INFLUENCE OF THE HYDROGEN-ION CONCENTRATION 
ON THE GROWTH AND FIXATION OF NITROGEN BY 
CULTURES OF AZOTOBACTER ‘ 
By P. h. GainSy, Professor of Bacteriology, and H. W. Batchelor, Laboratory Assist¬ 
ant in Bacteriologyj Kansas Agricultural Experiment Station 
Investigations in this laboratory have shown that the presence or 
absence of Azotobacter in natural soils is very closely associated with, if 
not dependent upon, the absolute reaction of the soil solution {4)? It 
has also been shown that in the laboratory the presence of this group of 
organisms in a soil can be controlled by varying the hydrogen-ion con¬ 
centration of the soil (5). 
In connection with these investigations it seemed desirable to study 
the influence of the hydrogen-ion concentration of laboratory culture 
media upon the growth and nitrogen fixing ability of pure cultures of 
Azotobacter.^ 
In their original investigations on Azotobacter, Beijerinck and Van 
Delden (r) failed to secure appreciable fixation of nitrogen by pure 
cultures. On the other hand, Lipman (6) experienced no difficulty in 
securing marked fixation by pure cultures, and he presents data to show 
that the ability of pure cultures to fix nitrogen in the media usually 
employed for their culture depended upon the neutralization of the 
acidity arising from the potassium phosphate present. Lipman further 
demonstrated that, quantitatively, the nitrogen fixed by pure cultures 
was inversely proportional to the titratable acidity of the media, and 
called attention to the probability that Beijerinck’s failure to secure 
nitrogen fixation was due to the unfavorable reaction of the medium 
employed. Since Lipman’s work appeared, it has been universally ac¬ 
cepted that these organizations will not function in a high concentra¬ 
tion of acid. 
With regard to the effect of different degrees of acidity, or hydrogen-ion 
concentrations, upon the growth of pure cultures of this group of organ¬ 
isms little information has been published. Fred (3) observed the growth 
of two different cultures in a medium of varying hydrogen-ion concentra¬ 
tions and noted no growth at Ph 6.4 to 6.6, while growth occurred at 
Ph 6.6 to 6.8. On the other hand, when grown in a medium of Ph 7-2 
the final hydrogen-ion concentration was found to be Ph 5.1. As already 
mentioned, previous investigations in this laboratory indicated that the 
hydrogen-ion concentration of the soil solution is the major factor in 
controlling the presence of this group of organisms in soils. Also, that 
the maximum concentration of hydrogen ions tolerated by this group of 
^ Accepted foi publication Oct. i6, 1922. Contribution No. 50, Department of Bacteriology, Kansas 
Agrietdture Experiment Station. 
* Reference is made by number (italic) to Literature cited,” p. 767. .... 
* The term “pure culture” is here used in a restricted sense. Owing to the extreme morphological 
variations exhibited by this group of organisms it is frequently very difficult to ascertain with certainty 
the purity of a given culture. All cultures used in these experiments conformed to the description given 
in the text of this paper. 
(759) 
Journal ci Agricultural Research 
Washington, D. C. 
ael 
Vol. XXIV, No. 9 
June 2, 1923 
Key No. ii^ns. -sJ 
