1. Energy metabolism in yeast

2. What is cellular respiration ?
Cellular respiration: is the set of the metabolic reactions and processes that take place in the cells of organisms to convert biochemical energy from nutrients into (ATP).
The reactions involved in respiration are catabolic reactions, which break large molecules into smaller ones, releasing energy.

3. Yeast (Saccharomyces cerevisiae)
Yeast: is a living organism that requires a warm, moist environment and a food source to grow and thrive.
Unicellular, facultative anaerobic fungi.

4. Under Microscope

5. Cellular respiration
In this lab, you will be using yeast to measure the rate of cellular respiration.
Yeast is a single-celled eukaryotic organism that utilizes carbohydrates for ATP production in the same way that plant and animal cells utilize carbohydrates.
Yeast will convert a carbohydrate into water and carbon dioxide during cellular respiration.
The greater the carbon dioxide production, the greater the rates of cellular respiration.

6. What are the Stages of Cellular Respiration?
STEP 1 – Glycolysis.
STEP 2 - The Krebs Cycle.
STEP 3 - The Electron Transport Chain (ETC).

7. STEP 1: Glycolysis
Glycolysis is takes place in the cytosol of cells in all living organisms.
This pathway can function with or without the presence of oxygen.
Process converts one molecule of glucose into two molecules of pyruvate (pyruvic acid), generating two net molecules of ATP.
Four molecules of ATP per glucose are actually produced. however, two are consumed as part of the preparatory phase.
The overall reaction can be expressed this way:
Glucose + 2 NAD+ + 2 Pi + 2 ADP pyruvate + 2 NADH + 2 ATP + 2 H+ + 2 H2O + heat

8. Step 2: The Fate of PYRUVATE
1. As pyruvate enters the mitochondrion, it is converted to acetyl CoA which enters the Krebs cycle in the matrix.
2. A carboxyl group is removed as CO2.
3. A pair of electrons is transferred from the NAD+ to form NADH
When oxygen is present, the mitochondria will undergo aerobic respiration which leads to the Krebs cycle.
However, if oxygen is not present, fermentation of the pyruvate molecule will occur.

9. In the presence of oxygen, acetyl-Co A is produced, then enters the citric acid cycle (Krebs cycle) inside the mitochondrial matrix, and gets oxidized to CO2.
While at the same time reducing NAD to NADH.
NADH can be used by the electron transport chain to create further ATP as part of oxidative phosphorylation.
The citric acid cycle is an 8-step process involving different enzymes and co-enzymes.

10. 2 molecules of pyruvate produce 2 NADH

11. Step 3: KREBS CYCLE or TCA
This is also called the citric acid or Tri-carboxylic acid cycle (TCA).
Takes place in matrix of mitochondria.
Requires Oxygen (Aerobic).
Occurs two times per one glucose molecule.
Each cycle produces 1 ATP, 3 NADH and 1 FADH.

12. Copyright Cmassengale

13. Substrate-Level Phosphorylation Oxidative Phosphorylation
Calculation of energy
Pathway
Substrate-Level Phosphorylation
Oxidative Phosphorylation
Total ATP
Glycolysis
2 ATP
2 NADH = ATP
6-8
CoA
2 NADH = 6 ATP 6
Citric Acid Cycle
6 NADH = 18 ATP 2 FADH2 = 4 ATP 24
TOTAL
4 ATP
32 ATP

14. Electron Transport
The mitochondria has two membranes: the outer one and the inner membrane.
The H+ which are brought to mitochondria accumulate between these two membranes.

15. The electrons move from molecule to molecule until they combine with oxygen and hydrogen ions to form water.
As they are passed along the chain, the energy carried by these electrons is stored in the mitochondrion in a form that can be used to synthesize ATP.
Electrons carried by NADH are transferred to the first molecule in the electron transport chain.
The electrons continue along the chain that includes several cytochrome proteins and one lipid carrier.
The electrons carried by FADH2 added to a later point in the chain.
Electrons from NADH or FADH2 ultimately pass to oxygen.
The electron transport chain generates no ATP directly.

17. Fermentation Anaerobic respiration.
In yeast called alcoholic fermentation.
The rate of fermentation can be affected by several factors:
Concentration of yeast.
Concentration of glucose.
Temperature.

18. For the yeast cell, this chemical reaction is necessary to produce the energy for life.
The alcohol and the carbon dioxide are waste products produced by the yeast.
The chemical reaction, known as fermentation can be watched and measured by the amount of carbon dioxide gas that is produced from the break down of glucose.
Yeast (Saccharomyces cerevisiae) can live in both aerobic as well as anaerobic conditions.
In the presence of oxygen, yeast can undergo aerobic respiration, where glucose is broken to CO2 and ATP is produced by protons falling down their gradient to an ATPase.

19. When oxygen is lacking, yeast only get their energy from glycolysis and the sugar is converted into ethanol, a less efficient process than aerobic respiration.
The main source of carbon and energy is glucose, and when glucose concentrations are high enough, gene expression of enzymes used in respiration are expressed and fermentation takes over respiration.
However, yeast can also use other sugars as a carbon source. Sucrose can be converted into glucose and fructose by using an enzyme called invertase, and maltose can be converted into two molecules of glucose by using the enzyme mannase.

21. Reagents and instrument
Respirometer: is a device used to measure the rate of respiration of a living organism by measuring the rate of exchange of oxygen and/or carbon dioxide.
Glucose solution.
Yeast solution.
Water.

22. Procedure
Obtain 5 test tubes and label them, then add solutions as follows to the appropriate tubes:
Glucose solution (ml)
Yeast suspension (ml)
Water (ml)
Tube 3 4 1 2 5

23. Attach a piece of aquarium tubing to the end of each 1 ml graduated pipette.
Then place the pipette with attached tubing into each test tube containing fermentation solutions.
Attach the pipette pump to the free end of the tubing on the first pipette.
Use the pipette pump to draw the fermentation solution up into the pipette.
Fill it past the calibrated portion of the tube but do not draw the solution into the tubing.
Fold the tubing over and clamp it shut with the binder clip so the solution does not run out.
Open the clip slightly and allow the solution to drain down to the 0 ml calibration line or slightly bellow.

24. Record your initial reading for each pipette(initial time).
Quickly do the same for the other four pipette.
Record your initial reading for each pipette(initial time).
2 mints after the initial readings for each pipette record the actual readings A in ml for each pipette in the actual A column.
Subtract I form A to determine the total amount of CO2 evolved A – I
Record this value in the CO2 evolved A-I column
From now on you will subtract the initial reading from each actual reading to determine the total amount of CO2 evolved
Continue taking readings every 2 minutes for each of the solutions for 20 minutes
Remember take the actual reading from the pipette and subtract the initial reading to get the total amount of CO2 evolved in each test tube.

25. Go To Work