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
Roles Played
Both Nitrosomonas europaea and Nitrosopumilus
maritimus convert ammonia (NH3) into nitrite (NO2-).
This project studies
ammonia-oxidizing
organisms.
Big Picture
Aim: To differentiate between ammoniaoxidizing 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 Oxidizing Bacteria (AOB)
Ammonia
monooxygenase
(amoABC)
Hydroxylamine
(NH2OH)
Nitrite
(NO2-)
Ammonia
(NH3)
Ammonia
monooxygenase
(amoABC)
Hydroxylamine
oxidoreductase
(HAO)
???
???
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.
Methylhydrazine Affect
on N. europaea
NO MH
1 µM MH
OD 600nm
2 µM MH
5 µM MH
Control - 10µM MH
MH
Added to
Control
Days of Incubation
Methylhydrazine Affect
Nitrite (µM)
on N. maritimus
NO
NOMH
MH
1µM MH
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)
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.
Nitrite Standard Curve
absorbancy at 540 nm
0.6
0.5
y = 0.1075x - 0.0081
R² = 0.9994
0.4
0.3
0.2
0.1
0
0
1
2
3
4
5
6
-0.1
Concentration of Nitrite (µg)
Standard curve is used to calculate the
nitrite concentration in the soil slurries.
µg of Nitrite per gm of Soil
Methylhydrazine Affect on Soil
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
NO
NO Methylhydrazine
MH
10 µMMethylhydrazine
MH
10µM
0
1
2
3
4
5
6
Days of Incubation
7
8
9
Conclusion
At the concentrations tested both ammoniaoxidizing 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 Oxidizing Bacteria (AOB)
Ammonia
monooxygenase
(amoABC)
Hydroxylamine
(NH2OH)
Nitrite
(NO2-)
Ammonia
(NH3)
Ammonia
monooxygenase
(amoABC)
Hydroxylamine
oxidoreductase
(HAO)
???
???
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