Dr6000 spectrophotometer

Biomass quantification was made by optical density (OD) at 600 nm using a UV-VIS DR6000 HACH spectrophotometer (Loveland, CO, USA) in a 1:10 dilution. Cell density was estimated using the standard curve of cell density (g/L) versus optical density (absorbance units) shown in Supplementary Figure S1.
Glucose, fructose, and ethanol concentrations were obtained by HPLC Agilent Technologies 1200 (Santa Clara, CA, USA) on an Aminex^® (Dublin, Ireland) HPX-87H+ column (300 mm × 7.8 mm) under the following conditions: column temperature 65 °C, refractive index detector temperature 60 °C, injection volume 1 µL, and mobile phase sulfuric acid 0.5 mM at a constant flow rate of 0.6 mL/min [40 (link)].
Minor compound analysis was performed by GC/MS using an Agilent 7890A gas chromatograph equipped with an Agilent 5975C mass spectrometric detector (Santa Clara, CA, USA) following that reported by Acosta-García et al. [39 (link)]. In brief, the samples (1 µL) were injected in split mode (70:1) in an HP-FFAP column (30 m length × 0.32 mm inner diameter × 0.25 μm thickness; Agilent Technologies) to separate the compounds. High-purity helium (99.999%) was used as a carrier gas at a 1 mL/min constant flow. The injector temperature was 180 °C. The oven temperature was set to 40 °C for 3 min, then increased to 52 °C at 3 °C/min for 1 min, then increased to 200 °C at 10 °C/min, and held at 200 °C for 15 min. On the other hand, the mass spectrometer was operated at 230 °C, with an ionization voltage of −70 eV and SCAN mode (1.6 scans per second). An alkane ladder was injected to calculate the Kovats retention index of each compound. AMDIS software (v. 2.73, build 149.31) was used to identify the compounds.

For food waste fermentation, TS, VS, and pH were measured according to the standard method [30 ]. Suspended solids were separated from the fermentation effluent via centrifugation at 5000 rpm for 10 min, the liquid supernatant was filtrated through a 0.45-μm membrane, soluble COD (SCOD), total nitrogen (TN), and VFAs were measured. The SCOD was analyzed following the HACH method. VFAs were determined using GC (Shimadzu, GC-2014) with a flame ionization detector and capillary column (Stabilwax-DA, 30 m × 0.25 mm × 0.25 μm). The temperature program followed that of previous methods [31 (link)].
For Y. lipolytica cultivation, a sterile pipette was used to periodically withdraw 1.5-mL samples from the batch cultures. To describe cell growth, cell concentrations were measured as the absorbance of the culture broth at 600 nm (OD600, DR-6000 spectrophotometer, HACH). Biomass production was expressed as DCW (dry cell weight). Cells were harvested from 10 mL of culture medium by centrifugation at 10,000 rpm for 10 min; washed with ethanol (95%) and hexanes to remove extracellular fatty acids [32 (link)]; and dried to constant weight at 105 °C in an oven, and then weighed.
Lipids were extracted in accordance with the method of Bligh and Dyer [32 (link)] with modifications [33 ]. Lipids were extracted from lyophilized biomass using a mixture of chloroform and methanol (2:1 v/v). The lipid mixture was centrifuged at 5000g for 25 min to completely dissolve the lipids in the solvent. Then, the organic phase was washed twice with 0.15% (w/v) NaCl solution. The purified chloroform layer was evaporated to dryness with nitrogen gas (Nitrogen Evaporation System). Lipid production was expressed as gram lipid per liter culture media. The lipid content was expressed as gram lipid per gram DCW (%).
The fatty acid composition of lipids was determined by GC analysis of FAMEs. FAMEs were converted from fatty acids via saponification followed by methylation in accordance with the method of [34 ]. FAMEs were analyzed by using a GC-8600 gas chromatograph (Tianpu Instrument Co., Ltd., Beijing, China) equipped with a flame-ionized detector and CP-Sil 88 capillary column (Agilent, USA, 60 m × 0.25 mm × 0.36 µm). The column temperature was maintained at 190 °C for 10 min, increased from 190 to 240 °C at a rate of 10 °C/min, and maintained at 240 °C for 15 min. Hydrogen was used as the carrier gas at 1.0 mL/min. The split ratio was 1:30 (v/v). The injector and the detector temperatures were set at 270 and 300 °C, respectively. Fatty acids were identified by comparing their retention times with those of standard solutions, then quantified based on their respective peak areas and normalized.
To further confirm the cell growth and lipid accumulation in Y. lipolytica, fluorescent testing was carried out with laser scanning fluorescence microscopy (LSCM). An amount of 10 μL cell sample from batch cultures was carefully collected at the end of the stationary phase, subsequently stained with 10 μL Nile Red, and imaged with laser scanning confocal microscopy (Leica TCS SP2) under an exciting wavelength of 543 nm.

The samples were collected from a beverage manufacturing factory located at Nilai, Negeri Sembilan, Malaysia and tested according to the method adopted from the Standard Methods for the examination of water and wastewater analysis [30 ]. COD was measured using an HACH Spectrophotometer DR6000 (Standard Method 5220 D for low range value and high range value) after being heated in COD reactor at 150 °C for 2 h. TSS measurement was carried out after filtration using a 0.45 µm filter on the vacuum pump, and the sample was heated in a drying oven at 103 °C. The gravimetric method was applied for TSS measurement.