Culturing your own Phytoplankton

[Shark Bait100]

Well I tested a new batch of cultures with increased light. They tested out with near zero on phosphates. I retested the original cultures I had in the frig, and they were off scale. This leads me to believe that I was not supplying enough light to fully eat up all the fertilizer, or I have to experiment with the shelf life of cultures in the frig.

[Moreta]

(nods at Shark Bait) I've found that higher/consistent light makes a huge difference.

[TheDeepSandBed]

Anthony,
I was wondering abotuyour comment of home grown quality versus lab grown quality of phytos. What exactly makes a "better" phytoplankto, and how would one go about measuring this quality to quantize exactly how much better these commercial products are?

I was also wondering, if there are anyways that you can store a culture (perhaps freeze it or something else) if you wanted to go on vacation for a couple of weeks that would be good if you wanted to start a culture back up from it in a few weeks?

[Anthony Calfo]

nutritional analysis... and expensive to have tested. But the same phyto on the same feeds, but grown at different temps (warmer/room temp is less favorable) produces a product of different nutritional value.

Hmm... I could be mistaken here, but recall reading this at length/often through the years as well as hearing the lab folks recite this.

I'll drop a line to some folks in the biz and see if we can''t find some data to clarify this.

My overall point though is:

lab/commercial cultured phyto is significantly more nutritious than home-grown

This is my belief. Now lets get some data and I'll reinforce that belief or learn something new

[Anthony Calfo]

well... that was fast: Mr Tagrin from DT's replied promtly to a request for information.

his comments are pasted below here.

He has also shared a reference from Guillard who developed the F/2 formula and is with the (CCMP) Center for Culture
of Marine Phytoplankton! (next posts)

interesting reading:

----------------------------

At DT's Plankton Farm; we use advanced cell separation technology that was developed to harvest live, undamaged cells. This is a long, tedious, and expensive process. This same process is used to wash the cells by repeatedly adding and extracting clean saltwater. This product was developed specifically for use in the closed system environment of reef aquariums.

In contrast, centrifuging phytoplankton is a process used by some companies to make algae paste for commercial aquaculture. This process was not designed to harvest live cells and in fact, the cells are scraped out like clay. This method is fast and cheap, but it damages a large percentage of cells. Several companies are purchasing these algal pastes as frozen concentrated phytoplankton to use as ingredients in their marine aquariums
products.

Another concern in any phytoplankton product is unwanted elements resulting from the growth medium. Our concentrating process utilizes a cell washing procedure to remove nutrients and metals that are necessary to culture phytoplankton. This procedure is done by repeatedly adding and extracting
clean saltwater to remove most of the residual culture media. Nutrient removal is necessary because high nutrient levels in packaged live phytoplankton will cause bacteria blooms that will kill off some of the live phytoplankton, and quickly degrade the product while in storage. Furthermore, high levels of dissolved nutrients that contribute to unwanted algal growth.

Metals are removed to keep them from building up in reef aquariums. While many growth media exist, most phytoplankton culture media contains; ferric chloride, EDTA, cobalt chloride, zinc sulfate, copper sulfate, manganese chloride, and sodium molybdate.

While the potential harm the buildup of these chemicals may have is debatable, not knowing their potential detriment is enough reason to avoid repeatedly adding them to the small closed
system volume of a reef aquarium.

Culturing phytoplankton may be a good option if you are using
phytoplankton to raise zooplankton, or for fish culture. The effectiveness and addition of growth media used may depend on the culture, and their result in a reef aquarium may be variable, depending on the culturist. However, our efforts at cleaning phytoplankton are done to make it as safe as possible for
long term use directly in a reef aquarium.

[Anthony Calfo]

A significant message/reminder that I take from the above comments by Mr Tagrin as it relates to the home culture of phyto harkens back to the "you are what you eat" issue as well as concerns with residuals from the medium/"fertilizers" used.

To me, this is akin to the dreadful habit that too many aquarists have of not thawing and decanting the pack juice of frozen foods. Just dumping it in (frozen or thawed) brings in nutrients with each feeding that are like rocket fuel for nuisance algae! Its such an important matter that some public aquariums/fisheries will rinse and aerate their thawing frozen food in cold water to strip proteins and nutrients that contribute unduly to diatom algae growth on the viewing panels of the display aquariums.

I did not realize that DTs rinsed their product. Very cool/interesting.

[Anthony Calfo]

the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP)

Recipes

f/2 Medium and Derivatives

(Guillard & Ryther 1962, Guillard 1975)

Below are recipes for f/2 medium, its derivatives (e.g, f/2 agar, f/2-Si, f/2 + Se, f/4, f/50) and related media (e.g., Black Sea). F/2 is listed first, followed by derivatives of f/2.

f/2 Medium
(Guillard & Ryther 1962, Guillard 1975)
To 950 mL filtered seawater add:
Quantity Compound Stock Solution Molar Concentration in Final Medium
1 mL NaNO3 75 g/L dH2O 8.83 x 10-4 M
1 mL NaH2PO4 · H2O 5 g/L dH2O 3.63 x 10-5 M
1 mL * Na2SiO3 · 9H2O* 30 g/L dH2O* 1.07 x 10-4 M*
1 mL f/2 trace metal solution (see recipe below) -
0.5 mL f/2 vitamin solution (see recipe below) -
Make final volume up to 1 L with filtered seawater and autoclave.
*Note: Autoclaved f/2 medium produces extensive silica precipitate. We delete silicate when it is not required by the alga (see f/2-Si medium below).


f/2 Trace Metal Solution
(Guillard & Ryther 1962, Guillard 1975)
To 950 mL dH2O add:
Quantity Compound Stock Solution Molar Concentration in Final Medium
3.15 g FeCl3 · 6H2O - 1 x 10-5 M
4.36 g Na2EDTA · 2H2O - 1 x 10-5 M
1 mL CuSO4 · 5H2O 9.8 g/L dH2O 4 x 10-8 M
1 mL Na2MoO4 · 2H2O 6.3 g/L dH2O 3 x 10-8 M
1 mL ZnSO4 · 7H2O 22.0 g/L dH2O 8 x 10-8 M
1 mL CoCl2 · 6H2O 10.0 g/L dH2O 5 x 10-8 M
1 mL MnCl2 · 4H2O 180.0 g/L dH2O 9 x 10-7 M
Make final volume up to 1 L with dH2O. Autoclave.


f/2 Vitamin Solution
(Guillard & Ryther 1962, Guillard 1975)
To 950 mL dH2O add:
Quantity Compound Stock Solution Molar Concentration in Final Medium
1 mL Vitamin B12 (cyanocobalamin) 1.0 g/L dH2O 1 x 10-10 M
10 mL Biotin 0.1 g/L dH2O 2 x 10-9 M
200 mg Thiamine · HCl - 3 x 10-7 M
Make final volume up to 1 L with dH2O. Autoclave and store in refrigerator. Note: Vitamin B12 and biotin are obtained in a crystalline form. When preparing the vitamin B12 stock solution, allow for approximately 11% water of crystallization (for each 1 mg of Vitamin B12, add 0.89 mL dH2O). When preparing the biotin stock solution, allow for approximately 4% water of crystallization (for each 1 mg of biotin, add 9.6 mL dH2O).

[TheDeepSandBed]

Anthony first of all, YOU ROCK! Thank you for the very interesting/informative posts (utilizing great contacts). I guess what I have taken from all this discussion, is that grow phytoplankton seems to be only a viable option if you are growing rotifers. In light of that I have decided to expand my experiment and try my hand at raising rotifers as well. However I still plan on trying to add a portion of my home grown greenwater to my tank - perhaps using a slightly modified methodology of doing a sort of water change on my phytos. Anyone have any idea how large of a filter I should use on the phyto? I am guessing around 20 um (cant do the nano symbol) since rotifers are around 52-56um.

Thanks a lot for all the info everyone - that is why there is no other better source for information than RC.

RESTECBA!!!

[Anthony Calfo]

very welcome my friend.

And indeed... this is not to say that home-grown phyto is bad. Its not a matter of good versus bad. But rather (IMO) good versus better. What is "better", though, is to be determined by the end user. For me... it is time savings and edge on quality that draws me to commercially produced phyto. If I needed (very) large quantities, or simply liked the challenge of tinkering, I may well indeed culture my own.

As for rotifers... yes, please! Do always culture zooplankton when possible. Copepods too (see the Marini article in Adbanced Aquarist e-zine, and the Rhodes article on copepods in the Conscientious Aquarist e-zine).

A majority of the corals we keep are carnivores and will benefit by such plankton.

FWIW... I culture my live copepods on bottled phyto

I will keep digging for the info on temp influence on nutritional value of cultured phyto.

[Moreta]

I use the phyto as a culture for both 'pods and to occasionally grow out brine shrimp, as well as dosing (some of) my tanks.

I really do like the DTs product, it's just too darn expensive.

Please do keep digging on the temp/nutrition aspect, as I culture in my basement which is usually 65 degrees or so. I get around that by putting mu culture bottles into shallow water in 10 gallon tanks and use a small heater to raise it to 75 degrees.

It's worked well, but I'd love to know how to tweak the nutritional content via temperature.

[Anthony Calfo]

another fab reply from Mr Tagrin/DT's (very good of him to share the tips/insights to phyto culture):

------------------

Most hobbyists who are culturing phytoplankton are growing
Nannochloropsis oculata. Nannochloropsis will be at its highest nutritional value if grown at a temperature of 20C ( 68F) or lower.

When grown at temperatures above 20C to 25C the EPA levels drop and the culture growth rate was unaffected.

Salinity is also important, a salinity between 25-30 ppt will produce the highest EPA levels. If it is cultured with a light dark cycle the algae divides fastest in the dark cycle and accumulates fatty acids better in the light cycle.

We grow it with 24 hour lighting because it will always have high EPA levels when harvested.

One other very important aspect is the potency of the vitamins. The B vitamins are very important in the production of EPA.

Vitamin potency is affected by temperature and different
vitamins have different minimal storage requirements. We use tissue culture grade vitamins that I get from Fisher Scientific. The B12 has to be stored refrigerated the Biotin is frozen and the Thiamine is at room temp. We make a stock solution of B12 and Biotin in doses to make up 2 liters for culture media that is kept in frozen storage. The daily dose of vitamins are mixed daily from frozen stock along with the Thiamine.

It is easy to culture green water but producing quality phytoplankton is not a simple process.

[TheDeepSandBed]

Does anyone know if there is a reliable way to make a preservable culture at home that you can use in case of crashes (rather than reordering a new culture)?

PS what does EPA stand for?

-Ben

[APFish]

OK, bottom line is that I believe some of the above information provided but w/o out the money to test the theories I have nothing to stand on. First of all, I believe a large amount growth rates, size of particulate matter and basically the genetic make up is just that genetics. When I started growing phyto I asked these same great questions to my local reef club with no response. I am finally glad to see an open discusssion. As generations increase, it would be my theory that several things would come into play. I would have to know the basic root of the chain and to where it was multiplying, I don't. DT's has done great research to find out how to get a range of different size phyto into their mixture. At the same time if one was to use their mixture would they not in fact end up with the same mixture or do they cross generate and therfore develop an entirely differnt breed of phyto. I hate little things. When a black cow breeds with a red bull, black is the dominate charecteristic and most of the time you end up with a black calf. My thought is, can a black pyhto of size 20 cross with a red bull of size 50 and end up with a size 20 calf most of the time. Will they even cross?


After all that, here is my two cents. I don't have the money to spend on DT's. So,I grow my own. I raise them at about 68 degrees. I place week salt water, almost brine, in a jar. I place some DT's where I break down once every couple of months and buy some, in the jar. Next, I add a squirt of fish emulsion and some trace minerls. Finally, I shake it up and put a air stone in the jar. I put it in front of a grow light, that is on for about 14 hours a day and shake it in the morning while I do the three S's of american manhood.

Could I be slowly killing my reef? I guess but it hasn't shown in any of my basic tests and I frag probably more than the average bear. Bottom line is, it is working for me. I am in this hobby because I like it and this works for me. I don't sell anything I raise and don't have the money for expensive theories.

[Samala]

What is this red bull calf question? Are you asking if different species of phyto grown together will result in hybrids? All I can say is most likely not. Sexual reproduction of phyto.. particularly the diatoms.. is a very badly understood process.. mostly because it is a fleeting occurrence in nature. Most of the time phyto's are reproducing vegetatively.. one cell splits into two daughter cells.

In diatoms this actually leads to a reduction in size because they are reproducing their silica shell, which is shaped like a petri dish. So, one daughter cell always works off the larger 'lid' of the petri and one of the smaller 'dish'. As they divide they get smaller.. most research points towards sexual reproduction in diatoms, which restores the cells to the 'normal' size, being triggered when the cells hit 35% of their original/normal size. That could be an easy way for DT's to assure you that you have a range of sizes in the mix if they are including diatoms like Thalassiora weissflogi or Chaetoceros gracilis. There are others.

The DSB - You could conceivably make your own algae plate. I routinely make up f/2 agar for algae plates in the lab using f/2 medium and adding an appropriate amount of agar on top, usually a 1.5% solution is good to keep the algae going. I'm wondering if perhaps you could substitute plain gelatin for agar at home. Heat the f/2 to get the gelatin to dissolve and wait until it has just start to set and inculate with the algae cells, they can take some heat without dying IME. Just an idea... kind of crazy but it might work. I stick the plates into a commercial growth chamber, I'm thinking you could leave the plates like this for awhile sitting near your lights if you have them on a timer. You'd certainly need to test it out first.

>Sarah

[Qwiv]

You guys raising phyto and rots, have you read Clownfishes, by Joyce D. Wilkerson
http://www.amazon.com/exec/obidos/tg...books&n=507846

I read it a while ago and she mentions keeping phyto and rots in a constant, low density culture outside in some sort of container. She does absolutly nothing to them and they grow. Anyone else tried this? I am going to try when I get my breeder setup done... one day. She says it does not make for production qty but is a great back up in case of a crash.

[TheDeepSandBed]

Sarah,

So you think in the gelatin idea that I should store them at room temp with lights? (I was thinking perhaps the freezer....Walt Disney style :P ). I guess I will have to do some tests once Im up and running.

[Samala]

DSB - Lemme try keeping some plates out this week and next at room temp and just adjacent to some of the timer-operated lights I have on my tank at home.. and I'll report back. I know that you can fridge them for a bit without them going bad.. so I'll have to try with the gelatin idea and see how long you can really push it. I was thinking of growing them a week and then fridging a week in a dark container so they dont get any light when I go to snack on something. That should mimic a vacation decently. I'll let you know - if you try it too we'll compare notes.

>Sarah

[TippyToeX]

Microfood culture: phytoplanktons, Rotifers, ciliates, Artemia, and copepods

[Anthony Calfo]

and more info from DT:

---------------

EPA is Eicosapentaenoic acid one of the most essential omega 3 fatty acids.

DHA is Docosahexaenoic acid, another essential omega 3 fatty acid.

Although animals can synthesize one from the other it is not as efficient and the goal from our continued research is to find a species of microalgae that would be a good source of DHA that survives refrigerated storage.

Note: Isochrysis and Pavlova are dead within 2 weeks of refrigerated storage.

People are being mislead by claims that they survive in
refrigerated storage.

[Kathy55g]

So one would need to replace their DT's 2 weeks after they go in the fridge. Does DT date the bottles as he refrigerates them before shipping, and being stored in the fridge at the LFS? Two weeks total, and then the bottle we buy loses 2 species!

My nannochloropsis survived quite well in my fridge, and began to grow again right away. I think I had them in there a month. Nanno may be different this way than Iso and Pavlo.

It's quite clear to me that DT's is a superior product, and one would need to pay for the expertise, skill, and research it took to get this superior product,

unless one does not need such finesse.

[Bugger]

Here's an overview. Nannochlorpsis oculata holds EDT best at 20 degrees and salinty 25-30. Dt's refridgegerates vitamin B12 and adds it separtly.
Can you find out anymore about there washing process? Do they make there own furtilzer from strach? How much does it cost to analize phytoplanton? Are there physical difference's between strains of the same phytoplanton or is chemical analyisis for the most part called for?

Great information I only wish I had more to share.

[Anthony Calfo]

A 'lil bit more info...

I will also follow up with Mr Tagrin for more info he can/will share relating to new questions. For now:

--------------------
(response to Andy's concern/question)
"DT's does not contain Isochrysis or Pavlova because they do not survive storage. DT's has been proven to maintain good nutritional value through 5-6 months and to stay alive for many months beyond. (Toonen 2002). The shelf life is based on the feeding trials published in the 2002 Marine Fish & Reef Annual. DT's must be maintained in refrigerated storage and will be highly nutritious for 5 months from harvest. Dennis"

[ezhoops]

No doubt that DT's has the science and technology to provide a very high quality product but.......if you spent about 10 minutes a day you too can grow a great phytoplankton like I've been doing for the past couple of years. I'll be the first to admit that my phytoplankton isn't as good as DT's but then it doesn't cost me an arm and a leg to feed my tank.

As far as contamination goes, IME as long as you don't over fertilize (which will kill your culture anyway) you should be fine. IME it is extremely difficult to poison your tank by overfeeding phyto. for experiemental purposes I've dumped a 2 litre bottle of phyto into my 29 gal and notice no changes in any params. I did have many filter feeding organisms in the tank (no sps) to consume it though.

I agree I would first purchase and read thoroughly the phytoplankton culture manual by Flordia aqua farms. before I began a home culture.

Also, if you want to deal with serious science and phytoplankton check out the university of texas phytoplankton dept.
here's a link to their site and many others.

http://www.bio.utexas.edu/research/utex/index.html

and others

http://www.botany.utoronto.ca/utcc/Links.html

Home culture is relatively easy and inexpensive, figure it this way (and I'm not knocking DT's at all, its a awesome product). for about 3 - 4 16oz bottles of DT's you can grow your own phytoplankton, probably more than one species, and feed your tank as often as you like, and give away extra to fellow reefers.
How can you go wrong?

One last thing, I wonder if Dennis can really explain the smell of DT's?

does anyone here who has grown their own phyto have that terrible smell to it? Mine doesn't even when it goes bad.

[Kathy55g]

What does it smell like? If it is sulphurous, it may be that sodium thiosulphate or some other sulphur compound is added to deactivate ammonia or chlorine.

[ezhoops]

it is a sulfurous smell but from what I figure phyto consume ammonia and so that would rule that out. Chlorine would kill phyto.