Manuel Zapata

SEPARATION OF CHLOROPHYLLS c1c2, AND c3 OF MARINE PHYTOPLANKTON BY REVERSED-PHASE-C18-HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY1

We separated chlorophylls c1 c2, and c3 of marine phytoplankton together with other pigments by a modification of the commonly applied reversed‐phase‐C18‐high‐performance liquid chromatography (RP‐C18‐HPLC) method. However, the chlorophyll c‐like pigment 2,4, Mg‐divinylpheoporphyrin as monomethyl ester, co‐eluted with chlorophyll c1. The method involves optimization of the mobile phase by using a very high ion strength solvent in combination with a high carbon loaded RP‐C18 column. Fingerprints of the various taxonomic groups of algae can thus be developed in a single run, including separation of the carotenoids lutein and zeaxanthin.

Separation of chlorophylls and their degradation products in marine phytoplankton by reversed-phase high-performance liquid chromatography

SummaryAn RP-HPLC procedure which separates chlorophylls and their degradation products is described. By employing an amonium acetate buffered mobile phase and a linear gradient elution, complex mixtures of chloroand phaeopigments are separated in less than 30 minutes. The method has been applied to the control of chlorophyll enzymatic degradation in cultures of two algal species, and has also been successfully used in the analysis of algal xanthophylls.

Characterization of NW Mediterranean Karlodinium spp. (Dinophyceae) strains using morphological, molecular, chemical, and physiological methodologies

Recurrent fish kills in the Spanish Alfacs Bay (NW Mediterranean) have been detected during winter seasons since 1994, and were attributed to an unarmored, ichthyotoxic, dinoflagellate, initially identified as Gyrodinium corsicum Paulmier, Berland, Billard, & Nezan. Several strains were isolated from the bay and their clonal cultures were compared by combined techniques, including light and electron microscopy, internal transcribed spacer and 5.8S rDNA nucleotide sequencing, and HPLC pigment analyses, together with studies of their photochemical performance, growth rates, and toxicity. Using phylogenetic analyses, all strains were identified as members of the genus Karlodinium, but they were separated into two genetically distinct groups. These groups, identified as Karlodinium veneficum (Ballantine) J. Larsen and K. armiger Bergholtz, Daugbjerg et. Moestrup, were also supported by the other techniques used. Detailed analyses of fine structural characteristics (including plug‐like structures in amphiesma and a possible layer of semi‐opaque material beneath the outer membrane) allowed discrimination of the mentioned two species. Specific differences in pigment patterns coincided with that expected for low‐ (K. veneficum) and high‐light (K. armiger) adapted relatives. The higher photosynthetic efficiency of K. veneficum and the longer reactivation times of the PSII reaction centers observed for K. armiger were in agreement with this hypothesis. The two species differed in toxicity, but the strains used always induced mortality when incubated with bivalves, rotifers, and finfish. Compared with K. armiger, strains of K. veneficum yielded higher cell densities, but had lower growth rates.

Losses of chlorophylls and carotenoids in aqueous acetone and methanol extracts prepared for RPHPLC analysis of pigments

SummaryRPHPLC methods for analysis of photosynthetic pigments (chlorophylls and carotenoids) usually require addition of water to methanol or acetone extracts to prevent distortion of early-eluting peaks corresponding to the more polar compounds. In this work we have investigated the short-(<2 min) and long-term (up to 48 h) effect of adding water to acetone and methanol extracts from two marine phytoplankton species,Emiliania hyxleyi andDunaliella tertiolecta. Solvent extracts were prepared and separated into fractions that were subsequently diluted with water to 90%, 80%, 70%, 60%, 50%, and 40% for methanol, and the same range extended to 30% and 20% for acetone. Changes in pigment concentration with time were followed spectrophotometrically and chromatographically. Losses of pigments as a result of precipitation were clearly observed immediately after dilution of acetone extracts to 60% or less and methanol extracts to 80% or less. For chlorophyll a the most substantial losses were recorded for 50% acetone (up to 27% decrease) and for 70% methanol (31% decrease). This effect increased considerably with time. Only for 90% and 80% acetone were the initial concentrations of all the pigments unchanged after 24h, and even up to 48 h. In contrast, more than 60% and 57% of the initial amounts of chlorophyll a were lost after 24 h in 50% acetone and 70% methanol extracts, respectively. These losses increased to 83% and 60% after 48 h. There was a clear correlation between the polarity of a pigment and the polarity of the solvent at which maximum precipitation occurred. Losses of pigment from pure acetone and methanol extracts with time were also observed, although we attribute these to pigment degradation rather than precipitation. Some of the losses occurring with time can be avoided by use of autosamplers in which the sample can be mixed with water immediately before injection.

THE MAIN NONPOLAR CHLOROPHYLL c FROM EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) IS A CHLOROPHYLL c 2-MONOGALACTOSYLDIACYLGLYCERIDE ESTER: A MASS SPECTROMETRY STUDY

The main nonpolar chlorophyll c ‐like pigment was extracted from Emiliania huxleyi (Lohm.) Hay et Mohler (strain CCMP 370) cultures and isolated by preparative column chromatography and HPLC. The pigment, whose visible spectrum closely resembled that of chlorophyll c 2, was studied by low‐resolution fast atom bombardment mass spectrometry, showing a very high mass molecular ion (m/z 1313). The fragment ions, either in the direct spectrum or obtained by tandem mass spectrometry with collision‐induced dissociation of the molecular ion, were compatible with the consecutive losses of two fatty acids (14:0 and 18:4), glycerol, and a hexose, leaving a chlorophyll c 2 backbone, suggesting the molecule consists of a chlorophyll c 2 residue linked, via an ester bond, to the sugar moiety of a monohexosyldiacylglycerol. The identities of the two fatty acid residues (14:0 and 18:4n‐3) were subsequently corroborated by gas chromatography of the corresponding methyl esters. Chemical hydrolysis–derivatization–gas chromatography–mass spectrometry demonstrated the occurrence of glycerol and that galactose is the constituent sugar. The porphyrin obtained on acid hydrolysis showed chromatographic and visible spectral properties identical to pheoporphyrin c 2. This evidence led us to propose a tentative structure whose molecular formula, C76H96O14N4Mg, was supported by the values of exact mass measurements by high‐resolution fast atom bombardment mass spectrometry. This novel structure represents the highest molecular weight natural chlorophyll described to date.

COOLIA CANARIENSIS SP. NOV. (DINOPHYCEAE), A NEW NONTOXIC EPIPHYTIC BENTHIC DINOFLAGELLATE FROM THE CANARY ISLANDS

A new photosynthetic dinoflagellate species, Coolia canariensis S. Fraga sp. nov., is described based on samples taken from tidal ponds on the rocky shore of the Canary Islands, northeast Atlantic Ocean. Its morphology was studied by LM and SEM. It is almost spherical and has a thick smooth theca with many scattered pores. Plate 1′ is the biggest of the epithecal plates, and 7″ is twice as wide as it is long. Phylogeny inferred from the D1/D2 regions of the LSU nuclear rDNA of three strains of C. canariensis and several strains of other Coolia species, C. monotis, C. sp., showed that C. canariensis strains clustered in a well‐supported clade distinct from the other species. No toxins were detected using mouse bioassay, liquid chromatography with Fluorescence detection (LC‐FLD) or liquid chromatography‐mass spectrometry (LC‐MS). Its pigment composition is of the peridinin type of dinoflagellates. Together with this new species, many other strains of C. monotis from the Atlantic Ocean and Mediterranean Sea have been analyzed for toxin presence, and no evidence of toxin production related to yessotoxins (YTXs) was found, as was previously suggested for C. monotis from Australia.

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 c3, 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.

Changes in fatty acids, amino acids and carbon/nitrogen biomass during nitrogen starvation of ammonium- and nitrate-grown Isochrysis galbana

Growth of cells ofIsochrysis galbana with either nitrate or ammonium as the N-source, and the effects of subsequent N-starvation of these cells, were compared. During exponential N-sufficient growth nitrate-grown cells had double the fatty acid content of the ammonium-grown cells but lower concentrations of a few amino acids. Following resuspension in N-free medium the fatty acid content of the ammonium-grown cells increased to that of the nitrate-grown cells, but there was no further increase in fatty acid content on a C-biomass or cellular basis during the following 4 days for either culture. Fatty acid synthesis was continuous during N-starvation, while it occurred during the light-phase only in exponential growth. The proportion of 18:1n9 fatty acid increased from 10 to 25% total fatty acids during N-starvation. Intracellular free amino acid content decreased in a similar manner in both cultures on N-starvation, the ratio of intracellular free amino-N/cell-C falling more rapidly than overall cellular N/C. It was concluded that optimal amino acid and fatty acid content would be attained by growth in the presence of excess nitrate. Measurements of chlorophyll and carotenoid content and ofin vivo fluorescence indicated that these parameters had potential for monitoring the C and N biomass in cultures grown under relatively constant (not necessarily continuous) illumination.

Carotenoids from further prasinophytes

Abstract The qualitative and quantitative carotenoid composition of seven prasinophytes (eight clones) have been examined by chromatographic (TLC and HPLC) and spectroscopic methods (VIS, CD and mass spectra). The prasinophytes studied fall into two pigment types: (A) those producing common green algal carotenoids (β,β-carotene, β,ϵ-carotene, lutein, zeaxanthin and the epoxides violaxanthin and neoxanthin) and (B) prasinophytes synthesising carotenoids peculiar to this algal class (prasinoxanthin, anhydroprasinoxanthin, uriolide, anhydrouriolide, micromonal, anhydromicromonal, micromonol, anhydromicromonol and dihydrolutein), where prasinoxanthin is a major carotenoid. Mantoniella squamata (clone 2) was grown under both low and high light intensity, revealing differences in carotenoid composition. Lutein together with lesser amounts of zeaxanthin and its epoxides were only detected at high light intensity. Three previously unidentified carotenoids were identified as prasinoxanthin (xanthophyll K), micromonal and dihydrolutein.

SPECTRAL CHARACTERIZATION OF NEW CHLOROPHYLL C PIGMENTS ISOLATED FROM EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) BY HIGH‐PERFORMANCE LIQUID CHROMATOGRAPHY1

Pigment extracts from the prymnesiophyte Emiliania huxleyi (Lohm.) Hay & Mohler (CCMP 370) were analyzed using a new chromatographic method based on the use of polymeric octadecylsilica columns, which split both the chlorophyll c3 peak and phytol‐substituted chlorophyll c‐like peak into two fractions. The pigments corresponding to each of these fractions were isolated. The spectra and chromatographic behavior suggest that chlorophyll c3 from E. huxleyi is a mixture of two different compounds whereas the splitting of the phytol‐substituted c‐like pigment could be the result of the equilibrium of two forms showing the same absorption spectrum. The method allowed, as well, the detection of magnesium 3,8‐divinylpheoporphy‐rin a, monomethyl ester in the prymnesiophytes E. huxleyi (CCMP 370) and Prymnesium parvum Carter (CCMP 708) and the chrysophyte Pelagococcus subviridis Norris (CCMP 1429).