On three separate occasions we investigated morphological and physiological aspects of bacterial associations with planktonic aggregates of the ubiquitous marine N2 fixing cyanobacterium Trichodesmium sp. Close associations generally characterized Trichodesmium blooms; associations were present during day‐ and night‐time. Colonization by both rod‐shaped and filamentous heterotrophic bacteria occurred on Trichodesmiun aggregates actively fixing N2 (acetylene reduction). Scanning electron and optical microscopy showed bacteria located both around and within aggregates. Microautoradiography demonstrated that associated bacteria largely mediated utilization of trace additions of 3H‐labeled carbohydrates (fructose, glucose, mannitol) and amino acids, whereas Trichodesmium utilized amino acids only. Oxygen measurements using microelectrodes revealed high localized oxygen consumption among aggregates, with rapid (within a minute) changes from supersaturated to subsaturated oxygen following the transition from photosynthetic illuminated to dark periods. Stab culturing techniques confirmed the presence of heterotrophic N2 fixers among aggregate‐associated bacteria. Parallel deployment of oxygen microelectrodes, the tetrazolium salt 2,3,5 triphenyl tetrazolium chloride (TTC) and acetylene reduction assays demonstrated microaerophilic requirements for expression of nitrogenase activity among cultured bacteria. Trichodesmium aggregates are characterized by dynamic nutrient and oxygen regimes, which promote and maintain simultaneous and contiguous oxygenic photosynthesis and N2 fixation. In part, the above‐mentioned consortial interactions with a variety of heterotrophic bacteria facilitate Trichodesmium biomass production and bloom formation in nitrogen depleted, oligotrophic tropical/subtropical waters.

Phytoplankton growth in many coastal and pelagic marine waters is chronically limited by nitrogen availability.Such conditions potentially favor the establishment of N,-fixing microorganisms (eubacteria and cyanobacteria). However, planktonic and benthic N, fixation is often either absent or present at ecologically insignificant rates. It has been proposed that deficiencies in inorganic nutrient (specifically molybdenum) availability could help explain this paradox. We examined both inorganic and organic nutrient limitations of marine N, fixation in nitrogen-deficient coastal North Carolina waters. Inorganic nutrient (phosphorus, iron, and molybdenum) availability consistently exceeded demands by N, fixers. In contrast, enrichment with the sugars fructose, glucose, sucrose, and maltose and the sugar alcohol mannitol either elicited N, fixation or enhanced existing rates of N, fixation. Supplementation with particles (organic detritus) also enhanced N2 fixation potentials; the combined addition of particles and organic compounds yielded maximum rates of N, fixation. This combination promotes the development of O,-reduced microenvironments (microzones) in which N, fixers can reside. A functional explanation for the observed stimulation of N, fixation is that it is an anaerobic process which, in aerobic marine waters, can only proceed in O,-poor microzones. Hence, deficiencies in organic matter rather than inorganic nutrient availability may play key roles in limiting and regulating marine N, fixation.

The nitrogen-deficient coastal waters of North Carolina contain suspended bacteria potentially able to fix N2. Bioassays aimed at identifying environmental factors controlling the development and proliferation of N2 fixation showed that dissolved organic carbon (as simple sugars and sugar alcohols) and particulate organic carbon (derived from Spartina alternifiora) additions elicited and enhanced N2 fixation (nitrogenase activity) in these waters. Nitrogenase activity occurred in samples containing flocculent, mucilage-covered bacterial aggregates. Cyanobacterium-bacterium aggregates also revealed N2 fixation. In all cases bacterial N2 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 02-Microelectrode 02 profiles revealed that aggregates had lower internal 02 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 02 was excluded. Triphenyltetrazolium chloride reduction also strongly inhibited nitrogenase activity. These findings suggest that N2 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 N2 fixation responses of these waters.

The nonheterocystous filamentous cyanobacterial genus Lyngbya is a widespread and frequently dominant component of marine microbial mats. It is suspected of contributing to relatively high rates of N2 fixation associated with mats. The ability to contemporaneously conduct 02-sensitive N2 fixation and oxygenic photosynthesis was investigated in Lyngbya aestuarii isolates from a North Caroli-na intertidal mat. Short-term (<4-h) additions of the photosystem II (02 evolution) inhibitor 3(3,4-dichlorephenyl)-1,1-dimethylurea stimulated light-mediated N2 fixation (nitrogenase activity), indicating potential inhibition of N2 fixation by 02 production. However, some degree of light-mediated N. fixation in the absence of 3(3,4-dichlorophenyl)-1, l-dimethylurea was observed. Electron microscopic immunocytochemical localization of nitrogenase, coupled to microautoradiographic studies of '4CO2 fixation and cellular deposition of the tetrazolium salt 2,4,5

Diel variations in N 2 fixation (acetylene reduction), CO 2 fixation, and oxygen concentrations were measured, on three separate occasions, in a marine microbial mat located on Shackleford Banks, North Carolina. Nitrogenase activity (NA) was found to be inversely correlated with CO 2 fixation and, in two of the three diel periods studied, was higher at night than during the day. Oxygen concentrations within the top 3 mm of the mat ranged from 0 to 400 μM on a diel cycle; anaerobic conditions generally persisted below 4 mm. NA in the mat was profoundly affected by naturally occurring oxygen concentrations. Experimentally elevated oxygen concentrations resulted in a significant depression of NA, whereas the addition of the Photosystem II inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea decreased oxygen concentrations within the mat and resulted in a significant short-term enhancement of NA. Mat N 2 -fixing microorganisms include cyanobacteria and heterotrophic, photoautotrophic, and chemolithotrophic eubacteria. Measured (whole-mat) NA is probably due to a combination of the NA of each of these groups of organisms. The relative contributions of each group to whole-mat NA probably varied during diel and seasonal (successional) cycles. Reduced compounds derived from photosynthetic CO 2 fixation appeared to be an important source of energy for NA during the day, whereas heterotrophic or chemolithotrophic utilization of reduced compounds appeared to be an important source of energy for NA at night, under reduced ambient oxygen concentrations. Previous estimates of N 2 fixation calculated on the basis of daytime measurements may have seriously underestimated diel and seasonal nitrogen inputs in mat systems.