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Fly Ash Influence on Microbial Growth
Jason Beiriger
CCHS, Grade 113rd Year in PJAS
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
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
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
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
• 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)
• 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
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 organismsSimple cell wallAntibiotics such as penicillin work against the formation of the cell wall
Objective/Purpose
• To determine if fly ash will significantly affect the survivorship of E. coli and Staphylococcus epidermidis
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.
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
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 108
cells/mL.
4. The culture was diluted in SDF to a concentration of approximately 103 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)
Table of Concentration
Concentrations(of leachate)
0% 1% 10% 50%
Leachate 0mL 0.1mL 1.0mL 5.0mL
SDF 9.9mL 9.8mL 8.9mL 4.9mL
Microbe 0.1mL 0.1mL 0.1mL 0.1mL
Total 10mL 10mL 10mL 10mL
Procedure Continued11. 0.1mL of cell culture was added to each test tube, yielding a final volume of 10.0mL and a cell density of approximately 103 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.
0.00% 0.10% 10.00% 50.00%0
20
40
60
80
100
120
140
Fly Ash Effects on E. coliP value= 2.827 E-09
% of Fly Ash Leachate in Solution
# of Surviving Colonies
0.00% 0.10% 10.00% 50.00%0
20
40
60
80
100
120
140
160
180
200
Fly Ash Effects on Staph
% of Fly Ash Leachate in Solution
# of Surviving Colonies
P value= 7.822E-12
0% 10% 50%0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
E. coliStaph
Survivorship Percentage Compared to Control
% of Fly Ash Leachate in Solution
Dunnett’s TestsVariable Comparison T value
compared tot critical value
Result
0.1% fly ash leachate to control (E. coli) 3.03>2.88 significant
10% fly ash leachate to control (E. coli) 4.70>2.88 significant
50% fly ash leachate to control (E. coli) 12.61>2.88 significant
0.1% fly ash leachate to control (Staph) 6.77>2.88 significant
10% fly ash leachate to control (Staph) 6.29>2.88 significant
50% fly ash leachate to control (Staph) 11.30>2.88 significant
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 Hypothesis Hypothesis
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
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
• 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.
• Madigan M, Martinko J (editors) (2006). Brock Biology of Microorganisms (13th ed.). Pearson Education. p. 1096. ISBN 0-321-73551-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.
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
