Biomass to Ethanol Procedure
A. Sample Preparation:
1. Label one or more 50mL falcon tubes and caps with name, date, sample description.
2. Measure 1.0g of you biomass samples and put the 1.0g sample into the corresponding 50mL
falcon tube
3. Test the initial glucose concentration using the blood glucose test monitor and test strips.
Describe the biomass (appearance? odor?). Record this value.
4. Test the initial ethanol concentration using the ethanol probe and record this data. Describe the
biomass (appearance? odor?).
B. Hot water pre-treatment:
1. Bring 400mL water to a gentle boil in a 600mL glass beaker. Use pre-heated water to fill your
beaker – heat water to boiling in a microwave. While heating proceed with the next steps.
2. Add 25mL distilled water to each of the 50mL falcon tubes.
3. Swirl to mix the biomass and water. Let it sit for 1 minute.
4. Loosely screw the cap onto the falcon tube.
5. Once the water is boiling, place 4 falcon tubes into the boiling water bath. Make sure the
biomass samples and the liquid are completely submerged below the surface of the boiling water
in the beaker.
6. Leave the tubes in the water for 10-25 minutes. The longer the time, the higher the potential yield
of ethanol will be.
7. Remove your samples and turn off the hot plate if finished with all samples. Allow samples to
cool to room temperature. Use a cold water bath to make the tubes cool more quickly.
8. Test the glucose concentration using the blood glucose test monitor and test strips. Record this
data. Describe any detectable changes in the biomass (appearance? odor?).
9. Test the ethanol concentration using the ethanol probe. Record this data. Describe any
detectable changes in the biomass (appearance? odor?).
C. Enzymatic Digestion (Hydrolysis)
1. Add 1.0mL of CellulastTM cellulose enzyme product to each falcon tube that is undergoing
hydrolysis.
2. Screw caps on tightly. Mix gently.
3. Place falcon tubes in 50°C water bath for 24 hours.
4. After 24-hour hydrolysis data collection, remove the tubes and allow to cool to room temperature.
5. Use the blood glucose test monitor and test strips. Record this data. Describe any detectable
changes in the biomass (appearance? odor?).
6. Test the ethanol concentration using the ethanol probe. Record this data. Describe any
detectable changes in the biomass (appearance? odor?).
7. At this point, all samples can be stored (frozen to prevent microbial contamination) until the next
lab.
D. Fermentation
1. After samples are at room temperature, add 1.0g yeast to each falcon tube and gently mix.
2. Loosely screw on the cop to the tubes. It is important that the tubes not be air-tight for the
fermentation. Yeast will produce CO2 and will build up pressure in the tube unless the gas is
allowed to escape.
3. Place falcon tubes in the racks in the 37°C water bath. Allow to ferment for 24 hours.
4. After 24 hours, remove falcon tubes from 37°C water bath and allow to cool to room temperature.
5. Take the final glucose readings. Use the blood glucose test monitor and test strips to test postenzyme glucose concentration of the sample. Record this data. Describe any detectable
changes in the biomass (appearance? odor?).
6. Take the final ethanol readings. Test the ethanol concentration using the ethanol probe. Record
this data. Describe any detectable changes in the biomass (appearance? odor?).