Activated TAML Effects on Microbial Survivorship

Ryan KramerGrade 10

Pittsburgh Central Catholic

Problem

• Water systems are polluted with:• Antibiotics• Pharmaceuticals (Estrogen, etc.)• Mercury• Sulfur• Sewage• Fertilizers• Organics• Cleaning projects• Dyes and other colored agents

Green Chemistry

• Chemistry designed to perform the same jobs as regular chemistry without the environmental hazards.

• Goals for Green Chemistry include:• Eliminating or reducing atmospheric emissions.• Finding an alternative to chlorine based water cleaners.• Finding an alternative energy sources.

TAML

• GREEN catalyst engineered by Dr. Terry Collins at CMU• Accelerates H2O2 oxidation reaction • Highly selective• Made of common elements

(C, H, O, N, Fe) • GREEN• Economical• Easy to synthesize

TAML Continued• Low working concentrations• Degrades quickly• Highly water-soluble• Produces non-toxic byproducts• Breaks down stable chlorine compounds among many

others• Practical uses include:

• Textile dye bleaching• Pulp and paper bleaching• Water cleaning• Laundry stain remover• Petroleum refining• Rapid destruction of bio-chem warfare

Hydrogen Peroxide

• Pale blue liquid• Used as disinfectant, antiseptic, and oxidizer• Works with TAML in oxidation chemistry processes• Weak acid• Is the ideal green reagent:

• Nontoxic• Inexpensive• Disposable• Fast acting

TAML and Hydrogen Peroxide

• Work together to clean water.• TAML catalyzes (activates) peroxide reaction.• Has been extensively tested and has

effectively degraded numerous chemical pollutants.

• Is a safe, easy-to-use, inexpensive water disinfection technology.

Food Coloring

• Studies have shown colors affect taste• For this reason companies add colors to enhance dining

experience

• It is a non-toxic dye.• Chosen because

• Its non-toxic• Easy to obtain• Serves as simple model of environmental color effluent problems

Saccharomyces Cerevisiae

• Used in baking and fermenting of alcoholic beverages.

• Most researched cell in the world.• Serves as a simple model of microorganisms in

aquatic ecosystems systems.

Catalase

• Common enzyme found in almost every living thing.

• Contains Porphyrin Heme, an iron group, that allows it to react with hydrogen peroxide.

• Functions as a catalyst for the decomposition of peroxide into water and hydrogen.

Purpose

• To determine if a quenched TAML/Peroxide reaction produces byproducts that are lethal to yeast cells.

Hypothesis

• Null Hypothesis: Quenched TAML/peroxide will have no significant effect on yeast survivorship.

• Alternative Hypothesis: Quenched TAML/peroxide will have a significant effect yeast survivorship.

Materials

• TAML• Hydrogen Peroxide• Red Food Coloring• Sterile Pipettes• 20mL Culture Tubes• 58 YEPD agar plates (1%

yeast extract, 2% peptone, 2% glucose (dextrose), 1.5% agar)

• YEPD Media (1% yeast extract, 2% peptone, 2% glucose (dextrose))

• Saccharomyces Cerevisiae• Sterile Filter • Stock Solution Tubes• Ethanol • Bunsen Burner• Spreader Bars• Vortex• Catalase• Sterile Filter• Distilled Water

Procedure

• A stock solution of red dye was generated (.015 uL dye per 10mL distilled water) in a stock solution tube.

• A 0.88M stock solution of hydrogen peroxide was generated in a stock solution tube.

• Food dye and water was sterilized with a 0.22 micron syringe and sterile filter.

Procedure Continued

Water and Yeast Control

Red Dye Control

2 Minute Quenched

20 Minute Quenched

40 Minute Quenched

Yeast .1mL .1mL .1mL .1mL .1mL

TAML 0mL 0mL .5mL .5mL .5mL

H202 0mL 0mL .5mL .5mL .5mL

Red Dye 0mL 5mL 5mL 5mL 5mL

Water 9.9mL 4.9mL 3.8mL 3.8mL 3.8mL

Catalase 0mL 0mL .1mL .1mL .1mL

Liquid Pulse Exposure

• Tubes were vortexed to mix the ingredients.• 100uL of the test subjects were spread on 8

agar plates per time period (4 per tube).• All plates were incubated for two days.• All colonies were counted.

Prolonged (agar) Exposure

• 0.5mL of the stock from the culture tube was mixed with 100uL of just water and yeast on 6 plates (3 per tube).

• All plates were incubated for two days.• All colonies were counted.

Water and Yeast Control

Red Dye Continuous Reaction

Yeast .1mL 0mL 0mL

TAML 0mL 0mL .5mL

H202 0mL 0mL .5mL

Red Dye 0mL 5mL 5mL

Water 9.9mL 5mL 3.9mL

Catalase 0mL 0mL 0mL

Prolonged Agar Exposure EffectP Value=.11

W+D Cont. Reaction W+Y0

1

2

3

4

5

6

T1T2

Extents of Time/ Contets of Tubes

Num

ber o

f Col

onie

s

Liquid Pulse ExposureW+D P Value= 4.6E-12

W+Y P Value=1.69E-10

2 min. 20 min. 40 min. W+D W+Y0

50

100

150

200

250

T1T2

Extent of Time/Contents of Controls

Num

ber o

f Col

onie

s

Dunett’s Test for Water and Dye

T Crit= 4.00

Extent of Exposure T Value Interpretation

2 min. 4.87 Significant

20 min. 7.75 Significant

40 min. 12.80 Significant

Dunett’s Test for Water and Yeast

T Crit= 4.00

Extent of Exposure T Value Interpretation

2 min. 10.73 Significant

20 min. 7.87 Significant

40 min. 2.86 Insignificant

Conclusion

• All the exposure times varied significantly from the water and dye control group.

• Null Rejected

• At 2 minutes and 20 minutes the growth was significantly less than the water and yeast control group’s growth.

• Null Rejected

• The 40 minute group did not vary significantly from the water and yeast control.

• Null Accepted

Conclusion Continued

• Very little growth happened during the Prolonged Exposure Test.

• Null Accepted • No significant variance from the control.

• Show that over a long period of time and increased concentration the byproducts of a quenched TAML/Peroxide reaction harms microbial life.

Extensions and Limitations

• Limitations:• TAML is not specifically designed for food coloring.• Yeast was just a model used for aquatic microbial life.

• Extensions:• Use chlorine or other chemicals that are problems in

aquatic ecosystems.• Use a form of microbial life present in an aquatic

ecosystem.

References

• http://www.chem.cmu.edu/groups/collins/about/about.html

• http://en.wikipedia.org/wiki/Food_coloring• http://en.wikipedia.org/wiki/Hydrogen_peroxi

de• http://en.wikipedia.org/wiki/Spectrophotome

ter• http://en.wikipedia.org/wiki/

Saccharomyces_Cerevisiae