Treatment of E. coli HB101 and the tetM gene by Fenton’s reagent and ozone in cow manure Murat Cengiz a , Merih Otker Uslu b , Isil Balcioglu b, * a Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Uludag University,16059, Bursa, Turkey b Institute of Environmental Sciences, Bogazici University, Bebek, 34342, Istanbul, Turkey article info Article history: Received 25 May 2009 Received in revised form 14 June 2010 Accepted 7 July 2010 Available online 31 July 2010 Keywords: Oxidation E. coli tetM Manure abstract The destruction of antibiotic-resistant microorganisms at the source of contamination is necessary due to their adverse effects and to their increasingly widespread occurrence in the environment. To address this problem, Fenton and ozone oxidation processes were applied to synthetically contaminated cow manure to remove the tetM gene and its host, Escherichia coli HB101. The efficiency of the processes was eval- uated by enumeration of E. coli HB101 and by PCR amplification of the tetM gene. The results of this study show that 56.60% bacterial inactivation (corresponding to a 0.36 log reduction) was achieved by a Fenton reagent dose of 50 mM H 2 O 2 and 5 mM Fe 2þ without acidifying the manure. Despite the high organic content of cow manure, 98.50% bacterial inactivation (corresponding to a 1.83 log reduction) was obtained by the ozonation process with an applied dose of 3.125 mg ozone/g manure slurry. The PCR study revealed that the band intensity of the tetM gene gradually decreased by increasing the Fenton reagent and the applied ozone dose. However, significantly high doses of oxidants would be required to completely eliminate bacterial pollution in manure. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction According to recent investigations (Kummerer, 2004; Schmitt et al., 2006), the use of manure as fertilizer and other manure disposal practices can lead to a high prevalence of antibiotic resistance genes in soil. It has been clearly shown that the number of antibiotic resistance genes increases in soil after application of manure sourced from a farm operation with low or regular use of antibiotics (Schmitt et al., 2006; Cengiz et al., 2010). In addition to already-resistant bacteria, manure can also contain antibiotics that lead to the devel- opment of resistance in the environment, and indigenous soil bacteria can create an undesirable reservoir for the spread of antibiotic resis- tance (Kummerer, 2004). Furthermore, a recent study has shown that bacteria from animal manure can transfer the tetM gene to natural soil bacteria (Haack and Andrews, 2000). Consequently, resistance genes are found in the environment as emergent contaminants along with various antibiotics (Chee-Sanford et al., 2001; Agerso et al., 2002). Among the various resistance genes, the diversity and occurrence frequency of tetracycline and sulfonamide resistance genes are considerably higher than others (Aarestrup et al., 2000; Aminov et al., 2001; Chopra and Roberts, 2001; Chee-Sanford et al., 2001; Agerso et al., 2002; Schmitt et al., 2006; Stine et al., 2007). Considering the role of manure in the spreading of resistance genes into the environment, there is an obvious need for a suitable treatment process to completely remove resistance genes and hosts of resistance genes from animal waste. Although various studies have been carried out on the removal of antibiotics from water and manure (Balcioglu and Otker, 2003; Ikehata et al., 2006; Uslu and Balcioglu, 2008, 2009a, b; Yalap and Balcioglu, 2009), no specific attempt has been made to destroy of resistance genes and bacteria in animal manure. On the other hand, chemical oxidation processes, including advanced oxidation processes (AOPs) wherein ozone, oxygen, and UV produce highly reactive oxidizing free radicals by combination with hydrogen peroxide, have been applied to water and wastewater to destroy various pollutants. Among the AOPs, Fenton and ozone oxidation are promising processes for the destruction of various microorganisms (Macauley et al., 2006; Petala et al., 2006). In the Fenton oxidation process, hydrogen peroxide is catalytically decomposed into reactive species mainly by hydroxyl radicals, which are able to oxidize a large number of cellular constituents (Ananthaswamy and Eisenstark, 1977; Wolff et al., 1986). Ozone treatment has been shown to be effective against both gram-positive and gram-negative bacteria and to produce sensorial and microbial effects in fish bacteria (da Silva et al., 1998). Moreover, its destructive effects on cell membranes and cytoplasmic components have been widely used for the inac- tivation of bacteria and fungi (Hinze et al., 1987; Pryor et al., 1991). Toxicity mechanisms of reactive species produced in Fenton and * Corresponding author. Tel.: þ90 212 359 70 35. E-mail address: balciogl@boun.edu.tr (I. Balcioglu). Contents lists available at ScienceDirect Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman 0301-4797/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jenvman.2010.07.005 Journal of Environmental Management 91 (2010) 2590e2593