5-1-7-required-practical-investigating-the-rate-of-photosynthesis

Investigating limiting factors in aquatic plants

• Investigations to determine the effect of environmental factors on the rate of photosynthesis can be carried out using aquatic plants, e.g.:

  • Elodea pondweed

  • algae, or algal beads

Investigating the effect of light intensity on photosynthesis in pond weed

Apparatus

• Aquatic plant, algae or algal beads

• Distilled water

• Test tube

• Beaker

• Lamp

• Ruler

• Sodium hydrogen carbonate

• Thermometer

• Test tube bung

• Syringe

Method

1. Prior to the experiment:

   • ensure that water is well aerated before use by bubbling air through

     • This will ensure that oxygen gas given off by the plant during the investigation forms bubbles and does not dissolve in the water

   • ensure that the plant has been well illuminated

     • This will ensure that the plant contains all the enzymes required and is carrying out photosynthesis

2. Set up the apparatus in a darkened room

   • This allows light from external light sources to be controlled

3. Add distilled water and sodium hydrogencarbonate to the boiling tube

   • This ensures that the pondweed has a controlled supply of dissolved carbon dioxide

4. Cut the stem of the pondweed at an angle before placing into the boiling tube

   • The angle increases the surface area for bubble formation

5. Position the lamp at a set distance from the tube, e.g. 10 cm

6. Measure the volume of gas collected in the gas-syringe over a set period of time, e.g. 1 minute

7. Repeat step 6 at least twice more

8. Alter the distance between the lamp and the pondweed, e.g. to 20 cm, and repeat steps 6-7

9. Repeat step 8 over a range of distances

10. Record the results in a table and plot a graph of volume of oxygen produced per minute against distance from the lamp

Alternative methods

• An alternative to using pond weed could be to produce immobilised algae beads; balls of alginate gel containing algae cells which can be used in place of pond weed

  • Advantage: beads have a known surface area and volume, so standardisation between repeats is easier

  • Disadvantage: algal balls can be less reliable in their oxygen production, and may be harder to look after than pond weed

Investigating other factors

• The effect of other limiting factors on the rate of photosynthesis can be investigated, e.g.:

  • carbon dioxide concentration: add sodium hydrogencarbonate (NaHCO₃) to the water surrounding the plant to produce different concentrations of dissolved CO₂

  • temperature: place the submerged plant in water baths of different temperatures

Required practical: factors affecting the rate of dehydrogenase activity

• During the light-dependent reactions high-energy electrons are emitted from chlorophyll

• These electrons eventually reduce NADP in a reaction catalysed by dehydrogenase enzymes

• When a redox indicator is added to photosynthesising material, the indicator will take up the electrons instead of the NADP; the indicator is said to become the electron acceptor

  • Examples of redox indicators include DCPIP and methylene blue

• Accepting electrons causes redox indicator to change colour

  • oxidised (blue) → accepts electrons → reduced (colourless)

• The rate at which a redox indicator changes colour from its oxidised (blue) state to its reduced (colourless) state can be used as a measure of the rate of dehydrogenase activity, and therefore the rate of the light-dependent reaction

Investigating the effect of light intensity on rate of dehydrogenase activity

Apparatus

• Leaves, e.g. spinach

• Pestle and mortar or food blender

• Isolation solution containing sucrose, potassium chloride and a pH 7 buffer

• Funnel

• Filter paper or cloth

• Beaker

• Centrifuge and centrifuge tubes

• Glass rods

• Ice-cold water bath

• Colorimeter and cuvettes

• Test tubes and rack

• Lamp

• DCPIP indicator

• Dropping pipette

Method

1. Grind up the leaves with 20 cm³ isolation medium in a pestle and mortar or blend them in a food blender for 10 seconds

   • This breaks apart the tissues of the leaf

   • The isolation medium will prevent cell damage due to osmosis or extreme pH

2. Filter the resulting liquid into a clean beaker using a funnel and some filter paper or cloth

   • This removes large pieces of leaf tissue

3. Transfer the filtered liquid into a centrifuge tube and centrifuge for 10 minutes

   • This will result in a pellet of chloroplasts forming at the bottom of the tube

4. Discard the liquid in the centrifuge tube and keep the pellet

   • The liquid here is known as the supernatant

5. Place 2 cm³ fresh isolation medium and the chloroplast pellet into a clean test tube, stirring with a glass rod to re-suspend the chloroplasts in the liquid; this is now the chloroplast extract

6. Transfer the chloroplast extract to an ice-cold water bath

   • The cold temperature of the water bath slows down the activity of the chloroplasts

7. Place a test tube containing 0.5 cm³ chloroplast extract into a test tube rack set up at a specified distance from a lamp, e.g. 10 cm

   • A beaker of water can be placed in between the lamp and the rack here to prevent a temperature increase due to heat from the lamp

8. Add 5 cm³ DCPIP solution to the chloroplast extract and mix together using a clean glass rod

9. Use a pipette to immediately remove a sample of the DCPIP-chloroplast mixture and place the sample into a clean cuvette

10. Place the cuvette into a colorimeter and take a reading for absorbance

11. Repeat steps 9-10 every minute for 10 minutes

12. Repeat steps 7-11 with the lamp at a series of different distances from the tube

13. Plot a line on a graph of absorbance against time for each distance from the lamp

Results

• The graph for each light intensity should show a decrease in absorbance over time as the DCPIP becomes fully reduced and the solution turns from dark blue to green

  • The colourless indicator results in a green solution due to the presence of green chloroplasts

• As the light intensity decreases the rate of photosynthesis will also decrease, and the time taken for the tube to turn green will increase

  • Fewer electrons are released by the chlorophyll, so the DCPIP accepts fewer electrons and it will take longer to turn from blue to colourless