RECIPE meeting Scheyern 17-20 May 2005

RECIPE Meeting Scheyern 17-19.05.2005RECIPE meeting Scheyern 17-20 May 2005

Progress report AR-WSL, EPFL ECOS

Andy SiegenthalerEmanuela Samaritani,

Edward Mitchell,Alexandre Buttler

RECIPE Meeting Scheyern 17-19.05.2005

Achievements since last meetingAndy (A), Edward (Ed), Emanuela (Em)

• A&Em: Methane emission in Chaux d’Abel, fall 2004• Em: Photosynthesis and respiration measurements in Chaux d’Abel

were started in May 2005• A&Em: CH4-CO2 depth profiles continues (peepers) - Data set now

complete for 2004.• A: Development of a new method for measuring soil respiration under

realistic conditions• A&Em: Soil respiration and methane production measurements in

WPIII to test the new method developed by Andy• A&Em: Soil respiration and methane production measurements in

WPI: Selective activities measured using specific inhibitors


Achievements - continuedAndy (A), Edward (Ed), Emanuela (Em)

• A&Em: Dendrochronological dating of the onset of regeneration

• A&Em: Testing probes and protocols for analysing methanogenic Archea using FISH and CARD-FISH

• Ed: WPI: Analyses of testate amoeba community structure in the Chaux d’Abel and Russey sites completed, ongoing for others sites

• Ed: Establishment of the phylogenetic position of the Arcellinida (one of the two groups of testate amoebae) - Protist, in press

• Ed, n.b. with Enrique Lara: Isolation of more Arcellinida species in view of continuing the phylogenetic work to increase the number of sequences in the data base. This will make it possible to analyse testate amoebae community profiles using molecular methods (c.f. also work by Antonis on the Euglyphid testate amoeba)


Dendrochronology

Collection and preparation of birch cores, December 2004

Microscopic counting and measurement of tree rings, March 2005


Birch recolonisation after abandonment La Chaux-d’Abel

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A B C

Regeneration stage

Yea

rs (

a) a

bc

Statistics: N=11, Kruskal-Wallis followed by Mann-Whitney

Method: tree cores collected from the oldest birch trees found at the shortest distance from the centre of a selected stage


Gas profiles measurements

Cylindrical peepers


Cylindrical peepersHT-Tuffryn membrane 50 mm (mesh: 0.2 µm) sealed to the PE cap with drinkable-water Coltgum

Cylinder (1m)

Acryl 50 ml pot with water-tight PE cap

Point 20 cm

Transparent PET 0.5 mm sheet to reduce possible leaching from the wood

Insertion for the pot and the the cap

PE cap

Pipe to let air get down when peepers are retrieved

Removable handle

Power-tape


Dissolved gases at different depths and stages (peepers)

Chaux d’Abel, December 2004

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H3 H4 H6 H7 H8

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A

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Dissolved gases at different depths and stages(peepers)

Le Russey, autumn 2004

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Stable isotopes in peeper’s water: method summary

1) Samples were taken from the headspace of the degassed peat solutions found in the dialysis chambers (peepers). We used very fine needles (0.55 mm diam.) to sample through the 1 cm thick septae.

2) Separation between CH4 and CO2 was made using a infrared gas chromatograph equipped with a Varian poraplot Q coated column together with plot fused silica (25m x 0,32 mm).

3) The isotopic (12C-13C)/12C ratios were measured separately for CH4 and CO2 with help of a continuous-flow isotope ratio mass-spectrometer coupled to the gas chromatograph (GC-MS).


Delta 13C at various depths and stages (peepers) - Analyses by Andreas

Chaux-d’Abel, autumn 2004

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H3 H4 H6 H7 H8

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A

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H3 H4 H6 H7 H8

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A

Note: the CH4 concentrations were too low to measure d13C at H3 and, for A and C at H4.


2D-clustering d13C-CH4 vs d13C-CO2

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-20.0 -18.0 -16.0 -14.0 -12.0 -10.0 -8.0 -6.0

d13C-CO2 (o/oo)

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A

B

C


Repartition of delta 13C from dissolved gases(peepers), pooled depths Chaux d’Abel - December 2004

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A B C

Stages

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Stages

ba c

Stats: N = 20, error bars = +/- 1 std error, Kruskal-Wallis followed by Mann-Whitney

D13

C-C

O2(

0/00

)


Soil respiration profiles :A new method of laboratory

incubation


Development of a new method to study soil respiration in mixed conditions

Some problems with standard basal respirations:Because of the difficulties to separate anaerobiosis from aerobiosis in cores that are an

assemblage of micro-sites and because it is also artificial to incubate peat in either N2 or air, we developed a method that allowed us to directly incubate the samples taken on-site.

We measured all replicates of Jura sites at three different depths over a period of 15 days at 4-6 days interval.

The advantages of this method are:1) reduced incubation times, 2) get as close as possible to peat atmosphere conditions, 3) no core disturbance to get reed of the roots, 4) no need of extreme incubations in either air or N2, 5) drastic reduction in the time of exposition to atmospheric conditions different than

in-situ ones, 6) increase of efficiency and cost effectiveness especially with further “selective”

respirations using various microbial inhibitors.


The Monovette system


New setup for basal respiration

1.7 cm peat cores sampling at 3 depths, from 3 replicates and in 3 stages

Transfer into a 9ml Monovette

leave no air space

Create artificial1 ml headspace and2 days incubation

Flushing 1 ml of the triggered

respiration

Create an artificial30% headspaceseal properly

Incubate the coresfor 2, 6, 12, 18 days

at 6°C

Take 1 ml of gas for GC analysis,

consider that loss

Short term, anaerobic and aerobic respirations

Inject 2 ml of water or of inhibitor

cocktail

Remove roots at one end of the

Monovette


Selective inhibition cocktails

Treatments BR M- M+ B- B+ F- E*+ MA+ ALL- HeatCyclohexamide (1000 mg/L) x x x x 105°C

Amphotericin B (25 mg/L) x x x x x x 105°CStreptomycin (1000 mg/L) + Penicillin G (106 U/L) x x x x x 105°CNa-2-bromoethanesulfonate and/or Na-fluoroacetate (H-) x x x Htroph x 105°C

Arguments for the choice of antibiotics

3. Well known antibiotics that have a maximal 20% inhibition of methanogens.

1. Synergetic effect between a beta-lactam and a aminoglycoside such as Penicillin and Streptomycin

2. Kanamycin and Gentamicin that have lower effect on methanogens but are known to ihibit eukaryotic ribonuclease P activity by aminoglycosides, although there is no data for streptomycin!


Preliminary tests for the WPIII basal respiration at depths in the control plots

Basal respiration throughout time

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CO2* (t0)/ppm

CO2* (t1)/ppm

CO2* (t2)/ppm

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CO2* (t4)/ppm

Nominal time (day: 2, 6, 10, 14, 18)

Evolved CO2 concentration (ppm)

A2CT25A2CT45A2CT7.5B2CT25B2CT45B2CT7.5C2CT25C2CT45C2CT7.5


WP1: Anaerobic respiration of methanogens Chaux-d’Abel

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Stages

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Satges

N.b. Interestingly, surface respiration measured in fall 2004 showed greater respiration in stage C (advanced) !

a

b b


Depth profiles: origin of the difference in the overall anaerobic respiration - Chaux-d’Abel

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H4 H6 H8

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Twice as much respiration in site A at 25 and 45 cm depths, very significant.


Trend for the autotrophic methanogensChaux-d’Abel (MA+)

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Stages

There seems to be less autotrophic methanogens activity in the intermediate stage.

°


Significantly lower basal respiration respiration in the

advanced stage

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Trend towards lower eukaryotic (E*) (all but fungi) respiration

in the advanced stage

Approx. -14%


Steady increase of the methanogens’ respiration - Le Russey

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Stages

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•Respiration is higher in the advanced stage and mainly occurring near the surface!•Not many significant results are found at le Russey, probably because of the reduced number of replicates (3x).

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WPIII. Basal respiration at various depths and for specific litters - preliminary results

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SEVEACT

Depths

CO2 respiration differs significantly among treatments only at one depth (H6)! Respiration is lower for Sphagnum as compared to either Eriophorum treatments

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CT EA EV S

Species


Litterbag experiment - species and water table effects on D13C-CO2

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1-LWT 2-MWT 3-HWT

Water table

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Species

Statistics: N = 9, error bars = +/- 1 std error(s), Kruskal-Wallis followed by Mann-Whitney

Method: surface gases collection with syringe after 8 min and 3h36 in the same dark chambers used for respiration. D13C analysed by A. Gattinger.

a b c a/b

Position


Description of microbial communities

•Because of the variability throughout time, we must terminate the spring 2004 re-sampling for WPI community counts.•As soon as our data-set is complete, we will link microbial diversity/abundance with physico-chemical and gas production measurements and integrate them in multivariate statistical analyses.•CARD-FISH measurements should be finished by end of July.•According to the preliminary C-balance results, I would look for the important nominal variables (treatments) and reduce the number of samples for the experimental sites.•Greetings from India !


Arcellinida phylogeny

• Illustrations of the seven testate amoebae taxa analyzed : a & b : Heleopera sphagni (small green spheres inside the cyst are endosymbiotic algae), c : Nebela tincta var. major, d & e : Hyalosphenia papilio , f : Bullinularia indica, g : Trigonopyxis arcula, h : Arcella artocrea, i : Centropyxis laevigata. Scale bars indicate 50 µm.


Partial SSU rRNA data

• MrBayes tree of partial SSU rRNA genes, showing the monophyly of Arcellinida. Numbers at nodes indicate posterior probabilities for MrBayes analysis (upper) and bootstrap values for ML analysis (lower).Nebela tincta major

Hyalosphenia papilio

Bullinularia indica

Trigonopyxis arcula

Centropyxis laevigata

Arcella artocrea

Heleopera sphagni


Tree of the Actin gene data

ArcellinidaHeleopera sphagni


Euglyphid testate amoebae

Arcellinida


Testate amoeba species richness in the Jura sites

Testate amoeba species richness

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CH-A CH-B CH-C CH-D RU-B RU-C


CA of testate amoebae samples


How will the community structure

of different taxonomic group

compare among the sites?

✩Finland

Britain

Vegetation

Netherlands

Switzerland

Axis 1

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✩Axis 2

Switzerland

Finland

Sweden

Netherlands

Britain

Axis 1

Axis 2

Testate amoebae


Publications

• In press:– Nikolaev SI, Mitchell EAD, Petrov NB, Berney C, Fahrni J,

Pawlowski J, 2005. The testate lobose amoebae (order Arcellinida Kent, 1880) finally find their home within Amoebozoa. Protist, 156(2): (In press).

• In prep, data available:– Emilie, Daniel, Hung, Andy, Edward: Microbial community

methodological paper– Andy: Respiration methodological paper– Andy: WPI - Respiration, CH4 measurements with inhibitors– Andy: WPIII - Respirations and methanogenesis profiles– P. Steinmann, Andy: WPI - Modelling of gas profiles in peat– Fatima, Edward, Alexandre, Daniel G, etc.: WPI preliminary -

peat OM, Bacteria, and testate amoebae in Jura sites


Publications (continued)• In prep, data partly available:

– Andy, Emanuela: WPI Peepers gas profiles, delta 13C– Andy, Emanuela: WPI Archaea community profiles FISH/CARD-

FISH– Emanuela, Andy, Mika: WPI Surface gas fluxes, photosynthesis,

respiration, methanogenesis– Andy, Daniel Gilbert, etc.: Microbial community structure in WPI

at all sites - Alone or with testate amoebae, vegetation and other data?

– Andy, Daniel Gilbert etc.: WPI Microbial communities in the Jura sites - relationship with other data gases, FISH, vegetation…

– Antonis, Edward: WPI Euglyphida diversity in Chaux d’Abel - comparison molecular and microscopic data


Publications (continued)

• Planned– Emanuela: WPI Growth of Sphagnum and vascular plant biomass

production at Chaux d’Abel

– Edward, Enrique???: Further phylogenetic data on the Arcellinida

• Possible / optional– Edward: WPI Paleoecology using testate amoebae


Does under-pressure have a differential effect on microbial activity?

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High pressure (0.51) Low P (0.33 atm)

Pressure modus

N=9. Peat samples were incubated in under-pressured 9 ml air tight syringes for a period of 22 days.

“Mont-Blanc”4808 m

“Nanda Devi”7828m

RECIPE meeting Scheyern 17-20 May 2005

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Transcript of RECIPE meeting Scheyern 17-20 May 2005