Caenimonas koreensis gen. nov., sp. nov., isolated from activated sludge

Footnotes

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain EMB320^T is DQ349098.

A transmission electron micrograph of cells and results of TLC of polar lipids of strain EMB320^T and maximum-likelihood and maximum-parsimony 16S rRNA gene sequence-based phylogenetic trees are available as supplementary material with the online version of this paper.

Activated sludge processes with cyclic changes of anaerobic and aerobic conditions have been used to remove phosphate from wastewater and are becoming more important for reducing eutrophication of lakes. An understanding of the microbial community responsible for phosphorus removal is a prerequisite for understanding the mechanism of enhanced biological phosphorus removal (EBPR). Therefore, efforts to isolate bacteria performing EBPR have been made in our laboratory (Lu et al., 2006) and here we describe the taxonomic characterization of a strain isolated from activated sludge that represents a novel genus belonging to the family Comamonadaceae.

Strain EMB320^T was isolated from activated sludge performing EBPR in a lab-scale sequencing batch reactor (SBR). Sodium acetate was supplied as a sole carbon source and the operation of the SBR has been described elsewhere (Jeon et al., 2003). A sludge sample was diluted serially with 1 % (w/v) saline solution and spread on R2A agar (Difco) and incubated at 20 °C for 7 days for isolation. Subcultivation was done on R2A agar at 30 °C for 5 days. The type strains of some other related taxa, Rhodoferax ferrireducens DSM 15236^T, Curvibacter delicatus DSM 11558^T, Xylophilus ampelinus DSM 7250^T, Ramlibacter tataouinensis DSM 14655^T, Variovorax paradoxus KCTC 12459^T and Polaromonas naphthalenivorans CJ2^T, were used as reference strains for biochemical tests.

Gram staining was performed using the bioMérieux Gram stain kit according to the manufacturer's instructions. Cell morphology and motility were studied using phase-contrast microscopy and transmission electron microscopy (JEM-1010; JEOL) as described by Jeon et al. (2005). Physiological characteristics of strain EMB320^T were examined by growing the isolate on R2A medium at different temperatures and pH. R2A medium was prepared with different pH values as described previously (Gomori, 1955). Oxidase activity was tested by oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck) and catalase activity was evaluated by production of oxygen bubbles in 3 % (v/v) aqueous hydrogen peroxide solution. The hydrolysis of compounds was assessed on R2A agar after 5 days of incubation according to methods described previously (Lányí, 1987; Smibert & Krieg, 1994). Nitrate reduction was performed according to the method of Lányí (1987) and acid production from carbohydrates was tested as described by Leifson (1963). Utilization of thiosulfate was tested in R2A broth supplemented with 10 mM Na₂S₂O₃ . 5H₂O as described by Spring et al. (2004) and the concentration of sulfate, the end product of thiosulfate oxidation, was quantified in spent R2A medium using anion chromatography (Dionex; ICS-1000). Carbon source utilization was tested in mineral medium as described by Kämpfer et al. (1991). The utilization of D-fructose, D-glucose, glycerol, malonate, D-mannitol, maleate and D- and L-tryptophan was tested. Chemolithoautotrophic growth of strain EMB320^T with hydrogen gas was tested on medium 81 (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) agar under the conditions described by Malik & Schlegel (1981). More enzyme activities and biochemical features were determined by using API kits (API ZYM, API 20E and API 20NE) as recommended by the manufacturer (bioMérieux).

Strain EMB320^T on R2A agar formed white, glistening, translucent, slightly sticky and slightly raised circular colonies when grown at 30 °C for 5 days. Growth was observed at temperatures between 10 and 35 °C, with an optimum growth temperature of 30 °C. The strain grew at the range of pH 6.0–9.0 with optimum growth between pH 7.0 and 8.0. Cells of the isolate were non-motile rods (0.4–0.6 µm wide and 0.8–2.0 µm long) without flagella (Supplementary Fig. S1, available in IJSEM Online), Gram-negative and oxidase- and catalase-positive. The isolate did not grow on R2A agar with the removal of yeast extract and peptone under aerobic conditions, but it grew well following the addition of vitamin mixture (Wolin et al., 1963), meaning that it was possible to replace these complex nutrients (yeast extract and peptone) with vitamins. Anaerobic growth was not observed after incubation for 10 days at 30 °C on R2A agar or on R2A agar containing 10 mM nitrate.

Analysis of fatty acid methyl esters was performed according to the instructions of the Microbial Identification System (MIDI Inc.) after cultivation for 5 days on R2A agar at 30 °C. Analyses of polar lipids and isoprenoid quinones were carried out using the methods described by Komagata & Suzuki (1987). Polyamines were analysed using an HPLC (Shimadzu LC-10A) equipped with a fluorescence detector (Shimadzu RF-10AXL) and a reversed-phase column [Akzo Nobel; Kromasil ODS (250x4.6 mm)] as described previously (Busse & Auling, 1988; Busse et al., 1997). The DNA G+C content of strain EMB320^T was determined using an HPLC fitted with a reversed-phase column [GROM; GROM-SIL 100 ODS-2FE (250x4.6 mm)] according to the method of Tamaoka & Komagata (1984). The major respiratory lipoquinone of strain EMB320^T was ubiquinone-8 (Q-8). The cellular fatty acids of the strain were summed feature 3 (C_{16 : 1}ω7c and/or iso-C_{15 : 0} 2-OH; 41.8 %), C_{16 : 0} (25.9 %), C_{18 : 1}ω7c (15.8 %), C_{18 : 1}ω7c 11-methyl (4.9 %), C_{10 : 0} 3-OH (3.5 %), C_{17 : 0} (2.0 %), C_{15 : 0} (1.9 %), C_{14 : 0} (1.5 %), C_{12 : 0} (0.9 %), C_{18 : 0} (0.9 %) and summed feature 7 (C_{19 : 0} cyclo ω10c and/or C_{19 : 1}ω6c; 0.9 %). The strain exhibited a polar lipid profile consisting of major lipid phosphatidylethanolamine, moderate amounts of phosphatidylglycerol and diphosphatidylglycerol and a small amount of an unknown amino group-containing lipid (Supplementary Fig. S2). The presence of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol and the lack of glycolipids were in agreement with the polar lipid characteristics reported for the related species Polaromonas naphthalenivorans, Polaromonas aquatica and Xenophilus azovorans. Strain EMB320^T was characterized by the presence of the betaproteobacteria-specific diamine 2-hydroxyputrescine [55 µmol (g dry weight)^–1] and putrescine [16 µmol (g dry weight)^–1]. The content of 2-hydroxyputrescine was a little higher than in any other species analysed so far, such as P. naphthalenivorans and P. aquatica (Busse & Auling, 1988; Kämpfer et al., 2006). Other polyamines were only detected in trace amounts. The G+C content of the genomic DNA of strain EMB320^T was 62.7 mol%. The phenotypic characteristics of strain EMB320^T are presented in the genus and species descriptions and are compared with those of closely related taxa in Table 1.

Table 1. Characteristics of strain EMB320T (Caenimonas koreensis gen. nov., sp. nov.) and related members of the family Comamonadaceae Data for reference genera were taken from Heulin et al. (2003) (Ramlibacter), Willems et al. (1991) and Yoon et al. (2006) (Variovorax), Hiraishi et al. (1991) and Finneran et al. (2003) (Rhodoferax), Ding & Yokota (2004) (Curvibacter), Irgens et al. (1996), Jeon et al. (2004), Kämpfer et al. (2006) and Sizova & Panikov (2007) (Polaromonas) and Willems et al. (1987) (Xylophilus) unless indicated. NA, No data available; RQ, rhodoquinone.

Cells are Gram-negative, strictly aerobic, non-motile rods, 0.4–0.6 µm wide and 0.8–2.0 µm long, at 30 °C on R2A agar. Catalase- and oxidase-positive. Nitrate is reduced to nitrite and thiosulfate is oxidized to sulfate. No aerobic chemoautotrophic growth with hydrogen as substrate. Contain phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol and an unknown amino group-containing lipid as polar lipids. The major isoprenoid quinone is ubiquinone-8 (Q-8). Major polyamines are 2-hydroxyputrescine and putrescine. The major fatty acids are C_{16 : 1}ω7c and/or iso-C_{15 : 0} 2-OH, C_{16 : 0} and C_{18 : 1}ω7c. The DNA G+C content of the type strain of the type species is 62.7 mol% (HPLC). Phylogenetically, the genus belongs to the family Comamonadaceae. The type species is Caenimonas koreensis.

Description of Caenimonas koreensis sp. nov.
Caenimonas koreensis (ko.re.en'sis. N.L. fem. adj. koreensis pertaining to Korea, where the type strain was isolated).

Displays the following properties in addition to those given in the genus description. Colonies are white, glistening, translucent, slightly sticky, raised and circular on R2A agar. Growth occurs optimally at 30 °C and pH 7.0–8.0. Negative for the production of indole, H₂S and acetoin and negative for citrate utilization (API 20E). Hydrolyses urea, gelatin and Tween 20 but does not hydrolyse casein, Tween 80, tyrosine, aesculin or starch. Acid is produced from raffinose, myo-inositol, D-lactose, L-arabinose and D-fructose, but not from D-glucose, sorbitol, sucrose, rhamnose, amygdalin, melibiose, D-galactose, D-mannose, D-mannitol, arbutin or salicin. Negative for assimilation of D-glucose, D-mannose, D-mannitol, N-acetylglucosamine, maltose, L-arabinose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid (API 20NE). Utilizes D-fructose, D-glucose, malonate and maleate as sole carbon sources, but does not utilize glycerol, D-mannitol or D- or L-tryptophan. Produces alkaline phosphatase, leucine arylamidase and urease, but not lipase (C14), cystine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase, arginine dihydrolase, lysine decarboxylase or ornithine decarboxylase. Weak activity is observed for esterase (C4), esterase lipase (C8), valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and tryptophan deamidase.

The type strain is EMB320^T (=KCTC 12616^T =DSM 17982^T), isolated from activated sludge that performed EBPR.