Cyanobacterial mat samples were collected from a shallow ephemeral freshwater near King Sejong Station (62° 13′S−58° 47′W) located on Barton Peninsula, King George Island, West Antarctica, in January 2010. Aliquots (1 ml) of mat samples were inoculated into 100 ml BG-11 medium (Rippka et al. 1979) containing 250 µg cycloheximide ml⁻¹ (Sigma, St. Louis, MO, USA) and incubated at 15°C with cool fluorescent light (approximately 70 µmole m⁻² sec⁻¹) in a light:dark cycle (16:8 h) until growth was apparent. Well-grown cyanobacterial cultures (1.5 ml) were centrifuged and streaked onto BG-11 agar. Emerging cyanobacterial filaments were then aseptically transferred to fresh BG-11 plates to separate cyanobacteria from contaminating bacteria. Cyanobacterial filaments were then streaked onto R2A and LB agar plates (Becton, Dickinson and Co., Sparks, MD, USA) and incubated for 14 days in the dark to check the axenic status of the culture. These steps were repeated until a pure culture of the cyanobacterium was obtained and the resulting pure culture was named as KNUA009 and the isolate was deposited at the Korean Collection for Type Cultures under the accession number KCTC AG40019.

Strain KNUA009 was grown in BG-11 medium for 10 days at 15°C. Live cells were harvested and suspended in a drop of immersion oil and inspected at 1000× magnification on a Zeiss light microscope equipped with differential interference contrast optics (Carl Zeiss, Standort Göttingen, Vertrieb, Germany). Bergey's manual of systematic bacteriology (Castenholz 2001) was used for taxonomic description and morphotypic identification. For 16S rRNA gene sequence analysis, DNA was extracted by using DNeasy plant mini kit (Qiagen, Hilden, Germany) and polymerase chain reaction (PCR) conditions and the primer sets, CYA106F, CYA781R(a) and CYA781R(b), were used as described by Nübel et al. (1997). Due to the highly conserved nature of the 16S rRNA gene, two other genetic markers, the ribosomal 16S–23S intergenic spacer (ITS) region and region RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) rbcLX, were employed. The ribosomal 16S–23S ITS region was evaluated using primers, 16S1407F and 23S30R (Taton et al. 2003). Region RuBisCO rbcLX was also amplified with primers, CW and CX, described by Rudi et al. (1998). Phylogenetic analysis was performed using the software package MEGA version 4.0 (Tamura et al. 2007) using the neighbour-joining method (Saitou & Nei 1987) with 1000 bootstrap replicates (Felsenstein 1985).

A 10-day-old seed culture of strain KNUA009 (1 ml) was inoculated into BG-11 medium in triplicate and incubated for 20 days. Survival and growth of KNUA009 were examined at 5–30°C (at intervals of 5°C) to identify the optimum growth temperature of the culture. Acidity tolerance tests were performed at 15°C ranging from pH 2.0 to 13.0 (at intervals of pH 1.0 unit). A carbon utilization test was carried out using glucose, sucrose, fructose, ribose, sodium acetate, glycerol and glycolic acid as described by Lambert & Stevens (1986) and Miura & Yokota (2006). The cell biomass was separated from the bottom of the flask and thoroughly mixed by pipetting and samples taken from each time point were homogenized by sonification for 15 s on an ultrasonic cell disruptor (Model 550, Fisher Scientific, Pittsburgh, PA, USA). Cyanobacterial density was determined by measuring the turbidity of a culture at 750 nm on an Optimizer 2120UV spectrophotometer (Mecasys, Daejeon, South Korea).

Fatty acid methyl esters were extracted from strain KNUA009 according to the standard protocol by Sasser (1990) and the fatty acid composition was determined by GC/MS (Jeol JMS700 mass spectrometer equipped with an Agilent 6890N GC, Agilent Technologies, Palo Alto, CA, USA) at Daegu Center, Korean Basic Science Institute. Peak identification and compound assignment were performed based on the electron impact mass spectrum. The National Institute of Standards and Technology mass spectral libraries were used as reference databases. The DNA G+C content of the isolate was determined by the high performance liquid chromatography method as described by Shin et al. (1996) at the Korean Culture Center of Microorganisms.

Molecular identification results are summarized in Table 1. Based on 16S rRNA gene sequence data, strain KNUA009 was most closely related to Antarctic cyanobacterial clones (JQ310327 & JQ310310) derived from Byers Peninsula with a sequence homology of 100%. The isolate also clustered with environmental clones from various extreme cryosphere around the world (Fig. 1). Its closest cultured organism was cyanobacterium cCLA-4 (HQ230226) with a sequence homology of only 90% (Table 1, Fig. 1). The 16S–23S ITS sequence analysis also showed that the closest sequence match for strain KNUA009 was an uncultured Antarctic cyanobacterial clone N184-8 (EU032395) recovered from a surface soil sample in Miers Valley with a sequence homology of 99%. It was also clustered together with other environmental clones obtained from either surface soil in Miers Valley or cyanobacterial mat in Lake Miers (Fig. 2). Leptolyngbya sp. GSE-PSE30-01B (HM018675) was the closest cultured relative for strain KNUA009, but the sequence homology was only 86%. Molecular characterization based on rbcLX region sequence comparison showed that uncultured bacterium clone ORU13 (X57359) was the closest match for strain KNUA009, but the query coverage was only 28%. This may be due to the lack of sequence data in GenBank for Antarctic cyanobacterial rbcLX genes, so no identification could be made with this data.

Strain KNUA009 was filamentous with straight and flexible trichomes composed of disc-shaped vegetative cells (Fig. 3). Cells were about 3 µm in width and >20 µm up to approximately >500 µm in length, exhibiting gliding motility. Its terminal cells were dome-shaped and the cross-walls were clearly visible. No akinetes and heterocytes were observed. Its colour ranged from brown to olive and densely aggregated trichomes readily formed macroscopically visible mats.

As shown in Fig. 4a, strain KNUA009 grew in a temperature range of 5–20°C whilst its optimal growth temperature was 15°C. However, this cyanobacterium could not survive at 25°C and above. Growth pH ranges were between pH 4.0 and 11.0 whereas optimal growth was attained at pH 7.0 (Fig. 4b). Since no alternative carbon sources tested here were utilized by strain KNUA009, this cyanobacterium appeared to be an obligate photoautotroph. The isolate did not show any growth in nitrogen-free BG-11 medium under its optimal growth conditions. Summarized characteristics for the strain are given in Table 2.

The major fatty acids of the isolate were C_16:0 (34.6±0.4%), C_16:1ω5c (24.9±0.7%), C_18:3ω3c (17.8±0.8%) and C_18:2ω7c (15.5±0.8%). In addition, C_18:1ω9c (5.2±0.7%), C_18:0 (1.2±0.2%) and C_16:1 (0.8±0.1%; double bond position unknown) were also detected. The G+C content of the genomic DNA of strain KNUA009 was 47.5 mol % which is within the mean DNA base composition (40–50 mol % G+C) of the genus Oscillatoria deposited at the Pasteur Culture Collection of Cyanobacteria (Castenholz 2001).