2.2 Respiration

Principles of Cellular Respiration and Energy Transfer

Respiration and photosynthesis are opposite forms of metabolism in living systems. Photosynthesis is anabolic: it uses light energy to build glucose from carbon dioxide and water, acting as a global carbon sink. Respiration is catabolic: it breaks glucose down into simpler substances to release energy, acting as a carbon release mechanism that returns carbon dioxide to the atmosphere.

Overall Equations for Aerobic Respiration

Aerobic respiration requires oxygen and breaks glucose down completely into carbon dioxide and water. You should know the process in both forms:

• Word equation: glucose + oxygen → carbon dioxide + water + energy (ATP)
• Balanced formula equation:

If an exam question asks for a balanced equation, write the chemical formulae with the numbers 6, 6, and 6. If it asks for a word equation, the chemical names are sufficient.

Transfer Molecules: ATP and NAD⁺

Two transfer molecules drive respiration:

ATP stands for adenosine triphosphate. It consists of the nitrogenous base adenine, a five-carbon ribose sugar, and three bonded phosphate groups. It functions as the universal energy transfer molecule in all living cells.

• When a cell needs energy to perform work, the terminal phosphate bond is broken: $\text{ATP} \rightarrow \text{ADP} + \text{P}_i + \text{energy}$.
• That released energy directly powers muscle contraction, active transport across membranes, nerve impulse transmission, cell division, and the synthesis of proteins and other biomolecules.
• Respiration releases energy from food to reattach a phosphate group to ADP, continuously recycling the cell's energy currency: $\text{ADP} + \text{P}_i + \text{energy} \rightarrow \text{ATP}$.

NAD⁺ (nicotinamide adenine dinucleotide) is a coenzyme that acts as a mobile carrier of electrons and protons. During the enzymatic breakdown of respiratory intermediates, NAD⁺ picks up high-energy electrons and protons (hydrogen), becoming reduced NADH. NADH transports these electrons to the inner mitochondrial membrane to drive the electron transport chain. By contrast, photosynthesis uses the related carrier NADP⁺ (reduced to NADPH); the extra 'P' is a helpful reminder for photosynthesis.

Stage 1: Glycolysis in the Cytoplasm

Respiration always begins with glycolysis, whether oxygen is present or absent. Glycolysis takes place in the cytoplasm—specifically in the cytosol, which is the fluid portion of the cytoplasm outside the organelles. It is entirely anaerobic and does not require oxygen.

During glycolysis, one six-carbon ($6\text{C}$) molecule of glucose is split through a sequence of enzyme-catalysed reactions into two three-carbon ($3\text{C}$) molecules of pyruvate (also called pyruvic acid):

• Energy investment and yield: Two molecules of ATP are initially consumed to phosphorylate and activate glucose. Four ATP molecules are then produced directly in the cytoplasm, giving a net gain of 2 ATP molecules per glucose molecule.
• Electron transfer: Enzymes strip high-energy electrons and protons from the carbohydrate fragments and transfer them to two NAD⁺ molecules, generating 2 NADH.
• Carbon balance: No carbon dioxide is released during glycolysis. All six carbon atoms from the original glucose are accounted for in the two $3\text{C}$ pyruvate molecules.

If oxygen is present, these two pyruvate molecules leave the cytoplasm and move into the mitochondrion to enter Stage 2. If oxygen is absent or limited, pyruvate remains in the cytoplasm and enters anaerobic fermentation pathways.

Fermentation: Anaerobic Energy Pathways

When oxygen is absent or in short supply, the second stage of respiration cannot operate. Without oxygen waiting at the end of the electron transport chain to receive electrons, NADH cannot shed its cargo, and cellular reserves of NAD⁺ run out. Without NAD⁺, glycolysis cannot proceed, and the cell produces no ATP at all.

Fermentation solves this blockage. Its biochemical purpose is to transfer electrons from NADH onto pyruvate or its derivatives. This regenerates oxidized NAD⁺, allowing glycolysis to continue producing its survival supply of energy. Anaerobic respiration gives a net gain of 2 ATP per glucose—all of it from glycolysis. The fermentation step itself makes no additional ATP.

1. Lactic Acid Fermentation

This pathway occurs in human skeletal muscle during strenuous exercise when the blood cannot supply oxygen fast enough to meet demand, as well as in certain anaerobic bacteria.

Pyruvate ($3\text{C}$) accepts electrons and protons directly from NADH, reducing pyruvate to lactic acid ($3\text{C}$):

No carbon dioxide is produced. In human muscle, accumulated lactic acid causes muscle fatigue and stiffness. The extra oxygen the body must consume after exercise to convert accumulated lactic acid back to pyruvate (and on to carbon dioxide and water, or to glucose in the liver) is called the oxygen debt. This oxygen debt explains why heavy breathing continues for several minutes after a sprint.

2. Alcoholic Fermentation

This pathway occurs in brewing and baking yeasts (Saccharomyces cerevisiae) and in certain plant tissues deprived of oxygen. Pyruvate loses one carbon atom as carbon dioxide to form ethanal, which is then reduced by NADH into ethanol ($2\text{C}$):

Applications Across Key Themes

• Technology and food: Yeast fermentation generates carbon dioxide bubbles that cause bread dough to rise, while commercial brewing uses alcoholic fermentation to produce beer and wine. Lactic acid bacteria such as Lactobacillus ferment lactose in milk, producing lactic acid that coagulates milk proteins to make yoghurt and cheese.
• Sustainability: Yeast fermentation converts plant sugars derived from crops or agricultural waste into bioethanol. Bioethanol is a renewable biofuel blended with petrol to reduce transport emissions and decrease reliance on fossil fuels.
• Health: Lactic acid accumulation during anaerobic muscle work serves as a clinical and athletic indicator of muscle fatigue and exertion thresholds. Anaerobic bacterial fermentation in the human large intestine also breaks down dietary fibre to generate beneficial short-chain fatty acids.

Stage 2: The Citric Acid Cycle in the Mitochondrial Matrix

When oxygen is available, the two pyruvate molecules formed in glycolysis cross the outer and inner mitochondrial membranes into the fluid interior, known as the matrix. Stage 2 completes the breakdown of the carbon skeleton.

The Transition Reaction (Acetyl CoA Formation)

Before entering the cycle, each three-carbon pyruvate molecule is modified in the matrix:

1. Pyruvate loses one carbon atom as carbon dioxide ($\text{CO}_2$).
2. It loses electrons and protons to NAD⁺, producing NADH.
3. The remaining two-carbon acetyl fragment joins with coenzyme A to form acetyl coenzyme A (acetyl CoA).

Because each glucose molecule produces two pyruvates, the transition reaction releases 2 $\text{CO}_2$ and forms 2 NADH per glucose.

The Citric Acid Cycle (Krebs Cycle)

Acetyl CoA delivers its two-carbon acetyl group into a cyclical sequence of reactions within the matrix:

• The $2\text{C}$ acetyl fragment combines with a four-carbon ($4\text{C}$) compound to form six-carbon ($6\text{C}$) citric acid, releasing coenzyme A to be reused.
• In a series of enzyme-catalysed steps, citric acid is oxidised. Two carbon atoms are stripped away and released as 2 $\text{CO}_2$.
• High-energy electrons and protons are transferred to coenzymes, yielding 3 NADH per turn of the cycle.
• One molecule of ATP is produced directly per turn.
• The starting $4\text{C}$ compound is regenerated at the end of the pathway, ready to receive another acetyl fragment.

Because one glucose molecule supplies two acetyl CoA molecules, the cycle turns twice per glucose, producing 4 $\text{CO}_2$, 6 NADH, and 2 ATP.

Products of Stage 2 as a Whole

Taken as a whole, Stage 2 produces carbon dioxide, water, and most of the ATP. The carbon dioxide is produced during the transition reaction and the citric acid cycle in the matrix. Water is formed at the inner mitochondrial membrane when spent electrons and protons combine with oxygen at the end of the electron transport chain.

Carbon Accounting: Tracking the Six Carbons of Glucose

Exam questions regularly ask where each carbon atom goes:

The Electron Transport Chain, Chemiosmosis, and ATP Synthase

The electron transport chain delivers the vast majority of ATP generated in aerobic respiration. It is located on the inner mitochondrial membrane, where the membrane folds inward to form cristae.

The Mechanism of Chemiosmosis

Chemiosmosis links electron transfer to ATP production in four steps:

1. Electron donation: NADH arriving from glycolysis and the citric acid cycle deposits high-energy electrons onto protein complexes embedded in the inner mitochondrial membrane, regenerating NAD⁺ for reuse in the cytoplasm and matrix.
2. Proton pumping: High-energy electrons pass along a series of electron carriers. The energy released during this electron transfer is used by the protein complexes to pump protons ($\text{H}^+$) from the matrix across the inner membrane into the intermembrane space.
3. Proton gradient and ATP synthesis: Because the inner membrane is impermeable to protons, they accumulate in the narrow intermembrane space, setting up a steep concentration gradient. Protons stream back into the matrix solely through the channel enzyme ATP synthase. ATP synthase harnesses this kinetic flow of returning protons to combine ADP and inorganic phosphate into ATP.
4. Terminal electron acceptor: At the very end of the chain, electrons must be removed so that carriers do not become blocked. Oxygen acts as the terminal electron acceptor, binding low-energy electrons and free matrix protons to form metabolic water:

Mitochondrial Structure and Function

When identifying parts on a diagram, be familiar with these structures:

• Outer membrane: A smooth boundary membrane containing channel proteins that permit pyruvate, oxygen, and ADP to enter from the cytoplasm.
• Inner membrane: A selectively permeable membrane containing electron carriers and ATP synthase enzymes.
• Cristae: Inward folds of the inner membrane. Make sure not to label the fold as a separate membrane—it is a fold of the inner membrane. Cristae increase the total surface area, allowing thousands of electron transport chains and ATP synthase enzymes to fit into a single organelle.
• Intermembrane space: The narrow compartment between the inner and outer membranes. Its small volume ensures that pumped protons build up a steep concentration gradient rapidly.
• Matrix: The fluid interior containing ribosomes, mitochondrial DNA, and the specific enzymes required for acetyl CoA formation and the citric acid cycle.

ATP Yield Comparison

Aerobic respiration yields around 30 to 38 ATP molecules per glucose molecule, whereas anaerobic respiration yields exactly 2 ATP molecules. Aerobic respiration is therefore roughly 18 times more efficient at extracting energy from fuel molecules.

Required Investigation: The Conditions Necessary for Fermentation

The specification requires you to be able to investigate the conditions necessary for fermentation and to use both primary data (your own laboratory measurements) and secondary data (published or class data) to support your conclusions. Expect this on the written paper as an experiment question. It is separate from the Biology in Practice Investigation coursework, which is based on an annual SEC brief.

Experimental Setup and Methodology

To investigate fermentation by yeast (Saccharomyces cerevisiae):

1. Boil a 10% glucose solution vigorously for five minutes to drive off all dissolved oxygen.
2. Allow the boiled glucose solution to cool to approximately 30 °C before introducing the yeast. Adding yeast to hot water would denature its enzymes and kill the cells.
3. Add a measured mass of dried baker's yeast and swirl gently to suspend the cells.
4. Carefully pipette a layer of vegetable oil or liquid paraffin over the mixture. The oil forms an impermeable physical barrier that stops atmospheric oxygen from dissolving back in, maintaining an anaerobic environment.
5. Fit the flask with a rubber stopper and delivery tube leading into a graduated gas syringe (to measure gas volume) or into a test tube of limewater.
6. Place the flask in a thermostatically controlled water bath at 30 °C for a timed period.

Fair Test, Reliability, and Safety

• Controlled variables: Keep all factors constant except the one being investigated. When testing the effect of temperature, use the same mass of yeast, the same volume and concentration of glucose solution, the same volume of oil layer, the same size conical flask, and the same gas-collection duration.
• Reliability: Set up each flask at least in duplicate or triplicate, calculate the mean volume of gas, and discard any clear anomalous readings before calculating the mean. Repeating the experiment under identical conditions checks repeatability; comparing your findings with another lab group's results or with published tables checks reproducibility.
• Safety: Use tongs or heat-resistant gloves when handling hot glassware and boiling glucose. Ensure the delivery tube remains unblocked so gas pressure does not build up inside the flask. Wash hands thoroughly after handling yeast suspensions.

Testing Environmental Variables and Controls

• Substrate test: Compare yeast in 10% glucose against a control of yeast in distilled water (no sugar). The sugar-free flask produces no gas, proving that sugar is the required respiratory substrate.
• Living biological catalyst test: Compare living yeast in glucose against a control containing boiled, denatured yeast in glucose. The boiled yeast flask produces 0 cm³ of gas, demonstrating that fermentation relies on active, functional biological enzymes.
• Temperature test: Set up identical anaerobic flasks at 10 °C, 25 °C, 35 °C, 45 °C, and 55 °C.

Chemical Confirmation of Products

Carbon dioxide production alone does not prove fermentation has occurred, because yeast also produces $\text{CO}_2$ during aerobic respiration. Demonstrating fermentation requires confirming ethanol formation:

• Carbon dioxide: Gas bubbled through limewater (calcium hydroxide) turns it from clear to milky/cloudy.
• Ethanol (the iodoform test): Filter the yeast mixture to obtain a clear, cell-free filtrate. Add potassium iodide (KI) solution followed by sodium hypochlorite ($\text{NaOCl}$) solution, and warm gently in a water bath. The formation of pale yellow crystals of triiodomethane (iodoform) with a distinct antiseptic smell confirms ethanol. If a chemical test is omitted, note the characteristic ethanol aroma of the filtrate or evaluate secondary data.

Worked Data Analysis: Calculating Fermentation Rate

A student collected carbon dioxide gas from yeast in 10% glucose across five temperatures over 20 minutes:

Rate is calculated as volume of gas divided by time:

• At 35 °C: $\text{rate} = 30\,\text{cm}^3 \div 20\,\text{min} = \mathbf{1.5\,\text{cm}^3\,\text{min}^{-1}}$
• At 10 °C: $\text{rate} = 4\,\text{cm}^3 \div 20\,\text{min} = 0.2\,\text{cm}^3\,\text{min}^{-1}$

Conclusion: The rate increases between 10 °C and an optimum near 35 °C because yeast enzymes and glucose molecules gain kinetic energy, resulting in more frequent collisions. Above 35 °C, the rate declines and drops to zero at 55 °C because high temperatures denature the active sites of yeast enzymes.

Key terms

Respiration

The controlled catabolic release of energy from food within living cells, transferring that energy into ATP.

Catabolism

Metabolic reactions that break down complex molecules into simpler ones, releasing energy.

Aerobic Respiration

The controlled release of energy from food using oxygen, completely breaking down glucose into carbon dioxide and water.

Anaerobic Respiration

The controlled release of energy from food without using oxygen, partially breaking down glucose in the cytoplasm.

Adenosine Triphosphate (ATP)

A nucleotide derivative made of adenine, ribose, and three phosphate groups that acts as the universal energy transfer molecule in cells.

Nicotinamide Adenine Dinucleotide (NAD+)

A coenzyme that acts as a mobile carrier of electrons and protons during respiration, becoming reduced to NADH.

Glycolysis

Stage 1 of respiration, occurring in the cytoplasm without oxygen, which breaks one 6C glucose into two 3C pyruvates with a net gain of 2 ATP and 2 NADH.

Pyruvate

The three-carbon product formed at the end of glycolysis.

Acetyl Coenzyme A

A two-carbon compound formed in the mitochondrial matrix when pyruvate loses a carbon atom as carbon dioxide before entering the citric acid cycle.

Citric Acid Cycle

A cyclical sequence of enzyme-controlled reactions in the mitochondrial matrix that oxidises acetyl fragments to carbon dioxide, producing NADH, ATP, and proton carriers.

Electron Transport Chain

A series of protein complexes and mobile carriers embedded in the inner mitochondrial membrane that transfer electrons and pump protons to establish a concentration gradient.

ATP Synthase

An enzyme embedded in the inner mitochondrial membrane that synthesises ATP from ADP and phosphate using the energy of protons flowing down their gradient.

Cristae

Infoldings of the inner mitochondrial membrane that provide a large surface area for electron transport chains and ATP synthase complexes.

Mitochondrial Matrix

The fluid-filled interior of the mitochondrion containing enzymes for acetyl CoA formation and the citric acid cycle.

Oxygen Debt

The extra volume of oxygen required after strenuous physical exercise to metabolise accumulated lactic acid back to pyruvate in the body.