lecture bsm vs dec11 slide share

11-12-2015

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www.anaerobic-microbiology.eu

“Impossible” anaerobic microbes

important in the global

Methane and Nitrogen Cycles

BSM December 11th 2015

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. Anaerobic oxidation of methane (AOM)

4. Latest sampling campaigns

5. Take home message

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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


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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Under explored microbial diversity

40,000 trains in DMSZ, LMGBC & ATCC

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” 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” Microbes

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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


Absolute prerequisite

Excellently Educated & Enthusiastic Team Members

www.ru.nl/microbiology/vacancies

www.ru.nl/masters/microbiology

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

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

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Prerequisite 2 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

Ammonium & Nitrate: toxicity & eutrophication

Nitrite & Nitric oxide : toxicity

Nitrous oxide : strong green house gas

ERC AG 2008

anammox

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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

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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

BLACK SEA Kuypers et al Nature 2003

Namibia OMZ Kuypers et al PNAS 2005

Peru OMZ Lam et al Nature 2007

Jettenia asiatica

Jettenia caeni

Jettenia moscovienisis

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


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

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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

- ?

Complete ammonium oxidiser (comammox)

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 anaerobic ammonia oxidizers

(Anammox)

After1 year good NH4+ plus NO2

- consumption


15N activity assays; N balance does NOT add up

• Labelled substrates: 15NH4+

• Anammox activity is higher than possible based on NO2- conversion

• Where is this additional NO2- coming from?

Increase in ratio 29N2

=

conversion of NH4+ in gas

Maartje van Kessel

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Fluorescence in situ hybridization

• Nitrospira is always present in flocs with anammox

• Stable coculture; cross feeding?

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

Januari 2012 November 2012


Metagenome sequencing, assembly and coverage binning

• Recovery of two high quality Nitrospira genomes

• Nitrospira nitrificans & Nitrospira nitrosa

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Metagenomic analyses: complete ammonium oxidiser

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


Comammox : 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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Specific AMO activity staining by fluorescent dye FTCP

red = Nitrospira

green = FTCP

blue= all bacteria

White = overlay


• Comammox does exist!

• Nitrospira species

• unusual/novel amoA gene

• Implications;

- N-cycle research

- Wastewater treatment plants

NO3- NO2

- NH4

+

N2

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TABLE OF CONTENT

Introduction anaerobic microbiology





4. Take home messages

ERC AG 2013

EcoMoM


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

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Schulze et al. (2010), Global Change Biology

Natural methane sources


Wetlands are important sources for methane

Methane Sinks:

aerobic and anaerobic methane munching microbes

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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Aerobic methane oxidation

Nitrite dependent anaerobic methane oxidation

Nitrate dependent anaerobic methane oxidation

Quest for Nitrate/Nitrite AOM


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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FISH: Archaea & Bacteria

Vaksmaa et al AEM in prep


Bacteria

Methylomirabilis

NO dismutase?

Archaea

Methanoperedens

Mechanism?

+ Nitrate + Nitrite

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Bacteria

Methylomirabilis

NO dismutase?

Archaea

Methanoperedens

Mechanism?

Extract DNA, RNA, Protein

Construct draft assemblies, RNAseq, proteomics

+ Nitrite


Ecophysiology Methylomirabilis oxyfera

• Doubling time of 2 weeks

• Ecophysiology Ks & Yield?

• Enrichment >80 % M. oxyfera

• Polygonal shape

• 2% cells have virus

Courtesy of Gambelli

Karin Stultiens

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Genome of Methylomirabilis oxyfera

2010 pathway of (aerobic) methane oxidation

Incomplete denitrification

Putative NO dismutase


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

lecture bsm vs dec11 slide share

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