Metabolic pathway for degradation of anthracene by halophilic Martelella sp. AD-3 Changzheng Cui a , Lei Ma b , Jie Shi a , Kuangfei Lin a , Qishi Luo c , Yongdi Liu a, * a State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, China b Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China c State Environmental Protection Engineering Center for Urban Soil Contamination Control and Remediation, Institute of Wastes and Soil Environment, Shanghai Academy of Environmental Science, Shanghai 200233, China article info Article history: Received 11 October 2013 Received in revised form 17 January 2014 Accepted 21 January 2014 Available online 11 February 2014 Keywords: Anthracene Biodegradation Moderate halophilic bacteria Metabolic pathway 3-Hydroxy-2-naphthoic acid abstract Anthracene (40 mg l 1 ) was completely depleted by Martelella sp. strain AD-3 under 3% salinity and a pH of 9.0 after 6 days of incubation. The metabolites were extracted and identified by high-performance liquid chromatography (HPLC) retention times, mass spectrometry, 1 H and 13 C nuclear magnetic reso- nance spectrometry, and comparison to authentic compounds or literature data. On the basis of the identified metabolites, enzyme activities and the utilization of probable intermediates, anthracene degradation by strain AD-3 is proposed via two distinct routes. In route I, metabolism of anthracene is initiated by the dioxygenation at C-1,2, then proceeds through 6,7-benzocoumarin, 3-hydroxy-2- naphthoic acid, salicylic acid and gentisic acid. In route II, anthracene is metabolized to 9,10- anthraquinone. The results suggest that strain AD-3 possesses efficient anthracene biodegradability in high salinity. To our knowledge, this work presents the first report of anthracene degradation by a halophilic PAH-degrading strain via two routes. The strain AD-3 may be a useful candidate for PAH- contaminated saline-alkali soil bioremediation. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction The petroleum industry generates a huge amount of oily and saline waste water (oily brines, production waters) after separation of crude oil from reservoir water, which has to be disposed. The main contaminants in this production water are aromatic and polycyclic aromatic hydrocarbons (PAHs) (Borgne et al., 2008; Zhuang et al., 2010). In addition, some marine and coastal sites are polluted by PAHs due to occasional accidents, such as oil spills during production, transportation or refining processes of the oil (Borgne et al., 2008). It is crucial to develop an efficient remedial process for both saline water and soil where natural attenuation is very slow. Anthracene (ANT), a tricyclic PAH, is found in high concentra- tions in PAH-contaminated sediments, surfaces soils, and waste sites (Wilson and Jones, 1993). It has been registered as a persistent compound that is toxic to marine life (Choi and Oris, 2003). In addition, ANT is considered a benchmark PAHs and serves as a signature compound to detect PAHs contamination, since its chemical structure is found in carcinogenic PAHs, such as benz[a] anthracene and benzo[a]pyrene. According to previous reports, biodegradation of ANT by Pseu- domonas (Evans et al., 1965; Menn et al., 1993), Mycobacterium (Moody et al., 2001; Herwijnen et al., 2003a), Rhodococcus (Tongpim and Pickard, 1996; Dean-Ross et al., 2001), Sphingomonas (Herwijnen et al., 2003b), Nocardia (Zeinali et al., 2008), and Bacillus (Ahmed et al., 2012) under non-halophilic conditions has been demonstrated to include two major types of metabolic pathways. One is via dioxgenation at the C-1 and C-2 ring positions, to form 1,2-dihydroxy-anthracene. This dihydroxylated intermediate is further metabolized by cleavage of the ring at the meta position to yield 4-(2-hydroxynaphth-3-yl)-2-oxobut-3-enoic acid. This com- pound is unstable and will spontaneously rearrange to form 6,7- Benzocoumarin, or can be further metabolized to 3-hydroxy-2- naphthoic acid. However, to our knowledge, all studies of this pathway since Evans et al. (1965), have been unable to detect 3- hydroxy-2-naphthoic acid from biodegradation of anthracene by wild-type strains. Subsequent metabolism proceeds via a pathway analogous to that of naphthalene biodegradation to yield salicylate or phthalic acid (Dean-Ross et al., 2001; Zeinali et al., 2008). The * Corresponding author. Tel./fax: þ86 21 64253988. E-mail addresses: cuichangzheng@ecust.edu.cn (C. Cui), ydliu@ecust.edu.cn (Y. Liu). Contents lists available at ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod 0964-8305/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2014.01.012 International Biodeterioration & Biodegradation 89 (2014) 67e73