James1

Understanding Photosynthesis and Cellular

Respiration using Enzymatic Reactions and the

Effects of pH on Proton-Motive Force.

Jhanielle James

3099800

BIOL- 075

April 2, 2020

Sherry Hebert
 James2

Abstract:

In this experiment, the relationship between photosynthesis and cellular respiration is

investigated along with factors that affect them such as temperature, enzyme activity. Also

ways in which they can be monitored and quantified such as cell pH and oxygen

consumption/ evolution are observed. This was done by using an oxygen machine to record

oxygen consumption/ evolution and photosynthesis rate in Euglena and yeast cells at varying

temperatures. Enzyme activity was then observed by measuring the maximum rate of

succinate dehydrogenase activity with a spectrophotometer under different conditions. The

generation of a proton- motive force by chloroplasts cells obtained from spinach leaves was

also observed by changes in pH in light and dark conditions using a pH meter. An artificial

electron acceptor was also used to understand the principle behind the topic.

The optimal temperature for respiration in yeast and Euglena cells are 50◦C and 40◦C

respectively, while the optimum temperature of photosynthesis in Euglena was found to be

40◦C. Succinate dehydrogenase catalyses a reaction that produces electrons that reduce DCIP

that changes from blue to colourless when reduced. The rate at which this reduction occurs is

decreased in the absence of substrate and increased in the presence of Sodium Azide. Finally,

it was determined that in photosynthesis, pH increases during light conditions and decreases

in dark conditions and also in the presence of CCCP.

Introduction:
 James3

Photosynthesis and cellular respiration are connected through an important relationship. This

relationship enables life to survive as we know it1. There are different factors that affect them

both and by studying these factors one can have an idea and therefore create ideal conditions

for further study of other cells. It is important to grasp the principle of photosynthesis and

cellular respiration so one can understand the smaller process that cascade together to carry

them out. These smaller biological processes operate around the purpose of maintaining

homeostasis, which is maintaining a balance or a constant internal environment. The general

purpose of this experiment is to understand the relationship between photosynthesis and

cellular respiration by depending on each other to provide energy and how the occur under

varying conditions.

Photosynthesis is the process by which green plants and other photosynthetic organisms

convert light energy to chemical energy in the form of sugars to use as a food source. It

consists of two processes, the Hill reactions and the Calvin cycle. In the light dependent stage

(Hill reactions) that occurs in the thylakoid membrane, photons from sunlight (light energy)

hit the chlorophyll molecules and excite them, eventually leading to the ejection of electrons

from what is called the reaction center of Photosystem II2. These electrons are replaced by

electrons generated by the photolysis of water (H20), which also results in the evolution of

oxygen (O2), which its rate is often equal to or similar to the rate of CO2 fixation2. The

electrons that are emitted from Photosystem II are picked up by an electron acceptor

molecule, which immediately transfers them to the electron transport system2. ATP is

generated (photophosphorylation) by chemiosmosis and ultimately, the cofactor NADP is

reduced to form NADPH after the electrons pass through a second reaction center,

Photosystem I.  ATP and NADPH provide chemical energy and reducing power that drives

the light-independent reactions (Calvin cycle)2. Photosythesis is described by the equation:

6CO2 + 6H2O → C6H12O6 + 6O2

 James4

In the Calvin cycle, the “dark” reactions take place in the stroma chloroplast. In this process,

carbon fixation occurs using ATP and NADPH as reactants that were generated from the

light-dependent stage3. They drive the chemical pathway using atmospheric carbon (CO2) to

form a three-carbon sugar called glyceraldehyde-3-phosphate (G3P)3. it is then used by cells

to make may sugars and other organic molecules. Most of the interconversions occur outside

the cell, therefore G3P is often transported outside the cell and used as raw material to make

food or more energy carrier molecules3. Respiration can be described by the equation:

C6H12O6 + 6O2  6CO2 + 6 H2O

Cellular respiration is a metabolic pathway that breaks down glucose and produces ATP. This

process starts with glucose as it enters glycolysis which occurs in the cytosol, where it

undergoes a series of chemical transformations to form a net result of two molecules of

pyruvate, 2 ATP and 2 NADH4. Each pyruvate is then transferred to the inner mitochondrial

membrane where they are oxidised to form Acetyl Coenzyme A4. This molecule then enters

the citric acid cycle where it ultimately forms ATP, NADH and FADH2; carbon dioxide is

also produced4. Finally, NADH and FADH2 that were previously made are deposited into the

electron transport chain where they are used to form ATP4. Oxygen is important as it is used

as the final electron acceptor at the end of the chain.

Photosynthesis and cellular respiration have a special relationship where they are opposites of

each other, i.e., the products of one reaction are the reactants of the other.  While

photosynthesis requires carbon dioxide and releases oxygen, cellular respiration requires

oxygen and releases carbon dioxide1. We use the oxygen released by photosynthesis for

cellular respiration1. When we inhale that oxygen, it is carried by our blood to all the cells in

our body1. In our cells, oxygen allows cellular respiration to proceed as cellular respiration

works best in the presence of oxygen1.

 James5

Cellular respiration is a metabolic process and is used to determine the metabolic rate of cells.

The rate of any reaction can be determined by measuring the rate at which the substrates

disappear, or the products appear5. Oxygen is consumed by respiring tissue, and its loss can

be used to measure the rate of aerobic respiration5. Similarly, because oxygen is given off by

photosynthesising cells, the rate at which it is given off can be used to quantify the rate of

photosynthesis. As oxygen is so deeply involved with these processes, they are affected by

the amount available. Low oxygen levels make it difficult for cellular respiration to take

place which can be fatal. Temperature also greatly affects cellular respiration and

photosynthesis. Both these processes use enzymes to carry out their purpose and they all have

an optimum temperature. This is the temperature at which the enzymes work best, however

any higher than this temperature will cause them to denature and die. As temperature

increases, so does the rate of cellular respiration and photosynthesis but up to a point. The

activity of certain enzymes can also affect these processes. For example, succinate

dehydrogenase is particularly important because it a membrane bound enzyme that catalyses

the oxidation of succinate to form fumarate along with forming two protons and an electron.

It is also participating in the citrate acid cycle and the electron transport chain. In lab 6 of this

experiment, pH was observed because it was expected to decrease in the light stage as ATP is

being made and hydrogen ions are being pumped into to thylakoid, and increase dark stage as

no ATP is being made so the hydrogen ions are flowing along their gradient into the stroma7.

In this experiment, the specific aim was to investigate how photosynthesis and cellular

respiration are related under varying conditions. This was done by measuring the rate of

oxygen consumption and evolution under varying temperatures, measuring the rate of

succinate dehydrogenase activity and observing the generation of proton-motive force by

changes in ph.

Methodology:
 James6

All the laboratory protocols were taken from the University of Winnipeg Cellular Biology

Lab Manual6.

Determination of rate and optimal temperature for aerobic respiration and photosynthesis:

A:500ml of tap water was prepared at 40 degrees Celsius and 1g of sucrose and 0.5g of yeast

was added to it. It was then allowed to sit at room temperature for 10 minutes. Set to 600nm

and blank with tap water, then determine the absorbance of yeast culture (should get an OD

of about 0.80 to 1.00). A culture was prepared using C1V1= C1V1 equation and the given

concentration. The oxygen machine was calibrated at 20 degrees Celsius, add 2ml of yeast

culture then the pen was repositioned to approximately 70 units by bubbling air into the

culture. The chamber was sealed then the rate of oxygen consumption was recorded for 4

minutes. When it was done running, the chart was stopped, the chamber stopper was

removed, and the chamber was cleaned three times with distilled water. The measurement

procedure was repeated at the same temperature fresh sample and stock culture. The bath was

adjusted to 30 degrees Celsius and monitored with a thermometer till it was reached. The

oxygen machine was recalibrated and charted at the new temperature. Fresh cells were used

for every run. With the help of the instructor, CCCP was added into the reaction chamber

with a syringe at the end of the second trial. The effects were monitored for 4 mins. The

procedure was repeated at 40, 50 and 60 degrees Celsius without CCCP and the trials for

each temperature were averaged and recorded.

B: A coverslip was placed on the haemocytometer and a drop of Euglena culture was added

and a bright field microscope was used for magnification. Refer to lab manual on how to read

the haemocytometer. A culture was prepared using the C1V1= C1V1 equation and the

information from the haemocytometer. The oxygen machine was run according to laboratory

protocols at 20 degrees Celsius with 2 ml of Euglena for 4 mins for a second trial. It was then
 James7

run at 30 degrees Celsius and CCCP was added towards the end of the second trial. The

procedure was repeated at 40, 50 and 60 degrees Celsius without CCCP and the trials for

each temperature were averaged and recorded.

C: Using the same culture that was prepared in part A the amount of cells/ml was determined

by using C1V1= C1V1. The oxygen machine was calibrated and run at 20 degrees Celsius with

2ml of Euglena for 3 mins in the light using a desk lamp, then run for 2 mins in the dark by

covering the reaction chamber with a black cloth. It was then run at 30 degrees Celsius and

the CCCP was added during the third trial according to laboratory protocols. The procedure

was repeated at 40, 50 and 60 degrees Celsius without CCCP and the trials for each

temperature were averaged and recorded.

D: A wet mount of Euglena and yeast cells were assembled and observed using a phase

contrast microscope at 400x magnification.

Understanding enzymatic reactions through the study of succinate dehydrogenase activity:

A: 40g of mushroom pieces were weighed and placed in a chilled mortar and grinded for 2

mins into a smooth paste with 5 grams of cold sand and 20ml of cold isolation buffer. An

additional 20ml of buffer was added then the mixture grinded for 2 more mins. The mixture

was then poured through four layers of cheesecloth and rinsed with more buffer to get out the

remaining. The extract was then balanced and centrifuged at 600 x g for 10 mins at 4 degrees

Celsius. After the centrifugation, the pellet was discarded, and the supernatant was balanced

and centrifuged at 12,000 g x g for 30 mins at 4 degrees Celsius. After the second centrifuge,

the supernatant was discarded, and the pellet was resuspended by vortexing it in 8ml of assay

mix. It was placed on ice and used as the mitochondrial pellet for the succinate

dehydrogenase assay. 1ml of this suspension was taken and placed in a glass tube, boiled for

3 mins and placed on ice without the lid. This is the boiled pellet to be used for part B.
 James8

B. i. DCIP: In five labelled cuvettes, dilutions of 50 micromolar stock solutions of DCIP

were prepared. Before mixing the contents, the absorbance readings were taken at 600nm for

cuvettes 2-5. Cuvette 1 was used as a blank.

ii. Succinate Dehydrogenase Assay: In ten labelled cuvettes, assay buffer, succinate, DCIP,

aizide and malonate were added. After swirling the centrifuge tube with the mitochondria, set

a timer and 0.3 ml of mitochondrial suspension to cuvettes 1 and 2. They were mixed then

using cuvette 1 as a blank, immediately read cuvette 2 at 600nm and record the value.

Readings were repeated every five minutes for 30 mins. Once those readings are done, 0.9ml

of the mitochondrial suspension was removed and added to cuvettes 3 and 4 and mixed while

using cuvette 3 as blank and taking the absorbance for cuvette 4. Readings were repeated

every five minutes for 30 mins. While taking reading cuvettes for 1-4 at appropriate time

points and blanking appropriately, start reading for cuvettes 5-10. 0.6 ml of mitochondrial

suspension was added to cuvettes 5, 6, 7, 8 and 9 and 0.6ml of boiled mitochondria was

added to cuvette 10. After blanking the machine with cuvette 5, readings of cuvette 6- 10

were taken. Readings were repeated every five minutes for 30 mins. All values were

recorded.

Observation of the generation of the proton-motive force through alterations in pH:

A: 35g of spinach leaves that were left in the cold and dark overnight, then placed in the light

for a few hours were ripped into small pieces then transferred to a chilled blender with 100ml

of homogenizing medium. It was then blended for three 10 second bursts then filtered with

cheesecloth into a chilled beaker. It was then distributed between 2 50ml centrifuge tubes,

balanced and centrifuged at 500 x g for 2 minutes at 4 degrees Celsius. After centrifugation,

discard the pellets and centrifuge the supernatant at 2000 x g for 10 mins at 4 degrees Celsius.

The supernatants were discarded, 1.5ml of 0.01 NaCl was added to each tube and
 James9

resuspended. 6ml of 0.01M NaCl was added then placed on ice to incubate for 10 mins. The

chloroplast suspension was centrifuged at 10000 x g for 5 mins at 4 degrees Celsius. The

supernatants were discarded and the pellets were resuspended by adding 2 ml of 0.01M NaCl,

recombined in one tube then stored on ice.

B: 0.1 ml of the swollen chloroplast suspension was added to 19.9 ml of 80% acetone, then

the mixture was poured into a glass spectrophotometer cuvette and the absorbance was taken

at 654nm. The absorbance value was multiplied by 5.6 to get the concentration of

chlorophyll. The chlorophyll suspension was diluted as needed using the C1V1= C1V1

equation. The tube was returned to the ice bath and aluminium foil was used to cover it.

C: A small magnetic stir bar, 1.1 ml 0.048 M NaCl, 0.6 ml PMS and 3.6 ml distilled water.

The beaker was placed on a stir plate and the pH electrode was lowered into it. 3.7 ml of the

chloroplast suspension was added, and the light source was turned on. The change in pH

every ten seconds for two mins was recorded. Then with the light off and a garbage bag used

to mage a cover the reaction, the pH was recorded for every ten seconds for two mins. The

change in pH was recorded again using another beaker with fresh chloroplasts with the same

time intervals with light on throughout, however after the first two mins, 20 ml of CCCP was

added.

Results:

Determination of rate and optimal temperature for aerobic respiration and

photosynthesis
 James10

Table 1: Showing the rate of oxygen consumption for each trial with its corresponding

temperature, their averages and standard deviation for yeast respiration. The highest rate was

observed at 50⁰C.

Rate of Oxygen/min/mL

Temperature Trial 1 Trial 2 Trial Trial 4 Trial 5 Trial 6 Averag Standard

3 e Deviation

20⁰C 2 5.3 1.8 2.6 3.7 4.2 3.267 1.251

30⁰C 6.2 8.04 3.8 4.7 6.4 6.3 5.907 1.35

40⁰C 9.8 9.4 5.6 5.1 8.2 12.1 8.367 2.429

50⁰C 9 9.8 5.9 8.4 13.2 12.8 9.85 2.528

60⁰C 3.9 3.3 2.8 4.2 3.2 2.4 3.3 0.611

Table 2: Showing the rate of oxygen consumption for each trial with its corresponding

temperature, their averages and standard deviation for Euglena respiration. The highest rate

was observed at 40⁰C.

Rate of Oxygen/min/mL

Temperature Trial 1 Trial 2 Trial Trial 4 Trial 5 Trial 6 Averag Standard

3 e Deviation

20⁰C 0 4 0 2.4 1 3 1.733 1.513

30⁰C 2 8 6 6.9 4.2 7.4 5.75 2.067

40⁰C 6 8 12 11.2 7.3 10.2 9.117 2.164

50⁰C 4 4 8 5.8 6.2 7.3 5.883 1.51

60⁰C 6 2 2 4 3 2 3.167 1.462

 James11

Figure 1. Shows the average respiration rate (Oxygen/min/mL) for Yeast and Euglena at the

corresponding temperatures (⁰C). Error bars showing the standard deviations were also

included.

Table 3. Showing the observed rate of oxygen evolution for each trial with its corresponding

temperature, their averages and standard deviation for Euglena photosynthesis. The highest

rate was observed at 40⁰C.

Observed rate of Oxygen/min/mL

Temperature Trial 1 Trial 2 Trial Trial 4 Trial 5 Trial 6 Averag Standard

3 e Deviation

20⁰C 15 10.6 18 11.5 14 14.3 13.9 2.41

30⁰C 26 16.1 27.4 18.2 26 21.6 22.55 4.257

40⁰C 30 19 33.6 26 35.2 27.4 28.533 5.336

50⁰C 17 16.5 10.8 20 14.3 12.7 15.217 3.011

 James12

Table 4. Showing the actual rate of oxygen consumption/ evolution for each trial with its

corresponding temperature, their averages and standard deviation for Euglena photosynthesis.

The highest rate was observed at 40⁰C.

Actual rate of Oxygen/min/mL

Temperature Trial 1 Trial 2 Trial Trial 4 Trial 5 Trial 6 Averag Standard

3 e Deviation

20⁰C 15 12.1 18 13.5 15 14.3 14.65 1.799

30⁰C 26.5 18.1 28.9 20.7 27.8 24.1 24.35 3.866

40⁰C 31.5 21 36.6 30 38.7 31.4 31.533 5.633

50⁰C 19 17.5 12.8 23.5 16.8 13.7 17.217 3.531

Figure 2. Shows the average observed rate of photosynthesis and the average actual rate of

photosynthesis for Euglena cells at the corresponding temperatures (⁰C). From the graph, the

maximum rate of photosynthesis occurred at 40⁰C.

 James13

Understanding enzymatic reactions through the study of succinate dehydrogenase

activity

Figure 3. Shows the optical density for cuvettes 2, 4 and 6 over a period of 30 mins.
 James14

0.5
0.45
0.4
Optical Density at 600nm

0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0 5 10 15 20 25 30 35
Time (mins)

Cuvette 6 Cuvette 7 Cuvette 8 Cuvette 9 Cuvette 10

Figure 4. Shows the optical density for cuvettes 6,7,8,9 and 10 over a period of 30 mins.

Cuvette 10 had the lowest rate of reaction.

Table 5: Showing the class data of the maximum rates of the reaction of the enzyme in tubes

6-9, their averages obtained from the multiple trials and the standard deviation for succinate

oxidation.

Tube Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 Average Standard

Deviation

6 0.5279 0.48 0.484 0.632 0.542 0.545 0.535 0.05

7 0.3858 0.245 0.326 0.335 0.325 0.315 0.322 0.041

8 0.4975 0.451 0.392 0.287 0.392 0.394 0.402 0.065

9 0.2944 0.296 0.216 0.134 0.326 0.253 0.253 0.064

 James15

Figure 5. Shows the average maximum rates for tubes 6-9 with the standard deviations. Tube

6 had the highest rate of the reaction.

Observation of the generation of the proton-motive force through alterations in pH

6.8

6.6

6.4
pH

6.2

5.8
0 50 100 150 200 250 300 350 400
Time (seconds)

Light/Dark/Light Light/CCCP

Figure 6. showing the average change in pH over time for the light/dark/light and light/CCCP

reaction conditions.
 James16

Discussion:

Photosynthesis and cellular respiration are processes that vital to cells and by extension, life.

They both produce energy that organisms need to carry out regular cellular processes and

greatly depend on each other to do so. Photosynthesis is essentially the opposite if respiration

as can be seen in their equations. The two are similar in that the both synthesise and utilize

ATP, electron carriers such as NADP, FAD, NADPH and FADH2, and they both use the

electron transport chain.

In this experiment, the rate of respiration and photosynthesis was investigated at different

temperatures. There are many enzymes that are needed to carry out these metabolic processes

which are greatly affected by temperature, therefore, if temperature affects these enzymes

then photosynthesis and cellular respiration will also be affected. The rate at which these

processes take place is quantified by oxygen consumption for respiration and oxygen

evolution for photosynthesis. Two species were investigated, Euglena and yeast. In table 1,

the rate of respiration (oxygen consumption) was measured using yeast cells at temperatures

of 20, 30, 40, 50 and 60 degrees Celsius. Six trials were done and then averaged to find a

single value. The highest rate was at 50 degrees which tells that this is the optimum

temperature of yeast. This means that at higher than this temperature, the enzymes will be

denatured, and their activity will drastically decrease along with metabolic rate. In table 2, the

rate of respiration was also observed, but in Euglena. According to the results, the optimum

temperature was seen at 40 degrees Celsius. Figure 1 shows the average respiration rate for

both species. Their optimum temperatures can be seen followed by the sudden decline in

respiration rate due t denaturation of the enzymes. The observed rate of photosynthesis was

also investigated in Euglena and according to table 3, the optimum temperature was seen at

40 degrees Celsius. This observed rate was obtained by conducting the experiment in light

conditions. However, the actual rate of photosynthesis was obtained by adding the rate of
 James17

oxygen consumption in the dark to the rate or oxygen evolution in the light. Figure 2

compares both rates of photosynthesis in Euglena and they both observe an optimum

temperature at 40 degrees Celsius.

Photosynthesis and cellular respiration are further investigated by understanding enzymatic

reactions through the study of succinate dehydrogenase activity with an artificial electron

acceptor. Succinate dehydrogenase is an enzyme that catalyse the oxidation of succinate to

fumarate in the citrate cycle. While doing this, it also produces two proton molecules and two

electrons that will be deposited into the electron transport chain by FADH2 to be ultimately

accepted by oxygen. In this experiment, the artificial electron acceptor used was DCIP which

becomes colourless when it is reduced. According to figure 3, cuvette had the lowest optical

density overtime, means that this cuvette has the optimal amount of substrate to react with the

enzyme. Sodium Azide was used in the study to ensure that the electrons did not go to the

ETC and only go to DCIP ensuring that it was the final electron acceptor. Its effect can be

seen in cuvette 8 from figure 4. The DCIP was not present in this cuvette and so resulted in a

higher OD or low rate or reaction. Cuvette 10 had the lowest rate of rection as it had the

boiled mitochondria which cannot take part in any reaction as it is destroyed.

Finally, observing the generation of proton-motive force through alterations in pH can be

used to quantify metabolic activity. During the electron transport chain, a proton motive

gradient is formed as ATPase uses hydrogen ions and protons to make ATP in the

intermembrane space of the thylakoid. The H+ ions are pumped across the membrane

resulting in changes in ph. During light conditions, hydrogen ions are pumped into the

thylakoid causing the pH to be increased in the stroma, while in dark conditions these ions

are pumped into the stroma resulting in a decrease in pH. This can be seen in figure 6 as the

pH increased in light conditions, decreased in dark conditions then increased again when the
 James18

light was turned back on. During the light/ CCCP conditions, there was an increase in pH

during the light stage then a decrease when the CCCP was added.

Some possible sources of error that are to be considered when carrying out the experiment are

to ensure proper calibration of oxygen machine and spectrophotometer. Also, proper timing

of the “light” and “dark” stages of the experiment. Failure to do these may result in the results

be skewed or not precise. This study could be improved to better carry out the aim by

ensuring that the procedures are meticulously timed when needed to be.

In conclusion, the results coincide with the expectations of this experiment. There is an

interdependent relationship between photosynthesis and cellular respiration, and they are both

affected by the cells environment such as temperature and the resources that are made

available to it, like oxygen and metabolic enzymes. The light and dark conditions of

photosynthesis affect the pH in chloroplasts. Due to the potentially fatal effects that could be

encountered by these processes, it is essential that ideal conditions are maintained so they are

not disrupted.

References:

1) Harwood, J., Wilkin, D., Kraus, D., Gray-Wilson, N., Brainard, J., Johnson, S., …

Karasov, C. (2019, November 20). Cellular Respiration and Photosynthesis. Retrieved

from https://www.ck12.org/biology/cellular-respiration-and-

photosynthesis/lesson/Connecting-Cellular-Respiration-and-Photosynthesis-MS-LS/

2) Results. (n.d.). Retrieved from http://www.users.miamioh.edu/smithhn/Photosynthesis

(new).htm
 James19

3) (n.d.). Retrieved from https://www.nature.com/scitable/topicpage/photosynthetic-

cells-14025371/

4) Steps of cellular respiration | Biology (article). (n.d.). Retrieved from

https://www.khanacademy.org/science/biology/cellular-respiration-and-

fermentation/overview-of-cellular-respiration-steps/a/steps-of-cellular-respiration

5) Saltveit, M. E. (n.d.). Measuring Respiration. Retrieved April 3, 2020, from

http://ucce.ucdavis.edu/files/datastore/234-20.pdf

6) University of Winnipeg. Cell Biology Lab manual, 2020. p. 45-56.

7) University of Winnipeg. Cell Biology Lab manual, 2020. p. 54-56