TEACHER REFERENCE PAGES - YEAST FERMENTATION LAB
Introduction
Most organisms, including yeasts, use oxygen in a process called
cellular respiration.
Cellular respiration is the controlled
breakdown of carbohydrate to carbon dioxide and water with capture
of some of the energy in the form of ATP. The rest of the energy is
lost in the form of heat. The first stage of the breakdown is
called glycolysis and the second stage is called the Krebs Cycle.
During this process, electrons are transferred from the
carbohydrates to oxygen in the process called electron transport
and water is formed as the final product of electron transport.
Electron transport produces a chemosmotic gradient of protons (H+)
and positive charges across a membrane and this gradient can drive
the formation of ATP. Cellular respiration produces approximately
38 ATP molecules from each molecule of the sugar glucose that is
broken down.
The carbon that was in the carbohydrate is fully
oxidized to form CO2 during respiration. For glucose, the 6 carbons
become 6 CO2 molecules.
Table 1.
yeast.
Comparison of respiration and fermentation of glucose in
PROCESS
CONDITION
S
PRODUCTS FROM GLUCOSE
AMOUNT OF ATP
RESPIRATION
AEROBIC
6 CO2 + 6 H2
38
FERMENTATION
ANAEROBIC
2 CO2 + 2 C2H6O
2
Fermentation, a process that can occur in the absence of oxygen,
partially breaks down carbohydrate by glycolysis to capture a small
amount of energy in the form of ATP.
The initial reactions of
fermentation and respiration are the same, but fermentation stops
after glycolysis whereas respiration continues into the Krebs
Cycle. The carbohydrate leftovers are different depending upon the
organism that performs the fermentation; usually one product is
more oxidized (electron-poor) than the starting molecule and the
other is more reduced (electron-rich).
In the case of yeast
fermentation, the products from one glucose (C6H12O6) molecule are
two molecules of ethanol (C2H5OH) and two molecules of CO2. Human
anaerobic (oxygen-free) muscle produces two molecules of lactic
acid (C3H6O3).
Even though the products are different, each
fermentation results in a limited, anaerobic breakdown of
carbohydrate with energy release.
Since the process does not
completely break down the carbohydrate, it does not release much
energy that can be captured in the form of ATP.
In yeast
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fermentation, there are 2 ATP molecules produced for each glucose
molecule that is fermented. This is a low yield compared to that
of respiration, but the ability to perform fermentation allows the
yeast to survive and grow in environments where no oxygen exists
(see Table 1).
Gas Chromatography
The major technique that is used to determine the type of
organic
molecules
produced
during
fermentation
is
gas
chromatography.
Gas chromatography (GC) is the separation of
compounds in the gas phase, depending on their relative ability to
adsorb onto the column packing and their volatility into the gas
phase at the temperature used. The gas chromatograph is a simple,
sensitive instrument which can be used to separate and identify
about 60% of all known organic compounds.
The compounds to be separated are injected into a gas stream
which passes through a column at a preset speed. Under a constant
set of conditions in terms of temperature, gas flow rate, and
column packing and size, repeated injections of a compound elute
from (come out of) the column at a nearly constant time from
injection.
Different compounds elute at different times.
One
factor which affects elution time is the molecular weight of the
compound; heavier compounds move more slowly through the column.
Elution time is also affected by polarity and other factors. The
column is first injected with known compounds called standards, and
their retention times are determined. Then, unknown mixtures of
compounds can be injected, and if the known compounds are in the
mixture, their peaks can be recognized by their characteristic
retention times.
A gas chromatograph detects the presence of a compound in its
eluate (exiting stream) by means of some property of the compound.
One common method used by GC detectors is to compare the
conductivity of a heated filament which is placed within a stream
of pure reference gas (helium in our lab) to a heated filament
placed in a stream of gas containing our sample molecules. When
molecules from our sample pass the detector filament, the changes
in conductivity caused by temperature changes are converted into
electrical signals which appear as peaks on a computer data screen.
Peaks seen in the eluate are plotted on a chart, and the
integrated peak area is proportional to the concentration of the
compound. Many GCs report the integral area of each of the peaks,
following the plotted graph of the peaks.
An approximate
proportionality between peak height and concentration can also be
seen (see sample printouts).
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SAMPLE GAS CHROMATOGRAPH PRINTOUTS
Peak #1
Water / Peak #2 Methanol / Peak #3 Ethanol / Peak #4 Isopropanol
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Equipment
Gas chromatograph
Micro syringes (4) for standards, control, aerobic, anaerobic
Hot plates
Standards for GC: ethanol; mix of ethanol, methanol, and
propanol; distilled water
Supplies
GROWING
3
1
2
1 set
600mL
45 gm
1/2pkg
YEAST CULTURES: (per class)
Plastic 1 L Erlenmeyer flasks
Aquarium pumps (2 flasks per pump) & gang valve
3' lengths tygon tubing
Rubber stoppers: 1 2-hole/1 1-hole/1 solid and
bent glass tubing to connect.
DI Water
Dextrose
Active dry Yeast
DEMONSTRATION (to detect carbon dioxide):
bottle Bromothymol blue indicator solution
2
100 ml beakers
1
Drinking straw
DISTILLATION:
3
600 mL Beakers labeled A/B/C
10
100 mL Graduated Cylinders
10
125 mL Glass Erlenmeyers flasks
10sets distillation tubing and stopper
10
100 mL Glass beakers
10
Thermometers
20
3-finger clamps
10
Ring stands
10
Screw cap vials (labeled A, B, or C)
10
Hot plates
Paper towels
Ice
MASS OF YEAST CELLS:
2
Top loading balances
10
Centrifuge tubes
1
Centrifuge
10
Bamboo skewers to clean centrifuge tubes.
GAS CHROMATOGRAPHY:
3
25 µL syringes
1 set
alcohol standards
2
Gas Chromatographs
Computer paper
Helium gas
Extension cords
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Laboratory procedures
Before the laboratory, flasks of yeast culture should be
prepared. We have found that five days in advance yields the
best results (greatest difference between aerobic and anaerobic
conditions.) You should have one aerobic/anaerobic flask set per
class. We have 10 flasks which usually go out with the van. This
will support 4 classes if you use two flasks for controls. If the
teacher supplies flasks for the controls, these 10 flasks will
support 5 classes. Additional flasks and pumps may be available
if needed. Set up each one-liter flask as follows: 500 ml water
(300 ml tap water for minerals and 200 ml distilled or deionized
water) + 28 g dextrose (another name for glucose). Label the
flasks: A = Aerobic, B = Anaerobic, C = Control. Add NO YEAST to
the (C) control flasks. Add 1.5 g of active dry yeast to each of
the other flasks.
Table 2.
Flask A (Aerobic)
Flask B (Anaerobic)
Flask C (Control)
300 mL Tap Water
300 mL Tap Water
300 mL Tap Water
200 mL DI Water
200 mL DI Water
200 mL DI Water
28 g Dextrose
28 g Dextrose
28 g Dextrose
1.5 g Yeast
1.5 g Yeast
NO Yeast
Air pumped in
NO Air added
NO Air added
The flask labeled A (aerobic) is stoppered with a 2-hole
rubber stopper with a long aeration tube that becomes submerged
below the liquid in the flask and a short escape tube for the air
in the flask, which should be bent over or attached to a piece of
Tygon (aquarium) tubing to prevent contamination. This flask
will be vigorously aerated for 4-5 days. Be careful to provide
sufficient aeration to prevent the aerobic flask from fermenting.
Either a compressed air line or an aquarium pump can be attached
to the long aeration tube in the flask. We have found that each
aerobic flask should have a separate air pump, except for the
large dual port pump which can support two flasks.
Insert a
cotton plug into the long aeration tube before connecting the
pump. Aeration stones and agitation may help increase aeration
efficiency. See Figure 1 for a diagram of the aerobic flask.
The flask labeled B (anaerobic) is stoppered with a 1-hole
rubber stopper containing a short tube that is attached to a long
piece of Tygon tubing. The end of this tubing outside of the
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flask is placed under water in a 600 mL beaker. The yeast in
this flask will use up the oxygen inside the flask and will
ferment glucose to produce carbon dioxide. The CO2 will displace
the remaining air, which will bubble out through the water. This
flask will not contain oxygen after the first hour or two of the
experiment. See Figure 2 for a diagram of the anaerobic flask.
The flask labeled C (control) contains no yeast and no air
circulation is needed.
A color coded system has been developed to reduce confusion
when distributing yeast cultures. Aerobic is red, anaerobic is
blue, control is green. The color coded system is used on the
graduated cylnders, dispensing beakers (400ml-plastic),
distillation vials, and culturing flasks and tubing. Also it may
be desirable to color code syringes and GC used in analysis of
products.
NOTE: Please note that there are no student directions in the
lab for Part I (demonstration) or for Part III (mass of yeast
cells). This is because many teachers opted not to have students
do these parts individually.
PART I: Demonstration
The purpose of the following demonstration is to verify that
the gas being produced in both yeast and human respiration is
carbon dioxide. If a gas sample is mixed with bromothymol blue
indicator solution, the color of the solution will change from
blue to green and finally to yellow as the carbon dioxide
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produces carbonic acid and drops the pH of the solution.
After 24 hours, the production of CO2 can be demonstrated in
the anaerobic yeast culture as follows:
1.
Place 50 mL of Bromothymol blue solution into a 125 mL
Erlenmeyer flask.
2.
Place the exit tubing from the anaerobic culture flask
into the solution and observe the results.
3.
Using a straw, exhale into a second beaker with
bromothymol blue solution and again observe the results.
Several students may demonstrate this as time permits.
Caution the students not to blow so hard as to splatter
the solution out of the flask (bromothymol blue will
stain).
PART II: Distillation
The products of the respiration or fermentation should be
collected by distillation. For each culture, including the
control, transfer the contents of the flask to a labeled 600 mL
beaker for convenience in dispensing. Each lab group should
obtain 50 ml of one of the cultures, noting which one they have,
and place it into a 125 mL Erlenmeyer flask and assemble the
distillation apparatus (see Figure 1 in student handout).
Ethanol can be separated by distillation because it boils at
78°C, while water boils at 100°C. The hot plates for
distillation should be turned on and the flask should be placed
on the hot plate. Be sure the thermometer does not extend into
the liquid. You want to measure the temperature of the vapors as
the product distills. Use the thermometer to maintain the
temperature of the vapors at 80-90° C. The flask should boil
vigorously (boiling point of 95% ethanol azeotrope is 78.15°C).
Do not overheat. Maintaining the temperature at the lower end of
this range will give a slower distillation and better (i.e.purer) results. Wrap the glass tube with cool, wet paper towels
to help condense the alcohol. Students should not add the cool
wet paper towel to their distillation flask until the temp is
above 70°C so that the wet paper does not heat up excessively
before boiling of the culture begins. Be careful that students
do not let excess water from their paper towel drip into the
collection vial.
When distillate begins to collect in the vial, turn off the
hot plate and continue collecting several more drops. Students
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need to get only the first ten drops of distillate which should
be the most concentrated alcohol mixture. If students collect
several mL of fluid, chances are that they are collecting a lot
of excess water.
Now the distillate will be tested by means of gas
chromatography. Please refer to the Gas Chromatograph
instructions beginning on page 19. First, as a teacher
demonstration, inject about one microliter of the ethanol
standard into the gas chromatograph. Observe the time that it
takes for the peak to appear. This should be done on both gas
chromatographs. Then, a standard containing a mixture of water,
methanol, ethanol, and propanol will be injected. Observe the
time it takes for each of the four peaks to appear. When running
standards, run a few samples through GC before you use the
printouts for instruction. The first few samples may not give
clear results. Have a print out for each GC available and
labeled for student reference. Make sure that students use the
standards for the same machine that they will be using for the
analysis of their sample (A for aerobic, B for anaerobic, C can
be on either-your choice)
Have the students record the retention times and names of
the standards onto their data tables. Notice that the retention
times will vary slightly with each machine due to the condition
of its columm and rate of gas flow. Remind students that they
must run their samples through the same machine for which they
are recording standard retention times. For convenience, the
machines will be labeled A and B.
Have students make predictions (Question #4) before
injecting their sample in GC. Explain that even the aerobic
culture will have some alcohol production due to incomplete
aeration of the flask.
Next, have students inject about one microliter of their
distillate in turn into the gas chromatograph. This will take 810 minutes per group. Have students record their culture code
letter (A, B, or C), the machine code number (A or B), and then
identify the peaks by name and the percentage yield of each
compound in their sample (see pages 7-8). This information will
be written on their computer printout first, and then transfered
to their data table. The students will need to refer to the data
from the standards to identify the components of their sample.
Doing examples on paper prior to the lab day will greatly
increase the students' understanding of these procedures. They
should see two peaks, one for water and one for ethanol. Any
other peaks are contaminants and can be ignored for this lab.
Have students average results from all the student samples
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before making conclusions about the results. Sloppy technique in
distillation will produce results which do not follow the theory
of the lab.
Note: In case no gas chromatograph is available, 2.5 ml of
each distillate can be mixed with 1 ml of concentrated I2/KI and
1.5 ml of 1.5 M NaOH. If ethanol is present, an iodoform
reaction occurs producing a yellow precipitate in about 10
minutes.
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SAMPLE AEROBIC CULTURE GAS CHROMATOGRAPH RESULTS
Yeast culture A
Gas Chromatograph B
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SAMPLE ANAEROBIC CULTURE GAS CHROMATOGRAPH RESULTS
Yeast Culture B
Gas Chromatograph B
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PART III: Mass of yeast cells
(Optional, if time permits. Can be done while waiting for
distillation to occur or for opportunity to inject sample) The
purpose of this experiment is to see how much growth was possible
for the aerobic versus the anaerobic yeast culture.
For this experiment, have students label and mass a centrifuge
tube on the top loading balance to .01 gms. Then have students
tranfer aproximately 10 ml of a yeast culture to their tube from
the culture remaining in the beakers from Part II. This volume
will fill up the tube to its white label.
Be sure that the
students stir up the culture in the beaker before transferring it
to their tubes, so as to get the yeast cells mixed evenly. This is
important if you want to get consistent results.
Have students centrifuge their sample for 5 minutes. Have
them pour off the supernatant (the liquid portion) into the sink.
The mass of the remaining pellet in the bottom of the centrifuge
tube can be estimated by massing the tube again on the top loading
balance. (If necessary, try to dry the inside of the tube with a
kimwipe before massing it). The tube mass should be subtracted
from the combined mass of the (tube + pellet) to get the wet mass
of the cells. This is a crude approximation but if means of the
masses for the class are compared, there should be a difference
between aerobic and anaerobic cultures. Aerobic cultures should
have larger masses due to increased energy utilization and
reproduction.
When performing this portion of the lab it may be better to
change the sequence of weighing the tubes to help you with clean
up. Have the students weigh their tubes with a yeast cell pellet
first and then again after they have rinsed out the pellet (using a
bamboo skewer or a bent paper clip helps remove the cells from the
tip of the centrifuge tube). The water which is inside the tube can
be ignored because it is present at both weighings. This is a crude
technique, but if you average all the data from the entire set of
classes or completely centrifuge the contents of the distribution
beakers (400ml plastic) you should find a difference in the weight
of the yeast cells between the two cultures. Again making
conclusions from a single sample does not yield good conclusions.
References
Warren D. Dolphin, Biology Laboratory Manual, Third Edition. Wm C
Brown Publisher, 1992.
Campbell, Neil. Biology, Second Edition. Benjamin Cummings, Menlo
Park, 1991.
Pietrzyk, D and Frank, C. Analytical Chemistry, An Introduction.
Academic Press, New York and London, 1974.
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