Primary production in the oceans is strongly limited by the availability of fixed nitrogen. In open ocean nutrient-impoverished areas, which make up ~50% of the global ocean surface, nitrogen is mainly provided through the process of biological atmospheric nitrogen (N2) fixation. N2 fixation is carried out by the so termed diazotrophs, marine microorganisms that may belong to the cyanobacteria, bacteria or archaea. For many years, autotrophic diazotrophs were thought to be the most abundant diazotrophs in the ocean. Autotrophic diazotrophs need light to fix carbon dioxide via photosynthesis, and therefore are constrained to the sunlit layer of the ocean, which is generally less than 100 m deep. Recent investigations have revealed that heterotrophic diazotrophs, which cannot photosynthesize, are present in greater abundance than autotrophic diazotrophs in the world’s oceans. Heterotrophic diazotrophs are not constrained by the availability of light and therefore are able to live in the dark ocean, the largest and less studied habitat on Earth. This discovery significantly expands the boundaries where N2 fixation was though to be possible and theoretically increases the inputs of fixed nitrogen to the ocean, which remain unaccounted for. Because they are not photosynthetic, heterotrophic diazotrophs need an external source of dissolved organic matter (DOM) for their nutrition. However, the nature of this DOM and how it influences their activity is largely unknown. This project aims to cover this gap by studying their relationship with DOM in the ocean. Through shipboard experiments and use of cutting-edge analytical techniques we will explore the spatial distribution of heterotrophic diazotrophs’ abundance, diversity and N2 fixation activity related to the in situ concentration and composition of DOM. The results will provide unique insights into the ecology of heterotrophic diazotrophs and their role in the oceanic nitrogen cycle.