Measurement of photosynthetic pigments fluorescence
Photoautotrophic organisms (cyanobacteria, algae, green plants) need energy for the production of organic compounds and therefore they employ the energy of light, which they absorb, into process called photosynthesis. However absorbed light is not entirely utilized for photochemical reactions. Part of the energy is dissipated as heat and another one is emitted as fluorescence. Fluorescence is a physical phenomenon in which the absorption of light photons triggers in the molecule of fluorescent compound an emission of photons with longer wavelength (but lower energy). Measurement of pigment fluorescence, especially chlorophyll fluorescence, may be used for the indirect quantification of phytoplankton in samples from an aquatic environment. Moreover, the chlorophyll fluorescence changes in the time in relation to photosynthetic activity and could be used for the characterization of the status of photosynthetic apparatus in assessed algae or cyanobacteria.
The respective part of photosynthetic apparatus of all photoautotrophic organisms, which is responsible of capturing light, is created by pigment-protein complexes containing chlorophyll-a and other accessorial pigments (chlorophyll-b, phycocyanine, phycoerythrine, fucoxantine, peridinine etc.) which help to absorb the energy of light and pass it to chlorophyll-a. Each of these pigments is specific for some group of phytoplankton and they differ in the ability to absorb different wavelengths of visible light. Based on the presence of the accessorial pigments, photoautotrophic organisms may be categorized to following groups: “blue” (cyanobacteria), “green” (green algae), “brown” (bacillariophytes – diatoms, dinophytes), “red” (rhodophytes) and “mixed” (cryptophytes). Due to the energy transfer, excitation by a light of appropriate wavelength triggers a fluorescence emission from chlorophyll-a molecule. Some of other photosynthetic pigments (e.g. phycocyanines) are fluorescent themselves. In case of an appropriate configuration of measuring instruments, pigment fluorescence may serve as an endpoint for the determination of total chlorophyll-a concentration and provides information about the presence of various groups of phytoplankton in measured sample. When the induced chlorophyll fluorescence technique is applied, even some information about physiological status of the phytoplankton, and especially about its photosynthetic activity can be obtained. Main advantage of fluorescence measurement is easiness and non-destructiveness of the method. Measurement of fluorescence can be done repeatedly on the same sample not only in a laboratory conditions, but also in situ.
According to the way of fluorescence measurement, the devices in the lab are distinguished into two groups: spectrofluorometers (see below), recording the fluorescence as a result of excitation by appropriate light wavelength, and fluorometers, which measure changes in induced chlorophyll fluorescence as a response to changes in photosynthetic activity of the organism.
Spectrofluorometers – FluoroProbe, BenthoFluor, BenthoTorch
At our department three kinds of fluorescence probes from bbe Moldaenke GmBH, Kiel, Germany) are used: submersible probe FluoroProbe for measurements in water column and probes BenthoFluor and BenthoTorch for measurements on surfaces. All these “fluorometric” probes use LED diodes of various wavelengths as excitation light sources (in case of FluoroProbe e.g. 370 nm, 470 nm, 525 nm, 570 nm, 590 nm and 610 nm respectively). Chlorophyll fluorescence emission is measured at fixed wavelength of 680 nm. Based on their excitation spectra, following phytoplankton groups can be discriminated in the measured sample: Chlorophyceae (green algae), Cyanophyceae (cyanobacteria), Bacillariophyceae (diatoms) together with Dinophyceae and Cryptophyceae. The amount of phytoplankton biomass is expressed as chlorophyll-a concentration (in µg per ml of sample). Detection limit as stated by manufacturer is 1 µg L-1 and according to our experience, upper limit for the detection, especially when cyanobacteria dominate in the measured sample, is approximately 50 µg L-1 (when higher concentration of chlorophyll-a in the sample is expected, it is necessary to dilute the sample appropriately).
FluoroProbe may be used in the laboratory (with the cuvette adapter) as well as in situ in entire profile of a dam (up to the depth of 100 meters depending on the length of measuring cable) or a stream. Due to built-in depth and temperature sensors it is able to record a vertical distribution of different groups of phytoplankton in the water column.
Probes BenthoFluor and BenthoTorch enable the determination of chlorophyll-a concentration in benthic mats – photoautotrophic communities on various surfaces, like sediments, stones or man-made materials both below and above water surface.
Physiological status and especially photosynthetic activity of cyanobacteria and algae can be assessed by fluorometers from Photon Systems Instruments (Brno, Czech Republic): fluorescence imaging system FluorCam MF700, cuvette lab fluorometer FL3500 FAST and pocket-sized field fluorometers AquaPen AP 100 in cuvette version or with submersible optical probe. More information about these machines and about induced chlorophyll fluorescence you can find here.
Selected publications to above mentioned issue:
- Gregor, J., Marsalek, B. (2004): Freshwater phytoplankton quantification by chlorophyll a: a comparative study of in vitro, in vivo and in situ methods. Water Research 38(3): 517-522.
- Gregor, J., Marsalek, B. (2005): A simple in vivo fluorescence method for the selective detection and quantification of freshwater cyanobacteria and eukaryotic algae. Acta Hydrochimica et Hydrobiologica 33(2): 142-148.
- Gregor, J., Marsalek, B., Sipkova, H. (2007): Detection and estimation of potentially toxic cyanobacteria in raw water at the drinking water treatment plant by in vivo fluorescence method. Water Research 41(1): 228-234.
- Gregor, J., Jancula, D., Marsalek, B. (2008): Growth assays with mixed cultures of cyanobacteria and algae assessed by in vivo fluorescence: One step closer to real ecosystems? Chemosphere 70(10): 1873-1878.