO₂/HCO₃^- balance.

Mixing of microalgal cultures may be necessary under certain circumstances: when cells must be kept in suspension in order to grow (particularly important for heterotrophic dinoflagellates); in concentrated cultures to prevent nutrient limitation effects due to stacking of cells and to increase gas diffusion. It should be noted that in the ocean cells seldom experience turbulence, and hence mixing should be gentle. Depending on the scale of the culture system, mixing is achieved by stirring daily by hand (test-tubes, erlenmeyers), aerating (bags, tanks), or using paddle wheels and jet pumps (ponds). Not all algal species can tolerate vigorous mixing. The following methods may be used: bubbling with air (may damage cells); plankton wheel or roller table (about 1 r.p.m.); and gentle manual swirling. Most cultures do well without mixing, particularly when not too concentrated, but when possible, gentle manual swirling (once each day) is recommended.