Journal of Life Science 2015 Vol. 25. No. 7. 733~739 ISSN (Print) 12259918 ISSN (Online) 22873406 DOI : http://dx.doi.org/.5352/jls.2015.25.7.733 Development of Axenic Culture and Astaxanthin Production in Microalgae Min Chang Son 1, DongJun Lee 1, Sejin Park 1, Min Sung Kim 2, Chul Won Lee 2 and Won Gun An 1,2 * 1 Division of Pharmacology, School of Korean Medicine, Pusan National University, Yangsan 626870, Korea 2 Institute of Marine BioTechnology Pusan National University, Busan 609735, Korea Received April 29, 2015 /Revised June 30, 2015 /Accepted July 13, 2015 Microalgae are a renewable natural resource that requires only sunlight, carbon dioxide, phosphorus, and nitrogen for rapid growth. They produce a broad variety of basic chemical substances such as vitamins, fatty acids and carotenoidsthat have high added value potential for the pharmaceutical and food industries. The aim of this study was to develop axenic culture and to establish a cell growth assay for microalgae. A further experiment was carried out to determine the yield of astaxanthin derived from microalgae. The axenic culture was developed using a mixture of antibiotics [ampicillin (0 μg/ml), streptomycin ( μg/ml), chloramphenicol ( μg/ml), penicillin ( μg/ml), neomycin (50 μg/ml), gentamycin (50 μg/ml), kanamycin ( μg/ml), and nystatin (1.5 μg/ml)] and then used to extract a variety of useful components from the microalgae. The optimal concentration for the antibiotic mixture was 13 percent. A spectrophotometric cell growth assay was also established. Astaxanthin was extracted from Haematococus lacustris with a yield of 1.9 3 μg /l per 1 ml of culture medium. In conclusion, the axenic culture method developed here allows extraction of highquality astaxanthin and other useful components from microalgae. Key words : Antibiotics, astaxanthin, axenic culture, cell growth assay, microalgae 서 현대사회는화석연료를주된에너지공급원으로사용하고 있으나화석연료는멀지않은장래에고갈될것으로예측되고 있으며, 그사용과정중에환경오염문제를발생시키고있다. 특히화석연료의연소과정에서발생하는이산화탄소는지구 온난화를유발하는것으로알려져있어대기중이산화탄소의 농도를감소시키기위한연구가다양한분야에서전세계적으 로폭넓게연구되고있다. 일례로, 화석연료가가지는문제점 을해결하기위한방안의하나로서빛에너지를이용할수 있는미세조류에관한연구가최근활발하게진행되고있다 [, 17, 23]. 미세조류는빛에너지를이용하여이산화탄소를고정화하는탄소동화능을보유하고있으며, 이를통해다양한유용물질을생산할수있다 [21]. 일부미세조류는에너지함량이높아연료로의사용가능성이타진되고있어이산화탄소의저감과유용물질및대체연료의생산이라는많은장점을가진다. 현재고부가가치의의약품, 색소, 탄수화물및정밀화학약품등 *Corresponding author *Tel : 825158455, Fax : 825158420 *Email : wgan@pusan.ac.kr This is an OpenAccess article distributed under the terms of the Creative Commons Attribution NonCommercial License (http://creativecommons.org/licenses/bync/3.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 론 유용성분을함유하고있는미세조류들을유전자조작, 생육환경조절및배양공정변수최적화를통해인위적대량배양이가능토록하는미세조류생명공학 (microalgal biotechnology) 이새로운개척분야로인식되고있다. 또한식품, 제약, 화장품등다양한분야에서천연물질의수요가급증하면서세균, 곰팡이등미생물유래유용물질외에천연물질생산원으로서미세조류에대한관심이고조되고있으며, 폐수처리와농업으로까지그이용범위가확대되고있다 [7]. 이러한미세조류가다양한유용물질을생산할수있음에도불구하고그생산량이극히적으며, 분리방법이이미정립된상태여서미세조류의상업적이용을저해하는요인으로작용하고있다. 따라서미세조류의무균순수분리및유용물질의검출방법을개발할필요성이있다. 미세조류는군체를형성하거나점액성물질에둘러싸여있는데세포외부의점액성물질에는많은세균이부착되어있다. 이러한점액성물질에부착되어있는세균들은쉽게분리되어제거되지않는다. 그러나유용물질들을함유하고있는미세조류를이용한유전학적연구및생리학적연구에서세균이제거된균주를요구하고있어미세조류를무균적으로순수분리시키는것은필수적이다 [35]. 무균적분리방법에는원심분리법 [28], 초음파법 [16], 희석법 [29], 자외선법 [27], 여과법 [11] 등의물리적방법과페놀 [22] 및항생제를이용하는생물화학적방법 [5, 33] 들이이용되고있으나항생제법이보편적으로널리사용되고있다. Isochrysis galbana H002, H007, H008, H009, H0은최근영양보충제로각광받고있는 docosahexaenoic acid (DHA),
734 생명과학회지 2015, Vol. 25. No. 7 eicosapentaenoic acid (EPA) 등의불포화지방산을합성하는미세조류로알려져있다 [1, 26]. 그리고미세조류 Haematococcus lacustris는생리활성물질인 astaxanthin (3,3'dihydroxyβ, β' carotene44'dione) 을생산하는것으로보고되고있다 [9, 15, 25]. Astaxanthin은자연계에널리분포되어있는 ketocarotenoid로 polysoprenoid와 oxygen quenching 기능을가진 benzenoid ring의결합체이며 [31], 물고기와동물의음식물속에들어있는색소의원료일뿐만아니라, vitamin E와 βcarotene 보다더강한 free radical 항산화활성을가진잠재력이있는물질이다 [8, 13]. 또한, astaxanthin은세포내환경이과산화상태에있을때막의 phospholipids와다른지질들을보호하는작용을가진물질로알려져있으며 [20, 24], astaxanthin 의항산화작용은 singlet oxygen 및 free radical을소거함으로써항암및면역기능활성에효과가있는것으로보고된바있다 [3, 4, 12, 14, 34]. 본연구에서는미세조류로부터바이오매스및생리활성물질의대량생산을최종목적으로설계되었으며, 이에따라먼저생리활성물질을생산하는미세조류의무균순수분리및미세조류배양에필요한균체생육도측정법을확립하고자하였으며, 또한생리활성물질인 astaxanthin의생성량을파악하고자하였다. Table 1. Microalgal species used for this study Microalga for astaxanthine production Haematococuslacustris 재료및방법 실험균주및배양 Astaxanthin과 DHA 및 EPA를동시에생산하는미세조류는 Table 1과같다. Haematococus lacustris는한국산업플랑크톤소재은행으로부터, Isochrysis galbana 균주들은한국해양미세조류은행으로부터공급받았으며, 이들미세조류들은모두순수분리되지않은단조주 (unialgal strain) 상태로공급받았다. 또한 H. lacustris는 BBM 배지로, I. galbana 균주들은 f/2si 배지로배양하여실험을진행하였다. 무균순수분리 단조주로부터오염되어있는세균을제거하기위하여항생제처리법을실시하여무균순수분리를시도하였다. 항생물질은 ampicillin (0 μg/ml), streptomycin ( μg/ml), chloramphenicol ( μg/ml), penicillin ( μg/ml), neomycin (50 Microalgae for production of DHA and EPA Isochrysisgalbana H2 Isochrysisgalbana H7 Isochrysisgalbana H8 Isochrysisgalbana H9 Isochrysisgalbana H μg/ml), gentamycin (50 μg/ml), kanamycin ( μg/ml), 그리고항진균제인 nystatin (1.5 μg/ml) 을사용하였으며, 각항생제를지시농도대로각각단독처리하거나항생제혼합물을조제하여처리하였다. 미세조류배양액을항생제에 12시간동안노출시킨후, 항생제를첨가하지않은배지에서배양하면서미세조류의생육도와배양액의무균성을조사하였으며, 무균성은일정량의배양액을 LB plate에접종하여 30 에서배양하면서미생물콜로니생성유무나현미경으로검경하여확인하였다. 아래 Table 2에표시한항생제를농도별로처리한후생균수측정법으로오염유무를평가하였다. 분광법을이용한측정미세조류의생성량, 성장속도등을정량적으로파악하기위해분광법을이용하여 absorbance와세포수의상관관계를조사하였다. Astaxanthin 생성을위한균주의배양조건 H. lacustris 순수균주를이용하여 0.1 vvm, 5% CO 2 조건에서 250 ml elernmeyer flask에 130 ml working volume으로전배양을실시하였으며, 배양 15일전후로성장상태가우수한균주를선발하여실험에사용하였다. 균주배양은 25 C, 3000 g, % inoculum, ph 7.0 조건에서실시하였다. H. lacustris의배양을위해 6070 μmol photon m 2 s 의조건에맞는형광등 개를장착하고, 일정광량을조절할수있게 shaking incubator를제작하여사용하였다. 또한이산화탄소농도의경우 air와고순도이산화탄소를일정비율로혼합하여조절하였으며, 유속은 flowmeter를이용하여조절하였다. Astaxanthin의유도배양된세포를원심분리하고질소원이배제된배지 (NIES N 배지 ) 에재현탁한다음, 2,000 g에서 5분간원심분리하였으며, 이과정을 3회반복하여이전의배지성분을세척하였다. 세척이완료된균주는 23, 150 rpm, 300 μmol photon m 2 s 1 의배지조건에서 astaxanthin induction 을실시하였다. Table 2. The concentrations of antibiotics used for this study Antibiotics Ampicillin Streptomycin Chloramphenicol Penicillin Neomycin Gentamycin Kanamycin Nystatin Target G(), G() G(), G() G(), virus, rickettsia G() G(), G() G(), G(), mycoplasma G(), G(), mycoplasma Yeasts, molds Concentration (μg/ml) 0 50 50 1.5
Journal of Life Science 2015, Vol. 25. No. 7 735 Astaxanthin의추출 Astaxanthin이유도된배양균체 (H. lacustris) 는 8,000 g, 4 로 분간원심분리하여상층액을제거하고균체는증류수로반복세척하였으며, 원심분리한균체에 acetone을첨가하여초음파파쇄기 (Branson 250, Dabbury, CT, USA) 로냉각추출을반복하였다. 그후, 원심분리를통하여침전물의균체찌꺼기는동결건조하여고형분회수율을계산하였으며, 상층액은감압, 농축하여용매를제거하고, nhexane을첨가하여용해시켰다. 또한분액깔때기에물과함께첨가하여흔들고, 수용액층을제거 (3회반복 ) 한후, sodium sulfate anhydrous 를이용하여잔류수분을제거시켜 astaxanthin을추출하였다 Astaxanthin의생성량측정 H. lacustris의세포벽을파쇄하여 astaxanthin을추출하였으며, 이를이용하여광범위한파장에걸쳐 UV spectrum을조사하였고, 또한확인된파장에서흡광도와 astaxanthin 농도의상관관계를조사하여생성량을측정하였다. Astaxanthin standard curve를그리기위해사용된표준물질은독일슈트트가르트 Subitec Gmbh 회사로부터공급받았다. 결과및고찰무균순수분리미세조류는세포벽에작은구멍들이존재하는데, 이곳에여러세균들이부착하여공생한다 [35]. 임과이는 Gyrodinium을순수분리하기위해 ampicillin, cephalosporin, neomycin 등의항생제처리와원심분리법을이용하였고 [33], 기등은 Alexandrium과 Peridinium의무균배양을위해항생제와미세필터를사용하였다 [11]. 또한, 조등은 ampicillin, gentamycin, kanamycin, neomycin, streptomycin 5종을혼합한항생제가 I. galbana의무균화에가장효과적이라고보고하였다 [6]. 본연구에서 6종의미세조류에대해혼합항생제 (ampicillin, streptomycin, chloramphenicol, penicillin, neomycin, gentamycin, kanamycin, nystatin) 을처리한후, 25 광조건에서 120시간정치배양한결과는 Table 3과같으며, 관찰결과모든미세조류가순수분리된것을확인할수있었다. 즉, 6종의미세조류순수분리를위해적합한혼합항생제의농도는 H. lacustris가 이었으며, I. galbanah8는 13% 이었고, I. galbanah7는 12% 이었으며, I. galbanah2, I. galbana H9, I. galbana H는각각 이었다. 이는 13% 혼합항생제의농도가미세조류의치사효과는줄이면서또한세균오염을막을수있는농도임을의미한다. 시간및초기접종농도에따른미세조류의증가량비교 Fig. 1은시간에따른미세조류의증가량을나타내었으며또한초기접종농도에따른미세조류의증가량을비교하였다. Table 3. The evaluation of bacterial contamination by treatment of antibiotics in the different microalgal species Microalgal species H. lacustris I. galbanah8 I. galbanah7 I. galbanah2 I. galbana H9 I. galbana H Mixture of antibiotics a 2% 3% 4% 5% 2% 3% 4% 5% 2% 3% 4% 5% Cell survival (%) 0.0 50.0 16.7 13.3 13.3 6.7 0.0 50.0 62.5 62.5 37.5 25.0 0.0 0.0 60.0 20.0 4.0 4.0 0.0 0.0 0.0 0.0 0.0 0.0 Bacterial contamination a Mixture of antibiotics were composed of 0 μg/ml ampicillin, μg/ml streptomycin, μg/ml chloramphenicol, μg/ml penicillin, 50 μg/ml neomycin, 50 μg/ml gentamycin, μg/ml kanamycin, and 1.5 μg/ml nystatin. Fig. 1의 CG에서알수있듯이미세조류량은초기접종의농도에대체적으로비례하였다. 분광법을이용한측정미세조류의성장속도생성량등을간편하게정량적으로측정하기위해 Melinda 등이사용한방법 [18] 에따라, UV spectrophotometer를이용하여세포수와 absorbance와의관계를알아보았다. Fig. 2에제시한바와같이 680, 750 nm의파장에서세포수와 absorbance 간에강한선형성이나타남을확인하였다. 680, 750 nm에서의흡광도는미세조류의 chlorophyll a를측정하는파장이며, P. subcapitata의경우 684 nm에서최대흡광도를나타낸다고알려져있는데종에따라최대흡광도의차이를보인다는보고와일치하였다 [19, 30]. 또한세포수와건조중량과의관계를 Fig. 3에나타내었다. 강한선형관계가관찰되었으며그비례상수는 2.0 8 이었다. 따라서 UV spectrometer를이용하여세포의생장속도를확인할수있었으며이렇게확립된방법을이용하여항산화제등의유용한
736 생명과학회지 2015, Vol. 25. No. 7 A H. lacustris B I. gabana C I. galbana H2 D I. galbana H7 E I. galbana H8 F I. galbana H9 G I. galbana H Fig. 1. Cell number changes for the time in the different microalgal species. Cell number changes in a variant of time was shown for each respective microalgae. From C to G, microalgae concentration of early stage is overall in a proportion to cell number.
Journal of Life Science 2015, Vol. 25. No. 7 737 Fig. 2. The relationship between cell number and absorbance intensity at 680 and 750 nm, was shown, respectively. Linear relationship was obtained. Katsuda's method Fig. 3. The relationship between dry weight and cell number was shown. Linear relationship was obtained. 용도로사용이가능한 astaxanthin을배양, 추출하는데에사용하였다. Fig. 5. Scanning for Haematococus lacustris and its medium (BBM) was shown. Astaxanthin의생성량측정 Astaxanthin은 vitamin E와 βcarotene 보다강력한 free radical 항산화활성을가진물질이며, 항암및면역기능활성에효과가있는것으로알려져있다. 앞서설명한대로 H. lacustris로부터생리활성물질인 astaxanthin을추출한후, 이를이용하여넓은파장에걸쳐 UV spectrum을얻었으며 472 nm 의파장주위로강한흡수피크를확인하였다 (Fig. 4A). 또한, 이렇게확인된 472 nm의파장에서 absorbance 양과 astaxanthin의농도는 Fig. 4B에서제시하는바와같이선형성을나타내었다. 이러한방법은 Ana 등 [2] 및 Strickland와 Parsons [32] 이사용한분광학적인방법에따른것이며, BBM 배지에서배양된 H. lacustris를이용하여 astaxanthin의축적정도를확인하기위하여 UV scanning을실시한결과, Fig. 5에서나타난바와같이 astaxanthin이축적된것을관찰하였다. 이는 Ana 등, 이등, 그리고박등이 H. lacustris로부터높은항산화효과를갖는천연색소인 astaxanthin의축적을확인한 A B Astaxanthin standard curve Fig. 4. UV scanning for astaxanthin and its relationship with the intensity at 475 nm was shown. Lineal relationship was observed.
738 생명과학회지 2015, Vol. 25. No. 7 후, 추출하였다는보고와일치하였다 [2, 15]. 또한배양액 1 ml 로부터얻은 astaxanthin의농도는 1.9 3 ±0.14 3 μg/l 이었다. 결 미세조류로부터유용성분추출을위한무균순수분리법을확립하기위하여항생제혼합물을이용하였으며, 그결과 13% 혼합항생제의농도가미세조류의치사효과는줄이면서또한세균오염을막을수있는최적농도임을확인하였다. 또한미세조류의양을간편하게측정하기위하여 UV spectrum 상의 680, 750 nm의파장에서흡광도와세포의양을비교한결과, 선형성이나타남을확인하고미세조류의생장량을평가하였다. 더나아가 H. lacustris 균주를배양하여천연항산화제등으로이용할수있는 astaxanthin을추출하였으며, H. lacustris 배양액 1 ml로부터얻은 astaxanthin의농도는 1.9 3 μg/l이었다. 따라서개발된무균순수분리법을이용하여미세조류로부터양질의 astaxanthin 및유용성분들을얻을수있을것으로사료된다. 론 감사의글 본연구는한국연구재단 (NRF2013R1A2009593) 과부산광역시신성장산업과의지원을받아수행하였음. References 1. Aarab, L., PerezCamacho, A., VieraToledo, M. dp., de Vicose, G. C., FernandezPalacios, H. and Molina, L. 2012. Embryonic development and influence of egg density on early veliger larvae and effects of dietary microalgae on growth of brown mussel Pernaperna (L. 1758) larvae under laboratory conditions. Aquacult. Int. DOI.07/s499 01296127. 2. Ana, C., Mariela, G., Silvia, V., Maritza, H. and Nelson, G. 2003. Optimization of biomass, total carotenoids and astaxanthin production in Haematococcus pluvialis Flotow strain Steptoe (Nevada, USA) under laboratory conditions. Biol. Res. 36, 343357. 3. Bendich, A. 1991. Non vitamin a activity of carotenoids: immuno enhancement. Food Sci. Technol. Res. 2, 127130. 4. Bennedsen, M., Wang, X., Willen, R., Wadstrom, T. and Andersen, L. P. 1999. Treatment of H. pylori infected mice with antioxidant astaxanthin reduces gastric inflammation, bacterial load and modulates cytokine release by splenocytes. Immunol. Lett. 70, 185189. 5. CampaCórdova, A. I., LunaGonzález, A., Ascencio, F., CortésJacinto, E. and CáceresMartínez, C. J. 2006. Effects of chloramphenicol, erythromycin, and furazolidone on growth of Isochrysisgalbana and Chaetocerosgracilis. Aquaculture 260, 145150. 6. Cho, J. Y., Choi, J. S., Kong, I. S., Park, S. I., Kerr, R. G. and Hong, Y. K. 2002. A procedure for axenic isolation of the marine microalga Isochrysisgalbana from heavily contaminated mass cultures. J. Appl. Phycol. 14, 385390. 7. Choi, S. P. and Sim, S. J. 2012. Microalgal bioconversion to organic resources form CO 2. KIC News 15, 1124. 8. Guerin, M., Huntley, M. E. and Olaizola, M. 2003. Haematococcusastaxanthin: applications for human health and nutrition. Trends Biotechnol. 21, 2216. 9. Hagen, C. H., Braune, W. and Greulich, F. 1993. Functional aspects of secondary carotenoids in Haematococcuslacustris [Girod] Rostafinski (Volvocales) IV.Protection from photodynamic damage.j. Photochem. Photobiol. 20, 153160.. Jo, B. H. and Cha, H. J. 20. Biodiesel production using microalgal marine biomass. KSBB J. 25, 9115. 11. Ki, J. S., Cho, S. Y. and Han, M. S. 2006. Axenic Culture Method: A filtration technique to produce axenic cultures of the armoured Dinoflagellates. In: Hur S.B. (ed), Culture and application of useful microalgal. Life Science Publishing Co., 131147. 12. Krinsky, N. I. 1989. Antioxidant functions of carotenoids. Free Radical Bio. Med. 7, 617635. 13. Kurashige, M., Okimasu, E., Inoue, M. and Utsumi, K. 1990. Inhibition of oxidative injury of biological membranes by astaxanthin. Physiol. Chem. Phys. Med. NMR 22, 2738. 14. Kurihara, H. 2002. Contribution of the antioxidative property of astaxanthin to its protective effect on the promotion of cancermetastasis in mice treated with restraint stress. Life Sci. 70, 25092520. 15. Lee, C. G. and Park, J. K. 2008. Immobilization of astaxanthin extracted from photosynthetic micro algae Haematococcus lacustris. J. Chitin Chitosan 13, 2214. 16. Lim, M., Ong, B. L. and Wee, Y. C. 1992. A method of obtaining axenic cultures of Trentepohlia spp. (Chlorophyta). J. Phycol. 28, 567569. 17. Mclaren, J. S. 2005. Crop biotechnology provides an opportunity to develop a sustainable future. Trends Biotechnol. 23, 339342. 18. Melinda, G., Clive, G., Robert, H. and Susan, H. 2011. Interference by pigment in the estimation of microalgal biomass concentration by optical density. J. Microbiol. Methods. 85, 119123 19. Millie, D., Schofield, O., Kirkpatrick, G., Johnsen, G. and Evens, T. 2002. Using absorbance and fluorescence spectra to discriminate microalgae. Eur. J. Phycol. 37, 313322. 20. Naguib, Y. M. A. 2000. Antioxidant activities of astaxanthin and related carotenoids. J. Agr. Food Chem. 48, 11501154. 21. Oh, H. M. and Ahn, C. Y. 2009. CO 2 Fixation and biodiesel production using microalgae. KIC News 12, 1220. 22. Olivier, S., Scragg, A. H. and Morrison, J. 2003. The effect of chlorophenols on the growth of Chlorella VT1. Enzyme Micro. Techno. 32, 837842. 23. Park, J. I., Woo, H. C. and Lee, J. H. 2008. Production of bioenergy from marine algae: Status and perspectives. Kor. Chem. Eng. Res. 46, 833844.
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