1. Introduction
Biotechnology in marine environments has led to the discovery of novel microorganisms in marine sediments, including actinomycetes, Gram-positive bacteria with a high G+C content that are significant for organic matter recycling, pharmaceutical production, and vitamin synthesis (Ravenschlag et al., 1999; Stach et al., 2003). Among actinomycetes, Streptomyces species are particularly notable, producing up to 80% of antibiotics (Williams et al., 1984) and contributing to various applications such as disease treatment, biocontrol, industrial enzymes, agrochemicals, and soil fertility (Hamza et al., 2013, 2015). As the largest genus within the Actinobacteria, Streptomyces is also responsible for approximately 70% of antibiotic production (El Hussein et al., 2014).
For precise bacterial identification, genotypic methods such as 16S rRNA gene sequence analysis are preferred over phenotypic methods, especially for rare or novel strains. High-performance thin-layer chromatography (HPTLC) enhances thin-layer chromatography (TLC) by automating processes and improving resolution and quantitative accuracy, overcoming manual application issues (Morlock et al., 2010). Two-dimensional chromatography further increases analytical capacity by rotating plates with different solvents (Nurok, 1989). Additionally, Fourier-transform infrared (FTIR) spectroscopy offers a non-invasive, label-free approach to rapidly characterise microbial strains by analysing the content, structure, and chemical modifications of biomolecules (Ami et al., 2013).
2. Materials and Methods
2.1. Isolation and Enrichment of Actinomycetes
Isolation of actinomycetes was carried out on Actinomycetes Isolation Agar using the serial dilution method (Nonomura & Ohara, 1969) and the spread plate technique (Collins et al., 1989). All pure colonies of marine actinomycetes were subcultured on agar plates and stored at 4 °C.
2.2. Characterisation of Actinomycete Isolates
2.2.1. Gram Staining
For Gram staining, a smear of the isolate was prepared, air-dried, and heat-fixed. It was then stained with crystal violet, washed with water, treated with iodine, decolourised with alcohol, counterstained with safranin, and washed again with water. Finally, the slide was dried and examined under an oil immersion microscope.
2.2.2. Biochemical Analysis of Actinomycete Isolates
Various biochemical tests were conducted following the guidelines provided in Bergey's Manual of Determinative Bacteriology (Buchanan & Gibbons, 1974; Holt et al., 2000).
2.3. Antagonistic Activity
The antagonistic activity of actinomycetes against food spoilage bacterial flora was assessed using the Kirby–Bauer disc diffusion method.
2.4. Molecular Characterisation
Molecular characterisation was carried out by 16S rRNA sequencing.
2.5. HPTLC Analysis
HPTLC was used for the chromatography-based purification of the antibacterial compound.
2.6. FTIR Analysis
FTIR spectroscopy was used to identify the functional groups of the bioactive compound.
3. Results and Conclusion
In this work, twelve different actinomycetes were isolated from the marine environment. In the present study, chalky white and grey isolates were noted. Such a dominance of members of the grey series has already been reported in different marine environments (Kim et al., 1998; Ndonde & Semu, 2000; Pridham & Tresner, 1974).
Various biochemical parameters of the Streptomyces isolates were used for their identification (Gottlieb, 1961; Jones & Bradley, 1964; Manfio et al., 2003). The production of citrase, urease, catalase, and oxidase is considered when characterising Streptomyces (Gotoh et al., 1982; Nitsch & Kutzner, 1969). Hydrogen sulphide and melanin production have been regarded as other important characters for the identification of actinomycetes (Shirling & Gottlieb, 1966).
Actinomycetes are a good source of antagonistic activity. The actinomycete isolates were tested for their antagonistic activity against food spoilage microorganisms, namely Bacillus megaterium, Bacillus licheniformis, Bacillus subtilis, Lactobacillus sp., and Lactobacillus lactis. The isolates showed high antagonistic activity against these food spoilage microorganisms. Apart from these, two biologically active metabolites produced by the actinomycetes were tested against organisms such as Vibrio haemolyticus and Enterococcus sp.
Based on the 16S rRNA analysis, isolate AMWM showed 100% similarity to Streptomyces lincolnensis. Analysis of 16S rRNA began with the amplification of the gene coding for 16S rRNA by the polymerase chain reaction (Hopwood et al., 1985).
HPTLC analysis was used to identify the biologically active metabolites produced by the marine water and marine soil actinomycetes. From marine water, loboporin was detected, while heronamide and desotamide were identified as secondary metabolites from marine soil. FTIR analysis complemented HPTLC by determining the functional groups present in these secondary metabolites.
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