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

Slavica Ilić1, Sandra Konstantinović

1, Vlada B. Veljković

1, Dragiša S. Savić and Gordana Đ. Gojgić-

Cvijović2

1Faculty of Technology, Bulevar oslobodjenja 124, 16000 Leskovac, Serbia 2ICTM, Department of Chemistry, Njegoševa 12, 11000 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 10.0 -30.0 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, CaCO3 3, NaCl 3, MgSO4 0.5, (NH4)2HPO4 0.5, K2HPO4 0.5

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 121oC 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 28oC. 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 λmax = 364 nm

(hexaene H-85) and λmax = 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 105oC to constant weight [9].

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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 (Xmax) and the maximum antibiotic

concentration (Cmax) and yield (Ymax)

Carbon source Xmax Hexaene H-85 Elaiophylin

CH

max YH

max CE

max YE

max

g/l µg/ml mg/gd.b µg/ml mg/gd.b

Glusoce 9.8 169 17.2 64 6.5

Fructose 9.2 119 12.9 56 6.1

Ribose 8.9 79 8.9 17 1.9

Lactose 8.6 156 18.1 83 9.6

Trehalose 9.5 115 12.1 35 3.7

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.

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Table 2. The influence of different nitrogen sources on the maximum dry biomass concentration (Xmax) the maximum antibiotic

concentration (Cmax) and yield (Ymax) (adopted from [8])

Nitrogen source Xmax Hexaene H-85 Elaiophylin

CH

max YH

max CE

max YE

max

g/l µg/ml mg/gd.b µg/ml mg/gd.b

SB (10 g/l) 8.9 212 23.8 56 6.3

SB (5 g/l) + ITC (5 g/l) 9.6 372 38.8 118 12.3

SB (5 g/l) + ISC (5 g/l) 9.3 293 31.5 92 9.9

SB (5 g/l) + IPH (5 g/l) 9.1 329 36.2 106 11.6

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.

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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)

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Table 3. The influence of CMC concentration on the maximum dry biomass concentration (Xmax) and the maximum antibiotics

concentration (Cmax) and yield (Ymax)

CMC Morphology description Xmax Hexaene Elaiophylin

CH

max YH

max CE

max YE

max

g/l g/l µg/ml mg/gd.b µg/ml mg/gd.b

0 Pellets: large, single 8.8 148 16.8 53 6.0

10 Pellets: small, dispersed

Filaments: single

8.4 169 20.1 95 11.3

20 Pellets: small

Filaments: short

9.2 157 17.0 58 6.3

30 Pellets: small, scattered

Filaments: single, slightly branched

9.4 213 22.6 67 7.1

50 Filaments: dispersed, short, branched, crossed 7.9 98 12.4 26 3.2

70 Filaments: short, branched, crossed 7.6 89 11.7 14 1.8

Fig. 4. Change of concentrations of hexaene H-85 and elaiophiline with increasing the CMC concentration (in g/l): -■- 0 (control); -

▼- 10; -◊- 20; -○- 30; -□- 50 and -♦- 70 [10,11]

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