Spectronic 601 spectrophotometer

ODs were measured at 600 nm in a Spectronic 601 spectrophotometer (Milton Roy, Ivyland, PA, USA). Samples were then centrifuged and filtered through a 0.2-μm syringe filter before placing them in high pressure liquid chromatography (HPLC) vials. Samples were analyzed for carbohydrates and organic acids (succinic, formic, acetic, and lactic acid) via HPLC using the Shodex SP0810 carbohydrate column and the Biorad Aminex HPX-87H organic acids column.
Furfural, HMF, furfuryl alcohol, and HMF-alcohol were separated and quantified on an Agilent 1100 series HPLC equipped with a diode array detector (DAD). The wavelengths monitored were 250 nm to detect furfural and 225 nm for furfuryl alcohol, HMF and HMFA. Samples and standards were analyzed using an Agilent Zorbax SB-C18 5 um 4.6 × 250 mm with a guard column. A mobile phase of acetate buffer (12.5 mM, pH = 4.5) and acetonitrile (4:1) was run isocratic at 1.0 mL/min for 10 min. Concentrations for the standards in acetate buffer were <1.0 mg/mL and standard curves were generated and used for quantitation. Samples were prepared by diluting 0.4 mL of culture supernatant with 0.4 mL of acetate buffer (25 mM, pH = 4.5) and 0.2 mL of acetonitrile. The flowrate was 1 mL/min, column compartment temperature of 25 °C, and injection volume was 10 µL.

Z. mobilis 8b and sub-strains #7, #18, and #21 were taken from cell stocks stored at -70°C. The pre-seed medium used was RMGX (8%:2%). The strains were revived by transferring 1 mL of the frozen stock into 9 mL of pre-seed medium in a 15-mL tube. The cultures were incubated at 33°C with no agitation and then sampled at 8 hours for an optical density reading at 600 nm using Spectronic 601 spectrophotometer (Milton Roy, Ivyland, PA, USA). The pre-seed culture was used to inoculate the seed flask with media composition of RMGX (8%:2%, w/v) or the batch seed fermentor, if needed, with media composition of RM (1×), 150 g/L glucose, 20 g/L xylose, and 1 g/L sorbitol. Temperature was controlled at 33°C and pH at 5.8 by 4 M KOH.

Zymomonas mobilis 8b and SS3 were taken from cell stocks stored at −70°C. The pre-seed medium consisted of 1X RM, supplemented with 80 g/L glucose and 20 g/L xylose. The culture was revived by transferring 1 mL of the frozen stock into 9 mL of pre-seed medium in a 15-mL tube. The cultures were incubated at 33°C with no agitation and then sampled at 8 h for an optical density reading at 600 nm using a Spectronic 601 spectrophotometer (Milton Roy, Ivyland, PA, USA). The pre-seed culture was used to inoculate the batch seed fermentor, if needed, with media composition of RM (1X), 150 g/L glucose, 20 g/L xylose, and 1 g/L sorbitol. Temperature was controlled at 33°C and pH at 5.8 by 4 M KOH.
Fermentations to evaluate the strains were done in BioStat-Q plus fermenters with a 300 mL working volume, 300 rpm, a temperature of 33°C, and pH 5.8 controlled with KOH (2 N). Saccharified liquor or hydrolysate used in fermentation was enriched with RM. The fermenters were inoculated with an OD 600 nm value of 1 using a direct transfer procedure (10% v/v). Ethanol yield was calculated based on initial sugar concentrations and differences between final and initial ethanol concentration.