Polyphasic bacterial community analysis of an aerobic activated sludge removing phenols and thiocyanate from coke plant effluent Tamás Felföldi a, * , Anna J. Székely a , Róbert Gorál a , Katalin Barkács b , Gergely Scheirich a , Judit András a , Anikó Rácz a , Károly Márialigeti a a Department of Microbiology, Eötvös Loránd University, Pázmány Péter stny. 1/c., H-1117 Budapest, Hungary b Department of Analytical Chemistry, Eötvös Loránd University, Pázmány Péter stny. 1/a., H-1117 Budapest, Hungary article info Article history: Received 23 July 2009 Received in revised form 16 October 2009 Accepted 14 December 2009 Available online 22 January 2010 Keywords: Coke wastewater Activated sludge Thiocyanate Phenols Microbial community structure abstract Biological purification processes are effective tools in the treatment of hazardous wastes such as toxic compounds produced in coal coking. In this study, the microbial community of a lab-scale activated sludge system treating coking effluent was assessed by cultivation-based (strain isolation and identifica- tion, biodegradation tests) and culture-independent techniques (sequence-aided T-RFLP, taxon-specific PCR). The results of the applied polyphasic approach showed a simple microbial community dominated by easily culturable heterotrophic bacteria. Comamonas badia was identified as the key microbe of the system, since it was the predominant member of the bacterial community, and its phenol degradation capacity was also proved. Metabolism of phenol, even at elevated concentrations (up to 1500 mg/L), was also presented for many other dominant (Pseudomonas, Rhodanobacter, Oligella) and minor (Alcalig- enes, Castellaniella, Microbacterium) groups, while some activated sludge bacteria (Sphingomonas, Rhodo- pseudomonas) did not tolerate it even in lower concentrations (250 mg/L). In some cases, closely related strains showed different tolerance and degradation properties. Members of the genus Thiobacillus were detected in the activated sludge, and were supposedly responsible for the intensive thiocyanate biodeg- radation observed in the system. Additionally, some identified bacteria (e.g. C. badia and the Ottowia- related strains) might also have had a significant impact on the structure of the activated sludge due to their floc-forming abilities. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Coke, produced by the pyrolysis of natural coals, is an indis- pensable material for most of the metallurgical facilities. During coking, coal decomposes into gases, liquid and solid organic com- pounds (Nowak et al., 2004). Coke wastewater is composed of large quantities of water used for the quenching of hot coke and for washing the produced gas. This liquid effluent contains high con- centration of ammonia, phenols, thiocyanate, cyanide and lower amounts of other toxic compounds, such as polyaromatic hydro- carbons (PAHs), e.g. naphthalene, and heterocyclic nitrogenous compounds, e.g. quinoline (Ghose, 2002; Toh and Ashbolt, 2002; Li et al., 2003; Vázquez et al., 2006a). The individual concentration of the contaminants depends on the quality of coal and the proper- ties of the coking process (Nowak et al., 2004). Coke wastewater handling usually consists of a series of phys- ico-chemical treatments reducing the concentration of ammonia, cyanide, solids and other substances (Ghose, 2002), followed by different biological treatments, mainly activated sludge process. The application of two or three consecutive activated sludge sys- tems is particularly favored as readily biodegradable substrates like phenol can be removed in the first step (Vázquez et al., 2006b). Phenols, which contribute to the greatest extent to the to- tal COD in coke wastewater, are not only highly toxic and carcino- genic compounds, but also inhibit advantageous biological processes like nitrification (Kim et al., 2008). Under optimal cir- cumstances, thiocyanate degradation can also be achieved in the first activated sludge step (Vázquez et al., 2006b). Only few studies have analyzed the microbiology of activated sludge adapted to treat the highly polluted coke wastewater. Whiteley et al. (2001) cultured and examined the Pseudomonad species, while others (Whiteley and Bailey, 2000; Manefield et al., 2005; Suenaga et al., 2007) used culture-independent meth- ods, which are considered to give a more realistic view of the microbial community composition (Hugenholtz et al., 1998). How- ever to assess the degrading potentials and physiological proper- ties of the most important members of these communities, a detailed polyphasic study, linking the results of culture-indepen- dent methods with isolated strains, is needed. This way, coke 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.12.053 * Corresponding author. Tel.: +36 13812177; fax: +36 13812178. E-mail address: tamas.felfoldi@gmail.com (T. Felföldi). Bioresource Technology 101 (2010) 3406–3414 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech