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Production of l -ornithine by arginine auxotrophic mutants of Brevibacterium ketoglutamicum in dual substrate-limited continuous culture
JO~RN~~.OPFE~NTATIONAND
BIOENCXNBERING
Vol. 81, No. 3, 216-219. 1996
Production of L-Ornithine by Arginine Auxotrophic Mutants of Brevibacterium ketoglutamicum in Dual Substrate-Limited Continuous Culture DAE KEON CHOI,’
WUK SANG RYU,’ CHA YONG CHOI,Z AND YOUNG HOON PARK’*
Bioprocess Technology Research Group, Korea Research Institute of Bioscience and Biotechnology, P. 0. Box 115, Yusong, Taejon 305-600’ and Department of Chemical Technology, College of Engineering, Seoul National University, Seoul 151-742,2 Korea Received 18 August 1995/Accepted 15 December 1995 Production of L-ornithine under dual limitation of arginine and phosphate was investigated in a chemostat culture of an arginine auxotroph of Brevibucterium ketoglutumicum. Under an arginine-iimited condition, culture instability, including the formation of revertant cells, could be a serious problem due to the inherent instability of the auxotrophic mutant strain. The difference in the growth rates between the auxotrophs and revertants was considered to be the main cause of the culture instability, and dual limitation with arginine and phosphate was found most appropriate to overcome this problem. When the ratio of the phosphate to arginine concentration in the feed medium was varied between 0.55 and 5.22 at a fixed dilution rate of 0.1 h-l, three distinct growth phases were recognized: phosphate-limited (0.55 to 2.01), dual arginine- and phosphate-limited (2.01 to 2.16) and a&nine-limited (above 2.16). In the dual arginine- and phosphate- limitation phase, the culture stability was markedly improved while the yield of L-ornithine was maintained at a high level. When the dilution rate was varied from 0.04 h-l to 0.17 h-l under the dual-limitation condition, maximum L-ornithine productivity was obtained at a value of 0.27 g/l/h at D=O.l h-l. [Key words: L-ornithine, Brevibacterium ketoglutamicum, arginine auxotroph, continuous substrate limitation]
culture, dual
More recently, it has been reported that under certain conditions the growth rate of an organism may be simultaneously limited by two or more substrates (10-16). It has also been indicated that a transition phase exists where the cell growth is limited by multiple nutrients (10-16). It is thus important to select a suitable pair for dual substrate limitation, since the physiological and metabolic activities of cells will differ depending on the choice of the two substrates (14). In this study, for the continuous production of Lornithine, phosphate was selected for dual substrate limitation along with arginine, since it is well known that under phosphate limitation bacterial cells exhibit higher steady-state cell viability (17) and metabolic activity (18) than under other substrate limitations. In addition, phosphate was considered to be one of the most appropriate limiting substrates for suppressing the overgrowth of revertant cells. A series of continuous culture experiments was carried out under dual phosphate- and arginine-limitation condition, and the culture behavior was analyzed with a view to process optimization for L-ornithine production.
L-Ornithine, an intermediate metabolite in arginine biosynthesis, and its derivatives have been reported to be effective for liver treatment (1). The first fermentative production of L-ornithine was in 1957 (2); L-ornithine was accumulated at a high yield (36% molar yield from glucose) using a citrulline-requiring mutant of Corynebacterium glutamicum. Since then, its biosynthetic pathway and regulatory mechanism have been well studied in glutamic acid-producing bacteria (3-8). N-Acetylglutamate kinase, the key enzyme in the biosynthetic pathway from glutamate, is known to be inhibited by L-arginine (6). Hence, to produce L-ornithine at a higher yield, the concentration of L-arginine should be controlled at low level during the fermentation (9). Although a higher product yield can be obtained with arginine limitation when auxotrophic mutants are used, culture instability often arises as a serious problem since it causes process instability, i.e., as the fermentation proceeds, the population of revertant cells increases and consequently the production of L-ornithine drops markedly. This is mainly due to the significant difference in the growth rates between the auxotrophic and revertant cells. Since revertant cells are more likely to grow faster than auxotrophic ones, once they appear in the culture they will eventually become dominant. Hence, to improve culture stability, it was necessary to find some means of controlling the growth rates of the two competing types of cells. Dual nutrient limitation was thus introduced to the continuous production of L-ornithine, one nutrient controlling cell growth and the other (arginine) product biosynthesis.
MATERIALS
AND METHODS
Microorganism The microorganism used in this study was Brevibacterium ketoglutamicum BK 533, an arginine auxotrophic mutant strain, which was derived from B. ketoglutamicum ATCC 21092 by mutagenesis using NTG and UV irradiation. The BK 533 strain lacks the enzyme ornithine carbamoyltransferase, which converts ornithine to citrulline, hence preventing arginine formation. Therefore, if the external supply of arginine
* Corresponding author. 216
L-ORNITHINE PRODUCTION
VOL. 81, 1996
is limited, its feedback inhibition is prevented, resulting in overproduction of L-ornithine. Media YPD medium containing 2% glucose, 1% yeast extract and 1% bacto peptone was used for the initial seed culture. The feed medium for the continuous culture consisted of 40 g glucose, 2 g yeast extract, 0.6 g KCI, 5 g (NH&Sod, 0.5 g MgS04.7Hz0, 50 mg L-arginine, O-2 g Na2HP04. 12Hz0, and 10 ml 100 x trace element solution per liter of distilled water. The 100 x trace element solution consisted of 500 mg MnS04.4Hz0, 200 mg NazMo04. 2H20, 500 mg ZnS04. 7H20, 10 mg H3B03, 40 mg CuS04.5H20, 10 mg CoC12.6H20, and 100 mg FeS04. 7Hz0 per liter of distilled water. Culture conditions A loopful of cells grown on a nutrient agar plate for 48 h at 30°C was inoculated into two 500-ml baffled flasks containing 50ml seed medium. After 13 h cultivation at 30°C and 170 rpm on a rotary shaker, the seed culture broth was transferred to a 2-f jar fermentor (Korea Fermentor Co., Inchon, Korea) containing 800 ml of the starting medium. After the cell growth reached the mid-exponential phase, continuous culture was started by supplying the feed medium and withdrawing the culture broth according to the specified dilution rates. The temperature was maintained at 30°C and the pH at 7.0 with 4N NH40H. The aeration rate was controlled in such a way that dissolved oxygen was not limited; the agitation speed was kept at 600rpm. Analytical methods Cell growth was monitored by measuring the optical density at 600 nm using a spectrophotometer (Uvicon 930, Kontron Instrument Co., Switzerland). The glucose concentration was measured using a YSI glucose analyzer (YSI 2000, Yellow Springs Instruments Co., USA). The L-ornithine, L-arginine and inorganic phosphate concentrations were determined calorimetrically (19-21). The total cell number and the number of revertant cells were determined by serially diluted plate counts on complex agar medium and minimal agar medium lacking arginine, respectively. Culture stability was defined as the ratio of the number of auxotrophic cells to the total cell number.
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Dual substrate- (arginine and phosphate) limited culture was thus tried. Under phosphate limitation, the growth rates of the two competing types of cells could be controlled equally well, so no growth advantage existed for either type. Hence, we combined phosphate limitation (to remove the growth advantage of revertant cells) with arginine limitation (for efficient production of Lornithine) in the culture. A series of continuous culture experiments was conducted at a constant dilution rate of 0.1 h-l to ascertain the optimum phosphate/arginine concentration ratio in the feed medium which would provide the dual limiting condition in the culture. Keeping the concentration of arginine in the feed medium lixed, the concentration of phosphate was varied and the steady-state behavior monitored. The results are summarized in Fig. 2. As the phosphate/arginine ratio increased from 0.55 to 5.22, three
RESULTS AND DISCUSSION Optimal feeding ratio of dual limiting substrates For efficient production of L-ornithine, an arginine-limited culture condition is essential. A typical time course of an arginine-limited chemostat culture at a dilution rate of 0.1 h-’ is shown in Fig. 1 (the phosphate/arginine concentration ratio in the feed medium is 5.22). It was noted that the total cell mass increased rapidly after about 90 h of chemostat operation. The population of auxotrophic cells was observed to decrease to less than 40% of the total cell mass, accompanied by a marked drop in the product concentration. This was obviously because of the rapid increase in the revertant population. Under such culture conditions, the growth of revertant cells could not be controlled by the dilution rate, while the auxotrophs could be controlled, since revertant cells could synthesize the additional arginine necessary for growth, while the auxotrophs could not. For this reason, a difference in the growth rates of the two competing types of cells was generated under the arginine-liited culture condition &,,. > pBux.). To improve the system performance, therefore, some measure was needed to repress the overgrowth of revertant cells during cultivation under the arginine-limited condition.
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218
J. FERMENT.BIOENG.,
CHOI ET AL.
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distinct growth phases were recognized in terms of the residual concentrations of phosphate and arginine-phosphate-limited (0.55 to 2.01), dual phosphate- and arginine-limited (2.01 to 2.16) and arginine-limited (above 2.16) phases. During the first phase, arginine was present in excess while phosphate was completely utilized. L-Omithine was scarcely produced due to the inhibition of N-acetylglutamate kinase by the high level of arginine in the culture broth. However, as the ratio increased, the steady-state level of arginine decreased as the cell mass increased, which, in turn, resulted in a higher steady-state level of L-ornithine. The linear increase in L-ornithine production as the phosphate/arginine ratio increased in the first phase could be ascribed to the increase in the steadystate cell mass concentrations of auxotrophs and to the decrease of feedback inhibition by arginine. It was also evidenced by the increase of the specific L-ornithine production rate (qp) and the yield on glucose (Y,,,). In this phase, chemostat operation continued for about 100-120 h and the proportion of revertants was negligible (1/104-1/105). In the dual phosphate- and argininelimited phase, the overgrowth of the revertants was still suppressed yet the qp values were maintained at high levels. The L-ornithine production pattern could be further extended to the arginine-limited phase, with phosphate/ arginine ratios higher than 2.16. At pho/arg=2.24, chemostat operation continued for 120 h and the ratio of revertants was also negligible (1/103). However, at pho/ arg=5.22 culture instability arose as a serious problem, as shown in Fig. 1. From the chemostat experiment, the optimal ratio of the dual limiting substrates (phosphateiarginine) was found to be located in a range from 2.01 to 2.16. Longterm continuous culture at a phosphate/arginine ratio of 2.01 was then carried out at the same dilution rate of 0.1 h-l. As shown in Fig. 3, the culture stability was significantly improved for longer than 250 h chemostat operation while maintaining a high level of L-ornithine productivity. These results indicated that optimally designed feeding of the dual limiting substrates could not only improve the culture stability significantly but also enhance the production of L-ornithine in a chemostat culture. Effects of dilution rate Further chemostat culture experiments were carried out at various dilution rates
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from 0.04 h-r to 0.17 h-l at a phosphate/arginine ratio of 2.01. Typical profiles of the continuous cultures at dilution rates of 0.04 hhl and 0.17 h-i are shown in Fig. 4. Residual concentrations of phosphate and arginine were found to be at a limiting level at all the dilution rates, clearly indicating that the cells were in a state of dual arginine- and phosphate-limitation over the whole range of dilution rates tested. However, with changes in dilution rate, considerable differences in the production of biomass and in L-ornithine production were observed. In Fig. 5, steady-state cell mass and L-ornithine concentrations along with some kinetic parameters are shown as a function of the dilution rate. The cell mass and Lornithine concentration increased 0.6- and 3-fold, respectively, as the dilution rate decreased from 0.17 h--I to 0.04 hh’. Similar observations were reported by Kiss et al. (22) in lysine production in threonine-limited continuous culture and by Park et al. (23) in phenylalanine production in phosphate-limited continuous culture. As can be seen in Fig. 5b, the product yields on glucose (Y,,,) and on cell mass (Y& decreased as the dilution rate increased, while the cell mass yield on glucose (Y,,,) increased. Since the cells were under the arginine- and phosphate-limited condition at all dilution rates tested, feedback inhibition by arginine would not have occurred and the biosynthetic activity for L-ornithine production remained fully active in all experiments. Therefore, the findings can be explained by the fact that, as the dilution rate decreased, a greater portion of the glucose was utilized for product formation rather than for cell mass production under the dual substrate limiting condition. It was also shown in the present study that the L-ornithine productivity had a maximum value of 0.27 g/l/h at D=O.l hh’, so D=O.l h-r was considered to be the optimal dilution rate.
L-ORNITHINE PRODUCTION
VOL. 81, 1996
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In conclusion, we have demonstrated that dual substrate limitation can be successfully applied to the bench-scale production of L-ornithine in a continuous mode, and we believe that the same principle should be applicable to large-scale industrial production of the amino acid. A pilot-scale (300 r) operation for the continuous production of L-ornithine is currently underway. The results will be reported shortly. REFERENCES 1. Salvatore, F., Cimino, F., Maria, C., and Cittadini, D.: Mechanism of the protection by L-ornithine-L-aspartate mixture and by L-arginine in ammonia intoxication. Arch. Biochem. Biophys., 107, 499-503 (1964). 2. Kinoshita, S., Nakayama, K., and Udaka, S.: The fermentative production of L-ornithine. J. Gen. Appl. Microbial., 3, 276277 (1957). 3. Udaka, S. and Kiooshita, S.: Studies on L-ornithine fermentation. I. The biosynthetic pathway of L-ornithine in Micrococcus glutamicus. J. Gen. Appl. Microbial., 4, 272-282 (1958). 4. Udaka, S. and Kinoshita, S.: Studies on L-ornithine fermentation. II. The change of fermentation product by a feedback type mechanism. J. Gen. Appl. Microbial., 4, 283-288 (1958). 5. Deken, R. H.: Pathway of arginine biosynthesis in yeast. Biochem. Biophys. Res. Commun., 8,462-466 (1962). 6. Yoshida, H., Araki, K., and Nakayama, K.: Mechanism of L-
14.
15.
16.
17. 18.
19. 20. 21.
22.
23.
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arginine production by L-arginine-production mutants of C. glutumicum. Agric. Biol. Chem., 43, 105-111 (1979). Yoshida, H., Araki, K., and Nakayama, K.: N-Acetylglutamate-acetylornithine acetyltransferasedeficient arginine auxotroph of C. glutamicum. Agric. Biol. Chem., 43. 18991903 (1979). Yoshida, H., Araki, K., and Nakayama, K.: N-Acetylornithine-o”-aminotransferase-deficient and N-acetylglutamokinase-deficient arginine auxotrophs of C. glutamicum. Agric. Biol. Chem., 44, 361-365 (1980). Udaka, S.: Pathway-specific pattern of control of arginine biosynthesis in bacteria. J. Bacterial., 91, 617-621 (1966). Hueting, S. and Tempest, D. W.: Influence of the glucose input concentration on the kinetics of metabolite production by Klebsiella aerogenes NCTC418, growing in chemostat culture in potassium- or ammonium-limited environments. Arch. Microbiol., 123, 189-194 (1979). Bader, F. B.: Kinetics of double substrate-limited growth, p. l32. In Bazin, M. J. (ed.), Microbial population dynamics. CRC series in mathematical models in microbiology. CRC Press, Boca Baton (1984). Egli, T. H. and Quayle, J. R.: Influence of the carbon: nitrogen ratio of the growth medium on the cellular composition and the ability of the methylotrophic yeast Hansenula polymorpha to utilize mixed carbon sources. J. Gen. Microbial., 132, 1779-1788 (1986). Grazer-Lampart, S. D., Egli, T. H., and Hammer, G.: Growth of Hyphomicrobium ZV620 in the chemostat: regulation of NH4+-assimilating enzymes and cellular composition. J. Gen. Microbial., 132, 3337-3347 (1986). BaItzIs, B.C. and Fredrickson, A. G.: Limitation of growth rate by two complementary nutrients: some elementary but neglected considerations. Biotechnol. Bioeng., 31, 75-86 (1988). Minkevich, I. G. and Eroshin, V. H.: A double substrate limitation zone of continuous microbial growth, p. 171-184. In Kyslik, P. and Novak, M. (ed.), Continuous culture. Academic Press, London (1988). Duchars, M. G. and Attwood, M. M.: The influence of C : N ratio in the growth medium on the cellular composition and regulation of enzyme activity in Hyphomicrobium X. J. Gen. Microbial., 135, 787-793 (1989). Postgate, J. R. and Hunter, J. R.: The survival of starved bacteria. J. Gen. Microbial., 29, 233-263 (1962). Neijssel, 0. and Tempest, D. W.: Bioenergetic aspects of aerobic growth of Klebsiella aerogenes NCTC 418 in carbon-limited and carbon-sufficient chemostat culture. Arch. Microbial., 107, 215-221 (1975). Chinard, F. P.: Photometric estimation of proline and ornithine. J. Biol. Chem., 199, 91-95 (1952). Rosenberg, H., Ennor, A. H., and Morrison, J. F.: The estimation of arginine. Biochem. J., 63, 153-159 (1956). Taussky, H. H. and Shore, E.: A microcaloric method for the determination of inorganic phosphorous. J. Biol. Chem., 202, 675-678 (1953). Kiss, R. D. and Stephanopoulos, G.: Metabolic characterization of a r.-lysine-producing strain by continuous culture. Biotechnol. Bioeng., 39, 565-574 (1992). Park, N. H. and Rogers, P. L.: L-Phenylalanine production in continuous culture using a hyper producing mutant of E. coli K 12. Chem. Eng. Commun., 45, 185-196 (1986).
BIOENCXNBERING
Vol. 81, No. 3, 216-219. 1996
Production of L-Ornithine by Arginine Auxotrophic Mutants of Brevibacterium ketoglutamicum in Dual Substrate-Limited Continuous Culture DAE KEON CHOI,’
WUK SANG RYU,’ CHA YONG CHOI,Z AND YOUNG HOON PARK’*
Bioprocess Technology Research Group, Korea Research Institute of Bioscience and Biotechnology, P. 0. Box 115, Yusong, Taejon 305-600’ and Department of Chemical Technology, College of Engineering, Seoul National University, Seoul 151-742,2 Korea Received 18 August 1995/Accepted 15 December 1995 Production of L-ornithine under dual limitation of arginine and phosphate was investigated in a chemostat culture of an arginine auxotroph of Brevibucterium ketoglutumicum. Under an arginine-iimited condition, culture instability, including the formation of revertant cells, could be a serious problem due to the inherent instability of the auxotrophic mutant strain. The difference in the growth rates between the auxotrophs and revertants was considered to be the main cause of the culture instability, and dual limitation with arginine and phosphate was found most appropriate to overcome this problem. When the ratio of the phosphate to arginine concentration in the feed medium was varied between 0.55 and 5.22 at a fixed dilution rate of 0.1 h-l, three distinct growth phases were recognized: phosphate-limited (0.55 to 2.01), dual arginine- and phosphate-limited (2.01 to 2.16) and a&nine-limited (above 2.16). In the dual arginine- and phosphate- limitation phase, the culture stability was markedly improved while the yield of L-ornithine was maintained at a high level. When the dilution rate was varied from 0.04 h-l to 0.17 h-l under the dual-limitation condition, maximum L-ornithine productivity was obtained at a value of 0.27 g/l/h at D=O.l h-l. [Key words: L-ornithine, Brevibacterium ketoglutamicum, arginine auxotroph, continuous substrate limitation]
culture, dual
More recently, it has been reported that under certain conditions the growth rate of an organism may be simultaneously limited by two or more substrates (10-16). It has also been indicated that a transition phase exists where the cell growth is limited by multiple nutrients (10-16). It is thus important to select a suitable pair for dual substrate limitation, since the physiological and metabolic activities of cells will differ depending on the choice of the two substrates (14). In this study, for the continuous production of Lornithine, phosphate was selected for dual substrate limitation along with arginine, since it is well known that under phosphate limitation bacterial cells exhibit higher steady-state cell viability (17) and metabolic activity (18) than under other substrate limitations. In addition, phosphate was considered to be one of the most appropriate limiting substrates for suppressing the overgrowth of revertant cells. A series of continuous culture experiments was carried out under dual phosphate- and arginine-limitation condition, and the culture behavior was analyzed with a view to process optimization for L-ornithine production.
L-Ornithine, an intermediate metabolite in arginine biosynthesis, and its derivatives have been reported to be effective for liver treatment (1). The first fermentative production of L-ornithine was in 1957 (2); L-ornithine was accumulated at a high yield (36% molar yield from glucose) using a citrulline-requiring mutant of Corynebacterium glutamicum. Since then, its biosynthetic pathway and regulatory mechanism have been well studied in glutamic acid-producing bacteria (3-8). N-Acetylglutamate kinase, the key enzyme in the biosynthetic pathway from glutamate, is known to be inhibited by L-arginine (6). Hence, to produce L-ornithine at a higher yield, the concentration of L-arginine should be controlled at low level during the fermentation (9). Although a higher product yield can be obtained with arginine limitation when auxotrophic mutants are used, culture instability often arises as a serious problem since it causes process instability, i.e., as the fermentation proceeds, the population of revertant cells increases and consequently the production of L-ornithine drops markedly. This is mainly due to the significant difference in the growth rates between the auxotrophic and revertant cells. Since revertant cells are more likely to grow faster than auxotrophic ones, once they appear in the culture they will eventually become dominant. Hence, to improve culture stability, it was necessary to find some means of controlling the growth rates of the two competing types of cells. Dual nutrient limitation was thus introduced to the continuous production of L-ornithine, one nutrient controlling cell growth and the other (arginine) product biosynthesis.
MATERIALS
AND METHODS
Microorganism The microorganism used in this study was Brevibacterium ketoglutamicum BK 533, an arginine auxotrophic mutant strain, which was derived from B. ketoglutamicum ATCC 21092 by mutagenesis using NTG and UV irradiation. The BK 533 strain lacks the enzyme ornithine carbamoyltransferase, which converts ornithine to citrulline, hence preventing arginine formation. Therefore, if the external supply of arginine
* Corresponding author. 216
L-ORNITHINE PRODUCTION
VOL. 81, 1996
is limited, its feedback inhibition is prevented, resulting in overproduction of L-ornithine. Media YPD medium containing 2% glucose, 1% yeast extract and 1% bacto peptone was used for the initial seed culture. The feed medium for the continuous culture consisted of 40 g glucose, 2 g yeast extract, 0.6 g KCI, 5 g (NH&Sod, 0.5 g MgS04.7Hz0, 50 mg L-arginine, O-2 g Na2HP04. 12Hz0, and 10 ml 100 x trace element solution per liter of distilled water. The 100 x trace element solution consisted of 500 mg MnS04.4Hz0, 200 mg NazMo04. 2H20, 500 mg ZnS04. 7H20, 10 mg H3B03, 40 mg CuS04.5H20, 10 mg CoC12.6H20, and 100 mg FeS04. 7Hz0 per liter of distilled water. Culture conditions A loopful of cells grown on a nutrient agar plate for 48 h at 30°C was inoculated into two 500-ml baffled flasks containing 50ml seed medium. After 13 h cultivation at 30°C and 170 rpm on a rotary shaker, the seed culture broth was transferred to a 2-f jar fermentor (Korea Fermentor Co., Inchon, Korea) containing 800 ml of the starting medium. After the cell growth reached the mid-exponential phase, continuous culture was started by supplying the feed medium and withdrawing the culture broth according to the specified dilution rates. The temperature was maintained at 30°C and the pH at 7.0 with 4N NH40H. The aeration rate was controlled in such a way that dissolved oxygen was not limited; the agitation speed was kept at 600rpm. Analytical methods Cell growth was monitored by measuring the optical density at 600 nm using a spectrophotometer (Uvicon 930, Kontron Instrument Co., Switzerland). The glucose concentration was measured using a YSI glucose analyzer (YSI 2000, Yellow Springs Instruments Co., USA). The L-ornithine, L-arginine and inorganic phosphate concentrations were determined calorimetrically (19-21). The total cell number and the number of revertant cells were determined by serially diluted plate counts on complex agar medium and minimal agar medium lacking arginine, respectively. Culture stability was defined as the ratio of the number of auxotrophic cells to the total cell number.
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;:
tt
:
z 60 L z : 4 40 M 0 20
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15og
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: 100 :g- 400
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0
600
9 a,
P 10
0
20
40
60 Time
FIG. 1. Chemostat dilution rate of 0.1 h-l.
experiment
80
100
50
z - 200
0
- 0
;; &
:
E
D
120
(h)
with arginine limitation
at a
Dual substrate- (arginine and phosphate) limited culture was thus tried. Under phosphate limitation, the growth rates of the two competing types of cells could be controlled equally well, so no growth advantage existed for either type. Hence, we combined phosphate limitation (to remove the growth advantage of revertant cells) with arginine limitation (for efficient production of Lornithine) in the culture. A series of continuous culture experiments was conducted at a constant dilution rate of 0.1 h-l to ascertain the optimum phosphate/arginine concentration ratio in the feed medium which would provide the dual limiting condition in the culture. Keeping the concentration of arginine in the feed medium lixed, the concentration of phosphate was varied and the steady-state behavior monitored. The results are summarized in Fig. 2. As the phosphate/arginine ratio increased from 0.55 to 5.22, three
RESULTS AND DISCUSSION Optimal feeding ratio of dual limiting substrates For efficient production of L-ornithine, an arginine-limited culture condition is essential. A typical time course of an arginine-limited chemostat culture at a dilution rate of 0.1 h-’ is shown in Fig. 1 (the phosphate/arginine concentration ratio in the feed medium is 5.22). It was noted that the total cell mass increased rapidly after about 90 h of chemostat operation. The population of auxotrophic cells was observed to decrease to less than 40% of the total cell mass, accompanied by a marked drop in the product concentration. This was obviously because of the rapid increase in the revertant population. Under such culture conditions, the growth of revertant cells could not be controlled by the dilution rate, while the auxotrophs could be controlled, since revertant cells could synthesize the additional arginine necessary for growth, while the auxotrophs could not. For this reason, a difference in the growth rates of the two competing types of cells was generated under the arginine-liited culture condition &,,. > pBux.). To improve the system performance, therefore, some measure was needed to repress the overgrowth of revertant cells during cultivation under the arginine-limited condition.
A 0
1
1
I
I
I
2
3
4
5
Phosphote/Arginine
6
ratio(w/w)
FIG. 2. Steady-state values of fermentation parameters at various ratios of phosphateiarginine in the feed medium.
218
J. FERMENT.BIOENG.,
CHOI ET AL.
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F
culture at a phosphatelarginine
distinct growth phases were recognized in terms of the residual concentrations of phosphate and arginine-phosphate-limited (0.55 to 2.01), dual phosphate- and arginine-limited (2.01 to 2.16) and arginine-limited (above 2.16) phases. During the first phase, arginine was present in excess while phosphate was completely utilized. L-Omithine was scarcely produced due to the inhibition of N-acetylglutamate kinase by the high level of arginine in the culture broth. However, as the ratio increased, the steady-state level of arginine decreased as the cell mass increased, which, in turn, resulted in a higher steady-state level of L-ornithine. The linear increase in L-ornithine production as the phosphate/arginine ratio increased in the first phase could be ascribed to the increase in the steadystate cell mass concentrations of auxotrophs and to the decrease of feedback inhibition by arginine. It was also evidenced by the increase of the specific L-ornithine production rate (qp) and the yield on glucose (Y,,,). In this phase, chemostat operation continued for about 100-120 h and the proportion of revertants was negligible (1/104-1/105). In the dual phosphate- and argininelimited phase, the overgrowth of the revertants was still suppressed yet the qp values were maintained at high levels. The L-ornithine production pattern could be further extended to the arginine-limited phase, with phosphate/ arginine ratios higher than 2.16. At pho/arg=2.24, chemostat operation continued for 120 h and the ratio of revertants was also negligible (1/103). However, at pho/ arg=5.22 culture instability arose as a serious problem, as shown in Fig. 1. From the chemostat experiment, the optimal ratio of the dual limiting substrates (phosphateiarginine) was found to be located in a range from 2.01 to 2.16. Longterm continuous culture at a phosphate/arginine ratio of 2.01 was then carried out at the same dilution rate of 0.1 h-l. As shown in Fig. 3, the culture stability was significantly improved for longer than 250 h chemostat operation while maintaining a high level of L-ornithine productivity. These results indicated that optimally designed feeding of the dual limiting substrates could not only improve the culture stability significantly but also enhance the production of L-ornithine in a chemostat culture. Effects of dilution rate Further chemostat culture experiments were carried out at various dilution rates
A .
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20
100
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0
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4
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0 0
20
40 Time
60
(h)
FIG. 4. Profiles of continuous cultures with a fixed phosphate/arginine ratio of 2.01. (a) D=O.O4 h-1; (b) D=O.17 h-l.
from 0.04 h-r to 0.17 h-l at a phosphate/arginine ratio of 2.01. Typical profiles of the continuous cultures at dilution rates of 0.04 hhl and 0.17 h-i are shown in Fig. 4. Residual concentrations of phosphate and arginine were found to be at a limiting level at all the dilution rates, clearly indicating that the cells were in a state of dual arginine- and phosphate-limitation over the whole range of dilution rates tested. However, with changes in dilution rate, considerable differences in the production of biomass and in L-ornithine production were observed. In Fig. 5, steady-state cell mass and L-ornithine concentrations along with some kinetic parameters are shown as a function of the dilution rate. The cell mass and Lornithine concentration increased 0.6- and 3-fold, respectively, as the dilution rate decreased from 0.17 h--I to 0.04 hh’. Similar observations were reported by Kiss et al. (22) in lysine production in threonine-limited continuous culture and by Park et al. (23) in phenylalanine production in phosphate-limited continuous culture. As can be seen in Fig. 5b, the product yields on glucose (Y,,,) and on cell mass (Y& decreased as the dilution rate increased, while the cell mass yield on glucose (Y,,,) increased. Since the cells were under the arginine- and phosphate-limited condition at all dilution rates tested, feedback inhibition by arginine would not have occurred and the biosynthetic activity for L-ornithine production remained fully active in all experiments. Therefore, the findings can be explained by the fact that, as the dilution rate decreased, a greater portion of the glucose was utilized for product formation rather than for cell mass production under the dual substrate limiting condition. It was also shown in the present study that the L-ornithine productivity had a maximum value of 0.27 g/l/h at D=O.l hh’, so D=O.l h-r was considered to be the optimal dilution rate.
L-ORNITHINE PRODUCTION
VOL. 81, 1996
7.
8.
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9.
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10.
0.4 2 0.2 0 0.4 _ f 0.3 7 i Z 0.2 P .? i t;
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Dilution
rote
0.2
11.
12.
1 -0 L?
a 0
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(h-‘)
FIG. 5. Relationship between steady-state parameters and dilution rate at a phosphatelarginine concentration ratio in the feed medium of 2.01.
In conclusion, we have demonstrated that dual substrate limitation can be successfully applied to the bench-scale production of L-ornithine in a continuous mode, and we believe that the same principle should be applicable to large-scale industrial production of the amino acid. A pilot-scale (300 r) operation for the continuous production of L-ornithine is currently underway. The results will be reported shortly. REFERENCES 1. Salvatore, F., Cimino, F., Maria, C., and Cittadini, D.: Mechanism of the protection by L-ornithine-L-aspartate mixture and by L-arginine in ammonia intoxication. Arch. Biochem. Biophys., 107, 499-503 (1964). 2. Kinoshita, S., Nakayama, K., and Udaka, S.: The fermentative production of L-ornithine. J. Gen. Appl. Microbial., 3, 276277 (1957). 3. Udaka, S. and Kiooshita, S.: Studies on L-ornithine fermentation. I. The biosynthetic pathway of L-ornithine in Micrococcus glutamicus. J. Gen. Appl. Microbial., 4, 272-282 (1958). 4. Udaka, S. and Kinoshita, S.: Studies on L-ornithine fermentation. II. The change of fermentation product by a feedback type mechanism. J. Gen. Appl. Microbial., 4, 283-288 (1958). 5. Deken, R. H.: Pathway of arginine biosynthesis in yeast. Biochem. Biophys. Res. Commun., 8,462-466 (1962). 6. Yoshida, H., Araki, K., and Nakayama, K.: Mechanism of L-
14.
15.
16.
17. 18.
19. 20. 21.
22.
23.
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arginine production by L-arginine-production mutants of C. glutumicum. Agric. Biol. Chem., 43, 105-111 (1979). Yoshida, H., Araki, K., and Nakayama, K.: N-Acetylglutamate-acetylornithine acetyltransferasedeficient arginine auxotroph of C. glutamicum. Agric. Biol. Chem., 43. 18991903 (1979). Yoshida, H., Araki, K., and Nakayama, K.: N-Acetylornithine-o”-aminotransferase-deficient and N-acetylglutamokinase-deficient arginine auxotrophs of C. glutamicum. Agric. Biol. Chem., 44, 361-365 (1980). Udaka, S.: Pathway-specific pattern of control of arginine biosynthesis in bacteria. J. Bacterial., 91, 617-621 (1966). Hueting, S. and Tempest, D. W.: Influence of the glucose input concentration on the kinetics of metabolite production by Klebsiella aerogenes NCTC418, growing in chemostat culture in potassium- or ammonium-limited environments. Arch. Microbiol., 123, 189-194 (1979). Bader, F. B.: Kinetics of double substrate-limited growth, p. l32. In Bazin, M. J. (ed.), Microbial population dynamics. CRC series in mathematical models in microbiology. CRC Press, Boca Baton (1984). Egli, T. H. and Quayle, J. R.: Influence of the carbon: nitrogen ratio of the growth medium on the cellular composition and the ability of the methylotrophic yeast Hansenula polymorpha to utilize mixed carbon sources. J. Gen. Microbial., 132, 1779-1788 (1986). Grazer-Lampart, S. D., Egli, T. H., and Hammer, G.: Growth of Hyphomicrobium ZV620 in the chemostat: regulation of NH4+-assimilating enzymes and cellular composition. J. Gen. Microbial., 132, 3337-3347 (1986). BaItzIs, B.C. and Fredrickson, A. G.: Limitation of growth rate by two complementary nutrients: some elementary but neglected considerations. Biotechnol. Bioeng., 31, 75-86 (1988). Minkevich, I. G. and Eroshin, V. H.: A double substrate limitation zone of continuous microbial growth, p. 171-184. In Kyslik, P. and Novak, M. (ed.), Continuous culture. Academic Press, London (1988). Duchars, M. G. and Attwood, M. M.: The influence of C : N ratio in the growth medium on the cellular composition and regulation of enzyme activity in Hyphomicrobium X. J. Gen. Microbial., 135, 787-793 (1989). Postgate, J. R. and Hunter, J. R.: The survival of starved bacteria. J. Gen. Microbial., 29, 233-263 (1962). Neijssel, 0. and Tempest, D. W.: Bioenergetic aspects of aerobic growth of Klebsiella aerogenes NCTC 418 in carbon-limited and carbon-sufficient chemostat culture. Arch. Microbial., 107, 215-221 (1975). Chinard, F. P.: Photometric estimation of proline and ornithine. J. Biol. Chem., 199, 91-95 (1952). Rosenberg, H., Ennor, A. H., and Morrison, J. F.: The estimation of arginine. Biochem. J., 63, 153-159 (1956). Taussky, H. H. and Shore, E.: A microcaloric method for the determination of inorganic phosphorous. J. Biol. Chem., 202, 675-678 (1953). Kiss, R. D. and Stephanopoulos, G.: Metabolic characterization of a r.-lysine-producing strain by continuous culture. Biotechnol. Bioeng., 39, 565-574 (1992). Park, N. H. and Rogers, P. L.: L-Phenylalanine production in continuous culture using a hyper producing mutant of E. coli K 12. Chem. Eng. Commun., 45, 185-196 (1986).