The Culture of Marine Phytoplankton

Introduction

The first step necessary to consider breeding any animal is the establishment of a reliable food source for the young. In general, hobbyists have little trouble providing for the needs of the adults, but frequently the care of the offspring requires more specific foods, and are more difficult to provide. This is especially true of most marine species, which have a biphasic life (there are two distinct life stages: larval and adult). Generally among species with a larval and adult stage, the larvae live in different habitats or feed on different prey items than do the adults.

Phytoplankton

Approximately 80% of marine organisms (about 90,000 species), both vertebrate and invertebrate, have such a biphasic life-cycle and produce planktonic larvae which spend some variable amount of time (ranging from minutes to months) developing in the water column before settling and metamorphosing into the adult life-form (Thorson 1964). True to form, these larvae are planktonic (live as freely-floating particles in the ocean), unlike the majority of the species in their adult forms, and generally tend to feed on entirely different foods than do the adults. As if the care of young with specialized feeding requirements were not enough of a hurdle to the hopeful home breeder, the majority of these marine larvae are microscopic and require suitably tiny prey items to ingest.
However, for those truly adventuresome (or foolhardy souls who don't consider asexual propagation of their tank inhabitants enough of a challenge, sexual propagation (involving the production and fusion of gametes through either copulation or free-spawning) of their reef inhabitants is the penultimate challenge.
Providing these suitably tiny particles is where the culture of phytoplankton (typically single-celled marine plants -- encompassing algae, diatoms, dinoflagellates, etc.) comes into play. Although this seems like an obvious an trivial thing, most people who have raised both phytoplankton and larvae consider the culture of marine invertebrate and fish larvae simple by comparison. If you plan to try breeding any of the critters in your aquarium, your first step should be to set up and successfully maintain a series of pure cultures of at least one, and hopefully several species of marine phytoplankton with which to feed any offspring produced. This sounds easy, but is generally much more difficult than it sounds. Many people have more trouble getting the algal cultures running well enough to start raising larvae than they do raising the larvae themselves.
First, let me start with some of the basics. There are two ways in which animals can reproduce: sexually and asexually. If the animals are asexual (no gamete production or fusion is involved), then a single individual can split itself into multiple copies (through various methods) and develop into a genetically identical colony to the original "parent" colony. In this case, there is obviously little special care required for propagating such animals at home -- if the parent colony can survive, then similarly good conditions and plenty of food usually suffice. Asexual reproduction broadly covers both the addition of units (such as cnidarian polyps or bryozoan zooids) to an existing colonial growth form, and the fission of individuals to produce two (or more) identical individuals that are not attached in any way (such as some cnidarians, echinoderms and polychaetes). The folks at GARF have dedicated a lot of time and energy to teaching hobbyists successful methods of asexual propagation of marine organisms. These web pages and newsletters provide a lot information regarding their techniques for successful asexual propagation of reef animals, and for most hobbyists, that is an admirable goal. However, for those truly adventuresome (or foolhardy) souls who don't consider asexual propagation of their tank inhabitants enough of a challenge, sexual propagation (involving the production and fusion of gametes through either copulation or free-spawning) of their reef inhabitants is the penultimate challenge. I give more detail on the breeding modes of marine invertebrates and larval culture in a series of articles for the Breeder's Registry periodical, The Journal of Maquaculture (Toonen, 1996a, 1997 a,b,c), and I discuss the biology and culture of rotifers in an article for Aquarium.Net (Toonen 1997d) -- but those subjects are not the focus of this article.
Instead, here I am going to concentrate on the most critical and often difficult step in the successful culture of marine invertebrate larvae: establishment of a successful and dependable phytoplankton culture. I assume that the average aquarist does not have the resources to purchase all their live food from a facility like Harbor Branch Oceanographic Institution (HBOI), so I will describe instead how to do it on your own at home. The first step to successfully culturing phytoplankton is obtaining both the nutrient supplement and the pure strain algal inoculant for your cultures. The supplement can come from a number of sources. First, you can buy sterile, premixed and measured aliquots of f/2 algal growth supplement from companies such as Carolina Biological Supply (1-800-334-5551), or Trans-Mississippi Biological Supply (1-800-544-5901). This is probably the easiest but also the most expensive way to feed your algal cultures.
Second you can buy bulk f/2 supplement from many aquaculture supply companies like Aquatic Ecosystems, Inc (1-800-422-3939), Fritz Aquaculture - now called Aquacenter (1-800-748-8921), Florida AquaFarms -- a division of HBOI's Aquaculture Facility -- (904) 567-8540, who provide a pre-mixed supplement which can be measured and added to culture vessels as needed. This second alternative is probably the best for people who want things as easy as possible, but still relatively inexpensive. The final method is the cheapest, but also the most work: you can mix your own nutrient supplement. Strathmann (1987) has a recipe based on Guillard's (1983) medium and the comments and adjustments of Bidwell and Spotte (1985), and Guillard (1975) is considered the authoritative source for all subsequent instructions and nutrient supplement recipes. The Botany Department at the University of Texas also sells media, and have an impressive list of the most up-to-date growth media recipes on their web site.

Alga-Grow Medium Recipe

• Filtered seawater: 950 ml +
• Deionized (Distilled or RO) water*: 50 ml +
• 1 M KNO3: 1 ml +
• IMR Metals Solution (see below): 1 ml

Sterilize the solution (sterilization techniques are discussed below) and then add:

• 12 mM KH2PO4: 5 ml +
• Vitamin Solution (see below)*: 1 ml +
• Antibiotic Solution (60-150 ppm - see below): 1-2.5 ml

*If culturing diatoms, add 7.5 g of silica to 250 ml of DI water to make a stock solution, then add 50 ml of this solution rather than the 50 ml of DI called for above; if this forms a precipitate (ppt), cut it in half and try again until no ppt forms - the amount possible to add will be dependent on the hardness of your water. Also add 5 ml of "diatom booster" (see below) along with the vitamin stock after sterilization.
Wrap the container completely in tinfoil and either freeze or store at 4C. I usually split the medium between several containers of useful size (e.g., 250 ml) and freeze those I am not using, and keep the one I am using in the fridge.

IMR Metals Solution

• Stock 1: 48 mg ZnSO47H2O (or 30 mg of ZnSO4H2O) +
• Stock 1: 25 mg CuSO45H2O +
• Stock 1: 30 mg CoSO47H2O +
• Stock 1: 3.0 g H3BO3 in 1 liter of DI.
• Stock 2: 25 mg Na2MoO42H2O in 1 liter of DI.
• Stock 3: 5.0 g FeCl36H2O +2.0 g MnSO4 in 100 ml DI (ignore ppt and use clear liquid on top).
• Stock 4: 50.0 g Na2EDTA2H2O in 1 liter of DI.

Again, all these stocks should be wrapped completely in tinfoil and stored in the fridge or frozen (it is not vital that these stocks be kept dark, but it does not hurt). To make up the IMR metals solution which is added to the medium, add 10 ml of each stock solutions 1,2 and 3 to 850 ml of DI water. Then add 100 ml of stock solution 4, and adjust the pH to 7.5 with dilute Nah. Finally, add enough DI to make the final volume 1.0 liters.

Vitamin Solution

• Cyanocobalamin (Vitamin B12) +: 0.5 mg
• Thiamine +: 0.025 mg
• Biotin in 250 ml of DI: 0.25 mg

It is essential that this stock be kept dark and cold for long-term storage. Make sure the tinfoil wrap is complete on this stock, and store at 4C or, preferably, frozen.

Antibiotic Solution

• Penicillin G +: 15.0 g
• Streptomycin in 250 ml of DI: 15.0 g

Again, it is essential that this stock be kept dark and cold for long-term storage. This stock needs to be frozen in tinfoil if you intend to keep it for more than a month or so. I simply take my frozen stocks out of the freezer and place them in the fridge to thaw overnight before I mix new medium, and then immediately replace them in the freezer when I am done. The amount of antibiotics added to your cultures is both a matter of preference and need. Mike Hadfield's group at the University of Hawaii use 60 ppm of Pen/Strep, whereas Dan Morse's group at UC Santa Barbara use 150 ppm. I personally use 60 ppm if I use any antibiotics at all, but IF you find it necessary to add antibiotics, I would suggest that you start with 60 ppm (1 ml of stock per liter), and increase that up to 150 ppm (2.5 ml of stock per liter) as necessary. I say IF you find it necessary, because I am personally opposed the superficial and ignorant use of antibiotics at home. With improper disposal - like simply dumping the stock down your sink or toilet - new bacteria are repeatedly exposed to the antibiotic, and eventually some individual that is resistant to that compound will be exposed to the antibiotics, have a huge selective advantage and rapidly spread. Chalk up another useless antibiotic to carelessness... If you are going to use antibiotics for treating your cultures, please do everyone (including yourself) a huge favor and boil your "waste" water before disposing of it.

Diatom Booster

• Sodium Glutamate: 1.7 g
• Sodium Glycerophosphate in 1 liter of DI: 0.2 g

Store in the dark and cold as suggested for the stock solutions above.
Once you have the nutrient supplement, you'll need an inoculant to start your cultures. Again, you have a number of options. First, you can simply the pure strain cultures to feed your larvae from a company such as HBOI. If you can afford to buy 20L flasks of culture from HBOI, you probably don't need to be reading this article, so the fact that you are suggests that you need to culture your own. The best place to obtain cultures is undoubtedly the Culture Collection of Algae at The University of Texas at Austin if you're looking for a specific alga, and you know the species, but there are other suppliers like Carolina Biological Supply, or Trans-Mississippi Biological Supply. Again this first option is the easiest and probably most expensive method of obtaining cultures, but in this case also by far the most reliable. Second, you can try to get inoculant from some local source (check with a University if there is one in your area, or try to contact other breeders in your area to "borrow" some of their culture). Third, you can try to isolate algal cells from natural seawater, or even possibly from well established reef tanks. This third option will again be the cheapest, but also the most work and by far the least reliable (you could as easily isolate a toxic dinoflagellate as a useful diatom). I would only recommend home isolation of algae if cost prohibits the purchase of known algal stocks. The easiest way to do this - if you have a good microscope handy - is to examine a droplet of water under a microscope and remove a single cell of an appropriate diatom or alga for culture (don't get greedy! One cell is really all you need, but set up several replicate cultures in case of contamination).
The species recommended and used by the folks at HBOI aquaculture facility include: Isochrysis galbana, Chaetoceros gracilis and Thalasiossira weissflogii. There are many other species of algae commonly used for larval culture (as anyone who has visited the Utex site will quickly discover), but I will only discuss these three here.

Isochrysis galbana

Gold-brown alga on the order of 5-8 micrometers which contain large lipid stores (this species produces many highly unsaturated fatty acids). An excellent food for most small planktotrophic species, especially in mixture with Chaetoceros or Thalassiosira. Especially recommended for mollusc larvae, copepods and rotifers.

Chaetoceros gracilis

A relatively small-diameter chain diatom, averaging about 20% lipid. A good supplemental food for most late stage or crustacean larvae, and an excellent food for adult bivalves.

Thalassiosira weissflogii fluviatilis

Large diatom, averaging about 20 or so micrometers in length. An excellent food for juvenile and adult bivalves, and a good food for echinoderms and crustaceans.
In order to be at all successful in culturing "pure" strain algal cultures, you will first need to sterilize the seawater in which algae are to be introduced. The best way to do this is, of course to autoclave it, but this is not always easy or desirable for the hobbyist at home. That leaves a couple of options. The first, if you have the equipment available or money and desire to buy it, is to Millepore filter your seawater to one micrometer (there aren't any cheap alternative filters I know of to get 1 micrometer filtered seawater - you have to bite the bullet and go for the Millepore stuff). This could be made cheaper (actually only by making each filter last longer before clogging and becoming useless) if you drip the water from your tank through a coffee filter (I HOPE coffee filters are not exposed to formalin in processing the way paper towels are!! I think they are oxygen whitened). The second method is much cheaper and more likely to be used at home. Microwaves have been found to kill most everything but fungal spores, so if you live in a relatively dry part of the country, and fungal spores are not a source of significant contamination, you need only microwave your culture medium. Guillard found that the medium need not even boil for the microwaves to kill most things in seawater. Generally if you place the medium in the microwave in the flask in which you intend to culture the algae for about 2 minutes on high, you'll have pretty sterile culture medium. If, however, you happen to live somewhere like Florida or the Carolinas, where fungal spores will definitely be a major problem, you'll need to get a special microwave pressure cooker. I have heard of them, and know people who use them, but have never tried it myself. The major concern for a pressure cooker is that you add some fresh water to the inside of the cooker (outside the flask) to prevent your algal growth medium from boiling over and making not only a mess, but wasting the medium.
The final method of sterilization is pasteurization. You can pasteurize filtered seawater by heating it to 60-65C for at least 30 minutes or to 85-90C for at least 2 minutes. Once heated, you should let the medium cool and sit at room temperature for 24 hours before a second pasteurization to kill any germinated spores (Jebram 1977). After the second round of heating, the medium should be moved directly to a fridge for cooling and equilibration, and allowed to sit with occasional gentle agitation for at least 24-48 hours. Pasteurized seawater should be stored at 4C until used (but be sure to always warm the water before using it). Of course, a combination of as many of these techniques as is available to you (e.g., pasteurization in a microwave pressure cooker) will increase your chances of obtaining a relatively sterile medium.
After sterilizing the medium (you should always sterilize the medium within the flask in which you plan to culture your algae to minimize the opportunity for contamination), you must aerate it for a while prior to adding the algal inoculant to remove excess carbon dioxide from the medium. Once the medium is sterilized (or nearly so), make sure that the stopper stays in the beaker, and that you never let anything come into contact with the medium or the edge of the stopper to go into the flask (that ESPECIALLY includes your hands - never touch the flask or stopper on the "inside" parts once sterilized). You can open the flask to add algal inoculant water and/or nutrient supplement, but make sure that you only lift the stopper enough to get whatever you're trying to add into the flask, and keep it directly over the culture flask to prevent anything settling into the flask while you're doing that. While the stopper is being held above the opening, make sure that neither the stopper nor the glass tubes touch anything, and replace the stopper immediately after adding new medium. If you plan to get into this seriously, I would suggest that you find a few glass 1 or 2L Erlenmeyer or Fernbach flasks (large flat-bottom, narrow-necked flasks) and get a good quality rubber stopper that tightly fits each flask. There are as probably as many methods of setting up your cultures as there are types of algae cultured, so I won't go into excruciating detail on the technique here, but will instead show you with how I maintain my algal cultures.
You basically want an airline tube to go into the culture and one to let air out of the culture (see the photograph of our setup here). Beyond that, you can add extras, like an outlet tube to "pour" culture from (see Toonen, 1996 for details), but although that is nice to have, it is not necessary to keep algae successfully. The main thing is to keep the algal cultures sterile -- everything else (feeding, lighting, water quality, etc.) is relatively forgiving by comparison, and much easier for the typical aquarist to control.
Once you have the thing set up, and are ready to add the algae, you can set the flasks on a shelf in front of a window that gets lots of sunlight, or set up a light specifically for it - which ever works best for your needs. Both GE and Phillips make cheap full-spectrum lights that are good alternatives to spending a mint on petshop aquarium bulbs (e.g., GE Chroma 50, Phillips color tone 50). I'd use at least 80 W of light directly above (or better yet around/beside -- you'll get better growth with a single bulb beside the cultures than two directly above it) the flasks if you want decent growth and final culture densities. I use 160 W of full spectrum light from the side (lights are mounted into the door of a light box which is sealed and with reflective Mylar all around the inside), and I don't start to feed the cultures to my larvae until they reach about 10,000,000 cells per ml (which takes about a week to ten days. Home use probably doesn't require anything so drastic, unless you plan to run a major breeding facility. Your cultures are probably fine if they look dark green or brown. The recommendation Guillard makes for lighting is to provide about 4000-6000 lux for about 14 hours per day. This is about the output I get at 10-14" away from the four 48" 6500K full spectrum lights (I have to use the old bulbs retired from my reef tank for something) in my light box door, just to make this archaic lux measurement into something tangible for most people (you'll get more photosynthetically active light from the cheap GE/Phillips bulbs mentioned above or even cool whites than from specialty aquarium bulbs, but I don't notice any difference in growth regardless of the brands/types of bulbs I tried).
Once you have a reliable supply of marine phytoplankton cultured, you're ready for the next step -- breeding tank inhabitants that produce feeding larvae, but that is another story for another time.

Literature Cited

Bidwell, J.P. and S. Spotte. 1985. Artificial seawaters: Formulas and methods. Jones and Bartlett Publ., Inc. Boston, Ms. 349pp. Guillard, R.R.L. 1975. Culture of phytoplankton for feeding marine invertebrates. In: Culture of Marine Invertebrate Animals. W.L. Smith and M.H. Chanley (eds.), Plenum Publishing Corp., New York, NY. pp. 29-60.

Guillard, R.R.L. 1983. Culture of Phytoplankton for feeding marine invertebrates. In: Culture of Marine Invertebrates: Selected Readings. C.J. Berg, Jr. (ed.), Hutchinson Ross Publ. Co., Stroudsburg, Pa. pp. 108-132.

Jebram, D. 1977. Experimental techniques and culture methods. In: Biology of Bryozoans. R.M. Woollacott and R.L. Zimmer (eds.), Academic Press, New York, NY. pp. 273-306.

Levin, L.A. and T.S. Bridges. 1995. Pattern and diversity in reproduction and development. In: Ecology of Marine Invertebrate Larvae. L. McEdward (ed.), CRC Press, Boca Raton, FL. pp. 1-48.

Peirson, W.M. 1983. Utilization of eight algal species by the Bay Scallop Agropecten irradians concentricus (Say). J. Exp. Mar. Biol. Ecol. 68:1-11.

Strathmann, M.E. 1987. Reproduction and Development of Marine Invertebrates of the Northern Pacific Coast: Data and methods for the study of eggs, embryos, and larvae. University of Washington Press, Seattle, WA. 670pp.

Thorson, G. 1964. Light as an ecological factor in the dispersal and settlement of larvae of marine bottom invertebrates. Ophelia 1:167-208.

Toonen, RJ. 1996. Invertebrate Culture, Part 1. The Journal of MaquaCulture 4(4):6-26.

Toonen, RJ. 1997a. Invertebrate Culture, Part 2. The Journal of MaquaCulture 5(1):4-13.

Toonen, RJ. 1997b. Invertebrate Culture, Part 3. The Journal of MaquaCulture 5(2):31-37.

Toonen, RJ. 1997c. Invertebrate Culture, Part 1. The Journal of MaquaCulture 5(3):41-51.

Toonen, RJ. 1997d. Reefkeeper's Guide to Invertebrate Zoology: Part 5, Phylum Rotifera. Aquarium.Net, March '97:7 pp.

By Rob Toonen