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.