5.1.7 Investigating the Rate of Photosynthesis

• Investigations to determine the effects of light intensity, carbon dioxide concentration and temperature on the rate of photosynthesis can be carried out using aquatic plants, such as Elodea or Cabomba (types of pondweed)
• The effect of these limiting factors on the rate of photosynthesis can be investigated in the following ways:
  • Light intensity – change the distance (d) of a light source from the plant (light intensity is proportional to 1/d²)
  • Carbon dioxide concentration – add different quantities of sodium hydrogencarbonate (NaHCO₃) to the water surrounding the plant, this dissolves to produce CO₂
  • Temperature (of the solution surrounding the plant) – place the boiling tube containing the submerged plant in water baths of different temperatures

• Whilst changing one of these factors during the investigation (as described below), ensure the other two remain constant
  • For example, when investigating the effect of light intensity on the rate of photosynthesis, a glass tank should be placed in between the lamp and the boiling tube containing the pondweed to absorb heat from the lamp – this prevents the solution surrounding the plant from changing temperature

Apparatus

• Distilled water
• Test tube
• Beaker
• Lamp
• Aquatic plant, algae or algal beads
• Ruler
• Sodium hydrogen carbonate solution
• Thermometer
• Test tube plug
• Syringe

Method

• Ensure the water is well aerated before use by bubbling air through it
  • This will ensure oxygen gas given off by the plant during the investigation form bubbles and do not dissolve in the water

• Ensure the plant has been well illuminated before use
  • This will ensure that the plant contains all the enzymes required for photosynthesis and that any changes of rate are due to the independent variable

• Set up the apparatus in a darkened room
  • Ensure the pondweed is submerged in sodium hydrogen carbonate solution (1%) – this ensures the pondweed has a controlled supply of carbon dioxide (a reactant in photosynthesis)

• Cut the stem of the pondweed cleanly just before placing into the boiling tube
• Measure the volume of gas collected in the gas-syringe in a set period of time (eg. 5 minutes)
• Change the independent variable (ie. change the light intensity, carbon dioxide concentration or temperature depending on which limiting factor you are investigating) and repeat step 5
• Record the results in a table and plot a graph of volume of oxygen produced per minute against the distance from the lamp (if investigating light intensity), carbon dioxide concentration, or temperature

The effect of light intensity on an aquatic plant is measured by the volume of oxygen produced

Results - Light Intensity

• The closer the lamp, the higher the light intensity (intensity ∝ 1/d²)
• Therefore, the volume of oxygen produced should increase as the light intensity is increased
• At a point, the volume of oxygen produced will stop changing even if the light is moved closer
  • This is when the light stops being the limiting factor and the temperature or concentration of carbon dioxide is limiting the rate of photosynthesis
  • The effect of these variables could then be measured by increasing the temperature of water (by using a water bath) or increasing the concentration of sodium hydrogen carbonate respectively

• The results should be displayed on a graph of light intensity vs. rate of photosynthesis
  • Rate of photosynthesis = volume of oxygen produced ÷ time elapsed

Limitations

• Algae is often used in experiments on photosynthesis and respiration rates but it can be very hard to maintain consistency in the number of algae and it can be hard to handle directly in the water
  • Immobilised algae beads are beads of jelly with a known surface area and volume that contain algae, therefore it is easier to ensure a standard quantity
  • Immobilised algae beads are easy and cheap to grow, they are also easy to keep alive for several weeks and can be reused in different experiments
  • The method is the same for algae beads though it is important to ensure sufficient light coverage for all beads

• The light-dependent reactions of photosynthesis take place in the thylakoid membrane and involve the release of high-energy electrons from chlorophyll a molecules
• These electrons are picked up by the electron acceptor NADP in a reaction catalysed by dehydrogenase
• However, if a redox indicator (such as DCPIP or methylene blue) is present, the indicator takes up the electrons instead of NADP
• This causes the indicator to change colour
  • DCPIP: oxidised (blue) → accepts electrons → reduced (colourless)
  • Methylene blue: oxidised (blue) → accepts electrons → reduced (colourless)
  • The colour of the reduced solution may appear green because chlorophyll produces a green colour

• The rate at which the 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 stage of photosynthesis
  • When light is at a higher intensity, or at more preferable light wavelengths, the rate of photoactivation of electrons is faster, therefore the rate of reduction of the indicator is faster

Light activates electrons from chlorophyll molecules during the light-dependent reaction. Redox indicators accept the excited electrons from the photosystem, becoming reduced and therefore changing colour.

Apparatus

• Leaves
• Isolation medium
• Pestel and mortar
• Lamp
• Test tubes
• Stopwatch
• Aluminium Foil

Method - Measuring light as a limiting factor

• Leaves are crushed in a liquid known as an isolation medium
  • This produces a concentrated leaf extract that contains a suspension of intact and functional chloroplasts
  • The medium must have the same water potential as the leaf cells so the chloroplasts don’t shrivel or burst and contain a buffer to keep the pH constant
  • The medium should also be ice-cold (to avoid damaging the chloroplasts and to maintain membrane structure)

• The experiment should be set up in a dark room so that the light source and intensity can be controlled
  • The room should be at an adequate temperate for photosynthesis and maintained throughout, as should carbon dioxide concentration

• Small tubes are set up with different intensities, or different colours (wavelengths) of light shining on them
  • If different intensities of light are used, they must all be of the same wavelength (same colour of light) - light intensity is altered by changing the distance between the lamp and the test tube
  • If different wavelengths of light are used, they must all be of the same light intensity - the lamp should be the same distance in all experiments

• DCPIP of methylene blue indicator is added to each tube, as well as a small volume of the leaf extract
• A control that is not exposed to light (wrapped in aluminium foil) should also be set up to ensure the affect on colour is due to the light
• The time taken for the redox indicator to go colourless (or green, as the chlorophyll may also colour the solution) is recorded
  • This is a measure of the rate of photosynthesis

Results

• A graph should be plotted of absorbance against time for each distance from the light
• As the light intensity decreases, the rate of photosynthesis also decreases
  • This is because the lowered light intensity will slow the rate of photoionisation of the chlorophyll pigment, so the overall rate of the light dependent reaction will be slower
  • This means that less electrons are released by the chlorophyll, hence the DCPIP accepts less electrons. This means that it will take longer to turn from blue to colourless

• When the DCPIP is blue, the absorbance is higher. The rate at which the absorbance decreases can therefore be used to determine the activity of the dehydrogenase enzyme
  • A higher rate of decrease, shown by a steep gradient on the graph, indicates that the dehydrogenase is highly active.

Limitations

• This experiment is not measuring the rate of dehydrogenase activity directly (through measuring the rate of substrate use or product made) but is instead predicting what the rate would be by measuring the rate of electron transfer from the photosystems
• The concentration of DCPIP will depend on the number of chloroplasts in a sample and therefore the number of light-dependent electron transport chains
  • It is therefore important to control the amount of leaf used to produce the chloroplast sample and also how much time is spent crushing the leaf to release the chloroplast
  • It is also a good idea to measure a specific wavelength absorption by each sample on the colorimeter before and after the experiment so you can get a more accurate change in oxidised DCPIP concentration
  • Results should also be repeated and the mean value calculated

• The time taken to go colourless is subjective to each person observing and therefore one person should be assigned the task of deciding when this is