Microorganisms and Organic Pollutants

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Microorganisms and Organic Pollutants

• Chapter 20
• Lecture 16

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Legacy Waste

• 430,000 confirmed cases of leaking underground
  storage tanks in U.S. as of 2003
• gt90 of the monitored stream and gt55 of shallow
  underground sites contaminated with pesticides
• gt1.4 million acres of chemical plumes in
  groundwater

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Dealing with the problem

• National Environmental Policy Act in 1970
• Environmental Impact Statements
• Applicant required to take a hard look at the
  environmental consequences of the proposed action
  (development).
• Environmental laws
• Clean Air Act
• Clean Water Act
• Comprehensive Environmental Response,
  Compensation and Liability Act (Superfund)
• Superfund Amendments and Reauthorization Act

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What are environmental contaminants?

• Pollutants
• naturally-occurring compounds in the environment
  that are present in unnaturally high
  concentrations.
• Examples
• crude oil
• refined oil
• phosphates
• heavy metals

• Xenobiotics
• chemically synthesized compounds that have never
  occurred in nature.
• Examples
• pesticides
• herbicides
• plastics

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Types of contamination

• Point source contamination
• contaminant emanating from a defined source
• discharge pipe from an industrial operation
• Non-point-source
• source of contaminant emanating from a large area
• fertilizers or pesticides applied to agricultural
  land

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Overall process of biodegradation
Electron donor
Carbon source
Organic C
Carbon dioxide
H2O
Electron acceptor
O2 aerobic respiration
NO3, Fe(III), Mn(IV), SO4, CO2 anaerobic
respiration
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Complete vs incomplete biodegradation
Incomplete
Glucose ? pyruvate ??CO2 H2O
TCA cycle
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Biological Reductive Dechlorination Pathway
PCE
TCE
cis-1,2-DCE
vinyl chloride
2e-, H
Cl
Vinyl chloride intermediate is more toxic than PCE
ethene
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Biological Reductive Dechlorination Pathway

• Hydrogen is preferred electron donor

VC ethene 93
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Cometabolism

• Bacterium uses some other carbon and energy
  source to partially degrade contaminant

degradation products
contaminant
bacterium
corn starch
CO2 H2O
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Consortium interactions

• Bacterium A uses some other carbon and energy
  source to partially degrade contaminant.
• Bacterium B metabolizes contaminant degradation
  products to carbon dioxide and water.

Bacterium B
CO2 H2O
O2
contaminant
degradation products
Bacterium A
O2
corn starch
CO2 H2O
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Combining cometabolism and consortium interactions
(TCE)
CH4 Cl2CCHCl
Methanotrophic bacterium
Methane Monooxygenase
O
CH3OH
Cl2-CHCl
Other populations of bacteria
H2CO
HCOOH
CO2
CO2 Cl-
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Carrying Capacity

• Although many chemical contaminants in the
  environment can be readily degraded because of
  their structural similarity to naturally
  occurring organic carbon, the amounts added may
  exceed the carrying capacity of the environment.
• Carrying capacity is the maximum level of
  microbial activity that can occur under the
  existing environmental conditions

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What limits carrying capacity?

• Physical-chemical factors
• pH, temperature, nutrients
• types of microbes present and their biomass

Low carrying capacity High carrying
capacity
No contaminant left
Contaminant breakthrough
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Determinants of extent and rate of contaminant
biodegradation

• Genetic potential of microbes to mutate key genes
  in such a way that gene product (enzyme) can
  catalyze step in contaminant degradation
• This requires period of time for such adaptation
  to occur (weeks, months, years?)

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Determinants of extent and rate of contaminant
biodegradation

• Bioavailability
• First step in biodegradation process is the
  uptake of the contaminant compound by the cell in
  order for intracellular enzymes to access the
  contaminant
• If contaminant is not water-soluble, it is
  difficult for cell to access and take up
  contaminant.

Low-density, non-aqueous phase liquid
(hydrocarbon, benzene
H2O
Dense, non-aqueous phase liquid (TCE, PCBs)
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Determinants of extent and rate of contaminant
biodegradation

• Bioavailability
• Production of surfactants
• Attachment to liquid-liquid interface

Low-density, non-aqueous phase liquid
(hydrocarbon, benzene
H2O
Dense, non-aqueous phase liquid (TCE, PCBs)
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Determinants of extent and rate of contaminant
biodegradation

• Bioavailability
• Production of surfactants
• Attachment to liquid-liquid interface
• Make cell surface more hydrophobic-nonpolar LPS
  or EPS

Low-density, non-aqueous phase liquid
(hydrocarbon, benzene
H2O
Dense, non-aqueous phase liquid (TCE, PCBs)
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Determinants of extent and rate of contaminant
biodegradation

• Bioavailability
• Sorption of contaminant to soil particles
• Diffusion of contaminant into soil matrix

Bacterial cell
Soil particles
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Determinants of extent and rate of contaminant
biodegradation

• Bioavailability
• Sorption of contaminant to soil particles
• Diffusion of contaminant into soil matrix

Contaminant no longer available to microbes
contaminant
Soil particles
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Determinants of extent and rate of contaminant
biodegradation

• Contaminant structure
• Steric effects

active site for enzyme blocked
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Determinants of extent and rate of contaminant
biodegradation

• Contaminant structure
• Electronic effects
• as electronegativity of substituents increased,
  biodegradation rates decreased

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Electonic Effects
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Determinants of extent and rate of contaminant
biodegradation

• Environmental factors
• organic matter (source of carbon and energy)
• subsurface, unsaturated zones have low organic
  matter concentrations
• oxygen availability
• nutrient (N,S, P) availability
• temperature
• pH
• Eh
• salinity
• water activity

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Most important factors controlling contaminant
biodegradation
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Bioaugmentation

• The addition of microorganisms with specific
  metabolic capabilities that are under-represented
  in the natural microbial populations that will
  promote degradation of the contaminant.
• This can increase carrying capacity of the system
  to degrade a contaminant

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• Bioaugmentation
• There are lots of companies around today that
  sell a variety of "formulations" to
• remove animal wastes
• keep ponds free of algae
• clean up gasoline leaked from underground storage
  tanks

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Hard to degrade contaminants

• Chlorinated hydrocarbons
• solvents
• lubricants
• plasticizers
• insulators
• herbicides and pesticides.

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Types of chlorinated compounds

• Aromatics
• Benzene
• Poly chlorinated biphenyls

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Meta-pathway for catechol degradation is often
used for degradation of chlorinated aromatics
Catechol is a common intermediate for
metabolizing many aromatic compounds and utilizes
enzymes encoded by the catA, catB, catC, and
catD genes
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CatR
cis, cis-muclonate
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• p ORF1 pheB pheA ORF2
• Promoter forms 2 complexes with catR
  in absence of inducer and 1
• complex with catR in presence of
  inducer
• catC catB p catR
  

plasmid
activation
CatR
chromosome
phenol, cis, cis-muconate are inducers
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The chlorocatechol degradative pathway is used to
degrade these chlorinated compounds.

• Similar to catechols, chlorocatechols are common
  intermediates of the degradation of
  chloroaromatics such as chlorobenzenes and
  chlorophenoxyacetates.
• Chlorocatechol-degrading genes that have been
  isolated from bacteria
• clc for chlorocatechol
• tcb for trichlorobenzene
• tfd for 2,4-dichlorophenoxyacetate

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The layout of the genes involved in
chlorocatechol-degradation on the plasmid is
similar to the layout of the catechol-degrading
genes on the chromosome
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• The clcABD operon is positively regulated by the
  clcR gene product, just as the catBC operon is
  controlled by the catR gene product.
• The clcA, tcbC and tfdC genes, all of which
  encode a similar chlorocatechol dioxygenase
  activity, have high nucleotide sequence identity.
• The clcB, tcbD and tfdD genes, all of which
  encode a similar chloromuconate cyclosomerase
  activity, have high nucleotide sequence identity.

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• Each of these chlorocatechol-degrading genes
  closely resembles the corresponding
  catechol-degrading cat genes, implying they
  evolved from common ancestral genes.
• CatR and ClcR cross-bind each others target
  promoter regions, indicating that the regulatory
  regions have considerable homology.
• CatR can regulate the clcABD operon but ClcR
  cannot regulate the catBC operon.

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Treatment strategies for subsurface contamination
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In situ
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Summary

• Many factors control biodegradability of a
  contaminant in the environment
• Before attempting to employ bioremediation
  technology, one needs to conduct a thorough
  characterization of the environment where the
  contaminant exists, including the microbiology,
  geochemistry, mineralogy, geophysics, and
  hydrology of the system