ORIGINAL ARTICLE Isolation of hexachlorocyclohexane-degrading Sphingomonas sp. by dehalogenase assay and characterization of genes involved in c-HCH degradation N. Manickam 1 , M. K. Reddy 2 , H. S. Saini 3 and R. Shanker 4 1 Environmental Biotechnology, Industrial Toxicology Research Centre, Mahatma Gandhi Marg, Lucknow, India 2 Analytical Chemistry, Industrial Toxicology Research Centre, Mahatma Gandhi Marg, Lucknow, India 3 Department of Microbiology, Guru Nanak Dev University, Amritsar, Punjab, India 4 Environmental Microbiology, Industrial Toxicology Research Centre, Mahatma Gandhi Marg, Lucknow, India Introduction Gamma-hexachlorocyclohexane (c-HCH; lindane) is a cyclic, saturated, chlorinated insecticide which had been used worldwide for crop protection and for the control of vector-borne diseases-like malaria. c-HCH is also used in pest management in vegetable crops and seed treatments. Mainly five isomers viz. a-HCH (53–70%), b-HCH (3– 14%), c-HCH (11–18%), d-HCH (6–10%) and e-HCH (3–5%) are formed during its synthesis as a technical mix- ture (Howard 1989). All isomers are persistent to varying levels in the environment (Feidieker et al.1994; Simonich and Hites 1995; Blais et al. 1998; Li et al. 1998, 2003; Breivik et al. 1999; Meharg et al. 1999) and substantial exposures can cause serious health problems like neuro- logical disorders, seizures, convulsions and various devel- opmental toxicities (ATSDR Agency for toxic substances and disease registry 1999). HCH is banned in 52 countries and it is expected that it will be banned globally under the international POPs Treaty (UNECE 1998), because of its categorization as a ‘persistent organic pollutant’. Selective enrichment techniques used earlier enabled the isolation of micro-organisms capable of degrading c- HCH (Senoo and Wada 1989; Sahu et al. 1990; Nagasawa et al. 1993; Manonmani et al. 2000). Micro-organisms such as Bacillus sp. (Gupta et al. 2000), Microbacterium sp. (Manickam et al. 2006a) and Pseudomonas aeruginosa (Kumar et al. 2005), have been identified to possess HCH Keywords dehalogenase, HCH, degradation, lin genes, Sphingomonas. Correspondence N. Manickam, Environmental Biotechnology, Industrial Toxicology Research Centre, Post Box 80, Mahatma Gandhi Marg, Lucknow, India. E-mail: nmanickam2005@yahoo.com 2007 ⁄ 0116: received 25 January 2007, revised 11 September 2007 and accepted 11 September 2007 doi:10.1111/j.1365-2672.2007.03610.x Abstract Aim: To screen and identify bacteria from contaminated soil samples which can degrade hexachlorocyclohexane (HCH)-isomers based on dechlorinase enzyme activity and characterize genes and metabolites. Methods and Results: Dechlorinase activity assays were used to screen bacteria from contaminated soil samples for HCH-degrading activity. A bacterium able to grow on a-, b-, c- and d-HCH as the sole carbon and energy source was identified. This bacterium was a novel species belonging to the Sphingomonas and harbour linABCDE genes similar to those found in other HCH degraders. c-Pentachlorocyclohexene 1,2,4-trichlorobenzene and chlorohydroquinone were identified as metabolites. Conclusions: The study demonstrates that HCH-degrading bacteria can be identified from large environmental sample-based dehalogenase enzyme assay. This kind of screening is more advantageous compared to selective enrichment as it is specific and rapid and can be performed in a high-throughput manner to screen bacteria for chlorinated compounds. Significance and Impact of the Study: The chlorinated pesticide HCH is a per- sistent and toxic environmental pollutant which needs to be remediated. Isola- tion of diverse bacterial species capable of degrading all the isomers of HCH will help in large-scale bioremediation in various parts of the world. Journal of Applied Microbiology ISSN 1364-5072 952 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 952–960 ª 2007 The Authors