Denitrification in Natural, Converted, and
Restored Wetlands of the Mid-Atlantic.
Jarrod O. Miller, Ph.D.
University of Maryland Extension
Lower Eastern Shore
What is denitrification?
Reduction of NO3- to gaseous N2
2NO3− → 2NO2− → 2NO → N2O → N2 (g)
Occurs in low oxygen (saturated) soils
Can be incomplete, producing N2O
NO3
Incomplete
Complete
N2O
N2
Who causes denitrification?
• Anaerobic bacteria
• Operate in low (<10%) O2 environments
• Mostly heterotrophs – obtain energy by
oxidizing carbon (organic matter)
• Oxygen released from N compounds is used
on organic matter
NO3
Incomplete
Complete
N2O
N2
Order of Preferred Reduction
Sudan et al., UC Davis
Denitrification and microsites
-Can occur side by side
Nitrate
Nitrification
Iron
Denitrification
Organic matter is a microbial energy
source
It can be simplified into three parts
Fresh organic matter has
more fulvic and humic acids
These have more N, P, K
Older organic matter
has more carbon, less
nutrients for microbes
Better for fungi
C/N ratio
Wood chips
C
N
If the C/N is above
25, the microbes
will have to steal N
from the soil to eat
Soil N
Why is C/N important to denitrification?
• In denitrification both NO3 and Carbon are
important
• When your soil C:N is greater than 25 (i.e. more
carbon), more N2 is produced
• When your soil C:N is less than 25 (i.e. more
nitrogen), increases N2O (incomplete)
NO3
Incomplete
Complete
N2O
N2
Temperature
Microbes are living things, both moisture and
temperature affect them
– Optimum temperature for denitrification is 35 to
35C
– Also occurs between 2 and 50C
pH
• Very strong pH (< 5.0) favors N2O
production
• Inhibits rapid denitrification
Denitrification Factors – In Summary
•
•
•
•
•
Oxygen depletion – may vary across porosity
Nitrate concentration – related to C/N
Quality and quantity of carbon – related to C/N
Temperature – regulated microbial activity
pH – affects rapid denitrification
Good luck predicting it
How do you measure denitrification?
Incomplete
Denitrification Enzyme Assay
– We measure N2O by gas
chromatography (incomplete)
– Block conversion of N2O to N2 with
acetylene (total)
Complete
Total
NO3
Incomplete
N2O
Complete
N2
How do you measure denitrification?
Incomplete
Denitrification Enzyme Assay
– We measure N2O by gas
chromatography (incomplete)
– Block conversion of N2O to N2 with
acetylene (total)
NO3
You may choose to add a nitrogen or
carbon source to maximize potential
N2O
Complete
N2
You can also measure in the field
But you may be
measuring N2O from
nitrification and
denitrification
Photoacoustic gas analyzer
•N2O and CO2
NO3
N2O
N2
Research Results
• CEAP – Mid-Atlantic States (Maryland,
Delaware, Virginia, North Carolina)
• Florida Everglades – Sugarcane/water table
management
This project was a part of CEAP
•
USDA Conservation Effects
Assessment Project (CEAP)
•
Observing the environmental
effects of conservation practices
•
Will guide future USDA efforts for
conservation policy
Multi agency effort
•
•
NRCS
ARS
• USGS
• University of Maryland
Agricultural Research Service
We were charged with denitrification
and gas emissions
Mid-Atlantic Region
Maryland
Eastern Shore
Delaware
Virginia
Middle Coastal Plain/Tidewater
Pocosin Region
North Carolina
Florence, SC
48 sites in 4 states:
i. 21 sites in Maryland
ii. 7 sites in Delaware
iii. 11 sites in Virginia
iv. 9 sites in North Carolina
Three site types:
1. Converted (drained ag land)
2. Restored
3. Natural (forested)
NRCS Conservation Practice Standards
• 657- Wetland Restoration
• 646 – Shallow Water Management
Converted
Natural
Depressional Coastal Wetlands
Restored Wetland
“Natural” wetland
Drainage Ditch
Pocosin Wetlands, Eastern NC
Soil Sampling (June 2009 - May 2011)
Used GIS to create 4
elevation zones in
each wetland.
Sampled each
wetland 3 times.
4
Sampling points follow the depression
1 = wettest
4 = driest
2
1
3
4
3
2
1
Denitrification analysis
Lab Denitrification:
•Denitrification enzyme assay (DEA)
•DEAi –Incomplete DEA
•nDEAi - Incomplete with NO3 addition
With acetylene
•DEA – Total DEA
•nDEA – Total DEA with NO3 addition
NO3
N2O
N2
NO3
N2O
N2
•Measure N2O by gas chromatography
Add NO3 to see maximum potential
Can also add glucose (carbon)
N2O Flux
Photoacoustic gas analyzer
•N2O and CO2
NO3
N2O
N2
DNA Extraction
nosZ gene
– Encodes the enzyme for N2O to N2
Results
Soil Properties
Management Elevation
Natural
Restored
Converted
Carbon (%)
Nitrogen (%)
C:N ratio
1 (wet)
7.5 a
0.48 a
13.8 d
2
7.1 b
0.42 b
15.2 c
3
6.1 c
0.35 c
16.7 b
4 (dry)
5.6 d
0.28 d
19.7 a
1
2.0 e
0.18 ef
9.3 e
2
2.1 e
0.19 ef
9.1 e
3
2.1 e
0.19 ef
9.3 e
4
1.8 ef
0.18 ef
9.3 e
1
2.0 e
0.20 e
9.3 e
2
1.9 ef
0.19 ef
9.0 e
3
1.6 fg
0.18 ef
8.0 f
4
1.5 g
0.17 f
7.5 g
Soil Properties
Management Elevation
Natural
Restored
Converted
pH
Moisture (%)
Soil Temp(C)
1
4.43 f
34.4 a
21.8 d
2
4.23 g
32.5 b
22.0 d
3
4.17 g
28.6 c
21.4 d
4
4.25 g
22.1 e
22.1 d
1
5.61 e
24.6 d
25.0 abc
2
5.78 d
19.5 f
24.6 bc
3
5.72 d
17.2 gh
24.3 c
4
6.12 a
13.5 i
24.7 bc
1
6.01 b
18.6 fg
25.3 ab
2
5.92 c
16.2 h
25.0 abc
3
6.02 b
14.0 i
24.9 abc
4
5.90 c
11.2 j
25.5 a
Soil Properties
Management Elevation
Natural
Restored
Converted
Ca (ppm)
Mg (ppm)
K (ppm)
1
111 f
58 f
75 f
2
55 g
41 g
62 g
3
47 g
35 gh
54 h
4
53 g
31 h
51 h
1
500 e
111 d
86 e
2
522 e
110 d
72 f
3
507 e
102 e
70 f
4
613 d
117 c
82 e
1
1086 a
178 a
149 a
2
890 b
143 b
132 b
3
778 c
123 c
119 c
4
624 d
106 de
108 d
Soil Properties
Management Elevation
Natural
Restored
Converted
NO3 (ppm)
SO4 (ppm)
PO4 (ppm)
1
4.81 ef
49.93 a
2.37 d
2
5.32 e
40.62 b
2.65 d
3
5.29 e
30.02 c
2.41 d
4
4.51 ef
17.21 de
2.59 d
1
6.13 e
14.68 ef
0.89 f
2
2.81 f
13.19 fg
1.25 e
3
6.92 e
12.84 fg
1.32 e
4
11.13 d
11.07 g
2.35 d
1
21.36 b
19.17 d
4.73 a
2
25.10 a
14.76 ef
4.12 b
3
22.15 b
11.11 g
4.64 a
4
17.81 c
7.51 h
3.74 c
Elevation
1
• No difference in incomplete
• Total DEA greater in ag land
• Due to NO3?
Plus NO3
• Adding nitrate erased
differences in
management
• Restored and natural
were nitrate limited
Elevation
2
• No difference in incomplete
• Total DEA greater in ag land
• Adding nitrate erased
differences in
management
• Restored and natural
were nitrate limited
Elevation
3
• Restored had greater
incomplete
• Natural had lowest total
• Adding nitrate erased
differences in
management
• Restored and natural
were nitrate limited
Elevation
4
• Natural had lowest total and
incomplete
• Adding nitrate increased
the incomplete in natural
• Natural did not catch up
in total DEA
Actual
Emissions
Natural has the lowest N2O, but
not statistically different
DNA
extraction
• Converted and
restored had greater
nosZ
• Higher nosZ in wetter
landscape positions
• As nosZ increased,
incomplete
denitrification
decreased
CEAP Conclusions
• Restored wetlands are similar to both natural and
converted (ag land)
– Retain Ca, Mg from liming
– Haven’t built up C and N yet
– Probably will continue to move towards natural state
• For DEA, restored wetlands are like natural in the
bottoms
– Act more like converted at their upper edges
When
exposed to
oxygen,
organic
matter is
broken down
by microbes
R. Reddy
ARS- Canal Point, FL
Adjusting water table
depth to observe:
1. Effects on sugarcane yield
2. Effects on soil properties
3. Effects on nutrient
leaching
Treatments included carbon sources
Greenhouse gas emissions from organic soils
Treat.
May
depth
July
depth
August
depth
Flood?
----------------(cm)----------------40a
40
40
40
No
4040
40
40
40
Yes
4016
40
40
16
Yes
1616
40
16
16
Yes
N2O
(mg m-2 d-1)
CO2
(g m-2 d-1)
40a
4040
4016
1616
p value
2.6
2.6
2.7
2.3
0.8237
27 b
34 a
26.7 b
25.7 b
0.0745
April
July
September
p value
6.6 a
0.7 b
0.4 b
<0.0001
24 b
31.7 a
29.4 ab
0.0366
ARS Research Station, Canal Point, FL
Control
Nitrate
Carbon
N and C
DEA Activity
• Glucose increased
potential DEA more
than nitrate
• Indicates that these
soils are carbon
starved
DEA Treatments
ugN /kg soil/ day
•No differences by water table
treatment
8
9
7
8
6
7
5
40a
4
4040
4016
3
1616
2
% Incomplete
% Incomplete
•Most differences came from
nitrate/glucose additions
1800
1600
1400
1200
1000
800
600
400
e
200
0
a
b
d
e
e
c
N2O
e
a
6
April
5
4
b
3
b
2
1
1
0
%DEA
%nDEA
%gDEA
%gnDEA
0
%DEA
%nDEA
%gDEA
Denitrification Enzyme Activity (DEA)
%gnDEA
July
September
Questions?