Biodegradation potential of pure and mixed bacterial cultures for removal of 4-nitroaniline from textile dye wastewater Azeem Khalid a , Muhammad Arshad b , David E. Crowley c, * a Department of Environmental Sciences, PMAS Arid Agriculture University, Rawalpindi 46300, Pakistan b Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad 38040, Pakistan c Department of Environmental Sciences, University of California, Riverside, CA 92521, USA article info Article history: Received 24 July 2008 Received in revised form 20 November 2008 Accepted 26 November 2008 Published online 11 December 2008 Keywords: Azo dyes Activated sludge Biodegradation Nitroaniline Nitrobenzene Wastewater abstract Environmentally toxic aromatic amines including nitroanilines are commonly generated in dye contaminated wastewater in which azo dyes undergo degradation under anaerobic conditions. The aim of this study was to develop a process for biological treatment of 4-nitroaniline. Three bacteria identified as Acinetobacter sp., Citrobacter freundii and Klebsiella oxytoca were isolated from enrichment cultures of activated sludge on 4-nitroaniline, after which the isolates and the mixed culture were studied to determine optimal conditions for biodegradation. HPLC analyses showed the mixed culture was capable of complete removal of 100 mmol/L of 4-nitroaniline within 72 h under aerobic conditions. There was an inverse linear relationship (R 2 ¼ 0.96) between the rate of degradation (V) and 4-nitraoaniline concentrations [S] over 100–1000 mmol/L. The bacterial culture was also capable of decol- orizing structurally different azo dyes (Acid Red-88, Reactive Black-5, Direct Red-81, and Disperse Orange-3) and also degraded nitrobenzene. Our findings show that enrichment cultures from activated sludge can be effective for the removal of dyes and their toxic intermediates, and that treatment may best be accomplished using an anaerobic–aerobic process. ª 2008 Elsevier Ltd. All rights reserved. 1. Introduction Nitroanilines are aromatic amines that are commonly gener- ated during the biodegradation of azo dyes under anaerobic conditions, where they are formed from the reductive cleavage of azo bonds (–N]N–) by microorganisms in waste- water streams (Dubrow et al., 1996; Pinheiro et al., 2004; van- der Zee and Villaverde, 2005). Depending on the individual compounds, many aromatic amine metabolites are consid- ered to be non-biodegradable or only very slowly degradable (Saupe, 1999) and have varying toxicities to aquatic life and higher organisms (Cartwright, 1983; Miller and Miller, 1983; Weisburger, 2002; Pinheiro et al., 2004; Ozturk and Abdullah, 2006). In studies aimed at improving biological treatment methods to remove these substances from azo dye waste- water, an activated sludge process is commonly used to treat the contaminants (Kudlich et al., 1999; Ekici et al., 2001; Worch et al., 2002). However, the microbial species that are involved are poorly characterized and the development of a degrader community depends on its adaptation conditions, and the availability of oxygen (Pinheiro et al., 2004). For example, Razo-Flores et al. (1997) investigated the biodegradability potential of aniline, aminophenols, aminobenzoates and 5- aminosalicylate by methanogenic sludge and found that all compounds, except aniline, were at least partially bio- degraded, but that the lag-phases to generate a degrader * Corresponding author. Tel.: þ1 951 827 3785; fax: þ1 951 827 3993. E-mail address: crowley@ucr.edu (D.E. Crowley). Available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/watres 0043-1354/$ – see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2008.11.045 water research 43 (2009) 1110–1116