PhD Defence: Vasiliki Papazachariou

Thesis title: Nitrogen fixation, diazotrophs and microbial communities associated with seagrass leaves and leaf debris in coastal temperate waters

Supervisor: Lasse Riemann, Professor, Marine Biological Section, Department of Biology, University of Copenhagen, Denmark

Assesment committee:
Michael Kühl (Chair), Professor, Marine Biological Section, Department of Biology, University of Copenhagen, Denmark
Nona Sheila R. Agawin, Professor, Department of Biology, University of the Balearic Islands, Palma de Mallorca, Spain
Kasper Elgetti Brodersen, Associate Professor, Environmental Dynamics, Department of Science and Environment, Roskilde University, Denmark

Abstract
Nitrogen fixation is a process extensively studied in the global ocean as it converts atmospheric N 2 gas into bioavailable ammonia, playing a critical role in supporting primary production. This process is also associated with marine eukaryotic hosts, such as seagrasses, indicating potential symbiotic interactions. Seagrass productivity, in particular, has been linked to nitrogen fixation, making it increasingly important to understand this connection as seagrasses face alarming declines. Although nitrogen fi fixation has been widely explored, the factors that regulate it, and the identity of the putative diazotrophs in various marine environments, remain not fully understood. This thesis examines coastal diazotrophy and its implications for nitrogen inputs in temperate waters, with a focus on host and substrate-associated interactions, specifically seagrass leaves and seagrass leaf debris. It explores epiphytic nitrogen fixation, the diazotrophic and general microbial communities present onZ ostera marina leaves, and the temporal and spatial factors influencing them. Further, this thesis investigates the hypothesis that seagrass leaf debris biomass serves as a niche for diazotrophs to colonize and fix nitrogen. To address these questions, measurements of nitrogen fixation activity were conducted, and putative and active diazotrophs, as well as general microbial community members, were analyzed using molecular techniques. The results presented here provide new insights into nitrogen fixation rates associated with temperate seagrass leaves and decomposing seagrass leaves. Living seagrass leaves were dominated by heterotrophic diazotrophs, with a strong seasonal influence. Additionally, certain diazotrophs emerged as core members of the microbial community. Nitrogen fixation rates under dark conditions suggest that heterotrophs, which comprised a significant portion of the microbial community, were also active. During seagrass leaf decomposition, nitrogen fixation was measured across all days, and the diazotroph community composition appeared substrate specific, with cyanobacteria being more prevalent in the first weeks and heterotrophs becoming dominant by the end. These findings provide novel insights into nitrogen inputs in temperate coastal waters via nitrogen fixation activity on seagrass debris. Together, these studies advance our understanding of diazotrophy associated with seagrass living leaves and leaf debris. This knowledge will serve as a critical basis for future studies aimed at better protecting and/or restoring threatened seagrass ecosystems and improving the management of seagrass debris.