A comparative study on the affinities for inorganic carbon uptake, nitrate and phosphate between marine diatoms and dinoflagellates Mr. T. (Thomas) Hofman - 11066938 Institute for Biodiversity and Ecosystem Dynamics (IBED) Supervised by: mw. dr. J.H.M. Verspagen Abstract: Eutrophication and increasing atmospheric carbon dioxide concentrations are water quality concerns threatening our drinking water and food supply due to a rise in harmful cyanobacterial and harmful algal blooms. Understanding which factors determine the species distribution of phytoplankton could help to prevent the increase of these blooms in the future. Growth is thought to be limited by the scarcest resource available. As eutrophic waters are, by definition, rich in macronutrients such as nitrate and phosphate, inorganic carbon limitation becomes more significant in population dynamics as a limiting factor. Moreover, due to increased growth rates in eutrophied oceans, inorganic carbon depletes faster. An in silico literature research on the the affinity for phosphate, nitrate and inorganic carbon in marine diatom and dinoflagellate species gave insights in species distribution, based on in vivo uptake kinetics, field measurements and uptake mechanisms of both taxonomic groups. The affinity for nitrate and inorganic carbon was significantly higher dinoflagellates. This difference could explain the species composition in marine environments. According to findings in this research, dinoflagellates are better adapted, based on their affinity for nutrients and inorganic carbon, to oligotrophic and Ci depleted environments. 1. Introduction Phytoplankton blooms can severely decrease water quality, threatening drinking water and food supply. Anthropogenic increase of atmospheric carbon dioxide (CO2) concentrations and nutrient enrichment alter hydrological patterns and strongly influence the duration, frequency and intensity of harmful cyanobacterial blooms (HCB’s) (Visser et al, 2016) and harmful algal blooms (HAB’s) (Smith and Schindler, 2009). Eutrophication has become one of the main water quality issues for aquatic ecosystems and despite being extensively researched in the past decades, many interactions remain to be understood (Smith and Schindler, 2009). The main causes for eutrophication are anthropogenic waste waters, manure and fertilizer use, and nitrogen emissions by industries. It is well established that both CO2 increase and nutrient increase positively affect growth in phytoplankton species (Visser et al, 2016). Acquisition of macronutrients and resistance to grazers and diseases are fundamental physiological processes in the distribution of phytoplankton (Litchman et al. 2007). Growth and reproduction for instance, are frequently limited by P and N in aquatic ecosystems (Elser et al. 2007). N and P availability are thought to be the major determinant in species composition in oligotrophic waters according to resource competition models (Burson et al. 2018). In eutrophic waters however where N and P availability are high, other factors such as CO2 and light availability are more likely to limit phytoplankton growth (Smith 1986, Hein 1997) and determine interspecific competition (Huisman et al. 1999, Ji et al. 2017). There is a negative correlation between the nutrient load and the CO2 availability in aquatic systems: at high nutrient load, CO2 concentrations are low, and vice versa (Balmer and Downing 2011). Therefore, it seems valid that species from oligotrophic systems have a high affinity for N and P, but a low affinity for Ci, while species from eutrophic systems have a low affinity for N and P, but a high affinity for Ci. Here we will study whether such a difference between nutrient and Ci affinty exists using published measurements. Inorganic carbon concentrations are generally not considered a limiting resource as carbon is relatively abundant with millimolar concentrations compared to nanomolar macronutrient concentrations and picomolar micronutrient concentrations (Millero, 2006). During intense phytoplankton blooms however, ocean surface Ci depletion does occur, relative to the deep ocean (Millero, 2006). Patches of Ci depleted ocean surface can last for several days to weeks during phytoplankton blooms, although they are replenished by physical exchange ratter quickly. The relatively low diffusion rate of CO2 limits the accumulation of inorganic carbon by phytoplankton. This limitation is aggravated by low affinity for CO2 in RubisCO and the oxygenase activities (Reinfelder, 2011). The ocean surface CO2 concentrations are below saturation levels of the RubisCO enzyme (Badger et al, 1998) and combined with the low diffusion rate of CO2, this can induce Ci limited growth. This limitation may however be overcome by motility, such as in dinoflagellates. 2 - Phytoplankton fix Ci as their carbon source as carbon dioxide (CO2) or bicarbonate (HCO3 ) (Verspagen et al. 2014). The uptake of inorganic carbon by phytoplankton can deplete the aquatic Ci concentrations. Depletion of the CO2 concentration can increase the pH of lakes. Consequently, CO2 depletion combined with high pH is often associated with algal blooms (Verspagen et al. 2014). This - - change in pH causes a shift in CO2: HCO3 ratio. As CO2 and HCO3 uptake are regulated by different membrane proteins with their own affinity, the pH of the system can greatly alter uptake kinetics for Ci. This study will look at marine species. As marine environments are less susceptible to change in pH due to a higher buffer capacity of the ocean, marine species are less likely to experience great - differences in CO2: HCO3 ratio, consequently this study looks at the Ci affinity, which is a - combination of both CO2 and HCO3 affinity. Recent research on the water quality in the Baltic sea uses the diatom: dinoflagellate ratio (Dia/Dino index) as an index for the quality of water (Wasmund et al. 2017). Diatoms and dinoflagellates are the major phytoplankton groups in marine environments. Diatoms are the only major group of phytoplankton that use silicate (Si) for their cell wall structure. Usually, Si is of no concern regarding eutrophic waters. However, high inputs of nitrogen (N) and phosphorus (P) cause the relative amount of Si available for diatoms to decrease (Wasmund et al. 2017). Presumably, according to Wasmund et al. (2017), this makes Si the limiting factor in diatom growth and prevents them from growing in severely eutrophied marine environments. Dinoflagellates will not experience limitation by Si availability as they do not require silicate for their cell structure and grow well in eutrophic waters. The Dia/Dino-index would thus decrease in eutrophic waters, while a high Dia/Dino-index indicates less eutrophied water (Wasmund et al. 2017). Indeed, dinoflagellates have a relatively low affinity for NO3 compared to other taxonomic groups according to Litchman et al. (2007), supporting their prevalence in eutrophic ecosystems. In contrast, the affinity for nitrate is high in diatoms (Litchman et al. 2007), which would be more beneficial in less eutrophic environments. Reinfelder (2011) however, reviewed carbon concentrating mechanisms (CCMs) in marine phytoplankton and found large diatoms prevailed in highly eutrophied water, which is low in Ci concentration. Reinfelder further suggested the dinoflagellate Ci affinity to be subordinate to that of diatoms, supported by their CCMs and field experiments. The absence of diatoms in eutrophied water and the abundance of dinoflagellates in eutrophied waters, as proposed by Wasmund et al. (2017) or the opposite, as argued by Reinfelder (2011), would make these groups relevant to test the for difference in nitrate (NO3), phosphate (PO4) and Ci affinity in highly eutrophied marine environments. Testing groups of organisms instead of species allows data on either the affinity for nitrate, phosphate or the affinity for Ci to be used as the average of the group is compared. Thus data on both the affinity for NO3/PO4 and Ci affinity per species, which is rarely available, is not essential. Clearly, affinity for PO4, NO3 and Ci differs among species in the groups. Compensation for those differences can be done based on cell size. Litchman et al. (2007) found that 3 the major parameters of nitrate uptake and growth, scale with cell size. The general relationship is a power function of the cell volume. Establishing the relations between PO4, NO3 and Ci affinity increases the understanding of the mechanisms determining the species composition in marine ecosystems. Affinity for N, P and Ci for many phytoplankton species has already been reported in a number of studies (Litchman, 2007; Edwards et al 2015, Schwaderer et al 2011, Clement et al 2017). Here, the research question: “Could a trait based approach on the Ci affinity, phosphate and nitrate affinity in marine diatom and dinoflagellate species explain part of the species distribution in marine environments under eutrophic conditions?” is answered using these data, and additional data collected from additional uptake kinetics experiments. The in silico quantification of Ci affinity using previous studies, could provide new insights in the phytoplankton uptake dynamics and species distribution. If differences in affinity are observed, this research aims to find the cause for these differences based on cell specific traits, and compare the differences to field observations in order to assess whether affinity is a good indication for species composition or not. This research predominately focused on