Betsy Leondar1,2, David Myrold2,3, Mary Santelmann4
1Bioresource
Research Interdisciplinary Program
2Subsurface Biosphere Initiative Undergraduate Research Program
3Department of Crop and Soil Sciences, Oregon State University
4Department of Geosciences, Oregon State University
Introduction
• Willamette Valley
• Wetland Restoration
• Significance
• Denitrification
Methods
• Plot design/soil sampling
• Percent Moisture
• Nitrate (NO3-), Extractable Organic Carbon
(EOC), Total Extractable Nitrogen (TEN)
• Denitrification Enzyme Assays (DEAs)
Betsy Leondar - Oregon State University (c) 2011
Results
• EOC, TEN, NO3• DEAs
• Two-Way ANOVA
• Correlations
Conclusions
• Future Research
Questions
Betsy Leondar - Oregon State University (c) 2011
Betsy Leondar - Oregon State University (c) 2011
Green Mountain
Hutchinson
Lovejoy
Knez
Zucker
Gotter Prairie
Westbrook
Betsy Leondar - Oregon State University (c) 2011
Wetlands
• Comprise ~1% of global landmass
• Organic-rich soils
• Creation or restoration
• Decreases loss of wetland functions due to
land use conversion
• Legislation
• Wetland Restoration Program
• Clean Water Act
Betsy Leondar - Oregon State University (c) 2011
Significance
• Wetlands important in delivery of
ecosystem services
• Water quality
• Flood protection
• Conservation of biodiversity
Betsy Leondar - Oregon State University (c) 2011
Denitrification
• Reduction of nitrate (NO3-) and nitrite
(NO2-) ions into gaseous nitrogen in the
form of nitric oxide (NO), nitrous oxide
(N2O), and dinitrogen (N2) gases
• Measured through Denitrification
Enzyme Assays (DEAs)
• Acetylene inhibition method
Betsy Leondar - Oregon State University (c) 2011
• Collect analytical data
• Assess productivity of ecosystem services
• Guide land use policy
• Denitrification
• NO and N2O
•
Greenhouse gases
•
•
Minimize off-gassing
Loss of inorganic nitrogen from root zone
•
•
Reduces crop productivity and efficacy of N
fertilizers
N budgets for management practices
Betsy Leondar - Oregon State University (c) 2011
Evaluate wetland restoration methods
• Compare different site types
• Restored sites
• Agricultural sites
• Natural wetlands
• Measure sites at different times of year
• November
• February
• April
Betsy Leondar - Oregon State University (c) 2011
Agricultural Sites
Restored wetlands
• Gotter Prairie Ag
• Hutchinson
• Westbrook
• Lovejoy
• Zugar
• Gotter Prairie North
Natural wetlands
• Green Mountain
• Gotter Prairie South
• Knez
Betsy Leondar - Oregon State University (c) 2011
In the Field
• Soil Sampling
Laboratory
• Nitrate
• Extractable Organic Carbon
• Percent Moisture
• Denitrification Enzyme Assays
Betsy Leondar - Oregon State University (c) 2011
Soil sampling
• Collected 5 eight-inch soil cores from plot
perimeter
• 27 plots from 9 sites
• 27 x 5 = 135 individual soil cores
• Collected once every 2.5 months
• Fall, Winter, Spring
Betsy Leondar - Oregon State University (c) 2011
In the Field
• Soil Sampling
Laboratory
• Percent Moisture (%H2O)
• Nitrate (NO3-)
• Extractable Organic Carbon (EOC)
• Total Extractable Nitrogen (TEN)
• Denitrification Enzyme Assays (DEAs)
Betsy Leondar - Oregon State University (c) 2011
20g of soil weighed before and after oven drying
Betsy Leondar - Oregon State University (c) 2011
• 10 g soil + 50 ml of
0.05 M Potassium
Sulfate (K2SO4)
• Shake for 1 hour
• Filter
• Run analysis on
autoanalyzer
Betsy Leondar - Oregon State University (c) 2011
• 20g soil/25ml C6H12O6-
KNO3
• Headspace air removed,
replaced with Argon
• Flasks shaken
• 500µl samples removed
for Gas Chromatograph
(GC)
• 12ml acetylene added
• Prohibits N2 formation
Betsy Leondar - Oregon State University (c) 2011
Results
• EOC, TEN, NO3• DEAs
• Two-Way ANOVA
• Correlations
Conclusions
• Future Research
Questions
Betsy Leondar - Oregon State University (c) 2011
40
35
Mg per kg soil
30
25
20
NatWet
ResWet
Ag
15
10
5
0
-5
C
N
NO3-
Nov '09
C
N
NO3-
C
Feb '10
Betsy Leondar - Oregon State University (c) 2011
N
April '10
NO3-
November
February
April
Ag – EOC
TEN
NO3-
16.0 mg C/kg soil
8.9 mg N/kg soil
3.4 mg NO3--N /kg
soil
27.8
16.1
2.7
28.1
14.2
11.8
Nat – EOC
TEN
NO3-
18.0
5.0
0.65
30.9
8.9
2.0
24.4
11.4
7.9
Res – EOC
TEN
NO3-
16.8
2.0
0.36
21.9
6.6
1.1
28.0
12.8
9.1
Betsy Leondar - Oregon State University (c) 2011
Results
• EOC, TEN, NO3• DEAs
• Two-Way ANOVA
• Correlations
Conclusions
• Future Research
Questions
Betsy Leondar - Oregon State University (c) 2011
ng N2O-N per gram of Soil per minute
0.025
0.02
0.015
Ag
NatWet
ResWet
0.01
0.005
0
BA
AA
November
BA
AA
February
Betsy Leondar - Oregon State University (c) 2011
BA
AA
April
November
February
April
Ag BA
0.00365
0.00256
0.00407
Ag AA
0.00747
0.00443
0.00555
Nat BA
0.0109
0.00595
0.0087
Nat AA
0.0100
0.0192
0.0130
Res BA
0.00470
0.00405
0.0104
Res AA
0.0110
0.00893
0.0109
Betsy Leondar - Oregon State University (c) 2011
Results
• EOC, TEN, NO3• DEAs
• Two-Way ANOVA
• Correlations
Conclusions
• Future Research
Questions
Betsy Leondar - Oregon State University (c) 2011
Treatment
Time
Treatment + Time
Before Acetylene
0.0456
0.158
0.4952
After Acetylene
0.0453
0.901
0.5031
BA:AA Ratio
0.0449
0.0006
0.6060
Percent Moisture
0.0033
0.1565
0.9316
Total Organic
Carbon
0.7258
0.0048
0.5081
Nitrate
0.0132
<0.0001
0.6357
Betsy Leondar - Oregon State University (c) 2011
Statistically significant p-values:
• BA + Treatment
• BA:AA Ratio + Treatment
• BA:AA Ratio + Time
• AA + Treatment
• Percent Moisture + Treatment
• EOC + Time
• Nitrate + Treatment
• Nitrate + Time
Betsy Leondar - Oregon State University (c) 2011
Results
• EOC, TEN, NO3• DEAs
• Two-Way ANOVA
• Correlations
Conclusions
• Future Research
Questions
Betsy Leondar - Oregon State University (c) 2011
BA
AA
BA:AA
%H2O
EOC
NO3-
BA
1
-
-
-
-
-
AA
0.425876
1
-
-
-
-
BA:AA
0.372848
-0.22712
1
-
-
-
%H2O
0.409928
0.745827
-0.48914
1
-
-
EOC
0.015959
0.22664
0.332972
-0.22119
1
-
NO3-
0.164751
-0.19945
0.75738
-0.6583
0.500112
1
Betsy Leondar - Oregon State University (c) 2011
Positive
• BA + AA
• BA + %H2O
• AA + %H2O
• BA:AA + NO3• EOC + NO3-
Negative
• BA:AA + %H2O
• %H2O + NO3-
Betsy Leondar - Oregon State University (c) 2011
• Positive correlation between %H2O and
DEA rates
• No correlation between NO3- and DEA
rate
• Positive correlation between EOC and
NO3• Negative correlation between %H2O and
NO3Betsy Leondar - Oregon State University (c) 2011
• Highest DEA rates occurred in February,
then November, then April
• Highest rates occurred in natural
wetlands, then restored wetlands, then
agricultural sites with some variation
Betsy Leondar - Oregon State University (c) 2011
• Higher nitrate means lower N2O
reduction activity
• Sufficient nitrate to accept e-
• Not as many e- left over to reduce N2O
• Nitrate production associated with
nitrification
• DEA rate is an effective restoration
evaluation method
• Compare microbial activity between sites
Betsy Leondar - Oregon State University (c) 2011
• Important to control N2O emissions due to its
greenhouse gas effects
• Wetland restoration
• Decrease use of N fertilizers
• Evaluation of wetland restoration practices
• Solid analytical data available between site
types
Betsy Leondar - Oregon State University (c) 2011
 Dr. David Myrold and Myrold Lab
 Dr. Mary Santelmann
The Natural Resources
Conservation Service
 Sara Taylor
 Portland Metro
 OSU Departments of Crop and Soil  Center for Genome Research and
Biocomputing
Science and Geosciences
 Private landowners for use of their
 Bioresource Research
property
Interdisciplinary Program
 Subsurface Biosphere Initiative
Undergraduate Research Program
Betsy Leondar - Oregon State University (c) 2011
Betsy Leondar - Oregon State University (c) 2011