Respiration Lab Biology I

Cellular respiration and fermentation are metabolic processes that organisms use to synthesize ATP using energy from the chemical bonds of organic molecules (food). All organisms need ATP to power endergonic metabolic reactions, move substances across membranes, power cell movement, and for myriad other processes. Many organisms use aerobic respiration, in which O2 serves as a final electron acceptor for the electron transport chain. Aerobic respiration can be subdivided into four metabolic pathways: 1) Glucose is partially oxidized during glycolysis in the cytoplasm 2) The product of glycolysis, pyruvate, is oxidized to produce acetyl-CoA in the matrix of mitochondria 3) Acetyl-CoA is oxidized to CO2 in the citric acid cycle in the matrix of mitochondria 4) Electron transport oxidizes NADH to set up a H+ gradient and chemiosmosis (by ATP Synthase) uses potential energy from that H+ gradient to power ATP synthesis at the inner mitochondrial membrane The ATP yield from aerobic respiration is approximately 32 ATP synthesized per molecule of glucose entering glycolysis (exact ATP yield varies depending on how much of the H+ gradient is used to power other work in the mitochondria). A summary of aerobic respiration is given below: Aerobic respiration:

C 6 H 12 O6 6O2 6CO2 6 H 2 O
(glucose)

Yield: ~32 ATP/glucose

Some bacteria that respire can use alternative electron acceptors (such as elemental sulfur) for the electron transport chain. Thus, they can take advantage of the high ATP yield of respiration in the absence of O2. Other organisms (and even human cells, such as muscle cells) are able to produce ATP in the absence of O2 by fermentation. During fermentation, NADH generated during glycolysis reduces (donates electrons to) pyruvate or some other organic molecule, so that NAD+ is made available again. This allows glycolysis to continue in the absence of events that occur in the mitochondria. The ATP yield from glycolysis alone is 2 ATP synthesized per molecule of glucose entering glycolysis. Although the ATP yield per glucose is much lower when fermentation is used than when respiration is used, fermentation involves many fewer steps than respiration, and so is much faster. Thus, organisms (cells) are able to generate enough ATP per unit time to meet their energy needs using fermentation. However, organisms (cells) that are capable of both aerobic respiration and fermentation will preferentially use aerobic respiration when O2 is available because the ATP yield per glucose is higher. Summary equations for two varieties of fermentation are given below:

Ethanol fermentation: C 6 H 12 O6 2C 2 H 5 OH 2CO2 (glucose) (ethanol)

Yield: 2 ATP/glucose (Yeast, plants, some microbes)

Lactic acid fermentation: C 6 H 12 O6 CH 3 CHOHCOOH (glucose) (lactic acid)

Yield: 2 ATP/glucose (Animal muscle, some microbes)

In Part I of this lab you will learn how to use a CO2 sensor to measure production of CO2 by organisms respiring aerobically or by fermentation. Then in Part II you will work with your team to design an investigation to discover something about respiration. Part I: using an O2 or CO2 probe to measure respiration or fermentation: Materials per team 25 germinating seeds (wet) 25 non-germinating seeds (dry) 1 respiration chamber (bottle or 250-ml glass Erlenmeyer flask) 1 LabQuest 1 CO2 gas sensor Paper towel 1. Set up the LabQuest and sensor. a. If the LabQuest battery is not charged, attach the LabQuest to a power cord and plug it in. b. Plug the CO2 sensor into channel 1. c. Note the CO2 reading in air and write this value down before beginning to measure increased CO2 levels due to respiration 2. Dry any water clinging to your seeds with a paper towel. Place the germinating seeds into the respiration chamber. Record the type of seed you are using and the number of seeds you used in the Lab Notes, question 3. Insert the shaft of the CO2 sensor into the flask after reading this entire step. There is a rubber stopper split down the side surrounding the sensor shaft. Squeeze the edges of the rubber stopper together with thumb and fingers as you push down on the stopper (not the sensor) to seal the gap so that no air can enter or exit the flask. You must seal this gap in order to get reliable data. 4. Wait 30 seconds so the sensor can stabilize after moving it. Then choose the green arrow button to begin data collection. The CO2 sensor will collect data for 10 minutes. (You will notice that data values oscillate up and down every four seconds. This doesnt mean that CO2 values are jumping around, just that the sensor is measuring values in within a range of error and you are seeing the high and low values for the range of potential values.) 5. Determine the rate of respiration for the germinating seeds: a. Drag the pointer from the point where the data value trend begins to increase to the end of the data, or when it reaches the maximum value and flatlines. b. Perform a linear regression to determine the respiration rate: i. Click the Analyze menu ii. Choose Curve fit and then click the box next to CO2 iii. On the Choose fit menu, choose Linear and then the OK button c. A box will appear on the right with the formula for a best fit line and values for m and b. The slope of the line, m, is the rate of respiration for the seeds. Record this value in the Lab Notes question 3. Be sure to include units (what are the units? it is the unit on the y-axis over the unit on the x-axis).

6. Remove the CO2 sensor from the flask. Use a few pieces of paper to form a stiff fan and fan air from the room into the holes in the sensor shaft to blow away CO2 molecules clinging to the sensor openings. (Dont blow on the sensor why not?) Continue fanning until the CO2 value returns to that measured in part c. of step 1. This will require a lot of vigorous fanning. Really, I mean a whole lot of fanning! Dont stop until the CO2 level returns to its original value. 7. To convince yourself that the increase in CO2 was due to respiration, repeat steps 3 6 with dry seeds that are not respiring. Record data from this trial in the Lab Notes, question 4. 8. Note any modifications that you had to make to measure respiration rates of respiring and non-respiring seeds, or comments about techniques in Lab Notes question 2. 9. In the interest of time, you may want to wait to produce a visual aid (Lab Notes question 5) until after you have completed Part II. Part II: Design an experiment to test some hypothesis about respiration Here are some questions to help you think about what hypothesis you might form and test around the process of respiration: 1) 2) 3) 4) 5) Why might different organisms have different respiration rates? Why might respiration rates differ for the same species at different times? How could you normalize respiration rates among different species? What kinds of abiotic (non-living) factors affect respiration or fermentation rates? What kinds of nutrients can organisms use for respiration or fermentation?

Your team will design a controlled or comparative experiment to test some hypothesis about respiration. Think of a question, hypothesis, and design the experiment, using the questions in the Lab Notes, Part II to guide your planning. You may do more than one simple experiment, but try to get things done during lab class. If you do controlled experiments, remember to control for all except the independent variable. Even if you do a comparative experiment, try to control for variables as much as possible. Here is a list of materials that you can use for your investigation. You will not use all of these materials, because this list includes materials for several very different questions and experiments. If you want something that isnt on this list, please ask, as it is likely we have it. Organisms that respire: (you can bring your own they must be able to live in air or a small volume of liquid and fit inside a 250-ml flask, such as snails, insects, goldfish, minnows or small crayfish from Lake Tahoe, small plants) One or more invertebrate animals Various types of respiring seeds Small plants (if you use a green plant, cover the respiration chamber with foil to stop photosynthesis) Yeast (a single-celled fungus that respires when O2 is available and uses alcohol fermentation when it is not) if you use yeast, you can dissolve active dry yeast in a small amount of water to make a culture (about 2 tsp per cup of water) like you do to make bread or pizza dough. Put a small amount of the culture in the flask and insert the CO2 probe, being careful that the probe is in the air above the liquid, and not submerged.

Reagents dH2O 5% Glucose (a monosaccharide that enters glycolysis directly) 5% Fructose (a monosaccharide plant sugar) 5% Lactose (a disaccharide milk sugar) 5% Sucrose (a disaccharide table sugar) Dry reagent sugars (in case you want to make solutions of different concentrations) 0.1 M NaF (NaF is an inhibitor of one enzyme of glycolysis) 0.1 M MgSO4 (Mg++ is a cofactor needed by one enzyme of glycolysis) pH buffers at pH 4, pH 7, and pH 10 Supplies and equipment: 250-ml Erlenmeyer flasks for respiration chambers 600-ml beakers in which to set up water baths of various temperatures Thermometers Ice Microwave to heat water Graduated cylinders Serological pipettes and transfer pipettes to measure amounts of liquid reagents Balances, weigh boats, and spatulas to weigh reagents or organisms Paper towels Other supplies readily available in the biology lab (ask for supplies that we used before or you think we should have) Clean-up: - Wipe off the CO2 sensor with a damp paper towel and then dry it. Return it to its box. - Return all Vernier equipment to the bench where you got it. - Return invertebrate organisms to their original container if they are alive. If they are dead, put them in the trash. - Return respiring seeds to the container where you got them. - Return any unused reagent stock bottles to the bench where you got them. Make sure the lids are tightened. - Wipe up any spills near balances or shared reagent bottles. - Collect any solutions with NaF in the NaF waste bottle in the waste beaker in the hood. - All other experimental solutions can be flushed down the drain with a few hundred ml of tap water. - Place used glassware (including graduated pipettes) in the basin of detergent water near the sink. - Throw away plastic transfer pipettes, gloves, and paper towels. - Once you have cleared your work space, spray it with cleanser (green liquid in spray bottles) and wipe it with damp paper towels to clean it.

Respiration Lab Notes: Write answers to these questions on your own paper or in a file on your computer. You can turn in one set of lab notes for your team with all of your names on it. Part I: 1. What kind of respiring seeds did you use to measure respiration rate? How many seeds did you use? 2. Did you have to make any adjustments from the printed procedure to measure the respiration rate of respiring seeds? If so, what exactly did you do differently? 3. What was the respiration rate for the respiring seeds? 4. What was the respiration rate for the dry (non-respiring) seeds? 5. Make some kind of visual aid (graph or chart) to compare the respiration rates of respiring and non-respiring seeds. You can do this by hand using a graph form from the back of your Inquiry Biology lab manual or use a software graphing tool, such as LoggerPro or MS Excel chart.

Part II: 6. As a team, brainstorm at least three questions that you could ask about respiration using the CO2 sensor to measure respiration rates. Try to think of questions that will have open-ended answers, rather than yes or no answers. 7. As a team, select one question. Brainstorm hypotheses that you could test to help answer this question. 8. Select one (or more than one closely-related) hypothesis and design an experiment to test it. a. What variable will you manipulate to test your hypothesis (or hypotheses)? This is your independent variable. How will you manipulate this variable? b. If you design a controlled experiment (for example, exploring how some factor affects respiration rate of a particular organism) what variables will you control? How will you control them? c. If you design a comparative experiment (for example, comparing respiration rates of different organisms) how will you normalize for differences among the organisms, such as differences in size? d. Will you measure anything or make any qualitative observations in addition to measuring respiration rate? If so, what will you measure or observe? e. Plan a data table or form that you can use to record data (measurements and/or observations) in an organized way. 9. What materials will you need to set up your experiment? 10. How will you carry out your experiment? Make sure that everyone in your team has a role. 11. Predict results that would support your hypothesis. 12. Predict results that would cause you to reject your hypothesis. 5

13. Once you have designed your experiment, talk through it with Suzanne to get feedback on your experimental design. Did you modify your design with this feedback? If so, how? 14. Carry out your experiment, recording qualitative observations and quantitative measurements in the lab notes. 15. Design some kind of visual aid (graphs or charts) to present your results in a way that helps you to form conclusions. (You can do a rough sketch in lab and polish this later for the lab notes.) 16. Do your results lead you to reject your hypothesis, or do your results support your hypothesis? Explain. 17. If you were to repeat this experiment, what might you do differently? 18. What additional questions or hypotheses are suggested by your results and conclusions? 19. Search Prim Library resources or the internet for information about respiration in your test organism(s) to see if you can find sources related to your results or conclusions. Briefly describe how information from the source(s) is related to your research. Cite any sources that you discuss, using the CSE citation-sequence system (number system) as described here: http://bcs.bedfordstmartins.com/resdoc5e/RES5e_ch11_o.html. 20. The Lab Notes are due one week from today (October 24).