MATERIALS AND METHODS

A. Water sampling and analysis

Four Lakes (Fig. 1) were selected based on the exploratory survey of 15 lakes during eight months (September 2009- April 2010) which includes water quality analysis of both Inlet and outlet channels. Water samples were collected from four lakes and one agricultural field in Bangalore during May 2010. They were selected based on the anthropogenic stress (industrial runoff, sewage runoff, unpolluted, high nutrient load) influencing on it. Triplicates were collected at each sampling point in 1L polythene bottle. On site physical parameters like pH, water temperature (WT), electric conductivity (EC), salinity and total dissolved solids (TDS) were analyzed using pH/EC probe. Dissolved oxygen (DO) was estimated following Wrinkler’s method. Samples were brought to Aquatic ecology laboratory for further analysis of chemical variables such as Nitrates, Phosphates, Alkalinity, Total hardness, Calcium hardness, Magnesium hardness, Chlorides, Sodium, Potassium, Biological oxygen demand (BOD) and Chemical oxygen demand (COD). These variables were estimated as per standard procedure^[27].

B. Microalgae sampling

Microalgae were sampled from aquatic plant at all sampling points by shaking vigorously and then squeezed in the plastic bag. The resulting brown suspension is transferred into a polythene sample bottle and preserved. Community structure analysis: 0.5 ml of the preserved microalgal sample was observed under light microscope (100X magnification). The entire coverslip was covered to record the presence/absence data of the taxa and photographed for identification.

C. Chlorophyll estimation

25 ml of the microalgal sample was centrifuged t 300 rpm and was filtered. The filtered sample was then processed for chlorophyll estimation following APHA method (APHA 10200 H).

D. Lipid characterization

25 ml of the microalgae sample was sonicated^[28] in water bath for 2 hours at room temperature in order to disrupt the cell membranes, chloroform: methanol (2:1) was added as the extraction solvent. The chloroform layer was evaporated using rotary evaporator (Eppendorf Vacuum Concentrator 5301) to obtain lipids. Thin layer chromatography: All samples were reconstituted in chloroform to make stock solutions. The stock solutions were 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)^[29]. 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 in -20 ºC until further analysis^[30].

E. Gas chromatography–mass spectrometry analysis

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 60 ºC 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 250 ºC while the initial column temperature was set at 40 ºC for 1 min. A 1µL sample volume was injected into the column and ran using a 50:1 split ratio. After 1 min, the oven temperature was raised to 150 ºC at a ramp rate of 10 ºC min^-1. The oven temperature was then raised to 230 ºC at a ramp rate of 3 ºC min-1, and finally the oven temperature was increased to 300 ºC at a ramp rate of 10 ºC 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).