KR100799065B1 - Medium composition obtained from fermented animal wastewater including a natural substitute chelator - Google Patents
Medium composition obtained from fermented animal wastewater including a natural substitute chelator Download PDFInfo
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Abstract
Description
도 1은 미세조류의 엽록소 a의 함량 변화를 나타내는 그래프1 is a graph showing the change in the content of chlorophyll a of microalgae
도 2a 내지 도 2d는 광합성 효율을 나타내는 그래프2A to 2D are graphs showing photosynthetic efficiency
본 발명은 생물활성수(Bacteria Mineral Water, BM)를 이용한 배지조성물에 관한 것으로, 보다 상세하게는 공지의 배지에 화학적 킬레이팅제(EDTA) 대신에 생물활성수를 첨가하여, 생물활성수를 천연 킬레이팅제로서 사용함으로서 미세조류의 생장이 우수할 뿐 아니라 독성으로 인한 부작용이 발생되지 않도록 한 킬레이팅제로서 생물활성수가 포함된 배지 조성물에 관한 것이다.The present invention relates to a medium composition using bioactive water (Bacteria Mineral Water, BM), and more specifically, to a known medium, bioactive water is added to the known medium instead of the chemical chelating agent (EDTA), thereby making the bioactive water natural. The present invention relates to a medium composition containing bioactive water as a chelating agent, which is used as a chelating agent so that the growth of microalgae is excellent and the side effects due to toxicity are not generated.
미세조류는 수질 판정을 위한 지표생물로서 연구되고, 비타민, 베타카로틴(β-carotene), 고도 불포화 지방산, 단백질, 기름(oil), 메탄가스 등 고부가가치 소재의 공급원으로, 건강식품, 향장, 사료, 먹이생물, 비료, 연료 등 생명공학적 연구개발 소재로 각광받고 있다.Microalgae are studied as indicators for water quality determination and are a source of high value-added materials such as vitamins, beta-carotene, polyunsaturated fatty acids, proteins, oils, and methane gas. It is being spotlighted as a biotechnological research and development material such as food, living organism, fertilizer and fuel.
특히 최근에는 생물적 환경정화(bioremediation)를 위해 미세조류를 응용하여 환경산업에 접목하여 환경친화적인 수질 오염원을 제거 및 대기 중에 CO2를 제거하는데 이용된다.Recently, microalgae have been applied for bioremediation and used in the environmental industry to remove environmentally friendly water pollutants and remove CO 2 from the atmosphere.
이러한 미세조류를 산업화하기 위해 1차적으로는 단위면적당 세포밀도를 증가시키는 기초연구가 필수적이며, 다양한 배양조건(배지종류, 광량, 광질, 온도, 특정 무기원소의 유무 등)에 따른 미세조류의 생장에 따른 기초연구가 다수 수행되었다(Kim and Giraud 1989; Kim and Dubacq 1997; Kim and Smith 2001).In order to industrialize these microalgae, basic research to increase cell density per unit area is essential, and the growth of microalgae according to various culture conditions (medium type, light quantity, mineral quality, temperature, presence of specific inorganic elements, etc.) A number of basic studies were conducted (Kim and Giraud 1989; Kim and Dubacq 1997; Kim and Smith 2001).
특히 폐수를 이용한 Chlorella, Scenedesmus와 Spirulina platensis의 대량배양연구는 수질을 정화하는 목적으로 응용되거나 고단백 사료원으로 개발되고 있다.Especially with wastewater, Chlorella , Scenedesmus and Spirulina Large scale culture studies of platensis have been applied for the purpose of water purification or as a high protein feed source.
이와 같이 미세조류를 대량생산하고 수질을 정화하는 기술로서 대한민국특허등록 제 500333호에 생물활성수를 상용배지에 3%첨가한 배지 조성물 및 그 제조방법이 공고되어 있다.As a technique for mass-producing microalgae and purifying water quality, Korean Patent Registration No. 500333 discloses a medium composition in which 3% bioactive water is added to a commercial medium, and a method of manufacturing the same.
상기에서 생물활성수(Bacteria Mineral Water)(참고문헌: 히로시 N. 1998. 세균이 지구를 구한다 - BMW 기술의 도전. 도서출판 푸른 평화, 216pp.)는 돼지의 뇨 등 축산폐수를 재활용한 활성수로서, 축산폐수가 유입된 용기에 암석과 부식토 펠레트를 투입하여 폭기시켜 발효시킨 최종처리수이다.In the above, Bacteria Mineral Water (Reference: Hiroshi N. 1998. Bacteria Saves the Earth-Challenges of BMW Technology. Blue Peace, 216 pp.) Is an active water recycled from livestock wastewater such as pig urine. As a final treatment, the livestock wastewater is introduced into a vessel into which the rock and humus pellets are aerated and aerated for fermentation.
그러나, 상기한 종래의 생물활성수를 이용한 배지조성물을 포함한 일반적인 미세조류 배양용 상용배지(f/2, ASW 등)에는 화학합성 킬레이팅제가 포함되는데, 이 킬레이팅제는 배지 조성물에 반드시 추가되는 미량의 중금속(Zn, Cu, Co 등)이 다른 성분과 용해되지 않고, 이들 이온의 독성 발현을 방지하는 기능을 한다.However, the conventional microalgae culture medium (f / 2, ASW, etc.) including a medium composition using the conventional biologically active water includes a chemical synthetic chelating agent, which is necessarily added to the medium composition. Traces of heavy metals (Zn, Cu, Co, etc.) do not dissolve with other components and function to prevent the toxic expression of these ions.
상기한 킬레이팅제로서는 통상 EDTA(etylenediaminetetraacetic acid)가 사용되고 있고, 이 EDTA는 난분해성 합성 아미노산으로 중금속 해독제, 방부제 등으로 이용되며, 배지 조성물 농도 변화에 따른 미세조류 생산성 향상을 위한 연구로서 EDTA농도 변화가 미세조류의 생장을 촉진한다는 연구보고가 있다(Sung et al. 1998).As the chelating agent, EDTA (etylenediaminetetraacetic acid) is usually used, and this EDTA is a hardly decomposable synthetic amino acid used as a heavy metal antidote and preservative. Has been reported to promote the growth of microalgae (Sung et al. 1998).
하지만 EDTA는 고가로 생산 판매되고 있는 화학합성 물질이므로, 미세조류를 대량생산 시에 EDTA를 사용할 경우, 생산단가의 상승으로 제품의 시장 경쟁력이 떨어지는 결점과, 과다 사용 시에는 EDTA 자체의 독성 때문에 생체에 미치는 부작용이 많은 단점이 있다.However, EDTA is a chemical synthetic material that is produced and sold at a high price. Therefore, when EDTA is used for mass production of microalgae, the production cost is increased, and the product's market competitiveness becomes inferior. There are many disadvantages to the side effects.
그 예로서, 상기한 킬레이팅제는 킬레이션 테라피(chelation theraphy)의 대체의학에서 만성피로증후군, 섬근육통, 루프스, 암, 관절염, 고혈압 등의 치료에 이용되나 혈중저칼슘상태, 알레르기, 저혈당 등과 같은 부작용이 대두되고 있고, 이러한 킬레이팅제는 유해금속에만 선택적으로 작용하는 것이 아니고 인간의 대사에 필요한 미량의 금속원소, 효소 등과도 결합하여 그 작용을 저해하여 생체에 극심한 부작용을 초래한다.For example, the chelating agent is used for the treatment of chronic fatigue syndrome, islet muscle pain, lupus, cancer, arthritis, high blood pressure, etc. in alternative medicine of chelation theraphy, but low blood calcium level, allergy, hypoglycemia, etc. The same side effects are emerging, and these chelating agents not only act selectively on harmful metals, but also bind to trace metal elements, enzymes, etc. necessary for human metabolism and inhibit their actions, resulting in severe side effects on the living body.
따라서 미세조류의 산업화를 위한 생산단가를 절감하고 대량생산 효율을 증가시키는 친환경 천연 킬레이팅제 개발이 요구될 뿐 아니라, 인체에 무해한 킬레이팅제의 개발이 요구된다.Therefore, the development of eco-friendly natural chelating agents to reduce the production cost and increase the mass production efficiency for the industrialization of microalgae is required, as well as the development of chelating agents harmless to the human body.
상기한 바와 같은 요구에 의하여, 본 발명의 목적은, 상용 배지에 화학합성 킬레이팅제 대신에 생물활성수을 첨가함으로서 천연 킬레이팅제로서 사용할 수 있도록 함에 있다.In view of the above demands, an object of the present invention is to add bioactive water to a commercial medium instead of a chemical synthetic chelating agent so that it can be used as a natural chelating agent.
본 발명의 다른 목적은, 상기 독성이 없는 천연 킬레이팅제를 사용함으로써 녹조류의 생산력이 최대화시킬 수 있는 배지조성률을 제공하여, 녹조류의 대량 생산이 가능하도록 하는데 있다.Another object of the present invention is to provide a medium composition rate that can maximize the productivity of green algae by using a natural chelating agent that is not toxic, to enable mass production of green algae.
상기한 목적을 달성하기 위한 본 발명의 특징은 화학합성 킬레이팅제가 포함되지 않은 공지의 상용배지에 생물활성수를 총배지의 중량에 대하여 3-50%첨가한 킬레이팅제로서 생물활성수가 포함된 배지 조성물에 있다.A feature of the present invention for achieving the above object is that the bioactive water is added as a chelating agent added 3-50% to the weight of the total medium in the known commercial medium containing no chemical synthetic chelating agent In the medium composition.
상기한 바와 같은 특징을 갖는 본 발명의 실시예를 하기에서 살펴본다.An embodiment of the present invention having the features as described above will be described below.
우선, 본 발명에서 사용된 재료와 배양 방법에 대하여 상세하게 살펴본다.First, look at in detail with respect to the materials and culture methods used in the present invention.
배양조건Culture condition
본 발명의 실시예에서 사용된 해산 미세조류는 녹조류(Chlorophyceae)인 크로렐라 오발리스(Chlorella ovalis)(KPCCI P-M-2)와 두나리엘라 파르바(Dunaliella parva)(KPCCI P-M-6)로서 영남대학교 해양과학연구센터의 한국산업플랑크톤소재은행(KPCCI)으로부터 분양을 받았다.The marine microalgae used in the embodiment of the present invention is Chlorella obalis (Chlorrophyceae). ovalis ) (KPCCI PM-2) and Dunaliella parva (KPCCI PM-6) were distributed by the Korea Industrial Plankton Material Bank (KPCCI) at the Youngnam University Marine Science Research Center.
대조구 배지는 표 1의 구성으로 구성된 f/2 배지고(Guillard 1975), 실험구 배지는 f/2 배지에 생물활성수(BM) 3%를 첨가한 배지(E+3), BM을 6% 첨가한 배지(E+6)와 f/2 배지 구성물질 중에 킬레이팅제(chelator)인 Na2EDTA를 제거한 배지(f/2-E)에 BM 3%를 첨가한 배지(-E+3)와 6%를 첨가한 배지(-E+6) 및 10%, 20%, 30%, 40%와 50% BM을 추가한 각각의 배지(-E+10, -E+20, -E+30, -E+40, -E+50)에서 상기한 녹조류를 접종하였다.The control medium was f / 2 medium composed of the composition of Table 1 (Guillard 1975), and the experimental medium was 6% of B + medium (E + 3) and BM added with 3% of bioactive water (BM) to f / 2 medium. BM 3% added to the medium (-E + 3) in which the chelator Na 2 EDTA was removed from the added medium (E + 6) and f / 2 medium constituents (f / 2-E) And 6% medium (-E + 6) and 10%, 20%, 30%, 40% and 50% BM medium (-E + 10, -E + 20, -E + 30) , -E + 40, -E + 50) were inoculated with the algae described above.
표 1은 화학 킬레이팅제로서 EDTA를 포함한 f/2 배지 구성( Composition of f/2 medium containing EDTA as a chemical chelator)을 나타낸다.Table 1 shows the composition of f / 2 medium containing EDTA as a chemical chelator.
접종된 미세조류는 일정한 명암주기(14h/L, 10h/D)와 24℃, 130μmol·m-2·s-1의 광량으로 쉐이킹 인큐베이터(shaking incubator)에서 세포분열이 왕성한 시기인 14일간 배양하고, 엽록소 a와 광합성효율을 측정하기 위해 세포가 배지에 완전히 순치되고, 세포분열 정체기에 접어드는 27일 동안 배양하였다.The inoculated microalgae were incubated for 14 days at a period of high cell division in a shaking incubator at a constant contrast cycle (14h / L, 10h / D) and light at 24 ° C and 130μmol · m −2 · s −1 . In order to measure the photosynthetic efficiency with chlorophyll a, the cells were completely incubated in the medium, and cultured for 27 days after entering the cell division plateau.
측정방법How to measure
미세조류의 생장률 측정Growth rate measurement of microalgae
두 녹조류를 배지에 접종한 날부터 2일마다 시료를 혈구계선반(hematocytometer)을 이용하여 미세조류의 단일세포를 측정하고, 세포분열율(K)을 수학식 1에 따라 계산하였다Every two days from the day when two green algae were inoculated into the medium, a single cell of microalgae was measured by using a hematocytometer, and the cell division rate (K) was calculated according to Equation 1.
No는 초기 개체수이며, N1는 최종 개체수, t0는 초기 개체수일 때 배양일, 그리고, t1은 최종 개체수일 때 배양일을 의미한다.N o is the initial population, N 1 is the final population, t 0 is the culture day when the initial population, and t 1 means the culture day when the final population.
엽록소 a의 정량 및 정성 분석Quantitative and Qualitative Analysis of Chlorophyll a
엽록소 a의 함량 측정은 배양된 녹조류 3mL를 유리섬유여과지(GF/C, 45 ㎜)로 여과시킨 후, 아세톤과 물을 9:1의 비율로 섞은 혼합액 10mL를 여과지에 추가한 후, 마쇄한 시료를 원심 분리관에 넣고, 밀봉하여 4℃ 어두운 곳에서 원심분리(4000rpm, 20min)하였다.The content of chlorophyll a was measured by filtering 3 mL of cultured green algae with glass fiber filter paper (GF / C, 45 mm), and then adding 10 mL of a mixture of acetone and water in a ratio of 9: 1 to the filter paper, and then grinding the sample. Was placed in a centrifuge tube, sealed, and centrifuged (4000 rpm, 20 min) in a dark place at 4 ° C.
상징액의 일부를 흡수셀에 옮겨 흡광광도계(Cary 50 Conc, Varian, USA)로 검액하였다.A portion of the supernatant was transferred to an absorbing cell and tested using an absorbance photometer (Cary 50 Conc, Varian, USA).
바탕시험액으로 아세톤:물을 9:1로 혼합한 혼합 용액을 취하여 대조액으로 하여, 663nm, 645nm, 630nm와 750nm에서 검액의 흡광도를 측정하고, 엽록소 a의 농도는 아래 수학식 2의 Standards Methods(APPA 1995)를 기준으로 하여 계산하였다.Take a mixed solution of acetone: water mixed with 9: 1 as a test solution, and use the control solution as the reference solution. Measure the absorbance of the sample solution at 663 nm, 645 nm, 630 nm and 750 nm, and the concentration of chlorophyll a is determined by the Standards Methods (APPA) 1995).
엽록소 a의 광합성효율은 형광 분석기(Phyto-PAM, Walz, Germany)에 의해 470㎚, 535㎚와 620㎚의 파장에서 PSII에서의 최대 광량자(quantum)를 수학식 3의 방법으로 측정하였다.The photosynthetic efficiency of chlorophyll a was measured by a fluorescence analyzer (Phyto-PAM, Walz, Germany) by measuring the maximum quantum at PSII at wavelengths of 470 nm, 535 nm and 620 nm by the method of Equation 3.
Fv/Fm은 PSII에서 발산하는 형광값의 역치로 이는 광계의 광합성효율을 의미하고, dF는 형광 효율 증가치, Ft 는 순간 형광률, Fm'는 최대 형광률을 의미한다.Fv / Fm is a threshold value of the fluorescence emitted from PSII, which means the photosynthetic efficiency of the light system, dF is the increase in fluorescence efficiency, Ft is the instantaneous fluorescence rate, and Fm 'is the maximum fluorescence rate.
돼지뇨 처리수(Porcine urine treated water ( 생물활성수Biologically active water , BM)의 수질 분석, Water quality analysis of BM)
본 발명에 사용된 생물활성수는 춘천 소양강 생물활성수(BM)농장에서 돼지뇨 정화 플랜트 시설에서 최종 정화된 처리수(BM)로서 미세조류의 배지로 이용하기 전에 돼지뇨의 원수와 처리수의 수질을 분석하였다.The biologically active water used in the present invention is the final purified water (BM) in the swine urine purification plant facility in Chuncheon Soyanggang bioactive water (BM) farm. Water quality was analyzed.
탁도는 디지털 탁도계(2100N, HACH, Korea)로 측정하였고, 수소이온농도, DO와 수온은 pH/DO Meter (D-55, Horiba, Japan), 전기전도도는 712 전기전도계(Metrohm, Switzerland)를 이용하여 측정하였다. 그리고 COD, SS, T-N와 T-P는 수질오염공정시험방법에 준하여 분석하였다.Turbidity was measured with a digital turbidity meter (2100N, HACH, Korea), hydrogen ion concentration, DO and water temperature were pH / DO meter (D-55, Horiba, Japan), and electrical conductivity was measured with 712 electrical conductivity meter (Metrohm, Switzerland). It measured using. COD, SS, T-N and T-P were analyzed according to the water pollution process test method.
BM의 원수와 처리수의 중금속(Fe, Mg, Cu, Ti, Mn) 변화량을 측정하기 위한 정량 및 정성 분석은 시료를 적당량 채취하여 4mL의 H2O2와 3mL의 HNO3를 첨가하여 마이크로웨이브(microwave)를 이용해서 분해한 후, 2~3% HNO3로 희석하여 Inductively Coupled Plasma Mass Spectrometer(ICP-MS)(PQ3, VG, UK)로 분석하였다.Quantitative and qualitative analysis to measure changes in heavy metals (Fe, Mg, Cu, Ti, Mn) of raw water and treated water of BM is performed by taking an appropriate amount of samples and adding 4 mL of H 2 O 2 and 3 mL of HNO 3 . After digestion using (microwave), it was diluted with 2-3% HNO 3 and analyzed by Inductively Coupled Plasma Mass Spectrometer (ICP-MS) (PQ3, VG, UK).
이상과 같은 본 발명의 실시예에 따른 결과를 살펴본다.Look at the results according to the embodiment of the present invention as described above.
BM의 수질 변화BM Water Quality Changes
토양미생물(방선균, 야생효모 등)에 의해 발효되기 이전의 원수(Wastewater), 10일간 발효된 시료(Fermentation for 10days), 40일간 발효된 시료(Fermentation for 40days)를 각각 적당량 채취하여 수질을 비교 분석하고 그 결과는 표 2 및 표 3과 같다.Comparative analysis of water quality by collecting appropriate amounts of wastewater, fermentation for 10days, and fermentation for 40days before fermentation by soil microorganisms (such as actinomycetes and wild yeast) The results are shown in Table 2 and Table 3.
* BM(Bactareia mineral water) originated from the BM Farm of Chunchon, * Bactareia mineral water originated from the BM Farm of Chunchon,
표 2는 BM의 물리화학적 특성(Physico-chemical characteristics of BM)을 나타내는 것으로, 총 질소(T-P)와 총 인(T-P)의 양은 원수에서보다 발효가 진행되면서 감소함을 알 수 있다.Table 2 shows the physico-chemical characteristics of BM, and it can be seen that the total nitrogen (T-P) and total phosphorus (T-P) levels decrease as fermentation proceeds than in raw water.
이는 원수가 토양미생물에 의해 발효되는 과정에서 미생물이 돼지뇨에 포함된 질소나 인 등으로 구성된 유기물질을 천연 킬레이팅제를 비롯한 또 다른 유기산으로 분해과정을 거치는 것임을 나타낸다.This indicates that in the process of raw water fermentation by soil microorganisms, microorganisms decompose organic substances composed of nitrogen or phosphorus contained in pig urine into another organic acid including natural chelating agent.
표 3은 원수 및 처리수에서 중금속 농도의 변화 (Variation of heavy metal concentrations in original swine urine and treated BM)를 나타내는 것으로, 원수와 40일간 발효된 시료인 처리수(BM)의 중금속 정량 및 정성 분석결과를 살펴보면, 구리(Cu)와 철(Fe) 함량은 원수에서 발효과정을 거치면서 감소하고, 마그네슘(Mg)과 망간(Mn)은 높아졌다. 특히 마그네슘함량이 원수보다 발효수에서 발효 10일과 40일에서 각각 15배와 12배로 증가하였다.Table 3 shows the variation of heavy metal concentrations in original swine urine and treated BM.The results of quantitative and qualitative analysis of heavy metals in raw water and 40 days fermented water (BM) Looking at, the copper (Cu) and iron (Fe) content was reduced as the fermentation process in raw water, magnesium (Mg) and manganese (Mn) was increased. In particular, magnesium content increased 15 and 12 times in fermented water than in raw water at 10 and 40 days of fermentation, respectively.
미세조류의 엽록소를 구성하는 마그네슘 이온은 4개의 피롤(pyrrole) 중 하나의 피롤 내에 구성된 질소와 착이온으로 킬레이팅된 중심 금속이온으로 광합성의 광화학 반응에서 산화환원 전위가 조절되어 명반응의 에너지 형성에 중요한 기능을 한다(Kim and Thomas 1991; 이 등 2004). Magnesium ions, which make up the chlorophyll of microalgae, are central metal ions chelated with nitrogen and complex ions in one of the four pyrroles, and the redox potential is controlled in the photochemical reaction of photosynthesis. It plays an important role (Kim and Thomas 1991; et al. 2004).
따라서 BM은 미세조류의 엽록소 합성을 유도할 수 있는 착이온이 다량 함유된 것이 확인되고, BM내에 마그네슘 이온의 증가는 미세조류가 엽록소 합성을 용이하게 하는 인자로 작용함을 알 수 있다.Therefore, it is confirmed that BM contains a large amount of complex ions capable of inducing chlorophyll synthesis of microalgae, and it can be seen that the increase of magnesium ions in BM acts as a facilitating factor for chlorophyll synthesis.
철(Fe) 이온은 돼지뇨인 축산폐수를 10일 발효 시에 41% 감소되었고, 40일간 발효된 BM은 78%로 감소되었다. 이는 철 이온이 미생물이 생산한 제2차 대사산물과 결합하여 EDTA의 기능을 대신하는 천연 킬레이팅제 합성에 사용된 것임을 알 수 있다. Iron (Fe) ions decreased 41% in pig urine livestock wastewater after 10 days of fermentation, and 78% of BM fermented in 40 days. It can be seen that iron ions are used in the synthesis of natural chelating agents to replace the function of EDTA by binding to secondary metabolites produced by microorganisms.
BM농도에 따른 세포의 생장률 변화Changes in Cell Growth Rate According to BM Concentration
해산 미세조류 Chlorella ovalis의 세포밀도를 표 4를 참조하여 살펴보면 EDTA가 첨가된 대조구 배지인 f/2 배지에 비해 f/2에 BM 3% 첨가한 배지(E+3)에서 생장한 세포분열 속도와 세포밀도가 증가(1.1배)한 반면에 f/2에 BM 6%를 첨가한 배지(E+6)에서 생장한 세포는 오히려 감소되었다(0.6배)Marine algae Chlorella The cell density of ovalis was increased with reference to Table 4, and cell growth rate and cell density increased in the medium added with BM 3% at f / 2 (E + 3), compared to the control medium added with EDTA. (1.1-fold), while cells grown in medium (E + 6) added BM 6% at f / 2 were reduced (0.6-fold).
반면에 f/2배지에서 EDTA를 제거한 -E+3, -E+6 배양액에서 배양 14일간 세포밀도는 대조구 배지인 f/2배지에 비해 높았다(각각 2.5배와 1.8배)On the other hand, the cell density of 14 days cultured in -E + 3 and -E + 6 cultures from which EDTA was removed from f / 2 medium was higher than that of f / 2 medium (2.5 and 1.8 times, respectively).
C. ovalis의 세포는 BM 추가농도가 높은 배지(30 - 50 %)에서 생장률이 훨씬 높았다(-E+30: 8배; -E+40: 9배; -E+50: 19배).Cells of C. ovalis had much higher growth rates (-E + 30: 8-fold; -E + 40: 9-fold; -E + 50: 19-fold) in medium with high BM concentration (30-50%).
반면에 Dunaliella parva는 표 4에서 BM 추가 농도가 3-5% 범위에서는 세포밀도의 큰 변화가 없었으나 EDTA가 제거된 -E+3 배지에서 생장한 세포밀도가 높게 나타났다. Dunaliella on the other hand Parva showed no significant change in cell density in the range of 3-5% of BM concentration in Table 4, but showed high cell density in -E + 3 medium without EDTA.
-E+10 배지에서는 두 미세조류가 f/2대조구 배지에 비해 각각 1.21배와 1.24배로 비슷한 증가율을 보였으나 -E+20 배지에서는 f/2대조구 배지에서 생장한 C. ovalis(1.3배)보다 D. parva (3배)의 세포 증가율이 훨씬 높았다. In the -E + 10 medium, the two microalgae showed a similar increase rate of 1.21 and 1.24 times, respectively, compared to the f / 2 control medium, but in -E + 20 medium, compared to C. ovalis (1.3 times) grown in the f / 2 control medium. The cell growth rate of D. parva (3 times) was much higher.
D. parva는 C. ovalis에 비해 BM 추가 농도 증가치에 따른 세포 분열율이 낮았으나 EDTA가 제거되고, BM의 추가농도가 증가할수록 C. ovalis의 세포와 같이 세포의 밀도가 증가되었다. D. parva showed lower cell division rate with increasing BM concentration than C. ovalis . However, as EDTA was removed and the concentration of BM increased, the cell density increased with C. ovalis cells.
이 결과는 f/2배지에 EDTA가 제거되고 50% BM을 추가한 -E+50 배지가 C. ovalis와 D. parva 배양의 최적 배지였고, BM이 화학합성 EDTA의 역할을 대신할 수 있다는 결과를 얻었을 뿐만 아니라 f/2 배지에서 배양된 두 녹조류에서 보다 최고 19배(C. ovalis)와 7배(D. parva)의 세포분열 속도를 증가시킴에 따라 미세조류의 산업화와 수질정화 및 대기의 CO2제어 실용화 기술 및 먹이생물 산업화를 위한 대량생산에 적용이 가능하다.These results indicate that -E + 50 medium with EDTA removed and 50% BM added to f / 2 medium was the optimal medium for C. ovalis and D. parva cultures, and BM could replace the role of chemical synthetic EDTA. In addition to increasing the cell division rate of up to 19-fold ( C. ovalis ) and 7-fold ( D. parva ) in both green algae cultured in f / 2 medium, microalgae industrialization, water purification and atmospheric It can be applied to the practical use of CO 2 control technology and mass production for the industrialization of food organisms.
표 4는 각종 배지에서 14일간 배양된 두 녹조류의 세포분열율 및 세포농도의 변화(Variation of cellular division rates and cell densities of Chlorella ovalis and Dunaliella parva cultured for 14 days in each medium)를 나타낸다.Table 4 shows the variation of cellular division rates and cell densities of Chlorella ovalis and Dunaliella of two green algae cultured in various media for 14 days. parva cultured for 14 days in each medium).
엽록소 a의 함량 변화Changes in Chlorophyll a Content
엽록소 a는 미세조류가 광합성 작용을 통해 빛에너지에서 생체에너지로 변환하는 매개체이고, 수계 소비자인 동물플랑크톤과 어류의 먹이가 되는 1차 생산자로서 수계 생태계의 흐름과 수환경을 파악할 수 있는 지표물질이 된다.Chlorophyll a is a medium through which microalgae converts light energy into bioenergy through photosynthesis and is a primary producer of animal plankton and fish, which is an aquatic consumer, and an indicator that can grasp the flow and aquatic environment of aquatic ecosystems. do.
미세조류의 1차 생산력을 분석하기 위해 도 1에 나타내는 엽록소 a의 함량 변화를 미세조류의 세포밀도 변화와 서로 비교 검토한다.In order to analyze the primary productivity of the microalgae, the change in the content of chlorophyll a shown in FIG. 1 is compared with the change in the cell density of the microalgae.
엽록소 a는 배양 27일째 색소 함량을 비교 분석한 결과, 배지에 EDTA유무에 따라 엽록소 a의 함량이 차별화되어 나타났다. Chlorophyll a was analyzed by comparing the pigment content on day 27 of the culture, and the content of chlorophyll a was different depending on the presence of EDTA in the medium.
C. ovalis의 엽록소 a의 농도는 -E+3 -> E+6 -> f/2 -> E+3 -> -E+6 배지 순으로 엽록소 a의 농도가 저하되었고, D. parva의 엽록소 a 함량이 -E+6 -> -E+3 -> E+6 -> E+3 -> f/2 배지 순으로 감소하였다.Chlorophyll a concentration in C. ovalis decreased in the order of -E + 3-> E + 6-> f / 2-> E + 3-> -E + 6 medium, and D. parva chlorophyll a. The a content was decreased in the order of -E + 6-> -E + 3-> E + 6-> E + 3-> f / 2 medium.
따라서 두 녹조류는 세포 밀도의 변화에서처럼 f/2배지에서 EDTA를 제거한 배지에서 엽록소 a의 합성률이 증가함을 알 수 있다Thus, both green algae showed the increased rate of chlorophyll a in the medium from which EDTA was removed from f / 2 medium as in the change of cell density.
BM의 농도를 증가시킨 C. ovalis의 엽록소 a의 함량은 -E+50에서 1,527 mg L-1로 가장 높았고, D. parva의 엽록소 a 함량도 -E+50에서 15,004 mg L-1로 가장 높았다.Chlorophyll a content of C. ovalis with increased BM concentration was the highest at -E + 50 (1,527 mg L -1 ) and D. parva was highest at -E + 50 (15,004 mg L -1 ). .
따라서 두 녹조류의 엽록소 a의 최적 합성 배지는 EDTA를 제거하고 50%의 BM을 추가한 -E+50 배지가 가장 효율성이 띄어난 BM 조성률로 나타났다. Therefore, the optimal synthesis medium of chlorophyll a of two green algae was -E + 50 medium which removed EDTA and added 50% of BM.
최대광합성 효율비교Maximum photosynthetic efficiency comparison
1차 생산력을 측정하기 위해 Phytoplankton Analyzer인 Phyto-PAM(식물플랑크톤 형광유도 측정기기)를 이용하여 470nm(blue filter를 통한 광수용색소가 엽록소 b일 경우), 535nm(green filter를 통한 광수용색소가 carotenoids일 경우), 620nm 파장(orange filter를 통한 광수용색소가 남색소일 경우)에서 형광값의 역치를 측정하여 C. ovalis와 D. parva의 광계II에서 최고 광합성 효율을 측정하였다.Phyto-PAM (phytoplankton fluorescence induction measuring instrument), Phytoplankton Analyzer, was used to measure the primary productivity. 470 nm (when photoreceptor pigment through blue filter is chlorophyll b), 535 nm (photoreceptor pigment through green filter) In the case of carotenoids), the threshold of fluorescence was measured at 620 nm wavelength (in case of photoreceptive dye through orange filter) and the highest photosynthetic efficiency was measured in photosystem II of C. ovalis and D. parva .
배양 27일 후 도 2a에 나타내는 바와 같이 C. ovalis는 f/2, E+3, E+6, -E+3, -E+6 배지에서 배양된 세포의 각 파장에서 동일한 유형을 나타냈으나, D. parva는 도 2b에 나타내는 바와 같이 배지의 종류에 따라 535nm와 620nm의 최대 광양자수가 증가하는 반면에 470nm는 감소하였다. C. ovalis showed the same type at each wavelength of cells cultured in f / 2, E + 3, E + 6, -E + 3, -E + 6 medium after 27 days of culture. As shown in FIG. 2B, the maximum photon counts of 535 nm and 620 nm increased with D. parva , while 470 nm decreased.
이는 D. parva의 세포가 배지 조성에 따라 총색소 함량 중에 보조색소인 엽록소 b에 의한 광수용 색소 안테나(light harvesting pigment antenna)보다 카로테노이드(carotenoid)에 의한 광수용 색소가 증가하여 광합성 효율이 증가한 것으로 판단된다. This indicates that the photosynthetic efficiency of carotenoids increased due to the increase in the number of D. parva cells, depending on the composition of the medium, compared to the light harvesting pigment antennas by chlorophyll b. Judging.
도 2c 내지 도 2d에서 BM이 더 많이 추가된 배지 그룹(f/2-E, -E+30, -E+40, -E+50)에서 C. ovalis는 도 2c에 나타내는 바와 같이 470nm에서는 -E+30 배지에서 생장한 세포가 광양자수(광합성효율)가 가장 높게 나타났고, 535nm와 620nm에서는 -E+40 배지에서 높게 나타난 것으로 보아 배지에 BM 추가 농도가 증가할수록 카로테노이드 광수용 색소가 증가하는 현상은 도 2d에 나타내는 D. parva와 동일한 결과를 나타냈다. C. ovalis in the medium group (f / 2-E, -E + 30, -E + 40, -E + 50) to which more BM was added in FIGS. 2C to 2D was obtained at 470 nm as shown in FIG. 2C. Cells grown in E + 30 medium had the highest photon number (photosynthetic efficiency), and 535nm and 620nm showed higher photoene counts in -E + 40 medium. As the concentration of BM added to the medium increased, the carotenoid photoreceptor pigment increased. The phenomenon showed the same result as D. parva shown in FIG. 2D.
이는 두 녹조류를 배양 시에 EDTA를 제거한 배지에 BM의 추가 농도를 증가시킬수록 엽록소 a함량의 증가와 보조색소 중에 엽록소 b보다 카로테노이드에 의한 광수용 색소 안테나가 더 증가하여 광합성 효율이 증가됨을 나타내는 것이다.This suggests that as the concentration of BM is increased in the medium without EDTA in the culture of two green algae, the photosynthetic efficiency is increased by increasing the chlorophyll a content and the photoreceptor pigment photoinduced by the carotenoid than chlorophyll b in the auxiliary pigment. .
따라서, EDTA의 유무와 BM의 배지조성률에 따른 두 녹조류의 세포밀도, 엽록소 a와 파장별(470nm, 535nm, 620nm) PSII의 광량자수의 결과들을 종합해 보면, f/2 대조구 배지 보다 BM이 추가된 조성률에 따라 생장률과 광합성률이 증가하였고, f/2배지 조성에 추가되는 화학성분의 킬레이팅제인 Na2EDTA를 제거시키고 BM을 50%를 추가한 배지가 가장 효율성이 높은 배지로 나타났다.Therefore, BM was added more than f / 2 control medium by combining the results of cell density of chlorophyll a and photon number of PSII by wavelength (470nm, 535nm, 620nm) according to the presence of EDTA and BM medium composition. The growth rate and photosynthesis rate increased according to the composition ratio, and the medium with the addition of 50% BM was removed as the most efficient medium by removing the chemical chelating agent Na 2 EDTA added to the f / 2 medium composition.
상기한 바와 같이 구성된 본 발명에 의하면, 화학합성 킬레이팅제를 사용하지 않고도 미세조류의 배양액으로서 사용이 가능하여, 화학합성 킬레이팅제의 독성에 의한 부작용이 발생하지 않을 뿐 아니라, 배양액의 비용이 대폭 절감되는 효과가 있다.According to the present invention configured as described above, it can be used as a culture medium of microalgae without using a chemical synthetic chelating agent, so that side effects due to the toxicity of the chemical synthetic chelating agent do not occur, There is a significant savings effect.
또한, 축산폐수를 미세조류 배양에 사용하므로, 환경정화의 효과와 함께 자원 재활용의 효과가 있다.In addition, since livestock wastewater is used for microalgae cultivation, there is an effect of resource recycling with the effect of environmental purification.
더욱이, 상기한 바와 같이 생물활성수를 배지에 50%첨가할 경우 녹조류의 생장률이 매우 높아서 생산성을 대폭 향상시키는 효과가 있다.Furthermore, when 50% of the biologically active water is added to the medium as described above, the growth rate of the green alga is very high, thereby greatly improving the productivity.
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US8679508B2 (en) | 2008-05-22 | 2014-03-25 | Ecophyco Tech, Ltd. | Microalgae with high-efficient ability to remove carbon dioxide and use thereof |
CN103960118A (en) * | 2014-05-26 | 2014-08-06 | 临沂大学 | Culture medium for cultivating platymonas helgolandica by waste water of starch factory and cultivation method thereof |
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KR20030095154A (en) * | 2002-06-10 | 2003-12-18 | 김미경 | Development of batch culture medium (KEPⅡ) of phytoplanktons (microalgae) for long term) |
JP2007008314A (en) * | 2005-06-30 | 2007-01-18 | Yokohama Rubber Co Ltd:The | Pneumatic run flat tire |
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US8679508B2 (en) | 2008-05-22 | 2014-03-25 | Ecophyco Tech, Ltd. | Microalgae with high-efficient ability to remove carbon dioxide and use thereof |
WO2012141596A1 (en) * | 2011-04-11 | 2012-10-18 | Institutt For Energiteknikk | Method for the production of aqueous nutrient source for macro and micro algae aquaculture farming, nutrient blend thus produced and method for feeding an algae aquaculture farm |
CN103960118A (en) * | 2014-05-26 | 2014-08-06 | 临沂大学 | Culture medium for cultivating platymonas helgolandica by waste water of starch factory and cultivation method thereof |
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