microbial survivorship in river fertilizer runoff pjas report by billy rielly central catholic high...
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Microbial Survivorship in River Fertilizer
Runoff
PJAS Report by Billy Rielly
Central Catholic high School
February 7th 2009
Surface Runoff Part of the water cycle and
describes the water that flows over a land surface.
It can have an effect on the river life in the various water sources it enters depending on the type of runoff.
Surface runoff pollutes lakes, streams, and rivers Affects water dwelling organisms
Types of Pollutants
Organic Pollutants Bacteria found in
animal feces, Tree and brush debris
from logging camps, Processed food from
landfills.
Inorganic Pollutants Silt runoff from
construction sites, Chemical waste from
industrial products, Heavy metals from acid
mine drainage.
Major containment affecting our water source due to runoff today: FERTILIZER
Contaminants affecting Pennsylvania’s water table today
Inorganic chemical contaminants, such as salts and metals,
industrial and/or domestic wastewater discharges from oil and gas production, mining, or farming.
Pesticides and herbicides which may occur from a variety of sources such as agriculture, urban storm water runoff, and residential uses.
Revitalization Today, the rivers of our
region seem to have recovered greatly from a once “deadly state.”
Recent initiatives have allowed significant remediation of aquatic areas
Great strides have been made in the restoration of the quality of Pittsburgh’s rivers, but some issues still occur.
The Clean Water Act Growing public awareness and
concern for controlling water pollution led the government to pass the Clean Water Act.
Gave EPA the authority to implement pollution control programs such as setting wastewater standards for industry.
Also continued requirements to set water quality standards for all contaminants in surface waters.
Unlawful for any person to discharge any pollutant from a point source into navigable waters, unless a permit was obtained.
Purpose To investigate the survivorship of yeast in
river water To investigate the survivorship of yeast in
fertilizer To determine if there are any synergistic
effects between the two variables
Null hypothesis
The varying concentrations of fertilizer will have no significant effect on the survivorship of yeast
Alternate (There will be no significant difference between the survivorship of yeast in river water or SDF.)
Miracle Grow Liquid Fertilizer(Fertilizer Used)
Commonly used lawn fertilizer
Inorganic, chemical fertilizer by-product of the petroleum
industry Salt-based Can be extremely harmful
to local watershed via runoff
Derived from Ammonium Phosphate, Potassium Nitrate, and Urea
Saccharomyces cerevisiae
scientific name for baker's yeast. (Fungus)
one of the most intensively studied eukaryotic model organism in molecular and cell biology
May be free living (Symbiont)
Past Studies involving yeast and water sources
Yeast survivorship was observed in the Tagus estuary in Portugal.
Yeast was still able to grow even with tides and river discharge.
Materials 54 YEPD agar plates YEPD Media (1% yeast extract, 2% peptone, 2% glucose) Sterile pipette tips Micropipettes Vortex Incubator Spreading platform, spreader bar, ethanol 20 mL Sterile capped test tubes with Sterile Dilution Fluid (SDF) (10
mM KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl)
Saccharomyces cerevisiae (Yeast) 0.22 micron syringe filters + 10 mL syringe 2 liters Allegheny River water 1 Container of miracle Grow all purpose liquid fertilizer Klett Spectrophotometer
Procedure
1. Saccharomyces cerevisiae was grown overnight in sterile YEPD media.
2. The culture was placed in incubator (30°C) until a density of 50 Klett spectrophotometer units were reached. This represents a cell density of approximately 107 cells/mL.
3. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.
4. Allegheny river water was sterile filtered using a (0.22 micron syringe filter)
5. 100 µL of cell culture was then added to the test tubes, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL.
TubesTube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6
yeast 0.1mL 0.1 0.1 0.1 0.1 0.1
River Water
8.9mL 8.9 8.9 1 0 0.9
Fertilizer 0 mL 1 0.1 0 1 0.1
SDF 1 mL 0 0.9 8.9 8.9 8.9
Total mLs
10mL 10 10 10 10 10
[fert] 0% 10 1 0 10 1
Procedure-Continued
6. The solutions were mixed by vortexing and allowed to sit at room temperature for 15 minutes.
7. After vortexing to evenly suspend cells, 100 µL aliquots were removed from the tubes and spread on YEPD agar plates.
8. The plates were incubated at 30 degrees for 48 hours.9. The resulting colonies were counted. Each colony is
assumed to have arisen from one cell.
0
20
40
60
80
100
120
140
160
0% 1% 10%
SDF
River Water
Concentration of Fertilizer
Col
onie
sFertilizer Influence on Yeast Survivorship
P=2.510-4
P=2.4910-4
P=1.810-2
Dunnett’s Test
10% Fertilizer vs. 0% Fertilizer
(River Water)
4.78 Significant
1% Fertilizer vs. 0%
Fertilizer
(River Water)
7.11 Significant
10% Fertilizer vs. 0% Fertilizer
(SDF)
3.76 Significant
1% Fertilizer vs. 0%
Fertilizer
(SDF)
3.89 Significant
α= .05
T-critical= 2.57
Key questions in the experiment
Does fertilizer have an effect on river water?
Does the fertilizer have an effect on the SDF?
Does water affect the survivorship of yeast?
Does fertilizer affect the survivorship of yeast differently in river water or SDF?
Was there an interaction between the fertilizer and the water?
Conclusions
Reject Null Hypothesis
The fertilizer appeared to have a significant effect on yeast survivorship in river water (P=2.4910-4)
The fertilizer appeared to have a significant effect on yeast survivorship in SDF (P=2.510-4)
Their appeared to be an interaction (synergy) between the variables of water type and fertilizer 2.96E-05 (2 Factor Anova)
Limitations and Extensions
When continuing this experiment, I will include testing the effect of fertilizer on the growth of E-coli, as well as yeast.
Also, I will use various fertilizers and compare the effect they had on the growth of the colonies as well.
Lastly, I will compare the growth of microbes in various water sources besides just the Allegheny River.