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Chemical Engineering Department RET Project
Enzymatic HydrolysisRET LABORATORY PROCEDURE
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
Chemical Engineering Department RET Project
Experiment BackgroundIn 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.
Chemical Engineering Department RET Project
Endo-glucanase enzymes break the cellulose molecules in the amorphous region. Cellobiohydrolase enzymes then break the cellulose into cellobiose, or dimers of glucose. Beta-glucosidase then finishes the conversion by separating the dimer into individual glucose molecules.
Procedural OutlineI. Glucose meter calibration
II. Solution preparationI. Pre-treated biomassII. Distilled waterIII. 1M Sodium citrate bufferIV. 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
Chemical Engineering Department RET Project
Glucose Meter Calibration
Chemical Engineering Department RET Project
I. Prepare a 0.05 M sodium citrate buffer solution from the stock 1 M solution:
+ =
1 mL of 1M sodium citrate buffer 19 mL of distilled water 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:
Dissolve 0.2 g of glucose in 50 mL 0.05M sodium citrate buffer
Chemical Engineering Department RET Project
Glucose Meter Calibration (3)III. Prepare the following calibration standards and measure glucose concentration using the
glucose meter:
Note: Shake the standards well
Chemical Engineering Department RET Project
Glucose Concentration (g/L)
4 g/L Glucose Solution Amount (µL)
0.05M Sodium Citrate Buffer Amount (µL)
Glucose Meter Reading
0 0 1000 1 250 750 2 500 500 3 750 250 4 1000 0
Glucose Meter Calibration (4)IV. Prepare a calibration curve and obtain equation for best fit line
Chemical Engineering Department RET Project
0 50 100 150 200 250 3000
0.5
1
1.5
2
2.5
3
3.5
4
4.5
f(x) = − 2.59368449753302E-05 x² + 0.0211058630182336 x + 0.0709395320967109R² = 0.99783432455685
Glucose Meter Reading
Glu
cose
Con
cent
ratio
n (g
/L)
Solution PreparationI. 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)
Chemical Engineering Department RET Project
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
Chemical Engineering Department RET Project
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
Chemical Engineering Department RET Project
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
Chemical Engineering Department RET Project
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
Chemical Engineering Department RET Project
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
Chemical Engineering Department RET Project
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
Chemical Engineering Department RET Project
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
Chemical Engineering Department RET Project
Glucose MeasurementI. 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
Chemical Engineering Department RET Project
Glucose MeasurementI. 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 hoursNote: 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.
Chemical Engineering Department RET Project
Data AnalysisI. 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
Chemical Engineering Department RET Project
Enzymatic Hydrolysis Data: 10:00 AM
SampleMeter
Readingg/L Net g/L
Meter Reading
g/L Net g/LMeter
Readingg/L Net g/L
Biomass #1 40 0.87 0.19 239 3.40 2.17 384 3.75 2.50Biomass #2 51 1.07 0.39 208 3.16 1.93 338 3.78 2.53
Control 30 0.68 - 60 1.23 - 61 1.25 -
Glucose Measurementt = 24 hrst = 0 hrs t = 48 hrs
Meter Calibration Curve: y = ax2 + bx + ca = -3.E-05b = 0.0211c = 0.0709
Data Analysis (1)II. The glucose concentration during the measured time period can now be plotted to view
how the hydrolysis progressed over time
Chemical Engineering Department RET Project
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. 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
Chemical Engineering Department RET Project
theoretical mass of glucose that could be produced
Data Analysis (3)VI. Determine the percent yield of glucose production
Chemical Engineering Department RET Project
Note: Moisture Content = 73%0 hrs 24 hrs 48 hrs
SampleMass of
Biomass (g)Sample Volume after
Enzymatic Hydrolysis (mL)Total Mass of Glucose
Produced (g)Theoretical Mass of
Glucose (g)Percent
YieldBiomass #1 0.19 2.17 2.50 1.00 20 0.050 0.1350 37.09Biomass #2 0.39 1.93 2.53 1.00 20 0.051 0.1350 37.47
Net Glucose Concentration (g/L)
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
III. Calculate the net glucose concentration in g/L
Chemical Engineering Department RET Project
Sample Calculations (1) IV. Calculate the amount of glucose produced in the flask
V. Calculate the theoretical mass of glucose that could have been produced
VI. Calculate the percent yield
Chemical Engineering Department RET Project
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