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1 PHYSIOLOGICAL STUDIES ON RHIZOBIUM V. THE EXTENT OF OXIDATION OF CARBONACEOUS MATERIALS' 0. R. NEAL2 AND R. H. WALKER' Iowa State College, Ames, Iowa Received for publication March 28, 1936 The earlier papers of this series (Walker, Anderson and Brown, 1933; Neal and Walker, 1935) dealt with the growth and respiration of Rhizobium under varying environmental conditions. In all cases, the organisms were provided with a sufficient source of energy to permit unhindered respiratory activity throughout the experiments. If only a small amount of the carbonaceous material is used in the substrate, on the other hand, the extent of oxidation of the compound caused by the organisms, may be determined within a reasonably short period. The procedure consists in comparing the volume of oxygen consumed by the culture with the volume that would be required for complete oxidation of the compound to carbon dioxide and water. The extent of oxidation of certain carbonaceous materials which can be effected by Rhizobium meliloti and R. japonicum, respectively, has been determined by this method and the results are reported in this paper. METHODS Warburg's (1926) respiration methods were employed in making continuous measurements of the oxygen consumed and the carbon dioxide produced by the organisms. In prelimn ary experiments, 1 Journal Paper J-337, Iowa Agricultural Experiment Station, Ames, Iowa. Project No Now with the Soil Conservation Service, in charge of the Soil Erosion Farm at Clarinda, Iowa. ' The authors are indebted to Dr. P. E. Brown for the suggestions and criticisms offered in the course of this work and in the preparation of the manuscript. 183

2 R. NEAL AND R. H. WALKER special Bunsen absorption coefficients were determined experimentally for the absorption of CO2 in the particular media employed and under the pressures of gas encountered in the later studies. These coefficients were substituted in Warburg's equation and used in calculating the vessel constant for each of the respiration vessels used, without alkali in the inner well. The following basic medium was employed throughout, K2HP04, 0.5 gram; MgSO4c7H20, 0.2 gram; NaCl, 0.1 gram; CaCO3, 3.0 grams; distilled water, 1,000 cc. Either sodium nitrate or ammonium chloride was added to the medium to provide 100 p.p.m. of nitrogen in each case. Glucose or other carbohydrate was added in varying minimum amounts as will be described later. All experiments were conducted in duplicate and the results given are averages of two closely agreeing determinations. The inocula were prepared from organisms grown for 3 to 4 days in a glucose-yeast extract liquid medium, then centrifuged and washed twice in a sterile per cent NaCl solution. A 1 cc. suspension of washed cells was placed in the respiration vessel which contained 1 cc. of a double concentration of the substrate. The vessels were immediately connected with the manometers and placed on the water bath to attain a temperature equilibrium before being closed to start the experiment. The temperature of the bath was maintained at 28.0C. a0.01o. The manometers were oscillated 90 to 95 times per minute, with an amplitude of approximately 3 cm. EXPERIMENTAL Rhizobium meliloti In the first experiment, Rhizobium meliloti was grown in media containing M/540, M/270, and M/180 concentrations of glucose. In order to determine the extent of the endogenous respiration by the organisms of the inocula, a fourth culture vessel was included in the experiment, in which the organisms were cultured as in the other three vessels except that there was no carbohydrate in the medium. Deductions were then made of the quantities of oxygen consumed in the controls from the amounts consumed in

3 PHYSIOLOGICAL STUDIES ON RHIZOBIUM the vessels containing glucose. The corrected amounts of oxygen consumed are shown by the graphs in figure 1. It is seen that the consumption of oxygen proceeded at about the same rate in each of the media for four hours. At that time there was an abrupt change in the rate of oxygen consumption in the medium, containing M/540 of glucose, and the total amount of oxygen consumed in that culture was not increased appreciably during the remainder of the experiment. At later periods, similar changes occurred in the rate of respiration of the organisms in the other media. The amount of oxygen consumed before these 4501 II 1.NP.A 41J) 300' Z25( FIG.~~~~~ 1.OYE1OSMTO1YRIOBU EIOII LCS EI 100 tionof17uos1i teedum 0. 4 a C. 1Z TiME (Hours) FIG. 1. OXYGEN CONSUMPTION By RHIZOBIUM MELILOTI IN GLUCOSE ME~DIA changes occurred was almost in direct proportion to the concentration of glucose in the medium. The amount of oxygen consumed per 2-hour interval, in each of the three media, is shown in figure 2. In each case the rate reached a maximum very quickly, then dropped to a low and fairly constant level. It is apparent that the maximum rate of oxygen consumption was a function of the concentration of glucose in the medium. Upon calculation of the theoretical amounts of oxygen required to oxidize completely the glucose in each of the media it is found that the M/540, M/270, and M/180 concentrations of the sugar 185

186160. R. NEAL AND R. H. WALKER would require, respectively, 497, 994, and 1491 c. mm. of oxygen.

4 R. NEAL AND R. H. WALKER would require, respectively, 497, 994, and 1491 c. mm. of oxygen. This calculation necessarily assumes that oxygen is the sole hydrogen acceptor in the dissimilation process. The actual amounts of oxygen consumed by the organisms during a 24-hour period in the three media, in increasing order of the glucose concentration, were 143, 306, and 488 c. mm. These values represent approximately 30 per cent of the theoretical amount of oxygen necessary for complete oxidation of the glucose ~~~~~ Glucos*u 0U 2: m.io E 0 I 0 c-a-o C 4 e - IU.. IL 14*A 1 IS K I lb ~ - tso ArcY1~~~ 1 Um 1LCS 14/4 ao I GLUCOE I ~LO - 9 agoxw_ 5am_; 7 _A X!.l.!~~~~~~~~~0 IA. TIME (Hours) TIME (Hours) FIG. 2 FIG. 3 FIG. 2. RATE OF OXYGEN CONSUMPTION BY RHIZOBIUM MELILOTI IN GLUCOSE MEDIA FIG. 3. RESPIRATORY QUOTIENT OF RHIZOBIUM MELILOTI IN GLUCOSE MEDIA It is possible that the organisms utilized only a fraction of the glucose, oxidizing it completely to carbon dioxide and water. It seems more probable, however, that all of the glucose was partially dissimilated with the production of intermediate or end products other than carbon dioxide. The abrupt drop in the rate of respiration as shown by the curves in figure 2 and which was observed in every experiment, would only be expected in a case where a drastic change occurred in the medium, such as a change in ph, temperature, aeration, or depletion of the food supply. I

5 PHYSIOLOGICAL STUDIES ON RHIZOBIUM Inasmuch as the temperature, aeration, and ph of the media were controlled it is logical to assume that there was a depletion of food supply at the point of deflection in the rate curve. Furthermore, when the total amounts of oxygen consumed by the organisms in the different concentrations of glucose are computed, as previously described, in percentage of the total amount of oxygen required for complete oxidation of the entire amount of sugar, it is found that the values obtained up to the inflection point in the curve in the different experiments agree rather closely. These values were 32.7, 30.7, and 28.9 per cent, respectively, for the M/180, M/270, and M/540 concentrations of glucose. The oxidation process is apparently stopped when it reaches a certain stage. Obviously this would not be the case if the organisms oxidized the dissimilated glucose completely. The data indicate, however, that the molecules of glucose are oxidized to a certain stage, whereupon the oxidation stops, leaving the residual material as intermediate or end products which are not utilized by the organism. The changes in respiratory quotient that occurred during the experiment substantiate this viewpoint. From the time the organisms were introduced into the various media until the rate of oxidation decreased abruptly, the respiratory quotient was considerably above unity in all cases and ranged from 1.16 to This is shown by the curves in figure 3. Furthermore, it may be noted that there was an abrupt decrease in respiratory quotient from above unity to 0.8 or lower and that this occurred simultaneously with the decrease in rate of oxygen consumption. This may be interpreted as indicating that the organisms were obtaining their energy for growth from the oxidation of glucose in the early part of the experiment. When the dissimilation of glucose had been carried as far as possible under the imposed conditions, it was no longer feasible for the organisms to obtain energy from that source, and as a result oxygen consumption was materially reduced. Furthermore, in order that the organisms 4 A more complete report on the significance of deviations of respiratory quotient from unity during the various growth phases will be presented for publication in the near future. 187

6 R. NEAL AND R. H. WALKER might continue to live, it became necessary to utilize the energy stored in the material of their cells, the proteins and fats. When these materials are burned in the air, their oxygen quotients are approximately 0.8 and 0.71, respectively. Thus, the low respiratory quotients of the organisms in the latter part of the experiment indicate that the organisms were oxidizing the stored proteins and fats for subsistence, and that they were not able to attack the intermediate or end products of the primary glucose oxidation, if there were any other outside the bacterial cell material itself. Undoubtedly, a large proportion of the original glucose not accounted for in the oxygen produced was transformed into cell material, and it is probably this material that was being oxidized, although slowly, in the latter part of the experiment. These results and interpretations are in strict agreement with those of Burk and Horner (1936), who concluded that the extracellular ammonia produced by Azotobacter is, in all probability, entirely derived from decomposition of normal cell nitrogen and not by direct synthesis from free nitrogen. They found that the formation of ammonia by Azotobacter never commenced until the concentration of utilizable organic substrate had reached a very low level of about 0.03 to 0.01 per cent so that the substrate had essentially disappeared. In the absence of a suitable substrate the Azotobacter carried on endogenous respiration by the oxidation of the stored proteins of the cell protoplasm and the excess nitrogen was given off in the form of ammonia as in the ammonification of other proteinaceous materials by microorganisms. Undoubtedly the same process occurred in the present experiments with Rhizobium, although no tests were made for the ammonia produced. The experiments reported here were completed before the appearance of the paper by Burk and Homer (1936). It is evident, therefore, that there was a complete change in the metabolism of the organisms which occurred at a definite time, and this, apparently, was determined by the disappearance of glucose from the substrate. In a second experiment using mannitol as the test substrate, results very similar to those with glucose were obtained. The rate of oxygen consumption, as shown in figure 4, increased rapidly

7 PHYSIOLOGICAL STUDIES ON RHIZOBIUM to a maximum early in the experimental period, then decreased abruptly to approximately the same level as that of the control. In this experiment the respiratory quotient was observed to range from 0.98 to 1.05 in the different media during the period of rapid oxygen consumption.. Simultaneously with the decrease in rate of oxygen consumption there was an abrupt decrease in respiratory quotient to approximately 0.8 or lower, just as was the case when the organisms were grown on a glucose substrate. This TIME (Hours) FIG. 4 FIG. 5 FIG. 4. RATE OF OXYGEN CONSUMPTION BY RHIZOBIUM MELILOTI IN MANNITOL MEDIA FIG. 5. RATE -OF OXYGEN CONSUMPTION BY RHIZOBIUM MELILOTI IN ERYTHRITOL MEDIA again indicates a change of metabolism from one of mannitol dissimilation to one of oxidation of stored proteins and fats. The maximum rate of respiration was apparently a function of the concentration of the material supplying energy and of time. The extent of oxidation of the mannitol, as determined by the total consumption of oxygen, will be discussed later. In erythritol ammonium-chloride media the rate of oxygen consumption by the alfalfa organism as shown in figure 5, again rose 189

8 R. NEMAL AND R. H. WALKER rapidly to a maximum, then decreased rather abruptly. The respiratory quotient decreased abruptly and simultaneously with the decrease in rate of oxygen consumption. The rate of respiration, however, did not drop to approximately the same level as that of the control, as in the previous cases, but continued at a considerably higher level throughout the experiment. Similarly the respiratory quotient did not drop to as low a value in this experiment and after the drop it varied between approximately 0.80 and Apparently, some constituent of the erythritol or some product of the early respiratory activity of the organisms was sufficiently available to permit this continued consumption of oxygen for a considerable period. Rhizobium japonicum The dissimilation of glucose by the soybean organism proceeds in a somewhat different manner. The rates of oxygen consumption by this organism when cultured in media containing 3, 23 and 1 gram per liter of glucose, are shown in figure 6. The rate increased slowly to a maximum considerably below that attained by Rhizobium meliloti, then there was an abrupt change; but the magnitude of the change was not as great as that shown in the experiments with the alfalfa organism. Simultaneously with the change in rate of oxygen consumption there was a decrease in the respiratory quotient; during the period of maximum rate of oxygen consumption the quotient varied from 1.10 to 1.20 in the various media, and when the rate of oxygen consumed decreased, the quotient fell to approximately 1.00 or below, ranging from 0.85 to 0.95 in most cases. In this connection R. japonicum differed somewhat from R. meliloti in that the respiratory quotient in cultures did not drop as low after the carbohydrate dissimilation had ceased. This may be explained, at least partially, by the fact that the soybean organism grew much more slowly on the glucose substrate than did the alfalfa organism, and the rate of oxygen consumption was never as high under similar conditions. The difference in rate of growth may be attributed to the fact that this carbohydrate is less easily attacked by this organism, or perhaps to a difference between the

PHYSIOLOGICAL STUDIES ON RHIZOBIUM 191 - --m-i---i--1 --- Z20 No a. _ s - I No G3lucose zoo 11~ Gluvos" M/540 m.. M/270 ISO ~ N - M/S 0 I c b i c c E 140 two organisms in redox potential requirements.

9 PHYSIOLOGICAL STUDIES ON RHIZOBIUM m-i---i Z20 No a. _ s - I No G3lucose zoo 11~ Gluvos" M/540 m.. M/270 ISO ~ N - M/S 0 I c b i c c E 140 two organisms in redox potential requirements. These two possibilities may be related to the conditions under which these experiments were conducted. It may be possible also that the dissimilation of glucose is brought about in a somewhat different manner under the conditions imposed here, but this hardly seems logical. It is important to note, however, that in spite of the difference in rate of oxygen consumption by the two organisms, there oc- zc- 41- u~~~~~~~~~u.1 _ N _ =-_ ZO ~ X- t48 6It ~ 1 o& j * 6 ' -M(ti ur L51 ckcloz 4 TIME Oi>X") TIME- (hours FIG. 6 FIG. 7 FIG. 6. RATE OF OXYGEN CONSUMPTION BY RHIZOBIUM JAPONICUM IN GLUCOSE MEDIA FIG. 7. RATE OF OXYGEN CONSUMPTION BY RHIZOBIUM JAPONICUM IN ARABINOSE MEDIA curred a similar decrease in rate at a definite stage in the dissimilation process, and that this change in rate was accompanied by a similar change in respiratory quotient. In arabinose media the rates of oxygen consumption by this organism, as shown in figure 7, were very similar to those exhibited by R. meliloti in media containing the various carbon compounds. The maximum rate attained in this experiment was practically the same as that reached by R. meliloti in a glucose

10 R. NEAL AND R. H. WALKER medium. The relatively high rate of oxygen consumption shown by R. japonicum is of particular interest inview of the fact that the soybean organisms are rather commonly considered as growing slowly. It seems that under favorable conditions, the rate of their activity in dissimilating carbonaceous materials might approach or even equal that of the more rapidly growing species of Rhizobium. It is also significant that in this experiment the changes in rates of oxygen consumption and respiratory quotient were very similar to those found in the earlier experiments and were as marked as those observed in experiments with R. meliloti. The extent of oxidation The extent of oxidation or of utilization of the various compounds by both R. meliloti and R. japonicum is shown in table 1. TABLE 1 Percentage oxidation of various carbonaceous materials by rhizobium GRAM ENERGY SOURCE PERR GRAM PER 1GRAM PER ENERGY SOURBCEC IUTER LITER IUTER Rhizobium meliloti: Glucose Mannitol Erythritol Rhizobium japonicum: Glucose Arabinose In the discussion of the first experiment, the agreement between the values obtained for different concentrations of the same carbon compound was pointed out in support of the hypothesis that the abrupt change which occurs in the rate of oxygen consumption is due to a change in the food supply of the organisms. At that point the first stage in the dissimilation process is reached, and it is probable that all of the original carbohydrate as such has been utilized, but that a comparatively large proportion of the carbonaceous material has not yet been completely oxidized to carbon dioxide and water. Neither the mechanism of the dissimilation process nor the

11 PHYSIOLOGICAL STUDIES ON RHIZOBIUM nature of the intermediate and end products remaining in the media have been investigated in this work. At present there is little evidence to permit answering these questions. Anderson, Peterson, and Fred (1928) demonstrated the production of pyruvic acid by two strains of Rhizobium meliloti grown in xylose, glucose, lactose, sucrose, and mannitol media. Fred, Baldwin and McCoy (1932) reported that only a limited growth with the production of a slightly alkaline reaction was secured with certain cultures of Rhizobium on a calcium pyruvate medium. Normally, this compound is thought to be very readily broken down by bacterial action. Georgi and Wilson (1933) concluded that most of the glucose carbon not appearing as carbon dioxide was transformed into gum. Hopkins, Peterson and Fred (1930) found this gum to consist largely of glucuronic acid. In anaerobic cultures of the root nodule bacteria, Virtanen, Nordlund, and Holle (1934) found butyric acid produced as an end product. It seems doubtful, however, whether this type of fermentation would occur in aerobic cultures. It is possible that all, or a part of, the sugar not accounted for by the consumption of oxygen was used in the formation of new cell tissue. This viewpoint is substantiated by the character of the change in respiratory quotient from one of carbohydrate metabolism to one of protein and fat metabolism which occurs simultaneously with the abrupt decrease in rate of oxygen consumption when a definite proportion of total oxygen required for complete oxidation has been consumed. SUMMARY Rhizobium meltiloti and Rhizobium japonicum were cultured in media containing comparatively small quantities of various carbonaceous materials and the oxygen consumed and carbon dioxide produced were measured at regular intervals during a 24-hour period. The rate of oxygen consumption increased consistently until approximately one-third of the theoretical amount required for complete oxidation of the carbohydrate was consumed by the organisms. At that stage in the dissimilation process, there was 193

12 R. NEAL AND R. H. WALKER an abrupt decrease in rate of oxygen consumption and also in the respiratory quotient of the organisms. This was interpreted as a change from carbohydrate metabolism to one of protein or fat metabolism. Although the character of the dissimilation process and the intermediate and end products have not been investigated, it seems possible that a large proportion of the carbohydrate not accounted for by the consumption of oxygen was used in the formation of new cell tissue. Rhizobium japonicum, which is usually looked upon as a slow growing organism, consumed oxygen, or grew, much more rapidly on a substrate containing arabinose than on one containing glucose, and the rate of growth on this medium compared favorably with that of Rhizobium meliloti on substrates containing glucose or mannitol. REFERENCES ANDERSON, J. A., PETERSON, W. H. AND FRED, E. B The production of pyruvic acid by certain nodule bacteria of the Leguminosae. Soil Sci., 25: BuRK, DEAN, AND HORNER, C. KENNETH 1936 The origin and significance of ammonia formed by Azotobacter. Soil Sci., 41: FRED, E. B., BALDWIN, I. L., AND McCoy, E Root nodule bacteria and leguminous plants. Univ. of Wisc. Studies in Sci., No. 5. GEORGI, C. E., AND WILSON, P. W The influence of the tension of oxygen on the respiration of rhizobia. Arch. Mikrobiol., 4: HOPKINS, E. W. PETERSON, W. H., AND FRED, E. B Composition of the gum produced by root nodule bacteria. Jour. Amer. Chem. Soc., 52: NEAL, 0. R., AND WALKER, R. H Physiological studies on Rhizobium. IV. Utilization of carbonaceous materials. Jour. Bact., 30: VIRTANEN, A. J., NORDLUND, M., AND HOLLO, E Fermentation of sugar by the root nodule bacteria. Biochem. Jour., 28: WALKER, R. H., ANDERSON, D. A., AND BROWN, P. E Physiological studies on Rhizobium. I. The effect of nitrogen source on oxygen consumption by Rhizobium leguminosarum Frank. Soil Sci., 37: WARBURG, Stoffwechsel der Tumoren. Julius Springer, Berlin.

volume and surface area. Walker and Winslow (1932) reported metabolic rates per cell being observed towards the end of the

volume and surface area. Walker and Winslow (1932) reported metabolic rates per cell being observed towards the end of the A COMPARISON OF THE METABOLIC ACTIVITIES OF AEROBACTER AEROGENES, EBERTHELLA TYPHI AND ESCHERICHIA COLI C. E. CLIFTON Department of Bacteriology and Experimental Pathology, Stanford University, California