TAML Remediation of Eutrophic Water Simon Sweeney Central Catholic High School 9th Grade

Problem Water pollution harms the ecosystem, a solution must be found to combat the pollution by degrading the pollutants in water to negate harmful effects on organisms. Pollution is a huge danger to the Earth's ecosystem. Harmful chemicals common in polluted water include Gasoline Pesticides Chlorine Fertilizers

TAMLs GREEN catalyst engineered by Dr. Terry Collins at Carnegie Mellon University Accelerates H2O2 oxidation reaction Highly selective Made of common elements (C, H, O, N, Fe) Economical Easy to synthesize

TAMLs (cont.) Low working concentrations (nM-low μM) Breaks itself down after reaction Highly water soluble Produces non-toxic byproducts Breaks down stable chlorine compounds Step towards a sustainable society Reduce exotic elements Think in terms of nature’s chemistry

Possible Applications of TAMLs ●Textiles ●Pulp and Paper ●Water Cleaning ●Petroleum Refining ●Biological Warfare

Isopropyl Alcohol Alcohol- the hydroxyl functional group is bound to a saturated carbon atom Isopropyl Alcohol A common rubbing alcohol, toxic Formula: C3H8O

Escherichia coli E. coli – very common, found in intestinal tract of most mammals There are many strains of E. coli, most are non-pathogenic Pathogenic strains can cause illness and death in humans Frequently studied in biology

Purpose Assess the success of TAML at degrading Isopropyl Alcohol by means of a biological assay

Hypotheses Null: TAML will have no significant effect on the lethality of the Isopropyl Alcohol. Alt: TAML will degrade Isopropyl Alcohol and negate the decrease in E. coli survivorship

Materials Isopropyl Alcohol (70%) Fe-TAML B catalyst H2O2 (.88 M) Lab-Grade Catalase (Ward’s) E. coli DH5α Sterile 250mL sidearm flask NaHCO3 (Store bought baking soda) Sterile Dilution Fluid (10mM KH2PO4, 10mM K2HPO4, 1 mM NaCl) Sterile test tubes Micropipette and sterile tips 20-200uL pipette and sterile tips Sterile Luria broth agar plates (1% Tryptone, 0.5% Yeast Extract, 1% NaCl) Tube racks permanent marker Ethanol Vortex Incubator Bunsen burner

Procedure A culture of E. coli DH5α was grown overnight in a sterile 250mL sidearm flask in LB media at 37°C. The culture was left to grow until it reached an absorbance reading of about 50 Kletts, which represents a cell density of about 108 cells/mL. The culture was serially diluted in SDF to a concentration of approximately 103 cells/mL. Petri dishes, poured with LB agar were labeled and left to warm on the lab desk. 0.0353g of solid Fe-TAML B was added to 10mL of sterile water to create a 5*10-3M stock solution. 0.89g of solid NaHCO3 was added to 10mL of sterile water to create a 1M stock solution. 0.1mL of solid lab-grade catalase was added to 30mL of sterile water to create a working concentration of catalase.

Procedure (cont.) 8. Concentrations of Isopropyl alcohol and H2O2 were added to 6 sterile test tubes containing SDF. 9. TAML activators were added to 3 of the tubes 10. Tubes were let sit for 20 minutes while the TAML oxidation reaction occurred 11. Catalase was added to all 6 of the tubes 12. Tubes were let sit for 15 minutes while catalase quenched the reaction 13. E. coli were added to all 6 of the tubes 14. Tubes were let sit for 20 minutes while the E. coli reacted to the solution 15. 0.1 mL of each concentration was plated onto LB agar plates 16. Plates were incubated at 37°C overnight, and the resulting colonies counted

Concentrations (in mL) 0% Alcohol, No TAML 1% Alcohol, No TAML 0% Alcohol, with TAML 1% Alcohol, with TAML Isopropyl Alcohol 0.1 SDF 9.1 9 8.9 E. coli TAML H2O2 0.5 Catalase 0.2 Sodium Bicarbonate Total 10

Average Colony Survivorship without Quenched Reaction Mix P-value= .00621

Average Colony Survivorship with Quenched Reaction Mix P-value= .19999

Stats Isopropyl Alcohol Effect on Survivorship Significant Quenched Reaction Mix and Alcohol Effect on Survivorship Not Significant

Interpretations The solution of TAML Activated H2O2 with Isopropyl Alcohol did not have a significant effect on the survivorship of E. coli. The Isopropyl Alcohol solution that did not include TAMLs did have a significant effect on the survivorship of E. coli. The TAMLs appeared to successfully degrade the Isopropyl Alcohol.

Conclusions REJECTED ACCEPTED Null: The Isopropyl Alcohol will have no significant effect on E. coli survivorship. REJECTED Null: The TAMLs will have no significant effect on the lethality of the Isopropyl Alcohol. Alt: The TAML activated H2O2 will have a significant effect on the degradation of the alcohol. ACCEPTED

Limitations Spread plating was inconsistent in timing and technique Low range of Alcohol Concentrations, TAML and Peroxide Only one exposure time Only one type of exposure (liquid pulse) Isopropyl alcohol not a common pollutant Only one microbial species Limited to one model Limited to one toxic substance

Extensions Greater range in exposures and time of exposure More microbial models Different type of exposure Chemically define the degradation products from the TAML reaction

References Carnegie Mellon University Green Science http://www.chem.cmu.edu/groups/Collins/ http://www.freedrinkingwater.com/water- contamination/water-contaminants-pollutants-list.htm http://www.cdc.gov/niosh/npg/npgd0359.html www.chem.cmu.edu/groups/collins/about/about.html http://www.cmu.edu/news/stories/archives/2015/june/catal ysts-remove-estrogenic-compounds-from-wastewater.html

Results (colony counts) Replicant 0% Alcohol, No TAML 1% Alcohol, No TAML 0% Alcohol, with TAML 1% Alcohol, with TAML 1 141 109 86 60 2 152 101 87 71 3 175 96 83 72 4 126 111 79 5 121 56 70

ANOVA (No Quenched Reaction Mix)

ANOVA (Quenched Reaction Mix)