Ureibacillus defluvii sp. nov., isolated from a thermophilic microbial fuel cell

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1 International Journal of Systematic and Evolutionary Microbiology (2014), 64, DOI /ijs Ureibacillus defluvii sp. nov., isolated from a thermophilic microbial fuel cell Shungui Zhou, 1 Jia Tang, 1 Dongxing Qin, 1,2 Qin Lu 1 and Guiqin Yang 1 Correspondence Shungui Zhou sgzhou@soil.gd.cn 1 Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou , PR China 2 Chemistry and Materials Institute, Sichuan Normal University, Chengdu , PR China A thermophilic bacterium, designated DX-1 T, was isolated from the anode biofilm of a microbial fuel cell (MFC). Cells of strain DX-1 T were oxidase-positive, catalase-positive and Gram-stainingnegative. The strain was found to be rod-shaped and non-motile and to produce subterminal spores. The strain was able to grow with NaCl at concentrations ranging from 0 to 6 %, at temperatures of C (optimum 55 6C) and ph (optimum ph 7.0). Phylogenetic analyses based on 16S rrna gene sequences showed that strain DX-1 T formed a cluster with Ureibacillus thermosphaericus DSM T (96.9 % 16S rrna sequence similarity), Ureibacillus composti DSM T (95.8 %), Ureibacillus thermophilus DSM T (95.7 %) and Ureibacillus terrenus DSM T (95.3 %). The G+C content of the genomic DNA was 40.4 mol%. The major quinone was MK-7, the peptidoglycan type was L-LysrD-Asp, and the major cellular fatty acids (.5 %) were iso-c 16 : 0 and iso-c 14 : 0. The polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol and phospholipids of unknown composition. Based on phenotypic characteristics, chemotaxonomic features and results of phylogenetic analyses, the strain was determined to represent a distinct novel species of the genus Ureibacillus, and the name proposed for the novel species is Ureibacillus defluvii sp. nov., with type strain DX-1 T (5CGMCC T 5KCTC T ). The genus Ureibacillus includes five species: Ureibacillus thermosphaericus (Andersson et al., 1995), Ureibacillus terrenus (Fortina et al., 2001), Ureibacillus suwonesis (Kim et al., 2006), Ureibacillus composti and Ureibacillus thermophilus (Weon et al., 2007). Bacteria belonging to the genus Ureibacillus are aerobic and thermophilic, and strains have been isolated from various environments, such as air (Andersson et al., 1995), soil (Fortina et al., 2001) and compost (Weon et al., 2007). In this study, a Gramstaining-negative bacterium, DX-1 T, was isolated from the anode biofilm of a microbial fuel cell (MFC). On the basis of polyphasic taxonomic studies following the minimal standards for describing novel taxa of aerobic, endosporeforming bacteria proposed by Logan et al. (2009), the strain is proposed to represent a novel species of the genus Ureibacillus. Strain DX-1 T was isolated from the anode biofilm of a sewage-fed MFC that had been continuously operated at 55 uc for 3 months. The biofilm was cut into pieces and one piece (about 5 cm 2 ) was suspended in a bottle Abbreviation: MFC, microbial fuel cell. The GenBank/EMBL/DDBJ accession number for the 16S rrna gene sequence of Ureibacillus defluvii DX-1 T is JX Three supplementary figures are available with the online version of this paper. containing 50 ml sterilized phosphate buffer solution (PBS; 140 mm NaCl, 2.7 mm KCl, 10 mm Na 2 HPO 4 and 1.8 mm KH 2 PO 4, ph 7.4). The bottle was incubated at 55 uc with shaking for one night, and 200 ml of culture was taken out, serially diluted, spread onto nutrient agar (NA) plates and incubated at 55 uc. After culture for 24 h, wellisolated colonies were picked using sterilized toothpicks and transferred to fresh NA plates. The colony isolation procedure was repeated until a single colony was obtained. Cell morphology was observed with a JEM 1400 transmission electron microscope (JEOL) after cultivation on NA plates at 55 uc for 12 h. In preparation for electron microscopy, the bacterial cells were suspended in PBS, dried on a nickel-coated mesh and negatively stained with phosphotungstic acid. The motility of cells was tested by observing the growth spread in a test tube containing semisolid NA medium. After cells of strain DX-1 T were cultivated on NB agar at 55 uc for 2 days, the presence of endospores was investigated using staining solution kit HB8300 (Qingdao Hope-Bio Technology) and observed with a light microscope (BX51, Olympus). The Gram staining reaction and hydrolysis of gelatin and casein were carried out according to the protocols of Dong & Cai (2001). The ph range (ph at intervals of 0.5 ph unit) for growth was determined in nutrient broth (NB) buffered with citrate/phosphate buffer or Tris/hydrochloride buffer G 2014 IUMS Printed in Great Britain 1617

2 S. Zhou and others (Breznak & Costilow, 1994). Tolerance of NaCl was determined in NB containing 0 7 % NaCl (w/v) with increments of 0.5 %. Growth was examined at various temperatures (20, 25, 30, 35, 40, 45, 50, 55, 60 and 65 uc) in NB. Growth on MacConkey agar was also tested. Catalase and oxidase activities were tested as described by McCarthy & Cross (1984). Other physiological and biochemical properties were determined according to the protocols of Dong & Cai (2001). Carbon metabolism, enzyme activities and acid production characteristics were examined with the ID 32GN and API 20E systems (biomérieux) according to the manufacturer s instructions. The isolate was thermophilic and the cells were Gramstaining-negative, catalase-positive and oxidase-positive. Gelatin and casein were not hydrolysed. Cells of strain DX- 1 T were non-motile and rod-shaped ( mm wide and mm long) (Fig. 1). Colonies were light brown and convex. The strain could hydrolyse aesculin and utilize valeric acid, 4-hydroxybenzoic acid, L-rhamnose, N- acetylglucosamine, D-ribose, inositol, sucrose and maltose. The results were negative for H 2 S and indole production, urease, b-galactosidase, arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase. The strain grew at uc (optimum 55 uc) and at ph (optimum ph 7.0). Strain DX-1 T could tolerate a higher concentration of NaCl (6 %, w/v) than closely related strains. Detailed morphological, physiological and biochemical characteristics are summarized in the description of the species and Table 1 and Fig. S1 available in the online Supplementary Material. Table 1. Distinctive characteristics of strain DX-1 T and its closest phylogenetic neighbours Strains: 1, Ureibacillus defluvii sp. nov. DX-1 T ;2,U. thermosphaericus DSM T ;3,U. composti DSM T ;4,U. thermophilus DSM T ;5,U. terrenus DSM T. +, Positive; 2, negative; ND, not determined. For spore position: ST, subterminal; T, terminal. Data were taken from this study unless indicated. Characteristic Spore position ST/T ST/T ST/T ND ST/T ph Range ph Optimum Growth Without NaCl With 6 % NaCl (w/v) Growth at: 30 uc uc Temperature optimum (uc) Urease Assimilation of: L-Arabinose L-Rhamnose N Acetylglucosamine D-Ribose Sucrose Maltose D-Mannose Acid production from: D-Glucose L-Rhamnose D-Mannitol Sucrose L-Arabinose DNA G+C content (mol%) * 42.4D 38.5D 39.6* *Data taken from Fortina et al. (2001). DData taken from Weon et al. (2007). Fig. 1. Transmission electron micrograph of a negatively stained cell of strain DX-1 T, which was characterized by a long rod shape. Bar, 1 mm. Genomic DNA was extracted according to standard procedures (Sambrook & Russell, 2001). The 16S rrna gene was PCR-amplified from genomic DNA using two bacterial universal primers (27f and 1492r; Baker et al., 2003). The PCR product was gel-purified using a Gel Extraction kit D (Omega Bio-tek), cloned into a plasmid vector using a TA cloning kit (TaKaRa) and sequenced by Sangon (Shanghai, China) using the 27f and 1492r primer pair as the sequencing primers. The pairwise sequence similarity was calculated using the EzTaxon-e server ( Kim et al., 2012). Phylogenetic analyses were carried out using the software package MEGA version 5.0 (Tamura et al., 2011) after multiple alignments of the sequences with CLUSTAL_X (Thompson et al., 1997). Non-homologous and ambiguous 1618 International Journal of Systematic and Evolutionary Microbiology 64

3 Ureibacillus defluvii sp. nov. nucleotide positions were excluded from the calculations, and a total of 1338 nt positions for 16S rrna were included in the further phylogenetic analyses. Distances were calculated according to Kimura s two-parameter model (Kimura, 1980) and clustering was performed with the neighbour-joining method (Saitou & Nei, 1987), minimum-evolution method (Rzhetsky & Nei, 1992) and maximum-likelihood method (Felsenstein, 1981). Statistical support for the branches of the phylogenetic trees was determined using bootstrap analyses (based on 1000 resamplings) (Felsenstein, 1985). A nearly complete 16S rrna gene sequence was determined for DX-1 T (1473 nt). Phylogenetic analyses based on 16S rrna gene sequences revealed that strain DX-1 T was most closely related to U. thermosphaericus DSM T (96.9 % 16S rrna sequence similarity), followed by U. composti DSM T (95.8 %), U. thermophilus DSM T (95.7 %) and U. terrenus DSM T (95.3 %), and consistently formed a cluster with them in the neighbour-joining tree, minimum-evolution tree and maximum-likelihood tree (Fig. 2, Fig. S2). Given that the levels of 16S rrna gene sequence similarity were all lower than 97 % between strain DX-1 T and its phylogenetic neighbours, DNA DNA hybridization studies were not carried out. It was clear from the phylogenetic analyses based on 16S rrna gene sequences that the novel isolate represented a member of the genus Ureibacillus and a distinct phyletic line that can be considered as a separate genomic species (Stackebrandt & Goebel, 1994). The G+C content of the genomic DNA was determined by HPLC according to the method of Mesbah et al. (1989). For cellular fatty acid analysis, cells were grown in NB at 55 uc for 24 h to exponential phase. Fatty acids in whole cells were saponified, methylated and extracted according to the standard protocol given by the Microbial Identification System (MIDI, version 6.0B, Microbial ID). The fatty acids were analysed with GC (model 6850, Agilent Technologies) and identified using the TSBA6.0 database of the Microbial Identification System (Sasser, 1990). Respiratory quinones were extracted according to the method of Collins et al. (1977) and analysed with HPLC as described by Tamaoka et al. (1983). Polar lipids were extracted, examined by two-dimensional TLC and identified using previously described procedures (Minnikin et al., 1979). Peptidoglycan structure was analysed according to the methods of Fortina et al. (2001). The G+C content of the genomic DNA of strain DX-1 T was determined to be 40.4 mol%. The fatty acid profile of Lysinibacillus macroides LMG T (AJ628749) Lysinibacillus boronitolerans 10a T (AB199591) Lysinibacillus xylanilyticus XDB9 T (FJ477040) Lysinibacillus sphaericus C3-41 (CP000817) Lysinibacillus fusiformis NBRC T (AB271743) Lysinibacillus massiliensis T (AY677116) 93 Lysinibacillus meyeri WS 4626 T (HE577173) Lysinibacillus odysseyi 34hs-1 T (AF526913) Caryophanon tenue DSM T (AJ491303) Sporosarcina newyorkensis 6062 T (GU994085) Planococcus plakortidis AS/ASP6(II) T (JF775504) Planococcus maitriensis S1 T (AJ544622) 100 Planomicrobium flavidum ISL-41 T (FJ265708) 71 Planomicrobium koreense JG07 T (AF144750) Ureibacillus defluvii DX-1 T (JX274433) 96 Ureibacillus thermosphaericus DSM T (AB101594) 97 Ureibacillus composti HC145 T (DQ348071) 96 Ureibacillus terrenus DSM T (AJ276403) 51 Ureibacillus thermophilus HC148 T (DQ348072) 73 Ureibacillus suwonensis DSM T (AY850379) Geobacillus stearothermophilus NBRC T (AB271757) Fig. 2. Phylogenetic tree reconstructed using the neighbour-joining method based on 16S rrna gene sequences, showing the position of strain DX-1 T and the closely related species of genera within the family Planococcaceae. Bootstrap values, generated from 1000 resamplings, at or above 50 % are indicated at the branching points. Geobacillus stearothermophilus NBRC T was used as the outgroup. Bar, 0.01 substitutions per nucleotide position. The minimum-evolution tree showed essentially the same topology (data not shown)

4 S. Zhou and others strain DX-1 T was similar to those of the type strains of the species of the genus Ureibacillus in that iso-c 16 : 0 predominates (65.4 %) (Table 2). However, the proportions of specific major fatty acids were different. The presence of C 16 : 1 v7c alcohol and iso-c 12 : 0 (Table 2), the relatively high percentage of iso-c 14 : 0, the relatively low percentage of anteiso-c 15 : 0 and the absence of C 16 : 0 indicated that strain DX-1 T represented a novel species within the genus Ureibacillus. The peptidoglycan was of the L-LysrD-Asp type (variation A4a). The polar lipids consisted of major amounts of diphosphatidylglycerol, moderate to minor amounts of phosphatidylglycerol and phospholipids of unknown composition (Fig. S3). The results for polar lipids and cell wall composition were in accordance with those given in the description of the genus Ureibacillus (Fortina et al., 2001). For respiratory quinones, strain DX-1 T possessed major amounts of MK-7, with MK- 8 as a minor component. With a G+C content of 40.4 mol%, strain DX-1 T contained MK-7 as the major respiratory quinone, L- LysrD-Asp as the peptidoglycan type and iso-c 16 : 0 and iso-c 14 : 0 as the predominant fatty acids. These and its position in the phylogenetic trees supported the affiliation of strain DX-1 T with the genus Ureibacillus. However, it could be differentiated clearly from closely related species of the genus Ureibacillus by characteristics such as temperature range and NaCl range for growth and the ability to assimilate L-rhamnose and N-acetylglucosamine. In detail, strain DX-1 T could grow at 30 uc or in presence of 6 % (w/v) NaCl, but the closely related strains could not. In addition, strain DX-1 T was negative for producing acid from D-glucose, L-rhamnose, D-mannitol, sucrose or L- arabinose. At least one of the reference strains was positive for acid production from these substrates. Strain DX-1 T had the ability to assimilate D-ribose, sucrose and maltose. At least one of the reference strains was negative for assimilation of these substrates. Moreover, strain DX-1 T showed less than 97 % 16S rrna gene sequence similarity to its closest relatives and other phenotypic and chemotaxonomic differences such as the proportions of some fatty acids, which also confirmed that this strain represented a species that differs from the species of the genus Ureibacillus with validly published names. Therefore, on the basis of the data presented, strain DX-1 T is considered to represent a novel species of the genus Ureibacillus for which the name Ureibacillus defluvii sp. nov. is proposed. Description of Ureibacillus defluvii sp. nov. Ureibacillus defluvii (de.flu9vi.i. L. gen. n. defluvii of sewage). Cells are thermophilic, Gram-staining-negative rods approximately mm wide and mm long. Cells are non-motile without flagella. Spores are ellipsoidal and occur subterminally and terminally in swollen sporangia. Colonies are round, light brown and convex after 24 h of aerobic growth on NA at 55 uc. Growth Table 2. Cellular fatty acid profiles of strain DX-1 T and closely related species of the genus Ureibacillus Strains: 1, Ureibacillus defluvii sp. nov. DX-1 T ;2,U. thermosphaericus DSM T ;3,U. composti DSM T ;4,U. thermophilus DSM T ;5,U. terrenus DSM T. Data were taken from this study. Values are percentages of the total fatty acids; 2, Not detected. Fatty acids (%) Saturated straight-chain C 14 : C 15 : C 16 : C 18 : C 20 : C 20 : 0 3-OH Unsaturated straight-chain C 16 : 1 v7c alcohol C 18 : 1 v9c C 20 : 2 v6,9c Summed feature 3* Summed feature 8* Saturated branched-chain iso-c 12 : iso-c 14 : iso-c 15 : iso-c 16 : iso-c 17 : iso-c 19 : anteiso-c 15 : anteiso-c 17 : Unsaturated branched-chain anteiso-c 17 : Summed features are groups of two or three fatty acids that cannot be separated by GLC using the MIDI system. *Summed feature 3 comprises C 16 : 1 v7c and/or C 16 : 1 v6c. *Summed feature 8 comprises C 18 : 1 v7c and/or C 18 : 1 v6c. occurs in 0 6 % (w/v) NaCl, at ph (optimum ph 7.0) and at uc (optimum 55 uc). The strain is catalase-positive and oxidase-positive. With the API 20E kit, results are negative for b-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and the Voges Proskauer reaction. Nitrate cannot be reduced. Indole and hydrogen sulfide are not produced. The strain can hydrolyse aesculin but not gelatin or casein. The following substrates are utilized: D- mannitol, valeric acid, 4-hydroxybenzoic acid, L-rhamnose, N-acetylglucosamine, D-ribose, inositol, sucrose, maltose, sodium malonate, sodium acetate, potassium 5-ketogluconate and 3-hydroxybenzoic acid. Acid is produced from D-sorbitol and amygdalin, but not from D-glucose, D- mannitol, inositol, L-rhamnose, sucrose, melibiose or L- arabinose. The peptidoglycan is of the L-LysrD-Asp type (variation A4a). The polar lipids consist of phosphatidylglycerol, diphosphatidylglycerol and phospholipids of unknown composition. The predominant fatty acids 1620 International Journal of Systematic and Evolutionary Microbiology 64

5 Ureibacillus defluvii sp. nov. (.5 %) are iso-c 16 : 0 and iso-c 14 : 0. The major quinone is MK-7. The type strain DX-1 T (5CGMCC T 5KCTC T ) was isolated from the anode biofilm of a sewagefed microbial fuel cell. The DNA G+C content is 40.4 mol%. Acknowledgements This work was supported by the Team Project of the Guangdong Natural Science Foundation (S and S ) and the Science and Technology Planning Project of Guangdong Province, China (2012B and 2012B ). References Andersson, M., Laukkanen, M., Nurmiaho-Lassila, E.-L., Rainey, F. A., Niemela, S. I. & Salkinoja-Salonen, M. (1995). Bacillus thermosphaericus sp. nov. a new thermophilic ureolytic: Bacillus isolated from air. Syst Appl Microbiol 18, Baker, G. C., Smith, J. J. & Cowan, D. A. (2003). Review and reanalysis of domain-specific 16S primers. J Microbiol Methods 55, Breznak, J. A. & Costilow, R. N. (1994). Physicochemical factors in growth. In Methods for General and Molecular Bacteriology, pp Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology. Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. (1977). Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100, Dong, X.-Z. & Cai, M.-Y. (editors) (2001). Determination of biochemical properties. In Manual for the Systematic Identification of General Bacteria, pp Beijing, China: Science Press (in Chinese). Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, Fortina, M. G., Pukall, R., Schumann, P., Mora, D., Parini, C., Manachini, P. L. & Stackebrandt, E. (2001). Ureibacillus gen. nov., a new genus to accommodate Bacillus thermosphaericus (Andersson et al. 1995), emendation of Ureibacillus thermosphaericus and description of Ureibacillus terrenus sp. nov. Int J Syst Evol Microbiol 51, Kim, B.-Y., Lee, S.-Y., Weon, H.-Y., Kwon, S.-W., Go, S.-J., Park, Y.-K., Schumann, P. & Fritze, D. (2006). Ureibacillus suwonensis sp. nov., isolated from cotton waste composts. Int J Syst Evol Microbiol 56, Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e: a prokaryotic 16S rrna gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, Logan, N. A., Berge, O., Bishop, A. H., Busse, H.-J., De Vos, P., Fritze, D., Heyndrickx, M., Kämpfer, P., Rabinovitch, L. & other authors (2009). Proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria. Int J Syst Evol Microbiol 59, McCarthy, A. J. & Cross, T. (1984). A taxonomic study of Thermomonospora and other monosporic actinomycetes. J Gen Microbiol 130, Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by highperformance liquid chromatography. Int J Syst Bacteriol 39, Minnikin, D. E., Collins, M. D. & Goodfellow, M. (1979). Fatty-acid and polar lipid-composition in the classification of Cellulomonas, Oerskovia and related taxa. J Appl Microbiol 47, Rzhetsky, A. & Nei, M. (1992). A simple method for estimating and testing minimum-evolution trees. Mol Biol Evol 9, Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI. Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA DNA reassociation and 16S rrna sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, Tamaoka, J., Katayama-Fujimura, Y. & Kuraishi, H. (1983). Analysis of bacterial menaquinone mixtures by high performance liquid chromatography. J Appl Bacteriol 54, Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, Weon, H.-Y., Lee, S.-Y., Kim, B.-Y., Noh, H.-J., Schumann, P., Kim, J.-S. & Kwon, S.-W. (2007). Ureibacillus composti sp. nov. and Ureibacillus thermophilus sp. nov., isolated from livestock-manure composts. Int J Syst Evol Microbiol 57,