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Michelle Land Neeraja Vajrala Luis Sayavedra-soto Daniel Arp Botany and Plant Pathology Department

Can You Differentiate the Ammonia Oxidizers in Soil?. Michelle Land Neeraja Vajrala Luis Sayavedra-soto Daniel Arp Botany and Plant Pathology Department. Nitrification Influences. Downside less than 30% of the applied N fertilizer is taken up by plants

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Michelle Land Neeraja Vajrala Luis Sayavedra-soto Daniel Arp Botany and Plant Pathology Department

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  1. Can You Differentiate the Ammonia Oxidizers in Soil? Michelle Land Neeraja Vajrala Luis Sayavedra-soto Daniel Arp Botany and Plant Pathology Department

  2. Nitrification Influences Downside • less than 30% of the applied N fertilizer is taken up by plants • increases loss of soil nitrogen through leaching and volatilization • increases nitrate pollution of ground waters • eutrophication • nitrifiers compete with plants for ammonia • The generation of nitrous oxide • A green house gas • Benefits • In terrestrial ecosystems: • nitrogen supply to plants • removal of high levels of ammonia in wastewater

  3. Roles Played Both Nitrosomonas europaea and Nitrosopumilus maritimus convert ammonia (NH3) into nitrite (NO2-). This project studies ammonia-oxidizing organisms.

  4. Big Picture Aim: To differentiate between ammonia-oxidizing archaea and bacteria and study their contributions to global nitrification. Prediction: There could be some inhibitors or inactivators that can specifically inhibit one of these communities.

  5. Metabolic Reaction of Ammonia-Oxidizing Bacteria and Archaea Ammonia Oxidizing Bacteria (AOB) Hydroxylamine (NH2OH) Ammonia monooxygenase (amoABC) Hydroxylamine oxidoreductase (HAO) Nitrite (NO2-) Ammonia (NH3) Ammonia monooxygenase (amoABC) ??? ??? Ammonia Oxidizing Archaea (AOA)

  6. Project Hypothesis: Methylhydrazine only effects the HAO enzyme in ammonia oxidizing bacteria. Prediction: Ammonia oxidizing bacteria will stop producing nitrite in the presence of methylhydrazine and ammonia oxidizing archaea will be unaffected. Methylhydrazine

  7. Methylhydrazine Affect on N. europaea NO MH 1 µM MH 2 µM MH 5 µM MH Control - 10µM MH OD 600nm MH Added to Control Days of Incubation

  8. Methylhydrazine Affect on N. maritimus Nitrite (µM) NO MH NO MH 1µM MH 2 µM MH Days of Incubation

  9. Soil Slurry Preparation • 20g of soil • phosphate buffer at PH=8 • 1.1 mM potassium chlorate solution • (to stop the conversion of nitrite to nitrate) • 0.4 mM ammonium sulfate (energy source)

  10. Analysis of Nitrite Formation Sulfanilamide + NNEQ + Nitrite The soil slurry is filtrated and then tested Reddish Purple Azo Dye This solution is then placed in a spectrophotometer at 540nm and the absorbency is taken.

  11. Standard curve is used to calculate the nitrite concentration in the soil slurries.

  12. Methylhydrazine Affect on Soil NO MH 10 µM MH µg of Nitrite per gm of Soil Days of Incubation

  13. Conclusion At the concentrations tested both ammonia-oxidizing archaea and bacteria were inhibited by methylhydrazine. Therefore these concentrations of methylhydrazine can not be used to differentiate the ammonia-oxidizing activity of AOB and AOA.

  14. Future Area of Study

  15. Metabolic Reaction of Ammonia-Oxidizing Bacteria and Archaea Ammonia Oxidizing Bacteria (AOB) Hydroxylamine (NH2OH) Ammonia monooxygenase (amoABC) Hydroxylamine oxidoreductase (HAO) Nitrite (NO2-) Ammonia (NH3) Ammonia monooxygenase (amoABC) ??? ??? Ammonia Oxidizing Archaea (AOA)

  16. Acknowledgements • Dr. Neeraja Vajrala • Dr. Luis Sayavedra-soto • Dr.Daniel Arp • Dr. Kevin Ahern • HHMI Undergraduate Internship Program • SBI Undergraduate Internship Program

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