BACTERIAL FORMATION OF L GLUTAMIC ACID FROM ACETIC ACID IN THE GROWING

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1 J. Gen. Vol. Appl. 7, No. Microbiol. 1, 1961 BACTERIAL FORMATION OF L GLUTAMIC ACID FROM ACETIC ACID IN THE GROWING CULTURE MEDIUM'' (II) GROWTH AND L GLUTAMATE ACCUMULATION IN A CHEMICALLY DEFINED MEDIUM AND SOME OTHER FERMENTATION PRODUCTS TOSHINAO TSUNODA, ISAMU SHIIO and KOJI MITSUGI From the Central Research Laboratory of Ajinomoto Co., Inc., Kawasaki Received for publication, September 12, 1960 In the preceding papers (1, 2), it was revealed that Brevibacterium flavum, No. 2247, and some other strains of genera Brevibacterium and Corynebacterium could grow on acetate as the sole carbon source and accumulated a considerable amount of L-glutamate in the growing culture medium. From these experiments, it was ascertained that the growth factors and the concentration of acetate and the ammonium ions were intimately related to the growth of the bacteria as well as to the L-glutamate accumulation. While Brevibacterium flavum, No grew well and accumulated L- glutamate in the glucose medium, which contained 100,ag. of thiamine hydrochloride, 2,g. of biotin and 2 g. of corn-steep liquor per liter as the growth factors, this strain also grew well but no L-glutamate was accumulated in a medium that contained acetate in stead of glucose, the rest being under the same condition. However, this strain grew well and accumulated L-glutamate in the acetate medium that contained only thiamine and cornsteep liquor as the growth factors. Therefore, it would be conceivable that this strain, which was reported to require biotin as an essential growth factor (3), may not be require biotin essentially for the growth and the accumulation of L-glutamate in the acetate medium. The present paper reports on some results on the formation of L-glutamate in a chemically defined medium and on the fermentation products in a complex medium. 1 A Chemical The DiNPH., Glutamic part of this study was presented at the monthly meeting of the Agricultural Society of Japan, held in Sept abbreviation used throughout this paper include a-kg., a-keto-glutaric acid; 2, 4-Dinitrophenylhydrazine or 2, 4-Dinitrophenylhydrazone; and Glut., L- acid. 30

1961 Bacterial Formation of L-Glutamic Acid (II) 31 MATERIALS AND METHODS Microorganism : Brevibacterium f avum, No. 2247 was used throughout the experiments.

2 1961 Bacterial Formation of L-Glutamic Acid (II) 31 MATERIALS AND METHODS Microorganism : Brevibacterium f avum, No was used throughout the experiments. Cultivation of bacteria was carried out by the same procedure as described in the preceding paper (2). Analytical methods : Methods for the determination of the bacterial growth, L-glutamic acid, aspartic acid, acetic acid, and x-ketoglutaric acid were described in the previous paper (2). Aspartic acid and glycine were also detected by paper chromatography (Solvent: Phenol-Water; 10 : 2, t- Butanol-Methylethylketone-Water-Dimethylamine; 40 : 40 : 20 : 4, (4)). Succinic acid was determined manometrically by a succinic oxidase preparation obtained from the pig heart muscle (5). Citric acid was determined by the colorimetric method of STERN (6) ; it was oxidized to pentabromoacetone, which in turn was converted with thiourea to a colored complex. RESULTS (I) Formation of L-glutamate in the chemically defined media In the preceding papers (1, 2), it has been shown that a considerable amount of L-glutamate is accumulated in the complex medium which contains thiamine and corn-steep liquor as the growth factors. In the preliminary experiment, therefore, the effects of vitamines such as contained in the corn-steep liquor, thiamine, folic acid, nicotinic acid, Ca-pantothenate, biotin, inositol, riboflavin, choline, pyridoxal, and p-aminobenzoic acid were studied. As it was ascertained from these results that thiamine was the most effective, and that seven vitamines cited in Table 1 were also seemed to be more or less effective on the L-glutamate formation, the effects of these vitamines were demonstrated by adding thiamine to the basal medium, as shown in Table 1. Tables 1 and 2 showed that the addition of biotin, l ~cg. per liter, brought the growth to increase, but brought the amounts of L-glutamate accumulated to drop, and also the main effect of folic acid was negative, while the effect of choline or the interaction, nicotinic acid x riboflavin x folic acid, or pyridoxal x inositol x folic acid was positive on the L-glutamate accumulation. Although it could not be concluded from these data, which vitamine was the most effective of these five, choline, nicotinic acid, pyridoxal, inositol, and folic acid, this strain grew in the chemically defined medium, which contained some of those together with thiamine, even if no biotin was added, to accumulate a considerable amount of L-glutamate, as shown in Exp. Nos. 10, 12, 14 and 16 of Table 1. The effect of biotin was further demonstrated in detail as shown in Fig. 1. It was observed that a reciprocal relationship existed between the cell growth and the L-glutamate accumulation, as shown in the experiment of glucose (7, 8), and that the level of biotin was extremely critical for the maximum accumu-

3 32 T. TSUNODA, I. Smio and K. MITSUGI VOL. 7 Table 1. Effects of vitamines. (Factorial design (216)and results) lation of L-glutamate, when no vitamine was added other than thiamine. For example, while the maximum growth required more than 2,ug. of biotin per liter, the maximum L-glutamate accumulation required ,ug. of biotin per liter. From Table 1 and Fig. 1, it was considered that the effects of biotin on the cell growth and the L-glutamate accumulation were not so strict as shown in the glucose medium. It is obvious that Brev. flavum, No can grow and accumulate a considerable amount of L-glutamate in the chemically defined media containing acetate as the sole carbon source. Finally, the effect of the concentration of inorganic salts was demonstrated

4 1961 Bacterial Formation of L-Glutamic Acid (II) 33 Table 2. The analysis of variance. Basal medium: N114-acetate, 15g.; Naacetate, 25g. (as acetic acid, respectively); KH2PO4, 2g.; MgSO4.7H2O, 0.4g.; thiamine, hydrochloride, 100,ig.; Fe++, Mn++, 2p.p.m., per liter. Seed : Saline cell suspension (O.D. = 0.6), 0.5 ml./50 ml. Cultured for 72 hours at 30. Total acetate (Initial acetate +added ace tate) was 55 g, per liter. Fig. 1. L-glutamate Effect of the concentration accumulation (- --). of biotin on the cell growth (-O-) and the

5 34 T. TSUNODA, I. Simo and K. MITSUGI VOL. 7 Table 3. Effects of inorganic acid. as shown in Table 3. These results showed that the concentration of MgSO4 was significant, in other words, 0.8 g. of MgSO4.7H2O per liter was better than 0.4 g, for both the growth and the L-glutamate accumulation. (II) Fermentation products The fermentation products in the broth were analyzed and were demonstrated in comparison with the results obtained in the glucose medium. Some of the fermentation products were identified paper-chromatographically and the quantitative estimation was followed by ion-exchange chromatography or by the individual method for the estimation. After Brev. flavum, No was cultured aerobically for 90 hours in the complex medium which

6 1961 Bacterial Formation of L-Glutamic Acid (II) 35 contained 35 g, of acetic acid, 2 g, of corn-steep liquor, 100 sag. of thiamine hydlochloride and salts, per liter, the broth was used for the following analysis. Amino acids Amino acids in the broth filtrate were identified as ninhydrin positive spots on the paper-chromatogram. There were two spots except for that of L-glutamate. The one was in accordance with aspartic acid in the Rf value by two solvent systems shown in Table 4, and also could response to Table 4. Rf Value of amino acids in the broth filtrate. the growth of Leuconostoc mesenteroides No. P-60 requiring aspartic acid. So aspartic acid was determined by microbioassay method using this organism. Another spot was in accordance with glycine in the Rf value, as shown in Table 4, but its amount in the broth was trace. Organic acids Organic acids in the broth were isolated by ion-exchange chromatography and identified. The acidic compounds in the broth filtrate were absorbed on the f ormate-form resin of Dowex-1 X-8 (200 mesh) and separated by the gradient elution method of BUSCx et. al. (9). The pattern of the gradient elution analysis was shown in Fig. 2. From this pattern, it is evident that there are more than four organic acids in the broth filtrate. These four fractions were identified as follows; Fraction I. The greater part of this fraction was identified as L-glutamic acid by paper chromatography.

7 36 T. TSUNODA, I. SmIO and K. MITSUGI VOL. 7 traction number Fig. 2. Chromatographic elution pattern of the organic acids in the broth filtrate. The organic acids were chromatographed on the ion-exchange column, Dowex-1 X-8 (200 mesh) (20 x 0.8 cm.), using formic acid as eluent. Frow rate was about 10 drops/min. Eluates, 2 ml. per one fraction, were dried up on CaCl2-NaOH (2: 1) and titrated by N/100 NaOH solution. Fraction II. This fraction contained at least three organic acids; a large amount of succinic acid, a small amount of aspartic acid and very small amount of of unknown acid (Table 5). Fraction III. Since the paper-chromatographic Rf value of the acid present in Fraction III. resembled that of citric, iso-citric or cis-aconitic acid (Table 5, (10)), it was identified by the formation of pentabromoacetdne-thiourea complex which was specific for citric acid. Characteristic absorption spectrum (Fig. 3) and melting point of its complex were in accordance with those of the complex of authentic citric acid.

8 1961 Bacterial Formation of L-Glutamic Acid (II) 37 Table 5. Paper chromatography of the ion-exchange chromatography fractions obtained from of the broth filtrate. Pentabromoacetonethiourea complex from authentic citric acid Thiourea complex from Fraction III. Mixture of above two complex melting point (uncorrected) Fraction IV. The carbonyl compound of Fraction IV was identified as a-ketoglutaric acid in the form of free acid (Table 5) and in the form of DiNPH by paper-chromatography (Solvent : n-butanol-ethanol- 0.5N NH44H; 7 : 1: 2) and the melting Fig. 3. Adsorption spectra of authentic Pentabromoacetone thiourea complexs (-0-) and of thiourea complex obtained from the fraction III (- x -). point of its DiNPH was; melting point (uncorrected) Authentic a-kg DiNPH Fraction IV comp. DiNPH Mixture of above two DiNPH Further more, organic acid present

9 38 T. TSUNODA, I. SHIIO and K. MITSUGI VOL. 7 in each fractions was titrated by N/100 NaOH, and also determined by the analytical method, specific for each acids (Table 6, A). In addition, the broth filtrate was directly applied to analyze by the individual methods (Table 6, B). These two data (A and B in Table 6) were Table 6. Estimation of organic acids in the broth filtrate. Table 7. Fermentation products of the broth filtrate.

10 1961 Bacterial Formation of L-Glutamic Acid (II) 39 approximately the same. Finally, the amounts of fermentation products in the broth cultured for 90 hours under the same condition as described in Fig. 6 of the preceding paper (2) were determined (Table 7). (The only difference between these two broth was the initial concentration of acetate). DISCUSSION It was shown that Brev. flavum, No could grow and accumulated a considerable amount of L-glutamate in the chemically defined medium, where acetic acid was the sole carbon source. Recently, it has been reported that L-glutamate accumulating bacteria require essentially biotin (7, 8), and as a matter of fact, Brev. flavum, No required biotin when it grew in the glucose medium (3). Although this strain required about sag. of biotin per liter for the maximum accumulation of L-glutamate in the acetate medium, which was only one-tenth as much as in the glucose medium, a considerable amount of L-glutamate was also accumulated in the medium that contained several vitamines with thiamine as the growth factors, even if no biotin was added. Therefore, the effects of biotin on the cell growth and on the L-glutamate accumulation are not so strict as shown in the glucose medium. Furthermore, aspartic acid, glycine, citric acid, succinic acid and a-ketoglutaric acid were detected as fermentation products with the exception of L- glutamic acid, but alanine, glutamine, pyruvic acid and lactic acid, which were reported to be contained in the broth from the glucose medium (12), were not detected. The amounts of succinic acid and aspartic acid in the acetate medium were more than those accumulated under the optimum condition for the L- glutamate accumulation from glucose, while it has been shown that a large amount of succinic acid was accumulated under the non-optimum condition for the L-glutamate accumulation from glucose (12). The accumulation of citric acid and glycine by L-glutamate forming bacteria has never been reported. Thus, it is obvious that there are remarkable differences in these two media, in which carbon sources were different from each other. It should be further studied in detail whether these organic acids would be accumulated substantially or not, and whether the condition described above for the L-glutamate accumulation in the acetate medium would be optimum or not. However, the relationship between the growth factor and the carbon source might be the most interesting one. SUMMARY 1) Brevibacterium flavum, N could grow in the chemically defined medium and accumulated a considerable amount of L-glutamic acid in its

11 40 T. TSUNODA, I. SHIIO and K. MITSUGI VOL. 7 growing culture medium. 2) It is likely that the effects of biotin on the cell growth and on the L-glutamate accumulation in the acetate medium are not so strict as shown in the glucose medium. 3) aspartic acid, glycine, succinic acid, citric acid and u -keto-glutaric acid were identified as by-products in the broth filtrate. ACKNOWLEDGEMENT The authors are indebted to Dr. H. OEDA and Mr. S. MOTOZAKI of our laboratory for the interest and encouragement during the course of this work. The authors are also indebted to Miss. K. NARUI, Miss. K. KANISAWA and Miss. S. HIROOKA of our laboratory for the microbioassay of aspartic acid. REFERENCES (1) SHHO, I., MITSUGI, K. and TsUNODA, T.: J. Biochem.; 46, 1665 (1959). (2) TSUNODA, T., SHIIO, I, and MITSUGI, K.: J. Gen. Appl. Microbiol. (Tokyo).; 7, 18 (1961). (3) OKUMURA, S., TSUGAWA, R., TSUNODA, T., MATSUI, T., KONO, K. and MIYAJI, N.: The monthly meeting of Agricultural Chemical Society of Japan, held in May (4) ROBERT, R. REDFIELD: Biochim. et Biophys. Acta., 10, 344 (1953). (5) UMBREIT, W. W., BURRIS, R. H, and STAUFFER, J. F.: Manometric techniques 1957, p (6) STERN, J. R.: Methods in Enzymology, Vol. III, p Academic Press, New York (7) TANAKA, K., IWASAKI, H. and KINOSHITA, S.: The Nippon Nogei-Kagaku Kaishi, 34, 593 (1960). (8) TANAKA, K., IWASAKI, H, and KINOSHITA, S.: ibid., 34, 600 (1960). (9) BUSCH, H., HURLBERT, R. B. and POTTER, V. R.: J. Bio. Chem., 196, 717 (1952). (10) LUGG, J. W. H. and OUERELL, B. T.: Nature, 160, 87 (1947). (11) CHEFTEL, R. I., MUNIEv, R. and MACHEBOEUF, M.: Bull. Soc. Chim. Biol., 33, 840 (1951). (12) OKADA, H., KAMEYAMA, I., OKUMURA, S. and TSUNODA, T.: The 1st Symposium of Amino Acid Fermentation, held in Nov

and the cells removed by centrifugation. These were resuspended in sterile 1949a), growth was measured in terms of acid production while dextran was

and the cells removed by centrifugation. These were resuspended in sterile 1949a), growth was measured in terms of acid production while dextran was THE NUTRITIONAL REQUIREMENTS OF LEUCONOSTOC DEXTRANICUM FOR GROWTH AND DEXTRAN SYNTHESIS1 VIRGINIA WHITESIDE-CARLSON AND CARMEN L. ROSANO Biochemistry Department, Medical College of Alabama, Birmingham,