ABSTRACT

Actinomycetes are the main microbial population in soil generating active secondary metabolites. However, the objectives of this research were to isolate, select a promising strain and improve the antibiotic production of actinomycetes. A prospective analytical study of several actinomycetes was performed from soil samples collected from different locations in Khartoum, Sudan. The crowded plate technique was used to isolate actinomycetes using different media. The strains were evaluated for their antimicrobial activity against certain Gram-positive, Gram-negative bacteria, yeasts, and fungi. Varied fermentation conditions such as temperature, pH, and light, agitation, aeration, and fermentation period have also been configured for the maximum production of antibiotics. From 62 isolates, 18% were active against at least one of the test organisms: Bacillus subtilis (ATCC 10400), Staphylococcus aureus (ATCC 289213), Escherichia coli (ATCC 13706), Klebsiella pneumoniae (ATCC 10031), Proteus vulgaris (ATCC 630), Pseudomonas aeruginosa (ATCC 15442), Salmonella typhimurium (ATCC 13311), Aspergillus niger (ATCC 16404), and Candida albicans (ATCC 10231). The strain (O-7) showed a very broader spectrum than other isolates. For the maximum production of antibiotics, suitable fermentation conditions were found to be as follows: Temperature 28°C, pH 7.0, Agitation 180 rpm, and fermentation duration 96 hours. It can be concluded that antimicrobial compounds formed by (O-7) isolated from the soil in Khartoum were efficient. The antibiotic generated exhibited the highest activity against various pathogens.

Key Words: Actinomycetes, Antimicrobial activity, Isolation, Soil.

Duration of fermentation

Processed fermentation batch containing optimized medium formulation and fermentation conditions was studied to determine the maximum duration of fermentation (Figure 6).

Based on the above-consolidated results, the fermentation medium and fermentation conditions have been optimized for the maximum production of antibiotics and are given as follows:

• Condition of fermentation: temperature (28°C), pH (7.0), agitation (180 rpm).
• Duration of fermentation (96 hours).
• Light and air condition.

Actinomycetes are known to be a producer of antibiotics, the most famous of which is Streptomycetes, to which this type (O-7) belongs, which has not been isolated in Sudan before, and antibiotics have not been extracted from it. The extracts of these isolated bacteria were tested on several pathogens after they were grown under different growth conditions and showed high efficacy.

Nutritional sources such as carbon, nitrogen, and minerals, as well as environmental factors, are known to have a profound effect on the production of antibiotics by actinomycetes [19]. In the present study, the effect of different carbon sources on the production of antibiotics by (O-7) strain is presented. Among the carbon sources, glucose has proved to be the best carbon source for both cell growth and strain-producing antimicrobial metabolites. The strain was found to produce high levels of antimicrobial metabolites in the medium supplemented with 5% glucose as the sole carbon source. Actinomycetes strains produced the highest biomass when glucose was added to the medium [18, 20]. In another strain that produces high levels of antimicrobial metabolites in the medium supplemented with glucose (2%) and reported that in the case of Streptomyces species, with regards to carbon sources species-specific variation may occur in cell growth and production of secondary metabolite [21]. It has been reported that the concentration of 3% of glucose was the optimum for maximum metabolite production [13]. On the other hand, this result disagreed with a previous study, which reported the chitin, starch was the appropriate carbon source to increase antimicrobial production [22, 23]. In the present study, there were significant differences between the (O-7) strain and the different glucose concentrations.

The effect of nitrogen sources on metabolite production was detected by fermentation media with the selected isolate (O-7). The results obtained showed that the optimal source of nitrogen for antibiotic production was a peptone for (O-7) isolate. This result was agreed with a previous study, which reported that the highest inhibition zone was recorded after peptone was used as a nitrogen source [24]. In other research, peptone is optimal for the production of bioactive agents [20]. It has been reported that, an inhibition zone as wide as 32 mm and 29 mm against E. coli FBFC-1407 and S. aureus ATCC-259233, in a medium supplemented with sodium nitrate [25]. In the current study, although the peptone is the best for the (O-7) strain, there were no significant differences in the statistical analysis using different nitrogen sources, but there were very significant differences in the diameter of the inhibition zone.

The role of pH in the production of antibiotics by (O-7) strain showed a high level of activity at neutral pH (7.0). This finding was an agreed study that reported an optimum pH of 7.0 [26, 27]. In another study, the optimum pH of the medium was adjusted to the basic scale of 9.0 for high activity of Streptomyces albolongus against S. aureus [28].

The environmental requirements and cultural conditions for the growth and production of antimicrobial agents have been studied by (O-7) isolate. The temperature of 28⁰C was found to be optimal for the highest growth as well as for the maximum production of the antimicrobial agent by the isolate. There was a very significant difference in the use of different temperatures for (O-7) strain, but there were no significant differences in the diameter of the inhibition zone. The same result with the strain S. sannanensis SU 118 has been reported [27]. The optimum temperature for antibiotic production of Streptomyces sp was found in other previous studies 45⁰C and 35⁰C [6, 25, 29].

The use of a wide-bottomed flask and agitated deep culture at a speed of 180 rpm increased the production of antibiotics by (O-7) isolate, particularly with a flask capacity of 750 ml. This result was agreed with a study that found the inhibitory rate reached a peak of about 90% when the speed was 180 rpm [30]. In a previous study, the agitation has shown a direct effect on the growth of Streptomyces sp. KOD1028. There were no significant differences in the use of different sizes of flasks in this study. In other research, agitation at 150 rpm was found to be the most appropriate for Streptomyces spp [31].

In the present research, an incubation period of up to 4 days was found to be optimal for maximum growth as well as for maximum production of antimicrobial agents. Similar results were reported in a previous study [11]. It has been reported that the seven days [27] and 12 days [13] were optimal for maximum growth and antimicrobial production. The results of the present study disagreed with the findings in a previous study, which reported a maximum antibiotic production in 30 days [6].

CONCLUSION

A promising strain was selected and carefully studied for maximum antimicrobial production, the suitability factors were optimized experimentally, carbon source (5% glucose), pH (7.0), temperature (28⁰C), agitation (180 rpm), and incubation period (96 hours).

Acknowledgments: This publication was supported by the Deanship of scientific research at Prince Sattam bin Abdulaziz University.

Conflict of interest: None

Financial support: None

Ethics statement: None

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