enzymatic hydrolysis

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Chemical Engineering Department RET Project Enzymatic Hydrolysis RET LABORATORY PROCEDURE

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Enzymatic Hydrolysis. RET 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 - PowerPoint PPT Presentation

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Page 1: Enzymatic Hydrolysis

Chemical Engineering Department RET Project

Enzymatic HydrolysisRET LABORATORY PROCEDURE

Page 2: Enzymatic Hydrolysis

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

Page 3: Enzymatic Hydrolysis

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.

Page 4: Enzymatic Hydrolysis

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

Page 5: Enzymatic Hydrolysis

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

Page 6: Enzymatic Hydrolysis

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

Page 7: Enzymatic Hydrolysis

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

Page 8: Enzymatic Hydrolysis

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)

Page 9: Enzymatic Hydrolysis

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

Page 10: Enzymatic Hydrolysis

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

Page 11: Enzymatic Hydrolysis

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

Page 12: Enzymatic Hydrolysis

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

Page 13: Enzymatic Hydrolysis

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

Page 14: Enzymatic Hydrolysis

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

Page 15: Enzymatic Hydrolysis

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

Page 16: Enzymatic Hydrolysis

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

Page 17: Enzymatic Hydrolysis

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

Page 18: Enzymatic Hydrolysis

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

Page 19: Enzymatic Hydrolysis

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

Page 20: Enzymatic Hydrolysis

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

Page 21: Enzymatic Hydrolysis

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

Page 22: Enzymatic Hydrolysis

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)

Page 23: Enzymatic Hydrolysis

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

Page 24: Enzymatic Hydrolysis

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

Page 25: Enzymatic Hydrolysis

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