Involvement of Dehalobacter strains in the anaerobic dechlorination of 2,4,6-trichlorophenol Zhiling Li, 1 Daisuke Suzuki, 1 Chunfang Zhang, 1 Naoko Yoshida, 2 Suyin Yang, 1 and Arata Katayama 1 , 3, * EcoTopia Science Institute, Nagoya University, Chikusa, Nagoya 464-8603, Japan, 1 Electronics-Inspired Interdisciplinary, Research Institute, Toyohashi University of Technology, Toyohashi 441-8580, Japan, 2 and Department of Civil Engineering, Graduate School of Engineering, Nagoya University, Chikusa, Nagoya 464-8603, Japan 3 Received 6 February 2013; accepted 7 May 2013 Available online 15 June 2013 A culture reductively dechlorinating 2,4,6-trichlorophenol (TCP) was enriched from a sediment contaminated with chlorinated aliphatic hydrocarbons. The culture dechlorinated 100 mM of 2,4,6-TCP to 4-chlorophenol within 15 days utilizing H 2 and a yeast extract as an electron donor and carbon source, respectively. Besides 2,4,6-TCP, the culture could also dehalogenate chlorophenols at ortho position and 2,4,6-tribromophenol at ortho and para positions, as well as chlorinated ethenes and ethanes. A 16S rRNA gene clone library analysis showed that the bacterial community was composed of members of the classes Clostridia, Bacteroidia, Spirochetes, and Epsilonproteobacteria. The phylogenetic and physiological characterization of the culture confirmed two novel Dehalobacter strains, TCP-5 and TCP-6, that were involved in the reductive dehalogenation of 2,4,6-TCP and other halogenated compounds. The study was significant as the first report to demonstrate the involvement of Dehalobacter in the reductive dehalogenation of both halogenated aromatic and aliphatic compounds. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: 2,4,6-Trichlorophenol; Halogenated phenols; Chlorinated aliphatic compounds; Wide dehalogenation activity; Organohalide- respiration; Dehalobacter strains] 2,4,6-Trichlorophenol (TCP) has been used extensively since the 1920s as a component of various pesticide formulations and as a wood preservative. It is, therefore, ubiquitously present in the environment (1). It is also formed during the disinfection of drinking water sources and during wastewater treatment, and is frequently detected in sewage sludge (2). Because of concern regarding the high toxicity, persistency, and carcinogenic proper- ties, 2,4,6-trichlorophenol (TCP) is listed as a priority pollutant by the US EPA, together with some other chlorophenol congeners (3). The important process in reducing the toxicity of the halogenated compounds is dehalogenation. The reductive organohalide-respiring process, which uses the chlorinated compounds as terminal electron acceptors via microbial respiration, was proven of paramount importance in the management of soil and groundwater sites (4). Previously, several studies had confirmed the reductive dechlorina- tion of 2,4,6-TCP in both enriched mixed culture and in anaerobic bioreactors (5e8). Meanwhile, organohalide-respiring bacteria that reductively dechlorinate various chlorinated aromatic compounds have been isolated and characterized. They belong to three phyla: Desulfito- bacterium spp. (9,10) in the phylum Firmicutes; Desulfomonile spp. (11,12), Anaeromyxobacter dehalogenans (13), Desulfovibrio dechlor- acetivorans (14), in the phylum Proteobacteria; and Dehalococcoides spp. in the phylum Chloroflexi (15). Among the species isolated, several Desulfitobacterium strains have been reported to possess dechlorinating activity for 2,4,6-TCP, including Desulfitobacterium chlororespirans strain Co23 (16), Desulfitobacterium dehalogenans JW/IU-DC1 (17), Desulfitobacterium hafniense strains DCB-2 (9), TCP-A (10), and PCP-1 (18), and Desulfitobacterium sp. PCE1 (19). Besides Desulfitobacterium, Dehalococcoides was also reported to utilize 2,4,6-TCP for growth (20). Other than that, there is no report of any other genus that reductively dechlorinates 2,4,6-TCP. Dehalobacter spp. have been widely regarded as obligate orga- nohalide-respiring species for the reductive dechlorination of chlorinated aliphatic hydrocarbons, including chloroethenes (21), chloroethanes (22), and chloromethane (23), whereas recent studies found Dehalobacter spp. also possessed the ability to dechlorinate chlorinated aromatic compounds (24,25). These studies suggested Dehalobacter spp. as a candidate for reductive dehalogenation of both halogenated aromatic and aliphatic compounds. In this study, we enriched a 2,4,6-TCP-dechlorinating culture where novel Dehalobacter strains were involved in the dechlori- nation process. Effects of putative electron donors and alternative electron acceptors for the dechlorination process, inhibitory treat- ment, and the dehalogenation spectrum were determined in the enriched culture. Analyses of a 16S rRNA gene clone library, dena- turing gradient gel electrophoresis (DGGE), and quantitative real- time PCR (qPCR) were conducted to determine the culture composition and to confirm the role of Dehalobacter strains. * Corresponding author at: EcoTopia Science Institute, Nagoya University, Chikusa, Nagoya 464-8603 Japan. Tel.: þ81 52 789 5856; fax: þ81 52 789 5857. E-mail address: a-katayama@esi.nagoya-u.ac.jp (A. Katayama). www.elsevier.com/locate/jbiosc Journal of Bioscience and Bioengineering VOL. 116 No. 5, 602e609, 2013 1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.05.009