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Respiration

Respiration is the process by which our bodies break down glucose to release energy. Energy is generated in the form of ATP to power processes such as muscle contraction and cell division.

Aerobic Respiration

Aerobic respiration is made of four stages: glycolysis , the link reaction , the Krebs cycle and oxidative phosphorylation . During aerobic respiration, glucose is effectively burned inside our bodies (it reacts with oxygen) to produce carbon dioxide, water and lots of energy in the form of ATP. The overall equation for aerobic respiration is:

The first stage of aerobic respiration is glycolysis , which takes place in the cytoplasm . Glycolysis converts glucose , a six-carbon molecule, into two smaller three-carbon molecules called pyruvate . This stage doesn’t require oxygen so it is an anaerobic process and is involved in both aerobic and anaerobic respiration pathways.

Glucose is phosphorylated using the phosphate groups from two molecules of ATP. ATP is hydrolysed into ADP and inorganic phosphate. This forms a molecule which is unstable and immediately breaks down into two three-carbon molecules called triose phosphate (TP). Hydrogen is removed from TP to convert it into pyruvate . The hydrogen is transferred to a coenzyme called NAD to form reduced NAD (NADH). The removal of hydrogen from TP oxidises it. The reduced NAD is used in the last stage of aerobic respiration, oxidative phosphorylation, whereas the pyruvate moves into the mitochondria for the next stage of respiration, the link reaction.

The conversion of triose phosphate to pyruvate produced four molecules of ATP. Since two molecules were used for the phosphorylation of glucose in the first step, this means there is a net gain of two ATP molecules in glycolysis.

The Link Reaction

The link reaction takes place in the mitochondrial matrix.

The link reaction takes place in the mitochondrial matrix and converts pyruvate into a molecule called acetyl coenzyme A (acetyl CoA). This stage does not produce any energy in the form of ATP but does produce reduced NAD and acetyl CoA. Reduced NAD will be used in oxidative phosphorylation while the acetyl CoA will be used in the next stage of aerobic respiration, the Krebs cycle.

During the link reaction, a carbon atom is removed from pyruvate, forming carbon dioxide . This converts pyruvate into a two-carbon molecule called acetate . Hydrogen is also removed from pyruvate in the conversion into acetate, which is picked up by the coenzyme NAD to form reduced NAD . The acetate is combined with coenzyme A (CoA) to form acetyl CoA .

Since one glucose molecule is converted into 2x pyruvate, the link reaction happens twice for every glucose molecule . This means that each molecule of glucose produces two molecules of acetyl CoA (along with 2x carbon dioxide and 2x NADH).

The Krebs cycle

The Kreb’s cycle (also known as the citric acid cycle) is a series of reactions which generate reduced NAD and a similar molecule called reduced FAD which are needed for oxidative phosphorylation. Acetyl CoA from the link reaction reacts with a four-carbon molecule called oxaloacetate . The coenzyme A portion of acetyl CoA is removed and returns to the link reaction to be reused. A 6-carbon molecule called citrate is produced. Carbon and hydrogen are removed from citrate, forming carbon dioxide and reduced NAD . The citrate is converted into a 5-carbon compound. Decarboxylation and dehydrogenation occur once more, which converts the 5-carbon compounds into the 4-carbon molecule oxaloacetate which we started with. ATP, 2 molecules of reduced NAD, one molecule of FAD and carbon dioxide are also formed in this step. This cycle takes place twice for each glucose molecule that is respired aerobically.

Oxidative Phosphorylation

Oxidative phosphorylation is the last stage of aerobic respiration and it is the part where most of the ATP is made . It uses the electrons that are being carried by reduced NAD and reduced FAD that have been generated in the first three stages. It takes place across the inner mitochondrial membrane and involves two processes - the electron transport chain and chemiosmosis .

The coenzymes reduced NAD and reduced FAD release hydrogen atoms which split into hydrogen ions and electrons. The electrons are passed onto electron carriers which are embedded within the inner mitochondrial membrane and travel along a series of electron carriers known as the electron transport chain . As they travel between the electron carriers, they lose energy . This energy is used by the carriers to pump hydrogen ions from the mitochondrial matrix across the inner membrane. Hydrogen ions accumulate in the intermembrane space and this generates a proton gradient (sometimes referred to as an electrochemical gradient) across the membrane. Hydrogen ions then flow back into the matrix through the enzyme ATP synthase which uses the movement of hydrogen ions (the proton motive force ) to add a phosphate group onto ADP to form ATP . The process by which the movement of hydrogen ions produces ATP is called chemiosmosis . Once the electrons reach the end of the electron transport chain, they are passed onto oxygen , which is referred to as the ‘final electron acceptor’. Oxygen combines with electrons and hydrogen ions to form water , one of the products of aerobic respiration.

Metabolic poisons , such as cyanide, disrupt oxidative phosphorylation by binding to electron carriers and inhibiting the movement of electrons along the electron transport chain. This reduces chemiosmosis since a proton gradient is not established and also inhibits the Krebs cycle since NAD and FAD are not regenerated. ATP production grounds to a halt so processes which require energy (such as the contraction of heart muscle) cannot take place, which can be deadly for the organism who has ingested the poison.

Total ATP production

Aerobic respiration produces a total of 38 ATP molecules per one molecule of glucose respired. Here’s a breakdown of the ATP production at each of the different stages. Each molecule of reduced NAD produces 3 ATP and each molecule of reduced FAD produces 2 ATP. Remember that the link reaction and Krebs cycle happen twice for each molecule of glucose, because it is converted into 2x pyruvate.

Glycolysis: direct production of 2 ATP

Glycolysis: 2 reduced NAD are converted into 6 ATP (2 x 3) in oxidative phosphorylation

Link reaction: 2 reduced NAD are converted into 6 ATP (2 x 3) in oxidative phosphorylation

Krebs cycle: direct production of 2 ATP

Krebs cycle: 6 reduced NAD are converted into 18 ATP (6 x 3) in oxidative phosphorylation

Krebs cycle: 2 reduced FAD are converted into 4 ATP (2 x 2) in oxidative phosphorylation

Total ATP = 2 + 6 + 6 + 2 + 18 + 4 = 38 ATP

Measuring the rate of respiration

The rate of respiration is measured using a piece of apparatus called a respirometer and works by measuring either the amount of oxygen used up by an organism or the amount of carbon dioxide produced . The faster the amount of oxygen consumed, the faster the rate of respiration.

You would set up the respirometer as shown in the diagram, with respiring organisms (such as woodlice) in one test tube connected to another test tube by a manometer . The manometer contains a coloured liquid which will move closer towards the respiring test tube as oxygen is consumed. The test tube on the right is a control test tube, containing a non-respiring substance, such as glass beads. The purpose of the control tube is to ensure that only respiration is causing the movement of liquid in the manometer. The control tube should be as similar as possible to the test tube e.g. the glass beads should be the same mass as the woodlice. In each test tube you need to add the same volume of potassium hydroxide solution which absorbs carbon dioxide - this ensures that the movement of the liquid is only affected by the decreasing levels of oxygen.

Once the apparatus has been set up, it is left for a certain period of time (e.g. 30 minutes). This will allow for the potassium hydroxide to absorb all of the carbon dioxide in the test tubes. You then record the distance moved by the liquid in the manometer in a given time , using the calibrated scale and a stopwatch. You then calculate the volume of oxygen taken in by the woodlice per minute . Repeat the experiment at least three times and calculate a mean.

Anaerobic respiration

Respiration can also occur in the absence of oxygen - this is called anaerobic respiration . In mammals, glucose can be converted into lactate (aka lactic acid) which releases a small amount of energy in the form of ATP.

The first step of anaerobic respiration is the same as aerobic respiration: glycolysis . Glucose is converted into pyruvate with the net release of 2 ATP molecules. 2 molecules of reduced NAD are also formed. In the second step, reduced NAD donates hydrogen (and electrons) to pyruvate, producing lactate and NAD . This regenerates more oxidised NAD for glycolysis. This enables anaerobic respiration to continue and ensures that small amounts of energy can still be made in the absence of oxygen, allowing biological reactions to keep ticking over.

Continued anaerobic respiration results in the build up of lactate , which needs to be broken down. Cells can convert lactate back into pyruvate , which is then able to enter aerobic respiration at the Krebs cycle . In addition, liver cells have the ability to convert lactate into glucose , which can then be respired aerobically (if oxygen is now present) or stored for later use.

Did you know…

Every day you produce and use your own body weight in ATP – some 200 trillion trillion molecules of it. Each ATP molecule releases a tiny amount of energy in one immediate burst which prevents our bodies from wasting resources by manufacturing more energy than we need.

Next Page: Forensics

Biodiversity, classification, conservation

• Cell structure
• Biomolecules
• Cell membrane
• Nuclear division
• Genetic control
• Transport in plants
• Transport in mammals
• Gas exchange
• Practical exam
• Energy & Respirtation
• Photosynthesis
• Homeostasis
• Coordination
• Inheritance
• Selection & Evolution
• Biodiversity
• Genetic Technology

27 August 2015

#96 using respirometers.

24 comments:

helped a lot!!!

the second organism in second tube also respires so doesnt intake of O2 affect this ?

Helped, thanks

After searching for hours futile, at 11:49pm the night before the lab write-up is due, this post was like heaven!! Thanks much.

this was great, thanks!

thanx . Very helpful .

really helpful!!

Your notes are simple understandable by everyone and they contain desirable content,, so thankyou for that.

Your notes are really good even they have made me to get the content .so thank you,,

very helpful, thanks a lot.

still helping, thank you!

Thank you :)

What are some limitations in the experiment?

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very clear explanation

Great notes! Thanks for sharing in neat and easy understand way! But it will be better if you put your name :) it helps with citation. Thanks for sharing <3

Thank you very much as it helped me a lot to connect my exams

hey, if I wanted to alter it to test anaerobic respiration, what should I use as an O2 absorbent?

Overview of Respiration

Respiration.

Respiration is a series of reactions that convert chemical energy stored in carbohydrates into ATP. It takes place in the mitochondria of eukaryotic cells.

Importance of respiration

• 6O 2 + C 6 H 12 O 6 (glucose) → 6CO 2 + 6H 2 O

Types of respiration

• Aerobic - respiration using oxygen.
• Anaerobic - respiration without oxygen.
• Both types of respiration start with the same stage: glycolysis (pictured).

Anaerobic respiration

• Anaerobic respiration does NOT use oxygen (e.g. short burst of intense exercise).
• Ethanol fermentation - takes place in plants and yeast.
• Lactate fermentation - takes place in animals.

Aerobic respiration

• Aerobic respiration uses oxygen (e.g. extended periods of exercise).
• The reaction and products are the same in plants, animal and yeast.
• Water is produced.
• More ATP is produced.
• Glucose is fully broken down.
• After glycolysis, there are more steps (the link reaction, the Krebs cycle and oxidative phosphorylation).

Respiration produces ATP and can be either aerobic or anaerobic. Glycolysis is the first stage in both these processes. Glycolysis happens in the cytoplasm and is an anaerobic process.

Phosphorylation

• The first step in glycolysis involves the phosphorylation of glucose to glucose phosphate using one molecule of ATP.
• Glucose phosphate is phosphorylated by another molecule of ATP to hexose bisphosphate (a six-carbon molecule)
• Hexose bisphosphate splits into two molecules of triose phosphate (TP).

• The two molecules of TP are oxidised to pyruvate (another three-carbon molecule) in a multi-step reaction.
• A single TP molecule produces two molecules of ATP and one molecule of reduced NAD (NADH) in this process.

• 2 ATP molecules.
• 2 NADH molecules.

1 Biological Molecules

1.1 Monomers & Polymers

1.1.1 Monomers & Polymers

1.1.2 Condensation & Hydrolysis Reactions

1.2 Carbohydrates

1.2.1 Structure of Carbohydrates

1.2.2 Types of Polysaccharides

1.2.3 End of Topic Test - Monomers, Polymers and Carbs

1.2.4 Exam-Style Question - Carbohydrates

1.2.5 A-A* (AO3/4) - Carbohydrates

1.3.1 Triglycerides & Phospholipids

1.3.2 Types of Fatty Acids

1.3.3 Testing for Lipids

1.3.4 Exam-Style Question - Fats

1.3.5 A-A* (AO3/4) - Lipids

1.4 Proteins

1.4.1 The Peptide Chain

1.4.2 Investigating Proteins

1.4.3 Primary & Secondary Protein Structure

1.4.4 Tertiary & Quaternary Protein Structure

1.4.5 Enzymes

1.4.6 Factors Affecting Enzyme Activity

1.4.7 Enzyme-Controlled Reactions

1.4.8 End of Topic Test - Lipids & Proteins

1.4.9 A-A* (AO3/4) - Enzymes

1.4.10 A-A* (AO3/4) - Proteins

1.5 Nucleic Acids

1.5.1 DNA & RNA

1.5.2 Nucleotides

1.5.3 Polynucleotides

1.5.4 DNA Replication

1.5.5 Exam-Style Question - Nucleic Acids

1.5.6 A-A* (AO3/4) - Nucleic Acids

1.6.1 Structure of ATP

1.6.2 Hydrolysis of ATP

1.6.3 Resynthesis of ATP

1.6.4 End of Topic Test - Nucleic Acids & ATP

1.7.1 Importance of Water

1.7.2 Structure of Water

1.7.3 Properties of Water

1.7.4 A-A* (AO3/4) - Water

1.8 Inorganic Ions

1.8.1 Inorganic Ions

1.8.2 End of Topic Test - Water & Inorganic Ions

2.1 Cell Structure

2.1.1 Introduction to Cells

2.1.2 Eukaryotic Cells & Organelles

2.1.3 Eukaryotic Cells & Organelles 2

2.1.4 Prokaryotes

2.1.5 A-A* (AO3/4) - Organelles

2.1.6 Methods of Studying Cells

2.1.7 Microscopes

2.1.8 End of Topic Test - Cell Structure

2.1.9 Exam-Style Question - Cells

2.1.10 A-A* (AO3/4) - Cells

2.2 Mitosis & Cancer

2.2.1 Mitosis

2.2.2 Stages of Mitosis

2.2.3 Investigating Mitosis

2.2.4 Cancer

2.2.5 A-A* (AO3/4) - The Cell Cycle

2.3 Transport Across Cell Membrane

2.3.1 Cell Membrane Structure

2.3.2 A-A* (AO3/4) - Membrane Structure

2.3.3 Diffusion

2.3.4 Osmosis

2.3.5 Active Transport

2.3.6 End of Topic Test - Mitosis, Cancer & Transport

2.3.7 Exam-Style Question - Membranes

2.3.8 A-A* (AO3/4) - Membranes & Transport

2.3.9 A-A*- Mitosis, Cancer & Transport

2.4 Cell Recognition & the Immune System

2.4.1 Immune System

2.4.2 Phagocytosis

2.4.3 T Lymphocytes

2.4.4 B Lymphocytes

2.4.5 Antibodies

2.4.6 Primary & Secondary Response

2.4.7 Vaccines

2.4.9 Ethical Issues

2.4.10 End of Topic Test - Immune System

2.4.11 Exam-Style Question - Immune System

2.4.12 A-A* (AO3/4) - Immune System

3 Substance Exchange

3.1 Surface Area to Volume Ratio

3.1.1 Size & Surface Area

3.1.2 A-A* (AO3/4) - Cell Size

3.2 Gas Exchange

3.2.1 Single-Celled Organisms

3.2.2 Multicellular Organisms

3.2.3 Control of Water Loss

3.2.4 Human Gas Exchange

3.2.5 Ventilation

3.2.6 Dissection

3.2.7 Measuring Gas Exchange

3.2.8 Lung Disease

3.2.9 Lung Disease Data

3.2.10 End of Topic Test - Gas Exchange

3.2.11 A-A* (AO3/4) - Gas Exchange

3.3 Digestion & Absorption

3.3.1 Overview of Digestion

3.3.2 Digestion in Mammals

3.3.3 Absorption

3.3.4 End of Topic Test - Substance Exchange & Digestion

3.3.5 A-A* (AO3/4) - Substance Ex & Digestion

3.4 Mass Transport

3.4.1 Haemoglobin

3.4.2 Oxygen Transport

3.4.3 The Circulatory System

3.4.4 The Heart

3.4.5 Blood Vessels

3.4.6 Cardiovascular Disease

3.4.7 Heart Dissection

3.4.8 Xylem

3.4.9 Phloem

3.4.10 Investigating Plant Transport

3.4.11 End of Topic Test - Mass Transport

3.4.12 A-A* (AO3/4) - Mass Transport

4 Genetic Information & Variation

4.1 DNA, Genes & Chromosomes

4.1.2 Genes

4.1.3 Non-Coding Genes

4.1.4 The Genetic Code

4.1.5 A-A* (AO3/4) - DNA

4.2 DNA & Protein Synthesis

4.2.1 Protein Synthesis

4.2.2 Transcription & Translation

4.2.3 End of Topic Test - DNA, Genes & Protein Synthesis

4.2.4 Exam-Style Question - Protein Synthesis

4.2.5 A-A* (AO3/4) - Coronavirus Translation

4.2.6 A-A* (AO3/4) - Transcription

4.2.7 A-A* (AO3/4) - Translation

4.3 Mutations & Meiosis

4.3.1 Mutations

4.3.2 Meiosis

4.3.3 A-A* (AO3/4) - Meiosis

4.3.4 Meiosis vs Mitosis

4.3.5 End of Topic Test - Mutations, Meiosis

4.3.6 A-A* (AO3/4) - DNA,Genes, CellDiv & ProtSynth

4.4 Genetic Diversity & Adaptation

4.4.1 Genetic Diversity

4.4.2 Natural Selection

4.4.3 A-A* (AO3/4) - Natural Selection

4.4.4 Adaptations

4.4.5 Investigating Natural Selection

4.4.6 End of Topic Test - Genetic Diversity & Adaptation

4.4.7 A-A* (AO3/4) - Genetic Diversity & Adaptation

4.5 Species & Taxonomy

4.5.1 Courtship Behaviour

4.5.2 Phylogeny

4.5.3 Classification

4.5.4 DNA Technology

4.5.5 A-A* (AO3/4) - Species & Taxonomy

4.6 Biodiversity Within a Community

4.6.1 Biodiversity

4.6.2 Index of diversity

4.6.3 Agriculture

4.6.4 End of Topic Test - Species,Taxonomy& Biodiversity

4.6.5 A-A* (AO3/4) - Species,Taxon&Biodiversity

4.7 Investigating Diversity

4.7.1 Genetic Diversity

4.7.2 Quantitative Investigation

5 Energy Transfers (A2 only)

5.1 Photosynthesis

5.1.1 Overview of Photosynthesis

5.1.2 Photoionisation of Chlorophyll

5.1.3 Production of ATP & Reduced NADP

5.1.4 Cyclic Photophosphorylation

5.1.5 Light-Independent Reaction

5.1.6 A-A* (AO3/4) - Photosynthesis Reactions

5.1.7 Limiting Factors

5.1.8 Photosynthesis Experiments

5.1.9 End of Topic Test - Photosynthesis

5.1.10 A-A* (AO3/4) - Photosynthesis

5.2 Respiration

5.2.1 Overview of Respiration

5.2.2 Anaerobic Respiration

5.2.3 A-A* (AO3/4) - Anaerobic Respiration

5.2.4 The Link Reaction

5.2.5 The Krebs Cycle

5.2.6 Oxidative Phosphorylation

5.2.7 Respiration Experiments

5.2.8 End of Topic Test - Respiration

5.2.9 A-A* (AO3/4) - Respiration

5.3 Energy & Ecosystems

5.3.1 Biomass

5.3.2 Production & Productivity

5.3.3 Agricultural Practices

5.4 Nutrient Cycles

5.4.1 Nitrogen Cycle

5.4.2 Phosphorous Cycle

5.4.3 Fertilisers & Eutrophication

5.4.4 End of Topic Test - Nutrient Cycles

5.4.5 A-A* (AO3/4) - Energy,Ecosystems&NutrientCycles

6 Responding to Change (A2 only)

6.1 Nervous Communication

6.1.1 Survival

6.1.2 Plant Responses

6.1.3 Animal Responses

6.1.4 Reflexes

6.1.5 End of Topic Test - Reflexes, Responses & Survival

6.1.6 Receptors

6.1.7 The Human Retina

6.1.8 Control of Heart Rate

6.1.9 End of Topic Test - Receptors, Retina & Heart Rate

6.2 Nervous Coordination

6.2.1 Neurones

6.2.2 Action Potentials

6.2.3 Speed of Transmission

6.2.4 End of Topic Test - Neurones & Action Potentials

6.2.5 Synapses

6.2.6 Types of Synapse

6.2.7 Medical Application

6.2.8 End of Topic Test - Synapses

6.2.9 A-A* (AO3/4) - Nervous Comm&Coord

6.3 Muscle Contraction

6.3.1 Skeletal Muscle

6.3.2 Sliding Filament Theory

6.3.3 Contraction

6.3.4 Slow & Fast Twitch Fibres

6.3.5 End of Topic Test - Muscles

6.3.6 A-A* (AO3/4) - Muscle Contraction

6.4 Homeostasis

6.4.1 Overview of Homeostasis

6.4.2 Blood Glucose Concentration

6.4.3 Controlling Blood Glucose Concentration

6.4.4 End of Topic Test - Blood Glucose

6.4.5 Primary & Secondary Messengers

6.4.6 Diabetes Mellitus

6.4.7 Measuring Glucose Concentration

6.4.8 Osmoregulation

6.4.9 Controlling Blood Water Potential

6.4.11 End of Topic Test - Diabetes & Osmoregulation

6.4.12 A-A* (AO3/4) - Homeostasis

7 Genetics & Ecosystems (A2 only)

7.1 Genetics

7.1.1 Key Terms in Genetics

7.1.2 Inheritance

7.1.3 Linkage

7.1.4 Multiple Alleles & Epistasis

7.1.5 Chi-Squared Test

7.1.6 End of Topic Test - Genetics

7.1.7 A-A* (AO3/4) - Genetics

7.2 Populations

7.2.1 Populations

7.2.2 Hardy-Weinberg Principle

7.3 Evolution

7.3.1 Variation

7.3.2 Natural Selection & Evolution

7.3.3 End of Topic Test - Populations & Evolution

7.3.4 Types of Selection

7.3.5 Types of Selection Summary

7.3.6 Overview of Speciation

7.3.7 Causes of Speciation

7.3.8 Diversity

7.3.9 End of Topic Test - Selection & Speciation

7.3.10 A-A* (AO3/4) - Populations & Evolution

7.4 Populations in Ecosystems

7.4.1 Overview of Ecosystems

7.4.2 Niche

7.4.3 Population Size

7.4.4 Investigating Population Size

7.4.5 End of Topic Test - Ecosystems & Population Size

7.4.6 Succession

7.4.7 Conservation

7.4.8 End of Topic Test - Succession & Conservation

7.4.9 A-A* (AO3/4) - Ecosystems

8 The Control of Gene Expression (A2 only)

8.1 Mutation

8.1.1 Mutations

8.1.2 Effects of Mutations

8.1.3 Causes of Mutations

8.2 Gene Expression

8.2.1 Stem Cells

8.2.2 Stem Cells in Disease

8.2.3 End of Topic Test - Mutation & Gene Epression

8.2.4 A-A* (AO3/4) - Mutation & Stem Cells

8.2.5 Regulating Transcription

8.2.6 Epigenetics

8.2.7 Epigenetics & Disease

8.2.8 Regulating Translation

8.2.9 Experimental Data

8.2.10 End of Topic Test - Transcription & Translation

8.2.11 Tumours

8.2.12 Correlations & Causes

8.2.13 Prevention & Treatment

8.2.14 End of Topic Test - Cancer

8.2.15 A-A* (AO3/4) - Gene Expression & Cancer

8.3 Genome Projects

8.3.1 Using Genome Projects

8.4 Gene Technology

8.4.1 Recombinant DNA

8.4.2 Producing Fragments

8.4.3 Amplification

8.4.4 End of Topic Test - Genome Project & Amplification

8.4.5 Using Recombinant DNA

8.4.6 Medical Diagnosis

8.4.7 Genetic Fingerprinting

8.4.8 End of Topic Test - Gene Technologies

8.4.9 A-A* (AO3/4) - Gene Technology

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A-A* (AO3/4) - Photosynthesis

Anaerobic Respiration

• Biology Article
• Experimentally Show That Carbon Dioxide Is Given Out During Respiration

Experiment To Prove That Carbon Dioxide Is Given Out During Respiration

One of the basic and fundamental life processes that are carried out by living entities is respiration. It is a catabolic process wherein complex organic molecules are broken down into simpler molecules. The process releases energy either in the absence or presence of oxygen, and hence respiration can be of two kinds:

• Aerobic respiration – This kind of respiration takes place in the presence of oxygen, hence it results in the complete glucose oxidation with the release of energy. It includes three stages – namely, Krebs cycle, ETS and Glycolysis. All events relating to ETS take place inside mitochondria while stages connected with glycolysis take place in the cytoplasm.
• Anaerobic respiration – In this type of respiration, oxidation of food takes place in an environment lacking oxygen supply. Less energy is released as a result of incomplete oxidation of glucose.

See Also: Differences Between Catabolism and Anabolism

To experimentally demonstrate that carbon dioxide is released during the process of respiration.

Principle/Theory

The process of respiration is biochemically carried out wherein food, glucose to be precise, is oxidized and energy is released. In this experiment, gram seeds (moistened) are used. The purpose of using these seeds is that they release carbon dioxide and are respiring actively. The released carbon dioxide is consumed by the solution of KOH.

Material Required

• Soaked gram seeds
• U-shaped delivery tube
• Conical flask
• Blotting paper (moist) /cotton wool
• Rubber cork with a single hole
• Freshly prepared KOH solution (20%)
• Germinate close to 25 seeds. This can be done by wrapping them in moist blotting paper or cotton wool for around 3 to 4 days.
• Set up the germinated or sprouted seeds in the conical flask. Spray some water into the flask to dampen the seeds.
• With the help of a thread, suspend the conical flask containing the test tube having a freshly prepared 20% KOH solution.
• Use the rubber cork to seal the opening of the conical flask.
• One edge of the U-shaped glass delivery tube present in the conical flask should be inserted through the hole in the rubber cork. The other edge should be placed into a beaker that is saturated with water.
• All attachments of the set-up should be sealed. This can be done using vaseline to create an air-tight environment.
• The initial water level present in the U-shaped delivery tube needs to be marked.
• Leave the experimental set-up uninterrupted for 1 to 2 hours. Observe the fluctuations in the water level in the tube.

Observation

Careful observation after a certain period of time reveals that the water level in the U-shaped delivery tube has risen in the beaker.

Conclusions

The rise in level water indicates that carbon dioxide is released as a result of germinating gram seeds during the process of respiration in the conical flask. The carbon dioxide that is released in the process is absorbed or consumed by the KOH solution that is suspended in the test tube in the conical flask, creating a vacuum or a void in the flask resulting in the upward water movement in the tube. Hence, the water level in the tube changes.

Precautions

• The seeds that are to be germinated need to be moistened
• Air-tight environment for all the connections in the experimental set-up
• The KOH solution that is used needs to be freshly prepared
• Care needs to be taken to ensure that one end of the delivery tube is placed in the conical flask. The other edge is submerged in the water of the beaker
• The tube that contains the KOH solution needs to be suspended carefully

Viva Questions

Q.1. Why is the energy output of the anaerobic respiration lesser than aerobic respiration?

A.1. The process of anaerobic respiration produces 2 ATP. Aerobic respiration, on the other hand, produces 38 ATP involving complete oxidation of glucose. In anaerobic respiration, glucose is partially broken down.

Q.2. List the levels of aerobic respiration.

A.2. The following are the levels in aerobic respiration:

• Krebs cycle
• Oxidative phosphorylation and ETS

Q.3. The cells’ energy currency is _________

A.3 Adenosine triphosphate (ATP)

Q.4. What happens when the photosynthesis rate is equal to the respiration rate?

A.4. When both are equal, it enters into a compensation point where there is no gross gas exchange taking place.

Q.5. Can plants respire and take part in photosynthesis?

A.5 Yes, plants can respire in addition to taking part in photosynthesis during the day time.

Q.6. What is the purpose of keeping the seeds moistened in the experiment?

A.6. Seeds are required to be moist as water is required for growth to germinate. If they are not moist enough, they may dry up resulting in a dip in the respiration rate.

Q.7. Can boiled seeds be used in place of moistened germinating seeds?

A.7. No, they cannot be used as boiled seeds cannot undergo respiration. The experiment will show no result.

Q.8. State the significance of using KOH solution in the experiment.

A.8. The solution is known to absorb carbon dioxide that is released during the process of respiration of germinating seeds, thereby creating a slight vacuum in the flask hence increase in the water level. The rise in water level indicates the occurrence of the process of respiration.

Q.9. List one circumstance under which there would be no rise in the water level in the apparatus.

A.9. If the test tube holding the KOH solution is discarded from the experimental setup, the carbon dioxide produced during the respiration process shall not be consumed hence there would be no inflation in the water level.

Q.10. In the experiment, what is the purpose of using Vaseline?

A.10. It is used because it is used to seal all the apparatus, hence securing the set-up air-tight.

Q.11. Can you think of an alternate method to depict the release of carbon dioxide during the respiration process?

A.11. In the same apparatus, water could be replaced by lime water as lime water tends to turn milky in the presence of carbon dioxide.

Q.12. What are respiratory gases?

A.12. Carbon dioxide and oxygen are involved in the process of respiration, and hence are known as respiratory gases.

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• Published: 17 August 2024

Experimental warming and drying increase older carbon contributions to soil respiration in lowland tropical forests

• Karis J. McFarlane   ORCID: orcid.org/0000-0001-6390-7863 1 ,
• Daniela F. Cusack   ORCID: orcid.org/0000-0003-4681-7449 2 , 3 , 4 ,
• Lee H. Dietterich   ORCID: orcid.org/0000-0003-4465-5845 2 , 5 , 6 ,
• Alexandra L. Hedgpeth 1 , 3 ,
• Kari M. Finstad   ORCID: orcid.org/0000-0002-3170-5877 1 &
• Andrew T. Nottingham   ORCID: orcid.org/0000-0001-9421-8972 4 , 7  

Nature Communications volume  15 , Article number:  7084 ( 2024 ) Cite this article

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Tropical forests account for over 50% of the global terrestrial carbon sink, but climate change threatens to alter the carbon balance of these ecosystems. We show that warming and drying of tropical forest soils may increase soil carbon vulnerability, by increasing degradation of older carbon. In situ whole-profile heating by 4 °C and 50% throughfall exclusion each increased the average radiocarbon age of soil CO 2 efflux by ~2–3 years, but the mechanisms underlying this shift differed. Warming accelerated decomposition of older carbon as increased CO 2 emissions depleted newer carbon. Drying suppressed decomposition of newer carbon inputs and decreased soil CO 2 emissions, thereby increasing contributions of older carbon to CO 2 efflux. These findings imply that both warming and drying, by accelerating the loss of older soil carbon or reducing the incorporation of fresh carbon inputs, will exacerbate soil carbon losses and negatively impact carbon storage in tropical forests under climate change.

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Introduction.

Tropical forests exchange more CO 2 with the atmosphere than any other terrestrial biome 1 , store nearly one-third of global soil carbon stocks 2 , and have the highest soil CO 2 efflux of any ecosystem 3 . Tropical terrestrial ecosystems also have the shortest mean residence time for carbon on Earth, as short as 6–15 years 4 , 5 , meaning that any change in carbon inputs or outputs could have large and relatively rapid consequences for tropical ecosystem carbon balance. Climate projections suggest a future that will be both warmer and drier for much of the tropics 6 with increasing drought intensity and dry season length for the Neotropics 7 , 8 . Despite the importance of tropical forests and their soils to the global carbon cycle and feedbacks to climate, uncertainty in predicting the response of tropical carbon cycling to future climate change remains high.

Soil CO 2 efflux is highly sensitive to temperature and moisture, which together have been shown to determine interannual patterns in emissions globally 9 . Even in tropical forests, where mean annual temperature is relatively high and temperature variability is relatively low, soil CO 2 efflux has been shown to increase with increasing temperature and peak at intermediate soil moisture content 10 , 11 . Meta-analyses of warming experiments across global terrestrial ecosystems have reported average increases in soil CO 2 efflux with warming of 9% 12 –12% 13 , and reported increases in soil CO 2 efflux following whole-profile warming are even higher (e.g., 34% 14 –55% 15 ). Thus, extrapolation of results from warming experiments suggests that climate warming will stimulate a net loss of global soil carbon to the atmosphere 16 . Importantly, none of the studies included in these meta-analyses were conducted in the tropics. Field warming experiments in tropical forests have only recently been instigated and early results show large increases in soil CO 2 efflux with increased temperature 15 as have laboratory incubations of tropical soils 17 , 18 . Soil moisture is also an important factor influencing soil microbial activity and respiration, and in the tropics, the seasonal variation in moisture is often greater than that of temperature 10 , 19 , 20 . However, field drying experiments in tropical forests have reported mixed responses of soil CO 2 efflux to drying, including increases 21 , decreases 22 , 23 and no responses 24 across forests of differing rainfall and seasonality. Field responses to drying have varied even among nearby forests, apparently related to baseline moisture and fertility 11 .

Most of the previous work in tropical forests only considered total CO 2 efflux rates, which are important for determining the overall carbon balance of tropical forests 25 , but are limited in their ability to uncover mechanisms behind observed change. Those mechanisms can be revealed by the determination of 14 C values, which indicate the average age of the carbon sources being metabolized and released as CO 2 26 , where in this context ‘new’ or ‘young’ carbon has been fixed from the atmosphere in the last few years, older ‘decadal-aged’ carbon is enriched in 14 C relative to the current atmosphere, and even older ‘century or millennial-aged’ carbon is depleted in 14 C relative to current atmosphere (see section “Methods” and Supplementary Fig.  1 ). These studies have provided valuable information on the response of the soil carbon cycle to climate change across a range of in situ experiments. For example, in thawing permafrost, 14 C signatures of soil CO 2 efflux revealed that warming and drying together caused an increase in the release of old carbon (depleted in 14 C relative to atmosphere) in soil carbon as CO 2 27 . In a temperate conifer forest, whole-profile soil warming increased soil CO 2 efflux and decreased soil carbon stocks by about 30%, changes that were attributed to increased decomposition of decadal-aged soil carbon pools 14 , 28 . In a temperate deciduous forest where experimental drying decreased soil CO 2 efflux by 10–30%, Δ 14 C of soil CO 2 efflux attributed this response to decreased microbial respiration near the soil surface 29 . In contrast, neither the rate nor the Δ 14 C of soil CO 2 efflux were affected by experimental drying in a tropical forest in Tapajos, Brazil 24 . We have an extremely limited understanding of how tropical forest soil CO 2 efflux (or its Δ 14 C) are affected by warming and drying in the same forest. Given that temperature and moisture are the major climatic drivers of soil CO 2 efflux 9 , 10 , 15 , 19 , 20 , and that in the tropics both significant warming and drying are predicted this century 30 , there is a critical need for studies that assess the impact of warming and drying together on both the magnitude and source (i.e., age) of soil CO 2 efflux in tropical forests.

In this study, we determined how warming and drying impact the amount and age of carbon released as soil CO 2 efflux in two distinct lowland tropical forest areas. We measured the Δ 14 C and δ 13 C of soil-respired CO 2 in Panamanian forests (Fig.  1 and Supplementary Table  1 ) that are subject to either in situ experimental soil warming (4 °C above ambient temperature to 1.2 m depth 15 , 31 ) or in situ experimental drying (50% throughfall exclusion 11 , 32 , 33 ). Our study sites are seasonally moist, semi-deciduous forests. The warming site and one drying site are both within the Barro Colorado Nature Monument in nearby and similar forests on similar soils, enabling a direct comparison of warming and drying effects on soil CO 2 efflux. A second drying experiment is on the northern side of the Panama Isthmus on infertile soils where mean annual precipitation (MAP) is greater, representative of a broad geographic area of the tropics. Given the seasonality of these forests, we performed measurements at stages of the seasonal cycle for which we expected the largest variation in CO 2 efflux between control and experimental plots based on previous studies 11 , 15 , 20 —the wet season and dry season or dry-to-wet season transition (see section “Methods”).

a SWT = the Soil Warming Experiment in Lowland Tropical Rainforest (SWELTR) and SL = San Lorenzo. SL and P12 are Panama Rainforest Changes with Experimental Drying (PARCHED) sites. Map made with Natural Earth. Free vector and raster map data @ naturalearthdata.com. b Experimental plot at SWT. c Soil CO 2 efflux sampling for 14 C analysis with heating cables visible on the soil surface. d A pair of experimental plots at P12 where 50% throughfall exclusion structures are on the left and a paired control plot (with no throughfall exclusion) is shown on the right.

We show that warming and drying both increase the relative contribution of older soil carbon to CO 2 efflux, but differences in total CO 2 efflux responses to warming versus drying suggest two different mechanisms. Specifically, warming stimulated the decomposition of older soil carbon by increasing overall soil CO 2 efflux, with our results indicating a microbial switch in resource use following the depletion of fresh organic matter under warmed conditions 34 . In contrast, drying reduced total soil CO 2 efflux, apparently limiting the mobility of fresh carbon in soils and delivery to decomposers. This restriction of microbial access to fresh carbon explains the shift towards increased contributions of older carbon in total soil CO 2 emissions. Thus, climatic warming and drying will likely increase the vulnerability of previously stored soil carbon in tropical forests by stimulating the decomposition of old carbon.

Results and discussion

Effects of experimental warming on the average age of respired co 2.

We investigated the effects of soil warming at the Soil Warming Experiment in Lowland Tropical Rainforest (SWELTR) 15 during two seasonal timepoints. Soil warming increased the Δ 14 C of respired CO 2 during the wet season, indicative of greater efflux of ‘bomb’ carbon under warmed and wet conditions (Fig.  2a ). Specifically, the mean Δ 14 C of respired CO 2 was 12 ± 5‰ higher in warmed plots than control plots in the wet season ( p   =  0.02). In the warmed plots, Δ 14 C of respired CO 2 was also 11 ± 5‰ higher in the wet season than in the dry season ( p   =  0.03).

a 14 C of respired CO 2 from single time-point measurements for n   =  2/plot (total n   =  10). The mean Δ 14 C value of air samples collected from the sites in this study in 2019 was −4 ± 3‰ and is indicated by the dotted reference line (mean) and gray shading (±standard error). Effects of experimental warming and season were tested using a three-way repeated measures ANOVA with collar type, treatment, and season (Supplementary Table  2 ). This test indicated a significant treatment by season interaction ( p  = 0.02), and a multiple comparisons test with a Holm adjustment indicated a significant effect of experimental warming in the wet season ( p  = 0.03) but not in the dry season ( p  = 0.33). b Monthly average total soil-respired CO 2 flux for March and October 2019 ( n  = 5 paired plots). Effects of experimental warming and season were tested using a two-way repeated measures ANOVA with treatment and season (Supplementary Table  3 ). This test indicated significant main effects for treatment ( p  = 0.03) and season ( p  = 0.03). The figures show plots warmed by +4 °C (WARM) and controls (CTRL). Solid lines indicate medians, dashed lines indicate means, ends of boxes show the upper (Q3) and lower (Q1) quartiles, whiskers indicate minimum and maximum ranges (calculated from quartiles), solid points are individual observations. Asterisks indicate statistically significant differences between control and warmed plots where p  ≤ 0.05 whereas NS indicates non-significant differences ( p  > 0.05).

The observed increase in the Δ 14 C of respired CO 2 indicated that carbon fixed nearer to the bomb spike (circa 1963; see Supplementary Fig.  1 for reference), i.e., decadal-aged carbon, contributed more to soil CO 2 flux under warmer and wetter conditions (with warming in the wet season) compared to cooler and drier conditions (without warming and in the dry season). Because roots typically respire CO 2 with Δ 14 C values close to the current atmosphere 26 , 35 , this result suggested that the shift was attributable to microbial (not live root) CO 2 flux. Thus, we found increased decomposition and loss of decadal-aged soil carbon under warmer and wetter conditions, while recently fixed carbon was the dominant source of carbon respired under drier or cooler conditions.

During the time periods of our study, total soil CO 2 flux rates were also higher in warmed and wet conditions (Fig.  2b ), with this difference in total flux likely driving increased utilization of older C. We found that total soil CO 2 flux increased from the dry season (March) to the wet season (October) by 60% ( p  = 0.03). Across seasons, experimental warming increased total soil CO 2 flux from 3.8 ± 0.4 μmol CO 2  m −2  s −1 in control plots to 6.0 ± 1.4 μmol CO 2  m −2  s −1 in warmed plots ( p  = 0.03). We partitioned the total soil CO 2 flux for the sampling periods in this study into heterotrophic (soil-derived) and autotrophic (root-derived) CO 2 flux using in situ root-exclusion and ingrowth cores (see section “Methods”) and found that 74 ± 7 % of total soil CO 2 flux was heterotrophic (Supplementary Fig.  2 ). These results were consistent with a published 2-year time series of soil CO 2 flux from this experiment 15 , which showed a 55% increase in soil CO 2 flux with warming attributed primarily to soil microbial (rather than live root) CO 2 flux.

Thus, our results strongly indicate that warming caused an increase in the emission of older carbon (with a higher Δ 14 C value) from soil organic matter (SOM) into the atmosphere, which can be explained by several mechanisms that are not mutually exclusive. First, warming-stimulated soil CO 2 efflux during the 18–24 months preceding our measurements 15 may have depleted the pool of fresh soil organic carbon (i.e., with a Δ 14 C value closer to 0‰) leading to a switch in microbial substrate use to older pools of carbon 36 . We found that bulk soils to 20 cm depth contained higher Δ 14 C than soil CO 2 efflux (Supplementary Fig.  3 ) and could have served as an older carbon source with a higher abundance of 14 C. Large increases in microbial enzymatic activity and a shift in the microbial community composition in surface soils were detected at our study site after 2 years warming, which may have coincided with a shift in substrate availability and use 31 . Second, warming may have increased the degradation of older carbon pools via priming, whereby the rapid metabolism of plant-carbon inputs provided the necessary energy for microbes to synthesize enzymes to access longer lived, more chemically complex, carbon pools 34 . This process was observed in response to additions of fresh plant litter at a forest near our warming site 37 . In support of this mechanism, during the wet season in warmed soil we observed increased soil CO 2 efflux (Fig.  2b ) 15 , increased activity of soil extracellular enzymes 15 , 31 , and increased variability in Δ 14 C of respired CO 2 (Fig.  2a ), which suggested an increased connectivity of soil organisms to a wider variety of carbon sources available for microbial metabolism.

The idea that priming effects contributed to increased soil CO 2 emissions with warming was further supported by our finding that the largest increase in CO 2 efflux with warming occurred during the seasonal peak in leaf litter decomposition 15 (when high decomposition rates could result in priming of older soil carbon). Importantly, in laboratory incubations (without the carbon inputs that could drive priming under field conditions), the Δ 14 C values of microbial CO 2 efflux were similar for soil collected from warmed and control plots (Supplementary Fig.  3 ). Under field conditions, the SOM contributing to the increase in CO 2 efflux with warming likely originates from shallow soils (to about 20 cm depth). Shallow soils are disproportionately affected by plant C inputs via litter decomposition and fine roots. In addition, the high abundance of 14 C in soils to 20 cm depth suggests that most of the carbon in the upper mineral soil layers is cycling on decadal timescales (Fig.  S3 ). Together, these results suggest that, under field warming, microbes dwelling in the upper soil layers may have primed 14 C-enriched SOM to increase the Δ 14 C in soil CO 2 efflux (Fig.  2a ), or they might have depleted younger stocks of C and shifted substrate utilization. Further studies are needed to elucidate the specific mechanisms.

Other experiments that warmed the soil profile (by 4–4.5 °C) have reported increases in annual soil CO 2 flux of ~35% in temperate forest 14 and 14% in boreal peat forest 38 , considerably lower than the 55% increase in soil CO 2 flux with whole-profile warming reported for our site 15 . No change in the Δ 14 C values of respired CO 2 was reported by warming experiments in two temperate forests where emissions were sustained by decadal-aged C 14 , 39 . However, more similar to our 14 C observations, soil warming increased Δ 14 C of respired CO 2 in a boreal forest, indicative of a greater contribution of decadal-aged carbon to the total flux 40 . Experimental warming also increased the age of CO 2 in porewater profiles in a boreal bog 41 , in soil pore spaces in tundra 42 , and in soil and ecosystem CO 2 flux in degraded permafrost 27 . These results suggest a variable, but potentially widespread, shift toward increased mobilization and loss of older soil carbon with climate warming.

Effects of experimental drying on the average age of respired CO 2

We investigated the effects of ecosystem drying at two sites that are part of the Panama Rainforest Changes with Experimental Drying (PARCHED) study 33 . We selected the P12 site for its similarity and proximity to the warming experiment, with equivalent MAP and similar soils at the two sites. We also included the San Lorenzo (SL) PARCHED site, which is also on infertile soils but receives about 800 mm more annual rainfall than the other two sites, increasing the representativeness of our study for wetter tropical forests.

We found that experimental drying led to an increase in the mean Δ 14 C of respired CO 2 by 8 ± 3‰ averaged across sites and sampling periods ( p  = 0.03, Fig.  3a ), consistent with a putative shift in microbial substrate use towards older, decadal-aged soil carbon. CO 2 Δ 14 C values also decreased by 6 ± 3‰ from the wet-to-dry season transition in May to the late wet season in November/December averaged across sites and treatment ( p  < 0.01). This is consistent with a depletion of the fresh carbon substrate delivered via dry season litterfall, over the course of the wet season, as supported by wet-season declines in total soil respiration and seasonal changes in soil biogeochemistry 11 , 33 . The Δ 14 C of respired CO 2 did not differ significantly between the drier (P12) and wetter (SL) sites (Fig.  4a , Supplementary Table  5 ), so sites were pooled for these analyses.

a 14 C of respired CO 2 for both P12 and SL for n   =  2/plot (total n  = 16). The mean Δ 14 C value of air samples collected from the sites in this study in 2019 was −4 ± 3‰ and is indicated by the dotted reference line (mean) and gray shading (±standard error). Effects of throughfall exclusion and season were tested using a four-way repeated measures ANOVA with collar type, site, treatment, and season (Supplementary Table  5 ). This test indicated significant main effects for treatment ( p  = 0.03) and season ( p  = 0.01). b Single time-point total soil-respired CO 2 efflux for both P12 and SL ( n   =  8 paired plots). Effects of throughfall exclusion and season were tested using a three-way repeated measures ANOVA with site, treatment, and season (Supplementary Table  6 ). This test indicated a significant treatment by season interaction ( p  = 0.05) and a multiple comparisons test with a Holm adjustment indicated a significant effect of throughfall exclusion in the dry-to-wet season transition ( p  = 0.02) but not in the wet season ( p  = 0.79). The figures show plots with 50% of throughfall excluded (DRY) and controls (CTRL). Solid lines indicate medians, dashed lines indicate means, ends of boxes show the upper (Q3) and lower (Q1) quartiles, whiskers indicate minimum and maximum ranges (calculated from quartiles), solid points are individual observations. Asterisks indicate statistically significant differences between control and throughfall exclusion plots where p  ≤ 0.05 whereas NS indicates non-significant differences ( p  > 0.05). Because 14 C values and rates of soil CO 2 efflux did not differ between P12 and SL, the sites were pooled for statistical analysis. The sites are shown separately in Fig.  4 .

a . Single time-point 14 C of respired CO 2 at SWELTR ( n  = 10/treatment and time point) and at P12 and San Lorenzo ( n  = 4/site, treatment, and time point). b Total soil-respired CO 2 flux rates are monthly averages for SWELTR ( n  = 5/treatment and time point) and single time-point measurements at P12 and San Lorenzo ( n  = 4/site, treatment, and time point). The figures show means as large symbols with standard errors and individual measurements as small symbols. Dark gray shading denotes the dry season, light gray shading denotes the dry-to-wet seasonal transition period, and no shading denotes the wet season.

During our study period, experimental drying led to a 27% decrease in total soil CO 2 efflux, from 7.8 ± 0.5 μmol CO 2  m −2  s −1 (control plots) to 5.7 ± 1.0 μmol CO 2  m −2  s −1 (throughfall exclusion plots), during the dry-to-wet season transition for both sites ( p  < 0.01, Fig.  3b ). This is consistent with longer term data over the first 5 years of this experiment, during which time these two sites had similar suppression of soil CO 2 flux in response to drying during the dry season and/or transitional seasons 11 . Soil CO 2 efflux partitioned into heterotrophic (soil-derived) and autotrophic (root-derived) components using field exclusion columns (see section “Methods”), suggested that this response was driven by a reduction in heterotrophic CO 2 flux rates ( p  < 0.01), with no apparent change in root-derived CO 2 efflux with drying (Supplementary Figs.  4 and  5 ). Overall, total, root, and heterotrophic soil CO 2 flux rates were higher during the dry-to-wet season transition compared to the wet season ( p  < 0.01), consistent with seasonal trends based on time series reported for these and other sites in the region 11 .

As with warming, our observed patterns could be explained by several mechanisms that are not mutually exclusive. First, drying decreased soil respiration as moisture limited microbial activity and the transport of soluble carbon substrates from the forest floor into mineral soils 43 as observed following throughfall exclusion in other tropical forests 22 , 23 , 44 . Our finding that the Δ 14 C of respired CO 2 increased under partial throughfall exclusion (Fig.  3a ) was consistent with reduced microbial access to fresh plant-carbon inputs. In our study, carbon in the 0–10 cm depth had higher Δ 14 C values than soil CO 2 efflux at both sites (Supplementary Fig.  6 ), suggesting that our observations may have been explained by a shift toward increased use of decadal-aged soil carbon (with a higher Δ 14 C value) by soil microbes. During the same period as our study, experimental drying caused a transition in the surface soil (0–10 cm depth) bacterial community composition toward a ‘drought microbiome’, which may have coincided with a microbial substrate shift. Indeed, other throughfall exclusion experiments reported decreased surface litter decomposition rates in Costa Rican forest 45 , decreased CO 2 efflux from the litter layer in the eastern Amazon 46 , and increased accumulation of forest floor material in temperate forest 29 .

Second, our observed patterns could have resulted from decreased fine root production, respiration rates, or turnover with experimental drying. A change in root turnover or exudation could occur even in the absence of changing root respiration rates, as indicated by our data (no change in root respiration apparent in exclusion columns), and might explain why the increased Δ 14 C of respired CO 2 with experimental drying during the dry-to-wet season transition persisted into the wet season, even as CO 2 efflux decreased. A shift in root growth toward greater depths to access available water is plausible as such a shift was observed in the Amazonian rainforest following experimental drying 23 , 47 , and is a general pattern across tropical forests during dry seasons and droughts 48 .

Third, our results could be explained by greater contributions of deeper, older soil carbon to surface soil CO 2 efflux as reported following throughfall exclusion in the eastern Amazon 23 . Deeper in the soil profile, soil carbon was older (Supplementary Fig.  6 ) and soil moisture was likely higher in dried plots 44 , such that root and microbial activities were potentially less affected by changes in soil moisture. As a result, CO 2 produced at depth may have comprised a larger component of surface soil CO 2 efflux with experimental drying. At P12, soil carbon pools to 50 cm depth that were enriched in Δ 14 C relative to in situ surface CO 2 efflux and could have contributed to the increase in Δ 14 C of respired CO 2 with seasonal or experimental drying, although older fractions of carbon in surface soils could also have contributed to this shift (Supplementary Fig.  6 ). At SL, however, subsurface particulate organic matter (originating mainly from roots) was depleted in 14 C relative to current atmosphere, indicating much older carbon, and could not have explained our observed increase in Δ 14 C of surface CO 2 efflux (Supplementary Fig  6 ). Thus, an increase in the decomposition of deep soil carbon pools does not consistently explain the patterns we observed across both sites, and we conclude that a shift in substrate utilization toward older surface soil carbon, and/or changes in root turnover or decomposition, most likely explain our 14 C data.

Seasonal effects on soil 14 C and CO 2 fluxes

The observed seasonal pattern in soil CO 2 efflux across sites (Fig.  4b ) likely reflected a combination of favorable conditions for microbial activity during seasonal rewetting: litter accumulated over the dry season provided ample carbon substrate 20 , 49 , dissolved organic carbon (DOC) production and transport facilitated microbial access to substrate 50 , and rewetting of soil following drought strongly stimulated microbial activity 23 , 44 , 51 . Then, the effect of throughfall exclusion attenuated and the effect of soil warming became more pronounced during the later wet season as soils at all sites became more uniformly wet. In these seasonally moist forests, litter and particulate organic matter build up over the dry season and fuel the sharp increase in respiration rates that we report during the dry-to-wet season transition period at P12 and SL 11 , 20 . Indeed, studies in nearby forests including the warming site (SWT) 15 and elsewhere in the tropics 23 , 47 have shown a similar increase in soil respiration rates during this dry-to-wet transition period, driven by seasonal patterns of moisture availability, microbial biomass 33 , and leaf-litterfall input 49 .

Potential combined effects of warming and drying

Climate change is expected to alter rates of soil carbon cycling in tropical forests through warming 52 and altered rainfall regimes 53 simultaneously. Unfortunately, no experiments have manipulated warming and drying together. Our results, based on individual responses to in situ experimental soil warming and soil drying in nearby forests, demonstrate that warming and drying not only change the emission of CO 2 from soils, but also the age of the carbon being emitted. Specifically, we found that both warming and drying increased the utilization of older soil carbon, even though warming increased but drying decreased soil CO 2 efflux. Thus, while further full factorial experiments are needed to elucidate the combined effects of warming and drying on soil carbon losses, our results indicate that warming and drying together will increase losses of older and previously stable soil carbon.

The consequences of our findings have wider implications when the impact of warming and drying on aboveground processes is considered alongside belowground effects. Both warming and drying have been shown to have detrimental effects on tropical forest productivity, based on in situ drying 53 and warming 54 experiments as well as observational studies during weather events 55 . Overall, these studies provide limited evidence for acclimation of photosynthetic activity. Thus, warming and drying together may decrease inputs of fresh carbon to soils, further exacerbating increased losses of older soil carbon observed here. Meanwhile increased CO 2 release from subsoil carbon is likely to continue under warming and drying, decreasing soil carbon storage throughout the soil profile, at least until drying extends to deeper soil horizons.

It is important to note that our reported increases in 14 C with experimental warming and drying reflected changes in the average age of carbon being respired (the equivalent of 2–3 years in the mean age of respired carbon, see Supplementary Fig.  1 ), but do not provide insight into the distribution of carbon ages contributing to these averages. Our ongoing work using Δ 14 C to study soil carbon storage and cycling in these and other sites along the Panama Isthmus rainfall gradient shows that soils in these forests store large proportions of young soil carbon compared with other ecosystem types, suggesting rapid turnover (Supplementary Figs.  3 and  6 ). Others have reported soil carbon turnover times of <10 years for numerous tropical forests 56 , 57 . While turnover times of decades to millennia were reported for clay-associated soil C at depth, the majority of soil C was found to have turnover times of less than a decade in Oxisols and Ultisols in Amazonian forest 58 . In forest in Puerto Rico, the mean carbon pool age for light density fractions (10–25% of the total C pool depending on depth) was 1–4 years to 60 cm depth 59 , demonstrating the presence of an important and very rapidly cycling soil carbon pool even at depth. Thus, we conclude that our observed shifts in the age of respired carbon are striking, indicating a substantial increase in contributions from older soil carbon fractions, especially considering that these results were observed following relatively short-term (1–3 year) experimental treatments.

In summary, we demonstrate how warming and drying affect the rate and age of soil carbon emission to the atmosphere in tropical forests, by determining the Δ 14 C of soil CO 2 efflux following experimental soil warming (whole-profile heating by 4 °C) and soil drying (50% throughfall exclusion). Experimental warming increased soil CO 2 efflux and, during the wet season, increased the age of respired soil carbon by roughly 2–3 years. In contrast, experimental drying decreased heterotrophic respiration rates, but also increased the age of respired soil carbon by roughly 2 years. Together, these results indicate an increase in the vulnerability of extant soil carbon, and a relative shift in microbial carbon use toward older sources: warming by depleting the pool of rapidly cycling carbon and stimulating the decomposition of old carbon; drying by reducing the mobility, accessibility, and subsequent decomposition of new carbon inputs. These findings imply a destabilization of old soil carbon under both warming and drying, which will have major implications for tropical forest–climate feedbacks. Our findings point to a need to study the effects of modified soil moisture and temperature together to capture and predict the net effects of climate change on tropical forest soil carbon storage now and in the future.

Study area and manipulation experiment descriptions

This study was conducted in three lowland tropical forest sites in central Panama. Two of the sites include experimental drying and one site includes experimental warming (Fig.  1 and Supplementary Table  1 ). Mean annual air temperature for all the sites is around 26 °C and air temperature is relatively constant over the year 60 . The region encompasses a precipitation gradient, with higher MAP to the north and lower MAP to the south, and has highly variable parent materials that influence available nutrients 61 , but all three sites in this study are on low-fertility soils as described below.

SWELTR 15 , 31 is located on Barro Colorado Island in the middle of the precipitation gradient, receiving just under 2600 mm MAP. Soils at the site are moderately weathered, clay-rich, Dystric Eutrudepts (Inceptisols) formed on the conglomerate parent materials of the Bohio formation, primarily basalt and graywacke sandstone. The experiment includes 5 paired warmed and control plots (ten plots total). Soil warming started in November 2016 and is achieved using resistance cables buried to 1.2 m depth to warm the entire soil profile by an average of 4 °C above ambient temperature.

The two experimental dying sites included in this study consist of throughfall reduction experiments and are part of the PARCHED study 11 , 33 . The drier site (P12) is located on Buena Vista Peninsula, at 51 m above sea level, and receives a similar MAP to SWELTR. Like SWELTR, soils at P12 are low-fertility Ultisols formed on the Bohio formation. The wetter site (SL) is located closer to the Caribbean coast, at 175 m above sea level, and receives about 3421 mm MAP. Soils at SL are low-fertility Oxisols formed on Chagres sandstone and contain more clay than soils at P12 33 contributing to overall higher soil moisture at SL than at P12. Each site includes four paired dry and control plots (eight plots total). SL has been previously referred to as Sherman Crane in the literature but has been renamed as the former evoked negative connotations associated with the legacy of colonialization. Throughfall reduction structures that exclude 50% of throughfall were installed over 10 × 10 m plots in June (P12) and July (SL) 2018 and remained fixed throughout the experimental period.

Field sampling and data collection

At all three sites, each plot has replicate soil respiration collars (20 cm diameter at SWELTR and 10 cm diameter at PARCHED) with fluxes measured regularly with an LI-8100 infrared gas analyzer (LI-COR Biosciences) along with soil temperature and moisture. In addition, root-exclusion and root-ingrowth cores were installed for all three experimental sites (10 cm diameter, 30 cm deep PVC tubes), which are used to partition heterotrophic and autotrophic respiration flux corrected for disturbance following Nottingham et al. 15 .

For SWELTR, we interpreted 14 C of CO 2 in the context monthly average total soil respiration rates, partitioned root and heterotrophic soil respiration rates, soil temperature, and soil moisture for March and October 2019. March was chosen rather than April because sampling for 14 C occurred the 1st week of April and early rains started later that month, marking the beginning of the transitional period from dry and wet season. Soil CO 2 efflux was measured biweekly using a Li-cor gas analyzer (IRGA Li-8100; LI-COR Biosciences), volumetric soil moisture at 0–10 cm depth was measured using a Thetaprobe (Delta-T sensor/Campbell) sensor and soil temperature at 0–10 cm depth was measured using an HI98509 thermometer probe (Hanna Instruments). To assess warming treatment effects on 14 C of bulk soil and CO 2 efflux in laboratory incubations, a soil core was collected from each plot in October 2019, after soil flux sampling, from the following depth increments: 0–10, 10–20, 20–50, and 50–100 cm. Soils were shipped immediately to Lawrence Livermore National Laboratory where they were processed and incubated in the laboratory. Soils were sieved to 2 mm and ground to a fine powder for 14 C measurement. Field-moist soils were picked free of large root fragments and incubated at field moisture at 26 °C until sufficiently high CO 2 concentrations were reached for 14 C analysis (4–7 days for 0–20 cm depths and 24–214 days for 20–100 cm depths).

For PARCHED, we interpreted 14 C of CO 2 in the context of instantaneous total soil CO 2 efflux, soil temperature, and soil moisture measured within 2.5 weeks of sampling for 14 C–CO 2 (≤3 days except for SC in May due to logistical constraints). For all respiration rate sampling points, soil CO 2 efflux was measured using a Li-cor gas analyzer (IRGA Li-8100; LI-COR Biosciences), volumetric water content at 0–10 cm depth was measured using a ML-3 ThetaProbe read by an HH2 Moisture Meter, and soil temperature at 0–10 cm depth was measured using a digital external soil temperature 5-inch probe (Forestry Suppliers Part 89102). Soil cores were collected at P12 and SL prior to the construction of throughfall exclusion structures but near the experimental plots. Soils from the 0–10 cm depth were collected in 2018 and shipped immediately to Lawrence Livermore National Laboratory where they were processed and incubated in the laboratory as described above for the SWELTR soils. We also performed 14 C measurements on bulk soils and density fractionations from soils that were sampled in 2015 in the following depth increments: 0–10, 10–25, and 25–50 cm depths. Soils from 0–10 cm and 20–50 cm depths were density fractionated into dense, free light, and occluded light fractions using low C/N sodium polytungstate (SPT-0, Poly-Gee) adjusted to a density of 1.7 g cm −3 . A detailed description of the density fractionation method can be found in the Supplementary Methods.

We collected gas samples from soil chambers placed over soil respiration collars and root-exclusion cores, for the determination of 13 C and 14 C in total soil respiration (soil + roots) and heterotrophic (root-free) soil respiration (we assumed that the CO 2 flux from the root-exclusion cores originated from SOM). Samples were collected from five sets of paired plots at SWELTR (ten plots total), from eight sets of paired plots at PARCHED (four control and four throughfall reduction plots at both P12 and SL), and during two sampling campaigns at each study site. At SWELTR, we collected samples during the dry season (April 6–7) and the wet season (October 2–3) of 2019, 2–3 years after the initiation of the warming treatment. At PARCHED, we collected samples during the dry–wet season transition (P12, May 22–24; SL, May 27–31) and the wet season (P12, November 21–23; SL, December 2–4) of 2019, roughly 1–1.5 years after the initiation of the drying treatment. Sampling occurred toward the end of each seasonal phase to represent when moisture limitation is at its lowest (late wet season) or greatest (late dry season). However, for PARCHED we sampled during the dry-to-wet season transition, which is when the difference in soil moisture between control and treatment plots is greatest—the dry season being effectively prolonged in treatment plots.

For gas collection, sampling collars were fitted with a static chamber lid constructed of PVC with two gas sampling ports. Chamber headspace was recirculated through a soda lime trap to scrub the headspace air of CO 2 for >4 times the chamber volume, using a battery-operated air pump. Chamber lid ports were closed to allow CO 2 to accumulate for long enough to ensure sufficient accumulation of CO 2 for 13 C and 14 C measurement (~1–4 h depending on flux rates). Headspace air was collected in an evacuated 1 L flask equipped with an 80 mL min −1 flow restrictor to minimize isotopic fractionation during sampling. During each sampling campaign at each site, we also collected a reference air sample into a 3 L flask equipped with a 6 mL min −1 flow restrictor.

Radiocarbon interpretation

The ability to use 14 C to indicate the C sources contributing to respiration stems from changes in atmospheric 14 CO 2 over the last century from atmospheric thermonuclear weapons testing 62 (Supplementary Fig.  1 ). Atmospheric thermonuclear weapons testing in the late 1950s and early 1960s doubled the amount of 14 C in the atmosphere. Following the atmospheric nuclear weapons test ban in 1963, the amount of 14 C in the atmosphere decreased as this so-called bomb C was taken up by vegetation and oceans allowing annual to sub-annual resolution of the time of carbon assimilation from the atmosphere. Fossil fuel emissions contribute to the sustained decline of atmospheric Δ 14 C values over recent decades. Thus, each year a unique Δ 14 C signature is assimilated by plants via photosynthesis, translocated, and allocated to biomass growth and metabolism. The Δ 14 C of respired CO 2 can indicate an average age, or mean time elapsed since that carbon was fixed from the atmosphere, although it is important to recognize that this carbon is not homogenous in source or age. In the absence of extreme stress (e.g., girdling or complete defoliation), plants, including roots, tend to respire carbon from recent photosynthates, with Δ 14 C of respired CO 2 close to the atmosphere in that year 35 , 63 . In contrast, the 14 C value of microbial respiration reflects the substrates the microbes are utilizing, which have an average age of several years or longer 26 . Studies have used the shift in the Δ 14 C value of soil-respired CO 2 to show changes in the source C pools supplying microbes over seasonal cycles 35 with disturbance including fire 64 , warming 40 , and drying 29 . Here, we interpreted shifts in the Δ 14 C value of soil-respired CO 2 with experimental warming or drying to reflect a shift in the average age of soil C substrates used for cellular metabolism and subsequent respiration as CO 2 . For reference, we determined an approximate age shift with experimental warming and drying by fitting data to estimate the average year of synthesis (fixation from the atmosphere) using an annual decline in atmospheric Δ 14 C of 4.6‰ calculated from 1995 to 2019 62 (see Supplementary Fig.  1 ).

CO 2 isotopic measurements

After collection, air samples were shipped to Lawrence Livermore National Laboratory’s Center for Accelerator Mass Spectrometry where CO 2 from the field and laboratory incubation headspace was purified using cryogenic separation. For each air sample, a split of extracted CO 2 was analyzed for 13 C at the Department of Geological Sciences Stable Isotope Laboratory at the University of California-Davis (GVI Optima Stable Isotope Ratio Mass Spectrometer). Each bulk soil and fraction sample was measured for δ 13 C at the Center for Stable Isotope Biogeochemistry at the University of California-Berkeley (IsoPrime100 mass spectrometer) and was combusted to CO 2 in the presence of CuO for radiocarbon analysis. All samples were reduced to graphite and analyzed for 14 C analysis on the Van de Graaff FN accelerator mass spectrometer. Measured δ 13 C values are reported relative to V-PDB and were used to correct 14 C values for mass-dependent fractionation. 14 C isotopic values are reported in Δ 14 C notation 65 , had an average AMS precision of 3‰, and were corrected for 14 C decay since 1950.

Statistical analysis

Statistical analyses were performed in R v. 4.3.2 66 using two-, three-, or four-way analysis of variance (ANOVA) with repeated measures using the nlme (v. 3.1.164) 67 and lme4 (v. 1.1.35.1) 68 packages at α  = 0.05. Statistical results are in Supplementary Tables  2 – 9 . Data were tested for normality and were not transformed. Δ 14 C values did not differ between total and root-free CO 2 flux (using root-exclusion columns; Supplementary Figs.  7 ,  8 and Supplementary Tables  2 ,   5 ), so root exclusion and total soil collars were pooled for tests of treatment and season effects. When present, interaction effects were investigated using the Phia (v. 0.3.1) 69 package with nlme or least squares means tests using the lsmeans (v. 2.30.0) 70 package with lme4 and with a Holm adjustment for multiple comparisons. Analyses were performed for SWELTR and PARCHED experiments separately and included effects of experimental treatment, season, and collar type (for isotopes only). The PARCHED ANOVA model included site to enable comparisons of the P12 and SL sites. In the text, results are reported as means followed by one standard error. Reported effect sizes are least squares means differences followed by one standard error.

Reporting summary

Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Data availability

The data generated and used in this study have been deposited at figshare [ https://doi.org/10.6084/m9.figshare.24240211 ] and at the US Department of Energy’s Environmental Systems Science Data Infrastructure for a Virtual Ecosystem (ESS-DIVE) [ https://data.ess-dive.lbl.gov/datasets/doi:10.15485/2425968 ].

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Acknowledgements

We thank Maria Jose Montero for help with field sampling at SWELTR. We thank Makenna Brown, Biancolini Castro, Lily Colburn, and Korina Valencia for help with field sampling at PARCHED. We thank the Smithsonian Tropical Research Institute (STRI) for their assistance in ensuring that samples were collected and exported in a responsible manner and in accordance with relevant permits and local laws. These sites are active ecological research sites maintained by the Smithsonian Tropical Research Institute. Samples were collected and exported in a responsible manner. Any disturbance associated with accessing field sites and collecting samples was performed consistent with directives from STRI. All experimental work, sample collection, and sample export to the United States was done in compliance with local, national, and international laws and regulations with the necessary research, export, and import permits. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344 (LLNL-JRNL-853569). This work was funded by the Office of Biological and Environmental Research in the U.S. Department of Energy Office of Science through award SCW1572 to K.J.M. and DE-SC0015898 to D.F.C. The study was further supported by a UK NERC Grant NE/T012226 to A.T.N. For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) license to any author accepted manuscript version arising from this submission.

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Contributions

K.J.M.: performed radiocarbon and statistical analyses, performed gas sampling and CO 2 purification, designed and funded the radiocarbon experiment with input from D.F.C. and A.T.N., performed data analysis, and wrote the manuscript. D.F.C.: provided data, led PARCHED, collected soil samples, helped write and edit the manuscript. L.H.D.: provided data, performed gas sampling, led field operations for PARCHED, collected soil samples, helped write and edit the manuscript. A.L.H.: prepared for and performed gas sampling at all study sites and helped edit the manuscript. K.M.F.: performed laboratory incubations and density fractionation, prepared samples for isotopic and elemental analysis, and helped write and edit the manuscript. A.T.N.: provided data, led SWELTR, collected soil samples, and helped write and edit the manuscript.

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McFarlane, K.J., Cusack, D.F., Dietterich, L.H. et al. Experimental warming and drying increase older carbon contributions to soil respiration in lowland tropical forests. Nat Commun 15 , 7084 (2024). https://doi.org/10.1038/s41467-024-51422-6

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Core Practical 15: Investigation of Respiration in Yeast ( Edexcel International A Level Biology )

Revision note.

Investigation of Respiration in Yeast

• A   redox indicator   is a substance that changes colour when it is reduced or oxidised
• They are used to investigate the effects of   temperature and substrate concentration   on the   rate of anaerobic respiration   in yeast
• These dyes can be added to a suspension of living   yeast cells   as they don’t damage cells
• Yeast can respire both aerobically and anaerobically, in this experiment it is their rate of anaerobic respiration that is being investigated
• Dehydrogenation   happens regularly throughout the different stages of aerobic respiration
• The hydrogens that are removed from substrate molecules are transferred to the final stage of aerobic respiration, oxidative phosphorylation, via the hydrogen carriers NAD and FAD
• The enzyme   dehydrogenase   catalyses the production of   reduced NAD   in   glycolysis
• When DCPIP or methylene blue are present they take up hydrogens from the organic compounds and get reduced instead of NAD
• Blue → colourless
• This means that the   rate of colour change   can correspond to the rate   dehydrogenase   would be working at and therefore,   the rate of respiration   in yeast
• The rate of respiration is inversely proportional to the time taken

Rate of respiration (sec -1 ) = 1 ÷ time (sec)

• Glucose solution

Method - Temperature

• Add a set volume of yeast suspension to test tubes containing a certain concentration of glucose
• Put the test tube in a  temperature-controlled water bath   and leave for 5 minutes to ensure the water temperature is correct and not continuing to increase or decrease
• Add a set volume of DCPIP to the test tube and start the stopwatch immediately
• This is subjective and therefore the same person should be assigned this task for all repeat experiments
• Repeat across a range of temperatures. For example, 30 o C, 35 o C, 40 o C, 45 o C
• For example, 0.1% glucose, 0.5% glucose, 1.0% glucose

Methylene blue or DCPIP is added to a solution of anaerobically respiring yeast cells in a glucose solution. The rate at which the solution turns from blue to colourless gives the rate of dehydrogenase activity

Controlling other variables

• Volume of dye added : if there is more dye molecules present then the time taken for the colour change to occur will be longer
• Volume of yeast suspension : when more yeast cells are present the rate of respiration will be inflated
• Type of substrate : yeast cells will respire different substrates at different rates
• Concentration of substrate : if there is limited substrate in one tube then the respiration of those yeast cells will be limited
• Temperature : an increase or decrease in temperature can affect the rate of respiration due to energy demands and kinetic energy changes. The temperature of the dye being added also needs to be considered
• pH : a buffer solution can be used to control the pH level to ensure that no enzymes are denatured

Interpretation of results

• A graph should be plotted of temperature against time
• This means hydrogens are released by the reactions more quickly, hence the DCPIP accepts electrons/hydrogens more quickly until all molecules of DCPIP are reduced. This means that it will take less time to turn from blue to colourless
• At extreme high temperatures, the enzyme may denature and meaning the colour change may not occur

Although the DCPIP and methylene blue undergo a colour change from blue to colourless it is important to remember that the  yeast suspension in the test tube may have a slight colour   (usually yellow). That means when the dye changes to colourless there may still be an overall yellow colour in the test tube. If this is the case it can be useful to have a control tube containing the same yeast suspension but with no dye added, then you can tell when the dye has completely changed colour.

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Author: Marlene

Marlene graduated from Stellenbosch University, South Africa, in 2002 with a degree in Biodiversity and Ecology. After completing a PGCE (Postgraduate certificate in education) in 2003 she taught high school Biology for over 10 years at various schools across South Africa before returning to Stellenbosch University in 2014 to obtain an Honours degree in Biological Sciences. With over 16 years of teaching experience, of which the past 3 years were spent teaching IGCSE and A level Biology, Marlene is passionate about Biology and making it more approachable to her students.