Can You Differentiate the Ammonia Oxidizers

in Soil?Michelle Land

Neeraja VajralaLuis Sayavedra-soto

Daniel ArpBotany 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 MH2 µM MH5 µM MHControl - 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

Nitr

ite (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 Methylhydrazine10µM Methylhydrazine

Methylhydrazine Affect on Soil

Days of Incubation

µg o

f Nitr

ite p

er g

m o

f Soi

l

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