Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory

Bio-surfactants are surface-active molecules which are produced by the wide range of microbes including bacteria, fungi, moulds, and yeast. This study was conducted to identify bio-surfactants by Bacillus subtilis combined with use of cheap substrates and industrial wastes (Mustard cake, Whey and Soya cake) which are found locally in Nepal. Bacillus subtilis, one of the most potential bio-surfactants producer; was isolated from soil sample of hydrocarbon contaminated site. Isolates were grown in a Minimal Salt Media (MSM) with 10% (v/v) mustard oil cake, whey and soya cake separately. The presence and potential of surfactant was determined by the oil spreading technique, emulsification index (%E24) and surface tension measurement. It was revealed that the surface tensions of cell free extract were 54.41, 60.02 and 56.64 mN/m for from mustard cake, whey and soya cake respectively as compared to distilled water (72.09) at 25oC. The emulsification index values was found to be highest in engine oil from the bio-surfactant extracted from mustard cake, soya cake and whey respectively. Similarly, mustard oil showed the lowest value of emulsification index. The highest emulsification activity was shown in mustard oil i.e. 1.13 from the cell free extract from mustard oil and lowest in engine oil i.e., 0.07, by the extract from soya cake medium, when measured in spectrophotometer at 540 nm. In conclusion, strain of Bacillus subtilis was found to be the potential surface active agent producers on the mustard oil cake, which can be useful medium for various environmental, food, medicinal and industrial processes

Screening and Molecular Characterization of Antibacterial Secondary Metabolite Producing Actinomycetes from Soils of Eastern Mountain Regions of Nepal

Antibacterial secondary metabolite is a bioactive compound like antibiotic that can be considered a substance, produced by one microorganism, which in low concentration inhibits the growth of other microorganisms. Actinomycetes, slow-growing gram-positive bacteria, are the major sources of bioactive compounds. This study aimed to screen and identify antibacterial secondary metabolite-producing actinomycetes by sequencing the 16S rRNA method (molecular identification) from the soils of the mountain region of eastern Nepal. Starch casein agar (SCA) medium was used for the isolation of actinomycetes which were confirmed by primary screening and secondary screening. Identification of presumptive genera was done based on macroscopic, microscopic, and biochemical characteristics and confirmed by sequencing their 16S rRNA genes. The antibacterial compound was produced by culturing the potential isolate in starch casein broth. Using organic solvents such as ethyl acetate, n-butanol, chloroform, dichloromethane, and methanol, the chemical was recovered from the fermented broth. TLC performed the antibacterial substance characterization. Only 9 (13.6%) of the 66 actinomycetes isolates showed antibacterial activity against test microorganisms. Only one of the nine isolates, M3, had antibacterial activity in primary screening against gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli, Salmonella Typhi, Shigella spp., and Pseudomonas aeruginosa) test bacteria. M3 was chosen for secondary screening due to its strong antibacterial activity. The minimum inhibitory concentration (MIC) of crude antibacterial substances was found to be 2.5 mg/mL against test organisms. According to the TLC chromatogram, the isolate produced only one compound with an Rf value of 0.81, completely distinct from the spot formed by gentamicin (standard), which had an Rf value of 0.89. The isolates were considered Streptomyces spp., a distinct taxonomic group based on characterization by macroscopic, microscopic, biochemical, physiological, and molecular techniques. This study concluded that Mountain regions are the reservoir of antibiotic- producing actinomycetes. Streptomyces is the most common genus