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?