Aerobic and anaerobic batch fermentations in 3 L bioreactors (Applikon Biotechnology) were conducted using 2.1 L of ACSH or 1.7 L of YPX or YPDX with 50 mM potassium phosphate medium. Vessels were sparged in the headspace with N
2 (150 sccm) for anaerobic fermentation or in the medium with air (200 sccm) for aerobic fermentation. Inocula were prepared from single colonies grown in YPD-Geneticin medium aerobically for ∼9 h, and then were diluted in ACSH or YPD medium with Geneticin at an initial OD
600 = 0.1 (approximately 0.08–0.1 g DCW/L), and then grown aerobically for approximately 20 h. Starter cultures were then inoculated at a starting OD
600 = 0.1 in bioreactor vessels maintained at 30°C and pH 5.0 with NaOH or HCl, and stirred at 500 rpm. For aerobic and anaerobic YPX growth assays, inoculum cultures were started from single colonies grown in YPD-Geneticin medium overnight and then diluted to OD
600 = 0.1 in YPX (no Tween-80 or ergosterol) at the start of the assay. Yeast cultures were grown in culture tubes containing 5–7.5 mL of medium shaken at 30°C, or in 30 mL of medium stirred in flasks placed in an anaerobic chamber (Coy) purged with hydrogen. For anaerobic experiments, bioreactors containing YPX, YPDX or ACSH were sparged with N
2 into the medium for at least 2 h. Flasks containing YPX were placed in the anaerobic chamber for at least 2 h prior to inoculation. Cell density measurements were determined by OD
600 measurements from cultures diluted 1∶10 or 1∶25 in water. All OD
600 measurements were blanked against uninoculated medium diluted in the same manner. Dry cell weight (DCW) was determined by vacuum filtering 50 mL of culture at 4 time points onto pre-weighed filters, washing with water and microwaving on 10% power for 15 minutes. Filtered cells were additionally dried by desiccant for 2–3 days and then weighed. DCW values in grams per 50 mL of culture included subtraction of the filter weight alone. Correlations between g DCW/L and OD
600 concentrations were calculated to provide cell density measurements based on cell mass/L. Medium glucose and xylose concentrations from aerobic tube and anaerobic flask experiments were determined by YSI instrument. Extracellular glucose, xylose, ethanol, glycerol and xylitol concentrations from aerobic and anaerobic bioreactor experiments were determined by high performance liquid chromatography (HPLC) and refractive index detection (RID) as published elsewhere [35] (
link).
Parreiras L.S., Breuer R.J., Avanasi Narasimhan R., Higbee A.J., La Reau A., Tremaine M., Qin L., Willis L.B., Bice B.D., Bonfert B.L., Pinhancos R.C., Balloon A.J., Uppugundla N., Liu T., Li C., Tanjore D., Ong I.M., Li H., Pohlmann E.L., Serate J., Withers S.T., Simmons B.A., Hodge D.B., Westphall M.S., Coon J.J., Dale B.E., Balan V., Keating D.H., Zhang Y., Landick R., Gasch A.P, & Sato T.K. (2014). Engineering and Two-Stage Evolution of a Lignocellulosic Hydrolysate-Tolerant Saccharomyces cerevisiae Strain for Anaerobic Fermentation of Xylose from AFEX Pretreated Corn Stover. PLoS ONE, 9(9), e107499.
