Gas chromatography

The neutral sugar content and composition was determined in duplicate according to Englyst and Cummings (1984) [16 ], using inositol as an internal standard. Samples were treated with 72% (w/w) H₂SO₄ (1 h, 30°C) followed by hydrolysis with 1 M H₂SO₄ for 3 h at 100°C, uronic acids released were analysed (section below) and the constituent sugars released were analysed as their alditol acetates using gas chromatography (ThermoScientific, Waltham, MA, USA). Total carbohydrate content was calculated as the sum of neutral carbohydrates and uronic acids.

Lipid samples were prepared from mouse liver tissue powders by a modified Bligh and Dyer method as previously described.⁵⁰ Internal standards for quantification of individual lipid molecular species were added before lipid extraction.⁵⁰ Shotgun lipidomics analyses were performed with a QqQ mass spectrometer (Thermo Fisher Scientific TSQ Vantage, San Jose, CA, USA) equipped with an automated nanospray device (TriversaNanomate, Advion Biosciences, Ithaca, NY, USA) and operated with the Xcalibur software (Thermo Fisher Scientific, Inc., Waltham, MA, USA) as previously described.^{51 (link)} Identification and quantification of triacylglycerol molecular species were performed as previously described.^{52 (link),53 (link)} Analysis of fatty acids was also carried out in 14-day-old male and female flies by gas chromatography (Microbial ID, Inc., Newark, DE, USA).

The cecal contents were extracted and placed in a −72 °C refrigerator. The water of cecal contents was removed using freeze-drying. Then, 500 μL of NaCl solution and 100 mg cecal contents were mixed and placed at ordinary temperature for 60 min, followed by 20 mL H₂SO₄ (10%, v/v) and 800 μL C₂H₅OC₂H₅ added to the sample and vibrated. After centrifugation (13,000 rpm, 20 min, 4 °C), the supernatant was collected and placed in new centrifuge tubes (containing 0.25 g Na₂SO₄). After centrifugation (13,000 rpm, 20 min, 4 °C), the supernatant was collected and analyzed using gas chromatography (Thermo Fisher Scientific, Carlsbad, CA, USA).

Fecal concentrations of short chain fatty acids (SCFAs) were determined as previously described [21 (link)], with slight modifications. In brief, approximately 0.1 g of fecal samples (n = 8–10/group) was placed into 1.5-mL centrifuge tubes, diluted with 1 mL 0.5% of phosphoric acid solution and homogenized. Then, the samples were centrifuged at 14,400 × g for 10 min to obtain the supernatant. The supernatant was extracted with equal volume of ethyl acetate, and precipitated in refrigerator at − 20 °C. Then, the samples were centrifuged at 14,400 × g for 10 min to obtain the supernatant. The concentrations of SCFAs in the supernatant were determined using gas chromatography (Thermo, Waltham, USA). All procedures were performed in duplicate.

Dry algal biomass was crushed and total lipid was estimated by adding chloroform and methanol (2:1 volume:volume) followed by heating in soxhlet apparatus for 6–7 cycles for extraction. The solvent was dried by rotary evaporator. The percentage of total lipid and lipid content was calculated by the formula described by Nag Dasgupta et al. [7 (link)]. Briefly, percentage of total lipid (Lipid%) in dry biomass was calculated by the following formula: $$\text{Lipid}\%=\text{weight}\left(\text{extracted}\text{lipid}\right)/\text{weight}\left(\text{biomass}\text{taken}\right)\times 100$$
The lipid content was calculated by the following formula: $$\text{Lipid content}\left(\text{mg}{\text{L}}^{-1}\right)=\text{Lipid}\%\times \text{Biomass content}$$
The lipid productivity was calculated by the formula given by Griffiths and Harrison [54 (link)]: $$\text{Lipid productivity}\left(\text{mg}{\text{L}}^{-1}{\text{day}}^{-1}\right)=\text{Lipid}\%\times \text{Biomass productivity}$$
The extracted lipid was refluxed for 5 h in round bottom flask at 50 °C in the presence of methanol and 2% sulphuric acid for transesterification. After removal of impurities the FAME mix was dissolved in hexane and analyzed by Gas-Chromatography (Thermo Fisher Scientific) and quantified against a standard FAME mix (Supelco, USA). Biodiesel properties of FAME were estimated from the percentage of fatty acids (weight/weight) obtained in a Gas-Chromatographic analysis using the online software “BiodieselAnalyzer© Ver. 2.2” (http://www.brteam.ir/biodieselanalyzer) [55 (link)].