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Anaerober Schadstoffabbau. Organismus des Tages Azoarcus tolulyticus.

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Präsentation zum Thema: "Anaerober Schadstoffabbau. Organismus des Tages Azoarcus tolulyticus."—  Präsentation transkript:

1 Anaerober Schadstoffabbau

2 Organismus des Tages Azoarcus tolulyticus

3 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!

4 Phylogenie von Azoarcus tolulyticus Domäne: Bakterien Phylum: Proteobacteria Klasse Betaproteobacteria Ordnung Rhodocyclales Familie Rhodocyclaceae Gattung Azoarcus

5 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 ? n = published species

6 Anaerobic bacteria using aromatics as sole source of energy and cell carbon Grampositive Proteobacteria Flavobacteria Cyanobacteria Aquifex Green Sulfurbacteria      Green Nonsulfurbacteria Thermotoga Archaea Eukarya Rhodopseudomonas Magnetospirillum Thauera aromatica Azoarcus Desulfococcus multivorans Geobacter metallireducens Synthrophobacterales Desulfotomaculum (Ferroglobus ?) Facultative Anaerobes Obligate Anaerobes

7

8 Flavonoide Phenole Tannine Lignane Quinone Abbau durch Mikroorganismen + O 2 - O 2 CO 2 Lignin Aromaten in der Natur Rohöl, Kohle Aminosäuren

9 Welche Schadstoffe sind wichtig?

10 Where has this picture been taken?

11 Wietze, Lüneburger Heide, around 1900

12 Natural oil seep in Wietze

13 Oil production 400 years ago

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

15 Why study biodegradation activities in contaminated aquifers? - generic processes in the subsurface - connecting function and structure of communities - novel biochemistry O 2, NO 3 -, SO 4 2-, Fe(III)

16 Prinzipielle Probleme des anaeroben Abbaus von Kohlenwasserstoffen Aktivierung – es fehlt der reaktive Sauerstoff Resonanzenergie des aromatischen Ringes Neue Chemie nötig

17 Funktionsweise der Benzylsuccinatsynthase, eine neues Radikalenzym

18 Benzylsuccinatsynthase gehört zu einer Familie von Radikalenzymen

19 Anaerobic catabolism of toluene CO- COSCoA COO- HO COSCoA COO- COSCoA COSCoA COO- Fumarate CH 3 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] H2OH2O CoASHSuccinyl-CoA 1

20 Anaerobic catabolism of toluene CO- COSCoA COO- COSCoA COO- CH 3 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 Benzylsuccinate Synthase Benzylsuccinate- CoA Transferase Benzylsuccinyl-CoA Dehydrogenase Phenylitaconyl- CoA Hydratase 3-Hydroxyacyl-CoA Dehydrogenase Benzoylsuccinyl CoA Thiolase

21

22 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

23 Unsaturated zone Saturated zone Depth [m bls] Sulfate + Toluene Sulfide [mg l -1 ] δ 18 O / δ 34 S [‰] δ 18 O δ 34 S 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

24 Tolueneδ 13 C Toluene ‰ (7.1 m) Toluene Isotope Analysis ‰ (6.9 m) Δ 13 C = -3.2 ‰  0.5 Significant fractionation at plume fringes! February 2006

25 ▼GW table plume core sulfidogenic gradient zone lower contaminated zone deep zone Bacterial 16S rRNA genes [cp g -1 ] F1 cluster bssA genes [cp g -1 ] Ratio bssA/16S rRNA genes Depth [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

26 ▼GW table plume core sulfidogenic gradient zone lower contaminated zone deep zone Shannon index (H‘) Depth [m] AB * 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) Depth-resolved bacterial community shifts [Winderl et al., in prep.] Sulfidogenic zone: bp T-RFs * = cloned

27 Ethylbenzol- abbau durch Denitrifizierer

28 Sulfatreduzierer und Ethylbenzol Nutzen auch den Angriff durch Fumarat wie bei Toluol

29 Anaerober Phenolabbau

30

31 Die anaerobe Ringöffnung

32 Frage! Wie würden Sie Crotonyl-CoA weiter abbauen?

33 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

34 Der Benzolring: Resonanzstabilisierung

35 Die Birch-Reduktion von Aromaten Chemie: e-Donor: Na 0 H-donor: X-OH. - e - H +, e -, H V H H C SCoAO AO C SCo -. e - C SCoAO H H Benzoyl-CoA Reduktase: e-Donor: Ferredoxin (ATP) H-donor: ? V H +, e -, H +

36 Benzoyl-CoA Reduktase aus Thauera aromatica 2 NH 3 + H 2 Nitrogenase 8 H+, 8 e- N 16 ATP + 16 H 2 O 16 ADP + 16 P i 2 ATP / e - Benzoyl-CoA Reduktase COSCoA COSCoA 2 ATP + 2 H 2 O 2 Fd(red) 2 Fd(ox) 1 ATP / e - 2 ADP + 2 P i

37 Energetics of benzoate degradation Denitrifyer: C 7 H 6 O HNO 3  7 CO H 2 O + 3 N 2  G’° = ~ kJ mol -1 Sulfate Reducer: C 7 H 6 O H 2 O SO 4 2-  7 HCO HS H +  G’° = -203 kJ mol -1 Fermenting bacteria: 4 C 7 H 5 O H 2 O  12 C 2 H 3 O 2 + CO CH H +  G’° = -48,5 kJ mol -1 Iron reducer: C 7 H 6 O H 2 O + 30 Fe(III)  7 HCO Fe(II) + 36 H +  G’° = <-1000 kJ mol -1

38 Frage! Welche Aktivierungsreaktionen für Kohlenwasserstoffe haben sie bis jetzt gelernt? Welche Zentralen Metabolite? Welche Schlüsselreaktionen für den weiteren Abbau nach der Aktivierung?

39 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

40 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)

41 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

42 Deltaproteobacteria Clostridia Betaproteobacteria Gammaproteobacteria

43 Meckenstock et al. (2000) Appl. Environ. Microbiol. 66, Substrate utilization of culture N47

44 I. 2-Methylnaphthalene degradation Annweiler et al. (2000) Appl. Environ. Microbiol. 66, V COOCH 3 COOCH 3 m/z Intensity VI COOCH 3 COOCH 3 m/z Intensity

45 The naphthylmethylsuccinate synthase reaction Annweiler et al. (2000) Appl. Environ. Microbiol. 66, COOH COOH HOOC COOH

46 Identification of the proteins involved in naphthalene- and 2-methylnaphthalene degradation M r (kDa) Naph 2MN enzymes in naphthalene- and 2- metylnaphthalene-grown cells?  comparison with the genome sequence of culture N47 Whole Genome Sequencing of culture N47 Genome size: 4,7 Mbp Bergmann et al., Environ. Microbiol. 2011

47 1 1,0002,0003,0004,0005,000 bp nmsBnmsC nmsDnmsA 2-Naphthylmethyl-succinate synthase (NMS) NMS activating enzyme CH 3 2-Methylnaphthalene COOH 2-Naphthylmethyl-succinate Fumarat COOH 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

48 10,000 12,00014,00016,00018,00020,000 bp bnsG bnsH bnsF bnsE bnsC bnsA COSCoA COOH COSCoA COOH COSCoA COOH OH COSCoA COOH O Naphthyl-2-methyl- succinate-CoA transferase Naphthyl-2-methyl- succinyl-CoA dehydrogenase Naphthyl-2-methyl- succinyl-CoA thiolase COSCoA BnsEFBnsG BnsH BnsCD BnsAB The bns genes from the genome (bns = beta-oxidation of naphthyl-2-methyl succinate) Naphthyl-2- methylen- succinyl-CoA dehydrogenase Naphthyl-2- hydroxymethyl- succinyl-CoA hydratase bnsD bnsB Selesi et al., J. Bacteriol. 2010

49 The upper 2- methylnaphthalene degradation pathway Addition of fumarate β-Oxidation Central intermediate 2-naphthoic acid Analogy to toluene degradation Succinyl-CoA

50 II. Naphthalene Putative degradation pathways CO-SCoA COOH COOH CH 3 2* 3* + HOOC COOH COOH CO-SCoA 4 * Succinyl-CoA COOH CO-SCoA COOH CO-SCoA 6 H 2 O OH 2 [H] COOH CO-SCoA 7 O HS-CoA 5* 8* 1* Succinat COO - ? ? CO 2 [CH 3 ] [CoA] 9 10 * Succinyl-CoA 2 [H]

51 Naph 2MN kDa 3 10NL pI NL pI Silver-stained 2-DGE gel of proteins from naphthalene- (left) and 2- methylnaphthalene-grown cells (right) Bergmann et al., Arch. Microbiol. 2011

52 Tab1 ORFs of N47 identified by MS/MS sequencing of selected overexpressed proteins of 2-DGE (figure 2). The up-regulation on naphthalene compared to 2-methylnaphthalene is given in the regulation factor column and the best enzyme match and corresponding function was retrieved from PEDANT database (Walter et al. 2009) Carboxylase-ORFs corresponding to sequenced spots only expressed with naphthalene Bergmann et al., Arch. Microbiol. 2011

53 A novel enzyme reaction in (bio)chemistry measuring naphthalene carboxylase activity Mouttaki et al., in review   With ATP ®  No ATP naphthalene 13 COOH 2-naphthoic acid 13 HCO - 3

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

55 Strong isotope exchange reaction of the carboxyl group 12 C-2-naphthoic acid (closed symbols), 13 C-2-naphthoic acid (open symbols) with (  ) and without (  ) addition of ATP. (  ) indicates the control assay in the absence of cell extract.

56 CO-SCoA COOH COOH CH 3 2* 3* + COOH COOH CO-SCoA 4 * uccinyl-CoA COOH CO-SCoA COOH CO-SCoA 6 H 2 O OH 2 [H] COOH CO-SCoA 7 O HS-CoA 5* 8* 1* Succinat COOH CO 2 [CoA] Succinyl-CoA 2 [H] CO-SCoACO-SCoA + 4 [H]+ 2 [H] ATPADP + Pi Activities measured in cell extracts HOOC II. Naphthalene proven degradation pathways

57 Investigation area ? N 100 meter Areas with NAPL- phase wells Groundwater flow Does anaerobic naphthalene degradation occur in the field? S1 S2 Contaminant source

58 Distribution of metabolites on a contaminated gas work site naphthalene 2-methyl- naphthalene S1 S2 [µg l -1 ] Griebler et al., Environ. Sci. Technol. 2004

59 2-methyl-naphthalene [µg l -1 ] Griebler et al., Environ. Sci. Technol Distribution of metabolites on a contaminated gas work site

60 2-methyl-naphthalene COOH COOH [µg l -1 ] Griebler et al., Environ. Sci. Technol Distribution of metabolites on a contaminated gas work site

61 2-methyl-naphthalene COOH [µg l -1 ] Griebler et al., Environ. Sci. Technol Distribution of metabolites on a contaminated gas work site

62 2-methyl-naphthalene COOH [µg l -1 ] Griebler et al., Environ. Sci. Technol Distribution of metabolites on a contaminated gas work site


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