Moisture and ash contents were determined according to analytical gravimetric methods [22]. Total nitrogen and crude protein content was determined using HR Test’n tube TN kit (HACH, US)
and protein content was calculated according to the Kjeldahl method with a conversion factor of 6.25. Starch content was determined using Megazyme’s total starch kit (Bray, Ireland).
Total carbohydrate content of FW was determined by two-step sulfuric acid hydrolysis according to NREL-LAP [23]. The lipid content was determined by hexane/isopropanol (3:2) method [24]. Reducing sugars were quantified by the dinitrosalicylic acid(DNSA) method using glucose as standard [25]. Free amino nitrogen (FAN) concentration was measured in hydrolyzates using
the ninhydrin reaction method [26]. The glucose and ethanol concentrations were determined using HPLC equipped with a refractive index (RI) detector. The separation was performed in
an Agilent column (H-plex H) operated at 65 C with a flow rate of 0.4 mL/min using 4 mM H2SO4 as the mobile phase. Dry cell weight was determined directly from the absorbance of the withdrawn
samples measured at 600 nm by a UV–visible spectrophotometer. The measured absorbances were converted to dry cell concentration using corresponding standard curve.