FRACKING CHEMICAL. Luke Deasy Central Catholic High School Grade 9 1st Year in PJAS. Effect on Microbial Life. Tuesday, January 29, 13

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1 FRACKING CHEMICAL Effect on Microbial Life Luke Deasy Central Catholic High School Grade 9 1st Year in PJAS

2 PROBLEM World s supply of natural gas is depleting A number of people believe that we must find new sources of natural gas to supply current energy demands. Many new techniques have arisen, one of which is hydraulic fracturing.

3 OBJECTIVE To determine whether or not fracking chemicals are harmful to the environment

4 CONTROVERSY Hydraulic Fracturing is effective, however it may be unhealthy for the environment. Many nature groups, such as EPA, have spoken against the practice of hydraulic fracturing. The root of the problem is the chemical solution used in mining the gas. Are living organisms at risk from fracking?

5 PREVIOUS STUDIES The U.S. Environmental Protection Agency is investigating the possible downsides to hydraulic fracturing. Studies indicate that livestock milk production and health are depleting near places where hydraulic fracturing and the making of fracking chemicals is taking place.

6 FRACKING CHEMICALS Substance used to pump natural gas out of the ground. Regulations prevented the obtaining of genuine fracking chemicals. Simulation (researcher approved) chemical mix substituted

7 FRACKING CHEMICALS SIMULATION Made of Sodium Chloride, Calcium Chloride, Magnesium Chloride, Potassium Chloride, Barium Chloride, Strontium Chloride Chemical Sodium Chloride Calcium Chloride Magnesium Chloride Potassium Chloride Barium Chloride Strontium Chloride Amount (mg/l)

8 SACCHAROMYCES cerivisiae Unicellular fungi (species of yeast) Round to ovoid in shape Reproduce asexually Commonly used as model organism Ubiquitous environmental microbe Reproduces quickly

9 HYPOTHESES Null Hypothesis The fracking chemicals will not significantly affect the survivorship of yeast. Alternative Hypothesis The fracking chemicals will significantly reduce the survivorship of yeast.

10 MATERIALS Fracking Chemicals Simulation (NaCl, CaCl, MgCl, BaCl, KCl, SrCl) Saccharomyces cerivisiae Micropipettes Micro rack Microtubes Turn table Spreader bars Ethanol Bunsen burner YEPD Media and agar plates (1% yeast extract, 2% peptone, 2% glucose (dextrose), 1.5% agar) 20 ml Sterile capped test tubes with Sterile Dilution Fluid (SDF) (10 mm KH2PO4, 10 mm K2HPO4, 1 mm MgSO4, 0.1 mm CaCl2, 100 mm NaCl) 0.22 micron syringe filters + 10 ml syringe Incubator Vortex Sidearm flask Klett Spectrophotometer

11 PROCEDURE I. Liquid Pulse Exposure 1. Saccharomyces cerivisiae was grown overnight in a sterile YEPD media. 2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3. The culture was incubated at 37 C until a density of Klett spectrophotometer units was reached (Cell density approximately 10 7 cells/ ml). 4. The cell culture was diluted in a sterile dilution fluid to a concentration of approximately 10 5 cells/ml. 5. The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations to a total of 9.9 ml.

12 PROCEDURE (CONTINUED) ml. of cell culture was then added to the test tubes, yielding a final volume of 10 ml. and a cell density of approximately 10 3 cells/ml. 7. The solution was mixed by vortexing and allowed to sit at room temperature for 15 minutes. 8. After vortexing to evenly suspend cells, 0.1 ml aliquots were removed from the tubes and spread on 64 YEPD agar plates. 9. The plates were incubated at 30 C for 48 hours. 10. The resulting colonies were counted. Each colony is assumed to have risen from one cell.

13 CONCENTRATION CHART Concentration of Fracking Chemicals 0% 1% 0.10% 0.01% Sterile Fluid 1 ml.9 ml.99 ml.999 ml Fracking Chemicals 0 ml.1 ml.01 ml.001 ml Yeast.1 ml.1 ml.1 ml.1 ml Sterile Fluid 8.9 ml 8.9 ml 8.9 ml 8.9 ml

14 FRACKING CHEMICALS EFFECT ON MICROBIAL Number of Yeast Colonies LIFE P Value: 1.1E-09 0% 0.01% 0.1% 1% Concentration of Fracking Chemical Average Yeast Colonies

15 SINGLE FACTOR ANOVA Analysis of variation Statistical test that assesses the differences between means of multiple groups. Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column Column Column Column ANOVA Source of Variation SS df MS F P-value F crit Between Groups E Within Groups Total

16 SIGNIFICANCE TEST (DUNNETT S TEST) If t>t crit significant T Critical=2.88 Alpha=.05 Fracking Chemicals Concentration T-Value Interpretation 0.01% 0.91 Not Significant 0.1% 2.23 Not Significant 1% 3.12 Significant

17 YEPD AGAR INFUSION PROCEDURE II. Infusion 1. Sterilized fracking chemicals were infused into YEPD agar media in one concentration (0.2 ml of the concentrated fracking fluid) and let sit in an incubator for 1 hour. 2. Yeast was grown overnight in sterile YEPD media ml from the control tube was spread onto each infused plate. 4. The plates were incubated at 37 C for 24 hours. 5. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

18 INFUSION Number of Yeast Colonies P Value: Number of Yeast Colonies 240 Control Infusion Type of Plate

19 CONCLUSIONS Reject the null hypothesis that fracking chemicals will not affect yeast survivorship in both types of exposures. The increased concentration of fracking chemical correlated with decreasing yeast survivorship Significant reduction and survivorship was found at concentrations of 1% and above.

20 LIMITATIONS Plating could have been slightly unsynchronized Only three concentrations of variable tested Only one experimental model used

21 FUTURE STUDIES Determine which specific compound in the simulation was the culprit for the killing of yeast Begin studying about possible green fracking solutions Test the effect of fracking chemical solution on different mediums such as E. coli or Staph Test synergistic effects Try to narrow the concentration range of affect Larger concentration range for infusion technique Test changes in growth rate

22 RESOURCES fracking-impacts-livestock_n_ html background-fracking

23 RESULTS Concentration % 0.1% 1% Average

24 INFUSION RESULTS Type of Plate Control Infused Average