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Effects of iron on the physiology of oceanic phytoplankton from the NE subarctic Pacific Ocean Muggli, Deborah Lynn 1995

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EFFECTS OF IRON ON THE PHYSIOLOGY OF OCEANIC PHYTOPLANKTON FROM THE NE SUBARCTIC PACIFIC OCEAN by DEBORAH LYNN MUGGLI B.Sc, The University of Minnesota, 1988 M.Sc, The University of Tennessee, 1990 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Oceanography) We accept this thesis as conforming J^pjthe reo j^red standard THE UNIVERSITVOF BRITISH COLUMBIA December 1995 ©Deborah Lynn Muggli, 1995 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. The University of British Columbia Vancouver, Canada Department DE-6 (2/88) 11 A B S T R A C T The physiology of 2 oceanic phytoplankton species freshly isolated from the subarctic Pacific (Station P, 145°W, 50°N) was examined, focusing on the effects of iron (Fe) on various physiological parameters. The two species, a coccolithophore Emiliania huxleyi and a large diatom Actinocyclus sp., represent different taxonomic groups and size classes of indigenous phytoplankton found at Stn P. The effect that nitrogen source ( N O 3 " vs. NH.4+) had on the physiology and metal nutrition of both E. huxleyi and Actinocyclus sp. under Fe-replete and Fe-stressed conditions was examined. In general, the diatom responded as expected based on.theoretical energy and Fe requirement predictions, with NH4"*"-grown cells exhibiting a physiological advantage over NC>3"-grown cells under Fe-stressed conditions. In contrast, E. huxleyi exhibited no physiological advantage when grown on N H 4 + compared to N O 3 " under Fe-stressed conditions, largely due to the decrease in cell volume of NO3"-grown cells under Fe-stress. Secondly, the effect of Fe on the sinking rate of both the small oceanic coccolithophore and the larger diatom was examined. Fe conditions drastically affected the sinking rate of the diatom, with a > 5 times increase in sinking rate under Fe-replete compared to Fe-stressed conditions. Fe conditions had no effect on the oceanic coccolithophore. EDTA concentrations of 100 jtM were found to inhibit cell division rates by 30 - 50%, as well as affecting carbon metabolism in both species. As this concentration of EDTA is used in many phytoplankton/trace metal studies, caution is warranted, especially when working with oceanic species. Lastly, the ecophysiology of the 2 oceanic species in natural Stn P water (no metals or artificial chelators added) and in the same seawater to which 5 nM Fe was added was examined. Under in situ conditions, E. huxleyi grew maximally while Actinocyclus sp. was unable to divide. When 5 nM of Fe was added, Actinocyclus sp. resumed cell division and Ill grew significantly faster than E. huxleyi. E. huxleyi's biochemical composition (chl a, C, N) did not change when Fe was added to the seawater, whereas the diatom exhibited signs of severe Fe-stress when grown on Stn P water with no additions. From the experiments conducted in this thesis, E. huxleyi appears to be better adapted to living in a low Fe environment than the diatom. These are the first laboratory data on phytoplankton species isolated from the NE subarctic Pacific (or any High Nutrient Low Chlorophyll region), and these data provide independent, physiological support for the Fe/grazing hypothesis in the NE subarctic Pacific. T A B L E O F C O N T E N T S Abstract ii Table of Contents iv List of Tables viii List of Figures xi Acknowledgments xiv General Introduction 1 Biological Oceanography of the Subarctic Pacific Ocean 1 Phytoplankton Assemblages at Station Papa 2 Phytoplankton Species used in Thesis 3 Role of Iron in Phytoplankton Metabolism 4 Nitrogen Metabolism 4 Photosynthesis and Respiration 5 Sinking Rate 6 Overview of Thesis 6 General Methods 8 Phytoplankton Isolates 8 Trace-Metal Clean Culture Techniques 13 Media Preparation 13 Culture Vessels , 13 Metal Quota Measurements 14 Culture Filtration and Filter Storage 14 Filter Digestion 15 Digestate Analysis 15 Filter Blanks 15 Polycarbonate vs. Cellulose Acetate Filters 16 Chapter 1: Effects of Iron and Nitrogen Source on the Physiology and Metal Composition of an Oceanic Diatom From the NE Subarctic Pacific 17 Introduction 17 Materials and Methods 18 Results 19 Cell Volume 19 Chlorophyll a Quotas 19 in vivo Fluoresence: Chlorophyll a 22 Carbon 22 Nitrogen 22 Carbon: Nitrogen 22 Metal Quotas 24 Iron 24 Manganese 24 Zinc 24 Effect of Ti(m) treatment 24 Discussion 27 Fe and Chlorophyll, Carbon, Nitrogen 27 Fe Quotas 28 Mn and Zn Quotas 29 Ecological Significance 30 Chapter 2: Effects of Nitrogen Source on the Physiology and Metal Nutrition of Emiliania huxleyi Grown Under Different Iron and Light Conditions 33 Introduction 33 Materials and Methods 34 Fe Experiments 35 Fe-Replete Experiments 35 Fe-Stressed Experiments 35 Irradiance Experiments 37 Results 38 Fe Experiments 38 Growth Rate 38 Cell Volume 38 Biochemical Composition 41 Metal Composition 43 Irradiance Experiments 48 Growth Rate 48 Cell Volume 48 Chlorophyll a 48 Discussion 50 Effect of N Source under Fe-Replete Conditions 50 Effect of N Source under Fe-Stressed Conditions 52 Growth Rate (Fe-stress vs low-light) 53 Cell Volume (Fe-stress vs low-light) 53 Chlorophyll a Quotas (Fe-stress vs low-light) 54 Metal Quotas 55 Ecological Relevance 56 Comparison of E. huxleyi Isolates from Different Locales ..56 The Subarctic Pacific 57 A Brief Summary of Chapters 1 and 2 59 Chapter 3: Effect of Iron on the Sinking Rate of an Oceanic Diatom and an Oceanic Coccolithophore from the NE Subarctic Pacific 61 Introduction 61 Materials and Methods 61 Results 62 Sinking Rate 62 Cell Volume 65 Nitrogen Quotas 65 Carbon Quotas 65 Chlorophyll a Quotas 65 Carbon:Chlorophyll a 67 Metal Quotas 67 Discussion 67 Large Oceanic Diatom vs Small Oceanic Coccolithophore 67 Sinking Rate of N H 4 vs. N03-Grown Cells of an Oceanic Diatom 68 Cell Volume, Fe, and Sinking Rates 68 Ecological Significance 70 Chapter 4: Effects of EDTA on Oceanic Phytoplankton 72 I. EDTA Suppresses the Growth of Oceanic Phytoplankton 72 Introduction 72 Materials and Methods 73 Results 75 Discussion 79 n. Possible Effects of the Presence of EDTA on the Physiology of an Oceanic Diatom Grown in Artificial Media 80 Introduction 80 Materials and Methods 80 Results 82 Growth Rate 82 Chlorophyll a Quotas 82 Carbon:Chlorophyll a 82 in vivo Fluorescence: Chlorophyll a 82 Carbon, Nitrogen Quotas 82 Iron Quotas 85 Discussion 85 Chapter 5: Ecophysiology of Two Oceanic Phytoplankton Species from the NE Subarctic Pacific 87 I. Effect of Iron Addition on Two Oceanic Phytoplankters Grown in Natural NE Subarctic Pacific Seawater with no Artificial Chelators Present 87 Introduction 87 Materials and Methods 88 Results 89 Growth Rate 89 Cell Volume 89 in vivo Fluoresence: Chlorophyll a 89 Chlorophyll a Quotas 89 Carbon and Nitrogen Quotas 89 Metal Quotas 92 Discussion 92 Growth Rates 92 in vivo Fluoresence: Chlorophyll a 93 Chlorophyll a, Carbon, and Nitrogen Quotas 94 Metal Quotas 94 n. Ecological Comparison of Emiliania huxleyi and Actinocyclus sp 95 Iron Requirements for Growth 95 E. huxleyi vs Actinocyclus sp 95 Comparison with Other Species 96 Mn and Zn Requirements 99 E. huxleyi and Actinocyclus sp. at Station P 100 General Conclusions 102 Future Research 104 Literature Cited 105 Appendix A: Maintenance Medium for Emilianina huxleyi and Actinocyclus sp. 114 Appendix B: Procedure for Acid-Cleaning Plastics 115 Appendix C: Programs used for the Determination of Fe, Mn, Zn, and Cu on the Varian Graphite Furnace AA 116 Vl l l LIST O F TABLES Table 1.1. Physiological parameters for nitrate and ammonium-grown cultures of Actinocyclus sp. under both Fe-replete and Fe-stressed conditions (+1 standard error in parenthesis; n=3). *indicates a significant difference at p< 0.05 between nitrate and ammonium-grown cells with the asterisk placed next to the greater value, """indicates a significant difference at p<0.05 between Fe-replete and Fe-stressed cells with the asterisk placed next to the greater value. CV=cell volume; /*=growth rate. 20 Table 2.1. Physiological parameters for nitrate and ammonium-grown cultures of Emiliania huxleyi under 3 Fe treatments: high Fe (100 nM Fe), low Fe (0.8 nM Fe), and Fe-stressed conditions (< <0.8 nM Fe). +1 standard error is given in parenthesis (n=4 for 100 nM Fe, n=3 for other Fe treatments). *indicates a significant difference at p<0.05 between N O 3 " and NH.4+-grown cells for a particular Fe treatment with the asterisk placed next to the greater value. ^Growth rates achieved over a 2 day period after 5 nM Fe was added to the Fe-stressed cultures, n =growth rate; CV=cell volume; C(t)=total cell carbon, including coccoliths; C(a)=cell carbon excluding coccoliths (coccoliths removed by acidification, see methods). 39 Table 2.2. Metal quotas normalized per cell and per cell volume for nitrate and ammonium-grown cultures of Emiliania huxleyi under 3 Fe treatments: high Fe (100 nM Fe), low Fe (0.8 nM Fe), and Fe-stressed (< <0.8 nM Fe) conditions. ± 1 standard error is given in parenthesis (n=4 for 100 nM Fe, n=3 for other Fe treatments). *indicates a significant difference at p< 0.05 between N O 3 " and NH4 + -grown cells for a particular Fe treatment with the asterisk placed next to the greater value. CV=cell volume; Fe(int) = intracellular Fe; Fe(t)=total Fe (both intra-and extracellular); amol=l x 10~18 moles; N/A=data not available. The Fe(int) values for the low Fe and Fe-stressed treatments are 75-90% of the measured Fe(t) (see methods). 44 Table 2.3. Metal quotas normalized to cell carbon for nitrate and ammonium-grown cultures of Emiliania huxleyi under 3 Fe treatments: high Fe (100 nM Fe), low Fe (0.8 nM Fe), and Fe-stressed (< <0.8 nM Fe) conditions. +1 standard error is given in parenthesis (n=4 for 100 nM Fe, n=3 for other Fe treatments). *indicates a significant difference at p< 0.05 between N O 3 " and NH.4+-grown cells for a particular Fe treatment with the asterisk placed next to the greater value. Fe(int)= intracellular Fe; C(t)= total cell carbon including coccoliths; C(a)=cell carbon excluding coccoliths (coccoliths removed by acidification, see methods); N/A=data not available. The Fe(int) values for the low Fe and Fe-stressed treatments are 75-90% of the measured Fe(t) (see methods) 45 ix Table 3.1. Physiological parameters for the oceanic diatom Actinocyclus sp. and the oceanic coccolithophore Emiliania huxleyi under both Fe-replete and Fe-stressed conditions (Jil standard error in parenthesis; n=4 for Fe-replete, n=3 for Fe-stressed cultures). •Indicates a significant difference at p< 0.05 between Fe-replete and Fe-stressed cells within a species with the asterisk placed next to the greater value. Metal values for E. huxleyi are from a similar experiment with Fe-replete cultures having a final Fe concentration of 100 nM Fe. $is an estimate based on the growth rate and Fig. 5.2. /n=growth rate; CV=cell volume; SR=sinking rate; C(t)=total carbon including coccoliths; C(a)=carbon excluding coccoliths 64 Table 4.1. Total metal concentrations added to the media and the calculated free ion metal concentrations determined using the chemical equilibrium program MINEQL. Units of total additions (e.g. Fej) are in molarity; units of calculated metal ions (e.g. Fe + 3) are in -log [molarity of ion] = p[M] 74 Table 4.2. Physiological parameters for Emiliania huxleyi grown with different EDTA concentrations under similar metal ion conditions in Aquil SOW. *indicates a significant difference (p< 0.05) of a particular EDTA treatment compared to the other EDTA treatments as determined by a 1-way ANOVA. ±1 standard error is given in parenthesis (n=3). n=growth rate; CV= cell volume; C(t)=total carbon including coccolith carbon; C(a)=carbon excluding coccolith carbon 77 Table 4.3. Physiological parameters for Actinocyclus sp. grown with different EDTA concentrations under similar metal ion conditions in Aquil SOW. *indicates a significant difference (p<0.05) of a particular EDTA treatment compared to the other EDTA treatments as determined by a 1-way ANOVA. ± 1 standard error is given in parenthesis (n=3). /t=growth rate; CV= cell volume 78 Table 4.4. Total metal concentrations added to the medium and the calculated ion metal concentrations determined using the chemical equilibrium program MINEQL. Units of total concentrations (e.g. Fe^ ) are in molarity; units of calculated metal ions (e.g. Fe + 3) are in -log[molarity of ion]=p[M] 81 Table 4.5. Physiological parameters for Actinocyclus sp. grown with and without EDTA under similar metal ion concentrations in Aquil SOW. *indicates a significant difference of a particular EDTA treatment compared to the other EDTA treatments as determined by a 1-way ANOVA. ± 1 standard error is in parenthesis (n=3). jw=growth rate; CV= cell volume 83 X Table 5.1. Physiological parameters for the oceanic diatom Actinocyclus sp. and the oceanic coccolithophore Emiliania huxleyi grown with 1 nM and 6 nM total Fe in Stn P water with no EDTA (+1 standard error in parenthesis; n=3). Carbon is total carbon, including coccolith carbon; cell volumes are spherical equivalents. I^ndicates 75% of Fe(t) as an estimate of Fe(int). *indicates a significant difference between Fe treatments for a given species with the asterisk placed next to the greater value. ^=growth rate; CV= cell volume; C(t)=total carbon including coccolith carbon; C(a)=carbon excluding coccolith carbon 91 xi L I S T O F F I G U R E S Fig. 1. Scanning electron micrograph of the oceanic coccolithophore Emiliania huxleyi. These calcifying cells were isolated from the NE subarctic Pacific Ocean. Samples of the cells were fixed for 15 min in 0.4% formaldehyde/disodium tetraborate solution, filtered onto polycarbonate filters, glued onto aluminum SEM stubs, and kept in a desiccator until completely dried (Holligan, pers. comm.). The samples were then sputter-coated with gold, and observed with a Cambridge 250T scanning electron microscope 10 Fig. 2. Scanning electron micrograph of the oceanic diatom Actinocyclus sp. from Stn P in the NE subarctic Pacific Ocean. Cell diameters vary from 15 to 60 fim. Frustules were acid-cleaned with saturated KMn04 and HC1 (Hasle 1978), filtered onto polycarbonate filters, rinsed with ethanol several times, glued to aluminum SEM stubs, and dried in a desiccator. Samples were then sputter-coated with gold and observed on a Cambridge 250T scanning electron microscope. The identification of genus for this diatom taxon was made using the following identification characteristics and the descriptions in Round et al. (1990): a large, single pseudonodulus; a ring of rimoportula inside the valve mantle; the external valve surface is finely porulated; the internal valve surface are foramina not obviously arranged in sectors. The identification of genus for this diatom was confirmed by Dr. F. J. R. Taylor 12 Fig. 1.1 A) Chlorophyll a per cell and B) in vivo fluorescence per chlorophyll a vs Fe condition for both ammonium and nitrate-grown cells of Actinocyclus sp. Error bars are +.1 standard error (n=3) 21 Fig. 1.2. A) Carbon per cell, B) Nitrogen per cell, and C) Carbon per nitrogen ratio vs Fe condition for both ammonium and nitrate-grown cells of Actinocyclus sp. Error bars are +1 standard error (n=3) 23 Fig. 1.3. Intracellular Fe per carbon ratio vs Fe condition for both ammonium and nitrate-grown cells of Actinocyclus sp. Error bars are ± 1 standard error (n=3) 25 Fig. 1.4. A) Fe per carbon, B) Mn per carbon, and C) Zn per carbon vs Fe conditions for both ammonium and nitrate-grown cells of Actinocyclus sp. Error bars represent ±1 standard error (n=3). 1 +Ti' indicates the Ti(III) reagent was used (see General Methods) 26 xu Fig. 2.1. Cell volume vs Fe treatment for both nitrate and ammonium-grown cells of E. huxleyi. Error bars represent ± 1 standard error (n=4 for 100 nM Fe, n=3 for other Fe treatments) 40 Fig. 2.2. A) Chlorophyll a per cell volume and B) Nitrogen per cell volume vs Fe treatment for both nitrate and ammonium-grown cells of E. huxleyi. Error bars represent +.1 standard error (n=4 for 100 nM Fe, n=3 for other Fe treatments) 42 Fig. 2.3. Carbon (acidified to remove coccoliths) per chlorophyll a vs Fe treatment for both nitrate and ammonium-grown cells of E. huxleyi. Error bars represent +.1 standard error (n=4 for 100 nM Fe, n=3 for other Fe treatments) 46 Fig. 2.4. A) Intracellular Fe per total carbon, B) Mn per total carbon, and C) Cu per total carbon vs Fe treatment for both nitrate and ammonium-grown cells of E. huxleyi. Error bars represent ± 1 standard error (n=4 for 100 nM Fe, n=3 for other Fe treatments) 47 Fig. 2.5. A) Specific growth rate, B) Cell volume, and C) Chlorophyll a per cell for both nitrate and ammonium-grown cells of E. huxleyi vs irradiance. Error bars represent + 1 standard error (n=3) 49 Fig. 3.1. Sinking rate vs Fe condition for A) Both ammonium and nitrate-grown cells of Actinocyclus sp., and B) Nitrate-grown cells of E. huxleyi. Error bars represent + 1 standard error (n=3) 63 Fig. 3.2. Cell volume vs Fe condition for A) Both ammonium and nitrate-grown cells of Actinocyclus sp., and B) Nitrate-grown cells of E. huxleyi. Error bars represent +1 standard error (n=3) 66 Fig. 3.3. Cell volume vs time for both ammonium and nitrate-grown cultures of Actinocyclus sp. grown in Fe-replete medium over a 40 day period. Error bars represent ± 1 standard error (n=3). The slope of the linear regressions are -45.2 (r2=0.8) and -24.7 (r2=0.8) for ammonium and nitrate-grown cells, respectively 69 Fig. 4.1 Specific growth rate vs EDTA concentration for A) E. huxleyi and B) Actinocyclus sp. Error bars represent +1 standard error (n=3) 76 Xll l 4.2. A) Carbon per chlorophyll a and B) in vivo fluorescence per chlorophyll a vs EDTA treatment for Actinocyclus sp. Error bars represent +.1 standard error (n=3). 84 5.1. Specific growth rate for both E. huxleyi and Actinocyclus sp. grown in both natural Station P water and the same water plus 5 nM Fe (no EDTA present in either treatment). Error bars represent +1 standard error (n=3) 90 5.2. Specific growth rate (measured by changes in cell #) vs intracellular Fe to total cell carbon for A) E. huxleyi and B) Actinocyclus sp. Error bars represent +1 standard error (n=3 or 4). Non-linear regressions were performed on both data sets to obtain the parameters Mmax ^  Qmin text f o r descriptions). The regression equation was y=Mmax*((x-Qmin)/x) • 9 7 xiv A C K N O W L E D G M E N T S Chronologically, the first person I would like to thank is Elaine Simons, for spending the time to teach me phytoplankton isolation techniques. Jay McNee also helped me early on, by again being generous with his time and teaching me trace-metal techniques and how to use the exploding microwave digestion apparatus. Graduate students in Kristin Orian's lab helped me with trace-metal problems, and let me use their equipment and space. The UBC JGOF's team (Hugh, Phil, Diane, Brad) were a tremendous help in obtaining water for me, which I needed to conduct experiments and to keep the cultures alive. Maureen Soon analyzed more than one tray of CN samples for me. I feel like I should thank Bert Mueller, but I'm not sure why. Mingxin Guo helped me more than once with experiments and baby-sitting cultures. Maude Lecourt also helped me with some experiments, and was a great office mate and friend. All of my office mates have been great. Chris Mewes and others were of tremendous help in the office, especially with really confusing paperwork. I would also like to generally thank all of the people who have been in the Harrison lab throughout the duration of my study (although I don't think that I can count them all!). Graduate students in the other biological labs (Lewis and Taylor) were also of great help. Thanks go to both mine and Murray's families, from the west coast to the midwest. Mom, Gary, Monique, and Cheryl have always been supportive and there when I needed them. Their occasional visits were wonderful and meant a lot to me. I may not see the rest of the Muggli's in Minnesota very often, but they are never very far from my thoughts, and are a part of me no matter where I go. Dad's long overdue visit was a highlight in my final year (as well as Beak's trek). Little did we know that we had only to dangle a fish in his sights to get him here. Lola and gang were our family just south of the border, and put up with our drop-in visits en route to Leavenworth or Yosemite. Never a dull moment. Warm thanks go to Ainsley and Dave, for their unending generosity and support of both Murray and I, and for making me feel like I have a home in a city where I was once a stranger. Murray of course gets enormous amounts of thanks, especially for his eternal good naturedness, patience, and passion for the outdoors, which helps keep life really in perspective. Paul Harrison was a wonderful advisor throughout my thesis. He is courageous enough to let his students roam at will, and was always more than supportive in all academic pursuits. Paul's strong commitment to teaching deserved admiration, and his support of myself (as well as other students) in teaching pursuits during summers was extremely appreciated. Thanks for everything. My supervisory committee members Drs. Al Lewis and Kristin Orians were great; always approachable and willing to help. I will always be indebted to Al Lewis for getting the indoor bike rack put up! Dr. Max Taylor also helped by taking the occasional peek through a microscope. The department was a very positive, productive environment in which to complete a thesis. Thanks everyone! 1 G E N E R A L I N T R O D U C T I O N The NE subarctic Pacific Ocean is one of 3 regions in the world's ocean currently described as an HNLC (High Nutrient Low Chlorophyll) region, where the trace metal iron (Fe) has been hypothesized and recently shown to limit phytoplankton biomass (Martin et al. 1989) as well as affect the species composition of the indigenous phytoplankton assemblages (Boyd et al. submitted). However, this evidence is based on experiments conducted in the field, where confounding factors are always present and unavoidable, making a study of the effects of Fe on individual species or taxonomic groups extremely difficult. This thesis examines ecophysiological aspects of indigenous phytoplankton species from the NE subarctic Pacific with respect to their Fe nutrition. As well, these laboratory results are extrapolated to field conditions where appropriate. The following topics will be discussed in this general introduction: the general biological oceanography of the subarctic Pacific including phytoplankton species composition, the phytoplankton isolates used in this thesis, the general role of Fe in phytoplankton metabolism, and finally, a general overview of the thesis. BIOLOGICAL OCEANOGRAPHY OF THE SUBARCTIC PACIFIC OCEAN The NE subarctic Pacific Ocean, specifically Station Papa (Stn P, 145°W, 50°N), is characterized by a constant, low (e.g. <0.4 H) concentration of chlorophyll a year round (e.g. no 'blooms'), despite the continual presence of macronutrients (Frost et al. 1983). Mixing brings macronutrients to the surface, and yearly euphotic zone concentrations of nitrate (NO3*) vary from 5 to 12 /xM (Martin et al. 1989, Varela, unpubl. data). Similarly, surface concentrations of silicic acid (Si(OH)4) and phosphate (PO^) are ca. 10-18 and 0.7-1.2 fjM, respectively (Martin et al. 1989, Varela, unpubl. data). Ammonium (NH4"*") concentrations range from 0.2 to 0.6 /xM (Varela, unpubl. data). The macronutrient concentrations remain relatively high within the upper mixed layer and are never fully utilized, suggesting that macronutrients do not control primary productivity in the NE 2 subarctic Pacific as they do in other oceanic regions. These observations were paradoxical to biological oceanographers until trace-metal clean techniques were developed, and true ambient concentrations of metals were measured in the ocean (Gordon et al. 1982). As a result of these new techniques, it was found that trace metals, especially Fe, were present in extremely low concentrations in the euphoric zone, with Fe concentrations being as low as 0.05 nmol kg seawater"! in surface waters at Stn P (Martin and Gordon 1988). This prompted field experiments designed to examine the possible effects of Fe on the indigenous phytoplankton assemblages (Martin et al. 1989). However, debate ensued regarding the results of such experiments, with criticisms revolving around the effect of containment on the indigenous organisms, possible contamination in control containers, and the under-representation of indigenous zooplankton in the experiments (Banse 1990, 1991). Researchers today are still faced with the same problems, although some specific physiological indicators are being developed and utilized in the field (e.g. flavodoxin/ferredoxin measurements; LaRoche et al. 1995). Wells et al. (1995) point out the absolute lack of studies on indigenous species from HNLC regions, and the need to examine isolates from the locales of interest. By studying indigenous phytoplankton from Stn P in the laboratory, much can be learned about their basic physiology (no isolates from Stn P have been studied to-date, and data on oceanic species in general are rare). As well, if ecological questions are to be asked, confounding field effects are avoided allowing true responses to be measured. These laboratory results can then help to interpret results obtained in the field (e.g. Boyd et al. submitted). PHYTOPLANKTON ASSEMBLAGES AT STATION PAPA As already mentioned, chlorophyll a concentrations at Stn P rarely fluctuate (although primary production varies seasonally as would be expected (McAllister 1969)), and the dominant autotrophs are small nanoflagellates and other small non-diatoms (Booth et al. 1982, Taylor and Waters 1982, Booth 1988, Boyd et al. submitted). Small cells are typically 3 numerically dominant, and can account for a majority of the phytoplankton carbon biomass, with cells < 5 in diameter accounting for two-thirds of the autotrophic carbon biomass at times (Booth 1988). However, large diatoms do exist at Stn P, and while not normally numerically abundant (ca. 10% of C biomass), they can account for up to 50% of phytoplankton carbon at times (Booth 1980, demons and Miller 1984, Booth et al. 1988). As well, large amounts of siliceous material does indeed sink to depth (Takahashi 1986, Takahashi et al. 1990, Wong, unpubl. data), indicating that diatom 'blooms' must be occurring, despite the lack of observational data from the upper water column. PHYTOPLANKTON SPECIES USED IN THESIS Two phytoplankton species, Emiliania huxleyi and Actinocyclus sp., were studied in the thesis, both representing different taxonomic groups and size classes of phytoplankton found at Stn P. The oceanic coccolithophore, E. huxleyi, is a very small cell (ca. 3-5 /im in diameter), and is a member of the numerically dominant size class indigenous to Stn P. As well, it is the most abundant coccolithophore found at Stn P (Honjo and Okada 1974), and is ubiquitous throughout the temperate regions of the world's ocean. This organism (although different isolates) has also been mentioned previously for its low Fe tolerance (Brand et al. 1983, Martin et al. 1989). Hence, not only is E. huxleyi representative of the small, non-diatom phytoplankton assemblage continually found at Stn P, but its ubiquitous nature also makes information on its physiology of general interest. The oceanic diatom, Actinocyclus sp., is a solitary centric diatom, with a diameter ranging from 20 to 60 pm. This is a very large cell for Stn P, and large diatoms are rarely found in typical phytoplankton sampling. However, demons and Miller (1984) found numerous large diatoms while sampling with nets at Stn P. As well, during a cruise to Stn P in August 1995, large diatoms were found to be numerically abundant in the mixed layer (e.g. a diatom 'bloom', Boyd pers. comm.), possibly due to atmospheric Fe supply. Because of 4 their large size relative to the numerically dominant fraction (<5 /*m), cell concentrations can be small and still amount to the same carbon or nitrogen biomass. Actinocyclus sp. must be identified by SEM techniques, as the distinguishing features of this genus cannot be detected by light microscopy, and therefore it may have been included within different genera (solitary centrics) in some studies. However, Actinocyclus curvatulus (the species of the isolate used in this thesis was never confirmed) is a major diatom species found in sediment traps at 1000 and 3800 m at Stn P, with the largest fluxes of this species occurring in the spring (Takahashi 1986, Takahashi et al. 1990). Hence, Actinocyclus sp. represents a large single-celled diatom, and similar diatoms sink to depth at Stn P. ROLE OF IRON IN PHYTOPLANKTON METABOLISM Fe plays a central role in many basic metabolic functions in phytoplankton cells, including photosynthesis, respiration, nitrogen metabolism, and detoxification processes. Because of its redox chemistry, Fe is involved in electron transport processes as well as enzymatic functions. The following is a more detailed look at the involvement of Fe in processes that are relevant to this thesis. In addition, the specific functions pertinent to a given chapter are included in the introduction of that chapter. Nitrogen Metabolism Nitrate and nitrite reductase catalyze the reduction of nitrate to nitrite, and nitrite to ammonium, respectively. Both enzymes contain Fe, in a cytochrome molecule in the former, and an Fe porphyrin molecule (and possibly ferredoxin as an electron donor) in the latter (Goodwin and Mercer 1983). As a result, cells utilizing nitrate should theoretically have a higher Fe requirement than cells utilizing ammonium, and the difference has been calculated by Raven (1988) to result in a 60% higher Fe requirement for cells grown on nitrate compared to ammonium to achieve the same growth rates. 5 In addition, a cell must theoretically expend more reductant to grow on nitrate than on ammonium, because nitrate must be reduced to first nitrite and then to ammonium before it can be incorporated into amino acids. It has been calculated that due to the extra reducing steps required for nitrate incorporation, cells growing on nitrate require 22% more reductant than cells growing on ammonium (Thompson et al. 1989). As well, the energetic cost for the actual transport of nitrate across the cell membrane has been suggested to be higher than that for ammonium (Falkowski 1975, Turpin and Bruce 1990). Therefore, regardless of the Fe conditions, cells utilizing ammonium should have a theoretical advantage based on energy considerations over cells utilizing nitrate. Due to these differences in Fe and energy requirements depending on nitrogen source, it would be expected that a cell grown on ammonium would have a physiological advantage over cells grown on nitrate. Under Fe-stress, cells growing on nitrate should theoretically be more severely 'stressed' than cells growing on ammonium. For example, nitrate-grown cells may not be able to maintain their normal nitrogen quotas under Fe-stress, whereas ammonium-grown cells may have normal nitrogen quotas. As well, the in vivo fluorescence relative to chlorophyll a can be used as an indicator of the inefficiency of photosynthetic electron transfer (Lawlor 1987), and could theoretically be higher for nitrate-grown cells under Fe-stress, due to the additional Fe and energy requirements of utilizing nitrate. Photosynthesis and Respiration Fe is involved in both chlorophyll synthesis and photosynthetic and respiratory electron transport processes. The synthesis of chlorophyll requires Fe, where it is eventually replaced by magnesium as the central metal in the porphyrin ring (Spiller et al. 1982), and hence these atoms of Fe may be recycled. The transport molecules involved in transporting electrons for photosynthesis and respiration that contain Fe include Fe-sulfur compounds such as ferredoxin, and cytochromes, which contain Fe as the central metal in a porphyrin ring (Goodwin and Mercer 1983). Hence, Fe-limitation can lead to a decrease in the efficiency of electron transport (which can be crudely measured by the amount of fluorescent light leaving the cell instead of being utilized by the photosytems; in vivo fluorescence:chlorophyll a (Lawlor 1987)), and possibly to a deficit of carbon skeletons within the cell. It is well documented that Fe-stress will impair both the photosynthetic and respiratory pathways in algae and higher plants (Glover 1977, Spiller & Terry 1980, Terry 1980, Terry 1983, Sandmann 1985, Greene et al. 1991, Greene et al. 1992), rendering them 'energy-stressed'. Sinking Rate Large diatoms have been found to regulate their buoyancy by energy-requiring processes, with energy derived from respiration as the principal source (Waite et al. 1992, Waite et al. submitted). It follows, then, that Fe-stress may affect phytoplankton sinking rates by stressing the energy-producing pathways needed by the cell to maintain its buoyancy. OVERVIEW OF THESIS In this thesis, I have isolated 2 representative phytoplankton species from the NE subarctic Pacific, and have examined basic physiological processes in the laboratory that may illuminate the organism's actual physiological response in the field (ecophysiology). Chapters 1 and 2 begin by asking the following question: what nitrogen source (nitrate vs ammonium) is more beneficial for these organisms to use, under both replete and limiting Fe conditions? These chapters were designed to examine theoretical predictions regarding nitrogen source and Fe conditions, and are largely laboratory-oriented, with short ecological applications sections at the end of each chapter. Chapter 3 examines the effect of Fe-status on the sinking rates of both species, something that has never previously been examined. An attempt is made to relate these findings to the ecological situation in the subarctic Pacific. Chapter 4 examines the effect of EDTA on oceanic phytoplankton species specifically, which questions the typical protocols used in laboratory studies when working on phytoplankton-trace metal interactions, and encourages more careful consideration of how oceanic species are cultured. Finally, Chapter 5 contains experiments that were designed to compare these 2 very different species under similar, 'Stn P-like' conditions. These results were intended to address in situ questions, and the discussion relates the results to the field situation at Stn P as much as possible. As well, Chapter 5 addresses other ecophysiological aspects of these 2 species, comparing and contrasting their differences. The general conclusions of all chapters are then given after Chapter 5. 8 G E N E R A L M E T H O D S PHYTOPLANKTON ISOLATES Both Emiliania huxleyi and Actinocyclus sp. were isolated from the NE subarctic Pacific (Station Papa, 145°W, 50°N). The coccolithophore, E. huxleyi (Fig. 1), was obtained from water samples brought back to the laboratory after collection at sea on 11/91, to which no Fe or nitrate was added. Numerous cells were isolated by mouth pipette, so the resulting culture was not from a single isolated cell. Cells from the field had multiple layers of coccoliths present, and coccolith formation was preserved by maintaining the cultures in microwave-sterilized Station P water. Originally 6, and later 4 different culture media were used to insure continued calcification (Appendix A). Cultures were maintained in acid-cleaned 250 ml polycarbonate flasks with screw-top lids. Actinocyclus sp. (Fig. 2) was isolated from water obtained from Stn P on 5/93, to which both Fe and nitrate had been added after collection at sea. Again, multiple cells were isolated to initiate cultures. This species is a solitary, centric diatom, whose original diameter upon isolation was ca. 20 /-cm. The medium in which Actinocyclus sp. was maintained is given in Appendix A. The cell diameter of this species gradually decreased while in culture, and approximately 1.5 years after isolation it reproduced sexually, resulting in cell diameters of nearly 60 jim. The larger cells were separated from the smaller cells, and cultures of pure large cells were obtained and used for the final experiment conducted for this thesis (second EDTA experiment in chapter 4). The large cells grew faster than the small cells initially, but the growth rate eventually slowed to the same rate as the maximal growth achieved for the smaller morphology. The nitrogen and carbon content of the large cells was of course much greater than that of the small cells. All maintenance cultures of Actinocyclus sp. were maintained in 50 ml glass tubes with screw-top lids. 9 Fig. 1. Scanning electron micrograph of the oceanic coccolithophore Emiliania huxleyi. These calcifying cells were isolated from the NE subarctic Pacific Ocean. Samples of the cells were fixed for 15 min in 0.4% formaldehyde/disodium tetraborate solution, filtered onto polycarbonate filters, glued onto aluminum SEM stubs, and kept in a desiccator until completely dried (Holligan, pers. comm.). The samples were then sputter-coated with gold, and observed with a Cambridge 250T scanning electron microscope. 11 Fig. 2. Scanning electron micrograph of the oceanic diatom Actinocyclus sp. from Stn P in the NE subarctic Pacific Ocean. Cell diameters vary from 15 to 60 /*m. Frustules were acid-cleaned with saturated KMn04 and HC1 (Hasle 1978), filtered onto polycarbonate filters, rinsed with ethanol several times, glued to aluminum SEM stubs, and dried in a desiccator. Samples were then sputter-coated with gold and observed on a Cambridge 250T scanning electron microscope. The identification of genus for this diatom taxon was made using the following identification characteristics and the descriptions in Round et al. (1990): a large, single pseudonodulus; a ring of rimoportula inside the valve mantle; the external valve surface is finely porulated; the internal valve surface are foramina not obviously arranged in sectors. The identification of genus for this diatom was confirmed by Dr. F. J. R. Taylor. 13 TRACE-METAL CLEAN CULTURE TECHNIQUES Media Preparation All culture media were microwave-sterilized in acid-cleaned (see Appendix B) 2 L Teflon® bottles with screw-top lids (Keller et al. 1988). An extra 2 minutes was added to the original protocol. Either natural seawater from Stn P or Aquil synthetic ocean seawater (Price et al. 1988/89) was used for all experiments. Seawater was Chelex-treated for some experiments following the Chelex-preparation procedure of Price et al. (1988/89), except that Ultra HC1 (Seastar Chemicals) was used for the final HC1 wash instead of reagent grade HC1 as called for in the original protocol. Glass sintered funnels were used in all washing steps, and the final titration was carried out in acid-cleaned HDPE (high density polyethylene) bottles. Chelex prepared for use with ocean seawater was titrated to pH 8; Chelex prepared for use with macronutrient stocks was titrated to pH 3 for better removal of Fe. The prepared Chelex-100 resin was packed in a Teflon®/HDPE column, and seawater was pumped through the column at <2 ml min"l. Seawater was collected in acid-cleaned carboys, microwave sterilized, and stored in acid-cleaned, sterilized (by boiling Nanopure water in the jugs in the microwave for 20 minutes), 4 L polycarbonate jugs, covered in at least 2 plastic bags until use. Macronutrient and metal additions were made after microwave sterilization. Chelex-treated macronutrient stocks were kept in acid-cleaned HDPE bottles in the refrigerator covered with multiple plastic bags. Culture Vessels All plastics coming into contact with the cultures or during metal quota analysis were rigorously acid-cleaned as outlined in Appendix B. For most experiments, either 3 L or 250 ml acid-cleaned polycarbonate flasks were used with silicon stoppers and Teflon® tubing. The black rubber part of plastic syringes were coated with SurfaSil (Pierce Chemicals) to avoid metal contamination. When stirring was required, acid-cleaned Teflon® stir bars were used. 14 All handling of cultures was conducted under class 100 flowhood conditions. Culture flasks were covered with 2 plastic bags and sealed for low Fe experiments. METAL QUOTA MEASUREMENTS The determination of metal quotas was done using non-radiotracer (or 'cold-metal'), trace-metal clean techniques. In general, cultures were filtered onto cellulose acetate filters, the filters were digested, and the resulting digestate was analyzed using a graphite furnace atomic absorption spectrometer. Culture Filtration and Filter Storage Cultures were filtered onto acid-cleaned cellulose acetate filters using acid-cleaned polycarbonate Nalgene filtration manifolds. The cellulose acetate filters were acid washed as follows: a soak in 5% reagent grade HC1 for 24 h, two rinses with Nanopure water (Barnstead Nanopure IIDDW system), a minimum of one week soak in 0.1 % Ultra HC1 (Seastar Chemicals). Filters were left in the final acid solution until time of use. The filtration rigs were acid-cleaned as in Appendix B. The filtration rig was set up in a class 100 laminar flowhood. Prior to filtration, the rigs were rinsed thoroughly with Nanopure water. Filters were placed on the rig with Teflon® forceps, and about 5 ml of Nanopure water was filtered through the filters. Cultures were then filtered onto the filters. A minimum of 1 L of culture was necessary to obtain satisfactory signals over blank values (3 times the standard error of the blanks). For total Fe measurements, the filters were then rinsed twice with 5 ml of Chelex-treated Stn P water. The filters were allowed to suck dry, carefully folded in half, and placed in an acid-cleaned locking petri dish. Petri dishes were put in plastic bags and frozen until the filters were to be digested. For intracellular iron measurements, the TifTfl) reagent of Hudson and Morel (1989) was used. Cultures were filtered as above, but filtration was halted when 5 to 10 ml of culture remained in the filtration manifold. The Ti(Tfl) reagent was then added to the 15 unfiltered culture remaining in the filtration apparatus. For cultures of E. huxleyi, coccoliths were removed prior to filtration by lowering the pH to 5 in an acid-cleaned vessel to avoid any complications involving adsorption of Fe onto the coccoliths, and to eliminate the extra barrier to the cell membrane. The Ti(III) reagent was always prepared fresh on the day of use. After 2 minutes of soaking with the Ti(UJ) reagent, the remaining culture/Ti(in) mixture was allowed to filter through and was followed by 2 rinses of 5 ml of Chelex-treated Stn P water. The filters were then handled as described above. For Mn, Zn, and Cu, the non-Ti(IQ) treated samples were used for the final quota measurements. Filter Digestion Cellulose acetate filters were digested in concentrated Ultra H N O 3 " (Seastar Chemicals) in acid-cleaned Teflon® jars with screw-top lids. Teflon® jars were allowed to sit at room temperature for 24 h. Subsamples were then taken and diluted appropriately (usually 10 times) in acid-cleaned 30 ml HDPE vials. Digestate Analysis Measurements of Fe, Mn, Zn, and Cu were carried out on a Varian graphite furnace atomic absorption spectrometer with Zeeman background correction. Platform tubes were used for all analyses, and all measurements were determined in a 5% H N O 3 " matrix. Programs used are given in Appendix C. A certified standard (SLRS-2 riverine water, certified standard, Environment Canada) was used during all runs to insure standard accuracy. Filter Blanks A minimum of 3 filter blanks were measured for each culture treatment. These were analyzed and subtracted from the sample filter results. For Ti(ITI) blanks, the filters were soaked as in the culture filters, and rinsed 2 times with 5 ml of Chelex-treated Stn P water. To mimic the acid-treatment of E. huxleyi cultures prior to filtration, an equivalent amount of acidified Nanopure water was filtered prior to Ti(ITJ) treatment. The Ti(IJJ) treatment (or acid-treatment plus Ti(IIT) treatment) did not significantly increase the Fe content of the filter blanks. 16 Polycarbonate vs. Cellulose Acetate Filters Acid-cleaned polycarbonate filters were initially used for metal quota determinations. The polycarbonate filters were digested using a CEM microwave digestion apparatus. As the digestion was time consuming and samples were occasionally lost, the possibility of using cellulose acetate filters was considered. Acid-cleaned cellulose acetate filters had a similar metal (Fe, Mn, Zn, Cu) content to acid-cleaned polycarbonate filters, and the cellulose acetate filters were much easier to digest. It was found that submersion in concentrated H N O 3 " was sufficient to completely digest the filter, and repeated measurements from the same sample were consistent, indicating that the digestate was homogeneous. Hence, cellulose acetate filters were used after the above information was determined. The use of either filter gave comparable results, as the filter blanks were subtracted from the samples in all cases. 17 CHAPTER 1: EFFECTS OF IRON AND NITROGEN SOURCE ON T H E PHYSIOLOGY AND M E T A L COMPOSITION OF AN OCEANIC DIATOM FROM THE NE SUBARCTIC PACIFIC INTRODUCTION This chapter examined the effect that nitrogen source had on basic physiological parameters of an oceanic diatom under both Fe-replete and Fe-stressed conditions. Based on the theoretical predictions discussed in the general introduction, it would be expected that cells grown on ammonium would have a physiological advantage over cells grown on nitrate under both Fe-replete and especially under Fe-stressed conditions. I present here the first report on the effect of Fe and nitrogen source on the physiology and metal composition of a freshly isolated oceanic diatom, Actinocyclus sp., from the NE subarctic Pacific. Since cells under Fe-stress were examined in which their intracellular Fe concentrations would be low, Mn and Zn quotas were also measured to obtain basic metal composition data for this organism and to see if either metal increased under Fe-stress, possibly indicating a physiological use of the metal when Fe becomes limiting to the cell. Metal quotas were measured by non-radiotracer techniques to directly determine the metal-status of the cells, allowing for the simultaneous measurements of multiple metals. Physiological parameters measured include growth rate, cell volume, in vivo fluorescence, carbon quota, nitrogen quota, chlorophyll a quota, and metal quotas for Fe (intracellular and total), Mn, and Zn. 18 MATERIALS AND METHODS Cultures of the oceanic diatom Actinocyclus sp. were maintained in natural Stn P water (with metals and nutrients added; see general methods for details), in an attempt to retain the organism's native physiology and morphology. Experiments were conducted with microwave-sterilized (Keller et al. 1988), Chelex-treated (Price et al. 1988/89), Stn P water. The total Fe in this chelexed Stn P water was measured as in Yang (1993) and found to be ca. 0.9 nmol kg"l. Macronutrient enrichments were made with Chelex-treated stocks (see Price et al. 1988/89). The final concentrations in the medium were 150 iiM Si(OH)4, 5 iiM P O 4 - 3 , and 50 /tM N O 3 " or N H 4 + . Cells did not grow well with ammonium concentrations of 75 itM or higher. Metals were added to a final concentration of 1.5 times the Aquil amounts (35 nM Mn, 12 nM Zn, 1.5 nM Cu, 150 nM Mo, 3.75 nM Co, 15 nM Se) (Price et al. 1988/89), and 10 fiM EDTA was also added. ESAW amounts of vitamins were used (Harrison et al. 1980), and 1000 nM Fe final concentration was used for the Fe-replete treatment. The EDTA and Fe were added first, and the medium was allowed to equilibrate for 48 h before use. Cultures were grown in 3 L acid-cleaned polycarbonate Fernbach flasks equipped with Teflon® tubing and Teflon® stirring bars and handled only under class 100 flowhood conditions. The average irradiance was 145 itmol m"2 s"l as measured by a Biospherical Instruments model QSL-100 light meter. This irradiance was saturating for growth. Flasks were rotated once a day to ensure the same light conditions between flasks. Cultures were grown on a 14:10 light:dark cycle with Vitalite fluorescent bulbs at 16°C. The cultures were maintained semi-continuously in either 'no Fe medium' (no Fe was added) or 'Fe-replete medium' (1000 nM Fe). These are referred to as the 'Fe-stressed' and 'Fe-replete' cells, respectively. Triplicate cultures of both nitrate and ammonium treatments were grown under both Fe conditions. Cultures were grown for over 10 generations before harvesting in log phase. Growth rates were calculated from linear regressions of plots of the natural log of cell number vs rime. Cell numbers and cell volumes were measured on a Coulter Counter model TAIL Volumes were calculated using the equation for a cylinder, with the radius calculated from the Coulter Counter, and the height obtained by microscopic measurements for Actinocyclus sp. Chlorophyll a concentrations were determined by in vitro fluorometry (Parsons et al. 1984). Samples for particulate organic carbon and nitrogen were collected on precombusted (450°C for 4 h) 13 mm Gelman A/E filters and analyzed on a Carlo Erba CNS analyzer. Metal quotas were measured using cold-metal techniques as described in the General Methods section. All statistical comparisons were made using a Student's t-test at the 95% confidence level (p<0.05). RESULTS Cell Volume The cell volume of Actinocyclus sp. decreased slightly but significantly from Fe-replete to Fe-stressed conditions (Table 1.1), with an 8% decrease occurring for nitrate-grown cells and an 18% reduction occurring for ammonium-grown cells. The cell volume of ammonium-grown cells under Fe-replete conditions was significantly greater than the cell volume of nitrate-grown cells under the same conditions. Chlorophyll a Quotas Under Fe-replete conditions, the chl a celH was significantly higher for nitrate compared to ammonium-grown cells (Fig. 1.1 A, Table 1.1). However, under Fe-stressed conditions, ammonium-grown cells had sigmficantly higher chl a celH than nitrate-grown cells (Fig. 1.1A, Table 1.1). Nitrate-grown cells decreased their chl a cell'l by jQ%f while 20 Table 1.1. Physiological parameters for nitrate and ammonium-grown cultures of Actinocyclus sp. under both Fe-replete and Fe-stressed conditions (+1 standard error in parenthesis; n=3). *indicates a significant difference at p< 0.05 between NO3" and NH4+ grown cells with the asterisk placed next to the greater value, **indicates a significant difference at p < 0.05 between Fe-replete and Fe-stressed cells with the asterisk placed next to the greater value. CV=cell volume; n=growth rate. Fe-Replete Fe-Stressed Parameters N H 4 + NCh" N H 4 + CV 4990*,** 4460** 4100 4100 0*m3) (116) (69) (26) (26) chl a cell"! 5.9** 8.7*,** 3.1* 2.7 (pg cell"1) (0.2) (0.5) (0.1) (0.2) chl a:C 0.012** 0.022*,** 0.006 0.009* (wt) (0.001) (0.001) (0.001) (0.001) C:chl a 84* 45 167*,** 106** (wt) (6) (3) (9) (10) in vivo Flrchl a 0.07 0.06 0.12** 0.30*,** (ng chl a - 1) (0.01) (0.01) (0.01) (0.02) C cell"1 496* 390** 519* 285 (pg cell-1) (39) (16) (26) U) N cell"1 82 70** 80* 38 (pg cell"1) (7.2) (2.3) (2.4) (0.4) C:N 7.1 6.5 7.6 8 8* ** (mol:mol) (0.5) (0.1) (0.3) (o.i) 0.0174 0.0192 0.0109 0.0149 (h"1) (0.0022) (0.0017) (0.0015) (0.0006)) 0.0061 0.0076 (0.0003) (0.0004) Fe(int):C 330** 370** 66 38 (/imol:mol) (85) (25) (31) (8) Mn(t):C 45** 38 29 46* (/imokmol) (3.3) (2.7) (0.2) (2.3) Zn(t):C 3.4 5.8** 4.4* 1.6 (/imol:mol) (0.7) (1.0) (0.3) (0.5) 21 10.0 Fe-Replete Fe-Stressed Fig. 1.1 A) Chlorophyll a per cell and B) in vivo fluorescence per chlorophyll a vs Fe condition for both ammonium and nitrate-grown cells of Actinocyclus sp. Error bars are ± 1 standard error (n=3). 22 ammonium-grown cells only decreased their chl a celH by 40% under Fe-replete compared to Fe-stressed conditions. The chl a:C ratio was significantly higher for Fe-replete cells than for Fe-stressed cells of Actinocyclus sp., with ammonium-grown cells showing a 50% reduction and nitrate-grown cells showing a 60% reduction in chl a:C under Fe-stressed conditions (Table 1.1). Nitrate-grown cells had greater chl a:C ratios than ammonium-grown cells under both Fe treatments. in vivo Fluoresence: Chlorophyll a The in vivo Fl:chl a ratio was significantly higher under Fe-stressed conditions for both nitrate and ammonium-grown cultures of Actinocyclus sp. (Fig. LIB, Table 1.1). However, in vivo Flxhl a increased 80% for nitrate-grown cells, but only 40% for ammonium-grown cells. Carbon The carbon content decreased significantly between Fe-replete and Fe-stressed cells of Actinocyclus sp. grown on nitrate (Fig. 1.2A, Table 1.1). However, the carbon content per cell remained constant for ammonium-grown cells under both Fe conditions. The carbon content was significantly greater for ammonium-grown cells than for nitrate-grown cells under both Fe treatments. Nitrogen Similar to carbon, the nitrogen content per cell decreased significantly for nitrate-grown cells, but not for ammonium-grown cells of Actinocyclus sp., under Fe-stressed conditions (Fig. 1.2B, Table 1.1). The nitrogen content decreased by about 45% for nitrate-grown cells. Again, ammonium-grown cells had more N cell"! m a n nitrate-grown cells under both Fe treatments. Carbon: Nitrogen The C:N ratio increased significantly for nitrate-grown cells of Actinocyclus sp. from Fe-replete to Fe-stressed conditions (Fig. 1.2C, Table 1.1). C:N values of ammonium-grown cells remained constant. 23 Fig. 1.2. A) Carbon per cell, B) Nitrogen per cell, and C) Carbon per nitrogen ratio vs Fe condition for both ammonium and nitrate-grown cells of Actinocyclus sp. Error bars are ± 1 standard error (n=3). 24 Metal Quotas Iron The intracellular iron to cellular carbon ratios, (Fe(int):C), decreased significantly between Fe-replete and Fe-stressed treatments, with about an 80% reduction in this ratio (Fig. 1.3, Table 1.1). There was no significant difference between nitrate and ammonium-grown cells for either Fe treatment. Manganese Mn:C ratios were not significantly different between nitrate and ammonium-grown cells under Fe-replete conditions (Table 1.1). However, under Fe-stressed conditions, nitrate-grown cells had significantly higher Mn:C ratios than ammonium-grown cells (Table 1.1). Ammonium-grown cells had significandy greater Mn:C ratios under Fe-replete vs Fe-stressed conditions; nitrate-grown cells had greater Mn:C ratios under Fe-stressed compared to Fe-replete conditions, although not significant at p< 0.05 (p<0.10) (Table 1.1). Z i n c There was no difference in Zn:C ratios between nitrate and ammonium-grown cells under Fe-replete conditions (Table 1.1). Under Fe-stressed conditions, ammonium-grown cells had higher Zn:C ratios than nitrate-grown cells (Table 1.1). Fe-replete cells had higher Zn:C ratios than Fe-stressed cells for nitrate-grown cultures (Table 1.1). Effect of TiaiD treatment The effect of the Ti(m) treatment on Fe, Mn, and Zn quotas is illustrated in Fig. 1.4. The Ti(LTf) wash significantly removed extracellular Fe for both nitrate and ammonium-grown cultures under Fe-replete conditions (Fig. 1.4A). However, when particulate Fe values were low (as in the Fe-stressed cells), the Ti(m) reagent did not statistically alter the Fe quotas at p < 0.05. As well, the Ti(m) treatment did not significantly alter the Mn content per carbon regardless of conditions (Fig. 1.4B). Under one circumstance, the use of the TifTfl) wash significantly increased the amount of Zn retained on the filters (Fig. 1.4C). 25 Fig. 1.3. Intracellular Fe per carbon ratio vs Fe condition for both ammonium and nitrate-grown cells of Actinocyclus sp. Error bars are +.1 standard error (n=3). Fig. 1.4. A) Fe per carbon, B) Mn per carbon, and C) Zn per carbon vs Fe conditions for both ammonium and nitrate-grown cells of Actinocyclus sp. Error bars represent +.1 standard error (n=3). 1 +Ti' indicates the Ti(IJJ) reagent was used (see General Methods). 27 DISCUSSION This is the first physiological study of an oceanic diatom isolated from the NE subarctic Pacific. As the subarctic Pacific is an HNLC region where Fe plays a key role in the physiology of the indigenous phytoplankton, it is important to study oceanic phytoplankton species from this region if we are to understand how Fe may affect the dynamics of phytoplankton assemblages in this region. As pointed out by Wells et al. (1995), published reports of laboratory studies on species from field sites in question (e.g. HNLC regions) are completely absent. In this study the effect of nitrogen source and Fe-status on Actinocyclus sp. are examined, and basic biochemical composition data on this oceanic diatom are reported. Fe and Chlorophyll. Carbon. Nitrogen Chl a quotas were lower for Fe-stressed cells, presumably due to the fact that Fe is required in the synthesis of chlorophyll (Spiller et al. 1982). This is in agreement with other researchers who found reductions of chl a quotas under Fe-stress for diatoms (Glover 1977, Greene et al. 1991, Greene et al. 1992), a dinoflagellate (Doucette and Harrison 1990), cyanobacteria (Gikema and Sherman 1983, Rueter et al. 1990, Rueter and Unsworth 1991), a chlorophyte (Greene et al. 1992), and a freshwater green alga (Rueter and Ades 1987). The oceanic coccolithophore E. huxleyi is an exception, since it does not appear to reduce its chl a quota under Fe conditions limiting to growth (Chapter 2). The chl a cell"! was significantly lower for nitrate-grown cells compared to ammonium-grown cells under Fe-stress (Table 1.1). This is what would be expected, as more Fe is required to utilize nitrate, rendering cells forced to utilize nitrate more Fe-stressed than cells able to utilize ammonium. The in vivo Flxhl a ratio was found to be higher under Fe-stress, similar to other studies (Sakshaug and Holm-Hansen 1977, Gikema and Sherman 1983, Doucette and Harrison 1990, 1991). However, the nitrate-grown cells exhibited an 80% increase in this ratio (compared to 40% for ammonium-grown cells), indicating that the nitrate-grown cells 28 suffered from inefficient photosynthetic electron transfer compared to ammonium-grown cells. Nitrate-grown cells of a dinoflagellate also exhibited higher in vivo Flxhl a compared to ammonium-grown cells under Fe-stress (Doucette and Harrison 1991). Nitrogen limitation can also increase in vivo Flxhl a (Kiefer 1973, Sakshaug and Holm-Hansen 1977), as nitrogen is required as well as Fe for the synthesis of cytochromes (Pushnik et al. 1984). The nitrate concentration in the medium at the time of harvesting the Fe-stressed cells was > 30 /xM nitrate (> 10 ammonium for ammonium-grown cultures). However, nitrate uptake rates were not measured, and it is possible that the nitrate-grown cells were partially 'N-stressed' as well, due to inability of nitrate-grown cells under energy restraints (i.e. Fe-stress) to transport and assimilate nitrate. The N cell"* value remained the same for ammonium-grown cells, but decreased significantly for nitrate-grown cells under Fe-stress (Fig. 1.2B, Table 1.1), indicating that the nitrate-grown cells were not able to maintain their optimal nitrogen quotas. This was also found to occur in the red tide dinoflagellate Gymnodiniwn sanguineum, with ammonium-grown cells containing 50% more nitrogen than nitrate-grown cells (Doucette and Harrison 1991). This is in agreement with theoretical expectations, and is reflected in the increased C:N ratio for nitrate-grown cells under Fe-stress, despite a significant decrease in C quota. The greater reduction in chl a cell"!, the greater increase in in vivo Flxhl a, and the lower N cell"! for nitrate-grown cells compared to ammonium-grown cells under Fe-stressed conditions all support the theoretical predictions that cells utilizing nitrate should be more Fe-stressed than cells utilizing ammonium. Fe Quotas The intracellular Fe to cellular C ratio of the cells decreased ca. 80% from Fe-replete to Fe-stressed conditions in this experiment. However, there was no difference in the Fe(int):C ratios between nitrate and ammonium-grown cultures for either Fe treatment (Fig. 1.3, Table 1.1). The C cell"! w a s lower for the nitrate-grown cells, and it is possible that the decrease in C resulting from energy constraints on the cells cancels out any necessary increase in Fe content. Based on theoretical calculations (Raven 1988), nitrate-grown cells should 29 contain more Fe that ammonium-grown cells (1.7 times), but this comparison can only be made if the cells are growing at identical growth rates (not the case in our study) and probably only under replete energy (Fe) conditions. Other researchers have measured cellular Fe to cellular C ratios in phytoplankton, but by radiotracer techniques. Measuring metal quotas directly by cold-metal techniques allows multiple metals to be measured simultaneously. However, limitations exist, especially when attempting to measure intracellular Fe only. The TifTfl) treatment was developed for use with radioisotopes of Fe; the method should not be used for other metals nor when working with dilute cultures of extremely Fe-stressed cells. When cells are Fe-stressed, they may increase the number of Fe uptake sites on their cellular membranes, thereby increasing the likelihood of any Fe in the Ti(III) solution binding to the cell surface. In these experiments Ti(LTJ) treated cells always resulted in lower Fe values than non-Ti(III) treated cells (although not always statistically significant), with the internal values of Fe-stressed cells being <_ 75% of the total Fe values, in agreement with previous experiments of Fe-stressed cells of a diatom using radiotracer techniques (Hudson and Morel 1990). As well, my Fe-stressed cells were not severely stressed, as cell division was still taking place. However, the possibility exists that some binding of contaminant Fe in the Ti(III) wash may exist, resulting in slightly elevated intracellular Fe quotas for the Fe-stressed cells. Again, I found no adsorption of Fe onto the cellulose acetate filters, but binding to Fe uptake sites on the cells may take place, with not all of this Fe being removed from the TifTfl) treatment. Mn and Zn Quotas Under Fe-replete conditions, there was no difference in Mn or Zn quotas with nitrogen source. Under Fe-stressed conditions, nitrate-grown cells had higher Mn quotas than ammonium-grown cells (Table 1.1). This may indicate a greater requirement for Mn under Fe-stressed conditions, as the nitrate-grown cells showed greater signs of Fe-stress than the ammonium-grown cells. As well, nitrate-grown cells had higher Mn quotas under Fe-stress than under Fe-replete conditions at a significance level of p < 0.10. An increase in Mn quota under Fe-stress has also been found by other researchers; Harrison and Morel (1986) observed 30 the same phenomenon while working with a coastal diatom. It appears that this response is not solely limited to diatoms, as increased Mn quotas under Fe-stressed conditions have also been found in an oceanic coccolithophore, Emiliania huxleyi when grown on nitrate (Chapter 2). Harrison and Morel (1986) suggested that Mn uptake may be inhibited at high Fe concentrations. There could also be a real, physiological requirement for Mn under low Fe conditions that has not yet been identified. The Zn:C ratio was higher for ammonium-grown cells under Fe-stressed conditions; the opposite of what was found for Mn. No reports of Zn quotas under different nitrogen sources have been made previously. Since the Zn quota for nitrate-grown cells under Fe-stress were significantly lower than ammonium-grown Fe-stressed cells and Fe-replete nitrate-grown cells, the transport of Zn into the cell may be impaired under energy-stressed conditions, with nitrate-grown cells being more adversely affected than ammonium-grown cells. Values for Mn and Zn quotas obtained from radiotracer methods have been previously reported in the literature for two diatom species (both Thalassiosira sp., one from the Sargasso Sea), and for three diatom species (all Thalassiosira sp., one from the Sargasso Sea), respectively (Sunda and Huntsman 1983, Sunda and Huntsman 1992). For Mn:C, the values range from 22 to 32 /tmol Mnrmol C as reported in Sunda (1988/89). Actinocyclus sp. values ranged from 30 to 45 /tmol Mn:mol C under all conditions tested, lying in the upper range of previously reported values. The Zn:C ratios (3 and 7 jtmol Zn:mol C) for Actinocyclus sp. under Fe-replete conditions with EDTA present are lower than previous reports for diatoms, which range from 11 to 14 jtmol Znrmol C (Sunda and Huntsman 1992). Ecological Significance From these experiments, it would appear to be more advantageous for Actinocyclus sp. to utilize ammonium for its nitrogen requirements under low Fe conditions, because nitrate-grown cells exhibited greater signs of physiological stress than ammonium-grown cells under the Fe-stressed conditions tested (lower chl a celH, higher in vivo Fl.xhl a, lower N and C 31 cell'l). This is in agreement with theoretical predictions made regarding nitrogen and Fe, as well as supporting field hypothesis. Price et al. (1991) found that the indigenous phytoplankton from the equatorial Pacific, another HNLC region, were indeed utilizing ammonium as their primary nitrogen source. This was a mixed assemblage, but some diatoms were present (19% by abundance). Assessing which nitrogen source diatoms exclusively utilize in the field is difficult, as it is impossible to separate diatoms from other phytoplankton groups in the field, and the larger diatoms are not usually numerically abundant at Stn P. However, nitrogen uptake measurements have been made at Stn P for nitrate, ammonium, and urea, and samples were size-fractionated, with the largest size (> 18 itm) being predominantly diatoms (Varela, unpubl. data). Results from these measurements are in preparation. Since ambient nitrate concentrations are never depleted at Stn P and ammonium is present at low concentrations but supplied constantly (0.2 - 0.6 iiM within the euphoric zone; Varela, pers. comm.), it is possible that this diatom is utilizing ammonium to meets its nitrogen requirements for at least part of the time. Given the possible addition of Fe via atmospheric deposition (Martin et al. 1989, Duce and Tindale 1991), the only physiological evidence to indicate that cells utilizing nitrate may be better off than cells utilizing ammonium was that nitrate-grown cells had higher chl a cell'l and lower in vivo Fl:chl a than ammonium-grown cells. This could indicate that nitrate-grown cells may be photosynthesizing faster or more efficiently, although it did not result in a significantly faster growth rate in our experiments. In order to determine if Fe-stressed cells switch from utilizing ammonium to nitrate when they are given Fe, the measurement of ammonium and nitrate uptake rates in the presence of both nitrogen sources at ecologically relevant concentrations is necessary. My data do not refute this possibility. Field experiments have indirectly supported the hypothesis that diatoms 'switch' from ammonium to nitrate utilization when Fe is added to natural Stn P water (Martin et al. 1989, Boyd et al. submitted). Experiments conducted in field conditions with enclosures can not distinguish whether the draw-down of nitrate upon addition of Fe is due to an increase in cell numbers (caused by an alleviation of Fe-stressed growth rates, or the elimination of grazers), or a switch from ammonium to nitrate utilization, as previously 32 mentioned by Price et al. (1991). Again, the best way to address this physiological question is via controlled laboratory experiments. Ideally, Stn P water with no artificial chelators would be used (see Chapter 4). My study provides a physiological basis to explain why an oceanic diatom from the subarctic Pacific should utilize ammonium rather than nitrate for its nitrogen requirements under low Fe conditions, as well as providing basic physiological and metal composition parameters of Actinocyclus sp. Actinocyclus curvatulus is a major diatom species that settles to depth at Stn P (Takahashi 1986, Takahashi et al. 1990), and studying the ecophysiology of large diatoms may help to understand the phytoplankton dynamics occurring in the subarctic Pacific. 33 CHAPTER 2: EFFECTS OF NITROGEN SOURCE ON THE PHYSIOLOGY AND M E T A L NUTRITION OF EMILIANIA HUXLEYI GROWN UNDER DIFFERENT IRON AND LIGHT CONDITIONS INTRODUCTION This chapter examines the same Fe and nitrogen interactions as in Chapter 1, but with an oceanic coccolithophore instead of an oceanic diatom. In this way, these two very different representative species from Stn P can be compared with respect to their Fe and nitrogen nutrition. Again, cells utilizing ammonium for their primary nitrogen source should theoretically have a physiological advantage over cells utilizing nitrate, under both Fe-replete and Fe-stressed conditions. As E. huxleyi was much more difficult to Fe-stress than the oceanic diatom, the protocol for achieving Fe-stressed cells was different, and there are three instead of two Fe treatments in these experiments. As well as being of interest in the NE subarctic Pacific, Emiliania huxleyi is quite ubiquitous, occurring in almost all of the world's ocean except polar waters. Most noted are the very large blooms of this calcifying organism in the North Atlantic, spanning areas of 250,000 km 2 or more (Holligan et al. 1993). This organism is receiving worldwide attention as a potential carbon sink due to its coccolith production and loss, as well as its influence on global climate via DMSP production and carbon sequestering (Keller et al. 1989, Flynn 1990). In the North Pacific, large blooms of this species (or any other phytoplankter) do not occur, but as mentioned in the General Methods section, E. huxleyi is a member of the most abundant size class present in the NE subarctic Pacific (Booth et al. 1982, Taylor and Waters 1982). As well, cells < 5.0 in diameter can be responsible for two-thirds of the phytoplankton carbon biomass at Stn P, with E. huxleyi belonging in this size class (Booth 1988). E. huxleyi is also the most abundant coccolithophore found at Stn P (Honjo and Okada 1974). 34 This is the first report on physiological, biochemical, and metal composition parameters obtained from an oceanic coccolithophore isolated from the NE subarctic Pacific. In this study, I examined both the effects of nitrogen source and Fe condition on E. huxleyi's physiological status, in an attempt to determine whether it is advantageous for this organism to utilize ammonium rather than nitrate under low Fe conditions. Experiments are also presented on the effects of irradiance on both nitrate and ammonium-grown cells, since it is under low irradiance levels that competition for photosynthetically derived energy may become crucial, similar to Fe-stressed conditions. Metal quotas were again measured using cold-metal, trace-metal clean techniques, allowing for the simultaneous measurement of multiple metals. Mn, Zn, and Cu were measured along with Fe to determine if their cellular levels varied with intracellular Fe levels, perhaps indicating additional utilization of these metals under Fe-stress. The physiological parameters measured were growth rate, cell volume, chlorophyll a quota, in vivo fluorescence per chlorophyll a, nitrogen quota, and carbon quota. MATERIALS AND METHODS Cultures of E. huxleyi were maintained in natural Stn P water to retain the organism's ability to form coccoliths. Laboratory data on this species were acquired shortly after isolation, with these particular experiments being conducted approximately one year after isolation. Experiments were carried out using microwave-sterilized (Keller et al. 1988), Chelex-treated Aquil synthetic ocean water, SOW, (Price et al. 1988/89). The dissolved Fe remaining in the SOW after it was passed through the Chelex column at a rate of < 1 ml min'i was <0.8 nmol kg~l, as measured by Yang (1993). Due to a limited supply of natural Stn P water, artificial seawater was used for these particular experiments. 35 Macronutrient stocks were made up in Nanopure water and Chelex-treated at pH 3.0 for 100% Fe removal (R. Chretien unpubl. data). Macronutrients were added to achieve a final concentration of 30 tiM of either N O 3 " or NH4"*", and 5 ttM PCty"3. This level of ammonium addition had no detrimental effect on the organism's growth rate or overall appearance. EDTA was added to yield a final concentration of 10 itM. Final metal concentrations added were: 8.0 nM Zn, 23 nM Mn, 1.0 nM Cu, 2.5 nM Co, 100 nM Mo, and 10 nM Se. 100 nM Fe final concentration was added for the Fe-replete cultures, and no Fe was added for the other treatments. Thiamine and biotin were added to give a final concentration of 1 x 10"^  g L"l and 5 x 10"^  g L~l , respectively. Fe Experiments Fe-Replete Experiments Semi-continuous cultures were grown in quadruplicate with either nitrate or ammonium as the nitrogen source in acid-cleaned 3 L polycarbonate Fernbach flasks. The flasks were equipped with Teflon® tubes and silicone stoppers. Cultures were not mechanically stirred as this knocked off many of the coccoliths, but they were mixed once a day by swirling the flasks by hand. Cultures were maintained at 16°C on a 14:10 light:dark cycle with Vitalite fluorescent lights. The irradiance, as measured by a Biospherical Instruments model QSL-100 sensor, was 150 |j,mol m~2 s"l which was saturating for growth. Cultures were grown for 10 generations and harvested in log phase. Fe-Stressed Experiments Cultures were grown in triplicate with either nitrate or ammonium as the nitrogen source in identical conditions as above. However, when E. huxleyi was semi-continuously diluted with 'no Fe' medium (<0.8 nmol kg"l), no physiological evidence of Fe limitation was observed. Therefore, I devised a culture protocol that resulted in Fe-limited cells after several transfers, similar to procedures used when comparing macronutrient deplete vs replete cells. Cultures were grown semi-continuously and sampled after 9 days (3 dilutions). These cells were in medium containing <0.8 nmol kg"l Fe with 10 tiM EDTA, but they showed 36 little or no signs of Fe-stress (no drastic decrease in growth rate). Cells from these same cultures were again diluted and allowed to grow up until early stationary phase was reached as determined by cell counts. At this time, all macro- and micronutrients except Fe were added directly to the culture flasks (no new medium was added), forcing the cell number to increase with minimal additions of Fe to the medium (via contamination). This was repeated until the addition of macronutrients caused no increase in cell numbers (2 additions with macro-and micronutrients, one addition with N and P only). These 'Fe-stressed' cultures represent cells whose cell division was halted due to Fe limitation. After sampling, a final concentration of 5 nM Fe was added to the cultures and cell division resumed. The final cell densities of the Fe-stressed cultures were ca. 6 x 10^  cells mH. Growth rates were calculated from linear regressions of plots of natural log of cell number vs time. Cell numbers and cell volumes were measured on a Coulter Counter model TAIL The pH was decreased to 5 with HC1 before counting to dissolve the coccoliths and prevent interference, as the coccoliths overlapped with some of the same channels as the cell counts. Chlorophyll a concentrations were determined by in vitro fluorometry (Parsons et al. 1984). Samples for particulate organic carbon and nitrogen were collected on precombusted 13 mm Gelman A/E filters and analyzed on a Carlo Erba CHN analyzer. Samples with and without coccoliths were measured. The carbon content of cells without coccoliths was determined by dissolving the coccoliths with HC1 (pH 5) and filtering. This procedure did not appear to lyse the cells, as the nitrogen per cell remained the same for both non-acidified and acidified samples. Metal quotas (Fe, Mn, Zn, Cu) were measured using non-radiotracer, trace-metal clean techniques, as described in the General Methods section. Acid-treated, Ti(IQ) washed filter blanks were subtracted from the identically treated culture samples. However, even though the acid and Ti(IU) treatment did not increase the filter blanks, the treatment did cause an increase in the Fe retained on the filters for culture samples of Fe-stressed cells. There are 3 possible reasons for this increase: 1) Any contaminating Fe present in the Ti(HI) reagent 37 could have bound to empty Fe-uptake sites on the cell surface of the Fe-stressed cells, 2) The extra particulate matter present on culture samples prevented complete rinsing with the Chelex-treated Stn P water, and 3) Fe-stressed cells may be producing organic compounds, possibly to aid in Fe acquisition and transport, which may cause retention of Fe onto the filters. Due to these complications at low particulate Fe concentrations (Fe-stressed cells), I have estimated the intracellular Fe using Hudson and Morel's (1990) finding that 75 - 90% of the total Fe in Fe-stressed diatom cultures was intracellular. As well, at external Fe concentrations below 100 nM, the Ti(]Tf) treatment does not appear to be necessary because it did not remove any extracellular Fe at very low external Fe concentrations (Sunda et al. 1991). All statistical comparisons were done using a Student's t-test at the 95% confidence level (p < 0.05). Comparisons were made between ammonium and nitrate-grown cultures for a given Fe treatment. Irradiance Experiments Cultures were grown in triplicate in Silinized-treated (SurfaSil Pierce Chemicals) 50 ml glass tubes (Price et al. 1988/89) with either 30 itM nitrate or ammonium as the nitrogen source. Medium preparation and additions were identical to the Fe-replete experiments (100 nM Fe, 10 iiM EDTA). Cultures were maintained in quasi-steady state (diluted at 1/3 of the maximal fluorescence) for a minimum of 6 generations using Vitalite fluorescent tubes, at irradiances of 24, 40, 70, 119, and 176 ttmol m~2 s~*. Growth rates were obtained by regressions of the natural log of in vivo fluorescence vs time. All other methods were the same as for the Fe experiments described above. 38 RESULTS Fe Experiments Growth rate The specific growth rates of nitrate and ammonium-grown cells were not significantly different under the high Fe (100 nM Fe) treatment (Table 2.1). Under the low Fe (0.8 nM Fe) treatment, nitrate-grown cells grew significantly faster than ammonium-grown cells (Table 2.1). For the Fe-stressed cultures, cell numbers were increased until the external Fe concentration reached levels limiting to growth, resulting in no cell division. After sampling, a final concentration of 5 nM Fe was added to the cultures, and cell division resumed at a growth rate of 0.0128 and 0.0095 h"* measured over a 2 day period for ammonium and nitrate-grown cultures, respectively (Table 2.1). Ammonium-grown cells grew significantly faster than nitrate-grown cells after this Fe addition. Cell volume The cell volume, CV, of nitrate and ammonium-grown cells was not statistically different under the high Fe treatment (Fig. 2.1, Table 2.1). As well, nitrate and ammonium-grown cells had similar cell volumes under the low Fe treatment (Fig. 2.1, Table 2.1). However, nitrate-grown cells had significantly smaller cell volumes than ammonium-grown cells under Fe-stressed conditions (p=0.007), with the mean cell volumes being 24.9 and 33.8 jtm3, respectively. In general, the CV of both nitrate and ammonium-grown cells were larger under the high Fe treatment than under the low Fe treatment (Fig. 2.1). However, the cell volume of ammonium-grown cells between the low Fe and Fe-stressed treatments remained identical, whereas the CV of nitrate-grown cells decreased 30% under Fe-stressed conditions (Fig. 2.1, Table 2.1). 39 Table 2.1. Physiological parameters for nitrate and ammonium-grown cultures of Emiliania huxleyi under 3 Fe treatments: high Fe (100 nM Fe), low Fe (0.8 nM Fe), and Fe-stressed conditions (< <0.8 nM Fe). +.1 Standard error is given in parenthesis (n=4 for 100 nM Fe, n=3 for other Fe treatments), "indicates a significant difference at p<0.05 between NO3" and NH4"1" grown cells for a particular Fe treatment with the asterisk placed next to the greater value. ^Growth rates achieved over a 2 day period after 5 nM Fe was added to the Fe-stressed cultures. /«=growth rate; CV=cell volume; C(t)=total cell carbon, including coccoliths; C(a)=cell carbon excluding coccoliths (coccoliths removed by acidification, see methods). High Fe Low Fe Fe-Stressed (lOOnMFe) (0.8nMFe) (<<0.8nMFe) Parameters N H 4 + NOV N H 4 + NOV N O V A* , 0.0175 0.0183 0.0134 0.0191* 0 0 (h-1) (0.0010) (0.0010) (0.0002) (0.0010) #[0.0128]* #[0.0095] (0.0006) (0.0007) CV 53.0 48.9 36.6 34.9 33.8* 24.9 GtnT3) (1.6) (2.5) (0.6) (0.7) (1.5) (0.9) chl a cell"1 120 120 110 110 130* 110 (fg celT1) (8) (3) (6) (13) (3) (4) chl a CV" 1 2.2 2.5 3.1 3.2 4.0 4.5* (fg urn'3) (0.2) (1.0) (0-3) (0.3) (0.2) (0.2) N cell"1 1.9 1.8 1.4 1.5 1.6* 1.3 (pg cell-1) (0.1) (0.1) (0.2) (0.1) (0.1) (0.1) NCV-1 36 37 38 43 47 52 (fg /tnT3) (2) (1) (6) (1) (2) (4) C(t) cell'1 31.9* 27.6 25.2 21.2 28.3* 13.4 (pg cell"1) (0.8) (1.1) (6.2) (3.4) (1.3) (0.1) C(t) C V 1 0.60 0.56 0.69 0.61 0.84* 0.52 (pg /tnT3) (0.01) (0.02) (0.17) (0.09) (0.02) (0.02) C(a) cell"1 13.9 13.9 17.7 16.5 19.8* 12.9 (pg cell'1) (0.3) (0.9) (2.6) (1.8) (1.1) (0.8) C(a) CV" 1 0.26 0.28 0.48 0.47 0.59 0.53 (pg /im"3) (0.01) (0.03) (0.07) (0.04) (0.02) (0.01) C(a):N 8.3 8.9 14.1 12.5 14.0 11.2 (mol:mol) (0.3) (0.7) (0.5) (1.3) (0.8) (1.2) in vivo Fl:chl a 0.30 0.28 0.32 0.35 0.35 0.36 (ng chl a - 1 ) (0.02) (0.01) (0.01) (0.03) (0.03) (0.01) C(a):chl a 120 120 160 150 150 120 (wt) (9) (11) (46) (5) (11) (5) 40 60 High Fe Low Fe Fe-Stressed (100 nM Fe) (0.8 nM Fe) (<<0.8 nM Fe) Fig. 2.1. Cell volume vs Fe treatment for both nitrate and ammonium-grown cells of E. huxleyi. Error bars represent ± 1 standard error (n=4 for 100 nM Fe, n=3 for other Fe treatments). 41 Biochemica l composition Chlorophyll a Chl a cell - 1 w a s n o t statistically different for nitrate and ammonium-grown cells under the high or low Fe treatment, but it was significantly higher for ammonium-grown cells under the Fe-stressed conditions (Table 2.1). However, when normalized to cell volume, chl a C V - 1 , m e c n | a content was significantly higher for nitrate-grown cells than for ammonium-grown cells under Fe-stressed conditions (Fig. 2.2A, Table 2.1). There was no significant difference in chl a CV"! between nitrate and ammonium-grown cells under the other Fe conditions (Fig. 2.2A, Table 2.1). Because of the large difference in cell volume between nitrate and ammonium-grown Fe-stressed cells, all physiological parameters were normalized to cell volume. in vivo Fluoresence: Chlorophyll a The in vivo Flxhl a, a measure of the efficiency of photosynthetic electron transport, was not significantly different between nitrate and ammonium-grown cells for any of the Fe treatments (Table 2.1). Nitrogen The nitrogen per cell volume, N CV"!, 0 f nitrate and ammonium-grown cells was not significantly different under any of the three Fe treatments, including the condition when Fe was limiting cell division (Fig. 2.2B, Table 2.1). As well, cells under Fe-stressed conditions had higher N CV"! values than cells under the other Fe treatments (Fig. 2.2B, Table 2.1). Carbon The total carbon content per cell volume, C(t) CV"!, w n i c h includes coccolith carbon, was not statistically different for nitrate and ammonium-grown cells under the high and low Fe treatments (Table 2.1). Similarly, cellular carbon excluding coccolith carbon (C(a) CV"!) was not different for ammonium and nitrate-grown cells under these Fe treatments. As well, the amount of coccolith carbon, estimated by subtracting C(a) from C(t), remained fairly constant regardless of nitrogen source or Fe treatment. The exception 42 (100 nM Fe) (0.8 nM Fe) (<<0.8 nM Fe) Fig. 2.2. A) Chlorophyll a per cell volume and B) Nitrogen per cell volume vs Fe treatment for both nitrate and ammonium-grown cells of E. huxleyi. Error bars represent +1 standard error (n=4 for 100 nM Fe, n=3 for other Fe treatments). 43 was nitrate-grown cells under Fe-stress. However, due to the very small size of these cells, most of the coccoliths probably were lost through the filter. Carbon: Chlorophyll a The C(a):chl a ratio was not statistically different for nitrate and ammonium-grown cells under any of the Fe conditions (Table 2.1, Fig. 2.3). The amount of chlorophyll a per carbon remained relatively stable for all cultures under the various Fe treatments, especially for the nitrate-grown cells (Table 2.1, Fig. 2.3). Metal composition Iron The intracellular Fe per CV, Fe(int) CV"!, was not significantly different between nitrate and ammonium-grown cells under any of the Fe treatments tested (Table 2.2). When Fe quotas were normalized to carbon, Fe(int):C(t) or Fe(int):C(a), there was still no significant difference between nitrate and ammonium-grown cells under the high Fe and the Fe-stressed treatments, but ammonium-grown cells had higher Fe(int):C(t) and Fe(int):C(a) than nitrate-grown cells under the low Fe treatment (Fig. 2.4A, Table 2.3). Manganese The Mn CV"! w a s m e same for nitrate and ammonium-grown cells under the high and low Fe treatments (Table 2.2). However, under Fe-stressed conditions, nitrate-grown cells had significantly higher Mn levels per CV than ammonium-grown cells (Table 2.2). When normalized to carbon, the same relationships held and were even more pronounced (Fig. 2.4B, Table 2.3). Zinc The Zn CV"! w a s m e same f o r nitrate and ammonium-grown cells under the high Fe treatment (Table 2.2). Similarly, when normalized to C, ammonium and nitrate-grown cells were not different under high Fe or under Fe-stressed conditions (Table 2.3). 44 Table 2.2. Metal quotas normalized per cell and per cell volume for nitrate and ammonium-grown cultures of Emiliania huxleyi under 3 Fe treatments: high Fe (100 nM Fe), low Fe (0.8 nM Fe), and Fe-stressed (< <0.8 nM Fe) conditions. +.1 standard error is given in parenthesis (n=4 for 100 nM Fe, n=3 for other Fe treatments), "indicates a significant difference at p<0.05 between NO3" and N H 4 + grown cells for a particular Fe treatment with the asterisk placed next to the greater value. CV=cell volume; Fe(int)=intracellular Fe; Fe(t)=total Fe (both intra-and extracellular); amol=1 x 10"18 moles; N/A=data not available. The Fe(int) values for the low Fe and Fe-stressed treatments are 75-90% of the measured Fe(t) (see methods). High Fe Low Fe Fe-S tressed (lOOnMFe) (0.8nMFe) (<<0.8nMFe) Parameters N H d + N C V N H 4 + NOV N H d + NO," Fe(int) cell"1 330 210 94-110 49-59 9-11 14-17 (amol cell"1) (38) (44) (26-32) (4-5) (3) (6-7) Fe(int) CV" 1 6.3 4.3 2.5-3.1 1.4-1.7 0.27-0.33 0.57-0.68 (amol ftm"3) (0.6) (1.0) (0.7-0.8) (0.08-0.09) (0.07-0.08) (0.02-0.03) Mn cell - 1 27 30 24 24 32 31 (amol cell"1) (1) (1) (2) (1) (0.4) (1.2) M n C V " 1 0.52 0.61 0.65 0.69 0.94 1.2* (amol fim' 3) (0.03) (0.05) (0.07) (0.03) (0.03) (0.1) Zn cell"1 45 41 N/A N/A 61 15 (amol cell"1) (4) (5) (3) (1) ZnCV" 1 8.4 8.3 N/A N/A 1.8 0.6 (amol /tm"3) (0.6) (0.1) (0.9) (0.04) Cu cell"1 4.4 2.8 2.2 3.6 3.1 2.7 (amol cell"1) (0.7) (1.2) (0.02) (0.80) (1.2) (0.3) C u C V " 1 0.08 0.06 0.06 0.10* 0.09 0.11 (amol /xm"3) (0.01) (0.02) (0.01) (0.02) (0.03) (0.01) 45 Table 2.3. Metal quotas normalized to cell carbon for nitrate and ammonium-grown cultures of Emiliania huxleyi under 3 Fe treatments: high Fe (100 nM Fe), low Fe (0.8 nM Fe), and Fe-stressed (< <0.8 nM Fe) conditions. +.1 standard error is given in parenthesis (n=4 for 100 nM Fe, n=3 for other Fe treatments), '"indicates a significant difference at p< 0.05 between NO3" and NH4 + grown cells for a particular Fe treatment with the asterisk placed next to the greater value. Fe(int)=intracellular Fe; C(t) = total cell carbon including coccoliths; C(a)=cell carbon excluding coccoliths (coccoliths removed by acidification, see methods); N/A=data not available. The Fe(int) values for the low Fe and Fe-stressed treatments are 75-90% of the measured Fe(t) (see methods). High Fe Low Fe Fe-Stressed (lOOnMFe) (0.8 nM Fe) (<<0.8nMFe) Parameters N H 4 + NOV N O , ' NHd  + NO," Fe(int):C(t) 130 90 56-68* 33-40 3.9-4.7 12-15 (/imol:mol) (14) (21) (1.3-1.5) (2.5-2.9) (1.0-1.2) (5.5-6.6) Fe(int):C(a) 280 160 66-80* 38^6 5.6-6.7 12-14 /x)j.mol:mol) (32) (54) (2.3-2.8) (3.6-4.3) (1.4-1.7) (5.2-6.2) Mn:C(t) 10 13 15 15 13 27* (/unol:mol) (0.7) (0-8) (5.7) (1.1) (0.5) (1.2) Mn:C(a) 25 26 17 17 19 27* (/imol:mol) (2.5) (1.5) (3.3) (1.0) (0.9) (2.3) Zn:C(t) 17 18 N/A N/A 26 13 (/imohmol) (1.5) (1.9) (14) (0.9) Zn:C(a) 41 30 N/A N/A 38 13 (/imohmol) (7.2) (5.6) (21) (0.7) Cu:C(t) 1.2 1.3 1.4 2.3 1.4 2.4 (/tmohmol) (0.4) (0.6) (0.3) (0.2) (0.6) (0.2) Cu:C(a) 3.8 1.4 1.3 2.3 2.0 2.4 (/tmohmol) (0.5) (0.5) (0.4) (0.5) (0.8) (0.1) 46 250 High Fe Low Fe Fe-Stressed (100 nM Fe) (0.8 nM Fe) (<<0.8 nM Fe) Fig. 2.3. Carbon (acidified to remove coccoliths) per chlorophyll a vs Fe treatment for both nitrate and ammonium-grown cells of E. huxleyi. Error bars represent +.1 standard error (n=4 for 100 nM Fe, n=3 for other Fe treatments). 47 High Fe Low Fe Fe-Stressed (100 nM Fe) (0.8 nM Fe) (<<0.8 nM Fe) Fig. 2.4. A) Intracellular Fe per total carbon, B) Mn per total carbon, and C) Cu per total carbon vs Fe treatment for both nitrate and ammonium-grown cells of E. huxleyi. Error bars represent ± 1 standard error (n=4 for 100 nM Fe, n=3 for other Fe treatments). 48 Copper The Cu CV"1 was not different between nitrate and ammonium-grown cells under high Fe or under Fe-stressed conditions (Table 2.2). However, nitrate-grown cells had higher Cu CV"1 values than ammonium-grown cells under the low Fe treatment (Table 2.2). The same relationships held true when normalized to carbon (Fig. 2.4C, Table 2.3). Irradiance Experiments Growth rate The growth rates of both nitrate and ammonium-grown cells increased with irradiance (Fig. 2.5A). At the lowest irradiance level which supported growth (24 ttmol m"2 s_l), nitrate-grown cells had significantly higher growth rates than ammonium-grown cells (Fig. 2.5A). At all other irradiance levels the growth rates of nitrate and ammonium-grown cells were not statistically different. Cell volume The cell volume of both nitrate and ammonium-grown cells was larger under high (and growth saturating) irradiances than under low irradiances (Fig. 2.5B). Under the two lowest irradiance levels there was no significant difference in cell volume between nitrate and ammonium-grown cells (Fig. 2.5B). However, ammonium-grown cells were significantly larger than nitrate-grown cells at growth saturating irradiances. Chlorophyll a Chl a CV"1 increased with decreasing irradiance, with the exception of the lowest irradiance level, where chl a CV" 1 decreased (Fig. 2.5C). At 47 and 70 itmol m"2 s"*, the chl a C V - 1 was significantly greater for nitrate-grown cells compared to ammonium-grown cells (Fig. 2.5C). Fig. 2.5. A) Specific growth rate, B) Cell volume, and C) Chlorophyll a per cell for both nitrate and ammonium-grown cells of E. huxleyi vs irradiance. Error bars represent +1 standard error (n=3). 50 DISCUSSION This is the first study to report basic physiological and multi-element metal composition parameters on an oceanic coccolithophore isolated from the NE subarctic Pacific. As well, the effect of nitrogen source on the basic physiology of an oceanic coccolithophore has not previously been examined, nor the interactions that Fe or irradiance may have in conjunction with nitrogen source. Specific results from the experiments will be discussed first, followed by discussions pertaining to the ecological scenario at Stn P. Effect of N source under Fe-replete conditions Under the Fe-replete treatment of 100 nM Fe in the Fe experiments, there appeared to be no physiological differences between nitrate and ammonium-grown cells in any of the parameters that were measured. Other researchers have previously found differences in growth rates between nitrate and ammonium-grown cells under saturating conditions. Thompson et al. (1989), using a marine diatom (Thalassiosira pseudonana) under saturating light and Fe-replete conditions, found that the growth rates of ammonium-grown cells were significantly higher (8%) than the growth rates of nitrate-grown cells. Similarly, Levasseur et al. (1993) found higher growth rates for ammonium-grown cells than for nitrate-grown cells under the same conditions. However, an oceanic diatom isolated from the NE subarctic Pacific was found to have no difference in the growth rates of nitrate and ammonium-grown cells under Fe-replete conditions (Chapter 1). Despite the possible energetic advantages, higher growth on ammonium does not always seem to be the case. Levasseur et al. (1993) also found that nitrate-grown cells had lower nitrogen quotas than ammonium-grown cells for the diatom Thalassiosira pseudonana under saturating light and Fe conditions. This may have been due to the lower energetic cost of ammonium transport and utilization compared to nitrate. In my Fe experiments, however, no such difference was observed under Fe-replete conditions; nitrate-grown cells were able to maintain similar nitrogen quotas compared to ammonium-grown cells. Thompson et al. (1989) found 51 that nitrate-grown cells had 21% lower carbon quotas than ammonium-grown cells, and they attributed this to the possibility that competition for reductant may reduce the carbon quotas (and growth rate) of nitrate-grown cells. However, again, nitrate-grown cells of E. huxleyi maintained the same carbon quotas, cell volume, chlorophyll, and nitrogen levels per cell volume as ammonium-grown cells under Fe-replete conditions (Table 2.1, Fig. 2.2). Similar to the physiological parameters, there was no difference in the metal composition of nitrate and ammonium-grown cells under Fe-replete conditions (Table 2.3). Despite the prediction that nitrate-grown cells should maintain higher Fe quotas than ammonium-grown cells under saturating conditions (Raven 1988), I did not find this in the Fe-replete cultures, even though the growth rates were similar. Sunda and Huntsman (1995) also did not find a difference in Fe(int):C ratios between nitrate and ammonium-grown cultures of E. huxleyi (their isolate was from the Gulf of Mexico). This expected relationship was also absent in Fe-replete cultures of an oceanic diatom from the NE subarctic Pacific (Chapter 1). I can compare my Mn, Zn, and Cu quotas with the few studies that have reported these parameters previously. This is the first study, however, to measure multiple metals simultaneously on an oceanic coccolithophore. The range of Mn:C(t) values that E. huxleyi maintained under replete conditions was between 10 to 14 p.mol Mn:mol C(t) (Fig. 2.4B, Table 2.3). The previously reported Mn:C ratios are for two diatom species, both Thalassiosira sp., and range from 22 to 32 jtmol Mn:mol C under saturating conditions maximal for growth (Sunda 1988/89). The oceanic diatom Actinocyclus sp. maintained a Mn:C ratio of 40 fimol Mn:mol C for Fe-replete conditions (Chapter 1). It appears then, that E. huxleyi has a lower requirement for Mn than any other previously reported species, including one diatom from the subarctic Pacific. This is in agreement with the finding of Brand et al. (1983); that oceanic coccolithophores have a low requirement of Mn for growth relative to other phytoplankton. The Zn:C ratio maintained by Fe-replete cells of E. huxleyi was ca. 17 /xmol Zn:mol C(t) (Table 2.3). Previous reports of Zn:C are from 3 diatom species (all Thalassiosira sp.) and 2 isolates of E. huxleyi (one from the Sargasso Sea, the other from the Gulf of Mexico), and range from 11 to 14 for the diatoms and as low as 1 timol Zn:mol C for the E. huxleyi isolates (Sunda and Huntsman 1992). An oceanic diatom from the NE subarctic Pacific maintained a ratio of 3 to 7 itmol Zn:mol C under Fe-replete conditions (Chapter 1). Our E. huxleyi from the NE subarctic Pacific appears to have a requirement for Zn that lies in the upper range of previously reported values, being at least double the requirement for the diatom from the same location. There have been no recently published estimates of Cu:C ratios in phytoplankton. However, Price and Morel (1994) report two studies on Chlorella sp. with the estimated values being 0.8 and 9 itmol Cu:mol C for 90% of growth (Walker 1953, Manahan and Smith 1973). My values for E. huxleyi are between 1 and 2 iimol Cu:mol C(t), lying within the range of previously reported values. The intracellular Fe:C ratios as a function of growth for this isolate of E. huxleyi will be reported in detail elsewhere (Chapter 5). Effect of N source under Fe-stressed conditions Theoretically, the competition for reductant should become more severe under Fe-stressed conditions, and therefore ammonium-grown cells should be able to maintain even higher growth rates, nitrogen quotas and carbon quotas, than nitrate-grown cells. Rueter and Ades (1987) examined the possibility of Fe-stressed cells being limited by reducing power using cultures of Scenedesmus quadricauda. They found that under Fe-stressed conditions, ammonium-grown cultures had higher carbon fixation rates than nitrate-grown cells under a range of irradiance levels (ca. 20 - 180 timol m~2 s"1). This observation along with other information allowed them to conclude that Fe-stressed cells can exhibit the same response as energy-limited cells (Rueter and Ades 1987). However, in my Fe experiments, ammonium-grown cells of E. huxleyi did not show signs of any energy advantage over nitrate-grown cells under Fe-stressed conditions. For example, nitrate-grown cells maintained the same or greater chlorophyll a, in vivo fluorescence per chlorophyll a, nitrogen and carbon levels as 53 ammonium-grown cells under Fe-stressed conditions (Fig. 2.2, Table 2.1). This was primarily the result of the nitrate-grown cells substantially reducing their cell volumes under Fe-stress (Fig. 2.1). G r o w t h Rate (Fe-stress vs low-light) Since Fe-stress could lead to energy-limited cells similar to light-limited cells, it is interesting to compare the effects of low irradiance levels on nitrate and ammonium-grown cells (Fig. 2.4). Under low irradiance levels where the competition for reductant would be most severe, nitrate-grown cells of E. huxleyi grew significantly faster than ammonium-grown cells, in contrast to what would be theoretically expected (Fig. 2.4A). In the Fe experiments, before the cessation of cell division by Fe-stress was achieved, nitrate-grown cells were also growing faster than ammonium-grown cells (data not shown) as was the case for the 0.8 nM Fe treatment. The smaller cell volume of nitrate-grown cells compared to ammonium-grown cells could have partially accounted for the faster division rate (lower requirement of C, N, metals, etc.), but why this was also found in the irradiance experiments when the cell volumes were identical under low light is puzzling. C e l l V o l u m e (Fe-stress vs low-light) The most contrasting finding between the low irradiance cultures and the Fe-stressed cultures was that no difference in cell volume was found between nitrate and ammonium-grown cells in the low irradiance cultures, whereas there was a large difference in cell volume between nitrate and ammonium-grown cultures under Fe-stress (Fig. 2.2; Fig. 2.4B). The decrease in cell volume of nitrate-grown cells and not ammonium-grown cells in response to severe Fe-stress in the Fe experiments indicates that somehow the nitrate-grown cells were more severely affected by Fe-stress than the ammonium-grown cells, and that more than just photosynthetically derived products and energy may have been the cause of this change. Fe-stress will also affect non-photosynthetic energy deriving processes such as respiration, as well as possibly hindering the synthesis of nitrate and nitrite reductase (although a difference on the scale of atoms of Fe would probably not cause a difference). Somehow the ammonium-grown cells under Fe-stress did not require a reduction in cell volume to meet their metabolic needs, whereas the nitrate-grown cells acclimate to Fe stress by reducing their cell volume. A decrease in cell volume supports the prediction made by Hudson and Morel (1990) that oceanic phytoplankton cannot increase their Fe transport kinetics, and consequently their only means of acclimation to Fe-stressed conditions is a reduction in Fe-requirement or a reduction in cell size. This reduction in cell volume in response to Fe-stress has also been found to occur in a dinoflagellate (Doucette and Harrison 1990), but nitrate was the only nitrogen source used. The only other study to-date that has examined the effects of both nitrate and ammonium on cell volume under Fe-stress is for an oceanic diatom, and no difference in cell volume was found under Fe-stress between nitrate and ammonium-grown cells (Chapter 1). C h l o r o p h y l l a quotas (Fe-stress vs low-light) The difference in chlorophyll a quotas between nitrate and ammonium-grown cultures in the irradiance experiments mimic the results obtained in the Fe experiments. Under two of the sub-saturating irradiance levels (45 and 70 ttmol m~2 s~l), nitrate-grown cells maintained higher chlorophyll a per cell volume ratios than ammonium-grown cells, similar to the case found under Fe-stress (Fig. 2.4C, Fig. 2.2A). Under low irradiance, where the competition for reductant is the most crucial, it is surprising to observe nitrate-grown cells mamtaining higher chlorophyll a levels than ammonium-grown cells. However, since nitrate-grown cells would need more reductant to transport and utilize their nitrogen source, they may require more chlorophyll a than ammonium-grown cells to meet their needs. Thompson et al. (1989) came to the conclusion that nitrate-grown cells may be more efficient in the production of reductant than ammonium-grown cells, and nitrate-grown cells could achieve this by being more efficient in light absorption or by improving their quantum yield in some other way. One of these ways may be to increase their chlorophyll levels. The same arguments can be applied to the fact that nitrate-grown cells of E. huxleyi under Fe-stress had higher levels of 55 chlorophyll than ammonium-grown cells. Apparently the expected additional stress of Fe limitation on nitrate-grown cells does not severely affect the cell's ability to synthesize chlorophyll relative to ammonium-grown cells. The generally expected sign of decreased chlorophyll quotas under Fe-stress that has been found in other studies (Glover 1977, Doucette and Harrison 1990, Greene et al. 1991, Rueter and Unsworth 1991, Greene et al. 1992) was not evident in E. huxleyi (Fig. 2.2A, Table 2.1). Despite a greater than 10 times reduction in the cellular Fe quota, Fe-stressed cells maintained relatively stable C:chl a ratios (Table 2.1). This oceanic coccolithophore appears truly unique in this regard. Comparing E. huxleyi to another phytoplankter from the subarctic Pacific, the oceanic diatom Actinocyclus sp., the chlorophyll levels decreased significantly under Fe-stress in the diatom (Chapter 1), whereas they did not decrease in the oceanic coccolithophore. E. huxleyi must have some unique metabolic characteristics which allow it to maintain chlorophyll synthesis under severe Fe-stress (when Fe was limiting cell division completely). This may be one of the ways in which this organism survives at Stn P continually. M e t a l quotas The only differences in metal quotas between nitrate and ammonium-grown cells when cell division had just ceased due to Fe-stress, was that nitrate-grown cells had significantly higher Mn:C ratios than ammonium-grown cells (Table 2.3). Both nitrate and ammonium-grown cultures in the Fe-stressed treatment were exposed to greater Mn levels than in the other two Fe treatments due to the addition of Mn in the process of inducing Fe-stress. However, ammonium-grown cells did not increase their cellular Mn levels, whereas nitrate-grown cells did (Fig. 2.4B, Table 2.2, Table 2.3). One factor that could possibly explain this, in part, was that the nitrate-grown cells decreased their cell volumes whereas the ammonium-grown cells did not. This reduction in cell volume would increase the surface area to volume ratio, possibly resulting in more surface uptake sites per volume than ammonium-grown cells. The phenomenon of increased cellular Mn levels under Fe-stress for 56 nitrate-grown cells has been found previously; Fe-limited cells of Thalassiosira weissflogii grown on nitrate had higher Mn quotas than Fe-replete cells at fixed concentration of free Mn ion (Harrison and Morel 1986). As well, an oceanic diatom from the NE subarctic Pacific also exhibited this response (Chapter 1). In my study, the nitrate-grown cells under Fe-stress contained more Mn and chlorophyll a per carbon than ammonium-grown cells, indicating that photosystem n may somehow be more active in Fe-stressed nitrate-grown cells than Fe-stressed ammonium-grown cells. Ecological Relevance C o m p a r i s o n of E. huxleyi isolates f r o m different locales It is known that genetic variation is prevalent among E. huxleyi isolates from different locations (Brand 1982, Young and Wesbrook 1991, van Bleijswijk et al. 1991), and this may explain some of the variation in physiological characteristics observed between isolates from different areas of the ocean. Growth rate can be an indicator of the overall physiology of a cell, and it is a parameter frequently measured. Although there are differences in culturing conditions, my E. huxleyi isolate from the subarctic Pacific has a much lower maximal growth rate than other reported isolates. Paasche and Klaveness (1970) found that the coccolith-forming cells of E. huxleyi from Oslo fjord, Norway had a maximum growth rate of 1.68 doublings per day (21°C, continuous light). Similarly, Brand (1982) examined 73 clones of E. huxleyi isolates from the Sargasso Sea and the Gulf of Maine, and found growth rates ranging from 1.5 to 1.7 divisions per day (20°C, 14:10 light:dark cycle). In contrast, my isolate of E. huxleyi from Stn P grows maximally at ca. 0.6 doublings per day at 16 °C with a 14:10 light:dark regime. This is also the maximal growth rate achieved when grown on ESAW under continuous illumination (Lecourt et al. in press). Since all other isolates reported here are from the Atlantic Ocean while mine is from the Pacific, genetic differences are to be expected. However, my isolate is the only one from an HNLC area. Brand (1994) hypothesized that some coccolithophores in oligotrophic areas may exhibit extreme k-57 selection; that is, grow extremely well at low nutrient concentrations and not grow any faster at higher nutrient concentrations. T h e Subarct ic Paci f ic Since dissolved Fe concentrations in the surface waters of the NE subarctic Pacific have been measured to be 0.05 - 0.4 nmol kg"1 (Martin and Gordon 1988, Boyd et al. submitted, Kudo, unpubl. data), our experimental conditions of 'low Fe' (total Fe = 0.8 nmol kg"1) may be representative of Stn P at some times. The external Fe concentration in the Fe-stressed culture flasks in which cell division had actually ceased was likely extremely small, as this organism has been found previously to be difficult to Fe-limit in the laboratory (Brand et al. 1983). However, in all of these experiments (as well as in all other previously reported trace metal-phytoplankton studies), EDTA was present in the culture medium (10 /nM EDTA in my case), making a direct comparison with entirely natural waters difficult. Not only does EDTA affect the speciation of trace metals, it may also interfere with naturally produced organic compounds that may serve as biological aids for metal acquisition. Results of an experiment conducted with both E. huxleyi and an oceanic diatom in natural Stn P water without EDTA present will be reported elsewhere (see Chapter 5). For the purpose of this study, then, I will focus my discussion on the possible effects of nitrogen source. Under the experimental low Fe conditions (0.8 nmol kg"1), ammonium-grown cells did not appear to have a physiological advantage over nitrate-grown cells. In fact, nitrate-grown cells had significantly higher growth rates than ammonium-grown cells under these conditions (Table 2.1). This would indicate that if fast cell division is ecologically favored and cells would acclimate to this, E. huxleyi may be utilizing nitrate in situ to meet its nitrogen requirements. ^ N experiments conducted at Stn P have indicated that the small size fraction (<5.0 /*m) is utilizing nitrate to some extent (30-50% of N requirement, Varela, unpubl. data). Similarly, Price et al. (1994) in the equatorial Pacific (another HNLC region), found that nitrate was being utilized by the indigenous small phytoplankton. Attributing nitrate uptake to single species in the field is difficult, as mixed assemblages are always present. As well, species assemblages can vary dramatically at a single locale, let alone in 58 different regions of the ocean. However, these field data would indicate that there is a possibility that E. huxleyi, and possibly other small prymnesiophytes like it, are indeed utilizing nitrate in situ. The irradiance experiments also support the notion that E. huxleyi is utilizing nitrate in situ, as faster growth was found for nitrate-grown cells under low light, and ammonium-grown cells never grew faster than nitrate-grown cells (Fig. 2.4A). Hence, results from my experiments would indicate that under normal, low Fe conditions in the subarctic Pacific, E. huxleyi is probably utilizing nitrate as its primary nitrogen source. In the event of Fe addition to the system (via atmospheric deposition, see Martin et al. 1989, Duce and Tindale 1991), our data does not indicate that one nitrogen source over the other is more beneficial to this phytoplankter. Assessing which nitrogen source is actually taken up under ecologically relevant concentrations in the laboratory is the next step in understanding the ecophysiology of this species at Stn P. Wells et al. (1995) point out the complete absence of laboratory studies on organisms from the field locations in question. Care must be taken especially when working with E. huxleyi, as there is much genetic variation between isolates (Brand 1982), and the origin of the isolate should be clearly identified in all reports (Paasche 1992). As well, if truly ecological questions are to be answered, the culture conditions in the laboratory need to be carefully considered (e.g. omitting artificial chelators; see Chapter 4). Physiological laboratory studies on organisms from HNLC regions need to be conducted if we are to truly understand the ecology of these systems, and how the individual organisms present would respond to perturbations to the system. 59 A BRIEF SUMMARY OF CHAPTERS 1 AND 2 U n d e r Fe-replete conditions: Actinocyclus sp.: *NH 4 +-grown cells had larger CV and C quotas than N03~-grown cells; *N03_-grown cells had higher chlorophyll quotas and lower in vivo Flxhl a than NH4+-grown cells, but not higher growth rates; •There was no difference in Fe(int):C, Mn:C, and Zn:C ratios between N O 3 " and NH4+-grown cells; Emiliania huxleyi: There were no differences in any of the parameters measured between N O 3 " and NH 4 +-grown cells, except that NH4+-grown cells produced more coccoliths than N03"-grown cells; Therefore, NH4+-grown cells of either species did not exhibit an obvious physiological advantage over N03"-grown cells under Fe-replete conditions. U n d e r Fe-stressed conditions: Actinocyclus sp.: *NH4+-grown cells had greater chl a, C, and N quotas than N03"-grown cells; *NH4+-grown cells had lower in vivo Flxhl a than N03"-grown cells; *N03"-grown cells had higher Mn:C and lower Zn:C ratios than NH4+-grown cells; Emiliania huxleyi: *N03"-grown cells had smaller CV than NH4+-grown cells, resulting in greater chl a CV" 1 and equal N CV" 1 and C(a) CV" 1 compared to NH4+-grown cells; *N03"-grown cells had the same in vivo Flxhl a as NH4+-grown cells; *N03"-grown cells had greater Mn:C ratios than NH 4 +-grown cells; 60 Therefore, NH4+-grown cells of the oceanic diatom appeared to have a physiological advantage over NC^-grown cells under Fe-stressed conditions, agreeing with theoretical predictions based on Fe and energy requirements. NC>3"-grown cells of the oceanic coccolithophore did as well or better physiologically as NH-4+-grown cells under Fe-stressed conditions, thus not agreeing with physiological predictions. As well, the diatom exhibited greater signs of physiological stress under Fe-stressed conditions compared to the oceanic coccolithophore, especially for N03"-grown cells. 61 C H A P T E R 3: E F F E C T O F I R O N O N T H E S I N K I N G R A T E O F A N O C E A N I C D I A T O M A N D A N O C E A N I C C O C C O L I T H O P H O R E F R O M T H E N E S U B A R C T I C P A C I F I C INTRODUCTION This chapter examines the possible effect that Fe may have on the sinking rate of two oceanic phytoplankton species from the NE subarctic Pacific. Because of the role of Fe in electron transport processes, and the finding that buoyancy in large diatoms is dependent on energy derived from respiration (Waite et al. 1992), Fe-stress may affect phytoplankton sinking rates by stressing the energy producing pathways needed by the cell to maintain its buoyancy. In addition, the nitrogen source utilized by a phytoplankter can affect its growth rate and cell volume (Thompson et al. 1989, Levasseur et al. 1993, see Chapters 1 and 2), both of which may have indirect effects on sinking rate. I present here the first report on the effect of Fe on phytoplankton sinking rates. The possible effect of Fe on sinking rates was examined for two oceanic phytoplankton species, an oceanic coccolithophore Emiliania huxleyi, and an oceanic diatom Actinocyclus sp., and the effect that nitrogen source may have on this relationship was examined for the oceanic diatom. MATERIALS AND METHODS The oceanic diatom, Actinocyclus sp., and the oceanic coccolithophore, Emiliania huxleyi, were grown under Fe-replete and Fe-stressed conditions. Experiments were conducted with microwave-sterilized (Keller et al. 1988), Chelex-treated (Price et al. 1988/89), Stn P water. The total Fe in this Chelex-treated Stn P water was measured as in Yang (1993) and found to be ca. 0.9 nmol kg - 1 . Macronutrient enrichments were made with 62 Chelex-treated stocks (see Price et al. 1988/89) to the final concentrations given in Chapters 1 and 2. Final vitamin and metal additions for the two species were the same as in Chapters 1 and 2 as well. Fe-replete cultures of the diatom had 1000 nM Fe added, and Fe-replete cultures of the coccolithophore had 100 nM Fe added. Fe-stressed cultures of both species were obtained as described in Chapters 1 and 2. Cultures were grown in acid-cleaned 3 L polycarbonate flasks on a 14:10 light:dark cycle with Vitalite fluorescent bulbs at 16°C. The irradiance was saturating for growth for both species (ca. 150 iimol m"2 s"l). Growth rates, cell volumes, chlorophyll a, and particulate carbon and nitrogen were all measured as in Chapters 1 and 2. Sinking rate was measured by the SETCOL method (Bienfang 1981) in motionless 1 m Plexiglas columns over a 3 h period under the same irradiance conditions as the cultures. The SETCOL columns were acid-cleaned prior to measurement and filled in a class 100 trace metal free room for the Fe-stressed experiments. Sinking rates were measured for both nitrate and ammonium-grown cultures of the diatom, and only for nitrate-grown cultures of the coccolithophore. Metal quotas were measured using non-radiotracer techniques, as described in the General Methods section. All statistical comparisons were made using a Student's t-test at the 95% confidence level (p< 0.05). RESULTS Sinking Rate Fe-stress had a dramatic effect on the sinking rate of the oceanic diatom (Fig. 3.1 A, Table 3.1). The sinking rate under Fe-stressed conditions was about 5 times greater than under Fe-replete conditions, with average sinking rates of 0.92 and 0.17 m d"1, respectively. This is in sharp contrast to the results obtained with the oceanic coccolithophore, Emiliania huxleyi (Fig. 3. IB). Fe had no effect on the sinking rate of this small coccolithophore, with the sinking rate under both Fe-stressed and Fe-replete conditions being about 0.12 m d"1. 63 CD -*-> CO •H a • i—( GO 1.00 0.80 l 0.60 >% £ 0.40 0.20 0.00 1.00 0.80 Actinocyclus sp. A NH 4 N0 Q • Erniliania huxleyi 0.60 - t J CO V K >, OX) T3 £ 6 0.40 -0.20 0.00 B NO, Fe-Replete Fe -S t r e s sed Fig. 3.1. Sinking rate vs Fe condition for A) both ammonium and nitrate-grown cells of Actinocyclus sp., and B) nitrate-grown cells of E. huxleyi. Error bars represent +1 standard error (n=3). 64 Table 3.1. Physiological parameters for the oceanic diatom Actinocyclus sp. and the oceanic coccolithophore Emiliania huxleyi under both Fe-replete and Fe-stressed conditions (+.1 standard error in parenthesis; n=4 for Fe-replete, n=3 for Fe-stressed cultures). *Indicates a significant difference at p< 0.05 between Fe-replete and Fe-stressed cells within a species with the asterisk placed next to the greater value. Metal values for E. huxleyi are from a similar experiment with Fe-replete cultures having a final Fe concentration of 100 nM Fe. $is an estimate based on the growth rate and Fig. 5.2. growth rate; CV=cell volume; SR=sinking rate; C(t)=total carbon including coccoliths; C(a) = carbon excluding coccoliths. E. huxleyi Actinocyclus s p . Parameters Fe-replete Fe-stressed Fe-replete Fe-stressed M , 0.0182* 0.0032 0.0192* 0.0149 (h-1) (0.0022) (0.0004) (0.0017) (0.0006) CV 34.5* 18.6 4460* 4100 (Mm"3) (1.1) (0.6) (69) (26) SR 0.12 0.10 0.16 0.93* (m day"1) (0.01) (0.02) (0.01) (0.01) Chl a cell'1 80 66 8700* 2700 (fg cell"1) (8) (5) (530) (220) C(a):chl a 210* 140 45 110* (wt) (21) (10) (4) (10) N cell"1 1.1* 0.82 70* 38 (pg cell"1) (0.1) (0.05) (2.3) (0.4) N CV" 1 31 44* 16* 9 (fg ntn3) (0.2) (0.2) (0.7) (0.2) C(t) cell - 1 18.7* 10.5 390* 285 (pg cell"1) (0.6) (0.6) (16.0) (1.0) C(a) cell"1 16.5* 9.3 not applicable not applicable (pg cell'1) (0.7) (0.3) C(t) C V 1 542 565 87.4* 69.5 (fg A""*3) (10) (19) (4.5) (0.1) C(a) CV" 1 478* 500 not applicable not applicable (fg Mm'3) (7) (3) Fe(int):C 90* 13$ 370* 38 0tmol:mol) (20) (est.) (25) (8) Mn:C(t) 13 27* 38 46 0*mol:mol) (0.8) (1.0) (2.7) (2.3) Zn:C(t) 18 13 5.8* 1.6 Oimoltmol) (2.0) (0.9) (1.0) (0.5) 65 For Actinocyclus sp., the sinking rate for ammonium-grown cells under Fe-replete conditions was significantly greater than the sinking rate of nitrate-grown cells (Fig. 3.1 A). There was no difference between the sinking rate of ammonium and nitrate-grown cells under Fe-stressed conditions. Cell Volume The cell volume of Actinocyclus sp. decreased slightly but significantly from Fe-replete to Fe-stressed conditions (Fig. 3.2A, Table 3.1), with an 8% decrease occurring for nitrate-grown cells and a 18% reduction occurring for ammonium-grown cells. The cell volume of ammonium-grown cells under Fe-replete conditions was significandy greater than the cell volume of nitrate-grown cells under the same conditions. In contrast, the cell volume of Emiliania huxleyi decreased substantially when grown on nitrate, with a decrease of 46% from Fe-replete to Fe-stressed conditions (Fig. 3.2B). Nitrogen Quotas The nitrogen per cell was significantly greater for Fe-replete than for Fe-stressed cultures of both the diatom and coccolithophore (Table 3.1). However, when normalized to cell volume, E. huxleyi actually had greater N CV_1 under Fe-stressed than under Fe-replete conditions. The diatom suffered from lower N quotas under Fe-stress regardless of normalization to CV (Table 3.1). Carbon Quotas The carbon per cell was greater for Fe-replete than for Fe-stressed cultures of both the diatom and the coccolithophore (Table 3.1). When normalized to cell volume, E. huxleyi maintained the same carbon quotas under Fe-replete and Fe-stressed conditions, whereas the diatom still had lower carbon per cell volume under Fe-stress (Table 3.1). Chlorophyll a Quotas E. huxleyi maintained the same chlorophyll a per cell regardless of Fe condition, while the diatom suffered from significantly lower chlorophyll a quotas under Fe-stress (Table 3.1). 66 6000 Actinocyclus sp. 40 30 Emiliania huxleyi CD o 6 20 CD u 10 -B N0C Fe-Replete Fe-S t ressed Fig. 3.2. Cell volume vs Fe condition for A) both ammonium and nitrate-grown cells of Actinocyclus sp., and B) nitrate-grown cells of E. huxleyi. Error bars represent +1 standard error (n=3). 67 Carbon:Chlorophyll a Fe-stress significantly increased the carbon:chlorophyll a ratio in the diatom, but actually decreased this ratio in the coccolithophore (using cellular carbon excluding coccoliths) (Table 3.1). E. huxleyi appears to be unique in that Fe-stress does not appear to affect its ability to synthesize chlorophyll (Chapter 2). Metal Quotas The decrease in Fe quotas from Fe-replete to Fe-stressed conditions was ca. 10 times for both species (Table 3.1). Mn quotas increased with Fe-stress for both species, although not significantly for the diatom (p<0.10) (Table 3.1). Zn quotas decreased with Fe-stress in the diatom (Table 3.1). In general, the diatom maintained higher Mn levels than the coccolithophore, whereas the coccolithophore maintained higher Zn levels than the diatom. For results and discussion on the metals quotas of both nitrate and ammonium-grown cultures of both species see Chapters 1 and 2. DISCUSSION Large Oceanic Diatom vs Small Oceanic Coccolithophore The sinking rate of the oceanic diatom, Actinocyclus sp., was drastically affected by the Fe-status of the cells, whereas the sinking rate of the coccolithophore, Emiliania huxleyi, was not affected by its Fe-status (Fig. 3.1). This different effect of Fe-status on sinking rate suggests that the relatively large diatom (20-60 itm diameter) may depend on energy requiring processes to maintain its buoyancy, while the small coccolithophore (5 itm diameter) does not. This supports the trend observed by Waite et al. (submitted), namely, that larger cells tend to actively control their sinking rates by energy requiring processes, whereas small cells do not. The fact that Fe-stress was able to induce such a large increase in the sinking rate of Actinocyclus sp. is another piece of evidence indicating that energy production from electron transport systems may somehow be involved in the maintenance of buoyancy of diatoms 68 (Waite et al. 1992). The sinking rate measured for Actinocyclus sp. under Fe-replete conditions is similar to the sinking rate maintained by Emiliania huxleyi, which would also indicate that the large diatom is somehow actively reducing its sinking rate to levels lower than would be expected based on the size of the cell. Sinking Rate of N H 4 vs. NO^-Grown Cells of an Oceanic Diatom Under Fe-replete conditions, the sinking rate of ammonium-grown cells was greater than the sinking rate of nitrate-grown cells of Actinocyclus sp. This could be due to the larger size of the ammonium-grown cells. However, the difference in sinking rate between ammonium and nitrate-grown Fe-replete cells was small compared to the observed difference between Fe-replete and Fe-stressed cultures. There was no difference between the sinking rate or cell volume of ammonium and nitrate grown cells of Actinocyclus sp. under Fe-stressed conditions. Because of the extra severity of energy stress upon nitrate-grown cells, the nitrate-grown cells might have been expected to have higher sinking rates. It is possible that the effect of Fe on sinking rate is a 'step response', where a threshold is reached above which the cells have no physiological control of their sinking rate. Hence, due to their identical cell volume, the ammonium and nitrate grown cells would be expected to sink at the same rate. The only way to test this possibility is to measure sinking rates under differing degrees of Fe-stress. Cell Volume. Fe. and Sinking Rates The change in cell volume observed for the diatom cannot explain the change in sinking rate observed between Fe-replete and Fe-stressed conditions. In fact, a decrease in the cell volume of Fe-stressed cells should produce a decrease in sinking rate. Therefore, the 5 times increase in sinking rate under Fe-stress would have been even higher if it were not for the small decrease in cell volume under Fe-stress. The decrease in cell volume of Actinocyclus sp. can be solely attributed to the natural decrease by asexual cell division (Fig. 3.3). The cell volume of Actinocyclus sp. for Fe-replete cultures grown on both nitrate and ammonium was monitored for 40 days, which was longer than the experiments reported here (25 days). A linear regression was used to predict 69 Actinocyclus sp. 5500 i — Time (days) Fig. 3.3. Cell volume vs time for both ammonium and nitrate-grown cultures of Actinocyclus sp. grown in Fe-replete medium over a 40 day period. Error bars represent ± 1 standard error (n=3). The slope of the linear regressions are -45.2 (r2=0.8) and -24.7 (r2=0.8) for ammonium and nitrate-grown cells, respectively. 70 the decrease in the cell volume expected during asexual division for the duration of the experiments. The cell volumes measured for the Fe-stressed cultures lie above the predicted cell volumes calculated using the regression equations and the initial cell volumes of the cultures. In contrast to the diatom, the cell volume of Emiliania huxleyi grown on nitrate decreased drastically under Fe-stress (46%), much lower than could be explained by normal daily variations in its cell volume. It is interesting to note, however, that E. huxleyi growing on ammonium did not decrease its cell volume under Fe-stress (Chapter 2). It appears that E. huxleyi can reduce its cell volume when growing on nitrate under low Fe conditions, helping it to maintain its low sinking rate, whereas the diatom cannot change its cell volume enough to compensate for the very large increase in sinking rate under Fe-stressed conditions. Reducing its cell volume is one way in which E. huxleyi may be able to adapt to its low Fe environment in the subarctic Pacific. A reduction in cell volume is particularly advantageous for cells living in a low Fe environment because it not only reduces sinking rates, but it reduces cellular requirements for N, C, and Fe (Chapter 2) and increases the surface area to volume ratio, enhancing the potential for cell surface transport of Fe and other nutrients. Therefore, the larger diatom which is unable to make large changes in its cell volume, may be more susceptible to Fe-stress in the subarctic Pacific than small cells that can reduce their cell volume and become even smaller. Ecological Significance Fe-status of the cell had an effect on the sinking rate of an oceanic diatom, but not on an oceanic coccolithophore from Stn P. Since the major source of Fe in the subarctic Pacific is thought to be aeolean (Martin et al. 1989), the low sinking rate of the coccolithophore may give it an advantage compared to other species of indigenous phytoplankton. However, the sinking rate of Fe-stressed cells of this diatom was < 1 m day"1, which is probably not fast enough to result in sinking to depth. This sinking rate is high for a physiologically active diatom cell, but most likely some other mechanism must be invoked for the occurrence of large fluxes of siliceous material to sink to depth. Clumping is one way in which cells can 71 sink to depth with greater speed, but no such phenomenon was observed in our cultures. However, cells were never allowed to go into senescence during our experiments, and stationary, unhealthy cells are more likely to clump and stick together. Smetacek (1985) proposed that once cells become severely stressed, aggregates may form, scavenging other minerals as well, and drastically accelerating sinking rates of the new particles (>_ 100 m d _ 1). Therefore, an increase in the sinking rate of a physiologically active cell may be an indicator of other processes about to occur if that cell is not able to return to a healthier state in a given amount of time. The time frame involved for senescent cells to recover or actually lyse is largely unknown and very variable between species (Berges, pers. comm.). We know that large pulses of siliceous material do indeed sink to depth (3800 m) at Stn P because of sediment trap data (Takahashi 1986, Takahashi et al. 1990, Wong, unpubl. data), and in fact, Actinocyclus curvatulus is a dorninant component of this material, exhibiting the greatest flux in the spring. However, the exact mechanism resulting in this flux has not been resolved. Since the sinking rate of the diatom is high under 'Fe-stressed' conditions (0.9 m d _ 1), this diatom would probably exist in a lower light environment. It has been found that Fe-deficient cells may be more susceptible to photochemical damage than Fe-replete cells (Geider and LaRoche 1994), implying that Fe-stressed cells may be better off in low light. As well, the lower light may allow the cell's metabolism to remain slow, decreasing its requirements for photosynthate and nutrients. However, low light is also theoretically expected to increase the Fe requirements of a cell (Raven 1988, 1990), although this has yet to be demonstrated in the laboratory. Any interactions between irradiance, Fe, and nitrogen source were not examined in this study. More detailed laboratory studies on the combined effects of irradiance and Fe-stress on sinking rates, along with examining senescent-phase cultures are needed to further understand the role of Fe in controlling the flux of phytoplankton to depth. 72 C H A P T E R 4: E F F E C T S O F E D T A O N O C E A N I C P H Y T O P L A N K T O N I. EDTA SUPPRESSES THE GROWTH OF OCEANIC PHYTOPLANKTON Introduction Currently, the synthetic chelator ethylenediaminetetraacetate (EDTA) is commonly added to marine phytoplankton culture medium to help alleviate toxic effects of some metals (e.g. Cd, Cu) and to increase the bioavailability of others (e.g. Fe) by preventing precipitation (Harrison et al. 1980, Price et al. 1988/89). Originally, EDTA was added to cultures because it was found to stimulate the growth of natural phytoplankton populations (Johnston 1963, 1964). The concentrations of EDTA used in the most common marine phytoplankton media today, range from 10 to 100 uM EDTA (15 /*M EDTA for ESAW (Harrison et al. 1980); 10 - 100 /*M EDTA for Aquil (Price et al. 1988/89); 100 /xM for 'K' medium (Keller et al. 1987)), with a precautionary note in Price et al. (1988/89) about possible toxic effects of EDTA concentrations of 100 or greater on some sensitive species. While EDTA is routinely used in trace metal-phytoplankton studies to control and define the speciation of metals, this synthetic chelator has been shown to have adverse effects on other biota, causing damage to cell membranes and lysing of bacteria (Cavard et al. 1989, Marvin et al. 1989, Temple et al. 1992). EDTA is also used to increase the permeability of membranes in bacteria (Ryan and Parulekar 1991). The effect of EDTA on algae has been examined previously, but precautions were not taken to insure that the high concentrations of EDTA tested were not in fact limiting the organisms for a required metal by drastically reducing the free metal ion concentrations (Marchyulenene et al. 1982, Dufkova 1984). I present here data on the effects of EDTA on the growth and biochemical composition of two oceanic phytoplankton species, in which I have varied the concentration of metals to achieve similar, replete, free ionic metal concentrations for each of the different EDTA 73 treatments. By ensuring that the free metal concentrations remain adequate for maximal growth, the effect of EDTA exclusively can be examined. Materials and Methods The oceanic coccolithophore, Emiliania huxleyi, and the oceanic diatom, Actinocyclus sp., were used for these experiments. Triplicate cultures were grown semi-continuously in acid-cleaned 250 ml polycarbonate flasks at 16°C under a 14:10 lighf.dark regime with saturating light for growth (180 itmol m"2 s"1, Vitalite fluorescent tubes). Cultures were grown for 10 generations and harvested in log phase. The synthetic ocean water recipe of Aquil was used (Price et al. 1988/89), with final macronutrient additions being: 50 itM N O 3 - , 5 itM P O 4 - 3 , 100 fiM Si(OH)4. ESAW amounts of vitamins were used (Harrison et al. 1980). Three different concentrations of EDTA were tested: 1, 10, and 100 itM final EDTA. The total amount of metals added and the resulting free metal ion concentrations as calculated by the chemical equilibrium program MINEQL (Westall et al. 1976) are given in Table 4.1. The metal additions made for the 10 tiM EDTA treatment are the same as for the marine phytoplankton culture medium Aquil (Price et al. 1988/89). Hence, the resulting metal ion concentrations should be replete for all species. As well, studies conducted by Brand et al. (1983) with 21 phytoplankton species reaffirms our replete metal conditions for iron, manganese, and zinc (requirements for maximal growth). EDTA and Fe were mixed first and allowed to stand for 20 minutes, and then added to the synthetic ocean water. The final medium was allowed to equilibrate for 48 h before use. Medium was sterilized by microwave sterilization (Keller et al. 1988), and all handling of cultures and medium was under class 100 flowhood conditions. Growth rates were determined by regressions of the natural log of cell number vs time. Cell numbers, chlorophyll a, and particulate carbon and nitrogen values were determined as in previous chapters. Cell volumes of the diatom in this study are reported as spherical equivalents. For Emiliania huxleyi, cellular carbon was measured both with and without coccoliths present. C(t) refers to total carbon including coccolith carbon, and C(a) refers to 74 Table 4.1. Total metal concentrations added to the medium Aquil and the calculated free ion metal concentrations determined using the chemical equilibrium program MINEQL. Units of total additions (e.g. Fet) are in molarity; units of calculated metal ions (e.g. Fe + 3) are in -log [molarity of ion] = p[M]. E. huxleyi and Actinocyclus sp. Parameters 1 fiM EDTA 10 /xM EDTA 100 itM EDTA TM1 TM1 Fet 8.23 x 10"8 8.23 x 10"7 8.23 x 10-6 Mnt 1.5 x 10-8 2.3 x 10"8 1.2 x lO"7 Zn t 1.5 x 10"8 2.3 x 10"8 8.0 x 10"8 Cu t 5.0 x 10- 1 0 5.0 x 10"10 2.0 x 10"9 Co t 1.0 x lO"9 1.0 x 10"8 1.0 x lO"7 Mot 1.0 x 10"7 1.0 x 10"7 1.0 x lO"7 Set 1.0 x 10'8 1.0 x 10"8 1.0 x 10"8 p[M] P(M1 prMi Fe+3 18.2 18.1 18.1 M n + 2 8.4 8.4 8.3 Zn+2 9.6 10.4 10.8 Cu+2 13.4 14.4 14.8 Co+2 10.6 10.6 10.6 M 0 O 4 -2 7.0 7.0 7.0 Se03*2 10.6 10.6 10.6 75 cellular carbon excluding coccoliths, where the coccoliths have been removed by an acid treatment (lowering the pH to 5.0). Statistical comparisons were performed using 1-way ANOVA and Student-Newman-Keul's test at a significance level of p < 0.05. The statistical program SIGMASTAT was used. Results The growth rates of both oceanic species were significantly reduced when 100 itM EDTA was present in the medium (Fig. 4.1, Table 4.2, Table 4.3). For the oceanic coccolithophore, the presence of 100 itM EDTA reduced its growth rate by 53 % compared to maximal growth (Table 4.2). The oceanic diatom was not quite as sensitive to EDTA as the oceanic coccolithophore, but its growth rate decreased nearly 30% compared to its maximum growth (Table 4.3). Cell volume, chlorophyll a, and nitrogen quotas were all unaffected by EDTA concentration (Table 4.2, Table 4.3). However, the carbon content per cell was affected by EDTA in both species. For E. huxleyi, the total carbon per cell (including coccolith carbon) was significantly greater for the 100 itM EDTA treatment compared to the 1 and 10 itM EDTA treatments (Table 4.2). The cellular carbon excluding coccoliths, C(a), did not change significantly with EDTA treatments, indicating that the excess carbon was due to coccolith carbon. An increase in cellular carbon also caused the significant increase of carbon to chlorophyll a ratios for the 100 itM EDTA treatment compared to the other two EDTA treatments (Table 4.2). Similar to E. huxleyi, the oceanic diatom had significantly higher carbon quotas under the highest EDTA treatment (Table 4.3). The presence of 100 itM EDTA caused an increase of 30% in the carbon per cell as compared to cells exposed to 1 itM EDTA (Table 4.3). This increase in cellular carbon resulted in the C:N ratio being significantly higher for 100 iiM EDTA cells than for cells exposed to 1 or 10 itM EDTA (Table 4.3). 76 CD PS o O o o CD CO 0 . 0 1 0 0 . 0 0 5 0 . 0 0 0 1 10 EDTA 100 Fig. 4.1 Specific growth rate vs EDTA concentration for A) E. huxleyi and B) Actinocyclus sp. Error bars represent ± 1 standard error (n=3). 77 Table 4.2. Physiological parameters for Emiliania huxleyi grown with different EDTA concentrations under similar metal ion conditions in Aquil SOW. *indicates a significant difference (p < 0.05) of a particular EDTA treatment compared to the other EDTA treatments as determined by a 1-way ANOVA. +1 standard error is given in parenthesis (n=3). ft=growth rate; CV= cell volume; C(t)=total carbon including coccolith carbon; C(a)= carbon excluding coccolith carbon. Emiliania huxleyi 1 fxM EDTA 10 /xM EDTA 100 /xM EDTA Parameters V- , 0.0340 0.0313 0.0159* (h-1) (0.0015) (0.0013) (0.0007) CV 38 39 41 (/xm"3) (!) (D (2) chl a cell - 1 140 120 120 (fg cell-1) (6) (5) (5) C(t) cell"1 8.2* 7.5* 12.1* (pg cell"1) (0.4) (0.2) (1.3) C(a) cell"1 7.5 7.1 8.1 (pg cell"1) (0.4) (0.2) (0.2) N cell"1 1.0 1.0 1.1 (pg cell"1) (0.1) (0.4) (0.1) C(a):chl a 54 59 68* (wt) (3) (2) (2) C(a):N 7.5 7.1 7.4 (wt) (0.4) (0.1) (0.1) 78 Table 4.3. Physiological parameters for Actinocyclus sp. grown with different EDTA concentrations under similar metal ion conditions in Aquil SOW. *indicates a significant difference (p<0.05) of a particular EDTA treatment compared to the other EDTA treatments as determined by a 1-way ANOVA. +.1 standard error is given in parenthesis (n=3). fi=growth rate; CV= cell volume. Actinocyclus sp. 1 /xM EDTA 10 itM EDTA 100 /xM EDTA Parameters 0.0254 0.0214 0.0187* (h-1) (0.0010) (0.0020) (0.0009) CV 21100 20500 21000 (xxm-3) (640) (635) (712) chl a cell"1 7.8 7.5 7.1 (pg cell"1) (0.2) (0.9) (0.9) C cell"1 409* 506* 589* (pg cell"1) (22) (16) (20) N cell"1 45 53 42 (pg cell'l) (3) (5) (2) C:chl a 52 67 83 (wt) (2) (9) (15) C:N 9.0 9.5 14* (wt) (0.3) (1.3) (0.7) 79 Discussion Cell division and carbon quotas were the physiological factors affected by increases in EDTA in these oceanic phytoplankton. The fact that the carbon quotas increased while the chlorophyll a quotas remained constant for both species indicates that EDTA may somehow uncouple photosynthesis from growth. Therefore, carbon acquisition and fixation would continue, and the carbon that would normally be allocated to cell growth processes would accumulate. In the coccolithophore, high EDTA concentrations caused an increase in coccolith carbon, perhaps providing a mechanism to rid the cell of excess carbon. Because of the effect that EDTA has on both membrane permeability and membrane electrical potential in bacteria and euryhaline algae (Marchyulenene et al. 1982, Ryan and Parulekar 1991, Pelletier et al. 1994), the possible affect on CO2 utilization was considered. If these phytoplankton species were utilizing free CO2 for their carbon source, an increase in the permeability of the cellular membrane could result in a greater flux of carbon into the cell, as free CO2 enters the cell via passive diffusion. As well, low-calcifying strains of E. huxleyi have been found to mainly use free CO2 as their carbon source (Dong et al. 1993). However, when the carbon assimilation rate is calculated for both species 0**C cell-!), a significant increase is not observed as would be expected. Therefore, EDTA probably did not affect the diffusion of CO2 into the cells of these two oceanic species. In this study I found that 100 fiM EDTA was enough to severely inhibit cell division of phytoplankton from the NE subarctic Pacific. Nirel et al. (unpubl. data) found _>500 fiM EDTA to be toxic to a coastal diatom Thalassiosira weissflogii, indicating that oceanic species may be much more sensitive to EDTA than coastal species. As some researchers use 100 jtM EDTA routinely (e.g. Sunda et al. 1991), and Keller et al. (1987) recommend using 100 iiM EDTA for their "K" medium for oceanic ultraplankton, caution is warranted, especially when working with newly isolated oceanic species. 80 H. POSSIBLE EFFECTS OF THE PRESENCE OF EDTA ON THE PHYSIOLOGY OF AN OCEANIC DIATOM GROWN IN ARTIFICIAL MEDIA Introduction In this experiment, the effect of the presence vs the absence of EDTA on the physiology of an oceanic diatom was examined. As in the above experiments, free metal ion concentrations were kept at replete levels for all treatments, including the treatment with no EDTA present. In this way, physiological effects due to the presence of EDTA could be examined. The highest concentration of EDTA tested in this experiment (10 fiM EDTA) was not as high as in the previous EDTA experiment (100 /xM EDTA), as the previous experiment was designed to test for toxicity, not the effect of the presence of EDTA. Basic physiological parameters were measured to determine if the presence or absence of EDTA affected the overall health of the cells. Measured parameters included growth rate, cell volume, chlorophyll a quota, in vivo flouresense: chlorophyll a (an indicator of photosynthetic electron transport efficiency, see Chapter 1), nitrogen and carbon quotas, and iron quota. Materials and Methods Actinocyclus sp. was grown semi-continuously in acid-cleaned 3 L polycarbonate flasks on a 14:10 lightrdark cycle with growth-saturating irradiance. The cultures of Actinocyclus sp. had not sexually reproduced yet (which resulted in much larger cells) when this experiment was conducted, and therefore the cells in this experiment are much smaller than in the previously discussed EDTA experiment. Cultures were harvested in early log phase after acclimation for 10 generations to the various EDTA treatments. Microwave-sterilized (Keller et al. 1988) Aquil synthetic ocean water (Price et al. 1988/89) was used (as opposed to natural Stn P water) in order to calculate metal speciation. Macronutrient and vitamin additions were the same as in Chapter 1 (nitrate was the nitrogen source), and metal and EDTA additions (0, 1, and 10 /xM) are given in Table 4.4. The resulting free metal ion concentrations were calculated using the chemical equilibrium 81 Table 4.4. Total metal concentrations added to the medium Aquil and the calculated free ion metal concentrations determined using the chemical equilibrium program MTNEQL. Units of total concentrations (e.g. Fet) are in molarity; units of calculated metal ions (e.g. Fe + 3) are in -logfmolarity of ion] =p[M]. *The confidence in this value is limited due to the possibility of precipitation in the absence of EDTA, and the unknown natural speciation of Fe in seawater. Parameters No EDTA Actinocyclus sp. 1 /iM EDTA 10 fiM EDTA [Ml Pvll n v n Fet 1.0 x 10-8 1.0 x 10-6 1.0 x 10-5 Mn t 2.3 x 10-8 2.3 x 10-8 2.3 x 10-8 Zn t 8.0 x 10-9 8.0 x 10-9 8.0 x 10-9 Cu t 1.0 x 10-9 1.0 x 10-9 1.0 x 10-9 Cot 2.5 x 10-9 2.5 x 10-9 2.5 x 10-9 Mot 1.0 x 10"7 1.0 x 10"7 1.0 x 10"7 Set 1.0 x 10-8 1.0 x 10-8 1.0 x 10-8 prMl P.M1 p[M] Fe+3 18.5* 16.9 16.3 M n + 2 8.2 8.2 8.2 Zn+2 8.2 9.5 10.1 Cu+2 12.2 12.8 13.3 Co+2 8.8 9.8 10.4 M0O4 -2 7.0 7.0 7.0 SeC-3-2 10.6 10.6 10.6 82 program MINEQL (Westall et al. 1976), and are also given in Table 4.4. All other methods are the same as in Chapter 1. Significant differences were determined using a 1-way ANOVA and a Student-Newman-Keul's test at a significance level of p< 0.05. The statistical program SIGMASTAT was used. Results G r o w t h Rate The growth rate of cultures with no EDTA present in artificial medium was significantly lower than the growth rate of cultures with either 1 or 10 itM EDTA present, resulting in a 40% decrease in maximal growth (Table 4.5). There was no statistical difference in growth rates between cultures grown in 1 or 10 itM EDTA. C h l o r o p h y l l a Quotas The chlorophyll a per cell was significantly different for all treatments, with the no EDTA cultures having the lowest chlorophyll a quotas, and the 1 tiM EDTA cultures having the highest chlorophyll a quotas (Table 4.5). C a r b o n : C h l o r o p h y l l a The carbon to chlorophyll a ratio was significantly greater for cultures with no EDTA present, with a 3 to 5 times greater ratio than cultures with EDTA present (Fig. 4.2A, Table 4.5). There was no statistical difference between cultures grown with 1 or 10 itM EDTA (Table 4.5). in vivo FluorescencerChlorophyll a The in vivo fluorescence per chlorophyll a was significantly greater for cultures with no EDTA present than for cultures with EDTA present (Fig. 4.2B, Table 4.5). There was no statistical difference between cultures with 1 or 10 itM EDTA present (Table 4.5). C a r b o n , Ni trogen Quotas There were no differences in carbon or nitrogen quotas and hence C:N ratios between any of the EDTA treatments (Table 4.5). 83 Table 4.5. Physiological parameters for Actinocyclus sp. grown with and without EDTA under similar metal ion concentrations in Aquil SOW. "Indicates a significant difference of a particular EDTA treatment compared to the other EDTA treatments as determined by a 1-way ANOVA. +.1 standard error is in parenthesis (n=3). growth rate; CV= cell volume. Parameters No E D T A Actinocyclus sp. 1 M M E D T A 10 nM E D T A M 0.0097* 0.0233 0.0215 (h-1) (0.0004) (0.0007) (0.0038) CV 1470 1350 1410 (/tm~3) (87) (49) (43) chl a cell'l 860* 3300* 2600* (fg cell-1) (170) (150) (170) Flxhl a"l 0.22* 0.09 0.10 (ng chl a"l) (0.01) (0.01) (0.01) N cell'l 33 26 39 (pg cell-1) (2) (3) (8) C cell-1 230 191 246 (pg cell-1) (34) (26) (76) C:chl a 270* 58 95 (wt) (81) (5) (27) C:N 8.2 8.5 7.4 (mol:mol) (1.5) (0.1) (0.7) C:N 7.0 7.3 6.3 (wt) (1.3) (0.1) (0.6) Fe(int):C 260* 1300* 22,000* (/xmolimol) (46) (210) (12,000) Fig. 4.2. A) Carbon per chlorophyll a and B) in vivo fluorescence per chlorophyll a EDTA treatment for Actinocyclus sp. Error bars represent ± 1 standard error (n=3). 85 Iron Quotas The Fe(int):C ratio increased (significantly) with the total amount of Fe added to the medium (for Fet ^ Table 4.4, for Fe(int):C see Table 4.5). Discussion The absence of EDTA adversely affected this oceanic diatom species when grown in artificial seawater, causing a decrease in growth rate and chlorophyll a quotas, and an increase in carbon per chlorophyll a and in vivo fluorescence per chlorophyll a ratios. The no EDTA cultures suffered from symptoms which could arise from Fe-limitation; a decrease in chlorophyll a quota, a decrease in the efficiency of photosynthetic electron transport, and a decrease in growth rate. However, the Fe(int):C ratio for the no EDTA cells was near the Fe-replete values for this species as reported in Chapter 1 (Table 1.1). As well, the calculated free ferric ion concentration (pFe=18.5) should be replete for this species (see Table 4.1). However, since no EDTA was added to the medium, the speciation of Fe in the medium is difficult to define. Debate still exists on whether certain Fe species exist in seawater, and the MINEQL program did not take into account the neutral ferric species. Because of the unknown natural speciation of Fe in seawater, the calculated free ferric ion concentration may be in error. As far as the Fe quota is concerned, the absence of EDTA may have resulted in an overestimate of the intracellular Fe quota (Price and Morel 1994). Because no EDTA was present in the medium, stable Fe hydroxides could have formed, although the Ti(IU) reagent was demonstrated to reduce >99% of ferric Fe precipitates (Hudson and Morel 1989). The resulting deleterious effects of no EDTA on cells may well have resulted from a combination of a low free ferric ion concentration, accompanied by higher C u + 2 (possibly Zn or Co as well) concentrations. The ratio of free metal ions has been found previously to affect phytoplankton growth rates and other basic physiological functions (Rueter and Morel 1981, Sunda and Huntsman 1983, Sunda 1988/89). When the relative proportion of a nutrient trace metal to a non-nutrient trace metal is decreased, the non-nutrient trace metal may compete and 86 actually replace sites and functions normally fulfilled by the nutrient trace metal (Sunda 1988/89). In this way, even though the nutrient trace metal (e.g. Fe) is present, other metals (e.g. Cu) may substitute and replace metabolic functions normally carried out by the nutrient trace metal, resulting in symptoms similar to the lack of the nutrient trace metal (Harrison and Morel 1983, Sunda 1988/89). Murphy et al. (1984) found that an oceanic diatom was much more susceptible to Cu toxicity when Fe concentrations were low. In my case, there may have been some interference with the normal function of Fe in chlorophyll synthesis, along with the function of electron transport components in the photosynthetic transport pathway in which Fe in a constitutive metal. There is the possibility that the adverse effects when no EDTA was present would not have occurred if natural Stn P water instead of artificial seawater was used. In Chapter 5, Actinocyclus sp. achieved a growth rate of 0.0226 h _ l (higher than the maximal growth rates achieved in this experiment) in un-Chelexed, Stn P water when 5 nM of Fe was added without any EDTA. Because of the need to calculate free metal ion concentrations, natural seawater could not be used in this experiment. The results of this experiment, then, pertain to circumstances when artificial seawater is used for trace-metal laboratory experiments. EDTA needs to be present in artificial medium for this diatom, either to increase bioavailability of Fe, or to decrease the free ion concentration of a competing metal (e.g. Cu, Cd). 87 C H A P T E R 5: E C O P H Y S I O L O G Y O F T W O O C E A N I C P H Y T O P L A N K T O N S P E C I E S F R O M T H E N E S U B A R C T I C P A C I F I C I. EFFECT OF IRON ADDITION ON TWO OCEANIC PHYTOPLANKTERS GROWN IN NATURAL NE SUBARCTIC PACIFIC SEAWATER WITH NO ARTIFICIAL CHELATORS PRESENT Introduction Iron is thought to affect not only the rate of primary production of HNLC (High Nutrient Low Chlorophyll) regions (Martin et al. 1989), but also to affect the phytoplankton species composition of these regions (Bruland et al. 1991, Miller et al. 1991, Morel et al. 1991, Price et al. 1991, 1994, Boyd et al. submitted). However, these hypothesis are based on field observations where confounding factors are always present, and on laboratory data derived from 'laboratory' species that are not representative of the regions in question. There is a real gap between laboratory and field data which is indeed difficult to fill, but attempts must be made if an understanding of the ecophysiology of organisms in HNLC regions is to be obtained. One of the largest differences between laboratory and field conditions is the addition of artificial chelators to phytoplankton growth medium in laboratory studies. The addition of artificial chelators may aid in maintaining laboratory maximal growth rates (see Chapter 4, Section II), but the presence of chelators such as EDTA completely alters the metal speciation of the medium, as well as potentially interfering with any natural organic 'chelators' produced by organisms. Trace metals such as Zn and Cu are known to be bound to organic complexes in natural seawater (Donat and Bruland 1990, Moffett et al. 1990), and recently it has been discovered that 99.9% of Fe in seawater is bound to organic compounds, possibly compounds produced by organisms themselves (Hudson 1995, Rue and Bruland 1995). The exact effect of the addition of EDTA to natural seawater is unknown (as the thermodynamics of the organic complexes are largely unknown). It is possible that EDTA may form a stronger bond 88 to metals than the 'natural' compounds or else outcompete natural chelators due to excess quantities typically added to culture medium, both instances interfering with any biological role that the natural metal-organic complexes may have. In this study, I have attempted to grow two phytoplankters from the NE subarctic Pacific in natural, unaltered Stn P water with no artificial chelators present in order to preserve the original chemistry and metal speciation of the seawater. In addition, the effect of a low level input of Fe was also examined, again with no artificial chelators added. This is the first study to compare two oceanic phytoplankton species actually isolated from an HNLC region, as well as comparing them under simulated field environmental conditions. Materials and Methods The oceanic coccolithophore Emiliania huxleyi, and the oceanic diatom Actinocyclus sp., were used for these experiments. Unialgal cultures of both species were grown in Stn P water with no metals or artificial chelators added, and in the same medium with 5 nM total Fe added without chelators. Though not all of the added Fe may have remained dissolved in the seawater, enough was bioavailable to result in a physiological response by the phytoplankton cells. Stn P seawater was collected in May 1994, and experiments were completed within 1 1/2 months of collection. Triplicate cultures were grown semi-continuously in acid-cleaned 3 L polycarbonate flasks under a 14:10 light:dark cycle at 16°C and 180 /xmol m_2 s"* irradiance. All handling of cultures was done under class 100 flowhood conditions, and all plastics coming into contact with the cultures were rigorously acid-cleaned as described in Appendix B. Chelex-treated (Price et al. 1988/89) macronutrients were added (10 NO3-, 1 fiM PO4-3, 10 /xM Si(OH)4) to increase cell numbers in order to obtain accurate cell counts. The total amount of Fe in the medium after all handling and macronutrient additions was measured to be 1.07 +. 0.13 nmol kg _l determined as in Yang (1993). Growth rate, chlorophyll a, particulate carbon, particulate nitrogen, and metal quotas were determined as in previous chapters. Statistical comparisons were made using a Student's t-test at a significant level of p<0.05. 89 Results G r o w t h R a t e In natural Stn P water with 1 nM total Fe present, the oceanic coccolithophore grew maximally, while the oceanic diatom was not able to divide (Fig. 5.1, Table 5.1). After the addition of 5 nM Fe, the oceanic diatom resumed cell division, while the growth rate of the coccolithophore significantly decreased (Fig. 5.1, Table 5.1). This resulted in the diatom growing significantly faster than the coccolithophore with the Fe addition (Table 5.1). The highest growth rate was achieved by E. huxleyi under the low Fe conditions (Fig. 5.1, Table 5.1). C e l l V o l u m e The cell volume of E. huxleyi remained constant regardless of the Fe treatment (Table 5.1). The cell volume of the diatom was significantly greater for the low Fe conditions (Table 5.1). in vivo F luoresencerChlorophyl l a The in vivo fluoresence per chlorophyll a was significantly higher for both species under the low Fe treatment compared to the 6 nM Fe treatment, being about 1.5 times greater under the low Fe treatment (Table 5.1). C h l o r o p h y l l a Quotas The chlorophyll a per cell did not change for either species with Fe treatment (Table 5.1). However, the carbon to chlorophyll a ratio was significantly higher under 1 nM Fe compared to 6 nM Fe for the diatom (Table 5.1). The carbon to chlorophyll a ratio remained constant for E. huxleyi regardless of Fe condition (Table 5.1). C a r b o n a n d Nitrogen Quotas The carbon per cell was significandy greater under the low Fe condition for the diatom, being almost 4 times greater than the carbon per cell under 6 nM Fe (Table 5.1). The carbon per cell remained constant for E. huxleyi regardless of Fe condition (Table 5.1). Similar to carbon, the nitrogen per cell was almost 4 times greater for Actinocyclus sp. grown under 1 nM Fe compared to 6 nM Fe (Table 5.1). Again, there was no difference in 90 CD -t-> cd o O 0.04 0.03 0.02 0.01 0.00 JE 1. huxleyi Actinocyclus sp. *cell division ceased S t n P (No E D T A ) S t n P +5 n M Fe ( N o E D T A ) Fig. 5.1. Specific growth rate for both E. huxleyi and Actinocyclus sp. grown in both natural Station P water and the same water plus 5 nM Fe (no EDTA present in either treatment). Error bars represent ± 1 standard error (n=3). 91 Table 5.1. Physiological parameters for the oceanic diatom Actinocyclus sp. and the oceanic coccolithophore Emiliania huxleyi grown with 1 nM and 6 nM total Fe in Stn P water with no EDTA (+.1 standard error in parenthesis; n=3). Carbon is total carbon, including coccolith carbon (C(t)), cell volumes are spherical equivalents. I^ndicates 75% of Fe(t) as an estimate of internal Fe (Fe(int)). *indicates a significant difference between Fe treatments for a given species with the asterisk placed next to the greater value. it=growth rate; CV= cell volume; C(t)= total carbon including coccolith carbon; C(a)=carbon excluding coccolith carbon. No EDTA Actinocyclus sp. E. huxleyi parameter 1 nMFe 6 nM Fe 1 nMFe 6 nM Fe A4 0 0.0226* 0.0305* 0.0120 (h"l) (0) (0.0018) (0.0071) (0.0007) CV 3080* 2530 44 44 (/xm3) (54) (200) (3) (1) chl a cell - 1 2900 2800 49 44 (fg cell-1) (570) (300) (5.2) (3.5) in vivo Flxhl a 0.17* 0.12 0.30* 0.19 (ng chl a-*) (0.01) (0.01) (0.01) (0.01) C(t) cell"1 514* 136 12.8 10.9 (pg cell - 1) (130) (13) (0.7) (3.1) NcelT 1 170* 45 3.6 2.6 (pg cell"1) (6) (5) (2.3) (1.3) C(t):chl a 180* 49 260 250 (wt) (H) (3) (5) (42) C(t):N 3.5 3.5 4.1 4.9 (molrmol) (0.7) (0.7) (0.4) (0.7) Fe(int):C #65 70 #40 50 (/xmol:mol) (16) (7) (20) (17) Mn:C 16* 9 12 3 (/xmol:mol) (2) (1) (10) (1) 92 the coccolithophore (Table 5.1). The C:N ratio was unaffected by Fe conditions for both species. M e t a l Quotas Despite differences in physiological parameters, the Fe quotas were not significantly different between species or between Fe treatments (Table 5.1). As well, there was much variation among cultures, as indicated by the standard errors. The Mn quotas reflect the general trends seen in Chapters 1 and 2, namely, that Mn quotas were higher for low Fe treatments compared to high Fe treatments (Table 5.1). However, the difference was only significant for the diatom (Table 5.1). Discussion G r o w t h rates In Stn P water with no added Fe, the diatom was not able to divide, whereas the coccolithophore grew at maximal rates (Fig. 5.1, Table 5.1). Dissolved Fe concentrations previously measured for surface values at Stn P vary between 0.05 - 0.4 nmol kg'l (Martin and Gordon 1988, Boyd et al. submitted, Kudo, pers. comm.), with 'dissolved' Fe measured in the field representing only Fe that has passed through a 0.45 /zm filter. The Stn P water (after all handling and additions were made) used in these experiments was not filtered so that the reported Fe values are total Fe. Therefore, the diatom would probably not be able to divide in situ without an additional input of Fe. When 5 nM Fe was added, cell division resumed at a maximal rate for the diatom, which was the same rate as the maximal growth rates achieved in any of the experiments conducted with this species in the laboratory. This maximal rate of growth, 0.023 h'l or 0.8 divisions per day, could be used in conjunction with measured grazing rates of indigenous macrozooplankton in order to determine whether this diatom and others like it could escape grazing pressure in the event of Fe input into the NE subarctic Pacific. Boyd et al. (submitted) estimated maximal growth rates of diatoms in the NE subarctic Pacific (by increases in cell numbers in Fe-enriched carboys) and found diatoms to be growing at a rate of 0.6 - 0.8 divisions per day, which agrees well with the 93 measurements found in the simulated Stn P laboratory experiment presented here. Since the growth rates measured in the experiments presented here are completely independent of confounding field factors (e.g. grazing), the similar rates measured between the laboratory experiment and the field estimate help validate the field estimate of diatom growth rates and indicate that grazing was not affecting the diatom biomass in the carboy. The coccolithophore grew maximally in Stn P water with no artificial chelators or additional metals added, and achieved a growth rate of 0.031 h"l or 1.1 divisions per day, again the maximum rate achieved in all laboratory studies on this species. This rate could be used as an estimate of the small, non-diatom phytoplankton fraction at Stn P (prymnesiophytes) under normal, ambient Fe levels, and used in conjunction with micrograzer estimates to determine if grazing can in fact keep these types of low-Fe tolerant species in check. Boyd et al. (submitted) estimated the growth rates of the indigenous phytoplankton at Stn P (10% diatom C biomass, the rest autotrophic nanofiagellates) by using dilution experiment techniques (Landry and Hassett 1982), obtaining an estimate of 0.7 divisions per day. The lower growth estimate achieved in the field compared to the laboratory may have been due to the presence of diatoms in the mixed assemblage of phytoplankton. However, the higher growth rate achieved with E. huxleyi in the laboratory during the simulated Stn P experiments may also indicate that some field techniques used to estimate growth rates of indigenous phytoplankton are underestimating true growth rates. in vivo Fluoresence:chlorophyll a The higher in vivo fluoresence per chlorophyll a under the low Fe treatment for both species indicates that photosynthetic electron transport was less efficient than in the 6 nM Fe seawater, as would be expected if the cells were in fact Fe-stressed (see Chapter 1). The diatom was indeed stressed to the point of not being able to divide. However, E. huxleyi was growing maximally in the low Fe seawater, despite the higher in vivo Fhchl a than when 6 nM Fe was present. Somehow, under low Fe conditions, E. huxleyi is able to grow maximally despite a reduction in electron transport efficiency. Perhaps the ability of this 94 species to maintain chlorophyll levels under low Fe helps compensate for the decrease in efficiency. Chlorophyll a, Carbon, and Nitrogen Quotas Due to the inability of the diatom to divide under the lower Fe condition (1 nM Fe), the carbon quotas, nitrogen quotas and cell volumes were all much greater than in the 6 nM Fe treatment. The chlorophyll a quota under 1 nM Fe was similar to the chlorophyll a quota under 6 nM Fe due to the cells not dividing, as otherwise the chlorophyll a quotas would have been much lower for the lower Fe treatment (see Chapter 1). Because the cells were deplete in chlorophyll a compared to carbon under the low Fe treatment, a significantly higher C:chl a ratio under the low Fe conditions was observed (Table 5.1). The C:chl a ratio of the diatom in 1 nM Fe was much greater than the ratio maintained by healthy, Fe-replete cells of this species (Table 1.1). E. huxleyi was able to maintain the same C:chl a ratio regardless of the Fe conditions (Table 5.1) with the ratios being similar to that maintained by Fe-replete cells of this species (Table 2.1), again indicating that E. huxleyi is able to maintain its chlorophyll synthesis at lower Fe levels than other phytoplankton. Metal Quotas Even though there were significant physiological differences between Fe treatments and species, the internal Fe quotas were not different regardless of species or Fe treatment. Price and Morel (1994) have noted the general similarity in metal composition of phytoplankton, but there are a number of reasons why my estimates of Fe quotas for these experiments are probably in error. First of all, there was great variability in the measurement of Fe(int):C for triplicate cultures. Even though EDTA is not an ecologically relevant substance to have in seawater, it does help to make the seawater more 'stable' in the sense that small perturbations in the metal concentrations will not likely alter the chemistry of the seawater substantially. In contrast, when no artificial chelators are present, there is a greater chance of the medium being more variable; organics produced by cultures could easily vary among flasks, and any contamination in one vessel could greatly affect the metal chemistry of 95 that vessel. As well as the absence of EDTA making the chemistry of the seawater more variable, Fe quota measurements made when no EDTA is present are thought to overestimate true intracellular Fe quotas by 10 to 100 times (Price and Morel 1994). Hence, although eliminating EDTA from seawater is much more ecological and necessary to address certain ecological questions, its absence makes the chemical definition of natural seawater extremely difficult and hampers the use of non-radiotracer techniques to estimate Fe (and perhaps other metal) quotas. The Mn quotas reflected the same trends found in Chapters 1 and 2, although the variability for E. huxleyi under the low Fe conditions was very high (Table 5.1). This could have in part been due to the use of total carbon instead of acidified carbon values, but acidified carbon values were not available for these experiments. The lower Mn:C ratios measured in this experiment compared to experiments when replete concentrations of Mn were present (Chapter 2) may indicate that Mn concentrations in the natural Stn P water may have been limiting. II. ECOLOGICAL COMPARISON OF EMILIANIA HUXLEYI AND ACTINOCYCLUS SP. Iron Requirements for Growth E. huxleyi vs Actinocyclus sp. The relationship between intracellular Fe (normalized to cellular C) versus specific growth rate for both oceanic species is given in Fig. 5.2. These data represent averages of triplicate or quadruplicate cultures grown in either Stn P water or Chelex-treated Aquil artificial water (Price et al. 1988/89) with nitrate or ammonium as the nitrogen source and 10 fiM EDTA present in all medium. A nonlinear regression was performed on both data sets, obtaining estimates of maximal growth rate (/*max) ^ d the minimum intracellular Fe quota required for cell division to occur (Qmin)- The Droop model (1973) was used for the curve fits. These curves illustrate how the oceanic coccolithophore is better adapted to low Fe 96 conditions than the oceanic diatom, with the minimum intracellular Fe:C required for cell division in the coccolithophore being 3 compared to 16 iimol Fe:mol C in the diatom. Other studies performed under various laboratory conditions have shown strains of E. huxleyi to be well adapted to low metal conditions (Brand et al. 1983). This oceanic strain of E. huxleyi is well adapted to the usual low Fe conditions existing at Stn P, whereas the diatom appears to be adapted to slightly elevated Fe conditions, such as could occur during an atmospheric dust event. Even though large diatoms are numerically rare at Stn P, pulses of particulate Si reach depth at Stn P (3800 m) with Actinocyclus curvatulus being a dominant species in the spring (Takahashi 1986, Takahashi et al. 1990, Wong, pers. comm.). This indicates that diatoms do indeed 'bloom' and sink to depth, and the mechanism is likely that the additional Fe allows larger diatoms to increase their growth rates (as demonstrated in the previous experiments) enough to escape grazing pressure by mezozooplankton, as has been hypothesized previously for the NE subarctic Pacific (Martin and Gordon, 1988, Miller et al. 1991) and recently demonstrated (Boyd et al. submitted). Comparison with other species There are 2 theoretical possibilities that could be useful in comparing the Fe-requirements of different species and results from other studies. These are 1) the minimum intracellular Fe quota required for cell division to occur, and 2) the minimum intracellular Fe quota required for maximal growth. However, in practice, these parameters may not be well defined. In the first case, cultures must be harvested immediately after cell division has ceased, or experiments must be conducted to obtain enough data to define the relationship between internal Fe quotas and growth. In the second case, a range of Fe concentrations needs to be tested, as there is a wide range of intracellular Fe quotas that will result in maximal growth. To be useful as a tool in comparing other species, the minimum intracellular Fe quota required for maximal growth should be used. Both of these parameters can be estimated for E. huxleyi and Actinocyclus sp. from the curves in Fig. 5.2, although not very accurately for the diatom. 97 O O 0 . 0 3 0 0 . 0 2 5 0 . 0 2 0 = 0.0179 max Q . =3.2 j 4 l * 0 . 0 1 5 0 : 0 1 0 1 43 ^ 0 . 0 0 5 <L) - £ o . o o o 43 0 . 0 3 0  0 . 0 2 5 -0 . 0 2 0 0 . 0 1 5 0 . 0 1 0 0 . 0 0 5 0 . 0 0 0 u =0.0204 ^ m a x Q . =15.6 100 "i r E. huxleyi A Z L Actinocyclus sp. ft B 300 400 J L 10000 20000 Fe( int ) :C( t ) ( /xmol :mol) Fig. 5.2. Specific growth rate (measured by changes in cell #) vs intracellular Fe to total cell carbon ratio for A) E. huxleyi and B) Actinocyclus sp. when EDTA (10 /xM) is present. Error bars represent + 1 standard error (n=3 or 4). Non-linear regressions were performed on both data sets to obtain the parameters n m s x Qmin ( s e e t e x t f ° r descriptions). The regression equation was y=Mmax*((x-Qmin)/x)-98 Existing reports for metal quotas (recent measurements only, which have used trace-metal clean techniques) are typically given as the metal:carbon ratio required for 90% of maximal growth. As well, a few authors have reported values of minimum requirements for cell division, although the state of the cultures and the time of sampling is usually not well defined. In general, cyanobacteria appear to have the highest Fe requirements for growth of any phytoplankton species, with marine and freshwater cyanobacteria ranging from 90 to 600 iimol Fe:mol C to maintain 90% of maximal growth (Rueter et al. 1990, Hutchins et al. 1991, Rueter et al. 1992). Only a single dinoflagellate has been examined to date, and the minimum Fe required for cell division to occur was found to be 110 itmol Fe:mol C (Doucette and Harrison 1991). If this one coastal dinoflagellate species (Gymnodinium sanguiniwn) is indicative of all dinoflagellate species, both the oceanic coccolithophore and diatom from the NE subarctic Pacific have far lower Fe requirements for growth. Diatoms are used most frequently in phytoplankton/trace-metal studies, with nearly all data obtained from Thalassiosira spp. The 2 coastal species studied, T. weissflogii and T. pseudonana, are reported to require 5-8 iimol Fe:mol C to maintain 90% of maximal growth (Harrison and Morel 1986, Sunda et al. 1991). An oceanic Thalassiosira sp., T. oceanica, has been reported to have the lowest Fe requirement for growth thus far, requiring only 2 itmol Fe:mol C for 90% of maximal growth (Sunda et al. 1991). An indication of error is not given in the estimates of these ratios. As well, large amounts of EDTA (100 iiM) were used in Fe-stressing the oceanic diatom, which I found to alter the physiology of the oceanic isolates from the NE subarctic Pacific (Chapter 4). From the curves in Fig. 5.2, the Fe:C ratio required for 90% of maximal growth is 20 for E. huxleyi and 76 itmol Fe:mol C for Actinocyclus sp. These values are higher than would be expected based on previous estimates and on the low Fe environment that these organisms came from. This study is the first to use non-radiotracer (or 'cold-metal') techniques to measure internal metal quotas, with all other studies using radiotracer techniques. It is quite possible that cold-metal techniques overestimate intracellular Fe 99 concentrations at low particulate Fe values, especially when cells are Fe-stressed. Because there is very little Fe in media when cells are Fe-stressed, the physical adsorption of Fe onto filters is minimal, and I found no significant contamination of filters by the Ti(UI) treatment. However, Fe-stressed cells will likely have empty uptake sites on their outer membrane, and the Ti(IU) reagent may not be able to 'pull' any bound Fe off. When radiotracers are used, any cold Fe bound to the exterior of the cell will not influence the Fe quota estimate. As well, Fe-stressed cells may be able to transport Fe across their membranes during the soaking step with the Ti(HT) reagent, although this should affect both radiotracer and cold-metal estimates. The lack of error estimates in previous reports on Thalassiosira sp. is unnerving, as there is always variability between cultures, and even if the Fe measurements are incredibly precise, carbon quotas vary between cultures as well, and sampling and filtration procedures add variability to both of these estimates. Even though my Fe:C ratios may be greater than previously reported diatom ratios, the difference between the oceanic coccolithophore and the oceanic diatom is still obvious. It is possible that a factor may be determined which could then be subtracted from Fe quotas below a given concentration, but this would involve performing radioisotope and cold-metal techniques on the same cultures under a range of Fe conditions. The aim of this study was primarily to compare and contrast the oceanic coccolithophore and the oceanic diatom within the framework of these experiments. Mn and Zn Requirements The Mn and Zn quotas for E. huxleyi and Actinocyclus sp. can be compared with other studies in which all metals were present at replete levels and all culture conditions were saturating for growth. Mn:C ratios measured for Actinocyclus sp. under replete conditions were ca. 40 /*mol Mn:mol C (Table 1.1), and for E. huxleyi were 10 - 15 /xmol Mn:mol C when normalized to total carbon, and 15 - 25 /*mol Mn:mol C when normalized to acidified carbon (coccoliths removed) (Table 2.1). For Zn:C ratios, Actinocyclus sp. maintained about 5 /xmol Zn:mol C while E. huxleyi maintained 17 - 35 jtmol Zn:mol C depending on whether 100 normalized to total carbon or acidified carbon (Table 1.1, Table 2.1). Comparing these 2 species, the diatom maintains up to 4 times more Mn than the coccolithophore, and the coccolithophore maintains 3 to 7 times more Zn than the diatom. Mn and Zn quotas have been measured for other species, but radiotracer techniques were used. However, the cold-metal determinations made here agree well with previous literature values. Mn:C ratios for 2 diatom species, both Thalassiosira sp., have been reported to be between 22 - 32 iimol Mn:mol C (Sunda 1988/89). That places Actinocyclus sp. slightly higher and E. huxleyi slightly lower than previous reports for Mn:C ratios. Zn quotas have previously been measured for 3 diatom species (all Thalassiosira sp.) and 2 isolates of E. huxleyi (one from the Sargasso Sea, and one from the Gulf of Mexico), and range from 11-14 for the diatoms and as low as 1 ttmol Zn:mol C for the E. huxleyi isolates (Sunda and Huntsman 1992). Hence, Actinocyclus sp. maintains a 2- 3 times lower Zn:C ratio than previously reported diatom requirements. However, the E. huxleyi isolate from the subarctic Pacific appears to have a higher requirement for Zn than both previously reported diatoms and other isolates of E. huxleyi. Isolates of E. huxleyi vary not only physiologically but genetically as well (Brand 1982, vanBleijswijk et al. 1991, Lecourt et al. in press), and my isolate from the NE subarctic Pacific is the only one from an HNLC region. Perhaps one of the ways E. huxleyi is able to grow maximally under such low Fe conditions in the subarctic Pacific, is by a replacement of normal Fe functions by Zn, resulting in higher Zn requirements. E. huxleyi and Actinocyclus sp. at Station P From the physiological experiments conducted here, E. huxleyi appears to be an organism able to survive and grow maximally under the low Fe (and other metal) concentrations typically found at Stn P. As well, this small oceanic coccolithophore may be utilizing nitrate to meet its nitrogen requirements. In contrast, the oceanic diatom Actinocyclus sp. is not as well adapted to typical low metal conditions at Stn P, but once Fe is added to the system, this diatom is able to grown maximally (and faster than the 101 coccolithophore, Fig. 5.1). This 20-60 /zm diatom is most likely utilizing ammonium to meet its nitrogen requirements, and may switch to nitrate utilization when Fe is available. The biomass of E. huxleyi at Stn P will therefore be dependent upon micrograzer rates at low Fe concentrations (since they are growing maximally in natural Stn P water), and may also be affected by Fe during atmospheric input events, with higher concentrations of Fe possibly slowing down its growth rate. The biomass of Actinocyclus sp., on the other hand, is likely controlled by Fe, as low Fe concentrations severely inhibited its growth. Upon addition of Fe to surface waters, Actinocyclus sp. could divide faster, and the resulting biomass would be largely determined by mezozooplankton grazing rates. Because of the large pulses of diatom material found in sediment traps at Stn P, Actinocyclus sp. and other large diatoms must be able to escape grazing pressure during times of accelerated growth. This scenario based on physiological observations in the laboratory supports the hypotheses that have been put forward based on fieldwork in the NE subarctic Pacific. Boyd et al. (submitted) conclude that small cells <5 /xm are primarily grazer controlled, and are most likely not limited by Fe and growing maximally. As well, they conclude that mezozooplankton are not able to keep up with larger diatom growth rates once Fe has been added to the system. Their conclusions confirm the Fe/grazing hypothesis stated previously by Miller et al. (1991). Field researchers have found that initially rare large diatoms grow up in carboys to which Fe has been added (Martin et al. 1989, Coale 1991, Boyd et al. submitted). It is difficult to interpret increases in biomass during field experiments because grazers are present, but my experiments indicate that the division rate of diatoms does in fact increase when Fe is added to natural Stn P water (Fig. 5.1). My experiments would also indicate that any increase in biomass of small non-diatoms found in carboy enrichment experiments is probably due to a detrimental effect on the micrograzers, as these cells are probably growing maximally and their biomass does not increase in situ. My physiological results from laboratory data have helped interpret phenomenon encountered in field studies, and provide independent, physiologically based support for the role of Fe in controlling phytoplankton assemblages at Stn P. 102 GENERAL CONCLUSIONS The general conclusions of this thesis will be given in the same order as the chapters. In Chapter 1, the effect of nitrogen source (nitrate vs ammonium) on the physiological health of an oceanic diatom under both Fe-replete and Fe-stressed conditions was examined. It was found that nitrate-grown cells of Actinocyclus sp. were indeed more physiologically stressed compared to ammonium-grown cells under low Fe conditions, as predicted by theoretical expectations based on Fe and energy requirements of nitrate vs ammonium utilization. The results indicate that the oceanic diatom is probably utilizing ammonium to meet its nitrogen requirements in situ at Stn P, as ammonium-grown cells maintained a 'healthier' physiological state than nitrate-grown cells under the low Fe conditions tested. Chapter 2 reports similar experiments conducted with an oceanic coccolithophore, and the results obtained with this species are in contrast to those which were found for the oceanic diatom. E. huxleyi appeared to gain no physiological advantage by utilizing ammonium rather than nitrate under low Fe conditions despite the theoretical calculations predicting otherwise. As well, this small coccolithophore was better off physiologically when it utilized nitrate rather than ammonium under the lowest Fe conditions tested, mainly due to the drastic decrease in cell volume exhibited by nitrate-grown but not ammonium-grown cells under Fe-stressed conditions. The results from Chapter 3 indicate that Fe affects the sinking rate of the large oceanic diatom but not the small oceanic coccolithophore, supporting the notion that electron transport processes are responsible for maintaining buoyancy regulation in large diatoms. When Fe conditions were replete, the large oceanic diatom and the small oceanic coccolithophore exhibited similar, low sinking rates. Under Fe-stressed conditions, the diatom's sinking rate increased five times, whereas the sinking rate of the coccolithophore remained constant. The different effect that low Fe conditions had on the sinking rates of these 2 species could have ecological implications for the sedimentation of siliceous and carbon material, where faster 103 sinking, unhealthy diatom cells may be more likely to sink to depth than smaller cells which maintain their physiological capabilities. Chapter 4 examined the effect of EDTA on oceanic phytoplankton, and it was found that the concentration of EDTA typically used in phytoplankton/trace-metal studies (100 tiM) was toxic to these 2 oceanic species, causing decreases in growth rates and interference with carbon metabolism. As well, when using artificial seawater medium, some EDTA appears to be necessary to achieve maximal growth for the oceanic diatom. Chapter 5 reported the results of an Fe addition experiment conducted in natural Stn P water with no artificial chelators or additional metals or vitamins added (and only slight amounts of macronutrients added). This simulated 'Stn P-like' experiment demonstrated that the oceanic coccolithophore E. huxleyi can grow maximally in natural Stn P water with no additions, while the oceanic diatom Actinocyclus sp. required the addition of Fe in order to divide. This direct evidence, along with other physiological evidence derived from more laboratory-oriented experiments, demonstrates that E. huxleyi is much better adapted to the 'typical' low-Fe conditions normally encountered at Stn P than the diatom. This is consistent with the observation that small non-diatoms are numerically the most dominant phytoplankton component found at Stn P. However, possible implications of these results are that in the event of Fe input via atmospheric deposition, the oceanic diatom could grow maximally and escape grazing pressure, form a short-lived 'bloom' until the added Fe was used up, and the resulting Fe-stressed cells would lose physiological control, increasing their sinking rates (perhaps forming clumps and accumulating other particles), which would result in their rapid flux to depth. The findings of this thesis demonstrate that different oceanic phytoplankton exhibit different physiological responses and adaptations to similar Fe conditions, and compares and contrasts the differences found between a small oceanic coccolithophore and a large oceanic diatom. As well as reporting the first basic physiological data on phytoplankton isolates from an HNLC region, the thesis also provides independent, physiological evidence supporting the role of Fe in controlling phytoplankton assemblages in the NE subarctic Pacific. 104 FUTURE RESEARCH This thesis examined some basic physiological responses of oceanic phytoplankton with respect to Fe nutrition, and a more in-depth look at specific metabolic processes would be the next step. Chapters 1 and 2 illustrated that E. huxleyi and Actinocyclus sp. respond differently to nitrogen sources, but the actual uptake of nitrogen was not measured. To really examine what nitrogen source these phytoplankton are utilizing in situ at Stn P, the uptake of both nitrate and ammonium should be measured under ecological concentrations and conditions. Both nitrogen sources should be present in the growth medium, which makes using l^N techniques impossible. Hence, the uptake would have to be measured by disappearance from the medium with an autoanalyzer. This would be especially difficult for ammonium, since the ambient concentrations are so low, so the concentration of ammonium in the medium would probably have to be elevated. The uptake of both nitrogen sources could also be measured in a 'transition1 experiment, where uptake is measured before and after the addition of Fe. This would indicate whether either species actually 'switches' from ammonium to nitrate utilization upon Fe addition, and would further help interpret field results (whether increased draw-down of nitrate upon Fe addition to carboys is due to cells growing up that use nitrate instead of ammonium, or whether the same species switch from ammonium to nitrate utilization). The other interesting physiological result that warrants further investigation is the increased Mn levels under low Fe conditions that was found for both species and for a coastal diatom previously (Harrison and Morel 1986). Because Mn is required for the splitting of water in the photosystems (PS IT), its increase under Fe-stress may indicate an increase in the activity of photosynthesis, or possibly a substitution of Mn for a 'normal' Fe function. The cellular Mn levels could be measured by the cold-metal techniques used in this thesis, and different aspects of photosynthesis could be examined with oxygen electrodes and other methods. Exarnining this phenomenon in more depth may illuminate an adaptation allowing some species to exist in low Fe environments. 105 LITERATURE CITED Banse K. 1990 Does iron really limit phytoplankton production in the offshore subarctic Pacific? Limnol. Oceanogr. 35:772-775 Banse K. 1991 Iron availability, nitrate uptake, and exportable new production in the subarctic Pacific. J. Geophys. Res. 96:741-748 Bienfang, P. K. 1981 SETCOL - A technologically simple and reliable method for measuring phytoplankton sinking rates. Can. J. Fish. Aquat. Sci. 38:1289-1294 Booth, B. C. 1980 Vernal phytoplankton community in the eastern subarctic Pacific: Predominant species. 6th Diatom Symposium, p. 339-358 Booth, B. C. , J. Lewin, R. E. Norris 1982 Nanoplankton species predominate in the subarctic Pacific in May and June 1978. Deep-Sea Res. 29:185-200 Booth, B. C. 1988 Size classes and major taxonomic groups of phytoplankton at two locations in the subarctic Pacific in May and August 1984. Mar. Biol. 97:275-286 Booth, B. C , J. Lewin, C. J. Lorenzen 1988 Spring and summer growth rates of subarctic Pacific phytoplankton assemblages determined from carbon uptake and cell volumes estimated using epifluorescence microscopy. Mar. Biol. 98:287-298 Boyd, P., D. L. Muggli, D. Varela, R. Chretien, R. Goldblatt, K. J. Orians, P. J. Harrison Concurrent iron enrichment and herbivory experiments in the NE subarctic Pacific. Mar. Ecol. Prog. Ser. submitted Boyd, P., R. Goldblatt, P. J. Harrison Mesozooplankton grazing manipulations during an in vitro iron enrichment in the NE subarctic Pacific. Nature submitted Brand, L . E. 1982 Genetic variability and spatial patterns of genetic differentiation in the reproductive rates of the marine coccolithophores Emiliania huxleyi and Gephyrocapsa oceanica. Limnol. Oceanogr. 27:236-245 Brand, L. E . , W. G. Sunda, R. R. L. Guillard 1983 Limitation of marine phytoplankton reproductive rates by zinc, manganese, and iron. Limnol. Oceanogr. 28:1182-1198 Brand, L . E. 1994 Physiological ecology of marine coccolithophores. In: Winter, A. , Siesser, W. G. (eds) Coccolithophores. Cambridge University Press, New York, p. 39-49 Bruland, K. W,. J. R. Donat, D. A. Hutchins 1991 Interactive influences of bioactive trace metals on biological production in oceanic waters. Limnol. Oceanogr. 36:1555-1577 106 Cavard, D., S. P. Howard, C. Lazdunski 1989 Functioning of the colicin A lysis protein is affected by triton X-100, divalent cations and EDTA. J. Gen. Microbiol. 135:1715-1726 demons, M . J. , C. B. Miller 1984 Blooms of large diatoms in the oceanic, subarctic Pacific. 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In: Lauchli, A. & Bielesky, R. K. (eds.) Encyclopedia of Plant Physiology, New Series, Vol 15: Inorganic Plant Nutrition. Springer Publ., Berlin, Heidelberg, pp. 63-96 Smetacek, V. S. 1985 Role of sinking diatom life-history cycles: ecological, evolutionary and geological significance. Mar. Biol. 84:239-251 Spiller, S. C , N. Terry 1980 Limiting factors in photosynthesis. II. Iron stress diminishes photochemical capacity by reducing the number of phyotosynthetic units. Plant Physiol. 65:121-125 Spiller, S. C , A. M . Castelfranco, P. A. Castelfranco 1982 Effects of iron and oxygen on chlorophyll biosynthesis. I. In vivo observations on iron and oxygen-deficient plants. Plant Physiol. 69:107-111 Sunda, W. G. , S. A. Huntsman 1983 Effect of competitive interactions between manganese and copper on cellular manganese and growth in estuarine and oceanic species of the diatom Thalassiosira. Limnol. Oceanogr. 28: 924-934 Sunda, W. 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Wilkinson 1992 Sensitivity of Pseudomonas stutzeri to EDTA: Solubilization of outer-membrane components. Microbios 72:109-118 Terry, N. 1980 Limiting factors in photosynthesis I. Use of iron stress to control photochemical capacity in vivo. Plant Physiol. 65:114-120 Terry, N. 1983 Limiting factors in photosynthesis IV. Iron stress-mediated changes in light-harvesting and electron transport capacity and its effects on photosynthesis in vivo. Plant Physiol. 71:855-860 Thompson, P. A. , M . E . Levasseur, P. J. Harrison 1989 Light-limited growth on ammonium vs. nitrate: What is the advantage for marine phytoplankton? Limnol. Oceanogr. 34:1014-1024 Turpin, D. H . , D. Bruce 1990 Regulation of photosynthetic light harvesting by nitrogen assimilation in the green alga Selenastrum minutum. FEBS 263:99-103 van Bleijswijk, J., van der Wal, P., Kempers, R., Veldhuis, M . , Young, J.R., Muyzer, G., deVrind-deJong, E . , P. Westbroek 1991 Distribution of two types of Emiliania huxleyi (Prymnesiophyceae) in the northeast Atlantic region as determined by immunofluoresence and coccolith morphology. J. Phycol. 27:566-570 Waite, A. M . , P. A. Thompson, P. J. Harrison 1992 Does energy control the sinking rates of marine diatoms? Limnol. Oceanogr. 37:468-477 Waite, A. M . , A. Fisher, P. A. Thompson, P. J. Harrison Energetic sinking rate control in marine diatoms: a spectrum of responses. Limnol. Oceanogr. submitted Walker, J. B. 1953 Inorganic micronutrient requirements of Chlorella. I. Requirements for calcium (or strontium), copper and molybdenum. Arch. Biochem. Biophys. 53:1-8 113 Wells, M . L . , N. M Price, K. W. Bruland 1995 Iron chemistry in seawater and its relationship to phytoplankton: a workshop report. Mar. Chem. 48:157-182 Westall, J. C , Zacharyl, L . , F. M. M . Morel 1976 MTNEQL: a computer program for the calculation of chemical equilibrium composition in aqueous systems. Tech. Note No. 18, R. M . Parsons Lab for Water Resources and Environmental Engineering. MIT, Cambridge Dept. of Civil Engineering, pp. 91 Yang, L . 1993 Determination of dissolved trace metals in the western North Pacific. M.Sc. Thesis, University of British Columbia, Vancouver, Canada, pp. 104 Young, J. R., P. Westbroek 1991 Genotypic variation in the coccolithophorid species Emiliania huxleyi. Mar. Micropaleontol. 18:5-23 114 A P P E N D I X A : M A I N T E N A N C E M E D I U M F O R EMILIANIA HUXLEYI A N D ACTINOCYCLUS S P . Following are the maintenance media for both Emiliania huxleyi and Actinocyclus sp. cultures. Cultures were kept in the NEPCC culture collection growth chamber at 16°C under low light (ca. 30 itmol m~2 s"l) until needed for experimentation. Medium was microwave-sterilized as in Keller et al. (1988). E. huxleyi: 1) Stn P water (no metals or EDTA added), 30 itM NO3-, 5 itM PO4- 3 2) Stn P water (no metals or EDTA added), 30 itM NH4+, 5 itM PO4- 3 3) Stn P water (no metals or EDTA added), 15 itM NO3-, 15 itM NH.4+, 5 ttM PO4-3 4) Stn P water plus metals* and 10 11M EDTA, 30 itM NO3-, 5 itM P04"3> ESAW vitamins (Harrison et al. 1980) 5) Stn P water plus metals* and 10 /xM EDTA, 30 itM NH4+, 5 itM P04"3) ESAW vitamins (Harrison et al. 1980) 6) Stn P water plus metals* and 10 itM EDTA, 15 itM NO3-, 15 itM NH4+, 5 /xM PO4- 3 ESAW vitamins (Harrison et al. 1980) Actinocyclus sp.: Stn P water plus metals* and 10 itM EDTA, 50 itM NO3-, 100 ttM Si(OH)4, 5 itM PO4- 3, ESAW vitamins (Harrison et al. 1980) *metal additions were made to final Aquil amounts (23 nM Mn, 8 nM Zn, 1 nM Cu, 2.5 nM Co, 100 nM Mo, 10 nM Se; Price et al. 1988/89), except Zn, which was doubled to reflect the higher requirement for Zn found in E. huxleyi. 100 nM Fe was added for E. huxleyi; 1000 nM Fe for Actinocyclus sp. 115 A P P E N D I X B : P R O C E D U R E F O R A C I D - C L E A N I N G P L A S T I C S This is a rigorous way to clean all plastics used for metal work, and any plastics coming into contact with cultures. Suitable plastics include HDPE, PC, PP, and Teflon®. Supplies routinely cleaned in this way include pipette tips, GFAA vials, 30 ml sample bottles, petri dishes, all PC culture vessels and accompanying Teflon® tubing and silicon stoppers. 1. Organic wash. Acetone or Sparkleen can be used. Do not use brushes or abrasives as this alters the surface layer of the plastic. 2. Soak in 25% reagent grade HC1 for at least 24 hours. For extra rigorous applications, heat as well. 3. Rinse twice with Nanopure water (or milliQ or equivalent). 4. Soak in 0.1 % Ultra HC1 (Seastar Chemicals) for 1 week. 5. Leave in Ultra-clean acid (e.g. filters), or pour off and dry carefully in drying oven. 116 A P P E N D I X C : P R O G R A M S U S E D F O R T H E D E T E R M I N A T I O N O F F E , M N , Z N , A N D C U O N T H E V A R I A N G R A P H I T E F U R N A C E A A Step Temp. Time Gas Flow Read Number (°C) (sec) (L/min) Command Fe, 4% H N O 3 , platform: 1 300 3.0 3.0 NO 2 300 35.0 3.0 NO 3 1000 5.0 3.0 NO 4 1000 5.0 3.0 NO 5 1000 1.0 0.0 NO 6 2550 0.8 0.0 YES 7 2550 3.0 0.0 YES 8 2550 2.0 3.0 NO 9 40 13.3 3.0 NO Mn, 4% H N O 3 , platform: 1 300 5.0 3.0 NO 2 300 30.0 3.0 NO 3 800 5.0 3.0 NO 4 800 3.0 3.0 NO 5 800 1.0 0.5 NO 6 2500 1.0 0.5 YES 7 2500 2.0 0.5 YES 8 2500 1.0 3.0 NO 9 40 13.3 3.0 NO Zn, 4% H N O 3 , platform: 1 300 3.0 3.0 NO 2 300 16.0 3.0 NO 3 700 5.0 3.0 NO 4 700 3.0 3.0 NO 5 700 1.0 0.1 NO 6 2100 0.7 0.1 YES 7 2100 2.0 0.1 YES 8 2300 1.0 3.0 NO 9 40 12.3 3.0 NO Cu 4% H N O 3 , platform: 1 300 5.0 3.0 NO 2 300 45.0 3.0 NO 3 1000 3.0 3.0 NO 4 1000 3.0 3.0 NO 5 1000 1.0 0.0 NO 6 2550 0.9 0.0 YES 7 2550 3.5 0.0 YES 8 2550 1.0 3.0 NO 9 40 13.3 3.0 NO 

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