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The effect of nitrogen deprivation on stable isotope 15n fractionation and preference of ammonium, nitrate,.. Sugimoto, Kugako 1998

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THE EFFECT OF NITROGEN DEPRIVATION ON STABLE ISOTOPE 15N FRACTIONATION AND PREFERENCE OF AMMONIUM, NITRATE, AND UREA BY MARINE DINOFLAGELLATES AND A RAPHIDOPHYTE by Kugako Sugimoto B. S., University of Hawaii at Manoa, 1994 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Earth and Ocean Sciences) We accept this thesis as conforming to the required standard THTfUNIVERSITY OF BRITISH COLMBIA OCTOBER 1998 © Kugako Sugimoto, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of /WZX O-Kcl Oc^an S^/>C0*L££<? The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ii ABSTRACT Nitrogen (N) preference and the uptake response of N-replete and N-starved dinoflagellate Amphidinium carterae and Prorocentrum micans, and the chloromonad (Raphidophyte) Heterosigma carterae grown on NH4+, N03", and urea were determined by N disappearance rates and uptake rates. N-replete cells preferentially took up NH4+ in the presence of three N forms. Urea was the second preference. NO3" was taken up last. N-replete Heterosigma carterae exhibited a strong preference for NH4+ uptake and its ratio of uptake preference of the three N sources relative to urea was 10 (NH4+):0.1 (N03"):1 (urea). These ratios suggest a strong influence of NH4+ on N03" uptake. However, the concentration of NO3" decreased rapidly when only NO3" and urea were present in a medium. This order of preference remained the same after N deprivation. However, N-deplete P. micans and A. carterae decreased their preference for N03" relative to both NH4+ and urea from 1.9 (NH4+):0.6 (N03"):1 (urea) to 1.5:0.5:1 and from 6.9 (NH4+):-0.2 (N03"):1 (urea) to 7.5:-1.1:1 respectively. On the other hand, N-deprived H. carterae increased its preference for N03" relative to both NH4+ and urea from 10 (NH4+):0.1 (N03'):1 (urea) to 7.5:0.2:1. The N uptake preference depended on the interactions of each N source. N-starved H. carterae in a batch culture grown on NH4+ showed an immediate NH4* uptake after the re-addition of NH4+ and ceased uptake for a short period and started to take up NH4+ again. This NH4+ uptake pattern existed, but was not evident in H. carterae grown on three N sources. iii Diel patterns of nitrate (NOV) uptake under a light:dark (14L10D) cycle were investigated in a batch culture of H. carterae which is known as a diel vertical migrator. The concentration of NOV in the culture decreased mostly during light periods, and hardly decreased or even increased (i.e. efflux) during dark periods. NOY-starved H. carterae showed N03 uptake during the dark period. NOY-starved cells grown under a lighfcdark cycle exhibited a longer period of no uptake after the re-addition of N03" than when grown under continuous light. The impact of N deprivation on N isotope fractionation by marine phytoplankton grown on NH4+, N03", and urea, or only N03" was examined by using the predicted model of 815N of particulate nitrogen (PN). The mechanism of isotope fractionation (s) for P. micans, A. carterae, and H. carterae changed rapidly with N availability. The best fit 8(NH4+) value for N-deprived A. carterae and H. carterae decreased from 20 and 24%o to 4 and < 4%o respectively following the re-supply of the three N sources while the e(NH4+) for P. micans increased from 12%o to 20%o. e(N03") for H. carterae grown on N03" also increased, probably due to the increased, efflux of dissolved organic N (DON) during the N deprivation. iv LIST OF CONTENT Abstract , ii List of Tables viList of Figures viiAcknowledgments xvIntroduction Overview 1 Ammonium (NH4+) 2 Nitrate (N03')Urea (CO(NH2)2)Physiology 3 UptakeAssimilation 5 Kinetic parameters 7 Starvation and rapid uptakeEcology 8 Interactions 1) NH4+and N03'2) Effect of starvation on interaction between NH4+ and N03" 9 3) Effect of light limitation on the interaction between NH4+ and N03" 10 4) Interactions between NH4+ and urea 15) Interactions between N03" and urea 1 6) Field study 12 Isotopic fractionation 3 Isotope expressionIsotope fractionations 4 Nitrogen isotope fractionation by phytoplankton 15 N utilization by dinoflagellates and flagellates 7 The species studied 18 1) Amphidinium carterae2) Heterosigma carterae 9 3) Prorocentrum micans 20 Objectives 22 Materials and Methods 3 Culture methodsBiomass, particulate N and nutrient analysis 26 Rate measurements 2Uptake rate 7 Nitrogen isotope analysisCalculations of the fractionation factor. 28 Multiple N source uptake model 9 Parti Results 30 Prorocentrum micans grown on and re-supplied with NH4+, N03", and urea (Experiment 1)Amphidinium carterae grown on and re-supplied with NH4+, N03", and urea (Experiment 2) 7 Heterosigma carterae grown on and re-supplied with NH4+, N03", and urea (Experiment 3) 46 Heterosigma carterae grown on and re-supplied with N03' (Experiment 4) 54 Heterosigma carterae grown on and re-supplied with N03" under LD cycle lights (Experiment 5) 8 Heterosigma carterae grown on and re-supplied with NH4+ under continuous light (Experiments) 63 Discussion 74 N Preference change 1) period 12) period H 7 Cellular physiological state 1) Surge uptake 79 2) Ammonium uptake in N-deplete phase 80 3) Nitrate uptake in N-deplete phase 2 4) Urea uptake in N-deplete phase 3 N interaction 84 Increased concentration and efflux of N 88 Ecology 9 Growth rate 91 Light: Dark cycle 1) Cell division and fluorescence number 93 2) Uptake diel periodicity 94 3) Heterosigma carterae and dark N03" uptake 96 Conclusions 98 Future studies 100 Part n Isotope fractionation Results 1) Prorocentrum micans grown on NH4+, N03", and urea 101 2) Amphidinium carterae grown on NH4+, N03', and urea 5 3) Heterosigma carterae grown on NH4+, NOV, and urea 109 4) Heterosigma carterae grown on N03" 10Sensitivity analysis 1) Prorocentrum micans grown on NH4+, N03", and urea 115 2) Amphidinium carterae grown on NH4+, N03", and urea 8 3) Heterosigma carterae grown on NH4+, N03", and urea 114) Heterosigma carterae grown on N03" 123 vi Discussion E(N03") 126 s(NH4+) 8 Effect of N deprivation 130 Conclusions 134 Future studies 5 References 6 Appendix 1 15LIST OF TABLES Table 1 Summary of experimental conditions. Nitrogen sources, species name, and average N starvation periods of triplicate batch cultures listed 24 Table 2 Comparison of the order of N preference before and after re-addition of NH4+, N03", and urea into triplicate batch cultures of P. micans, A. carterae, and H. carterae. Period I indicates the period when NH4+, N03", and urea were all present in the medium. Period II indicates the period when N03" and urea were present in the medium. Period in indicates the period when only urea was left in the medium 31 Table 3. Listing of variables and definitions used in the multiple N source uptake model 104 Table 4 Variables and their values used in the multiple N source uptake model. Prorocentrum micans, Amphidinium carterae, and Heterosigma carterae were grown on multiple N sources (N03", NH4+, and urea) under N-sufficient conditions. After 0 - 82 h of starvation, all three N sources were added simultaneously to the culture (i.e. N re-supply phase). Heterosigma carterae was grown on N03" under N03"-sufficient condition. After 52 h of N starvation, N03" was added to the culture (i.e. N re-supply phase). Symbols are defined in Table 3 106 VIII LIST OF FIGURES Figure 1 Growth of Prorocentrum micans grown on NH4+, N03", and urea under continuous light. Log plot of in vivo fluorescence versus time. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible 32 Figure 2 Nitrogen concentrations (urea as ng-at N L'1) during the growth of Prorocentrum micans grown on NH4+, N03', and urea under continuous light plotted against a) elapsed time and b) elapsed time since re-addition of NH4+, N03", and urea at t=0 to previously N-starved cultures for 56 h. Thus (b) is an expanded time version of the N concentrations during the re-addition phase shown in (a). Cultures were N-starved between 130 and 186.5 h and then 42 uM NH4+, 50 LIM N03> and 47 ug-at N L"1 urea were added at 186.5 h. Bars represent + 1 S.D. (n=2-3). Error bars are smaller than symbols where not visible 33 Figure 3 N uptake rates of triplicate, batch cultures of Prorocentrum micans grown on NH4+, N03", and urea and under continuous light plotted against a) average time between sampling and b) average time between sampling since re-addition of NH4+, N03", and urea at t=0 to previously N-starved cultures for 56 h. (b) is an expanded version of the re-addition phase of (a). Cultures were N starved between 130 and 186.5 h and NH4+, N03', and urea were added at 186.5 h. Bars represent ± 1 S.D. (n=2-3). Error bars are smaller than symbols where not visible 36 Figure 4 Relative N preference of NH4+ and N03' relative to urea for Prorocentrum micans during the period I before (•) and after (•) N starvation. The relative preference for urea is fixed at 1, both before and after N starvation 38 Figure 5 Relative N preference of urea relative to N03' for Prorocentrum micans during the period II before (•) and after (•) N starvation. The relative preference for urea is fixed at 1, both before and after N starvation 38 Figure 6 Growth of Amphidinium carterae grown on NH4+, N03", and urea under continuous light. Log plot of in vivo fluorescence versus time. Bars represent + 1 S.D. (n=2-3). Error bars are smaller than symbols where not visible 39 ix Figure 7 Nitrogen concentrations (urea as p.g-at N L"1) during the growth of Amphidinium carterae grown on NH4+, N03', and urea under continuous light plotted against a) elapsed time and b) elapsed time since re-addition of NH4+, N03", and urea at t=0 to previously N-starved cultures for 75 h. Thus (b) is an expanded time version of the N concentrations during the re-addition phase shown in (a). Cultures were N-starved between 119 and 193.5 h and then 29 uM NH4+, 30 uM N03', and 30 ug-at N L"1 urea were added at 193.5 h. Bars represent ± 1 S.D. (n=2-3). Error bars are smaller than symbols where not visible 41 Figure 8 N uptake rates of triplicate, batch cultures of Amphidinium carterae grown on NH4+, N03', and urea and under continuous light plotted against a) average time between sampling and b) average time between sampling since re-addition of NH4*, NOV, and urea at t=0 to previously N-starved cultures for 75 h. (b) is an expanded version of the re-addition phase of (a). Cultures were N-starved between 119 and 193.5 h and NH4+, N03", and urea were added at 193.5 h. Bars represent ± 1 S.D. (n=2-3). Error bars are smaller than symbols where not visible 43 Figure 9 Relative N preference of NH4+ and urea relative to N03" for Amphidinium carterae during the period I before (•) and after (•) N starvation. The relative preference for urea is fixed at 1, both before and after N starvation 45 Figure 10 Relative N preference of urea relative to N03" for Amphidinium carterae during the period n before (•) and after (•) N starvation. The relative preference for urea is fixed at 1, both before and after N starvation 45 Figure 11 Growth of Heterosigma carterae grown on NH4+, N03", and urea under continuous light. Log plot of in vivo fluorescence versus time. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible 47 Figure 12 Nitrogen concentrations (urea as ug-at N L"1) during the growth of Heterosigma carterae grown on NH4+, NOV, and urea under continuous light plotted against a) elapsed time and b) elapsed time since re-addition of NH4\ N03', and urea at t=0 to previously N-starved culture for 0 h. Thus (b) is an expanded time version of the N concentrations during the re-addition phase shown in (a). 31 uM NH4\ 41 uM N03", and 38 ug-at N L'1 were added at 175 h. Bars represent ± 1 S.D. (n=2-3). Error bars are smaller than symbols where not visible 48 X Figure 13 N uptake rates of triplicate, batch cultures of Heterosigma carterae grown on NH4+, N03', and urea and under continuous light plotted against a) average time between sampling and b) average time between sampling since re-addition of NH4+, N03", and urea at t=0 to previously N-starved cultures for 0 h. (b) is an expanded version of the re-addition phase of (a). NH4+, N03", and urea were added at 175 h. Bars represents 1 S.D. (n=2-3). Error bars are smaller than symbols where not visible 51 Figure 14 Relative N preference of NH4* and N03' relative to urea for Heterosigma carterae during the period I before (•) and after (•) N starvation. The relative preference for N03' is fixed at 1, both before and after N starvation 53 Figure 15 Relative N preference of urea relative to NOV for Heterosigma carterae during the period 11 before and after N starvation. The relative preference for urea is fixed at 1, both before (•) and after (•) N starvation 55 Figure 16 Growth of Heterosigma carterae grown on N03' under continuous light. Log plot of in vivo fluorescence versus time. Bars represent + 1 S.D. (n=2-3). Error bars are smaller than symbols where not visible 56 Figure 17 N03' concentrations during the growth of Heterosigma carterae grown on N03" under continuous light plotted against a) elapsed time and b) elapsed time since re-addition of N03" at t=0. Thus (b) is an expanded time version of the N concentrations during the re-addition phase shown in (a). Cultures were N03"-starved between 64 and 116 h and then 88 LIM N03" was added at 116 h. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible 57 Figure 18 N03* uptake rates of triplicate, batch cultures of Heterosigma carterae grown on N03* and under continuous light plotted against a) average time between sampling and b) average time between sampling since re-addition of N03" at t=0 to previously N-starved cultures for 51.5 h. (b) is an expanded version of the re-addition phase of (a). Cultures were N03" starved between 64 and 115 h and then N03" was added at 116 h. Bars represent + 1 S.D. (n=2-3). Error bars are smaller than symbols where not visible 59 Figure 19 Growth of Heterosigma carterae grown on N03" and under a 14:10 L:D cycle. Log plot of in vivo fluorescence versus time. Stippled xi areas indicate dark periods. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible 60 Figure 20 NOV concentrations during the growth of Heterosigma carterae on NOV and under a 14:10 LD cycle plotted against a) elapsed time and b) elapsed time after the re-addition of 73 uM N03' at 215 h to N-starved Heterosigma carterae for 96 h. Thus (b) is an expanded time version of the N concentrations during the re-addition phase shown in (a). Cultures were N03'-starved from 119 h to 215 h. Stippled areas indicate dark periods. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 62 Figure 21 N03" uptake rates of triplicate, batch cultures of Heterosigma carterae grown on N03' and under a 14:10 LD cycle plotted against a) average time between sampling and b) average time between sampling since re-addition of N03" at t=0 to previously N-starved cultures for 96 h. (b) is an expanded version of the re-addition phase of (a). Cultures were N03' starved between 119 and 215 h and then N03' was added at 215 h. Stippled areas indicate dark periods. Bars represent + 1 S.D. (n=2-3). Error bars are smaller than symbols where not visible 64 Figure 22 Growth of Heterosigma carterae on NH4+ under continuous light, culture 1. Log plot of in vivo fluorescence versus time 66 Figure 23 NH4+ concentrations during the growth of Heterosigma carterae grown on NH4+ under continuous light, culture 1, plotted against a) elapsed time and b) after the re-addition of NH4+ at t=0 to N-starved cells for 33 h. Thus (b) is an expanded time version of the N concentrations during the re-addition phase shown in (a). Culture was NH4*-starved between 65 and 98 h and then 77 uM NH4+ was added at 98 h 67 Figure 24 NH4+ uptake rates of a batch culture of Heterosigma carterae grown on NH4+ and under continuous light, culture 1, plotted against a) average time between sampling and b) average time between sampling since re-addition of NH4+ at t=0 to previously N-starved culture for 33 h. (b) is an expanded version of the re-addition phase of (a). Culture was NH4*-starved between 65 and 98 h and then NH4+ was added 98 h 68 Figure 25 Growth of Heterosigma carterae on NH4+ under continuous light, cultures 2 and 3. Log plot of in vivo fluorescence versus time. i J xii Bars represent ±1 S.D. (n=1-2). Error bars are smaller than symbols where not visible 70 Figure 26 NH4+ concentrations during the growth of Heterosigma carterae grown on NH4+ under continuous light, cultures 2 and 3, plotted against a) elapsed time and b) elapsed time after the re-addition of NH4+ at t=0, to N-starved cells for 18 h. Thus (b) is an expanded time version of the N concentrations during the re-addition phase shown in (a). Cultures were NH4+-starved between 88 and 106 h and then 74 uM NH4+ was added at 106 h. Bars represent + 1 S.D. (n=1-2). Error bars are smaller than symbols where not visible 1 Figure 27 NH4+ uptake rates of duplicate, cultures of Heterosigma carterae grown on NH4+ and under continuous light, culture 2+3, plotted against a) average time between sampling and b) average time between sampling since re-addition of NH4+ at t=0 to previously N-starved for 18 h. (b) is an expanded version of the re-addition phase of (a). Cultures were NH4+-starved between 88 and 106 h and then NH4* was added at 106 h. Bars represent + 1 S.D. (n=1-2). Error bars are smaller than symbols where not visible 73 Figure 28 Growth of Heterosigma carterae grown on various N sources and light conditions. Log plot of in vivo fluorescence versus time. ...78 Figure 29 Time series of [NCV] (•), [NH4+] (O) and [urea] (T) during the growth of Prorocentrum micans grown on NOV, NH4+, and urea under continuous light. Culture was N-starved between 105 and 186.5 h and then 36.6 ug-at N L'1 NH4+, 48.7 ug-at N L'1N03", and 49.4 ug-at N L'1 urea were added at 186.5 h. The best fits for the concentrations were determined by the dissolved N data using the equation: y = a*(1-EXP(b*(x-c))) where a is an initial N concentration, b is a co-efficient number, and c is the time when N reached 0, and are indicated by the solid lines 102 Figure 30 Time series of PN (A) and 515NPN (•) during the growth of Prorocentrum micans grown on N03', NH4+, and urea under continuous light. Culture was N-starved between 105 and 186.5 h and then 36.6 ug-at N L"1 NH4\ 48.7 ug-at N L"1 N03", and 49.4 ug-at N L"1 urea were added at 186.5 h. The 615NPN as predicted by the multiple N source uptake model is shown by the solid line. The 3 dotted lines indicate times when [NH4+], [NOV] and [urea] fits, determined by the dissolved N data to the concentration data in Fig. 29, reached 0, respectively 103 xiij Figure 31 Time series of [N03'] (•), [NH4+] (O) and [urea] (T) during the growth of Amphidinium carterae grown on NOV, NH4+, and urea under continuous light. Culture was N-starved between 119 and 193.5 h and then 29.6 ug-at N L1 NH4+, 34.3 ug-at N L"1 N03", and 31.2 ug-at N L"1 urea were added at 193.5 h. The best fits for the concentrations were determined by the dissolved N data using the equation: y = a*(1-EXP(b*(x-c))) where a is an initial N concentration, b is a co-efficient number, and c is the time when N reached 0, and are indicated by the solid lines 107 Figure 32 Time series of PN (A) and 815NPN (•) during the growth of Amphidinium carterae grown on N03\ NH4+, and urea under continuous light. Culture was N-starved between 119 and 193.5 h and then 29.6 ug-at N L"1 NH4+, 34.3 ug-at N L'1 NOV and 31.2 ug-at N L'1 urea were added at 193.5 h. The 815NPN as predicted by the multiple N source uptake model is shown by the solid line. The 3 dotted lines indicate times when [NH4+], [N03"] and [urea] fits, determined by the dissolved N data to the concentration data in Fig. 31, reached 0, respectively 108 Figure 33 Time series of [N03 ] (•), [NH4+] (O) and [urea] (T) during the growth of Heterosigma carterae grown on N03", NH4+, and urea under continuous light. Culture was added 32.2 ug-at N L"1 NH4+, 41.2 u.g-at N L"1 NOV and 37 ug-at N L"1 urea at 175 h. The best fits for the concentrations were determined by the dissolved N data using the equation: y = a*(1-EXP(b*(x-c))) where a is an initial N concentration, b is a co-efficient number, and c is the time when N reached 0, and are indicated by the solid lines 110 Figure 34 Time series of PN (A) and 815NPN (•) during the growth of Heterosigma carterae grown on N03", NH4+, and urea under continuous light. Nitrogen additions at 175 h to the culture were 32.2 ug-at N L"1 NH4+, 41.2 ug-at N L'1 N03", and 37 ug-at N L'1 urea. The 615NPN as predicted by the multiple N source uptake model is shown by the solid line. The 3 dotted lines indicate times when [NH4+], [N03"] and [urea] fits, determined by the dissolved N data to the concentration data in Fig. 33, reached 0, respectively. 111 Figure 35 Time series of [N03 ] during the growth of Heterosigma carterae grown on N03' under continuous light. Culture was N-starved between 64 and 115.5 h and then 85.6 ug-at N L'1 N03" was added at 115.5 h. The best fits for the concentrations were determined by the dissolved N data using the equation: y = a*(1-EXP(b*(x-c))) where a is an initial N concentration, b is a co-xiv efficient number, and c is the time when N reached 0, and are indicated by the solid lines 112 Figure 36 Time series of PN(A) and 815NPN(#) during the growth of Heterosigma carterae grown on N03* under continuous light. Culture was N-starved between 64 and 115.5 h and then 85.6 ug-at N L'1 NOV was added at 115.5 h. The 815NPN as predicted by the N source uptake model is shown by the solid line. The dotted line indicates times when [N03*] fits, determined by the dissolved N data to the concentration data in Fig. 35, reached 0 113 Figure 37 Response of the model to changes in a) e(NH/), b) e(N03"), and c) PNj in the N-sufficient condition for Prorocentrum micans grown on three N sources. The solid line represents the response of the model using the base values indicated in Table 4 116 Figure 38 Response of the model to changes in e(NH4+) in the N re-supply phase for Prorocentrum micans grown on three N sources. The solid line represents the response of the model using the base variables indicated in Table 4 117 Figure 39 Response of the model to changes in a) e(NH4+) and b) e(N03') in the N-sufficient condition for Amphidinium carterae grown on three N sources. The solid line represents the response of the model using the base values indicated in Table 4 119 Figure 40 Response of the model to changes in s(NH4+) in the N-sufficient condition for Amphidinium carterae grown on three N sources. The solid line represents the response of the model using the base values indicated in Table 4 120 Figure 41 Response of the model to changes in a) s(NH4+) and b) e(N03") in the N re-supply phase for Heterosigma carterae grown on three N sources. The solid lines represent the response of the model using the base variables indicated in Table 4 121 Figure 42 Response of the model to change in e(NH4+) in the N re-supply phase for Heterosigma carterae grown on three N sources. The solid line represents the response of the model using the base values indicated in Table 4 122 Figure 43 Response of the model to changes in s(N03") in the N-sufficient condition for Heterosigma carterae grown on three N sources. The solid line represents the response of the model using the base values indicated in Table 4 124 XV Figure 44 Response of the model to changes in e(N03") in the N re-supply phase for Heterosigma carterae grown on three N sources. The solid lines represent the response of the model using the base variables indicated in Table 4 125 xvi ACKNOWLEDGMENTS I would like to express my sincere gratitude to Dr. Paul J. Harrison for all his help in providing direction, encouragement, helpful advice, laboratory space, and funds for the experiments, as well as in reviewing this thesis. I would also like to thank Dr. Nathalie Waser for her direction on isotope fractionation analysis and helpful comments on this thesis. I would like to also thank Dr. F. J. R. Taylor for giving me a chance to study phytoplankton, providing cozy laboratory space with nice laboratory mates, such as Tonny Wagey and Juan Saldarriaga, a partial funding, and his comments on the thesis. I appreciate Dr. Al Lewis for helping me out from several emergencies and providing attention. Finally, I would like to thank Allen Milligan, Mingxing Guo, and Zhiming Yu for answering each of my questions in the laboratory and Bente Nielsen and Joe Needoba for helping with the isotope analysis. 1 INTRODUCTION overview Nitrogen (N) limitation determines the amount of primary production by phytoplankton in most coastal (Ryther & Dunstan 1971) and oceanic waters (Goldman et al. 1979). This has been expressed in terms of Liebig's "law of the minimum". The element in the lowest concentration relative to the other elemental requirements limits the production (Liebig 1840) and N is considered the principle limiting nutrient in the ocean. There are two kinds of primary production which are characterized by the form of N used. One is new production which is supported by allochthonous N sources such as N03" which is mixed into surface waters from the N03"-rich deep ocean, N2-fixation, riverine inflow and precipitation. Allochthonous N is defined as the encounter to the system for the first time. The other form of primary production is regenerated production. NH4+, urea, and amino acids are regenerated N sources. These N sources are released by death, cell lysis, grazing, viral attack (Suttle et al. 1990), and animal excretion. The concentration of regenerated N in coastal regions fluctuates. The maximum natural concentration is ~5 uM and concentrations < 0.5 uM are frequently found (McCarthy 1980; Sharp 1983; Antia et al. 1991). Among new and regenerated N, N03', NH4* and urea (CO(NH2)2) are the three most abundant forms of N in the ocean that are used by phytoplankton. Marine phytoplankton in the nutrient-depleted layer of the ocean may experience rapid N supply by upwelling of N03" (Walsh et al. 1978) or animal excretion of NH4+ and possibly urea (Dugdale 1967). Ammonium (NH4+) Atmospheric precipitation of NH4+ is important for N supply into the ocean (Buijsman etal. 1991). NH4+, however, can be released by biological activities such as zooplankton excretion, bacterial decomposition, and schooling fish. Regenerated NH4+ contributed a significant portion of the primary production in the study of the spring bloom in the New York Bight (Conway & Whitledge 1979). In this study NH4+ utilization as a percentage of NH4+ plus N03" utilization was 59% for the inshore areas and 70% at the shelf-break. The shelf-break had less N compared to the inshore water and primary production depended more on regenerated N. Nitrate (N03~) NOV is the most abundant form of N in coastal regions (Sharp 1983) and the most important N source in a highly productive ocean (Harrison et al. 1987). The abundance of N03' shows seasonal trends. Surface N03" concentrations of coastal waters increase during winter (> 20 ug-at N L"1) and gradually decrease due to the utilization by phytoplankton during the spring bloom. Urea (CO(NH2)2) Urea contains two atoms of N and therefore 1 uM urea = 2 ug-at L"1 or 2 umol L"1. The concentration of urea also largely depends on biological processes such as excretion by marine organisms, especially fish. Atmospheric precipitation also supplies urea to the marine system (Timperley et al. 1985). 3 Turley (1986) reported the highest urea concentration (23 uM) in the southern German Bight. Eutrophicated waters periodically exceed 3 uM urea. (Remsen 1971; Remsen et al. 1972: Berman 1974; Steinmann 1976; Satoh & Hanya 1981; Ignatiades 1986). Physiology There are two steps in the utilization of N by phytoplankton. The first step is uptake which is the actual transport of the particular form of N across the cell membrane. The second step is assimilation. In the second process, NOV, N02', and urea are reduced to NH4+ and are incorporated into amino acids during cellular metabolic processes (Lobban et al. 1985). The ambient N concentration, the relative abundance of different N forms, the physiological status of the phytoplankton, the availability of light, and temperature influence both uptake and assimilation of N by phytoplankton. Uptake NH4+ can be taken up by facilitated diffusion with the indirect expenditure of cellular metabolic energy. This diffusion needs carriers to bind NH4+ at the outer membrane and assist the passage across the membrane to the inner surface. Ion channels which are macromolecular pores that traverse the cell membrane, have been invoked to explain the high transport rates that occur down an electrochemical-potential gradient (Hedrich & Schroeder 1989). NH4+ may also be taken up by active transport processes. 4 NOV is actively taken up and hence this process requires energy. Carriers move N03" across the membrane against an electrochemical potential gradient. External concentrations of NOV are in the micromolar NOV range while intracellular concentrations are in the millimolar range. This large concentration difference between the inside and outside of the cell makes passive diffusion along an electrochemical gradient less important (Reed 1990). Active transport typically is much slower than channel-mediated transport because it is possible to transport only 103- 105 ions per second by active transport. In contrast, channels may allow 106 ions per second (Lobban & Harrison 1994). Urea is an uncharged molecule and therefore it diffuses through the cell membrane. However, phytoplankton have an active transport system and are capable of accumulating urea intracellularly. Light stimulates urea uptake and proton ionophores inhibit the uptake of urea. Thus, urea transport by phytoplankton requires ATP. Urea uptake does not directly require light (Syrett 1981). However, the production of ATP for membrane transport (Falkowski 1975) and the assimilation of urea occur during photosynthesis and this makes it appear that urea uptake depends on light. Light intensity and N uptake show a hyperbolic relationship with N uptake showing saturation at high light intensities (Eppley & Rogers 1970). In the ocean, light availability depends on the season, latitude, day/night cycle, and attenuation with depth. Phytoplankton in the ocean experience various light intensities in a daily periodic fashion. Malone et al. (1975) reported the periodicity of N uptake by various species in culture. The maximum N uptake 5 occurs during the day and the minimum uptake occurs at night (Maclsaac 1978; Fisher et al. 1982; Cochlan et al. 1991). Assimilation NH4* can be directly assimilated into amino acids, usually as glutamine. Incorporation of NH4+ into amino acids occurs in the chloroplast. Glutamate dehydrogenase (GDH) which has a low affinity for NH4\ is used when the internal pool of NH4* is high. On the other hand, glutamate synthetase (GS) has a high affinity for NH4+ and is used when the internal pool has low NH4+ concentration. Glutamine oxoglutarate aminotransferase (GDF) follows the reaction by GS. The control of NH4+ assimilation is often assumed to be rate-limiting for N incorporation (Syrett 1981). N03' must first be reduced to N02' by nitrate reductase using NADH or NAD(P)H as the electron donor. Nitrate reductase has been isolated and purified from many microalgae (Syrett 1981; Tischner etal. 1989; Solomonson & Barber 1990). The electron donor is usually NADH. However, NAD(P)H must be the electron donor in some phytoplankton (Lee 1980; Syrett 1981; Solomon & Barber 1990). Secondly, the product, N02", needs to be reduced to NH4+ by nitrite reductase with the help of ferredoxin as the electron donor. These steps follow the equation below: 2e" 6e' N03" > N02" > NH4+ 6 Therefore, growth on N03" requires extra energy. In addition to the high energy cost during the assimilation step, the energy cost for N03" uptake is higher than that for NH4+ (Falkowski 1975; Turpin & Bruce 1990). Since cells grown on N03" require an extra cost in reduction, the demand is increased for reducing power relative to ATP (Turpin & Bruce 1990). Both a higher photosynthetic quotient (Cleveland et al. 1989), and lower growth rate (Paasche 1971; Ward & Wetzel 1980; Rhee & Lederman 1983) occur. Several studies, however, reported that growth rates on NOV were not significantly different from those on NH4+ (Conover 1975; Syrett 1981; Thompson etal. 1989). Phytoplankton grown on N03" might compensate for their higher reductant requirements by adjustments in their biochemical composition, rather than by reducing their growth rates (Thompson et al. 1989). The reason why uptake of N03" is affected by photoperiod is due to the diel periodicity in activity and synthesis of the nitrate reductase enzyme (Syrett 1981). N03'-sufficient cells showed more pronounced diel periodicity of NOV uptake than N-limited cells. When urea is transported into the cell, it is broken down enzymatically using urease or urea carboxylase and converted to NH4+ as the final N product (Syrett 1981, 1988). The equation for this reaction is: CO(NH2)2 + H20 + 2H+ -> C02 + 2NH4+ This assimilation requires energy. In terms of reductant, urea assimilation is similar to NH4+. 7 Kinetic parameters A hyperbolic function of the concentration of the limiting nutrient (similar to the Michaelis-Menten equation for enzyme kinetics) expresses the rate of steady-state N uptake by phytoplankton (Eppley & Coatsworth 1968). Ks is the half-saturation constant that represents the substrate concentration at which the uptake rate is half its maximum. Spatial and seasonal abundance of a particular species partially depends on the value of these N uptake parameters (Maclsaac & Dugdale 1972). For example, species that possess a lower Ks have an advantage because they take up N faster than other species at low N concentrations. Frequently the uptake rate varies with time in the case of NH4+ limitation (Probyn & Chapman 1982; Rosenberg era/. 1984; Thomas & Harrison 1987) and NOV limitation (Thomas & Harrison 1987). A short incubation period can solve these problems by estimating the maximal uptake rate (Harrison et al. 1989)'. Starvation and rapid uptake Rapid uptake of NH4* by N-starved phytoplankton is an example of the effect of cellular physiological state on N uptake (Glibert & Goldman 1981). NH4+ uptake was facilitated by starvation and showed much more rapid uptake of NH4+ by N-starved phytoplankton than by the N-replete phytoplankton (Suttle & Harrison 1988). Rapid uptake of N03", however, did not consistently occur after N starvation (Price & Harrison 1988). Increased urea uptake rates were also not observed after N starvation (Bekheet & Syrett 1979; Price & Harrison 1988). 8 Generally, the effect of N starvation causes phytoplankton to take up NH4+ at rates in excess of those rates required to meet their ordinary N demands during balanced growth. Ecology Culture and field studies exhibit the uptake interactions between the various forms of N (Antia et al. 1991). The utilization of different forms might affect algal growth, species composition, and species succession (Harrison & Turpin 1982). This is because limiting nutrients have selective roles due to the nutrient uptake kinetics of the different species. For example, species that are capable of rapid uptake of a transient supply of NH4+ and urea (in a patch) would have an advantage because such cells need only to be exposed to an intermittent pulse of NH4+ in order to acquire their daily ration of N for growth (McCarthy & Goldman 1979). Interactions 1)NH4+and N03" The interaction of NOV and NH4+ in uptake has been well studied. Commonly, NH4* inhibits N03' uptake due to its preferential uptake over N03' as a N source. Chlorella vulgaris that was grown with NH4* and N03* ceased N03" assimilation after the addition of NH4* (Syrett & Morris 1963). These cells started to take up N03" immediately after NH4+ was used up in the medium. Chlamydomonas reinhardi also showed a similar phenomenon (Thacker & Syrett 1972a,b; Florencio & Vega 1983). Not only phytoplankton, but also macroalgae such as Gracilaria foliifera and Neoagardhiella baileyi preferred NH4+ over N03" as a N source (D'Elia & deBoer 1978). The presence of N03" did not decrease NH4+ uptake by these macroalgae. The NH4+ concentration affected the degree of inhibition in NOV uptake in the blue-green alga Anabaena cylindrica (Ohmori et al. 1977). In a lab study with Dunaliella tertiolecta and in a field investigation with Skeletonema costatum, NH4+ inhibited N03" uptake (Conway 1977). This inhibition of N03" uptake usually occurred above a certain range of NH4* concentration. Oyster-pond microalgae, however, showed a long-term elevated N03' uptake even in the presence of high NH4* concentration (Collos et al. 1989). The maximum N03" uptake by these microalgae occurred when NH4+ concentrations ranged between 10 and 30 uM and this maximum coincided with the maximum NH4+ uptake rate or with the start of a decrease in NH4* uptake rate. N03" uptake, then decreased and reached a secondary maximum rate again at low NH4+ concentrations. 2) Effect of the starvation on interaction between NH4+ and NOV N-deficient Skeletonema costatum showed strongly inhibited carrier-mediated N03' uptake without affecting diffusion (Serra et al. 1978). The N-deficient batch culture of the same species enhanced N03" uptake rate and accumulated a large N03" internal pool after the addition of a mixture of NH4+ and N03" (DeManche & Curl 1979). N03" uptake ceased following this initial N03" uptake and N03" assimilation was inhibited until the ambient NH4+ was 10 taken up. At this point, NOV uptake and assimilation commenced. In contrast, in the kelp, Laminaria groenlandica, N03* uptake was suppressed for 30 min after N03" and NH4+ were added back together to the N-starved macroalga (Harrison etal. 1986). 3) Effect of light limitation on the interaction between NH4+ and N03' Syrett and Morris (1963) used two different irradiances in investigating the preference of N03" and NH4+. The pattern of the inhibition of NOV uptake by NH4+, however, did not show any difference between high and low irradiance. However, the higher irradiance promoted the process because NH4+ disappeared faster and N03" uptake commenced earlier in the time course. 4) Interactions between NH4+ and urea The first study of the interaction between inorganic N (NH4+ and N03) and organic N (urea) was done by Lui and Roels (1970). NH4+ repressed the rate of urea uptake by the chrysmonad Ochromonas malhamensis in their study. Later, Horrigan and McCarthy (1982) showed that N-replete Thalassiosira pseudonana did not decrease urea uptake during the interaction with 10 uM NH4* for the first minute. In addition, urea uptake increased 5 min after these cells were exposed to NH4+. Skeletonema costatum, however, showed urea uptake inhibition by NH4+ (Horrigan & MaCarthy 1982). This inhibition required 30 min exposure to NH4+. According to another report by Lund (1987), inhibition of urea uptake occurred immediately after exposure to NH4+. Both Thalassiosira pseudonana 11 and Skeletonema costatum showed increased urea uptake inhibition by NH4+ under N-deplete conditions. The extent of inhibition of urea uptake may be a function of NH4* concentration, the N status of the cells and the species (Molly & Syrett 1988a; 1988b). NH4+ at 350 uM strongly inhibited urea uptake by Chlorella emersonii, but not by Phaeodactylum thcornutum. Urea uptake at NH4+ > 500 aM was strongly inhibited, particularly for Phaeodactylum thcornutum that was N-starved for 48 h. In contrast, 4 mM NH4+ was sufficient to inhibit urea uptake if the cells were N-replete. The addition of urea also depressed NH4+ uptake by Pheodactylum thcornutum (Molloy & Syrett 1988a). The uptake rate of urea-grown cells decreased to near zero when such cells were cultured and resuspended in 5 mM NH4+ for 24 h (Rees & Syrett 1979). Probably, NH4+ suppressed the development of the urea uptake mechanism in Phaeodactylum thcornutum. 5) Interactions between N03" and urea Urea inhibited NOV uptake in most experiments. In particular, such inhibition clearly occurred under dark and N-starved conditions (Molloy & Syrett 1988b). Nitrate reductase activity of the diatom, Cyclotella cryptica decreased due to the presence of urea (Liu & Hellebust 1974). 1 mM urea inhibited N03" uptake in the prasinophyte Platymonas (Ricketts 1988). However, 1 mM of NOV totally inhibited urea uptake (Ricketts 1988). These observations agree with the 12 general idea that urea suppresses N03' uptake, but at a lower level than NH4+ (Grand etal. 1967; McCarthy & Eppley 1972). Interestingly, NCV-sufficient cells showed enhanced uptake rates of both urea and NH4+ (Horrigan & McCarthy 1982). Later, however, Price and Harrison (1988) reported that N03'-replete Thalassiosira pseudonana did not exhibit an enhanced rate of urea uptake. Lund (1987), however, reported urea did not suppress NOV uptake by Skeletonema costatum. On the other hand, N03" suppressed urea uptake by the same species. In general, urea uptake is a constitutive property of phytoplankton growing on N03' as the sole N source. In the presence of NH4+, N03", and urea, NH4* are preferentially taken up, followed by N03"and urea (Dugdale & Goering 1967; Paasche & Kristiansen 1982; Levasseur et al. 1990). Phytoplankton prefer NH4+ (more reduced form) over NOV and urea (more oxidized forms) because NH4+ does not require a supply of electrons for reduction in the assimilation process which means less energy is required (Dugdale & Goering 1967; Eppley etal. 1973; McCarthy etal. 1977). 6) Field study In a field study of the interactions among NH4+, N03", and urea, all three N sources were taken up simultaneously in a natural spring phytoplankton assemblage from oyster ponds (Robert & Maestrini 1986). The urea uptake rate in this study, however, increased almost 10-fold with depletion of both NH4+ and NOV Interestingly, algal species composition changed within this assemblage. 13 Inorganic N (NH4+ and NOV) promoted diatoms. On the other hand, urea supported the growth of dinoflagellates. Simultaneous uptake of urea and other N sources by phytoplankton communities were reported in other field studies (McCarthy & Eppley 1972; Price etal. 1985). Isotopic fractionation N uptake and assimilation by phytoplankton is associated with N isotopic fractionation. The two naturally occurring stable isotopes of N are qualitatively similar in chemical behavior. Their reaction, however, occurs at slightly different rates and generally, 14N reacts more quickly than 15N due to lighter atomic mass. The isotopic composition of the product of a reaction could be affected by that of the substrate even though the difference in reaction rate is small. The product has the same isotopic composition as the original substrate if the reaction is complete. On the other hand, if the reaction is not complete, the resulting product pool is depleted in 15N relative to the original substrate. This is one of the important fractionation processes in the biogeochemical cycle of N in the ocean. Isotope expression The relative abundance of the stable isotopes of N is expressed as a ratio. For example, 15N/14N = 0.00677. The isotope ratio of a sample is measured relative to a standard. The del value expresses this relative difference and is defined as 14 8 in %o - ( R sample " R standard )/(R standard) X 1000 where R is the isotope ratio with the most abundant isotope in the denominator. Isotope fractionations Variations in relative isotope abundance are derived from the preferential reaction of one of the isotopes. Fractionation occurs during physicochemical processes. The fractionation factor (a) expresses the degree of segregation of one isotope as a result of a particular process and a is defined as: CL = R products reactants This equation can be expressed as: (a - 1 ) 1000 « 8Products - 8reactants and a is dependent on the environmental conditions. The isotopic composition of the product varies as a result of the extent of reaction. This process is referred to as Rayleigh distillation. The effect of Rayleigh distillation on the isotopic composition of the remaining reactant is expressed by Rf/R0 = F(a1) where f is the fraction of remaining reactant. Rf is the del value of the reactant at a particular f. Ro refers to the del value when f = 1, which indicates any reactant that is not removed. e (i.e. the per mil enrichment factor of the substrate relative to the product) is typically > 0 and expressed as: e = (a -1)x1000 15 Rayleigh equation (Mariotti etal. 1981) provides the magnitude of fractionation for a single-step, unidirectional reaction: 515Nproduct = 515NSubstrate, t=0 " E*- f/(1 - f)*ln f A positive e indicates that the light isotope accumulates more quickly than the heavy one. Montoya and McCarthy (1995) reported interesting results in which four flagellates, such as Isochrysis galbana, Pavlova lutheri, Dunaliella tertiolecta, and Chroomonas salina showed a lower fractionation factor (1 - 3%o) for NOV uptake and diatoms such as Skeletonema costatum and Thalassiosira weissflogii had higher fractionation factors (9 - 12%o). These authors suggested that the mobility of flagellates might result in a smaller diffusion zone around the cell, which would reduce the potential of a fractionating step during the uptake for N03" across the outer cell membrane. Nitrogen isotope fractionation by phytoplankton Recently the use of natural stable isotopes of N has provided a new way to examine the relationships between N sources and primary production (Francois etal. 1992; Calvert etal. 1992; Farrell etal. 1995). The isotope fractionation in various oceanic regimes can differ in terms of N availability, N source and species composition. The sedimentary 15N/14N ratio underlying nutrient-rich oceanic regimes can be used as a tracer of past relative N03" utilization and under certain assumptions, of past productivity (Calvert et al. 1992; Farrell et al. 1995). Low particulate 15N/14N ratios of bulk sedimentary 16 organic matter and N03'-rich waters and vice versa showed correspondence (Francois etal. 1992; Altabet & Francois 1994; Farrell etal. 1995). This correspondence results from the isotopic fractionation during the incorporation of NOV by phytoplankton. However, the factors which control fractionation by phytoplankton require further investigation (Goericke et al. 1994). The isotopic fractionation associated with the uptake of N03", N02~, and NH4+ has been studied in the laboratory (Wada & Hattori 1978; Wada 1980; Montoya & McCarthy 1995; Pennock et al. 1996). The degree of isotopic fractionation during N uptake by the diatom Phaeodactylum thcornutum clearly depended on the culture conditions (Wada & Hattori 1978). The isotope fractionation factor for N03" uptake by light-limited Phaeodacytylum thcornutum was inversely related to the specific growth rate of the cells (Wada & Hattori 1978). The same results were obtained by Wada (1980) in the study of fractionation factors for NH4+ uptake by the diatom Chaetoceros sp. Field studies on fractionation factors for the uptake of NH4+ and N03' by phytoplankton agreed with those from culture studies (Altabet & McCarthy 1985; Cifuentes et al. 1989; Montoya etal. 1991). The isotopic fractionation factor shows species differences. The diatom, Thalassiosira weissflogii exhibited a decline in the magnitude of the isotopic fraction factor for N03' uptake with growth rate (Montoya & McCarthy 1995). Other tested species did not vary consistently with growth rate. These results suggest that the net isotopic fractionation during growth on N03" is insensitive to growth rate. These authors concluded that the net isotopic fractionation may vary with photon flux density, which is supported 17 by the earlier studies by Wada (1980). Recently, Pennock et al. (1996) reported that fractionation was independent of the NOV concentration. The NH4+-grown diatoms, however, showed a dependency on the NH4+ concentration and fractionation increased as the NH4* concentration increased. N utilization by dinoflagellates and flagellates Like diatoms, dinoflagellates can use NH4+, N03", and urea. As in other phytoplankton, NOV and urea must be converted to NH4+ before incorporation into amino acids (Parsons & Harrison 1983). Dinoflagellates can store N03' internally. Internal concentrations of inorganic N were 1.8 mM N03' and 8.2 mM NH4+ in N03' and NH4+-grown Amphidinium carterae (Dortch et al. 1984). When the same species was grown on NH4+, the internal pool was 92 mM. The activity of nitrate reductase depends on the presence of N03' (Dortch & Maske 1982). Hersey and Swift (1976) reported the highest activity of nitrate reductase in Amphidinium carterae was twice as high at mid-day as at night. Some dinoflagellates did not exhibit this difference in laboratory and field studies (Packard & Blasco 1974; Eppley & Harrison 1975; Harrison 1976). Some dinoflagellates and flagellates migrate vertically downward to take up N03" at night below the nutricline and upward during the day for photosynthesis. These species possess high dark respiration activity that may account for this uptake activity. In addition, these cells used NADH (predominantly respiratory) rather than NADPH (chiefly from photosynthesis) for reduction of N03* (Eppley et al. 1969a). Nitrate reductase activity was less 18 under low light condition than under high light condition (Maclsaac 1978). The same authors reported little or no rhythmicity of nitrate reductase activity. However, migrating species such as Gymnodinium sanguineum (Dortch & Maske 1982) and Gonyaulax polyedra (Eppley et al. 1969b) did take up and reduce NOV in low light or darkness. Dinoflagellates possess both enzyme pathways, GDH and GS/GOGAT, for NH4+ incorporation. Amphidinium carterae, however, exhibited no GDH (Turpin & Harrison 1978). Such species might have an advantage in an NH4+-limited environment because GS has a higher affinity for NH4+ than GDH (Falkowski & Rivkin 1976). The species studied 1) Amphidinium carterae Amphidinium carterae Hulburt is a temperate naked marine dinoflagellate which occurs in shallow waters (Hulburt 1957). The average cell volume of Amphidinium carterae is 152 um3 (Needoba 1995). A growth rate of 0.76 d'1 was reported for this species grown under a 14:10 L:D cycle at 20 °C when 50 uM NH4+ was given to the culture at the beginning of the light period (Wheeler et al. 1983.) Some species of the genus Amphidinium have been associated with red tides and high concentrations have been reported to discolor sands in subtidal areas (Martin 1929; Herdman 1924a,b). Halstead (1965) described this species as a toxic dinoflagellate. Cells of Amphidinium carterae are toxic to fish and to mice (Thurberg & Sasner 1973). Because this toxin inhibited heart activities of 19 several marine animals, an acetylcholine analog was considered to be present in this organisms (Wangersky & Guillard 1960). Ikawa and Sasner (1975) identified toxins such as acrylycholine and choline o-sulfate in Amphidinium carterae. Seasonal fish kills by Amphidinium carterae blooms in the Sado estuary in Portugal was suspected (Sampayo 1985). Cyst formation has been reported and cycts remain healthy under a wide range of conditions (Sampayo 1985). Encysted stages survive conditions that would destroy the corresponding motile stage, enabling this species to repeatedly occur every year. 2) Heterosigma carterae The raphidophyte Heterosigma carterae is a marine naked unicellular biflagellate phototrophic flagellate (Yamochi 1983; Yokote et al. 1985). The classification of this organism was reviewed by Taylor (1992) and renamed from Heterosigma akashiwo. The cells were 8 - 25 um in length and 6 -15 pirn in width (Hara 1990). The growth rate of this species was < 0.8 d'1 under a 14:10 LD photoperiod of 160 umol photon m"2 s"1 at 20°C with 100 uM of each N source (Hoe Chang & Page 1995). A growth rate of 0.9 d'1 was reported when the culture was grown at 18°C on NH/ of 75% ESAW; NH4+ and N03" quotas grown on the same condition ranged from 46 to 47 and from 43.3 to 53 pg cell'1 respectively during the exponential phase (Wood & Flynn 1995). Heterosigma carterae has been identified as a major causative organism of noxious brown tide blooms in western Canada (Taylor & Haigh 1993), temperate and subtropical embayments in Japan (Yamochi 1989), Korea (Park 1989), 20 Singapore (Chang 1993), New Zealand (Larsen & Moestrup1992), England (Larsen & Moestrup 1989), western areas of North America (Tomas 1982) and Bermuda (Tomas 1978). Heterosigma carterae possesses toxins that causes massive fish kills such as salmon, yellowtails, sea bass and other fish in cages (Larsen & Moestrup 1989; Hallegraeff 1991; Honjo 1992). Four neurotoxic components of Heterosigma carterae were identified (Khan et al. 1997). This species is known to exhibit diel vertical migration (Nagasaki et al. 1996). There are several characteristics in the termination process of Heterosigma carterae brown tides. The population ceases diel vertical migration in situ and the brown tide disappears suddenly (Nagasaki etal. 1996). There is a size transition such that smaller-sized cells dominate toward the end of a simulated brown tide in outdoor tanks (Honjo & Tabata 1985). Lytic activities of the viruses suggest that they can be involved in regulating the bloom dynamics. Virus-like particles (VPLs) from infected Heterosigma carterae were isolated and showed strain-specific infection to Heterosigma carterae (Nagasaki & Yamaguchi 1997). Heterosigma carterae is known to possess benthic stages, including cysts, in the life cycle (Tomas 1978). This stage plays an important role as a seed population for brown tides. Cycts excyst between 10 and 15°C (French et al. 1995). 3) Prorocentrum micans Prorocentrum micans is a common constituent of coastal phytoplankton (Reid et al. 1985). Non-toxic blooms of Prorocentrum micans have been 21 reported in several coastal waters (Sweeney 1975; Cassie 1981; Avaria 1982; Hata et al. 1982; Pybus 1984). The cells were measured 35 - 70 um in length and 20 - 50 um in width (Horiguchi 1990). Growth rates of 0.59 - 0.99 d'1 were reported for this species grown in f/2 medium under continuous light and 19°C (Costas 1990). The minimum cell quota for N was estimated to be 0.74*10'12 mol cell"1 (Wang et al. 1996). A distinct phototactic response is characteristic to these species. The accumulation of Prorocentrum micans occurs in surface waters (Harvey 1966) and in the upper layer of the water column (Hattori et al. 1983). Therefore, they are exposed to a wide range in the intensity and quality of solar radiation. UV-absorbing compounds and yellow xanthophyll, and diadinoxanthin might protect cells by screening them from harmful radiation (Vernet et al. 1989). However, these UV absorbing compounds did not prevent photoinhibition and a decrease in chlorophyll per cell and a decrease in activities of Rubisco (Lesser 1996). Diel migration is another important characteristic of this species. Eppley and Harrison (1975) reported the ability to migrate through the thermocline on a diel basis. Prorocentrum micans migrated 6.3 m at a speed of 0.85 m h"1 (Edler & Olsson 1985). However, at this speed, Prorocentrum micans could not cross the halocline to reach NOV-rich water in Laholm Bay in Sweden. 22 Objectives The main objectives of this thesis were as follows: 1) Determine the impact of N deprivation on preference of three forms of N (NH4+, N03" and urea) by an ecologically important raphidophyte (Heterosigma carterae) and two dinoflagellates, Amphidinium carterae and Prorocentrum micans, isolated from British Columbia coastal waters. 2) Investigate responses by N-replete and N-deplete marine phytoplankton on N uptake. 3) Determine interactions between N03" uptake and light by Heterosigma carterae. 4) Predict 515N of particulate N using the multiple N source uptake model by assuming the sum of the Rayleigh contributions associated with the incorporation of each N source. 23 MATERIALS AND METHODS Culture methods - Amphidinium carterae Hulbert (NPCC 629), Heterosigma carterae Hulbert (NPCC 522R), Prorocentrum micans Ehrenberg (NPCC33) were obtained from the Northeast Pacific Culture Collection (NPCC), Department of Earth and Ocean Sciences, University of British Columbia. These cultures were grown on artificial seawater (ESAW) following the recipe of Harrison et al. (1980) as modified by Price et al. (1987). The medium contained a combination of three N sources, NO3", NH4+, and urea, which varied in concentration depending on the experiment (Table 1). The initial N03' and NH4+ concentrations in the medium ranged from 64 to 76 uM for Experiment 1 (Exp. 1), 2 (Exp. 2), and 3 (Exp. 3). Urea concentration ranged 14 to 22 uM since a molecule of CO(NH2)2 contains 2 atoms of N. The initial concentration of N for Experiment 4 (Exp. 4) and 5 (Exp. 5) ranged from 78 to 79 and from 77 to 83 uM respectively. The initial NH4+ concentration of Experiment 6 (Exp. 6) ranged from 70 to 78 uM. The N:P ratio in the medium was 4:1 to ensure that N was limiting biomass during stationary phase. Bicarbonate (NaHC03) was initially 2 mM and was added occasionally to the cultures to prevent C limitation and keep the concentration near 2 mM as in ESAW. The pH of the medium initially ranged from 8.2 to 8.4 and increased to 8.8 to 8.9 during the stationary phase for Exp. 1 and Exp. 4. HCI (6N) was occasionally added to prevent an increase in pH due to carbon uptake during growth for all experiments except Exp. 2. Culture medium was filter sterilized (0.22 um Millipore) and transferred into 1-liter 24 Table 1 Summary of experimental conditions. Nitrogen sources, species name, and average N starvation periods of triplicate batch cultures listed. Experiment Species N Source Light Condition Starvation Period (h) Added Back N Source 1 Prorocentrum micans NH4* continuous light 56 NH4* N03 N03" urea urea 2 Amphidinium carterae NH4* continuous light 75 NH4* N03" N03" urea urea 3 Heterosigma carterae NH4* continuous light 0 NH4* N03' N03-urea urea 4 Heterosigma carterae N03- continuous light 51.5 N03" 5 Heterosigma carterae N03" light:dark cycle light 96 N03-6 Heterosigma carterae NH4* continuous light 33 NH4* (culture 1) 18 NH4* (culture 2+3) 25 sterilized flasks, inoculated with the stock culture and placed in a cold room at 17.0±0.5°C. The cultures were gently stirred by hand twice a day. Autoclaving the flasks and inoculating the culture under the flow hood minimized bacterial contamination. The cultures were continuously illuminated with 170-185 umol photons m"2s'1 for all experiments except Exp. 5 which was grown on a 14L:10D cycle. The cells were acclimated to the N substrate in 1-L flasks for at least 6 days and when samples were first collected, the cells had been growing exponentially for a minimum of about three generations. After inoculation, the cultures were grown first in N-sufficient conditions. Then, they experienced N starvation for 0-101 h, and all three N sources were added back into the medium for Exp. 1, 2, and 3 (Table 1). Only N03" was added back for Exp. 4 and 5. Only NH4+ was added back for Exp. 6. Trace metals, vitamins, and macronutrients (Si, P, Fe, Bo, Se, and C) were added to the culture after N starvation so that their concentrations were the same as in ESAW. The experiment was designed to contrast a normal exponential growth (i.e. phase 1) and growth following the addition of N after a N starvation period, (i.e. phase 2). Growth conditions were designed to correspond to eutrophic and oligotrophic and/or temporarily N-depleted surface oceans. Therefore, dinoflagellates and the raphidophyte were grown under both-N-sufficient and N-depleted conditions. The N-sufficient phase simulated bloom conditions and coastal environments where the concentration of dissolved N is large relative to its biological uptake. On the other hand, the N re-supply phase was designed to 26 simulate oligotrophic oceans where new and regenerated N is supplied to the N-depleted surface ocean during episodic events. Biomass, particulate N and nutrient analysis - Samples for nutrients and particulate matter were mostly collected at relatively high cell densities at time intervals ranging up to 268.4 h. 815N analysis required a minimum of 1.5 umol N. The growth rate, u, was calculated from the following relationship: u = In (F2/F1) / (t2-t2), where F2 and F1 are the in vivo fluorescence values at time 2 (t2) and time 1 (t1) during log phase. Fluorescence was measured on Turner Designs model 10 fluorometer. Particulate nitrogen (PN) samples were collected by vacuum filtration at 0.5 atm onto pre-combusted (450 °C) glass-fiber filters (GF/F) measured with a Fisons™ automated CHN analyzer (model: NA 1500) on-line with the mass spectrometer which was used for 15N isotope analysis. The precision of each PN analysis was 1 - 2%. Aliquots of phytoplankton culture (30 to 480 ml) were withdrawn from the flasks at designated time intervals. The filtrate was used for NOV, NH/ and urea analyses. Manual N03" analyses were made using a spongy cadmium method (Jones 1984). NH4+ was analyzed according to Slawyk & Maclsaac (1972) and urea was measured manually according to the diacetyl monoxime method (Price & Harrison 1987). Rate measurements - Previously acid-washed polypropylene bottles were used to collect the samples and were rinsed once with the sample. The rate of 27 decrease of dissolved N concentration was calculated from the slope of external N concentration against time. This N disappearance rate is expressed as p.g-at urea N L*1 h'1 or uM h"1. To indicate the change in the preference for the three N sources, comparison of the phases of the uptake rate was calculated by using the ratio of each disappearance rate. Uptake rate - Uptake rate was calculated using the following equation: V=(C0-C1)/N*t when Co and Ci are the concentrations (ug-at N L*1) of N at the beginning and end of the time period. N is the cell density (cells L'1), t is the time period of uptake (h) and V is the uptake rate (ug-at 10"8 cells'1 h"1). Cell density was calculated from the fluorescence value which corresponded with the cell numbers at a particular time. Fluorescence and cell numbers were assumed to change in a linear relationship. Nitrogen isotope analysis - N isotope abundance was determined with a VG PRISM™ mass spectrometer. Particulate samples were prepared by rolling a GF/F filter in tin foil and compressing the foil into small pellets. Then, they were combusted in a stream of oxygen at an oven temperature of 1020° C. Results are expressed in the delta notation: 815N = (Rsample/Rstd-1)x1000 -(1) where R is the 15N/14N ratio and the standard (std) is N2 gas (NBS-14). The 815N of N1 and N2 standard, i.e. (NhUfeSO* was 1.34 and 20.85%o relative to NBS-14 28 respectively. It was 0.54 and 20.05%o relative to air. Internationally air is accepted as the standard. However, in the present studies, 815N relative to N6S-14 is used as a standard because absolute values are not critical for this study. The difference between the 815N of PN and the source is important other than the absolute value of either of them as explained below. The precision of 815N of the N source was 0.17%o (±SD of replicate pairs, n = 44 pairs). The 815N of the three N sources was: 3.82±0.18%o (mean ± 1SE, n = 12) for NaN03, -0.34±0.20%o (mean ± 1SE, n = 2) for NH4CI, and 0.06±0.12%o (mean ± 1SE, n = 3) for CO(NH2)2- Correction for carry-over of 15N-enriched N03" from the inoculum (0.5 mM NOV) to the culture medium was estimated to be small (i.e., < 0.2%o) due to the small volume of inoculum which was used. A small 15N enrichment might be introduced during culture dilution. Calculations of the fractionation factor - The isotope fractionation factor a was calculated using the accumulated product equation (Mariotti et al. 1981). Note that contrary to the definition adopted by Mariotti et al. (1981), a is typically > 1 and s (i.e. the per mil enrichment factor of the substrate relative to the product) is typically > 0; with e = (a -1) x 1000. In a closed system, as in a batch culture, the 815N of the first accumulated product is given by: 815NpN0 = 815NDNO - E  (2) 29 where PNo and DNo are the initial particulate N and dissolved N, respectively. Provided that s was constant during the consumption of the substrate, e was then derived from the accumulated product equation: 815NpN = 815N(DN)o - s • - f*ln f/(1 -f) - (3) where f is the fraction of unreacted substrate at any time during exponential growth. A curve of 815NPN versus F (F is defined as [-f/[1-f)] In f) was fitted with a linear function. In order to be applicable for equation (2), only data in log phase were analyzed for the calculation of s. The reason for this is it was only during this phase that N incorporation could be approximated by a one step unidirectional reaction. It means conversion of DN to PN. Once DN is exhausted, cell lysis will start and release various forms of N. Multiple N source uptake model - A model simulates the 815NPN which results from batch culture growth on multiple N sources. The model was designed to test if 815NPN can be described as the sum of the Rayleigh contributions associated with the incorporation of each N source. The dependent expressions for concentration of NH/, NOV, and urea are determined by the fits of the dissolved N data. The 815NPNt is described as follows: 615NPN, = (815NPN*ZPNX + 815NPNi*PNi)/(IPNx + PN,) 30 Parti RESULTS During batch culture experiments, N was used up and then cells were N-starved for up to 96 h before they were re-supplied with N. The first period when NH4+, NOV, and urea were all in the medium together is termed before I (bef. i) and the period after N re-supply is termed after I (aft. I). When only N03" and urea were present in the medium (i.e. NH4+ was already taken up), this period is termed before II (bef. n) and after N re-supply, after n (aft. II). When only urea was left in the medium, this period is termed before in (bef. in) and after in (aft. in). The order of N uptake preference was not changed in any experiments by N starvation (Table 2). However, the degree of preference of each N source changed. This will be illustrated in the following sections. Prorocentrum micans grown on and re-supplied with NH4+, N03+, and urea (Experiment 1) The growth curve of Prorocentrum micans is shown in Fig. 1. The growth rate, u calculated from 33 to 149 h was 0.30 d'1. This growth rate was the lowest among the three species used in the present study. The cell number was 940,000 cells L"1 at the fluorescence value of 9.4, which was used for the calculation of uptake rates (i.e. 1 fluorescence value corresponds to 100,000 cells L'1). The N concentration in the medium during the entire incubation period and after the re-addition of N sources is shown in Figs. 2a and 2b respectively. 31 Table 2 Comparison of the order of N preference before and after re-addition of NH4+, NOV, and urea into triplicate batch cultures of P. micans, A. carterae, and H. carterae. Period I indicates the period when NH4+, NOV, and urea were all present in the medium. Period n indicates the period when NOV and urea were present in the medium. Period in indicates the period when only urea was left in the medium. Period I Period n Period in NH4+ N sources in medium N03" Urea N03" Urea Urea Order Order 1st 2nd 3rd 1st 2nd Prorocentrum micans before N starvation NH, t+ N03- Urea N03" Urea Urea after N starvation NH ,+ NO3- Urea N03' Urea Urea Order Order 1st 2nd 3rd 1st 2nd Amphidinium carterae before N starvation NH : NO3- Urea N03" Urea Urea after N starvation NH : NO3- Urea N03" Urea Urea Order Order 1st 2nd 3rd 1st 2nd Heterosigma carterae before N starvation NH4+ N03' Urea N03* Urea Urea after N starvation NH4+ N03' Urea N03' Urea Urea 32 0 -I 1 r 0 50 100 150 200 250 Time (hours) Figure 1 Growth of Prorocentrum micans grown on NH4+, N03", and urea under continuous light. Log plot of in vivo fluorescence versus time. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 33 b 60 n Time (hours) Figure 2 Nitrogen concentrations (urea as ug-at N L"1) during the growth of Prorocentrum micans grown on NH4+, NOV, and urea under continuous light plotted against a) elapsed time and b) elapsed time since re-addition of NH4+, N03", and urea at t=0 to previously N-starved cultures for 56 h. Thus (b) is an expanded time version of the N concentrations during the re-addition phase shown in (a). Cultures were N-starved between 130 and 186.5 h and then 42 uM NH4+, 50 uM N03", and 47 ug-at N L"1 urea were added at 186.5 h. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 34 NH4+ disappeared from the medium within 49 h at a rate of 0.97 umol L"1 h"1. NH4+ showed a slow disappearance rate of 0.29 umol L"1 h"1 between 0 and 9 h, followed by a faster rate at 2.5 umol L"1 h'1 (9 -17 h) and 0.64 umol L"1 h"1 (17 -33 h). During that period (bef. I), urea was also taken up at a rate of 0.34 ug-at N L"1 h"1. Between 0 and 17 h, there was a slow uptake of 0.18 ug-at N L"1 h'1 followed by a much faster rate of 0.77 ug-at N L'1 h"1. However, NOV showed little uptake during this period. The disappearance rate of NOV during this period (I) was 0.21 umol L"1 h'1. In particular, N03" was taken up at a very slow rate of 0.07 umol L'1 h"1 for the first 41 h. After NH4+ was < 1 uM, the N03' disappearance rate increased to 0.57 umol L"1 h"1. Between 41 and 81 h, N03" disappeared at a rate of 0.81 umol L'1 h"1. During this period (bef. II) , the disappearance of urea was 0.27 ug-at N L"1 h"1. Between 33 and 65 h, very little urea was taken up and the disappearance rate was 0.06 ug-at N L'1 h'1. When N03" was < 1 uM, the disappearance rate of urea increased to 0.14 ug-at N L"1 h"1. Between 81 and 137 h, the urea disappearance rate was 0.30 ug-at N L"1 h"1. After 56 h of N starvation, the three N sources were re-supplied. For the first 2 h immediately after the re-supply of the three N sources, all three N sources remained almost at their initial concentrations. NH4+ disappeared from the medium within 13.5 h and the disappearance rate of NH4+ during the first 2 h was 2.2 umol L'1 h"1 and was followed by a faster rate of 5.5 umol L"1 h"1 (2-5 h). The concentration of urea again decreased together with NH4+, but at a rate of 35 65% of that of NH4+ during the period (aft. I). Between 1 -1.5 h after the re-supply of N, the disappearance rate of urea was negative (-1.3 ug-at N L'1 h"1). NOV was not used until the concentration of NH4* was < 1 uM Between 0 and 5 h, the disappearance rate of NOV was 1.1 umol L"1 h"1. Once NH4* disappeared from the culture, the disappearance of N03* exceeded that of urea for the following 18 h (aft. II). The disappearance rates of N03" and urea were 2.1 umol L"1 h"1 and 0.93 ug-at N L"1 h"1 respectively during this period (aft. II). Especially, between 13.5 and 25.5 h, the disappearance rate of N03" was 2.5 umol L'1 h'1. On the other hand, urea was taken up more slowly than N03" at 1.5 ug-at N L"1 h"1 between 4 and 25.5 h. Uptake rates are plotted versus the average time between sampling for the entire incubation period (Fig. 3a). The maximum NH4+ uptake rate (0.009 ug-at lO^cells'1 h"1) by N-replete cells occurred during the 9 -17 h interval. After the re-supply of the three N sources, the maximum NH4+ uptake rate (0.007 ug-at lO^cells'1 h'1) during the aft. I period occurred during the 3 - 4 h interval. (Fig. 3b). The maximum N03" uptake rate (0.003 ug-at fo^ceHs"1 h"1) occurred during the 41 - 49 h interval, which coincided with the exhaustion of NH4*. Subsequently, the average uptake rate during the 49 - 89 h interval was 0.002 ug-at lO^cells'1 h"1. N03" uptake rate was 0.009 ug-at lO^cells"1 h'1 for the aft. I period and 0.002 ug-at lO^cells'1 h"1 for the aft. II period. The maximum uptake rate of urea (0.004 ug-at lO^cells"1 h'1) by N-replete cells occurred during the 25 36 _P 0.020 -j 2 0.015 « o 0.010 a 0.005 | 0.000 a -coos Q. 3 z -0.010 -0 50 100 150 200 Time (hours) b — 0.012 -j •f 0.010 UL ^ 0.008 f° o 0.006 % 0.004 S 0.002 | 0.000 .£ -0.002 is a- -0.004 •} z -0.006 -I , , , . . . 0 5 10 15 20 25 30 Time (hours) Figure 3 N uptake rates of triplicate, batch cultures of Prorocentrum micans grown on NH4+, NOV, and urea and under continuous light plotted against a) average time between sampling and b) average time between sampling since re-addition of NH4+, N03", and urea at t=0 to previously N-starved cultures for 56 h. (b) is an expanded time version of the re-addition phase of (a). Cultures were N-starved between 130 and 186.5 h and NH4+, N03", and urea were added at 186.5 h. Bars represent + 1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 37 - 33 h interval during the bef. I period, and was 0.002 ug-at lO^cells"1 h"1. While NOV uptake was high during the bef. n period, the uptake rate of urea was 0.005 ug-at lO^cells'1 h'1 and 34% of N03" uptake. After N starvation and the re-addition of the three N sources, the maximum urea uptake (0.005 ug-at 10^cells"1 h"1) occurred during 25 - 33 h. Surge uptake of any of the N sources was not observed. The purpose of the present experiments was to determine the effect of N starvation on the preference for the three N sources, NH4+, N03~, and urea. Therefore, the relative disappearance rates were also calculated and expressed as ratios (Figs. 4 and 5). Before N starvation and while the three N sources (from 0 to 49 h) were present in the medium, the ratio of the disappearance and uptake rates of each N was NH4+:N03":urea = 1.9:0.6:1. Preference for urea over NH4+ and NOV during the aft. I period increased during N starvation. Preference for NH4+ and N03' relative to urea decreased and the ratio was 1.5:0.5:1 after N starvation. Period II was also compared. Before N starvation (from 49 to 97 h), the ratio of the disappearance rate of N03":urea was 2.1:1 compared to 2.0:1 after N starvation and not significantly different. Amphidinium carterae grown on and re-supplied with NH4+, N03", and urea (Experiment 2) The growth curve of Amphidinium carterae is given in Fig. 6. The growth rate (u) was 0.76 d"1 calculated from 59 to 143 h. When the fluorescence value 38 NOj N sources Figure 4 Relative N preference of NH4+ and NOV relative to urea for Prorocentrum micans during the period I before (•) and after (•) N starvation. The relative preference for urea is fixed at 1, both before and after N starvation. before N starvation i 1 after N starvation NO, urea N sources Figure 5 Relative N preference of urea relative to N03" for Prorocentrum micans during the period n before (•) and after (•) N starvation. The relative preference for urea is fixed at 1, both before and after N starvation. 39 2 i -1 -I , , 1 1— 0 50 100 150 200 Time (hours) Figure 6 Growth of Amphidinium carterae grown on NH4\ N03", and urea under continuous light. Log plot of in vivo fluorescence versus time. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 40 was 18, the cell number was 345,600 cells L"1, which was used for the calculation for the uptake rate (i.e. 1 fluorescence value corresponds to 19,200 cells L'1). N concentration in the medium is shown for the entire incubation period (Fig. 7a) and after the re-addition of N sources (Fig. 7b). Generally the values for separate cultures agreed well. First, NH4* disappeared from the medium within 71 h during the bef. I period at a rate of 0.48 umol L"1 h"1. During this period, the concentration of both NOV and urea remained at almost their initial concentrations. The disappearance rate for the period (bef. I) was -0.01 umol L"1 h"1 for NOV and 0.07 ug-at N L"1 h'1 for urea. The negative value for the NOV disappearance rate may be due to the efflux of NOV. The disappearance rates for NOV and urea were 0.01 umol L"1 h'1 and 0.11 ug-at N L"1 h"1 respectively between 0 and 77 h. When the concentration of NH4+ was < 1 uM, N03' and urea were both taken up at the rate of 0.77 umol L'1 h"1 and 0.46 ug-at N L'1 h"1 respectively during bef. n period. Between 15 and 21 h, the disappearance rates of N03" and urea were 0.79 umol L"V1 and 0.61 ug-at N L'1 h"1. Finally, urea was the only N source in the medium and disappeared within the following 11 h (bef. III) at a rate of 0.48 ug-at N L"1 h'1. After the cells were N-starved for 75 h and then re-supplied with the three sources of N, NH/ decreased quickly after the re-supply, and the disappearance rate was 10.1 umol L"1 h"1 during the first 2.7 h (aft. 1). Focusing only on the initial 0 to 15 min and 20 to 120 min periods, the rates were 29.8 and 9.2 1 41 a 50 -i Time (hours) Figure 7 Nitrogen concentrations (urea as ug-at N L'1) during the growth of Amphidinium carterae grown on NH4+, NOV, and urea under continuous light plotted against a) elapsed time and b) elapsed time since re-addition of NH4\ N03", and urea at t=0 to previously N-starved cultures for 75 h. Thus (b) is an expanded time version of the N concentrations during the re-addition phase shown in (a). Cultures were N-starved between 119 and 193.5 h and then 29 uM NH4+, 30 uM N03", and 30 ug-at N L'1 urea were added at 193.5 h. Bars represent + 1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 42 limol L'1 h'1 respectively. The concentration of N03" and urea increased during the first 5 min after the re-addition of the three N sources. N03" increased from 29.6 to 37.4 uM and urea increased from 30.4 to 34.5 ug-at N L"1. Between 0 and 40 min, efflux of NOV and urea occurred at rates of -12.3 umol L'1 h"1 and -5.3 ug-at N L'1 h'1 respectively. During the rapid decrease in NH4+ concentration (aft. I), N03" and urea concentrations both remained almost constant. However, urea started to decrease slightly before N03" started to decline. The decrease in urea started when NH4+ was 18 uM, while N03" started to decrease when NH4+ was 4 uM. When NH4* was present in the medium (aft. II), the disappearance rate of urea was 0.68 ug-at N L'1 h"1 and -0.74 ug-at N L"1 h"1 for NOV Between 50 min and 7 h , the observed rates were 3.7 umol L"1 h'1 for N03' and 2.7 ug-at N L"1 h"1 for urea. Once the concentration of NH4+ in the medium reached 1.7 uM, the disappearance rate of N03' was 6.6 umol L"1 h'1 and that of urea was 4.9 ug-at N L"1 h"1. After the concentration of N03" reached 5.6 uM, both N03" and urea decreased rapidly. The maximum uptake of NH4+ (0.08 ug-at lO^cells"1 h'1) for N-replete cells occurred during the 24 - 30 h interval (Fig. 8a). After the re-addition of three N sources, the maximum NH4* uptake rate (0.005 ug-at lO^cells"1 h"1) occurred during the initial 0 - 0.5 h interval (Fig. 8b). Uptake rates of N03" during the bef. I period fluctuated widely (-0.05 - 0.03 ug-at lO^cells"1 h'1) including negative uptake, indicating efflux. Uptake rate of N03" during the initial 0 - 5 h interval was -0.033 ug-at lO^cells'1 h"1. The maximum N03' uptake rates (0.027 43 0.2 <9 O If 0) I 3 0.0 -0.2 0 20 40 60 80 100 120 140 160 180 200 220 Time (hours) 10 15 Time (hours) Figure 8 N uptake rates of triplicate, batch cultures of Amphidinium carterae grown on NH4+, NOV, and urea and under continuous light plotted against a) average time between sampling and b) average time between sampling since re-addition of NH4+, NOV, and urea at t=0 to previously N-starved cultures for 75 h. (b) is an expanded version of the re-addition phase of (a). Cultures were N-starved between 119 and 193.5 h and NH4+, NOV and urea were added at 193.5 h. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 44 ug-at lO^cells"1 h'1) after the re-addition of N sources occurred during 6 - 8 h intervals when only NOV and urea were in the medium. Urea uptake during the bef. i period also fluctuated, and ranged from -0.031 to 0.055 ug-at lO^cells'1 h"1. Then, it became stable during the bef. n with an average urea uptake rate of 0.005 ug-at lO^cells"1 h"1 between 71 -113 h. Very low uptake (-0.030 ug-at lO^cells"1 h'1) occurred immediately after the re-addition of N sources (0-5 h), as the lowest NO3' uptake occurred during the same period. Urea uptake rate during the aft. 1 period was 0.004 ug-at lO^cells'1 h*1 and only 13% of NH4+ uptake. Uptake rate of urea during aft. II (2.3 - 8 h) was 0.011 ug-at lO^cells'1 h"1 and 76% of NOV uptake and reached a maximum uptake rate (0.031 ug-at lO^cells"1 h"1) during the last interval of aft. 11 (6 - 8 h). Surge uptake of any of the N sources was not observed. The ratio of the disappearance and uptake rates was calculated to find the N preference (Figs. 9 and 10). Before N starvation, the uptake ratio of NH4+:N03':urea was 6.9:-0.2:1, during the period that the three N sources existed in the medium (bef. 1). After N starvation, the uptake ratio for that same period (aft. 1) was 7.5:-1.1:1. The negative ratio for NOV indicates NOV efflux. The order of preference, NH4+, urea, and N03' remained the same after N deprivation. However, the relative preference of NH4* over NOV and urea increased after N deprivation. The preference of NH4+ relative to urea increased by 8.4%. The uptake of urea was faster than N03" after N starvation, which indicated that urea was preferably used by starved cells in period 1. The 45 before N starvation after N starvation NO,' N sources Figure 9 Relative N preference of NH4* and urea relative to NOV for Amphidinium carterae during the period I before (•) and after (•) N starvation. The relative preference for urea is fixed at 1, both before and after N starvation. Figure 10 Relative N preference of urea relative to NOV for Amphidinium carterae during the period n before (•) and after (•) N starvation. The relative preference for urea is fixed at 1, both before and after N starvation. relative preference of N03" over urea dropped dramatically by 5-fold. During the period when only N03' and urea were present in the medium (II), the ratios of disappearance rates of NOV and urea were 1.7:1 before N starvation and 1.3:1 after N starvation. Again, the order of preference was not changed. However, after N deprivation, the relative preference of N03" over urea decreased by 20%. N-starved Amphidinium carterae cells showed enhanced urea utilization ability over N03" in periods I and II. Heterosigma carterae grown on and re-supplied with NH4+, N03~, and urea (Experiment 3) The growth curve of Heterosigma carterae is given in Fig. 11. The growth rate was 0.56 d'1 calculated from 0 to 108 h. When the fluorescence values were 19.4 and 23.2, cell numbers were 457,080 and 520,640 cells L'1 respectively. The maximum cell density was 911,040 cells L"1 at the fluorescence value of 45. The calculation of cell density for Heterosigma carterae depended on these measured values in this study. The equation for the calculation of cell density was obtained from the change in the linear regression (cell density = 21755*[fluorescence value] - 27046; except for the fluorescence values < 1.24). The N concentration in the medium is shown for the entire incubation period (Fig. 12a) and after re-supply of the three N sources (Fig. 12b). Generally, the values for separate cultures agreed well. Before N starvation, NH4+ disappeared from the culture medium first. The disappearance rates of 47 2 i Figure 11 Growth of Heterosigma carterae grown on NH4+, NOV, and urea under continuous light. Log plot of in vivo fluorescence versus time. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 48 a 50 -i 20 40 60 80 Time (hours) Figure 12 Nitrogen concentrations (urea as ug-at N L"1) during the growth of Heterosigma carterae grown on NH4+, NOV, and urea under continuous light plotted against a) elapsed time and b) elapsed time since re-addition of NH4+, N03', and urea at t=0 to previously N-starved culture for 0 h. Thus (b) is an expanded time version of the N concentrations during the re-addition phase shown in (a). 31 uM NH4+, 41 uM N03', and 38 ug-at N L"1 were added at 175 h. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 49 NH4+ between 0 and 20 h and 20 and 62 h were only 0.09 umol L'1 h'1 and 0.57 umol L"1 h"1 respectively. While NH4+ was primarily used (bef. I), both NOV and urea remained near their initial concentrations. During this period (bef. I), the disappearance rates of NH4+, NO3", and urea were 0.47 umol L"1 h'1, 0.005 umol L"1 h"1, and 0.05 ug-at N L"1h"1 respectively. Especially low disappearance rates of 0.005 umol L"1 h"1 for N03" and 0.05 h"1 for urea were observed between 0 and 60 h and between 0 and 67.5 h respectively. The low N03" uptake was due to the increase in the N03" concentration during the first 12 h. Urea uptake showed less influence by NH4* in Exp. 1 and 2, and its concentration decreased more than NOV However, once the concentration of NH4+ reached 3.4 uM (bef. 11), N03" rapidly decreased within 63 h at a rate of 0.63 umol L"1 h"1. The disappearance rate of N03" between 67.5 and 107.5 h increased to 0.85 umol L'1 h'1. During the period when N03' and urea both existed in the medium (bef. 11), the disappearance rate of urea was 0.15 ug-at N L"1 h"1. Between 67.5 and 123 h, the rate of urea disappearance was 0.18 ug-at N L"1 h'1. Cells took up urea very slowly even after urea became the only N source in the culture medium. The disappearance rate of urea during bef. 111 period was 0.43 ug-at N L"1 h'1 and it was 0.44 ug-at N L"1 h"1 between 123 and 163 h. Since urea did not completely disappear from the culture medium, Heterosigma carterae did not experience true N starvation. After the re-supply of the three N sources, NH4+ was immediately used up within 6 h (aft. 1) at a disappearance rate of 5.1 umol L"1 h"1. At the end of NH4+ 50 uptake (6-13 h), the rate slowed to 0.17 umol L'1 h"1. During the same period (aft. i), NOV remained nearly constant and urea was slightly used at a disappearance rate of 0.68 ug-at N L"1 h'1. Again, in the presence of the three N sources, NH4+ strongly influenced on N03" uptake. The disappearance rates of N03" from 0 to 5 h, from 5 to 6 h, and from 11 to 15 h were -0.50, 3.2, and 5.1 umol L"1 h"1 respectively. Once NH4+ concentration became < 2.4 uM, N03" was quickly used up within the following 10.5 h (aft. II). Urea showed different phases of uptake. From 0 to 16.5 h (when N03" was primarily consumed) and from 16.5 to 27.5 h (after N03" was consumed), both rates were almost the same and they were 0.86 ug-at N L"1 h"1 and 0.89 ug-at N L"1 h'1 respectively. Then, urea disappeared gradually. The maximum NH4+ uptake rate (0.008 ug-at lO^cells"1 h'1) of N-replete cells occurred during 20 - 25 h interval, followed by a decrease in uptake before it reached a roughly constant rate of 0.008 ug-at lO^cells'1 h"1 (Fig. 13a). Uptake rate during the bef. I period was 96-fold higher than N03" and 10-fold higher than urea. In the N re-supply phase, the maximum uptake rate (0.003 ug-at lO^cells'1 h'1) occurred during the initial 0 - 0.5 h interval (Fig 13b). NH4+ uptake rate was 0.009 ug-at lO^cells"1 h"1 (4 - 5 h) from 0.007 ug-at lO^cells"1 h"1 (2 - 4 h). N03" uptake rate temporally decreased from 0.007 to -0.03 ug-at lO^cells"1 h"1 during the initial 0-20 h interval, followed by a rapid increase in uptake until it reached a roughly constant rate of 0.0008 ug-at lO^cells'1 h'1 during the bef. I period. After the re-addition of 51 i 0.10 -i = 0.08 <?° 0.06 O Z 0.04 t J 0.02 0.00 -0.02 0) JS a -0.04 z -0.06 NH4* Urea 50 100 150 Time (hours) 200 0.04 i J2 0.03 lOicel 0.02 JU 9 1 0.01 rate 0.00 « uptat -0.01 z -0.02 10 15 20 25 30 35 40 45 Time (hours) Figure 13 N uptake rates of triplicate, batch cultures of Heterosigma carterae grown on NH4+, NOV, and urea and under continuous light plotted against a) average time between sampling and b) average time between sampling since re-addition of NH4+, NOV, and urea at t=0 to previously N-starved cultures for 0 h. (b) is an expanded version of the re-addition phase of (a). NH4+, N03", and urea were added at 175 h. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 52 the three N sources, N03" uptake showed a negative value of-0.007 ug-at lO^cells h'1 during the initial 0 -1 h interval, followed by uptake rates which were close to zero. Toward the completion of NH4+ uptake, N03" uptake increased to 0.008 ug-at 10"6cells"1 h'1 (5 - 6 h) and maximum uptake rate (0.013 ug-at lO^cells"1 h"1) occurred during 7.5 -10 h interval. The maximum uptake rate of urea (0.046 ug-at lO^cells'1 h"1) by N-replete cells occurred during the 20 - 25.5 h interval, followed by a rapid decrease in uptake to roughly 0.002 ug-at lO^cells"1 h'1 in the next 25.5 -175 h. Interestingly, during bef. in (urea was the only available N source) urea uptake rate was very low, and roughly 0.001 ug-at lO^cells"1 h"1. In N re-supply phase, maximum urea uptake (0.005 ug-at lO^cells"1 h'1) occurred during 1 - 2 h interval. Evidence of any N surge uptake by N-deprived cells was not observed. Before the re-supply of N, the order of preference among the three N sources was NH4+, then urea, and lastly N03" in period I. The ratio of NH4+, NOV, and urea uptake during this period (bef. I) was 10:0.1:1 before the re-supply of N (Fig. 14). Heterosigma carterae showed a strong preference for NH4+ before N starvation. After N starvation and the re-supply of the three N sources, the ratio changed to 7.5 (NH4+):0.2 (N03'):1 (urea). During this period when three N sources existed in culture medium (aft. I), starved cells increased the relative preference of N03" to urea unlike the previous experiments (Exp. 1 and 2). The preference for N03" relative to NH4* also increased. As the N-replete culture decreased utilization of NH4+ and urea compared to Prorocentrum micans and Amphidinium carterae, N limitation might facilitate the ability to take 12 n NH/ NO," Urea N sources Figure 14 Relative N preference of NH4+ and N03' relative to urea for Heterosigma carterae during the period I before (•) and after (•) N starvation. The relative preference for NOV is fixed at 1, both before and after N starvation. 54 up N03". Otherwise, NH4* may influence on urea to a greater degree than N03". Following the 10.5 h period (aft. 11), the ratio of these N disappearance and uptake rates was N03':urea = 3.7:1 respectively (Fig. 15). The ratio of N03' and urea utilization before N limitation was 4.2 :1. The preference of urea relative to N03" was enhanced during this period (aft. 11). Heterosigma carterae grown on and re-supplied with N03' under continuous light (Experiment 4) The growth curve is shown in Fig. 16. The average growth rate, u, of the triplicate cultures was 1.0 d"1 calculated between 0 and 64 h. The relationship between fluorescence values and cell numbers is described in the results section of Exp. 3. The N03" concentration in the medium is shown for the entire incubation period (Fig. 17a) and after the re-addition of N03' (Fig. 17b). Generally the values for separate cultures agreed well. The N03" concentration in the culture medium remained quite constant during the first 16 h before N starvation. The disappearance rate was 0.24 umol L'1 h"1. This corresponded to the lag phase in growth rate. The concentration of N03' gradually decreased and reached near zero within 64 h. During this period, two evidently different uptake phases occurred. These two rates were 1.1 umol L"1 h"1 and 2.6 umol L'1 h'1 between 16 and 48 h, and 48 and 64 h respectively. After cells experienced 48 h of N starvation and N03" was re-added, N03' was immediately taken up by cells at a rate of 2.2 umol L'1 h"1 between 0 and 2.5 55 5 i NO," urea N sources Figure 15 Relative N preference of urea relative to NOV for Heterosigma carterae during the period n before and after N starvation. The relative preference for urea is fixed at 1, both before (•) and after (•) N starvation. 56 Figure 16 Growth of Heterosigma carterae grown on NOV under continuous light. Log plot of in vivo fluorescence versus time. Bars represent ± 1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 57 Figure 17 N03" concentrations during the growth of Heterosigma carterae grown on N03" under continuous light plotted against a) elapsed time and b) elapsed time since re-addition of NOV at t=0. Thus (b) is an expanded time version of the N concentrations during the re-addition phase shown in (a). Cultures were N03"-starved between 64 and 116 h and then 88 uM N03" was added at 116 h. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 58 h. This was immediately followed by the increased rate of 5.3 umol L'1 h'1 from 2.5 to 5.5 h and 11.8 umol L"1 h'1 from 5.5 to 10.5 h. NOV uptake rates are plotted versus average time between sampling for the entire incubation period in Fig. 18a. Maximum N03' uptake rate of 0.028 ug-at lO^cells"1 h"1 by N-replete cells occurred during 32 - 40 h interval, followed by an efflux of -0.006 ug-at lO^cells"1 h"1 (40 - 48 h). After the re-addition of N03", uptake rate was relatively low, and ranged from 0.002 to 0.013 ug-at lO^cells'1 h"1 during the initial 0 - 4.5 h interval (Fig. 18b). Then the N03" uptake rate increased to a maximum of 0.026 ug-at lO^cells"1 h'1 during the 8.5 -10.5 h interval, followed by a rapid decrease in uptake (0.006 ug-at 10"6cells'1 h"1, 10.5 -12.5 h) until the re-added N03" was completely utilized. The maximum N03" uptake rate of N-deplete cell was 91 % of N-replete cells. Heterosigma carterae grown on and re-supplied with N03" under L:D cycle lights (Experiment 5) The growth curve is shown in Fig. 19. The average growth rate under a L:D cycle was 0.72 d"1 calculated from 49 to 105 h, which was lower than under continuous light (1.0 d'1). Compared to continuous light, the growth curve was more variable, probably attributable to the L:D cycle. At most transit periods from light to dark, fluorescence increased except for the one transit period from 121 h (light) to 129 h (dark). On the other hand, a decrease in fluorescence 59 a 0.08 n O .0.04 -I . 1 • • • - ' 0 20 40 60 80 100 120 140 Time (hours) b 0.04 n 'tit a. -0.01 - 1 O -0.02 J • ' ' 1 ' 0 5 10 15 20 25 Time (hours) Figure 18 N03" uptake rates of triplicate, batch cultures of Heterosigma carterae grown on NOV and under continuous light plotted against a) average time between sampling and b) average time between sampling since re-addition of NOV at t=0 to previously N-starved cultures for 51.5 h. (b) is an expanded version of the re-addition phase of (a). Cultures were N03" starved between 64 and 115 h and then NOV was added at 116 h. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 60 0 50 100 150 200 250 300 Time (hours) Figure 19 Growth of Heterosigma carterae grown on N03" and under a 14:10 L:D cycle. Log plot of in vivo fluorescence versus time. Stippled areas indicate dark periods. Bars represent ± 1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 61 from the beginning to the end of dark period was observed between 57 - 66, 105 -114, and 152-161 h. During the dark period, the fluorescence increased, which indicated that they had divided. The relationship between fluorescence values and cell numbers is described in the results section of Exp. 3. The N03" concentration is shown for the entire incubation period (Fig. 20a) and after N limitation (Fig. 20b). Generally, the values for separate cultures agreed well. The disappearance rate of N03" was divided into three phases. They were 0.09 umol L'1 h"1, 0.77 umol L"1 h"1, and 1.1 umol L"1 h"1 between 0 and 42 h, 42 and 81 h, and 81 and 114 h respectively. Overall, the concentration of N03" fluctuated before the cells experienced N starvation. On the other hand, under continuous light, N03" decreased more smoothly and the growth curve was also smooth. N03' fluctuated for the first 42 h and then, when light was available, the concentration dropped rapidly from 76.9 to 63.4 uM during the following 15 h. Then, during the next dark period, the concentration of N03" increased from 53.6 to 62.5 uM during the following 9 h of darkness. During the next two light periods and one dark period, N03' decreased. This phenomenon was observed in all triplicate cultures and did not occur in cultures with continuous light before N starvation. After N re-supply to the medium, all triplicate cultures showed an increase in the N03" concentration. The average value of this increase was 11.7 uM (from 72.7 to 84.4 uM). This concentration remained above 80 uM for almost 3 h after the re-supply of N03". The efflux rate was -1.0 umol L'1 h'1 between 0 62 100 r 75 50 25 0 100 80 60 40 0 50 20 r-0 b 100 Time (hours) 200 250 ip^lllll o 1 1 \ I 1 J-^s« I 0 15 20 5 10 Time (hours) Figure 20 NOV concentrations during the growth of Heterosigma carterae on NOV and under a 14:10 LD cycle plotted against a) elapsed time and b) elapsed time after the re-addition of 73 uM NOV at 215 h to N-starved Heterosigma carterae for 96 h. Thus (b) is an expanded time version of the N concentrations during the re-addition phase shown in (a). Cultures were N03'-starved from 119 h to 215 h. Stippled areas indicate dark periods. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 63 and 4 h. Then the concentration of N03' started to decline and the disappearance rates between 5 and 8 h and 8 and 15.5 h were 3.8 umol L"1 h"1 and 6.0 umol L'1 h"1 respectively. Finally, N03" disappeared 62.5 h after the re-supply of N03". Compared to the continuous light culture, the concentration of NOV hardly decreased after the re-supply of N03", while N03" in the continuous light culture started to decrease immediately. N03" uptake rate in the N-replete phase fluctuated widely until it reached a maximum uptake rate (0.048 ug-at lO^cells'1 h'1, 42 - 49 h) (Fig. 21a). Then uptake rate reached a roughly constant rate of 0.004 ug-at 10^cells"1 h"1 in the next 66 - 97 h interval. Uptake rates at the end of dark periods always dropped from their previous values, and negative values (i.e. efflux) of -0.017 ug-at lO^cells1 h'1 (32.5 - 42 h), -0.013 ug-at lO^cells'1 h"1 (57 - 66 h) and very small values of 0.004 g-at lO^cells"1 h'1 (81 - 90 h), 0.001 ug-at lO^cells"1 h"1 (105 -114 h) occurred. The average N03" uptake rate after the re-addition of N03" was -0.006 ug-at lO^cells"1 h"1 during the first 5 h of the light period (Fig. 21b). The maximum uptake rate (0.023 ug-at lO^cells'1 h'1) occurred during the 9.5 -12 h interval in the light period. Maximum uptake rate of the N-deplete cell was 49% of that of N-replete cells. Heterosigma carterae grown on and re-supplied with NH4+ under continuous light (Experiment 6) Triplicate cultures were grown for this experiment. However, one culture had only 69.6 uM NH4* initially and consumed it very rapidly compared to the 64 Z 0 5 10 15 Time (hours) Figure 21 NOV uptake rates of triplicate, batch cultures of Heterosigma carterae grown on N03' and under a 14:10 LD cycle plotted against a) average time between sampling and b) average time between sampling since re-addition of N03* at t=0 to previously N-starved cultures for 96 h. (b) is an expanded version of the re-addition phase of (a). Cultures were N03"-starved between 119 and 215 h and then N03" was added at 215 h. Stippled areas indicate dark periods. Bars represent ±1 S.D. (n=2-3). Error bars are smaller than symbols where not visible. 65 other two cultures which had 78.4 uM for their average initial concentration. Therefore, the two results will be discussed separately. The growth curve for culture 1 is shown in Fig. 22. The growth rate, u, was 1.1 d"1 calculated from 0 to 58 h. The relationship between fluorescence values and cell numbers is described in the result section of Exp. 3. The NH4+ concentration for culture 1 is shown in Fig. 23a and after the NH4+ re-addition in Fig. 23b. The NH4+ in the first culture disappeared from the culture medium within 65 h. During the first 8 h, the disappearance rate of NH4+ was 0.41 umol L"1 h'1, which was followed by an increased rate of 1.6 umol L"1 h"1 between 14 and 45 h. Then cells experienced 33 h of N starvation. After the re-supply of NH4+, the concentration of NH4+ only decreased from 77.2 to 70.4 uM within the following 2 h at a rate of 3.4 umol L"1 h"1. Then, within the next 1.5 h, the concentration dropped from 70.4 to 56.1 uM and the rate during this period was 28.6 umol L"1 h*1. Interestingly, the NH4* concentration > 50 uM remained for the following 5 h and resulted in this very slow NH4+ disappearance. The disappearance rates during this period were 1.7 umol L'1 h'1 and 0.41 umol L"1 h"1 between 2.5 and 4.5 h and 4.5 and 7.5 h respectively. NH4+ gradually started to disappear again at the rate of 4.1 umol L"1 h'1 between 7.5 and 14.5 h, after the 5 h period where little uptake occurred. It took 32.5 h to consume all the added NH4+. The maximum NH4* uptake rate 0.031 ug-at lO^cells"1 h"1 of N-replete cells occurred during the 14 -18 h interval (Fig. 24a). In the N re-addition 66 Figure 22 Growth of Heterosigma carterae on NH4+ under continuous light, culture 1. Log plot of in vivo fluorescence versus time. 67 a Time (hours) b 100 -i Figure 23 NH4+ concentrations during the growth of Heterosigma carterae grown on NH4* under continuous light, culture 1, plotted against a) elapsed time and b) after the re-addition of NH4+ at t=0 to N-starved cells for 33 h. Thus (b) is an expanded time version of the N concentrations during the re-addition phase shown in (a). Culture was NH4*-starved between 65 and 98 h and then 77 uM NH4+ was added at 98 h. 68 0.05 n | 0.04 <» r 0.03 f 0.02 2 0.01 0.00 3 *X* -0.01 20 40 60 80 100 120 Time (hours) 10 Time (hours) 15 Figure 24 NH4+ uptake rates of a batch culture of Heterosigma carterae grown on NrV and under continuous light, culture 1, plotted against a) average time between sampling and b) average time between sampling since re-addition of NH4* at t=0 to previously N-starved culture for 33 h. (b) is an expanded version of the re-addition phase of (a). Culture was NH4+-starved between 65 and 98 h and then NH4+ was added at 98 h. 69 phase, maximum uptake (0.042 ug-at lO^cells"1 h'1) occurred during the 1 - 2.5 h interval, followed by a rapid decrease in uptake and very low NH4+ uptake period (2.5 - 6.5 h) with an average rate of 0.002 ug-at lO^cells"1 h'1 (Fig. 24b). Data from the other duplicate cultures agreed well with both growth 1.02 d"1 calculated 14 to 54 h and NH4+ disappearance rates (Figs. 25 and 26a). Before N starvation, the NH4+ concentration continued to decline without any lag period, which matched the log period of growth. The disappearance rates of NH4+ were 0.78 umol L'1 h"1 and 1.51 umol L'1 h'1 between 0 and 23 h and 23 and 54 h respectively. After the re-addition of NH4+ to N-starved cells, NH4+ concentration hardly decreased for the following 1.5 h (Fig. 26b), which also occurred in culture 1. The disappearance rate between 0 and 1.5 h was 0.96 umol L"1 h"1. Following the lag period, NH4+ concentration rapidly dropped from 72.1 to 59.4 uM at a rate of 25.4 umol L'1 h"1. As in culture 1, the concentration of NH4+ almost did not decrease after the rapid disappearance of NH4+. The disappearance rate from 2 to 4.5 h was 2.8 umol L'1 h"1. From 4.5 to 5.5 h, the rate increased to 13.0 umol L"1 h"1. Then, NH4+ started to disappear again gradually at a rate of 4.4 umol L'1 h'1 between 5.5 and 10.5 h and it took 24.5 h for the complete disappearance. NH4+ disappearance after the re-supply of NH4+ showed several steps. First, the concentration did not change and, then the concentration dramatically dropped followed by a slow decrease in NH4+. This phenomenon was more distinct than that observed in the same species grown on NOV and continuous light (Exp. 4). 70 0 -I 1 1 1 1 1 1 1 . 0 20 40 60 80 100 120 140 Time (hours) Figure 25 Growth of Heterosigma carterae on NH4* under continuous light, cultures 2 and 3. Log plot of in vivo fluorescence versus time. Bars represent ± 1 S.D. (n=1-2). Error bars are smaller than symbols where not visible. 71 a 100 n Time (hours) Figure 26 NH/ concentrations during the growth of Heterosigma carterae grown on NH/ under continuous light, cultures 2 and 3, plotted against a) elapsed time and b) elapsed time after the re-addition of NH/ at t=0, to N-starved cells for 18 h. Thus (b) is an expanded time version of the N concentrations during the re-addition phase shown in (a). Cultures were NH/-starved between 88 and 106 h and then 74 uM NH/ was added at 106 h. Bars represent ± 1 S.D. (n=1-2). Error bars are smaller than symbols where not visible. 72 A relatively high average N03* uptake (0.030 ug-at lO^cells'1 h'1) for N-replete cells occurred during the initial 0 -18 h interval (Fig. 27a). During the N-deplete phase, uptake rate fluctuated and even the NH/ concentration in the medium did not change much (2.5 - 4.5 h) (Fig. 27b). The maximum NH4+ uptake rate (0.034 ug-at lO^cells"1 h'1) occurred during the 4.5 - 5.5 h interval. 73 Figure 27 NH/ uptake rates of duplicate, cultures of Heterosigma carterae grown on NH4+ and under continuous light, culture 2+3, plotted against a) average time between sampling and b) average time between sampling since re-addition of NH4+ at t=0 to previously N-starved for 18 h. (b) is an expanded version of the re-addition phase of (a). Cultures were NH4+-starved between 88 and 106 h and then NH4+ was added at 106 h. Bars represent ± 1 S.D. (n=1-2). Error bars are smaller than symbols where not visible. 74 DISCUSSION N Preference change 1) period I In the present study, Prorocentrum micans, Amphidinium carterae, and Heterosigma carterae showed different degrees of N preference compared to the generally accepted pattern of preferring NH4+ and then N03", over urea (Dugdale & Goering 1967; Paasche & Kristiansen 1982; Levasseur etal. 1990). Urea uptake was higher than N03" uptake in the presence of NH4+. This result was different from previous field studies, where N utilization was 54% (NH4+), 26% (N03"), and 5% (urea) (Maguer et al. 1995) and 48% (NH4+), 32%(N03_), and 13% (urea) (L'Helguen etal. 1996). N-replete Prorocentrum micans preferred NH4+ over N03" and urea with an uptake ratio of 1.9:0.6:1 respectively. This species used the three N sources most evenly among the tested species. Urea utilization was seen at > 14 uM NH4+, which occurred in only Prorocentrum micans but not in the other species. N-depleted cells increased their urea preference relative to N03" and NH4+ and the N03" preference relative to NH4*. However, the increase in the preference for urea was greater than N03". The strong preference for NH4+ by Heterosigma carterae (Exp. 3) resulted in N preference ratios of 10 (NH4+):0.1 (N03'):1 (urea) by N-replete cells. Such a strong preference for a particular N source was found only in this species. N-75 replete cells did not use N03" when NH4+ > 1 uM. On the other hand, urea was utilized more than N03'. These results were in good agreement with the field study on several lakes in Brazil by Mitamura et al. (1995) who showed that the contribution of N03" as N source was negligible compared to NH4+ and urea. N-deplete cells of Heterosigma carterae decreased their preference for NH4+ relative to both N03' and urea. The preference for NH4+ relative to N03" showed a 59% decrease, and urea preference relative to N03" decreased 44%. This is the only species that decreased the preference for urea relative to N03* after N deprivation. Consequently, N-deplete cells used the three N sources more indiscriminately than N-replete cells. However, the preference for NH4+ was still very high compared to the other species, before and after N deprivation. Amphidinium carterae also showed a strong preference for NH4*. Interestingly, N-replete cells did not use N03" at all when NH4+ was available. N-deplete cells enhanced the relative preference of NH4+ over both N03" and urea and the consequence was a significant decrease in the relative preference for N03". This resulted in a wider range of the preferences for N sources than N-replete cells. In general, the present study showed that influence by NH4+ affected N03' to a greater extent than urea. For all three species, urea was more preferred than N03' in the presence of NH4+ (bef. and aft. I). The important new finding in the present study was the interactions of the three re-added N sources. Most reports of inhibition by N sources focused on only two N sources, NH4+ and N03". Therefore, previous reports of interactions 76 apply only partially to the present results. For example, the present results of the increased urea uptake relative to NH/ after the re-addition of three N sources (Exp. 1 and 2) were the opposite of the results by Horrigan and McCarthy (1982) and Lund (1987). This discrepancy is considered due to the interaction of N sources, culture conditions, and different species (Thalassiosira pseudonana and Skeletonema costatum). Furthermore, the previously reported increased NOV uptake by removing inhibition by NH4+ (Maclsaac & Dugdale, 1972) was not observed in my study, perhaps due to the enhanced urea uptake, when NH4+ was depleted in Exp. 1 and 2. However, the increased preference for urea over NH4+ and N03" (Exp. 1 and 2) and over NH4+ (Exp. 3) observed during aft. I periods agreed with the results by Rees and Syrett (1979). The increased urea preference by N-starved dinoflagellates in the present study is in agreement with the results of a recent study on a coccolithophore and a diatom by Waser et al. (1998a). In their study, using three N sources, NH4+ was utilized first, followed by the simultaneous uptake of N03" and urea by N-replete Thalassiosira pseudonana and Chaetoceros debilis. N-replete Emiliania huxleyi took up N in the following order NH4+, N03', and urea. However, N-deprived Thalassiosira pseudonana and Emiliania huxleyi used reduced N forms (NH4+ and urea) immediately after the re-supply of N sources and N03" was used last. Interestingly, N-starved Chaetoceros debilis took up only urea after the re-addition of three N sources, and NH4+ was not immediately utilized. These observations suggest that increasing preference for urea in the interaction of the three N sources is a general phenomenon in several groups of marine 77 phytoplankton. However, the evidence that the raphidophyte Heterosigma carterae did not increase urea preference after N deprivation in the present study also suggests increasing urea preference is group or species specific. 2) Period II During this period (n) of N uptake after N addition to N-starved cells, the preference for urea relative to NOV increased 20 and 12% in Amphidinium carterae and Heterosigma carterae respectively; there was no change in the urea preference in Prorocentrum micans. Further investigation is needed to determine why Heterosigma carterae developed a preference toward NOV utilization after N starvation. Heterosigma carterae grown on three N sources could not use urea before and after N deficiency. Heterosigma carterae did not grow well on urea (Fig. 28). The growth rate of Heterosigma carterae on urea was 0.90 d'1, (calculated from 0 to 3 d) and was not the lowest compared to growth on other N sources. However, maximum fluorescence of this culture with urea was 80% of Exp. 3 (three N sources), 78% of Exp. 4 (NOV and continuous light), 79% of Exp. 5 (NOV and LD cycle), and 78% of Exp. 6 (NH4+ and continuous light). Therefore, to compensate for N deficiency which was exacerbated by the incapability of using urea, Heterosigma carterae might have increased its capability to take up N03" during N deprivation. Overall, the order of preference of N was not changed for each period. However, it was revealed that the order of N preference really depended on the 78 Figure 28 Growth of Heterosigma carterae grown on various N sources and light conditions. Log plot of in vivo fluorescence versus time. 79 combinations of N sources. In addition, the N physiological state affected the degree of N preference, which was shown by a significantly increased preference for urea relative to NH4+ and N03" by N-starved Prorocentrum micans and Amphidinium carterae, and an increased N03' preference of N-deprived Heterosigma carterae. Cellular physiological state 1) Surge uptake N-starved cells did not exhibit evident of faster NH4+, NOV and urea uptake than non-starved cells for all three species, except for Heterosigma carterae grown on NH4+ (culture 1). In addition, the fastest uptake of N occurred some time after the re-addition of N sources, except NH4+ uptake by N-starved Amphidinium carterae and Heterosigma carterae grown on three N sources. Syrett (1953) and Harvey (1953) first demonstrated the effects of cellular physiological state on N uptake rates. Following these first results, a natural phytoplankton community and a batch culture were shown to exhibit rapid uptake of NH4+ after N deprivation (Conway et al. 1976; McCarthy & Goldman 1979; Goldman etal. 1981; Goldman & Glibert 1982; Dortch etal., 1982; Wheeler et al. 1982; Harrison 1983; Suttle & Harrison 1988; Syrett & Peplinska 1988). However, this enhanced uptake rate decreased rapidly after the cells overcame the N deficiency (Fitzgerald 1968). In general, species specificity (Conway & Harrison 1977) and the duration of N deprivation (Parslow et al. 1984a) determine the magnitude of the surge uptake response. This enhanced uptake 80 rate of N could be an adaptive response for phytoplankton to take up regenerated NH/ (McCarthy & Goldman 1979; Gilbert & Goldman 1981; Goldman & Glibert 1982). Consequently, it allows phytoplankton to maintain relatively high growth rates in oligotrophic waters (Goldman etal. 1979; Goldman & Glibert 1982). The apparent role of surge uptake for phytoplankton is to overcome the N deficiency and speed up the incorporation of N into macromolecules. Wheeler et al. (1982, 1983) suggested that growth is limited by the rate at which cellular metabolism can incorporate N into macromolecules. 2) Ammonium uptake in N-deplete phase During the N re-supply period, the fastest uptake rate (0.07 ug-at lO^cells"1 h"1) of NH/ was observed between 3 and 4 h in Prorocentrum micans. However, in Amphidinium carterae and Heterosigma carterae, the fastest rates occurred during the initial interval after the re-addition of N sources. The former species required an acclimation period to take up the re-added NH/. Waser et al. (1998a) also reported a similar phenomenon in which NH/ was taken up immediately after the three N sources by N-starved Emiliania huxleyi and Thalassiosira pseudonana, but not Chaetoceros debilis. Therefore, the capacity for surge uptake of NH/ must be species specific. Heterosigma carterae grown on NH/ showed the maximum uptake rate of 0.042 ug-at lO^cells"1 h"1 in culture 1 and 0.33 ug-at lO^cells'1 h'1 (culture 2+3). Following the rapid uptake of NH/, uptake rate slowed for a short period in the 81 Heterosigma carterae culture. The NH4+ uptake rates during this period were 0.006 (2.5 - 4.5 h) in culture 1 and 0.014 ug-at lO^cells"1 h"1 (2.5 - 4.5 h) in culture 2+3. The same phenomenon also occurred in Prorocentrum micans grown on three N sources. However, such a pattern was not as distinct as the pattern that occurred in cultures grown on only NH4+ (Exp. 6). Freshwater phytoplankton (Suttle & Harrison 1988) and a picoplankter, Micromonas pusilla (Cochlan ef al. 1991) exhibited the phenomenon of a short-term decrease in 15NH4+ uptake following surge uptake, which was similar to the results in the present study. This phenomenon could be explained by the short lag before the processing of NH4+ into amino acids, or a sudden loss of membrane potential due to the influx of cations (Suttle & Harrison 1988). Evidence that N-starved Lemna gibba, which was re-supplied with NH4+ suddenly lost membrane potential supports the later explanation (Ullrich ef al. 1984). Since approximately 100 ug-at N L"1 of N, with the combinations of NH4+ (cation), NOV (anion), and uncharged urea were re-supplied to Prorocentrum micans (Exp. 1) and Heterosigma carterae (Exp. 3), membrane potential might have been lost, although the magnitude of influx of cations is less than in Heterosigma carterae supplied with only 100 uM NH4+. The re-supply of NH4+ affects many cellular processes. During N deprivation, cultures continue accumulating polysaccharides by photosynthesis (Syrett 1981). Once these cultures are supplied with NH4+, they take up NH4+ very rapidly and convert it to organic N compounds. Simultaneously, the accumulated polysaccharide disappears because it is converted into organic N \ 82 compounds (Syrett 1953; Hattori 1958; Reisner et al. 1960). Also, these polysaccharides are used for producing energy due to increased respiration during the rapid assimilation of NH4+. 3) Nitrate uptake in N-deplete phase NOV was not rapidly taken up by N-starved Heterosigma carterae when it was the only N source (Exp. 4 and 5), and when it was one of three N sources with NH4+ and urea during period I (Exp. 1, 2, and 3). The concentration of N03" began to decline only after 3 h (Exp. 1), 6 h (Exp. 2), 11 h (Exp. 3), 6 h (Exp. 4), and 2.5 h (Exp. 5). Previous uptake rates were quite low and they were 0.001 (Exp. 1), 0.002 (Exp. 2), 0.004 (Exp. 3), 0.006 (Exp. 4), and -0.011 ug-at lO^cells'1 h"1 (Exp. 5). The difference in the period in which no N03' uptake occurs may be species dependent (Conway & Harrison 1977), and vary with the duration of N starvation (Parslow etal. 1984a), and other environmental factors. Overall, N-starved cells needed an acclimation period to start to take up N03" again. Maximum uptake rates ranged from 0.002 (Prorocentrum micans) to 0.025 ug-at lO^cells"1 h'1 (Heterosigma carterae grown on N03' under continuous light) and occurred some time after these low N03' uptake periods. In Exp. 1, 2, and 3, influence by NH4+ and urea could be responsible for the period of no N03" uptake. When NH4+ decreased to a certain concentration, then N03" started to decline. In Exp. 4 and 5, there must be another reason for Heterosigma carterae grown on NOV N03" was not taken up as readily by the N03'-starved culture of Heterosigma carterae compared to the reduced N form, NH4+ by NfV-starved Heterosigma carterae. A reduction in the ability of NOV uptake after N deprivation has been commonly reported (Collos 1980; Dortch et al. 1982). N03"-starved phytoplankton usually require previous exposure to NO3" before N03" uptake can occur (Dortch etal. 1982; Collos 1983; Parslow etal. 1984b). Starved cells achieve maximal N03" uptake after an acclimation period (Collos 1983; Harrison etal. 1989). One of reasons for a reduction in the initial N03" uptake is that long term N03" deprivation might reduce the ability of N03" uptake due to the loss of viability of cells. The decline in the ability to take up N03' after short term N deprivation is also attributable to the loss of an active uptake system (Falkowski 1975) and to inactivation of nitrate reductase (Syrett 1981). Inactivation of nitrate reductase alone did not interfere with the initial uptake of N03" since transient internal N03' pools were observed in even N-starved cells (Dortch et al. 1984). Details will be discussed in the following "Interaction" section. 4) Urea uptake in N-deplete phase In the present study, urea uptake after N-starvation and re-supply of urea was observed in the interaction of other N sources such as N03" and NH4+. Surge uptake of urea was not observed in any species. This agrees with the results of nb evidence of urea surge uptake by N-starved cells (Bekheet & Syrett 1979; Price & Harrison 1988). Maximum urea uptake rates were 0.044, 0.014, and 0.008 ug-at lO^cells'1 h"1 for Prorocentrum micans, Amphidinium carterae, and Heterosigma carterae respectively. However, there are many reports that 84 urea uptake is enhanced by N deprivation (Rees & Syrett 1979; Horrigan & McCarthy 1981; Price & Harrison 1988). The enhancement of urea uptake is attributed to the removal of NH4+ originating from urea in the cell (Rees & Syrett 1979). Increased N-specific urea uptake might be due to a reduction in the cell N quota and retention of all of the urea-N by the N-starved cells. N Interaction The present study demonstrated that NOV uptake by Prorocentrum micans, Heterosigma carterae, and Amphidinium carterae could not proceed in the presence of NH4+ concentrations as low as 1.6, 1.7, and 0.9 uM respectively before they experienced N deprivation. The three species started to take up N03" only after NH4+ reached near zero. An interesting finding was that urea uptake rate exceeded NH4+ uptake rate in Prorocentrum micans culture during a short-term decrease in NH4+ uptake followed a period of rapid uptake in the N-deplete phase. Generally, NH4+ influence on urea uptake was weaker compared to that on NOV and N03" influenced on urea uptake when only those two N sources co-existed in the medium for all three species. N-replete Prorocentrum micans (Exp. 1) showed less influence on N03' uptake by both NH4+ and urea than Amphidinium carterae (Exp. 2) and Heterosigma carterae (Exp. 3). In addition, Prorocentrum micans used urea more favorably than Amphidinium carterae and Heterosigma carterae during the period (I), both before and after N starvation. Thus, this species could utilize the three N sources indiscriminately. In contrast, in Amphidinium carterae, NH4+ 85 strongly influenced on both NOV and urea uptake before N starvation, and only NH4+ was taken up preferentially. Both N03" and urea remained near their initial concentration until the NH4* concentration < 1 uM. Then, urea and N03" were both taken up almost simultaneously. However, after they experienced N starvation, only urea started to decline in the medium when > 18 uM NH4+ existed. On the other hand, N03" was still not utilized at this NH4+ concentration. Therefore after N starvation, influence of urea uptake by NH4+ decreased but N03" uptake was still influenced. In Heterosigma carterae (Exp. 3), NOV was suppressed by NH4+ but not urea, before and after N starvation because once NH4+ disappeared from the culture medium, N03' was taken up much faster than urea. However, NH4+ influence on N03" uptake clearly decreased after N starvation. Focusing on the interaction between N03' and urea, N03" influence on urea uptake during the aft. II period was slightly strengthened in Prorocentrum micans. In contrast, such an influence was weakened in N-deplete Amphidinium carterae and Heterosigma carterae cultures. In general, species and nutritional state affect on interaction between N03' and NH4+ (Syrett 1981; McCarthy 1981). Inhibition of N03' uptake by NH4+ ranged from total suppression (Syrett & Morris 1963; McCarthy & Eppley 1972; Cresswell & Syrett 1979) to simultaneous and comparable rates of NH4+ and NOV uptake in cultures (Caperon & Ziemann 1976; Conway 1977) and natural communities (Price etal. 1985, Collos etal. 1989). Higher concentrations of 86 NH4+ inhibited N03' uptake, but stimulation of N03" uptake has been reported to occur at low NH4+ concentrations (Yin 1988). The mechanism of the inhibition of N03" utilization needs further investigation. Regulatory action at the level of N03" uptake (Eppley & Rogers 1970; Tischner & Lorenzen 1979) and N03' reduction (Syrett & Morris 1963; Hipkin et al. 1980) have been suggested to influence NOV uptake. To a certain extent, the effect of NH4+ on both mechanisms may be independent (Ullrich 1987). It is known that N03' uptake stops very quickly when NH4+ is added and starts when the NH4* has disappeared (Syrett & Morris 1963; Eppley et al. 1969a; Conway et al. 1976). This inhibition by NH4+ is too rapid to be accounted for by the inactivation of nitrate reductase. Probably, the primary and most rapidly acting effect of NH4+ on N03* utilization is attributed to an inhibition of the N03" uptake system. Then, this may be followed by the effects on N03" metabolism through inhibition of nitrate reductase activity, by irreversible proteolytic breakdown (Hipkin etal. 1980), reversible inactivation (Pistorius etal. 1978), or suppression of its synthesis (Morris & Syrett 1963; Amy & Garrett 1974). The rate of N03' uptake is modulated in response to changes in pools of some organic product of NH4+ assimilation (Syrett 1981; Guerrero et al. 1981). Highly C-deficient cells did not show inhibition of N03" uptake by NH4+ and these cells reduced N03" to NH4+ (Syrett & Morris 1963; Thacker & Syrett 1972a; Ullrich 1987). Therefore, NH4+ itself may not be an inhibitor and an organic product of NH4+ assimilation is suspected to be an inhibitor. 87 NH4+ repressed urea uptake in Thalassiosira pseudonana (Lui & Roels 1970). Lund (1987) found that NH4+ immediately inhibited urea uptake by Skeletonema costatum, while Horrigan and McCarthy (1982) reported that urea uptake inhibition occurred after 30 min. When these two diatom species were N-depleted, NH4+ inhibited urea uptake to an even greater extent. In general, urea suppresses NOV uptake but at a lower level than NH4+ (Grant et al. 1967; McCarthy & Eppley 1972; Molly & Syrett 1988b). However, simultaneous uptake of urea and other N sources by phytoplankton communities (McCarthy & Eppley 1972; Price et al. 1985) and a normal uptake rate of NOV in the presence of 10 ug-at N L"1 urea by Skeletonema costatum (Lund 1987) have been reported. Urea was taken up by a natural phytoplankton assemblage simultaneously with N03" and NH4+, with little influence on the uptake rates of NH4+ (Robert et al. 1986). Repression of nitrate reductase by urea, which was reported in studies on Cyclotella cryptica (Liu & Hellebust 1976) and Chlorella (Smith & Thompson 1968) is a possible explanation of the inhibition of N03" uptake by urea. Nutritional state affects the interaction between urea and other inorganic N sources. N03" uptake inhibition by urea weakened after N starvation in the study of Skeletonema costatum (Lund 1987). On the other hand, Molly and Syrett (1988a) reported that urea increased the suppression of N03" uptake. NH4+ suppression of urea uptake disappeared due to N starvation (Rees & Syrett 1979). Horrigan & McCarthy (1982) reported that urea and NH4+ uptake rates were enhanced even for N03'-sufficient cells. 88 To conclude, the magnitude of the effect of the interaction of three N sources on uptake rates was species specific, and influenced by N starvation. As it was mentioned in the N preference section, the interaction among N sources was variable and depended on the N sources in the medium. Since NH/ and urea are found in similar concentrations in the sea (Harrison 1992), it is important to consider urea in studies of N interactions. Increased concentrations and efflux of N An increase in the external N concentration during the initial interval after the re-addition of N sources occurred in the culture of Prorocentrum micans (three N), Amphidinium carterae (three N), Heterosigma carterae (three N), Heterosigma carterae (NOV), and Heterosigma carterae (NH/, culture 2+3). In the Amphidinium carterae culture, an increase in the concentration of both N03" and urea occurred. In Exp. 3 and 5, Heterosigma carterae excreted NOV. Urea efflux during the initial interval was -0.006 ug-at lO^cells"1 h'1 during 0 -1 h (Prorocentrum micans), -0.03 g-at lO^cells"1 h"1 during 0 - 0.5 h (Amphidinium carterae), and -0.0006 ug-at lO^cells"1 h'1 during 0 -1 h (Heterosigma carterae) when these three N-starved species were re-supplied with three N sources. Effluxes of N03" of -0.007 ^g-at lO^cells'1 h'1 (Exp. 3) and -0.022 ug-at lO^cells'1 h"1 (Exp. 5), during the initial interval of the N re-supplied phase were observed in N-starved Heterosigma carterae. The initial stage of urea uptake involves a rapid influx and probably an accumulation of an intracellular pool (Antia et al. 1991). Excretion of urea in the 89 form of dissolved organic N is not re-assimilated in the short term. Excretion into the culture medium of 14C-labelled organic products derived from urea-C could explain the lower urea uptake rates (Antia et al. 1991). Efflux of N03" in Exp. 3 coincided with the high uptake rate of NH4+. Therefore, NH4+ taken up by N-starved cells may be attributed to this efflux of N03'. The efflux of N03" of N-starved Heterosigma carterae in Exp. 5 occurred during the light period, although the N-replete culture showed several periods of efflux of N03' during dark periods. Pujo-Pay et al. (1997) reported < 10% of the N03" uptake was released as DON with the rate of 10.4 to 13.3 nmol N L"1 h"1. They mentioned the released DON was an important resource for some organisms. NH4+ excretion occurred in NH4+-grown Heterosigma carterae culture, and such a phenomenon has been reported often. NH4+ excretion was observed by axenic cultures of Thalassiosira pseudonana after the addition of 10 ug-at N L"1 of urea (Price & Harrison 1988). NH4+ excretion also occurred in urea-grown Phaeodactylum tricornutum (Rees & Syrett 1979). Urea appears to be one of the sources of NH4+ excretion. Ecology Amphidinium carterae showed an enhanced preference for NH4+ relative to both N03" and urea during the period I and preference for urea relative to N03" during period II. Prorocentrum micans also enhanced its preference for urea rather than N03" during N starvation, however, its preference for NH4+ 90 decreased. In general, species that increase their preference for NH4+ or urea have a better chance to take up a transit N supply of regenerated N, which is mainly NH4+ and urea. However, the increased preference for NOV relative to NH4+ and urea by Heterosigma carterae does not appear to offer an advantage for survival in a N-deplete environment. Nevertheless, N-deprived Heterosigma carterae still possessed a much stronger preference for NH4+ (NH4*:N03':urea = 39:1:5.2) compared to the other two species. The reduction in the ability to take up N03* and the increase in the ability to take up NH4+ may be an ecological acclimation by N-deprived phytoplankton in oligotrophic waters (Dortch et al. 1982). N03' is recycled over a much longer time period and it is usually supplied continuously at low rates by eddy diffusion from deeper N03'-rich waters (Dugdale 1967; Eppley et al. 1979). Sporadic N03" supply by upwelling, frontal mixing, and internal waves occurs in the euphotic zone at very slow rates (Walsh et al. 1978; Pingree et al. 1978; Parsons et al. 1981; Cullen etal. 1983). These physical events also dilute the phytoplankton community in the euphotic zone. Therefore, the demand for the nutrient by phytoplankton decreases and N03' remains in the euphotic zone for some time, which reduces the benefits for transient elevated uptake rates of N03" (Parslow et al. 1984b). Compared to NOV NH4* and urea are recycled rapidly within the euphotic zone and introduced to the system by animal excretion (Dugdale 1967) Maintaining a high uptake capability during N starvation results in metabolic costs (Raven 1986). Therefore, it is probably more beneficial for cells to have a high uptake capability for NH4+ and urea rather than N03' because NH4+ and 91 urea are recycled N sources. Interestingly, annual phytoplankton production expressed as N production in well mixed water (Western English Channel) was mainly based on recycling of N forms such as NH4+ (54%), but not new N forms (Maguer et al. 1995). To conclude, phytoplankton which are capable of assimilating NH4+ or urea rapidly after they are N starved may have an advantage for species succession and growth in the environment where N limitation is the primary stress. Therefore, Amphidinium carterae that increased the preference for NH4+ relative to NOV (period I), and the preference for urea relative to N03" (period II), can acclimate to N-deplete water, while Prorocentrum micans and Heterosigma carterae increased their preference for N03" during period 1 or 11. Growth rate Growth rates were 0.30 for Prorocentrum micans (three N sources), 0.76 for Amphidinium carterae (three N sources), 0.56 for Heterosigma carterae (three N sources), 1.0 for Heterosigma carterae (N03' + continuous light), 0.72 for Heterosigma carterae (N03' + LD cycle), 1.1 for Heterosigma carterae (NH4+) (culture 1), and 1.02 d"1 for Heterosigma carterae (NH4+) (culture 2+3). Prorocentrum micans had the lowest growth rate (0.30 d"1). Prorocentrum micans grown in f/2 medium under continuous light and 19±10°C showed growth rates ranging from 0.59 to 0.99 d'1 in the study by Costas (1990). The temperature used in the present study (17°C) was lower than the 19°C in Costas' study and could partially account for the lower growth rate in 92 Prorocentrum micans. Growth rates of Prorocentrum micans were measured in a culture containing a mixture of other dinoflagellates (Fedorov & ll'yash 1991). Prorocentrum micans and other dinoflagellates such as Glenodinium foliaceum, Excuviaella cordate, Gymnodinium kovalevskii, and Olisthodiscus luteus (Heterosigma carterae) were grown in Goldberg medium (Lanskaya 1971) under natural illumination at 20 - 22 °C. The growth rate of Prorocentrum micans was 0.2 d*1. When Prorocentrum micans was grown by itself without the interaction with other species, its growth rate was also low and < 0.5 d'1, even though a relatively high temperature was used. These results are in agreement with this study, and indicate that Prorocentrum micans possesses a low growth rate. Fedorov and ll'yash (1991) concluded that Prorocentrum micans was able to increase its population density slowly but steadily by utilizing limited amounts of resources and classified it as a "patient" species. Amphidinium carterae had the highest growth rate of 0.76 d"1 among the other three species grown on three N sources (Exp. 2). This species also exhibited the same growth rate grown under a 14:10 L.D cycle at 20°C when 50 LIM NH4+ was given to the culture at the beginning of the light period (Wheeler et al. 1983). However, if the same concentration of NH4+ was given at the beginning of the dark period, the growth rate increased to 1.4 d"1. Amphidinium carterae has been used for many studies because it grows well in various culture conditions. The growth rates of Heterosigma carterae ranged from 0.56 to 1.1 d"1 depending on culture conditions. Since this is the first study for Heterosigma carterae grown on three combined N sources, there are no comparable data on growth rates from the literature. However, growth rates on individual N sources are available. All growth rates were < 0.8 d"1 under 14:10 L.D photoperiod of 160 umol photon m'2 s'1 at 20°C with 100 uM of each N (Hoe Chang & Page 1995). Another study by Wood & Flynn (1995) reported 0.9 d"1 and 0.6 d"1 when grown on NH4+ and N03" of 75% ESAW (Harrison et al. 1980) respectively in a 12:12 L.D cycle at 18°C. The growth rates of N03" and NH4* grown cells in the present study were generally higher than those of literature values. Light: Dark cycle 1) Cell division and fluorescence number An expected step-wise increase in fluorescence, which indicates synchronicity and phasing of growth to the L:D cycle, was not evident in Heterosigma carterae under the 14:10 L:D cycle (Exp. 5). Therefore, the occurrence of cell division at night could not be detected. However, the fluorescence increased much more smoothly in cultures under continuous light (Exp. 4). The growth rates for Exp. 4 and 5 were 1.00 d"1 (0 - 64 h) and 0.72 d'1 (49 -105 h) respectively. Growth rate often varies with a 24 h periodicity and the timing of division depends on experimental conditions and species (Sweeney 1983). 94 2) Uptake diel periodicity Diel periodicity of NOV uptake occurred during log phase growth. N-replete Heterosigma carterae grown on NOV under a LD cycle showed high N03" uptake rate during the light period and at the beginning of the dark period. At the end of dark period, uptake rates were dramatically decreased to negative values or values close to zero. During early log phase, a large decrease in uptake rate was not observed in the culture under continuous light (Exp. 4). This observation agreed with findings by Cochlan et al. (1991) who found that NOV uptake rates were maximal during the light periods and decreased during the dark periods for Micromonas pusilla. The outdoor culture of Chaetoceros sp. which was grown on low N also exhibited cyclic variations in N03" uptake with the maximum at night and the minimum during the day (Malone et al. 1975). In the present study, after N starvation, N03" uptake occurred during the dark period. The uptake rate of N-deplete cells in the dark was 0.017 ug-at lO^cells"1 h"1 and higher than that of N-replete cells. This agrees with the finding that Pavlova lutheri grown on low N showed an increase in the relative importance of potential nighttime N uptake (Laws & Wong 1978). N availability has been considered a determining factor of the enhancement of nighttime uptake. When Gyrodinium aureolum was N-starved for 24 h, it exhibited an increase in nighttime N03' uptake (Paasche et al. 1984) even though it did not show nighttime N03" uptake before N starvation. N-starved dinoflagellates, Pyrocystis noctiluca and Dissodinium lunula were independent of the L:D cycle in their N03' uptake capacity (Bhovichitra & Swift 95 1977). N limitation may enhance the potential for dark N uptake more than light uptake. Consequently, diel periodicity of N uptake becomes less evident. The enhancement of light independence of N uptake might be an adaptive response to N limitation since N-starved phytoplankton could optimize their uptake capability under even low light. Cochlan et al. (1991) found that with increased N limitation, the relative dark uptake capacity increased four-fold for total (light + dark) N03" uptake, and two-fold for total NH4+ uptake. A consequence of enhanced dark N uptake by N starvation is the dampening of the diel N uptake periodicity. Laboratory studies by Harrison (1976) showed dark uptake by Gonyaulax polydra increased to ca. 40% of daytime uptake rate in N-starved cultures. On the other hand, in N-sufficient cultures, N uptake during nighttime was only ca. 20% of that during daytime. The absence of diel periodicity in N03" uptake occurred in axenic cyclostat cultures (Picard 1976). Dark NO3" uptake capacity of cyclostat cultures of the prymnesiophyte, Pavlova lutheri and the chlorophyte, Dunaliella tertiolecta exceeded the supply rate of N03" at high dilution rates, which suggested that the environment had less available N for cells (Laws & Caperon 1976; Laws & Wong 1978). The coccolithophore, Emiliania huxleyi, which was grown in both NO3-and NH4+-limited cyclostat cultures did not exhibit diel periodicity (Eppley et al. 1971). There are two contrasting examples in field studies. In N03"-depleted water off Peru with a dinoflagellate bloom dominated by Gymnodinium sanguineum, N03" uptake during nighttime was ca. 50% of that during the 96 daytime (Dortch & Maske 1982). On the other hand, in the bloom off Baja California, which was dominated by Gonyaulax polyedra, night time N03' uptake was 10 - 20% of daytime N03" uptake (Maclsaac 1978). The later case had a higher N03" concentration than the former case in the surface waters where phytoplankton resided. In addition, the nitracline off Baja California was shallower than off Peru and thus a shorter downward vertical migration in order to obtain N03". This result agreed with the field study by Harrison (1976), which revealed the dampening effect on N uptake by red tide populations dominated by Gonyaulax polyedra. 3) Heterosigma carterae and dark N03' uptake In the present study, high N03' uptake occurred during the light and not during the dark before N03' deprivation. Low N03" uptake (81 - 90 h, 105-114 h) and even efflux of N03" (32.5 - 42 h, 57 - 66 h) occurred at the end of dark periods. However, N03" uptake rate in the dark by N-starved cells was positive and higher than by N-replete cells. During diel migration of Heterosigma carterae, the cells stay close to the surface in the day time to carry out photosynthesis and migrate downwards to deeper water at night to take up nutrients (Watanabe et al. 1983; Yamochi & Abe 1984; Nagasaki etal. 1996). Considering that maximal nitrate reductase activity (Eppley & Harrison 1975) occurs during the day time, N03' uptake during night time via vertical migration is not efficient (Raven 1986). In addition, diel migration costs are metabolically high. Therefore, night-time N03" uptake does 97 not contribute to the entire supply of nutrients for Heterosigma carterae. However, considering that N limitation increased the SAN ratio (Cochlan et al. 1991) and increased nitrate reductase activity during the dark period (Eppley & Harrison 1975), vertical migration at night might be more beneficial for N-starved Heterosigma carterae than N-replete cells. 98 CONCLUSIONS This dissertation examined N uptake by the dinoflagellates, Prorocentrum micans and Amphidinium carterae, and the raphidophyte, Heterosigma carterae as a function of the photoperiod and N physiological states. The specific findings of the research are summarized below. 1. A change in the relative preference of the three N sources occurred during N deprivation. N-deplete Prorocentrum micans increased its preference for urea relative to both NH4+ and N03". N-deplete Amphidinium carterae enhanced its preference for NH4+ relative to both N03" and urea, and its preference for urea relative to N03' under the interaction of three N sources. The preference for N03" was enhanced by N-deprived Heterosigma carterae. 2. Strong NH4+ influence on N03" and urea uptake were observed in N-replete Amphidinium carterae and Heterosigma carterae. N-replete Prorocentrum micans only showed strong NH4+ influence on N03" uptake, but not on urea uptake. N deprivation weakened the influence of NH4+ on urea uptake in Heterosigma carterae. The N uptake preference depended on the interactions of each N source. 3. N03" uptake needed a longer acclimation period after the N03" pulse in N-starved Heterosigma carterae grown under a light:dark cycle than under continuous lights. A short acclimation period for N03' uptake occurred in 99 Prorocentrum micans, but not in Amphidinium carterae and Heterosigma carterae grown on three N sources. The duration of the acclimation periods appeared to be influenced by the light cycle and species specificity. 4. The uptake of N03" by Heterosigma carterae was phased by a light:dark cycle. N03" disappearance in the culture medium accompanied by a high uptake rate occurred mostly during the light periods and only a small disappearance accompanied by a low uptake rate and an efflux occurred during the dark periods. This uptake cycle may be due to nitrate reductase activity, which usually decreases at night. Diel vertical migration to take up N03" in deeper water at night seems less likely than previously thought for Heterosigma carterae. 100 FUTURE RESEARCH SUGGESTIONS The outstanding question is why N-deprivation changed the magnitude of N preference by marine phytoplankton. The impact of N deprivation on the mechanisms involved in the uptake of each N source needs further investigation. The change in the interaction among N sources during the N deprivation also needs to be examined under various conditions such as the availability of light, lower concentrations of N, and widely ranging periods of N starvation. An increase in the preference for urea as a N source during N starvation seems a general phenomenon in marine phytoplankton, which was observed in this study on dinoflagellates and other previous studies on a diatom and coccolithophores. However, the evidence for a decrease in the preference for urea by Heterosigma carterae (raphidophyte) suggests species or even group specificity. It was a surprise that N-replete Heterosigma carterae showed negative or very small N03" uptake rates during dark periods because it is known to vertically migrate downwards at night, presumably to take up nutrients. N-deplete Heterosigma carterae showed dark uptake of NO3'. However, unfortunately the dark period occurred when the cells had taken up most of the re-added N03", and therefore it was not possible to determine the effect of N starvation on dark uptake. Therefore, it is more interesting to re-add a higher concentration of N03" at the beginning of the dark period, which provides a longer period of observation to determine the influence of N deprivation on N03" uptake in the dark. 101 Partn RESULTS Isotope fractionation 1) Prorocentrum micans grown on NH/, N03', and urea In the N-sufficient phase (0-106 h), N03" and urea were used along with NH4+ (Fig. 29). However, N03" was completely consumed before urea. During growth mainly on NH4+, the apparent isotope discrimination was relatively large and decreased upon exhaustion of NH4+ (Fig. 30). At this time (31 h), NH4+ had been completely incorporated into PN and 815NPN was in good agreement with the expected value of -1.14%o of 615NAs (Table 3). N03' uptake was accompanied by a small change due to both a smaller e(N03") and a significant amount of preexisting PN produced during growth on NH4+. In the 65 -106 h time period, 815NPN showed a slight increase due to the uptake of urea. At stationary phase, the 815NPN was 0.24%o. This value was in good agreement with the 815Nmx of 0.26%o, which was expected when all three N sources were completely taken up. The model gave a good fit for the e(NH4) value of 12%o. However, during the drawdown of NOV and urea when the model used an e(urea) of 0%o, it systematically overestimated 815NPN. During the N re-supply phase, again, NH4* and urea were immediately utilized, and N03' uptake coincided with the complete consumption of NH4+. Using the same variables as in the N-sufficient phase, the model gave a good fit for e(NH4+). Again, the model systematically overestimated 815NPN during the 102 Time (hours) Figure 29 Time series of [NOV] (•), [NH4+] (O) and [urea] (•) during the growth of Prorocentrum micans grown on N03", NH/, and urea under continuous light. Culture was N-starved between 105 and 186.5 h and then 36.6 ug-at N L"1 NH4+, 48.7 ug-at N L"1N03", and 49.4 ug-at N L"1 urea were added at 186.5 h. The best fits for the concentrations were determined by the dissolved N data using the equation: y = a*(1-EXP(b*(x-c))) where a is an initial N concentration, b is a co-efficient number, and c is the time when N reached 0, and are indicated by the solid lines. 103 Figure 30 Time series of PN(A) and 815NPN(<1) during the growth of Prorocentrum micans grown on N03", NH4+, and urea under continuous light. Culture was N-starved between 105 and 186.5 h and then 36.6 ug-at N L'1 NH4+, 48.7 ug-at N L"1 N03', and 49.4 ug-at N L'1 urea were added at 186.5 h. The 815NPN as predicted by the multiple N source uptake model is shown by the solid line. The 3 dotted lines indicate times when [NH4+], [NOV] and [urea] fits, determined by the dissolved N data to the concentration data in Fig. 29, reached 0, respectively. 104 Table 3. Listing of variables and definitions used in the multiple N source uptake model. Variable Definition Value* PNM Measured PN concentration I PNX Calculated PN concentration from dissolved N drawdown PNj Initial PN concentration 815NNS 815N of the initial N03' source 3.04%o 815NAS 515N of the initial NH4+ source 1.14%815NUS 815N of the initial urea source 0.74%815NMX 815N of the initial mixed N sources (calculated from mass balance) 0.26-0.46%o 815NPNi 815N of PN, e(N03) Isotope fractionation for the overall reaction: NO3" -» PN e(NH4) Isotope fractionation for the overall reaction: NH4+ -» PN e(urea) Isotope fractionation for the overall reaction: Urea -» PN 0%o A Apparent isotope fractionation or discrimination, i.e. 815NPN - 815Nst *: constant values are given. 105 consumption of NOV and urea. All the base variables are summarized in Table 4. A sensitivity analysis is presented later. 2) Amphidinium carterae grown on NH4+, N03", and urea In the N-sufficient phase (0 -101 h), NH4+ was utilized first followed by the simultaneous uptake of N03' and urea (Fig. 31). Growth on NH4+ produced a large initial decrease in 815NPN to values of -8.94 to -12.1%o (Fig. 32). 515NPN then increased to about -0.6 %o during the simultaneous growth on urea and N03" (60 - 94 h) and finally reached 0.73%o at the stationary phase. The model gave a relatively good fit for the NH4+ uptake phase with e(NH4+) values of 20%o. However, the model systematically underestimated 815NPN during the simultaneous growth on urea and NOV 815NST of 0.76%o showed a relatively good agreement with the initial 815NMx (0.27%o). In the N re-supply phase, NH4+ was taken up immediately, followed by simultaneous uptake of urea and NOV 51SN was -0.24%o (193 h) and, then, increased to 0.33 and 0.04%o at 30 min and 1 h respectively after the re-addition of the three N sources. Positive 815NPN values indicate more incorporation of 15N into the cells than 14N. Using the same variables as in the N-sufficient phase, the model systematically underestimated the 815NPN during the growth on any N. A sensitivity study for Amphidinium carterae is also provided later. 106 Table 4 Variables and their values used in the multiple N source uptake model. Prorocentrum micans, Amphidinium carterae, and Heterosigma carterae were grown on multiple N sources (NOV, NH4\ and urea) under N-sufficient conditions. After 0 - 82 h of starvation, all three N sources were added simultaneously to the culture (i.e. N re-supply phase). Heterosigma carterae was grown on NOV under N03'-sufficient condition. After 52 h of N starvation, N03" was added to the culture (i.e. N re-supply phase). Symbols are defined in Table 3. Species e(NH4+) s(N03) PNi 815Nj* (%o) (%o) G*M) (%o) N-sufficient phase - 0.42 Prorocentrum micans 12 9 12 Amphidinium carterae 20 0 3.04 0.27 Heterosigma carterae 24 9 3.87 -1.36 Heterosigma carterae —» 5 5.1 1.08 N re-supply phase -0.54 Prorocentrum micans 18 9 107.4 Amphidinium carterae 4 0 77.1 -0.24 Heterosigma carterae 20 5 100 -0.01 Heterosigma carterae 9 92.9 0.78 *: 815Ni in the initial N source 107 50 i 0 20 40 60 80 100 180 185 190 195 200 Time (hours) Figure 31 Time series of [N03"] (•), [NH4+] (O) and [urea] (T) during the growth ol Amphidinium carterae grown on N03", NH4+, and urea under continuous light. Culture was N-starved between 119 and 193.5 h and then 29.6 ug-at N L'1 NH4+, 34.3 ug-at N L'1 N03", and 31.2 ug-at N L"1 urea were added at 193.5 h. The best fits for the concentrations were determined by the dissolved N data using the equation: y = a*(1-EXP(b*(x-c))) where a is an initial N concentration, b is a co-efficient number, and c is the time when N reached 0, and are indicated by the solid lines. 108 Figure 32 Time series of PN (•) and 815NPN (•) during the growth of Amphidinium carterae grown on N03", NH4+, and urea under continuous light. Culture was N-starved between 119 and 193.5 h and then 29.6 ug-at N L'1 NH4\ 34.3 ug-at N L'1 N03", and 31.2 ug-at N L"1 urea were added at 193.5 h. The 815NPN as predicted by the multiple N source uptake model is shown by the solid line. The 3 dotted lines indicate times when [NH4+], [N03] and [urea] fits, determined by the dissolved N data to the concentration data in Fig. 31, reached 0, respectively. 109 3) Heterosigma carterae grown on NH4+, NOV, and urea In the N-sufficient phase (0-175 h), the three N sources were utilized in sequence (Fig. 33). NO3" was taken up only when NH4+ decreased to < 1 uM. Large 815NPN values such as -8.16, -13.3, and -10.6%owere observed during the growth on NH4+ at 11, 36, and 49 h respectively (Fig. 34). The isotope discrimination decreased to a small value upon exhaustion of NH4+. At stationary phase, the 815NPN was -0.41 %o. This is a little lower than the value of 0.46%o for 815NMX- The model gave a relatively good fit. When the exhaustion of NH4+ occurred (68 h), 815NPN (-2.32%o) was in good agreement with the expected values of -1.14%o for 815NAS-Since urea was hardly consumed in the N-replete phase, cells were not N-starved completely. NH4+ was used first and followed by N03" and urea in the N re-supply phase. The 815NPN showed a relatively large decrease which was well predicted by the model using the same variables as in the N-sufficient phase. An s(NH4+) value of 20%o was used for the fit in N-deplete phase. 4) Heterosigma carterae grown on N03" Figure 35 shows the time series data for the nutrient fit. N03" was taken up without showing a distinct lag phase. In the N-sufficient phase (0 - 63 h), the model gave a relatively good fit with e(N03") values of 5%o (Fig. 36). However, 815NPN at the beginning of the experiment was lower (1.08%o) than the expected value (3.04%o). During the log phase, the 815NPN decreased to -1.04%o at 48 h 110 50 i 0 50 100 150 200 250 Time (hours) Figure 33 Time series of [N03"] (•), [NH4+] (O) and [urea] (T) during the growth of Heterosigma carterae grown on N03", NH4+, and urea under continuous light. Culture was added 32.2 ug-at N L'1 NH4+, 41.2 ug-at N L'1 NOV, and 37 ug-at N L'1 urea at 175 h. The best fits for the concentrations were determined by the dissolved N data using the equation: y = a*(1-EXP(b*(x-c))) where a is an initial N concentration, b is a co-efficient number, and c is the time when N reached 0, and are indicated by the solid lines. 111 Figure 34 Time series of PN (A) and 815NPN (•) during the growth of Heterosigma carterae grown on NOV, NH4+, and urea under continuous light. Nitrogen additions at 175 h to the culture were 32.2 ug-at N L"1 NH4+, 41.2 ug-at N L"1 N03', and 37 ug-at N L"1 urea. The 815NPN as predicted by the multiple N source uptake model is shown by the solid line. The 3 dotted lines indicate times when [NH4+], [N03] and [urea] fits, determined by the dissolved N data to the concentration data in Fig. 33, reached 0, respectively. 112 100 i Time (hours) Figure 35 Time series of [NOV] during the growth of Heterosigma carterae grown on NOV under continuous light. Culture was N-starved between 64 and 115.5 h and then 85.6 ug-at N L"1 N03"was added at 115.5 h. The best fits for the concentrations were determined by the dissolved N data using the equation: y = a*(1-EXP(b*(x-c))) where a is an initial N concentration, b is a co-efficient number, and c is the time when N reached 0, and are indicated by the solid lines. 113 Figure 36 Time series of PN (A) and 615NPN (•) during the growth of Heterosigma carterae grown on N03" under continuous light. Culture was N-starved between 64 and 115.5 h and then 85.6 ug-at N L"1 NOV was added at 115.5 h. The 815NPN as predicted by the N source uptake model is shown by the solid line. The dotted line indicates times when [N03'] fits, determined by the dissolved N data to the concentration data in Fig. 35, reached 0. 114 from the expected value for 3.04%o for the NOV source. During N starvation (100 h), the 815NpN was 0.90%o, which was lower than the expected value of 815NNS (3.04%O). In the N re-supply phase, NO3" was not taken up immediately. Positive 815NPN values (0.78 and 0.86%o) similar to the one in the stationary phase occurred when re-added NOV was not utilized (115.5-118 h). At the end of this period of N03" re-supply (119 h), 815N decreased again to -0.23%o. Then, negative values (-0.42 and -0.02%o) followed at 121 and 124 h respectively. During the exhaustion of NOV (128 h), 815NPN became positive (1.26%o) again. An e(NH4+) of 9%o was used for the model fit. 115 SENSITIVITY ANALYSIS Changes in various variables were made to study their effect on the 815NPN in N-replete and N re-addition conditions. The value of s(urea) was taken as a constant of 0%o. Changes in the values of e(NH4+), s(N03"), and PN, are presented in Table 3. 1) Prorocentrum micans grown on NH4+, N03", and urea In N-replete phase, variations in the values of e(NH4+) produced large changes in the 815NPN (Fig. 37a). The e(NH4+) that gave the best fit was 12%o. During this N-replete period, urea which has a lower s value than NH4+, was also taken up with NH4+. The values of e(N03') from various fits ranged from 5 to 9%o (Fig. 37b). Since 815NPN data during the drawdown of NOV were lacking, it was impossible to obtain the best e(N03"). Changes in PNf were made since PN, concentration seemed large in this culture (11.1 ug-at N L"1). PN, values used for the fit ranged from 11 to 13 ug-at N L'1 (Fig. 37c). A higher PN, value tended to improve the fit. Again, variations in the values of e(NH4+) produced a large change during the N-deplete phase (Fig. 38). Interestingly in the N-replete phase, the s(NH4+) that gave the best fit, increased to 18%o from 12%o. 116 e(NH4*) = 12 e(NH44) = 16 - e(NH4*) = 20 40 80 120 160 Time (hours) 200 Figure 37 Response of the model to changes in a) e(NH4+), b) e(N03"), and c) PNj in the N-sufficient condition for Prorocentrum micans grown on three N sources. The solid line represents the response of the model using the base values indicated in Table 4. 117 Figure 38 Response of the model to changes in s(NH4+) in the N re-supply phase for Prorocentrum micans grown on three N sources. The solid line represents the response of the model using the base variables indicated in Table 4. 118 2) Amphidinium carterae grown on NH4+, N03\ and urea Changing s(NH4+) values made a large difference in predicting 515NPN, however, it was not significant in the beginning (0 - 6 h) and the end (59 - 61 h) of NH4+ uptake (Fig. 39a). The model predicted the data well for growth on NH4+ for E(NH4+) values of 18 - 22%o. e(N03') decreased to a minimum value of 0%o (Fig. 39b). Lower s(N03") values tended to improve the fit. For the N-deplete phase, e(NH4+) ranged from 4 to 20%o (Fig. 40). None of the e(NH4+) values could fit the data during NH4+ drawdown. The model underestimated 515NPN in the entire N-deplete phase. 3) Heterosigma carterae grown on NH4+, N03\ and urea During the N-replete phase, the model predicted the data well for growth on NH4+ for e(NH4+) values of 22 - 26%o (Fig. 41 a). The s(NH4+) value that gave the best fit was about 24%o. PNj had a large effect on the response of the model during the initial growth on NH4+ (Fig. 41b). Variations in the values of s(NH4+) and PNj produced large changes in the 815NPN at low PN concentrations. The values of s(N03") ranged from 5 to 9%o (Fig. 41 c). The e(N03') value that gave the best fit was about 9%o. After the re-addition of three N sources, the model predicted well the data for values of e(NH4+) of 20%o (Fig. 42). This value was smaller in magnitude than the one derived in the N-sufficient phase. 119 Figure 39 Response of the model to changes in a) e(NH4+) and b) e(N03") in the N-sufficient condition for Amphidinium carterae grown on three N sources. The solid line represents the response of the model using the base values indicated in Table 4. 120 r -1 CO -2 \7 s(NH4*) = 4 E(NH/) = 12 s(NH/) = 20 180 182 184 186 Time (hours) 188 190 Figure 40 Response of the model to changes in e(NH4+) in the N-sufficient condition for Amphidinium carterae grown on three N sources. The solid line represents the response of the model using the base values indicated in Table 4. 121 0 50 100 150 200 Time (hours) Figure 41 Response of the model to changes in a) e(NH4+) and b) s(N03") in the N re-supply phase for Heterosigma carterae grown on three N sources. The solid lines represent the response of the model using the base variables indicated in Table 4. 122 1 i _ 0 o u> CO 170 180 • s(NH4*) = 4 — s(NH4*) = 12 — s(NH4*) = 20 190 200 Time (hours) 210 220 Figure 42 Response of the model to changes in e(NH4+) in the N re-supply phase for Heterosigma carterae grown on three N sources. The solid line represents the response of the model using the base values indicated in Table 4. 123 4) Heterosigma carterae grown on N03' Variations in the values of e(N03") produced large changes in the 815NPN at low PN concentrations in N-replete phase (Fig. 43). The values of e(N03) that fitted the data best was in the range of 5 - 7%o. The best fit value of e(N03') in the N-deplete phase clearly increased (Fig. 44). An e(N03") of 9 %o was used for the best fit model, although 815NPN values at 119 and 121 h were lower than the predicted 815NPN of e(N03') of 11 %o. The changes of e(N03") had a large effect on the fit for the 121 -125 h time period, but a small effect on the fit immediately after the re-addition of N03" (115.5 -116 h) and near the completion of NOV utilization (126-127 h). 124 Figure 43 Response of the model to changes in s(N03") in the N-sufficient condition for Heterosigma carterae grown on three N sources. The solid line represents the response of the model using the base values indicated in Table 4. 125 _2 -I -i 1 1 1 1 ' <——1 114 116 118 120 122 124 126 128 130 Time (hours) Figure 44 Response of the model to changes in e(N03") in the N re-supply phase for Heterosigma carterae grown on three N sources. The solid lines represent the response of the model using the base variables indicated in Table 4. 126 DISCUSSION The present study attempted to show that the 815NPN resulting from the growth of two dinoflagellates and a raphidophyte in a medium containing N03' or three N sources was predicted by the model. The model assumed 815NPN was described as the weighted sum of the changes in 815NPN resulted from the incorporation of each individual N source. Each N incorporation was treated as a unidirectional reaction and the total N incorporation was described as the sum of these reactions. The models fitted the data relatively well. However, during the simultaneous growth on two N sources, especially on NH4+ and urea, the prediction was difficult. For example, Prorocentrum micans had a much lower s(NH4+) than the expected value of 20%o. The data for Amphidinium carterae and Heterosigma carterae were relatively well predicted by using the expected e value of each N. e(N03) The e(N03') value for Amphidinium carterae in N-replete condition that gave the best fit was 0%o. This value was lower than e(N03") of 1.9+0.3%o obtained by Needoba (1997) for Amphidinium carterae grown on N03*. s(N03') of 0%o is also lower than previous estimates for diatoms, a coccolithophore and even dinoflagellates. e(N03") for the diatoms, Chaetoceros debilis, Chaetoceros simplex, Skeletonema costatum, Thalassiosira weissflogii, and Ditylum brightwellii were 4 - 5, 3.0±0.5, 2.2+0.4, 6.2±0.3, and 2.8+0.3%o respectively 127 (Needoba 1997; Waser et al. 1998a). The coccolithophore, Emiliania huxleyi had a N fractionation of 3.9±0.3%o (Needoba 1997). Dinoflagellates such as Prorocentrum minimum had e(N03') of 2.4±0.3%0 (Needoba 1997). Only Heterosigma carterae grown on N03' whose best fit e(N03") was 5%o, showed a good agreement with these values. On the other hand, s(N03") for Heterosigma carterae grown on three N sources appeared to be higher than 9%o. A high e(N03") was reported by Montoya and McCarthy (1995). These authors reported that diatoms such as Thalassiosira weissflogii and Skeletonema costatum had an e of 9.7 -15.4 and 6.4 -11.9%o respectively for a continuous culture experiment. Interestingly, 9.0%o for Skeletonema costatum reported by Pennock (1996) was very close to the e(N03") for Heterosigma carterae grown on three N sources in the present study. The discrepancy may be due to some process such as recycling of DON or NH4+ (Rees & Syrett 1979; Price & Harrison 1988; Pujo-Pay et al. 1997). This process was not accounted for the model and may produce discrepancies. At the moment, it is impossible to explain the reason of the discrepancy. The difference in isotope fractionation between dinoflagellates and flagellates on the one hand (i.e. low fractionation) (Montoya & McCarthy 1995) and coccolithophores and diatoms on the other hand (e.g. high fractionation) (Montoya & McCarthy 1995; Pennock et al. 1996) was not seen in this study. It has been hypothesized that the ability of dinoflagellates and flagellates to swim, decreases the zone of diffusion around the cell and results in the reduction of the potential of the fractionating step during the uptake of N03" across the outer cell membrane (Montoya & McCarthy 1995). This process could explain the low 128 s value observed. The silica shell of diatoms is considered to provide a barrier to NOV uptake and results in higher discrimination of isotopes (Montoya & McCarthy 1995). These morphological effects on isotope fractionation remain unclear. E(NH4+) Growth on NH4+ in N-replete conditions was accompanied by very large values of e(NH4+) ranging from 12 to 24%o. e(NH4+) for Heterosigma carterae (18 - 22%o) and Amphidinium carterae (20%o) were in good agreement with previously reported values such as 25 - 26%o for Skeletonema costatum (Pennock et al. 1996) and 18 - 20%o for Thalassiosira pseudonana (Waser et al. 1998b). However, Prorocentrum micans had a very low e(NH4+) of 12%o. This value is close to the estimates (6.5 - 9%o) of phytoplankton grown on NH4+ in coastal environments (Cifuentes etal. 1989; Montoya etal. 1991). Because of a very low e(NH4+) and a linear uptake of NH4+ and urea in N-replete conditions, it is suspected that the cell might have experienced N starvation at the beginning of the experiment. An e(NH4+) of 12%o is very close to the values of N-starved Emiliania huxleyi and a diatom assemblage (Waser et al. unpublished results). However, NOV concentration of the stock culture was 5.25 uM, thus Prorocentrum micans was not N-starved. One clue of this low s(NH4+) was that cells had a high PN. The calculated PN was 11.1 ug-at N L'1. This was the highest among other species in the present study. PNj of Amphidinium carterae (grown on three N sources), Heterosigma carterae (grown on three N sources), 129 and Heterosigma carterae (grown on N03') were 4.7, 3.9, and 5.1 ug-at N L"1 respectively. If the true PNj is higher than the calculated PNj, some of the discrepancy is explained. The PN of the first few points of Prorocentrum micans was 29.6 at 9 h and 45.2 ug-at N L'1 at 17 h. The calculated PNjS from these two measured PNs were 14.7 and 16.4 ug-at N L'1 respectively. These numbers are higher than 11.1 ug-at N L"1 of PNj that was used for the model and the true PNj is suspected higher than the calculated value. Another possibility for low e(NH4+), again, may be the excretion of NH4+ (Rees & Syret 1979; Price & Harrison 1988). The hypothesis that mobile cells may have a lower e value compared to non-mobile cells (Montoya & McCarthy 1995) could be another possibility for the low e(NH4+). This species has the ability to swim as fast as 0.85 m h"1 (Edler & Olson 1985). In contrast, it is known that high e(NH4+) values are found in chain-forming diatoms (Pennock et al. 1996; Waser et al. 1998a). Chaetoceros debilis and Skeletonema costatum had an e(NH4+) of 25 and 26%o respectively. These chain-forming diatoms discriminated against 15N during NH4+ incorporation than mobile cells. The mechanism of isotope fractionation for NH4+ and N03" incorporation is still unclear, and so is the different e values obtained (Waser et al. 1998a). Culture conditions such as NH4+ concentration, pH, and bubbling may affect e values of NH4+ relative to N03" because of efflux and influx of NH3 (Hoch et al. 1992) and the loss of gaseous NH3 from the culture medium at high pH (Waser etal. 1998b). 130 Effect of N deprivation Physiological state of dinoflagellates and raphidophytes affected the 815NpN. After the re-addition of N sources, the positive values of 815NPN were observed in the cultures of Amphidinium carterae grown on three N sources and Heterosigma carterae grown on N03". This phenomenon was observed in the study of coastal diatoms and the coccolithophore (Waser et al. 1998a). A positive number indicates that 15N was incorporated more quickly than 14N by the N-starved cells. One of the reasons for this inverse discrimination is the excretion of a 15N-depleted compound by the N-starved cells. An example of such an excreted compound is NH3 which is excreted by cells when it is incorporated into urea (Price & Harrison 1988). Amino acids can become enriched in 15N for a while when cells accumulate 15N-replete amino acids before the cells excrete some of these amino acids into the medium. Accumulating compounds indicate that the enzyme to aid the transformation to another compound may be rate limiting. Usually, intrinsic isotope fractionation (P) for the enzyme activity results in 15N-replete substrate and 15N-deplete products. However, the e value for membrane diffusion of NH3 is quite large (39%o) (Hermes et al. 1985), and results in excretion of 15N-deplete NH3. In fact, the NH4* concentration did not increase initially in Amphidinium carterae culture. Therefore, it is difficult to explain the inverse isotope discrimination by efflux of NH3 for this species. Another possibility is the excretion of isotopically light amino acids as explained above. It is not possible to determine what 15N-deplete compound was excreted in this study. 131 The e (NH/) of Prorocentrum micans grown on three N sources and the E(N03") of Heterosigma carterae grown on N03" increased after the cells became N-starved. Prorocentrum micans and Heterosigma carterae changed their best fit e values from 12 to 20%o and from 5 to 9%o respectively. One possible explanation for an increased e(NH/) has to do with a change in the transport system (active transport versus passive diffusion) during N starvation. The increase in e(N03) may be for a different reason than for e(NH/). Perhaps, the efflux of N03" increased after N starvation due to a slowed assimilation, which allowed for more 15N-replete N03" to efflux and resulted in high e values. Since Heterosigma carterae grown on N03" in the N-replete phase had much lower 515N of PNj (1.08%o) than the expected values of 3.04%o, the increased e(N03') value in the N-deplete phase was of a concern. It is not clear at present why there is such a discrepancy between the measured 815N and the fit by the model. It is possible that DON may have been released into the medium (Rees & Syrett 1979; Price & Harrison 1988; Pujo-pay 1997). The PN concentration (90.6 ug-at N L"1) at steady-state was in good agreement with the initial N03" concentration (78.5 ug-at N L'1) suggesting that the release of DON, if it occurred, was relatively small. Nonetheless, it is interesting that N-deprivation enhanced isotope fractionation and incorporated more 14N than 15N, which was the opposite of what was expected. Again, the hypothesis that mobile cells have lower e values (Montoya & McCarthy 1995) could be one possibility of higher e(NH/) of N-starved Prorocentrum micans and e(N03") of Heterosigma carterae. 132 During N starvation, cells tended to sink and stay at the bottom of the flask even though the flask was swirled by hand and after the re-addition of N sources, cells gradually dispersed in the medium again. The temporary loss of ability to move might result in the higher e values for these species. On the other hand, Amphidinium carterae decreased its e(NH4+) from 18 - 20%o to < 4%o. Previously, no change or decreased e values were reported to be due to N deprivation in the study of the open ocean clone of Emiliania huxleyi and the coastal clone of the same species grown on three N sources respectively (Waser et al. 1998a). Diatoms grown on three N sources also showed decreased e values after N deprivation (Waser et al. 1998a). All species changed the mechanism of isotope fractionation during N deprivation. This may be due to the rate limiting transport across the membrane and diffusion to the cell that occurred during N deprivation (Wada & Hattori 1978; Mariotti etal. 1982; Handily & Raven 1992; Evens etal. 1996). Otherwise, the rate-limiting step was the reduction of NO3" to NO2' by nitrate reductase which was affected by N deprivation. At this time, it is impossible to tell which step, either at the cell membrane or at the nitrate reductase level, is responsible for the isotope fractionation. If the cell did not change the mechanism of isotope fractionation, it indicates that the cell has recovered quickly from the impact of N deprivation. Such a species is able to live in the oligotrophic ocean because it takes up episodically supplied NH4+ (Dugdale 1967) with the same mechanism that occurs under N-replete conditions. 133 Difficulties in predicting 815NPN occurred during simultaneous uptake of two or three N sources. Since the purpose of this type of laboratory experiment involves the application for understanding N isotope fractionation in the ocean, interaction of each N is important for interpreting the 815NPN in the ocean. For example, if NH3 is released and recycled during the simultaneous uptake of NH4+ and urea, 815NPN was much lower than 815NPN grown on only NH4+. Therefore, not only e for each N source, but also the interaction of N sources needs to be considered in the model fit. N availability affected N isotope fractionation. It is interesting to compare the results of acclimation to the N-deplete condition obtained in Part. I of this study. Prorocentrum micans and Amphidinium carterae showed a response to N depletion by enhancing their ability to take up the reduced N form and episodically supplied urea (Dugdale 1967) rather than N03', although they decreased their ability to take up NH4*. However, none of species were able to keep the same isotope fractionation mechanism during the N deprivation. These observations indicate that marine phytoplankton have different methods and magnitudes of adaptation to N-deplete conditions. 134 CONCLUSIONS N availability had a great impact on 815N incorporation by marine phytoplankton. e (isotope fractionation) changed before and after N deprivation and following N re-supply. e(N03) of Heterosigma carterae grown on N03" increased during N starvation probably due to the increased efflux of 15N-deplete DON. For cultures grown on three N sources, E(NH4+) decreased in Amphidinium carterae and Heterosigma carterae and increased in Prorocentrum micans. 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Histochemical demonstration of a glycocalyx on the cell surface of Heterosigma akashiwo. Mar. Biol. (Berl.)88: 295-299. 156 APPENDIX 1 Equations used to calculate growth rates Growth rates (d"1) were calculated from fluorescence values between the beginning and the end of log phase according to the following equation: H = In (F2/F1) / (t2-t1) where F2 and F1 are the fluorescence values at time 2 (t2) and time 1 (t1), respectively and are reported as the mean (n = 3) ±1 S. D. for triplicate cultures. In vivo fluorescence was measured with a Turner Designs model 10 fluorometer. 

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