Abstract

    Eutrophication and algal blooms are extremely important topics to scientists, environmentalists, and fisheries.  In this experiment, the effects of nitrogen and nitrates on the production and growth of Euglena gracilis, a unicellular, photosynthetic eukaryote were explored.  Specifically, tests were performed to determine whether the addition of nitrates resulted in increased production of sugars, chlorophyll and proteins.  It was predicted that as the concentration of nitrates present in the euglena solution increased, the production of these various chemicals would also increase.  Four concentrations of sodium nitrate/euglena solution (0 mg/L NaNO₃, 500 mg/L NaNO₃, 1000 mg/L NaNO₃, and 1500 mg/L NaNO₃) were set up.  The tests performed on sugar production were Benedict’s, Barfoed’s, Selivanoff’s, Bial’s, and the iodine test.  The only test to give results was Selivanoff’s, which showed gradually increasing color intensity with increased nitrate concentration.  Hill’s Reaction, which measures the action spectrum of photosynthesis, could not detect any increased photosynthetic rates.  This test gave results that were not adequate for any proper conclusions.  The final test performed was the Bradford Assay, which detects increases/decreases in protein production.  The results of this test were inconclusive when compared against a standard curve.  Assays were conducted at the microscopic level for several sugar tests; however, the results were not completely definitive as to what they indicated.  It was also determined that many of these assays are not sensitive enough to detect minute amounts of the sugar molecules, as some of the tests were completely absent of reactivity. 



Discussion

   In this experiment, our main focus was to observe the effects of sodium nitrate on the major cell processes of euglena. Specifically, we focused on three processes: the production of sugars, the process of photosynthesis, and the production of proteins. Euglena have been shown to successfully process nitrates as a form of nitrogen (Cook, 1998).  Because of this, we predicted that experimental groups with added sodium nitrate would exhibit increased production of sugars, chlorophyll, and enzymes as a result of this increased amount of nitrogen in their environment. 

Sugars
    Of the five tests performed to detect various characteristics of the sugars, four tests produced no reaction.  Free aldehyde or ketone groups could not be detected in euglena after running Benedict’s test.  Monosaccharides also could not be detected by Barfoed’s test.  Negative results for Bial’s test and the iodine test were unable to identify furanoses and coiled polysaccharides, respectively.  Although this does not support our hypothesis, the lack of a reaction may not indicate a lack of sugars.  As stated in the introduction, one of our goals in this investigation was to see if the macroscopic tests performed previously can be applied to microscopic organisms.  Due to the euglena’s microscopic size, the sugars that they produce may not be present in significant amount to create visible color changes in the macroscopic tests that were run.  Because of this possible problem, certain microscopic tests were conducted on euglena, as will be discussed later.
    Unlike the results of the four tests mentioned above, the fifth test, Selivanoff’s test did seem to provide a reaction with the euglena solution.  The trial without the addition of sodium nitrate had a very light yellow color change. The three trials which contained sodium nitrate exhibited progressively stronger orange color changes. This reaction may indicate the presence of ketoses, considering the reaction time of three minutes. The darker orange color was observed increasingly in correspondence with the increase in sodium nitrates in the solution, and this may also indicate an increase in the concentration of these sugars, which is in support of our hypothesis.  To test this, our analysis of the absorption spectrum of the four trials of this test shows that an increase in the concentration of sodium nitrate corresponds with an increase in the absorption of the product.  This coincides well with the likelihood of an increase in the concentration of sugars mentioned above.
    Despite this evidence, it is conceivable that the reaction of Selivanoff’s test in this case does not indicate an increase in sugars.  There is evidence to suggest that major reagent found in Selivanoff’s reagent, resorcinol, can also be used as a reducing agent for nitrates (Velghe and Claeys, 1985).  If this redox reaction occurred, it would not correspond with an increase in the sugars produced by the euglena.
    A second possibility also may contradict our findings.  As stated previously, it has been found that euglena do have the ability to process nitrates as a form of nitrogen. However, a study by J. Wolken of the University of Pittsburgh School of Medicine suggests that euglena cannot process nitrates. Wolken suggests they feed on ammonia as a primary source of nutrition (Wolken, 1961).  One researcher has even commented that nitrogen metabolism in euglena has not been comprehensively studied, and that various strains and species of euglena can have differing nitrogen requirements (Buetow, 1968).  Nitrates, ammonium, or even amino acids may be used as nitrogen sources.  The inability of the researched species of euglena to process nitrates would clearly indicate an inconsistency in experimental techniques for the sugar assays as well as the Hill Reaction and Bradford Assay.


Microscopic Tests
    To adjust to the extremely small size of Euglena gracilis, our research group experimented with some techniques that we hoped would indicate increased sugar productions at the microscopic level.  With the aid of a microscope, some interesting changes were noticed.  At the cellular level, the three sugar tests run did show color changes on the membrane and inside the euglena.  This would indicate that the presence of sugars could be determined microscopically.  One problem still prevails.  It is extremely difficult to measure the relative changes in color in euglena at various nitrate concentrations, even at the microscopic level.  Sugars were detected in the euglena, but it is nearly impossible to determine how much sugar was present or whether there was an increase in sugar production.  If a spectrophotometer could be utilized at the microscopic level to reveal changes in absorbency, the problem would ideally be solved.

Photosynthesis
    The second test performed, The Hill Reaction, tested the action spectrum of the euglena.  A lower absorbance of a sample indicates a higher rate of photosynthesis.  Based on the experimental data, our results for the Hill Reaction were inconclusive and do not correlate to our hypothesis.  For two of the three trials ran, the absorbencies were lower for the 0 mg NaNO₃/L than for the higher concentrations of nitrates in euglena solution, and this was opposite of what was hypothesized.  Based on the inconclusiveness of the data, the hypothesis that photosynthesis would increase with nitrate concentration cannot be supported.
    Due to constraints in both equipment and lab techniques, it was hard to ensure an accurate reading of absorbance for this test.  The transport of the samples while keeping them in the dark (away from all light sources) was especially difficult given our classroom laboratory conditions.  It is possible that this had an effect on the outcome of this portion of our experiment.  More accurate tests in a more suitable environment would be needed to discern whether this was actually the case.

Proteins
    Our final test, the Bradford Assay, tested for protein concentration. The results for this assay are extremely hard to interpret.  Focusing solely on the absorption values for the euglena, there seems to be an increase in protein concentration when the nitrate concentration is increased.  However, when comparing these values to the standard curve made by measuring specific concentrations of Bovine Serum Albumin, the measured absorbency values for euglena/Bradford reagent would suggest that there were actually negative amounts of protein in the Euglena solution, which would be impossible.  This data is inconclusive and the hypothesis that total concentration of proteins produced by the euglena was increased with the addition of sodium nitrate into their environment cannot be supported.

Conclusion
    None of the three of the assays performed  (Sugar Tests, the Hill Reaction, and the Bradford Assay) offer support to our hypotheses that the amounts of sugar, chlorophyll, and protein produced by Euglena gracilis are increased with the addition of sodium nitrate to their living environment.  Our experimental data does not indicate that nitrates increase the rate of the cellular processes in euglena, if euglena processes nitrates at all.
    Throughout the experiment, it was difficult to suit our tests to fit the size of the euglena.  Our experimentation group had a limited supply of euglena, and thus many trials of the assays in this test were not possible.  It was also impossible to discern exactly how many euglena were in each vial that was received, as there was no standard count given.  Thus we were unable to determine if the addition of sodium nitrate had any effect on their reproduction or overall growth.
    When relating our experiment to the real life problem of algal blooms and eutrophication, many similarities can be found.  If an increased presence of nitrogen and/or phosphorus from fertilizers causes rapid growth and reproduction of algae, plankton, and euglenoids, then it is possible that it will have a similar effect on the cellular processes of Euglena gracilis as well.  Increased production of sugars, chlorophyll, and proteins, consistent with our test results, favor the overall well-being of the euglena.  These increases would lead to higher growth and reproduction rates as well.  By studying the beneficial effects of nitrogen on the euglena, it directly links the introduction of fertilizers in the area to the potentially harmful effects on an aquatic habitat.  Perhaps by manufacturing alternatives to high nitrogen/high phosphate fertilizers, algal blooms and eutrophication would no longer be such a pressing environmental issue. 
 


Sample Figure