DETECTION OF NEW PIGMENTS FROM EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY, LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY, VISIBLE SPECTROSCOPY, AND FAST ATOM BOMBARDMENT MASS SPECTROMETRY

Pigment extracts from Emiliania huxleyi (Lohm.) Hay et Mohler (strains CCMP 370, CCMP 373, and NIOZ CH 24) were analyzed using high-performance liquid chromatography (HPLC) on highly efficient monomeric and polymeric octadecylsilica columns using either ammonium acetate or pyridine containing mobile phases. Both systems showed chromatographic profiles with peaks corresponding to pigments of uncertain structure: those of the polar and nonpolar chlorophyll c forms and one peak whose on-line diode array spectrum resembled that of the fucoxanthin acyloxy derivatives. Liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry gave a molecular mass of 786 units for the unknown carotenoid. The pigments corresponding to each of these fractions were isolated and their visible spectra recorded in various solvents. Samples of the isolated pigments were subjected to analysis by fast atom bombardment mass spectrometry that confirmed a molecular mass of 786 for the unknown carotenoid and gave a mass of 654 units for the polar chlorophyll c ₃, compatible with the monovinylic structure previously suggested. The detection of these new pigments calls for attention on the use of correct methodologies when HPLC pigment signatures are used to study the taxonomic composition of natural phytoplankton populations.     

Breakdown of phytoplankton pigments in Baltic sediments: effects of anoxia and loss of deposit-feeding macrofauna

We examined the decay of chlorophyll a and the carotenoid fucoxanthin in oxic and anoxic sediment microcosms, with and without the deposit-feeding benthic amphipod Monoporeia affinis, over 57 days at 5 degrees C. Deep frozen phytoplankton from the Baltic Sea proper was added to all but a few microcosms. The range of chlorophyll a and fucoxanthin decay rate constants observed in microcosms with phytoplankton addition was 0.04-0.07 day(-1). The fastest pigment decay and build-up of chlorophyll breakdown products after phytoplankton addition were found in oxic treatments with amphipods. No effects of amphipods on pigment breakdown were found in anoxic treatments, or in treatments without phytoplankton addition. Greater losses of chlorophyll a in oxic (96%) than in anoxic (80%) treatments after 57 days indicates that preservation of sedimentary organic matter will be enhanced during periods of anoxia. Due to slow recruitment and recolonization in Baltic sediments, a single anoxic event may cause long-term (years) absence of significant macrobenthos. Anoxic events will thus not only reduce decay of plant pigments, and presumably other organic matter, while they last, but will also have longer-term effects, through elimination of macrofauna, which when present enhance organic matter decomposition.     

Phytoplankton Assemblages and Their Dominant Pigments in the Nervion River Estuary

In the Nervion River estuary surface samples were taken from March to September 2003 at six sites covering most of the salinity range with the aim to know the biomass and taxonomic composition of phytoplankton assemblages in the different segments. Nine groups of algae including cyanobacteria, diatoms, dinoflagellates, chlorophytes, prasinophytes, euglenophytes, chrysophytes, haptophytes, raphidophytes and cryptophytes were identified by means of a combination of pigment analysis by high-performance liquid chromatography (HPLC) and microscopic observations of live and preserved cells. Diatoms, chlorophytes and cryptophytes were the most abundant algae in terms of cells number, whereas fucoxanthin, peridinin, chlorophyll b (Chl b) and alloxanthin were the most abundant auxiliary pigments. Based on multiple regression analysis, in the outer estuary (stations 0, 1, 2 and 3) about 93% of the chlorophyll a (Chl a) could be explained by algae containing fucoxanthin and by algae containing Chl b, whereas in the rest of the estuary most of the Chl a (about 98%) was accounted for by fucoxanthin, Chl b and alloxanthin containing algae. The study period coincided with that of most active phytoplankton growth in the estuary and fucoxanthin was by far the dominant among those signature pigments. Several diatoms, chrysophytes, haptophytes and raphydophytes were responsible for fucoxanthin among identified species. Besides, dinoflagellates with a pigment pattern corresponding to chrysophytes and type 4 haptophytes were identified among fucoxanthin-bearing algae. Cryptophytes were the most abundant species among those containing alloxanthin. The maximum of Chl b registered at the seaward end in April coincided with a bloom of the prasinophytes Cymbomonas tetramitiformis, whereas the Chl b maxima in late spring and summer were accounted for by prasinophytes in the middle and outer estuary and by several species of chlorophytes in the middle and inner estuary. Other Chl b containing algae were euglenophytes and the dinoflagellate Peridinium chlorophorum. Dinoflagellates constituted generally a minor component of the phytoplankton.     

Biomass-pigment relationships in potamoplankton

During most of the growing season of 1994, pigment content,as determined by HPLC analysis of algal sample extracts, wasfollowed in the River Meuse (Belgium) potamoplankton. The concentrationof some algal pigments (chlorophylls a and b, fucoxanthin, lutein,echinenone and alloxanthin) was related to biomass estimatesof total phytoplankton and of major taxonomic components (diatoms,green algae, cyanobacteria and cryptomonads). Highly significantlinear regressions were obtained for chlorophyll a-total biomass,fucoxanthin-diatoms, lutein-green algae, chlorophyll b-greenalgae. However, no relationship was found for cyanobacteriaor cryptomonads and their specific pigments, which may be attributedto poor accuracy of biomass estimates for these non-dominantalgae. In conclusion, the good relationship found for dominantalgae and their specific pigments confirms the value of pigmentsas quantitative markers of phytoplankton, as detected in othermarine and freshwater environments.     

Study of phytoplankton characteristics in Tolo Harbour,Hong Kong,by HPLC analysis of chemotaxonomic pigments

Spatial and seasonal characteristics of phytoplankton in Tolo Harbour, Hong Kong, were studied by microscopic observation of phytoplankton samples and HPLC analysis of chemotaxonomic pigments. Diatoms dominated the phytoplankton. Common diatoms included Skeletonema costatum and species of Cerataulina, Leptocylindrus, Pseudo-nitzschia and Thalassiosira. Dinoflagellates occurred sporadically and mainly in the inner part of the harbour. The dinoflagellate Scrippsiella trochoidea was the causative organism for the red tide occurrences in March, April and September 2001. Significant positive correlations between fucoxanthin and diatoms and between peridinin and dinoflagellates suggested that fucoxanthin and peridinin were valuable chemotaxonomic markers for diatoms and dinoflagellates, respectively. Analysis of pigment ratios revealed that red tide events caused by dinoflagellates were marked by increase in the value of PERI:chl a and decrease in the value of FUCO:chl a. Increase in the value of FUCO:chl a also revealed the presence of a dense population of Pseudo-nitzschia that was not indicated by increase in chlorophyll a and fucoxanthin concentrations. Pigment analysis also revealed the presence of cyanobacteria, silicoflagellates, cryptophytes and green algae in the surface waters of Tolo Harbour.     

Response of Phytoplankton Photophysiology to Varying Environmental Conditions in the Sub-Antarctic and Polar Frontal Zone

Climate-driven changes are expected to alter the hydrography of the Sub-Antarctic Zone (SAZ) and Polar Frontal Zone (PFZ) south of Australia, in which distinct regional environments are believed to be responsible for the differences in phytoplankton biomass in these regions. Here, we report how the dynamic influences of light, iron and temperature, which are responsible for the photophysiological differences between phytoplankton in the SAZ and PFZ, contribute to the biomass differences in these regions. High effective photochemical efficiency of photosystem II (/ 0.4), maximum photosynthesis rate (), light-saturation intensity (), maximum rate of photosynthetic electron transport (1/), and low photoprotective pigment concentrations observed in the SAZ correspond to high chlorophyll and iron concentrations. In contrast, phytoplankton in the PFZ exhibits low / ( 0.2) and high concentrations of photoprotective pigments under low light environment. Strong negative relationships between iron, temperature, and photoprotective pigments demonstrate that cells were producing more photoprotective pigments under low temperature and iron conditions, and are responsible for the low biomass and low productivity measured in the PFZ. As warming and enhanced iron input is expected in this region, this could probably increase phytoplankton photosynthesis in this region. However, complex interactions between the biogeochemical processes (e.g. stratification caused by warming could prevent mixing of nutrients), which control phytoplankton biomass and productivity, remain uncertain.     

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.     

Photoadaptation in marine phytoplankton : changes in spectral absorption and excitation of chlorophyll a fluorescence

The optical properties of marine phytoplankton were examined by measuring the absorption spectra and fluorescence excitation spectra of chlorophyll a for natural marine particles collected on glass fiber filters. Samples were collected at different depths from stations in temperate waters of the Southern California Bight and in polar waters of the Scotia and Ross Seas. At all stations, phytoplankton fluorescence excitation and absorption spectra changed systematically with depth and vertical stability of the water columns. In samples from deeper waters, both absorption and chlorophyll a fluorescence excitation spectra showed enhancement in the blue-to-green portion of the spectrum (470-560 nm) relative to that at 440 nm. Since similar changes in absorption and excitation were induced by incubating sea water samples at different light intensities, the changes in optical properties can be attributed to photoadaptation of the phytoplankton. The data indicate that in the natural populations studied, shade adaptation caused increases in the concentration of photosynthetic accessory pigments relative to chlorophyll a. These changes in cellular pigment composition were detectable within less than 1 day. Comparisons of absorption spectra with fluorescence excitation spectra indicate an apparent increase in the efficiency of sensitization of chlorophyll a fluorescence in the blue and green spectral regions for low light populations.     

Association of fucoxanthin chlorophyll a/c-binding polypeptides with photosystems and phosphorylation in the centric diatom Cyclotella cryptica

Solubilization of thylakoid membranes of Cyclotella cryptica with dodecyl-beta maltoside followed by sucrose density gradient centrifugation or deriphate polyacrylamide gel electrophoresis resulted in the isolation of pigment protein complexes. These complexes were characterized by absorption and fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western immunoblotting using antisera against fucoxanthin chlorophyll a/c-binding proteins and the reaction center protein D2 of photosystem II. Sucrose density gradient centrifugation yielded four bands. Band 1 consisted of free pigments with minor amounts of fucoxanthin chlorophyll a/c-binding proteins. Bands 2, 3, and 4 represented a major fucoxanthin chlorophyll a/c-binding protein fraction, photosystem II, and photosystem I, respectively. Deriphate polyacrylamide gel electrophoresis gave rise to five bands, representing photosystem I, photosystem II, two fucoxanthin chlorophyll a/c-binding protein complexes, and a band mostly consisting of free pigments. In the Western immunoblotting experiments, the specific association of two fucoxanthin chlorophyll a/c-binding proteins, Fcp2 and Fcp4, to the photosystems could be demonstrated. In vivo experiments using antibodies against phosphothreonine residues and in vitro studies using [gamma-32P]ATP showed that fucoxanthin chlorophyll a/c binding-proteins of 22 kDa became phosphorylated.     

硅藻金色奥杜藻色素的HPLC分析与超临界CO2萃取研究

以硅藻金色奥杜藻(Odontella aurita)为实验材料,利用高效液相色谱法分析了其色素组成与含量,采取超临界CO2萃取技术研究了从干藻粉内提取岩藻黄素的条件。结果表明,该藻主要含有岩藻黄素、硅甲藻黄素、β-胡萝卜素、硅藻黄素等类胡萝卜素以及叶绿素a和叶绿素c1,其中岩藻黄素为该藻含量最高的类胡萝卜素。色素的萃取率与压强、温度、夹带剂含量以及萃取时间呈正相关,夹带剂含量对萃取率影响最大,CO2流速的影响最小;与有机溶剂法相比,超临界CO2萃取岩藻黄素效率略低,而更利于岩藻黄素的选择性萃取及分离提纯;岩藻黄素的SFE-CO2适宜条件为压强400 bar、温度50℃、CO2流速0.2 L/min、夹带剂比例10%、萃取时间2～3 h。     

Spectral fluorescence signatures in the characterization of phytoplankton community composition

Fluorescence spectral signatures from 28 algal cultures aredescribed.The cultures are split into four groups accordingto their accessory pigments. Phycocyanin and phycoerythrin,characteristic pigments of cyanobacteria, form groups I andII. The characteristic pigment found in group III is chlorophyllb (green and rasinophyte algae) and in group IV it is chlorophyllc (diatoms, dinophytes and some other algae).This preliminarycatalogue of spectral signatures was used to characterize fivenatural phytoplankton communities from brackish water environmentsas a comparison with phytoplankton species found in the samples.Accessory pigments such as phycocyanin and phycoerythrin, characterizinggroups I and II, can be used for identification without confusion.Distinguishing between groups III and IV is more complicated,because their accessory pigments do not have their own fluorescence.These groups can be characterized by increased fluorescenceof chlorophyll a induced by energy excited through chlorophyllb and c. The possibilities of applying the spectral fluorescencesignatures approach to the characterization of natural communitiesare discussed.     

Studies on phytoplankton pigments in Porto Novo waters (India). I. Mangrove

The annual variations of phytoplankton pigments were studied from January to December, 1971, at two stations of the local mangrove (Pichavaram) environment. At these two stations, chlorophyll a varied from 2.90 to 35.06; chlorophyll b from 0 to 10.02 and chlorophyll c from 0 to 18.12 μg/l. Plant carotenoids varied from 1.56 to 13.83 MSPU/m³ and phaeopigments from 0 to 12.28 μg/l. The main (primary) peak of chlorophyll a was recorded during March at Station 1, and during June at Station 2.Secondary maxima occurred during June and August at Station 1, and during September at Station 2. During the period studied chlorophyll a was the dominant pigment at both the stations, followed by chlorophyll c and chlorophyll b in that order. The increase in the concentration of pigments was mainly due to the presence of phytoplankton species belonging to the genera such as Coscinodiscus, Rhizosolenia, Thalassiothrix, Melosira, Chaetoceros and Biddulphia. During October, phytoplankton was less and the pigment concentration was also low.     

The Loch Eil project: Planktonic pigments in sediments from Loch Eil and the firth of Lorne

Photosynthetic pigments and their derivatives were measured in sediments in the fjordic Loch Eil and the Firth of Lome, Scotland, between November 1975 and November 1976. After acetone extraction from the top 10 mm of sediment cores, pigments were crudely separated, by fluorescence change on acidification, into (chlorophyll a + chlorophyllide a) and phaeopigments. The greatest pigment concentrations (mean 73 μg · g sediment dry wt^?1) were found in the most reducing sediments which also had a high average proportion (23%) of chlorophyll. The least mean pigment concentration (23 μg · g^?1) and proportion of chlorophyll (17%) were found in the most oxidizing sediments in the Firth of Lorne where there was a clear seasonal cycle, with a peak in sediment pigment concentration and chlorophyll proportion in May and June, just after the planktonic spring increase. The Loch Eil stations showed a less clear or no seasonal cycle; the station most affected by organic input was the most variable from month to month. It was concluded that redox status was the most obvious control of sediment pigment content, whereas the effect of sedimentation of phytoplankton was complex.     

Algal pigment diversity in coastal sediments from Kerguelen (sub-Antarctic Islands) reflecting local dominance of green algae,euglenoids and diatoms

Summary High Performance Liquid Chromatography analysis of algal pigments from inter- and subtidal (deep and shallow) sediments from the Kerguelen Islands showed clear differences in the pigment composition at the different stations. High concentrations of chlorophyll c and fucoxanthin were present at all locations, indicating significant diatom densities, chlorophyll b was detected at all sites. At one station the other green algal pigments were also present; here green algae contributed more to chlorophyll a concentrations than diatoms, as estimated by using pigment ratios and microscopic observations. At another location chlorophyll b was associated with a high concentration of diadinoxanthin, indicating an abundance of euglenoids. This indicates that chemotaxonomy can be powerful tool in microphytobenthos studies since enumeration of living cells are difficult as many algae are attached to sediment particles (epipsammic algae). Ways of diagenesis, carotenoid degradation and the role of grazing are briefly mentioned. Phaeophorbide a-like pigments were the most significant chlorophyll a degradation products, with concentrations up to 110 g · g^–1 dry weight sediment, i.e. 10 times the chlorophyll a concentration. Some taxonomic estimations, based on pigments ratios, and their limits, are discussed.     

EFFECTS OF IRON AND LIGHT STRESS ON THE BIOCHEMICAL COMPOSITION OF ANTARCTIC PHAEOCYSTIS SP. (PRYMNESIOPHYCEAE). II. PIGMENT COMPOSITION

A strain of Phaeocystis sp., isolated in the Southern Ocean, was cultured under iron- and light-limited conditions. The cellular content of chlorophyll a and accessory light-harvesting (LH) pigments increased under low light intensities. Iron limitation resulted in a decrease of all light-harvesting pigments. However, this decrease was greatly compensated for by a decrease in cell volume. Cellular concentrations of the LH pigments were similar for both iron-replete and iron-deplete cells. Concentrations of chlorophyll a were affected only under low light conditions, wherein concentrations were suppressed by iron limitation. Ratios of the LH pigments to chlorophyll a were highest for iron-deplete cells under both light conditions. The photoprotective cycle of diato/diadinoxanthin was activated under high light conditions, and enhanced by iron stress. The ratio of diatoxanthin to diadinoxanthin was highest under high light, low iron conditions.   Iron limitation induced synthesis of 19'-hexanoyloxyfucoxanthin and 19'-butanoyloxyfucoxanthin at the cost of fucoxanthin. Fucoxanthin formed the main carotenoid in iron-replete Phaeocystis cells, whereas for iron-deplete cells 19'-hexanoyloxyfucoxanthin was found to be the main carotenoid. This shift in carotenoid composition is of importance in view of the marker function of both pigments, especially in areas where Phaeocystis sp. and diatoms occur simultaneously. A hypothesis is presented to explain the transformation of fucoxanthin into 19'-hexanoyloxyfucoxanthin and 19'-butanoyloxyfucoxanthin, referring to their roles as a light-harvesting pigment.     

Bias in satellite-derived pigment measurements due to coccolithophores and dinoflagellates

Satellite-derived estimates of phytoplankton pigments are thoughtto be affected by the phytoplankton species composition. Measurementsof surface algal chlorophyll and satellite-derived pigment werecompared for waters containing coccolithophores and dinoflagellates.Satellite-derived chlorophyll concentration was underestimatedby a factor of 2–3 in a patch of the large coccolithophore,Umbilicosphaera sibogae, and also in a bloom of the dinoflagellate,Gonyaulax polyedra. Overall abundance and species-specific propertiessuch as light scatter and vertical migration probably causedthese results.     

Evidence for the existence of one antenna-associated, lipid-dissolved and two protein-bound pools of diadinoxanthin cycle pigments in diatoms

We studied the localization of diadinoxanthin cycle pigments in the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum. Isolation of pigment protein complexes revealed that the majority of high-light-synthesized diadinoxanthin and diatoxanthin is associated with the fucoxanthin chlorophyll protein (FCP) complexes. The characterization of intact cells, thylakoid membranes, and pigment protein complexes by absorption and low-temperature fluorescence spectroscopy showed that the FCPs contain certain amounts of protein-bound diadinoxanthin cycle pigments, which are not significantly different in high-light and low-light cultures. The largest part of high-light-formed diadinoxanthin cycle pigments, however, is not bound to antenna apoproteins but located in a lipid shield around the FCPs, which is copurified with the complexes. This lipid shield is primarily composed of the thylakoid membrane lipid monogalactosyldiacylglycerol. We also show that the photosystem I (PSI) fraction contains a tightly connected FCP complex that is enriched in protein-bound diadinoxanthin cycle pigments. The peripheral FCP and the FCP associated with PSI are composed of different apoproteins. Tandem mass spectrometry analysis revealed that the peripheral FCP is composed mainly of the light-harvesting complex protein Lhcf and also significant amounts of Lhcr. The PSI fraction, on the other hand, shows an enrichment of Lhcr proteins, which are thus responsible for the diadinoxanthin cycle pigment binding. The existence of lipid-dissolved and protein-bound diadinoxanthin cycle pigments in the peripheral antenna and in PSI is discussed with respect to different specific functions of the xanthophylls.     

DIFFERENCES BETWEEN IN VIVO ABSORPTION AND FLUORESCENCE EXCITATION SPECTRA IN NATURAL SAMPLES OF PHYTOPLANKTON

The fluorescence excitation spectrum of live phytoplankton cells represents the portion of light absorbed that has been effectively transferred to chlorophyll a of photosystem II, whereas light absorbed by photoprotective pigments will not lead to fluorescence. Therefore, the in vivo fluorescence excitation spectrum of phytoplankton has been used as a proxy for the action spectrum of phytoplankton in computations of primary production in the ocean. The distribution of chlorophyll a between photosystems, as well as variations in the pathway of energy inside the photosynthetic membrane, can also influence the fluorescence excitation spectrum. In this study, we investigated the contribution of photoprotective pigments to the differences found between in vivo absorption and fluorescence excitation spectra of phytoplankton measured during two cruises: one from Las Islas Canarias to Nova Scotia and another in the Labrador Sea. A comparison of normalized fluorescence excitation and absorption spectra showed high variability in the difference between absorption and fluorescence in the blue region of the spectrum for samples from the two cruises. This difference was not entirely correlated with the concentration of photoprotective carotenoids. In this paper, results are interpreted in terms of differences in pigment composition and known patterns of energy distribution in the photosystems of different algal groups.     

Phytoplankton pigments and community composition in Lake Tanganyika

1. A 2‐year (2002–2003) survey of chlorophyll and carotenoid pigments is reported for two off‐shore stations of Lake Tanganyika, Kigoma (Tanzania) and Mpulungu (Zambia), and from three cruises between those sites. Chlorophyll a concentrations were low (0.3–3.4 mg m^?3) and average chlorophyll a integrated through the 100 m water column were similar for both stations and years (36.4–41.3 mg m^?2). Most pigments were located in the 0–60 m layer and decreased sharply downward. Chlorophyll a degradation products (phaeophytins and phaeophorbides) were detected at 100 m depth, whereas carotenoids became undetectable. Temporal and seasonal variation of the vertical distribution of pigments was high. 2. The biomass of phytoplankton groups was calculated from marker pigment concentrations over the 0–100 m water column using the CHEMTAX software. On average for the study period, chlorophytes dominated in the northern station, followed by cyanobacteria T1 (type 1, or Synechococcus pigment type), whereas cyanobacteria T1 dominated in the south. Cyanobacteria T2 (type 2, containing echinenone), presumably corresponding to filamentous taxa, were detected in the rainy season. Diatoms (and chrysophytes) developed better in the dry season conditions, with a deep mixed layer and increased nutrient availability. Very large variation in the vertical distribution of algal groups was observed. 3. Our observations on phytoplankton composition are broadly consistent with those from previous studies. Our pigment data provide evidence for the lake‐wide importance of picocyanobacteria and high interannual variation and spatial heterogeneity of phytoplankton in Lake Tanganyika, which may render difficult assessment of long‐term changes in phytoplankton driven by climate change.