OPTIMIZATION OF PROCESS PARAMETERS FOR RIFAMYCIN B PRODUCTION UNDER SOLID STATE FERMENTATION FROM AMYCOLATOPSIS MEDITERRANEAN MTCC 14 Research Article BM VASTRAD 1 , SE NEELAGUND 2* 1 Department of Pharmaceutical Biotechnology, S. E. T`s College of Pharmacy, Dharwad, 2 Received: 18 February 2012, Revised and Accepted: 16 March 2012 Department of PG Studies and Research in Biochemistry, Jnana Sahayadri Kuvempu University, Shankarghatta, Karnataka, India. Email: neelgund@gmail.com ABSTRACT Solid-state fermentation (SSF) was carried out for the production of extra-cellular rifamycin B by Amycolatopsis Mediterranean MTCC 14 using four agro-industrial substrates, coconut oil cake, groundnut oil cake , ground nut shell and rice husk. Coconut oil cake and ground nut shell showed the highest antibiotic yield compared to groundnut oil cake and rice husk. Maximum rifamycin B titres were produced when SSF was carried out with 60% initial moisture content of the substrate, 8% (w/w) inoculums, substrate particle size 1.4 and 1.6 mm, initial pH 8, 32 o Keywords: Rifamycin B; Amycolatopsis Mediterranean MTCC 14; Solid state fermentation; Coconut oil cake; Optimization C as incubation temperature, and day 10 of incubation period in all four substrates. External supplementation of fermentation medium with various carbons, organic and inorganic nitrogen sources was benefit for antibiotic production. INTRODUCTION Rifamycins are clinically important ansamycin antibiotics, composed of a naphthalenic chromophore spanned by a long aliphatic ansa chain. The rifamycins derived from them exert their antibiotic activity by specific inhibition of bacterial DNA-dependent RNA polymerase of different microbial species 1,2,3 . At higher concentrations, these antibiotics also inhibit the RNA-dependent DNA polymerase of retroviruses as mono-therapy or in combination with other drugs 4,5,6 . Therefore, antibiotics of the rifamycin class such as rifampicin, rifabutin and rifapentine have been employed on a global basis in a number of well established combination regimes for the clinical treatment of tuberculosis, leprosy, AIDS-related mycobacterial infections and many other enteric infections 7, 8, 9,10 . Organisms known to produce rifamycins include Nocardia mediterranei 11 and Amycolatopsis mediterranei-CBS-42575 12 Solid-state fermentations(SSF) is generally defined as that in which microbial growth and product formation take place on solid substrate in absence of free water. Several reviews are available describing the techniques. In recent years, solid state fermentation technology has received increasing interest. This is partially because, as a fermentation technology, it has lower energy requirement and produces less waste water than alternative technology such as submerged technology. In addition, there are increasing environmental concern regarding the disposal of solid waste, and there use as substrate for the commercial production of microbial metabolites is becoming an alternative position . 13 The production of antibiotics by SSF has gained much attention in biotechnology studies for production of cephamycin . 14, 15 , oxytetracycline 16, 17 , iturin 18, 19 , neomycin 20, 21 , cephalosporin C 22 , penicillin 23 and rifamycins 24, 25 . The use of low cost agricultural solid residues, higher productivities, low energy requirements, lower wastewater production, extended stability of products and low production costs are some of the main advantages of SSF 13, 26 . The selection of a suitable microorganism is an important aspect of SSF for production of antibiotics 23 . The microorganism should be able to grow at low water activity, to be GRAS (“Generally Recognized as Safe”) 27 In the recent past, many agro-industrial byproducts such as wheat rawa . 14 , corn cobs 16 , okara 18 barley 28 etc. have been screened as low-cost solid substrates for production antibiotics in SSF. A perusal of literature shows that some of the solid substrates possess higher potential as compared to the other solid substrates for supporting/inducing the rifamycin production by Amycolatopsis sp. RSP 3. For example, wheat bran, corn husks and corn cobs are reported to be superior as compared to the other tested solid substrates for optimum rifamycin production by many microorganisms under the identical SSF condition 24 MATERIALS AND METHODS . However, the exact reason(s) for such a higher antibiotic production by microbes while growing on some specific substrates has never been explored and till date no logical explanation has been provided for the above observed effect. Microorganism maintenance and seed preparation Amycolatopsis mediterranei MTCC 14 29 used in the present study was obtained from the Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India. The culture was maintained on Q/2 agar slants. This medium consists of (g l -1 ): yeast extract, 4.0; malt, 10.0; glucose monohydrate, 4.0; oat flakes, 20.0and agar, 20.0. The pH was adjusted to 7.6 before sterilization and the slants were incubated at 28 °C for8 days. Surface growth of the selected slant was harvested in flasks containing 100 ml of vegetative medium. The flasks were incubated at 28°C in a rotary incubator shaker at 100 rpm for 56 to 72 h. Flasks showing the following parameters were selected for inoculation of the fermentation medium: pH, 7.6-7.9; percentage packed mycelial volume 30 Selection of the substrate 9 – 12%; color, intense orange to red; time for methylene blue decolorization, 50 – 70 s. Various oil cakes such as coconut oil cake (COC) and groundnut oil cake (GOC) was obtained from an local oil mill, Dharwad, India and traditional agro-industrial residues such as ground nut shell (GNS) and rice husk (RH) was obtained from the local market of Dharwad, India were screened as substrates for rifamycin B production. These were ground in a mill to a particle size between 0.8-2.0 mm and dried at 55-60°C for 12 h. Solid State Fermentation Initially, the fermentation conditions reported by Mahalaxmi et al 2011 were used. Solid substrates were dried in an oven at 50 o C for 5 h. Five grams of the dried substrate was taken in conical flask and supplemented with (in mg) soya bean meal 250, calcium carbonate 75, potassium nitrate 100, barbital 20 and magnesium sulphate 1 with predetermined quantity of water 24 . The initial moisture level of the substrate was adjusted by adding adequate amount of distilled water. The thoroughly mixed substrate was autoclaved at 121 o C for 20 min and cooled to room temperature (28 ± 2 o C) before inoculation. After cooling, the substrate was inoculated with 1 ml of seed culture (4 % w/w), thoroughly mixed with glass rod and incubated at 28 ± 2 o C for 15 days. International Journal of Current Pharmaceutical Research ISSN- 0975-7066 Vol 4, Issue 2, 2012 A Ac c a a d d e e m mi i c c S Sc c i i e e n n c c e e s s