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Sinking rates responses of oceanic phytoplankton to irradiance, nutrients, and iron stress Lecourt, Maude 1994

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SINKING RATES RESPONSES OF OCEAMC PHYTOPLANKTONTO IRRADIANCE, NUTRIENTS, AND IRON STRESSByMAUDE LECOURTB.Sc., Laval University, 1992A THESIS SUBMrn:’ED IN PARTIAL FULFILLMENT OF THEREQUIREMENTS FOR ThE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Oceanography)We accept this thesis as confirmingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIASeptember 1994®Maude Lecourt, 1994In presenting this thesis in partial fulfillment of therequirements for an advanced degree at the University of BritishColumbia, I agree that the Library shall make it freely availablefor reference and study. I further agree that permission forextensive copying of this thesis for scholarly purposes may begranted by the head of my department or by his or herrepresentatives. It is understood that copying or publication ofthis thesis for financial gain shall not be allowed without mywritten permission.(SignDepartment of_________________The University of British ColumbiaVancouver, CanadaDate 2(cfdj)J11ABSTRACTEmiliania huxleyi (Lohmann), a small oceanic coccolithophore,was isolated from the NE Subarctic Pacific Ocean. The sinking ratesof two strains of E. huxleyi, naked and coccolith-forming, weremeasured. The two strains were grown under saturating and light-limited conditions in media containing either NO3 or NH4 as theprimary nitrogen source. Sinking rates were measured during logand senescent growth phases. The naked strain grew significantlyfaster under saturating light than the coccolith-forming strain. Thecoccolith-forming strain of E. huxleyi had a significantly larger cellvolume and higher sinking rates than the naked strain under allconditions. The naked strain increased significantly its chi a contentunder light limitation. POC and PUN were significantly higher for thecoccolith-forming strain. During senescence, coccolith formation andcell aggregation increased for the coccolith-forming strain and thenaked strain, respectively, significantly increasing their sinking rates.No significant difference in physiological parameters was observedbetween NO3 and NH4+ grown cells, except that growth rate wasfaster under NH4 at saturating light. These results suggest thatcoccolith formation controls sinking rates, and therefore thecoccolith-forming strain has some advantages over the naked strain.This study also determined the sinking rates responses,physiological parameters, and iron quotas of E. huxleyi (coccolithforming strain) and an oceanic strain of the diatom Actinocyclus sp.(20 im diameter) under iron deficiency. Actinocyclus sp. was grownunder N03- and NH4+ to determine whether there was an advantagefor cells to grow on NH4 rather than NO3- under iron-depleteconditions. E. huxleyi was grown on NO3- only. Under iron-deficientconditions, Actinocyclus sp. increased its sinking rate 9 fold, despiteits 30% decrease in cell volume. E. huxleyi maintained its sinkingrates, but deceased its cell volume by 50%. Growth rates and ironquotas of Actinocyclus sp. and E. huxleyi significantly decreasedunder iron-deplete conditions. E. huxleyi maintained its POC and PUN111and increased its chi a content under iron-deplete conditions. On theother hand, Actinocyclus sp. significantly decreased its POC, PON andits chi a content when grown on N03. The results obtained underiron-deplete conditions indicate that E. huxleyi grown on N03 wasnot significantly affected by iron stress. However, Actinocyclus sp.was affected by iron deficiency, and was energetically affected bythe nitrogen source. Finally, small coccolithophores are betteradapted to their habitat (i.e. low iron concentrations in the NESubarctic Pacific) and can easily outcompete large diatoms in a lowiron environment.ivTABLE OF CONTENTSABSTRACT.iiACKNOWLEDGEMENTS viLIST OF FIGURES viiGENERAL INTRODUCTION 1Why study sinking rates7 1Factors affecting sinking rates 3Objectives 1 0CHAPTER 1. EFFECTS OF IRRADIANCE ON SINKING RATES OFNON-COCCOLITH AN]) COCCOLITH-FORMING STRAINS OFEMILIANIA HUXLEYI 11Introduction 11Materials and Methods 1 5Culture conditions 1 5Cell composition 1 6Analytical methods 1 7Results 1 9Growth vs irradiance 1 9Growth rate 1 9Cell volume 20Sinking rate 20Biochemical composition 23Scanning electron microscopy 29Discussion 3 1CHAPTER 2. SINKING RATES RESPONSES OF EMILIANIA HUXLEYIAND ACTINOCYCLUS SP.TO IRON STRESS 42Introduction 42Materials and Methods 46Culture conditions 46Cell composition 48Analytical methods 48Results 49Growth rate 49Cell volume 5 1Sinking rate 5 1VIron quota .5 1Carbon:nitrogen ratio .52Chlorophyll a 55Fluorescence:Chl a ratio 5 5Nitrogen 55Carbon 56Discussion 5 9General Conclusions 7 1Future Research 7 3References 74Appendix 86viACKNOWLEDGEMENTSI wish to express my thanks to my parents for their moral andfinancial support during these two years. I also thank the membersof the Oceanography Department, and the members of the Harrisonlab who helped and encouraged me during my stay. I express myappreciation to two friends: Anne Fisher for sharing with me all hersinking rate knowledge, and for chatting with me when I did not feellike working; Debbie Muggli for letting me use her isolate of E.huxleyi and her data. I also want to thank her for proposing projectideas.Finally I wish to express my sincere gratitude to my researchsupervisor, Dr. Paul 3. Harrison, for supporting my work, for hisadvice during the past two years and especially for all the help heoffered in the writing of this thesis.viiLIST OF FIGURESFIGURE1 Growth rate vs irradiance for E. huxleyi with and withoutcoccoliths grown on ammonium or nitrate media 202 Specific growth rate for E. huxleyi with and withoutcoccoliths vs nitrogen source and irradiance 203 Cell volume and sinking rates for E. huxleyi with and withoutcoccoliths vs nitrogen source and irradiance during log andsenescent phase 224 Chl a per cell volume and fluorescence:chl a for E. huxleyiwith and without coccoliths vs nitrogen source andirradiance during log and senescent phase 255 Nitrogen per cell volume and carbon per cell volume forE. huxleyi with and without coccoliths vs nitrogen sourceand irradiance during log and senescent phase 266 Carbon:nitrogen ratio for E. huxleyi with and withoutcoccoliths vs nitrogen source and irradiance duringlog and senescent phase 277 Carbohydrate per cell volume and lipid per cell volume forE. huxleyi with and without coccoliths vs nitrogen sourceand irradiance during log and senescent phase 2 88 Scanning electron micrographs of E. huxleyi naked andcoccolith-bearing strains 3 09 Scanning electron micrograph of Actinocyclus sp 45viii1 0 Growth rates for E. huxleyi in Fe-replete nitrate mediumand in three grow-ups (transfers) in Fe-deplete nitratemedium 5011 Growth rates for Actinocyclus sp. in Fe-replete medium and infive grow-ups (transfers) in Fe-deplete, nitrate orammonium media 501 2 Cell volume, sinking rates, natural and acidified-Ti wash Fequotas, and carbon:nitrogen ratio for E. huxleyi grown in nitratemedium under both Fe-replete and Fe-deplete conditions 5 31 3 Cell volume, sinking rates, natural and Ti(III) Fe quotas,and carbon:nitrogen ratio for Actinocyclus sp. grown innitrate and ammonium media under both Fe-repleteand Fe-deplete conditions 5 41 4 Chl a per cell volume, fluorescence:chl a ratio, nitrogen percell volume, and carbon per cell volume for E. huxleyigrown in nitrate medium under both Fe-replete andFe-deplete conditions 5 71 5 Chl a per cell volume, fluorescence:chl a ratio, nitrogenper cell volume, and carbon per cell volume forActinocyclus sp. grown in nitrate and ammonium mediaunder both Fe-replete and Fe-deplete conditions 5 81SINKING RATES RESPONSES OF OCEANIC PHYTOPLANKTON TOIRRADIANCE, NUTRIENTS, AND IRON STRESSGENERAL INTRODUCTIONMarine phytoplankton are the most abundant and widelydistributed form of plant life on Earth. Their annual net carbonproduction of 1010 tons represents at least one-half of the totalglobal plant production (Smayda, 1970). During the spring bloom,phytoplankton dominate the surface waters due to their high growthrate which is favored by high nutrients, warmer water andincreasing irradiance with longer daylength. Later in the seasonwhen surface nutrients are depleted, light levels are too high at thesurface and zooplankton populations have increased, it isadvantageous for phytoplankton to sink down to a cooler, nutrient-rich environment (Culver and Smith, 1989).The success of a phytoplankton population largely depends onits ability to remain suspended in the photic zone, where sufficientlight energy for photosynthesis is available. Motile cells can remainsuspended by swimming towards the light, but non-motile cellsgenerally have a specific weight that exceeds seawater, and thereforethey sink from the euphotic zone (Smayda, 1970). For example, thespecific weight of a diatom has been reported to be 2.07 g m11, andthat of seawater usually ranges from 1.020 to 1.028 g m11 (Smaydaand Boleyn, 1965). Elucidation of the mechanisms of phytoplanktonbuoyancy remains a classic problem in biological oceanography.Why study sinking rates?Marine phytoplankton sinking rates are potentially importantin determining the vertical distribution of phytoplankton biomassand productivity (Bienfang et al., 1982). The increase inphytoplankton biomass during a spring bloom represents anavailable food source that is essential to the recruitment success ofvarious zooplankton and fish. Subsequent sedimentation of the2bloom also constitutes a major input of biogenic material to thebenthos (Waite et al., 1992a). Another ecological reason to studysinking rates is to understand the species succession pattern in theupper mixed layer. For example, once a simply structured diatombloom sinks out of the euphotic zone, it can be replaced by a morecomplex recycling community consisting of various phytoplanktonspecies often dominated by flagellates and picoplankton (Guillardand Kilham, 1977; Smetacek, 1985).Recent interest in phytoplankton sinking rates is related to therole of the ocean as a possible sink for atmospheric C02 and itsimplication for global warming. The amount of carbon that can beabsorbed by the ocean is greatly augmented by the sinking ofbiogenic particulate organic matter and calcium carbonate into thedeep sea (Westbroek et al., 1993). As a result of this “biologicalpump”, the bottom waters contain a supersaturated concentration ofC02. The C02 is then trapped for up to 1000 years in the cold deepsea by vertical density stratification and cannot escape to theatmosphere until thermohaline circulation returns this deep water tothe sea surface (Libes, 1992). In addition, if phytoplankton reach thesediments, the carbon they contain might be buried for hundreds ofthousands of years (Martin and Gordon, 1988).A knowledge of the buoyancy phenomenon is not only vital fora proper understanding of the dynamics of phytoplankton retentionwithin the euphotic zone, but the vertical distribution of nonconservative substances and the general biochemical structure of theoceans are also influenced by the nutrient transport of sinkingplankton (Smayda and Boleyn, 1965). Sinking rates are even moreimportant because it should be possible to evaluate the carbonproduction that is negatively buoyant and its rate of downward flux,both of which are important parameters needed to quantify theglobal carbon cycle in the ocean (Smetacek, 1985). However, somemechanisms must permit periodic return of cells to the euphoticzone, since without this, the autotrophic phytoplankton species woulddecline over time in the surface layers.3Factors affecting sinking ratesCell size and shapeSinking rates are believed to be dependent both on thephysical characteristics of phytoplankton cells, such as size andshape, and on physiological phenomena which affect cell density(Bienfang et al., 1977). According to Stokes’ law, the sinking rate of aparticle greater than 5 p.m diameter is proportional to its size.However, large organisms appear to sink proportionally less rapidlythan is predicted by Stokes’ law (Smayda, 1970). The sinking rate ofa spherical particle can be changed by deforming its shape, withoutaltering its density or volume. The effect of shape on sedimentationis usually expressed in terms of its coefficient of form resistance(Walsby and Reynolds, 1980). Any deviation from a spherical shape(such as developing long spines and other protuberances) wouldincrease the surface area to volume ratio and so enhance thefrictional resistance of the cell and slow its passage through thewater (Smayda, 1970).Cell chain and aggregatesWhen two or more cells form a chain, the area where theytouch is no longer accessible to the suspending medium. Hence, thechain sinks more rapidly than the component cells (Walsby andReynolds, 1980). However, the effect of chain length on sinking rateseems to vary among species, depending on the nature of theintracellular connection, and chain morphology (Smayda, 1970).Aggregation can also be a way for phytoplankton to control theirsinking rate. The process by which aggregation occurs is commonlyunderstood to be an increase in cell surface stickiness, allowingclumping of cells as well as the incorporation of debris and mineralparticles in the water column, and eventually forming largeraggregates with much higher sinking rates than the individual cells(Smetacek, 1985). Finally, the transformation during the life cycle ofa cell from a non-motile to a motile stage can aid suspension.4Cell densityThe fact that phytoplankton have physiological control overtheir sinking rate followed the observation that sinking rates weremuch higher in preserved cells than live cells which obviously hadno metabolic control (Smayda, 1970). One of the ways proposed bywhich an active cell could change its sinking rate, was by changing itscell density. Cell density reflects the cell composition and theamount of the cell wall, cytoplasm and cell sap materials (Walsbyand Reynolds, 1980). The dominant forms of the several algal classesthat are represented in a phytoplankton assemblage usually havesurface deposits of silica, cellulose or calcium carbonate.i) Cell wall: Diatoms have a hydrated silicon dioxide cell wallsimilar to opal; coccolithophores have calcium carbonate scales(coccoliths) of calcite, aragonite or vaterite; and many dinoflagellateshave an internal surface of cellulose platelets (Smayda, 1970). Asignificant ballast results from this mineralization of silica andcalcium carbonate. For example, 40% of the dry weight of somecoccolithophores is due to calcium carbonate (Paasche, 1962), and thefrustule of the diatom can comprise up to 50% of their dry weight(Strickland, 1970).ii) Carbohydrates: Among the major cell constituents,carbohydrates have a density close to that of seawater.Carbohydrates are the first products of photosynthesis, and supplyenergy to the general metabolic cycles of the alga, as well as providecarbon skeletons for biosynthesis (Myklestad, 1988). Carbohydratecontent varies with nutrient status, irradiance, and growth phase.Relative intracellular carbohydrate concentration has been observedto increase markedly during senescent phase, including senescenceinduced by nitrate or phosphate limitation, for most phytoplankton(Mykiestad and Haug, 1972). Similarly, cells growing under highlight contain a large amount of carbohydrate, while cells at low lighthave little excess carbohydrate (Falkowski and LaRoche, 1991).5iii) Lipids: Most of the chemical components which make upthe protoplasm of living cells are heavier than water. Only lipidshave a density lower than that of seawater. Initially, it was thoughtthat the production of fats could compensate for the excess densityand allow suspension. The effect of environmental conditions on theproduction and storage of lipids in algae is not fully understood.Similar environmental factors have been shown to exert oppositeeffects. In general, fat production increases in a stationary orsenescent culture, suggesting that fat accumulation is a degenerativeprocess (Smayda, 1970). In most algae, enhancement of lipidaccumulation is caused by nitrogen-deficient conditions (Shifrin andChisholm, 1981). Also, high lipid content is typical of cells grown inhigh light intensities (Sukehik et al., 1989). However, the fataccumulation hypothesis, as a mechanism to control sinking rate, wasdiscarded by Smayda (1970), because lipids do not usually make upthe majority of the dry weight of a cell, and therefore cells willusually be heavier than water and will sink. Also, fat levels tend toincrease during senescence when sinking rates are highest (Smayda,1970).iv) Ionic pump: Amongst other changes in intracellulardensity, Ostwald (1902, quoted in Smayda, 1970) proposed that thesinking rate of phytoplankton is controlled by ionic regulation. Byselective accumulation of the lighter ions from seawater, or fromproducts of metabolism, it is possible to produce a vacuole that is lessdense than seawater and therefore it is able to provide buoyancy(Walsby and Reynolds, 1980). The size of the vacuole is importantfor this mechanism. Therefore, this ionic pump hypothesis has beenproposed as a buoyancy mechanism for some of the largerphytoplankton, such as diatoms, because they have a lower surfacearea to volume ratio than small phytoplankton (Gross and Zeuthen,1948; Smayda, 1970).For example, studies on the large diatom Ditylum brightwelliishowed that the density of the cell sap was lower than that ofseawater by excluding heavy ions (Ca2+, Mg2, and S042), over6lighter ions such as NH4 (Andersonand Sweeney, 1977, 1978). Also,high sinking rates associatedwith a high K/Na ratio (the atomicweight of potassium is about40% greater than that of sodium) havebeen reported (Smayda, 1970).Although, density is generally considered to be one of the mostimportant determinants of a cell’s sinking rate, others factorssuch asgrowth rate, irradiance, and nutrients will affect physiologicalprocesses and cause changes in sinking rates.It is generally accepted that thephysiological state ofpbytoplankton has a pronouncedinfluence on their sinking rates.Numerous laboratory and fieldexperiments indicate an associationbetween buoyancy regulation and physiological processes and,therefore, a connection to growth and photosynthetic processes(Eppley et al., 1967; Smayda, 1970; Anderson and Sweeney, 1977,1978; Bienfang, 1981 a,b). Becauseof this relationship,environmental variations in nutrients and irradiance, both of whichaffect production, may also influence buoyancy and ultimatelyvertical flux.Growth rateThe metabolic activity of phytoplankton makes them capable c,fvariable composition which cancause differences in particle densityand thereby influence sinkingrates (Bienfang, 1981a). Sinking ratesand growth rates are, in general, inversely correlated in the majorityof planktonic algae (Eppley et al., 1967; Smayda, 1970; Waisby andReynolds, 1980). As growth slows, individual cells seem to becomedenser, cease to divide, and eventually sink out. Steele and Yentscb(1960) reported that the “settling rate of actively dividing cells wasapproximately one-half the rateof slowly dividing senescent cells.”Decreases in sinking rates havealso been shown to occur insenescent cultures after nutrient enrichment (Smayda and Boleyn,1965; Smayda, 1970).7It is also a common observation that the surfaces of senescentcells are generally stickier than those of actively growing cells (Fogg,1966; Anderson and Sweeney, 1977; Smetacek, 1985; ). Thisphenomenon would tend to enhance aggregation, and thereforeincrease sinking rates. Because growth rate is a function of bothnutrients and irradiance (as well as other factors), the effect of asingle variable on sinking rates may not be as important as thecombined effects of all factors which influence growth (Culver andSmith, 1989).IrradianceThe hypothesis that sinking of phytoplankton cells is controlledby the factors which regulate photosynthesis was supported byseveral experiments (Steele and Yentsch, 1960; Anderson andSweeney, 1977, 1978; Bienfang, 1980; Bienfang et al., 1983). Anincrease in irradiance resulted in an increase in both growth andsinking rates for several species. The positive correlation suggeststhat in a low irradiance environment, sinking rates are lower becauseof lower growth and photosynthesis (Culver and Smith, 1989).Steele and Yentsch (1960) were the first to propose thatdiatoms might decrease their sinking rate in response to low lightand so contribute to the formation of the deep chlorophyll maximumlayer. From an adaptive point of view, it is an advantage for a cell toslow its sinking rate under low light conditions. However from anenergetic point of view (Waite et al., 1992b), a cell grown under highlight should be better able to maintain its buoyancy. According toWaite et al. (1992b), sinking rate control might be maintained as longas carbon stores were available to release energy, and a cell shouldreach its maximum sinking rate when all the energy sources wereexhausted.NutrientsAlthough a change in sinking rates has been seen to be highlyspecies-specific, and dependent on the cell’s previous lightconditions, it is well established that nutrient limitation also causes8an increase in sinking rate (Bienfang et al., 1982; Bienfang andHarrison, 1984). Smayda (1970) postulated that sinking faster undernutrient limitation could be a mechanism to reduce the small scalenutrient gradient caused by local uptake, and increase the diffusivesupply of nutrients to the cell. Optimal levels of light and nutrients,the two factors which most often limit phytoplankton growth, rarelycoincide in the open ocean. As a result, a cell with the ability toregulate its location within the water column by passive movementcould maximize its productivity and growth (Culver and Smith,1989).i) Nitrate vs ammonium: Amongst the various nutrients,nitrogen is the most abundant constituent of algal biomass (Syrett,1981). Ammonium (NH4+) and nitrate (NO3-), are generallyconsidered to be the most important sources of nitrogen forregenerated and new production, respectively (Dugdale and Goering,1967). In the presence of both nitrogen sources, NH4 ispreferentially taken up over N03, because nitrate must be reducedto first nitrite and then to ammonium before being incorporated intoamino acids (Syrett, 1981; Thompson et al., 1989; Levasseur et al.,1993).Also the energy cost for N03 uptake is higher than that forNH4 (Falkowski, 1975; Thompson et al., 1989; Turpin and Bruce,1990). Therefore, if the production of sufficient reductant limitsgrowth under conditions of low energy input (for example lightlimitation) then it should be possible to detect some indication of thislimitation in cells growing on nitrate vs ammonium (Thompson et al.,1989). In many cases however, the growth rates on NO3- were notsignificantly different from the growth rates on NH4 (Syrett, 1981;Thompson et al., 1989; Levasseur et al., 1993). Thompson et al.(1989) suggested that “phytoplankton growing on N03 maycompensate for their higher reductant requirements in other ways,such as adjustments in their biochemical composition, rather than byreducing their growth rates.”9How phytoplankton sinking rate is affected by factors such aslight, macro and micronutrients, and growth rate has been studiedfor several decades. However, most of the phytoplankton speciesexamined have been diatoms. The function of coccoliths incoccolithophores has been investigated, and some factors affectingcoccolith formation and mechanisms of calcification have beenelucidated (Paasche, 1964, 1965; Nimer and Merrett, 1992; Sikes andFabry, 1993). On the other hand, sinking rate studies oncoccolithophores are rare (Eppley et al., 1967; Bienfang, 1981 a,b).Thus far, the effect of nutrient limitation on sinking rates hasfocused on the macronutrients, but micronutrients such as iron, mayalso have a direct and immediate effect on the sinking rate ofphytoplankton. Iron is an essential micronutrient for phytoplanktonand exhibits some of the same characteristics of uptake asmacronutrients. Phytoplankton require more iron than any othertrace metal. Iron deficiencies affect several processes associatedwith photosynthesis in a variety of plant types (Morel et al., 1991a).Since the iron limitation hypothesis postulated by Martin et al.(1989), evidence has been obtained that iron is the factor limitingprimary production in some oligotrophic open ocean waters, whereiron concentrations and influx rates are low (Martin et al., 1990).Although the determination of the proportion of total iron inseawater that is bioavailable for uptake by phytoplankton is stilluncertain (Wells, 1988,89), many field enrichment experiments haveshown that low iron concentrations affect the physiology and speciescomposition in natural phytoplankton assemblages (Martin et al.,1989; Martin et al., 1990; Boyd et al., submitted). However,physiological studies on relevant oceanic species that could shed lighton some of the observed dynamics are rare.10The purpose of the present study was to increase ourknowledge of coccolithophore sinking rates and the physiology ofcoccolith vs non-coccolith forming strains. Sinking rates of Emilianiahuxleyi (naked vs coccolith-forming strains) were measured underdifferent irradiances in media containing either nitrate or ammoniumas the nitrogen source. Furthermore, this thesis examined for thefirst time sinking rates and physiological parameters of two oceanicphytoplankters, Emiliania huxleyi and a larger diatom, Actinocyclussp., under iron deficient conditions. Also, the effects of nitrogensource under iron deplete conditions was investigated forActinocyclus sp.OBJECTIVESThis thesis addresses the following five objectives:1. To measure the sinking rates of Emiliania huxleyi with andwithout coccoliths.2. To determine the effect of different irradiances on thesinking rates of E. huxleyi.3. To examine the effect of nitrogen source (N03 vs NH4) onthe sinking rates of E. huxleyi.4. To measure the sinking rates of E. huxleyi and the diatomActinocyclus sp. under both iron replete and iron deplete conditions.5. To examine the effect of nitrogen source under iron deficientconditions on the sinking rates of Actinocyclus sp.11Chapter 1EFFECTS OF IRRADIANCE ON SINKING RATES OF NONCOCCOLITH AND COCCOLITH-FORMING STRAINS OFEMILIANIA HUXLEYIINTRODUCTIONCoccolithophores are a group of phytoplankton speciesbelonging to the phylum Prymnesiophyta which is widely distributedin the world’s oceans. The importance of coccolithophores in thebiogeochemical cycling of carbon is based not only on their capacityfor photosynthetic carbon fixation, but also on their unique abilityamongst the phytoplankton to synthesize external plates of calcite,called coccoliths. These coccoliths may represent a large proportionof the total flux of particulate carbon to the deep ocean in some areas(Holligan et al., 1993). Thus coccolithophores are known tocontribute significantly to the fine fraction of oceanic sediments inthose areas where the sea floor lies above the lysocline (The depth atwhich shell dissolution starts to have a detectable impact on thecalcium carbonate content of the surface sediments) (Fernandez etal., 1993).Coccolithophores are also important because they producedimethylsulphonium propionate (DMSP). The oxidation products ofDMSP and dimethylsulfide (DMS) may accumulate as aerosols of nonseasalt sulfate above the ocean (Keller, 1989). In the atmosphere,these particles act as cloud condensation nuclei, enhancing cloudformation with potential influences on global climate, by changingheat transfer and absorption of the sun’s radiation (Shaw, 1983,quoted in Charison et al., 1987).Among coccolithophores, Emiliania huxleyi is presently themost widespread species in the world’s oceans, being found from thetropics to high latitude regions and from mid-ocean to inshorewaters, and it probably produces more calcite than any other single12phytoplankton species on Earth today (Holligan et al., 1993). Whilethe literature is extensive with studies on coccolith formation,comparatively little is known about the physiological andmorphological responses of E. huxleyi to environmental factors suchas light and nutrient variations.The life cycle of Emiliania huxleyi includes a motile uncalcified(S-cell type), a non-motile calcified form (C-cell type) and a naked(N-cell) type which, although possessing the basic apparatus forcoccolith synthesis, bears no coccoliths (Braarud, 1963; Paasche andKiaveness, 1970; Kiaveness, 1972). The rate of coccolith productionis variable. In natural populations, coccolith-bearing cells tend toaccumulate coccoliths at the cell surface resulting in self-shedding.However, cultured isolates tend to produce fewer coccoliths; oftenonly a proportion of the cell may be covered. With time, the abilityto produce coccoliths is often lost and the culture becomes dominatedby naked cells (Flynn, 1990).The carbon sources for photosynthesis and coccolith formationand the net inorganic reaction of deposition are important indetermining the extent to which carbon fixation by coccolithophoresmight be a sink for atmospheric C02. It has been shown by Paasche(1964), and confirmed many times since then, that coccolithformation in E. huxleyi is essentially a light-driven process. In theabsence of light, coccolith formation in this species proceeds veryslowly or not at all (Linschooten et al., 1991). However, neitherprocess is an absolute prerequisite for the other, as photosynthesistakes place unhindered in a calcium-free medium, whereascalcification may proceed at a reduced rate when photosynthesis isblocked by an inhibitor (Paasche, 1965). Therefore, the inorganicreactions of CaCO3 deposition and photosynthesis include both adirect uptake of external C02 as a substrate for photosynthesis andan influx of HCO3- from the sea that supplies both carbonatedeposition and C02 for photosynthesis through complementaryreactions (Sikes and Fabry, 1993).13Various hypotheses have been put forward to explain the roleplayed by coccoliths in the physiological ecology of coccolithophores.A number of laboratory experiments have suggested that factorssuch as temperature, nutrient supply, and the prevailing rates ofphotosynthesis and protein synthesis affect selection ordifferentiation between uncalcified and calcified forms (Flynn, 1990).For example, the coccolith production per cell appears to be enhancedunder nitrogen and phosphate depletion (Baumann et al., 1978).Also high temperatures appear to favour the growth of calcifiedforms of coccolithophores in nature, with uncalcified formspredominating in Arctic waters (Kiaveness and Paasche, 1979).There has been considerable speculation that coccolithproduction may allow cells to sink to the bottom of the euphotic zone,while the transformation of coccolith-bearing cells into naked cellsmay allow them to rise. Coccolith formation being light dependent, ithas been suggested that high light intensities and subsequentlylower nutrient concentrations may stimulate coccolith production.The increased calcification would increase the cell density, causingcells to sink into deeper, nutrient-rich waters which would increasethe rate of nutrient supply to their cell membrane by disturbing theunstirred layers around the cell (Munk and Riley, 1952). Once thecells are in nutrient-replete conditions, cell division would take placeand gives rise for the naked stage (motile or non-motile), and thencells would begin to migrate upward towards higher light intensitiesto undergo increased photosynthesis and growth until the cyclestarts again. This migration cycle in the photic zone is similar to thecycle proposed for large diatoms such as Rhizosolenia (Villareal et al.,1993) and Ditylum (Waite and Harrison, 1992).Another possible advantage of coccoliths is that when theybecome detached, they increase light scattering which raises theambient water temperature towards the growth optimum of —18°C(Westbroek et al., 1993). Also by scattering light, coccoliths mayprotect the cell against photodamage when they are near the surface(Braarud et al., 1952).14Although both cell types have been used in experimental work,there have been only a few direct comparisons between naked andcoccolith-bearing cells. The purpose of the present study was todetermine how physiological and physical conditions were associatedwith modifications in the buoyancy of this species. This wasachieved by comparing the sinking rate response of Emiliania huxleyiwith and without coccoliths under different irradiances in mediacontaining either nitrate or ammonium as the nitrogen source. Otherphysiological parameters, such as growth rates and cell compositionwere also measured.15MATERIALS AND METHODSCulture conditionsEmiliania huxleyi (Lohmann) was isolated in November, 1991from the Subarctic Pacific Ocean (Station Papa, 50°N, 145°W) by D. L.Muggli. Cultures of E. huxleyi were maintained in low macronutrientand micronutrient Stn P water to retain the organism’s ability toform coccoliths. The naked strain came from the same Station Papaisolate (originally bearing coccoliths), but was maintained in artificialseawater (Harrison et al., 1980) with high concentrations of nutrientsin the Northeast Pacific Culture Collection (NEPCC No. 732),Department of Oceanography, University of British Columbia, whereit lost the ability to form coccoliths after approximately one year.E. huxleyi, with or without coccoliths, was grown in semi-continuous batch cultures in enriched artificial seawater (ESAW)based on the recipe by Harrison et al. (1980), with the followingmodifications; ferrous ammonium sulphate and sodiumglycerophosphate were replaced with equimolar ferric chloride andsodium phosphate respectively, and selenite and molybdate wereadded to a final concentration of 1 nM. Macronutrient enrichmentwas reduced to 30 j.tM of either nitrate or ammonium and 2.0 j.iMphosphate. Silicate was omitted. Trace metal concentrations werereduced to ESAW/50 (Harrison et al., 1980).Cultures were grown in triplicate with either nitrate orammonium as the nitrogen source in 2 L glass flasks. Steriletechniques were used for all culture work in an attempt to minimizebacterial growth; the presence or absence of bacteria was notconfirmed. Temperature was maintained at 17°C and the cultureswere bubbled with air filtered through a 0.22 p.m membrane filter.The flasks were not mechanically stirred because stirring tended toknock off the coccoliths, but the cultures were gently swirled byhand twice daily. Continuous light was provided by Vita-lite16fluorescent tubes and attenuated by distance or neutral densityscreening to give a saturating (150 pmol photons rn-2 s1) and alimiting irradiance (20 imol photons rn-2 s1). The irradiance wasmeasured using a Biospherical Instruments model QSL-l00 lightsensor.Growth rates were followed by in vivo fluorescence, measureddaily using a Turner Designs model 10 fluorometer and cell countsusing a Coulter Counter model TAIl. Cell volume was also measuredon the Coulter Counter, although no correction was made for thepossible discrepancy between Coulter Counter volumes and volumesobtained microscopically (Montagnes et al., in press). The pH of theculture sample was decreased to 5 with HC1 before counting todissolve the coccoliths and prevent interference with cell counts; thispH did not affect the cell volume and the cells remained intact. Thesame treatment was conducted for the naked strain. All samplingwas conducted in mid-logarithmic growth phase and at senescentphase (2-3 days after reaching the cell density plateau).The irradiance vs growth rate experiment was carried outusing triplicate 50 ml glass tubes and the culture was grown underthe same conditions described above. Growth was monitored directlyin the culture tubes by reading in vivo chlorophyll a fluorescence atthe same time every day. The cultures were exposed to 10 differentphoton flux densities (PFDs) ranging from 10 to 350 imol photonsrn2 s-1 and supplied with either nitrate or arnmonium undercontinuous light. Three culture transfers were made prior to thebeginning of each experiment to ensure that the cells wereacclimated to the new irradiance.Cell compositionIn all experiments, samples for particulate organic carbon andnitrogen were determined by filtering samples onto pre-combusted13 mm Gelman A/E filters and analyzed on a Carlo Erba CHNanalyzer. Samples for chlorophyll a were filtered onto precombusted17Whatman GF/F filters and then extracted in 10 ml of 90% acetone,sonicated for 10 mm, and stored for 24 h in the dark at 4°C.Chlorophyll a concentrations were calculated from in vitrofluorescence measurements (Parsons et al., 1984).Carbohydrates were determined using the method of Kochert(1978) and using H2S04 instead of NaOH for the extraction asmodified by A. E. Fisher (pers. comm.). Carbohydrates in the cellswere extracted from a GF/F filter using 3N H2S04 and heated to100°C in a water bath for 2 h, quantified using the phenol-sulfuricacid method, and then measured using a LKP Ultrospec II UVspectrophotometer (wavelength = 490 nm).Subsamples for total lipids were extracted inchloroform:methanol:water (2:4:1) (Bligh and Dyer, 1959) andanalyzed by the lipid oxidation technique (Parsons et al., 1984) usingtripalmitin as a standard.Analytical methodsSinking rates were measured using the SETCOL method(Bienfang, 1981c). Cell number, measured on a Coulter Counter, wasused as a biomass index for the SETCOL calculations. Samples fromthe SETCOL were preserved in Lugol’s solution for one week prior tocounting. Sinking rates were measured over 3 h and were conductedunder the same conditions in which the cells were growing. Becausethe sinking rate measurements were made in the light, increases incell numbers often occurred during the experiment. Therefore theSETCOL method was modified according to Waite et al. (1992b).Instead of using (B00 + B0t)/2 as an estimate of initial biomass, theyaveraged final concentration of cells from all SETCOL fractions (top,middle, and bottom fractions of the column). Samples for lightmicroscope observations and pH were taken prior to the sinking ratetrial to estimate the health of the culture and assess carbonlimitation, respectively.18Morphological changes in coccoliths and cell surfaceultrastructure were monitored the day of the experiment usingscanning electron microscopy (SEM). Both strains of E. huxleyi werefirst fixed for 15 mm at room temperature with formalin in a boraxbuffer (pH 8.0) at a final concentration of 0.4%. The samples werethen concentrated by gentle filtration onto 0.60 pm Nuclepore filters,and rinsed with seawater following by DDW. For the coccolithbearing cells, the filters were air-dried and gold-coated. For thenaked strain, the filters were dehydrated in a graded ethanol seriesand dried in a Balzer’s Union CPD 020 Critical Point Dryer and goldcoated before insertion into a scanning electron microscope.All statistical comparisons were done using a Students t-test atthe 95% confidence level (p<O.05).19RESULTSGrowth vs irradianceCoccolith-bearing: Growth of E. huxleyi bearing coccolithssaturated at about 100 .tmol photons rn-2 s-1 for NO3-and NH4grown cells (Fig. 1). Although in the light-limited range (from 10 to35 j.trnol photons rn-2 s-1), growth rate on NO3-vs NH4 was notstatistically different (t-test P > 0.05), at PFDs > 282 .tmol photonsrn-2 s-1, NH4 grown cells grew statistically faster.Naked: Growth of the naked cells saturated at about 100 pmolphotons rn-2 s’ for nitrate grown cells, and closer to 200 pmolphotons rn2 s1 for ammonium grown cells (Fig. 1). At PFD 10 .jrnolphotons rn-2 s1 growth on N03 was significantly lower than on NH4,being 0.14 and 0.28 div day-’, respectively. The growth rate of thenaked amrnonium grown cells was significantly higher at PFDs > 116pmol photons rn-2 s1 than the naked NO3- grown cells. Underarnrnoniurn at PFDs > 116 j.imol photons rn-2 s-1, the naked strain ofE. huxleyi grew 40% faster than the coccolith-bearing cells. Undernitrate, the two strains showed no significant difference in theirgrowth rates.Growth rateThe specific growth rate of E. huxleyi was significantly higherfor the naked cells than for the coccolith-bearing cells under highlight (150 j.imol photons rn-2 s-i) for either nitrate or ammoniumgrown cells (Fig. 2). However, ammonium grown cells (naked andwith coccoliths) at high light, grew significantly faster (20%) thannitrate grown cells. Also, the growth rates of the low light (20 jimolphotons rn-2 s-i) cells were 4 times lower than the growth ratesachieved at saturating light. The low light grown cells showed nodifference in growth rate for either nitrogen source (N03 vs NH4) orthe type of cell (naked vs coccoliths).N nakedNH4naked N03A coccoliths NH40 coccoliths N03Figure 1. Growth rate vs irradiance for E. h uxleyi with andwithout coccoliths grown in ammonium or nitrate media. Barsrepresent standard error (÷/-1) from triplicate cultures.>-C)4-,2Figure 2. Specific growth rate for E. huxleyi with and without coccolithsvs nitrogen source and irradiance (high light =150 and low light = 2Opmolphoton m2 s-i). Bars represent standard error (+/-1) from triplicatecultures; where bar is not visible, triplicate cultures were identical.20I2100 100 200 300Irradiance(pmol photons rn-2 s-i)4000.80.60.40.20.0coccolithsD nakedN03 NH4 N03 NH421Cell volumeThe cell volume, CV, of the coccolith-bearing cells wasstatistically similar for all cells, regardless of the irradiance, nitrogensource, or growth phase (Fig. 3A). However, the CV of ammoniumgrown coccolith-bearing cells in log phase under high light wassignificantly higher than all the other cultures of coccolith-bearingcells. The cell volume of the naked cells tended to be more variable.Three trends were observed: CV decreased by 18% as the cultureentered stationary growth; there were a significant decrease in CVfor high light cultures compared to low light cultures, and ammoniungrown cells tended to be 1.2 times bigger than nitrate grown cells.The most noticeable difference in CV was that coccolith-bearing cellswere approximately 70% larger than naked cells and therefore allphysiological parameters were normalized to cell volume.(Parameters normalized per cell and other ratios are in Appendix5A-B).Sinking rateThe coccolith-forming cells sank significantly faster (by 40-400%) compared to the naked cells (Fig. 3B), except during log phasegrowth under light limitation, where there was no statisticaldifference between the two strains. For the coccolith-bearing cells,the sinking rates were significantly enhanced (2 to 6 times) when theculture entered stationary phase. A light intensity effect on sinkingrates was observed for coccolith-bearing cells at senescence and forthe naked cells during log phase. There was a 135 and 125%increase (for nitrate and ammonium, respectively) in sinking rates ofsenescent naked cells growing under high light compared to logphase cells under high light. The nitrogen source did not affect thesinking rates under any condition for either the coccolith or nakedstrains.40 0LOG-PHASEHighLightLowLightN03NH4N03SENESCENTNH4LOG-PHASEHighLightLowLightSENESCENTFigure3.Cellvolume(A) andsinkingrates(B)forE.huxieyi withandwithout coccolithsversusnitrogensource(NOçorNH4+)andirradiance(highlight=150andlowlight=20pmolphotonm2s-i)duringlogandsenescent phase.Barsrepresent standarderror(+/-1) fromtriplicatecultures;wherebar isnotvisible, triplicatecultureswereidentical.Icoccolithsnaked30 20 10 0 40 30 20 10-•coccolithsnakedE23Biochemical comnositionChlorophyll a: Coccolith-bearing cells showed no significantreduction in chi a/CV from log to senescent growth phase (Fig. 4A).There was no significant difference in chi a/CV between nitrate andammonium grown cells under either irradiance or the growth stage.However, the chl a content significantly increased for coccolithbearing cells under light limitation.The results were similar for the naked cells, but with a largerincrease (350%) in chl a/CV from high to low light during theexponential growth phase. In general, the coccolith-forming strainsunder high light contained 1.4 times more chl a than the nakedstrains.In vivo fluorescence per chl a was statistically different, forboth coccolith-bearing and naked cells, under either irradiance, fromlog to senescent growth phase (Fig. 4B). There was no significantdifference in fluorescence/chl a between the coccolith or nakedstrains. The only exception was a 150-200% increase influorescence/chi a from coccolith-bearing to naked cells at senescentphase under light limitation. The two strains of E. huxleyi exhibiteda 1.2 to 3.2 times decrease in fluorescence/chi a from high to lowlight during both growth phases. In vivo fluorescence per chl a alsoincreased when log phase cells became senescent.Nitrogen: During log phase, both naked and coccolith-bearingcells grown under low light had significantly higher nitrogen/CVcompared to high light (Fig. 5A). The nitrogen/CV tended to decreasewhen the culture entered stationary growth, but the difference wassignificant only for the naked cells. The nitrogen/CV did not varywith nitrogen source. The coccolith-bearing cells had significantlymore nitrogen/CV (1.4 to 4 times) than the naked cells.24Carbon: The total carbon (including coccolith carbon) contentper cell volume of the coccolith-forming strain, was significantlygreater (2 to 3 times) than the carbon/CV of the naked strain (Fig.5B). Carbon content tended to increase for cells in log phase underlow light, but the difference was significant only for the naked cells.There was no statistical difference between nitrate and ammoniumgrown cells under all the conditions.When carbon:nitrogen (C:N) ratios were examined (Fig. 6), nosignificant differences were observed under either light condition orthe nitrogen source. The only exception was for the coccolithbearing, ammonium grown cells at senescent phase under high lightwhich exhibited significantly larger values (32.7) than all the otherC:N ratios. The two strains of E. huxleyi exhibited a 50-100% higherC:N ratio when the log phase cells reached senescence.Carbohydrates: The coccolith-forming cells showed a smallincrease in their carbohydrate content per cell compare to the nakedcells (Appendix 5A-B). When normalized to cell volume (Fig. 7A), theresults tended to be more variable for naked and coccolith-bearingcells. Also there was no significant relationship betweencarbohydrate content/CV and nitrogen source, light condition, orgrowth phase.Lipids: There was no statistical difference in the lipidcontent/CV for nitrate and ammonium grown cells under all theconditions for either the naked or coccolith-bearing cells (Fig. 7B).The lipid/CV tended to increase when the culture entered stationarygrowth, but the difference was significant by 1.5 to 3 times only forthe coccolith-forming strain. There was a statistical difference in thelipid content between naked and coccolith-bearing cells under lowlight conditions. The naked cells tended to enhance their lipid/CV by60% and as high as one order of magnitude, when growing under lowlight.0 >c’—-C.) U14 6 4 2 0LOG-PHASEHighLightLowLightN03NH4N03SENESCENTNH40 0 0 I x CLOG-PHASEHighLightLowLightSENESCENTFigure4.ChIaper cellvolume(A)andfluorescence:chl a(B)forE.huxieyi withandwithout coccolithsversusnitrogensource(NO3-orNH4)andirradiance(highlight=150andlowlight=20pmolphotonrn-2s-i)duringlogandsenescentphase.Barsrepresent standarderror(+/-1)fromtriplicatecultures;wherebarisnotvisible,triplicatecultureswereidentical.•coccolithsDnaked12 10 8 6 4 2 0 14 12 10 8•coccolithsnaked-c UN03NH4N03NH4LOG-PHASEHighLightLowLightLOG-PHASEHighLightLowLight•coccolithsnakedSENESCENTSENESCENTFigures.Nitrogenpercellvolume(A)andcarbonpercellvolume(B)forE.huxleyiwithandwithout coccolithsversusnitrogensource(N03orNH4)andirradiance(highlight=150andlowlight=20jimolphotonm2s4)duringlogandsenescentphase.Barsrepresentstandarderror(+/-1)fromtriplicatecultures;wherebarisnotvisible,triplicatecultureswereidentical.r 2ci,CD,a) 0 I. z•coccolithsnakedr:3(V0 > C)0)C0.0‘-‘-Q I Cu UN03NH427C0)04-,zC0h..C-)LOG PHASESENESCENTFigure 6. Carbon:nitrogen ratio for B. huxleyi with and without coccolithsversus nitrogen source (NO3-or NH4)and irradiance(high light = 150 and low light= 20 pmol photon rn-2 s-1) during logand senescent phase. Bars represent standard error (+/-1) from triplicatecultures; where bar is not visible, triplicate cultures were identical.High Light Low Light• coccolithsnaked0E0E40302010040 -3020100NO3- NH4-INH4N03N03 NH4 NH4N03Figure7.Carbohydratepercellvolume(A)andlipidpercellvolume(B) forE.huxleyi withandwithoutcoccolithversusnitrogensource(NO3-orNH4)andirradiance(highlight=150andlowlight=20pmolphotonrn-2s-i)duringlogandsenescentphase.Barsrepresent standarderror(+/-1) fromtriplicatecultures;wherebarisnotvisible,triplicatecultureswereidentical.LOG-PHASEHighLightLowLightHighLightLowLightALOG-PHASE•coccolithsnaked0.50.40.30.20.1 0.00.50.40.3E 0 >InC I_•—>-c 0-o I... Ca Uii1N03NH4I•coccolithsnakedrE D 0 >IT Fa) C.)) 0.-•0.-J0.20.1 0.0ISENESCENTN03NH4N03NH4SENESCENTI’,.)0029Scanning electron microscopySEM was conducted on the coccolith and naked strains of E.huxleyi under the different conditions. Four scanning electronmicrographs are included to illustrate the major characteristics.Senescent cells formed more coccoliths than during log phase (Fig.8A). During exponential growth, high light cells formed one or twocomplete layers of coccoliths that stayed on the cell surface, whilelow light cells (Fig. 8B) usually formed only one fragile layer. Thenaked strain of E. huxleyi exhibited no significant differences in cellshape under the various conditions. Figure 8C and 8D shows a singlenaked cell and cells in division, respectively. The presence of flagellaor haptonema was not observed for either strain.30BFigures 8(A-D). Scanning electron micrographs of Emiliania huxleyi,naked and coccolith-bearing strains. A) Coccolith-forming cellsduring senescence and high light. B) Coccolith-forming cell during logphase and low light. C) Naked cell during log phase and high light.D) Naked cell during division.AC D31DISCUSSIONBasic studies on the cell composition and physiology ofEmiliania huxleyi are surprisingly rare in the literature, with moststudies focusing on calcification, carbon metabolism or growth rates.Paasche and Kiaveness (1970) compared cell morphology of thecoccolith-forming and naked cells of E. huxleyi, but failed to find anymajor dissimilarity between the two cell types. Eppley et al. (1967)measured the sinking rates of E. huxleyi with and without coccoliths,but they did not measure any other physiological parameters. Thecurrent work is the first to compare sinking rates, and physiologicaland morphological parameters of coccolith-forming and naked strainsof E. huxleyi, under different irradiances, growth phase (log orsenescence) and nitrogen source (N03 vs NH4+).Growth studiesLightMy results clearly demonstrated the genetic variabilitybetween the two strains. The naked strain of E huxleyi exhibitedsignificantly higher growth rates than the coccolith-bearing strainunder high irradiance. Paasche and Kiaveness (1970) found theopposite with a higher growth for the coccolith-forming cells underhigh light. They considered that the slower growth of the naked cellsmight result from a less favourable ratio between photosyntheticoutput and biomass. Brand (1982) also found different growth ratesin clones of E. huxleyi which was attributed to genetic variability, buthe did not test naked vs coccolith-forming clones. In the presentstudy, it might be possible that the naked cells divided faster underhigh irradiance due to their smaller cell volume and the fact thatthey did not spend energy on coccolith production. It seemsreasonable to assume that the formation of coccoliths involves anexpenditure of energy (Paasche, 1964), and therefore the coccolithforming strain grew slower because they have to reproduce the cell32content as well as the coccoliths (Klaveness, 1972; Hon and Gleen,1985).In the present study, a decrease in the growth rate of thecoccolith-forming strain under low light was also observed, but thedifference in growth rate between high and low light was lesspronounced than for the naked strain. Paasche (1968) reported thatcoccolith formation becomes light-saturated at a light intensitysomewhat lower than the amount of light required for saturation ofphotosynthesis, probably leading the low light cells to spend moreenergy on growth rate than on coccolith formation. This tendency ofthe cell to form less coccoliths under low light might enhance thelight reaching the cell surface, and increase photosynthetic efficiencyand therefore growth.Nitrate vs ammoniumThe growth rate was significantly different between nitrateand ammonium grown cells under saturating irradiance, for bothnaked and coccolith-bearing cells, with ammonium grown cellsachieving 20% higher growth rates than nitrate grown cells. This issimilar to the results obtained by Thompson et al. (1989) andLevasseur et al. (1993). Using a marine diatom (Thalassiosirapseudonana) under saturating light conditions, they found that thegrowth rates of ammonium grown cells were significantly higher(8%) than the growth rates of nitrate grown cells under high light.Because they found lower nitrogen and carbon quotas for nitrategrown cells than for ammonium grown cells, they suggested that thegreater energy required by NO3- grown cells for the reduction ofN03- was compensated by a reduction in growth rates (Thompson etal., 1989). However, in the present study, E. huxleyi did not showsigns of reductant competition because it maintained the samenitrogen and carbon per cell volume between nitrate and ammoniumgrown cells.Also Thompson et al. (1989) observed no difference in growthrate of T. pseudonana at low light between N03 and NH4grown cells.33The same pattern can be seen from the growth rate vs irradiancecurve of this study. At low light, both naked and coccolith-bearingcells grew at the same rates when they were grown on nitrate orammonium. However, above light saturation (>100 mol photons rn-2s1) growth rates of naked cells were higher than coccolith-formingcells, and ammonium grown cell grew faster than nitrate grown cells.Sinking rateNaked vs coccolith-bearing cellsThe main physiological comparison between the naked and thecoccolith-forming cells is the difference in cell volume. Once thecoccoliths were removed by acid treatment from the coccolithbearing cells, the cell itself was still 1.5 to 2 times bigger than thenaked cell, indicating again the possible genetic variation in strains ofE. huxleyi. This size difference was not reported before by eitherEppley et al. (1967) or Paasche and Kiaveness (1970). It can behypothesized that the size difference is due to the absence of thebasic internal apparatus (Kiaveness, 1972) for coccolith synthesis innaked cells and the presence of coccolith-forming vesicles in thecoccolith-forming cells (Kiaveness and Paasche, 1971). Also theaverage cell volume of the naked and coccolith-bearing cells tendedto decrease under low light. Presumably such variations reflectedchanges in the amount of organic matter present in a single cell(Paasche, 1967). These large differences in cell size, as well as thepresence of coccoliths around the cells should affect the density andtherefore the sinking rates.The average cell density calculated from Stokes’ law (seeAppendix 6) for the two strains with different cell volumes was 1.32g ml-1 for the naked cells and 1.44 g m11 for the coccolith-bearingcells after the coccoliths were removed with acid. This 10%difference in cell density is due to cell size (including possibleinternal coccoliths), but once the density of calcite, taken as 2.7(Smayda, 1970) and the coccolith carbon: total cell carbon ratio of 0.4(Paasche, 1962) is considered, then the difference in cell density34between the naked and the coccolith-forming cells with theircoccoliths on increases to 25%. This difference in cell density mayexplain the 1.5-2.0 times higher sinking rates for the coccolithbearing cells compared to the naked cells. Eppley et al. (1967)reported a cell density of 1.08 and 1.20 g mi-1 for the naked andcoccolith-bearing cells, respectively, accompanied by a 4.5 timeshigher sinking rate for the coccolith-forming cells. The higher valuesof sinking rates reported previously were measured using thefluorometric method that has frequently been showed tooverestimate sinking rates (Bienfang et al., 1977, 1981c). Also, theirexperiment was conducted using the same strain of E. huxleyi; theyjust removed the coccoliths from the cell surface with acid treatment,and measured the sinking rates as if they were naked cells. This acidtreatment may have affected the cell’s physiology prior to thesinking rate measurement.The number of coccoliths on the cell’s surface may be the mainway to control the sinking rate of the coccolith-forming strain.Although coccoliths were not counted during my experiments, manyobservations were made during both their growth phase and growthunder different irradiances. During log phase, the detachment ofcoccoliths or the cessation of their formation under low light, mightexplain the constancy in sinking rates of the coccolith-bearing cellsbetween both irradiances. This observation was reported manytimes by Paasche (1964, 1966, 1967, 1969). According to Paasche,coccolith formation is strongly light-dependent and connected withphotosynthetic processes. The loss of coccoliths would allow the cellsto maintain a low sinking rate (Baumann et al., 1978; Linschooten etal., 1991), whereas the naked cell, being more sensitive to lightlimitation would have less energy to control their buoyancy, and thusincreasing their sinking rates markedly. This is, in spite of thesmaller cell volume and lower metabolic requirements of naked cells.Linschooten et al. (1991) found that cells continued producingcoccoliths which were arranged in multiple layers, after growthstopped in a nutrient-impoverished medium. This observation was35previously reported by Kiaveness and Paasche (1979) and theyconcluded that cell division was shown not to be a prerequisite forthe formation of new coccoliths. This was also observed in thepresent experiment with a large increase in coccoliths, which formedmultiple layers when cells entered senescence due to the depletion ofnitrogen (Appendix 3). This increase in coccoliths made the cellsheavier leading to a large increase in sinking rates for the coccolithbearing senescent cells of E. huxleyi. Also, as division rates slow dueto nutrient depletion, biomass of a single cell can increase before itfinally divides into two new cells (Smayda, 1970). This accumulationof organic matter would increase sinking rates. Smayda (1970)reported a two-fold increase in sinking rate during senescence inseveral species.Cell aggregationThe weight and the cell volume may explain the higher sinkingrate of the coccolith-forming strain, but other factors need to beconsidered to explain the increase in sinking rate when naked cellsenter senescence. The excretion of surface mucus at senescence hasbeen observed by Fogg (1966). The production of extracellularcompounds such as carbohydrate, proteins, and lipids might increasethe cell surface stickiness and enhance cell aggregation (Andersonand Sweeney, 1977; Smetacek, 1985). Eppley et al. (1967) observedthat aggregate formation invariably results in sinking rates greaterthan those of individual cells composing the aggregate. Cellaggregation was observed when naked cells entered senescence inmy experiments, and this may explain the possible increase insinking rate.The coccolith-bearing cells also increased their sinking ratesduring senescence, however cell aggregation was not observed.Probably the presence of multiple layers of coccoliths made the cellsink, but also prevented surface contact. Surface stickiness incoccolith-bearing cells was reported by Paasche (1992), but insteadof forming aggregates between cells, it enhanced formation ofmultiple layers of coccoliths by sticking coccoliths to each other and36to the cell surface. Also, Smetacek (1985) observed thatcoccolithophores, on cessation of logarithmic growth, formed mucousagglomerations that sank to the bottom of the culture vessel.Nitrate vs ammoniumAccording to the hypothesis that energy controls sinking ratesin marine phytoplankton (Waite et al., 1992b), the competition forreductant should become more severe under low irradiance, andtherefore ammonium grown cells should be able to maintain lowersinking rates than nitrate grown cells. Although sinking ratesincreased under low light when photosynthetic energy was limiting,E. huxleyi did not appear to be energetically affected by the differentnitrogen sources. Similar results have been reported by Muggli andHarrison (submitted) for cells of E. huxleyi under iron stress. Nitrategrown cells maintained their normal physiological parameters,despite the theoretical advantage of using ammonium over nitrateunder iron limitation.Cell comnositionChlorophyll aThe coccolith-forming strain of E. huxleyi had significantlyhigher chlorophyll a than the naked strain. The same results werereported by Paasche and Kiaveness (1970) and Price (1992) whofound 10-13% less chl a for naked cells. One hypothesis to explainthis observation, is that the coccoliths may be reflecting light andthus shading the cells (Braarud et al., 1952). This would result in thechloroplasts being exposed to lower irradiances than the chioroplastsin the naked strain (Falkowski, 1980). To maintain maximumphotosynthesis, the coccolith-forming cells might possiblycompensate by increasing the density and/or size of thephotosynthetic units (Prezélin, 1981; Falkowski and LaRoche, 1991).This would be reflected by higher concentrations of chl a in coccolithbearing cells than in naked cells.37Chi a in coccolith-forming cells increased slightly withirradiance. On the other hand, chl a in the light sensitive nakedstrain was markedly increased by low light. Steemann Nielsen(1966) proposed that coccolith formation may be essential for themaintenance of maximum photosynthetic rates in coccolithophoresby providing C02 during the calcium carbonate precipitation reaction.This might explain the large increase in chi a by the naked cellsunder low light. By increasing the density and/or size of theirphotosynthetic units, the cell can increase its photosynthetic rate,and thus is able to maintain physiological parameters such as growthrate and sinking rate.Photosynthesis per cell in naked clones has been reported to behalf of that observed in coccolith-forming clones under comparableconditions (Paasche, 1968). However, Paasche (1968) concluded thatit was not necessarily an indication that photosynthesis is lessefficient in terms of carbon assimilated relative to carbon present incells, since the cell volume is normally smaller in the naked clones.In the results showed here, the in vivo fluorescence:chl a ratio wasnot statistically different for the naked and the coccolith-formingstrain regardless of the nitrogen source or the irradiance, indicatingthat the photosynthetic efficiency was similar for the two types ofcells. However, the photosynthetic efficiency decreased under lowlight, except for the naked senescent cells that maintained the samein vivo fluorescence:chl a ratio as the high light cells. Similar resultshave been reported by Price (1992).POC and PONThe coccolith-bearing strain of E. huxleyi had 3 times more POCand PON than the naked cells. The differences in carbon CV1 may beattributed to the presence of multiple layers of coccoliths, which arecomposed of calcium carbonate in an organic matrix. It is suggestedthat 40% of the carbon in the coccolith-forming strain is contained inthe coccoliths (Paasche, 1962). The 0.7 pg carbon CV1 found in thecoccolith-forming strain is similar to that found by Muggli andHarrison (submitted) which was approximately 0.65 pg carbon CV1.38However, the PON values found for the coccolith-bearing cellswere higher than those found by Muggli and Harrison (submitted).It was expected that the coccoliths and their organic matrix wouldcontain very little nitrogen, and therefore the level of PON should besimilar between the naked and the coccolith-bearing cells. However,the 1.4 to 4 times higher nitrogen/CV for coccolith-bearing cellsmight be due to the nitrogen associated with the coccolith that stayedon the cell’s surface and thereby increased the PON levels. PONresults for coccolith-forming cells from Muggli and Harrison(submitted) were closer to the PON obtained from the naked cells(0.045 pg nitrogen CV1 compared to 0.035 pg nitrogen CV1). Thishigher PON content for the coccolith-bearing cells accounted for theconstant carbon:nitrogen ratio under the different conditions. Duringexponential growth, both coccolith-forming and non-forming strainsincreased their POC, PON content under low light. This suggested thatcellular processes involved in the fixation of carbon and nitrogenwere affected by irradiance.CarbohydratesTheoretically, carbohydrate should be stored under high lightand increase when cells enter senescence (Mykiestad and Haug,1972; Falkowski and LaRoche, 1991). Furthermore, an increase incarbohydrate should increase the density of the cell and causesinking rates to increase. However, this was not observed in thepresent study. The only increase in carbohydrate that correspondedto an increase in sinking rate was for both naked and coccolithforming cells in exponential growth under low light. Thecarbohydrate content also tended to decrease for cells in senescentphase, although the sinking rate showed a major increase. Recently,A. E. Fisher (pers. comm.) failed to prove an association between highcarbohydrate content and high sinking rate in two marine diatoms.LipidsThe total lipid measured for the coccolithophore Hymenomonascarterae showed a decrease in the lipid fraction under nitrogen stress39(Shifrin and Chisholm, 1981). In the present study, the lipid contentof E. huxleyi (coccolith-forming and naked strains) increased underlow light and when cells ran out of nitrogen. The sinking ratechanges observed here were clearly not consistent with a fat-flotation hypothesis, because lipids were maximal when cell sinkingwas maximal. This was true of the data of Eppley et al. (1967) aswell, which indicated reduced buoyancy in the dark when theamount of lipid was declining. Also, Anderson and Sweeney (1977)followed sinking rate and cellular lipid over a diel cycle, and showedthat maximum lipid content corresponded to maximum sinking rates.Werner (1977) pointed out that diatoms under nitrogen-stresscommonly store carbohydrates initially and then shift to lipid storageonly after extended periods of nitrogen stress. If this is the case inthe present study, it can be hypothesized that an increase incarbohydrate followed by an increase in lipids during senescenceenhanced cell aggregation by excretion of those compounds, and leadto high sinking rates.Ecological stategiesOf the various possible functions of coccoliths, the increase insinking rates due to the added weight of one or more layers ofcoccolith scales seems the most credible. From the present study andthe literature (Eppley et al., 1967; Smayda, 1970; Bienfang, 1981 a,b)results show that coccolithophores, and especially E. huxleyi, havehigher sinking rates than most other groups of similar sizedphytoplankton, presumably because of their coccoliths.The advantage of coccoliths over diatom frustules anddinoflagellate thecae is that they are detachable and thus can beused as ballast. The resulting higher specific density could be usedto enhance their sinking to deeper, nutrient-rich water when surfacewaters become nutrient-depleted. My results showed thatcalcification from coccolith formation, was higher under lowernutrient conditions at senescence. Also the higher sinking rate can40reduce cell surface diffusion limitation of nutrient uptake by themovement of water around the cell. Results from this study confirmearlier observations that most phytoplankton sink faster whennutrients are limiting than when they are saturating (Smayda, 1970).An additional response of coccolithophores is to lose theircoccoliths (thus reducing their specific density) in the presence ofhigh nutrient concentrations (Kiaveness and Paasche, 1979). Thepresent results showed that a strain without coccoliths would becapable of more control over their sinking rate due to their lowdensity. However, it was not the objective of this study to determineif naked cells could be induced to form coccoliths under extremenutrient starvation. Although, Andersen (1981, quoted in Paasche,1992) induced coccolith production by phosphorus limitation, otherresearchers have failed to induced coccoliths in naked strains ofcoccolithophores (Paasche, 1966; Price, 1992).Coccolith formation was also found to be light dependent, withcalcification being higher under high light conditions. It is possiblethat coccoliths protect the cell against photodamage by reflecting thelight (Braarud et al., 1952), but it is also hypothesized that with morecoccoliths, the cell would sink to the chlorophyll maximum layerwhere nutrients are higher and light is lower. In this study, underlow light the coccolith-forming cells tended to lose their coccolithswhich decreased their sinking rates.The naked cells showed greater sensitivity to light. Thequestion from the present study is, why they did not form aflagellum to control their movement in the water and decrease theirsinking rates under low light? It is possible that the naked cells arenot part of the normal life cycle of this species and that theyrepresent a more permanent modification at the level of gene control(Paasche and Klaveness, 1970). Also because they are difficult todistinguish from the numerous other very small cells, we do notknow if they exist in nature. It was also argued by Smayda (1970)that the energy requirements for motility can be conserved and41flotation maintained by reducing motility and placing greaterreliance on passive flotation for suspension.Finally, this small oceanic phytoplankter can use nitrate orammonium equally well in a low light environment, and sinking ratewas not affected by the nitrogen source unlike previous expectations.Overall then, coccoliths can explain sinking rate responses to nutrientconcentrations, growth phase, and light variation. However,coccoliths cannot be the only reason for the success of this species. Ahigher potential growth rate than found in other oceaniccoccolithophores, combined with an extremely modest ironrequirement characteristic of the group as a whole (Brand, 1991 a,b),may give E. huxleyi a further edge over potential competitors in offshore bloom situations.42Chapter 2SINKING RATES RESPONSES OF EMILIANIA HUXLEYI ANDACTINOCYCLUS sp. TO IRON STRESSINTRODUCTIONThere are some regions of the ocean where macronutrient (N, P,Si) concentrations remain elevated all year round and chlorophyllconcentrations are low. These areas are called high nutrient, lowchlorophyll regions (HNLC). Clearly, factors other than macronutrientdeficiency limits phytoplankton growth in these HNLC regions suchas the Subarctic Pacific, the equatorial Pacific, and the SouthernOcean. The low iron concentrations in seawater, (surface open oceandissolved iron concentrations can be as low as 0.05 nmol kg-’ (Martinet al., 1989)) and the importance of this element for the physiologyof phytoplankton, prompted Martin and colleagues to postulate thatextremely low iron concentrations are responsible for limitingphytoplankton growth in these low aeolian iron input regions, andmore specifically, iron may influence the species composition of thephytoplankton assemblage. The addition of iron to samples from theSubarctic Pacific has been found to preferentially enhance thegrowth rate of large diatoms (>18 jim) rather than smallphytoplankters (Boyd et al., submitted).The composition of the phytoplankton assemblage found in theSubarctic Pacific is hypothesized to be a result of the nitrogen sourcethat a phytoplankter uses for its growth. The nitrogen source(nitrate vs ammonium) that a phytoplankter uses is thought toinfluence a species’ ability to survive in low iron environments basedon the theoretical calculations that nitrate utilization requires ironwhereas ammonium utilization does not (Bruland et al., 1991; Morelet al., 1991a; Price et al., 1991). Martin has demonstrated bothincreases in biomass (chlorophyll a) and enhanced nitrate utilizationwith iron addition experiments in the Subarctic Pacific and Southern43Ocean (Martin and Gordon, 1988; Martin and Fitzwater, 1988; Martinet al., 1989; Martin et al., 1990; Martin, 1991).Iron is a biologically and geologically important element. Itschemistry is very complex; in oxygenated waters Fe is oxidized fromthe soluble Fe (II) state to the highly insoluble Fe (III) state. Iron isalso essential for all life on Earth, being a constituent of manyoxidizing metallo-enzymes, pigments and proteins (Martin andGordon, 1988). Because of its oxidation-reduction properties, iron isimportant in catalyzing electron transfer reactions. Both nitrate andnitrite reductase contain iron to catalyse the reduction of nitrate tonitrite, and nitrite to ammonium. Iron is also involved in both thephotosynthetic and respiratory pathways of phytoplankton cells.Chlorophyll synthesis is dependent on iron nutrition, and Fe-limitedalgae have low concentrations of this pigment (Rueter and Ades,1987). Iron is also a component of cytochrome, porphyrin andferrodoxin molecules, which are involved in electron transport forphotosynthesis, respiratory processes, and nitrate reduction (Spilleret al., 1982).In general, iron limitation of phytoplankton causes a decreasein pigments and a decrease in iron-containing electron transportcompounds. The overall effect of these changes is less efficientenergy transfer during photosynthesis leading to increasedfluorescence in iron-limited cells (Glover, 1977; Rueter and Ades,1987; Doucette and Harrison, 1990).Under low iron conditions, cells utilizing nitrate shouldtheoretically have a higher iron requirement than cells utilizingammonium. Raven (1988) calculated that 1.6 times more Fe isnecessary for phytoplankton growing on nitrate than if ammonium isthe nitrogen source. Iron limitation could also affect nitrate uptakemore adversely than ammonium uptake, resulting in nitrate growncells having difficulty maintaining their normal nitrogen quotas.44Iron uptake rates of phytoplankton are controlled either by therate of coordination of dissolved Fe to surface transport ligands or,when ambient Fe is very low, by diffusion to the cell (Hudson andMorel, 1990). In addition to regulating short term uptake rates,organisms may respond to iron limitation by substituting anothermetal at a protein-active site or by replacing one iron-containingprotein with another (Harrison and Morel, 1986).Because of the increase in mass to surface area ratio in thelarge phytoplankton which makes both diffusion and surfacereactions relatively less efficient, large phytoplankton (>10 m)should be more easily Fe-limited than small ones when available Feconcentrations are < 0.05 nM (Price et al., 1991). To acquire the ironthey need for rapid growth, oceanic phytoplankton must decrease insize or reduce their Fe requirement (Hudson and Morel, 1990).While phytoplankton seem to be capable of modifying their ironrequirements and uptake efficiencies, iron availability to the cell iscontrolled to a large extent by the chemistry of iron in thesurrounding environment (Wells, 1988,89). Furthermore, a cell thatis iron-stressed could be analogous to one that is energy-stressed.As a result, under iron limitation, the cells may not have as muchenergy to expend on other functions, perhaps resulting in lowergrowth and higher sinking rates.In the past, the effect of nutrient limitation on sinking rate hasfocused on the macronutrients. The exact effect of nutrientlimitation on the sinking rate of a cell is usually indirect, anddepends on the mechanism used by that cell to regulate itsbuoyancy. Macronutrient limitation could take days to affect thesinking rate of a cell (Bienfang et at., 1982). In contrast, iron isintimately associated with the energy production of a cell, and ironlimitation could have an immediate affect on the cell’s ability toutilize stored energy by directly affecting electron transport. Ironlimited cells have been reported to be analogous to energy depletedcells (Muggli and Harrison, submitted). Iron could have a direct and45immediate effect on the sinking rate of phytoplankton, and to datethis has not been examined.The experiments presented here were conducted with twooceanic phytoplankton, a small oceanic coccolithophore, Emilianiahuxleyi (5 p.m diameter), and a larger diatom, Actinocyclus sp.(20 p.m diameter) (see Figure 1), both isolated from the SubarcticPacific Ocean. This study examined the effects of nitrogen source andiron stress on sinking rates and other physiological parameters ofthese two different phytoplankters.Figure 1. Scanning electron micrograph of Actinocyclus sp.46MATERIALS AND METHODSCulture conditionsThe coccolith-forming strain of Emiliania huxleyi (Lohmann)was the same isolate as in Chapter 1 and it was maintained in lowmacronutrient and micronutrient Stn P water (see Chapter 1 fordetails). The diatom Actinocyclus sp. was isolated from the SubarcticPacific Ocean (Station Papa, 50°N, 145°W) by D. L. Muggli in the fallof 1993. Stock cultures of Actinocyclus sp. were maintained innatural Stn P water which contained low macronutrients andmicronutrients.Experiments with E. huxleyi were carried out using chelexed0.22 m filtered Stn P water. The Chelex 100 resin was preparedaccording to Price et al. (1988, 1989), with the exception of usingUltra HC1 (Seastar Chemicals) for the final HC1 soak. Chelexed Stn Pwater was microwave-sterilized in acid-cleaned 2 L teflon bottles fora total of 12 mm (Keller et at., 1988). Stn P water was also used inexperiments with Actinocyclus sp., but only microwave-sterilized asdescribed above. All handling of the medium was done in a class100 laminar flow hood to prevent metal and bacterial contamination.Macronutrient stocks were made up in Nanopure water and chelexedat pH 3.0 for 100% iron removal (Chrétien, unpubi. data).Macronutrients for E. huxleyi were added to achieve a finalconcentration of 30 tM of nitrate, and 5.0 tM phosphate. ForActinocyclus sp., macronutrient concentrations were 50 j.tM nitrate orammonium, 150 pM silicate, and 20 iM phosphate. EDTA was addedto yield a final concentration of 10 tM for both species. Metal stockswere prepared in 0.01 M ultra HC1 in Nanopure water. Final metalconcentrations added were: 12 nM Zn, 35 nM Mn, 1.5 nM Cu, 3.75 nMCo, 150 nM Mo, 15 nM Se. Thiamine and biotin were added to give afinal concentration of 1 x iO g L1 and 5 x iO g L1, for bothspecies respectively. Vitamin B12 was added to the Actinocyclus sp.culture medium to yield a final concentration of 2.5 x iO g47E. huxleyi cultures were grown in quadruplicate with nitrateonly. Cultures of Actinocyclus sp. were grown in triplicate witheither nitrate or ammonium as the nitrogen source. Both specieswere grown in acid-cleaned 2.8 L polycarbonate Fernbach flasks,equipped with teflon tubing and silicone stoppers. Acid-cleantechniques were employed according to Muggli and Harrison(submitted). Cultures of E. huxleyi were not mechanically stirred, asdescribed in Chapter 1. Cultures were maintained at 16°C on a 14:10light:dark cycle with Vita-lite fluorescent lights. The irradiance, asmeasured by a Biospherical Instruments model QSL-100 sensor, was150 i.tmol photons m2 s1.Four cultures of E. huxleyi were grown with 100 nM iron addedto the medium. Four culture transfers were made prior to theexperiment to ensure that the cells were adapted to the cultureconditions. Iron-replete cells were obtained by sampling log phasecultures growing at maximal growth rate. Iron-deplete cultureswere obtained experimentally (Muggli, pers. comm.) as follows; fourcultures of E. huxleyi with no iron added were grown, and when thecells reached early senescence (presumably due to nitrogendepletion) nitrate, phosphate, Mn and Zn were added directly to thecultures. This was repeated until a slower growth rate was achieved.The iron-depleted cells were harvested when growth stopped.Cultures of Actinocyclus sp. were first grown in 1000 nM Fe,representing the iron-replete conditions. Since Actinocyclus sp. waslarger and had a higher iron requirement than E. huxleyi, it waspossible to iron stress the cells by simply transferring cultures intolow iron medium for four consecutive dilutions. When the growthrate slowed down to about 1/3 of the maximum growth rate, thecells were harvested. To speed up the achievement of iron-stressedconditions, the experiment was first conducted with 100 M EDTA forthe nitrate grown cells only. However the experiment was conductedagain with 10 J.LM EDTA (nitrate and ammonium) the sameconcentration that was used in the iron-replete conditions.48Growth rate was measured as described in Chapter 1, but acidtreatment (to remove coccoliths) was used on E. huxleyi only. Thecell volume of E. huxleyi was estimated by the Coulter Counter, andby microscope for Actinocyclus sp., assuming a cylindrical shape.Cell comnositionSamples for particulate organic carbon (POC) and nitrogen(PON), and samples for chlorophyll a were analyzed as described inChapter 1. The only difference was that POC and PON of E. huxleyiwere measured with and without (acid treated) the coccoliths.The iron internal quotas (natural and titanium-washed (Ti III))reported in the present study were measured by D. L. Muggli usingmicrowave digestion techniques and Varion graphite furnace atomicabsorption spectrometry. Details of these methods have beendescribed previously by Muggli and Harrison (submitted).Analytical methodsSinking rates were measured using the SETCOL method(Bienfang, 1981c) as described in Chapter 1, and modified accordingto Waite et al. (1992b). Sinking rates were measured over 3 hduring the beginning of light period of the light:dark cycle. TheSETCOL chambers were acid-cleaned (Muggli and Harrison,submitted) prior to the trial and soaked in Nanopure water until theday of the experiment. Light microscope observations were madeprior to the sinking rate trial to estimate the health of the culture.All statistical comparisons were done using a Students t-test atthe 95% confidence level (p < 0.05).49RESULTSGrowth rate1) Fe-replete: The specific growth rate, of E. huxleyi (Fig. 2) underFe-replete conditions in nitrate medium was higher than the specificgrowth rate of Actinocyclus sp. (Fig. 3) under the same conditions.The Fe-replete growth rate was not significantly different forActinocyclus sp. under either nitrate or ammonium as the nitrogensource. The growth rate of E. huxleyi during the first grow-up(transfer) in low Fe medium, was 50% lower than the growth rateachieved under Fe-replete conditions.ii) Fe-deplete: E. huxleyi maintained the same growth rate for thesecond grow-up under Fe-deplete conditions, but the growth rateduring the last grow-up decreased drastically, being 150 timesslower. E. huxleyi practically stopped growing.On the other hand, Actinocyclus sp. maintained the samegrowth rate when transferred from Fe-replete to Fe-depleteconditions. However the following grow-ups for both nitrogensources tended to vary. Nitrate grown cells of Actinocyclus sp.gradually decreased their growth rate from the first to the fourthgrow-up under Fe-deplete conditions, while the growth rate duringthe last grow-up increased and was as high as the maximum growthrate under Fe-replete conditions. Ammonium grown cells decreasedtheir growth rate by 50% during the second grow-up, but recoveredto the same growth rate as the first grow-up for the third grow-up.During the fourth grow-up under Fe-deplete conditions, Actinocyclussp. achieved 1/3 of its maximal growth rate, and finally the lastgrow-up was 2/3s of the maximum growth rate found under Fereplete conditions.50Figure 2. Growth rates for Emiiania huxleyi in Fe-replete (+Fe) nitratemedium and in three grow-ups (transfers) in Fe-deplete (-Fe) nitrate medium.Bars represent standard error (÷/-1) from four cultures.grow - upsI NOD NH4Figure 3. Growth rates for Actinocyclus sp. in Fe-replete (+Fe) and infive grow-ups (transfers) in Fe-deplete (-Fe) nitrate or ammoniummedia. Bars represent standard error (÷/-1) from triplicate cultures.0.6i-Fe=0.5-Fe0.40.30.20.10.0umax lastgrow-upsIumax 1st 2nd 3rd 4th last51Cell volumeThe cell volume, CV, of E. huxleyi (Fig. 4A) decreased by 50%under Fe-deplete conditions. Actinocyclus sp. also decreased its CV(Fig. 5A) by 30% under Fe-deplete conditions. CV between nitrateand ammonium grown cells was not significantly different undereither Fe-replete or Fe-deplete conditions. Also Fe-deplete cells with100 p.M EDTA had the same CV as the Fe-deplete cells with only 10p.M EDTA. CV of Actinocyclus sp. was 100 times larger than the CV ofE. huxleyi. Because of this difference in cell volume between thespecies, and for comparison, all physiological parameters werenormalized to cell volume. (Parameters normalized per cell andother ratios are given in Appendix 7).Sinking rateE. huxleyi (Fig. 4B) maintained the same sinking rate under Fe-replete and Fe-deplete conditions. Under Fe-replete conditions innitrate medium, the sinking rates were not statistically differentbetween E. huxleyi and Actinocyclus sp. (Fig. 5B), being 0.12 and0.15 m day-’, respectively. However, under Fe-deplete conditions,Actinocyclus sp. increased its sinking rate by 80% for either nitrateor ammonium grown cells. The nitrogen source did not affect thesinking rates under Fe-replete or Fe-deplete conditions forActinocyclus sp. The sinking rates under Fe-deplete conditions innitrate medium were the same, regardless of the EDTA concentration(10 p.M vs 100 p.M).Iron auotaThe iron quota per CV for E. huxleyi (Fig. 4C) was 9.6 amol p.m3under Fe-replete conditions, and 2.35 amol p.m3 under Fe-depleteconditions. Because there was no replication for the two Fe quotas,the 75% decrease in internal Fe under Fe-deplete conditions was notstatistically proven.52The study of iron quota for Actinocyclus sp. (Fig. 5C) was morecomplete, with natural and Ti (III) reagent wash values for both Fe-replete and Fe-deplete conditions. Fe-replete cells treated with Ti(III) showed a 75% reduction in iron quota per CV compared to theFe-replete quota. Fe-deplete quotas were not statistically differentbetween the two treatments, except for the Fe-deplete 100 j.iM EDTAcells which had a 40% reduction in their iron quota when they werewashed with Ti (III). Also, the 100 pM EDTA Fe-deplete samplescontained 2 to 3 times (including natural and Ti) more internal ironper CV than the 10 j.tM EDTA samples under nitrate. Ammoniumgrown cells had significantly higher iron quotas per CV than nitrategrown cells under Fe-replete (natural only) and Fe-deplete (naturaland Ti) conditions. The Fe-deplete quotas of Actinocyclus sp. weresignificantly lower (by 3 to 15 times) than the Fe-replete quotas.Carbon:Nitrogeri ratioThe carbon:nitrogen (C:N) ratio of E. huxleyi (Fig. 4D) decreasedsignificantly by 40% under Fe-deplete conditions. The C:N ratio forE. huxleyi was 2 times higher than the ratio for Actinocyclus sp. (Fig.5D). Nitrate grown cells of Actinocyclus sp. increased significantlytheir C:N ratio under Fe-deplete conditions for either 10 or 100 .tMEDTA. On the other hand, no significant difference in the C:N ratiowas observed for ammonium grown cells under the two Feconditions. Under Fe-deplete conditions the C:N ratio for the nitrategrown cells was significantly higher (13%) than the C:N ratio for theammonium grown cells. Also, for Actinocyclus sp. the C:N ratio forthe Fe-deplete 100 tM EDTA cells was 16% lower than the C:N ratiofor nitrate grown Fe-deplete 10 M EDTA cells.00I 4-, Co0 I c40 30A20 10I+Fe-Fe0.12—S0.100.080.060.040.020.0010308a) E’200L..>10u..—‘20’00(acidified+Ti)(natural)Figure4.Physiological parametersunderbothFe-replete(+Fe)andFe-deplete(-Fe)conditionsforE.huxleyigrowninnitratemedium.A)cellvolume;B)sinkingrates;C)natural andacidified-TiwashFequotas(10pMEDTA);D)carbon:nitrogenratio.Barsrepresent standarderror(+/-1)fromfourcultures;noreplicationforFequotas.1.2II•N03BDNH1+Fe-Fe-Fe10PM10PM100pMEDTAEDAEDA1.00.8 0.6 0.4 0.2 0.0 10+Fe-Fe-Fe10pM10pM100PMEDTABJAEDTA•N03,UNH4D+Fe+FenaturalTi10pMEI3IA-Fe-FenaturalTi10pMEI3TA308—Cr).)rILUfl40’00Figure5.PhysiologicalparametersunderbothFe-replete(+Fe) andFe-deplete(-Fe)conditionsforActinocyclussp.growninnitrateorammoniummedia.A)cellvolume;B)sinkingrates;C)naturalandTi(III)Fequotas(10 pMEDTAor100pMEDTA-nitrateonly);D)carbon:nitrogenratio.Barsrepresentstandarderror(+/-1)fromtriplicatecultures;wherebar isnotvisible, triplicatecultureswereidentical.-Fe-FenaturalTi100PMIOTA+Fe10PM IOTA-Fe10PM H3A-Fe100PMEIJTA55ChioroDhyll aE. huxleyi increased significantly (by 37%) its chi a per CV (Fig.6A) under Fe-deplete conditions. In contrast, Actinocyclus sp.decreased its chl a content per CV (Fig. 7A) by 50% for both nitrogensources under Fe-deplete conditions. Under Fe-replete conditions,chi a per CV was not statistically different between E. huxleyi andActinocyclus sp., however E. huxleyi had 3 times more chl a per CVwhen Fe-deplete and growing on nitrate. Ammonium grown cells ofActinocyclus sp. contained 60% less chi a per CV than the nitrategrown cells under Fe-replete conditions. Under Fe-depleteconditions, there was no significant difference in chl a regardless ofthe nitrogen source or the amount of EDTA in the culture medium(under N03 only).Fluorescence:ChI a ratioFor E. huxleyi, in vivo fluorescence per chi a increased by 4times under Fe-deplete conditions (Fig. 6B). Actinocyclus sp.(Fig. 7B) showed much lower in vivo fluorescence per chl a thanE. huxleyi. The ratio increased significantly (76% for nitrate growncells and 43% for ammonium grown cells) under Fe-depleteconditions. The nitrogen source did not affect the in vivofluorescence per chl a for Actinocyclus sp. under Fe-repleteconditions, but a large increase in fluorescence per chl a under Fedeplete conditions was observed for nitrate grown cells (2.4 timeslarger than NH4 grown cells). The Fe-deplete (100 pM EDTA)conditions showed 50% lower in vivo fluorescence per chi a as theFe-replete conditions, while the ratio was 8 times smaller than theFe-deplete (10 p.M EDTA) conditions under nitrate.56NitrogenNitrogen per CV for E. huxleyi (Fig. 6C) increased by 1.5 timesunder Fe-deplete conditions. Once the coccoliths were removed byacid treatment, cells under both Fe conditions increased significantlytheir nitrogen per CV, with the same difference (1.6 times) betweenFe-replete and Fe-deplete conditions. Nitrogen per CV forActinocyclus sp. (Fig. 7C) under Fe-replete conditions was similar tothe values for E. huxleyi growing on nitrate, however whenActinocyclus sp. became Fe-depleted it decreased significantly (by24%) its nitrogen per CV. Ammonium grown cells, and nitrate growncells with 100 .tM EDTA did not vary their nitrogen content underFe-deplete conditions. Finally, under both Fe conditions, ammoniumgrown cells had higher (1.2 to 1.5 times) nitrogen per CV than nitrategrown cells.CarbonThe total carbon content per CV for E. huxleyi (Fig. 6D), whichincludes coccolith carbon, and the carbon per CV associated with thecell only (after acid treatment), was not statistically different undereither Fe condition. The same pattern was observed for the carboncontent of Actinocyclus sp. (Fig. 7D) growing on nitrate; no statisticaldifference was obtained under Fe-replete or Fe-deplete (10 and 100pM EDTA) conditions. Ammonium grown cells of Actinocyclus sp. didnot vary their carbon per CV under Fe-stressed conditions, but theircarbon content was significantly higher (by 1.5 times) than thenitrate grown cells. Finally E. huxleyi had 5 times higher carbon perCV than Actinocyclus sp.Figure6.PhysiologicalparametersunderbothFe-replete(+Fe) andFe-deplete(-Fe)conditionsforE.huxleyigrowninnitratemedium.A)chiapercellvolume;B)fluorescence:chlaratio;C)nitrogenpercellvolume;D)carbonpercellvolume.CoccolithswereremovedinCandDbytreatingwithacid(acidified)andthennitrogenandcarbonweremeasured.Barsrepresentstandarderror(+/-1)fromfourcultures.-C C)0 0 0 I x CB‘a) oE>.a)c,Ci-c C) 2cn o > 2 z500400300200100 00.6+Fe-FeC0.100.080.060.040.020.00ml•coccolithso.sEr—. E0.4acidified>u0.3C)0) .0.20 0 L0.1( C)D+Fe•coccolithsQacidified-Fe——I0.0+Fe-Fevi33Figure7.PhysiologicalparametersunderbothFe-replete(+Fe)andFe-deplete(-Fe)conditionsforActinocyclussp.growninnitrateandammoniummedia.A)chiapercellvolume;B)fluorescence:chlaratio;C)nitrogenpercellvolume;D)carbonpercellvolume.Barsrepresentstandarderror(+/-1)fromtriplicatecultures;wherebarisnotvisible, triplicatecultureswereidentical.c0 0C.)‘x D).-...—..B2 1 0‘a) C.—.> o-c C)ro >0)Cc2 z+Fe-Fe10PM10PMEDTAEDTAUN03EJNH4+Fe10pMEDTA2 1 00.030.020.010.00-Fe 100pMEL3Anil’C•N03ONH4-Fe10pM EDTA-Fe100pMEDTA‘a) E D 0 > a) 0 C 0-o L. 00. 0.10) 0.0.0+Fe-Fe-Fe10PM10PM100PME13AEEWAWTA+Fe10PMwrA•--Fe-Fe10pM100pMEDTAEI3IAc.n Go59DISCUSSIONTo determine the role of Fe in natural phytoplanktoncommunities, it is necessary to determine whether phytoplanktonexhibit Fe-deficient characteristics, however, few studies haveexamined the physiological and biochemical effects of Fe deficiencyin algae. This was the first study to examine the interacting effectsof nitrogen source and iron stress conditions on the sinking rates andthe physiology of two newly isolated phytoplankton species from theSubarctic Pacific Ocean.Growth studiesGrowth represents the final outcome of all the cell’sphysiological processes. Obviously, iron is an integral component inmajor metabolic processes of a phytoplankton cell, and is thereforenecessary for growth to occur. It has been demonstrated that ironlimitation can cause decreased growth rates, and that therelationship between growth rate and iron nutritional availability isnot simply linear but that cells require geometrically more iron togrow at faster rates (Harrison and Morel, 1986; Rueter and Ades,1987).Emiliania huxleyiE. huxleyi was found to grow at maximal growth rates in Stn Pwater with no added iron (Muggli and Harrison, submitted). Also,Muggli and Harrison (submitted) found that E. huxleyi was notenergetically limited by different nitrogen sources under Fe-deficientconditions using artificial media. Because of its low Fe requirement,E. huxleyi would have been very difficult to get Fe-deficient if it wasgrown on ammonium as a nitrogen source, and therefore it wasgrown on nitrate only. E. huxleyi maintained half its maximumgrowth rate for two consecutive grow-ups in “no” Fe medium, butstopped growing after the last addition of macronutrients (N03 andP04) and trace metals (Mn and Zn). Brand et al. (1983) measured60E. huxleyi growth rates under Fe-replete (1.33 d-1), and Fe-deplete(1.16 d4) concentrations. However, other studies on thecoccolithophore Pleurochrysis carterae (Hudson and Morel, 1990) andon the diatom Thalassiosira pseudonana (Sunda et al., 1991) reported1/3 of maximum growth rate under Fe-deplete conditions.Actinocyclus sp.The growth rate of Actinocyclus sp. was slower than for E.huxleyi. The cell maintained its maximal growth rate after the firstdilution in “no” Fe medium, but slowly decreased to 1/3 of themaximum growth rate during the fourth grow-up. It was confirmedthat Fe-limited cells transported Fe faster than non-limited cells(Harrison and Morel, 1986). This may explain why Actinocyclus sp.was able to grow under Fe-deplete conditions for an extended period.Harrison and Morel (1986) found similar results with the diatomThalassiosira weissflogii when the specific growth rate decreasedwith decreasing Fe concentrations in the presence of a constantconcentration of free EDTA. Also, in a study conducted by Doucetteand Harrison (1990) the maximum specific growth rate of 10 neriticphytoplankton species under Fe limitation, ranged from 0.34 to1.99 &1, while the molar free ferric ion activity varied by two ordersof magnitude.However, in the present study, the last grow-up achieved halfthe maximum growth rate (see Fig. 3), suggesting contamination ofthe medium with dissolved Fe or the substitution for Fe-requiringmolecules by non-requiring ones in this organism (Hudson andMorel, 1990). The higher growth rate value could also be due to theregression estimate, which was based on only three cells count,because cell harvesting was done quickly to ensure health of theculture during the last grow-up. Also no growth rates were obtainedfrom the 100 .iM EDTA culture, but Muggli (pers. comm.) found thatActinocyclus sp. grown in Stn P water without EDTA grew faster thanwith EDTA added.61Nitrate vs ammoniumThere is no a priori reason to assume that cells grown in anenvironment providing an excess of energy (i.e. high light) shouldhave different maximal growth rates due to differences in theirnitrogen source in the growth medium (Thompson et al., 1989). Thiswas the case for Actinocyclus sp. grown on NO3- vs NH4+; cells grewas well or better on NO3- when Fe-replete. Thus, the results are incontradiction to the prevailing paradigm that NH4 is the favorednitrogen source of phytoplankton (Levasseur et aL, 1993).Because larger amounts of Fe are required for N03 reduction(Raven, 1988), inefficient N03 utilization was the expected result ofFe deficiency. However, under Fe-deplete conditions, Actinocyclussp. cells showed no significant difference in growth rate between thetwo nitrogen sources, and tended to grow better on NO3-. Muggli andHarrison (submitted) found that cells of E. huxleyi were growingfaster on NO3- than NH4 when Fe-deplete. Also, Morel et al. (1991b)observed that T. weissfloggi grew at the same rate with N03 or NH4as a source of N, under Fe deficiency and suggested that no additionalFe is required for growth on N03. It is possible that the ability ofcells to vary their maximum rate of nutrient uptake allows them tomaintain the same growth under NH4+ and N03 at very lowconcentrations of Fe (Harrison and Morel, 1986), and that the extraenergy cost of N03 reduction may be relatively small compared tothe total energy requirement of the cells (Levasseur et al., 1993).Sinking rateThis was the first study to report sinking rates of oceanicphytoplankton under their natural conditions (using Stn P water) andunder Fe-replete and Fe-deplete conditions. Therefore any sinkingrate comparison with the literature is impossible.Emiliania huxleyiThe main physiological response allowing the nitrate growncells of E. huxleyi to maintain their sinking rates was the marked62reduction in cell volume under Fe-deplete conditions. Reduction incell volume under Fe-deplete conditions was previously reported(Doucette and Harrison, 1990; Hudson and Morel, 1990; Rueter et al.,1990; Sunda et al., 1991; Morel et al., 1991a) and was also reportedfor E. huxleyi (Muggli and Harrison, submitted). This decrease in cellvolume was the result of Fe deficiency (as all other requirednutrients were present in saturating amounts). This supports theprediction made by Hudson and Morel (1990), namely, that oceanicphytoplankton can not have higher transport kinetics and that theonly available means of adaptation would be a reduction in cell sizeor Fe requirement. Obviously this 50% reduction in E. huxleyi’s cellvolume affected its sinking rates, and others physiologicalparameters.Actinocyclus sp.Actinocyclus sp. increased drastically its sinking rates (by 9times) under Fe-deficient conditions. This was the most significantdifference in all the physiological parameters between Fe-repleteand Fe-deplete conditions. Iron-limited cells function as if they areenergetically limited (Rueter and Ades, 1987), and can be comparedto light-limited cells. However, no studies have ever reported such alarge difference (generally 2 to 5 times increase) in sinking ratesbetween saturating and light-limited conditions (Steele and Yentsch,1960; Bienfang, 1980; Bienfang et al., 1983).In addition, Actinocyclus sp. decreased its cell volume underFe-deplete conditions, but was unable to maintain its buoyancy, likeE. huxleyi did between Fe-replete and Fe-deplete conditions.However, compared to E. huxleyi it was difficult to assume that thedecrease in cell volume of Actinocyclus sp. was due to Fe-depleteconditions only. Progressively decreasing cell size from division todivision, may also be another reason for the smaller cell volumeunder Fe-deplete conditions (see Appendix 9). It is possible that thelow Fe conditions (low energy) made sexual reproduction impossible,and that the cells were unable to maintain their normal size. Price(pers. comm.) reported elongation in a diatom under Fe limitation.63Lack of microscope observations in this study do not permit thisconclusion.The nitrogen source did not affect sinking rates or the cellvolume of Actinocyclus sp. for either Fe-replete and Fe-depleteconditions. Ammonium grown cells were previously reported to belarger in cell volume than nitrate grown cells by Thompson et al.(1989) for Thalassiosira pseudonana under high light conditions andby Muggli and Harrison (submitted) for E. huxleyi under Fe-depleteconditions.Rueter and Unsworth (1991), reported that iron-limited cells ofSynechococcus allocated a relatively higher amount of carbon intoprotein, which increased their cell density and protein concentrationby up to 7.8 times over a range of Fe limitation from the lowest(10-9 M) to the highest concentrations (10-6 M). It can behypothesized that Fe-deplete cells are heavier, and thereforedisadvantage over Fe-replete cells by this increased in density. Also,Actinocyclus sp. was so energetically limited by the low Feconcentrations that Fe-deplete cells were unable to maintain thesame buoyancy as Fe-replete cells even with a smaller cell size.Iron auotasThe concentration of iron that causes growth limitation andother changes in physiological parameters, is species-specific and areflection of the selective pressures of the species’ habitat (Morel etal., 1991b). Therefore, it was difficult to compare iron quotasmaintained by both oceanic phytoplankton with those alreadyreported, due to differences in growing conditions and culturemedium. An extended study of trace metal composition from thisexperiment will be made by D. L. Muggli.64Emiliania huxleyiDue to the very small size of E. huxleyi and its very low Ferequirement (Brand et al., 1983), cultures were mixed together toincrease the biomass available for the measurement of iron quota;this resulted in no replication for internal iron quota. However, adecrease in Fe quota per cell volume from Fe-replete to Fe-depleteconditions was observed. Also the difference could have been evenlarger if the Fe-replete samples were kept “natural” instead ofremoving the Fe associated with the coccoliths (acid treatment) andwith the cell surface (Ti (III) washed). The natural Fe quota per cellvolume for E. huxleyi decreased 3.5 times under Fe-deficientconditions; this is similar to the observations by Muggli and Harrison(submitted).Actinocyclus sp.The iron study conducted with Actinocyclus sp. was morecomplete and better results were obtained. The iron quotas per cellvolume revealed that a large part of the iron was bound to thefrustule of Actinocyclus sp. under Fe-deplete conditions. However,under Fe-deplete conditions all the iron was internal as shown by thelack of difference in the Fe quotas between natural and Ti(III)treated samples. Iron quotas decreased markedly under Fe-deficientconditions, especially for the natural treatment (by 10 times).Finally, the cells exposed to 100 .tM EDTA had higher internal iron asthe cells exposed to 10 p.M EDTA. It is possible that the addition ofEDTA disturbed the cells and suddenly bound all the Fe available inthe medium.Nitrate vs ammoniumUnder Fe-replete conditions, NH4 grown cells showed higherFe quotas when naturally treated, but once the cells were washedwith Ti (III), the Fe quotas were the same for both nitrogen sources.These results suggest that NH4 grown cells of Actinocyclus sp. hadmore uptake sites to bind extenal iron under Fe-replete conditions.The iron quota per cell volume was higher for NH4+ grown cellsunder Fe-deficient conditions. Theoretically, growth on NO3- should65require —40% more cellular Fe than growth on NH4 (Morel et al.,1991a). This elevated iron content in NH4 grown cells over N03grown cells did not reflect the expected higher Fe requirement forreductant for cells growing on N03, as produced by electrontransport processses (Thompson et al., 1989; Levasseur et al., 1993).Also, these results did not agree with Morel et al.’s (1991a)conclusion •that the low iron availability limits either the growth ofnitrate users or the ability of all algae to utilize nitrate.”The similar growth rate achieved under both nitrogen sourcessuggests that when Actinocyclus sp. grows on NO3, it maycompensate for the higher reductant requirement in other ways,such as adjustments in their biochemical composition (Thompson etal., 1989), and more particularly in this study, by decreasing itsinternal Fe quotas. Also, it is possible that both phytoplanktonspecies either increased their number of iron uptake molecules, orhave evolved substitutes for iron-containing molecules and whichwould further decrease their iron requirements when growing atvery low ambient Fe concentrations (Morel et al., 1991a).Carbon and NitrogenEmiliania huxleyiFe-deficient conditions did not affect E. huxleyi’s carbon CV1,as carbon content stayed the same under both Fe conditions. Thesame results were observed once the cells were acidified, showingthat the Fe-replete and deplete conditions did not cause the amountof coccoliths on the cell surface to vary. However nitrogen CV1under Fe-deficient conditions slightly increased causing thecarbon:nitrogen ratio to decrease by 40% under Fe-deficientconditions. The carbon:nitrogen ratio from acidified samples wereomitted because nitrogen CV1 samples were contaminated by theacid treatment. Previous results by Muggli and Harrison (submitted)showed that E. huxleyi grown on N03 artificial seawater mediumincreased its N CV-1 under Fe-deficient conditions, but also increasedits C CV-1, leading to a constant C:N ratio.66Actinocyclus sp.Carbon content per CV for Actinocyclus sp. was 5 times lowerthan for E. huxleyi, however the nitrogen content was similar.Therefore, Actinocyclus sp. had a smaller C:N ratio than E. huxleyi.Iron conditions (replete or deplete) and the addition of 100 tM EDTAdid not affect either the C:N ratio or the nitrogen and carbon contentof Actinocyclus sp.; however the nitrogen sources had a larger impacton POC and PUN.Nitrate vs ammoniumNitrate grown cells decreased significantly their nitrogencontent under Fe-deplete conditions, leading to an increase in C:Nratio. Nitrogen and carbon contents of ammonium grown cells didnot vary with the Fe conditions, but the values were alwayssignificantly higher than for cells grown on N03 for both Fe-repleteand Fe-deplete conditions.Similar results were previously reported for light experimentsby Rueter and Ades (1987); cultures incubated with N03 have lowercarbon fixation rates at all light intensities than those incubated withNH4. In addition, Thompson et al. (1989), using Thalassiosirapseudonana, found that N03 grown cells had 21% lower carboncontent and 24% lower nitrogen content per cell than NH4+ growncells. They concluded that cells growing on N03 could reduce theircarbon or nitrogen quotas such that constant amounts of reductantare needed to maintain growth rates equal to those of NH4+ growncells.Chlorouhvll aEmiliania huxleyiAs suggested by Levasseur et al. (1993), cells growing on N03can maintain equal POC and PUN and harvest more light energy(reductant) by increasing their chi a ce111. This increase in chl a perCV was observed for E. huxleyi growing on N03 in the present study.67Although, no comparison with ammonium grown cells was made,Muggli and Harrison (submitted) reported higher Chi a CV-1 andconstant C CV1 for E. huxleyi grown on NO3- compared to NH4 underFe-deficient conditions. They concluded that “the main physiologicalresponse allowing the N03 grown cells to maintain their normal cellcomposition was the marked reduction in cell volume.” In thepresent experiment, by reducing their cell volumes under Fe-deficient conditions, N03 grown cells of E. huxleyi reduced theirmetabolic requirements and were able to maintain normal nitrogenand carbon quotas and synthesize higher chorophyll per CV.An increase in the in vivo fluorescence:chl a ratio has beenreported as a good indicator for Fe-deficient cells (Rueter and Ades,1987; Rueter, 1988; Doucette and Harrison, 1990). Iron-sufficientcells, with higher chlorophyll concentrations are not only absorbingmore energy but they are loosing a lower proportion to fluorescence(Rueter and Ades, 1987). Although, in the present study, E. huxleyiincreased its chi a content under Fe-deficient conditions, and alsoincreased its in vivo fluorescence:chl a ratio by 75%. This largeincrease in fluorescence:chl a ratio, indicating lower photosyntheticefficiency, was not previously reported by Muggli and Harrison(submitted) for E. huxleyi.Actinocyclus sp.Contrary to E. huxleyi, Actinocyclus sp. decreased significantlyits chi a per CV under Fe-deplete conditions for both N03 and NH4grown cells. The same observation was also true for N03 grown cellswith the addition of 100 tM EDTA. However the in vivofluorescence:chl a ratio significantly increased, the same as reportedfor E. huxleyi in this study. The only exception was for cells grownunder N03 with 100 iM EDTA added; they decreased their in vivofluorescence:chl a ratio, after being disturbed by this high level ofEDTA. The increase in the fluorescence:chl a ratio associated withreduced nitrogen quotas has been suggested by Cleveland and Perry(1987) to result both from a rise in the specific absorption coefficient68of chi a and also from an uncoupling of photosynthesis in the diatomChaetoceros gracilis.Nitrate vs ammoniumThe nitrogen source did significantly affect the chl a CV1 underFe-replete conditions only. The higher content of chi a for N03grown cells, lead to a constant in vivo fluorescence:chl a ratioregardless of the nitrogen source under Fe-replete conditions.However the in vivo fluorescence:chl a ratio was statisticallydifferent for N03 and NH4 grown cells under Fe-deplete conditions,indicating that NH4 grown cells had better photosynthetic efficiencybetween the two types of cells.EDTAAll previous investigations with iron limitation, especiallywhen using radioactive methods, were done with 100 p.M EDTA(Harrison and Morel, 1986; Hudson and Morel, 1990; Sunda et al.,1991). Obviously, this very high concentration of EDTA is notnatural. Although the purpose of my study was not to compare theeffect of different concentrations of EDTA on cell physiology, someobservations were made during this study. A high concentration ofEDTA (100 jiM) compared to lower one (10 jiM) did not affect thegeneral physiology of the cell. One important advantage of a highEDTA concentration is that it can be used as a faster way to producean Fe-limited culture of phytoplankton. However, a highconcentration of EDTA may be toxic for some phytoplankton species,as reported by Muggli (pers. comm.) for Actinocyclus sp. andtherefore preliminary experiments or an acclimation period would bepreferable prior to experimentation.69Ecological considerationsIn a low Fe envinronment, it was previously found that oceanicspecies can survive much lower concentrations of iron than neriticspecies, due to their low Fe requirement (Brand et al., 1983). It wasalso hypothesized that small oceanic species will be able tooutcompete large phytoplankton species through adjustment of theirmaximum uptake rates, and that large species will be severely Fe-limited (Morel et al., 1991a).E. huxleyi, a small coccolithophore and Actinocyclus sp. a largerdiatom were isolated from the Subarctic Pacific Ocean. Actinocyclussp. was expected to be more growth-limited under low Fe conditionsthan E. huxleyi due to the size-dependent limit on uptake ratesimposed by diffusion (Hudson and Morel, 1990), and therefore itshould increase markedly its sinking rates under Fe-depleteconditions.Previous work by Hudson and Morel (1990) found that thediatom T. weissflogii has a greater maximal Fe uptake rate (by afactor of 8 on a per cell basis, and 4.5 on a per volume basis), thanthe coccolithophore Pleurochrysis carterae, but its half-saturationconstant was 4.4 times higher. They concluded that on a volumetricbasis, both species have similar Fe transport rates when transport isundersaturated. However they did not indicate which species wasfavoured by a higher Fe uptake rate under low Fe concentrations.In the present study, both E. huxleyi and Actinocyclus sp. didas well under Fe-replete conditions, but under a low Fe environment,E. huxleyi had an advantage because of its decrease in size and itsvery low Fe requirement. Although resolving the issue of relativefitness in low Fe environments requires a more carefuldetermination of the relative Fe requirements and influence of otherenvironmental parameters such as light, one can conclude that bymaintaining its buoyancy and physiological parameters such as POC,70PON, C:N ratio, and chi a, E. huxleyi is better adapted to survive in alow Fe habitat such as Stn P.Actinocyclus sp. was possibly energetically affected by iron-deplete conditions due to the reductant competition. However thecells maintained the same growth rates, sinking rates, andphotosynthetic efficiency under both nitrogen sources (N03 andNH4+), by decreasing their POC and PON content when grown onnitrate, under both Fe-replete and Fe-deplete conditions. Finally, theNH4 grown cells maintained higher Fe quotas under Fe-deficientconditions, opposite of what was theoretically expected.Given the major increase in sinking rates by Actinocyclus sp.under Fe-deplete conditions and the existing iron limitation for largecells at Stn P, this would suggest that it is the large phytoplanktonthat form the major component of the carbon flux out of the photiczone. Reflecting upon the biological carbon pump hypothesis ofMartin (1991), it is suggested that Fe fertilization would probablyenhance new production and growth rates, but would decreasesinking rates of individual phytoplankton cells (i.e. decrease the fluxof carbon out of the photic zone). However, once iron becomeavailable, other factors such as macronutrients, and trace metals,may limit growth rate and increase the sinking rates of oceanicphytoplankton.71GENERAL CONCLUSIONSVarious aspects of sinking rates responses and physiologicalparameters of oceanic phytoplankton were examined in this study.The major conclusions and contributions are summarized below.1. Coccolith formation affected the density of the cell, andmost importantly it increased the sinking rates of Emiliania huxleyi.The advantage of coccoliths as a ballast to vary sinking rates wasdemonstrated by the much higher sinking rates of the coccolithforming strain compared to the naked strain.2. Coccolith formation was found to be light dependent, andcoccoliths probably protect the cell against extreme high lightconditions.3. The naked strain of E. huxleyi was more sensitive to lightvariations than the coccolith-forming strain. The naked cells hadhigher growth rates under light saturation, while under low lightthey increased their sinking rates and chl a content compared to thecoccolith-forming strain.4. The growth phase affects sinking rates; the naked strainof E. huxleyi formed aggregates and coccolith-forming cells formedmultiple layers of coccoliths, leading to an increase in sinking ratesduring senescence.5. The nitrogen source (N03 vs NH4+) did not affect thesinking rates and other physiological parameters of the two strains,except growth rate, which was higher under NH4 for both naked andthe coccolith-forming cells.726. Actinocyclus sp. increased (by 9 times) its sinking rateunder Fe-deplete conditions, and E. huxleyi maintained the samesinking rates by decreasing its cell volume (by 50%) under Fe-deplete conditions.7. Growth rates of E. huxleyi and Actinocyclus species wereaffected by Fe-deplete conditions, and decreased gradually whentransferred to “no” iron medium.8. Actinocyclus sp. was affected by the nitrogen source, andobtained its maximum growth rate under nitrate (rather thanammonium) by decreasing its POC and PON.73FUTURE RESEARCHThere are a number of further experiments following from thisthesis:The role of coccoliths as a ballast to increase the cell densityand sinking rates of Emiliania huxleyi was demonstrated in thisstudy. However, before making generalities on the impact of thecoccolithophorid group on the carbon cycle, it is important tocompare the sinking rates responses of other coccolithophores with E.huxleyi under the same conditions. For example, fresh isolates (toensure no genetic variations and that the species retain their abilityto form coccoliths) of Criscosphaera carterae a coastal species, andCoccolithus neophelis an oceanic species (both available from theNEPCC), may be grown in nitrate or ammonium medium under two ormore light intensities, and sinking rates of naked vs coccolithforming strains of those species could be evaluated. This studywould increase our understanding of their possible role on thebiological pump and in the biogeochemical cycling of carbon.Futhermore, because many previous iron limitationexperiments used a very high concentration of EDTA compared to theconcentration of natural chelators in ocean, it would be important todetermine the effect of different concentrations of EDTA on cellphysiology as well as the possible toxicity of EDTA for somephytoplankton species. This experiment is important if one is toconfidently apply laboratory results to the natural environment.Therefore, a complete experiment should be conducted on the effectof various EDTA concentrations on cell physiology of E. huxleyi andActinocyclus sp. (e.g. growth rate, POC, PON, chi a, iron quotas, SEM)under Fe-replete and Fe-deplete conditions.74However, there are other areas amenable to future researchthat would add to our knowledge of factors affecting sinking rates.1. Genetic comparisons of the two strains of E. huxleyi(naked and coccolith-forming), using molecular techniques.2. Determine the effect of urea on growth rate, sinking ratesand cell physiology of E. huxleyi.3. Determine how the interdependence of Fe and nitrogennutrition is affected by light.4. Examine sinking rates of coastal species vs oceanicspecies under Fe-deplete conditions.5. 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Measurements were made at 6, 16, and24°C with both types of beads. Bars represent standard error (+1-1)from triplicate cultures; where bar is not visible, triplicate cultureswere identical.880.9 I ICALCULATED SR A0.8 -PMMA0.7V0.6 /. LATEX0.50.4 /•//0.3V/.o0.2-/° V 24°C— 0.10• 16°C0 6°C0.0 -—0.1 I0 5 10 15 20 25Particle Diameter (nm)09IBMEASURED SR0.8 -PMMA0.7T/LATEX I-l. 0.60.5 -0.4 -0.3 -/Y//) 0.1- • 16°Ct/ 7 24°C0 6°C0.0 -I I—0.10 5 10 15 20 25Particle Diameter (,um)890.0I IoPMMA-6°C0slope = 1.620I I I0.0 0.2 0.4 0.6 0.8Calculated SR (m day1)0.80.60.40.20.00.60.2 0.4 0.6CtlzxJ:2>..Cl)V0V:2Cl)a)(12a):20.60.40.20.00.60.40.20.01 .00.80.60.40.20.0LATEX 0oPe= 1.25Calculated SR (m day1)I I ILATEX16°Cslope = 1.140.0 0.2 0.4 0.6Calculated SR (rn day’)I I I0 /LATEX -24°Cslope = 1.21I I0.0 0.2 0.4 0.6 0.8 1.0>Cl)a)0a)>‘Cl)Vci)a)>.a)Cl)a)I I IPMMA V16°C t_///o0.4-2/slope = 1.01I I I I0.0 0.2 0.4 0.6Calculated SR (rn day1)1.0 I I I I0 /0.8 - PMMA24°C0.6-00.4 -00.2 -slope = 1.130.0_I0.0 0.2 0.4 0.6 0.8 1.0Calculated SR (m day’) Calculated SR (m day1)90EVUC-J.C.) UC C_JAppendix 2 (A-D). Semi-log plots showing growth curves as a functionof cell count over time forE. huxleyicoccolith-bearing cells. Cells weregrowing on either ammonium; A) and C); or nitrate; B) and D); undercontinuous light (high light =150 and low light = 20imol photon rn-2s1)Arrows represent day of harvesting for log and senescent phase cells.Bars represent standard error (÷/-1) from triplicate cultures.B20 21 22 23 24 2514‘I13EVU12-J1118 1914 -13121110 -15time (days)LtC14 16 18 20me (days)1413121110 -121413121110 -15 25-II, D .11’‘I.sJJ-4+Jy Li,t25 35me (days)NO3 -Lcm Light35time (days)91—a--— P04addeda NHaddeds— Blank—s—-— P0added• N03addedBlankAppendix 3 (A-B). Semi-log plots showing growth curves as a functionof in vivo fluorescence over time for E. huxleyi coccolith-bearing cells.When cells reached senescence (represented by arrow), nutrientswere added to the culture; phosphate (A-B), ammonium (A), nitrate (B),no nutrient was added to one culture (blank). These bioassays were usedto determine which nutrient was limiting when cells entered senescence.43200a,C)a,C)U)0z.4-Cl-JC)Ca)C)Cl)0C-J10time (days)20time (days)2092Appendix 4. pH for nitrate and ammonium grown cultures ofEmiliania huxleyi with (C) and without (N) coccoliths under high(150 iimol photons m2 s-i) and low (20 p.mol photons rn-2 s-i)irradiance, on the day of the SETCOL measurements (standard error(+1-1) in parenthesis; n=3).Log phase p HNNO3- 8.15 (0.04) 8.14 (0.03)High light8.20 (0.09) 8.12 (0.06)NO3- 7.95 (0.04) 8.05 (0.04)Low lightNH4 7.97 (0.01) 8.06 (0.01)Senescent pHNNO3- 8.01 (0.06) 8.09 (0.06)High lightNH4 8.05 (0.03) 8.03 (0.01)NO3- 7.98 (0.02) 7.80 (0.02)Low lightNH4 7.96 (0.03) 7.82 (0.01)93Appendix 5 (A-B). Physiological and biochemical compositionparameters for nitrate and ammonium grown cultures of Emilianiahuxleyi with (C) and without (N) coccoliths, under high (150 p.molphotons rn-2 s-1) and low (20 pmo1 photons m2 s1) irradiance duringlog (A), and senescent (B) phase (standard error (÷/-1) inparenthesis; n=3). Units are as follows: specific growth rate, p.(day-i); sinking rates (m day1); cell volume, CV (jim3); chi a per cell(ng chl a cell-i); chl a per CV (ng chl a j.tm-3); nitrogen per cell (pg Ncell-i); nitrogen per CV (pg N jim-3); nitrogen:chl a ratio (wt:wt);carbon per cell (pg C cell-i); carbon per CV (pg C j.tm3); carbon:chl aratio (wt:wt); carbon:nitrogen ratio (mol:mol); in vivo fluorescence (fi)per chl a * 1000 (ng chl a1); carbohydrate per cell (pg cell-1),carbohydrate per CV (pg .im3); lipid per cell (pg cell-1); lipid per CV(pg j.im3). See Materials and Methods in Chapter 1 for otherculturing details.94A) LOG PHASEPARAMETERS HIGH LIGHT LOW LIGHTNO NH4+ NO NH4+C N C N C N C N1.1. 0.503 0.564 0.592 0.690 0.168 0.143 0.147 0.157(0.004)(0.005) (0.005)(0.006) (0.02 1)(0.01 6) (0.01 5)(0.009)Sinking 0.071 0.041 0.116 0.052 0.099 0.101 0.083 0.135rates (0.008)(0.001) (0.025)(0.005) (0.02 1)(0.049) (0.018)(0.052)cell 25.7 19.1 32.3 25.6 24.9 15.9 25.2 19.2volume (0.3) (0.5) (1.1) (0.6) (0.2) (0.6) (0.9) (0.6)chia 69.1 42.3 121.0 58.4 112.0 151.2 125.3 207.5ce111 (6.9) (1.6) (13.5) (7.5) (5.1) (8.8) (13.1) (48.0)chi a CV4 2.68 2.22 3.78 2.28 4.49 9.55 4.94 10.7(0.23) (0.14) (0.56) (0.28) (0.20) (0.72) (0.34) (2.3)N ce111 1.68 0.59 2.26 0.87 2.64 1.07 2.64 1.33(0.17) (0.01) (0.16) (0.01) (0.15) (0.06) (0.36) (0.28)N C’?4 0.065 0.031 0.070 0.034 0.106 0.068 0.105 0.069(0.006)(0.001) (0.007)(0.001) (0.006)(0.005) (0.01 7)(0.01 3)N:chla 24.3 14.0 18.8 15.5 23.7 7.11 22.0 6.46(0.8) (0.4) (1.0) (2.3) (2.3) (0.02) (5.0) (0.16)C ceTh1- 11.6 4.07 16.1 5.25 18.9 6.03 17.5 7.14(1.7) (0.13) (1.1) (0.12) (0.8) (0.64) (2.3) (1.58)C CV1 0.45 1 0.2 13 0.502 0.205 0.759 0.380 0.700 0.370(0.063)(0.01 2) (0.053)(0.007) (0.03 8)(0.041) (0.1 10)(0.078)C:chl a 168.2 96.2 134.3 92.9 170.3 39.7 145.9 34.5(18.1) (1.5) (6.6) (11.7) (15.6) ‘(1.9) (32.5) (0.7)C:N 8.00 8.01 8.30 7.04 8.37 6.51 7.75 6.24(0.63) (0.24) (0.02) (0.24) (0.07) (0.33) (0.05) (0.08)fl:chl a 135.8 140.1 71.6 145.6 43.0 50.3 49.3 41.1(25.8) (2.2) (2.4) (20.0) (5.1) (9.8) (9.7) (7.7)Carboh. 3.24 2.80 6.20 3.69 5.51 5.33 5.07 3.85ce111 (0.16) (0.63) (1.07) (0.04) (0.23) (1.05) (0.67) (1.00)carboh. 0.126 0.083 0.194 0.144 0.221 0.337 0.200 0.199CV.1 (0.004)(0.030) (0.041)(0.002) (0.01 1)(0.069) (0.020)(0.050)lipid 3.62 0.61 2.61 2.09 2.74 2.90 1.01 9.17ceTh1 (0.23) (0.62) (0.34) (0.18) (1.56) (0.55) (0.26) (2.64)lipid CV1 0.140 0.098 0.082 0.081 0.110 0.182 0.041 0.469(0.006)(0.034) (0.013)(0.005) (0.063)(0.034) (0.01 1)(0. 124)95B) SENESCENTPARAMETERS HIGH LIGHT LOW LIGHTN03_ NH4+ NO_ NH4+C N C N C N C NSinkIng 0.138 0.096 0.307 0.117 0.456 0.134 0.485 0.284rates (0.034)(0.02 1) (0.038)(0.016) (0.065)(0.016) (0.215)(0.093)cell 25.4 15.5 26.7 20.2 26.1 14.5 22.8 14.9volume (0.2) (0.1) (1.9) (0.4) (1.2) (0.3) (0.3) (0.4)chia 46.9 17.8 32.2 18.6 99.0 47.2 100.4 51.0celP1 (16.8) (0.8) (4.1) (1.7) (9.8) (10.7) (2.6) (6.2)chi a CV4 1.86 1.15 1.20 0.92 3.78 3.22 4.46 3.40(0.68) (0.05) (0.05) (0.11) (0.21) (0.68) (0.07) (0.32)N ceJi1 1.50 0.35 0.66 0.35 2.08 0.39 2.20 0.36(0.27) (0.01) (0.09) (0.01) (0.18) (0.03) (0.13) (0.01)N CV1 0.05 9 0.023 0.024 0.0 17 0.079 0.027 0.098 0.024(0.01 1)(0.001) (0.001)(0.003) (0.003)(0.001) (0.005)(0.002)N:chl a 36.2 19.9 20.5 20.3 21.2 9.05 21.9 7.44(6.9) (1.0) (0.1) (2.5) (1.2) (1.45) (1.1) (1.01)C ceTh1 16.0 4.06 18.2 4.32 16.4 3.54 16.6 3.63(2.5) (0.05) (0.3) (0.28) (1.5) (0.16) (1.2) (0.13)C CV1 0.633 0.263 0.686 0.214 0.628 0.244 0.739 0.244(0. 104)(0.005) (0.042)(0.03 8) (0.040)(0.01 2) (0.055)(0.014)C:chl a 395.0 228.3 168.6 250.4 167.5 82.2 165.3 73.6(91.1) (11.1) (7.2) (23.4) (15.5) (17.1) (11.2) (10.2)C:N 12.5 13.3 32.7 14.5 9.19 10.4 8.78 11.5(0.4) (0.1) (3.2) (0.5) (0.34) (0.5) (0.17) (0.3)fl:chl a 278.9 370.6 301.6 360.2 131.4 322.2 93.2 289.7(83.9) (25.8) (44.6) (42.2) (13.1) (88.6) (11.2) (73.5)Carboh. 5.99 2.13 3.47 3.97 4.01 1.46 3.85 2.53ce111 (1.11) (0.14) (1.07) (0.31) (0.94) (0.05) (0.64) (0.22)carboh. 0.237 0138 0.129 0.197 0.152 0.101 0.171 0.168CV1 (0.046)(0.008) (0.005)(0.01 8) (0.032)(0.003) (0.027)(0.010)lipid 5.64 2.57 5.37 2.57 2.54 2.58 2.78 4.15celP1 (1.39) (0.40) (0.60) (0.32) (0.61) (0.40) (0.30) (1.13)lipid CV1 0.223 0.166 0.200 0.128 0.097 0.178 0.124 0.274(0.056)(0.025) (0.008)(0.017) (0.023)(0.028) (0.013)(0.066)96Appendix 6. Calculation of cell density from Stokes’ law equation.Stokes’ law equation:V = 2 g r2 (f’ - f) T’ Vs = velocity (m day1)9 g = gravitational acceleration Cm s2)r = radius of the cell (m)f = density of seawater (kg m3)f’ = density of the cell (kg rn-3)viscosity of seawater (kg m1 s1)Density of the cell isolated from Stokes’ law:f’ =f9 Vsi+fg r2 J-Radius from cell volume:_____Volume sphere= 4 it r3 r =\3[3 CV3 V4it-Density of seawater (f) and viscosity (‘9) from Table 11 and 25 inChemical Oceanography, 2nd edition, Riley and Skirrow Eds, 1975.-Velocity (Vs) from sinking rate data.Density of the cell with coccoliths:ftotal = Mtotal f = density (cell or coccolith)Vtotal M = mass (cell or coccolith)V = volume (cell or coccolith)= Mcocco + Meell = Mcocco + Mcell = fcell Pcocco (Mcocco + Mcell)Vcocco + VceIl Meocco + Mcell fcell Mcocco + fcocco Mcellfcocco fcellVariables:Mcocco = 0.4 cocco density = 2.7 cell density = from Stokes’ lawMcell (Smayda 1970)(Paasche 1962)Divided by Mcell = fcocco fcell (0.4 + 1’ Mccli[fceil(O.4) + fcocco (1)] MccliFinally = 1.4 fcocco fii0.4 fceii + fcocco97Appendix 7 (A-B). Physiological and biochemical compositionparameters in Fe-replete media (E. huxleyi = 100 nM Fe, Actinocyclussp. = 1000 nM Fe) and Fe-deplete media (transferred in “no” Femedia), under 14:10 light:dark cycle (150 .imol photons m2 s1):(A) For nitrate grown cultures of Emiliania huxleyi: sinking rates;cell volume; chi a per cell; total nitrogen per cell, N(t); acidified(coccoliths removed) nitrogen per cell, N(a); N(t) per chi a; N(a) perchi a; total carbon per cell, C(t); acidified carbon per cell, C(a); C(t) perchi a; C(a) per chi a; carbon(t):nitrogen(t); carbon(a):nitrogen(a); invivo fluorescence (fl) per chl a * 1000; chl a per Fe quota; carbon(t)per Fe quota; nitrogen(t) per Fe quota.(B) For nitrate and ammonium grown cultures of Actinocyclus sp:sinking rates; cell volume; chi a per cell; nitrogen per cell; nitrogenper chi a; carbon per cell; carbon per chi a; carbon:nitrogen; in vivofluorescence (fl) per chl a * 1000; chl a per Fe quota naturaltreatment; chi a per Fe quota Ti(III) washed; carbon per Fe quotanatural treatment; carbon per Fe quota Ti(III) washed; nitrogen perFe quota natural treatment; nitrogen per Fe quota Ti(III) washed.Standard error (+1-1) in parenthesis; n = 4 for E. huxleyi, n = 3 forActinocyclus sp. See Materials and Methods in Chapter 2 for otherculturing details.98A) EMILL4NIA HUXLEYIPARAMETERS Fe-Replete Fe-DepleteNO NOSinking (mdayfl 0.116 0.095rates (0.006) (0.024)cell (pm3) 34.48 18.63volume (1.10) (0.58)chia ce111 79.5 65.6(ngchlacell) (8.1) (5.5)N(t) ce111 1.06 0.82(pg N cell4) (0.09) (0.04)N(a) ceTh1 1.60 1.51(pgNcelll) (0.14) (0.06)N(t):chl a 13.59 12.78(wt:wt) (1.45) (1.26)N(a):chl a 20.63 23.35(wt:wt) (2.19) (1.39)C(t) ce111 18.71 10.72(pg C ceW1) (0.62) (0.56)C(a) cell-1 16.52 9.34(pg C cell-1) (0.65) (0.28)C(t):chl a 241.8 161.0(wt:wt) (22.5) (11.0)C(a):chla 213.5 144.7(wt:wt) (20.6) (9.7)C(t):N(t) 17.96 12.71(mol:mol) (1.08) (0.36)C(a):N(a) 12.15 7.22(mol:mol) (0.54) (0.14)fl:chl a 108.0 435.5(ngchla4) (14.0) (76.2)Chla:Fe 0.241 1.50(ng chi a amol)C(t):Fe 4715.4 20419.2(mol:mol)N(t):Fe 254.3 1411.7(mol:mol)99B) ACTINOCYCLUS sp.PARAMETERS Fe-Replete Fe-DepleteNO NH4÷ NO3 NH4÷Sinking 0.158 0.193 0.930 0.923rates (m day1) (0.010) (0.006) (0.011) (0.052)cell 3240.7 3425.2 2349.4 2365.4volume (jim3) (105.8) (116.4) (136.2) (95.1)chia cell-1 8.66 5.93 2.68 2.18(ng chi a ceW1) (0.53) (0.18) (0.22) (0.08)N cell-1 69.53 82.17 37.98 55.59(pg N cell1) (2.31) (7.24) (0.40) (3.16)N:chl a 8.09 13.92 14.40 25.50(wt:wt) (0.63) (1.50) (1.31) (1.03)C cell-1 390.1 495.9 285.1 360.6(pgCcell’) (16.1) (39.3) (1.4) (14.9)C:chla 45.40 83.66 108.13 165.88(wt:wt) (3.62) (6.17) (10.06) (9.32)C:N 5.61 6.07 7.50 6.50(mol:mol) (0.05) (0.38) (0.11) (0.26)fl:chl a 0.52 0.51 2.20 0.92(ng chi a .1) (0.05) (0.06) (0.20) (0.08)Chi a:Fe (natural) 0.182 0.088 2.48 0.470(ng chi a amol1) (0.018) (0.013) (0.26) (0.080)Chi a:Fe (Ti) 0.696 0.468 2.12 0.811(ngchlaamol1) (0.019) (0.104) (0.16) (0.215)C:Fe (mol:mol) 692.85 606.35 22692.1 6135.89(natural) (112.03) (56.89) (4245.5) (1040.79)C:Fe (mol:mol) 2622.1 3208.2 19396.7 10578.7(Ti) (174.8) (674.1) (3350.4) (2608.5)N:Fe (mol:mol) 106.06 85.36 2595.05 824.84(natural) (17.06) (3.68) (486.05) (105.28)N:Fe (mol:mol) 401.05 468.76 2214.83 1410.64(Ti) (27.30) (123.96) (377.52) (327.48)100Appendix 8 (A-F). Physiological parameters of Actinocyclus sp.grown under continuous light (ranging from 10-350 j.tmol photonsm2 s1) at 16°C and under two differents nitrogen sources (50 tMN03- or NH4+). A) Growth rate vs irradiance; B) Cell volume vsirradiance; C) Chi a per cell vs irradiance; D) Carbon:nitrogen ratio vsirradiance; E) Carbon per cell vs irradiance; F) Nitrogen per cell vsirradiance. Bars represent standard error (+1-1) from triplicatecultures; where bar is not visible, triplicate cultures were identical.Data were not expressed per cell volume because during theirradiance acclimation experiment with Actinocyclus sp., cells grown athigh light grew faster than cells grown at low light. Therefore, thereduction in cell volume at the high irradiance (Appendix 8B) wascaused by the increased number of cell generations of the high lightcells compared to the low light cells. Previously, the oppositeobservation was made by Thompson et al. (1989) for Thalassiosirapseudonana. They found that cell volume decreased as irradiancedecreased.160•_• 14080Z 601015000400030002000200irradiance irradiance(pmol photons nr2 s1) (LJrnoI photons m2 s-i)rI1412I .-420800700600Wa)C, C.50030012C 10a)II100 200 300 400irradiance(Limd photons rn-2 s-i)irradiance(pmd photons rn-2 s-i)0 100 200 300 400irradiance(pmd photons rn2 s-1)400 100 200 300 400irradia nce(trnoI photons m2 s-i)10240Appendix 9. Cell volume reduction of Actinocyclus sp. over time subjectedto a light:dark cycle (14:10), and grown in nitrate or ammonium media(see methods-Stn P water) enriched with 1000 nM Fe. Reduction in cellvolume is due to cell division only. Bars represent standard error (+/-1)from triplicate cultures. Lines show linear regression and the equationsare as follows:N03 y = 3135.8- 24.671x R2 = 0.764820000 10 20 30Time (days)NH4 y = 3762.7- 45.191x R2 = 0.758

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