Anaerober Schadstoffabbau

Organismus des TagesAzoarcus 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: Bakterien

Phylum: Proteobacteria

Klasse Betaproteobacteria

Ordnung Rhodocyclales

Familie Rhodocyclaceae

Gattung Azoarcus

The uncultured majority

• Black: 12 original Phyla (Woese 1987)many pure cultures

• White: 14 new phyla since 1987some isolates

• Gray: 26 candidate phylano 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

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

CO2CO2

Lignin

Aromaten in der Natur

Rohöl, Kohle Aminosäuren

CH2

CH

COOH

H2N

CH2

CH

COOH

H2N

OH

NH

CH2

CH

COOH

H2N

Welche Schadstoffe sind wichtig?

Where has this picture been taken?

Wietze, Lüneburger Heide, around 1900

Natural oil seep in Wietze

Oil production 400 years ago

Oil sand production (1950)height 60 m, 1 Mill m3

Why study biodegradation activities in contaminated aquifers?

A B

- generic processes in the subsurface

- connecting function and structure of communities

- novel biochemistryO2, NO3

-, SO4

2-, Fe(III)

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

COSCoA

COO-

Benzoyl-Succinyl-CoA

Benzoyl-CoA

Succinyl-CoA Succinate 2[H]H2O

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

COSCoA

COO-

Benzoyl-Succinyl-CoA

BenzylsuccinateSynthase

Benzylsuccinate-CoA Transferase

Benzylsuccinyl-CoADehydrogenase

Phenylitaconyl-CoA Hydratase

3-Hydroxyacyl-CoADehydrogenase

Benzoylsuccinyl CoA 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 10

6

6,5

7

7,5

8

8,5

9

0 10 20 30 40 50

6

6,5

7

7,5

8

8,5

9

0 20 40 60

6

6,5

7

7,5

8

8,5

9

0 100 200 300

Uns

atur

ated

zone

Sat

urat

edzo

ne

De

pth

[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 b

ls]

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 fractionation

at plume fringes!

February 2006

▼GW table

plume core

sulfidogenicgradient zone

lowercontaminatedzone

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 table plume core

sulfidogenicgradient zone

lowercontaminatedzone

deep zone

1 2 3 4

5

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

228137

159

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 Birch-Reduktion von Aromaten

Chemie:

e-Donor: Na0

H-donor: X-OH

.-

e - H+, e-, H+

- 3 VH

H

C

S C o AO AO

C

S C o

-.e -

C

S 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 H2O 16 ADP + 16 Pi

2 ATP / e-

Benzoyl-CoA Reduktase

C O S C o AC O S C o A

2 ATP + 2 H2O

2 Fd(red) 2 Fd(ox)

1 ATP / e-

2 ADP + 2 Pi

Energetics of benzoate degradation

Denitrifyer:

C7H6O2 + 6 HNO3 7 CO2 + 6 H2O + 3 N2

G’° = ~ -3000 kJ mol-1

Sulfate Reducer:C7H6O2 + 4 H2O + 3.75 SO4

2- 7 HCO3- + 3.75 HS- + 3.25 H+

G’° = -203 kJ mol-1

Fermenting bacteria:4 C7H5O2 + 18 H2O 12 C2H3O2+ CO2 + 3 CH4 + 8 H+

G’° = -48,5 kJ mol-1

Iron reducer:C7H6O2 + 19 H2O + 30 Fe(III) 7 HCO3

- + 30 Fe(II) + 36 H+

G’° = <-1000 kJ mol-1

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/Carboxylierung,

• Welche zentralen Metabolite? – Benzoat

• Welche Schlüsselreaktionen für den weiteren Abbau nach der Aktivierung?– Beta-Oxidation der Fettsäuren, – Ringreduktion durch Benzoyl-CoA-Reduktase

Andere wichtige Substanzen

• PAKs (Polycyclische Aromatische Kohlenwasserstoffe)– Naphthaline (Abbauwege teilweise beschrieben)– Phenanthren (nur ein Metabolit identifiziert,

Carbonsäure)– Biphenyl (nur ein Metabolit identifiziert, Carbonsäure)

• Benzol (Metabolite identifiziert, Benzoat, Phenol)

Why benzene and naphthalene?

Ecology:-Very recalcitrant in nature -Model system for PAHs (polycyclic aromatic hydrocarbons) degradation

Biochemistry:-The most stable C-H bond known (480 kJ/mol)-No such reaction known in chemistry or biology

Deltaproteobacteria

Clostridia

Betaproteobacteria

Gammaproteobacteria

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

Sul

fide

[m

M]

Time [d]

C O O H

Substrate utilization of culture N47

I. 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

VCOOC H3

COO CH3

m/z

Inte

nsity

50 100 150 200 250 300

252

224

284

165

VIC OOC H3

C O OC 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

Na

ph

tyl-

2-m

eth

yl-s

ucc

inic

aci

d [

µM

]

Time [hours]

+

COOH

COOH

HOOCCOOH

Identification of the proteins involved in

naphthalene- and 2-methylnaphthalene

degradationMr

(kDa) Naph 2MN

50

40

30

20

70

100

150 enzymes in naphthalene- and 2-metylnaphthalene-grown cells?

comparison with the genome sequence of culture N47

Whole Genome Sequencing of culture N47Genome size: 4,7 Mbp

Bergmann et al., Environ. Microbiol. 2011

1 1,000 2,000 3,000 4,000 5,000 bp

nmsB nmsCnmsD nmsA

2-Naphthylmethyl-succinate synthase (NMS) NMS activatingenzyme

CH3

2-Methylnaphthalene

COOH

COOH

2-Naphthylmethyl-succinateFumaratCOOH

HOOC

The nms genes from the genome

(nms= 2-naphthylmethyl succinate synthase)

NmsABC

(α-subunit)(β-subunit) (γ-subunit)

Selesi et al., J. Bacteriol. 2010

10,000 12,000 14,000 16,000 18,000 20,000 bp

bnsGbnsH bnsF bnsE bnsC bnsA

COSCoA

COOH

COSCoA

COOH

COSCoA

COOH

OH

COSCoA

COOH

OCOOH

COOH

Naphthyl-2-methyl-succinate-CoA

transferase

Naphthyl-2-methyl-succinyl-CoA

dehydrogenase

Naphthyl-2-methyl-succinyl-CoA

thiolase

COSCoA

BnsEF BnsG BnsH BnsCD BnsAB

The bns genes from the genome (bns =

beta-oxidation of naphthyl-2-methyl succinate)

Naphthyl-2-methylen-

succinyl-CoAdehydrogenase

Naphthyl-2-hydroxymethyl-succinyl-CoA

hydratase

bnsD bnsB

Selesi et al., J. Bacteriol. 2010

CO-SCoA

COOH

COOH

CH3

2*

3*

+HOOC

COOH

COOH

CO-SCoA

4*

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]

The upper 2-methylnaphthalene

degradation pathway

• Addition of fumarate• β-Oxidation• Central intermediate 2-naphthoic acid• Analogy to toluene degradation

Succinyl-CoA

II. NaphthalenePutative degradation pathways

CO-SCoA

COOH

COOH

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]

Naph 2MNkDa

3 10NLpI

116

66

45

35

25

18

14

3 10NLpI

20 20

3636

22 22

41 41

3030

35 3543 4321

21

2323

2626

25 2529 29

32 32

34 3440 40

4444

Silver-stained 2-DGE gel of proteins from naphthalene- (left) and 2-

methylnaphthalene-grown cells (right)

Bergmann et al., Arch. Microbiol. 2011

Carboxylase-ORFs corresponding to sequenced spots only expressed with naphthalene

Bergmann et al., Arch. Microbiol. 2011

A novel enzyme reaction in (bio)chemistry

measuring naphthalene carboxylase activity

Mouttaki et al., in review

0200400600800

10001200

0 20 40 60 80Time [min]

13C

-Na

ph

tho

ic a

cid

[nM

]

With ATP®No ATP

naphthalene

13COOH

2-naphthoic acid

13HCO-3

Clear dependence on cell extract

0 20 40 60 800

2

4

6

8

10A

cti

vit

y [

pm

ol

min

-1]

Protein amount [µg per assay]

Activity of naphthalene carboxylase determined within the first 10 min as a function of cell extract added.

Strong isotope exchange reaction of the carboxyl group

0

5

10

15

20

25

30

35

40

0 20 40 60

Time [min]

2-N

aph

tho

ic a

cid

[µM

]

12C-2-naphthoic acid (closed symbols), 13C-2-naphthoic acid (open symbols) with () and without () addition of ATP. () indicates the control assay in the absence of cell extract.

CO-SCoA

COOH

COOH

CH3

2*

3*

+ COOH

COOH

CO-SCoA

4*

uccinyl-CoA

COOH

CO-SCoA

COOH

CO-SCoA

6

H2O

OH

2 [H]COOH

CO-SCoA

7 O

HS-CoA

5*

8*

1*

Succinat

COOH

CO2

[CoA]

Succinyl-CoA

2 [H]

CO-SCoACO-SCoA

+ 4 [H]+ 2 [H]

ATPADP + Pi

Activities measured in cell extracts

HOOC

II. Naphthaleneproven degradation pathways

Investigation area

?

N100 meter

Areas with NAPL-phase

wells

Groundwater flow

B 14

B 2 7B 28

B 29

B 42

B 44

B 47

B 4 8

B 49

B 5 3

B 54

B 55B 56

B 57

B85

Does anaerobic naphthalene degradation occur in the field?

S1

S2

Contaminant source

1

5

10

50

100

500

1000

5000

10000

20000

30000

40000

50000

60000

70000

80000

85000

90000

B 14

B 2 7B 28

B 29

B 42

B 44

B 47

B 4 8

B 49

B 5 3

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 2 7B 28

B 29

B 42

B 44

B 47

B 4 8

B 49

B 5 3

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 2 7B 28

B 29

B 42

B 44

B 47

B 4 8

B 49

B 5 3

B 54

B 55B 56

B 57

B85

2-methyl-naphthalene

COOH

COOH

COOH

COOH

[µg l-1]

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

Distribution of metabolites on a contaminated gas work site

1

5

10

50

100

250

500

1000

1500

2000

3000

4000

5000

6000

B 14

B 2 7B 28

B 29

B 42

B 44

B 47

B 4 8

B 49

B 5 3

B 54

B 55B 56

B 57

B85

2-methyl-naphthalene

C O O H

C O O H

COOH

COOH

[µg l-1]

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

Distribution of metabolites on a contaminated gas work site

1

5

10

50

100

250

500

1000

1500

2000

3000

4000

5000

6000

B 14

B 2 7B 28

B 29

B 42

B 44

B 47

B 4 8

B 49

B 5 3

B 54

B 55B 56

B 57

B85

2-methyl-naphthalene

COOH

COOH

C O O H

COOH

[µg l-1]

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

Distribution of metabolites on a contaminated gas work site

1

5

10

50

100

250

500

1000

1500

2000

3000

4000

5000

6000

B 14

B 2 7B 28

B 29

B 42

B 44

B 47

B 4 8

B 49

B 5 3

B 54

B 55B 56

B 57

B85

2-methyl-naphthalene

COOH

COOH

COOH

C O O H

[µg l-1]

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

Distribution of metabolites on a contaminated gas work site