Anaerobic CO and formate oxidation

Transcript Anaerobic CO and formate oxidation

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

Georgy A. Zavarzin 1933-2011

Archaean biosphere

 -4.0 - -2.5 billion years  Temperature: +70 - +100 o C  Anaerobic  Reduced

Thermal habitats

Thermophiles on the Tree of Life

Methanogens, sulfur and sulfate reducers

CO 2 CH 4 H 2 SO 4 -2 S o H 2 S

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

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 59 102 o C with the optimum at 83 o C and in pH range 3.5-6.5 with the optimum at 5.2

Sulfate reduction: Vulcanisaeta moutnovskia

1 0,5 -20 0 3 2,5 2 1,5 30 80 Time, hours SO 4 130 H 2 S 2 1 180 0 4 3 6 5

V. moutnovskya

was found to be able to grow be sulfate reduction Substrates are yeast extract, ethanol and glycerol

Sulfate reduction: Vulcanisaeta moutnovskia

97 100 93

Pyrobaculum\Thermoproteus

99 61 100 91 99 100 100 100

dsrA Vmut_0501 Vulcanisaeta moutnovskia 768-28 Vulcanisaeta distributa DSM 14429 Caldivirga maquilingensis IC-167 Chlorobium Magnetococcus marinus MC-1

100

Archaeoglobus

100

Thermodesulfovibrio Desulfosporosinus Desulfitobacterium dichloroeliminans 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

CO 2 CH 4

Disproportionation of sulfur compounds

H 2 SO 4 -2 S o S o S 2 O 3 -2 H 2 O H 2 S SO 4 -2 H 2 S

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

  

4SO

3 2-

S

O

3 2 4

S

+ H

= 3SO

4 2 +

H

O

SO

4 2 + 4

H

O

SO

4 2 + 3

+ HS HS

HS

H

+ + 5

H

3:1 1:1 1:3

Δ G°’= -58.9 kJ mol -1

SO

3 2 Δ

’

= -22.3

kJ mol

-1

S

O

3 2 Δ

’

= +

1

0.3

kJ mol

-1

S

Thermosulfurimonas dismutans

5 4 7 6 3 2 1 0 0 10 20 30 Time ( h ) 40 50 Cells (x10exp7) per ml Sulfide (mM) Isolated from the hydrothermal chimney of Lau Spreading Center, Pacific Ocean, depth 2060 m Growth in the temperatures range from 50 to 92 o C, opt 74 o C Obligate anaerobe Obligate lithoautotroph Needs Fe(III) for H 2 S scavenging (growth up to 10 8 Capable to grow with H 2 reducing thiosulfate cells/ml

Thermosulfurimonas dismutans

New genus in

Thermodesulfobacteria

0.02

58 100 58 100 100 100

Thermodesulfobacterium hveragerdense

JSP T (X96725)

Thermodesulfobacterium thermophilum

DSM 1276 T (AF334601)

Thermodesulfobacterium commune

YSRA-1 T (AF418169)

Thermodesulfobacterium hydrogeniphilum

SL6 T (AF332514)

‘Geothermobacterium ferrireducens’

FW-1a T (AF411013)

Caldimicrobium rimae

DS T (EF554596)

Thermosulfurimonas dismutans S95 T (JF346116)

Thermodesulfatator indicus

CIR29812 T (AF393376) 100

Thermodesulfatator atlanticus

AT1325 T (EU435435)

Thermosulfidibacter takaii

ABI70S6 T (AB282756)

New thermophilic Deltaproteobacteria capable of sulfur disproportionation

Uzon Caldera, Kamchatka 52 29 59 97 100 69 70

‘ Dissulfurimicrobium hydrothermalis’ Sh68 Dissulfuribacter thermophilus S69 T (JQ414031)

100

Desulfobulbaceae Syntrophaceae

100

Desulfobacca acetoxidans

DSM 11109 T (CP002629)

Desulfomonile

100

Syntrophobacteraceae Deferrisoma camini

S3R1 T (JF802205)

Desulfuromonadaceae

100 Lau Spreading Center, Pacific Ocean 0.02

   Genome size – 2.20 Mb Carbon metabolism - autotrophic CO 2  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

CO 2 CH 4 H 2 SO

Anaerobic CO and formate oxidation

H 2 O 4 -2 S o H 2 S CO H 2 S o S 2 O 3 -2 H 2 O H 2 O H 2 S SO 4 -2 CO 2 H 2 HCOOH

Anaerobic CO and formate oxidation

CO + H

O = CO

+ H

250 200 150 100

CO Cells

9 7 5 15 13 11

H 2

3 50 1 0 0 50

Time, hours

100 -1 Growth of

Thermococcus barophilus

Ch5 on CO Tatyana Sokolova Tatyana Kochetkova (Svetlichny et al., 1991) Alexander Lebedinsky Daria Kozhevnikova 100% CO: phylogenetically diverse

Firmicutes

hyperthermophilic archaea of genus

Thermococcus

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

cooRa cooF cooS cooC 1/2 cooM cooK cooU+cooH cooX cooL

Carboxydothermus hydrogenoformans

Thermococcus sp. AM4 T. barophilus MPT and Ch5

T. onnurineus cooRa cooF cooS cooC 1/2 cooM cooU cooH cooY cooL cooK cooX “Thermofilum carboxydotrophus"

Anaerobic CO and formate oxidation

cooA cooC cooM cooK cooL cooX cooU cooH hypA cooF cooS

cooRa cooF cooS cooC 1/2 cooM cooK cooU+cooH cooX cooL

fdh

cooF 1/2 cooM 1/2 cooM 1/2 cooM cooK cooU+cooH cooX cooL

h f-tr cooRa cooF cooS cooC 1/2 cooM cooU cooH cooY cooL cooK cooX Carboxydothermus hydrogenoformans

Thermococcus sp. AM4 T. barophilus MPT and Ch5

T. onnurineus T. onnurineus T. gammatolerans

T. barophilus Ch5

“Thermofilum carboxydotrophus"

Anaerobic CO and formate oxidation

The energy of reaction:

HCOO

+ H

O → HCO

3 -

+ H

ΔG

' = +1.3 kJ/mol

was always considered to be insufficient to support microbial growth

In our experimental conditions ΔG 0 ‘ varied from -8 to -20 kJ/mol

Kim et al., Nature, 2010, 467:352-355

Anaerobic CO and formate oxidation

200 150 100 50 0 0

Cells Formate

H 2

2 1 20 40

Time, hours

60 0

Thermococcus T. barophilus

sp. able to grow on formate producing hydrogen:

T. gammatolerance T. onnurineus

three new isolates from different deep-sea hydrothermal areas

CO 2 CH 4 H 2 SO

Anaerobic CO and formate oxidation

H 2 O 4 -2 S o H 2 S CO H 2 S o S 2 O 3 -2 H 2 O H 2 O H 2 S SO 4 -2 CO 2 H 2 HCOOH

Radioisotopic tracing: detection of new metabolic groups

Uzon Caldera, Kamchatka

In situ

incubation Na 14 CO 3 14 C-acetate 14 C-products Micrograms C l(-1) day(-1) 10000 100 1 0,01 2 3 T, oC Lithotrophic methanogenesis Carbon assimilation Acetoclastic methaogenesis Acetate oxidation Acetogenesis

pH 8.5

Micrograms C l(-1) day(-1) 10000 100 1 0,01 65 70 85 T, oC Lithotrophic methanogenesis Carbon assimilation Acetoclastic methanogenesis Acetate oxidation Acetogenesis

pH 7.0

Micrograms C l(-1) day(-1) 10000 100 1 0,01 60 70 85 T, oC Lithotrophic methanogenesis Carbon assimilation Acetoclastic methanogenesis Acetate oxidation Acetogenesis

pH 3.5

Nikolay Pimenov

Radioisotopic tracing: detection of new metabolic groups

Uzon Caldera, Kamchatka

In situ

incubation Na 14 CO 3 14 C-acetate 14 C-products Micrograms C l(-1) day(-1) 10000 100 1 0,01

2 3 T, oC Lithotrophic methanogenesis Carbon assimilation Acetoclastic methaogenesis Acetate oxidation Acetogenesis

pH 8.5

Micrograms C l(-1) day(-1) 10000 100 1

0,01 65 70 85 T, oC Lithotrophic methanogenesis Carbon assimilation Acetoclastic methanogenesis Acetate oxidation Acetogenesis

pH 7.0

Micrograms C l(-1) day(-1) 10000 100 1

0,01 60 70 85

? ?

T, oC Lithotrophic methanogenesis Carbon assimilation Acetoclastic methanogenesis Acetate oxidation Acetogenesis

pH 3.5

CO 2 CH 4

Acetate

H 2 SO

Anaerobic CO and formate oxidation

H 2 O 4 -2 S o H 2 S CO H 2 S o S 2 O 3 -2 H 2 O H 2 O H 2 S SO 4 -2 CO 2 H 2 HCOOH

Radioisotopic tracing: detection of new metabolic groups

Uzon Caldera, Kamchatka

In situ

incubation Na 14 CO 3 14 C-acetate

Micrograms C l(-1) day(-1) 10000 100 1 0,01 65 70 85 T, oC Lithotrophic methanogenesis Carbon assimilation Acetoclastic methanogenesis Acetate oxidation Acetogenesis

pH 7.0

14 C-products Micrograms C l(-1) day(-1) 10000 100 1 0,01 2 3 T, oC Lithotrophic methanogenesis Carbon assimilation Acetoclastic methaogenesis Acetate oxidation Acetogenesis

pH 8.5

? ? ?

Micrograms C l(-1) day(-1) 10000 100 1 0,01 60 70 85 T, oC Lithotrophic methanogenesis Carbon assimilation Acetoclastic methanogenesis Acetate oxidation Acetogenesis

pH 3.5

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