Culture Journal, Species: Isochrysis galbana

General
Species:  Isochrysis Galbana
Species description:  microalgae that is an outstanding food source, however, it loves to crash.
Culture source (link if possible):  Ebay.com  (jdb0571)
If algae, CCMP # (Optional): 
http://ccmp.bigelow.edu/
Culture Establishment Date:  1/11/2013
Continuation Date: 

Culturing Vessel Details
Salinity:  2.023
Temperature:  ~70F. I will try and raise the temperature up to 27C (80.5C) as this was found to be an optimal temperature. I will post source soon.   
pH:   Unknown

Vessel description: 
3 -1 L mason jar with plastic lid drilled.
1 - 1 L erlenmeyer flask with 11.5 stopper drilled for 1 hole (hole is larger than airline)
Lighting description:  CFL, Will be switching out with 6000K-7000K 100W LED
Lighting cycle:  18/6
Aeration description:  aqualifter and a 4 way splitter.

Methodologies
Split methodology:

Culture medium description: 
Mason 1 - 1 mL guillard F/2 (1 mL per 2.5 L...oops)
Mason 2 - 1 mL guillard F/2 + 1 mL sodium silicate (~ 30 mg) (added 1 day after innoculation)
Mason 3 - 1 mL guillard F/2 + FeCl₃-6H₂O (haven't added yet, will find research with value)
Flask - 1 mL guillard F/2
 
Sterilized mason 1 and mason 2 by heating mason with ~.800mL saltwater in microwave for ~ 4 minutes, then added nutrients, and microwaved for ~7 minutes. Allowed to cool to slightly warm for hands.
 
Sterilized mason 3 by heating nutrients + non-sterilized water ~ 800 mL saltwater for ~8 minutes, allowed to cool to room temperature. The solution was cloudier than mason 1 and mason 2 when inocculanted  with 52 mL of starting culture.

Cell count:
 (if known)

Reference links:  
 
http://www.bashanfoundati...droraoptimalgrowth.pdf
Noteworthy information:
Optimal temperature 27C
Fe3+ supplementation without CO2 had a higher effect on cell count than without Fe3+ and with CO2
 
http://www.sciencedirect..../pii/S0146638001000419
Increasing temperature (up to 22C) had a corresponding increasing µ (growth rate)
Additional Information
(No Pictures or Videos in the Section Please)
Notes: 
innoculated with 52 mL of starting culture

pictures posted tomorrow.
You will be required to provide photographic evidence and as much detail as possible about your project in this thread.
If your thread does not contain detailed enough photos  and information the MBI Council will not be able to approve your reports.

 
 

 

** I don't use miracle grow! I used it to mix my guillards.
 
 

 

Mason 2 showed quite interesting precipitation effects upon addition of Na2SiO. 
Looks like most redissolved.
 
 
Day 2 - Timer was set to go off for only 3 hours. It is now set for 18/6. Cultures are looking lighter today. I will report how they look tomorrow.
 

Never got around to adding FeCl3 to Mason 3. (Article reports 1.2mg/L FeCl3-6H2O was added).
 
Mason 1 is looking pretty clear.
Mason 2 (w/ silicate) is looking pretty clear
Mason 3 and beaker 4 are looking great.
 
Mason 1 and Mason 3 should be replicates, but I think that Mason 1 and 2 have higher sparging rates.
Changed out the CFL bulb for my 100W ~6500K LED. It is dimmed down quite a bit right now, I need to get a fan blowing on the heat sink.

I took some pictures, I will link them soon. Currently, they can be found 
 
http://imgur.com/a/KciHX

You know, I thought I read somewhere on the Internet once that Isochrysis needed, or at least would perform better, with the addition of silicates.  I never could find that reference on the interwebs again, though, and I've grown Isochrysis for very long periods of time without any additives besides Guillard's F/2 (FAF Micro Algae Grow), FWIW.

I'm curious, do you have any links that discuss the benefit of adding silicate or Iron chloride to an Isochrysis culture?
 
Also, it sounds as though this is your first attempt at culturing Isochrysis.  Is that true?  If so, I'd suggest getting a stable culture using standard best practices first, and only then, start experimenting with interesting additives.  Just my $0.02.
 
Good luck!

I haven't read a single journal article that used silicate for their isochrysis. I was reading one article that was comparing growth rates (µ) for different media compositions, but I don't think they contained silicate. I'll post a link to that article shortly.
 
I have read in two locations that isochrysis needs silicate. One was a post from GreshamH and another was a blog post. I'll see if I can link to them.
 
This is my second time culturing iso, my first time it failed pretty miserably. 
 
Fe3+ has been known to cause phytoplankton blooms in the ocean; if you google "ocean fertilization", you will come upon a wealth of resources.
 
Isochrysis and FeCl3 :http://www.bashanfoundati...droraoptimalgrowth.pdf
 
FeCl3: http://en.cnki.com.cn/Art...OTAL-ZYZZ200508022.htm
 
I havent been able to access the above article. I think it is only 24 cents, (1.5 yuan) but I'm having problems since everything is in chinese.

3.9 mg/ L of Fe 3+ was determined to be optimal for Isochrysis galbana Parks 3011.
http://en.cnki.com.cn/Art...OTAL-SPKX200612058.htm
 
3.9 mg/L Fe 3+ is equivalent to ~7*10^-5 M (http://www.wolframalpha.c...3.9+mg+Fe+to+moles+Fe)
 
Guillards F/2 has a molar concentration of Fe3+ of 1.17*10^-5 M.
 
This is an approximate 6 fold increase of standard Guillard F/2. I know this is for a specific strain, but it would be interesting to investigate.
 
I'll try and access the full article and see if I can determine their methodology. 

Interesting stuff.  Keep the info coming!
 

 
That is common.  That is also why I'm suggesting to simply get a stable Isochrysis culture going with a simple, reproducable protocol first, and then start experimenting.  Getting a good, clean starter culture is important.  There are a few sources around, but the one I always use and the one I recommend is AlgaGen starters, which you can get from SeahorseSource.com  http://www.seahorsesource...p/detail.cgi?id=201125
 

i agree with jim i would either go with ALGEGEN's culture from seahorsesource.com or i got mine from florida aquafarms i got the bigger live culture they had and have never had any problems since ive had my iso cultures going for around 4 months now and only add 1.5ml per gallon F/2 from FAF and i add 1ml per gallon of kent marine essential elements 

Starters from carolina came in. They look quite dilute, I'll try and scale them up to 100 mL vessels first. Then I'll try 1000mL.
 
Some Updates:
 
Mason 3, and beaker 4 are doing well. Mason 1alpha (the 1.75L) isn't looking too hot. Mason 1 is still looking pretty dead.

Updates:
 
Mason 3 and beaker 4 have turned dark brown! They aren't opaque, but they are quite brown. I'll be splitting these cultures tomorrow.
 
Mason 1 has come back from the dead! It is a yellow-greenish color (stressed Iso looks green). Quite amazing to see it white for a week and then change colors today
 
Mason 1alpha (the new 1.75L mason) has darked up a bit. I thought this culture may have died as well.

Woot!  Brown is good.

More photos: http://imgur.com/a/uXTVa#KcdT4AK
 

 
 
Mason 3 was emptied and split into 2 -5000mL vessels. I can't find my rubber stoppers to use on these, anyone have a temporary suggestion until I can buy some (tomorrow)? I'd prefer not to drill the caps.
 
EDIT: I'll try some parafilm!

Parafilm is a good idea, but why not drill the caps? 

They are on the pricier side (~$3.50 each as they are autoclavable) and they are not mine. I'm going to see if I can buy some replacements, and, if so, I'll drill.
 
Camera died, so I'll have to upload the photos later of the 5L vessels.
Each of the 5L vessels were dosed with 3 mL guillard F/2. This will make them slightly higher than F strength. 
 
I'll also be able to upload a nannochloropsis, tetraselmis, and spirulina report later :-)

Large 5 gallon culture is looking great. However, my 5L cultures have turned green.  I recently added more lights, so I took them away yesterday. I split the 5L about a week or so ago and added more nutrients and sterilized salt water. Can anyone suggest anything to turn them brown again?

I've never been able to get them to go brown again after they go green on me.  I'd just re-start the 5L culture from the good looking 5 gallon culture, if it were me.
 
Is that 5 gallon culture a carboy?

Thanks for all the help Jim! I added some sodium metasilicate to one of the 5L vessels yesterday, I will let you know the results. If that doesnt work, I may try adding a bit of glycerol to supply an organic source. 
 
Does iso maintain its nutrition when it turns green? Would it be worthwhile to centrifuge it and save it? If not, I'll end up extracting the oils and making some biodiesel :-)
 
 
The 5 gallon vessel is not a carboy, I have modified several 5 gallon hdpe buckets into bioreactors.

Any update on the iron dosing? Has anyone else dosed it?

I would be interested in seeing pix and hearing more about this modification! Please expound!

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