hydrogen in inhibiting nitrogen fixation by Clostridium, an effect previously

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1 MOLECULAR HYDROGEN AND NITROGEN FIXATION BY CLOSTRIDIUM1 EUGENE D. ROSENBLUM AND P. W. WILSON Department of Agricultural Bacteriology, University of Wisconsin, Madison, Wisconsin Received for publication October 12, 1949 Although in recent years the mechanism of nitrogen fixation by Azotobacter and bythe symbiotic system of leguminousplantsplusrhizobium has been studied in detail, similar investigations with Clostridium are few. This apparent neglect arises not only from the relative inconvenience of handling an anaerobe but also from the discouragingly small and slow fixation with this agent. The common explanation of this relatively inefficient fixation (about 2 to 3 jig per mg carbohydrate) is that the energy obtained by fermentation is only about one-tenth that from oxidation. Since consideration of the energetics of fixation (Wilson and Burris, 1947) makes this reasoning unlikely, the possible role of metabolic hydrogen in inhibiting nitrogen fixation by Clostridium, an effect previously demonstrated for the aerobic systems, appeared to be worthy of investigation. METHODS In preliminary trials nitrogen fixation by Clostridium pasteurianum, strain W5, in a modified Winogradsky's N-free medium was studied using three methods of incubation: the conventional anaerobic jar, a circulating system, and shake flasks. Twenty-five ml of medium in large test tubes were used in the first two methods, the same quantity in a 500-ml Erlenmeyer flask in the third. In the circulating system the atmospheres were circulated by a Urey pump through a 10-liter reservoir bottle; although this method was superior for the removal of metabolic gases, it was neither so convenient nor consistent as the shake flasks. The anaerobic jar method, normally used in previous studies of fixation by this agent, gave the usual low and prolonged rate of fixation. The shake flask method was accordingly adopted for further studies that were concerned with the effect on nitrogen fixation of such factors as concentration and source of carbohydrate, concentration of calcium carbonate, additions of growth factors and reducing agents, and the like. From the results of these trials the following technique was adopted: the medium was the salt mixture of Jensen and Spencer (1947) plus 2 per cent sucrose and 1 per cent calcium carbonate; 25 ml in a 500-ml Erlenmeyer flask was inoculated with 0.5 ml of a 20-hour culture of Clostridium pasteurianum W5 grown on potato medium (Jensen and Spencer, 1947). The inoculum furnished not only necessary growth factors but sufficient combined nitrogen to give the organism a vigorous start. The cultures were shaken by a mechanical shaker and harvested 1 Supported in part by grants from the Rockefeller Foundation and from the Research Committee of the Graduate School from funds supplied by the Wisconsin Alumni Research Foundation. 83

2 QA EUGENE D. ROSENBLUM AND P. W. WILSON [VOL. 59 periodically. Combined nitrogen was added as 200 jig N per ml as ammonium sulfate which was sterilized separately. Chemical analysis. Total nitrogen was determined on an aliquot of 15 ml using a semimicro-kjeldahl method; sugar was estimated on an aliquot of 1 to 5 ml using an adapted Folin-Wu method (Folin, 1929). Initial and residual ammonia was determined by adding 3 ml of 1 N HCI to 15 ml of culture to dissolve residual calcium carbonate and removing the bacterial cells by centrifugation; ammonia was then estimated by distillation of an aliquot of the combined supernatants from triplicate samples. The cells were washed once (a small loss of nitrogen occurred at this step), and their total nitrogen was estimated by the semimicro- Kjeldahl method. Soluble nitrogen was calculated from the total nitrogen in the TABLE 1 Effect of hydrogen en uptake of atmospheric nitrogen by Clostridium GAS XPERIMENT TI:TME MTHOD NITROGEN 1mD pn. PH2 phe #g/mi days 1 8 Anaerobic jar Anaerobic jar Circulating sys tem Circulating sys tem Erlerneyer shake flask Erlenrmeyer shake flask supernatant less ammonia-n; all data were corrected for the volume of HCI added. RESULTS Effect of molecular hydrogen on nitrogen fixation. The results. from prelimin ry trials (Rosenblum, 1948) apparently supported the conclusion that molecular hydrogen inhibits nitrogen fixation by Clostridium, as had already been noted with Azotobacter (Wyss, Lind, Wilson, and Wilson, 1941), with the symbiotic system in red clover (Wilson, 1940), and with Nostoc (Burris and Wilson, 1946). For reasons originally emphasized by Burk (1934) this conclusion can be regarded only as tentative as it was based entirely on final total nitrogen content of the cells (see table 1 for typical data). Final decision had to await experiments in which the rates of fixation were compared. A number of shake flasks were grown under two atmospheres: 0.2 atm N atm He, and 0.2 atm N atm H2;

3 1950] HYDROGEN AND NITROGEN FIXATION BY CLOSTRIDIUM 85 triplicates of each treatment were harvested periodically and analyzed for total nitrogen and residual sucrose. Figure 1 summarizes the results from two typical experiments. As noted in earlier trials, the final total nitrogen fixed is higher in the presence of helium than in that of hydrogen, but no significant difference was observed in the rate of fixation under the two gas mixtures. An identical N2 to H2 N2 fixed I I I I I Experiment 7 / Experiment 8 r / ~~~/s ~~0' 00 0 Downloaded from 40L 20 L ol 20 (Expt 7) 20 (Expt 8) /1/@--4--pN 0.2+pHeO0.6atn / X / P-o-o--pN ph2 0.6atrIf // / / _ / 2' -Or-. p-ob- Figure 1. Influence of molecular HE on nitrogen fixation by Clostridium pasteurianum W5. ratio would cause a significant decrease in the rate of fixation by Azotobacter and the other aerobic agents. Hydrogen has no effect on the rate of uptake of combined nitrogen by the aerobic nitrogen fixers since its effect is specific for N2 fixation. For comparison, therefore, the effect of H2 on the uptake of NHs-N by Clostridium was studied. Rate experiments similar to those already described were made except that the medium contained about 200 ppm nitrogen as ammonium sulfate. The quantity a on October 15, 2018 by guest

4 86 EUGENE D. ROSENBLUM AND P. W. WILSON [VOL. 59 of nitrogen assimilated was calculated from the sum of the cell and soluble organic nitrogen less the initial cell and soluble organic nitrogen. The organism evidently excretes considerable soluble nitrogen during its growth. The data of a typical experiment are shown in figure 2; it is evident that no difference exists between the rate of uptake of combined nitrogen in the presence of the two gases. Efficiency of nitrogen assimilation. The efficiency of assimilation of free and combined nitrogen by Clostridium is given in table 2. Several observations are m m m NH3 - N uptake lug/ml --0 I / / OTAL NITROGEN _ // // // CELL NITROGEN -O-CpN O-o-pN phe 0.6 atm +ph20.6 atm TIME IN HOURS a a a I * I m 0 10 ZU 30 4U Figure S. Influence of molecular H2 on assimilation of NHs-N by Clostridium pasteurianum W5 (experiment 12). noteworthy. First, that nitrogen fixation by Clostridium as measured by extent rate, or efficiency, is not so markedly inferior to that of Azotobacter as is generally believed. Most of the apparent inferiority arises from the use of an inadequate technique that seriously limits the fixation process. Using the improved methods based on the modern studies of the organism's physiology, we have obtained uptake of both free and combined nitrogen of 150 to 200,ig per ml in 48 to 72 hours, values comparable at least to those observed with Azotobacter. Likewise, the efficiency of fixation by Clostridium, 6 to 7 ug per mg carbohydrate, compares

5 19501 HYDROGEN AND NITROGEN FIXATION BY CLOSTRIDIUM 87 favorably with the 10,ug per mg commonly obtained with Azotobacter. It is recognized that higher values for the efficiency of Azotobacter have been obtained, but it is emphasized that the values reported here for Clostridium need not represent the maximum. For example, because of the location of the shaking apparatus, incubation was at 27 C; this temperature may not be the optimum for fixation by Clostridium under the other experimental conditions used. Finally, in agreement with the results of the preliminary trials in which the rate and extent of fixation EXPERnINT TABLE 2 Efficiency of assimilation of free and combined nitrogen by Clostridium TiME 0.2 atm pns +0.6 atm ph2 0.2 atm pns atm phe l Nitrgetne ~ Efficiencyt Nitrogen Effidency h, pg/mi pg/mi Molecular nitrogen Combined nitrogen (NHs-N) (?) 11.2 (?) * TotaJ nitrogen minus initial nitrogen. t Micrograms nitrogen assimilated per mg sugar utilized. were decidedly lower, the efficiency is definitely and consistently less when 0.6 atm H2 is supplied in the gas phase. No consistent difference in the extent or efficiency of the uptake of ammonia in the presence of added hydrogen was observed. Effect of removal of carbon dioxide. In these studies metabolic hydrogen was not removed and might therefore be an important factor in any effect caused by the added molecular H2. The quantity of H2 evolved during fermentation was estimated by fastening to the rubber stopper of representative flasks a widemouth tube containing 6 ml of 40 per cent NaOH; the initial and final pressures

6 88 EUGENE D. ROSENBLUM AND P. W. WILSON [VOL. 59 were determined by an attached manometer. Typical data were: initial pressure (N2), 0.2 atm; final pressure with NaOH, 0.5 atm; final pressure without NaOH, 0.8 atm. Since the volume of N2 consumed is small, formation of both H2 and C02 during the fermentation was approximately 0.3 atm. This result means that each culture during growth was subjected to an average pressure of 0.3/2 or 0.15 atm of metabolic hydrogen. Thus the flasks under helium would have a gas phase of the following average composition: 0.2 atm N2, 0.6 He, 0.15 H2; those under H2 one of 0.2 atm N2, 0.75 H2. Such a difference in the ph2 produces a readily observed difference in the rate and efficiency of N-fixation by Azotobacter (Wyss et al., EXPERIMENT TABLE 3 Effect of removal of carbon dioxide on nitrogen assimilation by Clostridium SUCOF GAS* NaOH NITROGEN IFIZNY NMTROGEN ASSIMILATEDt EFFINCt pgb/ml 15 N2 He Present Absent H, Present Absent He Present Absent N2 H2 Present Absent He Present Absent N2 Hs Present Absent He Present Absent NH3 H, Present Absent * pn2, 0.2 atm atm of indicated gas. t Total nitrogen minus initial nitrogen. t Micrograms nitrogen assimilated per mg sugar used. Two hundred,ug NH,-N per ml. 1941). The fact that it does not affect the rate of fixation in Clostridium indicates either that H2 does not inhibit fixation in this organism or that the inhibition (evidenced by lower efficiency) is qualitatively and quantitatively different from that noted in the aerobic agents. When the contents of the flasks used in the estimation of metabolic gas production were analyzed for total nitrogen, it was found that the flasks from which C02 had been removed had fixed about 20 per cent more N2 than those to which no NaOH had been added. This unexpected finding was investigated further by repeating the procedure several times under N2 + He and N2 + H2 and using both N-free medium and medium containing NHa-N. Table 3 gives the results of

7 1950] HYDROGEN AND NITROGEN FIXATION BY CLOSTRIDIUM 89 several experiments. Removal of C02 causes an increase in total fixation and final efficiency when grown on the N-free medium, an effect seemingly independent of any effect due to H2. The assimilation of fixed nitrogen, however, does not appear to be influenced by C02 removal. The explanation for this effect of C02 is not known, but it might be attributed in part to a ph change, since the ph dropped from an initial value of 7.4 to 6.2 when C02 was not removed, but dropped only to 6.6 when removed. Excretion of nitrogen. It was mentioned previously that considerable quantities of soluble organic nitrogen are found in cultures of Clostridium pasteurianum W5 grown in a medium containing ammonium sulfate. As is evident from the data in table 4, organic compounds containing nitrogen are likewise excreted by the anaerobe when fixing molecular nitrogen. In contrast with Azotobacter, which TABLE 4 Nitrogen distribution during growth of Clostridium pasteurianum TIMZ ~~SUCROSE SOLUBLE ORGANIC CLNIRGN CONCENTRATION CELL NITROGEN NITROGEN NHa-N hr mg/mi pg/mi pg/mi pg/mi Nitrogen-free medium * Ammonia medium * * From inoculum. apparently does not liberate soluble organic nitrogen until the supply of carbohydrate is exhausted (Burk and Burris, 1941), Clostridium excretes organic nitrogen in young cultures before all sugar has disappeared. Separation of the cells from the culture by Seitz filtration established that the soluble organic nitrogen did not arise from extraction by the HCl used before centrifugation. The soluble nitrogen fraction from these cultures was low in ammonia nitrogen (except when added) and free of nitrite and nitrate; it was not precipitated by phosphotungstic acid. Its composition is being studied further. DISCUSSION Two facts established by this study have implications beyond the present work. First, the efficiency of nitrogen fixation by Clostridium is considerably higher than is generally recognized, and its rate of fixation is reasonably com-

8 90 EUGENE D. ROSENBLUM AND P. W. WILSON [VOL. 59 parable with that of Azotobacter. Results from investigations of enzyme mechanisms have firmly established the principle that the results are markedly influenced by the rate and extent of reaction. Because of this, many, if not all, of the significant physiological findings reported for Clostridium should be re-examined using cultural conditions that allow rates of nitrogen assimilation about ten times those observed in the majority of previously reported studies. The situation is comparable to the one obtaining in the early 1930's when it was recognized that much work with Azotobacter needed repeating because the limited knowledge of the organism's physiology had resulted in unbelievably poor fixation. Second, the effect of molecular hydrogen supplied in the atmosphere on nitrogen fixation by Clostridium differs markedly from that noted with the other agents previously tested. At least three alternative explanations may be suggested to account for the differences: (1) The mechanism of fixation by Clostridium is different from that of the other agents. This would not be too surprising since Clostridium fixes nitrogen anaerobically, whereas the other agents tested are aerobes. Studies of nitrogen fixation by the anaerobic photosynthetic bacteria (Gest and Kamen, 1949) would be informative on this point. (2) The mechanisms used by the various agents do not differ significantly, and molecular hydrogen does inhibit fixation in Clostridium. The explanation of the apparent lack of effect of H2 added to the atmosphere is that metabolic hydrogen released in the cell through breakdown of carbohydrate so dominates the contiguous fixation reaction that the effect of added H2 is restricted to the relatively minor one of reducing the efficiency. The lower extent, rate, and efficiency of nitrogen fixation by Clostridium in comparison with Azotobacter support this view. Although the efficiency of fixation by Clostridium on the basis of energy released is about 5 times that usually noted with Azotobacter, on an absolute basis (pg N fixed per mg carbohydrate used) its efficiency is only about one-half that of the aerobe. Elimination of this source of interference might prove to be extremely difficult unless a cell-free preparation capable of fixing N2 can be obtained. (3) The mechanism is similar in the various agents, but since Clostridium liberates molecular hydrogen it has developed a compensatory mechanism for preventing it from affecting its process of nitrogen fixation. Thus the organism avoids the metabolic error of producing in its carbohydrate breakdown an inhibitor of the reaction through which it assimilates molecular nitrogen. Decision as to the relative likelihood of these alternatives awaits the results from further studies of the mechanism of fixation by the species of this genus. SUMMRY Through improvement in technical details, the extent, rate, and efficiency of nitrogen fixation of Clostridium pasteurianum have been increased two- to threefold. These improvements include the use of large inoculum grown on a rich natural medium such as potato extract, the presence of a small quantity of com-

9 1950] HYDROGEN AND NITROGEN FIXATION BY CLOSTRIDIUM 91 bined nitrogen (20 to 30.ug per ml), and the use of shaken cultures. Based on the rate and efficiency of fixation, the optimum concentration of sucrose was about 1 per cent; however, as the efficiency at 2 per cent was only slightly lower, this concentration was ordinarily used because of the higher total fixation (0.5 grams of calcium carbonate should be added per gram of carbohydrate). Using these experimental conditions, we have obtained nitrogen fixation of 150 to 180 ug per ml in 48 to 72 hours with an efficiency of 6 to 8,ug per mg carbohydrate, values somewhat less but still reasonably comparable with those observed with Azotobacter. Alkaline absorption of liberated carbon dioxide during the fermentation likewise appeared to stimulate nitrogen fixation. The addition of molecular hydrogen to the atmosphere supplied Clostridium pasteurianum decreased the extent and efficiency of nitrogen fixation but did not affect the rate. This response differs significantly from that observed with the aerobic agents of fixation (Azotobacter, Nostoc, red clover). Three possible explanations of the difference are discussed. During assimilation of either free or combined nitrogen by C. pasteurianum, soluble organic nitrogen accumulates in the medium-up to 50 per cent of the total nitrogen assimilated may be found outside the cells. In contrast with similar results reported for cultures of Azotobacter, the appearance of the soluble organic nitrogen does not depend on the exhaustion of the supply of carbohydrate. REFERENCES BURK, D Azotase and nitrogenase in Azotobacter. Ergeb. Enzymforsch., 3, BuRK, D., AND BuRIas, R. H Biochemical nitrogen fixation. Ann. Rev. Biochem., 10, BuRms, R. H., AND WILSON, P. W Characteristics of the nitrogen-fixing system in Nostoc muscorum. Botan. Gaz., 108, FoLIN, Two revised copper methods for blood sugar determination. J. Biol. Chem., 82, GEST, H., AND KAmEN, M. D Studies on the metabolism of photosynthetic bacteria. IV. Photochemical production of molecular hydrogen by growing cultures of photosynthetic bacteria. J. Bact., 58, JENSEN, H. L., AND SPENCER, D The influence of molybdenum and vanadium on nitrogen fixation by Clostridium butyricum and related organisms. Proc. Linnean Soc. N. S. Wales, 72, ROSENBLUM, E. D Inhibition by hydrogen of anaerobic nitrogen fixation. Soc. Am. Bact., Proc. Meetings, 1948, 6-7. (See al8o Wisconsin Agr. Expt. Sta., Bull. 487, 2-3.) WILSON, P. W The biochemistry of symbiotic nitrogen fixation. University of Wisconsin Press, Madison. WILSON, P. W., AND BURRIS, R. H The mechanism of biological nitrogen fixation. Bact. Revs., 11, WYSS, O., LIND, C. J., WILsON, J. B., AND WILSON, P. W Mechanism of biological nitrogen fixation. VII. Molecular H2 and the pn2 function of Azotobacter. Biochem. J., 35,