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– 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

Roles PlayedBoth Nitrosomonas europaea and Nitrosopumilus maritimus convert ammonia (NH3) into nitrite (NO2

-).

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.

Metabolic Reaction of Ammonia-Oxidizing Bacteria and Archaea

Ammonia monooxygenase

(amoABC)

Ammonia (NH3)

Hydroxylamine (NH2OH)

Nitrite(NO2

-)

Hydroxylamineoxidoreductase

(HAO)

Ammonia Oxidizing Bacteria (AOB)

???Ammonia

monooxygenase(amoABC)

???

Ammonia Oxidizing Archaea (AOA)

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

Methylhydrazine Affect on N. europaea

NO MH1 µM MH

2 µM MH

5 µM MH

Control - 10µM MH

MH Added to Control

Days of Incubation

OD

600

nm

Methylhydrazine Affect on N. maritimus

NO MH

1µM MH

2 µM MH

Days of Incubation

Nit

rite

(µ

M)

NO MH

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)

Analysis of Nitrite Formation

Sulfanilamide+

NNEQ+

Nitrite

Reddish Purple Azo Dye

This solution is then placed in a spectrophotometer at 540nm

The soil slurry is filtrated and then tested

and the absorbency is taken.

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

0 1 2 3 4 5 6 7 8 90

102030405060708090

100110120130140

NO Methylhydrazine

10µM Methylhydrazine

Methylhydrazine Affect on Soil

Days of Incubation

µg

of N

itri

te p

er g

m o

f S

oil

NO MH10 µM MH

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

Metabolic Reaction of Ammonia-Oxidizing Bacteria and Archaea

Ammonia monooxygenase

(amoABC)

Ammonia (NH3)

Hydroxylamine (NH2OH)

Nitrite(NO2

-)

Hydroxylamineoxidoreductase

(HAO)

Ammonia Oxidizing Bacteria (AOB)

???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