Establishment and Maintenance of an Axenic Culture ... - Springer Link

Biotechnology and Bioprocess Engineering 20: 1056-1063 (2015) DOI 10.1007/s12257-015-0289-4

RESEARCH PAPER

Establishment and Maintenance of an Axenic Culture of Ettlia sp. Using a Species-specific Approach Hyung-Gwan Lee, Sang-Yoon Shin, Long Jin, Chan Yoo, Ankita Srivastava, Hyun-Joon La, Chi-Yong Ahn, Hee-Sik Kim, and Hee-Mock Oh

Received: 28 April 2015 / Revised: 14 July 2015 / Accepted: 3 August 2015 © The Korean Society for Biotechnology and Bioengineering and Springer 2015

Abstract The establishment of an axenic culture of microalgae is essential step in understanding its physiology, genetics, and ecology. However, culturing of microalgae is usually accompanied by complex and variable associated prokaryotic and eukaryotic microorganisms. Conventional approaches used for obtaining axenic cultures of microalgae are time-consuming and often involve difficulties in maintaining and preserving axenicity. In this study, we developed a procedure for establishing an axenic culture of Ettlia sp. YC001 and demonstrate that we maintained the axenic culture through subculture in the long term. Three sequential treatments, an antibiotic cocktail, serial dilution, and plate spreading, were applied to strain YC001 and we confirmed axenicity using molecular and physiological methods. The bacterial community associated with strain YC001 was investigated to select antibiotics for their specific elimination. The xenic culture (1 × 106 cells/mL) was treated with the antibiotic cocktail-5 (AC-5), carbendazim, chloramphenicol, imipenem, rifampicin, and tetracycline for 3 days, followed by serial dilution up to 1 × 102 cells and spreading on agar plates. The pure colonies were analyzed using denaturing gradient gel electrophoresis

Hyung-Gwan Lee, Hee-Sik Kim Sustainable Bioresource Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Korea Long Jin College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China Sang-Yoon Shin, Chan Yoo, Ankita Srivastava, Hyun-Joon La, Chi-Yong Ahn, Hee-Mock Oh* Bioenergy and Biochemical Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Korea Tel: +82-42-860-4321; Fax: +82-42-860-4594 E-mail: [email protected]

(DGGE), fluorescence-activated cell sorting (FACS), and scanning electron microscopy (SEM). The procedure we developed can be applied to other strains of microalgae for the establishment of axenic cultures. Keywords: antibiotic cocktail, axenic culture, Ettlia sp. YC001, serial dilution, plate spreading

1. Introduction In nature, microalgae are usually influenced by various prokaryotes and eukaryotes in the surrounding zone (phycosphere) [1]. Some associated bacteria promote algal growth mainly by generating a favorable environment through actions such as removing excess oxygen and supplying carbon dioxide, inorganic nutrients, vitamins, trace elements, chelators, and phytohormones [2-7]. However, other bacteria can be inhibitive; they modify the environment through their metabolism, algal lysis, or by competing with algae for limited nutrients [1,8-10]. Application of such interactions between microalgae and associated bacteria has a great potential for increased algal biomass production. Thus, an ecological approach might be crucial in large-scale microalgal cultivation and production of specific compounds such as lipid and carotenoids. The establishment of a microalgal axenic state remains an unsolved issue because of diverse and flexible interactions between microalgae and associated bacteria, requiring species-specific treatments, and long-term maintenance of axenicity. However, securing axenic strains of microalgae is essential in order to study their physiology, biochemistry, molecular, and cell biology [11]. Firstly, realizing the distinct physiological characteristics of microalgae, separately from

Establishment and Maintenance of an Axenic Culture of Ettlia sp. Using a Species-specific Approach

any interaction with associated bacteria, such as enzyme activity, carbon assimilation, and electron donor/acceptor, may provide taxonomic markers for identification of microalgae. Secondly, advanced molecular techniques for whole-genome sequencing, like NGS, require high-purity genomic fractions without contamination from any bacterial genome. Thirdly, microalgae-based food and pharmaceutical industries demand contaminant- and pathogen-free cultivation. Thus, the establishment and maintenance of axenic cultures is necessary for both biological and commercial applications. Axenic strains of microalgae can be obtained using various methods, such as sub-culturing, ultrasonication, phototaxis, ultraviolet radiation, micropicking, lysozymes, and antibiotic treatment [12-19]. While antibiotic treatment is frequently used because of its simplicity, antibiotics can be toxic to microalgae through processes such as vacuolization of the cytoplasm, destruction of chloroplasts, and cell death. Moreover, single antibiotic or a combination of several antibiotics are usually ineffective in eliminating associated bacteria because of low specificity, low cellular penetration, and the biosynthesis of extracellular polymeric substances (EPS). Various physical separation treatments have also been attempted, such as filtration, vortexing, sonication, or dilution and streak-plating, to eliminate tenaciously attached microorganisms before or after antibiotic treatment [20-23]. In previous studies, an antibiotic cocktail consisting of penicillin, tetracycline, chloramphenicol, aureomycin, and neomycin was shown to be effective in eliminating bacteria from cultures of Chlorella vulgaris, while imipenem, carbenicilin, and ciprofloxacin were successfully applied to obtain an axenic culture of benthic diatom [22-24]. Thus, the antibiotic treatment approach to establish axenic cultures should be applied with appropriate information regarding the type of microalgal species and the community of associated bacteria. Microalgae produce pharmaceutical and cosmetic substances such as DHA, EPA, and carotenoids and have also received attention as feedstock for biodiesel production; thus, research on microalgae has rapidly increased during the last decade [25]. Ettlia sp. YC001 grows well under high CO2 concentrations (up to 10%) and produces high volumes of lipids and various carotenoids [26]. Moreover, this strain has been used in a mass cultivation system in an outdoor raceway pond supported by real flue gas, making it a suitable candidate for producing biodiesel and highvalue compounds. Accordingly, the purpose of this study was to remove the bacterial population associated with Ettlia sp. YC001 based on sequential treatments with an antibiotic cocktail and to establish an axenic culture of strain YC001 for physiological and molecular studies and industrial use.

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2. Materials and Methods 2.1. Strain and cultivation Ettlia sp. YC001 was isolated from fresh water in Daejeon, South Korea [26]. The strain was deposited as AG50054 in the KCTC (Korean Collection for Type Cultures) and has been maintained in a steady state using a continuousculture system at an irradiance of 125 µmol photons/m2/sec and 25°C with 5% CO2 in a photo-bioreactor for over 6 months. This culture was used as the seed for this study. To measure its growth, the strain was also cultured in a 500-mL Erlenmeyer flask containing 200 mL of BG11 medium under continuous illumination with 125 µmol photons/m2/sec provided by white fluorescent lamps at 25°C and 120 rpm. Biomass was monitored via DCW and the number of cells was measured by microscopic analysis and FACS. 2.2. Analysis of associated bacteria To isolate the bacterial strains associated with YC001, aliquots of serially diluted xenic cultures up to 0.1 ~ 1.0 × 102 cells/mL were spread on R2A agar plates and incubated at 30°C for two weeks. Colonies were picked for sub-culturing until pure colonies were obtained. Genomic DNA was extracted from each culture and bacterial strain using a plant and bacterial DNA extraction kit (Intron, Korea) according to the manufacturer’s protocol. PCR amplification of bacterial 16S rRNA was performed using the bacterial universal primers 27F and 1492R. The PCR products were purified using a PCR Purification Kit (Solgent, Korea) and sequenced with an ABI 377 automated sequencer (Applied Biosystems, CA, USA). The sequences of about 1,400 bp were then aligned with published 16S rRNA sequences retrieved from the EzTaxon-e server and GenBank (http://www.ncbi.nlm.nih.gov) [27]. 2.3. Antibiotic susceptibility The paper disk diffusion method was used to determine the susceptibility of the associated bacteria to antibiotics [28]. Paper disks (6 mm in dia.) were placed on the surface of petri dishes that were spread with 1.0 × 103 cells of strain YC001. The following antibiotics were tested; carbendazim (Aldrich), chloramphenicol (Sigma), imipenem (Santa Cruz Biotech), rifampicin (Sigma), and tetracycline (Sigma) at concentrations of 1, 2, 5, 10, 20, 40, and 100 µg/mL. Antibiotic cocktail-3 (AC-3) consisted of three bacterialgrowth-inhibiting antibiotics (imipenem, chloramphenicol, and tetracycline), while antibiotic cocktail-5 (AC-5) also included rifampicin and carbendazim. The petri dishes were incubated for one week at 25°C under the light conditions described above, and bacterial growth inhibition

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Biotechnology and Bioprocess Engineering 20: 1056-1063 (2015)

was measured based on whether or not the disks were surrounded by a clear zone. To investigate the viability of strain YC001 and its associated bacteria after being treated with the antibiotic cocktails (ACs), growth was determined at AC concentrations of 5, 10, 20, 40, and 100 µg/mL. The ACs were applied to the cultures of 1.0 × 106 cells/mL in 15 mL R2A broth for 3 days at 25°C under dark conditions. The harvested cells were washed twice with BG11 medium and inoculated in 20 mL of BG11 broth at an initial concentration of 1.0 × 104 cells/mL. The cell state and number of bacteria and microalgae were measured by microscopy and FACS analysis for 2 weeks. The ratio of growth inhibition was calculated by comparing the number of cells between the control and each treatment.

controlled using BD FACS Diva software (version 5.0.2). The gate determining the microalgae and debris was set according to the values of the FSC (Forward Scatter), SSC (Side Scatter), and PerCP-Cy5.5 filter. Gates P1 (for debris) and P4 (for microalgae) were set using the values of the FSC and SSC. P1 was re-gated to P2 (for debris with fluorescence) and P3 (for debris without fluorescence) using the value of PerCP-Cy5.5 (Supplementary Fig. 1). While P3 included probable bacteria as well as cell debris, P2 only included microalgae selected by chlorophyll fluorescence. Thus, the value of gate P3 was considered as a factor for bacterial contamination in this study. To count the number of cells in each culture, the sorting was conducted up to 20,000 events or 30 seconds and the related events were recorded. The cells were sorted in 15 mL conical tubes containing 5 mL of sterile R2A medium to observe bacterial growth. The sorting, which included about 2,000 events in each gate, was conducted at least twice followed by cultivation at 25°C for 2 weeks.

2.4. Fluorescence-activated cell sorting (FACS) Flow-cytometry analysis was carried out using a BD FACS Aria II cell sorter from Becton Dickinson (CA, USA). The chlorophylls in the microalgae were excited using lowpowered, air-cooled, and solid-state lasers at 488 nm. The emitted fluorescence (EF) was measured using a 695/ 40 nm PerCP-cy5.5 filter, while the forward scatter (FSC) and side scatter (SSC) of the laser beam were used to determine the gate, distinguishing the microalgae from debris. All sorting by the BD FACS Aria II cytometer was

Fig. 1. Effect of antibiotic cocktail treatment on the viability of strain YC001 and growth inhibition of associated bacteria. Bacterial growth inhibition and algal viability were determined by estimating the number of cells on the corresponding microalgal gate and debris on the FACS analysis. Growth inhibition was calculated by comparing each treatment with the control. The debris of dead cell in the debris gate was not accurately excluded when estimating the inhibition of bacterial growth. The closed circles and triangles represent the treated with AC-3, while the open circles and triangles represent the treated with AC-5. Each result is the average of three biological replicates ± SD.

2.5. DGGE and SEM analysis Genomic DNA was extracted from the control and treated cultures using a Fast DNA Spin Kit (MPbio, USA). 16S rRNA gene fragments were partially amplified using the universal bacterial primers described above. DGGE was carried out using nested PCR primers: 341F-GC clamps (5'-CGC CCG CCGCGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCCTAC GGG AGG CAG CAG-3') and 907R. The PCR-amplified fragments were electrophoresed on a 12% polyacylamide gel using a 25 ~ 50% ureaformamide gradient for 20 h at 110 V and 60°C. DGGE was performed in a D-Code 16/16 cm gel system with a gel width of 1.0 mm (Bio-Rad). To observe the cells using SEM, the samples were fixed in a 2.5% paraformalehyde-glutaraldehyde mixture buffered with 0.1 M phosphate (pH 7.2) for 2 h, post-fixed in 1% osmium tetroxide in the same buffer for 1 h, dehydrated in graded ethanol, and substituted by isoamylacetae. The samples were then dried to the critical point in CO2, sputtered with gold using a sputter coater (SC502, Polaron), and observed using a HITACHI S4300N scanning electron microscope (Hitachi, Japan). The cultures were also stained with SYBR Green I (Invitrogen, USA) and observed under a fluorescence microscope (Microphot-FXA, Nikon). 2.6. Oxygen evolution To measure photosynthetic efficiency, oxygen evolution was measured by an Oxygraph (Hansatech, England) at 0, 35, 70, 100, 140, 180, 235, 350, and 410 µmol photons/m2/ s of irradiance. The photosynthetic parameters, such as the maximum photosynthetic rate (Pmax), photosynthetic efficiency (α), and initial saturation intensity of irradiance for


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photosynthesis (Ik), were all measured using a photosynthesisirradiance (P-I) curve [29,30]. P = Pmax(1 – e–αI/ Pmax) Ik= Pmax /α

3. Results and Discussion 3.1. Isolation and identification of associated bacteria The associated bacteria of Ettlia sp. YC001 were analyzed using culture-dependent and -independent methods based on a molecular biological approach. DNA was extracted from the xenic cultures of strain YC001 in the BG11 and R2A medium, respectively, on day 7 and the bacterial communities were analyzed based on the DGGE band patterns of the 16S rRNA fragments. Major bands were affiliated with Scenedesmus sp. chloroplast in the BG11 broth, while Brevundimonas sp. was dominant in the R2A medium. The other bands were similar in both cultures (Supplementary Fig. 2). Nine colonies were isolated based on their morphological characteristics and were then routinely maintained on R2A agar plates. To identify the isolated bacteria, DNA was extracted and a Blast search conducted using the 16S rRNA gene at the EZtaxon site. These bacteria mainly belonged to α, γ-Proteobacteria, Firmicutes, and Actinobacteria (Table 1). As the closest type strains were mostly isolated from soil, fresh water, and marine environments, the isolated associated bacteria seemed to have originated from both the collected water samples and the cultures. However, the precise roles of the isolated bacteria in the phycosphere of strain YC001 have not yet been defined. According to a previous report, most Brevundimonas sp., isolated from aquatic environments, have a growth-promoting effect on Chorella ellipsoidea [31]. Pseudomonas sp. is known to stimulate the growth of Asterionella glacialis, Scenedesmus bicellularis, and Chlorella sp. by supporting glycoproteins

Fig. 2. Schematic representation of a species-specific approach for establishment of axenic culture of Ettlia sp. YC001.

or the induction of more favorable environmental conditions [2,3,32]. Thus, further studies are needed to focus on the specific interaction between an axenic culture of strain YC001 and its associated bacteria in order to accomplish high-biomass productivity for biofuel production. 3.2. Antibiotic susceptibility and optimization of antibiotic cocktail treatment Antibiotics were selected using a species-specific approach according to the analyzed community of associated bacteria.

Table 1. Characterization of bacteria isolated from a culture of Ettlia sp. YC001, using a culture-based method Isolated strains 1 2 3 4 5 6 7 8 9 * **

Most close type strain* Brevundimonas naejangsanensis DSM7126T Blastomonas natatoria DSM 3183 T Psuedomonas toyotomiensis JCM15604T Bacillus niabensis DSM 17723T Bacillus herbersteinensis D-1-5aT Microbacterium ginsengisoli DSM18659T Microbacterium invictum DC-200T Blastococcus saxobsidensDSM44509 T Terrimonas lutea DYT

Similarity (%)** 98.5 99.8 99.8 99.2 99.0 98.5 98.6 99.7 96.6

Cell shape rod rod/ovoid straight rods rod rod short rod short rod rod, coccus rod

, : identified by blast search using 16S rRNA amplified with primers 27F and 1492R at the EZtaxon site. : cultured on an R2A agar plate for 7 days.

***

Colony color*** greyish yellow yellow yellowish white yellowish white cream-colored yellow violet pink pigmented yellow

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Table 2. Antibiotic susceptibility of bacteria associated with strain YC001

a low effect on the growth of microalgae, 10 µg/mL AC5 was selected as the optimal antibiotic concentration. The cultures treated with AC-5 were subcultured 2 times during 2 weeks in BG11 broth. The harvested cells were washed twice by vigorous vortexing with autoclaved deionized water and serially diluted microbial cells, up to 1.0 × 102, were spread on R2A agar plates. Moreover, the widelyused bacterial medium R2A was used for observing the growth of bacteria. Although it took time for detectable colonies to grow on the agar plates because of the small number of intial cells, the approach was successful in excluding any residual bacteria. Over sixty pure colonies were obtained and selected to investigate for axenicity.

Antibiotics Chloramphenicol Tetracycline Rifampicin Imipenem Carbendazim Antibiotic cocktail-3* Antibiotic cocktail-5**

Concentration (µg/mL) 1 R R R R R -

2 R R R R R -

5 R R R R R S S

10 R S S R S S S

20 R S S R S S S

40 S S S R S -

100 S S S R S -

R: resistant, S: sensitive, -: has not been performed. * : chloramphenicol, tetracycline, imipenem. ** : carbendazim, chloramphenicol, imipenem, rifampicin, tetracycline.

Firstly, three antibiotics, imipenem, chloramphenicol, and tetracycline, were selected to eliminate Proteobacteria and Firmicutes [33,34]. Rifampicin was also used to exclude actinomycetes which survived in a continuous culture after irradiation treatment with electro-beams (unpublished data) [35]. Finally, carbendazim was added to remove fungi which is frequently observed in liquid cultivars [36]. The associated bacteria were found to be sensitive to all the antibiotics at concentrations over 10 µg/mL, with the exception of chloramphenicol and imipenem, to which resistance was still evident at concentrations of 20 and 100 µg/mL, respectively (Table 2). However, the associated bacteria were much more sensitive to AC-3 and AC-5, even at a low concentration (5 µg/mL). Therefore, antibiotic cocktail treatment was selected for further study. While both cocktail treatments resulted in substantial bacterial growth inhibition, algal growth was decreased with antibiotic concentration up to 20 µg/mL (Fig. 1). However, at concentrations between 20 and 100 µg/mL, algal viability remained steady at about 55 ~ 58% in AC3 and 43 ~ 48% in AC-5. Thus, the inhibitory effect was similar after a certain antibiotic concentration threshold. The culture treated with AC-5 had fewer cells than the culture treated with AC-3, indicating that AC-5 had a more harmful effect on the growth of strain YC001. However, the cells recovered after 2 ~ 3 subcultures without antibiotics and showed no difference in cell size or growth when compared with the control, except for the cells treated at concentrations of 100 µg/mL. With regard to the growth inhibition of bacteria, AC-5 was able to reduce the debris gate up to 90% at a concentration of 10 µg/mL, whereas AC-3 was only able to do this at a concentration over 40 µg/mL. However, the reduction was never higher than 95%, even at 100 µg/mL, due to the limitation of FACS in accurately distinguishing the surviving bacteria from the dead cells in the debris gate. Because of high bacterial growth inhibition (~ 80%) and

3.3. Determination of axenicity using DGGE, FACS, and SEM A standard approach to confirm an axenic state is to spread an aliquot of the culture on an agar plate and check for the presence of bacteria during a fixed time period. While R2A medium provides sufficient nutrients to the bacteria, unculturable bacteria still pose some problems. Meanwhile, in the case of non-culture-based methods such as DGGE, PCR amplification is invariably limited because of an insufficient concentration of bacterial DNA or inaccurate primer annealing. Therefore, we used a series of phenotypic as well as genotypic experiments to verify axenicity. FACS

Fig. 3. DGGE analysis of controls and treatments. A-B, controls from subculture in BG11 (A) and R2A (B) medium over 3 months; C-E, axenic candidates in R2A medium.1, Scenedesmus sp. chloroplast 16S rRNA; 2, Pseudomonas toyotomiensis; 3-4, Blastomonas ursincola; 5, Brevudimonas halotolerans; 6, Catellibacterium nectariphilum; 7, Microbacterium paraoxydans.


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Fig. 4. Scanning electron microscopic images of control (A) and treated (B) cultures at 3,500X magnification; scale bar = 10 µm.

and SYBR Green I staining were used as visual methods to check for the presence of bacteria, while DGGE provided a higher resolution for detecting bacterial DNA based on a molecular approach (Supplementary Fig. 4). Finally, SEM images provided the highest resolution at a magnification of 3,500 ~ 8,000. All the procedures used in this study are presented in Fig. 2. Pure colonies were inoculated in 150 µL of R2A and BG11 broth, respectively, in 96-well plates and cultured for 2 weeks. The axenicity of cultures was confirmed by DGGE, FACS, and microscopy. The results of DGGE analysis revealed that some of the pure colonies still included bacteria like Brevudimonas sp., while some candidate cultures showed no bacterial bands except for that of Scenedesmus sp. chloroplast (Fig. 3). Twenty candidate axenic cultures were selected for FACS and analyzed at two-day intervals over 2 weeks. Based on 20,000 cells each from the controls and treated cultures, the occurrence of P3 events, which were assumed to be factors for bacterial contamination, occurred in less than 1% of all candidate axenic cultures, while they occurred in 24 ± 1.5% of the control cultures. These values were maintained over the 2 weeks. Furthermore, 2,000 cells from the P3 gate were inoculated in 5 mL of R2A broth to check for bacterial growth at 25°C under dark conditions during 2 weeks. While a layer of bacteria was observed in the control cells, no such growth was observed in the treated cells (data not shown). Two samples among the twenty candidate axenic cultures were selected for SEM analysis. Various species of bacteria, such as rods and coccuses, were observed in the controls, but the SEM images of the two selected cultures showed no bacteria (Fig. 4). Therefore, these were finally selected as axenic cultures of strain YC001. 3.4. Growth and preservation of an axenic culture To determine any damage done by the antibiotic cocktails,

Fig. 5. Photosynthetic rate curves for control and treated cultures. The circle and triangle represent the growth of control and treatmsent, respectively. Each result is the average of three biological replicates ± SD.

the microalgal cell growth and photosynthetic rate were measured. Growth was estimated by measuring the cell numbers and DCW every 2 days, while photosynthetic efficiency was determined by measuring oxygen evolution on day 12. The DCW was similar for both the control and the treated cultures at about 0.37 ~ 0.39 mg/mL, which is within the margin of error, while the number of cells in the treated cultures was 1.3 times more than that in the controls (Supplementary Fig. 3). In addition, a t-test confirmed a significant difference between the control and treated cultures (P < 0.05). FACS analysis showed that the control cells were 1.2 times larger than the treated cells. The O2 evolution rate was normalized using 106 algal cells. O2 evolution gradually increased with intensity of irradiance and reached saturation at approximately 140 and 235 µmol photons/m2/sec for the control and treated samples, respectively (Fig. 5). No inhibition of irradiance was shown up to 410 µmol/m2/sec, the strongest light used

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in this experiment. The maximum photosynthetic rate (Pmax), light saturation intensity, and photosynthetic efficiency (α) for the control and treated samples were 104.2 and 119.8 nmol O2/106 cells/h, 52.1 and 67.6 µmol photons/m2/sec, and 1.99 and 1.77 (nmol O2/106 cells/h)/(µmol photons/m2/sec), respectively. It seemed that the higher photosynthetic efficiency of the treated samples allowed supplementary energy to be allocated for rapid differentiation, thereby reducing the size of the algal cells. However, the growth and photosynthetic rate were not markedly different between the control and treated samples. Thus, the axenic cultures obtained in this study were not physiologically damaged and were suitable for further studies. While the maintenance of an axenic culture in liquid medium requires attention, this is easily accomplished on an agar plate with proper sealing. Pure colonies from these cultures were transferred to slant and plates of R2A agar every one month. New colonies from the 3rd sector of streaks were inoculated in 2 mL of BG11 and R2A broth and checked for any contamination through DGGE and FACS analysis. Consequently, axenic cultures of strain YC001 have been maintained using regular subcultures on bacterial agar plates for over 15 months.

Institute of Bioscience and Biotechnology) Research Initiative Program.

4. Conclusion The composition of AC-5 was specifically determined according to the population of associated bacteria and various modes of preventative action of broad-spectrum antibiotics. The optimal concentration and dosage of AC5 did not affect algal viability and only eliminated the associated bacteria. Serial dilution followed by spreaing on agar plates was effective in excluding any residual bacteria. Axenicity was confirmed using a series of physiological and molecular approaches, and has been maintained on plates by reglular subculture for over 15 months. The above developed procedure for establishment of axenic cultures and preservation of those cultures could be applied to other strains of microalgae as well. Moreover, axenic cultures can be used for whole-genome sequencing and ecophysiomic research, which hopefully will provide essential clues for the design of better industrial production systems for target compounds.

Acknowledgements This study was supported by the Advanced Biomass R&D Center (ABC) of the Global Frontier Project funded by the Ministry of Science, ICT, and Future Planning (20100029719) and a grant from the KRIBB (Korea Research

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