Background: As a limiting nutrient for algal growth, nitrogen (N) plays an essential role in the biological productivity of aquatic ecosystems [1,2]. However, the massive acceleration in the industrial production of reactive N (Nr) and its global use in artificial fertilizers have led to a host of environmental problems [3]. Nr includes inorganic (NH3, NH4+, NOx, HNO3, N2O, NO3-) and organic forms (e.g. urea, amines, peptides and proteins) that readily participate in various reactions of the global N cycle. Over the last 50 years, anthropogenic perturbations of the natural N cycle have led to an increasing accumulation of inorganic Nr in soil, water and air, intentionally through agriculture and unintentionally through fossil-fuel consumption and other activities, adversely affecting human health, biodiversity, environment and climate change [4].

 

The vast majority of studies of N in the marine environment have focused on dissolved inorganic N (DIN). However, the most recent compilation shows that dissolved organic N (DON) frequently comprises the largest component (60–69%) of total dissolved N in rivers, estuaries, coastal and surface ocean waters [5]. A variety of phytoplankton species can use DON directly to meet their N needs [6], and heterotrophic uptake of dissolved organic C (DOC) has been observed in a number of dinoflagellates [7] and chrysophytes [8,9]. 

Amino acids are essential biochemical components and account for 40-90% of the total N mineralized in coastal marine sediments [10,11]. They comprise the largest reservoir of ON compounds in most aquatic organisms, where they exist in free or combined (i.e. peptides and proteins) form. Recent studies have indicated that both bacterioplankton [12,13] and plytoplankton [14] can directly utilize oligopeptides (i.e. combined amino acid residues with a mass below 1000 Daltons). A study of peptide uptake by phytoplankton [14] demonstrated that both external hydrolysis and direct assimilation can occur, depending on molecular size, with highest rates measured in the size fraction containing the dominant phytoplankter. This suggests that phytoplankton contribute substantially to, or may even dominate, peptide uptake and observed extracellular peptide hydrolysis. Interestingly, terrestrial plants and microorganisms can directly utilize small peptides in soil and have been shown to choose peptides over amino acid monomers as substrates in a range of soils from different ecosystems [15]. Peptides may also have an unrecognized global importance in the marine N cycle, providing N to algae at an earlier stage of decomposition than previously acknowledged. This could enable certain algal species to gain an advantage over competitors, particularly if DIN is depleted. 

 

Increases in the occurrence of harmful algal blooms in the United States mid-Atlantic region have been attributed to elevated DON [16,17], while an invasion of the harmful alga Prorocentrum minimum into the Baltic Sea could have been enhanced by DON enrichment from anthropogenic sources [18]. Climate change scenarios predict both episodic conditions of elevated rainfall and extended periods of dry conditions [19], leading to variable riverine inputs to coastal areas, altered nutrient ratios, and likely the balance of inorganic to organic N in the N pool. Organic N can constitute the major fraction of the N pool but its dynamics are poorly understood relative to the well-characterized inorganic pool. It is crucial, therefore, to understand the role of organic N in coastal waters and how such changes impact on ecosystem function and health. This project will provide a basis for a more holistic understanding of the reactivity of organic N, and both its importance to algal productivity and potential contribution to environmental degradation and reduced biodiversity.

 

Hypothesis: Algal species that can directly access oligopeptides gain an advantage over competitors at low levels of DIN.

 

To test the hypothesis we have the following focussed aims and objectives:

 

 

 

Methods: Monospecific cultures of a variety of algal species will be selected from the Roscoff Culture Collection (http://www.sb-roscoff.fr/phyto/RCC) according to their importance both in terms of abundance in the Western English Channel ecosystem and potential to form harmful algal blooms (e.g. the coccolithophore Emiliana huxleyi, the dinoflagellates Prorocentrum minimum, Karenia brevis and Alexandrium minimum, the diatom Chaetoceros sp.). Growth of the selected species in culture media containing different N sources (inorganic vs organic) will be assessed at the Station Biologique de Roscoff during the first visit (Months 1-4). A sub-set of species able to use organic N will then be cultured at Plymouth University, where an oligopeptide (3-4 amino acid residues) tagged with Lucifer yellow anhydride will be added to the culture medium prior to innoculation so that the rate of peptide hydrolysis in each culture can be measured through the growth cycle, using HPLC with fluorescence detection14 (Months 5-14). The second visit to Roscoff will involve the co-culturing of algal species so that they are in competition for the oligopeptide N source, concentrations of which will be measured during the growth cycle by LC-MS (Months 15-18). All experiments will be conducted using replicate cultures. Cell numbers (and cell type in co-cultures) will be monitored by flow cytometry. Finally, rates of peptide hydrolysis will be measured in water samples collected during spring and summer algal blooms from coastal monitoring stations in the English Channel (L4, Plymouth and/or Astan, Roscoff). The quality and quantity of oligopeptides will be measured in these samples by LC-ESI-MS using a method developed at Plymouth [20] to: 1) place rates of peptide hydrolysis in context of peptide availability; 2) to measure levels of oligopeptides through a bloom to determine their significance as a source of N for algae (19-30 months). The thesis write-up will be the focus of the last 6 months of the project to enable a timely finish (30-36 months).

References

This studentship proposal has been designed to provide integrated multidisciplinary doctoral training across two leading European marine science research institutes. It takes advantage of existing links between the partners, particularly those developed during a recent (2013) project entitled “Abundance and speciation of organic nutrients during algal growth cycles” funded as a transnational access activity by the EU infrastructure program ASSEMBLE (www.assemblemarine.org). Published outcomes from the study will include articles in high-impact research journals and both groups involved have a strong track record of publications in marine science.

 

For the DON characterization component of this study we will employ a technique developed by the Plymouth group that combines solid phase extraction with HPLC-ESI-MS (High Performance Liquid Chromatography-Electrospray Interface-Mass Spectrometry) for the determination of low molecular weight DON in seawater samples. The method is sensitive at the 1x10-9 mol/L level and is appropriate for the DON concentration range reported for coastal waters (2-18 X 10-6 mol/L). ESI-MS has been used to monitor dissolved organic matter in river and saline water, the latter during a bloom of the harmful alga Chattonella cf. verruculosa, but structural characterization of the organic molecules was not achieved. Thus, our HPLC-ESI-MS technique represents a significant advance in analytical capability for DON characterization and cycling.  

    

The Biogeochemistry Research Centre (BGC) at the University of Plymouth was formed in 2009, bringing together two of the strongest research groups in the University with excellent reputations for the delivery of world-class research, teaching and enterprise activities (see http://www1.plymouth.ac.uk/research/bgc/). The BGC is a leading centre for nutrient analysis, based in accredited analytical laboratories (ISO 9001:2000) and hosted an international workshop on nutrient speciation and analysis in 2012. In September 2010, a €2.3 million upgrade of the research laboratories was completed. The BGC equipment suite includes a LC-ESI-MS, a HPLC with fluorimetric detector (purchased in 2013), a SKALAR nutrient analyser and a TOC V instrument (for analysis of dissolved organic nitrogen), all of which will be available for the study.         

 

The Plankton Group at the Marine Station in Roscoff (CNRS/UPMC) is the leading laboratory in France for the study of the biology of marine phytoplankton with an excellent track record of very high impact peer reviewed publications addressing various aspects of marine protist biodiversity, ecology, physiology, and evolution (see http://www.sb-roscoff.fr/Phyto/). The Marine Station hosts the Roscoff Culture Collection (RCC: http://www.sb-roscoff.fr/Phyto/RCC/), the national French service collection of marine microalgal cultures, that is among the 5 largest collections of its type in the world. The RCC maintains approximately 3000 culture strains with an extremely broad geographical and phylogenetic coverage. In 2014, the RCC will integrate 250m2 of dedicated laboratory space in the Biological Resource Centre building (€3 million renovation of the ex public aquarium), including 3 culture rooms, media preparation and culture transfer rooms, and state-of-the-art cryopreservation, flow cytometry and microscopy facilities.

The 2011 European Nitrogen Assessment analysis (http://www.nine-esf.org/ENA-Book) calculated that excess nitrogen in the environment costs the European Union between 70 billion and 320 billion € per year. It is the first time that an economic value has been placed on the threats posed by nitrogen pollution, including contributions to climate change and biodiversity loss. Results from the study will provide the basis for a more holistic understanding of the reactivity of organic nitrogen and its importance to algal productivity and potential contribution to environmental degradation. This maps directly on to the priority issues of water quality at a European level.  Furthermore, this new knowledge will provide a basis for much improved parameterisations for the cycling and transport of organic N in environmental models.  Ultimately, the concepts can be integrated into strategies for management of the production and use of N in Europe, co-ordinated by the European Nitrogen Centre (http://www.ini-europe.org), a component of the International Nitrogen Initiative (INI; http://www.initrogen.org), and the Scientific Committee on Problems of the Environment (SCOPE 54; http://www.icsu-scope.org).

We will apply to have the project adopted as an associated activity of the European Centre of the International Nitrogen Initiative. The INI operates at the global level and its Steering Committee directs the organisation of the tri-annual International Nitrogen Conferences, as well as targeted topic workshops. These develop the scientific basis for assessing the role and fate of reactive nitrogen, as well as engaging with industry, policy makers and community stakeholders. It also maintains an overview of global scale nitrogen related activities.

 

Data from the project will be made publicly available. Data on samples analyzed from stations associated with the Western Channel Observatory (WCO) will be displayed on the WCO website, which is publicly accessible at http://www.westernchannelobservatory.org.uk. Dr Fitzsimons has personal and Institute (Marine Institute) web pages in which the project will be publicized and explained. Dr Fitzsimons regularly offers expert comment in media features on water quality (television and print), while a study in which he was involved on dissolved organic phosphorus uptake by algae off the Plymouth coast was communicated in articles published locally and nationally (http://planetearth.nerc.ac.uk/news/story.aspx?id=334). Project outcomes will also be disseminated via the Roscoff Marine Station mediation department, notably through channels developed during the Interreg IV project Marinexus (www.marinexus.org) that involves both scientific and outreach partners in Roscoff and Plymouth.  

 

This project has the overarching aim of understanding the potential impact of changes in the availability of nitrogen species on phytoplankton productivity and biodiversity. As such, it is directly relevant to the EU biodiversity strategy to 2020.