Variovorax defluvii sp. nov., isolated from sewage Long Jin, 1 Kwang Kyu Kim, 2 Chi-Yong Ahn 1 and Hee-Mock Oh 1 Correspondence Chi-Yong Ahn cyahn@kribb.re.kr Hee-Mock Oh heemock@kribb.re.kr 1 Environmental Biotechnology Research Centre, Korea Research Institute of Bioscience & Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea 2 Korean Collection for Type Cultures, Biological Resource Centre, Korea Research Institute of Bioscience & Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea A polyphasic taxonomic study was carried out on 2C1-b T and 2C-21, two strains isolated from sewage flowing into River Geumho in Korea. Cells of the two strains were Gram-negative, non- spore-forming, motile and oval or rod-shaped. Comparative 16S rRNA gene sequence studies showed a clear affiliation of these two isolates with members of the Betaproteobacteria; they were most closely related to Variovorax boronicumulans KCTC 22010 T , Variovorax dokdonensis KCTC 12544 T , Variovorax ginsengisoli KCTC 12583 T , Variovorax paradoxus ATCC 17713 T and Variovorax soli KACC 11579 T showing 16S rRNA gene sequence similarities of 97.4–98.8 % with these strains and shared 100 % similarity with each other. The genomic DNA G+C contents of strains 2C1-b T and 2C1-21 were 65.5 and 65.2 mol%, respectively. Phenotypic and chemotaxonomic data [Q-8 as the major ubiquinone; C 16 : 0 , summed feature 4 (C 16 : 1 v7c and/or iso-C 15 : 0 2-OH), C 17 : 0 cyclo and summed feature 7 (C 18 : 1 v7c and/or v9t and/or v12t) as major fatty acids] supported the affiliation of strains 2C1-b T and 2C-21 to the genus Variovorax. Based on evidence derived from this polyphasic analysis, it is proposed that strains 2C1-b T and 2C1-21 represent a novel species for which the name Variovorax defluvii sp. nov. is proposed; the type strain is 2C1-b T (5KCTC 12768 T 5JCM 17804 T ). Chlorophenols belong to a group of toxic xenobiotics that are resistant to microbial attack and are widely used in industry as wood preserves, resulting in accumulation of these compounds in soils. Among the proteobacteria, members of the genera Alcaligenes, Arthrobacter, Azoto- bacter, Burkholderia, Pseudomonas, Ralstonia and Rhodo- coccus have been reported to degrade chlorophenols (Apajalahti & Salkinoja-Salonen, 1987; Ha ¨ggblom, 1990, 1992; Li et al., 1991; Martin-Le Garrec et al., 2001). The genus Variovorax within the family Comamonadaceae was proposed by reclassification of Alcaligenes paradoxus as Variovorax paradoxus, the type species of the genus (Willems et al., 1991). At the time of writing, the genus comprised five recognized species, Variovorax paradoxus (Willems et al., 1991), V. dokdonensis (Yoon et al., 2006), V. soli (Kim et al., 2006), V. boronicumulans (Miwa et al., 2008) and V. ginsengisoli (Im et al., 2010). Two strains, 2C1-b T and 2C1-21, were isolated from sewage flowing into River Geumho in South Korea, during screening for 2-chlorophenol-degrading bacteria. Sewage samples were initially stimulated with 100 p.p.m. 2-chlo- rophenol; the stimulated cultures were serially diluted in 0.85 % saline solution. Aliquots of each serial dilution were spread on R2A agar (Difco) and incubated at 25 u C for 7 days. Two yellow colonies, designated 2C1-b T and 2C1-21, were isolated. Both of the isolates were subculti- vated on R2A agar at 30 u C for 48 h. Based on phylo- genetic, phenotypic and chemotaxonomic analysis, the two strains should be placed in the genus Variovorax. The strains were further investigated using a polyphasic approach. For most experiments, all strains were cultivated on R2A agar or broth (Difco) at 30 u C for 48 h. V. boronicumulans KCTC 22010 T , V. dokdonensis KCTC 12544 T , V. ginsengisoli KCTC 12583 T , V. paradoxus ATCC 17713 T and V. soli KACC 11579 T were used as reference strains under the same conditions. The Gram reaction was performed with 2 % (w/v) crystal violet, iodine fixation and decolorization. Decolorization was accomplished using 95 % ethanol followed by a distilled water wash and counterstaining with safranin (Gerhardt et al., 1994). Cell morphology and motility were observed under a phase-contrast microscope (Nikon Optiphot; 10006 magnification) with cells grown on R2A agar for 1–3 days. Oxidase activity was tested using 1 % tetramethyl-p-pheny- lenediamine (Tarrand & Gro ¨schel, 1982) and catalase activity was tested using 3 % H 2 O 2 . Growth was investigated on R2A agar at different temperatures (4, 8, 10, 15, 20, 25, 30, 32, 37, 42 and 45 u C), NaCl concentrations (1, 2, 3 and 5 %) and The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains 2C1-b T and 2C1-21 are HQ385753 and HQ385754, respectively. A supplementary table is available with the online version of this paper. International Journal of Systematic and Evolutionary Microbiology (2012), 62, 1779–1783 DOI 10.1099/ijs.0.035295-0 035295 G 2012 IUMS Printed in Great Britain 1779