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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
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