(modified from Singh and Graeber 2005)

1. Grind Corn grain by using Foss water-cooled Knifetec 1095 sample mill (Tecator AB, Höganäs, Sweden) for 10 to 15s or until fine
2. Each sample is analyzed in triplicate
3. Corn grain fermentation is conducted following the 72 h fermentation
4. Label 125ml Erlenmeyer flasks
5. In to each flask add 25 g of ground corn grain
6. Add 75 mL deionized water
7. Add 27 uL alpha-amylase (9000-85-5, Sigma-Aldrich Inc., Allentown, PA) in to each flask fitted with a rubber cap and needle vent
8. Incubate the flasks for 90 min at 90°C
9. Meanwhile make 0.1% peptone water by adding 15gms of peptone in to 1L distilled water in a 2L flask. Close it with Aluminum foil. Autoclave the flask for 45 minutes on fluid cycle. This can be prepared on the previous day also
10. Bring the flasks out and let them cool to 60°C
11. Check the pH and adjust to 4.1 to 4.5
12. Make the yeast inoculum by adding 11gm of dry yeast (Fleischmann’s yeast, Fenton, MO) in to 99ml 0.1% peptone water (Wang et al. 1999)
13. Incubate the yeast at 38°C for 20 minutes
14. In to each flask add 20 mL glucoamylase (9032-08-0, Sigma-Aldrich Inc., Allentown, PA)
15. And then add 5 mL yeast inoculum and 4 mL 1M Ammonium sulfate (EM Science, Gibbstown, NJ)
16. Mix them properly and close them using rubber stoppers pierced with syringe needles
17. Take the before weights of each flask
18. Incubate the flasks at 60°C for 2 h
19. Check the in between weights
20. And then incubate at 32°C with 150 rpm for 72 h (Model C25, New Brunswick Scientific, Edison, NJ).
21. Check the final weights after incubation
22. The weight loss of the slurry after incubation is considered as the total amount CO₂ released from the fermentation. The ethanol yield is then computed using 1:1 molar ratio of ethanol to CO₂.

Reference

Singh, V., and J.V. Graeber. 2005. Effect of corn hybrid variability and planting location on ethanol yields. Trans. ASABE. 48:709-714.