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Variety of contaminants & bioremediation potential.

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Presentation on theme: "Variety of contaminants & bioremediation potential."— Presentation transcript:

1 Variety of contaminants & bioremediation potential

2 Environment-Chemical-Microbe Interactions in Biodegradation Environmental conditions affect the occurrance or type of biotransformation Some compound s degraded under aerobic & anaerobic conditions Others are degraded preferentially or solely in aerobic vs. anaerobic

3 Toluene: Aerobic degradation Degradation is initiated by either a mono- or di- oxygenase.

4 Toluene: Anaerobic degradation Conjugation of fumarate to toluene gives benzylsuccinate Benzylsuccinate via  -oxidation-like process to give benzoate (benzyl-CoA). Benzoate metaboized via  -oxidation-like process

5 Compounds with differential aerobic vs. anaerobic degradation potential Benzene, polynuclear aromatic hydrocarbons (PAH) Stable ring structures, aerobes utilize oxidizing power of oxygenases to initiate degradation Anaerobes lack similarly powerful oxidants Compounds are readily degraded aerobically, but persistent in anaerobic environments

6 Compounds with differential aerobic vs. anaerobic degradation potential Perchloroethylene (PCE) aka. tetrachloroethene Highly chlorinated, highly oxidized C atoms are good electron targets for anaerobes but not aerobes Cl atoms block activity of oxygenases. Readily degraded anaerobically, but persistent in aerobic environments

7 Biodegradation & biotransformation: Types of processes Compounds may be degraded by processes that may or may not support growth of the organism effecting the transformation Same for aerobic & anaerobic conditions: types of enzymes or biomolecules mediating transformations differ

8 Overview of metabolic processes

9 Catabolic enzymes: Characteristics key to growth support

10 Degradation mechanisms: Growth Supporting Pure culture model Good for detailed studies of metabolism and genetics May not be representative of activity in the environment

11 Consortium model Common for anaerobes May also be prevelant in aerobic environments, documented for degradation of some pesticides Complicates detailed studies of metabolism and genetics Degradation mechanisms: Growth Supporting

12 Pesticide-degrading consortium

13 Degradation mechanisms: Not growth supporting

14 Type I cometabolism Transformation of cosubstrate is dependent upon the presence of the substrate: acts to induce enzymes mediating transformation supports growth and activity of degraders Occurs naturally at low levels (low subtrate levels, population density) Effective use in bioremediation requires introduction of the substrate (inducer)

15 Example of Type I cometabolism Biphenyl degraders common in soil typically cannot grow on PCBs as Cl- products are not utilized Supplied with biphenyl, enzymes are induced that transform PCBs

16 Degradation linked to use as e- acceptor 1.Redox-active biomolecules involved in biosynthesis 2. Reductases (or other electron carriers) involved in respiration Oxidized compounds may be reduced by two kinds of interaction:

17 Degradation via use as e- acceptor: Growth supporting transformations Dehalorespiration Halogenated organics used by anaerobes as terminal electron acceptors Energy from electron transfer is captured Mediated by a specific reductase induced by growth with Cl-organic as electron acceptor Substrates include: chlorinated alkenes (PCE, TCE) chlorinated aromatics (chlorobenzenes, polychlorinated biphenyls)

18 Dehalorespiration: Scheme of electron transfer and energy conservation

19 Known dehalorespiring organisms All belong to Bacteria Many are related to SRB

20 Degradation via use as e- acceptor: Transformations not supporting growth Interaction with redox active cofactors Substrate oxidation electron carriers e- energy generation, biosynthesis Cl-organic H-organic + Cl-

21 Redox active cofactors in Type II cometabolism Compound serves as electron acceptor Energy is not conserved, does not support growth Incidental contact of oxidized Cl-organic with a reduced e- transfer molecule Growth substrate has no direct affect on the occurance or rate of transformation No specific inducer is involved = Type II cometabolism

22 Types of reductive dehalogenation reactions Two categories: are hydrogenolysis and vicinal reduction Hydrogenolysis: displacement of a single chlorine atom by hydrogen. occurs with both aryl and alkyl compounds. Vicinal reduction: displacement of two chlorine atoms from two adjacent carbon atoms and formation of a carbon-carbon double bond. occurs only with alkyl compounds.

23 Types of organisms mediating alkyl reductive dehalogenation Physiologically diverse Eucarya, Bacteria, Archaea aerobes, anaerobes, fac. anaerobes Activity identified in many culture collection organisms (isolation not associated with ability to dechlorination)

24 Factors affecting rates of biodegradation in soil Environmental Temperature, moisture, pH, etc. Microbial Acclimation of a population of degraders Chemical-Microbial Interactions Levels of chemical (high, toxicity; low, subthreshold) Chemical structure (aerobic vs. anaerobic degradation) Environmental-Chemical Interactions Bioavailability

25 Acclimation in “History Soils” Soils with a history of use of a pesticide may exhibit accelerated degradation of this compound or related compounds Reflects enrichment of microbes that grow on the pesticide History effect

26 Factors affecting rates of biodegradation in soil Environmental Temperature, moisture, pH, etc. Microbial Acclimation of degraders Chemical-Microbial Interactions Chemical structure (aerobic vs. anaerobic degradation) Levels of chemical (high, toxicity; low, insufficient energy) Environmental-Chemical Interactions Bioavailability

27 Effects of chemical concentration amount of chemical degradation rate growth support threshold growth support Increasing toxicity toxicity threshold

28 threshold Non-growing, no change in biomass, constant degradation rate Growing, increase in biomass, increasing degradation rate

29 Toxicity is dose (concentration)-dependent Log(P) = 3.77 PCP: biocidal, a wood treatment to supress microbial degradation Low concentrations:a growth substrate (e- donor) for aerobes High concentrations: toxicity

30 Solvent-effect toxicity: Relation to Kow Hydrophobicity assayed by octanol-water partitioning (Kow) Increasing hydrophobicity = increasing Kow Values of Kow are large, expressed as a log, Log(P) = Log(Kow) In general: Gm+ more tolerant than Gm- Toxicity becomes significant at ca. Log(P) < 3 Log(P) values TCE = 2.42 PCE = 3.14

31 Factors affecting rates of biodegradation in soil Environmental Temperature, moisture, pH, etc. Microbial Acclimation of a population of degraders Chemical-Microbial Interactions Levels of chemical (high, toxicity; low, subthreshold) Chemical structure (aerobic vs. anaerobic degradation) Environmental-Chemical Interactions Bioavailability

32 Bioavailability and the “weathering” phenomenon Definition: Occurrence of a compound in a state that is accessible to microbes Bioavailable = dissolved in aqueous phase Bioavailability (biodegradation potential) dec. with inc. residence time in soil = “weathering”

33 Mechanisms in weathering of organic chemicals Time-dependent entry of compound into a state or location that is inaccessible to microbes. Nature of sites or states hypothesized to be small pores that restrict entry of cells regions within SOM that strongly retain the compound Microbes can’t enter aggregate Access to compound requires: 1.desorption from sorbed (solid) phase 2.diffusion to reach cells

34 Bioremediation Biodegradation: Biochemically catalyzed transformation of a compound to one or more metabolites of lower molecular weight, May directly support growth or occur as to a growth so Bioremediation: Focused or applied aspects of biodegradation to effect removal of pollutants or transformation to nontoxic products Most bioremediation applications involve natural activities of microbes, either indigenous or introduced

35 Types of Bioremediation Intrinsic Enhanced aka, Natural attenuation “Let nature take its course” Degradative activities effected by indigenous microbes under ambient conditions No intervention to alter aspects of the environment affecting microbial activity aka, Engineered remediation Biostimulation Alteration of the environment to enhance activities effected by indigenous microbes Bioaugmentation Inoculation of organisms to introduce a type of catalysis not displayed by the indigenous community

36 Natural attenuation: Examples Chlorinated solvents (PCE, TCE): environment: anaerobic process: reductive dechlorination (e- acceptor) products: vinyl chloride, ethylene Polychlorinated biphenyls (PCBs): environment: anaerobic process: reductive dechlorination products: dechlorinated PCBs (6 Cl -> 4 Cl) Benzene, toluene, ethylbenzene, xylene (BTEX): environment: anaerobic process: C assimilation, e- donor products: biomass, mineralized BTEX

37 Natural Attenuation of PCBs PCBs in anaerobic environments transformed by reductive dehalogenation Mediated by anaerobes; both growth-supporting and cometabolic mechanisms Key process in the environmental fate of PCBs in sediments Highly chlorinated PCBs are transformed preferentially End products are PCBs, but with fewer Cl atoms than parent compound

38 Enhanced bioremediation Biostimulation Needed degradative activities are possessed by indigenous community Environmental factor(s) limit expression to a degree satisfactory for remediation Environmental manipulations differ by type of compound and environment = operative biodegradative mechanisms Examples: Hydrocarbons (oil, fuels) - aerobic - growth - use as C source/e-donor TCE - aerobic - cometabolic PCE/TCE - anaerobic - growth/cometabolic

39 Biostimulation of aerobic hydrocarbon degradation Oil & fuel spills; Many constituents of mixtures are used as C source = sufficient indigenous activity potential Large amounts of C, growth limited by nutrient availability (N, P) Moisture levels & temp. also affect activities

40 Biostimulation of aerobic hydrocarbon degradation Inducer needed to induce oxygenase mediating this step spontaneous chemical decomposition

41 Inducers and enzymes in aerobic degradation of TCE

42 Comparative biostimulation of aerobic TCE degradation

43 Enhanced bioremediation: Biostimulation of anaerobic degradation of PCE/TCE Nature of PCE/TCE contamination Used as degreasing agents Wide-spread groundwater contaminants DNAPL sink into groundwater Removal by physical means difficult PCE/TCE often present with hydrocarbons Utilization of hydrocarbons drives aquifers to be anaerobic Dense NonAqueous Phase Liquids

44 PCE/TCE Reductive dechlorination pathway PCE/TCE used as electron acceptor, often by sulfate-reducing bacteria Dechlorination enhanced by increased e- donor supply May also be effected by levels of natural e- acceptors (SO 4 -2 )

45 Using organic substrates to promote reductive dechlorination of PCE/TCE Acids and alcohols are fermented to yield hydrogen Hydrogen serves as e- donor in reductive dechlorination The fermenting organism may or may not also effect dechlorination NADH + H+ ----hydrogenase--> NAD+ + H2

46 Enhanced bioremediation: Bioaugmentation Motivation: Activity is absent/not effectively selectively targeted by biostimulants alone Example: Dehalorespirer Dehalococcoides ethenogenes may be important in effecting efficient transformation of PCE/TCE to ethene D. ethenogenes may not be present in all contamianted aquifers

47 Field test of bioaugmentation to enhance reductive dechlorination of PCE/TCE Injection of: consortium containing D. ethenogenes methanol, acetate bromide Monitoring points Extraction, closed loop

48 acetate + methanol microbes + acetate + methanol PCE DCE

49

50 Not used

51 Clays (aluminosilicates); sorb ions, sorption greates by swelling types Soil matrix components affecting weathering: Clays Swelling clay: Layers not linked Layers separated by H2O Interlayers exposed Non-swelling clay: Layers joined Prevents entry of molecules Entry and sorbtion to interlayer reduces availability for degradation

52 NOC Sorption to SOM Postulated nature of sorption sites: 1. Hydrophobic clefts & voids within humics? 2. Highly condensed, rigid (glass-like) vs. flexible (rubber-like) regions Void Cleft

53 The Scientist 15[6]:1, Mar. 19, 2001 NEWS PCB Dilemma Government, industry, and public debate dredging vs. bioremediation in the Hudson River Natural Bioremediation-- Too Slow? At the crux of the debate seems to be the natural process of bioremediation that is taking place in the river. Both sides--EPA and GE--agree that this is happening, but questions arise over the pace and degree of degradation. The EPA's summary report and poster displays at the meeting conclude, "PCBs in sediments will not be naturally 'remediated' via dechlorination. The extent of dechlorination is limited, resulting in probably less than 10 percent mass loss of PCBs." Sound Science, Or Overkill? An environmental dredging project of this magnitude is unprecedented. "There have been projects, although not of this scale, proposed, but not yet implemented. However, all of the technologies have been used; this isn't anything new. What we are doing is putting them all together into one large package," says Douglas Tomchuk, EPA's Remedial Project Manager for the Hudson River PCB site.

54 Acclimation of degrader populations in soil Defination The period preceding the on-set of biodegradation, which the chemical is not degraded, and after which biodegradation is rapid

55 Abundance/activity of degraders affects apparent length of acclimation phase Degrader population initially low and/or inactive (non-induced) Initial exposure to substrate allows populationgrowth Supports more rapid degradation of subsequent introduction of substrate Apparent acclimation phase is shortened or absent

56 Chemical & microbial factors in apparent acclimation periods Chemical Small amounts of degradation are more apparent with low substrate levels Microbial Substrate depletion is proportional to growth At high substrate levels, small amounts of growth may not be detected = apparent acclimation

57 Carbamate History Soils Carbamates are a widely used class of insecticide All are variations of a core chemical structure

58 Accelerated degradation of carbamates in history soils Prior exposure to one carbamate acclimates the community to degrade other carbamates

59 Chemical-Environmental affects on biodegradation The chemical may not be in a physical form that is accessible to the organisms (bioavailability) The concentration of the target chemical may be too low to support replication of the degrading organism (threshold) The concentration of the chemical or other compounds may be high enough to inhibit degradation specifically (competition) or have a general inhibitory effect on microbial metabolism (toxicity). One or more accessory nutrients may be inadequate (nutrient imbalance)

60 Toxicity of Organic Compounds Membrane damage: Solvent effect associated with hydrophobic chemicals (hydrocarbons, chlorinated hydrocarbons) compounds partition into membranes membranes are disrupted electrochemical gradients are dissipated Physiological Disruption: Uncoupling e- transfer from ATP synthesis (chloro- and nitro-phenols) carry protons across membrane, dissipate  pH Damage to Macromolecules; Enzymes, DNA Usually associated with reactive intermediates, not the original compound TCE --Moase--> TCE-epoxide (reactive) ----> Protein/DNA binding

61 Solvent-effect toxicity: Hydrophobicity and water solubility Relative toxicity related to hydrophobicity/water solubility Inc. hydrophob = inc. tendency to lodge in membranes, also = dec. water solubility = dec. exposure to microbes Most toxic are hydrophobic compounds with significant aqueous solubility

62 Soil matrix components affecting weathering: SOM Sorbtion of ions & neutral organic compounds (NOC) Inc SOM = Inc. sorption capacity for NOC Reduction of SOM reduces sorption capacity for NOC

63 Weathering: Combined effects of minerals & SOM Minerals & SOM combine to form aggregates NOC sorption to SOM on surface NOC sorption to SOM in aggregate interior Access to a chemical is limited by diffusion of the compound to the microbial cell

64 Energy needs & Growth support threshold Total cell energy need = growth (biosynthesis to produce new cells) + maintenance (retain solute gradients, repair DNA, etc.) Energy for growth >>> Energy for maintenance Growing cell energy = biosynthesis to produce new cells Non-growing (but alive) = maintenance

65 Corrinoid cofactors implicated in reductive dehalogenation

66 Reductive dechlorination of organochlorine pesticides Reductive dechlorination affects fate, behavior, toxicity Occurs commonly in soils usually under anaerobic conditions Possibly a result of Type II cometabolism

67 Mechanism for Type II cometabolism by methanogens

68 Reductases in dehalorespiration Proteins mediating the final stage of electron transfer specifically induced May be integrated into membranes or peripherally associated with these

69 Solvent-effect toxicity in Bacteria Organisms vary in solvent tolerance, reflecting differences in cell surface or membrane composition In general: Gm+ more tolerant than Gm- Toxicity becomes significant at ca. Log(P) < 3


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