1. Microbial Survivorship in River Fertilizer Runoff PJAS Report by Billy Rielly Central Catholic high School February 7 th 2009

2. 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

3. 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

4. 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.

5. 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.

6. 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.

7. 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

8. 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.)

9. 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

10. 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)

11. 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.

12. 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 KH 2 PO 4, 10 mM K 2 HPO 4, 1 mM MgSO 4, 0.1 mM CaCl 2, 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

13. 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 10 7 cells/mL. 3.The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 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 10 3 cells/mL.

14. Tubes Tube 1Tube 2Tube 3Tube 4Tube 5Tube 6 yeast0.1mL0.1 River Water 8.9mL8.9 100.9 Fertilizer0 mL10.101 SDF1 mL00.98.9 Total mLs 10mL10 [fert]0%1010 1

15. 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.

16. Concentration of Fertilizer Colonies Fertilizer Influence on Yeast Survivorship P=2.5 10-4 P=2.49 10-4 P=1.8 10-2

17. Dunnett’s Test 10% Fertilizer vs. 0% Fertilizer (River Water) 4.78Significant 1% Fertilizer vs. 0% Fertilizer (River Water) 7.11Significant 10% Fertilizer vs. 0% Fertilizer (SDF) 3.76Significant 1% Fertilizer vs. 0% Fertilizer (SDF) 3.89Significant α=.05 T-critical= 2.57

18. 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?

19. Conclusions Reject Null Hypothesis The fertilizer appeared to have a significant effect on yeast survivorship in river water (P=2.49 10-4) The fertilizer appeared to have a significant effect on yeast survivorship in SDF (P=2.5 10-4 ) Their appeared to be an interaction (synergy) between the variables of water type and fertilizer 2.96E-05 (2 Factor Anova)

20. 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.