Algal Photosynthesis

BioSolar Team G.C. Dismukes

Princeton University

Dept of Chemistry &

Princeton Environmental Institute

Cyanobacteria Green alga

Arthrospira m. Chlamydomonas r.
 Complex Utilization of Algal Biomass

Respiration
Photosynthesis
Fermentation

Algal
Biomass

Carbohydrates Proteins
Lipids/TAG Minerals

Plant
H2, ethanol Biodiesel Food
 COMPARISON BETWEEN CROP EFFICIENCIES:

THE BIODIESEL EXAMPLE

Area required to match Area required as a

Biodiesel
Plant Source current global oil demand percentage of global
L/Hect/Year
million hectares land mass
Soybean 446 10932 72.9
Rapeseed 1190 4097 27.3
Mustard 1300 3750 25.0
Jatropha 1892 2577 17.2
Palm Oil 5950 819 5.5
Algae Low 1% 45000 108 0.7
Algae High 4% 137000 36 0.2

• LOW ALGAE ESTIMATES BASED ON GREENFUELS TUBULAR BIOREACTOR DESIGN; EQUAL TO

1% OF AVERAGE SUNLIGHT ENERGY CONVERSION TO BIODIESEL.

• HIGH ALGAE ESTIMATES BASED ON NRELS PEAK ALGAE PERFORMANCE, THIS WOULD BE
EQUAL TO 4% OF AVERAGE SUNLIGHT ENERGY CONVERTED TO BIODIESEL.

• ONLY 13.3% OF THE WORLDS LAND MASS IS ARABLE, ALGAE BIOREACTORS OR RACEWAYS DO
NOT REQUIRE ARABLE LAND.

• Source data Chisti 2007

• www1.eere.energy.gov/biomass/pdfs/biodiesel_from_algae.pdf
• http://www.greenfuelonline.com/technology.htm
Slide credit , B. Hankamer
 Light Curve of Photosynthesis

2H2O Æ O2 + 4H+ + 4e­

150 Pmax
CO2 Photoinhibition
PO4
3­
σ
2
Oxygen production, mmol/min/m
“N”
100 PSII

50

Carbohydrates
Proteins 0
α
500 1000 1500
Lipids Irradiance, W/m
2
Blue Light LED
Chlorella
Ananyev & Falkowski Light compensation point
Acclimation of Plants occurs via σPSII

Increasing of Solar Irradiation

Antenna
Antenna
LHCII Antenna
LHCII
LHCII
RC RC RC
PSII PSII PSII

σPSII is high σPSII is moderate σPSII is low

 Optimizing algal biomass production in an

outdoor pond: a simulation model*

Model consisting of photoadapation, gross photosynthesis

& respiration under wide irradiance levels.
Model gives reliable predictions of:
• yearly averaged production rate = 10 g C m-2 d– 1
• yearly averaged chlorophyll areal density = 0.65 g m-2 for
the maximal production rate.
• under optimal operational conditions, the diurnal
respiration losses averaged 35% of gross
photosynthesis

*Sukenik , Levy , Levy, Falkowski , Dubinsky;

Journal of Applied Phycology 3: 191-201, 1991

Marine alga Isochrysis galbana

 Light-saturated photosynthesis –
limitation by electron transport or carbon fixation?
Central Paradox of plant and algal photosynthesis
(Emerson & Arnold, J. Meyer ):
The greater the Chl content (eg. growth at low light) the slower the e- transfer rate
of photosynthesis
Explanation*
• As cells adapt to lower growth irradiance levels, the minimal turnover time
of photosynthesis τ, H2OÆCO2, increases from 3.5 to 14.5 ms, in parallel
with increases in the thylakoid surface density and the contents of the
photosynthetic units (all pigments, Photosystem II, PQ, cytochrome b-6-f,
Photosystem I).
• Thus, at all growth irradiance levels, the relative proportion of these
membrane-bound electron-transport components remains constant.
• By contrast, the cellular pool size of ribulose-l,5-bisphosphate
carboxylase/oxygenase is independent of growth irradiance.
• Hence, ratio of [RUBISCO]/[ET-chain] varies between 4.8 and 1.2 as a
function of growth irradiance levels. Identical to τ!
Conclusion
• under nutrient saturated conditions the absolute rate of light-saturated
photosynthesis is limited by carbon fixation rather than electron transport

*Sukenik, Bennett & Falkowski et al. BBA, 1986

 Light Curves of Photochemical and

non-Photochemical Quenching
Occurs in the Occurs in the PSII
LHC antenna NPQ 1.0 qP Reaction center
0.18
Non-Photochemical Quenching

0.16 0.8

Photochemical Quenching
Open
0.14
0.6
0.12 PSII

0.10 0.4 closed

0.08
0.2
0.06

0.0
0.04

0 50 100 150 200 250 0 50 100 150 200 250

Chlorella,
-2 -1
Ananyev & Falkowski Actinic Light (PFD), μE m s
Decreasing Chlorophyll Antenna Size and Improving Light Utilization Efficiency

RNAi technology to down-regulate the entire LHC gene family in Chlamydomonas r. *

*Mussgnug et al Plant Biotechnology Journal (2007) 5, pp. 000–000

Mutant Stm3LR3 had significantly reduced levels of LHCI and LHCII mRNAs and
proteins while chlorophyll and pigment synthesis was functional.

Stm3LR3 also exhibited …reduced sensitivity to photoinhibition, resulting in an

increased efficiency of cell cultivation under elevated light conditions.
Photoinhibition Mixotrophic growth rates under high-light
at high light in TAP medium.
Conclusions from multiple studies
On truncated antennas:
•The reduced optical cross section
provides better growth at high
light only.
•Reduced energy conversion to
ATP + NADPH limits cell growth at
ambient light intensity.
 Low Photosynthetic Quantum Efficiency at High Light Intensity

PSII Quantum Efficiency is Limiting

(1)
Photosystem II Quantum Efficiency at Zero Light Flux at Maximum Solar Flux in Green Algae

0.5
0.8 Duck weed (hydroponic) Chlamydomonas
HisTAG pf3 Open

Fluorescence Yiled Fv/Fm

Plants, eg. Inga, spinach 0.4
0.6
Microalgae: Chlorella, Euglena, Chlamydomonas
& carbonate-requiring cyanobacteria 0.3 PSII
Fv/Fm

Most cyanobacteria and hydrated lichens WT

0.4
0.2
Closed
Exolithic cyanobacteria, eg. Gloeobacter violaceus,
0.2 Chl-d-containing phototrophs, eg. Acaryochloris m.
0.1 cyt b-559
mutant

0.0
Dehydrated lichens, germ cells-etiolated leaves 0.0
(1)
Note: for studied species t1 of QA
-
reoxidation has range from 160 to 250 μs.
0 100 200 300 400 500 600 700
-2 -1
Photon Flux Density, μE m s
Ananyev, Hamilton, Nixon, Dismukes (200
Solution:

•Reduce the size of the antenna (less Non-photochemical Quenching)

•Increase the number of the plastoquinone electron acceptors in the pool
eg., Plants and algae have 6-8 PQ/pool vs. Cyanobacteria at 3-5x larger pool
Ananyev & Dismukes
 PSII turnover in vivo can be monitored via Fv/Fm :

Period-four oscillations reveal WOC cycle

S1 α
μs 10
0μ
α 30 s
30s

8
β
period 0.5
S0 S2
0.52
4.16 β α
2

FFT amplitude
O2

μs
10s
0.4

350
0.50
1
Fv/Fm (Experiment)

S4 S3

Fv/Fm (DCMU)

2 H2O 1 ms
α
0
0.48
0.00 0.25 0.50 0.3

Experiment Cycles, 1/s

Kok model of the
CaMn4 redox cycle
0.46 0.2
photochemical
Fitting misses: α
0.44
α = 0.085 0.1

β = 0.035
double hit/misses: β

2 μM DCMU
0.42
0 10 20 30 40 50
Single Turnover Flash Number
Ananyev & Dismukes, 2005
 In vivo Cyanobacterial PSII turn over time (2-3
ms) approaches the maximun in vitro rate!

8
Arthrospira
0.20 A α - Chlorella

Quality factor, 1/(α+β)

cyanobact
β- 7
α - Arthrospira
0.15 β- 6
α and β

5 Chlorella
0.10 (alga)
4
previous
studies
0.05 3

2
1 10 100 1000 1 10 100 1000
dark time between flashes, ms dark time between flashes, ms
Conclusions:
Arthrospira maxima cells have the fewest misses and double turnovers

Ananyev & Dismukes, 2005

 Arthrospira m. Conclusions
• Highest PSII photochemical quantum efficiency of all
cyanobacteria: light stored as charge separation

• The fastest WOC yet observed in vivo with fewest

misses: highest turnover efficiency

• has the largest PQ pool 3-5x vs green algae

• Bicarbonate is an essential cofactor for fastest WOC

 O2 pressure does NOT slow or reverse PSII turnover

Native Cells –maple leaves 1 bar air
3.0
Chl Variable Fluorescence Yield: 10.3 bar O2

H2O Æ PSII Æ PQ

Fourier Amplitude
2.5
0.60
1 bar air
10.3 bar O2 2.0
43 bar
0.55

Fv/Fm
Baseline and 0.50 1.5
normalization
0.45 1.0
0.04

1 bar air 0.40 0.5

0 10 20 30 40 50
0.02 10.3 bar O2
Flash Number 0.0
Fv/Fm

0.00
1 bar air
-0.02 10.3 bar O2
5.5

-0.04
5.0 Plants algae+ 43 bar
4.5
0 10 20 30 40 50
4.0
Flash Number
3.5

Period
4.4 3.0
2.5
Fourier Amplitude

2 2.0
1 bar air 1.5
Fourier 10.3 bar O2
1.0
Transformation
0.5
1 0.0

Clover

A. maxima
G. biloba

Dunaliela

A. maxima
Maple

thylakoids

C. reinhardtii
Eunimus
Spinach
2 4 6 8 10

Period (Flash Number)

Kolling, Brown, Ananyev & Dismukes, submitted

 Athrospira maxima
Illumination at elevated O2 pressure
kills PSII due to the PSI Mehler reaction Effect of Light Exposure on Variable Fluorescence
0.46

Protect against O2, Strain dependent

0.44 1.0 bar air

Integrated Fv/Fm
2
35 mE/m
0.42
0.40 10.3 bar O2
P* 0.38 7 mE/m
2

respiration
0.36
0.34
0.32 10.3 bar O2
Fd
35 mE/m
2

NAD+ 0.30

P* 0 5 10 15 20 25 30
ndh Time (hours)
QB O2 → O2•-
PQ-pool
Effect of DBMIB on Variable Fluorescence

X
cytb6f 0.45
1 bar air

DBMIB

Integrated Fv/Fm
0.40 10.3 bar O2
P
0.35

P PSI
2 H2O 0.30

40 μM DBMIB
Mn4Ca
0.25

PSII 0 5 10 15 20 25 30 35

O2 + 4 H+ Time (hours)

Kolling, Brown, Ananyev & Dismukes, in prepn

 Death by O2 Induced Photo-inactivation

Lifetimes at 10 bar O2
65
60
55
50
50% loss of Fv/Fm
90% loss of Fv/Fm
* Conclusions:
45 •Protect against O2 during daylight
*extrapolated
Time (hours)

40
35 %showed slow decay under 1 bar air •Eukaryotes, including algae, are very
30
plants algae sensitive
25 *
20 •Arthrospira maxima is far more
15 tolerant (other cyanobacteria?)
10
5
0
Clover
%
G. biloba

Dunaliela

A. maxima
C. reinhardtii
Eunimus

Spinach
Maple
 GMO of fermentation pathway improves dark H2 yield:
Bryant (Penn State) and Dismukes (Princeton)

Bottom Line: 2-3 X H2 yield

WT
Succinate
Malate Succinate
H2 Malate
11uM Fumarate
51uM Lactate
H2
Acetate
Acetate
 Algal culturing do list

• Micro- & macro-nutrients requirements for

photosynthesis, respiration & fermentation
differ
• Mixing w/o shearing is critical
• CO2 fixation is rate limiting
• Protect against O2 during daylight
• Reduce antenna size for light efficiency
• Metabolic GMOs help fermentation yields

 single turnover flash cluster

G. Ananyev 50 ns to CW

Princeton Fast Repetition Rate 0.1 µs to ∞

dark
Fluorometer
• Solid-state lasers enable:
• λ = UV, blue, green, red, NIR
• pulse trains of variable duration & rep. rate: ≥ 50
ns
• digital noise suppression at the pulse rep. freq.
0-20 MHz