michelle land neeraja vajrala luis sayavedra-soto daniel arp botany and plant pathology department
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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