How does CO₂ concentration affect photosynthesis?

How does CO₂ concentration affect photosynthesis?

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I have heard the theory that with the increase of CO2 in the air, the speed of the photosynthesis would increase, thereby limiting the increase of CO2 levels.

What is currently the rate limiting factor of photosynthetic reactions in plants? Is it really CO2, or rather the limited energy that a plant can absorb from sunlight?

The rate-limiting step of photosynthesis is the CO2 assimilating enzyme Rubisco (short for ribulose-1,5-bisphosphate carboxylase/oxygenase) (Jensen, 2000). It uses ribulose-1,5-bisphosphate and CO2 as substrates to generate glucose.

Given that Rubisco is the rate limiting step in photosynthesis, an increase in its substrate CO2 would expectedly lead to an increase in photosynthesis. However, the regulation of Rubisco is complex and is influenced not only by CO2, but also by O2 (which competes with CO2 for the active site), Mg2+ and a regulating enzyme called Rubisco activase (Jensen, 2000). Hence, the effects of an increase in atmospheric CO2 may be more complex than simply enhancing photosynthesis by increasing Rubisco activity.

Indeed, a review by Poorter (1993) showed that a doubling of CO2 lead to an average increase in photosynthesis of only 37% across more than 150 plant species. He describes various factors that determine photosynthesis rates other than CO2:

  1. One factor that limited photosynthesis under high CO2 was nitrogen (N). Since Rubisco is an enzyme, it has to be synthesized from amino acids. As Rubisco constitutes about 30% of the total protein in a plant leaf, Rubisco is probably the most abundant protein on earth and a major sink for plant nitrogen (Jensen, 2000). For example, C3 species capable of symbiosis with N2-fixing organisms had higher growth increases compared to other C3 species under high CO2;
  2. Certain plants utilize CO2 better than others. CAM species were the least responsive, followed by C4 plants, while C3 plants increased their photosynthesis rates the most under high CO2. Examples of C3 plants are herbaceous crop plants (Poorter, 1993);
  3. Plants with a large intrinsic growth rate may benefit more from high CO2. These plants are said to have a high sink strength, as they rapidly convert additional photosynthesis products into outgrowth.

- Jensen, PNAS (2000); 97(24): 12937-38
- Poorter, Veg (1993);104/105: 77-97

Impacts of increased atmospheric CO2 concentration on photosynthesis and growth of micro- and macro-algae

Marine photosynthesis drives the oceanic biological CO(2) pump to absorb CO(2) from the atmosphere, which sinks more than one third of the industry-originated CO(2) into the ocean. The increasing atmospheric CO(2) and subsequent rise of pCO(2) in seawater, which alters the carbonate system and related chemical reactions and results in lower pH and higher HCO(3) (-) concentration, affect photosynthetic CO(2) fixation processes of phytoplanktonic and macroalgal species in direct and/or indirect ways. Although many unicellular and multicellular species can operate CO(2)-concentrating mechanisms (CCMs) to utilize the large HCO(3) (-) pool in seawater, enriched CO(2) up to several times the present atmospheric level has been shown to enhance photosynthesis and growth of both phytoplanktonic and macro-species that have less capacity of CCMs. Even for species that operate active CCMs and those whose photosynthesis is not limited by CO(2) in seawater, increased CO(2) levels can down-regulate their CCMs and therefore enhance their growth under light-limiting conditions (at higher CO(2) levels, less light energy is required to drive CCM). Altered physiological performances under high-CO(2) conditions may cause genetic alteration in view of adaptation over long time scale. Marine algae may adapt to a high CO(2) oceanic environment so that the evolved communities in future are likely to be genetically different from the contemporary communities. However, most of the previous studies have been carried out under indoor conditions without considering the acidifying effects on seawater by increased CO(2) and other interacting environmental factors, and little has been documented so far to explain how physiology of marine primary producers performs in a high-CO(2) and low-pH ocean.

Dynamic photosynthesis in different environmental conditions

Incident irradiance on plant leaves often fluctuates, causing dynamic photosynthesis. Whereas steady-state photosynthetic responses to environmental factors have been extensively studied, knowledge of dynamic modulation of photosynthesis remains scarce and scattered. This review addresses this discrepancy by summarizing available data and identifying the research questions necessary to advance our understanding of interactions between environmental factors and dynamic behaviour of photosynthesis using a mechanistic framework. Firstly, dynamic photosynthesis is separated into sub-processes related to proton and electron transport, non-photochemical quenching, control of metabolite flux through the Calvin cycle (activation states of Rubisco and RuBP regeneration, and post-illumination metabolite turnover), and control of CO₂ supply to Rubisco (stomatal and mesophyll conductance changes). Secondly, the modulation of dynamic photosynthesis and its sub-processes by environmental factors is described. Increases in ambient CO₂ concentration and temperature (up to

35°C) enhance rates of photosynthetic induction and decrease its loss, facilitating more efficient dynamic photosynthesis. Depending on the sensitivity of stomatal conductance, dynamic photosynthesis may additionally be modulated by air humidity. Major knowledge gaps exist regarding environmental modulation of loss of photosynthetic induction, dynamic changes in mesophyll conductance, and the extent of limitations imposed by stomatal conductance for different species and environmental conditions. The study of mutants or genetic transformants for specific processes under various environmental conditions could provide significant progress in understanding the control of dynamic photosynthesis.

Keywords: CO2 assimilation Carbon dioxide fluctuating irradiance light transients lightfleck sunfleck temperature vapour pressure deficit..

The Effect of Oxygen Concentration on Photosynthesis in Higher Plants

The influence of oxygen concentration in the range 0–21% on photosynthesis in intact leaves of a number of higher plants has been investigated.

Photosynthetic Co2 fixation of higher plants is markedly inhibited by oxygen in concentrations down to less than 2%. The inhibition increases with oxygen concentration and is about 30% in an atmosphere of 21% O2 and 0.03% Co.2. Undoubtedly, therefore, oxygen in normal air exerts a strong inhibitory effect on photosynthetic Co2 fixation of land plants under natural conditions.

The inhibitory effect of oxygen is rapidly produced and fully reversible.

The degree of inhibition is independent of light intensity.

The quantum yield for Co2 fixation, i.e. the slope of the linear part of the curve for Co2 uptake versus absorbed quanta, is inhibited to the same degree as the light saturated rate at all oxygen concentrations studied.

Diverse species of higher plants, varying greatly in photosynthetic response to light intensity and Co2 concentration, and with light saturated roles of Co2 fixation differing by a factor of more than 10 times, show a remarkable similarity in their response to oxygen concentration. By contrast, when studied under the same conditions as the higher plants, the green algae Chlorella and Ulva did not show-any measurable inhibition of photosynthetic Co2 fixation. Similarity, the increase in fluorescence intensity with increasing oxygen concentrations found in higher plants also was not seen in Chlorella. The present results, together with previous data on the photosynthetic response of algae to oxygen concentration, indicate that the photosynthetic apparatus of higher plants differs considerably from that of algae in its sensitivity to oxygen.

The inhibitory effect of oxygen on photosynthetic Co2 fixation in higher plants is somewhat higher at wavelengths which excite preferentially photosystem I. Also, the Emerson enhancement of Co2 fixation measured when a far red beam of low intensity is imposed on a background of red light is greater under low oxygen concontrution than under air. Measurements of reversible light-induced absorbance changes reveal that the change at 591 nm, probably caused by pla.stocyanin, is affected by oxygen concentration only if photosystem II is excited. the reducing effect on plastocyanin, caused by excitation of this system, decreases with increasing oxygen concentration. From these results it is suggested that a possible site of the inhibition by oxygen is in the electron carrier chain between the two photosystems. Oxygen might act as an electron acceptor at this site, causing reducing power to react back with molecular oxygen. However, this hypothesis does not account for equal inhibitions of the quantum yield and the light saturated rate of photosynthetic CO2 uptake.

Through the photosynthetic process plants take up carbon dioxide and evolve oxygen. The present high concentration of molecular oxygen in the atmosphere is generally considered to have arisen from the activity of photo-synthetic organisms. The effect of oxygen concentration would seem, therefore, to he a problem of great interest, not only in the field of the biophysics and biochemistry of photosynthesis, but in ecology and other branches of biology as well.

It was discovered by Warburg (1920) that high concentrations of oxygen inhibit the rate of photosynthetic oxygen evolution in the unicellular alga Chlorella. Since then, it has been confirmed by various authors that oxygen cconcentrations in the range 21–100 per cent have a marked inhibitory effect on photosynthesis, particularly at saturating light intensities. There is some evidence that under conditions when carbon dioxide concentration limits photosynthesis, the inhibition may become obvious even in 21 per cent oxygen. The inhibition has not been considered to operate at low light intensities. A review on the subject has been given by Turner and Brittain (1962).

Various hypotheses have been put forward to explain the inhibitory effect of oxygen, commonly referred to as the Warhurg effect. Some authors favor the idea of enzyme inhibition Turner et al. (1958) that one or more enzymes of the carbon reduction cycle are inactivated by oxygen: lirianlals (1962) that enzymes of the oxygen-evolving complex are inhihited. Other hypotheses concern back-reactions in which molecular oxygen is taken up, thus reversing the photosynthetic process. These reactions include photo-oxidation, photorespiration, and the Mehler reaction (Tamiya et al., 1957). At present, there is no generally accepted hypothesis explaining the effect.

The often conflicting results on which these hypotheses were based have been obtained mostly on algae. The first observation of an inhibitory effect on photosynthesis in a higher plant was made hy McAlister and Myers (1940) in wheat leaves. They found that the photosyntlietic CO2 uptake was markedly lower in air than in an atmosphere of about 0.5 per cent oxygen. At the CO2 concentration used (0.03%) the inhibition was present both at high and moderate light intensities. No data were obtained at low light intensities.

Although the study of the effect of oxygen concentration on photosynthesis in higher plants would seem to be of great interest, particularily since the natural environment of most land plants is an atmosphere with an oxygen content of 21 per cent, it has attracted very little attention. To the author's knowledge no thorough investigation on the subject has been published.

The present investigalion is directed toward elucidatirng the photosynthetic response of higher plants to oxygen concentrations up to that of normal air. Data are presented showing that the photosynthetic CO2 fixation in intact leaves of higher plants, regardless of light intensity, is strongly inhibited by oxygen in normal air, and that the pholosynthetic response to oxygen differs considerably from that of green algae. The present investigalion is directed toward elucidatirng the photosynthetic response of higher plants to oxygen concentrations up to that of normal air. Data are presented showing that the photosynthetic CO2 fixation in intact leaves of higher plants, regardless of light intensity, is strongly inhibited by oxygen in normal air, and that the pholosynthetic response to oxygen differs considerably from that of green algae.

The forest in the future

In woodland in Staffordshire, six towering metal structures bathe the area in the CO₂ concentrations expected on Earth in 2050. The experiment aims to find out how forest ecosystems will cope with our planet’s changing atmosphere, writes Anna Gardner

Opening image by Peter Miles

Covering more than 30% of the terrestrial biosphere, forests are essential for a plethora of ecosystem services. One of these is the carbon sequestration of up to 20–30% of all anthropogenic CO₂. Since the 1750s, atmospheric CO₂ has almost doubled due to a combination of burning fossil fuels (causing around 90% of the rise) and deforestation (responsible for around 10% of the rise).

Currently, the CO₂ concentration in our atmosphere is around 407ppm, but climate models predict CO₂ will continue to rise and far exceed this figure in the near future[1]. Understanding how the terrestrial environment will respond to rapidly rising CO₂ levels is therefore vital in order to plan how society can adapt to climate change.

Woodland wonder

No single laboratory experiment – or even an infinite series of such experiments – can test the response of a complex ecosystem such as a forest to changes in atmospheric composition . Whole ecosystem tests are required to study system-level responses. This is where the high-tech ‘sci-fi forest’ at the Birmingham Institute of Forest Research (BIFoR) comes in. BIFoR’s free-air carbon enrichment (FACE) facility, which is part of the University of Birmingham, is undertaking a unique outdoor experiment to expose whole, intact patches of temperate deciduous forest to the CO₂ concentrations that will pervade the planet by about 2050.

Matched by only one other forest-scale facility in the world (Western Sydney University’s EucFACE in Australia[2]) and stepping forward from previous FACE facilities – built on agricultural fields and into which juvenile trees were planted – BIFoR FACE has been built into existing ‘old growth’ forest. In this way, an existing plant and soil community in its own hydrological and climate setting is the experimental subject.

From the roadside, the facility looks like typical British woodland, but once you’re through the gates you can see this is no ordinary woodland walk . The FACE facility has been ‘smuggled’ into mature oak-and-hazel woodland in the heart of Staffordshire, taking particular care not to disturb the existing woodland canopy and the delicate soil structure.

FACE experiments allow for large areas of ecosystems to be exposed to enriched levels of carbon dioxide while maintaining other biotic and abiotic processes. It is essentially a laboratory experiment that has been taken outside and applied not to a leaf, or even a tree, but to half-a-dozen large trees, a dozen smaller trees, hundreds of ground plants, tens of thousands of invertebrates, and many billions of microbes in the soil. From above, these striking 25m-tall steel structures, arranged in circles, look like something from a sci-fi movie.

The BIFoR FACE facility seen from above, showing the 25m-high steel towers which pump 'enriched air' onto 30m-wide plots of forest. Image courtesy of Norbury Estate

At BIFoR FACE, we are exposing areas of woodland to CO₂ concentrations of roughly 550ppm, which is 150ppm above ambient levels. This is the CO₂ concentration expected by the middle of the century, so we are effectively simulating the future atmosphere around these trees to study how they may respond.

To do this, CO₂ gas is delivered through pipes into the forest, run through mixing fans to combine the CO₂ with regular air and pumped out through vertical cylindrical pipes as ‘enriched air’.

The BIFoR FACE experiment will run for a minimum of 10 years , which is essential for the study of long-lived species such as oak trees. This facility will enable the much-needed real-world experiments to improve our climate projections and evaluate risks to forest ecosystems and the services they provide.

This year marked the second year of experimental research at FACE, with multidisciplinary work occurring in each layer of the forest ecosystem. There are currently more than 20 research projects running simultaneously, from investigations of underground root systems and soil microbiology to understanding canopy leaf physiology – but much more remains to be studied.

The woodland has been divided into nine circular experimental plots, each 30m in diameter, which are grouped into the three categories. Treatment plots are areas of woodland exposed to enriched levels of carbon dioxide (550ppm). Ambient plots are areas identical to the treatment plots, with the same infrastructure, but the towers release unenriched air in the same way as the treatment plots release enriched air. Finally, control plots are areas of woodland left to their natural state that contain no infrastructure beyond walkways to prevent compaction of the soil. These plots act as controls on the effect of building the infrastructure into the forest.

FACE experiments require bespoke engineering to ensure a stable supply of elevated CO₂ to the treatment plots throughout the UK’s highly changeable weather conditions. To do this, process-control software determines the amount and direction of CO₂ release, responding rapidly to changes in wind speed and direction, so that CO₂ is always introduced into the ring on the upwind side and in just sufficient quantity to maintain the target concentration.

The woodland itself has been fully instrumented with a range of scientific equipment and automatic sensors installed to monitor ecosystem form and function. Unusually for a forest research area, mains power and data lines are available throughout the site. This allows for a good deal of automation in the measurements, which, in many cases, can be accessed remotely. In addition to the fieldwork research at the FACE facility, laboratory-based research on collected samples is conducted at the University of Birmingham.

A closer view of one of the six FACE arrays. Image courtesy of BIFoR

A head for heights

With a forest’s enormous structures and overhanging canopies, it is easy to feel dwarfed by and even apprehensive of its apparent stillness. However, you soon realise how busy and alive forest ecosystems are and how much there is still left to discover about them.

My PhD project involves studying the physiology of 150-year-old oaks, specifically their carbon uptake under elevated CO₂, so my research focuses on photosynthesis and transpiration. I will be observing how carbon uptake may change daily and seasonally, but also how different environmental factors such as temperature and rainfall may affect these processes.

This, excitingly, requires me to have my head in the clouds and feet off the ground as often as possible. I use a special canopy rope-access system that enables me to spend most of my days during the growing season 25m high at the top of the oak tree canopy .

From my perch, I particularly enjoy spring mornings when the nuthatches are hopping up and down the trunks and the woodpeckers are busy hammering away on a neighbouring tree. Yet at this height in the canopy, you see so much more than just trees and birds you see all the layers of the ecosystem working together as one.

My fieldwork involves taking sunrise-to-sunset measurements, known as diurnal sampling, from spring to autumn. The measurements I take are non-destructive and instantaneous, largely based on one piece of equipment strapped to me as I am hauled up into the canopy. I use a portable photosynthesis machine with a gas-exchange analyser unit and a head attachment that carefully attaches on to a leaf to make the measurements.

This cutting-edge technology calculates a wide variety of parameters based on the movement of carbon dioxide and water into and out of the leaf. These include rates of photosynthesis and transpiration, and stomatal conductance. The instrument also monitors environmental variables such as temperature, humidity and sunlight in order to interpret the photosynthetic parameters. I can track changes in the leaves’ physiology throughout the day.

BIFoR’s meteorological station monitors and records weather data. Combined with my measurements, this will enable me to observe the interactions between CO₂ enrichment and climatic data such as temperature and weather events.

I have completed my first season’s data collection, so I will be spending the ‘leaf-off’ period interpreting and analysing this dataset. (The great thing about studying deciduous trees is that they let you have a fieldwork rest period.) I will need at least another year’s dataset before I can start seeing robust patterns in the results.

After many CO₂ enrichment experiments in laboratories and at other FACE facilities, our knowledge base has been built on how crop plants and young tree plantations may be affected by increasing atmospheric CO₂. However, the response in mature, complex forest ecosystems may be different, and BIFoR FACE is the only facility in the northern hemisphere to address the impact of climate and environmental change on mature woodlands.

Regarding my research into leaf physiology, I expect that the photosynthesis process will be altered under elevated CO₂, but that the response will vary across the timeline of the longer-term project. There are many other projects at BIFoR FACE, including the study of insect physiology, fungal microbiology and root development, all of which will produce data essential to understanding the functioning of our future mature woodlands.

Future work

Studying long-lived species such as oak trees requires time, so the research at BIFoR FACE has only just begun. Over the next few years, the facility will generate large quantities of data from experiments inside the woodland, and in the laboratories, to help answer questions related to the uncertainty of our future ecosystems both in the UK and globally.

You can follow the research developing at BIFoR at

Anna Gardner is a PhD student at the University of Birmingham and her PhD is funded by the John Horseman Trust. The BIFoR FACE experiment is funded by donations from the JABBS Foundation, and the University of Birmingham has a number of other collaborating institutions and organisations.

Factors affecting rates of Photosynthesis (part 2): Grade 9 Understanding for IGCSE Biology 2.20 2.23

In the previous post on photosynthesis, you revised how there were four environmental factors that can affect rates of photosynthesis in a plant:

  • light intensity
  • light wavelength
  • temperature
  • carbon dioxide concentration

This post will explain the results from experiments with Elodea in which one factor is altered (the independent variable) and the other three are kept exactly the same (control variables)

Light intensity

The independent variable (light intensity) is on the x axis and the dependent variable (number of bubbles per minute) is on the y axis.

How do we explain the pattern in this graph?

As the light intensity increases the rate of photosynthesis increases. This is because a higher light intensity gives more energy to the chloroplasts and so more reactions can happen per second and the rate goes up. But beyond the orange dot on the graph, the increases in rate slows down until at around 12 units of light, adding more light has no effect on the rate. At these high light intensities some other factor is now the limiting factor as opposed to light intensity. The limiting factor remember is the factor in the shortest supply. So perhaps above 12 units of light photosynthesis is limited by the concentration of carbon dioxide. The only way to find the limiting factor is to repeat the experiment with more carbon dioxide and see whether the rate is higher above 12 units.

Light wavelength

Although this graph is not perfect, it does show how the rate of photosynthesis varies at different light wavelength.

Rates of photosynthesis peak in the blue-violet and red parts of the visible spectrum with a much lower rate in green light. The reason for this is that chlorophyll pigments do not absorb green light well.

Carbon Dioxide concentration

The pattern is similar to the light intensity relationship. When carbon dioxide concentrations are low, it is the limiting factor for photosynthesis and so increasing the concentration will increase the rate. As the graph levels off, some other factor is now the limiting factor – perhaps light intensity or temperature.


Temperature is a factor that affects photosynthesis because of enzymes. Many reactions in photosynthesis are catalysed by enzymes and enzymes all have an optimum temperature.

This pattern is not explained by limiting factors. At low temperatures the rate is low because the enzymes and the substrate molecules are moving really slowly. This means there are few collisions between the substrate and the active site of the enzyme. As temperature increases, the rate increases as there are more collisions and more enzyme-substrate complexes are formed per second. But high temperatures denature enzymes: the bonds that hold the enzyme in its precious 3-D shape are broken and the enzyme molecule unravels. So the active site may either change shape or may be lost as a catalyst. This slows the rate down to an extremely low rate.

How does CO₂ concentration affect photosynthesis? - Biology

The purpose of this experiment was to determine the effect of carbon dioxide concentration on the rate of photosynthesis in spinach leaves. Small circular disks were cut out of the spinach leaves using a standard hole puncher. Then solutions of differing concentrations of carbon dioxide, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0%, were all prepared, and each solution was spread equally among five cups. There was also a control solution that contained only water. Any gases within the leaves were then sucked with a syringe, using a specific technique to create a vacuum. For each solution of differing carbon dioxide concentration, there were five cups of solution with ten leaf disks per cup, so there was a total of 50 leaf disks per concentration. The cups were then exposed to light for 20 minutes, and the number of disks floating in each cup was measured every minute. Results were calculating by finding the ET50 for each concentration. The ET50 is the time it takes for 50% of the leaf disks to float and is a good indicator of the rate of photosynthesis. It was hypothesized that if the concentration of carbon dioxide was increased, then the rate at which photosynthesis occurs will also increase. The null hypothesis was that the concentration of carbon dioxide will have no effect on the rate of photosynthesis. The results of the experiment supported the hypothesis. So, the experiment suggests that there is a direct relationship between CO2 concentration and the rate of photosynthesis.

Lesson Plan: Impact of Climate Change on Photosynthesis

As a high school or undergraduate Biological Sciences teacher, you can use this set of computer-based tools to help you in teaching photosynthesis, factors affecting photosynthesis, and the impact of increasing levels of atmospheric CO2 and some climate-related factors on photosynthesis.

This lesson plan enables students to understand the process of photosynthesis in plants and the various factors that influence it. Students will explore how changes in climate related factors such as CO2 levels, temperature, water availability can influence photosynthesis, thereby impacting plant growth and agricultural yields. A hands-on lab activity will enable students to assess the change in the rate of photosynthesis when CO2 concentration changes.

Thus, the use of this lesson plan allows you to integrate the teaching of a climate science topic with a core topic in the Biological Sciences.

Use this lesson plan to help your students find answers to:

  1. What is photosynthesis?
  2. What are the key differences between C3, C4, and CAM plants?
  3. How do plants respond to climate change?
  4. Discuss the impact of increasing global CO2 levels on photosynthesis.
  5. Is global food security at risk due to climate change? Explain.

This is a Teacher-submitted Lesson Plan.

Teacher-submitted lesson plan, Contributed by: Dr. Aditi Kothari-Chhajer and Dr. Neeti Mehla, Department of Botany, Sri Venkateswara College (University of Delhi), Delhi.

A Level Biology Project

I plan to investigate how different factors affect the rate of photosynthesis. I will be changing the levels of light and CO2 and then measuring the photosynthetic rate.


This is a A-level biology project. It helped me get an A grade for biology many years ago. The whole project is reproduced here for your reference.

The rate of photosynthesis is affected by a number of factors including light levels, temperature, availability of water, and availability of nutrients. If the conditions that the plant needs are improved the rate of photosynthesis should increase.

The maximum rate of photosynthesis will be constrained by a limiting factor. This factor will prevent the rate of photosynthesis from rising above a certain level even if other conditions needed for photosynthesis are improved. This limiting factor will control the maximum possible rate of the photosynthetic reaction.

For instance, increasing the temperature from 10ºC to 20ºC could double the rate of photosynthesis as the plant's enzymes will be closer to their optimum working temperature. As the temperature is increased, molecules in the cells will be moving at a faster rate due to kinetic theory. If the temperature is raised above a certain level, the rate of photosynthesis will drop as the plant's enzymes are denatured. They will therefore be more likely to join onto the enzymes and react.

The amount of water available to the plant will affect the rate of photosynthesis. If the plant does not have enough water, the plant's stomata will shut and the plant will be deprived of CO². It is difficult in normal lab conditions to prove that water directly affects photosynthesis unless a heavy isotope is used to trace the path of water.

Chlorophyll is needed for photosynthesis. This can be proved by studying a variegated leaf. It is however very difficult to study how different levels of chlorophyll in the plant will affect it's photosynthesis rate. This is because in a variegated leaf the cells either contain chlorophyll or they don't.

Carbon dioxide concentration will directly affect the rate of photosynthesis as it is used in the photosynthesis reaction. It is also easy to change the amount of carbon dioxide that the plant receives.

Light is also directly used in the photosynthesis reaction and is easy to change in normal lab conditions. Carbon Dioxide and Light are the factors that I will change in the experiment as they are easy to change and measure.

  1. Elodea
  2. 20mm² syringe
  3. Capillary tubing
  4. Stand
  5. Stopwatch
  6. Ruler
  7. NaHCO³ Solution
  8. Bench lamp
  9. Distilled water

I could measure the decrease in the substances needed for photosynthesis, such as how much the amount of CO2 decreases over time. This is however difficult in normal lab conditions. I will instead measure how one of the products of photosynthesis (oxygen) increases over time. I am planning to use the following method for my experiment.

  1. The apparatus is set up as below with the syringe full of the 0.01M solution of NaHCO3 solution. Two marks 10cm apart are made on the capillary tubing.
  2. The syringe is placed 0.05m away from the lamp.
  3. Using the syringe plunger the meniscus of the NaHCO3 is set so that it is level with the first mark.
  4. A stopwatch is then started. The meniscus should gradually move down the capillary tube as the elodea produces oxygen as a by-product of photosynthesis. As the oxygen is produced it increases the pressure in the syringe and so the meniscus is pushed down the tube.
  5. When the meniscus reaches the level of the bottom mark the stopwatch should be stopped and the time should be noted in a table such as the one below.

The light intensities have been worked out using the following equation

Light Intensity = 1 / Distance² (m)

6. Using the same piece of elodea and the same distance between the lamp and the syringe the experiment (steps 1 to 5) should be repeated for the other concentration of NaHCO3.
7. The experiment (steps 1 to 6) should then be repeated at each different distance between the syringe and the light for all the NaHCO3 concentrations. The remaining distances are 0.05m, 0.06m, 0.07m, 0.08m, 0.1m, 0.2m, 0.3m, and 0.5m.
8. The entire experiment should then be repeated three times in order to obtain more accurate data and to get rid of any anomalies that may occur in a single experiment.

Measuring the volume of oxygen is more accurate than counting the number of bubbles produced as each bubble could be a different size. In order to make this experiment as accurate as possible a number of steps must be taken.

  • The experiment should be carried out in darkness with only the light from the bench lamp reaching the elodea.
  • The same piece of elodea should be used each time in order to make sure that each experiment is being carried out with the same leaf surface area.
  • The amount of NaHCO3 solution should be the same for each experiment. 20mm² should be used each time.
  • The lamp should be at the same height for each experiment. It should be level with the syringe each time.
  • The distance should be measured from the front of the lamp to the syringe. Although taking these steps will make the experiment more accurate, it's accuracy is still limited by several factors.
  • Some of the oxygen will be used for photosynthesis by the plant.
  • Some of the oxygen will dissolve into the water.

From these recorded times I will work out the rate of the reaction using the following equation.

Rate Of the Reaction = 1 / Time (s)

Using these rates I plan to plot a graph of the rate of reaction against light intensity.


I predict that if the light intensity increases the rate of the reaction will increase at a proportional rate until a certain level is reached, the rate of increases will then go down. Eventually a level will be reached where increasing the light intensity will have no more effect on the rate of reaction as there is some other limiting factor.

Light is needed for photosynthesis in plants. When chloroplasts in the leaf's cell are exposed to light they synthesise ATP from ADP. Oxygen is produced as a by-product of the photosynthesis reaction. Therefore increasing the concentration of light will increase the amount of ATP being synthesised from ADP and so more oxygen will be released as a by product.


I predict that as the concentration of NaHCO3 increases the rate of the reaction will increase at a proportional rate. Eventually increasing the NaHCO3 concentration more will have no effect as other limiting factors will be limiting the rate of photosynthesis. Carbon dioxide is needed for the photosynthesis reaction. It is used to make the organic products of photosynthesis. If the elodea is able to absorb more CO2 then the rate of photosynthesis will increase as the plant is able to make more of the organic compounds. The plant is given CO2 in the form of NaHCO3.

Pooled results from the group were used. They were taken over a 2 day period.

Molarity of NaHCO3
Light Intensity 1/d² (m) 0.00
(Distilled water)
0.01 0.02 0.05 0.07 0.1
400 3571 1666 1099 523 200 243
278 1670 5183 988 600 375 262
204 4998 4485 1175 1005 473 351
156 5590 2300 1770 1445 621 550
100 9990 3150 2900 2552 1224 645
25 4762 3984 2850 1640 1408
11 5945 4348 3780 2830 2564
4 16480 11904 5196 6578 3226

Using these results I worked out the rate

Rate Of the Reaction = 1 / Time(s) x 1000

The rate was multiplied by 1000 to make the numbers easier to handle.

Molarity of NaHCO3
Light Intensity 1/d² (m) 0.00
(Distilled water)
0.01 0.02 0.05 0.07 0.1
400 0.28 0.60 0.91 1.91 5.00 4.12
278 0.60 0.19 1.01 1.67 2.67 3.82
204 0.20 0.22 0.85 1.00 2.11 2.85
156 0.18 0.43 0.56 0.69 1.61 1.82
100 0.10 0.32 0.34 0.39 0.82 1.55
25 0.21 0.25 0.35 0.61 0.71
11 0.17 0.23 0.26 0.35 0.39
4 0.06 0.08 0.19 0.15 0.31

A graph of the rate of reaction against light intensity was drawn. It shows how the amount of CO2 and light affect the rate of photosynthesis. Lines of best fit were drawn for each CO2 concentration to make up for any inaccuracy in any individual result. The line of best fit gives a good picture of how the overall rate of reaction is affected by the light and CO2.

I will analyse the results for how the amount of light and CO2 affects the rate of photosynthesis.

My prediction that the rate of photosynthesis would go up if the light intensity and NaHCO3 levels were increased proved correct. As the elodea absorbed the light and CO2 it produced oxygen gas which increased the pressure in the syringe. This pushed the air bubble in the capillary tube down. The chloroplasts produce ATP and reduce NADP to NADPH2 when exposed to light. It is at this stage of the reaction that oxygen is produced as a waste product.

As predicted when the light intensity increases so does the rate of photosynthesis. I predicted that a level would be reached where increasing the light intensity would have no more effect on the rate of reaction as there would be some other limiting factor which limits the rate of the reaction. The rate increases at a steady rate as the light intensity increases until near the end of each line where the rate of increase decreases. This is either because the photosynthesis reaction has reached it's maximum rate of reaction or another factor is limiting the rate. As 6 different CO2 concentrations were used I can see that the first five reactions are not occurring at their maximum rate as there is the 0.1M NaHCO3 rest which is occurring at a faster rate then the other 5. The photosynthesis reactions of the other five test must therefore be limited by the concentration of CO2 to the plant.

As predicted when the NaHCO3 concentration is increased the plant in able to get more CO2 which causes the rate of reaction to go up. I predicted that once the NaHCO3 had been raised above a certain level increasing the rate further would have no effect as there would be other limiting factors limiting the rate of the reaction. As the NaHCO3 concentration in the water was increased the rate of photosynthesis was able to go up. The plant therefore made more oxygen as a waste product. At a NaHCO3 concentration of 0.1M once the light intensity gets above 300 the rate of reaction slows down very quickly. This could be because photosynthesis is occurring at it's maximum possible rate or because another limiting factor is limiting the rate of reaction.

Distilled Water

With the distilled water the rate of reaction went up from 0.1 to 0.4 when the light intensity was increased from 100 to 400. This is a 4 times rise which is quite large. The curve on the graph does however level out quite soon showing that the rate is being limited by the lack of NaHCO3 in the water.

0.01M NaHCO3

At a light intensity of 4 the rate is 0.06 but this rises to 0.6 when the light intensity is brought up to 400. The curve is very shallow and levels off towards a light intensity of 350 - 400.

0.02M NaHCO3

The amount of NaHCO3 is double that of the 0.01M NaHCO3 experiment. The rate also finishes off twice that of the 0.01M experiment. This would surgest that there was a directly proportional relationship between the amount of NaHCO3 and the rate of reaction.

0.05M NaHCO3

The curve for the 0.05M NaHCO3 is steeper than the previous curves. The rate rises to 1.9 at a light intensity of 400.

0.07M NaHCO3

The 0.07M NaHCO3 test produces a line which is steeper than all the previous curves. The plant is using the extra CO2 to photosynthesise more. As the plant has more CO2 the limiting factor caused by the lack of CO2 is reduced. This test did produce a big anomaly. The rate for a light intensity of 400 is 5. By following the line of best fit I can see that this result should be more like 3.5. The elodea for this test was very close to the light source. It is possible that it had been left here for a while which caused the lamp to heat the elodea up. This would have increased the rate of reaction of the plant's enzymes which would have increased the photosynthesis rate.

0.1M NaHCO3

The 0.1M NaHCO3 produced the steepest line. Near the end of the line it looks as if the rate of reaction is hit by another limiting factor. The line goes up steadily but then between a light intensity of 300 and 400 levels off very quickly. This would surgest that at a 0.1M NaHCO3 is sufficient for the plant to photosynthesise at it's maximum rate with it's current environmental conditions. Increasing the NaHCO3 concentration after this level would therefore have no effect unless the next limiting factor was removed.

The fact that the curve levels off so quickly indicates that there is another limiting factor limiting the photosynthesis. It could be temperature. These tests are being carried out at room temperature so the temperature would have to be raised another 15ºC before the enzymes in the plant's cells were at their optimum working temperature. More tests could be done by using water that was at a higher temperature to see what effect this would have on the photosynthesis rate. It is however impossible to raise the plant's temperature without affect other factors. For instance the actual amount of oxygen released by the plant is slightly more than the readings would surgest as some of the oxygen would dissolve into the water. At a higher temperature less oxygen would be able to dissolve into the water so the readings for the photosynthesis rate could be artificially increased.

It is also possible that the photosynthetic reactions in the plant are occurring at their maximum possible rate and so can not be increased any more.

The light is probably not a limiting factor as all but one of the curves level off before the maximum light intensity of 400 is reached. The maximum light intensity that the plants can handle is therefore just below 400.

Water will not be a limiting factor as the plants are living in water. They therefore have no stomata and absorb all their CO2 by diffusion through the leaves.

The accuracy of this experiment is limited by a number of factors.

  1. Some of the oxygen give off is used for respiration by the plant.
  2. Some of the oxygen dissolved into the water.
  3. Some was used by small invertebrates that were found living within the pieces of elodea.
  4. The higher light intensities should be quite accurate but the smaller light intensities would be less accurate because the light spreads out. the elodea will also get background light from other experiments.
  5. The lights are also a source of heat which will affect the experiments with only a small distance between the light and the syringe. this heating could affect the results.
  6. Using the same piece of elodea for each experiment was impractical as the elodea's photosynthesis rate decreased over time. By using a different piece of elodea for each experiment did create the problem of it being impossible for each piece to have the same surface area.
  7. As the tests took place over a two day period there will be some inaccuracy caused by factors such as temperature. There was no practical way for the long tests to be kept at a totally constant temperature for the two day period and they will probably have cooled down at night and then warmed up in the day leading to a slight inaccuracy.

This experiment could be improved in a number of ways.

  1. It could be repeated more times to help get rid of any anomalies. A better overall result would be obtained by repeating the experiment more times because any errors in one experiment should be compensated for by the other experiments.
  2. Each person should have done their experiments in a different room to cut out all background light.
  3. All the experiments should be done sequentially.
  4. A perspex screen could have been placed between the light and the syringe to reduce any heating effect that the light may have.
  5. The experiment could have been carried out with higher NaHCO3 to see if increasing the concentration would increase the rate of photosynthesis, or if a concentration of 0.1M NaHCO3 produces the maximum rate of photosynthetic reaction.


This is a real A-level school project and as such is intended for educational or research purposes only. Extracts of this project must not be included in any projects that you submit for marking. Doing this could lead to being disqualified from all the subjects that you are taking. You have been warned. If you want more help with doing your biology practicals then have a look at 'Advanced Level Practical Work for Biology' by Sally Morgan. If you want more detailed biology information then I'd recommend the book 'Advanced Biology' by M. Kent.


This entry was posted on Tuesday, June 3rd, 2008 at 8:14 pm and is filed under Life. You can follow any responses to this entry through the RSS 2.0 feed. You can leave a response, or trackback from your own site.


Growth chamber experiments

We bought soybean seeds from the Wotu seed company in Hebei Province of China. We grew three plants in each pot (30 cm diameter × 50 cm long), then set up five pots in each of the seven walk-in environmental growth chambers for 90 days CO2 treatment, where the CO2 concentration was regulated to ambient concentration (400 ppm) or elevated concentrations (600, 800, 1000, 1200, 1400 and 1600 ppm). The ambient and elevated CO2 concentrations within the chambers were maintained through a CO2 tank containing high purity CO2 gas (99.99%) to avoid any hurt or pollution on winter wheat plants. All of the seven growth chambers were maintained with the same other environmental factors including relative humidity of 65%, photosynthetic photon flux density (PPFD) of 1000 μmol m − 2 s − 1 , temperature of 25/21 °C (day/night), and 12-h photoperiod for the 90 days treatment. These winter wheat plants were fertilized with half-strength Hoagland’s solution twice weekly (150 mL per pot) and irrigated once daily with plain tap water (200 mL per pot) during the establishment and treatment periods of soybean plants under elevated CO2 concentrations.

Measuring leaf gas exchange

We performed the measurements of leaf gas exchange at the end of the CO2 treatment period. We randomly selected one fully expanded leaf from each pot for leaf gas exchange measurement (n = 5) with a portable photosynthesis system (LI-6400XT LICOR, Inc.). These selected leaves were firstly equilibrated at the corresponding growth CO2 levels with saturating PPFD of 1500 μmol photon m − 2 s − 1 and growth temperature of 25 °C. The portable photosynthesis system automatically controlled the CO2 concentrations in the cuvette using an injector system combined with a CO2 mixer. All of the measurements on leaf gas exchange were performed with the vapor pressure deficit (VPD) lower than 1.5 kPa to avoid moisture limitation. Then, the photosynthesis vs intercellular CO2 (An-Ci) curves were measured at cuvette chamber CO2 of 50, 100, 150, 200, 300, 400, 600, 800, 1000, 1200, 1400, and 1600 ppm. Data from An-Ci curves were used to compare treatment effects on the light-saturated net photosynthetic rates (An) at ambient or elevated CO2 of their growing condition. An estimation method was used to obtain the maximum carboxylation rate of Rubisco (Vcmax), and the maximum capacity of electron transport mediated ribulose bisphosphate (RuBP) regeneration (Jmax) for each observed An-Ci curve. Meanwhile, stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (Tr), and dark respiration rate (Rd) were also determined with the portable photosynthesis system (LI-6400XT LICOR, Inc.). In addition, the leaf-level water use efficiency (WUE) was determined by the values of the net photosynthetic rate (An) and transpiration rate (Tr) according to the formula WUE = An / Tr.

Measuring morphological traits of individual stoma and spatial distribution pattern of stomata

We randomly selected five fully expanded ear leaves at the heading stage in each of the ambient and elevated CO2 concentration plots to determine the stomatal characteristics. We sampled impressions of stomata with colorless nail polish from the middle section of the adaxial and abaxial leaf surfaces. Firstly, the adaxial and abaxial leaf epidermis were carefully cleaned with degreased cotton balls and then smeared with nail varnish from the mid-area between the leaf edge and the central vein for half an hour. The thin film with stomatal impression (approximately 5 mm × 15 mm) was peeled off from the leaf surface and mounted on a glass slide, and immediately covered with a cover slip and lightly pressured with a fine-point tweezer [47, 63]. We photographed the stomatal features with a microscope (DM2500, Leica Corp, Germany) equipped with a digital camera (DFC 300-FX, Leica Corp, Germany), and then analyzed thirty separate fields of 0.16 mm in each leaf section. We also combined and counted the stomata on each surface for calculating stomatal density (SD) of the adaxial and abaxial surface, respectively [47]. Moreover, we randomly selected six digital photographs of the adaxial and abaxial surfaces to measure the stomatal length (SL), stomatal width (SW), stomatal area (SA) and stomatal perimeter (SP) using AutoCAD 2010 software. In addition, we calculated stomatal shape index (SSI), which is calculated by the function that shape index= ( frac>>< mathrm> imes 100\% ) , where SA is the stomatal area and SP is the stomatal perimeter. The stomatal area index (SAI) is defined as the total stomatal area per unit leaf area calculating as stomatal average density × stomatal area per stoma × 100%. In addition to stomatal density and pore traits, we also characterized the spatial distribution pattern of stomata for each image by digitizing the stomatal positions into a shape file in GIS with the ArcMap software [47]. The spatial distribution pattern of stomata on leaves was quantified using the Ripley’s K-function with generating the x and y coordinates of stomata for each image in GIS and then calculating the Lhat (d) value (the transformed K value) based on these stomatal coordinates using the R statistic software. We compared the Lhat (d) values at different scales (distances) for detecting the spatial distribution pattern of stomata with the upper and lower boundaries generated by the 95% confidence level with the Monte Carlo simulations of 100 replicates [47, 76]. In the current study, we only reported the spatial distribution patterns of stomata on the middle section of the leaves due to the large number of stomatal images of winter wheat leaves.

We snapped three leaf pieces (2 mm × 2 mm) from the middle section of each leaf and fixed them with 2.5% (v/v) glutaraldehyde (0.1 M phosphate buffer, pH 7.0) to visualize the changes in stomatal morphology among different CO2 concentrations. Firstly, we washed these leaf samples several times with buffer and fixed them in 1% (v/v) osmium tetroxide for three hours and these samples were dehydrated with an ethanol series. Then, these leaf samples were carefully coated with gold in a high-vacuum evaporation unit. Finally, we examined and photographed the morphological traits of stomata with a scanning electron microscopy (FEI Corp, USA).

Measuring leaf anatomical structures

Changes in the leaf internal anatomy of the winter wheat plants exposed to different CO2 concentrations were examined with leaf cross-sections under a light microscopy [77]. These images of leaf cross-sections were collected from the middle section of leaves to observe and measure leaf anatomical features using Image J software (NIH, USA). We estimated leaf mesophyll thickness between epidermal layers at five points in each cross-section [78]. We also randomly selected 20 clear palisade layer cells and 20 sponge layer cells from each leaf cross-section image to measure cell length, cell width, cell area, and cell perimeter with an Auto CAD software.

Analyzing leaf non-structural carbohydrates and nitrogen

We collected leaf samples from each pot as a replicate (n = 5 pots) for analyzing the non-structural carbohydrates. These sampled leaves were dried with an oven at 75 °C for 48 h to consistent weight, and then these samples were ground to fine powder for spectrophotometrically analyzing glucose, fructose, sucrose, and starch with a glucose kit [79]. Similarly, we also sampled plant tissues from each pot (n = 5 pots) for analyzing the total carbon (C) and nitrogen (N) in different plant tissues (leaf, stem, and root) with an elemental analyzer [80]. All of the analyses were expressed on a percentage dry matter basis.

Analyzing data

We used the one-way ANOVA to analyze the effects of CO2 on the stomatal traits, soluble sugar and starch concentrations, carbon and nitrogen contents, as well as morphological and anatomical features. Two-way ANOVA was employed to test the effects of CO2 concentration and leaf surface position (abaxial vs. adaxial) on the morphological traits of stomata with statistically significant differences at p < 0.05 level. We also employed linear and non-linear regressions for estimating the relationships between CO2 concentration and other variables. The raw data from the leaf photosynthesis measurements were processed in Excel spreadsheets where the non-linear An-Ci curve fitting was performed [81]. The net assimilation rate (An) versus intercellular CO2 concentration (An-Ci curve), was fitted to estimate the maximum carboxylation rate (Vcmax), maximum electron transport rate (Jmax) based on the measurements of An-Ci curves. In addition, linear and non-linear (quadratic equations) regressions were employed to examine the relationships between CO2 concentration and other variables.

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