Isolation of heterotrophic diazotrophic bacteria from estuarine surface waters

TY - JOUR

T1 - Isolation of heterotrophic diazotrophic bacteria from estuarine surface waters

AU - Farnelid, Hanna

AU - Harder, Jens

AU - Bentzon-Tilia, Mikkel

AU - Riemann, Lasse

N1 - Special Issue: Nitrogen Cycle Ecology

PY - 2014/10/1

Y1 - 2014/10/1

N2 - 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 N2-fixers (diazotrophs) in aquatic nitrogen (N) cycling. However, the lack of cultivated representatives currently precludes an evaluation of their N2-fixing capacity. In this study, microoxic or anoxic N-free media were inoculated with estuarine Baltic Sea surface water to select for N2-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 Gammaproteobacterium affiliated with Pseudomonas and one Alphaproteobacterium affiliated with Rhodopseudomonas were shown to represent established members of the indigenous diazotrophic community in the Baltic Sea, with abundances of up to 7.9×104 and 4.7×104nifH 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.

AB - 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 N2-fixers (diazotrophs) in aquatic nitrogen (N) cycling. However, the lack of cultivated representatives currently precludes an evaluation of their N2-fixing capacity. In this study, microoxic or anoxic N-free media were inoculated with estuarine Baltic Sea surface water to select for N2-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 Gammaproteobacterium affiliated with Pseudomonas and one Alphaproteobacterium affiliated with Rhodopseudomonas were shown to represent established members of the indigenous diazotrophic community in the Baltic Sea, with abundances of up to 7.9×104 and 4.7×104nifH 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.

U2 - 10.1111/1462-2920.12335

DO - 10.1111/1462-2920.12335

M3 - Journal article

C2 - 24330580

SN - 1462-2912

VL - 16

SP - 3072

EP - 3082

JO - Environmental Microbiology

JF - Environmental Microbiology

IS - 10

ER -