Algal Photosynthesis_dismukes

Algal Photosynthesis G.C. Dismukes BioSolar Team

Princeton University

Dept of Chemistry &

Princeton Environmental Institute

Cyanobacteria Green alga Arthrospira m. Chlamydomonas r.

Complex Utilization of Algal Biomass

Algal Biomass

Carbohydrates Lipids/TAG

Proteins Minerals

H2, ethanol Biodiesel Plant Food

Photosynthesis Respiration Fermentation

COMPARISON BETWEEN CROP EFFICIENCIES: THE BIODIESEL EXAMPLE

Plant Source Biodiesel L/Hect/Year

Area required to match current global oil demand

million hectares

Area required as a percentage of global

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

Oxy

gen

prod

uctio

n, m

mol

/min

/m2

2H2O Æ O2 + 4H+ + 4e

3CO2

150

PO4

“N” 100

50

Carbohydrates

Proteins 0

Lipids

σPSII

α

Photoinhibition Pmax

500 1000 1500

Irradiance, W/m2

Blue Light LED Chlorella

Light compensation point Ananyev & Falkowski

Acclimation of Plants occurs via σPSII

Antenna LHCII

RC PSII

Antenna LHCII

RC PSII

Antenna LHCII

RC PSII

σPSII is lowσPSII is high σPSII is moderate

Increasing of Solar Irradiation

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 diurnalrespiration losses averaged 35% of grossphotosynthesis

*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 parallelwith 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 afunction 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

Chlorella, Ananyev & Falkowski

0 50 100 150 200 250

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Non

-Pho

toch

emic

al Q

uenc

hing

Actinic Light (PFD), μE m-2 s -1

NPQ

0 50 100 150 200 250

0.0

0.2

0.4

0.6

0.8

1.0

Phot

oche

mic

al Q

uenc

hing

qP Occurs in the LHC antenna

Occurs in the PSII Reaction center

Open

PSII

closed

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

0.0

0.2

0.4

0.6

0.8

Fv/F

m

Dehydrated lichens, germ cells-etiolated leaves

Exolithic cyanobacteria, eg. Gloeobacter violaceus, Chl-d-containing phototrophs, eg. Acaryochloris m.

Most cyanobacteria and hydrated lichens

Microalgae: Chlorella, Euglena, Chlamydomonas & carbonate-requiring cyanobacteria

Plants, eg. Inga, spinach

Duck weed (hydroponic)

0.0

0.1

0.2

0.3

0.4

0.5

Fluo

resc

ence

Yile

d Fv

/Fm

Chlamydomonas HisTAG pf3

WT

cyt b-559 mutant

Open

PSII

Closed

0 100 200 300 400 500 600 700 (1)Note: for studied species t1 of QA- reoxidation has range from 160 to 250 μs.

-1Photon Flux Density, μE m-2 s Ananyev, Hamilton, Nixon, Dismukes (200Solution:

•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

Fv/F

m (E

xper

imen

t)

S1

S2

S3S4

S0

30 μs 100 μs

350μs

1 ms

30s

10s

2 H2O

8 β

β

α

α

α

α

Kok model of the CaMn4 redox cycle

0 10 20 30 40 50

0.00 0.25 0.50 0

1

2

FFT

ampl

itude

Experiment Cycles, 1/s

period 4.16

2 μM DCMU

Fitting α = 0.085 β = 0.035

0.20.46

0.5 0.52

O2

0.40.50 Fv/Fm (D

CM

U)

0.3 0.48

photochemical misses: α0.1 0.44 double hit/misses: β

0.42

Single Turnover Flash Number

Ananyev & Dismukes, 2005

1 10 100 1000

0.05

0.10

0.15

0.20

α a

nd β

dark time between flashes, ms

α - Chlorella β -α - Arthrospira β -

A

previous studies

1 10 100 1000 2

3

4

5

6

7

8

Qua

lity

fact

or, 1

/(α+β

) dark time between flashes, ms

Chlorella (alga)

Arthrospira cyanobact

Conclusions: Arthrospira maxima cells have the fewest misses and double turnovers

In vivo Cyanobacterial PSII turn over time (2-3 ms) approaches the maximun in vitro rate!

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

2 4 6 8 10

1

2

Four

ier A

mpl

itude

Period (Flash Number)

1 bar air 10.3 bar O2

4.4

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Four

ier A

mpl

itude


43 bar

0 10 20 30 40 50 0.40

0.45

0.50

0.55

0.60

Fv/F

m

Flash Number


Native Cells –maple leaves Chl Variable Fluorescence Yield: H2O Æ PSII Æ PQ

Baseline and normalization

Fourier Transformation

0 10 20 30 40 50

-0.04

-0.02

0.00

0.02

0.04

Fv/F

m

Flash Number


0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

Per

iod


A. m

axim

aA

. max

ima

C. r

einh

ardt

ii D

unal

iela

thyl

akoi

ds

Spi

nach

Eun

imus

Map

leC

love

rG

. bilo

ba

43 barPlants algae+

Kolling, Brown, Ananyev & Dismukes, submitted

Illumination at elevated O2 pressure kills PSII due to the PSI Mehler reaction

Athrospira maxima

Effect of Light Exposure on Variable Fluorescence 0.46

1.0 bar air 35 mE/m2

0.44

0.42

0.40 10.3 bar O2 0.38 7 mE/m2

Protect against O2, Strain dependent

Inte

grat

ed F

v/Fm

P*

0.30 NAD+

respiration 0.36

0.34

10.3 bar O20.32

ndh

PSII

P* QB

PQ-pool

P

Mn4Ca

DBMIBX 0 5 10 15 20 25 30

Time (hours) O2 → O2•-

Fd 35 mE/m2

Effect of DBMIB on Variable Fluorescence

0.45 cytb6f

40 μM DBMIB

10.3 bar O2

1 bar air

P

Inte

grat

ed F

v/Fm

0.40

0.35

0.30

0.25

PSI2 H2O

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

0 5

10 15 20 25 30 35 40 45 50 55 60 65

*

*

Tim

e (h

ours

)

50% loss of Fv/Fm 90% loss of Fv/Fm

A. m

axim

a

C. r

einh

ardt

ii

Dun

alie

la

Spi

nach

Eun

imus

Map

le%

Clo

ver

G. b

iloba

*extrapolated %showed slow decay under 1 bar air

plants algae

Conclusions: •Protect against O2 during daylight •Eukaryotes, including algae, are very sensitive •Arthrospira maxima is far more tolerant (other cyanobacteria?)

WT

51uM 11uM

Succinate

Malate

Fumarate

H2

Acetate

Succinate Malate H2

Lactate

Acetate

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

Bottom Line: 2-3 X H2 yield

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

• 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

single turnover flash cluster 50 ns to CW

0.1 µs to ∞

darkPrinceton Fast Repetition Rate Fluorometer

G. Ananyev

Algal Photosynthesis_dismukes

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