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?