SGM Journals
Bacteroidetes

Flavobacterium haoranii sp. nov., a cypermethrin-degrading bacterium isolated from a wastewater treatment system

Jun Zhang, Rui-Bo Jiang, Xiao-Xia Zhang, Bao-Jian Hang, Jian He, Shun-Peng Li

  • 1Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Life Sciences College of Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China
  • 2Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China
  • Correspondence
    Shun-Peng Li
    lsp{at}njau.edu.cn

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(12):2882–2886 · https://doi.org/10.1099/ijs.0.020776-0

    View at publisher PubMed

    Abstract

    A Gram-negative, non-spore-forming, yellow-pigmented bacterium, strain LQY-7T, was isolated from activated sludge treating synthetic pyrethroid-manufacturing wastewater. The taxonomic status of the strain was determined using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain LQY-7T was a member of the genus Flavobacterium but had low similarities with other species of this genus (95.0 % similarity with Flavobacterium indicum GPTSA100-9T and <94 % similarities with other Flavobacterium species). On the basis of phenotypic, genetic and phylogenetic data, strain LQY-7T should be classified as a representative of a novel species of the genus Flavobacterium, for which the name Flavobacterium haoranii sp. nov. is proposed; the type strain is LQY-7T (=ACCC 05409T =KCTC 23008T).

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain LQY-7T is GQ988780.

    The genus Flavobacterium was proposed by Bergey et al. (1923) and emended by Bernardet et al. (1996) to include Gram-negative, aerobic, rod-shaped, yellow-pigmented bacteria that are usually motile by gliding and have a DNA G+C content of 30–41 mol% (Bernardet & Bowman, 2006; Park et al., 2006). At the time of writing, the genus comprised about 60 recognized species isolated from diverse habitats such as fresh- and salt-water, diseased fish, soil, sediment and micromats.

    Cypermethrin is one of the most important synthetic pyrethroid pesticides and is widely used to control pests in cotton and vegetable crops. However, cypermethrin affects the central nervous system, causes allergic skin reactions, lymph node and spleen damage, and eye irritation. In addition, cypermethrin is highly toxic to fish and other aquatic organisms, as well as to bees. It has been classified as ‘moderately hazardous’ (Class II) by the World Health Organization and considered as a possible human carcinogen by the US Environmental Protection Agency. Microbes play significant roles in degrading and detoxifying cypermethrin residues in the environment (Kaufman et al., 1981; Roberts & Standen, 1981). In this paper, a cypermethrin-degrading bacterial strain, designated LQY-7T, was isolated from activated sludge in a synthetic pyrethroid-manufacturing wastewater treatment facility (Yangnong Chemical Group Co., Jiangsu Province, China). The taxonomic status of this strain was determined using a polyphasic taxonomic approach. The data obtained suggest that the isolate represents a novel species of the genus Flavobacterium.

    For investigation of morphological features, strain LQY-7T was cultivated aerobically on trypticase soy agar (TSA; Difco) at 30 °C. Cell morphology and dimensions were examined by light microscopy (BH-2; Olympus) and transmission electron microscopy (H-7650; Hitachi) using cells from an exponentially growing culture. Gram-staining was performed according to the classical Gram procedure (Buck, 1982). Gliding motility, production of flexirubin-type pigments and adsorption of Congo red by colonies were investigated by the methods of Bernardet et al. (2002). Growth at various temperatures (4, 10, 15, 20, 25, 30, 35, 37 and 40 °C) and pH (4.0–10.0 at intervals of 0.5 pH units) was assessed on trypticase soy broth (TSB) after 3 days of incubation. Salt tolerance was tested on TSB supplemented with 0–8 % (w/v) NaCl after 3 days of incubation. Growth on nutrient agar, R2A agar, cetrimide agar, Simmons' citrate agar and MacConkey agar was also evaluated at 30 °C. Sensitivity to antibiotics was tested on TSA plates using discs containing the following antibiotics: erythromycin (15 μg), tetracycline (30 μg), gentamicin (10 μg), chloramphenicol (30 μg), kanamycin (30 μg), streptomycin (10 μg), rifampicin (5 μg), ampicillin (10 μg), polymyxin B (30 μg), penicillin G (1 μg), cefradine (30 μg), roxithromycin (15 μg), lincomycin (2 μg), carbenicillin (100 μg), spectinomycin (100 μg), amoxicillin (10 μg), bacitracin (0.04 μg) and vancomycin (30 μg). Standard physiological tests were carried out according to the methods described by Lányí (1987). Additional physiological characteristics were determined with the API 20NE, API 50CH and API ZYM (bioMérieux) systems according to the manufacturer's instructions.

    For analysis of fatty acids, the strain was grown at 30 °C on TSB. Cells were harvested during the exponential phase by centrifugation, washed with distilled water and freeze-dried. The cellular fatty acids in the freeze-dried cells were identified by the Identification Service of the DSMZ (Braunschweig, Germany) as described by Kämpfer & Kroppenstedt (1996), Kuykendall et al. (1988) and Miller (1982).

    Extraction and purification of total DNA was carried out according to standard procedures (Sambrook & Russell, 2001). The DNA G+C content was determined by thermal denaturation (Marmur & Doty, 1962) using Escherichia coli K-12 as a standard. Amplification of the 16S rRNA gene was performed by PCR using a bacterial universal primer set (27F and 1492R; Lane, 1991). The PCR product was purified using the AxyPrep PCR Purification kit (AxyGen) and sequenced using an automated sequencer (Applied Biosystems model 3730). Similarity searches were performed using the NCBI program blast () and the sequence match program of the Ribosomal Database Project (). A phylogenetic tree of strain LQY-7T and closely related strains was reconstructed using the software package mega version 3.1 (Kumar et al., 2004) after multiple alignment of data using clustal_x (Thompson et al., 1997). Distances were calculated according to the Kimura two-parameter model (Kimura, 1980, 1983) and clustering was performed by the neighbour-joining method (Saitou & Nei, 1987). Bootstrap analysis of 1200 resamplings was used to evaluate tree topology (Felsenstein, 1993).

    An almost complete 16S rRNA gene sequence (1438 bp) was obtained for strain LQY-7T. Similarity search results in the Ribosomal Database Project and GenBank indicated that strain LQY-7T was a member of the genus Flavobacterium. It shared 95.0 % similarity with Flavobacterium indicum GPTSA100-9T and <94 % similarities with other Flavobacterium species. A tree (Fig. 1⇓) depicting the phylogenetic relationships of strain LQY-7T with closely related strains demonstrated that the strain formed a subclade with F. indicum GPTSA100-9T with a high bootstrap value (98 %), also indicating that this isolate probably represents a novel member of the genus Flavobacterium.

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining tree showing the phylogenetic relationship between strain LQY-7T and related species based on 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 1200 replications) are indicated at the branch-points. Bar, evolutionary distance (Knuc) of 0.01.

    Chemotaxonomically, the major fatty acids (>5 %) of the strain included iso-C15 : 0 (39.5 %), iso-C15 : 1 G (26.3 %), iso-C17 : 0 3-OH (7.9 %), anteiso-C15 : 0 (6.3 %) and iso-C15 : 0 3-OH (5.6 %), which is in agreement with the profiles for most species of the genus Flavobacterium. Detailed fatty acid profiles are given in Table 1⇓.

    Table 1.

    Cellular fatty acid composition (%) of strain LQY-7T and closely related species of the genus Flavobacterium

    Strains: 1, strain LQY-7T (this study); 2, F. indicum GPTSA100-9T (Saha & Chakrabarti, 2006); 3, F. cucumis R2A45-3T (Weon et al., 2007); 4, F. columnare ATCC 23463T (Weon et al., 2007); 5. F. terrae R2A1-13T (Weon et al., 2007); 6, F. suncheonense KACC 11423T (Kim et al., 2006); 7, F. saliperosum S13T (Wang et al., 2006); 8, F. cheniae NJ-26T (Qu et al., 2008). −, <1 % or not detected; nr, not reported.

    The novel strain was Gram-negative, non-spore-forming and rod-shaped (0.3–0.7×1.3–2.0 μm); gliding motility was not observed, Congo red was not absorbed and flexirubin-type pigments were not produced. Colonies on TSA were yellow, glistening, sticky, convex and circular with entire margins. Growth was observed at 15–37 °C (optimum, 30 °C), at pH 6.0–8.5 (optimum, pH 7.0) and in 0–5 % NaCl (optimum, 1 %). Good growth was observed on nutrient agar and TSA. Weak growth occurred on R2A agar. No growth occurred on cetrimide agar, Simmons' citrate agar or MacConkey agar. The G+C content of the genomic DNA was 34 mol%, which fell within the range of 30–42 mol% reported for the genus Flavobacterium (Bernardet & Bowman, 2006). Phenotypic characteristics that differentiate strain LQY-7T from related Flavobacterium species are listed in Table 2⇓.

    Table 2.

    Differential phenotypic characteristics of strain LQY-7T and type strains of closely related species of the genus Flavobacterium

    Strains: 1, strain LQY-7T (this study); 2, F. indicum GPTSA100-9T (Saha & Chakrabarti, 2006); 3, F. cucumis R2A45-3T (Weon et al., 2007); 4, F. columnare ATCC 23463T (Bernardet & Grimont, 1989; Bernardet et al., 1996); 5, F. terrae R2A1-13T (Weon et al., 2007); 6, F. suncheonense KACC 11423T (Kim et al., 2006); 7, F. saliperosum S13T (Wang et al., 2006); 8, F. cheniae NJ-26T (Qu et al., 2008). +, Positive; −, negative; w, weakly positive; nr, not reported; y, yellow; gy, greenish yellow; yo, yellowish orange.

    Therefore, on the basis of its phenotypic and phylogenetic characteristics, strain LQY-7T should be classified as a representative of a novel species of the genus Flavobacterium, for which the name Flavobacterium haoranii sp. nov. is proposed.

    Description of Flavobacterium haoranii sp. nov.

    Flavobacterium haoranii (hao.ran′i.i. N.L. gen. masc. n. haoranii of Hao-Ran, in honour of Hao-Ran Jian, a respected Chinese microbiologist, for his enormous contributions to the establishment and development of environmental microbiology in China).

    Cells are Gram-negative rods, 0.3–0.7 μm in width and 1.3–2.0 μm in length, non-spore-forming and non-motile. Congo red is not absorbed and flexirubin-type pigments are not produced. Colonies on TSA are yellow, circular and convex with entire margins. Grows at 15–37 °C, at pH 6.0–8.5 and in 0–5 % NaCl. Good growth occurs on nutrient agar and TSA; weak growth occurs on R2A agar. No growth occurs on cetrimide agar, Simmons' citrate agar or MacConkey agar. Starch, gelatin and aesculin are degraded, but casein, cellulose, agar, pectin, chitin, DNA, tyrosine and egg yolk are not. Positive for catalase and oxidase. The Voges–Proskauer test is negative. In API 20NE tests, nitrate reduction, indole production, glucose fermentation, arginine dihydrolase and β-galactosidase are negative, but urease and aesculin hydrolysis are positive; does not assimilate all carbohydrates tested. In API ZYM tests, alkaline phosphatase, esterase C4, esterase lipase C8, cystine arylamidase, trypsin, α-chymotrypsin, leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and α-glucosidase activities are present. Lipase C14, α-galactosidase, β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, β-glucosidase, α-mannosidase and α-fucosidase activities are absent. In API 50CH tests, acid is produced from aesculin, maltose, inulin and starch. Sensitive to erythromycin, chloramphenicol, rifampicin, spectinomycin, cefradine, streptomycin, ampicillin, polymyxin B, tetracycline, penicillin G, lincomycin, carbenicillin, amoxicillin and vancomycin; resistant to roxithromycin, gentamicin, kanamycin and bacitracin.

    The type strain is LQY-7T (=ACCC 05409T =KCTC 23008T), isolated from activated sludge in a synthetic pyrethroid-manufacturing wastewater treatment facility (Yangnong Chemical Group Co., Jiangsu Province, China).

    Acknowledgments

    We are grateful to Dr Susanne Verbarg and Dr B. J. Tindall (German Collection of Microorganisms and Cell Cultures) for analysis of the cellular fatty acids. This work was supported by the National Natural Science Foundation of China (30970099), the National High Technology Research and Development Program of China (2006AA10Z402) and the Social Development Program Fund of Jiangsu Province (BS2007056).

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