Aerobic Respiration

 

Quick Introduction

 

Aerobic respiration is the metabolic process that provides usable energy for most cells (including plants) by breaking the chemical bonds in glucose (or some other substrate). The summary equation is:

 

glucose (C6H12O6) + oxygen (O2) à carbon dioxide (CO2) + water (H2O) + energy

 

Note that in this process, glucose is oxidized to carbon dioxide (and electrons and hydrogen lost) and oxygen is reduced to water (by gaining those electrons and hydrogen). Some of the energy released is lost as heat, but (fortunately) some of it is also conserved to power the formation of the high-energy molecule, ATP.

 

We will be studying aerobic respiration in yeasts, single-celled fungi classified in the species, Saccharomyces cerevisiae. Yeasts are good organisms to investigate because they respond quickly to changes in the nutrient composition of their environment and are small enough to easily fit in a reaction cuvette.

 

Remember from previous labs that chemical processes may be investigated in closed systems (cuvettes) by measuring the rate of change in the amounts of key products and/or reactants. Thus, respiration rates can be studied by measuring the decrease of glucose or oxygen or the formation of carbon dioxide. Today, we are going to measure the formation of carbon dioxide using a CO2 sensor interfaced to our computers.

 

What to do:

 

There are two RQ’s to be considered in today’s lab:

 

1)      Is glucose required for aerobic respiration?

2)      Does respiration rate change with yeast concentration?

 

(Half the class (groups NS1, NS2, NS3) will work on RQ 1 and the other half (SS1, SS2, SS3) on RQ 2.)

 

Pose a hypothesis from this RQ and design an experiment to test your hypothesis.

 

For RQ1, use the following concentrations of glucose: 0, 20, 40, 60, 80 and 100mM

For RQ2, use the following concentrations of yeast: 0, 10, 20, 30, 40 and 50 g/L

 

Answer the following questions about your experiment: What are the controls? What are the experimental groups? Independent variable? Dependent variable? Replications? Remember to construct an appropriate data collection table in your lab notebook and check with the instructor before you proceed.

 

Tools:

 

CO2 probe set up

  1. Make sure the CO2 probe is connected to the LabPro interface and the computer.
  2. Make sure the tip of the probe is in air before you continue. Note: this probe will be ruined by immersion in liquid. Make sure that it never comes in contact with the yeast solution.
  3. On the right hand of the screen, click on the “Logger Pro” icon.
  4. Check the boxed display. It should read approximately 400-500 ppm. If it is not, try to bring the reading down using the portable fan at your lab table. Remember that you exhale a lot of CO2. If you breathe directly on the sensor, it will be difficult to get the probe to an operating condition. If all else fails, your instructor can do a re-calibration of the probe.
  5. You are now ready to begin readings. In subsequent experiments, make sure the starting CO2 level is the same.

Setup for Measuring Yeast Respiration

1.      Place a small stir bar in the reaction cuvette (125 ml flask).

2.      Position the flask in the center of your stir-plate.

3.      To the flask, add 25 ml of yeast stock solution (50 g/L) and 25 ml of glucose (500 mM). Remember that you will use varying amounts of yeast and/or glucose in your subsequent experiments, but make sure that the final volume is always kept at 50 ml. Also, if you are doing experiments modifying the amount of glucose, dilute your glucose stock with a solution of mannitol (a non-reactive sugar) to maintain similar osmolarity in each of your experimental groups.

4.      Start the stir-bar so that you can see visible movement of the solution.

5.      Wait 5 minutes and then transfer the yeast to a new 125 ml flask with stir bar.

6.      Using the rubber stopper assembly, insert the CO2 sensor in the flask.

  1. Click on "collect" on the computer. It has been preset to collect CO2 data for 300 seconds (at 1 sample every 2 seconds). The computer will plot the change in CO2 concentration per time on the screen as you watch.
  2. Once the data gathering is complete, hold down the left hand button on the mouse and select the interval on the line representing a linear change in CO2 concentration. The interval should be for at least 60 seconds. The selected region should be highlighted. Select “analyze” on the menu across the top of the screen and then chose “linear fit”. A box should appear on the graph with the equation of the line and the correlation coefficient (goodness of fit for the data to the line). Record the slope in your data collection table. What are the units?

How to save data after a run:

  1. Select "experiment" on the menu across the top of the screen and then chose "store latest run".
  2. If you want to hide the data from a run, select “data” from the top menu and chose "hide data set".  NOTE: Always choose “run 1” (or whatever number is appropriate) on the menu that appears. DO NOT CHOOSE “LATEST RUN”. If you do, on the next assay, this will cause the program to lock up and give the message, “waiting for data”. (If you make this mistake anyway, go to the “data” menu and select “show data set” and the problem will be resolved.)

 

Assignment:

1.      Sketch the final figure from today’s results on the board

2.      Create a computer generated figure of your data using Excel:

http://homepage.mac.com/haleyub/Students/PhotoAlbum31.html

 

1.      What was the relationship between glucose or yeast concentration and CO2 evolution in your experiment? Was it linear? Nonlinear? Explain.

2.      Did the results support your hypothesis?