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Inorganic carbon acquisition by natural phytoplankton assemblages and marine diatoms Martin, Cheryl Lynne 2005

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INORGANIC CARBON ACQUISITION BY NATURAL PHYTOPLANKTON ASSEMBLAGES AND MARINE DIATOMS By CHERYL LYNNE MARTIN B.Sc, University of British Columbia, Vancouver, 2002 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Botany) THE UNIVERSITY OF BRITISH COLUMBIA April 2005 © Cheryl Lynne Martin, 2005 Thesis Abstract Marine phytoplankton (microalgae and cyanobacteria) play a vital role in the global carbon cycle. It is therefore essential to understand the mechanisms involved in inorganic carbon (C) acquisition by these organisms. I examined C uptake strategies of natural phytoplankton assemblages in the Bering Sea and numerous individual marine diatom species in the laboratory. This study employs the isotope disequilibrium method to quantitatively determine the C uptake strategies of phytoplankton, and the degree to which they utilize carbon dioxide (CO2) and/or bicarbonate (HCO3") as their C source for photosynthesis. The first part of this study investigated C acquisition strategies of natural phytoplankton communities in the Bering Sea. HCO3" seemed to be the predominant C source for all phytoplankton assemblages, and the results suggest that HCO3" utilization occurred mainly through a direct transport system. Although species composition and ambient CO2 concentration did not appear to influence direct HCO3" transport, these parameters did appear to influence indirect HCO3" utilization by the enzyme external carbonic anhydrase (eCA). Ship-board CO2 manipulation incubations were performed and there was a statistically significant CO2 effect on the pathways of C assimilation. The second part of this study examined C uptake strategies of several diatom species in the laboratory. To my knowledge, no studies have thoroughly investigated an array of marine diatom species in relation to their C acquisition strategies. The majority of the diatom species utilized HCO3" as their key C source, and it appears that most of the HCO3" utilization occurred through a direct transport process. The C acquisition strategies of the diatoms in this study appear to be independent of diatom size (i.e. surface area: volume ratio) and growth rate. iii TABLE OF CONTENTS THESIS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES vi LIST OF FIGURES viii ACKNOWLEDGEMENTS xi CHAPTER 1: GENERAL INTRODUCTION 1 How Phytoplankton Affect Atmospheric CO2 Concentrations 1 How CO2 Concentrations Affect Phytoplankton 3 How Phytoplankton Deal With a Low CO2 Environment 5 Carbon Acquisition Strategies of Natural Phytoplankton Communities 9 Thesis Objectives 12 CHAPTER 2: INORGANIC CARBON ACQUISITION BY NATURAL PHYTOPLANKTON ASSEMBLAGES IN THE BERING SEA 14 Introduction 14 Materials and Methods 17 Sampling locations 17 Isotope disequilibrium experiments 18 CO2 incubation experiments 19 Data analysis and modeling 20 Results 24 In situ stations 24 iv Isotope disequilibrium experiments 25 CO2 incubation experiments 26 Discussion 37 Direct HCOi transport 38 External carbonic anhydrase activity 39 Conclusions 43 C H A P T E R 3 : I N O R G A N I C C A R B O N A C Q U I S I T I O N B Y M A R I N E D I A T O M S 4 5 Introduction 45 Materials and Methods 48 Algal strains and culturing conditions 48 Microscope analysis 49 Isotope disequilibrium procedure 50 Results 52 Discussion 61 Carbon acquisition strategies 61 Carbon acquisition in relation to diatom size 64 Carbon acquisition in relation to diatom growth rate 66 Conclusions 68 F U T U R E R E S E A R C H 6 9 R E F E R E N C E S 7 1 v List of Tables Table 2.1: Nutrient concentrations and biological characteristics of the Bering Sea sampling stations (CO2 concentrations are only reported for membrane inlet mass spectrometry). Error bars represent ± standard error (n=3) 30 Table 2.2: Isotope disequilibrium results from the Bering Sea sampling stations. The /HC03-value represents the fraction of direct HCO3" transport (see Materials and Methods in Chapter 2 for a description of the analysis). Error bars represent ± standard error (n=2). Phytoplankton were collected for experiments using 2.0um filters with the exception of stations 5 and 6 where 20u.m filters were also used 32 Table 2.3: Isotope disequilibrium results from CO2 manipulation experiments. The /HC03-value represents the fraction of direct HCO3" transport. Error bars represent ± standard error (n=2). Phytoplankton were collected for experiments using 2.0um filters 36 Table 3.1: A list of marine diatom species (and their corresponding culture identifications) used in isotope disequilibrium experiments during summer 2002 through winter 2003 54 Table 3.2: Isotope disequilibrium results from marine diatom species. The/HC03-value represents the fraction of direct HCO3" transport. Error bars represent ± standard error (n=2-4). Diatom species that were investigated in both 2002 and 2003 are distinguished with (yr=2) being noted beside the Culture ID 57 vi Table 3.3: Surface area: volume (SA/V) ratio and b) growth rate (u) (day"1) for each marine diatom species. Error bars for growth rates are ± standard error (n=10-15). Diatom species that were investigated in both 2002 and 2003 are distinguished with (yr=2) being noted beside the Culture ID 59 vii List of Figures Figure 2.1: Bering Sea sampling locations for isotope disequilibrium experiments 28 Figure 2.2: a) Expected versus predicted a-values (i.e. modeled eCA activity) for hypothetical/HC03-values. b) Expected versus predicted/HC03-values for hypothetical a-values. The integral equation (4) (chapter 2) was applied to generate predicted Gl-and /HC03-values 29 Figure 2.3: Time course of 1 4 C incorporation during isotope disequilibrium experiments for stations (a) 3, (b) 4, (c) 7, and (d) 8. The best-fit curves were obtained by applying equation (4) (see Materials and Methods, Chapter 2). The dashed curves represent a CGvonly uptake model fit. Error bars represent ± standard error (n=2) and are smaller than the symbol when not visible 31 Figure 2.4: Instantaneous 1 4 C uptake rates for stations (a) 3 and (b) 4 during isotope disequilibrium experiments. Best-fit curves were obtained by applying equation (1) (see Materials and Methods, Chapter 2), Error bars represent ± standard error (n=2) and are smaller than the symbol when not visible 33 Figure 2.5: Apparent enhancement of the H C O 3 7 C O 2 exchange (%) (external carbonic anhydrase activity) in relation to station CO2 concentrations measured with (a) CO2 SYS program and (b) Membrane inlet mass spectrometry (MIMS) 34 viii Figure 2.6: Effects of low (150ppm) and high (750ppm) CO2 conditions on I 4 C incorporation (normalized to chlorophyll) during isotope disequilibrium experiments for the (a) first and (b) second CO2 incubation experiments. The best-fit curves were obtained by applying equation (4) (see Materials and Methods, chapter 2). Error bars represent ± standard error (n=2) and are smaller than the symbol when not visible...35 Figure 3.1: Time course of 1 4 C incorporation during isotope disequilibrium experiments for a) Thalassiosira weissflogii (TW), b) Leyanella arenaria (694), c) Pseudo-nitzschia turgidula (SPI), and d) Cylindrotheca fusiformis (425). The best-fit curves were obtained by applying equation (4) (see Materials and methods, chapter 2). The dashed curves represent a CCVonly uptake model fit. These time courses display a high level of direct HCO3" transport. Error bars represent ± standard error (n=2) and are smaller than the symbol when not visible 55 Figure 3.2: Time course of 1 4 C incorporation during isotope disequilibrium experiments for a) Nitzschia thermalis (608), b) Chaetoceros socialis (653), c) Phaeodactylum tricornutum (640), and d) Pseudo-nitzschia grand (PG1). These time courses display a relatively higher amount of CO2 transport, (see Fig. 3.1 for further details) 56 Figure 3.3: Time course of 1 4 C incorporation during isotope disequilibrium experiments for Thalassiosira pseudonana (TP) in a) 2002 and b) 2003. The best-fit curves were obtained by applying equation (4) (see Materials and methods, chapter 2). The dashed curves represent a CCVonly uptake model fit. Error bars represent ± standard error (n=2) and are smaller than the symbol when not visible 58 ix Figure 3.4: /Hco3-values (fraction of direct HCO3" transport) over a range of a) surface area: volume (SA/V) ratios and b) growth rates (p day"1). Apparent enhancement of the HCO37CO2 exchange (%) (external carbonic anhydrase activity) over a range of c) SA/V ratios and d) growth rate. Error bars represent ± standard error (n=2-4) and are smaller than the symbol when not visible 60 x Acknowledgements It is a pleasure to thank the many people who made this thesis possible. I would like to gratefully acknowledge the enthusiastic supervision of Philippe Tortell. He provided encouragement, guidance and support throughout the course of this work. He was persistent in driving me to excel with my research and writing, and I thank him tremendously for that. Drawing on his endless knowledge of oceanography, he enriched my acquaintance with the subject through work in the laboratory, coaching sessions and three research expeditions at sea. There are also many more people at UBC I want to acknowledge. I would like to thank my supervisory committee, Tony Glass, Robert Guy, and Robert DeWreede for providing guidance in the early stages of my thesis. Julie Granger has been abundantly helpful and encouraging by providing me with sound advice and excellent ideas for my work. She will make a phenomenal supervisor for some lucky students in the future. Miranda Corkum and Nina Nemcek have been great friends to have on this journey. I have learned so much from all our experiences. Maite Maldonado, Tawnya Peterson, Adrian Marchetti, and Shannon Harris have also provided help with experimental setup, general advice, and good company. Darren Tuele was extremely valuable during two research cruises to St. Papa. I would like to thank him for building anything AND everything for my onboard experiments. I am also grateful to Carol Leven (Oceanography Office), Lebby Balakshin and Veronica Oxtoby (Botany Office) for providing assistance with a number of bureaucratic matters. Lebby and Veronica were keen to answer my questions and always reminded me of important academic procedures and deadlines. I am also especially grateful to my friends and family. My two best friends, Anna and Carolyn, have always been so supportive of me. In addition, it has always been a pleasure to ensure my knowledge of the Fashion industry and the Arts are up-to-date. I am grateful to my parents for 'repeatedly' asking what I do so I can 'repeatedly' explain my research to them. These explanations have helped me understand the importance of my research on a broader scale. Finally, I am forever indebted to my fiance, Andrew. I am very grateful to Andrew for his understanding, endless patience and encouragement when it was most required. He has never complained about reading my writing and has been so helpful every step of the way. Thank you Andrew. On top of the knowledge gained in doing my thesis, I have learned so much about my abilities, my goals and myself. This has been a stimulating adventure that will, for years to come, encourage me to expand the body of oceanographic knowledge in our community. This work was supported by the Natural Sciences and Engineering Research Council of Canada. xii Chapter 1: General Introduction How Phytoplankton Affect Atmospheric C 0 2 Concentrations Years of research in oceanography have expanded our understanding of the oceans' contribution to the global carbon cycle. The oceans are a sink for substantial amounts of naturally occurring and anthropogenically released carbon dioxide (CO2), and thus, play a fundamental role in regulating global CO2 concentrations. It has been suggested that the oceans have regulated atmospheric CO2 levels throughout glacial-interglacial cycles for the past 500,000 years (Petit et al. 1999). Levels of atmospheric CO2 have historically been between 180ppm during glacial times, and 280ppm during interglacial times (Petit et al. 1999). However, since the Industrial Revolution, the concentration of CO2 in the atmosphere has risen at an unprecedented rate to a present-day value of ~360ppm (Falkowski et al. 2000). It is estimated that in the absence of phytoplankton in the world's oceans, CO2 concentrations would be 150 to 200ppm higher than present day (Falkowski et al. 2000). Marine phytoplankton (microscopic algae and cyanobacteria) utilize CO2 for photosynthesis in the sunlit surface waters (the euphotic zone). Once CO2 in the surface ocean is utilized by the phytoplankton, roughly 25% of the organic carbon produced sinks from the surface water into the deep ocean (Falkowski et al. 1998). The transfer of carbon from the upper to the deep ocean increases dissolved inorganic carbon (DIC) concentrations below the euphotic zone (Raven and Falkowski 1999) due to the oxidation of organic carbon by heterotrophic bacteria. This transfer of carbon enables the surface ocean to absorb additional CO2 from the atmosphere, hence, increasing oceanic carbon 1 uptake. This process is referred to as the 'biological pump' (Volk and Hoffert 1985). The world's oceans provide a huge reservoir for carbon with an estimated "50 times more soluble inorganic carbon than the atmosphere" (Raven and Falkowski 1999). The CO2 that is sequestered into the deep ocean reservoir will not exchange with the atmosphere for approximately 1,000 years (Bearman, ed. 2001). The biological pump is essential for regulating atmospheric CO2, and therefore, it is equally important to understand what regulates the efficiency of this process. The availability of essential nutrients, such as nitrate, phosphate, silicate, iron, and the amount of light, have a significant impact on the strength of the biological pump given that phytoplankton growth can be limited if one nutrient is in short supply. The availability of nutrients also influences phytoplankton community structure (Chisholm 1992), which in turn contributes to the efficiency of the biological pump. For instance, a community dominated by diatoms will be very efficient at exporting carbon into the oceans' interior (Raven and Falkowski 1999) as a result of the diatoms' large size and fast-sinking rates. It is well known that these variables impact the strength of the biological pump, and thus, phytoplankton growth. However, surprisingly little information is available regarding pathways of carbon (C) uptake of phytoplankton and their responses to changing atmospheric CO2 concentrations. It is therefore essential to develop our understanding of the mechanisms of C uptake and how various CO2 concentrations affect these processes. My thesis will contribute significantly towards understanding mechanisms of C uptake in both the field and laboratory, and will reveal how ambient CO2 concentrations affect C uptake. 2 How C 0 2 Concentrations Affect Phytoplankton The interaction between the ocean and the atmosphere (described in the previous section) emphasizes how phytoplankton play an important role in regulating global CO2 concentrations. However, it is also necessary to understand how CO2 concentrations affect phytoplankton. Presently, atmospheric CO2 and O2 concentrations are 0.03% and 21%, respectively (Laing 1991). As many as 3.5 billion years ago, atmospheric CO2 concentrations were one thousand times greater than present day values, with little or no free oxygen (Kasting 1993). At this time, the first oxygenic photoautotrophs, the cyanobacteria, are thought to have evolved (Beardall and Raven 2004). The evolution of these organisms caused dramatic change on a global scale as they gradually diminished the high atmospheric CO2 concentrations (through photosynthetic uptake), and released oxygen as a metabolic waste product. Remarkably, the organisms' photosynthetic biochemical pathways did not evolve as the composition of the atmosphere slowly changed. These pathways that evolved during anaerobic conditions have been highly conserved throughout geological time and are thus, poorly adapted to the modern atmosphere (Stryer 1988). An important, highly conserved component of photosynthetic metabolism is the primary C-fixing enzyme, ribulose-l,5-bisphosphate carboxylase/oxygenase (RubisCO). RubisCO is responsible for two biochemical pathways, photosynthesis and photorespiration (involving carboxylase and oxygenase reactions, respectively). Whereas photosynthesis utilizes inorganic C and converts it into organic C using CO2 as a substrate, photorespiration is the oxidation whereby O2 is utilized and CO2 is released. 3 CO2 and O 2 must therefore compete antagonistically to bind to the active site of RubisCO. The anaerobic conditions present in the early atmosphere allowed the biochemical pathways of photosynthesis to evolve in the absence of photorespiration. In contrast, the I0W-CO2 and high-02 environment of present day cause RubisCO to be catalytically inefficient due to the under saturation of CO2 at its active site and its propensity for photorespiration. These I0W-CO2 conditions are not only problematic to RubisCO in the terrestrial biosphere, but in marine ecosystems as well. The CO2 concentration in the modern ocean reflects the low C O 2 concentration in the atmosphere. CO2 accounts for less than 1% of the total inorganic C in seawater, which could prove problematic given that CO2 is the only C species RubisCO can utilize (Cooper et al. 1969). The half-saturation constant (Km) of RubisCO ranges between 20-200um (Kaplan et al. 1991), depending on the phytoplankton species. The K m of RubisCO is substantially higher than the CO2 concentration found in surface seawater (10/xM) (Stumm and Morgan 1981). From this standpoint, photosynthetic efficiency is likely to be sub-optimal in the aerobic environmental conditions of present day. Although the concentration of CO2 is low, other forms of inorganic C are abundant with bicarbonate/carbonate ( H C O 3 7 C O 3 2 " ) accounting for the remaining 99% of inorganic C in seawater (Stumm and Morgan 1981). This is due to the reaction between CO2 and water to form a weak acid (carbonic acid) that dissociates to form HCO3" and C O 3 2 " (Falkowski et al. 2000). Although the interconversion between CO2 and HCO3" occurs spontaneously, it is exceptionally slow relative to other chemical reactions. The speciation of C in seawater could potentially limit marine phytoplankton growth given that RubisCO can only utilize CO2 (Riebesell et al. 1993). In the past, 4 however, the general conception has been that, "Carbon will rarely, if ever, be limiting in marine environments" (Goldman et al. 1972). This assertion serves as an example of 'carbon' including all three carbon species, and not exclusively CO2. Truly understanding the potential for CO2 limitation in photosynthesis depends upon understanding which of the C species are available for transport. My thesis examines ways in which natural phytoplankton assemblages and various laboratory diatom species deal with the low CO2 concentration found in surface seawater, and determines which C species are transported. My thesis will add to a large body of research that has already thoroughly examined particular model species such as Chlamydomonas reinhardtii (green algae) and Synechococcus sp. (cyanobacteria). How Phytoplankton Deal With a Low C 0 2 Environment Numerous phytoplankton taxa have developed strategies to overcome the low CO2 affinity of RubisCO and the low CO2 concentration that exists in the external aquatic medium. These "carbon-concentrating mechanisms" (CCMs) enable phytoplankton to accumulate internal C pools 5-75 times higher than the concentration of the surrounding medium (Colman et al. 2002). CCMs facilitate the carboxylation reaction of RubisCO by maximizing the CO2 concentration at the active site of the enzyme and thus, enhancing the rate of photosynthetic C fixation. The CCM consists of three basic components. These are: 1) the active uptake of HCO3", 2) the active uptake of CO2, and 3) the activity of external (periplasmic, 5 membrane-bound) carbonic anhydrase (Colman et al. 2002). This enzyme plays a vital role in C acquisition by catalyzing the otherwise slow interconversion of HCO3" to CO2 (Huertas et al. 2000). One or a combination of all three strategies enhance C acquisition. Another important component that many phytoplankton possess is internal carbonic anhydrase, an enzyme whose function is to elevate the CO2 concentration around RubisCO (Husic et al. 1989). There is discrepancy among phytoplanton species, however, in the C uptake strategies and extent to which the CCM is relied upon. It appears that the dependency of a phytoplankton group on CCMs increases as the CO2 affinity of their RubisCO decreases (Tortell 2000). In spite of the variation in CCMs among phytoplankton species, one common characteristic shared by most species is their ability to exploit the abundant HCO3" pool available in seawater either via external carbonic anhydrase (eCA) or through direct HCO3" transport. The expression and activity of eCA is perhaps the best studied aspect of the CCM. External CA functions as part of an indirect HCO3" transport system. The enzyme maintains a chemical equilibrium between HCO3" and CO2 in the diffusive boundary layer by converting the abundant pool of HCO3" to CO2. This achieves the highest possible concentration of CO2 on the external surface of the plasma membrane (Williams and Turpin 1987). CO2 is then the preferred C species actively or passively taken up. In the absence of eCA, cellular CO2 uptake rates can exceed replenishment of CO2 in the surrounding medium because of the low rate of HCO3" dehydration and low diffusion rate of CO2 (Huertas et al. 2000). Studies have shown that, even within a few hours, eCA activity greatly increases when cells are exposed to low CO2 concentrations (Badger and 6 Price 1992; Burkhardt et al. 2001). External carbonic anhydrase activity is just one of the physiological aspects examined in C uptake systems. There are two model phytoplankton species in which physiological aspects of C uptake systems have been most thoroughly studied. An in-depth examination of CCMs in the model cyanobacterial Synechococcus sp. has been achieved due to the simple cellular structure of this organism and its remarkable ability to increase intracellular DIC concentrations under low CO2 conditions (Raven 1985; Aizawa and Miyachi 1986). Some of the most recent studies have identified genes for four distinct C uptake systems in Synechococcus (Price et al. 2002). There are two essential, active uptake systems (one each for CO2 and HCO3"), and two additional transporters (one each for CO2 and HCO3") that are only induced under CO2 limitation (Price et al. 2002). While one or a combination of the C uptake systems can be utilized, HCO3" is the C species that accumulates internally to as much as 1000-fold higher than the surrounding medium (Price et al. 2002). A microcompartment, known as the carboxysome, contains RubisCO and internal CA. Carboxysomal CA functions to elevate the CO2 concentration around RubisCO (Reinhold et al. 1991). Although external carbonic anhydrase is not found in cyanobacteria (Aizawa and Miyachi 1986), the other components of this CCM allow for maximum photosynthetic C-fixation. Chlamydomonas reinhardtii (green algae), another model microalgae species, is also able to accumulate CO2 around RubisCO. This organism has been shown to possess direct uptake transport systems for both HCO3" and CO2 (Sultemeyer et al. 1991; Raven 1997). However, unlike cyanobacteria, nearly all green algae possess eCA (Kimpel et al. 1983; Colman et al. 1984), and thus a large fraction of HCO3" use is indirect. Regardless 7 of the C species transported, HCOyis the C species that accumulates internally. Internal CA is then able to saturate RubisCO's active sites with CO2 within a sub-chloroplast compartment, known as the pyrenoid (Moroney and Mason 1991). Genetic work has recently begun to identify genes that may be involved in C acquisition. It has been suggested that the gene ccml (or cia5) acts as a primary regulator when the cell detects C limitation (Fukuzawa et al. 2001). For example, ccml, in Chlamydomonas, operates by upregulating the expression of other genes that are linked to CCM functions (Price and Badger 2002). The physiological data available for cyanobacteria and green algae allow for a thorough understanding of CCMs. Both the structurally complex green alga group and the archaic cyanobacteria group show similar tactics for managing C limitation. The information obtained from these model species has been extrapolated to other phytoplankton species, even though they do not represent the full range of taxonomic variability. It is therefore important that studies focus on other phytoplankton groups that have not been extensively examined. Diatoms, for instance, are an important group of marine phytoplankton because of their dominant role in primary production and organic C export to the deep sea (see previous section). Studies on C acquisition by diatoms have revealed features similar to those found in green algae and cyanobacteria. Diatoms have been shown to take up HCO3" directly (Nimer et al. 1997; Tortell et al. 1997; Burkhardt et al. 2001) and employ eCA activity (Colman et al. 2002; Morel et al. 2002) to acquire C. However, the understanding of their C uptake mechanisms is limited. Hopefully, the recent sequencing of genomes of two diatoms, Thalassiosira pseudonana and Phaeodactylum tricornutum 8 (Armbrust et al. 2004; Montsant et al. 2005) will provide further insight into the specific transport systems of diatoms. In the interim, my thesis provides an extensive comparative analysis of numerous diatom species by quantitatively determining the relative proportions of CO2 and HCO3" that contribute to their C acquisition strategies. Carbon Acquisition Strategies of Natural Phytoplankton Communities "It is perhaps unsatisfactory that in this field, as in many others, assessment of algal behavior in nature often depends upon the extrapolation of that obtained from cultures in laboratory systems. Although the latter are clearly the foundation for the relevant physiology, very few natural populations and sequences of events have been directly studied using an array of the techniques now available. The ecological significance of possible "adaptation" phenomena in CO2 uptake has yet to be established for any growing natural population." -Tailing (1985) While the examination of C acquisition systems in laboratory phytoplankton cultures has increased, limited information remains available on C acquisition systems in phytoplankton communities in natural environments. Recent work in the coastal and eastern Subtropical and Equatorial Pacific Oceans has confirmed the existence of CCMs in natural marine phytoplankton assemblages (Tortell et al. 2000; Tortell and Morel 2002). Coastal diatom-dominated phytoplankton communities have been shown to possess internal C pools 3-5.5 times greater than the external seawater (Tortell et al. 2000), and CA activity has been observed at many sampling stations (Tortell et al. 2000; Tortell and Morel 2002). Direct HCO3" uptake appears to be an essential part of the CCM 9 of natural phytoplankton communities accounting for half of the C acquired by diatom-dominated phytoplankton communities in the Southern Ocean (Cassar et al. 2004). Whether HCO3" is utilized directly or indirectly via eCA, the data obtained from a very limited number of field sites suggest that HCO3" is a key C source for natural phytoplankton assemblages. However, the widespread use of HCO3" as a C source in many other oceanic regions is unknown. As a result, further field studies are needed before generalizations can be made about C acquisition strategies by natural marine phytoplankton communities. A further important question is the potential affect of ambient CO2 concentrations on C acquisition systems in natural phytoplankton assemblages. Increased CCM activity has been observed under low CO2 conditions in incubation experiments with marine phytoplankton communities. In coastal and equatorial Pacific waters, low CO2 concentrations (150ppm) induced HCO3" utilization via eCA activity (Tortell et al. 2000; Tortell and Morel 2002). Conversely, high-C02 conditions favored C02-only uptake for the phytoplankton communities (Tortell and Morel 2002). Lake Kinneret, in Israel, has been an ideal location to explore how ambient CO2 concentrations affect blooms of the dinoflagellate Peridinium gatunese. During the P. gatunese annual bloom, there was a notable increase in CCM activity as the inorganic C concentration in the lake decreased by 40% (Berman-Frank et al. 1998). Intracellular C concentrations increased 5-70 times, and CA activity by 5-50 times at the end of the bloom when CO2 levels were low (Berman-Frank et al. 1998). Changes in C acquisition strategies in response to ambient C0 2 concentrations have also been reported in many laboratory studies. These studies suggest the increase in CCM activity is associated with a rise in the affinities for CO2 and 10 HCCVuptake activities during I0W-CO2 conditions (Badger and Price 1992; Price et al. 1998; Kaplan and Reinhold 1999). It is becoming increasingly important to determine how natural phytoplankton assemblages respond to various CO2 levels as atmospheric CO2 concentrations continue to rise at an unprecendented rate. In addition to examining C acquisition strategies by natural phytoplankton assemblages, my thesis will also investigate how these communities respond physiologically to low and high-C02 conditions. 11 Thesis Objectives (Laboratory Objectives) My research did contribute to the understanding of inorganic carbon (C) acquisition by marine diatoms. My objective was to examine the sources of inorganic C utilized by a wide variety of diatom species maintained in laboratory cultures. No studies have thoroughly surveyed an array of marine diatom species in relation to their C utilization through such a comparative analysis, and my intent was to generate new insights on this topic. My specific goals were to quantitatively examine the relative contributions of HCO3" and CO2 to C uptake in different diatoms in relation to cell size and growth rates of species, and to examine the importance of extracellular carbonic anhydrase (eCA). I hypothesized that most marine diatoms utilize HCO3" as their primary source of carbon and use eCA to convert HCO3" to CO2 (indirect HCO3" utilization) for RubisCO. In addition, I hypothesized that the contribution of HCO3" and CO2 to C uptake is related to cell size and growth rates of different diatom species. I expected that larger cells will satisfy a greater fraction of their C demands by direct HCO3" uptake and indirect HCO3" utilization (via extracellular CA) because their surface area to volume ratio (nutrient supply: demand) is much lower than that of small cells. Therefore, HCO3" utilization would prevent large cells from becoming limited by CO2. Smaller cells have a higher surface area to volume ratio and may be able to meet their C demands with minimal use of HCO3". In addition, species with a higher growth rate will demand more C to meet their photosynthetic requirements compared to slower growing diatoms. As a result, their dependency on direct HCO3" uptake and eCA activity might be greater. 12 (Field Objectives) My oceanographic field research did contribute to the small number of existing field studies. My objective was to further examine the importance of HCO3" and CO2 as carbon sources for coastal and oceanic phytoplankton communities. I quantitatively examined the relative proportions of HCO3" and CO2 taken up by natural phytoplankton assemblages, and considered the extent to which eCA plays a role. I monitored phytoplankton biomass, species composition, primary productivity, nutrients, CO2 concentrations, and determined whether any of these chemical or biological properties influence C acquisition strategies in natural phytoplankton communities. My second objective in the field was to examine the carbon acquisition by natural phytoplankton assemblage in responses to low and high-C02 conditions. I hypothesized that phytoplankton communities subjected to low CO2 concentrations will show higher eCA activity and a higher fraction of direct HCO3" uptake. This will allow the communities to meet their C demands, and therefore, be able to maintain their growth rates even at low CO2 concentrations. I monitored growth rate, primary productivity, and species composition in both the low and high CO2 environments to verify if any changes occur due to the difference in CO2 concentration. 13 Chapter 2: Inorganic Carbon Acquisition by Natural Phytoplankton Assemblages in the Bering Sea Introduction In recent years, there has been increasing interest among the oceanographic community in the physiological mechanisms of inorganic carbon acquisition by marine phytoplankton. This stems from a need to better understand the potential responses of phytoplankton to changing atmospheric CO2 concentrations (Houghton et al., 1995, 2001), and the controls on the C isotope signatures of marine organic matter (Hinga et al. 1994). Previous work (Riebesell et al. 1993) suggested the possibility of CO2 limitation of marine phytoplankton growth. However, numerous laboratory physiological studies with a wide variety of marine phytoplankton species have documented the existence of carbon concentrating mechanisms (CCMs) through which cells saturate C-fixation by RubisCO, the rate-limiting carboxylase enzyme in the Calvin cycle (Badger and Price 1992; Badger et al. 1998; Colman et al. 2002). These CCMs enable phytoplankton to accumulate internal C concentrations 5-75 times higher than those of the surrounding medium (Colman et al. 2002), thus, maximizing the CO2 concentration at the active site of RubisCO and repressing the enzyme's oxygenase activity. The CCM has three individual components that contribute to C acquisition. The C transport system can involve the active transport of HCO3" (the predominant form of inorganic C in the oceans) and/or CO2, as well as the extracellular enzyme carbonic anhydrase, which catalyzes the otherwise slow interconversion between these carbon species (Huertas et al. 2000; Colman et al. 2002). The extracellular carbonic anhydrase (eC A) functions as part of an indirect HCO3" system, allowing cells to access the large 14 HCCVpool in seawater by catalyzing its conversion to CO2. CO2 can be subsequently taken up either actively or passively. The net effect of eCA is to maintain the maximum potential supply of CO2 at the cell surface. The CCM is essential for various algal taxa to obtain their C source, but the exact mechanisms of the CCMs vary significantly among species. In laboratory studies, different phytoplankton species have been shown to depend on one, or a combination of the three individual components of CCMs (Raven 1997; Colman etal. 2002). While CCMs have been characterized using laboratory cultures of many phytoplankton species, relatively little information is available on C uptake physiology by natural phytoplankton assemblages. Part of the difficulty inherent in field studies is the co-existence of many species whose physiological mechanisms of C uptake have not been examined. Nonetheless, recent field work has documented the existence of CCMs in natural phytoplankton assemblages (Tortell et al, 1997, 2000; Tortell and Morel 2002; Cassar et al. 2004). Tortell et al. (1997, 2000) demonstrated that natural phytoplankton communities had the capacity to concentrate inorganic carbon, and were able to maintain rapid growth rates over a wide range of CO2 concentrations. These early field studies also showed that CCM activity and cell biochemistry were modulated by the external CO2 concentration, and suggested that HCO3" was an important source of inorganic C for phytoplankton. The experiments did not, however, provide strict quantitative estimates for the relative proportions of HCO3" and CO2 taken up by cells. Two recent field studies (Tortell and Morel 2002; Cassar et al. 2004) have employed the isotope disequilibrium method (Espie and Colman 1986) to estimate the contribution of HCO3" to total C uptake by natural phytoplankton assemblages. This method allows for the quantitative 15 determination of the HCO3" and CO2 contributions to C uptake based on the slow kinetic exchange between these C species in seawater (Espie and Colman 1986; Elzenga et al. 2000). The results obtained from these experiments demonstrate that HCO3" contributes significantly to phytoplankton C uptake in both the Equatorial Pacific and Southern Ocean (Tortell and Morel 2002; Cassar et al. 2004). The field data also demonstrated that HCO3" utilization increases in I0W-CO2 conditions (Tortell et al. 2000; Tortell and Morel 2002). While these field studies on C acquisition provide an important step forward, they only represent a few distinct oceanic environments (Southern Ocean, Subtropical/Equatorial Pacific Ocean). The extent to which the results apply to natural phytoplankton assemblages in many other regions of the oceans is unclear. In this study, we present new results from isotope disequilibrium experiments conducted in the southeastern (SE) Bering Sea. Although the impact of phytoplankton growth on the carbonate system of the Bering Sea has been examined (Codispoti et al. 1982, 1986), no information is available on the physiological mechanisms of C acquisition by indigenous phytoplankton in these waters. We examined inorganic C uptake by phytoplankton in relation to large spatial gradients in primary production and CO2 concentrations in the Bering Sea. Our results show that FJCO3" is the predominant C species taken up by phytoplankton communities in both the offshore and coastal regions of the Bering Sea, and suggest that a large fraction of HCO3" uptake occurs through a direct transport mechanism. In addition, CO2 incubation experiments demonstrate that HCO3" utilization by phytoplankton can be tightly regulated by ambient CO2 concentrations. 16 Materials and Methods Sampling Locations Field experiments were conducted during August and September, 2003 aboard the R/V Kilo Moana. Sampling sites were located across three regions of the southeastern Bering Sea; the open-ocean domain (western gyre), the continental shelf break, and the continental shelf (Fig.2.1) (Loughlin and Ohtani, eds. 1999). The continental shelf break divides the deep, open-ocean domain from the outer continental shelf (Kinder and Coachman 1978). The outer edge of the continental shelf receives large fluxes of water from the nutrient-rich slope. This supply of nutrients makes the shelf edge vicinity one of the most biologically productive areas in the Bering Sea (Hansell et al. 1989). The large continental shelf experiences spatially and temporally variable hydrography which impacts the biological productivity of the entire region (Overland et al. 1999). Field studies in the SE Bering Sea have documented some of the lowest pCOi in the world's oceans with large variability over small spatial scales (Codispoti et al. 1982). During our cruise, nutrient concentrations, chlorophyll a, CO2 concentrations, primary productivity and calcification rates were measured at most stations. Nutrient concentrations were determined with an on board autoanalyzer, and chlorophyll a was measured by fluorometric analysis (Parsons et al. 1984). Concentrations of CO2 at most stations were obtained from a membrane-inlet mass spectrometer (MEMS) (Tortell 2005), but in some cases, CO2 concentrations were derived from potentiometric pH measurements and salinity-derived alkalinity using algorithms of Lewis and Wallace (1998) (CO2SYS Program). For each station, phytoplankton community composition was qualitatively examined with an inverted microscope using concentrated 17 phytoplankton samples preserved w i t h 2 % formaldehyde. P lank ton ic ca lc i f i ca t ion and pr imary produc t iv i ty were measured i n short-term 1 4 C uptake experiments. Ca lc i f i ca t ion rates were measured i n order to determine i f the coccol i thophore , Emiliania huxleyi w h i c h possesses a c a l c i u m carbonate ce l l cover ing, was a significant contributor to the phytoplankton c o m m u n i t y structure. Th i s species has been responsible for massive b looms i n the continental she l f surface waters o f the B e r i n g Sea since 1997 ( M e r i c o et a l . 2004). Tr ip l ica te sample bottles and one dark bottle conta ining 2 0 0 m l o f seawater, col lected from the c h l o r o p h y l l a m a x i m u m (10-30m), were incubated for 8 hours w i t h 1 4 C i (25/iCj) to assess the amount o f C f ixed and the amount contr ibut ing to ca lc i f ica t ion (see L a m et al . 2001). Incubations were conducted i n a ship-board f low through chamber maintained at ambient temperature, w i t h two layers o f neutral-density screening to provide 3 0 % o f surface irradiance. F o r isotope d i s equ i l i b r ium experiments, phytoplankton assemblages were col lected from the c h l o r o p h y l l a m a x i m u m (10-30m), w h i c h was determined at each station from C T D casts. A p p r o x i m a t e l y 10-20 litres o f seawater f rom the C T D N i s k i n bottles were gravi ty filtered onto 2 or 2 0 u m polycarbonate membrane filters. Phytoplankton col lected o n filters were immedia te ly resuspended i n p H 8.5 seawater (buffered w i t h 2 0 m M B i c i n e ) , and isotope d i sequ i l ib r ium experiments begun w i t h i n 15 minutes. 18 Isotope Disequilibrium Experiments To determine the relative contributions of CO2 and HCO3" to C uptake by phytoplankton assemblages, we used the isotope disequilibrium technique (Espie and Colman 1986; Elzenga et al. 2000) following the protocol of Tortell and Morel (2002) with a few minor modifications. Briefly, cells were resuspended in a seawater buffer (pH 8.5, 20mM Bicine) and acclimated for 5 minutes at 10°C in an oxygen electrode chamber illuminated with a slide projector at ~300pmol m"2sec"'. To initiate the experiments, a 20/uCi 1 4 C spike (pre-equilibrated at pH 7.0, buffered with 50mM Hepes) was injected into the concentrated cell suspension (in pH 8.5 seawater buffer). After the injection of the 1 4 C spike, subsamples of 200/xL were withdrawn at short intervals, between 5 and 300 seconds, and dispensed into 1ml of 50% HC1. Acidified samples were placed in a fume hood and degassed for 4 hours by purging air into each sample vial. This degassing period allowed for removal of residual inorganic 1 4 C that had not been incorporated into photosynthetic products. C O 2 Incubation Experiments To examine the COvdependent regulation of C acquisition, we carried out ship-board CO2 manipulation experiments following the protocol of Tortell et al. (2002). Seawater was collected in the oceanic domain (high nutrient, low chlorophyll waters) and 5nM iron was added to promote phytoplankton growth. We used triplicate bottles that were continuously bubbled with 150ppm or 750ppm CO2 in air supplied from commercially prepared gas mixtures. Nutrient concentrations, chlorophyll a, species 19 composition, primary productivity and calcification rates were measured in incubation samples as described above for the discrete in situ samples. The chlorophyll a data were used to derive growth rates for each CO2 incubation treatment. We carried out two separate CO2 manipulation experiments, during which isotope disequilibrium experiments were performed for each CO2 treatment. Samples from each of the replicate incubation bottles were pooled together and filtered onto 2uxn polycarbonate filters. Isotope disequilibrium experiments were performed as described above. Data Analysis and Modeling The isotope disequilibrium method was designed to quantify the relative contributions of HCO3" and CO2 to C uptake by photosynthetic cells (Espie and Colman 1986). This technique is based upon the slow (uncatalyzed) interconversion between HCO3" and CO2 (Johnson 1982) which enables these chemical species to be differentially labeled with 1 4 C over time periods of several minutes. In the initial radiolabeled C spike solution, at p H 7.0, 14CO*2 accounts for ~ 20% of the total D I C (dissolved inorganic carbon) whereas in the cell suspension, at p H 8.5, 0.3% of the total D I C exists as CO2 (Morel and Herring 1993). Following the addition of the 1 4 C spike, the specific activity of CO2 in the seawater buffer is initially high, decaying exponentially to an equilibrium value over a period of several minutes. In contrast, the initial specific activity o f H , 4 C 0 3 7 1 4 C 0 3 2 " in the seawater buffer is close to its equilibrium value (Elzenga et al. 2000), and therefore, changes relatively little during the time course. It should be noted that the 1 4 C tracer addition does not significantly affect the bulk concentration or chemical speciation o f the unlabeled DIC in solution. 20 The instantaneous rate of 1 4 C uptake by phytoplankton is affected by the changes in the specific activities of CO2 and HCO3" during the experimental time-course. This instantaneous rate is equal to the sum of CO2 and HCO3" uptake rate (modified from Elzenga et al. 2000): d(DPMt)/dt=Vt(l-/Hco3)*S.A.co2, + Vt(/Hco3)*S.A.Hco3t (1) (DPM=disintegrations per minute) Where d(DPMt)/dt is the instantaneous rate of C uptake at time t, V t is the total rate of C uptake, and /Hccois the fraction of direct HCO3" uptake. S.A.coztand S.A.HC03tare the specific activities of CO2 and HCO3", respectively, at any particular time, t. During steady-state photosynthesis, Vtand fHcm are assumed to be constant so that changes in the instantaneous 1 4 C uptake rate reflect only changes in the specific activity of the two C species. The changes in the specific activity of CO2 and HCCVwith time follow an exponential rate law (Elzenga et al. 2000): S.A.C02, = S.A .Dic + AS .A . co 2 e a l t (2) S.A.Hco3t = S.A.Dic + A S . A . Hcose"21 (3) S.A.DIC is the specific activity of the total dissolved inorganic carbon species at equilibrium, A S . A . C02 and A S . A . HCO3 are the differences between the initial and equilibrium values of the specific activity of CO2 and HCGVjespectively. Values of cd and cY2 represent the temperature, salinity, and pH-dependent first order rate constants for CO2 and HCO3" hydration and dehydration, respectively, as described by Espie and Colman (1986). Under our experimental conditions (temperature=10°C, salinity = 34psu, 21 pH values 8.5), al and ce2 are 0.0152 and 0.0173 (seconds"1), respectively. These values were calculated from the equations of Espie and Colman (1986) using temperature and salinity corrections from Johnston (1982). Integrating equation (1) yields the time-course of 1 4 C accumulation (modified from Elzenga et al. 2000): DPM t = V T (l-/Hco3)(alt+(AS.A.co2/ S.A.Dic)*(l - e"alt))/ al+... +Vt(/'Hco3)(Q2t+(AS.A. HCXB/ S . A . D I C (DPM=disintegrations per minute) The values of A S . A . C 0 2 / S . A . D I C and A S . A . HC03/ S . A . D I C are set by the difference in pH between the 1 4 C spike and seawater buffer, with the values of 49 and -.24, respectively. Previous studies have used various numerical approaches to quantify the proportions of HCO3" and CO2 taken up in isotope disequilibrium experiments. Elzenga et al. (2002) fit data with the integral equation (eqn (4)), while Tortell and Morel (2002) and Cassar et al. (2004) derived the ratio of initial and final uptake rates. This latter method is an approximation of the instantaneous analysis (eqn (1)). Both of these approaches are quantitatively valid, but the previous analyses have not fully addressed the statistical error associated with the calculated fractions of HCO3" and CO2 utilized. In the case of the integrated equation, we have found that there can be large errors in the parameter estimates, even when the overall curve fit appears to be excellent. We have used two quantitative approaches to analyze our Bering Sea results. The integral equation (4) can be used to quantify the fraction of HCO3" utilized and the extent of eCA expression. We fit the integrated equation (4) to our 1 4 C accumulation data using the Marquand-Levenberg non-linear regression algorithm (SigmaPlot®). As part of this 22 analysis, we obtained best fit parameter estimates for V t and/^C03 as well as standard errors for these parameters. In order to estimate eCA activity, the rate constants (al and o2) were allowed to vary as parameters, constrained to be equal to or greater than the uncatalyzed rates. In this manner, we allowed for the possibility of eCA expression that would act to increase the rate constant of CO2/HCO3' equilibration. To confirm that our analysis is accurate, we created 25 hypothetical time courses of 1 4 C incorporation using five different a- (i.e. modeled eCA activity) and five different/HC03-values. We applied the integrated equation (4) to the curves to obtain predicted values for these parameters. The expected versus predicted a- and/^ C03-values from our analysis are shown in Figure 2.2. The mean deviation between the predicted and expected was 4.7% and 7.7% for the a- and/HCo3-values, respectively. The second quantitative approach was to transform the integrated time-course data into a differential form. The transformation was used to approximate instantaneous uptake rates across the entire time course, and was calculated by taking the difference between 1 4 C accumulation in successive time points and dividing by the time interval (i.e. finding the slope between successive time points). This transformed data was then fit using equation (1), and an instantaneous uptake rate was estimated from the model output at any given time point. I then took the ratio between the derived 1 4 C uptake rate at t=7.5 (P7.5) and t=275 seconds (P275) and calculated an/HC03-value (the fraction of direct H C O 3 " transport) from this statistic. This calculation was based upon a standard curve relating the fraction of HCO3" and CO2 uptake to P7.5/P275. A lower P7.5/P275 ratio specifies a smaller difference between the initial and equilibrium specific activity values, and therefore, a greater fraction of direct HCO3" transport. 23 Results In Situ Stations The sampling sites in the open ocean (western gyre), shelf break, and continental shelf domains in the southeastern Bering Sea showed spatial variability in their chemical and biological properties. Details of nutrient concentrations, chlorophyll a, CO2 concentrations, productivity, calcification rates and species composition at each station are given in Table (2.1). In the western gyre, nutrient concentrations were generally high (>10pM NO3"), while chlorophyll a values were typically low (<lpg/L), and CO2 concentrations were close to atmospheric equilibrium (~370ppm). In contrast, the continental shelf and shelf break areas were typically characterized by low nutrient concentrations, high chlorophyll a values, and lower CO2 concentrations. The highest productivity was recorded in the coastal areas and shelf break domain while the lowest productivity was observed in the western gyre. In general, calcification rates were less than 1% of primary productivity at all stations with no apparent relationship between these two parameters. In contrast, phytoplankton species composition showed general differences across sampling domains, with nanoflagellates dominating in the western gyre and large diatoms predominant in the continental shelf break and coastal areas. Dominant diatom species included Chaetoceros sp., Pseudonitzchia sp., Fragilariopsis sp., Cylindrotheca sp. Rhizoselenia sp., and some Thalassiosira species. The chemical and biological properties observed at the sampling sites were comparable to previous studies carried out in the Bering Sea {see Loughlin and Ohtani, eds. 1999) 24 Isotope Disequilibrium Experiments Figure 2.3 shows some of the results of isotope disequilibrium experiments, conducted with phytoplankton assemblages collected in-situ. In all cases, direct HCO3" transport accounted for the majority of C uptake, with fuco3- (the fraction of direct H C O 3 " transport) ranging from 65-90%. Error estimates for/HCO3- ranged from 1-6.3% (Table 2.2). Given that our model fits included a variable rate constant (a), the modeled C uptake also included the possibility of indirect H C O 3 " utilization via CA-catalyzed dehydration at the cell surface. The apparent enhancement of the HCO37CO2 exchange ranged from 32-466%, implying that eCA activity was present and that indirect HCO3" utilization also occurred (Table 2.2). The data from all stations could not be adequately fit using a CCVonly model (dashed curve), even when the rate constants (a) were allowed to vary. This demonstrates that direct HCO3" transport is an essential component in C acquisition for all the phytoplankton assemblages. To confirm the results derived from the integral equation fit, we also analyzed the isotope disequilibrium data using a differential fit (see Materials and Methods). Figure 2.4 gives an example of results from stations 3 and 4 following data transformation into differential form. The analysis of the data using a differential fit showed that direct HCO3" transport comprised 78-90% of the total C uptake. The quantitative results from both the differential and integral analyses agree well (Table 2.2), with the average absolute deviation between the two varying by only 5.2%. We examined the relationships between C uptake by the phytoplankton assemblages and the environmental properties of the sampling sites. There were no significant relationships observed between the fraction of direct HCO3" transport and any 25 of the chemical and biological properties we measured at each station. Additionally, there were no significant associations between the apparent enhancement of the uncatalyzed H C O 3 7 C O 2 exchange (eCA activity) and nitrate, phosphate, silicate, chlorophyll a, or primary productivity. Although we did not obtain good correspondence between the MEVIS and the CO2SYS program for seawater C O 2 concentrations, we observed higher eCA activity at stations with lower CO2 concentrations (Figure 2.5). The phytoplankton community composition also had no apparent affect on the amount of direct HCO3" utilization. In contrast, eCA activity appeared to be higher at stations dominated by diatoms, with lower eCA activity characteristic for stations dominated by nanoflagellates. This outcome is supported by results from the size-fractionated isotope disequilibrium experiments (>2pm and >20pm), which revealed a significantly higher eCA activity in the diatom dominated >20pm size-category (Table 2.2). C O 2 Incubation experiments CO2 manipulation experiments were conducted to examine the CC>2-dependent regulation of the C uptake system. No significant differences in phytoplankton biomass, nutrient concentrations, growth rates, calcification rates, or primary productivity were observed between the low (150ppm) and high (750ppm) CO2 incubations. Growth rates for the low and high CO2 incubations were 0.81 ± 0.14 d"1 and 0.88 ±0.1 d"', respectively. Calcification rates were <1% of primary productivity. A qualitative examination of the phytoplankton assemblages showed no significant differences in species composition between the low and high CO2 incubations. Both experiments had a mixture of 26 nanoflagellates, dinoflagellates, and thin pennate diatoms, with Cylindrotheca sp. being the most prevalent diatom. Large centric diatoms were also found in both CO2 treatments of the second incubation experiment. Although productivity, growth rate, and biomass did not vary between the low and high CO2 treatments, we observed significant differences in the relative proportions of HCO3" and CO2 uptake by the phytoplankton assemblages. There was a significant increase in the fraction of direct HCO3" transport in low CO2 treatments for both incubations (Table 2.3). In addition to the increase of direct HCO3" transport, total C uptake rates were also higher in the I0W-CO2 conditioned cells. Moreover, the apparent enhancement of the HCO37CO2 exchange (eCA activity) was higher for the 750ppm treatments in both experiments. These results show a clear CO2 effect on the C uptake system of phytoplankton assemblages. 27 Figure 2 .1: B e r i n g Sea s ampl ing locations for isotope d i sequ i l i b r ium experiments 28 Figure 2.2: a) Expec t ed versus predicted a-values (i.e. mode led e C A act ivi ty) for hypothetical/ H co3-values. b) Expec ted versus predicted/ H co3-values for hypothetical a-values. The integral equation (4) (chapter 2) was appl ied to generate predicted a- and /HC03-values 29 Table 2.1: Nutrient concentrations and biological characteristics of the Bering Sea sampling stations ( C 0 2 concentrations are only reported for membrane inlet mass spectrometry). Error bars represent ± standard error (n=3). Station calcification dominant Nitrate Phosphate Silicate Chi a co 2 Productivity (MgC L'1 hf1) rates group (MM) (MM) (MM) (M9 I-"1) (ppm) 1 2 3 - - - 0.68 - 0 . 2 2 ± 0 . 0 1 3 <1% nanoflagellates 14.7 1.10 16.7 0.73 386 0.14+0.009 <1% nanoflagellates 1.33 0.32 - 3.7 - 1.9±0.17 <1% diatoms 4 0.02 0.04 0.81 4.0 159 2 .2±0 .14 <1% diatoms 5 (2pm filter) 0.10 0.20 1.32 2.4 - 1.6±0.05 <1% nanoflagellates 5 (20Lim filter) 0.10 0.20 1.32 2.4 - 1.6±0.05 <1% diatoms 6 (2pm filter) 0.12 0.05 3.58 1.4 256 0.51 ±0 .006 <1% nanoflagellates 6 ( 2 0 u m filter) 0.12 0.05 3.58 1.4 - 0 .51±0 .006 <1% diatoms 7 3.36 0.20 10.1 0.97 202 - diatoms 8 9 2.41 0.09 8.6 0.97 221 - - nanoflagellates 0.04 0.05 0.85 6.0 - - - diatoms 10 11 15.7 1.23 39.1 5.7 - - - nanoflagellates - - - - - - diatoms 30 5000 8000 6000 4000 2000 100 200 300 5000 4000 3000 2000 1000 300 1 — 1 — 1 — 1 — 1 — 1 — 0 100 1 ' I I 200 300 d --• A ^ T C — • — 1 i i 0 100 1 1 i 1 200 300 Seconds Seconds Figure 2.3: Time course of 1 4 C incorporation during isotope disequilibrium experiments for stations (a) 3, (b) 4, (c) 1, and (d) 8. The best-fit curves were obtained by applying equation (4) (see Materials and Methods, Chapter 2). The dashed curves represent a C02-only uptake model fit. Error bars represent ± standard error (n=2) and are smaller than the symbol when not visible. 31 Table 2.2: Isotope disequilibrium results from the Bering Sea sampling stations. The /HC03-value represents the fraction of direct HC03" transport (see Materials and methods in chapter 2 for a description of the analysis). Error bars represent ± standard error (n=2). Phytoplankton were collected for experiments using 2.0pm filters with the exception of stations 5 and 6 where 20pm filters were also used. Station /Hco3-value Integral Fit /Hco3-value Differential Fit Apparent enhancement of the H C 0 3 7 C 0 2 exchange (%) (eCA activity) Integral Fit 1 0.83 ± 0.024 - 91 ± 19 2 0.80 ±0.037 0.78 58 ± 10 3 0.87 ±0.010 0.89 163 ± 2 4 4 0.77 ±0.027 0.88 466 ± 70 5 (2 pm) 0.77 ± 0.022 0.78 111 ± 14 5(20pm) 0.85 ±0.017 0.90 242 ± 42 6(2pm) 0.67 ±0.055 - 45 ± 4 6(20pm) 0.65 ± 0.063 - 104 ± 17 7 0.87 ±0.019 0.88 170 ± 4 1 8 0.79 ±0.031 0.83 71 ± 1 1 9 0.90 ± 0.013 0.83 170 ± 4 1 10 0.83 ±0.031 0.88 32 ± 5 11 0.90 ±0.028 - 157 ± 8 0 32 <u CL, Q ID -4-» C3 C* <u 3 D in 3 O <u C nt c CO ID OH Q CL, D 3 O <D C CO a 100 0 50 100 150 200 250 300 0 50 100 150 200 250 300 Seconds Figure 2.4: Instantaneous 1 4 C uptake rates for stations (a) 3 and (b) 4 during isotope disequilibrium experiments. Best-fit curves were obtained by applying equation (1) (see Materials and methods, chapter 2). Error bars represent ± standard error (n=2) and are smaller than the symbol when not visible. 33 > 00 c C3 500 400 n 300 i 200 ] 100 o a o O 150 200 250 300 350 C 0 2 Concentration (ppm) 400 Figure 2.5: Apparent enhancement of the HC0 37C0 2 exchange (%) (external carbonic anhydrase activity) in relation to station C0 2 concentrations measured with (a) C0 2 SYS program and (b) Membrane inlet mass spectrometry (MEMS). 34 0 100 200 300 Seconds Figure 2.6: Effects of low (150ppm) and high (750ppm) C 0 2 conditions on 1 4 C incorporation (normalized to chlorophyll) during isotope disequilibrium experiments for the (a) first and (b) second C0 2 incubation experiments. The best-fit curves were obtained by applying equation (4) (see Materials and methods, Chapter 2). Error bars represent ± standard error (n=2) and are smaller than the symbol when not visible. 35 Table 2.3: Isotope disequilibrium results from C O 2 manipulation experiments. The /HC03-value represents the fraction of direct HCO3" transport. Error bars represent ± standard error (n=2). Phytoplankton were collected for experiments using 2.0um filters. Incubation CO2 cone, (ppm) ./kcoa-value Apparent enhancement of the H C 0 3 7 C 0 2 exchange (%) (eCA activity) 1 150 0.74 ±0.017 163 ± 16 750 0.53 ±0.066 222 ± 32 2 150 0.85 ± 0.019 275 ± 48 750 0.73 ±0.063 624±170 36 Discussion The purpose of this study was to investigate the importance of HCO3" and CO2 as carbon sources for natural phytoplankton assemblages, and contribute much needed data to a handful of existing field studies. We were able to quantitatively examine C acquisition by phytoplankton communities at 11 stations in the Bering Sea across a range of phytoplankton biomass, species composition, productivity, and CO2 concentrations. We also examined the effects of experimental CO2 manipulations on phytoplankton C acquisition systems. Our key observation is that HCO3" was the predominant C source for all phytoplankton assemblages, and our results suggest that H C 0 3 " utilization occurred mainly through a direct transport system. By comparing the results across stations, we found that direct HCO3" use was not related to phytoplankton species composition or CO2 concentration. In contrast, species composition and ambient CO2 concentrations appeared to influence eCA activity. In the direct CO2 manipulation incubations, we observed a statistically significant CO2 effect on the pathways of C assimilation. Overall, the combined integral/differential analysis clearly indicated that HCO3" was the dominant source of C taken up by the phytoplankton assemblages, with the majority of this uptake apparently resulting from a direct transport process. Phytoplankton also appeared to utilize H C 0 3 " indirectly through the CA-catalyzed dehydration of HCO3". The apparent enhancement of the uncatalyzed H C 0 3 7 C 0 2 exchange (eCA activity) (Table 2.2), showed a modest enhancement of extracellular dehydration of HCO3" compared to the uncatalyzed rate. Our results are consistent with field studies which have also reported HCO3" as being the primary source of C taken up 37 by phytoplankton communities (Tortell and Morel 2002; Cassar et al. 2004). Cassar et al. (2004) reported direct HCO3" transport accounted for over 50% of the DIC uptake by Southern Ocean phytoplankton assemblages. Although we have used a different quantitative approach for determining the fraction of HCO3" and CO2 uptake, we have obtained similar findings in natural phytoplankton assemblages. This suggests that HCO3" uptake by phytoplankton communities is widespread across many oceanic regions. Direct HCO3" Transport Direct HCO3" transport appears to be an important C acquisition strategy of phytoplankton assemblages as it is prevalent across the variable environmental conditions of the SE Bering Sea (Table 2.2). We looked for relationships between direct HCO3" transport by the phytoplankton assemblages and the various biological/chemical parameters. We suggest that direct HCO3" transport is constitutive given that it occurs independently of species composition, phytoplankton size fractions, CO2 concentrations, and all other environmental parameters. The fact that direct H C 0 3 " transport facilitates the majority of carbon acquisition in Southern Ocean phytoplankton assemblages, regardless of environmental conditions, supports our suggestion. (Cassar et al. 2004). Contrasting the results from our in situ experiments are the results from our CO2 manipulation experiments. These results provide evidence that CO2 concentrations regulate direct HCO3" transport by natural phytoplankton assemblages. In two independent incubation experiments, phytoplankton assemblages acclimated to the low-CO2 conditions showed a greater proportion of direct HCO3" utilization than the high-C02 grown phytoplankton assemblages (Table 2.3). In addition, the total biomass-normalized 38 C uptake rate was higher for I0W-CO2 grown cells (Fig.2.6). These results demonstrate that low CO2 conditions act to increase direct HCO3" transport and upregulate the C uptake system. This upregulation of transport rates may also be associated with a rise in the affinities for CO2 and HCO3" uptake as observed in laboratory experiments (Kaplan et al. 1994; Price et al. 1998; Kaplan and Reinhold 1999), but our data do not allow us to assess this directly. The regulation of C uptake rates by phytoplankton may explain why phytoplankton assemblages are able to maintain constant growth rates across a range of CO2 concentrations. External Carbonic Anhydrase Activity Although we did not observe any relationships between the in situ environmental parameters and direct HC03" transport, it is clear that species composition, and possibly ambient CO2 concentrations affect the level of external carbonic anhydrase (eCA) activity by the phytoplankton assemblages. The level of eCA activity was dependent upon the dominant phytoplankton group present at each station. We must note that all stations contained a mixture of nanoflagellates, dinoflagellates, and diatoms, and thus, the comparison of species composition to the level of eCA activity is qualitative. Nonetheless, the stations dominated by nanoflagellates and/or dinoflagellates (Table 2.1) tended to have the lowest apparent enhancement of the uncatalyzed HCO37CO2 exchange (eCA activity) (Table 2.2). Most notably, these were the open-ocean stations (1,2), two coastal stations (5 (>2um),6 (>2um)) as well as stations 8 and 10. In the remaining stations, the abundance of diatoms appeared to increase along with the activity of eCA. The stations along the continental shelf-break (3,7,10) and one coastal station (11) were 39 all dominated by diatoms such as Chaetoceros sp., Cylindrotheca sp., Fragilariopsis sp., Pseudonitzchia sp., and other unidentified pennate and centric diatoms. The highest eCA activity was observed at station 4 where the diatom, Rhizoselenia sp., was the dominant species. The results of the size-fractionated isotope disequilibrium experiments (>2um and >20um) provided further evidence for a taxonomic dependence of eCA activity. The data reveal a significant difference in eCA activity between the size fractions. The smaller size class that was dominated by nanoflagellates and small dinoflagellates, had lower eCA activity. By comparison, diatoms and larger dinoflagellates were dominant, and higher eCA activities were observed. Our observations of eCA expression are consistent with results from the Eastern Subtropical and Equatorial Pacific Ocean, where diatom dominated communities appeared to possess high eCA activity relative to phytoplankton assemblages dominated by other taxonomic groups (Tortell and Morel 2002). In contrast, no significant differences in eCA activity were found amongst Southern Ocean phytoplankton communities (Cassar et al. 2004). Higher eCA activity observed in many diatom-dominated communities may reflect the lower surface area to volume ratio of these large cells in comparison to nanoflagellates and small dinoflagellates. Together, the higher eC A activity and the use of a direct HCO3" uptake system would allow diatoms to satisfy a greater fraction of their C demands by way of HCO3" utilization. While many taxa appear to rely heavily on direct HCO3" transport, elevated eCA activity may be a particular feature of diatoms. Most laboratory studies that have focused on nano- and dinoflagellates suggest little or no eCA activity in dinoflagellates (Dason et al. 2004), 40 eustigmatophytes (Huertas et al. 2002), and Emiliana huxleyi (haptophyte) (Elzenga et al. 2000). However, a few studies have detected eCA activity in dinoflagellates (Berman-Frank et al. 1998; Nimer et al. 1997) and a haptophyte (Burns and Beardall 1987; Elzenga et al. 2000). It is therefore presently difficult to make generalizations about carbon acquisition in the group as a whole. Another environmental parameter that may affect eCA activity is the ambient CO2 concentration. Although we did not obtain good correspondence between the membrane-inlet mass spectrometer (MEMS) and the CO2SYS for seawater CO2 concentrations, we observed higher eCA activity at stations with lower CO2 concentrations (Figure 2.5). Correspondingly, Tortell and Morel (2002) observed the lowest apparent eCA activity at the station with the highest CO2 concentration. The most substantive field evidence for a CO2 effect on eCA activity has come from Lake Kinneret (Israel), where low-C02 conditions have been clearly shown to enhance eCA activity in natural phytoplankton assemblages (Berman-Frank et al. 1994,1998). This situation is rather unique in that the phytoplankton assemblage was completely dominated by a single species. However, due to the lack of reliable environmental CO2 data, we are unable to conclude that the availability of CO2 in the environment influences the C uptake systems of Bering Sea phytoplankton assemblages. Regardless, our CO2 manipulation experiments demonstrate CO2 concentrations regulate C uptake strategies in Bering Sea phytoplankton communities. Our incubation experiments show that CO2 concentrations regulate the expression of eCA activity. We observed lower eCA activity in low C0 2 conditions in both incubation experiments (Table 2.3). Why Bering Sea phytoplankton assemblages in our 41 CO2 incubations decreased eCA activity in the I0W-CO2 environment is unclear. We realize this contradicts previous studies that show CA activity is an essential component in the supply of CO2 to phytoplankton in I0W-CO2 grown laboratory cultures (Aizawa and Miyachi 1986; Sueltemeyer 1998; Lane and Morel 2000). Field studies have also observed an increase in CA expression in low CO2 conditions (Berman-Frank et al. 1994, 1998; Tortell et al. 2000). However, it is with some difficulty that our results of eCA activity are compared to these studies given that "CA" represents both internal and external CA activity. Nonetheless, experiments with Phaeocystis sp. (Elzenga et al. 2000), Phaeodactylum sp. (John McKay and Colman 1997), and several other phytoplankton species (Nimer et al. 1997; Morel et al. 2002) have demonstrated an increase in external CA activity in low CO2 conditions. Higher eCA activity has also been observed in Southern Ocean phytoplankton assemblages exposed to I0W-CO2 conditions (Cassar et al. 2004) where eCA activity increased at the expense of direct HCO3" transport (Cassar et al. 2004). However, it is unclear why Southern Ocean phytoplankton assemblages would decrease direct HCO3" transport in order to increase eCA activity. Nonetheless, the available evidence suggests that CO2 concentrations regulation certain aspects of C uptake systems of phytoplankton assemblages. We suggest the differences in C acquisition strategies found in the CO2 incubations are due strictly to physiological responses of the phytoplankton communities given that no obvious differences in growth rate, productivity, or species composition were found. A similar lack of C02-dependent growth rate effects has been observed in previous CO2 manipulation experiments with natural phytoplankton communities (Tortell et al. 2000, 2002; Tortell and Morel 2002). Over the time course of the experiments, we 42 also observed no obvious change in species composition between the high and I0W-CO2 conditions. Our results are analogous to experiments carried out in the coastal and eastern Subtropical and Equatorial Pacific Oceans (Tortell et al. 2000; Tortell and Morel 2002). Contrary to our results, other studies have observed an increase in phytoplankton growth and productivity due to elevated CO2 concentrations (Hein and Sand-Jensen 1997), and a decreased growth rate in I0W-CO2 conditions (Berman-Frank et al. 1998). Longer term CO2 manipulation experiments have also detected taxonomic shifts in Equatorial Pacific communities. The taxonomic shift involved a 50% decrease in diatoms and a 60% increase in Phaeocystis sp in low CO2 conditions (Tortell et al. 2002). Species-specific responses, light, iron, and other essential nutrients may all contribute to the varying results in phytoplankton growth rate, productivity, and C02-induced changes in species composition. Conclusions This field study determined C acquisition strategies of natural phytoplankton assemblages in the southeastern Bering Sea and examined the effects of CO2 manipulations on phytoplankton C acquisition systems. The following conclusions can be drawn: • HCO3" was the dominant C source utilized by the phytoplankton assemblages. • This study suggests that HCO3" utilization occurred mainly through a direct HCO3" transport system. 43 Direct HC03" transport was constitutive given that there were no associations with species composition, phytoplankton size fractions, CO2 concentrations and all other environmental parameters. External carbonic anhydrase (eCA) showed a modest enhancement of extracellular HCO3" dehydration of 45-465% compared to the uncatalyzed rate. Diatom-dominated stations show higher eCA activity than stations dominated by nanoflagellates. Higher eCA activity was observed at stations with lower CO2 concentrations. The CO2 manipulation experiments demonstrated that I0W-CO2 grown phytoplankton assemblages showed greater direct HCO3" utilization than high-CC>2 grown phytoplankton assemblages. The total biomass-normalized C uptake rate was higher for I0W-CO2 grown cells. Lower eCA activity was observed in I0W-CO2 grown phytoplankton assemblages in the CO2 incubation experiments. There were no obvious differences in growth rate, species composition, or productivity between low- and high-CCh grown phytoplankton assemblages. Chapter 3: Inorganic Carbon Acquisition by Marine Diatoms Introduction Marine phytoplankton play a fundamental role in the regulation of atmospheric CO2 concentrations, and hence, the global carbon cycle. Approximately 40% of the worldwide carbon fixation is due to photosynthesis by marine phytoplankton (Falkowski 1994). Specifically, diatoms (bacillariophyceae), are responsible for almost half of the oceans' primary production (photosynthesis) (Treguer et al. 1995). These microalgae are not only essential for fixing CO2 to organic C, they are perhaps the most important phytoplankton taxa for exporting C from the surface to the deep waters by means of the biological pump (Raven and Falkowski 1999). Diatoms are ubiquitous in marine and freshwater habitats (Raven and Waite 2004), although they are particularly successful in nutrient-rich coastal and upwelling regions (Thomas et al. 1978). Traditionally, scientists have examined how limiting nutrients, such as nitrate, phosphate, and iron affect marine phytoplankton growth and physiology (Greene et al. 1991; Geider et al. 1993). However, inorganic carbon is seldom considered as a limiting nutrient for marine phytoplankton growth due to the high concentration of DIC (dissolved inorganic carbon) in seawater (~2mM). The majority of inorganic C is in the form of bicarbonate (HCO3") and carbonate (CO32"), while CO2 accounts for less than 1% of the total DIC (Stumm and Morgan 1981; Morel and Herring 1993). This is problematic for RubisCO, the central C fixing and rate-limiting enzyme in photosynthesis, given that the enzyme is only able to use CO2 as its carbon substrate (Cooper et al. 1969). Even though CO2 could potentially limit diatom growth (Riebesell et al. 1993), several lines of 4 5 evidence suggest that diatoms are able to concentrate CO2 around RubisCO to alleviate such potential limitation. The strategies used by phytoplankton to increase the CO2 concentration around RubisCO, and thus, favor the carboxylation reaction for efficient C-fixation (see Raven 1997) are collectively called carbon concentrating mechanisms (CCMs). The direct, active uptake of HCO3", and CO2, and the use of extracellular carbonic anhydrase (eCA) have all been documented as CCMs for diatoms (Korb et al.1997; Tortell et al. 1997; Elzenga et al 2000; Burkhardt et al. 2001). The role of eCA in C acquisition is to catalyze the otherwise slow interconversion of FKXVto CO2 (Huertas et al. 2000). There is still much debate over the C acquisition strategies used by diatoms (Tortell et al. 1997; Cassar et al. 2002). Nonetheless, it is known that diffusive CO2 uptake is not the sole mechanism for C uptake as previously believed (Riebesell et al. 1993). While a few diatom species do apparently rely on diffusive C0 2 uptake (Cassar et al. 2002), these species appear to be rare, and are thus poor predictors of the behaviour of most other marine diatom species. Determining C uptake strategies of diatoms, and the degree to which they utilize CO2 and/or HCO3", is important for understanding the oceans' response to future increases in CO2 concentration. To determine C uptake strategies for all 200,000 existing diatom species (Mann and Droop 1996) would be unreasonable. However, this present study intended to determine C uptake mechanisms for a variety of diatom species that differ in their size and growth rate. I quantitatively determined the fraction of HCO3" and CO2 contributing to C uptake. Concurrently, I derived the apparent external carbonic anhydrase activity of each diatom species. The isotope disequilibrium technique was 46 employed to determine each of these variables (see chapter 2) . My results show that HCO3" is the predominant C species utilized by the majority of the diatom species, regardless or their size or growth rate. The data suggest that a large fraction of HCO3* uptake occurs through a direct transport mechanism. 47 Materials and Methods Algal Strains and Culturing Conditions Eleven different diatom species were obtained from the Canadian Center for the Culture of Microorganisms (CCCM) (Vancouver, Canada). Two additional species, Pseudo-nitzschia grand and Pseudo-nitzschia turgidula, were kindly supplied by Adrian Marchetti (UBC) while Thalassiosira weissflogii, T. oceanica, and T. pseudonana were kindly supply by Maite Maldonado (UBC). Cells were grown in artificial seawater medium, Aquil (Price et al. 1988/89). The major salts were dissolved in Milli-Q® water (Millipore Corp.) and enhanced with filter-sterilized (0.2 um Gelman Acrodisc PF) nutrients (Price et al. 1988/89) and chelexed ESAW vitamins (Harrison et al. 1980). All chemicals used were purchased from Sigma-Aldrich Canada Ltd (Oakville, ON). The cultures were maintained in iron-replete conditions by the addition of 56.5nM total Fe (free ferric ion activity of 10e"19,4M (pFel9.4)). Before diatom cultures were grown, cultureware was acid-washed and microwave-sterilized (Keller et al. 1988) with Milli-Q® water in order to minimize bacterial contamination. The cultures were subsequently grown in 28mL polycarbonate centrifuge tubes (Naglene) under continuous light at a photon density flux of 150umol m" s" m a 20°C walk-in incubator. The exceptions were Pseudo-nitzschia grand and Pseudo-nitzschia turgidula which were kept at low temperature and light (12°C, 50umol photons m'V 1 ) and cultured with HNLC water (high nutrients low chlorophyll) from Ocean St Papa (OSP; 45°N, 145°W) (supplied by A. Marchetti). Semi-continuous batch cultures were maintained by transferring 50uL of late exponential 48 phase culture into a new centrifuge tube with fresh Aquil medium. All transfers were executed at a sterile Class-100 laminar flow bench. To determine growth rates, chlorophyll a fluorescence (in vivo chlorophyll) was monitored daily using a Turner Designs® model 10-AU fluorometer (Brand et al. 1981). The slope of the natural log of fluorescence over time provided an estimate of the growth rate (p, day"1) (Guillard 1973). Acclimation to the Aquil medium was complete when growth rates between successive transfers were ±10% of each other (approximately one month). Microscope Analysis Live diatom cultures were examined using an inverted microscope and stage micrometer. Length and width were determined for approximately 25 randomly selected cells (per culture). When live cells were not available, cultures fixed with acid Lugol's iodine (Throndsen 1978), and CCCM size data were used as an alternative. However, the preserved cultures were used with caution given that fixatives can potentially cause cells to shrink (Montagnes et al. 1994). Surface area (SA) and Volume (V) were estimated based on a simple geometric shape (i.e. cylinder) following the suggestions from Montagnes and Franklin (2001) and Menden-Deur and Lessard (2000). It has been suggested that the difference in volume based on a standard geometric shape or composite shapes (e.g., cylinder plus two cones) is minimal (Menden-Deur and Lessard 2000). A surface area: volume ratio (SA/V ratio) was determined for each diatom. 49 Isotope Disequilibrium Procedure A list of diatom species (and their corresponding culture identifications) used in isotope disequilibrium experiments during summer 2002 through winter 2003 is provided in Table 3.1. In preparation for isotope disequilibrium experiments, 250mL polycarbonate containers with fresh Aquil medium were inoculated with 2-5mL of an exponential phase batch culture. The containers were grown under the same conditions (described above). Once late exponential growth phase was reached (approx. 5 days), the diatom culture was gently filtered onto a 2um polycarbonate filter. The diatoms were resuspended off of filters into pH 8.5 Aquil medium (buffered with 20mM Bicine), and isotope disequilibrium experiments began immediately. The isotope disequilibrium procedure, data analysis, and modeling were nearly identical to the Materials and Methods in chapter 2, with the exception of a few minor modifications. The amount of 1 4 C spike injected into the concentrated cell suspension was 10u.Ci 1 4 C , and not 20uCi as with the natural phytoplankton assemblages. The experiments were carried out at 15°C, instead of 10°C. Due to the effect of temperature on the chemical kinetics of the carbon species, experiments were run for 180 seconds instead of 300 seconds. The rate constants, al and a2, were calculated to be 0.0272 and 0.0315 (seconds'1), respectively, given the increase in temperature. Once the experiments were complete, sample vials were degassed on a shaker table over night rather than purged with air. Both degassing techniques are reliable for the removal of residual inorganic 1 4 C . Subsequent to degassing, 5ml of scintillation fluid (ScintiSafe™) was added to each vial and a scintillation counter was used to detect the disintegrations per minute (dpm) of organic 1 4 C in each vial. Analysis and modeling to 50 determine the relative proportions of C0 2 and HC03" utilized were completed as described in Chapter 2, however, only the integral fit (eq.4, chapter 2) was used for analysis. Results Time courses of 1 4 C incorporation during isotope disequilibrium experiments are shown for several diatom species. Results for species that exhibited a significant amount of direct HCO3" transport (high /HCO3 values) are shown in Fig. 3.1 while species that utilized relatively large quantities of CO2 in comparison to the other diatoms in this study are shown in Fig. 3.2. The fraction of HCO3" and CO2 utilized, in addition to the enhancement of a (i.e. apparent enhancement of eCA activity) were assessed by applying equation 4 (chapter 2) to the time courses of 1 4 C incorporation. The best-fit curves (solid lines) represent equation (4) and the dashed curves represent a CCVonly uptake model fit. Fractions of direct HC03" uptake (/HCO3-values) for all diatom cultures are shown in Table 3.2. /Hco3-values ranged from 0.41-0.94. Direct HCO3" transport accounted for the majority of C uptake in all the diatom species with the exception of Phaeodactylum tricornutum and Pseudo-nitzschia grand. Although these were exceptions, direct HCO3" uptake still accounted for nearly half of the total C uptake. While the apparent enhancement of the HCO37CO2 exchange (eCA activity) varied from 3% to 1200% (Table 3.2), the majority of diatom species only showed an enhancement that was <200%. For a few species, isotope disequilibrium experiments were conducted multiple times over an extended period. C uptake results for Asterionella glacialis (809), Chaetoceros socialis (653), Thalassiosira weissflogii (TW), and Thalassiosira oceanica (TO) were all similar between 2002 and 2003 (<5% difference between the years). In contrast, Chaetoceros decipiens (537), Skeletonema costatum (782), and Thalassiosira pseudonana (TP) showed changes in/Hco3-values that varied between 6-16% during the 52 same time frame. Chaetoceros decipiens (537) also had a significant change in the apparent enhancement of the HCO37CO2 exchange (eCA activity) when this value went from 84% to 1240% between 2002 and 2003. Thalassiosira pseudonana (TP) had the largest change in C uptake strategies for all of the diatoms in this study (Fig. 3.3) as the level of direct HC03" transport increased by 16% and eCA activity increased from ca. 100% to 635% between the years. Cell surface area: cell volume ratios (SA/V) and growth rates were calculated for each diatom species (Table 3.3). SA/V ratios varied between 0.4-1.7 while growth rate (p) varied between 0.37-1.94 day"1. Asterionella glacialis (809) was the only diatom to decrease its growth rate from 1.6d"' to l.ld"1 between the 2002 and 2003. When SA/V ratios and growth rates were compared to C acquisition (/HCO3 and eCA activity) by the diatoms, no relationships were observed (Fig. 3.4). 53 Table 3.1: A list of marine diatom species (and their corresponding culture identifications) used in isotope disequilibrium experiments during summer 2002 through winter 2003. Culture ID Species Name 553 Odontella aurita 608 Nitzschia thermalis 643 Thalassiosira rotula 809 Asterionella glacialis 626 Grammatophora oceanica 782 Skeletonema costatum 653 Chaetoceros socialis 537 Chaetoceros decipiens TW Thalassiosira weissflogii TO Thalassiosira oceanica 425 Cylindrotheca fusiformis 640 Phaeodactylum tricornutum 694 Leyanella arenaria PG1 Pseudo-nitzschia granii SP1 Pseudo-nitzschia turgidula TP Thalassiosira pseudonana 54 a 20000 T — , 1 4 o che-Seconds T i m e ( s e c o n d s ) Figure 3.1: Time course of 1 4 C incorporation during isotope disequilibrium experiments for a) Thalassiosira weissflogii (TW), b) Leyanella arenaria (694), c) Pseudo-nitzschia turgidula (SPI), and d) Cylindrotheca fusiformis (425). The best-fit curves were obtained by applying equation (4) (see Materials and methods, Chapter 2). The dashed curves represent a C02-only uptake model fit. These time courses display a high level of direct HCO3" transport. Error bars represent ± standard error (n=2) and are smaller than the symbol when not visible. 55 3500 3000 2500 2000 1500 1000 500 j 7000 6000 4000 3000 2000 50000 30000 o CL 20000 10000 H Seconds Figure 3.2: Time course of 1 4 C incorporation during isotope disequilibrium experiments for a) Nitzschia thermalis (608), b) Chaetoceros socialis (653), c) Phaeodactylum tricornutum (640), and d) Pseudo-nitzschia grand (PG1). These time courses display a relatively higher amount of C0 2 transport, (see Fig. 3.1 for further details). 56 Table 3.2: Isotope disequilibrium results from marine diatom species. The/Hco3-value represents the fraction of direct HC03" transport. Error bars represent ± standard error (n=2-4). Diatom species that were investigated in both 2002 and 2003 are distinguished with (yr=2) being noted beside the Culture ID. Culture ID fHC03 values Apparent enhancement of the HCO3/CO2 exchange (%) (eCA activity) 553 0.70±0.03 83±5.0 608 0.60±0.11 36±3.9 643 0.69±0.24 -809 (yr=2) 0.85±0.02 165±25 626 0.76±0.04 54±10 782 (2002) 0.80±0.01 157±11 782 (2003) 0.70±0.05 135±25 653 (yr=2) 0.60±0.05 95±3.6 537 (2002) 0.70±0.03 84±6.7 537 (2003) 0.64±0.10 1238±374 TW (yr=2) 0.94±0.01 128±21 TO (yr=2) 0.75±0.02 69±11 425 0.84±0.02 411±68 640 (2002) 0.51±0.09 10±0.7 640 (2003) 0.44±0.15 14±2.3 694 0.87±0.01 69±11 PG1 0.41±0.21 62±7.0 SP1 0.89±0.01 157±18 TP (2002) 0.76±0.02 102±7 TP (2003) 0.92±0.02 635±143 57 Q_ Q •o CD O 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 a J/ // J O 10000 CL o o •— 8000 6000 4000 H 2000 50 100 Seconds 150 200 Figure 3.3: Time course of 1 4 C incorporation during isotope disequilibrium experiments for Thalassiosira pseudonana (TP) in a) 2002 and b) 2003. The best-fit curves were obtained by applying equation (4) {see Materials and methods, Chapter 2). The dashed curves represent a C02-only uptake model fit. Error bars represent ± standard error (n=2) and are smaller than the symbol when not visible. 58 Table 3.3: Surface area: volume (SA/V) ratio and b) growth rate (u) (day"1) for each marine diatom species. Error bars for growth rates are ± standard error (n=10-15). Diatom species that were investigated in both 2002 and 2003 are distinguished with (yr=2) being noted beside the Culture ID. Culture ID SA/V ratios M (day1) 553 0.41 1.10±0.020 608 0.41 0.37±0.015 643 0.48 0.86±0.018 809 (2002) 0.50 1.60±0.030 809 (2003) 0.50 1.10±0.030 626 0.55 0.68±0.011 782 (yr=2) 0.73 0.87±0.020 653 (yr=2) 0.80 1.17±0.021 537 (yr=2) 0.91 1.15±0.024 TW (yr=2) 0.96 1.18±0.023 TO (yr=2) 1.04 1.22±0.022 425 1.05 1.14±0.022 640 (yr=2) 1.21 1.44±0.019 694 1.22 1.94±0.054 PG1 1.70 1.0±0.030 SP1 1.70 1.2±0.030 TP (yr=2) 1.71 1.56±0.028 1.0 0.8 w CD .2 0.6 CD > i g 0.4 0.2 0.0 ;i4oo f i 3 s 1 - i 1 r~ 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Surface areaA/olume ratio Growth rate (day"'') Figure 3.4: /Hco3-values (fraction of direct HC03" transport) over a range of a) surface area: volume (SA/V) ratios and b) growth rates (u day"1). Apparent enhancement of the HCO37CO2 exchange (%) (external carbonic anhydrase activity) over a range of c) SA/V ratios and d) growth rate. Error bars represent ± standard error (n=2-4) and are smaller than the symbol when not visible. 60 Discussion The goal of this laboratory study was to determine C uptake strategies of a variety of diatom species, and the degree to which they utilize CO2 and/or HCO3". All species were grown in light-saturated and nutrient replete conditions in order to examine how C acquisition strategies operate under optimal conditions. A significant observation is that the majority of the diatom species utilized HCO3" as their predominant C source. It appears that most of the HCO3" utilization occurred through a direct transport system with only a few species relying greatly on eCA activity (indirect HCO3" utilization). Our results suggest that CO2 was also a C source taken up in addition to HCO3". Whereas previous studies have reported similar carbon-concentrating mechanisms for diatoms (see Colman et al. 2002 for review), to my knowledge, no studies have thoroughly examined an array of marine diatom species in relation to C acquisition. After quantitatively determining the fraction of HCO3" and CO2 utilized, results were compared to diatom size (i.e. cell surface area: cell volume ratio) and growth rate. No relationships were found when diatom size and growth rate were compared to C acquisition results. Carbon Acquisition Strategies Based on the integral model fits (eq.4, chapter 2) from the isotope disequilibrium data, the quantitative analysis of C acquisition indicates that HC03" was the dominant C species utilized by the majority of diatoms, and direct HCO3" transport appears to be widespread (Table 3.2). Direct HCO3" uptake by diatoms has been reported in a number of previous studies (Nimer et al 1997; Tortell et al. 1997; Burkhardt et al. 2001; Cassar et 61 al. 2004). While indirect HCO3" utilization via eCA activity for C acquisition by diatoms has also been well documented (Burkhardt et al. 2001; Siiltemeyer 1998; Morel et al. 2002), our results suggest that eCA activity is not a major contributor to total C uptake as previously believed. External CA activity rarely shows an enhancement of over 200% versus the uncatalyzed rate (Table 3.2). Although HCO3"utilization is widespread among the diatom species studied, CO2 is clearly still a part of the C acquisition process. Our findings are consistent with previous studies that observe the simultaneous uptake of both C0 2 and HCO3" (Burkhardt et al. 2001; Colman et al. 2002). In spite of the simultaneous utilization of CO2 and HC03~by most of the diatom species, it is obvious that there are species-specific differences in the relative proportions of CO2 and HCO3" utilized. These differences can be observed in the time courses of 1 4 C incorporation (Fig.3.1 and 3.2) and are best represented in Table 3.2. The species-specific differences can be attributed to variations in specific transporters and C transport systems. For instance, there is the possibility that some diatoms have a functional C4 photosynthetic pathway (Reinfelder et al. 2000). It appears that Thalassiosira weissflogii may be able to use HC03" directly to make C4 compounds (Reinfelder et al. 2000). The C4 compounds would be transported into the chloroplast and decarboxylated to liberate CO2 for RubisCO (Morel et al. 2002). In spite of decades of research on C acquisition by diatoms, specific transporters and transport systems are still unknown. There is, however, the potential for separate, independent transport systems for CO2 and HCO3" like those found in cyanobacteria (Price et al. 2002) as well as C4 photosynthetic pathways in other marine diatoms. With the recent sequencing of the genomes for Thalassiosira pseudonana (Armbrust et al. 2004) and Phaeodactylum tricornutum 62 (Monsant et al. 2005), hopefully an in-depth analysis of the specific C transport systems will be performed. In addition to species-specific differences in C acquisition, this study also found time to time variation within a few identical species. The largest variation in C uptake was observed in Thalassosira pseudonana, although HCO3" remained the predominant C source. This variation can be observed in the time courses of 1 4 C incorporation during 2002 and 2003 (Fig.3.3). Both direct HCO3" transport and apparent enhancement of the uncatalyzed HCO37CO2 exchange (eCA activity) increased during the year. One possible explanation is that an isotope disequilibrium experiment was carried out during sexual reproduction of Thalassosira pseudonana. Although this species is regularly asexual, every spring Thalassiosira sp. undergoes sexual reproduction that is independent of size, and nutrient limitation (M. Maldonado, pers. comm.). Another possible explanation is that Thalassosira pseudonana may be sensitive to long-term culturing conditions, and therefore, subject to physiological and/or biochemical change. These physiological and/or biochemical changes could also be due to selection of variant strains from within the batch culture over the time period (1 year). Although this study focused on C acquisition by diatoms under optimal conditions and steady-state growth, had this not been the case, potential variability in C acquisition results could have been generated by various stages in the life cycle. For example, Chlamydomonas reinhardtii has been shown to have diurnal variations in the functioning of its carbon-concentrating mechanisms (Marcus et al. 1986). Due to differences in C acquisition even within identical species, it is challenging to compare the results of this study with those from many others. This is particularly 63 difficult given that quantitative proportions of HCO3" and CO2 utilization for a number of diatoms have never been assessed. Cassar et al. (2002) quantitatively determined that Phaeodactylum tricornutum takes up little or no HCO3", whereas this study finds HCO3" utilization accounts for approximately 50% of the total C acquisition. However, a comparison of data from diatoms studied here with data from those reviewed by Colman et al. (2002) reveals that similar C uptake strategies are employed by the identical diatom species in both studies. Carbon Acquisition in Relation to Diatom Size Although HCO3" was a dominant C source for most diatoms examined, variation in the relative proportion of CO2 and HCO3" utilized was observed nonetheless. This study investigated if the variation in C acquisition strategies is affected by diatom size. A cell surface area: cell volume ratio (SA/V ratio) seems appropriate to use as a size parameter given that it is a good comparison for the supply/demand ratio for nutrient acquisition. As a diatom's size increases, the SA/V ratio decreases. This induces a decrease in nutrient flux per volume and a potential limitation on the number of nutrient transporters (Raven and Kiibler 2002). Therefore, surface area may act as a constraint for C uptake (Chisholm 1992). Furthermore, an increase in size also increases the thickness of the diffusive boundary layer which may also create a problem for C uptake by decreasing the diffusive flux of nutrients to the cell surface (Raven 2003). Although CO2 is less than 1% of the total dissolved inorganic C (DIC) in seawater, this study speculates that smaller cells might rely more on CO2 given that they have a high SA/V ratio, a smaller diffusive boundary layer, and therefore, a better 64 diffusive CO2 supply in relation to their demand for C. There is also an energetic cost of running C transport systems. It has been suggested that HCO3" has a higher energy cost given that this negatively charged molecule must cross a negative electric potential difference (Burkhardt et al. 2001). Accordingly, smaller diatoms appear to have less of a requirement for active C uptake strategies energized by photosynthesis. In contrast, the physical limitations of a thicker diffusive boundary layer and smaller SA/V ratio of larger diatoms ought to impede CC -^uptake, encouraging them to rely on HCO3". Consequently, the need for active HC03" utilization increases as a diatom increases in size. However, this study demonstrates that even small diatoms utilize HCO3" as a dominant C source. HC03" was the predominant C source for most diatoms which ranged in SA/V ratios from 0.4 -1.7 (Fig.3.4a). Contrary to expectations, the highest SA/V ratios were coupled to/HC03-values of 76-92%. Additionally, no relationship was observed between eCA activity and SA/V ratios (Fig.3.4c). Evidence from the literature supports the lack of a relationship between C acquisition strategies of diatoms and their size. Numerous phytoplankton with even larger SA/V ratios in comparison to the smallest diatom in this study possess direct HCO3" uptake systems and/or rely on eCA activity. Supporting evidence comes from the prevalence of CCMs in haptophytes (Burns and Beardall 1987; Elzenga et al. 2000) and other picoplankton (Colman et al. 2002). Even the smallest cyanobacteria employ C concentrating mechanisms (Price et al. 2002). In addition, Raven (2003) reviews numerous studies in order to summarize CCMs in relation to the biology of algae. Although algae size and C acquisition is not a significant theme, it is mentioned that HCO3" utilization is common among the smallest phytoplankton and therefore, no additional exploration into C acquisition and algae size is discussed (Raven 2003). It 65 seems as though the coexistence of CO2 and HCO3" transport systems is beneficial to a diatom even if it is small. A HCO3" transport system would enable diatom species of all sizes to exploit the abundant HCCVpool in the ocean in addition to utilizing CO2. In spite of the energy costs associated with active C uptake, cost-benefit examinations propose that algae with active C acquisition strategies are at no disadvantage in comparison to species that depend on CC^by diffusion (Raven et al. 2000). Apparently, the energy costs of the CCM balance the costs of photorespiration and glycolate metabolism (Raven et al. 2000). Although this study found no relationships between C uptake strategies and SA/V ratios, it is possible that certain diatoms have the ability to alter C acquisition strategies as their size increases or decreases. For instance, size has been implicated as a factor in C acquisition strategies of Diatoma moniliformis Kiitzing (Potapova and Snoeijs 1997). It was shown that this species changes its cell proportion during its life cycle. This fits well with seasonal cycles of population growth, with high surface area to volume ratios during the period of optimal growth in spring (Potapova and Snoeijs 1997). Further investigation should be completed on C acquisition strategies in relation to changes in cell size for individual species. C acquisition in relation to diatom growth rate Having determined that no relationship exists between C acquisition and diatom size, this study shifted its focus to examine the effects of diatom growth rates on C uptake. However, one complication arises from the fact that growth rate and cell size (i.e. volume) are not independent variables (Sarthou et al. 2005). This study investigated the relationship between cell size (i.e. volume) and growth rate of 16 diatoms and 66 compared results to those from 67 diatoms (Sarthou et al. 2005). Although Sarthou et al. (2005) observed a low coefficient of determination (r2=0.48), they described the relationship between growth rate (|a.max) and cell volume (V) with the allometric relation: U m a x - 3.4 V" 0 1 3 . This study demonstrated an identical coefficient of determination (r2=0.48, p<0.01) and related growth rate (u) to cell volume (V) with the following equation: u=3.9V"0 2 2 . Intuitively, diatoms with higher growth rates will demand more C to meet their photosynthetic requirements and hence, should have a higher demand for HCO3". Thus, their dependency on direct HCO3" uptake and eCA activity should be greater. Earlier, this study also proposed that large diatoms (i.e. low SA/V) should depend on HC03" utilization. However, a literature review reveals that smaller cells have the highest growth rate (see e.g. Geider et al. 1986; Raven and Kubler 2002). This fact complicates the study's predictions regarding the relationships between C acquisition strategies, diatom size, and growth rate. Accordingly, it can be concluded that no relationship exists between C acquisition strategy and growth rate (Fig. 3.4b,d). No significant difference in /HCO3 or eCA activity was observed in Asterionella glacialis (809), even with a decrease in its growth rate from 2002 to 2003. Supporting evidence comes from the Southern Ocean where it was found that increases in growth rate of natural phytoplankton assemblages did not elicit any significant changes in C acquisition (Cassar et al. 2004). Given that no relationships were found between C acquisition and diatom size, it is reasonable to have observed no relationships between C uptake and growth rate. 67 Conclusions This laboratory study employed the isotope disequilibrium technique to quantitatively determine the fraction of HCO3" and CO2 utilized by a variety of marine diatoms. The conclusions of this study are as follows: • HCO3" was the predominant C species utilized by most of the diatoms in this study. • The data suggest that the majority of HCO3" utilization occurred though a direct transport process. • Although eCA activity was present, it did not appear to be a major contributor of total C uptake by the diatom species. • CO2 was also an important element in the transport process. • There were species-specific differences in the fraction of HCO3" and CO2 uptake, but these differences were not related to diatom size (i.e: surface area: volume) or growth rate. 68 FUTURE RESEARCH This thesis has provided information on inorganic carbon acquisition by natural phytoplankton assemblages and marine diatom species. C acquisition by natural phytoplankton assemblages was assessed across a range of phytoplankton biomass, species composition, productivity, and CO2 concentrations. The second part of the thesis provided a comparative analysis of C uptake strategies of a variety of marine diatoms in relation to diatom size and growth rate. The studies in this thesis open up further questions in relation to C acquisition by phytoplankton. Below is a list of questions that could potentially be addressed in future studies, however, it is by no means exhaustive and represents but a few of the many directions in which future research may proceed. Is HCO3" the predominant C source utilized by natural phytoplankton assemblages in other regions of the ocean? Does the availability of micronutrients, such as iron, affect C acquisition? Does C acquisition by natural phytoplankton assemblages change as the succession of species composition changes? How do natural phytoplankton assemblages in other regions of the ocean respond to varying CO2 concentrations? What are the effects of C acquisition strategies when light availability changes? 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