- Email: [email protected]
Use of a triiodide resin for isolation of axenic cultures of microalgal Nannochloropsis gaditana
Accepted Manuscript Use of a triiodide resin for isolation of axenic cultures of microalgal Nannochloropsis gaditana Kibok Nam, Won-Sub Shin, Byeong-ryool Jeong, Min S. Park, Ji-Won Yang, Jong-Hee Kwon PII: DOI: Reference:
S0960-8524(15)00414-9 http://dx.doi.org/10.1016/j.biortech.2015.03.082 BITE 14772
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
27 January 2015 16 March 2015 17 March 2015
Please cite this article as: Nam, K., Shin, W-S., Jeong, B-r., Park, M.S., Yang, J-W., Kwon, J-H., Use of a triiodide resin for isolation of axenic cultures of microalgal Nannochloropsis gaditana, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.03.082
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Use of a triiodide resin for isolation of axenic cultures of microalgal Nannochloropsis gaditana
Kibok Nam1, Won-Sub Shin1, Byeong-ryool Jeong1, Min S. Park1,2, Ji-Won Yang1,2 Jong-Hee Kwon3,* 1
Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehakno,
Yuseong-gu, Daejeon 305-701, Republic of Korea 2
Advanced Biomass R&D Center, KAIST, 291 Daehakno, Yuseong-gu, Daejeon 305-701,
Republic of Korea 3
Department of Food Science & Technology, Gyeongsang National University, Jinju
660-701, Republic of Korea
*
Corresponding authors
Jong-Hee Kwon, Tel: +82-55-772-1901, Fax: +82-55-772-1909, E-mail: [email protected] 1
Abstract Triiodide resin (TR) was used to generate axenic cultures of microalgae by employing the antibacterial capability of triiodide. A Nannochloropsis gaditana culture contaminated with bacteria was passed through a column filled with TR using the gravity flow. Based on analyses of flow cytometry and vital staining using a fluorescent dye SYTOX Green, three cycles of TR treatments remarkably reduced the number of viable bacteria but had little effects on the microalgae. This novel approach is a simple, rapid, and cost-effective method that can be used to isolate axenic cultures of microalgae.
Keywords: Microalgae; Triiodide Resin; Axenic Culture; Nannochloropsis gaditana 2
1. Introduction Microalgae are the most promising feedstock for the sustainable production of commodities such as food, animal feed, chemicals, industrial materials, and biofuels. In natural ecosystems, microalgae usually occur with other microorganisms, such as fungi and bacteria (Shishlyannikov et al., 2011). However, microalgae used for production of high-value products and bioenergy are typically grown in monoculture cultures to maintain reliable productivity (Wilkie et al., 2011). Furthermore, axenic cultures are essential for basic molecular genetics research and for biological and physiological investigations under mixotrophic and heterotrophic conditions (Hyka et al., 2013). Previous studies have used different approaches to generate axenic cultures of microalgae (Divan & Schnoes, 1982; Doan et al., 2011; Hyka et al., 2013; Suga et al., 2011; Sykora et al., 1980; Watanabe et al., 2005; Wiedeman et al., 1964). The most common method is to prepare cultures from a single cell by serial dilution, which consists of streaking on selective agar plates and repeated sub-culturing (Levy et al., 2009; Timmins et al., 2009). This method is simple and cost-effective, but is not possible when cultures are screened from environmental samples, such as wastewater that contains large amounts of bacteria or bacteria attached to algal cell walls. In addition, the relatively low colony-forming rate of microalgae is a difficulty with this method (Cho et al., 2013; Kuo & Lin, 2013). Previous research indicated that a strongly basic quaternary ammonium anion exchange triiodide resin, (TR) complex (resin-I3), is an effective disinfectant against diverse bacteria (Taylor et al., 1970), although the exact mechanism of this effect is unknown. The most common application of contact TR is for purification of water for domestic and industrial use (Taylor et al., 1970). Interestingly, preliminary tests 3
indicated that Nannochloropsis gaditana was more resistant to TR than their bacterial contaminants. The high tolerance of microalgae to TR could be due to the unique chemistry and structure of their cell walls. The present research was designed to overcome the limitations of conventional methods for the generation of axenic cultures of microalgae. In particular, this novel method combines existing screening methods with a simple process that uses TR columns. This strategy was evaluated by assaying for bacterial colony forming units (CFUs), DAPI staining of broth culture, and cytometric analysis using SYTOX Green staining. No previous research has used a TR complex for the isolation of axenic cultures of microalgae. This process has great potential for the isolation of pure microalgal strains from bacteria-contaminated cultures because it does not require antibiotic treatment or expensive flow cytometry equipment used for cell sorting.
2. Materials and methods 2.1. Preparation of TR columns Triiodide ion (I3-) was prepared by combining crystalline I2 and aqueous potassium iodide (KI), according to the protocol of Taylor et al. (1970). The macroporous strong base anion resin, A-520 E (Purolite Co), was submerged in triiodide solution. Then, the resulting TR complexes were extensively rinsed with distilled water until colorimetric iodimetry showed that these rinses were free of iodide. Resin-I3, under the trade name A605, can be purchased from Purolite. A cylindrical tube (3-cm diameter, 8-cm height) was prepared, and its conical bottom was sealed with a steel mesh to prevent efflux of TR. After filling the column with TR, the resin was rinsed with sterilized distilled water prior to treatment with bacteria-contaminated cultures of the microalgae. 4
2.2. Treatment of N. gaditana cultures with a TR column N. gaditana cultures that were contaminated with several bacteria were prepared (OD ~1.5 at 680 nm). The formation of colony forming units (CFUs) was used to quantify and identify viable bacteria. Tryptone-yeast extract-glucose (TYG) agar medium and LB agar medium (10-fold diluted) were prepared to accommodate the broad nutrient requirements of diverse bacteria. Then 50 mL of a bacteria-contaminated culture of N. gaditana was passed over the TR column by gravity flow. The TR columntreated and untreated N. gaditana cultures were then plated onto TYG and LB agar at 100-fold dilution and incubated for 48 h at 37 °C and at room temperature. All treatment and plating procedures were performed in triplicate. After this short incubation period, the number of bacterial colonies was counted; microalgal colonies were not detected because they cannot generate visible colonies on TYG or LB agar within 48 h.
2.3. Screening for bacteria-free cells in TR-treated cultures For screening of TR-treated microalgae, 10 µL aliquots of TR column-treated N. gaditana cultures were added to a 96-well plate (SPL, Korea), with 200 µL of LB per well, and cultivated for 24 h under light. The growth of bacteria was measured by optical density at 600 nm. Bacteria-free algal cultures were screened from these plates. The percentage of axenic cultures was calculated as: (Number of bacteria-free wells) / (Total number of total wells) × 100 % Each bacteria-free culture of N. gaditana was inoculated into a culture flask that contained 200 mL of f/2 (N 5×) medium, and cultivated with 3 % CO2 under 50 µmol photons m-2 s-1 of light. Growth of N. gaditana was measured by cell concentration and optical density at 680 nm. 5
2.4. Identification of bacteria in contaminated N. gaditana cultures The colony PCR method with the iProof DNA polymerase kit (BioRad 172-5302) was used for the identification of bacteria, with primers for a universal bacterial 16S rDNA. The forward primer was 9F (5`- GAGTTTGATCCTGGCTCAG-3`), and reverse primer was 1492R (3`- GCTTACCTTGTTACGACTT-5`). The PCR protocol consisted of initial denaturation (95 °C for 5 min), denaturation/annealing/extension (30 cycles at 95 °C for 1 min, 58 °C for 1 min 72 °C for 1 min), and a final extension (72 °C for 5 min). Amplified bacterial 16S rDNA was confirmed by electrophoresis. The gel extraction method (QIAquick Gel Extraction Kit, Cat. No. 28706) and the BLAST library were used for identification of extracted 16S rDNAs. In particular, 50 µL of bacterial 16S DNA was loaded onto 0.5 % agarose gels. After electrophoresis, the desired region was excised and transferred into a 1.7 mL Eppendorf tube. Then, 500 µL of QC buffer was added, and the tubes were incubated for 10 min at 50 °C after mixing. Then, 200 µL of isopropanol was added and the mixture was transferred into 2 mL collection tubes. These tubes were centrifuged (13,000 rpm × 1 min at room temperature) and the flow-through was discarded. For washing, 750 µL of PE buffer was added and centrifuged (13,000 rpm × 1 min). For elution, 30 µL of elution buffer was added and the solution was centrifuged again (13,000 rpm × 1 min). This purified DNA was used for sequencing and strain identification.
2.5. SYTOX Green staining and flow cytometry analysis SYTOX Green, a green-fluorescent stain that permeates the membranes and stains the nucleic acids of dead or inactive prokaryotes and eukaryotes, was used to determine the number of bacteria and microalgae damaged by TR treatment. Contaminated N. 6
gaditana cultures, before and after TR treatment, were stained with 5 mM SYTOX Green and the mixture was incubated for 10 min at room temperature without light. Before analysis, spotting points of pure bacteria and pure N. gaditana were measured without staining. A high speed flow cytometer, MoFlo XDP (Beckman Coulter, Fullerton, CA) was used to identify cells of different sizes and for separation of cells of the same size but different fluorescence. The excitation wavelength was 488 nm (argon laser) and emission was measured at 530–540 nm.
2.6. Fluorescence microscopy DAPI (4′-6-diamidino-2-phenylindole), a fluorescent stain that permeates the membranes of live cells and stains A-T rich regions in DNA, was used to identify live cells. Fluorescence was measured under a fluorescence microscope (Microphot-FXA, Nikon), with excitation at 365 nm. The DNA-DAPI complex in bacterial cells fluoresces bright blue, and the red autofluorescence of chlorophyll in microalgae masks this blue fluorescence.
3. Results and discussion 3.1. Effect of triiodide resin on xenic cultures of N. gaditana The number of total bacteria in each N. gaditana culture was measured indirectly by determining CFU mL-1 using LB and TYG agar plates. Prior to treatment, the test culture contained 2.99 ± 0.12 × 107 cells mL-1 of N. gaditana (determined with a Neubauer Improved DHC-N01 hemocytometer), and had bacterial counts of 5.87 ± 0.62 × 106 CFU mL-1 (LB medium) and 2.63±0.19 × 106 CFU mL-1 (TYG medium). There were 5 main bacterial strains detected by the 16S rDNA assay: Pseudomonas sp., 7
Stappia sp., Labrenzia sp., Microbacterium sp., and Tistrella mobilis. After 3 times TR treatments, 0.29 CFU mL-1 (LB medium) and 0 CFU mL-1 (TYG medium) were present in the culture (Table 1). Flow cytometry analyses with SYTOX Green staining were used to confirm the impact of TR treatment on the viability of N. gaditana cells and bacteria in the xenic culture. SYTOX Green only stains inert or dead cells, so the fluorescence will increase if there are more dead cells after TR treatment (du Plessis & Hamman, 2014; Rychtecký et al., 2014). Analysis of bacteria (Fig. 1a) indicated that the fluorescence increased significantly after TR treatment, suggesting that most bacteria were damaged or killed by TR treatment. On the other hand, analysis of N. gaditana (Fig. 1b) indicated only a small change in fluorescence, suggesting that N. gaditana cells were not severely damaged by this treatment. These results indicate that most viable bacteria in the xenic N. gaditana culture were selectively screened-out by TR treatment.
3.2. Screening xenic cultures of N. gaditana Although TR treatment remarkably decreased the number of bacteria in the xenic N. gaditana culture, TR treatment alone may not necessarily yield pure cultures of N. gaditana. Thus, to obtain an axenic culture, TR-treated N. gaditana cells were added into 96 well plates that contained LB medium to test for the presence of bacteria. Each well was observed for 48 h, with untreated samples as the negative control. This process was repeated with five times. About 80 % of the samples reached a threshold OD600nm value, indicating the presence of bacteria. The average probability of obtaining an axenic culture by screening following TR treatment was 19.25 %. Bacterial growth was observed in all untreated culture samples (data not shown). 8
One of the bacteria-free aliquots was inoculated into fresh f/2 medium to confirm the purity of the TR-treated culture of N. gaditana. After 4 days of cultivation, this culture was subjected to DAPI staining and observed by fluorescence microscopy; an untreated culture was the positive control. The untreated culture had abundant bacteria indicated by blue and green fluorescent spots (Supplemental Fig. 1a), but the TRscreened culture only had red fluorescence due to auto-fluorescence of chlorophyll from N. gaditana (Supplemental Fig. 1b). These results indicate that TR treatment dramatically reduced the number of bacteria in the N. gaditana culture, although they do not prove that the resulting N. gaditana was absolutely pure immediately after TR treatment. Nonetheless, these results indicate that TR treatment dramatically and selectively reduced the bacterial population and therefore greatly increased the probability of selecting an axenic part within whole culture.
3.3 Growth of xenic and axenic cultures of N. gaditana The growth of xenic N. gaditana cultures differed from that of axenic cultures (Fig. 2). In particular, based on measurement of chlorophyll concentration (680 nm), growth of the axenic culture showed considerably lower maximum concentration than the xenic culture (Fig. 2). Previous research reported similar results with other species (Covarrubias et al., 2012; Kim et al., 2014). For instance, a xenic culture of Chlorella sp. grew much better than an axenic culture in terms of dry cell weight and lipid content (Cho et al., 2015; Lee et al., 2013), and the authors suggested an interaction of Chlorella sp. with certain bacteria enhanced the accumulation of microalgal biomass. However, the mechanism of this effect in N. gaditana and Chlorella sp. are unknown because 9
there are only limited studies of the symbiotic interactions between microalgae and bacteria. In the present study, the main bacterial contaminants were Pseudomonas sp., Stappia sp., Labrenzia sp., and Microbacterium sp. Tristrella mobilis. Investigation of the mechanism of the interactions between N. gaditana and these bacteria also requires starting with an axenic microalgal culture. The present study describes an easy method for isolation of axenic N. gaditana cultures. Further optimization of this method will allow additional applications in fundamental research, including studies of the interaction between microalgae and bacteria.
4. Conclusions Isolation of axenic cultures of microalgae is very important for basic and applied studies, but most current methods are laborious, time consuming, and expensive. Passage of a xenic culture of N. gaditana through a triiodide resin (TR) column effectively enables to screen microalgae from bacteria, because this substrate damages or kills bacteria but has little effect on microalgae. The results of the present study suggest that use of a TR column for isolation of axenic cultures of microalgae is a simple, rapid, and cost-effective approach for the isolation of axenic cultures of microalgae.
Acknowledgements This work was supported by a grant from the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Science, ICT & Future Planning (ABC-2010-0029728). 10
References 1. Cho, D.-H., Ramanan, R., Heo, J., Lee, J., Kim, B.-H., Oh, H.-M., Kim, H.-S. 2015. Enhancing microalgal biomass productivity by engineering a microalgal–bacterial community. Bioresour. technol. 175(0), 578-585. 2. Cho, D.H., Ramanan, R., Kim, B.H., Lee, J., Kim, S., Chan, Y., Choi, G.G., Oh, H.M., Kim, H.S. 2013. Novel approach for the development of axenic microalgal cultures from environmental samples. J. Phycol. 49(4), 802-810. 3. Covarrubias, S., de-Bashan, L., Moreno, M., Bashan, Y. 2012. Alginate beads provide a beneficial physical barrier against native microorganisms in wastewater treated with immobilized bacteria and microalgae. Appl. Microbiol. Biotechnol. 93(6), 2669-2680. 4. Divan, C.L., Schnoes, H.K. 1982. Production of axenic Gonyaulax cultures by treatment with antibiotics. Appl. Environ. Microbiol. 44(1), 250-254. 5. Doan, T.T.Y., Sivaloganathan, B., Obbard, J.P. 2011. Screening of marine microalgae for biodiesel feedstock. Biomass Bioenergy. 35(7), 2534-2544. 6. du Plessis, L.H., Hamman, J.H. 2014. In vitro evaluation of the cytotoxic and apoptogenic properties of aloe whole leaf and gel materials. Drug Chem. Toxicol. 37(2), 169-177. 7. Hyka, P., Lickova, S., Pribyl, P., Melzoch, K., Kovar, K. 2013. Flow cytometry for the development of biotechnological processes with microalgae. Biotechnol. Adv. 31(1), 216. 8. Kim, B.-H., Ramanan, R., Cho, D.-H., Oh, H.-M., Kim, H.-S. 2014. Role of Rhizobium, a plant growth promoting bacterium, in enhancing algal biomass through mutualistic interaction. Biomass Bioenergy. 69(0), 95-105. 9. Kuo, R.C., Lin, S.J. 2013. Ectobiotic and Endobiotic Bacteria Associated with Eutreptiella sp. Isolated from Long Island Sound. Protist. 164(1), 60-74. 10. Lee, J., Cho, D.-H., Ramanan, R., Kim, B.-H., Oh, H.-M., Kim, H.-S. 2013. Microalgaeassociated bacteria play a key role in the flocculation of Chlorella vulgaris. Bioresour. 11
Technol. 131(0), 195-201. 11. Levy, J.L., Stauber, J.L., Wakelin, S.A., Jolley, D.F. 2009. The effect of bacteria on the sensitivity of microalgae to copper in laboratory bioassays. Chemosphere. 74(9), 12661274. 12. Rychtecký, P., Znachor, P., Nedoma, J. 2014. Spatio-temporal study of phytoplankton cell viability in a eutrophic reservoir using SYTOX Green nucleic acid stain. Hydrobiologia. 740(1), 177-189. 13. Shishlyannikov, S.M., Zakharova, Y.R., Volokitina, N.A., Mikhailov, I.S., Petrova, D.P., Likhoshway, Y.V. 2011. A procedure for establishing an axenic culture of the diatom Synedra acus subsp. radians (Kütz.) Skabibitsch. from Lake Baikal. Limnol. Oceanogr. 9, 478-484. 14. Suga, K., Tanaka, Y., Sakakura, Y., Hagiwara, A. 2011. Axenic culture of Brachionus plicatilis using antibiotics. Hydrobiologia. 662(1), 113-119. 15. Sykora, J.L., Keleti, G., Roche, R., Volk, D.R., Kay, G.P., Burgess, R.A., Shapiro, M.A., Lippy, E.C. 1980. Endotoxins, Algae and Limulus Amebocyte Lysate Test in DrinkingWater. Water Res. 14(7), 829-839. 16. Taylor, S., Fina, L., Lambert, J. 1970. New water disinfectant: an insoluble quaternary ammonium resin-triiodide combination that releases bactericide on demand. Appl.Microbiol. 20(5), 720-722. 17. Timmins, M., Thomas-Hall, S.R., Darling, A., Zhang, E., Hankamer, B., Marx, U.C., Schenk, P.M. 2009. Phylogenetic and molecular analysis of hydrogen-producing green algae. J. Exp. Bot. 60(6), 1691-1702. 18. Watanabe, K., Takihana, N., Aoyagi, H., Hanada, S., Watanabe, Y., Ohmura, N., Saiki, H., Tanaka, H. 2005. Symbiotic association in Chlorella culture. FEMS Microbiol. Ecol. 51(2), 187-196. 19. Wiedeman, V.E., Walne, P.L., Trainor, F.R. 1964. A new technique for obtaining axenic 12
cultures of algae. Can. J. Bot. 42(7), 958-959. 20. Wilkie, A.C., Edmundson, S.J., Duncan, J.G. 2011. Indigenous algae for local bioresource production: Phycoprospecting. Energy Sustainable Dev. 5(4), 365-371.
13
Figure Legends
Fig. 1. Effect of TR treatment on bacteria and N. gaditana cells based on SYTOX Green staining followed by flow cytometry. a) Fluorescence of bacteria. TR treatment significantly shifted the fluorescence peak, indicating that treatment damaged or killed these cells. b) Fluorescence of N. gaditana. TR treatment had little effect on the fluorescence peak, indicating that treatment had little or no effect on these cells. Red lines: before TR treatment; green lines: after TR treatment.
Fig. 2. Cellular growth of xenic and axenic N. gaditana cultures grown with f/2 medium. Growth was measured by change in optical density at 680 nm, a measure of chlorophyll content. Indicated values are the averages of three independent measurements with standard errors.
Table Table 1. Effect of TR treatment on the number of bacteria present in xenic cultures of N. gaditana. CFU (cells mL-1)
No Treatment
1 Treatment
2 Treatments
3 Treatments
LB medium
5.87±0.62 ×106
4.01±0.10 ×104
4.00±0.10 ×103
0.29
TYG medium
2.63±0.19×106
4.30±0.10×104
1.00±0.40 ×103
0
14
15
16
Highlights Triiodide resin has a disinfectant effect on bacteria. Contaminated Nannochloropsis gaditana culture was treated with triiodide resin. TR treatment can be an effective tool for obtaining axenic microalgal culture. Growth kinetic of axenic and xenic N. gaditana was quite different.
17
S0960-8524(15)00414-9 http://dx.doi.org/10.1016/j.biortech.2015.03.082 BITE 14772
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
27 January 2015 16 March 2015 17 March 2015
Please cite this article as: Nam, K., Shin, W-S., Jeong, B-r., Park, M.S., Yang, J-W., Kwon, J-H., Use of a triiodide resin for isolation of axenic cultures of microalgal Nannochloropsis gaditana, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.03.082
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Use of a triiodide resin for isolation of axenic cultures of microalgal Nannochloropsis gaditana
Kibok Nam1, Won-Sub Shin1, Byeong-ryool Jeong1, Min S. Park1,2, Ji-Won Yang1,2 Jong-Hee Kwon3,* 1
Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehakno,
Yuseong-gu, Daejeon 305-701, Republic of Korea 2
Advanced Biomass R&D Center, KAIST, 291 Daehakno, Yuseong-gu, Daejeon 305-701,
Republic of Korea 3
Department of Food Science & Technology, Gyeongsang National University, Jinju
660-701, Republic of Korea
*
Corresponding authors
Jong-Hee Kwon, Tel: +82-55-772-1901, Fax: +82-55-772-1909, E-mail: [email protected] 1
Abstract Triiodide resin (TR) was used to generate axenic cultures of microalgae by employing the antibacterial capability of triiodide. A Nannochloropsis gaditana culture contaminated with bacteria was passed through a column filled with TR using the gravity flow. Based on analyses of flow cytometry and vital staining using a fluorescent dye SYTOX Green, three cycles of TR treatments remarkably reduced the number of viable bacteria but had little effects on the microalgae. This novel approach is a simple, rapid, and cost-effective method that can be used to isolate axenic cultures of microalgae.
Keywords: Microalgae; Triiodide Resin; Axenic Culture; Nannochloropsis gaditana 2
1. Introduction Microalgae are the most promising feedstock for the sustainable production of commodities such as food, animal feed, chemicals, industrial materials, and biofuels. In natural ecosystems, microalgae usually occur with other microorganisms, such as fungi and bacteria (Shishlyannikov et al., 2011). However, microalgae used for production of high-value products and bioenergy are typically grown in monoculture cultures to maintain reliable productivity (Wilkie et al., 2011). Furthermore, axenic cultures are essential for basic molecular genetics research and for biological and physiological investigations under mixotrophic and heterotrophic conditions (Hyka et al., 2013). Previous studies have used different approaches to generate axenic cultures of microalgae (Divan & Schnoes, 1982; Doan et al., 2011; Hyka et al., 2013; Suga et al., 2011; Sykora et al., 1980; Watanabe et al., 2005; Wiedeman et al., 1964). The most common method is to prepare cultures from a single cell by serial dilution, which consists of streaking on selective agar plates and repeated sub-culturing (Levy et al., 2009; Timmins et al., 2009). This method is simple and cost-effective, but is not possible when cultures are screened from environmental samples, such as wastewater that contains large amounts of bacteria or bacteria attached to algal cell walls. In addition, the relatively low colony-forming rate of microalgae is a difficulty with this method (Cho et al., 2013; Kuo & Lin, 2013). Previous research indicated that a strongly basic quaternary ammonium anion exchange triiodide resin, (TR) complex (resin-I3), is an effective disinfectant against diverse bacteria (Taylor et al., 1970), although the exact mechanism of this effect is unknown. The most common application of contact TR is for purification of water for domestic and industrial use (Taylor et al., 1970). Interestingly, preliminary tests 3
indicated that Nannochloropsis gaditana was more resistant to TR than their bacterial contaminants. The high tolerance of microalgae to TR could be due to the unique chemistry and structure of their cell walls. The present research was designed to overcome the limitations of conventional methods for the generation of axenic cultures of microalgae. In particular, this novel method combines existing screening methods with a simple process that uses TR columns. This strategy was evaluated by assaying for bacterial colony forming units (CFUs), DAPI staining of broth culture, and cytometric analysis using SYTOX Green staining. No previous research has used a TR complex for the isolation of axenic cultures of microalgae. This process has great potential for the isolation of pure microalgal strains from bacteria-contaminated cultures because it does not require antibiotic treatment or expensive flow cytometry equipment used for cell sorting.
2. Materials and methods 2.1. Preparation of TR columns Triiodide ion (I3-) was prepared by combining crystalline I2 and aqueous potassium iodide (KI), according to the protocol of Taylor et al. (1970). The macroporous strong base anion resin, A-520 E (Purolite Co), was submerged in triiodide solution. Then, the resulting TR complexes were extensively rinsed with distilled water until colorimetric iodimetry showed that these rinses were free of iodide. Resin-I3, under the trade name A605, can be purchased from Purolite. A cylindrical tube (3-cm diameter, 8-cm height) was prepared, and its conical bottom was sealed with a steel mesh to prevent efflux of TR. After filling the column with TR, the resin was rinsed with sterilized distilled water prior to treatment with bacteria-contaminated cultures of the microalgae. 4
2.2. Treatment of N. gaditana cultures with a TR column N. gaditana cultures that were contaminated with several bacteria were prepared (OD ~1.5 at 680 nm). The formation of colony forming units (CFUs) was used to quantify and identify viable bacteria. Tryptone-yeast extract-glucose (TYG) agar medium and LB agar medium (10-fold diluted) were prepared to accommodate the broad nutrient requirements of diverse bacteria. Then 50 mL of a bacteria-contaminated culture of N. gaditana was passed over the TR column by gravity flow. The TR columntreated and untreated N. gaditana cultures were then plated onto TYG and LB agar at 100-fold dilution and incubated for 48 h at 37 °C and at room temperature. All treatment and plating procedures were performed in triplicate. After this short incubation period, the number of bacterial colonies was counted; microalgal colonies were not detected because they cannot generate visible colonies on TYG or LB agar within 48 h.
2.3. Screening for bacteria-free cells in TR-treated cultures For screening of TR-treated microalgae, 10 µL aliquots of TR column-treated N. gaditana cultures were added to a 96-well plate (SPL, Korea), with 200 µL of LB per well, and cultivated for 24 h under light. The growth of bacteria was measured by optical density at 600 nm. Bacteria-free algal cultures were screened from these plates. The percentage of axenic cultures was calculated as: (Number of bacteria-free wells) / (Total number of total wells) × 100 % Each bacteria-free culture of N. gaditana was inoculated into a culture flask that contained 200 mL of f/2 (N 5×) medium, and cultivated with 3 % CO2 under 50 µmol photons m-2 s-1 of light. Growth of N. gaditana was measured by cell concentration and optical density at 680 nm. 5
2.4. Identification of bacteria in contaminated N. gaditana cultures The colony PCR method with the iProof DNA polymerase kit (BioRad 172-5302) was used for the identification of bacteria, with primers for a universal bacterial 16S rDNA. The forward primer was 9F (5`- GAGTTTGATCCTGGCTCAG-3`), and reverse primer was 1492R (3`- GCTTACCTTGTTACGACTT-5`). The PCR protocol consisted of initial denaturation (95 °C for 5 min), denaturation/annealing/extension (30 cycles at 95 °C for 1 min, 58 °C for 1 min 72 °C for 1 min), and a final extension (72 °C for 5 min). Amplified bacterial 16S rDNA was confirmed by electrophoresis. The gel extraction method (QIAquick Gel Extraction Kit, Cat. No. 28706) and the BLAST library were used for identification of extracted 16S rDNAs. In particular, 50 µL of bacterial 16S DNA was loaded onto 0.5 % agarose gels. After electrophoresis, the desired region was excised and transferred into a 1.7 mL Eppendorf tube. Then, 500 µL of QC buffer was added, and the tubes were incubated for 10 min at 50 °C after mixing. Then, 200 µL of isopropanol was added and the mixture was transferred into 2 mL collection tubes. These tubes were centrifuged (13,000 rpm × 1 min at room temperature) and the flow-through was discarded. For washing, 750 µL of PE buffer was added and centrifuged (13,000 rpm × 1 min). For elution, 30 µL of elution buffer was added and the solution was centrifuged again (13,000 rpm × 1 min). This purified DNA was used for sequencing and strain identification.
2.5. SYTOX Green staining and flow cytometry analysis SYTOX Green, a green-fluorescent stain that permeates the membranes and stains the nucleic acids of dead or inactive prokaryotes and eukaryotes, was used to determine the number of bacteria and microalgae damaged by TR treatment. Contaminated N. 6
gaditana cultures, before and after TR treatment, were stained with 5 mM SYTOX Green and the mixture was incubated for 10 min at room temperature without light. Before analysis, spotting points of pure bacteria and pure N. gaditana were measured without staining. A high speed flow cytometer, MoFlo XDP (Beckman Coulter, Fullerton, CA) was used to identify cells of different sizes and for separation of cells of the same size but different fluorescence. The excitation wavelength was 488 nm (argon laser) and emission was measured at 530–540 nm.
2.6. Fluorescence microscopy DAPI (4′-6-diamidino-2-phenylindole), a fluorescent stain that permeates the membranes of live cells and stains A-T rich regions in DNA, was used to identify live cells. Fluorescence was measured under a fluorescence microscope (Microphot-FXA, Nikon), with excitation at 365 nm. The DNA-DAPI complex in bacterial cells fluoresces bright blue, and the red autofluorescence of chlorophyll in microalgae masks this blue fluorescence.
3. Results and discussion 3.1. Effect of triiodide resin on xenic cultures of N. gaditana The number of total bacteria in each N. gaditana culture was measured indirectly by determining CFU mL-1 using LB and TYG agar plates. Prior to treatment, the test culture contained 2.99 ± 0.12 × 107 cells mL-1 of N. gaditana (determined with a Neubauer Improved DHC-N01 hemocytometer), and had bacterial counts of 5.87 ± 0.62 × 106 CFU mL-1 (LB medium) and 2.63±0.19 × 106 CFU mL-1 (TYG medium). There were 5 main bacterial strains detected by the 16S rDNA assay: Pseudomonas sp., 7
Stappia sp., Labrenzia sp., Microbacterium sp., and Tistrella mobilis. After 3 times TR treatments, 0.29 CFU mL-1 (LB medium) and 0 CFU mL-1 (TYG medium) were present in the culture (Table 1). Flow cytometry analyses with SYTOX Green staining were used to confirm the impact of TR treatment on the viability of N. gaditana cells and bacteria in the xenic culture. SYTOX Green only stains inert or dead cells, so the fluorescence will increase if there are more dead cells after TR treatment (du Plessis & Hamman, 2014; Rychtecký et al., 2014). Analysis of bacteria (Fig. 1a) indicated that the fluorescence increased significantly after TR treatment, suggesting that most bacteria were damaged or killed by TR treatment. On the other hand, analysis of N. gaditana (Fig. 1b) indicated only a small change in fluorescence, suggesting that N. gaditana cells were not severely damaged by this treatment. These results indicate that most viable bacteria in the xenic N. gaditana culture were selectively screened-out by TR treatment.
3.2. Screening xenic cultures of N. gaditana Although TR treatment remarkably decreased the number of bacteria in the xenic N. gaditana culture, TR treatment alone may not necessarily yield pure cultures of N. gaditana. Thus, to obtain an axenic culture, TR-treated N. gaditana cells were added into 96 well plates that contained LB medium to test for the presence of bacteria. Each well was observed for 48 h, with untreated samples as the negative control. This process was repeated with five times. About 80 % of the samples reached a threshold OD600nm value, indicating the presence of bacteria. The average probability of obtaining an axenic culture by screening following TR treatment was 19.25 %. Bacterial growth was observed in all untreated culture samples (data not shown). 8
One of the bacteria-free aliquots was inoculated into fresh f/2 medium to confirm the purity of the TR-treated culture of N. gaditana. After 4 days of cultivation, this culture was subjected to DAPI staining and observed by fluorescence microscopy; an untreated culture was the positive control. The untreated culture had abundant bacteria indicated by blue and green fluorescent spots (Supplemental Fig. 1a), but the TRscreened culture only had red fluorescence due to auto-fluorescence of chlorophyll from N. gaditana (Supplemental Fig. 1b). These results indicate that TR treatment dramatically reduced the number of bacteria in the N. gaditana culture, although they do not prove that the resulting N. gaditana was absolutely pure immediately after TR treatment. Nonetheless, these results indicate that TR treatment dramatically and selectively reduced the bacterial population and therefore greatly increased the probability of selecting an axenic part within whole culture.
3.3 Growth of xenic and axenic cultures of N. gaditana The growth of xenic N. gaditana cultures differed from that of axenic cultures (Fig. 2). In particular, based on measurement of chlorophyll concentration (680 nm), growth of the axenic culture showed considerably lower maximum concentration than the xenic culture (Fig. 2). Previous research reported similar results with other species (Covarrubias et al., 2012; Kim et al., 2014). For instance, a xenic culture of Chlorella sp. grew much better than an axenic culture in terms of dry cell weight and lipid content (Cho et al., 2015; Lee et al., 2013), and the authors suggested an interaction of Chlorella sp. with certain bacteria enhanced the accumulation of microalgal biomass. However, the mechanism of this effect in N. gaditana and Chlorella sp. are unknown because 9
there are only limited studies of the symbiotic interactions between microalgae and bacteria. In the present study, the main bacterial contaminants were Pseudomonas sp., Stappia sp., Labrenzia sp., and Microbacterium sp. Tristrella mobilis. Investigation of the mechanism of the interactions between N. gaditana and these bacteria also requires starting with an axenic microalgal culture. The present study describes an easy method for isolation of axenic N. gaditana cultures. Further optimization of this method will allow additional applications in fundamental research, including studies of the interaction between microalgae and bacteria.
4. Conclusions Isolation of axenic cultures of microalgae is very important for basic and applied studies, but most current methods are laborious, time consuming, and expensive. Passage of a xenic culture of N. gaditana through a triiodide resin (TR) column effectively enables to screen microalgae from bacteria, because this substrate damages or kills bacteria but has little effect on microalgae. The results of the present study suggest that use of a TR column for isolation of axenic cultures of microalgae is a simple, rapid, and cost-effective approach for the isolation of axenic cultures of microalgae.
Acknowledgements This work was supported by a grant from the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Science, ICT & Future Planning (ABC-2010-0029728). 10
References 1. Cho, D.-H., Ramanan, R., Heo, J., Lee, J., Kim, B.-H., Oh, H.-M., Kim, H.-S. 2015. Enhancing microalgal biomass productivity by engineering a microalgal–bacterial community. Bioresour. technol. 175(0), 578-585. 2. Cho, D.H., Ramanan, R., Kim, B.H., Lee, J., Kim, S., Chan, Y., Choi, G.G., Oh, H.M., Kim, H.S. 2013. Novel approach for the development of axenic microalgal cultures from environmental samples. J. Phycol. 49(4), 802-810. 3. Covarrubias, S., de-Bashan, L., Moreno, M., Bashan, Y. 2012. Alginate beads provide a beneficial physical barrier against native microorganisms in wastewater treated with immobilized bacteria and microalgae. Appl. Microbiol. Biotechnol. 93(6), 2669-2680. 4. Divan, C.L., Schnoes, H.K. 1982. Production of axenic Gonyaulax cultures by treatment with antibiotics. Appl. Environ. Microbiol. 44(1), 250-254. 5. Doan, T.T.Y., Sivaloganathan, B., Obbard, J.P. 2011. Screening of marine microalgae for biodiesel feedstock. Biomass Bioenergy. 35(7), 2534-2544. 6. du Plessis, L.H., Hamman, J.H. 2014. In vitro evaluation of the cytotoxic and apoptogenic properties of aloe whole leaf and gel materials. Drug Chem. Toxicol. 37(2), 169-177. 7. Hyka, P., Lickova, S., Pribyl, P., Melzoch, K., Kovar, K. 2013. Flow cytometry for the development of biotechnological processes with microalgae. Biotechnol. Adv. 31(1), 216. 8. Kim, B.-H., Ramanan, R., Cho, D.-H., Oh, H.-M., Kim, H.-S. 2014. Role of Rhizobium, a plant growth promoting bacterium, in enhancing algal biomass through mutualistic interaction. Biomass Bioenergy. 69(0), 95-105. 9. Kuo, R.C., Lin, S.J. 2013. Ectobiotic and Endobiotic Bacteria Associated with Eutreptiella sp. Isolated from Long Island Sound. Protist. 164(1), 60-74. 10. Lee, J., Cho, D.-H., Ramanan, R., Kim, B.-H., Oh, H.-M., Kim, H.-S. 2013. Microalgaeassociated bacteria play a key role in the flocculation of Chlorella vulgaris. Bioresour. 11
Technol. 131(0), 195-201. 11. Levy, J.L., Stauber, J.L., Wakelin, S.A., Jolley, D.F. 2009. The effect of bacteria on the sensitivity of microalgae to copper in laboratory bioassays. Chemosphere. 74(9), 12661274. 12. Rychtecký, P., Znachor, P., Nedoma, J. 2014. Spatio-temporal study of phytoplankton cell viability in a eutrophic reservoir using SYTOX Green nucleic acid stain. Hydrobiologia. 740(1), 177-189. 13. Shishlyannikov, S.M., Zakharova, Y.R., Volokitina, N.A., Mikhailov, I.S., Petrova, D.P., Likhoshway, Y.V. 2011. A procedure for establishing an axenic culture of the diatom Synedra acus subsp. radians (Kütz.) Skabibitsch. from Lake Baikal. Limnol. Oceanogr. 9, 478-484. 14. Suga, K., Tanaka, Y., Sakakura, Y., Hagiwara, A. 2011. Axenic culture of Brachionus plicatilis using antibiotics. Hydrobiologia. 662(1), 113-119. 15. Sykora, J.L., Keleti, G., Roche, R., Volk, D.R., Kay, G.P., Burgess, R.A., Shapiro, M.A., Lippy, E.C. 1980. Endotoxins, Algae and Limulus Amebocyte Lysate Test in DrinkingWater. Water Res. 14(7), 829-839. 16. Taylor, S., Fina, L., Lambert, J. 1970. New water disinfectant: an insoluble quaternary ammonium resin-triiodide combination that releases bactericide on demand. Appl.Microbiol. 20(5), 720-722. 17. Timmins, M., Thomas-Hall, S.R., Darling, A., Zhang, E., Hankamer, B., Marx, U.C., Schenk, P.M. 2009. Phylogenetic and molecular analysis of hydrogen-producing green algae. J. Exp. Bot. 60(6), 1691-1702. 18. Watanabe, K., Takihana, N., Aoyagi, H., Hanada, S., Watanabe, Y., Ohmura, N., Saiki, H., Tanaka, H. 2005. Symbiotic association in Chlorella culture. FEMS Microbiol. Ecol. 51(2), 187-196. 19. Wiedeman, V.E., Walne, P.L., Trainor, F.R. 1964. A new technique for obtaining axenic 12
cultures of algae. Can. J. Bot. 42(7), 958-959. 20. Wilkie, A.C., Edmundson, S.J., Duncan, J.G. 2011. Indigenous algae for local bioresource production: Phycoprospecting. Energy Sustainable Dev. 5(4), 365-371.
13
Figure Legends
Fig. 1. Effect of TR treatment on bacteria and N. gaditana cells based on SYTOX Green staining followed by flow cytometry. a) Fluorescence of bacteria. TR treatment significantly shifted the fluorescence peak, indicating that treatment damaged or killed these cells. b) Fluorescence of N. gaditana. TR treatment had little effect on the fluorescence peak, indicating that treatment had little or no effect on these cells. Red lines: before TR treatment; green lines: after TR treatment.
Fig. 2. Cellular growth of xenic and axenic N. gaditana cultures grown with f/2 medium. Growth was measured by change in optical density at 680 nm, a measure of chlorophyll content. Indicated values are the averages of three independent measurements with standard errors.
Table Table 1. Effect of TR treatment on the number of bacteria present in xenic cultures of N. gaditana. CFU (cells mL-1)
No Treatment
1 Treatment
2 Treatments
3 Treatments
LB medium
5.87±0.62 ×106
4.01±0.10 ×104
4.00±0.10 ×103
0.29
TYG medium
2.63±0.19×106
4.30±0.10×104
1.00±0.40 ×103
0
14
15
16
Highlights Triiodide resin has a disinfectant effect on bacteria. Contaminated Nannochloropsis gaditana culture was treated with triiodide resin. TR treatment can be an effective tool for obtaining axenic microalgal culture. Growth kinetic of axenic and xenic N. gaditana was quite different.
17