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Factors affecting l -arginine production in the continuous culture of an l -arginine producer of Corynebacterium acetoacidophilum
[J. Ferment. Technol., Vol. 66, No. 3, 285-290. 1988]
Factors Affecting L-Arginine Production in the Continuous Culture of an L-Arginine Producer of Corynebacterium acetoacidophilum t TOMOKI AZUMA* a n d TOSHIHIDE NAKANISHI
Technical Research Laboratories, Hofu Plant, Kyowa Hakko Kogyo Co., Ltd., Kyowa-machi, Hofu, Yamaguchi 747, Japan The effects of culture conditions on L-arginine production by continuous culture were studied using a stable L-arginine hyperproducing strain of Corynebacterium acetoacidophilum, SC-190. Strain SC-190 demonstrated a volumetric productivity of 1.35 g. l - l ' h -1 at a dilution rate of 0.083 h -1 and feeding sugar concentration of 8%, and a product yield of 29.2% at a dilution rate 0.021 h -1 and feeding sugar concentration of 15%. The corresponding values for fed-batch culture are 0.85 g'l-X'h-1 and 26%. However, the product yield decreased with an increase in the volumetric productivity. To achieve stable L-arginine production, aeration and agitation conditions sufficient to maintain an optimal level ofredox potential (>-100 mV) were necessary. The addition of phosphate to the feeding medium led to a decrease in L-arginine production. It was confirmed in the steady state that growth and L-arginine formation were inhibited by a high concentration of L-arginine.
I n o u r p r e v i o u s p a p e r , l) w e d e s c r i b e d t h e results o f c o n t i n u o u s c u l t u r e u s i n g a n La r g i n i n e h y p e r p r o d u c i n g s t r a i n o f Corynebacterium acetoacidophilum, M C - 13. L - A r g i n i n e p r o d u c t i o n d e c r e a s e d d u e to t h e a p p e a r a n c e o f v a r i o u s m u t a n t s w i t h less or n o L - a r g i n i n e production capability after shifting from f e d - b a t c h to c o n t i n u o u s c u l t u r e . Strain SC190, i s o l a t e d f r o m t h e c o n t i n u o u s c u l t u r e broth of strain MC-13, produced L-arginine i n a s t a b l e m a n n e r for m o r e t h a n 250 h. A c q u i r i n g this s t r a i n e n a b l e d us to c a r r y o u t continuous culture. Industrially,
increasingly
efficient
pro-
d u c t i o n o f a m i n o a c i d s is a l w a y s d e s i r e d . A l t h o u g h c o n t i n u o u s c u l t u r e has b e e n r e p o r t ed to b e a n e f f e c t i v e m e t h o d for i m p r o v i n g o v e r a l l productivity,2~ it has o n l y r a r e l y b e e n a p p l i e d to a m i n o a c i d p r o d u c t i o n , a-6) p a r t i c u l a r l y for L - a r g i n i n e . 7) I n this p a p e r , w e e x a m i n e d t h e factors t L-Arginine Production by Continuous Culture (II) * Corresponding author
affecting L-arginine production by the continuous culturing of strain SC-190. Materials and M e t h o d s Microorganism Strain SC-190, used throughout this work, was isolated as a strain having stable L-arginine production from the continuous culture broth of L-arginine producing mutant MC-13 as previously described. I) Media and cultivations The compositions of the seed, starting and feeding media have been described previously. 1) The feeding medium for continuous culture contained 8-25% cane molasses and 3% (NH4),SO4. The pH was not adjusted. The fed-batch and continuous cuhures were carried out as reported in a previous paper.l) To ensure a steady state, the culture was continued for a period of at least 3 retention times under specific continuous conditions before sampling. Analysis The amount of L-arginine accumulated, growth (OD660) and sugar concentration were measured by methods described previously. 1) LLactate which accumulated was assayed by an enzymatic method using L-lactate dehydrogenase, s) The redox potential (ORP) of the culture broth was
286
AZUMA and NAKANISHI
measured using a combined platinum and AgCla (reference) electrode system.
[J. Ferment. Technol.,
1.5 A
30 A
Results
~ I.O
Effect o f f e e d i n g sugar concentration The effects of feeding sugar concentration on the various steady state parameters were examined under the condition of a constant sugar feeding rate per unit volume (3.65 g'/-l'h -1) by changing the dilution rate at feeding sugar concentrations ranging from 8% to 25%. The results are shown in Fig. 1. The concentrations of L-arginine and cells (DCW) increased with the increase in feeding sugar concentration. The maximum values of L-arginine (62.3g./-1) and DCW (32.5 g./-1) were obtained at a feeding sugar concentration of 25 %. The volumetric productivity (P) and the product yield (Yp/,) showed maximum values of 1.02 g./-~.h-~ and 28.3%, respectively, at a feeding sugar concentration of 15%. Cell yield (Yxzs) decreased and product yield per unit cell (Yp/.) increased as the sugar concentration increased. Effect o f dilution rate In order to further improve volumetric productivity and product yield, the effects of dilution rate on t h e s t e a d y state p a r a m e t e r s w e r e e x a m i n e d at
-%
2o o N.
~" Q5
I
0
I
I
3O
60
~ 40 E
"~ Z0 ..I
0
5
,b
;o
,'5
50
a. 05
io
0
30
60
20 .~ to N
~ 20 i
0.02
0.04
0.06 008 Dilution rote (h -I )
i
i
0.1 0
0
Feeding sugor conc. (%)
~=. LO L.
0
2'5
Fig. 1. Effect of feeding sugar concentration on the steady state parameters. (A): O, P; ©, Yp/s; A, Yp/,,; A, Yx/s. (B): 0 , L-arginine concentration; O, DCW. The sugar feeding rate per unit volume was kept constant at 3.65g.l-Z.h -x at all feeding sugar concentrations by varying the dilution rate. The dilution rates were 0.046, 0.037, 0.024, 0.018 and 0.015 h -1 at the feeding sugar concentrations of 8, 10, 15, 20 and 25%, respectively.
1.5
0
I
B
0.02
0.04
i
0.06
i
0.08 Dilution rote (h -I )
i
0.1
Fig. 2. Effect of dilution rate on the steady state parameters. Feeding sugar concentration: O, 20%; 0, 15%; A, 10%; ,L, 8%.
Vol. 66, 1988]
Factors Affecting L-ArginineProduction
various feeding sugar concentrations (Fig. 2). The volumetric productivity at lower feeding sugar concentrations increased when the dilution rate was high. L-Arginine production decreased due to the wash-out at the lower dilution rate as the feeding sugar concentration increased. Volumetric productivity reached a m a x i m u m value of 1.35 g./-1.h-1 at a dilution rate of 0.083 h - I and a sugar concentration of 8%. This value represents an improvement of more than 50% compared with the value for batch culture (0.85 g.l-l.h-1). The m a x i m u m of the product yield was 29.2% at a dilution rate of 0.021 h-x and sugar concentration of 15%. The product yield decreased inversely with an increase in volumetric productivity. Effect o f o x y g e n s u p p l y The effects of limited oxygen supply on the various parameters were studied. A limited oxygen supply condition was set up by varying the agitation rate from 6 0 0 r p m to 4 0 0 r p m
287
under aeration at 3/.min-1. As shown in Fig. 3, L-arginine concentration, product yield and volumetric productivity dropped with a decrease in the agitation rate, with substantial decreases being observed at the agitation rate of 4 0 0 r p m . The redox potential, which might reflect the dissolved oxygen concentration in the broth, also decreased with a decrease in the agitation rate, finally reaching a minimal value (--210 mV) at the agitation rate of 400 rpm. O n the contrary, the accumulation of Llactate increased below 500 rpm and reached a m a x i m u m value of 7.0 g.l-i at the agitation rate of 4 0 0 r p m . The highest value for L-arginine production was obtained at 600 rpm, a control condition.
Effect
of
phosphate
concentration
The feeding medium for continuous culture contained only molasses and ammonium sulfate. Since a m m o n i u m sulfate is an 1.5
1.5
A
A
3O
so .~ -
= 1.0
L) r~
.2'
20
1.0
20 -
b~
..f 0..
o£
05
I
,
a
t
0
B
6O
_
B
.-100
..a
_
4c c E o~
- -
~
~ 2c
n
~1
_1
0
'
400
"---. .......... 500 Agitotion
I0
600
6O
5O
40
40
"rU
o
io%
(rpm)
Fig. 3. Effect of agitation on the steady state parameters. (A): O, P; O, Yp/.; A, DCW. (B): O, L-
30 c~ J i
i
i
o
0.05
o. I
K H2
P04 (%)
Fig. 4. Effect of phosphate concentration on the steady state parameters. arginine concentration; A, ORP; A, L-lactate (A): @, P; ©, Yp/,. (B): @, L-arginine concentration; ©, DCW. accumulated. KH~PO4 was added to the feeding medium. The The culture was carried out using a feeding sugar culture was carried out using a feeding sugar concentration of 20% at a dilution rate of 0.018 h-1 concentration of 20% at a dilution rate of 0.018 h-1. with aeration at 3/.rain -1.
288
AzuM~ a n d NAKANISHI
essential nitrogen source for L-arginine production, its limitation inhibits the formation of L-arginine. On the other hand, it has already been confirmed that phosphate could be a limiting factor of growth in batch culture experiments (data not shown). Thus, the effect of phosphate was examined by varying the concentration of KH2PO4 added to the feeding medium. As shown in Fig. 4, the cell concentration increased as the concentration of KH2PO4 increased. In contrast, the L-arginine concentration, product yield and volumetric productivity decreased. It was found that the amount of phosphate supplied by cane molasses was sufficient for maximum production. Effect of L-arglnlne addition in the s t e a d y state In batch cultures, the culture conditions such as the cell, nutrient and product concentrations change continually. On tile other hand, constant culture conditions can be maintained in continuous cultures. Therefore, the effect
[J. Ferment. Technol.,
of a change in a single factor on cell metabolism can be detected by maintaining the other factors constant. Thus, the effect of Larginine addition was examined in order to elucidate whether regulation by L-arginine operates or not in the presence of accumulated L-arginine. Sixty grams of L-arginine dissolved in 200 ml of water were added at steady state where the L-arginine concentration was maintained at approximately 40 g.1-1. As a control, an equal volume of sterile water was added. The results are presented in Fig. 5. Immediately after the addition of L-arginine, growth was inhibited and the residual sugar concentration increased. The dotted line in Fig. 5 indicates the theoretical curve of the decrease in Larginine concentration calculated from the dilution rate. The actual curve observed, however, was constantly below the theoretical curve. This result suggests that product inhibition by L-arginine functions when a large amount of L-arginine (approximately 60 g'/-1) accumulates. Discussion
60
0.5
4(]
0.4
4
Q3
3 8
o~
c
0
;02 ~3 o"
c
2 m
q
,Go ,;o 2Go 2;o Culture time (h)
Fig. 5. Effect of L-arginine on L-arginine production in the steady state. ©, 0 , L-arginine concentration; D, I , growth; A, A, residual sugar. O p e n a n d closed symbols indicate control a n d arginine addition experiments, respectively. T h e arrow indicates the point of L-arginine (60 g/ 200 ml) addition. As a control experiment, a n equal volume of sterile water (200 ml) was added. T h e culture was carried out u n d e r a feeding sugar concentration of 15% at a dilution rate of 0.021 h -1.
Factors affecting L-arginine production in continuous culture have been studied. As shown in Fig. 1, both volumetric productivity (P) and product yield (Y~/s) indicated maximum values at a feeding sugar concentration of 15% and a constant sugar feeding rate per unit volume (3.65 g'/-1.h-1). The decrease in product yield at the lower feeding sugar concentration was assumed to be caused by the elevated cellular yield because of the higher dilution rate (higher specific growth rate). On the other hand, it seemed that the decrease in product yield at the higher feeding sugar concentration was due to the inhibition of L-arginine formation by the accumulated L-arginine. The result obtained for Larginine addition in the steady state also supports this explanation. In Table 1, L-arginine concentration, volumetric productivity and product yield are compared for L-arginine t~rmentations using various microorganisms published in the literature. No data for continuous
Vol. 66, 1988] Table 1. Strain C. acetoacidophilum
Comparison of productivities among various L-arginine producing mutants. Arg
Batch Yp / 8
P
Arg
(g.l-x)
(%)
(g'/-l'h-l)
(g.l-X)
55
26
0.85
---
B. flavum a S. marcescens b S. marcescens c
289
Factors Affecting L-Arginine Production
40 40 __
(31) (29)
Continuous Yp/ s P
(%)
(g.l-X'h-~)
D
(h-l)
16
20
1.35
--
62.3
24. 9
0. 9 3
0.
--
43.7
0. 9 2
0.
1.0
0. 1
(0. 67 ) (0. 28) --
. . 10
29. 2
. .
. . --
0. 083 015 021
. .
Culture conditions: a jar fermentor, b flask, ¢ immobilized cells. The value underlined indicates the maximum value for each parameter in continuous culture by strain SC190. The value bracketed was calculated by the authors using the L-arginine concentration, the initial sugar concentration and the culture time described in the report.
culture experiments with Brevibacterium flavum Nomenclature are available. 9) With Serratia marcescens, batch culturO 0) and continuous culture using P : volumetric productivity (g-l-l"h-1) immobilized cellsT~ have been reported. Yp/s : yield of L-arginine per unit weight Strain SC-190 exhibited the highest values of sugar (%) of L-arginine concentration and volumetric Yxzs : y i e l d of cells per unit weight of productivity in both batch culture and sugar (%) continuous culture. In particular, the L- Yp/x : yield of L-arginine per unit weight arginine concentration and product yield of cells (%) were maintained at high values, 16 g-I-~ and DCW : dry cell weight (g.l-1) 20%, respectively, when maximum volumet- D : dilution rate (h-~) ric productivity (1.35 g./-1.h-1) was obtained. Acknowledgments This suggests that the process can be applied to industrial production. We are grateful to Mr. Sadao Noguchi for his Strain SC-190 required sufficient aeration encouragement throughout this work. and agitation ( > - - 1 0 0 m V as ORP) for References stable L-arginine production. It was found that the critical level of the redox potential 1) Azuma, T., Nakanishi, T., Sugimoto, M.: J. was --150 rnV. Measurement of O R P has Ferment. Technol., 66, 279 (1988). been reported in a batch culture experiment 2) Srinivasan, V . R . , Summers, R . J . : Continuous using a mutant of B. flavum. 8) However, Cultures of Cells (Calcott, P.H.), Vol. 1, p. 97, since O R P changes with time in batch culture CRC Press, Inc., Florida (1981). where both the culture environment and cell 3) Mimura, A., Yoshida, T., Matsuno, T., Hashida, W., Taguchi, H., Otake, T., Teramoto, S.: metabolism constantly change, it appears J. Ferment. Technol., 41,275 (1963). that the culture phase for measurement is 4) Choi, Y.J., Tribe, D . E . : Biotechnol. Lett., 4, important when interpreting the value. 223 (1982). The results of the phosphate addition 5) Park, N. H., Rogers, P . L . : Chem. Eng. Commun., experiment indicate that the phosphate con45, 185 (1986). centration may be a limiting factor for cell 6) Toma, M., Svinka, J., Ruklisa, M . P . , Sakse, A., growth and product formation in continuous Baburin, L.A.: Prikl. Biokhim. Mikrobiol., 20, culture. The control of concentrations of 95 (1984). critical nutrients was found to be effective for 7) Fujimura, M., Kato, J., Tosa, T., Chibata, I.: Appl. Microbiol. Biotechnol., 19, 79 (1984). achieving higher productivity.
290
AZUMAand NAKAmSHt
8) Gaucehm, K. : Methoden der Enzymatischen Analyse, p. 1945, Verlag Chemie, Weinheim (1970). 9) Akashi, K., Shibai, H., Hirose, Y.: J. Ferment. Technol., 57, 321 (1979).
10) Takagi, T., Kisumi, M., Chibata, I.: Appl. Microbiol. Biotechnol., 21, 378 (1985). (Received November 30, 1987)
Factors Affecting L-Arginine Production in the Continuous Culture of an L-Arginine Producer of Corynebacterium acetoacidophilum t TOMOKI AZUMA* a n d TOSHIHIDE NAKANISHI
Technical Research Laboratories, Hofu Plant, Kyowa Hakko Kogyo Co., Ltd., Kyowa-machi, Hofu, Yamaguchi 747, Japan The effects of culture conditions on L-arginine production by continuous culture were studied using a stable L-arginine hyperproducing strain of Corynebacterium acetoacidophilum, SC-190. Strain SC-190 demonstrated a volumetric productivity of 1.35 g. l - l ' h -1 at a dilution rate of 0.083 h -1 and feeding sugar concentration of 8%, and a product yield of 29.2% at a dilution rate 0.021 h -1 and feeding sugar concentration of 15%. The corresponding values for fed-batch culture are 0.85 g'l-X'h-1 and 26%. However, the product yield decreased with an increase in the volumetric productivity. To achieve stable L-arginine production, aeration and agitation conditions sufficient to maintain an optimal level ofredox potential (>-100 mV) were necessary. The addition of phosphate to the feeding medium led to a decrease in L-arginine production. It was confirmed in the steady state that growth and L-arginine formation were inhibited by a high concentration of L-arginine.
I n o u r p r e v i o u s p a p e r , l) w e d e s c r i b e d t h e results o f c o n t i n u o u s c u l t u r e u s i n g a n La r g i n i n e h y p e r p r o d u c i n g s t r a i n o f Corynebacterium acetoacidophilum, M C - 13. L - A r g i n i n e p r o d u c t i o n d e c r e a s e d d u e to t h e a p p e a r a n c e o f v a r i o u s m u t a n t s w i t h less or n o L - a r g i n i n e production capability after shifting from f e d - b a t c h to c o n t i n u o u s c u l t u r e . Strain SC190, i s o l a t e d f r o m t h e c o n t i n u o u s c u l t u r e broth of strain MC-13, produced L-arginine i n a s t a b l e m a n n e r for m o r e t h a n 250 h. A c q u i r i n g this s t r a i n e n a b l e d us to c a r r y o u t continuous culture. Industrially,
increasingly
efficient
pro-
d u c t i o n o f a m i n o a c i d s is a l w a y s d e s i r e d . A l t h o u g h c o n t i n u o u s c u l t u r e has b e e n r e p o r t ed to b e a n e f f e c t i v e m e t h o d for i m p r o v i n g o v e r a l l productivity,2~ it has o n l y r a r e l y b e e n a p p l i e d to a m i n o a c i d p r o d u c t i o n , a-6) p a r t i c u l a r l y for L - a r g i n i n e . 7) I n this p a p e r , w e e x a m i n e d t h e factors t L-Arginine Production by Continuous Culture (II) * Corresponding author
affecting L-arginine production by the continuous culturing of strain SC-190. Materials and M e t h o d s Microorganism Strain SC-190, used throughout this work, was isolated as a strain having stable L-arginine production from the continuous culture broth of L-arginine producing mutant MC-13 as previously described. I) Media and cultivations The compositions of the seed, starting and feeding media have been described previously. 1) The feeding medium for continuous culture contained 8-25% cane molasses and 3% (NH4),SO4. The pH was not adjusted. The fed-batch and continuous cuhures were carried out as reported in a previous paper.l) To ensure a steady state, the culture was continued for a period of at least 3 retention times under specific continuous conditions before sampling. Analysis The amount of L-arginine accumulated, growth (OD660) and sugar concentration were measured by methods described previously. 1) LLactate which accumulated was assayed by an enzymatic method using L-lactate dehydrogenase, s) The redox potential (ORP) of the culture broth was
286
AZUMA and NAKANISHI
measured using a combined platinum and AgCla (reference) electrode system.
[J. Ferment. Technol.,
1.5 A
30 A
Results
~ I.O
Effect o f f e e d i n g sugar concentration The effects of feeding sugar concentration on the various steady state parameters were examined under the condition of a constant sugar feeding rate per unit volume (3.65 g'/-l'h -1) by changing the dilution rate at feeding sugar concentrations ranging from 8% to 25%. The results are shown in Fig. 1. The concentrations of L-arginine and cells (DCW) increased with the increase in feeding sugar concentration. The maximum values of L-arginine (62.3g./-1) and DCW (32.5 g./-1) were obtained at a feeding sugar concentration of 25 %. The volumetric productivity (P) and the product yield (Yp/,) showed maximum values of 1.02 g./-~.h-~ and 28.3%, respectively, at a feeding sugar concentration of 15%. Cell yield (Yxzs) decreased and product yield per unit cell (Yp/.) increased as the sugar concentration increased. Effect o f dilution rate In order to further improve volumetric productivity and product yield, the effects of dilution rate on t h e s t e a d y state p a r a m e t e r s w e r e e x a m i n e d at
-%
2o o N.
~" Q5
I
0
I
I
3O
60
~ 40 E
"~ Z0 ..I
0
5
,b
;o
,'5
50
a. 05
io
0
30
60
20 .~ to N
~ 20 i
0.02
0.04
0.06 008 Dilution rote (h -I )
i
i
0.1 0
0
Feeding sugor conc. (%)
~=. LO L.
0
2'5
Fig. 1. Effect of feeding sugar concentration on the steady state parameters. (A): O, P; ©, Yp/s; A, Yp/,,; A, Yx/s. (B): 0 , L-arginine concentration; O, DCW. The sugar feeding rate per unit volume was kept constant at 3.65g.l-Z.h -x at all feeding sugar concentrations by varying the dilution rate. The dilution rates were 0.046, 0.037, 0.024, 0.018 and 0.015 h -1 at the feeding sugar concentrations of 8, 10, 15, 20 and 25%, respectively.
1.5
0
I
B
0.02
0.04
i
0.06
i
0.08 Dilution rote (h -I )
i
0.1
Fig. 2. Effect of dilution rate on the steady state parameters. Feeding sugar concentration: O, 20%; 0, 15%; A, 10%; ,L, 8%.
Vol. 66, 1988]
Factors Affecting L-ArginineProduction
various feeding sugar concentrations (Fig. 2). The volumetric productivity at lower feeding sugar concentrations increased when the dilution rate was high. L-Arginine production decreased due to the wash-out at the lower dilution rate as the feeding sugar concentration increased. Volumetric productivity reached a m a x i m u m value of 1.35 g./-1.h-1 at a dilution rate of 0.083 h - I and a sugar concentration of 8%. This value represents an improvement of more than 50% compared with the value for batch culture (0.85 g.l-l.h-1). The m a x i m u m of the product yield was 29.2% at a dilution rate of 0.021 h-x and sugar concentration of 15%. The product yield decreased inversely with an increase in volumetric productivity. Effect o f o x y g e n s u p p l y The effects of limited oxygen supply on the various parameters were studied. A limited oxygen supply condition was set up by varying the agitation rate from 6 0 0 r p m to 4 0 0 r p m
287
under aeration at 3/.min-1. As shown in Fig. 3, L-arginine concentration, product yield and volumetric productivity dropped with a decrease in the agitation rate, with substantial decreases being observed at the agitation rate of 4 0 0 r p m . The redox potential, which might reflect the dissolved oxygen concentration in the broth, also decreased with a decrease in the agitation rate, finally reaching a minimal value (--210 mV) at the agitation rate of 400 rpm. O n the contrary, the accumulation of Llactate increased below 500 rpm and reached a m a x i m u m value of 7.0 g.l-i at the agitation rate of 4 0 0 r p m . The highest value for L-arginine production was obtained at 600 rpm, a control condition.
Effect
of
phosphate
concentration
The feeding medium for continuous culture contained only molasses and ammonium sulfate. Since a m m o n i u m sulfate is an 1.5
1.5
A
A
3O
so .~ -
= 1.0
L) r~
.2'
20
1.0
20 -
b~
..f 0..
o£
05
I
,
a
t
0
B
6O
_
B
.-100
..a
_
4c c E o~
- -
~
~ 2c
n
~1
_1
0
'
400
"---. .......... 500 Agitotion
I0
600
6O
5O
40
40
"rU
o
io%
(rpm)
Fig. 3. Effect of agitation on the steady state parameters. (A): O, P; O, Yp/.; A, DCW. (B): O, L-
30 c~ J i
i
i
o
0.05
o. I
K H2
P04 (%)
Fig. 4. Effect of phosphate concentration on the steady state parameters. arginine concentration; A, ORP; A, L-lactate (A): @, P; ©, Yp/,. (B): @, L-arginine concentration; ©, DCW. accumulated. KH~PO4 was added to the feeding medium. The The culture was carried out using a feeding sugar culture was carried out using a feeding sugar concentration of 20% at a dilution rate of 0.018 h-1 concentration of 20% at a dilution rate of 0.018 h-1. with aeration at 3/.rain -1.
288
AzuM~ a n d NAKANISHI
essential nitrogen source for L-arginine production, its limitation inhibits the formation of L-arginine. On the other hand, it has already been confirmed that phosphate could be a limiting factor of growth in batch culture experiments (data not shown). Thus, the effect of phosphate was examined by varying the concentration of KH2PO4 added to the feeding medium. As shown in Fig. 4, the cell concentration increased as the concentration of KH2PO4 increased. In contrast, the L-arginine concentration, product yield and volumetric productivity decreased. It was found that the amount of phosphate supplied by cane molasses was sufficient for maximum production. Effect of L-arglnlne addition in the s t e a d y state In batch cultures, the culture conditions such as the cell, nutrient and product concentrations change continually. On tile other hand, constant culture conditions can be maintained in continuous cultures. Therefore, the effect
[J. Ferment. Technol.,
of a change in a single factor on cell metabolism can be detected by maintaining the other factors constant. Thus, the effect of Larginine addition was examined in order to elucidate whether regulation by L-arginine operates or not in the presence of accumulated L-arginine. Sixty grams of L-arginine dissolved in 200 ml of water were added at steady state where the L-arginine concentration was maintained at approximately 40 g.1-1. As a control, an equal volume of sterile water was added. The results are presented in Fig. 5. Immediately after the addition of L-arginine, growth was inhibited and the residual sugar concentration increased. The dotted line in Fig. 5 indicates the theoretical curve of the decrease in Larginine concentration calculated from the dilution rate. The actual curve observed, however, was constantly below the theoretical curve. This result suggests that product inhibition by L-arginine functions when a large amount of L-arginine (approximately 60 g'/-1) accumulates. Discussion
60
0.5
4(]
0.4
4
Q3
3 8
o~
c
0
;02 ~3 o"
c
2 m
q
,Go ,;o 2Go 2;o Culture time (h)
Fig. 5. Effect of L-arginine on L-arginine production in the steady state. ©, 0 , L-arginine concentration; D, I , growth; A, A, residual sugar. O p e n a n d closed symbols indicate control a n d arginine addition experiments, respectively. T h e arrow indicates the point of L-arginine (60 g/ 200 ml) addition. As a control experiment, a n equal volume of sterile water (200 ml) was added. T h e culture was carried out u n d e r a feeding sugar concentration of 15% at a dilution rate of 0.021 h -1.
Factors affecting L-arginine production in continuous culture have been studied. As shown in Fig. 1, both volumetric productivity (P) and product yield (Y~/s) indicated maximum values at a feeding sugar concentration of 15% and a constant sugar feeding rate per unit volume (3.65 g'/-1.h-1). The decrease in product yield at the lower feeding sugar concentration was assumed to be caused by the elevated cellular yield because of the higher dilution rate (higher specific growth rate). On the other hand, it seemed that the decrease in product yield at the higher feeding sugar concentration was due to the inhibition of L-arginine formation by the accumulated L-arginine. The result obtained for Larginine addition in the steady state also supports this explanation. In Table 1, L-arginine concentration, volumetric productivity and product yield are compared for L-arginine t~rmentations using various microorganisms published in the literature. No data for continuous
Vol. 66, 1988] Table 1. Strain C. acetoacidophilum
Comparison of productivities among various L-arginine producing mutants. Arg
Batch Yp / 8
P
Arg
(g.l-x)
(%)
(g'/-l'h-l)
(g.l-X)
55
26
0.85
---
B. flavum a S. marcescens b S. marcescens c
289
Factors Affecting L-Arginine Production
40 40 __
(31) (29)
Continuous Yp/ s P
(%)
(g.l-X'h-~)
D
(h-l)
16
20
1.35
--
62.3
24. 9
0. 9 3
0.
--
43.7
0. 9 2
0.
1.0
0. 1
(0. 67 ) (0. 28) --
. . 10
29. 2
. .
. . --
0. 083 015 021
. .
Culture conditions: a jar fermentor, b flask, ¢ immobilized cells. The value underlined indicates the maximum value for each parameter in continuous culture by strain SC190. The value bracketed was calculated by the authors using the L-arginine concentration, the initial sugar concentration and the culture time described in the report.
culture experiments with Brevibacterium flavum Nomenclature are available. 9) With Serratia marcescens, batch culturO 0) and continuous culture using P : volumetric productivity (g-l-l"h-1) immobilized cellsT~ have been reported. Yp/s : yield of L-arginine per unit weight Strain SC-190 exhibited the highest values of sugar (%) of L-arginine concentration and volumetric Yxzs : y i e l d of cells per unit weight of productivity in both batch culture and sugar (%) continuous culture. In particular, the L- Yp/x : yield of L-arginine per unit weight arginine concentration and product yield of cells (%) were maintained at high values, 16 g-I-~ and DCW : dry cell weight (g.l-1) 20%, respectively, when maximum volumet- D : dilution rate (h-~) ric productivity (1.35 g./-1.h-1) was obtained. Acknowledgments This suggests that the process can be applied to industrial production. We are grateful to Mr. Sadao Noguchi for his Strain SC-190 required sufficient aeration encouragement throughout this work. and agitation ( > - - 1 0 0 m V as ORP) for References stable L-arginine production. It was found that the critical level of the redox potential 1) Azuma, T., Nakanishi, T., Sugimoto, M.: J. was --150 rnV. Measurement of O R P has Ferment. Technol., 66, 279 (1988). been reported in a batch culture experiment 2) Srinivasan, V . R . , Summers, R . J . : Continuous using a mutant of B. flavum. 8) However, Cultures of Cells (Calcott, P.H.), Vol. 1, p. 97, since O R P changes with time in batch culture CRC Press, Inc., Florida (1981). where both the culture environment and cell 3) Mimura, A., Yoshida, T., Matsuno, T., Hashida, W., Taguchi, H., Otake, T., Teramoto, S.: metabolism constantly change, it appears J. Ferment. Technol., 41,275 (1963). that the culture phase for measurement is 4) Choi, Y.J., Tribe, D . E . : Biotechnol. Lett., 4, important when interpreting the value. 223 (1982). The results of the phosphate addition 5) Park, N. H., Rogers, P . L . : Chem. Eng. Commun., experiment indicate that the phosphate con45, 185 (1986). centration may be a limiting factor for cell 6) Toma, M., Svinka, J., Ruklisa, M . P . , Sakse, A., growth and product formation in continuous Baburin, L.A.: Prikl. Biokhim. Mikrobiol., 20, culture. The control of concentrations of 95 (1984). critical nutrients was found to be effective for 7) Fujimura, M., Kato, J., Tosa, T., Chibata, I.: Appl. Microbiol. Biotechnol., 19, 79 (1984). achieving higher productivity.
290
AZUMAand NAKAmSHt
8) Gaucehm, K. : Methoden der Enzymatischen Analyse, p. 1945, Verlag Chemie, Weinheim (1970). 9) Akashi, K., Shibai, H., Hirose, Y.: J. Ferment. Technol., 57, 321 (1979).
10) Takagi, T., Kisumi, M., Chibata, I.: Appl. Microbiol. Biotechnol., 21, 378 (1985). (Received November 30, 1987)