Biodegradation of phenol by oxygenase producing thermophilic microorganisms Neetika Mathur1*, Vinod Kumar Nigam2, Ashwani Kumar1 and Purnendu ghosh2 1 Department of Botany, University of Rajasthan, Rajasthan- 302 004 2 Department of Biotechnology, Birla Institute of Scientific Research, Jaipur, Rajasthan-302 001 Abstract: Screening and characterization of phenol degrading bacterial isolates from the soils and sewage samples of desert regions of Rajasthan was performed under batch cultivation. The degradation studies were monitored by the consumption of NADH and it was found that maximum degradation was achieved at 500C. Out of nine phenol adapted oxygenase producing cultures, four cultures BISR 00T1, BISR 00T3, BISR 00T5 and BISR 00T8 showed maximum enzymatic activity. Various biochemical tests were also performed for their characterization. Key words: biodegradation, oxygenases, thermophilic, phenol, pollutants. Introduction: Rapid industrial and economic development, human exploitation of fossil fuels and production of many synthetic compounds have introduced many compounds that are not naturally present in the environment. Many of these xenobiotics substances are toxic to the living system and their presence in the aquatic and terrestrial habitats often have serious ecological consequences. Bioremediation is emerging as most ideal technology for removing pollutants from the environment by the action of microbes or other _______________________________________ * Corresponding author: Neetika Mathur, Department of Botany, University of Rajasthan, Jaipur 302 004; Phone No: 0141-2546381; Email: biological systems, restoring contaminated sites and preventing further pollution (Caplan, 1993; Dua et al., 2002). Phenols and chlorophenols are introduced in the environment in the waste stream of several industrial operations, through its use as biocides or as by-product of other industrial operations, such as pulp bleaching with chlorine, water disinfection or even waste incineration and as degradation product of other chlorinated xenobiotics (Bollag et al., 1986). Other sources of phenol can be oil refineries, chemical plants, explosives manufacturing, resin manufacturing, rubber reclamation, textile mills and plants etc. Phenols have a relatively high oxygen demand (2.4 mg of O2/mg of phenol) for degradation. These are toxic to fish at a level of above 2 mg/L and as little as 0.005mg/L of phenol can impart objectionable tastes and odour to the drinking water (S. N. Jogdand). Because of their toxic effects, phenols and chlorophenols tend to accumulate and in some cases, the contamination of soil and water is of concern (Keither and Tellard 1979, Moos et al., 1983; Borthwick and Schimmel 1978). Several decontamination techniques are available for the removal of contaminants from water such as adsorption, ion exchange, incineration etc., but all are unable to destroy the contaminants. Biodegradation is a technique which could potentially degrade these contaminants to innocuous products (Murialdo et al., 2003). Microbial degradation of phenol and chlorophenols has been reported by several groups (Baker et al., 1980; Pignattelo et al., 1983; Saber and Crawford, 1985; Rozich and Colvin, 1986; Apajalahti and Salkinoja-Salonen, 1986; Radehaus and Schmidt, 1992; Ramos et al., 1995; Lee et al., 1998; Reddy and Gold, 2000; Cortes et al., 2002; Kirchner et al., 2003). Biological methods can reduce phenol down to 0.5-1 mg/L level. Phenol hydroxylase (also known as monooxygenase) catalyses the efficient ortho-hydroxylation of phenol and ultimately its degradation (Kirchner et al., 2003). Phenols are first converted into more reactive dihydroxylated intermediates and then subjected to ring cleavage by molecular oxygen in presence of NAD (P) H dependent flavoprotein monooxygenases (Moonen et al., 2003). The ability of oxygenase to incorporate oxygen into organic compounds is important because many of the hydrophobic pollutants such as polycyclic aromatic hydrocarbons are high in carbon and hydrogen content but low in oxygen content. It is reported that enzyme catechol 2, 3-dioxygenase catalyzes the phenol degradation by meta-cleavage pathway in aerobic, sporulating, motile, rod shaped thermophilic bacteria characterized as Bacillus species with growth temperature optima of 500-600C (Ali Saiqa et al., 1998). These thermophilic bacteria can be used in treatment of phenolic waste water. The biotechnological significance and the benefits of the use of thermophilic bacteria in enhancing hydrocarbon removal under different environmental conditions were investigated. Mesophilic sources of phenol have been investigated but due to thermal instability of the enzymes the attention is being made to search for thermostable enzymes (Moiseeva et al., 2002). Many of the organic compounds are first degraded to catechol or protocatechuate by oxygenases (both dioxygenases and monooxgenases). The intermediates are metabolized by a ring cleavage type of dioxgenases to either beta-ketoadipate or 2-keto-4-hydroxyvalerate and finally metabolized by TCA cycle. In the present study, the organisms isolated from the hot desert region of Rajasthan are selected for evaluating their phenol degrading capabilities. The enzymes (whole cells) have also been characterized at various physico chemical parameters.