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Microorganisms and Organic Pollutants. Chapter 20 Lecture 16. Legacy Waste. 430,000 confirmed cases of leaking underground storage tanks in U.S. as of 2003 >90% of the monitored stream and >55% of shallow underground sites contaminated with pesticides

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legacy waste
Legacy Waste
  • 430,000 confirmed cases of leaking underground storage tanks in U.S. as of 2003
  • >90% of the monitored stream and >55% of shallow underground sites contaminated with pesticides
  • >1.4 million acres of chemical plumes in groundwater
dealing with the problem
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
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

What are environmental contaminants?
types of contamination
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
overall process of biodegradation
Overall process of biodegradation

Electron donor

Carbon source

Organic C

Carbon dioxide

H2O

Electron acceptor

O2aerobic respiration

NO3, Fe(III), Mn(IV), SO4, CO2anaerobic respiration

complete vs incomplete biodegradation

Complete

(mineralization)

Glucose→ CO2 + H2O

TCA cycle

Complete vs incomplete biodegradation

Incomplete

Glucose → pyruvate →→CO2 + H2O

TCA cycle

slide8

2e-, H+

2e-, H+

2e-, H+

H

Cl

Cl

H

Cl

Cl

Cl

H

Cl

Cl

H

H

Cl

H

H

H

H

Cl

Cl

H

Cl

Cl

Cl

Biological Reductive Dechlorination Pathway

PCE

TCE

cis-1,2-DCE

vinyl chloride

2e-, H+

Cl

Vinyl chloride intermediate is more

toxic than PCE

ethene

biological reductive dechlorination pathway
Biological Reductive Dechlorination Pathway

Hydrogen is preferred electron donor

VC ethene 93

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

degradation

products

contaminant

bacterium

corn

starch

CO2 + H2O

consortium interactions
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

combining cometabolism and consortium interactions
Combining cometabolism and consortium interactions

(TCE)

CH4 Cl2C=CHCl

Methanotrophic

bacterium

Methane Monooxygenase

O

CH3OH

Cl2-CHCl

Other populations

of bacteria

H2CO

HCOOH

CO2

CO2 + Cl-

carrying capacity
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
what limits carrying capacity
What limits carrying capacity?
  • Physical-chemical factors
    • pH, temperature, nutrients
  • types of microbes present and their biomass

Low carrying capacity High carrying capacity

Contaminant breakthrough

No contaminant left

determinants of extent and rate of contaminant biodegradation
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?)
determinants of extent and rate of contaminant biodegradation17
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)

determinants of extent and rate of contaminant biodegradation18
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)

determinants of extent and rate of contaminant biodegradation19
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)

determinants of extent and rate of contaminant biodegradation20

Contaminant

Particle

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

determinants of extent and rate of contaminant biodegradation21
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

determinants of extent and rate of contaminant biodegradation22
Determinants of extent and rate of contaminant biodegradation
  • Contaminant structure
    • Steric effects

active site for enzyme blocked

determinants of extent and rate of contaminant biodegradation23
Determinants of extent and rate of contaminant biodegradation
  • Contaminant structure
    • Electronic effects
      • as electronegativity of substituents increased, biodegradation rates decreased
determinants of extent and rate of contaminant biodegradation25
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
bioaugmentation
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
slide29
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
hard to degrade contaminants
Hard to degrade contaminants
  • Chlorinated hydrocarbons
    • solvents
    • lubricants
    • plasticizers
    • insulators
    • herbicides and pesticides.
types of chlorinated compounds

Cl

Cl

Cln

Cln

Types of chlorinated compounds
  • Aromatics
    • Benzene
  • Poly chlorinated biphenyls
meta pathway for catechol degradation is often used for degradation of chlorinated aromatics
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

slide34

CatR

cis, cis-muclonate

slide36
p ORF1pheBpheA 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

the chlorocatechol degradative pathway is used to degrade these chlorinated compounds
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
slide38

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

slide39
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.
slide40
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 other’s target promoter regions, indicating that the regulatory regions have considerable homology.
  • CatR can regulate the clcABD operon but ClcR cannot regulate the catBC operon.
summary
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