Discussion

Materials and Methods

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Algal and Bacterial Strains

The algal strain used in this work was the Chlamydomonas reinhardtii metE mutant obtained by Helliwell et al. [ Helliwell ref.] and which is vitamin B12-dependent. The bacterial strain used in this work is the vitamin B12-producing soil bacterium Mesorhizobium loti, which was shown to be able to support the growth of the C. reinhardtii metE mutant, implying an exchange of vitamin B12 [Helliwell ref.]. 

Culture Growth

All cultures were grown at 25⁰C, with shaking at 120rpm and in a 12h/12h light dark cycle with a light intensity of approximately 70 µmol m-2 s-1 during the light period. The culturing workflow is depicted in Figure … The Chlamydomonas reinhardtii metE mutant was grown in 600ml of TRIS minimal media supplemented with 1000ng/L B12 in a 1L conical flask for 48 hours with an approximate starting cell density of 1x10cells/ml. Subsequently 300ml was resuspended in 1L of fresh TRIS minimal media, this time supplemented with 100ng/L B12 and 5mM NaH13CO3. This culture was incubated for 48 hours with samples taken at 6, 24, 30 and 48 hours. 
A 400ml culture of Mesorhizobium loti was grown in TRIS minimal media supplemented with 0.1% glycerol for 21 hours starting with an optical density of 0.09 at wavelength 600nm. 90ml from this culture was then sampled, centrifuged at 3220g for 20 minutes to obtain a pellet of bacteria which was resuspended in 750ml TRIS minimal media supplemented with 5mM NaH13CO3 and 250ml of the labelled algal culture was added to initiate the co-culture. The co-culture was then incubated for 72 hours with samples taken at 6, 24, 30, 48, 52 and 72 hours. The same procedure was followed for the control cultures of axenic bacteria in different concentrations of glycerol. 90ml was taken from the bacterial pre-culture and resuspended in 1L of TRIS minimal media supplemented with 5mM NaH13CO3 and either no, 0.001%, 0.01% or 0.1% glycerol.