Nitrate reductase activity of soils

Dissimilatory nitrate reductase in soils is the enzyme that catalyzes the reduction of NO₃3⁻ to NO₂⁻ under anaerobic conditions. The detection of this enzyme in soils is reported, and a simple, sensitive and precise method to assay its activity is described. The method involves determination of the NO₂⁻-N produced when soil. 2,4-dinitrophenol (DNP), and KNO₃ are incubated under waterlogged conditions at 25°C for 24 h. At a certain concentration, depending on the soil type, DNP inhibits nitrite reductase but not nitrate reductase. The DNP concentration required for optimum NO₂⁻ production in five soils ranged from 5 to 300 μg DNP g⁻¹ soil. The nitrate reductase activity of six soils studied ranged from 18 to 80 μg NO₂⁻-N produced g⁻¹ soil 24 h⁻¹. Optimum activity was found at 5 mM KNO₃ and nitrate reductase was inhibited at >5 mM KNO₃. Nitrate reductase activity in soils is inactivated at temperatures above 40°C and is completely destroyed by steam sterilization. The relationship between duration of incubation and the amount of NO₂⁻-N produced showed a lag of about 10 h, but in general, thereafter, this relationship was linear for a certain period of incubation, which varied among the soils studied. The duration of the lag was reduced, but not completely eliminated, either by previous incubation for 10 h or by bubbling N₂ gas in the soil-water mixture for 3 min to remove the dissolved O₂ in the soil-water mixture before addition of NO₃⁻. The relationship between the amount of soil used and the NO₂⁻-N produced was linear unless the substrate concentration was limiting the reaction rate. Application of the Lineweaver-Burk transformation of the Michaelis-Menten equation indicated that the K_m values for nitrate reductase in Ames and Okoboji soils were 3.7 and 2.9, respectively, and the V_max values were 122 and 126μg NO₂⁻-N produced g⁻ soil 24 h⁻.