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PERGAMON Workshop CH4OX Kiel, June 6-8, 2012. Establishing standard protocols for the quantification of microbial methane oxidation rates in sediments and in the water column Tina Treude & Ingeborg Bussmann. Introduction. Why a workshop on methane oxidation methods?. Because….

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PERGAMON Workshop CH4OX Kiel, June 6-8, 2012

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Pergamon workshop ch4ox kiel june 6 8 2012

PERGAMON Workshop CH4OX

Kiel, June 6-8, 2012

Establishing standard protocols for thequantification of microbial methane oxidation rates in sediments and in the water columnTina Treude & Ingeborg Bussmann


Pergamon workshop ch4ox kiel june 6 8 2012

Introduction

Why a workshop on methane oxidation methods?

Because…..

…community working on methane oxidation rates is increasing.

…experiences need to be exchanged.

…methods should be optimized/streamlined to reach maximum comparability.

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

Introduction

1. Aerobic oxidation of methane by methanotrophic bacteria at the sediment-water interface or in the water column

CH4 + 2O2  CO2 + 2H2O

methane oxygen carbon dioxide water

ΔG°= -204 kJ mol-1

2. Anaerobic oxidation of methane by methanotrophic archaea and sulfate-reducing bacteria in deeper zones of the sediment

CH4 + SO42-  HCO3- + HS- + H2O

methane sulfate bicarbonate sulfide water

ΔG°= -16.6 kJ mol-1

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

Introduction

1. Aerobic oxidation of methane by methanotrophic bacteria at the sediment-water interface or in the water column

14CH4 + 2O2 14CO2 + 2H2O

methane oxygen carbon dioxide water

ΔG°= -204 kJ mol-1

2. Anaerobic oxidation of methane by methanotrophic archaea and sulfate-reducing bacteria in deeper zones of the sediment

14CH4 + SO42-  H14CO3- + HS- + H2O

methane sulfate bicarbonate sulfide water

ΔG°= -16.6 kJ mol-1

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

Introduction

1. Aerobic oxidation of methane by methanotrophic bacteria at the sediment-water interface or in the water column

C3H4 + 2O2 CO2 + 23H2O

methane oxygen carbon dioxide water

ΔG°= -204 kJ mol-1

2. Anaerobic oxidation of methane by methanotrophic archaea and sulfate-reducing bacteria in deeper zones of the sediment

14CH4 + SO42-  H14CO3- + HS- + H2O

methane sulfate bicarbonate sulfide water

ΔG°= -16.6 kJ mol-1

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

Introduction

Final Goal of the Workshop

Minimum: Method protocols published on the PERGAMON website

Optimum: "Best practice" Publication

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

Introduction

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

Introduction

Participants:Tina Treude (GEOMAR),Stefan Krause (GEOMAR),Philip Steeb (GEOMAR),Ingeborg Bussmann (AWI),Roman Osudar (AWI),Helge Niemann (Uni Basel),Lea Steinle (Uni Basel)Jens Kallmeyer (Uni Potsdam),Susan Mau (MPI Bremen),Anna Matousu (University of South Bohemia)

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

Overview

Workshop Overview

Presentations

Treude, T.: General introduction into methods for anaerobic oxidation of methane (AOM)

Bussmann, I.: General introduction into methods for aerobic oxidation of methane (MOx)

Kallmeyer, J: Relevance of blanks in radiotracer measurements

Mau, S: Comparison of the tracers [3H]-CH4 and [14C]-CH4 to measure methane oxidation rates in the water column

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

Overview

Workshop Overview

Discussions

Discussing pros/cons and necessary improvements/streamlining of AOM rate measurements in sediments.

Discussing pros/cons and necessary improvements/streamlining of MOx rate measurements in the water column.

Tasks

Preparation of optimized method protocols for "AOM" and "MOx".

Planning of method guidelines

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

AOM

Anaerobic oxidation of methane (AOM)

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

AOM

  • AOM in sediments(only with sulfate)

  • Introduction

  • Approaches and methodologies

    • Sampling equipment

    • Incubation

      • Vials/tubes

      • Amount of replicates (depending on vial/tube)

      • Tracer amounts (liquid volume and activity)

      • Time

      • Temperature, Light

      • How to stop

      • High vs. low active sediments (time, tracer)

    • Controls

      • Minimum amount

      • Corrections

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

AOM

  • AOM in sediments(only with sulfate)

    • Storage

      • Different types of vials

      • Length of storage

    • Analyses

      • GC

      • radioactive CH4

      • radioactive CO2

      • Volume determination

      • Scintillation counting

    • Calculations

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

AOM

  • AOM in sediments(only with sulfate)

    • Potential pitfalls and suggestions for

    • improvements

      • Degassing/loss of methane gas

      • Smearing

      • Sediment compression

      • Core slicing

      • Ex situ vs in situ

      • Heterogeneity (extrapolations)

      • Error in the vial headspace volume

      • Re-oxidation and loss of methane in

      • glass tube incubations

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

AOM

  • AOM in sediments(only with sulfate)

  • Data reporting

    • Volumetric rates vs. rates per weight

    • cm2 vs. m2

    • Day vs. year

    • Replicates vs. means (and standard deviations)

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

MOx in thewater column

Aerobic oxidation of methane (MOx)

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

General

- methane concentrations range from 1 to 5 nM in open ocean to < 1000 nM in freshwater

- rather low consumption rates, compared to the sediment

- but the volume of the water column(s) is immense, thus MOXrate in the water column have to be taken into account

MOx in thewater column

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

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Principle of tracer measurements

*educt = * product

total educt total product

MOx in the

water column

incubation time

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

MOx in thewater column

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

Incubation:

- bottle size (20 - 160 ml)

- stoppers (different materials, also toxic ones!!)

- incubation time

- bubble size & syringe

MOx in thewater column

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

freshwater controls

Incubation:

bottle size

stoppers

incubation time

bubble size & syringe

stopping the incubation

controls

storage of samples

MOx in the

water column

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

Sample processing

MOx in the

water column

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

Sample processing:

- immediate counting of *CH4 containing fraction

- *H2O containing fraction is stable over time

Sample storage

- as short as possible

- keep cool

MOx in the

water column

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

Calculation:

- turnover time (days) = 1 / k‘ = [CH4] / rox

- methane oxidation rate (nmol L-1 d-1)

= k‘ / [CH4]

- effective first orde rate k‘ = fraction of tracer oxidized per unit time

MOx in the

water column

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

MOx in the

water column

Results from off Spitzbergen 2011

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

MOx in the

water column

Results from Elbe transects

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


Pergamon workshop ch4ox kiel june 6 8 2012

Thank you for your attention

PERGAMON Workshop CH4OX, Kiel, June 6-8, 2012


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