Diverting Photosynthetic Electrons From Suspensions

of Chlamydomonas Reinhardtii Algae

Dr Manon GUILLE-COLLIGNON

Photosynthesis : Our link to the Energy of the Cosmos

What is Photosynthesis?

+

-

• Conversion energy of light into chemical energy

Formation of oxygen and fixation of atmospheric carbon dioxide

• Light absorption – Formal charge separation in PS - Electron transfer steps

- H2O oxidation and CO2 reduction

6H2O + 6CO2 + photons = C6H12O6 + 6O2

"Flanged" mechanism.

Context and Goals of the Project

• Saturation of the photoconversion is related to rate limiting electron transfer steps

occurring downstream of the Photosystem II.

• Upon illumination, most excited PSII can not release their charge

Limitation of the photosynthetic activity but also cell damages, photoinhibition.

✓ Idea of “Taking advantage" of Photosynthesis

✓ Producing a current by Harvesting Electrons at the Rate limiting

electron transfer Step

• Producing a Current ? Get a Good Electron Transfer

➢ Immobilization of photosynthetic systems at the electrode surface (loss of biological

integrity of organisms)

➢ Suspension of intact algae (long timeframes, photoreparation, cell growth)

« Natural » photosynthesis :

we adapt our toolbox to the biological target

• Which scale for the Biological targets ? Preserve Cell Integrity

Photosynthetic units : isolated photosystems

Sub-structures : thylakoid membranes

Whole organisms : leaf, algae, cyanobacteria…

Badura et al. Energy Environ. Sci. 2011, 4, 3263.

How to build the Project?

« Artificial » photosynthesis :

the biological system is built with

the diversity of the chemistry toolbox

Which Tool(s) ?• Using an electrode…

ii

+ e-

e-

MOx

MRed

• Using an electrode alone will be too invasive

An electron relay/cargo (= mediator) is required under two molecular forms : MRed and Mox

(no direct electron transfer)

+

-

• Be a (quasi-)reversible redox couple - fast electron transfer -

• Harvest electrons between the charge separation and the rate determining steps

Properties of the Redox Mediator

• Endogenous PSII acceptors: plastoquinone

Exogenous PSII acceptors + electron carriers: Quinones

✓ Downstream of PSII

✓ Upstream of b6f complex

Electrochemical Studies

Evaluating the QH2 ability to be Oxidized at an Electrode surface

Principle : control variation of the working electrode potential vs. a reference electrode

• i = f(E) recordings (cyclic voltammetry)

DiffusionElectron

transfer

MRed

MOx

ERef

e-

Working

electrode

MRed = MOx + ne-

MOx + ne- = MRed

Electron

transfer

Electron transfer

kinetic control

Diffusion controli

E

• What data can be provided by electrochemistry?

Electron transfer rate

Potential values at which a species can be oxidized or reduced

Mechanism(s), Thermodynamic data…

Benzoquinone

(BQ / BQH2)

Phenylbenzoquinone

(PPBQ / PPBQH2)

Naphtobenzoquinone

(NBQ / NBQH2)

2,6-dimethylbenzoquinone

(2,6-DMBQ / 2,6-

DMBQH2)

2,5-dimethylbenzoquinone

(2,5-DMBQ / 2,5-

DMBQH2)

2,6-dichlorobenzoquinone

(2,6-DCBQ / 2,6-DCBQH2)

2,5-dichlorobenzoquinone (2,5-

DCBQ / 2,5-DCBQH2)

Listing of QH2 / Q forms investigated

Cargo Mediator=Q Ea (mV/ Ag/AgCl)

pH 7.4

Solubility

Benzoquinone +250 >=5mM

2,5-dichloroquinone +130 0,5 <s< 1 mM

2,6-dichloroquinone +135 0,75mM

2,5-diméthylquinone +365 >=1mM

2,6-diméthylquinone +190 >=1mM

Naphtoquinone +35 0,85mM

Phenyl-quinone +180 >=0,5mM

Results of Electrochemical Measurements

• All QH2 forms can be considered since voltage values at which they can be oxidized

are convenient for electrolysis experiments. Solubility is not an issue.

• Electrochemical properties won’t be the dominant parameter to choose the best cargo

Fluorescence Studies

PhotoSystemII

PhotoSystemII*

hν

Electron flow

Fluorescence

A fluorescence decrease in presence of quinone represents the electron flux diverted due to

quinones

A lot of fluorescence equivalent to a little electron flow and viceversa

Evaluating the Quinones Ability to Divert Electrons from PSII

Principle : the electron charge released by PSII can be undirectly monitored through

the fluorescence relaxation.

• A very sophisticated set-up of fluorescence measurements does exist in

Time (ms)

0 1000 2000 3000 4000 5000

Flu

ore

scence (

a.u

.)

0

2000

4000

6000

8000

F0

Fstat

Fmax

Zone 1 : Low 2,6-DCBQ effects → Lost quinones

Zone 2 : Increase in ϕ = f(CQ) → Electron harvesting by quinones

Zone 3 : Saturation → Excess of quinones has no longer any effect

Longatte et al. Biophys. Chem. 2015, 205, 1.

Fluorescence Studies of Algae with Quinones

Concentration (µM)

0 10 20 30 40

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Representative 𝜙 = (CQ) curve

Effect of 2,6-DCBQ concentration

𝜙 =𝐹𝑚𝑎𝑥 − 𝐹𝑠𝑡𝑎𝑡𝐹𝑚𝑎𝑥 − 𝐹0

𝛟 Electron extraction yield = % Photons converted to Electron flux

Longatte et al. Biophys. Chem. 2015, 205, 1; Photochem. Photobiol. Sci. 2016, 15, 969.

Quantification of Quinones Ability to Harvest Electrons

• Best quinones: DCBQ et PPBQ.

• Globally consistent with highest redox potentials and electrowithdrawing groups

E1/2 (mV vs SHE)

-200 -100 0 100 200 300 400

D

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

NBQ

2,5-DCBQ

PPBQ

2,6-DCBQ

2,5-DMBQ

2,6-DMBQ

BQ

D∞

D

0

QC

Extraction of an Infinite Derivation value : D∞

Diagram zones show conditions where :

The maximum harvesting occurs

The harvesting is controlled by electron transfer rate or mass transport

Performance Predictions : 3D Diagrams

Longatte et al. Photochem. Photobiol. Sci. 2016, 15, 969.

Zone B : Harvesting is controlled

by mass transport of quinones

= D

Zone E : Harvesting is controlled by

electron transfer rate

0 2 4

-6

-4

Log I° (Light flow; µE.m-2.s-1)

0,2

0,6

-8

Zone A : No Harvesting

0 =

E

CB

D

A

Zone diagrams can be built to predict performances

Longatte et al. Electrochim. Acta 2017, 236, 337 ; Fu et al. Nat. Commun. 2017, 8, 15274.

Preparative scale : → algae suspension (20 mL; 107 algae.mL-1) under forced convection

→ 2,6-DCBQ (100 µmol.L-1)

Experimental Photocurrents : Experimental Set-Up

Spectrometer : illumination

Spectroelectrochemical cuvette

→ WE = carbon gauze 1 cm2

→ CE = Pt

→ REF: Ag/AgCl

• Steady state photocurrents (over one hour) : 10 – 100 µA.cm-2

• Experimental parameters : quinone concentration, illumination

Experimental Photocurrents : Results at Preparative Scale

Longatte et al. Electrochim. Acta 2017, 236, 337 ; Fu et al. Nat. Commun. 2017, 8, 15274.

.

Time / s

0 1000 2000 3000 4000 5000 6000

Ph

oto

curr

ent

/ µ

A.c

m-2

0

10

20

30

40

50

60

70

100 µM

40 µM

Illumination

340 µE.m-2.s-1

• Electron harvesting by Quinones + Electrochemical Quinone regeneration by Electrode

Limitation of Steady State Photocurrents

Longatte et al. Chem. Sci. 2018, 9, 8271.

Time / s

0 2000 4000 6000 8000

Photo

curr

ent / µ

A.c

m-2

-10

0

10

20

30

40

50

60

70

Photoinactivation under strong light ?

• Photoinhibition

• Reduced species accumulation

• Alterations of the biological system

during experiments ?

Side-effects of quinones ? Poisoning ?

• Reactions with macromolecules

• Degradation of proteins, lipids, DNA

A Miniaturized Set-up for Rationalization

Sayegh et al. Electrochim. Acta. 2019, 304, 465-473.

→ Cyclic voltammetry analysis « in situ »

→ « Fast » recording of photocurrents

1 cm

Time (s)

0 200 400 600 800 1000

Photo

curr

ent (µ

A)

0

5

10

15

20

25

30

Equilibrationtime

The preparative set-up is not suitable for systematic analyses and optimisations

→ New « miniaturized » set-up to reduce duration of experiences

E (V vs Ag/AgCl)

-0.2 0.0 0.2 0.4 0.6

Curr

en

t (µ

A)

-4

-2

0

2

4

6

8

10

12

14

16

No light

Under illumination

Limitation of Steady state Photocurrents with Miniaturized Set-up

Longatte et al. Chem. Sci. 2018, 9, 8271; Sayegh et al. Electrochim. Acta. 2019, 304, 465-473.

→ Poisoning seems to be related to the oxidising power of quinones

Oxidising quinones are both able to extract electrons and poison the system

Log(Time Stability)

2.8 3.0 3.2 3.4 3.6 3.8

Ste

ady s

tate

photo

curr

ent

(µA

)

0

5

10

15

20

25

30

2,6-DCBQ

PPBQ

2,6-DMBQ

2,5-DMBQ

NBQ

Limitation of Steady state Photocurrents with Miniaturized Set-up

Longatte et al. Chem. Sci. 2018, 9, 8271; Sayegh et al. Electrochim. Acta. 2019, 304, 465-473.

→ Poisoning seems to be related to the oxidising power of quinones

Oxidising quinones are both able to extract electrons and poison the system

Log(Time Stability)

2.8 3.0 3.2 3.4 3.6 3.8

Ste

ady s

tate

photo

curr

ent

(µA

)

0

5

10

15

20

25

30

2,6-DCBQ

PPBQ

2,6-DMBQ

2,5-DMBQ

NBQ

Quinones = « double agents » = harvesting BUT also poisoning…

Conclusion : Two General Issues

R = ?

"Artificial" photosynthesis

Yield ~ 15%

Experimental Current density ~ 1-10 mA.cm-²

"Natural" photosynthesis

Yield ~ 5%

Experimental Current density ~ 100 µA.cm-²

Modeling : > 1 mA.cm-²

Perspectives

Limit poisoning by Quinones

Acknowledgements

UMR 8640

CNRS-ENS-Sorbonne Université

« PASTEUR », ENS, Paris

Dr. Guillaume LONGATTE

Dr. Adnan SAYEGH

Dr. Léna BEAUZAMY

Marc ARDERIU ROMERO

Pr. Frédéric LEMAITRE

Dr. Jérôme DELACOTTE

Pr. Eric LABBE

Dr. Olivier BURIEZ

Dr. Christian AMATORE

UMR 7141, Institut de Biologie

Physico-Chimique, Paris

Dr. Han-Yi FU

Dr. Sandrine BUJALDON

Dr. Fabrice RAPPAPORT

Dr. Francis-André WOLLMAN

Dr. Yves CHOQUET

Dr. Daniel PICOT

Dr. Benjamin BAILLEUL

Pr. Pierre JOLIOT