Experiment Summary

Report
Enzymatic Hydrolysis
RET LABORATORY PROCEDURE
Chemical Engineering Department RET Project
Safety to Consider
 Broken glassware
 Be careful when handling glassware
Chemical hazards
 Tetracycline and cycloheximide are antimicrobials and have low hazard properties
 However, ALWAYS wear laboratory gloves
Hot plate
 Do not touch the hot plate
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Experiment Background
In this lab we will be using pretreated biomass and
completing enzyme hydrolysis to breakdown the cellulose
into glucose for use in fermentation. We are adding
antimicrobials to the mix in order to prevent the growth of
any bacteria that would potentially ferment the glucose
right away. This will allow us to measure the amount of
glucose that is produced. However due to the fact that
antimicrobials are in this mix we cannot use this mixture in
the fermentation process. This lab is to show students that
cellulose is being broken down into glucose. In the real
world production setting antimicrobials would not be used
in enzyme hydrolysis. The mixture would be sterile from
the pretreatment process and after the enzymatic
hydrolysis flow directly into fermentation.
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Endo-glucanase enzymes break the cellulose molecules in the
amorphous region. Cellobiohydrolase enzymes then break
the cellulose into cellobiose, or dimers of glucose. Betaglucosidase then finishes the conversion by separating the
dimer into individual glucose molecules.
Procedural Outline
I.
II.
Glucose meter calibration
Solution preparation
I.
Pre-treated biomass
II. Distilled water
III. 1M Sodium citrate buffer
IV. Cycloheximide (antimicrobial)
V. Tetracycline (antimicrobial)
III.
Prepare 50 °C water bath in pressure cooker
IV.
Enzyme addition: Accellerase BG and Accellerase 1500
V.
Glucose concentration measurement every 24 hours
VI.
Data Analysis
VII. Sample Calculations
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Glucose Meter Calibration
I.
Prepare a 0.05 M sodium citrate buffer solution from the stock 1 M solution:
1 ∙ 1 = 2 ∙ 2
1  ∙ 1.0 = 2 ∙ 0.05 →  =  
+
1 mL of 1M sodium citrate buffer
=
19 mL of distilled water
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20 mL of 0.05M sodium citrate buffer
Glucose Meter Calibration (2)
II.
Prepare a 50 mL 4 g/L glucose solution in 0.05 M sodium citrate buffer solution:
4
1000 
=

50 
→  = 0.2 
1 ∙ 1 = 2 ∙ 2 → 1 ∙ 1 = 50  ∙ 0.05
→  2.5  1    ℎ 47.5  2
Dissolve 0.2 g of glucose in 50 mL 0.05M sodium citrate buffer
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Glucose Meter Calibration (3)
III.
Prepare the following calibration standards and measure glucose concentration using the
glucose meter:
Note: Shake the standards well
Glucose Concentration 4 g/L Glucose Solution 0.05M Sodium Citrate
(g/L)
Amount (µL)
Buffer Amount (µL)
0
0
1000
1
250
750
2
500
500
3
750
250
4
1000
0
Glucose Meter
Reading
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Glucose Meter Calibration (4)
IV. Prepare a calibration curve and obtain equation for best fit line
4.5
Glucose Concentration (g/L)
4
y = -3E-05x2 + 0.0211x + 0.0709
R² = 0.9978
3.5
3
2.5
2
1.5
1
0.5
0
0
50
100
150
Glucose Meter Reading
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200
250
300
Solution Preparation
I.
Transfer 1 gram of Aspen pre-treated wood chips to a 50 mL Erlenmeyer flask using 19 mL of
distilled water (record the exact mass added)
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Solution Preparation (2)
II.
It is advisable to prepare two biomass solutions in order to compare the measurements
taken from essentially duplicate solutions
III.
A control may also be prepared to determine if and how the chemical additions affect the
glucose measurements. The control contains all chemical additions except for the biomass,
or wood chips
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Solution Preparation (3)
IV. Add 1 mL of 1M sodium citrate buffer solution to all flasks
V.
Add 80 μL of tetracycline (10 mg/mL in 70% ethanol) to all flasks
VI. Add 60 μL of cycloheximide (10 mg/mL in dH2O) to all flasks
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Prepare 50 °C Water Bath in Pressure
Cooker
I.
Place the pressure cooker on top of the hot plate
Note: pressure cooker should have a thin layer of water (≈ ½”) at its bottom to allow
even heating on all sides of each flask
II.
Set the hot plate to a temperature of 50 °C, assuring correct temperature by placing the
thermometer tip into the pressure cooker
Note: this may take some practice. It is advised to be able to maintain 50 °C in the
pressure cooker before beginning the experiment
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Enzyme Addition
I. Determine the dry weight of biomass added to flask using
the following formula
Dry mass = wet mass * (1 – % moisture content)
Note: Biomass % moisture content is written on each biomass bag
For example, if the wet mass added was 1.0 g and the moisture content was 73%:
Dry mass = 1.3 g * (1 – 0.73) = 0.27 g
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Enzyme Addition (2)
II.
Add enzymes to the woodchip flask in the following loadings:
Accellerase 1500: 250 μL per 1 gram of dry biomass
Accellerase BG: 90 μL per 1 gram of dry biomass
From the previous slide, the dry mass was 0.27 g:
Add 250 μL · 0.27 = 67.5 μL of Accellerase 1500
Add 90 μL · 0.27 = 24.3 μL of Accellerase BG
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How to use the Glucose Meter
For best results, use the dip method:
 Dispense 10 µL of the solutions onto a piece of wax paper using the pipetter. Allow the sample to sit for
30 seconds to reach room temperature
 Insert a strip into the glucose meter
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How to use the Glucose Meter (2)
 “Dip” the end of the test strip into the solution on the wax paper
 Record the results in your laboratory notebook
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Glucose Measurement
I.
After each glucose measurement cover each flask with tin/aluminum foil and wrap a strip of parafilm
around the top of each flask to prevent evaporating material from escaping
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Glucose Measurement
I.
Measure the initial glucose concentration using the glucose meter at t = 0 hours
II.
Place the flask back into the pressure cooker for 24 hours (at 50 °C)
III.
Measure the glucose concentration using the glucose meter at t = 24 hours
IV. Repeat steps II and III at t = 48 hours and t = 72 hours
Note: The meter may read “HI” when measuring. If so, use a 1.5 mL microcentrifuge tube to dilute a
small portion of the biomass solution with the 0.05 M sodium citrate buffer solution to obtain
readable results.
For example, make a 1:1 diluted solution by mixing 0.5 mL of the biomass solution with 0.5 mL of the
0.05 M sodium citrate buffer solution, then test that. If this measurement is still outside the range of
the glucose meter, continue diluting the solution to lesser concentrations until a readable
measurement can be made.
If the meter reads “Lo”, assume the glucose concentration is 0 g/L.
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Data Analysis
I.
Using the glucose measurement data that was collected and the calibration curve that was
obtained for this batch of test strips, determine the glucose concentration of each glucose
meter measurement in g/L
2
Enzymatic Hydrolysis Data: 10:00 AM
Sample
Biomass #1
Biomass #2
Control
Meter Calibration Curve: y = ax + bx + c
a = -3.E-05
b = 0.0211
c = 0.0709
Glucose Measurement
t = 0 hrs
t = 24 hrs
Meter
Meter
g/L
Net g/L
g/L
Net g/L
Reading
Reading
40
0.87
0.19
239
3.40
2.17
51
1.07
0.39
208
3.16
1.93
30
0.68
60
1.23
-
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t = 48 hrs
Meter
Reading
384
338
61
g/L
Net g/L
3.75
3.78
1.25
2.50
2.53
-
Data Analysis (1)
II.
The glucose concentration during the measured time period can now be plotted to view
how the hydrolysis progressed over time
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Data Analysis (2)
III.
Assume the volume in each flask is 20 mL
IV.
Using the volume and final (48 or 72 hrs) net glucose concentration, calculate the amount of
glucose produced in each flask

  ∙
V.
 
1000 

=  
Determine the theoretical mass of glucose that could have been produced from a dry biomass
basis
Note: assume biomass is 50% cellulose and 50% lignin
  
2
∙ 1 − %   = theoretical mass of glucose
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that could be produced
Data Analysis (3)
VI. Determine the percent yield of glucose production

  =
∙ 100%
ℎ
Note: Moisture Content = 73%
0 hrs
24 hrs 48 hrs
Net Glucose
Mass of
Sample
Concentration (g/L)
Biomass (g)
Biomass #1
0.19
2.17
2.50
1.00
Biomass #2
0.39
1.93
2.53
1.00
Sample Volume after
Enzymatic Hydrolysis (mL)
20
20
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Total Mass of Glucose Theoretical Mass of Percent
Produced (g)
Glucose (g)
Yield
0.050
0.1350
37.09
0.051
0.1350
37.47
Sample Calculations
I.
The sample calculations will be performed on Biomass #1 at the 48 hour measuring period
II.
Calculate the glucose concentration in g/L using the calibration curve
  = 384


III.
= −3 ∙ 10−5 ∙ 3842 + 0.0211 ∗ 384 + 0.0709 = . 
Calculate the net glucose concentration in g/L



= #1 −  = 3.75
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− 1.25


= . 




Sample Calculations (1)
IV. Calculate the amount of glucose produced in the flask
2.50
V.


∙
20 
1000 

= .   
Calculate the theoretical mass of glucose that could have been produced
1.00  
2
∙ 1 − 0.73 = .   
VI. Calculate the percent yield
  =
0.05 
0.135 
∙ 100 % = .  %
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Acknowledgements
Funding from the National Science Foundation
“RET Site: “Wood-to-Wheels” – Research Experiences for High School Teachers in Sustainable
Transportation Technologies”
Grant no. EEC-1009617
Chemical Engineering Department RET Project

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