MATERIALS AND METHODS

1. Microorganisms and Culture Conditions

The freshwater algae used in the present study were collected using plankton net from the Centenary pond, Indian Institute of Science, Bangalore. Chaetocerossp. was isolated from a marine sample and cultured. The medium used to culture microalgae viz., Chlorococcumsp. and Microcystis sp. was Bold’s basal media (Bold 1949, Bischoff and Bold 1963) and for Chaetoceros sp. was f/2 media (Guillard and Ryther, 1963). Initial stock cultures of Chlorococcumsp., Microcystis sp. were maintained in BBM and Chaetocerossp. was cultured in f/2 media and incubated for 10 days at 250C under 16:8 light:dark (L:D) photoperiod in culture room. The halotolerance was determined using different NaCl concentrations i.e., 0.2 ppt, 1 ppt and 2 ppt for freshwater algae and 35 ppt, 175 ppt and 350 ppt for marine alga. To study the effect of salinity on the three different algae, experiments were carried out in 250 ml Erlenmeyer flaskseach containing 100 ml of appropriate medium incubated at 250C under 16:8 light:dark (L:D) photoperiod. Control cultures were also maintained under similar culture conditions. The experiments were set up for 7 days. The cell count and extraction of total lipid of all the three microalgae at three different salinities were determined every day. All the experiments were carried out in duplicates.

2. Extraction of Total Lipids

The total lipids were extracted by mixing chloroform – methanol (4:2 v/v) with the algal samples using slightly modified version of Bligh and Dyer’s method (Bligh and Dyer, 1959). Algal biomass was collected by centrifuging the algal culture at 3000 rpm for 10 minutes. The supernatant was discarded in case of Chlorococcumsp. and Chaetocerossp. whereas cells of Microcystis sp. were collected from the upper part of the centrifuge tube. The algal biomass was suspended in 4 ml chloroform and 2 ml methanol and shaken well. The cells were then, subjectedto sonication for the complete disruption of cells for 1 hour. The chloroform – methanol forms a biphasic layer. The lower lipid layer was separated carefully using the eppendorf micropipette and transferred into a centrifuge tube. About 2 ml of distilled water was added and vortex well for further purification. The total lipid was transferred to clean dried and weighed glass centrifuge tube. The weight of total lipid wasdetermined gravimetrically.