Batch and fed-batch fermentation

Both XF3XP and XF3XP07 yeast strains were first grown in 100 mL SC medium including all the appropriate nucleotides and amino acids, with 20 g/L glucose for 2 days. Then, cells from 5 mL of culture were centrifuged, washed twice with double-distilled water, and inoculated into 5 mL fresh SC medium with 40 g/L xylose in glass disposable tubes overlaid with 0.5 mL dodecane for batch fermentation. The initial ODs were similar, i.e., 2.38 ± 0.05 and 2.45 ± 0.06, with no significant difference (p > 0.05). Samples were taken at various time points to measure the 1-hexadecanol concentration, OD₆₀₀, and xylose concentration. At each time point, the glass tubes of yeast cultures were allowed to sit for 2 min until the organic layer could be clearly visualized. To measure the 1-hexadecanol concentration, 3 μL of dodecane was withdrawn from the organic layer and then diluted by 100 times using ethyl acetate followed by the analysis using the GC–MS protocol mentioned above. To monitor OD₆₀₀, 20 μL of yeast culture was taken from the water layer and mixed with 180 μL of double-distilled water, followed by measuring the absorbance at 600 nm using a Biotek Synergy 2 Multi-Mode Microplate Reader (Winooski, VT). To measure the concentration of xylose, 100 μL of yeast culture was taken from the water layer and mixed with 900 μL of double-distilled water, which was then centrifuged at 13,000 rpm for 5 min. The supernatant was taken and analyzed by Shimadzu HPLC (Columbia, MD) equipped with an Aminex HPX-87H column (Bio-Rad, Hercules, CA) and Shimadzu RID-10A refractive index detector. The column was kept at 50 °C, and 5 mM sulfuric acid solution was used as a mobile phase with a constant flow rate of 0.6 mL/min. Each data point represents the mean of triplicate samples. In this discontinuous fed-batch fermentation, additional xylose (0.5 mL with concentration of 200 g/L) and dodecane (0.05 mL) were fed every 12 h. Samples were taken after the replenishment to measure 1-hexadecanol concentration, OD₆₀₀, and xylose concentration using the similar methods as that for batch fermentation. The biological triplicates were implemented in both batch and fed-batch fermentation for all the strains.