I am trying to grow Arthrospira platensis using Zarrouk’s medium for research purposes (not for commercial use or consumption by any animal). Growth conditions are set at 30C continuously, with continued agitation using a shaker at 140rpm. Light source is a basic 40W lamp on a cycle of 12hrs on, 12hrs off. Samples are in sets of 50ml in 250ml conical flasks with foil to allow some aeration. However, I have come across a few potential problems that I would appreciate some help with. If you could provide credible sources for your answers that would be gratefully appreciated.
1. Are all the micronutrients required for Zarrouk’s medium? Many protocols appear to differ in the make-up of the micronutrients used. Some appear to completely exclude the "micro-nutrients B" section altogether. What affect would this have on the growth? I have not been able to collect Manganese Chloride, Molybdenum Trioxide, Ammonium Metavanadate, Sodium Tunsgstate, Cobalt Nitrate, so being able to exclude some of these would be helpful. Of these, Manganese Chloride and Molybdenum Trioxide belong to the "micronutrients A" portion. How crucial are these nutrients to the growth of spirulina? for the purposes of this experiment the (exotic) nutrient content of the spirulina is not important - only that it proliferates and is healthy.
2. To avoid precipitates forming when autoclaving chemicals with phosphates with magnesium and calcium, several components were autoclaved separately: sodium bicarb with dipotassium hydrogen phosphate, the rest of the main nutrients, and the micro-nutrients A (minus the molybdenum trioxide and manganese chloride dichloride). However, after autoclaving, the first portion (Sodium bicarb & dipotassium hydrogen phosphate) has large white, crystalline precipitates, the second portion (Rest of main nutrients) has a small quantity of light brown precipitate, and the final portion of micro-nutrients has some slight grains left at the bottom. When these are then combined and used a medium for Spirulina growth, the precipitate persists and has remained there for several days. It is then worrying that many of the nutrients are not available for use by the cyanobacteria and are instead locked up in non-dissolving crystal structure at the bottom of the conical flasks. images have been included.
NaHCO3
K2HPO4
Rest of main nutrients:
NaNO3
K2SO4
NaCl
MgSO4
CaCl2
FeSO4
EDTA-Na2
Micro-nutrients A:
H3BO3
ZnSO4
CuSO4
is it possible that I should have altered the pH first, before autoclaving/combining the reagents? unfortunately my background is in biomedical science, not chemistry. perhaps there is a simple explanation? in many protocols the bicarbonate was filtered rather than autoclaved. is this also a potential culprit?
I would like to recommend only addition of biotin (vitamin H; at 20 μM), which may be the oldest growth factor.
Effect of biotin has recently reported to accelerate the growth of Caco-2 cells (please see file; SEC fucoidan determination). In order to prevent the contamination by fungi and yeast, addition of Amphotericin B may be required.
Serum of 12mo (female, common cold) has UPF0284 protein Tery_1555 (Trichodesmium erythraeum IMS101; sea sawdust; cyanobacteria; carbon fixation and nitrogen fixation) at 21.1 μg/mg of serum protein.
Serum of 1y11mo (female, biotin therapy 44w; excess of biotin) has 4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase, chloroplast at 6.0 μg/mg of serum protein. and NAD(P)H oxidoreductase chain H, chloroplast (Arabidopsis thaliana (Mouse-ear cress)) at 13.6 μg/mg of serum protein, and Nodulation protein Z (Azorhizobium caulinodans; nitrogen fixation bacterium possibly has photosynthetic activity) at 5.1 μg/mg of serum protein.
Serum of 7mo (male; biotin therapy 13w; possibly excess of biotin) has Glycine dehydrogenase (Bradyrhizobium japonicum; nitrogen fixation bacterium, possibly has photosynthetic activity) at 2.0 μg/mg of serum protein.
I am always wondering why such photosynthetic and nitrogen fixation bacteria have been invaded into human serum, but serum of excess of biotin seems to be infected by Cyanobacteria and Rhizobium. Therefore, I have considered that a Cyanobacterium of Spirulina (Arthrospira platensis) may also require biotin as a growth factor.
This is modification of Zarrouk’s medium which we are using:
http://sagdb.uni-goettingen.de/culture_media/02%20Spirulina%20Medium.pdf
I am not sure but I think it was modified to prevent precipitation.
Zarrouk's medium is based on distilled water. However, in my experience, such a white crystalline precipitates occurs when using seawater. I don't know why, but the cause of white crystals is sodium bicarbonate.
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