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Respiration Experiments

Respiration is the enzymatic-controlled release of energy from organic compounds in a living cell.

Aerobic Respiration: the enzymatic-controlled release of energy from organic compounds in a living cell using oxygen.

Anaerobic Respiration: the enzymatic-controlled release of energy from organic compounds in a living cell without using oxygen.

The energy released by respiration is of very little value unless it is used to produce ATP.


ATP

Adenosine triphosphate (ATP) is the most abundant short-term energy store and immediate source of energy for cell work.

ATP: adenine + ribose sugar + three phosphate groups. Adenine + Ribose is called Adenosine.
ATP = A + P + P + P A: adenosine        P: phosphate

ATP releases energy when the last phosphate is removed.
A + P + P + P ATP
»»
A + P + P + energy + P (free) ADP + energy + P (free)
ADP: adenosine diphosphate

ATP can be remade by the addition of a phosphate onto ADP i.e. by the phosphorylation of ADP.
The phosphorylation of ADP requires energy. Respiration is one source of energy to produce ATP.

Living cells use up ATP at a very fast rate - a human cell needs about 2 million a second.
In order to maintain constant energy, a supply of ATP must be replaced as it is used.



Note: Light is the energy source to make ATP in the light phase of photosynthesis.
ATP is made during cyclic and non-cyclic photophosphorylation.
The ATP from the light phase is used to drive the dark phase reactions in the production of glucose.

Aerobic Respiration: Glucose + Oxygen Carbon Dioxide + Water + Energy (38 ATP)
C6H12O6 + 6O2
»»
6CO2 + H2O + Energy (38 ATP)
Anaerobic Respiration:
 
Plants and Fungi   Glucose »» Ethanol + Carbon Dioxide + Energy (2 ATP)
Animals and some Bacteria    Glucose »» Lactic Acid + Energy (2 ATP)
  (Lactic acid is a colourless liquid miscible with water.)

In anaerobic respiration the glucose is only partially broken down.
A lot of energy is still available in ethanol and lactic acid.

Note: ethanol is one member of the family of chemicals called the alcohols; ethanol is the alcohol of beer, wine and spirits.

Anaerobic respiration is also known as fermentation.

Aerobe: an organism lives and grows only in the presence of free oxygen; it respires aerobically.

Anaerobe: an organism that can live and grow in the absence of free oxygen, it can respire without free oxygen.
Obligate Anaerobe: an organism that is not capable of aerobic respiration (free oxygen is toxic to some of these).
Facultative Anaerobe: an organism that is usually respires aerobically but can survive by anaerobic respiration in the absence or shortage of free oxygen.


Proofs for Respiration

  1. Production of carbon dioxide.
  2. Consumption of oxygen.
  3. Production of heat.

Experiment (a): To Show That Carbon Dioxide is Given Off During Respiration

  1. Use a vacuum pump to draw air through the apparatus.

  2. Experimental Procedure.
    1. Bottle 1. Solution of sodium hydroxide to remove carbon dioxide from the air.
    2. Bottle 2. Limewater solution - to test air for CO2. Stays clear - CO2 completely removed from air.
    3. Bell jar with small live animals.
    4. Bottle 3. Limewater solution - to test air for CO2. Turns milky - CO2 is in the air.

  3. Precaution: use a sterilising solution to kill any micro-organisms on the inside of the bell jar so that the animals are the only live organisms and they alone are responsible for the production of CO2.
  4. Control.
    Same set-up but living organisms are not present in the bell jar.
    Both limewater bottles stay clear - no carbon dioxide in the air emerging from the bell jar.

    Conclusion:
    CO2 is only produced when living organisms are present.
    Therefore, the living organisms produced the CO2.
    Therefore the living organisms respired.

Modification of the experiment, checking a plant for respiration.
In normal light photosynthesis is faster than respiration.
Therefore all the CO2 produced by respiration will be used for photosynthesis - the plant will not release CO2.
Photosynthesis has to be stopped without damaging the plant - so keep the plant in darkness.

  1. Cover the bell jar with a light-proof material - plant cannot photosynthesise without light.
  2. Cover the plant pot with an air-tight plastic bag to prevent CO2 from the respiration of soil organisms escaping into the air of the bell jar.

Experiment (b): To Show Aerobic Respiration By Living Organisms - demonstrate the consumption of O2

  1. Experimental Procedure:
    1. Test tube with soda lime - soda lime will remove CO2 from the air.
    2. Place a small animal, e.g., a beetle, in the test tube with a perforated partition between it and the soda lime.
    3. Seal the test tube with a one-hole stopper with a right angle glass tube leading into a beaker of coloured water.
    4. Keep the temperature constant at room temperature, 20°C - thermostatically regulated heating system.

  2. Precaution: use a sterilising solution to kill any micro-organisms on the inside of the glassware - so that the animal is the only live organism and it alone is responsible for the consumption of O2.
  3. Control Procedure
    Same set-up but there is no animal in the test tube.

    Results:
    Control: no change in the level of water in the glass tube - same level as the water in the beaker.
    Experiment: water has risen up the tube towards the test tube containing the animal, the air remaining in the test tube has reduced by 20% and does not allow anything to burn in it.
    Therefore the air has lost oxygen.
    Oxygen was only removed from the air when the live animal was present.
    The animal is responsible for the loss of oxygen from the air - therefore the animal respired aerobically.

Why is the temperature kept constant ?
Gases expand when heated and contract when cooled. Volume changes due to temperature changes would upset the true results of the experiment. Any volume change must only be due to the animal.

But CO2 is produced and excreted into the air as fast as O2 is used up and extracted from the air.
So how is the volume of the air reduced -
The soda lime absorbs the CO2 from the air.
The mass of the air reduces because it is losing O2 to the animal.
This reduces the air pressure in the test tube.
There is now a pressure difference - higher air pressure outside.
The liquid in the beaker is pushed up the tube towards the animal. This keeps the air pressure in the test tube constant at
atmospheric pressure.

Plant Usage Of O2
The experiment above depends on the uptake of oxygen from the air.
The air must be the sole source of oxygen for the plant.
Photosynthesis produces oxygen. Plants use oxygen from their photosynthesis for respiration.
To remove this source of oxygen the plant must be kept in darkness - no light, no photosynthesis.
Alternatively, non-green germinating seeds can be used.

Experiment (c): To Show That Respiring Organisms Produce Heat Energy

  1. Soak pea seeds in water overnight.
    With oxygen and a suitable environment these seeds will germinate.
  2. Kill half of the seeds by placing them in boiling water.
  3. Sterilise the surface of the seeds with a sterilising solution to kill all surface micro-organisms.
  4. Wash the insides of two vacuum flasks with a sterilising solution to kill all micro-organisms.
  5. Place the live seeds in one flask (experiment) and the dead seeds in another (control).
  6. Insert a thermometer into each flask, record the temperature and seal the flasks with cotton wool.
  7. Record the temperature daily over the next five days.

    Results:
    Control: no temperature change.
    Experiment: steady rise in temperature each day.
    Conclusion: only the living seeds caused a temperature rise, therefore respiring organisms produces heat.

Experiment (d) To Demonstrate Anaerobic Respiration by Living Organisms

  1. Boil water for 15 minutes to remove all the dissolved oxygen.
  2. Almost fill two flasks with the water, allow them to cool to 25°C in the sealed flasks - sealed to prevent re-oxygenation.
  3. Dissolve glucose in each flask.
  4. Add live yeast to one - the experiment. No yeast in the other - the control.
  5. Place a thin layer of oil on the top of the water in each - the water remains deoxygenated by preventing contact with air.
  6. Insert a thin glass tube from each stoppered flask into a test tube of limewater.
  7. Maintain the temperature at 25°C in a water bath or heating tray.

    Results:
    Control: no change in flask or limewater.
    Experiment: bubbles of colourless gas escape from the solution and pass through the limewater, which goes milky. The gas is therefore CO2.
    Conclusion: the yeast produced CO2 in the absence of oxygen and, therefore, the yeast respired anaerobically.


Links

Another look at the biochemistry of respiration.
http://www.lcusd.net/lchs/mewoldsen/BCellularRespiration.htm

A site dealing with cellular respiration.
http://www.ultranet.com/~jkimball/BiologyPages/C/CellularRespiration.html

This page takes a simplified approach to the study of biochemistry and may be good if you're still unsure of its basic idea.
http://www.geocities.com/j31645/22.html

Glossary of biochemistry terms.
http://www.nadh.com/site3/glossary.htm#sectA

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