J. Exp. Biomed. Sci. 14 (2008) 91 96 Effect of Growth Improvement in Photosynthetic Bacteria as a Function of 880 nm Light Emitting Diode Luminosity Daesik Kim 1, So Young Chang 2 and Jin Chul Ahn 2, 1 Department of Clinical Laboratory Science, Dongnam Health College, 2 Medical Laser and Device Research Center, Dankook University, Cheonan 330-714, Korea Light Emitting Diode (LED) of 880 nm was used as a function of luminosity in culture of the photosynthetic bacteria including Rhodobacter sp.. An array of 880 nm LED was driven with an energy density of 6.0 mw/cm 2. In processing time, we were able to show that the cell growth were gained of significant changes in the pigment and in the dry weight. And we also showed that photosynthetic bacteria had the resonable relativity of optical density to dry weight. LED-880 nm is of great significance for the potential use of photo-bioreactor construction. Key Words: Photosynthetic bacteria, Light emitting diode, Energy density, Cell growth, Luminosity, Rhodobacter sp., Bacteria growth 서 무기물은물론각종유기물을영양원으로이용하여광합성산물을생산하는광합성세균은고온및저온에서도잘견디고, 내염성도강하며담수상태인곳에서는거의대부분존재하는미생물 (Lee, 1971) 로이세균의생리화학적, 생태학적기능에관한기초연구를기반으로한농업적이용에관한연구와미생물상품개발연구들이활발히진행되어왔다. 광합성세균은녹색유황세균 (green sulfur bacteria) 과홍색유황세균 (purple sulfur bacteria), 홍색비유황세균 (purple non sulfur bacteria) 으로그게구별되며농축수산물및폐수처리, 단일단백질 (Sasikala and Ramana, 1995), 항미생물제재 (Mundt et al., 2003), 치료물질구성성분 (Suwanto, 1999; Sasaki et al., 2002) 등유용물질생산에광범위하게이용되고있다. 특히, 황색비유황세균의로도박터종 (Rhodobacter species) 은무기물보다는유기물을광합성반응의수소공여체로우선이용하는특성이있어 (Lee, 1971; Cheong et al., 1997) 유기물폐수의정화처리및양식, 축산사료첨가제, 유기질비료의기능개선제분야에서폭넓게사용되는등농업적이용측면에서연구가이 * 논문접수 : 2008 년 6 월 11 일수정재접수 : 2008 년 6 월 23 일 교신저자 : 안진철, ( 우 ) 330-714 충남천안시안서동산 29 번지, 단국대학교의과대학 329 호 ( 단국대학교의학레이저연구센터 ) Tel: 041-550-1786, e-mail: jcahn@dankook.ac.kr 론 미활발히진행되고왔고 (Matsunaga et al., 1991; Halon et al., 1998; Rajasekhar et al., 1999) 또한비타민 B12, Coenzyme Q10, ALA (aminolevulinic acid), porphyrin과같은의약분야에이용가능한제품들을생산하고, 식품산업에서향증강제로이용되면서인간면역기능을위한식이재료로이용가능한 RNA를생산할수있어고부가가치상품생산을위한재료로도주목받고있다 (Ken et al., 2005). 산업에사용할광합성세균의대량배양의필요성이대두되면서, 광원으로자연빛을활용한미생물대량배양에서야기되었던시간, 장소및계절적문제점 (Pulz, 2001) 을해결하기위해인공배양기를고안하여형광등, 삼파장램프등의인공조명 (artificial light) 을새로운광원으로대체하여미생물의대량생산및소량생산이가능하게되었다 (Kim, 2002). 광원으로쓰이는인공조명의전기적인효율 (electrical efficiency), 열소산 (heat dissipation), 신뢰성 (reliability), 내구성 (durability), 수명 (lifetime), 비용 (cost), 스펙트럼출력 (spectral output) 면을개선하여보다효과적인광합성세균배양을위해 Laser diode (LD) 와 Light emitting diode (LED), Vertical cavity surface emitting laser (VCSEL) 과같은다양한광원을이용한미생물배양연구들이시도되었다 (Bertling et al., 2005). Rhodobacter sp. 를비롯한황색비유황세균에들어있는대표적인광합성색소인 bacteriochlorophyll (Bchl) 은종류에따라 a, b, c, d, e, f로나뉜다. Bchl을함유하는 chromatopore의흡수곡선을살펴봤을때여러파장에서흡수대를나타내는데아세톤이나에탄올과같은유기용 - 91 -
매추출을사용하지않은미생물부유액상태에서 805 nm와 875 nm의장파장쪽에서도흡수극대를나타내는것으로보고된바있으며 (Kim and Kim, 1981) Chromatium tepidum과같은균종에서는 917, 855, 800 nm에서흡수극대가나타나는것으로보고되었다 (Tsunenori et al., 1986). 이와같이황색비유황세균이적외선 (Infra red) 파장빛부분에서흡수극대를나타내며광합성을하는것에착안하여본실험에서는스펙트럼분포 (Spectral distribution) 가 840~920 nm의특성을나타내고있는 880 nm LED를광원으로사용했을때광합성세균의생장증진효과를알아보고자하였다. 재료및방법 1. 균주및배양배지본실험에사용된균주는 Rhodobacter sphaeroides 및 Rhodobacter capsulata. 로우일환경산업에서판매중인홍균-PSB를사용하였으며, 제품구입배지에서계대배양하여사용하였다. 기본배지조성은증류수 liter 당 KH 2 PO 4 0.33 g, MgSO 4 7H 2 O 0.33 g, NaCl 0.33 g, NH 4 Cl 0.5 g, CaCl 2 2H 2 O 0.05 g, Sodium succinate 1.0 g, Yeast extract 0.02 g, Trace salt solution 1 ml, FeSO 4 7H 2 O 0.02% solution 0.5 ml, ph 7.0이다. Trace salt solution 조성은증류수 liter 당 ZnSO 4 7H 2 O 10 mg, MnCl 2 4H 2 O 3.0 mg, H 3 BO 3 30 mg, CoCl 2 6H 2 O 20 mg, NaCl 2 6H 2 O 2.0 mg, Na 2 MoO 4 3.0 mg 이다. 배지를조성하여고압습윤멸균후파이렉스캡배양튜브 (Pyrex tube, Culutre, 25 150, Screw cap with Rubber Liner) 에접종하여 30±1 에서배양하였다. 2. 광원장치본연구에사용된광원은 880 nm LED (Photron Co., PI-3A521) 로스펙트럼분포 840~920 nm 사이의빛의강도 (Luminous intensity) 를나타내고있으며에너지의세기는 6.0 mw/cm 2 로공급하였다. 실험대조군으로삼파장램프 ( 형광등 ) 와 630 nm LED (U-Jin LED Co., LTD, ULP- H5R1101 A) 를사용하였다. 880 nm LED와 630 nm LED의경우 12 16개의 LED ( 에너지세기 6 mw/cm 2 ) 판넬을제작하고, 높이 14 cm의기둥을세워 30±1 항온기에장착하여사용하였다 (Fig. 1). 삼파장램프의경우 2,025 Lux를유지한항온기를사용하였다. (A) 630 nm LED 3. 균주의생장및균체량 본연구에사용된광합성세균은혐기성조건을필요로하므로모든광합성세균배양은혐기적인조건하에서이루어졌으며, 시간에따른균체의생장정도는균체가분비하는색소의변화양상을육안으로관찰하고, 배양액의일정량을취하여분광광도계 (Spectrophotometer, Optizen 2120 UV) 를이용하여 600 nm에서흡광도를측정하여양상정도를파악하였다. 균체량측정을위해시간별로배양된균체액을 5,000 rpm에서 15분간원심분리 (Hanil Science Co., Ltd, Combi-514R, Rotor No. 6) 한후상등액을제거하고침전물을배지 1 ml에부유시켜초고속원심분리기를이용하여 11,000 rpm에서 2분동안재차원심분리 (Hanil Science Co., Ltd, Micro-12) 하여얻은침전물을진공동결건조기 (Hanil Science Co., Ltd, Ecospin 3180C) 로 -86, 1,800 rpm에서 over-night시켜건조균체량을측정하였다. 결 과 1. 880 nm LED 의균체생장효과 (B) 880 nm LED Fig. 1. 630 nm LED and 880 nm LED panel array. They had the same energy density (6 mw/cm 2 ) but their colors were different. The 630 nm LED was reddish and the 880 nm LED was no colored. 본연구에서실험한 LED 효과를비교하기위해 880 nm LED 및 630 nm LED의에너지세기를 6.0 mw/cm 2 로고정하고, 삼파장램프는 2,025 Lux 조건에서 24 h, 48 h 및 72 h 동안 30±1 에서배양된광합성세균의시간별생장정도를흡광도를이용하여확인한결과는 Fig. 2와같다. 접종 24시간후 OD 600 에서측정한흡광도는각각 1.0, 0.8 및 0.7로에서측정값이별차이를나타나지않았고, 접종 48시간후측정한흡광도는각각 3.3, 2.3 및 1.6 로나타났으며, 접종 72시간후측정값은각각 4.9, 3.9 및 3.2로대조군에비해서 880 nm LED 처리배양균이높은흡광도수치를보이고있음을확인하였다 (Fig. 2). - 92 -
Fig. 2. Effect of cell growth as a function of LED luminosity. 880 nm LED-treated group had a significantly higher absorbance compared to 630 nm LED and light-treated groups. Fig. 4. Dry weight of cell growth as a function of LED luminosity. 880 nm LED-treated group had a significantly higher the amount of dry weight compared to the other groups. 도달함을알수있었다. Fig. 2에서보여주는결과는통상적으로배양되는광합성세균의배양기간이 5~10일임을감안하면대조구로쓰인광원들의효능이떨어진다기보다는 880 nm LED 가 bacteriochlorophyll을가진광합성세균의광합성작용을촉진시킴으로써단시간에효과적인생장이가능하게할수있었음으로사료된다. 또한시간에따른균체의생장정도는균체가분비하는색소의변화양상을육안으로관찰하였는데 Rhodobacter 종을포함한황색비유황세균의색소가자색을나타내는특성대로본연구에서실험한균주도시간에따른흡광도에비례해서색을나타냈다 (Fig. 3). 2. 건조균체량 Fig. 3. Pigmented aspect of purple bacteria in culture medium. In processing time, 880 nm LED significantly enhanced the pigment compared to the other light sources. Fig. 2에서보는바와같이 880 nm LED를광원으로하여광합성세균을배양한경우가 630 nm LED 및삼파장램프경우보다훨씬효과적인결과를나타내고있음을알수있다. 접종후 72시간이되었을때실제로산업및농가에서미생물대량및소량배양시접종되는종균의흡광도 OD 600 4.2보다도높은 OD 600 4.9를나타낸반면대조구로실험한 630 nm LED와삼파장램프의경우접종후 72시간일때흡광도 OD 600 는각각 3.9, 3.2를나타냈으며, 접종후 4~5일이지난후에야 OD 600 4.2에 880 nm LED 광원을이용하여시간에따른미생물배양액의균체량을알아보기위해접종후 24 h, 48 h 및 72 h 동안배양된균체액을원심분리후진공동결건조시켜얻은건조균체량을측정하여대조구와비교하였다 (Fig. 4). 접종 24시간후측정한건조균체량은각각 9.0 mg, 8.0 mg 및 5.9 mg으로흡광도와마찬가지로많은차이를나타나지않았고, 접종 48시간후측정한건조균체량은각각 25.7 mg, 20.0 mg 및 14.6 mg였으며, 접종 72시간후건조균체량은각각 32.3 mg, 25.5 mg 및 21.2 mg로 880 nm LED에서배양된균체가접종 48시간이후부터는가장많은건조균체량을나타냄을확인하였다 (Fig. 4). - 93 -
Fig. 4에서보는바와같이 880 nm LED를광원으로이용하여배양한광합성세균의건조균체량이 630 nm LED 및삼파장램프경우보다많음을알수있다. 접종후 72시간동안배양한건조균체량을비교한결과 880 nm LED 처리군의경우가 630 nm LED에비해 26% 정도, 삼파장램프에비해 52% 정도높은균체량을나타내었다. 이결과는 Fig. 2의흡광도측정치와마찬가지양상을보였으며, 본실험결과로부터 880 nm LED 처리배양액의흡광도와배양액의건조균체량에상관성이있음을확인할수있다. 고찰자연환경에광범위하게서식하고있는광합성세균은균의생리, 생태적특성을이용하여하수나생활폐수의정화, 유기물이오염되어침전된진흙의생물적환경정화등유기물폐수처리공정등에적용시켜왔고 (Ken et al., 2005), 어류양식, 축산사료첨가제, 유기질비료의기능개선제분야 (Nah et al., 1997) 에서폭넓게사용되는등농업적이용에관한연구와단일단백질 (Sasikala and Ramana, 1995), 항미생물제재 (Mundt et al., 2003), 치료물질구성성분 (Suwanto, 1999; Sasaki et al., 2002) 등의유용물질을생산하는미생물상품개발연구들이활발히진행되어왔다. 황색비유황세균의로도박터종 (Rhodobacter species) 은무기물보다는유기물을광합성반응의수소공여체로우선이용하는특성이있어 (Lee, 1971; Cheong et al., 1997) 유기물폐수의정화처리및농업적이용측면에서연구가이미활발히진행되고왔다 (Matsunaga et al., 1991; Halon et al., 1998; Rajasekhar et al., 1999). Rhodobacter speroids의경우비타민 B 12, Coenzyme Q10, ALA (aminolevulinic acid), porphyrin과같은의약분야에이용가능한고부가가치물질들을생산할수있음이보고되고있고, Rhodovulum sp. PS88의경우식품산업에서향증강제로이용되고, 인간면역기능을위한식이재료로이용가능한 RNA 및 drug delivery system (DDS) 에적용할수있는 ESP와같은물질들을생산할수있어고부가가치여러가지산업의상품재료로주목받고있으며활발히연구개발중에있다 (Ken et al., 2005). 광합성세균의산업적대량배양의필요성이대두되면서기존자연광만을이용한미생물배양에서야기되는여러문제점들 (Pulz, 2001) 을해결하기위한방편으로 인공조명을새로운광원으로대체하여미생물의대량생산및소량생산이가능하게되었다 (Kim, 2002). 전기적인효율, 열소산, 신뢰성, 내구성, 수명, 비용, 스펙트럼출력면등을개선한고효율광합성세균배양을위한인공광원으로 LD, LED, VCSEL 등의빛소자를적용한연구들이진행되었고, 성과들이보고되었다 (Bertling et al., 2005). Magnesium porphyrin 화합물인 bacteriochlorophyll (Bchl) 은홍색비유황세균의대표적인광합성색소로 Bchl을함유하는 chromatopore의흡수곡선을살펴봤을때여러파장에서흡수대를나타내는데아세톤으로추출한이나에탄올과같은유기용매추출을사용하지않은미생물부유액상태에서 805 nm와 875 nm의장파장쪽에서도흡수극대를나타내는것으로보고된바있으며 (Kim and Kim, 1981), Chromatium tepidum 균종의경우 chromatopore 의흡수곡선이 917, 855, 800 nm에서흡수극대가나타나는것으로보고되었고 (Tsunenori et al., 1986), Rb. capsulatus 37B4 균종의경우 860 nm에서흡수극대가나타나는것으로보고되었다 (Bertling, 2005). 이와같이황색비유황세균이적외선파장빛부분에서흡수극대를나타내며광합성을하는것에착안하여 880 nm LED를이용한광합성세균의생장증진효과를확인한본실험에서는 880 nm LED가부착된배양기를사용하여광합성미생물을배양했을때기존에통상적으로 5~10일정도이던배양시간을 2배정도단축시키는결과를확인할수있었다. Hans 등이 660 nm LED가장착된광합성세균용 bioreactor 를고안하여클로렐라배양실험을실시하였을때열소비손실이적어에너지효율면에서효과가있다는보고를한바있어 (Hans et al., 1996) 고가의레이저다이오드 (LD) 나비교적짧은수명의형광등보다는 LED 광원의이용이경제적측면에서보다효과적임을알수있었다. 기존논문에서는특정광원을이용한광합성세균배양실험보다는 5,000 Lux 이상의형광등을이용한홍색비유황세균의배양법이나 (Kim and Lee, 2000), 3,000 Lux 정도의형광등을광원으로이용하여 ALA를생산하는 Rhodobacter capsultus 의배양특성 (Cheong et al., 1997) 을연구하는등의세균자체에관심을가진연구들이많아 880 nm LED를이용한다른연구결과들과비교할수없었다. 또한, 실험군으로사용되었던적색광을나타내는 630 nm LED를실험에사용된에너지세기인 6 mw/cm 2 로하였을때의빛의밝기를조도계로측정했을때약 8,000 Lux 정도의밝기를나타내었고, 똑같은조건으로적외선 - 94 -
파장을나타내는 880 nm LED의경우약 25 Lux의빛의밝기를나타내었는데, 본실험결과에서는두실험군모두대조군인 2,025 Lux의형광등보다미생물생육기간을단축시킴을알수있었으며, 두실험군이같은에너지세기를나타내지만조도측정시빛의밝기가현저하게적게나타나는 880 nm LED를광원으로사용한실험군이미생물생육기간이훨씬효과적으로단축되는것을확인하였는데이는빛의밝기가빛의세기가비례하지는않는것으로사료되며마찬가지로 880 nm LED를이용한다른연구결과들과비교할수없었지만저비용, 고효율면에서 LED 광원의가치는확인할수있었다. 농업적이용측면의한일환으로유기재배농가에서화학적유기질비료를대신하여생물학비료로광합성세균이광범위하게사용되고있고그효능의탁월함또한보고되고있지만 (Nah et al., 1997), 기존의통상 5~10일정도소요되던미생물배양시간은실수요자들의경제적부담일수밖에없다고사료되어수행한본실험은 880 nm LED를광원으로사용하여단시간에효과적으로고농도광합성세균을배양할수있음을확인할수있었다. 농축수산업및수질정화분야에 880 nm LED가부착된광합성세균배양기를사용함으로써미생물배양에숙련되지않은실수요자들도오염의위험부담과유지비부담을줄이면서품질면에서도우수한광합성세균을효과적으로배양하여사용할수있을것으로사료되며, single cell protein이나항미생물제재, drug delivery system에적용될수있는물질들을생산하는광합성세균의배양에도 880 nm LED를광원이사용한다면산업경제적인측면에서많은효과를볼수있을것으로사료된다. REFERENCES Bertling K, Hurse TJ, Kappler U, Rakic AD. Lasers-an effective artificial source of radiation for the cultivation of anoxygenic photosynthetic bacteria. Biotechnol Bioeng. 2005. 94: 337-345. Cheong DY, Choi YM. Isolation and cultural characteristics of δ-aminolevulinic acid producing photosynthetic bacteria. Agr Chem Biotech. 1997. 40: 561-566. Halon SP, Graham DL, Hogan PJ, Holt RA, Reeve CD, Shaw AL, McEwan AG. Asymmetric reduction of racemic sulfoxides by dimethyl sulfoxide reductases from Rhodobacter capsulatus, Escherichia coli and Proteus species. Microbiology 1998. 144: 2247-2253. Hans CPM, Hans B, Udo M, Van H, Bernd MAK, Luuc RM, Roger AB. Application of light emitting diodes in bioreactors: flashing light effects and evergy economy in algal culture (Chlorella pyrenoidosa). Biotech Bioeng. 1996. 50: 98-107. Ken S, Masanori W, Yoshito S, Akihiro I, Napavarn N. Application of photosynthetic bacteria for medical fields. J Biosci Bioeng. 2005. 100: 481-488. Kim YU, Kim KS. Studies on the isolation and the application of photosynthetic bacteria. J Kor Agr Chem Soci. 1981. 24: 132-138. Kim JK, Lee BK. Mass production of Rhodopseudomonas palustris as diet for aquaculture. Aquacul Eng. 2000. 23: 281-293. Lee CG, Palsson BO. High density algal photobioreactors using light emitting diodes. Biotechnol Bioeng. 1994. 44: 1161-1167. Lee KW. General characters and application of photosynthetic bacteria. Kor J Microbiol. 1971. 9: 130-138. Matsunaga T, Takeyama H, Sudo H, Oyama N, Ariura S, Takano H, Hirano M, Burgess JG, Sode K, Nakamura N. Glutamate production from CO 2 by marine cyanobacterium Synechococcus sp. using a novel biosolar reactor employing lightdiffusing optical fibers. Appl Biochem Biotechnol. 1991. 28: 157-167. Matthijs HCP, Balke H, Van Hes UM, Kroon BMA, Mur LR, Binot RA. Application of light-emitting diodes in bioreactros: Flashing light effects and energy economy in algal culture (Chlorella pyrenoidosa). Biotechnol Bioeng. 1996. 50: 98-107. Mundt S, Kreitlow S, Jansen R. Fatty acids with antibacterial activity from the cyanobacterium Oscillatoria redekei HUB 051. J Appl Phycol. 2003. 15: 263-267. Nah KC, Cho JY, Chung SJ. Effects of compost supplemented with cultured solution of photosynthetic bacteria (Rhodopseudomonas capsulatus) on the early growth of plug seedings of tomato. Kor J Organic Agr. 1997. 5: 105-115. Pulz O. Photobioreactors: Production systems for phototrophic microorganisms. Appl Microbiol Biotechnol. 2001. 57: 287-293. Rajasekhar N, Sasikala C, Ramana CV. Photoproduction of L-tryptophan from indole and glycine by Rhodobacter sphaeroides OU5. Biotechnol Appl Biochem. 1999. 30: 209-212. Sasaki K, Watanabe M, Tanaka T. Biosynthesis, biotechnological production and applications of 5-aminolevulinic acid. Appl Microbiol Biotechnol. 2002. 58: 23-29. Sasikala C, Ramana CV. Biotechnological potentials of anoxygenic - 95 -
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