Investigating Photosynthesis and Respiration using Algal BallsTeaching Notes

Introduction and contextPhotosynthesis, a chemical reaction, is dependent on several factors. These are known as rate limiting factors and most 14-16 students need to be able to identify/discuss these factors.

In this protocol, the green alga Scenedesmus quadricauda is immobilised in alginate beads. Changes in the colour of hydrogen carbonate indicator solution (also called bicarbonate indicator) can then be used to investigate the rate of photosynthesis under different environmental conditions.

Like flowering plants, S. quadricauda cells contain the photosynthetic pigments chlorophyll a and b. This enables the green algae to photosynthesis and means that they are currently classified in the Plant Kingdom1 (along with angiosperms, conifers, ferns and mosses), although there is still debate amongst biologists on this.

Scenedesmus are described as colonial, normally being found in colonies of 2 or 4 cells. The end cells of the colonies typically have 2 long spines protruding from the outer corners. Each cell is approximately 15µm long and contains a single, plate-like chloroplast. There are approximately 80 recognised species of Scenedesmus1.

Scenedesmus species have recently been investigated for production of biodiesel and have shown to be promising in terms of high lipid and oleic acid content2.

References

1. M.D. Guiry in Guiry, M.D. & Guiry, G.M. 2012. AlgaeBase. World-wide electronic publication, National University of Ireland, Galway. http://www.algaebase.org, searched on 10 November 2012(specific page: http://www.algaebase.org/search/genus/detail/?genus_id=43474)

2. Pandian Prabakaran and A. David Ravindran, Current Science, Vol. 102, NO. 4, 25 February 2012 online at: http://www.currentscience.ac.in/Volumes/102/04/0616.pdf

Video

For a video demo showing how to create the algal balls and how best to use this in the classroom, see our YouTube channel:www.youtube.com/watch?v=fI3x68CkKW0

OverviewThis document will explain how to use the immobilised algae to demonstrate / investigate:

1. Photosynthesis only proceeds in the light2. Light intensity affects the rate of photosynthesis3. The compensation point between photosynthesis and respiration4. Wavelength (colour of light) affects the rate of photosynthesis

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Scenedesmus , a green alga, contains chlorophyll a and b. They are often found in small colonies of 2-4 cells.

5. Concentration of algae will affect the rate of photosynthesis

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How photosynthesis is measured in these investigations

Using an indicator solution to obtain quantitative resultsHydrogen carbonate indicator solution is sensitive to changes in pH caused by CO2 from the air dissolving in water to form carbonic acid. When hydrogen carbonate indicator solution is equilibrated to atmospheric CO2 levels, it is red. When CO2 levels increase it changes from red through orange to yellow. When CO2 levels decrease, it changes from red through magenta to deep purple. This makes it an ideal indicator to use with photosynthesis investigations. When photosynthesis is proceeding more quickly than respiration, CO2 will be removed from the solution and it will change from red to magenta/deep purple. When respiration is proceeding more quickly than photosynthesis, the solution will change from red to orange/yellow.

This colour change can be measured using a colorimeter measuring absorbance at 550nm or by comparing the colour of the indicator with a set of standard buffered solutions.

NB By getting students to gently breathe through a straw into a test tube of the indicator solution, you can demonstrate that CO2 causes the solution to change colour from red to yellow (eye protection should be worn, and care taken to ensure students don’t suck up the indicator).

Indicator colour / pHStandard buffered solutions or indicator colour charts can be used to allow students to match the indicator in their bijou bottles to a pH value.

Colour changes that may occur during the investigation, and the corresponding pH values.← Increasing CO2 in indicator Atmospheric level of CO2 (0.04%)(pH 8.4) Decreasing CO2

in indicator →

yellow orange red magenta purplepH 7.6 pH 7.8 pH 8.0 pH 8.2 pH 8.4 pH 8.6 pH 8.8 pH 9.0 pH 9.2

(Download full colour chart from http://www.saps.org.uk/attachments/article/235/Colour%20chart%20showing%20hydrogencarbonate%20indicator.pdf)

Hydrogen carbonate indicator can vary in colour depending on supplier. You can prepare your own standard solutions for comparison rather than using this chart. These solutions can be made up using standard boric acid/borax buffer solutions (see technical notes). In this way, a range of desired colours / pH values can be displayed in the classroom for students to check their bijou bottles against. Students should be supplied with indicator that has been equilibrated to atmospheric CO2 concentration (see technical notes) for their investigations. This continuous pH data can be plotted on a line graph against the independent variable that is being investigated. Each investigation can lead to good discussions about how science works and the scientific method.

Absorbance of indicator at 550nmThe indicator can be removed from the bijou bottles using clean pipettes, placed into cuvettes, and the absorbance at 550nm (wavelength of green light) measured using a colorimeter.

The relationship between absorbance and pH is linear at 550nm with this range of pH values. The absorbance of green light increases with increased pH of bicarbonate indicator.

Using a colorimeter will give absorbance data accurate to one or two decimal places, whereas using colour charts or buffer solutions will give colour changes that are more subjective.

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Investigation 1: Light and Dark

Using algal balls to show that light is needed for photosynthesis

Approximately 20 algal balls are placed into a bijou bottle filled to the top (to reduce the buffering effect of the air trapped inside the bottle) with hydrogen carbonate indicator solution equilibrated to atmospheric CO2 concentration.

If near a suitable lamp (see technical notes), the indicator will change to purple within 30 minutes. This occurs as CO2 has been removed from the indicator solution during the process of photosynthesis. If in the dark, the indicator will change to yellow as carbon dioxide produced in respiration enters the solution.

Placing bijou bottles at different distances from a lamp, leaving them for differing times, changing the number of algal balls are all methods that demonstrate photosynthesis in action and can produce colour changes in the indicator solution from red through to purple.

Typical simple qualitative results

Start point In the dark(covered with black sugar paper - paper removed for photograph)

Near a lamp

pH 8.4 (cherry red);0.04% CO2 in atmosphere

pH 8.0 (yellow); more yellow because of increased CO2 produced in respiration (creates carbonic acid).

This CO2 is not removed as photosynthesis does not proceed in the dark.

pH 9.2 (purple); more purple because CO2 is being removed from the indicator for use in photosynthesis.

This reduces the amount of carbonic acid in the solution so it becomes more alkaline

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Investigation 2a: Light intensity (distance from a lamp)

Using algal balls to show that the rate of photosynthesis increases with increasing light intensity

Setting up algal balls at different distances from a suitable lamp for a set amount of time can show that light intensity affects the rate of photosynthesis. The colour change is slower further from the lamp as photosynthesis proceeds more slowly at lower light levels.

Students who have compared their colour change to charts or standard indicator solutions can plot line graphs of pH value vs. distance from the lamp.

If using a colorimeter, students can plot absorbance at 550nm against distance from the lamp.

With high ability students, you may prefer to convert distance to an approximate value for light intensity by using the inverse square law, 1/D2, where D is distance from the lamp.

As distance from the lamp/light intensity increases the absorbance and pH values decrease showing that the rate of photosynthesis decreases as light intensity becomes a rate limiting factor. This experiment works best in a darkened room so that other illumination sources are reduced as much as possible. However with very bright light sources this effect is more limited.

Typical data obtained (when using a 150W halogen lamp in a darkened room)Data will vary according to the type of lamp used and the concentration of algae in the algal balls. However, the trends and patterns should remain the same. Distances as large as those noted below are not needed if using an energy saving compact fluorescent lamps, as light levels drop more quickly over shorter distances than Halogen lamps (see technical notes re lighting).

Distance from lamp -(mm)

(optional column)Relative light intensity (1/D2) (x10-5)*

(if using colourimeter)Absorbance of indicator (550nm)

pH value

250 1.60 0.81 8.8350 0.81 0.74 8.6500 0.40 0.56 8.4780 0.16 0.39 8.21250 0.006 0.25 8.0

*1/D2 produces numbers with many decimal places so that adjustment may be required to give a sensible number for analysis/plotting on a graph. For instance in this example, at a distance of 250cm, 1/D2 is equal to 0.000016 and so multiplying this by 105 gives a suitable number (1.6) for analysis.

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Investigation 2a: Light intensity (neutral density filters)

Using algal balls to show that the rate of photosynthesis increases with increasing light intensity. Neutral density filters reduce transmittance of all wavelengths of light and so can be used to demonstrate the effects of light intensity on photosynthetic rates. As the ND rating increases, the indicator solution containing the algal balls goes from red to yellow as respiration is proceeding more quickly than photosynthesis due to decreasing light intensity (light is becoming a rate limiting factor). Neutral density filters are obtainable from the NCBE (www.ncbe.reading.ac.uk).

If you are using halogen lamps that give off a lot of heat, using neutral density filters allows you to choose a distance from the lamp where temperature effects will not adversely affect the investigation. Any heating effect that occurs, will therefore affect all bottles of algae equally.

We suggest using filters with these values: 0.15, 0.3 and 0.6. (This is because they can also be used to find the compensation point between photosynthesis and respiration (see investigation 3). Leaving one bottle unwrapped is a ND value of 0.0, and wrapping one bottle in thick black sugar paper will effectively produce a ND value of 1.0.

Typical Data Obtained

Absorbance after 50 mins in front of a 42W CFL portable floodlamp (column two shows how much light gets through each type of filter).Filter on bottle Amount of light transmitted

into the bottle (%)(if using colourimeter)Absorbance of indicator (550nm)

pH value

None (0.0 ND) 100 0.34 9.00.15 ND 71 0.30 8.60.3 ND 50 0.17 8.40.6 ND 25 -0.03 8.2Black paper (1.0 ND) 0 -0.15 7.8

Line graphs can be drawn of ND filter value/light transmitted into the bottle against absorbance at 550nm/pH value of indicator solution. Lines (curves) of best fit can then be added and discussed.

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Investigation 3: Compensation point between photosynthesis and respiration

Neutral density (ND) filters can be used to find the compensation point between photosynthesis and respiration. Respiration produces CO2 and photosynthesis uses CO2. When the two processes are in balance there is no net production of CO2 and we call this point the compensation point. We can use ND filters to find the light level at which this compensation point is reached.

Neutral density filters reduce transmittance of all wavelengths of light. As the ND rating increases, the amount of light transmitted into the bottle decreases and so the indicator solution containing the algal balls goes more yellow.

We suggest using filters with the values 0.15, 0.3 and 0.6ND that allow 71, 50 and 25% of light, respectively, to transmit through the filter. Leaving one bottle unwrapped (ND value 0.0) will equate to 100% transmittance, and wrapping one bottle in thick black sugar paper (ND value of 1.0) will equate to 0% light transmittance.

When a graph of absorbance at 550nm is plotted against % light transmitted, the compensation point for photosynthesis (expressed as the % of light transmitted into the bijou bottle) can be estimated by reading the % light transmitted value at an absorbance value of 0.

Typical data obtained and example graph showing how to work out the compensation pointAbsorbance after 50 mins in front of a 42W CFL portable lamp (equivalent to a 200W Halogen lamp)(column two shows how much light gets through each type of filter)

In the table, the negative values for absorbance show that there has been a colour change towards the more yellow/acidic end of the spectrum caused by an increase in CO2. This shows that at these values for light transmitted into the bijou bottles (0 and 25%), respiration was the dominant process.

In the bottles with positive results for absorbance of indicator, there has been a colour change to the more purple/alkaline end of the spectrum because photosynthesis is the dominant process.

The point at which there is no net change in the concentration of dissolved CO2 is the compensation point.

From the graph, this point is estimated to be when the % transmitted light into the bottle is 29%.

Similar results can be obtained by plotting absorbance against pH although this will not produce as accurate or precise results if pH is being estimated based on indicator colour rather than a pH meter.

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Filter on bottle

Amount of light transmitted into the bottle (%)

Absorbance of indicator (at 550 nm)

pH value

None (0.0 ND)

100 0.34 9.000.15 ND 71 0.30 8.950.3 ND 50 0.17 8.750.6 ND 25 -0.03 8.35Black paper (1.0 ND)

0 -0.15 8.00

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Investigation 4: Wavelength of light

Using acetate / photographic filters to show that the wavelength (colour) of light affects the rate of photosynthesis

The wavelength range for visible light is 400 (blue) – 750 nm (red). The green part of the spectrum is 510 – 555nm.

Plants do not use all wavelengths of light equally in photosynthesis. Plants appear to be green because they are reflecting green light which shows that little green light can be used in photosynthesis. The Scenedesmus algae used in this investigation, like flowering plants, ferns and mosses, contain chlorophyll a and b and so appear green.

Most plants prefer red and blue light for photosynthesis, although this will vary from species to species according to the environment in which they are found. If plants don’t receive enough light of a suitable wavelength then this could become a rate limiting factor for photosynthesis.

Acetate filtersGreen, red, blue or clear (control) acetate filters wrapped round the bijou bottles can be used to show which colour/wavelength of light is used by the algae in photosynthesis. For instance, if a filter does not transmit any light that can be used in photosynthesis, then photosynthesis will stop and the indicator solution will change towards the more yellow/acidic end of the spectrum. If photosynthesis can proceed at a rate that exceeds respiration, the indicator solution will change towards the more purple/alkaline end of the spectrum showing that the wavelength being tested is sufficient for photosynthesis to proceed and overtake the rate of respiration.

More accurate colour filters can be used (Primary Red, Primary Green and Bray Blue) (see suppliers details on technical notes pages)

Typical data obtained After 30 mins in front of a 42W portable CFL floodlamp. Column two shows which light in the range 400-680nm gets through each filter.Type of filter on bottle

Colour of light allowed into the bottle

(if using colourimeter)Absorbance of indicator (550nm)

pH value

Clear acetate All (400-680nm) 0.94 9.0Primary red Red (600-680 nm) 0.74 8.6Bray Blue Blue (425-500 nm) 0.56 8.4Primary Green Green (475-590 nm) 0.32 7.8Black paper None 0.24 7.6

These data are best represented as categoric data using a bar chart of filter colour against absorbance at 550nm or pH value.

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Scenedesmus contain chlorophyll a and b

Investigation 5: Number of algal balls / concentration of algae in the balls

Students can vary the number of algal balls in their bijou bottles. The more algal balls in a bottle, the more algal cells there will be and the more photosynthesis can occur.

Students can also vary the concentration of the algal culture in the balls by diluting the concentrated algal culture that they start with.

The number of algal balls/concentration of algae can be plotted against absorbance at 550nm/pH value to create a line graph with a line/curve of best fit.

When there are more algal balls or when the algae are more concentrated, the time taken for the the indicator to change to the purple/ alkaline end of the spectrum will decrease.

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Questions / discussions for after the practical

Questions that apply to all of the investigations

1. Which of your algal balls will be getting the most light? Which will be getting the least?

This indicator solution goes purple when carbon dioxide is removed from it

2. What process in plants uses carbon dioxide?3. In which of your bottles will this process be taking place most quickly? Explain.

This indicator solution goes yellow when carbon dioxide is added to it

4. What process in living things (including plants) produces carbon dioxide?5. In which of your bottles will this process be taking place?

6. What are your overall predictions? (Use the terms photosynthesis and respiration)7. What do your results show? (Do they fit predictions, trends or patterns, explain anomalies)8. Can you answer the question that you were investigating?9. How will you show your results on a graph/chart?10. Was this a fair test? What were your variables (dependent, independent, control)?11.Can you think of a method that would have enabled you to collect more reliable data?

Questions that apply to specific investigations:

Investigation 4. Wavelength of light

1. Which colour of light appears to be best for photosynthesis? Explain.2. Was photosynthesis occurring in the bottle with the green filter? Explain.

(It should not proceed quickly, although the Primary Green filter does let some blue light through.)

3. Students could be asked to look up the absorbance of chlorophyll a and b and relate these to the investigation

Acknowledgements

This protocol was developed by Debbie Eldridge from King Ecgbert School (Sheffield) during a secondment as a SAPS/Robinson College Schoolteacher Fellow.

These resources were updated and extended by Vicki Cottrell of Didcot Girls School during a secondment as Nuffield Education Fellow.

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