Another Phytoplankton Culture Tutorial

[Chris_from_SoCal]

This is a day 14 update. Both tanks are lighter since I just doubled the amount of salt water. I did the same last week. I also doubled the amount of F2 each time. I did remove some to feed the copepods. One more week and each tank will be full then I can use a larger amount to feed them and keep it going if all goes well.

Splitting into two seems to be the right thing since one tank is definitely doing better than the other. I did have to increase the airflow. The deeper it gets the more air is needed. I have made it a habit to shake each tank every other day so I do not get any growth stuck to the walls.

This is using cheap 1 gallon plastic containers from Walmart, bottom LED lighting for growing and a 3D printed base. I also 3D printed a holder for the solid airline to connect it to the lid that is easily removable. At the top of the solid airline is a valve to adjust the air.

 

[EnterName]

This is a day 14 update. Both tanks are lighter since I just doubled the amount of salt water. I did the same last week. I also doubled the amount of F2 each time. I did remove some to feed the copepods. One more week and each tank will be full then I can use a larger amount to feed them and keep it going if all goes well.

Splitting into two seems to be the right thing since one tank is definitely doing better than the other. I did have to increase the airflow. The deeper it gets the more air is needed. I have made it a habit to shake each tank every other day so I do not get any growth stuck to the walls.

This is using cheap 1 gallon plastic containers from Walmart, bottom LED lighting for growing and a 3D printed base. I also 3D printed a holder for the solid airline to connect it to the lid that is easily removable. At the top of the solid airline is a valve to adjust the air.

Yepp, always split in multiple smaller batches and always keep a backup for a few days. Preferably in the fridge.

Just to show how lazy you can get with Nannochlorpsis: The batch I started is still running, no water added, no fertilizer, culture still pure and dense. I'm currently trying to get a Tetra culture running from a crashed starter I received. It will take a while but I might get there... We will see. If it worked I will post here and explain what I did (it isn't too much different or complicated).

I might also soon show how you can use certain substances to stain phyto cultures to see if the cells are still alive or if you are basically dealing with dead particles. I will have to see if I can find the time.

 

[martin.a]

Is it a very bad idea to premix 25 L of sea water, add hypochlorite and store in a dark place?
It would make staring a new culture so much quicker. Just fill the vessels, squirt F2 and sodium thiosulfate via sub micron filter and good to go.
I am writing this in the middle of the night when it was 2 days I simply had no time to mix and sterilise.

 

[huthain]

Hey there so today I harvested my secknd batch of phyto using some of the first batch. I used 2 seperate containers one came out darker than the other however there is some bubbling/foaming at the top of the lighter one has this gone bad? Is it safe to dose and or use as another starter culture?

 

[EnterName]

Is it a very bad idea to premix 25 L of sea water, add hypochlorite and store in a dark place?
It would make staring a new culture so much quicker. Just fill the vessels, squirt F2 and sodium thiosulfate via sub micron filter and good to go.
I am writing this in the middle of the night when it was 2 days I simply had no time to mix and sterilise.

Good question... I know that chlorine will gas out over time so it's possible that after a while it loses potency, but if the container is air tight, doesn't have much headroom, and can withstand a potential pressure build up (I don't think there will be much pressure though) it should work.

I would dechlorinate first before adding F2 to be honest. The chlorine might react with the fertilizer otherwise.

 

[EnterName]

Hey there so today I harvested my secknd batch of phyto using some of the first batch. I used 2 seperate containers one came out darker than the other however there is some bubbling/foaming at the top of the lighter one has this gone bad? Is it safe to dose and or use as another starter culture?

I would use the darker one as starter culture, but it's still safe to dose in my opinion, as long as it doesn't smell like absolute death

I keep backup cultures that I often neglect and after they crash, I still use them to feed my corals. They would catch detritus in the wild as well, I don't think they mind.

 

[martin.a]

Good question... I know that chlorine will gas out over time so it's possible that after a while it loses potency, but if the container is air tight, doesn't have much headroom, and can withstand a potential pressure build up (I don't think there will be much pressure though) it should work.

I would dechlorinate first before adding F2 to be honest. The chlorine might react with the fertilizer otherwise.

I will do and report.

 

[martin.a]

Question about Tetraselmis: The cells clump and drop each time I start a new culture. Last 2 times I was really careful to match salinity and temperature, bubbled air for 12h before inoculation, and it still ended up at the bottom.
The cells re-suspend and partially de-clump when I manually mix the new culture, and all is fine after 2 days of babysitting. I am not sure if the cells permanently re-suspend though, as some green 'sediment' remains. But the culture takes off and all is good without any further manual agitation. Is this normal? I checked the culture under a scope and I could see only Tetra cells, nothing else.

 

[EnterName]

Question about Tetraselmis: The cells clump and drop each time I start a new culture. Last 2 times I was really careful to match salinity and temperature, bubbled air for 12h before inoculation, and it still ended up at the bottom.
The cells re-suspend and partially de-clump when I manually mix the new culture, and all is fine after 2 days of babysitting. I am not sure if the cells permanently re-suspend though, as some green 'sediment' remains. But the culture takes off and all is good without any further manual agitation. Is this normal? I checked the culture under a scope and I could see only Tetra cells, nothing else.

This is a common "issue" as far as I know. Usually Tetraselmis are flagellated cells that move very fast on their own, but when introduced into a new media they might still end up reacting with a stress response where they become non-motile and drop to the bottom. Non-motile cells are relatively large and dense so they are hard to keep suspended. Shaking the culture in the first few days from time to time should get the job done until the so called "lag phase" where reproduction seems to stall is over. As long as the culture increases in density afterwards it should be fine. Maybe the sudden change in nutrients when entering a new culture vessel is already enough for the stress response, I don't know.

 

[DmitryB]

I've had good results with Nanno. Today started Tetra in 2 separate small pods. So let's see how that goes. My air bubbles for Nanno go wild, but Tetra requires a gentle bubbling as I understand. Probably closer to what copepods would get? But otherwise it's the same process as Nanno, right? Same amount of F/2?

What's the next culture to try after Tetra so I can start looking into that? I'd like to start mixing colors.

 

[EnterName]

I've had good results with Nanno. Today started Tetra in 2 separate small pods. So let's see how that goes. My air bubbles for Nanno go wild, but Tetra requires a gentle bubbling as I understand. Probably closer to what copepods would get? But otherwise it's the same process as Nanno, right? Same amount of F/2?

What's the next culture to try after Tetra so I can start looking into that? I'd like to start mixing colors.

F/2 works for Tetraselmis.

Synechococcus (a "good" cyanobacterium) is quite easy to keep and often recommended to get rid of cyano problems in tanks (I don't know if it's an effective treatment though).

Then there is Rhodomonas salina and "Isochrysis galbana" which has been reclassified to Tisochrysis lutea I believe but you will most likely find it as Isochrysis.

 

[DmitryB]

F/2 works for Tetraselmis.

Synechococcus (a "good" cyanobacterium) is quite easy to keep and often recommended to get rid of cyano problems in tanks (I don't know if it's an effective treatment though).

Then there is Rhodomonas salina and "Isochrysis galbana" which has been reclassified to Tisochrysis lutea I believe but you will most likely find it as Isochrysis.

I'm intrigued by Rhodomonas salina. You think it's a good match for Nanno and Tetra? In terms of balancing the nutrients each offers. Are Nanno and Tetra - for all intents and purposes - interchangeable? (I'm basing this question on nothing but their color!)

 

[EnterName]

F/2 works for Tetraselmis.

Synechococcus (a "good" cyanobacterium) is quite easy to keep and often recommended to get rid of cyano problems in tanks (I don't know if it's an effective treatment though).

Then there is Rhodomonas salina and "Isochrysis galbana" which has been reclassified to Tisochrysis lutea I believe but you will most likely find it as Isochrysis.

I'm intrigued by Rhodomonas salina. You think it's a good match for Nanno and Tetra? In terms of balancing the nutrients each offers. Are Nanno and Tetra - for all intents and purposes - interchangeable? (I'm basing this question on nothing but their color!)

I like Rhodomonas because I see a chance of it actually surviving in reef tanks for a little bit. I find Cryptophyceae specimens from time to time in my tank even though I never dosed Rhodomonas: https://www.reef2reef.com/threads/microscopy-thread.1138845/post-14468506 (page 7, post #136).

It would be the next on my list to test because Tetraselmis seems to be a great success judging from the number of cells that resemble Tetraselmis I keep finding weeks after dosing. Before dosing I never saw them.

In contrast: Nannochloropsis and Synechococcus seem to vanish much faster. To be fair they are smaller and harder to find, but they also don't swim much on their own so the skimmer is probably highly efficient in removing them.

This is all just a hypothesis as I am not able to actually verify this with multiple tanks and scientific methods, but at least in my tank it appears to apply.

I would have to research the nutrient composition of Tetra and Nanno to answer your question. I hope I remember and come back for an answer

 

[EnterName]

Sooo.... I'm back as promised with some web-scraped data

Parameter	Nannochloropsis (various sp.)	Rhodomonas salina	Tetraselmis suecica
Protein (% DW)	35 (N. oculata) [1] 50-55 (sp., nutrient-replete) [2]	40.0 [6] 29 (as Chroomonas salina) [1]	31 [1]
Lipid (% DW)	18 (N. oculata) [1] 40-40.2 (N. salina, f/2 and Miquel's media) [3] Total fatty acids up to 28.3 (N. sp., nutrient starvation) [2]	12.0 [6] 12 (as Chroomonas salina) [1]	10 [1]
Carbohydrate (% DW)	7.8 (N. oculata) [1]	9.1 (as Chroomonas salina) [1]	12 [1]
EPA	25.8 % TFA (N. salina) [4] Up to 44.2 % TFA (N. salina, day 21, large scale) [5] 4.2-4.9 % DW (N. sp.) [2]	4 % TFA [6] 8 % TFA [7]	3.10 mg/g DW (strain CCAP 66/38) [8] 2.8 - 4.2 % TFA (Tetraselmis sp., 10 - 30 °C) [9] 4 - 10 % TFA (prasinophyte class average) [10]
DHA	Deficient [12] Outlier: 4.18 % TFA (N. salina) [4]	4.7 % TFA [6] 3-7 % TFA [7]	Deficient [11]
ARA	9.5 % TFA (N. salina) [4]	?	?
Other	α-tocopherol 431 µg/g DW (N. salina) [5]	PUFA 47 % TFA [6] PUFA > 60 % TFA [7]	β-carotene 7.17 mg/g DW Lutein 0.39 mg/g DW (strain CCAP 66/38) [8]

EPA	Eicosapentaenoic Acid (an Omega-3 fatty acid)
DHA	Docosahexaenoic Acid (an Omega-3 fatty acid)
ARA	Arachidonic Acid (an Omega-6 fatty acid)
PUFA	Polyunsaturated Fatty Acids
DW	Dry Weight
TFA	Total Fatty Acids
?	No verified value found

I don't know too much about the nutritional benefits of these different fatty acids and especially not with respect to corals, so I can't tell you which one is the best food source. But if you want to provide all three (ARA, DHA and EPA), which seems to be the go-to approach for aqua-culturing various organisms, you could go for Rhodomonas for DHA and Nannochloropsis for ARA and EPA.

I still like Tetraselmis because, unlike Nannochloropsis, I can actually observe it in my tank for a while. It might also have some antibacterial benefits, which I find very interesting. Do those benefits apply to corals? I don't know. But in a "worst case" scenario, I manage to bring food to the copepods where they actually live: on the substrate, the glass and the rockscape, not the open water column. Nannochloropsis apparently just floats around until the skimmer takes care of it (my observations so far).

Can I prove any of this to be beneficial in some way? No, unfortunately not.
But it's the closest I'll get to actually observing any benefit at all from dosing phytoplankton. Rhodomonas will definitely be next on my list as I expect a similar outcome to Tetraselmis and actually like to see that I'm not pouring dead organic matter into my tank

Sources:

[1]	https://www.fao.org/4/W3732E/w3732e07.htm
[2]	https://doaj.org/article/3045d80512594f9698330174049edc0a
[3]	https://drs.cift.res.in/items/6c39fa37-d637-4fa2-b4d9-5d24833b5d0b
[4]	https://krishi.icar.gov.in/ohs-2.3.1/index.php/record/view/61027
[5]	https://pmc.ncbi.nlm.nih.gov/articles/PMC4999905
[6]	https://revistas.ucr.ac.cr/index.php/rbt/article/view/3415
[7]	https://link.springer.com/10.1007/s10811-022-02730-1
[8]	https://pure.atu.ie/en/publications...-fatty-acid-profiling-of-marine-species-wi-3/
[9]	https://pmc.ncbi.nlm.nih.gov/articles/PMC5121895/table/t1
[10]	https://acnpsearch.tweb-dev.unibo.it/singlejournalindex/7648635
[11]	https://digital.csic.es/handle/10261/71870
[12]	https://www.jstage.jst.go.jp/article/jos1956/42/4/42_4_272/_pdf/-char/ja

 

[DmitryB]

Sooo.... I'm back as promised with some web-scraped data

Parameter	Nannochloropsis (various sp.)	Rhodomonas salina	Tetraselmis suecica
Protein (% DW)	35 (N. oculata) [1] 50-55 (sp., nutrient-replete) [2]	40.0 [6] 29 (as Chroomonas salina) [1]	31 [1]
Lipid (% DW)	18 (N. oculata) [1] 40-40.2 (N. salina, f/2 and Miquel's media) [3] Total fatty acids up to 28.3 (N. sp., nutrient starvation) [2]	12.0 [6] 12 (as Chroomonas salina) [1]	10 [1]
Carbohydrate (% DW)	7.8 (N. oculata) [1]	9.1 (as Chroomonas salina) [1]	12 [1]
EPA	25.8 % TFA (N. salina) [4] Up to 44.2 % TFA (N. salina, day 21, large scale) [5] 4.2-4.9 % DW (N. sp.) [2]	4 % TFA [6] 8 % TFA [7]	3.10 mg/g DW (strain CCAP 66/38) [8] 2.8 - 4.2 % TFA (Tetraselmis sp., 10 - 30 °C) [9] 4 - 10 % TFA (prasinophyte class average) [10]
DHA	Deficient [12] Outlier: 4.18 % TFA (N. salina) [4]	4.7 % TFA [6] 3-7 % TFA [7]	Deficient [11]
ARA	9.5 % TFA (N. salina) [4]	?	?
Other	α-tocopherol 431 µg/g DW (N. salina) [5]	PUFA 47 % TFA [6] PUFA > 60 % TFA [7]	β-carotene 7.17 mg/g DW Lutein 0.39 mg/g DW (strain CCAP 66/38) [8]

EPA	Eicosapentaenoic Acid (an Omega-3 fatty acid)
DHA	Docosahexaenoic Acid (an Omega-3 fatty acid)
ARA	Arachidonic Acid (an Omega-6 fatty acid)
PUFA	Polyunsaturated Fatty Acids
DW	Dry Weight
TFA	Total Fatty Acids
?	No verified value found

I don't know too much about the nutritional benefits of these different fatty acids and especially not with respect to corals, so I can't tell you which one is the best food source. But if you want to provide all three (ARA, DHA and EPA), which seems to be the go-to approach for aqua-culturing various organisms, you could go for Rhodomonas for DHA and Nannochloropsis for ARA and EPA.

I still like Tetraselmis because, unlike Nannochloropsis, I can actually observe it in my tank for a while. It might also have some antibacterial benefits, which I find very interesting. Do those benefits apply to corals? I don't know. But in a "worst case" scenario, I manage to bring food to the copepods where they actually live: on the substrate, the glass and the rockscape, not the open water column. Nannochloropsis apparently just floats around until the skimmer takes care of it (my observations so far).

Can I prove any of this to be beneficial in some way? No, unfortunately not.
But it's the closest I'll get to actually observing any benefit at all from dosing phytoplankton. Rhodomonas will definitely be next on my list as I expect a similar outcome to Tetraselmis and actually like to see that I'm not pouring dead organic matter into my tank

Sources:

[1]	https://www.fao.org/4/W3732E/w3732e07.htm
[2]	https://doaj.org/article/3045d80512594f9698330174049edc0a
[3]	https://drs.cift.res.in/items/6c39fa37-d637-4fa2-b4d9-5d24833b5d0b
[4]	https://krishi.icar.gov.in/ohs-2.3.1/index.php/record/view/61027
[5]	https://pmc.ncbi.nlm.nih.gov/articles/PMC4999905
[6]	https://revistas.ucr.ac.cr/index.php/rbt/article/view/3415
[7]	https://link.springer.com/10.1007/s10811-022-02730-1
[8]	https://pure.atu.ie/en/publications...-fatty-acid-profiling-of-marine-species-wi-3/
[9]	https://pmc.ncbi.nlm.nih.gov/articles/PMC5121895/table/t1
[10]	https://acnpsearch.tweb-dev.unibo.it/singlejournalindex/7648635
[11]	https://digital.csic.es/handle/10261/71870
[12]	https://www.jstage.jst.go.jp/article/jos1956/42/4/42_4_272/_pdf/-char/ja

This is awesome, thank you! Might be worth doing all 3 to match nutrients, but also delivery to various parts of the tank.

I'm on day 2 of Tetra and not sure about the color. I read here it settles on the bottom at first before, so perhaps this color is the result of that?

 

[BlueBayouReefHTX]

I like Rhodomonas because I see a chance of it actually surviving in reef tanks for a little bit. I find Cryptophyceae specimens from time to time in my tank even though I never dosed Rhodomonas: https://www.reef2reef.com/threads/microscopy-thread.1138845/post-14468506 (page 7, post #136).

It would be the next on my list to test because Tetraselmis seems to be a great success judging from the number of cells that resemble Tetraselmis I keep finding weeks after dosing. Before dosing I never saw them.

In contrast: Nannochloropsis and Synechococcus seem to vanish much faster. To be fair they are smaller and harder to find, but they also don't swim much on their own so the skimmer is probably highly efficient in removing them.

This is all just a hypothesis as I am not able to actually verify this with multiple tanks and scientific methods, but at least in my tank it appears to apply.

I would have to research the nutrient composition of Tetra and Nanno to answer your question. I hope I remember and come back for an answer

Rhodomonas is found in the ocean naturally so I would expect the only barriers to it surviving in the tank is that tanks specific ecosystem and other factors such as UV, skimmers etc. That being said, this type of phytoplankton is generally hard to grow and i've found the effort given is not worth it. However it is beautiful to look at under the light.

 

[DmitryB]

Rhodomonas is found in the ocean naturally so I would expect the only barriers to it surviving in the tank is that tanks specific ecosystem and other factors such as UV, skimmers etc. That being said, this type of phytoplankton is generally hard to grow and i've found the effort given is not worth it. However it is beautiful to look at under the light.

Is it contamination that kills it? What are the barriers to growing it successfully at home? (I've started triple disinfecting: first vinegar/water, then spraying with alcohol, then sterilizing the saltwater with bleach. If that's not enough, I don't know what else to do! LOL. My Ninja Pressure Cooker doesn't do enough pressure to fully sterilize, as I understand it.)

 

[BlueBayouReefHTX]

Is it contamination that kills it? What are the barriers to growing it successfully at home? (I've started triple disinfecting: first vinegar/water, then spraying with alcohol, then sterilizing the saltwater with bleach. If that's not enough, I don't know what else to do! LOL. My Ninja Pressure Cooker doesn't do enough pressure to fully sterilize, as I understand it.)

Autoclaving is generally the best way to go however that's not feasible for most hobbyists. Unless you're using smaller glassware. I use 1 gallon and 5 gallon carboys and unless you have a larger autoclave, its not going to work or fit inside a standard pressure cooker. I also don't like using chlorine to sterilize my seawater and resort to boiling. Its not going to kill absolutely everything, but crashes are few and far between. You just have to be careful about overboiling and precipitating your salt solution.

You can look at Rhodomonas wrong and it crashes. It doesn't refrigerate well either, you'll have to split on the day of harvest if you want to keep it going long term. I wash glassware with Alconox, and it it with either 91% alcohol or Star-san and let air dry.

 

[EnterName]

Is it contamination that kills it? What are the barriers to growing it successfully at home? (I've started triple disinfecting: first vinegar/water, then spraying with alcohol, then sterilizing the saltwater with bleach. If that's not enough, I don't know what else to do! LOL. My Ninja Pressure Cooker doesn't do enough pressure to fully sterilize, as I understand it.)

Autoclaving is generally the best way to go however that's not feasible for most hobbyists. Unless you're using smaller glassware. I use 1 gallon and 5 gallon carboys and unless you have a larger autoclave, its not going to work or fit inside a standard pressure cooker. I also don't like using chlorine to sterilize my seawater and resort to boiling. Its not going to kill absolutely everything, but crashes are few and far between. You just have to be careful about overboiling and precipitating your salt solution.

You can look at Rhodomonas wrong and it crashes. It doesn't refrigerate well either, you'll have to split on the day of harvest if you want to keep it going long term. I wash glassware with Alconox, and it it with either 91% alcohol or Star-san and let air dry.

Well, I'm actually autoclaving all my culture vessels and medium anyway so the sterilization isn't the issue. I will see if Rhodomonas is worth the effort sterile filtering the three components of my fertilizer though.

Thanks for the heads up either way, I'm curious how it will turn out. The hardest part will probably be to source a decent source that doesn't cost a few hundred bucks to be actually sterile.

 

[EnterName]

Sooo.... I'm back as promised with some web-scraped data

Parameter	Nannochloropsis (various sp.)	Rhodomonas salina	Tetraselmis suecica
Protein (% DW)	35 (N. oculata) [1] 50-55 (sp., nutrient-replete) [2]	40.0 [6] 29 (as Chroomonas salina) [1]	31 [1]
Lipid (% DW)	18 (N. oculata) [1] 40-40.2 (N. salina, f/2 and Miquel's media) [3] Total fatty acids up to 28.3 (N. sp., nutrient starvation) [2]	12.0 [6] 12 (as Chroomonas salina) [1]	10 [1]
Carbohydrate (% DW)	7.8 (N. oculata) [1]	9.1 (as Chroomonas salina) [1]	12 [1]
EPA	25.8 % TFA (N. salina) [4] Up to 44.2 % TFA (N. salina, day 21, large scale) [5] 4.2-4.9 % DW (N. sp.) [2]	4 % TFA [6] 8 % TFA [7]	3.10 mg/g DW (strain CCAP 66/38) [8] 2.8 - 4.2 % TFA (Tetraselmis sp., 10 - 30 °C) [9] 4 - 10 % TFA (prasinophyte class average) [10]
DHA	Deficient [12] Outlier: 4.18 % TFA (N. salina) [4]	4.7 % TFA [6] 3-7 % TFA [7]	Deficient [11]
ARA	9.5 % TFA (N. salina) [4]	?	?
Other	α-tocopherol 431 µg/g DW (N. salina) [5]	PUFA 47 % TFA [6] PUFA > 60 % TFA [7]	β-carotene 7.17 mg/g DW Lutein 0.39 mg/g DW (strain CCAP 66/38) [8]

EPA	Eicosapentaenoic Acid (an Omega-3 fatty acid)
DHA	Docosahexaenoic Acid (an Omega-3 fatty acid)
ARA	Arachidonic Acid (an Omega-6 fatty acid)
PUFA	Polyunsaturated Fatty Acids
DW	Dry Weight
TFA	Total Fatty Acids
?	No verified value found

I don't know too much about the nutritional benefits of these different fatty acids and especially not with respect to corals, so I can't tell you which one is the best food source. But if you want to provide all three (ARA, DHA and EPA), which seems to be the go-to approach for aqua-culturing various organisms, you could go for Rhodomonas for DHA and Nannochloropsis for ARA and EPA.

I still like Tetraselmis because, unlike Nannochloropsis, I can actually observe it in my tank for a while. It might also have some antibacterial benefits, which I find very interesting. Do those benefits apply to corals? I don't know. But in a "worst case" scenario, I manage to bring food to the copepods where they actually live: on the substrate, the glass and the rockscape, not the open water column. Nannochloropsis apparently just floats around until the skimmer takes care of it (my observations so far).

Can I prove any of this to be beneficial in some way? No, unfortunately not.
But it's the closest I'll get to actually observing any benefit at all from dosing phytoplankton. Rhodomonas will definitely be next on my list as I expect a similar outcome to Tetraselmis and actually like to see that I'm not pouring dead organic matter into my tank

Sources:

[1]	https://www.fao.org/4/W3732E/w3732e07.htm
[2]	https://doaj.org/article/3045d80512594f9698330174049edc0a
[3]	https://drs.cift.res.in/items/6c39fa37-d637-4fa2-b4d9-5d24833b5d0b
[4]	https://krishi.icar.gov.in/ohs-2.3.1/index.php/record/view/61027
[5]	https://pmc.ncbi.nlm.nih.gov/articles/PMC4999905
[6]	https://revistas.ucr.ac.cr/index.php/rbt/article/view/3415
[7]	https://link.springer.com/10.1007/s10811-022-02730-1
[8]	https://pure.atu.ie/en/publications...-fatty-acid-profiling-of-marine-species-wi-3/
[9]	https://pmc.ncbi.nlm.nih.gov/articles/PMC5121895/table/t1
[10]	https://acnpsearch.tweb-dev.unibo.it/singlejournalindex/7648635
[11]	https://digital.csic.es/handle/10261/71870
[12]	https://www.jstage.jst.go.jp/article/jos1956/42/4/42_4_272/_pdf/-char/ja

This is awesome, thank you! Might be worth doing all 3 to match nutrients, but also delivery to various parts of the tank.

I'm on day 2 of Tetra and not sure about the color. I read here it settles on the bottom at first before, so perhaps this color is the result of that?

Your culture looks like it didn't settle much at all. Usually the water is nearly clear and you only see green on the bottom.