Abstract
A new and extremely sensitive method for measuring nitrogenase activity through acetylene reduction is presented. Ethylene produced by nitrogenase-mediated reduction of acetylene is detected by using laser photoacoustics (LPA). This method possesses a detection limit making it 3 orders of magnitude more sensitive than traditional gas chromatographic analysis. Photoacoustic detection is based on the strong and unique absorption pattern of ethylene in the CO(inf2) laser wavelength region (9 to 11 (mu)m). The high sensitivity allowed on-line monitoring of nitrogenase activity in a culture of the heterocystous cyanobacterium Nodularia spumigena, which was isolated from a water bloom in the Baltic Sea. This setup makes it unnecessary to take subsamples from the culture and avoids long incubations in sealed vials. The fast response of the LPA technique allows measurement of real-time dynamic changes of nitrogenase activity. The method was used to analyze in vivo saturation of nitrogenase by acetylene in N. spumigena. It is demonstrated that 20% acetylene does not saturate nitrogenase and that the degree of saturation depends on light intensity. With concentrations of acetylene as low as 2.5% it is possible to assess the degree of saturation and to extrapolate to total nitrogenase activity. In N. spumigena nitrogenase activity becomes independent of light intensity above 20 to 80 (mu)mol of photons m(sup-2) s(sup-1) at 20% O(inf2).