Degrading PCE ( Tetrachloroethylene ) In Sulfide Conditions using Bacteria

1. Degrading PCE (Tetrachloroethylene) In Sulfide Conditions using Bacteria By: Zach Steinbaugh and Greg Ancmon Monarch High School

2. Introduction • Tetrachloroethylene (PCE), is a man-made liquid that is widely used in dry cleaning and for removing grease from metal surfaces. • Tetrachloroethylene is a colorless liquid that has a sweet odor detectable by most people at 1 ppm. • Tetrachloroethylene is also an excellent solvent of organic materials. • PCE is an organic compound that is part of a class of chemicals called chloinated solvents.

3. Research Question • Will PCE be degraded into a less harmful form in different sulfide concentrations by using a naturally occurring bacteria enriched with natural microbial consortium containing species of Dehalococcoides sp.(DHC) • The degradation of PCE is a multi stepped process that requires a livable environment for the bacteria to function properly if we are to have a successful project what types of sulfide environments will prove to work the best for the bacteria?

4. Hypothesis • If the bacterium is not inhibited at certain sulfide concentrations and has the right conditions to live, they should break down the chemical PCE into its less harmful forms such as TCE, DCE, VC (Vinyl Chloride) and eventually Ethane.

5. Background • The chemical PCE is a known human carcinogen along with its by products TCE, DCE, VC. Vinyl Chloride is the most harmful to humans. Drinking Water Regulations by the USEPA (U.S. Environmental Protection Agency) states PCE and TCE must be no greater than 5 mg/L. DCE and can’t exceed 70 mg/L. VC can’t exceed 2 mg/L.

6. Background cont. • The samples were taken from a dry cleaning site in Thornton, Colorado. Different wells were drilled around the dry cleaning site to take samples of ground water to test for all concentrations of the PCE, TCE, etc.

7. Visual Breakdown of PCE

8. Procedure • Set up twenty bottles with various amounts of solutions. • Mix will need to consist of PCE, emulsified vegetable oil (carbon source), sulfide (bi-product of breakdown), sulfate (naturally occurring), and artificial ground water. • Monitor changes in the activity of PCE. The bottles need to be kept in a nitrogen container. • Take weekly samples to test for PCE, TCE, DCE, VC, Sulfate, Sulfide, Ph, carbon and gas levels. The bottles will turn various colors depending on the rate of chemical degradation.

9. Experimental Setup

10. Methods (Sulfide/Sulfate) • Ph will be measured by litmus paper tests. • Sulfide and Sulfate will also be measured by spectrophotometer. • Measuring Sulfide: First: Make a solution of sulfide by using 100ml of water mixed with .7314g of Sulfide. Stir thoroughly. • Dilute into 6 bottles 1) .8mg/L sample 2) .4mg/L Sample 3) .2mg/L Sample 4) .1 mg/L Sample 5) .05 mg/L Sample 6) .025mg/L • Fill the rest of the bottles with milli Q water and make them all equal to 10 ml even. • Next drip 1ml of the sulfide reagent into each bottle. • The solution will turn an aqua/lavender when there is sulfide present. When you have waited five minutes for each bottle you may measure the concentrations for each bottle using a spectrophotometer at a set wave length of 664 alphas. • Record Data. • Lastly clean up all materials and dispose properly of all the hazardous chemicals. • Measuring Sulfate: Same as last example. Dilutions: 1) 70mg/L 2) 35 mg/L 3) 17.5mg/L 4) 8.75 mg/L 5) 4.375 mg/L 6) 2.1875

11. Pre Sulfate Calibration Curve

12. Pre Sulfide Calibration Curve

13. TCE Extraction • Weigh each of the 20 syringes. • Extract 3 ml of Pentane and weigh needles again. • Next Extract 1 ml of sample from each bottle into each needle. • Shake the needles for about 2 minutes each so PCE, TCE, DCE should separate. • Bend each needle so the chemicals will float to the top and squirt them into little 1 ml bottles to be sent of for testing. • Weigh each needle again. Clean up all needles and materials and dispose properly of all the hazardous chemicals.

14. Total Organic Carbon • First: Extract 4mL of solution per bottle. • Second: Use small filters to get out the unnecessary items from the solution. • Third: Dilute 5 extracted solutions into milliliters of your choice. • Fourth: Measure using the TOC Machine. Lastly clean up you lab area and any hazardous chemicals.

15. General Conclusions • Initially, we planned to have results by early February. We ran into several sources of error. The main source of error included miscalculating our sulfide concentrations to be added into each of our bottles, resulting in ¾ of our bottles to have no results because the high pH caused all of the bacteria to die. • We hope to have preliminary results by early April which include all of our extraction data. (PCE/TCE, TOC, pH) • We expect our pH to remain constant (ph 7) throughout the whole experiment. We expect PCE concentration to decline and see an increase in TCE and DCE concentrations. Along with these tests, we will continue to test gas production by the bacteria.

16. Bibliography • Angela R Bielefeldt; Environmental Engineering Students Helping Clean-up Sites Contaminated By Dry Cleaning Activities In Colorado; Engineering Excellence Fund Committee, University of Colorado, Boulder; Feb. 28, 2008. • Angela R Bielefeld; Remediation Of Sites Contaminated By Dry Cleaning Activities In Colorado; Outreach Committee, University of Colorado, Boulder; May 2, 2007. • Samuel Fogel; Bioremediation of Carbon Tetrachloride and PCE: Field Results; Bioremediation Consulting Inc. Report, Watertown, MA. • Stephen H. Zinder and J. M. Gossett ; Environmental Health Issues ; Environmental Health Perspectives Supplements Vol. 117 No. 12 Dec. 2009. • DiStefano TD, Gossett JM, Zinder SH. Reductive dechlorination of high concentrations of tetrachloroethene to ethene by an anaerobic enrichment culture in the absence of methanogenesis. Appl Environ Microbiol 57:2287-2292 (1991). • Habeck, Micheal. "Tetrachloroethylene." Eco-USA. United States Public Health Service, 1996. Web. 13 Feb. 2010. <http://www.eco-usa.net/toxics/chemicals/tetrachloroethene.shtml>.

17. Thank you! • Angela R. Beilefeldt, Ph.D, Director of Environmental Engineering, University of Colorado, Boulder. • Meredith Berlin, Graduate Student of Environmental Engineering, University of Colorado, Boulder. • Eva Kraus, Graduate Student of Environmental Engineering, University of Colorado, Boulder.