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All rights reserved Biofuels and Environmental Biotechnology Biotechnology and Bioengineering DOI 10.1002/bit.26212 Anaerobic Reduction of 2,6-dinitrotoluene by Shewanella oneidensis MR-1: Roles of Mtr Respiratory Pathway and NfnB# Dong-Feng Liu1, ? , Di Min1, ? , Lei Cheng1 , Feng Zhang1 , Dao-Bo Li1 , Xiang Xiao2,* , Guo-Ping Sheng1 , Han-Qing Yu1 ,*

1 CAS Key Laboratory of Urban Pollutant Conversion, University of Science and Technology of China, Hefei, 230026, China

2 School of Environment and Safety Engineering, Jiangsu University, Zhenjiang, 212013, China ? These authors contributed equally. *Corresponding authors: Dr. Xiang Xiao, Fax: +86

551 63601592;

E-mail: [email protected] Prof. Han-Qing Yu, Fax: +86

551 63601592;

E-mail: [email protected] # This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/bit.26212] Additional Supporting Information may be found in the online version of this article. This article is protected by copyright. All rights reserved Received August 4, 2016;

Revision Received October 22, 2016;

Accepted October 31,

2016 This article is protected by copyright. All rights reserved ABSTRACT Dinitroluene (DNT) is a widely present pollutant in aquatic environments, and its biodegradation is an economically attractive way to effectively removal. In aquatic environments, the presence of electrochemically active bacteria (EAB) could contribute to the anaerobic bioreduction of DNT. However, the mechanism behind such a biodegradation process at gene level remains to be further elucidated. In this work, the anaerobic reduction of 2,6-dinitrotoluene (2,6-DNT) by Shewanella oneidensis MR-1, a typical EAB in aquatic environments, was investigated. S. oneidensis MR-1 was found to be able to obtain energy for growth through the anaerobic respiration on 2,6-DNT. Experimental results show that the Mtr respiratory pathway, a transmembrane electron transport chain, was involved in the 2,6-DNT bioreduction. Knockout of cymA or nfnB resulted in a substantial loss of its 2,6-DNT-reducing ability, indicating that both CymA and NfnB were the key proteins in the microbial electron transfer chain. The genetic analysis further confirms that the Mtr respiratory pathway and NfnB are mainly responsible for the anaerobic reduction of 2,6-DNT by S. oneidensis MR-1. This work is useful to better understand the anaerobic bioreduction of nitroaromatic compounds in aquatic environments and remediate the environments contaminated by nitroaromatic compounds. This article is protected by copyright. All rights reserved KEYWORDS: 2,6-dinitrotoluene;

anaerobic reduction;

Shewanella oneidensis MR-1;

Mtr respiratory pathway;

NfnB This article is protected by copyright. All rights reserved Introduction Dinitrotoluenes (DNTs) are synthesized by successive nitration of toluene and used as explosives and raw materials for manufacture of diisocyanate and dyestuffs. As a main isomer of DNT, the production and usage practices, discharges, and accidental spills of 2,6-dinitroluene (2,6-DNT) have resulted in contamination of water and groundwater (Hashimoto et al., 1982). 2,6-DNT exhibits acute toxicity and low level carcinogenicity (Rickert et al., 1984). Thus, effective degradation of 2,6-DNT is essential. Various physicochemical methods have been used to degrade 2,6-DNT. However, the high costs of the above methods limit their applications. Comparatively, microbial degradation of 2,6-DNT offers a more cost-effective way to remediate DNT-contaminated environments. Many microbial strains, such as Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii strain JS863, can grow on 2,6-DNT as their sole carbon and nitrogen source (Nishino et al., 2000). Some mixed enrichment cultures in freshwater and biofilm reactors have also been reported to biodegrade 2,6-DNT (Lendenmann et al., 1998;