Marine Biology

, Volume 150, Issue 1, pp 17–28 | Cite as

Chlorophyll fluorescence as a proxy for microphytobenthic biomass: alternatives to the current methodology

  • B. JesusEmail author
  • R. G. Perkins
  • C. R. Mendes
  • V. Brotas
  • D. M. Paterson
Research Article


Pulse amplitude modulated (PAM) fluorescence has been used as a proxy of microphytobenthic biomass after a dark adaptation period of 15 min to stabilise the minimum fluorescence yield (F o 15 ). This methodology was investigated for in situ migratory and ex situ engineered non-migratory biofilms, comparing dark adaptation to low (5% ambient) and far-red light treatments over different emersion periods. Far-red and low light reduced potential errors resulting from light history effects, by reversal of non-photochemical quenching after 5 min of treatment, compared to over 10 min required by conventional dark adaptation. An in situ decline of minimum fluorescence yield over 15 min was observed during the dark adaptation for migratory biofilms, but was not observed in the non-migratory biofilms indicating that the major cause of decline was downward vertical migration of cells into the sediment. This pattern occurred in far-red light after 10 min, but not for the low light treatment, indicating that low light maintained the biomass at the surface of the sediment. It is therefore concluded that low light treatment is a better option than conventional dark adaptation for the measurement of minimum fluorescence as a proxy of microphytobenthic biomass.


Dark Adaptation Fluorescence Yield Dark Treatment Pulse Amplitude Modulate Downward Migration 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Fo, Fo5, Fo10, Fo15

Minimum fluorescence yield after 10 s and after 5, 10 and 15 min of treatment, respectively

Fm, Fm5, Fm10, Fm15

Maximum fluorescence yield after a saturating pulse, after 10 s and after 5, 10 and 15 min of treatment, respectively

Fv/Fm, Fv5/Fm5, Fv10/Fm10, Fv15/Fm15

PSII photochemical efficiency after a saturating pulse, after 10 s or 5, 10 and 15 min of treatment, respectively


Non-photochemical fluorescence quenching


Photosynthetic photon flux density


Photosystem II



B. Jesus was funded by a PhD grant from FCT (Praxis XXI BD21634/99) and a Post Doctoral grant (POCI BPD/20993/2004). This work was also funded by the HIMOM project (Contract n° EVK3-2001-00043 I). The authors thank L. Ribeiro for the valuable help with diatom identification.


  1. Barranguet C, Kromkamp J (2000) Estimating primary production rates from photosynthetic electron transport in estuarine microphytobenthos. Mar Ecol Prog Ser 204:39–52CrossRefGoogle Scholar
  2. Blanchard GF, Chrétiennot-Dinet MJ, Dinet A, Robert J-M (1988) Methode simplifiée pour l’extraction du microphytobenthos des sédiments marins par le gel de silice Ludox. Compt Rend Acad Sci Paris 307:569–576Google Scholar
  3. Brotas V, Cabrita T, Portugal A, Serôdio J, Catarino F (1995) Spatio-temporal distribution of the microphytobenthic biomass in intertidal flats of Tagus estuary (Portugal). Hydrobiologia 300/301:93–104CrossRefGoogle Scholar
  4. Caron L, Berkaloff C, Duval JC, Jupin H (1987) Chlorophyll fluorescence transients from the diatom Phaeodactylum tricornutum: relative rates of cyclic phosphorylation and chlororespiration. Photosynth Res 11:131–139CrossRefGoogle Scholar
  5. Casper-Lindley C, Bjorkman O (1998) Fluorescence quenching in four unicellular algae with different light-harvesting and xanthophyll-cycle pigments. Photosynth Res 56:277–289CrossRefGoogle Scholar
  6. Consalvey M, Jesus B, Perkins RG, Brotas V, Underwood GJC, Paterson DM (2004) Monitoring migration and measuring biomass in benthic biofilms: the effects of dark/far-red adaptation and vertical migration on fluorescence measurements. Photosynth Res 81:91–101CrossRefGoogle Scholar
  7. Consalvey M, Perkins RG, Underwood GJC, Paterson DM (2005) PAM Fluorescence: A beginners guide for benthic diatomists. Diatom Res 20:1–22CrossRefGoogle Scholar
  8. Dau H, Hansen UP (1988) The involvement of spillover changes in State 1-State 2 transitions in intact leaves at low light intensities. Biochim BiophysActa 934:156–159CrossRefGoogle Scholar
  9. Defew EC, Perkins RG, Paterson DM (2004) The influence of light and temperature interaction on a natural estuarine microphytobenthic assemblage. Biofilms 1:21–30CrossRefGoogle Scholar
  10. Eaton JW, Moss B (1966) The estimation of numbers and pigment content in epipelic algal populations. Limnol Oceanogr 11:584–595CrossRefGoogle Scholar
  11. Folk RL (1954) The distinction between grain size and mineral composition in sedimentary-rock nomenclature. J Geol 62:344–359CrossRefGoogle Scholar
  12. Ford RB, Honeywill C (2002) Grazing on intertidal microphytobenthos by macrofauna: is pheophorbide a a useful marker. Mar Ecol Prog Ser 229:33–42CrossRefGoogle Scholar
  13. Forster RM, Kromkamp JC (2004) Modelling the effects of chlorophyll fluorescence from subsurface layers on photosynthetic efficiency measurements in microphytobenthic algae. Mar Ecol Prog Ser 284:9–22CrossRefGoogle Scholar
  14. Genty B, Briantais JM, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990:87–92CrossRefGoogle Scholar
  15. Hagerthey SE, Defew EC, Paterson DM (2002) Influence of Corophium volutator and Hydrobia ulvae on intertidal benthic diatom assemblages under different nutrient and temperature regimes. Mar Ecol Prog Ser 245:47–59CrossRefGoogle Scholar
  16. Honeywill C, Paterson DM, Hagerthey SE (2002) Instant determination of microphytobenthic biomass using fluorescence. Eur J Phycol 37:485–492CrossRefGoogle Scholar
  17. Jakob T, Whilhelm C (1999) Activation of diadinoxanthin de-epoxidase due to a chlororespiratory proton gradient in the dark in the diatom Phaeodactylum tricornutum. Plant Biol 1:76–82CrossRefGoogle Scholar
  18. Jakob T, Goss R, Wilhelm C (2001) Unusual pH-dependence of diadinoxanthin de-epoxidase activation causes chlororespiratory induced acumulation of diatoxanthin in the diatom Phaeodactylum tricornutum. J Plant Physiol 158:383–390CrossRefGoogle Scholar
  19. Jesus B, Brotas V, Marani M, Paterson DM (2005) Spatial dynamics of microphytobenthos determined by PAM fluorescence. Estuar Coast Shelf Sci 60:30–42CrossRefGoogle Scholar
  20. Jesus B, Perkins RG, Consalvey M, Brotas V, Paterson DM (2006) How does migrations by benthic microalgae affect fluorescence measurements of photophysiology? Mar Ecol Prog Ser (in press)Google Scholar
  21. Krammer K, Lange-Bertalot H (1986) Bacillariophyceae. 1. Teil: Naviculaceae. Vol. 2/1 of Süsswasser flora von Mitteleuropa. Gustav Fischer Verlag, Stuttgart, New YorkGoogle Scholar
  22. Krammer K, Lange-Bertalot H (1988) Bacillariophyceae. 2. Teil: Bacillariaceae, Epithemiaceae, Surirellaceae. Vol. 2/2 of Süsswasserflora von Mitteleuropa. Gustav Fischer Verlag, Stuttgart, JenaGoogle Scholar
  23. Krammer K, Lange-Bertalot H (1991a) Bacillariophyceae. 3. Teil: Centrales, Fragilariaceae, Eunotiaceae. Vol. 2/3 of Süsswasserflora von Mitteleuropa. Gustav Fischer Verlag, Stuttgart, JenaGoogle Scholar
  24. Krammer K, Lange-Bertalot H (1991b) Bacillariophyceae. 4. Teil: Achnanthaceae, Kritische Erg¨anzungen zu Navicula (Lineolatae) und Gomphonema, Gesamtliteraturverzeichnis. Vol. 2/4 of Süsswasserflora von Mitteleuropa. Gustav Fischer Verlag, Stuttgart, JenaGoogle Scholar
  25. Kromkamp J, Barranguet C, Peene J (1998) Determination of microphytobenthos PSII quantum efficiency and photosynthetic activity by means of variable chlorophyll fluorescence. Mar Ecol Prog Ser 162:45–55CrossRefGoogle Scholar
  26. Lavaud J, Rosseau B, Etienne A (2002) In diatoms, a transthylakoid proton gradient alone is not sufficient to induce a non-photochemical fluorescence quenching. FEBS Lett 523:163–166CrossRefGoogle Scholar
  27. Lavaud J, Rousseau B, Etienne A-L (2004) General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae). J Phycol 40:130–137CrossRefGoogle Scholar
  28. Lorenzen CJ (1967) Determination of chlorophyll and pheopigments: spectrophotometric equations. Limnol Oceanogr 12:343–346CrossRefGoogle Scholar
  29. MacIntyre HL, Geider RJ, Miller DC (1996) Microphytobenthos: the ecological role of the “secret garden” of unvegetated, shallow-water marine habitats. i. distribution, abundance and primary production. Estuaries 19:186–201CrossRefGoogle Scholar
  30. Middelburg JJ, Barranguet C, Boschker HTS, Herman PMJ, Moens T, Heip CHR (2000) The fate of intertidal microphytobenthos carbon: an in situ 13C-labelling study. Limnol Oceanogr 45:1224–1234CrossRefGoogle Scholar
  31. Mouget J-L, Rosa P, Tremblin G (2004) Acclimation of Haslea ostrearia to light of different spectral qualities—confirmation of ‘chromatic adaptation’ in diatoms. J Photochem Photobio B 75:1–11CrossRefGoogle Scholar
  32. Müller P, Li X, Niyogi K (2001) Non-photochemical quenching. A response to excess light energy. Plant Physiol 125:1558–1566CrossRefGoogle Scholar
  33. Olaizola M, Laroche J, Kolber Z, Falkowski PG (1994) Non-photochemical fluorescence quenching and the diadinoxanthin cycle in a marine diatom. Photosynth Res 41:357–370CrossRefGoogle Scholar
  34. Oxborough K, Hanlon ARM, Underwood GJC, Baker NR (2000) In vivo estimation of the photosystem II photochemical efficiency of individual microphytobenthic cells using high-resolution imaging of chlorophyll a fluorescence. Limnol Oceanogr 45:1420–1425CrossRefGoogle Scholar
  35. Paterson DM (1989) Short-term changes in the erodibility of intertidal cohesive sediments related to the migratory behavior of epipelic diatoms. Limnol Oceanogr 34:223–234CrossRefGoogle Scholar
  36. Perkins RG, Underwood GJC, Brotas V, Snow GC, Jesus B, Ribeiro L (2001) Responses of microphytobenthos to light: primary production and carbohydrate allocation over an emersion period. Mar Ecol Prog Ser 223:101–112CrossRefGoogle Scholar
  37. Perkins RG, Oxborough K, Hanlon ARM, Underwood GJC, Baker NR (2002) Can chlorophyll fluorescence be used to estimate the rate of photosynthetic electron transport within microphytobenthic biofilms? Mar Ecol Prog Ser 228:47–56CrossRefGoogle Scholar
  38. Round FE (1981) The ecology of the algae, 1st edn. Cambridge University Press, CambridgeGoogle Scholar
  39. Ruban A, Lavaud J, Rosseau B, Guglielmi G, Etienne A (2004) The super-excess energy dissipation in diatom algae: comparative analysis with higher plants. Photosynth Res 82:65–175CrossRefGoogle Scholar
  40. Serôdio J (2003) A chlorophyll fluorescence index to estimate short-term rates of photosynthesis by intertidal microphytobenthos. J Phycol 39:33–46CrossRefGoogle Scholar
  41. Serôdio J (2004) Analysis of variable chlorophyll fluorescence in microphytobenthos assemblages: implications of the use of depth-integrated measurements. Aquat Microb Ecol 36:137–152CrossRefGoogle Scholar
  42. Serodio J, Catarino F (2000) Modelling the primary productivity of intertidal microphytobenthos: time scales of variability and effects of migratory rhythms. Mar Ecol Prog Ser 192:13–30CrossRefGoogle Scholar
  43. Serôdio J, Silva JM, Catarino F (1997) Non destructive tracing of migratory rhythms of intertidal benthic microalgae using in vivo chlorophyll a fluorescence. J Phycol 33:542–553CrossRefGoogle Scholar
  44. Serôdio J, Silva JM, Catarino F (2001) Use of in vivo chlorophyll a fluorescence to quantify short-term variations in the productive biomass of intertidal microphytobenthos. Mar Ecol Prog Ser 218:45–61CrossRefGoogle Scholar
  45. Snoeijs P (1993) Intercalibration and distribution of diatom species in the Baltic Sea, vol 1. Opulus Press, UppsalaGoogle Scholar
  46. Snoeijs P, Balashova J (1998) Intercalibration and distribution of diatom species in the Baltic Sea. Opulus Press, UppsalaGoogle Scholar
  47. Snoeijs P, Kasperoviciene J (1996) Intercalibration and distribution of diatom species in the Baltic Sea, vol 4. Opulus Press, UppsalaGoogle Scholar
  48. Snoeijs P, Potapova M (1995) Intercalibration and distribution of diatom species in the Baltic Sea, vol 3. Opulus Press, UppsalaGoogle Scholar
  49. Snoeijs P, Vilbaste S (1994) Intercalibration and distribution of diatom species in the Baltic Sea, vol 2. Opulus Press, UppsalaGoogle Scholar
  50. Ting CS, Owens TG (1993) Photochemical and nonphotochemical fluorescence quenching processes in the diatom Pheodactylum tricornutum. Plant Physiol 101:1323–1330CrossRefGoogle Scholar
  51. Underwood GJC (2002) Adaptations of tropical marine microphytobenthic assemblages along a gradient of light and nutrient availability in Suva Lagoon, Fiji. Eur J Phycol 37:449–462CrossRefGoogle Scholar
  52. Underwood GJC, Kromkamp J (1999) Primary production by phytoplankton and microphytobenthos in estuaries. Adv Ecol Res 29:93–153CrossRefGoogle Scholar
  53. Underwood GJC, Nilsson CN, Sundbäck K, Wlff A (1999) Short-term effects of UVB radiation on chlorophyll fluorescence, biomass, pigments, and carbohydrate fractions in a benthic diatom mat. J Phycol 35:656–666CrossRefGoogle Scholar
  54. Witkowski A, Lange-Bertalot H, Ditmar M (2000) Diatom flora of marine coasts. Iconographia Diatomologica, vol 7. Verlag K.G. RugellGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • B. Jesus
    • 1
    • 2
    Email author
  • R. G. Perkins
    • 3
  • C. R. Mendes
    • 1
  • V. Brotas
    • 1
  • D. M. Paterson
    • 2
  1. 1.Faculdade de Ciências, Instituto de OceanografiaUniversidade de LisboaLisboaPortugal
  2. 2.Sediment Ecology Research Group, Gatty Marine labsUniversity of St. AndrewsScotlandUK
  3. 3.School of Earth, Ocean and Planetary SciencesCardiff UniversityCardiffUK

Personalised recommendations