RESPIRATION AND

FERMENTATION: AEROBIC

AND ANAEROBIC OXIDATION

OF ORGANIC MOLECULES

Bio 171 – Week 6

Procedure

Label test tubes well, including group name

1) Add solutions listed to small test tubes

2) For 1-6, fill remaining volume with yeast suspension; for 7 fill remaining volume with water.

3) Slide larger test tube over the smaller tubes; hold smaller tube against the bottom of the larger tube and invert. There should be no air trapped at the top of the tube. It may help to practice this with water.

4) Label test tubes well, including group name

5) Incubate tubes at 37˚C for 40 minutes.

6) After 40 minutes, measure the height (in millimeters) of the bubble of accumulated CO2. Record results in table 12.1

Procedure

Wear gloves – NaF is corrosive and toxic

Shake yeast bottles before using

Absolutely NO eating in lab

Clean benches immediately when done

Rinse test tubes well and put upside-down in rack

near sink.

Cellular Respiration

Cellular Respiration: oxidation of organic molecules

into energy in the form of ATP

ATP = Adenosine Triphosphate: organic molecule

containing high-energy phosphate bonds

Cellular Respiration - Summary

C6H

12O

6 + 6O

2 6CO

2 + 6H

20 + e- + 36-38ATP

Glucose is oxidized [removes electrons], O2 is reduced (oxidation-reduction reaction or REDOX)

Remember OIL RIG – Oxidized Is Loss (of electrons) and Reduction Is Gain)

Remember that adding/removing a hydrogen is a way of adding/removing an electron

One glucose yields 36-38 ATP

Electrons (as H) moved by coenzymes NAD+ and NADH2

Steps of Cellular Respiration

Glycolysis

Prep Reactions

Krebs Cycle

Electron Transport

Chain

Respiration: Glycolysis

Energy Investment Step

Two ATP used to split glucose into two 3-carbon molecules

Energy-Harvesting Step

1) 3-carbon molecules oxidized by NAD+, resulting in two NADHs

2) Phosphate group added to each.

3) Substrate-level ATP synthesis or substrate-level phosphorylation: enzyme passes high-energy phosphate to ADP, and ATP results (adenosine diphosphate triphosphate (+2 ATP, 3PG (3-phosphoglycerate))

4) 3PG is oxidized by the removal of water (+2H2O, 2 PEP)

5) Substrate-level ATP synthesis again. (+2 more ATP, 2 pyruvate)

NET GAIN: 2 ATP, (because we used two in energy investment) + 2 pyruvate.

Respiration: Glycolysis

Glycolysis: “Sugar-splitting” or “Energy investing” step

Occurs in cytoplasm

Requires 2ATP

Glucose split into 2 Pyruvate

Respiration: First Set of Reactions

Glycolysis: “Sugar-splitting” or “Energy investing” step

Occurs in cytoplasm

Requires 2ATP

Glucose split into 2 Pyruvate

Final Products:

2 NET ATP (4 produced, but 2 were used)

2 NADH

2 Pyruvate

If Oxygen is Present…

Prep Reactions

Pyruvate oxidation into acetyl-CoA

One NADH produced

Citric Acid Cycle

Occurs in matrix of mitochondria

Acetyl-CoA oxidized into two CO2

Produces 1 ATP per turn

Store energy in electron carries such as NAD+ and FAD+

Electron Transport Chain

Electrons from NADH and FADH2 move through a series of proteins called the ETC

Potential energy released during these redox reactions creates proton gradient across a membrane; flow of protons across the membrane generates ATP

Anaerobes – organisms that live without oxygen

Some use nitrate, sulfate or other inorganic compounds

as electron acceptors instead of oxygen.

Some use glycolysis reduce the pyruvate

If No Oxygen is Present…

NADH reduces Pyruvate

C6H12O6 2CO2 + 2C2H5OH + ATP

C6H12O6 2CH3CHOHOCOOH + ATP

Occurs in anaerobic organisms (anaerobes)

Occurs temporarily in plants and animals

Roots in anaerobic soils

In muscles for rapid bursts of energy

Glucose

Glycolysis

Pyruvate

Animals, some

microbes

Plants, some

microbes

CO2

Lactate Ethanol

NADH

NAD+

Advantages/Disadvantages?

Disadvantages:

Less ATP produced (2 VS 36/38 in aerobic respiration)

Produces toxins (lactic acid or ethanol)

Advantages:

Can produce ATP without oxygen

Byproducts used in many foods (economic value)

Back to the lab

Pyruvate: product of glycolysis; reduced to ethanol or

lactic acid during anaerobic fermentation

Magnesium Sulfate (MgSO4): Provides Mg2+ , a

cofactor that activates some enzymes of glycolysis

Sodium fluoride (NaF) – an inhibitor of some enzymes

of glycolysis – inhibits phosphorylation

Glucose – a common organic molecule used as an

energy source for respiration

Lab Results

Table 12.1: Experimental Treatments of CO2 Production During Anaerobic

Fermentation Tube 3M Na

Pyruvate

(Activator)

0.1 M

MgSO4

(Activator)

0.1 M

NaF

(Inhibitor)

5.0%

Glucose

(Activator)

Water Fill With CO2 Produced

After 40 Min (mm)

1 - - - - 7.5 mL Yeast Suspension 0

2 - - - 2.5 mL 5.0 mL Yeast Suspension 25

3 - 5.0 mL - 2.5 mL - Yeast Suspension 30

4 - - 0.5 mL 2.5 mL 4.5 mL Yeast Suspension 10

5 - - 5.0 mL 2.5 mL - Yeast Suspension 5

6 2.5 mL - 2.5 mL 2.5 mL - Yeast Suspension 40

7 - - - 2.5 mL 2.5 mL Water

Predictions

Tube 1: Water and yeast suspension.

What purpose does this tube serve?

Tube 2: Glucose (activator), Water, Yeast suspension

What process does glucose activate?

Tube 3: MgSO4, Glucose, and Yeast suspension.

Tube 4: NaF (inhibitor), Glucose, Water, Yeast suspension.

What happened to CO2 production here?

Tube 5: NaF (inhibitor), Glucose, Yeast suspension

This tube had 10x as much NaF as Tube 5

Tube 6: Pyruvate, NaF (inhibitor), Glucose, Yeast suspension

What happened to CO2 production here?

What process do you think NaF inhibits?

Tube 7: Glucose, water.

What is the purpose of this tube?