Microbial diversity in the anaerobic tank of a full-scale produced water treatment plant Ruyin Liu a , Dong Li a , Yingxin Gao a , Yu Zhang a , Song Wu b , Ran Ding a , Abd El-Latif Hesham a,c , Min Yang a, * a State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, Beijing 100085, China b Jidong Oilfield, PetroChina, China c Genetics Department, Faculty of Agriculture, Assiut University, Assiut, Egypt 1. Introduction Produced water, characterized by containing petroleum hydro- carbons, is concurrently recovered during crude oil and natural gas exploration, as well as production in oilfields. Biological processes have been used for the removal of contaminants from produced water [1–3]. In the Jidong Oilfield of China, a full-scale biological system, which consists of successive anaerobic and aerobic biofilm reactors, has been utilized to treat produced water. The anaerobic treatment tank was responsible for the removal of approximately 45% of the polycyclic aromatic hydrocarbons (PAHs) [4] and 25% of the total petroleum hydrocarbons (TPH) (this study) from the influent produced water. Although it has been assumed that the biomass in the anaerobic tank plays a key role in the removal of petroleum hydrocarbons, little is known about the microbial populations in the biofilm. Microbes that utilize petroleum hydrocarbons under aerobic conditions are well known. In the late 1980s, bacteria were isolated and shown to degrade hydrocarbons under anaerobic conditions. Hydrocarbons, including benzene, toluene, ethylbenzene, xylenes, alkanes, and PAHs, could be degraded by coupling them to sulfate reduction, nitrate reduction, or methanogenesis [5–7]. Currently, many anaerobic hydrocarbon-degrading bacteria, which are mainly sulfate-reducing (SRB) and denitrifying bacteria, have been isolated, and the steps in anaerobic metabolism that mediate hydrocarbon degradation have been elucidated in many representative micro- organisms. Meanwhile, many library experiments have demon- strated that methanogenic archaea are involved in the anaerobic mineralization of petroleum hydrocarbons. Anderson and Lovley showed that rapid alkane decay via methanogenesis occurred in oil- bearing sediments [8], suggesting an important role for methano- genic hydrocarbon degrading organisms in petroleum-rich anaero- bic environments. Other investigations have proved the existence of indigenous archaea and bacteria in reservoirs [9], and metabolites indicative of anaerobic hydrocarbon degradation have been isolated from many oil samples [10], suggesting that hydrocarbon-degrading microbial communities are present in oil reservoirs. Thus, whether the microbes from the oil reservoir play a role in the process of anaerobic hydrocarbon degradation in the anaerobic treatment tank in the produced water treatment system is an important question. Process Biochemistry 45 (2010) 744–751 ARTICLE INFO Article history: Received 15 October 2009 Received in revised form 10 December 2009 Accepted 16 January 2010 Keywords: Produced water Anaerobic tank Biofilm Archaea Bacteria Community ABSTRACT Microbial characteristics in the anaerobic tank of a full-scale produced water treatment plant capable of anaerobic hydrocarbon removal were analyzed and compared to those in the influent produced water using cultivation-independent molecular methods. Clones related to methanogens including the methylotrophic Methanomethylovorans thermophila and hydrogen- and the formate-utilizing Methano- linea tarda were in abundance in both samples, but greater numbers of M. tarda-like clones were detected in the biofilm library. Both DGGE and cloning analysis results indicated that the archaea in the biofilm were derived from the influent produced water. Bacterial communities in the influent and biofilm samples were significantly different. Epsilonproteobacteria was the dominant bacterial group in the influent while Nitrospira and Deltaproteobacteria were the predominant groups in the biofilm. Many clones related to syntrophic bacteria were found among the Deltaproteobacteria. One Deltaproteobacteria clone was related to Syntrophus, which is commonly found in methanogenic hydrocarbon-degrading consortia. A number of Deltaproteobacteria clones were assigned to the clone cluster group TA, members of which predominate in various methanogenic consortia that degrade aromatic compounds. These results suggest that a microbial community associated with methanogenic hydrocarbon degradation may have been established in the biofilm. ß 2010 Elsevier Ltd. All rights reserved. * Corresponding author. Tel.: +86 10 62923475; fax: +86 10 62923541. E-mail address: yangmin@rcees.ac.cn (M. Yang). Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio 1359-5113/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.01.010