& Bioch ial em b ic ro a c l i T M e f c Venkata Ratna Ravi Kumar et al., J Microbial Biochem Technol 2011, 3:5 h o Journal of l n o a l n o r DOI: 10.4172/1948-5948.1000058 g u y o J ISSN: 1948-5948 Microbial & Biochemical Technology Research Article Article OpenOpen Access Access Screening of Antagonistic Marine Actinomycetes: Optimization of Process Parameters for the Production of Novel Antibiotic by Amycolatopsis Alba var. nov. DVR D4 Venkata Ratna Ravi Kumar Dasari*, Murali Yugandhar Nikku and Sri Rami Reddy Donthireddy Center for Biotechnology, College of Engineering, Andhra University, Visakhapatnam- 530 003, India Abstract Screening of six marine sediment samples near NTPC of the Visakhapatnam (India) Coast of Bay of Bengal resulted in the isolation of 72 isolates of actinomycetes. Among these, Amycolatopsis alba var. nov. DVR D4 showed broad antibacterial activity spectra against Gram-positive and Gram-negative bacteria; and produced antibacterial metabolite extracellulary under submerged fermentation conditions. The chemical and physical process parameters affecting the production of the antibiotic were optimized. The maximum antibiotic activity was obtained with the optimized production medium containing D-glucose, 2.0 %w/v; malt extract, 4.0 %w/v; yeast extract, 0.4 %w/v; dipotassium hydrogen phosphate, 0.5 %w/v; sodium chloride, 0.25 %w/v; zinc sulphate, 0.004 %w/v; calcium carbonate, 0.04 %w/v; with inoculum volume of 5.0 %v/v at 6.0 pH, 28°C incubation temperature, 220 rpm and for 96 h incubation. Keywords: Screening; Optimization; Submerged fermentation; Apart from the cultural conditions (inoculum age, inoculum Amycolatopsis alba; Antibacterial activity; Minimum inhibitory volume) of the organism, fermentation medium has profound effect concentration on product formation directly or indirectly. Lower concentration of nutrients may lead to poor growth and poor yields because of under Introduction nutrition. High concentrations may have a deleterious effect on The microorganisms especially bacteria and actinomycetes are growth. Catabolite repression and toxicity may be observed. To satisfy virtually unlimited sources of novel compounds with many therapeutic such requirement, medium should contain a judicious combination applications. Actinomycetes among them hold a prominent position of various nutrients (mainly few inorganic salts, carbon and nitrogen due to diversity and proven ability to produce new structures. source, water, etc.) at concentrations that favors the growth of organism Actinomycetes are widely distributed in terrestrial and aquatic and product formation. ecosystems. Especially in soil, actinomycetes play a crucial role in the recycling of refractory biomaterials by decomposing complex mixtures In the present study, it is decided to screen marine sediment samples of polymers in dead plant, animal and fungal materials and capable of from shore area (Bay of Bengal: an arm of Indian Ocean) at NTPC, producing several secondary metabolites [1]. But the rate of discovery Visakhapatnam, India for the isolation of antagonistic actinomycetes of new compounds from terrestrial actinomycetes has decreased, and to optimize the process parameters for the maximum production whereas the rate of re-isolation of known compounds has increased of antibacterial metabolite under submerged fermentation. [2].Thus, it is crucial that new groups of actinomycetes from pristine Materials and Methods habitats need to be explored as sources of novel bioactive secondary metabolites. Collection of samples Actinomycetes play a significant role among the marine bacterial A total of six sediment samples from Bay of Bengal (NTPC area, communities, because of its diversity and ability to produce novel Visakhapatnam, India) were collected by grab sampler and stored in bioactive compounds of high commercial and therapeutic value [3,4] sterile containers for the systematic screening of actinomycetes. About Marine ecosystem still an untapped source of microbial diversity and 50 g of each sample was collected from different places (at different marine microbes are particularly attractive because they have not distances and different depths) at NTPC area. been extensively exploited as their terrestrial counterparts; and high potency required for bioactive compounds to be effective in the marine environment, due to the dilution effect of seawater. Most of the marine *Corresponding author: Venkata Ratna Ravi Kumar Dasari, Center for bioactive compounds that have been successfully screened and isolated; Biotechnology, College of Engineering, Andhra University, Visakhapatnam- 530 and structurally elucidated so far originated from microorganisms, 003, India, E-mail: [email protected] especially from bacteria. It is estimated that less than 1% of potentially Received November 07, 2011; Accepted December 07, 2011; Published useful chemicals from marine environment has been screened so far, December 09, 2011 with microbial products especially bioactive compounds representing Citation: Venkata Ratna Ravi Kumar D, Murali Yugandhar N, Sri Rami Reddy approximately 1% of the total number [5]. D (2011) Screening of Antagonistic Marine Actinomycetes: Optimization of Process Parameters for the Production of Novel Antibiotic by Amycolatopsis Alba As innovative methodologies in current screening programmes, var. nov. DVR D4. J Microbial Biochem Technol 3: 092-098. doi:10.4172/1948- actinomycetes with unique morphology and / or physiology have 5948.1000058 been targeted and isolation methods for them have been developed. Copyright: © 2011 Venkata Ratna Ravi Kumar D, et al. This is an open-access These isolated have then been exposed to non-conventional as well as article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, conventional cultural conditions to induce antibiotic production. provided the original author and source are credited J Microbial Biochem Technol ISSN:1948-5948 JMBT, an open access journal Volume 3(5): 092-098 (2011) - 092 Citation: Venkata Ratna Ravi Kumar D, Murali Yugandhar N, Sri Rami Reddy D (2011) Screening of Antagonistic Marine Actinomycetes: Optimization of Process Parameters for the Production of Novel Antibiotic by Amycolatopsis Alba var. nov. DVR D4. J Microbial Biochem Technol 3: 092-098. doi:10.4172/1948-5948.1000058 Screening and isolation of production studies. Five ml of sterile water was transferred aseptically into each slant and the growth of the isolate on the surface of the Marine sediment samples were stored at 4°C until isolation. medium was scrapped with sterile inoculating needle and transferred Actinomycetes are isolated by plating out the samples in proper each into 45 ml of production medium (composition: sucrose, dilutions. Actinomycetes colonies can easily be distinguished on the 20.0g; malt extract, 10.0g; yeast extract, 4.0g; dipotassium hydrogen plate from those of fungi and true bacteria. They are often compact, phosphate, 5.0g; sodium chloride, 2.5g; zinc sulphate, 0.04g; calcium leathery giving a conical appearance and have a dry surface. carbonate, 0.4g; 1.0L sterile distilled water with pH 7.0) and incubated About 1 gm of each of the above sample was taken in a 250 ml at 28°C on a rotary shaker at 220 rpm for 5 days. Then the samples conical flask containing 100 ml of sterile water and was kept on a were collected into sterile centrifuge tubes and centrifuged at 4000 rpm rotary shaker for 15 minutes. The suspension was serially diluted up for 10 min, at 8°C and clear culture filtrate was separated. The clear to 10-6 level. Isolation was carried out on starch casein agar (SCA- supernatant was used for antibiotic assay using cup-plate method. soluble starch, 10.0g; vitamin free casein, 0.3g; KNO3, 2.0g; NaCl, The antibacterial activity against the bacterial organisms was tested on 2.0g; K2HPO4, 2.0g; MgSO4.7H2O, 0.05g; CaCO3, 0.02g; FeSO4.7H2O, nutrient agar medium. 0.01g; agar, 20.0g; sterilized natural aged sea water, 1.0L; pH, 7.2; ° supplemented with rifampicin 2.5µg/ml and cycloheximide 75µg/ml to The molten sterile nutrient agar medium was cooled to 40-45 C, inhibit bacterial and fungal contamination, respectively) plates, which inoculated with test organism, mixed thoroughly, poured into sterile had been seeded with a sediment sample suspension of 1.0 ml each and petri plates and allowed to settle. Cups were made using sterile cork incubated at 28°C for 14 days [6]. After 14 days, actinomycete colonies borer. The clear supernatant fermentation broth was added to each cup were carefully isolated from different plates avoiding any bacterial or (50 µl) by using micropipette. The procedure was repeated for all the fungal contamination. The actinomycete colonies, which appeared promising isolates and each isolate was tested for antibacterial activity. different from one another to the naked eye (surface texture), were The plates were kept in the refrigerator for about 2 h for antibiotic ° transferred and incubated at 28°C for 2 weeks; and maintained on yeast diffusion and then incubated at 37 C. After 24 h the inhibition zones extract malt extract (YEME – yeast extract, 4.0g; malt extract, 10.0g; were recorded. Experiments were conducted in triplicate and results D-glucose, 4.0g; agar, 20.0g; distilled water, 1.0L; pH, 7.2) slants at 4°C were the average of the three default trails. ° and as a glycerol suspension (20%v/v) at -20 C [7]. Taxonomy and genotypic characterization The isolates were pooled together and cultures which appeared The cultural and morphology properties were examined by using identical to naked eye in respect of color of aerial mycelium, reverse standard procedures [8] and procedures of Williams et al. [9] after color, soluble pigment and colony texture were eliminated. A total of growth on YEME Agar for 7-10 days at 28°C. Phenotypic tests were 72 actinomycetes were isolated from the above samples. carried out following the procedures of Goodfellow et al. [10] and Antimicrobial activity studies Gordon et al. [11]. Chemotaxonomic characters were determined as described previously [12,13-17].