Duarte and Maugeri (2014) studied lipid production by Candida sp. LEB-M3 cultivated in pure and raw glycerol. The feasibility of biodiesel production by the yeast Candida sp. LEB-M3 was indicated by predicting FAME properties for pure and raw glycerol respectively, including cetane number (56–53), heat of combustion (37–39 kJ/g), oxidative stability (8.58 h), kinematic viscosity (3.82–3.79 mm²/s), density (807–872 kg/m³), and iodine index (74–115.5 gI₂/100 g). Leiva-Candia et al. (2015) estimated biodiesel properties produced from SCO derived from Rhodosporidium toruloides, Lipomyces starkey, and Cryptococcus curvatus cultivated on biodiesel by-product streams. More specifically, cetane number (62.39–69.74), lower calorific value (37,393.49–37,561.68 kJ/kg), cold-filter plugging point (4.29–9.58°C), flash point (158.73–170.34°C), and kinematic viscosity (4.6–34.87 mm²/sat 40°C) were determined.

Research has also focused on direct transesterification of SCO for biodiesel production. A successful direct transesterification method will eliminate the need for lipid extraction, reducing the use of solvents and avoiding the potential lipid loss during the extraction phase. Two issues must be considered at this point, namely the high water content of the microbial mass and the presence of lipases at the end of the fermentation, which hydrolyse the intracellular triglycerides, increasing the content of free-fatty acids in the SCO. During the procedure of alkaline catalysis, soap formation through neutralization of free-fatty acids and saponification of triglycerides is triggered due to the presence of FFAs and water, while in the case of acid catalysis, this problem is avoided through esterification of FFAs into their corresponding esters. Liu and Zhao (2007) reported a direct acid-catalyzed methanolysis method that uses oleaginous microbial mass from Lipomyces starkeyi, Rhodosporidium toruloides, and Mortierella isabellina as feedstock for biodiesel production with FAME yield up to 90% and a CN of 59.9, 63.5, and 56.4, respectively. The optimum reaction conditions applied by Liu and Zhao (2007) were 0.2 mol/L H₂SO₄ at 70°C for 20 h with a biomass-to-methanol ratio of 1:20 (w/v). Vicente et al. (2009), compared the efficiency of direct transesterification with indirect transesterification (lipid extraction was carried out by three solvent systems including chloroform:methanol, chloroform:methanol:water, and n-hexane) for biodiesel production from SCO produced by the fungal strain Mucor circinelloides. The direct transesterification method produced FAME with higher purities (>99%) than those from the indirect process (91.4–98.0%) and a significantly higher yield due to a more efficient lipid extraction when the acid catalyst was present (Vicente et al., 2009). The reaction conditions applied by Vicente et al. (2009) were 8% (w/w relatively to the microbial oil) BF3, H₂SO₄, or HCl for 8 h at 65°C with a methanol-to-oil molar ratio of 60:1. Thliveros et al. (2014) introduced a direct alkali-catalyzed methanolysis method using the yeast Rhodosporidium toruloides Y4. Under the conditions of 4 g/L NaOH, 1:20 (w/v) dried biomass to methanol ratio for a 10-h reaction duration at 50°C, the FAME yield was 97.7%. Koutinas et al. (2014) reported that the production of biodiesel from SCO via indirect transesterification of extracted SCO is a more cost-competitive process than direct conversion of dried yeast biomass.