Diazotrophs and N₂-Fixation Associated With Particles in Coastal Estuarine Waters

Putative heterotrophic bacteria carrying out N₂-fixation, so-called non-cyanobacterial diazotrophs (NCDs), are widely distributed in marine waters, but details of how the O₂-inhibited N₂-fixation process is promoted in the oxic water column remains ambiguous. Here we carried out two experiments with water from a eutrophic temperate fjord to examine whether low-oxygen microenvironments within particulate organic matter could be loci suitable for N₂-fixation. First, water enriched with natural particles or sediment showed higher N₂-fixation rates than bulk water, and nitrogenase genes (nifH) revealed that specific diazotrophs were affiliated with the particulate matter. Second, pristine artificial surfaces were rapidly colonized by diverse bacteria, while putative diazotrophs emerged relatively late (after 80 h) during the colonization, and phylotypes related to Pseudomonas and to anaerobic bacteria became dominant with time. Our study pinpoints natural particles as sites of N₂-fixation, and indicates that resuspension of sediment material can elevate pelagic N₂-fixation. Moreover, we show that diverse natural diazotrophs can colonize artificial surfaces, but colonization by "pioneer" bacterioplankton that more rapidly associate with surfaces appears to be a prerequisite. Whereas our experimental study supports the idea of pelagic particles as sites of N₂-fixation by heterotrophic bacteria, future in situ studies are needed in order to establish identity, activity and ecology of particle associated NCDs as a function of individual particle characteristics.