Effects of oxygen,mannitol and ammonium concentrations on nitrogenase [C2H2] activity in a marine skeletal carbonate sand

Following a lag of 3 to 18 h, acetylene reduction in mannitol-amended sand systems proceeded at approximately constant and high rates for periods up to 4 days. Carbon dioxide production and O₂ consumption were low in these systems in comparison to similar systems additionally amended with ammonium, indicating N-limitation of growth in the former. Thus, long-term acetylene assays of mannitol-amended sand and suspensions from the sand incubated at various partial pressures of oxygen could be used to characterize the O₂-sensitivity of the N₂-fixing bacterial population as a whole, in batch-type systems with a minimal degree of enrichment or change in pO₂ during the course of the assays. Results of various studies suggested that aerobic or microaerophilic N₂-fixing bacteria were absent or scarce in the sand, and that nitrogenase activity occurring in aerobically incubated systems occurred in anaerobic microenvironments. Hydrogen stimulated acetylene-reducing activity, but the time course differed from that of mannitol-supported activity, and proceeded with shorter lags in systems incubated at 0.2 and 0.05 atm O₂ than in systems incubated anaerobically. Efficiency of N₂ fixation [C₂H₂] increased with decreasing initial mannitol concentration. For sand washed with seawater to remove native combined inorganic nitrogen, and amended with 0.015% mannitol, 374 μmoles added NH₄-N/kg wet sand caused almost complete repression of nitrogenase activity, while concentrations as low as 12 μmoles added NH₄-N/kg wet sand appeared to cause at least partial repression of nitrogenase activity. Some implications of these results for the existence of anaerobic microenvironments in the cavities of skeletal carbonates, and for N₂-fixation in the seagrass rhizosphere are discussed.