Can You Differentiate the Ammonia Oxidizers in Soil? Michelle Land Neeraja Vajrala Luis...
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Transcript of Can You Differentiate the Ammonia Oxidizers in Soil? Michelle Land Neeraja Vajrala Luis...
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