SGM Journals
Bacteroidetes

Flavobacterium caeni sp. nov., isolated from a sequencing batch reactor for the treatment of malachite green effluents

Ying Liu, Jing-Hua Jin, Yu-Guang Zhou, Hong-Can Liu, Zhi-Pei Liu

  • 1State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
  • 2Environment Protection Research Institute of Light Industry, Beijing 100089, PR China
  • Correspondence
    Zhi-Pei Liu
    liuzhp{at}sun.im.ac.cn

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(2):417–421 · https://doi.org/10.1099/ijs.0.010603-0

    View at publisher PubMed

    Abstract

    A Gram-stain-negative, heterotrophic, aerobic, non-spore-forming and non-motile bacterial strain, designated LM5T, was isolated from activated sludge from a sequencing batch reactor for the treatment of effluents contaminated by malachite green. The taxonomy of strain LM5T was studied by phenotypic and phylogenetic methods. Strain LM5T formed orange colonies on R2A and YP plates. Cells were rods, 0.4–0.6 μm in diameter and 0.8–1.2 μm in length. Growth occurred at 10–35 °C (optimum, 20–25 °C), at pH 5.5–9.5 (optimum, pH 6.5–7.5) and in the presence of 0–2 % (w/v) NaCl (optimum, 0.5 %). Oxidase and catalase activities were present. Flexirubin-type pigments were present, but extracellular glycans were absent. MK-6 was the major respiratory quinone. The major fatty acids were iso-C15 : 0 (28.3 %) and iso-C17 : 1ω9c (13.8 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain LM5T was a member of the genus Flavobacterium with highest sequence similarity to Flavobacterium soli DS-6T (93.2 %) and Flavobacterium lindanitolerans IP-10T (92.9 %). Together with F. lindanitolerans IP-10T, strain LM5T formed a distinct lineage in the phylogenetic tree. The DNA G+C content was 52±0.6 mol% (HPLC), which is significantly higher than that of other species of the genus Flavobacterium (30–41 mol%). Based on phylogenetic and phenotypic evidence, strain LM5T is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium caeni sp. nov. is proposed; the type strain is LM5T (=CGMCC 1.7031T=NBRC 104239T).

    • ↵†These authors contributed equally to this work.

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain LM5T is EU313814.

    The genus Flavobacterium belongs to the family Flavobacteriaceae in the phylum ‘Bacteroidetes’ (Ludwig & Klenk, 2001). It was proposed by Bergey et al. (1923) and emended by Bernardet et al. (1996). Recently, Bernardet & Bowman (2006) provided an overview of the taxonomy and ecology of members of the genus Flavobacterium. Species of the genus are widespread in nature and have been isolated from many freshwater and soil habitats. In recent years, many novel species belonging to the genus Flavobacterium have been identified. At the time of writing, the genus Flavobacterium comprises about 50 species with validly published names. A Flavobacterium strain, designated LM5T, was isolated from activated sludge from a sequencing batch reactor for the treatment of malachite green effluents and subjected to a polyphasic taxonomic study; the results are presented in this report.

    Malachite green is an N-methylated diamino-triphenylmethane dye used for dyeing silk, leather, cotton, wool and jute. Traditionally, malachite green has also been used to treat fungal infections in fishes (Alderman, 1985). Malachite green can be toxic to human cells and promotes liver tumour formation in rodents (Srivastava et al., 2004). Due to its potentially harmful effects, the US Food and Drug Administration nominated malachite green as a priority chemical for toxicity and carcinogenicity testing in 1993. Strain LM5T was isolated from activated sludge in an anaerobic/aerobic sequential batch reactor used for the treatment of malachite green-contaminated effluents. The sludge sample was suspended in normal saline by vigorous vortexing and part of the suspension was spread onto low-organic Luria–Bertani [containing (l−1): 1.0 g tryptone (Difco), 0.5 g yeast extract and 10.0 g NaCl, pH 7.0] agar plates and incubated at 30 °C for 1 week. Single colonies on the plates were picked up and strain LM5T was purified by repeated streaking on new plates. Subcultures were performed on R2A agar (Difco) and the strain was stored in R2A broth (Difco) supplemented with 15 % glycerol (v/v) at −80 °C.

    The following tests were performed in parallel on strain LM5T and on its closest phylogenetic neighbours, Flavobacterium soli DS-6T, Flavobacterium lindanitolerans IP-10T and Flavobacterium terrae R2A1-13T. Growth was tested on R2A agar, marine 2216 agar (MA; laboratory prepared), nutrient agar (NA; laboratory prepared), trypticase soy agar (TSA; Difco), 0.1× TSA and YP agar [containing (l−1): 3.0 g tryptone (Difco), 3.0 g yeast extract (Difco), 0.5 g MgSO4 . 7H2O, 0.3 g NaCl, 15 g agar, pH 7.0]. Cell morphology was examined using phase-contrast microscopy and transmission electron microscopy (H600; Hitachi). Gram staining was performed as described by Gerhardt et al. (1994). Catalase and oxidase activities and H2S production were determined as described by Dong & Cai (2001). Gliding motility was assessed as described by Bernardet et al. (2002). Growth under anaerobic conditions was determined after incubation in an anaerobic chamber with anaerobically prepared R2A broth. Salt tolerance was tested in R2A broth supplemented with 0–2.5 % (w/v) NaCl (at intervals of 0.5 %). The pH range for growth was tested in R2A broth adjusted to pH 5.0–10.0 (at intervals of 0.5 pH). The temperature range for growth was tested in R2A broth at 5–40 °C (at intervals of 5 °C). Hydrolysis of casein [50 % (v/v) skimmed milk], l-tyrosine (0.5 %, w/v), starch (0.2 %, w/v), Tweens 20, 40, 60 and 80 (1 %, v/v), carboxymethylcellulose (0.5 %, w/v), egg yolk (5 %, w/v), pectin (0.5 %, w/v), agar (1.5 %, w/v) and DNA (0.1 %, w/v) was tested according to Gerhardt et al. (1994) using R2A agar as a basal medium.

    The presence of flexirubin-type pigments and extracellular glycans was assessed by using the KOH and Congo red tests, respectively (Fautz & Reichenbach, 1980; Bernardet et al., 2002).

    Carbon source assimilation was studied by using the GN2 MicroPlate (Biolog) according to the manufacturer's instructions. Acid production from various substrates was assessed by using API 50CH test strips (bioMérieux) and 0.5× CHB/E medium (bioMérieux) according to Yoon et al. (2006). Additional biochemical properties and enzyme activities were detected by using API 20NE and API ZYM strips (bioMérieux). Susceptibility to antibiotics was determined using filter-paper discs (Beijing Pharmaceutical Company) containing various antibiotics as specified in the species description. The phenotypic properties of strain LM5T are given in Table 1⇓ and in the species description. F. terrae R2A1-13T was unable to utilize any GN2 MicroPlate substrates.

    Table 1.

    Phenotypic characteristics of strain LM5T and closely related species of the genus Flavobacterium

    Strains: 1, LM5T; 2, F. soli DS-6T; 3, F. lindanitolerans IP-10T; 4, F. terrae R2A1-13T. All data are from this study (except for the DNA G+C content of the three reference strains). All strains are negative for absorption of Congo red, DNA degradation, H2S production and nitrate reduction. In GN2 MicroPlate assays, all strains (except F. terrae R2A1-13T, which does not utilize any GN2 MicroPlate substrates) utilize dextrin, α-d-glucose, maltose, α-ketovaleric acid, l-alanyl glycine, l-proline, l-threonine and uridine. In the API ZYM system, all strains are positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but negative for lipase (C14), α-galactosidase, β-galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. +, Positive; −, negative; w, weakly positive.

    Genomic DNA extraction, PCR and sequencing of the 16S rRNA gene were performed according to Kim et al. (1998). The sequence of the amplified fragment (1387 nt) was determined by direct sequencing and compared with available 16S rRNA gene sequences in GenBank using the blast program (Altschul et al., 1990) available at NCBI. Strain LM5T showed highest 16S rRNA gene sequence similarity to F. soli DS-6T (93.2 %), F. lindanitolerans IP-10T (92.9 %), F. terrae R2A1-13T (92.7 %) and Flavobacterium flevense ATCC 27944T. Strain LM5T shared 91.2 % sequence similarity with the type strain of the type species of the genus Flavobacterium, Flavobacterium aquatile ATCC 11947T. A multiple alignment with closely related members of the genus Flavobacterium was performed by using the clustal w program (Thompson et al., 1994). Ambiguous and unalignable bases were removed manually and a neighbour-joining phylogenetic tree was constructed from the evolutionary distance matrix calculated using the mega program version 3.1 (Kumar et al., 2004). The stability of the relationships was assessed by means of bootstrapping (based on 1000 replicates). Strain LM5T, together with F. lindanitolerans IP-10T, formed a distinct phylogenetic lineage within the genus Flavobacterium (Fig. 1⇓), indicating that it probably represents a novel member of the genus.

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of Flavobacterium caeni LM5T and representative members of the genus Flavobacterium. Numbers at nodes are bootstrap values >50 % (1000 replications). Cellulophaga lytica ATCC 23178T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.

    Isoprenoid quinones were extracted and analysed as described by Komagata & Suzuki (1987). For fatty acid analysis, cell mass of strain LM5T, F. soli DS-6T, F. lindanitolerans IP-10T and F. terrae R2A1-13T was obtained from R2A plates after cultivation for 2 days at 30 °C. Cellular fatty acids were extracted, methylated and analysed following the instructions of the Sherlock Microbial Identification System (MIDI). The major respiratory quinone was MK-6, as in all members of the family Flavobacteriaceae. The dominant fatty acid of strain LM5T was iso-C15 : 0 (28.3 %), in line with the three most closely related Flavobacterium type strains grown under the same conditions (Table 2⇓). It differed from them, however, by a significantly higher content of iso-C17 : 1ω9c.

    Table 2.

    Cellular fatty acid profiles of strain LM5T and closely related species of the genus Flavobacterium

    Strains: 1, LM5T; 2, F. soli DS-6T; 3, F. lindanitolerans IP-10T; 4, F. terrae R2A1-13T. All data are from this study. Values are percentages of the total fatty acid content. Only fatty acids representing at least 1 % of the total fatty acids in all strains are shown. −, Not detected; tr, trace (<1.0 %).

    The DNA G+C content of strain LM5T was determined by the thermal denaturation (Sly et al., 1986) and HPLC (Mesbah et al., 1989) methods using DNA from Escherichia coli K-12 as a control. Both methods gave almost the same results, i.e. 52 mol% (Tm) and 52±0.6 mol% (HPLC; mean and sd of five experiments), values that are significantly higher than those of other species of the genus Flavobacterium (30–41 mol%; Bernardet & Bowman, 2006; Park et al., 2006; Wang et al., 2006).

    The phylogenetic tree clearly showed that strain LM5T belongs to the cluster comprising Flavobacterium columnare and several recently described species of the genus Flavobacterium that share rather low levels of 16S rRNA gene sequence similarity with the type species of the genus, F. aquatile, and with the Flavobacterium species that form the main cluster (Fig. 1⇑). Moreover, the DNA G+C content of strain LM5T clearly fell outside of the range reported for the genus (Bernardet & Bowman, 2006). Yet, as the overall phenotypic characteristics of strain LM5T were in accordance with those of the genus Flavobacterium, it was decided to describe it as a novel species in this genus, Flavobacterium caeni sp. nov., pending further taxonomic research. Strain LM5T could be differentiated from other species of the genus by means of the characteristics listed in Tables 1⇑ and 2⇑.

    Description of Flavobacterium caeni sp. nov.

    Flavobacterium caeni (cae′ni. L. gen. n. caeni of sludge).

    Strictly aerobic and heterotrophic. Cells are Gram-stain-negative, non-motile, non-flagellated and non-spore-forming rods, 0.4–0.6 μm in diameter and 0.8–1.2 μm in length. Abundant growth occurs on YP agar, R2A and 0.1× TSA; weak growth occurs on TSA, MA and NA. Colonies are orange, circular, smooth with entire margins and about 2–3 mm in diameter after incubation on R2A at 30 °C for 2 days. Growth occurs at 10–35 °C (optimum, 20–25 °C), at pH 5.5–9.5 (optimum, pH 6.5–7.5) and in the presence of 0–2 % (w/v) NaCl (optimum, 0.5 %). Oxidase and catalase activities are present. Arginine dihydrolase activity is absent. Acid is not produced from glucose. l-Tyrosine, starch and Tweens 20 and 80 are hydrolysed. Tweens 40 and 60 are weakly hydrolysed. Casein, pectin, carboxymethylcellulose, agar, DNA and urea are not hydrolysed. A brown pigment is produced on tyrosine agar. No precipitate is formed on egg yolk agar. Flexirubin-type pigments are present, but extracellular glycans are absent. In the API ZYM test, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, acidic phosphatase, naphthol-AS-BI-phosphohydrolase and α-glucosidase activities are present, but lipase (C14), α-galactosidase, β-galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase activities are absent. In GN2 MicroPlate assays, α-cyclodextrin, dextrin, glycogen, d-galactose, α-d-glucose, α-d-lactose, maltose, d-mannose, turanose, succinic acid monomethylester, formic acid, α-ketovaleric acid, propionic acid, glycyl l-glutamic acid, l-threonine, uridine, l-alanyl glycine and l-proline are utilized; all other substrates in the MicroPlate are not utilized. Acid is produced from aesculin and produced weakly from maltose, d-lactose, starch and glycogen in the API 50CH strip; acid is not produced from the other substrates in the strip. Sensitive (μg per disc unless otherwise stated) to vancomycin (30), gentamicin (10), carbenicillin (100), streptomycin (10), tetracycline (30), ampicillin (10), erythromycin (15), novobiocin (30), novobiocin (5), chloramphenicol (30), ciprofloxacin (5), corfloxacin (10), rifampicin (5) and penicillin (10 U); intermediate resistance is observed for polymixin B (300 U) and kanamycin (30). The major respiratory quinone is MK-6. The major fatty acids (>10 % of the total fatty acids) are iso-C15 : 0 and iso-C17 : 1ω9c.

    The type strain, LM5T (=CGMCC 1.7031T=NBRC 104239T), was isolated from activated sludge from a sequencing batch reactor for the treatment of malachite green effluents. The DNA G+C content of the type strain is 52±0.6 mol% (HPLC).

    Acknowledgments

    This work was supported by grants from the high-tech development program of China (863 program no. 2006AA06Z316).

    References

    1. Alderman, D. J. (1985). Malachite green: a review. J Fish Dis 8, 289–298.
    2. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403–410.
    3. Bergey, D. H., Harrison, F. C., Breed, R. S., Hammer, B. W. & Huntoon, F. M. (editors) (1923). Bergey's Manual of Determinative Bacteriology. Baltimore: Williams & Wilkins.
    4. Bernardet, J.-F. & Bowman, J. (2006). The genus Flavobacterium. In The Prokaryotes: a Handbook on the Biology of Bacteria, 3rd edn, vol. 7, pp. 481–531. Edited by M. Dworkin, S. Falkow, E. Rosenberg, K. H. Schleifer & E. Stackebrandt. New York: Springer.
    5. Bernardet, J.-F., Segers, P., Vancanneyt, M., Berthe, F., Kersters, K. & Vandamme, P. (1996). Cutting a Gordian knot: emended classification and description of the genus Flavobacterium, emended description of the family Flavobacteriaceae, and proposal of Flavobacterium hydatis nom. nov. (basonym, Cytophaga aquatilis Strohl and Tait 1978). Int J Syst Bacteriol 46, 128–148.
    6. Bernardet, J.-F., Nakagawa, Y. & Holmes, B. (2002). Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. Int J Syst Evol Microbiol 52, 1049–1070.
    7. Dong, X.-Z. & Cai, M.-Y. (2001). Determinative Manual for Routine Bacteriology. Beijing: Scientific Press (English translation).
    8. Fautz, E. & Reichenbach, H. (1980). A simple test for flexirubin-type pigments. FEMS Microbiol Lett 8, 87–91.
    9. Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology.
    10. Kim, S. B., Falconer, C., Williams, E. & Goodfellow, M. (1998). Streptomyces thermocarboxydovorans sp. nov. and Streptomyces thermocarboxydus sp. nov., two moderately thermophilic carboxydotrophic species from soil. Int J Syst Bacteriol 48, 59–68.
    11. Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in bacterial systematics. Methods Microbiol 19, 161–207.
    12. Kumar, S., Tamura, K. & Nei, M. (2004). mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
    13. Ludwig, W. & Klenk, H.-P. (2001). Overview: a phylogenetic backbone and taxonomic framework for procaryotic systematics. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 1, pp. 49–65. Edited by D. R. Boone, R. W. Castenholz & G. M. Garrity. New York: Springer.
    14. Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
    15. Park, M., Lu, S., Ryu, S. H., Chung, B. S., Park, W., Kim, C.-J. & Jeon, C. O. (2006). Flavobacterium croceum sp. nov., isolated from activated sludge. Int J Syst Evol Microbiol 56, 2443–2447.
    16. Sly, L. I., Blackall, L. L., Kraat, P. C., Tian-Shen, T. & Sangkhobol, V. (1986). The use of second derivative plots for the determination of mol% guanine plus cytosine of DNA by the thermal denaturation method. J Microbiol Methods 5, 139–156.
    17. Srivastava, S., Sinha, R. & Roy, D. (2004). Toxicological effects of malachite green. Aquat Toxicol 66, 319–329.
    18. Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
    19. Wang, Z.-W., Liu, Y.-H., Dai, X., Wang, B.-J., Jiang, C.-Y. & Liu, S.-J. (2006). Flavobacterium saliperosum sp. nov., isolated from freshwater lake sediment. Int J Syst Evol Microbiol 56, 439–442.
    20. Yoon, J.-H., Kang, S.-J. & Oh, T.-K. (2006). Flavobacterium soli sp. nov., isolated from soil. Int J Syst Evol Microbiol 56, 997–1000.