Culturing Phytoplankton

Discussion in 'Do It Yourself (DIY)' started by CJO, Apr 9, 2011.

  1. CJO

    CJO Well-Known Member

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    A Step-by-Step Guide to Culturing Phytoplankton

    Background

    It all started when I first saw a mandarin dragonet (Synchiropus splendidus) and decided that I wanted a pair for my 65 gallon tank. My profession is an engineer, so I tend to do a lot of research before jumping into something new. The research quickly led me to realize that my tank wasn’t large enough to support two mandarins on its own and that I would have to supplement the available copepods in the aquarium somehow. After a search at the local LFS, I recognized that I couldn’t afford to buy bottles of copepods every week and, to make a mandarin cost efficient, I would have to culture copepods on my own. I found some good news during the initial research- copepods are very hardy and live on detritus and other plankton found in the water column and settled on the substrate. Further research led to some bad news, it’s very hard to culture copepods on just detritus as the ammonia and nitrates build up quickly in a small culturing vessel, which leads to the culture’s crash. In addition, the most nutritious copepods are cultured on phytoplankton.

    Introduction to phytoplankton

    So, what is phytoplankton? Phytoplankton is a single-cell creature that makes up the base of the food chain in the ocean. It is responsible for half of the photosynthesis that takes place in the entire world! The next step up in the food chain is zooplankton, which include rotifers, copepods, Mysis shrimp, etc. These tiny organisms eat phytoplankton and are in turn eaten by corals, anemones, sea horses, fish and even the blue whale.
    There are many types of phytoplankton. The most commonly cultured one in the marine aquarium hobby is Nannochloropsis oculata, usually referred to as Nanno. Nanno is very nutritious. However, it also has a very hard outer membrane and is hard for some zooplankton to digest. Rotifers have no problem with it, but copepods do. Another common type of phytoplankton used is Isochrysus galbana (Tahitian Strain), also referred to as T. Iso. T. Iso is another nutritious form of phytoplankton and balances well with Nanno. I also culture a diatom, Thalassiosira weissflogii (TW), since diatoms make up a large portion of a copepods diet in nature. Others use Tetraselmus (Tet) or Pavlova, which are supposed to be good and somewhat easy to culture, but I have never tried them myself. Nannochloris is another type of phytoplankton that is sometimes measured. I would recommend not using it for culturing zooplankton since it is not as nutritious as the other ones mentioned.

    As I mentioned before, phytoplankton can be a very nutritious food. Specifically, it is the high amounts of EPA and DHA that make them so beneficial. Nanno and T. Iso are commonly cultured and fed to zooplankton because Nanno is high in EPA (30%) but low in DHA (0%) and T. Iso is low in EPA (3%) and relatively high in DHA (10%) and they are both fairly easy to culture. However, for the phytoplankton to be nutritious, it needs to grow on a nutritionally based diet.

    I’ve seen several well written guides that talk about growing phytoplankton with common household fertilizer. While easy and convenient, after talks with several phycologists, they all said that it results in phytoplankton that is green (or brown), but nutritionally deficient. All of them recommended using fertilizer based on Guillard’s F/2. The one I use is Micro Plankton Grow from Florida Aqua Farms.

    All phytoplankton grows in a certain pattern. The first part of the cycle is the initial cycle where the cells are just starting to divide. This is a relatively slow period of growth and lasts around a day (depending on the initial culture concentration). Next begins the exponential growth period. This is where the phytoplankton has the most nutrition and usually lasts between 6 and 10 days. After the growth curve is a period of no growth where the phytoplankton is basically using up its stored energy and nutrients because it has already consumed all the nutrients from the water. This period varies in time but can last several days for most phyto, but is relatively quick for T. Iso.

    This growth curve is why it is important to harvest your culture during a set period of time when it is most nutritional and before it crashes. Normally, this will be between 7 and 10 days from the time the culture is started. It is particularly important with T. Iso because of the short period of time from its exponential growth period until it crashes.
     
    Last edited: Apr 9, 2011
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  2. CJO

    CJO Well-Known Member

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    Phytoplankton Culture Procedure

    Materials needed

    Nannochloropsis only
    • Source of clean saltwater at 1.019 SG
    • Flexible airline
    • Hard airline
    • Culture container(s) and stopper(s)
    • Phytoplankton Inoculant
    • Micro Algae Grow
    • Measuring syringe
    • Airflow adjuster
    • Light source
    • Timer

    Other phytoplankton- all the above plus
    • Airline splitter if culturing several vessels (recommend the metal ones for fresh water)
    • 0.2 micron air filter (only if culturing other than just Nannochloropsis)
    • Silicate (if growing diatoms)
    • TW only- source of clean saltwater at 1.025-1.026 SG
    If reusing culturing vessels and tubing

    • Cleaning brush
    • Alconox or other residue-free soap (if not acid washing)
    • Chlorine (recommend spa or pool granulated chlorine)
    • Chlorine test strips
    • De-chlorinator (sodium thiosulfate or commercial product)
    • Muriatic or hydrochloric acid (optional, but recommended)
    • Quick disconnects (nice to have, but don’t need)
    [​IMG]

    From left to right- muriatic acid, sodium thiosulfate, granulated chlorine, chlorine test strips (on top of chlorine container), measuring pipette, liquid micro algae grow, culture vessel, Alconox soap


    Setup

    Culture Vessel Setup

    [​IMG]

    The setup is fairly simple, especially if you are only culturing Nannochloropsis. The first step is getting your culture vessel(s) ready. Drill two 7/32†holes in the top and insert the rigid airline tubing. Put the top on the culture vessel and push down the rigid airline tubing until it hits the bottom of the culture vessel. Cut the tubing off about 2-1/2†above the top of the vessel. You can use some heat (I used a soldering torch) to bend the airline. This is your inlet tube. Then put in a short piece of hard airline tubing for the outlet vent and bend it in a U shape so that nothing can fall in. Raise inlet airline tubing a little from bottom of vessel so that it’s not touching the bottom.


    Next, connect flexible airline tubing between inlet airline and airflow adjuster and connect another piece between airflow adjuster and air pump. Optional- add quick disconnect between airflow adjuster and rigid airline to make the removal of the culture vessel easier. If culturing several types of phyto, add .2 micron air filter between airflow adjuster (or quick disconnect if you have it) and the rigid airline to help prevent cross contamination.


    Culturing Area Setup

    [​IMG]

    Set up a 5 gal white bucket
    Attach lighting fixture into scrap 2x4
    Screw in a bright light bulb (I use 150W comparable fluorescent light)


    Culture Setup


    The base culture water is simply salt water like what you make for your tank. For all of the cultures I listed, except TW, the culture water should be made to a specific gravity of 1.019. For TW, it needs to be around 1.025 to 1.026.

    If you are reusing the culture vessels, you need to add chlorine until it is between 3-5 ppm. I make my culture water up in a 5-gallon water jug about a month and add chlorine at that time. You don't need much chlorine. It takes approximately 1/8 teaspoon of granulated chlorine to chlorinate 5 gallons of culture water.

    Let the chlorinated culture water sit in the culture vessels for at least 3 hours. Add dechlorinator to dechlorinate the water. It will take some testing with the test strips and adding a small amount of dechlorinator until you find the proper amount. Try not to add way too much dechlorinator or it will strip vital minerals from the water.

    Next, add proper amount of Micro Algae Grow or Guillard’s F/2. For the Micro Algae Grow, this is approximately 0.38 ml per liter of culture water. If culturing diatoms, add proper amount of silicate (a few drops for 1 liter of culture water).

    Fill culture vessel to proper level- the amount of culture water should be approximately 3x-5x the size of the amount of inoculant used. I use 800ml water bottles and fill it 4/5 the way up with culture water and then most of the rest of the way up with inoculant. Be sure to leave a small air gap so that it doesn’t overflow when you turn on the air.


    Procedure

    Once you have the inoculated culture water, place it in the 5 gallon bucket and attach the airline. Attach the light source to the timer and set it to be on for 16 hours per day. That’s pretty much it. In about a week, you should have a fully grown culture of your phytoplankton. Use a portion of it to start the next culture and use the rest to feed your tank or zooplankton. It’s also a good idea to keep a little bit in reserve in a dark, cool place to restart your culture in case it crashes.


    Cleaning

    If you are reusing your materials, it is very important to keep your vessels cleaned and sanitized. The best way to clean the vessels is to acid wash them. However it is less dangerous to simply wash them out with water and Alconox and a scrub brush. Be sure to clean the stopper and rigid airline tubing. Rinse it out and store it with the chlorinated culture water with the stopper and airline until it’s time to use again.

    I acid wash mine. After I empty a culture container, I fill it up with a dilute solution of hydrochloric acid. You can use muriatic acid from Home Depot or similar to make your solution as it’s the same thing. Be careful! This is an acid that can burn. Always add acid to the water and never the other way around. I usually fill the culture container about 90 percent full with tap water and then add the acid. Also be sure to have proper ventilation. The acid contains chlorine and can be very dangerous to breathe in.

    I keep the acidic solution in the vessels with the airline and stopper until it’s time to reuse the vessel. Then, I rinse the vessel, rigid airline tubing and stopper thoroughly before adding the chlorinated culture water.


    Sources for materials

    There are many sources for phytoplankton and materials to be used for culturing them. I get most of the basic supplies from Home Depot or Lowe’s Home Improvement. The culture vessels I used are simply empty glass Voss water bottles from the grocery store. Most of the rest can be sourced from Florida Aqua Farms. They carry flexible airline tubing, rigid airline tubing, airline filters, Micro Algae Grow and the phytoplankton cultures. If you are just going to culture the Nanno, the algae disk is fine.

    If you are going to grow any of the other kinds, spend a little more and use the liquid cultures, they start much easier. Billgax says that many of the clam and mussel hatcheries will give you starter cultures for free. That sounds like a great option for those in coastal areas.

    Florida aqua farms also has a book, the Plankton Culture Manual. This is considered the Bible of phytoplankton (and zooplankton) culturing and is a great resource. Although it is somewhat written from the perspective of growing commercially, I highly recommend it if you are interested in learning more about culturing phytoplankton and zooplankton.
     
    Last edited: Apr 29, 2011
  3. Tabasco1

    Tabasco1 Well-Known Member

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    CJO-nice write up. Thank you for putting the time in. How much time would you say that it takes to culture as described above? After you culture, these are used to feed zooplankton, correct? I would be interested to hear how the zooplankton or pods are then cultured. IF you have the time and when you get a chance. :)
     
  4. CJO

    CJO Well-Known Member

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    Thanks! It takes me about 1/2 hour to an hour a week to culture the phytoplankton. I plan on writing up a copepod culture guide next. They are actually pretty simple.

    CJ
     
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  5. Tabasco1

    Tabasco1 Well-Known Member

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    Sweet thanks! From the write up it sounded like it took alot more time than that! :) Looking forward to the next installment!
     
  6. Billgax

    Billgax Well-Known Member

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    If one wanted to culture other strains (for clams and other phyto feeders), there are clam/mussel hatcheries around coastal areas (a ton around here) that will often give people starter cultures of chaetoceros, isocrisis, etc. The culturing process essentially stays the same. Good way to sources free, pure and clean cultures!!!
     
  7. Tabasco1

    Tabasco1 Well-Known Member

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    BTW- I am not sure the process, but I vote that you submit this for a sticky.
     
  8. CJO

    CJO Well-Known Member

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    That's a great idea. I don't have that benefit, but I'll add it to the writeup.

    CJ
     
  9. CJO

    CJO Well-Known Member

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    I appreciate it! I'll probably wait a little to see what kind of feedback I get. If it's good, I'll send Skinz a PM.

    CJ
     
  10. Billgax

    Billgax Well-Known Member

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    A guy could probably be convinced (i.e. asked) to pull some of his Fisheries strings for a fellow phyto culturist! I have some connections at a couple shellfish farms and could get my hands on some goodies in the event you'd have the want/need. :)
     
  11. CJO

    CJO Well-Known Member

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    I appreciate it, I've been thinking about starting Tet as well, but calling me a phyto culturist is a little too strong for what I do! Please let me know if you see anything else in the writeup to change. I plan to add pictures in the future, but I'm sure I missed some things as well.

    CJ
     
  12. Spek

    Spek Active Member

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    awesome job!! this is a great write up
     
  13. CJO

    CJO Well-Known Member

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    Thanks man, I appreciate it.

    CJ
     
  14. Electrobes

    Electrobes Moderator Staff Member Team R2R R2R Excellence Award Partner Member Article Contributor

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    Nice write up! Why is Nano your main culture CJ? I thought they discovered others which made for a better food source?
     
  15. CJO

    CJO Well-Known Member

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    Thanks, Christian. Nanno isn't my main culture, I culture all three equally. I sourced my Nanno from Dr. Adelaide Rhodes. She had said that she had found a strain of the Nanno that copepods could digest. Also, Nanno seems to do a better job of keeping my copepod culture water clean than the others.

    CJ
     
  16. Electrobes

    Electrobes Moderator Staff Member Team R2R R2R Excellence Award Partner Member Article Contributor

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    Oh cool! I used to culture back in the dark days when very few cultures types existed! I stopped right around I changed from a clam tank to a softy tank.. I was usually culturing Isocrysis. I loved the crazy lab look you get with the set up!
     
  17. CJO

    CJO Well-Known Member

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    Nice. Did you use a similar procedure? It seems like most people are just lining up 2-liter bottles of Nanno in front of 48" fluorescent tubes. I like that the bucket is contained and reflects the light back onto the phyto. I still need to add some pics. Also, let me know if you have any comments, tip or tricks to add to the writeup.

    CJ
     
    Last edited: Apr 11, 2011
  18. Reggie

    Reggie unregistered

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    thanks for the writeup CJO!

    At MAX, I got to talk to the folks at Phyto2 and they said that if you plan to culture your own, just contact them when ordering and specify that they will be used for culturing.
     
  19. Reggie

    Reggie unregistered

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    Under 'Culture Setup':

    You meant 1.019 and 1.025, right?
     
  20. CJO

    CJO Well-Known Member

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    Thanks, Reggie. This was the first I had heard of Phyto2. It's good to hear that they don't seem threatened by hobbyists culturing their own. Their website looks interesting, but there was one piece of information that I saw on there that concerns me a little. They said "When Phytoplankton encounters other species of Phytoplankton, it reacts by releasing poisons to kill the other species which in turn kills itself." I've heard a few other people mention this, but when I asked a couple of phycologists, they said that they had never heard that before. I've never had an issue combining them before either and most commercial phtyoplankton supplements contain a mixture of different types.

    CJ
     

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