biology 121 lab

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Objectives • Measure carbon dioxide evolution and

uptake in plants and animals.

• Study the effect of temperature on cell respiration.

• compare respiration rates in germinating and non-germinating peas.

Introduction Energy is required by living organisms for movement, transport, and growth. Nothing happens without energy! The Sun is the ultimate source of virtually all energy on the planet Earth. Solar energy is captured by plants through the process of photosynthesis. The glucose molecules holding this energy are broken down by metabolic processes, creating usable energy for living systems. Cellular respiration is a series of reactions in which glucose molecules are broken down, releasing stored chemical bond energy (Figure 6.1). The released energy is used to make the energy rich molecule ATP (adenosine triphosphate). Carbon dioxide is released as a by-product of the breakdown of glucose. It is a crucial by-product from the perspective of plants, because they need CO2 to perform photosynthesis. Glycolysis is the first step in cellular respiration, and it results in the net production of two ATP molecules. In glycolysis, the 6- carbon glucose molecules are “split” into two, 3-carbon pyruvate (pyruvic acid) molecules.

LAB TOPIC 6: RESPIRATION

Pyruvate has two potential routes – aerobic respiration or anaerobic respiration [as either lactate fermentation or alcohol fermentation] (Figure 6.1).

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In laboratory today, you will be examining respiration in organisms that use aerobic respiration, which makes use of oxygen. In this pathway, pyruvate is broken down completely, and h igh-energy electrons are stripped away and passed through a series of electron carriers. Energy is released at each transfer, and is used to make a net 34 ATP molecules. Oxygen is the final electron acceptor in the electron transport system, hence the name aerobic cellular respiration. In lecture you will compare this process to anaerobic respiration, which occurs in the absence of oxygen or under low oxygen conditions. The equation below summarizes the process of aerobic respiration: C6H12O6  +  6  O2  à 6  CO2  +  6  H2O  +  ATP  +  Heat   Glucose      Oxygen          Carbon        Water                                                                        Dioxide     Considering the equation for aerobic respiration what variables could you measure to monitor respiration rate?  

 

Figure  6.1     Glycolysis  and  the  potential  fates  of  pyruvate  during  cellular  respiration.  

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Oxygen Consumption during Aerobic Respiration Aerobic respiration uses oxygen as the terminal electron‐acceptor in the electron transport chain and produces carbon dioxide (see equation above). You can, therefore, monitor the respiration rate of an organism by measuring its uptake of oxygen or evolution of carbon dioxide. Here, we will measure the respiration rate of the crickets, Gryllus sp., and English peas, Pisum sativa, using a gas‐phase CO2 probe and meter. This equipment can be used to measure the level of CO2 in the atmosphere of a closed chamber in units of parts per million (ppm) or mg/L.

Respiration rate is dependent on a variety of factors including the size and the level of activity of an organism, temperature, etc. At a given temperature, would you expect a crickets or peas to have a higher respiration rate on a per mass basis?

Exercise 6.1 Evolution of Carbon Dioxide by a Plant

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Here we will measure respiration in germinating peas. We will also address the questions “Do peas undergo cell respiration before germination?” and “What is the effect of temperature on the cell respiration of peas?” Using your collected data, you will be able to answer these questions.

Hypothesis Construct null and alternative hypotheses (e.g. for the effect of germination and temperature on respiration). Remember, your hypotheses must be testable.

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Prediction Predict the result of the experiment based on your hypotheses. Your prediction would be what you expect to observe as a result of this experiment (if/then).

Plants undergo both respiration and photosynthesis (at least during the day). As these two processes offset each other to some degree or another, the amount of CO2 evolved or consumed depends on how active photosynthesis is relative to respiration. To eliminate this problem from the determination we will make today, we will use pea seeds. Since these are not significantly photosynthetic, changes we measure in CO2 concentration in the chamber will be due to respiration alone. These peas undergo cell respiration during germination as they begin the process of seedling growth.

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PROCEDURE – Part 1 Effects of Germination (You will be using Logger Pro as you did for the spectrophotometer)

1. Obtain 25 germinating peas and blot

them dry between two pieces of paper towel.

2. Place the germinating peas into the

respiration chamber. 3. Place the shaft of the CO2 Gas Sensor in

the opening of the respiration chamber. 4. Wait one minute, then begin measuring

carbon dioxide by clicking the green arrow button (Experiment/ Start Collection). Collect data for 5 minutes then Stop Collection.

5. Remove the CO2 Gas Sensor from the

respiration chamber. Remove and weight the peas (record the weight below).

6. Place the peas on ice for Part 2 of the

experiment. 7. Use a notebook or notepad to fan air

across the openings of the probe shaft of the CO2 Gas Sensor for 1 minute. Make sure the CO2 readings return to 300-400 ppm.

8. Fill the respiration chamber with water,

empty, dry the inside thoroughly with a paper towel.

9. Determine  the  rate  of  respiration:    

a. Move  the  mouse  pointer  to  the   point  where  the  data  values  begin  to   increase.  Hold  down  the  left  mouse   button.  Drag  the  mouse  pointer  to  the   end  of  the  data  and  release  the  mouse   button.  

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b. Click  “Analyze”  and  the  “Linear   Fit”  button    to  perform  a  linear   regression.  A  floating  box  will   appear  with  the  formula  for  a  best   fit  line.  

c. Record  the  slope  of  the  line,  m,   as  the  rate  of  respiration  for   germinating  peas  at  room   temperature  in  Table  6.1.  

  d. Close  the  linear  regression  box   by  clicking  on  it  and  deleting.  

  e. Move  your  data  to  a  stored  run   by  choosing  “Store  Latest  Run”  from   the  Experiment  menu.  

  10. Obtain  25  non-­‐germinating  peas  and  

place  them  in  the  respiration  chamber  

11.      Repeat  Steps  3–9  for  the  non-­‐ germinating  peas  and  record  the  rate  in   Table  6.1.  

 

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Part 2 – Effects of Temperature

1. Remove  the  peas  from  the  ice  and   blot  them  dry  between  two  paper   towels.  

2. Repeat  Steps  3–9  from  Part  1  to   collect  data  with  the  cold   germinating  peas  and  record  the   rate  in  Table.6.1.  

TABLE  6.1    

Peas   Temperature  (ºC) Rate  of  respiration     Mass-­‐Corrected   Respiration  Rate   (ppm/g/min)  

Germinating,  Room  Temperature   20 Non-­‐germinating,  Room  Temperature   20  

Germinating,  Cool  Temperature   4

QUESTIONS

1. Do  you  have  evidence  that  cell  respiration   occurred  in  peas?  Explain.  

2.What  is  the  effect  of  germination  on  the   rate  of  cell  respiration  in  peas?  

3.What  is  the  effect  of  temperature  on  the  rate   of  cell  respiration  in  peas?  

4.Why  do  germinating  peas  undergo  cell   respiration?  

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Exercise 6.2 Evolution of Carbon Dioxide by an Animal

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Animals, like plants and other eukaryotes obtain energy for growth and day to day metabolism through metabolism of sugars through the glycolytic pathway of respiration and produce CO2 as a result of this activity. Thus, we can measure the rates of respiration as we have done above for peas. However, because they do not photosynthesize, any changes in CO2 concentration

are due to respiration alone. Table 6.2

Hypothesis Construct null and alternative hypotheses (e.g. for the effect temperature on respiration and a comparison of peas and crickets). Remember, your hypotheses must be testable. Prediction Predict the result of the experiment based on your hypotheses. Your prediction would be what you expect to observe as a result of this experiment (if/then).

PROCEDURE

1. Obtain  5  crickets  from  your   instructor.  

2. Place  the  crickets  into  the  respiration   chamber.  

3. Start  a  new  File.    Perform  the   experiment  as  outlined  in  steps  3-­‐9   above  at  room  temperature  and  4°C   (pre-­‐  incubate  in  the  refrigerator  and   then  held  on  ice).  

4. Record  your  results  in  Table  6.2.    

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. Crickets   Temperature  (ºC) Rate  of  respiration  

  Mass-­‐Corrected   Respiration  Rate   (ppm/g/min)  

Room  temperature   20 Cool  temperature   4  

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Questions for

1. What is the importance of cellular respiration to living organisms?

2. The concentration of oxygen in an environment can affect the respiration rate of an organism. With this in mind, why would you need to limit the length of time of the experiments we ran today?

3. You are designing an experiment to examine the effect running speed has on the respiration rate of cross‐ country runners. State null and alternative hypotheses for this experiment:

Vocabulary