microbial photosynthesis - kubioold.science.ku.dk/mkuhl/ambio/materials/michael... · microbial...
TRANSCRIPT
1
Microbial Photosynthesis
Michael KühlMarine Biological Laboratory
Check: www.mbl.ku.dk/mkuhlfor publication downloads etc.
2
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
3
4
Benthic diatoms
5
6
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
7
Different algal groups have different antenna pigments
Eukaryotic phototrophsevolved via endosymbiosisfrom prokaryoticoxygenic phototrophs.
The only known oxygenicprokaryotes are cyanobacteria.
8
Foto: Lucas Stal5 mm
”Farbstreifen Sandwatt”
9
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
10
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
11
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.
12
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 !!
13
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
14
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.
15
The color of anoxygenic phototrophsis strongly affected by their carotenoids
Type 1 Type 2Type 1
16
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
17
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
18
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
19
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
20
-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”
21
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
22
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
23
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
24
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
25
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
26
”Algography”
27
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
28
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
29
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
30
UV-induced migration of cyanobacteria
UV-induced migration of cyanobacteria
1 mm+ UV
- UVcyanobacteria
Cyanobacteria