An overview of cultivation methods and the reasons for cultivating microalgae.

Marine Aquaculture April 22, 2014 Print Friendly and PDF

Microalgae are, literally, “little seaweeds.”  They are living microorganisms that are single-celled or colonial, like bacteria and protozoa.  They are different, however, from most other microbes in that they use the energy of sunlight to make their own food by photosynthesis.  Microalgae are the primitive ancestors of plants, and microalgae “invented” photosynthesis billions of years ago.  Microalgae are enormously diverse, in terms of their physical appearance and internal chemistry.  They can be found in the sea, in freshwater, in soil, on rocks, and even in snow, providing evidence of the sustainability of microalgae in the earth’s sunlit ecosystems.  In fact, approximately half of the oxygen put into the earth’s atmosphere each day comes from microalgae, with the rest coming from land plants.

Just as plants are the base – the beginning – of the food webs on land, microalgae, also termed phytoplankton, provide the base of aquatic food webs.   Fueled by the sugar produced by photosynthesis, microalgae absorb mineral elements, mainly the nitrogen and phosphorus found in garden fertilizers, to increase in numbers. As single-celled organisms, when they grow, they don’t get bigger, you get more of them.  Their biomass is a mixture of proteins, carbohydrates, and fats, providing nutritious food for tiny animals such as copepods as well as oysters, clams, and mussels.  The smorgasbord of thousands of microalgal species in natural waters somehow satisfies the nutritional needs of all the animals that live there, but as humanity makes the transition from hunter to farmer in the sea and freshwater, just as we have on land, it makes sense to select only the most nutritious species of microalgae to cultivate as feeds for aquatic and marine animals that eat microalgae.

History of Microalgae Cultivation

The isolation, selection, and ultimately mass-cultivation of microalgae for use in aquaculture are ongoing steps in our transition from hunter to farmer.  Domestication of microalgae has required higher levels of technology than were available 10,000 years ago when land farming began.  Because microalgae are too small to see without a microscope, we did not even know what they looked like until the microscope was invented, although we could see the water discolored green, brown, or red when they “bloomed.”  As the description and naming of microalgae by microscopists proceeded, there was a natural inclination to “capture” these tiny organisms, many of which swim actively, and keep them alive in containers to observe their activity, growth, and etc.  Near the end of the 19th Century, methods using tiny pipettes, agar plates, and various dilution techniques were developed, and pure cultures of algae propagated from single cells were established (Pringsheim, 1912).  The single cell isolated in a container of water with nutrients and light divided, eventually giving rise to a population of genetically-identical individuals; this is the technical definition of a “clone.”  Many of the algal strains used in aquaculture are clones.

Once clonal cultures of microalgae were obtained, scientists sought to propagate these cultures, mainly for scientific study.  Observations in nature had revealed that many tiny animals eat microalgal cells, so some research was directed toward describing and even quantifying feeding rates of the so-called “zooplankton” that eat phytoplankton.  For feeding studies to be possible, larger and larger quantities of microalgae were required, so larger culture vessels were employed allowing successful mass-cultivation of microalgae.  It was scientific enquiry, not farm efficiency, which drove the early development of microalgal culture during the early 20th Century.

The mid-20th Century saw the beginning of the “modern” aquaculture era (Nash, 2011); this recent, science-based development is differentiated from ancient practices, mainly in Asia, that encouraged natural aquatic ecosystems to produce fish and other human foods.  Building upon scientific knowledge of the feeding habits of both finfish and shellfish, entrepreneurs established commercial farming operations. Cultured microalgae figure heavily in shellfish farming (Korringa, 1976) and also are used in live-feed production for larvae of some marine finfish larval rearing (Stottrup and McEvoy, 2003).  In all cases, it is the young stages, generally larvae and early post-larval stages, which feed on microalgae.

As mentioned, scientific research on the food habits of animals that eat microalgae drove the development of culture methods for the microalgae themselves.  The molluscan shellfish sector led these advances, with scientific reports appearing in the 1950s-1970s that laid the groundwork for microalgal culture practices in use today (Davis and Guillard, 1958; Ukeles, 1976).  As aquaculture’s contribution to seafood production increases, and with the recent interest in microalgal-based biofuel production, engineers have been hard at work designing bioreactors and pond-management schemes employing automated monitoring and control equipment borrowed from other industries.  These advances, however, have been slow to infiltrate hatcheries that mainly continue to rely upon mid-20th Century technologies that, while not optimized, are somewhat reliable and are not dependent upon advanced technologies.

Microalgal Culture Techniques

So then, how are microalgae cultivated in a typical hatchery?  First, strains of pure cultures are obtained from research institutions or commercial suppliers.  These strains must be maintained alive at the hatchery to be available for inoculating the large-scale cultures fed to animals.  Most hatcheries maintain stock cultures just as a research laboratory does – by subculturing, or transplanting, a small volume from a small flask culture (125 -250 milliliters) that has grown up into a new flask containing sterilized culture medium.  The most common microalgal enrichment added to salt or brackish water is referred to as the “f/2” recipe; pre-mix products available commercially have largely replaced “home-made” recipes in the past few decades.  Flasks of media generally are sterilized in a pressure-cooker because an autoclave that would be used in a research laboratory is too expensive for a hatchery. 

Transplanting stock cultures to maintain a “seed bank” in the hatchery generally is done every 2 weeks or so.  When it is time to inoculate a larger-scale culture, the contents of the stock flask is poured into a larger flask (1-3 liters) that may or may not be steam-sterilized.  Such intermediate flasks grow between one and three weeks before being used to inoculate the next-larger stage.  Eventually, 20-liter bottles generally are inoculated; at this scale, or smaller, most hatcheries do not have the capacity to steam-sterilize. 

Methods used to eliminate unwanted microorganisms from natural water used to culture microalgae – the unwanted guests include other microalgae, predatory protozoans, molds, and most bacteria – include filtration, pasteurization, and bleaching (chlorination-dechlorination).  Once pests have been removed from water and chemical nutrients have been added, the other resources needed by microalgae to grow are light and carbon dioxide.  In many hatchery settings, artificial light (light bulbs) can be used to supply energy for photosynthesis in microalgal cultures.  This does have the advantage of predictability, but it is difficult to supply sufficient artificial light to dense algal cultures from bulbs, and the cost of electricity to light microalgal cultures accounts for over 90% of the cost of growing algae.  More recently, greenhouses have been integrated into hatcheries to take advantage of natural sunlight for microalgal cultures.  Carbon dioxide is also an essential reactant in photosynthesis. Carbon dioxide is fed to the algae cultures through the air supply. Physical mixing with propellers or paddlewheels is common in industrial algal cultures grown for extractable chemicals and is being seen more often in aquaculture.

In some hatcheries, sufficient quantities of microalgae are produced in 20-liter bottles.  Generally, though, larger containers are needed to produce sufficient quantities.  The most common microalgal culture containers in hatcheries are 2-4-meter tall tubes made of transparent fiberglass sheeting (Kalwall tubes) or plastic bags in wire-mesh frames that contain 200-500 liters of culture.  Some of the relatively recent innovations in microalgal culture containers include round, shallow tanks that can take advantage of natural sunlight or be lit by cheaper-to-operate vapor or halide bulbs, “raceway” systems that circulate shallow cultures around an oval “racetrack,” and horizontal, partially-inflated bag cultures.  Round tank cultures tend to not be shallow enough for gas exchange or light penetration, so these are bubbled or stirred with a foil to mix. 

Once the container has been decided upon, one has three options for culture management.  By far, most aquaculture hatchery microalgal cultures are operated as batch cultures.  In a batch culture, a tank is filled with medium, inoculated, allowed to grow, harvested completely, washed, and re-filled for the next inoculation.  Batch culture management is easy to understand and it allows flexibility in when in the culture cycle one harvests, but it is very labor intensive and prone to deadly gaps in feed availability if there are problems in the inoculum chain.  Many of the newer, “bag systems” employ continuous culture, in which medium is pumped steadily into a culture unit, which overflows finished algal culture at the same rate as the inflow of medium.  This management scheme saves labor by minimizing daily attention needed and should yield a dependable quantity and quality of algal feed over time.  One downside of continuous culture is that the systems generally are run at much slower flow rates than could be sustained as a hedge against variable growth rates arising from changes in light, temperature, etc., so the hatchery operator devotes more space than necessary to algal feed cultures.  Another disadvantage is that recovery time from a catastrophic failure can be long because there is not a line of inoculum cultures being produced.  A compromise culture-management scheme that combines aspects of both batch and continuous culture is semi-continuous culture.  In this approach, once an algal culture is established, portions are harvested periodically, medium is added to replace harvested volume, and the culture is given sufficient time to grow before the next harvest.  Think of semi-continuous culture as “cutting the grass.”  This strategy reduces the daily labor of washing and refilling and it allows the operator to optimize production according to culture performance.  Increasingly, hatcheries are adopting semi-continuous culture management.

As implied above, microalgae are cultivated for use in several types of hatcheries.  In molluscan shellfish hatcheries, larvae are fed mixtures of diatoms, prymnesiophytes (the ‘golden-brown’ flagellates), and some prasinophytes, known commonly as the “scaly green algae.” until they undergo metamorphosis.  Thereafter, diatoms and prasinophytes generally are used for the short time between metamorphosis and transfer of post-set to nursery systems where natural plankton provides the nutrition.  In shrimp hatcheries, first-feeding larvae are fed prymnesiophytes and diatoms, and zooplankton feeds for later developmental stages of shrimp, such as rotifers and brine shrimp, may be enriched with cultured microalgae, although artificial, prepared products have largely replaced microalgae as enrichments.  In marine finfish hatcheries growing species with feeding larval stages, a method called “the greenwater technique” often is used.  Prey for larval fish, rotifers and brine shrimp, are introduced to larval tanks along with a sufficient quantity of microalgae that the water becomes discolored.  The presumable reason for the success of this method, compared to clear-water culture, is that the zooplankton are feeding continuously on microalgae, making them more nutritious and easily visible to the predatory larval fish.  There may also be water-quality benefits to greenwater, as the microalgae consume respiratory carbon dioxide from the fish, add oxygen to the water, and absorb nitrogenous wastes, mainly ammonia, from the fish.  These hatchery applications are the most widespread uses of microalgal cultures.

Biofuels

As mentioned, application of existing knowledge in commercial shellfish and finfish hatcheries has lagged behind new scientific findings about the limitations of current methods.  The expansion of hatchery-base aquaculture will rely upon improvements in dependability and economy of microalgal feed cultures.  A recent explosion of investment in microalgal biofuel research has the potential to yield unanticipated breakthroughs that may benefit hatchery operation.  To date, this has not occurred or has not been recognized; therefore, continued, incremental improvement in farming methods, informed by existing scientific knowledge, seems likely in the near future.

The aforementioned spike in interest in microalgal biofuel production is in the research phase now, and transition to commercial production remains uncertain.  Existing, industrial applications of microalgal culture mainly depend upon high-value, extractable chemical components.  Large, raceway systems growing the marine chlorophyte Dunaliella in several countries supply most of the world’s demand for the antioxidant pigment beta-carotene.  Astaxanthin, the pigment responsible for the pink color of salmon flesh, is extracted from mass cultures of the freshwater chlorophyte Haematoccus.  A number of companies have established mass production of the freshwater cyanobacterium Spirulina, but a high-value commercial market for this organism has been slow to develop.  Because microalgal culture is relatively demanding technically, compared to land plant cultivation, future applications of microalgal culture most likely will be driven by discovery of new chemical components of very high value unique to certain microalgae.  Otherwise, expansion of seafood aquaculture and need for hatchery seed will be the main drivers of technological innovation and application in microalgal cultivation in the near future.

 

References Cited

Davis, H.C. and Guillard, R. R. 1958. Relative value of ten genera of micro-organisms as food for oyster and clam larvae. U. S. Fish & Wildl. Serv., Fish. Bull. 58, 293-404.

Korringa, P.  1976.  Farming Marine Organisms Low in the Food Chain.  Elsevier Scientific Publishing Company, New York.  264 pp.

Nash, C.E.  2011.  The History of Aquaculture.  Wiley-Blackwell, Ames, Iowa, USA.  227 pp.

Pringsheim, E.G.  1912.  Kulturversuche mit chlorophyllfuhrenden mikroorganismen.  Beitr. Biol. Pft. 11, 305-332.

Stottrup, J.G and McEvoy, L.A. (Eds.).  2003.  Live Feeds in Marine Aquaculture.  Blackwell Science, Oxford, UK.  318 pp.

Ukeles, R.  1976.  Cultivation of plants. Mar. Ecol. 3, 367-466.

 

Useful Resources

 

Anderson, R.A.  2005.  Algal Culturing Techniques.  Academic Press, Cambridge, MA, USA.  578 pp.

Kirk, J.T.O.  1983.  Light and photosynthesis in aquatic ecosystems.  Cambridge University Press, UK.  401 pp.

Richmond, A. (Ed.).  2008.  Handbook of Microalgal Culture: Biotechnology and Applied Phycology.  John Wiley and Sons, New York.  584 pp.

Richmond, A.  1986.  CRC Handbook of Microalgal Mass Culture.  Taylor and Francis Group, New York.  528 pp.  

Van Patten, P., Li, J.Y., and Wikfors, G.H.  2012.  A Student’s Guide to the Common Phytoplankton of Long Island Sound.  Connecticut Sea Grant web page:  http://seagrant.uconn.edu/publications/marineed/phytoplankton/phytoplank...

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This work is supported by the USDA National Institute of Food and Agriculture, New Technologies for Ag Extension project.