Compound-specific stable isotope analysis as a tool to characterize the role of microbial community structure in C cycling
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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 Ecosystem Management Global Change (climate, elevated GHG)? Microbial community Fungi Bacteria Soil organic carbon
3 Ecosystem Management Global Change (climate, elevated GHG) Microbial community Fungi Bacteria Soil organic carbon
4 Possible reasons for fungalinduced 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) Microbial communities distinguished Gram + bacteria Gram bacteria Actinomycetes Fungi saprotrophic Fungi mycorrhizal Bacterial residues Fungal residues Molecular marker Phospholipid fatty acids (PLFA): living structures i14:0, i15:0, a15:0, i16:0, i17:0, a17:0 Monounsaturated (16:1w7, 18:1w7, cy17:0, cy19:0) 10Me-FAs 18:1w9c, 18:2w6,9 16:1w5 Amino sugars (AS): microbial residues Galactosamine Muramic Acid Glucosamine From Glaser, 2006; Drissner et al. (2006)
6 Research Objective Grassland management intensity Elevated CO 2 STRUCTURE (CSA) I. METHODOLOGY II. APPLICATIONS Microbial community FUNCTION (CSSIA) GC-c-IRMS ( 13 C-PLFA) LC-c-IRMS ( 13 C-AS) Carbon cycling
7 II. APPLICATIONS: CO 2 pulse-labeling approach 13 CO 2 Roots + exudates 13 C Soil biota 13 C 13 C-PLFA GC-c-IRMS I. Impact of elevated CO 2 (Giessen FACE, Germany since OBJECTIVES 1998) 1. Ambient CO 2 (350 ppm) Investigate 2. Elevated elevated CO CO 2 (450 ppm) 2 and grassland management impacts on root-c II. Impact of grassland management assimilating microbial communities (Merelbeke, Belgium since 2000): Activity 1. N-fertilization niche differentiation level Link 1. stimulated 450 kg N fungal ha -1 yr -1 pathways to C kg N ha -1 yr stabilization mechanisms -1 (aggregation; 3. 0 kg N ha -1 yr -1 fungal-derived 2. Mowing frequency OM) 1. 5 times per year 2. 3 times per year
8 Measurements (ongoing) 24 h after pulse-labeling Several times during/after pulse-labeling Expected Aboveground stimulated plant fungal/mycorrhizal material: 13 C pathways: Roots: * less 13 C intense management Root-associated * elevated COsoil: bulk 13 C & 13 2 C-PLFA Expected Bulk soil: niche bulk dominance 13 C & 13 C-PLFA of fungal activity: * macroaggregates Physical fractions (aggregate size fractions): Expected preferential stabilization of fungal products: 13 C * fractions microaggregates within macroaggregates 13 C-PLFA AS concentrations
9 First results FACE pulse-labeling Mol% PLFA-C (0-7.5 cm) - 10h after start pulse-labeling Gram+ Gram- Ambient CO2 Elevated CO i-c14:0 i-c15:0 a-c15:0 i-c16:0 i-c17:0 a-c17:0 C16:1w7c C17:0 cy C18:1w7c C19:0 cy 10-MeC16:0 10-MeC18:0 C16:1w5c C18:1w9c C18:2w6,9c mol% PLFA-C 6 Act Enhanced saprotrophic fungal abundance Fungi In collaboration with Müller et al
10 First results FACE pulse-labeling Ambient CO 2 treatment Elevated CO 2 treatment 3 hours during pulse-labeling 10 hours after start pulse-labeling G+ G- Act Fungi G+ G- Act Fungi i-c14:0 i-c15:0 a-c15:0 i-c16:0 i-c17:0 a-c17:0 C16:1w7c C18:1w7c 10-MeC16:0 10-MeC18:0 C16:1w5c C18:1w9c C18:2w6,9c i-c14:0 i-c15:0 a-c15:0 i-c16:0 i-c17:0 a-c17:0 C16:1w7c C18:1w7c 10-MeC16:0 10-MeC18:0 C16:1w5c C18:1w9c C18:2w6,9c δ 13 C enrichement (relative to time 0 PLFA) -10 In collaboration with Müller et al
11 First results FACE pulse-labeling Root-derived mol% PLFA-C (0-7.5 cm) - 10h after pulse-labeling Ambient CO2 Elevated CO2 G+ G- Act Fungi Saprotrophic fungi AM fungi i-c14:0 i-c15:0 a-c15:0 i-c16:0 i-c17:0 a-c17:0 C16:1w7c C18:1w7c 10-MeC16:0 10-MeC18:0 C16:1w5c C18:1w9c C18:2w6,9c Root-derived mol% PLFA-C 0 In collaboration with Müller et al
12 First results FACE pulse-labeling Ambient CO 2 treatment hours during pulse-labeling 10 hours after pulse-labeling 11 months after pulse-labeling i-c14:0 i-c15:0 a-c15:0 i-c16:0 i-c17:0 a-c17:0 C16:1w7c C18:1w7c 10-MeC16:0 10-MeC18:0 C16:1w5c C18:1w9c C18:2w6,9c i-c14:0 i-c15:0 a-c15:0 i-c16:0 i-c17:0 a-c17:0 C16:1w7c C18:1w7c 10-MeC16:0 10-MeC18:0 C16:1w5c C18:1w9c C18:2w6,9c δ13c enrichement (relative to time 0 PLFA) G+ G- Act Fungi Elevated CO 2 treatment G+ G- Act Fungi C-assimilating community shifts over time? Different preferential OM sources? In collaboration with Müller et al
13 Possible reasons for fungalinduced 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 2. 13 C-substrate incubation approach + 13 C substrate (uniformly labeled) Wheat substrate C/N δ 13 C 90% sand 4% POM 4% silt 2% clay Soil biota 13 C Grains Leaves Roots 13 C-CO ± ± ± ± ± ± C-PLFA Gas-IRMS Stems HWE Leaves 57.2 ± ± ± ± 2.6 OBJECTIVES 13 C-Amino sugars Determine formation rates of fresh plant-residue-derived fungal vs. bacterial amino sugars Investigate impact of substrate GC-c-IRMS quality GC-c-IRMS on fungal and bacterial activity and turnover LC-c-IRMS Determine inherent biochemical stability of fungal vs. bacterial amino sugars
15 3. 13 C-substrate incubation approach Expected results 1. Fungal:bacterial activity ( 13 C-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
16 Summary 13 C-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?) 13 C-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
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