Photosynthesis Research

, Volume 83, Issue 3, pp 343–361

Estimation of chlorophyll content and daily primary production of the major algal groups by means of multiwavelength-excitation PAM chlorophyll fluorometry: performance and methodological limits

  • Torsten Jakob
  • Ulrich Schreiber
  • Volker Kirchesch
  • Uwe Langner
  • Christian Wilhelm


The performance and methodological limits of the Phyto-PAM chlorophyll fluorometer were investigated with laboratory grown algae cultures and natural phytoplankton from the rivers Saar and Saale. The Phyto-PAM is a 4-wavelength chlorophyll fluorometer with the functional combination of chlorophyll (Chl) estimation and assessment of photosynthetic activity, both differentiated into the main algal groups. The reliability of fluorescence-based Chl estimation strongly depends on the group specific calibration of the instrument and the resulting chlorophyll/fluorescence (Chl/F) ratios in reference algal cultures. A very high reliability of the Chl estimation was obtained in the case of constant Chl/F-ratios. Algae grown at different light intensities showed marked differences in Chl/F-ratios, reflecting differences in pigment composition and Chl a specific absorption (a*). When the Phyto-PAM was calibrated with laboratory grown diatoms, the Chl a in river grown diatoms was underestimated, due a lower content of accessory pigments and stronger pigment packaging. While this aspect presently limits the application of PAM fluorometry in limnology, this limitation may be overcome by future technical progress in the detection of dynamic changes in Chl/F-ratio via fluorescence-based measurements of the functional PS II absorption cross-section. Practically identical Chl/F-ratios were found for the diatom-dominated waters of the rivers␣Saar and Saale, suggesting that the same instrument calibration parameters may be applied for hydrographically similar surface waters. For this particular case, despite of the present methodological limitations, the potential of PAM fluorometry in limnology could be demonstrated. Light response curves were measured to estimate primary production with a spectrally resolved model in daily courses at two sampling sites. Fluorescence based primary production was closely correlated with measured oxygen evolution rates until midday. In the afternoon, at the water surface the fluorescence approach gave higher␣rates than the measured oxygen evolution. Possible explanations for the observed differences are discussed.


chlorophyll estimation chlorophyll fluorescence light adaptation light response curves modelling Phyto-PAM phytoplankton primary production 



chlorophyll aspecific absorption;




relative electron transport rate


fluorescence yield


maximal fluorescence yield of illuminated sample


Photosytem II quantum yield


PAR-value characteristic for light saturation


light-emitting diode


low light


medium light


pulse amplitude modulation


photosynthetically active radiation


maximal rate of photosynthesis


primary production




absorbed pho-tosynthetically active radiation


rapid light curves


functional absorption cross section of Photosystem II


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  1. Aalderink, RH, Jovin, R 1997Estimation of the photosynthesis/irradiance (P/I) curve parameters from light and dark bottles experimentsJ Plankton Res1917131742Google Scholar
  2. Allen, JF 1992Protein phosphorylation in regulation of photosynthesisBiochim Biophys Acta1098275335PubMedGoogle Scholar
  3. Asada, K 1996

    Radical production and scavenging in chloroplasts

    Baker,  NR eds. Photosynthesis and the Environment,Kluwer Academic PublishersDordrecht, The Netherlands123150
    Google Scholar
  4. Asada, K 1999The water-water cycle in chloroplasts: scavenging of active oxygen and dissipation of excess photonsAnn Rev Plant Physiol Plant Mol Biol50601639CrossRefGoogle Scholar
  5. Badger, MR, Price, GD 1992The CO2 concentrating mechanism in cyanobacteria and green algaePhysiol Platarum84606615CrossRefGoogle Scholar
  6. Badger, MR, Schreiber, U 1993Effects of inorganic carbon accumulation on photosynthetic oxygen reduction and cyclic electron flow in the cyanobacterium Synechococcus PCC7942Photosynth Res37177191CrossRefGoogle Scholar
  7. Barone, R, Naselli-Flores, L 1994Phytoplankton dynamics in a shallow, hypertrophic reservoir (lake Arancio, Sicily)Hydrobiologia289199214CrossRefGoogle Scholar
  8. Behrenfeld, MJ, Falkowski, PG 1997A consumer’s guide to phytoplankton primary productivity modelsLimnol Oceanogr4214791491Google Scholar
  9. Beutler, M, Wiltshire, KH, Meyer, B, Moldaenke, C, Lüring, C, Meyerhöfer, M, Hansen, U-P, Dau, H 2002A fluorometric method for the differentiation of algal populations in vivo and in situPhotosynth Res723953CrossRefGoogle Scholar
  10. Beutler, M, Wiltshire, KH, Arp, M, Kruse, J, Reineke, C, Moldaenke, C, Hansen, U-P 2003A reduced model of the fluorescence from the cyanobacterial photosynthetic apparatus designed for the in situ detection of cyanobacteriaBiochim Biophys Acta16043346PubMedGoogle Scholar
  11. Bischoff, HW, Bold, HC 1963Some soil algae from Enchanted Rock and related algal species. Phycological studies IVUniversity Texas Publication64171213Google Scholar
  12. Bricaud, A, Bedhomme, AL, Morel, A 1988Optical properties of diverse phytoplanktonic species: experimental results and theoretical interpretationsJ Plankton Res10851873Google Scholar
  13. Büchel, C, Wilhelm, C, Lenartz-Weiler, I 1988The molecular analysis of the light adaption reactions in the yellow-green alga Pleurochloris meiringensis (Xanthophyceae)Bot Acta4306310Google Scholar
  14. Campbell, D, Hurry, V, Clarke, AK, Gustaffson, P, Öquist, G 1998Chlorophyll fluorescence analysis of cyanobacterial photosynthesis and acclimationMicrobiol Mol Biol R62667683Google Scholar
  15. DeEll , JRToivonen , PMA eds. 2003Practical Applications of Chlorophyll Fluorescence in Plant Biology.Kluwer Academic Publishers,Dordrecht, The NetherlandsGoogle Scholar
  16. Dubinsky, Z, Falkowski, PG, Wyman, K 1986Light harvesting and utilization by phytoplanktonPlant Cell Physiol2713351349Google Scholar
  17. DuRand, MD, Olson, RJ 1998Diel patterns in optical properties of the chlorophyte Nanochloris sp.: relating individual-cell to bulk measurementsLimnol Oceanogr4311071118Google Scholar
  18. Eilers, PHC, Peeters, JCH 1988A model for the relationship between light intensity and the rate of photosynthesis in phytoplanktonEcol Model42199215CrossRefGoogle Scholar
  19. Falkowski, PG, Kolber, Z 1995Variations in chlorophyll fluorescence yields in phytoplankton in the world oceansAust J Plant Physiol22341355Google Scholar
  20. Falkowski, PG, Wyman, K, Ley, AC, Mauzerall, DC 1986Relationship of steady state photosynthesis to fluorescence in eucaryotic algaeBiochim Biophys Acta849183192Google Scholar
  21. Figueroa, FL, Conde-Alvarez, R, Gomez, I 2003Relations between electron transport rates determined by pulse amplitude modulated chlorophyll fluorescence and oxygen evolution in macroalgae under different light conditionsPhotosynth Res75259275CrossRefGoogle Scholar
  22. Friedman, AL, Alberte, RS 1986Biogenesis and light regulation of the major light harvesting chlorophyll-protein of diatomsPlant Physiol804351Google Scholar
  23. Geel, C, Versluis, W, Snel, JFH 1997Estimation of oxygen evolution by marine phytoplankton from measurement of the efficiency of Photosystem II electron flowPhotosynth Res516170CrossRefGoogle Scholar
  24. Geider, RJ 1987Light and temperature dependence of the carbon to chlorophyll a ratio in microalgae and cyanobacteria: implications for physiology and growth of phytoplanktonNew Phytol106134Google Scholar
  25. Geider, RJ, Platt, T, Raven, JA 1986Size dependence of growth and photosynthesis in diatoms: a synthesisMar Ecol Prog Ser3093104Google Scholar
  26. Genty, B, Briantais, JM, Baker, NR 1989The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescenceBiochim Biophys Acta9908792Google Scholar
  27. Gilbert, M, Domin, A, Becker, A, Wilhelm, C 2000Estimation of primary productivity by chlorophyll a in vivo fluorescence in freshwater phytoplanktonPhotosynthetica38111126CrossRefGoogle Scholar
  28. Gilbert, M, Wilhelm, C, Richter, M 2000Bio-optical modelling of oxygen evolution using in vivo fluorescence: comparison of measured and calculated photosynthesis/irradiance (P-I) curves in four representative phytoplankton speciesJ Plant Physiol157307314Google Scholar
  29. Harris, GP 1984Phytoplankton productivity and growth measurements: past, present and futureJ Plankton Res6219235Google Scholar
  30. Jakob, T, Goss, R, Wilhelm, C 1999Activation of diadinoxanthin de-epoxidase due to a chlororespiratory proton gradient in the drak in the diatom Phaeodactylum tricornutumPlant Biol17682Google Scholar
  31. Jeffrey, SW, Humphrey, GF 1975New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplanktonBiochem Physiol Pflanzen167191194Google Scholar
  32. Kashino, Y, Kudoh, S, Hayashi, Y, Suzuki, Y, Odate, T, Hirawake, T, Satoh, K, Fukuchi, M 2002Strategies of phytoplankton to perform effective photosynthesis in the North WaterDeep-Sea Res II4950495061CrossRefGoogle Scholar
  33. Kautsky, H, Hirsch, A 1931Neue Versuche zur KohlensäureassimilationNaturwissenschaften19964CrossRefGoogle Scholar
  34. Kirk, JTO 1994Light and Photosynthesis in Aquatic Ecosystems.Cambridge University PressNew YorkGoogle Scholar
  35. Kolber, Z, Falkowski, PG 1993Use of active fluorescence to estimate phytoplankton photosynthesis in situLimnol Oceanogr3816461665Google Scholar
  36. Kolbowski, J, Schreiber, U 1995

    Computer-controlled phytoplankton analyzer based on a 4-wavelengths PAM chlorophyll fluorometer

    Mathis, P eds. Photosynthesis: from Light to Biosphere (V)Kluwer Academic PublishersDordrecht, The Netherlands825828
    Google Scholar
  37. Krause, G, Weis, E 1991Chlorophyll fluorescence and photosynthesis: The basicsAnnu Rev Plant Phys42313349CrossRefGoogle Scholar
  38. Larkum, AW 2003

    Light-harvesting systems in algae

    Larkum,  AWDouglas, SERaven,  JA eds. Photosynthesis in AlgaeKluwer Academic PublishersDordrecht, The Netherlands277304
    Google Scholar
  39. Lavaud, J, Gorkom, H, Etienne, A 2002Photosystem II electron transfer cycle and chlororespiration in planktonic diatomsPhotosynth Res745159CrossRefGoogle Scholar
  40. Ley, AC, Mauzerall, D 1998Absolute absorption cross sections for PS II and the minimum quantum requirement for photosynthesis in Chlorella vulgarisBiochim Biophys Acta68095106Google Scholar
  41. Lohr, M, Wilhelm, C 1999Algae displaying the diadinoxanthin cycle also possess the violaxanthin cycleProc Natl Acad Sci USA9687848789CrossRefPubMedGoogle Scholar
  42. Lorenzen, CJ 1966A method for the continuous measurement of in vivo chlorophyll concentrationDeep-Sea Res13223227Google Scholar
  43. Mi, H, Endo, T, Schreiber, U, Ogawa, T, Asada, K 1992Electron donation from cyclic and respiratory flows to photosynthetic intersystem chain is mediated by pyridine nucleotide dehydrogenase in the cyanobacterium Synechocystis PCC 6803Plant Cell Physiol3312331237Google Scholar
  44. Mimuro, M, Akimoto, S 2003

    Carotenoids of light harvesting systems: energy transfer processes from fucoxanthin and peridinin to chlorophyll

    Larkum,  AWDouglas, SERaven,  JA eds. Photosynthesis in Algae,Kluwer Academic PublishersDordrecht, The Netherlands335349
    Google Scholar
  45. Ort, DR, Baker, NR 2002A photoprotective role for O2 as an alternative sink in photosynthesisCurr Opin Plant Biol5193198CrossRefPubMedGoogle Scholar
  46. Owens, TG 1986Light-harvesting function in the diatom Phaeodactylum tricornutum. II. Distribution of excitation energy between photosystemsPlant Physiol80739746Google Scholar
  47. Provasoli, L, Mc Laughin, JJA, Droop, MR 1975The development of artificial media for marine algaeArch Mikrobiol25392428CrossRefGoogle Scholar
  48. Reinfelder, JR, Kraepiel, AML, Morel, FMM 2000Unicellular C4 photosynthesis in a marine diatomNature407996999CrossRefPubMedGoogle Scholar
  49. Rotatore, C, Colman, B, Kuzma, M 1995The active uptake of carbon dioxide by the marine diatoms Phaeodactylum tricornutum and Cyclotella spPlant Cell Environ18913918Google Scholar
  50. Schnitzler-Parker, M, Armbrust, EV, Piovia-Scott, J, Keil, RG 2004Induction of photorespiration by light in the centric diatom Thalassiosira weissflogii (Bacillariophyceae): Molecular characterization and physiological consequencesJ Phycol40557567Google Scholar
  51. Schreiber U (1998). Chlorophyll fluorescence: new instruments for new applications. In Garab G (ed) Photosynthesis: Mechanisms and Effects (V), pp 4253–4258. Kluwer Academic Publishers, Dordrecht, The NetherlandsGoogle Scholar
  52. Schreiber U (2004) Pulse-Amplitude-Modulation (PAM) fluorometry and saturation pulse method: an overview. In: Papageorgiou GC and Govindjee (eds) Chlorophyll Fluorescence: a Signature of Photosynthesis, pp 279–319. Kluwer Academic Publishers, Dordrecht, The NetherlandsGoogle Scholar
  53. Schreiber, U, Neubauer, C 1990O2-dependent electron flow, membrane energization and the mechanism of non-photochemical quenching of chlorophyll fluorescencePhotosynth Res25279293CrossRefGoogle Scholar
  54. Schreiber, U, Bilger, W, Schliwa, U 1986Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometerPhotosynth Res105162CrossRefGoogle Scholar
  55. Schreiber U, Bilger W and Neubauer C (1994). Chlorophyll fluorescence as a noninvasive indicator for rapid assessment of in vivo photosynthesis. In Schulze E-D and Caldwell MM (eds) Ecophysiology of Photosynthesis, pp 49–70. Springer, BerlinGoogle Scholar
  56. Schreiber, U, Hormann, H, Neubauer, C, Klughammer, C 1995aAssessment of Photosystem II photochemical quantum yield by chlorophyll fluorescence quenching analysisAust J Plant Physiol22209220Google Scholar
  57. Schreiber, U, Endo, T, Mi, H, Asada, K 1995bQuenching analysis of chlorophyll fluorescence by the saturation pulse method: Particular aspects relating to the study of eucaryotic algae and cyanobacteriaPlant Cell Physiol36873882Google Scholar
  58. Schreiber, U, Gademann, R, Ralph, PJ, Larkum, AWD 1997Assessment of photosynthetic performance of prochloron in Lissoclinum patella in hospite by chlorophyll fluorescence measurementsPlant Cell Physiol38945951Google Scholar
  59. Selig, U, Hübener, T, Michalik, M 2002Dissolved and particulate phosphorus forms in a eutrophic shallow lakeAquat Sci6497105CrossRefGoogle Scholar
  60. Smith, RC, Prézelin, BB, Bidigare, RR, Baker, KS 1989Bio-optical modeling of photosynthetic production in coastal watersLimnol Oceanogr3415241544Google Scholar
  61. Staehr, PA, Henriksen, P, Markager, S 2002Photoacclimation of four marine phytoplankton species to irradiance and nutrient availabilityMar Ecol Prog Ser2384759Google Scholar
  62. Stramski, D, Shalapyonok, A, Reynolds, RA 1995Optical characterization of the oceanic unicellular cyanobacterium Synechococcus grown under a day-light cycle in natural irradianceJ Geophys Res10013295308CrossRefGoogle Scholar
  63. Trissl HW (2003). Modeling the excitation energy capture in thylakoid membranes. In Larkum AW, Douglas SE and Raven JA (eds) Photosynthesis in Algae, pp 245–276. Kluwer Academic Publishers, Dordrecht, The NetherlandsGoogle Scholar
  64. Tuji, A 2000The effect of irradiance on the growth of different forms of freshwater diatoms: implications for succesion in attached diatom communitiesJ Phycol36639661CrossRefGoogle Scholar
  65. White, AJ, Critchley, C 1999Rapid light curves: A new fluorescence method to assess the state of the photosynthetic apparatusPhotosynth Res596372CrossRefGoogle Scholar
  66. Wilhelm, C, Krämer, P, Lenartz-Weiler, I 1989The energy distribution between the photosystems and light-induced changes in the stoichiometry of system I and II reaction centers in the chlorophyll b-containing alga Mantoniella squamata (Prasinophyceae)Photosynth Res20221233Google Scholar
  67. Wilhelm C, Bida J and Domain A (1995a). Is the measure of PSII quantum yield by means of in vivo chl a-fluorescence really a direct measure of phytoplankton primary production? In Mathis P (ed) Photosynthesis: from Light to Biosphere (V), pp 809–812. Kluwer Academic Publishers Dordrecht, The NetherlandsGoogle Scholar
  68. Wilhelm, C, Volkmar, P, Lohmann, C, Becker, A, Meyer, M 1995The HPLC-aided pigment analysis of phytoplankton cells as a powerful tool in water quality controlJ Water Supply Res T44132141Google Scholar
  69. Yentsch, CS, Phinney, DA 1985Spectral fluorescence: a taxonomic tool for studying the structure of phytoplankton populationsJ Plankton Res7617632Google Scholar
  70. Zhang, Y, Prepas, EE 1996Regulation of the dominance of planktonic diatoms and cyanobacteria in four eutrophic hardwater lakes by nutrients, water column stability and temperatureCan J Fish Aqua Sci53621633CrossRefGoogle Scholar
  71. Ziegler, R, Egle, K 1965Zur quantitativen Analyse der Chloroplastenpigmente. I. Kritische Überprüfung der spektralphotometrischen ChlorophyllbestimmungBeitr Biol Pfl411137Google Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Torsten Jakob
    • 1
  • Ulrich Schreiber
    • 2
  • Volker Kirchesch
    • 3
  • Uwe Langner
    • 1
  • Christian Wilhelm
    • 1
  1. 1.Biology I/Plant PhysiologyUniversity of LeipzigLeipzigGermany
  2. 2.Julius-von-Sachs Institute of Biosciences, Botany IUniversity of WuerzburgWürzburgGermany
  3. 3.German Federal Institute of HydrologyKoblenzGermany

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