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MCB 3020, Spring 2005
Chapter 15:Microorganisms in the Environment
2Today:I. Microbial impact on environmentII. PhotosynthesisIII. MethanogenesisIV. Nitrogen fixation
3I. Microbial Impact on the Environment Some examples:
Nitrogen fixation (N2 --> NH3)Nitrification, denitrification
Methane production:sewage treatment, landfills;cow rumen; greenhouse gas
Biodegradationwastewater treatmentlandfill and toxic waste degradation
Photosynthesis
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Primary producers(plants, photosynthetic microbes)
Solar energy (ultimate source of energy)
Consumers(herbivores,carnivores)
Decomposers(nonphotosynthetic
bacteria, fungi)
Interaction of organisms on earth
5II. Photosynthesis
The synthesis of chemical compoundslike glucose using energy from light.
hv C6H12O66 CO2 + 6 H2O + 6 O2
6A. 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
7Two sets of reactions are involved in photosynthesis
Light reactions: light energy is converted to chemical energy in theform 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
•
•
8
Light reactions6 H2O + hv 6 O2
ATP,reductant
6 CO2Dark reactions C6H12O6
Light reactions generate ATP and NADPH. Dark reactions use ATP and NADPH to reduce CO2 to carbohydrate (glucose).
12 H+ + 12 e-
Oxygenic photosynthesis
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How do plants and microbes capture the energy of light?
Using pigments like chlorophyll
alga
cyanobacterium
B. Light reactions of photosynthesis
10Chlorophyll
The main pigment for harvestinglight energy by photosynthesis
Located in photosynthetic membranes
11
R
N
N N
N
Mg
O
R
R
Chlorophyll
porphyrin or “magnesium tetrapyrrole”
TB
N
pyrrole
[cf. cytochromes (Fe), vitamin B12 (Co)]
12Arrangement of chlorophyll in membranes
photosynthetic membrane
200-300light harvesting
chlorophyll moleculesreaction center (RC)
TB
13Anoxygenic photosynthesis• does not produce O2
• “purple” and “green” bacteria
Oxygenic Photosynthesishv C6H12O66 CO2 + 6 H2O + 6 O2
• cyanobacteria (prokaryotic)• photosynthetic algae (eukaryotic)• plants (eukaryotic)
14C. Anoxygenic photosynthesis1. overview2. components3. electron flow4. membrane arrangement and ATP synthesis
TB
151. Overview (anoxygenic PHS)
Light ATPPMF +
Used by purple and green bacteria
+ reductant
Photophosphorylation use of light energy to make proton gradient for ATP synthesisTB
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Quinone poolCytochromes (Cyt)
TB
2. ComponentsReaction center
ChlorophyllBacteriopheophytin (Bph)Quinones (Q)
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P870
P870*
Q pool
BphQAQB
cyt. bc1
cyt. c2
3. Electron flow in anoxygenic PHS
lightenergy
reactioncenter (RC)
NAD(P)+
NAD(P)H
-1.0V
+0.5VTB
Midpoint potential
H2S, SO
18Sometimes 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!)
TB
19
LHRCBphQ
QQQQ bc1
c2c2
4. Membrane arrangement
H+
H+
H+
H+ADPPi
ATPTB
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• proton motive force (PMF) for ATP synthesis is generated when electrons are transferred from the Q pool to cytbf.
How is ATP made by photophosphorylation?
• ATP is made when PMF is dissipated using ATP synthase.
21D. Oxygenic photosynthesis1. overview2. components3. electron flow4. photophosphorylation
TB
221. Oxygenic photosynthesis: overview
Algae, cyanobacteria, higher plants
Light ATP
NAD(P)H
O2
+H2O
+
+NAD(P)++
PMF+
TB
232. ComponentsPhotosystem II (P680)Photosystem I (P700)Plastocyanin (PC)Quinone poolCytochromes (Cyt)
TB
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FeS
P680*Ph
P700PC
QAQB
Cyt bf
P680
P700*
Fd
NADPHNADP+
3. Electron flow in oxygenic PHS
H2O2e- + 2 H+ + 1/2 O2
Q pool
+1V
-1V
cyclicelectron flow
TBwater-splitting reaction
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Note:
4. Photophosphorylation use of light energy to make proton gradient for ATP synthesis
The membrane organization and ATP synthesis are generally similar to anoxygenic photosynthesis
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H+ H+ H+
stroma
thylakoid
ATP
ADP + Pi
In eukaryotes, photophosphorylationoccurs in the chloroplast
27E. 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.
28Autotrophs
Organisms that use CO2 as theirprimary carbon source.
Many are primary producers inecosystems.
29Calvin cyclereductive pentose phosphate pathwayuses ATP and NADPH to fix CO2
6 CO2 + 12 NADPH + 18 ATP fructose 6-P + 12 NADP+ + 18 ADP + 17 Pi
TB
30Key enzyme of the Calvin cycle:Ribulose bisphosphate carboxylase(RubisCo)first enzyme in the Calvin cycle
CO2 + ribulose bisphosphate two 3-phosphoglyceric acids
TB
31Subsequent reactions (after RubisCo):
In a series of reactions requiring ATP,NADPH, and molecular rearrangements,fructose 6-phosphate is produced fromphosphoglyceric acid
ultimately, glucose can be made
TB
32Photosynthesis 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
33III. Methanogenic Archaeaa diverse group of strict anaerobes that produce methane as a catabolic end-product.
CH4
34A. Methanogenic ecosystemswastewater treatment facilities,landfills, sediments, rumen, digestive tracts,anaerobic microenvironments
B. Methanogenic growth substratesH2 + CO2 formate, methanol, methylamines, acetate
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Polymers(polysaccharides, lipids, proteins)
Monomers(sugars, fatty acids, amino acids)
polymer degrading microbes
acetate
H2 + CO2
fermentation by microbes
CH4CH4+ CO2
The anaerobic food chain (landfill, rumen, etc.)
TB
methanogens
36C. The unusual coenzymes of methanogenesis.
1. Methanofuran
OCH2 ORCH2NH2
a formyl group carrier
372. Methanopterin
O
HN
NH2N N
H
NH
CH3
CHCH3
HN R
C1 carrier functionally analogous to folate
Carriers C1 groups at several oxidation states
383. Factor F430
N N
NNNi
a nickel tetrapyrrol
Methyl carrier
394. Factor F420
HO N N
NH
O
O
R
a 5-deazaflavin
functions as an electron carrier
405. Coenzyme B
HOPOCHCHNHCCH2CH2CH2CH2CH2CH2SHO=
HO
CH3 O=COOH
an electron carrier with anactive sulfhydryl group
416. Coenzyme M
HS-CH2-CH2 SO3–
a methyl carrier
CH3-S-CH2-CH2 SO3–
methyl-CoM
42D. The pathway of methanogenesis from CO2
CO2H2
MF-CHO
MP-CHO
MP-CH2OH
H2F420
MF = methanofuran
MP = methanopterin
43H2
MP-CH3
CoM-CH3
CH4
F420
CoB-SH
CoB-SS-CoM +H2
CoB-SH + HS-CoM
44IV. Nitrogen fixation• Use of nitrogen gas (N2) as a nitrogen source.• Occurs in prokaryotes only• Some prokaryotes enter into symbiotic relationships with leguminous plants
TB
45A. NitrogenaseEnzyme that catalyzes the reduction of N2 to NH3.
Fe protein
MoFe protein
461. Overall reaction of nitrogenase
N2 + 8H+ + 8e- +16 ATP
The formation of H2 is a by-reaction
2NH3 + H2 + 16 ADP +16 Pi
Nitrogenase
TB
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N2
NO3-NH3
nitrogenfixation
nitrification
denitrification
The microbial nitrogen cycle
48Study objectives1. 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.
498. Name the unusual coenzymes of methanogenesis and their general functions.9. 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.)
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MCB 3020 Spring 2004
Chapter 11: Industrial and
Environmental Microbiology
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I. Industrial production of antibiotics II. Other microbial products III. Biodegradation
A. Wastewater treatmentB. LandfillsC. Bioremediation
Industrial and Environmental Microbiology
52I. Antibiotic production
A. Genera known for productionB. DiscoveryC. Production
53A. Genera known for production
1. Streptomyces2. Penicillium3. Bacillus
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1. spread petri dish with soil dilution2. overlay with indicator organism3. incubate
bacterial colonies
zones of inhibition
B. Discovery
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4. isolate the organism5. purify the antibiotic6. eliminate known antibiotics7. assign structure8. improve yield9. improve purification10. animal testing11. clinical trials
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1. Penicillin
a. natural penicillinb. semi-syntheticc. biosynthetic
C. Production of antibiotics
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grow cells in large fermentor
remove cells
extract antibiotic
crystallize
a. natural penicillin
58b. semi-synthetic penicillinRemove R-group and add new side-chains by chemical synthesis.
natural penicillin R = CH2-CO-
HN
ON
S CH3
CH3H
COO-H
R
59c. biosynthetic penicillins
Add excess R-group precursor to the fermentor.
602. Streptomycin production
A-factor is an inducer of streptomycinbiosynthetic genes
O
O
OH CH3
CH3
O
A-factor is added to the fermentor
613. Tetracycline production
Avoid glucose in the growth medium
Use low phosphate growth medium
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A. VitaminsB. Amino acidsC. CortisoneD. EnzymesE. VinegarF. Citric acid
II. Other microbial products
G. YeastH. Beer and WineI. Distilled beveragesJ. Commodity ethanolK. Food
63A. Vitamins
1. Vitamin B12
2. Riboflavin
64B. Amino acids
glutamateaspartatephenylalaninelysine
65C. Cortisone (steroid)Produced by bioconversion
Bioconversion: the use of microbes to catalyze specific chemical reactions
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O
C
CH3
O
cortisonechemicalsynthesis
progesterone
O
C
CH3
O
bioconversion
HOhydroxy-progesterone
67D. Enzymes
1. Proteaseslaundry detergents
2. Glucose isomerasefructose production
68extremozymes
enzymes resistant to extreme conditions
extremophiles
organisms that grow in extreme environments
extremophiles are the source of extremozymes
69E. Vinegar
Acetic acid bacteria
ethanol acetaldehyde
acetic acid(vinegar)
Produced mainly from wine and cider
70F. Citric acid
Used to acidify foods and add tartness
Produced by Aspergillus niger (fungus)
A. niger uses citrate to obtain iron fromlow-iron environments
especially soft drinks
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citrateTCA citrate
Fe3+
Fe3+ citrate
chelation (strong noncovalent binding)
citrateFe3+
A. niger
iron limitation increases citrate production
72G. YeastSaccharomyces cerevisiae
Grow aerobicallyCollect cells
Baker's yeastNutritional yeast
73H. Beer and wineSaccharomyces spp.
Winefermented grapes
Beer fermented malt(made from germinated barley)
anaerobic growth
74I. Distilled alcoholic beverages
Wine BrandyFermented molasses RumFermented potatoes Vodka
juniper berriesFermented grain and Gin
Fermented malt Whiskeydistillation
75J. Commodity ethanolSolventGasohol
90% gasoline10% ethanol
76K. Food from microorganisms
1. single-celled organisms
yeast for protein
2. mushrooms
fungal fruiting bodies
77Microbial Impact on the Environment Some examples:
PhotosynthesisNitrogen fixation (N2 --> NH3)Nitrification, denitrification
Methane production:sewage treatment, landfillin cows; greenhouse gas
Biodegradationwastewater treatmentlandfill and toxic waste degradation
78III. Biodegradation
biological degradation of wastes or pollutants
A. Wastewater treatmentB. LandfillsC. Bioremediation
79A. Waste water treatment1. Treatment stages2. Details of secondary treatment3. Overview of treatment
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Primary treatmentremoval of sediment and debris
Secondary treatmentremoves organic matter
Tertiary treatmentremoves inorganic compounds
1. Treatment stages
TB
812. Details of secondary treatmenta. anaerobic
sludge digestor
b. aerobictrickling filteractivated sludge treatmentaerobic sludge digestor
TB
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wastewateranaerobic
sludge digestor(closed tank)
recalcitrant solidscells + CH4 + CO2
a. Anaerobic
TB
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Polymers(polysaccharides, lipids, proteins)
Monomers(sugars, fatty acids, amino acids)
polymer degrading microbes
acetate
H2 + CO2
fermentation by microbes
CH4CH4+ CO2
Inside the anaerobic digestor
TB
methanogens
84b. Aerobic secondary treatmenti. Trickling filterwastewater
open tank containingcrushed rocks
cells + CO2
recalcitrant solids
TB
85Inside the trickling filter
Microbes attached to rocks growby consuming the organic matter inthe wastewater.
"biological solids" are shed from the rocks
TB
86
wastewater
open, aerated, tank
activated sludge (flocs)
ii. activated sludge treatment
flocswastewater is held for a short time
TB
87Aerobic sludge digestor
Place flocs in an aerobic tank fora longer time period.
TB
88
Organic matter and microbes in thewastewater bind to the flocs.
Flocs consist of microbes and organicmatter
Zoogloea produces a slime that is theglue of the flocs.
About 10% of flocs is Zoogloea ramigera
TB
89
activated sludge, or trickling filter solids
anaerobic or aerobic sludge digestor
drying, composting,pasteurization, irradiation
spread on land, add to landfill, dumpin ocean,or incinerate
3. Overview
TB
90B. Landfills: anaerobic and aerobic biodegradation Polymers
(polysaccharides, lipids, proteins)
Monomers(sugars, fatty acids, amino acids)
polymer degrading microbes
organic acids; acetate H2 + CO2
fermentation by microbes
aerobic degradation
CO2
aerobic degradation
anaerobicdegradation
CH4CH4+ CO2
methanogens
91C. Bioremediationuse of microorganisms to enhancethe removal or detoxification of unwanted chemicals in the environment
e.g. petroleum spills chlorinated solvents pesticides heavy metals
921. Some strategies for enhancing biodegradation in nature:
a. identify organisms that naturally degrade pollutantsb. add whatever nutrient is the "limiting factor" for biodegradationc. genetically engineer better organisms (?)
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• chlorinated solvent• common contaminant in drinking water• suspected carcinogen
C=CClCl
Cl H
Trichloroethylene (TCE) sample
2. Example: bioremediation of chlorinated solvents
?
What organismsdegrade TCE?
94Trichloroethylene can be degraded aerobically or anaerobically.
C=CClCl
Cl H
CO2, 3 Cl-, H2O
O2
aerobic degradation
(e.g. ammonia-oxidizingbacteria)
O2
3 Cl-, H2C=CH2
(methanogens)
anaerobic degradation
95In contrast, tetrachloroethylene (PCE) can be degraded ONLY by anaerobic organisms.
C=CClCl
Cl Cl
O2
4 Cl-, H2C=CH2
(methanogens)
anaerobic degradation
How might you enhancethe biodegradation of TCE and PCE in pollutedenvironments?