HOW TO CULTURE PHYTOPLANKTON

What is phytoplankton?

Phytoplankton is a collective term for single-cell algae (microalgae). These live in both marine (salt water) and limnic (fresh water) habitats. In the Baltic Sea alone, over 800 different types of phytoplankton can be found. Like higher plants, microalgae have the ability to synthesize organic compounds from carbon dioxide (CO ₂ ) and water using sunlight. Molecular oxygen is produced as a byproduct. This biochemical reaction is called photosynthesis and is one of the most important biological processes on earth, as it directly or indirectly supplies all organisms with food. In terms of process technology, microalgae are used to produce fuels and food supplements, among other things. The following is mainly about marine phytoplankton.

What is phytoplankton used for in (marine) aquariums and aquaculture?

Phytoplankton is primarily used as feed in (marine) aquariums and aquaculture. It can either be taken up directly by organisms (e.g. soft corals, sponges, tube worms, etc.) or indirectly through the food chain.

This is particularly useful in the breeding of marine invertebrates and fish, as their larval stages often feed exclusively on zooplankton. Before the zooplankton is fed to them, however, it can be enriched with the help of phytoplankton. Microalgae have a high content of unsaturated fatty acids, vitamins, amino acids, minerals and trace elements, which, after being absorbed by the zooplankton, are in turn available to the larvae and have a positive effect on larval development.

In addition, the dosage of phytoplankton has an influence on the bacterial composition and the microfauna and flora in aquariums as well as the water chemistry (nutrient concentrations, pH value, oxygen saturation, etc.).

Phytoplankton in shrimp farming: on the one hand as an enrichment of the feed organisms, on the other hand for the greenwater method, which reduces cannibalism among the larvae and at the same time improves water quality by absorbing excess nutrients

Which phytoplankton species are best suited for breeding?

There are many different types of phytoplankton, which differ greatly in their properties and cultivation requirements. So before you breed a specific type of phytoplankton, you should consider what goals you are pursuing with phytoplankton breeding:

• Feeding filter feeders, corals and microorganisms in your aquarium
• Enrichment of zooplankton for rearing larvae
• Stabilization of pelvic biology and nutrient balance
• Increase competitive pressure on unwanted algae and bacteria
• other

If, for example, you want to raise fish larvae, the cultivated plankton should have a high content of omega-3 fatty acids, as these play an important role in the larval development of fish, but unfortunately cannot be synthesized by the larvae themselves and therefore must be ingested with food.

For direct feeding of corals and filter feeders, however, the size of the algae cells and their cell wall properties are important, as this affects absorption and digestibility. Since you usually don't keep just one type of coral in your aquarium, it can make sense to feed a mixture of different microalgae.

If, on the other hand, an unwanted algae or bacteria is to be displaced with the help of phytoplankton, the phytoplankton you cultivate should have very similar or the same requirements as the organism to be displaced.

In addition, the individual phytoplankton species are sensitive to different levels of contamination, density and temperature fluctuations, and nutrient availability. This means that the choice of phytoplankton species to cultivate also depends on how much effort you can or want to put into breeding. Phytoplankton concentrates, pastes, or freeze-dried powders that are now available on the market may be a better choice for you.

You can find out about the cultivation requirements of the individual species (lighting intensity and duration, salinity, temperature, fertilizer, etc.) in advance on the Internet or in relevant specialist literature, or you can simply ask the breeder from whom you get your starter culture under what conditions he or she cultivates the corresponding microalgae. This has the great advantage that the algae do not have to get used to new culture conditions first.

Each phytoplankton species has different properties (in terms of content/proportion of unsaturated fatty acids, cell size, etc.) and cultivation requirements. While Nannochloropsis is relatively easy to cultivate, Isochrysis, for example, is much more demanding.

What do you need for successful phytoplankton cultivation?

To breed phytoplankton in your own home, you usually only need a few things:

• Translucent culture vessel (aquariums, bottles, glasses, buckets or professional plankton reactors)
• Light source (fluorescent tubes, LEDs)
• Diaphragm air pump (optional with air filter) or magnetic stirrer (to keep the medium or algae moving)
• Phytoplankton fertilizer
• Starter culture

When choosing your culture vessel, you should make sure that it has a volume large enough for your needs. The material from which the container is made should let in as much light as possible, be easy to clean and have a certain resistance to acids and alkalis. A large container opening makes cleaning easier. However, the containers should be able to be closed at the top with a lid or cover to reduce the risk of contamination. In practice, we have found that transparent food-safe buckets with a tap (volume 5, 10, 20 and 30 liters) and 5-liter glass balloons have proven to be particularly effective. These containers are very easy to clean and offer an unbeatable price-performance ratio. We use a highly concentrated citric acid solution (600 g/l) to clean the containers. We also use silicone brushes and dirt erasers for the plastic containers (these tools do not scratch plastic) and stainless steel balls for the glass balloons (e.g. for cleaning glass carafes). Of course, you can also use a professional (and often somewhat more stylish) phytoplankton reactor for your breeding.

Both fluorescent tubes and various LED bulbs are suitable as light sources. A higher blue and red portion of the spectrum (cold white, daylight white) is advantageous when cultivating phytoplankton. There are (almost) no limits to your creativity here, and you can use almost anything from LED construction spotlights to light strips to light bulbs. However, the light source should have sufficient power or intensity, depending on the type of phytoplankton to be cultivated and the culture vessel you have chosen. If you subsequently find that the intensity of your lighting is not sufficient or that the "maturation" of the culture is too slow, you can try to compensate for this deficit by extending the lighting phase. Especially with plastic containers, you should also make sure that the light source is a sufficient distance from the container wall.

Diaphragm air pumps are the means of choice for supplying the culture medium with sufficient oxygen and keeping the algae cells suspended so that they do not settle on the bottom of the container. The higher the culture vessel you use, the more power or pressure the pump must be able to deliver. The total volume of the culture medium to be moved also plays a role and you may even want to ventilate several culture vessels at the same time. In this case, it is very advisable to use a sufficiently large air pump in conjunction with a suitable pressure distribution box, as otherwise it will be very difficult to ventilate the individual culture vessels evenly. In addition, you should always pass the air in front of the culture vessel through a hydrophobic sterile air filter (e.g. PES filter with a pore size of 0.2 µm) to avoid contamination with bacteria.

Over the years, many different fertilizers have been developed for the cultivation of phytoplankton. In practice, the so-called Guillard or F/2 fertilizer (Guillard & Rhyter, 1962) has proven to be particularly effective. With a few modifications, this fertilizer is suitable for the cultivation of numerous phytoplankton species. We ourselves use this fertilizer for all the plankton species we cultivate. When cultivating diatoms (diatoms), we also add silicate to the fertilizer or medium. You should find out in advance which fertilizer is suitable for which plankton or which modifications need to be made. Under no circumstances should you use ordinary flower or plant fertilizer! Its composition is tailored to the needs of land plants, so that the entire fertilizer or the individual fertilizer components may not be used up/absorbed evenly by the microalgae. This means that heavy metals and fertilizer residues can get into your aquarium when you feed the plankton. The nutrients, trace elements and vitamins contained in the special phytoplankton fertilizers (e.g. F/2) are present in a certain ratio, so that the individual components are consumed evenly (in a fixed ratio) by the microalgae. In practice, this is used to check whether the culture is "ripe" for harvesting. You should only feed or use phytoplankton when the fertilizer has been completely used up. You usually measure the nitrate value of your phytoplankton batch to do this. If no nitrate can be detected, the fertilizer has been completely used up and you can feed the plankton. Both seawater-compatible nitrate test strips and normal droplet tests are suitable for measuring nitrate.

Our tip: Cut the test strips in half lengthwise using scissors, this will allow you to take twice as many measurements. With droplet tests, you can halve the sample volume to do this. It's really just about checking that no more nitrate can be detected in the mixture.

The "ripeness test" - nitrate measurement is used to check whether a crop is ready for harvest.

How do you prepare the culture medium?

The specialist literature often refers to the so-called medium. This is actually just freshly prepared sea water and a certain amount of phytoplankton fertilizer.

In order to avoid the risk of contamination with bacteria or other single-cell organisms, it is recommended to sterilize the salt water before use.

Sterilization can be done mechanically (filtration), physically (UV radiation, microwaves, heat) or chemically (ethylene oxide, sodium hypochlorite). Since very few of us have our own autoclave and boiling large amounts of medium is a bit more complicated, we want to show you here how we sterilize the salt water that we use for our medium inexpensively and without much effort.

For this we use the chemical sodium hypochlorite. This is contained in chlorine-containing cleaning products, for example, which you can easily buy in the supermarket. When buying, please make sure that it does not contain any surfactants or other additives. The branded product DanKlorix©, for example, contains no other additives apart from sodium hypochlorite and can therefore be used without hesitation. Warning: When handling such chemicals or cleaning products, we strongly recommend that you wear gloves and safety goggles! You should also make sure that the room in which you are working is well ventilated. And please remember to protect your clothing, because chlorine is a very effective bleaching agent and creates unsightly stains when it comes into contact with colored textiles.

Depending on the contamination of the source water, you should use between 0.5 and 5 ml of chlorine cleaner per liter of salt water. We usually add 0.75 to 1 ml of chlorine cleaner to our medium, as the (germ) contamination of tap water in Germany is very low and it is best to use osmosis water to prepare the salt water anyway. We only add fertilizer to the salt water after sterilization with sodium hypochlorite so that vitamins, for example, are not damaged by oxidation. Theoretically, the sterilization of the medium is complete after about 30 minutes. However, it is recommended that you leave the mixture to stand for 12 hours or, ideally, overnight.

Before the phytoplankton fertilizer and starter culture can be added, the chlorine must be completely neutralized. For this purpose, a sodium thiosulfate pentahydrate solution with a concentration of 60 g/l is used. The chemical sodium thiosulfate pentahydrate is used in photography, for example, as a fixing salt and can therefore be ordered online relatively easily. Now add one ml of the sodium thiosulfate pentahydrate solution for each ml of the chlorine cleaner previously used.

To be on the safe side, we add 2 ml of the sodium thiosulfate pentahydrate solution per ml of chlorine cleaner, as the sulfate in these concentrations has no effect on the phytoplankton - but excess chlorine does. 4 hours after adding the sodium thiosulfate, all of the chlorine should have been converted to harmless hydrogen sulfate. This can be easily checked with chlorine test strips. If you don't have any chlorine test strips to hand, your nose will do - it is very sensitive to chlorine.

Here is a short example: You want to sterilize a total of 5 liters of salt water. To do this, you add 5 ml of chlorine cleaner. After a waiting time of >12 hours, you add 10 ml of the sodium thiosulfate pentahydrate solution and wait another four hours. You can now use the sterilized salt water for your phytoplankton culture and add phytoplankton fertilizer and starter culture.

In our opinion, the sterilization step is essential for the successful and long-term cultivation of very sensitive plankton species (e.g. Isochrysis sp. or Rhodomonas sp. )!

Tip from us: To make your work easier, you can also get more buckets, canisters or glass bottles than you actually need for your culture. You can fill these with salt water of the right salinity and add chlorine cleaner - but not sodium thiosulfate straight away. This way you can store the salt water for a few weeks. It is only dechlorinated when it is to be used for a fresh culture. This saves you a lot of time in everyday life, as you do not have to constantly clean, fill, chlorinate and dechlorinate a single container (long waiting time), but can simply access your supply. You can collect the used culture vessels and clean and refill/chlorinate them together on your phytoplankton cleaning and care day.

Part of the phytoplankton cultivation at Coralaxy: the self-built pressure distribution boxes ensure even ventilation. Air filters and sealing plugs reduce the risk of contamination. Some of the backup cultures are in the Erlenmeyer flasks.

What should be the salinity of the salt water used to produce the medium?

How high the salinity of the salt water should be for producing the medium depends primarily on the requirements of the plankton to be cultivated. However, you should make sure that you always stay two to three PSU below the optimum stated in the literature, as water evaporates through the aeration of the culture and the salinity increases continuously during the cultivation process. It may even be the case that so much water evaporates that you have to refill with fresh osmosis water. If you want to play it safe with regard to possible contamination, you can also sterilize the osmosis water beforehand with sodium hypochlorite.

How much fertilizer and starter culture do you need for a successful plankton culture?

As previously described, Guillard's F/2 fertilizer is the most commonly used fertilizer for the cultivation of phytoplankton.

To prepare the finished culture medium with this fertilizer, you usually add 1 ml of the F/2 fertilizer to 1 liter of fresh (preferably previously sterilized) sea water. Please be sure to follow the instructions of the respective manufacturer (!), as modifications to the original recipe may have been made, which in turn lead to different dosage amounts. The recommended dosage amount of 1 ml per liter is also a guideline that works for the cultivation of most phytoplankton species, but may need to be reduced or increased (depending on the phytoplankton species to be cultivated).

How quickly the fertilizer is consumed depends primarily on the setup of your culture (culture vessel used, lighting intensity/duration, strength of aeration, additional use of CO ₂ , etc.) as well as on the amount and type of phytoplankton used for inoculation.

We recommend that you start with a phytoplankton:medium ratio of 1:5. This means that if you have a culture vessel with a volume of 5 liters, you will need 1 liter of phytoplankton and 4 liters of medium. In general, the more phytoplankton you use to start your culture, the lower the risk of contamination with bacteria or another type of phytoplankton.

With a 1:5 approach, the time until the approach matures is usually 7 to 14 days. If you are not satisfied with the density of your culture after this time, even though all the fertilizer has already been used up, you can "re-fertilize" the culture without any problems.

Batch, semi-continuous or continuous cultures – what are they?

In practice, there are three different cultivation methods regarding the establishment and harvesting of the crop.

In the so-called batch culture (also discontinuous culture), the mixture is completely harvested after the fertilizer has been completely used up, the culture vessel is completely cleaned and then a new mixture is made with a new starter culture. The batch process is a little more complex and less efficient than the other two cultivation methods. However, the risk of contamination during the cultivation process is significantly reduced. In addition, with a little experience, it is easy to predict when the mature culture can be harvested (planability). This cultivation method is particularly suitable for "home use" and the cultivation of particularly sensitive plankton species ( Rhodomonas sp., Isochrysis sp. ). We also use this cultivation method and the procedure described in the previous text primarily refers to it.

In semi-continuous culture, only a certain amount of plankton is harvested after ripening (usually between 25 and 50%). The harvested plankton is then replaced with fresh medium. Since this is an ongoing culture and the phytoplankton:medium ratio is sometimes 1:1, the culture can be harvested again after only a short time. The process is repeated until the culture collapses. This saves both work and time. However, the risk of contamination increases. Semi-continuous cultures are well suited to phytoplankton species that are less sensitive (e.g. Tetraselmis sp. ).

Continuous culture is the most demanding type of cultivation, as only as much fertilizer (or other resources) is added as can be immediately consumed by the microalgae. This means that theoretically the culture can be harvested at any time. A lot of (measuring) technology is required for this method, and it is definitely too expensive and time-consuming for the average hobby grower.

Hygiene and clean working in phytoplankton cultivation is essential!

Hygiene plays a very important role in the cultivation of phytoplankton. That's why we want to give you a few practical tips for dealing with and handling plankton cultures:

• If possible, use a hydrophobic sterile filter (e.g. PES with a pore size of 0.2 µm) to ventilate your cultures. This greatly reduces the risk of bacteria entering the air.
• Before working on or with the cultures, wash your hands thoroughly and then use a hand disinfectant solution. You can get this at the drugstore or pharmacy.
• Disinfect all surfaces and equipment that you work on/with. A 70% isopropanol or ethanol solution is suitable for this. You can get this from a pharmacy, for example. The exposure time is at least 60 seconds. It is also advisable to permanently prepare a disinfection bath with chlorine cleaner (500 ml cleaner to 5 liters of water) in a separate tub or bucket, in which work equipment, cups, flasks, etc. can be placed and disinfected.
• If you want to work with several types of plankton at the same time, always start with the most sensitive type. For example, if you want to start new cultures of Isochrysis sp. , Tetraselmis sp. and Nannochloropsis sp. , you should work in exactly this order, as Isochrysis sp. is the most sensitive algae in terms of the risk of contamination and Nannochloropsis sp. is the least sensitive algae in this list. When switching to a different type of microalgae, carefully disinfect all surfaces, tools and your hands.
• All cultures should always be labeled with the most important information: name of the plankton, date of starter culture, amount of starter culture used, amount of fertilizer used, salinity, etc. This will help you keep track of things when working with multiple cultures and batches. If something does go wrong and a batch collapses, proper labeling will help you troubleshoot later.
• Ideally, you should get your own tools for each type of plankton (e.g. measuring cups, pipettes, etc.). You can either buy them in different colors, mark them in different colors, or label them accordingly.
• If you cultivate zooplankton in addition to phytoplankton, it is advisable to place the cultures at a certain spatial distance or even in different rooms to prevent contamination. Here, too, extra tools are essential. If your phytoplankton and zooplankton cultures are on the same shelf for reasons of space, the phytoplankton should always be placed above the zooplankton. If some phytoplankton drips into the zooplankton cultures while you are working, it is less of a problem than the other way around.
• Buying a microscope is definitely a worthwhile investment for plankton cultivation. This is the only way you can safely check your cultures for impurities and contamination. A simple student microscope is sufficient for this.
• Sooner or later you will contaminate your cultures with other microalgae, bacteria, single-celled organisms or zooplankton, or the cultures will collapse. In this case you should create back-up cultures in advance that can be "started up" again if necessary. There are various ways of creating back-up cultures. In general you should touch the back-up cultures as little as possible, i.e. it is best not to ventilate them, only open them when necessary, etc. In addition, these cultures should be started with a "better" phytoplankton:medium ratio (1:1 or even higher). In some circumstances it may even be sensible to start the back-up cultures with a higher salinity. The microalgae may then only multiply slowly, but the risk of contamination is significantly reduced. Smaller glass vessels are ideal for creating back-up cultures. You can use Erlenmeyer flasks or a jam jar for this. We ourselves create three different back-up cultures for each phytoplankton species:

• Storage in the refrigerator, volume 500 ml, phytoplankton:medium ratio at least 1:1, no ventilation (regular shaking), closure with cotton plug, aluminum foil or a sponge
• Storage on the windowsill (so that the cultures get some daylight), volume 500 ml, phytoplankton:medium ratio at least 1:1, no ventilation (regular shaking), closure with cotton plug, aluminum foil or a sponge
• Storage in the phytoplankton shelf, lighting via fluorescent tubes/LEDs, volume 500 ml, phytoplankton:medium ratio at least 1:1, ventilation via sterile filter, closure with cotton plug or sponge

By setting up different backup cultures, you significantly reduce the risk of contamination. The refrigerator cultures naturally have the lowest risk of contamination. However, these may take some time to reactivate, as the microalgae first have to get used to the cultivation conditions again. Therefore, it is always advisable to first check the ventilated/illuminated backup cultures for impurities/contamination using a microscope, and if they are clean, to start a new batch with these cultures, as the microalgae are already used to the cultivation conditions and a new culture can be started up more quickly.

Back-up cultures should always be restarted at a set interval. We restart our ventilated/lit back-up cultures every 7 days, and the cultures that we keep in the fridge or on the windowsill every 14 days.

Tip from us: If you only have a small amount of starter culture available for the approach or if you have to start a new culture from a backup culture, it is recommended that you do this in several steps (in aquaculture this is called upscaling). This means that you continuously double the volume of the culture (500 ml à 1000 ml à 2000 ml à 5000 ml). By using a phytoplankton:medium ratio of 1:1, you significantly reduce the risk of contamination and avoid the culture collapsing.