Reduction of Acetylene to Ethylene by Soybean Root Nodules

By use of the 15N technique it has been firmly established (1) that excised soybean nodules fix atmospheric nitrogen for a period of about 2 hours. The rate of fixation is greatly reduced in sliced nodules, and fixation is virtually lost when nodules are crushed (2). More recently, Bergerson (3) has investigated the effects of partial pressures of oxygen on N2 fixation in excised soybean nodtules and has devised a method (4) for the demonstration of N2 fixation in breis of nodules. In this procedure it was necessary to exclutde all traces of oxygen while nodules were crushed in a press, but oxygen was an essential component for No fixation in reaction mixttures containing nodule breis. No reproducible fixation of N2 gas by cell-free extracts of leguminous nodules has been accomplished. Recently Hardy and Knight (7) (also manuscript kindly supplied by Dr. R. W. Hardy) have utilized cell-free extracts of Azotobacter vinelandii and Clostridium pasteurianuim in a study of the specificity of the nitrogen fixing enzyme for electron acceptors. Extracts from these organisms not only catalyze the reduction of N2 to ammonia, but also catalyze the reduction of N2O to ammonia, of azide to N2 and ammonia and of cyanide to methane and ammonia. In the latter redtuction methylamine has been identified as an intermediate. Furthermore Shollhorn and Burris (8), and Dilworth (6) have shown that extracts of Clostridium pasteurianum catalyze the reduction of acetylene to ethylene. The evidence indicating that the nitrogen fixing enzyme is non-specific for electron donor, and catalyzes the reduction of several compounds other than nitrogen gas is convincing. The purpose of this investigation was t3 determine whether soybean nodules would reduice acetylene to ethylene and to establish the optimum conditions for the reaction if it were demonstrated. Soybean plants (Glycine max. Merr., var. Chipewa) were inoculated with a commercial preparation of Rhizobiumt japonicum, and cultured in a glasshoutse in pots of perlite supplied with a nitrogen-free nutrient solution. After a period of abotut 43 days, plants were removed from the culture vessels, nodules were harvested in a cold room at 40, washed with distilled water, and tutilized for acetylene reduction within a period of 1 hour. The prodtuction of ethylene from acetylene was measutred by uise of a Beckman GC-2A gas chromatograph equipped with a hydrogen flame detector and a recorder and integrater. The conditions employed in the chromatography were as follows. An aluminum column 183 cm in length and 0.63 cm. in diameter was packed with 41 cc of 60 to 80 mesh alumina. Helitum was tused as the carrier gas and was passed through the coluimn at a flow rate of 88 ml per minute. The temperature of the column was 130? and the attentuation of the gas chromatograph was 100. The ethylene and acetylene peaks from the column were identified by comparison of retention times with those of known standard samples of gas. The identity of the gases also was indicated by the demonstration that the addition of bromine water to a typical reaction mixture containing nodules resulted in the disappearance of peaks attribtuted to ethylene and acetylene. Further details of the experimental procedure are presented in the legends of figures. The conditions for optimum ethylene production by soybean nodules are presented in figures 1 to 3. A time coturse for the production of ethylene from acetylene is illustrated by the data presented graphically in figure 1. Ethylene production proceeded almost linearly for a period of about 1 hour then the rate diminished rapidly and virtually ceased after an incubation period of 1.5 hours. Control reaction mixtures containing nodtules that previously had been boiled for 3 minutes prodtuced no ethylene from acetylene. The time course for acetylene reduction is similar to the time course of nitrogen fixation by soybean nodules reported by Aprison and Burris (1). The data in figuire 2 show that oxygen is indispensible for the reduction of acetylene to ethylene. The rate of acetylene reduction proceeded almost linearly at oxygen concentrations ranging between 0.0 and 0.2 atmosphere. The addition of oxygen at concentrations greater than 0.2 of an atmosphere resuilted in no further increase in the rate of acetylene reduction. The concentration of oxygen required for the satturation of the acetylene reduction system is considerably less than that reported for the maximum rate of N2 reduction in sliced nodules (5) btut considerably greater than the value of about 0.06 atm which resulted in maximum N2 fixation by soybean nodule breis (4). Data showing the effect of acetylene concentration on the production of ethylene are presented in figure 3. A concentration of about 0.1 of an