Identification and quantification of phytoplankton groups in lakes using new pigment ratios – a comparison between pigment analysis by HPLC and microscopy

1. Pigment analysis by high‐performance liquid chromatography (HPLC) combined with data analysis using the CHEMTAX program has proven to be a fast and precise method for determining the abundance of phytoplankton groups in marine environments. To determine whether CHEMTAX is applicable also to freshwater phytoplankton, 20 different species of freshwater algae were cultured and their pigment/chlorophyll a (Chl a) ratios determined for exponential growth at three different light intensities and for stationary growth at one light intensity. 2. The different treatments had a relatively insignificant impact on the absolute values of the diagnostic pigment/Chl a ratios, with the exception of cyanobacteria and cryptophytes for which the zeaxanthin/Chl a and alloxanthin/Chl a ratios varied considerably. 3. The pigment ratios were tested on samples collected in six different eutrophic Danish lakes during two summer periods using the CHEMTAX program to calculate the biomass of the phytoplankton groups as Chl a. The CHEMTAX‐derived seasonal changes in Chl a biomass corresponded well with the volume of the microscopically determined phytoplankton groups. More phytoplankton groups were detected by the pigment method than by the microscopic method. 4. Applying the pigment ratios developed in this study, the pigment method can be used to determine the abundance of the individual phytoplankton groups, which are useful as biological water quality indicators when determining the ecological status of freshwater lakes.     

An HPLC analysis of the summer phytoplankton assemblage in Lake Baikal

1. The enormous size and spatial heterogeneity of Lake Baikal require rapid methods for large sample sets. We therefore tested the applicability of a novel, high‐performance liquid chromatography (HPLC)‐based, combination of methods for analysing phytoplankton. In July 2001, samples were collected in a transect across the lake at various depths down to 30 m. Phytoplankton (>3 μm) and autotrophic picoplankton (APP) were counted under light and epifluorescence microscopes, respectively. Pigments were analysed with HPLC. 2. The pigment data allowed the contributions of the dominant phytoplankton groups to the total chlorophyll a (Chl a) in the lake to be estimated by multiple linear regression and by the CHEMTAX matrix factorisation program. Three marker pigments, fucoxanthin, lutein and zeaxanthin, were shown to be useful indicators of the abundance and spatial distribution of certain phytoplankton groups. The relative contributions of the various phytoplankton groups to the total Chl a in the lake determined using these marker pigments were similar, but not identical, to those determined by cell counts. 3. Pigment analyses of isolated strains from Lake Baikal and some European lakes confirmed that phycoerythrin‐containing Cyanobacteria with very high amounts of zeaxanthin were responsible for the low Chl a/zeaxanthin ratios of the water samples. A picoplanktonic species of Eustigmatophyceae was isolated from the lake. Its high violaxanthin content, responsible for very low Chl a/violaxanthin ratios of some water samples, can be used to estimate the contribution of this group to total Chl a.     

Comparing microscopic counts and pigment analyses in 46 phytoplankton communities from lakes of different trophic state

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Does pigment composition reflect phytoplankton community structure in differing temperature and light conditions in a deep alpine lake? An approach using HPLC and delayed fluorescence techniques1

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 β‐carotene:chl a ratio derived from HPLC.     

Does the chlorophyll a content of phytoplankton vary with trophic status in lakes on the New Zealand central volcanic plateau?

Recently, it has been shown that ratios of chlorophyll a toparticulate phosphorus (Chl a/PP) and chlorophyll a to particulatenitrogen (Chl a/PN) were significantly higher in eutrophic thanoligo/mesotrophic waters in 17 lakes on the central volcanicplateau, North Island, New Zealand. This difference was thoughtto be due to an increase in the chlorophyll a content of phytoplanktonin these eutrophic lakes. Corresponding measurements of chlorophylla and phytoplankton cell volume made during this study do notsupport this hypothesis. However, ratios of chlorophyll a toadenosine triphosphate and estimates of percentage phytoplanktonbiomass were significantly higher (P<0.05) in our eutrophicthan oligo/mesotrophic samples, suggesting that Chl a/PP andChl a/PN may be high in eutrophic waters simply because phytoplanktoncomprise more of the total microbial biomass. This hypothesisis supported by a strong linear relationship (r=0.88, P<0.001)between Chl a/PP and percentage phytoplankton biomass in sixof our study lakes where corresponding measurements were made.     

A comparison of HPLC pigment analyses and biovolume estimates of phytoplankton groups in an oligotrophic lake

Assessment of the contribution of distinct algal groups to phytoplanktonbiomass in oligotrophic lakes by marker pigments is comparedwith assessment by cell-counting biovolume estimates. Seasonalsamples from an oligotrophic alpine lake (Redon, Pyrenees) mostlyincluded species of chrysophytes, dinoflagellates, cryptophytesand chlorophytes. The chlorophyl a (Chl a) corresponding toeach algal group was estimated using HPLC pigment analyses andthe CHEMTAX program. Chl a estimates and biovolume showed asignificant correlation for all the groups during the ice-freeseason except for chlorophytes. However, some of the samplesfrom the initial phase of the ice cover presented a clear departurefrom the relationship during the ice-free period in most groups.On the other hand, the ratios between a specific marker pigmentand the biovolume of the marked algal group were significantlyconstant within the photic zone (>1% surface irradiance)for most of the pigments and groups, including chlorophytes.Nevertheless, the ratios increased and showed a large variabilityfor samples below the photic depth or below the ice cover. Theviolaxanthin-chrysophyte biovolume ratio presented an opposedtendency to other pigment-biovolume ratios, which increasedin inverse proportion to the depth of the sample. The resultsare discussed in terms of methodological limitations, acclimationresponses and species composition.     

Pigment ratios and phytoplankton assessment in northern Wisconsin lakes

Nine lakes in northern Wisconsin were sampled from February through September 1996, and HPLC analysis of water column pigments was carried out on epilimnetic seston. Pigment distributions were evaluated throughout the water column during summer in Crystal Lake and Little Rock Lake. The purpose of our study was to investigate the use of phytopigments as markers of the main taxonomic groups of algae. As a first approach, multiple regression of marker pigments against chlorophyll a (chl a) was used to derive the best linear combination of the main xanthophylls (peridinin, fucoxanthin, alloxanthin, lutein, and zeaxanthin). A significant regression equation (r²= 0.98) was obtained for epilimnion data. The good fit indicates that the chl a:xanthophyll ratios were fairly constant in the epilimnion of the nine lakes over time. Chlorophyll a recalculated from the main xanthophylls in each sample showed good agreement with measured chl a in epilimnetic waters. A second approach used the CHEMTAX program to analyze the same data set. CHEMTAX provided estimates of chl a biomass for all algal classes and allowed distinction between diatoms and chrysophytes, and between chlorophytes and euglenophytes. These results showed a reasonably good agreement with biomass estimates from microscope counts, despite uncertainties associated with differences in sampling procedure. Changes of pigment ratios over time in the epilimnetic waters were also investigated, as well as differences between surface and deep samples of Little Rock Lake and Crystal Lake. We found evidence that changes in the ratio of photoprotective pigments to chl a occurred as a response to changes in light climate. Changes were also observed for certain light‐harvesting pigments. The comparison between multiple regression and CHEMTAX analyses for inferring chl a biomass from concentrations of marker pigments highlighted the need to take account of variations in pigment ratio, as well as the need to acquire additional data on the pigment composition of planktonic algae.     

Phytoplankton of an eutrophic tropical reservoir: comparison of biomass estimated from counts with chlorophyll-a biomass from HPLC measurements

The seasonal variation of phytoplankton in an eutrophic tropical reservoir was evaluated through photosynthetic pigments analyzed by HPLC. The contributions of algal classes to total chlorophyll a (TChl-a) were estimated by two procedures. The first one used fixed marker pigment/chlorophyll a ratio available from culture studies of the major species of each class. In the second procedure, a matrix factorization program (CHEMTAX) was used to analyze the pigment data. The pigment data were compared with carbon biomass estimated from microscope analysis. A significant correlation between total chlorophyll a (measured by HPLC) and total biomass was obtained, indicating only a slight variation in the content of algal chlorophyll a when compared to its fluctuations in carbon biomass. The interpretation of pigment data with CHEMTAX resulted in a good agreement with biomass. Although displaying some differences, the general pattern of the phytoplankton community dynamics and the major shifts in composition, biomass and the cyanobacterial bloom were evidenced. In contrast, Chl-a biomass estimates from fixed Xan/Chl-a ratios presented poor agreement with microscope data and did not register the principal changes in phytoplankton. Our results also highlighted the needs of better understanding of the relationships between marker pigments, chlorophyll-a and algal biomass.     

QUANTIFICATION OF THE RELATIVE ABUNDANCE OF THE TOXIC DINOFLAGELLATE,KARENIA BREVIS (DINOPHYTA), USING UNIQUE PHOTOPIGMENTS1

Diagnostic photopigment analysis is a useful tool for determining the presence and relative abundance of algal groups in natural phytoplankton assemblages. This approach is especially useful when a genus has a unique photopigment composition. The toxic dinoflagellate Karenia brevis (Davis) G. Hansen & Moestrup comb. nov. shares the diagnostic pigment gyroxanthin‐diester with only a few other dinoflagellates and lacks peridinin, one of the major diagnostic pigments of most dinoflagellate species. In this study, measurements of gyroxanthin‐diester and other diagnostic pigments of K. brevis were incorporated into the initial pigment ratio matrix of the chemical taxonomy program (CHEMTAX) to resolve the relative contribution of K. brevis biomass in mixed estuarine phytoplankton assemblages from Florida and Galveston Bay, Texas. The phytoplankton community composition of the bloom in Galveston Bay was calculated based on cell enumerations and biovolumetric measurements in addition to chl a‐specific photopigment estimates of biomass (HPLC and CHEMTAX). The CHEMTAX and biovolume estimates of the phytoplankton community structure were not significantly different and suggest that the HPLC–CHEMTAX approach provides reasonable estimates of K. brevis biomass in natural assemblages. The gyroxanthin‐diester content per cell of K. brevis from Galveston Bay was significantly higher than in K. brevis collected from the west coast of Florida. This pigment‐based approach provides a useful tool for resolving spatiotemporal distributions of phytoplankton in the presence of K. brevis blooms, when an appropriate initial ratio matrix is applied.     

Phytoplankton chemotaxonomy in waters around the Svalbard archipelago reveals high amounts of Chl b and presence of gyroxanthin-diester

Phytoplankton pigment signatures from a cruise in 2005 are herein presented and used as a chemotaxonomic tool for phytoplankton diversity in the Svalbard marine archipelago. Studies from these waters have until recently reported only a few groups of phytoplankton, and while this paper is the first to show that the diversity around Svalbard includes all major phytoplankton pigment groups, the results are seen in relation to other similar studies from the Arctic. We present two potentially important marker pigments: prasinoxanthin, originating from prasinophytes, and gyroxanthin-diester, possibly originating from the temperate- and bloom-forming coccolithophore Emiliania huxleyi. Pigment identification by HPLC revealed a significant amount of Chlorophyll b-containing chlorophyceae, euglenophyceae and prasinophyceae. Prasinoxanthin was present at 50% of the examined stations, typically at Chl a maximum (15–25 m depth), in both Atlantic and Arctic water masses. Gyroxanthin-diester, in contrast to prasinoxanthin, was found only in Atlantic water masses and at low concentrations. Our data may be important for the identification and verification of remotely sensed images of different pigment groups of phytoplankton and their corresponding biomass, typically estimated from Chl a. Remotely sensed presence of coccoliths, indicating E. huxleyi at sea surface, is discussed in relation to water masses and pigment signatures at sea surface and Chl a maximum depths.     

Pigment-Based Chemotaxonomy - A Quick Alternative to Determine Algal Assemblages in Large Shallow Eutrophic Lake?

Pigment-based chemotaxonomy and CHEMTAX software have proven to be a valuable phytoplankton monitoring tool in marine environments, but are yet underdeveloped to determine algal assemblages in freshwater ecosystems. The main objectives of this study were (1) to compare the results of direct microscopy and CHEMTAX in describing phytoplankton community composition dynamics in a large, shallow and eutrophic lake; (2) to analyze the efficiency of the pigment-based method to detect changes in phytoplankton seasonal dynamics and during rapid bloom periods; (3) to assess the suitability of specific marker pigments and available marker pigment:chlorophyll a ratios to follow seasonal changes in eutrophic freshwater environment. A 5-year (2009-2013) parallel phytoplankton assessment by direct microscopy and by CHEMTAX was conducted using published marker pigment:chlorophyll a ratios. Despite displaying some differences from microscopy results, the pigment-based method successfully described the overall pattern of phytoplankton community dynamics during seasonal cycle in a eutrophic lake. Good agreement between the methods was achieved for most phytoplankton groups - cyanobacteria, chlorophytes, diatoms and cryptophytes. The agreement was poor in case of chrysophytes and dinoflagellates. Our study shows clearly that published marker pigment:chlorophyll a ratios can be used to describe algal class abundances, but they need to be calibrated for specific freshwater environment. Broader use of this method would enable to expand monitoring networks and increase measurement frequencies of freshwater ecosystems to meet the goals of the Water Framework Directive.     

Discrimination of marine algal taxonomic groups using delayed fluorescence spectroscopy

We present a method for in situ monitoring of phytoplankton composition changes in a marine environment. The method is based on delayed fluorescence excitation spectra analyzed with CHEMTAX software, which is generally used for determination of phytoplankton communities with HPLC pigment data. Delayed fluorescence (DF) is a photosynthetic parameter that can only be measured in living cells. Algal DF excitation spectra are group-specific, based on their composition of photosynthetic pigments.DF excitation spectra of 14 marine algal species from different families were measured with a delayed fluorescence spectrometer. Mixtures were prepared from northern Adriatic algal species representing six taxonomic groups: dinoflagellates (Prorocentrum minimum), diatoms (Skeletonema costatum), cyanobacteria (Synechococcus sp.), prasinophytes (Micromonas sp.), cryptophytes (Teleaulax sp.), and prymnesiophytes (Isochrysis galbana). The DF excitation spectra (DFS) and HPLC pigment compositions of the mixtures were analyzed with CHEMTAX software. The prediction power of DFS–CHEMTAX method was comparable to HPLC–CHEMTAX.     

Use of marker pigments and functional groups for assessing the status of phytoplankton assemblages in lakes

Phytoplankton is a key biological quality element for the establishment of the Water Framework Directive (WFD) ecological status in reservoirs and lakes. In freshwaters, inverted microscope examination is the traditional standard method for estimating phytoplankton and assessing taxonomic composition. Based on the enumeration of algal units and measurements for biovolume calculation, this technique is cumbersome and time-consuming. In large monitoring programmes, such as the application of the WFD in lakes and reservoirs, chemotaxonomy (HPLC pigment analysis and CHEMTAX treatment) is ideally suited as an alternative method because it allows the rapid processing of large numbers of samples from numerous locations and depths, thereby providing ideal temporal and spatial resolution. The low taxonomical detail obtained by HPLC and CHEMTAX (phytoplankton classes or phyla) can easily be overcome by a rapid inverted microscope screening with identification of the dominant species. Combining HPLC and microscopy provides a useful method for monitoring phytoplankton assemblages, which can be used to implement the WFD with respect to phytoplankton. Here, we present the application of a method combining marker pigments and microscopy to phytoplankton samples from 12 Belgian reservoirs. This method substantially reduced the workload and enabled us to assess the status of the phytoplankton assemblage in these lakes. The method complies with the WFD, as it takes into account taxonomic composition, assesses abundance and biomass of the phytoplankton taxa, and easily detects blooms. Additionally, a set of templates of probability of occurrence of phytoplankton functional groups at the maximal ecological potential for reservoirs from the Central/Baltic region is presented, based on reference conditions defined for natural lakes from other regions.     

Contrasting responses of intertidal microphytobenthos and phytoplankton biomass and taxonomic composition to the nutrient loads in the Jiulong River Estuary

Microphytobenthos (MPB) and phytoplankton are important primary producers in the estuarial ecosystem, and their functions are critical to the ecosystem's biodiversity and environmental safety. The aim of this study was to compare the response of MPB and phytoplankton to the nutrient loads in a eutrophic estuary, which has seldom been studied. We used high‐performance liquid chromatography (HPLC) and CHEMTAX software to examine the biomass and taxonomic composition of both MPB and phytoplankton at Da‐yu Island (DYI) and Ji‐yu Island (JYI) in the Jiulong River Estuary from July 2010 to March 2012. The results showed that MPB chlorophyll a was low in the summer and high in the winter at both DYI and JYI, indicating a unimodal pattern. However, the phytoplankton chlorophyll a showed a mirrored pattern. Diatoms were the dominant class in both benthic and pelagic environments. Although redundancy analysis indicated that the effects of different environmental factors could not be easily separated, it is likely that phosphate and temperature were the most important factors regulating the seasonal patterns of MPB and phytoplankton diatoms, respectively. MPB and phytoplankton cyanobacteria was co‐limited by salinity and temperature. The high N/P ratio and low phosphate favored chlorophytes and cyanobacteria. Our study demonstrates the use of HPLC and CHEMTAX in an integrated survey of the spatial and temporal distribution patterns of MPB and phytoplankton in an estuarial ecosystem. The contrasting responses of MPB and phytoplankton to nutrient loads indicate the critical role of MPB in subtropical estuarial ecosystem function. The relationship between nutrients and MPB may indicate a significant contribution to carbon and nutrient cycling.     

Factors influencing the variability of pigments in the surface sediments of mountain lakes

1. Surface sediment biofilm samples from 82 Pyrenean lakes were analysed for marker pigment composition using high performance liquid chromatography (HPLC). 2. Variability in the pigment composition among lakes was investigated by multivariate statistical analyses using a large data set of factors describing lake chemical, physical, morphological and catchment characteristics. 3. Due to the widely varying light penetration in the lakes, the most significant gradient of pigment composition extended from a benthic to a planktonic signal. The most important pigments in the gradient were alloxanthin (cryptophytes marker pigment, planktonic signal) and diatoxanthin (diatoms marker pigment, benthic signal). The molar ratio between these two marker pigments was positively correlated with lake depth. 4. Chlorophyll‐a preservation was found to be positively related to light penetration and the development of an autothrophic biofilm on the surface sediment and negatively related to decreasing pH and the percentage of alpine meadows in the lake catchments. 5. Zooplankton marker pigments in the surface sediment, including grazing by‐products (e.g. phaeophorbides) and carotenoids (astaxanthin, canthaxanthin, echinenone) incorporated into their tissues, were correlated with the areal abundance of zooplankton. 6. Marker pigments for photosynthetic bacteria, BChl‐e and okenone, were found mainly in relatively shallow lakes with large catchments that are forested, probably because of their higher loading of allochthonous organic matter. 7. The evaluation of a preservation index (Chl‐a expressed as a percentage of a‐phorbins) and the alloxanthin/diatoxanthin ratios throughout the sediment record of mountain lakes can provide evidence of historical changes in the relative importance of planktonic versus benthic primary production and might ultimately be interpreted in terms of climatic or environmental changes.     

Dynamics of phytoplankton pigment characteristics in the Biya head water

Concentrations of phytoplankton photosynthetic pigments were measured at the Biya head water (in the immediate vicinity of Lake Teletskoe) every 10 days from July 1998 through December 2001. Comparison of phytoplankton pigment characteristics at the river’s head waters and at the surface of the lake’s pelagic zone for the ice-free period shows that the monitoring data give a clear picture of the formation and functioning of the lake phytoplankton. Analysis of seasonal fluctuations of pigment characteristics and their proportions revealed characteristic stages in the development of lake algal cenoses. We have demonstrated similarities and differences in the seasonal dynamics of the phytoplankton between Lake Teletskoe, a deep oligotrophic lake in the south of West Siberia, as compared with the general pattern observed in temperate lakes. According to the concentration of chlorophyll a, the trophic status of the lake’s pelagic zone and the Biya head water is ultraoligotrophic-oligotrophic. Yellow:green pigment ratio ranks Lake Teletskoe among “carotenoid lakes“. Low coefficients of linear correlation between chlorophyll a, water level and biogenic matter concentration shows that the relationships analyzed are far more complex than has been inferred from the combined effect of abiotic and biotic factors.     

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

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.     

Spatial and temporal photosynthetic compartments during summer in Antarctic Lake Kitiesh

Summary Four autotrophic compartments were recognised in Lake Kitiesh, King George Island (Southern Shetland) at the beginning of the summer in 1987: snow microalgae, ice bubble communities, phytoplankton in the water column and benthic communities of moss with epiphytes. Chlorophyll a concentration and pigment absorption spectra were obtained in these four compartments before and/or after the thawing of the ice cover. During the ice free period, carbon fixation and biomass was measured in the phytoplankton and in the benthic moss Campyliadelphus polygamus. From these measurements we conclude that the benthic moss is the most significant autotrophic component in this lake in terms of biomass, chlorophyll a content and primary productivity. The integral assimilation number (The ratio of carbon fixation per unit area to biomass per unit area) values were similar for both phytoplankton and the moss, ranging from 3.6 to 5.4 mg C (mg Chl a)^–1h^–1in phytoplankton and from 4.0 to 6.4 mgC (mg Chl a)^–1h^–1 in the benthic moss. This approach allows comparisons of carbon fixation efficiency of the chlorophyll a under a unit area between compartments in their different light environments.     

Pigment Content of Selected Planktonic Algae in Response to Simulated Natural Light Fluctuations and a Short Photoperiod

The lipophilic photosynthetic pigments in Limnothrix redekei, Planktothrix agardhii (cyanobacteria), Stephanodiscus minutulus, Synedra acus (diatoms), Scenedesmus acuminatus, and Scenedesmus armatus (chlorophycean) all isolated from an eutrophic lake were quantitatively determined by HPLC. The algae were grown semi-continuously under nutrient sufficient conditions at 20°C at a 12/12 h light/dark cycle with constant irradiance or with simulated natural light fluctuations as well as at a 6/18 h light/dark cycle with constant irradiance, all at the same daily light exposure. The zeaxanthin and the myxoxanthophyll contents of cyanobacteria were not influenced by fluctuating light, a short photoperiod or a different sampling time. The chlorophyll b/a ratio, the lutein/chlorophyll a ratio, and the neoxanthin content of chlorophycean as well as the chlorophyll c/a and the fucoxanthin/chlorophyll a ratio of diatoms were only slightly influenced by these factors. Therefore in some cases marker pigment contents and in other cases marker pigment/chlorophyll a ratios may be more useful for quantifying the relative importance of different taxonomic groups in natural phytoplankton. Simulated natural light fluctuations or the length of the photoperiod only slightly influenced the pigment content or the marker pigment/chlorophyll a ratio.