Carbon Dosing Phyto Cultures

[chimbo84]

I came across this research paper last night. TLDR, it explores the hypothesis that typical phototrophic culturing methods (light with aeration) are carbon and light limited. The argument here is that as cell density increases it blocks the light more and more and you eventually reach a plateau where the culture cannot continue to divide because it is blocking the light from penetrating into the culture.

To remedy this, the authors added a source of organic carbon and found that the addition of 25-50mmol of glycerol (aka., glycerine/glycerin) resulted in a 2x improvement in cell density and growth rate for T-Iso when combined with the typical light and aeration culture method (referred to in this paper as phototrophy). I am going to experiment with this and post my results to this thread but I think this could be really useful since we often have organic carbon sources handy from vodka and vinegar dosing.

FYI, 50mmol of glycerine equates to roughly 4.6g of glycerine per 1L of culture so its a pretty small addition for such a dramatic improvement. Most of the benefit was observed at half that concentration as well so even just 2.5g of glycerine per liter should provide a drastic boost to productivity.

 

[Subsea]

Is not Guilford f/2 fertilizer a carbon source?

 

[chimbo84]

Is not Guilford f/2 fertilizer a carbon source?

It is not.

The formula outlined by Guillard in 1975 is just nitrate, phosphate, silica, and trace vitamins and minerals.

https://shellfish.ifas.ufl.edu/wp-content/uploads/Phytoplankton-Culture-for-Aquaculture-Feed.pdf

 

[Subsea]

It is not.

The formula outlined by Guillard in 1975 is just nitrate, phosphate, silica, and trace vitamins and minerals.

https://shellfish.ifas.ufl.edu/wp-content/uploads/Phytoplankton-Culture-for-Aquaculture-Feed.pdf

Is carbon a mineral? Does a mineral have carbon in it?

Your experiment will add a specific carbon source. I assume you are also adding some fertilizer.

 

[fryman]

Interesting, following along.

I found that tiso, tetraselmis, and nannochloropsis are CO2 limited. I inject CO2 (similar to planted tank setup). I measured a very significant increase in density using CO2.

CO2 injection increased phyto culture density

Just for the heck of it I got a planted tank-style CO2 diffuser setup and ran a couple of otherwise identical phyto cultures side-by- side with one using injected CO2 and the other regular air pumps. After 7 days the CO2-infused nanno culture reached a density of 10.1 million cells/ml, while...

www.reef2reef.com

 

[chimbo84]

Is carbon a mineral? Does a mineral have carbon in it?

Your experiment will add a specific carbon source. I assume you are also adding some fertilizer.

The only components of f/2 that contain any form of carbon are the vitamins b7, b12, and thiamin. However, it is very likely that these are not broken down into available forms through the metabolic processes of the phyto and even if they were, they are in such low concentration as to be negligible in this context. The dosing of glycerine to provide a carbon source provides a vastly different concentration of organic carbon outside of anything provided by f/2 alone.

Yes, my experiment protocol includes the addition of Guillard's f/2 fertilizer. The cultivation method is the same as typical phototrophy except with the addition of Glycerine at 50mmol concentration.

 

[chimbo84]

Interesting, following along.

I found that tiso, tetraselmis, and nannochloropsis are CO2 limited. I inject CO2 (similar to planted tank setup). I measured a very significant increase in density using CO2.

CO2 injection increased phyto culture density

Just for the heck of it I got a planted tank-style CO2 diffuser setup and ran a couple of otherwise identical phyto cultures side-by- side with one using injected CO2 and the other regular air pumps. After 7 days the CO2-infused nanno culture reached a density of 10.1 million cells/ml, while...

www.reef2reef.com

Very interesting! Yes, this would support the theory that the culture is in fact carbon limited. This is one of the two reasons we aerate cultures (to provide a supply of organic carbon via CO2 and to circulate the culture and prevent settling). By providing a direct carbon source in solution with Glycerine, I suspect we may get vastly greater cell density and doubling time as indicating in the research paper (which also supports your results). A 2.5x increase in the Nanno culture is insane. It must have looked black.

I am going to check out your method for measuring cell density. I was going to do it via dry weight but I don't really want to kill the phyto to measure it.

 

[sixty_reefer]

I’ve heard that some types of phytoplankton can become mixotrophs although I would imagine that dosing vodka or vinegar into a culture could cause a bacterial bloom, in a way the bacterial bloom could cause co2 to increase in the vessel but it could also limit the culture by striping nitrates and nitrogen I personally find the easier way to increase carbon by using natural salt. It mixes at a very low ph allowing for more co2 to become available for the culture.
nevertheless I will be following curious to see your results