Istituto per lo Studio degli Ecosistemi

Torzillo Giuseppe
 Area di ricerca
CNR
Sesto Fiorentino (Firenze)
 Research activities
(1) Selection and characterisation of strains for renewable
energy production (e.g. bio-hydrogen, biodiesel).

2) The effect of environmental stress on photosynthesis and

growth of microalgae.

3) Development of photobioreactor designs for mass

cultivation of different microalgal strains.

4) Role of cyanobacteria in improving soil structure and

fertility.

5) Application of phototrophic micro-organisms to

environmental bioremediation;.

6) Application of molecular techniques to the identification,

taxonomy and phylogenetic analysis of several microbial groups.

Total 7 researchers;3 technicians; 3 PhD students; Students 3 -4; Fellow 2-3

Light saturated Photosynthesis rates recorded with
1200
region, with P 1100 Arthrospira platensis strain M2
independent 1000
from irradiance 900 Pmax

P ( molO2mg-1Chla h-1)
800 A
700
600  = initial slope= AB/CB
500
400
300
Ik = Pmax / 
Light limited 200
part, P Ik Saturation irradiance (µmol photons /m2/s)

100

increases with 0

irradiance -100
C 0 400 B 800 1200
-2 -1
1600 2000 Compensation point where
PFD ( mol m s )
net photosynthesis is zero
P/I curve
150 The parameter Ik represents an optimum on the
(µM O2/mg Chl a+b/h)

120 photosynthetic irradiance curve indicating the irradiance at

90
60 which control of photosynthesis passes from light absorption
30 and photochemical energy conversion to reductant utilization.
Oxygen

0 Also, the predominant fluorescence quenching mechanism (see

-30
-60 Chlamydomonas later) at PFDs < Ik for O2 evolution is photochemical, i.e.
-90 photosynthetic, whereas above Ik it is nonphotochemical,
-120 reinhardtii
-150 involving thermal dissipation.
-180
-210
0 100 200 300 400 500 600 700
Handbook of Microalgal Culture: Applied Phycology and Biotechnology, Second Edition. Edited by Amos
Light intensity Richmond and Qiang Hu. 2013 John Wiley & Sons, Ltd. Published 2013 by Blackwell Publishing Ltd.
µmoles of photons/m2/s 90. Chapter 6 Torzillo and Vonshak)
 Pmax
Photoinhibition

Pmax

Photoinhibition

α Ik Ik

Torzillo et al., (unpublished)

 IMPORTANT PARAMETERS

 = Initial slope

 mol O2 (mg Chl a) -1 s-1

 Microalgae
= Only proportional
 mol photons m-2 s-1 to quantum yield
of Photosynthesis

 mol O2 m-2 s-1 For Leaves, we get

= directly the quantum
 mol photons m-2 s-1 yield of photosynthesis
 mol O2
= QUANTUM YIELD (ɸ )
mol photons
6CO2 + 12H2O + (8 moles photons ) x 6 = C6 H12 O6 + 6 O2 + 6 H2O

6 mol O2 = 0.125 (that is: 0.125

=
QUANTUM
YIELD
8 moles photons x 6 moles of O2 are produced
with one mole of photons)

1/ 0.125 = 8 (Quantum requirement)

8 photons (quanta) are required per one molecule of O2 produced,
i.e. 8 moles of photons (quanta) per 1mole of O2 produced.
The number of photons (quanta) required to reduce a
molecule of CO2 (or oxidise 1 molecule of H2O) is called
QUANTUM REQUIREMENT
 Photosynthesis Efficiency based on solar light
6CO2 + 6 H2O + 6 (9.4 moles Photons (quanta)) = C6 H12 O6 + 6O2

2808 KJ/ mole of hexose

PE = = 23.8 % (of PAR)
209 KJ x 9.4 (mole photons) x 6

23.8 X 0.45 = 10.7% of Solar light

1) 2808 KJ (Energy content of a mole of hexose)

2) 209 KJ is the average energy content of a mole of photons of visible light

3) 9.4 mole of photons (quanta) are required to reduce 1 mole of CO2

(ref. D. Walker)

4) Time 6 because it is necessary to reduce 6 moles of CO2 to

form a mole of hexose

5) Time 0.45 because the PAR is approximately 45% of visible light

 Maximum biomass output per Ha and square meter
1) Assuming an average global daily irradiance of 17.8
MJ/m2/day (average value for 360 days, e.g. in Italy Florence)

2) 17,8 MJ/day x 360 days= 6408 MJ/m2/year

3) Assuming the caloric of combustion of biomass to be 22 MJ/Kg

4) 6408 MJ/m2/year x 0.107

= 31 kg/m2/year
22 MJ/Kg

5) 31 kg m-2 year-1 x10.000 m2 =310 ton/Ha/year

31 kg/m2/year = 31.000 g/m2/year; 31.000 g/m2/year/ 360 days = 86 g/m2/day

6) 86 g/m2/day (Maximum theoretical yield per square meter per day)

 Maximum biomass output per Ha and square meter
1) Assuming an average global daily irradiance of 17.8
MJ/m2/day (average value for 360 days, e.g. in Italy Florence)

2) 17,8 MJ/day x 360 days= 6408 MJ/m2/year

Joule = 0.239 cal
3) 6.408.000.000J/m2/year x 0.234 = 1.531.512.000
cal /m2/year = 1.531.512 Kcal

Assuming the caloric of combustion of biomass to be 5.2 Kcal/g

(1.531. 512Kcal m-2 year-1 ) x 0.107

4) = 31514 g m-2 year-1
5.2 Kcal g-1
31.514 g m-2 year-1 x10.000 m2 =315 ton/Ha/year
((315 x1000000)/360)/10.000= 87.5 g/m2/day
5) 87.5 g/m2/day (Maximum theoretical yield per square meter per day)
 110
100 Reflection/scattering:
Incident
sunlight
about 10 % of PAR is
90 energy
Incident reflected/scattered by
(100%)
Energy (% of initial) 80
light
at the
cultures.
earth
surface
70 (90%)

60
50
40 Available
as PAR
(41%)
30 Available
for charge
separation
20 (32.8 %) Biom ass
9.4% Biomass
10 (5%)

0
1 2 3 4 5 6
Process
 110
Radiation outside PAR Photons above
100 700 nm and below 400 nm are not used
Incident
sunlight by microalgae and thus about 55% of
90 energy
(100%)
Incident incident solar light is unavailable to
light
drive photosynthesis.
Energy (% of initial)
80 at the
earth
surface
70 (90%)

60
50
40 Available
as PAR
(41%)
30 Available
for charge
separation
20 (32.8 %) Biom ass
9.4% Biomass
10 (5%)

0
1 2 3 4 5 6
Process
 110
Loss of useful PAR energy at
100
Incident 680 nm (PSII) and 700 nm (PSI)
due to non-photochemical
sunlight
90 energy
Incident
(100%)
light
processes= 20.4% .
Energy (% of initial)
80 at the
earth
surface
70 (90%)

60
50
40 Available
as PAR
(41%)
30 Available
for charge
separation
20 (32.8 %) Biom ass
9.4% Biomass
10 (5%)

0
1 2 3 4 5 6
Process
 110
Conversion of energy to biomass: Assuming
100 that for light conversion to biomass (i.e., 1
Incide nt
sunlight
mol of hexose yields 2808 KJ mol -1, and that
90 e ne rgy
Incide nt
a minimum of 9.4 mol of quanta are
(100%)
light required to release 1 mole of O2, (2808
Energy (% of initial)

80 at the KJmol-1/(173.5 KJ mol-1 x 6 (9.4 mol quanta)

e arth
surface x 100 = 28.7 % (i.e. an energy loss of 71.3%)
70 (90%)

60
50
40 Available
as PAR
(41%)
30 Available
for charge
se paration
20 (32.8 %) Biom ass
9.4% Biomass
10 (5%)

0
1 2 3 4 5 6
Process
 110
Photoinhibition and Photosaturation:
100 Assuming that 45% of the remaing energy
Incide nt

90 sunlight is lost because of photosynthesis

e ne rgy
(100%)
Incide nt saturation.
light
Energy (% of initial)
80 at the
e arth
surface
70 (90%)

60
50
40 Available
as PAR
(41%)
30 Available
for charge
se paration
20 (32.8 %) Biom ass
9.4% Biomass
10 (5%)

0
1 2 3 4 5 6
Process
After Tredici, 2010
PHOTOSYNTHESIS (ETR) VS LIGHT IRRADIANCE CURVE

ETR = ∆F/F′m x PFD x 0.5 x a*

Chlorella sorokiniana Acclimated to high

light

Acclimated to low
light
But it depends on the
species !

Es.= 900 µmol m-2 s-1L/D (50%-

50%) = 450 µmol m-2 s-1

450/3 = 150 µmol m-2 s-1

 LIGHT
LIGHT

Absorption

D
F
Heat

P=  x d
P= yield (g/m2/day)
PSII CORE
 = growth rate (day-1)
d = culture depth (m)

Acclimation to high light P

Electron Transport
Electron Transport
 LIGHT
LIGHT

Absorption

D
F
Heat

P=  x d
P= yield (g/m2/day)
PSII CORE
 = growth rate (day-1)
d = culture depth (m)

P
Acclimation to low light
Electron Transport
Electron Transport
S0= ground singlet state; S1, S2 = excited singlet states; V”= vibronic states;  = life- time
of electrons in the individual quantum states. t= time required for the transition of the electron from
ground state to individual excited states.
Florescence
 Relationship between excited states, absorption spectra
and fluorescence emission in photosynthetic pigments.

S2

S1

S0
The emission of the photon from the decay of the lowest singlet excited state is
fluorescence. The peak of fluorescence emission occurs at a slightly longer
wavelength (e.g. Lower energy level) that the absorption maxima. In vivo
fluorescence emission is centered around 685 nm in algae
 LIGHT

Photosystem II

Photochemistry

P680 e- QA

Chlorophyll
fluorescence
Heat
Photochemical Non-photochemical
quenching (qP) quenching (NPQ)
CHL FLUORESCENCE ANALYSIS:

1) The saturation pulse method

(Quenching analysis), Slow
fluorescence, sec.

2) Chlorophyll fluorescence transient

(OJIP test) (Fast fluorescence, s)
CHL FLUORESCENCE ANALYSIS:
THE SATURATION PULSE METHOD
(Quenching analysis)
 The basics of the chlorophyll fluorescence technique
DCMU
Flash

Photochemistry (photochemical quenching)

1Chla* Heat (non-photochemical quenching)

Fluorescence
Midpoint
potential
Em7 (Volt)

P700* 3 ps
- 0.9
A0 40-200 ps

A1
15-200 ns
Fx

- 0.6
FA/ FB
~ 2µs
P680* 3 ps
Fd
Pheo
200 ps
- 0.3 QA 150-600 s

QB 1 ms
1 ps
0.0
PQ h

f
Cytochrome b6/
Com plex
1-15 ms 1 ms
200 µs
PC
+ 0.3 P700
˜ 1 ms
PSI
H2 0 100-800 s h
+ 0.6 4Mn
200 ns
Yz
O2 P680
+0.9
PSII

Donor side of PSII Acc eptor side of PSII (donor side of PSI) Acceptor side of PSI
 Maximum (potential )
Fv/Fm = (Fm-Fo)/Fm Photochemical quantum
yield of PSII. It is a
DARK-ADAPTED
measure of the proportion of
Fm the light absorbed by PSII
LIGHT - ADAPTED that is potentially utilizable in
photochemistry. QA fully oxided

F´m
Fv

F

Fs

Fo
Fo´
0
SP Saturating Pulses Sequence
ML ML AL FR AL OFF
 Fm’ – Fo’ / Fm’ = Fv’/Fm’ Maximum (potential )
Photochemical quantum
yield of PSII in the light.
DARK-ADAPTED
It is a measure of the
Fm proportion of the light
LIGHT - ADAPTED absorbed by PSII that is
potentially utilizable in
photochemistry in the light.

F´m
Fv

F

Fs

Fo
Fo´
0
SP Saturating Pulses Sequence
ML ML AL FR AL OFF
 F/ F′m = (F′m- Fs )/F′m Effective Quantum yield of
PSII: It is a measure of the proportion
DARK-ADAPTED of the light absorbed by PSII that is
effectively utilized in photochemistry.
Fm

LIGHT - ADAPTED

F´m
Fv

F

Fs

Fo
Fo´
0
SP Saturating Pulses sequence
ML ML AL FR AL OFF
 The Effective quantum yield (or PSII operating
efficiency) is the product of Qp and Fv/Fm’

∆ F Fq’
Fv’ Fm’ – Fs =
X
=
Fm’– F0’ Fm’ Fm’
Fm’

qP
qP : is related to the fraction of PSII reaction centres with Q
A oxidised
i.e. indicates (expresses) the ability of the photosynthetic apparatus to
maintain QA in the oxidised state.

FV/Fm x qP (but in the dark qP =1) = Fv/Fm

= F

= Fs
 The relation between quantum yield of PSII (F/F´m) and quantum yield of CO2
fixation (CO2) in leaves (Genty et al. 1989, BBA).

10 photons/CO2
0.7
12 photons /CO2

F/F´m

8 photons/CO2

0.35

0.043

Baker 2008

CO2 ( mol CO2/mol photons)

K = 4 e-
CO2 = (F/Fm’ x fraction PSII x (1/k)
Fraction PSII = 0.5
The relation between Oxygen evolution and Electron transfer rate
in Chlorella sorokiniana (Torzillo et al. unpublished)

400

(moles O2/mg Chl-a/h)

Evoluzione di ossigeno
300

200

100
Y = 173.3 X r 2 = 0.997

0
0.0 0.5 1.0 1.5 2.0
ETR ( PFD x F/F'm x a* x 0.5)
(moles electrons . s-1. mg-1 Chl-a)
 Comparison of fluorescence parameters according to nomenclature reported by Snel (Photosynthesis Res.) and Baker (2008). Annu.,
Rev., Plant Biol. 59: 89-113.

Parameter Parameter Definition Physiological relevance

(Snel) (Baker)

Fo, Fo’ Fo, Fo’ Minimal fluorescence
From dark (Fo) and light
adapted algae (leaf)
Fs F’ Fluorescence from light adapted
algae at the steady-state
Level of fluorescence when QA is maximally
oxidized (PSII centres open)
Fs at the steady state gives an indication of the
degree of reduction of the PQ.

Fm, Fm’ Fm, Fm’ Maximal fluorescence from Level of fluorescence when QA is maximally
dark- and light adapted algae, reduced (PSII centres closed)
respectively
Fv, Fv’ Fv, Fv’ Variable fluorescence from Demonstrate the ability of PSII to perform
dark- and light adapted algae, photochemistry (QA reduction)
respectively
F= Fm’-Fs Fq’ = Fm’- Difference in fluorescence Photochemical quenching of fluorescence by
F’ between Fm’ and Fs or Fm’ and open PSII centres.
F’
Fv/Fm Fv/Fm Maximum quantum efficiency Maximum efficiency at which light absorbed
of PSII photochemistry (in the by PSII is used for reduction of QA.
dark)
F/Fm’= Fq’/ Fm’ Effective (actual, apparent) Estimates the efficiency at which light
= (F‘v/F‘m) x F‘m-Fs)/F‘m- F0) quantum yield of PSII: Also absorbed by PSII is used for QA reduction. At
PSII operating efficiency a given PFD this parameter provides an
qP
estimate quantum yield of linear electron flux
If qP =1 , then ΔF/F'm = F'v/F'm through PSII.
Continued
Fv’/Fm’ Maximum PSII Provides an estimate of the maximum efficiency of PSII
Fv’/Fm’ efficiency (in photochemistry at a given PFD, which is the PSII
(Fv’ = Fm’-Fo’) the light) operating efficiency if all the PSII centres were “open”
(QA oxidized), that is, qP=1 (see previous pag. ΔF/F‘m).
Fv’/Fm’ estimates the maximum quantum yield of PSII
photochemistry that can be achieved in the light-adapted
state when QA is maximally oxidated.

qP = Fq’/Fv’ PSII efficiency Relates the PSII maximum efficiency to the PSII operating
(Fm’-Fs) /(Fm’ –Fo’) factor. efficiency Nonlinearity related to the proportion of PSII centres that
are “open” QA oxidized. Mathematically identical to the coefficient
Photochemical
of photochemical quenching qP. Redox state of QA, i.e. the fraction
quenching. of PSII reaction centers that are open and capable of
photochemistry. qP is used to estimate the redox state of the QA,
i.e. the fraction of of the PSII reaction centers that are open and
capable of photochemistry.
NPQ = (Fm/Fm’) - 1 NPQ = Non-photoche Estimates the non-photochemical quenching from Fm to
(Fm/Fm’)-1 mical Fm’. Monitors the apparent rate constant for heat loss from
quenching PSII.
qE qE Energy- Associated with light-induced proton transport into the
dependent thylakoids lumen. Regulates the rate of excitation of PSII
quenching reaction centres.
qI qI Photoinhibitory Results from photoinhibition of PSII photochemistry
quenching
qT qT State transition Results from phosphorylation of light-harvesting
quenching complexes associated with PSII.

qL = (F'm-Fs)/F'm-F'o) qL= (Fq’/Fv’)x(Fo’/F’) Fraction of Estimates the fraction of “open” PSII centres (with QA oxidized) on
x (F'o/Fs) qP PSII centres the basis of the lake model for the PSII photochemistry apparatus. It
allows a more accurate estimates of the redox state of the QA
that are “open”.
compared to the use qP.
ΔF/Fm’ = (F'v/F'm) x (F'm-Fs/F'm-F'0) ( Effective photochemical efficiency of PSII).
qP

PSII efficiency factor

qP

F/F’m

qL = (Fm’-Fs)/(F’m – F’0) x F’0/Fs This parameter is linearly

related to the redox state of QA
qP

qL= The fraction of PSII reaction centers that are open

 Fv/Fm ratios in different Class of algae after dark adaptation

CLASS CH ACCESSORY PSII/PSI Fv/Fm

L PIGMENTS ratio

Chlorophyta (e.g. higher a, b Cartenoids, xanthophylls 1.2-1.6 0.83 -0.85

plants, Chlorella)
Rodophyta (red algae) a, d Phycobilins, carotenoids 0.6-0.7

Pheophyceae (brown a, c Fucoxanthin 0.7- 0.8

algae)
Bacillariophyceae a, c Fucoxanthin 0.6-0.7
(Diatoms)
Xanthophyceae (Yellow a, c Carotenes, xanthophylls 0.67
green algae )
Prasinophyceae (e.g Carotenes, xanthophylls 0.37
Tetraselmis) a, b

Cyanobacteria Phycobilins, β-carotene 0.60 - 0.65

- Arthrospira a and xanthophylls 0.5-0.9
Synechocystis 6803 0.40 – 0.45
 ELECTRON TRANSPORT RATE

ETR = F/F′m x PFD x 0.5 x IA

0.5 = Transport of one electron requires of two For
quanta, as two photosystems are involved leaves

IA = Absorbed irradiance (e.g. 0.85 for leaves)

ETR = ∆F/F′m x PFD x 0.5 x a* For

microalgal
suspensions

a* = Chlorophyll optical cross-section (m2/mg Chl)

 Measurement of the Chlorophyll absorption cross-section (or
optical absorption cross section, extinction coefficient ). This
parameter describes the ability of a molecule to absorb light
at a given wavelength.

a* = (m2 (mg chl)-1

a* = [A x 1/0.01 m x ln (10) ]/ [Chl]
Where:

- A = ABS (In-vivo absorption of the sample) (dimensionless)

- [Chla]= mg m-3 A = log (Io/I)

- 100 To transform cm in m

- Ln (10) to tranform log in ln lnA = 2.3 log A

Chlorella sorokiniana grown under low
(20 µmol photons m-2 s-1) and high
PFD (200 µmol photons m-2 s-1)
 ELECTRON TRANSFER RATE IN MICOALGAL
SUSPENSIONS

ETR = (F′m- Fs)F´m x PFD x 0.5 x a*

a* (m2 (mg chl)-1 = Chlorophyll specific absorption cross-section

(or optical absorption cross section)

P (photosynthesis) =  x PFD x a*

1 mole O2 = 4 mol electrons

NON PHOTOCHEMICAL QUENCHING

NPQ = (Fm-F′m) / F′m

 NPQ = Fm -F´m/ F´m
DARK-ADAPTED
Fm

LIGHT - ADAPTED

F´m
Fv

F

Fs

Fo
Fo´
0
SP Saturating Pulses sequence
ML ML AL FR AL OFF
 NONPHOTOCHEMICAL QUENCHING OF CHL
FLUORESCENCE (NPQ)

qE qT qI

CAUSE  pH LHCII 1)Numerours

detachment Zeaxanthin
transfer to PSI 2)Dark stable  pH
3) PSII inactivation
RELAXATION 5-10 s 5-10 min 30 min-hours
(usually) (days)
INHIBITORS antimicin A none (DTT)
(DTT)
STIMULATORS zeaxanthin (Fluoride) 1)Photosynthesis
(ascorbate) anaerobiosis inhibitors
2) Stress conditions
 The Zeaxanthin Cycle

Epoxidation
De-epoxidation under
under excess of limiting
light. light or
dark
conditions

HO

1Chl* H+
+ Zea or Anth Chl + 1Zea* or 1Anth*
Singlet energy
transfer
 AquaFluo, mai 2007

The current ‘green’ NPQ model

NPQ is characterised by a change in energy

transmission capacity of the LHC to the reaction center
light
H+
* Absorption Stroma
mode Vx
Thylakoid
membrane
PS II Photochemistry
PsbS
Lumen
VDE
H+
* Absorption
mode 3.
light heat (NPQ)
+ H+
Dissipation
mode Vx
2. VDE Ax PS II Photochemistry
Zx PsbS
H+
1. protonation pH
Lavaud J.
 Time course of the Photon Flux Density (PFD)
during the day in summer.

2000

s -1)
1600
-2
PFD(  mol photons m
1200

800

400

0
5 7 9 11 13 15 17 19 21
Time of day (h ou rs)
(Torzillo et al. J.Appl. Phycol. 2003)
e- e- e- e- e-

e- e-
State transitions monitored with Chlrophyll
fluorescence (Faraloni et al.,2013)

120 0.300
110 0.275

O2 concentration (mol ml-1)

100 0.250

Fluorescence a.u. 90 0.225

80 0.200
70 0.175
60 0.150
50 0.125
40 0.100
30 0.075
20 0.050
10 0.025
0 0.000
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
Time (min)
 ABSORPTION AND CHLOROPHYLL
FLUORESCENCE SPECTRA OF LEAVES
(Redrawn from CRC Critical Review in Analytical Chemistry vol. 19
(1988), Lichtenthaler and Rinderle).

Arbitrary Units

Detection > 710 nm

Wavelength (nm)
PAM101/102/103 WALZ
(Germany)
APPLICATION OF CHLOROPHYLL
FLUORESCENCE
MEASUREMENTS IN
MICROALGAL CULTURES
APPLICATIONS OF CHLOROPHYLL FLUORESCENCE
ANALYSIS IN ALGAL BIOTECHNOLOGY

1) In photosynthesis and photoinhibiotion

studies

3) For rapid screening of mutants

4) To quantify the effect of stress on

photosynthesis

5) To study the relation between chlorophyll

fluorescence and pigment changes (e.g.
xanthophyll cycle pigments)

7) To estimate primary productivity

MAIN ADVANTAGES OF CHL FLUORESCENCE APPLICATION

Non-invasiveness

High Sensitivity (compared to conventional measurements,

e.g. O2 evolution)

Rapidity
 Deactivation scheme of excited Chl a (P680) in PSII

Physiological conditions

Photochemistry (photochemical quenching)

1Chla* Heat (non-photochemical quenching)
Fluorescence

Stress conditions
Photochemistry (photochemical quenching)
1Chla*
Heat (non-photochemical quenching)

Fluorescence
 MODULATED VERSUS NON-MODULATED
FLUOROMETERS

Non-modulated
Modulted (Pulsed)
(continuous) Fluorometer
Fluorometer
 Diurnal changes in the Fv/Fm ratio of Spirulina platensis
cultures grown outdoors in photobioreactors at two temperatures.

0.8 1.5

0.7

PFD (mmol m-2 s-1)

1.0
0.6
Fv /Fm

0.5
0.5
35o C
0.4 25o C
PFD
0.3 0.0
9 10 11 12 13 14 15 16 17
Time of day (h)
Daily time courses of Fv/Fm ratio in Arthrospira platensis (Spirulina) cultures grown
under sunlight in tubular reactors (Czech Rep. Reactor)
0,7

0,6

0,5

0,4
Fv/Fm

0,3
420
730
0,2
1170
2310
0,1

0,0
8 10 12 14 16 18
Daytime
Daily time courses of NPQ in Arthrospira platensis (Spirulina) cultures grown
under sunlight in tubular reactors (Czech Rep. Reactor)

1,0

420
730
1170
0,8 2310

0,6
NPQ

0,4

0,2

0,0
8 10 12 14 16 18
Daytime
 Diurnal changes in the Fv/Fm ratio of Spirulina cultures grown
outdoors in photobioreactors under different stress conditions

0.8 0.8
HLAC 35 °C Low O2 HLAC 25 °C Low O2
0.7 0.7
35 °C High O2 25 °C High O2
0.6 0.6
Fv/Fm

Fv/Fm
0.5 0.5

0.4 0.4

0.3 0.3
0.2 0.2
8 10 12 14 16 18 8 10 12 14 16 18 20
Time of day (h)
Time of day (h)

35 °C low O2 35 °C high O2 25 °C Low O2 25 °C High O2

0.8 2000 0.8 2000
LLAC LLAC
0.7 0.7
1600
1500 0.6
0.6
Fv/Fm

0.5

Fv/Fm
1200
PFD
0.5 1000 0.4
0.3 800
0.4
500 0.2
0.3 400
0.1
0.2 0 0.0 0
7 9 11 13 15 17 19 8 10 12 14 16 18
Time of day ( h) Time of day (h)
 CONCLUDING REMARKS

1) Although chlorophyll fluorescence represents a

powerful technique for the detection of stress in
algae and higher plants, it must be always
accompanied by measurement of other physiological
parameters (e.g. photosynthesis and growth)

2) Indeed, with fluorescence it is easy to generate

data, but it is also easy to generate large amounts of
meaningless data. This is particulartly true when
dealing with non-homogenous algal suspensions)

3) Special attention is required when dealing with

cyanobacteria, which have a very different light
harvesting complex, and a much higher PSI/PSII
ratio.
FINE
LEZIONE 1.