Effects Of Fertilizer on Yeast Cell and E. Coli Survivorship By, Alexander Nese, Pittsburgh Central Catholic, 9th grader

Problem Fertilizers are used worldwide from home gardens to industrial farms, but little is known about they're effects on micro life. What effects do fertilizers have on these essential micro life?

Yeast Saccharomyces cerevisiae. Easy to manipulate in laboratories. Most commonly studied cell. Has similar reproduction, metabolism, and chemistry as other more advanced eukaryotic cells, like human cells.

Escherichia coli (E. coli) Gram-negative, facultatively anaerobic, rod-shaped bacterium Strains are usually harmless; present in lower intestine of warm-blooded organisms The most widely studied prokaryotic model organism and an important species in the fields of biotechnology and microbiology

Common IndustriAL Fertilizer A chemical or natural substance added to soil or land to increase its fertility. Three main chemicals in fertilizers. Nitrogen promotes overall grass growth. Phosphate strengthens root growth. Soluble Potash helps grass withstand stress, drought, or disease.

Fertilizer Being Tested "Miracle-Gro" Indoor Plant Food

Fertilizer Ingredients Total Nitrogen- 1% 0.5% Urea Nitrogen 0.25% Ammoniacal Nitrogen 0.25% Nitrate Nitrogen Phosphate- 1% Soluble Potash- 1%

Purpose The purpose of this experiment is to test the effects of fertilizer on microbial survivorship.

Hypotheses Null hypothesis: The fertilizer will have no effect on microbial survivorship. Alternate hypothesis: The fertilizer will have a significant effect on microbial survivorship.

Materials Escherichia coli cells Micropipettes Micro Rack Micro Tubes Vortex Incubator Fertilizer (Miracle Grow) Ethanol Bunsen Burner Sterile water

Materials (Con't) Sterile Dilution Fluid [SDF] (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl) Sterile test tubes Racks for tubes Turn tables Saccharomyces cerevisiae 80 YEPD Agar plates (1% yeast extract, 2% glucose, 1.5% agar) Sterile pipette tips Sterile spreader bars

Procedure (Liquid Pulse) Yeast and E. coli were grown overnight in sterile LB media. Samples of the overnight culture were added to fresh media in a sterile sidearm flask. The cultures were incubated until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10^8-10^9 cells/ml. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/ml. The Fertilizer was diluted with sterile dilution fluid to concentrations of 0%, 0.1%, 1%, and 10% to total 9.9 m

Test Tube Concentrations 0% 0.1% 1% 10% Sterile Water 9.9 ml 9.89 ml 9.8 ml 8.9 ml Yeast/E. Coli 0.1 ml Fertilizer 0 ml 0.01 ml 1 ml

Procedure (Liquid Pulse Cont'd) 6) 0.1 ml. 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. 7) The tubes were allowed to incubate at room temperature for 5 minutes. 8) After vortexing to evenly suspend cells, 0.1 ml. aliquots were removed from the tubes and spread on LB agar plates. The plates incubated at 37°C overnight. 10) The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

Procedure (Agar Infusion) Plates were infused as follows: Low- 20 ml. of fertilizer, and 180 ml. of sterile fluid was added to 8 different plates (4 yeast, 4 E. Coli) High- 200 ml. of sterile fluid was added to 8 different plates (4 yeast, 4 E. Coli) Plates were allowed to incubate for 1 hour to promote infusion 0.1 ml. were transferred from the control tube to the plates , and then spread with sterile spreader bars The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

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ANOVA Statistical test that allows for the comparison of means of different groups, to determine significant variation Utilizes p-values as measure of significance p>0.05: not significant p<0.05: significant P-value= <10^-6

Dunnett’s Test A test used to find out which variable groups produced significant variation compared to a control. If T-value is greater than the T critical, variations are considered significant.

Dunnett’s Test Results T Value Result 0.1% Yeast 6.67 Significant 1% Yeast 2.98 10% Yeast 18.08 Agar High Yeast 16.32 Agar Low Yeast 36.44 0.1% E. Coli 6.19 1% E. Coli 11.69 10% E. Coli 7.59 Agar High E. Coli 14.2 Agar Low E. Coli 4.05 T-value must be above 2.88 to have significant variation

Interpretation of Results Null hypothesis can be rejected because my p-value was less than 0.05 Alternate hypothesis can be accepted In both tests all concentrations had significant variation

Limitations Desynchronized plating Limited concentration exposures Only one exposure time was tested Synergistic effects were not tested

Extensions Different types of fertilizer Different bacterias More exposure times

Bibliography https://nces.ed.gov/nceskids/createagraph/default.aspx?ID=f5d4a8154eda4 311ac714b40fe5faa96 http://www.miraclegro.com/smg/goprod/miracle-gro-indoor-plant-food- plant-food/prod11050114 https://www.greenviewfertilizer.com/articles/understanding-N-P-K