Kein Folientitel

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)
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1. Extraction and analysis of phospholipid
biomarker in peat (bog) samples (W P04 D12-D14)
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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
(i200) all archaea - saturated long chain
(i400) all archaea - cyclic long chain
(i400-cy) Crenarchaeota- unsaturated short
chain (i201) methanogens
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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)
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PLEL-derived isoprenoids (archaeal/methanogenic
marker)
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MUFAs (methanotrophic marker)
161,8 362/203/159/171
161,5 362/161/201/129
161,7 362/189/173/157
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SATFAs (general bacterial marker)
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2. Extraction and analysis of DNA in peat (bog)
samples (W P04 D12-D14)
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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

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DNA 217a (bacterial primer 16S Juretschko et
al., AEM, 1998)
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217b
13
218a
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218b
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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
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
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Rescaled Distance Cluster Combine

Bin2 thresh300
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3. Production of 13C/15N labelled plant litter
for field experiment (WP 04 D13 WP 05 D19)
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Production of 13C/15N labelled plant litter from
Sphagnum fallax
Sampling in Swiss Jura (Le Creux de lEpral) 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
(172O 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 60O 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
Sampling of 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
(172O 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 80O 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
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Results of 13C labelling
E. vaginatum(shoots)50.2
Capitulum(from 200g dm)22.6
Stems (5 plants) 19.1
Stems (5 plants) 7.8
Sphagnum
StemsLeaves(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)
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Results of 15N labelling
E. vaginatum(shoots)16.9
Sphagnum
Capitulum(from 200g dm)1.85
Leaves(2x5 plants)1.64
StemsLeaves(from 200g dm)1.48
Stems (2x5 plants)2.25 and 1.77
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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

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