Biology 002 Fall Quarter 2010

Lab 5 page 1

LABORATORY 5: CELLULAR METABOLISM

A. INTRODUCTION All living organisms must continuously expend energy to maintain themselves and to carry out life activities. The rate of energy utilization is known as the metabolic rate. The metabolic rate of any organism varies within wide limits, but is always greater than zero. Heterotrophic organisms utilize chemical energy to drive energy-requiring life processes. This is the energy that is liberated when a molecule is changed to another form which contains less energy. For example, oxidation of glucose to form carbon dioxide and water liberates energy as shown below. Glucose C6H12O6 180 grams + + + Oxygen 6 O2 192 grams Carbon Dioxide 6 CO2 264 grams + + Water 6 H2O + + + + Energy Energy Energy Energy

+ 108 grams

372 grams

372 grams

The chemical energy contained in nutrients such as sugars is derived from the radiant energy of the sun and is converted to chemical form by green plants through the process of photosynthesis. These molecules (e.g., sugars) provide a relatively stable form in which energy can be stored and transported. However, before this energy can be used, it must be converted to another chemical form; a complex organic molecule known as adenosine triphosphate (ATP). When the terminal phosphate is split off ATP, considerable energy is made available for cellular work, and the resultant molecule is known as adenosine diphosphate (ADP). ADP is then reconverted to ATP with energy derived from sugars or other nutrients. The flow of energy from the solar radiation captured by plants (via photosynthesis) to its use by both plants and animals is shown diagrammatically to the right. This energy drives many fundamental life processes such as movement, growth, and reproduction
RESPIRATION (plants and animals) ATP (chemical potential energy)

O2 + sugar

CO2 + H20

The release of energy from food Light energy molecules occurs by two, multi-step Photosynthesis biochemical pathways. The first of these is (plants and plant-like protists) called fermentation. Fermentation usually occurs in the absence of oxygen and is thus referred to as being anaerobic. The other biochemical mechanism of energy release is called cellular respiration. Cellular respiration is referred to as being aerobic because it can only occur in the presence of oxygen. In todays exercise we will measure metabolic rate in both an anaerobic and an aerobic organism by measuring the amount of gasses either produced or consumed by these organisms. The tool that we will use for these measurements is called a manometer which is designed to monitor changes in gas volume within a closed system.

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B. FERMENTATION Following is a balanced summary equation for the multi-step process of fermentation: C6H12O6 + 2 Pi + 2 ADP Glucose 2 CH3CH2OH + 2 CO2 + 2 ATP + heat. Ethanol

This equation shows that for every molecule of glucose consumed: two moles each of ethanol, carbon dioxide, and ATP are produced as well as a small quantity of heat. In todays lab you will begin by measuring the rate of CO2 production by yeasts undergoing alcoholic fermentation. This rate will then be related to the rate of ATP production which is a reflection of the metabolic rate of the yeasts. This part of todays lab will address the following questions: Question #1B: How will increasing the number of yeast cells influence the metabolic rate of the yeast suspensions? Question #2B: How will substituting galactose for glucose influence the metabolic rate of the yeast suspensions
should have the opportunity to set-up at least one experiment).

Procedure: All experiments in today's exercise will be done in groups of four (each group member All students will be responsible for all data collected by the group. Preparation of yeast samples. 1. Set up the three fermentation samples in test tubes according to the values specified in the following table: Tube 1 2 3 Label glu - 0.3 glu - 0.6 gal - 0.6 Suspending solution: volume, %age, and type 6 ml - 20% - glucose 6 ml - 20% - glucose 6 ml - 20% - galactose Weight Yeast (grams) 0.3 0.6 0.6

2. Seal each of the tubes with PARAFILM and set them aside to activate. (During this time the yeast cells hydrate and the cells metabolic machinery becomes functional). During this period you should swirl the cells as demonstrated by your TA to speed the activation process. Be careful to avoid placing the tubes in an excessively cool spot such as beneath an air conditioning duct; this would slow the activation process. 3. During activation, set up a ring-stand, manometer, and the connecting tubing as shown in Figure 1. Use an empty tube on the ring-stand to get it set up correctly. The manometer loop contains liquid that will move as the total gas volume within the tube changes. Measurement of CO2 evolution by yeast. The following steps will be carried out separately for each of the 3 samples/tubes listed above. You are now ready to begin measuring gas production in your samples. What you need to do at this point is determine whether or not your samples are ready to measure. Measurement can proceed when the sugar/yeast cell mixture begins to bubble and the PARAFILM bulges upward.

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1. Unclamp the empty set-up tube (or the measured sample tube) from the ring-stand and remove the syringe from the 3-way valve if there is one present. 2. Turn the valve arm toward the manometer loop (Fig. 1) and insert rubber stopper attached to the manometer into the sample tube containing the most active sugar-yeast sample. Clamp the tube onto the ring stand. Do not move anything (except the valve arm) until you are finished measuring the sample! Do not touch the sample tube because this may raise the temperature the manometer will respond to even small temperature changes. 3. When your are ready to begin your trial, record the level of liquid in the manometer and use a syringe to remove 1.5 ml of air from the yeast test tube via the 3-way valve. Start the timer. 4. Quickly turn the valve arm towards the syringe (this will cause the fluid level in the manometer to be displaced) and record new level of the liquid in manometer. 5. As CO2 is produced, the gas volume in the sample tube and manometer tubing will increase and return the liquid to its starting level. At this point, turn the valve arm towards the loop and record the time it took the liquid to return to its original level. This represents the time it took the yeast cells to produce 1.5 ml CO2!! 6. Repeat this measurement twice, by removing 1.5 ml air for each repetition as you did above. Average the three time intervals, to determine time required to produce 1.5 ml CO2. 7. Uncouple the yeast sample tube, and place it in the hot water bath on the side bench for 15 minutes while you proceed with the next experiment. At the end of 15 minutes, remove the tube, and place in a beaker of fresh tap water to cool to room temperature. When cool, close the tube with PARAFILM and set aside. 8. Repeat steps 1 -7 with the next tube to be measured. 9. After completing your measurements on the room temperature tubes, proceed to measure the heat-treated (control) tubes. Make sure that these tubes have cooled to room temperature before measurement.
Flow Path

Valve arm

Flow Path

Flow Path

Valve arm

Syringe

3 - way Valve, detail shown above. Manometer Fluid Levels Manometer Loop Sample Tube Manometer Base Figure 1. The manometer set-up with detail showing the paths of air flow with different positions of valve arm. Ring Stand

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C. CELLULAR RESPIRATION Cellular respiration is the main process of ATP production used by plants, animals and many microorganisms. The use of oxygen in the conversion of sugars to CO2 and water liberates much more of the energy stored in the sugar than does fermentation. Thereby, about 18 times as much ATP is generated than would be from fermentation of the same amount of sugar. The overall reaction is shown below. C6H12O6 + 6 O2 + 36 ADP* + 36Pi* 6 CO2 + 6 H2O + 36 ATP* + heat *As reported in your text, this value can be as high as 38 under ideal conditions.
Measurement of the rate of oxygen consumption of an aerobic organism provides a useful measure of its rate of energy production that also can be related to the rate of ATP production. In this exercise you will measure the consumption of oxygen by some corn seedlings. Again, we will use the manometer. The data collected here will be used to address the following question: Question #1C: In terms of the mass specific rates you calculated (g ATP / gram of tissue x hour), did the yeast suspensions or the corn seedlings have a higher metabolic rate?

Procedure: 1. Obtain and weigh a 17 x 150 mm test tube and record the weight. Tilt the tube on its side and add about 16 corn seedlings (your TA will tell you how many) with radicles (the big white things that stick out of the corn kernel). Be careful not to lump them at the bottom of the tube. Instead, they should be spread out in a single layer along the lower 2/3's of the tube. This arrangement is necessary to allow all of the seeds to have adequate gas exchange with the air inside the tube. 2. Once the seeds are in the tube, weigh the tube plus the seeds. The weight of the seeds can then be calculated and recorded. 3. Place a small wad of cotton over the corn in the tube and add about a 1/2 of Ascarite Drierite pellets over the cotton. The pellets will remove the CO2 and H2O vapor from the tube as it is given off by the respiring seeds. Note: (The Ascarite contains NaOH; therefore, do not allow
the brown ascarite pellets to touch your skin!!)

4. With the syringe removed, turn the arm of the 3-way valve on the manometer towards the manometer and connect the tube to the manometer, as in the previous exercise. You should tilt the tube in the ring stand to avoid having the seeds settle to the bottom of the tube. Do not touch the sample tube because this may raise the temperature the manometer will respond to even small temperature changes. 5. To begin the experiment, note and the level of the liquid in the manometer. Next, fill the syringe with 1.5 ml of air. Replace the syringe into the 3-way valve and inject the 1.5 ml air into the seed reaction tube. Displace the fluid level in the manometer by turning the valve arm towards the syringe. Record the time and note the new level of the fluid in the manometer. 6. At this point, you should calculate the difference, in units, between the level of the manometer fluid before and after the injection of 1.5 ml of air. Once you have this value, you can determine the volume (in ml) contained by one length ml 1.5 ml unit of the tubing on your manometer. In other words: = # of units moved manometer unit

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7. Allow the seeds to respire for 20-25 minutes. At the end of this period, record the elapsed time and the new manometer level. Determine how many units the manometer fluid moved during that time. If you multiply the number of units moved by the number of ml/unit you will arrive at the volume of O2 consumed in the time interval that you used. 8. Carefully remove the ascarite/drierite material, and place tube in the hot water bath for 15 minutes. 9. Cool tube with the seeds, and repeat steps 2-7 to examine oxygen consumption in this control preparation. Before beginning, make sure tube is at room temperature. Limit your readings to 5 minutes. D. DATA ANALYSIS: As mentioned previously, metabolic rate is defined as the rate at which an organism uses energy. A useful and fairly accurate expression of metabolic rate is the rate at which an organism produces ATP. The connection between the synthesis of ATP the use of energy is simple: The immediate energy source for cells is ATP which is produced from food. Cells cannot store large quantities of ATP, so, in order for cells to convert the energy in food to ATP, they must be using ATP (and thus making more ADP available for further ATP synthesis). So, how do we connect our measurements of the rate CO2 production by yeast cells or the rate of O2 consumption by corn seedlings to their respective rates of ATP production? The basis for converting these measurements lies in the summary equations that exist for these metabolic pathways: Fermentation: 1 C6H1206 (glucose) + 2 ADP + 2 Pi 2 CH3CH2OH (ethanol) + 2 CO2 + 2 ATP + heat Cellular respiration: 1 C6H1206 + 6 O2 + 36 ADP + 36 Pi 6 CO2 + 6 H2O + 36 ATP + heat The important part of these equations is that the coefficients of the different components represent the proportional quantities of the different components to each other. In fermentation, for example, two molecules of ethanol are produced for each molecule of glucose consumed. It is more useful to make these coefficients relate to a specific number of molecules rather than individual molecules. This specific number is 6.02 x 1023 molecules. This many molecules of any chemical is known as a mole. The concept of a mole is useful for two reasons: 1. It allows us to know the actual weight (in a measurable range) of a mole of molecules of any factor in the above equations. For example, a mole of ATP weighs 589 grams and a mole of glucose weighs 180 grams. 2. For gasses, it is also known that a mole of any gaseous molecule occupies a fixed volume (under the same conditions) which is 22.4 liters (or 22,400 milliliters). Now we have all the tools needed for converting the rate of gas production or consumption to the rate of ATP synthesis. Our goal is to convert the rates of gas production or consumption that we have measured in lab to the rate of ATP production (expressed as grams ATP/hour).
Please see next page for calculation guide.

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Calculations: 1. Determine how many moles of gas were produced or consumed: (remember that 1 mole of gas occupies 22.4 liters or 22,400 milliliters.) ml gas produced or consumed (from your data) x 1 mole gas 22,400 ml = moles gas produced or consumed
(use this value for part 2)

2. Calculate the rate of gas production/consumption;

(ml gas / hour): = moles gas produced or consumed hr

(use this value for part 3)

moles gas produced or consumed (from step 1) x 3600 sec total time it took to move (sec) (from your data) 1 hr 3. Calculate rate of ATP production in moles. FERMENTATION
Remember that for every one mole of CO2 produced, there is one mole of ATP produced. Therefore: Rate of CO2 production (from step 2)

CELLULAR RESPIRATION
Remember that for every one mole of O2 consumed, there are 6 moles of ATP produced. Therefore: Rate of O2 consumption (from step 2) x 6 = Rate of ATP prod. in moles.
(use this value for part 4)

= Rate of ATP
prod. in moles.
(use this value for part 4)

4. Calculate the absolute rate of ATP production in grams. Remember that a mole of ATP weighs 589 grams. moles ATP produced (from step 3) x 589 grams = grams ATP hour mole hour 5. For comparisons between the yeast trials, the absolute rate of ATP production calculated in box #4 is sufficient. For comparisons between the yeast trials and the corn seedling results a mass specific value is needed. Here the rate of ATP production (grams ATP/hour) must be expressed relative to the respective weights of the organisms measured. In other words, the mass specific rate is obtained by dividing the absolute rate of ATP production by the weight of the organisms giving: _____grams ATP __ gram organism x hour.

Please see next page for date collection sheet.

Biology 002 Fall Quarter 2010

Lab 5 page 7

Table for Collection of Raw Data

Time for manometer column to return to original level (sec.). Trial # 1. 2. (if practical) 3. (if practical) heat treated Cellular Respiration - Seeds: Weight of seeds: ___________________ g. Duration of measurement: ___________ sec. Manometer Calibration: ___________ ml/unit # of units moved (live seeds): ____________ # of units moved (heat treated): __________ 0.3 g yeast & 20% Glu 0.6 g yeast & 20% Glu 0.6 g yeast & 20% Gal