Léane Dépret

Marie Vandaele
Group 241

Report of practical courses on photosynthesis

1) Introduction

Plants are organisms autotrophic to carbon capable of performing photosynthesis. Photosynthesis is a

transformation of light energy into chemical energy. It consists of the water oxidation thanks to the energy from
solar radiations leading to a synthesis of O2 and the assimilation of CO2 from the atmosphere in order to reduce
it into carbohydrates such as glucose.
Photosynthesis takes place in the chloroplasts which are specific organelles of plant cells. To be more precise, it
occurs in the thylakoids membrane (for the light phase) where the cytochromes, photosystems I and II, and a set
of protein complexes associated with pigments are found and capable of converting light energy into chemical
energy. The dark phase involving the synthesis of carbohydrates occurs in the stroma of chloroplasts.
We learned during our lectures that photosynthesis depends on many different parameters.
Therefore, we can wonder what internal and environmental factors allow an optimal photosynthesis.
To do so, we did two different tutorials in order to answer our problem. The objectives of the practical
courses were to determine which pigments are involved in photosynthesis, to demonstrate the process of starch
synthesis and to identify the different environmental parameters that impact photosynthesis. We used different
plant organs according to their properties. Indeed we took Petroselinum crispum and Elodea canadensis leaves
because they contain photosynthetic pigments and are the place where photosynthesis occurs. We also used
Solanum tuberosum tuber since it is the place where starch is stored and it contains lots of it.

2) Material used and method

A) Photosynthetic pigments experiment

Material used

We would like to determine which pigments are involved in photosynthesis. Therefore we made a
chromatography on Whatman paper.

- Solution (solvant) made of 85% of petroleum ether, 10 % of acetone and 5% of cyclohexane

- Whatman paper
- A shredded material composed of a mix of parsley (Petroselinum crispum) and acetone
- A large glass tube with a cap and a hook fixed on it
- A spectrophotometer

Method:

Ten minutes before starting the chromatography, we prepared a tube which contained 15 ml of solvent in
order to saturate the tube's internal atmosphere. During this time, we drew three different landmarks on the
Whatman paper : the first line is where the solvent reaches the paper, the second is where the sample is placed
and the third is where the solvent reaches its limit and which will indicate the end of the experiment. We drilled a
hole on the top of the paper to hang it on the hook. We deposited around 20 drops of sample on line 2. Once the
paper is ready, it is held by the hook of the stopper of the glass tube and immersed in the solvent. The tube
should be capped tightly and placed in a tube rack under a fume hood. After about an hour, when the transfer
was complete, we removed the paper from the solution and underlined the end of each dye transfer. After
validation by the teacher, we cutted the last spot of each pigment and deposited it in different tubes in common
with the pigments of the other groups in acetone. It will allow us to create the absorption spectrum by
spectrophotometry.

B) Starch synthesis experiment

We would like to determine if the starch catalysis is due to enzymes or due to a chemical reaction.
Material used:

- Lugol
- A starch dispersion made from a solution of a potato tuber (Solanum tuberosum) and water
- A solution at 10mg/mL of glucose 1-phosphate
- Water
- Glass tubes
- Spectrophotometer

Method:

We used potato tuber because the tuber stores the most starch and the solution was already prepared.
First we needed to determine the maximum absorption of our sample and to do that we took 5mL of it
which we mixed with 0.2mL of lugol. Then, we made an absorption spectrum between 400 and 700 nm with a
spectrophotometer to determine the maximum that we will use for the rest of the experiment.
Second we prepared 6 different tubes containing each one 1 mL of the starch dispersion centrifuged at
12000 g, 4°C for 5 minutes and 1mL of the glucose 1-phosphate solution. These tubes correspond to the group
A. We took another tube in which we added 1mL of the starch dispersion which was incubated at 100°C for 5 min
and we mixed it with 1mL of the glucose 1-phosphate solution. The unique tube is the group B. The tubes are
maintained in the ice during the process.
Finally we incubated the 7 tubes into water at 38°C during different amounts of time (from 0 for A0 to 30
min for A30 and B30). When the tube is removed from the incubator we placed it in ice to stop the reaction and
lastly we added 7,5 mL of water and 0,5mL of lugol, a solution that we prepared before which allows the
highlighting of the starch that was catalyzed in each tube. When we finished mixing the two solutions we made
the absorbance’s measurement quickly at the maximum lambda to deduce a curve thanks to the mean of the
group’s value.

C) Environmental parameters experiment

We would like to determine the influence of different environmental parameters such as light intensity, CO2
concentration and light quality on photosynthesis.

Material used

- A bioreactor containing Elodea (Elodea canadensis) fragments

- The ExAO interface
- A computer with the associated software
- An oximeter probe
- A light meter
- LED source (green, red, blue and white)
- A reflective mirror
- Light source (lamp)
- Water
- 1%, 0.5% and 0.1% potassium bicarbonate buffered solution

Method

To begin we added the 0.5% potassium bicarbonate buffered solution in the central tank of the
bioreactor. Then we cutted 532.1 mg of Elodea from the aquarium in order to put it in the bioreactor. The
bicarbonate solution must have a final volume of 20 mL. We also filled the thermostatic enclosure with water to
prevent the plant material from overheating. We finally introduced the oximeter probe inside the central tank.
Then we varied the light intensity by making measurements at three different distances between the
light source and the bioreactor : 30 cm, 20 cm and 10 cm. About the CO2 concentration, we used the 1%, 0.5%
and 0.1% potassium bicarbonate buffered solution. And finally for the light quality, we placed the LED source on
the bioreactor and the reflective mirror in front of it. We took off the oximeter probe to replace it with a light meter
and we measured four different light colors (green, red, blue and white). We then measured the photosynthetic
activities by using the oximeter probe.
 3) Results

A) Photosynthetic pigments experiment:

We know that in order to determine a type of pigment we need three factors : its color on the chromatography, its
Rf (frontal rapport) and its absorption spectrum. First, we were able to identify the different colors of the pigments
on the chromatography (Fig.0). We were not able to see the pigment n°3 on our chromatography.

Figure 0 : Results of the chromatography on Whatman paper

Second we calculated the Rf (solvent migration distance/pigment migration distance) for each of the 4 pigments.
We obtained these results :

Rf1 = 5.9 cm /18.5 cm = 0,319 Rf2 = 0,481 Rf3 (missing) Rf4 = 0,703 Rf5 = 0,934

And finally we obtained 5 absorption spectra thanks to the spectrophotometer (Fig.1).

Pigment 1 Pigment 2 Pigment 3

Pigment 4 Pigment 5

Figure 1 : Absorption spectra of the five pigments

B) Starch synthesis experiment

Thanks to the spectrophotometer, we obtained a graph of the maximum absorption of the dispersion sample
between 400 and 700 nm (Fig.2). Therefore we could use our maximum of absorption (591 nm) for the
continuation of the experience.

Figure 2 : Graph of the maximum of absorption of the dispersion sample between 400 and 700 nm
 Then we measured the absorbance of the seven samples at 591 nm (Fig.3). We also were able to make the
average between every group's values in order to get more precision. We finally drew a graph of those mean
absorbances (Fig.4).

Enzymatic extract Extract A Extract B

Incubation time (in minutes) 0 5 10 15 20 30 30

Absorbances at λmax (591 0,182 0,293 0,341 0,520 0,817 1,391 -0,021
nm)

Averages absorbances at 0,137 0,2045 0,314 0,488 0,762 1,311 -0,0025

λmax

Figure 3 : Table of absorbances at 591 nm according to the incubation time

Figure 4 : Graph of mean absorbances of the starch dispersion according to the incubation time

C) Environmental parameters experiment

Light intensity variation

During the three experiments we noted at each time our leading coefficient in light and in dark according to the
distance. We obtained different values (Fig.5).

Distance between the bioreactor and the Leading coefficient a in light (in Leading coefficient a in dark (in
lamp mgO2.L-1.min-1) mgO2.L-1.min-1)

30 cm 190e-6 -488e-6

20 cm 646e-6 -189e-6

10 cm 785e-6 -59.5e-6

Figure 5 : Table of the leading coefficient in dark and light (in mgO2.L-1.min-1) according to the distance (in cm)

In order to draw the graph PNI=f(1/d2) (Fig.7) we needed to convert our leading coefficient in mgO2.L-1.min-1
into the Photosynthetic Net Intensity (PNI) in mgO2.min-1.g-1FM. To do so, we multiplied our precedent a by our
volume (20 mL) and divided it by our quantity of Elodea (532.1 mg) (Fig.6). For example, for the leading
coefficient at 30 cm we have :
PNI = 190e-6*20e-3/0.5321 = 7.14e-6 mgO2.min-1.g-1FM.

In order to calculate the Photosynthetic Gross Intensity we used the formula : PGI = RI + PNI
For example, PGI at 10 cm = -2.24e-6 + 2.95e-5 = 2.73e-5 mgO2.s-1.g-1FW

Distance between the PNI : Photosynthetic RI : PGI : Photosynthetic Light intensity : 1/d2
bioreactor and the lamp Net Intensity Respiratory Intensity Gross Intensity (cm-2)
(mgO2.s-1 (mgO2.s-1 (mgO2.s-1
.g-1FW) .g-1FW) .g-1FW)

30 cm 7.14e-6 -1.83e-5 -1.12e-5 0.01

 20 cm 2.43e-5 -7.10e-6 1.42e-5 0.0025

10 cm 2.95e-5 -2.24e-6 2.73e-5 0.0011

Figure 6 : Table of the PNI, RI and PGI (in mgO2.min-1.g-1FM) according to the distance (in cm)

Figure 7 : Graph of the PNI (in mgO2.min-1.g-1FM)

according to the light intensity (in cm-2)

CO2 concentration variation

During the three experiments we noted at each time our leading coefficient in light and in dark according to the
concentration of the potassium bicarbonate buffered solution (CO2). We did the same calculations as before in
order to get our PNIs, RIs and PGIs. We obtained the values below (Fig.8).

Concentration of the PNI : RI : PGI :

potassium bicarbonate Photosynthetic Net Intensity Respiratory Intensity Photosynthetic Gross
buffered solution (in %) (mgO2.min-1.g-1FM) (mgO2.s-1.g-1FW) Intensity (mgO2.s-1.g-1FW)

1% 2.63e-5 -1.98e-5 6.5e-6

0.5% 2.95e-5 -7.10e-6 2.24e-5

0.1% 3.95e-6 -1.27e-5 1.67e-5

Figure 8 : Table of the PNI, RI and PGI (in mgO2.min-1.g-1FM) according to the concentration of CO2 (in %)

Therefore we were able to draw the graph PNI=f([CO2]) (Fig.9).

Figure 9 : Graph of the PNI (in mgO2.min-1.g-1FM)

according to the concentration of CO2 (in %)

Light quality variation

We are searching for the optimal wavelengths for photosynthesis. Therefore we did the same calculations as
above but using four types of lights and we obtained the values below (Fig.10).
In order to calculate PGIc we used the formula : PGIc = PGI*(White Light Lux / Coloured Light Lux).
For example : PGIc in blue = − 2,35e-4*(1226/627) = −4,59e-4 mgO2.s-1.g-1FW

Light quality Lux measure RI PNI PGI PGIc

(nm) (lux) (mgO2.s-1.g-1FW) (mgO2.s-1.g-1FW) (mgO2.s-1.g-1FW) (mgO2.s-1.g-1FW)

White 1226 - - - -
 Blue (470 nm) 627 -892e-6 657e-6 -2.35e-4 -4.59e-4

Green (527 nm) 462.44 -1.45e-3 189e-6 -1.261e-3 -3.34e-3

Red (624 nm) 697.34 -1.03e-3 738e-6 -3.32e-4 -5.84e-4

Figure 10 : Table of the PNI, RI, PGI and PGIc (in mgO2.min-1.g-1FM) according to the light quality (in nm)

We were thus able to build the histogram PGIc=f(Light quality) (Fig.11)..0

Figure 11 : Graph of the PGIc (in mgO2.min-1.g-1FM) according to the light quality (in nm)

4) Discussion

A) Photosynthetic pigments experiment:

Thanks to the results we obtained, we are able to identify the pigments implied in photosynthesis. About
the color, pigments n°1 and 2 are both green so we can suppose that they are different types of chlorophyll.
Pigments n°4 and 5 are orange/yellow allowing us to deduce that perhaps they are carotenoids.
First, we will compare pigment 1 and pigment 2 since they both have quite the same color. Since they
are green, we hypothesize that they are chlorophyll but we don’t know which one is chlorophyll a and which one
is chlorophyll b. When we look at their absorption spectrums, we observe in both cases two peaks.The first peak
is in blue between 420 and 480 nm and the second one is in red around 660 nm. The two peaks are more
internal for pigment 1 and more external for pigment 2. Furthermore, we know that chlorophyll b is more polar
than chlorophyll a because of the CHO group present on the pyrol B instead of a CH3 in chlorophyll a. Therefore
chlorophyll b being more polar will migrate less than chlorophyll a and then have a Rf smaller than chlorophyll a.
Therefore we can conclude that pigment 1 is chlorophyll b and pigment 2 is chlorophyll a. Chlorophyll b is known
to absorb between 430 and 660 nm as we can relate on the graph and chlorophyll b absorbs between 450 and
650 nm.
Second, pigments 3, 4 and 5 are quite similar in terms of the color (yellow/orange) and of the absorption
spectrum. Indeed, they all have three peaks between 400 and 500 nm characteristic of carotenoids. However,
they don’t have the same migration on the chromatography. Indeed, we know that according to their structure,
xanthophylls are more polar than beta-carotenes since they possess OH groups. Therefore xanthophylls will
migrate less than beta-carotene. We can conclude that pigment 3, 4 and 5 are carotenoids and pigment 4 is
xanthophyll and 5 is beta-carotene. Because we could not get our pigment 3 on our chromatography, we can
hypothesize that it is a xanthophyll since it migrates less than pigment 4 and 5. Carotenoids absorb between 420
and 460 nm.

B) Starch synthesis experiment

We made the starch synthesis experiment in order to measure the appearance of the lugol-starch
complex in the solutions. First we know that there is a linear relation between the absorbance A and the
concentration of the complex in solution. This relation is the Beer-Lambert law written as being : A = ε*c*l
with A the absorbance, ε the molar extinction coefficient (in M-1.cm-1), c the chromogen concentration
(in M-1) and l the optical path (in cm). Because ε and l are both constant, only the chromogen concentration can
vary the absorbance. Therefore if the concentration of the lugol-starch complex increases, the absorbance will
increase too.
We used a control solution (solution B) made of the starch solution but boiled at 100°C in order to
determine if the starch catalysis is biochemical or chemical. Indeed boiling the solution allowed the possibility to
denature proteins. If there is no starch production in the control solution, the reaction would be possible thanks to
enzymes.
When we look at the graph A=f(time) (Fig.4) we can first see that the absorbance at 0 min is not equal to
0 because starch was already present in the potato tuber but it was not synthesized during the reaction. Second
we can note that the absorbance depends on time. When time increases, the absorbance increases too. Thus
the concentration of starch increases over time. And third we notice that the starch production is zero in the
boiled solution allowing us to deduce that the absence of enzymes could not catalyze any starch.
To conclude, the starch formation of the potato tuber is a spontaneous reaction due to proteins because
the more time we waited, the more starch was created and a boiled solution could not synthesize any starch.
Thus the starch synthesis is a biochemical reaction and not a chemical one.

C) Environmental parameters experiment

Three different environmental parameters have been tested in order to optimize photosynthesis : light
intensity, CO2 concentration and light quality.
For the light intensity we can see that the more light there is, the more O2 will be released. First It
means that light is essential for the O2 release and it is a limiting factor. Second, the quantity of O2 depends on
the intensity of light. However we can suppose that if the light intensity is too strong, it might saturate the
photosystems, PNI will form a tray, and the PNI might go down if they are getting degraded. We can define the
compensation point as being the point where all O2 produced by photosynthesis is consumed by respiration. We
can deduce it where the curve crosses the x axis. Since our curve is always above zero, we found our
compensation point by extending it. Thus the compensation point is nearly 5.45e-4 cm-2.
For CO2 concentration, we can observe on the graph that the more CO2 there is in the medium, the
more O2 production there will be. However after a certain point (when the concentration is superior to 0.5%) the
concentration of O2 decreases. Therefore the concentration of CO2 has an impact on photosynthesis as it
increases the photosynthetic activity but also decreases it if too concentrated.
Finally about the quality of light, we can observe that the plant reacted differently to the different
wavelengths. Indeed as we can notice on the action spectrum or histogram, photosynthesis intensity is much
stronger when exposed to blue and red light than to green light. Furthermore, photosynthesis is a bit better when
there is blue light instead of red light. This can be correlated to the absorption spectrum of the pigments we saw
earlier. As a matter of fact, chlorophylls and carotenoids absorb more in blue light meaning that photosynthesis is
more efficient in blue. Then, chlorophylls absorb also in red thus photosynthesis also works in red. However no
absorption in green or just a few can be noted. This is the reason why plants are green since they reflect green
light instead of absorbing it. Therefore it is important for plants to absorb blue and red light in order to optimize
their photosynthesis.

5) Conclusion

The goal of the practical courses was to determine the internal and environmental factors allowing an optimal
photosynthesis. We noticed that photosynthesis depends on the composition of pigments and the presence of
enzymes. The most important pigments performing photosynthesis are chlorophylls (a and b) and carotenoids
(xanthophylls and beta-carotene). Enzymes catalyze the starch production essential for the storage of glucose for
the plant’s growth. Those are internal factors. For the environmental factors, we first realized that light is essential
for the O2 synthesis. The increase of its intensity allows it to perform better photosynthesis. Second, we
discovered that the concentration of CO2 also has an impact because it increases the O2 release but if it is
present in a huge quantity, it will be harmful for the plant. And finally the light wavelengths have an impact on
photosynthesis. Blue and red wavelengths will increase the photosynthetic activity while green light is not really
absorbed by plants.

6) Bibliography

- Mr Hilbert’s courses (light and dark phase of photosynthesis)

- Mr Neutelings tutorials (Relation between pigments and photosynthesis activity, photosynthesis energy)