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;Sproër 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 chemical 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). The acid, D-malic acid, L-malic acid, bromo-succinic acid, cellular fatty acid composition was determined by a-hydroxy butyric acid, b-hydroxy butyric acid, a- using the Sherlock Microbial Identification System keto-butyric acid, acetoacetic acid, propionic acid and (MIDI) according to the manufacturer’s instructions. acetic acid as sole carbon source. Nitrate reduction can The fatty acids were analyzed by GC (Agilent be an important index to distinguish Ma-20T to H. G6890N) and identified by using the Microbial ganghwensis FR1050T and M. hydrocarbonoclasticus Identification software package (Sherlock Version sp. 17T, too. Among type strains of Hahella and M. 6.0). The G?C content of the DNA of strain Ma-20T hydrocarbonoclasticus sp. 17T, only Ma-20T cannot was determined by using the HPLC method (Mesbah reduce nitrate (result for H. ganghwensis FR1050T et al. 1989). from API 20NE was different to that of Baik et al. 2005). Other cultural, physiological and biochemical Phylogenetic analysis characteristics of strain Ma-20T are given in the species description and in Table 1 in detail. The near complete 16S rRNA gene sequence of strain The polar lipids of strain Ma-20T were composed of Ma-20T was obtained by PCR amplification with the phosphatidylethanolamine, three unidentified phospho- universal primers 27F and 1492R (Lane 1991). lipids and three unidentified polar lipids; that of H. Alignment of 16S rRNA gene sequences of members ganghwensis FR1050T included phosphatidylethanol- in the Gammaproteobacteria was performed by using amine, one unidentified phospholipid and four uniden- the SINA software package (Pruesse et al. 2012)in tified polar lipids; while M. hydrocarbonoclasticus sp. SILVA rRNA database. Phylogenetic tree was con- 17T contained phosphatidylethanolamine and four structed using the neighbour-joining (Saitou and Nei unidentified phospholipids (Supplementary Fig. S2). 1987), maximum-parsimony (Swofford 1993), and The major respiratory quinone of strain Ma-20T was maximum likelihood (Felsenstein 1981) methods in ubiquinone 8 (Q-8), while which in both genera Hahella the software package MEGA version 5.0 (Tamura and Marinobacter (Sproër et al. 1998;Wangetal.2009) et al. 2011). The genetic distance matrices were were Q-9 except for H. chejuensis 96CJ10356T (mate- estimated by the Kimura two-parameter model (Kim- rial and data unavailable). The major fatty acids were ura 1980). The topology of the phylogenetic tree was C18:1x6c/C18:1x7c (summed feature 8), C16:1x6c/ evaluated by using the bootstrap resampling method of C16:1x7c (summed feature 3), C16:0,C12:03-OH and Felsenstein (1985) with 1,000 replicates. C12:0 (Table 2). The high abundance of summed feature 123 936 Antonie van Leeuwenhoek (2013) 104:933–939

Table 1 Phenotypic characteristics that distinguish strain Ma- Table 2 Fatty acid content (%) of strain Ma-20T and the type 20T to close related type strains strains of related taxa Characteristics 1 2 3 Fatty acid 1 2 3

a Nitrate reduction – ? ? C10:0 1.11 – 2.08

Oxidase – ?? C12:0 4.09 3.74 6.59

Temperature for growth (°C) 10–43 10–43 10–45 C14:0 – 2.48 2.62

NaCl tolerance % (w/v) 0.5–11 1–9 0.5–20 C16:0 13.54 18.94 20.47

Acid production from glucose – – ? C17:0 – – 2.12

Utilization of C18:0 1.41 – 2.15

D-Maltose ? –– C10:03-OH 2.78 – –

D-Glucose – ? – C12:03-OH 4.63 6.36 12.16

Mannose – ? – C16:0N alcohol – 8.52 –

Quinone composition Q-8 Q-9 Q-9 C17:010-methyl – – – b c DNA(G?C) (mol%) 58 44 57.5 Iso-C17:0 1.38 – – Strains: 1, Ma-20T;2,Hahella ganghwensis FR1050T;3, C16:x9c – 10.59 8.94 T Marinobacter hydrocarbonoclasticus sp. 17 C17:1x8c 1.10 – 3.17 a Result from API 20NE was different to that of Baik et al. C18:3x6c(6,9,12) – 4.73 –

(2005) C18:1x9c – 36.74 26.32 b Data from Baik et al. (2005) Summed feature 3a 30.76 4.1 5.28 c Data from Spro¨er et al. (1998) Summed feature 8a 32.84 – 1.26 Summed feature 9a – – 1.62

8 and 3 was an important feature in distinguishing T T T T Strains: 1, Ma-20 ;2,Hahella ganghwensis KCTC 12277 ;3, Ma-20 to H. ganghwensis FR1050 and M. hydrocar- Marinobacter hydrocarbonoclasticus sp. 17T T bonoclasticus sp. 17 ,meanwhile,therewasnoC16:0N a Summed feature 3 comprised C x6c and/or C x7c. T 16:1 16:1 alcohol present in Ma-20 , which was a typical Summed feature 8 comprised C18:1x7c and/or C18:1x6c. component in the fatty acids profile of genus Hahella Summed feature 9 comprised iso-C17:1x9c and/or C16:010- (Lee et al. 2008). The DNA G?C content of strain methyl Ma-20T was 58 mol%. Preliminary sequence comparisons with 16S rRNA gene sequences deposited in the GenBank/EzTaxon-e On the basis of phenotypic, chemotaxonomic and (Kim et al. 2012) database indicated that strain Ma- phylogenetic difference to its close related type 20T (1,421 bp, GenBank accession No. JX981925) strains, we suggest that strain Ma-20T represents a belonged to the Gammaproteobacteria. On the basis novel species of a new genus in Gammaproteobacte- of 16S rRNA gene sequence similarity, the closest ria, for which the name Nonhongiella spirulinensis relative was H. ganghwensis FR1050T. Ma-20T shared gen. nov., sp. nov. is proposed. 91.55 and 91.45 % 16S rRNA gene sequence similarity with the type strains of H. ganghwensis FR1050T Description of Nonhongiella gen. nov. and H. chejuensis 96CJ10356T, respectively; meanwhile shared 89.21–91.26 % sequence similarity with Nonhongiella (Non.hong.i.el’la, L. adv. non, not; N.L. type strains in the genus of Marinobacter (results from dim. fem. n. Hongiella, a name of a bacterial genus; EzTaxon-e 16S rRNA gene sequence blast). In the N.L. fem. n. Nonhongiella, not Hongiella, in recog- maximum likelihood phylogenetic tree (Fig. 1) based nition of the event that Ma-20T had been replaced for on 16S rRNA gene sequences, strain Ma-20T formed a about 1 month by a Hongiella strain in the course of distinct branch within the monophyletic clade com- new taxon identification, both of these two strains prising of Ma-20T and a subclade of the genus were red pigmented at that time). Hahella, the topology was also supported by the Cells are Gram-negative, aerobic, non-spore-forming, maximum-parsimony tree and neighbor-joining tree catalase-positive, oxidase-negative, and motile rods with (Supplementary Figs. S3, S4). single polar flagellum. Major fatty acids are 123 Antonie van Leeuwenhoek (2013) 104:933–939 937

100 Haliea salexigens 3X/A02/235T(AY576769) 62 T Chromocurvus halotolerans EG19 (AM691086) Dasania marina KOPRI 20902T(AY771747) Pseudomonas aeruginosa DSM 50071T(HE978271) Cellvibrio mixtus subsp. mixtus ACM 2601T(AF448515) T 56 Marinimicrobium koreense M9 (AY839869) T 81 Gilvimarinus chinensis QM42 (DQ822530) 56 Marinimicrobium haloxylanilyticum SX15T(GQ920839) Simiduia agarivorans SA1T(EF617350) Oceanospirillum linum ATCC 11336T(M22365) T 74 Marinobacter hydrocarbonoclasticus SP.17 (X67022) T 51 Zooshikella ganghwensis JC2044 (AY130994) 87 Kistimonas asteriae KMD 001T(EU599216) Nonhongiella spirulinensis Ma-20T(JX981925) T 52 Hahella chejuensis KCTC 2396 (CP000155) 100 Hahella ganghwensis FR1050T(AY676463) Marinobacterium lutimaris AN9T(FJ230839) Escherichia coli ATCC 11775T(X80725)

0.02

Fig. 1 Maximum likelihood phylogenetic tree based on 16S bootstrap resamplings, only values above 50 % are shown. Bar rRNA gene sequences showing the relationship between strain 0.02 substitutions per site. Escherichia coli ATCC11775T was Ma-20T and type species in related genera from Gammaprote- used as outgroup obacteria. Numbers at nodes indicate percentages of 1,000

C18:1x6c/C18:1x7c, C16:1x6c/C16:1x7c, C16:0,C12:03- (w/v) NaCl, and grows optimally at 30 °C, pH 7.5–9.0 OH and C12:0. The polar lipids include phosphatidyleth- in natural seawater medium. anolamine, three unidentified phospholipids and three Production of H2S or indole doesn’t occur. unidentified polar lipids. The major respiratory quinone Negative for Arginine dihydrolase, b-galactosidase, is Q-8. The DNA G?C content of the type species is b-glucuronidase, utilization of urea, and reduction of 58 mol%. Member of the Gammaproteobacteria.The nitrate. Tween 40,D-maltose, L-alanine, L-aspartic type species is N. spirulinensis. acid, L-glutamic acid, L-histidine, L-serine, a-keto- glutaric acid, D-malic acid, L-malic acid, bromo- Description of Nonhongiella spirulinensis sp. nov. succinic acid, a-hydroxy butyric acid, b-hydroxy butyric acid, a-keto-butyric acid, acetoacetic acid, Nonhongiella spirulinensis (spi.ru.lin.en’sis, L. fem. propionic acid and acetic acid can be utilized. While n. spirula-ae, a coil; N.L. fem. dim. n. Spirulina,a pectin, dextrin, D-trehalose, D-cellobiose, gentiobiose, small coil; N.L. fem. adj. spirulinensis, referring to sucrose, D-turanose, starchyose, D-raffinose, a-D-lac- Spirulina, a cyanobacterium). tose, D-melibiose, b-methyl-D-glucoside, D-salicin, The description is as for the genus with the N-acetyl-D-glucosamine, N-acetyl-b-D-mannosamine, following additional properties. Cells are usually N-acetyl-D-galactosamine, N-acetyl neuralminic acid, 0.73–0.8 lm in diameter and 1.24–4.26 lm long. D-glucose, D-mannose, D-fructose, D-galactose, Colonies on MA are pale-yellow, non-translucent, and 3-methyl glucose, L-fucose, L-rhamnose, inosine, D- circular with regular edges. Growth occurs at pH sorbitol, D-mannitol, D-arabitol, myo-inositol, glyc- 6.0–10.0 and 10–43 °C in the presence of 0.5–11 % erol, D-glucose-6-PO4, D-Fructose-6-PO4, D-aspartic 123 938 Antonie van Leeuwenhoek (2013) 104:933–939 acid, D-serine, glycyl-L-prolin, L-arginine, L-pyroglu- Gauthier MJ, Lafay B, Christen R, Fernandez L, Acquaviva M, Bonin P, Bertrand JC (1992) Marinobacter hydrocarbo- tamic acid, D-galacturonic acid, L-galactonic acid noclasticus gen. nov., sp. nov., a new, extremely halotol- lactone, D-gluconic acid, D-glucuronic acid, glucuro- erant, hydrocarbon-degrading marine bacterium. Int J Syst namide, mucic acid, quinic acid, D-saccharic acid, p- Bacteriol 42(4):568–576 hydroxy-phenylacetic acid, methyl pyruvate, D-lactic Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) (1994) Methods for general and molecular bacteriology. American acid methyl ester, L-lactic acid, citric acid, c-amino Society for Microbiology, Washington, DC butryric acid, and formic acid were not utilized. This Giovannoni SJ, Foster RA, Rappe´ MS, Epstein S (2007) New isolate is sensitive to sodium lactate, fusidic acid, cultivation strategies bring more microbial plankton spe- guanidine hydrochloride, niaproof, lithium chloride, cies into the laboratory. Oceanography 20(2):62–69 sodium bromate, potassium tellurite. Gutierrez T, Green DH, Whitman WB, Nichols PD, Semple KT, T T Aitken MD (2012a) Algiphilus aromaticivorans gen. nov., The type strain, Ma-20 (=KCTC 32221 =LMG sp. nov., an aromatic hydrocarbon-degrading bacterium T 27470 ), was isolated from pool of S. platensis isolated from a culture of the marine dinoflagellate cultivation, Sanya, China. Lingulodinium polyedrum, and proposal of Algiphilaceae fam. nov. 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