Acid Rain Effects on Microbial Survivorship Ramsey Lewand Pittsburgh Central Catholic PJAS 2017 Grade 9

Purpose and Problem 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 pH: 4.0

Escherichia coli Commonly known as E. coli Gram-negative(-) bacterium Found in the lower intestines of warm- blooded 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 as a lab subject Used 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’ pH 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 Sterile Dilution Fluid Staphylococcus epidermidis 100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl 10 Test tubes (5x E. coli, 5x Staph) Sterile Spreader Bars Micro-pipettes Vortex Micro-pipette tips Bunsen burner Test tube racks Ethanol 0.5 M sulfuric acid Incubator YEPD Agar Petri Dishes 1% yeast extract, 2% peptone, 2% dextrose

Procedures 1. A culture of E. coli C600 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 (mL) Molarity 10-3 Of 10-2 substock 10-4 10-5 Of 10-4 substock 10-6 Control 0 Sulfuric Acid 1 0.1 Sterile Fluid 8.9 9.8 9.9 Microbe (Either E. coli or Staph) Total Volume 10

3.0 5.0 6.0 6.5 Tube concentrations (cont.) 10-3 10-4 10-5 10-6 Molarity 10-3 Of 10-2 substock 10-4 10-5 Of 10-4 substock 10-6 Control 0 pH 3.0 5.0 6.0 6.5 7.0 *Acid rain has an average pH value of 4.0

Procedures (cont.) 5. The test tubes were then mixed through inversion for 15 minutes to emulsify the solution. 6. 0.1ml 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: 0.05 If the p-value is below the alpha, then the effects are significant

P-value: 2.48-8 Control 0 77 10^-6M 72.2 10^-5M 70.8 10^-4M 66 10^-3M Average Colony Count Control 0 77 10^-6M 72.2 10^-5M 70.8 10^-4M 66 10^-3M 40.4 P-value: 2.48-8

P-value: 3.33-9 Control 0 183.6 10^-6M 169.6 10^-5M 161.6 10^-4M 140.2 Average Colony Count Control 0 183.6 10^-6M 169.6 10^-5M 161.6 10^-4M 140.2 10^-3M 106.6 P-value: 3.33-9

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 10-3 10-4 10-5 10-6 E. coli T-value 9.991 3.003 T-crit=3.48 10-3 Sulfuric Acid 10-4 10-5 10-6 E. coli T-value 9.991 3.003 1.692 1.310 Significance Significant Not Sig. S. epidermidis T-Value 11.375 6.411 3.250 2.068

Average Colony Count of Concentration ____________ 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 10-3M. S. epidermidis- significant effect at 10-3M and 10-4M S. epidermidis was more greatly affected at the lower concentrations; E. coli colonies had decreased more in higher concentrations

Limitations and Extensions Health of Microbes? Use more species of bacteria More replicates could have supplied more conclusive evidence Use more replicates Use more concentrations of sulfuric acid More bacteria species could have been used Use concentrations of nitric acid The synergistic effects of sulfuric acid and other chemicals More concentrations of sulfuric acid could have been used

Works cited National Center for Biotechnology Information. PubChem Compound Database; CID=1118, https://pubchem.ncbi.nlm.nih.gov/compound/1118 (accessed Nov. 1, 2016). "E. Coli Infections: MedlinePlus." MedlinePlus. 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 Count Sum Average Variance Row 1 5 385 77 32.5 Row 2 361 72.2 31.7 Row 3 354 70.8 29.2 Row 4 330 66 56.5 Row 5 202 40.4 17.8 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 4176.24 4 1044.06 31.12880143 2.47835E-08 2.866081402 Within Groups 670.8 20 33.54 Total 4847.04 24  

Staphylococcus epidermidis Anova: Single Factor SUMMARY Groups Count Sum Average Variance Row 1 5 918 183.6 162.3 Row 2 848 169.6 124.3 Row 3 808 161.6 137.3 Row 4 701 140.2 100.7 Row 5 533 106.6 48.3 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 18001.84 4 4500.46 39.27788445 3.32799E-09 2.866081402 Within Groups 2291.6 20 114.58 Total 20293.44 24