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Solid state cultivation of Streptomyces clavuligerus for cephamycin C production
Process Biochemistry 34 (1999) 325 – 328
Solid state cultivation of Streptomyces cla6uligerus for cephamycin C production Krishna Prasad Kota, Padma Sridhar * Microbiology Department, Osmania Uni6ersity, Hyderabad, Andhra Pradesh, India Received 17 June 1997; received in revised form 21 May 1998; accepted 24 May 1998
Abstract Solid state cultivation of Streptomyces cla6uligerus for cephamycin C production was carried out in a system consisting of wheat rawa 5 g; cotton seed deoiled cake 5 g; sunflower cake 0·5 g; corn steep liquor 1 g; MgSO4.7H2O 0·06 g; CaCO3 0·1 g; K2HPO4 4·4 g; with initial moisture content of 80%, initial pH 6·5 and a fermentation temperature in the range 28 – 30°C. The fermentation cycle was about 5 days. Streptomyces cla6uligerus growth was observed on the 2nd day and production of cephamycin C was initiated on 3rd day. Abundant mycelial growth was observed from the 3rd day and reached stationary phase by the 5th day. Cephamycin C was produced maximally at a rate of 15 mg/g substrate on the 5th day and was stable until the 30th day with only marginal decrease in titre. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Solid state fermentation; Wheat rawa; Streptomyces cla6uligerus; Cephamycin C
1. Introduction Cephamycin C is a broad spectrum, b-lactam antibiotic produced by a variety of organisms including Streptomyces cattleya, Streptomyces cla6uligerus and Nocardia lactamdurans [1,2]. Submerged fermentation is generally employed for cephamycin C production but requires high energy [3]. While in search for cheaper fermentation processes with a high yield of antibiotic, solid state fermentation (SSF) was found to be more attractive. This paper describes the conditions suitable for a S. cla6uligerus hyper producing strain to produce cephamycin C through SSF.
2. Material and methods Wheat bran, wheat rawa (wheat grains broken into small pieces of size of 0·1 – 1 mm) rice bran, sago, soybean, brick powder, coconut coir, rice straw, rice husk, sugarcane baggasse, cotton fibers, ground nut deoiled cake, coconut deoiled cake, corn steep liquor, * Corresponding author.
NaNO3, NH4NO3, urea, MgSO47H2O, CaCO3 and K2HPO4 were all obtained locally.
2.1. Cultures Streptomyces cla6uligerus was used for cephamycin production and E.coli strain ESS was used as the indicator organism for bioassay.
2.2. Culture media and condition S. cla6uligerus was grown in M1 Medium (starch 1%; Sunflower cake 0·5%; corn steep liquor 1%; MgSO4.7H2 0·1%; K2HPO4 4·4%). The spores were then inoculated into solid state medium in 250 ml flasks, mixed thoroughly and incubated at 28–30°C with intermittent shaking. All experiments were done in triplicate and average values taken.
2.3. Antibiotic extraction Antibiotic was extracted directly in water by adding three times the weight of solid medium and by shaking for 1 h.
0032-9592/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 9 8 ) 0 0 0 7 8 - 8
K. Prasad Kota, P. Sridhar / Process Biochemistry 34 (1999) 325–328
326
2.4. Antibiotic assay
Table 2 Additional nutrient supports for cephamycin C production
Antimicrobial activity of culture broth was tested by the agar diffusion method as described earlier using E.coli ESS as indicator organism [4].
Additional nutrient supports
Weight (g)
Cephamycin C titres (mg/g)
Ground nut deoiled cake Coconut deoiled cake Cotton seed deoiled cake Sunflower deoiled cake NaNO3 NH4NO3 Urea Corn steep liquor Mustard deoiled cake Cotton seed deoiled cake +corn steep liquor +sunflower deoiled cake
1 1 1 1 1 1 1 1 1 5+1+1
7·0 7·0 8·8 8·0 Nil Nil Nil 7·5 7·0 10·0
2.5. High performance liquid chromatography Culture extract was filtered and antibiotic was estimated as described earlier [5]. HPLC was used with a reverse phase column Shimpak ODS (C18 octadecyl silane). The mobile phase was 0·05 M KH2PO4 adjusted to pH 3·0 with concentrated phosphoric acid. A 60% 0·05 M KH2PO4 (pH 3·0) and 40% acetonitrile mixture was used for fine resolution of peaks. 20 ml of fermentation broth was injected and eluted at a flow rate of 1·5 ml/min. Cephamycin C was detected at 253 nm.
(7–10 mg/g of weight of substrate). Above 80% moisture concentration, further increases resulted in decreases of cephamycin C concentrations.
3. Results Among the different solid supports used (Table 1) only some could support the growth of S. cla6uligerus and production of cephamycin C. Wheat rawa gave the highest titre value of cephamycin C. Addition of sunflower deoiled cake, cotton seed deoiled cake, corn steep liquor, enhanced production (Table 2) and at 0·5, 1·0 and 50% weight of support respectively gave highest titres of cephamycin C (10 mg/g).
3.2. Inoculum le6el When solid supports were mixed with 1× 102 cell/g of solid support cephamycin C production was very low (1 mg/g) but as the concentration reached 108 cell/g the cephamycin C concentration reached a maximum of 10 mg/g of substrate. A further increase in cell concentration did not affect the concentration of cephamycin C.
3.1. Physical parameters The effect of initial moisture content on cephamycin C production is shown in Fig. 1. Below 50% moisture there was no appreciable growth of S. cla6uligerus. In the range from 60 to 80% moisture content there was substantial growth and cephamycin C production
3.3. Initial pH The effect of initial pH on cephamycin C production is shown in Fig. 2. When the initial pH was 5·0 there was very little growth and no production of
Table 1 Solid supports for the cephamycin C production Solid supports
Weight (g)
Cephamycin C titres (mg/g)
Wheat bran Wheat rawa Rice husk Rice straw Sago Soy bean Sugarcane baggasse Cotton fibres+rice bran Coconut Coir+rice bran Brick powder+rice bran
10 10 10 10 10 10 10 5+5
5·2 7·0 Nil Nil 5·0 3·1 Nil 6·0
5+5
4·2
5+5
3·7 Fig. 1.
K. Prasad Kota, P. Sridhar / Process Biochemistry 34 (1999) 325–328
327
Fig. 4. Fig. 2.
cephamycin C. As the pH was increased, production of cephamycin C increased and reached a maximum (15 mg/g) at 6·5. A further increase in pH, resulted in a decrease in cephamycin C production and there was no production at 8·0 (Fig. 3).
wards with a concentration of 6 mg/g and reached a maximum on day 5 (15 mg/g) and then decreased slightly up to day 30 (9·5 mg/g). Thereafter there was no cephamycin C production.
3.4. Incubation temperature
4. Discussion
The effect of temperature on cephamycin C production is shown in Fig. 4. At 30°C cephamycin C production was 15 mg/g and then decreased to zero when the incubation temperature was 37°C. A low concentration of cephamycin C was produced at 20°C (6 mg/g).
The largest single class of drugs made entirely through biological synthesis are antibiotics. At least 70 of the approximately 100 antibiotics used for treatment of human infections are derived from substances produced by Streptomyces [6]. Cephamycin C is a broad spectrum b-lactam antibiotic which is a basic bulk drug for semisynthetics like cefoxitin, cefametadozole [7]. Submerged fermentation (SmF) is usually employed for commercial production of cephamycin C but, an alternative economical method involving SSF for cephamycin C production is described. During the course of cephamycin C production by SSF, wheat rawa was found to be most suitable for production by S. cla6uligerus. Cephamycin C was detected on the 3rd day of fermentation and reached a maximum on the 5th day. Cephamycin C production was almost stable until the 30th day which was not the case in SmF. The initial moisture content of substrates was critical for cephamycin C production by SSF. At less than 50% moisture there was no growth of S. cla6uligerus. As the initial percentage of water was increased the production of cephamycin C increased until it reached a maximum at 80%. The ideal pH for cephamycin C production was 6·5. The pH (6·5) and temperature (28°C) were optimal both for SSF and SmF for cephamycin C production. It may be concluded that the process of cephamycin C production through SSF is potentially comparable to that of SmF.
3.5. Fermentation period The production of cephamycin C in solid substrate was observed from the 3rd day of fermentation on-
Fig. 3.
328
K. Prasad Kota, P. Sridhar / Process Biochemistry 34 (1999) 325–328
Acknowledgements The authors would like to acknowledge Department of Biotechnology, Government of India, for the financial support vide letter no. BT/02/08/92 to PS and University Grants Commission, Government of India vide letter no. 98/UGC-CSIR/94 for fellowship to KPK. References [1] Nagarajan, R, Boeck, L D, Gorman, M, Hamill, R L, Higger, D E, Hoehn, M M, Stark, W M and Whitney, J G, Journal of the American Chemical Society 1971;93:2308–12.
.
[2] Stapley, E O, Jackson, M, Hernandez, S, Zimmerman, S B, Cirria, S A, Mochale, S A, Mochale, S, Mata, J M, Woodreff, H B and Henlti, D, Antimicrobial Agents and Chemotherapy 1972;2:122 – 35. [3 ] Manual of Industrial Microbiology and Biotechnology, eds Arnold L Demain and Nadine A Solomen. American Society for Microbiology, Washington, D.C 1986, pp. 66 – 83. [4] Kavanagh, F, In Analytical Microbiology, Vol. I. Academic Press, New York, 1984, pp. 5 – 18. [5] Brana, A F P and Demain, A L, Journal of General Microbiology 1989;132:305 – 1317. [6] Yang, Shang-Shyng and Ling, Meei-Yuch, Biotechnology and Bioengineering 1989;33:1021 – 8. [7] Aharonowitz, Y and Demain, A Archives of Microbiology 1977; 115:169 – 73.
Solid state cultivation of Streptomyces cla6uligerus for cephamycin C production Krishna Prasad Kota, Padma Sridhar * Microbiology Department, Osmania Uni6ersity, Hyderabad, Andhra Pradesh, India Received 17 June 1997; received in revised form 21 May 1998; accepted 24 May 1998
Abstract Solid state cultivation of Streptomyces cla6uligerus for cephamycin C production was carried out in a system consisting of wheat rawa 5 g; cotton seed deoiled cake 5 g; sunflower cake 0·5 g; corn steep liquor 1 g; MgSO4.7H2O 0·06 g; CaCO3 0·1 g; K2HPO4 4·4 g; with initial moisture content of 80%, initial pH 6·5 and a fermentation temperature in the range 28 – 30°C. The fermentation cycle was about 5 days. Streptomyces cla6uligerus growth was observed on the 2nd day and production of cephamycin C was initiated on 3rd day. Abundant mycelial growth was observed from the 3rd day and reached stationary phase by the 5th day. Cephamycin C was produced maximally at a rate of 15 mg/g substrate on the 5th day and was stable until the 30th day with only marginal decrease in titre. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Solid state fermentation; Wheat rawa; Streptomyces cla6uligerus; Cephamycin C
1. Introduction Cephamycin C is a broad spectrum, b-lactam antibiotic produced by a variety of organisms including Streptomyces cattleya, Streptomyces cla6uligerus and Nocardia lactamdurans [1,2]. Submerged fermentation is generally employed for cephamycin C production but requires high energy [3]. While in search for cheaper fermentation processes with a high yield of antibiotic, solid state fermentation (SSF) was found to be more attractive. This paper describes the conditions suitable for a S. cla6uligerus hyper producing strain to produce cephamycin C through SSF.
2. Material and methods Wheat bran, wheat rawa (wheat grains broken into small pieces of size of 0·1 – 1 mm) rice bran, sago, soybean, brick powder, coconut coir, rice straw, rice husk, sugarcane baggasse, cotton fibers, ground nut deoiled cake, coconut deoiled cake, corn steep liquor, * Corresponding author.
NaNO3, NH4NO3, urea, MgSO47H2O, CaCO3 and K2HPO4 were all obtained locally.
2.1. Cultures Streptomyces cla6uligerus was used for cephamycin production and E.coli strain ESS was used as the indicator organism for bioassay.
2.2. Culture media and condition S. cla6uligerus was grown in M1 Medium (starch 1%; Sunflower cake 0·5%; corn steep liquor 1%; MgSO4.7H2 0·1%; K2HPO4 4·4%). The spores were then inoculated into solid state medium in 250 ml flasks, mixed thoroughly and incubated at 28–30°C with intermittent shaking. All experiments were done in triplicate and average values taken.
2.3. Antibiotic extraction Antibiotic was extracted directly in water by adding three times the weight of solid medium and by shaking for 1 h.
0032-9592/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 9 8 ) 0 0 0 7 8 - 8
K. Prasad Kota, P. Sridhar / Process Biochemistry 34 (1999) 325–328
326
2.4. Antibiotic assay
Table 2 Additional nutrient supports for cephamycin C production
Antimicrobial activity of culture broth was tested by the agar diffusion method as described earlier using E.coli ESS as indicator organism [4].
Additional nutrient supports
Weight (g)
Cephamycin C titres (mg/g)
Ground nut deoiled cake Coconut deoiled cake Cotton seed deoiled cake Sunflower deoiled cake NaNO3 NH4NO3 Urea Corn steep liquor Mustard deoiled cake Cotton seed deoiled cake +corn steep liquor +sunflower deoiled cake
1 1 1 1 1 1 1 1 1 5+1+1
7·0 7·0 8·8 8·0 Nil Nil Nil 7·5 7·0 10·0
2.5. High performance liquid chromatography Culture extract was filtered and antibiotic was estimated as described earlier [5]. HPLC was used with a reverse phase column Shimpak ODS (C18 octadecyl silane). The mobile phase was 0·05 M KH2PO4 adjusted to pH 3·0 with concentrated phosphoric acid. A 60% 0·05 M KH2PO4 (pH 3·0) and 40% acetonitrile mixture was used for fine resolution of peaks. 20 ml of fermentation broth was injected and eluted at a flow rate of 1·5 ml/min. Cephamycin C was detected at 253 nm.
(7–10 mg/g of weight of substrate). Above 80% moisture concentration, further increases resulted in decreases of cephamycin C concentrations.
3. Results Among the different solid supports used (Table 1) only some could support the growth of S. cla6uligerus and production of cephamycin C. Wheat rawa gave the highest titre value of cephamycin C. Addition of sunflower deoiled cake, cotton seed deoiled cake, corn steep liquor, enhanced production (Table 2) and at 0·5, 1·0 and 50% weight of support respectively gave highest titres of cephamycin C (10 mg/g).
3.2. Inoculum le6el When solid supports were mixed with 1× 102 cell/g of solid support cephamycin C production was very low (1 mg/g) but as the concentration reached 108 cell/g the cephamycin C concentration reached a maximum of 10 mg/g of substrate. A further increase in cell concentration did not affect the concentration of cephamycin C.
3.1. Physical parameters The effect of initial moisture content on cephamycin C production is shown in Fig. 1. Below 50% moisture there was no appreciable growth of S. cla6uligerus. In the range from 60 to 80% moisture content there was substantial growth and cephamycin C production
3.3. Initial pH The effect of initial pH on cephamycin C production is shown in Fig. 2. When the initial pH was 5·0 there was very little growth and no production of
Table 1 Solid supports for the cephamycin C production Solid supports
Weight (g)
Cephamycin C titres (mg/g)
Wheat bran Wheat rawa Rice husk Rice straw Sago Soy bean Sugarcane baggasse Cotton fibres+rice bran Coconut Coir+rice bran Brick powder+rice bran
10 10 10 10 10 10 10 5+5
5·2 7·0 Nil Nil 5·0 3·1 Nil 6·0
5+5
4·2
5+5
3·7 Fig. 1.
K. Prasad Kota, P. Sridhar / Process Biochemistry 34 (1999) 325–328
327
Fig. 4. Fig. 2.
cephamycin C. As the pH was increased, production of cephamycin C increased and reached a maximum (15 mg/g) at 6·5. A further increase in pH, resulted in a decrease in cephamycin C production and there was no production at 8·0 (Fig. 3).
wards with a concentration of 6 mg/g and reached a maximum on day 5 (15 mg/g) and then decreased slightly up to day 30 (9·5 mg/g). Thereafter there was no cephamycin C production.
3.4. Incubation temperature
4. Discussion
The effect of temperature on cephamycin C production is shown in Fig. 4. At 30°C cephamycin C production was 15 mg/g and then decreased to zero when the incubation temperature was 37°C. A low concentration of cephamycin C was produced at 20°C (6 mg/g).
The largest single class of drugs made entirely through biological synthesis are antibiotics. At least 70 of the approximately 100 antibiotics used for treatment of human infections are derived from substances produced by Streptomyces [6]. Cephamycin C is a broad spectrum b-lactam antibiotic which is a basic bulk drug for semisynthetics like cefoxitin, cefametadozole [7]. Submerged fermentation (SmF) is usually employed for commercial production of cephamycin C but, an alternative economical method involving SSF for cephamycin C production is described. During the course of cephamycin C production by SSF, wheat rawa was found to be most suitable for production by S. cla6uligerus. Cephamycin C was detected on the 3rd day of fermentation and reached a maximum on the 5th day. Cephamycin C production was almost stable until the 30th day which was not the case in SmF. The initial moisture content of substrates was critical for cephamycin C production by SSF. At less than 50% moisture there was no growth of S. cla6uligerus. As the initial percentage of water was increased the production of cephamycin C increased until it reached a maximum at 80%. The ideal pH for cephamycin C production was 6·5. The pH (6·5) and temperature (28°C) were optimal both for SSF and SmF for cephamycin C production. It may be concluded that the process of cephamycin C production through SSF is potentially comparable to that of SmF.
3.5. Fermentation period The production of cephamycin C in solid substrate was observed from the 3rd day of fermentation on-
Fig. 3.
328
K. Prasad Kota, P. Sridhar / Process Biochemistry 34 (1999) 325–328
Acknowledgements The authors would like to acknowledge Department of Biotechnology, Government of India, for the financial support vide letter no. BT/02/08/92 to PS and University Grants Commission, Government of India vide letter no. 98/UGC-CSIR/94 for fellowship to KPK. References [1] Nagarajan, R, Boeck, L D, Gorman, M, Hamill, R L, Higger, D E, Hoehn, M M, Stark, W M and Whitney, J G, Journal of the American Chemical Society 1971;93:2308–12.
.
[2] Stapley, E O, Jackson, M, Hernandez, S, Zimmerman, S B, Cirria, S A, Mochale, S A, Mochale, S, Mata, J M, Woodreff, H B and Henlti, D, Antimicrobial Agents and Chemotherapy 1972;2:122 – 35. [3 ] Manual of Industrial Microbiology and Biotechnology, eds Arnold L Demain and Nadine A Solomen. American Society for Microbiology, Washington, D.C 1986, pp. 66 – 83. [4] Kavanagh, F, In Analytical Microbiology, Vol. I. Academic Press, New York, 1984, pp. 5 – 18. [5] Brana, A F P and Demain, A L, Journal of General Microbiology 1989;132:305 – 1317. [6] Yang, Shang-Shyng and Ling, Meei-Yuch, Biotechnology and Bioengineering 1989;33:1021 – 8. [7] Aharonowitz, Y and Demain, A Archives of Microbiology 1977; 115:169 – 73.