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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
63
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
83
Stress identification
84
Biological feedback system
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90
Rewitalizacja Rotundy PKO Banku Polskiego
(Pracownia architektoniczna Gowin & Siuta, kraków)
