Ryegrass Analyzed for Protein, Carbohydrate, Pigment, Height and Mass After Being Treated with Liquid, Water Soluble, Pellet, and Spike Forms of Miracle Gro®,.





Rebecca Andrews, Peter Howard, and Alejandra Ramirez



LBS 145: Biology II Cell and Molecular Biology

Monday 7pm10pm

Dr. Fata-Hartley

February 27, 2006




Written by Alejandra Ramirez

Revised by Rebecca Andrews (first) and Peter Howard (second)


            The question addressed in this study was whether or not the form of fertilizer, liquid, spikes, water soluble, or pellets, had an effect on the growth of Ryegrass. We hypothesized the liquid form of Miracle Gro® fertilizer would produce grass with the highest amounts of protein (due to the highest amounts of nitrogen present), chlorophyll A and B, and mass, while not affecting the types of carbohydrates present. The experiment had five treatments: a liquid form, spike form, pellet form, water soluble form of Miracle Gro® brand fertilizer, and a control treatment of only distilled water, with four replications per treatment. To determine the effectiveness of the fertilizers the following tests were performed; Barfoed’s carbohydrate test for the presence of monosaccharides, Selivanoff’s test for the presence of ketoses or aldoses, a Bradford Assay determined the concentration of protein in the grass, and Thin Layer Chromatography (TLC) was used to determine concentrations of Chlorophyll A and B as well as to determine the presence of β-Carotene and Xanthophylls. Observations were noted for growth, height, and overall appearance. The results showed that no fertilizer influenced the type of carbohydrates present in the grass; but liquid fertilizer did, as hypothesized, increase the amount of protein compared to the other fertilizer forms (0.078mg/μL for the liquid versus 3.71 mg/μL for the control). The control produced the highest average growth (5.50cm). The treatment with the most plant mass (root and leaf together) was the spike treatment (5.698g). The liquid treatment had the most chlorophyll A (396.3μg/g) and total chlorophyll (778.8μg/g), and interestingly the pellet treatment had the most chlorophyll B (322.9μg/g), but not the highest total chlorophyll.



Written By Rebecca Andrews

Revised by Peter Howard and Rebecca Andrews

Re-revised by Alejandra Ramirez and Peter Howard


            Fertilizers are essential to food production for the expanding human population. Because of this there is much research in how to make fertilizers more efficient and productive (Miner and Lee, 2003). The market for fertilizers is huge in the U.S., and even larger from the perspective of the word market. Research on how to make more efficient and productive fertilizers is carried on by governments, non-governmental organizations, companies, and educational institutions (Anonymous-2, 2006). The market for commercial consumer fertilizers has been expanding as the boom in home gardening as a hobby has expanded. There is a lot of money to be made by a company if the company can produce the highest quality and most efficient fertilizer at a price attractive to consumers. There are many brands of consumer fertilizers on the market, and the most identifiable brand is Miracle Gro®, sold by The Scotts Company®. Miracle Gro® comes in many different forms, each with the claim to be as good as any other form. The question then arises, which form is really the best when held up to scientific scrutiny? In previous studies it has been shown that nitrogen and phosphorus fertilizers are capable of affecting the growth and overall health of plants (Clarkson and Warner 1979).  The experiments that were performed sought to determine which form of Miracle Gro® really outperforms the others. It is hypothesized that the liquid form of Miracle Gro® would promote the most protein, chlorophyll pigment, average height, and total mass in L. perenne, perennial ryegrass. The fertilizers are not expected to change the carbohydrate composition of the grass.

            After the grass was grown for forty one days and treated with the different forms of fertilizers according to the instructions from the Miracle Gro® packaging, different macromolecule assays along with an independent assay were performed. For carbohydrates, a iodine test for starches, a Selivanoff’s test for ketoses and aldoses, and a Barfoed’s test for monosaccharides, disaccharides, and polysaccharides were performed. The results from these three tests showed that the different forms of the fertilizers did not change the composition of the carbohydrates when compared to the control treatment (no fertilizer). This was predicted in the hypothesis. More research could be done in this area by analyzing the amounts of carbohydrates in each treatment. Each carbohydrate performs many different duties in the plant, and the fertilizers may or may not have increased or decreased amounts of specific carbohydrates, depending on their function in the plant.

            When the amount of protein was analyzed (Bradford assay), the liquid fertilizer had the highest protein content, at 77.6μg of protein per gram of sample. The next highest amount of protein was found in the control treatment, 74.2μg/g. The next highest amount of protein was in the water soluble treatment, at 72.4μg/g. The other treatments, the pellets and the spikes, had a much lower amount of protein (57.4μg/g and 50.6μg/g respectively). This supports the hypothesis; the liquid fertilizer did increase the amount of protein present in the ryegrass by a small amount, but these results also raise another very interesting question. Why did the pellet and spike fertilizers decrease the amount of protein present in the ryegrass, when compared to a treatment with no fertilizer?

By fertilizing the ryegrass with the pellets and the spikes, this lowered the amounts of protein present. This goes against the thinking that if there is more nitrogen available to the plant, as it was provided by the fertilizer, that there would be more production of amino acids, and thus more protein formation. The control treatment, with no added nitrogen, was able to produce more protein than the pellet and the spike treatments. This suggests that there may be more than one factor in increasing protein production, other than just adding more nitrogen to the soil that the plant is in. There may be an unknown factor that these treatments may have lacked. Also, another suggestion that this finding creates is that maybe these treatments did not lack a factor for protein production, but instead had some ingredient that inhibited the creation of new protein. One explanation of how these factors may have decreased protein creation is the fact these fertilizers are always present in the soil. The pellets sit on the surface of the soil and break down and release nutrients into the soil, and the spikes are inserted into the soil, and slowly degrade and release nutrients. The constant presence of these concentrated amounts of chemicals may inhibit the production of protein in the plant, but the mechanism for inhibition is unknown. Further experimentation on the effects of solid fertilizers that remain present during all times of growth would be a good avenue to explore to discover the mechanism that inhibited protein manufacture.

            Amounts of photosynthetic pigments chlorophyll A and chlorophyll B were measured after amounts of protein were determined. The liquid fertilizer treatment produced the most total amount of chlorophyll A and total chlorophyll. This partially supports the hypothesis. Combined with the data for the liquid fertilizer treatment from the Bradford assay, the increased amount of protein may have resulted in the increased amount of chlorophyll found in the liquid treatment. Protein is essential in the catalyzing of reactions, proteins act as enzymes, which speed up reactions or allow reactions that would not occur spontaneously to react (Stanford et. al., 2005)

. This lead to the suggestion that the increased amount of protein that was created in the liquid treatment helped catalyze reactions that produce chlorophyll and other photosynthesizing pigments. A counter argument to this would be that the pellet fertilizer treatment contained a larger amount of chlorophyll B than the liquid fertilizer treatment did. This is the part of the hypothesis that is not supported by the data. As discussed above, the pellet fertilizer treated grass had much less protein than the liquid fertilized grass did. So if the mechanism for pigment formation is linked to levels of protein, this argument suggests that the lower levels of protein present in the pellet fertilizer treated grass would produce lower levels of chlorophyll B. The data suggests that this trend is not present, or that this was a fluke in the data. One last suggestion is that perhaps the protein that was produced in the pellet treated grass is that the proteins that this fertilizer produced higher amounts of enzymes that are specific for the production of chlorophyll B, while not producing as high amounts of other enzymes that are specific for chlorophyll A. More research could be done on this subject, perhaps by varying the amounts of components for production of enzymes that catalyze reactions that produce chlorophyll A or B.

            The heights, masses, and NTEP ratings for each treatment were performed and gave interesting results. The hypothesis predicted the tallest average growth to come from the liquid fertilizer treatment, but instead the tallest treatment was the water soluble treatment, followed by the control and pellet treatments at the same height, then the spike treatment, and lastly the liquid treatment. This was surprising, since the liquid had the highest amounts of protein and chlorophyll; the hypothesis predicted that this treatment would also be the tallest. The liquid treatment was on pace to end up being the tallest, but around day 30 the leaves of the grass started to fray and thin. From day 30 to the last day of the experiment, the plants got progressively worse and stopped growing upwards, but instead started to fall over and grow outwards. The other treatments grew normally, and the water soluble treatment grew the highest. It is interesting that the pellet treatment grew as high as the control, and raises the question about the amount of protein present and how that relates to general health. As discussed above, protein is used in providing structure to the plant. Since the pellet treatment’s amount of protein was significantly less than that of the control treatment, why did they grow to the same average height? This suggests that there may be other factors beside protein concentration in determining how high ryegrass will grow.

            The masses of the plants also showed a trend contrary to the predictions of the hypothesis. The liquid fertilizer treatment weighed the least when compared to the other treatments. The spike fertilizer treatment weighed the most, followed by the control, then the water soluble treatment, and then the pellet fertilizer treatment. The most mass was produced by the spike fertilizer treatment, which, as stated above, had the least amount of protein, which is used for creating structure in the plant. This raises another question, if the grass with the most mass has much lower amounts of protein present; perhaps another macromolecule is providing structure to the plant. Monosaccharide carbohydrates can link up using glycosidic linkages to form cellulose, chitin, or peptidoglycan, which are long polysaccharides that form parallel strands that are held together by hydrogen bonds or peptide bonds (Freeman 2005). This may explain why the sample with the most mass did not have the most protein as predicted. This may also explain why there was no trend relating height to protein concentration. Although the experiments performed did not show the presence of these polysaccharides, this is an interesting point that could be extended to other types of plants. Further experimentation could determine if carbohydrates played a role in plant mass or height.

            NTEP ratings gave an overall picture to how well the plant grew. The poor ratings for leaf texture for the liquid were due to the fraying of the plant’s leaves from day 30 to day 41. Why this treatment started to act in this manner was unknown, but the general health of the plant was noticeably reduced in the last eleven days of the experiment. For all of the other treatments, scores were very average and in the range predicted by the hypothesis. An average score is a acceptable score, scores close to very good or very bad are more important that variation in the middle of the range of scores (Anonymous-1, Unknown).

            Further experimentation that would increase the knowledge of how the fertilizers work would include; determining the concentrations of carbohydrates in the ryegrass, testing specifically for cellulose, chitin, or peptidoglycan, determining the amino acids present in the protein samples, and extending the amount of time allowed for growing to see long term effects (Minner and Lee, 2003). Also, tests on the effects of solid fertilizers when left in the soil continuously may shed light on the interesting results obtained from the pellet and spike treatments.

            Sources of error in this experiment could have contributed to errors in the data, which could have skewed results. Human error might have affected this experiment in every aspect. Not following specific watering instructions, fertilizing instructions, or procedures for the tests may have produced skewed data. The spectrometer used in determining amounts of chlorophyll and protein was very old and prone to breaking. This factor may have distorted the data, which may explain for why some results differed from the predictions. The soil that the grass was grown in could have contained unknown factors that could have influenced the growth of the plants. Other sources of error may include the exposure of the grass to unknown factors that may have affected growth when growing in the Holmes Hall dorm room.

            In conclusion, no fertilizer seemed to provide an edge over the other. Each fertilizer had its own merits and its own faults. For the average consumer who whishes to increase the general health of his or her lawn, it could be suggested that no fertilizer be used, since the control treatment was generally healthy and while not the best in any category, a consumer could get good looking healthy grass without the time and money spent fertilizing. If a consumer wishes to fertilize his or her lawn, the water soluble fertilizer seemed to perform the best. The liquid fertilizer would have been a good recommendation if it had not started to fray around day 30 to the end of the experiment.




Figure 2:  Graph showing the average heights of all trials.  These values are from the measurements obtained 41 days into the experiment before the grass was trimmed for experimentation. 
































By: Rebecca Andrews


Anonymous-1.  Unknown.  National Turfgrass Evaluation Program.  http://www.ntep.org Accessed 1/28/06.


Anonymous-2. 2006. The Fertilizer Institute. http://www.tfi.org Accessed 1/18/05.


Bunnel, B. T., McCarty, L. Bridges, W.C. 2005. TifEagleBermudagrass Response to Growth Factors and Mowing Height when Grown at Various Hours of Sunlight.  Crop Science 45: 575-582.


Clarkson, D and Warner, A.  1979.  Relationships between Root Temperature and the Transport of Ammonium and Nitrate Ions by Italian and Perennial Ryegrass.  Plant Physiology  64:557-561.


Freeman, S. 2005. Biological Science. Upper Saddle River, NJ. Pearson Education Inc. 1283p.


Krha, M., Maleszewski, J., Cooper, S., Wilterding, J., Sayed, M., Luckie, D., and Fata-Hartley, C. 2006. LBS 145 Spring 2006 Cell and Molecular Biology Lab Manual. East Lansing: MSU Printing Services. 117.


Minner, D.D., Lee, S.K. 2003.  Evaluating the Influence of a Liquied Organic Polymer (Turf2Max) on Soil Aggregation and Growth of Perennial Ryegrass.  Crop Science 43: 664-669.


Stanford, R.L., White, R.H., Krusz, J.P., and Thomas, J.C. 2005. Temperature, Nitrogen, and Light Effects on Hybrid Bermudagrass Growth and Development.  Crop Science 45: 2491-2497