RESEARCH FOR BIOLOGY IA

Question: To investigate the effect of different variables on photosynthesis using the floating leaf disk.
nvestigations to determine the effects of light intensity, carbon dioxide concentration
and temperature on the rate of photosynthesis can be carried out using aquatic
plants, such as Elodea or Cabomba (types of pondweed)

Introduction

Every living organism needs energy to grow and reproduce. Humans and animals eat foods with
carbohydrates, proteins, and fats to produce the energy they need to survive. But plants do not eat.
They make their own energy source in the form of energy-rich carbohydrates (sugars) through a
process called photosynthesis. Photosynthesis is a multi-step, enzyme-mediated process that converts
light energy into chemical energy. During photosynthesis, plant cells use light energy (such as light
emitted from the sun), water (H2O), and carbon dioxide (CO2) as reactants to produce sugar
molecules (C6H12O6) and oxygen (O2).

Photosynthesis takes place in the chloroplasts within the plant's cells. The chloroplasts contain special
pigments that react to light. Chlorophyll is one of the pigments that can absorb light in the blue and
red spectrum from the visible light spectrum. Chlorophyll does not absorb light in the green spectrum
of light but reflects it instead. This is why leaves with chlorophyll usually appear green. During the
first part of photosynthesis—the light-dependent reaction—chlorophyll and other pigments harness
the light energy to produce NADPHand ATP, which are two types of energy-carrier molecules. At the
same time, water is split into oxygen (O2) and protons (H+). The next stage is light-independent and
is often referred to as the dark reaction. In this step, the two energy-carrier molecules, NADPH and
ATP, are utilized in a series of chemical reactions called the Calvin cycle. In the Calvin cycle, the
plants take carbon dioxide (CO2) from the air and use it to ultimately make sugars such as glucose or
sucrose. These sugars can be stored for later use by the plant as an energy source to fuel its
metabolism and growth.

Photosynthesis is responsible for replenishing Earth's atmosphere with oxygen that we breathe. Thus,
it is not only crucial for plants, but also for all organisms that rely on oxygen for their survival. Many
factors affect how quickly plants are able to conduct photosynthesis. Without enough light or water,
for example, a plant cannot photosynthesize very quickly. Similarly, the concentration of carbon
dioxide—another reactant in photosynthesis—affects how fast photosynthesis can occur. Temperature
also plays a significant role, as photosynthesis is an enzyme-mediated reaction. This is because at high
temperatures, enzymes can get damaged and thus become inactivated. Other factors that affect the rate
of photosynthesis are the light intensity, the amount of chlorophyll and other color pigments in a plant,
and the color of light.

At low light intensities, as light intensity increases, the rate of the light-dependent reaction,
and therefore photosynthesis generally, increases proportionately (straight line relationship).
The more photons of light that fall on a leaf, the greater the number of chlorophyll molecules
that are ionised and the more ATP and NADPH are generated. Light dependent reactions
use light energy and so are not affected by changes in temperature.

Intensity of light

As light intensity is increased further, however, the rate of photosynthesis is eventually limited by
some other factor. So the rate plateaus. At very high light intensity, chlorophyll may be damaged and
the rate drops steeply (not shown in the graph).

Chlorophyll a is used in both photosystems. The wavelength of light is also important. PSI absorbs
energy most efficiently at 700 nm and PSII at 680 nm. Light with a higher proportion of energy
concentrated in these wavelengths will produce a higher rate of photosynthesis.

Light Intensity

● The higher the light intensity, the higher the rate until a certain point. If you increase the intensity of the light, more
light can be trapped by the chloroplasts to provide energy to drive the photosynthetic reaction.
● At high light intensity, rate levels off. Again, this is up until a certain point, when the maximum amount of light has
already been trapped. When you reach this point, light is no longer a limiting factor.

Limiting factors Affecting the

Rate of Photosynthesis diagram

● You can prove the need for light by testing leaves of a plant left in the dark for 48 hours. You can test the leaves for
starch in the same way as described above and will find that they won’t turn blue-black as no starch is present. The
plant would have used up its starch stores without being able to make more due to the lack of light.

Investigating the Effect of Light Intensity

 ● Vary light intensity. Place a lamp at differing distances from the plant and observe the oxygen production through
the movement of the bubble. This will give you an opportunity to see the effect of light intensity on the rate of
photosynthesis.
● Control the other variables, and repeat for reliability. Control the temperature of the room and the time taken for the
experiment. Make sure to repeat the experiment three times at each distance, then take a mean value in order to
attain more reliable results.

Limiting factors Affecting the

Rate of Photosynthesis

Preparation of the leaf disks:

1) Use the cork borer (to cut out the number of disks needed for your experiment).

2) Put disks in a syringe and suck up 5 cc (5 ml) of .2% sodium bicarbonate (baking
soda)

3) Put finger over end of syringe, pull back on plunger to about the 35 cc mark (on a 60
cc syringe) and hold this position for 30 seconds. You should see air coming out the sides
of the disks. As this is done, the oxygen is being removed from the spongy layer of the
leaf and the .2% sodium bicarbonate is entering the spongy layer. This is the source of
carbon dioxide needed for the plant to carry out photosynthesis

4) Carefully squirt out the .2% sodium bicarbonate. Suck up about 10 cc's of water.
Check to see if the plant disks sink in the water. If they don't, remove the water and try
steps 2 and 3 again.

5) Choose the disks that sink. Make sure enough disks are available to properly complete
a controlled experiment. They are now ready to be used in your experimental set up. The
disks will float when they have produced a measured amount of oxygen through
photosynthesis. The time needed for the disks to float is an indirect measure

of the rate of photosynthesis occurring in the leaf disks.

Materials and Equipment

● Transparent cups, 6 or more (large enough to hold 300 mL)

● Measuring cup

● Water

● 1/8 or 1/4 teaspoon

 ● Baking soda

● Dish soap

● Plant leaves (spinach or ivy work best)

● Single hole puncher or sturdy straw

● Plastic syringe, 10-mL or bigger (without the needle)

● Permanent marker

● Light source (a bright light works best)

● Paper towels

● Timer

● Pencil or pen

Apparatus

● Distilled water

● Test tube

● Beaker

● Lamp

● Aquatic plant, algae or algal beads

● Ruler

● Sodium hydrogen carbonate solution

● Thermometer

● Test tube plug

● Syringe
● To investigate the following:

○ Different light sources: LED, CFL, incandescent, halogen, sunlight

○ Different light intensities: Ruler or measuring tape

○ Different light colors: Colored cellophane sheets, tape

○ Water temperature: Thermometer, microwave, ice cubes

○ Different leaf colors: Yellow, red, and green leaves

○ Different plant types: Leaves from different plant species

● Lab notebook
Experimental Procedure

The following instructions are for three trials:

1. Fill three cups with 300 mL water. Add about 1/8 teaspoon baking soda and 1 drop of liquid
dish soap to each of the cups. Gently stir the solution until everything has dissolved. Try not
to create too many bubbles. Note: The baking soda provides the plant leaves with carbon
dioxide for photosynthesis.
2. Punch out 30 leaf disks from the spinach or ivy leaves using the hole puncher or a straw.
Avoid cutting through major leaf veins.
3. Remove the plunger of the syringe and place 10 leaf disks into the syringe barrel.
4. Place the plunger back into the syringe and push it down until only a small volume of air is
left in the syringe. Be careful not to crush the leaf disks.
5. Suck up a small volume of the baking soda solution from one of the cups into the syringe with
the leaf disks and hold it vertically. The leaf disks should all float on the surface of the
solution.
6. Remove the air pockets within the leaves to make the leaf disks sink, as follows.
7. Carefully push out all the air from the syringe.
8. Close the opening of the syringe with a finger and pull back on the plunger to create a
vacuum. Hold the vacuum for 10–15 seconds and swirl the leaf disks to suspend them in the
solution.
9. Stop pulling on the plunger and remove your finger from the syringe opening to release the
vacuum.
10. Repeat step 6 until all leaf disks have sunk to the bottom of the solution.
11. Remove the plunger from the syringe and pour all 10 leaf disks with the solution into a fresh,
clear plastic cup. Fill the cup with baking soda solution up to a depth of about 3 cm. Label
this cup "1."
12. Repeat steps 3–8 twice more, with 10 leaf disks each, to prepare the other two cups. Label the
other cups "2" and "3."
13. Place all three cups with the leaf disks under your light source. Make sure the light shines
straight onto the cups from above. Each cup should be positioned so that they get an equal
amount of light.
14. Start a timer. Observe closely what happens to the leaf disks. Write your observations down in
your lab notebook.
15. At the end of each minute, record the number of floating disks for each cup in a data table like
Table 1 in your lab notebook. Briefly swirl the cups to prevent the leaf disks from getting
stuck to the bottom or sides of the cups.
16. Continue the experiment until all of the leaf disks are floating.

Method
● Ensure the water is well aerated before use by bubbling air through it
○ This will ensure oxygen gas given off by the plant during the
investigation form bubbles and do not dissolve in the water
● Ensure the plant has been well illuminated before use
○ This will ensure that the plant contains all the enzymes required for
photosynthesis and that any changes of rate are due to the
independent variable
 ● Set up the apparatus in a darkened room
○ Ensure the pondweed is submerged in sodium hydrogen carbonate
solution (1%) – this ensures the pondweed has a controlled supply of
carbon dioxide (a reactant in photosynthesis)
● Cut the stem of the pondweed cleanly just before placing into the boiling tube
● Measure the volume of gas collected in the gas-syringe in a set period of time
(eg. 5 minutes)
● Change the independent variable (ie. change the light intensity, carbon dioxide
concentration or temperature depending on which limiting factor you are
investigating) and repeat step 5
● Record the results in a table and plot a graph of volume of oxygen produced
per minute against the distance from the lamp (if investigating light intensity),
carbon dioxide concentration, or temperature

Results - Light Intensity

● The closer the lamp, the higher the light intensity (intensity ∝ 1/d2)
● Therefore, the volume of oxygen produced should increase as the light
intensity is increased
● At a point, the volume of oxygen produced will stop changing even if the light
is moved closer
○ This is when the light stops being the limiting factor and the
temperature or concentration of carbon dioxide is limiting the rate of
photosynthesis
○ The effect of these variables could then be measured by increasing the
temperature of water (by using a water bath) or increasing the
concentration of sodium hydrogen carbonate respectively
● The results should be displayed on a graph of light intensity vs. rate of
photosynthesis
○ Rate of photosynthesis = volume of oxygen produced ÷ time elapsed

Limitations

● Algae is often used in experiments on photosynthesis and respiration rates

but it can be very hard to maintain consistency in the number of algae and it
can be hard to handle directly in the water
○ Immobilised algae beads are beads of jelly with a known surface area
and volume that contain algae, therefore it is easier to ensure a
standard quantity
○ Immobilised algae beads are easy and cheap to grow, they are also
easy to keep alive for several weeks and can be reused in different
experiments
○ The method is the same for algae beads though it is important to
ensure sufficient light coverage for all beads