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

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."

Short History of Nitrous Oxide

Short History of Nitrous Oxide

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)

NO3-

N2

NO2-

NH4+

NH2OH

N2O

NO

DENITRIFICATION

NITRIFICATION

The Nitrogen Cycle

The Nitrogen CycleNO3

-

N2

NO2-

NH4+

NH2OH

N2O

NO

DENITRIFICATION

FIXATION

The Nitrogen CycleNO3

-

N2

NO2-

NH4+

NH2OH

N2O

NO

DENITRIFICATION

NITRIFICATION

FIXATION

Cellular toxin

Greenhouse gas

The Nitrous Oxide Reductase is dependent on copper

N2O + 2e- + 2H+ N2 + H2O

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

N2 CuNO3-

Complete denitrification

Other factors to consider:pH

organic carboninfluence of the plant

nature of the microbial community

N2NO3-

Field & lab experimentation

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