Biofuels: Producing Ethanol from Cellulosic Material

1. Sample Preparation

1. Select cellulosic material to be used as feedstock. This can be corn stalks, grasses, leaves, pet bedding, or paper. 
2. Using a ball mill grinder (or coffee grinder if ball mill grinder is not available), grind feedstock into a fine powder with no large pieces remaining.
3. Measure 1.0 g of feedstock and place in a 50-mL centrifuge tube. Label the tube with the feedstock chosen.
4. Label a second 50 mL centrifuge tube as “Control”. Do not put any feedstock in this tube.

2. Pretreatment

1. Set up a 500-mL beaker with approximately 400 mL of water on a hotplate and heat to a gentle boil.
2. Add 25 mL of distilled water to the 2 centrifuge tubes. Swirl to mix. Put the cap on the centrifuge tubes loosely.
3. Put the centrifuge tubes in the beaker full of gently boiling water. Be sure that water from the water bath does not leak into the tubes. Let boil for 30 min.
4. Let tubes cool to room temperature.

3. Enzymatic Digestion

1. Add 1 mL of cellulase enzyme to both tubes.
2. Put tubes in an incubator at 50 °C for 24 h.
3. Allow the tubes to cool to room temperature.

4. Fermentation

1. Add 1.0 g of active yeast (regular grocery store yeast is ok) to each of the centrifuge tubes. Swirl to mix.
2. Put an airlock on top of the centrifuge tube. The airlock allows CO₂ to escape, keeping the pressure low in the centrifuge tube.
3. Place the centrifuge tubes in a rack and put in an incubator at 37 °C for 24 h.
4. Using an ethanol sensor, measure the ethanol concentration in the control tube (Figure 1). Ethanol sensors can be purchased through Vernier or PASCO for approximately $100 each.
   
   Figure 1. Ethanol probe measuring the ethanol concentration in the control tube.
5. Using an ethanol sensor, measure the ethanol concentration in the sample tube (Figure 2).
   
   Figure 2. Handheld tablet displaying % of ethanol.