Tag 5

Anaerober Schadstoffabbau

Organismus des Tages Azoarcus

tolulyticus

Warum ist Azoarcus

tolulyticus spannend?

•

Kann Toluol und Phenol abbauen•

Wurde aus einem kontaminierten Aquifer

isoliert in Michigan•

Denitrifizierer

sind praktisch alle fakultativ

anaerob und können aerob atmen!

Phylogenie von Azoarcus

tolulyticus

Domäne: BakterienPhylum: ProteobacteriaKlasse BetaproteobacteriaOrdnung RhodocyclalesFamilie RhodocyclaceaeGattung

Azoarcus

The uncultured majority

•

Black: 12 original Phyla (Woese

1987) many pure cultures

•

White: 14 new phyla since 1987 some isolates

•

Gray: 26 candidate phyla no isolates

Rappé

& Giovannoni

(Annu

Rev Microbiol, 2003)

Keller & Zengler

(Nat Rev Microbiol, 2004)

What are they all doing ?

1205

1367

220

1808

91

8

4

9

13

1124

25

n = published species

Anaerobic bacteria using aromatics as sole source of energy and cell carbon

Grampositive

Proteobacteria

Flavobacteria

Cyanobacteria

Aquifex

Green Sulfurbacteria

α

βγ

δ

ε

Green Nonsulfurbacteria

Thermotoga

Archaea Eukarya

RhodopseudomonasMagnetospirillum

Thauera aromaticaAzoarcus

Desulfococcus multivoransGeobacter metallireducensSynthrophobacterales

Desulfotomaculum

(Ferroglobus ?)

Facultative Anaerobes Obligate Anaerobes

Flavonoide

Phenole

Tannine

Lignane

Quinone

Abbau

durch Mikroorganismen

+ O2 - O2

CO2 CO2

Lignin

Aromaten in der Natur

Rohöl, KohleAminosäuren

CH2

CHCOOH

H2N

CH2

CHCOOH

H2N

OH

NH

CH2

CHCOOH

H2N

Welche Schadstoffe sind wichtig?

Prinzipielle Probleme des anaeroben Abbaus von Kohlenwasserstoffen

•

Aktivierung –

es fehlt der reaktive Sauerstoff

•

Resonanzenergie des aromatischen Ringes

•

Neue Chemie nötig

Funktionsweise der Benzylsuccinatsynthase, eine neues Radikalenzym

Benzylsuccinatsynthase

gehört zu einer Familie von Radikalenzymen

Anaerobic catabolism of toluene

C O-COSCoA

COO-

HO

COSCoA

COO-COSCoA

COSCoACOO-

Fumarate

CH3

Toluene

COO-O

Benzyl-succinate

Benzyl-succinyl-CoA

E-Phenylita-conyl-CoA

2-Carboxymethyl-3-Hydroxy-

Phenylpropionyl-CoA

O

COSCoACOO-

Benzoyl-Succinyl-CoA

Benzoyl-CoA

Succinyl-CoA Succinate 2[H]H2

O

2[H]CoASHSuccinyl-CoA

1

Anaerobic catabolism of toluene

C O-COSCoA

COO-COSCoA

COO-CH3

Toluene

COO-O

Benzyl-succinate

Benzyl-succinyl-CoA

E-Phenylita-conyl-CoA

2-Carboxymethyl-3-Hydroxy-

Phenylpropionyl-CoA

HO

COSCoA

COO-ACOSCo

Benzoyl-CoA

2

O

COSCoACOO-

Benzoyl-Succinyl-CoA

BenzylsuccinateSynthase

Benzylsuccinate-CoA Transferase

Benzylsuccinyl-CoADehydrogenase

Phenylitaconyl-CoA Hydratase

3-Hydroxyacyl-CoADehydrogenase

BenzoylsuccinylCoA Thiolase

Construction of the

multi-level

well

hochauflösendes Modul

4 Module vorgefertigt

Kabel- und Kapillarstränge

Bereit zur Abfahrt

Installation of a high resolution

multi-level well in Düsseldorf-Flingern

6

6,5

7

7,5

8

8,5

9

-5 0 5 106

6,5

7

7,5

8

8,5

9

0 10 20 30 40 506

6,5

7

7,5

8

8,5

9

0 20 40 606

6,5

7

7,5

8

8,5

9

0 100 200 300Uns

atur

ated

zone

Satu

rate

dzo

ne

Dep

th[m

bls

]

Sulfate + Toluene Sulfide [mg l-1] δ18O / δ34S [‰]

δ18O

δ34S

Sulfate Isotope Analysis

1)

The

plume

fringe

concept

holds!

2)

Steep

geochemical

gradients

at the

fringes

3)

Biodegradation and sulfate

reduction

take

place

in the

sulfidogenic

zone

of overlapping

gradients

of

toluene

and sulfate

Tolueneδ

13C Toluene

0 5 10 15 20 25 30 35 40 45 506

6,5

7

7,5

8

8,5

Dep

th[m

bls

]

Toluene

[mg l-1]

-25,0-24,5-24,0-23,5-23,0-22,5-22,0-21,5-21,0-20,5

δ

13C [‰]

-21.8 ‰ (7.1 m)

Toluene

Isotope Analysis

-24.5 ‰

(6.9 m)

Δ13C = -3.2 ‰

±

0.5

Significant

fractionationat plume

fringes!

February

2006

▼GW tableplume

core

sulfidogenic

gradient

zone

lower

contaminated

zone

deep

zone

103 105 107 109

5

6

7

8

9

10

11

12

13

0.0 0.5 1.0 1.5

Bacterial

16S rRNA genes [cp

g-1]

F1 cluster

bssA genes [cp

g-1]

Ratio bssA/16S rRNA genes

Dep

th[m

]• Highly specialized

degrader community in sulfidogenic zone

• Distribution correlates to different zones

• Biomass does not reflect specific degraders

[Winderl et al., in prep.]

Quantitative distribution

of bacterial

16S rRNA and bssA genes

150 300 450 600 750 900

▼GW tableplume

coresulfidogenic

gradient

zone

lower

contaminated

zone

deep

zone

1 2 3 45

6

7

8

9

10

11

12

13

Shannon index

(H‘)

Dep

th[m

]

A B

* 6.3 m

6.65 m

7.2 m

* 7.6 m

8.7 m

9.8 m

* 11.7 m

* 6.8 m

T-RF length

(bp)

130

228137159

228159

130 149

177

Depth-resolved bacterial community shifts

[Winderl et al., in prep.]

Sulfidogenic zone:

• 130

• 137

• 149

• 159

• 177

• 228 bp T-RFs

* = cloned

Ethylbenzol- abbau durch Denitrifizierer

Sulfatreduzierer

und Ethylbenzol

•

Nutzen auch den Angriff durch Fumarat wie bei Toluol

Anaerober Phenolabbau

Die anaerobe Ringöffnung

Frage!

•

Wie würden Sie Crotonyl-CoA

weiter abbauen?

Frage!

•

Wie würden Sie Crotonyl-CoA

weiter abbauen?

•

Antwort: Beta-Oxidation der Fettsäuren–

Hydratisierung

zum Alkohol

–

Dehydrogenase

zum Keton–

Spaltung mit HS-CoA

zu zwei Acetyl-CoA

Der Benzolring: Resonanzstabilisierung

Die Schlüsselenzyme:

ΔG<<0

Monooxygenasen

C H 3 C H 2 O H

O2

+2 [H]

+ H2

O

DioxygenasenOH

OHC O O H

C O O HO H

O H

O2 O2

ΔG<<0

+2 [H] -2 [H]

Aromatenstoffwechsel von Aerobiern

Anaerobe Aromatenabbauer

Grampositive

Proteobacteria

Flavobacteria

Cyanobacteria

Aquifex

Green Sulfurbacteria

α

βγ

δ

ε

Green Nonsulfurbacteria

Thermotoga

Archaea Eukarya

PhototropheNitrat-Atmer

Nitrat-atmer

Sulfat-AtmerEisen AtmerFermentierer

Sulfat Atmer

Thauera aromatica

Fakultative Anaerobier Obligate Anaerobier

Anaerober Aromatenstoffwechsel: zentrale Rolle von Benzoyl-CoA

CO-SCoA

Benzoyl-CoA

Phenylalanin

Phenylacetat

Phenol

Ethylbenzol

Anthranilsäure

Benzoate

Toluol

Naphtalin

Xylole

Cresole

Salicylsäure

ReduktiveDearomatisierung

Ringöffnung

β-oxidation

Acetyl-CoA

CO2

?

e-

NO3 -

N2

SO42-

H2 S Fe(II)

Fe(III)

ATP ATP ATP

Die Birch-Reduktion von Aromaten

Chemie:

e-Donor: Na0

H-donor: X-OH

.-

e - H+, e-, H+

- 3 VH

H

CS C o AO AO

CS C o

-.e -

CS C o AO

H

H

Benzoyl-CoA Reduktase:

e-Donor: Ferredoxin (ATP)

H-donor: ?- 1.9 V

H+, e-, H+

Benzoyl-CoA Reduktase aus Thauera aromatica

2 NH3 + H2

Nitrogenase

8 H+, 8 e-

N N

16 ATP + 16 H2 O 16 ADP + 16 Pi

2 ATP / e-

Benzoyl-CoA Reduktase

C O S C oAC O S C o A2 ATP + 2 H2 O

2 Fd(red) 2 Fd(ox)

1 ATP / e-

2 ADP + 2 Pi

Energetics of benzoate degradation

Denitrifyer:C7

H6

O2

+ 6 HNO3 7 CO2 + 6 H2O + 3 N2ΔG’° = ~ -3000 kJ mol-1

Sulfate Reducer:C7

H6

O2

+ 4 H2

O + 3.75 SO42- 7 HCO3

- + 3.75 HS- + 3.25 H+

ΔG’° = -203 kJ mol-1

Fermenting bacteria:4 C7

H5

O2

+ 18 H2

O 12 C2H3O2+ CO2 + 3 CH4 + 8 H+

ΔG’° = -48,5 kJ mol-1

Iron reducer:C7

H6

O2

+ 19 H2

O + 30 Fe(III) 7 HCO3- + 30 Fe(II) + 36 H+

ΔG’° = <-1000 kJ mol-1

Central phloroglucinol/resorcinol pathways of anaerobic aromate degradation

OH O

O

COOH

O

OH

COOH

O O

OO

CO-SAoC

OH

OHOH H2

O

NADP+

NADPH

Acetyl-CoA

Acetate

2 CoASH

3 Acetyl-CoA

Phloroglucinol

O

COOH

O

O O

COOH

OH

OH

H2

O

2

Central phloroglucinol/resorcinol pathways of anaerobic aromate degradation

Resorcinol

H2

O2 [H]

2 Acetyl-CoA +1/2 Butyryl-CoA

3 Acetyl-CoA

Frage!

•

Welche Aktivierungsreaktionen für Kohlenwasserstoffe haben sie bis jetzt gelernt?

•

Welche Zentralen Metabolite?•

Welche Schlüsselreaktionen für den weiteren Abbau nach der Aktivierung?

Frage!

•

Welche Aktivierungsreaktionen für Kohlenwasserstoffe haben sie bis jetzt gelernt? –

Fumarataddition

radikalisch, direkte Oxidation,

Phosphorylierung, direkte Spaltung mit HS-CoA•

Welche zentralen Metabolite? –

Benzoat, Phloroplucinol, Resorcinol

•

Welche Schlüsselreaktionen für den weiteren Abbau nach der Aktivierung?–

Beta-Oxidation der Fettsäuren,

–

Ringreduktion durch Benzoyl-CoA-Reduktase

Anaerober Abbau von Naphthalinen

Meckenstock et al. (2000) Appl. Environ. Microbiol. 66, 2743-2747.

0 20 40 60 80 1000.0

0.5

1.0

1.5

2.0

2.5

Sulfi

de [m

M]

Time [d]

C O O H

Metabolites

in anaerobic

2- methylnaphthalene degradation

Annweiler et al. (2000) Appl. Environ. Microbiol. 66, 5329-5333.

50 100 150 200 250 300

115

141

167

195

226

286

VC OOC H3

C OOC H3

m/z

Inte

nsity

50 100 150 200 250 300

252

224

284

165

VIC OOC H3

C OOC H3

m/z

Inte

nsity

The

naphthylmethylsuccinate

synthase reaction

Annweiler et al. (2000) Appl. Environ. Microbiol. 66, 5329-5333.

0 1 2 3 4 5 6

0.0

0.2

0.4

0.6

0.8

Nap

htyl

-2-m

ethy

l-suc

cini

c ac

id [µ

M]

Time [hours]

+

COOH

COOH

HOOCCOOH

Activation

of naphthylmethylsuccinate

with co-enzyme

A

Safinowski and Meckenstock FEMS Microbiol. Lett. 2004

Succinyl-CoA

Succinate

COOH

CO-SCoA

COOH

COOH

0 10 20 30 400

100

200

300

400 Succinyl-CoA NMS-CoA NMS

Con

cent

ratio

n [µ

M]

Time [min]

β-Oxidation of naphthylmethylsuccinyl-CoA

2[H]

COOH

CO-SCoA

COOH

CO-SCoA

0 10 20 30 400.0

0.1

0.2

0.3

0.4

0.5

0.6

NMeS

NM

S-co

ncen

tratio

n [µ

M]

Time [min]

Safinowski and Meckenstock FEMS Microbiol. Lett. 2004

The

upper

2-methylnaphthaline degradation pathway

•

Addition of fumarate

•

β-Oxidation•

Central intermediate

2-

naphthoic acid

+1*2*

3*

4*

5*

Succinyl-CoA

2[H]

HS-CoA

H2O

2[H]

6

7

Succinyl-CoA

Succinate

8*CO-SCoA

COOH

CO-SCoA

COOH

CO-SCoA

COOH

COOH

HOOCCOOH

OH

COOH

CO-SCoAO

COOH

CO-SCoA

Safinowski and Meckenstock FEMS Microbiol. Lett. 2004

COOH

COOH

COOH

COOH

COOH

COOH

HOOCCOOH

+

COOHO

COOHOH

COOH

COOHor

COOH

COOH

CO2

Upper degradation pathways

to 2-

naphthoic acid Reduction

of 2- naphthoic acid

Ring cleavage

and degradation

via

cyclohexane

ring structures

CH3

How

is

naphthalene

activated?

Methylation

of an aromatic

hydrocarbon?

DD

DDDD

D

DD

D

DD D

D

DD

D

DD D

D

DCOOH

COOH

CH3

CH3

D

HOOCCOOH

Deuterated

naphthalene

as substrate

for

culture

N47

Product

should

have

the

mass of naphthyl-2-methylsuccinate

plus 7 mass

units

Postulated degradation

pathway

for

anaerobic naphthalene degradation

CO-SCoA

COOHCOOH

CH3

2*

3*

+HOOC COOH

COOH

CO-SCoA

4*

Succinyl-CoA

COOH

CO-SCoA

COOH

CO-SCoA

6

H2O

OH

2 [H]COOH

CO-SCoA

7 O

HS-CoA

5*

8*

1*

Succinat

COO-

?

?

CO2

[CH3]

[CoA] ?

9

10*

Succinyl-CoA

2 [H]

How

to assess

the

degradation

activity

in the environment

A B

-Adsorption

-Dilution/dispersion

-Microbial degradation

COOH

COOH

COOH

COOH

COOH

COOH

HOOCCOOH

+

COOHO

COOHOH

COOH

COOHor

COOH

COOH

Upper degradation pathways

to 2-

naphthoic acid Reduction

of 2- naphthoic acid

Ring cleavage

and degradation

via

cyclohexane

ring structures

CH3

Detection

of anaerobic

naphthalene degradation

in the

environment

Investigation area

?

N100 meter

Areas with NAPL-phase

wells

Groundwater flow

B 14

B 27B 28

B 29

B 42

B 44

B 47

B 48

B 49

B 53

B 54

B 55B 56

B 57

B85

A former

coal

gasification

site

near

Stuttgart, Germany

S1

S2

Contaminant source

1510501005001000500010000200003000040000500006000070000800008500090000

B 14

B 27B 28

B 29

B 42

B 44

B 47

B 48

B 49

B 53

B 54

B 55B 56

B 57

B85

Distribution of metabolites

on a contaminated gas work

site

naphthalene

1

5

10

50

100

250

500

1000

1500

2000

3000

4000

5000

6000

B 14

B 27B 28

B 29

B 42

B 44

B 47

B 48

B 49

B 53

B 54

B 55B 56

B 57

B85

2-methyl- naphthalene

S1

S2

[µg l-1] [µg l-1]

Griebler et al., Environ. Sci. Technol. 2004

1

5

10

50

100

250

500

1000

1500

2000

3000

4000

5000

6000

B 14

B 27B 28

B 29

B 42

B 44

B 47

B 48

B 49

B 53

B 54

B 55B 56

B 57

B85

Distribution of metabolites

on a contaminated gas work

site

2-methyl-naphthalene

COOH

COOH

COOH

COOH

[µg l-1]

Griebler et al., Environ. Sci. Technol. 2004

1

5

10

50

100

250

500

1000

1500

2000

3000

4000

5000

6000

B 14

B 27B 28

B 29

B 42

B 44

B 47

B 48

B 49

B 53

B 54

B 55B 56

B 57

B85

Distribution of metabolites

on a contaminated gas work

site

2-methyl-naphthalene

C O O H

C O O H

COOH

COOH

[µg l-1]

Griebler et al., Environ. Sci. Technol. 2004

1

5

10

50

100

250

500

1000

1500

2000

3000

4000

5000

6000

B 14

B 27B 28

B 29

B 42

B 44

B 47

B 48

B 49

B 53

B 54

B 55B 56

B 57

B85

Distribution of metabolites

on a contaminated gas work

site

2-methyl-naphthalene

COOH

COOH

C O O H

COOH

[µg l-1]

Griebler et al., Environ. Sci. Technol. 2004

1

5

10

50

100

250

500

1000

1500

2000

3000

4000

5000

6000

B 14

B 27B 28

B 29

B 42

B 44

B 47

B 48

B 49

B 53

B 54

B 55B 56

B 57

B85

Distribution of metabolites

on a contaminated gas work

site

2-methyl-naphthalene

COOH

COOH

COOH

C O O H

[µg l-1]

Griebler et al., Environ. Sci. Technol. 2004

Andere wichtige Substanzen

•

PAKs

(Polycyclische

Aromatische Kohlenwasserstoffe)–

Naphtaline

(Abbauwege teilweise beschrieben)

–

Phenanthren

(nur ein Metabolit

identifiziert, Carbonsäure)

–

Biphenyl

(nur ein Metabolit

identifiziert, Carbonsäure)

•

Benzol (Metabolite

identifiziert, Benzoat, Phenol)