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Biodegradation and Detoxification

Biodegradation and Detoxification. June 3, 2014 Joonhong Park. Definitions. Terms. Disappearances of a polluting chemical Abiotic transformation of a chemical Biotic transformation of a chemical (Biodegradation) Detoxification Activation Cometabolic vs. Metabolic Biodegradation.

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Biodegradation and Detoxification

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  1. Biodegradation and Detoxification June 3, 2014 Joonhong Park

  2. Definitions

  3. Terms • Disappearances of a polluting chemical • Abiotic transformation of a chemical • Biotic transformation of a chemical (Biodegradation) • Detoxification • Activation • Cometabolic vs. Metabolic Biodegradation

  4. Detoxification

  5. Detoxification The most important role of microorganisms in the transformation of pollutants Detoxification refers to the change in a molecule that renders it less harmful to one ore more susceptible species (human, animals, plants and microorganisms) CO2 Toxin Metab. Seq. A Detoxification Reaction Mineralization Inactive Product Toxin Metab. Seq. B Typical Product Atypical Product

  6. Transformation processes resulting in detoxification • Hydrolysis: addition of water • Hydroxylation: addition of OH • Dehalogenation: removal of Cl or halogens; (i) reductive dehalogenation, (ii) hydrolytic dehalogenation, and (iii) dehydrodehalogenation • Demethylation or other dealkylation • Methylation: R-OH => R-O-CH3 • Nitro reduction: R-NO3 => R-NH2 • Deamination: metamitron, Figure 4.5 • Ether cleavage: 2,4-D, Figure 4.5 • Conversion of nitrile to amide: R-C≡N => R-C(=O)-NH2 • Conjugation: A product of a toxicant combined with a natural metabolite

  7. Activation

  8. Activation One of the more surprising, and possibly the most undesirable, aspects of microbial transformations in nature is the formation of toxicants. Often, by-products of biodegradation are more toxic than their parental compounds Important to know about the pathway of biodegradation including activation and its degradation products (intermediates)

  9. Mechanisms of Activation (I) a. Dehalogenation:PCE => TCE => DCE => VC => ethane b. Halogenation: fungi, algae and bacteria add chloride or bromine to organic compounds c. Nitrosamine formation R R NH + NO2-= N-N=O + OH- R’ R’ (nitrosamine) d. Epoxidation O -HC=CH- => -HC-CH- (ex. Aldrin to Dieldrin) e. Conversion of phosphorothinate to phosphate RO S RO O RO -P-OX => RO - P – OX (cf. phosphorodithoates)

  10. Mechanisms of Activation (II) f. Metabolism of Phenoxyalkanoic Acids: beta-oxidation (2,4-D, Figure 5.5; 4-nonlyphenol Figure 5.6) g. Oxidation of Thioethers O O -C-S-C- => -C-S-C => -C-S-C- O h. Hydrolysis of Esters O O RCOR’ + H2O => RCOH + HOR’ • Other Activations - Production of TCDD by peroxidases - Methylation (organics, mercury, arsenic, tin etc.) - Demethylation - Anaerobic degradation of RDX into 1,1-and1,2-dimethylhydrazine

  11. Discussion: Risks of Biodegradation The products of microbial biodegradation of a pollutant may or may not be more toxic to human, animals, plants or other ecosystems. Is biodegradation always good? What should be the ultimate goal of bioremediation? What measurements are required for answering the above questions?

  12. Effect of Chemical Structure on Biodegradation

  13. Chemical Factors Affecting Biodegradation Many possible chemical reactions are foreign to microorganisms (xenobiotics) Biodegradation rates vs. Xenobiotic metabolism (growth-linked metabolism vs. cometabolism)

  14. Chemical Factors Affecting Biodegradation Xenophores(substituents): Table 11.2 in B&B • Cl, Br, NO2, SO3H, CN or CF3 (strong xenophores) • CH3, NH2, OH and OCH3 (often acting as xenophores) • OH, COOH, amide, anhydride etc. (stimulaitng biodegradation) The addition of xenophore: Table 11.2 in B&B - The more addition, the less biodegradable. The position of xenophore: Table 11.3 in B&B

  15. Chemical Factors Affecting Biodegradation Methylbranching vs. beta-oxidation -Linear ABS vs. ABS with a quaternary C Molecules containing an aromatic and alky/aliphatic acid moiety PAH biodegradability (Figure 11.5 in B&B): 3 rings (biodegradable) vs. 4 and more rings (little biodegradable) Chlorinated Hydrocarbon Biodegradability

  16. Degree of Halogenation vs. Biodegradation 가장 산화됨 Cl H Cl Cl Cl H C C C C C C Cl Cl H H Cl H Tetrachloroethene (Perchloroethene, PCE) cis-Dichloroethene (cDCE) monochloroethene (vinyl chloride, 발암물질) Cl H H H Cl C C H C C C C Cl Cl Cl H H H Trichloroethene (TCE, Cs = 1,100 mg/L) ethene trans-Dichloroethene (tDCE) 가장 환원됨

  17. Aerobic degradation Sorption Reductive dechlorination Sorption onto Subsurface Material Degradation Rate Monochlorinated Polychlorinated 0.25 4 Degree of Chlorination

  18. Biodegradability The apliphatic and aromatic hydrocarons are readily biodegradable by a range of aerobic bacteria and fungi. The key is that molecular O2 is needed to activate the molecules via initial oxygenation reactions. Evidence of anaerobic biodegradation of aromatic hydrocarbons is growing. Anaerobic biodegradation rates are slower than aerobic rates, but they can be important when fast kinetics are not essential. Most halogenated aliphatics can be reductively dehalogenated, although the rate appears to slow as the halogen substituens are removed. Highly chlorinate aromatics, including PCBs, can be reductively dehalogenated to less halogenated species. Lightly halogenated aromatics can be aerobically biodegraded via initial oxygenation reactions. Many of the common organic contaminants show inhibitory effects on microorganism growth and metabolism. Due to their strongly hydrophobic nature, many of the inhibitory responses are caused by intereactions with the cell membrane. In some cases, intermediate products of metabolism can be more toxic than the original contaminant.

  19. Predicting Biodegrability? Structure-Activity Relationship (SAR) or Structure-Biodegradation Relationship (SBR) Biodegradation rate of a compound = f(waster solubility, melting/boiling points, molecular weight, molecular volume, molar refractivity, density, logKow, van der Waals radius, van der Waals volume, Hammett’s sigma, pKa, dipole moment, etc.) Microbial Physiology Microbial Ecology Environmental Abiotic Factors

  20. Acclimation

  21. Factors Affecting Acclimation Environmental Factors (temperature, pH and aeration) The concentration of N, P or both (nutrients) (cf. the degradation of P- and N-containing organic compounds is inhibited in the presence of nutrients) The concentration of the compound that is being metabolized • Threshold • Detection limit Site specific microbial communities • Biodegradation population • Interaction with other population members

  22. Explanations for the Acclimation Phase (a) Proliferation of small populations (Figure 3.3 in B&B) (b) Presence of toxins (Figure 3.4 in B&B) (c) Predation by protozoa (Figures 3.5 and 3.6 in B&B) (d) Appearance of new genotypes (mutation or gene transfer) (e) Enzyme Induction and Lag Phase (the regulations of gene expression and enzyme induction, diauxie, catabolic repression, etc.)

  23. Predicting Biodegradation Intermediates (Pathways)

  24. Biodegradation Pathway Databases Biocatalysis/Biodegradation Database (Univ. of Minnesota) http://umbbd.ethz.ch/index.html List of 219 pathways, 1503 reactions, 1396 compounds 993 enzymes, 543 microorganism entries, 249 biotransformation rules KEGG Pathway Database – GenomeNet http://www.genome.jp/kegg/pathway.html

  25. Mono-cyclic Aromatics (BTEX): Upstream Hydroxylation via mono- and di-oxygenases Ketone formation Ring cleavage Johnson, Park, Kukor and Abriola, 2006, Biodegradation

  26. Mono-cyclic Aromatics (BTEX): Upstream Carboxylation (anaerobic conditions)

  27. Mono-cyclic Aromatics (BTEX): Downstream

  28. Mono-cyclic Aromatics (BTEX): Reduction of Double Bonds Benzene Cyclohexene COOH COOH COOH Benzoic acid 1-cyclohexene carboxylic acid cyclohexane carboxylic acid CH3 HO CH3 4-methycyclohexanol Toluene

  29. Cycloalkanes: oxidation OH -CH2-CH2- Hydroxylation -CH-CH2- O Ketone formation -C-CH2- -HC=CH- Dehydrogenation

  30. Methyl Groups (RCH3): (terminal) oxidation OH R-CH2 Alcohol R-CH3 O Aldehyde R-CH O Carboxylic acid R-C-OH R could be alkanes and aromatics in oil products, surfactants etc.

  31. Alkanes (CH3(CH2)nCH3): Dehydrogenation R-CH=CH-R’ R-CH2-CH2-R’ Ex. The conversion of eicosane (C20H42) to eicos-9-ene in soil The formation of 1-dodecene from tetradecane by anaerobes

  32. Alkyl Groups (R(CH2)nCH3): Sub-terminal Oxidation (hydroxylation or keto formation) O Ar-CH2-CH3 Ar-CH-CH3 ethylbenzene O Ar-CH2-Ar Ar-CH-Ar

  33. Alkenes and Others with Double Bonds: Reduction, Oxidation, Hydration and Epoxide Formation R-CH2-CH2-R’ R-CH=CH-R’ R-COH-CH2-OH R-CH=CH2 R’ R’ R-CHCH2-OH R-CH=CH2 R’ R’ O R-CH=CH-R’ R-CH-CH-R’

  34. Alkynes and Others with Triple Bonds: Reduction R-CH=CH2 R-C≡CH HC≡CH H2C=CH2 acetylene ethene (ethylene) The acetylene reduction is done by bacterial nitrogen fixation

  35. Carboxylic Acids (R-COOH): Decarboxylation and Reduction ArCOOH ArH R-CH2CH3 R-CH-COOH CH3 Decarboxylation R-CHO R-CH2OH R-COOH Reduction

  36. Carboxylic Acids and Alcohols: Ester Formation O R-CH2OH + R’-COOH R-CH2OCR’ Ester Esters: Hydrolysis O R-CH2OCR’ R-CH2OH + R’-COOH Microbial hydrolysis of malathion, bromoxynil, etc.

  37. Alkanoic Acids [R(CH2)nCOOH], Alkanes [H(CH2)nCH3], and Alkyl Groups [R(CH2)nCH3]: Beta-oxidation R-(CH2)nCH2CH2COOH R-(CH2)nCH=CHCOOH β-position R-(CH2)nCHCH2COOH OH R-(CH2)nCOOH R-(CH2)nCCH2COOH + O CH3-COOH

  38. Hydroxyl Groups (ROH): Methylation and Ether (ROR’) Formation R-OH R-OCH3 Ar ArOH ArOCH3 PAH Phenoxy herbicides ArOCH2COOH ArOH ArOCH3 ArOCH2CH3 ArOH O-ethylation Ar Ar Ar diphenylmetahne to 1,1,1’1’-tetra phenyl-dimethyl ether CH3 CH-O-CH Ar Ar Ar Ether (ROR):Cleavage Table 12.9

  39. Halogenated Aromatics: Reductive Dehalogenation Cl Cl Cl Cl Cl Cl + H+ + Cl- Cl H + [2H] Cl Cl Cl Cl Cl Cl H Cl Cl Cl Cl Cl Cl Cl H Cl H Cl Cl H H H isomers ArCl6 => HArCl5 => H2ArCl4 => H3ArCl3 => H4ArCl2 => H5ArCl

  40. Halogenated Alkanes and Alkenes: Reductive Dehalogenation CCl4 + [2H] HCCl3 + H+ + Cl- CCl4 => HCCl3 => H2CCl2 Cl2CHCHCl2 => Cl2CHCH2Cl => ClCH2CH2Cl => ClCH2CH3 Cl Cl cDCE PCE H Cl MCE (VC) C C H C C Cl Cl H Cl Cl C C H H Cl H H Cl C C C C Cl Cl H TCE Cl H H ethene tDCE C C H H

  41. Halogenated Compounds: Hydrolytic Dehalogenation, Dehydrodehlogenation, Halogen Migration Hydrolytic Dehalogenation RCl + H2O ROH + H+ + Cl- Ex. Trans-1,3-dichloropropen into trans-3-chloroallyl alcohol R2CHCCl3 R2C=CCl2 + H+ + Cl- Dehydro- Dehalogenation + H+ + Cl- R2CHCHCl2 R2C=CHCl R2CHCH2Cl R2C=CH2 + H+ + Cl- Ex. DDT metabolism (DDT=>DDE=>DDMU) Halogen Migration Cl H Cl H C C C C OH Cl Cl Cl TCE 1,1,1-trichloro-ethanol Cl H

  42. Other Halogenated Compounds RCCl3 RCOOH From THM (trihalomethyl)-containing compounds ArCl ArSCH3 To methylthio derivatives

  43. Amines Reductive deamination NH2 RCH2CHCOOH RCH2CH2COOH Dehydro- deamination OH Hydrolytic deamination RCH2CHCOOH RCH=CHCOOH N-methylation N-acylation O Aniline ArNH2 ArNHCCH3 N heterocycle N-oxidation RNH2 RNO RNO2 RNHOH 2-methylquinoline

  44. Amines Reduction Dimerization ArNH2 ArNH2 ArN=NAr’ ArN=NAr + Ar’NH2 N-nitrosation R-NH + NO2- R-N-NO + OH- R’ R’ O S-addition ArNH2 ArNHSCH3 Dealkylation RN-Alk2 RN-Alk RNH2

  45. Carbamates and Amides: Cleavage Carbamate Cleavage O RCNHR’ RCOOH + H2NR’ Amide Cleavage O RCNH2 RCOOH + H3N

  46. Nitriles (RC≡N) Dihalogenated benzonitriles used as pesticides O O RCNH2 RCOH RC≡N N-Nitroso Compounds (Nitrosamines): R-N-NO R-N-H Denitrosation R’ R’ Azobenzenes: Reduction to Amines ArN=NAr’ ArNH2 + H2NAr’

  47. Nitro Compounds (RNO2) Reduction ArNHOH ArNH2 ArNO2 ArNO (nitroso-) (hydroxylamino-) (amino-) Hydrolytic Denitration RNO2 + H2O ROH + NO2- + H+ Reductive Denitration ArNO2 ArH Nitrate Esters (RONO2) RONO2 ROH Cleavage R(ONO2)3 HOR(ONO2)2 (HO)2RONO2 (OH)3R

  48. C-S Bond: Cleavage R R RSR’ RSH (+ HR’) RSR’ ROH CHSR’’ C=O RSO3H ROH R’ R’ Sulfate Esters (ROSO3H):Cleavage ROSO3H ROH Thiols (RSH): Dimerization Methylation ArSH ArSCH3 RSH RSSR Thioethers (RSR’): Oxidation Disulfides(RSSR): Cleavage O O = = RSR’ RSR’ RSR’ RSSR RSH = O

  49. Phosphate Esters O O O HO AlkO AlkO O AlkO = = = = P-OR P-OR P-OR POH AlkO HO AlkO HO Phoshorothioates Cleavage S S AlkO AlkO = = P-OH P-OH S AlkO HO AlkO = P-OR AlkO O O AlkO AlkO = = P-OH P-OH HO AlkO Degradation O O O AlkO AlkO AlkO = = = P-OH PSR PSH AlkO AlkO AlkO

  50. Phoshoro-Di-thioates Cleavage S AlkO = P-SH S AlkO S AlkO AlkO = = P-SR PSR AlkO HO O AlkO = P-OH AlkO

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