The impact of different carbon and nitrogen sources on antibiotic production by Streptomyces hygroscopicus CH-7

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1 The impact of different carbon and nitrogen sources on antibiotic production by Streptomyces hygroscopicus CH-7 Slavica Ilić 1, Sandra Konstantinović 1, Vlada B. Veljković 1, Dragiša S. Savić and Gordana Đ. Gojgić- Cvijović 2 1 Faculty of Technology, Bulevar oslobodjenja 124, Leskovac, Serbia 2 ICTM, Department of Chemistry, Njegoševa 12, Belgrade, Serbia The effects of the composition and rheology of the fermentation medium on the antibiotic production by Streptomyces hygroscopicus CH-7 were studied. To increase the yields of hexaene H-85 and elaiophylin, the basic fermentation medium was reformulated using different carbon (glucose, lactose, ribose, fructose and trehalose) and nitrogen (isatin-schiff bases) sources. The maximum yields of hexaene H-85 and elaiophylin were obtained with lactose (10 g/l) and soybean (10 g/l) as well as with glucose (10 g/l), soybean (5 g/l) and isatin-3-thiosemicarbazone (5 g/l). The morphology of S. hygroscopicus CH-7 changed with addition of sodium salt of carboxymethylcellulose (CMC) to the basic nutrition medium at the initial concentration between 1.0 and 70.0 g/l, but the microbial growth was dominantly as small dispersed pellets. The production of hexaene H-85 and elaiophylin was stimulated by CMC in the range between g/l. Keywords: Streptomyces, antibiotics, Schiff base, β-cyclodextrine, complex, carboxymethylcellulose, morphology. 1. Introduction The genus Actinomyces are capable of producing different secondary metabolites [1-4]. Streptomyces hygroscopicus, a member of this group, produces a range of polyenic antibiotics depending on environmental and nutritional conditions [2,5]. Production of the antibiotics by this genus can be improved by optimizing both the nutrition medium formulation and operating conditions. The use of new carbon and nitrogen sources can lead to the better microbial growth and antibiotics production [1,6-8]. S. hygroscopicus CH-7, a strain isolated from a soil sample, was shown to produce three antibiotics: polyenic hexaene H-85, polyetheric nygericine and macrodiolide elaiophylin [2,5]. This work presents the important results of the authors on studying antibiotic production by S. hygroscopicus CH-7 related to the optimization of the nutrition medium for improving the antibiotic yields [2,5,9-11]. The basic nutrition medium contained glucose and soybean as carbon and nitrogen sources, respectively. Lactose, ribose, fructose and trehalose were used instead of glucose and isatin-schiff bases were employed instead of soybean in the modified nutrition media. The effect of the rheological behavior of the nutrition medium on the morphology of S. hygroscopicus CH-7 was also studied. The rheology of the nutrition medium was changed from Newtonian to pseudoplastic by varying the initial concentration of sodium salt of carboxymethylcellulose (CMC). The main goal was to determine the best microbial growth conditions for the maximum production of hexaene H-85 and elaiophylin. 2. Material and methods Microorganism. The strain S. hygroscopicus CH-7 was isolated from a soil sample from Vojvodina, Serbia. Isatin- Schiff bases were synthesized and characterized as described elsewhere [12]. Growth media and conditions. The producing microorganism was grown on the basic and modified nutrition media. The basic nutrition medium contained (in g/l): glucose 15, CaCO 3 3, NaCl 3, MgSO 4 0.5, (NH 4 ) 2 HPO 4 0.5, K 2 HPO and soybean 1.0. Lactose, ribose, fructose and trehalose (15 g/l) were also used as carbon source instead of glucose. The modified nutrition medium had the same composition as the basic one except that the nitrogen source was a mixture of soybean (0.5 g/l) and the isatin-schiff base (0.5 g/l). CMC was added to the basic nutrition medium between 1.0 to 70.0 g/l. The nutrition medium was autoclaved at 121 o C before use. The nutrition medium (100 ml) was poured in the sterile Erlenmeyer flasks (1 l). The medium was inoculated with a 48 h culture (5 ml) under sterile conditions. The culture flasks were fixed on a rotary shaker (200 rpm; rotation diameter: 2.0 cm) placed in a thermostated cabinet at 28 o C. Samples (10 ml) were taken periodically during the seven days fermentation process. Analytical methods. Hexaene H-85 and elaiophylin were first extracted from the fermentation broth by n-butanol and ethyl acetate, respectively. Their concentrations were determined by measuring the absorbance at λ max = 364 nm (hexaene H-85) and λ max = 252 nm (elaiophylin) with a Perkin-Elmer Lambda 15 UV/VIS spectrophotometer [2,5,9]. The microbial growth was followed by measuring the dry biomass weight. The fermentation broth was centrifuged at 4000 rpm for 15 min to separate the mycelial biomass. The biomass was then dried at 105 o C to constant weight [9].

2 3. Results and discussion The influence of different carbon source on the antibiotic production. The concentration of dry biomass increased until the third or forth day of the fermentation and then decreased. The highest dry biomass concentration was achieved in the basic nutrition medium (9.8 g/l), as can be seen in Table 1. The variations of hexaene H-85 and elaiophylin concentrations with the progress of the microbial growth are shown in Fig. 1. The impact of different carbon sources on the maximum antibiotics yield in the basic and modified nutrition media can be estimated from Table 1. The maximum production of hexaene and elaiophylin was in the nutrition media with glucose and lactose, while the minimum antibiotic production was observed in the nutrition medium with ribose. Table 1 The influence of different carbon sources on the maximum dry biomass concentration (X max ) and the maximum antibiotic concentration (C max ) and yield (Y max ) Carbon source X max Hexaene H-85 Elaiophylin C H max Y H max C E max Y E max g/l µg/ml mg/g d.b µg/ml mg/g d.b Glusoce Fructose Ribose Lactose Trehalose Fig. 1. Variations of hexaene H-85 and elaiophilin concentrations during the S. hygroscopicus CH-7 growth in the nutrition media containing different carbon sources: - - glucose, - - fructose, - - lactose, - - ribose, and - - trehalose The influence of different nitrogen source on the antibiotic production. To study the effect of different nitrogen sources on the antibiotic production, a part of soybean (SB) (5 g/l) was replaced with isatin-schiff bases (isatin-3- thiosemicarbazone - ITC, isatin-3-semicarbazone - ISC and isatin-3-phenylhydrazone IPH; 5 g/l). Table 2 shows the impact of nitrogen source on the maximum concentration of dry biomass and antibiotics yield on basal and modified media is shown. The addition of isatin Schiff bases to the basic nutrition medium slightly increased the microbial growth, the maximum dry biomass concentration being achieved in the medium with ITC. Isatin-Schiff bases stimulated the production of hexaene H-85 and elaiophylin, compared to the basic medium. The best results were obtained with ITC as can be seen in Table 2.

3 Table 2. The influence of different nitrogen sources on the maximum dry biomass concentration (X max ) the maximum antibiotic concentration (C max ) and yield (Y max ) (adopted from [8]) Nitrogen source X max Hexaene H-85 Elaiophylin C H max Y H max C E max Y E max g/l µg/ml mg/g d.b µg/ml mg/g d.b SB (10 g/l) SB (5 g/l) + ITC (5 g/l) SB (5 g/l) + ISC (5 g/l) SB (5 g/l) + IPH (5 g/l) Fig. 2. Variations of hexaene H-85 and elaiophilin concentrations during the S. hygroscopicus CH-7 growth in the nutrition media containing different nitrogen sources: - - SB (control), - - IPH, - - ITC, and - - ISC (adopted from[8]) The influence of CMC on the strain morphology. Different morphological shapes of filamentous microorganisms can result with varying rheological behavior of fermentation broth [13]. Filamentous growth and dispersed pellets-like growth are frequently observed in fermentation broths having high viscosity and non-newtonian rheological characteristics [14]. The morphology of S. hygroscopicus CH-7 depended on the initial CMC concentration, as it can be seen in Table 3 and Fig. 3. At lower CMC concentrations the growth was dominantly in the pellet form, while at higher CMC concentrations, the growth was dominantly filamentous. In addition, the CMC concentrations between 10.0 to 30.0 g/l stimulated the production of hexaene H-85 and elaiophylin.

4 Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) Fig. 3. Morphology of S. hygroscopicus CH-7 on media with CMC (in g/l): a) 0.0 (control), b) 10.0, c) 20.0, d) 30.0, e) 50.0 and f) 70.0 (magnification 5X) 1340 FORMATEX 2010

5 Table 3. The influence of CMC concentration on the maximum dry biomass concentration (X max ) and the maximum antibiotics concentration (C max ) and yield (Y max ) CMC Morphology description X max Hexaene Elaiophylin C H max Y H max C E max Y E max g/l g/l µg/ml mg/g d.b µg/ml mg/g d.b 0 Pellets: large, single Pellets: small, dispersed Filaments: single 20 Pellets: small Filaments: short 30 Pellets: small, scattered Filaments: single, slightly branched 50 Filaments: dispersed, short, branched, crossed Filaments: short, branched, crossed Fig. 4. Change of concentrations of hexaene H-85 and elaiophiline with increasing the CMC concentration (in g/l): (control); ; ; ; and [10,11] References [1] Abbas A, Edwards C. Effects of metals on Streptomyces coelicolor growth and actinorhodin production. Applied Environmental Microbiology. 1990; 56: [2] Vučetić J, Karadžić I, Gojgić-Cvijović G, Radovanović E. Improving hexaene H-85 production by Streptomyces hygroscopicus. Journal of Serbian Chemical Society. 1994;59: [3] Okami and Hotta, Search and discovery of new antibiotics. In: Goodfellow M, Williams ST, Mordarski M (eds) Actinomycetes in biotechnology. Academic Press, San Diego, CA, 1988: [4] Prosser JI, Tough AJ. Growth mechanisms and growth kinetics of filamentous microorganisms. Critical Review of Biotechnology. 1991; 10: [5] Karadžić I, Gojgić-Cvijović G, Vučetić J. Hexaene H-85, a hexaene H-85 macrolide complex. Journal of Antibiotics. 1991; 12: [6] Lee MS, Kojima I, Demain AL. Effect of nitrogen source on biosynthesis of rapamycin by Streptomyces hygroscopicus. Journal of Industrial Microbiology and Biotechnology. 1997; 19: [7] de Queiroz Sousa MFV, Lopes CE, Pereira Junior NA. Chemically defined media for production of actinomycin D by Streptomyces parvulus. Brazillian Archive of Biology and Technology. 2001; 44:

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