Stability of a mixed microbial population in a biological reactor during long term atrazine degradation under carbon limiting conditions Chaitanyakumar Desitti * , Michael Beliavski, Sheldon Tarre, Michal Green Faculty of Civil and Environmental Engineering, Technion- Israel Institute of Technology, Haifa 32000, Israel article info Article history: Received 25 June 2017 Received in revised form 16 July 2017 Accepted 16 July 2017 Available online xxx Keywords: Atrazine Biological reactor Carbon limitation Archaea Nitrification abstract The stability of a mixed bacterial population in a fixed bed reactor for atrazine biodegradation under carbon limiting conditions was examined. A one litre reactor was filled with volcanic scoria and operated as a sequencing batch reactor (SBR) for 66 batches over a period of 230 days. The reactor was inoculated with a mixed atrazine degrading bacterial population and given an atrazine growth medium for start-up, followed by batches of 20 mg/L atrazine in 5 mM phosphate buffer with no additional carbon. Complete (100%) atrazine biodegradation was observed in all batches. Until the 10th batch after start-up, traces of cyanuric acid were observed and 66e70% of the theoretical nitrogen available from atrazine was recovered in the form of ammonium. From the 10th batch onwards, 100% of the theoretical nitrogen available from atrazine was recovered in the form of nitrate with no trace of cyanuric acid observed. After reactor start-up the reactor contained 98% bacteria with the relative abundance of the dominant bacteria comprised of Xanthobacter (25.6%), Variovorax (23%), Ralstonia (12.2%), and Comamonas (8.1%). At the end of reactor operation, 70.8% bacteria and 29.2% archaea were found with Xanthobacter, Ralstonia and Variovorax (1.4%) in much smaller amounts, replaced mainly by Candidatus Nitrososphaera (29.2%), Phyllobacterium (5.5%) and Nitrospira (5.4%). © 2017 Elsevier Ltd. All rights reserved. 1. Introduction Atrazine (2-chloro-4-ethylamino-4-isopropylamino-s-triazine) is a well-known and extensively used herbicide to control weeds among crops. Atrazine residues have been observed in soil and aquatic environments due to extensive use and a relatively long half-life (Thurman et al., 1992). Atrazine has been shown to cause sexual abnormalities in frogs (Hayes et al., 2003) and reduced testosterone production in rats (Trentacoste et al., 2001). The Environmental Protection Agency (USEPA) has found short-term atrazine exposure above the drinking water maximum contami- nant level (MCL) to potentially cause heart, lung, and kidney congestion, low blood pressure, muscle spasms, weight loss, and damage to the adrenal glands (Graziano et al., 2006; Bethsass et al., 2006). The current USEPA drinking water MCL for atrazine is 3 mg/L (set in 1991), while the European Union has a maximum permis- sible concentration of 0.1 mg/L for atrazine and an allowed maximum of 0.5 mg/L for the combined total concentration of atrazine and its degradation products. Physicochemical methods like chemical oxidation, coagulation/ flocculation, electrolysis, adsorption, membrane filtration, ion ex- change, volatilization and irradiation have been examined for atrazine removal. Most of these methods are quick in process, and transform atrazine into a less toxic form, but complete minerali- zation is not possible, leaving hazardous sludge, requiring safe disposal (Ghosh et al., 2001). Biological treatment methods are more reliable because they are cost effective and eco-friendly in nature. The basic assumption prior to the 1990s was that the halogen and amine substitutions on the s-triazine ring make most herbicidal s-triazines resistant to biodegradation (Cook, 1987). However, some traces of de-ethylated atrazine (DEA) and de- isopropylated (DIA) metabolites were observed in soil (Sirons et al., 1973). After 1990, the discovery of a bacterial isolate, Pseudomonas sp. strain ADP (P. ADP), changed the current understanding of atra- zine's biodegradation in the environment. Much research has been carried out on pure cultures of atrazine degraders and has provided substantial knowledge about the taxonomic diversity of atrazine- degrading microbes and individual metabolic pathways (Table 1). * Corresponding author. E-mail addresses: kumar.enviengineer@gmail.com (C. Desitti), michaelb@tx. technion.ac.il (M. Beliavski), shelly@tx.technion.ac.il (S. Tarre), agmgreen@tx. technion.ac.il (M. Green). Contents lists available at ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod http://dx.doi.org/10.1016/j.ibiod.2017.07.007 0964-8305/© 2017 Elsevier Ltd. All rights reserved. International Biodeterioration & Biodegradation xxx (2017) 1e9 Please cite this article in press as: Desitti, C., et al., Stability of a mixed microbial population in a biological reactor during long term atrazine degradation under carbon limiting conditions, International Biodeterioration & Biodegradation (2017), http://dx.doi.org/10.1016/ j.ibiod.2017.07.007