Nitrogenase Activity (Acetylene Reduction) of Root-Associated, Cold-Climate Azospirillum, Enterobacter, Klebsiella , and Pseudomonas Species During Growth on Various Carbon Sources and at Various Partial Pressures of Oxygen

A comprehensive view of the diazotrophic bacterial flora of plants requires that attention be paid to the appropriate carbon and oxygen requirements during isolation of the bacteria. Twenty compounds (monosaccharides, disaccharides, polyols, and organic acids) were therefore examined as carbon and energy sources for nitrogenase activity in semisolid stab cultures at pO2 values of 0.21, 0.02, and ≤0.002 with 12 strains of diazotrophic root-associated bacteria. With the facultatively anaerobic bacteria of the generaKlebsiella andEnterobacter , the best substrate was sucrose, followed by fructose and mannitol, whereas among the organic acids, only malic and fumaric acids supported any activity. With the obligately aerobic bacteria of the generaAzospirillum andPseudomonas , disaccharides were not utilized for nitrogen fixation, but several organic acids were accepted in addition to monosaccharides and polyols; malate and glucose were the best substrates. The patterns of the carbon sources utilized for nitrogen fixation were coherent within the species, with the exception of oneKlebsiella pneumoniae and oneEnterobacter agglomerans strain, both isolated from the same individual grass plant, which were unable to utilize lactose. Anaerobic conditions (pO2 value of ≤0.002) were required for maximum nitrogenase activity with the facultatively anaerobic bacteria, with the exception of one strain ofE. agglomerans , which required atmospheric oxygen (pO2 value of 0.21). Also, the obligately aerobic diazotrophs required atmospheric oxygen for maximum nitrogenase activity. The maximum specific nitrogenase activities (expressed as micromoles of C2 H4 · milligram of bacterial protein−1 · hour−1 ) noted during the exponential growth phase of the bacteria were the following: 2.68 withAzospirillum lipoferum on malate, 2.41 withK. pneumoniae and 1.58 withE. agglomerans on sucrose, and 0.95 withPseudomonas sp. on malate.