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

Nitrification Influences Benefits In terrestrial ecosystems: nitrogen supply to plants removal of high levels of ammonia in wastewater 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 A huge part of the nitrogen cycle is completed by microorganisms Nitrification the oxidation of ammonia to nitrite and then to nitrate Benefits: Plants take up ammonia and nitrates, prefer nitrates Plants can absorb nitrates and ammonium ions NH4+, prodinated form of ammonia NH3, change back and forth depend on PH the relatively slow growth and sensitivity of ammonia oxidizing bacteria often limits throughput in waste managment AOB and AOA are essential for the removal of ammonia in waste water Downside when too much fertilizer is added: The gain of, often less than 30% of the applied N fertilizer is recovered due to losses associated with and following nitrification. Nitrite and Nitrate are much more volatile or mobile compounds and become increasingly more volatile with each step volatilization : evaporation of nitrogen Nitrite and nitrate can cause an excessive enrichment of the water (eutrophication), leading to a rapid growth of algae and reduces its oxygen content in aquatic ecosystems Dead zones in the Gulf of Mexico at the Mississippi delta are increasing evidence that N pollution has reached a global level of impact. Denitrification produces nitrous oxide All soils do as well as burning of fossil fuels A more potent green house gas than CO2, just a smaller amount

Roles Played Both Nitrosomonas europaea and Nitrosopumilus maritimus convert ammonia (NH3) into nitrite (NO2-). Ammonia oxidation: thought to be catalyzed only by Bacteria. Several AOB’s isolated and studied in past decades Metagenomic studies suggested members of archaea also capable of ammonia oxidation and these members are ubiquitous in terrestrial, marine enviornments . Recently an ammonia oxidizing archaea (Nitrosopumilus maritimus) has been isolated. Explain both parts of nitrification This project studies ammonia-oxidizing organisms.

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. The big picture is to try and find a compound that eliminates just one of the two colonies. So to try and find out how much the archaea and how much the bacteria are contributing to the nitrogen cycle. 4

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 oxidizing archaea do convert ammonia to nitrite but do not have HAO gene homologes The big picture is to try and find a compound that eliminates just one of the two colonies. So to try and find out how much the archaea and how much the bacteria are contributing to the nitrogen cycle. Methylhyderazine has been tested on just the HAO enzyme and a inactivater of that enzyme N. Europaea isolated almost a hundred years and archaea have recently been discovered and more recently put into their own Domain. Part of the reason is because they are very very difficult to isolate and analyze, they grow in very low densities Ammonia monooxygenase (amoABC) ??? ??? Ammonia Oxidizing Archaea (AOA)

Project Methylhydrazine 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. The big picture is to try and find a compound that eliminates just one of the two colonies. So to try and find out how much the archaea and how much the bacteria are contributing to the nitrogen cycle. 6

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 Mehthylhydrazine on the third day for the control 10µM All the other ones from day 1 Days of Incubation 7

Methylhydrazine Affect on N. maritimus Nitrite (µM) NO MH NO MH 1µM MH Trying 5µM on N. Maritimus, because there was an affect at 5µM on N. Europaea  they grow in very low densities and that is why nitrite formation is used instead of OD 2 µM MH Days of Incubation

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) Extending this Knowledge to Soils Soils have ammonia oxidizing bacteria (AOB) and archaea (AOA) Soils have different percents of Archaea and bacteria living in the soil

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

A standard curve with known concentrations of sodium nitrite wefrand the absorbancies were taken With this standard curve absorbancies of unknow concetrations can be caluated to find out their known concentrations Standard curve is used to calculate the nitrite concentration in the soil slurries.

Methylhydrazine Affect on Soil NO MH 10 µM MH µg of Nitrite per gm of Soil Both archaea and bacteria were affected by methylhydrazine There was one artifact one of the three controls did not produced nitrite A little increase in the 10 µM MH could be from the soil system or the MH started to degrade Days of Incubation

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.

Future Area of Study Mehthylhydrazine on the third day for the control 10µM All the other ones from day 1 14

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 oxidizing archaea do convert ammonia to nitrite but do not have HAO gene homologes The big picture is to try and find a compound that eliminates just one of the two colonies. So to try and find out how much the archaea and how much the bacteria are contributing to the nitrogen cycle. Methylhyderazine has been tested on just the HAO enzyme and a inactivater of that enzyme N. Europaea isolated almost a hundred years and archaea have recently been discovered and more recently put into their own Domain. Part of the reason is because they are very very difficult to isolate and analyze, they grow in very low densities Ammonia monooxygenase (amoABC) ??? ??? Ammonia Oxidizing Archaea (AOA)

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