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Effect of dilution rate in continuous production of l -ornithine by an arginine auxotrophic mutant
JOURNALOF FERMENTATION ANDBIOENGINEERING Vol. 80, No. 1, 97-100. 1995
Effect of Dilution Rate in Continuous Production of L-Ornithine by an Arginine Auxotrophic Mutant DAE KEON CHOI, WUK SANG RYU, BONG HYUN CHUNG, SOON OOK HWANG, AND YOUNG HOON PARK* Bioprocess Technology Research Group, Genetic Engineering Research Institute, KIST, Taedok Science Town, Taejon 305-600, Korea Received 20 January 1995/Accepted 6 April 1995
Continuous production of L-omitbine by an arginine auxotroph of Brevibacterium ketoglutamicum was carried out in a 2-Zjar fermentor. The effects of the dilution rate on the fermentation parameters were investigated under L-arginine limited conditions. The maximum L-omithine yield and volumetric productivity obtained were 0.213 g/g and 0.313 g/l/h, respectively. The specific L-ornithine production rate increased with the dilution rate, with a maximum value of 0.084 g/g/h at a dilution rate of 0.125 h-l. [Key words: L-ornithine, Brevibacterium ketoglutamicum, arginine auxotroph,
L-Omithine belongs to the glutamic acid family (1) and is an intermediate metabolite in arginine biosynthesis. It is known to be effective in the treatment of liver disorders (2). Its fermentative production was first reported in 1957 (3). L-Ornithine was accumulated with a high yield (36% molar yield from glucose) using a citrullinerequiring mutant of Corynebacterium glutamicum. Since then, its biosynthetic pathway and regulatory mechanism have been well studied in Escherichia coli (4, 5) and also in glutamic acid-producing bacteria (6-10). N-Acetylglutamatekinase, a key enzyme in the biosynthetic pathway from glutamate, is known to be inhibited by L-arginine (11). Hence, to produce r.-ornithine with a higher yield, the concentration of L-arginine should be controlled at a low level during the fermentation (12). The growth rate of the microorganism should also be carefully maintained within an optimal range, since the cellular metabolic activity is highly dependent on the specific growth rate. The effects of the specific growth rate on the production of L-ornithine should therefore be analyzed first to optimize the fermentation process. In the present study, continuous culture experiments were carried out with an L-ornithine overproducing mutant, strain BK533, to investigate the effects of the dilution rate (specific growth rate) on the specific substrate uptake rate, L-ornithine production rate, production yield and volumetric productivity under L-arginine limited conditions. The metabolic activity of the strain was also ana-
continuous
culture]
(NH&S04, 0.5 g MgS04.7H20, 100 mg L-arginine, lOm1 100X trace element solution per liter of distilled water. This was also used as the feed medium for the continuous operation. YNB was used to supplement the intrinsically required cofactors such as biotin, thiamine and other vitamins. The 100X trace element solution consisted of 500 mg MnS04. 4H20, 200 mg NazMo04. 2H20, 500mg ZnS04.7Hz0, 10 mg H3B03, 40 mg CuS04. 5H20, 10mg CoC12.6Hz0, and 1OOmg FeS04.7Hz0 per liter of distilled water. A loopful of cells grown on a nutrient agar plate for 48 h at 30°C was inoculated into 500-ml baffled flask containing 30 ml 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-l jar fermentor (Korea Fermentor Co., Inchon, Korea) containing 11 of the defined medium. At the mid-exponential phase, continuous culture was started by supplying the feed medium and withdrawing the culture broth continuously. The temperature was maintained at 30°C and the pH at 7.0 with 4 N NH40H. Agitation speed was kept at
lyzed .
The microorganism used in this study was BrevibacteriBK533, an L-arginine auxotrophic mutant, which was derived from B. ketoglutamicum ATCC 21092. B. ketoglutamicum ATCC 21092 is not a wild-type strain but is reported to be an ornithine producer (arginine-requiring mutant). It was further treated by UV irradiation and NTG to derive a strain of higher yield. YPD medium containing 2% glucose, 1% yeast extract and 1% Bacto Pepton was used for the seed cultures. A chemically defined medium which was used for batch and fed-batch cultures consisted of: 40g glucose, 6.7 g YNB (yeast nitrogen base w/o amino acids), 0.5 g KH2POI, 1 g Na2HP0.,. 12H20, 5 g urn ketoglutamicum
0
5
10
15
20
Time
Ih\
25
30
35
FIG. 1. Growth response of BK 533 to arginine concentrations in shake-flask culture experiments. BK 533 was cultivated at 30°C and 180 rpm for 33.5 h. Arginineconcentrations (mg/C): 0( 0), 12.5 (v), 25 ( q), 50 (A),75 (O), 125 (v), 250 ( l).
* Corresponding author. 97
98
CHOI ET AL.
J. FERMENT.BIOENG., 50
10
40
30
20
10
i BOO Time (h)
Time (h)
FIG. 2. Time courses of L-ornithine production in (a) batch and (b) fed-batch cultures. The initial concentration of arginine was 100 mg/l in both cases. (a) Without additional feeding of arginine; (b) with continuous feeding of arginine (started at 11 h when the initially added arginine was almost exhausted). Glucose (40 g/l) and ammonium sulphate (5 g/f) were supplemented at 27 h.
600rpm. Air was supplied at a rate of 1 wm. Cell growth was monitored by measuring the optical density at 600 nm spectrophotometrically using a spectrophotometer (UVICON 930, Kontron Instrument Co., Switzerland). Glucose was measured by a glucose & lactate Analyzer (YSI 2000, Yellow Springs Instrument Co., USA). L-0rnithine and L -arginine were determined calorimetrically (13, 14). In the course of the selection process for high yield strains, the strains obtained were very unstable during the cultivation due to loss of their amino acid producing activities. However, as the selection process was repeated, more stable L-ornithine producer strains could be obtained, though it was very difficult to prevent the formation of revertant (amino acid non-producing) cells completely. Strain BK 533 was one of the stable and highyielding strains, in which the proportion of revertant cells at the start of the continuous culture was below l/ 106-l/10’, which was very low compared to that (1/103l/l@) of unstable strains. The proportion of revertant cells was found to be negligible even after 95 h of cultivation with arginine limitation. This culture stability could be maintained at all experiments. The growth response of strain BK533 to the arginine concentration in the
shake-flask culture experiments is shown in Fig. 1. The cell concentration appeared to increase linearly with the increase in the arginine concentration. To characterize the kinetic relationship between growth and L-ornithine production, batch and fed-batch culture experiments were conducted in a jar fermentor prior to continuous culture (Fig. 2). It was interesting to note that although cell growth stopped with the exhaustion of arginine, a steady accumulation of L-ornithine could be observed (Fig. 2a). On the other hand, when arginine was continuously supplied to the fermentor in a limited amount, cell growth continued and L-ornithine was produced in parallel with the cell growth at a specific production rate of 0.02 g/g/h, which was 1.6 times higher than that of the culture without arginine feeding (Fig. 2b). In these experiments, it was noted that L-ornithine was produced according to a growth-associated pattern, but that a considerable amount of the amino acid was also accumulated although cell growth ceased. In order to study the effects of the dilution rate on the production of L-ornithine, chemostat experiments with arginine as a limiting nutrient were carried out. A typical set of data for the continuous production of L-ornithine 40 5
14 12(-
(b)
9
10 Y&_____
-30
.c P - 20 ;
I)6 -
Time FIG.
3.
(h)
i
ti
Time
(h)
Continuous culture of r.-ornithine overproducing mutant strain BK533. (a) Dilution rate=0.078 h-r; (b) 0.153 h-l.
NOTES
VOL.80, 1995
J 0.0 &
0.4 ? ;
;:
-(O)
*
:;;;
< n fi
cl
' 0.1
-
I
0.0
0.1
- 0.4 - 0.2
0.0
FIG. 4. tion rate.
0.0 0.2
Dilution rote (h’l Relationship between steady state parameters and dilu-
at dilution rates of 0.078 h-l and 0.153 h-l is given in Fig. 3. The cultures were allowed to grow in batch mode for about 5 h prior to the start of continuous operation. A steady state was assumed after about 3-6 residence time, when the variables (biomass, glucose and L-ornithine concentration) became nearly constant. As can be seen from the figure, production of L-ornithine reached a steady state level about 20 h after the onset of arginine limitation in the culture. The effects of the dilution rate on the biomass and Lornithine production are summarized in Fig. 4a. The biomass concentration decreased as the dilution rate increased, which is similar to the case of L-lysine fermentation under the condition of threonine limitation (15). This can be explained by the fact that continuous culture under the limitation of an essential amino acid (in the present case, arginine) is, as was pointed out by Kiss et al. (15), analogous to the nitrogen-limited continuous culture of a prototrophic microbe in that cell growth is limited by the limitation of protein synthesis. Under nitrogen-limited growth, the cell concentration increased as the specific growth rate decreased due to the accumulation of carbon-energy reserve compounds and showed an apparent increase in cell yield (16, 17). L-Ornithine production was largely dependent on the dilution rate, and the highest titer of L-ornithine was 3.2 g/Z at the dilution rate of 0.078 h--l. In Fig. 4b, it can be seen that the specific uptake rates of glucose (qs) rose as the dilution rate increased. The specific L-ornithine production rate (qp) and volumetric productivity also increased, and L-ornithine production was thus considered to be growth-associated in a dilution rate range from 0.056 to 0.125 h-l. However, it was observed that at dilution rates higher than 0.125 h- l, the specific L-ornithine production rate decreased. A similar observation was previously reported in the case of L-lysine production (15). As the cell growth rate increases, more precursor metabolites are drawn off at various points in the metabolic pathways
99
for biomass production. Hence, the amount of carbon source utilized for the synthesis of L-ornithine decreases as the specific growth rate increases, which in turn results in the reduction of the specific production rate of L-ornithine. This was also reflected in the biomass and L-ornithine production yields, as shown in Fig. 4c. With increasing dilution rate, the biomass yield on glucose (YXjS)increased, while the product yields on glucose (Y& and on cell mass (Y+) showed a decreasing pattern, with maximum values of 0.213 and 0.853 g/g, respectively, at a dilution rate of 0.078 h.-‘. These results show that a larger portion of glucose was used for the production of L-ornithine than for the biomass production at this dilution rate. At lower dilution rates, the decrease in the L-ornithine yield could be attributed to the requirement for energy maintenance. The above kinetic parameter values could not be directly compared with other results because no other continuous-culture data on L-ornithine production have so far reported. However, the maximum L-ornithine yield of 0.213 g/g (29% molar yield from glucose) was not so much lower than that (36% molar yield from glucose) reported by Kinoshita et af. (3), and this strain is considered to have a potential for the practical production of L-ornithine. It was found that under arginine limitation, there existed a different optimal specific growth rate which gave the highest production yield or productivity. That is, while the greatest productivity was obtained in a higher specific growth rate range, the highest L-ornithine yield and titer were obtained in a lower range. It is well known that the fermentative production of amino acids is typically a recovery cost-intensive process (18), in which the product concentration is a critical factor for development of the process. Hence, the results of the present study provide the important observation that the practical production of L-ornithine can be realized through the fed-batch culture of this organism in a lower growth rate range, which will give the highest L-ornithine yield and titer. An improved strategy for fed-batch culture operation based on this observation resulted in a much higher productivity of up to 1 g of L-ornithine/I/h for 3-d operation (data not shown). REFERENCES 1. Davis, B. D.: Intermediates
2.
3. 4. 5. 6.
in amino acid biosynthesis, p, 257268. In Meister, A. (ed.), Advances in enzymology. Interscience Publishers Ltd., London (1955). Selvatore, F., Chino, 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). Kinoshita, S., Nakayama, K., and Udaka, S.: The fermentative production of L-ornithine. J. Gen. Appl. Microbial., 3, 276277 (1957). Vogel, H. J.: Path of ornithine synthesis in E. coli. Proc. Natl. Acad. Sci. USA, 39, 578-583 (1953). Vogel, H. J., McElroy, W. D., and Glass, B.: Amino acid metabolism, p. 335. Johns Hopkins Press, Baltimore (1955). Udaka, S. and Kinoshita, S.: Studies on L-ornithine fermentation. I. The biosynthetic pathway of L-ornithine in Micrococcus glutamicus. J. Gen. Appl. Microbial., 4, 272-282 (1958). Udaka, S. and Kinoshita, S.: Studies on L-ornithine fermentation. IL The change of fermentation product by a feedback type mechanism. J. Gen. Appl. Microbial., 4, 283-288 (1958). Deken, R. H.: Pathway of arginine biosynthesis in yeast. Biochem. Biophys. Res. Commun., 8, 462-466 (1962). Yoshida, H., Araki, K., and Nakayama, K.: N-Acetyl-
100
10.
11.
12. 13.
CHOI ET AL. glutamate-acetylornithine acetyltransferase-deficient arginine auxotroph of C. glutamicum. Agric. Biol. Chem., 43, 18991903 (1979). Yoshida, H., Araki, K., and Nakayama, K.: iV-Acetylornithine-&aminotransferase-deficient and N-acetylglutamokinasedeficient arginine auxotrophs of C. glutamicum. Agric. Biol. Chem., 44,361-365 (1980). Yoshida. H.. Araki. K.. and Nakavama. K.: Mechanism of Larginine’ production by’ t.-arginine-production mutants of C. glutamicum. Agric. Biol. Chem., 43, 105-111 (1979). Udaka, S.: Pathway-specific pattern of control of arginine biosynthesis in bacteria. J. Bacterial., 91, 617-621 (1966). Chinard, F.P.: Photometric estimation of proline and ornithine. J. Biol. Chem., 199, 91-95 (1952).
J.
FERMENT. BIOENG.,
14. Rosenberg, H., Enoor, A. H., and Morrison, J. F.: The estimation of arginine. Biochem. J., 63, 153-159 (1956). 15. Kiss, R. D. and Stephanopoolos, G.: Metabolic characterization of a L-lysine-producing strain by continuous culture. Biotechnol. Bioena.. 39. 565-574 (1992). 16. Senior, P. J.: Regilation of nitrogen metabolism in Escherichia co/i and Klebsielia aerogenes: studies with the continuous culture technique. J. Bacterial., 123, 407-418 (1975). 17. Wang, D. I. C., Cooney, C. L., Demain, A. L., Donnill, P., Humphrey, A. E., and Lilly, M. D.: Fermentation and enzyme technology, p. 98-137. John Wiley & Sons, New York (1979). 18. Cooney, C. L.: Bioreacters: design and operation. Science, 219, 728-733 (1983).
Effect of Dilution Rate in Continuous Production of L-Ornithine by an Arginine Auxotrophic Mutant DAE KEON CHOI, WUK SANG RYU, BONG HYUN CHUNG, SOON OOK HWANG, AND YOUNG HOON PARK* Bioprocess Technology Research Group, Genetic Engineering Research Institute, KIST, Taedok Science Town, Taejon 305-600, Korea Received 20 January 1995/Accepted 6 April 1995
Continuous production of L-omitbine by an arginine auxotroph of Brevibacterium ketoglutamicum was carried out in a 2-Zjar fermentor. The effects of the dilution rate on the fermentation parameters were investigated under L-arginine limited conditions. The maximum L-omithine yield and volumetric productivity obtained were 0.213 g/g and 0.313 g/l/h, respectively. The specific L-ornithine production rate increased with the dilution rate, with a maximum value of 0.084 g/g/h at a dilution rate of 0.125 h-l. [Key words: L-ornithine, Brevibacterium ketoglutamicum, arginine auxotroph,
L-Omithine belongs to the glutamic acid family (1) and is an intermediate metabolite in arginine biosynthesis. It is known to be effective in the treatment of liver disorders (2). Its fermentative production was first reported in 1957 (3). L-Ornithine was accumulated with a high yield (36% molar yield from glucose) using a citrullinerequiring mutant of Corynebacterium glutamicum. Since then, its biosynthetic pathway and regulatory mechanism have been well studied in Escherichia coli (4, 5) and also in glutamic acid-producing bacteria (6-10). N-Acetylglutamatekinase, a key enzyme in the biosynthetic pathway from glutamate, is known to be inhibited by L-arginine (11). Hence, to produce r.-ornithine with a higher yield, the concentration of L-arginine should be controlled at a low level during the fermentation (12). The growth rate of the microorganism should also be carefully maintained within an optimal range, since the cellular metabolic activity is highly dependent on the specific growth rate. The effects of the specific growth rate on the production of L-ornithine should therefore be analyzed first to optimize the fermentation process. In the present study, continuous culture experiments were carried out with an L-ornithine overproducing mutant, strain BK533, to investigate the effects of the dilution rate (specific growth rate) on the specific substrate uptake rate, L-ornithine production rate, production yield and volumetric productivity under L-arginine limited conditions. The metabolic activity of the strain was also ana-
continuous
culture]
(NH&S04, 0.5 g MgS04.7H20, 100 mg L-arginine, lOm1 100X trace element solution per liter of distilled water. This was also used as the feed medium for the continuous operation. YNB was used to supplement the intrinsically required cofactors such as biotin, thiamine and other vitamins. The 100X trace element solution consisted of 500 mg MnS04. 4H20, 200 mg NazMo04. 2H20, 500mg ZnS04.7Hz0, 10 mg H3B03, 40 mg CuS04. 5H20, 10mg CoC12.6Hz0, and 1OOmg FeS04.7Hz0 per liter of distilled water. A loopful of cells grown on a nutrient agar plate for 48 h at 30°C was inoculated into 500-ml baffled flask containing 30 ml 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-l jar fermentor (Korea Fermentor Co., Inchon, Korea) containing 11 of the defined medium. At the mid-exponential phase, continuous culture was started by supplying the feed medium and withdrawing the culture broth continuously. The temperature was maintained at 30°C and the pH at 7.0 with 4 N NH40H. Agitation speed was kept at
lyzed .
The microorganism used in this study was BrevibacteriBK533, an L-arginine auxotrophic mutant, which was derived from B. ketoglutamicum ATCC 21092. B. ketoglutamicum ATCC 21092 is not a wild-type strain but is reported to be an ornithine producer (arginine-requiring mutant). It was further treated by UV irradiation and NTG to derive a strain of higher yield. YPD medium containing 2% glucose, 1% yeast extract and 1% Bacto Pepton was used for the seed cultures. A chemically defined medium which was used for batch and fed-batch cultures consisted of: 40g glucose, 6.7 g YNB (yeast nitrogen base w/o amino acids), 0.5 g KH2POI, 1 g Na2HP0.,. 12H20, 5 g urn ketoglutamicum
0
5
10
15
20
Time
Ih\
25
30
35
FIG. 1. Growth response of BK 533 to arginine concentrations in shake-flask culture experiments. BK 533 was cultivated at 30°C and 180 rpm for 33.5 h. Arginineconcentrations (mg/C): 0( 0), 12.5 (v), 25 ( q), 50 (A),75 (O), 125 (v), 250 ( l).
* Corresponding author. 97
98
CHOI ET AL.
J. FERMENT.BIOENG., 50
10
40
30
20
10
i BOO Time (h)
Time (h)
FIG. 2. Time courses of L-ornithine production in (a) batch and (b) fed-batch cultures. The initial concentration of arginine was 100 mg/l in both cases. (a) Without additional feeding of arginine; (b) with continuous feeding of arginine (started at 11 h when the initially added arginine was almost exhausted). Glucose (40 g/l) and ammonium sulphate (5 g/f) were supplemented at 27 h.
600rpm. Air was supplied at a rate of 1 wm. Cell growth was monitored by measuring the optical density at 600 nm spectrophotometrically using a spectrophotometer (UVICON 930, Kontron Instrument Co., Switzerland). Glucose was measured by a glucose & lactate Analyzer (YSI 2000, Yellow Springs Instrument Co., USA). L-0rnithine and L -arginine were determined calorimetrically (13, 14). In the course of the selection process for high yield strains, the strains obtained were very unstable during the cultivation due to loss of their amino acid producing activities. However, as the selection process was repeated, more stable L-ornithine producer strains could be obtained, though it was very difficult to prevent the formation of revertant (amino acid non-producing) cells completely. Strain BK 533 was one of the stable and highyielding strains, in which the proportion of revertant cells at the start of the continuous culture was below l/ 106-l/10’, which was very low compared to that (1/103l/l@) of unstable strains. The proportion of revertant cells was found to be negligible even after 95 h of cultivation with arginine limitation. This culture stability could be maintained at all experiments. The growth response of strain BK533 to the arginine concentration in the
shake-flask culture experiments is shown in Fig. 1. The cell concentration appeared to increase linearly with the increase in the arginine concentration. To characterize the kinetic relationship between growth and L-ornithine production, batch and fed-batch culture experiments were conducted in a jar fermentor prior to continuous culture (Fig. 2). It was interesting to note that although cell growth stopped with the exhaustion of arginine, a steady accumulation of L-ornithine could be observed (Fig. 2a). On the other hand, when arginine was continuously supplied to the fermentor in a limited amount, cell growth continued and L-ornithine was produced in parallel with the cell growth at a specific production rate of 0.02 g/g/h, which was 1.6 times higher than that of the culture without arginine feeding (Fig. 2b). In these experiments, it was noted that L-ornithine was produced according to a growth-associated pattern, but that a considerable amount of the amino acid was also accumulated although cell growth ceased. In order to study the effects of the dilution rate on the production of L-ornithine, chemostat experiments with arginine as a limiting nutrient were carried out. A typical set of data for the continuous production of L-ornithine 40 5
14 12(-
(b)
9
10 Y&_____
-30
.c P - 20 ;
I)6 -
Time FIG.
3.
(h)
i
ti
Time
(h)
Continuous culture of r.-ornithine overproducing mutant strain BK533. (a) Dilution rate=0.078 h-r; (b) 0.153 h-l.
NOTES
VOL.80, 1995
J 0.0 &
0.4 ? ;
;:
-(O)
*
:;;;
< n fi
cl
' 0.1
-
I
0.0
0.1
- 0.4 - 0.2
0.0
FIG. 4. tion rate.
0.0 0.2
Dilution rote (h’l Relationship between steady state parameters and dilu-
at dilution rates of 0.078 h-l and 0.153 h-l is given in Fig. 3. The cultures were allowed to grow in batch mode for about 5 h prior to the start of continuous operation. A steady state was assumed after about 3-6 residence time, when the variables (biomass, glucose and L-ornithine concentration) became nearly constant. As can be seen from the figure, production of L-ornithine reached a steady state level about 20 h after the onset of arginine limitation in the culture. The effects of the dilution rate on the biomass and Lornithine production are summarized in Fig. 4a. The biomass concentration decreased as the dilution rate increased, which is similar to the case of L-lysine fermentation under the condition of threonine limitation (15). This can be explained by the fact that continuous culture under the limitation of an essential amino acid (in the present case, arginine) is, as was pointed out by Kiss et al. (15), analogous to the nitrogen-limited continuous culture of a prototrophic microbe in that cell growth is limited by the limitation of protein synthesis. Under nitrogen-limited growth, the cell concentration increased as the specific growth rate decreased due to the accumulation of carbon-energy reserve compounds and showed an apparent increase in cell yield (16, 17). L-Ornithine production was largely dependent on the dilution rate, and the highest titer of L-ornithine was 3.2 g/Z at the dilution rate of 0.078 h--l. In Fig. 4b, it can be seen that the specific uptake rates of glucose (qs) rose as the dilution rate increased. The specific L-ornithine production rate (qp) and volumetric productivity also increased, and L-ornithine production was thus considered to be growth-associated in a dilution rate range from 0.056 to 0.125 h-l. However, it was observed that at dilution rates higher than 0.125 h- l, the specific L-ornithine production rate decreased. A similar observation was previously reported in the case of L-lysine production (15). As the cell growth rate increases, more precursor metabolites are drawn off at various points in the metabolic pathways
99
for biomass production. Hence, the amount of carbon source utilized for the synthesis of L-ornithine decreases as the specific growth rate increases, which in turn results in the reduction of the specific production rate of L-ornithine. This was also reflected in the biomass and L-ornithine production yields, as shown in Fig. 4c. With increasing dilution rate, the biomass yield on glucose (YXjS)increased, while the product yields on glucose (Y& and on cell mass (Y+) showed a decreasing pattern, with maximum values of 0.213 and 0.853 g/g, respectively, at a dilution rate of 0.078 h.-‘. These results show that a larger portion of glucose was used for the production of L-ornithine than for the biomass production at this dilution rate. At lower dilution rates, the decrease in the L-ornithine yield could be attributed to the requirement for energy maintenance. The above kinetic parameter values could not be directly compared with other results because no other continuous-culture data on L-ornithine production have so far reported. However, the maximum L-ornithine yield of 0.213 g/g (29% molar yield from glucose) was not so much lower than that (36% molar yield from glucose) reported by Kinoshita et af. (3), and this strain is considered to have a potential for the practical production of L-ornithine. It was found that under arginine limitation, there existed a different optimal specific growth rate which gave the highest production yield or productivity. That is, while the greatest productivity was obtained in a higher specific growth rate range, the highest L-ornithine yield and titer were obtained in a lower range. It is well known that the fermentative production of amino acids is typically a recovery cost-intensive process (18), in which the product concentration is a critical factor for development of the process. Hence, the results of the present study provide the important observation that the practical production of L-ornithine can be realized through the fed-batch culture of this organism in a lower growth rate range, which will give the highest L-ornithine yield and titer. An improved strategy for fed-batch culture operation based on this observation resulted in a much higher productivity of up to 1 g of L-ornithine/I/h for 3-d operation (data not shown). REFERENCES 1. Davis, B. D.: Intermediates
2.
3. 4. 5. 6.
in amino acid biosynthesis, p, 257268. In Meister, A. (ed.), Advances in enzymology. Interscience Publishers Ltd., London (1955). Selvatore, F., Chino, 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). Kinoshita, S., Nakayama, K., and Udaka, S.: The fermentative production of L-ornithine. J. Gen. Appl. Microbial., 3, 276277 (1957). Vogel, H. J.: Path of ornithine synthesis in E. coli. Proc. Natl. Acad. Sci. USA, 39, 578-583 (1953). Vogel, H. J., McElroy, W. D., and Glass, B.: Amino acid metabolism, p. 335. Johns Hopkins Press, Baltimore (1955). Udaka, S. and Kinoshita, S.: Studies on L-ornithine fermentation. I. The biosynthetic pathway of L-ornithine in Micrococcus glutamicus. J. Gen. Appl. Microbial., 4, 272-282 (1958). Udaka, S. and Kinoshita, S.: Studies on L-ornithine fermentation. IL The change of fermentation product by a feedback type mechanism. J. Gen. Appl. Microbial., 4, 283-288 (1958). Deken, R. H.: Pathway of arginine biosynthesis in yeast. Biochem. Biophys. Res. Commun., 8, 462-466 (1962). Yoshida, H., Araki, K., and Nakayama, K.: N-Acetyl-
100
10.
11.
12. 13.
CHOI ET AL. glutamate-acetylornithine acetyltransferase-deficient arginine auxotroph of C. glutamicum. Agric. Biol. Chem., 43, 18991903 (1979). Yoshida, H., Araki, K., and Nakayama, K.: iV-Acetylornithine-&aminotransferase-deficient and N-acetylglutamokinasedeficient arginine auxotrophs of C. glutamicum. Agric. Biol. Chem., 44,361-365 (1980). Yoshida. H.. Araki. K.. and Nakavama. K.: Mechanism of Larginine’ production by’ t.-arginine-production mutants of C. glutamicum. Agric. Biol. Chem., 43, 105-111 (1979). Udaka, S.: Pathway-specific pattern of control of arginine biosynthesis in bacteria. J. Bacterial., 91, 617-621 (1966). Chinard, F.P.: Photometric estimation of proline and ornithine. J. Biol. Chem., 199, 91-95 (1952).
J.
FERMENT. BIOENG.,
14. Rosenberg, H., Enoor, A. H., and Morrison, J. F.: The estimation of arginine. Biochem. J., 63, 153-159 (1956). 15. Kiss, R. D. and Stephanopoolos, G.: Metabolic characterization of a L-lysine-producing strain by continuous culture. Biotechnol. Bioena.. 39. 565-574 (1992). 16. Senior, P. J.: Regilation of nitrogen metabolism in Escherichia co/i and Klebsielia aerogenes: studies with the continuous culture technique. J. Bacterial., 123, 407-418 (1975). 17. Wang, D. I. C., Cooney, C. L., Demain, A. L., Donnill, P., Humphrey, A. E., and Lilly, M. D.: Fermentation and enzyme technology, p. 98-137. John Wiley & Sons, New York (1979). 18. Cooney, C. L.: Bioreacters: design and operation. Science, 219, 728-733 (1983).