chrysogenum in Submerged Culture

Transcription

1 APPLIED MICROBIOLOGY, Sept., 1965 Copyright 1965 American Society for Microbiology Vol. 13, No. 5 Printed in U.S.A. Production of Calcium Gluconate by Penicillium chrysogenum in Submerged Culture G. R. AMBEKAR, S. B. THADANI, AND V. M. DOCTOR Research Laboratories, Hindustan Antibiotics Limited, Pimpri, India Received for publication 12 April 1965 ABSTRACT AMBEKAR, G. R. (Hindustan Antibiotics Ltd., Pimpri, India), S. B. THADANI, AND V. M. DOCTOR_ Production of calcium gluconate by Penicillium chrysogenum in submerged culture. Appl. Microbiol. 13: The waste mycelium of Penicillium chrysogenum HA-1 (obtained at the end of penicillin fermentation), or a 24- hr-old freshly grown vegetative inoculum of this organism, was found to utilie glucose for the production of calcium gluconate by submerged fermentation in shake flasks. After 72 to 96 hr of fermentation at 24 C, 9 to 95% of the reducing sugar from the 15%o glucose medium was converted to calcium gluconate. Reuse of the mycelium for successive experiments reduced the fermentation period to 72 hr or less because of an enhancement of glucose utiliation. Ten successive batches of 15% glucose medium were fermented by the reuse method. Fermentation media containing up to 3% glucose could be used, provided boric acid was added to prevent the precipitation of calcium gluconate formed. We found that 3% hydrol (a by-product of glucose manufacture containing 5 to 55% reducing sugar), when used in place of glucose in the fermentation medium, inhibited the rate of glucose utiliation. However, this effect was partially reversed by pretreatment of hydrol with 2 to 4% activated charcoal before addition to the fermentation medium. Shortly after Molliard (1922) reported the synthesis of gluconic acid by a fungus, Butkewitsch (1923) found a strain of Aspergillus niger which, in the presence of calcium carbonate, formed gluconic acid almost to the exclusion of other acids. A few years later, May et al. (1927) initiated the work of a U.S. Department of Agriculture group, and tested fungi, especially those belonging to the genera Aspergillus, Mucor, Penicillium, and Monilia. They first reported the production of gluconic acid by P. luteum purpurogenum group. After studying (Herrick and May, 1928) the conditions for the production of gluconic acid by this organism in surface culture, with the use of shallow pans, Moyer, May, and Herrick (1936) reported that this strain of Penicillium lacked biochemical and vegetative vigor and investigated a large number of Penicillium species, which led to the discovery of a culture of P. chrysogenum (534-11) having a greater ability to synthesie gluconic acid, than other Penicillium strains. Using this culture of P. chrysogenum (534-11) and carrying out fermentations at 3 C in shallow aluminum pans with a surface-volume ratio of.4 to.5, these workers obtained a 6% conversion of glucose to gluconic acid at the end of 8 to 1 days. They also investigated the production of gluconic acid by P. chrysogenum when cultivated in submerged culture, and reported yields of 8 to 87% (based on the quantity of glucose supplied) in 8 days. The purpose of the present study was (i) to investigate the use of P. chrysogenum (HA-1) mycelium (obtained at the end of penicillin fermentation) in the calcium gluconate fermentation, (ii) to determine the fermentation conditions for this organism which will produce high yields of calcium gluconate under submerged conditions, and (iii) to investigate the use of hydrol (a byproduct of glucose manufacture containing 5 to 55% reducing sugar) as the starting raw material in place of glucose for calcium gluconate fermentation. A preliminary report on this study has appeared (Doctor and Ambekar, 1964). MATERIALS AND METHODS Cultures. P. chrysogenum strain Thom HA-1, strain HA-9 (a mutant of a Russian strain), and strain Q 176 were used in these investigations. The strains were maintained on agar slants of the following composition: 1% glycerol, 1% glucose, 1% NaCl,.5% yeast extract,.5% MgSO4-7H2,.1% KH2PO4, and 2% agar. Spore inoculum. Spores of P. chrysogenum were obtained by the following procedure (Thirumalachar and Gopalkrishnan 1963). A 1-g 713 Downloaded from on January 3, 219 by guest

2 714 AMBEKAR, THADANI, AND DOCTOR APPL. MICROBIOL. ẕ J CI) LJ - 7 D LLJ i: V -j LA ce LLJ ' l H U R S FIG. 1. Rate of glucose utiliation and calcium gluconate formation during fermentation of glucose (15%) and hydrol (2%) with Penicillium chrysogenum HA-1, 1% vegetative growth (24 hr) inoc- c ulum. (1) Glucose concentration in grams per 1 c ml of fermentation solution. (2) Glucose concentration (in hydrol) in grams per 1 ml of fermenta- 5 tion solution. (1') Calcium gluconate concentration, t in grams per 1 ml offermentation solution (formed _ from corresponding amount of glucose utilied in 1). (2') Calcium gluconate concentration in grams per, 1 ml of fermentation solution (formed from corresponding amount of glucose (in hydrol) utilied C R in 2). <U) o Z U g.d LiU- 6 o 4-4* L E 3O r-w amount of barley was weighed in 5-ml Erlen- L meyer flasks and sterilied by autoclaving at 12 C for 6 min. In separate tubes, 2 ml of sterile solution of 6% honey and 1% peptone in water were prepared; 1 ml of the spore inoculum from an c agar slant was taken and mixed with 2 ml of honey-peptone solution aseptically. The mixed suspension was poured in barley flasks and shaken vigorously to disperse the spores evenly on barley grains. The flasks were kept at 24 C for 8 days. At the end of this time, a spore suspension was prepared by adding sterile distilled water to the barley flask and decanting the supernatant aseptically. Vegetative growth inoculum. Inoculum medium of the following composition was used: 2% sucrose, 1% corn steep liquor (5% solids),.3% CaCO3,.25% NaNO3,.5% MgSO4-7H2, and.25% KH2PO4. The ph of the medium was adjusted to 5.5 Ėrlenmeyer flasks (5 ml) containing 1 ml of medium were sterilied by autoclaving at 12 C for 3 min. The flasks were inoculated with the spore suspension of P. chrysogenum and incubated with shaking on a rotary shaker (25 rev/min, describing a circle of 2-inch (5.1 cm) diameter) at 24 C for 24 to 48 hr. Mycelium of Penicillium chrysogenum HA-. The mycelium was obtained by aseptic centrifugation of the fermented broth (obtained at the end of penicillin fermentation), washing the mycelial residue with sterile distilled water, and resuspending it in the same volume of water as the original broth. One of the following methods was investigated for reusing the mycelium: (i) inoculation of the fresh medium with a sample of the previously fermented mycelium, or (ii) filtration of the mycelium under aseptic conditions and resuspending it in the fresh production medium. Fermentation medium. The fermentation medium reported by May, Herrick, and Wells (1934) was modified to include 15% glucose or 2 to 3% hydrol, and.3% NaNO3,.15% KH2PO4, Q II Q Downloaded from on January 3, 219 by guest FIG. 2. Effect of inoculum on calcium gluconate fermentation of glucose (15%) with Penicillium chrysogenum HA-J. Symbols:, spore inoculum; A, mycelium from penicillin fermentation as inoculum, 5%; El, mycelium from penicillin fermentation as inoculum, 1 to 3%; *, vegetative growth inoculum (24 or 48 hr), 1%.

3 VOL. 13, 1965 CALCIUM GLUCONATE PIRODUCTION BY P. CHRYSOGENUM CL 11 I LU. w j 7 ow 12 / 2* 3 2 I t ~ '6 FIG. 3. Reuse of mycelium in calcium gluconate fermentation of glucose (J5C,1) with Penicillium chrysogenum (HA-1). (1) Mycelium from penicillin fermentation as inoculum, 1%7. (2) Mycelium from 1 as inoculum. (3) Mycelium from 2 as inoculum..125% M1gSO47112, and 2.5% CaCO3. The constituents, with the exception of CaCO3, were dissolved in water and autoclaved at 12 C for 3 min. Calcium carbonate was autoclaved separately and mixed with the rest of the solution before inoculation. When higher concentrations of glucose were used, the concentrations of the other constituents of the medium were proportionately increased. Boric acid was added (after dry steriliation) in the solid form, 24 hr after inoculation. The quantities of boric acid added were.28,.57, and.85% for 2, 25, and 3% glucose media, respectively. These amounts of boric acid were reported (Moyer, Umberger, and Stubbs, 194) to keep in solution the calcium gluconate formed during fermentation. Hydrol was diluted 1:1 with water, filtered to remove suspended impurities, neutralied to ph 7. with NaOH, and then was added to the medium in place of glucose. Fermentation. The fermentations were carried out in 5-ml or 1-liter Erlenmeyer flasks on a rotary shaker (25 rev/min, describing a circle of 2-inch diameter) at 24 or 28 C. Results obtained by changing fermentation conditions, including media variation, were compared with the results obtained with the standard medium. The use of 5-ml and 1-liter Erlenmeyer flasks, and a variation in the volume of the fermentation medium in the flasks, allowed a range of oxygen availability to be obtained, depending upon the degree of aeration desired t 28 / a 23. VI) 22 Z 21/ < 2 I 4 19O tf 17-6J6 ~u 14- a. 12,, o 13 N O. Lf 7. 7 Lfl H U R S FIG. 4. Fermentation of concentrated solutions of glucose (2, 25, 3%) with Penicillium chrysogenum HA-J. Mycelium from penicillin fermentation as inoculum (1%). (1) 2% glucose +.28% boric acid; (2) 25% glucose +.57% boric acid; (3) 3% glucose +.85% boric acid; (4) 2% glucose, no boric acid; (5) 3% glucose, no boric acid; (1') ph in 1; (3') ph in 3; (4') ph in 4; (5') ph in 5. Downloaded from on January 3, 219 by guest

4 716 AMBEKAR, THADANI, AND DOCTOR APPL. MICROB'IOL. TABLE 1. Boric acid tolerance of Penicillium chrysogenum HA-J as shown by the growth at 72 hr from the time of addition of boric acid to the medium Boric acid added* Boron Glucose Growtht concn % ppm % , , , 35.5 * Boric acid was added 24 hr after inoculation with the vegetative growth inoculum. t Growth in the absence of boric acid is represented by 1.. In the experiments concerning the reuse of the mycelium, a fresh batch of shake flasks containing sterile fermentation medium were inoculated with 1% of the fermented broth (with its proportionate amount of fungal growth) from the previous experiment. Analytical methods. The samples for analysis were taken at 24-hr intervals until the fermentation was completed. A 5-ml amount of the fermentation broth was centrifuged, and the supernatant liquid was used for analysis of glucose and calcium gluconate. The rate of glucose utiliation during fermentation was measured by estimating the reducing sugar by the Somogyi (1952) method. For calcium gluconate assay, the supernatant liquid obtained after centrifugation, was neutralied to ph 7. by adding Ca(OH)2, and the calcium gluconate formed was estimated by the method given in the U.S. Pharmacopiea. The formation of gluconic acid in the fermented media was confirmed by paper chromatographic separation of organic acids on Whatman no. 1 filter paper. A descending technique was used, and the solvent system was butanol-acetic acid-water (4:1:5, v/v; Partridge, 1948). The chromatograms were developed with a slightly alkaline.4% alcoholic solution of bromophenol blue. Recovery of calcium gluconate. Calcium gluconate was isolated from the fermented broth by the following procedure. The broth was filtered, and the ph of the filtrate was adjusted to 7. by adding Ca(OH)2. The neutralied filtrate was concentrated in vacuo to one-third the original volume and cooled to 5 C. Calcium gluconate precipitated out on cooling. The precipitate was filtered and washed thoroughly with ice-cold water and then with acetone, and was dried at 6 C. When hydrol was used as the starting raw material, the calcium gluconate precipitate was thoroughly washed with ice-cold water by making a slurry in water, and was refiltered under suction. A yield of 75 to 8% of calcium gluconate based on the initial concentration in the fermented broth was obtained. The crude product was treated with 1 to 2% activated charcoal and recrystallied from water to obtain the pure white compound. RESULTS AND DISCUSSION The rates of glucose utiliation and calcium gluconate formation during fermentation of glucose (15 %) or hydrol (2 %) medium by P. chrysogenum HA-1 are plotted in Fig. 1. The results show that with 15% glucose medium 95% of the reducing sugar was utilied for calcium gluconate production in 2 days, and with 2% hydrol medium the rate of utiliation of glucose was slower, requiring 3 days to complete the fermentation. Further, a comparison of the plot for glucose utiliation with that for calcium gluconate formation shows that a direct relationship exists between utiliation of glucose from the medium and the formation of calcium gluconate. Therefore, in all subsequent experiments, only the rate of glucose utiliation was measured. The effect of sie and age of inoculum on the rate of glucose utiliation by P. chrysogenum is shown in Fig. 2. It is apparent that when the fermentation medium was inoculated with spores of P. chrysogenum HA-1 the utiliation of glucose was slower because of the initial time required by the spores to germinate. However, with 1% of the 24- to 48-hr-old vegetative growth inoculum, there was no initial lag period, 1 8o o H O U P S FIG. 5. Effect of aeration on calcium gluconate fermentation of glucose (15%) with Penicillium chrysogenum HA-J; 1% vegetative growth (24 hr) inoculum. Symbols:, 1 ml of medium in 5-ml Erlenmeyer flask; A, 1 ml of medium in 1,-ml Erlenmeyer flask; l, 5 ml of medium in 1,-ml Erlenmeyer flask. Downloaded from on January 3, 219 by guest

5 VOL. 13, 1965 CALCIUM GLUCONATE PRODUCTION BY P. CHRYSOGENUM *1 UR 8Z L _ur 24 ;i, 8C b 12 FIG. 6. Effect of temperature on calcium gluconate fermentation of glucose (15%) with Penicillium chrysogenum HA-JO; 1% mycelium from penicillin fermentation as innoculum. Symbols:, temperature 24 C; A, 28 C. and the fermentation was complete within 3 days. On the other hand, if the penicillin waste mycelium (obtained at the end of penicillin fermentation) was used as the inoculum, the fermentations were slower than in those inoculated by the freshly grown vegetative inoculum. Increasing the inoculum sie of the penicillin waste mycelium from 5 to 1% increased the rate of fermentation. However, a further increase in the inoculum sie from 1 to 3% did not enhance the rate of glucose utiliation. The effect of reuse of the penicillin waste mycelium for several runs of calcium gluconate production are reported in Fig. 3. The results show that the rates of glucose utiliation were enhanced with each subsequent reuse of the mycelium, up to the third experiment. After this, the mycelium could be reused for seven additional experiments without significant loss of potency. Since the two procedures (see Materials and Methods for details) employed for the preparation of the mycelium for the reuse experiments gave identical results, the first one was adopted for all subsequent experiments because of its simplicity. During the first run of calcium gluconate production, the rate of glucose utiliation was faster with the 24- to 48-hr vegetative growth inoculum of P. chrysogenum in comparison with that of penicillin waste mycelium (compare Fig. 2 and 3). However, in subsequent experiments where the mycelium was reused, the rate of glucose utiliation was the same as when inoculated by the freshly grown vegetative inoculum. Results of the reuse experiments with the latter type of inoculation, however, did not show any enhancement of the rate of fermentation. The effect of borate addition to the glucose medium on the utiliation of higher concentrations of glucose by P. chrysogenum is plotted in Fig. 4. It was found that addition of.28,.57, and.85% boric acid to the fermentation media was sufficient to keep calcium gluconate formed from 2, 25, and 3% glucose, respectively, in solution, without adversely influencing the rates of glucose utiliation. However, if boric acid was omitted from the fermentation, the ph of the fermented broth was lowered owing to the free gluconic acid formation, which in turn may retard LLU XL 12- J -i I Z 1 g O LL a 9- (:) <K < O I1 w FIG. 7. Use of different strains and combinations of strains in calcium gluconate fermentation of glucose (15%); 1% vegetative growth (24 hr) inoculum. Symbols:, strain Q 176; A, strain HA-9, combination of HA-9 and Q 176; O, strain HA-J, combination of HA-9 and HA-J. Downloaded from on January 3, 219 by guest

6 718 ANIBEKAR, THADANI, AND DOCTOR APPL. MICROBI'OL P 13-/ ,/, 1- / OU s-o/ u. 62 / o 7 - o H U R S FIG. 8. Effect of hydrol concentration on calcium ui gluconate 13-- fermentation of hydrol with Penicillium chrysogenum HA-1; 1%1/ vegetative growth (24 hr) inoculum. Symbols: i\, 2% hydrol; O3, 25%, hydrol; O, 3% hydrol. 5i 15-, B Ld D FIG Effect ofhyrotrconcentrtooncadrlcwium atvtdcacaonclimgluconate fermentationo yrlwt eiilu cfhrysrogeu 8 HA-JO;1w eeaive growthll(u4 hr)sy HAInouu. 9- Symbols: A, 2% hydrol; A, 25% hydrol; 8-9-~~~~~ the G.at.effof pretreioatmglcoen ofinthydo witer actiated chrca onclcuhluoae fermentation. tde eeas ofmydrlce%ih eiiliumofp chrysogenumha1ofadtn th vaiutliatioof aeof gluccose duinthe cacu late conate fermentation medium. The results (Table 1) show that the addition of boric acid at levels higher than.85/% significantly retarded the growth of the mycelium, as shown by a 5% reduction of the mycelium dry weight. Figure 5 shows the effect of increased aeration on the rate of glucose utiliation by P. chrysogenum HA-1 during gluconate production, It is apparent from the results that an increased rate of glucose utiliation was obtained by an increase in aeration which was caused either by using larger flasks or by reducing the volume of the fermentation medium. Studies on the effect of temperature on the glucose utiliation by P. chrysogenum HA-1 showed that the rate of glucose utiliation was higher at 24 C than at 28 C (Fig. 6). This is in contrast to results with P. chrysogenum strains used by the Russian workers (Pokrovskoya, Novikova, Finogenova, 1963) for glucose oxidase or by the American workers (Moyer, May, and Herrick, 1936) for gluconic acid production, which were grown between 27 and 3 C. Munk, Paskova, Hanus (1963) reported stimulation of glucose oxidase production by A. niger when the fermentation medium was supplemented by KCl or FeSO4*7H2. Similar stimulation of glucose exidase synthesis by these minerals was also reported by Pokrovskaya et al. (1963), with Penicillium strains. In the present studies, the addition of.3% KCI or.1% FeSO4 *7H2 to the fermentation medium did not stimulate calcium gluconate production by P. chrysogenum HA o 11-- v 1- / 9 Ni 8. P 7-- D O 3- ~Z FIG. 1. Effect of inoculum on calcium gluconate fermentation of hydrol (3%) with Penicillium chrysogenum HA-J. Symbols:, mycelium from penicillin fermentation as inoculum, 1%; A, vegetative growth (24 hr) inoculum, 5%; U, vegetative growth (24 hr) inoculum, 1 or J5%. Downloaded from on January 3, 219 by guest

7 VOL. 13, 1965 CALCIUM GLUCONATE PRODUCTION BY P. CHRYSOGENUM 719 A 5% increase in glucose oxidase activity was reported by Munk et al. (1963), when they compared the fermentation in submerged culture of mixed strains of A. niger with single culture of a strain. The results of studies on calcium gluconate production with mixed submerged cultivation of two P. chrysogenum strains showed no enhancement of glucose utiliation (Fig. 7). Hydrol, a by-product of glucose manufacture containing 5 to 55% reducing sugar, is a cheaper source of glucose. Therefore, studies were conducted on the use of hydrol in place of glucose for calcium gluconate production. Effects of using various concentrations of hydrol in the fermentation medium on the rate of glucose utiliation by P. chrysogenum are reported in Fig. 8. The results show that an increase in the hydrol concentrations from 2 to 3% significantly decreased the rate of glucose utiliation. However, this inhibitory effect of hydrol was partially reversed by pretreatment of hydrol by 2 or 4% activated charcoal, as shown by the results in Fig. 9. The effect of sie and age of the inoculum on the rate of glucose utiliation (from the hydrol medium) by P. chrysogenum HA-1 also showed that the rate of glucose utiliation is optimal with 1% of the 24-hr-old vegetative growth inoculum (Fig. 1). ACKNOWLEDGMENTS We wish to express our appreciation to M. J. Thirumalachar for his interest and encouragement during the course of the work. LITERATURE CITED BUTKEWITSCH, W Uber die "Citronensauregarung." Biochem. Z. 142: DOCTOR, V. M., AND G. R. AMBEKAR Production of calcium gluconate by Penicillium chrysogenum (HA-1) in submerged fermentation. Hindustan Antibiot. Bull. 6:179. HERRICK, H. T., AND. E. MAY The production of gluconic acid by the Penicillium luteum purpurogenum Group II. Some optimal conditions for acid formation. J. Biol. Chem. 77: MAY,. E., H. T. HERRICK, G. THOM, AND N. B. CHURCH The production of gluconic acid by Penicillium luteum purpurogenum Group I. J. Biol. Chem. 75: MAY,. E., H. T. HERRICK, AND P. A. WELLS Gluconic acid production by submerged mold growths under increased air pressure. Ind. Eng. Chem. 26: MOLLIARD, M Sur une nouvelle fermentation acide produite par le sterigmatocystis nigra. Compt. Rend. 174: MOYER, A. J.,. E. MAY, AND H. J. HERRICK The production of gluconic acid by Penicillium chrysogenum. Zentr. Bakteriol. Parasitenk. Abt. II. 95: MOYER, A. J., E. J. UMBERGER, AND J. J. STUBBS Fermentation of concentrated solutions of glucose to gluconic acid. Ind. Eng. Chem. 32: MUNK, V., I. POSKOVA, AND J. HANUS Factors influencing glucose oxidase activity in submerged cultivation of Aspergillus niger. Folia Microbiol. 8:23. PARTRIDGE, S. M Partition chromatography of sugars. Biochem. J. 42:238. POKROVSKAYA, N. V., L. M. NOVIKOVA, AND T. V. FINOGENOVA Production of glucose oxidase by some molds. Microbiology (USSR) 31:639. SOMOGYI, M Notes on sugar determination. J. Biol. Chem. 195: THIRUMALACHAR, M. J., AND K. S. GOPAL- KRISHNAN Method for obtaining large scale sporulation of Filamentous fungi. Hindustan Antibiot. Bull. 5:69. Downloaded from on January 3, 219 by guest