Using Real-time PCR to assess changes in the crude oil degrading microbial community in contaminated seawater mesocosms Mehdi Hassanshahian a, * , Michail M. Yakimov b , Renata Denaro b , Maria Genovese b , Simone Cappello b a Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran b Istituto per l’Ambiente Marino Costiero (IAMC), CNR of Messina, Messina, Italy article info Article history: Received 4 December 2013 Received in revised form 24 April 2014 Accepted 9 June 2014 Keywords: Biodegradation Crude-oil Expression Mesocosm Quantification abstract The real-time polymerase chain reaction (RT-PCR) was used to follow changes in the proportion of hydrocarbonoclastic bacteria in the marine microbial community in oil polluted mesocosms during bioremediation field trial. Assay for alk-B1 of Alcanivorax borkumensis and alk-BT of Thalassolituus olei- vorans were validated and found to be both sensitive and reproducible. Quantification of alk-B1 from A. borkumensis SK2 in mesocosms show that in single bioaugmentation mesocosm (M1) this gene has high quantity in fifth day of sampling but in biostimulating mesocosm (M2) and consortium bio- augmentation mesocosm (M3) the high quantity of this gene was in tenth day of sampling. The com- parison between expression of alk-BT and alk-B1 in M3 mesocosm show that alk-B1 copy number was more than alk-BT. The proportion of alk-B1 or alk-BTcontaining bacteria was positively correlated to the concentration of crude oil in the mesocosms. After the concentration of crude oil in the mesocosms decreased the gene copy number of alkane monooxygenase genes also decreased. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Oil spills occur frequently and are a major cause of marine pollution. Crude oil is composed mainly of hydrocarbons, such as saturated hydrocarbons, aromatics, resins and asphaltenes (Harayama et al. 1999; Hassanshahian et al., 2013). Saturated hydrocarbons and aromatics are easily biodegradable; for efficient remediation after oil spills; we need to understand the behavior of microbial pop- ulations responsible for degrading crude oil (Hassanshahian et al., 2012a, b, 2014a). In bioremediation studies, the quantitative analysis of functional genes have provided a valuable tool for studying the relationship between specific microbial populations and the performance of the degradation processes (Ringelberg et al., 2001; Piskonen et al., 2005). In the environment, however, wide ranges of bacteria participate in the degradation of organic contaminants. Since diverse bacteria are associated with different phases of pollutant degradation (Watanabe et al., 2002; Katsivela et al., 2004) better interpretation of the microbial community dynamics occurring during the progression of decontamination is important in the development of more efficient remediation processes (Hassanshahian et al., 2010 ; Tebyanianet al., 2013). The detection of functional catabolic genes, for evaluating po- tential micro-organisms capable to biodegrade hydrocarbons, has been attempted much less often (Chandler and Brockman, 1996; Joshi and Walia, 1996). The ability to accurately quantify functional genes in the envi- ronment is an important step in understanding many ecological processes. Existing techniques include hybridization methods such as dot blots, and end-point polymerase chain reaction (PCR) methods such as competitive PCR and most probable number (MPN-PCR). Although these methods are useful, hybridization methods are often only semi-quantitative and end-point PCR methods require many reactions and a post-PCR analysis step, making them both labor- intensive and expensive. Real-time PCR is fast becoming the most popular alternative originally developed for gene expression studies; it has found many applications in microbial ecology (Heid et al., 1996; MacKay, 2004; Hassanshahian et al., 2014b). These include the measurement of 16S rRNA genes as a measure of total and specific cell populations (Smits et al., 2004), the measurement of catabolic genes such as the bssA gene, coding for benzylsuccinate synthase, the key enzyme of anaerobic toluene degradation (Beller et al., 2002) and atrazine-degrading genes (atz) (Devers et al., 2004). The advantages * Corresponding author. Tel.: þ98 9132906971; fax: þ98 3413202032. E-mail addresses: mshahi@uk.ac.ir, hasanshahi@gmail.com (M. Hassanshahian). Contents lists available at ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod http://dx.doi.org/10.1016/j.ibiod.2014.06.006 0964-8305/© 2014 Elsevier Ltd. All rights reserved. International Biodeterioration & Biodegradation 93 (2014) 241e248