A new psychrophilic, strictly aerobic bacterium, strain 40-3, was isolated from  seawater samples collected at Spitsbergen in the Arctic. The cells are gram negative,  straight or curved rod shaped and non-spore forming (2-3 µm long and 0.4-0.6 µm  wide). Colonies on agar medium are slightly orange, circular, smooth and convex.  40-3 strain grows optimally over the temperature range of 10-15 °C and a pH range  of 7-8 in media containing 8 to 9 % NaCl (w/v). Growth occurs with α cyclo-dextrin,  dextrin, tween 80, N-acetyl-D-glucosamine, α-D-glucose, maltose, sucrose, methyl  pyruvate, D,L-lactitate, succiniate , bromo succinic acid, inosine, esculin ferric citrate,  L-arabinose, potassium gluconate, malic acid and trisodium citrate. In the presence of  glucose H2 S was produced and nitrates are reduced to nitrites. The fatty acid methyl  ester (FAME) are composed of 17.89 % straight chain saturated FAMEs, 14,85 %  terminally branched saturated FAMEs and 17.73 % monounsaturated FAMEs. The DNA  base ratio is 48 mol % G + C. Phylogenetic analysis reveals as close relationship to 
Shewanella putrefaciens with 99 % 16S rDNA composition identity and 50 % DNADNA similarity. The phylogenetic evidence, together with phenotypic characteristics,  show that this psychrophilic strain constitute a novel species of the genus Shewanella.  The name Shewanella arctica is proposed. Interestingly, when grown on glucose as a  carbon source Shewanella arctica produced numerous industrially important enzyme  systems including amylase, pullulanase, protease, ornithine decarboxylase, alkaline  phospatase, esterase (C4), lipase (C8), leucine arylamidase, valine arylamidase,  naphthol-AS-BI-phosphohydrolase and N-acetyl-β–  . Due to the 
functional new physochrophilic Shewanella strainresource for the isolation of new  enzyme system, which may operate at low reaction thereby increasing the energy  efficiency of industrial processes. 
 

• Spitsbergen

• Psychrophile

• Shewanella arctica

• Industrial enzymes

Qoura F, Brueck T, Antranikian G (2014) The Psychrophile Shewanella arctica sp. Nov: A New Source of Industrially Important Enzyme Systems. JSM Biotechnol Bioeng 2(1): 1026.

FAME: Fatty Acid Methyl Ester

Recently, increasing attention has been directed to coldadapted microorganisms able to grow at/or close to the freezing point of water, namely psychrophiles [1]. Cold adapted microorganisms are found in both permanently and temporarily cold habitats, which comprise more than 80 % of the Earth’s biosphere. The genus Shewanella MacDonell and Colwell 1985 comprises a ubiquitous group of gram negative, aerobic and facultatively anaerobic δ-Proteobacteria. This genus comprises more than 25 species inhabiting a wide range of environments including spoiled food, oil field wastes, redox interfaces in marine and freshwater, cold water and sediments of the deep sea [2-6]. During the last decade, the bacteria of this genus have received a significant amount of attention due to their important roles in cometabolic bioremediation of halogenated organic pollutants [7], destructive souring of crude petroleum [8] and the dissimilatory reduction of magnesium and iron oxides [9].

Recently, a comprehensive study of the phylogenetic relationships and taxonomy of the genus provided an improved approach for identification of newly isolated wild strains [6]. In this study a novel psychrophilic bacterium was isolated from seawater samples obtained from the area of Spitsbergen, in the Arctic. The novel isolate was found to be similar to, but distinct from in a number of characteristics, a previously described genus, Shewanella. Taxonomic and physiological analysis of the newly isolated strain demonstrated that the represent is a novel specie of the genus Shewanella, and we propose the name Shewanella arctica sp. nov. For the species represented by the strain 40- 3. Further, the isolated Shewanella strain secreted numerous industrially relevant enzyme systems including hydrolyases such, as amylase, protease and, esterases and lipases. Enzymes derived from physchiphilic organism have the potential to operate at low temperatures thereby improving the energy efficiency of biotechnological process [10-12]. Further, many physochophilic enzymes have shown the propensity to operate over a wider temperature profile than those derived from mesophilic organism [13,14]. More recently, various Shewanella sp. have been flagged as excellent sources for novel industrially relevant enzyme systems [15-17]. Therefore, the enzymes systems that can be produced using the current bacterial strain have potential use in the biofuels and food (amylase, lipase) as well as the chemical industry (pullulanase, alkaline phospatase, leucine arylamidase, valine arylamidase).

Sample collection, media and culturing conditions

Seawater samples were collected in 1998 during an expedition at Spitsbergen, Norway. Samples were transported to the laboratory at temperature between 2 to 10 °C. A 1 ml volume of liquid sample was incubated in a complex marine liquid medium. The complex marine medium consisted of a basal medium supplemented with a solution of different carbon sources. The basal medium contained (volume g ): NaCl 28.13 g; KCl 0.77 g;  x  0.02 g;  x  0.5 g;  1.0 g; iron-ammonium-citrate 0.02 g; yeast extract 0.5 g; 10-fold concentration trace element solution (DSM 141) 1 ml; 10-fold concentration vitamin solution (DSM 141) 1 ml;  2.3 g;  x  2.9 g. The carbon source mixture solution (volume g ): NA-acetate 0.5 g;  -succinate 0.5 g; Na-pyruvate 0.5 g; DL-malate 0.5 g; D-mannitol 0.5 g and glucose 2 g. The final pH of the complex medium was 7. Incubation was carried out at 4 °C for about 4-7 days, before growth and colonies on agar plates were observed. Colonies were selected on the basis of morphological differences. For the isolation of a pure culture, serial dilution and plating techniques were applied. The pure isolates were routinely cultivated on complex marine medium agar plates at 15 °C for 4 days.

Cellular characterization

Gram staining test was performed by staining cells using the Hucker method [18]. For the sporulation test cells were grown for up to 6 days in medium containing no carbon source other then 0.1 % (w/v) yeast extract. The presence of spores and cell morphology were determined by phase-contrast microscope (Zeiss/Axioskop).

Optimal temperature, pH and salt requirement for growth

The optimum growth temperature was tested between 4-37 °C and pH 7. The pH optimum for growth was tested between pH 2-10 at 15 °C. The salt requirement was determined on different NaCl concentration between 0 and 10 % (w/v), with no change of the other salts concentrations at pH 7 and 15 °C. Growth was measured by determining the optical density at 600 nm (1 – cm path length) using Schimadzu UV spectrophotometer.

Substrate spectrum

Substrates utilization from the new strain was tested on api 20 NE strips (20050) (Bio Merieux. Inc), api 20 E strips (20 100/20 160) (Bio Merieux. Inc) and Biolog GN2 micro-plates. api 20 NE strips (20050) (Bio Merieux. Inc) and api 20 E strips (20 100/20 160) were also used for testing the ability of the new strain to produce indol (tryptoPhane), acetoin and H2 S (from sodium thiosalfate) and the ability of the strain to reduce nitrates to nitrites and nitrites to nitrogen. The strips and micro-plates were inoculated with the new strain cells suspension (cells were resuspended in NaCl 0.85 % (w/v) medium to a final OD of 0.5 nm) and incubated at 15 °C overnight.

A large number of carbon source were tested: α cyclodextrin, dextrin, Tween 80, Tween 40, N-acetyl-D-glucosamine, α-D-glucose, maltose, sucrose, methylpyruvate, D,L-lactic acid, succinic acid, bromo succinic acid, inosine, esculin ferric citrate, L-arabinose, potassium gluconate, malic acid, trisodium citrate, glycogen, N-acetyl-D-galacto-samine, adonitol, capric acid, D-arabitol, cellobiose, L-erythol, D-fructose, L-fructose, D-galactose, gentiobiose, m-inositol, α-D-lactose, lactulose, D-mannitol, D-mannose, D-mellobiose, β-methyl-D-glucose, D-psicose, D-raffinose, L-rhamnose, D-sorbitol, D-trehalose, turanose, Xylitol, mono-methyl succinate, 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-hydroxy-butyric acid, β-hydroxybutyric acid, γ-hydroxy-butyric acid, p-hydroxyphenylacetic acid, itaconic acid, α-keto butyric acid, α-keto glutaric acid, α-keto valeric acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, seabacic acid, succinamic acid, glucuronamide, alaninamide, D-alanine, L-alanine, L-alanyl-glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl-L-aspartic acid, glycylL-glutamic acid, L-histidine, hydroxy L-proline, L-leucine, L-ornithine, L-phenyl-alanine, L-proline, L-pyroglutamic acid, D-serine, L-serine, L-threonine, D,L-carnitine, γ-amino butyric acid, urocanic acid, uridine, thymidine, phenylthylamine, putrescine, 2-aminoethanol, 2,3-butanediol, glycerol, D,L α-glycerol phosphate, glucose 1-phosphate, glucose 6-phosphate, phenylacetic acid.

Screening for enzyme production

The ability of the new strain to produce: alkaline phospatase, arginine dihydrolase, esterase (C4), esterase lipase (C8), lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α chymotrpsin, acid phosphatase, omithine decarboxylase, lysine decarboxylase, urease, tryptophane deaminase, naphthol-AS-BI-phosphohydrolase, α and β – galactosidase, β – glucoronidase, α and β – glucosidase, N-acetyl-β – glucosaminidase, α – mannosidase and α – fucosidase was tested on api zym (25 200) strips (bio Merieux, Inc) by inoculation the strips wells with cell suspension of the new strain (cells were resuspended in water to a final OD of 5 nm). The strips were incubated at 15 °C for 6 hours.

Amylase, arabinase, arabinoxylanase, protease, HE-cellulase, glucanase, dextranase, galactanase, galactomannanase, βglucanase, pullulanase, curdlanase xylanase and xyloglucanase production from the new strain was tested on diffusion agar plates containing the base medium and 0.1 % (w/v) of one of the following substrates: red pullulan (pullulanase), azo-casien (protease) , AZCL – pullulan (pullulanase), AZCL – HE – cellulose (HE – celulase), AZCL – arabinan (arabinase), AZCL – arabinoxylan (arabinoxylanase), AZCL – curdlan (curdlanase), AZCL – amylose (amylase), AZCL – dextran (dextranase), AZCL – galactan (galactanase), AZCL – galactomannan (galactomannanase), AZCL – β - glucan (β – glucanase) AZCL – xylan (xylanase) and AZCL – xyloglucan (xyloglucanase). Substrate degradation was detected by clearing zone/color diffusion halo around the colonies after the new stain was grown on these substrates agar plates at 10 °C for 2-4 days. AZCL – polymers were purchased from Megazume, Bray, Ireland.

Fatty acids analysis

Cells of the new strain were harvested from a 2 l culture sample by centrifugation and were used for fatty acids analysis. Fatty acids methyl ester (FAME) was performed according to the modified method of Lepage and Roy (1984) [19]. Total lipids were extracted according to Bligh and Dyer (1959) [20]. The FAMEs were analyzed by capillary gas chrompack [21]. A fused silica capillary column D23, 40 m (Fisons) was used for the separation of fatty acid species.The chromatographic conditions were as follows: injector (PTV): 65 °C – 270 °C split ratio 15:1; carrier gas: helium at a 40 cm s-1 flow. Column oven: initial temperature : 60 °C for 0.1 min; from 60 °C to 180 °C at 40 °C min-1; 180 °C for 2 min; from 180 °C to 210 °C for 3min; from 210 °C to 240 °C at 3 °C min-1; 240 °C for 10 min. The spectra were recorded by a flame ionization detector at 280 °C.

16S rDN A amplification and sequencing

Cells of the new strain were harvested from 500 µl a cell culture sample by centrifugation and resuspended in 100 µl of water. A sub-sample of 1 µl was used as template for the amplification of the 16S rDNA. PCR-mediated amplification of the 16S rDNA was carried out according to Stakebrandt & Gloebel (1994) [22]. PCR products were purified using the QIA quick PCR purification kit (Qiagen). Purified PCR products were directly sequenced using the Tag Dye Deoxy Terminator Cycle sequencing kit (Applied Biosystems). Sequence reactions were electrophoresed using Applied Bio systems model 373S DNA sequence. Both strands of amplification product were sequenced using primers 8F, 518F and 1504R [23]. The complete 16S rDNA sequence of the new strain was discovered by the assembly of all sequence products using Pregap4 version 1.4bl and Gap4 version 4.8bl software.

Phylogenetic analysis

BLAST analysis was performed by NBCI online database on the new strain 16S rDNA sequence to determine the phylogenetic grouping to which the new strain was most closely related. Reference sequences utilized in phylogenetic analysis were retrieved from NCBI database and aligned with the newly determined sequence of the new strain by using CLUSTAL W (1.83) software. The phylogenetic and molecular evolutionary analyses was performed with neighbour joining method by using software from PHYLIP, version 3.57c [24]; the DNADIST program with Kimura-2 factor was used to compute the pairwise evolutionary distances for the above aligned sequences [25], the topology of the phylogenetic tree was evaluated by performing a bootstrap (algorithm version 3.6 b) with 1000 bootstrapped trials. The phylogenetic tree was drawn using Tree View 32 software. As out group the 16S rDNA of Bacillus subtilis was used.

The 16S rDNA sequence data was compared with all currently available sequences of organisms belonging to the genus Shewanella: Shewanella putrefaciens (U91552.1), Shewanella affinis (AF500080.1), Shewanella alga (U91544.1) Shewanella denitrificans (AJ457093.1), Shewanella amazonensis (AF005248.1), Shewanella aquimarina (AY485225.1), Shewanella baltica (AJ000214.1), Shewanella benthica (X82131.1) Shewanella colwelliana (AY653177.1), Shewanella decolorationis (AJ609571.1), Shewanella fidelia (AF420313.1), Shewanella frigidimarina (AJ300833.1), Shewanella gaetbuli (AY190533.1) Shewanella gelidimarina (U85907.1), Shewanella hanedai (X82132.1), Shewanella japonica (AF500079.1), Shewanella kaireiae (AB094598.1), Shewanella livingstonis (AJ300834.1), Shewanella marinintestina (AB081759.1), Shewanella marisflavi (AY485224.1), Shewanella massilia (AJ006084.1) Shewanella pacifica (AY366086.1), Shewanella pealeana (AF011335.1), Shewanella saccharophilus (AF033028.1), Shewanella sairae (AB081762.1), Shewanella schlegeliana (AB081761.1), Shewanella surugaensis (AB094597.1), Shewanella violacea (D21225.1), Shewanella waksmanii (AY170366.1), Shewanella woodyi (AF003548.1) and Shewanella olleyana (AF295592.1).

G + C content of genomic DNA

Cells of the new strain were harvested from a 2 l culture sample by centrifugation and were used for the determination of G + C content of the genomic DNA. The cells were disrupted with a cell French presser and purified by chromatography hydroxyapatit [26]. The mol % G + C of genomic DNA was determined by high performance liquid chromatography (HPLC) (Shimadzu corp, Japan) [27]. The analytical column was a VYDAC 201 SP 54, C18 5 µm (250 x 4.6 mm) equipped with a guard column 201 GD 54H (Vydac, Hesperia, USA).

DNA – DNA hybridization

DNA – DNA hybridization of the new strain with the strain Shewanella putrefaciens (DSM 6067 = ATCC 8071) was carried out from 3 g cell mass of each strain. DNA was isolated using a French pressure cell (Thermo spectroic) and was purified by chromatography on hydroxyapatite as described by Cashion. DNA – DNA hybridization was carried out as described by De Ley [28], with the modification described by Huss [29], using a model 2600 spectrophotometer equipped with a model 2527-R thermopropgrammer and plotter (Gilford Instrument Laboratories). Renaturation rates were computed with the TRANSFER. BAS program of Jahnke [30].

Physiological and morphological characteristics

Enrichment cultures (pH 7) containing glucose inoculated with seawater sample from Spitsbergen showed bacterial growth after one week of incubation at 4 °C. Microscopy revealed the presence of straight rod cells. After a number of transfers on marine medium agar plates at 15 °C, one culture was shown to exhibit the same characteristics and was selected as the culture for the strain 40-3. Cells of the strain 40-3 were found to be gram negative. They were straight or curved rod - shaped (0.4-0.6 µm wide and 2-3 µm long). They occurred singly. Spores could never be detected. The strain 40–3 showed high similarity in morphological characterization to the strains belonging to the genus Shewanella identified as: straight or curved rod–shaped, 2-3 µm length, 0.4- 0.7 wide, gram negative and non–spore forming [5,6,31-45]. The morphological characteristics of the strain 40-3 and the related strains are listed in Table 1.

Table 1: Comparative characteristics of the isolated strain 40-3 with related Shewanella sp. strains. 1. Strain 40-3, 2. Shewanella putrefaciens [date reproduced from Venkateswaran]; 3. Shewanella baltica [date reproduced from Ziemke]; 4. Shewanella frigidimarina [date reproduced from Bowman]; 5. Shewanella pacifica [date reproduced from Ivanova]; 6. Shewanella gaetbuli [date reproduced from Yoon et al (2004)]; 7. Shewanella waksmanii [date reproduced from Ivanova]. +, positive; -, negative; ND, not determined.

No growth was observed under anaerobic condition with glucose. Growth occurred only under aerobic conditions between 4 and 25°C. Optimal growth was optioned at 10-15°C where the growth rate reached its maximum. No growth was observed above 25°C (Data not shown). The strain 40-3 required seawater and grew well at salt (NaCl) concentration of 0–10 % (w/v) with optimum at 8-9 % NaCl (w/v) (Data not shown). The pH range for growth was 6–9, with an optimum at pH 7–8 (Data not shown). Under the optimum conditions using 2 g-1glucose, the growth rate was 0.48 h-1. The optimal growth temperature and pH of the strain 40–3 (10–15 °C and pH 7–8) were similar to the most related Shewanella species: Shewanella putrefaciens (25–35 °C and pH 7-8), Shewanella frigidimarina (20–22 °C), Shewanella pacifica (20–25 °C), Shewanella gaetbuli (30 °C and pH 7-8) and Shewanella waksmanii (20–22 °C and pH 7.5) [6,31,34,36,45]. However, Shewanella baltica grew at 4 °C [46]. The strain 40–3 grew in the presence of a wide range of NaCl concentration from 0 to 10 % (w/v) (optimum 8 to 9 % (w/v)) unlike the related Shewanella species: S. putrefaciens, S. frigidimarina, S. pacifica, S. gaetbuli and S. waksmanii, where no growth was observed above 6 % NaCl (w/v) [6,31,34,36,45]. The growth conditions of the strain 40-3 and the related strains are listed in Table 1

Substrate spectrum

The strain 40-3 grew on a variety of substrates. Good growth was observed on: α-cyclodextrin, dextrin, tween 80, N-acetyl-Dglucosamine, α-D-glucose, maltose, sucrose, methylpyruvate, D,Llactic acid, succinic acid, bromo succinic acid, inosine, esculin ferric citrate, L-arabinose, potassium gluconate, malic acid or trisodium citrate. No growth was observed on glycogen, tween 40, N-acetylD-galacto-samine, adonitol, capric acid, D-arabitol, cellobiose, L-erythol, D-fructose, L-fructose, D-galactose, gentiobiose, m-inositol, α-D-lactose, lactulose, D-mannitol, D-mannose, D-mellobiose, β-methyl D-glucose, D-psicose, D-raffinose, L-rhamnose, D-sorbitol, D-trehalose, turanose, xylitol, monomethyl succinate, acetic acid, cis-aconitic acid, citric acid, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, α-hydroxy-butyric acid, β-hydroxy-butyric acid, g-hydroxy-butyric acid, β-hydroxy phenylacetic acid, itaconic acid, α-keto butyric acid, α-keto glutaric acid, α-keto valeric acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, seabacic acid, succinamic acid, glucuronamide, alaninamide, D-alanine, L-alanine, L-alanylglycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycylL-aspartic acid, glycyl-L-glutamic acid, L-histidine, hydroxy L-proline, L-leucine, L-ornithine, L-phenyl-alanine, L-proline, L-pyroglutamic acid, D-serine, L-serine, L-threonine, D,Lcarnitine, g-amino butyric acid, urocanic acid, uridine, thymidine, phenylthylamine, putrescine, 2-aminoethanol, 2,3-butanediol, glycerol, D,L α-glycerol phosphate, glucose 1-phosphate, glucose 6-phosphate or phenylacetic acid. The strain 40–3 was able to produce . Indole (Tryptophane) and aceton were not produced. The strain was also able to reduce nitrates to nitrites but not nitrites to nitrogen. Shewanella putrefaciens and the strain 40–3 shared the ability to utilize: maltose, sucrose, succiniate, D–galactose, fumarate and lactose [6]. Shewanella baltica and the strain 40–3 on the other hand, shared the ability to utilize maltose, sucrose, malic acid and citrate [46]. The strain 40–3 and the related strains Shewanella putrefaciens, Shewanella baltica, Shewanella frigidimarina and Shewanella waksmanii were able to produce H2 S (from sodium thiosulfate) and reduce nitrates to nitrites [6,31,36,46]. And finally the strain 40-3 and all previous Shewanella species were unable to produce indole or acetoin [6,31,36,45]. The ability of the strain 40–3 and related strains to grow on various substrates is listed in Table1.

Fatty acid analysis

The FAMEs profiles display only those fatty acids comprising ≥ 0.05 % of the total. Straight chain saturated FAMEs were 17.89 % total, terminally branched saturated FAMEs were 14.85 % total and monounsaturated FAMEs were 17.73 % total. 16:0 straight chain saturated FAME was the most abundant FAME found in the strain 40-3 (11.24 %) as well as, in the related strains: Shewanella putrefaciens (19.1 %), Shewanella baltica (4.3 %), Shewanella frigidimarina (11.8 %), Shewanella gaetbuli (8.4 %) and Shewanella waksmanii (6.2 %) [6, 31, 36, 45, 46]. 15:0- iso terminally branched saturated FAME was the most abundant FAME found in the strain 40-3 (9.4 %) and in the related strains: Shewanella putrefaciens (21.1%), Shewanella baltica (14.3 %), Shewanella frigidimarina (9 %), and Shewanella waksmanii (32.5 %) [6, 31, 36, 45, 46]. However, 16:1 ω7c was the most abundant FAME in all Shewanella species mentioned above, but not in the strain 40–3 where, 18:1 ω7c was the most abundant FAME (Table 2).

Table 2: Comparative fatty acids composition (%) of the new isolated strain 40-3 and related Shewanella species. 1. Strain 40-3, 2. Shewanella putrefaciens [date reproduced from Venkateswaran]; 3. Shewanella baltica [date reproduced from Ziemke]; 4. Shewanella frigidimarina [date reproduced from Bowman]; 5. Shewanella gaetbuli [date reproduced from Yoon]; 6. Shewanella waksmanii [date reproduced from Ivanova]. Both the strain 40 – 3 and Shewanella frigidimarina produce eicosapentaenoic acid, 20:5ω3. ND, not determined.

DNA base composition and DNA - DNA hybridization

The G + C content of the DNA from the strain 40-3 was 48 mol %. High similarity was shown between the G + C content of strain 40-3 (48 mol %) and related strains: Shewanella putrefaciens (47 mol %), Shewanella baltica (46 mol %), Shewanella frigidimarina (40-43 mol %), Shewanella pacifica (40 mol %), Shewanella gaetbuli (42 mol %) and Shewanella waksmanii (42 mol %) [6,31,34-36,45,46]. The 16S rDNA sequence of the strain 40-3 (1.447 kb) was analyzed and compared to all currently available 16S rDNA sequences of organisms belonging to the genus Shewanella (Figure 1). The closest relationship was to Shewanella putrefaciens (U91552.1) with an identity on 16S rDNA level of 99 %. However, Shewanella massilia (AJ006084.1) and Shewanella baltica (AJ000214.1), showed 98% identity, Shewanella decolorationis (AJ609571.1) and Shewanella frigidimarina (AJ300833.1) showed 97 % identity, Shewanella pacifica (AY366086.1) and Shewanella gaetbuli (AY190533.1) showed 96% identity and Shewanella marisflavi (AY485224.1) and Shewanella waksmanii (AY170366.1) showed 95 % identity, on the 16S rDNA level.

Figure 1: Phylogenetic dendrogram based on 16S rDNA gene sequence comparison indicating the position of the new strain Shewanella arctica sp. nov. 40-3 within the genes Shewanella. performed by the neighbor-joining method using software from PHYLIP, version 3.57c; the DNADIST program with Kimura-2 factor was used to compute the pair wise evolutionary distances for the above aligned sequences , the topology of the phylogenetic tree was evaluated by performing a bootstrap (algorithm version 3.6 b) with 1000 bootstrapped trials. The tree was draw using Tree View 32 software. Bar correspond to 2 nucleotide substitutions per 100 nucleotides.

Phylogenetic treeing placed the strain 40-3 (according to its 16S rDNA composition) among the species of the genus Shewanella. The strain 40-3 showed a stable relative branching order with Shewanella putrefaciens and was placed in between Shewanella putrefaciens and Shewanella massilia (Figure 1). DNA-DNA hybridization of the strain 40-3 and the most identical strain Shewanella putrefaciens (DSM 6067) showed only 50 % DNA–DNA similarity despite the fact that the 16S rDNA identity between both, the new strain and Shewanella putrefaciens was very high (99 %). This strongly indicates that the newly isolated strain (40-3) is a new species within the genus Shewanella.

All data mentioned previously demonstrate clearly that the new isolate 40-3 is a new species within the genus Shewanella. This fact was also supported by the DNA-DNA hybridization, phylogenetic, G+C content, morphological and fatty acid analysis data. Based on the phylogenetic analysis by 16S rDNA and DNA-DNA hybridization homology, the new isolated strain 40-3 was found to be closely related to Shewanella putrefaciens, but represents a new species within the genus Shewanella. we propose to assign the newly isolate to the genus Shewanella as Shewanella arctica sp. nov.

Screening for enzymes production The strain 40–3 was able to produce several enzymes including amylase, pullulanase, protease, esterase (C4), esterase/ lipase (C8), omithine decarboxylase, alkaline phospatase, , leucine arylamidase, valine arylamidase, naphthol-AS-BIphosphohydrolase and N-acetyl-β–glucosaminidase. No activities for arabinase, arabinoxylanase, HE-cellulase, glucanase, dextranase, galactanase, galactomannanase, β-glucanase, curdlanase xylanase, xyloglucanase, α- and β–galactosidase, β–glucoronidase, α and β–glucosidase, α–mannosidase, α– fucosidase, acid phosphatase, cystine arylamidase, arginine dihydrolase, lysine decarboxylase, urease and tryptophane deaminase were detected. Amylase activities have also been reported for Shewanella pacifica and Shewanella gaetbuli, while protease activities are reported for Shewanella pacifica [34,45] (Table 1). This is the first time that a phsychrophilic Shewanella strain is reported to secrete hydrolyases with specific pullulanase (E.C. 3.2.1.XX), esterase (C4, E.C. 3.1.1.1), lipase (C8, E.C.3.1.1.3), phosphohydrolase (E.C. 3.1.3.XX) and arylamidase (E.C. 3.4.14.XX) activities. These specific enzyme activates are of industrial interest for the conversion of biomass to biofuels, food additives and platform chemicals [47-49]. Therefore, the new Shewanella strain represents an excellent resource to isolate new enzyme activities with potential industrial relevance. Since enzymes derived from phychrophilic organisms often operate at low temperatures, they can contribute sigficantly to enhynce the energy efficiency of an industrial process. The most prominent example is the use of phychrophilic protease in laundry powder formulation, which enable textile cleaning at temprastures as low as 15°C therby significantly contributing to household energy saving measures [50]. The same features will contribute to energy efficiency in starch based bioethanol production or the lipase catalysed transesterfication of plant oil derived fatty acids [13]. We have recently isolated and characterised the genetic elements encoding Shewanella arctica pullulanase and protease activities. Additionally we have purified and characterized the recombinant protein products, expressed in an E. coli system. The detailed description of these enzyme systems will be published elsewhere.

Description of Shewanella arctica sp. nov. and conclusion

Shewanella arctica (arc’ ti.ca. L. fem. adj. arctica from the Arctic, referring to the site were the type strain was isolated). Cells are straight or curved rod- shaped, gram negative, 2-3 µm long and 0.4–0.6 µm wide, occur singly, non spore forming and strictly aerobic. Colonies on agar medium are slightly orange, circular, smooth and convex. Temperature range for growth is 4–25 °C, with an optimum between 10 and 15 °C. No growth above 25 °C was detected. Range of pH for growth 6–9, with an optimum at pH 7–8. Growth from 0-10 % NaCl (w/v) concentrations with an optimum between 8 and 9 % (w/v). Growth is observed with α-cyclo-dextrin, dextrin, tween 80, N-acetyl-D-glucosamine, α-D-glucose, maltose, sucrose, methylpyruvate, D,L-lactitate, succiniate , bromo succinic acid, inosine, esculin ferric citrate, L-arabinose, potassium gluconate, malic acid and trisodium citrate. In the presence of glucose, H2 S is produced and nitrates are reduced to nitrites. The fatty acid methyl esters (FAME) are composed of 17.89 % straight chain saturated FAMEs, 14,85 % terminally branched saturated FAMEs and 17.73 % monounsaturated FAMEs. Phylogenetic analysis reveals as close relationship to Shewanella putrefaciens with 99 % 16S rDNA composition identity and 50 % DNA-DNA similarty.

The DNA base ratio is 48mol % G + C. Habitat: artic seawater. The strain 40-3 (DSM 16509) is isolated from seawater samples taken from Spitsbergen. The Shewanella arctica strain was capable of secreting numerous industrially relevant enzyme systems when grown on glucose as the sole carbon source. The applied enzyme screening procedure could detect amylase, pullulanase, protease, esterase (C4), lipase (C8), ornithine decarboxylase, alkaline phospatase, leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase and N-acetyl-β– glucosaminidase activities. This is the first time that a Shewanella strain is been reported to secrete hydrolases with specific pullulanase, alkaline phospatase, esterase (C4), lipase (C8), arylamidase, phosphohydrolase and glucosaminidase activities. The extreme functional diversity of the secreted enzyme systems provides a rich resource for the isolation industrially relevant biocatalysts, which can potentially optimize the energy efficiency of biotechnological processes producing biofuel, food additives and/or platform chemicals.

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