Lecture impossible early prokaryotes handouts

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www.anaerobic-microbiology.eu

Discovery and properties of early

“impossible”

anaerobic micro-organisms

MIKE JETTEN

GRAVITATION CENTER OF EXCELLENCE

SOEHNGEN INSTITUTE OF ANAEROBIC MICROBIOLOGY

(SIAM)


CV Prof. Dr. Ir. Mike Jetten

Year Position University

1980 1986 Molecular Sciences WUR NL

1987 1991 PhD, Anaerobic Microbiology WUR, NL

1991 1994 Post doc, Molecular Microbiology MIT, USA

1994 2000 Assistant Professor in Microbiology TU Delft, NL

2000 now Full Professor in Microbial Ecology RU Nijmegen

TEAM EFFORTS & AWARDS

2008 ERC ADVANCED GRANT anammox

2012 SPINOZAPREMIE

2013 KNIGHTHOOD

2013 ERC ADVANCED GRANT ecomom

2013 ZWAARTEKRACHT SUBSIDIE Siam/nessc

../../Downloads/MSM Jetten - Science Cafe Enschede.avi

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Discovery and properties of early

“impossible”

anaerobic micro-organisms

MIKE JETTEN

GRAVITATION CENTER OF EXCELLENCE

SOEHNGEN INSTITUTE OF ANAEROBIC MICROBIOLOGY

(SIAM)


TABLE of CONTENT

Introduction (anaerobic) microbiology

1. Anaerobic oxidation of ammonium (anammox)

2. Complete ammonium oxidation (comammox)

3. Historical prespective

4. Anaerobic oxidation of methane (AOM)

5. Latest sampling campaigns

6. Take home message

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“The Earth is a microbial planet, on which macro-organisms are recent

additions, highly interesting and extremely complex, but in the final analysis

relatively unimportant in a global context.”

Wheelis et al. (1998) PNAS 95:11043-11046


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Many very useful (anaerobic) microbes

–W……………

–O……………

–N……………..

–F……………..

–A…………….

–X………….

Very few pathogens

Earth = Microbial planet


Many very useful (anaerobic) microbes

–Wastewater treatment

–Oxygen production

–Nitrogen fixation

–Food and fermentation

–Drugs and Antibiotics

–Degradation of xenobiotics

Very few pathogens

Earth = Microbial planet

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Life would not long

remain possible in

the absences of

microbes


Life would not long

remain possible in

the absences of

microbes

Gnotobiotic mice

Life in a bubble

Less bowel movement

Reduced immune system

Reduced organs

Severe Nutritional requirements

Sudden exposure to pathogens would be lethal

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http://www.techinsider.io/what-would-happen-if-bacteria-

disappeared-2015-12


Earth as Microbial Planet

Healthy Air & Oxygen:

50% O2 production by cyanobacteria

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Healthy Air & Oxygen:

50% O2 production by cyanobacteria since 2.7 Gy ago



w/o cyanobacteria

How long would aerobes continue to thrive?

100-1000y

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Human Microbiome



Human Microbiome

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Human Microbiome

Stomach

(pH 2, 104

cells/g)

Small intestine

(pH 4–5, up to

108 cells/g)

Large intestine

(pH 7, about

1011 cells/g)

Gut microbiome essential for digestion, protection against pathogens,

and supply of vitamins and nutrients


http://news.discovery.com/videos/why-we-cant-live-without-bacteria.htm

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Our brain contains ≥ 30 bacterial metabolites


-4Gy -3Gy -2Gy -1Gy 0

Bacteria

Plants

Animals

Small in size: 100 nm – 2 mm

Large in numbers: 1030 microbial cells on Earth (50% of biomass)

OXYGEN

. Humans

ANAEROBIC MICROBIOLOGY ROCKS!

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Prebiotic

chemistry

Precellular life Early cellular

life

LUCA Evolutionary

diversification

3.8–3.7 bya

Bacteria

Archaea

- Components of DNA replication,

transcription, and translation all

in place

Divergence of Bacteria and Archaea

- Cellular compartments - Early cells likely had high rates of HGT

Lipid bilayers

- Replication

- Transcription

DNA

- RNA-templated translation

Protein synthesis

- Catalytic RNA - Self-replicating RNA

RNA world

- Amino acids - Nucleosides - Sugars

Biological

building blocks

RNA

DNA

Protein

mRNA

HGT between cells

4.3–3.8 bya

A

U G

C

G A C

U

G U U

G G

C

T

A G

C U

G A

G G

C G A

C

A G

C

A

U

G

C

C G

T A

G C

A U

C G

A U

A U

C G

G C

T A

G C

A U

C G

T

C

C

G

C

T A

G

C

G

G

A

G

T

C

A

C

G

T

T

G

T

C

A

G

C G

G C

T A

A T

C G

A T

G C

C G

Early metabolism : methanogenesis & acetogenesis

4H2 + CO2 CH4 + 2H2O

4H2 + 2CO2 CH3COOH + 2H2O


The acetyl-CoA pathway for CO2 fixation

Methanogens T = tetrahydromethanoptrein

Acetogens T = tetrahydrofolate

Nickel iron sulfur protein CODH/ACS

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Early metabolism : methanogenesis & acetogenesis

4H2 + CO2 CH4 + 2H2O

4H2 + 2CO2 CH3COOH + 2H2O

What happens with acetate?

What is the fate of CH4 ?

Fermentation back to CH4

Oxidation to CO2

Oxidation

what do you need to oxidize CH4?

electron acceptors


How much do we know about the microbes on our planet?

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Under explored microbial diversity

40,000 strains in culture collections

3,224,600 16S rRNA genes in RDP

10,000,000,000,000,000,000,000,000,000,000 Nonillion microbial cells on Earth

Terra incognita


Microbial Metabolic Diversity

CH4

NH4+ & CH4

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The Quest for the “Impossible” Anaerobic Microbes

H2 CH4 H2S NH4+ Fe2+

O2

NO3- ??? ???

Fe3+

SO42-

CO2

After 40 years of searching in vain

They were being called

“impossible” microbes

ELECTRON DONORS E

LE

CT

RO

N A

CC

EP

TO

RS

OXIC

ANOXIC

??? ???

???


H2 CH4 H2S NH4+ Fe2+

O2

NO3- ??? ???

Fe3+

SO42-

CO2

After 40 years of searching in vain

They were being called

“impossible” microbes

ELECTRON DONORS

EL

EC

TR

ON

AC

CE

PT

OR

S

OXIC

ANOXIC

CS2

???

The Quest for the “impossible” Anaerobic Microbes

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https://www.youtube.com/watch?v=va6D6Na0PRM


HOW TO DISCOVER THESE “IMPOSSIBLE” MICROBES?

• Survey of selected ecosystems

• Bring Best Samples to Lab

• Design optimal bioreactors

• Enrichment under optimal conditions

• Grow enough cells

• Use of the molecular toolbox to

unravel their secrets

• Back to the ecosystem

• Application of the new microbes

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Absolute prerequisite

Excellently Educated & Enthusiastic Team Members

www.ru.nl/microbiology/vacancies

www.ru.nl/masters/microbiology


(Inter)national Collaboration & Funding

Gravitation Center of Excellence

Soehngen Institute of Anaerobic Microbiology

http://www.ru.nl/microbiology/vacancies

http://www.ru.nl/masters/microbiology

../../Downloads/MSM Jetten - Science Cafe Enschede.avi

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Prerequisite State-of-the-Art Methods

Bioreactors Bioreactors Bioreactors

Bioreactors Bioreactors Bioreactors

Metagenomics, bioinformatics, new experiments

illumina, minion

pacbio


TABLE OF CONTENT

Introduction anaerobic microbiology





5. Take home messages

ERC AG 2008

anammox

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N2

NO2-

NO

N2O

denitrification

nitrification

NH4+

NH2OH

NO3-

Nir

NOR

Nar

N2OR

HAO

AMO

NAOR

(N)O2

N2 fixation N2-ase

Nrf

1886

Gayon & DuPetit

1894

Winogradsky

1899

Beijerinck

The Nitrogen cycle till 1995

35


ANTHROPOGENIC ALTERATION OF THE NITROGEN CYCLE

NITROGEN FERTILIZERS

NITROGEN DEPOSITION

Ammonium & Nitrate: toxicity & eutrophication

Nitrite & Nitric oxide : toxicity

Nitrous oxide : strong green house gas

ERC AG 2008

anammox

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FAO 2008, Canfield et al. 2010 Nature, Rockström et al. Nature 2009

2008

proposed safe alteration boundary

anthropogenic N deposition

60% BN2F

vs

40% AN2F


kg

N. h

a−

1. y

−1

Galloway et al. 2008, Science

Global N deposition

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Global N eutrophication


N2

NO

N2O

NH2OH

NO2-

N2H4

-III -II -I 0 I II III IV V

Oxidation state

NH4+ NO3

-

Anammox?

Microbial N cycle : 2 missing microbes

anammox & comammox

denitrification

comammox?

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Calculations in the N cycle

NH4+ + NO2

- N2 + 2H2O ΔG’0 = -358 kJ/mol

Engelbert Broda 1910-1983


Anaerobic pilot plant, TU Delft, the Netherlands

Influent

Effluent

Mulder , van de Graaf et al FEMS Ecology 1995

Mulder et al FEMS 1995; van de Graaf et al AEM 1995

NH4+ + NO2

- N2 + 2H2O

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ANAMMOX MILESTONES

ANAEROBIC PILOT PLANT Mulder et al FEMS 1995

SBR ENRICHMENT CULTURES Strous et al AMB 1998

PHYLOGENETIC IDENTITY Strous et al Nature 1999

LADDERANE LIPIDS Damste et al Nature 2002

Jettenia asiatica

Jettenia caeni

Jettenia moscovienisis

© Jetten et al 2009


Anammox in the Ocean’s oxygen minimum zones

In collaboration with Kuypers Woebken et al

44

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ANAMMOX MILESTONES

BLACK SEA Kuypers et al Nature 2003

Namibia OMZ Kuypers et al PNAS 2005

Peru OMZ Lam et al PNAS 2007

OMZs: 50% N loss


ANAMMOX MILESTONES

GENOME ASSEMBLY Strous et al Nature 2006

CELL BIOLOGY Van Niftrik & Jetten MMBR 2012

METABOLISM Kartal et al Nature 2013

CHINESE WETLANDS Zhu et al Nature Geoscience 2013

Zhu et al Sci report 2015

PEPTIDOGLYCAN DETECTED van Teeseling et al Nature comm 2015

PROTEIN STRUCTURES Dietl et al Nature 2015

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3-step anammox pathway

2H+

N2H4 HZS

NH4+

2H+

HDH N2

4H+

1e

3e

4e

NO2-

nirS NO


3-step anammox pathway

2H+

N2H4 HZS

NH4+

2H+

HDH N2

4H+

1e

3e

4e

NO2-

? NO

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How does ANAMMOX make the rocket fuel hydrazine?

Protein purification

15N14N

Protein activity Hydrazine and N2 production

Protein crystalisation Immunogold labelling hzsABC


From discovery to application

PROOF OF

CONCEPT

HYPOTHESISANAMMOX

BACTERIA DO EXIST1995 DISCOVERYWWTP & Ocean

1996 PATENTAPPLICATION

1998 LICENSE AGREEMENT

2002 FULL SCALE IMPLEMENTATION

2006 EXPORT TO CHINA

From Discovery to Application

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ANAMMOX APPLICATION the Added Value

Less oxygen demand

No COD use

Less biomass production

No emission of CO2 and N2O


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https://www.youtube.com/watch?v=NJmOjJ87X68


HOW DOES ANAMMOX GET NITRITE?

NH4+ + NO2

- N2 + 2H2O

COOPERATION WITH OTHER NITROGEN CYCLE MICROBES

AOB NH4+ + O2

- NO2

AOA NH4+ + O2

- NO2

NOB ?

DENITRIFIERS NO3 + ORG NO2

DNRA NO3 + ORG NO2

METHANE OXIDIZERS NO3 + CH4 NO2

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Microbial interactions of ANAMMOX bacteria

High NH4+

Low O2 AOB Sliekers et al 2002

NO2-

Low NH4+

Low O2 AOA Yan et al 2012

Low NO3-

Low O2 Nitrospira Van Kessel et al

Nature 2015

High S2- NO3-

No O2 DNRA Lam et al 2008

Russ et al 2014

Low NO3-

No O2 Denitrifiers Russ et al 2015

High CH4

No O2

AAA/Moxyfera Luesken et al 2012

Haroon et al 2013

Anammox


NH4+ + 1.5 O2 NO2

- AOB/AOA

NO2- + 0.5 O2 NO3

- NOB

NH4+ + 2 O2 NO3

- ?

Does a Complete ammonium oxidiser

(comammox) exist?

ERC AG 2008

anammox

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• Inoculum: Biofilm from aquaculture biofilter

• Medium: Aquaculture water, plus low [NH4+, NO2

-,

NO3-] ; No extra carbon source; No O2 supply

Conditions for anammox & comammox

After1 year good NH4+ plus NO2

- consumption


Fluorescence in situ hybridization

• Nitrospira is always present in flocs with anammox

• Stable coculture; cross feeding?

• What does Nitrospira do?

pink = anammox; green = Nitrospira; blue= all bacteria

Januari 2012 November 2012

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What does Nitrospira do in this culture?

Extract DNA

Sequence DNA by high trough put

Assemble contigs & bin genomes

Analysis of genomes

Design new experiments


Metagenome sequencing, assembly and coverage binning

• Recovery of two high quality Nitrospira genomes

• Nitrospira nitrificans & Nitrospira nitrosa

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Metagenomic analyses: Nitrospira has amoA and hao!

Only in ammonium oxidizers

Only in nitrite oxidizers

• Both Nitrospira spp. genomes contain genes for

- Ammonia monooxygenase

- Hydroxylamine dehydrogenase

- Nitrite oxidoreductase

Experimental validation

Test with ATU = amo inhibitor

Test with FISH MAR

Test with specific AMO labeling

Sebastian Luecker


Aerobic batch incubation assays

NH4+ oxidation w/o ATU

NH4+ oxidation with ATU

Shows full inhibition

NO2- oxidation

Maartje van Kessel

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AMO staining: specific fluorescent dye for active AMO

red = Nitrospira

green = FTCP

blue= all bacteria

White = overlay


Conclusions

• Comammox does exist!

• Nitrospira species

• unusual/novel amoA gene

• Implications;

- N-cycle research (especially nitrification)

- Waste water treatment plants

NO3- NO2

- NH4

+

N2

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N2

NO2-

NO

N2O

denitrification nitrification

NH4+

NH2OH

NO3-

Nir

NOR

Nar

N2OR

HAO

AMO

NAOR

(N)O2

N2-fixation

Nrf

N2-ase

The nitrogen cycle & anammox

1999 Anammox 2015 Comammox


Modular evolution of the N cycle Stein & Klotz 2016 Current Biology

Primordial

Abiotic: NOx and NH3

NOx >>> NH3 leading to molybdinum iron sulfur narG &

to iron nirS or nrfA

Coupling of electrons via quinones

Key invention: hydrazine synthesis! Closing the N cycle

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Key invention: hydrazine synthesis! Closing the N cycle

Addition of N2 fixation before GOE

With Cu, NO and N2O reductase full denitrification evolved

With Cu, amo and nitrification evolved

Cu

becomes

available


TABLE OF CONTENT

Introduction anaerobic microbiology





4. Take home messages

ERC AG 2013

EcoMoM

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Theoretical considerations Also with nitrate

3 CH4 + 8 NO2- + 8 H+ 3 CO2 + 4 N2 + 10 H2O

∆G0’ = -928 kJ mol-1 CH4

Energetically feasible, should exist in nature

• important greenhouse gas, global warming potential

about 25x that of CO2

• atmospheric concentrations have doubled since

industrialization

• one of the main products of anaerobic

decomposition of organic material

• energy-rich, but high activation energy (for a long

time only aerobic degradation was known)

Significance of methane


Schulze et al. (2010), Global Change Biology

Natural methane sources

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Wetlands are important sources for methane

Methane Sinks:

aerobic and anaerobic methane munching microbes

https://www.youtube.com/watch?v=oa3M4ou3kvw


Aerobic methane oxidation

Nitrite dependent anaerobic methane oxidation

Nitrate dependent anaerobic methane oxidation

Quest for Nitrate/Nitrite AOM

http://www.youtube.com/watch?v=oa3M4ou3kvw&feature=player_detailpage

http://www.youtube.com/watch?v=oa3M4ou3kvw

http://www.youtube.com/watch?v=oa3M4ou3kvw&feature=player_detailpage

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https://www.youtube.com/watch?v=bHdS0UjncZE


HIGH NO3- due to agricultural run-off /ground water

HIGH CH4 production in the sediment

sampling sites

Twente

kanaal Brunsummerheide

Ooij

polder

Where do we find nitrate/nitrite-AOM?

Vercelli Paddy fields

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Suitable counter gradient profiles of nitrate & methane

-250

-200

-150

-100

-50

0

-100 0 100 200 300 400 500

NO3-

NH4+

CH4d

ep

th [

cm]

concentration[µmol/L]

Nutrient profile

Activity tests

qPCR

Enrichment

FISH

Metagenome

Stable isotopes


Activity assays of soil samples

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

2,0

0 20 40 60

um

ol/

g d

ry w

eigh

t

Time (days)

NO3

NO3

ControlCH4

ControlCH4

Annika

Vaksmaa

Vaksmaa et al FEMS Microbiology Ecology submitted

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0

1000

2000

3000

4000

5000

6000

7000

8000

0 20 40 60 80

um

ol

Time (hours)

13CH4

15NO3-

Annika Vaksmaa

AOM reactor 13C labelled CH4 and 15N-NO3

Vaksmaa et al AEM in prep


FISH: Archaea & Bacteria

Vaksmaa et al AEM in prep

Microbe_journaal_14_4_2006

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Bacteria

Methylomirabilis

NO dismutase?

Archaea

Methanoperedens

Mechanism?

+ Nitrate + Nitrite


Bacteria

Methylomirabilis

NO dismutase?

Archaea

Methanoperedens

Mechanism?

Extract DNA, RNA, Protein

Construct draft assemblies, RNAseq, proteomics

+ Nitrite

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Methylomirabilis oxyfera

• Doubling time of 2 weeks

• Ecophysiology Ks & Yield?

• Enrichment >80 % M. oxyfera

• Polygonal shape

• 2% cells have virus

Courtesy of Gambelli

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0 50 100 150 200

[Nit

rite

] (m

M)

Time (min)

Karin Stultiens


Genome of Methylomirabilis oxyfera

2010 pathway of (aerobic) methane oxidation

Incomplete denitrification

Putative NO dismutase

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Possible mechanisms of M. oxyfera

Can oxygen be released? Use of suicide substrates, inhibitors and 15N18O nitrite

MMO enzyme

C3H6

15N18O2- NO

15N18O2-

NO N2

O2

Unknown enzyme

C3H6O

acetylene

O2

O2

O2 O2

O2 O2

formate

83


Methylomirabilis oxyfera putative NO dismutase 2NO N2 +O2

15N18O experiments show: Oxygen Production

0

10

20

30

40

50

60

70

80

0 60 120 180 240 300 360 420 480

time [min]

O2 r

ele

as

ed

[n

mo

l/e

xe

tain

er]

nitrite & propylene

nitrite, propylene & acetylene

nitrite & methane

nitrite, propylene & oxygen

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Bacteria

Methylomirabilis?

NO dismutase?

Archaea

Methanoperedens?

Mechanism?

Extract DNA, RNA, Protein

Construct draft assemblies, RNAseq, proteomics

+ Nitrate + Nitrite


Nitrite is formed from nitrate by AOM

increase in Archaea to 70%

→

NO2-

NO3-

Up to 2 mM NH4+ (10% of total N)

can be observed

Baoli Zhu Simon Guerrero Cruz

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Arslan Arshad Cornelia Welte

Genome inventory

Complete reverse CH4 pathway

HDR complex

FQO complex

Many cytochrome c genes

BC1 complex

Nitrate reductase narGH

Nitrite reductase nrfAH

Menaquinone

Cytochrome c

F420 co factor


New impossible microbes can discovered

• Metagenomics is powerful method to unravel

metabolic diversity & secrets

• Hydrazine synthase in anammox

• M oxyfera makes O2 from NO

• Novel nitrate reducing AOM archaea

• Remaining : The Quest for iron-AOM

ERC AG 2013

EcoMoM

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Sampling site: Gulf of Bothnia

216 m water depth


Sediment biogeochemistry

• iron-AOM

CH4 + 8Fe(OH)3 + 7CO2 → 8 FeCO3 + 14H2O

Olivia

Rasigraf

Egger, Rasigraf et al EST 2014

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Incubation set up

Sediment slurry in mineral medium

Fe(OH)3 or MnO2 nanoparticles

13CH4 (~50%)

12CO2 (~5%)

Headspace gas analysis: GC-MS Ion measurements: ICP-OES

Anaerobic set-up



13CH4 oxidation to 13CO2 coupled to Fe3+ reduction

0

1

2

3

4

5

0 20 40 60 80 100 120

13C

O2 [

µm

ol]

control

days


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0

1

2

3

4

5

0 20 40 60 80 100 120

13C

O2 [

µm

ol]

only 13CH4

control

days




0

1

2

3

4

5

6

0 20 40 60 80 100 120

13C

O2 [

µm

ol]

only 13CH4

13CH4 & Fe3+

control

days



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0

1

2

3

4

5

6

0 20 40 60 80 100 120

13C

O2 [

µm

ol]

only 13CH4

13CH4 & Fe3+

control

Fe3+ injection

days



Anammox, Comammox, Moxyfera, AAA and other new

(an)aerobic microbes could save the world

Unique bacteria hiding out in a witches’ brew of anoxic

water not only thrive in cold wetlands and oceans but

also chow down its ammonium and methane

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The Soehngen Institute of

Anaerobic Microbiology

Wishes you a happy