Large-Scale Production of the Azotobacter for Enzyme-s

Transcription

1 PRODUCTION OF AZOTOBACTER FOR ENZYMES 135 using the artificial rumen technique. J. Animal Sci., 10, HARTLEY, P The value of Douglass medium for the preparation of diphtheria toxin. J. Pathol. Bacteriol., 25, HOFLUND, S., QUIN, J. J., AND CLARK, R The influence of different factors on the rate of cellulose digestion (a) in the rumen and (b) in the ruminal ingesta as studied in vitro. Onderstepoort J. Vet. Sci. Animal Ind., 23, HUFFMAN, C. F Ruminant nutrition. Ann. Rev. Biochem., 22, HUNGATE, R. E The anaerobic mesophilic cellulolytic bacteria. Bacteriol. Revs., 14, MAcLEOD, R. A Further mineral requirements of Streptococcus faecalis. J. Bacteriol., 62, MARSTON, H. R Fermentation of cellulose in vitro by organisms from the rumen of sheep. Biochem. J., 42, PEARSON, R. M., AND SMITH, J. A. B The utilization of urea in the bovine rumen. 3. The synthesis and breakdown of protein in rumen ingesta. Biochem. J., 37, VILES, F. J., AND SILVERMAN, L Determination of starch and cellulose with anthrone. Anal. Chem., 21, WILLIAMS, V. J., AND MOIR, R. J Ruminal flora studies in the sheep. III. The influence of different sources of nitrogen upon nitrogen retention and upon the total number of free microorganisms in the rumen. Australian J. Sci. Research Ser. B4, Large-Scale Production of the Azotobacter for Enzyme-s MARTIN ALEXANDER AND P. Department of Bacteriology, Lee and Burris (1943) obtained sufficient azotobacter cells, using a 200-gallon fermentor, to demonstrate the presence of several enzymes in cell-free preparations. Research of the past decade has increased considerably our knowledge of how to prepare bacterial enzymes and how specific enzymes vary with experimental conditions and with physiological age of the organism. It is evident that enzymes of these earlier preparations from physiologically old cells possessed low specific activities. Because of the usefulness of the azotobacter for examination of many aspects of. bacterial respiration (Stone and Wilson, 1952), the large-scale production of the organism was re-examined in detail with the emphasis on the yield of cells with high enzymatic activity (end of the logarithmic phase of growth) rather than the total crop. Although the particular solution obtained in many of the problems applies only to the azotobacter, the method of study illustrates the type of investigation that should be made with any organism to be used for extensive investigations of bacterial enzymes. Consequently, it is significant for this general problem in bacterial physiology. EXPERIMENTAL METHODS Azotobacter vinelandii strain 0 was grown at 30 C in Burk's N-free liquid medium (unless otherwise specified) prepared according to Wilson and Knight (1952, 1 Supported in part by grants from the Atomic Energy Commission, the Rockefeller Foundation, and the Research Committee of the Graduate School from funds provided by the Wisconsin Alumni Research Foundation. W. WILSON University of Wisconsin, Madison, Wisconsin Received for publication December 14, 1953 p. 53). Shake flask experiments were made with 25 m of medium in 500-ml Erlenmeyer flasks incubated on a 400-rpm rotary shaker.2 Aerated cultures were grown in 9-liter bottles essentially as described by Wilson and Knight (1952, p. 42). Humidified air sterilized by passage through sterile glass wool was supplied at a rate of 12 liters per minute, being dispersed by two gas dispersion tubes (Corning extra coarse) with fritted cylinders (maximum pore size of ,u), a total sparging surface of 2.7 square inches per bottle. The 200-gallon copper pilot plant fermentor used was described by Lee and Burris (1943); it contained 300 L of the basal medium, the solution of iron and molybdenum salts being sterilized separately by filtration. Thirty-six L of a 24-hour culture grown in six 9-liter serum bottles was the inoculum. The air supplied from a 30-lb line was dispersed through four cylindrical Aloxite diffuser tubes, 40,3 at the bottom of the tank. Each sparger had an outside -diameter of 1j% in and an over-all length of 9 in, a total 198 square inches of sparging surface in the fermentor. A secondary air line from a motor-driven blower supplying nonsterile air (Connersville Blower %35,4 0.1 cu ft per rev) was used late in the fermentation when the oxygen requirement was the greatest, and the possibility of overgrowth by contaminants was the least. Bottle and pilot plant cultures were routinely harvested in a refrigerated 2 New Brunswick Scientific Company, New Brunswick, N. S.- 3 The Carborundum Company, Perth Amboy, N. J. 4 Roots-Connersville Blower Corp., Connersville, Indiana.

2 136 MARTIN ALEXANDER AND P. W. WILSON Sharples supercentrifuge, although for rapid harvesting of the entire 300 L, a Westphalia yeast separator was more useful. For studies requiring extremely high rates of effective aeration, 1.5 L of medium (6 per cent sucrose) plus 10 per cent inoculum was added to the 3.5-L fermentor fitted with a variable speed agitator described by Olson and Johnson (1949). The agitator was operated at 1850 rpm, and air saturated with water was supplied at a rate of 6.0 liters per minute providing an effective aeration (Cooper, Fernstrom, and Miller, 1944) of 10 mmol oxygen per liter per minute. Nitrogen was determined by the Kjeldahl procedure, sucrose, by a modification of the method of Folin and Wu (1920), and turbidities expressed as K-S units were measured in a Klett-Summerson colorimeter using a 660-m,u filter. Dry weights were estimated by centrifuging the cells in 40 ml of culture medium and drying in a tared tube to constant weight in a vacuum desiccator containing sulfuric acid. RESULTS Small-Scale Fermentations Factors limiting the total yield of cells as well as causing the early break in the logarithmic phase of growth were investigated by varying the concentration of each constituent. No effect was noted by increasing the concentration of the potassium, magnesium, calcium, iron, molybdenum, phosphate, or sulfate ions over that in the basal medium. Several mono- and disaccharides substituted for sucrose gave no better yields. Increase in the sucrose concentration from zero to about 3 per cent resulted in an increase in yield (as measured by nitrogen fixed) directly proportional to TABLE 1. Effect of sucrose concentration on the yield and growth rate of Azotobacter vinelandii* SUCROSE IN MEDIUM k VALUEt FINAL YIELD (72 HR) per cent ug N/ml 0 _ * Grown in 500-ml shake flasks. t The velocity constant of growth in the logarithmic phase, the k value, is defined by the equation: k = 2.3 lo.3final growth time in hours initial growth In these experiments the k value has been evaluated turbidimetrically. the sugar concentration with an over-all fixation efficiency of 12.6 /hg N per mg sucrose. With 3 to 7 per cent sucrose, the yield of cells increased although the amount of N2 fixed was no longer directly proportional to the sugar concentration; sugar concentrations greater than 7 per cent had no effect on yield (table 1). The greatest quantity of nitrogen fixed resulting from an increase in the sugar supply was 670 gg N per ml, a value more than 2.5 times that obtained in the basal medium (2 per cent sucrose). The high yields did not result from some stimulatory substance in the commercial sucrose since the yield could be duplicated using CP sucrose. The increase in total fixation with greater sugar concentrations was accompanied by a dlecrease in the growth rate, undoubtedly arising from the higher osmotic pressures; the velocity constant of growth k (Wilson and Knight, 1952) decreased from with 1 to 3 per cent sucrose to with 6 per cent. The inability of the organism to utilize all the available carbohydrate indicates that some nutrient has become deficient for optimum growth. Increased conicentrations of all minerals known to be required by the azotobacter (potassium, phosphorus, magnesium, sulfur, calcium, ferric iron, and molybdenum) and also elements possibly needed in trace amounts (zinc, copper, manganese, boron, and cobalt) were individually incorporated into the basal medium modified to contain excess sugar (8 per cent). Of the substances tested, iron alone exerted a stimulatory effect on the total N2 fixed. Maximum fixation was obtained using 8 ppm iron as FeCl3. After applying corrections for evaporation, the amount of N2 assimilated after 72 hours in this modified medium was 1,050 Mg N per ml, a yield four times that obtained in the basal medium. This fixation is, as far as we know, considerably higher than any reported previously in the literature. The efficiency of fixation was 13.1 jig N per mg sucrose. Another variable that markedly influenced the yield of azotobacter was aeration or, more precisely, oxygenation since the demand of these organisms for N2 is very small compared to that for O2. Altering the volume of medium in the shake flask varied the effective aeration, TABLE 2. Relationship of effective aeration to yield of cells of Azotobacter vinelandii* MMOL 02/L/MIN K-St wgn/mlt * The medium initially contained 4 per cent sucrose. Grown in shake flasks. t Klett-Summerson units: end of logarithmic phase of growth. t Final harvest (72 hr).

3 TABLE 3. Growth of Azotobacter vinelandii under conditions of high aeration* PRODUCTION OF AZOTOBACTER FOR ENZYMES HOURS AG N/ML C02 IN EFFLUENT GASt 137 per cent * Grown in 3.5-L stainless steel fermentors. Effective aeration rate, 10 mmol 02/L/min. t Determined by method of Maxon and Johnson (1952). that is, the rate at which oxygen dissolves in the medium, as measured by the sulfite oxidation method (Cooper, Fernstrom, and Miller, 1944). Increase in effective aeration not only increased the total yield of cells but also gave a greater yield of bacteria in the active, logarithmic phase of growth (table 2). The effect of 02 supply was investigated further by employing the 3.5-L stainless steel fermentor designed to give high rates of aeration. The major effect is prolongation of the period of logarithmic growth. At the peak of the logarithmic phase, about 550,ug N2 were fixed per milliliter of medium (table 3). Thus, by increasing the O2 supply, the yield of cells in the exponential phase of growth has been increased to a level more than three times that obtained in the large-scale fermentations (reported in the next section). At the time of harvest, and before the maximum fixation had been attained, 710,ug nitrogen were found per ml of medium in a fermentation time of 10 hours; the yield of dry cells was 6.33 g per L. In the ninth hour alone, 175 Ag N were fixed per ml, a value 70 per cent of the total fixation previously obtained in the complete culture cycle of the organism in the basal medium. The efficiency of fixation was 13.4,ug N per mg sucrose and the efficiency of conversion of sugar to cells was 12 per cent. Large-Scale Fermentations The studies described in the preceding section were designed to determine maximum yields under optimum conditions, but the scale was small. The next stage was to determine how close these yields could be approached using available apparatus sufficiently large-scale to produce enough cells for routine use in preparation of cell-free enzymes. A typical growth curve of a culture grown in aerated 9-liter bottles (figure 1) indicates that the 02 supply soon becomes a limiting factor, but even so, satisfactory yields of active cells can be obtained. In various experiments, the yield of cell paste from the Sharples centrifuge varied from 5.8 to 12.0 grams per liter, depending upon the age of the culture. Similar studies with Azotobacter agile 4.4, an organism with a more rapid growth rate in both flasks and in bottles and HOURS FIG. 1. Growth curve of Azotobacter vinelandii in aerated bottle culture. (6 liters medium in 9-L bottle). which produces large amounts of gum, gave yields of cell paste varying from 6.8 to 19.6 g per L, depending upon the age at harvest. The great demand for cells, especially of young organisms, required for the study and purification of the enzymes extracted from these bacteria led to the pilot plant culture of azotobacter. Although higher growth rates were obtained using ammonium rather than molecular nitrogen, the latter source was used since this laboratory is primarily concerned with the study of the N2-fixing enzyme system. Typical data obtained from pilot plant culture of A. vinelandii are shown in figure 2 and table 4. Yields of cells at the peak of the logarithmic phase were as much as 5.1 g cell paste per liter of medium. In the experiment illustrated in figure 2, the blower was turned on at 14 hours; more than 160 jig nitrogen were fixed per ml before the end of the logarithmic phase. In a similar experiment made, however, without the benefit of increased aeration from the blower, the exponential phase ended at a level of fixation of about 100 /Aog N per ml, re-emphasizing the role of oxygen for the maintenance of active growth for this organism. At the 20-hour harvest in the experiment cited, the

4 138 MARTIN ALEXANDER AND P. W. WILSON FIG. 2. Growth curve of Azotobacter vinelandii in pilot plant. (300 L medium in 200-gallon copper fermentor). total yield was 1,530 g of a young active cell paste (68 per cent moisture) or 492 g of dry cells. In the process 3,060 g of sucrose were consumed with a net fixation of 51.0 g of N2, an over-all efficiency of fixation of g N per mg sugar. The efficiency of the conversion of sucrose to cells was 14.1 per cent. Two additional observations made on these cells appear to be noteworthy: a) the unusually long period of lag (figure 2) possibly arising from the presence of copper; and b) the fat content of the cells, so high that a definite layer separated during centrifugation when preparing the enzymes. TABLE 4. Pilot plant culture of Azotobacter vinelandii* HOURS K-St jsg N/ML SUCROSE/MGL CMGDRY _ t * Grown in 200-gallon copper pilot plant fermentor. t Klett-Summerson units. t Auxiliary blower turned on at 14 hours. Preparation of Enzymes Azotobacter vinelandii offers many advantages for enzyme production: it is easily grown in a simple medium using molasses, a cheap carbohydrate source, and requiring the addition of neither growth factors nor fixed nitrogen (Lee and Burris, 1943). The enzymes of this bacterium are of special interest because of the organism's very high respiration rate, its almost complete oxidation of substrate, and its ability to utilize molecular nitrogen. The enzymes can, as a rule, be readily prepared cell-free by any one of many methods of grinding or by disruption in a sonic oscillator. Frozen cells may be stored for extended periods; for example, cells obtained from the pilot plant fermentor were stored at -10 C until they were used. Enzymatic activity of such organisms remained essentially unchanged for a period of several months. With A. agile, however, a rapid loss of activity occurred in frozen material, so that all cells that could be conveniently handled at one time could be supplied from bottle cultures, and no pilot plant experiments were made. Extracts, made usually in a refrigerated 10-kc Raytheon magnetorestrictive oscillator (Wilson and Knight, 1952) of cells grown in 9-L bottles or in the 200-gallon fermentor have been prepared, and the following enzymes and enzyme systems have been studied by various workers in this laboratory: A system catalyzing the individual steps as well as the complete tricarboxylic acid cycle (Stone and Wilson, 1952; Repaske and Wilson, 1953), a-ketoglutaric oxidase (Lindstrom, 1953), hydrogenase (Green and Wilson, 1953), a soluble succinoxidase (Repaske, Wilson, and Mahler, 1953), and the system responsible for fixation of N2 (Hamilton et al., 1953). In addition, as yet unpublished studies are under way dealing with fumarase (R. M. Bock an(d R. A. Alberty), the cytochrome system (T. G. G. Wilson), and the system concerned with preliminary breakdown of carbohydrates (L. E. Mortenson). DISCUSSION The chief conclusion from this investigation of production of azotobacter cells for preparation of enzymes is that the yield of suitable cells will, under ordinary circumstances, be a function of the supply of oxygen. Under the conditions used in these experiments the major factor limiting prolongation of the logarithmic period (at which stage the cells possess the highest specific activity of the enzymes that we have been studying) depends almost solely on the effectiveness of aeration. The importance of oxygen supply to nitrogen fixation has been previously commented on (Wilson and Burris, 1947), but its significance for maximum production of specific enzyme systems has been appreciated only qualitatively. The experiments described

5 PRODUCTION OF AZOTOBACTER FOR ENZYMES 139 here provide data on aeration requirements which must be provided for in the design of apparatus for largescale production of this organism if future needs exceed the present one. It is clear that with the large-scale equipment used in this study only a fraction of the possible yield of suitable cells was obtained. Study of the growth curves together with estimation of the effective aeration made it evident that the limiting factor was ability to supply the cells with sufficient oxygen. The laboratory studies with the fermentor designed to overcome this handicap (Olson and Johnson, 1949) suggest that with apparatus already designed for plant-scale operation, for example, growth of yeast (Inskeep et al., 1951), greatly increased yields of suitable azotobacter cells would be obtained. A second point of interest is that once the limitation of oxygen supply is met, the medium previously believed to be optimum for growth is deficient. First, the energy supply must be greatly increased, and when this is done, the supply of minerals, initially iron, becomes inadequate. When aeration is adequate, Burk's medium should be modified to contain at least 6 per cent sucrose and 8 ppm Fe++. With a technique of continuous feeding so that osmotic effects would not limit the maximum concentration of nutrients, it is likely that both the over-all yield as well as the total nitrogen fixed could be increased to a level even greater than those reported in this study (which are among the highest, if not the highest, in the literature). Finally, a calculation of some theoretical interest for the mechanism of nitrogen fixation can be made from the data of the run in the 3.5-L fennentor. Between the 8th and 9th hours the effluent gas contained approximately 3 per cent C02; since air was being supplied at the rate of 6.0 L per min, this corresponds to a rate of CO2 production of 10.8 L per hr. Assuming a mole for mole conversion of the O2 consumed by the organism to the CO2 evolved, a good approximation in the azotobacter (Fife, 1943a, b; Lineweaver, 1933), the 02 consumed by the cell in respiration is 10.8 L at 30 C or 0.43 moles O2. In the same time interval, 175,ug nitrogen were fixed per ml at 30 C or a total of moles N2 in the fermentor. Thus the ratio of moles N2 fixed per mole O2 consumed is 0.022, a value comparable to those obtained in manometric trials by Meyerhof and Burk (1928). SUMMARY The total quantity of molecular nitrogen fixed by Azotobacter vinelandii is proportional to the concentration of sugar in the medium with an over-all fixation efficiency of about 12.6,g N2 assimilated per mg sucrose. At high carbohydrate levels the total yield is apparently independent of the sucrose concentration, but this effect arises from a deficiency of iron. The yield of young cells which are the ones of chief interest for preparation of many enzymes is primarily dependent upon effective aeration. With high rates of aeration in a 3.5-L fermentor, a yield of 6.3 g dry weight of young cells per liter of medium was reached. In an investigation of growing the cells for production of cell-free enzymes, comparatively large-scale cultures were grown either in 9-L bottles or in a pilot plant fermentor with 300 L of medium. Although the yields, expressed as jug N per ml, in these large-scale experiments were much lower than those obtained in the laboratory under more optimal conditions (1,050 Ag N per ml), they were satisfactory for present requirements. The total yield was as much as 1500 grams of active cell paste in a single fernentation. Cells harvested from these cultures could be stored easily and possessed high enzymatic activity. Physiologically active extracts of such cells were prepared with little difficulty, and several of these cell-free enzymes have been studied and partially purified. REFERENCES COOPER, C. M., FERNSTROM, G. A., AND MILLER, S. A Performance of agitated gas-liquid contactors. Ind. Eng. Chem., 36, FIFE, J. M. 1943a An apparatus for studying respiration of Azotobacter in relation to the energy involved in nitrogen fixation and assimilation. J. Agr. Research, 66, FIFE, J. M. 1943b The effect of different oxygen concentrations on the rate of respiration of Azotobacter in relation to the energy involved in nitrogen fixation and assimilation. J. Agr. Research, 66, FOLIN, O., AND WU, H A simplified and improved method for determination of sugar. J. Biol. Chem., 41, GREEN, MARGARET, AND WILSON, P. W Hydrogenase and nitrogenase in azotobacter. J. Bacteriol., 65, HAMILTON, P. B., MAGEE, W. E., AND MORTENSON, L. E Nitrogen fixation by Aerobacter aerogenes and cellfree extracts of the Azotobacter vinelandii. Soc. Am. Bacteriologists, Bacteriol. Proc., August 10 to August 14, p. 82. INSKEEP, G. C., WILEY, A. J., HOLDERBERRY, J. M., AND HUGHES, L. P Food yeast from sulfite liquor. Ind. Eng. Chem., 43, LEE, S. B., AND BURRIS, R. H Large-scale production of azotobacter. Ind. Eng. Chem., 35, LINDSTROM, E. S The a-ketoglutaric oxidase of azotobacter. J. Bacteriol., 65, LINEWEAVER, H Characteristics of oxidation by Azotobacter. J. Biol. Chem., 99, MAXON, W. D., AND JOHNSON, M. J Continuous photometric determination of carbon dioxide in gas streams. Anal. Chem., 24, MEYERHOF, O., AND BURK, D tber die Fixation des Luftstickstoffs durch Azotobakter. Z. physik. Chem., A, 139, OLSON, B. H., AND JOHNSON, M. J Factors producing high yeast yields in synthetic media. J. Bacteriol., 57,

6 140 H. PIVNICK, W. E. ENGELHARD AND T. L. THOMPSON REPASKE, R., AND WILSON, P. W Oxidation of intermediates of the tricarboxylic acid cycle by extracts of Azotobacter agile. Proc. Natl. Acad. Sci. U. S., 39, REPASKE, R., WILSON, T. G. G., AND MAHLER, H. R Soluble succinoxidase from azotobacter. Soc. Am. Bacteriologists, Bacteriol. Proc., August 10 to August 14, p STONE, R. W., AND WILSON, P. W Respiratory activity of cell-free extracts from azotobacter. J. Bacteriol., 63, WILSON, P. W., AND BURRIS, R. H The mechanism of biological nitrogen fixation. Bacteriol. Revs., 11, WILSON, P. W., AND KNIGHT, S. G Experiments in Bacterial Physiology. Burgess Publishing Co., Minneapolis. The Growth of Pathogenic Bacteria in Soluble Oil Emulsions HILLIARD PIVNICK, W. E. ENGELHARD, AND T. L. THOMPSON Department of Bacteriology, University of Nebraska, Lincoln, Nebraska Mineral oils, when emulsified with materials such as soaps of petroleum sulfonates, fatty acids, abietic acid or resin, are referred to as soluble oils. These oils, mixed with water, form stable emulsions which are universally employed as coolants and lubricants for drilling, cutting, and grinding of metals. In addition to the emulsifying agent, soluble oils may also contain fatty oils, disinfectants and emulsion stabilizers. The oils supplied by the manufacturer are sterile, but when mixed with water in the machine shop they support microbial growth (Duffet et al., 1943; Fabian and Pivnick, 1953; Lee and Chandler, 1941; Page and Bushnell, 1921). In fact, contaminants from soil, floor sweepings, air, river water, and feces grow readily in these emulsions (Fabian and Pivnick, 1953). The C. B. Dolge Company (undated pamphlet) has reported that feces and other body discharges are found in soluble oil emulsions. Duffet et al. (1943), Page and Bushnell (1921), and Pivnick and Fabian (unpublished data) found coliform bacteria in samples obtained from factories in the United States. Recently, Pivnick (unpublished data) has found that emulsions from factories in Great Britain, Canada, and the United States contained between 103 and 105 coliform bacteria per ml. The relationship of fecal pollution to the spread of enteric diseases suggested that an investigation of enteric pathogens in soluble oils should be undertaken. Preliminary experiments by Okawaki (1953) showed that enteric pathogens grew well in this medium. This report is concerned with the growth of some representative enteric pathogens and Klebsiella pneumoniae. MATERIALS AND METHODS Oil emulsion. The oil sample was obtained from a local depot of a nationally recognized petroleum company. This sample contained mineral oils emulsified Received for publication December 18, 1953 with soaps of petroleum sulfonate and resin. The sample was mixed with tap water to give an emulsion containing 2 per cent oil, and sterilized by autoclaving for 15 minutes at 121 C. Cultures. Cultures of Salmonella, Shigella and Klebsiella were generously supplied from the stock culture collections of the University of Nebraska, University of Michigan, The Ohio State University, Dr. E. Cope of the Michigan State Department of Health, and Dr. P. R. Edwards of Chamblee, Georgia. Cultures were also obtained from the American Type Culture Collection (A.T.C.C.). The name and source of the individual cultures are given in table 1. Inoculum. Cultures were grown in nutrient broth for 18 to 24 hours at 37 C, washed with physiological saline and inoculated into the oil emulsions. Inoculum size was varied to yield from 104 to 106 cells per ml of emulsion. The gram reaction, motility, and sugar fermentations of each culture showing growth in the emulsions were checked 4 days after inoculation. Culture methods. The inoculated emulsions were incubated at 30 C. Aliquots were removed after 0, 4 and 8 days incubation and plated on nutrient agar. Agar plates were incubated at 30 C and colonies counted after 72 hours. EXPERIMENTAL RESULTS Thirty different strains of enteric pathogens and two strains of Klebsiella pneumoniae were tested for their ability to grow in soluble oil emulsions. Representative data from these experiments are shown in table 1. Genus Salmonella. All strains of S. schottmuelleri, S. typhimurium, S. oranienburg, and S. pullorum grew readily in oil emulsions. Conversely, all six strains of S. paratyphi, and three of the four strains of S. typhosa tested failed to grow. The remaining strain of S. typhosa (obtained from Dr. P. R. Edwards) grew readily when