K. Denef, P. Boeckx, O. Van Cleemput Laboratory of Applied Physical Chemistry (ISOFYS)

1. Compound-specific stable isotope analysis as a tool to characterize the role of microbial community structure in C cycling K. Denef, P. Boeckx, O. Van Cleemput Laboratory of Applied Physical Chemistry (ISOFYS) Ghent University (Belgium)

2. Fungi Bacteria Ecosystem Management Global Change (climate, elevated GHG) ? Microbial community Soil organic carbon

3. Ecosystem Management Global Change (climate, elevated GHG) Microbial community Fungi Bacteria Soil organic carbon

4. 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

5. Molecular markers for fungi vs. bacteria (Compound-specific analysis: CSA) From Glaser, 2006; Drissner et al. (2006)

6. Grassland management intensity Elevated CO2 STRUCTURE (CSA) Microbial community GC-c-IRMS (13C-PLFA) II. APPLICATIONS FUNCTION (CSSIA) LC-c-IRMS (13C-AS) Carbon cycling Research Objective I. METHODOLOGY

7. II. APPLICATIONS: 1. 13CO2 pulse-labeling approach 13CO2 • I. Impact of elevated CO2 (Giessen FACE, Germany since 1998) • 1. Ambient CO2 (350 ppm) • 2. Elevated CO2 (450 ppm) 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) Roots + exudates 13C Soil biota13C • II. Impact of grassland management (Merelbeke, Belgium since 2000): • N-fertilization level • 450 kg N ha-1 yr-1 • 225 kg N ha-1 yr-1 • 0 kg N ha-1 yr-1 • Mowing frequency • 5 times per year • 3 times per year 13C-PLFA GC-c-IRMS

8. Measurements (ongoing) 24 h after pulse-labeling Several times during/after pulse-labeling • 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

9. First results FACE pulse-labeling Mol% PLFA-C (0-7.5 cm) - 10h after start pulse-labeling Gram+ Gram- Enhanced saprotrophic fungal abundance Act Fungi In collaboration with Müller et al

10. First results FACE pulse-labeling G+ G- Act Fungi G+ G- Act Fungi In collaboration with Müller et al

11. Root-derived mol% PLFA-C (0-7.5 cm) - 10h after pulse-labeling Saprotrophic fungi G+ G- AM fungi Act Fungi First results FACE pulse-labeling In collaboration with Müller et al

12. First results FACE pulse-labeling G+ G- Act Fungi G+ G- Act Fungi C-assimilating community shifts over time? Different preferential OM sources? In collaboration with Müller et al

13. 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

14. + 13C substrate (uniformly labeled) 90% sand 4% POM 4% silt 2% clay Soil biota 13C 13C-CO2 13C-Aminosugars Gas-IRMS 13C-PLFA GC-c-IRMS LC-c-IRMS GC-c-IRMS 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

15. Expected results • Fungal:bacterial activity (13C-PLFA) greater for lower quality substrate soils • Different fungal vs. bacterial AS formation rates  estimates for fungal vs. bacterial turnover rates • No differences in “inherent” stability of fungal vs. bacterial AS; stability controlled by clay-OM interactions & physical protection 3. 13C-substrate incubation approach

16. 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

17. 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