1

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

4

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

9

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

16

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

20

• 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

25

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

26

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

47

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

62

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

66

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

71

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

TB

80

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

82

wastewateranaerobic

sludge digestor(closed tank)

recalcitrant solidscells + CH4 + CO2

a. Anaerobic

TB

83

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 (?)

93

• 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?