Isolation and identification of a nitrile hydrolyzing bacterium and simultaneous utilization of aromatic and aliphatic nitriles I. Mukram, Anand S. Nayak, B. Kirankumar, T.R. Monisha, Pooja V. Reddy, T.B. Karegoudar * Department of Biochemistry, Gulbarga University, Gulbarga 585106, Karnataka, India article info Article history: Received 9 December 2014 Received in revised form 28 February 2015 Accepted 1 March 2015 Available online Keywords: Benzonitrile Benzamide Rhodococcus sp. MTB5 NHase Amidase abstract Rhodococcus sp. MTB5, a bacterial strain capable of utilizing benzonitrile as the sole source of carbon and nitrogen was isolated from a nitrile-contaminated agricultural soil sample by selective enrichment culture technique. This bacterial strain is capable of utilizing both aliphatic and aromatic nitriles. The production of ammonia was noticed during the growth of this strain in the medium supplemented with benzonitrile. The enzyme assay and GC studies revealed that benzonitrile degradation by Rhodococcus sp. MTB5 follows a NHase/amidase pathway involving benzamide and benzoic acid as metabolic in- termediates. The resting cells grown on benzonitrile showed NHase and amidase activities of 0.155 and 0.261 units, respectively. The strain utilized 30 mM benzonitrile completely and could tolerate and grow in benzonitrile at concentration up to 60 mM. The cells grown on glucose and ammonium nitrate did not show the above enzymatic activities indicating the inducible nature of these enzymes. In addition to benzonitrile, the isolate was also able to utilize indole-3-acetonitrile and aliphatic nitriles, namely, propionitrile, butyronitrile and valeronitrile. Furthermore, the bacterium simultaneously utilizes different combinations of aromatic and aliphatic nitriles. These novel features make the bacterium a suitable candidate for the in-situ application of sites contaminated with both aliphatic and aromatic nitriles. © 2015 Elsevier Ltd. All rights reserved. Introduction Nitriles are cyano group (-C^N) containing organic compounds, with the general formula RC≡N. These are found naturally in plants, bone oils, insects, and microorganisms (DiGeronimo and Antoine, 1976). They are also synthesized and the synthetic nitriles have been used extensively in the manufacture of solvents, extractants, pharmaceuticals and drug intermediates (Banerjee et al., 2002). Among all other synthetic nitriles, benzonitrile (C 6 H 5 C^N) is considered the most important nitrile because of its wide appli- cation as a solvent and intermediate in the manufacture of drugs, perfumes, dyes, rubber, textiles, agrochemicals, resins, and spe- cialty lacquers. It is also used in the production of melamine, pro- tective coatings, molding resins, as an additive in jet fuel, nickel- plating, cotton bleaching baths, for drying acrylic fibre and in the preparation of benzoguanamine under alkali conditions (Pollak et al., 1991; Mckinney and DeVito, 1996). Apart from all those ap- plications, benzonitrile is also a major component in widely used herbicides like dichlobenil (2,6-dichlorobenzonitrile), bromoxynil (3,5-dibromo- 4-hydroxybenzonitrile), and ioxynil (4-hydroxy-3,5- diiodobenzonitrile) (Nawaz et al., 1992). The direct discharge of wastewater containing these nitrile compounds poses severe health hazard as most of them are highly toxic and some mutagenic and carcinogenic in character (Nawaz et al., 1991; Pollak et al., 1991). The release of nitriles into water bodies results in letting cyanide, which persists in the soil or sur- face water causing severe environmental pollution. The indis- criminate use of benzonitrile as agricultural herbicide and its uncontrolled release as waste water by chemical industries leads to their increasing distribution throughout the environment (Cantarella et al., 2006) and hence requires its detoxification. Benzonitrile is known to cause health hazards like decrease in motility, muscular force (Cheav et al., 1990) and methemoglobi- nemia (Divyani and Natasha, 2012). Thus, the removal of benzoni- trile from industrial effluents and environment has become imperative. Bioremediation is one such mechanism used for the detoxification of contaminated sites. Several physical and chemical methods like photo-oxidation and combustion are available for the treatment of benzonitrile containing wastes. However, these methods are not preferred because of their high operational cost, * Corresponding author. Tel.: þ91 8472 263289; fax: þ91 8472 245632. E-mail address: goudartbk@gmail.com (T.B. Karegoudar). Contents lists available at ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod http://dx.doi.org/10.1016/j.ibiod.2015.03.002 0964-8305/© 2015 Elsevier Ltd. All rights reserved. International Biodeterioration & Biodegradation 100 (2015) 165e171