Production of an antifungal antibiotic by Streptomyces aburaviensis IDA-28

Production of an antifungal antibiotic by Streptomyces aburaviensis IDA-28

Microbiol. Res. (200 I) 155, 315 - 323 http://www.urbanfischer.de/joumals/microbiolres Itt~. _ ' .. .... . © Urban & Fischer Verlag Production of a...

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Microbiol. Res. (200 I) 155, 315 - 323 http://www.urbanfischer.de/joumals/microbiolres

Itt~. _ ' .. .... . ©

Urban & Fischer Verlag

Production of an antifungal antibiotic by Streptomyces aburaviensis IDA-28 S. Raytapadar, A. K. Paul Microbiology Laboratory, Department of Botany, Calcutta University, Calcutta, India Accepted: March 31, 2000

Abstract A broad-spectrum antifungal Streptomyces isolate, IDA-28, from Indian soil has been characterized and identified as Streptomyces aburaviensis var. ablastmyceticus (MTCC 2469). Nutritional and cultural conditions for the production of antibiotic by this organism under shake-flask conditions have been determined. Antibiotic production in synthetic medium reached the maximum on the 5th day of incubation at 30°C. Glucose and starch were found to be the best carbon sources while NH 4 N0 3 was preferred as nitrogen source. Optimum temperature and pH for antibiotic production were 32°C and 7.4, respectively. Phosphate at a concentration sub-optimal for growth enhanced antibiotic production. Supplementation of medium with casein hydrolysate improved both growth and antibiotic titre but yeast extract exhibited marked inhibition. Key words: Streptomyces - antifungal actinomycetes - antimicrobial spectrum - nutritional requirements - cultural conditions - antibiotic production

Introduction Streptomycetes have established themselves as the most potent group of microorganisms capable of forming a wide variety of antibiotics (Berdy 1989). The majority of these antibiotic substances are antibacterial in nature and are indispensable for the treatment of bacterial infections of plants and animals including the human beings. On the contrary, antifungal antibiotics effective against fungal disorders are relatively few mainly because of their solubility and toxicity problems. (Bushell 1982; Berdy 1986). The ever increasing incidence of Corresponding author: A. K. Paul e-mail: [email protected] 0944-5013/01/155/04-315

$15.00/0

fungal infections in plants, animals and human being has directed the attention towards the search for actinomycetes producing novel antifungal antibiotics having a broad spectrum of activity and lesser toxicity (Woodruff and Burg 1986; Georgiev 1988). Systematic screening of antifungal actinomycetes from Indian soil (Srivastava et af. 1978; Gupta and Gupta 1982; Paul and Banerjee 1984; Sharma 1989; De and Gupta 1991, 1993; Haque et at. 1992; Raytapadar and Paul 1995, 1996) has been carried out for the production of antibiotics effective against pathogenic fungi. Such screening programs have led to the discovery of a number of antibiotics effective against fungi (Thirumalachar et af. 1961, 1964; Thirumalachar and Menon 1962; Gopalkrishnan et af. 1968).

A substantial amount of information is available on the regulation of antibiotic production by actinomyctes (Hopwood 1978; Vandamme 1984; Berwick 1988). Antibiotic production by a strain is profoundly influenced by the nutritional, physiological and environmental factors prevailing during the growth of the organism. Attainment of high antibiotic production, therefore, requires optimal concentration of the components in the fermentation medium, optimum pH and viscosity. Other factors include the levels of inorganic phosphorus, metal ions, organic nitrogen sources and metabolic carbon, and in several cases substrate precursors are also important for high titres of the desired antibiotics (Srinivasan et at. 1991). In the course of a survey of antifungal actinomycetes from soils of West Bengal (Raytapadar and Paul 1995) a Streptomyces strain IDA-28 was found to show a broad spectrum of antimicrobial activity as determined by cross-streak and agar-cup assay against fungi, yeast and bacteria. The present communication deals with the deMicrobiol. Res. 155 (200 I) 4

315

tailed characterization, identification and optimization of conditions for the production of antibiotic by this organism in shake-flask culture.

Materials and methods Bacterial culture and maintenance. Streptomyces IDA-28 was isolated from a soil sample of Darjeeling, West Bengal, India following enrichment (Tsao et al. 1960) and dilution plating on glucose-asparagine agar. The strain was purified on glucose-asparagine agar and maintained on the same medium by subculturing at monthly intervals. Characterization of the Streptomycete. Morphological characteristics were studied following the agar-cylinder culture technique of Nishimura and Tawara (1957) while cultural and physiobiochemical characteristics were determined according to the methods of Shirling and Gottlieb (1966), Kutzner (1981) and Locci (1989). Nature of the spore surface was studied in a Hitachi Scanning Electron Microscope S2360N according to the method of Williams and Davies (1967). Antimicrobial activity of the strain was determined by the conventional cross-streak method (Waksman and Lechevalier 1962), while the sensitivity of the isolate to antibiotics was determined following the standard antibiotic disk sensitivity testing method (DIFCO Manual, 10th ed., DIFCO Laboratories Inc., Detroit, MI, 1984). Cultural conditions. To determine the nutritional and cultural conditions for antibiotic production Pridham and Gottlieb's inorganic salts medium (Pridham and Gottlieb 1948) was used as the base. The medium (20 ml per 100 ml Erlenmeyer flask) was inoculated with 1.5 ml of homogenous spore suspension (O.D = 0.2) in 0.1 % Tween 80 solution and incubated at 30°C in a rotary shaker ( 120 rpm). Measurement of growth and antibiotic. Growth of the producer strain was measured as dry weight of the mycelium. Antibiotic titre of the culture filtrate was measured by agar-cup assay using Curvularia lunata as the test organism. Assay plates were prepared by inoculating 20 ml of potato dextrose agar with 0.5 ml of freshly prepared spore suspension. Agar-cups (8 mm diameter) were filled with 0.1 ml of mycelia-free culture filtrate in triplicate and the plates were incubated at 30°C for 48 h. The zone of inhibition surrounding the cups were measured to nearest mm and the concentration of the antibiotic was determined from the standard curve prepared following the same method using the purified antibiotic. The amount of glucose in the medium was measured by di-nitro salicylic acid method (Bemfeld 1955). 316

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Results The isolate IDA-28 possesses a well-developed branched substrate mycelium not fragmented into bacillary or coccoid forms, the aerial mycelium bears rectusflexibilis type of sporophores containing long chains of nonmotile spores wih smooth surface (Fig. 1). Physiological and biochemical characteristics as described in Table I showed that the organism is strictly aerobic, Gram-positive, does not produce melanoid

Fig.I. Scanning electron micrograph of Streptomyces abura-

viensis IDA-28 showing sporophore morphology and the spore surface.

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Table 1. Physiological and biochemical characteristics of Streptomyces IDA-28. Character

Response

Growth under anaerobic conditions Gram reaction Production of melanoid pigment Production of diffusible pigment Range of temperature for growth Optimum temperature for growth Range of pH for growth Optimum pH for growth Growth on MacConky Agar Haemolysis on blood agar Growth in presence of Phenol (0.1 %, 1%) CoCI 2 (0.5%, 5%) Crystal violet (0.0001 %) Sodium azide (0.01 %,0.02%) Potassium tellurite (0.001 %,0.01 %) NaCI tolerance LD so of spores at 55°C Enzyme activity Catalase production Hydrolysis of casein Liquifaction of gelatin Hydrogen sulphide production Nitrate reduction Hydrolysis of fat Hydrolysis of cellulose Degradation activity Adenine Arbutin DNA Guanine Hypoxanthine Starch Testosterone Tween-80 L-tyrosine Xanthine

+ ± 20°C-37°C 30°C 6.4-7.8 7.2 +

± + ~7%

5.5 min

+ + ± + + + + +

+

+, positive response; -, negative response; ±, feeble response.

pigment but produces light yellow diffusible pigment in some media and shows limited degradative activities. The organism appeared to be highly flexible towards the utilisation of carbon and nitrogen sources (Table 2). Lactose, propionate, acetate, malonate as C source and NaN0 2 as N source were not utilized by the strain. The antibiotic sensitivity profile of the strain IDA-28 revealed that the organism is sensitive to streptomycin, kanamycin, gentamycin, vancomycin, novobicin, norfloxacin, ciprofloxacin, chlortetracycline and rifampicin but is resistant to penicillins, erythromycin and bacitracin. Strain IDA-28 was inhibitory to a wide variety of filamentous and yeast-like fungi, but bacteria were comparatively less affected (Table 3). Among fungi, Botrytis and Fusarium were not inhibited while Klebsiella and Staphylococcus among bacteria were unaffected. Time course of growth, extracellular accumulation of antibiotic, and changes in the amount of glucose and pH of the medium clearly revealed that growth attained its maximum on the 3rd day of incubation. During growth glucose was rapidly utilized and was almost exhausted at the end of fermentation. Concomitant with growth, the pH of the medium decreased to 6.4 followed by a slow rise to 7.7. Extracellular antibiotic accumulation was initiated from the 2nd day of incubation, increased steadily to attain its maximum on the 5th day and sharply declined thereafter (Fig. 2). Of the 20 different culture media used, good antibiotic yield was obtained with complex organic media like soyabean meal broth, beef extract glucose peptone medium and casein hydrolysate peptone medium. Production was moderate in glucose-asparagine broth, cornmeal broth, soyabean meal glucose broth and soyabean meal com steep liquor dextrin broth (Fig. 3). Effects of 14 different carbon sources on growth and antibiotic yield are shown in Table 4. Glucose followed by starch and rhamnose favoured growth and antibiotic production but lactose, sucrose, meso-inositol and mannitol were feebly utilised. The optimum concentration of

Table 2. Carbon and nitrogen source utilization pattern of Streptomyces IDA-28 Response

Carbon source a

Nitrogen source b

Well utilised

D-glucose, D-galactose, L-rhamnose, mannose, L-arabinose, raffinose, maltose, D-fructose, salicin, cellobiose, inulin, starch D-xylose, glycerol, sucrose mannitol, meso-inositol, Na-citrate D-Iactose, Na-propionate, Na-acetate, Na-malonate

L-arginine, L-asparagine, L-alanine, L-glycine, L-histidine, L-methionine, L-serine, L-threonine, NaNO" KNO" NH 4N01, NH 4C1, (NH4hS04 DL-tryptophan, L-hydroxyproline, L-cysteine, L-phenylalanine, L-glutamic acid, L-valine NaN0 2

Weakly utilised Not utilised

Pridham and Gottlieb's mineral medium was used as the base. Citrate, propionate, acetate, and malonate were added to the medium at 0.1 % (w/v) levels while other carbon sources were tested at 1.0% (w/v) level. b Pridham and Gottlieb's mineral medium with 1% (w/v) glucose was used as the base and nitrogenous compounds were added at 0.1 % (w/v) level.

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glucose and starch were found to be 2% and 4% (w/v), respectively (Fig. 4). Thirteen organic and six inorganic nitrogen sources were tested at 0.42 g N2 I-I. Organic nitrogen sources supported growth and antibiotic production better compared to inorganic ones (Table 5). The most suitable organic nitrogen sources were L-asparagine and L-glycine while NH 4N0 3 and (NH 4h S04 were chosen from among the inorganic ones. The optimum concentrations 318

Microbiol. Res. 155 (200 I) 4

of L-asparagine and NH 4N0 3 were 0.63 and 0.42 g

N z 1-1 respectively (Fig. 5).

The optimum temperature for growth and antibiotic production was found to be 32°C (Fig 6). The pH optimum for growth and antibiotic production were 7.0 and 7.4 respectively (Fig. 7). Antibiotic production was maximum at 7.5% (v/v) inoculum dose while a dose of 12.5% (v/v) was optimum for growth (data not shown).

Table 3. Antimicrobial spectrum of Streptomyces IDA-28

Test Organism

Length of inhibition zone, mm

Fungi Alternaria alternata Aspergillus niger Botrytis alli Colletotrichum dematium Curvularia lunata Curvularia pallescens Fusarium sp. Helminthosporium oryzae Penicillium perpenogenum Phytophthora sp. Saccharomyces cerevisiae Bacteria Bacillus subtilis B. cereus Citrobacter sp. Escherichia coli Klebsiella pneumoniae Micrococcus flavus Pseudomonas fluorescens Staphylococcus aureus

II 5 6 19 18 10 15 5 7

2

I 9

3 7 2

Table 5. Effect of nitrogen sources on growth and antibiotic production by S. aburaviensis IDA-28.

Nitrogen source 0.42 g N2 1- 1

Growth dry wt., g I-I

Ilg ml- I

L-alanine L-arginine L-asparagine L-cysteine L-glutamic acid L-glycine L-hydroxyproline L-methionine L-phenylalanine L-serine L-threonine DL-tryptophan L-valine NaNO) KNO) NH4C1 NH 4N0 3 (NH4)S04 NaN0 2

0.95 1.15 1.87 0.87 0.67 1.17 0.45 0.95 0.8 1.02 1.12 0.45 0.70 0.92 0.9 0.9 1.3 1.0

21.55 34.0 54.95 12.59 6.0 39.81 5.21 17.23 12.59 12.59 17.23 7.76 9.77 10.97 10.97 12.59 41.62 22.23

Antibiotic yield

Each value represents average of triplicate sets.

Table 4. Effect of carbon sources on growth and antibiotic production by S. aburaviensis IDA-28.

Carbon source 1% (w/v)

Growth dry wt., g I-I

Ilg ml- I

Arabinose Fructose Galactose Glucose Glycerol Lactose Maltose Mannitol Mannose Meso-inositol Rhamnose Starch Sucrose Xylose

0.87 1.15 1.31 1.46 0.62 0.30 0.81 0.51 0.88 0.16 1.20 1.36 0.48 0.70

21.55 21.55 31.62 77.43 7.94 6.30 16.98 6.30 16.98 5.10 49.86 50.12 5.01 10.97

Antibiotic yield

Each value represents average of triplicate sets.

Phosphate at a concentration sub-optimal for growth favoured antibiotic production. The optimum concentration of K2 HP04 for growth and antibiotic production was 0.135 and 0.675 g I-I of P0 4- respectively (Table 6). When the basal medium was supplemented with casein hydrolysate both growth and antibiotic production were stimulated. Maximum growth was found at a

Table 6. Effect of phosphate on growth and antibiotic production by S. aburaviensis IDA-28. K 2HP 04 g I-I

Growth dry wt., g I-I

Ilg ml- I

Antibiotic yield

0.0167 0.0338 0.0675 0.135 0.27 0.54 1.08

1 1.415 1.865 2.2 1.83 1.75 1.65

9.66 18.29 79.43 63.09 48.68 36.30 16.98

Each value represents average of triplicate sets.

Table 7. Effect of casein hydrolysate and yeast extract supplementation on growth and antibiotic production by S. aburaviensis IDA-28.

Cone.

Casein hydrolysate

% (w/v)

A

B

Yeast extract A

B

0 0.2 0.4 0.6 0.8 1.0

1.82 2.87 3.4 3.95 4.12 4.0

54.95 63.09 78.29 114.12 77.43 68.48

1.65 2.35 2.5 2.9 3.3 3.62

61.60 31.62 25.98 20.92 17.14 15.14

Each value represents average of triplicate sets. A =Growth, dry wt. g I-I; B =Antibiotic yield, !lg ml- I Microbiol. Res. 155 (200 I) 4

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Microbiol. Res. 155 (200 I) 4

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concentration of 0.8 % (wIv) but optimum concentration for antibiotic production was 0.6% (w/v). Yeast extract on the other hand supported growth but was inhibitory to antibiotic production (Table 7).

The gray colour of the aerial mycelium, rectiflexibilis type of sporophore with smooth spores (Fig. I), inability to produce melanoid pigment (Table I) and pattern of carbon source utilization (Table 2) confirmed the inclusion of the strain in the non-chromogenic gray series of the genus Streptomyces (Kutzner 1981; Locci 1989; Buchanan and Gibbons 1974). When the characteristics of the isolate were compared with those already described in Bergeys Manual of Systematic Bacteriology (Vol. IV), it appeared that the isolate IDA-28 belonged to S. aburaviensis (Nishimura et al. 1957), with a similarity value (SSM x 100) of 90%. Comparison of characteristics of Streptomyces I DA-28 with those of S. aburaviensis (Nishimura et al. 1957), S. aburaviensis var. ablastmyceticus (Hashimoto et al. 1968) and S. aburaviensis var. tuftiformis (Shoji et al. 1970) have indicated closest similarity of the isolate with that of S. aburaviensis var. ablastmyceticus. Identity of the isolate was further confirmed by the Institute of Microbial Technology, Chandigarh, India, and the culture has been deposited in the Microbial Type Culture Collection and Gene Bank, Chandigarh as Streptomyces aburaviensis I DA-28 (MTCC No. 2469). S. aburaviensis I DA-28 do not follow the typical trophophase-idiophase relationship in synthetic medium (Fig. 2). Growth and the release of antibiotic in the medium were simultaneous but antibiotic production continued for at least 2 days even after the cessation of growth. A similar relationship has also been reported in the production of chloramphenicol using proline as nitrogen source (Malick 1972; Vining 1986). The sharp decline of biomass may possibly be due to autolysis of the mycelia. The composition of the fermentation medium may be an important factor in manipulating growth and antibiotic production by microorganisms (Okami and Hotta 1988). Complex natural media were more suitable compared to other synthetic media (Fig. 3). The high antibiotic titre in these media may be due to their content of glucose or starch and also the presence of soluble amino acids and peptides. The carbohydrate utilization pattern reveals a close relationship between the carbohydrate metabolism and the antibiotic production by the organism (Table 4). Optimum growth and antibiotic production occurred at 2% glucose and 4% starch, but biomass accumulation was greater with starch than with glucose. Other than carbon, the source of nitrogen is important for the production of antibiotic substances. The present strain produced the antibiotic with most of the organic and inorganic nitrogen sources, but ammonium nitrogen was best suited for antibiotic production (Table 5). Microbiol. Res. 155 (200 I) 4

321

Tereshin (1976) also recommended ammonium salts for production of mycoheptin. The environmental requirements and tolerance of streptomycetes have been surveyed in detail (Kutzner 1981; Srinivasan 1991). In terms of its optimum temperature (32 0c) for growth the organism appeared to be mesophilic in nature while its optimum pH (7.0) for growth suggested its inclusion in the neutrophilic actinomycetes group. Inorganic phosphate levels that are sub-optimal for growth in general favoured good antibiotic production. These results corroborate the findings of Martin (1977) on the control of antibiotic synthesis by inorganic phosphate. The molecular mechanism of the phosphate effect on antibiotic biosynthesis, however, is largely unknown. The enhancement of growth and antibiotic production by casein hydrolysate supplementation suggests the requirement of some amino acids by the strain (Table 4). Addition of yeast extract on the other hand stimulated growth but significantly inhibited extracellular antibiotic production. The reason for such inhibition has not been investigated properly but the role of some vitamins on the extracellular accumulation of secondary metabolites have been reported.

Acknowledgement We are grateful to the Institute of Microbial Technology for identification of the strain. Our thanks are also due to Prof. A. Sengupta, Department of Geology, Calcutta University for Scanning Electron Microscopic studies. This work was supported by the University Grants Commission, Delhi.

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