EXTRACELLULAR POLYMERIC SUBSTANCE (EPS) FROM KOCURIA SP. BRI 36: A KEY COMPONENT IN HEAVY METAL RESISTANCE Original Article ANURADHA MULIK, RAMA BHADEKAR Department of Microbial Biotechnology, Rajiv Gandhi Institute of IT and Biotechnology, Bharati Vidyapeeth Deemed University, Pune 411046, India Email: neeta.bhadekar@gmail.com Received: 07 Nov 2017 Revised and Accepted: 28 Mar 2018 ABSTRACT Objective: Evaluation of Extracellular Polymeric Substance (EPS) induced heavy metal tolerance in Kocuria sp. BRI 36. Methods: Initially, the effect of different concentrations of glucose (1-10 %) on EPS production by BRI 36 was examined. At optimum glucose concentrations, EPS levels were measured by varying heavy metal concentrations (10-50 ppm) of Pb 2+ , Cd 2+ and Cr 3+ Results: Kocuriasp. BRI 36 produced maximum EPS (1g/l) at 5% glucose. Increase in EPS production up to 89% (considering 1g/l as 100%) with an increase in concentrations of heavy metals up to 40 ppm. MTC levels of BRI 36 for heavy metals increased up to 700 ppm when it was cultivated in presence of 5% glucose indicating a major role of surface polymer in metal adsorption. The function of EPS as a protective cover was also evident from an increase in survival percentage of BRI 36 up to 39.4 at MTC. Comparative analysis of extracted crude EPS by FTIR revealed the involvement of O-H, C=O, and C-O-C groups in metal adsorption . Maximum tolerable concentration (MTC) and survival percentage of BRI 36 were determined under conditions that support EPS synthesis. Comparative analysis of extracted crude EPS was performed by Fourier Transform Infrared Spectroscopy (FTIR) to establish functional groups involved in the metal interaction. Conclusion: Antarctic oceanic isolate Kocuria sp. BRI 36 has an ability to produced EPS under stress conditions of heavy metals. Simultaneously, its MTC values increased due to increase in EPS levels. These observations suggest the possibility to develop gentle, environmentally safe and cost- effective method for heavy metal removal. Keywords: Exopolysaccharides, Fourier Transform Infrared Spectroscopy (FTIR), heavy metals, Maximum Tolerable Concentration (MTC) © 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) DOI: http://dx.doi.org/10.22159/ijpps.2018v10i5.23535 INTRODUCTION Many bacteria produce biopolymers with varied chemical properties through the utilization of simple to complex substrates. They could either be intracellular or extracellular. The extracellular polymeric substances (EPS), also known as exopolysaccharides, have a high molecular weight and consist mainly of carbohydrates. However, the intracellular biopolymers are few and have very limited use [1]. EPS may be grouped into four major classes; polysaccharides, inorganic polyanhydrides (such as polyphosphates), polyesters, and polyamides [2]. Most marine bacteria can produce EPS, which occur in two forms viz. capsular polysaccharides in which the polymers are covalently bound to the cell surface; and slime polysaccharides that either remain attached (loosely bound) to the cell surface or are released into the environment [3, 4]. EPS produced by marine bacteria are thought to play a key role in the protection against the marine environment characterized by extreme physicochemical conditions such as low or high temperature, high pressure, low nutrient concentration, high salinity, and heavy metal presence [5]. Occurrence of heavy metals in soil and water are found to be important micronutrients, provided that the levels are in a limit [6, 7] or else they contaminate natural resources which in turn has an adverse effect on the ecosystem [8, 9]. BRI isolates from Antarctic oceanic region have an ability to grow under extreme environmental conditions [10]. Among them red-orange pigment producing Kocuriasp. BRI 36 was observed to tolerate and accumulate heavy metals [11]. The response of BRI 36 to different heavy metals at different concentrations in terms of amount and color of pigment produced might prove useful in the detection of heavy metal contamination [12]. Our recent studies showed the possible role of chromosomal genetic elements in heavy metal tolerance of BRI 36 [13]. The isolate under study was also found to produce EPS at high sugar concentration. Interestingly, heavy metal tolerance of BRI 36 in terms of maximum tolerable concentration (MTC) was found to increase considerably with an increase in EPS produced by the isolate. Role of EPS in metal resistance has been previously documented by various researchers [14-17]. These observations prompted us to undertake studies on EPS production, and its effect on heavy metal tolerance of Kocuria sp. BRI 36. MATERIALS AND METHODS Chemicals and reagents All the media components were purchased from HiMedia Laboratories Pvt. Ltd. (Mumbai, India). The stock solutions of cadmium, lead and chromium at the concentrations of 1000 ppm each were purchased from Sigma-Aldrich. All chemicals and reagents used were of analytical grade. Analysis of EPS Kocuriasp. BRI 36 was cultivated EPS production at different sugar concentrations on Mineral Salt Medium (MSM) at 30±2 °C for 48 h with 10% inoculum. The isolate was then grown in MSM containing 5 % glucose at 30±2 °C for 48 h. The occurrence of slimy colonies at the end of incubation period indicated EPS production by BRI 36. The isolate was then grown in MSM containing 5 % glucose at 30±2 °C for 48 h with shaking at 120 rpm. The contents were centrifuged (7826 xg for 20 min) and the culture cell-free broth was used to estimate EPS as carbohydrates following phenol-sulphuric acid method [18]. The production of Extracellular Polymeric Substance (EPS) was studied by growing the isolate at different concentrations of glucose (1–10%) and EPS as carbohydrates were estimated as mentioned above. International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 10, Issue 5, 2018