Plankton and Plankton Surrogates for Reef Aquaria — Cryopreserved Phytoplankton

Aquarists can make their systems more closely resemble natural reefs through the addition of a phytoplankton surrogate, cryopreserved phytoplankton.

It seems that the reef aquarium hobby has taken the metaphor that reefs are “nutrient deserts” too literally. We have achieved a good deal of success by emulating many of the physical and chemical factors of the water flowing over the reef. In terms of dissolved nutrients, wild reefs are indeed nutrient deserts (Sorokin 1989). However, this ignores very important non-dissolved nutrients. The water flowing over wild reefs is rich in food in little packages. The living portion of these packages consists of planktonic organisms, plus a portion of copepod fecal pellets and other “things.” The purpose of this column is to point out one way aquarists can make their systems more closely resemble natural reefs: through the addition of a phytoplankton surrogate, cryopreserved phytoplankton.

Why Should You Care About Phytoplankton?
Not as much attention as one might like has been given to the exact prey particle types and sizes required by organisms of interest to aquarists. Rather, attention has been given to species of interest to aquaculturists.

Some literature reports describe the particle sizes preferred by sponges. For example, Wyeth et al. (1996) examined feeding responses of fragments of sponge tissue to bacteria, phytoplankton and latex spheres of various sizes using microscopy. The smaller phytoplankton are in the size range efficiently captured by sponges.

It has recently been appreciated that several species of soft corals take up phytoplankton. Fabricius et al. (1995a,b) have recently demonstrated convincingly that Dendronephthya and three other types of soft corals from the Red Sea feed on phytoplankton, gaining sufficient carbon from that phytoplankton to sustain themselves. This was demonstrated from examination for food organisms in the gut contents of corals and also by detection of chlorophyll autofluorescence in intact animals. (See also Sprung and Delbeck 1997 for earlier suggestions and demonstration of herbivory in cnidarians.)

The details of the work of Fabricius, of the Australian Institute of Marine Science in Townsville, Australia, the Zoological Institute at the University of Munich, Germany, and Benayahu and Genin of Tel Aviv University and The Hebrew University (Fabricius et al. 1995a,b) respectively are extremely important. This tri-continental trio has demonstrated that Dendronephthya hemprichi obtains most of its nutrition from small cells of phytoplankton. Very few cyanobacteria were present, which is significant because cyanobacteria are reported to outnumber eukaryotic (larger, nucleated) algae in these waters, both in numbers and in biomass. On average, only 0.02 animals were recovered from the gut contents of each polyp. Most were weakly swimming mollusk larvae and copepods. The biomass from phytoplankton is two orders of magnitude greater.

They also studied the rate of decrease in chlorophyll autofluorescence in intact animals. As this organism is completely azooxanthellate, the only chlorophyll present is from ingested prey. Over the first 14 hours, the chlorophyll autofluorescence decreased by 3.5 percent per hour. This may be telling us something about the rate at which these organisms digest food and suggests to me that daily feedings with phytoplankton would be beneficial. 

They starved a few Dendronephthya for a few days in the laboratory and then flowed water containing phytoplankton over them at various rates. The animals rapidly obtained phytoplankton from the water, and the rate was flow dependent — fastest at the fastest flow rate evaluated, which was 8 to 10 centimeters per second. In the second paper (Fabricius et al. 1995b) in which higher flow rates were examined, the optimal flow rate for phytoplankton capture was found to be 15 centimeters per second, although the size of the polyps increased linearly with flow rates to 32 centimeters per second.

It seems clear that if we want to keep this species in captivity, we need to provide it with an ample supply of phytoplankton and adjust the flow rate to the animal to 10 to 15 centimeters per second (4 to 6 inches per second). People who have been attempting to culture Dendronephthya with only Artemia nauplii seem to have been barking up the wrong tree.

In addition to these octocorals, it is thought that phytoplankton are an essential part of the diets of many tubeworms, bryozoans, tunicates and, perhaps, some sponges. Indeed, if a sessile filter-feeder does poorly in captivity, a reasonable first guess is that phytoplankton might be an important part of its diet. It is interesting to note that the aquaculturists raising bivalves, such as oysters, in flow-through systems with microalgal enrichment often have to do brine dips on the oysters to remove fouling organisms, including tunicates, competing for food. To me, bryozoans, tunicates, sponges and tubeworms are every bit as fascinating and attractive as small-polyped stony corals. I’m anxious to find a way to keep them alive and healthy in my aquarium. Hence my involvement in this question.

The State of Our Systems
Through highly efficient foam fractionation or protein skimming, we clear organic molecules and particulate matter from our systems. Whether the parcels are desirable or undesirable, the efficient physicochemical machinery of foam fractionators whisks those particles and molecules out of the system.

This is desirable in one respect, because it allows us to continue to feed the saltwater fish and other reef creatures without long-term accumulation of nutrients in the system. Continued feeding without a nutrient export mechanism would ultimately convert the aquarium from an oligotrophic (nutrient poor) state to a eutrophic (nutrient rich) state. Skimmers are undesirable in that they rapidly clear some small particles from the system that we might like to keep…those being the various planktonic creatures and even various organic molecules that can be adsorbed directly by some marine creatures.

Several alternatives to foam fractionators have been proposed in the hobby. They include totally “natural” systems like Eng’s, the exclusive use of activated carbon and fast-growing soft corals championed by Bob Stark and others, the use of mangrove filters, algae scrubbers (Adey and Loveland 1991), the use of chemical sorbents for nutrients, among others. All of these systems have their merits, but in terms of ejecting large quantities of nutrients from a system, a powerful foam fractionator is unmatched, particularly in a short-term crisis.

Moreover, the champions of these various alternative systems have not demonstrated that they have greater holo-planktonic and phytoplanktonic populations than skimmed systems. Indeed, in 1991 Adey and Loveland flatly commented that it was impossible to keep an appreciable population of holo-plankton in algal turf scrubber systems. It is not clear to me why the more recent amateur advocates of these systems believe otherwise.

There are two immediate “drains” on putative phytoplankton pools that come immediately to mind: capture via physicochemical mechanisms, like protein skimming or mechanical filtration, and capture via filter-feeding organisms. Even without any drain on phytoplankton pools via protein skimming, it seems likely that the very high benthic surface area to volume ratios that characterize small closed systems will confront any phytoplankton with a veritable “wall of mouths,” even if no skimmer is present. The ocean isn’t between 1 and 3 feet deep, or at least most of it isn’t. But our aquariums are. So, the bottom is much closer to the surface in our systems than in much of the world’s oceans.

In the open ocean, plankton merely have to contend with other planktonic predators over most of the ocean’s surface. But, as the water washes over the shallows, they are confronted by efficient epibenthic filter feeders that are in a very good position to sample large volumes of water. So, our systems are not very “ocean-like” in terms of their high benthic area to volume ratios. I believe this has a critical impact on the inability of our systems to support stable phytoplankton populations, above and beyond the obvious damage done by protein skimming.

Adey and Loveland (1991) briefly describe this as a “scaling problem” with small captive systems and point to large volumes of overflowing water necessary to support filter feeders. They have felt it necessary to resort to additions of phytoplankton and zooplankton surrogates (Isochrysis and brine shrimp nauplii) to their systems at various times. This is within systems that are arguably as plankton-friendly as they get.

It has been observed that the phytoplankton content of oceanic water is reduced by passing over a reef (Fabricius 1995b.) Clearly, the ocean is able to maintain stable phytoplankton populations, but this is only because the ocean is huge and is on average about a mile deep. It has a low benthic surface area to volume ratio. Truly enormous systems, like Biosphere II, are certainly in a better position to maintain a semi-normal holo-plankton than the tiny closed systems in your living room. But, it has yet to be demonstrated that any private-scale marine system can maintain a stable and appreciable standing stock of phytoplanktonic organisms.

In last month’s “Without a Backbone” column, Ron Shimek evaluated the planktonic creatures in one of his reef aquaria. Aside from being a pioneering look at this important question, the results are significant in that there was effectively a zero population of holo-planktonic life or life that spends its entire life adrift in the ocean. Effectively, only larval forms of creatures that live in the live sand or in the rocks were found. Phytoplankton was effectively absent, although the mesh of the plankton net was too coarse to definitively settle that issue.

Shimek makes a statement that I would like disagree with in a courteous manner: “We presently lack any real way to culture sufficient amounts (of bacteria or microplankton) or to provide sufficient diversity within each type.” It would not be a major effort to set up bacterial and phytoplankton cultures in one’s home, although a certain amount of knowledge is required to do so and opportunities exist in the aquarium industry for servicing this important niche. I also believe that the cryopreserved phytoplankton I will describe later in this article are an important step forward in this direction.

Open the System!
If a closed system is unable to emulate one aspect of natural systems, then we have two choices: 1) ignore the deficiency and plod along with that model or 2) open the system and supply the component the system is unable to supply. I’ll have more to say about this concept later, but for now, let’s imagine that there is a flow of nutrients across the system — into the system through additions of aquarium foods of all kinds, and out largely through the foam fractionator. In between these endpoints, organisms are fed, they make wastes, stay alive and are beautiful and fascinating to watch. Such a system is quite different than other “natural” models in that we are not asking the system to produce “enough” endogenous phytoplankton to support all the filter feeders we might like to keep. Rather, we are continuously managing the system via import of plankton and plankton surrogates and export of nutrients.

Cryopreserved Marine Phytoplankton
Over the past few months, I’ve been corresponding with Inland Seafarm regarding the potential of artificially grown microalgae for the reef aquarium hobby. Owners Randy and Tim Reed got into the microalgae business as a necessary component of their inland oyster culture system. Their goal is to grow extremely clean, pathogen-free oysters for the human food market. Because oysters are filter feeders and do very well on a diet of phytoplankton, they got into phytoplankton culture in a big way. I became aware of them from one of the aquaculture mailing lists on the Internet, and obtained some samples of their products for evaluation by myself and several other reef aquarists in my area.

More information on the nutritional properties of microalgae can be found at the Seafarm website and in Brown et al. 1989. Thompson et al. (1994) have shown that diatoms cultured at higher irradiance are more nutritious than diatoms cultured at lower irradiance, so generalizing the exact nutritional quality of phytoplankton from a compilation is slightly dangerous and certainly imprecise.

Phytoplankton isn’t inexpensive. It is important to note that the paste is about 3000 times as concentrated as a culture of that species grown to the limits of the modified F2 media the Reeds use. So, 100 milliliters is equivalent to 300 liters of microalgae culture. That is about 75 gallons of dense algal culture. Even factoring in the cost of overnight shipping, this seems like a reasonable price.

I’ve had experience with the 100-milliliter squeeze tube so far and it seems to be a fairly reasonable way to handle the paste. I have not seen the 250-milliliter pump bottle yet. The format that microalgae are presented in is important from an end user’s perspective. Remember the grass stains on the jeans of your youth? Well, if you get some microalgae on your clothes you can have a “seagrass” stain from hell. So, be cautious when you handle it and remember that if you mess up, the garment you are wearing may have spots of the same color as the microalgae in question.

When I started talking with the Reeds, they were selling only “natural” microalgae, which were pelleted in their continuous-flow centrifuges. The problem with the natural presentation is that it has a very short half life on refrigeration — very optimistically, a month. With cryopreservation, the marine algae can be stored at -20 degrees Celsius (-4 degrees Fahrenheit), typical of a freezer, rather than at refrigerator temperatures of 4 degrees Celsius (39 degrees Fahrenheit). This vastly extends the lifetime of the product. I’ve examined the cryopreserved algae under a microscope at about two weeks storage and they are relatively intact (as judged by light microscopy), but few viable cells could be recovered.

The Reeds chose propylene glycol as a cryopreservative. There are several options for cryopreservation of biological samples. Cells can be cryopreserved in substances like glycerol, ethylene or propylene glycol, dimethylsulfoxide (DMSO) and some other candidates. From that list glycerol would be my first choice and propylene glycol would be my second. Propylene glycol was probably chosen based on its physical properties. The viscosity of pure propylene glycol is lower than pure glycerol and that makes it easier to resuspend the microalgae paste in the latter preservative. I do have some reservations about propylene glycol. Although it is used as a solvent for coloring and flavors in food and as an emulsifier, it can also cause contact dermatitis in susceptible individuals (for one example, see Nater et al. 1977, and additional references in these safety sheets.

Propylene glycol is superior to ethylene glycol because its LD50 (50 percent lethal dose) in mammalian systems is substantially lower than ethylene glycol. You may be familiar with both compounds, because ethylene glycol was the antifreeze of choice in cars for many years. Propylene glycol is gradually gaining favor in that market because it is regarded as less toxic than ethylene glycol (LD50 for ethylene glycol for rats is 4.7 grams per kilogram, and for propylene glycol is 20 grams per kilogram — Merck Index.) Propylene glycol is metabolized to lactic and pyruvic acids through aldehyde intermediates. One oxidation product of ethylene glycol is oxalic acid, which certainly isn’t good for you in large quantities.

Feeding Instructions
It is interesting to note that we have been feeding our systems with suspended microalgae every time we knock the diatoms off the front glass with a cleaning magnet. Some individuals (Daniel Knop and Terry Siegel, personal communications) have noted positive responses from corals when the front glass is cleaned and the benthic diatoms are released into the water column.

Several fellow aquarists (Terry Siegel, Doug Robbins and Greg Schiemer) and I evaluated four samples of microalgae from Seafarm. The first set of samples was not cryopreserved and consisted of a diatom and Isochrysis. The second set of samples was cryopreserved and consisted of two types of flagellates (Isochrysis galbana and Tetraselmis chuli). I’m using “flagellates” as a term of convenience here. It is not a valid taxonomic division of algae.

I’ve noted feeding responses in various invertebrates to all of these algae, as has Terry Siegel. Typically, a small amount (a few milliliters for my 75 gallon system) is taken and dispersed in seawater from the aquarium in a small container and then the microalgae is added to the aquarium in an area of high water flow. My tangs all attempt to feed on any chunks of material, so they seem to recognize it as food.

Terry Siegel’s observations proved to be quite interesting: “Craig Bingman provided me with samples of the four algae mentioned in this article, including the cryopreserved Isochrysis galbana and the Tetraselmis chuli. Sporadic feeding of my main reef systems (650 gallons of water total) with these algae proved entirely beneficial, with the gorgonians and sponges responding especially favorably.

“I have also been feeding these algae to a small 12-gallon experimental aquarium that is not skimmed. It has a small outside filter box that contains a small amount of carbon. I change about 50 percent of the water every three to four days with water taken from the main system. A small powerhead provides water circulation and three power-compact tubes provide the lighting.

“I have been maintaining three types of hard-to-keep corals: Alveopora, Dendronephthya and Lemnalia. As can be seen from the photos in this article, the Alveopora and Lemnalia are thriving. In fact, the Lemnalia has reproduced by some means that is unclear. The new shoots are 18 inches from the mother animal.

“The condition of the four Dendronephthya is problematic. Two specimens thrived for months with this feeding regime, then died almost overnight. I doubt they starved in a dozen hours. Perhaps they died of some type of infection. The remaining two specimens are still holding their own. Because I have been randomly using the four types of algae provided to me, at this point I cannot tell which is the most beneficial.”

Doug Robbins has been using a glass rod to pick up a bit of paste on the end of it and dispere this into his system. Again, there is a lot of commotion among the saltwater fish and attempts to eat the paste every time the microalgae is fed. Robbins believes that several of the invertebrates in his aquarium are doing better now, especially one orange ball sponge that seemed to be getting smaller before he started feeding microalgae. It now appears to be growing.

Greg Schiemer made an interesting observation when he fed cryopreserved microalgae to his system. It seems that several of the small-polyped scleractinian corals retracted their tentacles and some soft corals reacted in a similar fashion. It is not clear if this was a feeding response or avoidance on the part of the corals. Terry Siegel and Doug Robbins have not described any reaction that could be interpreted as avoidance in their systems.

More quantitative guidance on the amount of plankton that might be required by a captive reef system comes from Adey and Loveland (1991). They observe that about 2 grams (dry weight) per square meter of holo-plankton passed over a well-developed reef in St. Croix. Because phytoplankton is the most abundant type of plankton, on a mass basis, this number gives an upper limit for how much phytoplankton our systems might require.

The optimum way to feed phytoplankton would be continuously. However, once or twice a day may be as often as aquarists are willing to add phytoplankton to their systems. Terry Siegel and Doug Robbins have been adding phytoplankton to their systems every few days. I believe smaller daily additions are more appropriate. They also have not been coming close to the upper limit figure of 2 grams of dry weight per square meter per day. At the more sparing and cautious rates we have been using, none of us has noticed an excess of algal growth. This is presumably because the foam fractionators in the systems are efficiently exporting the excess nutrients.

To get the most out of phytoplankton or any type of plankton or plankton surrogate feeding, it is useful to be able to isolate the display aquarium from the protein skimmer for at least an hour. If the skimmer is running in a sump, accomplishing this is as simple as just turning off the sump return pump. It is important that there be some powerhead or other auxiliary pumps operating in the display aquarium to provide water motion to keep the microalgae suspended during this time. The microalgae are intact, but non-viable, and even the flagellates cannot move on their own. Diatoms are always reliant on water movement to keep them suspended. Some are highly adapted to staying in the water column. This example shows three diatoms and one dinoflagellate caught in a plankton net at Low Isles Research Station, The Great Barrier Reef, Queensland Australia. One of the diatoms in the figure is highly adapted to staying in the water column. The long pinnae keep the diatom suspended with even a whisper of water motion.

For general use in feeding a reef aquarium, I would suggest glycerol cryporeservation and the use of a mixture of all the flagellates that Inland Seafarm is currently culturing. The mixture of sizes is along the theory that we should “shoot them all and let the filter feeders sort them out.” By providing a range of sizes, we are likely to be able to support a diverse assemblage of filter feeders. I personally do not have any reservations about adding diatoms to my system, but some people may be concerned about the additional load of biogenic opal, which may dissolve to some extent to form free silica species in the water.

Other Uses
There are several other possible uses for these cryopreserved algae, in addition to directly feeding organisms in the display aquarium. We have used them as an enrichment to “store bought” Artemia, which are often pale and starved by the time you get them. One imagines these starved Artemia to be devoid of nutritional value. When a small amount of microalgal paste is added to the culture water, they immediately begin to turn green as they feed on the phytoplankton. So, these products could be used to maintain brine shrimp in good condition in a store, and they might also be used by aquarists to gut load the brine shrimp with a carotenoid-rich, unsaturated fatty acid- and protein-rich substance prior to feeding them to the creatures in their display systems.

For individuals who maintain zooplankton cultures, which are usually dependent on a continuous supply of living phytoplankton, contamination of those phytoplankton cultures with rotifers can be a devastating event, as the rotifers rapidly consume their future food supply. A stock of cryopreserved phytoplankton might be very useful in keeping the zooplankton cultures going while fresh, uncontaminated phytoplankton cultures grow.

“Do It Yourself” Phytoplankton
Algal culture involves choice of the correct medium, a source of light of appropriate intensity and some water motion, which on a small scale can be achieved with an airstone. Carbon dioxide injection and pH control can be useful for larger-scale systems. This site gives a glimpse of an intermediate scale lab culture facility.

For individuals who would like to start growing their own phytoplankton, there is a wealth of valuable information available on the web and in books. Perhaps the best reference is the Plankton Culture Manual (Hoff and Snell 1996). For individuals who prefer to get their information for free from the web, there are several web resources out there.

Important web resources for individuals wishing to obtain cultures of microalgae can be found at the American Type Culture Collection. Their cultures tend to be quite expensive, but this is a valuable collection for research purposes.

The Culture Collection of Algae at The University of Texas at Austin (UTEX) has an extensive collection of microalgae that are suitable for growth as food. UTEX charges $15 per culture for academic users and $50 for all others. They also have an invaluable collection of media recipes on-line that has been extremely useful to me.

Still more media recipes can be found at The Culture Collection of Algae and Protozoa (CCAP). They have an extensive list of media recipes. More media recipes can be found at the North East Pcific Culture Collection of the University of British Columbia’s Department of Oceanography, and at the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP).

By far the least expensive place to obtain microalgae cultures is from Carolina Biological Supply. They charge approximately $5 per culture. Although they have a website, unfortunately, their list of living organisms is not on-line. If you purchase algal cultures from them, a small but useful booklet describing some media and small-scale culture techniques is included with the purchase.

Another source of relatively inexpensive cultures and materials for culturing microalgae and rotifers is Florida Aqua Farms, Inc.’s Aquaculture Supply. They have cultures of the algae most common in aquaculture, some in stable and storable form, as well as resting cysts of two size classes of marine rotifers. They also have algal growth media, culture aquariums and a host of other interesting equipment.

Acknowledgments — I would like to thank Terry Siegel, Doug Robbins and Greg Schiemer for testing the phytoplankton products in their systems and sharing their observations with me. Further, I would also like to thank Inland Seafarm for their generosity with samples. I am not financially connected with Inland Seafarms in any way.

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