METHODS

1. Microalgae sample collection: Natural population of Microcystis sp. was obtained from Vaderahalli Lake situated in Bangalore, India. Microcystis sp. formed more than 95 % of the total micro algal community due to algal bloom.

2. Cell disruption: An aliquot of 6.71g of the dry cell biomass was mixed with 250 ml of distilled water and was disrupted using following methods

1. Sonication disruption method: This includes
1. Sonication using a sonicator (KIKA Labortechnik Staufen, Germany) for a time interval of 10 (10), 20 (20), 30 (30), 40 (40), 50 (50) and 60 (60) minutes
2. Sonication - hexane solvent (hexane: 2-propanol, 2:3) for a time interval of 30 (VS 30) and 60 (VS 60) minutes, and
3. Sonication – supercritical nitrogen for a time interval of 30 (VS 30n) and 60 (VS 60n) minutes.
2. Bead beating disruption method: Variants used here are
1. Using 1mm glass beads for a time interval of 10 (10), 20(20), 30(30), 40(40), 50(50) and 60 minutes (60),
2. Using hexane solvent (hexane: 2-propanol, 2:3) for a time interval of 60  minutes (VB3H), and
3. Using supercritical nitrogen for a time interval of 60 minutes (VB3Hn).
3. Manual disruption methods are:
1. Supercritical nitrogen – manual for a time interval of 60 minutes (VHM), and
2. Hexane solvent (hexane: 2-propanol, 2:3) – manual for a time interval of 60 minutes (VNM).

3. Lipid extraction: The protocol for lipid extraction from micro algae was as follows:

1. Pre-thin layer chromatography: The extraction of lipids was done by mixing in chloroform: methanol (2:1) using a modified Bligh and Dyer’s methods (Bligh & Dyer 1959). The chloroform layer was evaporated using rotary evaporator (Eppendorf Vacuum Concentrator 5301) to obtain lipids. This step was important since lipids are highly sensitive to hydrolysis and oxidation processes during storage (Sasaki & Capuzzo 1984).
2. Thin layer chromatography: Samples were reconstituted in chloroform to make stock solutions. These were later spotted in bands onto silica gel TLC plates (Merck KGaA). The mobile phase consisted of a solvent system of hexane/diethyl ether/acetic acid (70:30:1 by volume) (Maloney 1996). The plates were developed by exposing the vapors of iodine crystals to stain the plates for visualizing neutral lipids. The samples were extracted and stored at -20 0C until further analysis (Mansour et al. 2005).
3. Gas chromatography-mass spectrometry analysis (GC-MS): After the initial thin layer chromatography (TLC) lipid screening, the extracts were converted into fatty acid methyl esters (FAME) using Boron trifluoride-methanol and was heated in water bath at a temperature of 600C for 1 hour. The methylated sample was then purified further for GC-MS. The main focus of using GC-MS was purely for lipid identification rather than quantification. The injector and detector temperatures were set at 2500C while the initial column temperature was set at 400C for 1 min. A 1 µL sample volume was injected into the column and ran using a 50:1 split ratio. After 1 min, the oven temperature was raised to 1500C at a ramp rate of 10 0C min-1. The oven temperature was then raised to 2300C at a ramp rate of 30C min-1, and finally the oven temperature was increased to 3000C at a ramp rate of 100C min-1 and maintained at this temperature for 2 min. The total run time was programmed for 47.667 min. The mass spectra were acquired and processed using Agilent Chem Station (5975 C; Agilent, USA).

4. Data Analysis: Hierarchical cluster analysis of the fatty-acid composition was performed for sonication, bead beating and hand crushing using PAST Version 2.08 (Hammer et al. 2004). Hierarchical clustering was performed based on a Bray-Curtis similarity coefficient applied to untransformed percentage composition data. The SIMPER (SIMilarity PERcentage—species contribution) in PAST (Version 2.08) was used to investigate the percentage of the fatty acids of the clusters formed. One way ANOVA was used to check the significance level for manual method, a significance level of p < 0.05 was used in all tests.