EXPERIMENT 3: Converting Cellulose Biomass to Ethanol (CB2E)

Transcription

1 EXPERIMENT 3: Converting Cellulose Biomass to Ethanol (CB2E) Modified for this workshop from: Activity CB2E, Great Lakes Bioenergy Research Center BBSRC Practice Biofuel Activities, Activity 2A and Activity 2C Fermentation of lignocelluloses Overview In this lab you will investigate the challenge of converting various cellulosic biomass into ethanol. Each team will use a different source of cellulose biomass. There will be a friendly completion to see which team can produce the most ethanol per gram of dry cellulosic biomass. Each team will pick a cellulosic biomass sample, break it down, convert it into sugars, and then into ethanol through the processes described below. You will track the conversion process by measuring glucose and ethanol levels at key stages. This data collection by the various teams will help determine which biomass source (feedstock) and pretreatment methods (heat, ph) and enzymes are most effective at producing sugars and ethanol. You will use this information and information gathered from the other lab activities to develop explanations for why some samples produced more sugar and ethanol than others. The three steps required to convert cellulosic biomass into ethanol are: 1. Pretreatment mechanical, chemical (alkali) and heat treatments to loosen cell wall structure and expose cellulose (explosion, boiling, autoclaving, pressure-cooking) 2. Hydrolysis (enzymatic digestion) break the long cellulose molecules into individual glucose molecules 3. Fermentation yeast consume glucose and oxygen and produce ethanol and CO 2 In this activity you can compare the effectiveness of a variety of pretreatments on a variety of feedstocks, including straw, maize and rapeseeds, or others as you choose. The popcorn mimics the process of steam explosion that can be used to open up plant cell walls to allow enzymes access to polysaccharides, but other methods such as autoclaving, pressure-cooking and boiling can be employed. Each team will assess the effects of different variables. You will have only a day to set up/run the experiment. In school the experiment can be run for up to a week to allow for sufficient levels of fermentation for measurable levels of CO 2 to be produced. In this activity you will also compare the effectiveness of enzymes at hydrolyzing a variety of feedstocks. Background: Large amounts of sugar molecules are present in the lignocellulose of plant material and current research aims to unlock the fermentable sugars in agricultural or forestry wastes and residues from cereal production such as straw, bran, brewer s grain and wood. To enable yeast to carry out fermentation the sugars trapped in plant cell wall lignocellulose, must be released by pretreatment with steam or chemicals followed by hydrolytic breakdown of the released polysaccharides with enzyme cocktails. Currently chemical treatment involves either strong acid or mild alkali but due to the requirement for

2 specialized equipment to carry out the procedure as well as the treatment of waste chemicals before disposal, research is focusing on steam treatments at present. Pretreatments change the structure of cell walls and polymers by disrupting intermolecular forces holding them together, allowing greater access by enzymes and water. Enzymatic digestion of exposed polysaccharides can produce mono-, di- and tetrasaccharides. Enzymes are expensive and need to be recovered from industrial processes. Immobilizing enzymes enables easier recovery and development of more efficient continuous processing. However, it restricts the ability of the enzymes to carry out cleavage of the insoluble polysaccharides. Sustainable liquid biofuels can be produced from lignocellulosic biomass such as wood and straw. These materials contain polysaccharides that can be converted through enzymatic hydrolysis into simple sugars, which can then be fermented to produce liquid biofuels. Bioethanol produced on a large scale in Brazil and the USA is made from sugar cane or maize respectively. Sugars from sugar cane can be fermented by Saccharomyces cerevisiae without prior treatment as they are already disaccharides, but starch polymers from maize or wheat need conversion to di- or monosaccharides, by a hydrolysis reaction known as saccharification, prior to fermentation. The enzyme mixtures used in saccharification of starch are amylases, enzymes also found in human saliva and secreted by the pancreas. In plants the majority of sugars are locked into the cell walls in ways that are poorly understood, preventing effective digestion by enzymes. Scientists are actively searching for enzymes that can rapidly release sugars from these indigestible woody materials. Lignocellulose and hemicelluloses are broken down by the actions of a range of enzymes including cellulases and hemicellulases. Amylases are not able to hydrolyze the polysaccharides found in cell wall. Three enzymes are compared for their ability to produce fermentable sugars from the feedstock. Cellulase breaks down accessible cellulose molecules whereas pectinases break down the pectin in cell walls that holds the cellulose molecules in place. Pectin is predominantly found in non-woody parts of plants (as it is associated with the primary cell wall found around all plant cells) and holds cells together. Amylases will allow comparison of the effectiveness of saccharification versus breakdown of the cellulose The following is for reference only, and designed to fit the time constraints of this workshop. Procedures follow this schedule. Day 1: Monday Morning: Experimental Design and planning Teams select a biomass sample and cutting treatment You must prepare both an experimental and a control treatment. Consider running two experimental set-ups for replication purposes. Afternoon Sample preparation and organization

3 Set up experiment Cut, grind and/or boil biomass Measure initial sugar levels Start Pretreatment if ready Day 2: Tuesday Pretreatment /start Hydrolysis (Enzyme Digestion) (1 day) Add cellulose enzymes (Cellulclast) Place experimental and control tubes in water bath for 24 hours (overnight) Measure sugar levels (after 24 hours = Wednesday morning) Day 3: Wednesday Finish Hydrolysis/ start Fermentation (1 day) (Measure the 24-hour sugar levels from the Hydrolysis set-up) Measure the initial ethanol levels Add yeast and allow to incubate. Measure ethanol and glucose levels after 30 minutes. Return to incubator (37 C) overnight Measure the final ethanol and glucose levels (after 24 hours = Thursday morning) Day 4: Thursday End Fermentation Analysis, Conclusions and Discussion Measure the final ethanol and glucose levels from Fermentation set-up Graph the final results Summarize conclusions and communicate findings to class Write up final results based on evidence from your other lab group results Procedure Step 1: Sample Preparation and Pretreatment (Day 1) *Goal: Break down plant cell walls to release the cellulose fibers* Every group will have 2 tube-setups with the same foodstock and biomass. If any pre-treatment is required do so (cutting, grinding, drying, etc.) Label two 50mL falcon tubes and caps with your team initials, date, and sample description (biomass source and any pre-treatment). Every group will have 2 tube setups with the same biomass (or more if you wish) If any pre-treatment is required do so (cutting, grinding, drying, etc.) Measure 1.0 gram of your biomass samples for each set-up. Put into the corresponding 50mL falcon tube

4 Hot water PRETREATMENT. (boiling, pressure cooking, autoclaving, ) The procedure given below is for boiling. You will need to develop a protocol for using a pressure cooker, autoclave or other device) Start the hot plate to bring approximately 400mL water to a gentle boil in a 500 ml glass beaker. Use pre-heated water to fill your beaker. Set up a falcon test tube holder (i.e. chicken-wire screen or aluminum foil) for your 500 ml beaker as directed. Add 25 ml of distilled water to all three of your labeled Add 50mL falcon tubes. Swirl to mix the biomass and the water. Let it sit for 1 minute. Loosely screw the cap onto the falcon tube. Wait for water in your beaker to come to a gentle boil on your hot plate. Make sure that the biomass samples and the liquid are completely submerged below the surface of the boiling water in the beaker. Leave tubes in the water for 10 to 25 minutes depending on how much time you have. The longer the time period, the higher the potential yield of ethanol will be. Turn off the hot plate and remove your samples. Allow them to cool to room temperature. Use a cold-water bath to make the tubes cool more quickly. Test the initial glucose concentration using the blood glucose test monitor and test strips. Record this data. Describe any detectable changes in the biomass (appearance? odor?). Test the initial ethanol concentration using the ethanol probes. Record this data. Describe any detectable changes in the biomass (appearance? odor?). If samples will not be used in the next 2 days, refrigerate or freeze them immediately. This will suppress microbial growth. Step 2: Enzymatic digestion (hydrolysis) Day 2 fibers into glucose (sugar)* *Goal: Digest the cellulose Three enzymes can be compared for their ability to produce fermentable sugars from the feedstocks. Cellulase breaks down accessible cellulose molecules whereas pectinases break down the pectin in cell walls that holds the cellulose molecules in place. (Cellucast) Run at 50 C, Pectin is predominantly found in non-woody parts of plants (as it is associated with the primary cell wall found around all plant cells) and holds cells together. Run at 35 C. Amylases will allow comparison of the effectiveness of saccharification versus breakdown of the cellulose. Run at 35 C. Preparing Enzyme Solutions: Celluclast -- 1 ml from the bottle Amylase Preparing 100 ml solution of 1% enzyme solution Measure out 1 g of the enzyme.

5 Pectinase: Add the solid to about 70 ml of water or buffer (if needed for the activity) in a beaker Stir to dissolve (do not warm the solution). Pour the solution into an appropriate measuring cylinder/volumetric flask. Dilute to the final volume with pure water. Pour into a labeled bottle and mix well. Store in the refrigerator or on ice during use. Carry out the procedure that the students will undertake and consult with the teacher to confirm that the results are satisfactory. It may be necessary either to dilute the solution further with more water or add more of the enzyme *Goal: Digest the cellulose fibers into glucose Remove samples from refrigerator or freezer and bring to room temperature. Make sure the common water bath or the incubator is at 37 C. Add 1.0 ml of enzyme product (cellulase, pectinase, amylase or other enzyme) to each test tube that is undergoing hydrolysis. The control will not have any enzyme added. Screw caps on tightly. Mix gently. Place both falcon tubes in a common water bath or incubator at 37 C. Leave the tubes in the water bath for 24 hours. After 24-hour hydrolysis, collect data. Use the blood glucose test monitor and test strips to test post-enzyme glucose concentration of the sample. Record this data. Describe any detectable changes in the biomass (appearance, odor?). Test the ethanol concentration using the calibrated ethanol sensors. To calibrate sensors use ethanol concentrations ranging from 0.1% to 3 %. (0.1, 0.5, 1, 2 and 3 %) Record this data. Describe any detectable changes in the biomass (appearance, odor?). **Note: for more accurate ethanol readings, allow samples to reach room temperature before taking measurements, and calibrate the Ethanol sensors with two different concentrations of ethanol. If fermentation will not begin at this stage, freeze or refrigerate samples to prevent microbial contamination. Step 3: Fermentation *Goal: convert glucose (sugar) into ethanol (fuel).* *Goal: convert glucose (sugar) into ethanol (fuel).* Make sure the common water bath or incubator is at 37 C. Add ¼ teaspoon or 1.0 gram of active yeast to each tube. These measurements are roughly equivalent. Gently mix in the yeast. The yeast will grow more quickly if evenly mixed. Loosely screw on the cap to the tubes. It is important that the tubes not be airtight for the fermentation. Yeast will produce CO 2 and will build up pressure in the tube unless the gas is allowed to escape.

6 Place falcon tubes upright in the 37 C water bath or incubator. Use a test tube rack or similar apparatus to keep falcon tubes upright. **OPTIONAL: After 30 minutes measure ethanol and glucose concentration. Record data and other observations about changes. Return your falcon tubes to the 37 common water bath or incubator for 24 hours of fermentation. After 24 hours, remove your falcon tubes from the 37 C water bath. **Note: If 24-hour measurement does not fit with class schedule, instructor can remove samples from water bath and refrigerate or freeze until final measurements can be taken. Take final glucose readings: Use the blood glucose test monitor and test strips to test postenzyme glucose concentration of the sample. Record this data. Describe any detectable changes in the biomass (appearance, odor?). Take final ethanol readings: Test the ethanol concentration using the ethanol probes. Record this data. Describe any detectable changes in the biomass (appearance, odor?). **Note: for more accurate ethanol readings, allow samples to reach room temperature before taking measurements. Clean tubes and lab area after experiments are complete. Analysis, Discussion, and Conclusions To organize and draw conclusions from your data, it is helpful to compare changes in glucose and ethanol levels over time using bar graphs. Using a computer program such as Microsoft Excel (or by hand), create two bar graphs to summarize your results. The empty graphs below can serve as a guide. Discuss the graphs with your lab group. Do these results match your initial prediction? Why or why not? How can you explain your results? Explain, summarize and communicate your results as instructed DATA CHART SEPARATE DOCUMENT in Google Drive Clean tubes and lab area after experiments are complete.