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To investigate the effects of different types of  substrates on the rate of respiration of Saccharomyces Cerevisiae by measuring the volume of CO2 produced using a gas syringe

Background information

What is respiration?

Respiration is the name given to the biological process which occurs in all living cells. It involves the release of energy, by oxidising glucose. Glucose is a carbohydrate, specifically a monosaccharide, which undergoes a series of reactions releasing energy which the cell uses. Every cell needs energy and thus every cell respires to release the energy that it needs. However, respiration consists of many stages. These stages are glycolysis, the link reaction, the Krebs cycle and finally, the electron transport chain. These are explained in detail below:

C6H2O6(aq) + 6 O2 (g)     →   6 CO2 (g)    +     6H2O(l) + energy

Glucose     + Oxygen   →   Carbon dioxide +  water   + energy

Glycolysis: a process in which a 6C sugar, usually glucose, is split into 2 pyruvate molecules, which are a 3C acid. The process takes place in the cytoplasm of the cell and is summarised in detail below.

Glucose, like many sugars is very unreactive and therefore needs to be activated. Therefore it is converted into a more reactive hexose sugar, fructose bisphosphate by adding 2 phosphate molecules that have come from 2 molecules of ATP. ATP is converted to ADP as shown in the diagram. This process is called phosphorlyation.

The phosphorylated 6VC sugar, Fructose Bisphosphate is split into 2 molecules of glyceraldehyde – 3 – phosphate, which consists of 3C each.

Each GAL3P then undergoes an oxidation reaction forming 2 molecules of GP, which are soon converted into 2 molecules of the 3C acid, pyruvate. This oxidation reaction releases 2 electrons and 2 hydrogen ions (H+) from each GAL3P, which are transferred to coenzyme NAD (nicotinamide adenine dinucleotide) to form 2 reduced NAD (NADH + H+). The energy released during the production of pyruvate from GAL3P is used to produce 4 molecules of ATP as shown.

At the end of glycolysis, there is a net yield of 2 molecules of ATP, 2 molecules of reduced NAD, (which have the potential to produce more ATP) and 2 molecules of pyruvate. The reduced NAD goes to the electron transport chain while pyruvate enters the link reaction.

Link reaction: a process that links glycolysis to the Krebs cycle. It oxidises pyruvate into AcetlyCoenzyme A and occurs in the mitochondrial matrix, as this site contains enzymes needed for the process.

Pyruvate, from glycolysis is actively transported to the matrix of the mitochondria, for the link reaction. The pyruvate is oxidised to an acetyl group releasing 2 H+ (dehydrogenation) which are picked up by NAD+ forming reduced NAD. (This is a redox reaction since pyruvate is oxidised by the NAD+, which itself, is reduced.) Also, since a carbon atom is removed (decarboxylation) in the form of CO2, this reaction is called oxidative decarboxylation. A 2C Acetyl group is formed as a result of this reaction.

The 2C molecule combines with a carrier molecule, Coenzyme A to form AcetlyCoenzyme A or AcetylCoA.

However, for every glucose molecule that enters glycolysis, 2 pyruvates are made therefore the link reaction must happen twice for every glucose molecule.

So, at the end of the link reaction, 2 molecules of CO2 have been removed as a waste product of respiration, 2 molecules of reduced NAD have been formed (which go to the electron transport chain) and 2 molecules of AcetylCoA have been formed, which both enter the kreb cycle. No ATP is made.

Krebs cycle: Cyclic series of reactions by which CO2 molecules and electrons are removed from AcetylCoA forming reduced carried molecules i.e. reduced FAD NAD for the electron transport chain. The Krebs cycle also occurs in mitochondrial matrix.

The 2C acetyl group in AcetylCoA combines with a 4C molecule called oxaloacetate forming a 6C molecule called citrate. The CoA removed is recycled and is used in another link reaction.

Citrate undergoes oxidative decarboxylation to form a 5C molecule called oxoglutarate. So one molecule of CO2 is lost alongside a H+ ion and 2 electrons. The H+ and 2 electrons are picked up by oxidised NAD (NAD+) converting it to its reduced form, NADH.

Finally, the oxoglutarate is dehydrogenated and decarboxylated forming a 4C oxaloacetate molecule. Dehydrogenation by dehydrogenase enzymes reduces coenzyme FAD and NAD, while decarboxylation removes a C atom in the form of CO2. During the regeneration of the oxaloacetate, energy released by the oxidation reaction is used to synthesise 1 molecule of ATP, as shown below.

Again, like the link reaction, the Krebs cycle happens once for each pyruvate molecule. Since glucose produces 2 pyruvate molecules in glycolysis, the Krebs occurs twice for each glucose molecule that undergoes aerobic respiration.

So at the end of the Krebs cycle, the following has been produced for each glucose:

  • 2 molecules of ATP
  • 6 molecules of reduced NAD
  • 2moelcules of reduced FAD
  • 4 molecules of CO2 - which like CO2made in the link reaction diffuses out of the mitochondria, then out of the cell eventually out of the organism as a waste product.

The electron transport chain – oxidative phosphorylation: a mechanism by which electrons are transported down electron carriers transferring energy used to synthesis ATP that cells can use. It takes place in the inner mitochondrial membrane (on cristae), which is attached to the proteins involved in the electron transport chain (ETC).

NADH dehydrogenase, the first carrier protein, catalyses the oxidation of reduced NAD removing H atoms from it forming oxidised NAD. The H atoms are split into the H+ ions and electrons.

The electrons are passed to the next protein, cytochrome-complex via electron carriers. The cytochrome complex contains a haem group which has a Fe3+ ion. As electrons enter the cytochrome complex, the Fe3+ is reduced to Fe2+ and then immediately oxidised back into Fe3+ releasing the electrons. These reactions are catalysed by oxidoreductase enzymes. Energy released by the exothermic oxidation reaction is used to power the hydrogen pump, pumping H+ from mitochondrial matrix to the intermembranal space.

The electrons are released from the cytochrome complex are transported by another carrier to cytochrome oxidase, the final protein involved. Within the protein, the same process occurs as in the cytochrome complex with the Fe3+ becoming reduced then oxidised to release electrons. Energy released is used to pump more hydrogen ions out of the mitochondrial matrix.

The electrons released from cytochrome oxidase are then accepted by oxygen. This is the terminal electron acceptor and it combines with H+ and electrons to form water, another waste product.

Each of the 3 carrier proteins acts as a proton pump. They use energy from the electrons to pump H+ ions out of the matrix to the intermembranal space. Since the concentration of H+ is higher in the intermembranal space, an electrochemical gradient exists. Therefore H+ diffuses down their concentration gradient through the ATP synthase enzyme releasing energy. The energy is used to synthesise ATP by phosphorylation, i.e. adding Pi to ADP.

Since oxygen is needed to act as a terminal electron acceptor, this process is also called oxidative phosphorylation.

For each reduced NAD that enter the electron transport chain and is oxidised by NADH dehydrogenase, 3 molecules of ATP are made, while for each reduced FAD that enters the electron transport chain, 2 molecules of ATP are made.

The table below summarises the total amount of ATP produced by respiration of one glucose molecule.

However, the table above is only appropriate to aerobic (with oxygen) respiration. So if oxygen is present, the four stages summarised above occur, synthesising 38 molecules of ATP but what happens when no oxygen is present? I.e. what happens in anaerobic (without oxygen) respiration?

In the absence of oxygen, neither the Krebs cycle nor the electron transport chain can take place. This is because oxygen is no longer available to act as the terminal electron acceptor, therefore, electrons are not removed from the final carrier protein, cytochrome oxidase. As a result, the electrons do not leave the cytochrome complex as the cytochrome oxidase is effectively blocked. So electrons accumulate on the carriers which remain in their reduced states. The initial carrier protein, NADH dehydrogenase cannot therefore oxidise the reduced NAD to form NAD+. No oxidised NAD forms so oxidation reactions (such as the oxidation reaction that converts the 6C Citrate to 5C oxoglutarate,) which occur in the Krebs, cease. This is because these reactions require oxidised NAD to remove and accept electrons/hydrogen ions from the molecules, causing the molecules to become oxidised while becoming reduced themselves. Respiration thus comes to a halt.

So anaerobic respiration is simply classified as glycolysis that goes no further. The 6C glucose is converted to 3C pyruvate like normal using the small amount of NAD+ present.

Regeneration of oxidised NAD from the production of ethanol is highly vital in order to keep glycolysis going. Without it, all the supply of oxidised NAD in the cell will be used up leaving none to oxidise anymore glucose to pyruvate. This would mean that glycolysis would stop too.

So pyruvate is converted to another compound. It first loses CO2 (decarboxylation) forming ethanal. The ethanal then accepts hydrogen from reduced NAD, produced in glycolysis becoming reduced. This is called fermentation. Ethanol forms and oxidised NAD is resynthesised ensuring that glycolysis continues.


Enzymes are biological catalysts, which have many functions depending on the type of enzyme. Enzymes can increase the rate of a reaction by lowering the activation energy needed for the reaction, without undergoing a chemical change themselves. They have a specific tertiary shape that determines the shape of there active site. The shape of the active site is highly specific and can only bind onto its complementary substrate.

There are three types of enzymes I need to know about; digestive enzymes, transport enzymes and respiratory enzymes.

Digestive enzymes: are enzymes which work to break down larger molecules into smaller molecules.

The ‘induced fit theory’ explains how enzymes can catalyse the breakdown of substrate molecules in a reaction. As I have mentioned before, enzymes have an active site that has a shape highly specific to that of the substrate. This means that they can usually act on one type of substrate only. When the substrate enters the active site of the enzyme, it causes the enzyme to change its shape slightly to fit snugly with the shape of the substrate. This is called ‘induced fit.’ So the enzyme is considered to be flexible and can alter its shape in the presence of a substrate locking the substrate more tightly to it. The substrate is held within the active site by temporary bonds that form between the R groups of the amino acids in the active site and the substrate. An enzyme bound to its substrate is called an enzyme-substrate complex. It puts a strain onto the bonds of a substrate molecule breaking the peptide bonds within it. The products formed are released and the active site is free therefore it can accept another substrate molecule.

The breaking of the peptide bond mentioned above is a hydrolysis reaction. The hydrolysis of a disaccharide, maltose is illustrated below.

The disaccharide maltose, splits into 2 monosaccharide molecules of alpha glucose. Water is added, which reacts with the glycosidic bond that exists between the 2 glucose molecules of maltose. The bond breaks as shown. Maltose is said to be hydrolysed into its monomers.

Digestive enzymes include carbohydrase, protease and lipase, which catalyse the hydrolysis of sugars, proteins and lipids respectively.

Carbohydrase: digests polysaccharide sugars into disaccharides / monosaccharides.

Protease: digests proteins into dipeptides and amino acids

Lipase: digests lipids into fatty acids and glycerol.  

The enzymes involved in the respiration in saccharomyces cerevisiae are:

  • Maltase - catalyses the hydrolysis of the disaccharide, maltose into 2 monosaccharides of glucose.
  • Sucrase – catalyses the hydrolysis of the disaccharide, sucrose into the 2 monosaccharides that it is made up of; glucose and fructose.
  • Lactase – an enzyme that catalyzes the hydrolysis of the disaccharide, lactose into the 2 monomers, glucose and galactose.
  • Isomerase – an enzyme which catalyses the conversion of one isomer into another, i.e. fructose into glucose. In respiration, the main respiratory substrates are carbohydrates (specifically glucose) which are oxidised to release energy. Therefore it is vital that the saccharomyces cerevisiae contains carbohydrase enzymes to digest large carbohydrates into smaller monosaccharides.
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Transport enzymes: are enzymes which move substances from one region to another.

Such enzymes are usually found on the membrane of cells or organelles. The cell membrane controls the entry and exit of substances from the cell with the aid of these enzymes. It is made up of a phospholipid bilayer which contains proteins that span the membrane. These proteins transport large molecules such as amino acids and glucose, and ions such as sodium ions in and out of the cell via the cell membrane.

These membrane-bound proteins transport substances across via facilitated diffusion and active transport.


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