Abstract
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.
Highlights
N2-fixation by bacterioplankton is an important external input of nitrogen to the ocean (Karl et al, 2002; Carpenter and Capone, 2008; Benavides and Voss, 2015)
We carried out two experiments with water from the eutrophic Roskilde Fjord (RF, Denmark) to examine whether the presence of natural particles would stimulate N2-fixation by non-cyanobacterial diazotrophs (NCDs)’s, and whether NCD communities could efficiently colonize and proliferate on artificial surfaces
Bacterial Abundances and Rates of N2-Fixation Bacterial abundance did not differ between bulk water, particleenriched, and filtrate treatments despite the 10-fold enrichment with particles (p = 0.1869), but was overall slightly lower after the 24 h incubation (p = 0.0196, Supplementary Figure S2)
Summary
N2-fixation by bacterioplankton (diazotrophy) is an important external input of nitrogen to the ocean (Karl et al, 2002; Carpenter and Capone, 2008; Benavides and Voss, 2015). One current enigma is how single-celled heterotrophic bacteria are able to fix N2 in the presence of dioxygen (O2), as it inhibits nitrogenase activity presumably due to the toxicity of O2 to the enzyme (Goldberg et al, 1987). Heterotrophic N2-Fixation on Particles formation or temporal segregation of photosynthetic O2 production and N2-fixation (Reddy et al, 1993; BermanFrank et al, 2007), little is known about the strategies employed by NCDs. Some non-marine NCDs protect nitrogenase by surrounding themselves by a layer of O2-impermeable extracellular polymers (Sabra et al, 2000) – and a somewhat similar strategy was recently observed in an oxygenated culture of a free-living marine Pseudomonas strain (Bentzon-Tilia et al, 2015a). Suggests that this strategy is energetically costly to the cell, even more than N2-fixation itself (Inomura et al, 2016), conceivably constraining the utility of this strategy among free-living bacterioplankton, especially in oligotrophic waters. Heterotrophic bacteria may thrive and fix N2 in low-oxygen microenvironments associated with naturally occurring nutrient- and carbon rich particles (Ploug et al, 1997; Klawonn et al, 2015)
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