8.2 Applications and skills

8.2.3 Bioenergetics and the lollipop experiment

Bioenergetics is the study of how energy is transformed in living systems. Experiments in bioenergetics require the design of elegant protocols, and painstaking repetitions. 

Application: Use of radioactive isotopes to elucidate the Calvin cycle

  • In Calvin’s protocol, unicellular photosynthetic algae, Chlorella, are cultured in a round glass flask (the lollipop).  Brief pulses of carbon dioxide labelled with radioactive carbon-14 are bubbled through the flask.

Figure 8.2.3a – The lollipop apparatusFigure 8.2.3a – The lollipop apparatus

  • Samples of the culture are removed at timed intervals ranging from a few seconds to a few minutes. The samples are fixed in heated ethanol, concentrated, and analysed using two-dimensional chromatography.

Figure 8.2.3b Principle of two-dimensional chromatographyFigure 8.2.3b Principle of two-dimensional chromatography

  • The chromatograms are dried and placed against photographic film in the dark. The radiation from the 14C-labelled compounds creates dark spots on the film. The images created are called autoradiographs.

Figure 8.2.3c – Autoradiograph showing stable intermediates from the Calvin cycleFigure 8.2.3c – Autoradiograph showing stable intermediates from the Calvin cycle

  • From a series of autoradiographs, Calvin was able to determine when each of the metabolites is produced during the light-independent reactions.

The Hill reaction

  • We know that the light-independent reactions occur in the stroma of the chloroplast, while the light-dependent reactions occur on the thylakoids.
  • Robin Hill (1899-1991) was the first to demonstrate that oxygen production (light-dependent) occurs independently from sugar production (light-independent). Using Hill’s reagent, dichlorophenolindophenol (DCPIP) we can replicate his experiment.
  • In the light-dependent reactions, oxygen is reduced by electrons from the transport chain. DCPIP is a redox indicator. When it accepts electrons, it will change colour. DCPIP is blue when oxidised, and colourless when reduced.

Try it!

Note: Keep all materials chilled or bathed in ice in order to prevent lysis of membranes.

1. Mash a good quantity of soft leaves (spinach works well) in a chilled mortar and pestle, with a small amount of sand and isotonic buffer. Filter through muslin cloth into a centrifuge tube.

Figure 8.2.3d – Centrifuge protocol for Hill reactionFigure 8.2.3d – Centrifuge protocol for Hill reaction

2. Place the filtrate in a centrifuge at low speed for ten minutes. Decant the supernatant into a new tube, and discard the pellet.

3. Centrifuge the suspension at high speed for ten minutes. Discard the supernatant and keep the pellet, which contains concentrated chloroplast.

4. Resuspend the chloroplast pellet in isotonic buffer.

5. Prepare two test tubes – one exposed to light, and one covered in dark paper. Place a 0.5cm3 sample of the chloroplast suspension in each. Add 4.5cm3 DCPIP solution.

Figure 8.2.3e – DCPIP changes colour to indicate that electrons are transferred during the light-dependent reactionsFigure 8.2.3e – DCPIP changes colour to indicate that electrons are transferred during the light-dependent reactions

6. After about 5 minutes, you should see a colour-change in the sample exposed to light, while the other sample remains dark blue.

Learn about a prototype of the 'artificial leaf'.

Figure 8.2.3f – ChlorellaFigure 8.2.3f – Chlorella
Chlorella is a good model organism because the algal cell has only one large chloroplast.

Nature of Science

Developments in research follow improvements in apparatus. The Calvin cycle was determined using 14C and autoradiography.

TOK

  • The lollipop experiment was a creative protocol. To what extent is the creation of an elegant protocol similar to the creation of a work of art? What evidence is there that scientific discovery is inevitable rather than the result of individual curiosity and creativity?
  • Many metabolic pathways have been described following a series of carefully controlled and repeated experiments. To what degree can looking at component parts give us knowledge of the whole?

Figure 8.2.3g – Centrifuge tubeFigure 8.2.3g – Centrifuge tube
After centrifuging, heavier components end up in the pellet, while the supernatant contains lighter components in suspension.

Figure 8.2.3h – SpectrophotometerFigure 8.2.3h – Spectrophotometer
Classroom spectrophotometer

IA

  • How could you modify the Hill protocol to get quantitative data? (Hint: does your school have a spectrophotometer?)

Figure 8.2.3i – Colour changeFigure 8.2.3i – Colour change
Colour-change over time.

International mindedness

The Global Artificial Photosynthesis (GAP) project requires international cooperation. Its aim is to create an artificial 'leaf' that can create oxygen and hydrogen from water and sunlight. Consider the impact that the success of this project would have on public health, and on energy and food security.

Further reading

Faunce, T. 'Global artificial photosynthesis project: a scientific and legal introduction' in Journal of Law and Medicine (19) 2:275-281, December 2011.

Course links

  • Principles of chromatography 2.2.9.