Post on 15-Apr-2017
11-12-2015
1
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
Department of 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
11-12-2015
2
Department of Microbiology
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
Department of Microbiology
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
11-12-2015
3
Department of Microbiology
Microbial Metabolic Diversity
CH4
Department of Microbiology
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
??? ???
11-12-2015
4
Department of Microbiology
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
Department of Microbiology
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
11-12-2015
5
Department of Microbiology
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
Department of Microbiology
Pre requisite 2
Excellently educated & enthusiastic team members
www.ru.nl/microbiology/vacancies
www.ru.nl/masters/microbiology
www.anaerobic-microbiology.eu
11-12-2015
6
Department of Microbiology
Pre requisite 3 State-of-the-art methods
Bioreactors Bioreactors Bioreactors
Bioreactors Bioreactors Bioreactors
Metagenomics, bioinformatics, new experiments
Sanger, illumina,
454, pacbio
Department of 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 messages
ERC AG 2008
anammox
11-12-2015
7
Department of Microbiology
Calculations in the N cycle
NH4+ + NO2
- N2 + 2H2O ΔG’0 = -358 kJ/mol
Engelbert Broda 1910-1983
Department of Microbiology
Anaerobic pilot plant, TU Delft, the Netherlands
Influent
Effluent
Mulder , van de Graaf et al FEMS Ecology 1995
11-12-2015
8
Department of Microbiology
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
Department of Microbiology
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
11-12-2015
9
Department of Microbiology
3-step anammox pathway
2H+
N2H4 HZS
NH4+
2H+
HDH N2
4H+
1e
3e
4e
NO2-
nirS NO
Department of Microbiology
3-step anammox pathway
2H+
N2H4 HZS
NH4+
2H+
HDH N2
4H+
1e
3e
4e
NO2-
? NO
11-12-2015
10
Department of Microbiology
How does ANAMMOX make the rocket fuel hydrazine?
Protein purification
15N14N
Protein activity Hydrazine and N2 production
Protein crystalisation Immunogold labelling hzsABC
Department of Microbiology
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
11-12-2015
11
Department of 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 messages
ERC AG 2013
EcoMoM
Department of Microbiology
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
11-12-2015
12
Department of Microbiology
Schulze et al. (2010), Global Change Biology
Natural methane sources
Department of Microbiology
Wetlands are important sources for methane
Methane Sinks:
aerobic and anaerobic methane munching microbes
11-12-2015
13
Department of Microbiology
Aerobic methane oxidation
Nitrite dependent anaerobic methane oxidation
Nitrate dependent anaerobic methane oxidation
Quest for Nitrate/Nitrite AOM
Department of Microbiology
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?
11-12-2015
14
Department of Microbiology
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
Department of Microbiology
Consortium of Archaea
& Bacteria ?
11-12-2015
15
Department of Microbiology
Bacteria
Methylomirabilis
NO dismutase?
Archaea
Methanoperedens
Mechanism?
Extract DNA, RNA, Protein
Construct draft assemblies, RNAseq, proteomics
+ Nitrate + Nitrite
Department of Microbiology
Bacteria
Methylomirabilis
NO dismutase?
Archaea
Methanoperedens
Mechanism?
Extract DNA, RNA, Protein
Construct draft assemblies, RNAseq, proteomics
+ Nitrite
11-12-2015
16
Department of Microbiology
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
Department of Microbiology
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
11-12-2015
17
Department of Microbiology
Genome of Methylomirabilis oxyfera
2010 pathway of (aerobic) methane oxidation
Incomplete denitrification
Putative NO dismutase
Department of Microbiology
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
11-12-2015
18
Department of Microbiology
Bacteria
Methylomirabilis?
NO dismutase?
Archaea
Methanoperedens?
Mechanism?
Extract DNA, RNA, Protein
Construct draft assemblies, RNAseq, proteomics
+ Nitrate + Nitrite
Department of Microbiology
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
11-12-2015
19
Department of Microbiology
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
Department of Microbiology
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
11-12-2015
20
Department of 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 messages
ERC AG 2013
EcoMoM
Are the nitrate CH4 archaea important in the environment?
Department of Microbiology
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
11-12-2015
21
Department of Microbiology
Sampling site: Gulf of Bothnia
216 m water depth
Department of Microbiology
Sediment biogeochemistry
• iron-AOM
CH4 + 8Fe(OH)3 + 7CO2 → 8 FeCO3 + 14H2O
Olivia
Rasigraf
11-12-2015
22
Department of Microbiology
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
Department of Microbiology
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
11-12-2015
23
Department of Microbiology
0
1
2
3
4
5
0 20 40 60 80 100 120
13C
O2 [
µm
ol]
only 13CH4
control
days
13CH4 oxidation to 13CO2 coupled to Fe3+ reduction
Department of Microbiology
0
1
2
3
4
5
6
0 20 40 60 80 100 120
13C
O2 [
µm
ol]
only 13CH4
13CH4 & Fe3+
control
days
13CH4 oxidation to 13CO2 coupled to Fe3+ reduction
11-12-2015
24
Department of Microbiology
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
13CH4 oxidation to 13CO2 coupled to Fe3+ reduction
Department of Microbiology
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