Microorganisms - ENVIS Centre

A total of 137 different isolates of marine actinomycetes were isolated from deep sea sediment collected from the Bay of Bengal. All the 137 marine actinomycetes isolates exhibited antibacterial activity against human bacterial pathogens, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus spp. Based on the screening results, 10 potential marine actinomycetes were selected and tested for their antifungal activity against two plant pathogenic fungi Rhizoctonia solani and Fuzarium oxysporum. Of which, four isolates showed remarkable antibacterial activity, one isolate showed good antifungal activity and remaining isolates showed moderate activity.

Searching of novel antimicrobial secondary metabolites from marine actinomycetes is gaining momentum in recent years. The Indian marine environment is rich in biodiversity, especially microorganisms. However, the wealth of marine micro-flora has not been fully investigated (Ramesh, 2009). Searching for previously unknown microbial strains is an effective approach, which would yield biologically novel active substances. It is known that the antimicrobial activity of the metabolic products of aquatic bacterial strains is not weaker than the corresponding activity of soil strains (Sponga et al., 1999). In addition, the limited attempts have been made on marine organisms and their metabolites in India (Sivakumar et al., 2007; Ramesh and Mathivanan 2009; Ramesh et al., 2009). Importantly investigation of marine actinomycetes with reference to bioactive molecule production in India is still at its infancy. Therefore, exploration of marine actinomycetes for secondary metabolites production is worthy task. The bioactive molecules derived from these actinomycetes could be used as therapeutic drugs for the treatments of various ailments in human and animals and as agrochemicals for the management of insect pests, diseases and weeds in agriculture, etc. as suggested by Lange and Sanchez Lopez (1996) and Prabavathy et al. (2009).

Materials and methods

Collection of marine sediments and isolation of marine actinomycetes
Deep sea soft sediment samples were collected from the Bay of Bengal during the Cruise programmes organized by the National Institute of Ocean Technology (NIOT), Chennai. The research vessel has equipped with sediment collection unit and it was actively utilized for the sediment collection. The sediment samples were pre-treated with 60ºC in a water bath for 5 min to minimize the bacterial growth (Goodfellow and Haynes, 1984). The resultant sediment suspension was serially diluted. From the required dilutions, 100 µL of suspension was taken and plated on aged seawater amended starch casein agar (SCA) medium in Petriplates and incubated for 21 days at room temperature (28 ± 2°C). The development of powdery, chalky and leathery marine actinomycetes colonies were observed periodically. Prominent marine actinomycetes colonies were picked and subcultured on SCA slants. After 7 days of growth pure colonies were selected and maintained in SCA slants as well as glycerol stocks. All the isolated marine actinomycetes were designated with three letters MML followed by four digits Arabic numericals. The culture characteristics of all the marine actinomycetes were documented.

Extraction of extracellular and intracellular metabolites

The marine actinomycetes were grown in antibiotic production medium at room temperature (28±2ºC) for 10 days. The cultures were harvested and filtered through cheesecloth to separate the mycelial biomass. The culture broths were centrifuged at 12000×g for 15 min and the supernatants were collected. The cell-free supernatants and mycelial biomass were used separately for the extraction of bioactive principles. Extraction of extracellular metabolites was done by adding ethyl acetate to the cell-free supernatant in the ratio of 1:1 (v/v). The solvent layer (upper) was collected and concentrated using a rotoevaporator. The mycelial biomasses were used for the extraction of intracellular bioactive metabolites. Acidified acetone (Acetone : HCl at 99:1 ratio) was added to the mycelial biomass in the ratio of 2:1 (v/w) and kept under shaking at 200 rpm in a rotary shaker. After 12 h, the acetone extracts were separated by vacuum filtration and concentrated using a rotoevaporator.

Screening the crude metabolites against human bacterial pathogens

The concentrated crude extracts of extra and intracellular metabolites were dissolved in ethyl acetate and double distilled water at 1:9 (v/v) ratio respectively, and filter sterilized using 0.2 µm filter. The antibacterial activity was determined according to the method of Peela et al., (2005) using Mueller Hinton agar (MHA). To each sterile Petriplate, 20 ml of MHA was poured and allowed to solidify under aseptic condition. Three human pathogenic bacteria, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus spp. were spread inoculated onto sterile MHA in separate plates using sterile cotton swabs. After which, wells were made in each plate using sterile 5 mm diameter cork borer. The filter sterilized extra and intracellular metabolites were added separately at 100 µL in each well and incubated at 37ºC. Whole culture filtrates and heat killed culture filtrates were also tested in this study along with streptomycin as control. After 24 h, the bacterial growth was observed and the zone of inhibition was measured.

Testing of selected actinomycetes against plant pathogenic fungi

Ten marine actinomycetes were selected based on their antibacterial activity against human pathogens in the primary screening. The antagonistic potential against Rhizoctonia solani and Fuzarium oxysporum of all the 10 selected actinomycetes were determined by dual culture technique on Potato Dextrose agar (PDA). Mycelial discs of 6 mm in diameter cut out from the culture of pathogens were placed at the centre point of 90 mm Petriplates separately under aseptic condition. The each actinomycetes streaks were made exactly 3 cm away from the pathogen and the plates were incubated at room temperature for three days and the antagonistic activity was observed.

A total of 137 marine actinomycetes have been isolated from 40 different marine sediment samples collected from the Bay of Bengal during the Cruise programme organized by the NIOT, Chennai and are being maintained at Biocontrol and Microbial Metabolites Lab, Centre for Advanced Studies in Botany, University of Madras. Among the 137 isolated marine actinomycetes tested for antibacterial activity against three human pathogenic bacteria. All the 137 isolated marine actinomycetes were active against all the three bacterial pathogens namely S. aureus, P. aeruginosa and Bacillus spp. Intra and extracellular, live and heat killed culture filtrates of marine actinomycetes exhibited antibacterial activity against the test organisms (Fig. 1). Based on the screening results, 10 marine actinomycetes were selected for further studies. Interestingly, nine out of 10 isolates were isolated from the estuary sediments. Of which, the isolates MML1703, MML1737, MML1767 and MML1778 showed remarkable activity against bacterial test pathogens.

Fig. 1 Antibacterial activity of actinomycetes isolated from marine sediments against human bacterial pathogens in well plate assay.

A: Extracellular; B: Intracellular;
C: Control (Streptomycin); D: Culture filtrate (Live);
E: Culture filtrate (Heat killed)

The selected 10 marine actinomycetes were further tested for antifungal activity against two plant pathogenic fungi Rhizoctonia solani and Fuzarium oxysporum. Among the 10 actinomycetes, the isolate MML1715 showed maximum antifungal activity against R. solani and F. oxysporum, (Fig .2).

Plates showing the growth of control R. Solani (left) and inhibted by isloated actinomycetes MML1715 (right)

Plates showing the growth of control F.oxysporum (left) and inhibted by isloated actinomycetes MML1715 (right)

Fig. 2 Antifungal activity of isolated marine actinomycetes (MML1715) against plant pathogenic fungi on dual culture technique.

Marine-derived actinomycetes are potent source of novel bioactive compounds and the treasury of Indian marine micro-flora has not been realized so far. The cruise programme organized by the NIOT, Chennai has enabled us to collect 40 deep sea sediment samples from different locations of the Bay of Bengal. Interestingly, we could isolate 137 marine actinomycetes from these samples, which indicate the richness of microbial diversity in the Bay of Bengal.

All the tested 137 isolates showed good antibacterial activity against human bacterial pathogens. Further, the marine isolates obtained from the estuary sediment samples were more active than the others that were isolated from the normal sea bed. Sponga et al., (1999) suggested that the actinomycetes inhibiting the growth of antibiotic-resistant microorganisms produce more active antimicrobial substances than the conventional antibacterial sources. The selected 10 isolates showed good antibacterial activity, this characteristic features not remains with antibacterial alone and also added antifungal activity too. This wide spectrum activity nature is very common in marine actinomycetes (Ramesh, 2009). Based on the present study, we conclude that the actinomycetes of Bay of Bengal have tremendous scope on identifying novel bioactive metabolites. Further study is in progress for isolating the potential novel antimicrobial compounds.

The authors are grateful to Prof. R. Rengasamy, Director, CAS in Botany, University of Madras, Chennai, India for providing lab facility. We thank the National Institute of Ocean Technology (NIOT), Chennai for helping us in the collection of marine sediments. We thank the Department of Biotechnology (DBT), GOI, New Delhi for the financial assistance.

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