Regeants used for the experimental work, were analytical grade purchased from Sigma-Aldrich (Sigma‐Aldrich Trading Co., Shanghai, China). The renewable nutrient substrate was SWCO obtained from households (Cluj-Napoca, Romania). SWCO was used by itself as a carbon source for microbial growth, and it was also used as a lipidic substrate for transesterification reaction to obtain CG fraction.
CG obtaining process (transesterification)
CG fraction was obtained from SWCO in our laboratory using alkali transesterification, as follows: 1000 mL of SWCO were mixed with 240 mL of anhydrous methanol 99.8% and 7 g of NaOH (reagent grade, ≥ 98%, anhydrous pellets), for 24–48 h at room temperature. Two independent phases were obtained: 1030 mL of upper phase - fatty acids methyl esters (biodiesel fraction), and 100 mL of lower phase - aqueous phase (CG fraction) [30, 43, 44]. The content of glycerol, pH, and fatty acids profile were analyzed further for the CG phase. Before adding to the cultivation media, the pH of CG was adjusted to 6 with 2M HCl, the methanol content was removed by evaporation at 70°C for about 40 min (Heidolph Laborata 4000 rotary evaporator, Heidolph Instruments, Schwabach, Germany), and it was sterilized separately at 120°C for 20 min.
Microorganism and culture media:
Lyophilized Y. lipolytica ATCC 20177 purchased from American Type Culture Collection (Manassas, Virginia, USA) was used for the experimental trials. The cells were maintained on yeast malt extract agar plates (yeast extract 3 g/L, malt extract 3 g/L, dextrose 10 g/L, peptone 5 g/L, agar 20 g/L) at 4°C, and re-activated periodically at every 2–3 months.
Culture media components and cultivation conditions were similar to those mentioned by Anastassiadis and Rehm . The inoculums representing 10% of the culture were prepared by transferring 107 CFU/mL (corresponding to 20 mL cellular suspension) into 500 mL shake flask with buffers containing 200 mL of fresh culture media with the following components: 6 g/L NH4Cl, 1.05 g/L KH2PO4, 1.48 g/L MgSO4 x 7H2O, 0.33 g/L MnSO4 x 4H2O, 0.14 g/L FeSO4 x 7H2O, 4 mg/L CuSO4 x 5H2O, 0.08 g/L ZnSO4 x 7H2O, 8 mg/L CoSO4 x 7H2O, 0.08 g/L H3BO3, 0.2 g/L CaCl2, 0.2 g/L NaCl, 0.2 mg/L KI, 0.4 mg/L Na2MoO4 x 2H2O, 0.5 g/L citric acid, 4 mg/L Thiamine-HCl, 0.5 mg/L Biotin, 1.25 mg/L Pyridoxine-HCl, 1.25 mg/L Ca-D-Pantothenate, 1 mg/L Nicotinic acid. The carbon sources used for the inoculums were 50 g/L of PG, CG, and SWCO. The shake flasks were incubated for 2 days at 30oC, pH 5, and 200 rpm (Innova 44 Incubator Shaker, New Brunswick, Eppendorf, Hamburg, Germany).
For the batch fermentations at the bioreactor level (2 L working volume) was used cultivation media consisting of the following components: 6 g/L NH4Cl, 100 g/L carbon source (PG, CG, SWCO), 1.05 g/L KH2PO4, 1.48 g/L MgSO4 x 7H2O, 0.33 g/L MnSO4 x 4H2O, 0.14 g/L FeSO4 x 7H2O, 4 mg/L CuSO4 x 5H2O, 0.08 g/L ZnSO4 x 7H2O, 8 mg/L CoSO4 x 7H2O, 0.08 g/L H3BO3, 0.2 g/L CaCl2, 0.2 g/L NaCl, 0.2 mg/L KI, 0.4 mg/L Na2MoO4 x 2H2O, 2.5 g/L citric acid, 4 mg/L Thiamine-HCl, 0.5 mg/L Biotin, 1.25 mg/L Pyridoxine-HCl, 1.25 mg/L Ca-D-Pantothenate, 1 mg/L Nicotinic acid. Sterile silicon oil was periodically added as antifoaming agent. Vitamins (thiamine-HCl, biotin, pyridoxine-HCl, Ca-D-Pantothenate, and nicotinic acid) and the ammonium source (NH4Cl), for both inoculums and batch fermentation media, were added separately after the medium autoclaving, by filtration through 0.20 µm sterile filters (Macherey-Nagel, Düren, Germany).
Bioreactor batch cultivation:
All experiments were conducted in a 5 L Eppendorf bioreactor (model: BioFlo® 320, one unit, Eppendorf, Hamburg, Germany) filled with a 2 L work volume of medium culture connected and equipped with pH and temperature sensors. Temperature, pH, and rotation were maintained at 30°C, 5, and 200 rpm, and the pH was adjusted automatically by adding 40% NaOH. The inoculums were added in sterile conditions. The fermentation process ran for 192 h in aerobic conditions, maintained by continuous addition of filtered air (0.20 µm filters - Macherey-Nagel) into the fermentation broth through a peristaltic pump (Watson Marlow 520 S, Cornwall, England) settled at 10 rpm and 200 mL/min. Periodically, samples were collected to perform specific tests.
Fatty acids profile from SWCO and CG
The fatty acids concentration from SWCO and CG was established by Gas Chromatography - Mass Spectrometry (GC-MS). The profile of the fatty acids from the total lipids was determined through transesterification by using 1% H2SO4 in methanol [30, 46]. The methylated fatty acids content was determined with a gas chromatograph coupled to a mass spectrometer, model PerkinElmer Clarus 600 T GC-MS (PerkinElmer, Inc., Shelton, CT, USA). A volume of 0.5 µL sample was injected into a 60 m × 0.25 mm i.d., 0.25 µm film thickness SUPELCOWAX 10 capillary column (Supelco Inc., Darmstadt, Germany). The procedure was conducted under the following conditions: injector temperature 210°C; as carrier gas was used helium at a flow rate of 0.8 mL/min; the split ratio of 1:24; oven temperature 140°C (hold 2 min) to 220°C at 7°C/min (hold 23 min); electron impact ionization voltage was 70 eV; trap current 100 µA; ion source temperature 150°C; mass range 22–395 m/z (0.14 scans/s with an intermediate time of 0.02 s between the scans). The fatty acids content was identified by comparing their retention times with known standards (37 components FAME Mix, Supelco no. 47885-U, Darmstadt, Germany) and the resulting mass spectra to those in the database (NIST MS Search 2.0). The amount of each fatty acid was expressed as a percentage of total fatty acid content.
Fourier Transform Infrared Spectrophotometry (FT-IR) was used to observe the fingerprint for the molecular vibrations of PG, CG, and SWCO. In this regard, an FT-IR spectrophotometer (model: Shimadzu IR Prestige − 21) was equipped with an attenuated total reflectance module, and as background, petroleum ether was used. The spectra were recorded on a wavelength range of 600–4000 cm− 1 at a resolution of 4 cm− 1, and 64 scans for a spectrum .
Biomass, cellular viability, viability staining:
Biomass formation and cellular viability were determined as in our previous work . Biomass as the quantity of Cell Dry Weight (CDW) was established by filtering 10 mL of the fermentation broth through 0.20 µm filters, washing them twice with double distilled water, and drying them at 104°C for 8 h until a constant mass was observed.
The cellular viability was determined by diluting 1 mL of fermentation sample to 9 mL of a sterile saline solution of 0.8% NaCl. A volume of 100 µL from different dilutions was passed on Petri dishes with yeast malt extract agar and incubated at 30°C for about 2 days. The plate counting method  established the viability of Y. lipolytica cells (log10 CFU/mL). The cellular viability was also observed under the microscope light as methylene-blue colored cells, while colorless cells were considered unviable .
Succinic and citric acids production, and substrate consumption (PG, CG, SWCO)
The succinic and citric acids content and the consumption of glycerol from the cultivation media were identified through HPLC . The HPLC unit was equipped with a quaternary pump, solvents degasser, and manual injector coupled with a refractive index detector (RID) (Agilent 1200, Santa Clara, CA, USA). The compounds separation was performed using a Polaris Hi-Plex H column, 300 × 7.7 mm (Agilent Technologies, CA, USA). As mobile phase was used 5 mM H2SO4 with a 0.6 mL/min flow rate. The column temperature was 80°C, and the RID temperature was maintained at 35°C. The elution of the compounds runs for 20 min. The data acquisition and the interpretation of the results were performed using OpenLab CDS ChemStation Edition software (Agilent Technologies, CA, USA).
The consumption of fatty acids from the cultivation media consisting of SWCO was monitored by GC-MS, through the method described previously [30, 46].