WO2011027925A1 - Outdoor perfusion process for culturing marine microalgae utilizing thermal effluents from a nuclear power plant - Google Patents

Outdoor perfusion process for culturing marine microalgae utilizing thermal effluents from a nuclear power plant Download PDF

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WO2011027925A1
WO2011027925A1 PCT/KR2009/005042 KR2009005042W WO2011027925A1 WO 2011027925 A1 WO2011027925 A1 WO 2011027925A1 KR 2009005042 W KR2009005042 W KR 2009005042W WO 2011027925 A1 WO2011027925 A1 WO 2011027925A1
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nuclear power
microalgae
power plant
culture
outdoor
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PCT/KR2009/005042
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French (fr)
Korean (ko)
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이현용
오성호
한재건
김영
이신영
조정섭
김나영
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강원대학교 산학협력단
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Priority to KR1020107017778A priority Critical patent/KR101184974B1/en
Priority to PCT/KR2009/005042 priority patent/WO2011027925A1/en
Publication of WO2011027925A1 publication Critical patent/WO2011027925A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/02—Photobioreactors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M43/00—Combinations of bioreactors or fermenters with other apparatus
    • C12M43/08—Bioreactors or fermenters combined with devices or plants for production of electricity

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  • the present invention relates to a perfusion culture process of marine microalgae, and more particularly, by utilizing a warm thermal effluent discharged from a nuclear power plant, culturing the microalgae,
  • the present invention relates to a technology capable of cultivating the marine microalgae that is required, thereby enabling mass cultivation outdoors, and at the same time, helping to solve environmental problems caused by warm water drainage.
  • the present invention utilizes the thermal power of the nuclear power plant, which was used as the pentagonal water of the conventional nuclear power plant and brought into the sea, and used the outdoor nuclear cultivation of marine microalgae, which effectively caused various environmental problems. There is a characteristic in solving the problem.
  • Batch culture, fed-batch culture, etc. are known as a culture process of seawater microalgae.
  • the strain cannot grow to a certain concentration because nutrients are easily depleted and toxic by-products are accumulated by putting a culture medium in the incubator.
  • the method of culturing strains using a perfusion culture process as in the present invention is effective.
  • the conventional batch culture and The fed-batch cultivation process is easy to cultivate, but it is difficult to obtain high concentration of cells in batch culture, and the fed-batch cultivation can obtain high concentration of cells. It causes their severe imbalance.
  • perfusion culture has the advantage that high concentration culture is possible by continuously supplying fresh culture solution and continuously removing the used culture solution.
  • Korea located in the mid-latitude, has a low temperature due to cold winter northwest winds, making it difficult to carry out outdoor culture of marine microalgae in warm regions. There are also similar difficulties in other countries where sea temperature is low.
  • An object of the present invention is to provide a method for culturing a large amount of microalgae outdoors in a seawater environment.
  • the present invention having the above object as a method for culturing the marine microalgae in a perfusion type outdoors using a nuclear power plant,
  • Fresh nutrients necessary for the cultivation of microalgae are continuously injected into the photo-bioreactor along with the nuclear wastewater, and the pump is continuously pumped from the photo-bioreactor using a pump.
  • the microalgae contained in the cultured medium are filtered through a level filter and continuously fed back into the photo-bioreactor and overflowed. Part), and the remaining culture solution is mixed with the nuclear power source wastewater to circulate outside the photo-bioreactor and then discarded.
  • the means for circulating to the outside of the photo-bioreactor is characterized in that the transparent tube.
  • the present invention is characterized by the above-described culture method or the culture system itself, and is not characterized in the kind of nutrients. Nutrients necessary for the cultivation of microalgae may be appropriately selected and used according to circumstances.
  • the marine microalgae are cultivated outdoors in an optical-bioreactor, and the used medium (spent medium) is drawn out of the optical-bioreactor. Add fresh medium to continuously supplement nutrients.
  • the microalgae go out together with the culture medium, and the microalgae contained in the discharged medium are filtered through a level filter and re-introduced into the incubator.
  • the level filter is not a special structure, but means a filter for filtering submerged microalgae. Filtering by level filter is based on the difference in weight, so that the overflow of the upper layer where the heaviest microalgae in the spent medium does not sink and the remaining light impurities are discharged out .
  • the overflow of the culture medium used in the above process is discarded and the remainder is mixed with the new nuclear power plant wastewater to pass around the incubator.
  • This is to utilize the warmth of the culture medium used to maintain the temperature of the photo-bioreactor.
  • the mixing of the used culture with the new plant warm water drainage is due to the rather low temperature of the used culture.
  • the means for circulating some of the culture medium used in the nuclear power plant wastewater and circulating to the outside of the photo-bioactor is preferably a transparent rib, so as not to block sunlight from penetrating into the photo-bioreactor. .
  • the temperature inside the photo-reactor or after the mixing of some of the culture medium with the nuclear power plant is a separate temperature. It can be adjusted according to the culture environment of the microalgae by checking using a pedometer. The matters related to this may be selected by those of ordinary skill in the practice of the present invention, and thus detailed description thereof will be omitted.
  • the present invention by cultivating marine microalgae using the hot effluent discharged from the nuclear power plant, as a result of stratifying the conditions necessary for the cultivation of microalgae that requires warm temperature, even in an environment where the sea water is not warm Outdoor mass cultivation of marine microalgae is possible without the use of additional energy. In addition, it is possible to cultivate a high concentration of marine microalgae, which was difficult in the conventional fed-batch culture method.
  • the energy stored in the hot water drainage is not only directly used for culturing microalgae, but also used to maintain the incubator's warmth by circulating it in a tube wrapped around the incubator without throwing away the used culture solution. Not only can they be used completely, but they can also help solve environmental problems caused by warm water.
  • FIG. 1 illustrates an example of implementing the present invention, which shows an example of an outdoor perfusion culture process using thermal effluent of nuclear energy plant.
  • the marine microalgae are cultivated outdoors in the light-bioreactor.
  • new medium containing nutrients necessary for microalgae cultivation fresh medium
  • new medium is added to the inlet continuously with nuclear wastewater to supplement nutrients.
  • fresh medium is added to the inlet continuously with nuclear wastewater to supplement nutrients.
  • the culture medium is continuously discharged, and a means such as a pump can be used (2).
  • the discharged culture may contain spent medium and microalgae.
  • the present invention can use the same hot water drainage as the general seawater, while still utilizing the advantages of the existing seawater culture technology, while improving energy utilization and contributing to the problem of environmental pollution caused by warm water drainage.
  • an experimental example of an outdoor perfusion light cultivation method using nuclear power plant wastewater is introduced.
  • the culture solution was prepared using Enrichment Solution 20in / i (Pasteurized thermal plume, NaN0 3 4.7g / 2L, in 1H of nuclear power plant 1 «autoclave sterilized at 121 ° C for 15 minutes using a autoclave (HK-AC120, Korea).
  • Enrichment Solution 20in / i Pulsteurized thermal plume, NaN0 3 4.7g / 2L, in 1H of nuclear power plant 1 «autoclave sterilized at 121 ° C for 15 minutes using a autoclave (HK-AC120, Korea).
  • the incubator was also autoclaved at 121 ° C for 15 minutes using a high pressure sterilizer and then sprinkled with alcohol around the inlet to ignite the inlet, and then cultured and microalgae Chlorella minut / ss / maWTEX, LB2341, USA). Inoculate and stir the strain at 150 rpm. And the same culture medium is added continuously to supplement the nutrients.
  • the used culture solution is continuously taken out.
  • the microalgae like the culture medium used, will go out.
  • the microalgae strain is separated and sent back into the incubator using the level filter contained in the discharged medium, while the overflow is discarded and the remaining culture is around the incubator. Circulate in a transparent tube wrapped.
  • the cell weights of outdoor cultures using warm water and outdoor cultures using general seawater were compared.
  • 30 ⁇ culture medium and cells were taken from the 14 «photo-bioreactor at 5 days intervals and measured in the wavelength range of 540 nm using a UV / Visible spectrophotometer (Kontron Instruments). Samples collected for dry cell weight measurement were filtered with 0.45 ⁇ filter paper, dried at 80 ° C. for 24 hours, and then dried.
  • lipid production was compared.
  • dry cells were lyophilized and pulverized, and then pre-treated.
  • 20 times of solvent CHC1 3 : methanol (2: 1 v / v) was added to lg, which was pulverized by Folch method. Stir for 90 minutes.
  • the mixture was centrifuged, the supernatant was taken, 0.9% NaCl solvent was added, vortexed for several seconds, and centrifuged again. The supernatant was discarded, and the extract containing lipids (lower layer) was dried and weighed to obtain a fat content.
  • thermo effluent thermo effluent

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Abstract

The present invention relates to an outdoor perfusion process for culturing marine microalgae, utilizing thermal effluents from a nuclear power plant. An aim of the present invention is to provide a method for mass-culturing microalgae outdoors, even in environments in which seawater is not warm. In the process of the present invention, fresh nutrients needed for culturing microalgae are continuously fed, together with thermal effluents from a nuclear power plant, into a photobioreactor, while a culture medium is continuously pumped out of the photobioreactor using a pump, wherein microalgae included in the pumped out culture medium is filtered by means of a level filter and is continuously fed back into the photobioreactor, overflow is discarded, and the remaining culture medium is mixed with thermal effluents from the nuclear power plant and circulated outside the photobioreactor, after which the culture medium is discarded.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
원전 온배수를 활용한 해양 미세조류의 옥외에서의 관류식 배양 방법  Perfusion Culture Method of Marine Microalgae Using Nuclear Power Plant
【기술분야】 Technical Field
본 발명은 해양 미세조류 (microalgae)의 관류식 배양 방법 (perfusion culture process)에 관한 것으로， 더욱 상세하게는 원자력발전소에서 배출되는 더 운 온배수 (thermal effluent)를 활용하여 미세조류를 배양함으로써 따뜻한 온도를 요하는 해양 미세조류의 배양에 필요한 조건을 층족시켜 옥외 대량 배양이 가능하 게 하는 동시에 종래에 온배수로 인해 야기돼온 환경 문제 해결에 일조할 수 있는 기술에 관한 것이다.  The present invention relates to a perfusion culture process of marine microalgae, and more particularly, by utilizing a warm thermal effluent discharged from a nuclear power plant, culturing the microalgae, The present invention relates to a technology capable of cultivating the marine microalgae that is required, thereby enabling mass cultivation outdoors, and at the same time, helping to solve environmental problems caused by warm water drainage.
본 발명은 종래 원자력 발전소의 넁각수로 쓰이고 바다로 홀러들어가 여러 환경 문제점을 야기하였던 원전 온배수를 해양 미세조류의 옥외 배양에 활용함으로 쎄 원전 온배수에 내장된 열에너지를 효율적으로 활용하는 동시에 온배수로 인한 환경 문제를 해소시킨 데에 특징이 있다.  The present invention utilizes the thermal power of the nuclear power plant, which was used as the pentagonal water of the conventional nuclear power plant and brought into the sea, and used the outdoor nuclear cultivation of marine microalgae, which effectively caused various environmental problems. There is a characteristic in solving the problem.
【배경기술】 Background Art
해양 미세조류를 바이오연료로서 산업적으로 이용하기 위해서는 고농도의 배 양이 필수적이다.  High concentrations of culture are essential for the industrial use of marine microalgae as a biofuel.
해수 미세조류의 배양 공정으로는 회분식 배양 (batch culture), 유가식 배양 (fed-batch culture) 등이 알려져 있다.  Batch culture, fed-batch culture, etc. are known as a culture process of seawater microalgae.
먼저 회분식 배양의 경우 배양기 내에 일정한 배양액을 넣고 배양하는 방식 으로 쉽게 영양분이 고갈되고 독성 부산물이 축적되기 때문에 균주가 일정 농도 이 상으로 자라지 못한다.  First, in the case of batch culture, the strain cannot grow to a certain concentration because nutrients are easily depleted and toxic by-products are accumulated by putting a culture medium in the incubator.
위와 같은 회분식 배양의 단점을 극복한 것이 유가식 배양인데， 이는 세포의 성장에 맞추어 새로운 배양액을 계속 공급해 주는 방식으로서 배양액 공급 시 농축 된 배양액을 사용하여 지속적으로 영양분을 공급해 줌으로써 균주의 농도를 높게 올릴 수 있다. 반면， 배양 말기에 노폐물의 축적 및 영양소들의 심각한 불균형에 의해 생육이 저하된다.  It is a fed-batch culture that overcomes the shortcomings of batch culture as described above. This is a method of continuously supplying a new culture solution in accordance with the growth of cells, and it is possible to increase the concentration of the strain by continuously supplying nutrients using the concentrated culture solution when supplying the culture solution. Can be. On the other hand, growth is reduced by the accumulation of waste products and severe imbalance of nutrients at the end of the culture.
따라서 본 발명과 같이 관류식 배양 (perfusion culture) 공정을 이용한 균주 의 배양 방법이 효과적이라고 생각된다. 위에서 살핀 것처럼 기존의 회분식 배양과 유가식 배양 공정은 배양 방식이 쉽기는 하나 회분식 배양의 경우 고농도의 세포를 얻기 어려우며， 유가식 배양은 고농도의 세포를 얻을 수 있지만 배양액 공급 시 농 축된 배양액을 사용하여 배양 말기에 노폐물의 축적 및 영양소들의 심한 블균형을 초래한다. 이에 비해 관류식 배양은 신선한 배양액을 연속적으로 공급하고 사용 배 양액을 연속적으로 제거해 줌으로써 고농도 배양이 가능하다는 장점이 있다. Therefore, it is considered that the method of culturing strains using a perfusion culture process as in the present invention is effective. As with the above, the conventional batch culture and The fed-batch cultivation process is easy to cultivate, but it is difficult to obtain high concentration of cells in batch culture, and the fed-batch cultivation can obtain high concentration of cells. It causes their severe imbalance. In comparison, perfusion culture has the advantage that high concentration culture is possible by continuously supplying fresh culture solution and continuously removing the used culture solution.
한편 중위도에 위치한 우리나라 (대한민국)는 추운 겨울 북서풍의 영향으로 온도가 낮은 편이어서 따뜻한 지역에서 이루어지는 해양 미세조류의 옥외 배양을 수행하는 것이 곤란하다. 또한 해수의 온도가 낮은 다른 나라에서도 비슷한 어려움 이 있다.  Meanwhile, Korea (South Korea), located in the mid-latitude, has a low temperature due to cold winter northwest winds, making it difficult to carry out outdoor culture of marine microalgae in warm regions. There are also similar difficulties in other countries where sea temperature is low.
본 발명에서는 이러한 문제점을 해결하기 위해 원자력 발전소에서 배출되는 온배수를 활용하고자 하였다. 이러한 시도는 최초로서， 그동안 바다 생태계를 파괴 하는 등 생태계에 나쁜 영향을 줌에도 불구하고 달리 관리하기가 어려웠던 원전 온 배수는 본 발명에 의해 해양 미세조류의 옥외 배양에 활용됨으로써 에너지 활용과 환경오염 해결에 기여할 수 있게 되었다. 원자력 발전소가 연안에 위치한 경우 막 대한 양의 온배수가 끊임없이 바다로 배출되고, 온배수의 높은 열에너지는 원전 주 변 해역의 바다 생태계에 다양한 영향을 주어 궁극적으로 수산업에 악영향을 미쳤 는바， 본 발명에서는 기존 해수보다 평균 7~8°C 높은 온도를 나타내는 온배수의 특 징을 역으로 이용하였다. 기존에 해양 미세조류를 옥외 배양하는 것은 기온이 높은 지역에서만 가능하였는데， 본 발명에 따르면 원전 온배수를 공급받을 수 있는 지역 이라면 기온이 낮은 지역이라 하더라도 옥외 광배양이 가능하다. In the present invention, to solve this problem, it was intended to utilize the hot water discharged from the nuclear power plant. This is the first time that nuclear power plant drainage, which had been difficult to manage despite the adverse effects on the ecosystem such as destroying the marine ecosystem, was utilized for outdoor cultivation of marine microalgae by the present invention, thereby solving energy utilization and environmental pollution. To contribute. When a nuclear power plant is located on the coast, a huge amount of hot water is constantly discharged to the sea, and the high heat energy of the hot water has various effects on the marine ecosystem around the nuclear power plant, which ultimately adversely affects fisheries. The characteristic of the warm-water drainage, which shows an average higher temperature of 7-8 ° C, was used in reverse. Conventionally, the outdoor microculturing of marine microalgae was possible only in a high temperature region, and according to the present invention, even if the region can be supplied with nuclear power plant, outdoor light cultivation is possible even in a low temperature region.
【발명의 상세한 설명】 [Detailed Description of the Invention]
본 발명은 해수가 따뜻하지 않은 환경에서도 미세조류를 옥외에서 대량으로 배양할 수 있는 방법을 제공하는 데 목적이 있다.  An object of the present invention is to provide a method for culturing a large amount of microalgae outdoors in a seawater environment.
위와 같은 목적을 가진 본 발명은 원전 온배수를 활용하여 해양 미세조류를 옥외에서 관류식으로 배양하는 방법으로서，  The present invention having the above object as a method for culturing the marine microalgae in a perfusion type outdoors using a nuclear power plant,
미세조류의 배양에 필요한 신선한 영양물질을 원전 온배수와 함께 광-바이오 리액터에 연속적으로 주입하는 동시에， 펌프를 이용하여 광-바이오리액터 (photo- bioreactor)에서 배양액을 연속적으로 퍼내고,  Fresh nutrients necessary for the cultivation of microalgae are continuously injected into the photo-bioreactor along with the nuclear wastewater, and the pump is continuously pumped from the photo-bioreactor using a pump.
퍼내진 배양액 중 그에 포함된 미세조류는 레벨 필터 (level filter)로 걸러 내 광-바이오리액터에 다시 연속하여 투입하고， 오버플로우 (overflow, 넘쳐흐르는 부분)는 버리며, 그 나머지 배양액은 원전 온배수와 흔합하여 광-바이오리액터의 외부를 순환한 다음에 버려지도록 한 것을 특징으로 한다. The microalgae contained in the cultured medium are filtered through a level filter and continuously fed back into the photo-bioreactor and overflowed. Part), and the remaining culture solution is mixed with the nuclear power source wastewater to circulate outside the photo-bioreactor and then discarded.
또한 이때, 퍼내진 배양액 중 일부를 원전 온배수와 흔합하여 광 -바이오리액 터의 외부에 순환시키는 데에 있어서, 광-바이오리액터의 외부로 순환시키는 수단 은 투명한 튜브인 것을 특징으로 한다. 본 발명은 위와 같은 배양 방법 내지 배양 시스템 자체에 특징이 있는 것이 지， 영양물질의 종류에 특징이 있는 것이 아니다. 미세조류의 배양에 필요한 영양 물질은 상황에 따라 적절히 선택하여 사용할 수 있다. 본 발명의 원전 온배수를 이용한 해양 미세조류의 관류식 광배양 방법에서 는， 광 -바이오리액터에서 해양 미세조류를 옥외 배양하되， 사용한 배양물 (spent medium)은 광-바이오리액터의 바깥으로 빼내면서 새로운 배양물 (fresh medium)을 투입하여 계속적으로 영양분을 보층한다. 사용한 배양물 (spent medium)을 밖으로 배출할 때 미세조류가 배양액과 같이 바깥으로 나가게 되므로， 배출되는 배양액에 들어있는 미세조류는 레벨 필터 (level filter)로 여과하여 다시 배양기 안으로 들 여보낸다. 레벨 필터 (level filter)는 특별한 구조를 가진 것은 아니고 가라앉은 미세조류를 거르기 위한 필터를 의미한다. 레벨 필터에 의한 거름 방식은 무게 차 에 의해 이루어지는 것으로 사용한 배양물 (spent medium) 속의 가장 무거운 미세조 류가 밑에 가라않고 나머지 가벼운 불순물 등이 존재하는 상위층의 오버플로우 (over flow)는 밖으로 배출되게 된다.  In addition, at this time, in circulating a part of the culture medium to be discharged to the outside of the photo-bio reactor by mixing with the nuclear power plant waste water, the means for circulating to the outside of the photo-bioreactor is characterized in that the transparent tube. The present invention is characterized by the above-described culture method or the culture system itself, and is not characterized in the kind of nutrients. Nutrients necessary for the cultivation of microalgae may be appropriately selected and used according to circumstances. In the perfusion type optical culture method of marine microalgae using the nuclear power plant of the present invention, the marine microalgae are cultivated outdoors in an optical-bioreactor, and the used medium (spent medium) is drawn out of the optical-bioreactor. Add fresh medium to continuously supplement nutrients. When the spent medium is discharged out, the microalgae go out together with the culture medium, and the microalgae contained in the discharged medium are filtered through a level filter and re-introduced into the incubator. The level filter is not a special structure, but means a filter for filtering submerged microalgae. Filtering by level filter is based on the difference in weight, so that the overflow of the upper layer where the heaviest microalgae in the spent medium does not sink and the remaining light impurities are discharged out .
또한， 위 과정에서 사용된 배양액 증 오버플로우 (overflow)는 버리고 나머지 는 새로운 원전 온배수와 흔합하여 배양기 주변을 통과하게 한다. 이는 사용된 배 양액의 온기를 광-바이오리액터의 온도 유지에 활용하기 위함이다. 사용된 배양액 을 새로운 원전 온배수와 흔합하는 것은, 사용된 배양액의 온도가 다소 낮아져 있 기 때문이다.  In addition, the overflow of the culture medium used in the above process is discarded and the remainder is mixed with the new nuclear power plant wastewater to pass around the incubator. This is to utilize the warmth of the culture medium used to maintain the temperature of the photo-bioreactor. The mixing of the used culture with the new plant warm water drainage is due to the rather low temperature of the used culture.
본 발명에서， 사용된 배양액 중 일부를 원전 온배수와 흔합하여 광-바이오리 액터의 외부로 순환시키는 수단은 투명한 류브인 것이 바람직한데， 이는 햇빛이 광 -바이오리액터 내부로 투과되는 것을 차단하지 않도록 하기 위해서다.  In the present invention, the means for circulating some of the culture medium used in the nuclear power plant wastewater and circulating to the outside of the photo-bioactor is preferably a transparent rib, so as not to block sunlight from penetrating into the photo-bioreactor. .
본 발명의 본질적인 특징은 아니지만 참고로 밝히면, 광-바이오리액터 내부 의 온도나， 사용된 배양액 중 일부가 원전 온배수와 흔합된 후의 온도는 별도의 온 도계를 사용하여 체크함으로써 미세조류의 배양 환경에 맞게 조절할 수 있다. 이와 관련된 사항은 본 발명을 실시하는 단계에서 통상의 기술자에 의해 선택될 수 있는 내용이므로 자세한 설명은 생략한다. 본 발명에 따르면， 원자력발전소에서 배출되는 더운 온배수 (thermal effluent)를 활용하여 해양 미세조류를 배양함으로써 따뜻한 온도를 요하는 미세조 류의 배양에 필요한 조건을 층족시키는 결과， 해수가 따뜻하지 않은 환경에서도 추 가적인 에너지의 사용 없이 해양 미세조류의 옥외 대량 배양이 가능하게 된다. 또한 기존의 유가식 (fed-batch) 배양 방법에서는 어려웠던 해양 미세 조류의 고농도 배양을 할 수 있다. Although not an essential feature of the present invention, it is to be noted that the temperature inside the photo-reactor or after the mixing of some of the culture medium with the nuclear power plant is a separate temperature. It can be adjusted according to the culture environment of the microalgae by checking using a pedometer. The matters related to this may be selected by those of ordinary skill in the practice of the present invention, and thus detailed description thereof will be omitted. According to the present invention, by cultivating marine microalgae using the hot effluent discharged from the nuclear power plant, as a result of stratifying the conditions necessary for the cultivation of microalgae that requires warm temperature, even in an environment where the sea water is not warm Outdoor mass cultivation of marine microalgae is possible without the use of additional energy. In addition, it is possible to cultivate a high concentration of marine microalgae, which was difficult in the conventional fed-batch culture method.
또한 그간 문제시 되어온 원전 온배수를 미세조류의 배양에 직접 사용할 뿐 아니라， 사용된 배양액을 그대로 버리지 않고 다시 모아서 배양기 주위를 감싸고 있는 튜브에 순환시켜 배양기의 보온 유지에 사용되도록 함으로써， 온배수에 저장 된 에너지를 완전히 사용함은 물론 온배수로 인한 환경 문제 해결에 일조할 수 있 다.  In addition, the energy stored in the hot water drainage is not only directly used for culturing microalgae, but also used to maintain the incubator's warmth by circulating it in a tube wrapped around the incubator without throwing away the used culture solution. Not only can they be used completely, but they can also help solve environmental problems caused by warm water.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 1은 본 발명을 실시하는 하나의 예를 도시한 것으로서, 원자력 발전소의 온배수를 이용한 옥외 관류식 배양 (Outdoor Perfusion Culture Process Using Thermal Effluent of Nuclear Energy Power Plant)의 예를 보인 것이다.  1 illustrates an example of implementing the present invention, which shows an example of an outdoor perfusion culture process using thermal effluent of nuclear energy plant.
① 투입 흐름 ： 온배수 및 영양물질 (Input flow from into photo-bioreactor ： Thermal Effluent with others nutrients)  ① Input flow from into photo-bioreactor: Thermal Effluent with others nutrients
② 배출 흐름 ： 온배수， 사용된 배양물질 및 미세조류 (Out flow from photo- bioreactor ： Thermal Effluent with spent medium and microalgae)  ② Outflow from: on-water drainage, used culture materials and microalgae (Out flow from photo-bioreactor: Thermal Effluent with spent medium and microalgae)
③ 온배수와 사용된 배양물질의 오버플로우 (Over flow of thermal effluent and spent medium)  ③ Overflow of thermal effluent and spent medium
④ 온배수와 사용된 배양물질 (thermal effluent and spent medium) ④ Warm effluent and cultured material (thermal effluent and spent medium)
⑤ 걸러진 미세조류 (filtered microalgae) ⑤ filtered microalgae
⑥ 사용된 배양물질을 포함한 온배수와 새로 투입되는 온배수의 흔합물 (Mix of constant temperature thermal effluent and spent medium) 【실시예】 ⑥ Mix of constant temperature thermal effluent and spent medium EXAMPLE
도 1과 함께 본 발명의 실시예를 설명한다.  An embodiment of the present invention will be described with reference to FIG. 1.
본 발명에서는 광 -바이오리액터에서 해양 미세조류를 옥외 배양한다. 광-바 이오리액터에는 미세조류 배양에 필요한 영양물질을 함유한 새로운 배양물 (fresh medium)을 원전 온배수와 함께 주입구에 연속적으로 투입하여 영양분을 보층해준다 (①)ᅳ 원전 온배수를 사용하므로 추운 조건에서도 옥외 배양을 할 수 있다.  In the present invention, the marine microalgae are cultivated outdoors in the light-bioreactor. In the photo-bio reactor, new medium containing nutrients necessary for microalgae cultivation (fresh medium) is added to the inlet continuously with nuclear wastewater to supplement nutrients. (①) Can be grown outdoors.
그러는 한편, 광-바이오리액터에서는 연속적으로 배양액을 배출시키는데 이 때 펌프와 같은 수단을 이용할 수 있다 (②) . 배출되는 배양액에는 사용된 배양물질 (spent medium)과 미세조류가 포함돼 있을 수 있다.  On the other hand, in the photo-bioreactor, the culture medium is continuously discharged, and a means such as a pump can be used (②). The discharged culture may contain spent medium and microalgae.
배출되는 배양액의 오버플로우는 버리고 (③)， 그에 포함된 미세조류는 레벨 필터 (level filter)로 여과하여 다시 배양기 안으로 보낸다 (⑤) . 또한 그와 같이 하고 남은 나머지 배양액 [④, 여기에도 사용된 배양물질 (spent medium)이 포함돼 있을 수 있다]은 새로운 원전 온배수와 흔합하여 (⑥) 배양기 주변을 감싼 투명한 튜브를 통과시킨 후 버린다. 이로써 사용된 배양액은 마지막까지 배양기의 온도 유 지에 기여하게 된다. 이처럼 본 발명에서는 새 배양액이 들어오고 사용된 배양액은 나가는 과정을 반복하여 미세조류에 필요한 최적의 요건을 만족시키는 동시에 노폐물의 축적올 방 지하며 , 또한 그 과정에서 배양기 밖으로 배양액과 함께 나가는 미세조류는 여과기 로 걸러 다시 배양기로 되돌리기 때문에 배양기 내의 세포수는 계속 증가한다.  (3) Discard the overflow of the discharged culture medium, and the microalgae contained therein are filtered through a level filter and sent back into the incubator (⑤). In addition, the remaining culture solution [4, which may contain a spent medium (spent medium)] is then mixed with a new nuclear power plant (6) and passed through a transparent tube wrapped around the incubator. The broth used thereby contributes to maintaining the temperature of the incubator until the end. As described above, in the present invention, the new culture solution enters and the used culture solution is repeated to satisfy the optimum requirements for microalgae and at the same time prevents the accumulation of waste products. The number of cells in the incubator continues to increase because it is filtered off and returned to the incubator.
상기와 같은 본 발명은 일반 해수와 동일한 성분의 온배수를 사용하기 때문 에 기존의 해수 배양 기술의 장점을 그대로 활용할 수 있으면서도, 에너지 활용성 을 높이고 온배수에 따른 환경오염 문제 해소에 일조할 수 있다. 다음으로， 원전 온배수를 이용한 옥외 관류 광 배양 방법에 대한 실험예를 소개한다.  As described above, the present invention can use the same hot water drainage as the general seawater, while still utilizing the advantages of the existing seawater culture technology, while improving energy utilization and contributing to the problem of environmental pollution caused by warm water drainage. Next, an experimental example of an outdoor perfusion light cultivation method using nuclear power plant wastewater is introduced.
[제 1공정 ： 배양액 멸균 공정] [Step 1 ： Culture Sterilization Step]
배양액은, 고압 멸균기 (한국기기 제작， HK-AC120, Korea)를 이용해 121°C에 서 15분간 가압 살균한 원전 온배수 1«에 Enrichment Solution 20in / i (Pasteurized thermal plume, NaN034.7g/2L, Na2glycerophosphate · 5H20 0.7g/2L , ES Fe solution 325 /2L, Ρ—Π Metal solution 325 /2L, HEPES buffer 6.5g/2L, Vitamin B12 3m^/2L, Biotin Vitamin Solution 3m^/2L, Thiamin Vitamin Solution 3 /2L)을 가한 것을 이용한다. The culture solution was prepared using Enrichment Solution 20in / i (Pasteurized thermal plume, NaN0 3 4.7g / 2L, in 1H of nuclear power plant 1 «autoclave sterilized at 121 ° C for 15 minutes using a autoclave (HK-AC120, Korea). Na 2 glycerophosphate5H 2 0 0.7g / 2L, ES Fe solution 325 / 2L, Ρ—Π Metal solution 325 / 2L, HEPES buffer 6.5g / 2L, Vitamin B 12 3m ^ / 2L, Biotin Vitamin Solution 3m ^ / 2L, Thiamin Vitamin Solution 3 / 2L) .
[제 2공정 ： 접종 및 교반 공정] [2nd process: inoculation and stirring process]
배양기 역시 고압 멸균기를 이용하여 121°C로 15분간 가압 살균한 뒤 주입구 주위에 알코올을 뿌려 입구 주위를 점화한 뒤， 입구를 통하여 배양액 및 미세조류 인 Chlorella minut/ss/maWTEX, LB2341, USA)를 접종하고 균주를 150rpm으로 교반 한다. 그리고 같은 배양액을 계속적으로 투입하여 영양분올 보층한다. The incubator was also autoclaved at 121 ° C for 15 minutes using a high pressure sterilizer and then sprinkled with alcohol around the inlet to ignite the inlet, and then cultured and microalgae Chlorella minut / ss / maWTEX, LB2341, USA). Inoculate and stir the strain at 150 rpm. And the same culture medium is added continuously to supplement the nutrients.
[제 3공정 ： 관류식 배양을 통한 균주 분리 공정] [Step 3: Strain Separation Through Perfusion Culture]
배양액의 투입과 동시에 사용된 배양액을 계속하여 바깥으로 빼낸다. 이때 사용된 배양액과 같이 미세조류도 바깥으로 나가게 되는데， 배출되는 배양액에 들 어 있는 레벨 필터를 이용하여 미세조류 균주를 분리해 다시 배양기 안으로 보내는 한편， 오버플로우 (overflow)는 버리고 나머지 배양액은 배양기 주변을 감싼 투명한 튜브로 순환시킨다.  Simultaneously with the addition of the culture solution, the used culture solution is continuously taken out. At this time, the microalgae, like the culture medium used, will go out. The microalgae strain is separated and sent back into the incubator using the level filter contained in the discharged medium, while the overflow is discarded and the remaining culture is around the incubator. Circulate in a transparent tube wrapped.
[제 4공정 ： 온배수 이용 공정] [4th process ： Hot water drainage process]
제 3공정에서 투명한 튜브로 들어온 배양액은 새로운 원전 온배수와 흔합되어 튜브 안으로 홀려보내 배양기의 보온성을 유지하게 한다. 실험예 1.  In the third process, the culture solution introduced into the transparent tube is mixed with the new nuclear power plant wastewater to be introduced into the tube to maintain the warmth of the incubator. Experimental Example 1.
본 실험을 위하여 원전 온배수가 흘러나오는 울진 원자력 발전소 근처에서 기온이 낮은 1월과 2월에 걸쳐 실험을 진행하였다. 14 짜리 옥외 관류 광-배양장 치 (광 -바이오리액터 )를 이용하였으며， 일반 해수와 원전 온배수를 이용하여 도 1과 위에서 설명한 공정에 따라 실험을 하였다. 그래프 1은 이에 따른 배양기 내부 배 양액의 온도 변화를 비교 분석한 것이다. For this experiment, the experiment was conducted in January and February when the temperature was low near Uljin Nuclear Power Plant where the nuclear power plant drained water. Fourteen outdoor perfusion photo-cultivation devices (optical-bioreactors) were used, and experiments were carried out using the general seawater and the nuclear power plant. Graph 1 is a comparative analysis of the temperature change of the culture medium in accordance with this.
Figure imgf000009_0001
Figure imgf000009_0001
Time(day)  Time (day)
— ^Seawater ~■ -- Thermal effluent  — ^ Seawater ~ ■-Thermal effluent
그래프 1. 울진군에서의 1~2월의 일반 해수와 원전 온배수의 온도 비교 위와 같이 온배수를 통한 관류 광 배양 장치를 이용한 것이 울진의 온도에 비해 상당히 높은 온도를 유지함을 알 수 있다. 따라서 온배수를 이용한 관류 광배 양 방법을 통해 일정한 보온성이 유지되므로 낮은 온도 지역에서의 옥외 배양이 가 능함을 알 수 있다. 실험예 2.  Graph 1. Comparison of temperature of general seawater and nuclear power plant in Jan-February in Uljin-gun It can be seen that the use of perfusion light cultivation apparatus through the warm-drainage maintains a considerably higher temperature than that of Uljin. Therefore, it can be seen that it is possible to cultivate outdoor in low temperature area because constant heat retention is maintained through the perfusion photoculture method using warm water. Experimental Example 2.
온배수를 이용한 옥외 배양과 일반 해수를 이용한 옥외 배양에 따른 균체량 을 비교해 보았다. 균체 생육을 측정하기 위해 14«짜리 광 -바이오리액터에서 5일 간격으로 30^의 배양액과 균체를 취한 후， UV/Visible spectrophotometer (Kontron Instruments)를 이용하여， 540nm의 파장 영역에서 측정하였다. 건조 균체량 측정을 위해 채취한 시료를 0.45μηι 여과지로 여과한 후， 80°C 항온기에 24시간 정도 두어 건조한후， 건조무게를 정확하게 측정하였다. The cell weights of outdoor cultures using warm water and outdoor cultures using general seawater were compared. In order to measure the cell growth, 30 ^^ culture medium and cells were taken from the 14 «photo-bioreactor at 5 days intervals and measured in the wavelength range of 540 nm using a UV / Visible spectrophotometer (Kontron Instruments). Samples collected for dry cell weight measurement were filtered with 0.45μηι filter paper, dried at 80 ° C. for 24 hours, and then dried.
Figure imgf000010_0001
Figure imgf000010_0001
— · ~ Seawater -■— Thermal effluent — · ~ Seawater-■ — Thermal effluent
그래프 2. 일반 해수와 온배수에서의 Chlorella minutissima엑 50일간 세포 성장 비교 그래프 2에 나타난 바와 같이， 보온성이 유지된 온배수를 이용한 관류 배양 이 아무런 작용을 하지 않은 옥외 관류 배양에 비해 상대적으로 높은 균체 생성량 을 보였다. 이로써 온배수를 통한 배양이 해수를 이용한 배양액에 비해 불리하게 작용하지 않으며 오히려 온배수의 높은 열에너지로 인하여 옥외 대량 배양이 효율 적으로 이루어질 수 있음을 확인할 수 있다. 실험예 3.  Graph 2. Comparison of Chlorella minutissima ex 50-day Cell Growth in Normal Seawater and Warm Drainage As shown in Graph 2, perfusion cultures with warmed drainage showed relatively higher cell production than outdoor perfusion cultures without any action. Seemed. As a result, it can be seen that the cultivation through the hot drainage does not adversely affect the culture solution using the sea water, but rather, the outdoor mass cultivation can be efficiently performed due to the high thermal energy of the hot drainage. Experimental Example 3.
온배수를 이용한 배양과 일반 해수를 이용한 옥외 배양을 통한 바이오 디젤 의 원료로서 가능성을 알아보기 위해， 지질 생성량을 비교하여 살펴보았다. 균체 내 지질 측정을 위해 건조 균체를 동결 건조한 후 분쇄하는 전처리를 한 다음， Folch법을 이용해 분쇄된 시료 lg에 20배의 용매 CHC13: methanol (2:1 v/v)를 넣고 상온에서 30~90분 동안 교반하였다. 그 다음 이를 원심분리하고 상등액을 취하여 0.9% NaCl 용매를 넣고 수초간 볼텍싱한 후 또다시 원심분리하여 상등액은 버리고 지질이 포함된 추출액 (하층)을 건조해 무게를 칭량하고 이를 지방함량으로 하였다. In order to examine the possibility of biodiesel as a raw material of the cultivation using warm drainage and the outdoor cultivation using general seawater, lipid production was compared. For the measurement of lipid in cells, dry cells were lyophilized and pulverized, and then pre-treated. Then, 20 times of solvent CHC1 3 : methanol (2: 1 v / v) was added to lg, which was pulverized by Folch method. Stir for 90 minutes. Then, the mixture was centrifuged, the supernatant was taken, 0.9% NaCl solvent was added, vortexed for several seconds, and centrifuged again. The supernatant was discarded, and the extract containing lipids (lower layer) was dried and weighed to obtain a fat content.
Figure imgf000011_0001
Figure imgf000011_0001
— ^-Seawater -■— Thermal effluent — ^ -Seawater-■ — Thermal effluent
그래프 3. 일반 해수와 온배수에서의 ChloreJIa minutissima 50일간 세포 지질 비교 그 결과 위와 같이 온배수를 사용하는 경우 바이오 디젤의 유용 물질인 지질 함량이 크게 증가됨을 알 수 있다. 이로써 낮은 온도의 영향을 받는 지역의 옥외 배양 시 바이오 디젤용 지질을 생산하기 위한 온배수를 이용한 관류식 광 배양 공 정을 통하여 풍부한 바이오 디젤용 지질을 생산할 수 있는 가능성을 확인하였다. ' Graph 3. Comparison of ChloreJIa minutissima 50-day Cell Lipids in Normal Seawater and Warm Wastewater As a result, it can be seen that the lipid content, which is a useful material of biodiesel, is significantly increased when the warm wastewater is used. As a result, the possibility of producing abundant biodiesel lipids was confirmed through a perfusion type light culture process using on-water drainage to produce lipids for biodiesel during outdoor culture in an area affected by low temperature. '
【산업상 이용가능성】 Industrial Applicability
본 발명에 따르면， 원자력발전소에서 배출되는 더운 온배수 (thermal effluent)를 활용함으로써 추운 지역에서도 해양 미세조류를 옥외에서 대량으로 배 양할 수 있다. 그리하여 바이오 연료의 활용에 직접적으로 이용될 수 있다.  According to the present invention, by utilizing the hot effluent (thermal effluent) discharged from the nuclear power plant, it is possible to cultivate a large amount of marine microalgae outdoors in cold areas. Thus it can be used directly for the utilization of biofuels.

Claims

【청구의 범위】 [Range of request]
【청구항 1】  [Claim 1]
미세조류의 배양에 필요한 신선한 영양물질을 원전 온배수와 함께 광-바이오 리액터에 연속적으로 주입하는 동시에， 펌프를 이용하여 광 -바이오리액터에서 배양 액을 연속적으로 퍼내고，  Fresh nutrients necessary for the cultivation of microalgae are continuously injected into the optical-bioreactor together with the nuclear power plant wastewater, and the culture liquid is continuously pumped out of the optical-bioreactor using a pump.
퍼내진 배양액 중 그에 포함된 미세조류는 레벨 필터 (level filter)로 걸러 내 광-바이오리액터에 다시 연속하여 투입하고, 오버플로우 (overflow)는 버리며， 그 나머지 배양액은 원전 온배수와 흔합하여 광-바이오리액터의 외부를 순환한 다 음에 버려지도록 한 것을 특징으로 하는,  The microalgae contained in the cultured medium are filtered through a level filter and continuously fed back into the photo-bioreactor, the overflow is discarded, and the remaining culture is mixed with the warm water drainage of the nuclear power plant. Circulating the outside of the reactor and then discarded,
원전 온배수를 활용한 해양 미세조류의 옥외에서의 관류식 배양 방법.  Perfusion culture method of marine microalgae using nuclear wastewater from nuclear power plants.
【청구항 2] [Claim 2]
제 1항에 있어서，  The method of claim 1,
퍼내진 배양액 중 일부를 원전 온배수와 흔합하여 광-바이오리액터의 외부에 순환시키는 데에 있어서， 광-바이오리액터의 외부로 순환시키는 수단은 투명한 튜 브인 것을 특징으로 하는,  In circulating a part of the culture broth to the outside of the optical-bioreactor in combination with the nuclear power plant wastewater, the means for circulating to the outside of the photo-reactor is a transparent tube,
원전 온배수를 활용한 해양 미세조류의 옥외에서의 관류식 배양 방법.  Perfusion culture method of marine microalgae using nuclear wastewater from nuclear power plants.
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