Temperate southern Australian coastal waters are characterised by surprisingly high rates of nitrogen fixation and diversity of diazotrophs

Biological dinitrogen (N(2)) fixation is one mechanism by which specific microorganisms (diazotrophs) can ameliorate nitrogen (N) limitation. Historically, rates of N(2) fixation were believed to be limited outside of the low nutrient tropical and subtropical open ocean; however, emerging evidence suggests that N(2) fixation is also a significant process within temperate coastal waters. Using a combination of amplicon sequencing, targeting the nitrogenase reductase gene (nifH), quantitative nifH PCR, and (15)N(2) stable isotope tracer experiments, we investigated spatial patterns of diazotroph assemblage structure and N(2) fixation rates within the temperate coastal waters of southern Australia during Austral autumn and summer. Relative to previous studies in open ocean environments, including tropical northern Australia, and tropical and temperate estuaries, our results indicate that high rates of N(2) fixation (10–64 nmol L(−1) d(−1)) can occur within the large inverse estuary Spencer Gulf, while comparatively low rates of N(2) fixation (2 nmol L(−1) d(−1)) were observed in the adjacent continental shelf waters. Across the dataset, low concentrations of NO(3)/NO(2) were significantly correlated with the highest N(2) fixation rates, suggesting that N(2) fixation could be an important source of new N in the region as dissolved inorganic N concentrations are typically limiting. Overall, the underlying diazotrophic community was dominated by nifH sequences from Cluster 1 unicellular cyanobacteria of the UCYN-A clade, as well as non-cyanobacterial diazotrophs related to Pseudomonas stutzeri, and Cluster 3 sulfate-reducing deltaproteobacteria. Diazotroph community composition was significantly influenced by salinity and SiO(4) concentrations, reflecting the transition from UCYN-A-dominated assemblages in the continental shelf waters, to Cluster 3-dominated assemblages in the hypersaline waters of the inverse estuary. Diverse, transitional diazotrophic communities, comprised of a mixture of UCYN-A and putative heterotrophic bacteria, were observed at the mouth and southern edge of Spencer Gulf, where the highest N(2) fixation rates were observed. In contrast to observations in other environments, no seasonal patterns in N(2) fixation rates and diazotroph community structure were apparent. Collectively, our findings are consistent with the emerging view that N(2) fixation within temperate coastal waters is a previously overlooked dynamic and potentially important component of the marine N cycle.