Fermentation: How
Yeast Obtain Energy

In this lab, we will be studying the process of fermentation as a source of energy for cells, specifically yeast cells, and will relate this to “everyday life” by making “lab brew.” This process, and the steps that are needed to prevent the brew from turning to vinegar also illustrate one of the classic principles in the field of microbiology.

Yeast Cells Do Alcohol Fermentation

Many microorganisms (micro = small), notably yeasts and bacteria, extract energy from their food (glucose) by fermentation. One of the best-known types of fermentation is alcohol fermentation in which the overall chemical reaction is:

C₆H₁₂O₆ (glucose) → 2CO₂ + 2CH₃CH₂OH (ethyl alcohol)

or, starting from sucrose or maltose,

C₁₂H₂₂O₁₁ + H₂O → 4CO₂ + 4CH₃CH₂OH (ethyl alcohol)

Humans have known about and utilized the process of fermentation for many thousands of years. CO₂ liberated by yeast cells doing alcohol fermentation causes bread to rise, while the liberated alcohol gives it its wonderful smell. The Egyptians and many subsequent civilizations have fermented grains such as barley (or wheat — but note that both are “off-limits” for someone with a gluten sensitivity) to break the starch down to malt (maltose), then glucose, and finally alcohol. Since pretty-much the same ingredients go into bread and beer, in the past, beer has actually been referred to as “liquid bread.”

For about the same length of time, people have also known that various fruits, especially grapes, could also be fermented to produce alcoholic beverages. Thus, alcoholic fermentation is the process which is responsible for the production of wine, beer, and other fermented products. It is the toxic nature of ethanol which acts to preserve these brews, and which leads to intoxication upon consumption. In fact, yeasts cannot generally survive in alcohol concentrations in excess of approximately 12 to 14%.

In this lab, we will be studying the process of alcohol fermentation as performed by the yeast, Saccharomyces cerevisiae (sacchar = sugar; myces = fungus; Ceres = goddess of grain; vis = to see; -ia = state of, condition of, disease). It was this process of alcohol fermentation which Louis Pasteur studied, leading to a biochemical understanding of biological processes.

Within the yeast cells, the actual chemical reactions that turn sugar into alcohol are catalyzed by a number of enzymes (en = in; zym = yeast) — biological catalysts that help other chemicals to react. Although numerous enzymes are found in living organisms, each one with its own specialized function, the very first such chemicals to be studied were those in yeast which are involved in the process of fermentation. Since they were found in yeast, they were called “enzymes.” We now know of many more enzymes, most of which are not found in yeast, yet the name is still used.

Stoichiometry of Fermentation

Today’s lab can be used to demonstrate a number of important principles relating to alcoholic fermentation including the stoichiometry (the study of the definite proportions in which chemicals will interact with each other — stoichi = an element, metr = measure) of the conversion of sugar to alcohol. The following conversion factors will be important, here:

Molecular Weight of Sucrose/Maltose	342.17 g/m
Moles of EtOH per Mole of Sucrose/Maltose	4 m
Molecular Weight of Ethanol	46.05 g/m
Grams of EtOH per Mole of Sucrose/Maltose	4 × 46.05 = 184.20 g
Grams of EtOH per Gram of Sucrose/Maltose	184.20 ÷ 342.17 = 0.538 g
Density of Pure EtOH at 20° C	0.789 g/mL
Density of Pure H2O at 20° C	0.99823 g/mL
Weight of One Can of Malt Syrup	1.5 kg (3.3 lb)
Percentage of Weight of Maltose in Malt Syrup*	80%
Percentage of Weight of H2O in Malt Syrup*	20%
Weight of 1 C of Granulated Sucrose†	213.97 g
Density of Malt Syrup§	1.426 g/mL
*These percentages were found on another Web site †This was weighed here in our lab §This number was found on another Web site as 0.084 gal/lb, and since 0.084 gal     = 318.04 mL and 1 lb = 453.59 g, therefore 453.59 g/318.04 mL = 1.426 g/mL

Putting all that together, if the fermentation reaction is 100% efficient (which is rare for most biological reactions), for every gram of sucrose or maltose that is put in, 0.538 g (= 0.682 mL) of ethanol should be produced. Thus, it would be of interest to examine the recipe(s) and calculate the hypothetical stoichiometry involved.

For our lab brew, the amount of sugar going in depends on which recipe will be followed. The original, Fankhauser recipe calls for 1¼ C of sucrose + ⅓ C of canned malt syrup per 1-gal batch. For the sucrose in the original, Fankhauser recipe,

1.25 C sucrose	×	213.97 g sucrose	×	0.538 g EtOH	=	35.97 g EtOH
4 L of lab brew		1 C sucrose		1 g sucrose		L of lab brew

and for the maltose,

0.33 C syrup	×	236.64 mL syrup	×	1.426 g syrup	×	0.80 g maltose	×	0.538 g EtOH
4 L		1 C syrup		1 mL syrup		1 g syrup		1 g maltose

=	11.98 g EtOH
	L of lab brew

which can be combined and converted to milliliters:

35.97 + 11.98 g EtOH	×	1 mL EtOH	=	59.51 mL EtOH	= 5.95% v/v
L of lab brew		0.789 g EtOH		L of lab brew

Alternatively, the lab brew may be made with ⅞ to 1 C of malt, and no added sucrose per 1-gal batch. In that case, the calculations would be:

1 C syrup	×	236.64 mL syrup	×	1.426 g syrup	×	0.80 g maltose	×	0.538 g EtOH
4 L of lab brew		1 C syrup		1 mL syrup		1 g syrup		1 g maltose

=	36.31 g EtOH
	L of lab brew

36.31 g EtOH	×	1 mL EtOH	=	46.02 mL EtOH	= 4.60% v/v
L of lab brew		0.789 g EtOH		L of lab brew

Interestingly, when we’ve tested the alcohol content of aged “lab brew” via distillation and calculation of specific gravity or via refractive index, we typically have obtained results in the vacinity of 5%. Thus, a) we know we’re “in the right ballpark,” and b) this must be a fairly efficient chemical reaction.

Calculating these numbers for the mead would require looking up the density of honey and what percentage of it is actually sugar (also, note that the sugar in honey is “invert syrup” — a 50:50 mixture of glucose and fructose). For the wine, in addition to the sucrose that is added, we’d need to know how much sugar is in the grapes.

Acetobacter Can Turn Alcohol into Vinegar

Louis Pasteur was a famous French microbiologist who lived in the 1800s. People involved in the wine industry of that day asked him to research why some bottles of wine were OK while others spoiled and turned into vinegar (vin = wine; aigre = sour). Pasteur discovered that the whitish bloom on the skins of grapes contained a number of small, oval cells which he identified as yeast. These are the yeast that turned the smashed grapes into wine.

In wine that had turned to vinegar, Pasteur also found small, rod-shaped bacteria (this shape is called bacillus) as a “contamination” or “infection” in the wine. These were found to belong to the genus Acetobacter (aceto = vinegar; bacter = rod). Pasteur discovered that if the wine was heated to 63° C and held at that temperature for 30 min, the Acetobacter would be killed. This process is named in his honor: pasteurization. Some people object to treating wine in this way, claiming that the flavor is changed. Today, by law, all wine sold in the United States must have sulfites added to kill anything living in it (and some highly allergic humans).

There is, however, another way to inhibit growth of Acetobacter. It has been discovered that while yeast do not need the presence of oxygen (O₂) to do fermentation (fermentation is an anaerobic process), Acetobacter do need O₂ to turn tthe alcohol produced by the yeast into vinegar, more specifically acetic acid (CH₃COOH). Thus, if O₂ can be eliminated, the Acetobacter cannot grow. However, since the process of fermentation is evolving CO₂, the fermentation vessel cannot be sealed or it will explode. This necessitates the use of an airlock which allows the CO₂ produced to bubble out through a water barrier which simultaneously prohibits O₂ from entering. We will, therefore, be making use of Pasteur’s discoveries to keep our brew from turning into malt vinegar.

Background on Hops or Other Added Herbs

For centuries, beer has been flavored by the addition of various bitter herbs, for example, meadowsweet (Spiraea latifolia), alehoof (which is another name for Gill-over-the-Ground, Glecoma hederacea), and/or alecost or costmary (Chrysanthemum balsamita). In the eleventh century, Bavarians started adding hops (Humulus lupulus) to the brew to act as a preservative (it helps extend the “shelf-life” of the beer) and to flavor the beer, as a replacement for the bitter herbs previously used. This practice was borrowed by the British in the sixteenth century.

Modern breweries start with a cooked “mash” of sprouted barley which is fermented by a special strain of yeast. Added to this is a water-extract or “tea” from hops. For home-brewing, cans of barley malt with (or without) hops extract added can be purchased to add to sugar water to make beer. This is what we will use in this experiment.

A slight digression, but an interesting, related topic. While the size of 1 in. is now defined in terms of the equivalent length in millimeters, initially, way back in about 1066, an inch was defined as the length of three barleycorns (grains of barley) end-to-end.

Hops (Humulus lupulus) is in the family Cannabinaceae (The plant in this photo is another member of family Cannabinaceae.). Its native habitat includes damp areas where it can be found twining tightly around willow (and other) trees. Because of this, the Greek philosopher Pliny called it lupus salictarius which means “willow wolf.” Interestingly, apparently it always twines in a clockwise direction. “Humulus” is a Medieval latinization of an Anglo-Saxon word, “humule.” In the U. S. today, most hops are grown in Washington, Oregon, California, and Idaho.

Like this other member of the Cannabinaceae, the part of the hops plant that is used is the female strobilus (a part of the female plant surrounding the flowers). This has also been used medicinally for centuries. The primary constituents responsible for the medicinal properties and bitter flavor are two chemicals called humulone and lupulone. These are unstable in the presence of light and air, thus dried hops rapidly lose their flavor and medicinal effectiveness (and must be used for brewing within a relatively short time after harvest). Hops is a well-known sedative and has long been used as a sleep aid. Animal and human research has shown that, indeed, it is a CNS depressant. Hops is also used herbally as a diuretic and antibiotic.

Supplies and Ingredients for One Gallon

Ingredients are listed for a 1-gal recipe per lab section.” Your class will need:

Needed for All Recipes

• one 1-gal. jug — a clean, left-over, cider jug works well



• airlock (made from rubber stoppers and glass tubing)



• hot pads, cooking pot(s), hot plate or stove, measuring cup(s) and spoons, wooden spoon
• can opener or blender, depending on which recipe you will be making
• 2 CLEAN 2-liter soft-drink bottles with their plastic lids (If you have some, please wash them thoroughly, let them air dry, and bring them in when we are ready to bottle.)

• 1 tsp live yeast (Brewer’s, ale, or wine yeast is preferable, but regular baking yeast will work with a slightly different final product.)
• ¼ tsp table sugar (sucrose)
• ¼ C warm water

Recipe A1 — “Lab Brew”

Additional ingredients are listed for Dr. Fankhauser’s “original” recipe, scaled down to a 1-gal batch of “lab brew.”

• ⅓ C canned, hopped malt extract (When making a larger batch, we have also used unhopped malt + a packet of dried hops, but because of the way the hops are packaged, that would be difficult to do with a 1-gal. batch. Both the malt and hops are available from a beer/wine supply store.)
• 1¼ C white sucrose (⅓ to ½ C brown sugar may be substituted for an equal amount of the white for darker beer. Dextrose/glucose or maltose may also be substituted for part/all of the sucrose or the ratio of malt extract to sugar may be increased.
• cool tap water: about 5 to 5½ C + ~ ½ gal. + more

Recipe A2 — “Lab Brew”

The ingredients and procedure for this recipe are similar to recipe A1, above, with one significant change:

• The sugar called for in the above recipe may totally be replaced by malt extract. Thus about ⅞ to 1 C of malt extract would be used in place of the amounts of malt and sugar listed above.

Recipe B — Wine

• a “bunch” of grapes — you may wish to weigh how many are used, and do remember to record in your lab notebook what kind of grapes are used
• 1⅓ to 1½ C sugar
• cool tap water: about 5 to 5½ C + more

Recipe C — Mead

This recipe was adapted from one published in Country Commune Cooking by Lucy Horton.

• 3 C honey
• 1 oz (~3 to 4 in. long piece) fresh gingerroot, chopped
• 2 tsp whole cloves
• 2 tsp ground nutmeg or 1 whole nutmeg, smashed with a hammer
• cool tap water: about 5 to 5½ C + ~ ½ gal. + more

(Someone who tasted a batch of this mead commented that he thought the spices were too strong. We’re experimenting with decreasing the amounts of the various spices.)

Procedure

For All Recipes

1. So that we don’t waste supplies, your class will make one of the following recipes as a whole-class project. Various people from the class are encouraged to volunteer to do different parts of the procedure so everyone can be involved in some way. Everyone should watch what’s going on and take notes.
2. Add 1 tsp yeast and ¼ tsp sugar to about ¼ C water at about 37° C (warm but not hot). Let the mixture sit for about 15 min to “proof” (while you continue with other steps). It should, then, be bubbly or frothy. If nothing happens, it’s dead, so you’ll need to try again.

Recipe A1 — “Lab Brew”

3. If not already opened, open a can of malt extract and carefully remove the label in such a way that it can be taped back on for identification. In a pot, place a few inches of water and the can containing the malt extract. Heat the can of malt until it can be poured.
4. When the malt is softened and can be poured/measured, in a separate pot, measure and mix the ⅓ C hopped malt, the 1¼ C sucrose, and about 5 to 5½ C water.
5. Slowly heat to boiling but do not let it boil over. Stir slowly to prevent burning. Have hot pads ready to remove the pot from the heat if/when needed.
6. Put a little under ½ gal cold water into the jug, then add the hot sugar solution with a funnel. Avoid running the hot solution down the sides of the jug or it may crack from the heat. Pour some down the funnel directly into the cold water, then swirl to warm the bottle. Repeat the pour-and-swirl until all the hot liquid has been transferred.
7. Fill the jug to the “hip” with cold water. In pharmaceutical jargon, one would say, “Q. s. to the hip,” where “q. s.” stands for “quantum sufficit” in Latin which means as much as suffices, as much as is necessary. This “final” mixture is called the “wort.”
   

Recipe A2 — “Lab Brew”

C-G.    Follow recipe A1, except in step D, in place of the malt extract plus sugar, use ⅞ C (= ¾ C + 2 T) of just malt (OK to round to ~1 C).

Recipe B — Wine

3. In a blender, grind a “bunch” of grapes (weigh first?) with about 5 to 5½ C of water.
4. Add about 1⅓ to 1½ C sugar and blend to mix it all together.
5. Note: this mixture will not be cooked because the natural yeasts on the grape skins aid the fermentation process, but would be killed by the heat.

F-G.   Pour the mixture into the jug and q. s. to the hip of the jug.

Recipe C — Mead

3. In a pot, mix 3 C honey and 5 to 5½ C water. Slowly heat to boiling but do not let it boil over. Stir slowly to prevent burning. Have hot pads ready to remove the pot from the heat if/when needed.
4. Add 1 oz (~3-4 in long piece) chopped, fresh gingerroot, 2 tsp whole cloves and either 2 tsp ground nutmeg or 1 whole nutmeg that has been smashed with a hammer.
5. Boil another 5 min, making sure it doesn’t boil over.
6. Put a little under ½ gal cold water into the jug.
7. Then, add the hot sugar solution with a funnel. Avoid running the hot solution down the sides of the jug or it may crack. Pour it down a funnel directly into the cold water. Q. s. to the hip with cold water.

Then, for All Recipes

8. Assuming the yeast solution is bubbly, add it to the jug. This is called pitching the wort, with “pitching” used here to refer to setting up something (like pitching a tent) — the wort is being set up to ferment. If the yeast mixture is not bubbly, mix up a new batch (with different yeast). It is very important to add all the cold water first, before adding the yeast, because if the solution is too hot when the yeast is added it will kill the yeast.

9. Place an airlock on top of your bottle and add water to the correct level. This will allow CO₂ from the fermentation process to escape without permitting entrance of outside air that contains O₂ needed by Acetobacter bacteria that could cause the wort to spoil or turn to vinegar. Label the jug with the lab section, date, and contents (type of malt, sugar, honey, grapes, and/or yeast.
10. After 1 to 2 days (next lab period?), the beer should have a good head on it (called the “high kreusenen” stage), or the wine or mead should be vigorously bubbling. Check the airlock to make sure its water level is OK.
11. After about 4 days, the foam should subside, at which point, fill the jug up to the bottom of the neck with water. In pharmaceutical jargon, one would say, “Q. s. to the neck,” where q. s. stands for quantum sufficit in Latin which means “as much as suffices, as much as is necessary.” Replace the airlock. Note: depending on which days your lab meets, you may be able to do this step as soon as the next lab period.
12. After about 8 to 10 days or so (may take longer if the room is cooler), bubbling should have decreased to a very slow rate. Check the airlock to make sure its water level is OK.
13. About two to three weeks after the brew was made, it will be ready for bottling. In each of the CLEAN 2-L bottles (check to make sure they have their caps), add 2 tsp. sugar using a funnel. Carefully decant — pour off the liquid or supernatant — into a large, clean beaker or bucket (Optionally, the brew may be siphoned into the beaker or bucket), avoiding the sediment on the bottom, and from that container, pour the brew into the bottles. If it foams up, you may need to partially-fill the bottles, then come back to add more when the foam subsides. Assuming you’ve been able to successfully decant the brew without leaving too much behind with the sediment, it should work to divide the brew between the two 2-L bottles, leaving about 1½ inch of head-room in each bottle, so at the end, you should look at how much is in each bottle and use the last of the brew that’s in the bucket to top off whichever appear to be a bit “lower” than the other.
14. Tightly cap the bottles and check for leaks by gently squeezing each bottle. Store in a 60 to 70° F (16 to 21° C) location for a minimum of 10 to 15 days to allow the residual yeast to ferment the added sugar. This is what forms the carbonation in the beer (wine, mead). Note that if you bottle the beer (wine, mead) too soon and/or add too much sugar and/or get too much of the yeast sediment into the bottles, too much CO₂ may be formed and the bottle(s) may/will explode!
15. As soon as possible, swirl the sediment in the jug to loosen it and immediately and thoroughly wash out the jug, making sure to scrub off any yeast stuck to it, then place it upside-down in a sink or a rack to dry. Note that the yeast mixture is difficult to impossible to remove once it has dried, so it is important to quickly and thoroughly clean the jug.
16. Again, rinse out the carboy as soon as possible, before the left-over yeast dries. The flavor improves with age, so it is recommended to age the beer (wine, mead) for at least two weeks before consumption. Do not let the bottles freeze or get too warm or they may explode. It is a good idea to store the bottles in a location that will not be damaged if one does explode — it does occasionally happen.
17. While current University policy does not allow us to let you take any of this lab brew home, if you later decide to make some on your own, after the beer (wine, mead) has aged for at least two weeks, to taste it, refrigerate the bottle upright for at least 12 hr. Do not shake or disturb the sediment. When you open it, carefully decant it all into a pitcher without stopping, so that the sediment is not stirred up, and avoid pouring out the sediment when you get to the bottom. You may try to decant it into glasses, but you must pour it all out and not stop midway, or the sediment will be agitated. While the beer or wine may keep a bit longer, it is recommended that the mead be consumed within a year or less.

Note that the left-over yeast, especially the strain used by commercial breweries, is processed to remove some of the bitter flavor, killed, and purified, then dried and sold as brewer’s yeast in the health-food stores or used as a supplement in livestock feed. It is an excellent source of the B vitamins and other nutrients. Since the yeast in the sediment at the bottom of the jug and/or bottles is still alive, some of it could, potentially, be used to ferment another batch of brew or to make some bread.

Remember to Take Notes

Throughout the process, take notes on any information provided by your instructor and on what, specificaly, your class did (including what kind of malt, what kind of yeast, etc.). Make sure to record any deviations from the written protocol (did you use a different type of sugar?). Make sure to include all observations and data (you could even observe how many bubbles per minute are coming through the airlock on various days as the brew ferments). Draw pictures, especially of any new equipment (airlock), and/or take notes where needed. Optionally, if available, a “souvenir” piece of the yeast packet or piece of malt label (only if the can is empty and the label no longer needed) may be included.

Optional, Related Activities

Go Visit a Brewery or Winery

As time and interest allow, you may wish to visit a local brewery or winery to see how they make beer or wine. Take notes. Are there any “souvenirs” that you could include in your lab notebook?

Bake Some Bread

As time and interest allow, you may further study the process of alcohol fermentation and production of CO₂ by yeast by baking a batch of bread at home. One possible, simple recipe is included here (also adapted from Country Commune Cooking), but you may have another personal favorite that you like to use.

Supplies and Ingredients Needed

• ½ C honey
• 2 T or 2 pkg yeast
• 2 C warm (not hot) H₂O
• whole wheat flour (approx. 6 C or so)
• ¼ C oil (safflower is highest in vitamin E, olive has a good taste, others will work, too)
• big mixing bowl
• measuring cup(s) and spoons
• wooden spoon (and/or electric mixer)
• 2 loaf pans or cookie sheet (coated with oil and dusted with whole wheat flour)

Procedure

1. In a large bowl, mix the honey and warm water. If the water is too hot, it will kill the yeast. Warm tap water is OK.
2. Sprinkle in the yeast and gently stir to dissolve. Wait about 10 min to make sure the yeast is alive: the mixture should start to get frothy/foamy.
3. Add the oil and 2 C of flour. Beat approx. 1000 strokes by hand or for a while with the electric mixer to develop the gluten, a protein that makes the structure of the bread that holds in the CO₂.
4. Gradually, add approx. another 4 C of flour. The dough should stick together but not be too stiff. Optionally, you may add raisins, sunflower seeds, chopped nuts, cinnamon, or chopped onion and various herbs such as basil and sage, or whatever sounds like a good bread ingredient. You may also substitute soy, rye, oat, or other flour or meal for 1 to 2 C of the whole wheat flour.
5. Knead the dough about 10 min until it becomes elastic.
6. Coat the dough lightly with oil and place in an oiled bowl. Cover with a damp (clean) cloth or kitchen towel (not terry cloth). Allow to rise in a warm place (a gas oven that’s turned off is good) for about one hour until doubled in size.
7. If not already done, coat the loaf pans/cookie sheet with oil and dust with flour.
8. “Punch down” the dough (deflate it somewhat), and form into two loaves. Place into the loaf pans or form round loaves on the cookie sheet.
9. Cover lightly with the towel and let rise until double again (maybe another hour although it shouldn’t take as long this time).
10. Bake at 350° F for about ½ to 1 hr or until done (check it after ½ to ¾ hr). Signs of doneness are a) if the sides have shrunken away from the pan and the crust is browned and b) when you turn it out of the pan and tap on the bottom, it sounds “hollow.” If not, put it back in the pan and back into the oven for a while. Baking time may vary depending on a number of factors.
11. Turn the bread out onto a wire rack to cool. To keep for more than a day or two, store in the refrigerator in a plastic bag. May be frozen.

Variations: You may add from 1 to 2 C up to not more than half the total of a different kind(s) of flour. Unbleached white flour has a lot less nutrients, so will make a lighter-colored, higher-rising bread but lower in vitamins and fiber. Rye bread can be made by adding up to half rye flour and some caraway seeds. A couple tablespoons of unsweetened cocoa and instant coffee will make it dark like pumpernickel (using molasses in place of honey will help darken it, too). A cup of soy flour plus a cup of wheat germ will increase the protein content of the bread. Instead of loaves, you could braid the bread by forming three strands and braiding them (bake on cookie sheet). Dinner rolls could be made by forming into small balls and baking on a cookie sheet or in muffin tins (will not take as long to bake). Cinnamon bread can be made by, after punching down the dough, rolling or patting it out into two rectangles. Each rectangle should then be sprinkled with powdered cinnamon, and optional raisins and/or chopped nuts. Then, starting at one end, roll up the rectangle to form a loaf, and place in a loaf pan to bake. After baking, when the bread is sliced, the slices will have a spiral of cinnamon in them.

Fankhauser Popcorn Seasoning

In the distant past, a Biology lab tradition, after Friday volleyball, was a taste of lab brew accompanied by popcorn seasoned with Fankhauser popcorn seasoning. As time allows, we may still be able to sample the popcorn.

Fankhauser Popcorn Seasoning Ingredients

• 4 T brewer’s yeast
• 2 T powdered kelp
• 1 T salt
  

Note: Larger batches may be made by using the same proportions of ingredients (like 4 C + 2 C + 1 C, etc.).

Mix thoroughly (in blender if the yeast is in flakes). Store in a tightly-sealed jar.

To Make a Batch of Popcorn

1. Place about 10 mL (= 2 T, walnut-sized piece) of coconut oil into a heavy pot and place the pot on the largest burner on the stove. Turn the burner on high.
2. When the oil is melted and hot (a corn kernel will sizzle), add 400 mL (= 1⅔ to 1¾ C) corn kernels and cover. Shake until all the corn is popped.
3. Pour the popped corn into a clean paper grocery sack (with top turned over twice to stiffen it).
4. While shaking the bag to stir and mix, slowly drizzle on 30 to 40 mL (2 to 2¾ T) of a 1:1 butter/oil mixture (provides butter flavor with less cholesterol). Then, dust with 30 to 40 mL of seasoning mixture.

In this popcorn, the brewer’s yeast provides a number of the B vitamins. Kelp provides iodine, needed by your thyroid gland. The kelp, brewer’s yeast, and popcorn are complementary protein sources, thus form a complete protein when combined. By mixing the butter 50:50 with vegetable oil, it is possible to have the good buttery taste, yet reduce the amount of cholesterol (cholesterol is found only in animal products, thus would be in the butter, but not vegetable oil).

Things to Include in Your Notebook

Make sure you have all of the following in your lab notebook:

• all handout pages (in separate protocol book)
• all notes you take during the introductory mini-lecture
• all notes and data you gather as you perform the experiment, both the day we mix the brew and the day we bottle it
• detailed drawing (yours!) of an airlock
• drawings of any other “new” equipment used and any other brewing supplies used
• optional sample(s) of label from can of hops, yeast packet, etc.
• answers to all discussion questions, a summary/conclusion in your own words, and any suggestions you may have
• optionally, all notes and data you gather if you are able to visit a brewery or winery or look up related information online
• optionally, all notes and data you gather if you make bread at home
• any returned, graded pop quiz