Biodegradation 11-19-07.pdf lecture by adelaide17madette


									                                                       Terms in Biodegradation
                                               • Biotransformation = biological
                  ESM 219                        modification of parent compound
                                               • Biodegradation = biological destruction
                                               • Biomineralization= biological
        Introduction to Biodegradation
                                                 transformation with CO2 and H2O as end

                                                Biodegradation: what do you want
      Terms in Biodegradation
                                                            to know?
                                               • What is the compound?
• catabolic transformations yield energy          – Name(s), forumula, properties?
                                               • Does it biodegrade?
                                               • What are the requirements for biodegradation?
• cometabolic transformations don’t yield         – Nutrients? Environmental conditions?
  energy to the catalysts; primary substrate   • Who does it?
                                                  – Single strain? Consortia?
  required. example: oxidation of TCE by       • What’s the pathway?
  methanotrophs                                   – Intermediates? End products?
                                               • What gene(s) are involved?
                                               • How fast does it occur?
                                                  – Under what conditions?

     Biodegradation/ Biocatalysis
                                                What about chemicals not in BBD?
•                    • Example: APEOs: alkylphenol
• Examples                                       polyethoxylates (Science, 1984, v225).
  – Atrazine: very-well studied, described        – Detergents, widely used
     • What’s known / not known?                  – Being phased out (see JohnsonDiversey, 2006)
  – Osmium: metal                                 – Biodegrades to endocrine disrupters (See
• Other attributes of the database                  Science, above) in WWTPs

                Related Research                                          Related Research (cont.)
• Chen et al. (2006): kinetics for one isolated                  • Porter and Hay (AEM, 2007).: finding the
  bacterial strain, sole carbon source OPEOn                       responsible gene (monoxygenation) in OP
   –   enriched from rice paddy (recycle drainage, rice field)     degradation
   –   μ vs. S (Fig. 2)                                               – Enrichment, C&E = octyphenol (OP), activated
   –   Form & amount of N (Fig. 3)                                      sludge—the polyethoxylate is gone
   –   pH (Fig. 4)                                                    – i.d. by 16S rRNA = Sphingomonas
   –   Models of growth, degradation (Fig. 5)                         – What can you do with the result? (i.e.
   –   End products not quite identified…. (Fig. 7)                     identification of the opdA gene and its activity,
                                                                        Fig. 3)

                        Toxicity                                                     Acclimation
                                                                 •   delay that precedes onset of biodegradation
• Pollutant substrates are also toxic, with effects              •   concentration dependent
  observable in
   – Lag period
                                                                 •    reduced after exposure
   – Slow growth rate                                            •    explanations:
   – Low yield / Low growth efficiency                               – small starting population
• metabolites can affect other community members                     – toxins: slow growth, are removed, selective pressure,
• effects on other processes: e.g., Ni inhibits                      – predation
  nitrification, carbon mineralization and activities                – genetic change + gene transfer + growth
  of specific enzymes                                                – diauxie (preferential substrate)
                                                                     – enzyme induction (minor), lag phase (minor)

       Biotransformation Rxns
• Detoxification
  – Hydrolysis: e.g. ester bond (C-O-C) in
    malathion; (activation of some herbicides (e.g.,
  – Hydroxylation: e.g. replacing H with OH in
  – Dehalogenation: reductive = replacement with
    H; hydrolytic = replacement with OH, for

       Biotransformation Rxns
• Detoxification (continued)
  – Dealkylation: e.g. demethylation of chloroneb
  – methylation: e.g, pentachlorophenol; conversion of
    2,4, dinitrophenol to 2-amino-4 and 4-amino-2-
  – nitro reduction
  – Deamination
  – ether cleavage: e.g., 2,4-D loss of phytotoxicity
  – nitrile to amide conversion
  – conjugation: e.g. pyrene + glucose = glucoside

       Biotransformation Rxns
• Activation
  – Hydrolysis: activation of some herbicides e.g.
  – Dehalogenation, .e.g. vinyl chloride formation
    during anaerobic TCE metabolism
  – Hg methylation, As methylation, methylated tin
  – nitrosamine formation

                                                      Genes in biodegradation are often encoded on plasmids.

        Biotransformation Rxns
• Activation (continued)
    – Epoxidation, e.g. aldrin to dieldrin
    – conversion of phosphorothionate to phosphate,
      e.g. conversion of parathion to oxygen analog
      (S on molecule replaced with O)
    – analog compounds: e.g. 6-(2,4-
      dichlorophenoxy)hexanoic acid to 4-(2,4-DB)
      then to 2,4-D (the herbicide)

      Petroleum Biodegradation                                     Refined petroleum
• Petroleum (crude, refined, structural
• Pathways (aerobic, anaerobic)
• Environmental factors

       Petroleum microbiology
• Zobell (1940’s) biodegradation shown
• Atlas & Bartha (1970s--) marine systems
• Gibson, Ratledge (1970s) pathways
• Chakrabarty (1970s--) specificity, long chains,
  engineered strains
• Grbic Galic, Lovely (1980s) anaerobic
• Bordon, Tiedje (1980s--) e- delivery
• 1990’s----: engineered systems, bioaugmentation,
  bioavailability, MTBE

                                                            most well-studied for toluene under denitrifying or
                                                            sulfate-reducing conditions.
                                                            mechanism is nucleophilic attack on the
                                                            methyl-carbon by
                                                                                                                   Oxidation products of aromatics
                                                            succinyl CoA, resulting in two “dead end”
                                                            benzylsuccinic acid, benzyl fumaric acid.
                                                            Is there a “productive” pathway?

mono- or dioxygenase dependent
Aromatic, polyaromatic, substituted aromatic
intermediates prior to ring cleavage (above)
 ring cleavage by ortho or meta
ring cleavage products feed into the CAC
multiple pathways for complex molecules

          two common oxidation products of aromatic compounds

Hydroxylation of benzene to catechol by a monooxygenase in which NADH is an electron donor.

                                                                                                                          Factors influencing
                                                                                                                        biodegradation of petrol
                                                                                                                  • sufficient numbers of microbes with genetic
                                                                                                                  • available electron acceptor and nutrients
                                                                                                                  • temperature and water availability
                                                                                                                  • bioavailability
                                                                                                                  • contaminant structure (steric, electronic)
                                                                                                                  • toxicity

                                                                                      Compartment generalizations
                                                                                   • Surface soil = aerobic and rapid
                                                                                   • Vadose zone = aerobic and rapid once
                             Steps in oxidation of an aliphatic hydrocarbon,
                             the first of which is catalyzed by a monooxygenase.
                                                                                     population sizes have increased
                                                                                   • Groundwater = slow due to low population
                                                                                     sizes; turns anaerobic due to slow resupply
                                                                                     of oxygen

       Modes of petrol uptake
• Liquids
  – uptake of solubilized form
  – direct contact with NAPL
  – dispersion and direct contact with
• Solids (PAHs)
  – direct contact
  – uptake of solubilized form

        Chlorinated Solvents                                                                       Pesticides
• aerobic co-metabolic, e.g. TCE oxidation                                         • undergo similar reactions as other pollutants
  by methanotrophs with methane, formate or                                          under either anaerobic or aerobic conditions
  methanol as primary substrate                                                          • example of community activity and possible
• anaerobic reductive dechlorination may                                             importance of sequential anaerobic / aerobic
  result in toxic end product                                                        episodes: DDT

                                                                                          Pathway of atrazine catabolism by
                                                                                           Pseudomonas sp. ADP and other

Pathway of aerobic 2,4,5-T biodegradation. The final products, succinate and acetate,
are catabolized in the citric acid cycle. The pathway involves a dioxygenase.

    Global distribution of bacteria isolated
   for their ability to dechlorinate atrazine.                                                                      Metals
                                                                                        • Ag and Hg are most toxic to microbes, then Cd,
                                                                                          Zn, Cu, Cr, Pb, Ni
                                                                                        • Tolerance is often observed (plasmid encoded
                                                                                        • Hg (methylation, demethylation)
                                                                                        • Cd: may be accumulated
                                                                                        • Se, As subject to oxidation state changes by
                                                                                          microorganisms, depending on redox conditions
                                                                                        • Cr: (VI) from oxidation of (III) can occur with
                                                                                          Mn as electron acceptor

      Rules of Thumb: Hydrocarbon
                                                                                           Rules of Thumb:Halogenated
 • C10 to C18 used more readily than longer or shorter chain lengths                    • on aromatics: ο and p positions more resistant than m
 • <C8 aliphatics are toxic to many bacteria (more soluble, few can
   degrade)                                                                             • anaerobic dehalogenation followed by aerobic ring
 • long chain (MW 500-600) slow to degrade                                                cleavage and mineralization
 • branching increases recalcitrance                                                    • TCE, TCA, PCE:
 • fungi can grow on up to 34 Cs                                                           – reductive dechlorination to vinyl chloride
 • cycloparafins (alicylclics) slow to degrade                                             – aerobic cometabolism
 • recalcitrance of aromatics is f(MW, substitution)
 • anaerobic aromatic degradation does occur, mineralization is still
   under study
 • saturated and alkenes degrade similarly
 • branching decreases biodegradability
 • halogen substitution decreases biodegradability

           Rules of Thumb:PAHs                                                    Rules of Thumb:Explosives
•   2 to 3 rings degrade
•   4 rings + recalcitrant                                              • Nitroaromatics TNT, Dinoseb, dinitro-o-cresol
•   intermediate of oxidation may be carcinogenic                             – aerobic by oxidative cleavage of nitro group
                                                                              – anaerobic conversion of nitro group to amino group with formation
•   solubility may limit
                                                                                of toxic hydroxylamine
•   cometabolism for higher MWs

                   Rules of Thumb                                                      Biodegradation Kinetics
• PCBs                                                                  Rates: how fast do transformations proceed?
    – more Cls, more resistant
    – aerobic: mostly degrades to chlorobenzoate, then transformed by
      other organisms to                                                depends on catalysts and environmental factors
        • a) chlorocatechol then dechlorination                         generally, aerobic in hours, anaerobic in days or months
        • b) dechlorination and dioxygenation,
        • c) dehalogenation.                                            sources for rates:
    – anaerobic: reductive dechlorination then aerobic degradation      Handbooks (Howard) CAUTION
                                                                          primary literature (journal articles)
• Dioxins                                                                 measure in the laboratory
    – can be cometabolized: TCDD: 2,3,7,8 tetrachlorodibenzo-p-dioxin
      (TCDD)                                                              measure in the field… to is another story

      Biodegradation: How do we
         know it is happening?
• Loss of parent material
• Production of metabolites
    – CO2 (labeled or not)
    – Intermediates
    – Best if direct relationship to pollutant
• Increase in biomass
    – Degraders
    – Predators (e.g. protozoa)
                                                                        Growth of Burkholderia (formerly Pseudomonas) cepacia on 2,4,5-T as sole source of carbon and energy.
                                                                        The strain was enriched from nature using a chemostat to keep the concentration of herbicide low.
                                                                        Growth here is aerobic on 1.5 g/l of 2,4,5,-T. The release of chloride from the molecule is indicative of

       Threshold / Persistence
• Biodegradation can stop at low
  – Inadequate substrate to induce enzyme
  – Physicochemical binding (sorption)
     • Leads to low bioavailability
     • Most compounds need to be inaqueous phase for
       biodegradation to occur.

                                                                          Ongoing Concerns
                                                       • Contaminants of concern
                                                          – E.g. MTBE, perchlorate, nitrate
                                                       • New “contaminants”
                                                          – Nanoparticles?
                                                       • Conferences:

                                                       • Tools: proving that biodegradation is occurring in


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