Growth curves for Chlamydomonas noctigama, Chlorella vulgaris, Anabaena variabilis and Nostoc spongiaeforme were made using growth as µg chlorophyll per mL (chl a and chl b for green algae, and chl a for cyanobacteria) at respective optimum conditions to define the different phases of growth as described by Vonshak and Maske (1982). Various optimum conditions considered as baseline for growth at different phases.
Growth rate and doubling time determination: Growth rate has been expressed in terms of the relative growth constant or specific growth constant (µ) (Fogg 1975):
µ = log10 Nt – log10 N0 / t
where, t = Time in the hour
Nt = Biomass after t hour
N0 = Biomass at “0” time
The maximum growth rate is defined as the maximum growth rate under light saturation at a specified temperature. The mean generation time or doubling time (g) has been calculated from a specific growth constant, µ:
Doubling time, g = 0.0301 / µ (Fogg 1975)
Estimating O.D. (Optical Density): Growth was also estimated by measuring the optical density at 750 nm using a Shimadzu digital spectrophotometer (model UV-120-01) as described by Rodolfi et al. (2009).
Production of green algae and cyanobacteria biomass at the 3 phases of growth under optimum conditions: All the respective optimum conditions (i.e., pH, light intensity, temperature, aeration, nutrient element concentration in medium and vitamin supplement) were provided to produce both green algae (Chlamydomonas noctigama and Chlorella vulgaris) and cyanobacteria (Anabaena variabilis and Nostoc spongiaeforme) biomass at their 3 phases of growth (i.e., logarithmic phase, stationary phase, and in between of stationary and death phases) where unialgal cultures were used.
Harvesting and processing of microalgal biomass: The microalgae were harvested by centrifugation at 5000 rpm for 10 minutes using a Kokusan refrigerated centrifuge (model H-103N).
The wet algal biomass (harvested on petri dish) was dried in an oven at 60oC for at least 24 hours. Then the dried biomass was scrapped and stored in a plastic jar.