Microbial communities of
thermal environments possible analogues of early
Earth ecosystems?
E.A. Bonch-Osmolovskaya
Winogradsky Institute of Microbiology Russian Academy of
Sciences
Summary
 Archaean biosphere
 Thermal habitats
 Electron donors and acceptors
 Metabolic diversity of thermophilic prokaryotes
 Evidence for new metabolic groups
 Carbon cycle in thermal ecosystems – is it closed?
Summary
 Archaean biosphere
 Thermal habitats
Georgy A. Zavarzin
1933-2011
 Electron donors and acceptors
 Metabolic diversity of thermophilic prokaryotes
 Evidence for new metabolic groups
 Carbon cycle in thermal ecosystems – is it closed?
Archaean biosphere
 -4.0 - -2.5 billion years
 Temperature: +70 - +100oC
 Anaerobic
 Reduced
Thermal habitats
Thermophiles on the Tree of Life
Thermophiles on the Tree of Life
Methanogens, sulfur and sulfate reducers
CO2
CH4
SO4-2
H2
So
H2S
Methanogens, sulfur and sulfate reducers
New methanogens in terrestrial hot springs
Alexander
Merkel
Geyser Valley, Kamchatka
Hot spring 2012 (Т 58˚C, pH 5.7)
108 clones
Methanogens, sulfur and sulfate reducers
Methanogens, sulfur and sulfate reducers
Sulfate reduction: Vulcanisaeta moutnovskia
Isolated from the hot
springs of Moutnovsky
Volcano, Kamchatka
Maria
Prokofeva
Nikolai
Chernyh
Evgeny
Frolov
Nikolay
Pimenov
Grows in the temperature range from 59102oC with the optimum at 83oC and in
pH range 3.5-6.5 with the optimum at 5.2
Sulfate reduction: Vulcanisaeta moutnovskia
3
6
Growth
5
2
4
SO4
mM
Cells, 107/ml
2.5
1.5
3
1
2
H2S
0.5
1
0
-20
0
30
80
Time, hours
130
180
V. moutnovskya was found to
be able to grow be sulfate
reduction
Substrates are yeast extract,
ethanol and glycerol
Sulfate reduction: Vulcanisaeta moutnovskia
93
Pyrobaculum\Thermoproteus
97
dsrA Vmut_0501 Vulcanisaeta moutnovskia 768-28
100
Vulcanisaeta distributa DSM 14429
Caldivirga maquilingensis IC-167
100
Chlorobium
100
Magnetococcus marinus MC-1
99
Archaeoglobus
100
100
99
100
100
61
Thermodesulfovibrio
Desulfosporosinus
Desulfitobacterium dichloroeliminans
91
Desulfotomaculum
100
0.1
Crenarchaeal genes encoding sulfate reduction enzymes make a separate
cluster, while those of Archaeoglobus are related to bacterial ones
Sulfate reduction: Vulcanisaeta moutnovskia
H2O
Disproportionation of sulfur compounds
CO2
H2S
CH4
SO4-2
H2
So
S2O3-2
So
H2S
SO4-2
Alexander
Slobodkin








Galina
Slobodkina
Disproportionation - redox reaction in which compound with an intermediate
oxidation state is simultaneously reduced and oxidized to form two different
products
Electron donor and electron acceptor
Inorganic sulfur fermentation
Disproportionation of sulfur compounds: sulfite, thiosulfate, elemental sulfur
Formation of sulfate and sulfide
4SO32- + H+ = 3SO42- + HS3:1
S2O32- + H2O = SO42- + HS- + H+ 1:1
4S0 + 4H2O = SO42- + 3HS- + 5H+ 1:3
ΔG°’= -58.9 kJ mol-1 SO32ΔG°’= -22.3 kJ mol-1 S2O32ΔG°’= +10.3 kJ mol-1 S0
Cells (x10exp7) per ml, sulfide (mM), sulfate
(mM)
Thermosulfurimonas dismutans
7
6
5
4
3
Cells (x10exp7)
per ml
Sulfide (mM)
2
1
0
0
10
20
30
40
50
Time (h)
Isolated from the
hydrothermal chimney
of Lau Spreading
Center, Pacific Ocean,
depth 2060 m
Growth in the temperatures range from 50 to 92 oC, opt 74 oC
Obligate anaerobe
Obligate lithoautotroph
Needs Fe(III) for H2S scavenging (growth up to 108 cells/ml
Capable to grow with H2 reducing thiosulfate
Thermosulfurimonas dismutans
New genus in Thermodesulfobacteria
100
100
58
Thermodesulfobacterium hveragerdense JSPT (X96725)
Thermodesulfobacterium thermophilum DSM 1276T (AF334601)
Thermodesulfobacterium commune YSRA-1T (AF418169)
Thermodesulfobacterium hydrogeniphilum SL6T (AF332514)
100
‘Geothermobacterium ferrireducens’ FW-1aT (AF411013)
100
Caldimicrobium rimae DST (EF554596)
Thermosulfurimonas dismutans S95T (JF346116)
Thermodesulfatator indicus CIR29812T (AF393376)
58
100
Thermodesulfatator atlanticus AT1325T (EU435435)
Thermosulfidibacter takaii ABI70S6T (AB282756)
0.02
New thermophilic Deltaproteobacteria capable of sulfur disproportionation
Uzon Caldera, Kamchatka
‘Dissulfurimicrobium
97
hydrothermalis’ Sh68
Dissulfuribacter thermophilus S69T (JQ414031)
100
Desulfobulbaceae
70
100
59
Syntrophaceae
Desulfobacca acetoxidans DSM 11109T
(CP002629)
Desulfomonile
100
100
Syntrophobacteraceae
69
52
29
100
0.02
Deferrisoma camini S3R1T
(JF802205)
Desulfuromonadaceae
Lau Spreading Center,
Pacific Ocean


Genome size – 2.20 Mb
Carbon metabolism - autotrophic CO2 fixation via reductive acetyl-CoA pathway
 Identified genes: CO dehydrogenase/acetyl-CoA synthase, acetyl-CoA synthase subunit,
 Acetyl-CoA synthase corrinoid iron-sulfur protein, large subunit;
 Acetyl-CoA synthase corrinoid activation protein
 NAD-dependent formate dehydrogenase alpha subunit
 5,10-methylenetetrahydrofolate reductase
 Carbon monoxide dehydrogenase CooS subunit
 Methylenetetrahydrofolate dehydrogenase
 Formate--tetrahydrofolate ligase

Hydrogen metabolism – uptake [Ni/Fe] hydrogenase
 Identified genes:
[Ni/Fe] hydrogenase, group 1, large subunit
 [Ni/Fe] hydrogenase, group 1, small subunit
 Uptake hydrogenase large subunit
 Ni,Fe-hydrogenase I cytochrome b subunit
 Hydrogenase maturation protease
 [NiFe] hydrogenase metallocenter assembly protein HypC
 [NiFe] hydrogenase nickel incorporation protein HypA
 [NiFe] hydrogenase nickel incorporation-associated protein HypB
 [NiFe] hydrogenase metallocenter assembly protein HypF
Genome of Thermosulfurimonas dismutans

Sulfur metabolism – complete pathway of sulfate reduction
 Identified genes:
 Thiosulfate sulfurtransferase, rhodanase
 Dissimilatory sulfite reductase (desulfoviridin), alpha and beta subunits
 Tetrathionate reductase subunit A
 Sulfite reduction-associated complex DsrMKJOP protein DsrP (= HmeB)
 Sulfite reduction-associated complex DsrMKJOP iron-sulfur protein DsrO (=HmeA)
 Sulfite reduction-associated complex DsrMKJOP multiheme protein DsrJ (=HmeF)
 Sulfite reduction-associated complex DsrMKJOP protein DsrK (=HmeD)
 Sulfite reduction-associated complex DsrMKJOP protein DsrM (= HmeC)
 Tetrathionate reductase subunit C
 Tetrathionate reductase subunit B
 Anaerobic dimethyl sulfoxide reductase chain B
 Anaerobic dimethyl sulfoxide reductase, A subunit
 Polysulphide reductase, NrfD
 Adenylylsulfate reductase alpha-subunit
 Adenylylsulfate reductase beta-subunit
 Sulfate adenylyltransferase, dissimilatory-type
 Sulfite reductase, dissimilatory-type gamma subunit
 Sulfite reductase alpha subunit
 Sulfite reductase beta subunit
 Dissimilatory sulfite reductase clustered protein DsrD
 Octaheme tetrathionate reductase
Anaerobic CO and formate oxidation
CO2
H2O
H2O
H2S
CH4
SO4
H2
CO
-2
H2
So
SO4-2
S2O3-2
So
H2O
CO2
H2S
H2
HCOOH
Anaerobic CO and formate oxidation
CO + H2O = CO2 + H2
15
Tatyana
Sokolova
13
CO
200
Alexander
Lebedinsky
11
9
150
Cells
7
100
5
H2
50
Cells, 107/ml
СО, Н2, µmol/ml of mediumл
250
(Svetlichny et al., 1991)
-1
0
50
Daria
Kozhevnikova
3
1
0
Tatyana
Kochetkova
100% CO:
phylogenetically diverse Firmicutes
hyperthermophilic archaea of genus Thermococcus
100
Time, hours
Growth of Thermococcus barophilus Ch5
on CO
45% CO:
hyperthermophilic archaea of genus Thermofilum
5% CO:
Thermophilic bacteria of genus Dictyoglomus
Anaerobic CO and formate oxidation
cooA cooC
cooM
cooK cooL cooX cooU cooH hypA cooF cooS
Carboxydothermus hydrogenoformans
cooRa cooF
cooRa cooF
cooS
cooS
cooC
cooC
1/2 cooM
1/2 cooM
cooK cooU+cooH cooX cooL
Thermococcus sp. AM4
T. barophilus MPT and Ch5
T. onnurineus
cooU cooH cooY cooL cooK cooX
“Thermofilum carboxydotrophus"
Anaerobic CO and formate oxidation
cooA cooC
cooM
cooK cooL cooX cooU cooH hypA cooF cooS
Carboxydothermus hydrogenoformans
cooRa cooF
fdh
cooF
cooRa cooF
cooS
1/2 cooM
cooS
cooC
1/2 cooM
cooC
1/2 cooM
cooK cooU+cooH cooX cooL
1/2 cooM
1/2 cooM
cooK cooU+cooH cooX cooL h f-tr
Thermococcus sp. AM4
T. barophilus MPT and Ch5
T. onnurineus
T. onnurineus
T. gammatolerans
T. barophilus Ch5
cooU cooH cooY cooL cooK cooX
“Thermofilum carboxydotrophus"
Anaerobic CO and formate oxidation
The energy of reaction:
HCOO- + H2O → HCO3- + H2
ΔG0' = +1.3 kJ/mol
was always considered to be insufficient to support microbial growth
In our experimental conditions ΔG0‘ varied from -8 to -20 kJ/mol
Kim et al., Nature, 2010, 467:352-355
Anaerobic CO and formate oxidation
3
200
Cells
2
H2
100
Formate
1
50
0
0
0
20
40
Time, hours
60
Cells, *107/ml
H2, HCOOH, mkmol/ml
150
Thermococcus sp. able to grow on
formate producing hydrogen:
T. barophilus
T. gammatolerance
T. onnurineus
three new isolates from different
deep-sea hydrothermal areas
Anaerobic CO and formate oxidation
CO2
H2O
H2O
H2S
CH4
SO4
H2
CO
-2
H2
So
SO4-2
S2O3-2
So
H2O
CO2
H2S
H2
HCOOH
Radioisotopic tracing: detection of new metabolic groups
Uzon Caldera, Kamchatka
Micrograms C l(-1)
day(-1)
10000
In situ incubation
Na14CO3
14C-acetate
100
1
14C-products
0,01
651
702
853
T, oC
Lithotrophic methanogenesis
Acetoclastic methaogenesis
Carbon assimilation
Acetate oxidation
Acetogenesis
pH 8.5
Micrograms C l(-1)
Micrograms C l(-1)
day(-1)
day(-1)
10000
10000
100
100
1
1
0,01
0,01
65
70
85
60
Lithotrophic methanogenesis
Acetoclastic methanogenesis
Carbon assimilation
Acetate oxidation
pH 7.0
70
Nikolay
Pimenov
85
T, oC
T, oC
Acetogenesis
Lithotrophic methanogenesis
Acetoclastic methanogenesis
Carbon assimilation
Acetate oxidation
pH 3.5
Acetogenesis
Radioisotopic tracing: detection of new metabolic groups
Uzon Caldera, Kamchatka
Micrograms C l(-1)
day(-1)
?
10000
In situ incubation
Na14CO3
14C-acetate
100
1
14C-products
0,01
651
65
702
70
853
85
T, oC
Lithotrophic methanogenesis
Acetoclastic methaogenesis
Carbon assimilation
Acetate oxidation
Acetogenesis
pH 8.5
Micrograms C l(-1)
Micrograms C l(-1)
day(-1)
day(-1)
?
10000
100
100
1
1
0,01
0,01
65
70
85
60
Acetoclastic methanogenesis
Carbon assimilation
Acetate oxidation
70
85
T, oC
T, oC
Lithotrophic methanogenesis
pH 7.0
?? ?
10000
Acetogenesis
Lithotrophic methanogenesis
Acetoclastic methanogenesis
Carbon assimilation
Acetate oxidation
pH 3.5
Acetogenesis
Anaerobic CO and formate oxidation
CO2
H2S
CH4
SO4
Acetate
H2O
H2O
H2
CO
-2
H2
So
SO4-2
S2O3-2
So
H2O
CO2
H2S
H2
HCOOH
Radioisotopic tracing: detection of new metabolic groups
Uzon Caldera, Kamchatka
Micrograms C l(-1)
day(-1)
10000
In situ incubation
Na14CO3
14C-acetate
100
1
14C-products
0,01
651
702
853
T, oC
Lithotrophic methanogenesis
Acetoclastic methaogenesis
Carbon assimilation
Acetate oxidation
Acetogenesis
pH 8.5
?
? ??
Micrograms C l(-1)
Micrograms C l(-1)
day(-1)
day(-1)
10000
10000
100
100
1
1
0,01
0,01
65
70
85
60
Lithotrophic methanogenesis
Acetoclastic methanogenesis
Carbon assimilation
Acetate oxidation
pH 7.0
70
85
T, oC
T, oC
Acetogenesis
Lithotrophic methanogenesis
Acetoclastic methanogenesis
Carbon assimilation
Acetate oxidation
pH 3.5
Acetogenesis
Conclusions
• Microbial communities of thermal environments contain anaerobic
lithoautotrophic microorganisms capable to use electron donors and
acceptors of volcanic origin, and to assimilate inorganic carbon in cell
material.
• C1 compounds of abiogenic origin can also fuel microbial ecosystems; no
electron acceptor is required.
• Anaerobic thermophilic lithoautotrophs able to disproportionate sulfur
compounds are phylogenetically diverse, widely spread and also could act as
the primary producers in primary ecosystems of the Archaean Earth.
• New anaerobic lithotrophic thermophiles are still to be discovered.
• Microbial communities of thermal habitats are able to perform both primary
production and complete mineralization of organic matter, thus, closing the
carbon cycle in these environments.
Acknowledgements:
Collaboration:
Institute of Volcanology and Seysmology RAS (expeditions)
IFREMER, France (expeditions)
University of Portland, USA (expeditions)
Center «Bioengineering» RAS (sequencing and annotation of genomes)
KORDI, Republic of Korea (the genomics of formate-utilizing archaea)
Financial support:
Programs of RAS
Russian Foundation of Basic Research