Effects of Citrus Fruits as Fertilizers Using Photosynthetic Testing, Growth Rate and Leaf Area Ratio of Hedera helix

By Zimin Zhao, Katrina Shedd, David Hartfield and Sonia Rahangdale

LBS145 Cell and Molecular Biology

Section 1 M2

Dan Gutteridge, James Hardie, Professor Luckie

3/3/05

Figure 1: Orange extract, orange peel extract, grapefruit peel extract, and grapefruit extract. These are the four extracts that we ran our carbohydrate tests and protein test on. We also applied them to our Hedera Ivy plants.

Abstract

(Authored by Katrina Shedd)

It is known that Citrus fruits contain sugars and proteins but not much is known about how they differ in oranges, grapefruits, and their peels. The purpose of this study is to discover which of these fruits, and which part, is most beneficial to plants. Using Benedict’s test, Barfoed’s test, Selivanoff’s test, and Bial’s test on extracts of the inside of oranges and grapefruits, and peels of the fruit we found that they all contain reducing sugars in the form of disaccharides and polysaccharides, ketoses and aldoses, and hexose furanoses. The Bradford protein assay determined the amount of protein in our orange extract to be the highest. Citrus fruit peels are often used in fertilizers, it is important to find out how peels and fruit affect photosynthesis and growth of plants. We first tested the pH of the four extracts, finding the grapefruit to be the most acidic and the grapefruit peel the most basic. After five weeks, we measured the growth of our Hedera helix, and found their leaf area ratios (LAR). Using a chi-squared goodness of fit test we found that there was no significant difference in the growth of our plants or in the LAR, based on a p-value of 0.05. Paper chromatography was performed and the Rf values we found did not match any pigments. The Hill Reaction was also used and we found that most photosynthesis occurred in the control plants and white light and the least occurred with the orange plant under red light.

Discussion

(Written by Zimin Zhao, revised by Sonia Rahangdale, Kati Shedd, David Hartfield)

In this experiment we compared the kinds of carbohydrates, the protein concentrations, the photosynthetic properties and the acidity of different citrus fruits and citrus fruit parts to help determine which would be the best fertilizer for the Hedera Ivy, Hedera helix. The citrus fruits we studied are oranges and grapefruits, and these fruits’ insides and peels were studied separately. The average increase in height, the photosynthetic properties, and the leaf area ratio of the plants applied with different extracts of the citrus fruits and citrus fruit parts were also examined. We predicted that both oranges and grapefruits contain reducing sugars, disaccharides, ketoses, aldoses, hexose furanoses (Spiegel-Roy and Goldschmidt, 1996) and proteins (Sinclair, 1972). We hypothesized that the orange peel extract would be the best fertilizer for plants because studies have shown that the juice of citrus fruits are much more acidic than their peels (Spiegel-Roy and Goldschmidt, 1996), and we suspected that grapefruit extracts from both the peel and the fruit would be too acidic for the plants (Sinclair, 1972). Furthermore, studies have shown that orange peels contain potassium, which is necessary for plants to grow (James et al., 2004). From this, we predicted that a chi-squared test on both the average height the plants grew and the leaf area ratio would show that there is a significant difference between the control plants, the treatment with orange extract, the treatment with orange peel extract, the treatment with grapefruit extract and the treatment with grapefruit peel extract. We expect to find this because if the orange peel is the most effective fertilizer then it should cause the most growth and thus producing the tallest plants and plants with the highest leaf ratio (Spiegel-Roy and Goldschmidt, 1996). It was also predicted that the plants applied with all four extracts would contain the photosynthetic pigments -- chlorophyll a and chlorophyll b because both pigments are green and the color of the plants is green (Krha et al., 2005). It is known that chlorophyll is “responsible for the green color of plants” (Freeman, 2002). The orange peel and grapefruit peel extracts are predicted to contain carotene, which is orange-yellow, and xanthophylls, which is pale yellow because the peels are yellowish orange in color. Finally, we predicted that the Hill Reaction would show that white light is the best for photosynthesis of all the plants because white light consists of many different colors or wavelengths of light, while blue and red light only consist of single wavelengths of light (Krha et al., 2005).

In the Benedict’s test, which tests for the presence of a free aldehyde or ketone group, our results showed that all four extracts: orange, orange peel, grapefruit, and grapefruit peel, produced a red precipitate. This indicates that there is a free aldehyde or ketone group present in the sugars in all four extracts. Since sugars that contain a free aldehyde or ketone are reducing sugars, it can be concluded that all four extracts contain reducing sugars. Barfoed’s test, which distinguishes monosaccharides from di- and polysaccharides showed a precipitate in all four extracts. This result shows the presence of monosaccharides in all four extracts. Selivanoff’s test, which differentiates ketoses and aldoses showed that all four extracts contain monosaccharide ketoses because all four extracts turned red in less than one minute. Bial’s test, which is used to detect furanoses, showed the presence of pentose-furanoses in all four extracts since all four extracts turned either green/olive or green after heating. (Krha et al., 2005) For each extract in all four of the sugar tests three replications were performed, the replications showed the same results most of the time, so our results are reliable. Moreover, our results support the prediction that oranges and grapefruits contain reducing sugars and ketoses, but contradict the prediction that they contain disaccharides, aldoses, and hexose furanoses. Bradford protein assay was performed to find and compare the protein concentration in each extract. The results supports our predictions and show that the orange peel extract contains the highest concentration of protein, the next highest is the orange extract. The grapefruit peel contains a slightly lower protein concentration than the orange extract, and the grapefruit contains the lowest concentration of protein.

Paper chromatography of the orange peel extract and the grapefruit peel extract showed no separation of pigments. This result indicates that the orange peels and grapefruit peels may not have any photosynthetic pigments in them. Paper chromatography on the extracts of plants from the control group and the orange fruit treatment showed the presence of a green pigment with an Rf value of 0.087 and 0.065 respectively. Paper chromatography on the extracts of plants from the orange peel treatment, the grapefruit treatment, the grapefruit peel treatment showed the presence of a green pigment with an Rf value of 0.098, 0.100, and 0.103 respectively; and also showed the presence of a yellow pigment with an Rf value of 0.523, 0.655, and 0.259 respectively. The yellow pigment with an Rf value of 0.655 found in the plants from the grapefruit treatment is close to the Rf value of carotene, which is about 0.622. Indicating that the plants applied with grapefruit extract contain carotene. The results observed for the plants from the control group and the orange, orange peel, grapefruit peel treatments contradicts our prediction because their Rf values were not close to the previously found Rf values of carotene, xanthophylls (0.416), chlorophyll a (0.326), or chlorophyll b (0.192). This indicates that these plants contain photosynthetic pigments other than these four pigments, but the plants in the orange peel treatment, grapefruit treatment and grapefruit peel treatment may have more photosynthetic pigments than the plants in the control group and the orange fruit treatment.

The Hill Reaction on Hedera Ivy chloroplasts showed that plants applied with orange fruit extract had the highest absorbance when exposed to no light, second highest absorbance when exposed to red light, third highest absorbance when exposed to white light, and lowest absorbance when exposed to blue light. This contradicts our prediction and indicates that in this plant extract, indophenol was reduced the most when exposed to blue light. Indophenol turns clear from the original blue color when it is reduced; the more reduced indophenol is the lower the absorbance should be. Thus, the Hedera Ivy applied with orange fruit extract utilizes blue light the most for photosynthesis. The result for the plants applied with orange peel extract showed the highest absorbance when exposed to blue light, second highest absorbance when exposed to no light, third highest absorbance when exposed to red light, and lowest absorbance when exposed to white light. This supports our prediction and indicates that in this plant extract, indophenol was reduced the most when exposed to white light. So white light is the best for photosynthesis for the Hedera Ivy applied with orange peel extract. The results for the plants applied with grapefruit extract, grapefruit peel extract and the control group all showed the highest absorbance when exposed to no light, second highest absorbance when exposed to blue light, third highest absorbance when exposed to red light, and lowest absorbance when exposed to white light. This also supports our prediction and indicates that white light is the best for photosynthesis for these plants. From eye observation, no difference was observed in the darkness of the color of the plant extracts and indophenol solutions although the solutions with more reduced indophenol should appear light. This may be due to the human eye’s inability to observe a slight color change.

Furthermore, pH tests on the extracts showed that the grapefruit has a lower pH value than the orange fruit, but the grapefruit peel has a higher pH value than the orange peel. Overall, the fruits had lower pH values than the peels. These results support our prediction that fruits are generally more acidic than the peels and grapefruit extract might be too acidic for the plants, but contradicts our prediction in that the grapefruit peel had the highest pH value of all the fruit parts.

The final examination of the growth of the Hedera Ivy due to the application of the extracts included a chi-squared test on the average increase in height of the plants in each treatment, and a comparison between the leaf area ratios of the plants in each treatment. Results indicate that the plants in the grapefruit peel extract treatment had the most increase in height. Chi-squared test on the increase in height of the plants in each treatment produced a p-value of 0.5330, indicating that there is no significant difference in the height increase of the plants in the different treatments. Comparison between the leaf area ratios of the plants in each treatment showed the highest leaf area ratio for the plants in the grapefruit extract treatment. Chi-squared test on the leaf area ratios produced a p-value of 0.93162, indicating there is no significant difference between the leaf area ratios.

Since there is no significant difference in the increase in height and the leaf area ratios of the plants in the different treatments, our experimental results do not support our hypothesis that orange peels are the best fertilizer for plants. Using orange fruit, orange peels, grapefruit or grapefruit peels as fertilizer for the Hedera Ivy will not cause a big difference in its growth.

There are many possible sources of error in this experiment. In the sugar tests, it is possible that the original yellowish color of the extracts affected the color of the solutions after heating and the concentration of extract we used might not be the best concentration for the sugar tests. More accurate and reliable tests for sugar will reduce this error. Also, the spectrophotometer might not be very accurate in reading the absorbance in the protein assay and for the Hill reaction because there are a few instances when the machine altered the zero point. A more accurate spectrophotometer will produce better results. Possible sources of error for paper chromatography might be that the petroleum ether solution was not an effective enough solvent for the separation of the pigments in our extracts. Other solvents can be used to run the paper chromatography or other methods: such as thin layer chromatography and column chromatography may be performed to improve the results (Lehman, 1999). In addition, since there were difficulties in obtaining pellets through centrifugation of the peel extracts to run the paper chromatography, the solutions of extracts used might not be effective for paper chromatography. For further experimentations, a more high-powered centrifuge can be used to obtain the pellets.

Exposure to light while making and aliquoting the indophenol solutions, and inaccuracies in the timing of the reaction are all possible sources of error in the Hill Reaction. And inefficiency of the pH meter and contaminations of the pH meter probe many produce erroneous pH values of the extracts. Other possible sources of error in our experiment include: not enough replications on the tests we ran, the way in which our plants were fertilized, and how much fertilizer was used. Our experiment had only 3 replications of each different treatment, which may not be an effective sample size to yield reliable results. Perhaps more replications could improve the reliability of this experiment. The probability of this being a source of error is low as the test results we obtained from the 3 replications did not vary from one another. It was also difficult to determine how much extract should be used for each of the controls to actually affect the results of the experiment. Giving too little would not affect the plants’ growth and too much may actually do the plant harm rather than aiding it in growth. In this experiment, we may have applied too small amounts of extracts to each plant, causing there to be no significant difference in the overall growth of the plants in the different treatments.