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carbon ratio on the specific production rate of spiramycin by Streptomyces ambofaciens
Process Biochemistry Vol. 31, No. 1, pp. 13-20, 1996 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. AU rights reserved 0032-9592/96 $9.50 + 0.00 0032-9592(95)00008-9
ELSEVIER
Effect of Nitrogen/Carbon Ratio on the Specific Production Rate of Spiramycin by Streptomyces ambofaciens Anissa Low&, Ahmed Lebrihi,” Chouki Benslimane, G&ard Lefebvre & Pierre Germain Laboratorie de Microbiologic Industrielle et Alimentaire, For& de Haye, 54500 Vandoeuvre, France (Received 18 November
ENSAIA,
Institut National Polytechnique
de Lorraine, 2 avenue de la
1994; accepted 20 February 1995)
Streptomyces ambofaciens was grown on a synthetic medium with glycerol (log litre-‘) and ammonium (20 mM) as carbon and nitrogen sources, respectively. Batch fermentation produced a specific production rate of spiramycin (Q)
of O-3
mg h - ’g- ’DC W. Improvement of spiramycin production was achieved by fedbatch culture. The strong influence of the initial uptake rates of glycerol (qpJ and ammonium (q,,& on the speci@c production rate of spiramycin (qSP) was demonstrated by conducting several fermentations with continuous feeding of glycerol and ammonium, in different ratios. The specific production rate of spiramycin was increased 10 fold. A low specific growth rate was also necessary for spiramycin biosynthesis.
phate12 were in excess. In this fermentation is shown to result spiramycin biosynthesis when batch cultures. Using fed-batch ence of initial ycerol specific production rate of studied.
INTRODUCTION Antibiotic production is influenced by the nitrogen,1-4 carbonsT6 and phosphate sources738 used in the fermentation medium. Additional feeds of medium ingredients, precursors, or complete medium using fed-batch culture are often found to extend biosynthesis and result in greater productivity of the desired metabolite.” Production of the 16-membered macrolide antibiotic tylosin was increased several fold in fed-batch fermentation.‘” Streptomyces ambofaciens produces a 16-membered macrolide antibiotic, spiramycin. In batch biosynthesis was greatly culture, spiramycin reduced when ammonium,” glycerol or phos*To whom correspondence
paper, fed-batch in stimulation of compared with cultures the influ-
Yankninm/gl ( YNFI:,gy)on the spiramycin
was
MATERIALS AND METHODS Microorganisms These studies were carried out with Streptumyces ambofaciens RP 18 1110 (Rhone-Poulenc, Vitrysur-Seine, France) producer of spiramycin and Bacillus subtilis ATCC 6633 as test microorganism.
should be addressed.
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Spiramycin production in fed-batch
Equation (3) is used for the determination of the specific production rate of spiramycin (q,,). The maximum specific production and volumetric production of spiramycin are expressed in (mg g-l DCW) and (mg litre- l), respectively. Yammonium/glycerol is calculated from ~NH: and qd, and expressed in mmols g- ’ ( YNHr/qgly= qNH$/ %Y )*
RESULTS Characteristics of growth and spiramycin production in batch culture on basal medium with glycerol 10 g litre - * and ammonium 20 mM The results from a typical spiramycin batch culture on the synthetic medium are shown in Fig. 1. This fermentation was characterized by a maximal specific growth rate of 0.05 5 h- ’ and specific uptake rate of glycerol and ammonium of 0.2 g h-l g-’ DCW and 0.5 mm01 h-l g-’ DCW, respectively. Spiramycin production began after 50 h of fermentation when the nitrogen and carbon sources were completely consumed. Spiramycin was produced with a global specific production rate (q,J of 0.3 mg h-l g-r DCW and reached 35 mg litre- ’ and 14 mg gg * DCW of final volumetric production and a maximum specific production of spiramycin, respectively.
15
At the end of fermentation, phosphate were detected.
7 mM of residual
Characteristics of growth and spiramycin production in fed-batch cultures To prevent the negative effect on spiramycin biosynthesis of an eventual accumulation of phosphate, I2 the feed medium for fed-batch cultures did not contain phosphate. About 1 mu of phosphate was detected constantly during the course of each fed-batch. Fed-batch culture (1) with 40 g litre- ’glycerol and 180 mM ammonium at a flow rate of 0.019 litre h-l
Figures 2 and 3 show the results obtained from a fed-batch fermentation with a continuous feed of glycerol (40 g litre-‘) and ammonium (180 mM). The fed-batch was started at 43 h. The flow rate used (0.019 litre h- ‘) led to initial specific consumption rates of glycerol (q@,) and ammonium (qNH:)of 0.1 g h-l g-l DCW and 0.45 mm01 h-’ g-’ DCW, respectively. Feeding
Begining
Eyd
10
0
48
96
144
192
240
288
336
Time (h)
‘0
48
96
Tie
144
192
(h)
Fig. 1. Batch culture. Kinetics of growth (O), consumption of glycerol (m), consumption and accumulation of ammonium ions ( ?),? consumption of phosphate (0) and production of spiramycin (0) in batch culture on the basal chemical defined medium with glycerol at 10 g litre-’ and ammonium at 20 mM as carbon and nitrogen sources, respectively.
Fig. 2. Fed-batch culture no. 1. Kinetics of growth (O), consumption of glycerol (?? ),consumption and accumulation of ammonium ions ( ?),? consumption of phosphate (0) and production of spiramycin (0) in fed-batch culture on initial volume of 3 litres of the basal chemical defined medium with glycerol at 10 g litre-’ and ammonium at 20 mM as carbon and nitrogen sources, respectively, with continuous feeding medium containing 40 g litre- ’ of glycerol and 180 mM of ammonium, at a constant flow rate of 0.019 litre h- l. - : Indicates the beginning of the feeding. --D: Indicates the end of the feeding.
16
A. Lou&, A. Lebrihi, C. Ben&mane, G. Lefebvre, P. Germain
0.06 .
0.05 g h-’ g-’ DCW and 0.1 mm01 h-’ g-l DCW, respectively. The maximal specific production rate of spiramycin was observed when 30 mM of ammonium were present in the medium. The accumulation of ammonium observed after cessation of feeding was probably due to proteolysis. Fed-batch culture (2) with 40g litre-’ glycerol and 66 rn~ ammonium at a flowrate of 0.019 litre h-’
40
90
140
190
240
Time(h)
Fig. 3. Evolution of the kinetic parameters of fed-batch culture no. 1. Evolution of specific growth rate (0), specific uptake rate of glycerol ( ?? ), specific uptake rate of ammonium (0) and specific production rate of spiramycin (0) in fedbatch culture on initial volume of 3 litres of the basal chemical defined medium with glycerol at 10 g litre-’ and ammonium at 20 mM as carbon and nitrogen sources, respectively, with continuous feeding medium containing 40 g litre- ’of glycerol and 180 mM of ammonium, at a constant flow rate of 0.0 19 litre h - I.
The results showed that glycerol was completely consumed; less than 0.5 g litre- ’ was detected during the fermentation suggesting growth limitation by carbon source. In contrast, ammonium ions were found in excess. Ammonium was largely accumulated at the end of fedbatch (70 mM) probably causing a cessation of spiramycin production. Production of spiramycin began at 100 h and a final volumetric production of 60 mg litre-’ and a maximum specific spiramycin production of 14 mg gg’ DCW was achieved. The maximum specific production rate of spiramycin (0.5 mg h- ’ g- ’ DCW) was increased by 60% with respect to batch culture. Spiramycin production occurred when the specific growth rate and the specific uptake rates of glycerol and ammonium fell to about 0.01 h- ‘,
In an attempt to bypass the ammonium effect occurring at high initial specific ammonium uptake rates and to prevent the accumulation of these ions during batch feeding, a second fedbatch was carried out. The feed medium contained glycerol at 40 g litre- l but the ammonium concentration was reduced to 66 mM. These concentrations given with a flow rate of 0.019 litre h-’ corresponded to initial specific uptake rates of glycerol and ammonium of O-1 g h-l g-’ DCW and 0.16 mmol h-l g-l DCW respectively. Feeding commenced at 60 h. Figures 4 and 5 show the results obtained from the fermentation. In these conditions, less than 0.5 g litre- ’ of glycerol were detected during the fed-batch. Ammonium was not detected at all. These observations could indicate conditions of limitation in nitrogen source. In Figs 4 and 5, maximal qsp occurred at 96 h (1.5 mg h- ’ gg ’ DCW) when the specific uptake rate of glycerol and ammonium was lowered to 0.05 g h-’ gg ’ DCW and 0.082 mmol h-’ g- ’ DCW respectively and the specific growth rate to O-035 h- ‘. q,r was doubled by comparison with the first fed-batch in which ammonium ions were accumulated. The maximum specific production of spiramycin was 21 mg g- ’ DCW and the final volumetric production reached 110 mg litre-‘. We noted that S. ambofaciens sporulated under these fermentation conditions. This observation may imply extreme conditions of limitation, particularly in ammonium. Previous studies with Streptomyces griseus showed that sporulation occurred in submerged culture limited by carbon and nitrogen sources.” Fed batch culture (3) with 27.5 g litre- ’glycerol and 100 mM ammonium at aflow rate of 0~027litre h-’
In order to improve spiramycin production, ammonium feeding was performed so as to obtain an intermediate initial specific uptake rate of ammonium between the first and the second fedbatch experiments (O-36 mm01 h- ’ g-’ DCW).
Spiramycin production in fed-batch Begining
Feeding
17 0.06
End
1
J 5
0.4
0.2
0
48
96
144
192
240
288
Time (h)
Fig. 4. Fed-batch no. 2. Kinetics of growth (O), consumption of glycerol (m), consumption and accumulation of ammonium ions ( ?),?consumption of phosphate (0) and production of spiramycin (0) in fed-batch culture on initial volume of 3 litres of the basal chemical defined medium with glycerol at 10 g litre-’ and ammonium at 20 mM as carbon and nitrogen sources, respectively, with continuous feeding medium containing 40 g litre-’ of glycerol and 66 mM of ammonium, at a constant flow rate of 0.019 litre h-‘. -: Indicates the beginning of the feeding. + : Indicates the end of the feeding.
Glycerol was always fed to obtain an initial specific uptake rate of 0.1 g h- ’ gg ’DCW. Ammonium and glycerol were fed with a flow rate of O-027 litre h- ’ at a concentration of glycerol and ammonium of 27.5 g litre- ’ and 100 mM, respectively. Feeding was started at 47.5 h. Figures 6 and 7 show the results obtained from the fermentation which was characterized by a high initial qsr value of 3 mg h- ’g- ’DCW which was maintained at O-5 mg h- ’ gg ’ DCW after a sudden decrease. The final volumetric concentration of spiramycin was 125 mg litre- ’ and the specific spiramycin production was 28 mg g- ’ DCW. During the fed-batch, glycerol and ammonium were detected at less than 0.5 g litree’ and 0.3 mM, respectively. This could imply an equilibrium between nitrogen and carbon sources. Production of spiramycin occurred when a decrease in ,u was observed and the specific uptake rates of glycerol
40
140
90
‘90
Tune (h)
Fig. 5. Evolution of the specific growth rate (O), specific uptake rate of glycerol ( ??), specific uptake rate of ammonium (0) and specific production rate of spiramycin (0) in fedbatch culture on initial volume of 3 litres of the basal chemical defined medium with glycerol at 10 g litre- 1 and ammonium at 20 mM as carbon and nitrogen sources, respectively, with continuous feeding medium containing 40 g litre- l of glycerol and 66 mM of ammonium at a constant flow rate of 0.0 19 litre h- I.
and ammonium were about 0.05 g h-’ gg ’DCW and O-18 mmol h- ’ g- ’ DCW, respectively. At the end of the feeding, accumulation of 13 mM of ammonium was observed, probably due to the proteolysis.
DISCUSSION The data presented in this paper are summarised in Table 1. They show that in batch culture on the chemically defined medium, spiramycin was produced with a global qsl, value of O-3 mg h- ’g- ’ DCW when ammonium and glycerol became limiting. This result confirmed many other studies which reported that in batch culture, antibiotic
18
A. Lount?s, A. Lebrihi, C. Benslimane, G. Lefebvre, P. Germain
biosynthesis began as a result of a nutritional limitation.16 In order to maintain conditions conducive to spiramycin production for an extended time,
0.06
Feeding End
0
48
96 Time (h)
125
Time (h)
144
Fig. 7. Evolution of kinetic parameters of fed-batch culture no. 3. Evolution of specific growth rate (o), specific uptake rate of glycerol (w), specific uptake rate of ammonium (0) and specific production rate of spiramycin (0) in fed-batch culture on initial volume of 3 litres of the basal chemical defined medium with glycerol at 10 g litre- I and ammonium at 20 mM as carbon and nitrogen sources, respectively, with continuous feeding medium containing 27.5 g litre-’ of glycerol and 100 mM of ammonium, at a constant flow rate of 0.027 litre h- ’.
Fig. 6. Fed-batch of no. 3. Kinetics of growth (O), consumption of glycerol (m), consumption and accumulation of ammonium ions ( ?),?consumption of phosphate (0) and production of spiramycin (0) in fed-batch culture on initial volume of 3 litres of the basal chemical defined medium with glycerol at 10 g litre-’ and ammonium at 20 mM as carbon and nitrogen sources, respectively, with continuous feeding medium containing 275 g litre- 1 of glycerol and 100 mM of ammonium, at a constant flow rate of 0.027 litre h-‘. -: Indicates the beginning of the feeding. --D : Indicates the end of the feeding.
Table 1. Summary of results obtained under different conditions of batch and fed-batch cultures Initial feeding conditions
Fermentation process
qNHf
(mmol h-’ g-’ DCW) Control Fed-batchbatch culture no. 1 Fed-batch culture no. 2 Fed-batch culture no. 3
q5,, (max) (mg h-l Y NH;/&+
Spiramycin production
g-’ DCW)
(mm01g- ‘)
0.1
0.45
4;
0.3 05
14
35 60
3;
0.1
0.16
1.6
1.5
21
110
0
0.1
0.36
3.6
3
28
125
“Present at maximum spiramycin production.
0.3
Spiramycin production in fed-batch
determination of the parameters which influenced antibiotic biosynthesis was investigated using fedbatch culture with controlled initial kinetic parameters qglY and qNH: ( YNH:,giy). Continuous feeding of two key nutrients: glycerol and ammonium with different ammonium/glycerol ratios, simulating conditions of limitation, excess of ammonium or equilibrium between carbon and nitrogen sources, were performed. The highest specific production rate of spiramycin (qsP= 3 mg h- ’ g- ’ DCW) was obtained when the initial YNHf/glywas 3.5 mm01 g - ‘. Analysis of ammonium and glycerol indicated that the conditions allowing the best spiramycin production corresponded to an equilibrium between nitrogen and carbon sources. Under these conditions, it was possible to improve the specific production rate of spiramycin 10 fold in comparison to batch culture. A strong influence of the initial specific uptake rates of ammonium and glycerol on spiramycin biosynthesis was demonstrated. Since low spiramycin productivities occurred at high initial uptake rates of ammonium, the observed stimulation of spiramycin production following the restricted feeding of the nutrients suggests that catabolite inhibition/repression of antibiotic biosynthesis could be overcome by conditions generated at appropriate uptake rates of rapidly metabolisable carbon and nitrogen sources. The specific uptake rate of ammonium seemed to have a great effect on spiramycin production via the concentration of ammonium generated in the medium and present at the beginning of spiramycin production. This view was supported by the accumulation of ammonium ions, originated from high initial feeding or proteolysis, stopping of spiramycin biosynthesis. Indeed, the presence of 30 mM (first fed-batch) or strict limitant conditions of ammonium (second fed-batch) at the onset of spiramycin production, resulted in specific production rates of spiramycin of 83 and 50% respectively lower than those obtained in the presence of low concentration ammonium. In addition to the regulatory role, the nitrogen source seemed necessary for spiramycin biosynthesis since the chemical structure of spiramycin contains two amino sugars. The negative effect of ammonium on 16-membered macrolide antibiotic biosynthesis was reported in previous studies.’ ‘1I7 The onset of spiramycin synthesis was also influenced by the specific growth rate since qsP
19
increased when p decreased. A low specific growth rate appeared to be necessary to allow spiramycin production. The slow down in growth suggests a reduction in primary metabolic activities which would induce metabolic differentiation resulting in cells with a physiological state able to synthesise spiramycin. Antibiotic biosynthesis has been observed to begin when the specific growth rate reached a minimal levellx and the importance of specific growth rate on antibiotic production was studied in several Streptomyces sp. Spiramycin production occurred during the growth phase when the specific growth rate was limited by initial phosphate.” It has also been reported that an increase in the growth rate could repress key enzymes of antibiotic biosynthesis. Thus, tylosin production reached a maximum when the growth rate was 0.017 h- ’ and decreased at higher values.20 Vu-Trong and Gray6 showed an increase in the specific growth rate repressed the biosynthesis of propionyl-CoA carboxylase and methylmalonyl-CoA carboxyltransferase (enzymes necessary for the interconversion of propionyl-CoA and methylmalonyl-CoA, major precursors of tylonolide biosynthesis), in Streptomyces fradiae. In Streptomyces clavuligerus,21 cephamycin C production and expandase activity (an important enzyme of cephamycin C biosynthesis) reached a maximum at a very low specific growth rate equal to O-01 h-i and became practically nil when the growth rate was increased to 0.05 h-l. A considerable enhancement in the qsP value was obtained in fed-batch cultures especially in cultures with equilibrium conditions between nitrogen and carbon sources. However the dynamic pattern of qs,, in all the fed-batch cultures was characterized by high initial values that decreased steadily as the fermentation progressed. A lack in nutrient elements, particularly carbon and nitrogen sources could not be the cause of this phenomenon. Indeed, during each fed-batch qgly and &H,+ were maintained at the minimal basal level necessary for basal metabolism. Under these fermentation conditions, the specific growth rate was not maintained at an appropriate value and fell to zero or negative, reflecting bacterial death. This could be attributed to the irreversible loss of the metabolic differentiation of the cells required to produce spiramycin. The imposed conditions of feeding, although ensuring nutritional requirements, probably could not permit metabolic
20
A. Lou&s, A. Lebrihi, C. Be&mane,
differentiation which is an important signal to stimulate spiramycin biosynthesis. Correlation between expression of antibiotic biosynthesis and differentiation genes was reported by Hopwood. To confirm the relationship between y and qSp, continuous cultures with imposed low specific growth rate are required. A continuous cultivation system with a low constant growth rate was found to extend the time of maximal nikkomycin production of Streptomyces tendae.23
REFERENCES 1. Flores,
M. E. & Sanchez, S., Nitrogen regulation of erythromycin formation in Streptomyces erythreus.
FEMS Microbial. Lett., 26 (1985) 191-4. 2. Omura, S., Tanaka, Y., Mamada, H. & Masuma,
R., Effect of ammonium ion, inorganic phosphate and amino acids on the biosynthesis of protylonolide, a precursor of tylosin aglycone. J. Antibiot., 37 (1984)
494-502. 3. Shen, Y. Q., Heim,
J., Solomon, N. A., Wolfe, S. & A. L., Repression of B-lactam production in Cephalosporium acremonium by nitrogen sources. J. Antibiot., 37 (1984) 503-l 1. 4. Vu-Trong, K. & Gray, P. P., Influence of ammonium on the biosynthesis of the macrolide antibiotic tylosin. Enz. Demain,
Microbial. Technol., 9 (1987) 590-3. 5. Lebrihi, A., Lefebvre, G. & Germain, P., Carbon catabo-
lite regulation of cephamycin C and expandase biosynthesis in Streptomyces clavuligerus. Appl. Microbial. Biotechnol., 28 ( 1988) 44-5 1. 6. Vu-Trong, K. & Gray, P. P., Continuous culture studies on the regulation of tylosin biosynthesis. Biotechnol. Bioeng., 24 (1982) 1093-103. 7. Lebrihi, A., Lefebvre, G. & Germain, P., Phosphate repression of cephamycin and clavulanic acid production by Streptomyces clavuligetus. Appl. Microbial. Biotechnol., 26.( 1987) 130-5. 8. Vu-Trong, K., Bhuwapathanapun,
S. & Gray, P. P., Metain tylosin-producing Streptomyces control of tylosin biosynthesis. Antimicrob. Agents Chemother., 19 (1981) 209-12. 9. Court, J. R. & Pirt, S. J., The application of fed-batch ??culture to the penicillin fermentation, Abstract papers, bolic
regulation fradiae: phosphate
G. Lefebvre, P. Gerrnain
5th International Fermentation Symposium, ed. H. Dellweg. Verlag Versuchs, Berlin, 1976, p. 127. 10. Gray, P. P. & Vu-Trong, K., Production of the macrolide antibiotic tylosin in cyclic fed-batch culture. Biotechnol. Bioeng., 29 (1987) 33-40. 11. Lebrihi, A., Lamsaif, D., Germain, P. & Lefebvre, G., Effect of ammonium ions on spiramycin biosynthesis in Streptomyces ambofaciens. Appl. Microbial. Biotechnol., 37 (1992) 382-7.
12. Lou&, A., Rlgulation de la biosynthkse de la spiramytine chez Streptomyces ambofaciens par les sources azoties, carbonCes et les ions phosphate. Contr6le du catabolisme de la valine. PhD thesis, INPL-Nancy, France, 1994. 13. Bok, S. H. & Demain, A. L., Calorimetric assay for polyols. Anal. Biochem., 8 1 ( 1979) 18-20. 14. Kuzdzal-Savoie, S. & Lebon, F., Extraction of butterfat from liquid or dried milk. Tech. Lair., 690 (1971) 12-13. 15. Kendrick, K. E. & Ensign, J. C., Sporulation of Streptomyces griseus in submerged cultures. J. Bacterial., 155 (1983) 357-66. 16. Demain, A. L., Aharonowitz, Y. & Martin, J. F., Metabolic control of secondary biosynthetic pathways. In Biochemistry and Genetic Regulation of Commercially Important Antibiotics, ed. L. C. Vining, Addison Wesley,
London, 1983, pp. 49-72. 17. Omura, S., Tanaka, Y., Mamada, H. & Masuma, R., Effect of ammonium ion, inorganic phosphate and amino acids on the biosynthesis of protylonolide, a precursor of tylosin aglycone. J. Antibiot., 37 (1984) 494-502.
18. Martin, J. F., Biosynthesis of polyene macrolide antibiotics. Ann. Rev. Microbial., 31 (1977) 13-38. 19. Untrau-Taghian, S., Lebrihi, A., Germain, P. & Lefebvre, G., Influence of growth rate and precursors availability on spiramycin production, in Streptomyces ambofaciens. Can. J. Microbial., 41 (1995) 157-62.
20. Gray, P. P. & Bhuwapathanapun, S., Production of the macrolide antibiotic tylosin in batch and chemostat cultures. Biotechnol. Bioeng., 22 (1980) 1785-804. 21. Lebhrihi, A., Lefebvre, G. & Germain, P., A study on the regulation of cephamycin C and expandase biosynthesis by Streptomyces clavuligerus in continuous and batch culture. Appl. Microbial. Biotechnol., 28 (1988) 39-43. 22. Hopwood, D. A., Understanding the genetic control of antibiotic biosynthesis and sporulation in Streptomyces. In Biology of Actinomycetes ‘88. Japan Scientific Society Press, 1988, pp. 3-10. 23. Schi.iz, T. C., Piedler, H. P. & Z&ner, H., Optimized nikkomycin production by fed-batch and continuous fermentation. Appl. Microbial. Biotechnol., 39 (1993) 433-7.
ELSEVIER
Effect of Nitrogen/Carbon Ratio on the Specific Production Rate of Spiramycin by Streptomyces ambofaciens Anissa Low&, Ahmed Lebrihi,” Chouki Benslimane, G&ard Lefebvre & Pierre Germain Laboratorie de Microbiologic Industrielle et Alimentaire, For& de Haye, 54500 Vandoeuvre, France (Received 18 November
ENSAIA,
Institut National Polytechnique
de Lorraine, 2 avenue de la
1994; accepted 20 February 1995)
Streptomyces ambofaciens was grown on a synthetic medium with glycerol (log litre-‘) and ammonium (20 mM) as carbon and nitrogen sources, respectively. Batch fermentation produced a specific production rate of spiramycin (Q)
of O-3
mg h - ’g- ’DC W. Improvement of spiramycin production was achieved by fedbatch culture. The strong influence of the initial uptake rates of glycerol (qpJ and ammonium (q,,& on the speci@c production rate of spiramycin (qSP) was demonstrated by conducting several fermentations with continuous feeding of glycerol and ammonium, in different ratios. The specific production rate of spiramycin was increased 10 fold. A low specific growth rate was also necessary for spiramycin biosynthesis.
phate12 were in excess. In this fermentation is shown to result spiramycin biosynthesis when batch cultures. Using fed-batch ence of initial ycerol specific production rate of studied.
INTRODUCTION Antibiotic production is influenced by the nitrogen,1-4 carbonsT6 and phosphate sources738 used in the fermentation medium. Additional feeds of medium ingredients, precursors, or complete medium using fed-batch culture are often found to extend biosynthesis and result in greater productivity of the desired metabolite.” Production of the 16-membered macrolide antibiotic tylosin was increased several fold in fed-batch fermentation.‘” Streptomyces ambofaciens produces a 16-membered macrolide antibiotic, spiramycin. In batch biosynthesis was greatly culture, spiramycin reduced when ammonium,” glycerol or phos*To whom correspondence
paper, fed-batch in stimulation of compared with cultures the influ-
Yankninm/gl ( YNFI:,gy)on the spiramycin
was
MATERIALS AND METHODS Microorganisms These studies were carried out with Streptumyces ambofaciens RP 18 1110 (Rhone-Poulenc, Vitrysur-Seine, France) producer of spiramycin and Bacillus subtilis ATCC 6633 as test microorganism.
should be addressed.
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Spiramycin production in fed-batch
Equation (3) is used for the determination of the specific production rate of spiramycin (q,,). The maximum specific production and volumetric production of spiramycin are expressed in (mg g-l DCW) and (mg litre- l), respectively. Yammonium/glycerol is calculated from ~NH: and qd, and expressed in mmols g- ’ ( YNHr/qgly= qNH$/ %Y )*
RESULTS Characteristics of growth and spiramycin production in batch culture on basal medium with glycerol 10 g litre - * and ammonium 20 mM The results from a typical spiramycin batch culture on the synthetic medium are shown in Fig. 1. This fermentation was characterized by a maximal specific growth rate of 0.05 5 h- ’ and specific uptake rate of glycerol and ammonium of 0.2 g h-l g-’ DCW and 0.5 mm01 h-l g-’ DCW, respectively. Spiramycin production began after 50 h of fermentation when the nitrogen and carbon sources were completely consumed. Spiramycin was produced with a global specific production rate (q,J of 0.3 mg h-l g-r DCW and reached 35 mg litre- ’ and 14 mg gg * DCW of final volumetric production and a maximum specific production of spiramycin, respectively.
15
At the end of fermentation, phosphate were detected.
7 mM of residual
Characteristics of growth and spiramycin production in fed-batch cultures To prevent the negative effect on spiramycin biosynthesis of an eventual accumulation of phosphate, I2 the feed medium for fed-batch cultures did not contain phosphate. About 1 mu of phosphate was detected constantly during the course of each fed-batch. Fed-batch culture (1) with 40 g litre- ’glycerol and 180 mM ammonium at a flow rate of 0.019 litre h-l
Figures 2 and 3 show the results obtained from a fed-batch fermentation with a continuous feed of glycerol (40 g litre-‘) and ammonium (180 mM). The fed-batch was started at 43 h. The flow rate used (0.019 litre h- ‘) led to initial specific consumption rates of glycerol (q@,) and ammonium (qNH:)of 0.1 g h-l g-l DCW and 0.45 mm01 h-’ g-’ DCW, respectively. Feeding
Begining
Eyd
10
0
48
96
144
192
240
288
336
Time (h)
‘0
48
96
Tie
144
192
(h)
Fig. 1. Batch culture. Kinetics of growth (O), consumption of glycerol (m), consumption and accumulation of ammonium ions ( ?),? consumption of phosphate (0) and production of spiramycin (0) in batch culture on the basal chemical defined medium with glycerol at 10 g litre-’ and ammonium at 20 mM as carbon and nitrogen sources, respectively.
Fig. 2. Fed-batch culture no. 1. Kinetics of growth (O), consumption of glycerol (?? ),consumption and accumulation of ammonium ions ( ?),? consumption of phosphate (0) and production of spiramycin (0) in fed-batch culture on initial volume of 3 litres of the basal chemical defined medium with glycerol at 10 g litre-’ and ammonium at 20 mM as carbon and nitrogen sources, respectively, with continuous feeding medium containing 40 g litre- ’ of glycerol and 180 mM of ammonium, at a constant flow rate of 0.019 litre h- l. - : Indicates the beginning of the feeding. --D: Indicates the end of the feeding.
16
A. Lou&, A. Lebrihi, C. Ben&mane, G. Lefebvre, P. Germain
0.06 .
0.05 g h-’ g-’ DCW and 0.1 mm01 h-’ g-l DCW, respectively. The maximal specific production rate of spiramycin was observed when 30 mM of ammonium were present in the medium. The accumulation of ammonium observed after cessation of feeding was probably due to proteolysis. Fed-batch culture (2) with 40g litre-’ glycerol and 66 rn~ ammonium at a flowrate of 0.019 litre h-’
40
90
140
190
240
Time(h)
Fig. 3. Evolution of the kinetic parameters of fed-batch culture no. 1. Evolution of specific growth rate (0), specific uptake rate of glycerol ( ?? ), specific uptake rate of ammonium (0) and specific production rate of spiramycin (0) in fedbatch culture on initial volume of 3 litres of the basal chemical defined medium with glycerol at 10 g litre-’ and ammonium at 20 mM as carbon and nitrogen sources, respectively, with continuous feeding medium containing 40 g litre- ’of glycerol and 180 mM of ammonium, at a constant flow rate of 0.0 19 litre h - I.
The results showed that glycerol was completely consumed; less than 0.5 g litre- ’ was detected during the fermentation suggesting growth limitation by carbon source. In contrast, ammonium ions were found in excess. Ammonium was largely accumulated at the end of fedbatch (70 mM) probably causing a cessation of spiramycin production. Production of spiramycin began at 100 h and a final volumetric production of 60 mg litre-’ and a maximum specific spiramycin production of 14 mg gg’ DCW was achieved. The maximum specific production rate of spiramycin (0.5 mg h- ’ g- ’ DCW) was increased by 60% with respect to batch culture. Spiramycin production occurred when the specific growth rate and the specific uptake rates of glycerol and ammonium fell to about 0.01 h- ‘,
In an attempt to bypass the ammonium effect occurring at high initial specific ammonium uptake rates and to prevent the accumulation of these ions during batch feeding, a second fedbatch was carried out. The feed medium contained glycerol at 40 g litre- l but the ammonium concentration was reduced to 66 mM. These concentrations given with a flow rate of 0.019 litre h-’ corresponded to initial specific uptake rates of glycerol and ammonium of O-1 g h-l g-’ DCW and 0.16 mmol h-l g-l DCW respectively. Feeding commenced at 60 h. Figures 4 and 5 show the results obtained from the fermentation. In these conditions, less than 0.5 g litre- ’ of glycerol were detected during the fed-batch. Ammonium was not detected at all. These observations could indicate conditions of limitation in nitrogen source. In Figs 4 and 5, maximal qsp occurred at 96 h (1.5 mg h- ’ gg ’ DCW) when the specific uptake rate of glycerol and ammonium was lowered to 0.05 g h-’ gg ’ DCW and 0.082 mmol h-’ g- ’ DCW respectively and the specific growth rate to O-035 h- ‘. q,r was doubled by comparison with the first fed-batch in which ammonium ions were accumulated. The maximum specific production of spiramycin was 21 mg g- ’ DCW and the final volumetric production reached 110 mg litre-‘. We noted that S. ambofaciens sporulated under these fermentation conditions. This observation may imply extreme conditions of limitation, particularly in ammonium. Previous studies with Streptomyces griseus showed that sporulation occurred in submerged culture limited by carbon and nitrogen sources.” Fed batch culture (3) with 27.5 g litre- ’glycerol and 100 mM ammonium at aflow rate of 0~027litre h-’
In order to improve spiramycin production, ammonium feeding was performed so as to obtain an intermediate initial specific uptake rate of ammonium between the first and the second fedbatch experiments (O-36 mm01 h- ’ g-’ DCW).
Spiramycin production in fed-batch Begining
Feeding
17 0.06
End
1
J 5
0.4
0.2
0
48
96
144
192
240
288
Time (h)
Fig. 4. Fed-batch no. 2. Kinetics of growth (O), consumption of glycerol (m), consumption and accumulation of ammonium ions ( ?),?consumption of phosphate (0) and production of spiramycin (0) in fed-batch culture on initial volume of 3 litres of the basal chemical defined medium with glycerol at 10 g litre-’ and ammonium at 20 mM as carbon and nitrogen sources, respectively, with continuous feeding medium containing 40 g litre-’ of glycerol and 66 mM of ammonium, at a constant flow rate of 0.019 litre h-‘. -: Indicates the beginning of the feeding. + : Indicates the end of the feeding.
Glycerol was always fed to obtain an initial specific uptake rate of 0.1 g h- ’ gg ’DCW. Ammonium and glycerol were fed with a flow rate of O-027 litre h- ’ at a concentration of glycerol and ammonium of 27.5 g litre- ’ and 100 mM, respectively. Feeding was started at 47.5 h. Figures 6 and 7 show the results obtained from the fermentation which was characterized by a high initial qsr value of 3 mg h- ’g- ’DCW which was maintained at O-5 mg h- ’ gg ’ DCW after a sudden decrease. The final volumetric concentration of spiramycin was 125 mg litre- ’ and the specific spiramycin production was 28 mg g- ’ DCW. During the fed-batch, glycerol and ammonium were detected at less than 0.5 g litree’ and 0.3 mM, respectively. This could imply an equilibrium between nitrogen and carbon sources. Production of spiramycin occurred when a decrease in ,u was observed and the specific uptake rates of glycerol
40
140
90
‘90
Tune (h)
Fig. 5. Evolution of the specific growth rate (O), specific uptake rate of glycerol ( ??), specific uptake rate of ammonium (0) and specific production rate of spiramycin (0) in fedbatch culture on initial volume of 3 litres of the basal chemical defined medium with glycerol at 10 g litre- 1 and ammonium at 20 mM as carbon and nitrogen sources, respectively, with continuous feeding medium containing 40 g litre- l of glycerol and 66 mM of ammonium at a constant flow rate of 0.0 19 litre h- I.
and ammonium were about 0.05 g h-’ gg ’DCW and O-18 mmol h- ’ g- ’ DCW, respectively. At the end of the feeding, accumulation of 13 mM of ammonium was observed, probably due to the proteolysis.
DISCUSSION The data presented in this paper are summarised in Table 1. They show that in batch culture on the chemically defined medium, spiramycin was produced with a global qsl, value of O-3 mg h- ’g- ’ DCW when ammonium and glycerol became limiting. This result confirmed many other studies which reported that in batch culture, antibiotic
18
A. Lount?s, A. Lebrihi, C. Benslimane, G. Lefebvre, P. Germain
biosynthesis began as a result of a nutritional limitation.16 In order to maintain conditions conducive to spiramycin production for an extended time,
0.06
Feeding End
0
48
96 Time (h)
125
Time (h)
144
Fig. 7. Evolution of kinetic parameters of fed-batch culture no. 3. Evolution of specific growth rate (o), specific uptake rate of glycerol (w), specific uptake rate of ammonium (0) and specific production rate of spiramycin (0) in fed-batch culture on initial volume of 3 litres of the basal chemical defined medium with glycerol at 10 g litre- I and ammonium at 20 mM as carbon and nitrogen sources, respectively, with continuous feeding medium containing 27.5 g litre-’ of glycerol and 100 mM of ammonium, at a constant flow rate of 0.027 litre h- ’.
Fig. 6. Fed-batch of no. 3. Kinetics of growth (O), consumption of glycerol (m), consumption and accumulation of ammonium ions ( ?),?consumption of phosphate (0) and production of spiramycin (0) in fed-batch culture on initial volume of 3 litres of the basal chemical defined medium with glycerol at 10 g litre-’ and ammonium at 20 mM as carbon and nitrogen sources, respectively, with continuous feeding medium containing 275 g litre- 1 of glycerol and 100 mM of ammonium, at a constant flow rate of 0.027 litre h-‘. -: Indicates the beginning of the feeding. --D : Indicates the end of the feeding.
Table 1. Summary of results obtained under different conditions of batch and fed-batch cultures Initial feeding conditions
Fermentation process
qNHf
(mmol h-’ g-’ DCW) Control Fed-batchbatch culture no. 1 Fed-batch culture no. 2 Fed-batch culture no. 3
q5,, (max) (mg h-l Y NH;/&+
Spiramycin production
g-’ DCW)
(mm01g- ‘)
0.1
0.45
4;
0.3 05
14
35 60
3;
0.1
0.16
1.6
1.5
21
110
0
0.1
0.36
3.6
3
28
125
“Present at maximum spiramycin production.
0.3
Spiramycin production in fed-batch
determination of the parameters which influenced antibiotic biosynthesis was investigated using fedbatch culture with controlled initial kinetic parameters qglY and qNH: ( YNH:,giy). Continuous feeding of two key nutrients: glycerol and ammonium with different ammonium/glycerol ratios, simulating conditions of limitation, excess of ammonium or equilibrium between carbon and nitrogen sources, were performed. The highest specific production rate of spiramycin (qsP= 3 mg h- ’ g- ’ DCW) was obtained when the initial YNHf/glywas 3.5 mm01 g - ‘. Analysis of ammonium and glycerol indicated that the conditions allowing the best spiramycin production corresponded to an equilibrium between nitrogen and carbon sources. Under these conditions, it was possible to improve the specific production rate of spiramycin 10 fold in comparison to batch culture. A strong influence of the initial specific uptake rates of ammonium and glycerol on spiramycin biosynthesis was demonstrated. Since low spiramycin productivities occurred at high initial uptake rates of ammonium, the observed stimulation of spiramycin production following the restricted feeding of the nutrients suggests that catabolite inhibition/repression of antibiotic biosynthesis could be overcome by conditions generated at appropriate uptake rates of rapidly metabolisable carbon and nitrogen sources. The specific uptake rate of ammonium seemed to have a great effect on spiramycin production via the concentration of ammonium generated in the medium and present at the beginning of spiramycin production. This view was supported by the accumulation of ammonium ions, originated from high initial feeding or proteolysis, stopping of spiramycin biosynthesis. Indeed, the presence of 30 mM (first fed-batch) or strict limitant conditions of ammonium (second fed-batch) at the onset of spiramycin production, resulted in specific production rates of spiramycin of 83 and 50% respectively lower than those obtained in the presence of low concentration ammonium. In addition to the regulatory role, the nitrogen source seemed necessary for spiramycin biosynthesis since the chemical structure of spiramycin contains two amino sugars. The negative effect of ammonium on 16-membered macrolide antibiotic biosynthesis was reported in previous studies.’ ‘1I7 The onset of spiramycin synthesis was also influenced by the specific growth rate since qsP
19
increased when p decreased. A low specific growth rate appeared to be necessary to allow spiramycin production. The slow down in growth suggests a reduction in primary metabolic activities which would induce metabolic differentiation resulting in cells with a physiological state able to synthesise spiramycin. Antibiotic biosynthesis has been observed to begin when the specific growth rate reached a minimal levellx and the importance of specific growth rate on antibiotic production was studied in several Streptomyces sp. Spiramycin production occurred during the growth phase when the specific growth rate was limited by initial phosphate.” It has also been reported that an increase in the growth rate could repress key enzymes of antibiotic biosynthesis. Thus, tylosin production reached a maximum when the growth rate was 0.017 h- ’ and decreased at higher values.20 Vu-Trong and Gray6 showed an increase in the specific growth rate repressed the biosynthesis of propionyl-CoA carboxylase and methylmalonyl-CoA carboxyltransferase (enzymes necessary for the interconversion of propionyl-CoA and methylmalonyl-CoA, major precursors of tylonolide biosynthesis), in Streptomyces fradiae. In Streptomyces clavuligerus,21 cephamycin C production and expandase activity (an important enzyme of cephamycin C biosynthesis) reached a maximum at a very low specific growth rate equal to O-01 h-i and became practically nil when the growth rate was increased to 0.05 h-l. A considerable enhancement in the qsP value was obtained in fed-batch cultures especially in cultures with equilibrium conditions between nitrogen and carbon sources. However the dynamic pattern of qs,, in all the fed-batch cultures was characterized by high initial values that decreased steadily as the fermentation progressed. A lack in nutrient elements, particularly carbon and nitrogen sources could not be the cause of this phenomenon. Indeed, during each fed-batch qgly and &H,+ were maintained at the minimal basal level necessary for basal metabolism. Under these fermentation conditions, the specific growth rate was not maintained at an appropriate value and fell to zero or negative, reflecting bacterial death. This could be attributed to the irreversible loss of the metabolic differentiation of the cells required to produce spiramycin. The imposed conditions of feeding, although ensuring nutritional requirements, probably could not permit metabolic
20
A. Lou&s, A. Lebrihi, C. Be&mane,
differentiation which is an important signal to stimulate spiramycin biosynthesis. Correlation between expression of antibiotic biosynthesis and differentiation genes was reported by Hopwood. To confirm the relationship between y and qSp, continuous cultures with imposed low specific growth rate are required. A continuous cultivation system with a low constant growth rate was found to extend the time of maximal nikkomycin production of Streptomyces tendae.23
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M. E. & Sanchez, S., Nitrogen regulation of erythromycin formation in Streptomyces erythreus.
FEMS Microbial. Lett., 26 (1985) 191-4. 2. Omura, S., Tanaka, Y., Mamada, H. & Masuma,
R., Effect of ammonium ion, inorganic phosphate and amino acids on the biosynthesis of protylonolide, a precursor of tylosin aglycone. J. Antibiot., 37 (1984)
494-502. 3. Shen, Y. Q., Heim,
J., Solomon, N. A., Wolfe, S. & A. L., Repression of B-lactam production in Cephalosporium acremonium by nitrogen sources. J. Antibiot., 37 (1984) 503-l 1. 4. Vu-Trong, K. & Gray, P. P., Influence of ammonium on the biosynthesis of the macrolide antibiotic tylosin. Enz. Demain,
Microbial. Technol., 9 (1987) 590-3. 5. Lebrihi, A., Lefebvre, G. & Germain, P., Carbon catabo-
lite regulation of cephamycin C and expandase biosynthesis in Streptomyces clavuligerus. Appl. Microbial. Biotechnol., 28 ( 1988) 44-5 1. 6. Vu-Trong, K. & Gray, P. P., Continuous culture studies on the regulation of tylosin biosynthesis. Biotechnol. Bioeng., 24 (1982) 1093-103. 7. Lebrihi, A., Lefebvre, G. & Germain, P., Phosphate repression of cephamycin and clavulanic acid production by Streptomyces clavuligetus. Appl. Microbial. Biotechnol., 26.( 1987) 130-5. 8. Vu-Trong, K., Bhuwapathanapun,
S. & Gray, P. P., Metain tylosin-producing Streptomyces control of tylosin biosynthesis. Antimicrob. Agents Chemother., 19 (1981) 209-12. 9. Court, J. R. & Pirt, S. J., The application of fed-batch ??culture to the penicillin fermentation, Abstract papers, bolic
regulation fradiae: phosphate
G. Lefebvre, P. Gerrnain
5th International Fermentation Symposium, ed. H. Dellweg. Verlag Versuchs, Berlin, 1976, p. 127. 10. Gray, P. P. & Vu-Trong, K., Production of the macrolide antibiotic tylosin in cyclic fed-batch culture. Biotechnol. Bioeng., 29 (1987) 33-40. 11. Lebrihi, A., Lamsaif, D., Germain, P. & Lefebvre, G., Effect of ammonium ions on spiramycin biosynthesis in Streptomyces ambofaciens. Appl. Microbial. Biotechnol., 37 (1992) 382-7.
12. Lou&, A., Rlgulation de la biosynthkse de la spiramytine chez Streptomyces ambofaciens par les sources azoties, carbonCes et les ions phosphate. Contr6le du catabolisme de la valine. PhD thesis, INPL-Nancy, France, 1994. 13. Bok, S. H. & Demain, A. L., Calorimetric assay for polyols. Anal. Biochem., 8 1 ( 1979) 18-20. 14. Kuzdzal-Savoie, S. & Lebon, F., Extraction of butterfat from liquid or dried milk. Tech. Lair., 690 (1971) 12-13. 15. Kendrick, K. E. & Ensign, J. C., Sporulation of Streptomyces griseus in submerged cultures. J. Bacterial., 155 (1983) 357-66. 16. Demain, A. L., Aharonowitz, Y. & Martin, J. F., Metabolic control of secondary biosynthetic pathways. In Biochemistry and Genetic Regulation of Commercially Important Antibiotics, ed. L. C. Vining, Addison Wesley,
London, 1983, pp. 49-72. 17. Omura, S., Tanaka, Y., Mamada, H. & Masuma, R., Effect of ammonium ion, inorganic phosphate and amino acids on the biosynthesis of protylonolide, a precursor of tylosin aglycone. J. Antibiot., 37 (1984) 494-502.
18. Martin, J. F., Biosynthesis of polyene macrolide antibiotics. Ann. Rev. Microbial., 31 (1977) 13-38. 19. Untrau-Taghian, S., Lebrihi, A., Germain, P. & Lefebvre, G., Influence of growth rate and precursors availability on spiramycin production, in Streptomyces ambofaciens. Can. J. Microbial., 41 (1995) 157-62.
20. Gray, P. P. & Bhuwapathanapun, S., Production of the macrolide antibiotic tylosin in batch and chemostat cultures. Biotechnol. Bioeng., 22 (1980) 1785-804. 21. Lebhrihi, A., Lefebvre, G. & Germain, P., A study on the regulation of cephamycin C and expandase biosynthesis by Streptomyces clavuligerus in continuous and batch culture. Appl. Microbial. Biotechnol., 28 (1988) 39-43. 22. Hopwood, D. A., Understanding the genetic control of antibiotic biosynthesis and sporulation in Streptomyces. In Biology of Actinomycetes ‘88. Japan Scientific Society Press, 1988, pp. 3-10. 23. Schi.iz, T. C., Piedler, H. P. & Z&ner, H., Optimized nikkomycin production by fed-batch and continuous fermentation. Appl. Microbial. Biotechnol., 39 (1993) 433-7.