weight, cell volume, and cell number. BACTER, RHIZOBIUM, AND SACCHAROMYCES'

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1 PHYSICAL CHARACTERISTICS OF CELLS OF AZOTO- BACTER, RHIZOBIUM, AND SACCHAROMYCES' HANS LINEWEAVER Bureau of Chemistry and Soils, U. S. Department of Agriculture, Washington, D. C. Received for publication October 25, 1937 Recent investigations concerning the mechanism of nitrogen fixation by Azotobacter have been concerned with kinetics (Burk, 1934; Endres, 1934a; Lineweaver, Burk and Deming, 1934), essential elements for fixation as distinguished from growth (Burk, 1934; Burk and Horner, 1935b), chemical intermediates and products of fixation (Burk and Horner, 1935a and c; Endres, 1934b and 1935; Roberg 1935; Winogradsky, 1932), and fixation in vitro (Bach, Yermolieva and Stepanian, 1934; Roberg, 1936). The present communication reports general physical characteristics of Azotobacter, and for comparison, of Rhizobium and Saccharomyces obtained during direct examination of the enzymic mechanism of nitrogen fixation previously proposed on the basis of kinetic data (Lineweaver, 1938; Lineweaver, Burk and Deming, 1934). The physical characteristics of the cells determined were the density, water content, and relation between dry weight, cell volume, and cell number. MATERIALS AND METHODS Azotobacter cells. A. vinelandii, A. chroococcum (Burk strain B8), and A. beijerinckii (Burk strain B6) were grown in large scale amounts using methods employed by members of this laboratory working with Dr. G. E. Hilbert. The bacteria were grown at 31'C. in pyrex bottles containing 17 liters of culture medium with 1 or 2 per cent sucrose. The bottles were inoculated with 100 cc. of a 2- to 4-day heavy growth of Azotobacter. The cul- 1 These investigations were carried out in the Biochemical Nitrogen Fixation Section of the Fertilizer Research Division. 501

2 502 HANS LINEWEAVER tures were aerated with air for 1 to 2 days and then with 1: 1 oxygen-nitrogen mixture for 1 to 3 days more. The bacteria were harvested by centrifugation in a Sharples super-centrifuge, the 17 liters of culture yielding from 40 to 90 grams wet weight of bacteria. For study, the centrifuged cells were re-suspended in culture medium, generally containing 1 per cent sucrose, and consisting of the clear liquid obtained after the following mixture had been thoroughly shaken, allowed to stand, and settle: 0.8 gram of K2HPO4, 0.2 gram of KH2PO4, 0.2 gram of NaCl, 0.2 gram of MgSO4c7H20, 0.1 gram of CaSO4c2H20, 1000 grams of HO. Fe as iron humate, about 0.5 mgm. per liter, and Mo as Na2MoO4, about 0.1 mgm. per liter, were generally added when the suspension was made. Heat-inactivated cells were prepared by rapidly heating a suspension to 900C., and, after 5 to 15 minutes, cooling rapidly to 250C..Rhizobium meliloti, alfalfa strain 131, University of Wisconsin, was grown by the same technique used for Azotobacter and suspended in medium containing 0.2 per cent sucrose. Baker's yeast cells were obtained from Fleishmann's Yeast Company in commercial starch-free, one pound cakes, and were suspended in medium or in distilled water. The per cent total solids by weight, was determined by weighing samples before and after drying to constant weight at 1100C. The error was estimated to be about :4:0.15 weight per cent. The densities of the suspensions and centrifugates were determined by weighing in a flask ( cc. at 250C.) that had a flat ground glass lip for sealing with a ground glass plate. Duplicate determinations generally agreed to within gram per cubic centimeter. The cell volumes of the suspensions were determined as illustrated in figure 1 (cf. cell volume of blood, Peters and Van Slyke, 1932, p. 73). The cell suspensions, followed by bromobenzene, were drawn by suction into the lower opening of the tube. The rubber seal, cut from rubber tubing, is adequate since the pressure on the outside of the seal is greater than that on the inside due to the CaCl2 solution. The samples were centrifuged repeatedly at

3 CELLS OF AZOTOBACTER AND RHIZOBIUM 503 about 3500 r.p.m. and the per cent volumes read at intervals until they were found to be essentially constant; the total time required was of the order of 30 to 60 minutes. The dry weight was determined by drying once-washed centrifuged cells to constant weight at 110'C. The error was estimated to be from 43 to :i:5 per cent. The count, made microscopically, was accurate to i10 per cent. _ Cork _-Centrifugate - l -rsaturated CaClp -.Cells Bromobenzene Rubber Seal Rubber Seat FIG. 1 CALCULATIONS The water content and total solids by volume of the various liquids are given by the equations: Water content (grams per cc.) = Density X per cent water by weight *. 100 Total solids (grams per cc.) = Density X per cent total solids by weight.-100 The density of the cells and the water content of the cells are given by the following equations: D, = [D. - Db(1 - c.v.)]/c.v., and Water content of cells (grams per cc.) = D- Dry weight (grams per cc.) *. c.v. JOURNAL OF BACTERIOLOGY, VOL. 35, NO. 5

4 504 HANS LINEWEAVER where Dc, D. and Db are the densities of the cells, suspension and centrifugate respectively, and c.v. is the cell volume in cubic centimeter per cubic centimeter of suspension. RESULTS AND DISCUSSION Although an approximately constant relation between cell volume and dry weight per cubic centimeter for any one organism was observed (table 1), there was some slight variation, as indicated in figure 2. This may be attributed to a number of varying factors, such as size of inoculum, rate of aeration, and duration of growth, that are always encountered when growing microorganisms. The water contents for the cells, reported in table 2, agree with data for various bacteria and yeasts obtained by other investigators upon drying centrifuged cells at 100 to 1100C. (Buchanan and Fulmer, 1928, p. 68). The water content of bacteria generally lies between 75 and 85 per cent (Buchanan and Fulmer, 1928, table VI). The values found for A. vinelandii are 80, 78 and 74, for A. chroococcum 85, for A. beijerinckii 86 and for R. meliloti 68 per cent. The two values for the yeast, 79 and 80 per cent, although slightly higher than the average value reported in the literature, 73 per cent, fall well within the wide limits, 68 and 83 per cent. The density and water content of one species of Azotobacter, A. vinelandii, were observed to vary between comparatively narrow limits, to 1.106, and 74 to 80 per cent. The other two species yielded the values 1.036, 1.04 and 85 and 86 per cent, which appear to be significantly different from those of the first. The explanation for this difference may depend upon the wellknown fact that different species produce different amounts of gum-like material, which may intimately surround the cells. Such material would almost certainly have both a different and more variable density and water content than the more opaque cell material. The absolute accuracy of both the density and water content values, as well as the volume per cell and cell diameter values, depend on the accuracy of the observed centrifugal cell volume, which, in turn, depends on the extent of packing that takes

5 CELLS OF AZOTOBACTER AND RHIZOBIUM TABLE 1 Dry weight, cell volume, total solids, density, and water content of experimental material at 250C. MATERIAL AND RECORD NUMBER Medium Medium (1 per cent sucrose) Azotobactert vin. 60 vin. 64 vin. 68 chr. 68 beij. 68 vin. 46 vin. 38 vin. 24-1t vin. 24-2t vin. 24-3t Rhizobium Yeast** DRY WEIGHT PER CENT CELL VOLUME gram cc. per cc. per cc. X t: (2.3)tt (4.5)tt (9.0)tt PER CENT TOTAL SOLIDS grams per gram X 100 (0.18) DENSITY H20 Liquid Liud Centrif- CONTENT OF LIQUID ugate grams grams gram. per cc. per cc. per cc * 0.966* 0.992* 0.989* 0.981* * 0.947* 0.899* * 0.930* 0.865* 43 per cent.-505 * Water content (46f) (c.v.) (1 -c.v.) = (c.v.); water content (38 and 24) = (c.v.); water content (42 and 43) = (c.v.); where c.v. is the cell volume in cubic centimeter per cubic centimeter. t The diluent was medium (1 per cent sucrose), exception see footnote 1. X The diluent was medium (no sucrose). The diluent was medium (0.2 per cent sucrose). ** The diluent was H20 except for the 42 series where medium was used. tt The cell volumes of these heat-inactivated cells were calculated by the equation: (c.v.) = 435 (dry weight). tt Somewhat less accurate than other figures,

6 506 HANS LINEWEAVER place. If the cells are assumed to be rigid spheres packed horizontally, one sphere directly upon another, per cent of the total volume would be filled with water and similarly if the spheres were packed obliquely per cent of the space would be filled with water. The cells, however, are seldom exact 0 2 2C x Q 10 L0 =60 0 0) E 80 ~-60,o o0 Yeast Yeast/ Slope = 412 I I,, I I I I I I MZ.16.2 Dry Weight mg. per cc. FIG. 2. THE RELATION BETWEEN CELL VOLUME, DETERMINED BY CENTRIFUGATION, AND DRY WEIGHT OF AZOTOBACTER, LEGUME BACTERIA AND YEAST CELLS spheres and almost certainly are not rigid so that we would not expect a 25 per cent error in the water content figures on this account. Some water will, of course, always be held in the interstices by capillarity, and be a source of error. Another picture of the situation may be obtained from the observation that the volumes per cell calculated from the microscopically estimated

7 CELLS OF AZOTOBACTER AND RHIZOBIUM 507 cell diameters were about 50 per cent of the volume per cell obtained by dividing the observed per cent cell volume by the count per 100 cc. (table 2, column 5). These diameters, however, were only about 30 per cent less than those calculated from the observed per cent cell volume (table 2). This order of agreement while not entirely satisfactory, is about as good as can be expected TABLE 2 Size, density and water content of Azotobacter, Rhizobium and yeast cells at 260C. ORGANISM Azotobacter vin vin vin. 68 chr. 68t beij. 68t vin. 60 vin. 64 Rh. meliloti Yeast (commercial) * COUNT millions per cc. 10,000 3,200 30,000 3,000 10,000 33,600 12,000 DRY WEIGHT gram per cc (57) PER CENT CELL VOL- UME cc. per cc. X VOL- UME PER CELL c.mm. X 10' (57) CALCU- DENSITY LATED 0I DlAME- CEOPS TER EL mm. X * grams per cc , Corresponding ellipsoidal diameters, ratio 1.5 to 1, are 3.4, 2.2. WATER CONTENT OF CELLS gram per nt per cc. bi t These strains contain from 2 to 3 per cent nitrogen while the A. vinelandii, under these culture conditions contain 12 to 18 per cent nitrogen. without detailed microscopic work involving shape and size of the cells, both stained and unstained, as well as counts. SUMMARY The relations between dry weight, cell volume, and count per cubic centimeter have been determined for three species of

8 508 HANS LINEWEAVER Azotobacter, for Rhizobium meliloti and for baker's yeast. The densities and water contents of the various cells determined were: Azotobacter vinelandii cells 1.09 and 0.84 gram per cubic centimeter (average); Azotobacter chroococcum 1.04 and 0.87 gram per cubic centimeter; Azotobacter beijerinckii 1.04 and 0.88 gram per cubic centimeter; Rhizobium meliloti 1.10 and 0.75 gram per cubic centimeter; and Saccharomyces and 0.85 gram per cubic centimeter. The writer is much indebted to Dr. Dean Burk for his encouragement and suggestions during the pursuit of this work. He is also indebted to Professor J. C. W. Frazer of Johns Hopkins University for suggestions and criticisms. The valuable criticisms offered by Drs. F. E. Allison and C. A. Ludwig as well as Dr. Burk in the preparation of the manuscript are greatly appreciated. REFERENCES BACH, A. N., YERMOLIEVA, Z. V., AND STEPANIAN, M. P Fixation de l'azote atmosph6rique par l'interm6diaire d'enzymes extraites de cultures d'azotobacter. Compt. rend. Acad. Sci. U. S. S. R., 1, 1, BUCHANAN, R. E., AND FULMER, E. I Physiology and Biochemistry of Bacteria. Baltimore, Vol. I. BURK, D Azotase and Nitrogenase in Azotobacter. Ergebnisse der Enzymeforschung (Nord and Weidenhagen), Leipzig, III, BURK, D., AND HORNER, C. K. 1935a The production of ammonia by Azotobacter and its relation to the mechanism of nitrogen fixation. Trans. A, Third Intern. Cong. Soil Sci., 1, BURK, D., AND HORNER, C. K. 1935b The specific catalytic r6le of molybdenum and vanadium in nitrogen fixation and amide utilization by azotobacter. Ibid., 1, BURK, D., AND HORNER, C. K. 1935c tvber Hydroxylamin, Hydrazin und Amide als Intermediarprodukte bei der N2-Fixation durch Azotobacter. Naturwiss., 23, ENDRES, G. 1934a Zur Kenntnis der stickstoffassimilierenden Bakterien I. Ann. Chem. I, 512, ENDRES, G. 1934b tber ein Zwischenprodukt der N2-Assimilation. Naturwiss., 22, 662. ENDRES, G Zur Kenntnis der stickstoffassimilierenden Bakterien II. tmber die Bindung des Luftstickstoffes durch Azotobacter. Ann. Chem. II, 518,

9 CELLS OF AZOTOBACTER AND RHIZOBIUM 509 LINEWEAVER, H The Solubility and Chemical and Physical Absorption of Nitrogen Gas in Azotobacter Cells. Jour. Biol. Chem., 122, LINEWEAVER, H., BURK, D., AND DEMING, W. E The Dissociation Constant of Nitrogen-Nitrogenase in Azotobacter. Jour. Amer. Chem. Soc., 56, PETERS, J. P., AND VAN SLYKE, D. D Quantitative Clinical Chemistry. Baltimore, 2, ROBERG, M Beitrage zur Biologie von Azotobacter. II. Der Stickstoffgehalt der Filtrate von Azotobakterkulturen. Jahrb. f. Wiss. Bot., 82, ROBERG, M Beitrage zur Biologie von Azotobacter. III. Zur Frage eines ausserhalb der Zelle den Stickstoff bindenden Enzyms. Jahrb. f. Wiss. Bot., 83, WINOGRADSKY, S Sur la synthese de l'ammoniaque par les Azotobacters du sol. Ann. Inst. Pasteur, 48, Downloaded from on January 23, 2019 by guest