Determination of chlorophylls and carotenoids of marine phytoplankton: separation of chlorophyll a from divinylchlorophyll a and zeaxanthin from lutein

A rapid reverse-phase HPLC method is presented for the identificationand quantification of most of the phytoplankton pigments. Thismethod yields the resolution of divinyl-chlorophyll a and chlorophylla, as well as the partial resolution of lutein and zeaxanthin,and of divinyl-chlorophyll b and chlorophyll b. In addition,chlorophylls c_1,2 and c₃ are well resolved. The analysis timefor one sample is 20 mm, which makes this method particularlysuited when large numbers of samples have to be processed.     

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.     

Characterization of genes encoding the light-harvesting proteins in diatoms: biogenesis of the fucoxanthin chlorophylla/c protein complex

In chromophytic algae the major light-harvesting complex is the fucoxanthin chlorophylla/c protein complex. Recently, we have cloned several highly related cDNA and genomic sequences encoding the fucoxanthin chlorophylla/c proteins from the diatomPhaeodactylum tricornutum. These genes are clustered on the nuclear genome. The sequences of the fucoxanthin chlorophylla/c proteins as deduced from the gene sequences have some similarity to the chlorophylla/b proteins associated with light-harvesting complexes of higher plants and green algae. Like the chlorophylla/b proteins of higher plants, the fucoxanthin chlorophylla/c proteins are synthesized as higher-molecular weight precursors in the cytoplasm of the cell and are transported into the plastids. However, the mode of transport into diatom plastids is very different from the mechanism involved in transporting proteins into the chloroplasts of higher plants and green algae. We focus here on the characteristics of the fucoxanthin chlorophylla/c proteins, the mode of transport of these proteins into plastids, the arrangement of the genes encoding these proteins, and efforts to utilize these genes to develop a DNA transformation system for diatoms.     

In vivo fluorescence excitation and absorption spectra of marine phytoplankton: I. Taxonomic characteristics and responses to photoadaptation

Absorption and fluorescence excitation spectra were measuredfor batch cultures of five species of marine phytoplankton grownunder high and low light. These spectra were examined for propertiescharacteristic of taxonomic position and of photoadaptive response.While regions of absorption and excitation of chlorophyll afluorescence diagnostic of pigment composition were identifiable,photoadaptive response had greater influence on spectral variability.Although reduced growth irradiance caused changes in both theabsorption and fluorescence excitation spectra, the fluorescenceexcitation spectrum appears to be more sensitive to alterationsin the ambient light field for growth than does the absorptionspectrum. For a single species. the fluorescence excitationspectrum for a sample grown at low irradiance showed greaterstructure than that for the sample grown at a high irradiance.Under low light conditions, the excitation of chlorophyll afluorescence by accessory pigments increased relative to theexcitation by chlorophyll a itself The highest fluorescenceyields occur in the blue-green region of the spectrum, correspondingto bands of peak absorption by the accessory pigments. Changesin absorption spectra are less marked, but two features recur.First. in the blue-green region of the spectrum from -500–560nm. absorption is enhanced in the low-light cells relative tothat of the high-light cells. Second, the ratio of absorptionat 435 nm to that at 676 nm was greater for the high-light cells.Correlating changes in pigment concentrations were observed.The influence of photoadaptation on the properties of fluorescenceexcitation spectra is as great or greater than the influenceof pigment complements characteristic of specific algal taxa.     

Immunological Quantification of Proteins Related to Light-Harvesting Chlorophyll a/b Protein Complexes of the Two Photosystems in Rice Mutants Totally and Partially Deficient in Chlorophyll b

Ten rice chlorina mutants of Type I, which totally lack chlorophyllb and hence are unable to synthesize light-harvesting chlorophylla/b protein complexes of photosystem II (LHC-II), containedmRNA for proteins related to LHC-II. Immunoblotting with anantiserum, which had been raised against the 24 and 25 kDa apoproteinsof LHC-II and found to cross-react with the 26 kDa protein ofLHC-II and the 20 and 21 kDa apoproteins of light-harvestingchlorophyll a/b protein complexes of photosystem I (LHC-I),revealed that all the five proteins related to LHC-Iand LHC-IIwere present in reduced amounts in the Type I mutants. ThreeType HA mutants, which have a chlorophyll a/b ratio of 10, weremore abundant in the apoproteins, while three Type IIB mutantswith the ratio of 15 were heterogeneous in terms of the apoproteincontent. All the chlorina mutants contained less P700 comparedwith the wild type rice, but were relatively more abundant inthe LHC-I proteins than the LHC-II proteins. The results showthat all the rice chlorina strains are mutants of chlorophyllb synthesis and the deficiency of chlorophyll b differentlyaffects accumulation of the apoproteins of LHC-I and LHC-II.To balance light absorption between the two photosystem, lossof LHC-II is partly counter-balanced by a decrease in the numberof PSI complexes in the mutants. (Received January 21, 1988; Accepted April 28, 1988)     

HPLC analysis of phytoplankton pigments from Lake Kinneret with special reference to the bloom-forming dinoflagellate Peridinium gatunense (Dinophyceae) and chlorophyll degradatio products

Photosynthetic pigments extracted from the paniculate materialof the water column of Lake Kinneret were studied throughoutthe periods of May 1988-June 1989, and November 1993-November1994, by means of HPLC. The temporal and vertical variationof the pigment suite found agreed with the microscopically determinedphytoplankton record. The regression calculations of taxon-specificbiomass with the corresponding signature pigments suggest thatpigment analysis may be a useful tool for the monitoring ofbloom-forming species, e.g. the dinoflagellate Peridinium gatunenseNygaard. The HPLC pigment analysis permitted the identificationand quantification of chlorophyll degradation products, providingfor the first time information about their composition in LakeKinneret. Chlorophyllide a was the major detectable degradationproduct of chlorophyll a, varying between 1 and 9% of the chlorophylla concentration. Other chlorophyll a derivatives appeared mostlyin minor quantities. Pheophytin a was virtually lacking in allthe samples. Removal rates of pigments, measured by sedimentationtraps, indicated that the degradation of chlorophyll a via chlorophyllidea is a dynamic process that continues during the sedimentationof the phytoplankton particles.     

BENTHIC AND PLANKTONIC ALGAL COMMUNITIES IN A HIGH ARCTIC LAKE: PIGMENT STRUCTURE AND CONTRASTING RESPONSES TO NUTRIENT ENRICHMENT1

We investigated the fine pigment structure and composition of phytoplankton and benthic cyanobacterial mats in Ward Hunt Lake at the northern limit of High Arctic Canada and the responses of these two communities to in situ nutrient enrichment. The HPLC analyses showed that more than 98% of the total pigment stocks occurred in the benthos. The phytoplankton contained Chrysophyceae, low concentrations of other protists and Cyanobacteria (notably picocyanobacteria), and the accessory pigments chl c₂, fucoxanthin, diadinoxanthin, violaxanthin, and zeaxanthin. The benthic community contained the accessory pigments chl b, chl c₂, and a set of carotenoids dominated by glycosidic xanthophylls, characteristic of filamentous cyanobacteria. The black surface layer of the mats was rich in the UV‐screening compounds scytonemin, red scytonemin‐like, and mycosporine‐like amino acids, and the blue‐green basal stratum contained high concentrations of light‐harvesting pigments. In a first bioassay of the benthic mats, there was no significant photosynthetic or growth response to inorganic carbon or full nutrient enrichment over 15 days. This bioassay was repeated with increased replication and HPLC analysis in a subsequent season, and the results confirmed the lack of significant response to added nutrients. In contrast, the phytoplankton in samples from the overlying water column responded strongly to enrichment, and chl a biomass increased by a factor of 19.2 over 2 weeks. These results underscore the divergent ecophysiology of benthic versus planktonic communities in extreme latitudes and show that cold lake ecosystems can be dominated by benthic phototrophs that are nutrient sufficient despite their ultraoligotrophic overlying waters.     

Photosynthetic efficiency of marine plants

Multicellular marine plants were collected from their natural habitats and the quantum efficiency of their photosynthesis was determined in the laboratory in five narrow wave length bands in the visible spectrum. The results along with estimates of the relative absorption by the various plastid pigments show a fairly uniform efficiency of 0.08 molecules O₂ per absorbed quantum for (a) chlorophyll of one flowering plant, green algae, and brown algae, (b) fucoxanthol and other carotenoids of brown algae, and (c) the phycobilin pigments phycocyanin and phycoerythrin of red algae. The carotenoids of green algae are sometimes less efficient while those of red algae are largely or entirely inactive. Chlorophyll a of red algae is about one-half as efficient (_o2 = 0.04) as either the phycobilins, or the chlorophyll of most other plants. These results as well as those of high intensity and of fluorescence experiments are consistent with a mechanism in which about half the chlorophyll is inactive while the other half is fully active and is an intermediate in phycoerythrin- and phycocyanin-sensitized photosynthesis.     

Pigment patterns in suspended matter from the Elbe estuary,Northern Germany

In the Elbe hardly anything is known about the actual fate of phytoplankton and the resultant pigment composition of suspended matter. As part of a longterm study on the role of suspended matter in the cycles of nitrogen and associated oxygen consumption processes in the Elbe estuary in northern Germany (‘Sonderforschungsbereich 327 Elbe’ project) and the characterization of estuarine and coastal water bodies with regard to the transport of pollutants such as heavy metals, we have started to characterize suspended matter with regard to the pigments present therein. This will allow us to study phytoplankton turnover and to determine the role of phytoplankton decay mechanisms in the mobilization, binding, and transport of pollutants associated with particulate matter. Our first data on the pigment composition of suspended matter from the Elbe and particularly over the region of Hamburg harbour, obtained using a High Performance Liquid Chromatography (HPLC) method, is presented. The pigment concentrations varied considerably over the summer months. Values up to 250 mg l⁻¹ of chlorophyll a were observed upstream of Hamburg. We found that on its course through Hamburg the concentrations (μg g⁻¹ of particulate matter) of all pigments in the Elbe drop to under half those found upstream of Hamburg. The pigment concentrations in the turbidity maximum were significantly lower than in the rest of the estuary due to the decline of algae as a result of inadequate light conditions and the salinity gradient. The highest particulate matter concentrations were found in the turbidity maximum and just downstream of Hamburg. The highest particulate organic carbon values were found just downstream of Hamburg. The presence of of fucoxanthin and the chlorophyllsc andb, and the changes in pigment patterns were indicative for the dominant algal classes and the phytoplankton succession in the suspended matter. The ratio of chlorophylla to lutein was found to be a possible indicator of phytoplankton breakdown.     

In situ quantum yield of phytoplankton in a subalpine lake

The percent of light absorbed by algae, estimated from a regressionof attenuation of downwelling photosynthetic photon flux densityon chlorophyll a concentration, was used in conjunction withmeasurements of phytoplankton photosynthesis to estimate quantumyields () of Castle Lake phytoplankton populations during the1980 ice-free season. The maximum values occurred during mid-summerin the deep hypolimnion (<1% of surface photon flux) andwere {small tilde} 78% of the theoretical maximum. However,later in the season (September) the deep-water values decreasedsignificantly to {small tilde}27% of the theoretical maximumin conjunction with a decrease in quantum flux density to thesewaters. The values for epilimnetic phytoplankton populationswere never >15% of the theoretical maximum. The presenceof relatively high concentrations of peridinin and chlorophyllc in the deep-water algae enabled these organisms to absorbthe predominantly green light in this region of the lake which(i) allowed the use of a single extinction value per unit ofchlorophyll (k_c) at all depths when calculating , and (ii) increasedthe of the deep-water algae, relative to those in the near-surfacewaters. These data indicate that the deep-water algae in CastleLake were able to utilize their ambient light field, particularlyin the early summer, and suggest that the deep-chlorophyll layerin this lake is maintained primarily by in situ algal growthrather than sinking and accumulation of organisms from nearsurface waters.     

The presence of chlorophyll b and the estimation of phaeopigments in marine phytoplankton

A reverse-phase h.p.l.c. technique was used to estimate theconcentration of chlorophyll b in phytoplankton cultures, fecalpellets of Calanus pacificus, and suspended paniculate matterfrom the Central North Pacific, Oregon coastal waters, and DabobBay (a temperate fjord in Puget Sound, WA, USA). The purposewas to assess the distribution of this pigment in the euphoticzone and its effect on the fluorometnc estimation of phaeopigments.Analyses of natural waters confirm high chlorophyll b concentrations(median mass ratio of b:a > 0.3) at the depth of the chlorophylla maximum in tropical waters while values for temperate planktonare relatively low (median mass ratio of chl b:a = 0.05) andpatchy. Zooplankton fecal pellets showed a significant enrichmentin chlorophyll b, suggesting grazing as a mechanism to explainhigh concentrations of this pigment at the bottom of the euphoticzone. It is estimated that the presence of chlorophyll b couldcause an average overestimation of phaeopigment concentrationby the fluorometnc technique of 38% between 0 and 200 m in theCentral North Pacific. This effect is more pronounced at thelayer of chlorophyll b maximum (120–140 m). ¹Present address: Marine Biology Research Division, A-002, ScrippsInstitution of Oceanography, La Jolla, CA 92093, USA     

CHLOROPHYLL PIGMENTS IN METHANOL EXTRACTS FROM TEN AXENIC CULTURED DIATOMS AND THREE GREEN ALGAE AS DETERMINED BY REVERSE PHASE HPLC WITH FLUOROMETRIC DETECTION 1

Chlorophyll pigments in 100% mrthanol extracts from ten axenic cultured diatoms and three green algae were examined using reverse phase HPLC with Jluoromelric detector and field desorption mass spectrometry to identify same unknown peaks tn HPLC chromatograms from natural phytoplankton samples. Characteristics of the HPLC chromatograms were the presence of chlorophyllide and two unknown peaks. Field desorption mass spectrometry of the oxidation products of authentic chlorophyll a suggested that the peaks were due to 10-hydroxy and 10-hydroxy lactone chlorophylls a and 10-methoxy and 10-methoxy lactone chlorophylls a fanned during each process of filtration, storage at -20°C and extraction in methanol. The present results indicated that a new type of chlorophyll c derivative may exist and, as far as we use filtration to collect phytoplankton and store the sample under air even at -20°C, the formation of oxidation products of chlorophyll pigments may he unavoidable.     

Phytoplankton community and succession in a newly man-made shallow lake, Shanghai, China

We studied phytoplankton community and succession in Lake Dishui, the largest man-made coastal lake in China. The lake experienced drastic changes in physicochemical conditions since its creation in 2003. Monthly phytoplankton communities were characterized between 2006 and 2011. A two-dimensional solution of nonmetric multidimensional scaling clearly delineated four groups of distinct phytoplankton community structure. Indicator species analysis showed that Group I (2006–2008) was characterized by mainly nanoplankton including Chromulina pygmaea. Group II (2009) was characterized by nonmotile, unicellular, elongated, or filamentous taxa, which are resistant to grazing pressure due to their large size. Group III (winter–early spring since 2010) was characterized by many motile taxa. The most characteristic taxa in Group IV (summer–autumn since 2010) were the flagellate algae. Seasonal variation in phytoplankton community was highest after 2010. Changes in phytoplankton communities may closely reflect rapid changes in lake environmental conditions such as desalination and nutrient enrichment.     

High performance liquid chromatography detection of phototrophic bacterial pigments in aquatic environments

Pigment extracts of phototrophic bacteria isolated from Lake Kinneret (Rhodopseudomonas palustris, Thiocapsa roseopersicina, Prosthecochloris aestuaris andChlorobium phaeobacteroides) were studied by means of high performance liquid chromatography (HPLC). An absorption wavelength of 360 nm provided the best resolution among the pigments of the species tested and between them and chlorophylla. Signature pigments were identified for each of these species, and their presence was thereby monitored in lake water samples.C. phaeobacteroides, which was observed in the anaerobic hypolimnion and predominated in the metalimnion, was recognized by a characteristic cluster of major chlorophyllous pigment peaks. The spectral qualities of these pigments were close but not identical to published data on bacteriochlorophylle, presumably due to the use of different solvents for extraction. The intensity of these pigment peaks was employed to determine the depth of the greatest phototrophic bacterial biomass, which was not related to that of algae.     

Properties of phycobilins from Porphyra naiadum

The phycobilin pigments were freshly extracted from Porphyra naiadum in the cold. At least two types of phycoerythrin (I and II) can be distinguished by electrophoresis, chromatography, and spectral characteristics. At pH 5.0 phycoerythrin II has a relatively large negative charge, while phycoerythrin I is nearly iso-electric. At pH 7.0, however, phycoerythrin I has the larger negative charge. Mobilities have been calculated by visual measurement of electrophoresis. Phycoerythrin II can be converted to phycoerythrin I by storing at pH 7.0. Chromatography indicates at least two types of phycocyanin as well.     

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.     

Freeze fracture immunocytochemistry of light-harvesting pigment complexes in a cryptophyte

Summary Immunocytochemical techniques using colloidal gold as the marker have been used to examine the location of the two light harvesting pigment-protein complexes in cryptophyte chloroplasts. A comparison of post-embedding thin section labelling and freeze fracture labelling has been carried out onRhodomonas salina using polyclonal antibodies to a chlorophylla/c ₂ light-harvesting complex, phycoerythrin and the -subunit of phycoerythrin. The effect of different fixation procedures on the intensity of labelling and ac curacy of antigen location have been examined and the effectiveness of uranyl acetate and tannic acid in improving both the preservation of thylakoid structure and labelling density of phycoerythrin has been demonstrated. Freeze fracture labelling gives better spatial res olution of the different antigens than post-embedding labelling, as well as better definition of thylakoid membranes. It confirms the location of phycoerythrin in the thylakoid lumen and the location of the chlorophylla/c ₂ LHC in both appressed and unappressed thylakoid membranes.Abbreviations PE phycoerythrin - chl chlorophyll - LHC light-har-vesting complex     

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.     

Photosynthetic action spectra of marine algae

A polarographic oxygen determination, with tissue in direct contact with a stationary platinum electrode, has been used to measure the photosynthetic response of marine algae. These were exposed to monochromatic light, of equal energy, at some 35 points through the visible spectrum (derived from a monochromator). Ulva and Monostroma (green algae) show action spectra which correspond very closely to their absorption spectra. Coilodesme (a brown alga) shows almost as good correspondence, including the spectral region absorbed by the carotenoid, fucoxanthin. In green and brown algae, light absorbed by both chlorophyll and carotenoids seems photosynthetically effective, although some inactive absorption by carotenoids is indicated. Action spectra for a wide variety of red algae, however, show marked deviations from their corresponding absorption spectra. The photosynthetic rates are high in the spectral regions absorbed by the water-soluble "phycobilin" pigments (phycoerythrin and phycocyanin), while the light absorbed by chlorophyll and carotenoids is poorly utilized for oxygen production. In red algae containing chiefly phycoerythrin, the action spectrum closely resembles that of the water-extracted pigment, with peaks corresponding to its absorption maxima (495, 540, and 565 mµ). Such algae include Delesseria, Schizymenia, and Porphyrella. In the genus Porphyra, there is a series P. nereocystis, P. naiadum, and P. perforata, with increasingly more phycocyanin and less phycoerythrin: the action spectra reflect this, with increasing activity in the orange-red region (600 to 640 mµ) where phycocyanin absorbs. In all these red algae, photosynthesis is almost minimal at 435 mµ and 675 mµ, where chlorophyll shows maximum absorption. Although the chlorophylls (and carotenoids) are present in quantities comparable to the green algae, their function is apparently not that of a primary light absorber; this role is taken over by the phycobilins. In this respect the red algae (Rhodophyta) appear unique among photosynthetic plants.     

Diel activity of Gammarus pulex (Crustacea) in a South Swedish stream: comparison of drift catches vs baited traps

The benthos of a perennially ice-covered Antarctic lake, Lake Hoare, contained three distinct signatures of lipophilic pigments. Cyanobacterial mats found in the moat at the periphery of the lake were dominated by the carotenoid myxoxanthophyll; carotenoids: chlorophyll a ratios in this high light environment ranged from 3 to 6.8. Chlorophyll c and fucoxanthin, pigments typical of golden-brown algae, were found at 10 to 20 m depths where the benthos is aerobic. Anaerobic benthic sediments at 20 to 30 m depths were characterized by a third pigment signature dominated by a carotenoid, tentatively identified as alloxanthin from planktonic cryptomonads, and by phaeophytin b from senescent green algae. Pigments were not found associated with alternating organic and sediment layers. As microzooplankton grazers are absent from this closed system and transformation rates are reduced at low temperatures, the benthos beneath the lake ice appears to contain a record of past phytoplankton blooms undergoing decay.