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

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

4. 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 Bacterial & eukaryotic diversity Analysis of Bacterial & eukaryotic diversity Analysis of esterlinked fatty acids (PLFA): - saturated (SATFA): Gram-positives, sulfate reducer - monounsaturated (MUFA): Gram- negatives, methanotrophs - polyunsaturated (PUFA): fungi, protozoa 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:

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)) 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…) 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

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

7. MUFAs (methanotrophic marker) 16:1,8362/203/159/171 16:1,5362/161/201/129 16:1,7362/189/173/157

8. SATFAs (general bacterial marker)

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

10. 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 Extraction and analysis of DNA in peat (bog) samples (WP 04: D12-D14)

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

12. 217b

13. 218a

14. 218b

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

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

17. 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 13 C/ 15 N labelled plant litter from Sphagnum fallax Sampling in Swiss Jura (Le Creux de l‘Epral) in November 2003; ca 3 m 2 Sphagnum carpets Growth conditions indirect irrigation (onto cotton fleece) with demin. water, no additional light; 20-25 0 C temperature; 85-95% humidity, surplus water box irrigation cotton fleece

20. Production of 13 C/ 15 N labelled plant litter from Sphagnum fallax 15 N Labelling aquous solution of NH 4 Cl (99% 15 N; 0.1g L -1 m 2 ) was sprayed on Sphagnum carpets prior to 13 C labelling 13 C Labelling for 2 month with 13 C enriched CO 2 (+172% O PDB = 1.3 atom%) in an air tight tent; prior to labelling ambient CO 2 was pumped off; 3-5 labelling pulses per day, when CO 2 concentration was below 300 ppm; during the night CO 2 was pumped off; average labelling in the tent was between 20 and 60% O PDB Harvesting only the upper 2/3 of the plants were harvested to collect only the (labelled) newly grown parts

21. Production of 13 C/ 15 N labelled plant litter from Eriophorum vaginatum & Eriophorum angustifolium Sampling of plants in French Jura (Le Russey) in October 2003 Growth conditions in hydroponic culture (clay marbles), 120 plants/m 2 ; additional light for 14h; 20-25 0 C temperature; 70-90% humidity Nutrient solution and 15 N labelling a modified Hoagland solution (pH 5.6) was applied Nutrients (mg L -1 )

22. 13 C Labelling for 2 month with 13 C enriched CO 2 (+172% O PDB = 1.3 atom%) in an air tight tent; prior to labelling ambient CO 2 was pumped off; 3-5 labelling pulses per day, when CO 2 concentration was below 300 ppm; during the night CO 2 was pumped off; average labelling in the tent was between 30 and 80% O PDB Harvesting only 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 Production of 13 C/ 15 N labelled plant litter from Eriophorum vaginatum & Eriophorum angustifolium

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

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

25. labelling in Sphagnum (esp. capitulum) seems to be suffient for field experiment in 2003 for compound specific isotope analysis ( 13 C- PLFA), 13 C 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 13 C studies is required, the use of whole plants is not recommendable, as it leads to high variations less variation in 15 N among single plants and plant compartments Conclusions/suggestions from plant labelling

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 12 C/ 13 C ratios by GC/MS-C-IRMS MS (DSQ) IRMS (DeltaPlus Advantage ) 20% of the analyte 80% of the analyte

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