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
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
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,
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.
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.
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.
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
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.
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
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: