by Thomas OConnor Central Catholic High School Grade Run Off and Pollution Risk Pesticides include any chemical, antibacterial, biological agent, or other similar substance used to kill or repel unwanted species. Pesticides can easily get into water supplies Toxic substances in water supplies kill life and disrupt ecosystems What technologies can we develop to treat this problem? Tetra Amido Macrocyclic ligand (TAML) GREEN catalyst engineered through research led by Dr. Terry Collins of Carnegie Mellon Universtiy Accelerates H 2 O 2 oxidation reaction Highly selective Broad Range Made of common elements found in nature (C, H, O, N, Fe) GREEN Economical Easy to synthesize TAML (continued) Structure Central iron atom, specific location of reactivity. Site to which hydrogen peroxide binds while transforming to a highly reactive form Attached to ringed carbon structure(macrocycles) by 4 nitrogen atoms (tetra-amido) Possible Applications Textiles Pulp and Paper Water Cleaning Petroleum Refining Biological Warfare Paper Mill releasing bleaches and other pollutants into water supplies Carbaryl Pesticide used was 1- naphthyl N-methylcarbamate, colloquially known as Carbaryl Used as insecticide Works by inhibiting acetylcholinesterase (AchE) Acetylcholinesterase degrades acetylcholine from the synaptic cleft between an affecter nerve and a muscle. Acetylcholine is the neurotransmitter that propagates the contraction signal from the central nervous system to muscle It binds to receptors on muscle cell, stimulating the cascade of reactions resulting in contraction Inhibition of AchE results in excessive acetylcholine in synaptic cleft, causing muscle to continually contract. E. Coli Escherichia coli (E. coli) very common, found in intestinal tract of most mammals One of the most Frequently studied prokaryotic organisms in biology. Can be easily manipulated, makes good subject for experimentation There are many strains of E. coli, most are non- pathogenic, although Pathogenic strains are quite dangerous and can cause illnesses. Frequently studied in biology ubiquitous, simple structure, easily manipulated in the laboratory Yeast Saccharomyces cerevisiae Single most studied eukaryotic cell Eukaryotic model, has similar biochemistry to other eukaryotes Easily manipulated Frequently studied Purpose Do the ingredients of the TAML reaction significantly alter microbial survivorship? Does Carbaryl hurt microbial cell survivorship? Do products of 2 min reaction between activated TAML and pesticide affect microbial survivorship? Do products of 30 min reaction between activated TAML and pesticide affect microbial survivorship? Hypotheses Null Carbaryl will have no significant effect on Yeast or E. Coli survivorship Null - Activated TAML mix will not have a significant effect on E. coli or Yeast survivorship. Null- Activated TAML can not safely break down carbaryl Materials Garden-Tech Sevin-5 pesticide 2.5 mM stock TAML solution H 2 O 2 (.88 M) Lab-Grade Catalase (Wards) E Coli DH5 Saccharomyces cerevisiae Sterile 250mL sidearm flask Sterile Dilution Fluid (SDF) Sterile Water Sterile Polystyrene conical Sterile test tubes Micropipette and sterile tips Pipette pump and sterile tips Sterile filters 60 sterile Petri dishes, poured with LB media 60 sterile Petri dishes, poured with YEPD Media Tube racks Fine tip permanent marker Klett Spectrophotometer Metler Scale Weigh Boats Ethanol Vortex SDF (per 1 liter) 100mM KH 2 PO 4 100mM K 2 HPO 4 10mM MgSO 4 1mM NaCl Procedure Preliminary Study 1. Cultures of E Coli DH5 and Saccharomyces cerevisiae were grown overnight in a sterile 250mL sidearm flask in LB media or YEPD media at 37C. 2. The cultures were left to grow to 50 Kletts units, which represents a cell density of about 10 8 cells/mL or 10 7 cells/mL. 3. The cultures were serially diluted in SDF to a concentration of approximately 10 3 cells/mL. 4. LB agar and YEPD agar plates were labeled and left to warm in incubator 5. 4g of Garden-Tech Sevin-5 pesticide was added to 20 mL of sterile water to create a stock solution 100 times as strong as recommended for home use. Procedure (cont.) 8. Amounts (in mL) of the various stock solutions and SDF were added to 8 tubes (2 tubes per variable): 9. Tubes were vortexed and then 0.1mL from each tube was plated 5 times, yielding a total of 20 plates each for yeast and for E. Coli 10. Plates were then placed in an incubator set at 37C overnight. 11. Colonies were then counted, each colony was assumed to have arisen from one cell. Control 10x Concen- tration1x.1x Yeast or E.Coli.1 Pesticide Stock SDF for E.Coli SW for Yeast Total10.0 Procedure (cont.) 9. Tubes were vortexed and then 0.1mL from each tube was plated 5 times, yielding a total of 20 plates each for yeast and for E. Coli 10. Plates were then placed in an incubator set at 37C overnight. 11. Colonies were then counted, each colony was assumed to have arisen from one cell. Procedure (Focus Study) 1. Cultures of E Coli DH5 and Saccharomyces cerevisiae were grown overnight in a sterile 250mL sidearm flask in LB media or YEPD media at 37C. 2. The cultures were left to grow to 50 Kletts units, which represents a cell density of about 10 8 cells/mL or 10 7 cells/mL. 3. The cultures were serially diluted in SDF to a concentration of approximately 10 3 cells/mL. 4. LB agar and YEPD agar plates were labeled and left to warm in incubator 5. 4g of Garden-Tech Sevin-5 pesticide was added to 20 mL of sterile water to create a stock solution 100 times as strong as recommended for home use mg of solid lab-grade catalase was added to 10 mL of sterile water to create a working concentration of catalase. Procedure (cont.) 8. Amounts (in mL) of the various stock solutions and SDF were added to 8 tubes (2 tubes per variable): Control Activated TAML onlyCarbaryl only2 minute30 minute H2O20.50 TAML0.50 Carbaryl00111 SDF for E.Coli SW for Yeast Total Procedure (cont.) 9. The tube with TAML mix only and 2 minute experimental tube was left to sit for 2 min (and the 30 min tube for 30 min) while the TAML oxidation reaction occurred mL of catalase was added to each TAML containing tube to quench the reaction, and degrade any residual H 2 O 2. Tubes were then left to quench for 2 min mL of the E. Coli or Yeast were then added to each tube. 12. Tubes were vortexed and then 0.1mL from each tube was plated 8 times, yielding a total of 40 plates each for yeast and for E. Coli 13. Plates were then placed in an incubator set at 37C overnight. 14. Colonies were then counted, each colony was assumed to have arisen from one cell. Yeast ReplicateControl.1xx10x Average E.Coli Control.1xx10x Average Preliminary study data Preliminary Study Yeast ANOVA Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Control x x x ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups E Within Groups Total F value> F-crit Interpretation: at least two means vary significantly. Preliminary Study E.Coli ANOVA Anova: Single Factor SUMMARY GroupsCountSumAverageVariance Control x x x ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups E Within Groups Total F value> F-crit Interpretation: at least two means vary significantly. Preliminary study ANOVA results Yeast ANOVA P=4.17*10 -5 E.Coli ANOVA yields P=1.73* P value cutoff : alpha =.05 Interpretation: At least two means vary significantly in both the yeast and E. Coli data. Dunnetts Test (prelim study) If t > t-crit, then data significant, reject null ConcentrationT-ValueT-CritIntepretation.1x Not significant 1x Not significant 10x Significant ConcentrationT-ValueT-Crit.1x Not Sgnificant 1x Significant 10x Signficant Yeast E. Coli Replicate No Pesticide and No TAMLTAML + No Pesticide Pesticide +No TAML2 minute exposure 30 minute exposure Average Yeast Data Yeast ANOVA F value> F-crit Interpretation: at least two means vary significantly. Anova: Single Factor SUMMARY GroupsCountSumAverageVariance No Pesticide or TAML TAML + No Pesticide Pesticide +No Taml minute exposure minute exposure ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups E Within Groups Total484239 E.Coli Data Replicate No Pesticide or TAMLTAML + No Pesticide Pesticide +No Taml 2 minute exposure 30 minute exposure average Anova: Single Factor SUMMARY GroupsCountSumAverageVariance No Pesticide or TAML TAML + No Pesticide Pesticide +No Taml minute exposure minute exposure ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups E Within Groups Total E.Coli ANOVA F value> F-crit Interpretation: at least two means vary significantly. ANOVA results Yeast ANOVA yields P=2.69* E.Coli ANOVA yields P=2.04* P value cutoff : alpha =.05 Interpretation: At least two means vary significantly in both the yeast and E. Coli data. Dunnetts Test If t > t-crit, then data significant, reject null Variable T-ValueT-CritIntepretation Activated TAML Only Not significant Pesticide Only Significant 2 minute exposure Not significant 30 minute exposure Not significant Yeast E. Coli Variable T-ValueT-CritIntepretation Activated TAML Only Not significant Pesticide Only Significant 2 minute exposure Not significant 30 minute exposure Not significant Conclusions Carbaryl is harmful to microbial life Activated TAML alone does not harm microbial life Break down products of Carbaryl are safe to microbes. Limitations/ Extensions Difficulty in synchronous plating One exposure time Could be done with other species A field study could be conducted Different reaction mixes References Dr. Terry Collins, Thomas Lord Professor of Chemistry at Carnegie Mellon University Dr. John Wilson, Assistant Professor, Biostatistics at the University of Pittsburgh Mr. Mark Krotec, PTEIlinkinghub.elsevier.com/retrieve/pii/S X