16S rdna-based phylogenetic analysis. The first 443 bp of the 16S rrna gene were

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1 1 Supporting Information 2 16S rdna-based phylogenetic analysis. The first 443 bp of the 16S rrna gene were 3 briefly amplified from the bacterial genomic DNA by PCR using a PCR Master Mix including 4 AmpliTaq Gold DNA Polymerase and 0005F (5`-TGGAGAGTTTGATCCTGGCTCAG-3`) and R (5`-TACCGCGGCTGCTGGCAC-3`) primers (TaKaRa, Shiga, Japan). 1 PCR reaction 6 was performed on a T1 Thermocycler (Biometra, Goettingen, Germany) using an Ex Taq 7 polymerase (Takara-shuzo) under the condition recommended by the manufacturer. The PCR 8 cycle included an initial denaturation period of 10 min at 95 C and 30 cycles at 95 C for 30 9 sec, 60 C for 30 sec, and 72 C for 45 sec, followed by 72 C for 10 min and incubation at 4 10 C until processed further. The PCR amplicon was purified with the Wizard PCR Preps DNA kit 11 (Promega, Medison, MI). 12 Effect of culture conditions on growth and carotenoids production. In all tests, cells 13 were pre-cultured in an appropriate medium (4 ml) at 30 ºC in a rotary shaker (150 rpm) for 1 14 day. To test the effect of medium composition, five commercial media were tested. Twenty ml 15 of each medium was inoculated with 1% pre-culture in a 150-mL Erlenmeyer flask and 16 incubated at 30 C in a rotary shaker (150 rpm) for 2 days. To investigate the effect of glucose 17 concentration, NB medium was supplemented with 1% (w/v) glucose. To test the effect of 18 aeration, three aeration conditions were established by varying the media volume, vessel and

2 19 agitation rate. For low aeration condition, cells were inoculated in 4m L NB in a culture tube 20 (φ18 mm), then incubated in a rocking shaker (120 rpm). For medium aeration condition, cells 21 were inoculated in 20 ml NB in 150 ml volume Erlenmeyer flask, then incubated in arbitrary 22 shaker (150 rpm). For high aeration condition, cells were inoculated in 50 ml NB in 500 ml 23 volume baffled flask, then incubated in arbitrary shaker (200 rpm). Growth of strain N-5 was 24 determined by measuring the optical density values determined at 660 nm using a Hitachi 25 U2000 spectrophotometer

3 37 38 Table S1. Pattern of sole carbon source utilization by strain N-5. Result Positive (17) Borderline (12) Carbon source α-cyclodextrin, dextrin, tween 80, α-d-glucose, maltose, pyruvic acid methyl ester, β-hydroxybutyric acid, succinic acid, L-alaninamide, D- alanine, asparagine, L-glutamic acid, glycyl-l-aspartic acid, glycyl-l glutamic acid, L-proline, L-serine, α-d-glucose-1-phosphate glycogen, tween 40, D-mannose, L-rhamnose, turanose, succinic acid mono-methyl ester, itaconic acid, α-keto valeric acid, bromosuccinic acid, L-alanyl- L- glycine, L-ornithine, L-threonine, D- glucose-6-phosphate 39 Negative (66) N-acetyl-D- galactosamine, N-acetyl-D- glucosamine, adonitol, L- arabinose, D-arabitol, D-cellobiose, i-erythritol, D-fructose, L-fucose, D-galactose, gentiobiose, m-inositol, α-d-lactose, lactulose, D- mannitol, D-melibiose, β-methyl-d-glucoside, D-psicose, D-raffinose, D-sorbitol, sucrose, D-trehalose, turanose, xylitol, acetic acid, cisaconitic acid, citric acid, formic acid, D-galactonic acid Lactone, D- galacturonic acid, D-gluconic acid, D-glucosaminic acid, D- glucuronic acid, α-hydroxybutyric acid, γ-hydroxybutyric acid, p- hydroxy phenlyacetic acid, α-keto butyric acid, α-ketoglutaric acid, D,L-lactic acid, malonic acid, propionic acid, quinic acid, D- saccharic acid, sebacic acid, succinamic acid, glucuronamide, L- alanine, L-aspartic acid, L-histidine, hydroxy-l-proline, L-leucine, L- phenylalanine, L-pyroglutamic acid, D-serine, D,L-carnitine, γ-amino butyric acid, urocanic acid, inosine, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, glycerol, D,L,αglycerol phosphate Total 95 carbon substrates were tested using BIOLOG GN2 microplate

4 Table S2. Differential characteristics between strain N-5 and the type strains of the closely related species of Brevundimonas spp. 1, N-5; 2, Brevundimonas vesicularis DSM 7226 T, and 3, Brevundimonas nasdae DSM T (W1-2B T ). Characters are scored as: +, positive; -, negative. The data are obtained from this study, Segers et al and Li et al., All strains were positive for catalase, oxidase, hydrolysis of aesculin and assimilation of glucose, maltose and malate. Characteristic N Source of isolation marine water medicinal leech space station Colony pigmentation orange orange creamy β-galactosidase Hydrolysis of gelatin Indole production Acid from glucose Assimilation of L-arabinose Gluconate Adipic acid Citrate Phenyl acetate Capric N-acetyl D-glucosamine

5 µm Figure S1. Light microscope micrograph of strain N-5 cells. Cells are rods ( µm in 66 diameter and µm in length), which grew singly, in pairs and chains. Cells were grown 67 for 2 days at 30 C on nutrient agar (NA). Bar, 5.0 μm

6 Figure S2. Chiral HPLC for separation and identification of all-trans-astaxanthin produced by 80 strain N-5. Astaxanthin synthetic (Asx-S) contains all three trans isomers 3S,3 S; 3R, 3 S and 81 3S,3 S (bottom diagrams A & B). Total carotenoids (TC) and purified astaxanthin (Asx) 82 produced by strain N5 contain 3S,3 S (top diagrams A & B). Mixed astaxanthin (TC or purified 83 astaxanthin of N5 and Astaxanthin synthetic) contain three isomers (middle diagrams A & B). 84 Separation of carotenoids was performed using method D on Sumichiral OA-2000 column 85 (4.0 x 250mm, Sumitomo Chemicals) with n-heptane:methylenchloride:isopropanol (70:25:5)

7 86 at a flow rate of 1 ml min -1 (A) or 0.8 ml min -1 (B), and detected at 480 nm Figure S3. Growth and production of carotenoids by strain N-5. (A) Growth curve of strain N-5 92 in LB broth. Effect of NB broth supplementation with glucose (1%) on (B) growth (OD 660 ) and 93 (C) carotenoids production (OD 480 ) of strain N-5. Data and error bars represent the average 94 and standard deviation of three measurements, respectively

8 References: 101 (1) Brosius, J.; Palmer, M. L.; Kennedy, P. J.; Noller, H. F., Complete nucleotide sequence of a S ribosomal RNA gene from Escherichia coli. PNAS 1978, 75 (10), (2) Segers, P.; Vancanneyt, M.; Pot, B.; Torck, U.; Hoste, B.; Dewettinck, D.; Falsen, E.; 104 Kersters, K.; De Vos, P., Classification of Pseudomonas diminuta Leifson and Hugh 1954 and 105 Pseudomonas vesicularis Büsing, Döll, and Freytag 1953 in Brevundimonas gen. nov. as 106 Brevundimonas diminuta comb. nov. and Brevundimonas vesicularis comb. nov., respectively. 107 Int. J. Syst. Evol. Microbiol. 1994, 44 (3), (3) Li, Y.; Kawamura, Y.; Fujiwara, N.; Naka, T.; Liu, H.; Huang, X.; Kobayashi, K.; Ezaki, T., 109 Sphingomonas yabuuchiae sp. nov. and Brevundimonas nasdae sp. nov., isolated from the 110 Russian space laboratory Mir. Int. J. Syst. Evol. Microbiol. 2004, 54 (3),