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.
7
”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
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UV radiation and it’s effects on organisms UV-C (