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

2. 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.

3. 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

4. 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

5. 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

6. 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

7. 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.

8. 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.

9. 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

10. 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.

11. 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

12. 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

13. Procedures (cont.)
5. The test tubes were then mixed through inversion for 15 minutes to emulsify the solution ml samples were removed from tubes and plated onto YEPD agar 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.

14. 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

15. 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

16. 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

17. 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

18. 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

19. Average Colony Count of Concentration
____________
Average of
Control

20. 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

21. 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

22. Works cited
National Center for Biotechnology Information. PubChem Compound Database; CID=1118,
(accessed Nov. 1, 2016).
"E. Coli Infections: MedlinePlus." MedlinePlus. N.p., n.d. Web. 30 Nov
Bukhari, Mohammad. "Staphylococcus Epidermidis." Staphylococcus Epidermidis. N.p., 27
"What Is Acid Rain?" EPA. Environmental Protection Agency, 31 Mar Web. 30 Nov.
Mark Krotec, PTEI

23. 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 4
E-08
Within Groups
670.8 20
33.54
Total 24

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 4
E-09
Within Groups
2291.6 20
114.58
Total 24