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Fly Ash Influence on Microbial Growth Jason Beiriger CCHS, Grade 11 3rd Year in PJAS.

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Presentation on theme: "Fly Ash Influence on Microbial Growth Jason Beiriger CCHS, Grade 11 3rd Year in PJAS."— Presentation transcript:

1 Fly Ash Influence on Microbial Growth Jason Beiriger CCHS, Grade 11 3rd Year in PJAS

2 Fly Ash Product of the burning of finely ground coal in a boiler to create electricity Commonly used in cement and concrete applications Industries claim that fly ash is neither toxic nor poisonous The EPA classifies Fly Ash as a non-hazardous material even though some of the components are considered to be harmful

3 Government Policy India- “notification” against improper use of fly ash and cement components Netherlands – Acceptable as long as the concentration of each carcinogenic component does not exceed 0.1% U.S.- Little regulation – California Department of Transportation requires that mineral admixtures like fly ash comprise at least 25% of the cementitious material in any concrete used in state- funded paving project – Montana- provides tax incentives for companies who install equipment to begin utilizing material like fly ash

4 Carcinogenic Components Quartz Exposure to quartz can lead to “black lung” pneumoconiosis or silicosis Prolonged irritation of lung tissue can cause lung fibrosis, leading to the development of tumors unlikely that the quartz in pulverized fuel ash is carcinogenic Chromium VI Chromium (VI) accounts for about 6% of all chromium in fly ash Used as pesticide to preserve wood Chromium leaching – Mild conditions, roughly 1% of all chromium present leached out – Extreme conditions, roughly 5% of all chromium present leached out Relatively low compared to the acceptable concentrations

5 Carcinogenic Components Continued Radioactive substances Found throughout the earth’s crust – Emit a certain amount of radioactive radiation naturally background radiation – Use of these substances can result in the concentration of radiation Still a small concentration – Unlikely to harm unless directly inhaled Dioxins Incomplete combustion of fossil fuels and waste can lead to the production of hydrocarbons – Atoms of chlorine, fluorine or bromine can replace some of the hydrogen atoms in these hydrocarbons to form dioxins Dangerous to animals – Fly ash contains small amounts

6 One of the most common forms of bacteria found in many environments Symbiont in intestinal tracts of many mammals Gram negative, rod shaped bacillus Most non-pathogenic Pathogenic strains can lead to life threatening infections Escherichia coli (E. coli)

7 Gram positive coccus Common surface symbiont in many mammals (human) Most forms considered non-pathogenic Pathogenic forms can be life threatening Forms biofilms Staphylococcus Epidermidis

8 Gram Bacteria Stain Categories Gram Positive (Staph)Gram Negative (E. coli)  Cell wall is thin extra layer of lipopolysaccharide which adds extra level of protection  If the toxin enters the circulatory system it causes a toxic reaction  This outer membrane protects the bacteria from several antibiotics  Most pathogenic bacteria in humans are gram-positive organisms  Simple cell wall  Antibiotics such as penicillin work against the formation of the cell wall

9 Objective/Purpose To determine if fly ash will significantly affect the survivorship of E. coli and Staphylococcus epidermidis

10 Null Hypothesis Fly ash will not significantly affect the survivorship of E. Coli and Staphylococcus Epidermidis. Hypothesis Fly ash will significantly affect the survivorship of E. Coli and Staphylococcus Epidermidis.

11 Materials LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) Sterile dilution fluid [SDF] (10mM KH2PO4, 10mM K2HPO4, 1mM MgSO4,.1mM CaCl2, 100mM NaCl) Klett spectrophotometer Sterile pipette tips and Micropipettors Vortex Sidearm flask Spreader bar Ethanol Micro burner Escherichia Coli bacteria Staphylococcus Epidermidis bacteria Rubber Gloves Test tubes Test Tube Rack SDF Test Tubes Scale Weigh boat 0.2 micron sterile filter Fly Ash Incubator

12 Procedure 1. Bacteria (E. coli and Staph) was grown overnight in sterile LB media 2. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3. The cultures were placed in a shaking water bath until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 8 cells/mL. 4. The culture was diluted in SDF to a concentration of approximately 10 3 cells/mL. 5. 5.00 g of fly ash was weighed out and was then added to 50.0mL of SDF, creating a 10% fly ash extract. 7. The extract was vortexed for 15 minutes and left to settle for 48 hours. 8. The leachate was pipetted out and sterilized using a 0.2 micron sterile filter. The remaining pellet was disposed of. 9. The leachate was diluted with sterile dilution fluid to the chosen concentrations to a total of 9.9 ml. For example: 1 ml. of 10% leachate + 8.9 ml. of SDF = final concentration of almost 1% leachate. (the addition of 0.1 ml. of cell culture will result in a total of 10 ml. and a 1% concentration)

13 Table of Concentration Concentrations (of leachate) 0%1%10%50% Leachate0mL0.1mL1.0mL5.0mL SDF9.9mL9.8mL8.9mL4.9mL Microbe0.1mL Total10mL

14 Procedure Continued 11. 0.1mL of cell culture was added to each test tube, yielding a final volume of 10.0mL and a cell density of approximately 10 3 cells /mL. 12. The solution in each tube was mixed by vortexing and allowed to sit at room temperature for 15 minutes. 13. After vortexing to evenly suspended cells, 0.1mL aliquots were removed from the tubes and spread on LB- Agar plates. 14. The plates were incubated at 37 degrees Celsius for 24 hours. 15. The resulted colonies were counted. Each colony is assumed to have arisen from 1 cell.

15 % of Fly Ash Leachate in Solution # of Surviving Colonies

16 % of Fly Ash Leachate in Solution # of Surviving Colonies P value= 7.822E-12

17 Survivorship Percentage Compared to Control % of Fly Ash Leachate in Solution

18 Dunnett’s Tests Variable ComparisonT value compared to t critical value Result 0.1% fly ash leachate to control (E. coli)3.03>2.88significant 10% fly ash leachate to control (E. coli)4.70>2.88significant 50% fly ash leachate to control (E. coli)12.61>2.88significant 0.1% fly ash leachate to control (Staph)6.77>2.88significant 10% fly ash leachate to control (Staph)6.29>2.88significant 50% fly ash leachate to control (Staph)11.30>2.88significant

19 Conclusion Fly ash will not significantly affect the survivorship of E. Coli and Staphylococcus Epidermidis. Rejected by analysis Fly ash will significantly affect the survivorship of E. Coli and Staphylococcus Epidermidis. SUPPORTED by analysis Null HypothesisHypothesis

20 Limitations Synchronizing the exact times of plating. Limited amount of materials Further Testing More replicates Different microbes – Yeast Different harmful substance Infuse Fly Ash into the plate

21 Sources Managing Coal Combustion Residues in Mines, Committee on Mine Placement of Coal Combustion Wastes, National Research Council of the National Academies, 2006 Human and Ecological Risk Assessment of Coal Combustion Wastes, RTI, Research Triangle Park, August 6, 2007, prepared for the U.S. Environmental Protection Agency Research Triangle ParkU.S. Environmental Protection Agency American Coal Ash Association www.acaa-usa.org "Is fly ash an inferior building and structural material". Science in Dispute. 2003. http://findarticles.com/p/articles/mi_gx5204/is_2003/ai_n19124302/?tag =content;col1. "Is fly ash an inferior building and structural material" http://findarticles.com/p/articles/mi_gx5204/is_2003/ai_n19124302/?tag =content;col1 Madigan M, Martinko J (editors) (2006). Brock Biology of Microorganisms (13th ed.). Pearson Education. p. 1096. ISBN 0-321-73551-X.ISBN0-321-73551-X Rybicki EP (1990). "The classification of organisms at the edge of life, or problems with virus systematics". S Aft J Sci 86: 182–6. ISSN 0038-2353.ISSN0038-2353

22 ANOVA Abbreviation for analysis of variance Statistical test comparing variation within and between experimental groups If the P- value is lower than the alpha value (.05), then the result is significant (a result of the variable influence) Sample ANOVA


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