New pathogenic microorganisms and new diseases which are multidrug-resistant have been emerging and controlling such diseases with therapeutic measures has become a global concern. Actinomycetes have been targeted because of their ability to produce various new compounds to treat pathogenic and deadly diseases (Genilloud 2018). Different habitats have been explored for isolation of actinomycetes, and marine habitats are still being explored due to their vastness. In this research, three coastal regions of Chennai, Tamil Nadu, India have been explored and a total of 15 soil sediment samples were collected and the dried samples were used for the marine actinomycetes isolation. The five actinomycetes were isolated in CSPY-ME agar medium based on colony morphology, which were named as BN01, BN12, BN13, BN14, and GB15. All the actinomycetes isolates grew well on the starch casein agar medium. Similarly, Valan et al(2012) isolated 210 actinomycetes from marine sediment samples from the Andhra Pradesh coast of India and they have used starch casein agar medium for antibacterial and antifungal studies (Valan et al. 2012).
The isolated cultures were characterized by morphological and various physicochemical properties adopting standard methods (Shirling and Gottlieb 1966b). The spore morphology is considered as an important characteristic of the actinomycetes and in this study, the isolates BN13 and GB15 have oval-shaped short-chain spores. The colour of aerial and substrate mycelium, as well as the production of soluble pigment and melanin was observed in six different media, namely M2, M3, M4, M5, M6 and M7, and it revealed that the colour varied from white, grey, yellow, brown, red, pink, green, and black. Similar results were shown by (Ramachandran et al. 2019) as the mycelial colour varies from brown, yellowish brown, light grey, dark grey, and white. It also showed that the actinomycetes isolate had not produced any soluble pigments and melanin.
The isolate BN01 showed excellent growth at 4% NaCl, whereas BN12 showed at 2% NaCl. At concentrations 2, 4, and 6% NaCl, the isolates BN13 and BN14 have moderate and maximum growth tolerance, respectively. The isolate GB15 has good growth at 10% NaCl. These results were compared with (Valan et al. 2012) where all the marine actinomycetes isolated from sediment showed good growth at 2% NaCl, moderate growth at 4%, less growth at 6%, and no growth at 8% NaCl. All the isolates have degraded the organic compounds hypoxanthine and tyrosine except BN12, and except BN13, all isolates have degraded casein. But no isolates have degraded xanthine or adenine. Most of the isolated actinomycetes were dependent on glucose, fructose, lactose, galactose, maltose, sucrose, and raffinose as a carbon source. The isolate GB15 does not utilize maltose, galactose, inositol or rhamnose. Similar to this study, (Sarika et al. 2021) reported that the actinomycetes have utilized various carbon sources such as dextrose, arabinose, maltose, and lactose.
Since enzymes have been explored and used in the pharmaceutical industry, actinomycetes have been used in the production of enzymes. The amylase enzyme is used to hydrolyze starch and is applied in food, textile, paper and textile industries (Kafilzadeh and Dehdari 2015) whereas the protease enzyme is used in breaking down proteins into peptides and amino acids. The protease enzymes are produced by animals, plants, bacteria, and fungi. The enzymes protease and amylase were produced by all the isolates and it was confirmed by the formation of clear hydrolytic zones. This result was consistent with the report of (Chakraborty et al. 2021), where 61.42% of total isolates produced amylase and 42.85% produced protease. Cellulolytic bacteria have been used in the degradation of lignocellulosic biomass. The isolates BN01, BN13, and BN14 have produced cellulase by using the substrate carboxymethylcellulose and a similar result was reported by (Prasad et al. 2013), where the cellulose hydrolysis was found to be optimum by the isolates. Oxidase enzyme production was observed only in isolate BN14 and was confirmed by the formation of a dark blue colour in the oxidase disk after the addition of culture broth. The catalase enzyme was highly produced in isolates BN12 and BN14 whereas moderately produced in isolates BN01 and BN12. The isolates BN01 and BN12 have the ability to reduce nitrates, whereas others do not have nitrate-reducing capacity. It was confirmed by the formation of pinkish red colour after the addition of nitrate reagents to culture broth.
Based on the BLAST analysis, the isolate BN01 showed high similarity with Streptomyces rochei strain, the isolates BN12, BN13, and BN14 shared high similarity with Streptomyces sp. whereas the isolate GB15 displayed high similarity with Saccharopolyspora sp. Similarly, (Chakraborty et al. 2021) have characterized actinomycetes from marine sediments as Streptomyces levis strain KS46 by 16s rRNA molecular sequencing with 97.97% of sequence similarity.
The secondary metabolites were produced by initially growing the isolates in ISP-2 broth and transferred into the production medium. The crude secondary metabolites were extracted by the liquid-liquid extraction process by using ethyl acetate as a solvent. Further, crude secondary metabolites were fractioned using chloroform ethyl acetate and methanol. Antimicrobial, antioxidant, and anticancer assays were performed for the crude fractions. It shows that both gram positive and gram negative bacterial and fungal pathogens were susceptible to atleast one of the actinomycetes strain. The maximum zone of inhibition was exhibited by Saccharopolyspora sp. strain GB15. The gram-negative bacteria were found to be more resistant when compared with gram-positive bacteria due to the presence of lipopolysaccharide in gram-negative bacteria.. In contrast to this, (Rajivgandhi et al. 2019) showed methanol to be an excellent solvent for the antibacterial extract with a 29 mm zone of inhibition against E. coli followed by ethyl acetate and chloroform extracts.
The antioxidant radical scavenging activities of the extracts were analyzed by the change in colour from purple to yellow. It is observed that the ethyl acetate and methanol fractions of BN01 and BN12 have rapid colour changes from purple to yellow shades which show increased radical scavenging activity. But, in chloroform fractions, no colour changes were observed, and hence, they do not have radical scavenging activity. The ethyl acetate and methanol fractions of BN13, and chloroform and methanol fractions of BN14 show gradual colour changes from purple to yellow; hence they concluded to have medium radical scavenging activity, whereas the methanol and ethyl acetate fractions of GB15 have rapid colour changes from purple to yellow, hence having high radical scavenging activity. since no colour change was observed in all the isolates. (Krishnamoorthy et al. 2020) reported that coral-reef associated actinomycetes Saccharopolyspora sp.IMA1 showed maximum radical scavenging activity of 65.3% at the concentration of 100 µg/mL. Similarly, 33% of radical scavenging activity at the concentration of 5 µg/mL was observed in Streptomyces sp. Al-Dhabi-100 isolated from marine environment reported by (Abdullah Al-Dhabi et al. 2020).
Since, the ethyl acetate fraction of Streptomyces sp. strain BN12 has excellent radical scavenging activity, the MTT assay was performed using the same fraction. The anticancer activity for the ethyl acetate fraction of Streptomyces sp. strain BN12 against human breast cancer cell line MCF-7 showed better cytotoxicity. The microscopic images showed that the treated MCF-7 cell line has less number of cells compared with the MCF-7 control. The inverted microscopic images show the changes in cell morphology of the MCF-7 cell line after treatment with the crude extract and a smaller number of cells observed compared with control. (Davies-Bolorunduro et al. 2019) reported the highest cytotoxicity against the AGS cell line by the crude extracts of S. fulvissimus isolated from the Lagos Lagoon sediment with the IC50 value of 0.030 mg/mL at the crude extract concentration of 0.01-5 mg/mL.
The GC-MS profiling showed presence of ten compounds in the ethyl acetate crude extract of Streptomyces sp. strain BN12 such as fatty acid esters, alcohols, flavonoids and ketones. Among 10 compounds, Isosativene, Flavone, Phytol, Morin, Octadecanoic acid 3-oxo- methyl ester and Oleic acid were found to have antioxidant, antibacterial, anti-inflammatory and anti-cancer activity (Kumar; Cushnie and Lamb 2005; Chen et al. 2013; Caselli et al. 2016; Park et al. 2016; Islam et al. 2018; Chenniappan et al. 2020; Kopustinskiene et al. 2020). Beekwilder et al reported that the compound 1-Butanone, 1-(2-hydroxyphenyl) have been used as food additive (Beekwilder et al. 2007). The compounds 2,4-Monoethylidene-I-xylitol, 2,6-Bis(1,1-dimethyl)-4-phenylmethylenecyclohexa-2,5-dien-1-one and 4,7-Methanoazulene decahydro-1,4,9,9-trtramethyl were not reported any biological activity. This result was compared with (Chakraborty et al. 2021), where 42 bioactive compounds were produced from ethyl acetate extract of Streptomyces sp. such as fatty acid esters, steroids, alcohols, fatty alcohols and ketone.