Carbon Monoxide Inhibition of Azotobacter in Microrespiration Experiments1
Journal of Bacteriology
Extremely small quantities of carbon monoxide in air inhibit nitrogen fixation by red clover plants inoculated with Rhizobium trifolii . In preliminary trials with Azotobacter vinelandii Wyss (1941) found a similar inhibition at carbon monoxide concentrations about 10 times those required for the symbiotic system. We have made a detailed study of the inhibition of this azotobacter using several sources of combined nitrogen to determine specificity for the nitrogen fixation process. Two methods
... ocess. Two methods have been used, each of which possesses certain advantages: a macro method which periodically measures directly the nitrogen fixed and a micro method which indirectly estimates nitrogen fixation by determining the increase in rate of respiration with time. The macro method is relatively longtime, 30-40 hr in comparison with the 4-7 hr required in the respiration studies. The results of the macro experiments have been described by Lind and Wilson (1942) ; in this paper the micro-respiration data are discussed. METHODS Young cultures of Azotobacter vinelandii (18-24 hr) are diluted with Burk's medium so that the initial respiration is about 30-50 mm8/hr/ml. Respiration is measured in the Warburg micro-respirometer on duplicate or triplicate flasks; readings are made every 30 minutes for a period of 5 hr. The rate of fixation is measured by the specific rate constant, k, defined by the equation k 2.303 log respiration rate in mm_/hr at t2 t2k-= l respiration rate in mm3/hr at t2 It is conveniently determined through multiplying by 2.303 the slope of the line obtained when the logarithm of the rate of oxygen uptake is plotted against time. Thus, in figure 1 , the slope of the line for the organisms fixing N2 in the absence of CO is 0.140. The k value is therefore equal to 0.323. Usually in the estimation of the slopes of the lines by the usual statistical procedure, only the hourly rates are included since this gives sufficient accuracy and reduces materially the statistical calculations. Details of the methods for preparing the cultures, medium, and gas mixtures, and for the statistical analysis of the data are given in our previous reports Wilson, 1941, 1942; Wyss, Lind, Wilson and Wilson, 1941) .