1

Microbial Photosynthesis

Michael KühlMarine Biological Laboratory

mkuhl@bi.ku.dk

Check: www.mbl.ku.dk/mkuhlfor publication downloads etc.

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

Energy consumptionEnergy generation

Red membrane areaswith aerobe respiration

Purple ”photosynthetic”membrane areas

Energy generation and consumption of Halobacteria

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4

Benthic diatoms

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Chlorophyll a is the key photopigmentin oxygenic photosynthesis

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

Antenna pigments

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Different algal groups have different antenna pigments

Eukaryotic phototrophsevolved via endosymbiosisfrom prokaryoticoxygenic phototrophs.

The only known oxygenicprokaryotes are cyanobacteria.

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Foto: Lucas Stal5 mm

”Farbstreifen Sandwatt”

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

Not all N2-fixing cyanobacteriahave heterocysts!

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

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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Pigments:Chlorophyll-a, ß Carotene,c-Phycoerythrin, Allophycocyanin, c-phycocyanin, 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

Phenotypic characteristics of Cyanobacteria

Phycoerythrin and Phycocyanin are import antenna pigments of Cyanoabacteria

PhycoerythrinPhycocyanin

400 450 500 550 600 650 7000

50

100

150

200

Lysets bølgelængde (nm)

Foto

synt

ese

(µm

ol il

t l-1 m

in-1)

Kiselalger

Cyanobakterier

Karotenoider

KlorofylKlorofyl

Fykobiliner

Aktions-spektrum

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Ascidian with Prochloron symbionts

Kühl et al. in prep.

Lissoclinum patella

Prochloron spp.

10 µm

300 400 500 600 700

5

10

15

0.0

0.2

0.4

0.6

0.8

Rel

ativ

e ab

sorb

ance

Wavelength (nm)

Prochloron

Abs

orba

nceChl a

•One of 3 separate lineages ofprochlorophytes in cyanobacteria.•Contains Chl a & b as majorphotopigments. No phycobilins.

•Not cultivated – many attemptswithout success.

•Discovered in 1975, lives in symbiosiswith ascidians.

•Prochlorothrix•Prochlorococcus (late 1980’s)(special Chl a2 & b2)

10 µm

Kühl & Larkum 2002

Chl b

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.

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

Flagella Absent

Phenotypic characteristics of Prochlorophytes Prochlorophytes as the missing link?

Based on phenotypic characteristicslike pigmentation and organization of thylakoids.

Based on genotypic characteristicsof 16S rRNA genes.

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Acaryochloris marina

10 µm

400 500 600 700 800A

bsor

banc

e (a

.u.)

Wavelength (nm)

Chlorophyll d

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

•Assumed a symbiont, but recently also found epiphytic on red algae, but niche unknownuntil recently... Can use NIR Kühl et al. 2005 Murakami et al. 2004

Habitat of Acaryochloris marina

a

b c

Prochloron

Acaryochloris-like cells

400 500 600 700 8000

50

1000.0

0.5

1.0

Flu

ores

cenc

e (a

.u.)

0.0

0.5

1.0

Abs

orba

nce

(a.u

.)S

cala

r irr

adia

nce

(% o

f inc

iden

t irr

adia

nce)

Wavelength (nm)

d

e

Habitat of Acaryochloris marina

Kühl et al. 2005

Inorganic carbon is fixed in the Calvin cyclein oxygenic phototrophs

Rubisco !!

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Photosynthesis: 2 Types• Oxygenic

• Anoxygenic

– Plants, algae, cyanobacteria

–Other types of photosynthetic bacteria

– Light energy to generate ATP and reduce CO2 to synthesize carbohydrates and release molecular oxygen

– 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 + 2H2O + light energy -> [CH2O] (carbohydrate) + O2 + H2O

CO2 + 2H2A + light energy -> [CH2O] + 2A + H2O

e.g. e.g.2H2S 2S

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Two types of reaction center are involved in photosynthesis

Chlorophyll-based

Type 1 Type 2

Bacteriochlorophyll a is the key photopigmentin anoxygenic photosynthesis

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

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The color of anoxygenic phototrophsis strongly affected by their carotenoids

Type 1 Type 2Type 1

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Chlorosomes areefficient light collectors in green photosynthetic bacteria

prob. none?H2OChl a & b-Prochlorophytes

some speciessome species?H2OChl a (&d)Cyanobacteria

Oxygenic Photosynthetic Bacteria

probably all speciesall species? (photoautotrophy?)one or more of Bchl a, c, d

MulticellularFilamentous Green Bacteria (including family Chloroflexaceae)

nonepotentially all speciesS– or So

(So globules formed outside cell from S–)

mainly Bchl c, d or e

Green Sulfur Bacteria (including family Chlorobiaceae*)

probably all speciesall speciesProb. all: H2 . Some:

low levels of S–, S2O3–

, SoBchl a & b

Purple Non-Sulfur Bacteria (family Rhodospirillaceae*)

some speciespossibly all speciesS– or H2

(So globules formed outside cell from S–)

Bchl a & bPurple Sulfur Bacteria of the family Ectothiorhodospiraceae

some speciessome speciesS– or So or H2

(So globules formed inside cell from S–)

Bchl a & bPurple Sulfur Bacteria of the family Chromatiaceae

Anoxygenic Photosynthetic Bacteria

Chemotrophy?Photoheterotrophy?Electron donor for photoautotrophyChlorophyllsGroup of bacteria

Anoxygenic photo-litho-autotrophType IIBchl a & b + car(Intracell. Membranes)

Purple Sulfur BacteriaChromatiaceae (31)Ectothirhodospiraceae(9)

Anoxygenic photo-organo-heterotrophAerobic chemo-organo-heterotrophType II

Bchl a + car(Intracell. Membranes)b-proteobacteria (4)

Oxygenic photo-litho-autotrophType I + II

Chl a +phycobilins + car(thylakoid membranes)

Chl a & b + car

Chl a2 &b2 + car (+PBS)

Chl d, a + car (+PBS)

Cyanobacteria (>1000)

Prochloron, Prochlorotrhix (2

Prochlorococus (1)

Acaryochloris (1)

Oxygenic Photosynthetic Bacteria

Anoxygenic photo-organo-heterotrophType IBchl g + carHeliobacteriaceae (5)

Aerobic chemo-organo-heterotrophType IIBchl aAerobic a-proteobacteria(23)

Anoxygenic photo-organo-heterotrophAerobic chemo-organo-heterotrophType IIBchl a , b + car

(Intracell. Membranes)a-proteobacteria (31)

Anoxygenic photo-litho-autotrophType IBchl a, c, d, e + car(Chlorosomes)

Green Sulfur Bacteria (15)

Anoxygenic photo-organo-heterotrophicAerobic chemo-organo.heterotrophicType IIBchl a & c + car

(Chlorosomes)

Green filamentous bacteria Chloroflexus-subdivision (3)

Anoxygenic Photosynthetic Bacteria

Preferred growth modeReaction centerLight harvestingGroup of bacteria

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Inorganic carbon is fixed in the Calvin cycleby anoxygenic purple bacteria

Rubisco !!

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

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

Classification

Antenna

Chlorophyll

Electron flow

Rubisco

Carbon fixing

Purple Bacteria(Proteobacteria)

Carotenoids in spirilloxanthin, okenone, or rohodopinal groupsLH1 & LH2 complexes

Bacteriochlorophylla & b

Reverse e- flow (reverse Krebs Cycle)

+Rubisco

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

Green SulfurBacteria

Chlorosomesfunnel light to RC, carotenoids (& bachl c, d, e) in isorenieratene & chlorobactenegroups

Bacteriochlorophyllc, d or e, (sm. amt. of a)

cyclic

None

Reverse TCA

Green Filamentous(nonsulfur) bact.

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

Bacteriochlorophyllsc or d (sm. amt. of a)

Reverse e- flow (reverse Krebs Cycle)

None

Hydroxy-propionatepathway.Some reverse TCA

Heliobacteria(gram + bact.)

Carotenoids –neurosporene

Bacteriochlorophyll g

cyclic

None

None, no Calvin Cycle, no reverse TCA, photoheterotrophs

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

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Classification

Ecology

Produce O2?

Photosynth. Type

Electron Donor

Electron acceptor

Reaction Center

Purple Bacteria(Proteobacteria)

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

No, anoxygenic

Like photosystemII, quinone-type

H2S, H2 & other

Quinone, Fe between quinones

P870, Bchl a

Green SulfurBacteria

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

No, anoxygenic

Like photosystem IFe-S

Sulfide & organic hydrogen donors

Ferredoxin

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

Green Gliding(nonsulfur) bact.

Facultativelyaerobic: aerobic- live heterotrophically, not photosynth., anaerobic-photosynthetic, do not fix N

No, anoxygenic

Like photosystemII, quinone-type

H2S, organics

Quinone, Mnbetween quinones

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

Heliobacteria(gram + bact.)

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

No, anoxygenic

Like photosystem IFe-S

Organic donors

FeS

P789, homodimeric

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

Sulfuretum in Nivå Sulfuretum in Nivå

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Foto: Lucas Stal5 mm

”Farbstreifen Sandwatt”

400 500 600 700 800 9000.0

0.2

0.4

0.6

0.8

1.0

400 500 600 700 800 900

Cyanobacterial layer

Carotenoids

Bacteriochlorofyll a

Phycobilins

Chlorofyll a

Chlorofyll a

Wavelength (nm)

Abs

orba

nce

Purple bacterial layer

Carotenoids

Bacteriochlorofyll a & c

Bacteriochlorofyll c

Differential light utilisation governs coexistence

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-365, 405, 566, 762

375, 419, 575, 788

Bchl g

738459, 648460-462, 710-725

Bchl e763425, 654450, 715-745Bchl d

775433, 663457-460, 745-755

Bchl c

1040368, 407, 582, 795

400, 605, 835-850, 986-1035

Bchl b

907-915358, 579, 771

375, 590, 805, 830-911

Bchl a

740-760400, 697714-718Chl d-455, 645655Chl b

680-685435, 663670-675Chl aCellsextractCells

Fluorescence maxima (nm)Absorption maxima (nm)Pigment

Foto: Lucas Stal5 mm

”Farbstreifen Sandwatt”

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Microscale light measurementsMicroprobes (A-D) for:

- radiance, irradiance,scalar irradiance (UV-

NIR light)- Surface detection- Pigment fluorescence- Diffusivity/Flow

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

All based on multimodegraded index optical fibers100/140 µm core/claddingN.A. = 0.22

Fiber-optic Microsensors

Kühl & Revsbech 2001

Field radiance measurements

Collimated light

48o

60o

0o140o

Downwelling light Forward scattered light Back scattered light

Microscale light measurements

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A

-180 -120 -60 0 60 120 1800

20

40

60

80

100

120

140

160

Angle of incident light

%of

aver

age

resp

onse

B

-180 -120 -60 0 60 120 1800

20

40

60

80

100

Angle of incident light

Scalar irradiance probe Irradiance probe

Light-collecting properties offiber-optic microprobes

Microscale light measurements

Kühl et al. 1997 Kühl & Jørgensen 1992.

2.0

1.5

1.0

0.5

0.0

0.01 0.1 1 10 100

0 50 100 150 PAR (% of Ed)

Dep

th (m

m)

A

0 5 10

B

K0 (mm-1)

Strong light attenuation due toabsorption and scattering

[ ]12

20

10

00

)()(ln)(ln)(

zzE

E

dzEdK

−⎥⎦⎤

⎢⎣⎡

−=−=λ

λλλ

Kühl et al. in prep.

Mapping the spatial light distribution

Collimated light

48o

60o

0o140o

Downwelling light Forward scattered light Back scattered light

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Collimated light

48o

60o

0o140o

Downwelling light Forward scattered light Back scattered light

Directional vs. Diffuse lightFrom one direction Integral from all directions

400 500 600 700 8000

50

100

150

200

Klorofyl

1.00.80.6

0.2

0.4

Ska

lar i

rradi

ans

(% a

f ind

fald

ende

lys)

Lysets bølgelængde (nm)

0.0

BakterieklorofylKlorofyl

Fykobilin

Spektral lysnedtrængningLight source

O2 microsensor

Experimental set-up for O2-measurements

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Cyanobacteria

Diatoms

400 450 500 550 600 650 7000

50

100

150

200

Lysets bølgelængde (nm)

Foto

synt

ese

(µm

ol il

t l-1 m

in-1)

Kiselalger

Cyanobakterier

Karotenoider

KlorofylKlorofyl

Fykobiliner

Aktions-spektrum

3

2

1

0

-10 100 200 300

3

2

1

0

-1

0 2 4 6 8

Dep

th (m

m)

Photosynthesis(nmol O2 cm-3 s-1)

Photosynthesis

Oxygen

WaterBiofilm

0 200 400 600 800

Oxygen(µmol O2 l

-1) Scalar irradians

(µmol photons m-2 s-1)

Light

Light and Photosynthesis

Kühl & Jørgensen 1992

Photosynthesis in steep light gradients

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Carotenoids protect against photooxidation

Chl + radiation → Chl*

Chl* + O2 → Chl + O2*

Chl* + carotenoids → Chl + carotenoids*

O2* + carotenoids → O2 + carotenoids*

Carotenoids* → carotenoids + heat

Activated chlorophyll and oxygenforms radicals that can breakdown proteins, lipids and otherkey components of cells

Absorption vs. Action spectrum

Photokinesis

PhotophobicResponse

Phototaxis

BehaviourMeasuredQuantity

Single CellEffect

EcologicalSignificance

positive

negative

lightintensity

I

Intensity

Speed

Intensity

Speed

ColonyEffect

Accumulation indark areas

Accumulation inilluminated areas

step-up

step-down

change inlight

intensitydIdt

reversal ofdirection

Trapping indark areas

Trapping inilluminated areas

posi-tivenegative

bimodal

direction oflight

I

Moving towardslight source

Moving away fromlight source

Avoiding photo damage

(Optimizing photosynthesis)

Avoiding photo damage

Positioning in benthic systems

Optimizing photosynthesis

Positioning in benthic systems

Optimizing photosynthesisMoving to surfacein pelagic systems

Moving to the bottomin pelagic systems

Moving perpendicularto light direction

Keeping depthin pelagic systems

Types of photobehaviour

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”Algography”

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Different light optima for different phototrophs

Gradient-capillary-cell-tracking-setup

inverted microscopeoxygen-microelectrode with Picoammeter

videocamera

videorecorder

sulfidic�agar plug

medium with�bacteria

gas space

microsensors for

end of tubing

oxygen�sulfide�

pH

pH reference�electrode

Gradient Capillary Setup

flat glass capillary�(40 x 8 x 0.8 mm3)

Motility of Microorganismsin Response to Light, Oxygen, and Sulfide

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

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Marichromatium gracile

6.6

6.8

7.0

0 1 2 3 4 5 60

100

200

300

400

500

d istance (mm)

pH

pH

O2

H2Srel. ce lldensity

D arkness

Cell distribution in relation tooxygen, sulfide, and pH gradients

Thar & Kühl 2001

M. gracile: dark-light transitionanoxic oxicca. 500µm

Thar & Kühl 2001

Marichromatium gracilePhobic responses towards

increasing oxygen concentrations and darkness

Thar & Kühl 2001

Response to light-dark border Response to oxygen gradient

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UV radiation and it’s effects on organismsUV-C (<280 nm)• Strongly absorbed in the atmosphere, no ecological relevance.

UV-B (280-320 nm)• Direct damage on biological chromophores due to absorption in:

DNA, (thymin-dimers)EnzymesLipidsPhotosystems.

UV-A (320-400 nm)• indirect damage via photodynamic reactions caused by free radicals, especially reactive oxygen species•Similar damage is induced by high levels of visible light.

4.60 x 10-3-93 x 1031 x 103Wadden Sea

0.26 x 10-3-3120 x 10317 x 103Southern Ocean

0.12 x 10-3-6150 x 10325 x 103Sargasso Sea

Water

21.6103330.450.23Muddy sediment

10.5105250.950.50Cyanobacterial mat

17.2103240.720.36Sandy sediment

6.5131232.400.98(dry)

4.1127153.101.25Beach sand (wet)

Sediment

K(UV-B)mm-1

Max E0(UV-B)

%incident

Ē0(UV-B)

%incident

Z(1%)Visible

mm

Z(1%) UV-Bmm

Habitat

Importance of UV radiation

UV light is present in a larger part of the photic zone in sedimentsthan in the photic zone of natural waters

Net productivity

Gross productivity

UV radiation effect onphotosynthetic microbial mat

UV protection – sunscreen pigments, Scytonemin and MAA’s

Cell+sheath

Sheath

scytonemin

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UV-induced migration of cyanobacteria

UV-induced migration of cyanobacteria

1 mm+ UV

- UVcyanobacteria

Cyanobacteria