Isolation of heterotrophic diazotrophic bacteria from estuarine surface waters

The wide distribution of diverse nitrogenase (nifH)genes affiliated with those of heterotrophic bacteria inmarine and estuarine waters indicates ubiquity and anecologically relevant role for heterotrophic N₂-fixers(diazotrophs) in aquatic nitrogen (N) cycling. However,the lack of cultivated representatives currently precludesan evaluation of their N₂-fixing capacity. In thisstudy, microoxic or anoxic N-free media were inoculatedwith estuarine Baltic Sea surface water to selectfor N₂-fixers. After visible growth and isolation ofsingle colonies on oxic plates or in anoxic agar tubes,nifH gene amplicons were obtained from 64 strainsand nitrogenase activity, applying the acetylenereduction assay, was confirmed for 40 strains. Twostrains, one Gammaproteobacterium affiliated withPseudomonas and one Alphaproteobacterium affiliatedwith Rhodopseudomonas were shown torepresent established members of the indigenousdiazotrophic community in the Baltic Sea, with abundancesof up to 7.9 × 10⁴ and 4.7 × 10⁴ nifH copies l⁻¹respectively. This study reports media for successfulisolation of heterotrophic diazotrophs. The appliedmethodology and the obtained strains will facilitatefuture identification of factors controlling heterotrophicdiazotrophic activity in aquatic environments,which is a prerequisite for understanding and evaluatingtheir ecology and contribution