Abstract
Summary The wide distribution of diverse nitrogenase ( nifH ) genes affiliated with those of heterotrophic bacteria in marine and estuarine waters indicates ubiquity and an ecologically relevant role for heterotrophic N 2 ‐fixers (diazotrophs) in aquatic nitrogen ( N ) cycling. However, the lack of cultivated representatives currently precludes an evaluation of their N 2 ‐fixing capacity. In this study, microoxic or anoxic N ‐free media were inoculated with estuarine B altic S ea surface water to select for N 2 ‐fixers. After visible growth and isolation of single colonies on oxic plates or in anoxic agar tubes, nifH gene amplicons were obtained from 64 strains and nitrogenase activity, applying the acetylene reduction assay, was confirmed for 40 strains. Two strains, one G ammaproteobacterium affiliated with P seudomonas and one A lphaproteobacterium affiliated with R hodopseudomonas were shown to represent established members of the indigenous diazotrophic community in the B altic S ea, with abundances of up to 7.9 × 10 4 and 4.7 × 10 4 nifH copies l −1 respectively. This study reports media for successful isolation of heterotrophic diazotrophs. The applied methodology and the obtained strains will facilitate future identification of factors controlling heterotrophic diazotrophic activity in aquatic environments, which is a prerequisite for understanding and evaluating their ecology and contribution to N cycling at local and regional scales.