Ammonia Oxidizer Enrichment Sarah Douglas Shelbi Russell
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Ammonia Oxidizer Enrichment
Sarah DouglasShelbi Russell
Ammonia oxidation
NH3+ NH2OH NO2
-
AmoABCHAO
Bacteria & Archaea Bacteria
Archaeal pathway unknown, hydroxylamine may be oxidized by the nitroxyl pathway (Walker et al., 2010)
Phylogenetic diversity of ammonia oxidizers
Bacteria β-Proteobacteria (Nitrosomonas & Nitrospira) γ-Proteobacteria (Nitrosococcus)
Crenarchaeota
Sampling
Monterey Bay Aquarium activated sand 19°C and 20°C
Hopkins sand by aquarium outflow and coast
Enrichment procedure
• Crenarchaeota medium inoculated with 50 mL of 10 mM filtered slurry water sample– NH4Cl– Cofactors:
• 7-vitamin, selenite/tungstate, and SL12 trace metal solution
– Bicarbonate carbon source– Phosphate buffer
• Store in the dark • Sample every 2 days for 2 weeks
Spectrophotometric determination of substrate and product concentration
0 2 4 6 8 10 12 14 16 18 20 220
50
100
150
200
250
Hopkins Aquarium Sand
Aisle AmmoniaAisle NitriteWindow AmmoniaWindow Nitrite
Days
Conc
entr
ation
(uM
)
0 2 4 6 8 10 12 14 16 18 20 220
50
100
150
200
250
Dead Seal Beach Sand
Dead Seal Beach Sand (Aisle) Am-moniaDead Seal Beach Sand (Aisle) NitriteDead Seal Beach (Window) AmmoniaDead Seal Beach (Window) Nitrite
Days
Conc
entr
ation
(uM
)
0 2 4 6 8 10 12 14 16 18 20 220
50
100
150
200
250
Monterey Bay Aquarium Sand
21 degrees C (Aisle) Ammonia21 degrees C (Aisle) Nitrite21 degrees C (Window) Ammonia21 degrees C (Window) Nitrite18 degrees C (Aisle) Ammonia18 degrees C (Aisle) Nitrite18 degrees C (Window) Ammonia18 degrees C (Window) Nitrite
Days
Conc
entr
ation
(uM
)
Monterey Bay Aquarium Bradley’s experiment
0 2 4 6 8 10 12 14 15 17 19 210
20
40
60
80
100
120
140
160
180
200
KJ - Nitrite
KJ - Ammo-niaBS - Nitrite
BS - Ammo-niaBS Add - Ni-trite
BS Add - Ammonia
BS Trans - Nitrite
BS Trans - Ammonia
Time (days)
Co
nce
ntr
atio
n (
uM
)
Added 200 uM Ammonia
PCR amplification of bacterial and archaeal amoA
AOA amoA AOB amoA
Monterey Bay Aquarium sand 21°
Monterey Bay Aquarium sand 18°
Hopkins beach sand
Hopkins aquarium sand
Positive control
Conclusion
• Bacterial ammonia oxidizers appear to be more prevalent
• Archaea at competitive disadvantage in high ammonia environments?
• Lack of correlation between ammonia depletion and nitrite generation: complex ecosystem?
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