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Anaerober Schadstoffabbau

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!.

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Anaerober Schadstoffabbau

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  1. Anaerober Schadstoffabbau

  2. Organismus des TagesAzoarcus 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 13 9 1205 4 n = published species 1367 220 • Black: 12 original Phyla (Woese 1987)many pure cultures • White: 14 new phyla since 1987some isolates • Gray: 26 candidate phylano isolates 8 1808 91 • What are they all doing ? 11 24 25 Rappé & Giovannoni (Annu Rev Microbiol, 2003)Keller & Zengler (Nat Rev Microbiol, 2004)

  6. Anaerobic bacteria using aromatics as sole source of energy and cell carbon Facultative Anaerobes Obligate Anaerobes Desulfococcus multivorans Geobacter metallireducens Thauera aromatica Azoarcus Synthrophobacterales g d b e Rhodopseudomonas Magnetospirillum a Proteobacteria Desulfotomaculum Grampositive Cyanobacteria Flavobacteria (Ferroglobus ?) Eukarya Archaea Green Sulfurbacteria Green Nonsulfurbacteria Thermotoga Aquifex

  7. Rohöl, Kohle Aminosäuren Abbau durch Mikroorganismen - O2 + O2 CO2 CO2 Aromaten in der Natur Lignin Flavonoide Phenole Tannine Lignane Quinone

  8. Welche Schadstoffe sind wichtig?

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

  10. Funktionsweise der Benzylsuccinatsynthase, eine neues Radikalenzym

  11. Benzylsuccinatsynthase gehört zu einer Familie von Radikalenzymen

  12. C O S C o A COO- C O S C o A C O O - O C O O - C H 3 Benzyl- succinate Toluene Anaerobic catabolism of toluene C O - Benzyl- succinyl-CoA E-Phenylita- conyl-CoA 2[H] Succinyl-CoA Succinate Fumarate H2O C O S C o A C O S C o A C O O - C O O - O H O C O S C o A Benzoyl-CoA Benzoyl- Succinyl-CoA 2-Carboxymethyl- 3-Hydroxy- Phenylpropionyl-CoA 1 2[H] Succinyl-CoA CoASH

  13. COO- C O S C o A O C O O - C H 3 Benzyl- succinate Toluene C O S C o Benzoyl-CoA Anaerobic catabolism of toluene C O S C o A C O O - C O - Benzylsuccinate Synthase Benzylsuccinyl-CoA Dehydrogenase Benzylsuccinate- CoA Transferase Benzyl- succinyl-CoA E-Phenylita- conyl-CoA Phenylitaconyl- CoA Hydratase C O S C o A C O S C o A C O O - C O O - H O O A 3-Hydroxyacyl-CoA Dehydrogenase Benzoylsuccinyl CoA Thiolase Benzoyl- Succinyl-CoA 2-Carboxymethyl- 3-Hydroxy- Phenylpropionyl-CoA 2

  14. Installation of a high resolution multi-level well in Düsseldorf-Flingern Construction of the multi-level well Kabel- und Kapillarstränge hochauflösendes Modul 4 Module vorgefertigt Bereit zur Abfahrt

  15. δ18O δ34S Sulfate Isotope Analysis Unsaturated zone Sulfide [mg l-1] Sulfate + Toluene δ18O / δ34S [‰] Saturated zone Depth [m bls] • The plume fringe concept holds! • Steep geochemical gradients at the fringes • Biodegradation and sulfate reduction take place in the sulfidogenic zone of overlapping gradients of toluene and sulfate

  16. Toluene [mg l-1] 10 15 20 25 30 35 40 45 50 0 5 6 6,5 7 Depth [m bls] 7,5 Toluene 8 δ13C Toluene 8,5 -25,0 -24,5 -24,0 -23,5 -23,0 -22,5 -22,0 -21,5 -21,0 -20,5 δ13C [‰] Toluene Isotope Analysis February 2006 -24.5 ‰ (6.9 m) -21.8 ‰ (7.1 m) Δ13C = -3.2 ‰ 0.5 Significant fractionation at plume fringes!

  17. 103 105 107 109 5 Bacterial 16S rRNA genes [cp g-1] F1 cluster bssA genes [cp g-1] 6 ▼GW table plume core sulfidogenicgradient zone 7 8 lowercontaminatedzone 9 Depth [m] 10 11 deep zone 12 13 0.0 0.5 1.0 1.5 Ratio bssA/16S rRNA genes Quantitative distribution of bacterial 16S rRNA and bssA genes • Highly specialized degrader community in sulfidogenic zone • Distribution correlates to different zones • Biomass does not reflect specific degraders [Winderl et al., in prep.]

  18. Depth-resolved bacterial community shifts A Shannon index (H‘) B T-RF length (bp) 1 2 3 4 5 150 300 450 600 750 900 * 6.3 m • Sulfidogenic zone: • 130 • 137 • 149 • 159 • 177 • 228 bp T-RFs 6 ▼GW table 6.65 m plume core sulfidogenicgradient zone 7 130 149 * 6.8 m 228 159 177 8 130 7.2 m 159 228 137 lowercontaminatedzone Depth [m] 9 * 7.6 m 10 8.7 m 11 9.8 m deep zone 12 * 11.7 m 13 * = cloned [Winderl et al., in prep.]

  19. Ethylbenzol-abbau durch Denitrifizierer

  20. Sulfatreduzierer und Ethylbenzol • Nutzen auch den Angriff durch Fumarat wie bei Toluol

  21. Anaerober Phenolabbau

  22. Die anaerobe Ringöffnung

  23. Frage! • Wie würden Sie Crotonyl-CoA weiter abbauen?

  24. 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

  25. Der Benzolring: Resonanzstabilisierung

  26. O2 + H2O Monooxygenasen +2 [H] C H C H O H 3 2 DG<<0 O2 O2 OH Dioxygenasen O H C O O H -2 [H] +2 [H] OH C O O H O H DG<<0 Aromatenstoffwechsel von Aerobiern Die Schlüsselenzyme:

  27. . H+, e-, H+ - O S C o A O S C o A C C - . H+, e-, H+ Die Birch-Reduktion von Aromaten H Chemie: e-Donor: Na0 H-donor: X-OH - e - 3 V H O S C o A C Benzoyl-CoA Reduktase: e-Donor: Ferredoxin (ATP) H-donor: ? - e - 1.9 V H H

  28. Benzoyl-CoA Reduktase 2 ATP + 2 H2O 2 ADP + 2 Pi C O S C o A C O S C o A 1 ATP / e- 2 Fd(red) 2 Fd(ox) Benzoyl-CoA Reduktase aus Thauera aromatica Nitrogenase + 16 H2O 16 ATP + 16 Pi 16 ADP 2 NH3 + H2 N N 2 ATP / e- 8 H+, 8 e-

  29. Iron reducer: C7H6O2 + 19 H2O + 30 Fe(III)  7 HCO3- + 30 Fe(II) + 36 H+ DG’° = <-1000 kJ mol-1 Sulfate Reducer: C7H6O2 + 4 H2O + 3.75 SO42- 7 HCO3- + 3.75 HS- + 3.25 H+ DG’° = -203 kJ mol-1 Fermenting bacteria: 4 C7H5O2 + 18 H2O 12 C2H3O2+ CO2 + 3 CH4 + 8 H+ DG’° = -48,5 kJ mol-1 Energetics of benzoate degradation Denitrifyer: C7H6O2 + 6 HNO3 7 CO2 + 6 H2O + 3 N2 DG’° = ~ -3000 kJ mol-1

  30. H O O H O H O H O O H O H2O C O O H Acetyl-CoA O Acetate O O H O O C O O C o A S - O C Central phloroglucinol/resorcinol pathways of anaerobic aromate degradation NADP+ Phloroglucinol NADPH 2 CoASH 3 Acetyl-CoA

  31. O H Central phloroglucinol/resorcinol pathways of anaerobic aromate degradation O H O 2 [H] H2O C O O H O O H2O C O O H O Resorcinol 3 Acetyl-CoA 2 2 Acetyl-CoA +1/2 Butyryl-CoA

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

  33. 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

  34. 2.5 2.0 1.5 Sulfide [mM] 1.0 C O O H 0.5 0.0 0 20 40 60 80 100 Time [d] Anaerober Abbau von Naphthalinen Meckenstock et al. (2000) Appl. Environ. Microbiol. 66, 2743-2747.

  35. C O O C H 3 V C O O C H 3 141 167 226 115 286 195 Intensity 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 m/z C O O C H 3 VI C O O C H 3 165 284 224 Intensity 252 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 m/z Metabolites in anaerobic 2-methylnaphthalene degradation Annweiler et al. (2000) Appl. Environ. Microbiol. 66, 5329-5333.

  36. H O O C + C O O H C O O H C O O H The naphthylmethylsuccinate synthase reaction Annweiler et al. (2000) Appl. Environ. Microbiol. 66, 5329-5333.

  37. Succinyl-CoA C O O H Succinate C O O H C O - S C o A C O O H Activation of naphthylmethylsuccinate with co-enzyme A Safinowski and Meckenstock FEMS Microbiol. Lett. 2004

  38. 2[H] C O - S C o A C O O H C O - S C o A C O O H β-Oxidation of naphthylmethylsuccinyl-CoA Safinowski and Meckenstock FEMS Microbiol. Lett. 2004

  39. 2* 1* H O O C + C O O H C O O H 3* C O O H Succinyl-CoA Succinate C O - S C o A 4* C O O H 2[H] C O - S C o A 5* C O O H H O 2 7 8* O H C O - S C o A 6 C O O H HS-CoA Succinyl-CoA 2[H] O C O - S C o A C O O H C O - S C o A The upper 2-methylnaphthaline degradation pathway • Addition of fumarate • β-Oxidation • Central intermediate 2-naphthoic acid Safinowski and Meckenstock FEMS Microbiol. Lett. 2004

  40. H O O C C O O H CH3 C O O H C O O H C O O H C O O H C O O H O H C O O H O C O O H C O O H C O O H C O O H C O O H C O O H How is naphthalene activated? + Ring cleavage and degradation via cyclohexane ring structures CO2 or Upper degradation pathways to 2-naphthoic acid Reduction of 2-naphthoic acid

  41. D D C O O H D H O O C D C O O H D D D C O O H Methylation of an aromatic hydrocarbon? CH3 D D D D CH3 D D D D D D D D D D D D Deuterated naphthalene as substrate for culture N47 Product should have the mass of naphthyl-2-methylsuccinate plus 7 mass units

  42. Postulated degradation pathway for anaerobic naphthalene degradation

  43. How to assess the degradation activity in the environment • Adsorption • Dilution/ • dispersion • Microbial degradation

  44. H O O C C O O H C O O H C O O H C O O H C O O H C O O H O H C O O H O C O O H C O O H C O O H C O O H C O O H C O O H Detection of anaerobic naphthalene degradation in the environment + Ring cleavage and degradation via cyclohexane ring structures CH3 or Upper degradation pathways to 2-naphthoic acid Reduction of 2-naphthoic acid

  45. A former coal gasification site near Stuttgart, Germany Investigation area ? Areas with NAPL-phase S2 wells Groundwater flow N S1 Contaminant source 100 meter

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

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

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