Post on 13-Apr-2017
The Production of Nitrous Oxide by Bacteriain Chemostat and Soil
David RichardsonUEA, Norwich
Liz BaggsUniversity of Aberdeen
Short History of Nitrous Oxide
Joseph Priestley1775
N2O
Short History of Nitrous Oxide
N2O
Sir Humphry DavyPresidente 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 soundYet are my limbs with inward transports fill'd And clad with new-born mightiness around."
Nitrous Oxide is a Potent Greenhouse Gas
20 years 100 years 500 years
1 1 162 23 7
275 296 156
Carbon Dioxide CO2
Methane CH4
N2ONitrous Oxide
Atmospheric nitrous oxide has increased by 20% over the last 100 years
N2O concentrations (IPCC Fourth Assessment Report, 2007)
Soil is a significant source of N2OIPCC 2007: ‘Land surface properties and land-atmosphere interactions
that lead to radiative forcing are not well quantified’.
SoilOceanCattle & feedlotsIndustryAtmosphereBiomass burning
10.2 Tg N y-1
Upturn in N2O production due toincreases in soil N availability:
•N deposition•N fertilization
Source: IPCC (2007)
The Nitrogen CycleNO3
-
N2
NO2-
NH4+
NH2OH
N2O
NO
DENITRIFICATION
NITRIFICATION
FIXATION
Cellular toxin
Greenhouse gas
Continuous culture studies with bacteria
feed sample
effluent
pH control (1 M NaOH, 0.1 M H2SO4) DO2 monitoring
air in
air out
temperature control
• minimal medium (succinate, nitrate)
• pH 7.0
• Temp: 37°C
aerobic anaerobic
P.denitrificans chemostat cultureThe effect of oxygen
0 20 40 60 80 100 1200.00.20.40.60.81.01.21.41.61.82.0
Dry
mas
s m
g/m
l
Time h
0 20 40 60 80 100 1200
200400600800
10001200140016001800200022002400
N2O
µM
Time
aerobic anaerobic
P.denitrificans chemostat cultureThe effect of oxygen
What is the effect of copper on denitrification?
NO2-NO3
- N2O
N2
NOCyt Cd1
Haem dependientes
Cu dependientes Nitrous oxide reductase
‘copper replete’ (20 µM)
‘copper limited’ (0.8 µM)
‘copper deplete’ (copper not detectable)
aerobic anaerobic
P.denitrificans chemostat cultureThe effect of Copper
0 20 40 60 80 100 1200.00.20.40.60.81.01.21.41.61.82.0
Dry
mas
s m
g/m
l
Time h
Cu 18 µM Cu 0.8 µM Cu 0 µM
aerobic anaerobic
0 20 40 60 80 100 1200
5
10
15
20
25
30N
O- 3 m
M
Time h
Cu 18 µM Cu 0.8 µM Cu 0 µM
P.denitrificans chemostat cultureThe effect of Copper
aerobic anaerobic
0 20 40 60 80 100 1200
200400600800
10001200140016001800200022002400
N2O
µM
Time
Cu 18 µM Cu 0.8 µM Cu 0 µM
P.denitrificans chemostat cultureThe effect of Copper
What is the effect of copper on denitrification?
NO3- NO2
- N2O
N2
Cu Replete:
<1% of NO3- N2O
Cu limited:
11% of NO3- N2O
Cu deplete:
40% of NO3- N2O
0.4% of NO3- NO2
-
Cu dependent Nitrous oxide reductase
NOIron dependent
Nitrite reductase
some agricultural soils are copper deplete< 10 micromolar bioavailable copper
Incomplete denitrification
N2ONO3-
Complete denitrification
Other factors to consider:pH
organic carboninfluence of the plant
nature of the microbial community
N2NO3-
M iscan thus W illow
N2O
-N (µ
g N
m-2
)
0
50
100
150
200
250
300
D enN itr
M iscan thu
N2O
-N (µ
g N
m- 2
)
0
20
40
60
80
100
120
Gross nitrification mg N kg-1 d-1
10 days
nitrificationnitrate reduction
Nitrification versus nitrate reduction
Sandy loamNH4NO3 at 12 g N m-2
Miscanthus 7.3 ± 0.3SRC Willow 12.0 ± 0.6
Denitrifier-N2O & N2
Day of year (2001)
170 175 180 185 190 195
Den
itri
fied
15 N
-N2O
+ 1
5 N-N
2 fl
ux
(mg
N m
-2 d
-1)
0
10
20
30
40
50
36 Pa60 Pa
Rain
fall
(mm
)
1612840
Air
tem
p (o
C)
12162024
15N10 atom %
Day of year (2001)
170 175 180 185 190 195
N2
:N2O
rat
io
0
100
200
300
400
500
36 Pa60 Pa
Lab soil columns
AirO
2gr
adie
nt
C exudate A C exudate BD
iffer
ent d
enitr
ifier
ge
ne c
opy
num
bers
?
15N-N2O15N-N2
CuNir, cdNir, NosZ
pump
Different denitrifier
gene copy numbers?
N2O/O2
CuNir, cdNir, NosZ
CuNir, cdNir, NosZ
O2 analyser
flow controller
mg
15N
-N2O
m-2
41d
-1
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Nitrification Denitrification
pH 4.5 pH 7.00
1
2
3
4
5
6
7
15 mg Cu kg-1 soil60 mg Cu kg-1 soil
µ g N
2O-N
m-2
7 d-1
Potential for enhancing N2O reduction
N2O:N2?
Alleviation of Cu-limitation at pH 7?
Does C influence N2O reduction?
Common exudation compounds from Ectomycorrhizal fungi.
K15NO3, 5 g N m-2, 10 atom % excess 15N.3.6 g C l-1 70% WFPS
0.0
0.1
0.2
0.3
0.4
0.5
mg
15N
-N2O
m-2
14d
0
20
40
60
80
100controlglucosemannitoloxalic acid
mg
15N
-N2 m
-2 1
4d
14 days N2O N2
Differences in regulation of NO & N2O reductases?Preference for different C compounds in rhizosphere denitrifier community?
Where is C flowing in the rhizosphere?
On root surfaceColonising root tip
In situ visualisation of pseudomonads marked with unstable gfp in the rhizosphere of a barley seedling
Where is N2O produced in the rhizosphere?
carbon
N2O Source partitioning ?
nutrient depletion zone
N - lux fusion
C - reporting
Den
itri
fica
tion
Distance
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
Mapping location of active microbes
13C
15N-NO3 applied to soilAir filled poreWater filled poreAnoxic zoneOxic zone
SOM
15N & 13C in denitrifieror 15N-N2O
15N in denitrifieror 15N-N2O
Hotspots of denitrifier activity (e.g. with C quantity, quality & O2 availability)N2O production & source partitioning in situ.
Manipulating the rhizosphere for function
Lower N application
13C SOM
High N application NetCH4
N2O N2O:N2
Nitrification + DenitrificationNitrifier denitrification
Inhibition of CH4 oxidationCH4 oxidation
Denitrification
Lowered nitrifier denitrification
Lower N2O:N2
Plant breeding for exudate C compounds which enhance reduction of N2O to N2
Denitrification
SOM management to alleviate Cu-limitation to enhance reduction of N2O to N2
Distance from root/time
Future challenge: Resolving issues of scale
gene
plant
field
landscape
10-8 m
10-2 m
102 m
105 m
Bug to big
Modelling
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
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.
http://www.nitrousoxide.org/index.htmld.richardson@uea.ac.uke.baggs@abdn.ac.uk