Ann Microbiol (2016) 66:895–905 DOI 10.1007/s13213-015-1174-2 ORIGINAL ARTICLE Bacillus pseudoflexus sp. nov., a moderately halophilic bacterium isolated from compost Piyush Chandna1 & Shanmugam Mayilraj3 & Ramesh Chander Kuhad1,2 Received: 28 May 2015 /Accepted: 20 October 2015 /Published online: 14 November 2015 # Springer-Verlag Berlin Heidelberg and the University of Milan 2015 Abstract A Gram-stain-positive, motile, rod-shaped, phosphatidylglycerol, diphosphatidylglycerol, phosphatidyleth- endospore-forming moderately halophilic bacterium (RC1T) anolamine, and five unidentified phospholipids, as well as one was isolated from compost. The strain, designated RC1T,was unidentified aminophospholipid) supported the affiliation of grown at 2–11 % (w v−1)NaCl[optimum5%(wv−1)], 15– RC1T to the genus Bacillus. The polyphasic taxonomic investi- 42 °C (optimum 30 °C), and pH of 5.0–11.0 (optimal pH 7.0). gation (phenotypic and chemotaxonomic characteristics, phylo- The G+C content of DNAwas 40.4±0.2 mol %. The 16S rRNA genetic data, and genomic distinctiveness) indicated that RC1T gene sequence of RC1T showed 97 % similarity with Bacillus represents a novel species within the genus Bacillus,forwhich paraflexus MTCC 9831T and99%similaritywithB. flexus the name B. pseudoflexus sp. nov. is now proposed. The type DSM 1320T. Phylogenetic analysis based on 16S ribosomal strain B. pseudoflexus is RC1T (= MTCC 9830T=KCTC RNA gene sequences showed that RC1T was a member of the 13723T=CCM 7753T). genus Bacillus and most closely related to B. paraflexus MTCC 9831T and B. flexus DSM 1320T. Further, DNA-DNA hybridi- Keywords Bacillus pseudoflexus sp. nov. zation revealed that the relatedness between RC1T and phyloge- Gram-stain-positive . Moderately halophilic . Mesophilic netically closely related species, B. paraflexus MTCC 9831T and bacteria . Compost B. flexus DSM 1320T, was 41 and 58 %, respectively. Chemo- taxonomic data (peptidoglycan type, meso-diaminopimelic acid; predominant isoprenoid quinone, MK-7; predominant fatty Introduction acids, anteiso-C15:0 (28.9 %), iso-C15:0 (15.9 %), iso-C17:1E (7.8 %), and iso-C18:1H (5.4 %); major polar lipids, Composting is a complex biological process, involving many species of bacteria and fungi that convert low-value materials The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene into high-value products. A large number of strains, belonging to sequence of RC1T is FN999944. The type strain Bacillus pseudoflexus is the genera Bacillus, Micrococcus, Clostridium, Staphylococcus, T RC1 has been deposited in the Microbial Type Culture Collection and Citrobacter, Serratia, Klebsiella, Pseudomonas, Enterobacter, Gene Bank (MTCC 9830T), Korean Collection for Type Cultures (KCTC 13723T), and Czech Collection of Microorganisms (CCM 7753T). and Escherichia, were isolated from compost (Herrmann and Shann 1997; Ryckeboer et al. 2003a, b;Ashrafetal.2007; * Ramesh Chander Kuhad Yamamoto et al. 2009;Dangetal.2011;Sharmaetal.2013; [email protected] Jurado et al. 2014; Guo et al. 2015). Amongst the aerobic pro- karyotes isolated from compost, the majority belong to the genus 1 Lignocellulose Biotechnology Laboratory, Department of Bacillus. The genus Bacillus was first established by Ferdinand Microbiology, University of Delhi South Campus, New Delhi 110 Cohn and included three species of rod-shaped bacteria, 021, India B. subtilis, B. anthracis,andB. ulna (Cohn 1872). Presently, 2 Department of Microbiology, Central University of Haryana, the genus Bacillus comprises more than 295 species, including Mahendergarh District, Pali, Haryana 123 029, India seven subspecies (http://www.bacterio.net/). Members of this 3 Microbial Type Culture Collection (MTCC), Institute of Microbial genus are able to form endospore which resists adverse Technology (IMTECH), Chandigarh 160 036, India conditions. These were isolated from various sources, 896 Ann Microbiol (2016) 66:895–905 including volcanic ash (Logan et al. 2004), fresh and marine motility was observed using both light microscopy (Cayol water (Yoon et al. 2004;Baiketal.2010), hypersaline sites et al. 1994) and in motility agar medium (Farmer 1999). The (Hong et al. 2012), and different kinds of soils (Madhaiyan presence of flagella was determined by negative staining. et al. 2010;Chenetal.2011). Halophilic and/or halotolerant Growth at different temperatures (4.0, 15.0, 25.0, 30.0, 37.0, bacilli have been isolated from the Dead Sea (Arahal et al. 40.0, 42.0, 45.0, 50.0, 55.0, 60.0, and 65.0 °C) was 1999), sea water (Yoon et al. 2003), deep sea sediments (Bae ascertained in the trypticase soya medium agar (TSA) slants et al. 2005), solar saltern (Lim et al. 2006), and salt lake (Xue for 48 h. Growth under anaerobic conditions was also evalu- et al. 2008). Moderately halophilic/halotolerant bacteria that ated after incubating in an anaerobic chamber (BBL, USA) on grow optimally in media containing 3–15 % (w v−1)NaClare 0.1× TSA supplemented with 0.5 % (w v−1) glucose or 0.1 % widely distributed among many hypersaline habitats (Ventosa (w v−1) potassium nitrate. Growth at different pH 4.0, 5.0, 6.0, et al. 1989). Taxonomically, the genus Bacillus represents phys- 7.0, 8.0, 9.0, 10.0, and 11.0 was assessed in trypticase soya iologically a very heterogeneous group of Gram-stain-positive, (TS) broth at 30 °C; the pH was adjusted to acidic, neutral, and aerobic, or facultative anaerobic endospores-forming, rod- alkaline pH by sodium acetate, Tris–HCl, and sodium hydrox- shaped bacteria widely distributed in nature. Very few reports ide (NaOH), respectively. Growth at different NaCl concen- are available on the isolation of moderate halophilic Gram-stain- trations (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 %, positive bacterium from compost. The present article describes wv−1) was examined in nutrient broth medium at pH 7.3±0.1. the isolation of an aerobic, Gram-stain-positive, mesophilic, Growth was monitored by measuring the turbidity at optical moderately halophilic, and endospore-forming rod-shaped bac- density (OD)600 by using a spectroscopic method terium, RC1T, from compost. (SPECORD, Analytik Jena, Germany). These physiological The aim of this study was to determine the taxonomic sta- tests were carried out according to the methods described pre- tus of an isolate RC1T by using the polyphasic approach. The viously in the literature (Gordon et al. 1973;Barrowand minimal standards required for characterization of an isolate Feltham 1993; Logan et al. 2009). Other physiological char- were performed as recommended by Logan et al. (2009). acteristics, including methyl red tests and Voges–Proskauer, resistance to lysozyme, and utilization of citrate, were exam- ined as described by Smibert and Krieg (1994). Biochemical Materials and methods characteristics, such as: activity of catalase, oxidase, decar- boxylation of ornithine and lysine, nitrate reduction, hydroly- During the process of composting, various mesophilic and sis of starch, gelatin, casein, aesculin, ortho-Nitrophenyl-β- thermophilic bacteria were isolated and characterized, as de- galactoside (ONPG), and Tween 20, 40, 60, 80, and 100; scribed by Chandna et al. (2013a). A bacterium designated as assimilation of carbon source, production of hydrogen sulfide T RC1 isolated from the compost sample was grown on nutri- (H2S) and indole; susceptibility to different antibiotics ent agar (NA) (HiMedia M 012, India), pH 7.3±0.1 and tem- employing the disk diffusion method using commercially perature 30±2 °C, and was screened for various enzymes like available disks (HiMedia, India), were tested according to xylanase, cellulase, amylase, and protease. The pure culture the methods of Smibert and Krieg (1994). The urease test was maintained in 20 % (v v−1) glycerol suspension at −20 °C. was performed according to Kim et al. (2006). For comparison, a reference (type) strain was obtained from Utilization of various carbon sources, enzyme activity, and the Deutsche Sammlung von Mikroorganismen und acid production from different substrates were tested with the Zellkulturen, Braunschweig, Germany (DSMZ) [B. flexus commercially available Biolog (GEN III, USA) and 1320T (ABO21185)]. Unless otherwise indicated, morpholog- bioMérieux systems (Hazelwood, USA), as per the manufac- ical, physiological, phenotypic, chemotaxonomic, and molec- turers’ instructions. ular studies were performed with the cells grown on NA (pH 7.3±0.1) at 30±2 °C. Chemotaxonomic characteristics Morphology, physiological, and biochemical For quantitative analysis of cellular fatty acid, the isolated characteristics RC1T and reference strains {B. flexus DSM 1320T and B. paraflexus MTCC 9831T) were grown at 30 °C for 24 h The colony morphology of an 18-h-old culture of RC1T was (late logarithmic phase) on TSA plates. The cells of both examined, employing light microscopy (Olympus DP70, Ja- strains were harvested, washed thrice with distilled water to pan), scanning electron microscopy (SEM; LEO435VP, remove traces of media, and freeze-dried. The fatty acid meth- USA), and transmission electron microscopy (TEM; yl esters (FAME) were extracted, saponified, and methylated Morgagni 268D, USA), as described previously (Chandna according to the protocol of the MIDI Sherlock Microbial et al. 2013b). Reaction to Gram stain was examined using Identification System (MIS) system (MIDI; Microbial ID, light microscopy according to Gerhardt et al. (1981). Cell USA). The fatty acids were analyzed by gas chromatography Ann Microbiol (2016) 66:895–905 897