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

Scientists: Andreas Gattinger, Michael SchloterTechnicians: 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 EcologyD- 85764 Neuherberg

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

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

HO

O

CH2

C

O

C

O

O P O

O

O

NH3+

H O

CH2

O

O P O

O

O

NH3+

Archaeal diversityAnalysis 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 diversityBacterial & eukaryotic diversityAnalysis of 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:

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

RT: 0.00 - 70.00

0 10 20 30 40 50 60 70

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

13.59

16.31

20.7212.29

24.58

39.33

28.41

28.91

30.74

31.9539.9835.28

43.05

44.47 53.6157.1211.60 64.02

NL:2.95E8

TIC MS PLEL_261

RT: 19.24 - 23.01

19.5 20.0 20.5 21.0 21.5 22.0 22.5 23.0

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

115

Rela

tive A

bundance

20.72

20.47

21.92

19.81 22.60

22.04

21.4322.51 22.84

19.90 20.9819.71 21.14

21.8122.2819.60 21.6520.21

NL:1.53E8

TIC MS PLEL_261

PLEL-derived isoprenoids (archaeal/methanogenic marker)

RT: 0.00 - 86.67

0 10 20 30 40 50 60 70 80

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

58.43

52.15

41.02

47.2614.95 34.44 60.23 70.16 78.4832.9725.0110.93

NL:1.66E7

TIC MS MUF_203

MUFAs (methanotrophic marker)

RT: 50.34 - 63.16

51 52 53 54 55 56 57 58 59 60 61 62 63

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive A

bundance

58.43

58.60

52.15

53.98

51.9558.7752.66

55.26 60.23 60.45 61.3755.6052.8750.70 58.08

NL:1.66E7

TIC MS MUF_203

MUF_203 #5103 RT: 51.96 AV: 1 NL: 3.73E4T: + c Full ms [ 50.00-650.00]

160 180 200 220 240 260 280 300 320 340 360

m/z

0

10

20

30

40

50

60

70

80

90

100

110

120

Rela

tive A

bundance

156.6

173.0

172.3200.4

201.0188.5

161.1

202.7

214.8

361.2173.9

362.7234.2 280.3252.7 314.9 367.1278.4 281.8215.4 337.0 360.1

16:1,8 362/203/159/171

16:1,5 362/161/201/129

16:1,7 362/189/173/157

RT: 0.00 - 86.65

0 10 20 30 40 50 60 70 80

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

ela

tive A

bundance

30.27

24.80

40.76

37.4157.4641.05

15.0363.27

22.2151.16

46.9168.78

20.8173.14 84.6814.23

NL:6.51E7

TIC MS SAT_201

SATFAs (general bacterial marker)

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

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

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

0

5000

10000

15000

20000

25000

30000

0 100 200 300 400 500 600 700

0 4 1 0 2 8 _ ib 0 3 _ G 0 5 _ 0 4 1 0 2 8 1 5 F H

Size (nt)

Dye

Sig

nal

64,65

66,55

74,74

76,83

78,08

80,35

97,10

98,63

100,47

101,83

161,57

163,73

164,78

176,34

188,47

203,89 250,26

427,49

429,68

431,93

434,35

436,13

438,23

440,59

217b

0

1000

2000

3000

4000

5000

6000

7000

8000

0 50 100 150 200 250 300 350 400 450 500 550 600 650 700

0 4 1 0 2 8 _ ib 0 3 _ H 0 5 _ 0 4 1 0 2 8 1 5 F C

Size (nt)

Dye S

ignal 64,71

66,53

74,74

76,87

78,15

80,30

97,12

98,63

100,43

101,77 164,84

176,44

203,90 250,43

427,41

431,87

434,35

436,14

438,26

440,65

218a

0

2500

5000

7500

10000

12500

15000

0 100 200 300 400 500 600 700

0 4 1 0 2 8 _ ib 0 3 _ B 0 6 _ 0 4 1 0 2 8 1 5 G 4

Size (nt)

Dye S

ignal

66,50

74,73

77,11

78,20

82,17

96,96

98,35

176,07

436,16

438,47

440,88

218b

0

5000

10000

15000

20000

25000

0 100 200 300 400 500 600 700

0 4 1 0 2 8 _ ib 0 3 _ C 0 6 _ 0 4 1 0 2 8 1 5 F Z

Size (nt)

Dye S

ignal

66,55

74,73

77,00

78,16

96,93

98,37

176,10

436,10

438,55

440,86

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 +---------+---------+---------+---------+---------+

Bin1thresh200

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 +---------+---------+---------+---------+---------+

B-4-3 B-4-1

B-4-2 E-3-1

Bin2thresh200

Rescaled Distance Cluster Combine C A S E 0 5 10 15 20 25 Label Num +---------+---------+---------+---------+---------+

A-3-2 E-3-3 A-3-3 B-3-3 B-3-2 A-4-3 B-3-1 A-4-2 E-3-2

A-4-1 B-4-3 B-4-1 A-3-1 B-4-2

E-3-1

Bin2thresh300

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

Production of 13C/15N labelled plant litter from Sphagnum fallax

Sampling in Swiss Jura (Le Creux de l‘Epral) in November 2003; ca 3 m2 Sphagnum carpets

Growth conditionsindirect irrigation (onto cotton fleece) with demin. water, no additional light; 20-250C temperature; 85-95% humidity,

surplus water

box

irrigation

cotton fleece

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

Production of 13C/15N labelled plant litter from Eriophorum vaginatum & Eriophorum angustifolium

CaCl2 233,1MgSO4 90,3K2SO4 87,2NaH2PO4 36,8Na2HPO4 4,6H3BO3 3,1FeC6H5O7 2,4MnSO4 1,2ZnSO4 0,3CuSO4 0,2NH4Cl (50% 15N) 80,2

Sampling of plants in French Jura (Le Russey) in October 2003

Growth conditionsin hydroponic culture (clay marbles), 120 plants/m2; additional light for 14h; 20-250C temperature; 70-90% humidity

Nutrient solution and 15N labellinga modified Hoagland solution (pH 5.6) was applied

Nutrients (mg L-1)

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

Production of 13C/15N labelled plant litter from Eriophorum vaginatum & Eriophorum angustifolium

Results of 13C labelling

-10

0

10

20

30

40

50

60

d1

3C

(p

er

mil

PD

B)

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 biomassSphagnum: 5.5 kg (fresh matter) 0.5 kg (dry matter)E. vaginatum: 50 g (fresh matter) 20 g (dry matter)

SphagnumSphagnum

0,00

2,00

4,00

6,00

8,00

10,00

12,00

14,00

16,00

18,00

d1

5N

(%

)

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 15N labelling

SphagnumSphagnum

• 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

Conclusions/suggestions from plant labelling

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

Simultaneous identification and quantification of PLFA/PLEL from environmental samples and their corresponding 12C/13C

ratios by GC/MS-C-IRMS

MS(DSQ)

IRMS(DeltaPlusAdvantage)

20% of the analyte

80% of the analyte

13C/12C

-29

-28

-27

-26

-25

-24

-23

-22

-21

ungedüngt mineralisch biologischdynamisch

organischbiologisch

konventionell

Kartoffel Mais

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

aa

a

a

b

b b

b a

a

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