Previous methods of performing aquatic acetylene-reduction assays are described and several problems associated with them are discussed. A refinement of these older techniques is introduced and problems that it overcomes are also discussed. A depth profile of nitrogen fixation (C2H4 production), obtained by the refined technique, is shown for a fertilized Canadian Shield lake in the Experimental Lakes Area of northwestern Ontario.

A radiotracer method which measures rates

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A medium is described on which selected Rhizobium japonicum strains express hydrogenase (H2 uptake) activity under free-living conditions. Low concentrations of carbon substrates, decreased oxygen tension, and the quantity of combined nitrogen in the medium were major factors influencing hydrogenase expression. Hydrogenase activity was dependent upon a preincubation period in the presence of H2 under conditions such that the cels did not exhibit nitrogenase activity. H2 uptake rates were easily measured amperometrically in aerobically or anaerobically prepared suspensions from free-living cultures. Six R. japonicum strains that formed nodules with the ability to utilize H2 oxidized this gas when grown in free-living cultures. In comparison six randomly chosen strains forming nodules that lost H2 in air either showed no or low capacity to take up H2 under ree-living conditions. The reduction of triphenyltetrazolium chloride in an agar medium was used to detect strains capable of oxidizing H2. This method has enabled us to isolate a spontaneous R. japonicum mutant strain that has lost the ability to utilize H2. This mutant strain forms nodules that evolve H2 but other symbiotic characteristics appear normal. This strain will be useful in evaluating the importance of the hydrogenase system in the nitrogen-fixing process of legumes.In addition to catalysis of N2 reduction, nitrogenases from all known sources catalyze ATP-dependent H2 evolution. Apparently, H2 evolution during N2 fixation is an inherent property of the nitrogenase reaction. Energy loss through nitrogenase-dependent H2 evolution is important because four or five ATP molecules are consumed per pair of electrons transferred (1, 2) regardless of whether N2 or H+ is the electron acceptor. The significance of ATP-dependent evolution of H2 by nodules as an energy-wasteful process during N2 fixation by legumes has been pointed out by Dixon (3), Schubert and Evans (4) The H2-uptake medium is a further modification of the modified LNB5 medium used to detect acetylene reduction activity in free-living cultures of R. japonicum (11). The H2-uptake medium contains the following in 1 liter of distilled water: NaH2PO4-H20, 150 mg; CaCl2-2H20, 150 mg; MgSO4-7H20, 250 mg; iron EDTA, 28 mg; MnSO4-H20, 10 mg; H3BO3, 3 mg; ZnSO4-7H20, 2 mg; NaMoO4-2H20, 0.25 mg; CuSO4-5H20, 0.04 mg; CoC12-6H20, 0.025 mg; KI, 0

Specific rates of mercury methylation and demethylation were determined for water and surficial sediment samples taken from several lakes located in the Experimental Lakes Area, northwestern Ontario. Specific rates of mercury methylation were found to increase with decreasing pH in epilimnetic water samples in which pH was adjusted prior to incubation and in epilimnetic water samples taken from lakes of different pH. Reduction of pH also increased methyl mercury production at the sediment surface. Both increases and decreases in pH reduced specific rates of mercury demethylation. However, these changes were smaller than for methylation. Proportionally, specific rates of methylation increased faster than increasing concentrations of Hg2+, while specific rates of mercury demethylation increased linearly with increasing concentrations of methyl mercury. Overall, this study predicts that the net rate of methyl mercury production in the water column and at the sediment–water surface will increase as a result of lake acidification, and this may at least partially explain why the mercury concentration of fish appears to increase during lake acidification.