10th international swimming pond conference 9th … 2019...prof. hazem m. kalaji warsaw university...

Fluorescence of chlorophyll as a tool to

assess the degree of eutrophication of

aquatic systems

Prof. Hazem M. KalajiWarsaw University of Life Sciences -

SGGW1

10th International Swimming Pond Conference9th and 10th September 2019 in Warsaw, Poland

Amazement and Surprise at Swimming Pool and Natural Pool

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Ecosystem components and interactionsAnisWebster- https://slideplayer.com/slide/13585533/

3Eutrophication process representation (Feem re-elaboration from Arpa Umbria, 2009)

Eutrophication, or hypertrophication, is when a body of water becomes overlyenriched with minerals and nutrients which induce excessive growth of algae. This process may result in oxygen depletion of the water body.

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Types of Eutrophication

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Algal bloom in 2010 along the coast of Qingdao, eastern China (nationalgeographic.it/)

Need for non-invasive and fast methodsNOT ONLY to monitor water ecosystemsbut…..

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http://bioenergy.asu.edu/photosyn/education/experiments/protein_exp/rcreq4.htm

Photosyntheticmachinery

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Photosynthesis

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Kautsky Effect

Czas w min

Jedn

ostk

i wzg

lędn

e

0-1 0 1 2 3

Photosynthesis

Chlorophyll fluorescence

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IR

ChlorophyllFluorescence FR

Photochemical reactions

E = Dissipation + Chlorophyll Fl + Photosyntheis = 1

Photosynthesis

Fluorescence +

Heat

100%

Fluorimeters (stress meters)

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After dark adaptation

Fo - fluorescence level when plastoquinone electron acceptor pool (Qa) is fully oxidised.

Fm - fluorescence level when Qa is transiently fully reduced.

Fv - variable fluorescence (Fm-Fo).

Fv/Fm - maximum quantum efficiency of photosystem II.

Tfm - time at which Fm occurs.

Area - area over the curve between Fo and Fm, relates to the pool size of

PSII electron transport acceptors.

OJIP analysis (Strasser R.J., Srivasatava A. and Govindjee,1995 Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria, Photochemistry and Photobiology, 61, 32-34.).

After light adaptation

20Kalaji et al. 2014

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Electrocardiograph

Stethoscope

Pulse = 60-80/minute

Fluorimeter

Maximal quantum yield Fv/Fm = 0.83-0.85 r.u.

Chlorophyll fluorescence induction curve

Heart PSII

Chl

Flu

ores

cenc

e

0 2 4 6 8 10 12 14 16 18 20 30 100 min

F0

measuring light(weak red light pulses)

Fm

saturating pulse(1 s, 3000 μmol m-2 s-1)

F0‘

Fs

Fp

actinic light(continuous, non-saturating)

Fm‘Fm‘1 Fm‘5 Fm‘20 Fm‘90

Chlorophyll Fluorescence• Bioindicator, Biomarker, Biosensor, Physiological

fingerprinting

• WHY ?

• Sensitive• Reliable• Non invasive• Fast with reasonable cost• Applicable in all living organisms with chlorophyll

(plants, algae, mosses, lichens, animals etc.)

fs

FromPhotons

to aPlant

ps ns µµµµµs ms s hr wk yr

EXCITED STATE

CHROMOPHORE

REACTION CENTER

MEMBRANE

CHLOROPLAST

LEAF

PLANT

GOV 92

4e

C2H5

VH

C

H

H

CH3

CH3

NNMg

N N

HC

H3CH

Light absorption

Primary photochemistry

Electron transport

O2 evolution

CO2 fixation

Metabolism

Productivity

?

?

Time-scales : understanding plant response to stress

Chlorophyll Fluores

cence

Modified after Osmond and Chow, Aust J Plant Physiol 1988

Prof. Reto Strasser-

Geneva University, Bioenergetics Lab.

09/09/2019 FCha 29

(O-J) phase corresponds to a complete reduction of the primary electron acceptor QA of PSII,

the release of fluorescence quenching during the (J-I) phase is controlled by the PSII donor side (water splitting activity).

(I-P) corresponds to the release of fluorescence quenching by the oxidised plastoquinone pool

Fluorescence Rise

Srivastava A, Strasser RJ (1999) in: Crop Improvement for Food Security(Behl RK et al. eds.) SSARM, HISAR, pp 60-71

(1)Haldimann P, Strasser RJ (1999) Photosynthesis Research 62: 67-83

0,1 1 10 100 1000 ms light

O

J

I

P

Chl

Flu

ores

cenc

e in

rel.

u.

200 µM DCMU(1)

30 min 750 Wm-2

30 min 42°CK

maximum quantumconversion: Fv / Fm or Fv / F0

Fv

F0

Fm

0.05 0.2 0.5 1.0 2 5 10 20 50 100 200 500 1000 2000 5000 1.0E+4 2.0E+4 5.0E+4 1.0E+5Time [ms]

0

500

1000

1500

2000

2500

3000Fl

uore

scen

ce [m

V]Fluorescence Raw Curv es

0.05 0.1 0.3 2 30

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Control

Mg deficient

Necrotic

Technical parameters

Slope at the origin of the fluorescence rise MO = (F300µs-FO) / (FM-FO)Relative variable fluorescence at 2 ms VJ = (F2ms/FO) / (FM-FO)

The specific fluxes (expressed per RC - reaction center)

Absorption, per RC ABS/RC = (MO/VJ) / ((1-FO/FM))Trapping at time zero, per RC TRo/RC = MO/VJDissipation at time zero, per RC DIo/RC = (ABS/RC) - (TRo/ABS)Electron transport at time zero, per RC ETo/RC = (MO/VJ) (1-VJ)

The phenomenological fluxes (expressed per CS – cross section of the leaf tissue)

Absorption, per CS ABS/CS = (TRO/ABS) / (ABS/CS)Trapping at time zero, per CS TRo/CS = (TRo/ABS) (ABS/CS)Dissipation at time zero, per CS DIo/CS = (ABS/CS) - (TRo/CS)Electron transport at time zero, per CS ETo/CS = (MO/VJ) (1-VJ)

The yields (or fluxes ratios)

Maximum quantum yield of primary photochemistry

Probability that a traped exciton moves an electron further thanQA-

Probability that an absorbed photon moves an electron furtherthan QA-

Vitality Indexes

Density RCs per chlorophyll

Conformation term for primary photochemistry

Conformation term for the thermal reactions (non lightdepending reactions)

Performance Index

Driving force on a chlorophyll basis

jPo = TRo/ABS = (FM-FO) / FM

Yo = ETo/TRo = 1 - VJ

jEo= jPo Yo = (TRo/ABS) (ETo/TRo)= ETo/ABS = (1-FO/FM) (1-VJ)

RC/ABS

(jPo/(1 - jPo)) = TRo/DIo = FO/FM

(Yo /(1 - Yo)) = ETo/(dQA-/dt0)

PIABS = [RC/ABS] [jPo /(1 - jPo] [Yo /(1 - Yo]

DFABS = log [PIABS]

Derived JIP-test parameters table

Siper-plot representation. Variations of the normalised JIP-test parameters by the respective control. More precisely, the nutritional stress linked to a lack of B and Mg is regarded as a deviation of the reference state and considered as non stress (for which the control values turn on a circle with a radius of 100%).Boron deficiency first appears on the youngest leaves whereas magnesium deficiency can be detected on the oldest leaves.

These graphics present the constellation of selected JIP-test

parameters which quantify the behaviour of plants exposed to

different stress treatment.

-1,50

-1,00

-0,50

0,00

0,50PICSM

RC/ABS

jPo/(1-jPo)

Yo/(1-Yo)

PIABS

PICSo

DIo/CSM

ETo/CSM

TRo/CSM

ABS/CSM

RC/CSM

DIo/CSo ETo/CSo TRo/CSoABS/CSo

RC/CSo

DIo/RC

ETo/RC

TRo/RC

ABS/RC

jDo

jEo

Yo

jPo

VIVJ

Respective controlB deficiency (youngest leaves)Mg deficiency (mature leaves)

ETo/RC

ABS/RC

DIo/RC

TRo/RC

per reaction centre

ABS/RC

TRo/RC

DIo/RC

ETo/RC

ABS/CS

TRo/CS

ETo/CS

DIo/CS

per cross section

per reaction centre

ABS/CS

ETo/CS

DIo/CS

TRo/CS

per cross section

control plants

Mg deficient plants

ETo/RC

ABS/RC

DIo/RC

TRo/RC

per reaction centre

ABS/RC

TRo/RC

DIo/RC

ETo/RC

ABS/CS

TRo/CS

ETo/CS

DIo/CS

per cross section

per reaction centre

ABS/CS

ETo/CS

DIo/CS

TRo/CS

per cross section

control plants

Mg deficient plants

Some examples of recent applications

Plant stress prediction - University of Geneva, Switzerland

chlorophyll fluorescence in terrestrialvegetation- trees physiological state

European Space Agency (http://www.esa.int/esaLP/SEM2VSBE8YE_index_0.html)

Remote Sensing of Vegetation Fluorescence

Fruit quality estimation

Fruit quality estimation

Fruit quality estimation

Water Quality_Oak

Seed quality

SORTING

Wageningen University and Research Centre

Jalink, Hendrik (NL); Schoor, Rob van der (NL); Bino, Raoul John (NL).

SPORTS FACILITIES (Euro 2012, Poland / Ukraine)

India- Himalaya

Chlorophyll Fluorescence

Airborne Remote Sensing

Quality assessment of urban trees:A comparative study of physiologicalcharacterisation, airborne imaging andon site fluorescence monitoring bythe OJIP-test

Hermans C et al. Journal of Plant PhysiologyVolume 160, Issue 1, 2003, Pages 81-90

Leopold Park, Brussels - Belgium

Precision farming and agriculture

First-of-its-kind fluorescence map offers a newview of the world's land plants

Scientists from NASA's Goddard Space Flight Center in Greenbelt, Md. (physorg.com)

SPACE STATIONS

Discovering life on Mars

Water Ecosystems Applications

Water Quality: lake, rivers and oceans

Biofilm on boats and ships

Jebel Ali Power and Desalination Station

Algae

http://daac.gsfc.nasa.gov/oceancolor/scifocus/oceanColor/warming.shtmlhttp://oceancolor.gsfc.nasa.gov/

National Alarm System (Modis – NASA)

Dr. Sonia Cruz

Sea slug vitality assessmentDe Aveiro University, Portugal

ELYSIA VIRIDIS

PLACIDA DENDRITICA

High and low light, with and without Codium

Kalaji H.M., Sytar O., Brestic M., Samborska I.A., Cetner M.D., Carpentier C. (2016) Risk Assessment of Urban Lake Water Quality Based on in-situ Cyanobacterial and Total Chlorophyll-a Monitoring. Pol. J. Environ. Stud. Vol. 25, No. 2 (2016), 1-7. DOI: 10.15244/pages/60895

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SPIRODELA OLIGORRHIZA

Spirodela oligorrhiza without (0) and with toxin MC-LR (T)

Spirodela oligorrhiza with toxins: MC-RR and MC-LR

Toxins

0,640,660,680,7

0,720,740,760,780,8

0,820,84

0 1 5 10 25 50 100 1 5 10 25 50 100

Control RR=LR RR<LRConcentration of toxins (ug/l)

Fv/F

m

Control 5µg L-1 25 µg L-1 100 µg L-1

• JIP-Test technique as biosensor for early detection of heavy metals effects on water plants (Spirodela oligorrhiza)

Hazem M. Kalaji, Z. Romanowska-Duda, Reto J. Strasser

BIOLOGICAL LETT. 2005, 42(2): 191

Spirodela oligorrhiza plants were grown under optimal conditions on a growth medium with or without the addition of heavy metals (Cu,Pb)ions during 24 hours to growth medium in the range from 0 to 10 ppm

Heavy metals

Spirodela oligorrhiza with different concentrations of Cd

Spirodela oligorrhiza with different concentrations of Cu

Minimalna Fluorescencjia Chlorofilu Fo

0100200300400500600

0,5 1 2 5 10

Stężenie Metali ppm

F 0 kontrola

CuPb

Czas uzyskania maksymalnej Fluorescencji Chlorofilu Fm

0

100

200

300

400

500

0,5 1 2 5 10

Stężenie Metali ppm

Tfm (m

s) kontrolaCuPb

Maksymalna wydajność kwantowa fotoukładu II (PS II)

0

0,2

0,4

0,6

0,8

1

0,5 1 2 5 10

Stężenie Metali ppm

Fv/Fm

kontrola

Cu

Pb

Minimal Fluorescence (Fo)

Heavy metals concentration (ppm)

Heavy metals concentration (ppm)

Heavy metals concentration (ppm)

Time to reach maximal fluorescence (Tfm)

Maximal quantum efficiency of photosystem II (Fv/Fm)

Nikodem Szymanski, Irena Burzyńska, Hazem Mohamed Kalaji, Grażyna Mastalerczuk. (2018) Fluorescencja chlorofilu jako narzędzie do oceny stopnia eutrofizacji ekosystemów wodnych na przykładzie stawów na obszarze gminy Raszyn. INŻYNIERIA EKOLOGICZNA 19, 2, 73-80.

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Handy PEA fluorimeter (Handy Plant Efficiency Analyzer) Hansatech Instruments Ltd.

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Kalaji HM, Sytar O, Brestic M, Samborska IA, Cetner MD, Carpentier C.

Risk Assessment of Urban Lake Water Quality Based on in-

situ Cyanobacterial and Total Chlorophyll-a Monitoring. Polish Journal of

Environmental Studies. 2016;25(2):655-661. doi:10.15244/pjoes/60895.

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AlgaTorch bbe moldaenke - Germany

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The Dream

Stress Identifying

IF:

Tf(max) 280 - 294Area > 37968.00PHI(Po) 0.79 - 0.77Kn < 0.58Kp < 2.13ABS/RC > 2.48TRo/RC 1.81 - 1.95ETo/RC > 1.13DIo/RC < 0.53PI >20.47

Water Stress

IF:

Tf(max) 290 - 299Area 37968.00PHI(Po) 0.79 - 0.77Kn 0.58 – 0.66

Kp < 2.13ABS/RC < 2.48TRo/RC 1.81 - 1.95ETo/RC 1.13- 1.25DIo/RC < 0.53PI 20 - 30

Nitrogen deficiency

Instrument Development

I am Thirsty.

Please give me water

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Stress identification

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Biological feedback system

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Continuous measurementshttp://wykres-alt.esence-soft.pl/

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Rewitalizacja Rotundy PKO Banku Polskiego

(Pracownia architektoniczna Gowin & Siuta, kraków)