compound-specific stable isotope analysis as a tool to characterize the role of microbial community...

Compound-specific stable isotope analysis as a tool to

characterize the role of microbial community structure

in C cyclingK. Denef, P. Boeckx, O. Van Cleemput

Laboratory of Applied Physical Chemistry (ISOFYS)

Ghent University (Belgium)

Soil organic carbon

Ecosystem Management

Global Change (climate, elevated GHG)

?Microbial community

Fungi Bacteria

Soil organic carbon

Ecosystem Management

Global Change (climate, elevated GHG)

Microbial community

Fungi Bacteria

Possible reasons for fungal-induced C sequestration

• Fungal alteration of soil physical structure– Aggregate formation (Bossuyt et al., 2001)

– Aggregate stabilization: Glomalin (Wright et al., 1999)

– Fungal-induced macroaggregate-C protection (Frey et al., 2003)

– Preferential protection of fungal-derived C in microaggregates within macroaggregates (Simpson et al., 2004)

• Differences in “physiology”: more uncertainties– C utilization efficiency? (Thiet et al., 2006)

– Stability of fungal- vs. bacterial-derived OM: unknown

Molecular markers for fungi vs. bacteria(Compound-specific analysis: CSA)

Microbial communities distinguished

Molecular marker

Phospholipid fatty acids (PLFA): living structuresGram + bacteria i14:0, i15:0, a15:0, i16:0, i17:0, a17:0

Gram – bacteria Monounsaturated (16:1w7, 18:1w7, cy17:0, cy19:0)

Actinomycetes 10Me-FAs

Fungi saprotrophic 18:1w9c, 18:2w6,9

Fungi mycorrhizal 16:1w5

Amino sugars (AS): microbial residuesBacterial residues Galactosamine

Muramic Acid

Fungal residues GlucosamineFrom Glaser, 2006; Drissner et al. (2006)

STRUCTURE (CSA)

Microbial community

FUNCTION (CSSIA)

Research Objective

I. METHODOLOGY

Carbon cycling

Grassland management intensity

Elevated CO2

II. APPLICATIONS GC-c-IRMS (13C-PLFA)

LC-c-IRMS (13C-AS)

II. APPLICATIONS: 1. 13CO2 pulse-labeling approach

Soil biota 13C

13C-PLFA

GC-c-IRMSGC-c-IRMS

13CO2

Roots + exudates 13CI. Impact of elevated CO2 (Giessen FACE,

Germany since 1998)1. Ambient CO2 (350 ppm)2. Elevated CO2 (450 ppm)

II. Impact of grassland management (Merelbeke, Belgium since 2000):1. N-fertilization level

1. 450 kg N ha-1 yr-1

2. 225 kg N ha-1 yr-1

3. 0 kg N ha-1 yr-1

2. Mowing frequency1. 5 times per year2. 3 times per year

OBJECTIVES

• Investigate elevated CO2 and grassland management impacts on root-C assimilating microbial communities

• Activity niche differentiation• Link stimulated fungal pathways to C

stabilization mechanisms (aggregation; fungal-derived OM)

Measurements (ongoing)

• Aboveground plant material: 13C• Roots: 13C• Root-associated soil: bulk 13C & 13C-PLFA• Bulk soil: bulk 13C & 13C-PLFA• Physical fractions (aggregate size fractions):

– 13C fractions– 13C-PLFA– AS concentrations

Expected stimulated fungal/mycorrhizal pathways:* less intense management* elevated CO2

Expected niche dominance of fungal activity:* macroaggregates

Expected preferential stabilization of fungal products:* microaggregates within macroaggregates

24 h after pulse-labeling Several times during/after pulse-labeling

First results FACE pulse-labeling

Mol% PLFA-C (0-7.5 cm) - 10h after start pulse-labeling

i-C

14

:0

i-C

15

:0

a-C

15

:0

i-C

16

:0

i-C

17

:0

a-C

17

:0

C1

6:1

w7

c

C1

7:0

cy

C1

8:1

w7

c

C1

9:0

cy

10

-MeC

16

:0

10

-MeC

18

:0

C1

6:1

w5

c

C1

8:1

w9

c

C1

8:2

w6

,9c

mol%

PLF

A-C

0

2

4

6

8

10

12

14

16

18

Ambient CO2Elevated CO2

In collaboration with Müller et al

Gram+ Gram-

Act

Fungi

Enhanced saprotrophicfungal abundance

i-C

14

:0

i-C

15

:0

a-C

15:0

i-C

16

:0

i-C

17

:0

a-C

17:0

C16:1

w7

c

C18:1

w7

c

10-M

eC1

6:0

10-M

eC1

8:0

C16:1

w5

c

C18:1

w9

c

C18:2

w6

,9c

- 10

- 5

0

5

10

15

20

25

30

3 hours during pulse- labeling10 hours after start pulse- labeling

Ambient CO2 treatment

i-C

14

:0

i-C

15

:0

a-C

15:0

i-C

16

:0

i-C

17

:0

a-C

17:0

C16:1

w7

c

C18:1

w7

c

10-M

eC1

6:0

10-M

eC1

8:0

C16:1

w5

c

C18:1

w9

c

C18:2

w6

,9c

13C

enri

chem

ent

(rel

ativ

e to

tim

e 0

PLF

A)

- 10

- 5

0

5

10

15

20

25

30

Elevated CO2 treatment



G+ G- Act Fungi G+ G- Act Fungi



i-C

14:0

i-C

15:0

a-C

15:0

i-C

16:0

i-C

17:0

a-C

17:0

C16:1

w7c

C18:1

w7c

10-M

eC16:0

10-M

eC18:0

C16:1

w5c

C18:1

w9c

C18:2

w6,9

c

Root-

der

ived

mol%

PLF

A-C

0

5

10

15

20

Ambient CO2Elevated CO2

Root-derived mol% PLFA-C (0-7.5 cm) - 10h after pulse-labeling

G+ G-

Act Fungi

Saprotrophicfungi

AM fungi



i-C

14

:0

i-C

15

:0

a-C

15

:0

i-C

16

:0

i-C

17

:0

a-C

17

:0

C1

6:1

w7

c

C1

8:1

w7

c

10

-M

eC1

6:0

10

-M

eC1

8:0

C1

6:1

w5

c

C1

8:1

w9

c

C1

8:2

w6

,9c

- 5

0

5

10

15

20

25

30

3 hours during pulse- labeling10 hours after pulse- labeling11 months after pulse- labeling

Ambient CO2 treatment

i-C

14

:0

i-C

15

:0

a-C

15

:0

i-C

16

:0

i-C

17

:0

a-C

17

:0

C1

6:1

w7

c

C1

8:1

w7

c

10

-M

eC1

6:0

10

-M

eC1

8:0

C1

6:1

w5

c

C1

8:1

w9

c

C1

8:2

w6

,9c 1

3C

enri

chem

ent

(rel

ativ

e to

tim

e 0 P

LFA

)- 5

0

5

10

15

20

25

30

Elevated CO2 treatment

G+ G- Act Fungi

G+ G- Act Fungi

C-assimilating community shifts over time?Different preferential OM sources?

Possible reasons for fungal-induced C sequestration

• Fungal alteration of soil physical structure– Aggregate formation (Bossuyt et al., 2001)

– Aggregate stabilization: Glomalin (Wright et al., 1999)

– Fungal-induced macroaggregate-C protection (Frey et al., 2003)

– Preferential protection of fungal-derived C in microaggregates within macroaggregates (Simpson et al., 2004)

• Differences in “physiology”: more uncertainties– C utilization efficiency? (Thiet et al., 2006)

– Stability of fungal- vs. bacterial-derived OM: unknown

2. 13C-substrate incubation approach

OBJECTIVES

• Determine formation rates of fresh plant-residue-derived fungal vs. bacterial amino sugars

• Investigate impact of substrate quality on fungal and bacterial activity and turnover

• Determine inherent biochemical stability of fungal vs. bacterial amino sugars

+ 13C substrate (uniformly labeled)

90% sand4% POM4% silt2% clay

Soil biota 13C

Wheat substrate C/N 13C

Grains 12.7 ± 0.3 651.9 ± 3.3

Leaves 37.5 ± 0.3 716.9 ± 2.3

Roots 41.0 ± 6.6 634.8 ± 6.9

Stems 57.2 ± 1.4 730.9 ± 1.0

HWE Leaves 81.3 ± 4.6 731.1 ± 2.6

13C-CO2

GC-c-IRMSGC-c-IRMS

13C-PLFA

13C-Amino sugars

GC-c-IRMSGC-c-IRMS

LC-c-IRMSLC-c-IRMS

Gas-IRMSGas-IRMS

3. 13C-substrate incubation approach

Expected results

1. Fungal:bacterial activity (13C-PLFA) greater for lower quality substrate soils

2. Different fungal vs. bacterial AS formation rates estimates for fungal vs. bacterial turnover rates

3. No differences in “inherent” stability of fungal vs. bacterial AS; stability controlled by clay-OM interactions & physical protection

Summary

• 13C-PLFA analysis:– Structure of the active C-metabolizing community

and how affected by land-use/management/global change

– Trace C sources (roots vs. residue/fresh vs. native OM)

– But limited to group-level (species?)

• 13C-Amino Sugar analysis:– Fate of microbial residues– Quantify formation/turnover rates– Investigate stabilization mechanisms

Funding Agency

FWO – Fund for Scientific Research Vlaanderen (Belgium)

Collaboration (FACE research)

Dr. Christoph Müller (Justus-Liebig University, Giessen, Germany)

Masters students

Mihiri Wilasini (Physical Land Resources, UGent)

Undergraduate thesis students

Heike Bubenheim (Justus-Liebig University, Giessen, Germany)

Charlotte Decock (UGent)

IRMS technicians

Jan Vermeulen

Katja Van Nieuland

compound-specific stable isotope analysis as a tool to characterize the role of microbial community...

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