Materials and Methods

1.    Algal screening and selection

Algal samples were collected as a part of biomonitoring programme from sewage fed lake and sewage treatment plants. Spirogyra sp. and Phormidium sp. (macroalgae) were predominant in Varthur lake, Bangalore. Euglena sp. (microalgae) dominated in the sewage treatment plants at Mysore STP. Algal species were identified using standard keys [29] based on their external appearance, colour, morphological characteristics, size, habitat, orientation of chloroplast, cellular structure and pigments etc. Wastewater samples collected were concentrated by centrifuging 15 ml volume. Algae were enumerated using 3 replicates of 20 µl of the concentrated sample where it was placed over the slides with cover slips for microscopy observations. The numbers of algae present were calculated as per equation 1:

2.    Characterisation of the growth environment and water quality

Water samples (1 lt.) in triplicates were collected from the chosen sampling locations between 9 and 12 a.m. Sampling was done always from the sun facing side of the banks/shore avoiding shaded areas. pH, water temperature, electrical conductivity (APHA method 205), total dissolved solids (TDS), salinity, dissolved oxygen (DO), dissolved free carbon-dioxide (free CO₂) and turbidity were measured on-site using the standard methods. 1-litre sub-sample was analysed according to standard methods  [30], total biochemical oxygen demand over 5 days (BOD5) (APHA method 507), filtered BOD5 (APHA method 507, GF/C filtered (1.2 µm pore) with subsequent addition of 1 ml of unfiltered sample), chemical oxygen demand (COD) (APHA, 5220 C), suspended solids (SS) (APHA method 209c), SAR (APHA 1206), turbidity (Hach turbidity meter, APHA method 214a), ammoniacal nitrogen (APHA method 417a), total Kjeldahl nitrogen (TKN) (APHA method 420a, total nitrogen (TN by calculation of TKN + TON), Phosphates (APHA, 4500-P D). All sample bottles were acid-washed. The samples were stored in a cool place until transfer (within few hours) to the laboratories. In addition, visual clarity/transparency of the lake/STP wastewater was measured as with secchi disk.

3.    Algal density and harvesting of algal biomass

Samples collected from field were weighed for total dry wt. Quantification for unit area/volume was done taking 1m X 1m quadrate for microalgae and 10 l volume for microalgae. The macroscopic algal biomass was collected from a quadrate of 1m X 1m from the lakes. These floating algae were carefully washed at site and were transferred to the laboratory for further separation. After microscopic analysis, the samples were washed thoroughly with deionised water and were concentrated by centrifuging at 3292 g for 20 mins for further lipid extraction. The pellet was scrapped carefully using spatula and was exposed to drying at room temperature. The samples were preserved for further use.

4.    Algal Oil Extraction

4.1    Cell disruption: Algal cells after concentration were disrupted using techniques such as (1) Bead beating (bead diameter: 0.75-1 mm) using magnetic stirrer (Shalom Instruments), (2) Sonication using a ultrasonic bath (frequency 35 kHz) for 30mins, and (3) Mortar-pestle Maceration (macerated for 10 mins, manually).

4.2    Lipid Extraction: Lipids were extracted from solvent mixture as per modified Bligh and Dyer’s method (1959) [31]. 0.5gm of dry algal biomass was mixed with a solvent mixture of chloroform and methanol in the ratio of 2:1 (v/v). The organic chloroform layer was separated and was evaporated using a rotary evaporator with a water bath temperature of 60°C. The lipid classes were separated by one-dimensional thin-layer chromatography (TLC) using TLC plates (10 X 10cm, 0.25mm thickness, Merck, Darmstadt, Germany) coated with silica gel. The solvent system used for elution of lipids was a combination of petroleum ether: diethyl ether: acetic acid in a ratio of 70:30:1 (v/v) [32]. The bands were visualized after staining the TLC plates with iodine vapours as per standard TLC protocols [33]. The triacylglyceride (TAG) layer was immediately and carefully scrapped out and was processed for total lipid extraction.

4.3    Methylation: The fatty acids were analysed by methylation of lipid samples using Boron trifluoride-Methanol (BF₃-MeOH) as per established FAME protocols, where BF₃-MeOH converts fatty acids to their methyl esters in 2 minutes. BF₃-MeOH is the most commonly used catalyst used for FAME preparation and has also been adopted by American Oil Chemist’s Society (AOCS) in 1969 [34] (Method Ce 2-66). The extracted samples were heated at 60°C for 15 minutes. It was then cooled in ice-bath for 5 minutes followed by the addition of 1 ml water and hexane respectively. After settling, the top hexane layer was removed and washed using anhydrous sodium sulphate for further purification. The sample was then injected into the gas chromatography column using hexane as a carrier gas. The methyl esters formed were assessed via gas chromatography (GC) and identified using mass spectroscopy (MS) on the basis of their retention time and abundance.

4.4    Fatty Acid Composition using GC-MS: The component of fatty acids was assessed through gas chromatograph (Agilent Technologies 7890C, GC System) using detection by Mass spectrometry (Agilent Technologies 5975C insert MSD with Triple-Axis Detector). The injection and detector temperature were maintained at 250 °C and 280 °C respectively (ASTM D 2800). 1µl volume of sample was injected into the column, whose initial temperature was maintained at 40 °C. After 1min the oven temperature was raised to 150 °C at a ramp rate of 10 °C min^-1. The oven temperature was then raised to 230 °C at a ramp rate of 3 °C min^-1 and finally it was raised to 300 °C at a ramp rate of 10 °C min^-1 and this temperature was maintained for 2 minutes. The methylated sample was loaded onto silica column with helium gas as carrier in splitless mode. The total run time was calculated to be 47.667 min. Fatty acids were identified by comparing the retention time obtained to that of known standards.