FRACKING CHEMICAL Effect on Microbial Life Luke Deasy Central Catholic High School Grade 9 1st Year in PJAS
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.
OBJECTIVE To determine whether or not fracking chemicals are harmful to the environment
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?
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.
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
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) 75.65 16.65 0.475 0.3846 0.1857 0.20
SACCHAROMYCES cerivisiae Unicellular fungi (species of yeast) Round to ovoid in shape Reproduce asexually Commonly used as model organism Ubiquitous environmental microbe Reproduces quickly
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.
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
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 50-60 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.
PROCEDURE (CONTINUED) 6. 0.1 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.
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
FRACKING CHEMICALS EFFECT ON MICROBIAL Number of Yeast Colonies 130 97.5 65 32.5 0 LIFE P Value: 1.1E-09 0% 0.01% 0.1% 1% Concentration of Fracking Chemical Average Yeast Colonies
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 1 8 949 118.625 89.41071429 Column 2 8 1028 128.5 167.4285714 Column 3 8 751 93.875 43.26785714 Column 4 8 677 84.625 80.55357143 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 10151.09375 3 3383.697917 35.55605073 1.0978E-09 2.946685266 Within Groups 2664.625 28 95.16517857 Total 12815.71875 31
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
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. 3. 0.1 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.
INFUSION Number of Yeast Colonies 300 285 270 255 P Value: 0.004 Number of Yeast Colonies 240 Control Infusion Type of Plate
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.
LIMITATIONS Plating could have been slightly unsynchronized Only three concentrations of variable tested Only one experimental model used
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
RESOURCES http://www.huffingtonpost.com/2012/12/06/ fracking-impacts-livestock_n_2251055.html http://www.dosomething.org/tipsandtools/ background-fracking http://www.propublica.org/special/hydraulicfracturing-national http://www.epa.gov/hydraulicfracture/
RESULTS Concentration 0 0.01% 0.1% 1% 120 132 87 82 108 118 84 95 130 142 102 92 128 122 88 68 121 120 96 83 126 124 97 93 106 117 99 78 110 153 98 86 Average 114.7 128.5 97.5 89.2
INFUSION RESULTS Type of Plate Control Infused 274 257 314 242 285 229 297 204 263 264 234 280 262 251 Average 292.5 248.6