Download Microbial Photosynthesis

Microbial Photosynthesis
Michael Kühl
Marine Biological Laboratory
[email protected]
Check: www.mbl.ku.dk/mkuhl
for publication downloads etc.
1
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
8
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
9
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
11
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
12
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
13
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.
14
The color of anoxygenic phototrophs
is strongly affected by their carotenoids
Type 1
Type 2
Type 1
15
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)
16
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
17
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
18
”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)
19
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
20
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
21
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.
22
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)
23
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
24
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
25
”Algography”
26
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
27
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 (<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)
Enzymes
Lipids
Photosystems.
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.
Importance of UV radiation
Habitat
Z(1%)
UV-B
mm
Z(1%)
Visible
mm
Ē0
(UV-B)
%incident
Max E0
(UV-B)
%incident
K
(UV-B)
mm-1
Sediment
Beach sand (wet)
1.25
3.10
15
127
4.1
(dry)
0.98
2.40
23
131
6.5
Sandy sediment
0.36
0.72
24
103
17.2
Cyanobacterial mat
0.50
0.95
25
105
10.5
Muddy sediment
0.23
0.45
33
103
21.6
Sargasso Sea
25 x 103
150 x 103
6
-
0.12 x 10-3
Southern Ocean
17 x 103
120 x 103
3
-
0.26 x 10-3
1 x 103
3 x 103
9
-
4.60 x 10-3
Water
Wadden Sea
UV light is present in a larger part of the photic zone in sediments
than in the photic zone of natural waters
UV protection – sunscreen pigments, Scytonemin and MAA’s
UV radiation effect on
photosynthetic microbial mat
Gross productivity
scytonemin
Cell+sheath
Net productivity
Sheath
29
UV-induced migration of
cyanobacteria
UV-induced migration of
cyanobacteria
+ UV
- UV
1 mm
cyanobacteria
Cyanobacteria
30