T H E FIXATION OF ATMOSPHERIC NITROGEN BY YEAST AS A FUNCTION OF T H E HYDROGEN ION CONCENTRATION” BY ELLIS I. FULMER AND LEO M. CHRISTEXSEN
It has been previously statedl that yeast will grow continuously and in an apparently good state of nutrition on a synthetic medium with atmospheric nitrogen as the sole source of that element. I n omitting the ammonium salt not only was the yeast deprived of its usual source of nitrogen but the physico-chemical environment of the medium was seriously disturbed2. Rhile continued growth is maintained by yeast on the synthetic medium devoid of all but atmospheric nitrogen the quantitative estimation of the nitrogen fixed in that medium mas not feasible because of the relatively small crop of yeast obtained. I n order to obtain quantitative results on the nitrogen fixation by the yeast it seemed advisable to add some of the growth stimulant, bios, with the minimum addition of nitrogen. It had been shown previously2 that bios does not play the same r81e as ammonium salts in yeast nutrition. I n the work here described molasses was used in the medium as the source of bios. Data are here presented showing that the fixation of atmospheric nitrogen by yeast in this medium is markedly affected by the hydrogen ion concentration. In dealing with Azotobacter Lipman3recognized the fact that the reaction of the medium is important. Fred4, in studying two different cultures of nitrogen-fixing bacteria, noted that while no growth occurred at pH6.4-6.6 that growth did occur a t pH6.6-6.8. However in the above work the pH changed from 7.2 to 5.1 during the growth of the organism. Gainey5 came to the conclusion khat the presence of Azotobacter in the soil is correlated with the absolute reaction of the soil solution and that the maximum hydrogen ion concentration tolerated by the organisms is a t about pH 5.9 to 6.0. Later Gainey6 made a thorough study of the growth and nitrogen fixation by Azohobacter in a synthetic medium as a function of pH. He concluded that the data presented “point very definitely to a limiting hydrogen ion concentration of pH 3.9 to 6.0 for the various cultures of Azotobacter employed when grown under the conditions of the experiments. Vigorous growth and nitrogen fixation took place at pH 6.1 to 6.5, the optimum for nitrogen fixation apparently being somewhat higher than the optimum for growth.” *From the laboratory of biophysical chemistry. Chemistry department, Iowa State College. 1Fulmer: Science, (2) 57, 645 (1923). 2Fulmer, Shcrwood and Nelson: Ind. Eng. Chem. 16, 921 (1924); Fu1mc.r: Colloid Symposium Monograph, 2, 204 (1925). 325th Ann. Report New Jersey Agr. Exp. Sta., 237 (1904). 4J.4gr. Res. 14, 317 (1918). jJ. Agr. Res. 14, 265 (1918); Abs. Bact., 6 , 14 (1922). J. Agr. Res. 24, 759 (1923).
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ELLIS I. FULNER A N D LEO M. CHRISTENSEN
In our general studies of yeast nutrition we have maintained two types of yeast designated in this laboratory as Nos. 11 and 12 both of which came originally from a Fleischmann yeast cake. When plated on synthetic agar medial No. I I forms small circular colonies with regular edge while the colonies of No. 1 2 are larger with radiate edge, In beer wort No. 11 grows on the bottom of the medium while No. 12 grows on the surface. In mediiim E2 containing ammonium salt No. 1 1 grows more readily than No. 12, the former growing entirely on the bottorr, of the medium while No. 1 2 at certain stages of its growth shows a tendency to “creep” up the sides of the flask. These iwo types resemble those described by Eddy, Kerr and Williams3. It was soon apparent that No. 1 2 grew much more readilythan No. I I in an ammonia free medium and it was used in the studies here described. The medium contained per one hundred cubic centimeters 6 grams of cane molasses and 0.50 grams of dipotassium phosphate, the latter acting as buffer. The pH of the media was adjusted after sterilization in order to obviate any changes in hydrogen ion concentration that might take place during the sterilization process, For this adjustment of reaction under sterile conditions a special apparatus and method was devised which have been previously described4. After inoculation several of the flasks were heated to kill the yeast and these flasks were used as blanks. The flasks were then sealed with paraffin leaving a capillary vent for pressure equalization. Incubation took place a t 30°C. MThilegrowth is much more rapid under aeration the danger of contamination in a large series is considerable in spite of all precautions so the method outlined above was adopted. At specified intervals the flasks were removed for examination and analysis. The cultures were examined for n-old growth and a gram stain was made. The yeast count was also determined. The nitrogen in the medium was determined by the Gunning modification of the Kjeldahl method. After the digestion had become clear crystalline potassium permanganate was added and the digestion continued for four hours. After the usual distillation the ammonia was determined colorimetrically by Nessler’s reagent.
Discussion of Results In Table I will be found data from typical series of experiments. The amount of fixation at various time intervals at a given pH was not determined from a single flask but each experiment represents a separate culture. It is a t once apparent that the medium according to the method of analysis used lost nitrogen in the beginning and that the actual gain did not show until after six to eight weeks at which time the cells had begun to break up. It is ‘Fulmer and Grimes: J. Bact. 8, 585 (1923). *Fiilmer, Nelson and Sherwood: J. Am. Chem. SOC., 43, 191 (1923). J. Am. Chem. Soc., 46, 2846 (1924). 4Christensen and Fulmer: Ind. Eng. Chem. 17, 93j (1925).
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FIXATION OF NITROGEN BY YEAST
also notable that the maximum gain takes place at the same pH in which there is maximum loss. This phenomenon has been observed many times in our work and after considering all phases of the matter our hypothesis is as follows. Yeast is known to be rich in ring nitrogen compounds and in the early stages of growth the nitrogen may be thrown into a compound not to be analyzed by usual methods. Later in the growth these compounds may be transformed into materials amenable to the analysis as used. h detailed analytical study is being made in an attempt to account for all of the nitrogen of the yeast.
FIG.I
FIG.2
TABLE I PH I
Gain in nitrogen in mgms/roo cc of culture Time in weeks 2 3 4 6
8
4.0 4.5
-0.92
5.0
-2.10
5.5 6.0 6.2 6.4 6.j 6.8
-1.19 -0.51
7 .o 7.2
7.4 7.6 7.8 8.0 8.5 9.0
9.5
-1.52
___
-3.60 -2.60 -3.20
__ -3.40 -3.50 -4.50
-1.00
-1.20
-0.90
-3.20
__
-__
__ -
__ -4.72 -2.56 -3.63 -5.35 -7.00 __ ___
__
-_ -4. I9 -2.46 -3.36 -3.63 -3.71
__
-3.22 -2.48 -1.85 + I . 19 (+2.01) +2.31 (+ 1.54) -0.84 -1.80
--
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ELLIS 1 . FULMER AND LEO M. CHRISTENSEN
At any rate it seems probable that the failure of several investigators to observe fixation with microorganisms may have been due to this time element, Lipman’ in his experiments on the fixation of atmospheric nitrogen by yeasts and molds allowed his cultures to stand for a month. The data show conclusively that the yeast used does fix atmospheric nitrogen in the medium employed and that the fixation is a function of the hydrogen ion concentration. There are two optimal concentrations at pH of 6.0 and 7.9 respectively. The optimum on the alkaline side is much more potent that the acid optimum concentratjon. The results of the sixth and the eighth week analysis are plotted in Fig. I and 2 . The effects of the composition of the medium and of temperature are being studied as they influence the fixation as a function of pH.
Conclusion The amount of nitrogen fixed by yeast in molasses a t 3ooC is a function of hydrogen ion concentration there being two optimal concentrations one at pH = 6.0 and the other at pH = 7.9 with the latter concentration the more potent. J. Riol. Chem. 10, 169 (1911).
