John Osborne

February 2018
1. INTRODUCTION
This is more than just the preamble to a lab investigation; it is a route to understanding what an APES
student needs to know about photosynthesis and the linear flow of energy through biological systems.

1.1 WHAT IS PHOTOSYNTHESIS?

Photosynthesis is the metabolic process used by all autotrophs to capture light energy and convert it to
chemical energy in carbohydrates. Low energy compounds, water and carbon dioxide are combined to
form high energy compounds, carbohydrates. Although numerous intermediary reactions are involved, the
overall photosynthetic reaction is simple enough to describe:

6CO2 + 6H2O --------> C6H12O6 + 6O2

The carbon and oxygen in carbon dioxide (CO2) combine with the hydrogen gained from splitting
water (H2O) yielding a carbohydrate (CHO), the 6-carbon sugar glucose, and oxygen gas.
Photosynthesis in chloroplasts occurs in two stages:

1. LIGHT DEPENDENT STAGE,

when photons of light energy are captured by
chlorophyll and other pigments in chloroplasts,
and used to split water into hydrogen and
oxygen. The oxygen is given off (a waste
byproduct) and the hydrogen is carried into the
second stage. Some ATP is also generated and
is used to help drive the second stage.

2. LIGHT INDEPENDENT STAGE,

when the hydrogen is reacted with carbon
dioxide to synthesise energy-rich sugars. This
http://hyperphysics.phy- is the Calvin cycle.
astr.gsu.edu/hbase/Biology/calvin.html

1.2 THE EFFICIENCY OF PHOTOSYNTHESIS & PLANT ADAPTATIONS

1.2.1 Optimising the rate of photosynthesis

Surprisingly photosynthesis is a remarkably inefficient reaction and manages to convert a maximum of
2% of available, solar, light energy into chemical energy. Less efficient than solar panels! Why is this?
Only 45% of solar light falls within the necessary wavelengths; considerable light is reflected from
plants; inefficiency of photosynthetic reactions - heat loss; and some light is transformed into long
wavelength heat.

Plants have evolved a wide variety of adaptations to help them optimise photosynthesis. Some of these
adaptations include leaf shape, arrangement and orientation, the presence of satellite pigments to capture
a broader spectrum of light wavelengths, different photosynthetic chemistry, means of growing upwards
faster and higher to obtain more light, and the use of toxins or mechanical devices to prevent leaves being
eaten by herbivores. Conversely some plants need special adaptations to reduce the damaging risks
present in intense or prolonged light. U/V wavelengths can be just as damaging to plant cells and DNA as
it is to human skin cells. Desert plants, for instance often have highly reflective leaves and can orientate
their leaves away from the sun.

1.2.2 The Law of Limiting Factors in Photosynthesis

Photosynthesis and the rate at which it occurs, is a fine example of the law of limiting factors at work.
The reactants necessary for photosynthesis are water and carbon dioxide; light is also necessary.
Whichever of the three is in shortest supply, will determine the speed at which the reaction can occur.
You could shine all the light you wanted on the leaves of a plant but, if they are not sufficiently irrigated
or not surrounded by sufficient carbon dioxide, the rate of photosynthesis will not be optimum. Water is
seldom an issue for photosynthesis - in water shortages, plants quickly become non-functional. Although
not a reactant, temperature plays an important part in determining the rate of photosynthesis.
Horticulturalists and plant growers are careful to manipulate temperature, carbon dioxide, light and water,
so that optimum photosynthesis is achieved.

Look carefully at the graph on the next page and see if you can interpret exactly what it is showing.
1.2.3 The Importance of Photosynthesis
Photosynthesis is not something plants just do for no particular reason! Every single organism that now
inhabits the planet Earth and every single organism that ever existed has/had a fundamental need for
energy in a chemically stored form which can later be exploited by cell respiration, to allow the organism
to function.

Because energy cannot be created, all of Earth's organisms were or are, either directly dependent upon
photosynthesis for their method of harvesting the sun's light energy and transforming it into chemical
energy, or indirectly dependent upon photosynthesis because they are consumers of photosynthesisers
(green plants or producers or autotrophs), which have stores of chemical energy in their cells.

Another essential understanding is that

energy flows through systems. It does not
cycle. And, as it flows from one level to
another of a system, considerable amounts of
energy are 'lost', usually as heat, so the
available quantity of energy diminishes.
Energy does not and cannot cycle; you
cannot create more energy along the way and
you cannot recover 'lost' energy. What this
means to us is that energy in a natural system
is finite and must be replaced by capturing
more of the sun's energy in photosynthesis.
1.2.4 The Laws of thermodynamics and photosynthesis
So we must think also of the efficiency of photosynthesis and the linear flow of energy through biological
systems in the context of the LAWS OF THERMODYNAMICS:

1. LAW 1: CONSERVATION OF ENERGY: Energy cannot be created nor destroyed but it can be
transformed from one form to another.
So, in photosynthesis, light energy is being transformed from light to chemical energy. In cellular
respiration, the transformation is from chemical to chemical (energy in glucose to energy in ATP)

2. LAW 2: TRANSFORMATION OF ENERGY: Energy transformations always lead to the 'loss' of

energy, usually in the form of heat. This can also be thought of as increased entropy.
As energy moves through a system (eg an ecosystem), less and less energy is available. Ecologists
talk about the 10% rule - approximately just 10% of the energy in one trophic level is available to
the next level.

3. BECAUSE OF ENTROPY: Photosynthesis requires a constant input of light energy, and all
organisms need a constant supply of chemical energy.

2. MEASURING THE RATE OF PHOTOSYNTHESIS

Scientists like to measure the rate of photosynthesis in different conditions. This may have economic
implications, for instance to help plant growers understand better how crop yields can be maximised. To
measure the rate of photosynthesis, there are three possibilities:

1. Measure the rate at which biomass increases - assuming new biomass is being made as a direct
result of photosynthesis.
2. Measure the rate at which carbon dioxide is taken up by the plant - assuming that the carbon
dioxide absorption is directly related to the rate of photosynthesis.
3. Measure the rate at which oxygen is given off by the plant - the oxygen results from the splitting of
water in the light dependent stage of photosynthesis.

Research labs will most likely measure carbon dioxide uptake but this is way too complex to do in a
school lab. Small increments of biomass are also tricky to assess. So we are left with trying to measure
oxygen release.

A conventional way of measuring oxygen release is to

count bubbles being given off by an aquatic plant, such as
commonly available Canadian pondweed (Elodea). This is
certainly quantitative but is fraught with errors and
uncertainties. Results are seldom reliable. You cannot
compare photosynthesis in terrestrial plants by this method.

https://www.newcollegeworcester.co.uk/photos
ynthesis-practical

A viable and now, much used alternative is THE FLOATING LEAF DISK ASSAY. Fun to do, easy to
conduct, uses readily available materials and equipment, and is quantitative.
2.1 THEORY OF THE FLOATING LEAF DISK ASSAY

Let's start by looking at the structure of a typical leaf:

https://www.slideshare.net/RichardsonBio100/lecture-23-25104906

Plant leaves consist of wide areas of photosynthesising cells, mostly in the palisade and spongy
mesophyll layers of the leaves. These cells are loosely packed and are surrounded by air spaces, so that
they can easily exchange carbon dioxide and oxygen in the air, and allow water to move around the leaf.
The air spaces fill with oxygen when photosynthesis is going on, which explains why a freshly picked
leaf will float on the surface of water.

But what would happen if all the air is forced out of the air spaces in the leaf? What will the leaf do then
when it is put on water? Sink! It sinks because its density, without the air in the spaces in the leaf, is more
than the density of water. If the leaf is again allowed to photosynthesise, it will again fill the air spaces
with oxygen. The leaf will float back up to the surface of the water, as its density drops. This gives us a
magical way to compare rates of photosynthesis in different conditions or in different plants - after
getting leaves to sink, let them photosynthesise. The faster the leaves rise to the surface, the faster is the
rate of photosynthesis.

If we supply the leaves with the necessary requirements for photosynthesis, including carbon dioxide and
light, the oxygen which the leaves produce will form gas bubbles and the leaves will re-float. In essence
this is our experimental method. Rather than whole leaves we use small disks cut from leaves to perform
the floating leaf disk assay. This assay of photosynthesis may be used to quantitatively investigate many
questions, including those factors which affect the rate of photosynthesis such as light intensity and
wavelength, CO2 concentration, plant adaptations, respiration, and pigment content.
 2.2 WHAT EXACTLY WILL BE MEASURED? THE RELATIONSHIP BETWEEN
PHOTOSYNTHESIS AND RESPIRATION

https://www.ck12.org/biology/cellular-respiration-and-
photosynthesis/lesson/Connecting-Cellular-Respiration-and-Photosynthesis-MS-LS/

The metabolic life in a leaf is of course not as simple as just whether it photosynthesises or not. While it
may photosynthesise for the hours when there is daylight, and so produce oxygen (and carbohydrates), we
must not forget that all cells in a leaf are, all the time, respiring, and thus using oxygen (and glucose).

Our investigation will be measuring net production of oxygen during photosynthesis, assuming that some
of the oxygen produced will be used for cell respiration. We will not be measuring gross production of
oxygen.

In the bigger picture of energy use and storage, this interaction between respiration and photosynthesis is
hugely important. Photosynthesis enables green plants to capture solar energy and transform this light
energy into chemical energy. The photosynthesis reactions are said to be anabolic - they use energy to
react low-energy chemicals together to form a high-energy product (glucose) which retains energy in the
form of chemical bonds. Respiration is the reverse - the reactions are catabolic, meaning a high-energy
d-
compound (glucose) is broken down to release its stored energy, with the release also of low-energy,
- 'waste' products, such as carbon dioxide and water. You should also know the terms endothermic
(endergonic) and exothermic (exergonic) - using and releasing energy respectively.
2.3 BASIC PROCEDURE FOR THE FLOATING LEAF DISK ASSAY

Materials (Fig. 1)

 300 ml 0.2% Sodium bicarbonate solution. (This is about 1/8 tsp baking powder in 300 ml water)
 Liquid soap
 Plastic syringe, without needle - 10ml or bigger
 Freshly picked leaves. Some leaves also do well if they are kept in water for a few hours
beforehand. Avoid heavyweight leaves, or leaves with a thick waxy protection or hairs. Spinach is
good.
 Cork borer or hole puncher. Even a strong, plastic, drinking straw will do.
 Small paint brush
 Aluminium foil or similar to keep already cut leaf disks in the dark until you are ready to start
timing
 Plastic cups
 Marker pen
 Glass, stirring rod
 Timer
 Light source - low heat, high intensity, daylight bulb. If you can get a 'grow' bulb, even better.

Fig. 1
1. Make a solution of about 0.2% sodium bicarbonate. This is about 1/8 tsp sodium bicarbonate in
300 ml distilled water. Add 1, maximum 2 drops of liquid soap to the solution. (The soap helps
the solution move into the leaf and replace the oxygen in the air spaces.)

2. Cut 10 leaf disks from a fresh leaf, using a cork borer or

hole puncher. You could also use a strong straw. (Fig. 2)

Fig. 2

3. Remove the plunger from a clean 10-ml syringe. Blow, tip or

gently brush the 10 disks into the body of the syringe. Be sure
the leaf disks are near the tip of the syringe as you re-insert the
plunger so as not to damage the disks. (Fig. 3)

Fig. 3

4. Insert the tip of the syringe into a beaker of 0.2% sodium bicarbonate solution and draw about 8 ml
of solution into the syringe. The leaf disks should be floating at this time.

5. Hold the syringe tip upward and expel the air by depressing the plunger carefully. Don't squash the
disks.

6. Seal the tip of the syringe with a finger. Pull back on the plunger,
creating a partial vacuum within the syringe. If you have a good
seal, it should be hard to pull on the plunger and you should see
bubbles coming from the edge of the leaf disks. This is how you
will replace the air with water, in the spaces of the leaf disks.
(Fig. 4)

Fig. 4

7. After about 5 seconds, simultaneously release your finger and the

plunger. Some of the leaf disks should start to sink because now,
without gas in the air spaces, they will be more dense than the
water around them. Tap the side of the tube to dislodge bubbles on
the edges of the disks. (Fig. 5)
Fig. 5

8. Repeat steps 5 and 6 until all disks sink - maybe 3 or 4 times. Do not overdo these steps!! It is easy
to damage the cells of the leaves.

9. Gently pour the disks and solution into a clear plastic cup
that has been labelled and filled with 100 ml of
bicarbonate solution. The disks should sink to the
bottom of the cup. (Figs. 6 & 7)

Fig. 6 Fig. 7
10. Repeat this procedure until you have as many experimental and control groups and repeats as
you require. Keep them in the dark until all are ready for you to put them in the light at the same
time, and start timing your investigations.

11. Place under the light source and start the timer. At the end of each
minute, record the number of floating disks. (Fig. 8) Then gently
swirl the disks with a glass rod to dislodge any that are stuck
against the sides of the cups. Continue until all of the disks are
floating. The time required for a leaf disk to float is an index of
the net rate of photosynthesis in that leaf disk. However, since
some leaf disks will be "early floaters" and others will be "late
floaters", you need to devise a method to quantify the results more
accurately. The mean time? The time for the middle one to
rise? Think what is best. Fig. 8

12. Theoretically, if the light is now removed, the discs will sink again, as the oxygen is used up in
respiration. This seldom happens easily. If it does happen, the figures can be used to compare
respiration and photosynthesis rates.
2.4 USING THE BASIC PROCEDURE TO INVESTIGATE SOME FACTOR WHICH
AFFECTS THE RATE OF PHOTOSYNTHESIS

Trial Run
Run a trial of the investigation, to figure out the experimental technique and the timing necessary, and
ensure that you can generate useful results.

Choosing Other Variables to Test

Repeat this experiment to test some variable that may affect photosynthetic rate. Use the Planning
Format, to generate a hypothesis, decide upon the experimental variables and to outline the
procedure you will follow.
[Points <2]

Recording Results
Collect as much useful data together in a results table. Make sure that you make repeats, so that there
is sufficient date to analyse with some validity.
[Points <2]

Analysis of Results
Find some way, probably a graph or simple statistics, to analyse your results.
[Points <2]

Conclusion and Evaluation

State a conclusion which supports or rejects your hypothesis. Justify the conclusion by referring to the
results and the analysis.

Identify a minimum of three sources of error and uncertainty and make recommendations about how to
reduce or eliminate these errors.
[Points <2]

Communication
Create a video of your investigation and procedure. Post this on the Blog.
[Points <2]

REFERENCES
Main source: https://blogs.cornell.edu/cibt/files/2015/09/Floating-Leaf-Disk-Brad-Williamson.pdf

There are several YouTube videos to watch how the lab is done. Paul Anderson/Bozeman Science is
a good one: https://www.youtube.com/watch?v=ZnY9_wMZZWI

Relationship between photosynthesis and cell respiration:

http://photosynthesiseducation.com/photosynthesis-and-cellular-respiration/

Limiting factors in photosynthesis (YouTube video):

https://www.youtube.com/watch?v=1curtzL8rUM

The Laws of Thermodynamics and living systems (incl. Photosynthesis):

https://www.thoughtco.com/laws-of-thermodynamics-373307