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MCB 3020, Spring 2005
Chapter 15:
Microorganisms in
the Environment
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Today:
I. Microbial impact on environment
II. Photosynthesis
III. Methanogenesis
IV. Nitrogen fixation
I. Microbial Impact on the
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Environment
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Some examples:
Photosynthesis
Biodegradation
wastewater treatment
landfill and toxic waste degradation
Methane production:
sewage treatment, landfills;
cow rumen; greenhouse gas
Nitrogen fixation (N2 --> NH3)
Nitrification, denitrification
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Interaction of organisms on earth
Solar energy (ultimate source of energy) 4
Primary producers
(plants, photosynthetic microbes)
Decomposers
(nonphotosynthetic
bacteria, fungi)
Consumers
(herbivores,
carnivores)
II. Photosynthesis
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The synthesis of chemical compounds
like glucose using energy from light.
hv
6 CO2 + 6 H2O  C6H12O6 + 6 O2
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A. Overview of photosynthesis
• occurs in plants, algae (eukaryotic),
and cyanobacteria (prokaryotic)
• makes organic carbon (also called
reduced carbon or “fixed” carbon)
• makes ATP and NAD(P)H
(reductant) to synthesize organic
carbon
Two sets of reactions are involved in
photosynthesis
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• Light reactions: light energy is
converted to chemical energy in the
form of ATP and reductant [NAD(P)H]
• Dark reactions (light-independent):
chemical energy is used to reduce CO2,
usually to the level of a sugar
Light reactions generate ATP and NADPH.
Dark reactions use ATP and NADPH to
reduce CO2 to carbohydrate (glucose).
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Oxygenic photosynthesis
6 H2O + hv
Light reactions
6 O2
12 H+ + 12 e-
ATP,
reductant
6 CO2
Dark reactions
C6H12O6
B. Light reactions of photosynthesis
How do plants and microbes
capture the energy of light?
alga
cyanobacterium
Using pigments like chlorophyll
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Chlorophyll
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The main pigment for harvesting
light energy by photosynthesis
Located in photosynthetic membranes
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Chlorophyll
R
N
N
Mg
N
N
N
pyrrole
R
R
O
porphyrin or “magnesium tetrapyrrole”
[cf. cytochromes (Fe), vitamin B12 (Co)]
TB
Arrangement of chlorophyll in membranes 99
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200-300
light harvesting
chlorophyll molecules
reaction center (RC)
photosynthetic membrane
TB
Anoxygenic photosynthesis
• does not produce O2
• “purple” and “green” bacteria
Oxygenic Photosynthesis
hv
6 CO2 + 6 H2O  C6H12O6 + 6 O2
• cyanobacteria (prokaryotic)
• photosynthetic algae (eukaryotic)
• plants (eukaryotic)
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C. Anoxygenic photosynthesis
1. overview
2. components
3. electron flow
4. membrane arrangement
and ATP synthesis
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TB
1. Overview (anoxygenic PHS)
Used by purple and green bacteria
Light
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PMF + ATP + reductant
Photophosphorylation
use of light energy to make
proton gradient for ATP synthesisTB
2. Components
Reaction center
Chlorophyll
Bacteriopheophytin (Bph)
Quinones (Q)
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Quinone pool
Cytochromes (Cyt)
TB
3. Electron flow in anoxygenic PHS
Midpoint potential
-1.0V
P870*
light
energy
Bph
QA
QB
reaction
center (RC)
NAD(P)+
Q pool
cyt. bc1
+0.5V
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P870
cyt. c2
NAD(P)H
H2S, SO
TB
Sometimes reductant (i.e.NAD(P)H)
is made using some of the electron
carriers of anoxygenic
photosynthesis.
In this case, electrons must be
supplied by an outside source like
H2S (but not water!)
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TB
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4. Membrane arrangement
+
H
c2
LH
TB
RC
c2
QQ
QQ
Bph
Q
H+
H+
bc1
ATP
H+
Pi
ADP
How is ATP made by
photophosphorylation?
• proton motive force (PMF) for
ATP synthesis is generated
when electrons are transferred
from the Q pool to cytbf.
• ATP is made when PMF is
dissipated using ATP synthase.
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D. Oxygenic photosynthesis
1. overview
2. components
3. electron flow
4. photophosphorylation
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TB
1. Oxygenic photosynthesis:
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overview
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Algae, cyanobacteria, higher plants
Light
+
H2 O
+
+
NAD(P)
TB
ATP
+
PMF
+
NAD(P)H
+
O2
2. Components
Photosystem II (P680)
Photosystem I (P700)
Plastocyanin (PC)
Quinone pool
Cytochromes (Cyt)
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TB
3. Electron flow in
oxygenic PHS
P680*
Ph
P680
+1V
FeS
Fd
cyclic
QA
QB electron flow
Q pool
Cyt bf
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-1V P700*
PC
2e- + 2 H+ + 1/2 O2
NADP+
NADPH
P700
H2O
water-splitting reaction
TB
4. Photophosphorylation
use of light energy to make
proton gradient for ATP synthesis
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Note:
The membrane organization and
ATP synthesis are generally similar
to anoxygenic photosynthesis
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photophosphorylation
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In eukaryotes,
occurs in the chloroplast
ADP + Pi
H+ H+ H+
ATP
stroma
thylakoid
E. Dark reactions of photosynthesis
(light-independent reactions)
CO2 reduction (CO2 fixation)
to form organic matter
uses ATP and NADPH made in
light reactions to reduce CO2
Dark reactions can occur in the light, but
do not require light.
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Autotrophs
Organisms that use CO2 as their
primary carbon source.
Many are primary producers in
ecosystems.
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Calvin cycle
reductive pentose phosphate
pathway
uses ATP and NADPH to fix CO2
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6 CO2 + 12 NADPH + 18 ATP 
+
fructose 6-P + 12 NADP
+ 18 ADP + 17 Pi
TB
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Key enzyme of the Calvin cycle:
Ribulose bisphosphate carboxylase
(RubisCo)
first enzyme in the Calvin cycle
CO2 + ribulose bisphosphate 
two 3-phosphoglyceric acids
TB
Subsequent reactions (after
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RubisCo):11
In a series of reactions requiring ATP,
NADPH, and molecular rearrangements,
fructose 6-phosphate is produced from
phosphoglyceric acid
ultimately, glucose can be made
TB
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Photosynthesis review
• energy from sunlight
• chlorophyll (captures light energy)
• ATP made by photophosphorylation
• NADPH
• CO2 reduced to carbohydrates
via RubisCo and Calvin cycle
Result: sugar from light, water, air
III. Methanogenic Archaea
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a diverse group of strict anaerobes that
produce methane as a catabolic end-product.
CH4
A. Methanogenic ecosystems
wastewater treatment facilities,
landfills, sediments, rumen,
digestive tracts,
anaerobic microenvironments
B. Methanogenic growth substrates
H2 + CO2
formate, methanol, methylamines,
acetate
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The anaerobic food chain (landfill, rumen, etc.)10
Polymers
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(polysaccharides, lipids, proteins)
polymer degrading microbes
Monomers
(sugars, fatty acids, amino acids)
fermentation by microbes
acetate
H2 + CO2
methanogens
CH4+ CO2
CH4
TB
C. The unusual coenzymes of
methanogenesis.
1. Methanofuran
R
OCH2
CH2NH2
O
a formyl group carrier
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2. Methanopterin
O
HN
NH2
N
H
N
N
H
R
HN
CH
CH3
CH3
C1 carrier functionally analogous to folate
Carriers C1 groups at several oxidation states
3. Factor F430
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Ni
a nickel tetrapyrrol
Methyl carrier
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4. Factor F420
R
HO
N
N
O
NH
O
a 5-deazaflavin
functions as an electron carrier
5. Coenzyme B
=
=
O CH3
O
HOPOCHCHNHCCH2CH2CH2CH2CH2CH2SH
HO
COOH
an electron carrier with an
active sulfhydryl group
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6. Coenzyme M
HS-CH2-CH2 SO3–
a methyl carrier
CH3-S-CH2-CH2 SO3–
methyl-CoM
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D. The pathway of methanogenesis from CO2 10
H2
H2
F420
CO2
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MF-CHO
MF = methanofuran
MP-CHO
MP = methanopterin
MP-CH2OH
H2
F420
CoB-SH
MP-CH3
CoM-CH3
CoB-SS-CoM + CH4
H2
CoB-SH + HS-CoM
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IV.
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Nitrogen fixation
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• Use of nitrogen gas (N2) as a nitrogen source.
• Occurs in prokaryotes only
• Some prokaryotes enter into symbiotic
relationships with leguminous plants
TB
A. Nitrogenase
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Enzyme that catalyzes the reduction of N2 to NH3.
Fe protein
MoFe protein
1. Overall reaction of nitrogenase
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N2 + 8H+ + 8e- +16 ATP
Nitrogenase
2NH3 + H2 + 16 ADP +16 Pi
The formation of H2 is a by-reaction
TB
The microbial nitrogen cycle
nitrogen
fixation
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N2
denitrification
NO3
NH3
nitrification
-
Study objectives
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1. Know the details of photosynthesis and be able to compare and
contrast oxygenic and anoxygenic photosynthesis.
2. Compare and contrast photophosphorylation, oxidative phosphorylation, and
substrate level phosphorylation. How is the proton motive force made?
Where does photosynthesis occur in eukaryotes?
3. What is the role of water in oxygenic photosynthesis? Does water play the
same role in anoxygenic photosynthesis?
4. Define autotroph. What is the purpose of the Calvin cycle? What types of
organisms use this cycle? Know the reaction catalyzed by Rubisco.
How is glucose made in the dark reactions of photosynthesis?
5. Be able to describe how a photosynthetic cell makes sugar from air, water,
and light. What is the purpose of the ATP and NADPH? How are they made?
How are they used in the production of sugars from CO2?
6. What are methanogenic Archaea? Where are they found?
What are the substrates for methanogenesis?
7. Understand the role of methanogens in the anaerobic food chains of rumen,
landfills, wastewater treatment facilities, and other anaerobic ecosystems.
8.
9.
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Name the unusual coenzymes of methanogenesis and their general functions.
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Define nitrogen fixation. What organisms are capable of nitrogen fixation?
What is the reaction of nitrogenase? Note that it requires reductant and ATP.
10. Distinguish between nitrification and denitrification. (See last slide.)
MCB 3020 Spring 2004
Chapter 11:
Industrial and
Environmental Microbiology
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Industrial and Environmental Microbiology31
I. Industrial production of antibiotics
II. Other microbial products
III. Biodegradation
A. Wastewater treatment
B. Landfills
C. Bioremediation
I. Antibiotic production
A. Genera known for production
B. Discovery
C. Production
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A. Genera known for production
1. Streptomyces
2. Penicillium
3. Bacillus
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B. Discovery
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1. spread petri dish with soil dilution
2. overlay with indicator organism
3. incubate
bacterial colonies
zones of inhibition
4. isolate the organism
5. purify the antibiotic
6. eliminate known antibiotics
7. assign structure
8. improve yield
9. improve purification
10. animal testing
11. clinical trials
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C. Production of antibiotics
1. Penicillin
a. natural penicillin
b. semi-synthetic
c. biosynthetic
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a. natural penicillin
grow cells in large fermentor
remove cells
extract antibiotic
crystallize
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b. semi-synthetic penicillin
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Remove R-group and add new
side-chains by chemical synthesis.
R
H
N
S
CH3
H
O
N
natural penicillin R =
H
CH3
COOCH2-CO-
c. biosynthetic penicillins
Add excess R-group precursor
to the fermentor.
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2. Streptomycin production
A-factor is added to the fermentor
A-factor is an inducer of streptomycin
biosynthetic genes
OH
O
O
O
CH3
CH3
3. Tetracycline production
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Avoid glucose in the growth medium
Use low phosphate growth medium
II. Other microbial products
A. Vitamins
B. Amino acids
C. Cortisone
D. Enzymes
E. Vinegar
F. Citric acid
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G. Yeast
H. Beer and Wine
I. Distilled beverages
J. Commodity ethanol
K. Food
A. Vitamins
1. Vitamin B12
2. Riboflavin
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B. Amino acids
glutamate
aspartate
phenylalanine
lysine
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C. Cortisone (steroid)
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Produced by bioconversion
Bioconversion:
the use of microbes to catalyze
specific chemical reactions
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CH3
C O
progesterone
O
bioconversion
HO
O
CH3
C O
hydroxy-progesterone
chemical
synthesis
cortisone
D. Enzymes
1. Proteases
laundry detergents
2. Glucose isomerase
fructose production
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extremozymes
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enzymes resistant to extreme conditions
extremophiles
organisms that grow in extreme
environments
extremophiles are the source
of extremozymes
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E. Vinegar
Produced mainly from wine and cider
Acetic acid bacteria
ethanol
acetaldehyde
acetic acid
(vinegar)
F. Citric acid
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Used to acidify foods and add tartness
especially soft drinks
Produced by Aspergillus niger (fungus)
A. niger uses citrate to obtain iron from
low-iron environments
A. niger
TCA
3+
Fe
citrate
citrate
citrate
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Fe3+ citrate
3+
Fe
chelation
(strong noncovalent binding)
iron limitation increases citrate production
G. Yeast
Saccharomyces cerevisiae
Grow aerobically
Collect cells
Baker's yeast
Nutritional yeast
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H. Beer and wine
Saccharomyces spp.
anaerobic growth
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Wine
fermented grapes
Beer
fermented malt
(made from germinated barley)
I. Distilled alcoholic beverages
distillation
Fermented malt
Wine
Fermented molasses
Fermented potatoes
Fermented grain and
juniper berries
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Whiskey
Brandy
Rum
Vodka
Gin
J. Commodity ethanol
Solvent
Gasohol
90% gasoline
10% ethanol
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K. Food from microorganisms
1. single-celled organisms
yeast for protein
2. mushrooms
fungal fruiting bodies
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Microbial Impact on the Environment
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Some examples:
Biodegradation
wastewater treatment
landfill and toxic waste degradation
Methane production:
sewage treatment, landfill
in cows; greenhouse gas
Photosynthesis
Nitrogen fixation (N2 --> NH3)
Nitrification, denitrification
III. Biodegradation
biological degradation of wastes
or pollutants
A. Wastewater treatment
B. Landfills
C. Bioremediation
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A. Waste water treatment
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1. Treatment stages
2. Details of secondary treatment
3. Overview of treatment
TB
1. Treatment stages
Primary treatment
removal of sediment and debris
Secondary treatment
removes organic matter
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Tertiary treatment
removes inorganic compounds
TB
2. Details of secondary treatment
a. anaerobic
sludge digestor
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b. aerobic
trickling filter
activated sludge treatment
aerobic sludge digestor
TB
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a. Anaerobic
wastewater
anaerobic
sludge digestor
(closed tank)
cells + CH4 + CO2
recalcitrant solids
TB
Inside the anaerobic digestor
Polymers
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(polysaccharides, lipids, proteins)
polymer degrading microbes
Monomers
(sugars, fatty acids, amino acids)
fermentation by microbes
acetate
H2 + CO2
methanogens
CH4+ CO2
CH4
TB
b. Aerobic secondary treatment
i. Trickling filter
wastewater
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open tank containing
crushed rocks
cells + CO2
recalcitrant solids
TB
Inside the trickling filter
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Microbes attached to rocks grow
by consuming the organic matter in
the wastewater.
"biological solids" are shed from the
rocks
TB
ii. activated sludge treatment
wastewater
activated sludge (flocs)
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open, aerated, tank
flocs
wastewater is held for a short time
TB
Aerobic sludge digestor
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Place flocs in an aerobic tank for
a longer time period.
TB
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organic
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Flocs consist of microbes and
matter
Organic matter and microbes in the
wastewater bind to the flocs.
About 10% of flocs is Zoogloea ramigera
Zoogloea produces
a slime that is the
glue of the flocs. TB
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3. Overview
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activated sludge, or trickling filter solids
anaerobic or aerobic sludge digestor
drying, composting,
pasteurization, irradiation
spread on land, add to landfill, dump
in ocean,or incinerate
TB
B. Landfills: anaerobic and aerobic
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biodegradation
Polymers
(polysaccharides, lipids, proteins)
polymer degrading
microbes
Monomers
aerobic
(sugars,
fatty
acids,
amino
acids)
degradation
CO2
fermentation by
microbes
organic acids; acetate H2 + CO2 anaerobic
methanogens
aerobic
degradation
CH4+ CO2
CH4
degradation
C. Bioremediation
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use of microorganisms to enhance
the removal or detoxification of
unwanted chemicals in the environment
e.g. petroleum spills
chlorinated solvents
pesticides
heavy metals
1. Some strategies for enhancing
biodegradation in nature:
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a. identify organisms that naturally
degrade pollutants
b. add whatever nutrient is the
"limiting factor" for biodegradation
c. genetically engineer better
organisms (?)
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2. Example:
bioremediation of chlorinated solvents
Cl
Cl
C=C
Cl
What organisms
degrade TCE?
H
Trichloroethylene (TCE)
sample
• chlorinated solvent
• common contaminant in drinking water
• suspected carcinogen
?
Trichloroethylene can be degraded
aerobically or anaerobically.
Cl
aerobic
degradation
(e.g. ammoniaoxidizing
bacteria)
CO2, 3
Cl-,
Cl
O2
H 2O
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Cl
C=C
H
O2
3
anaerobic
degradation
(methanogens)
Cl ,
H2C=CH2
In contrast, tetrachloroethylene (PCE)
can be degraded ONLY by anaerobic
organisms.
Cl
Cl
C=C
How might you enhance
the biodegradation of
TCE and PCE in polluted
environments?
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Cl
Cl
O2
4
anaerobic
degradation
(methanogens)
Cl ,
H2C=CH2