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					The Photosynthesis Rates of Euglena gracilis
Biology Extended Essay
Vivian Rhee

Photosynthesis is a chemical reaction, which usually happens in plant cells. However, one protist, called Euglena gracilis, shares the same ability as the plant. Scientists have done much research to find out if the euglena’s photosynthesising ability is as effective as a plant’s. As part of my research, I conducted my on experiment using the Euglena gracilis as subjects. To find out under which coloured light the euglena photosynthesises the best in, I put each sample under white, red, blue, and green lights for three days and measured the light absorbance, using a Colorimeter. After considering the results I obtained, as well as other research, I conclude that the euglena’s photosynthesising rate is most effective when under the blue light.

Table of Contents

Introduction ……………………………………………………………………………….…3 Photosynthesis and Pigments …………..…………………………………………………….3 Euglena ………………………………………………………………………………………6 Other studies …………………………………………………………………………………8 Photosynthesis Experiment ………………………………………………………………….11 Conclusion …………………………………………………………………………………...12 Bibliography & Works Cited ………………………………………………………………..14 Appendix A ………………………………………………………………………………….16 Appendix B ……………………………………………………………………………….....17


When people mention photosynthesis, they generally think of the process occurring in plants. The reason is because photosynthesis is how the chloroplasts, which are in plant cells, make food. However, the protists in the genus Euglena are unique; they are both autotrophs and heterotrophs. This means that the protist can make its own food from photosynthesis and gain food from other sources as well. The photosynthetic process is possible for the euglena because it has chloroplasts in its body. Scientists did much research on the photosynthesis rates of plants in different coloured lights. Therefore, if the Euglena is similar to plants, then how would the Euglena’s photosynthesis rate be affected when it is under different coloured lights?

Photosynthesis and Pigments
Photosynthesis is a chemical reaction where light energy is converted to carbohydrate energy: 6 H2O + 6 CO2 + light energy → C6H12O6 + 6 O2 (water) (carbon dioxide) (glucose) (oxygen)

The reaction occurs in the chloroplasts of plant cells and two of the main stages in photosynthesis are called light reactions and dark reactions. In the light reactions, electrons from water are “excited” or raised to higher energy levels in two steps called photosystems I and photosystems II (Mader, 133-136). In both procedures, chlorophyll absorbs light energy that is used to excite the electrons. Normally, these electrons are passed to a cytochromecontaining electron transport chain (Mader, 133-136). In photosystem II, these electrons are used to generate ATP (adenosine triphosphate). In photosystem I, high-energy electrons are used to produce the reduced coenzyme NADPH (nicotinamide adenine dinucleotide 3

phosphate). Both ATP and NADPH are then used in the dark reactions to produce glucose (Mader, 133-136). In the chloroplasts, the main pigments are chlorophyll a (blue-green) and chlorophyll b (yellow-green), and the accessory pigments are carotene (orange) and xanthophylls (yellow). Furthermore, each pigment has its maximum light absorption because pigments have their own absorption spectra (Farabee). Chlorophyll a absorbs wavelengths at 400 - 450 nm and at 650 Figure 1 Absorption spectrum of several plant pigments

700 nm well, chlorophyll b absorbs best at 450 - 500 nm and at 600 - 650 nm, and xanthophylls absorbs well at 400 - 530 nm ("Photosynthetic pigment"). The reason why humans see plants in green is because most of the pigments have the lowest absorption in the green-yellow region of the visible light spectrum; therefore, the pigments reflect the green light (Mader, 129). However, carotenes and chlorophyll b absorb some of the energy in the green wavelength as well as in the orange-red end of the spectrum (Farabee). This is mysterious because the orange and red lights have longer wavelengths and thus they are lower in energy. According to Farabee, the reason to this mystery is due to the origins of the photosynthetic organisms in the sea. Shorter wavelengths, which have more energy, cannot permeate more than five metres into the sea water (Farabee). Therefore, the longer wavelengths were an advantage to the early photosynthetic algae as the longer wavelengths were able to penetrate deeper into the water, giving some energy to the organisms that lived in the bottom of the ocean (Farabee). In this way, the action spectrum of photosynthesis is the relative


effectiveness of different wavelengths of light at generating electrons (Farabee). Farabee also says that if a pigment absorbs light energy, one of the three things will occur:

1. Energy is dissipated as heat. 2. The energy may be emitted immediately as a longer wavelength, a phenomenon known as fluorescence. 3. Energy may trigger a chemical reaction, as in photosynthesis.

In addition, a chemical reaction only occurs only when chlorophyll is related to proteins embedded in a membrane or the membrane infolding found in photosynthetic prokaryotes, such as cyanobacteria and prochlorobacteria (Farabee).

There are three main factors which affect photosynthesis: light irradiance and wavelength, carbon dioxide concentration, and temperature (“Photosynthesis”). In the early 1900’s, Frederick Frost Blackman and Gabrielle Matthaei performed two experiments to find out the effects of light intensity and temperature on the rate of carbon assimilation, which is the incorporation of carbon from atmospheric carbon dioxide into organic molecules (“Carbon assimilation”):

At constant temperature, the rate of carbon assimilation varies with irradiance, initially increasing as the irradiance increases. However at higher irradiance this relationship no longer holds and the rate of carbon assimilation reaches a plateau. 2. At constant irradiance, the rate of carbon assimilation increases as the temperature is increased over a limited range. This effect is only seen at high irradiance levels. At low irradiance, increasing the temperature has little influence on the rate of carbon assimilation.



From these experiments, Blackman and Matthaei noticed two important concepts. They found out that temperature affects the rate of carbon assimilation, meaning that there must be two sets of reactions in the full process of carbon assimilation, the light and dark reactions (“Photosynthesis”). This was a significant finding because it is known from research that photochemical reactions are not affected by temperature (“Photosynthesis”). Secondly, Blackman discovered the concept of limiting factors, one of them being the wavelength of light (“Photosynthesis”).

In addition, the rate of sugar production from light-independent reactions increases as carbon dioxide concentrations increase. RuBisCO is an enzyme, which captures carbon dioxide in the light-independent reactions; it has a “binding affinity” for carbon dioxide and oxygen (“Photosynthesis”). RuBisCO “fixes carbon dioxide,” or converts carbon dioxide gas into a solid form, when the carbon dioxide concentration is high. However, if the carbon dioxide concentration is low, RuBisCO will bind oxygen instead of carbon dioxide in a process called photorespiration, where sugar is not produced (“Photosynthesis”).

Euglenoids are freshwater, unicellular organisms. When scientists first discovered these protists, they were not sure whether to call the euglenoids plants or animals. The two factors that caused this confusion were that the protists could photosynthesise, the characteristic of an autotroph, and they could also absorb food from other sources like a heterotroph. Near the base of the euglenoid’s flagellum, there is an
Figure 2 Anatomy of a Euglena


eyespot, which shades a photoreceptor for detecting light (Mader, 553). The eyespot, which is about 6 μm2 in cross-section, is a cluster of numerous orangered pigmented globules that vary from 0.1 to 0.3 μm in diameter (Wolken). The movement of a euglena is characterised as a pulsation, sideways rotation, and forward swimming (Wolken). While the sideways rotation and forward swimming are made by the whipping of the flagellum, the direction of the movements are decided by eyespot’s attraction to a light source. In this way, the eyespot is related to phototaxis, the movement of an organism toward or away from a light source, where light acts as the stimulus. Its reaction of swimming toward the light source classifies it as positively phototactic (Wolken). However, when it is in a very high light intensity environment, such as under a microscope, the euglena tends to swim away from the light to avoid the high temperature.

Figure 3 Swimming pattern of Euglena gracilis in response to light. (a, b) Paths of motion; (c) orientation and degree of left-right turning.

In addition, the chloroplasts of the euglena are surrounded by three membranes instead of two. In this way, the euglenoid chloroplasts are much more complex than those in plants, suggesting that the protists evolved twice in an endosymbiotic relationship: they


advanced, once, from cyanobacteria to the chlorophyte nucleus, and then to the nucleus of the Trypanosome-like host (Leegood). As a result, precursor import, the import of a compound that participates in the chemical reaction to produce another compound, is more advanced (Leegood). The pigments that are in the euglena’s chloroplasts are β-carotene, chlorophyll a, diadinoxanthin, chlorophyll b, and neoxanthin (Giddings). Since most of the protist’s pigments are the same as a plant’s, it is possible to explain why the two’s photosynthesising habits are similar.

Other Studies
Many experiments and observations have been performed on both plants and euglenas. In a journal called “Regulation of the Photosynthesis Rhythm in Euglena gracilis”, Thomas A. Lonergan and Malcolm L. Sargent say that they found a five-day circadian rhythm of oxygen in Euglena gracilis. They believed, as William G. Walthers and Leland N. Edmunds Jr. once said, that changes in the activity of glyceraldehyde-3-P dehydrogenase might generate the photosynthetic oscillations in Euglena. Additionally, they found out that “carbonic anhydrase activity [was] correlated with photosynthetic rates in several algae” (Lonergan and Sargent). For the experiment, they maintained a controlled environment for the euglena by using sterile air bubbles and a temperature of 25℃. The photosynthesis rates were measured from oxygen evolution measurements in a system using a light intensity of 5×106 ergs∙cm-2∙sec-1. After putting the protists under constant bright lights, they were then transferred to constant dim lights of 2×103 ergs∙cm2

∙sec-1, allowing the rhythm to continue for five more cycles. However, whilst under the

constant bright light, the rhythm was quickly dulled after just one cycle. Therefore, contrary to Walther and Emund’s results, Lonergan and Sargent found out that glyceraldehyde-3-P 8

dehydrogenase activity is not rhythmic, and they were able to conclude that the photosynthetic rhythm continues under constant conditions. Lonergan and Sargent also believe that the difference between the two reports might have been caused by the dissimilar culture growth conditions, and by the fact that “[their] enzyme preparation was more active and required three orders of magnitude more NADPH to reach saturation” (Lonergan and Sargent). Another study, which was done on spinach, was a spectrophotometric analysis of the absorption of green light and red light. The essay proved that green light has a low absorbency, while red light has a high absorbency (“A Spectrophotometric Analysis”). First, the observer separated the pigments from a spinach leaf by using paper chromatography. The four pigments, in order from the origin to the solvent front, were chlorophyll b, chlorophyll a, xanthophylls, and carotene. The separated pigments were then analysed for their absorption spectrum, a graph that shows a pigment’s light absorption against wavelengths, using a spectrographometer. In the graph, there were peaks at the red and blue wavelengths, showing maximum absorbency, while the dramatic drop at the green wavelengths represented low absorbency. Furthermore, this experiment showed that chlorophyll a and b are the primary pigments in photosynthesis. It confirmed that chlorophyll a is the only pigment that can directly participate in light reactions, whereas the accessory pigments absorb a slightly different set of wavelengths, allowing plants to have an absorbance of a wider spectrum of colours in photosynthesis (“A Spectrophotometric Analysis”). On the other hand, a research, "Effects of Blue and Red Light on the Rate of Photosynthesis," written by B. Braddock, S. Mercer, C. Rachelson, and S. Sapp was quite fascinating. They, too, hypothesised that the photosynthesis rate would be “positively correlated” to the different wavelengths of light (Braddock et al.). Although they were using juniper needles for their experiment, they expected that the results would conclude with the 9

blue light giving a higher photosynthesising rate than a red light. In addition, like Lonergan and Sargent, they measured the rate of change of carbon dioxide concentration, alternating for ten minutes under each of the red and blue lights. Then, they measured the mass of the juniper needles in each sample, calculating the change of carbon dioxide concentration per gram of juniper needles. During observation, it was noted that the plants in red light produced less carbon dioxide than when in blue lights. After three trials of the experiment under each light, they were able to get mean values of 2.36 ppm/g/min under the blue light and -0.27 ppm/g/min under the red light (Braddock et al.). Although there was no significant difference between the two mean values, Braddock and the others said that the photosynthesis rate was still always slightly higher when the juniper needles were under the red light rather than the blue. Consequently, they were able to reject their hypothesis as “blue light does not make plant needles photosynthesise faster than red light” (Braddock et al.). The four scientists also say that through their research, they found a study that found a decreasing rate of photosynthesis in blue light and another study found that the rate of photosynthesis occurred fastest in red light. They believe that the cause for this was because xanthophylls were dissipating the excess energy associated with blue light (Braddock et al.). Explaining their results, the four scientists said,

“… due to the high-energy nature of blue light, some of the blue light shining onto the juniper needles is absorbed by plant pigments other than the chlorophylls and is not transferred to the photosynthetic reactions.” (Braddock et al.)

They also said that because xanthophylls and carotenes absorb only in the blue spectrum, the two pigments are possibly dissipating the high-energy blue light (Braddock et al.). Therefore,


with more trials of their experiment, they believe that they could come up with a significantly faster rate of photosynthesis under red light compared to blue light.

Photosynthesis Experiment
As part of my investigation, I conducted my own photosynthesis experiment to determine how the euglena behaves under different coloured lights. Using the species Euglena gracilis as subjects, I tested the light absorbance of three coloured lights: red, blue, and green. To control the experiment, I included the white light as one of the variables. Also, I controlled the samples of euglena by retrieving the euglena until the drawn line of a liquid dropper. To maintain similar photosynthesising conditions as the one in plant cells, DPIP (2,6-dichlorophenol-indophenol), which is a blue dye, was used to replace NADPH in the light reactions. When the dye is oxidised, it is blue; however, when it is reduced, it turns colourless. Since DPIP replaces NADPH in the light reactions, it turns from blue to colourless when reduced during photosynthesis. The samples of euglena were put in a 250 mL beaker with a ratio of phosphate buffer to distilled water to DPIP, which equals 1:3:1. Then, they were placed behind a 1000 mL beaker of water and under the light. The beaker of water acted as a heat shield, protecting the chloroplasts from warming by the flood lamp. After each day of the three-day experiment, I observed a small sample of euglena under the microscope and measured the absorbance of light by using a Colorimeter. The experiment was also kept in an isolated room at all times so that the changing lights from the sun and ceiling lights could be controlled and limited. (See Appendix A for complete procedure.) I hypothesised that the euglena would have the highest photosynthesis rate when it is under blue light. This is because, according to physics, the colour blue of the visible light


spectrum has a shorter wavelength than red light, and thus it also has more energy. I also thought the euglena would start trying to survive by consuming the algal cells in the liquid or even die because they would not be able to photosynthesis for a long time in a coloured light. One of the findings during observation was that the euglena looked slightly darker when it was under the white and blue light. Other findings include the euglena moving quickly, spinning, folding, and squashing itself on Day 3 under the blue light. However, these behaviours seemed normal for the protist. The protists also tended to move away from the microscope’s spotlight as mentioned before. Furthermore, on Day 3 of observation under the red and green lights, I found another photosynthetic protist, which was round with green chloroplasts and moved in a circular path- my source of euglena had been contaminated. In the end, the calculated results for the rate of photosynthesis showed as predicted. The photosynthesis rate was highest under the blue light with 0.2285 A/day, the red light came second with 0.2110 A/day, and then it was the white light with 0.1490 A/day. The euglena had the lowest photosynthesis rate under the green light with 0.1245 A/day. (See Appendix B for complete results.)

After exploring how Euglena gracilis behave in coloured lights and examining the journals of many scientists, it is clear that the euglena photosynthesises the best under blue lights because of the euglena’s pigments are very similar to the ones in plant cells and the short blue wavelength has the most energy. Through my results, I found out that the euglena always photosynthesised slightly more effectively under the blue light compared to the red light, contradicting the lab results of Braddock et al. on juniper needles. The reason for this difference could be due to the fact that my experiment was too basic; however, it could also


be because the euglena is still also a heterotrophic protist while the juniper needle is only an autotroph. On the other hand, my results agreed to the journal “A Spectrophotometric Analysis of the Absorption of Green Light Versus Red Light Absorption in Spinach Leaves”. Although the subject of this experiment was spinach leaves, our results both came to the conclusion that red light is more effective than green light in light absorption. Nevertheless, my experiment was rudimentary and I realised that many improvements could have been made. I could have considered the other factors of photosynthesis and vary the concentration of carbon dioxide, temperature, and brightness of light. Also, if possible, I could have weighed the mass of the algal matter that was in the liquid mixture and find out if a difference in mass was made. With further investigation, I believe that I can find out the reason to the contradicting results of my experiment and Braddock and his associates’ experiment.


Bibliography & Works Cited
“A Spectrophotometric Analysis of the Absorption of Green Light Versus Red Light Absorption in Spinach Leaves”. 29 Mar 2009. <http://www.> Bloch, Konrad and George Constantopoulos. “Effect of Light Intensity on the Lipid Composition of Euglena gracilis.” J. Biol. Chem. 242: 3538-3542. JBC Online. 1967. 25 Mar 2009. <> Braddock, B., S. Mercer, C. Rachelson, and S. Sapp. "Effects of Blue and Red Light on the Rate of Photosynthesis". CU Boulder, 2001. 29 Mar 2009. <http://spot.color /~basey/bluer. htm> “Carbon assimilation”. High Beam Research. 2009. 4 May 2009. <http://www.highbeam. com/doc/1O6-carbonassimilation.html> Epstein, H. T., Jerome A. Schiff, and Arthur I. Stern. “Studies of Chloroplast Development in Euglena. V. Pigment Biosynthesis, Photosynthetic Oxygen Evolution and Carbon Dioxide Fixation during Chloroplast Development.” Plant Physiol. 39: 220-226. Plant Physiology. 1964. 25 Mar 2009. <> "Euglena." Wikipedia, The Free Encyclopedia. 30 Apr 2009, 17:39 UTC. 4 May 2009. <>. Farabee, M. J. "Photosynthesis". 2007. 29 Mar 2009. < faculty/farabee/biobk/BioBookPS.html#Table%20of%20Contents> Giddings, John Calvin and Roy A. Keller. “Pigments of Euglena”. Advances in chromatography. U.S.: CRC Press, 1989. Google Book Search. 10 Apr 2009. < glena%2Bpigment&source=bl&ots=9AOQef5uCG&sig=lQpEO0sCizDROtaTZErjYI 9OTBM&hl=en&ei=vBPLSbnNIYHasAP87vm0Cg&sa=X&oi=book_result&resnum =6&ct=result#PPA161,M1> Leegood, Richard C., Thomas D. Sharkey, and Susanna Von Caemmerer. “Calvin Cycle Enzymes and Expression in Euglena gracilis.” Photosynthesis: Physiology and Metabolism. U.S.: Springer, 2000. Google Book Search. 7 Apr 2009. <http://books. asts+in+euglena+surrounded+by+three+membranes&source=bl&ots=LYFjQgofDt&s ig=5xgSz0WF1OO9GL9Yfqb6lJlQ20&hl=en&ei=GCzcSeqGDpD2MKn0mNsN&sa =X&oi=book_result&ct=result&resnum=8#PPA27,M1> Lonergan, Thomas A. and Malcolm L. Sargent. “Regulation of the Photosynthesis Rhythm in Euglena gracilis: I. Carbonic Anhydrase and Glyceraldehyde-3-Phosphate 14

Dehydrogenase Do Not Regulate the Photosynthesis Rhythm.” Plant Physiol. 61: 150153. Plant Physiology. 1978. 25 Mar 2009. < /61/2/150> Mader, Sylvia J., Inquiry into Life. 8th ed. Carol J. Mills. U.S.: WCB/McGrawHill, 1997. "Photosynthesis." Wikipedia, The Free Encyclopedia. 4 May 2009, 13:26 UTC. 4 May 2009. <>. "Photosynthetic pigment". Wikipedia, The Free Encyclopedia. 13 Mar 2009, 00:27 UTC. 29 Mar 2009. < oldid =276870380> Walther, William G. and Leland N. Edmunds Jr. “Studies on the Control of the Rhythm of Photosynthetic Capacity in Synchronized Cultures of Euglena gracilis (Z)1, 2, 3, 4”. Plant Physiol. 51:250-258. Plant Physiology. 1973. 18 Apr 2009. <http://www. /cgi/content/abstract/51/2/250> Wolken, Jerome J. “The photoreceptor system for phototaxis”. Light Detectors, Photoreceptors, and Imaging Systems in Nature. U.S.: Oxford University Press, 1995. Google Book Search. 29 Mar 2009. < C&pg=PA77&lpg=PA77&dq=euglena%2Bpigment&source=bl&ots=hDcB8BQ PxI&sig=WY1L9MUui_oY5BfBsCafQ1iBdh8&hl=en&ei=vBPLSbnNIYHasAP87v m0Cg&sa=X&oi=book_result&resnum=3&ct=result#PPA76,M1>


Appendix A: Experiment Procedure
1. Fill one 250mL beaker with a ratio of phosphate buffer to distilled H2O to DPIP, which equals 1:3:1. Place a few Euglenas in it. 2. Obtain a 600mL beaker filled with water and a flood lamp. Arrange the beakers and light so that the lamp shines on the 600mL beaker, and behind it is the 250mL beaker with the euglena. The 600mL will act as a heat shield, protecting the chloroplasts from warming by the flood lamp. 3. Place the beaker under a normal coloured light bulb for 3 days. This will be the control. 4. After each day, observe one euglena under the microscope by preparing a wet mount slide and by measuring the light absorbency of the euglena. 5. To prepare a wet mount slide, add a drop of methyl cellulose to the culture before placing the cover slip on the slide. Methyl cellulose will slow down the euglena’s movement. 6. Mount the slide onto the microscope and focus under low power. Record down any observations, such as colour and size. 7. To measure the light absorbency, place two euglenas in a cuvette filled with the chemical solution that was in the beaker. 8. To correctly use a Colorimeter cuvette, remember:  All cuvettes should be wiped clean and dry on the outside with a tissue.  Handle cuvettes only by the top edge of the ribbed sides.  All solutions should be free of bubbles.  Always position the cuvette with its reference mark facing toward the white reference mark at the top of the cuvette slot on the Colorimeter. 9. Calibrate the Colorimeter. a) Open the Colorimeter lid. b) Holding the blank cuvette by the upper edges, place it in the cuvette slot of the Colorimeter. Close the lid. c) Press the < or > button on the Colorimeter to select a wavelength of 565nm (Red) for this experiment. d) Press the CAL button until the red LED begins to flash, then release. When the LED stops flashing, the calibration is complete. 10. Repeat steps 1 to 9 using the different coloured light bulbs.


Appendix B: Experiment Results
Data Absorbance (A) Red Blue 0.161 0.154 0.279 0.316 0.583 0.611

Time (day) 1 2 3

Normal 0.202 0.336 0.500

Violet 0.145 0.233 0.394

Colour White Red Blue Green

Rate of photosynthesis (A/day) 0.1490 0.2110 0.2285 0.1245

Observations (colour, size, movement, etc.) Day 1        Day 2  Dark green/black  Transparent  Black green  Transparent   Day 3  Dark green/black  transparent  Light green  Moves quickly, spins, folds, “squishes”  Green  Found another photosynthetic protist (round, green chloroplasts, moves in circular motion)  Jellybean shape  Green  Found another photosynthetic protist (round, green chloroplasts, moves in circular motion)

White light

Blue light

Dark green Transparent Moves quickly Black green Transparent

Red light

Green Moves quickly

Green Moves quickly

Green light



 

Green Size seems quite large


Graph of Euglena gracili’s photosynthesis rate


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