Photosynthesis with algal balls:
Post-16 investigations
Teaching notes
Introduction and context
All post-16 biology specifications cover the basics of photosynthesis including the concept that
electrons in chlorophyll absorb light energy that is then converted to ATP.
Most specifications suggest that students should be able to plan, carry out, analyse and interpret
data from investigations into the rate limiting factors affecting photosynthesis.
The WJEC and SQA also expect students to have an understanding of the wavelengths of light
absorbed by chlorophyll a, chlorophyll b and other photosynthetic pigments. SQA specifically
expect students to understand the terms transmission and reflection with respect to light that is not
absorbed in photosynthesis.
This resource has been produced to support educators where students can investigate
photosynthesis to obtain quantitative data that can be analysed.
In the suggested investigations that follow, the 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.
The details of how to immobilise the algae in alginate beads are described in documents for
technicians and for students.
See ‘Photosynthesis with algal balls: general students sheet’ and ‘Photosynthesis with algal balls:
Technical details’ available here: http://www.saps.org.uk/secondary/teaching-resources/235
Details of how to use a colorimeter to collect quantitative changes in a hydrocarbon indicator
solution to find the rate of photosynthesis are described in the document ‘Photosynthesis with algal
balls: teachers notes’ available here: http://www.saps.org.uk/secondary/teaching-resources/235
Contents
This document will explain how to use the immobilised algae to demonstrate / investigate:
1) Light intensity affects the rate of photosynthesis
a) by moving the algae set distances from a light source
b) by using neutral density filters
2) The compensation point between photosynthesis and respiration
including more information on the use of neutral density filters
3) Wavelength (colour of light) affects the rate of photosynthesis
including information on absorbance and transmittance of known filters
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Photosynthesis with algal balls - post-16 teaching notes p. 1
1a.
Light intensity (distance from a lamp)
In this investigation, algal balls in hydrogen carbonate indicator solution are set at different
distances from a suitable lamp for a set amount of time. At the end of this time period, the indicator
solution is removed from the algal ball bottles and placed in a cuvette for use in a colorimeter.
Absorbance of the indicator solution at 550nm is noted at each distance (compared with a
control/reference sample of indicator that is in equilibrium with the surrounding air).
The further the distance from the lamp, the slower the change in absorbance, as photosynthesis
proceeds more slowly at lower light levels.
Students can plot graphs of absorbance at 550nm against distance from the lamp/light intensity.
Distance from the lamp can be converted 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 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 lamp
(For more info on choice of lights see ‘Photosynthesis with algal balls: Technical details’ available
here: http://www.saps.org.uk/secondary/teaching-resources/235 )
Distance from
lamp (cm)
Relative light intensity
(1/D2) (x10-5)*
Absorbance of indicator (550nm)
250
350
500
780
1250
1.60
0.81
0.40
0.16
0.006
0.81
0.74
0.56
0.39
0.25
*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/D 2 is equal
to 0.000016 and so multiplying this by 105 gives a suitable number (1.6) for analysis.
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Photosynthesis with algal balls - post-16 teaching notes p. 2
1b.
Light intensity (neutral density filters)
Investigating light intensity by using the distance from a lamp as the independent
variable can introduce uncontrollable variables into an investigation. For example if
incandescent or halogen bulbs are being used these can introduce a heating effect
that affects the rate of photosynthesis. If desk lamp fittings are being used,
maintaining the same angle of light can also be difficult to control. Neutral density (ND) filters allow
light intensity investigations to take place without the needs to move the samples.
ND filters reduce transmittance of all wavelengths of light. As the ND rating increases, the amount
of light transmitted through the filter decreases. If these filters are wrapped round a sample of algal
balls, the amount of light entering the bottle will decrease as will the rate of photosynthesis. As a
result the indicator solution containing the algal balls goes more yellow/acidic as carbon dioxide
levels increase.
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.
This experiment can be extended to include calculation of the compensation point between
photosynthesis and respiration. See notes on experiment 2.
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Photosynthesis with algal balls - post-16 teaching notes p. 3
More notes on Neutral Density Filters and their absorption / transmission spectra
The name of each filter includes an approximate absorbance of the filter at all wavelengths.
For example, the filter 210(0.6 ND) has an absorbance of approximately 0.6-0.7 across the
spectrum as shown below (although the absorbance starts to fall off above 680 nm).
You can see from the transmission spectrum that the filter will allow approximately 20-22% of all
light in the spectral range 400 – 680 nm to pass – i.e. light intensity would be reduced by 78-80 %.
Other neutral density filters are available and the transmission spectra show a similar shape, with
higher or lower transmittance depending on the rating.
Neutral density filters offer an alternative way of reducing light intensity across the spectral range.
Notionally the amount of light transmitted in the spectral range 400-680 nm by each of the neutral
density filters is summarised in the table below:
Filter name
Light
transmitted (%)
298(0.15 ND)
209(0.30 ND)
210(0.6 ND)
211(0.9 ND)
299(1.2 ND)
71
50
25
12.5
6.3
The neutral density filters can be used in combination with colour filters or with each other. For
example, it has been found that the rate of photosynthesis in the presence of 4 layers of Filter 298
is the same as that found in the presence of one layer of Filter 210.
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Photosynthesis with algal balls - post-16 teaching notes p. 4
2.
The compensation point between photosynthesis and respiration
Compensation point is not specifically mentioned in all post-16 specifications,
although students are expected to understand that both photosynthesis and
respiration occur in plants cells and that carbon dioxide concentration is a ratelimiting factor in photosynthesis.
Most specifications also suggest that students should be able to carry out
qualitative investigations into the rate of photosynthesis, and should be able to
interpret and analyse data. This investigation provides an excellent opportunity
to combine investigative skills with understanding how photosynthesis and
respiration are linked.
Respiration produces CO2 and photosynthesis uses CO2. When the two processes are in balance
there is no net production of CO2, we call this point the compensation point. We can use neutral
density (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 so the rate of photosynthesis should
decrease.
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.
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Photosynthesis with algal balls - post-16 teaching notes p. 5
2.
The compensation point between photosynthesis and respiration (contd.)
Typical data obtained and example graph showing how to work out the compensation point
Absorbance after 50 mins in front of a 42W CFL portable lamp.
(column two shows how much light gets through each type of filter)
Filter on bottle
Amount of light transmitted into the bottle (%)
None (0.0 ND)
100
0.15 ND
71
0.3 ND
50
0.6 ND
25
Black paper (1.0 ND)
0
Absorbance of indicator (at 550 nm)
0.34
0.30
0.17
-0.03
-0.15
Graph of absorbance against % light transmitted into the bottle by the ND filter
Compensation point in this example
occurs when approximately 29% of light
is transmitted into the bottle.
(A zero value for absorbance indicates
no net change in the indicator solution)
In the data table, the negative values for absorbance show that the colour change is towards the
more yellow/acidic end of the spectrum caused by increasing CO2. Therefore, 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, 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%.
If not using a colorimeter, 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 with a colour chart.
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Photosynthesis with algal balls - post-16 teaching notes p. 6
3.
The colour / wavelength of light
Some specifications require students to understand that different photosynthetic pigments absorb
different wavelengths of light, and so increase the range of useful wavelengths.
A convenient way to investigate the effect of light wavelength / colour on the rate of photosynthesis
in algae is to place a filter between the light source and the algal balls by wrapping it around the
bottle.
Here, a blue filter absorbs all wavelengths of
light besides those in the blue part of the
spectrum.
Blue light is transmitted through the filter and into
the bottle containing algal balls.
Using different filters offers scope for a large
number of potential investigations.
Absorbance Spectra for the main photosynthetic pigments
It can be seen from these spectra that chlorophylls, the
main photosynthetic pigments, absorb most light in the
violet/blue and red ends of the visible light spectrum.
Filters that absorb light at these parts of the spectrum will
therefore slow down the rate of photosynthesis in a sample
where these filters are used.
(source: http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookPS.html)
How to choose which filters to use
The filters of one supplier, Lee Filters, have been investigated to find out which are best for
photosynthesis investigations using algal balls in hydrogen carbonate indicator. A separate excel
spreadsheet is available that contains absorption and transmission data for several filters. These
files can be used to generate absorption / transmission spectra so that individual filters (or
combinations of filters) can be chosen to suit the investigation. Choosing filters that prevent
transmission of light of a suitable wavelength for one or more of the main photosynthetic pigments
should produce reduced photosynthetic rates compared to a control sample in white light.
The filters included are: 104 Deep Amber, 106 Primary Red, 119 Dark Blue, 124 Dark Green, 139
Primary Green, 182 Light Red, 344 Violet, 716 Mikkel Blue and 722 Bray Blue.
If you need more information about what absorbance and transmission mean and how to use the
data in the appendix, a section on using the spectrum data follows.
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Using the filter data to produce absorption and transmission spectra
To produce absorption and/or transmission spectra for individual filters use the accompanying
spreadsheet. Find the tab for the filter of interest and use the data to plot graphs for either
absorbance or transmittance.
Below are absorption and transmission graphs produced using the data for filter 124 Dark Green.
Absorbs light strongly between 400–460nm and between 600–
700nm. These wavelengths will not pass through the filter.
Transmits (allows through) light of between 460560nm. Also transmits light in excess of 750nm.
When these data are compared to the absorption spectra of the main photosynthetic pigments, it
can be seen that the main wavelengths of light absorbed by both chlorophyll a and b, as well as βcarotene, will not be transmitted to a sample wrapped in a 124 Dark Green filter, and we would
expect a low photosynthetic rate for such a sample.
Combining filters
If you want to use two filters, e.g., 124 (Dark Green) and 182 (Light Red), you can use the filter
data to predict the effect of combining the two. You would need to add the absorbance values (at
any given wavelength) and the resulting sum will be the absorbance of the new filter combination.
Sample data
Wavelength
(nm)
402
406
410
414
418
422
Absorbance
Filter 124
1.79
1.85
1.91
1.98
2.05
2.10
Absorbance
Filter 182
2.41
2.39
2.39
2.39
2.39
2.41
Combined
absorbance
4.20
4.24
4.30
4.37
4.44
4.50
It can be difficult to look at absorption spectra and understand what they mean in terms of light that
will be transmitted through the filter. You can see that the absorbance scale has a maximum of
about 4.5 in this particular case. In fact an absorbance value above 2.0 is very high. So very little
light will be passing through this combination of filters. A more useful way of presenting the data
would be to produce a transmission spectrum.
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Photosynthesis with algal balls - post-16 teaching notes p. 8
Transmittance spectra with combined filters
You can use the spreadsheets to calculate the transmittance of different filter combinations.
You can convert the combined absorbance value to transmittance using the following calculation
for each wavelength value.
Transmittance of combined filters = 1/(10 ^ summed absorbance value)
In excel it is best to set up a formula to do this for you, as using the ^ operator (‘to the power of’)
makes this a complicated calculation. If, for example, you wanted the combined transmittance
value to appear in cell E2 of your spreadsheet, and the combined sum was in cell D2, then you
would type the following formula into cell E2: =1/(10^D2).
1
2
3
4
A
B
C
D
E
Wavelength
(nm)
402
406
410
Absorbance
124
1.79
1.85
1.91
Absorbance
182
2.41
2.39
2.39
Combined
absorbance
4.20
4.24
4.30
Combined
Transmittance
0.00
0.00
0.00
For this combination,
transmittance is 0 for
these 3 wavelengths
This would not be a very useful filter combination for photosynthesis
experiments.
None of the main photosynthetic pigments absorb light above 700nm
More about Absorbance and Transmission
This shows a blue filter exposed to green light.
The amount of light falling on the filter (also called the incident light) is
defined as I0, and the amount of light that passes through the filter is
defined as I
The amount of light transmitted in this case to be quite small as blue
filters will tend to absorb most of the green incident light.
The transmittance, T, of the sample is defined as the ratio of transmitted light to incident light: T = l / l0
T will be equal to 1.00 when all of the incident light is allowed through, and if any light is absorbed at all, T
will be less than 1.00. If all of the light is absorbed, then T will be equal to zero.
Most colorimeters, spectrometers and spectrophotometers (devices that measure the amount of light
absorbed or transmitted by a sample) provide data in terms of absorbance (often given the symbol A). The
absorbance of a sample is a measure of the amount of light absorbed by the sample. It follows that
absorbance and transmittance are related to one another but the exact relationship is not simple. The
absorbance of a sample is defined as: A = log10 (l0 / l)
By combining the above 2 expressions, transmittance can be calculated as: T = 1 / 10A
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