Acid Rain Effects on Microbial Survivorship Ramsey Lewand
Acid Rain Effects on Microbial Survivorship Ramsey Lewand Pittsburgh Central Catholic PJAS 2017 Grade 9
Purpose and Problem: How does acid rain affect life, especially that of microbes in Earth's soil? Purpose: To determine if sulfuric acid has significant effects on the survivorship of Escherichia coli and Staphylococcus epidermidis.
Acid Rain Contains high amounts of nitric and sulfuric acids Created by emissions of sulfur dioxide and nitrogen oxides Emissions come from both natural and man made factors Can be deposited onto the earth in two forms: dry and wet deposition Average p. H: 4. 0
Escherichia coli Commonly known as E. coli Gram-negative(-) bacterium Found in the lower intestines of warmblooded organisms Used routinely in microbiology Found in ecosphere and used as a model for gram(-) bacteria found in soil
Staphylococcus epidermidis Commonly known as Staph Gram-positive(+) bacterium Part of normal human flora, especially the skin flora Commonly Used used as a lab subject as a model for gram(+) bacterium found in soil
Past studies High concentrations of sulfuric acid have been shown to be a hindrance to the growth and survivorship of microbes Lessened growth/survivorship can affect microbes’ ability to perform nitrogen fixation Lack of nitrogen fixation can kill surrounding organisms
Interaction of Components Microbes are commonly affected by acid rain, which can have devastating effects on the surrounding environment The sulfuric acid affects the organisms’ p. H levels, possibly causing enzymes to malfunction and eventually kill cells.
Hypothesis Null hypothesis- the sulfuric acid will not have significant effects on the survivorship of Escherichia coli and Staphylococcus epidermidis. Alternative hypothesis- the sulfuric acid will have significant effects on the survivorship of Escherichia coli and Staphylococcus epidermidis.
Materials Escherichia coli Staphylococcus epidermidis 10 Test tubes (5 x E. coli, 5 x Staph) Micro-pipettes Micro-pipette tips Test tube racks 0. 5 M sulfuric acid YEPD Agar Petri Dishes 1% yeast extract, 2% peptone, 2% dextrose Sterile Dilution Fluid 100 m. M KH 2 PO 4, 100 m. M K 2 HPO 4, 10 m. M Mg. SO 4, 1 m. M Na. Cl Sterile Spreader Bars Vortex Bunsen burner Ethanol Incubator
Procedures 1. A culture of E. coli C 600 was grown overnight. 2. The culture of cells was diluted to a concentration of 108 cells/ml. 3. Staphylococcus epidermidis was grown until a density of 50 klett spectrophotometer density was reached. This was approximately 108 cells/ml. 4. The following ingredients were used to create the following exposures.
Tube concentrations (m. L) Molarity 10 -3 10 -4 10 -5 10 -6 Control 0 Of 10 -2 Of 10 -4 Sulfuric Acid 1 0 Sterile Fluid Microbe (Either E. coli or Staph) Total Volume 8. 9 9. 8 9. 9 0. 1 0. 1 10 10 10 substock
Tube concentrations (cont. ) Molarity 10 -3 10 -4 10 -5 10 -6 Of 10 -2 Of 10 -4 3. 0 5. 0 6. 5 substock p. H substock *Acid rain has an average p. H value of 4. 0 substock Control 0 substock 7. 0
Procedures (cont. ) 5. The test tubes were then mixed through inversion for 15 minutes to emulsify the solution. 6. 0. 1 ml samples were removed from tubes and plated onto YEPD agar. 7. 5 replicates were plated for each concentration. 8. The plates were incubated at 37°C for approximately 18 hours. 9. The resulting colonies were counted visually. Each Colony was assumed to have arisen from one cell.
Anova: Single Factor Analysis of data that compares variation within groups to variation between the groups Alpha: If 0. 05 the p-value is below the alpha, then the effects are significant
E. coli Survivorship Curve Number of E. coli Colonies 90 80 70 60 Average Colony Count Control 0 10^-6 M 10^-5 M 10^-4 M 10^-3 M 77 72. 2 70. 8 66 40. 4 50 40 P-value: 2. 48 -8 30 20 10 0 Control 0 10^-6 M 10^-5 M 10^-4 M Concentration of Sulfuric Acid (M) 10^-3 M
Number of S. Epidermidis Colonies S. epidermidis Survivorship Curve 250 200 150 Average Colony Count Control 0 10^-6 M 10^-5 M 10^-4 M 10^-3 M 183. 6 169. 6 161. 6 140. 2 106. 6 100 P-value: 3. 33 -9 50 0 Control 0 10^-6 M 10^-5 M 10^-4 M Concentration on Sulfuric Acid (M) 10^-3 M
Dunnett's Test Compares experimental groups with the control Determines which experimental groups produced significant variation If T value > T-crit then the results were significant
Dunnett's Test Results T-crit=3. 48 10 -3 10 -4 10 -5 10 -6 Sulfuric Acid E. coli T-value 9. 991 3. 003 1. 692 1. 310 Significance Significant Not Sig. S. epidermidis T-Value Significance 11. 375 6. 411 3. 250 2. 068 Significant Not Sig.
Average Colony Count of Concentration ______ Sulfuric Acid Comparative Effects Percentage of Surviving Microbes 100 92. 5 93. 8 90 88 92 85. 7 _ 76. 3 80 70 58 60 52. 5 50 40 30 20 10^-6 10^-5 10^-4 Concentration of Sulfuric Acid (M) Staph E. coli 10^-3 Average of Control
Conclusion The ANOVA for Escherichia coli and Staphylococcus epidermidis presented a p-value lesser than 0. 05, so the null hypothesis was rejected. E. coli- significant effect at 10 -3 M. S. epidermidis- significant effect at 10 -3 M and 10 -4 M S. epidermidis was more greatly affected at the lower concentrations; E. coli colonies had decreased more in higher concentrations
Limitations and Extensions Limitations- Extensions- Health of Microbes? More replicates could have supplied more conclusive evidence More bacteria species could have been used More concentrations of sulfuric acid could have been used Use more species of bacteria Use more replicates Use more concentrations of sulfuric acid Use concentrations of nitric acid The synergistic effects of sulfuric acid and other chemicals
Works cited National Center for Biotechnology Information. Pub. Chem Compound Database; CID=1118, https: //pubchem. ncbi. nlm. nih. gov/compound/1118 (accessed Nov. 1, 2016). "E. Coli Infections: Medline. Plus. " Medline. Plus. N. p. , n. d. Web. 30 Nov. 2016. Bukhari, Mohammad. "Staphylococcus Epidermidis. " Staphylococcus Epidermidis. N. p. , 27 "What Is Acid Rain? " EPA. Environmental Protection Agency, 31 Mar. 2016. Web. 30 Nov. Mark Krotec, PTEI
Escherichia coli Anova: Single Factor SUMMARY Groups Row 1 Row 2 Row 3 Row 4 Row 5 ANOVA Source of Variation Between Groups Within Groups Total Count Sum 5 5 5 SS Average 385 361 354 330 202 df Variance 32. 5 31. 7 29. 2 56. 5 17. 8 77 72. 2 70. 8 66 40. 4 MS 4176. 24 4 670. 8 20 4847. 04 24 F P-value F crit 1044. 06 31. 12880143 2. 47835 E-08 2. 866081402 33. 54
Staphylococcus epidermidis Anova: Single Factor SUMMARY Groups Row 1 Row 2 Row 3 Row 4 Row 5 ANOVA Source of Variation Between Groups Within Groups Total Count Sum 5 5 5 SS 918 848 808 701 533 df Average Variance 183. 6 162. 3 169. 6 124. 3 161. 6 137. 3 140. 2 100. 7 106. 6 48. 3 MS 18001. 84 4 2291. 6 20 20293. 44 24 F P-value F crit 4500. 4639. 27788445 3. 32799 E-092. 866081402 114. 58
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