LBS 145 Cell and Molecular Biology

"The Carbs"

Combs, E., Mauch C., Smith, K., Wayo D.

Lab T2, Brent and Jeff

 

Analysis of Festuca rubra using Bradford, Hills, Height and Sugar Tests to Determine the Effects of Added Fertilizer

 

Abstract

Our lab group will determine whether adding fertilizer to grass plants alters the rate of growth. Starting with seeds, we will conduct 60 treatments in total using plastic planters. We’ll plant seeds in each compartment using general plant soil. Varying the amount of fertilizer administered to the planters, using 0.1-gram, 0.25 grams, 0.5 grams, and 1.0 gram, two 4-pack containers will be our control, having no fertilizer added.  The variation in amount of fertilizer administered will allow us to see whether the potentially beneficial effects of fertilizer plateau at some amount. Every treatment including the control will be watered daily using 25mL of tap water. After two weeks of fertilization, we’ll test the plants for any increase in height, protein content, sugar amounts, and photosynthesis. Using Bradfords’ Assay, we expect that fertilizer will have a direct effect on the protein content of the plants. The increase in nutrients available to the plant will cause the plant to produce more protein (VanWieringen et. al. 2005).  The Hill Reaction will be used to test the photosynthesis of the plants. We expect that the more fertilizer given will result in an increase in rate of photosynthesis (www.Science Projects.com, 2004). With more fertilizer we expect more plant cells, increasing the amount of chloroplasts, thus increasing the rate of photosynthesis.  We’ll be performing the Iodine test and Glucose Diabetes testing strips to monitor the sugar in the plants. We expect that the increase in nutrients available will cause the plant to produce more sugars (Wang and Zhang. 2004).

 

Figure 5:  Glucose levels in grass remain about the same despite any increase in fertilizer.  Here we increased the fertilizer from 0g to 0.1g, 0.25g, 0.5g, and 1g respectively.  Measurements were taken with a diabetes glucose testing device to see if the measurements increased when the fertilizer increased.  Three trials at each level of fertilizer and each control were taken and this is the average amount of glucose derived from the three trials.

 

Discussion

The initial question that we set out to answer was to find how beneficial fertilizer was on grass plants, or even if it could be harmful to the plants.  The belief that fertilizer helps the growth of plants is well known, and supported by the research of VanWieringen et. al.  Despite this research, our group set out to determine what the fertilizer actually does to the internal components of the plant, when administered at different quantities.  We also know that nitrogen in the soil helps produce healthy leaves, phosphorus helps produce a strong root system, and potassium helps a plant to be, in general, healthier (Kerrigan and Nagel. 1998).  We believed that the fertilizer would increase the grass plants protein content, glucose content, photosynthetic ability, and height, and therefore the plants overall health, and then it will also continue to increase those numbers as the treatments increased with amount of fertilizer.  We also know that over fertilizing can result in nutrient deficiencies because the nutrients bind to excess elements (Kerrigan and Nagel. 1998 ).  After performing the Bradfords assay, Hills analysis, Benedicts test, Iodine test, Glucose tests, and simple height measurements, we found our predictions to be partially inaccurate.  The protein, glucose, and height increased from the control for the 0.1g and 0.25g treatments, but declined for the 0.5g and 1.0g treatments.  The Hill’s reaction showed us that all of the plants were able to perform photosynthesis better for white light, but were less able to perform photosynthesis when provided with the red or blue light.  This shows us that the fertilizer has no real change on the ability of photosynthesis.

The first tests that we performed on the plants were Benedicts and Iodine tests.  These gave us solid qualitative data, showing us that the plants definitely had a free Ketone sugar and very low, if any, amount of starch inside of them.  Although these tests do not report any actual amount of sugar, it is still important to note that the fertilizer does not hurt the plant and stop it from producing carbohydrates.  Benedicts test shows us if there is a free Ketone or Aldehyde group in the solution.  If there is a presence of a free sugar group, the reaction will produce a red precipitate (Krha et. al. 14).  We found that all treatments tested positive for a free sugar group (Figure 3).  When we performed the Iodine test, which tests for the presence of starch in a solution (Krha et al 15), we found that all of the tests were negative, or inconclusive (Figure 2).  The Iodine test gives us a dark color for a positive result and no color change for a negative result.  From these results, we know that the fertilizer does not effect the plant enough to stop it from performing photosynthesis, and therefore still allowing the grass to create energy to use.

We then preformed the Bradfords assay on the plants.  Bradfords assay allows us to determine the total protein concentration in a substance (Krha et al 117).  To run this assay, we need to use a spectrometer to find the absorbance of solutions.  The more protein in the solution, the higher the absorbance will read. After running the assay, we found that the control had 0.17ug/ul, the plants given 0.1grams of fertilizer had 0.049ug/ul, the plants given 0.25 grams had 0.042ug/ul, and the plants given 0.5 and 1.0 grams had 0ug/ul (Table 1, Figure 4).  All of those results were found when using a 15ul sample of the grass matter. These results show us that the fertilizer was detrimental to the treatments with the higher levels of fertilizer.  The lower levels were able to produce more protein.  The control had more total protein than any of the fertilized treatments.  The fertilizer therefore must have done something to the plant that stunted, if not stopped, the production of protein.

We then performed the Hills reaction on the grass plants.  Hills reaction shows us how the photosynthesis is changed by different variables in plants.  It determines the action spectrum of photosynthesis by testing the plant matter with different types of light. The Hills reagent, when reacted with photosynthesis bleaches the solution, making it less absorbent.  A lower absorbance indicates that a greater amount of photosynthesis has occurred (Krha et al 126).  We found that as we increased the amount of fertilizer, photosynthesis was almost unaffected.  All treatments were able to perform photosynthesis more effectively than the control, when given white light.  Only the 0.1g treatment was able to perform photosynthesis better than the control when given red light, and no treatment performed photosynthesis better than the control when given blue light (Table 2).  These results are not very continuous, making our group believe that no major change to photosynthesis occurs when given fertilizer.

When we tested the plant matter with diabetes monitoring glucose test strips, we found that the control group registered an average glucose reading of 19.66 mg/dl. The 0.1gram fertilizer group showed a reading of 20.33 mg/dl, the 0.25gram showed 21 mg/dl, the 0.5gram showed 21 mg/dl and the 1.0gram treatment showed an average reading of 17 mg/dl (Figure 5).  This illustrates to us that the fertilizer actually aided the plants in producing glucose.  As with all of the other tests, it also shows that when too much fertilizer is added, it harms the plant.

We also monitored the growth of the grass.  We found that the grass given 0.1g or 0.25g fertilizer grew quicker than the grass given no fertilizer.  This supports our theory that the fertilizer helps the plant produce more nutrients, aiding it in survival and growth.  However, too much fertilizer proved to slow the growth, as seen in the 0.5 gram and 1.0 gram treatments.  The control averaged a height of 7cm; while the 0.1gram treatment averaged 9cm and the 0.25gram treatment averaged 10cm (Figure 1).  This shows an increase in height in accordance with the fertilizer, but we can not find a direct correlation because of the 0.5gram treatment averaging 5cm and the 1.0g treatment averaging only 4cm.  Again, this leads us to believe that too much fertilizer harms the plant growth.

Our results show us that plants given too much fertilizer are actually much less healthy than plants given a lower amount of fertilizer.  The plants given low amounts of fertilizer are the healthiest and most nutrient rich of all of the treatments we tested.  The 0.25 gram treatment was the healthiest and most nutrient rich of all of the treatments.  The lower fertilizer treatments had higher protein, glucose, grew taller, and also looked healthier than the treatments with a higher amount of added fertilizer.  The 0.1 gram treatment was the second healthiest, followed by the control, 0.5 gram, and 1.0 gram treatments, respectively. This order shows that the use of fertilizer does benefit plants, but only in low doses.  The higher amount of fertilizer causes the nutrients to bind together, prohibiting the plants the use of any of the nutrients from the soil (Kerrigan and Nagel. 1998).  For that reason, I believe that the plants in the higher treatments did not grow as well.  Even though more fertilizer and nutrients were added, there were fewer nutrients actually there for the absorption by the plants. 

One of the biggest problems we found with our experimental method, was that the fertilizer initially blocked the grass seed from sprouting.  This allowed the control group and groups with less fertilizer to grow more quickly, allowing them to get a head start on nutrient production, thus, skewing the results.  We therefore started the experiment over, allowing all of the treatments to sprout, before applying the first dose of fertilizer. After later finding the results of all of our assays, we recognized that the fertilizer blocking the initial sprouting of the grass is most likely directly correlated to the poor health of the higher dosed fertilized treatments.  Another weakness we found with our experiment is that the light boxes we placed the grass plants in may have given different amounts of light to each plant or even an entire treatment.  This could have lead our experiment to incomplete or even inaccurate results. 

The results of our assays and other experiments was able to verify our hypothesis and predictions that an increased amount of fertilizer is able to increase the amount of protein, glucose, photosynthetic ability and height, in low enough doses.  Higher amounts of fertilizer harm all of the tested treatments.  The results support the previous research we found, as well as extended the findings to include a correlation between the amount of fertilizer to crop yield and nutritional value of the plants (VanWieringen et. al. 2005).  VanWieringen et. al. used a different type of fertilizer that may have released nutrients differently or at slower rates.  We originally believed that the more fertilizer added would increase the plants protein, glucose, photosynthesis, and height, but this believe has been proved wrong by our results.  We found that when the amount of fertilizer gets too large, the plants will not produce as many nutrients and possibly eventually die.