Transcript Fly Ash

Fly Ash Influence on
Microbial Growth
Jason Beiriger
CCHS, Grade 11
3rd 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 statefunded 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
Escherichia coli (E. coli)
• 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
Staphylococcus Epidermidis
• Gram positive coccus
• Common surface symbiont in many mammals
(human)
• Most forms considered non-pathogenic
• Pathogenic forms can be life threatening
• Forms biofilms
Gram Bacteria Stain Categories
Gram Positive (Staph)
Most pathogenic bacteria in
humans are gram-positive
organisms
Simple cell wall
Antibiotics such as penicillin
work against the formation of
the cell wall
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
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
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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 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 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 LBAgar 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.
Fly Ash Effects on E. coli
140
P value= 2.827 E-09
120
# of Surviving Colonies
100
80
60
40
20
0
0%
0.10%
10%
% of Fly Ash Leachate in Solution
50%
Fly Ash Effects on Staph
200
P value= 7.822E-12
180
160
# of Surviving Colonies
140
120
100
80
60
40
20
0
0%
0.10%
10%
% of Fly Ash Leachate in Solution
50%
Survivorship Percentage Compared to Control
90%
80%
70%
60%
50%
E. coli
40%
Staph
30%
20%
10%
0%
0.10%
10%
% of Fly Ash Leachate in Solution
50%
Dunnett’s Tests
Variable Comparison
T value
compared to
t 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
Null Hypothesis
• Fly ash will not
significantly affect the
survivorship of E. Coli
and Staphylococcus
Epidermidis.
• Rejected by analysis
Hypothesis
• Fly ash will
significantly affect the
survivorship of E. Coli
and Staphylococcus
Epidermidis.
• SUPPORTED by
analysis
Limitations
Further Testing
• Synchronizing the
exact times of
plating.
• More replicates
• Different microbes
– Yeast
• Limited amount of • Different harmful
substance
materials
• 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