Nitrogen Fixation by Members of the Tribe Klebsielleae.

Mahl, M. C. (University of Wisconsin, Madison), P. W. Wilson, M. A. Fife, and W. H. Ewing. Nitrogen fixation by members of the tribe Klebsielleae. J. Bacteriol. 89:1482-1487. 1965.-Strains of species of the tribe Klebsielleae were tested for nitrogen fixation by inoculating actively growing cultures into side-arm flasks containing a medium with a growth-limiting quantity of combined nitrogen (25 mug of nitrogen per ml as ammonium sulfate). The flasks were evacuated, filled with pure N(2), sealed, and placed on a shaker at 30 C. Growth was followed by optical-density measurements; maximal growth was obtained in 9 to 10 hr. Yeast extract was then added as a source of amino acids to shorten the induction time for the nitrogen-fixing enzymes. Fixation was determined either by estimating total nitrogen with a semimicro Kjeldahl technique or by exposing 20- to 22-hr-old samples to an atmosphere of N(2) (15) and helium for 5 hr and then analyzing the digested sample for N(15) in a mass spectrometer. None of the 22 strains of the two Enterobacter (formerly Aerobacter) species fixed nitrogen; neither did any of eight strains of Serratia species that were tested. Of 31 strains of Klebsiella pneumoniae, 13 incorporated atmospheric nitrogen. Net nitrogen fixed ranged from 17 to 65 mug/ml. It is concluded that these facultative anaerobic, gram-negative, nitrogen-fixing rods should be placed in the genus Klebsiella pneumoniae. The nitrogen-fixing organism tentatively classified as Achromobacter N-4 should also be changed to K. pneumoniae strain N-4, because it has been found to be an anaerogenic strain of K. pneumoniae.