METHODOLOGY (overview)

• Pretreatment – The loosening of the cell walls to expose cellulose using

mechanical, chemical, and heat treatments (boiling) that sterilizes the exposed
cellulose.
• Hydrolysis (enzymatic digestion) – It breaks the long cellulose molecules and
separates them into individual glucose molecules.
• Fermentation – Yeast consumes the glucose and oxygen creating ethanol and
carbon dioxide (CO2).

METHODOLOGY (stepwise)

REMINDERS
 Measure specific gravity of distilled water, sample solution after pre-treatment,
and sample solution after enzymatic digestion, and sample solution after
fermentation.
 Document EVERY(lol) step, especially once you begin to do readings.
 Ensure that glassware is clean and rinsed with distilled water before use

Step A: Sample Preparation and Pretreatment (day 1) : release the cellulose fibers by
breaking down plant cell walls.

1. Prep the incubator to 50 C o

2. Pre-heat 3.5 liters distilled water in a pot that has been washed and rinsed with
distilled water.
3. Prepare all glassware/equipment that need to be washed
*what needs to be washed is anything that might come in contact with the sample
a. 9x flasks/beakers
b. 2x graduated cylinder
c. Measuring spoons
d. Hydrometer
e. Thermometer
f. Extra beakers (4x)
2. Wash all glassware with soap and tap water. Rinse.
3. Rinse all glassware with distilled water
*Applying distilled water is done to prevent contamination of the sample/chemical by whatever the
glassware may have come in contact with previously
6. Once dry, label the flasks/beakers as follows:
a. 3x flasks → Control A, Control B, Control C
b. 3x flasks → Cellulase A, Cellulase B, Cellulase C
c. 3x flasks → Amylase A, Amylase B, Amylase C
7. Wash 300g monggo beans with tap water. Rinse with distilled water.
8. Place 10g monggo beans in every flask. Add 150mL of pre-heated distilled water.
Swirl to mix. Let rest for 1 minute.
9. Heat a ll set-ups on hot plate for 30 minutes (or more if time allows) at almost
boiling temperature. Keep the flasks sealed with foil/parafilm.
10. Cool all set-ups to room temperature. Use a cool water bath to hasten the
process.
11. Test the initial glucose concentration by using the blood glucose test monitor and
test strips.
. Take a dropper’s worth of solution from the sample and apply it dropwise on the
test strip.
a. Once absorbed, insert the strip on the device
b. Record the reading as well as its corresponding units
c. Throw away the used test strip and get a new one for another sample
12. Test the initial ethanol concentration by using the hydrometer
. Make sure that the water is no longer warm. Pour each set-up’s solution (at least
90mL) in a 100mL graduated cylinder.
a. Place the hydrometer VERY CAREFULLY in the graduated cylinder.
b. Record the specific gravity as indicated on the side of the hydrometer (the units
that range from 1.000, 0.990, 0.980,...)
c. Return the solution to its respective container and wash the graduated cylinder
before pouring in another set-up’s solution.
13. Observe and describe any changes in the biomass such as appearance, odor,
etc. Record the data.
14. Seal the Amylase and Control set-ups and refrigerate in 5 C for future use. Prep
o

the Cellulase set-ups for the next step.

Step B: Enzymatic digestion (hydrolysis) (day 1&2)

1. Cellulose hydrolysis
a. Verify incubator temperature is at 50 C ± 2 .
o o

b. Add 5mL cellulase solution to each of the Cellulase set-ups -- measure these
using the small graduated cylinder. Seal and swirl gently to mix.
c. Place Cellulase set-ups in incubator and let incubate for 48 hours.
d. After 48 hours, remove the set-ups from the incubator and let cool to room
temperature.
*At this point, one of you may do Step B.1.e. while the other may move on to Step B.2.a.
Or you may choose to do all the steps together so as to prevent mistakes. Your choice.
e. Test post-hydrolysis glucose and ethanol concentrations → follow Steps
A.11 and A.12
f. Once returned to their respective containers, seal the samples and refrigerate at
5 C.
o

2. Amylose hydrolysis
 a. Remove Amylase and Control set-ups from refrigerator and let warm to room
temperature.
b. Right after removing the Cellulase set-ups from the incubator, the incubator may
immediately then be prepped to 37 C.
o

c. Add the following to each of the Amylase and Control set-ups:

i.Control (A, B, C) -- nothing
ii.Amylase (A, B, C) -- 5mL glucoamylase solution
d. Seal and swirl gently to mix.
e. Verify that the temperature inside the incubator is 37 C ± 2 .
o o

f. Place the Amylase and Control set-ups in the incubator and let incubate for 24
hours.
g. After 24 hours, remove the set-ups from the incubator and let cool to room
temperature.
h. Test post-hydrolysis glucose and ethanol concentrations → follow Steps A.11
and A.12
i. Once returned to their respective containers, seal the samples and refrigerate at
o
5 C.

Step C: Fermentation: conversion of glucose (sugar) into ethanol (fuel). (day 2&3)
1. Prep incubator to 37 Co

2. Remove set-ups from the refrigerator and let warm to room temperature
3. To each of the set-ups, add 10g yeast. Swirl gently to mix.
4. Seal, but leave a small space for the produced CO to escape.
2

5. Verify that the temperature inside the incubator is 37 C.o

6. Place all the set-ups in the incubator and let ferment for 24 hours.
a. OPTIONAL: After 30 minutes measure ethanol and glucose concentration.
Observe and describe any changes in the biomass such as appearance, odor, etc.
7. After 24 hours, remove set-ups from incubator.
8. Test post-hydrolysis glucose and ethanol concentrations → follow Steps A.11
and A.12