235 USHA et al: DEGRADATION OF H-ACID BY ALCALIGENES LATUS Journal of Scientific & Industrial Research Vol. 71, March 2012, pp. 235-240 *Author for correspondence E-mail. bg.ushams@gmail.com Study on degradation of H-acid by Alcaligenes latus isolated from textile industrial effluent M S Usha 1 *, M K Sanjay 2 , S M Gaddad 3 and C T Shivannavar 3 1 Department of Microbiology, Jain University, Bangalore 560 011, India 2 Department of Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India 3 Department of Microbiology, Gulbarga University, Gulbarga 585 106, India Received 05 July 2011; revised 04 January 2012; accepted 06 January 2012 This study presents 100% degradation of H-acid under optimized conditions using Alcaligenes latus, isolated from textile industrial effluent. Gene/s responsible for H-acid degradation was/were found to be present on plasmid DNA. Addition of bipyridyl to incubated medium resulted in accumulation of terminal aromatic compound, suggesting that catechol may be terminal aromatic compound in degradation pathway of H-acid by A. latus. SDS-PAGE of cell free extracts showed two prominent bands close to molecular weight of catechol 1,2-dioxygenase. Keywords : Alcaligenes latus, Catechol 1,2-dioxygenase, H-acid degradation Introduction Significant quantities of synthetic azo dyes are discharged in effluents that not only colour the receiving water bodies, but also lead to the formation of more hazardous intermediate chemicals by microorganisms or through photo oxidation. Dye intermediates (amino- hydroxyl- naphthalene sulphonic acids) are important building blocks for the large scale production of azo dyes. Sulphonic acid group confers a xenobiotic character to this class of chemicals 1 . Some studies 2-7 are available on biodegradation of naphthalene derivatives. H-acid (1-amino 8-hydroxy naphthalene 3,6- disulfonic acid), a precursor of several azo dyes 4 , is listed under Toxic Substances Control Act (TSCA). Few attempts have been made to treat H-acid by physicochemical methods 8-10 . Reports on biodegradation of H-acid are limited 11,12 . Alcaligenes latus, isolated from a textile industrial effluent, has been used to study effect of carbon and nitrogen sources as well as the effect of immobilization on H-acid degradation efficiency of culture 13-15 . This study presents role of protein and plasmid in H-acid degradation by A. latus. Experimental Section Chemicals and Media H-acid and other chemicals were obtained from Hi- media Pvt Ltd, Mumbai. Mineral salts medium 16 (Na 2 HPO 4 . 2H 2 O, 12 g; KH 2 PO 4 , 2 g; NH 4 NO 3 , 0.5 g; MgCl 2 . 6H 2 O, 0.1 g; Ca (NO 3 ) 2 . 4H 2 O, 50 mg; FeCl 2 . 4H 2 O 7.5 mg; and a trace elements solution, 0.1 ml), containing 100 ppm each of H-acid, glucose and yeast extract, was used for the growth of bacteria. A. latus culture was maintained on a solid medium prepared by adding 1.5% agar to medium with H-acid. Degradation of H-acid by A. latus under Optimized Conditions H-acid degradation by A. latus under standardized optimum conditions was carried out in 100 ml of mineral salts medium (pH 7) containing H-acid, yeast extract and dextrose with 10 ml of A. latus inoculum incubated at 37°C for 24 h at 150 rpm. Decrease in H-acid concentration was measured by OD at 390 nm. Growth was monitored by measuring OD at 660 nm. COD (Chemical Oxygen Demand) Estimation Estimation of COD after 24 h of incubation was carried out according to standard methods 17 . Reduction in COD was calculated as Reduction% in COD = [(COD 0 -COD t )/COD 0 ]×100 where COD 0 = initial COD and COD t = COD at time t.