Analysis of Phytoplankton Pigments by Excitation Spectra of Fluorescence (original) (raw)
Fluorescent screening of phytoplankton and organic compounds in sea water
Journal of Environmental Monitoring, 2000
The¯ow-through spectro¯uorometers, FLUO-IMAGER 2 , were developed to measure the abundance of phytoplankton, including the analysis of pigment composition and concentration of organic pollution and dissolved organic matter (DOM), in continuous mode. The measurements can be carried out without the timeconsuming pretreatment of water samples. The analytical concept uses the technique of spectral¯uorescent signatures (SFS), based on the systematized spectral library comprising the SFS of major phytoplankton species and chemical pollution. The SFS technique has been applied for several years in qualitative and quantitative screening of organic compounds and phytoplankton in the Baltic, North and Norwegian Seas. The results of the analysis of phytoplankton pigments, the dynamic processes of bloom development, DOM and oil pollution are presented.
Freshwater Biology, 2005
1. This study introduces delayed fluorescence (DF) excitation spectroscopy as an on-line tool for in situ monitoring of the composition and biomass of various colour classes of phytoplankton when they are photosynthetically active (cyanobacteria, chlorophytes, chromophytes and cryptophytes). The DF data are validated by comparison with those from conventional methods (weekly microscopic counts and the measurement of chlorophyll concentration).2. The composition of phytoplankton as assessed by DF agreed reasonably well with the results from microscopic counts, particularly when differences in chlorophyll-specific DF integrals of the various colour classes were taken into account.3. Integrals of DF spectra were converted into concentration of chlorophyll a using empirical factors derived from field data. The value of the conversion factor was nearly twice as high when the relative abundance of cyanobacteria was low (<15%) than when it was high. The converted DF-chl time series agreed well with chlorophyll measurements particularly when blooms were developing. As the DF method is inherently free of the interference caused by pigment degradation products, the discrepancy between the two data sets increased during the collapse of blooms and when sediment resuspension was intense.4. Fourier spectrum analysis of the time series of DF-chl indicated that samples must be taken, at a minimum, every 2–3 days to capture the dynamics of phytoplankton. As a consequence, the dynamics of various algal blooms, including their timing, duration and net growth rate, could be estimated with greater confidence than by using conventional methods alone.5. On-line DF spectroscopy is an advanced technique for monitoring daily the biomass and composition of the photosynthetically active phytoplankton in aquatic environments, including turbid shallow lakes. At present, the detection limit is around 1 mg DF-chl a m−3 in terms of total biomass but confidence in estimates of phytoplankton composition declines sharply below about 5 mg chl a m−3.6. On-line DF spectroscopy represents a promising approach for monitoring phytoplankton. It will be useful in water management where it can act as an early-warning system of declines in water quality. In basic ecological research it can supplement manual methods. While default calibration spectra may be acceptable for routine monitoring, we suggest a careful individual calibration of the DF spectrometer for basic research. The statistical methods developed here help to assess the adequacy of various calibration sets.
Journal of Marine Systems, 2017
This study presents the results of the first field application of a flow-through multi-wavelength Fast Repetition Rate fluorometer (FRRF) equipped with two excitation channels (458 and 593 nm). This device aims to improve the measurement of mixed cyanobacteria and algae community's photosynthetic parameters and was designed to be easily incorporated into existing ferrybox systems. We present a spatiotemporal analysis of the maximum photochemical efficiency (F v /F m) and functional absorption cross section (σ PSII) recorded from April to August 2014 on a ship-of-opportunity commuting twice per week between Helsinki (Finland) and Travemünde (Germany). Temporal variations of F v /F m and σ PSII differed between areas of the Baltic Sea. However, even though the Baltic Sea is characterized by several physico-chemical gradients, no gradient was observed in F v /F m and σ PSII spatial distribution suggesting complex interactions between biotic and abiotic controls. σ PSII was sensitive to phytoplankton seasonal succession and thus differed according to the wavelength used to excite photosystems II (PSII) pigments. This was particularly true in summer when high σ PSII (593) values were observed later and longer than high σ PSII (458) values, reflecting the role of cyanobacteria in photosynthetic light uptake measured at community scale. In contrast, F v /F m variations were similar after excitation at 458 nm or 593 nm suggesting that the adjustment of F v /F m in response to environmental factors was similar for the different groups (algae vs. cyanobacteria) present within the phytoplankton community.
Use of fluorescence information for automated phytoplankton investigation by image analysis
Journal of Plankton Research, 2008
Automated identification and quantification of algae in microscopic images is a tool that allows high taxonomic resolution with reasonable technical efforts. However, in samples containing various non-algal objects, this is still not a satisfactorily solved problem. We show that autofluorescence information improves discrimination of algae from non-algal objects as well as phycoerythrin (PE) containing algae from others. We analyse the stability of the autofluorescence to estimate its constraints. Cold and dark storage of glutaraldehyde fixed samples maintains autofluorescence sufficiently for 3 weeks. Under repeated excitations, chlorophyll a (Chl a) or PE autofluorescence show an exponential decrease followed by an intermediate maximum. A peak also occurs in emission wavelength ranges without chlorophyll and PE fluorescence. The unspecific autofluorescence causing the peaks is at least partly identical with the blue -green fluorescence (BGF) in plant cells. BGF interferes with identification of algae, thus correction of pigment autofluorescence with such unspecific fluorescence allows a more reliable algal discrimination procedure. A classification scheme for discrimination of Chl a and PE-containing algae shows a high performance in a test with natural samples. Integration of fluorescence and bright-field image information provides a powerful tool for phytoplankton analysis in complex samples.
Estuarine, Coastal and Shelf Science, 2002
We studied the temporal variability in the composition of plant pigments in water-column particulate matter and in surface sediments, with specific emphasis on bloom events. The pigment concentration of sinking and suspended particles was used to evaluate pre-and post-depositional decomposition of bloom-derived phytodetritus in the Baltic proper. Water, sediment, and sediment trap samples were collected in 1994 at a long-term coastal monitoring station with fine sediments and a water depth of 36 m, situated near the Askö Laboratory in the northwestern part of the Baltic Sea proper. Annual phytoplankton succession showed an early spring bloom dominated by diatoms, followed by dinoflagellates in late spring and early summer. Later in summer, this dinoflagellate community was gradually replaced by a filamentous, nitrogen-fixing cyanobacterial bloom; late summer-early autumn was characterized by diatoms. The highest chlorophyll a flux occurred in spring during peak diatom bloom conditions, which resulted in peak concentrations of chlorophyll a in sediments. Assuming that all chlorophyll a in sediments in early May was derived from the current bloom, it was estimated that approximately half (48%) the bloom-derived chlorophyll a was decomposed in two months. The highest total phaeopigment/chlorophyll a ratios (an index of the amount of chlorophyll a decay), were found in water column particulate matter (PM) during the spring bloom and occasional high-energy winter periods. The latter higher ratios were likely due to wind-induced resuspension of surface sediments into the water column and trap materials. The settling rate of pigments in the water column indicated rapid sedimentation of diatoms and slow sedimentation of dinoflagellates during the spring bloom. In summer, unicellular cyanobacteria, including picocyanobacteria, appeared to have higher sedimentation rates than filamentous nitrogen-fixing cyanobacteria, which seemed to decompose largely in the water column. Thus, this study demonstrated that plant pigments were useful biomarkers of the taxonomic composition of phytoplankton blooms and their sedimentation and subsequent decomposition in the Baltic Sea.
Field Analytical Chemistry & Technology, 2000
A two-channel fluorometric sensor system for the detection of the waterbloom phytoplankton Microcystis aeruginosa has been developed. Excitation wavelengths of 620 and 440 nm were used, the former for detecting the cyanobacteria themselves, and the latter for subtracting the interference due to eukaryotic algae present in the sample. The fluorescence of the cyanobacteria and eukaryotic algae was measured at 645 and 680 nm, respectively. A linear relationship between phycocyanin fluorescence was established for the species examined in the range of to 10 0 g/ml ؊1 chlorophyll a.
Journal of Phycology, 2007
In vivo delayed fluorescence (DF) and HPLC ⁄ CHEMTAX pigment analyses were used to investigate seasonal and depth distributions of phytoplankton in a deep alpine mesotrophic lake, Mondsee (Austria). Using chl a equivalents, we determined significant relationships with both approaches. Community structure derived from pigment ratios of homogenous samples was compared with microscopic estimations using biovolume conversion factors. An advantage of the HPLC ⁄ CHEMTAX method was that it gave good discrimination among phytoplankton groups when based on a pigment ratio matrix derived from multiple regression analysis. When a single algal group was dominant, such as epilimnetic diatoms or hypolimnetic cyanobacteria in the deep chl maxima, HPLC ⁄ CHEMTAX results were significantly correlated with microscopic estimations (diatoms: r = 0.93; cyanobacteria: r = 0.94). Changes in the composition of photosynthetically active pigments were investigated with DF and benefited from excitation spectra that considered all light-harvesting pigments, which made it possible to assess the enhancement of accessory photosynthetically active pigments relative to active chl a (chl a DF672). Changes in similarity index, based on normalized DF spectra, confirmed compositional shifts observed by microscopy. At chosen wavelengths of DF spectra, 534 and 586 nm, we generally observed a significantly inverse relationship between normalized DF intensities and temperature and light along both seasonal and depth gradients. The relative increase in photosynthetically active pigments other than chl a DF672 under low light and temperature was caused by an increasing dominance of diatoms and ⁄ or phycobilin-rich cyanobacteria and Cryptophyta. DF spectra provided a more accurate picture of community pigments acclimated to light and temperature conditions than the b-carotene:chl a ratio derived from HPLC.
Optimization of variable fluorescence measurements of phytoplankton communities with cyanobacteria
Photosynthesis Research, 2012
Excitation-emission fluorescence matrices of phytoplankton communities were simulated from laboratory-grown algae and cyanobacteria cultures, to define the optical configurations of theoretical fluorometers that either minimize or maximize the representation of these phytoplankton groups in community variable fluorescence measurements. Excitation sources that match the photosystem II (PSII) action spectrum of cyanobacteria do not necessarily lead to equal representation of cyanobacteria in community fluorescence. In communities with an equal share of algae and cyanobacteria, inducible PSII fluorescence in algae can be retrieved from community fluorescence under blue excitation (450-470 nm) with high accuracy (R 2 = 1.00). The highest correlation between community and cyanobacterial variable fluorescence is obtained under orange-red excitation in the 590-650 nm range (R 2 = 0.54). Gaussian band decomposition reveals that in the presence of cyanobacteria, the emission detection slit must be narrow (up to 10 nm) and centred on PSII chlorophyll-a emission (*683 nm) to avoid severe dampening of the signal by weakly variable phycobilisomal fluorescence and non-variable photosystem I fluorescence. When these optimizations of the optical configuration of the fluorometer are followed, both cyanobacterial and algal cultures in nutrient replete exponential growth exhibit values of the maximum quantum yield of charge separation in PSII in the range of 0.65-0.7.
Journal of Phycology, 2007
In vivo delayed fluorescence (DF) and HPLC ⁄ CHEMTAX pigment analyses were used to investigate seasonal and depth distributions of phytoplankton in a deep alpine mesotrophic lake, Mondsee (Austria). Using chl a equivalents, we determined significant relationships with both approaches. Community structure derived from pigment ratios of homogenous samples was compared with microscopic estimations using biovolume conversion factors. An advantage of the HPLC ⁄ CHEMTAX method was that it gave good discrimination among phytoplankton groups when based on a pigment ratio matrix derived from multiple regression analysis. When a single algal group was dominant, such as epilimnetic diatoms or hypolimnetic cyanobacteria in the deep chl maxima, HPLC ⁄ CHEMTAX results were significantly correlated with microscopic estimations (diatoms: r = 0.93; cyanobacteria: r = 0.94). Changes in the composition of photosynthetically active pigments were investigated with DF and benefited from excitation spectra that considered all light-harvesting pigments, which made it possible to assess the enhancement of accessory photosynthetically active pigments relative to active chl a (chl a DF672). Changes in similarity index, based on normalized DF spectra, confirmed compositional shifts observed by microscopy. At chosen wavelengths of DF spectra, 534 and 586 nm, we generally observed a significantly inverse relationship between normalized DF intensities and temperature and light along both seasonal and depth gradients. The relative increase in photosynthetically active pigments other than chl a DF672 under low light and temperature was caused by an increasing dominance of diatoms and ⁄ or phycobilin-rich cyanobacteria and Cryptophyta. DF spectra provided a more accurate picture of community pigments acclimated to light and temperature conditions than the b-carotene:chl a ratio derived from HPLC.