Lecture dsmz 19 nov slide share

11-12-2015

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Department of Microbiology

How to discover & unravel

the secrets of impossible

anaerobic microbes

(by metagenomic analysis)

DSMZ November 19th 2015

MIKE JETTEN

SOEHNGEN INSTITUTE OF ANAEROBIC MICROBIOLOGY


TABLE of CONTENT

Introduction (anaerobic) microbiology

1. Anaerobic oxidation of ammonium (anammox)

2. Anaerobic oxidation of methane (AOM)

3. Latest sampling campaigns

4. Take home message

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Many very useful microbes

–Wastewater treatment

–Oxygen production

–Nitrogen fixation

–Food and fermentation

–Drugs and Antibiotics

–Degradation of xenobiotics

Very few pathogens

Earth = Microbial planet


Under explored microbial diversity

40,000 strains in DMSZ & 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

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Microbial Metabolic Diversity

CH4


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

EL

EC

TR

ON

AC

CE

PT

OR

S

OXIC

ANOXIC

??? ???

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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


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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Pre requisites : Complementary & Synergy of staff How can we discover

new microbial

(anaerobic) processes

with global importance?

Which roles do

microbes play in

volcanic

environments?

How does microbial

cell structure

influence function?

How do nitrogen

cycle bacteria

cope with oxygen

limitation?

Can we apply microbes

through understanding

ecophysiology?

Which roles do

microbes play in

(an)aerobic oxidation of

methane in wetlands?

How do microbes

interact with

wetland plants

and animals?

H OpdenCAMP L VAN NIFTRIK B KARTAL C WELTE S LUECKER C LUEKE


Pre requisite 2

Excellently educated & enthusiastic team members

www.ru.nl/microbiology/vacancies

www.ru.nl/masters/microbiology

www.anaerobic-microbiology.eu

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

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

http://www.anaerobic-microbiology.eu/



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Pre requisite 3 State-of-the-art methods

Bioreactors Bioreactors Bioreactors

Bioreactors Bioreactors Bioreactors

Metagenomics, bioinformatics, new experiments

Sanger, illumina,

454, pacbio


TABLE OF CONTENT

Introduction anaerobic microbiology




4. Take home messages

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

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ANAMMOX MILESTONES

1995 ANAEROBIC PILOT PLANT Mulder et al FEMS Ecol

1998 SBR ENRICHMENT CULTURES Strous et al AMB

1999 PHYLOGENETIC IDENTITY Strous et al Nature

2002 LADDERANE LIPIDS Damste et al Nature

2003 BLACK SEA Kuypers et al Nature

2005 Namibia OMZ Kuypers et al PNAS

2007 Peru OMZ Lam et al Nature

Jettenia asiatica

Jettenia caeni

Jettenia moscovienisis

OMZs: 50% N loss


ANAMMOX MILESTONES

2006 GENOME ASSEMBLY Strous et al Nature

2008 CELL BIOLOGY Van Niftrik et al Mol Mic

2011 METABOLISM Kartal et al Nature

2013 CHINESE WETLANDS Zhu et al Nature Geoscience

2015 PEPTIDOGLYCAN DETECTED van Teeseling et al & Jeske et al Ncomm

PROTEIN STRUCTURES Dietl et al Nature

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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 NOB Van Kessel et al

Nature 2015 accepted

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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TABLE OF CONTENT






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

Twentekanaal Brunsummerheide Ooijpolder

Where do we find nitrate/nitrite-AOM?

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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


Consortium of Archaea

& Bacteria ?

Microbe_Journaal_14-04-2006.wmv

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Bacteria

Methylomirabilis

NO dismutase?

Archaea

Methanoperedens

Mechanism?

Extract DNA, RNA, Protein

Construct draft assemblies, RNAseq, proteomics

+ Nitrate + Nitrite


Bacteria

Methylomirabilis

NO dismutase?

Archaea

Methanoperedens

Mechanism?



+ Nitrite

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Physiology of 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


Assembly

2.7 Mb; 58% GC; 40-fold

Metagenome Approach

Community DNA

GS-Flex 90 Mb in 242 bp reads

Complemented with Sanger

Sequencing of WGS ; Fosmids

2010

MetaGenomics of M. oxyfera bacteria

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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

Katharina

Ettwig

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Bacteria

Methylomirabilis?

NO dismutase?

Archaea

Methanoperedens?

Mechanism?



+ 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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Genome inventory Complete reverse CH4 pathway

HDR complex

FQO complex

Many cytochrome c genes

BC1 complex

Nitrate reductase narGH

Nitrite reductase nrfAH

Arslan Arshad Cornelia Welte


Nitrate generally is more abundant than nitrite, so large potential

Many surveys do contain related mcrA and 16S rRNA genes

Are the nitrate CH4 archaea important in the environment?

ERC AG 2013

EcoMoM

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TABLE OF CONTENT






ERC AG 2013

EcoMoM

Are the nitrate CH4 archaea important in the environment?


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

• AOM present in paddy fields

• Remaining : The Quest for iron-AOM

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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

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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, Moxyfera, AAA and other new anaerobic microbes

could save the world

Thank you !

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

Science

Lecture dsmz 19 nov slide share