1. RECIPE meeting May 17-20 Th Munich,GERMANY « Earth Science Institute of Orleans », France Group « Organic Matter in water, sols, sediments and rocks » Laure Comont, Fatima Laggoun-Défarge, Jean-Robert Disnar Pascale Gautret, Nathalie Lottier, Marielle Hatton, Li Huang

2. Munich progress meeting May 17-20th WP5 work progress since Carentan meeting II- Sugar analyses on the bulk peat compared to the fine-grained fraction (Le Russey sites on FRC & FRD) => identification of biomarkers (OM degradation processes) III- Tests of aminoacid analyses on 2 samples from CH (solid and soluble fractions) : => interesting preliminary results obtained for identification of neoformation markers => results are correlated with cryo-scanning electron microscopy (cryo-SEM) observations IV – Microtexture of peat (2 distinct scales):  cryo-SEM  X-ray computed tomography I – Exploration of physico-chemical data (PCA) on peat OM from the five studied sites (WP01) (correlations between C, N, organic constituent countings; sugar analyses in both bulk peat and fine-grained fraction)

3. I – DETERMINATION OF VARIOUS CORRELATIONS Munich progress meeting May 17-20th

4. Principal component analyses (PCA) CH : 1963 (~40 yrs) and FR : 1984 (~20 yrs) FB (less than 5 yrs ?) FI : 1975 - 1978 (~30 yrs) SC : 2000 (~5 yrs) => 1995 (~10 yrs) => 1955 (~50 yrs) Peat harvesting ended :

5. Munich progress meeting May 17-20th Principal component analyses (PCA) 3 distinct trends starting from the « new » regen. peat of all sites: - Jura sites -Scotish sites -Finnish sites Schematic model of peat evolution - Similar evolution at 1st steps of regen. for Jura & Scotish sites => similar plant compositions of new peat (mainly Sphagna) - Distinct evolution for FI => the composition of « litter » from Finish sites is quite different (C.rostrata, E.vaginatum) - The old peat evolution of SC & FI sites converge with the same variables which characterise a more humified peat

6. II - SUGAR ANALYSES ON BULK PEAT (comparison with fine-grained fractions) LE RUSSEY FRC (2 nd regeneration stage) FRD (intact area) Munich progress meeting May 17-20th

7. Le Russey FRC,2 nd regen.stage Sugar analyses on the BULK peat (compared to the FINE fraction <200µm) Total sugar contents (mg/g): - Bulk ≈ 432 (top) => 137 (bottom) - Fine ≈ 206 (top) => 80 (bottom) sugars derived from inherited plants tissues (Tot. Cell., Ara & Xyl) sugars derived (partly) from microbial syntheses (Hem.Gluc, Fuc, Rham, Man & Rib) - High sugars decrease well correlated with %fine-grained fraction increase - Tot.Hem.sugars increase well correlated with % of Muc. -progressive Hem.Gluc. increase is certainly due to cellulose destructuration - High difference, between both fractions, of Rham & Rib (relative %)

8. Le Russey FRD intact area Sugar analyses on the BULK peat (compared to the FINE fraction <200µm) Total sugar contents (mg/g): - Bulk ≈ 295 (top) => 170 (bottom) - Fine ≈ 227 (top) => 125 (bottom) sugars derived from inherited plants tissues (Tot. Cell., Ara & Xyl) sugars derived (partly) from microbial syntheses (Hem.Gluc, Fuc, Rham, Man & Rib) - Progressive decrease of sugars (compared to FRC) & no correlation with % fine fraction - increase at depth corresponding to preservation processes - Less contrast between bulk and fine peats compared to FRC - progressive increase of H. cell. Glucose

9. I-surface: II-depth: FRC FRD Tot. Sugars: high degradation Bulk ≈ 432 (top) => 137 (bottom) Fine ≈ 206 (top) => 80 (bottom) Tot. Sugars: low degradation Bulk ≈ 295 (top) => 170 (bottom) Fine ≈ 227 (top) => 125 (bottom) Degradation of sugar parallels destructuration of plant tissues Degradation of sugars ≠ destructuration of plant tissues Higher contribution of Rib in the fine-grained fraction => Degradation? High Cellulosic sugars, Xyl in the bulk fraction => Primary input preservation Munich progress meeting May 17-20th Sugar analyses, preliminary conclusions - Surface processes: evolution kinetics strongly differ in the « new » regenerating peat and in the intact peat.

10. Munich progress meeting May 17-20th III - AMINOACID ANALYSES LA CHAUX D’ABEL FEN SITUATION CHA43 CHA73

11. Aminoacid analyses, experiment Munich progress meeting May 17-20th Acid hydrolysis HPLCquantification & distribution of AA Living plants : Mosses: S.fallax & Polytrichum (steams & rhizoids) Cyperaceae: E.vaginatum (leaves & roots) E.angustifolium 3 4 5 6 7 8 Test of identification of total aminoacids: La Chaux d’Abel,fen situation CHA peat profil Soluble fraction, ultrafiltration: fraction (>3kDa) F1 fraction (<3kDa) F2 Solid bulk peat Soluble fraction, ultrafiltration: fraction (>3kDa) F1 fraction (<3kDa) F2 Solid bulk peat 3kDa ~ 400 Å

12. Chaux d’Abel, CHA situation, aminoacid results Munich progress meeting May 17-20th - 2 compartments: soluble and solid fractions In soluble fraction: high % of basic AA (Lys & Arg) ➱ Preferential accumulation of basic AA in acidic environment ? ➱ Another source (microorg. groups rich in basic AA)? -Gly: smallest and simplest AA => index of degradation living plants peat soluble OM

13. Munich progress meeting May 17-20th Chaux d’Abel, CHA situation, aminoacid results Solid bulk Solid fraction: All AA are well represented including di-carboxylic AA (Asp, Glu..) soluble (> 3kDa) Soluble fraction: mainly composed of basic AA (Arg & Lys) + Free ammonia => degradation markers soluble (< 3kDa) Example: chrommatogram of level 4 (5-10 cm) Non proteinic AA (Orn & unknown peaks) => neosynthetic AA from abiotic or biotic activity ? ? ? ? ? ?

14. => 2 hypotheses - important and widespread microbial population (either active or dead) ? to be correlated with microbial groups WP03 - selective preservation of some plant proteins and accumulation due to specific environmental conditions ? Chaux d’Abel, CHA situation, aminoacid results Munich progress meeting May 17-20th At 5-10 cm (regenerated peat): high diminution of total AA compared to living plants => degradation of plant proteins in this level. That’s confirmed by the total AA quantity in soluble fraction. At 35 cm (old peat): amounts of AA are 20 times higher than those at the surface and also higher than those of living plants. => 20% of Gly  96% of free NH3  42% of Gly Hydrolysable AA YIELD (mg/g) or (mg/l)

15. Frequency histograms of individual AA examplify these correlations Munich progress meeting May 17-20th Chaux d’Abel, CHA situation, aminoacid results Good correlation between peat samples and some living plants Microbial production or selective preservation ?

16. Chaux d’Abel, sugar results on soluble fraction Munich progress meeting May 17-20th level 4 (5-10 cm)level 7 (32,5-37,5 cm) Total sugar contents ≈ 5,5 mg/l 58% of unknowns (neoformed ?) Total sugar contents ≈ 8,3 mg/l 23% of unknowns (neoformed ?) Higher Total sugar contents in the soluble fraction at depth than at the sub-surface (level 7 vs. 4)  are these soluble compounds autochthonous or not (i.e. brought by diffusion from the surface or elsewhere); Microbial production or selective preservation ? Higher proportions of xylose (glucose?) and fucose at depth => inheritance (xylose?) and/or in situ microbial production (fucose?)?

17. Munich progress meeting May 17-20th Cryo-scanning electron microscope (cryo-SEM) The cryo-SEM is equipped with a freeze-drying sample preparation system. It allows studing the composition and the micromorphology of hydrous materiel (peat) and spatial relationships between organic components. FRC, 2 nd regeneration stage level 3 (0-5 cm) level 5 (12,5 - 17,5 cm) level 8 (42,5 - 47,5 cm) Tests were carried out on samples from Le Russey site: Microbial production or selective preservation ?

18. The cryo-SEM, results, FRC 2 nd regeneration stage level 3 Microbial production or selective preservation ? 3D polysaccharidic network Cross section of Sphagna steam Microorganisms inside well-preserved cell walls level 5 Biofilm Eps (extracellular polymer secretion) level 8 3D polysaccharidic network degraded cell walls Bacterial colony 50µm12µm2,3µm 5µm3µm

19. Munich progress meeting May 17-20th Preliminary conclusions distinct degradation processes between these 2 peat samples: Solid fractionSoluble fraction Total AA ≈ 5 µg/mgTotal AA ≈ 43,51µg/ml Total sugars ≈ 5.5 mg/l level 4 : - High living plant proteins degradation in the solid fraction - High sugars degradation in soluble fraction Solid fractionSoluble fraction Total AA ≈ 13,14 µg/ml Total sugars ≈ 8,3 mg/l Total AA ≈ 134 µg/mg level 7 : - High hydrolysable AA yield in the solid fraction  ≠ materiel sources  lower degradation - Higher Tot. sugar contents in the soluble fraction 2 processes are determined : - Selective preservation - Microbial production Which of these 2 processes is dominant ?

20. IV – PHYSICAL MICROTEXTURE OF THE PEAT Munich progress meeting May 17-20th FRA, bare peat level 3 (0-5 cm) FRC, 2 nd regeneration stage level 3 (0-5 cm) level 5 (12,5 - 17,5 cm) level 8 (42,5 - 47,5 cm)

21. Munich progress meeting May 17-20th Physical microtexture of the peat I- Cryo-SEM observations: bare peat, FRA2 nd reg. stage, FRC more compact, dense peat analyses are in progress distinct µmorphologies in the bare peat & the transition « old »/« new » peat II- X-ray computed Tomography : Tests carried out on 2 samples (Coll. R. Swennen from Leuven University, Belgium): RyI = FRC level 7-12 cm RyI = FRC level 40-50 cm Visualisation of the 3D porous network on different peat types under increasing range of wetness (re-wetting process) level 3level 5 5µm

22. Munich progress meeting May 17-20th Conclusions - Association of AA analyses + sugar analyses in soluble fraction => interesting approach to better identify the markers of OM degradation to be applied in WPO2… - Exploration of physico-chemical data (PCA) on peat OM from the five sites (WP01) => distinct evolution trends for Jura peatlands and SC/FI peatlands => climatic influence ? Initial vegetation types ? -2 important problems arise: => datations of both «new» peat horizons and transitional peat layer => insufficient numbers of peat samples along the profiles of the new regenerating horizon (only 2 to 3) For WPO2 => adopt a better strategy of sampling !!!!!