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:

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

Humans have known about and utilized this fact, this process, for many thousands of years. CO2 liberated by yeast helps our 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. In this lab, we will be studying this 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).

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.


Acetobacter Can Turn Alcohol into Vinegar

Yeast Yeast with Acetobacter 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.

barley 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.

a plant 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.

hops 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 a 5-Gal Batch

Ingredients are listed for Dr. Fankhauser’s “original” recipe. Notes are included on some of the variations we have previously, successfully tried.


Procedure

So that we don’t waste supplies, one batch of this recipe will be made as a class. Everyone is encouraged to participate in helping to mix up the lab brew.

  1. Add 1 pkg (or 1 T) yeast to ½ C water at about 37° C (warm but not hot) with 1 tsp sugar. Let sit for about 15 min (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.
  2. 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.
  3. Into a separate, large pot, place approximately ½ gal. (= 8 C) of water, and begin to heat it.
  4. ***If unhopped malt is being used, heat this water, then put in the hops and let it steep to make “tea” (technically, this is called an infusion). You could, optionally, pour the hops tea/infusion through a strainer to attempt to remove some of the hops pieces before adding the warmed malt extract, but the pieces are so small, and settle out, anyway, as the brew ferments, so it probably isn’t worth the effort to try to strain them out.
  5. Measure the malt (1 C for the “original” recipe) and dissolve in the water. If sugar (sucrose, glucose/dextrose, etc. is being used, add the sugar (6 C for the “original” recipe) and stir to dissolve.
  6. Slowly heat the mixture to boiling but do not let it boil over. Stir slowly to prevent burning. Have hot pads ready to remove it from the heat.
  7. Add about 2 gal. cold tap water to the jug, then slowly add the hot sugar solution with a funnel, such that the solution goes straight down the funnel and directly into the cold water. ***Avoid running the hot solution down the sides of the jug or it may crack from the heat.
  8. After all the sugar solution has been added, then fill the jug to the “hip” (about 4½ gal. in the 5-gal. carboy) with cold water. This mixture, this sugar solution, is called the wort.
  9. 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.
    10. carboy with airlock
  10. Place an airlock on top of your bottle and add water to the airlock. 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 beer to spoil or turn to vinegar. Label the jug with the lab section, date, type of malt, (hops,) sugar, and yeast.
  11. After 1 to 2 days (next lab period?), the beer should have a good head on it (called the “high kreusenen” stage). 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. 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 all have their caps), add 2 tsp. sugar using a funnel. Carefully decant (carefully pour off the liquid or supernatant [super = over, above; natant = swimming], leaving the sediment on the bottom) 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. As it foams up, you may need to partially-fill several 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 among 10 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 those which appear to be a bit “lower” than the rest.
  13. 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. Note that if you bottle the beer 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!
  14. 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.
  15. 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 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.
  16. Again, rinse out the carboy as soon as possible, before the left-over yeast dries. The flavor improves with age. 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.

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.


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

As time and interest allow, you may wish to visit a local brewery or winery to see how they make beer or wine.


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 recipe is included here, but you may have another personal favorite that you like to use.

Supplies and Ingredients Needed

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.


popcorn In the past, a Biology lab tradition has been 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

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 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 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 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).


Other Things to Include in Your Notebook

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


Copyright © 2010 by J. Stein Carter. All rights reserved.
Based on printed protocol Copyright © 1982 D. B. Fankhauser
and © 1988 J. L. Stein Carter.
This page has been accessed Counter times since 18 Dec 2010.