Physiology and Cultivation of Algae and Cyanobacteria 3. Photosynthesis • light • structure & function – membrane structure – pigments – photosystems • photosynthesis – reactions – light dependent – light independent • energy transfers Light • sunlight vs. PAR • units; W m-2 s-1; mmol m-2 s-1 • spectrum • absorption, transmissivity, reflection, scattering, interference • environmental accessibility (spectrum, int..) Beer’s law Light attenuation with depth (z) REFRACTION: SNELL’S LAW Structure & function • thylakoid membrane structure • pigments • photosystems Structure of the main pigments of the thylakoid membrane. • general equation: » nCO2 + nH2O + light (CH2O)n + nO2 • light dependent reactions • light interception, l. energy transfer • excitation, charge separation • ETR » linear » cyclic • O2 evolution • 4NADP+ + 2H2O + 4ADP + 4Pi 4NADPH + 4ATP + O2 • light independent reactions • CBB – Calvin Benson Bassham Cycle • RuBisCO • CA Photosynthetic reactions Chl a 8 photons + 4e- Role of Carbon Concentrating Mechanism • Carbonic Anhydrase • periplasmic space, carboxysomes (b-g algae), pyrenoid (algae) • CA vs. RuBisCO • mechanisms of HCO3 - uptake – active transp. via CMP (enough ATP) – symport Na+ HCO3 - - via icpB complex – Na+/H+ antiport help – difusion • CO2 uptake - difusion Energy transfers & regulations • excitation energy (excess) dissipation pathways – photosynthesis – state stransitions – heat production (Xanthophyll cycle) – fluorescence • photoaclimation – light dependent motions; state transitions; non-photochemical processes • photoinhibition – photomodification – photodamage • photorespiration & chlororespiration • RuBisCo – generation of glycolate • enigma; PQ pool reducion Photosynthesis–Irradianceresponsecurve (Pvs.Ecurve) Measurement of photosynthetic production Methods: – gravimetric / voluntometric – turbidimetric / nephelometric – fluorometric – gasometric – IRGA; O2 measurement – Light response curve – Pn = GP - R Gravimetry / Voluntometry • Dry weight, fresh weight • PCV (packed cell volume) • AFDW (Ash free dry weight) • Content of primary compound – chl a, Total chl, proteins Turbidimetry / Nephelometry • Turbidimetry – amount of absorbed light vs. number & size of particles (cells) – spectrophotometers (750, 735, 680nm,..) • Nephelometry – amount of scattered light (90°, 70°, 37°) vs. number & size of particles (cells) – amount of scattered light is far greater than the transmitted light >> offers higher sensitivity than turbidimetry Turbidimetry & Nephelometry • blank • selection of wavelenght (750, 735, 680nm,..) • standard curves must be plotted – similar cell size • precipitation and settlement may occur – mix the sample well prior to measurement – keep the timing • Kinetic reaction – provides additional information Gasometry • Manometry, Formation of titratable compounds (NO>NO2) • measure CO2, O2 • IRGA cuvette – aerophytic algae, soil crusts – water vapor • CO2 electrode, O2 electrode, optode – Fast & sensitive, dimension vary – Cathode vs. background consumption !! » Membrane type – Clark » Bare type – ZrO2 – Optode – luminescence quenching Carbon dioxide electrode Clark type oxygen electrode Oxygen optode Chlorophyll fluorescence • What is chlorophyll fluorescence? • Basic methods of Chl fluorescence measurements • Signals, kinetics of Chl fuorescence – fast Chl fluorescence kinetics (parameters) – slow Chl fluorescence kinetics (parametrs) – saturation pulse method, quenching analysis • Chl fluorescence imaging Fluorescence emission Lichtenthaler et Miehe 1997 Basic de-excitation mechanisms of Chl a Chl a* Photosynth.process (linear transport e- ) Thermal dissipation Regulation between PSII and PSI Chlorophyll fluorescence 3Chla* Chla* Reversible transport Qa 3(P680+ Pheo-) 1(P680+ Pheo-) 3 P680* 1 P680* 3 B carotene* P 680 Fluorescence O2* 3 B carotene* heat heat Singlet Recombin. Charge separation Triplet Recomb. Antennae q. Chlorophyll fluorescence techniques • Chl fluorescence signal • Saturation pulse method • Chl fluorescence kinetics (transients) • Quenching analysis • Rapid light/response curves • Emission/absorption spectra • Chl fluorescence imaging Temperature response curve Kuropatwa 1994 Fast Chl f. induction curve 0 0.2 0.4 0.6 0.8 Time ( s ) Fv(mV) O I D P 2 s Fv / 2 t (Fv/2) DCMU effect on fast kinetics Effect of temperature on fast kinetics 5 oC 20 oC O I D P Fast Chl transient [R. Strasser] OJIP taken after dark adaptation OJIP taken after % light pretreatment (1-64 %) Parameters derived from fast kinetics • Signals at O,I, D P points, O(K)JIP • Ratios Fo/Fm Fv/Fm • Half-times • Rates of Chl fluorescence increase • Area over rising part of curve • Estimation of Active / inactive RC (Qb-reducing) Parameters are related to structure and function of photosystem II Slow Chl fluorescence kinetics (log) Slow kinetics of Chl fluorescence +SP saturation puls actinic ligh measuring l. Parameters derived from slow kinetics • Fv / Fm (“potential” photochemistry) • Yield PS II (FII) (“actual” photochemistry) • Rdf (“vitality”) • Photochemical, non-photochemical quenching – qP, qN, NPQ, qPrel, qNrel, qFo – Components of qN: qE, qT, qI Parameters are related to whole photochemistry of photosynthesis Rapid light reponse curves • Actinic light (AL) is step-increased after 30 sec. • At each AL level, saturation pulse is applied • For each AL level, Yield of PS II is calculated • Pn = (Yield * PAR * abs * 0.5) / c c = 8-14 • Light reponse curves of Pn Chlorophyll fluorescence imaging • Variable Chl fluorescence – Amount of Chl, mechanical load – Growing/active zones on crusts • Chl fluorescence parameters – Stress (HL, heavy metals…etc) – Water status Schematic diagraph of CCD camera