Isolation of heterotrophic diazotrophic bacteria from estuarine surface waters

Hanna Farnelid, Jens Harder, Mikkel Bentzon-Tilia, Lasse Riemann

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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
Original languageEnglish
JournalEnvironmental Microbiology
Issue number10
Pages (from-to)3072-3082
Number of pages11
Publication statusPublished - 2014
Externally publishedYes


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