Isochrysis culture regime...thoughts?

[LordJoshaeus]

Hi everyone! I am working on devising a mixotrophic Isochrysis culture regime for if I culture copepods for marine fish/invert larvae. How does this sound?

CULTURE CONTAINERS; Two 1 gallon jars. I would restart one jar each week, saving 1/4th of the jar for restarting the culture and putting the rest in the fridge to feed the pods over the course of the week.
LIGHTING; A 12 inch, 5000K LED bar, on 12 hours a day. Shooting for about 100 mmol at the surface of the cultures.
WATER: Salt water derived from water change water on a saltwater tank. The water would be passed through a 1 micron filter sock to prevent competitors or predators from entering the cultures, and I may also use bleach after the filter sock to be particularly thorough (obviously the water would have to be dechlorinated after the bleach has done its job); I would then add F/2 fertilizer, .5 ml glycerol/glycerin (which Isochrysis and many other microalgae can use as a carbon source) and some baking soda (the bicarbonates would make photosynthesis easier for the Iso, which could convert it to CO2).
CIRCULATION; An air line running full bore in each jar.
HEATING; Both cultures would be situated in a container of water heated to the high 70's fahrenheit with a 25 watt heater.

Two more questions...how do I tell when the Iso is ready to be harvested, so I can adjust the culture restart times as needed? And roughly how many gallons of Parvocalanus culture could this setup support? Thanks

EDIT: Forgot a question! I was going to use 2 drops bleach per quart old saltwater (coming to about .4 ml standard household bleach per gallon of the saltwater) to disinfect the water. How much seachem prime would I subsequently have to use to neutralize the bleach?

 

[ichthyogeek]

I guess if you have it on hand, you might as well try with glycerol/glycerin and baking soda, but my question is why? Don't microalgae already use CO2 as a carbon source? And doesn't F/2 already contain sodium bicarbonate (baking soda)?

Iso is ready to be harvested...when Iso is ready to be harvested. It depends on what density of microalgae you're aiming for. If you've got a microscope, then great. If not, you're going to be reliant on the color test. Some microalgaes are pickier than others For example, I'v worked with Rhodomonas and Dunaliella before. Rhodo is a picky phytoplankton, it wants higher nutrients than most, and will crash if you don't harvest frequently. Dunaliella on the other hand is okay with being neglected for quite a while. It may be worthwhile to pick up a copy of the Plankton Culture Manual.

Here's a paper that describes what density to of iso cells to stock with for Parvo.

Bleach vs saltwater vs. Prime:
First, calculate how much actual bleach (sodium hypochlorite) is present in one mL of bleach solution (the stuff in the bottle). call this X
Next, figure out how much bleach a drop of prime is supposed to neutralize. Call this Y
Finally, take X and multiply it by the number of mL you're dosing. This gives you the total amount of bleach you're adding. Divide this by Y, to get the number of drops of prime you're adding to neutralize it.

 

[LordJoshaeus]

I guess if you have it on hand, you might as well try with glycerol/glycerin and baking soda, but my question is why? Don't microalgae already use CO2 as a carbon source? And doesn't F/2 already contain sodium bicarbonate (baking soda)?

Iso is ready to be harvested...when Iso is ready to be harvested. It depends on what density of microalgae you're aiming for. If you've got a microscope, then great. If not, you're going to be reliant on the color test. Some microalgaes are pickier than others For example, I'v worked with Rhodomonas and Dunaliella before. Rhodo is a picky phytoplankton, it wants higher nutrients than most, and will crash if you don't harvest frequently. Dunaliella on the other hand is okay with being neglected for quite a while. It may be worthwhile to pick up a copy of the Plankton Culture Manual.

Here's a paper that describes what density to of iso cells to stock with for Parvo.

Bleach vs saltwater vs. Prime:
First, calculate how much actual bleach (sodium hypochlorite) is present in one mL of bleach solution (the stuff in the bottle). call this X
Next, figure out how much bleach a drop of prime is supposed to neutralize. Call this Y
Finally, take X and multiply it by the number of mL you're dosing. This gives you the total amount of bleach you're adding. Divide this by Y, to get the number of drops of prime you're adding to neutralize it.

I did not realize the F/2 already had baking soda as an ingredient...my bad. As for the glycerin, a study found that glycerin can be used as a carbon source (for heterotrophic, rather than phototrophic, growth...they directly eat it rather than using it for photosynthesis) by Isochrysis, and the same study found that this species grew better when capable of both photosynthesizing and eating the glycerin (growth was impeded if they were only allowed to grow heterotrophically, eg with glycerin but no light). Here's the study in question; https://www.hindawi.com/journals/bmri/2013/983465/

I have no microscope currently, alas. I was not able to find a direct "Quantity of prime neutralizes X amount of bleach" formula, though purigen's recharging directions list a rather roundabout formula. Going to take a better look at it now...

EDIT: While I was not able to get the formula for neutralizing bleach with prime, I WAS able to find the equation for neutralizing bleach with plain ol' sodium thiosulfate; 4 NaOCl (bleach) + Na2S2O3 (dechlor) + 2 NaOH → 4 NaCl + 2 Na2SO4 + H2O

As household bleach is generally 5.25% bleach by mass, it would take about .015 grams thiosulfate to neutralize .5 ml bleach - roughly the amount I would be using per gallon here.

 

[LordJoshaeus]

As household bleach is generally 5.25% bleach by mass, it would take about .015 grams thiosulfate to neutralize .5 ml bleach - roughly the amount I would be using per gallon here.

There was an error to the above calculation...sodium thiosulfate is almost always sold as the pentahydrate, which has a higher molar mass of 248.18 grams per mol (I had used 158.11 grams/mol, which is the value for anhydrous sodium thiosulfate) ; plugging this 'new' value in revealed that it takes about .024 grams thiosulfate to neutralize .5 ml standard household bleach. Also, the bleach disinfecting method I was planning to use is derived from drinking water protocols, which recommended leaving the water for half an hour to let the bleach do its job...I forgot to mention that part above.

 

[ichthyogeek]

That's true, but remember you're using sodium thiosulfate in a hydrated form (i.e. Prime, which is liquid). Isn't chemistry fun???

And I'm fairly certain that thiosulfate itself does not have any negative effects on micralgal growth as well, so you're allowed to overdose. In addition, some of that bleach is going to be decomposed due to sunlight, temperature, and reactions with organic materials (like bacteria!).

 

[LordJoshaeus]

That's true, but remember you're using sodium thiosulfate in a hydrated form (i.e. Prime, which is liquid). Isn't chemistry fun???

And I'm fairly certain that thiosulfate itself does not have any negative effects on micralgal growth as well, so you're allowed to overdose. In addition, some of that bleach is going to be decomposed due to sunlight, temperature, and reactions with organic materials (like bacteria!).

Since I was not able to find the equation for dechlorinating bleach with prime, I was just going to buy some thiosulfate powder (which has a density of about 1.2-1.3 grams per ml as a powder...I was going to assume the lower value to be safe) and make my own solution with DI water to do the job. Good to know that there would be no serious consequence to overdosing somewhat, though

Is there any reason using the old aquarium water would not work so long as I disinfect the water before using it, by the way? I understand that the reason given for not using old aquarium water for live food cultures like this is the risk of other organisms coming in from the old water and outcompeting or eating the desired organism, and if the water is completely sterile when added to the culture that would not seem to be an issue.

EDIT; Further calculation found that a mere quarter teaspoon of thiosulfate powder would be sufficient to neutralize 60 gallons of seawater 'bleached' in the above fashion. If this works, it would seem a lot more viable than having to make new seawater every week for the algae (I calculated it, and for me using a DI resin filter my setup would cost twice as much if I had to make new water for the algae...people with RODI units would not find it much cheaper).

EDIT 2; I sure like to blab a lot on trivial things like growing algae! Apparently carbonate levels as high as one part per thousand (or 1 gram per liter) can improve isochrysis growth (for comparison, a teaspoon of baking soda per gallon would add about 922 ppm carbonates). See here; https://link.springer.com/article/10.1007/s10811-019-01888-5

 

[ThRoewer]

I'm planning on setting up a few pure algae cultures as well, probably Nannochloropsis, Tetraselmis, and Isochrysis to have a better mix. But I'm probably going with 5 gallon water bottles or Kalwall tubes to get the volumes I need.

I was recommended to use 0.5 ml/L of regular household bleach to sterilize the water, wait 24 hours and then use thiosulfate to neutralize the bleach, and then check with a pool chlorination test kit that it is properly dechlorinated.

 

[ichthyogeek]

I'm so jealous of y'all. My future setup's going to have to be 2L bottles and maybe a few 1 gallon containers to maximize space. No fancy kalwalls or 5 gallon water bottles for me...

 

[LordJoshaeus]

I'm planning on setting up a few pure algae cultures as well, probably Nannochloropsis, Tetraselmis, and Isochrysis to have a better mix. But I'm probably going with 5 gallon water bottles or Kalwall tubes to get the volumes I need.

I was recommended to use 0.5 ml/L of regular household bleach to sterilize the water, wait 24 hours and then use thiosulfate to neutralize the bleach, and then check with a pool chlorination test kit that it is properly dechlorinated.

Never heard of a Kalwall tube before...wouldn't gas exchange be an issue with such a tall container?

I was basing my sterilization figures on emergency drinking water guidelines. I read that, during an emergency, 2 drops (.1 ml) of bleach should be added per quart of water to be disinfected (if done right, making the water smell like chlorine), stirred in, and allowed to sit for 30 minutes before drinking the water. Confusingly, I just did further research in response to your reply and found that 6% and 8.25% bleaches are also common (in which case it may be safer for me to double the above figure I gave for the sodium thiosulfate dose...). The chlorination kit would definitely be a good idea.

EDIT: Could this 'bleached' old tank water also be used for copepods, or would a residue from the thiosulfate harm them?

 

[ThRoewer]

Never heard of a Kalwall tube before...wouldn't gas exchange be an issue with such a tall container?

You would need to aerate them. You need agitation anyway for almost all algae cultures or they crash or at a minimum never reach their maximal possible density.

EDIT: Could this 'bleached' old tank water also be used for copepods, or would a residue from the thiosulfate harm them?

That is precisely what I plan on doing, use sterilized old tank water for algae and pod cultures.

 

[LordJoshaeus]

You would need to aerate them. You need agitation anyway for almost all algae cultures or they crash or at a minimum never reach their maximal possible density.

That is precisely what I plan on doing, use sterilized old tank water for algae and pod cultures.

True...I was just wondering whether even aeration could overcome the very low surface area that the kalwall tubes seem to possess. Being able to reuse the old tank water would save an awful lot of money, especially on salt (which, at least from my research and past experiences, seems to be the largest expense with saltwater)

 

[LordJoshaeus]

EDIT 2; I sure like to blab a lot on trivial things like growing algae! Apparently carbonate levels as high as one part per thousand (or 1 gram per liter) can improve isochrysis growth (for comparison, a teaspoon of baking soda per gallon would add about 922 ppm carbonates). See here; https://link.springer.com/article/10.1007/s10811-019-01888-5

Sorry...got my math wrong again! I had calculated the above carbonate figure with bicarbonate's molar mass. Using the molar mass for carbonates (and thus removing the hydrogen in the bicarbonates from the equation) gives a slightly smaller (but still substantial) 907 ppm, or .907 grams per liter.

 

[ThRoewer]

Here's a paper that describes what density to of iso cells to stock with for Parvo.

...See here; https://link.springer.com/article/10.1007/s10811-019-01888-5

You guys got access to those papers?

 

[ichthyogeek]

Whoa...sorry about that, I didn't realize that my university access was still available...which means I have to download a bunch of papers to save them for later....I thought they were publicly accessible!

Here's the relevant bit about Iso cultures from the paper I sent: 150 000 cells isochrysis galbana (tahitian iso) + 150 000 cells Chaetoceros muelleri per mL were fed to Parvocalanus cultures. The papers make it unclear as to how much of said 150K cells/mL were added to the culture, but I assume it was either fed to 150K cells/mL, or an amount of 150k cells/mL was added to each parvo culturing container.

 

[ThRoewer]

Whoa...sorry about that, I didn't realize that my university access was still available...which means I have to download a bunch of papers to save them for later....I thought they were publicly accessible!

Here's the relevant bit about Iso cultures from the paper I sent: 150 000 cells isochrysis galbana (tahitian iso) + 150 000 cells Chaetoceros muelleri per mL were fed to Parvocalanus cultures. The papers make it unclear as to how much of said 150K cells/mL were added to the culture, but I assume it was either fed to 150K cells/mL, or an amount of 150k cells/mL was added to each parvo culturing container.

Yeah, I had access to many papers on my previous job but not enough time to find everything of interest...

 

[LordJoshaeus]

Even with a microscope, I suspect that it may be difficult to count the number of algae cells in a culture...there's just so many of them, even just per milliliter. I was just planning on feeding the copepods enough Iso to slightly tint the culture, and not feed again until the water is not tinted (I would check once in the morning and once in the evening).

Also, I found an interesting idea while researching phytoplankton culture; https://www.melevsreef.com/articles/culture-your-own-phytoplankton

He mentions that he rotates his bottles of active phyto cultures daily to put sediment in the bottom back into suspension, and he said that this resulted in his cultures getting quite dark and lasting 3 weeks or so. I think I am going to restart my main cultures once a week, but I may still set up an experimental culture and see how long it lasts with the rotating treatment.

 

[taricha]

Even with a microscope, I suspect that it may be difficult to count the number of algae cells in a culture...there's just so many of them, even just per milliliter.

secchi stick

 

[ichthyogeek]

Even with a microscope, I suspect that it may be difficult to count the number of algae cells in a culture...there's just so many of them, even just per milliliter. I was just planning on feeding the copepods enough Iso to slightly tint the culture, and not feed again until the water is not tinted (I would check once in the morning and once in the evening).

A secchi stick does work, but it tends to be used for big volumes of culture (like with ponds). I prefer using hemocytometers. Far easier to use with a microscope, and you count a much smaller volume and multiply up.

 

[LordJoshaeus]

secchi stick

A secchi stick does work, but it tends to be used for big volumes of culture (like with ponds). I prefer using hemocytometers. Far easier to use with a microscope, and you count a much smaller volume and multiply up.

Cool! Looking into both of those options. I sent Florida aqua farms a message asking about how big a microalgae culture needs to be for the secchi stick (which they refer to as a 'microalgae density measurer') to work properly.

 

[LordJoshaeus]

Hi everyone! Florida Aqua farms got back to me about the microalgae density measure. They replied, on quote;

"It can only be as tall as the stick the width does not matter. It is like 35 cm long."

Clearly this device is small enough to be hobbyist friendly...