Lecture ngw 2 feb 2016 handouts

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www.anaerobic-microbiology.eu
De ontdekking van “onmogelijke”
anaërobe micro-organismen
NGW 2 Februari 2016
Wageningen Forum Building C222 1945h
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

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De ontdekking van “onmogelijke”
anaërobe micro-organismen
NGW 2 Februari 2016
Wageningen ForumBuilding C222 1945h
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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-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!
“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
Nitrogen Fixation & Crop Protection
w/o N2 fixation 50% of crops would fail
Compensate by Increased fertilizer production/use
Massive increase CO2 and global warming
What would happen w/o N2 fixation?

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Healthy Air & Oxygen:
50% O2 production by cyanobacteria
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
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

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

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Our brain contains ≥ 30 bacterial metabolites
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+
SO4
2-
CO2
After 40 years of searching in vain
They were being called
“impossible” microbes
ELECTRON DONORS
ELECTRONACCEPTORS
OXIC
ANOXIC
??? ???
???
H2 CH4 H2S NH4
+ Fe2+
O2
NO3
-
??? ???
Fe3+
SO4
2-
CO2
After 40 years of searching in vain
They were being called
“impossible” microbes
ELECTRON DONORS
ELECTRONACCEPTORS
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

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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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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
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

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kgN.ha−1.y−1
Galloway et al. 2008, Science
Global N deposition
Global N eutrophication

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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?
Calculations in the N cycle
NH4
+ + NO2
-  N2 + 2H2O ΔG’0 = -358 kJ/mol
Engelbert Broda 1910-1983

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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
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 PNAS 2007
Jettenia asiatica
Jettenia caeni
Jettenia moscovienisis
OMZs: 50% N loss

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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
3-step anammox pathway
2H+
N2H4HZS
NH4
+
2H+
HDH N2
4H+
1e 3e 4e
NO2
-
nirS NO

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3-step anammox pathway
2H+
N2H4HZS
NH4
+
2H+
HDH N2
4H+
1e 3e 4e
NO2
-
? NO
How does ANAMMOX make the rocket fuel hydrazine?
Protein
purification
15N14N
Protein activity
Hydrazine and N2 production
Protein crystalisationImmunogold labelling hzsABC

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From discovery to application
PROOF OF
CONCEPT
HYPOTHESIS
ANAMMOX
BACTERIA DO EXIST
1995 DISCOVERY
WWTP & Ocean
1996 PATENT
APPLICATION
1998 LICENSE
AGREEMENT
2002 FULL SCALE
IMPLEMENTATION
2006 EXPORT TO
CHINA
From Discovery to Application
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

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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
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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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
• 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

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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
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

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Metagenome sequencing, assembly and coverage binning
• Recovery of two high quality Nitrospira genomes
• Nitrospira nitrificans & Nitrospira nitrosa
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

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Aerobic batch incubation assays
NH4
+ oxidation w/o ATU
NH4
+ oxidation with ATU
Shows full inhibition
NO2
- oxidation
Maartje van Kessel
AMO staining: specific fluorescent dye for active AMO
red = Nitrospira
green = FTCP
blue= all bacteria
White = overlay

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Conclusions
• Comammox does exist!
• Nitrospira species
• unusual/novel amoA gene
• Implications;
- N-cycle research (especially nitrification)
- Waste water treatment plants
NO3
-
NO2
-
NH4
+
N2
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

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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
kanaalBrunsummerheide
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+
CH4
depth[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
umol/gdryweight
Time (days)
NO3
NO3
Control
CH4
Control
CH4
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
umol
Time (hours)
13CH4
15NO3
-
Annika Vaksmaa
AOM reactor 13C labelled CH4 and 15N-NO3
Vaksmaa et al AEM in prep
FISH: Archaea & Bacteria
After assembly and
coverage vs GC% based contig binning
diagnostic genes present in metagenome
Archaea mcrA, acs/codh, narG, nrf
Moxyfera: pmoA, mdh, nirS, nod1, nod2
Vaksmaa et al AEM in prep

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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
[Nitrite](mM)
Time (min)
Karin Stultiens
Genome of Methylomirabilis oxyfera
2010
pathway of (aerobic) methane oxidation
Incomplete denitrification
Putative NO dismutase

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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]
O2released[nmol/exetainer]
nitrite & propylene
nitrite, propylene & acetylene
nitrite & methane
nitrite, propylene & oxygen
Bacteria
Methylomirabilis?
NO dismutase?
Archaea
Methanoperedens?
Mechanism?
Extract DNA, RNA, Protein
Construct draft assemblies, RNAseq, proteomics
+ Nitrate + Nitrite

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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 ZhuSimon Guerrero Cruz
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

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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
Sampling site: Gulf of Bothnia
216 m water depth

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Sediment biogeochemistry
• iron-AOM
CH4 + 8Fe(OH)3 + 7CO2 → 8 FeCO3 + 14H2O
Olivia
Rasigraf
Egger, Rasigraf et al EST 2014
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

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13CH4 oxidation to 13CO2 coupled to Fe3+ reduction
0
1
2
3
4
5
0 20 40 60 80 100 120
13CO2[µmol]
control
days
0
1
2
3
4
5
0 20 40 60 80 100 120
13CO2[µmol]
only 13CH4
control
days

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0
1
2
3
4
5
6
0 20 40 60 80 100 120
13CO2[µmol]
only 13CH4
13CH4 & Fe3+
control
days
0
1
2
3
4
5
6
0 20 40 60 80 100 120
13CO2[µmol]
only 13CH4
13CH4 & Fe3+
control
Fe3+ injection
days

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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
The Soehngen Institute of
Anaerobic Microbiology
Wishes you a happy

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