Inquiry Investigation: Factors Affecting Photosynthesis Background Photosynthesis fuels ecosystems and replenishes the Earth's atmosphere with oxygen. Like all enzyme-driven reactions, the rate of photosynthesis can be measured by either the disappearance of substrate, or the accumulation of products. The equation for photosynthesis is: 6CO 2 + 6H 2 O C 6 H 12 O 6 + 6O 2 What could you measure to determine the rate of photosynthesis? 1) the production of oxygen 2) The consumption of carbon dioxide The difficulty related to measuring the production of oxygen is compounded by the complementary process of aerobic respiration consuming oxygen as it is produced. Therefore, measuring oxygen production is equivalent to measuring net photosynthesis. A measurement of respiration in the same system allows one also to estimate the gross production. Figure 1 The cells of a leaf are arranged in layers. Learning Objectives To design and conduct an experiment to explore the effect of a factor that affects photosynthesis. To connect and apply concepts, including the relationship between cell structure and function, strategies for capture and stores of energy, and the diffusion of gases across membranes. Floating Leaf Disk Assay In this investigation, you will use a system that measures the accumulation of oxygen in the leaf. The leaf is composed of layers of cells. The spongy mesophyll layer is normally infused with gases, oxygen and carbon dioxide. Leaves (or disks cut from leaves) will normally float in water because of these gases. If you draw the gases out from the spaces, then the leaves will sink because they become more dense than water. If this leaf disk is placed in a solution with an alternate source of carbon dioxide (in the form of bicarbonate ions) then photosynthesis can occur in a sunken leaf disk. As photosynthesis proceeds, oxygen accumulates in the air spaces of the spongy mesophyll and the leaf becomes buoyant and floats. Oxygen and carbon dioxide are exchanged through openings in the leaf called stoma. Figure 2
Basic Procedure for Measuring the Rate of Photosynthesis Materials: baking soda, liquid soap, plastic syringes, leaves (spinach or ivy; something smooth and not too thick; avoid plants with hairy leaves), hole punch, cups or beakers, timer, light source 1. The bicarbonate serves as an alternate dissolved source of carbon dioxide for photosynthesis. Prepare 3 ml of a.2% solution. (This is not very much - it is only about 1/8 of a teaspoon of baking soda in 3 ml of water.) 2. Add 1 drop of dilute liquid soap to this solution. The soap wets the hydrophobic surface of the leaf, allowing the solution to be drawn into the leaf. It s difficult to quantify this since liquid soaps vary in concentration. Avoid suds. If your solution generates suds then dilute it with more bicarbonate solution. 3. Use a hole puncher to cut uniform leaf disks 1 for each trial. Take care to avoid major veins. 4. Place 1 leaf disks into the syringe and pull in a small volume of the bicarbonate and soap solution. Replace the plunger and push out most of the air, but do not crush your leaves. 5. Create a vacuum by covering the tip of the syringe with your finger. Draw back on the plunger. 6. Release the vacuum so that the solution will enter the disks. You will probably have to repeat this procedure 2-3 times in order to get the disks to sink. You may need to gently tap the syringe to dislodge discs from the sides. If you have difficulty getting your disks to sink after about 3 evacuations, it is usually because there is not enough soap in the solution. Add a few more drops of soap to the bicarbonate solution, and try again with new leaf disks. 7. Pour the disks and solution into a clear plastic cup. Add bicarbonate solution to a depth of about 3 centimeters. Use the same depth for each trial. Shallower depths work just as well. 8. Place under the light source and start the timer. At the end of each minute, record the number of floating disks. Then swirl the disks to dislodge any that are stuck against the sides of the cups. Continue until all of the disks are floating. Figure 3 Figure 4
Analyzing Data In this experiment, the disks floating are counted at the end of each time interval. The time required for 5% of the leaf disks to float is represented as Effective Time = ET 5. Repeated testing of this procedure has shown that the ET 5, or median is a reliable and repeatable point of reference, to make comparisons between experiments. The median will generally provide the best estimate of the central tendency of the data because, on occasion, a disk fails to rise or takes a very long time to do so. These data are from a demonstration investigation using grape ivy leaf disks. Table 1. Sample results Minutes Disks floating 1 2 3 4 5 6 7 1 8 1 9 1 1 1 11 4 12 7 13 8 14 1 Leaf Disks Floating 1 9 8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8 9 1 11 12 13 14 Time (min) Figure 5 By interpolating from the graph, the 5% floating point is about 11.5 minutes. Using the 5% point provides a greater degree of reliability and repeatability for this procedure. As Steucek, et. al. (1985) described this term is referred to as the ET 5. Therefore, the ET 5 for this experiment is 11.5 minutes. The problem with ET5 is that it goes down as the rate of photosynthesis goes up--it is an inverse relationship and creates the following type of graph (data from Steucek, et al. 1985.): 25 2 ET 5 (min) 15 1 5 2 4 6 8 1 12 14 Light Intensity (FT C) Figure 6. Plot of ET5 over a range of light intensities. As light intensity increases, ET5 decreases, indicating an increase in photosynthetic rate.
To correct for this representation of the data and present a graph that shows increasing rates of photosynthesis with a positive slope the ET 5 term can be modified by taking the inverse or 1/ET 5. This creates a graph like this (data from Steucek, et al. 1985.):.15 1/ET 5 (min 1 ).1.5 2 4 6 8 1 12 14 Light Intensity (FT C) Figure 7 Designing an Experiment Choose one factor to test. Consider the following: Environmental Variables light intensity light colour temperature bicarbonate concentration direction of incoming light ph of solution Plant or Leaf Variables leaf colour (amount of chlorophyll) leaf size stomata density stomata distribution light-starved leaves vs. leaves kept in bright light type of plant leaf age leaf variegation Assignment It is your task to design and carry out a controlled experiment that tests the effect of one factor, on net oxygen production. Factors to consider: What are my variables independent, dependent? How many different conditions (values) for my independent variable will I be testing? What factors need to be controlled? How exactly will I carry out my experiment? What should my data table look like? You will only be provided with the following:.2% sodium bicarbonate solution (if you want a higher concentration, you will have to prepare this yourself), hole punch, spinach leaves, dish soap, beakers or cups. You must supply everything else.
Evaluation You must submit a formal lab report with the following components: Abstract - Concisely summarize the entire experiment. Include the purpose, a brief overview of the methods used, and the conclusions obtained. It should be no more than a few sentences long. Introduction - Include the goal of your experiment, and all background information and scientific concepts on which your experiment is based. This includes the general technique you will be using to accomplish your goal. You must cite all information internally. Concepts should be clearly tied together. You should address the following in some order: o photosynthesis, and the factors that can affect its rate o the factor you have chosen to investigate, and its hypothesized effects o background information on the leaf disk assay o the purpose of your experiment Procedure - In a paragraph or two, briefly summarize the procedure you followed in the lab. Include only the steps that are relevant to the experiment (i.e., don't mention putting on goggles, cleaning up glassware, etc.). Do not include steps you used to analyze your data or observations. Remember that you want to provide enough information that your reader has a clear idea of how you carried out the experiment, but you also do not want to provide so much detail that it is overwhelming. Materials List the substances and equipment used during this lab experiment. These may not be exactly the same as the ones on the lab handout. For chemicals, include relevant chemical formulas and quantities. Results Re-copy your observation table(s). One sample plot of Floating Disks vs. Time. This is the plot you use to determine ET 5. You will have to construct one for each of your experimental conditions, but you need only include one in your report. A table showing the value of ET 5 and 1/ET 5 for each experimental condition. A figure that clearly compares 1/ET 5 for each of your conditions. Scatter plot For continuous (quantitative) variables, like temperature Bar graph For categorical (qualitative) variables, like leaf colour Be sure to include table headings (number and title) and figure captions (number and description) where necessary. Discussion - Write your Discussion in paragraph form. You will need to cite any information obtained by research. Address the following topics, in some order. In 2-3 sentences, summarize the steps you took to analyze your raw data/observations (i.e., describe any calculations you did, or interpretation of data), and any trends you observed. Explain the relationship you observed (if any). Did you see what you expected to? If yes, briefly remind the reader why. If no, account for this. Relate your results to a real-world application How can this knowledge be applied in a useful way? (Do some research here.) Make reference to what you observed in your experiment. Comment on the accuracy of the experiment: Are the results are accurate? Explain why or why not, by commenting on three possible sources of error. Suggest two possible changes to the procedure of this lab that would produce more accurate results. Suggest a possible extension for this lab experiment. Conclusion - Refer back to the goals of your experiment, and summarize the outcome. Did you achieve your goal? If you did not achieve your purpose, or your hypothesis was not correct, state why. If there were any major sources of error, be sure to include them. Citations - Properly cite all sources both internally, and in a works cited list at the end of your report. Use APA citation style. You should have at least three reliable sources.