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Functional and Molecular Diversity in Nitrogen Cycle Enzymes under Contrasting Agricultural Management Systems
Jeanette Norton John Stark
Jennifer ReeveGraduate Students:
Yang OuyangAvneet Kakkar
Mussie HabteselassieGraduate Student:
Lisa Woodruff
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On a global basis reactive N inputs related to human activities now exceeds natural N inputs
From Fowler, D., M. et al. 2013. The global nitrogen cycle in the twenty-first century. Philosophical Transactions of the Royal Society B-Biological Sciences 368.
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Why study nitrogen mineralization and nitrification in agricultural systems?
• Typical fertilizer N use efficiencies are often less than 50% due to N loss.
• N mineralization and nitrification are key N-transformations that largely determine the availability and mobility of N in soils
• Nitrification leads to denitrifcation , N2O emissions and nitrate leaching
• Decrease the impacts of N loss and increase N fertilizer use efficiency in agricultural ecosystems, aim to match soil N supply with plant demand
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Nitrification oxidizes ammonium to nitrate
NH4+ NO2
- NO3-
Ammonia monooxygenaseAMO
Ammonia oxidizers
Nitrite oxidoreductaseNXR
Nitrite oxidizers
• Both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) have the enzyme ammonia monooxygenase.
• Our understanding of the relative contribution of AOA and AOB to nitrification in different environmental conditions is limited
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Utah State University SiteStrongly calcareous Millville silt loam (Typic Haploxeroll) pH 8.0, irrigated Field plots planted to silage corn, started 2011CRB 4 N source treatments x 4 repsControl (no additional N)Ammonium sulfate (AS100 & 200 kg N ha-1) Composted steer manure (200 kg N ha-1)
*
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University of Georgia SiteCecil sandy loam (Fine, kaolinitic, thermic Typic Kanhapludults, pH 5.31:1 Field plots planted to corn, started 2012CRB 4 N source treatments x 4 repsControl: No Nitrogen fertilizerAmmonium sulfate (AS100), 112 kg N ha-1
Ammonium sulfate (AS200), 224 kg N ha-1
Poultry litter (PL) and cover crop N, 112 kg N ha-1
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•AOA numbers higher than AOB but unchanged by treatment •AOB numbers affected by treatment and year, significant after 1 year
•AOB increase with repeated treatments of AS fertilizers
Abundance of ammonia oxidizers- Utah
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Georgia- Abundance of ammonia oxidizers
•AOA numbers higher or similar to AOB, AOA unchanged by treatment •AOB numbers affected by treatment and year, significant in year 2 year
•AOB increase with repeated treatments of AS fertilizers at AS200 level
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UtahAre there changes in AO community composition?
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GeorgiaAre there changes in AO community composition?
Changes observed for AOB communities by treatment and year, diversity (Shannon Index) decreased for AS treatments
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Nitrification potential in August-Utah
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UtahHow do these changes in community relate to process rate ?Nitrification potentials in soil slurries 2014 – seasonal rates
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GeorgiaHow do these changes in community relate to process rate ?Nitrification potentials in soil slurries 2014 – seasonal rates
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How to evaluate the relative contribution of AOA and AOB?
(Taylor et al 2013)
AOA are resistant to octyne, while AOB are sensitive to octyne.
Octyne
Nitrification potential
Octyne-resistant nitrification potential
AOA
Octyne-sensitive nitrification potential
AOB
1-Alkyne C8
Fresh soil
NH4+
The differential inhibitor: 1-Octyne
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Potential nitrification rates (~1 mM NH4
+)
compared to
Gross nitrification rates from isotope pool dilution (at ambient ammonium).
Note
scal
e 20
XAug 2014
Aug 2014
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AOB and AOA activities change with increasing ammonium concentration Note scale 10X
AOB respond to additional NH4+ while AOA’s
capacity is saturated at relative low NH4+
concentration.
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Nitrification kinetic parameters from Michaelis-Menten model
0 5 10 15 20 25 300.0
0.2
0.4
0.6
0.8
1.0 AOB
NH4+ (μM )
Nitr
ifica
tion
rate
(mm
ol N
kg-
1d-1
)
0 5 10 15 20 25 300.0
0.2
0.4
0.6
0.8
1.0 AOB
NH4+ (μM )N
itrifi
catio
n ra
te(m
mol
N k
g-1d
-1)
AS200 Compost
AOB: Vmax= 4.8 mmol N kg-1 d-1; km=161 μMAOA: Vmax= 0.3 mmol N kg-1 d-1; km=3.6 μM
AOB: Vmax= 0.7 mmol N kg-1 d-1; km=44 μMAOA: Vmax= 0.2 mmol N kg-1 d-1; km=2.9 μM
Vmax is the maximum rate.
Km is the half-saturation constantV=Vmax*S/(Km+S)
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How AOA and AOB activities respond to temperature?
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Activity and abundance of nitrite oxidizers (all Nitrospira) in 2015
Ammonium fertilizers increase nitrite oxidation, but not abundance
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UtahN mineralization enzyme activities in 2014
Arginase Urease
Protease β-glucosaminidase
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Take home messages• Ammonia-oxidizing bacteria were more responsive than archaea to
N source. AOB abundance and community structure were changed significantly by ammonium fertilizers.
• Ammonium availability and temperature controlled the relative contribution of AOA and AOB to nitrification.
• Controlling the activity of AOB immediately after the application of
mineral N fertilizers would be an effective strategy to reduce and delay nitrification and therefore improve N use efficiency.
• Understanding the differential response of AOB and AOA to substrate and temperature may allow the nitrifier community to be included into simulation models as dynamic components driving N flux, linking microbial communities and function
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Acknowledgements
FundingAgriculture and Food Research Initiative Competitive Grants Program Grant no. 2011-67019-30178 from the USDA National Institute of Food and Agriculture. Utah Agricultural Experiment Station
Graduate Students:Yang OuyangLisa WoodruffAvneet Kakkar
Field and lab work:Danielle BarandiaranCory OrtizCraig Marlen RiceJeremiah MooreHenry Linford
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Rates TreatmentsVmax (mmol N/kg/day) Km (µM)
Jun Aug Jun Aug
Octyne-sensitive
Nitrification
Control 0.41 ±0.18 a 0.32 ±0.15 a 19.4 ±23.6 a 13.58 ± 5.4 a
AS100 2.47 ±0.45 b 1.41 ±0.43 b 86.2 ± 9.0 b 58.90 ±26.1 b
AS200 4.77 ±1.33 c 2.15 ±0.39 c 160.9 ±34.7 c 89.25 ±19.6 b
Compost 0.67 ±0.09 a 0.61 ±0.16 a 44.0 ±13.8 a 31.42 ±7.80 a
Octyne-resistant
Nitrification
Control 0.20 ±0.02 0.22 ±0.01 4.1 ±3.3 3.15 ±0.66AS100 0.22 ±0.04 0.25 ±0.07 11.2 ±11.1 3.30 ±1.66AS200 0.27 ±0.12 0.33 ±0.09 3.6 ±2.46 2.88 ±1.25Compost 0.20 ±0.03 0.23 ±0.01 2.9 ±0.84 3.07 ±0.31
Nitrification kinetic parameters from the Michaelis-Menten model results
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Seasonal dynamics of activity of ammonia oxidizers
Fertilizers
Fertilizers
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Are there changes in nitrifying community composition?
The nitrifying community was not altered after the first year of fertilization, but was significantly shifted by four years of repeated application of ammonium fertilizers.
Treatment: p=0.001 Year: p=0.001