CONTRIBUTION OF AQUATIC BIOAGGREGATES TO HETEROTROPHIC DIAZOTROPHY: DIRECT OBSERVATIONS AND N₂ FIXATION RATES

Previous studies indicated that heterotrophic diazotrophs can actively fix N₂ in aquatic environments with adverse conditions for diazotrophy such as oxygenated and nitrogen-rich waters. Indirect observations suggested that heterotrophic diazotrophy may be promoted by planktonic and transparent exopolymer particles (TEP). These particles comprise polysaccharides, contain high carbon to nitrogen ratio (~20:1) and found as hotspots of intense microbial activity. In this study, we developed a direct approach that enables to link between heterotrophic diazotrophs and bioaggregates that comprise TEP using a new microscopical method which is based on immunolocalization of the nitrogenase enzyme. This new observational technique was accompanied by a suite of complementary measurements such as N₂ fixation rates, bacterial activity, and bacterial abundance. Initially, we developed our direct immunolabeling approach under controlled conditions using Vibrio natriegens as a model heterotrophic diazotroph, before running microcosm experiments in the Qishon estuary on natural populations. Immunolabeling the nitrogenase enzyme by a florescent fluorophore localized clusters of heterotrophic diazotrophs on bioaggregates that comprise TEP, even when unfavorable conditions prevailed. We also found that N₂ fixation rates and bacterial activity were higher (40 % and 73 %, respectively) following the addition of TEP compared to the unamended controls. Our results suggest that bioaggregates provide favorable microenvironments for N₂ fixation by heterotrophic diazotrophs. We surmise that this newly developed method may provide new insights which will enhance the understanding of planktonic heterotrophic N₂ fixation in marine and freshwater environments.