1. Effects of TAML Treated Water on Yeast Survivorship
Michael Gemmell
9th Grade
Pittsburgh Central Catholic High School

2. Problem
Modern technology such as factories and water refineries have posed potential environmental risks. Scientist have searched for means of degrading these risks into harmless substances.

3. TAML GREEN catalyst engineered by Dr. Terry Collins at CMU
Accelerates H2O2 oxidation reaction
Made of common elements found in nature.
(C, H, O, N, Fe)
GREEN
Easy to synthesize

4. TAML Low working concentrations 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

5. Possible Uses Textiles Water Cleaning Petroleum Refining
Biological Warfare
TAML could be effective in degrading triclosan.

6. Triclosan Antibacterial and anti-fungal agent
Increasing use in consumer products (soaps, textiles, kitchen utensils)
Binds to ENR enzyme in cell, which increases affinity for NAD+ (nicotineamide- adenine-dinucleotide), which is a stable conformation of the 3 chemicals which inhibits fatty acid synthesis.

7. Triclosan Health Concerns
Can be degraded in water by UV radiation to form several highly-toxic dioxins that act as potent endocrine disruptors.
Only partially degraded by current water treatment methods
Significant amount is released into environment
Increases the risk of infertility, early puberty, and other hormone related problems in humans.

8. Purpose
Determine the ability of TAML + H2O2 to degrade Triclosan, and test that reaction's residue on the survivorship of yeast.

9. Yeast Eukaryotic, unicellular fungi.
Used in genetics and cell biology, in light of the fact that the cell cycle of a yeast cell is similar to the cell cycle of humans.
Saccharomyces cerevisiae- common yeast.
Similar genetic makeup, biochemistry, and cell cycle to other eukaryotes, including human cells.
Most studied cell in the world.

10. Hypothesis
Null: The residue of TAML-Activated H2O2 and Triclosan reaction will not have a significant effect on the survivorship of yeast.
Null: Triclosan by itself will not have a significant effect on the survivorship of yeast cells.
Null: TAML-Activated H2O2 will not have a significant effect on the survivorship of yeast cells.
Alternate: The residue of TAML-Activated H2O2 and Triclosan will have a significant effect on the survivorship of yeast cells.

11. Materials Tube Racks Permanent Marker Ethanol Matches Vortex
Triclosan (99% pure, powdered)
Fe-TAML B catalyst
H2O2 (.88 M)
Lab-Grade Catalase (Ward’s)
Saccharomyces cerevisiae
Sterile 250mL sidearm flask
NaHCO3
Sterile Water
Test tubes
Micropipette and sterile tips
Pipette pump and sterile tips
60 Sterile YEPD Agar Petri Dishes
Tube Racks
Permanent Marker
Ethanol
Matches
Vortex
YEPD Media
Bacto peptone 20 g 2%
Yeast extract 10 g 1%
Dextrose 20 g 2%
H2O to 1L

12. Procedure
A culture of yeast 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 107 cells/mL.
The culture was serially diluted to a concentration of approximately 103 cells/mL.
0.0353g of solid Fe-TAML B was added to 10mL of sterile water to create a 5*10-3M stock solution.
0.0289g of solid triclosan (99%) was added to 1mL of alcohol and 9mL of sterile water to create a 1*10-2M stock solution.
0.89g of solid NaHCO3 was added to 10mL of sterile water to create a 1M stock solution.
0.025g of solid lab-grade catalase was added to 50mL of sterile water to create a working concentration of catalase.

13. Tube Contents Control 0.01% Triclosan 0.1% 1% 10% RXM 0.1% 0mL 0.01mL
CRXM 0.1%
10%
RXM 0.1%
Variable
0mL
0.001mL
0.01mL
0.1mL
1mL
Sterile Water
9.8mL
9.79mL
9.7mL
8.8mL
NaHCO3
Yeast
Total
10mL

14. Procedure TAML activators and H2O2 were mixed with Triclosan
Tubes were let sit for 20 min while the TAML oxidation reaction occurred.
0.1mL of catalase was added to each tube to quench the reaction, and degrade any residual H2O2. Tubes were then left to quench for 20 min.
0.1mL of yeast (at 103 cells/mL) was then added to each tube.
Tubes were vortexed and then 0.1mL from each tube was plated 5 times, creating a total of 60 plates.
Plates were then placed in an incubator set at 37°C overnight.
Colonies were then counted, each colony was assumed to have arisen from one cell.

15. Triclosan Effects on Yeast Survivorship
P=2.33E-05
Colonies
Concentration

16. TAML+ H2O2 Effects on Yeast Survivorship
Colonies
Concentration

17. TAML+ H2O2 +Triclosan (Reaction Mix) Effects on Yeast Survivorship
Colonies
Concentration

18. Summary of ANOVA Data Analysis
Variable
P-Value
Significance
TAML+H2O2+Triclosan P=
Insignificant
Triclosan
P=2.33E-05
Significant
TAML+H2O2 P=

19. Dunnett’s Test for Triclosan
Concentration
t-value
T-crit value
Significance
0.01%
0.0136
2.78
Insignificant
0.1%
5.16
Significant 1%
5.17

20. Interpretations
TAML+ H2O2 +Triclosan did not have a significant effect on the survivorship of yeast.
The TAML accelerated the oxidation of the H2O2 and degraded the Triclosan.
The Triclosan only significantly affected the survivorship of yeast when the concentration exceeded 0.1%.
TAML+ H2O2 by itself did not have a significant effect on the survivorship of yeast.
Next slide is limitations and further study

21. Conclusions
Null: The residue of TAML-Activated H2O2 and Triclosan reaction will not have a significant effect on the survivorship of yeast.
Accepted
Null: Triclosan by itself will not have a significant effect on the survivorship of yeast cells.
Rejected
Null: TAML-Activated H2O2 will not have a significant effect on the survivorship of yeast cells.
Alternate: The residue of TAML-Activated H2O2 and Triclosan will have a significant effect on the survivorship of yeast cells.

22. Extensions/Limitations
Spread plating limited not only by human error, but by unequal staggering.
Limited to only one model.
Limited to one anti-bacterial substance.
Extensions
Testing higher concentrations of Fe-TAML and lower concentrations of Triclosan could improve results.
Using different bacteria could improve results.
Test TAML on other anti-bacterial substances.
Vary the length of the reaction and compare results.

23. References f
antoine.frostburg.edu/chem/senese/101/consumer/faq/triclosan.shtml
linkinghub.elsevier.com/retrieve/pii/S X

24. TAML+ H2O2 +Triclosan P=

25. Triclosan
P=2.33E-05

26. TAML+ H2O2 P=

27. TAML+ H2O2
Colonies
Concentration

28. Triclosan
Colonies
Center title
Concentration

29. TAML+ H2O2 +Triclosan Reaction Mix
Colonies
LABEL AXES
Concentration