1466-1476, 2012 ISSN 1819-544X This Is a Refereed Journal and All Articles Are Professionally Screened and Reviewed

1466 Journal of Applied Sciences Research, 8(3): 1466-1476, 2012 ISSN 1819-544X This is a refereed journal and all articles are professionally screened and reviewed

ORIGINAL ARTICLES

Investigation on some Streptomyces species produce antibiotic with immobilized cells by using calcium alginate

Mona Ibrahim Mabrouk

Microbiology Department, National Organization for Drug Control and Research, Egypt.

ABSTRACT

In the present work, fifty isolates of actinomycetes were isolated from soil from different governments in Egypt and only 32 isolates have been selected for their ability to the antimicrobial production but they differ in their levels of activities and there were 5 isolates gave the highest antimicrobial activity and all of them belong to Streptomyces genus by studying morphological and physiological characteristics. one isolate was chosen for their highest broad spectrum activity, sensitivity antibiotic of free and immobilized cells of Streptomyces showed that immobilized cells was more effective against tested organisms. This paper refers to the application of calcium alginate with immobilized cells as biocatalyst for 6 days, with starch as carbon and calcium nitrate as nitrogen The increased antibiotic production of in immobilized cells was observed when compared with that in free cells..

Key words: Streptomyces, antibiotic production, immobilization, Immobilized cells

Introduction

A large number of pathogenic bacteria have become resistant to antibiotics in common use, also the emerging disease and the toxicity of currently used compounds, which had a major health problem and most of raw materials of antibiotics were imported from other countries lead to an argue need for new bioactive compounds. Streptomyces generally synthesis a sizeable number of diverse natural secondary metabolites (Onaka et al., 2001), such as antibiotics, insecticides, herbicides, immunosuppressive actions (Mao et al., 2007), vitamins, alkaloids, plant growth factor, enzymes and enzyme inhibitors ( Augustine et al., 2005 and Ben-Fguira et al., 2005).Streptomyces is the largest antibiotic producing genus in the microbial world discovered so far (Anderson & Wellington, 2001) where produce about 75% of the commercially and medically useful antibiotics and approximately 60% of antibiotics developed for agricultural use ( El-Naggar et al., 2006) Immobilization of cells is now a useful technique to investigate the potential of new industrial production processes. This technique has been used to increase production of several kinds of antibiotics and may improve, occasionally, the yield of these antibiotics. The application of immobilized cells to study microbial processes is one of the main trends in modern biotechnology (El-Naggar et al., 2003 Bandyopadhyay et al., 1993). The potential of immobilized cell is to maintain a high concentration of cells and provide the required condition for the continuous production of the antibiotic without the need to grow more cells (Sarrá et al., 1997).Cell Immobilization can provide many operational and economic advantages such as prolonged metabolic activities, reuse of biocatalyst and preventing washing out of cells at higher flow rates ( Koller et al., 200 Gauatam et al., 2002). Immobilization of Streptomyces S-34 cells in calcium alginate using different concentration of calcium chloride, different beads diameter and curing time in order to improve the antimicrobial production was studied, therefore, the present study was to study the entrapment of Streptomyces S-34 cells in calcium alginate gel for antibiotic production, the effect of alginate concentration, bead diameter

Material and methods

Chemicals:

All chemicals and medium constituents in the study were procured from OXOID (England) and sodium alginate was procured from Sigma (USA)

Microorganisms:

The Streptomyces used in this study were isolated from the agricultural soils of different Egyptian government regions, El-Giza, El- Dakahlia, El-Kalubeia.

Corresponding Author: Mona Ibrahim Mabrouk, Microbiology Department, National Organization for Drug Control and Research, Egypt. 1467 J. Appl. Sci. Res., 8(3): 1466-1476, 2012

Media used:

Different media were used throughout this investigation. The composition of each media is given in gram per liter distilled water as follows:

I. Media used for isolation and purification of Streptomyces isolates from soil:

Starch nitrate agar medium (Waksman, 1962 )

II. Media used for growing bacteria and fungi respectively:

Nutrient agar medium (Jacobs & Gerstein, 1960 Saubaroud dextrose agar medium (Jacobs & Gerstein, 1960)

III. Media used for studding the antimicrobial activities of Streptomyces isolates:

Glycerol aspargine agar medium (Pridham & Lyons, 1961): Glycerol nitrate agar medium (Waksman, 1962) Oatmeal extract agar medium ( Kuster, 1959b) Fishmeal extract agar medium (Hussein & El- Gamal, 1978) Gouze No.1 agar medium (Gauze et al., 1959 ) Inorganic salts starch agar medium (Kuster, 1959a)

IV.Media used for identification of Streptomyces isolates

Czapex- Dox agar medium (Waksman, 1967) Tyrosine agar medium (Shinobu, 1958) Peptone-yeast iron agar medium (Tresner & Danga, 1958). Tryptone –yeast extract broth medium (Pridham & Gottlieb,1948) Mineral salts agar medium (Shirling & Gottlieb, 1966)

1-Isolation and purification of Streptomyce from soil:

Soil samples collection:

Soil samples were taken from 10-20 cm depth below the soil surface and the soil of the top region was excluded. Each sample consists of a mixture of three samples from different spots in the same area. Samples were air- dried at room temperature for 7-10 days and then passed through 0.8mm mesh sieve and were preserved at room temperature until use. (Anisuzzaman et al., 2001; Sultan et al., 2002; Hayakawa et al., 2004; El-Naggar et al., 2006).

Isolation of Streptomyces from soil:

Samples (10g) of air dried soil were mixed with 100mL sterile saline solution. The mixtures were shaken vigorously for 1h and then allowed to settle for 1h. Portions (1mL) of soil suspensions (dilution 10-1) were transferred to 9mL of sterile saline solution and subsequently diluted to 10-6 under sterile condition. One mL of each dilution (10-3-10-6) was spread over the surface of sterile plates of starch nitrate agar medium supplemented with nystatin (50μg mL-1) in order to minimize fungal contamination. Three replicates were considered for each dilution. The inoculated plates were incubated at 28°C for 14 days (El-Naggar et al., 2006).

Purification of Streptomyces isolates:

The Streptomyces colonies obtained after 14 days were picked up and re-cultivated several times (by streaking technique) under the same conditions of isolation for purity. The purified Streptomyces isolates were grouped according to the color of aerial mycelium and maintained on starch nitrate agar medium in a refrigerator at 4°C until used (Kutzner, 1981; Abdel-Fatah, 1985; Ahmed et al., 2006 and Taddei et al., 2006).

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For long storage, it was grown in starch-casein broth for 7 days. To it glycerol was added to a final concentration of 15% (v/v) and stored at -20°C (El-Naggar et al., 2006). For screening studies fifty Streptomyces isolates were collected.

2-Screening for antimicrobial activity of Streptomyces isolates against different test organisms on different media:

Agar disk method:

Each Streptomyces isolates inoculated on different media, namely starch nitrate agar (SNA), glycerol asparagine agar (GAA), glycerol nitrate agar (GNA), Gouze No.1 agar (G1A), inorganic salts starch agar (ISSA), fishmeal extract agar (FMEA), oatmeal extract agar (OMEA) and yeast malt extract agar (YMEA) media and incubated at 28°C for 7 days, from well-grown of Streptomyces colony mass, 8mm agar disks were prepared by using sterile cork borers.Disks were then aseptically transferred to nutrient agar and saubaroud dextrose agar media plates seeded with test Gram negative, Gram positive bacteria, yeast and fungi respectively (Table 1). Controls included plain disks from media without inoculation. Plates were incubated at 37°C for 24h for bacteria and 28°C for 48-72h for yeast and fungi and bioactivity was evaluated by measuring the diameter of inhibition zone (DIZ, mm) against different test microorganisms (Sultan et al., 2002; Augustine et al., 2004; Boudemagh et al., 2005; Shahrokhi et al., 2005 and Shahidi Bonjar et al., 2006). This method used to select the best media for antagonistic activity and the most sensitive test organism.

Well diffusion method:

The previous selected Streptomyces isolates were growing in suitable previous liquid media in rotary shaker at 180 rpm at 28°C for 5 days and the activity was evaluated by well diffusion method. Filtrate was assayed using 200μL of culture filtrate to fill agar wells (8mm) punched in nutrient agar and saubaroud dextrose agar plates which had been seeded with test microorganisms (Table 1). Petri dishes were maintained for 2h in a refrigerator at 4C to allow the diffusion of the bioactive compound then incubated at 37°C for 24 h for bacteria and 28°C for 48-72 h for yeast and fungi and bioactivity was evaluated by measuring the diameter of inhibition zone (DIZ, mm), where the degree of activity depending on the mean diameter of inhibition zone in mm (DIZ, mm), to the following groups very week activity (DIZ ≤ 10 mm), weak activity (DIZ, 11 – 20 mm), moderately active group (DIZ 21 – 30 mm), and good activity (DIZ ≥ 30 mm) (; Shahrokhi et al., 2005; Sujatha et al., 2005; El-Naggar et al., 2006; Shahidi Bonjar et al., 2006 and Yu et al., 2007 ).This method used to select the best media for antagonistic activity and the most sensitive test organism.

Table 1: Test microorganisms used for antimicrobial activity. Source Test organism No. Microbiological Laboratory of National Salmonella typhi 1 Organization of Drug Control and Research, Shigella sp 2 Egypt. Klebsiella pneumonia 3 Escherichia coli(CAIM-1350 4 Staphylococcus aureuSCAIM-1352 5 Bacillus subtili CAIM-1007 6 Micrococcuslutu CAIM-1246 7 Bordetell bronchiseptia ATCC-4617 8 Candida albicans CAIM-22 9 Saccharomyces cervice(CAIM-14) 10 Salmonella typhi 11 Center of Agriculture Research, Egypt Rhizoctonia solani 12 Fusarium oxysporum 13

3.Identification of the selected isolates:

Identification of Streptomyces species was carried out according to the standard methods and media adapted by Shirling & Gottlieb (1966). The key proposed by Locci (1989) were consulted and International Streptomyces Project (ISP) introduced by Shirling & Gottlieb (1966, 1968a & b, 1969, 1972), Kuster (1972) and Holt et al. (1994) were also used. Identification including the studying of cultural, morphological and physiological characteristics of the selected isolate for their highest broad spectrum activity,.

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Cultural properties:

The growth, color of mature sporulating aerial mycelia and color of substrate mycelium as view from the reverse side as well as the diffusible soluble pigments other than melanin pigment were determined on inorganic salt starch agar medium, glycerol asparagine agar medium, oatmeal extract agar medium and yeast-malt extract agar medium after 7, 14 and 21 days of incubation

Morphological properties:

The morphological characteristics of spore chain of the isolates were determined by direct microscopic examination of the cultural spore chain after 3 days of inorganic salt starch agar medium using 400X magnification. Microphotographs were produced using microscope film camera device Karl-Zeiss Jena and Image analysis system in Microbiology laboratory, National Organization for Drug Control and Research (NODCAR). For direct microscopic examination, culture medium sample were first diluted to approximately 0.25g/L,becauseThis prevented the occurrence of overlapping hyphae when the sample was placed on a microscope slide (Jonsbu et al., 2002). Examination of spore surface ornamentation was carried out after 14 days by Transmission Electron Microscopy in Electron Microscopy unit, JEM Cairo University, Egypt. Spore print was taken by gently pressing the grids with collodion film over the sporulating surface of starch nitrate agar culture (Tresner et al., 1961).

Physiological properties:

These characteristics comprise carbon utilization test, melanin production, cellulolytic activity, sodium chloride tolerance and streptomycin sensitivity. The selected isolates were studied for its ability for the utilization of different carbon source, D-glucose, D- fructose, D-xylose, D-mannitol, D-galactose, L-rhamnose, raffinose, cellulose, sucrose, L-arabinose, L-Inositol using mineral salts agar medium (Shirling & Gottlieb, 1966) and the same medium without carbon source was served as a control. The Streptomyces isolates were investigated for the ability of production of melanin pigments on peptone yeast extract iron agar, tyrosine agar or tryptone extract broth media after 2-4 days of incubation at 28°C. In addition, this isolates were examined for their sensitivity to streptomycin (100 g mL-1) using starch nitrate agar medium (Waksman, 1967)and tolleranceto different sodium chloride concentrations was studied using starch nitrate agar medium by adding different sodium chloride concentrations(1-15%)

Immobilization of Streptomyces speceis cells for antimicrobial production:

Preparation of cell suspension:

The isolate Streptomyces speceis was grown on starch nitrate agar medium at 28˚C for 7 days for complete sporulation, then 5mL of sterile distilled water was added and spores removed by scraping then transferred to a 250mL Erlenmeyer flask containing 50mL of inoculum medium and incubated on a rotary shaker at 180 rpm at 28˚C. After 48h of incubation, the culture broth was centrifuged, the cell pellet harvested and washed with sterile 2% KCl and 0.9% NaCl solution and the cells were resuspended in 25mL sterile saline solution. This cell suspension was used as inoculum for the immobilization of whole cells (Sridevi & Sridhar, 1999 &2000; Srinivasulu et al., 2002 & 2003 and Adinarayana et al., 2004).

Immobilization of cells in calcium alginate:

Cells were immobilized using sodium alginate byionotropic method (inclusion in polymeric gels) (Marek et al., 1985; Kierstan & Coughlan, 1985; Sridevi & Sridhar, 1999 & 2000 and Adinarayana et al., 2004). Sodium alginate solution (3%wt/vol) was prepared by dissolving sodium alginate in 10 mL hot water. The contents were stirred vigorously for 10min. to obtain thick uniform slurry without any undissolved lumps and then sterilized by autoclaving The concentration of the cationic solution calcium chloride (CaCl2) has a significant effect on the gelling behavior of alginate, so the immobilization procedure was carried out with different concentration of various cationic solutions (0.125M, 0.25M & 0.5M) and with various curing time (20 min. & 1h) with different bead diameter by using different sizes of needles and the diameter of beads was calculated using image analysis system. Both alginate slurry and cell suspension (equivalent to 0.03g dry cell weight) were mixed and stirred for 10 min. to get a uniform mixture.

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The slurry was taken into a 1mL (0.13 mm) sterile syringe and 3 mL (0.22 mm) sterile syringe then added drop-wise into different concentration of CaCl2 solution from 5cm height and kept for curing at 4˚C for 20min. and 1hour. The cured beads were washed with sterile water 3 or 4 times. All of those operations were carried out aseptically under laminar flow unit (Farid et al., 1994). The immobilized beads were transferred into 50mL optimized production medium(starch as carbon and calcium nitrate as nitrogen)The increased production of streptomycin in 250mL Erlenmeyer flasks and incubated on a rotary shaker at 180 rpm at 28˚C for different incubation period then bioassay evaluated by measuring the diameter of inhibition zone (DIZ, mm). For free cultures, each 50mL medium was inoculated with Streptomyces spores equivalent to those used in the immobilized cultures.The antibiotic bioassay was carried out at different time intervals (1, 2, 3, 4, 5and 6 days) and compared to those obtained from the free cultures.

Results:

1.Preliminary screening of the actinomycetes isolates for antimicrobial production:

In the present study, fifty actinomycetes were isolated from different soils at various localities in Egypt, which are El-Giza 28%, El- Dakahlia 44%, El-Kalubeia 28% and were purified using starch nitrate agar medium. Data in Table (2) reveal that Streptomyces isolates were divided into 4 groups according to their color of aerial mycelium (Locci, 1989) which are gray, white, green and yellow, where gray Streptomyces isolates represent the major group (60%) followed by white group (32%), but yellow and green group (4%) represent the minor group. Antimicrobial activity exhibited by 42 isolates of the total 50 isolates against different test microorganisms (Table 1) where antagonistic gray isolates exhibited 85.7%, white isolates exhibited 11.9% and 2.38% green group, while yellow group dos not showed any activity.Also, results in Table (3) show that degree of antimicrobial activity of the antagonistic isolates was evaluated according to Ilić et al. (2005), where Staphylococcus aureus, Bacillus subtilis, Salmonella typhi, Micrococcus lutes, Shigella sp., Rhizoctonia solani, Bordetella sp., Candida albicans, Fusarium oxysporum and Klebseila pneumonia are the sensitive test organisms and Staphylococcus aureus, Salmonella typhi, Candida albicans and also Rhizoctonia solani are used as test organisms in subsequent experiments as Gram positive, Gram negative bacteria, yeast and fungi respectively, because they are inhibited by higher numbers of Streptomyces isolates

Table 2: Number of the antagonistic isolates in each actinomycetes group from the total number of isolates: Groups of Antagonistic

actinomycetes (%) isolates Number of isolates Isolates (%) Gray 30 60 % 26 85.71 % White 16 32 % 5 11.91 % Green 2 4 % 1 2.38 % Yellow 2 4 % 0 0 Total 50 100 % 32 100 %

Table 3: Degree of activity of Streptomyces isolates against each test organism: Streptomyces isolates

Test microorganisms S-25 S-20 S-20 S-21 S-24 S-26 S-29 S-31 S-32 S-33 S-34 S-35 S-38

Salmonella typhi W - W - M W W M M MW W Shigella sp. W - - - W WM M M MM W Klebseila pneumonia W - - - W - W M W M - - Escherichia coli - - - - W - W W M W W W Staphylococcus aureus W W M M M W W M M M W W Bacillus subtilis M W W WM M W M M M W M Micrococcus lutes W W W WW W W M M MM M Bordetella sp. - - M - M - W M W M W - Candida albicans W W- - - W W M W M W W Saccharomyces cervicea W M W - - W W W M W W W Rhizoctonia solani W W WM M W - M - M W - Aspragillus niger W W W- - W W W- W - W Fusarium oxysporum - - W W W - W W M M W W Degree of activity according to Ilić et al., 2005 on inorganic salt starch agar medium: Very weak activity (DIZ ≤ 10 mm) (V) Weak activity (DIZ, 11-20 mm) (W) Moderately active group (DIZ, 21-30 mm) (M) Good activity (DIZ ≥ 30) (G)

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Table 4: Antimicrobial activity of selected isolates on starch nitrate agar medium against different test organisms using disk method: Isolates

Test icrococcus lutes lutes icrococcus lepsilles pneumonia pneumonia lepsilles scherichia scherichia acillus subtilis sp. ordetella solani hizoctonia nige spragillus usarium

Salmonella Salmonella typhi sp. Shigella K E B M B R A F org. coli aureus Staphylococcus Candida albicans Sacharomyc cervicea oxysporum Diameter of Inhibition zone (mm) S-1 12.0 00.0 00.0 26.0 20.0 00.0 00.0 19.5 00.0 20.0 00.0 00.0 26.0 S-2 00.0 00.0 00.0 00.0 22.0 20.0 00.0 20.0 15.0 18.0 00.0 12.0 00.0 S-3 20.0 12.0 00.0 15.0 12.0 12.0 20.0 22.0 00.0 00.0 00.0 00.0 15.0 S-4 00.0 12.5 00.0 00.0 00.0 00.0 00.0 20.0 00.0 00.0 00.0 00.0 00.0 S-5 20.0 18.5 20.0 00.0 30.0 22.0 20.0 00.0 00.0 00.0 00.0 00.0 00.0 S-6 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 20.0 20.0 00.0 00.0 00.0 S-7 00.0 00.0 00.0 00.0 20.0 18.5 30.0 15.0 00.0 00.0 00.0 00.0 00.0 S-8 26.0 22.0 00.0 18.0 00.0 20.5 18.5 12.5 12.5 18.5 00.0 00.0 18.0 S-9 00.0 22.5 22.5 25.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 25.0 S-10 00.0 00.0 00.0 00.0 22.0 00.0 15.0 00.0 00.0 00.0 00.0 00.0 00.0 S-11 25.0 20.0 15.0 00.0 21.0 15.5 20.0 00.0 20.0 18.0 00.0 00.0 00.0 S-12 00.0 17.5 11.0 12.0 20.0 12.5 22.5 12.0 25.0 20.0 00.0 00.0 12.0 S-13 12.0 15.0 00.0 00.0 00.0 00.0 00.0 18.5 00.0 00.0 00.0 00.0 00.0 S-14 00.0 00.0 12.0 00.0 22.0 00.0 22.0 00.0 25.0 00.0 00.0 12.0 00.0 S-15 22.0 22.0 21.0 00.0 22.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 S-16 22.0 22.0 15.0 21.0 00.0 00.0 00.0 12.0 00.0 20.0 00.0 00.0 21.0 S-17 00.0 00.0 00.0 00.0 25.0 24.5 29.0 00.0 00.0 00.0 00.0 00.0 00.0 S-18 30.0 14.5 00.0 12.0 00.0 00.0 00.0 00.0 20.0 12.5 00.0 00.0 12.0 S-19 20.0 22.5 00.0 00.0 22.0 24.0 25.5 25.0 20.0 23.0 00.0 00.0 00.0 S-20 20.0 15.0 27.0 12.0 16.0 15.0 15.0 17.5 12.0 20.0 00.0 12.0 12.0 S-21 12.0 00.0 15.0 15.0 15.0 00.0 00.0 00.0 21.0 13.0 00.0 19.0 15.0 S-22 00.0 00.0 00.0 00.0 00.0 17.0 00.0 00.0 24.0 14.0 00.0 15.0 00.0 S-23 13.5 15.0 21.5 18.5 18.0 13.0 21.5 22.0 14.5 00.0 00.0 00.0 18.5 S-24 24.5 25.0 00.0 28.0 22.0 23.0 23.5 00.0 20.0 00.0 00.0 16.0 18.0 S-25 24.0 27.0 00.0 25.0 20.0 21.5 29.0 00.0 25.0 14.0 00.0 15.0 25.0 S-26 00.0 00.0 00.0 00.0 18.0 22.0 12.0 22.0 00.0 00.0 00.0 00.0 00.0 S-27 23.0 23.0 22.0 00.0 30.0 20.0 24.0 00.0 22.5 00.0 00.0 15.0 00.0 S-28 00.0 00.0 00.0 00.0 20.0 18.0 22.0 00.0 00.0 00.0 00.0 00.0 00.0 S-29 00.0 00.0 00.0 00.0 16.0 22.5 00.0 00.0 00.0 00.0 00.0 00.0 00.0 S-30 00.0 00.0 00.0 00.0 14.0 20.0 00.0 00.0 22.0 00.0 00.0 00.0 00.0 S-31 22.0 12.0 15.0 20.0 20.0 22.0 20.0 12.0 20.0 00.0 00.0 00.0 20.0 S-32 15.0 12.5 18.0 22.0 20.0 22.0 20.0 20.0 23.0 12.0 00.0 15.0 22.0 S-33 12.0 12.5 15.0 22.0 18.5 00.0 20.0 15.0 22.0 12.0 12.0 12.0 22.0 S-34 12.0 25.0 25.0 12.0 28.0 20.0 20.0 18.5 22.0 11.0 14.5 14.0 12.0 S-35 18.5 00.0 21.0 15.0 21.0 12.0 12.5 21.0 12.0 00.0 00.0 00.0 15.0 S-36 00.0 00.0 00.0 00.0 00.0 00.0 20.0 00.0 26.5 00.0 00.0 00.0 00.0 S-37 00.0 00.0 00.0 00.0 31.0 00.0 24.0 00.0 30.0 00.0 00.0 00.0 00.0 S-38 16.0 19.5 00.0 14.0 25.0 12.5 00.0 12.0 00.0 00.0 00.0 15.0 14.0 S-39 00.0 00.0 00.0 00.0 21.0 24.5 22.5 00.0 30.0 00.0 00.0 00.0 00.0 S-40 00.0 00.0 00.0 00.0 19.0 17.0 22.0 00.0 25.5 00.0 00.0 00.0 00.0 S-41 00.0 00.0 00.0 00.0 30.0 18.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 S-42 00.0 00.0 00.0 00.0 21.0 16.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 *Diameter of disc 8mm

Activity of the selected isolates:

Data record in Tables (5,6) clearly indicate that 12 isolates exhibited a very strong antimicrobial activity, especially against Staphylococcus aureus, Bacillus subtilis, Micrococcus lutes, Salmonella typhi, Escherichia coli, Candida albicans, Rhizoctonia solani, Fusarium oxysporum by using agar diffusion method and by.using well diffusion method. Among these isolates, five isolates S-31, S-32, S-33, S-34 and S-35 showed the best antagonistic activity against many test organisms as compared to the other selected isolates.

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Identification of Streptomyces isolates:

Isolates S-31, S-32, S-33, S-34 and S-35 which were proved to be the most active isolates in antimicrobial production were identified according to the key proposed by Locci (1989), (ISP) introduced by Shirling & Gottlieb (1966, 1968a & b, 1969, 1972), Kuster (1972) and Holt et al., 1994 was also used. The purified isolates were morphologically investigated. Results revealed that they are belonging to the genus Streptomyces as they form well developed branching, non-septate, non-fragmented aerial mycelia bearing a long spore chains and non-motile spores, tolerate NaCl concentrations up to 7% and S –32 up to 5%. All 5 strains grow well in pH range between 6.5 and 8.0.The strains are capable of utilizing several carbonsources, including arabinose, fructose, Dgalactose, D-glucose, insitol, mannitol,D-mannose, D-melibiose, raffinose, L- rhamnose, salicin, and xylose. they.This isolates was also characterized by good growth on Czapek's agar medium and no growth in the presence of streptomycin The strains differ in som morphological and biochemical characteristics which shows that are not identical but could be affiliated to the same genus.

Table 5: Antimicrobial activity of selected isolates on shaking starch nitrate medium using well diffusion method: Test org.

Isolates icrococcus lutes lepsilles pneumonia lepsilles pneumonia scherichia scherichia acillus ordetella sp. hizoctonia spragillus usarium ubtilis ubtilis olan Staphylococcus aureus aureus Staphylococcus Shigella sp. sp. Shigella

Salmonella Salmonella typh K E B s M B R s A F col Candida albicans Sacharomyces cervicea niger oxysporum Diameter of Inhibition zone (mm) S-20 20.0 15.0 27.0 12.0 17.5 15.0 16.0 00.0 12.0 13.0 00.0 00.0 13.0 S-21 00.0 00.0 00.0 00.0 32.0 30.0 24.5 00.0 22.5 00.0 00.0 00.0 00.0 S-24 00.0 21.0 00.0 00.0 27.5 00.0 00.0 18.5 22.0 22.5 00.0 15.0 22.5 S-25 00.0 12.0 00.0 12.5 24.0 17.0 17.0 00.0 18.5 22.0 15.0 17.0 22.0 S-26 00.0 20.0 25.0 00.0 21.0 00.0 15.5 20.0 19.0 00.0 00.0 00.0 00.0 S-29 00.0 00.0 00.0 22.0 00.0 00.0 22.0 00.0 17.5 00.0 00.0 00.0 00.0 S-31 20.0 15.0 12.0 20.0 30.0 28.0 28.0 20.0 12.0 14.0 00.0 12.0 00.0 S-32 15.0 20.0 15.0 15.0 20.0 20.0 18.0 18.5 12.0 15.0 12.0 12.0 12.0 S-33 21.0 22.0 12.5 20.0 20.0 30.0 25.0 20.0 18.0 18.5 00.0 00.0 18.5 S-34 20.0 26.0 15.5 12.0 28.0 25.0 20.0 20.0 15.0 18.0 20.0 15.0 16.0 S-35 16.0 20.0 14.0 15.0 25.0 21.5 20.0 12.0 20.0 12.5 00.0 12.0 12.5 S-38 16.0 20.0 00.0 00.0 25.0 25.0 17.5 00. 20.0 00.0 00.0 00.0 00.0 *Diameter of well 8mm

Immobilization of Streptomyces cells for antimicrobial production:

Cell immobilization using ionotropic gelation of macromolecules with multivalent cations by using calcium alginate. Results in Table (6,7) clearly indicated that in shaken cultures, immobilized cells are the better for antimicrobial production than free cells and also indicate that immobilized cells shifted the day of antimicrobial production from 5th day in free cells to 4th day in immobilized cells.Results revealed that in shaken cultures, immobilized Streptomyces cells showed better antimicrobial production than free cells. The enhanced production of the secondary metabolites by immobilized cells can be explained by the stabilization of the biosynthetic factors and activities of the enzymes involved on the secondary metabolism due to the immobilization constraints, this constraints created inside polysaccharide gels can modify the physiological behavior of a microorganism compared to that of the free cell cultures. It has been frequently observed that the immobilized cells can preserve their biosynthetic activities over long period than free cells do (Nava Saucedo et al., 1996 and Asanza et al., 1997)Results also indicate that antimicrobial production by immobilized cells shifted the day of production from 5th day in free cells to 4th day in immobilized cells. Data also showed that the smaller bead diameter with 0.13mm have a better effect on the antimicrobial production than the larger one (0.22mm). Data showed the effect of curing time indicated that the beads prepared with 20 min. curing time were found to be better for antimicrobial production. Results represented that 0.25M was the most effective concentration of CaCl2 than 0.125& 0.5M CaCl2 this may be because that concentration of ionotropic cation have a pronounced effect on the stability of gel beads Results also showed that the 0.13mm beads diameter prepared with 20min. of curing time were found to be better for antimicrobial production than the 0.22mm bead diameter prepared with 20min. & 1h of curing time and Also 0.25M CaCl2.was the most effective concentration of CaCl2 for antimicrobial production than 0.125M & 0.5M.

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Table 6: Effect of immobilized Streptomyces cells on antimicrobial activity (staphycoccus aureus )at 20 min. using differentconc. of CaCl2 & diameter of beads at fourth day: CaCl 0.125M 0.25M 0.5M 2 Free cells conc. Diameter of beads Diameter of beads Diameter of beads (Control) Incubation period 0.13 0.22 0.13 0.22 13.0 15.0 1st 00.00 18.0 20.0 20.0 11.0 18.0 20.0 2nd 20.0 22.0 20.0 25.0 20.0 25.0 25.0 3rd 25.0 30.0 30.0 30.0 30.0 29.0 23.0 4th 30.0 32.0 30.0 37.0 32.0 32.0 28.0 5th 35.0 30.0 32.0 35.0 30.0 25.0 25.0 6th 30.0 30.0 30.0 30.0 25.0 13.0 15.0

Table 7: Effect of immobilized Streptomyces cells on antimicrobial activity (staphycoccus aureus ) at 1h using different conc. of CaCl2 & diameter of beads at fourth day:

CaCl2 0.125M 0.25M 0.5M conc. Free cells Diameter of beads Diameter of beads Diameter of beads Incubation period (Control) 0.13 0.22 0.13 0.22 0.13 0.22 1st 00.00 16.0 20.0 18.0 18.0 13.0 15.0 2nd 20.0 21.0 22.0 20.0 20.0 18.0 20.0 3rd 25.0 24.5 22.0 30.0 30.0 25.0 25.0 4th 30.0 30.0 28.0 33.0 28.0 29.0 26.0 5th 35.0 30.0 28.0 30.0 26.0 22.0 28.0 6th 30.0 27.0 28.0 30.0 30.0 25.0 25.0

Discussion:

A large number of pathogenic bacteria have become resistant to antibiotics in common use. This antibacterial resistance is presently an urgent focus of research and new antibiotics are necessary to combat these pathogens. Also, new bioactive compounds are permanently needed due to the emerging disease and the toxicity of currently used compounds (Demain, 1998; Bull et al., 2000 and Mao et al., 2007). Filamentous soil bacteria belonging to the genus Streptomyces are widely recognized as industrially important microorganisms because of their ability to produce many kinds of secondary metabolites such as antibiotics currently used as pharmaceutical (75%) and agrochemical products (60%) (; Tanaka & Omura, 1993; Lazzarini et al., 2000; El-Nagaar et al., 2003; Mellouli et al., 2003; Pamboukian & Facciotti, 2004 and Ben- Fguira et al., 2005). In the present study, the isolation of Streptomyces species from Egyptian soil was obtained, which produce broad spectrum antimicrobial product. Data revealed that fifty actinomycetes isolates were isolated from different soils at various localities in Egypt and were purified using starch nitrate agar medium. Data also revealed that Streptomyces isolates were divided into 4 groups according to their color of aerial mycelium (Locci, 1989) and 42 isolates showing activity against different test microorganisms on different media. Mellouli et al. (2003) isolated a new actinomycete strain identified as Streptomyces caelestis US24 producing antibacterial activities against Gram negative and Gram positive bacteria from Tunisian soil and Boudemagh et al. (2005) isolated twenty-seven strains of actinomycetes from Saharan soils in Algeria and tested for their antifungal activity. The degree of antimicrobial activity of the antagonistic isolates was evaluated according to Ilić et al. (2005) who classified the antagonistic activity, depending on the mean diameter of inhibition zone in mm (DIZ, mm). In this respect, only 12 isolates had high antimicrobial activity against different test microorganisms on different media.El-Leithy (1958) and Mahmoud et al. (1983) reported that, the strain showing antagonistic action on solid medium may not produce antibiotic in liquid medium. In this respect, data showed that 5 isolates S-31, S-32, S-33, S-34 and S-35 have the highest activity as compared to the other selected isolates and S-32 and S-34 have high broad spectrum antimicrobial activity and inorganic salt starch medium was the most favorable one for antimicrobial production.The search for new, safer and broad spectrum antimicrobial with greater potency has been progressing slowly (Gupte et al., 2002), which may be as a result of that fungi are eukaryotic and have machinery for protein and nucleic acid synthesis similar to that of higher animals (Georgopapadakou & Tkacz, 1994). It is, therefore, very difficult to find out compound that selectivity inhibit fungal metabolism without toxicity to humans (Glazer & Nikaido, 1995; Georgopapadakou & Tkacz, 1996; Dasgupta, 1998 and Augustine et al., 2005). Fungal infections have been gaining prime importance because of the morbidity of hospitalized patients (Beck-Sague & Jarvis, 1993 and Gupte et al., 2002). Although synthetic drugs contribute to be a major proportion of the antifungal used, natural antifungal have their own place in the actimycotic market (Cragg et al., 1997).Data revealed that five isolates showed the highest antimicrobial activity against different test organisms were identified using key proposed by Locci (1989), ISP (Shirling & Gottlieb, 1968a&b, 1969 and 1972), Kuster (1972) and Holt et al. (1994) as follows: resembles Streptomyces speceis Also this result can be explained by that the cell wall membrane system interacts strongly with the gel matrix network. Since this network can present different structural presentations, unconventional three-dimensional adjustments between the cell wall and the matrix are possible; therefore, these altered spatial organizations at discrete molecular levels induce the cells at determined specific conditions to enhance the

1474 J. Appl. Sci. Res., 8(3): 1466-1476, 2012 biosynthesis of secondary metabolism and increase the permeability mechanisms (Asanza et al., 1997). Entrapment of cells in non-toxic alginate is one of the simplest, cheapest and most frequently used methods for immobilization (Nilsson, 1987; Palmieri et al., 1994; Park & Chang, 2000 and Adinarayana et al., 2004)Srinivasulu et al. (2002) suggested that neomycin production by free and alginate immobilized Streptomyces marinensis cells were increased two and a half times with immobilized cells.Immobilization of the cells retarded the growth rate of S. rimosus but increased the length of the growth period and improves the oxytetracycline production from 153µg to 252µg while by free cells oxytetracycline production from 121µg to 124µg (Yang & Yueh, 2001). Data also showed that the smaller bead diameter with 0.13mm have a better effect on the antimicrobial production than the larger one (0.22mm) and Srinivasulu et al. (2003) agreed with these results, reporting that the effective neomycin production was achieved with 3.24mm bead diameter than 1.48, 2.16, 4.46 & 5.44mm diameter where oxytetracycline production decreased with increasing bead diameter. Also Mussenden et al. (1992) and Yang & Yueh (2001) reported that the beads with a diameter of 1.5mm had a 3- fold higher penicillin production in Penicillum chrysogenum than 3.5mm beads diameter.This might be due to increased surface area of the bead, which enhances the mass transfer. At the same time, cell leakage was increased with a decrease in a bead diameter (Srinivasulu et al., 2003) and may be because that gel conformation prevented the exchange of oxygen and substrate between cell and the environment (Veelken & Pape, 1982).Data showed the effect of curing time indicated that the beads prepared with 20 min. curing time were found to be better for antimicrobial production. This result are in line with those obtained by Asanza et al. (1997) who approved that the best curing time for formation of beads by using potassium carrageenan was 20 min. for chlortetracycline and tetracycline production with K-carrageenan immobilized S. aureofaciens. This might be due to increased surface area of the bead, which enhances the mass transfer. At the same time, cell leakage was increased with a decrease in a bead diameter (Srinivasulu et al., 2003) and may be because that gel conformation prevented the exchange of oxygen and substrate between cell and the environment (Veelken & Pape, 1982). This result is in accordance with Adinarayana et al. (2004) who reported that neomycin production was started at 24h of the fermentation and increased to a maximum level by 96h in case of free cells whereas the maximum yield was attained at 72h for immobilized cells This result is in accordance with Srinivasulu et al. (2003) who reported that better neomycin production was achieved with 0.25M CaCl2 than other different concentration of cation (0.01M, 0.125M, 0.375M and 0.5M). Also Yang & Yueh (2001) reported that the optimum concentration for oxytetracycline production by Streptomyces rimosus was 2% calcium chloride solution.This result may be explained on the bases that at a higher concentration of cation, the gelling was instantaneous and a casehardening effect was noticed and the micro porous structure of the bead was altered. The pore size and number of pores might have decreased. The beads prepared with 0.5M and higher concentrations of cations were spherical in shape where beads prepared with 0.125M cation were irregular in shape (Srinivasulu et al., 2003).Also high concentration of calcium alginate led to tight crosslinking between cells and alginate, which reduced nutrient transport and microbial activity, where at low alginate concentration, resulted in loose texture between cells and alginate so facilitated nutrient leakage through stirring treatment during cultivation (Yang & Yueh, 2001) Results also showed that the 0.13mm beads diameter prepared with 20min. of curing time were found to be better for antimicrobial production than the 0.22mm bead diameter prepared with 20min. & 1h of curing time and Also 0.25M CaCl2.was the most effective concentration of CaCl2 for antimicrobial production than 0.125M & 0.5M.Srinivasulu et al. (2003) reported that the immobilized cells of S. marinensis NUV-5 in calcium alginate are more effective for neomycin production with optimized parameters, such as alginate at 2% wt/vol, 0.25M CaCl2, 1h curing time and 3.24 mm bead diameter than free cells. Adinarayana et al. (2004) mentioned that the productivity of neomycin (72.97 mg/L/h) by calcium alginate immobilized cells was greater than with free cells (45.05 mg/L/h), where the total neomycin produced with continuous fermentation was 62% greater than with that of free cells. These results are in agreement with Kopp et al. (1984) who found that the immobilization of S. aureofaciens with calcium alginate gel beads was found to improve the tetracycline production. mobilized cells of Actinomycetes were found to be more efficient for the production of antibiotics with batch fermentation. The possible routes of antibiotic synthesis and its biological efficiency are currently under investigation( V. Dhananjeyan et al., 2010)

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