Impact of Agriculture on the Soil CH4 Sink:

Use of 13CH4 stable isotope probing to investigate unculturable soil methanotrophic bacteria

Richard EvershedBristol Biogeochemistry Research Centre,

Organic Geochemistry Unit,

School of Chemistry,

University of Bristol

OH

OH

OH

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O

Exploring soil processes through building block biomolecules

OH

OH

Bacteria

Animals

Fungi

Bacteria

Ubiquitous

• Sources of CH4

– Wetlands– Agriculture– Landfill Sites– Energy production and use – Natural gas venting– Biomass burning– Domestic Sewage

• Sinks of CH4

– Chemical reactions in the atmosphere with OH (ca. 90%)

Soils via methane oxidising bacteria (5%; methanotrophic bacteria: methane eating bacteria)

Sources and sinks of methane

• An enhanced understanding of the methanotrophic bacteria would help in managing methane sinks e.g. fertilizer effects, tillage, etc.

–

Low affinity = ‘high capacity’ methanotrophic bacteria

• Adapted for growth in elevated concentrations of CH4 (% range) i.e. where methanogenesis is occurring, e.g. Landfills, wetlands etc.

• More than 90% of methane produced in methanogenic environments is reoxidised by methanotrophs.

• Can be grown in the laboratory

• The total biological sink is comparable to the atmospheric sink

CH4 CH3OH HCHO HCO2H CO2

Biosynthesis and growth of new organisms

Energy

High affinity = ‘low capacity’ methanotrophs

• Found in well-aerated soils, i.e. grasslands, forests, agricultural land, etc.

• Consume CH4 directly from the atmosphere

• Survive on trace levels of atmospheric CH4, i.e. 2 ppmv

• Vitally important methane sink

• As yet cannot be cultured in the laboratory hence very difficult to study

Methane oxidising bacteria

CH4 CO2

Culture independent methods: phospholipid fatty acid analysis (PLFA) of soil microbes

•Phospholipids are major components of cell membranes

O

OH

OH

PO

O

O

O

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O

O

O

OH

O

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• Phospholipids short-lived on cell death •Thus, PLFAs represent living microbes

Phospholipid

fatty acids (PLFAs)

_

Phospholipid

Rationale

Evershed et al. Current Opinion Biotechnol. (2006) 17, 72–82

None of the PLFAs is ONLY produced by methanotrophs

Gas chromatography ‘fingerprints’ of PLFAs of soil microbes

•Wide range of microbial groups represented but impossible to say which PLFAs come from methanotrophs

No need to lie!No need to lie!No need to lie!

Selective 13C- labelling of subset of microbial population

Stable isotope probing (‘FISHING’ ) for methane eating bacteria’

13CH4

Incubate

Highly complex soil microbial community

Only bacteria consuming 13CH4

become 13C-labelled

• Challenge: detection of very low concentrations of 13C- labelled PLFA amongst 10,000 x the concentration of unlabelled PLFAs

Synthetic air + 13CH4

Soil incubations

Flow through chamber

Soils

Chamber flushed every 24 hSyn

the

t ic

air

+ 1

3C

H4

Maxfield et al. Environ. Microbiol. (in press)P.J. Maxfield et al. Appl. Environ. Microbiol. (2006) 72, 3901–3907.

Crossman et al. Environ. Sci Technol. (2004)

Exposure of soils to different concentrations of 13CH4 to look forhigh and low affinity methanotrophs

• Analyse PLFAs by GC-IRMSto locate 13C-label

• Only the PLFAs from the methanotrophic bacteriatake up 13 CH4

• Dominance of 18:1 PLFAindicates same type of methanotrophic bacteria oxidising methane at both high and low concentrations

• Concentrations of 13C-labelledPLFAs indicates methanotrophsare ca. 0.01% of all soil bacteria

δ13

C v

alue

s13

C-

PLF

A c

once

ntra

tion

PLFA Crossman et al. Org. Geochem. (2005)

• How do fertiliser treatments impact on methanotrophic bacteria in soils?

• Run by IGER (Institute of Grassland & Environmental Research)

• Long term study: 6 different soil treatments over 14 years

• Wider investigation the impact of grazing and other agricultural practices on uplands

Effects of low input farming in the Brecon Beacons, South Wales

P.J. Maxfield et al. Environ. Microbiol. 2008 10, 1917–1924

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Methanotroph PLFAs

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N Ca P K

Ca P K

Total Microbial Biomass PLFAs

Methanotrophs supressed by inorganic

fertiliser use

No fertiliser No fertiliser

1.22 nmol g-1 d-1

0.46 nmol g-1 d-1

0.75 nmol g-1 d-1

Conclusions

• 13CH4 stable isotope probing links soil methane oxidation capacity of soils to the microorganisms involved.

• Methanotropic bacteria are minor components of the soil microbial biomass (ca. 106 cells g-1 soil, i.e. 0.01 %) and are sensitive to disturbances.

• Inorganic fertiliser additions supress CH4 oxidation linked to reductions in the methanotropic bacterial populations.

• Work at Rothamsted also points to tillage as a major factor in affecting methanotroph populations (ca. 105 cells g-1 soil).

• More work needed to understand the potential capacity of the soil CH4 sink nationally and globally.

• 15N-probing providing new insights into the fate of N-fertilisers.

• NERC and BBSRC thanked for financial support.