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Summary of the current status of the work of TUM-BO

Summary of the current status of the work of TUM-BO. Scientists: Andreas Gattinger, Michael Schloter Technicians: Franz Buegger (IRMS, plant labelling), Alexandra Hagn, Conny Galonska (DNA) Christine Kollerbaur (Lipids) Voluntary worker (Environmental Protection): Matthias Weiss

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Summary of the current status of the work of TUM-BO

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  1. Summary of the current status of the work of TUM-BO Scientists: Andreas Gattinger, Michael Schloter Technicians: Franz Buegger (IRMS, plant labelling), Alexandra Hagn, Conny Galonska (DNA) Christine Kollerbaur (Lipids) Voluntary worker (Environmental Protection): Matthias Weiss Technical University of Munich(at the campus of GSF-Research Center for Environment & Health)Chair of Soil Ecology D- 85764 Neuherberg

  2. Summary of the current status of the work of TUM-BO 1. Extraction and analysis of phospholipid biomarker in peat (bog) samples (WP 04: D12-D14) 2. Extraction and analysis of DNA in peat (bog) samples (WP 04: D12-D14) 3. Production of 13C/15N labelled plant litter for field experiment (WP 04: D13; WP 05: D19) 4. Adaptation of the analytical device GC/MS-c-IRMS for compound specific isotope analysis (WP3) 5. Socioeconomical appraisal for German peatlands (WP 01: D3)

  3. 1. Extraction and analysis of phospholipid biomarker in peat (bog) samples (W P04: D12-D14)

  4. Side chain analysis of phospholipids biomarker to describe bacterial, eukaryotic and archaeal diversity with particular emphasis on methanogenic archaea and methanotrophic bacteria; the following fractions (biomarker) are analysed: Bacterial & eukaryotic diversityAnalysis of esterlinked fatty acids (PLFA):- saturated (SATFA): Gram-positives, sulfate reducer- monounsaturated (MUFA): Gram- negatives, methanotrophs - polyunsaturated (PUFA): fungi, protozoa Archaeal diversity Analysis of etherlinked isoprenoids (PLEL):- saturated short chain (i20:0): all archaea - saturated long chain (i40:0): all archaea - cyclic long chain (i40:0-cy): Crenarchaeota- unsaturated short chain (i20:1): methanogens

  5. Extraction and analysis of phospholipid biomarker in peat (bog) samples (W P04: D12-D14) From the peat samples investigated within work programme 1, 208 samples were selected for PLFA analysis; from layer 6 and 8 only duplicate samples were analysed to reduce sample amount for PLFA and DNA analysis (59 from Finland (FI), 40 from France (FR), 46 from Switzerland (CH), 43 from Scotland (SCO), 20 from France (FB)) Up to now only the 59 Finnish samples could be analysed: 236 GC/MS runs because of 4 different PLFA fractions, in average 20-30 PLFA compounds per run are to be identified and quantified Problems with solid-phase extraction of the Finnish samples encountered, hence modified extraction procedure has to be applied onto the following peat samples (CH, FR…)

  6. PLEL-derived isoprenoids (archaeal/methanogenic marker)

  7. MUFAs (methanotrophic marker)

  8. SATFAs (general bacterial marker)

  9. 2. Extraction and analysis of DNA in peat (bog) samples (W P04: D12-D14)

  10. Extraction and analysis of DNA in peat (bog) samples (WP 04: D12-D14) • The same 208 peat samples were selected for DNA analysis as for PLFA • From all 208 peat samples DNA was extracted (DNA extraction kit soilBio101 following test analysis with MLURI) • MLURI (Rebekka) received all DNA extracts (apart from FB samples) for fungal community fingerprints • EPFL/UfZ (Antonis) received DNA extracts (only CH samples) for protozoan diversity studies • first DNA analysis by TUM-BO: bacterial communities using 16S primer and subsequent t-RFLP analysis

  11. DNA 217a (bacterial primer 16S; Juretschko et al., AEM, 1998)

  12. 217b

  13. 218a

  14. 218b

  15. Label Num + --------- + --------- + --------- + --------- + --------- + òûòòòø ò÷ ùòø òòòòò÷ ùòòòø òûòòòø ó ó ò÷ ù ò÷ ùòòòø òòòòò÷ ó ùòø òòòòòòòòòòò÷ ó ùòòòòòòòòòòòòòòòø òòòòòòòòòòòòòòò÷ ó ó òòòòòòòòòòòòòòòòò÷ ùòòòòòòòòòòòòòø òòòòòòòòòòòòòûòòòòòòòòòòòòòòòø ó ó òòòòòòòòòòòòò÷ ùòòò÷ ùòø òòòòòòòòòòòòòòòòòòòòòòòòòòòòò÷ ó ó òòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòò÷ ó òòòòòòòòòòòòòòòòòòòòòòòòòò òòòòòòòòòòòòòòòòòòòòòòòú òòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòò÷ C A S E 0 5 10 15 20 25 Bin1 thresh200 B-3-1 B-3-3 B-3-2 A-3-2 E-3-3 A-3-3 A-4-3 A-4-2 E-3-2 A-4-1 B-4-3 B-4-1 A-3-1 E-3-1 B-4-2

  16. C A S E 0 5 10 15 20 25 Label Num + --------- + --------- + --------- + --------- + --------- + òûòòòòòø ò÷ ùòø òòòòòòò÷ ùòø òòòòòòòòò÷ ó òò òòòòòòòòòôòòòø òòòòòòòûòòò÷ ùòòòòòø òòòòòòò÷ ó ùòòòòòòòòòòòø òòòòòòòòòòòòòòò÷ ó ó òòòòòòòòòòòòòòòòòòòòò÷ ùòòòòòòòòòòòòòø òòòòòòòòòòòòòòòòòûòòòòòò òòòòòòòø ó ó B-4-3 òòòòòòòòòòòòòòòòò÷ ùò÷ ùòø B-4-1 òòòòòòòòòòòòòòòòòòòòòòòòòòòòòòò÷ ó ó òòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòò÷ ó B-4-2 òòòòòòòòòòòòòòòòòò òòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòú E-3-1 òòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòò÷ A-3-3 E-3-3 Bin2 thresh200 A-3-2 A-4-3 B-3-2 B-3-1 B-3-3 A-4-2 E-3-2 A-4-1 A-3-1

  17. C A S E 0 5 10 15 20 25 Label Num + --------- + --------- + --------- + --------- + --------- + A-3-2 òûòø E-3-3 ò÷ ùòø A-3-3 òûò÷ ùòø B-3-3 ò÷ ó ùòø B-3-2 òòòòò÷ ó ùòòòø A-4-3 òòòòòòò÷ ó ùòòòø B-3-1 òòòòòòòòò÷ ó ùòòòòòòòòòòòòòø A-4-2 òòòòòòòòòòòòò÷ ó ó E-3-2 òòòòòòòòòòòòòòòòò÷ ùòòòòòòòòòòòòòø A-4-1 òòòòòòòòòòòòòòòûòòòòòòòòòòòòòø ó ó B-4-3 òòòòòòòòòòòòòòò÷ ùò÷ ùòø B-4-1 òòòòòòòòòòòòòòòòòòòòòòòòòòòòò÷ ó ùòø A-3-1 òòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòò÷ ó ó B-4-2 òòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòò ÷ ó E-3-1 òòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòòò÷ Rescaled Distance Cluster Combine Bin2 thresh300

  18. 3. Production of 13C/15N labelled plant litter for field experiment (WP 04: D13; WP 05: D19)

  19. Production of 13C/15N labelled plant litter from Sphagnum fallax Samplingin Swiss Jura (Le Creux de l‘Epral) in November 2003; ca 3 m2 Sphagnum carpets Growth conditions indirect irrigation (onto cotton fleece) with demin. water, no additional light; 20-250C temperature; 85-95% humidity, irrigation cotton fleece box surplus water

  20. Production of 13C/15N labelled plant litter from Sphagnum fallax 15N Labellingaquous solution of NH4Cl (99% 15N; 0.1g L-1 m2) was sprayed on Sphagnum carpets prior to 13C labelling 13C Labellingfor 2 month with 13C enriched CO2(+172%O PDB = 1.3 atom%) in an air tight tent; prior to labelling ambient CO2 was pumped off; 3-5 labelling pulses per day, when CO2 concentration was below 300 ppm; during the night CO2 was pumped off; average labelling in the tent was between 20 and 60%O PDB Harvestingonly the upper 2/3 of the plants were harvested to collect only the (labelled) newly grown parts

  21. Production of 13C/15N labelled plant litter from Eriophorum vaginatum & Eriophorum angustifolium Samplingof plants in French Jura (Le Russey) in October 2003 Growth conditions in hydroponic culture (clay marbles), 120 plants/m2; additional light for 14h; 20-250C temperature; 70-90% humidity Nutrients (mg L-1) Nutrient solution and 15N labelling a modified Hoagland solution (pH 5.6) was applied

  22. Production of 13C/15N labelled plant litter from Eriophorum vaginatum & Eriophorum angustifolium 13C Labellingfor 2 month with 13C enriched CO2(+172%O PDB = 1.3 atom%) in an air tight tent; prior to labelling ambient CO2 was pumped off; 3-5 labelling pulses per day, when CO2 concentration was below 300 ppm; during the night CO2 was pumped off; average labelling in the tent was between 30 and 80%O PDB Harvestingonly the upper 2/3 of the plant shoots were harvested to collect only the (labelled) newly grown parts, poor growth of E. angustifolium, which was not harvested

  23. Results of 13C labelling E. vaginatum(shoots)+50.2 Capitulum(from 200g dm)+22.6 Stems (5 plants) +19.1 Sphagnum Stems (5 plants) +7.8 Stems+Leaves(from 200g dm)+2.9 Leaves(2x5 plants)-3.2 Yield of labelled biomassSphagnum: 5.5 kg (fresh matter) 0.5 kg (dry matter)E. vaginatum: 50 g (fresh matter) 20 g (dry matter)

  24. Results of 15N labelling E. vaginatum(shoots)+16.9 Sphagnum Capitulum(from 200g dm)+1.85 Leaves(2x5 plants)+1.64 Stems (2x5 plants)+2.25 and +1.77 Stems+Leaves(from 200g dm)+1.48

  25. Conclusions/suggestions from plant labelling • labelling in Sphagnum (esp. capitulum) seems to be suffient for field experiment in 2003 for compound specific isotope analysis (13C-PLFA), 13C values in PLFA/PLEL from test core samples varied between -26 and -30 %0 (natural abundance) • only 1 labelled plant species avaible in sufficient amounts for field experiment in 2003; for getting more labelled Eriophorum plant material, seeds will be purchased and grown in sandy/mineral soil substrate (problems with the hydroponic culture after transplantation!) • a better homogenisation of the plant litter for 13C studies is required, the use of whole plants is not recommendable, as it leads to high variations • less variation in 15N among single plants and plant compartments

  26. 4. Adaptation of the new analytical device GC/MS-c-IRMS for compound specific isotope analysis

  27. Simultaneous identification and quantification of PLFA/PLEL from environmental samples and their corresponding 12C/13C ratios by GC/MS-C-IRMS 20% of the analyte MS(DSQ) IRMS(DeltaPlusAdvantage) 80% of the analyte

  28. 13C measurements of archaeal PLELat the natural abundance level(5 treatments, 4 replicate field plots each) b b b a b a a a a a

  29. 5. Socioeconomical appraisal for peatlands in Germany (WP 01: D3) • survey was originally planned for this spring but has been postponed to September 2004, because of manpower • in parallel a German group (among others M. Drösler, University of Bayreuth) is generating a new peatland inventory, as the current data is of poor quality (quite old, patchy, wrong, etc.) • TUM-BO will use the new basic data on German peatlands, which will be available in autumn 2004 as well as inquiring for the socioeconomics information as suggested in Charquemont 2003

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