1. Chemical Engineering Department RET Project Enzymatic Hydrolysis RET LABORATORY PROCEDURE

2. 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

3. 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. 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.

4. Procedural Outline I.Glucose meter calibration II.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 Chemical Engineering Department RET Project

5. Glucose Meter Calibration Chemical Engineering Department RET Project

6. Glucose Meter Calibration (2) Chemical Engineering Department RET Project

7. 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 001000 1250750 2500 3750250 410000

8. Glucose Meter Calibration (4) IV.Prepare a calibration curve and obtain equation for best fit line Chemical Engineering Department RET Project

9. 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) Chemical Engineering Department RET Project

10. 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

11. 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 dH 2 O) to all flasks Chemical Engineering Department RET Project

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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 Chemical Engineering Department RET Project

18. 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. Chemical Engineering Department RET Project

19. 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 Chemical Engineering Department RET Project

20. 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

21. Data Analysis (2) Chemical Engineering Department RET Project theoretical mass of glucose that could be produced

22. Data Analysis (3) Chemical Engineering Department RET Project

23. Sample Calculations Chemical Engineering Department RET Project

24. Sample Calculations (1) Chemical Engineering Department RET Project

25. Acknowledgements Chemical Engineering Department RET Project