Investigating photosynthesis using immobilised algae
This procedure offers a method for measuring the rate of photosynthesis which depends directly on the rate of uptake of carbon dioxide by the photosynthetic organism. Hydrogencarbonate indicator changes colour with pH, which is determined by the concentration of carbon dioxide in solution.
The photosynthetic organism is a fast-growing green alga – such as Scenedesmus quadricauda – immobilised in alginate beads. These algal balls make it easy to standardise the amount of photosynthetic tissue in any investigation.
There is scope for students to develop the protocol to investigate a range of factors.
This protocol is adapted with permission from information on the Science and Plants for Schools (SAPS) website (see www.saps.org.uk).
You can make up the algal balls in one lesson, discuss how to set up the main procedure, and carry it through in the next lesson. A preliminary investigation by teacher/ technician will allow you to estimate the amount of algal material and indicator to use to get a result with your equipment in the time available.
Apparatus and Chemicals
For each group of students:
Transparent containers (glass bottles are ideal) with sealable lids, around 10 cm3, 6–12
Clamp stand, boss and clamp
Syringe barrel, 10 cm3
Beaker, 100 cm3, 2
Cocktail stick to stir alginate
150 W lamp (Note 5)
Container of water as heat filter (Note 6)
Ruler/ tape measure
For the class – set up by technician/ teacher:
Colorimeter (Note 1)
Algal suspension, 2.5 cm3 concentrated for each group (Note 2)
Sodium alginate, 2–3%, 100 cm3 for a class of 30 students (Note 3)
Calcium chloride solution, 2%, 50 cm3 per group
Hydrogencarbonate indicator, 50–100 cm3 per group (Note 4)
Hydrogencarbonate indicator, standard colour scale, if colorimeter not available (Note 4)
Light meters, if available
Health & Safety and Technical notes
Do not look directly into the lamps.
Do not touch the lamps while hot.
Keep flammable material away from the lamps in use.
1 Colorimeter. There is a linear relationship between absorbance of the indicator (at 550 nm – bright green filter) and pH over the range studied in this procedure.
2 Growing your alga. Prepare a culture of green alga such as unicellular Scenedesmus quadricauda. Make up a solution of algal enrichment medium, and subculture the alga into this. Aerate gently and keep at temperatures between 18–22 °C. Constant illumination ensures faster growth of the alga. After 3–4 weeks, the culture should have a green ‘pea soup’ colour. Subculture the alga again to maintain a healthy culture. You could use other algae, but Scenedesmus should produce 2 to 3 litres of dark green ‘soup’ in about 4 weeks from 50 cm3 of original culture. (Details from SAPS Sheet 23).
3 Preparing solutions to make alginate beads (Refer to Recipe card 2):
- Dissolve 3 g of sodium alginate in 100 cm3 of cold, pure water. Stir with a spatula every half hour or so. Leave overnight and stir in the morning.
- Dissolve 4 g of calcium chloride-6-water in 200 cm3 of pure water in a 250 cm3 beaker.
4 Hydrogencarbonate indicator. Refer to Recipe card 34 and Hazcard 32. Low hazard once made; must be made fresh by qualified staff using fume cupboard. The indicator is very sensitive to changes in pH, so rinse all apparatus with the indicator before use. Avoid exhaling over open containers of the indicator. Make up a ‘standard colour scale’ of reaction bottles containing buffers from pH 7.6- pH 9.2 with hydrogencarbonate indicator if students will not have access to a colorimeter.
5 Lamps. You need a brighter light than a standard 40 W or 60 W bench light. Low energy bulbs produce too limited a spectrum of light for full activity. 150 W tungsten or halogen lamps are best. 150 W portable halogen lamps have a stand and handle separate from the body of the lamp which makes them safer to handle. But they do produce heat, so you will need a heat filter for the investigation (Note 6).
6 Heat filter. Use a large flat-sided glass vase (available from Ikea or Homebase or other domestic suppliers) or a medical ‘flat’ filled with water. With a high power lamp, the small volume in a medical ‘flat’ may get too hot for comfort.
7 Making alginate beads:
- When making up the alginate or diluting the algal culture it is essential to use pure water; otherwise calcium ions in the water will cause the alginate to 'set' prematurely.
- If your beads are not the size and texture you want, try different mixes with your active material, or different concentrations of sodium alginate (around 2–3%), or make the syringe nozzle narrower (with glass capillary tubing) or wider (by sawing off and adding a plastic tube). Different brands of alginate have different consistencies. You need a viscous mixture that will drip steadily through the syringe. Keep a note of what worked for this supply and keep your syringe barrels with nozzles for next time.
8 Neutral density filters: can be sourced as a film from photographic suppliers (for example, Lee filters www.leefilters.com. A neutral density filter reduces the amount of light transmitted by the same amount at all wavelengths.
There are no ethical issues associated with this procedure.
SAFETY: Take precautions to avoid burns, fires and dazzle caused by hot, bright lamps.
Do not leave the apparatus unattended overnight as the lamps are so hot.
Preparation of algal beads
a Concentrate your active algal culture (Note 2). Do this by leaving 50 cm3 to settle for 30 minutes in a measuring cylinder until you have a darker green sediment. Carefully pour off the pale green suspension to leave just 5 cm3 of concentrated culture.
b Make up the solutions you need to prepare alginate beads (Note 3).
c Mix 5 cm3 of the algal culture with 5 cm3 of the 3% sodium alginate solution (Note 3) in a very small beaker.
d Clamp a syringe barrel above a beaker of calcium chloride solution (see diagram and Note 7) – making sure the tip of the syringe is well above the solution in the beaker.
e Pour the alga/alginate mixture through the syringe barrel so it drips through and forms beads in the beaker. Swirl the beaker gently as the drops fall. (Note 7.)
f Allow the beads to harden for a few minutes before straining them out of the beaker through a tea strainer.
g Rinse the beads in distilled water. The algae in the beads will stay alive for several months in a stoppered bottle of distilled water in the refrigerator.
h Make up hydrogencarbonate indicator (see Note 4 and Recipe card 34). It is important to make up enough for the whole investigation as the depth of colour of this indicator is so variable. Make a few litres and keep for the duration of the investigations, aerating before each lesson. Make up a ‘standard colour scale’ of reaction bottles containing buffers from pH 7.6- pH 9.2 with hydrogencarbonate indicator if students will not have access to a colorimeter.
Procedure using the algal beads – investigating light intensity
a Rinse 6 translucent bottles with hydrogencarbonate indicator solution.
b Add equal numbers of algal balls to each container – around 20.
c Add a standard volume of indicator to each container (7–10 cm3).
d Replace the lid.
e Note the colour of the solution against a standard set of bottles of indicator, and/or measure the absorbance at 550nm using a colorimeter.
f Put one container in the dark. Set up the other containers at different light intensities – either by placing at different distances from the lamp, or by wrapping different neutral density filters (Note 8) around the bottles.
g Place a heat filter between the light and the containers to absorb heat from the lamp. (Note 6.)
h After 30 minutes, note the colours of the solution at each distance.
i Once the bottles cover a range of colours, which will probably take more than 60 minutes, take samples from each bottle and measure the absorbance at 550 nm using a colorimeter. Start with the bottle that has changed most – the one at highest light intensity.
j Plot absorbance against light intensity.
k You could measure light intensity at each distance if a light meter is available, or calculate light intensity (1 ÷ distance2).
It takes at least an hour for colour changes to happen – so students will need to return to the lab at break or after lessons to ‘read’ the results.
Plotting absorbance against light intensity for the above procedure will show a proportional relationship at low light intensities, levelling off at higher intensities which indicates another limiting factor. The method with neutral density filters (rather than changing distance from the lamp) is reportedly more accessible for some students.
Carbon dioxide is unlikely to be the limiting factor here as the indicator contains a relatively high concentration of hydrogencarbonate ions.
Other factors to investigate include:
- amount of algal material: Shake 1 litre of algal culture, and leave 500 cm3, 250 cm3 or 100 cm3 to settle in measuring cylinders, saving the bottom 5 cm3 each time. Make up into balls as above – each batch will have a different amount of algae, but the same surface area to volume ratio for the balls.
- surface area to volume ratio: Make smaller balls using a narrower bore to drip the alginate through.
- wavelength of light: Use coloured filters wrapped around the containers, or try different lamps (if you can identify the spectrum of light produced). Coloured filters will also alter the light intensity.
This practical reportedly works well for coursework investigations as students can suggest improvements or developments after some preliminary work with the technique.
Set aside a container of indicator and algal beads to keep in the lab for a few days. Students will be able to see changes to the colour of indicator according to the time of day – algae use up more carbon dioxide than they produce during the day and release carbon dioxide at night when they are not taking up any.
There is a linear response between absorbance of the indicator (at 550nm) and pH over the range studied in this procedure. Using a colorimeter (at 550nm) is one way to quantify this procedure. Alternatively you could set up a ‘standard colour scale’ with buffer solutions and indicator at the same concentration as the reagent in the investigation. Students can then compare their reaction vessels with the standards and interpolate to estimate pH.
You can also use this technique of immobilisation in an alginate bead to study enzymes or yeast cells. The beads can be packed into a column (for example, a syringe barrel) and a suitable substrate passed over the beads. Collect the products at the bottom of the column and use the immobilised enzymes or cells again and again without the need to separate them from the reactants.
Health and safety checked, September 2008
Download the student sheet Investigating photosynthesis using immobilised algae (62 KB) with questions and answers.
http://www.saps.org.uk/secondary/teaching-resources This is the SAPS teaching resources webpage which includes links to photosynthesis experiments (accessed October 2011).
Adapted with permission from information on the Science and Plants for Schools (SAPS) website (see www.saps.org.uk).