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The following pages are excerpts from:
Plankton Culture
Manual
Fifth Edition
Frank H. Hoff & Terry W. Snell
Florida Aqua Farms, Inc. 1999
ISBN: 0-9662960-0-1
Culturing Phytoplankton
Biomass Production
Phytoplankton or microalgae, are the synthesizers of organic matter in aquatic
environments. Microalgae utilize photosynthesis to construct complex carbon molecules like
the more familiar land plants. Dissolved nutrients from various sources are absorbed by
microalgae and, in the presence of light, combined into more complex molecules necessary for
survival. Microalgae are major contributors to the production of biomass in oceans, estuaries,
lakes, and reservoirs. Although small, the combined effects or billions of these cells provide
most of the plant material consumed by animals higher in the food web. The entire food web
receives its energy from the biomolecules synthesized by these microscopic plants. For
example, production on the surface of the open ocean is about 50 grams or approximately one
cup of carbon per square meter per year, almost all of which is due to phytoplankton
photosynthesis (carbon atoms are the building blocks of all complex organic molecules). With
this kind of productivity, it is no wonder the open ocean and phytoplankton with are referred to
as the pastures of the sea.
Nutrient recycling
In addition to supplying food for animals, phytoplankton play a central role in nutrient
cycling in aquatic habitats. Microalgae absorb primary nutrients such as ammonia, urea,
nitrates, phosphates, and potassium, as well as metals such as iron, copper, manganese, zinc,
molybdenum, and vanadium. Certain vitamins such as B12, thiamin, and biotin also have been
found to be essential or beneficial to many algae species and are selectively absorbed or
produced by some species. Removal and alteration of nutrients by phytoplankton in the ocean
is not permanent, since nutrients are slowly being regenerated as microalgae cells die and
decompose. Other sources of dissolved nutrients include terrestrial runoff, rain, and coastal
up-welling. In marine and freshwater aquaria or culture tanks, phytoplankton help maintain
water quality through their removal of excess nutrients and the regulation of pH. In effect,
each algae cell is a living bio filter.
Source of various products
Microalgae are capable of rapid population growth and play a major role in nutrient
recycling. In addition, they help balance the pH of aquatic systems by removing excess carbon
dioxide and adding oxygen. They have been cultured to produce oils, chemicals,
pharmaceuticals, and polysaccharides. At least 40 food manufacturers in the USA are currently
utilizing Spirulina as food supplements or ingredients.
As the effects of fossil fuel use become of increasing concern, scientists are looking to
microalgae as a high yield, carbon neutral source of biodiesel. Extensive research is being
conducted using many species including Chlorella protothecoides, Botyrococcus braunii, and
Chlamydomonas reinhardtii.
Nutrient Uptake
Most species of algae are photoautotrophs that derive energy from light and carbon dioxide to
obtain carbon atoms. Some are also true heterotrophs that do not requires light and can acquire energy
and carbon from organic compounds such as sugars and organic acids. Yet, many other species are
mixotrophic and can reproduce in light or darkness. Heterotrophic and mixotrophic microalgae are
found in virtually every taxonomic class. Photoautotrophic algae that also have ability to obtain energy
heterotrophically often grow at increased rates by simultaneously utilizing light and carbon dioxide as
well as organic substances. Under commercial pharmaceutical applications densities 1000 times higher
can be achieved then under phototrophic conditions. Some algae have been labeled as “obligate
photoautotrophs” based on experimentation at ambient nutrient levels. Even some of these species can
become heterotrophic when nutrient levels are substantially above or below natural conditions.
Examples are Brachiomonas submarina and some strains of Haematococcus pluvialis which can reduce
nitrate when growing photosynthetically, but are unable to do so in the dark. When supplied with a
reduced nitrogen source such as ammonia, they were found to grow heterotrophically. Other examples
are Prymnesium parvum and Chroomonas (Pyrenomonas) salina which are unable to grow
heterotrophically on low natural levels of glycerl, but can do so when supplied with very high
concentrations (.25M). Many microalgae are grown heterotrophically to produce pharmaceutical
products.
Algal Growth Media
Chemical features of the aquatic environment play an important role in determining algae
growth rates and quality. Microalgae and macroalgae derive nutrients from the surrounding water.
They have no roots like land plants, but are able to absorb nutrients directly across their cell
membranes.
Since the objective of culturing algae is to obtain the highest density in the shortest possible
time, the utilization of natural seawater or freshwater with limited enrichment will not yield good
results. In natural waters, trace metal concentrations are usually sufficient, but either nitrogen or
phosphorus is usually limiting. Phosphorus limitation is more common in freshwater, whereas nitrogen
limitation is more common in the sea. Cultured microalgae grow best in media with quite different
primary and trace nutrient composition than natural waters. Nitrate levels in intensive microalgae
cultures are commonly 100 to 1000 times higher than that found in nature.
Microalgae growing in intensive cultures are therefore quite different from those growing in
nutrient-limited natural waters. Natural algae populations are subject to marked changes in
temperature, light intensity and duration, leading to unpredictable growth rates. It is usually possible
for aquaculturists to improve both predictability of microalgae growth and biomass yield by using
controlled intensive culture methods. Higher cell densities hand more consistent nutritional quality
frequently the results of intensive culture. Generally, smaller culture vessels offer more control over the
culture environment and algae yields. Upscaling from small flasks to large tanks or even outdoor ponds
cannot be done without culture modifications, sacrifice of control, loss of nutrient balance, and yield
loss per unit volume. Algae densities are usually lower in larger culture vessels, but what is most
important is the number of alga cells per unit volume. For example, in a 100-liter vessel you consistently
reach 20 million cells/ml, but in a 1000 liter vessel you may only be able to achieve 5 million cells/ml in
the same period of time. Based on the fact that smaller vessels are easier to control, it is usually better
to use five 100-liter culture vessels rather than one 500-liter vessel. Problems in upscaling are one of
the areas most needing research.
Physical Requirements
Microalgae are hardy plants, yet still require specific environmental conditions to grow well. As
with any living organism, physical conditions have a large influence on the growth of microalgae. Each
species has a particular range of temperatures, light intensities, spectral preferences, salinities, and
oxygen/carbon dioxide ratios that yield maximum growth. Some microalgae grow at temperatures
below 10oC, while some blue-greens grow best around 60oC. Optimal growth temperatures vary with
alga species, light intensity, nutrient concentration and composition, as well as acclimation temperature.
Continuous agitation may be required for species found living in swift currents. Other species live in
seafoam at salinities as high as 85 ppt (parts of salt per thousand parts of water). It takes a great deal of
controlled experimentation to determine the optimal growth conditions for a particular species, but
fortunately, this information is known for many species valuable to aquaculturalists.
Light
A light intensity of 2500-5000 lux is optimal for microalgae. Light output from a bulb is often
listed in lumens which is synonymous with the term lux. The spectral qualities or wavelengths of the
common “cool white” bulb are poor for intense microalgae culture. Most algae respond to red and blue
light. Bulb aging is very important since its output and spectral proportions change over time. Yearly
bulb replacement is essential.
Fluorescent light fixtures should ideally be mounted about six inches away, either horizontally or
vertically, depending on the height of the culture vessels. Lighting can be controlled by a simple light
timer and set for a minimum of 16 to 24 hours of light. Depending on your particular conditions you
may get better results using continuous light. However, photoinhibition is possible if the light is too
intense for too long. Small containers, high temperatures, and nutrient deficiencies increase the
chances of causing photoinhibition.
Light shading by algae cells may become limiting as density increases. At high densities,
incoming light is shaded from all but the cells currently at the outer surface of the container. Shaded
cells become light-limited as their photosynthetic capacity is diminished. Therefore, the configuration of
the culture vessel, light intensity and duration, coupled with ideal circulation patterns become more
important as cell densities increase. Utilizing algae that grow mixotrophically may have a distinct
advantage in these intense cultures.
Temperature Requirements
Microalgae that most aquaculturalists are interested in culturing are tropical strains which
respond best to temperatures between 16o-27oC, with an optimum of about 24oC. Small daily
flucturations within this range present no serious problems. Lower temperatures will not kill the algae,
but will drastically slow growth, whereas sustained temperatures above 35oC will kill most microalgae.
Chemical Requirements
Good growth of a microalgae culture is dependent on a proper balance between specific major
and minor nutrients. Imbalances in these nutrients usually lead rapidly to cessation of culture growth.
Exhaustion of nutrients is one of the most important factors limiting growth and controlling nutritional
quality in mass culture. Many fertilizers contain inadequate amount of nitrogen or unstable metals,
espe3cially iron, which can lead to retarded microalgae growth. In alkaline solutions, iron and other
metals often precipitate over time. Chelating agents such as EDTA are used to maintain metals in
solution, making them available to microalgae.
In addition to macronutrients, micronutrients consisting of trace metals and vitamins are usually
required for optimal growth. Most microalgae are auxotrophic, so they are unable to synthesize all the
necessary vitamins and must assimilate them from their environment. Thiamin (B1) , cobalamin (B12
and biotin are considered essential vitamins for may microalgae. In fact, it has been estimated that
about 70% of all planktonic algae require vitamin B12.
Inorganic elements essential to many species of algae include: nitrogen, phosphorus, potassium,
magnesium, calcium, sulfur, iron, copper, manganese, zinc, molybdenum, sodium, cobalt, vanadium,
silicon, chlorine, boron and iodine. Of these, nitrogen, phosphorus, magnesium, iron, copper
manganese, zinc, and molybdenum are required by all algae.
pH
Excessively high or low pH depresses microalgae growth by disrupting many cellular processes.
If nitrogen is supplied to cultures as an ammonium salt, most microalgae will selectively take up the
ammonium ion, thus lowering the pH. Conversely, if nitrate ions are removed by algae, pH tends to
increase, but is usually buffered by carbon dioxide supplied to the cultures. However if carbon dioxide
becomes limiting, culture pH can climb as 11, eventually killing the microalgae. If the culture is
maintained under ambient or a controlled light/dark cycle, pH elevates during the light period and drops
during the dark period, tending to provide pH balance.
Alternative carbon sources like glucose, glycerol, lactic and acetic acid have been used with
mixed results. Acetic acid not only lowers pH, it dissociates to provide a carbon source. Use of sugars
often leads to excessive bacteria growth if microalgae densities are low. Since many microalgae have
the ability to grow mixotrophically, utilization of mixed inorganic and organic nutrients are often used.
Bacterial and microalgae associations
The associations between microalgae and bacteria are becoming better understood. It ahs been
suggested that the best algae growth occurs in bacteria-free (axenic) cultures, conditions that are
difficult and expensive to maintain. However, as more experience with microalgae culture accumulates,
it appears that many microalgae species grow best in association with bacteria. This is perhaps the
result of nutrient recycling or nutrient complementation between algae and bacteria. An example is
found in Asterionella japonica, which grows satisfactorily in culture as long as bacteria are present, but
ceases to grow axenically, even when several organic supplements are provided.
Ionic Composition of Local Waters
The ionic composition of local water, especially hardness (calcium and magnesium levels) can
affect microalgae growth. Newly inoculated cultures may take a few days to adapt to the characteristics
of local water and grow slowly at first. Other indications of a water problem are algae clumping,
settling, or failing to achieve exponential growth. Often these problems can be overcome by mixing
deionized or distilled water with local water to start your cultures, then gradually introducing them to
full strength local water. Vigorous stirring several times a day helps prevent clumping and settling.
Clumping also can occur if phosphate concentrations are too high.
Aquarium Salts
If you are culturing marine microalgae, it is important to select a good quality aquarium salt
mixture that is well balanced, like Instant Ocean TM, Forth Fathoms TM, or Reef Crystals TM. Rock salt
commonly used in water softeners, or table salt are not suitable because their ionic compositions do not
resemble seawater. Since most microalgae are found in bay systems and coastal waters the salinity
should be in the range of 12-27 ppt (1.015-1.020 density). A small box that can be resealed is best, or
you can store a portion of the newly opened salts in an airtight plastic container. Most brands contain
sodium thiosulfate for removing chlorine which is found in most city tap water. All salt mixtures go
through chemical changes when mixed, and must be stabliz3ed and balanced before starting a culture.
To be on the safe side, mix salts and heavily aerate for at least three to four hours before adding the
fertilizer and microalgae. Best results with all artificial salts are obtained by mixing salts in distilled
water. However, standard sterilized bottled water is suitable.
Fertilizer
We strongly advise you not to use common terrestrial plant fertilizers, especially those
containing ammonia or urea as a nitrogen source. Residual ammonia or urea from over-fertilized
cultures or those that are harvested prematurely may contain toxic levels of ammonia. In addition,
common agricultural fertilizers usually do not contain essential trace metals and vitamins suitable for
good microalgae growth and are often high in phosphates. The best advice when starting an algae
culture is to have patience. Many factors affect algae growth including: physiological conditions of
inoculant cells, water quality, light, temperature, fertilizer, and pH. Algae must acclimate to the
prevailing conditions before rapid cell division commences.