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Nonhongiella Spirulinensis Gen. Nov., Sp. Nov., a Bacterium Isolated from a Cultivation Pond of Spirulina Platensis in Sanya, China

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Nonhongiella Spirulinensis Gen. Nov., Sp. Nov., a Bacterium Isolated from a Cultivation Pond of Spirulina Platensis in Sanya, China Antonie van Leeuwenhoek (2013) 104:933–939 DOI 10.1007/s10482-013-0012-4 ORIGINAL PAPER Nonhongiella spirulinensis gen. nov., sp. nov., a bacterium isolated from a cultivation pond of Spirulina platensis in Sanya, China Guanghua Wang • Jiewei Fan • Hualian Wu • Xiaoyong Zhang • Guangyu Li • Hao Zhang • Xi Yang • Fangfang Ye • Wenzhou Xiang • Xiang Li Received: 30 May 2013 / Accepted: 19 August 2013 / Published online: 27 August 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract A Gram-negative, aerobic, motile rod strain, OH (4.63 %), and C12:0 (4.09 %). The DNA G?C designated Ma-20T, was isolated from a pool of marine content of strain Ma-20T was 58 mol %. Phylogenetic Spirulina platensis cultivation, Sanya, China, and was analyses based on 16S rRNA gene sequences showed subjected to a polyphasic taxonomy study. Strain Ma- that strain Ma-20T belonging to Gammaproteobacteria, 20T can grow in the presence of 0.5–11 % (w/v) NaCl, it shared 88.46–91.55 and 89.21–91.26 % 16S rRNA 10–43 °C and pH 6–10, and grew optimally at 30 °C, pH gene sequence similarity to the type strains in genus 7.5–9.0 in natural seawater medium. The polar lipids Hahella and Marinobacter, respectively. In addition to were composed of phosphatidylethanolamine, three the large 16S rRNA gene sequence difference, Ma-20T unidentified phospholipids and three unidentified polar can also be distinguished from the reference type strains lipids. The respiratory quinone was ubiquinone 8 (Q-8) Hahella ganghwensis FR1050T and Marinobacter T and the major fatty acids were C18:1x6c/C18:1x7c hydrocarbonoclasticus sp. 17 by several phenotypic (summed feature 8, 32.84 %), C16:1x6c/C16:1x7c characteristics and chemotaxonomic properties. On the (summed feature 3, 30.76 %), C16:0 (13.54 %), C12:03- basis of phenotypic, chemotaxonomic and phylogenetic properties, strain Ma-20T is suggested to represent a novel species of a new genus in Gammaproteobacteria, Electronic supplementary material for which the name Nonhongiella spirulinensis gen. The online version of T this article (doi:10.1007/s10482-013-0012-4) contains supple- nov., sp. nov. is proposed. The type strain is Ma-20 mentary material, which is available to authorized users. (=KCTC 32221T=LMG 27470T). G. Wang Á J. Fan Á H. Wu Á X. Zhang Á H. Zhang Á X. Yang Á F. Ye Á W. Xiang (&) Á X. Li (&) Keywords Nonhongiella spirulinensis Á Key Laboratory of Marine Bio-resources Sustainable Spirulina platensis Á Chemotaxonomy Á 16S Utilization (LMB), Guangdong Key Laboratory of Marine rDNA based phylogeny Materia Medica (LMMM-GD), South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China e-mail: [email protected] Introduction X. Li e-mail: [email protected] The most efficient way to gather information about a microorganism is to study it in culture and relate that G. Li knowledge to field observations (Giovannoni et al. Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, 2007), while it was estimated that only 0.01–0.1 % of Xiamen 361005, China the marine microorganism was cultivable (Kogure et al. 123 934 Antonie van Leeuwenhoek (2013) 104:933–939 1979; Porter and Feig 1980; Ferguson et al. 1984). New May, 2012, Sanya, China, and then diluted to 1,000 cultivation strategies have brought much more micro- fold and plated on SMA plates (simplified from marine bial plankton species into the laboratory, which also agar 2216, contains peptone 0.5 g, yeast extract 0.1 g, hold good promise in the future (Giovannoni et al. ferric citrate 0.1 g, agar powder 18 g, natural seawater 2007). Phytoplankton are known to release up to 25 % 1 L, pH 8.0). The S. platensis cultivation pool was of the total organic carbon fixed by photosynthesis into running in a continuous style, and the medium was the surrounding ‘‘phycosphere’’ or region immediately natural seawater amended with NaHCO3, the natural surrounding and influenced by algal cells (Doucette seawater was just filtered to remove bigger particles, 1995). The extracellular products of phytoplankton in which marine bacteria was preserved, then domes- include simple amino acids, organic acids, peptide, ticated and enrichment in the process of S. platensis sugars, polyalcohols, vitamins, enzymes, and so on. cultivation. Strain Ma-20T was isolated from the SMA These products may play important role in marine food plate and was further cultivated at 30 °C on MA plates chains, especially as potential nutrients for bacteria (peptone 5 g, yeast extract 1 g, ferric citrate 0.1 g, (Fogg 1966; Riquelme et al. 1989; Whittaker and potassium nitrate 0.5 g, agar powder 18 g, natural Feeney 1971;Williams1975). They were uptake in a seawater 1 L, pH 8.0), and maintained as glycerol rate twice of glucose by heterotrophic bacteria suspensions (glycerol/natural seawater = 20/80, v/v) (Strasˇkrabova´ and Fuksa 1982), in which amino acids at -80 °C. and organic acids were more frequently utilized than sugars and other derivatives (Berland et al. 1970). So far, Morphological and biochemical characterization several new bacterial taxons were isolated and identified from phytoplankton environments, for example, Alg- For comparative studies, the type strains Hahella iphilus aromaticivorans and Porticoccus hydrocarbo- ganghwensis FR1050T (=KCTC 12277T, Baik et al. noclasticus were isolated from dinoflagellate 2005) and Marinobacter hydrocarbonoclasticus sp. Lingulodinium polyedrum (Gutierrez et al. 2012a, b), 17T (=MCCC 1A03297T=ATCC 49840T, Gauthier Polycyclovorans algicola was isolated from diatom et al. 1992;Spro¨er et al.1998), obtained from the Skeletonema costatum (Gutierrez et al. 2013), Pelagi- Korean Collection for Type Cultures (KCTC) and monas varians and Planktotalea frisia were from Marine Culture Collection Center of China (MCCC), phytoplankton bloom (Hahnke et al. 2012, 2013), respectively, were used as control. Data for other type Marivita cryptomonadis was isolated from Cryptomon- strains in Hahella was from Lee et al. (2001) and as sp. (Hwang et al. 2009), Lentibacter algarum, (2008). Cell morphology was examined by transmis- Algibacter lectus and Mesonia algae were isolated from sion electron microscopy (Hitachi TEM System- green algae (Nedashkovskaya et al. 2003, 2004;Lietal. H7650) after negative staining with 1 % (w/v) phos- 2012). Spirulina platensis (SP) was a good bioactive photungstic acid. Mobility was tested using the additive and hold good promise as components of hanging drop technique (Bernardet et al. 2002). The microbial culture media (Blinkova et al. 2001), and Gram reaction was determined by using cells grown which had been verified to have good performance in the on MA at 30 °C for 72 h, according to the method growth of Rhodospirillum rubrum (Vatsala et al. 2011). described by Gerhardt et al. (1994). Catalase activity In this study, a novel marine bacterial taxon, strain was investigated via bubble production in 3 % (v/v) Ma-20T was isolated from a pool of marine S. platensis hydrogen peroxide solution; oxidase activity was cultivation, Sanya, China, and was subjected to determined by oxidase test strips (Huankai, China). polyphasic identification. Substrate utilization (sole carbon source) and chem- ical sensitivity were determined by using BIOLOG GEN III MicroPlate and API 20NE according to the Materials and methods manufacturer’s protocol. Growth in the presence of 0, 0.5, 1, 3, 5, 7, 9, 11, 15 and 20 % (w/v) NaCl was Isolation and culture conditions tested in MA without agar powder, growth at pH 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 and 11 was tested in MA Aquatic sample was collected from the pool of S. without agar powder (pH 5.5–8, 0.1 M KH2PO4/ platensis cultivation in a form of algae broth on 14th K2HPO4 buffer; pH 8.5–10, 0.1 M Glycine/NaOH 123 Antonie van Leeuwenhoek (2013) 104:933–939 935 buffer; pH 11, 0.1 M Na2CO3/NaOH buffer), too. Results and discussion Growth at 4, 10, 22, 30, 37, 43 and 48 °C was tested on MA. Endospore formation and other phenotypic test Colonies of strain Ma-20T on MA were pale-yellow, were determined as described by Dong and Cai (2001). non-translucent, and circular with regular edges. Cells Insecticide resistant was performed accidentally in the were 0.73–0.8 lm wide and 1.24–4.26 lm long course of the incubator drosophila extinguishing by (Supplementary Fig. S1), which was the widest cell beta cypermethin and imiprothin mixture spraying. among Ma-20T, H. ganghwensis FR1050T and M. hydrocarbonoclasticus sp. 17T. The organism can Chemotaxonomic characterization grow at pH 6.0–10.0 and 10–43 °C in the presence of 0.5–11 % (w/v) NaCl, and grew optimally at 30 °C, Cell biomass for DNA extraction, polar lipids and pH 7.5–9.0 in natural seawater medium. The range of quinone analyses was obtained from MA broth after NaCl tolerance was wider than type strains in Hahella 4 days incubation at 30 °C, and that for cellular fatty (0.5–9 %, Lee et al. 2008) and narrower than M. acid composition analysis was from MA in the same hydrocarbonoclasticus sp. 17T (0.5–20 %). conditions. Respiratory lipoquinones were extracted Strain Ma-20T was Gram-negative, aerobic, non- according to Collins (1994) and analyzed by reversed- spore-forming, catalase-positive, oxidase-negative, phase HPLC as described by Komagata and Suzuki nitrate not reduction, and motile rods with single (1987). Polar lipids were extracted as described by polar flagellum. The organism can hydrolyse Tween Kamekura (1993), and identified by two-dimensional 40, utilize D-maltose, L-alanine, L-aspartic acid, L- thin-layer chromatography followed by spraying with glutamic acid, L-histidine, L-serine, a-keto-glutaric appropriate detection reagents (Tindall 1990).
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