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

International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1617 1621 DOI 10.1099/ijs.0.056655-0 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 510650, PR China 2 Chemistry and Materials Institute, Sichuan Normal University, Chengdu 610068, 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 25 60 6C (optimum 55 6C) and ph 6.0 8.0 (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 10633 T (96.9 % 16S rrna sequence similarity), Ureibacillus composti DSM 17951 T (95.8 %), Ureibacillus thermophilus DSM 17952 T (95.7 %) and Ureibacillus terrenus DSM 12654 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 1.12358 T 5KCTC 33127 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 JX274433. 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 5.0 10.0 at intervals of 0.5 ph unit) for growth was determined in nutrient broth (NB) buffered with citrate/phosphate buffer or Tris/hydrochloride buffer 056655 G 2014 IUMS Printed in Great Britain 1617

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 (0.3 0.7 mm wide and 1.5 2.5 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 25 60 uc (optimum 55 uc) and at ph 6.0 8.0 (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 10633 T ;3,U. composti DSM 17951 T ;4,U. thermophilus DSM 17952 T ;5,U. terrenus DSM 12654 T. +, Positive; 2, negative; ND, not determined. For spore position: ST, subterminal; T, terminal. Data were taken from this study unless indicated. Characteristic 1 2 3 4 5 Spore position ST/T ST/T ST/T ND ST/T ph Range 6.0 8.0 7.0 9.0 6.0 8.0 6.0 8.0 7.0 9.0 ph Optimum 7.0 7.0 8.0 7.0 7.0 7.0 8.0 Growth Without NaCl + 2 + + 2 With 6 % NaCl (w/v) + 2 2 2 2 Growth at: 30 uc + 2 2 2 2 65 uc 2 2 2 + + Temperature 55 50 55 55 55 50 55 optimum (uc) Urease 2 + 2 2 + Assimilation of: L-Arabinose 2 2 + + 2 L-Rhamnose + 2 2 2 2 N- + 2 2 2 + Acetylglucosamine D-Ribose + 2 + + 2 Sucrose + 2 2 + + Maltose + 2 2 + 2 D-Mannose 2 + 2 2 2 Acid production from: D-Glucose 2 + 2 2 + L-Rhamnose 2 + 2 2 + D-Mannitol 2 + 2 2 + Sucrose 2 + 2 2 + L-Arabinose 2 + 2 + + DNA G+C content (mol%) 40.4 37.0* 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 D2500-01 (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 (http://eztaxon-e.ezbiocloud.net/; 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

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 10633 T (96.9 % 16S rrna sequence similarity), followed by U. composti DSM 17951 T (95.8 %), U. thermophilus DSM 17952 T (95.7 %) and U. terrenus DSM 12654 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 0.01 54 97 Lysinibacillus macroides LMG 18474 T (AJ628749) 61 100 Lysinibacillus boronitolerans 10a T (AB199591) Lysinibacillus xylanilyticus XDB9 T (FJ477040) Lysinibacillus sphaericus C3-41 (CP000817) 58 100 Lysinibacillus fusiformis NBRC 15717 T (AB271743) Lysinibacillus massiliensis 4400831 T (AY677116) 93 Lysinibacillus meyeri WS 4626 T (HE577173) Lysinibacillus odysseyi 34hs-1 T (AF526913) Caryophanon tenue DSM 14152 T (AJ491303) Sporosarcina newyorkensis 6062 T (GU994085) 79 99 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 10633 T (AB101594) 97 Ureibacillus composti HC145 T (DQ348071) 96 Ureibacillus terrenus DSM 12654 T (AJ276403) 51 Ureibacillus thermophilus HC148 T (DQ348072) 73 Ureibacillus suwonensis DSM 16752 T (AY850379) Geobacillus stearothermophilus NBRC 12550 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 12550 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). http://ijs.sgmjournals.org 1619

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 0.3 0.7 mm wide and 1.5 2.5 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 10633 T ;3,U. composti DSM 17951 T ;4,U. thermophilus DSM 17952 T ;5,U. terrenus DSM 12654 T. Data were taken from this study. Values are percentages of the total fatty acids; 2, Not detected. Fatty acids (%) 1 2 3 4 5 Saturated straight-chain C 14 : 0 0.6 0.8 0.6 0.5 2 C 15 : 0 2 3.6 2 2 3.5 C 16 : 0 2 0.7 2.8 2.8 6.2 C 18 : 0 2 3.6 0.4 0.3 4.7 C 20 : 0 2 0.5 2 2 1.2 C 20 : 0 3-OH 2 2 0.6 2 2 Unsaturated straight-chain C 16 : 1 v7c alcohol 3.3 2 2 2 2 C 18 : 1 v9c 2.5 3.8 2 2 2 C 20 : 2 v6,9c 2 2 2 0.2 0.8 Summed feature 3* 2.3 2.7 2 2 2 Summed feature 8* 2.7 5.3 2 2 2 Saturated branched-chain iso-c 12 : 0 1.0 2 2 2 2 iso-c 14 : 0 13.4 0.2 1.0 0.8 2 iso-c 15 : 0 1.5 3.2 4.3 5.8 5.8 iso-c 16 : 0 65.4 52.3 63.6 64.3 58.7 iso-c 17 : 0 0.2 7.8 12.1 12.5 10.8 iso-c 19 : 0 2 2 2 2 0.6 anteiso-c 15 : 0 0.6 11.2 9.7 9.9 6.5 anteiso-c 17 : 0 0.4 2.3 3.1 3.4 0.8 Unsaturated branched-chain anteiso-c 17 : 1 0.1 1.2 2 2 2 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 6.0 8.0 (optimum ph 7.0) and at 25 60 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

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