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The Calvin Cycle, Practice Problems and FAQs

The Calvin Cycle, Practice Problems and FAQs

We all depend on plants directly or indirectly for food. We know that plants prepare their own food through the process of photosynthesis. But do you know what form of food they are preparing? Yes, you are correct. They prepare carbohydrates. But do you know how they prepare carbohydrates? The first carbohydrate formed in the plants during photosynthesis is glucose. This will be then converted to sucrose and then used for many purposes. Since glucose is a carbohydrate, the main raw material plants will utilise for making this is the atmospheric CO2. So basically, the carbon atom (C) from the atmosphere is entering the plants to form the glucose. This is very clear from the stoichiometric equation of the photosynthesis given below.

                                     Fig: Stoichiometric equation of photosynthesis

How will atmospheric CO2 get converted into glucose? Can we also make glucose like that? Do they need any more ingredients for this process? Obviously they need energy, right?. Where do they get this energy then? They get all the energy for making food from the chain of reactions happening before fixing the CO2 called light reactions. The reactions that happen in the presence of light in photosynthesis are called light reactions. They occur at the chloroplast of plant cells. The reactions to fix the CO2 and to make the glucose will be happening after the light reactions. So what do you think, is light required for the synthesis of glucose? Can CO2 be fixed in the chloroplast itself? How the CO2 fixed gets converted into glucose? How much energy is needed by the plants to synthesise one molecule of glucose? Answers to all these questions are explained through a group of cyclic reactions called the Calvin cycle. Let’s discuss more about the Calvin cycle in this article. 

Table of contents:

Dark reaction

The phase of photosynthesis that happens independent of sunlight is called the dark reaction. It is also known as light-independent reaction, biosynthetic phase, Blackman’s reaction or CO2 fixation. Dark reaction does not mean that it occurs only at night.

The process of photosynthesis will only be completed when a dark reaction takes place along with the light reaction. The energy molecules like ATP and NADPH formed as a result of light reaction are used in the dark reaction. The electron transport systems in the light reaction are arranged in the thylakoid membrane in such a way that the byproducts formed will be available for the dark reactions to take place. 

                              Fig: Connection between light and dark reaction

CO2 is fixed in different pathways in different plants. CO2 can be fixed through three types of cyclic reactions, like the Calvin cycle (C3 cycle), Hatch and Slack cycle (C4 cycle) and CAM cycle (Crassulacean Acid Metabolism). Here we are going to discuss how CO2 is fixed through the Calvin cycle in depth.

Calvin cycle

The Calvin cycle is the most widespread CO2 biofixation pathway among the autotrophs. It is also known as the reductive pentose phosphate cycle. It is the first CO2 fixation cycle discovered by Calvin, Benson and Bassham. The organisms where the Calvin cycle takes place are the plants, microalgae and also the photoautotrophic and chemoautotrophic bacteria.

In this cycle the Ribulose-1, 5-bisphosphate carboxylase - oxygenase (RuBisCO), which is a key enzyme of this process will catalyse the process of capturing the CO2 molecule from the atmosphere and the conversion of the Ribulose-1,5-bisphosphate (RuBP) to 3-phosphoglycerate (PGA). Here the 3-PGA formed is a three-carbon compound which is the first stable product of this pathway, hence this cycle is known as the C3 cycle. Those plants which follow the C3 cycle are called C3 plants. Examples of C3 plants include wheat, soybean, oat etc. 

                                                            Fig: C3 plants 

In this cycle the carbon from the atmosphere is fixed into a sugar molecule (glucose) and this sugar molecule is further reduced to form carbohydrates. Hence the Calvin cycle is also known as the Photosynthetic Carbon Reduction (PCR) Cycle of Photosynthesis. 

                             Fig: Reduction of carbon dioxide into sugar

History of calvin cycle

Calvin was awarded the Nobel Prize in 1961 for the discovery of the C3 cycle. Calvin and his coworkers discovered the C3 pathway by using the following methods:

  • Radioactive tracer technique
  • Paper chromatography
  • Autoradiography

Radioactive tracer technique

They used radioactive 14C as a tracer, to find the fate of carbon dioxide in the dark reaction. They injected 14CO2 into an illuminated suspension of organisms like green algae, Chlorella and Scenedesmus. These organisms were carrying out the photosynthesis by using normal CO2. Now they will use this radioactive CO2 (14C) to carry out the photosynthesis and whatever carbon products formed during the photosynthesis will have the radioactive CO2.

They used hot methanol for killing the algal cells at different intervals and stopped their enzymatic reactions. Now they made an extract of algal cells which have the radioactive CO2 (14C).

Paper chromatography

By using the two dimensional paper chromatography, they extracted the radioactive compounds from the algal cells. 


In this method the paper chromatogram from the previous step is pressed with an X-ray film and as a result some spots will develop on the X-ray. These spots represent the location of radioactive compounds. By comparing the position of standard compounds, the radioactive compounds were identified.

They found the first stable product as 3-phosphoglycerate (PGA), which contained one radioactive carbon. Later they found radioactivity in many compounds which include tetroses, pentoses, hexoses and heptoses. In the later stages, three carbons were radioactive. Using all this available information, Calvin constructed the cycle until the formation of glucose. 

RuBisCO was identified by Bassham. Benson discovered the path of carbon assimilation in the plants. Hence the Calvin cycle is called the Calvin-Benson-Bassham or CBB cycle. 

Site of calvin cycle

We already saw that the Calvin cycle takes place by using the energy molecules of light reaction, which is taking place in the thylakoid membranes of the chloroplast. The ATP and NADPH formed as a result of light reactions are present in the stroma of chloroplast. So for utilising this energy, the dark reaction should take place in the stroma. 

Stroma is the aqueous fluid that surrounds the thylakoid of the chloroplasts. Hence the site of the Calvin cycle is the stroma of chloroplast. 

                                                 Fig: Structure of the chloroplast

Raw materials of Calvin cycle 

The raw materials required for the dark reaction by the Calvin cycle are as follows:

  • ATP (Formed by light reaction)
  • NADPH (Formed by light reaction)
  • CO2 (Enters through the stomata present in the leaf surface)

                                                       Fig: Stomata

Steps of Calvin cycle

The Calvin cycle occurs in all the photosynthetic plants (both C3 and C4). It has three phases as follows:

  • Carboxylation
  • Reduction
  • Regeneration

                                                                Fig: Steps of calvin cycle

Carboxylation - Carbon dioxide fixation

Carboxylation is the process by which a carboxylic acid group is produced by treating a substrate with carbon dioxide. It is the most crucial step in the Calvin cycle. It is done by using the substrate Ribulose-1,5-bisphosphate (RUBP), which is a 5 carbon ketose sugar. It is the primary acceptor of CO2. RuBP that is being used for carbon fixation has to be replenished by the end of this cycle for the RuBP to accept more CO2 to continue this cycle.

                                                      Fig: Structure of RuBP

When one molecule of CO2 enters the Calvin cycle, then the fixation of CO2 by the RuBP occurs. It produces an unstable, intermediate molecule 2-Carboxy-3-keto-1,5-biphosphoribotol. This reaction is catalysed by the enzyme RuBP carboxylase-oxygenase. This enzyme can show the oxygenase activity too, hence called Ribulose-1,5-bisphosphate carboxylase oxygenase or RuBisCO. 

The intermediate molecule formed will then split in the presence of water to form 2 molecules of 3-phosphoglyceric acid. Hence one carbon atom reacts with RUBP to form 2 molecules of 3PGA (phosphoglyceric acid) at the end. 

                                                       Fig: Carboxylation


It is the most abundant enzyme on the Earth. It possesses active sites for both CO2 and O2. But has greater affinity for CO2 when CO2: O2 is nearly equal. So there will be a competition between CO2 and O2. Hence RuBisCO shows both carboxylation and oxygenation activity. 

                         Fig: Structure of RuBisCO

Reduction of PGA

The reactions involved in this second phase of the Calvin cycle are the reversal of the reactions found in the glycolysis part of respiration. Hence this phase is also called glycolytic reversal. 

In this phase the two molecules of 3PGA are converted to two molecules of glyceraldehyde-3-phosphate (G3P). ATP delivers energy and NADPH attaches one hydrogen to each of the PGA chains. NADPH donates electrons to, or reduces, 3PGA to make G3P. So for two molecules of G3P, two molecules of ATP and two molecules of NADPH are used. 

                                            Fig: Reduction and structure of G3P

The G3P molecule formed has two fates as follows:

  • Regeneration of RUBP
  • Production of glucose

Regeneration of RuBP

We were talking about the reactions when a molecule of CO2 is fixed. So when one molecule of CO2 is fixed, plants get two molecules of G3P which is a three carbon molecule (total six carbon atoms). From this we have to make glucose and also the regeneration of RuBP (5 carbon atoms) for the next cycle. 

Glucose is a six carbon molecule (6 carbons) and through fixing one molecule of CO2 we get only one carbon. G3P will provide the carbon atom of fixed CO2 for the production of glucose. Now from the six carbon atoms of two molecules of G3P, five carbon atoms are left, which can go for the regeneration of RuBP (5C molecule). One molecule of ATP is utilised for the regeneration of RuBP.

                                        Fig: Calvin cycle fixing one CO2 molecule

After the entry of one molecule of CO2, plants could regenerate one molecule of RuBP. But, they got only one C (carbon) atom to make the glucose molecule and needed five more carbon atoms. 

Hence plants take a total of six turns of the Calvin cycle to make one glucose molecule. So, 6 carbon atoms to be fixed for the formation of one glucose molecule. 

Energy required to fix one molecule of carbon dioxide 

The total energy taken and released by the plants to fix one molecule of carbon dioxide is as follows: 



1 CO2

1 Carbon atom is fixed in C6





Now let us see how the Calvin cycle will be when plants fix 6 carbon dioxide molecules.

Six Calvin cycles

After the fixation of one CO2, only 1 carbon atom is fixed for the synthesis of glucose. We will discuss now how the number of molecules changes in each of the three phases of the Calvin cycle. 

Carbon dioxide fixation 

We know that one RuBP and one CO2 molecule gives two molecules of 3 PGA. Hence, plants need 6 RuBP (6X5 = 30 carbons) to fix 6 molecules of CO2 (6 C)which will give 12 molecules of 3PGA (12X3 = 36 C). So here there will be a total of 36 carbon atoms.

                                          Fig: Carbon fixation in six Calvin cycles


The 12 molecules of 3PGA will use 12 ATP and 12 NADPH molecules to form 12 molecules of G3P. Two of the 12 molecules G3P will be used to form a molecule of glucose which is a 6 carbon molecule. The remaining 10 G3P molecules will not be wasted. They will be used in the next phase of the Calvin cycle, which is known as regeneration.

So here the 12 molecules of G3P (12X3) also have 36 C atoms. From this, 6 C atoms will go for the synthesis of glucose.


10 (30 carbon) of the 12 molecules of G3P are converted to 6 molecules of RuBP (6X5 =30C). One molecule of G3P has 3C. So, 10 molecules of G3P have 30 carbon atoms. 

One RuBP molecule consists of five carbon atoms. So from 10 molecules of G3P, plants can make 6 molecules of RuBP (6X5 = 30C). The process begins with 6 RuBP initially. So, all the borrowed RuBP are given back to the plant cell. The regeneration steps require one ATP for phosphorylation to form one RuBP. So for 6 molecules of RuBP, plants utilise 6 molecules of ATP. 

                               Fig: 6 cycles of Calvin cycle to make one glucose molecule

Even though all the three phases of the Calvin cycle have been shown in a simplified form with the formation of molecules like PGA, G3P and RuBP, there are many intermediate molecules that are formed to complete the cycle. Some of them are Dihydroxyacetone phosphate, Fructose-6-phosphate, Ribose-5-phosphate etc. Along with the RuBisCO, each step or reaction in the Calvin cycle is catalysed by different enzymes like triose phosphate dehydrogenase, transketolase, epimerase, isomerase etc. 

Energy required to fix six molecules of carbon dioxide 

The total energy taken and released by the plants to fix one molecule of carbon dioxide is as follows: 



6 CO2

6 Carbon atom is fixed in C6

18 ATP

18 ADP


12 NADP+

Summary of the reactions in the Calvin cycle

The reactions of the Calvin cycle can be summarised as follows:

                      Fig: Summary of the reactions in the Calvin cycle

Significance of Calvin cycle 

The following are the major significances of the Calvin cycle:

  • The Calvin cycle is included in the biosynthetic phase of the photosynthesis. So the major function of the Calvin cycle is to produce carbohydrates through fixing carbon dioxide.
  • Carbohydrate is the main source of energy in every food chain as plants occupy the first trophic level.
  • Glucose, fructose and sucrose are formed mainly through this pathway. 
  • The Calvin cycle also produces the reaction intermediates like glucose, sedoheptulose etc., which are required for other physiological reactions (respiration) that occur in the plant body. 
  • It is a universal pathway and even present in C4 plants. 
  • It is required for the growth and development in plants. 

Practice Problems

Q 1. What happens to the biosynthetic process, immediately after light becomes unavailable?

a. It will continue for some time and then stops
b. It will stop and start again if the light is made available
c. It will stop permanently if the light is not made available in future
d. all of the above 

Answer: The chlorophyll absorbs light during the light-dependent process, which produces ATP and NADPH. Glucose synthesis happens during the light independent reaction, also known as the biosynthetic phase. In the stroma of the chloroplasts, the biosynthetic phase takes place. These processes rely on light reaction byproducts like ATP and NADPH rather than directly depending on light. As a result, after a while, ATP and NADPH are no longer available in the absence of light. Hence the Calvin cycle (biosynthetic phase) halts in the absence of ATP and NADPH, and at that time there is no synthesis of glucose. After a while, when the light is reintroduced, light reactions occur that result in the production of ATP and NADPH. The light-independent process resumes activity when ATP and NADPH are available. So, the light independent reaction halts in the absence of light and resumes when light is again accessible. Hence the correct option is d. 

Q 2. ATP and NADPH are the products of light reactions. These products are first used in which of the following steps of the Calvin cycle?

a. Reduction
b. Regeneration
c. Carboxylation
d. All of these

Answer: The Calvin cycle occurs in the stroma of the chloroplast and is responsible for fixing carbon dioxide molecules to synthesise glucose. The three stages of the Calvin cycle are carboxylation, reduction, and regeneration. Two molecules of ATP are used for phosphorylation during the reduction stage, along with two molecules of NADPH. Each RuBP molecule that is rebuilt during the regeneration phase requires one ATP molecule. There is no utilisation of an ATP or NADPH molecule during the carboxylation process. Hence the correct option is a. 

Q 3. Which of the following is required for the synthesis of one molecule of glucose in the Calvin pathway?

a. 3 CO2 + 18 ATP + 18 NADPH
b. 6 CO2 + 12 ATP
c. 6 CO2 + 18 ATP + 12 NADPH
d. 6 CO2 + 18 ATP + 24 NADPH

Answer: Energy molecules created during the light reaction of photosynthesis include ATP and NADPH. ATP and NADPH are not necessary for carboxylation. The reduction and regeneration phases of the Calvin cycle require ATP and NADPH. 6 CO2, 18 ATP, and 12 NADPH are needed to synthesise one molecule of glucose. In order to complete the reduction phase of the Calvin cycle, 12 ATP and 12 NADPH are used. During the regeneration stage, 6 ATP is needed. Hence the correct option is c. 

Q 4. For the regeneration of one RuBP molecule, how many molecules of ATP and NADPH are utilised in the Calvin cycle?

a. 1 ATP and 1 NADPH
b. 1 NADPH only
c. 1 ATP only
d. 2 ATP and 2 NADPH

Answer: When one molecule of CO2 is fixed, plants get two molecules of G3P which is a three carbon molecule (total six carbon atoms). From this we have to make glucose (6 carbon atoms) and also the regeneration of RuBP (5 carbon atoms) for the next cycle. Glucose is a six carbon molecule and through fixing one molecule of CO2 we get only one carbon. G3P will provide the carbon atom of fixed CO2 for the production of glucose. Now from the six carbon atoms of two molecules of G3P, five carbon atoms are left, which can go for the regeneration of RuBP (5 carbon molecules). One molecule of ATP is utilised for the regeneration of RuBP. Hence the correct option is c. 

Q 5. Which of the following enzymes are required for the carboxylation in C3 plants?

a. Kinase
b. RuBisCo
c. Nitrogenase
d. Alpha - ketoglutarate dehydrogenase

Answer: The Calvin cycle begins with carboxylation, or fixing carbon dioxide. Ribulose-1,5-bisphosphate serves as the main carbon dioxide acceptor. When RuBP and carbon dioxide react, an unstable six-carbon molecule is formed. The enzyme RuBisCO (ribulose-1,5-bisphosphate carboxylase-oxygenase) is responsible for catalysing this reaction. The unstable six-carbon complex splits up into two 3-PGA molecules (3-phosphoglycerate). An enzyme called kinase catalyses the addition of phosphate groups from high energy phosphate-donating molecules to particular substrates. A Fe-Mo protein called nitrogenase is involved in the biological fixation of nitrogen. The citric acid cycle or Krebs cycle has an enzyme known as alpha-ketoglutarate dehydrogenase complex . Hence the correct option is b. 


Q 1. What is carbon refixation?
Answer: The ability to recapture CO2 produced during photorespiration which results in the improvement in efficiency of C3 plants like rice and bamboo is called carbon refixation. Around the stroma in the mesophyll cells, there will be the growth of chloroplast extensions called stromules. The plants achieve refixation with the help of stromules. This will result in the passage of photorespired CO2 (from mitochondria) through the chloroplast filled with RuBisCO. 

Q 2. How do C4 plants differ from C3 plants in the location of RuBisCO?
Answer: The Calvin cycle is observed in both C3 and C4 plants. But the location of RuBisCO is different in them. RuBisCO occurs in mesophyll cells in C3 plants and in bundle sheath cells in C4 plants. 

Q 3. Who discovered light reaction?
Answer: Jan Ingenhousz was the scientist who first identified the function of light in photosynthesis. He established the location of photosynthesis by demonstrating the significance of light in the absorption of CO2 and the release of oxygen by green plants.

Q 4. Is there any difference between 3 PGA and G3P?
Answer: The chief difference between G3P and 3-PGA is that although 3-PGA has a carboxylic acid functional group at the carbon-3 position, G3P has an aldehyde functional group at that same location. Glyceraldehyde 3-phosphate is referred to as G3P, and 3-PGA is referred to as 3-phosphoglyceric acid.

YOUTUBE LINK: https://www.youtube.com/watch?v=5wGbMUUdUys

Related Topics

Light reaction, Practice Problems and FAQs 

Cyclic and Non-cyclic photophosphorylation, Practice Problems and FAQs 

Photorespiration, C2 Cycle, Factors affecting photorespiration, Difference between dark respiration and photorespiration, Practice Problems and FAQs 

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