Analyzing the metabolic process of

photosynthesis

Katharine Lahmann
322442 Biology 199 (L5)
February 17, 2010 Chris Hall
Katharine Lahmann
322442
February 17, 2010

Analyzing the metabolic process of photosynthesis

In order to understand the process of photosynthesis we must first look at the small, green
colored molecule, chlorophyll, and the sites of photosynthesis. These molecules are found within the
grana of the chloroplasts in organisms such as plants, green algae and cyanobacteria (Dolphin 2008).
The process of photosynthesis enables plants to utilize light energy and convert it in to a more usable
form of carbohydrate molecules (ATP). This is a common energy currency between virtually every
organism in the ecosystem.

6 CO2(g) + 12H2O + Chlorophyll → C6H12O6 + 6O2(g)

The leaves on plants are appendages that increase the organism's amount of light received
and thus increasing the amount of ATP, NADPH and Oxygen produced. Oxygen is released from the
photosynthetic reaction and can be found within the intercellular spaces (W. Worrall and H.Zandberg,
2008).

The process of photosynthesis takes place in two steps. The first is when the light energy is
converted in to chemical energy in the chlorophyll; the chlorophyll then transfers the energy to the
electrons for cellular work such as carbon dioxide to create organic molecules and used to make ATP.
The higher energy electrons can be made into NADPH from NADH and the by-product is oxygen
(Dolphin, 2008).
This is also known as the light reactions. The second step is carbon fixation (Calvin cycle). This is
there is a usage of energy and hydrogen to attach Carbon dioxide molecules to already existent
organic molecules from the light reactions (Dolphin, 2008). They then break off in to sugar molecules
for the organisms to use.

They also help to facilitate gas exchange between Carbon dioxide and Oxygen.
The purpose of these experiments is to make an indirect measurement of the overall photosynthetic
rate (W. Worrall and H. Zanberg, 2008). This measurement can be done by looking at the amount of
Oxygen produced. This experiment analyzes the cellular respiration that occurs within the leaves
when exposed to a particular light intensity. The questions that one might attempt to answer in this lab
are, will the photosynthetic rate increase/decrease when one uses a light source with a higher
wavelength? What will happen to the oxygen production? Will the photosynthetic rate
increase/decrease with respect to the distance of the light source in the experiment? One would
hypothesize that the as the intensity of the light increases, so does the photosynthetic rate and thus
making the production of oxygen greater.
Materials and Methods:

Extraction procedure:

60g of frozen spinach leaves and 150ml of water containing 0.2g of MgO powder was placed
into a blender to homogenize the plant tissue to extract Chlorophyll. It was then placed in to a 500 ml
beaker along with 150 ml acetone and covered with watch glass and foil. The mixture was then stirred
for 10-15 minutes in a magnetic stirrer (Dolphin, 2008). The solution was then separated by pouring
the mixture into a beaker layered with cheesecloth. This is called solvent partitioning where
contaminating compounds are partially removed. If we wanted make the extract contain less
contaminants then the process would be performed a couple of times over but for this experiment it
will only be done once.

50 ml of filtrate, 50 ml of petroleum ether and 25 ml of 10% NaCl was added in to a funnel for
further separation (Dolphin, 2008). The mixture is then shaken so that chlorophyll will be retrieved
(Dolphin, 2008). The funnel was then inverted after capped and vented so that the pressure doesn't
build up. The funnel was placed on a ring and let to sit so to allow both solvent solutions to undergo
separation. (Dolphin, 2008). At the bottom of the funnel there is a polar aqueous solvent and should
is released periodically by loosening the stopper (Dolphin, 2008). This was so one could obtain the
top portion which is the non-polar chlorophyll solution. It took approximately 15 minutes.

Draining the separated chlorophyll solution

30 ml of 80% methanol and 15 ml of NaCl was added in to the flask and shaken. The content
of the lower level were drained off (Dolphin, 2008). Then 30 ml of NaCl was added to the flask,
shaken once more and the remaining acetate and methanol was removed and discarded of (Dolphin,
2008). The result the past procedures was a bright green solution, which was concentrated
chlorophyll.

Any traces of water were removed by adding 2g of Na2(S04) to the concentrate (Dolphin,
2008).. It was then filtered off before the wavelength readings were done in the spectrophotometer
(Dolphin, 2008).

Absorption Spectrum

The spectrophotometer was then zeroed using pure petroleum ether (Dolphin, 2008).
The concentrate chlorophyll extract was then put into a tube and read in absorbance intervals of 20
nm zeroing after each time a new wavelength is calculated and recorded according to Table 26.1
(Dolphin, 2008). The light color was found by the insertion of a paper strip in to a test tube and then
placed in to the spectrophotometer. The wavelength was then changed and the procedure was
repeated.

Light Intensity and Photosynthetic Rate

The measurement of aerobic respiration was taken indirectly. The procedure in the lab manual
(Dolphin, 2008) was not followed in analyzing the photosynthetic rate in comparison to light intensity.

Preparation of solutions

A solution of 0.2% sodium bicarbonate with a few drops of concentrated detergent and a
control solution (deionized water with the addition of a few concentrated detergent drops) were
prepared in advance.
Preparation of the Leaf Disks

Three plastic cups were labeled numbers 1 through 3. A 40 ml beaker was labeled as thes
control. 40 disks were then cut out of a single spinach leaf by using the bottom end of a pencil so that
they were all the same size (W. Worrall and H.Zandberg, 2008).

Control

10 disks were then placed at the bottom of the syringe and 8.0 ml of control solution was drawn
in to the syringe (W. Worrall and H.Zandberg, 2008). The air at the top of the syringe was removed
and a finger was placed over top of the opening of the syringe and created a vacuum (W. Worrall and
H.Zandberg, 2008). If the leaves do not sink at first try lightly tapping on the syringe or performing the
step again. If leaves are still buoyant then add another drop of bicarbonate solution mixing well (W.
Worrall and H.Zandberg, 2008).

This was shaken and held for 10 seconds and then released in to a 40 ml beaker. It was then
placed in to a drawer so that no light would be in contact with the contents within the beaker.

Experimental

The procedure was repeated 3 more times only placing each new set in to a different cup and
drawing up 10 ml of the designated solution (bicarbonate solution instead of the control solution) (W.
Worrall and H.Zandberg, 2008). If the leaves do not sink at first try lightly tapping on the syringe or
performing the step again. If leaves are still buoyant then add another drop of bicarbonate solution
mixing well (W. Worrall and H.Zandberg, 2008).

Measuring the Effect of Light Intensity on the Photosynthetic Rate

The control beaker and cup #1 were placed beside each other in in the middle of the white
sheet of paper which covered the metal base of the light source (W. Worrall and H.Zandberg, 2008).
The remaining two cups where then placed in the drawer for later use. The light was then
placed 20 cm above the stand of the lamp to the bottom of the light bulb (W. Worrall and
H.Zandberg, 2008).

A stop watch began counting every 30 seconds from the point where the light was
turned on (W. Worrall and H.Zandberg, 2008). Three tests were taken each time altering the
distance between the cup and the light source. The first test was run at a distance of 20 cm.
The second was 40 cm, and the third was 55cm. The 20 cm test was with cup #1, the test of
40 cm with cup #2, and the 55 cm test was done using cup #3 (W. Worrall and H.Zandberg,
2008). The number of disks in both the control and the experimental cup were recorded in
table 26.2, 26.3 and in table 26.4.
Results:

Table 26.1: Light Absorption Characteristics of Chlorophyll

Wavelength (nm) Light Color Absorbance

420 Violet 1.054
440 Violet 0.941
460 Indigo 0.687
480 Indigo 0.315
500 Blue 0.061
520 Blue 0.028
540 Green 0.041
560 Green 0.057
580 Yellow 0.087
600 Yellow 0.0112
620 Orange 0.145
640 Orange 0.246
660 Red 0.651
680 Red 0.137

Table 26.2: Results from Suha Handal

Time (Minutes)

Figure 1: Number of disks floating at a Light Intensity of 20 cm

Exact time 5 floated: 3.45 minutes

Table 26.3: Results from Suha Handal

Time (minutes)

Figure 1: Number of disks floating at a Light Intensity of 40 cm

Exact time 5 floated: 8.04 minutes

Table 26.4: Results from Suha Handal

Time (minutes)

Figure 1: Number of disks floating at a Light Intensity of 55 cm

Exact time 5 floated: 6.43 minutes
Discussion:

Figure 4: The cross sectional anatomy of a leaf

The figure above (Figure 4) gives us a visual of the inside of the intercellular space
where gas exchange/ carbon fixation occurs.

The leaves are the photosynthetic organs of plants where energy is created to be used
by the plants itself and other organisms. The fascinating process of photosynthesis is the
conversation of light energy into chemical energy, or, ATP which is converted into sugars;
fructose and glucose. Within the intercellular spaces within a leaf, the exchange of carbon
dioxide and oxygen take place leaving the conclusion that leaves stay afloat because their
intercellular spaces are filled with oxygen. By vacuuming the leaves, the oxygen is drawn out
of the intracellular spaces and the leaves sink.

In the experiments, the bicarbonate solution was used to replace the carbon dioxide that
occurs normally during this process; and the soap to allow the access of the ionic solution
past the hydrophobic layer (Dolphin, 2008). Light was then added to initiate photosynthesis at
the molecular level. Water is essential component to performing photosynthesis because the
hydrogen ions that are found within the water are ripped off when energized and absorbed into
the electron transport chain. These electrons are then used to create the energy molecule, ATP.

The results that were obtained proved the hypothesis to be true. ‘As the light intensity
increases; the production of oxygen increases’ and thus saying overall that the photosynthetic
rate increases. Table 26.2 shows the light intensity at a distance of 20 cm. If one compared the
results from that table to the results in table 26.3 ( at 40 cm) and table 26.4 ( at 55 cm) it is
shown that the number of leaves that are able to float to the top, for example, at 4.5 minutes,
decrease the farther away the light source is. The more distance that there is between the
autotrophic organism and the light source the lesser the rate of oxygen production will be.

For an individual investigation I wanted to alter the light source to a higher wavelength
to see if this too would increase the photosynthetic rate. My results confirmed this hypothesis
and the results for this experiment performed with a blue light at a light intensity of 20 cm can
be found on table 4a and table 4b. Two trials were performed to ensure that the results
obtained were accurate.

According to the graphed results for the photosynthetic rate versus distance, there is a
light compensation point for spinach at approximately 8.80 minutes. At this time there would
be no excess, or, no net oxygen being produced by the organism. That is, there is only enough
oxygen produced to suffice the plants aerobic processes. We can then say then that the graph
is a visual for when the oxygen production is equal to the rate of consumption (Dolphin, 2008).

In all of the experiments one might notice that there was a control tube that was used
alongside each of the experiments. The purpose of using the control tube was for any other
factors that might have affected the experiment. Some factors would include the surrounding
temperature and the way that the disks were prepared. If they were all taken from the same leaf
and things like that. In the process of photosynthesis, the factor of heat would have increased
the rate of oxygen production and thereby causing more of the leaves to rise. The control was
there to ensure that heat would not be a contributing factor for if it was; the leaves within the
control tube would rise also and the experiment would have to be performed again.
Table 4a: Open Inquiry Trial 1: Light intensity at 20cm Table 4b: Open Inquiry Trial 2: Light
intensity at 20cm

Time # Of disks # Of disks

(Minutes) floating in floating in
control experimental
0.5 0 0
1 0 0
1.5 0 0
2 0 0
2.5 0 0
3 0 0
3.5 0 0
4 0 1
4.5 0 1
5 0 3
5.5 0 5
6 0 6
6.5 0 7
7 0 9
7.5 0 10

Exact time 6.31 minutes

where 5
floated:
 Time # Of disks # Of disks
(Minutes) floating in floating in
control experimental
 0.5 0 0
1 0 0
1.5 0 0
2 0 0
2.5 0 0
3 0 0
3.5 0 0
4 0 1
4.5 0 2
5 0 2
5.5 0 2
6 0 4
6.5 0 6
7 0 8
7.5 0 8
8 0 9
8.5 0 10

Exact time 6.31 minutes

where 5
floated:

Literature Cited:

Campbell, N.A 2009 Biology

Eighth Ed. 2008
Pearson Education, Inc. Publishing as Pearson Benjamin Cummings.

Dolphin, W.D 2008 Biological Investigations: Form, Function, Diversity & Process
Eighth Ed. McGraw-Hill New York

Tompkins, S. and Hewitson, J. (2007). What happens to leaf disks at the compensation point? Science
and Plants for Schools; Questions and Answer Archive. Accessed online August 2007 at http://www-
saps.plantsci.cam.ac.uk/records/rec316.htm

Williamson, B. (2007). The Floating Leaf Disk Assay for Investigating Photosynthesis. Exploring Life
Community. Accessed online August 2007 at http://www.elbiology.com/labtools/Leafdisk.html