Over 90% of commercial algae biomass production is currently with large-scale open-pond systems. Such systems usually suffer from low production rates during the cold seasons (Sheehan et al. 1998, Benemann 2008). Hence, cyanobacteria isolated from the polar regions offer interesting potential for the production of biomass and biofuel due to their psychotrophic characteristics as Tang et al. (1997) previously reported that many high-latitude cyanobacterial strains remain active at low temperatures.

However, obtaining axenic cultures is essential for genetic, physiological and taxonomic research. Although numerous methods to produce axenic cultures of cyanobacteria have been suggested (Rippka 1988, Ferris and Hirsch 1991, Bolch and Blackburn 1996, Choi et al. 2008), it is still very difficult to eliminate all contaminating bacteria from environmental samples. Since cyanobacteria are a very diverse group, exhibiting enormous variations in growth, morphology and metabolic capabilities, any particular approach cannot guarantee success of purification.

The objectives of the current study were to axenically obtain cyanobacteria from an Arctic freshwater bloom sample by using a combination of antibiotics and physical separation techniques and to test the isolate’s cold-tolerance capability. Our eventual goal is to discover the mechanisms of cold tolerance in Arctic cyanobacteria, identify and isolate genes with high activity. These genes will then be incorporated into indigenous cyanobacteria for consistent biofuels production during the low-temperature seasons.

Arctic cyanobacterial bloom samples were taken from the temporal water runoff region, located approximately 10 km east of Dasan Station in Ny-Alesund, Svalbard Islands in August 2009 (Table 1, Fig. 1). Samples were then taken to the laboratory and 1 mL of bloom samples were inoculated into 100 mL BG-11₀ medium (Rippka et al. 1979) with cycloheximide (Sigma, St. Louis, MO, USA) at a concentration of 250 μg mL^-1. The flasks were incubated on an orbital shaker (Vision Scientific Co. Ltd., Bucheon, Korea) at 160 rpm and 15°C until cyanobacterial growth was apparent.

Well-grown cyanobacterial cultures (1.5 mL) were centrifuged at × 3,000 g for 15 min. Resulting pellets were streaked onto BG-11 agar supplemented with imipenem (100 μg mL^-1) (Choongwae Pharma Corporation, Seoul, Korea) and cycloheximide (20 μg mL^-1) and incubated in the dark for 24 hours to eliminate bacterial contamination. Plates were then incubated in a light : dark cycle (16 : 8 hours) at 15°C and filamentous growth was observed daily. When visible to the naked eye, emerging cyanobacterial filaments were 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 Company, Sparks, MD, USA) and incubated in the dark to check the axenic status of the culture for 7 to 14 days. Contaminating bacteria that survived the imipenem treatment were identified by 16S rRNA gene sequencing (Lane 1991). Non-axenic (bacterially contaminated) cyanobacterial culture was further incubated on BG-11 agar with kanamycin (100 μg mL^-1) (Duchefa Biochemie, Haarlem, The Netherlands) for another 24 hours in the dark and the rest of the purification steps were repeated until a pure culture of the cyanobacterium was obtained (Fig. 2).

[Table 1.] Description of sampling point

Description of sampling point

[Fig. 1.] Cyanobacterial bloom in temporal water runoff from melting glacier ice in Ny-Alesund, Svalbard Islands.

[Fig. 2.] Schematic of obtaining axenic cultures from environmental samples.

The strain was grown in BG-11₀ medium (without nitrogen) for 10 days. Live cells were harvested and suspended in sterile dH₂O and inspected at × 400 magnification on a Zeiss Axioskop 2 light microscope (Carl Zeiss, Korea Co. Ltd., Seoul, Korea) equipped with differential interference contrast optics.

PCR conditions and the primer sets CYA106F, CYA781R(a), and CYA781R(b) were used for 16S rRNA sequence analysis as described by Nubel et al. (1997). The phycocyanin encoding operon intergenic spacer (PC-IGS) region was amplified using the primer pair, PCβF and PCαR specific for cyanobacteria (Neilan et al. 1995). Region ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) rbcLX was amplified with primers CW and DW described previously (Rudi et al. 1998). Synthesis of the primers used and DNA sequencing were performed at the Macrogen facility (Macrogen, Seoul, Korea).

A seed culture of Nodularia spumigena KNUA005 was inoculated into both BG-11₀ and BG-11(+) media in triplicate and incubated in a light : dark cycle (16 : 8 hours) on an orbital shaker at 160 rpm and 10, 15, 20 and 25°C for 21 days. Cyanobacterial density was determined by measuring the optical density of a culture at 750 nm on an Optimizer 2120UV spectrophotometer (Mecasys Co. Ltd., Daejeon, Korea). Then, growth curves were drawn to reveal the effect of temperature on N. spumigena KNUA005.

After imipenem treatment and physical separation by streaking onto BG-11 agar plates, two strains of contaminating bacteria still survived and co-existed with the cyanobacterial culture. These bacteria, CB1 and CB2, were identified as Rhizobium sp. and Brevundimonas sp., respectively (Table 2). However, kanamycin treatment successfully removed the contaminants from the cyanobacterium. The purity of the culture was verified when no bacterial growth was observed after incubating for 14 days after streaking the culture onto R2A and LB agar plates.

Molecular characterization showed that the Arctic cyanobacterium was N. spumigena and all results inferred from 16S rRNA, PC-IGS and rbcLX sequence analyses were in agreement (Table 3). The isolate’s morphological features also suggested that the isolate was N. spumigena (Fig. 3). The trichomes were straight or slightly sinuous, the vegetative cells were discoid-shaped and the heterocysts were both at intercalary and terminal.

As shown in Fig. 4A, N. spumigena KNUA005’s optimal growth temperature in BG-11₀ was 20°C, but it was also able to grow well at 15°C. However, this heterocystous organism showed a tendency of growing slower in BG-11(+) medium than in nitrogen-free BG-11₀ medium (Fig. 4B). There was little or no cyanobacterial growth in either BG-11₀ or BG-11(+) media at 10°C, but the cyanobacterium remained alive and subsequently grew well when placed under favourable conditions (data not shown).

[Fig. 3.] Light microscope images of Nodularia spumigena KNUA005. Scale bars represent 20 μm.

[Table 2.] Identification of contaminating bacteria isolated from the cyanobacterial culture

Identification of contaminating bacteria isolated from the cyanobacterial culture

[Table 3.] Results from BLAST searches using 16S rRNA, PC-IGS, rbcLX sequences of Nodularia spumigena KNUA005

Results from BLAST searches using 16S rRNA, PC-IGS, rbcLX sequences of Nodularia spumigena KNUA005

[Fig. 4.] Growth curves of Nodularia spumigena KNUA005 grown in BG-110 (A) and BG-11(+) (B) media. OD, Optical density.

Many research groups have successfully isolated cyanobacterial cultures from a variety of environmental samples. Nevertheless, cultures maintained in laboratories are mostly unialgal, not axenic cultures, which are not suitable for understanding genetic, biochemical or physiological properties of a particular taxon. In this study, we have developed a solid medium-based isolation method for effective axenic culture production of filamentous cyanobacteria.

Imipenem is a β-lactam antibiotic derived from Streptomyces cattleya with a broad spectrum against aerobic and anaerobic Gram positive bacteria as well as Gram negative ones through inhibiting peptidoglycan biosynthesis. Incubation in the dark for 24 hrs may have killed the majority of contaminating bacteria while the cyanobacterium remained unaffected. However, two contaminants, Rhizobium sp. CB1 and Brevundimonas sp. CB2, belonging to Gram negative bacteria survived the imipenem treatment. They were not physically separated by streaking technique either, but were finally eliminated by the application of kanamycin, which prevents mRNA translation by interacting with the 30S ribosomal subunit.

This method uses a combination of two antibiotics with different mechanisms of action in an attempt to prevent any contaminating bacterial growth. This approach may provide an effective way of axenic culture production for filamentous cyanobacteria from heavily contaminated environmental samples.

N. spumigena is known as one of the major bloom formers in late summer throughout the world (Horne and Galat 1985, Carmichael et al. 1988, Codd et al. 1994). It was also reported that many polar cyanobacteria generally show optimal growth at 15°C or above (Tang et al. 1997, Chevalier et al. 2000). According to the temperature tolerance test, N. spumigena KNUA005’s maximal growth was attained in the range of 15 to 20°C which is consistent with the previous reports. However, the isolate exhibited a tolerance to low temperatures ranging from 10 to 15°C which suggests that there are cold-tolerance genes present in this Arctic cyanobacterium. Hence, screening and identification of these useful genes using differentially expressed gene tag profiling and the Solexa^® (Illumina, Inc., San Diego, CA, USA) massive parallel se-ACKNOWLEDGEMENTS This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0075777). This research was also funded by KOPRI Project No. PE10060 (Research on Culturable Polar Organisms and their Applications). REFERENCES Benemann, J. R. 2008. Opportunities and challenges in algae biofuels production. Available from: http://www.fao.org/uploads/media/algae_positionpaper.pdf. Accessed 2 Apr 2010. Bolch, C. J. S. & Blackburn, S. I. 1996. Isolation and purification of Australian isolates of the toxic cyanobacterium Microcystis aeruginosa Kutz. J. Appl. Phycol. 8:5-13. Carmichael, W. W., Eschedor, J. T., Patterson, G. M. L. & Moore, R. E. 1988. Toxicity and partial structure of a hepatotoxic peptide produced by the cyanobacterium Nodularia spumigena Mertens emend. L575 from New Zealand. Appl. Environ. Microbiol. 54:2257-2263. Chevalier, P., Proulx, D., Lessard, P., Vincent, W. F. & de la Noue, J. 2000. Nitrogen and phosphorus removal by high latitude mat-forming cyanobacteria for potential use in tertiary wastewater treatment. J. Appl. Phycol. quencing approach are needed. Cold-tolerance genes will then be incorporated into native cyanobacteria for consistent production of algae-based biofuels in the next research stage.

Our research group has already obtained four potential candidates for biofuel production that were isolated from cyanobacterial bloom samples in Lake Daecheong, Korea in late summer 2009 (unpublished data). It is hoped that new genetic strains may have potential for sustainable biofuel production under unfavourable weather conditions such as those experienced in the autumn and winter seasons.

However, it should be noted that N. spumigena produces Nodularin, a potent hepatotoxin which may pose a health risk for humans and animals (Runnegar et al. 1988, Sivonen et al. 1989). N. spumigena is also responsible for a large part of nitrogen input into water bodies (Huber 1986) due to their nitrogen-fixing ability. Thereby, care should be taken when working with this isolate to prevent release into environment.