Nitrate removal from the effluent of a fertilizer industry using a bioreactor packed with immobilized cells of Pseudomonas stutzeri and Comamonas testosteroni Swati L. Zala 1 , Jayalexmy Ayyer 2 and Anjana J. Desai 1, 1 Department of Microbiology and Biotechnology Centre, Faculty of Science, M.S. University of Baroda, 390 002 Gujarat, India 2 Gujarat Narmada Valley Fertilizer Company Ltd., Narmadanagar, Bharuch, Gujarat, India *Author for correspondence: Tel.: þ91-265-2794396, Fax: þ91-265-2792508, E-mail: desai_aj@yahoo.com Received 6 August 2003; accepted 27 January 2004 Keywords: Bioreactor studies, Comamonas testosteroni, denitrification rate, immobilization, Pseudomonas stutzeri Summary A bioreactor for the removal of nitrate nitrogen (NO 3 -N) from industrial effluent is described which is comprised of a glass column (60 cm · 6 cm) packed with alginate beads containing denitrifying organisms Pseudomonas stutzeri and Comamonas testosteroni. The effluent containing high concentrations of nitrate (600–950 mg l )1 ) from the fertilizer industry and fusel oil (methanol as a major component) as organic carbon were used in the process. The reactor is operated in the continuous mode by injecting the pretreated nitrate-containing effluent at the top of the column. The Hydraulic retention time (HRT) was adjusted by changing the flow rates. When nitrate-containing wastewater was treated with immobilized cells, the nitrate removal rate reached a maximum 1.66 ± 0.07 Kg NO 3 -N m )3 d )1 at an influent NO 3 -N concentration of 850 mg NO 3 -N l )1 within 12 h. The denitrification activity of the immobilized cells was compared with that of the free cells. Introduction Elevated levels of nitrate in drinking water are a hazard to humans and animals. The permissible limit of nitrate in potable water is not more than 10 mg l )1 NO 3 -N. Microbiological contribution offers a radical solution, since bacteria can effectively reduce nitrate to molecular nitrogen (N 2 ) (Tiedje 1994). Up to 95–100% nitrate removal was achieved by using a consortium of Pseu- domonas stutzeri and Comamonas testosteroni by the suspended growth technique (Zala et al. 1999). Immo- bilized bacterial bioreactors have a higher productivity per unit biomass and are ideal for small manufacturers and commercial laboratories, which lack space for a conventional free cell treatment plant (Chibata et al. 1983). A number of studies (Santos et al. 1993; Uemoto & Saiki 1996, 1999; Kesseri et al. 2002) have shown an effective denitrification with attached and immobilized cells on a different supporting matrix. Uemoto & Saiki (2000) had achieved nitrogen removal from wastewater by use of a tubular gel containing Nitrosomonas euro- paea and Paracoccus denitrificans. Gas-permeable sili- cone tubing was used as the basis of a bioreactor for autotrophic denitrification (Ho et al. 2001). A hollow fibre membrane bioreactor (HFMB) was operated for hydrogenotrophic denitrification (Sarina & Andreas 2001). The matrix used in an immobilized bioreactor should be able to take sufficient organic load with effective mass transfer, which increases the efficiency of the process. Here we report nitrate removal from an industrial effluent using a consortium of bacteria immobilized on to alginate beads. Immobilized cells are convenient to handle, appear to be less susceptible to microbial contamination and permit easy separation of products from the biocatalyst. The denitrification efficiency of an immobilized bacterial consortium is compared here with the free cell culture. Materials and methods Cell immobilization procedure The bacterial isolates used for this experiment, P. stutzeri and C. testosteroni, were isolated from the denitrifying reactor of a fertilizer company (Zala et al. 1999). Cell cultures were separately grown aerobically in peptone nitrate broth; peptone, 5 g l )1 ; beef extract, 3gl )1 ; potassium nitrate, 1 g l )1 , incubated on a shaker for 24 h at 37 °C ± 2. Following growth, cells were collected by centrifugation, washed twice with saline and a 10% (v/v) cell suspension (0.55 g l )1 dry weight of P. stutzeri and 0.34 g l )1 dry weight of C. testosteroni) added into a solution of 3% (w/v) sodium alginate. The mixture was extruded drop wisevia syringe into 0.1 M World Journal of Microbiology & Biotechnology 20: 661–665, 2004. 661 Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands.