Yeast Fermentation Lab

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Yeast Fermentation
incorporating many possible variables
(temperature, inhibitors, substrates, osmolarity, etc.)

Devised by the Biology 101 class
Paul D. Camp Community College
Smithfield, Virginia USA
(Fall 2009)

    Respiration* consists of the linkage of three major metabolic pathways - glycolysis and the combination of the Krebs Cycle and ETOP (electron transport/oxidative phosphorylation) - all shown here in the entirety:

    Unfortunately the whole of respiration is run by complicated higher organisms, which do not lend themselves to peeking inside the process to see how it works. A classic example of this is the respiration exhibited by germinating pea seeds. The substrates cannot be manipulated in either kind or quantity because the starch substrate is already in place in the seeds; interference by toxins cannot be studied because the seed is a self-contained and nearly impenetrable entity. The only parameter that can be changed is the temperature. Perhaps we might do better by separating these various pathways, "divide and conquer," and study only one.

    Fermentation,** which is glycolysis plus a couple of closing steps (the yellow portion of the above figure) is carried out not only by most higher organisms but also by microorganisms such as ordinary bakers yeast. These single cells, unlike pea seeds, can be stirred into making suspensions of different concentration to be handled as if they were simple reagents. What is more is that since yeast don't not store up significant amounts of substrate, we can alter the amounts and kinds of substrates, or of toxins, or of temperature to see what impact those might have on the ultimate fermentation reaction products - ethanol and most significantly because it is easily measured - carbon dioxide, CO2, by volume or merely by counting the rate at which bubbles are produced.

    One last important item to note is that yeast dislike using oxygen, and thus even in the presence of air will ferment.*

Basic Materials per group per run

Preparing Your Yeast Culture

  1. Make a liter of room temperature tap-water. This is to be used in all instances.
    1. Take a thermometer from your drawer without touching the bulb. Read its temperature. That's room temperature.
    2. To cold tap water stir in enough hot water to bring it up to room temperature.
  2. Measure out 150 mL of the water and pour it into the 250 mL beaker
  3. Weigh out 30 gm of the dried yeast and add it to the beaker and swirl until suspension is homogeneous.
  4. This is now your "source culture"

Preparing Your Equipment

  1. Assemble the flexible tubing to the copper tube and insert the copper tube through the stopper (use a water-soluble libricant such as lanolin or a KY product!). Insert the stopper in the flask.
  2. Place the open end of the flexible tubing into the half-full beaker of water so that bubbles can be counted if and when they are produced.
  3. Cautions:
    1. You should count the number of bubbles in each of at least three separate minutes. The reaction might be speeding up, and, if so, the later minutes will have higher counts. Record the average of the higher counts.
    2. Wait at least three minutes before beginning to count the first minute as CO2 is water soluble and must saturate the solution before reliable counts can be made.

Preparing Your CONTROL

  1. Pour 225 mL water into the 500 mL flask
  2. Add 10 gm sucrose; swirl to dissolve completely
  3. Add 25 mL of the "source culture"; swirl to achieve homogeneity
  4. Insert the stopper and place the plastic tubing down into the bubble beaker.
  5. CO2 production will achieve a steady rate in about 5 minutes, at which time you will start taking your three 1-minute readings.

Experimental Runs
(For each set, make a graph! Bubbles/min [vertical]/dose [horizontal])

  1. EVERY group does a CONTROL run plus their assignment (one of A through F).

  2. Variable Substrate Concentration: GROUP 1
    1. CONTROL: 10 grams of sucrose is dissolved into the reaction chamber and the run is made.
    2. 5 grams of sucrose
    3. 15 grams of sucrose
    4. 20 grams of sucrose

  3. Variable Yeast GROUP 2
    1. Make three runs using 12.5 mL, 25 mL (CONTROL) and 50 mL of "source culture", respectively.

  4. Variable Substrate Type GROUP 3
    1. CONTROL: 10 grams of sucrose
    2. 10 grams of glucose (dextrose)
    3. 10 grams of galactose
    4. 10 grams of ["pink"] Sweet'n'Low

  5. Variable Inhibitors GROUP 4
    Do only two of below: the ethanols or two of the others.
    1. CONTROL: NO inhibitor added
    2. 7.5 mL of ethanol
    3. 15 mL of ethanol
    4. 25 mL of ethanol
    5. "smidgeon" of penicillin
    6. "smidgeon" lead nitrate
    7. 10 mL of clorox

  6. Variable Temperature GROUP 5 (Hardest!)
    (Suggested tricks: [1] Before you add the yeast to your flask, heat the 225 mL of water to about 3°c above your final desired temperature. Add your sucrose, and then your yeast. Meanwhile a partner is making a waterbath to the desired temperature. Put the flask in the waterbath; make your "run"; at the end, measure the temperature INSIDE the flask. This procedure will speed thermal equilibration. [2] While shaking the flask, do so with as little hand-contact as possible - perhaps by holding the flask up by the stopper. Remember you have warm hands and they can heat the flask.)
    1. Figure out the temperature regulating devices you'll employ
    2. Run temperatures of approximately 15°c, 25°c and 35°c (record the actual temperatures used - measure the liquid INSIDE of the flask!)
    3. Caution: before plugging in the stopper allow the flask to become equilibrated to the temperature you want OR ELSE you will be measuring the expansion of a gas (Boyle's Law) that is heating up.
    4. Upon graphing your results using the true temperatures, draw a best-fit line and extrapolate/interpolate what the temperatures would be at 15, 25, 35°c.

  7. Variable pH GROUP 6
    1. Measure the pH of the CONTROL
    2. Make runs of pH's of approximately 2-pH units below and above that of the control (record the actual ones used by testing with pH paper.)
    3. To do: make up CONTROL flask, add 10 mL of an appropriate buffer to it; test with pH paper; add more buffer if needed.
    4. Make your "run"

R E S U L T S ***

  1. Each group is to report their work including drawing a graph of their results on the board.
  2. Each person in every group is responsible for copying down the results of the other groups.

Author List: Giovanni Albino, Kenton Andrews, Adrian L. Daughtrey, Courtney Moody, Kecia L. Taliaferro, C. Michael Hall, Lauren A. Hogge, Kirk D. Smith, Tiffany W. Robertson, Elizabeth M. Moore, JonAlan Williams, Cindy C. Jones, Halie C. Repka, Varia F. McCloud, Jenny Nelson, Lashena Spratley, and Shaina D. James

* Respiration: "aerobic" metabolism - one that makes use of atmospheric oxygen, O2. Usually only CO2 is produced.
E.g.: 1 sucrose → 12 CO2

** Fermentation: anaerobic metabolism without the use of atmospheric oxygen. Usually alcohols, lactic acid or other acids are formed. Some fermenters, such as yeast, produce some CO2.
E.g.: yeast: 1 sucrose → 6 ethanol + 6 CO2

*** Results from two lab sections