This presentation formed part of the Farming Futures workshop 'Cropping Climate Change: Making business sense of nitrous oxide and the nitorgen cycle' March 5th 2010

1. The Production of Nitrous Oxide by Bacteria
in Chemostat and Soil
David Richardson
UEA, Norwich
Liz Baggs
University of Aberdeen

2. Short History of Nitrous Oxide
N2O
Joseph Priestley
1775

3. Short History of Nitrous Oxide
N2O
Joseph Priestley
1775

4. Short History of Nitrous Oxide
N2O
Sir Humphry Davy
Presidente de la Royal Society 1820-27
Ode to Nitrous Oxide
"Yet are my eyes with sparkling lustre fill'd
Yet is my mouth replete with murmuring sound
Yet are my limbs with inward transports fill'd
And clad with new-born mightiness around."

5. Short History of Nitrous Oxide

6. Short History of Nitrous Oxide

7. Nitrous Oxide is a Potent Greenhouse Gas
20 years 100 years 500 years
Carbon Dioxide 1 1 1 CO2
Methane 62 23 7 CH4
Nitrous Oxide 275 296 156 N2O

8. Atmospheric nitrous oxide has increased by
20% over the last 100 years
N2O concentrations (IPCC Fourth Assessment Report, 2007)

11. Soil is a significant source of N2O
IPCC 2007: ‘Land surface properties and land-atmosphere interactions
that lead to radiative forcing are not well quantified’.
Soil
Ocean
Cattle & feedlots
Upturn in N2O production due to Industry
increases in soil N availability: Atmosphere
•N deposition Biomass burning
•N fertilization
10.2 Tg N y-1
Source: IPCC (2007)

12. The Nitrogen Cycle
NO3-
NO2-
D
E N
N I
I T
T R
R I
I
NO NH2OH F
F I
I C
C A
A T
T N2O NH4+ I
I O
O N
N
N2

13. The Nitrogen Cycle
NO3-
NO2-
D
E
N
I
T
R
I
NO NH2OH
F
I
C
A
T N2O NH4+
I
O
N
N
T IO
A
N2 FIX

14. The Nitrogen Cycle
NO3-
NO2-
D
E N
N I
I T
T R
R
I Cellular toxin NH2OH I
F
NO F
I
I C
C A
A
Greenhouse
T
T N2O NH4+ I
gas
I O
O N
N
N
T IO
A
N2 FIX

15. The Nitrous Oxide Reductase is
dependent on copper
N2O + 2e- + 2H+ N2 + H2O

16. Continuous culture studies with bacteria
air
out
air in pH control (1 M NaOH, 0.1 M
H2SO4) DO2 monitoring
feed sample
effluent
• minimal medium
(succinate, nitrate)
• pH 7.0
• Temp: 37°C temperature
control

17. P.denitrificans chemostat culture
The effect of oxygen
2.0
1.8
1.6
aerobic anaerobic
1.4
Drymass mg/ml
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0 20 40 60 80 100 120
Time h

18. P.denitrificans chemostat culture
The effect of oxygen
2400
2200
2000 aerobic anaerobic
1800
1600
1400
N2O µM
1200
1000
800
600
400
200
0
0 20 40 60 80 100 120
Tim e

19. What is the effect of copper on denitrification?
‘copper replete’ (20 µM)
‘copper limited’ (0.8 µM)
‘copper deplete’ (copper not detectable)
N2
NO3- NO2- N2O
NO Cu dependientes
Cyt Cd1 Nitrous oxide reductase
Haem dependientes

20. P.denitrificans chemostat culture
The effect of Copper
Cu 18 µ M
2.0 Cu 0.8 µ M
1.8 Cu 0 µ M
1.6
1.4
Drymass mg/ml
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0 20 40 60 80 100 120
aerobic anaerobic
Time h

21. P.denitrificans chemostat culture
The effect of Copper
30
Cu 18 µM
Cu 0.8 µM
25
Cu 0 µM
20
NO3 mM
15
-
10
5
0
0 20 40 60 80 100 120
aerobic anaerobic
Time h

22. P.denitrificans chemostat culture
The effect of Copper
2400
C u 18 µ M
2200
C u 0.8 µ M
2000
Cu 0 µM
1800
1600
1400
N2O µM
1200
1000
800
600
400
200
0
0 20 40 60 80 100 120
aerobic anaerobic
Tim e

23. What is the effect of copper on denitrification?
Cu Replete:
<1% of NO3- N2O
Cu limited:
11% of NO3- N2O
Cu deplete: N2
40% of NO3- N2O
0.4% of NO3- NO2-
NO3- NO2- N2O
NO Cu dependent
Iron dependent Nitrous oxide reductase
Nitrite reductase

24. NO3- N2O
some agricultural soils are copper deplete
< 10 micromolar bioavailable copper
Incomplete denitrification

25. NO3- N2 Cu
Complete denitrification

26. NO3- N2
Complete denitrification
Other factors to consider:
pH
organic carbon
influence of the plant
nature of the microbial community

27. Field & lab experimentation

28. Nitrification versus nitrate reduction
10 days
300 120
250 100
N2O-N (µg N m ) nitrification
N2O-N (µg N m )
nitrate reduction
-2
-2
200 80
D en
N itr
150 60
100 40
50 20
0 0
M is c a n th u s W illo w M is c a n th u
Sandy loam
NH4NO3 at 12 g N m-2
Gross nitrification mg N kg-1 d-1
Miscanthus 7.3 ± 0.3
SRC Willow 12.0 ± 0.6

29. Denitrifier-N2O & N2
0
Rainfall
4
(mm)
8
12
16
Air temp (oC)
24
20
16
12
50
Denitrified 15N-N2O + 15N-N2 flux
40
36 Pa
30 60 Pa
(mg N m-2 d-1)
20
500
10
400 36 Pa
N2 :N2O ratio
0 60 Pa
300
170 175 180 185 190 195
200
15N Day of year (2001)
100
10 atom %
0
170 175 180 185 190 195
Day of year (2001)

30. Lab soil columns
15
N-N2O
15
N-N2
Air
p
pum
C exudate A C exudate B
gene copy numbers?
N2O/O2
Different denitrifier
CuNir,
gene copy numbers?
Different denitrifier
O2 gradient
cdNir,
NosZ
CuNir, CuNir,
cdNir, cdNir,
NosZ NosZ
O2 analyser
flow controller

31. Potential for enhancing N2O reduction
3.5
3.0 Nitrification
mg 15N-N2O m-2 41d-1
Denitrification
2.5
2.0
1.5
1.0
0.5
0.0
7
6 N2O:N2?
15 mg Cu kg-1 soil
5 60 mg Cu kg-1 soil
µg N2O-N m-2 7 d-1
4
Alleviation of Cu-limitation at pH 7?
3
2
1
0
pH 4.5 pH 7.0

32. Does C influence N2O reduction?
Common exudation compounds from Ectomycorrhizal fungi.
3.6 g C l-1 K15NO3, 5 g N m-2, 10 atom % excess 15N. 70% WFPS
100 14 days N2O 0.5 N2
control
mg N-N2O m 14d
mg N-N2 m 14d
80 0.4 glucose
-2
mannitol
-2
60 0.3 oxalic acid
40 0.2
15
15
20 0.1
0 0.0
Differences in regulation of NO & N2O reductases?
Preference for different C compounds in rhizosphere denitrifier community?

33. Where is C flowing in the rhizosphere?
Colonising root tip On root surface
In situ visualisation of pseudomonads marked with
unstable gfp in the rhizosphere of a barley seedling

34. Where is N2O produced in the rhizosphere?
N2O Source partitioning ?
nutrient depletion zone
C - reporting
Denitrification N - lux fusion
Distance
carbon

35. Mapping location of active microbes
Blue = 28Si- Green = 12C14N- (represents organic matter)
Red = 15/14N ratio images (distribution of 15N enriched P. fluorescens)
Herrmann et al 2007
Rapid Comm Mass Spec 21, 29-34

36. Mapping location of active microbes
15N-NO applied to soil
3
Air filled pore
Water filled pore
Anoxic zone
Oxic zone
15N & 13C in denitrifier
13C
or 15N-N2O
15N in denitrifier
SOM 15N-N O
or 2
Hotspots of denitrifier activity (e.g. with C quantity, quality & O2 availability)
N2O production & source partitioning in situ.

37. Manipulating the rhizosphere for function
LowerN application
High N application Net
CH4 N2O N2O:N2
13C
SOM Denitrification
Nitrification + Denitrification
Nitrifier denitrification
Inhibition nitrifier denitrification
Lowered of CH4 oxidation
CH4 oxidation
Lower N2O:N2
Plant breeding for exudate C
Denitrification compounds which enhance
reduction of N2O to N2
SOM management to alleviate
Distance from root/time
Cu-limitation to enhance
reduction of N2O to N2

38. Future challenge: Resolving issues of scale
Bug to big 10-8 m gene
10-2 m plant
Modelling
102 m field
105 m landscape

39. How can we constrain the soil-N2O budget?
Advancing techniques & adopting interdisciplinary approaches to
quantify and understand controls on N2O.
Tackling issues of scale. Integrating chemostat and soil studies to field/landscape.
Understanding control of microsite structures on
microbial community composition & processes.
Greater understanding of regulation of the N2O reductase:
mitigation by reducing N2O to N2?
Greater understanding of interactions with C cycle.
Enhance quantification and understanding of N2O production
informing targeted and sustainable management for mitigation

40. The Nitrous Oxide Focus Group is a
consortium-based research initiative
established to explore the action of
the greenhouse gas, Nitrous Oxide;
its role in climate change, the role of
bacteria in the greenhouse gas
emissions and to develop techniques
to mitigate its effect.
Ultimately the Group will work toward solutions for the wider
community and commercial and non-academic partners are being
sought to inform and enable the development of opportunities arising
from the Nitrous Oxide Focus Group’s research.
d.richardson@uea.ac.uk http://www.nitrousoxide.org/index.html
e.baggs@abdn.ac.uk