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In this lab, students will learn about ethanol and its important role in our world’s ever-increasing demand for energy. Students will go through the process of fermenting and distilling corn for ethanol production.
There are many variables that can affect ethanol production. This lab may be used as a stand-alone lab, with a prescribed procedure for producing ethanol, or as a follow-up after performing Kansas Corn: Fermenting Fuel – Designing a Procedure for Fast Fermentation. When using this approach to the lab, students use their data to produce their own procedure and compete to see which group can produce the most efficient fermentation. This is determined by comparing the largest volume of flammable alcohol or the most CO2 collected during fermentation.
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The importance of ethanol and how it relates to today’s energy needs.
The process of anaerobic respiration for yeast and how they produce ethanol.
The increasing demand for liquid fuels for transportation, increased world-demand for oil (gasoline), and the negative consequences of global warming have all contributed to the increased use of corn-based sugar to produce ethanol. Ethanol can be used as a substitute for gasoline, as it can be burned in many of today’s passenger cars and trucks. Most gas stations currently use 10% ethanol in their gasoline. However, it has also been used as 85% ethanol to 15% gasoline at some gas pumps, and this blend is called “E85” or “flex fuel”. Running this fuel in the gasoline engine typically does not require any mechanical modification. Not all gasoline motors are manufactured to run on E85, so it is best to check the vehicle owner’s manual before fueling up with E85.
In the United States, commercial production of fuel ethanol involves breaking down the starch present in corn into simple sugars, like glucose, and feeding these sugars to yeast for fermentation. Next they recover ethanol and other byproducts, such as animal feed, corn oil, and carbon dioxide. Ethanol is an alcohol produced by yeast during fermentation. Fuel ethanol is ethanol that has been highly concentrated and blended with gasoline to render the alcohol undrinkable.
For each pound of simple sugars, yeast can produce approximately 0.5 pounds (0.15 gallons) of ethanol and an equivalent amount of carbon dioxide. The value of corn for ethanol production is due to its large volume of carbohydrates, specifically starch. Starch can be easily processed to break down into simple sugars, and then fed to yeast to produce ethanol. Modern ethanol production can produce approximately 2.8 gallons to 3 gallons of fuel ethanol for every bushel of corn.
Ethanol production uses only the starch portion of the corn, which is about 70% of the kernel. All the remaining nutrients: protein, fat, minerals, and vitamins, are concentrated into distillers dried grains, which is used as feed for livestock. Some ethanol plants also remove the corn oil from distiller’s grain to create renewable diesel. About 40% of the United States’ corn crop is used to produce ethanol.
Groups will be competing to see which group can produce the most ethanol in the time frame allotted by the teacher.
Limitations on materials may be made or provided, or a cost applied to each step of the procedure (for example: heating sample for 10 minutes = 100 dollars, each ml of enzyme or buffer solution = 50 dollars, each g of yeast = 100 dollars, etc.), and have students stay within a budget for their production. A price per ml of CO2 or flammable ethanol produced could change the competition to the most profitable procedure.
Student procedures that rapidly produce CO2 may need to be transferred to a larger 500 ml flask and leave some air in the flask to prevent popping their stopper apparatus.
Prescribed Preparation of Corn Mash (1 class period):
Add 100 ml distilled water to a 500 ml beaker and heat between 80°C to 90°C to near boiling.
Weigh out 50 g of ground corn. Add ground corn to the 500 ml beaker and stir.
Boil for 10 minutes, being careful not to burn mixture.
After boiling is completed, remove the beaker from the hotplate and allow it to cool to 50°C or below.
While the corn mash is cooling, measure 50 ml of distilled water and pour into a 250 ml beaker. Shake the amylase solution, then measure 5 ml of the amylase solution into a small graduated cylinder and add to the 250 ml beaker of water. Stir the resulting mixture and add it to the cooled corn mash. Stir the mixture occasionally with a stirring rod throughout the next 10 minutes.
At the end of the 10-minute period, measure 20 ml of the pH 5 buffer. Shake the buffer solution and add it to the corn mash to maintain a slightly acidic pH.
Shake the glucoamylase solution, then measure 5 ml of glucoamylase solution. Add it to the corn mash.
Add 5.0 g of yeast to the corn mash and stir the entire mixture well.
Part 1: Student Designed Procedures (1 class period)
Student Procedures developed in Kansas Corn: Fermenting Fuel– Designing a Procedure for Fast Fermentation can be used to prepare corn mash. After corn mash is prepared, the rest of the lab should be conducted as written.
Part 2: Fermentation While Collecting CO2 by Water Displacement (allow to sit overnight)
This procedure will allow tracking of the CO2 production of the yeast. Knowing how much gas is produced will allow calculation of how much fermentation has taken place and to be sure adequate fermentation has occurred before distillation is attempted. The direct measurement of CO2 allows students to compare the rate of fermentation of their procedures.
Insert glass tubing into a single-holed stopper large enough to fit your corn mash/yeast mixture to a 500 ml Erlenmeyer Flask.
Fill a tub half full of water (food coloring may be added for increased visibility).
Fill a large graduated cylinder completely full with water from the tub.
Cover the top of the graduated cylinder with a glass square and quickly invert it so the opening is under the surface of the water in the tub.
Use a ring stand and utility clamp to hold the cylinder in this position.
Attach rubber tubing to the glass tubing in your flask. Run the tubing under the water and up into the opening of the suspended graduated cylinder.
As the yeast metabolize and produce ethanol, they also produce carbon dioxide. There is a direct correlation between the amount of carbon dioxide produced and the amount of ethanol. Over time, the graduated cylinder may fill up with gas and displace the water inside. You may need to refill the cylinder with water several times depending on the size.
Record the total amount of CO2 produced. The amount of ethanol produced can be calculated using the procedure outlined in Calculating the amount of ethanol produced from carbon dioxide. (Note: If 750 ml of CO2 has not been produced, see Trouble Shooting Fermentation below.)
The experiment pictured was set up to test the effect of the amount of yeast in the otherwise identical samples. The sample shown on the right had the amount of yeast written in an unsuccessful lab procedure; the sample on the left had five times as much yeast solution. (Note: A time-lapse video of this experiment is available at kscorn.com.)
Trouble Shooting Fermentation:
750 ml of CO2 is the amount given off to produce 2.0 ml of ethanol. Less than 750 ml of CO2 would indicate incomplete fermentation, or a problem with fermentation.
Possible problems in fermentation, if less than 750 ml of CO2 is produced:
If bubbles are still forming fermentation may not be complete.
Allow more time if bubbles are still forming.
If bubbles are not forming, the enzymes may have been added when mash was too warm, or insufficient yeast was added:
Glucoamylase may be added.
Add 1 g (or more) of dry yeast, mix well and allow fermentation to continue.
Calculating the Amount of Ethanol Produced from Carbon Dioxide
During fermentation, glucose is converted ethanol and carbon dioxide according to the following equation:
This means that for every molecule of carbon dioxide produced, there is a molecule of ethanol produced as well. By calculating the amount of carbon dioxide molecules, the amount of ethanol can also be determined.
Because the carbon dioxide is a gas, moles/liter of a gas can be used to calculate moles of CO2.
If a simpler calculation is preferred, this can be combined into:
To calculate mass of ethanol:
To calculate volume of ethanol:
Part 3: Filtering the Solids (15 minutes)
Use a large strainer with a large bowl/beaker underneath to strain out any large solids from your fermented corn mash. Repeat this step two to three times.
Collect the solids from the strainer. Place the solids in a large piece of cheese cloth. Wrap up the solids, then hand squeeze the liquid out of the solids into your beaker.
Line your strainer with a new piece of cheesecloth. Pour your liquid through the cheesecloth lined strainer into a bowl/beaker below. Collect the cheesecloth with the strained solids, and again hand squeeze the liquid out of the solids into your bowl/beaker. You should have removed most of the solids from this mixture at this time.
Part 4: Distillation of Ethanol from Corn Mash (30-45 minutes)
Set up the distillation apparatus as shown in the image above. Make sure to either grease or wet the ground glass joints before connecting them. This helps to prevent any vapor from escaping the joints and to keep the joints from freezing together.
Pour the strained solution into the distillation flask. Use a heating mantle to heat the liquid and control the temperature. The best separation of alcohol will occur if the distillation is done slowly. Ethanol’s boiling point is 78.37°C and water’s is 100°C; therefore, be careful to keep the temperature between these two boiling points.
Collect the ethanol distillate samples into a small flask to be used for the Alcohol Flame Test. Wrap the opening of the flask and end of the condensing tube with aluminum foil to help prevent evaporation of the ethanol. Pour the distillate samples into capped vial until ready to do the flame test.
Part 5: Density Test (5-10 minutes)
Using an appropriate graduated cylinder, measure the total volume of distillate collected.
Record the mass of a 10 ml graduated cylinder.
Pour distillate into graduated cylinder. If more than 10 ml was collected, add approximately 9 ml.
Record the volume of the distillate.
Measure and record the mass of the distillate and graduated cylinder.
Determine the mass of the distillate by subtracting the graduated cylinder mass from the measurement recorded in step 4.
Determine the density of the distillate by dividing the mass by the volume.
Use the graph provided to estimate the percentage of ethanol in the distillate.
Multiply the total volume of the distillate by the percentage as a decimal to determine the total volume of ethanol in distillate.
Part 6: Alcohol Flame Test (5-15 minutes)
Use a pipette to remove a 2 ml sample of your distilled ethanol and place the ethanol on a watch glass or in a ceramic evaporating dish. Light the ethanol with a lighter. A quality sample will light with a pale blue flame. Time how long the flame burns. The longer the flame burns, the greater the alcohol concentration. If the distillate does not burn, the water concentration is too high.
If No Flame is Produced:
Ethanol’s boiling point is 78.37°C and water’s is 100°C; therefore, be careful to keep the temperature between these two boiling points. If distillation ran with temperature close to 100°C, the mixture may contain too much water.
There are two possible solutions:
Distillate may be run through the distillation process again.
Add 4 ml of distillate with several drops of food coloring to a test tube containing 1 g of potassium carbonate, K2CO3, and insert stopper. Shake vigorously and allow layers to form. If none form, add more potassium carbonate and repeat. Water in the distillate is attracted more to the potassium carbonate and leaves the ethanol in a concentrated layer that will contain the food coloring. The ethanol layer will form above the salt water layer due to its lower density. Carefully pipette or decant the ethanol off of the salt water layer.
Potassium carbonate has saturated the water and forced the ethanol out of solution. The food coloring stays in the ethanol layer. This should be nearly pure ethanol.
Ethanol is a part of the agricultural industry that has job openings from corn farming, ethanol production, to government policy jobs in Washington, D.C. Ethanol product jobs are readily available, and so are jobs in biofuel research. Typically, you do not need a degree to work in an ethanol production plant, but for higher salaries, consider a degree in agriculture, chemistry, biology, or a related field. Workers in ethanol plants transport the fermented corn to distillers, monitor the dehydration process, and package the final ethanol product safely. Car companies are increasingly advancing their research departments to deal with the growing trend of renewable energy. The government also hires workers for the research and development of ethanol products.
Any educator electing to perform demonstrations is expected to follow NSTA Minimum Safety Practices and Regulations for Demonstrations, Experiments, and Workshops, which are available at http://static.nsta.org/pdfs/MinimumSafetyPracticesAndRegulations.pdf, as well as all school policies and rules and all state and federal laws, regulations, codes and professional standards. Educators are responsible for abiding appropriate legal standards and better professional practices under a duty of care to make laboratories and demonstrations in and out of the classroom as safe as possible. If in doubt, do not perform the demonstrations.
Make Learning Fun
Investing in teachers is a priority therefore the Kansas Corn Commission is committed to providing free materials and training to help teachers excel in the classroom. Distillation kits and funding for ethanol plant tours are available to teachers who teach Kansas Corn’s ethanol labs. Teachers who seek to expand their knowledge and skill of these labs are encouraged to seek out a training opportunity.