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.     

Grazing experiments with two freshwater zooplankters:fate of chlorophyll and carotenoid pigments

In order to evaluate the validity of the gut pigment methodto assess grazing and diet in two freshwater zooplankters, experimentswere carried out to check chlorophyll a and xanthophyll conservationduring feeding. For both animals, two sets of experiments wereconducted by incubating animals in the laboratory, either isolatedfrom a reservoir (the calanoid copepod, Eudiaptomus gracilis)or cultured under high-food conditions (the cladoceran, Daphniagaleata). For both animals, gut pigments and clearance rateson different types of algae were determined from the same incubations.Chlorophyll a and derivatives, as well as major algal carotenoids,were analysed by High Performance Liquid Chromatography (HPLC).In copepods, the pigment profiles from the gut extracts reflectedthe diet of the animals poorly. The animal extracts containedalmost exclusively alloxanthin (or an alloxanthin-like pigment)in large amounts, whereas the other pigments were lost in highproportions (>70% for lutein and fucoxanthin; 57 and 78%for a-phorbins). The cladocerans fed on the main types of algaeabundant in the suspensions, with a preference, however, forsmall cells. Although the main xanthophylls from these algaewere detected in the Daphnia extracts, some destruction of luteinand fucoxanthin may have occurred (18.7 and 30%). The loss ratefor alloxanthin seemed more variable (0 and 68%), possibly dependingon food concentration. As for the transformation of a-phorbins,E.gracilis and D.galeata behaved quite differently. The HPLCprofiles of copepod extracts always showed a very small chlorophylla peak, along with phaeophytin a and pyrophaeophytin a. Thosefrom the cladoceran exhibited a large phaeophorbide a peak,along with some chlorophyll a and phaeophytin a. In fact, D.galeatadid not destroy a-phorbins under our experimental conditionsbut converted chlorophyll a mainly into phaeophorbide. Froma comparison of our results with data from other studies, itseems that in these two zooplankters, use of gut pigment datafor quantitative grazing assessment should be considered withcaution.     

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.     

Chlorophyll analysis by high-performance liquid chromatography

The separation and determination of chlorophylls by high-performance liquid chromatography (HPLC) is described. Chlorophylls and their derivatives were separated by reversed-phase HPLC based on hydrophobic interaction between solute and support, using an octadecyl silica column and elution with 100% methanol. Separated pigments were detected fluorometrically with a sensitivity in the picomole range: the fluorescence response was linear over a wide pigment concentration range. Resolution of five chlorophylls a and four protochlorophyll species esterified with different alcohols was achieved within 22 min in a single experiment. This method can be used for the determination of chlorophyll b, bacteriochlorophyll a esters and products synthesized from chlorophyll, but not for nonesterified pigments, i.e., chlorophyllide, protochlorophyllide and chlorophyll c. The chromatographic mobility of chlorophyll a esterified with different alcohols increases with increasing number of carbon atoms in the esterifying alcohols. The plots obtained from the logarithm of the capacity factor (k′) of these pigments versus the numbers of carbon atoms of the alcohol molecule gave a straight line, thus permitting the estimation of the chain length of unknown pigment esterifying alcohols. This HPLC separation technique did not cause the formation of artifacts. The deviation of the individual retention time for each pigment is less than ±0.5%, thus making this method suitable for the rapid identification and quantification of unknown pigments.     

Pigment Composition of a Novel Oxygenic Photosynthetic Prokaryote Containing Chlorophyll d as the Major Chlorophyll

The principal pigment found in the majority of oxygenic photosyntheticorganisms is known to be chlorophyll a. However, we isolateda new oxygenic photosynthetic prokaryote that contained chlorophylld as a predominant pigment with chlorophyll a being a minorpigment. Chlorophyll d had previously been noted but its naturaloccurrence and function remained unclear. Cells of the new prokaryotehad an absorption maximum at red region of 714–718 nmdue to chlorophyll d absorption, but no characteristic absorptionpeak of chlorophyll a around 680 nm was observed. Chlorophylld of the new organism was identified spectrophotometricallyin several solvents and its chemical structure was confirmedby NMR and FABMS analysis. The cell also contained a chlorophyllc-like pigment, zeaxanthin and a-carotene but not chlorophyllb and ß-carotene. The content of chlorophyll d accountedfor more than 2% of the cell dry weight, while the content ofchlorophyll a was less than 0.1%. The chlorophyll a/d ratioremained between 0.03 and 0.09 under different culture conditions.The light absorption characteristics and the high content ofchlorophyll d along with the small content of chlorophyll aindicated the existence of a new light utilization mechanisminvolving chlorophyll d. (Received October 7, 1996; Accepted December 16, 1996)     

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.     

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.     

Study of selective feeding in the marine cladoceran Penilia avirostris by HPLC pigment analysis

Food selection by the marine cladoceran Penilia avirostris was studied in the field by HPLC analysis of phytoplankton marker pigments and in the laboratory by microscopic measurement of cell removal. Comparison between pigment composition in natural phytoplankton and in P. avirostris showed that P. avirostris preferred diatoms, cryptophytes and chlorophytes, and ignored prymnesiophytes and dinoflagellates. Peridinin, the marker pigment for dinoflagellates was found in P. avirostris only when the dinoflagellate populations were dominated by Prorocentrum. Pigment degradation rates ranged from 13.73% for alloxanthin to 36.62% for chlorophyll a. Clearance rates measured in the laboratory provided further evidence of strong preference for diatoms and cryptophytes, and avoidance of dinoflagellates. Microscopic counts suggested that P. avirostris was feeding on prymnesiophytes, although ingestion of prymnesiophytes could not be confirmed by pigment analysis.     

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.     

Alterations in Pigment Content in Leaves of Pisum sativum After Exposure to Supplementary UV-B

Pea plants were either illuminated with visible light supplementedwith ultraviolet-B (UV-B) radiation for five days, or transferredback to control light after short exposures (hours) to UV-B.Spectra of NaOH-extracted pigments from UV-B-exposed plantsshowed a decrease in absorption in the visible region and anincrease in the UV region: the former a consequence of the lossof chlorophyll, the latter probably due to induced synthesisof protective pigments. The decrease in chlorophyll absorptionwas an earlier event than the increase in UV absorption. Inextracts from plants which had recovered, the increase in UVabsorption was greater than in leaves subjected to three daysof UV-B. This stresses the importance of recovery from UV-Bfor extensive synthesis of protective pigments. Analysis oftetrapyrrolic pigments showed that UV-B treatment caused noallomerization of chlorophylls. Formation of chlorophyllidesa and b was greatly enhanced during the degradation of chlorophylls;however, the concentrations of chlorophyllides were three ordersof magnitude lower than those of the chlorophylls and did notincrease greatly as chlorophyll degradation progressed. No protoporphyrinIX, uro- or copro-porphyrins III were detected, which suggeststhat early steps in chlorophyll synthesis are not affected byUV-B light. (Received May 15, 1992; Accepted August 6, 1992)     

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.     

Gut pigment destruction by the copepod Acartia clausi

The extent of chlorophyll (Chi) a degradation into colorlessproducts by the copepod Acartia clausi was determined by measuringgut fluorescence prior to fecal pellet production. The efficiencyof pigment degradation of animals fed at different concentrationsof Thalassiosira weiss-fiogii varied between 30.56 and 94.05%,and the quantity of pigments lost during gut passage was directlyrelated to total ingestion. The role of feeding history in pigmentdestruction was tested. The fraction of ingested Chi a degradedby copepods acclimated to a high food concentration (15 ng Chla ml^–1) was greater than the fraction degraded by thoseacclimated to a low food concentration (1.5 ng Chl a ml^–1).The percentage of ingested Chl a that A.clausi transformed intofluorometrically undetected compounds was not constant. At present,knowledge of the pigment destruction process indicates thatChi a and Chi --derived pigments are not considered useful quantitativetracers of the feeding activity of copepods. The results ofthis study suggest a reassessment of the application of thegut fluorescence method to evaluate grazing activity of thecopepod A.clausi     

CHANGES IN EMISSION SPECTRA OF PHOTOSYNTHETIC PIGMENTS IN VIVO

1. The effects of 3-(4'-chlorophenyl)-1, 1-dimethylurea (CMU)onthe fluorescence of photosynthetic pigments in vivo wereinvestigatedin blue-green, red and brown algae and in isolatedspinach chloroplasts.CMU caused an increase in steady statelevel of fluorescenceof chlorophyll a, but did not influencethe fluorescence ofphycobilins. The spectrum of the fluorescenceincrement hada peak at 685 m/µ and a shoulder at 730–740mµ.These two bands probably arise from chlorophyll a(C_f684) belongingto pigment system II.
2. On excitation of chlorophylla in a red alga, Porphyra yezoensis,a fluorescence band witha peak at 720 mµ was observedbesides a shoulder at 685mµ. The 720 m band is inferredto arise from chlorophylla (probably, C_f-1) in pigment systemI.
3. On addition of CMUto the algal cells, the induction of fluorescencewas modifiedto take a simple time course. The induction wasobserved onlywith respect to the fluorescence of chlorophylla, but not inthe fluorescence of phycobilins. The spectrumof the "transient"fluorescence showed two emission bands ofchlorophyll a at 685mµ and 740 mµ, and was quitesimilar in form tothe spectrum of the CMU-caused increase insteady state fluorescence.
4. These facts were interpreted in terms of the correlation offluorescence of chlorophyll a and the photochemical reactionsof photosynthesis

(Received July 20, 1967; )     

Reversed-phase high-performance liquid chromatography of chlorophylls and carotenoids

Reversed-phase high-performance liquid chromatography with octadecyl- or octylsilylated silica gel as the stationary phase provides a powerful tool in the analysis of chloroplast pigments from higher plants and green algae. Chromatographic columns packed with 10 μm chemically bonded silica gel particles allow the simultaneous separation of chlorophylls a and b, chlorophyll isomers, pheophytins a and b, α-carotene, β-carotene, lutein, violaxanthin, lutein-5,6-epoxide, antheraxanthin, neoxanthin and several minor carotenoids from a single sample within a short analysis time. The quantitative analysis requires a minimum of 1–5 pmol for carotenoids and 5–10 pmol for chlorophylls. Pigment degradation products, formed on polar stationary phases, are not found in reversed-phase high-performance liquid chromatography due to the weak hydrophobic forces on which the separation mechanism is based. The production of altered pigments however, either induced by various treatments or generated during the isolation, can be monitored as the reversed-phase system is selective enough to separate cis-isomers and oxidation products from their parent compounds. The reproducibility of the individual retention time for each pigment is better than ±1.5% which facilitates the identification of unknown pigments. The method is applied to the analysis of the pigment composition of Chlorella fusca, spinach (Spinacia oleracea) chloroplasts, and to the rapid determination of the ratio of chlorophyll a to chlorophyll b.     

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.     

Can phaeopigments be used as markers for Daphnia grazing in Lake Constance?

The formation of chlorophyll a degradation products was measuredwith natural phytoplankton from Lake Constance and Daphnia magnaand native Daphnia as grazers in grazing experiments duringspring bloom conditions using high-pressure liquid chromatography(HPLC). Chlorophyll a start concentrations were between 1.2and 16.3 µg l^–1; phaeopigment weights constituted5% of chlorophyll a weight. Only phaeophorbide a was a markerfor Daphnia grazing; concentrations of other phaeopigments (phaeophytina, chlorophyllide a and two unidentified phaeopigments) didnot increase during Daphnia grazing. Conversion efficiencies(chlorophyll a to phaeophorbide a) were between 0 and 43% ona weight basis, and between 0 and 65% on a molar basis. Conversionefficiencies were highest at high grazer density (40 Daphnial^–1) and after a 24 h exposure time. Grazing by microzooplanktonprobably led to the formation of the two unidentified phaeopigments.In Lake Constance, Daphnia density was significantly positivelycorrelated with the phaeophorbide a/chlorophyll a ratio whenit was <5000 Daphnia m^–3. However, when higher Daphniadensities were included in calculations, then Daphnia densitywas positively, but insignificantly, correlated with the phaeophorbidea/chlorophyll a ratio. This suggests that when the level offood per Daphnia is low, then grazing is more efficient withless production of phaeophorbide a and a higher production ofcolourless products.     

Digestion of chlorophylls and carotenoids by the marine protozoan Oxyrrhis marina studied by h.p.l.c. analysis of algal pigments

Algal pigments were measured, by reverse-phase h.p.l.c., duringgrazing experiments with the protozoan Oxyrrhis marina Dujardinon Rhodomonas sp. Chlorophylls and carotenoids were degradedto colourless residues; the rate of degradation of pigmentswas highest in the light. Very little chlorophyll a (5%) wasdegraded to phaeophytin and none to phaeophorbide during grazing.This suggests that phaeopigment concentrations cannot be usedas a measure of algal mortality due to grazing when heterotrophicProtozoa are a component of the grazer community.     

In situ diel variation in gut pigment contents of Ceriodaphnia sp. in stratification and destratification periods

The short-term, in situ diel grazing of Ceriodaphnia sp. duringperiods of stratification and mixing was investigated usingthe technique of fluorimetric analysis of the gut pigments.There were considerable seasonal differences in feeding behaviourIn mixing, when the concentration of chlorophyll a in the watercolumn was high and Ceriodaphnia abundance was low, gut pigmentcontents showed clear diel variation patterns, probably dueto diel variations of the high values of feeding activity observedin the 24 hour cycle The maximum values were found at dawn.On the other hand, no diel variations in gut pigment were observedduring periods of stratification and while the amounts of pigmentsin the water and in the gut were very low, species abundancewas high. Taking into account the ambient conditions, the authorsdiscuss the possibility that the change of feeding of the Ceriodaphniasp. observed when the environment changed from a mixing periodto one of stratification represents a change from herbivorousto detritivorous behavior.     

Modulation of Chlorophyll, Carotene and Xanthophyll Formation by Penicillin, Benzyladenine and Embryonic Axis in Mung Bean (Phaseolus aureus L.) Cotyledons

Penicillin stimulated the synthesis of pigments in the cotyledonsof intact embryos and excised cotyledons of mung bean (Phaseolusaureus L.) and enhanced benzyladenine-induced accumulation ofchloroplast pigments. The presence of the embryonic axis duringlight exposure proved to be beneficial for chlorophyll synthesisby the cotyledons whereas its presence in dark germination producedan adverse effect. The possible involvement of nucleic acidand protein synthesis in light-regulated chlorophyll formationis suggested. The stimulating effect on pigment synthesis providedby penicillin in this system seems to involve a maintenanceof nucleic acid and protein synthesis. Phaseolus aureus L., mung bean, pigment synthesis, cotyledons     

Pigment destruction by Calanus pacificus: impact on the estimation of water column fluxes

Measurements of gut pigment made prior to fecal pellet productionin Calanus pacificus females and CV copepodites suggest that(i) chlorophyll a and/or its pheopigment derivatives are degradedinto molecules that are not detectable by the standard fluorometrictechnique; and (ii) the percentage of ingested chlorophyll awhich degrades into fluorometrically undetected molecules isnot constant. Thus, measurements of chlorophyll and pheopigmenta in the guts of zooplankton can only yield minimum estimatesof in situ grazing rates. Estimates of the vertical flux ofprimary particulates based on chlorophyll and pheopigment abudgets may also be underestimated.