Oxygen-poor microzones as potential sites of microbial n(2) fixation in nitrogen-depleted aerobic marine waters.

The nitrogen-deficient coastal waters of North Carolina contain suspended bacteria potentially able to fix N(2). Bioassays aimed at identifying environmental factors controlling the development and proliferation of N(2) fixation showed that dissolved organic carbon (as simple sugars and sugar alcohols) and particulate organic carbon (derived from Spartina alterniflora) additions elicited and enhanced N(2) fixation (nitrogenase activity) in these waters. Nitrogenase activity occurred in samples containing flocculent, mucilage-covered bacterial aggregates. Cyanobacterium-bacterium aggregates also revealed N(2) fixation. In all cases bacterial N(2) fixation occurred in association with surficial microenvironments or microzones. Since nitrogenase is oxygen labile, we hypothesized that the aggregates themselves protected their constituent microbes from O(2). Microelectrode O(2) profiles revealed that aggregates had lower internal O(2) tensions than surrounding waters. Tetrazolium salt (2,3,5-triphenyl-3-tetrazolium chloride) reduction revealed that patchy zones existed both within microbes and extracellularly in the mucilage surrounding microbes where free O(2) was excluded. Triphenyltetrazolium chloride reduction also strongly inhibited nitrogenase activity. These findings suggest that N(2) fixation is mediated by the availability of the appropriate types of reduced microzones. Organic carbon enrichment appears to serve as an energy and structural source for aggregate formation, both of which were required for eliciting N(2) fixation responses of these waters.