Dear Keith,

i am honestly not so familiar with the gravimetric qunatification of lipids, since I usually use a phosphate assay to "quantify" lipid content. I am also not familiar with the model system you are using but maybe a few considerations of mine can help you.

The first question which came to my mind is, how sure are you that you have an increase in lipids? Could it be that you have "just" an increase in hydrophobic metabolites or even multispanning transmembrane proteins (e.g. transportproteins of certain flux complexes) which potentially could be extracted alongside your lipids and by that artifically increase the "lipid content". Did you ever tryied to check on a simple TLC if you see an increase in lipids when normalized to cell count or wet weight of culture?

Since i am not familiar with Chlorella i am just spit balling here a bit, but is Chlorella known to form lipid droplets?

Also how certain are you, that you look at an intracellular increase in lipid content? Can it be that Chlorella just increases in size /swells under those conditions which in return would result in a higher demand of lipids to ensure proper membrane properties.

If you truly have an increase in lipids i would maybe check if you can see any changes in the cells morphology like increase in ER due to ER-stress, or increase in mitochondria to cope with nutrient / energy stress? So basically can you see changes in intracellular structures which would explain a higher demand of lipids?

Can you clearly state that the increase in lipids is on the side of chlorella or could it be that Rhizobium is the one contributing stronger to that observation when in co-culture?

Regarding the pH i cant make any statements but i would not be supprised if your modell organisms copes with the drastic pH shifts in a way that would require more lipids and by that adjust their membranes in terms of fluidity etc.

So if you have the tools you should check your "lipid extract" on a TLC and check the morphology of your cells for any striking changes in any of the compartments, this way you might be able to narrow down what the origin of the higher lipid demand might be.

I am sorry i could not give a concrete answer for your questions but i hope that i could spark at least some sort of ideas.

Cheers

Daniel

Hi Dr. Daniel,

Thank you very much for your insights.

I already performed TLC and GC to further confirm the results from the gravimetric quantification of total lipids and TAGs. Our results supported the data obtained from the gravimetric method of lipid measurement. 

Yes, chlorella can form lipid droplets, too. I will conduct Nile Red Fluorescence Assay this week to measure intracellular lipid accumulation.

I tried extracting lipids from Rhizobium as well, and subjected it to TLC, but there were no visible bands in comparison to my control.

I know about ER stress, but I'm not sure if the increase in pH is enough to induce ER stress and unfolded protein response since I'm assuming that the intracellular pH of Chlorella is not affected by the increase of pH in the medium.

But I will check the morphology and check for any significant changes... 

Thanks again!

- Keith