Cultivation and isolation of N2-fixing bacteria from suboxic waters in the Baltic Sea

Nitrogenase genes (nifH) from heterotrophic dinitrogen (N2)-fixing bacteria appear ubiquitous in marine bacterioplankton, but the significance of these bacteria for N cycling is unknown. Quantitative data on the N2-fixation potential of marine and estuarine heterotrophs are scarce, and the shortage of cultivated specimens currently precludes ecophysiological characterization of these bacteria. Through the cultivation of diazotrophs from suboxic (1.79 μmol O2 L(-1)) Baltic Sea water in an artificial seawater medium devoid of combined N, we report the cultivability of a considerable fraction of the diazotrophic community in the Gotland Deep. Two nifH clades were present both in situ and in enrichment cultures showing gene abundances of up to 4.6 × 10(5) and 5.8 × 10(5) nifH gene copies L(-1) within two vertical profiles in the Baltic Sea. The distributions of the two clades suggested a relationship with the O2 concentrations in the water column as abundances increased in the suboxic and anoxic waters. It was possible to cultivate and isolate representatives from one of these prevalent clades, and preliminary analysis of their ecophysiology demonstrated growth optima at 0.5-15 μmol O2 L(-1) and 186-194 μmol O2 L(-1) in the absence of combined N.