Abstract—Biodegradation of dimethyl phthalate (DMP), diethyl phthalate (DEP) and dibutyl phthalate (DBP) and their mixture by a bacterial strain is reported. The strain was isolated by enrichment technique from garbage dumped soil nearby Indian Institute of Technology Bombay. The isolate had the ability to utilize mixture of DMP, DEP and DBP in the growth medium as well as the individual phthalate esters as the sole source of carbon and energy. The bacterial isolate was identified as Variovorax sp. based on 16S r-DNA sequence analysis. Variovorax sp. degraded >99% of 300 mg/l of DMP, DEP, DBP or a mixture containing 100 mg/l of each within 30 h. Metabolites or end products were not detected by HPLC analyses. Biodegradation of phthalate esters followed first-order kinetics. The rates of biodegradation of individual esters followed the order: DEP>DBP>DMP while in the mixture, rates followed the order: DEP>DMP>DBP. Crude cell free extracts prepared from bacterial isolates contained cocktail of inducible enzymes responsible for the degradation of DMP or DEP. Index Terms—Biodegradation, enzyme, phthalate esters, variovorax. I. INTRODUCTION Phthalate esters (PAEs) are a group of compounds, which include dialkyl or alkyl aryl esters of 1, 2 -benzendicarboxylic acid (phthalic acid). Among phthalates esters, dimethyl phthalate (DMP), diethyl phthalate (DEP) and dibutyl phthalate (DBP) are mostly used for a variety of commercial purposes and have been listed as priority pollutants by agencies such as U.S. Environmental Protection Agency [1]. Humans are regularly exposed to PAEs through food, medicines, plastics, dairy products, cosmetics, etc. PAEs and their metabolites have been reported to cause reproductive and developmental disorders. PAEs have also been reported to be hepatotoxic, teratogenic and carcinogenic [2]. PAEs are not tightly bonded to the polymers (such as plastics), hence leach out into the environment and pose serious risks to ecosystem components. DMP is used in cellulose ester based plastics, paints, adhesives, printing inks and coatings. Due to its wide applications in industries, DMP has been recognized as a significant environmental contaminant. DMP is a relatively stable compound in the natural environment with a half life of ≈20 years [3]. DMP promotes chromosomal injuries in human leucocytes, also known to be an endocrine disrupting Manuscript received February 10, 2012, revised February 28,2012. B. Prasad and S. Suresh are with the Centre for Environmental Science and Engineering (CESE) at the Indian Institute of Technology Bombay (IITB), Powai, Mumbai, India. (e-mails: sumathis@iitb.ac.in; bablup@iitb.ac.in) chemical (EDC) that interferes with the reproductive system and normal development of animals and humans [4]. DEP is used as a solvent vehicle for fragrance and in cellulose ester based plastics. DEP has been reported to cause abnormality in sexual differentiation and also act as an endocrine disrupting chemical [5], [6]. DBP is used in cosmetics, children's toys and child care products. DBP has been reported to reduce human sperm production and its motility, decrease rates of pregnancy and promote miscarriages [2], [7], [8]. Removal and degradation of PAEs by abiotic processes such as hydrolysis and photodecomposition has been reported to be slow and insignificant. However, breakdown by microorganisms is considered to be one of the major routes of environmental degradation for these widespread pollutants [9]. Number of investigators has demonstrated successful degradation of several PAEs by microbes under aerobic conditions in soil, water and wastewater [10]. Although there are number of reports on the biodegradation of individual PAEs, there is scanty literature available on degradation of mixture of PAEs. Industrial wastes frequently contain mixture of PAEs and therefore it is important to investigate the biodegradation of their mixtures rather than individual PAEs. The goal of present investigation was to demonstrate biodegradation of individual phthalate esters and their mixture using whole cells of a newly isolated bacterial strain as well as cell free crude enzyme preparations from the isolate. II. ATERIALS AND METHODS A. Chemicals Dimethyl phthalate, diethyl phthalate and dibutyl phthalate were procured from Sigma-Aldrich, USA. HPLC grade methanol and acetonitrile were purchased from Merck India Limited, Mumbai. All chemicals were of high purity (>99%). All experiments were performed in duplicate unless otherwise specified. B. Isolation of PAE Degrading Bacterial Strain and Culture Conditions Microorganisms capable of utilizing the three phthalate esters as a mixture (DMP, DEP and DBP) were isolated via enrichment culture technique. Samples were taken from soil contaminated with electronic and plastic wastes situated near Powai Lake, Indian Institute of Technology Bombay (IITB). The initial enrichment culture was started by inoculating 100 ml of sterile mineral salts medium (MSM) with 1 g of fresh soil sample and 100 mg/l each of DMP, DEP and DBP in a 250 ml Erlenmeyer flask. The MSM consisted of: 3.5 g Biodegradation of Dimethyl Phthalate, Diethyl Phthalate, Dibutyl Phthalate and Their Mixture by Variovorax Sp. B. Prasad and S. Suresh International Journal of Environmental Science and Development, Vol. 3, No. 3, June 2012 283