describe and carry out investigations using redox indicators, including DCPIP and methylene blue, to determine the effects of temperature and substrate concentration on the rate of respiration of yeast

Respiration – Investigating the Effects of Temperature and Substrate Concentration Using Redox Indicators

Learning‑Outcome Alignment (Cambridge International AS & A Level Biology 9700)

• AO1: Describe aerobic and anaerobic respiration, locate each stage of metabolism (glycolysis, link reaction, Krebs cycle, oxidative phosphorylation) and calculate the respiratory quotient (RQ).
• AO2: Design, carry out and evaluate investigations; analyse data using enzyme‑kinetics concepts (initial rate, V_max, K_m, Arrhenius equation).
• AO3: Demonstrate practical skills – planning, data collection, use of spectrophotometry, statistical treatment, critical evaluation of methodology.
• Specific syllabus outcome 12.2 Respiration – use of DCPIP and methylene‑blue to monitor yeast respiration.

Context – Where the Assay Fits in Cellular Respiration

In Saccharomyces cerevisiae glucose is first broken down by glycolysis in the cytoplasm, producing pyruvate, ATP and NADH. Under anaerobic (fermentative) conditions pyruvate is converted to ethanol and CO₂; under aerobic conditions pyruvate enters the mitochondrion where it is oxidised (link reaction), the Krebs cycle runs, and the NADH/FADH₂ generated feed the electron transport chain (ETC) on the inner mitochondrial membrane. The redox indicators used in this investigation accept electrons from the same NADH/FADH₂ pool that normally reduces the ETC, so the rate of colour loss is a proxy for the overall rate of respiration.

Location of Each Stage (AO1)

• Glycolysis – cytoplasm; produces 2 ATP (net), 2 NADH and 2 pyruvate per glucose.
• Link reaction (pyruvate decarboxylation) – mitochondrial matrix; converts pyruvate to acetyl‑CoA, yields 1 NADH per pyruvate (2 NADH per glucose) and releases CO₂.
• Krebs cycle – mitochondrial matrix; per acetyl‑CoA generates 3 NADH, 1 FADH₂, 1 GTP (≈1 ATP) and 2 CO₂.
• Oxidative phosphorylation (ETC + ATP synthase) – inner mitochondrial membrane; uses NADH and FADH₂ to produce ≈30 ATP per glucose and consumes O₂.

Background Theory

• Aerobic respiration (overall equation):

  \[

  \mathrm{C6H{12}O6 + 6\,O2 \;\longrightarrow\; 6\,CO2 + 6\,H2O + \approx30\,ATP}

  \]

• Anaerobic (fermentative) respiration:

  \[

  \mathrm{C6H{12}O6 \;\longrightarrow\; 2\,C2H5OH + 2\,CO2 + \approx2\,ATP}

  \]

• Respiratory Quotient (RQ) – ratio of CO₂ produced to O₂ consumed.

  \[

  \mathrm{RQ = \frac{V{CO2}}{V{O2}}}

  \]

  For glucose, the theoretical RQ = 1.0. In this investigation RQ will be measured for every temperature and substrate‑concentration series.

Redox Indicators

Principles of the Assays

• The indicator acts as an artificial electron acceptor. The faster it is reduced, the faster electrons are being transferred through the metabolic pathway.
• Colour loss is monitored spectrophotometrically; the change in absorbance (ΔA) per unit time is proportional to the rate of respiration.
• DCPIP accepts electrons early (mainly from NADH generated in glycolysis and the link reaction), whereas methylene blue can be reduced later in the chain and also by fermentative pathways. Consequently, the two assays together give insight into the balance between aerobic and anaerobic metabolism.

Experimental Design

Two parallel series of experiments are performed – one with DCPIP, the other with methylene blue. Each series investigates:

1. The effect of temperature (15 °C, 25 °C, 35 °C, 45 °C) at a fixed glucose concentration (5 % w/v).
2. The effect of glucose concentration (0 %, 2 %, 4 %, 6 % w/v) at a fixed temperature (25 °C).

All trials are carried out in triplicate. A sealed fermentation tube (graduated cap) is included in every reaction to allow mandatory measurement of CO₂ volume for RQ calculation.

Variables

Materials

• Active dry yeast (S. cerevisiae)
• Glucose stock solution 10 % w/v (prepare fresh)
• DCPIP solution 0.20 mM (amber bottle)
• Methylene‑blue solution 0.10 mM (dark bottle)
• Distilled water
• Water baths (15 °C, 25 °C, 35 °C, 45 °C) with calibrated thermometers
• Spectrophotometer or colourimeter (600 nm for DCPIP, 660 nm for methylene blue)
• 1 cm path‑length cuvettes, pipettes, disposable tips, 15 mL conical tubes, timer, vortex mixer
• Sealed fermentation tubes with graduated caps (for CO₂ measurement)
• Dissolved‑oxygen probe (optional, for direct O₂ consumption)

Procedure

Common steps for both indicators

1. Label three tubes for each temperature (or each glucose level) – one tube per replicate.
2. Add 5 mL distilled water to each tube.
3. Add the required volume of glucose stock to obtain the desired % w/v. Mix thoroughly.
4. Weigh 0.50 g wet yeast and add to the tube. Vortex for 5 s to disperse.
5. Add the appropriate indicator:

   • DCPIP series – 1 mL of 0.20 mM DCPIP.
   • Methylene‑blue series – 1 mL of 0.10 mM methylene blue.

6. Insert a sealed fermentation tube with a graduated cap into the reaction mixture (ensure the cap is upright and the gas‑collecting chamber is empty).
7. Place the tubes in the pre‑set water bath and allow 2 min for temperature equilibration.
8. Immediately record the initial absorbance (A₀) at the appropriate wavelength using a blank that contains all reagents except the indicator.
9. Take absorbance readings every 30 s for 5 min (or until the colour is essentially lost). Record as A₁, A₂ … A₁₀.
10. After the final reading, measure the volume of CO₂ displaced in the graduated cap (read to the nearest 0.1 mL). Record this value for RQ calculation.

Specific notes

• Keep the cuvette temperature as close as possible to the water‑bath temperature (use a thermostated cuvette holder if available).
• For methylene blue, protect the solution from ambient light to avoid photoreduction.
• Zero the spectrophotometer with a blank containing everything except the indicator before each set of readings.

Data‑Recording Template

Data Analysis

1. Initial rate of colour loss

• Plot ΔA versus time for each trial.
• Identify the linear portion (usually the first 2–3 min) and calculate the slope (ΔA min⁻¹) by linear regression.

2. Convert absorbance change to moles of indicator reduced

Beer‑Lambert law:

\[

\Delta c = \frac{\Delta A}{\varepsilon \, l}

\]

• \(\varepsilon\) = 21 000 L mol⁻¹ cm⁻¹ (DCPIP) or 6 500 L mol⁻¹ cm⁻¹ (methylene blue).
• \(l\) = 1 cm (standard cuvette).
• Multiply \(\Delta c\) by the reaction volume (10 mL) to obtain moles of indicator reduced per minute – a quantitative proxy for the respiration rate.

3. Kinetic interpretation

• Temperature effect – Plot the initial rates (or \(\ln\) rate) against 1/T (K) to obtain an Arrhenius plot. The gradient = \(-E_a/R\) gives the activation energy (E_a).
• Substrate‑concentration effect – Plot initial rate versus glucose concentration. Fit the data to the Michaelis‑Menten equation:

  \[

  v = \frac{V{\max}[S]}{Km + [S]}

  \]

  Use a Lineweaver‑Burk (double‑reciprocal) plot to estimate V_max and an apparent K_m for glucose in yeast.

• Compare the kinetic parameters obtained with DCPIP and methylene blue; differences reflect the point at which each indicator intercepts electron flow (early vs late in the chain) and the contribution of fermentative pathways.

4. Respiratory Quotient (mandatory)

1. Calculate the volume of CO₂ produced (V_CO₂) from the graduated cap reading (corrected for water vapour if needed).
2. Estimate O₂ consumption (V_O₂) in one of two ways:

   • Stoichiometric method: 1 mol glucose consumes 6 mol O₂. Convert the amount of glucose added (mass/180 g mol⁻¹) to moles, then to the theoretical O₂ volume at STP.
   • Direct method (optional): Use a dissolved‑oxygen probe to record the drop in dissolved O₂ over the 5‑min assay; convert the Δ[O₂] to volume (using solubility data at the assay temperature).

3. Compute RQ:

   \[

   \mathrm{RQ = \frac{V{CO2}}{V{O2}}}

   \]

   Compare the experimental RQ with the theoretical value of 1.0 for pure aerobic respiration. Values < 1.0 indicate a fermentative contribution.

5. Statistical treatment

• Calculate mean ± standard deviation for each set of replicates.
• Use a t‑test (two‑sample) or one‑way ANOVA to assess whether differences between temperatures or substrate levels are statistically significant (p < 0.05).

Evaluation – Limitations, Sources of Error & Improvements (AO3)

• Indicator concentration – Excess indicator can compete with natural electron carriers, lowering the true respiration rate; insufficient indicator yields low absorbance and poor signal‑to‑noise. Optimise concentration in a preliminary trial.
• Oxygen availability – In sealed tubes O₂ may become limiting, especially at high temperatures. Consider using a larger headspace or a gentle stream of air for aerobic runs, and a separate sealed set for strictly anaerobic conditions.
• Temperature control – Water‑bath temperature can drift; a circulating digital thermostat with ±0.1 °C accuracy minimises this error.
• Yeast viability – Age and storage of dry yeast affect metabolic activity. Re‑hydrate fresh yeast 10 min before the assay and record the time since re‑hydration.
• Spectrophotometer drift – Re‑zero before each series and use a fresh blank each time.
• Mixing – Incomplete mixing leads to uneven distribution of yeast and indicator. Vortex briefly after each addition and again after the temperature equilibration step.
• Replicates & randomisation – Perform at least three independent replicates and randomise the order of temperature or concentration trials to avoid systematic bias.
• CO₂ measurement accuracy – Ensure the graduated cap is free of air bubbles before insertion; correct for water vapour pressure at the assay temperature.
• Extension suggestions – Test the effect of pH (buffers pH 4–8), compare alternative carbon sources (sucrose, maltose), or replace the colourimetric assay with a dissolved‑oxygen probe for a direct measurement of O₂ consumption.

Safety and Waste Disposal

• Wear lab coat, nitrile gloves and safety goggles at all times.
• DCPIP is a mild irritant – avoid skin contact and wash hands after use.
• Methylene blue stains skin and fabric – handle with care and clean spills promptly.
• Dispose of yeast suspensions down the sink with plenty of running water.
• Collect spent indicator solutions in labelled waste bottles and dispose of them according to school chemical‑waste procedures.

Possible Extensions (Further Investigation)

• Effect of pH on respiration rate (use buffered glucose solutions pH 4–8).
• Comparison of different carbon sources (sucrose, maltose, galactose).
• Use of a dissolved‑oxygen probe to obtain a direct, continuous O₂ consumption curve.
• Investigation of the impact of enzyme inhibitors (e.g., cyanide, antimycin A) on the colour‑loss kinetics.
• Modelling the data with non‑linear regression software to obtain more accurate kinetic parameters.