Materials and Methods

Collection of Wastewater Algae
Algae growing in wastewaters were collected from sewage fed freshwater lakes (Bellandur and Varthur, South Bangalore) of Bangalore and Sewage treatment plant (Rayan kere STP) at Mysore, Karnataka, India. Samples were collected during the mid day as algal densities are expected to be higher at peak photosynthetic period in such open waters. Water samples were also collected for laboratory analysis.

Isolation and Culturing
Water samples (200 ml) containing algae were centrifuged (5000 rpm) repeatedly and washed with de-ionized water. The near pure populations of select algae were then serially diluted and spread over nutrient agar plates. After their growth in agar plates they were inoculated in 500 ml Erlenmeyer flask containing artificial wastewater media prepared as per Yujie et al. ¹⁶ and were incubated at 25°C under cool white fluorescent light (~4000 lux) and a light to dark period of 16:8 hour was maintained.. The monitoring of algal growth was carried out through an optical microscope every two days. Identification, enumeration and counting of algal samples were done using standard keys and protocols^17,18. Isolated microalgae were then concentrated to a cell density of 10⁷cells/ml and inoculated to filtered-sterilized wastewaters¹⁹ (collected from STP). Later algae were cultivated in batch mode for 10 days in I lt. Erlenmeyer flasks with a working volume of 1 lt. Every day the algal cells were harvested, centrifuged and were checked for growth, compositional variability’s and were dried at 60 ºC for dry wt. measurements. All experiments were conducted in triplicates; the data presented are expressed as mean ± std.dev (µ ± SD). The physico-chemical parameters and nutrients in the wastewater used for culturing is provided in Table 1.

Table 1.   Physicochemical Characteristics of domestic wastewater used for experiments

Lipid Extraction
The total lipids content were determined as per Bligh and Dyer ²⁰. The cell disruption was performed by ultra-sonication (frequency 35 kHz) for 30 minutes in continuous mode at 25^oC and then the lipids were extracted with chloroform-methanol (2:1, v/v), and then separated into chloroform and aqueous methanol layers by the addition of methanol and water to give a final solvent ratio of chloroform:methanol:water as 2:1.1:0.9. The chloroform layer was then evaporated using a rotary vacuum evaporator with a water bath temperature of 60°C and lipids were concentrated.

Lipid extraction and processing
Lipid classes were separated through 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 (mobile phase; v/v). The bands were visualized after staining the TLC plates with iodine vapours as per standard TLC protocols. The triacylglyceride (TAG) layer was carefully removed and collected immediately and was processed for lipid extraction. The presence of lipid compounds was detected by brown bands against a white background, which were compared with the coconut oil standard.

Spectral Signatures and Biochemical composition transition by ATR-FTIR
Composition of algal biomass and the type of functional groups of the algal extract is assessed through FTIR spectroscopy study.  Attenuated total reflectance ATR-FTIR spectra were collected on a Bruker Alpha Spec Instrument. Dried algal cells were pressed against the diamond cell prior to scanning.  The extracts from Chlorococcum sp., Microcystis sp. and Phormidium sp. with Coconut oil (standard) were observed for their functionalities in the spectrogram. The spectra were collected in the Mid IR range (64 scans) from 4000 to 800 cm^-1 (at a spectral resolution of 2 cm^-1) and data were analyzed using Origin Pro 8 SR0, v8.0724 (B724) with an initial base line correction, and scaled upto Amide I_max. The peaks with each of the spectral curves were carefully fitted and calculated²¹. The biochemical transitions in the algal cells were monitored by the analysis of Carbohydrate to Protein (C/P); Lipid to Protein (L/P) and Lipid to Phosphate (L/Phos.) ratio^22,23,24.

Analysis of Fatty acid Methyl Ester Profile
Methylation of lipids was performed by converting all fatty acids to their corresponding Methyl esters (transesterification) using Boron trifluoride-Methanol (BF₃-MeOH) and H₂SO₄ as the catalyst.  BF₃-MeOH converts fatty acids to their methyl esters and has been used as most common catalyst for FAME preparation. The extracted samples were heated at 60°C for 15mins. It was then cooled in ice-bath for 5 mins 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 methylated sample was loaded onto silica column with helium gas as carrier in split-less 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. The composition of the FAME was assessed through gas chromatograph (Agilent Technologies 7890C, GC System) using detection by Mass spectrometry (Agilent Technologies 5975C insert MSD with Triple-Axis Detector). HP-5% Phenyl Methyl Siloxane: 756-42744 column was used, max temperature 325^oC; with column dimensions 30 m X 250 µm X 0.25 µm that was purged with helium gas.

The injection and detector temperature were maintained at 250°C and 280°C respectively (ASTM D 2800). 1 µl volume of sample was injected into the column, whose initial temperature was maintained at 40°C. After 1 min the oven temperature was raised to 100°C at a ramp rate of 10°C min^-1. The oven temperature was then raised to 150°C at a ramp rate of 2°C min^-1 and then it was raised to 230°C at a ramp rate of 3°C min^-1 and finally the oven temperature was raised to 300°C at a ramp rate of 10°C min^-1 this temperature was maintained for 2 minutes. The components were identified based on their retention times, abundance and fragmentation patterns.