Biodiesel

The S. tonkinensis plants (from Jishui, Jiangxi Province) used in this study were purchased by Jiangsu Guoxing Co., Ltd., in 2011, and planted in the Styracaceae Germplasm Repository, Luhe District, Nanjing, China (32°32′ N, 118°50′ E), where they grew under natural conditions. In early May, 2017, 15 trees were tagged for sampling. Beginning July 15th (50 DAF), fresh fruits were randomly collected every 10 days. The kernels stripped from fruits and seed coats were immediately frozen in liquid nitrogen and stored at − 70 °C until use. After drying in a 65 °C oven for 72 h and weighing, oil content and FA composition, including proportions of saturated, monounsaturated and polyunsaturated FA, were determined by extraction on a Soxhlet apparatus followed by gas chromatography-mass spectrometry (GC/MS), using methods previously described by Zhang et al. [15 (link)]. Biodiesel fuel properties including density (ρ), kinematic viscosity (η), cetane number (CN), iodine value (IV), and cold filter plugging point (CFPP) were predicted from the FA composition according to Wang et al. [40 (link)] and expressed using a triangular prediction model based on the percentages of saturated, monounsaturated, and polyunsaturated acids [40 (link)].
Kernels from four representative time points were used for comparative deep transcriptome analysis. There were three biological replicates for each time point, with each replicate consisting of material pooled from five different trees. Total RNA was extracted using Plant RNA Kit (Omega Bio-Tek, Doraville, GA, USA) according to the manufacturer’s instructions. The quantity and quality of total RNA were assessed using 1% agarose gels and a Nanodrop ND 2000 Spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). The integrity and concentration of total RNA were assessed using a Bioanalyzer 2100 RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA).

BIodIESEL from algal lipids was derived by acidic trans-esterification method. Algae lipid was mixed with methanol with 1:56 ratio (weight ratio) and the reaction was carried out at 45 °C for 4 h in the presence of sulfuric acid (H₂SO₄) as catalyst with1:1 weight ratio of catalyst to lipids. The upper biodiesel layer was separated by a separating funnel, washed several times with 5% NaCl solution to remove any traces of methanol and glycerol. Then, the biodiesel was dried over anhydrous sodium sulfate and collected and evaporated at 45 °C to constant weight. The biodiesel yield was determined gravimetrically by the following equation, Biodiesel yields (%) = [biodiesel mass (g) / algae mass (g) X lipid content %] × 100%

A algae was cultivated in BBM containing a combination of limited two element ( 0.125 g/L Nitrogen and 0.13 g/L phosphorus) and optimal levels of CO₂ and 10 mg/L Fe as the most suitable nutrient levels, in 2000 L (large scale) tubular photobioreactor for enhance lipid accumulation for biodiesel production. The PBR was continuously aerated by compressed-air from an air pump through the static sparger and air flow rate was controlled by a flow meter. The cultivation system was maintained at 24-h photo-period via twenty cool-white fluorescent lights (Philips, TL-D 36 W/54–765) that was illuminated with an intensity of 130–170 µmol m⁻² s⁻¹_.