Sodium alginate (~250 cps) from brown algae was purchased from sigma (USA). The plasmid pMD-18-T (Takara) and Escherichia coli DH5α were used for cloning and sequencing of 16S rRNA genes. The clones were grown on LB agar or liquid medium containing 100 μg/mL of ampicillin at 37 °C. Algal samples were collected from six locations in the intertidal zone of the Kings Bay in Ny-Ålesund, Svaldbard during the Chinese Arctic Yellow River Station Expedition in July, 2011. The numbers and locations of the six samples were shown in Table 1. The algal samples were stored in airtight sterile plastic tube at 4 °C until returning to the laboratory. The six algal samples were all identified as Laminaria sp.

Each Laminaria sample was cut into small pieces using sterile scissors. One gram of every sample was put in 100 mL of the enrichment medium containing marine broth 2216 (Difco) plus 1% sodium alginate. After incubated at 15 °C, 200 rpm for 8 h, the culture broths were serially tenfold diluted to 10⁻⁶ dilution with sterile artificial sea water. Aliquots of 100 μL diluted samples (10⁻²–10⁻⁶ dilution) were spread on screening plates with a medium composed of 0.1% yeast extract, 0.5% tryptone, 1% sodium alginate, 1.5% agar powder and artificial seawater (pH 7.0). The plates were then incubated at 15 °C for a proper time to form detectable colonies. Morphologically different colonies were selected and further purified by repeatedly streaking on the same medium. The purified strains were stored in 10% glycerol at −80 °C.

The purified isolates were, respectively, streaked on the plate with a medium containing 0.1% yeast extract, 0.5% tryptone, 0.4% sodium alginate, 1.5% agar powder and artificial seawater (pH 7.0). After being incubated at 15 °C for 4 days, the plates were stained by flooding with 10% (weight/volume) cetylpyridiniun chloride. If a strain excreted alginate lyase into the medium, a clear halo of depolymerization around the strain colony on a white background could be observed. Therefore, the isolates with a clear halo were selected as alginate lyase-excreting bacteria.

The genomic DNA of each isolate was extracted by using the BIOTEKE genomic DNA purification kit. The 16S rRNA genes were amplified from the genomic DNA by PCR with two primers (1492r: 5′-GGTTACCTTGTTACGACTT-3′, 27f: 5′-AGAGTTTGATCCTGGCTCAG-3′) [13]. The amplified DNA fragments of the 16S rRNA genes were ligated into pMD-18-T cloning vectors (Takara) and sequenced by Biosune Inc. (Shanghai, China). Sequence alignment was performed using CLUSTAL X (v 1.83). Neighbor-joining trees were constructed using MEGA version 3.1 with neighbor-joining method and Kimura two parameter model [14].

Alginate lyase activity was quantitatively determined by measuring the amount of reducing sugars released from alginate by using the Somogyi-Nelson method [15]. Bacterial culture was centrifuged at 12,000× g, 4 °C for 12 min. The supernatant was properly diluted with 20 mM phosphate buffer (pH 7.0) if needed. Then, 20 μL diluted culture supernatant was mixed with a 180 μL solution composed of 20 mM phosphate buffer (pH 7.0) and 0.5% sodium alginate. The mixture was incubated at an indicated temperature for 30 min. After incubation, 200 μL Nelson copper reagent was added, and then the mixture was bathed in boiling water for 15 min, followed by a fast cool in ice bath. Then 100 μL arsenomolybdate was added. Enzyme activity was determined by monitoring the absorbance increase at 680 nm. A standard curve was prepared using mannose at concentrations ranging from 0.1 to 1 mM. One unit of enzyme activity was defined as the amount of protein that generates 1 nmol reducing sugars per min. A blank control was set by mixing Nelson copper reagent and the substrate solution followed by an addition of culture supernatant to inactiviate the alginate lyase activity before incubation.

The alginate lyase-excreting bacteria were cultivated in the liquid screening medium at series temperatures, from 10 °C to 35 °C with an interval of 5 °C, and 200 rpm for 4 days. Timing sampling was done during the cultivation. The bacterial density in each sample was measured at a wavelength of 600 nm (OD₆₀₀) to draw a growth curve. Each sample was centrifuged at 12,000× g, 4 °C for 12 min, and the supernatant was collected to measure the alginate lyase activity at its optimal temperature. The optimal temperature for alginate lyase activity was determined by measuring the enzyme activity from 10 °C to 60 °C with an interval of 10 °C.

After cultivation of the bacteria enriched from the Arctic Laminaria on screening plates, a lot of colonies appeared on the plates with 10⁻²–10⁻⁵ diluted samples. More than 70 isolates were purified and subjected to 16S rRNA gene amplification. Isolates with the same (or only one base difference) 16S rRNA gene sequence were considered as the same strain. According to an alignment of the 16S rRNA gene sequences, a total of 65 different strains were isolated from the Arctic Laminaria samples. A phylogenetic analysis based on the 16S rRNA gene sequences showed that the recovered Laminaria-associated bacteria belonged to nine genera, namely Cobetia, Psychrobacter, Winogradskyella, Psedoalteromonas, Psychromonas, Polaribacter, Shewanella, Flavobacterium and marinomonas (Figure 1). Psychrobacter (33/65) were the most predominant group. Psychromonas (10/65) and Polaribacter (8/65) also showed some preponderance.

Alginate lyase-excreting bacteria were further screened from the recovered Laminaria-associated isolates by screening plates. Twenty one strains showed clear haloes after 10% cetylpyridiniun chloride was flooded on the plates (Supplementary Materials, Figure S1), indicating that these strains could excrete alginate lyase. The 21 strains were taken as alginate lyase-excreting bacteria for further study. These strains belonged to five genera, namely Psychrobacter, Winogradskyella, Psedoalteromonas, Psychromonas and Polaribacter. Psychromonas (8/21), Psedoalteromonas (6/21) and Polaribacter (4/21) were the predominant genera (Figure 2). Therefore, it seemed that the predominant genera of the Laminaria-associated bacteria and the alginate lyase-excreting bacteria showed some difference.

A distance-based neighbor-joining tree was constructed with the 16S rRNA gene sequences of the alginate lyase-excreting bacteria in this study and reference sequences from the GenBank database (Figure 3). There were three predominant branches, namely Psychromonas, Pseudoalteromonas and Polaribacter. Strains related to Psychromonas were the most frequently recovered (recovered from three Laminaria samples) and formed the largest group in terms of abundance (8 of 21 strains, Figure 3). These Psychromonas strains showed high similarity (more than 98.3%) with Psychromonas arctica Pull 5.3^T that was isolated from a seawater sample collected from Spitzbergen in the Arctic (Supplementary Materials, Table S1). Four strains, 2-2, 2-5, 2-9 and 2-17, showed high similarity (more than 98.5%) with Polaribacter butkevichii KMM 3938^T that was isolated from the seawater collected from the Sea of Japan, Russia. Strain 2-15 exhibited a distant relationship with any of the previously identified species. It may represent a potentially new species, which merits further study.

Among the 21 alginate lyase-excreting strains, 20 strains had the optimal temperatures for growth at 10–25 °C, indicating that most alginate lyase-excreting strains are cold-adapted bacteria (Table 2). The optimal growth temperature was just 10 °C for strains 0-7 and 2-1, and 15 °C for strains 0-4, 2-17 and 3-12, showing that these five strains are psychrophiles. The other stains with an optimal growth temperature of 20 °C, 25 °C or 30 °C are psychrotrophiles. Correspondingly, the optimal temperatures for the strains to produce alginate lyases were also low. Ten strains showed the same optimal temperature for growth and alginate lyase production. However, the optimal temperature for some strains to produce extracellular alginate lyases was lower than that for growth, such as strains 0-8, 2-11 and 3-12, while others showed higher optimal temperatures for alginate lyase production than for growth. In general, the strains showed low optimal temperatures for growth and extracellular enzyme production, reflecting their adaptation to the cold Arctic Ocean.

The optimal temperatures for the extracellular alginate lyases produced by the 21 strains were ranged from 20 °C to 50 °C (Table 2). The alginate lyases produced by 11 strains showed the highest activity at 20–30 °C, indicating the good cold-adaptation of these alginate lyases. The alginate lyases produced by the other 10 strains showed the highest activity at 40–50 °C. The differences in the optimal temperatures for enzyme activity suggest that the alginate lyases excreted by these strains may be different kinds of alginate lyases. The results indicate that a majority of bacterial extracellular alginate lyases from the Arctic Ocean are cold-adapted enzymes.

The extracellular alginate lyase activity level of every strain was determined by measuring the alginate lyase activity in the culture at its optimal temperature (Figure 4). Among the 21 alginate lyase-excreting strains, strain 2-2 had the highest extracellular alginate lyase activity level, more than 200 U/mL, while strain C-1 had the lowest extracellular alginate lyase activity level, lower than 60 U/mL. Strains 0-4 and C-11 also had a level of near 200 U/mL. This indicated that these strains may be good candidates for isolation of novel alginate lyases.