original scientific paper UDK 58.084(262.3 Tržaški z.) 582.27(262.3 Tržaški z.) GROWTH AND ECOLOGICAL ROLE OF THE SELECTED CLASSES OF MARINE PHYTOPLANKTON Barbara ŠVACELJ B.St., CŠOD , Fiesa Home, SI-6330 Piran, fies a 60 dipl. bid , CŠOD , Dom Fiesa, SI-6330 Piran, Fiesa 60 Patricija MOZETIČ M.Sc., Marine biological station Piran, SI-6330 Piran, Fornače 41 mag., Morska biološka postaja Pitan, Sf-6330 Piran, Fornače 41 Senka TERZIČ Ph.D., institute Ruder Boškovič, Center for Marine Research-Division Zagreb, HR-1000 0 Zagreb, Bijcnieka 54 dr., Institut Ruder Boškovlč, CIM-Zavod Zagreb, HK-tOOOO Zagreb, Bijenička 54 ABSTRACT Growth and some biochemical characteristics were followed in six monocultures from four classes of marine phytoplankton, The growth and size parameters, as well as pigment composition of selected species were examined. The approach of chemotaxonomic pigment biomarkers was used to interpret: the pigment fingerprints of natural phytoplankton from the Gulf of Trieste. The presence and concentrations of pigments analysed by HPLC method were supported by microscopic observations of the same samples. Key words: phytoplankton, monocultures, growth, biomarkers, HPLC, Gulf of Trieste Ključne besede: fitoplankton, monokuiture, rast, biomarkerji, HPLC, Tržaški zaliv INTRODUCTIO N Phytoplankton monocultures have been largely used to determine the species/group characteristics and dy­namics. Controlled conditions are usually the only pos­sible way to study the morphology, ecophysiology, bio­chemical composition and therefore the taxonomic po­sition of algae. For example, a large number of small or rare organisms, as well as toxic or potentially toxic spe­cies have been identified and their characteristics have become known only in monocultures. The past and current efforts to identify phytoplankton from natural waters rely largely on microscopic evalu­ation. This requires a high level of taxonomic skill, but it-can be also significantly variable among researchers, and time consuming work. However, by using a light microscope it is not always possible to identify an or­ganism not only at the species but also at a higher, class level. This is the case of the non-taxonomic phytoplank­ton group, commonly denominated as a group of mi­croflagellates. Microflagellates are small (approx. 10 pm) naked flagellates, which belong to different algal classes. Because of their small size, fragile structure and use of aggressive fixatives (for example formaldehyde) for storage and counting procedures, microflagellates can be often overlooked, wrongly identified and their number underestimated. Besides classical microscopic techniques, either op­tical or electronic, new methods using different biomarkers as a tool to assess phytoplankton diversity have emerged lately. Among several biomarkers, photo­synthetic pigments have proved to be effective for pro­viding information about the phytoplankton chemotax­onomic composition, physiological status, primary pro­duction and trophic state (Millie et ai, 1993), High-performance liquid chromatography (HPLC) was suc­cessfully applied to the determination of chlorophylls (a, b, c, d, e), phycobilins of cyanobacteria and the red al­ 8. ŠVAGEIJ, P. MOZETIČ, S. TERZIČ: GROWT H AN D ECOLOGICAL ROLE O F THE SELECTED ..., 157-106 PIGMENTS PHYLOGENETIC CROUPS j Chlorophylls a ail groups (the only chlorophyll in Cyanophyta and Eustigmatopbyi.a) b Chlorophyta, Eugl eno phyla, Prasirso phyta, ProChlorophyta C] Bacili ariopbyta, Chrysophyta, Prymnesiophyta, Raphidophyta, Xat> thopbyta c2 Baciliariophyta, Cryptophyta, Dyno­phyta, Prymnesiophyta, Raphido­phyta, XanthophySa Bacil lariophyta, Chrysophyta, Dyno­pliyta, Prymnesiophyta 8-ciesethyl, 8-vinyi a ProChlorophvta fl-desethyl, 8-vinyi b ProChlorophyta Mg 2,4-diviniJpheopor-Prasinoph yta phyrin monomethyl es­ter Carotenoids alloxanthin Cryptophyta 19' butanoyioxyfucoxan-Dynophyta, Prymnesiophyta, ihin Raphidophyta crokoxanthin Cryptophyta dinoxanthin Dynophyta, Prymnesiophyta echinenone Cyanophyta, ProChloropl'iyta fucoxanthin Bacilianophyta, Chrysophyta, Dyno­ phyta, Prymnesiophvta 1 9'~hexanoyloxyfucoxan-Dynophyta, Prymnesiophyta ihin lutein Chlorophyta, Prasinophyta monadoxanthin Cryptophyta myxoxanthophyll Cyanophyta osdllaxanthin Cyanophyta peridinin Dynophyta prasinoxanthin Prasinophyta pyrrhoxanthin Dynophyta siphonaxanthin Chlorophyta, Euglenophyta, Prasino­ phyta vaucheriaxanthin Etistigmatophyta, Xanthophyta zeaxanthin Cryptophyta, Cyanophyta, Prasino­ phyta, Prochlorophyta Phycobilins allophycocyanirs Cyanophyta phycocyanin Cyanophyta, Cryptophyta phycoerythcin Cyanophyta, Cryptophyta, Rhoclo­ pliyta Table 1: Photosynthetic pigments present in pbylo­genetic algal groups (after Millie et al., 1993). Tabela 1: Zastopanost posameznih fotosintetskih pig­mentov pri filogenetskih skupinah alg (povzeto po Mil­lie et al., 1993). gae, and a wide range of the oxidised carotenoids - the xanthophylis, To date, more than 400 compounds are known, and many of them are highly specific only for one taxortomic group (Table 1). H PLC determination of biomarker pigments is a high­ ly sensitive and accurate method which sometimes enables the identification and detection of groups that have been overlooked by the standard microscopic method (Cieskes & Kraay, 1983). The greater part of the pigment composition studies have been done in the oceanic waters (Wright & Jeffrey, 1987; Bidigare et al., 1990; Buma et al., 1990; Barlow et al., 1993), while coastal areas and estuaries have received less attention (Denant eta!., 1991; Male] et al., 1995; Terzici, 1996). Besides the taxonomic identification it is essential, from the ecological point of view, to characterise the flow of organic matter through the pelagic ecosystem (Verity ef al,, 1992). The basic parameter of living or­ganic matter is the biomass of organisms in terms of or­ganic carbon. Estimation of organic or cell carbon (C) from the chlorophyii a (Chi a) concentration is com­monly used. However, the C:Ch! a ratio can vary greatly among different: phytopfankion groups and seasons (Booth et al., 1988; Sieracki et al., 1992), therefore the estimates of carbon are not very accurate. For example, the C:Ch! a ratio of 50 (Strickland & Parsons, 1972) is frequently used. A common but time consuming alterna­tive is the estimation of cell carbon from the ceil volume using appropriate conversion factors or formulas as shown in Table 2. The aim of this work was to determine the growth and some biochemical characteristics (pigment com­position, cell carbon) of six monocultures. The species were chosen from the most important - dominant groups of the phytoplankton community in the Gulf of Trieste. The predominant groups in this shallow bay are diatoms and microflagellates, the latter being the most abundant group in the greater part of the year (Fanuko, 1981). The other important groups are dinofiagellates, cocco­lithophores, and silicoflagellates. Phytoplankton suc­cession is strongly influenced by the riverine and urban freshwater inputs and the seasonal stratification of the water column (Smetacek, 1991). Diatom peaks occur in early spring and autumn, which are characterised by the freshwater inputs and mixing of the water column, and occasionally during the summer following episodic storms and consequently nutrients1 input (Maiej et ah, in prep.). MATERIALS AN D METHODS Ctslturing and sampling procedure The isolated species were: Isochrysis galbana (class Prymnesiophyceae =Haptophyceae), Emiliania huxleyi (class Prymnesiophyceae), Nitzschia closterium (class Baciilariophyceae), Phaeodactykim tricomutum fclass Bacillariophyceae), Prorocentrum micans (class Dino­phyceae) and Tetraselmis suecica (ciass Prasinophv­ceae). These species originated from the Culture collec­tion of Plymouth Marine Laboratory, except for the spe­ B IVAGEU, P. MOZETIČ, S. TERZIC: GROWT H AN D ECOLOGICAL ROLE OF THE SELECTED ..., 1.5?. 166 cies N. closterium and E. huxieyi, which were isolated from the Cuff of Trieste. The cultures were grown in 20­litre polycarbonate containers at a temperature between 16 and 17°C. Light was provided at a 12 hour light/dark interval by neon Hcool white" bulbs, with the intensity varying from 20 to 50 pE nr2s-1 . Guillard's f/2 medium {Cuillard, 1975) was used for most of the cultured spe­cies, with the addition of silicate only for the diatoms. For the species E. huxieyiwhich requires lower nitrogen content, Keller's medium was used (Keller ef at., 1987). All media were prepared with filter-sterilised natural sea water from the Gulf of Trieste. immediately after the inoculation (60-120 ml culture in the stationary phase was added to 10 I medium) subsamples for cell counts, pigments' analyses and Chi a concentration were taken. Cell number and volume were determined in neutralised formaldehyde preserved subsamples (1.5% final concentration). The growth of monocultures based on the cell counts was followed daily, while the cell volume measurements and bio­chemical analyses were performed again during expo­nential and stationary phase. Natural sea water samples for phytoplankton and pigment composition were taken monthly from January till December 1993 at a station in the southern part of the Gulf of Trieste. A 5-litter Niskin bottle was used for sampling at five depths: 0, 5, 10, 15 and 21 m (bottom). 800 ml phytoplankton samples were preserved with 1.5% neutralised formaldehyde. Analyses The ceil number was counted on a Fuchs-Rosenthal haemocytometer using a light microscope at a lOOx and 400x magnification. The growth rate (k in division day 1 ) was calculated daily from the cell number using the equation (Guiilard, 1973): k = ln(N-]/N{))/ (0,6931 x (trt0)) where N-j and NQ are cell numbers at times t-] and to, respectively, and tj -to is the time difference in days. Phytoplankton from natural samples was identified and counted on an inverted microscope using the tech­nique of Utermohl (1958), where 50 or 100 fields of the bottom chamber were examined at 200x and 4G0x magnification. The ceil volume was determined as an average vol­ume of 15 cells using cells dimensions and the best fit­ting geometric formula based on the cell shape (Edler, 1979). The carbon content was calculated from the cell volume using conversion factor 0.13 pg (pm)"3 for P. micans, and 0.11 pg (pm)-3 for other species. The car­bon to chlorophyll a ratio (C:Ch! a) was based on the cell carbon calculation and Chi a concentration. Chlorophyll a concentration was determined fluorometrically on a Turner 112 Fluorimeter (Holm-Hansen ef a/., 1965), 15-25 mi subsamples were filtered conversion group/size ciass reference factor/formula ( C CMm>-3) 0.13 armoured Smetacek, 1975 dinoflagellates 0.11 eukaryotic autotrophs Strathman, 1967 except armoured clinofl. 0,35 10-100 (|.im):l Verity et al., 1992 0,24 100-1000 {41m)3 0.16 >1000 (!imp " 0,075 flagsllat.es Hegmeier, 1961 log C--0.SM+0.85 7 diatoms x logV log C=(-0.24xiogV)­ general Mullin ef a/., 1966 0.29 C=aV'> general Montagnes et it/., 1994 Table 2: Conversion factors and formulas for cal­culating the cell carbon (C) based on cell volumes (V) for different classes and size groups of phytoplankton. Tabela 2: Pretvorbni faktorji in enačbe za prera­čunavanje celičnega ogljika (C) iz celičnih volumnov (V) za posamezne skupine in velikostne razrede fito­planktona. onto 0.22 [irrt Millipore filters, extracted in 90% acetone and the fluorescence of extracts measured. The qualitative and quantitative analyses of pigments in the monocultures as well as in the natural samples were determined using a reverse-phase H PLC method (Mantoura & Llewellyn, 1983; Barlow et ai., 1993). Water samples (30 ml to 1 I) were filtered through 47 mm Whatman GF/F filters and immediately frozen until analysed. Frozen samples were extracted in 4 to 10 ml of 90% acetone using sonication and cenirifugecl to re­move cellular debris. Chlorophylls and carotenoicls were detected by absorbance at 440 nm in the UV/Vis spec­trophotometry detector. Data collection and processing utilised Spectra Physics PC1000 software, The phytoplankton abundance and concentration of pigments from disciete natural samples were depth-inte­grated over the whole water column. RESULTS Monocultures The growth and size parameters of the monocultures determined in the exponential and stationary phase are presented in Table 3. At the beginning of the experiment, the highest cell number was counted in /. galbana culture (9.3xl04 cells mh1), which reached also the highest cell density in the whole experimental period (1.0x107 cells ml"1). Only 2.0x103 cells mL1 were counted in P. micans culture at S. IVAGtLS, P. MOZETIČ, S. TERZIČ: GROWTH AND ECOLOGICAL ROLE OF THE SELECTED .., 157-1 Mi species growth cell k Chi a ceil OCh l a phase volume (divisions (pgcell-t) carbon {(Mm)3) day' } (PR celt' ) L. huxfeyf exp 157/9 0.5') 0.04 15.7 392 5 iS? 153,1 0,03 20.2 673 1. galbana exp <(4.5 0,3.3 0.10 4.9 49 slat 'ffj .3 0.20 5.1 25 N. ctosterium exp 50.9 0.49 0.2B 5.6 19 stal SB.3 0.70 6.4 9 P. tricornutum exp 118.9 0.49 0,12 13.1 109 slai 192.1 0.13 21.1 162 t', m !cans exp 19 m O.tG 0.1 S 2486,0 165 73 ssat 23903 0.22 3! 07,0 14522 T. suecica exp 170.2 0.26 0.88 18.7 21 sut 153.0 o. s a 16.8 29 Table 3: The size and growth parameters chlorophyll a (Chi a) and cell carbon (C) content and the C:Chl a ra­tio of the monocultures in (he exponential (exp) and stationary (stat) growth phase. Tabela 3: Parametri velikosti in rasti, vsebnost klorofila a (Chl a) in celičnega ogljika (C) ter razmerje C:Chl a v monokulturah v eksponentni (exp) in stacionarni fazi rasti (stat). 1.0E+1 -a ig l.OE-tO-s « u • M 1.0E-1 J + i I.OE-2"i -Î lag phase phase Mal phrtsv 1.0E- ! -a »1XIE-2": m ta St« i.oe-3--= i S * S 1.0E-5-' lag phage exp phase stal phase • T. suecica, • N. closterium, A P. tricornutum, + I. galbana, • E. huxleyi, X P. micaris Fig. 1: Chlorophyll a content (log10 pg Chl a) calculated per (a) cell and (b) volume unit in the lag, exponential (exp) and stationary (stat) phase of the monocultures. (Note the different units o n y axes.) Slika 1: Vsebnost klorofila a (log^o pg Chl a), prera­čunana na (a) celico in (b) volumsko enoto v lag, eks­ponentni (exp) in stacionarni (stat ) fazi rasti mono­kultur. (Upoštevaj različne enote na oseh y.) the beginning and 4.Txl 0 4 cells mi-1 at the end of the experiment. This species had also the longest lag phase (10 days), while diatoms and E. huxleyi passed over a 4 days lag phase. The growth rates differed substantially between the monocultures. The fastest growing species were diatoms N. closterium and P. tricornutum, the lat­ter reaching the highest growth rate of 1.1 divisions day1 in the exponential phase. Other species had a growth rate about 1 division day1 in the exponential phase, whereas P. micans did not exceed 0.73 division day1 . Except for T. suecica, the ceil volumes increased with ageing of the monocultures. The largest cell volume was measured in P. micans (23903 (jimp), while other species had a cell volume below 200 (pmp. During the stationary phase, the highest Chl a con­centration was measured in I. galbana (1678.4 pg H ) and N. closterium culture (787.9 pg H) , white the low­est in P. micans (8.9 ug H ) and E. huxleyi culture (35.1j.ig I"1). Chl a biomass was expressed as Chl a con­centration per cell (pg cell"1) and per unit of cell volume (pg (pm)-3; Fig. 1) and in all cultures the latest concen­trations were lower than the former. In both exponential and stationary phases the highest C.hl a concentration per cell and per (!.imp was found for N. closterium and Fig. 2: A HPtC chromatogram showing characteristic pigment pattern of the species E. huxleyi grown in the monoculture (exponential phase). Legend of the absor­bance peaks: (1)~chlorophyll c$, (3)=chlorophyll c7+ t'2, (6)~fucoxanthin, (8)=19'-hexanoyloxyfucoxanthin, (9)-diadinoxanthin, (13)= chlorophyll a. Slika 2: H PLC kromatogram in značilni pigmenti vrste E. huxleyi, gojene v monokulturi v eksponentni fazi. Legenda absorbcijskih viškov: (1)=klorofil Cj, (3)­klorofil cj+c2, (6)=fukoksantin, (8)=19'-heksanoiloksi­fukoksantin, (9)~diadinoksantin, (13)= klorofil a. 0. iVACfc'LJ. P. MOZETIČ, S.TCRZIC': GROWTH AND ECOLOGICAL KOL L O f THf SELECTED ..., ÎS7-I66 T. 5uecica. The largest differences between pg Chi .j cell"1 and pg Chi a (pm)~3 were observed in P. micans culture, due to very low biomass as compared to cell number and large ceil volume. The discrepancy be­tween high cell number and low biomass was observed also for E. huxleyi with the lowest Chi a concentrations per cell. With the ageing, the Chi a concentration de­creased in some species, while in others increased. The highest carbon content, based on the cell vol­ume, was calculated for the largest species - P. micans. The cell carbon increased in all species during the ex­periment, except for T, suecica in the stationary phase. Here, the carbon content decreased following the change of cell volume. The pigment composition was determined by the HPLC method, Besides Chi a, many accessory pigments were present in monocultures. Different biomarkers were selected according to the presence and concentra­tion of the dominant accessory pigments. A typical chromatogram of a monoculture is shown in Fig. 2. Be­sides Chi a, the diatom monocultures were characterised 2000 1500 1000 i 500 days "> ^ •=> V? # # ^ # ^ ^ days by the presence of Chi c-f+cx, diadinoxanthin, ft-caro­tene, and a characteristic biomarker fucoxanthin. P. mi-cans was characterised by the presence of Chi cj+C2, diadinoxanthin, and peridinin as the biomarker for di­noflagellates. 19'-hexanoyloxyfucoxanthin (19'hex), Chi Cj+C2, Chi cj , and diadinoxanthin were found in E. huxleyi culture, and 19'-hex was defined as a biomarker for this species as welt as for the group of prymnesio­phyates. However, the other prymnesiophyte I. galbana showed untypical pigment composition which re­sembled that of the diatoms. The chromatogram of T. suecica revaled the presence of Chi b, zeaxanthin/lutein and K-carotene, the typical prasinophycean as well as green algae pigments. In some monocultures, the concentration of biomarkers incrased in such pro­portions in the stationary phase that exceeded Chi a content. Natural samples Over the whole sampling period (January -Decem- Ch i isIM i b F" I iuc W-Ue x Mil ! mm per mm zca/Eut SK5S T 1'— âJjcoftegell. difioftage-Ii. coccoliihoph. nniiin] dialoms [ i mkroftagttlL E « of- & ^ 200 [.!g I'1) with approximately the same cell numbers. Pigment biomarkers in the monocultures and natural samples Pigments detected in the monocultures were used as biomarkers to determine the presence of the respective phytoplankton groups in natural samples. However, un­certainties arise because some of the accessory pigments are present in many groups or, on the contrary, some of them are not typical of all the species of the specific group. For example, the diatom biomarker fucoxanthin is present also in prymnesiophyte I. galbana and unar­moured dinoflagellates (Jeffrey et al., 1975). Knowing the ratios between the concentration of Chi 0. ŠVAGELJ, P, MOZETI Č S. TERZLC: GROWT H AN D ECOLOGICAL ROI E OF THE SELECTED ..., 1.5?-T6F> a and typical accessory pigment, one can calculate the relative contribution of a specific group to the total Chi a biomass (Everitt et al., 1990). Peridinin as a selective biomarker for dinoflagellates {Whittle & Casselton, 1968) is suitable for the estimation of their biomass in a natural sample. The Chi a: peridinin ratio obtained from the monoculture P. micans is 1.4 compared to 2.6 found in literature (Everitt etal., 1990). The relative abundance of a phytoplankton group (X in percentage) is calculated using the formula: X = K x (Cpjg/Cchfa) where K is the known ratio between the concentration of Chi a and accessory pigment of the same spe­cies/group, Cpjg and Cchia are concentrations of the ac­cessory pigment and Chi a in natural sample. Using the formula and Chi a: peridinin ratio of 1.4, relative abun­dance of dinoflagellates in natural samples were calcu­lated (Table 4), These percentages are compared with the percentages based on the microscopic counts. Some discrepancies appear when comparing the results, since peridinin concentration does not always follow din­oflagellate cell numbers. In fact, in June the highest cell number was counted, while the peridinin concentration was very low. Again, in May dinoflagellate abundance was low, but peridinin was not even detected. After a more accurate examination of the sample it showed up that in late spring a group of dinoflagellates - Gymno­dimales dominated, which contain fucoxanthin as the main carotenoid {Jeffrey et al., 1975), and the genus Gy­rodsnlum that contains 19'-hex (Tangen & Bjornland, 1981) was also present. In October, however, a very high concentration of peridinin was detected and the amount of the pigment per ceil was 22-times higher than in June (6,35 pg cell"1 in October and 0.29 pg cel H in June). In this period armoured dinoflagellates were more abundant and the ones containing fucoxanthin or 19'­hex were almost missing. The amount of peridinin cal­culated per cell reached a maximum of 0.0614 pg celM in P. micans monoculture, which is in an astonishing disagreement with the estimate made in field conditions. Since the concentration of pigment per cell varies sig­nificantly not only seasonally but also during the life­time of a population and in relation to the abiotic factors in the environment, the quantitative estimations of the cell abundance using biomarker pigment concentrations should be performed with a great care. Fucoxanthin is present also in other algal classes. Consequently, without a support of microscopic obser­ date % drnofiageli. % dinoilagell. 1 (Chi a biomass) (toiai ce!I No.) j San 21 12 14 May 1 2 0 4 Km 9 4 6 lui 14 13 3 Sept 8 10 5 1 Oct 12 8 1 1 Table 4: The relative abundances (%) of dinoflagellates of the total Chi a biomass and total cell numbers in natural samples, based on peridinin and Chi a con­centration and microscopic cell counts respectively. Tabela 4: Relativni deleži (%) dinoflagelatov pri skupni ktorofilni biomasi in skupnem številu celic v naravnih vzorcih, izračunani iz koncentracije peridinina in klo­rofila a ter iz števila celic. vations the HPLC fingerprint can be misunderstood. In January, in contrast to October and July, the high con­centration of fucoxanthin cannot be related to a diatom bloom, since this group was almost missing as revealed from the microscopic observations, An explanation cOLild be that some unidentified species of prymnesio­phytes with a pigment composition similar to /, galbana or other algal classes from the group of microflagellates are responsible for the high fucoxanthin level. On the other hand, it was shown that classic microscopy is sometimes insufficient especially in the case of mi­croflagellates. A January the 19'-hex peak coincides with the elevated number of coccolithophores, while in May, june and September other unidentified prymnesio­phytes or some dinoflagellates contribute to the high concentrations of this pigment. A minor zeaxanthin/ lutein peak is detected in June and it might again repre­sent unidentified green algae or even cyanobacteria. In conclusion, the pigment composition roughly fol­lowed the taxonomic composition determined with mi­croscope. The greatest discrepancies were present in the case of fucoxanthin and peridinin, but even with other pigments it was impossible to assess the exact contribu­tion of a certain group, since many pigments are not highly specific. HPLC method on the other side con­tributes largely to the identification of the taxa that are commonly placed within the microflagellates, which is phylogenetically a very heterogenic group. Also, to im­prove the knowledge of the pigment composition in re­lation to the physiology of the phytoplankton and abi­otic factors in the environment, more experiments on monocultures have to be done. 8. ŠVAGEU, p, MOZETIČ, 5. TERZSČ: GROVVTH AN D ECOt.OCSCAL ROIE O F THE SEI.ECTEE) ..., T 57-166 POVZETEK Avtorice so raziskovale rast in nekatere biokemične značilnosti monokultur šestih vrst štirih najpogostejših razre­dov morskega fitoplanktona iz Tržaškega zaliva. Predstavljeni so bili rastna hitrost, celični volumen, vsebnost celičnega ogljika, preračunana iz celičnega volumna, koncentracija klorofila a in pigmentna sestava. Vrste so se med seboj razlikovale v hitrostih rasti in trajanju začetne lag faze. Najhitreje rastoče monokulture so bile diatorneji P. Sricornutum in N. closterkim ter kokolitoforida E. huxieyi. Njihova lag faza je trajata 4 dni, medtem ko je bila pri drugih monokutturah 8-10 dni. Največje Število celic in največjo biomaso, izraženo kot koncentracijo klorofila a, je dosegla i. gafbana v stacionarni fazi. Največji celični volumen je imela vrsta P. micans, pa tudi tudi vsebnost celičnega ogljika, preračunana iz volumna ter razmerje C:Chl a, sta bila pri tej vrsti največja. Analiza pigmentne sestave posameznih monokultur je bila narejena s tekočinsko kromatografijo visoke ločljivosti {HPLC metoda). Diatomejski biomarker fukoksantin je bil najden tudi pri netipični primnezioficeji !. galbana, peridinin pa je bil najden le pri dinoflagelatu P, micans. 79'-heksanoiloksifukoksantin, značilen pomožni pigment primnezioficej, je bil najden v kulturi E. huxleyi, pri zeleni algi T. suecica pa biomarkerja klorofil b in zeaksantin/lutein. Značilni pomožni pigmenti -biomarkerji so bili uporabljeni pri razlagi pigmentnih spektrov naravnih vzorcev iz Tržaškega zaliva. Na osnovi koncentracije biomarkerjev so avtorice določile pojavljanje in zastopanost posameznih razredov fitoplanktona. Kemot.aksonomska sestava je bila potrjena z mikroskopskimi pregledi istih vzorcev. Analiza pigmentnih spektrov se je v večini primerov ujemala z rezultati mikroskopskih pregledov, največja neskladja pa so bila pri fukoksantinu in peridininu. Raziskava je pokazala, da je metoda biomarkerjev zelo uporabna za določevanje taksonomske sestave naravnih vzorcev. Vendar je zaradi nekaterih pomanjkljivosti, na katere so avtorice naletele pri obeh metodah, za taksonomsko analizo fitoplanktona priporočljiva uporaba obeh. REFERENCES Banse, K. 1977. Determining the carbon to chlorophyll ratio of natural phytoplankton. Marine Biology, 41, 199­ 212. Barlow, R.C., Manioura, R.F.C., Cough, M.A. & File-man, T.W. 1993. Pigment signatures of the phytoplank­ton composition in the northeastern Atlantic during the 1990 spring bloom. Deep-Sea Research 40, (1/2), 459­ 477. 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