Enhancing bioenergetics curricula for laboratory courses in plant and animal physiology and ecology.

 

This site is dedicated to describing a National Science Foundation (NSF) funded project (to Drs. J.S. Roden and C.T. Oswald at Southern Oregon University's, Department of Biology) to enhance bioenergetics education at the undergraduate level. The mission of the Course Curricular and Laboratory Improvement (CCLI, award # 0311363). program at NSF is to improve the quality of science, technology, engineering, and mathematics education for all undergraduate students. The main focus of this project is to improve laboratory experiences in bioenergetics for introductory plant and animal physiology courses as well as upper division plant and animal ecophysiology courses. Below we will describe the equipment and techniques utilized as well as the results of the first 2 years of implementation. We hope that through this web site other physiologists can benefit from our experiences.

INTRODUCTION
The biologists of the future need to be broadly trained in bioenergetics in order to meet the many ecological, environmental and economic challenges our world will face. Bioenergetics is an aspect of biological systems that touches nearly all areas of living and even nonliving components of the biosphere. Laboratory exercises that help students learn how organisms capture and utilize energy are a critical component to any physiology course. Since bioenergetics is a key linkage between organisms and their environment, students interested in ecology and environmental studies will also benefit from curricula that expose them to modern techniques and allow them to address questions relating to adaptations, global change, ecological relationships, biogeochemical cycles and metabolic diversity.

Exercises where students do experiments in a laboratory environment aid student understanding of difficulty concepts. Opportunities for "active learning" in bioenergetics are often hindered by the lack of suitable equipment. Many undergraduates view research grade instruments as a "black box," too complicated to understand. In addition, research grade instruments are too expensive to have multiple work stations so few students actually get hands-on experience. New equipment has been developed that utilizes a similar platform and component parts that, at a relatively low cost, allows students to perform a variety of different classroom experiments in plant and animal bioenergetics. Our goal is to test whether student learning of plant and animal bioenergetics is enhanced through the use of low cost instrumentation and newly developed inquiry based labs that stimulate active learning.

 

METHODS and MATERIALS

New lab exercises in bioenergetics were developed for the 2003/2004 plant and animal physiology courses at Southern Oregon University. Most exercises utilized the same measurement platform (Qubit Systems Inc., Kingston, Ontario, Canada) and many of the same component parts (the same pump, flow meter, cuvettes, infra-red gas analyzers, oxygen sensors, humidity sensors etc.) making them quite cost effective to set up multiple work stations (6 per lab). One exercise also utilized a bomb calorimeter (model 1281, Parr Instrument Co., Moline, Ill. USA).

Three new labs were developed for the introductory plant physiology course (Bi 331). The first new lab (PDF file of the lab exercise is available here) involved the measurement of O2 evolution and the action spectra of photosynthesis. Students exposed fully induced leaves (most often pole bean, but other species also worked well) to red, green and blue light of the same intensity and measured the slope of the increase in O2 concentration inside a closed cuvette. This experiment was done at the same time as TLC extraction and identification of chloroplast pigments, nicely clarifying the distinction between action spectra and absorption spectra. The second lab (PDF file of the lab exercise is available here) involved estimation of FV/FM, relative quantum yield of photochemistry (FPSII) and electron transport rates using chlorophyll fluorescence. The students generated a light response curve of electron transport to study both the linear and saturable features of the light reactions. In the final exercise (PDF file of the lab exercise is available here), students determine the response of carbon assimilation to CO2 concentration using gas exchange techniques. A CO2 response curve is a powerful tool to understand the Calvin cycle, photorespiration, Rubisco kinetics, stomatal conductance dynamics and issues (global change) related to increased atmospheric CO2 concentrations.

Three new labs were also developed for the animal physiology class including (1) effects of diet on digestive efficiency in mice using bomb calorimetry (2) metabolic rates and respiratory quotients in mice and frogs using oxygen and carbon dioxide exchange and (3) metabolic responses to temperature in frogs and mice (conductance and Q10) by monitoring oxygen consumption.

In 2002/2003 surveys and examinations on the principles of and methods used in bioenergetics were conducted at the end of the term for students in plant and animal physiology classes who had not gone through the new exercises (the "PRE" group). The same bioenergetics surveys and examinations were given to the 2003/2004 and 2004/2005 classes after having used the new instrumentation and gone through the new exercises (the "POST" group). Comparisons between PRE and POST groups were used to assess the effectiveness of the new exercises and equipment to enhance student learning in bioenergetics principles, procedures and calculations.

 

RESULTS

Plant Physiology laboratory

The result for 2004 and 2005 students were pooled (n = 25, PRE group n = 10) as there were only a few differences in the responses between the two classes. Students exposed to the new instrumentation and exercises (the POST group) were more confident in their ability to answer plant bioenergetics questions (Figure 1). The increase was an average of 1.7 units higher (on a 1-5 scale, > 40% increase, p < 0.001). Two questions were added as a test of the survey. Question #1 (* in Fig. 1) was a question that all students, PRE and POST, should have known, and clearly they were all confident. Question #4 (** in Fig. 1) is one that none of the students should have known since it was not covered in either the PRE or POST classes. Although, many students felt that they could give an educated guess for question 4, there was no significant difference between PRE and POST groups.

Figure 1. Student self-assessment of ability to answer plant bioenergetics questions. Values are mean (+ se). See below for questions.

Have the laboratory experiences prepared you to answer these kinds of questions? The rating scale is from 1 = I have no clue how to even begin to answer this question, to 3 = I could provide an educated guess, but I am not overly confident to 5 = I am confident that I know the answer.

1. Why is it important to calculate leaf area in order to measure photosynthetic rates using gas exchange systems?
2. Why is it important to measure flow rates in order to calculate photosynthetic rates when using a gas exchange apparatus?
3. What do we mean by photosynthetic capacity and what experimental conditions would you use to estimate it?
4. Why does leaf transpiration rate need to be calculated in order to estimate internal CO2 concentration?
5. Describe how one could separate photosynthetic O2 flux from respiratory O2 flux in the same leaf.
6. What is meant by the term carboxylation efficiency and how would you go about calculating it?
7. What procedure would you use to estimate the action spectra of photosynthesis?
8. What factors are limiting photosynthetic carbon uptake when the CO2 saturation point is reached?
9. What is meant by the term chlorophyll fluorescence and why can we use it to probe the light reactions of photosynthesis?
10. Why does the fluorescence parameter Fv/Fm directly relate to photosynthetic quantum yields?
11. How can chlorophyll fluorescence be used to calculate electron transport rates?
12. Why is chlorophyll fluorescence a powerful tool for probing plant responses to stressful conditions?

When asked to actually answer multiple choice questions, the POST group did markedly better than the PRE group (Figure 2). Although we discussed chlorophyll fluorescence in lecture for the PRE group, it is clear that hands-on experience with fluorometers is essential for students to grasp the difficult concepts and methods involved. Again, a question was added that neither group should have known to test the exam (* in Fig. 2) and although the POST group did better that PRE group their scores were still not much better than guessing.

The multiple choice exam had an option for students to profess ignorance ("I don't know the answer") rather than simply guess (Figure 3). Many more students in the PRE group professed ignorance than in the POST group. Only in the test assessment question (* in Fig. 3) did the POST group show a substantial number of students professing ignorance.

Figure 2. Mean test scores for multiple choice questions in plant energetics.

Figure 3. Mean % of students who professed ignorance regarding multiple choice questions.

An attitudinal survey demonstrated (Figure 4) that students felt new exercises and equipment were beneficial to their understanding of plant bioenergetics. All together, the increase in ratings was an average of 2 units (on a 1-5 scale, 50% increase, p < 0.001).

Figure 4. Attitudinal survey of bioenergetics curricula. Values are mean (+ se).

Rate each item from 1 = strongly disagree to 5 = strongly agree.
Gas exchange
1. I feel I have a good understanding of the principles of gas exchange (photosynthetic CO2 and O2 exchange).
2. I feel current lab experiences were valuable for improving my understanding of the principles of gas exchange.
3. I feel I am adept at calculations involved in gas exchange.
4. I feel current lab experiences were helpful in improving my ability to do calculations involved in gas exchange techniques.
Chlorophyll fluorescence
5. I feel I have a good understanding of the principles of chlorophyll fluorescence.
6. I feel current lab experiences were valuable for improving my understanding of the principles of chlorophyll fluorescence.
7. I feel I am adept at calculations involved in chlorophyll fluorescence including electron transport rates.
8. I feel current lab experiences were helpful in improving my ability to do calculations involved in chlorophyll fluorescence.
General
9. Overall, I feel I have a good understanding of the methods currently utilized in studies of plant bioenergetics.
10. Overall I feel I have a good understanding of the biology of bioenergetics (photosynthesis, respiration, e- transport).
11. I feel that the lab techniques utilized were up-to-date and enhanced my understanding of plant bioenergetics.

 

Animal Physiology laboratory

In animal physiology labs both multiple choice and essay questions were utilized to test student understanding of the theory, application, procedures and calculations associated with bomb calorimetry and open-flow respirometry with respect to metabolism, digestion efficiency, energy balance and endothermy /ectothermy. Students from the POST group scored significantly better on both essay tests and multiple choice test (p < 0.001). Multiple choice scores increased from 35 (% correct) to 71% and essay scores increased from 21 to 55% for students from 2003/2004 (POST) as compared to students from the previous year (PRE) who did not have the benefit of the new equipment and exercises (Figure 5).

Figure 5. Test scores of animal physiology classes that did (POST) and did not (PRE) use bioenergetics instrumentation. Tests were given the last day of the term without advance notice. Students received bonus points commensurate with their test scores. Values are mean (+ sd). (PDF file of test questions)

Students' self-assessments in animal physiology showed increased confidence in their understanding of bioenergetics compared to the previous year (Figure 6, p < 0.001 for first 4 categories, p < 0.005 for the last). Among students who had used the new equipment, 77% to 88% agreed or strongly agreed that they had a good understanding of the principles and calculations involved in calorimetry and respirometry. Among those who had not used the equipment, only 5% to 26% agreed with those statements. Thus, their self-assessment is consistent with knowledge tests.

 

Figure 6. Self-assessment of understanding of animal energetics in physiology classes that did (POST) and did not (PRE) use bioenergetics instrumentation. The surveys were conducted the last day of the term without advance notice. A score of 5 indicated strong agreement with the statement that the student had a good understanding of each topic listed below. A score of 0 indicated strong disagreement.

SAMPLE SURVEY QUESTIONS ANIMAL ENERGETICS
Rate each item from 1 = strongly disagree to 5 = strongly agree.

Bomb calorimetry
1. I feel I have a good understanding of the principles of bomb calorimetry.
2. I feel lab experience was (or would have been, prior to equipment purchase) valuable in improving my understanding of the principles of bomb calorimetry.
3. I feel I am adept at calculations involved in bomb calorimetry.
4. I feel lab experience was (or would have been, prior to equipment purchase) valuable in improving my ability to do calculations involved in bomb calorimetry.
Respirometry
5. I feel I have a good understanding of the principles of measuring metabolic rate by carbon dioxide and oxygen exchange (respirometry).
6. I feel lab experience was (or would have been, prior to equipment purchase) valuable in improving my understanding of the principles of respirometry.
7. I feel I am adept at the calculations involved in respirometry.
8. I feel lab experience was (or would have been, prior to equipment purchase) valuable in improving my ability to do respirometry calculations.
General
9. Overall, I feel I have a good understanding of the methods in animal energetics.
10. Overall, I feel I have a good understanding of biology of animal energetics.
11. I feel the lab exercises in animal energetics contributed to my understanding of the biology of animal energetics.
Equipment use
12. The bomb calorimeter was fairly easy to learn how to use.
13. I enjoyed using the calorimeter in lab.
14. The respirometry system was fairly easy to learn how to use.
15. I enjoyed using the respirometry system in lab.

 

CONCLUSIONS

Student understanding of bioenergetics with regard to concepts and applications, as well as the procedures and calculations used to study bioenergetics, was enhanced when inquiry-based exercises that promote active learning were incorporated into undergraduate plant and animal physiology laboratories.

The low-cost, bioenergetics equipment utilized in this study performed quite satisfactorily and allowed students to work on their own instrument in small groups rather than crowd around a single research grade instrument. The instruments produced reasonable results and were tractable to undergraduates, which allowed students to gain knowledge of the methods used in bioenergetics research.

 

Overall assessment and recommendations for more advanced courses.

COST
The Qubit systems are ideal for undergraduate labs especially in primarily teaching institutions although larger universities would find them useful as well. One principle benefit is the shared measurement platform and component parts. Since, at our university (SOU), animal and plant physiology labs share the same physical space, sharing component parts is not problematic as lab periods never coincide. The sharing of components allowed us to obtain multiple work stations (up to 6) for 6 different instruments. Thus we obtained equipment to set up 36 different instruments for about the cost of 2 research grade gas exchange systems! If animal and plant physiology labs coincide and sharing is impossible, a bit more money is required. However, within a lab like plant physiology, sharing components within the 3 different setups utilized in this project (O2 evolution, chlorophyll fluorescence and CO2 gas exchange) would require the purchase of only 6 of the shared components rather than 18.

PEDAGOGY

There are two main educational benefits that non-research grade instruments provide. The first is that they are tractable to students. The Qubit systems are not designed to be compact, but to be educational. For example, all the tubing and sensors for a gas exchange system are out on the bench so that student can follow the flow of gases from input to pump to flow meter to chamber to humidity sensor to drying column to IRGA. The reference values are obtained by manually connecting tubing to different ports and thus, after a few repetitions, students understand better the importance of the difference between reference and sample measurements. Research grade instruments are often the proverbial black box to undergraduates. You push a button and the instrument does all the work and spits out a number. I (JR) was fortunate enough to learn gas exchange techniques in Bob Pearcy's lab at UC Davis where most of the best equipment was home made. Building and fixing home-made gas exchange devises was invaluable to me when using, say a LiCor 6400, because I understood what was happening inside the black box. Students utilizing the Qubit systems get a similar benefit because of the open and less automated nature of the instrument.

The second principle educational benefit is that with reduced cost (see above), more stations can be purchased and thus a more hands-on experience can be provided for the students. Often labs have 18-20 students and since most faculty cannot afford much more than 1 or 2 research grade instruments, 10 or more students would have to crowd around one instrument to learn how to use it. With 6 stations groups of 2, 3 or at the most 4 work together on an instrument of their own, making for an active learning environment.