either ammonium or gaseous nitrogen as the sole nitrogen source. For some of the growth studies and for growing inocula for larger cultures 10 to

Transcription

1 PHYSIOLOGY OF NITROGEN FIXATION BY AEROBACTER AEROGENES' ROBERT M. PENGRA AND P. W. WILSON Department of Bacteriology, University of Wisconsin, Madison, Wisconsin Received for publication July 15, 1957 Skinner (1928) tested 23 strains of Bacteriumn aerogenes (Aerobacter aerogenes) and reported that two, and possibly a third, assimilated gaseous nitrogen when cultured in liquid medium, a claim confirmed 27 years later by Hamilton and Wilson (1955) using the N" isotopic method. Their experiments indicated that A. aerogenes incorporated significant amounts of tracer and that anaerobically, in a well buffered medium, fixed sufficient nitrogen to be measured by a semimicro-kjeldahl method. We have extended this result to a total of 7 strains. Jensen (1956) independently obtained fixation by 2 of 3 strains of the organism isolated from water. For obvious reasons the numerous studies that deal with the physiology of the aerobacter have not included investigations concerned with this only recently established characteristic. The work reported in this paper deals with this aspect of the physiology of this organism. MATERIALS AND METHODS Of the several strains tested, the strain M5aL was chosen for study as its almost entire freedom of capsular gum made it much easier to handle. Both Skinner and Hamilton used media containing calcium carbonate to buffer against the acid produced by the fermenting organism, but for this investigation a medium was required that contained a soluble buffer to facilitate harvesting clean cells and reading turbidity of growing cultures. A modification of the medium proposed by Monod and Wollman (1947) for growing Escherichia coli was satisfactory. Its composition is: Na2HPO4, 12.5 g; KH2PO4, 1.5 g; MgSO4.- 7H2O, 0.2 g; CaCl2, 0.01 g; sucrose, g; Fe-Mo solution (Wilson and Knight, 1952), 1.0 ml; in 1 L of distilled water. Excellent growth of A. aerogenes was obtained in this medium using either ammonium or gaseous nitrogen as the sole nitrogen source. For some of the growth studies and for growing inocula for larger cultures 10 to 30,ug of ammonium acetate-nitrogen was added to the medium. When one or more liters of medium were prepared, the phosphates were dissolved in 70 per cent of the water and the balance of the medium in the remaining 30 per cent. The two solutions were autoclaved separately and combined just before inoculation. Growth in shake flasks took place in 50 mnl of the described medium placed in 250-ml Erlenmeyer flasks. For studies where turbidity was used as a measure of growth, the flasks had fused to them pyrex test tubes that fit the Klett- Summerson colorimeter; these were placed about two in below the flask rim and at an angle about 20 degrees below horizontal. Turbidity measurements of a growing culture were made by tipping a sample of the contents of the flask into the "side-arm" tube without exposing the culture to contamination. Each flask was closed with a rubber stopper fitted with a glass tube vent on which was placed a short length of rubber pressure tubing closed with a screw type pinch clamp. The flasks were evacuated with a water pump to about 0.05 atmospheres of pressure and refilled with high purity (99.99 per cent) Linde tank nitrogen. This was repeated three times and after the fourth evacuation the desired atmosphere supplied. In the growth studies 0.8 atm of N2 was admitted, the other 0.2 atm remaining as negative pressure to be replaced by fermentation gases. The flasks were then placed on a Brunswick rotary shaker and incubated at 30 C. Growth was followed using a no. 66 filter in the Klett-Summerson colorimeter. One liter cultures were grown in a 2-L Erlennmeyer flask equipped with a three hole rubber stopper which held an inlet tube ending in a l Supported in parts by grants from the Atomic Energy Commission Contract No. AT(11-1)-64 sintered glass sparger, a tube for sampling the and the National Science Foundation Grant culture and a gas outlet tube. The nitrogen gas NSF-C sparging slowly through the culture was first 21

2 22 PENGRA AND WILSON [VOL. 75 filtered through a cartridge filter containing glass wool. Six liter cultures were grown in 9-L serum bottles with similar gassing and sampling arrangements. The organisms were carried on nutrient agar slants. Transfers were made into shake flasks as have been described, with or without the colorimeter side-arm tube, and a N2 atmosphere placed over the culture. When the culture was well into the exponential phase of growth, sufficient gas pressure had accumulated so that it had to be vented periodically. For 50 ml cultures in shake flasks, 0.5 to 2.5 ml of inoculum from the flask culture was used, for 1-L cultures the entire 50 ml, and for 6-L mass cultures 2 such inoculum flasks were used. The two gaseous inhibitors studied (hydrogen and oxygen) were added to the atmosphere above growing cultures and their effects determined by measuring turbidity for a short time and by initial and final nitrogen determinations. These test cultures were prepared by growing a liter culture as has been described until it was in the early exponential phase of growth, placing 50-ml aliquots into 250-ml flasks and taking an initial 10-ml sample for Kjeldahl nitrogen determination. The atmosphere for the hydrogen inhibition studies was measured with a mercury manometer as it was placed in the flasks. Such small amounts of oxygen were required to inhibit fixation that this gas was added to the flasks with a hypodermic syringe by inserting the needle into the rubber vent tube after clamping it off, and allowing the partial vacuum left in the flask to empty the syringe. Kjeldahl nitrogen determinations were made by the semi-micro-procedure described by Wilson and Knight (1952). Sample digestion for N15 determination was done using a mercury catalyst. A Consolidated-Nier isotope ratio mass spectrometer was employed for the mass analysis. RESULTS AND DlSCUSSION Growth. Figure 1 is a growth curve of 4erobacter aerogenes strain M5aL grown in shake flasks as has been described. The nitrogen source for curve NH4+ was about 150,ug ammonium acetate-nitrogen per ml of medium, and in curve N2 about 20,ug ammonium acetate-nitrogen per ml of medium. The atmosphere over the cultures for curve NH4+ was helium, and for curve F2.6 Log Hours Figure 1. Growth of Aerobacter aerogenes on free and combined nitrogen. Curve NH4+ is growth on 150,g NH,+-N/ml under helium. Curve N2 is growth on 20,ug NH4+-N/ml under N2. K-S = Klett-Summerson readings. N2 tank nitrogen. Final nitrogen determinations at 30 hr were 160 and 80,ug per ml for cultures NH4+ and N2, respectively. Curves for mass cultures are similar; however, as much as 150,ug/ml of nitrogen may be fixed in these cultures. Curves for cell nitrogen parallel optical density curves sufficiently well in these cultures that optical density may be used in most experiments as a measure of nitrogen fixed. Adaptation to N2. The second lag period and the two exponential increases in curve N2 of figure 1 were observed in all growth curves when a small amount of ammonium nitrogen was used in the medium but never when the medium was free of fixed nitrogen. The length of the initial exponential phase as well as the second lag phase is directly related to the initial ammonium content of the medium. This second lag phase suggested a period of adaptation to N2, i. e., induced formation of nitrogenase. To test this interpretation two types of experiments were made.

3 19581 PHYSIOLOGY OF NITROGEN FIXATION BY A. AEROGENES 23 The first was to determine if ammonia utilization had completely stopped and nitrogen fixation not y-et started during the second lag phase and w-hether fixation and the second exponential phase of growth started concurrently. A 6-L culture was used but instead of flushing tank nitrogen through it, an atmosphere consisting of 20 per cent N2 with ca. 11 atom per cent N'5 excess and 80 per cent helium wvas recirculated through it for most of the growth period. The gas was placed over the culture by evacuation and refilling as described in Methods and was circulated with an automobile fuel pump run by a } hp electric motor. The rate of flow was controlled by a screw clamp on the pressure tubing connecting the pump and the culture. The gas was cleared of moisture and CO2 by passing it successively through tubes containing CaCl2 and I Hours Figure 2. Adaptation of Aerobacter aerogenes to N2. Up to 50 hr the atmosphere containing N2,5 was circulated through the culture as described in the text. At 50 hr tank nitrogen was bubbled through it for the remainder of growth. The medium initially contained 20,ug of combined nitrogen as ammonium acetate. Between the 16th and 28th hr sufficient nitrogen was fixed to be detected by the isotope method but not enough to be detected by Kjeldahl analysis. TABLE 1 Adaptation to N2 by Aerobacter aerogenes Age of Parent Culture hr Length of Subculture Lag KOH pellets and finallv filtered through sterile glass wool. Turbidity, total culture nitrogen, cell nitrogen, residual sugar, ph and atom per cent N" excess were determined on aliquots of the culture taken every 2 hr. Figure 2 presents the data for culture nitrogen, cell nitrogen and atom per cent N" excess. As can be seen, no significant amount of N21, was incorporated into the cells for a period after the ammonia was exhausted from the medium. After this lag, incorporation took place rapidly until tank nitrogen was started through the culture at 50 hr at which time fixation of nitrogen containing virtually no N" (normal nitrogen contains about 0.36 atom per cent N1") diluted the isotope in the cells. This lag, from about the 8th to the 16th hr, before N" was taken up suggests that the cells were "adapting" to N2. The second type of experiment was one in which aliquots were taken aseptically from a parent culture during the adaptive lag and early exponential phases, adjusted to a common turbidity by dilution with sterile medium and used to inoculate flasks of nitrogen-free medium. The length of the lag phase of these subcultures was measured; table 1 shows the results of one such experiment. These data suggest that the "nitrogenase" system or some component of it is induced in A. aerogenes; to date, no such induction has been reported for the genus Azotobacter or Clostridium. Inhibition by H2. Wilson and his collaborators demonstrated that hydrogen is a specific competitive inhibitor of nitrogen fixation in the aerobic nitrogen-fixing systems (See Wilson, 1951 for review of literature). Rosenblum and Wilson (1950) pointed out the difficulty in attempting to demonstrate an inhibition by hydrogen of nitrogen fixation in the anaerobic Clostridium pasteuir- hr

4 24 PENGRA AND WILSON [VOL. 75 HOURS Figure 3. Effect of H2 on assimilation of N2 and NH4+ by Aerobacter aerogenes. * ph2=4 0 ianum because this organism produces gaseous hydrogen during growth. They did find a small decrease in the total nitrogen fixed but no lessening of the rate of fixation under an atmosphere containing 60 per cent H2. Under the conditions we have used, our strains of A. aerogenes fix nitrogen only anaerobically and the same difficulty is met, i. e., production of H2 during growth. The effect of metabolically produced hydrogen was minimized by growing a 1-L culture under flowing nitrogen (see Methods) until it was in the exponential phase of growth; then aliquots were measured into flasks, samples taken for initial nitrogen determinations, and the desired experimental atmospheres placed over TABLE 2 Fixation of N2 by Aerobacter aerogenes presence of yeast extract* in the Mg NI Oxygen Supply Strain Treatment Flaskt (20 MI) Aerobic UWi Autoclaved and 7.8 incubated Aerobic UWi Incubated 8.1 Anaerobic UW1 Incubated 30.0 Aerobic M5aL Autoclaved and 8.2 incubated Aerobic M5aL Incubated 7.4 Anaerobic M5aL Incubated 43.0 * 0.1 g/l of yeast extract in medium. t Each value an average of 3 flasks. I/pN2 Figure 4. Lineweaver-Burk double reciprocal plot showing competitive inhibition by hydrogen of nitrogen fixation by Aerobacter aerogenes. l/k is the reciprocal of the growth rate constant and 1/pN2 is the reciprocal of the substrate concentration. Figure 5. Oxygen inhibition of nitrogen fixation by Aerobacter aerogenes. Optical density increases in the first hour are probably from growth before combined nitrogen becomes limiting.

5 1958] PHYSIOLOGY OF NITROGEN FIXATION BY A. AEROGENES 25 them. These cultures were grown on a rotary shaker to give maximum gas exchange between the culture and the atmosphere. Turbidities were read during growth and total nitrogen was determined at the end of 3 to 4 hr of growth. The quantity of hydrogen produced in this short time did not greatly alter the H2-N2 ratios in the flasks. Figure 3 illustrates the effect of atmospheric hydrogen upon the assimilation of gaseous and ammonia nitrogen by A. aerogenes. In experiments using various partial pressures of hydrogen and nitrogen, it was observed that although growth was limited by hydrogen it was still exponential, the growth rate constant being decreased by hydrogen. Figure 4 is a Lineweaver- Burk double reciprocal plot, as described by Wilson (1949), of the data from these experiments. The growth rate constant k was determined using the formula k = (2.3/t) log (final total N/initial total N) in which t is time in hours. The significantly different slopes of the lines and the coincidence of the lines with and without inhibitor on extrapolation to infinite substrate concentration indicate that the hydrogen inhibition is competitive. Inhibition by 02. Species of Aerobacter grow well either aerobically or anaerobically when supplied with ammonium nitrogen. Hamilton and Wilson (1955) noted that only a small amount of nitrogen was fixed when the cells were grown aerobically but increased inarkedly upon lowering the PO2 of the atmosphere over the culture. Jensen (1956), however, demonstrated good fixation either aerobically or anaerobically and reported that his medium had to contain a small amount of yeast extract (Difco) for fixation to occur. These results contrast markedly with those observed using strains UW1 and M5aL (table 2). With these strains and under the conditions described oxygen is a specific inhibitor of fixation (figure 5). As is shown in the right hand portion of figure 5, the facultative nature of this organism permits it to use up the small amount of oxygen placed over the culture; the exhaustion of 02 allows fixation to begin. Even at low P02, however, yeast extract did not reverse the inhibition. From this survey of nitrogen fixation by A. aerogenes it appears that this organism has many desirable characteristics that will make it useful for studies on the mechanism of biological nitrogen fixation. These include: a readily demonstrable "adaptation" to N2, anaerobic inhibition by H2, and sensitivity to 02. Explanation of the discrepancy in the results of Jensen (1956) and ourselves regarding 02 sensitivity may yield significant information with respect to the comparative physiology of anaerobic and aerobic fixation. SUMMARY A medium containing a soluble buffer lias been devised for studying the growth of Aerobacter aerogenes on free and combined nitrogen using turbidity as a measure of growth. It appears that the "nitrogenase" system in this organism is induced by N2. Molecular hydrogen is a competitive inhibitor of nitrogen fixation by the aerobacter. Such competition has been previously demonstrated for the aerobic agents of fixation but not for the anaerobic agents, the clostridia and the photosynthetic bacteria. Under the conditions used in these experiments molecular oxygen inhibits nitrogen fixation specifically although Jensen could detect no such inhibition in his trials. REFERENCES HAMILTON, P. B. AND WILSON, P. W Nitrogen fixation by Aerobacter aer ogenes. ln Biochemistry of nitrogen, pp A. I. Virtanen homage volume. Ann. Acad. Sci. Fennicae, II A. Chemica. JENSEN, V Nitrogen fixation by strains of Aerobacter aerogenes. Physiol. Plantarum, 9, MONOD, J. AND WOLLMAN, E L'inhibition de la croissance et de l'adaptation enzymatique chez les bacteries infect6es par le bacteriophage. Ann. inst. Pasteur, 73, ROSENBLUM, E. D. AND WILSON, P. W Molecular hydrogen and nitrogen fixatiorn by Clostridium. J. Bacteriol., 59, SKINNER, C. E The fixation of nitrogen by Bacterium aerogenes. and related species. Soil Sci., 25, WILSON, P. W Kinetics and mechanisms of enzyme reactions. In Respiratory enzymes, rev. ed., Ch. 2. Edited by H. A. Lardy. Burgess Publishing Co., Minneapolis. WILSON, P. W Biological nitrogen fixation. In Bacterial physiology, chapter 14. Edited by C. H. Werkman and P. W. Wilson. Academic Press, Inc., New York. WILSON, P. W. AND KNIGHT, S. G Experiments in bacterial physiology. Burgess Publishing Co., Minneapolis.