Kolmen merellisen dinoflagellaatin glukoosinottopotentiaal

Iliash, Ljudmila V.

Kaitala, Seppo

Tamminen, Timo

Helsingin yliopiston digitaalinen arkisto

VESIENTUTKIMUSLAITOKSEN JULKAISUJAPUBLICATIONS OF THE WATER RESEARCH INSTITUTETimo Tamminen & Jorma Kuparinen: On the measurement of heterotrophic activity inthe aquatic environment using iabeiied substratesTiivistelmä: Vesistöjen bakteeriplanktonin aktiivisuuden mittauksesta radioaktiivistenmerkkiaineiden avulla 4Timo Tamminen: Linear transformations of the Michaeiis-Menten kinetic equation innaturai microbiai communities researchTiivistelmä: Michaelis-Menten -kinetiikan lineaarimuunnokset luonnon mikrobiyhteisöjentutkimuksessa 11Timo Tamminen, Seppo Kaitala & Ludmila V. lliash: The heterotrophic glucose uptakepotential of three marine dinofiageliatesTiivistelmä: Kolmen merellisen dinoflagellaatin giukoosinottopotentiaali 21Jorma Kuparinen, Kirsti Lahti, Tuija Taisi, Timo Tamminen & Anneli Virtanen:Determination of the Michaelis-Menten kinetic parameters with singie concentrationassaysTiivistelmä: Bakteeriplanktonin glukoosinottoa kuvaavien kineettisten parametrien määritysyhden substraattipitoisuuden menetelmällä 26Jorma Kuparinen, Kirsti Lahti, Ari Mäkelä, Seppo Rekoiainen, Tuija Taisi, TimoTamminen, Anneli Virtanen & Antti Uusi-Rauva: A practicai approach to themeasurement of microbiai heterotrophic activity by the single concentration methodTiivistelmä: Vesistöjen bakteeriplanktonin aktiivisuuden määritys yhden substraattipitoisuudenmenetelmällä: käytännön suoritus 35Tuija Taisi, Timo Tamminen & Jorma Kuparinen: Variability in pianktonic heterotrophicactivity and primary productivity assays in reiation to sampiing strategiesTiivistelmä: Perustuotannon ja bakteeriplanktonin aktiivisuuden mittauksen tilastollinenluotettavuus näytteenoton suunnittelun perustana 42VESIHALLITUS— NATIONAL BOARD OF WATERS, FINLANDHelsinki 1984Tekijä on vastuussa julkaisun sisällöstä, eikä siihen voidavedota vesihallituksen virallisena kannanottona.The author is responsible for the contents of the publication.It may not be referred to as the official view or policyof the National Board of Waters.IS BN 951-46-8080-4ISSN 0355-0982Helsinki 1984. Valtion painatuskeskus21THE HETEROTROPHIC GLUCOSE UPTAKEPOTENTIAL OF THREE MARINEDINOFLAGELLATESTimo Tamminen1),Seppo Kaitala2)& Ljudmila V. Iliash2)TAMMINEN, T., KAITALA, S. &‘ILIASH, L.V. 1984. The heterotrophicglucose uptake potential of three marine dinofiagellates. Publications of theWater Research Institute, National Board of Waters, Finland, No. 56.The heterotrophic potential of the marine dinofiagellates Prorocentrummicans Ehr., Gymnodinium splendens Lebour. and Gyrodinium Jissum (Lev.)was studied in unialgai cultures as net assimilation of 14C-glucose during iightand dark incubations. The heterotrophic potential of aigal cells was highest nthe early stages of culture growth. Light and dark incubations did not differsignificantiy in heterotrophy. G. splendens had the highest heterotrophicpotential according to both maximum velocity per cell and the half-saturationconstant (K) of glucose. The kinetic parameters of ali cultures indicated lowaffinity for glucose. Half-saturation constants (140—1000 pg glucose . 1-1)exceeded those usually measured in natural planktonic communities by one tothree orders of magnitude. It therefore seems unlikely that measurernents ofthe heterotrophic activity or potential of natural microplankton would beaffected by these mixotrophic algae to any significant extent.Index words: Marine dinofiagellates, algal heterotrophy, heterotrophicpotential, glucose assimilation.1. INTRODUCTIONSeveral fractionation studies have indicated that inmeasurements of the heterotrophic activity orpotential of natural microplankton, the sizefraction corresponding mainly to free-livingbacterial cells (appr. 0.2—2 pm) is responsible forthe major portion of heterotrophy (e.g. Williams1970, Allen 1971, Berman 1975, Gocke 1975, Azamand Hodson 1977). Wright and Hobbie (1966)presented data on net assimilation of glucose andacetate by natural ommunities of bacterioplankton and phytop1ankton. They postulated that inheterotrophic assim1ation measurements at relatively low substrate concentrations (<300 pg 1-1),bacteria completely dominate uptake because oftheir higher affinity or the substrate. Therefore it1) Tvärminne Zoological Station, University of Helsinki,SF-10850 Tvärminne, Finlandcould be safely assumed that assimilation measurements with low substrate concentrations describethe heterotrophic metabolism of bacterioplankton.These results have also been supported byautoradiographical studies (e.g. Hoppe 1976).However, recent literature on the physiology ofseveral groups of phytoplankton has presentedaccumulating evidence of the heterotrophic capacity of these organisms. In particular nano- andpicoplankters belonging to the Chrysophyceae,bluegreen algae (Cyanobacteria) and dinofiagellates, as well as microzooplankton species ofZoomastigophora, have been thought to utilizedissolved organic substrates in their nutrition.Owing to the lack of sufficient experimentaldata, these considerations are stiil speculative withregard to natural conditions, in which concentra2) Department of General Ecology and Hydrobiology,Moscow State University, Moscow V-234, USSR22tions of dissolved organic compounds of lowmolecular weight are in the order of microgramsper litre. Fieid evidence for active aigal heterotrophy (McKinley 1977, Vincent and Goldman1980) is scarce and partly ambiguous (for discussion see Tamminen 1984).In this study, heterotrophic potential wasmeasured in uniaigal cultures of Prorocentrummicans Ehr., Gymnodinium splendens Lebour. andGyrodinium fissum (Lev.). Three phases of cuituregrowth were assayed with P. micans and two phaseswith G. splendens in order to evaluate the effect ofthe physioiogical state of celis on their heterotrophy. The heterotrophic potential of the algaewas measured as net assimilation of 14C—glucosein both light and dark incubations.2. MATERIALS AND METHODSThe algae were cultured in a luminostat wjth a14 + llhourphotopperiodicity(light + dark).Lightintensity was 8 000 lux and temperature 19°C. Thecultivation medium is presented in Table 1. Theceli density of each cuiture was determinedmicroscopicaily at the time of incubation.Incubations (18 mi) were performed in sterile 20ml test tubes. The incubation time was 4 hours,and it was terminated by adding Lugol solution.Blanks were prepared by adding Lugoi solution totest tubes before additions of radioactivity.D—(’4C U) -glucose (specific activity 396pCi mmoi1, Techsnabexport, Moscow, USSR)was added into subsamples at final concentrationsof 12.3, 123 and 1270 pg giucose . 11. Eachconcentration was added in duplicate and a blanksampie was used. After termination of theincubations, sampies were fiitered on 2.5 pmmembrane filters (Synpor, Czechoslovakia). Thefiitrates were filtered on 0.45 pm membrane filters(Gelman, USA). Ali fiiters were washed with 15 mlof fiitered sea water of salinity equal to that of themedia.Sampies were measured with Mark II and MarkIII liquid scintillation counters (Packard, USA) bythe external channei ratio method. The scintillation cocktail consisted,pf a filter, 100 pl of H20and 10 ml of Reahim ZS-7A scintillation cocktaii(Harkovskyi zavod himreaktivov, USSR). Themeasured dpm (disintegrations per minute) vaiueswere caiculated into turnover times (T = C twhere C = added radioactivity, t = incubationtime, c = measured radioactivity).Table 1. Goldberg medium for(Kabanova 1961). The salinity of theand the pH 8.0.KNO3 202 mgNa2HPO4 7.1 pgFeC136H20 270 pgMnCI2 4H20 198 pgCoC12 . 6HO 238 pgsynthetic seawater 1.0 1Kinetic parameters describing the uptake ofglucose were calcuiated according to Wright andHobbie (1965, 1966) for both <2.5 pm and >2.5pm size fractions. Since the media contained noglucose, the sum parameter (K + 5) reduced to K,the haif-saturation constant for the substrate.3. RESULTSThe uptake kinetics of different size fractionsdiffered qualitatively from each other in ali thecultures (Fig. 1). Measurements of the algalfraction (>2.5 pm) followed closely the lineartransformation of Michaeiis-Menten kinetics overthe assayed concentration range. It was obvious,however, that the 1270 pg . 1-1 addition exceededthe saturation concentration for bacteria, resultingin two different phases of giucose uptake. Beiowthe 123 ,g . 11 addition, bacteria showed verysignificantiy greater affinity for glucose than didalgae, with K vaiues in the order of only a fewh100 - .——.-T50—I:1000 100K AFig. 1. Glucose uptake kinetics in different fractions ofdark incubations of Gyrodinium fissum.aigal cultivationsmedium was 260/001000 ,ug1’ ,tg11 1000 150023micrograrns per litre. It was therefore concludedthat the fractionation between algae and bacteria inthe cultures had succeeded satisfactorily, and thatthe measured glucose uptake in the >2.5 pmfraction described the heterotrophic metabolism ofalgae.The relationship between aigal heterotrophy inlight and dark incubations varied between 0.83 and1.20, with a mean of 1.00 (Table 2). No stimulativeor inhibitive effect of light on aigal heterotrophywas thus observed. The heterotrophic potential(maximum uptake velocity of glucose, V) wastransformed into per cell values to investigate theeffect of the physiological state of cells on theirheterotrophic potential (Table 2). It appeared thatwith both P. micans and G. splendens, whencultures of different ages were assayed, theheterotrophic potential decreased with age, beinghighest during the logarithmic growth phase of thecultures.When different species were compared G.splendens, which grew in the lowest densities, hadclearly the highest maximum uptake velocity percell. Old P. micans cultures had the lowestheterotrophic potential values (Table 2).4. DISCUSSIONValues of the half-saturation constant (K) arelinear extrapolations of uptake measurements,when the so-called Lineweaver-Burk transformation is used in the calculations (see Fig. 1). Whenthe extrapolation distance increases, the precisionof the constant naturally decreases. This isreflected in the values in Table 2, as the halfsaturation constant should be a more or less stableproperty of the culture — affinity for the substrate—so that the values in light and dark incubationswould be expected to agree closely. However, thevalue of the constant shows reasonable variation inthe experimental data. The smallest values — thusindicating the greatest affinity for glucose — werecalculated for G. splendens cultures (140—240pgDirect comparison between the results obtainedfrom laboratory cultures and those from naturalcommunities is meaningless, because the physiological state of the cells is not equal. The halfsaturation constant (K) in natural communities isalso a somewhat problematic parameter, as it is notin fact a constant, but necessarily a temporaryweighted mean of different species, fluctuatingduring the course of succession within thecommunity. Furthermore, the separation of thecomponents of the kinetic sum value (K + S)obtained in the substrate uptake analysis of naturalcommunities is laborious, and the sum value canusually be considered only as an upper limit forboth parameters.With these constraints in mmd, the halfsaturation constant can be used as a generalindicator of the utility of the substrate forplanktonic community. The K values in Table 2are one to three orders of magnitude higher thanthose usually measured with natural microplankton(see for example Sepers 1977, Hoppe 1978,Kuznetsov and Romanenko 1979). This differencecan hardly be attributed only to the effects oflaboratory conditions.Table 2. Kinetic parameters of glucose uptake in the aigal fraction.Species Density Light V V per cell K V (dark)cell . mP1 pg . h pg h1 . ce111 pg . 1-’ V (light)Prorocentrum micans 3040 0 57.6 18.9 1000P.micans 3040 100 50.2 16.5 530 1.15P.micans 5880 0 37.2 6.3 290P.micans 5880 100 38.9 6.6 260 0.95P.micans 8220 0 55.4 6.7 480P.micans 8220 100 60.8 7.4 620 0.91Gymnodinium sp]endens 320 0 19.1 59.7 210G.splendens 320 100 15.9 49.7 140 1.20G.splendens 600 0 17.5 29.2 240G.splendens - - 600 100 18.1 - 30.2 - 210 - 0.97Gyrodinium fissum 6420 0 64.3 10.0 810G.fissum 6420 100 78.0 12.1 910 0.83Mean 1.0024Therefore, it can be assumed that thesemixotrophic dinofiagellates are likely to play only aminor role in the cycling of dissolved organiccompounds of low molecular weight in nature. Ourresults are in agreement with the paradigmpreviously presented for the heterotrophy ofnatural bacterio- and phytoplankton (Wright andHobbie 1966), as the glucose uptake kinetics of thedinoflagellates were qualitatively less efficient thanthose of both natural bacterioplankton andbacteria in the laboratory cultures. Concentrationsof glucose — or of other components of the labiledissolved organic carbon pool — in natural waterbodies hardly ever reach the range in which aigalheterotrophic capacity would overcome that ofbacterioplankton (hundreds of micrograms perliter), with the possible exception of extremelyeutrophic conditions.This conclusion of the quantitatively minorimportance of dinoflagellate heterotrophy for thecycling of the labile fraction of dissolved organiccarbon does not neglect the possible qualitativesignificance of heterotrophy for algal nutrition. Itis here considered mainly with regard to the carboncycle and the utilization of the single concentrationmethod for determination of the heterotrophicpotential of natural bacterioplankton communities.If substrate additions are in the range of thosepresented by Kuparinen et al. (1984), theseexperiments support the assumption that therneasurements describe the heterotrophic metabolism of bacterioplankton.LOPPUTIIVISTELMÄMerellisten dinofiagellaattien Prorocentrum micansEhr., Gymnodinium splendens Lebour. ja Gyrodinium fissum (Lev.) heterotrofista potentiaalia mitattiin‘4C-glukoosin nettoassimilaation avulla valossa ja pimeässä. Leväsolujen heterotrofinen potentiaali oli korkeimmillaan nuorissa viljelmissä.Valo ei vaikuttanut heterotrofiaan. G. splendensosoittautui tehokkaimmaksi glukoosin käyttäjäksisekä solukohtaisen maksimaalisen ottonopeudenettä puolikyllästysvakion (K) perusteella.Kaikkien viljelmien glukoosin käyttö oli tehotonta. Puolikyllästysvakiot (140—1000 pg glukoosia 1-1) olivat kertaluokkia korkeampia kuin luonnon planktonyhteisöissä tavallisesti mitatut. Onepätodennäköistä, että nämä miksotrofiset dinoflagellaatit vaikuttaisivat merkittävästi vesistöissä mitattaviin heterotrofisiin parametereihin.ACKNOWLEDGEMENTSThis study was carried out in the Moscow StateUniversity and was made possible by the mutualscientific exchange program between the MoscowState University and the University of Helsinki.The authors wish to thank the following personsfor kind cooperation: professors V.D. Fyodorovand V.N. Maximov of the Department of GeneralEcology and Hydrobiology, MSU; Ms. R.D.Ozrina of the Isotope Laboratory, Faculty ofBiology, MSU; Mr. V.M. Chromov of theDepartment of General Ecology and Hydrobiology, MSU; Mr. A.N. Puchkov of the Department of General Ecology and Hydrobiology,MSU; professor R. Ryhänen of the Department ofLimnology, University of Helsinki; Ms. E. Melkas,University of Helsinki; Ms. S. Hällfors and Mr. G.Hällfors of the Tvärminne Zoological Station,University of Helsinki; the collective of theaspirant room of the Department of GeneralEcology and Hydrobiology, MSU.Helsinki, September 1982Timo Tamminen, Seppo Kaitala, Ljudmila V. Iliash.REFERENCESAllen, H.L. 1971. Dissolved organic carbon utilization insizefractionated aigal and bacterial communities. Int.Rev. ges. Hydrobiol. 56: 73 1—749.Azam, F. & Hodson, R. 1977. Size distribution andactivity of marine microheteroprophs. Limnoi. Oceanogr. 22: 492—501.Berman, T. 1975. Size fractionation of natural aquaticpopulations associated with autotrophic and heterotrophic carbon uptake. Mar. Biol. 33: 215—220.Gocke, K. 1975. Untersuchungen iiber die Aufnahme vongelöster Glukose unter natiirlichen Verhältnissendurch grössenfraktionierter Nano- und Ultrananoplankton. Kieler Meeresforschungen 31: 87—94.Hoppe, H-G. 1976. 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The heterotrophic glucose uptake potential of three marine dinoflagellates

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