Nocardioides Daeguensis Sp. Nov., a Nitrate- Reducing Bacterium Isolated from Activated Sludge of an Industrial Wastewater Treatment Plant

International Journal of Systematic and Evolutionary Microbiology (2013), 63, 3727–3732 DOI 10.1099/ijs.0.047043-0

Nocardioides daeguensis sp. nov., a nitrate- reducing bacterium isolated from activated sludge of an industrial wastewater treatment plant

Yingshun Cui,13 Sung-Geun Woo,2,33 Jangho Lee,2 Sahastranshu Sinha,3,4 Myung-Suk Kang,5 Long Jin,6 Kwang Kyu Kim,7 Joonhong Park,2 Myungjin Lee8 and Sung-Taik Lee1

Correspondence 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Sung-Taik Lee 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea [email protected] 2School of Civil and Environmental Engineering, Yonsei University, Seoul 120-749, Myungjin Lee Republic of Korea [email protected] 3Research and Development Division, H-Plus Eco Ltd, BVC #301, KRIBB, Eoeun-dong, Yuseong-gu, Daejeon 305-333, Republic of Korea 4Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, West Bengal, India 5National Institute of Biological Resources, Environmental Research Complex, Gyeongseo-dong, Seo-gu, Incheon 404-708, Republic of Korea 6Environmental Biotechnology Research Center, Korea Research Institute of Bioscience & Biotechnology, 52 Eoeun-dong, Yuseong-gu, Daejeon 305-806, Republic of Korea 7Korean Collection for Type Cultures, Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-dong, Yuseong-gu, Daejeon 305-806, Republic of Korea 8WIZCHEM Co., Ltd. Inno-Biz Park No. 403 HNU, 461-6 Jeonmin-dong, Yuseong-gu, Daejeon, 305- 811, Republic of Korea

A Gram-reaction-positive, rod-shaped, non-spore-forming bacterium (strain 2C1-5T) was isolated from activated sludge of an industrial wastewater treatment plant in Daegu, South Korea. Its taxonomic position was investigated by using a polyphasic approach. On the basis of 16S rRNA gene sequence similarity, the closest phylogenetic relatives were the type strains of Nocardioides nitrophenolicus (98.6 % similarity), N. kongjuensis (98.5 %), N. caeni (98.4 %), N. simplex (98.3 %), N. aromaticivorans (98.1 %) and N. ginsengisoli (97.5 %); the phylogenetic distance from other species with validly published names within the genus Nocardioides was T greater than 3 %. Strain 2C1-5 was characterized chemotaxonomically as having LL-2,

6-diaminopimelic acid in the cell-wall peptidoglycan, MK-8(H4) as the predominant menaquinone

and iso-C16 : 0,C16 : 0 and C17 : 1v6c as the major fatty acids. The G+C content of the genomic DNA was 74.9 mol%. These chemotaxonomic properties and phenotypic characteristics supported the affiliation of strain 2C1-5T to the genus Nocardioides. The results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain 2C1-5T from existing species with validly published names. Therefore, strain 2C1-5T represents a novel species of the genus Nocardioides, for which the name Nocardioides daeguensis sp. nov. is proposed, with the type strain 2C1-5T (5JCM 17460T5KCTC 19799T).

The genus Nocardioides was proposed by Prauser (1976) 3These authors contributed equally to this work. with Nocardioides albus as the type species. Since then, The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene species affiliated with the genus Nocardioides have been sequence of strain 2C1-5T is HQ246164. actively isolated and studied taxonomically. At the time of Two supplementary tables are available with the online version of this writing, the genus comprises 57 species with validly paper. published names, including the recently described species

047043 G 2013 IUMS Printed in Great Britain 3727 Y. Cui and others

Nocardioides caricicola (Song et al., 2011), N. hungaricus of Nocardioides nitrophenolicus (98.6 % similarity) (Yoon (To´th et al., 2011), N. ginsengisegetis (Im et al., 2011), N. et al., 1999), N. kongjuensis (98.5 %) (Yoon et al., 2006), N. maradonensis (Lee et al., 2011), N. iriomotensis (Yamamura caeni (98.4 %) (Yoon et al., 2009), N. simplex (98.3 %) et al., 2011), N. ultimimeridianus (Lee et al., 2011), N. (Yoon et al., 1997), N. aromaticivorans (98.1 %) (Kubota alpinus (Zhang et al., 2012), N. daejeonensis (Woo et al., et al., 2005) and N. ginsengisoli (97.5 %) (Cui et al., 2009). 2012) and N. ginsengagri (Lee et al., 2012). Relationships between strain 2C1-5T and other members of the genus Nocardioides are shown in the phylogenetic tree in Polychlorinated phenols have been released into the Fig. 1, which was reconstructed by the neighbour-joining environment as a result of their wide usage as biocides, and maximum-parsimony methods. Strain 2C1-5T formed a wood preservatives and organic precursors in the synthesis monophyletic group with these six species of the genus of chlorophenoxyacetate herbicides. They are frequently Nocardioides with a high bootstrap value (99 %). The isolate transformed by anaerobic micro-organisms to various was clearly differentiated from other species in the genus lower-chlorinated phenols, which can be further dechlori- Nocardioides. On the basis of phylogenetic inference, these nated to chlorophenol or phenol (Boyd & Shelton, 1984; six species were the closest neighbours of strain 2C1-5T; N. Cole et al., 1994; Mikesell & Boyd, 1986). Since aromaticivorans DSM 15131T, N. caeni KCTC 19600T, N. chlorophenol cannot be degraded easily under anaerobic ginsengisoli Gsoil 1124T, N. kongjuensis KCTC 19054T, N. conditions, chlorophenol and phenol may constitute two nitrophenolicus KCTC 0457BPT and N. simplex DSM 20130T major byproducts of anaerobic polychlorophenol degrada- were therefore obtained from culture collections, grown tion (Madsen & Aamand, 1992; Woods et al., 1989). under the same conditions and used as reference strains in Therefore, a micro-organism that can degrade both most phenotypic tests. compounds simultaneously would be very useful. Gram-reaction testing was performed by the non-staining In this study, we describe the physiological, biochemical method as described by Buck (1982). Cell morphology and and phylogenetic analysis of a Nocardioides-like strain, motility were observed with an Olympus light microscope 2C1-5T. We also report briefly on its utilization of phenolic (61000 magnification) using the hanging drop technique compounds (phenol, 2-chlorophenol, 3-chlorophenol, 4- with cells grown for 2 days at 30 uC on R2A agar and nutrient chlorophenol, N-nitrophenol and dibenzofuran) on agar agar. Catalase activity was determined by using bubble plates and in liquid medium. production in 3 % (v/v) H2O2 and oxidase activity was Strain 2C1-5T was isolated from activated sludge of an determined using 1 % (w/v) tetramethyl p-phenylenedia- industrial wastewater treatment plant in Daegu, South Korea, mine. Carbon source utilization tests, acid production tests during screening of 4-chlorophenol-degrading bacteria. A 1 % and additional physiological tests were performed using the suspension of this activated sludge was initially stimulated API20NEandAPIID32GNkits(bioMe´rieux) according to with 50 p.p.m. 4-chlorophenol for 2 weeks and was then the instructions of the manufacturer. Growth at 4, 10, 15, 20, diluted serially in 0.85 % saline solution. Aliquots of each serial 25, 30, 37, 42 and 45 uC was assessed on R2A agar after 5 days dilution were spread on 1/2 R2A agar (Difco) and incubated at of incubation. Salt tolerance was determined on R2A agar 30 uC for 2 weeks. Single colonies that appeared on the 1/2 supplemented with 1, 2, 3, 4, 5, 6, 7, 8 and 10 % (w/v) NaCl, R2A plates were then purified by transferring onto fresh 1/2 and observed after 5 days of incubation at 30 uC. Growth on R2A plates and incubating under the same conditions. The LB agar, nutrient agar, trypticase soy agar and MacConkey almost-complete sequence of the 16S rRNA gene of strain agar (all from BD) was also evaluated after 7 days of T 2C1-5 was compiled using the SeqMan software (DNASTAR). incubation at 30 uC. Growth at pH 5.0–11.0 (at intervals of 16S rRNA gene sequences of related taxa were obtained from 0.5 pH units) was tested by incubating the strain in R2A the GenBank database. Multiple alignments were performed broth adjusted to the appropriate pH for 5 days at 30 uC. by using the CLUSTAL X program (Thompson et al., 1997) and Anaerobic growth was tested in serum bottles after adding gaps were edited in the BioEdit program (Hall, 1999). thioglycolate (1 g l21) and resazurin oxygen indicator to R2A Evolutionary distances were calculated using Kimura’s two- broth and replacing the upper air layer with nitrogen gas. parameter model (Kimura, 1983). Phylogenetic trees were For measurement of the G+C content of the chromosomal reconstructed using the neighbour-joining (Saitou & Nei, DNA, genomic DNA was extracted and purified as described 1987) and maximum-parsimony (Fitch, 1971) algorithms in by Moore & Dowhan (1995), and the G+C content was the MEGA4 program (Tamura et al., 2007) and by using the determined as described by Mesbah et al. (1989) using maximum-likelihood algorithm (Felsenstein, 1981) in PHYLIP, reversed-phase HPLC. Cellular fatty acids were analysed version 3.69 (Felsenstein, 2009), based on bootstrap values from organisms grown on R2A agar for 48 h at 30 uC. The from 1000 replications (Felsenstein, 1985). cellular fatty acids were liberated, methylated and extracted The obtained 16S rRNA gene sequence of strain 2C1-5T was according to the protocol of the Sherlock Microbial a continuous stretch of 1393 bp. The isolate was identified Identification System (MIDI) version 3.0 (Sasser, 1990). using the EzTaxon-e server (http://eztaxon-e.ezbiocloud. The fatty acid methyl esters were then analysed by GC net/; Kim et al., 2012) on the basis of 16S rRNA gene (Agilent 6890; Hewlett Packard) using the Aerobe database sequence data. Sequence similarity calculations after align- (TSBA6, version 6.0) of the Sherlock Microbial Identification ment indicated that the closest relatives were the type strains software package. For chemotaxonomic analysis (isoprenoid

3728 International Journal of Systematic and Evolutionary Microbiology 63 Nocardioides daeguensis sp. nov.

67 Nocardioides kongjuensis A2-4T (DQ218275) Nocardioides nitrophenolicus NSP41T (AF005024) 0.005 50 Nocardioides daeguensis 2C1-5T (HQ246164) 99 Nocardioides caeni MN8T (FJ423551) Nocardioides aromaticivorans H-1T (AB087721)

71 Nocardioides ginsengisoli Gsoil 1124T (AB245396) Nocardioides simplex DSM 20130T (Z78212) 77 Nocardioides maradonensis RP-B30T (FM998000) 99 Nocardioides ultimimeridianus RP-B26T (FM997998) 78 Nocardioides humi DCY24T (EF623863) 50 Nocardioides panacisoli Gsoil 346T (FJ666101) Nocardioides tritolerans MSL-14T (EF466107) Nocardioides fonticola NAA-13T (EF626689) 100 Nocardioides salarius CL-Z59T (DQ401092) 82 Nocardioides basaltis J112T (EU143365) Nocardioides dokdonensis FR1436T (EF633986) Nocardioides kribbensis KSL-2T (AY835924) 62 Nocardioides ginsengisegetis Gsoil 485T (GQ339901) Nocardioides dubius KSL-104T (AY928902) 87 Nocardioides alkalitolerans KSL-1T (AY633969) Nocardioides hankookensis DS-30T (EF555584) T 99 Nocardioides pyridinolyticus OS4 (U61298) 80 Nocardioides aquiterrae GW-9T (AF529063) Nocardioides terrigena DS-17T (EF363712) 97 Nocardioides sediminis MSL-01T (EF466110) 64 Nocardioides ganghwensis JC2055T (AY423718) 99 Nocardioides oleivorans DSM 16090T (AJ698724) 100 Nocardioides exalbidus RC825T (AB273624) Nocardioides furvisabuli SBS-26T (DQ411542) T 63 100 Nocardioides albus DSM 43109 (X53211) Nocardioides luteus KCTC 9575T (AF005007) 80 Nocardioides marinus CL-DD14T (DQ401093) 90 Nocardioides panacihumi Gsoil 616T (AB271053)

Fig. 1. Neighbour-joining phylogenetic tree reconstructed from a comparative analysis of 16S rRNA gene sequences showing the relationships between Nocardioides daeguensis sp. nov. 2C1-5T and type strains of related species of the genus Nocardioides. Filled circles at nodes indicate generic branches that were also recovered by using the maximum-parsimony and maximum-likelihood algorithms. Bootstrap values (expressed as percentages of 1000 replications) ¢50 % are shown at branch points. Bar, 0.005 substitutions per nucleotide position.

quinones and cell-wall peptidoglycan type), strain 2C1-5T v/v). The crude n-hexane–quinone solution was purified was grown on R2A for 5 days at 30 uC. Collected cells were using a Sep-Pak Vac silica cartridge (Waters) and subse- lyophilized for 24 h. Isoprenoid quinones were extracted quently analysed by HPLC as described previously (Hiraishi with chloroform/methanol (2 : 1, v/v), evaporated under et al., 1996). The isomer type of the diamino acid of the cell- vacuum conditions and re-extracted in n-hexane/water (1 : 1, wall peptidoglycan was determined by using TLC after http://ijs.sgmjournals.org 3729 Y. Cui and others hydrolysis with 6 M HCl at 100 uC for 18 h, as described by Table 1. Physiological characteristics of Nocardioides dae- Komagata & Suzuki (1987). guensis sp. nov. 2C1-5T and type strains of related species of the genus Nocardioides DNA–DNA hybridization experiments were performed between strain 2C1-5T and the reference strains with the Strains: 1, 2C1-5T;2,N. aromaticivorans DSM 15131T;3,N. caeni method described by Ezaki et al. (1989) using photobiotin- KCTC 19600T;4,N. ginsengisoli Gsoil 1124T;5,N. kongjuensis KCTC labelled DNA probes and microdilution wells. Hybridiza- 19054T;6,N. nitrophenolicus KCTC 0457BPT;7,N. simplex DSM tion was performed reciprocally with five replications for 20130T. All experimental results are from the present study. API 20NE each sample. The highest and lowest values obtained for and API ID 32 GN (bioMe´rieux) were used according to the each sample were excluded and the means of the remaining instructions of the manufacturer. All strains were negative for three values were converted to percentage DNA–DNA assimilation of L-fucose, inositol, itaconic acid, lactic acid, potassium relatedness values. 2-ketogluconate, potassium 5-ketogluconate, sodium malonate, suberic acid and D-sorbitol. All strains were negative for arginine A wide variety of catechol 1,2-dioxygenase (C12O) and dihydrolase activity, indole production, reduction of nitrate to nitrogen catechol 2,3-dioxygenase (C23O) genes were detected as and urease activity. +, Positive; –, negative; W, weakly positive. described previously (Sei et al., 1999). Utilization of aromatic compounds as sole carbon sources was determined Characteristic 1234567 by using a minimum salt medium (Bae et al., 1996) with 2 % Noble agar (Difco). After sterilization, aromatic compounds Assimilation of: (400, 500, 600, 700, 800 and 1000 p.p.m. phenol, 50, 100, Adipic acid 2 W + W 2 W 2 200, 300, 400 and 500 p.p.m. 2-chlorophenol, 50, 100, 200, Capric acid 22222+ 2 250, 300, 350 and 400 p.p.m. 3-chlorophenol, 50, 100, 150, Gluconic acid W ++++++ 200, 250, 300 and 400 p.p.m. 4-chlorophenol, 50, 100, 150 3-Hydroxybenzoic acid 222+ 2 + 2 + +++ and 200 p.p.m. N-nitrophenol and 50, 100, 150 and 4-Hydroxybenzoic acid 2 2 2 ++ + + 200 p.p.m. dibenzofuran) were added separately. We also 3-Hydroxybutyric acid 2 W 2 + ++++ detected the ability of 2C1-5T to use aromatic compounds in Malic acid W W Phenylacetic acid W 222222 liquid medium with the same components without Noble Propionic acid + 22+ W + 2 agar. To minimize evaporation of phenolic compounds, Sodium acetate + 2 W +++2 these tests were performed in 160 ml serum bottles with Trisodium citrate 222W 22W butyl robber stoppers. After sterilization, aromatic com- Valeric acid + 22+++2 pounds (phenol, 2-chlorophenol, 3-chlorophenol, 4-chlor- L-Arabinose 2 + 2 + 22+ ophenol, N-nitrophenol and dibenzofuran, each at 10, 25 D-Glucose W + 2 ++++ and 50 p.p.m.) were added separately. The change of Maltose 2 + 2 + WW2 aromatic compounds in liquid medium was detected as Mannose 222+ W 2 + described previously (Bae et al., 1996). Melibiose 2 + 2222+ L 2 +++2 + 2 Cells of strain 2C1-5T were Gram-reaction-positive, -Rhamnose D-Ribose 2 + 222++ aerobic, non-spore-forming, short rods. Colonies of the Sucrose +++ W +++ isolate cultured on R2A agar, nutrient agar, LB agar and N-Acetylglucosamine W 22+ 22+ trypticase soy agar plates were pale yellow, circular, opaque L-Alanine 222++ W + and convex, with a diameter of 1–2 mm after 2 days at L-Histidine 222+ 2 W 2 u T 30 C. The phenotypic characteristics of strain 2C1-5 are L-Proline WW2 + 2 + W summarized in the species description and compared with Salicin 222222+ those of the closest reference strains in Table 1. Utilization L-Serine 2 W 22W 22 of phenolic compounds as sole carbon sources on agar Glycogen WW22222 plates is described in the species description. Aromatic D-Mannitol 2 + 2 + 22+ compounds in liquid medium were not used by strain 2C1- Reduction of nitrate to nitrite + 222222 5T after 1 month. This might be because the washed Noble Acidification of glucose 222+ 222 agar still contained some nutrients that allowed bacterial b-Galactosidase ++2 ++++ growth on agar plates but not in liquid medium. Catechol b-Glucosidase ++22+++ dioxygenase C23O was detected in strain 2C1-5T by PCR Protease 22+ 22+ 2 amplification, but C12O was not detected. The DNA G+C content of strain 2C1-5T was 74.9 mol%, a little higher than the DNA G+C contents reported for N. 1997). The respiratory quinone system supported the aromaticivorans (72.0–72.4 mol%; Kubota et al., 2005), N. affiliation of strain 2C1-5T to the genus Nocardioides,asthe caeni (71.5 mol%; Yoon et al., 2009), N. ginsengisoli majority of species in the genus Nocardioides have MK- T (70.2 mol%; Cui et al., 2009), N. kongjuensis (72.1 mol%; 8(H4) as the predominant quinone. Strain 2C1-5 and the Yoon et al., 2006), N. nitrophenolicus (71.4 mol%; Yoon four closest strains contained LL-2,6-diaminopimelic acid et al., 1999) and N. simplex (72.0–73.5 mol%; Yoon et al., in the cell-wall peptidoglycan. The fatty acid profiles of

3730 International Journal of Systematic and Evolutionary Microbiology 63 Nocardioides daeguensis sp. nov.

T 2C1-5 and the type strains of the most closely related melibiose, L-rhamnose, D-ribose, L-alanine, L-histidine, species of the genus Nocardioides are summarized in Table salicin, L-serine and D-mannitol. Utilizes the following S1, available in IJSEM Online. According to the results of aromatic compounds as carbon sources on agar plates at fatty acid detection, iso-C16 : 0,C16 : 0 and C17 : 1v6c made the concentrations given: phenol (400–700 p.p.m.), 2- up about 50–70 % of the total fatty acids in all compared chlorophenol (50–500 p.p.m.) and 4-chlorophenol (50– strains, again supporting the conclusion that 2C1-5T 150 p.p.m.). Does not utilize the following aromatic belonged to the genus Nocardioides (Yoon et al., 1999; compounds as carbon sources on agar plates at the Cui et al., 2009). Strain 2C1-5T exhibited relatively low concentrations given: phenol (800–1000 p.p.m.), 3-chloro- levels of DNA–DNA relatedness with respect to the type phenol (50–400 p.p.m.), 4-chlorophenol (200–300 p.p.m.), strains of N. nitrophenolicus (35.2 %), N. simplex (30.8 %), N-nitrophenol (50–200 p.p.m.) and dibenzofuran (50– N. kongjuensis (28.9 %), N. aromaticivorans (19.6 %), N. 200 p.p.m.). MK-8(H4) is the predominant menaquinone caeni (11.2 %) and N. ginsengisoli (8.5 %); these values are and iso-C16 : 0,C16 : 0 and C17 : 1v6c are the major cellular fatty low enough to assign strain 2C1-5T to a novel species of the acids. genus Nocardioides (Wayne et al., 1987; Stackebrandt & The type strain, 2C1-5T (5JCM 17460T5KCTC 19799T), Goebel, 1994). Detailed DNA relatedness results are given was isolated from activated sludge of an industrial waste- in Table S2. water treatment plant in Daegu, South Korea. The G+C In summary, the characteristics of strain 2C1-5T are content of genomic DNA of the type strain is 74.9 mol% consistent with the description of the genus Nocardioides (as determined by HPLC). with regard to morphological, biochemical and chemotaxonomic properties. However, the phylogenetic distance Acknowledgements between strain 2C1-5T and recognized species of the genus Nocardioides, its unique phenotypic characteristics (Table This work was supported by the Mid-career Researcher Program 1) and the low levels of DNA–DNA relatedness (Wayne through an NRF grant funded by MEST (no. 2009-0081153). et al., 1987) warrant the assignment of strain 2C1-5T to a novel species of the genus Nocardioides, for which the name References Nocardioides daeguensis sp. nov. is proposed. Bae, H. S., Lee, J. M., Kim, Y. B. & Lee, S.-T. (1996). Biodegradation of Description of Nocardioides daeguensis sp. nov. the mixtures of 4-chlorophenol and phenol by Comamonas testosteroni CPW301. 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Utilizes the Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a following compounds as sole carbon sources: 3-hydro- maximum likelihood approach. J Mol Evol 17, 368–376. xybutyric acid, malic acid, propionic acid, sodium acetate, Felsenstein, J. (1985). Confidence limit on phylogenies: an approach valeric acid and sucrose. Utilizes the following compounds using the bootstrap. Evolution 39, 783–791. weakly as sole carbon sources: gluconic acid, phenylacetic Felsenstein, J. (2009). PHYLIP (phylogeny inference package), version acid, D-glucose, N-acetylglucosamine, L-proline and gly- 3.69. Distributed by the author. Department of Genome Sciences, cogen. Does not utilize the following compounds as sole University of Washington, Seattle, USA. carbon sources: inositol, itaconic acid, suberic acid, sodium Fitch, W. M. (1971). Toward defining the course of evolution: malonate, lactic acid, potassium 5-ketogluconate, L-fucose, minimum change for a specific tree topology. 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