ANTIBIOTIC PRODUCTION BY STREPTOMYCES HYGROSCOPICUS D1.5: CULTURAL EFFECT

ANTIBIOTIC PRODUCTION BY STREPTOMYCES HYGROSCOPICUS D1.5: CULTURAL EFFECT Barun K. Bhattacharyya, Sushil C. Pal, Sukanta K. Sen^*

Microbiology Laboratory, School of Life Sciences, Department of Botany, Visva-Bharati, Santiniketan, India

Submitted: April 09, 1997; Returned to authors for corrections: October 23, 1997;

Approved: July 23, 1998

ABSTRACT

In an attempt to screen out new potent antibiotic producers from soil, Streptomyces hygroscopicus D_1.5 was isolated and was found antagonistic to both bacteria and fungi. It could utilise arginine as nitrogen source and glycerol as carbon source at 0.75 g/l and 11.5 g/l level, respectively for maximum antibiotic yield.

Key words: Antibiotics, fermentation, Streptomyces

With the increasing misuses of antibiotics, the serious problem of antibiotic resistance is comming up very fast. Therefore, intensive search for new antibiotics is going on world wide (1,4,11) from Streptomyces which is the greatest source of antibiotics. To make the production of the antibiotic feasible, it is necessary to develop the optimum production conditions. Several scientists have attributed considerably in this field (1,7). The strain S. hygroscopicus D_1.5 (2) is a producer of nonaromatic ester group of antibiotic having carbonyl and more than one methyl and ethyl groups. This communication reports the influence of cultural conditions for antibiotic production by S. hygroscopicus D_1.5.

The antibiotic producer strain, S. hygroscopicus D_1.5 is a soil isolate (2). The test organisms (Bacillus subtilis & Escherichia coli) are from laboratory culture collection. The spores were used as inoculum (3.75x10⁶ spores/ml) at 2.5% level. The recommended media were used for nitrogen source (12) and carbon source (10) and the arginine glycerol salt (3) for all other experiments. The fermentation was carried out in pH 7.0 at 30^oC temperature for seven days, if not stated. The growth was measured in terms of dry weight of biomass (mg/ 50 ml). The antibiotic yield was determined in terms of diameter (mm) of inhibition zone.

To determine the best suitable nitrogen, both organic and inorganic nitrogenous compounds were tested for better antibiotic yield. All nitrogenous compounds were added as nitrogen equivalent (0.032 g/l nitrogen) of basal medium. Among, the nitrogen sources arginine showed its superiority for growth and antibiotic yield (Table 1). All other sources showed almost equal yield but much less. As the arginine was the most suitable nitrogen source, it was obligatory to judge its actual concentration for optimum production. It was found that optimum antibiotic production was at 0.75 g/l (Fig. 1).

Table 1:
Effect of nitrogen and carbon source on growth and antibiotic production by D_1.5

Dry weight mg / 50 ml ; ** Diameter of inhibition zone, mm.

Figure 1:_1.5

To find out the most suitable carbon source for growth and antibiotic production, different carbon compounds (0.626% of carbon, w/v) were tested with optimum level of nitrogen (0.75 g/l). It was observed that glycerol was most suitably utilised as carbon source (Table 1). But the strain responded poorly in all the sources. However, maximum yield was recorded at the concentration of 11.50 g/l (Fig. 1).

Apart from the above nutritional conditions, physical parameters were also worked out. It was found that change of pH affect positively for both cell growth and antibiotic yield and pH 7.0 remained as optimum. The best antibiotic yield was recorded when fermentation was carried out for 7 days at 30^oC.

Streptomyces as chemoautotrophic organism, requires synthetic medium for their cultivation. Among all synthetic media, the medium of El-Nakeeb and Lechevalier (3) was found most suitable for the strain S. hygroscopicus D_1.5 (2). Arginine (0.75 g/l) and glycerol (11.5 g/l) was found to support growth and antibiotic production most convincingly. Hobbs et al. (5) reported the carbon and nitrogen source for actinorhodin production by S. coelicolor. Legator and Gottlieb (9) observed that 1% glycerol supported better chloramphenicol production by S. venezuelae.

S. hygroscopicus D_1.5 being a mesophilic organism, grew and produced antibiotic best at 30^oC. Suetsuna and Osajima (11) showed that 28^oC is the optimum temperature for griseorhodin production by S. californicus JCM6910. It was found that the pH 7.0 was the best for antibiotic production by S. hygroscopicus D_1.5. James et al. (6) reported that the optimum pH for production of antibiotic, granaticin by S. thermoviolaceus was 6.5-7.0. The optimum incubation period was 7 days. However optimum antibiotic production was reported even on 4th day of fermentation in S. paulus UC8560 (8).

The production of antibiotic, as secondary metabolite in excess is controlled by the genetic make up that imparts fullest expression and is profoundly influenced by the kind and quality of the nutritional elements and environmental factors. This is also substantiated by the above experimental results. The working strain S.hygroscopicus D_1.5 may be developed to be an ideal one for industrial exploitation, having less nutritional, temperature (mesophilic) and pH (neutrophilic) control, hence less energy input for antibiotic yield.

ACKNOWLEDGEMENT

Bergey’s Manual of Determinative Bacteriology

2. Charkrabarty,D.; Mondal.B.; Pal,S.C., Sen,S.K. Characterisation and identification of broad spectrum antibiotic producing Streptomyces hygroscopicus D1.5. Hind. Antibiot. Bull., 37: 37-43, 1995.

3. El-Nakeeb, M.A. and Lechevalier,H.A. Selective isolation of aerobic actinomycetes. Appl. Microbiol., 75-77, 1963.

4. Haque,S.F.; Sen,S.K.; Pal,S.C. Nutrient optimisation for production of broad spectrum antibiotics by Streptomyces antibioticus Sr15.4. Acta Microbiol. Hung. 42: 155-162, 1995.

5. Hobbs,G.; Frazer,C.M.; Gardner, D.C.J.; Flett, F.; Oliver,S.G. Pigmented antibiotic production by Streptomyces coelicolor A3(2): Kinetics and the influence of nutrients. J. Gen. Microbiol., 136: 2291-2296, 1990.

6. James,P.D.A.; Iqbal,M.; Edwards,C.; Millar, P.G.G. Extracellular protease activity in antibiotic producing Streptomyces thermovilaceous. Curr. Microbiol., 22: 377-382, 1991.

7. Korn-Wendisch, F. and Kulzner, H.J. The family Streptomycetaceae. In: The Prokaryotes, Springer-Verlag, New York, 1991, p.921-995.

8. Labrode,A.,L.; Cialdella,J.I.; Fox,J.A.; Marshall,V.P. Directed biosynthesis of paulomycine-A the effect of L-methionine, L-threonine and alpha ketobutyric acid. J. Antibiot., 38: 1426-1428, 1985.

9. Legator,M.; and Gottlieb,D. The dynamics of chloramphe-nicol synthesis. Antibiot. Chemother., 3: 809-817, 1953.

10. Pridham,T.G. and Gottlieb,D. The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bacteriol., 56: 107-114, 1948.

11. Suetsuna,K. and Osajima,Y. Cultural conditions for production of griseorhodin by a culture of Streptomyces californicus JCM6910. Suisan Diagakkakenkyn Hokoku, 38: 17-22, 1990.

Streptomyces J.Gen. Microbiol.,

• ¹
  Buchanan,R.E. and Gibbons, N.E. Bergey’s Manual of Determinative Bacteriology (8th edition). The Williams and Wilkins Co., Baltimore, U.S.A., 1974, p.657-845.
• ²
  Charkrabarty,D.; Mondal.B.; Pal,S.C., Sen,S.K. Characterisation and identification of broad spectrum antibiotic producing Streptomyces hygroscopicus D1.5. Hind. Antibiot. Bull., 37: 37-43, 1995.
• ³
  El-Nakeeb, M.A. and Lechevalier,H.A. Selective isolation of aerobic actinomycetes. Appl. Microbiol., 75-77, 1963.
• ⁴
  Haque,S.F.; Sen,S.K.; Pal,S.C. Nutrient optimisation for production of broad spectrum antibiotics by Streptomyces antibioticus Sr15.4. Acta Microbiol. Hung. 42: 155-162, 1995.
• ⁵
  Hobbs,G.; Frazer,C.M.; Gardner, D.C.J.; Flett, F.; Oliver,S.G. Pigmented antibiotic production by Streptomyces coelicolor A3(2): Kinetics and the influence of nutrients. J. Gen. Microbiol., 136: 2291-2296, 1990.
• ⁶
  James,P.D.A.; Iqbal,M.; Edwards,C.; Millar, P.G.G. Extracellular protease activity in antibiotic producing Streptomyces thermovilaceous. Curr. Microbiol., 22: 377-382, 1991.
• ⁷
  Korn-Wendisch, F. and Kulzner, H.J. The family Streptomycetaceae In: The Prokaryotes, Springer-Verlag, New York, 1991, p.921-995.
• ⁸
  Labrode,A.,L.; Cialdella,J.I.; Fox,J.A.; Marshall,V.P. Directed biosynthesis of paulomycine-A the effect of L-methionine, L-threonine and alpha ketobutyric acid. J. Antibiot., 38: 1426-1428, 1985.
• ⁹
  Legator,M.; and Gottlieb,D. The dynamics of chloramphe-nicol synthesis. Antibiot. Chemother., 3: 809-817, 1953.
• ¹⁰
  Pridham,T.G. and Gottlieb,D. The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bacteriol., 56: 107-114, 1948.
• ¹¹
  Suetsuna,K. and Osajima,Y. Cultural conditions for production of griseorhodin by a culture of Streptomyces californicus JCM6910. Suisan Diagakkakenkyn Hokoku, 38: 17-22, 1990.
• ¹²
  Williams,S.T.; Goodfellow,M.; Alderson,G.; Wellington,E.M.H.; Seneath,P.H.A.; Sackin,M. Numerical classification of Streptomyces and related genera. J.Gen. Microbiol., 129: 1743-1813, 1983.

*

Corresponding author. Mailling address: Microbiology Laboratory, School of Life Sciences, Department of Botany, Visva-Bhorati, Santiniketan - 731235, India.

Publication Dates

• Publication in this collection
  26 Feb 1999
• Date of issue
  Sept 1998

History

• Accepted
  23 July 1998
• Received
  09 Apr 1997
• Reviewed
  23 Oct 1997