Microbial Photosynthesis

Microbial Photosynthesis Michael Kühl Marine Biological Laboratory [email protected] Check: www.mbl.ku.dk/mkuhl for publication downloads etc. 1 Halob...
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Microbial Photosynthesis Michael Kühl Marine Biological Laboratory [email protected] Check: www.mbl.ku.dk/mkuhl for publication downloads etc.

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Halobacteria live at >20% salinity in places like the Dead Sea and in Salterns and salt lakes.

Energy generation and consumption of Halobacteria

Purple ”photosynthetic” membrane areas

Energy consumption

Red membrane areas with aerobe respiration

Energy generation

2

3

Benthic diatoms

4

5

Chlorophyll a is the key photopigment in oxygenic photosynthesis

Antenna pigments

Other chlorophylls have the same structure but different side groups and protein-associations cause different spectral absorption properties.

6

Different algal groups have different antenna pigments

Eukaryotic phototrophs evolved via endosymbiosis from prokaryotic oxygenic phototrophs. The only known oxygenic prokaryotes are cyanobacteria.

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”Farbstreifen Sandwatt”

5 mm

Foto: Lucas Stal

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The key enzyme in N2-fixation is nitrogenase which is inhibited/destroyed by oxygen

Types and characteristics of nitrogen-fixing cyanobacteria Type I Heterocystous (e.g. Anabaena, Nostoc, Nodularia, Calothrix, Scytonema) • Exclusively filamentous species with heterocysts • Strategy: Spatial separation of N2 fixation and photosynthesis and protection of nitrogenase in the heterocyst • Diazotroph growth under fully oxic conditions • Occurence; Lakes and brackish water, paddy fields, microbial mats, in symbiosis with plants and animals Type II Anaerobic N2-fixing non-heterocystous (e.g. Plectonema, Oscillatoria, Synechococcus, many more) • Both filamentous and unicellular species • Strategy: Avoidance of oxygen. Only induction and maintenance of nitrogenase when no or low O2 • Occurence: In many different environments but unclear if always growing diazotrophically

Not all N2-fixing cyanobacteria have heterocysts!

Type III Aerobic N2-fixing non-heterocystous (e.g. Oscillatoria, Trichodesmium, Lyngbya, Microcoleus) • Both filamentous and unicellular species • Strategy: Not precisely known. Temporal separation of N2 fixation and oxygenic photosynthesis? Spatial organization and behavioral oxygen protective mechanisms? • Diazotrophic growth under fully oxic conditions • Occurence: Tropical ocean (Trichodesmium), paddy fields, microbial mats

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Phenotypic characteristics of Cyanobacteria Pigments: Chlorophyll-a, ß Carotene, c-Phycoerythrin, Allophycocyanin, cphycocyanin, other chlorophylls absent Nuclear material: DNA is free in the central region of the cell (nucleoplasm) and is not enclosed in a membrane Food reserves: Cyanophycean starch, cyanophycin granules (argenine and aspartic acid) Thylakoid features: Chloroplast absent; the thylakoids are free in the cytoplasm and unstacked; phycobilisomes present Cell wall: Four-layered peptidoglycan wall in which murein is the principal component Flagella Absent

Aktions-spektrum

Phycoerythrin and Phycocyanin are import antenna pigments of Cyanoabacteria

Fykobiliner Karotenoider Klorofyl

Phycoerythrin

Phycocyanin

-1

-1

Fotosyntese (µmol ilt l min )

Klorofyl

200 Kiselalger

150

100

50

Cyanobakterier

0 400

450

500

550

600

650

700

Lysets bølgelængde (nm)

10

Prochloron spp.

Ascidian with Prochloron symbionts Lissoclinum patella

10 µm 0.8 15

•One of 3 separate lineages of prochlorophytes in cyanobacteria. •Contains Chl a & b as major photopigments. No phycobilins.

•Not cultivated – many attempts without success. •Discovered in 1975, lives in symbiosis with ascidians.

Chl a

0.6

10 0.4

Chl b

Absorbance

10 µm

Relative absorbance

Prochloron

0.2

5

0.0 300

400

500

600

700

Wavelength (nm)

•Prochlorothrix •Prochlorococcus (late 1980’s)

(special Chl a2 & b2)

Kühl et al. in prep.

Phenotypic characteristics of Prochlorophytes Pigments: Chlorophyll-a + Chlorophyll-b, ß Carotene, Zeaxanthan, Cryptoxanthin, no phycobiliprotein pigments Nuclear material: DNA is free in the cytoplasm and is not enclosed in a membrane, it is not central as in Cyanophyta but is rather diffuse throughout the cell.

Kühl & Larkum 2002

Prochlorophytes as the missing link? Based on phenotypic characteristics like pigmentation and organization of thylakoids.

Food reserves: Cyanophycean starch; no cyanophycin granules Thylakoid features: Chloroplast absent; the thylakoids are free in the cytoplasm and stacked in groups of two or more; phycobilisomes absent Cell wall: Four-layered peptidoglycan wall in which murein is the principal component

Based on genotypic characteristics of 16S rRNA genes.

Flagella Absent

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Habitat of Acaryochloris marina

Acaryochloris marina

•Cyanobacterium isolated from didemnid ascidians. •Contains Chl d as the major photopigment – also in the reaction centers! •Minor amounts of Chl a and phycobilins.

Absorbance (a.u.)

10 µm

Chlorophyll d

400

500

600

700

800

Wavelength (nm)

•Assumed a symbiont, but recently also found epiphytic on red algae, but niche unknown until recently... Can use NIR

Kühl et al. 2005

Inorganic carbon is fixed in the Calvin cycle in oxygenic phototrophs

b

c

1.0

d

0.5

1.0

0.5

0.0 100

0.0

Fluorescence (a.u.)

Acaryochloris-like cells

Scalar irradiance (% of incident irradiance)

Prochloron

Absorbance (a.u.)

Habitat of Acaryochloris marina a

Murakami et al. 2004

Rubisco !!

e

50

0 400

500

600

700

800

Wavelength (nm)

Kühl et al. 2005

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Photosynthesis: 2 Types • Oxygenic – Plants, algae, cyanobacteria – Light energy to generate ATP and reduce CO2 to synthesize carbohydrates and release molecular oxygen CO2 + 2H2O + light energy -> [CH2O] (carbohydrate) + O2 + H2O

• Anoxygenic –Other types of photosynthetic bacteria – Light energy used to create ATP and reduced organic/inorganic compounds to generate reducing power for carbon fixation. Does not release oxygen, does not use water CO2 + 2H2A + light energy -> [CH2O] + 2A + H2O e.g. 2H2S

e.g. 2S

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Two types of reaction center are involved in photosynthesis Type 1

Type 2

Chlorophyll-based

Bacteriochlorophyll a is the key photopigment in anoxygenic photosynthesis

Other bacteriochlorophylls have the same structure but different side groups and protein-associations cause different spectral absorption properties.

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The color of anoxygenic phototrophs is strongly affected by their carotenoids

Type 1

Type 2

Type 1

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Chlorosomes are efficient light collectors in green photosynthetic bacteria

Group of bacteria

Electron donor for photoautotrophy

Chlorophylls

Photoheterotrophy?

Chemotrophy?

Anoxygenic Photosynthetic Bacteria Purple Sulfur Bacteria of the family

Chromatiaceae

Purple Sulfur Bacteria of the family

Ectothiorhodospiraceae Purple Non-Sulfur Bacteria (family Rhodospirillaceae*) Green Sulfur Bacteria (including family Chlorobiaceae*)

Bchl a & b

S– or So or H2 (So globules formed inside cell from S–)

some species

some species

Bchl a & b

S– or H2 (So globules formed outside cell from S–)

possibly all species

some species

Bchl a & b

Prob. all: H2 . Some: low levels of S–, S2O3– , So

all species

probably all species

or (So globules formed outside cell from S–)

potentially all species

none

? (photoautotrophy?)

all species

probably all species

S–

mainly Bchl c, d or

Multicellular Filamentous Green Bacteria (including family Chloroflexaceae)

e

one or more of Bchl a, c, d

So

Oxygenic Photosynthetic Bacteria Cyanobacteria

Chl a (&d)

H2O

some species?

some species

-Prochlorophytes

Chl a & b

H2O

?

prob. none

Group of bacteria

Light harvesting

Reaction center

Preferred growth mode

Anoxygenic Photosynthetic Bacteria Green filamentous bacteria Chloroflexus-subdivision (3)

Bchl a & c + car (Chlorosomes)

Type II

Anoxygenic photo-organo-heterotrophic Aerobic chemo-organo.heterotrophic

Green Sulfur Bacteria (15)

Bchl a, c, d, e + car (Chlorosomes)

Type I

Anoxygenic photo-litho-autotroph

a-proteobacteria (31)

Bchl a , b + car (Intracell. Membranes)

Type II

Anoxygenic photo-organo-heterotroph Aerobic chemo-organo-heterotroph

Bchl a

Type II

Aerobic chemo-organo-heterotroph

Type II

Anoxygenic photo-organo-heterotroph Aerobic chemo-organo-heterotroph

Aerobic a-proteobacteria (23) b-proteobacteria (4)

Purple Sulfur Bacteria Chromatiaceae (31)

Ectothirhodospiraceae(9) Heliobacteriaceae (5)

Bchl a + car (Intracell. Membranes) Bchl a & b + car (Intracell. Membranes) Bchl g + car

Type II

Type I

Anoxygenic photo-litho-autotroph Anoxygenic photo-organo-heterotroph

Oxygenic Photosynthetic Bacteria Cyanobacteria (>1000)

Prochloron, Prochlorotrhix (2 Prochlorococus (1) Acaryochloris (1)

Chl a +phycobilins + car (thylakoid membranes) Chl a & b + car

Type I + II

Oxygenic photo-litho-autotroph

Chl a2 &b2 + car (+PBS) Chl d, a + car (+PBS)

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Inorganic carbon is fixed in the Calvin cycle by anoxygenic purple bacteria

Inorganic carbon is fixed in the reverse citric acid cycle by anoxygenic green sulphur bacteria

Rubisco !!

Inorganic carbon is fixed in the hydroxy-propionate pathway by anoxygenic green non-sulphur bacteria.

Classification Purple Bacteria (Proteobacteria)

Green Filamentous (nonsulfur) bact.

Green Sulfur Bacteria

Heliobacteria (gram + bact.)

Antenna

Carotenoids in spirilloxanthin, okenone, or rohodopinal groups LH1 & LH2 complexes

Bchl c arranged in chlorosomes to harvest light, Carotenoids gamma or beta carotene (isorenieratene & chlorobactene groups), LH1

Chlorosomes funnel light to RC, carotenoids (& bachl c, d, e) in isorenieratene & chlorobactene groups

Carotenoids – neurosporene

Chlorophyll

Bacteriochlorophyll Bacteriochlorophylls c or d (sm. amt. of a) a&b

Bacteriochlorophyll Bacteriochlorophyll g c, d or e, (sm. amt. of a)

Electron flow Reverse e- flow (reverse Krebs Cycle)

Reverse e- flow (reverse Krebs Cycle)

cyclic

cyclic

Rubisco

None

None

None

Hydroxy-propionate pathway. Some reverse TCA

Reverse TCA

None, no Calvin Cycle, no reverse TCA, photoheterotrophs

+Rubisco

Carbon fixing Calvin Cycle (but can use reverse TCA, tricarboxylic acid cycle {citric acid cycle}, fixes C into organic molecules used for metabolites or cellular components)

Believed to have common ancestor Lateral transfer of phototrophy from one to the other, or from common ancestor to descendents of both lines

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Classification

Purple Bacteria (Proteobacteria)

Green Gliding (nonsulfur) bact.

Green Sulfur Bacteria

Heliobacteria (gram + bact.)

Ecology

Nonsulfur bact.: grow aerobically by respiration on organic source of carbon in dark, Sulfur bact: must fix CO2

Facultatively aerobic: aerobic- live heterotrophically, not photosynth., anaerobicphotosynthetic, do not fix N

Photolithotrophic, CO2 as sole C source (can use acetate), strict anaerobes, obligate phototrophs

Obligate anaerobe, sensitive to O2, photoheterotrophic Can’t tolerate sulfide, rarely aquatic, fix N

Produce O2?

No, anoxygenic

No, anoxygenic

No, anoxygenic

No, anoxygenic

Photosynth. Type

Like photosystem II, quinone-type

Like photosystem II, quinone-type

Like photosystem I Fe-S

Like photosystem I Fe-S

Electron Donor

H2S, H2 & other

H2S, organics

Sulfide & organic hydrogen donors

Organic donors

Electron acceptor

Quinone, Fe Quinone, Mn between quinones between quinones

Ferredoxin

FeS

Reaction Center

P870, Bchl a

P840, Bchl a Heterodimeric, adequate to reduce ferredoxin, can reduce NAD+ to NADH directly

P789, homodimeric

P840, Bchl a, carotenoids not in RC, homodimeric, lacks H subunit

Sulfuretum in Nivå

Dense blooms of anoxygenic phototrophs occur in stratified waters with a thermo- and/or halocline and anoxic bottom water.

Sulfuretum in Nivå

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”Farbstreifen Sandwatt”

5 mm

Foto: Lucas Stal

Differential light utilisation governs coexistence Cyanobacterial layer

Purple bacterial layer

1.0 Carotenoids

Carotenoids

Chlorofyll a

0.8

Absorbance

Chlorofyll a

0.6

Bacteriochlorofyll a

0.4

Bacteriochlorofyll c

Phycobilins

0.2

Bacteriochlorofyll a & c

0.0

400

500

600

700

800

900 400

500

600

700

800

900

Wavelength (nm)

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Pigment Chl a

Absorption maxima (nm)

Fluorescence maxima (nm)

Cells

extract

Cells

670-675

435, 663

680-685

Chl b

655

455, 645

-

Chl d

714-718

400, 697

740-760

Bchl a

375, 590, 805, 830-911

358, 579, 771

907-915

Bchl b

400, 605, 835-850, 986-1035

368, 407, 582, 795

1040

Bchl c

457-460, 745-755

433, 663

775

Bchl d

450, 715-745

425, 654

763

Bchl e

460-462, 710-725

459, 648

738

Bchl g

375, 419, 575, 788

365, 405, 566, 762

-

”Farbstreifen Sandwatt”

5 mm

Foto: Lucas Stal

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Microscale light measurements

Fiber-optic Microsensors Microprobes (A-D) for:

- radiance, irradiance, scalar irradiance (UVNIR light) - Surface detection - Pigment fluorescence - Diffusivity/Flow

Micro-opt(r)odes (E) for: - O2, pH, CO2, temperature

All based on multimode graded index optical fibers 100/140 µm core/cladding N.A. = 0.22 Kühl & Revsbech 2001

Field radiance measurements

Microscale light measurements

Collimated light

48o 60o 0o

Downwelling light

Forward scattered light

140o

Back scattered light

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Light-collecting properties of fiber-optic microprobes Scalar irradiance probeA

160

Irradiance probe

B

100

140 120

80

100 60

80 60

40

40 20 20 0 -180

-120

-60

0

60

120

180

0 -180

-120

Angle of incident light

-60

0

60

120

180

Angle of incident light

Kühl et al. 1997

Strong light attenuation due to absorption and scattering PAR (% of Ed) 0 0.01

50 0.1

1

100

150

10

100 0

K 0 (λ ) = −

d ln[E0 (λ )] =− dz -1

Kühl & Jørgensen 1992.

Mapping the spatial light distribution ln ⎡ ⎢⎣

E 0 ( λ )1

⎤ E0 (λ ) 2 ⎥⎦

z 2 − z1

Collimated light

K0 (mm ) 5

48o

10

60o 0o

140o

0.0 Downwelling light

Depth (mm)

% of average response

Microscale light measurements

Forward scattered light

Back scattered light

0.5 1.0 1.5 2.0

A

B Kühl et al. in prep.

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Directional vs. Diffuse light From one direction

Integral from all directions

Collimated light

48o 60o 0o

Downwelling light

Spektral lysnedtrængning Fykobilin

Back scattered light

Experimental set-up for O2-measurements Light source

Bakterieklorofyl

Klorofyl

Klorofyl

Skalar irradians (% af indfaldende lys)

Forward scattered light

140o

200

150

0.0

O2 microsensor

0.2 100 0.4 50

0.6 0.8 1.0

0 400

500

600

700

800

Lysets bølgelængde (nm)

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Aktions-spektrum Diatoms

Fykobiliner Karotenoider Klorofyl

-1

-1

Fotosyntese (µmol ilt l min )

Klorofyl

200

Cyanobacteria

Kiselalger

150

100

50

Cyanobakterier

0 400

450

500

550

600

650

700

Lysets bølgelængde (nm)

Light and Photosynthesis Oxygen (µmol O2 l-1) -1

0

200

400

600

Scalar irradians (µmol photons m-2 s-1) 0 100 200 300

800

Water Biofilm

Depth (mm)

0 Photosynthesis

Oxygen

2

-1

0

Light

1

Photosynthesis in steep light gradients

1

2

3

3 0

2

4 Photosynthesis (nmol O2 cm-3 s-1)

6

8 Kühl & Jørgensen 1992

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Carotenoids protect against photooxidation

Absorption vs. Action spectrum

Activated chlorophyll and oxygen forms radicals that can break down proteins, lipids and other Chl* + O2 → Chl + O2* key components of cells Chl + radiation → Chl*

Chl* + carotenoids → Chl + carotenoids* O2* + carotenoids → O2 + carotenoids* Carotenoids* → carotenoids + heat

Types of photobehaviour Behaviour

Measured Quantity

Speed

Photokinesis positive

negative

Single Cell Effect

light intensity

I

Colony Effect

Ecological Significance

Accumulation in dark areas

Avoiding photo damage

Accumulation in illuminated areas

(Optimizing photosynthesis)

Intensity Speed

Intensity

Photophobic Response step-up

step-down

change in light intensity

dI dt

Phototaxis positive

direction of light

negative

I bimodal

Trapping in dark areas reversal of direction

Trapping in illuminated areas

Moving towards light source Moving away from light source Moving perpendicular to light direction

Avoiding photo damage Positioning in benthic systems Optimizing photosynthesis Positioning in benthic systems Optimizing photosynthesis Moving to surface in pelagic systems Moving to the bottom in pelagic systems Keeping depth in pelagic systems

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”Algography”

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Different light optima for different phototrophs

Gradient-capillary-cell-tracking-setup

Motility of Microorganisms in Response to Light, Oxygen, and Sulfide Gradient Capillary Setup

video camera

flat glass capillary (40 x 8 x 0.8 mm3)

end of tubing microsensors for

pH reference electrode

video recorder

oxygen sulfide pH

gas space

sulfidic agar plug oxygen-microelectrode with Picoammeter inverted microscope

medium with bacteria

The setup is mounted on a light microscope which allows computer-aided cell tracking via digital video recordings

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M. gracile: dark-light transition

Marichromatium gracile Cell distribution in relation to oxygen, sulfide, and pH gradients

anoxic

ca. 500µm

oxic

pH 7 .0

50 0

D arkness

pH 6 .8

40 0 6 .6 rel. ce ll de n sity

30 0 20 0

H2S

O2

10 0 0 0

1

2

3 4 d ista nc e (m m )

5

6

Thar & Kühl 2001

Thar & Kühl 2001

Marichromatium gracile Phobic responses towards increasing oxygen concentrations and darkness

Response to light-dark border

Response to oxygen gradient Thar & Kühl 2001

28

UV radiation and it’s effects on organisms UV-C (