Biodegradation Microbiology

1
Biodegradation Microbiology
Biodegradation Microbiology

• Introduction
• Naturally occurring compounds
• Hydrocarbons
• Pesticides
• Insecticides
• Herbicides
• Fungicides
• Use patterns, transport and circulation in
  the environment

2
Molecular Models
1,4-dichlorobenzene
DNA molecule
P-cresol
Molecular Models
Molecular models of many of the compounds
mentioned in this section are available on the
course website To view these models, you need
to use a plug-in such as RASMOL After
downloading and installing plug-in, you can look
atthree-dimensional representations of molecules,
rotate them freely using the mouse, change the
colour scheme, zoom in and out, change the view
from basic stick representations to stick and
ball, etc. Note that some of the molecule files
are very large and may cause errors in display on
slower computer systems or slow internet
connections. Also note that when you first
click on the name of molecule from the list, it
may appear as a small window. Click on the
molecule name again and it should appear
full-size.
3
General Introduction

• Introduction
• Microbial degradation of chemicals in the
  environment is an important route for removal.
• The types of compounds range from plastics
  through organic chemicals (both industrial
  chemicals used in large quantities and trace
  chemicals such as pesticides) to organometallics
  such as methylmercury.
• The biodegradation of these compounds is often a
  complex series of biochemical reactions and is
  often different when different microorganisms are
  involved. The particular details of each
  biodegradation scheme are not particularly
  important, but the general series of reactions
  and enzyme types involved are relatively
  straightforward.
• The biodegradation schemes and pathways for
  biodegradation of many chemicals can best be
  understood by considering the same pathways for
  natural chemicals such as hydrocarbons, lignins,
  cellulose and hemicellulose. Many of the
  pathways, particularly the later stages of
  metabolism, are similar.

4
Biodegradation - Definitions
Biodegradation    -    Definitions Overview   
Biodegradation is the partial or complete
conversion of the compound of interest to its
elements. It usually mediated by microorganisms
but many macro-organisms can also carry out
biodegradative processes.  The term
"biodegradation" is usually applied to compounds
that are xenobiotic - compounds manufactured or
used by humans in the course of their activities
and thereby introduced as a "foreign"  substance
(xeno foreign) into an environment. It is also
often applied to the study of the
"biodegradation" of naturally occurring compounds
such as lignin or cellulose - typically, in that
case the compounds studied are those that are
more resistant to decomposition.  You will see
the terms "biotransformation", "partial
biodegradation" and "complete biodegradation" 
used in some literature. These terms are used
to distinguish between the complete decomposition
of a compound to its elemental form (complete
biodegradation) and an intermediate stage of
"partial biodegradation" to less complex
molecules. This is somewhat of an artificial
distinction. To restrict the use of the term
"biodegradation" to mean "complete
biodegradation" is  too limiting.
"Biotransformation" has also come to mean the
changing of a compound to another reasonably
stable molecule (often one that is useful or one
that is less or more toxic than the original)
5
Biodegradation - Interactions
Biodegradation of a given compound is a complex
result of the interacting factors listed in this
diagram. It is a function of the chemical
structure of the compound, the environmental
conditions, the organisms present and their
quantities, the adsorption, release and
solubility of the compound, the general
bioavailability of the compound, interactions
with other compounds present in the environment,
kinetics of growth and metabolism, threshold
effects, co-metabolic processes, acclimation
effects,  and others.
6
Biodegradation Interactions 2
Biodegradation of a given compound is a complex
result of the interacting factors listed in this
diagram. It is a function of the chemical
structure of the compound, the environmental
conditions, the organisms present and their
quantities, the adsorption, release and
solubility of the compound, the general
bioavailability of the compound, interactions
with other compounds present in the environment,
kinetics of growth and metabolism, threshold
effects, co-metabolic processes, acclimation
effects,  and others. Some factors appear in
more than one of these columns since they involve
more than one of the columns (Biological,
Chemical and Environmental) Remember that all of
these features are inter-related and that it is
often impossible to separate them neatly as we
have done here Some of the interacting factors
are summarised in the diagram. Note that these
same features and factors will be used in the
module on Applications/Bioremediation
7
Biodegradation - Concepts

• Some of the concepts in the diagram that are
  important in looking a biodegradation activities
  (and which may not be as familiar to you as the
  others) are
• Bioavailability
• Toxicity
• Thresholds
• Sorption (see Module on Groundwater)
• Acclimation (see 2,4-D herbicides)
• Recalcitrance (dealt with in this module)

8
Lack of Biodegradation

• There can be many reasons why a particular
  compound, although biodegradable in testing, is
  not biodegraded
• Required nutrients are missing
• Environmental conditions are unsuitable
• Toxic substance concentration is too high
• Compound may be at too low a concentration
• Compound may not be bioavailable

9
Biodegradation 2

• There can be many reasons why a particular
  compound, although biodegradable in testing, is
  not biodegraded
• Required nutrients are missing
• Environmental conditions are unsuitable
• Toxic substance concentration is too high
• Compound may be at too low a concentration
• Compound may not be bioavailable
• Required nutrients are missing - One or more
  nutrients required for growth are missing. The
  "limiting" nutrient is the one that is exhausted
  first in the growth cycle. This assumes that
  growth is required for biodegradation this may
  not be true if sufficient biomass is already
  present, or if the compound is degraded by
  enzymes already present.

10
Biodegradation 3

• There can be many reasons why a particular
  compound, although biodegradable in testing, is
  not biodegraded
• Required nutrients are missing
• Environmental conditions are unsuitable
• Toxic substance concentration is too high
• Compound may be at too low a concentration
• Compound may not be bioavailable
• Required nutrients are missing - One or more
  nutrients required for growth are missing. The
  "limiting" nutrient is the one that is exhausted
  first in the growth cycle. This assumes that
  growth is required for biodegradation this may
  not be true if sufficient biomass is already
  present, or if the compound is degraded by
  enzymes already present.
• Environmental conditions are unsuitable - There
  are many environmental conditions that can
  inhibit growth or metabolism high or low pH
  levels, high or low Eh (redox potentials), high
  or low temperatures, etc. A good example is in
  some peat bogs where organic materials may be
  over 20,000 years old.  

11
Biodegradation 4

• There can be many reasons why a particular
  compound, although biodegradable in testing, is
  not biodegraded
• Required nutrients are missing
• Environmental conditions are unsuitable
• Toxic substance concentration is too high
• Compound may be at too low a concentration
• Compound may not be bioavailable
• Required nutrients are missing - One or more
  nutrients required for growth are missing. The
  "limiting" nutrient is the one that is exhausted
  first in the growth cycle. This assumes that
  growth is required for biodegradation this may
  not be true if sufficient biomass is already
  present, or if the compound is degraded by
  enzymes already present.
• Environmental conditions are unsuitable - There
  are many environmental conditions that can
  inhibit growth or metabolism high or low pH
  levels, high or low Eh (redox potentials), high
  or low temperatures, etc. A good example is in
  some peat bogs where organic materials may be
  over 20,000 years old.  
• Toxic substance concentration is too high - Some
  conditions (high hydrogen sulfide levels, high
  acid concentrations, etc. may inhibit growth or
  metabolism.
• Compound may be at too low a concentration If a
  compound is degraded through the growth of
  microorganisms on that compound, there will be a
  minimum concentration below which growth will not
  occur. There will be an even lower concentration
  that will not even provide maintenance energy
  levels sufficient for the organism.

12
Bioavailability

• Bioavailability can be affected by
• Sorption to some solid materials in the
  environment (see Groundwater Module)
• Presence in a NonAqueous Phase Liquid (NAPL)
• Confinement or entrapment in physical soil or
  aquifer matrix
• Complexation
• Solubility (often translated to Octanol/Water
  Partition Coefficient)

13
Octanol-water partition coefficient
Octanol Water Partition Coefficient This measure
is the partition coefficient derived when a
compound is mixed with water and octanol and
allowed to equilibrate. It is supposed to
represent the degree of bioconcentration that
compound would undergo in animal tissues because
octanol is a surrogate for the lipids in animal
tissues. A compound that is very slightly
soluble in water (hydrophobic) is usually soluble
in lipids (or octanol). Compounds soluble in
water (hydrophilic) are not soluble to the same
extent in octanol or lipids.
14
Toxicity - Overview

• Toxicity is too large a field to cover adequately
  in this course. Briefly, toxicological analyses
  attempt to measure the effects of chemicals on
  different types of organisms.
• The dose-response of chemicals applied to these
  organisms is used to calculate a toxicity
  concentration that is then applied to regulate
  the allowable concentration of that chemical in
  the field.
• Toxicology tends to be divided into four main
  areas
• Hazard identification
• Exposure assessment
• Dose-response measurements - Quantitative risk
  characterization by estimation.
• Risk Assessment is the process of calculating,
  by integrating all of the available data from
  different sources, the risk that an individual
  will develop symptoms by a given time for a
  specified set of exposure conditions. The risk is
  the probability (from 0 to 1) of this happening
  to an individual.

15
Toxocity 2

• Some other points
• Toxicologists generally believe there is a
  threshold dose below which there are no toxic
  effects in any individual in a population.
• This is thought to be be true for most biological
  effects except carcinogenicity where a single
  molecule could in theory cause a deleterious
  mutation. The basis for this belief is that are
  doses of toxic chemicals where it is impossible
  to find differences between treated and untreated
  organisms.
• Unfortunately, every test has a statistical
  "resolving power" or limit of detection based on
  the number in the sample population exposed and
  the statistical analysis of the results. It also
  ignores the effect of a very small dose on
  pre-existing toxicity from other chemicals. Even
  that small addition may then cause a small
  increase in effects. 

16
Thresholds - Overview

• Since many organic pollutants are present only in
  very low concentrations in the environments, the
  concept that there might be a minimum
  concentration below which they would not be
  biodegraded is and important and interesting one.
  The concept of a threshold concentration that is
  below that needed for microorganisms to take in,
  assimilate and biodegrade is therefore very
  important.
•   

17
Thresholds 2

• Since many organic pollutants are present only in
  very low concentrations in the environments, the
  concept that there might be a minimum
  concentration below which they would not be
  biodegraded is and important and interesting one.
  The concept of a threshold concentration that is
  below that needed for microorganisms to take in,
  assimilate and biodegrade is therefore very
  important.
• Note Even though the concentrations of compounds
  in the field can be low (at the threshold
  concentration for biodegradation), they still can
  have important effects. Such as
• Very low levels of a compound, when applied to
  an entire population, may cause significant risk
  to some of that population.
• Compounds can be bio-concentrated to higher
  levels in target organisms and cause effects (see
  DDT)
• Even at these low concentrations, some compounds
  are toxic, especially to aquatic organisms
• Some standards for human and ecosystem health
  are set very low because the risk analysis
  procedures tell us that the risk is only
  acceptable at these low acceptable
  concentrations.
•   

18
Naturally occurring materials
Naturally Occurring Compounds
There are some compounds which are
naturally-occurring and are also recalcitrant
they are resistant to biodegradation in the sense
that they are not rapidly degraded in nature.
One of the most important of this group is
lignin. Lignin is a complex polymeric compound in
plants that is degraded only very slowly when it
enters the soil.
19
Biodegradation

• Microorganisms respond to the presence of
  organic materials by growing rapidly and using
  the easily available parts of the organic
  material.
• Different populations of microorganisms then use
  more resistant parts until a recalcitrant or
  resistant fraction remains. The order of use is
  often
• carbohydrates
• proteins
• cellulose and hemicellulose
• lignins and associated compounds.
• There are distinct populations of microorganisms
  able to degrade each of these categories of
  organic materials.
• The environmental conditions play a large role
  in determining the speed at which the materials
  are used.
• The physical form of the materials (finely
  divided versus granular, crystalline cellulose
  versus amorphous cellulose, etc.) also plays a
  large part in determining the rate of reaction.

20
Factors

• The most important environmental factors are
• Temperature
• pH
• water content
• oxygen content
• There are therefore three main determinants of
  the fate of organic materials added to a soil or
  water system
• Microbial activities
• Environmental conditions
• Chemical structure of the compound.
• Each of these factors works by affecting the
  microbial colonization and breakdown of the
  materials. Two examples will be used cellulose
  breakdown and lignin breakdown

21
Cellulose

• Cellulose breakdown
• Cellulose is a carbohydrate consisting of b 1-4
  linked glucose units in a linear chain. It is
  degraded by a very wide range of bacteria and
  fungi and is degraded under a variety of
  different environmental conditions.
• Typically bacteria of the genera Streptomyces,
  Cytophaga, Cellulomonas, Nocardia, and Vibrio are
  involved in cellulose breakdown while Clostridium
  is an important anaerobic cellulose degrading
  organism.
• Some bacteria can degrade it at very high
  temperatures (C. thermocellum).
• Anaerobic metabolism results in low molecular
  weight fatty acids and carbon dioxide while
  carbon dioxide is the main metabolite under
  aerobic conditions.
• Aerobic decomposition is thus more efficient
  than anaerobic more cellulose-carbon is
  transformed to CO2 and biomass.
• Many fungi are also extremely active in
  cellulose decomposition Trichoderma, Chaetomium,
  Aspergillus, Fusarium, and Phoma are particularly
  active in soils.

22
Cellulose

• The enzymatic processes are similar in most
  microorganisms
• extracellular cellulase enzymes are produced.
• There are several different compounds in the
  cellulase enzyme system.
• The normal cellulase enzyme complex contains
  three types of enzymes
• a Cl enzyme which acts primarily on native
  cellulose and not on partially degraded
  cellulose,
• a Cx enzyme which acts upon partially degraded
  cellulose molecules in two ways the endo 1-4
  glucanases break the cellulose internally at
  random while the exo 1-4 glucanases attack the
  end of the chains resulting in cellobiose
  molecules.
• The degradation of the cellobiose and other
  small fragments is by the enzyme b-glucosidase
  that forms glucose from these fragments.

23
Lignin
Lignin

• Lignin is a complex polymer found in plants,
  especially in woody plants.
• It is an amorphous, three-dimensional aromatic
  polymer composed of oxyphenylpropane units.
• It is formed by polymerization in the plant of
  cinnamyl alcohols p-coumaryl alcohoI, coniferyl
  alcohol and sinapyl alcohol

24
Lignin
There are three main types of lignin, depending
on the type of plant   1. guaiacyl lignin from
conifers, lycopods, horsetails and ferns. Mainly
coniferyl alcohol units with small amounts of
coumaryl and sinapyl alcohol units.  2.
guaiacyl-syringyl lignin from dicotyledonous
angiosperms and some gymnosperms. Equal amounts
of coniferyl and sinapyl units with minor amounts
of coumaryl units.  3. guaiacyl-syringyl-p-hydr
oxyphenyl lignin in the highly evolved plants and
woody tissues of conifers. Equal amounts of all
three types of units.
25
Partial structure of a hypothetical lignin
molecule from European beech (Fagus sylvatica).
The lignin of beech contains units derived from
coniferyl alcohol, sinapyl alcohol, and
para-coumaryl alcohol in the approximate ratio
100707 and is typical of angiosperm lignin.
26
Lignin

• O - (ether links) are very common
• Phenyl-C-C-C structure is common
• Highly aromatic nature of material
• Large number of methoxy groups
• Large number of - O - cross links between
  subunits

27
Lignin Biodegradation

• Lignin biodegradation
• The lignin molecules are degraded by the
  "white-rot fungi" and some bacteria.
• There are several hundred species of white rot
  fungi including members of the Agaricaceae,
  Hydnaceae, Polyporaceae, and the Thelephoraceae.
• The white rot basidiomycetes are probably the
  most efficient microorganisms at degrading
  lignin. The soft rot fungi include Chaetomium
  spp., Paecilomyces andAllescheria spp.
• Bacteria such as Pseudomonas, Xanthomonas,
  Acinetobacter, Bacillus, Arthrobacter,
  Micrococcus, Aeromonas, Chromobacterium, and
  Flavobacterium, have all been implicated in
  lignin breakdown.
• Good evidence has been given for Streptomyces,
  Bacillus, Nocardia, and Pseudomonas spp. in that
  they have released 14CO2 from radiolabelled
  lignin molecules.

28
Hydrocarbons
Biodegradation
Hydrocarbons

• Importance of hydrocarbon microbiology
•  
• Oil spill problems (marine and freshwater)
• Degradation of stored oil supplies
• Use of hydrocarbons as carbon source for
  microbial food production (?)
• Oil prospecting using microbial distributions in
  soils and air
• Use of bacteria to release oil from oil sands
• Waste disposal - methane production, etc.
• Soil gases and groundwater evolution

29
Hydrocarbon Biodegradation 1
Hydrocarbon biodegradation The main types of
hydrocarbons are Alkane C-C single
bond Alkene CC double bond Alkine CC
triple bond Alicyclic Aromatic Complex
Aromatic
30
The United States is only able to domestically
extract 36 of the crude oil consumed.
31

• Canada holds the second largest oil reserves in
  the world, with over 178 billion barrels of oil.
  Over the next decade, Canadas importance as a
  leading oil producer is expected to increase, as
  oil sands production is projected to triple.
  Other key non-OPEC producers include Russia, the
  United States, Mexico, China and Norway.

32
Gas Chromatography
Natural sources of hydrocarbons tend to be
complex mixtures of the different types above.
The particular composition varies so greatly that
it is often possible to analyze a particular
hydrocarbon mixture with such discrimination that
the oil well and certainly the oil field, where
it originated can be identified. This is usually
done by gas chromatographic techniques, yielding
"fingerprints" of the particular oil
Analysis by gas chromatographic techniques are
essentially fractional distillation methods
higher boiling point hydrocarbons are delayed in
the GC column and so elute later in the process.
In other words, different fractions of the oil
"boil off" at different temperatures. This is
the basis of oil refining, where the crude
hydrocarbon is heated and the different
components are collected at different
temperatures. After refining, the gasoline,
diesel fuel, kerosene, etc., have a much less
complex series of different chain lengths of
hydrocarbons. Octane is a component of gasoline
and it is the standard by which the gasoline is
rated - the "octane rating". Diesel fuel is less
volatile (higher chain lengths). The crude
hydrocarbon also contains other components such
as metals (vanadium, nickel) and sulfur as well
as many different aromatic compounds.
33
Comparison of hydrocarbon distribution from
different types of oil Top NIST Standard oil
1582 Middle Gulf of Mexico oil May
2010 Bottom NIST standard 1494
34
Mechanisms General 1

• Hydrocarbons
• Mechanisms of degradation
• General
• Hydrocarbons are only degraded in water.
• Bacteria and fungi do not grow in hydrocarbons,
  they grow on water in or surrounding the
  hydrocarbons.
• All hydrocarbons are soluble to some extent.
• This is why the process of degradation is able
  to start and then continue. Without the presence
  of water, oil is not degraded.
• Emulsion formation is an important part of
  hydrocarbon degradation. There are two kinds of
  emulsion
• Oil-in-water emulsion are common and occur when
  small quantities of oil are present in large
  quantities of water. Small droplets of oil are
  suspended in water.

35
Mechanisms General 2

• Water-in-oil emulsion are less common and occur
  when large quantities of oil are present in
  water.
• These are droplets of water suspended in a matrix
  of oil.
• The damage caused by oil spills in marine or
  freshwater systems is usually caused by the
  water-in-oil emulsion.
• A thick layer of crude oil on the surface of
  water will take up about 50 water by weight and
  remain a free-flowing, oily liquid.
• It can spread over the water surface to form an
  oily film and will eventually disperse and be
  degraded.
• When the water content reaches about 80 by
  weight, the consistency of the emulsion changes -
  it becomes a thick semi-solid mass with a
  grease-like consistency. It is often called
  "chocolate mousse" at this stage because of both
  its consistency and its brown colour. This
  process does not readily occur with fuel oils
  such as diesel oil and kerosene, but occurs
  easily with light and heavy crude oils - the
  usual cargoes of oil tankers.

36
Microorganisms
Many types of microorganisms can degrade
hydrocarbons. Bacteria, yeasts, and filamentous
fungi all have taxa that degrade some types of
hydrocarbon molecules. Bacteria - The
heterotrophic bacteria utilize carbon from the
hydrocarbons as a source of carbon and energy for
biomass production. There are many genera of
bacteria which carry out these reactions on
different hydrocarbons. They include Bacillus,
Pseudomonas, Mycobacteria, and actinomycetes
(especially Nocardia spp.) The autotrophic
bacteria Thiobacillus and Desulfovibrio can both
metabolize the sulfur component of crude oil.
Thiobacillus can metabolize S to H2SO4 (sulfuric
acid) and Desulfovibrio metabolizes this under
anaerobic conditions to sulfide. Both sulfide and
sulfuric acid can damage metal containers and
fuel systems. This is not true utilization of
hydrocarbons as carbon sources, but is important
in practical terms any water present in oil
storage tanks may lead to the growth of these
organisms and subsequent damage. Fungi - Many
yeasts are active in soils and water systems in
hydrocarbon degradation, as are some very common
genera of mycelial fungi such as Trichoderma,
Aspergillus and Cladosponum.
37
Rates of Biodegradation
None of these microorganisms degrade all of the
possible hydrocarbon molecules at the same rate
if at all. There is a definite preferential use
of certain chain lengths of aliphatic
hydrocarbons and each organism may have a
different spectrum of activity. In general
terms, the utilization of n-alkanes (non-branched
alkane hydrocarbons) is reasonably well
established
Gases
Liquids
Solids
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 ------
C19 C20 C21C22 C23 C24..
Not susceptible tomicrobial attack
Attacked by manymicroorganisms
Solid at room temperaturenot susceptible to
attack
Methane is attacked only by very specific groups
of bacteria
This means that a crude oil subjected to
microbial action will lose some of the
hydrocarbon chain lengths and retain others it
will be "enriched" in those hydrocarbons not
susceptible to microbial decompositlon.
38
Oil releases
The graph below shows how many millions of
gallons of oil each source puts into the oceans
worldwide each year                           
                                    
Routine Maintenance 137 Million
Gallons Every year, bilge cleaning and other
ship operations release millions of gallons of
oil into navigable waters, in thousands of
discharges of just a few gallons each. 137
million gallons Down the Drain 363 Million
Gallons Used engine oil can end up in waterways.
An average oil change uses five quarts one
change can contaminate a million gallons of fresh
water. Much oil in runoff from land and municipal
and industrial wastes ends up in the oceans. 363
million gallons. Road runoff adds up Every year
oily road runoff from a city of 5 million could
contain as much oil as one large tanker spill
http//www.ddluk.com/oilspillteachers/weblinkInfo.
html
39
Oil Releases 2
Up in Smoke 92 Million Gallons Air pollution,
mainly from cars and industry, places hundreds of
tons of hydrocarbons into the oceans each year.
Particles settle, and rain washes hydrocarbons
from the air into the oceans . Offshore
Drilling 15 Million Gallons Offshore oil
production can cause ocean oil pollution, from
spills and operational discharges
. Natural Seeps 62 Million Gallons Some
ocean oil "pollution" is natural. Seepage from
the ocean bottom and eroding sedimentary rocks
releases oil. Big Spills 37 Million
Gallons Only about 5 percent of oil pollution in
oceans is due to major tanker accidents, but one
big spill can disrupt sea and shore life for
miles
Gulf of Mexico Deepwater Horizon spill 2010
40
Oil Spills
A large recent spill was the deliberate emptying
of 250 million gallons into the Persian Gulf by
the Iraq government. This was in addition to 80
million gallons spilled into the Gulf during the
Iran-Iraq war in 1983
In Module 5- Background Materials -
http//wvlc.uwaterloo.ca/biology447/modules/module
5/5_back1.htm
41
Marine Oil Spills
Marine Oil Spills and Remediation of the Exxon
Valdez Oil spill 
WebLink
Go to the Exxon Valdez Spill WWW site
to get updates on Cleanup The Exxon Valdez
accident spilled 11,000,000 gallons of crude oil
in Prince William Sound, Alaska on March 24,
1989. About 2.5 billion has been spent by Exxon
plus about 160 million by state and the federal
governments in clean up efforts. Exxon's efforts
were concentrated on beach cleanup and oil
recovery. The Exxon Valdez was only one of
numerous oil spills occurring around the world. 
 


42
Marine Oil Spills and Remediation of the Exxon
Valdez Oil spill 
WebLink
Go to the Exxon Valdez Spill WWW site
to get updates on Cleanup The Exxon Valdez
accident spilled 11,000,000 gallons of crude oil
in Prince William Sound, Alaska on March 24,
1989. About 2.5 billion has been spent by Exxon
plus about 160 million by state and the federal
governments in clean up efforts. Exxon's efforts
were concentrated on beach cleanup and oil
recovery. The Exxon Valdez was only one of
numerous oil spills occurring around the world. 
 

• Tanker accidents contribute only about 5 percent
  of the total oil entering the seas each year. In
  1985 it was 12.5 percent.
• The total entering the seas may be as large as
  2.5 million gallons per year (U.S. Coast Guard
  estimates).
• There were 16,000 accidents in 1988 - spilling
  four times the amount of the Exxon Valdez oil
  spill into US waters.
• About 35 of the oil entering the seas each year
  comes from normal ballasting and washing
  operations of tankers and 36 comes from urban
  and industrial run-off. 

43
Fate
What happened to the oil released into the
environment? A 1992 National Oceanic and
Atmospheric Administration (NOAA) study provided
some insight, estimating that the great majority
of the oil either evaporated, dispersed into the
water column or degraded naturally. Cleanup
crews recovered about 14 percent of the oil and
approximately 13 percent sunk to the sea floor.
About 2 percent (some 216,000 gallons) remained
on the beaches
44
Exxon Valdez 1997
In 1997, eight years after the oil spill,
villagers from Chenega Bay returned to nearby
beaches to clean some of the most heavily-oiled
sites. The crew of mostly-local residents
applied a chemical agent to the weathered oil at
five sites, along about one-half mile of beach on
LaTouche and Evans islands. They used PES-51, a
citrus-based product from the oil of oranges and
lemons. PES-51 binds to the oil and floats,
allowing both the chemical agent and the oil to
be collected through the use of oil-absorbent
pads.  
45
Exxon Valdez 2001

• The most recent survey of lingering oil was
  conducted in the intertidal zone of Prince
  William Sound in Summer 2001 by NOAA. The survey
  covered roughly 8,000 meters of shoreline.
  Ninety-six sites were randomly selected from the
  total number of oiled beaches assessed during
  previous surveys he survey results indicate a
  total area of approximately 20 acres of shoreline
  in Prince William Sound are still contaminated
  with oil.
• Oil was found at 58 percent of the 91 sites
  assessed and is estimated to have the linear
  equivalent of 5.8 km of contaminated shoreline.
• Buried or subsurface oil is of greater concern
  than surface oil.
• Subsurface oil can remain dormant for many years
  before being dispersed and is more liquid, still
  toxic, and may become biologically available.
• A disturbance event such as burrowing animals or
  a severe storm reworks the beach and can
  reintroduce unweathered oil into the water.
• Results of the 2001 survey showed that the oil
  remaining on the surface of beaches in Prince
  William Sound is weathered and mostly hardened
  into an asphalt-like layer.
• The toxic components of this type of surface oil
  are not as readily available to biota, although
  some softer forms do cause sheens in tide pools.

46
Exxon Valdez 1
Whatever else the Exxon Valdez spill did, it led
to a massive cleanup operation and the first
large scale testing of some bioremediation
technologies in a reasonably controlled
experimental environment. Some problems were
that the initial cleanup was by high-pressure hot
water sprays onto the beaches. Very little oil
was actually recovered by the "skimmers" and boom
flotation devices that were used to try to
contain and recover the raw oil. Estimates range
up to a recovery of 5 to 7 of the spilled oil.
Many different treatment systems were tried
ranging from some new low toxicity dispersants to
steam cleaning to addition of fertilizers to
promote bioremediation. The dispersants have to
be applied within the first 36 hours to be
effective at all and they were not used after
that time.
Exxon Valdez
   
47
Aerial photo of Exxon Valdez oil coating Alaska
beach
The addition of nutrients was tested to see if
they stimulated or promoted biodegradation of oil
on beaches contaminated with oil. The types of
nutrients ranged from water soluble mixtures
(232 NP garden fertilizer formulation) through
slow release fertilizers (isobutylenediurea) and
oleophilic (oil soluble - Inipol EAP 22 oleic
acid, urea, lauryl phosphate). Each was tested
in the laboratory and in small scale field
experiments. Application rates were adjusted to
minimize eutrophication effects and fish
toxicity. The most effective in the tests was the
oleophilic group. Two (Inipol and Customblen)
were eventually approved for use in the field.
Initial claims were that the beaches were
"significantly cleaner" after application of the
oleophilic fertilizers. 
48
Exxon Valdez 2
Exxon Valdez Some researchers thought that using
dispersants could transport the oil into deeper
waters and cause more damage to marine organisms.
Another method that was NOT tried at Prince
William Sound, but that is often effective if
used within the first few hours, is burning the
floating oil slick About 90 can be removed by
this method but it causes air pollution. The oil
layer must be more than 3 mm thick and should not
have formed the oil-in-water emulsion that
prevents burning. High wave action promotes
emulsion formation. One of the major problems
with the science at Prince William Sound was that
it was driven by urgency and controlled
experiments were very infrequent. Much of the
data is also sealed - to be used in any upcoming
law suits. Because of this, data of recovery
percentages and sub-lethal toxic effects are
essentially unavailable . The secrecy
surrounding the data even on the extent,
frequency and temperature of water during the hot
water washing sessions makes meaningful
comparisons with other methods very difficult to
carry out. This is coupled with a very low
baseline data level about Prince William Sound in
the first place almost no data on the situation
existing before the spill is available. Only 10
or 12 very short stretches of beach were set
aside to act as control sections. The
scientists from NOAA (National Oceanic and
Atmospheric Administration) had to fight the oil
company and their consultants and operating
cleanup companies as well as the local people to
get even that much.
Biodegradation Scientists from Exxon and the EPA
tried to speed up the natural rates of
biodegradation by hydrocarbon-utilizing bacteria
and yeasts by adding nutrients to the system. The
best that can be said about the results is that
they are still in doubt some scientists say they
achieved an increase (Exxon says 5 to 10 fold,
Ron Atlas and the EPA say about 2 fold) of the
rate of biodegradation and others say the natural
process would have been just as fast.
49
Biochemistry 1
There are many different reactions involved with
the degradation of hydrocarbons by
microorganisms. We will deal only with the more
common reaction types. A) Hydroxylation at
C1. B) Hydro-peroxidation C) Dehydrogenation
reactions D) Subterminal reactions
50
Biochemistry 2
Note molecular oxygen
51
Biochemistry 3
Note No molecular oxygen is required
52
Biochemistry 4
Note that the first three processes deal with a
terminal -CH3 group while subterminal oxidation
process "splits" the alkane at a subterminal
site.
Note molecular oxygen is required
53
Generalizations

• There are some generalizations that can be made
  concerning the biodegradation of aliphatic
  hydrocarbons (alkanes, alkenes and alkines
  alkynes ).
• The chain length has a significant effect on
  biodegradability it is almost the same for
  alkanes, alkenes and alkines alkynes. Not a
  simple relationship to number of carbon atoms.
• Aliphatic compounds are more easily degraded with
  decreasing saturation and increasing reactivity
  Thus
• Degradation rates
• Alkines alkenes
  alkanes
• The degree and type of branching in a structure
  has a marked effect on the biodegradability of
  aliphatic compounds. In general terms, branching
  decreases biodegradability.
• In aliphatic compounds with very large numbers of
  carbons, the presence of branching may bring the
  branch chain length down to the number of carbon
  atoms which can be degraded this would promote
  biodegradability compared to the compound with
  the same number of carbon atoms but in a
  non-branched linear chain.

Faster than
Faster than
54
Aromatic HC
Aromatic hydrocarbons  The aromatic ring
structure and the convention for naming various
substituent groups is given below.
If a phenol is considered, then
other compounds with groups at the different
carbon position can be named either by the ortho,
meta, para convention, or by the 1,2- 1,3- and
1,4-, etc. convention. For instance, a
chlorophenol with a chlorine atom at position
number 3 would be 3-chlorophenol or
meta-chlorophenol Benzene itself is quite
susceptible to decomposition in some ecosystems.
55
Aromatic - Groups
Adding another substituent group has a
significant effect on the rate of degradation of
the new compound. For example, adding different
substituent groups to benzene (producing mono
substituted benzenes) produces the following
result
Group added to benzene
Persistence in soil.(half-life) - Days
56
Phenol Substitution
OH
-Cl -Br Substituent groups
57
Summary of Substitution

•  
• The effects of these substituent groups may be
  summarized
• Single substituent groups on benzene affect the
  degradation in the order (COOH or OH) -gt NH2
  -gt OCH3 -gt SO3H -gt NO2 (increasing
  persistence).
• Meta substitution for halogens on phenol causes
  the greatest persistence.
• Ortho- and para-substitution of halogens has
  less effect.
• Increasing amounts of chlorination or
  bromination in a molecule increase persistence.

Metabolic pathways for aromatic hydrocarbon
decomposition There are many pathways of
breakdown for aromatic compounds. Many of them
have catechol as a central intermediate.
The next stage is ring fission of catechol,
followed by incorporation into the normal
biochemical pathways of the cell. This can be
by two distinct mechanisms ORTHO or META
cleavage
OH
OH
catechol
58
Generalized aromatic metabolism
Generalized aromatic metabolism
59
Ortho and Meta pathways
60
Dehalogenation
Dehalogenation

• One irnportant mechanism in the breakdown of
  many compounds is the removal of the halogen
  atoms, which confer much of the resistance to
  decomposition of such diverse compounds as the
  insecticide DDT, polychlorinated biphenyl (PCB),
  pentachlorophenol (PCP), halogenated benzenes
  (e.g. hexachlorobenzene), etc.
• This is often a process of dehalogenation
  involving several mechanisms, by which halogen
  atoms are removed from the molecules.
• These processes can occur under both anaerobic
  (reductive dehalogenation) and aerobic conditions
• Some compounds (such as the chlorinated
  benzenes) appear to be only dehalogenated under
  aerobic conditions, whereas other compounds can
  be dehalogenated under either anaerobic or
  aerobic conditions.
• Yet other compounds (some pesticides and
  halogenated 1 and 2 carbon compounds) undergo
  only reductive dehalogenation.

61
Benzene utilization
Utilization of benzene Surprisingly little
information was available before 1968. Early
observations showed that bacteria grown on
benzene were able to metabolize catechol (see
above). Experiments with Pseudomonas putida by
Gibson and his colleagues showed two routes to
catechol from benzene.                       
  
62
Routes from benzene to catechol
Generalized metabolic routes from benzene to
catechol. The enzymes involved have been
extracted from a number of bacteria including
Pseudomonas, Moraxella and Arthrobacter. A three
component enzyme system was found to be
operative 
The conversion from cis-benzenediol to catechol
was by a dehydrogenase enzyme - cisbenzenediol
dehydrogenase - coupled with NAD.
63
PAHs
Polyaromatic hydrocarbons

• These compounds are fused ring aromatic
  hydrocarbons - common in coal products and coal
  byproducts.
• They are ubiquitous pollutants in the atmosphere
  and are relatively resistant to biodegradation
  they can therefore accumulate to substantial
  levels in the environment.
• Since some of the larger species are
  carcinogenic, they can pose a significant health
  hazard.
• There are more than 70 compounds classed as PAHs
  (or Polynuclear Hydrocarbons PNAs) and they have
  from 2 to 7 rings.
• They have been detected in a wide range of soils
  and sediments, including some ancient sediments.
  Significant quantities are present in both
  industrial and domestic effluents and sometimes
  cause problems in waste water treatment.
• Another source is from hydrocarbon spills, the
  more biodegradable aliphatic and aromatic
  fractions are removed leaving the more resistant
  fractions these often include PNA's which can
  sink to the bottom of water bodies and become
  permanent pollutants of oceans, lakes and rivers.
  

64
PAHs (contd)
Polyaromatic hydrocarbons (contd)

• They are produced in large quantities from
  coking operations and gasification processes
  using coal and from other sources of incomplete
  combustion such as automobile exhausts, power
  generation plants, refuse burning and industrial
  emissions.
• They can also have a natural origin in coal
  deposits, from natural aromatics such as
  terpenes, sterols and quinones from plants which
  volatilize and can become condensed to PAHs
  (PNAs).
• Plant lignins also may become progressively
  decomposed to humic substances which can become
  larger during maturation of the pest or coal
  deposits and can eventually produce PAHs (PNA's)
• Several species of bacteria have produced PAHs
  in agar devoid of hydrocarbons but containing
  glycerol.
• Algae can also synthesize PHAs.
• In summary, PHAs are mainly of anthropogenic
  origin, but also have geological and natural
  biological origins.

65
Examples of PAHs
Examples of common polyaromatic hydrocarbons
A listing of more than 650 PAHs and their
chemical names, properties and CHIME molecular
diagrams from NIST (Special Publication 922)
66
PAH Biodegradation

• Biodegradation of PAH's
• Factors affecting biodegradability include
• The number of fused rings
• Number and position of substituents of the
  rings.
• Degree of ring saturation.

• In a typical experiment
• There was no significant oxidation of compounds
  with more than three rings. Four-membered ring
  systems are oxidized at negligible rates.
  Five-membered ring systems underwent no
  significant oxidation
• Naphthalene nuclei with a small alkyl group
  (methyl, vinyl or ethyl) were oxidized rapidly
  while those with phenyl were extremely resistant.
  
• A substituent at position 2 allowed faster
  oxidation than at position 1 (less steric
  hindrance)
• Other methyl-substituted ring systems were
  oxidized slowly.
• Substituted naphthalene with more than 1 methyl
  group on one ring had at least a small amount of
  oxidation.
• Methyl substitution of methyl groups on both
  rings led to decreased rates of oxidation.
• Increased ring saturation) led to decreased
  oxidation.
• As long as one aromatic ring was available, some
  measurable oxidation rate was achieved.
• The perhydro compounds (cis- and trans-decaline,
  perhydrophenanthrene, hexahydroindane and
  perhydrofluorene) were resistant to
  decomposition.

End of Section Continued in Next Module
67
Pesticide Biodegradation
Biodegradation - Pesticides

• Pesticides including
• Insecticides
• Herbicides
• Fungicides
• Use patterns, transport and circulation in the
  environment

68
Overview
The factors that influence biodegradation are the
same for pesticides as for other compounds We
will concentrate on the biochemistry, the
chemical and environmental factors and
recalcitrance in this Module 5 and deal with the
kinetics and other aspects in Module 7.
69
Insecticides Chlorinated hydrocarbons
Insecticides Chlorinated hydrocarbons

• DDT and analogues  
• The outline below gives a general picture of the
  many interconversions that DDT can undergo in the
  environment.
• Many of the analogues of DDT are breakdown
  products of the DDT itself.
• The main reaction route in microorganisms is
  through TDE (DDD) and TDEE.
• It is a direct reductive dechlorination process
  and is carried out by bacteria in the soil and
  water.
• Studies on the metabolic fate of the other
  products of DDT metabolism are limited.
• The importance of DDT is emphasized when
  examining the distribution of the material in the
  environment.

70
DDT Biodegradation
Biodegradation of DDT
dechlorination
dechlorination
71
DDT - details
From the University of Minnesota
Biocatalysis/Biodegradation Database -- LINK
72
DDT and analogues
Note increasing levels at higher levels in the
food chain - Bioaccumulation
73
Cyclodienes

• BHC (lindane)  
• The gamma isomer of BHC was extensively used as
  an insecticide studies on its biodegradation are
  limited. It does not accumulate to the same
  extent as DDT in animal tissues.
• Chlorinated cyclodiene insecticides  
• These compounds represent one of the more
  persistent groups of pesticides. Examples are
  aldrin, dieldrin, heptachlor, isodrin and endrin.
  Aldrin and dieldrin have been banned in many
  jurisdictions and are only used for particular
  purposes in many other jurisdictions.
• In soil and water, degradation is probably
  microbial but very often leads to the formation
  of an epoxide ring structure in the molecules.
•  

Aldrin
Dieldrin
Heptachlor
74
Cyclodienes

• This is formed from the CHCH group of the least
  chlorinated ring. This process of epoxidation
  converts aldrin to dieldrin, isodrin to endrin,
  and heptachlor to heptachlor epoxide. All the
  epoxide forms are more stable than the parent
  form.
•  
• Oxidation processes of cyclodiene compounds
  yield more stable toxic intermediates
• The rates of disappearance of the various
  cyclodiene insecticides are as follows on the
  next slide

Stable toxic intermediates
75
Stable Toxic Intermediates
The so called "stable toxic intermediates" are
not completely inert (as can be seen by their
eventual breakdown by microorganisms).
Heptachlor
Heptachlor epoxide
76
Organophosphates
Organophosphorous compounds
The group contains many well known insecticides
such as malathion, parathion, methyl parathion,
mevinphos, sevin, and diazinon. Their persistence
is of a lower order of magnitude than the
organochlorine insecticides above. They are not
particularly persistent pesticides, with half
lives in soils measured in weeks or months.
They are metabolised by many different
microorganisms, particularly members of the
Pseudomonas, Arthrobacter, Streptomyces, and
Thiobacillus genera and by the fungi in the
Trichoderma genus. The formula of the
organophosphorus insecticides is relatively
simple Malathion
Parathion
77
Organophosphates
There are many sites of attack for microorganisms
in these molecules. There are therefore many
different possible breakdown patterns. For
example, in malathion, the sites of attack are
marked A,B,C, and D and can be attacked by
phosphatase, mixed function oxidase, and carboxyl
esterase enzymes A phosphatase and
mixed function oxidases B mixed function
oxidase C phosphatase D carboxyl
esterase Many different products can be formed.
It has been shown that autoclaving soil destroys
about 90 of its ability to degrade malathion.
However, a treatment with sterilizing levels of
gamma irradiation does not affect the soils
ability to degrade malathion. It was possible to
extract a fraction of the soil with 0.2N NaOH
that activity degraded malathion. It is therefore
possible that some organic compound in soils is
able to chemically degrade some organophosphorus
insecticides.
methyl parathion
Diazinon
78
Carbamates
Carbamates 

• Carbaryl is an example of the carbamate
  insecticides. Its formula is
•                                                
                 
• Other examples are 3-keto carbofuran,
  carbanolate, propoxur, carbofuran and landrin.
  They are moderately persistent insecticides.
  Typical mechanisms of biodegradation are by
• Ring hydroxylation
• 2) N-methyl hydroxylation

79
Insecticides - Summary
Insecticides - Summary

• Insecticides range in persistence from
  organophosphates (1 week to 16 months), through
  carbamates (2 months to 14 months), to
  organochlorines (18 months to 25 years).
• In most cases, the persistence can be explained
  by the chemical structure and by the degree of
  water solubility.
• "Stable toxic intermediates" can be produced,
  but these are eventually degraded in most cases.
• Bioaccumulation can occur with the more lipid
  soluble and persistent insecticides such as DDT.
• In certain cases, chemical breakdown of the
  organophosphorus insecticides is possible

80
Herbicides - Phenoxyacetic
Herbicides - Phenoxyalkanoic acids and
derivatives
The three main groups are the phenoxyacetic,
phenoxypropionic, and 4-phenoxybutyric
derivatives. Many of them are the acids of these
groups, but more recently, forms such as esters
and amines are becoming more common. The basic
structure is
Phenoxyacetic group
1 CI 2 CI 2,4-dichlorophenoxyacetic (2,4-D)
3 H   1 CI 2 CI 2,4,5-trichlorophenoxyaceti
c acid (92,4,5-T) 3 CI   1 CH 2 CI MCPA 3
H
81
Herbicides - Phenoxypropionic
Phenoxypropionic group
1 CH3 2 CI mecoprop 3 H   1 CI 2 CI
2(2,4,5-trichlorphenoxy) propionic acid -
silvex 3 CI
82
Herbicides - Phenoxybutyric
Phenoxybutyric group
1 CH3 2 CI MCPB (4-(2-methyl-4-chloro-phenoxy)
butyric acid 3 H   1 CI 2 CI
2,4-DB4-(2,4-dichlorophenoxy)butyric acid  
1 CI 2 CI 2,4,5-DB4-(2,4,5,trichlorophenoxy)
butyric acid 3 CI
83
Herbicides - Sesone
A closely related compound to the phenoxy group
Is Sodium 2,4-dichlorophenoxy
ethyl sulfate (Sesone herbicide)
84
Herbicides - Degradation of phenoxy-alkanoics
Degradation of Phenoxyalkanoic Herbicides
Plants are able to degrade these herbicides in
their tissues, but the rates of degradation are
very low. The rates of degradation vary
considerably in soils. Most of the available
results are from the phenoxyacetic - particularly
2,4-D itself. The patterns of microbial breakdown
may be summarised as follows 1). 2,4-D is
rapidly degraded in soil. MCPA is more persistent
and 2,4,5-T is even more resistant to
decomposition. 2). Microorganisms in soils can
become "adapted" in two separate ways a). by
enzyme induction b). by selection for
2,4-D-degrading organisms 3). Such adaptation
often leads to the rapid degradation of other
members of the group of phenoxy herbicides. 4).
Many microorganisms can degrade more than one of
the groups. 5). There appear to be two main
pathways of biodegradation a). Via
hydroxyphenoxyacetic acid b). Via phenol
85
Herbicides - 2,4-D Biodegradation
2,4-D and MCPA Biodegradation
86
2,4D Biodegradation
A more complete version of 2,4-D biodegradation
From The University of Minnesota
Biocatalysis/Biodegradation Database
87
Herbicides - S-triazine group

• s-triazine group
• The most commonly used a-triazines are the
  herbicides atrazine, prometryne, simazine, and
  ametryne. They are used in very large quantities
  (especially atrazine and simazine) in weed
  control in field crops.
• Atrazine
  2. Simazine
• The triazines are moderately persistent in soils
  (atrazine about 12 months, simazine about 12-14
  months).

88
Herbicides - Triazine biodegradation

• General formula for triazine herbicides  
• Biodegradation of Triazine Herbicides
• Attack can occur at C2 by hydroxylation
• Attack can occur at C4 or C6 by oxidative
  removal
• Attack by cleavage of triazine ring to produce
  straight chain compound. Reaction is slow in
  soil.

89
Herbicides - Substituted ureas
Substituted ureas
There are many different substituted urea
herbicides being used. Typical examples include
monuron, linuron, diuron, neburon, chlorbromuron,
etc. Most are phenyl substituted ureas
                                         
Monuron The phenyl
substituted urea herbicides resist chemical
breakdown in soils and are degraded by
microorganisms. Typical persistence is of the
order of 4-18 months in most soils. Typical
schemes of biodegradation include dealkylation

90
Herbicides - Bipyridilium group
Bipyridylium group

• Diquat and paraquat are the most commonly used
  bipyridylium herbicides.  
•                                                   
       
• Diquat dibromide (Reglone)                       
                                        

91
Herbicides - Others
Thiolcarbamates
Typical examples are CDEC, pebulate and
diallate. Vapourisation and biodegradation are
the most significant factors in the losses from
soils. Very little information is available on
the metabolic pathways involved, or on the
products formed.
Chlorinated aliphatic acids
The most important are TCA (trichloracetic acid)
and dalapon (2,2-dichloropropionic acid). The
persistence is of the order of  3 - 6 months.
Microbial decomposition is the most important
factor. Hydrolytic processes are the most common
biodegradative pathways.
Miscellaneous
The other chemicals used as herbicides include
the trifluaralin group, the chloracetamides,
amitrole, phenyl, carbamates, and
pentachlorophenol.
92
Fungicides - Aliphatics
Fungicides - Aliphatic compounds
These compounds (carbon disulfide, ethylene,
dibromide, chloropicrin, formaldehyde, allyl
alcohol, etc.) are often used as soil fumigants
to kill fungi and nematodes. They are degraded
at various rates by the microorganisms in soil.
Chloropicrin and allyl alcohol are probably the
most persistent. They are often degraded by soil
fungi, even though they are fungicidal!
Quinones - dichlone
Chloranil and dichlone are effective fungicides.
They are SH enzyme inhibitors and are usually
degraded by hydrol