ISOLATION, SCREENING AND IDENTIFICATION OF CEFDINIR DEGRADING YEASTS FOR THE TREATMENT OF PHARMACEUTICAL WASTEWATER Original Article SELVI A, NILANJANA DAS* Nilanjana Das, Environmental Biotechnology Division, School of Bio Sciences and Technology, VIT University, Vellore 632014, India. Email: nilanjana00@lycos.com Received: 27 Jun 2014 Revised and Accepted: 29 Jul 2014 ABSTRACT Objective: The aim of the present study was to isolate, screen and identify the cefdinir degrading yeasts for the treatment of pharmaceutical wastewater. Methods: The steps include isolation of yeasts from pharmaceutical wastewater, screening of yeasts for cefdinir degradation, studies on effects of pH, carbon and nitrogen sources on yeast growth, treatment of pharmaceutical wastewater using yeast. Results: Out of five yeast isolates, four were screened for cefdinir degradation and identified by molecular techniques. Yeast isolates were found to utilize cefdinir as sole carbon and energy source at an optimum pH 6.0. Addition of extra carbon and nitrogen sources in the growth medium did not show any improvement of yeast growth. Maximum cefdinir degradation by four yeast isolates was found to be 72%, 78%, 81% and 84% respectively and significant reduction in BOD, COD, TSS and TDS was noted after treating the pharmaceutical wastewater with the yeast isolates. Conclusion: The isolated yeasts may serve as potential remediation agents for the treatment of pharmaceutical wastewater containing cefdinir, a semi-synthetic cephalosporin derivative. Keywords: Biodegradation, Cefdinir, Pharmaceutical wastewater, Yeasts. INTRODUCTION Pharmaceutical industries involved in the production of antibiotics discharge their wastes openly which contains some quantity of active compounds [1]. These industries often require appropriate microorganisms to reduce its COD load efficiently [2]. Wastewater from cephalosporin production units contain complicated components such as organic substances, soluble or colloid solid substances along with toxic non-biodegradable and bacteriostatic antibiotics which are matters of concern. The presence of these toxic antibiotics in the environment cause great harm to the human beings [3]. Cefdinir is an advanced third generation semi-synthetic cephalosporin antibiotic, characterized by a vinyl group at C-3 and a (Z)-2- (2-amino -4 thiazolyl ) -2- ( hydroxyimino) acetyl moiety at C- 7 and is used for the treatment of acute respiratory related disorders and mild skin infections [4]. The effluents released from cephalosporin production units are reported to release harmful compounds which are resistant to biodegradation, photo- transformation and natural degradation [5]. The presence of high concentration of cephalosporin in the environment leads to very high chemical oxygen demand (COD) thus by increasing the toxic strength of the effluent [3]. The physico-chemical methods used for the removal of the antibiotic compounds have various limitations like high operating cost, huge labour, production of toxic metabolites etc. [6]. Hence, developing a low cost technology for wastewater treatment becomes necessary and bioremediation technology involving microorganisms can be a better option as a less expensive and more environmentally friendly alternative to the conventional treatment methods [7]. There are reports on the use of bacteria viz. Pseudomonas putida and Pseudomonas. fluorescens MATERIALS AND METHODS , Bacillus and Bacteriods for degradation of cephalosporin derivatives [8,9]. However, no report is available on cefdinir degradation by microorganisms. Yeasts have been found to be efficient in treating high strength organic wastewaters from pharmaceutical industries [2,10,11,12]. Therefore, preliminary study was conducted to isolate the yeasts from pharmaceutical wastewater and screen them based on their cefdinir degradation efficiencies. Furthermore, pharmaceutical wastewater was treated with the yeast isolates to test their efficiencies in terms of reduction of BOD, COD, TSS and TDS from wastewater. Sample collection Pharmaceutical wastewater was obtained from the cephalosporin production unit, Chennai and Ranipet, India. Samples collected from the point of discharge into the environment were kept in clean sterile 5L plastic bottles, transferred immediately to the laboratory and stored in 4℃. Various physico-chemical characteristics of pharmaceutical waste water were analyzed using standard methodologies [13]. Chemicals Cefdinir (<99% pure) was kindly donated by Orchid Pharmaceuticals, Chennai. Dimethyl sulphoxide (DMSO) procured from SRL Chemicals, India Ltd., was used to prepare a stock solution of cefdinir (10 4 Yeast isolation procedure mg/L ). All other chemicals were of analytical grade and procured from Himedia Ltd, India and SRL chemicals, India Ltd. The yeasts were isolated by serial dilution method by culturing in yeast extract peptone dextrose agar (YEPD) medium containing (g/L): yeast extract-10 g, peptone-20 g, dextrose-20 g, agar-20 g. Streptomycin antibiotic was added to the YEPD medium in order to prevent bacterial contamination. The YEPD agar was autoclaved at 121 ºC for 15 min. After cooling to 50º C, the media was poured onto the Petri dishes with immediate swirling of the Petri dishes to ensure adequate mixing and uniform surface. 100 μL of various dilutions are plated onto YEPD agar by spread plate technique. Triplicates were prepared for each dilution and the sampled plates were incubated at 28 ± 2 °C for 48 h. After incubation, all the probable yeast colonies were observed by visual identification and confirmed by staining procedure through microscopy. The isolated yeasts were further streaked onto YEPD agar plates to obtain pure culture. The pure cultures were stored at 4 ºC and subcultured at regular intervals. Acclimatization procedure The isolated yeast species were grown in YEPD broth containing cefdinir (100 mg/L) for a period of 6 days and the cefdinir International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 6, Issue 8, 2014 Innovare Academic Sciences