KR20190022967A - Mixed strain having nitrogen fixation activity, microbial agent containing the same and biofertilizer containing the same - Google Patents
Mixed strain having nitrogen fixation activity, microbial agent containing the same and biofertilizer containing the same Download PDFInfo
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- KR20190022967A KR20190022967A KR1020170106793A KR20170106793A KR20190022967A KR 20190022967 A KR20190022967 A KR 20190022967A KR 1020170106793 A KR1020170106793 A KR 1020170106793A KR 20170106793 A KR20170106793 A KR 20170106793A KR 20190022967 A KR20190022967 A KR 20190022967A
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- enterobacter ludwigii
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 title claims abstract description 86
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- 241001217893 Enterobacter ludwigii Species 0.000 claims abstract description 28
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
Abstract
Description
본 발명은 질소 고정능을 갖는 혼합 균주, 이를 포함하는 미생물 제제 및 상기 미생물 제제를 포함하는 생물 비료에 관한 것으로, 보다 상세하게는 세데세아 라파게이(Cedecea lapagei) 균주 및 엔테로박터 루드위기(Enterobacter ludwigii)의 혼합 균주를 이용한 기술에 관한 것이다.The present invention relates to a mixed strain having nitrogen fixation ability, a microbial agent containing the microbial agent, and a biological fertilizer including the microbial agent. More particularly, the present invention relates to a Cedecea lapagei strain and an Enterobacter ludwigii ). ≪ / RTI >
세계적으로 지난 수십 년간 곡류, 채소, 과수 등 농작물의 수량을 증가시키기 위하여 해마다 많은 양의 화학 비료와 함께 병, 해충 및 잡초 방제를 위한 화학 농약을 사용해 왔다. 이와 같은 계속적인 화학 비료 및 농약 사용에 의하여 토양 산성화, 지력 손실, 환경오염과 생태계 파괴, 인축독성, 병해충 및 잡초의 농약에 대한 저항성 유발 등의 문제가 지속적으로 제기되고 있다. Globally, over the past decade, we have been using chemical pesticides for disease, pest and weed control, along with a large amount of chemical fertilizer each year to increase the yield of crops such as grains, vegetables, and fruit trees. Such continuous use of chemical fertilizers and pesticides has continuously raised problems such as soil acidification, loss of power, environmental pollution and ecosystem destruction, lethal toxicity, pest resistance and resistance to pesticides in weeds.
국내외에서는 이러한 문제들을 해결하기 위하여 화학 비료 및 농약의 사용량을 감축하고자 노력하고 있으며, 특히 우리나라에서는 1999년 친환경농업 육성법을 제정하여 2013년까지 약 40% 정도의 화학 비료 및 농약 사용량 감축 노력을 계속해오고 있다. 이와 동시에 사회적으로는 생활수준 향상으로 인한 안전 식품에 대한 국민의 요구가 증가되고 있고, 농업 생산 환경 및 자연 생태계의 보존을 지향하는 유기농업이나 자연농법 등의 친환경농업이 급속히 확대되고 있어, 화학 비료 및 농약을 대체할 수 있는 미생물을 이용한 비료 및 생물 농약 개발에 대한 연구가 급격히 증가되고 있다. 이렇게 개발된 미생물제는 대량 배양 후 제제화하여 엽면 살포 또는 입제로 토양 처리함으로써 병해충을 방제하거나 미생물 비료로 이용되기도 한다.In order to solve these problems at home and abroad, efforts are being made to reduce the use of chemical fertilizers and pesticides. In particular, in 1999, the Korean government enacted the Environmentally Friendly Farming Act in order to reduce the use of chemical fertilizers and pesticides by about 40% have. At the same time, the public is increasingly demanding safer food due to the improvement of living standards. Environment-friendly agriculture such as organic farming and natural farming methods that are aiming to preserve agricultural production environment and natural ecosystem are rapidly expanding. And studies on the development of fertilizers and biopesticides using microorganisms that can replace pesticides have been rapidly increasing. The microbial agents thus developed are formulated after large-scale cultivation, and are used as a microbial fertilizer or as a pest control agent by treating the soil with a foliar spray or a granule.
한편, 중요한 식물 영양분인 질소(N)는 화학 비료의 사용에 의해 대부분 유지되며, 질소의 결핍 시 식물 생장에 어려움을 초래한다. 수확량을 높이기 위해 화학 비료를 과잉 이용하는 것은 저투입으로 높은 생산성을 추구하는 지속가능한 농업의 원리에 반하며, 환경적으로도 문제가 될 수 있다(Shantharam, S. and A.K. Mattoo. 1997. Plant Soil 194, 205-216). 화학 비료의 단점이 인식되었음에도 불구하고, 최대 작물 수확에 대한 충분한 유전적 잠재력은 거의 인식되지 않고 있다(Kennedy et al. 2004. Soil Biol . Biochem . 36, 1229-1244). 질소 고정세균 및 고등식물 사이의 복잡한 상호작용 중의 하나인 대기 중 질소를 고정하여 이용하는 생물학적 질소 고정(Biological Nitrogen Fixation, BNF)은 양분공급, 특히 질소의 공급으로 수확량이 최적으로 유지되는 것을 도울 수 있다.On the other hand, nitrogen (N), an important plant nutrient, is largely retained by the use of chemical fertilizers, and it causes difficulties in plant growth when nitrogen is deficient. Over-exploitation of chemical fertilizers to increase yields is contrary to the principle of sustainable agriculture that seeks high productivity with low input, and can also be an environmental problem (Shantharam, S. and AK Mattoo, 1997. Plant Soil 194 , 205-216). Despite the recognition of the shortcomings of chemical fertilizers, there is little known genetic potential for maximum crop harvesting (Kennedy et al., 2004. Soil Biol . Biochem ., 36, 1229-1244). Biological Nitrogen Fixation (BNF) using fixed atmospheric nitrogen, one of the complex interactions between nitrogen-fixing bacteria and higher plants, can help maintain optimum yields with nutrient supply, especially nitrogen supply .
질소 고정 세균은 식물이 이용할 수 있는 암모니아로 대기 중의 N2를 전환하는 능력이 있어 C는 충분하지만 N이 부족한 환경에서 식물 생장을 촉진시키는 능력이 있다. 질소 고정 세균은 식물 생장 및 수확량에 유리한 효과를 나타내는 세균 그룹인 식물 생장 촉진 근권 세균(Plant Growth Promoting Rhizobacteria, PGPR) 하에 위치할 수 있는데, 이는 질소 고정 외에 또 다른 기작에 의해 식물 생장을 촉진하기 때문이다. 질소 비료의 사용을 줄이는 것 외에 식물과 질소 고정 세균의 상호작용을 이용하는 것은 사용된 비료의 효율을 증가시킬 수 있다(Okon, Y. and C.A. Labandera-Gonzolez. 1994. Soil Biol . Biochem . 26, 1591-1601). Nitrogen-fixing bacteria are capable of promoting plant growth in environments where nitrogen is not available, although C is sufficient because plants have the ability to convert N 2 in the atmosphere to ammonia available. Nitrogen-fixing bacteria can be placed under the Plant Growth Promoting Rhizobacteria (PGPR), a group of bacteria that has beneficial effects on plant growth and yield, because it promotes plant growth by another mechanism besides nitrogen fixation to be. In addition to reducing the use of nitrogen fertilizer using the interaction of plants and nitrogen-fixing bacteria, it can increase the efficiency of fertilizer use (Okon, Y. and CA Labandera- Gonzolez. 1994. Soil Biol. Biochem. 26, 1591 -1601).
한편, 저관리 경량형 도시 녹화 시스템 연구에 있어서 가장 고려해야 할 사항은 경량형 소재의 개발과 효율적인 식물 재배를 위해 필요한 영양분을 공급해 주는 것이므로, 효과적인 질소고정 혼합 균주가 제공되는 경우 옥상녹화에서 저관리 경량형 시스템의 효율의 증대를 도출할 수 있을 것으로 기대된다. On the other hand, the most important consideration in the study of low-management lightweight urban greening system is that it provides the necessary nutrients for the development of lightweight materials and efficient plant cultivation. Therefore, It is expected that the efficiency of the system will be increased.
본 발명의 일 측면은, 화학제를 이용하지 않아 화학 농약 및 화학 비료 사용으로 인한 환경 오염의 발생을 줄이고, 친환경 식물 재배 및 농작물 재배가 가능한 질소 고정능을 갖는 혼합 균주를 제공하는 것이다. One aspect of the present invention is to provide a mixed strain having nitrogen fixing ability capable of reducing the occurrence of environmental pollution due to the use of chemical pesticides and chemical fertilizers without using chemicals, and cultivating environmentally friendly plants and cultivating crops.
본 발명의 다른 측면은 이를 포함하는 미생물 제제를 제공하는 것이다.Another aspect of the present invention is to provide a microorganism preparation containing the same.
본 발명의 또 다른 측면은 이를 포함하는 생물 비료를 제공하는 것을 목적으로 한다. Another aspect of the present invention is to provide a biocidal fertilizer containing the same.
본 발명의 일 견지에 의하면, 세데세아 라파게이(Cedecea lapagei) 균주 및 엔테로박터 루드위기(Enterobacter ludwigii) 균주로 이루어진, 질소 고정능을 갖는 혼합 균주가 제공된다. According to one aspect of the present invention, there is provided a mixed strain having nitrogen fixability, which comprises a strain of Cedecea lapagei and an strain of Enterobacter ludwigii.
본 발명의 다른 견지에 의하면, 질소 고정능을 갖는 세데세아 라파게이(Cedecealapagei) 균주 및 엔테로박터 루드위기(Enterobacter ludwigii) 균주의 혼합 균주, 또는 이의 배양물을 유효 성분으로 포함하는, 미생물 제제가 제공된다. According to another aspect of the present invention, there is provided a microbial agent comprising, as an active ingredient, a mixed strain of a strain of Cedecealapagei having nitrogen fixing ability and an Enterobacter ludwigii strain, or a culture thereof do.
본 발명의 또 다른 견지에 의하면, 상기 미생물 제제를 포함하는 생물 비료가 제공된다.According to another aspect of the present invention, there is provided a biological fertilizer comprising the microbial agent.
본 발명의 질소 고정능을 갖는 혼합 균주는 고효율의 질소 고정능을 나타냄으로써, 화학 비료 등을 대신하여 생물 비료로 활용이 가능하며, 따라서 친환경 식물 재배 및 농작물의 재배가 가능하고, 나아가 옥상 녹화에 있어서 저관리 경량형 시스템의 효율을 증대시킬 수 있다. The nitrogen-fixing ability of the mixed strain of the present invention exhibits a high efficiency of nitrogen fixation so that it can be used as a biomass fertilizer in place of a chemical fertilizer. Therefore, it is possible to cultivate environment-friendly plants and cultivate crops, Thus increasing the efficiency of the low management lightweight system.
도 1은 아세틸렌 환원법분석 결과로, 5일차에 GC-FID를 이용하여 에틸렌(ethylene) 생성여부를 확인한 결과를 나타낸 것이다.
도 2는 식물 성장 촉진 평가 결과를 나타낸 것이다. FIG. 1 shows the result of confirming ethylene production using GC-FID on the 5th day as a result of acetylene reduction analysis.
Fig. 2 shows the evaluation result of plant growth promotion.
이하, 본 발명의 바람직한 실시 형태들을 설명한다. 그러나, 본 발명의 실시형태는 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 이하 설명하는 실시 형태로 한정되는 것은 아니다. 또한, 본 발명의 실시형태는 당해 기술분야에서 평균적인 지식을 가진 자에게 본 발명을 더욱 완전하게 설명하기 위해서 제공되는 것이다.Hereinafter, preferred embodiments of the present invention will be described. However, the embodiments of the present invention can be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below. Further, the embodiments of the present invention are provided to more fully explain the present invention to those skilled in the art.
본 발명에 의하면, 세데세아 라파게이(Cedecea lapagei) 균주 및 엔테로박터 루드위기(Enterobacter ludwigii) 균주로 이루어진, 질소 고정능을 갖는 혼합 균주가 제공된다. According to the present invention, there is provided a mixed strain having nitrogen fixability, which comprises a strain of Cedecea lapagei and an strain of Enterobacter ludwigii.
이때, 상기 세데세아 라파게이(Cedecea lapagei) 균주는 국립농업과학원 농업유전자원센터에 수탁번호 KACC 81003BP로 기탁된, 세데세아 라파게이(Cedecea lapagei) MK7 균주(KACC 81003BP)인 것이 바람직하며, 또한, 상기 엔테로박터 루드위기(Enterobacter ludwigii) 균주는 국립농업과학원 농업유전자원센터에 수탁번호 KACC92184P 로 기탁된, 엔테로박터 루드위기(Enterobacter ludwigii) Y8 균주(KACC92184P)인 것이 바람직하다. At this time, the above-mentioned Cedecea lapagei) strain, the sede Seah Rapa Gay (Cedecea lapagei), it is preferable in MK7 strain (KACC 81003BP), also, the Enterobacter Ruud crisis (Enterobacter ludwigii deposited as accession No. KACC 81003BP in agricultural genetic Center National Academy of Agricultural Sciences) The strain is preferably Enterobacter ludwigii strain Y8 (KACC92184P) deposited under accession number KACC92184P in the National Institute of Agricultural Science and Technology.
본 발명의 상기 세데세아 라파게이(Cedecea lapagei) 균주 및 엔테로박터 루드위기(Enterobacter ludwigii) 균주는 질소 고정을 통해 식물체에 양분 공급, 특히 질소의 공급으로 수확량이 최적으로 유지되는 것을 도울 수 있는 것으로, 질소 고정능을 통해 대기 중의 질소(N2)를 암모니아로 전환할 수 있어 탄소원(C)은 충분하지만 질소원(N)이 부족한 환경에서도 식물의 생장을 촉진시킬 수 있다. The above-mentioned Cedecea lapagei strain and Enterobacter ludwigii strain can help nutrient supply to the plant through nitrogen fixation, especially nitrogen supply, to optimize the yield and nitrogen nitrogen (N 2 ) can be converted to ammonia, so that the carbon source (C) is sufficient, but the plant growth can be promoted even in an environment where the nitrogen source (N) is insufficient.
특히 본 발명의 세데세아 라파게이(Cedecea lapagei) 균주 및 엔테로박터 루드위기(Enterobacter ludwigii) 균주의 혼합 균주는 이와 같은 식물의 생장을 촉진 효과를 현저하게 증대시킬 수 있으며, 그 결과 옥상녹화에서 저관리 경량형 시스템의 효율의 증대를 도출할 수 있다.In particular, the Cedecea lapagei strains and Enterobacter ludwigii strains can significantly enhance the growth of such plants and result in an increase in the efficiency of the low control lightweight system in rooftop greening .
구체적으로, 상기 세데세아 라파게이(Cedecea lapagei) MK7 균주(KACC 81003BP) 및 엔테로박터 루드위기(Enterobacter ludwigii) 균주는, 질소 고정능을 나타내는 유전자로 NifH 유전자를 포함하는 것이 바람직하고, 이로부터 질소 고정이 가능한 균주인 것이 바람직하다.Specifically, the above-mentioned Cedecea lapagei ) MK7 strain (KACC 81003BP) and Enterobacter ludwigii strain preferably contain NifH gene as a gene showing nitrogen fixation ability, and it is preferable that it is a strain capable of fixing nitrogen.
본 발명의 다른 견지에 의하면 질소 고정능을 갖는 세데세아 라파게이(Cedecealapagei) 균주 및 엔테로박터 루드위기(Enterobacter ludwigii) 균주의 혼합 균주, 또는 이의 배양물을 유효 성분으로 포함하는, 미생물 제제가 제공된다.According to another aspect of the present invention, there is provided a microbial agent comprising, as an active ingredient, a mixed strain of Cedecealapagei strain and Enterobacter ludwigii strain having nitrogen fixation ability, or a culture thereof .
이때, 상기 세데세아 라파게이(Cedecea lapagei) 균주는 국립농업과학원 농업유전자원센터에 수탁번호 KACC 81003BP로 기탁된, 세데세아 라파게이(Cedecea lapagei) MK7 균주(KACC 81003BP)인 것이 바람직하며, 또한, 상기 엔테로박터 루드위기(Enterobacter ludwigii) 균주는 국립농업과학원 농업유전자원센터에 수탁번호 KACC KACC92184P 로 기탁된, 엔테로박터 루드위기(Enterobacter ludwigii) Y8 균주(KACC92184P)인 것이 바람직하다. At this time, the above-mentioned Cedecea lapagei) strain, the sede Seah Rapa Gay (Cedecea lapagei), it is preferable in MK7 strain (KACC 81003BP), also, the Enterobacter Ruud crisis (Enterobacter ludwigii deposited as accession No. KACC 81003BP in agricultural genetic Center National Academy of Agricultural Sciences) The strain is preferably Enterobacter ludwigii strain Y8 (KACC92184P) deposited with the National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology, under accession number KACC K91184P.
또한, 상기 미생물 제제는, 화학 비료를 대체하기 위한 생물 비료 등에 적용 가능한 것으로, 생물 비료용 미생물 제제인 것이 바람직하고, 상기 미생물 제제는 통상적인 방법으로 제형화할 수 있으며, 예를 들어 건조분말 형태 또는 액상비료 형태로 제조할 수 있다. 그러나 그 제형에 특별히 한정되지는 않는다. In addition, the microorganism preparation is preferably a microorganism preparation for biological fertilizer that can be applied to biological fertilizer to replace chemical fertilizer, and the microorganism preparation can be formulated in a conventional manner, for example, in the form of a dry powder It can be produced in liquid fertilizer form. However, the formulation is not particularly limited.
상기 액상비료 형태의 경우, 토양에 뿌려서 토양과 혼합하여 사용가능하고, 건조분말 형태의 경우 비료 형태로, 토양에 뿌려 사용이 가능하다. 또한 상기 미생물 제제는 담체, 부식토 등을 혼합하여 사용할 수 있으나, 이에 한정되는 것은 아니다.In the case of the liquid fertilizer type, it can be used by being mixed with the soil by spraying on the soil, and in the form of a dry powder, it can be sprayed on the soil in the form of fertilizer. In addition, the microbial agent may be mixed with a carrier, a corrosive soil, and the like, but is not limited thereto.
한편, 상기 미생물 제제는 다양한 식물에 적용되어, 생물 비료로 활용 가능하며, 이때 상기 식물은 예를 들어 잔디, 옥수수, 당근, 양배추, 감자 등으로, 상기 미생물 제제를 적용함으로써, 화학 비료 등의 사용 없이 친환경적 농법에 의하여 대기 중의 질소를 고정하여 암모니아성 질소를 식물체에 공급가능하며, 수확물의 양을 증가시킬 수 있고, 잔디 등의 식물 생장을 촉진시킬 수 있다. On the other hand, the microorganism preparation is applied to various plants and can be utilized as a biomass fertilizer. In this case, the plant can be used as a fertilizer, for example, by applying the microorganism preparation such as grass, corn, carrot, cabbage, potato, It is possible to supply ammonia nitrogen to the plant by fixing the nitrogen in the atmosphere by the environmentally friendly farming method, to increase the amount of the harvest, and to promote the growth of plants such as lawn.
본 발명의 다른 견지에 의하면, 상기 본 발명의 미생물 제제를 포함하는 생물 비료를 제공한다. 이때, 상기 세데세아 라파게이(Cedecea lapagei) 균주는 국립농업과학원 농업유전자원센터에 수탁번호 KACC 81003BP로 기탁된, 세데세아 라파게이(Cedecea lapagei) MK7 균주(KACC 81003BP)인 것이 바람직하며, 또한, 상기 엔테로박터 루드위기(Enterobacter ludwigii) 균주는 국립농업과학원 농업유전자원센터에 수탁번호 KACC KACC92184P 로 기탁된, 엔테로박터 루드위기(Enterobacter ludwigii) Y8 균주(KACC92184P)인 것이 바람직하며, 상기 혼합 균주의 우수한 잘소 고정능에 의하여 대기 중의 질소를 고정하여 암모니아성 질소를 식물체에 공급하여 생장을 촉진시킬 수 있다.According to another aspect of the present invention, there is provided a biological fertilizer comprising the microbial agent of the present invention. At this time, the above-mentioned Cedecea lapagei) strain, the sede Seah Rapa Gay (Cedecea lapagei), it is preferable in MK7 strain (KACC 81003BP), also, the Enterobacter Ruud crisis (Enterobacter ludwigii deposited as accession No. KACC 81003BP in agricultural genetic Center National Academy of Agricultural Sciences) The strain is preferably Enterobacter ludwigii Y8 strain (KACC92184P) deposited with Accession No. KACC92184P at the National Institute of Agricultural Science and Technology, Center for Agricultural Genetic Resources, and the nitrogen of the atmosphere is fixed And ammonia nitrogen is supplied to the plant to promote the growth.
이하, 본 발명을 실시예에 의거하여 구체적으로 설명하는 바, 본 발명이 다음 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited by the following Examples.
실시예Example
1. 미생물 시료 채취 및 균주 분리1. Microbial sample collection and strain isolation
문경의 부식토와 연천의 농지에서 토양을 채취하여, 토양 1g을 이용해 시리얼 다일루션(serial dilution) 기법과 평면도말법에 의해 균들을 분리하고, 이들 균들을 질소원이 소량 첨가된 배지(mannitol 5.0 g/L, malic acid 3.2 g/L, KH2PO4 3.0 g/L, monosodium glutamate 0.1 g/L, MgSO4 0.1 g/L CaCl2 2H2O 0.005 g/L, H3BO3 0.15 g/L, FeSO4 7H2O 0.13 g/L, Na2MoO4 2H2O 0.013 g/L, ZnSO4 7H2O 0.11 g/L, CoSO4 7H2O 5 mg/L, Cuso4 2H2O 4 mg/L, MnCl2 4H2O 4 mg/L, inositol 0.12 mg/L, riboflavin 0.02 mg/L, p-amino benzoic acid 0.02 mg/L, nicotinic acid 0.02 mg/L, biotin 0.2 mg/L, thiamine HCl 0.02 mg/L, pyridoxine HCL 0.02 mg/L, calcium pantothenate 0.02 mg/L,)에서 점액질이 형성되고 다른 균들에 비해 높은 성장을 보인 58개의 균과 환경미생물은행(KEMB)에서 분양 받은 44개의 균을 도 1에 개시된 리스트와 같이 확인하였다. 도 1에서 R1 내지 L20-1은 환경미생물은행(KEMB)에서 분양받은 균을 이용한 결과를 나타낸 것이고, MK4 내지 Y12는 토양에서 분리된 균을 이용한 결과를 나타낸 것이다. Soils were collected from Munkyeong 's soil and Yeoncheon' s farmland, and the bacterial isolates were separated by serial dilution method and planarization method using 1 g of soil. These bacteria were added to a medium supplemented with a small amount of nitrogen source (mannitol 5.0 g / L , malic acid 3.2 g / L, KH2PO4 3.0 g / L, monosodium glutamate 0.1 g / L, MgSO4 0.1 g / L CaCl2 2H2O 0.005 g / L, H3BO3 0.15 g / L, FeSO4 7H2O 0.13 g / L, Na2MoO4 2H2O 0.013 g / L, ZnSO4 7H2O 0.11 g / L, CoSO4 7H2O 5 mg / L, Cuso4 2H2O 4 mg / L, MnCl2 4H2O 4 mg / L, inositol 0.12 mg / L, riboflavin 0.02 mg / L, p-amino benzoic acid 0.02 mg L, nicotinic acid 0.02 mg / L, biotin 0.2 mg / L, thiamine HCl 0.02 mg / L, pyridoxine HCL 0.02 mg / L and calcium pantothenate 0.02 mg / Of the 58 isolates and 44 isolates from the Environmental Microbial Bank (KEMB) As shown in the list shown in Fig. In FIG. 1, R1 to L20-1 show the results of using the microorganisms cultured in the environmental microbial bank (KEMB), and MK4 to Y12 show the results of using the bacteria isolated from the soil.
2. 아세틸렌 환원법분석(Acetylene reduction assay, 2. Acetylene reduction assay (Acetylene reduction assay, ARAARA ))
질소원이 소량 첨가된 배지에서 높은 성장을 보인 상기와 같은 102 개의 균들의 질소고정 능력을 확인하기 위해 아세틸렌 환원법분석(Acetylene reduction assay, ARA)을 수행하여 니트로게나아제(nitrogenase)의 활성을 간접적으로 확인하였다. Acetylene reduction assay (ARA) was performed to confirm the nitrogen fixation ability of the above-mentioned 102 bacteria which showed high growth in a medium containing a small amount of nitrogen source to indirectly confirm the activity of nitrogenase Respectively.
상기 아세틸렌 환원법은 질소 고정 미생물이 공통적으로 가지고 있고, N2 가스 또는 아세틸렌과 같은 3중 결합을 가진 물질을 환원시킬 수 있는 효소인 질소 고정효소(nitrogenase)의 활성을 간접적으로 측정하는 방법으로 질소 고정효소에 의해 아세틸렌이 에틸렌으로 환원되었을 때 생성되는 에틸렌의 양을 측정함으로써 질소 고정능을 측정할 수 있다.The acetylene reduction method is a method of indirectly measuring the activity of a nitrogenase, which is an enzyme capable of reducing a substance having a triple bond such as N 2 gas or acetylene, commonly having nitrogen fixing microorganisms, The nitrogen fixation ability can be measured by measuring the amount of ethylene produced when acetylene is reduced to ethylene by the enzyme.
상기 아세틸렌 및 에틸렌의 변화량은 GC-FID(Gas Chromatography-Flame Ionization Detector)로 측정하였으며, 이때 상기 GC-FID의 조건은 하기 표 1과 같다.The amount of change of acetylene and ethylene was measured by a GC-FID (Gas Chromatography-Flame Ionization Detector). The conditions of the GC-FID were as shown in Table 1 below.
ARA는 배양용기 (serum bottle)를 이용하여 수행하였다. 상기 배양용기 (serum bottle) 내에 상기 질소가 소량 함유된 배지 100ml을 넣고, 알루미늄 씰(aluminum seal)과 고무 캡(cap)을 이용해 닫힌계를 만들었다. ARA was performed using a serum bottle. 100 ml of a medium containing a small amount of the nitrogen was placed in the serum bottle, and a closed system was made using an aluminum seal and a rubber cap.
고무 캡을 이용해 배양용기 (serum bottle) 내의 기상부를 99% 질소와 1% 산소로 조성하고, 상기 1.에서 분리하여 획득된 균을 각각 1 g/L 양으로 접종하였다. 상기 균을 2일간 28℃에서 적응시킨 후, 적응된 균이 들어있는 배양용기 (serum bottle)의 기상부를 89% 아르곤과 10% 아세틸렌 및 1% 산소로 조정하였다.The gaseous phase in the serum bottle was composed of 99% nitrogen and 1% oxygen using a rubber cap, and the bacteria obtained in the above step 1 were each inoculated in an amount of 1 g / L. The bacteria were adapted for 2 days at 28 ° C and the gaseous phase of the serum bottle containing the adapted bacteria was adjusted to 89% argon, 10% acetylene and 1% oxygen.
그 후 0일, 3일, 5일에 각각 GC-FID를 이용하여 에틸렌(ethylene) 생성여부를 하기와 같이 확인하였다. 그 결과, 5일차에 확인한 결과를 도 1에 나타내었으며, 이 중 아세틸렌(acetylene)을 에틸렌(ethylene)으로 환원시킨 8개의 균주, L20, MK4, MK7, Y4, Y5, Y6, Y7, Y8을 확인하였으며, 그 중 MK7과 Y4, Y8 균주가 아세틸렌(acetylene)을 대부분 에틸렌(ethylene)으로 환원시킨 것으로 확인하였다.Then, on the 0th day, the 3rd day and the 5th day, the generation of ethylene was confirmed using GC-FID as follows. As a result, the results confirmed on the 5th day are shown in FIG. 1, and eight strains, L20, MK4, MK7, Y4, Y5, Y6, Y7 and Y8, in which acetylene was reduced with ethylene, Among them, MK7, Y4 and Y8 strains were found to have reduced acetylene to ethylene.
이에, 상기 3개의 균주 MK7과 Y4, Y8에 대하여 재현성 실험을 실시 하였다. 재현성 실험은 상기 ARA를 통해 수행하였다. 그 결과, 재현성 실험에서 MK7과 Y8 균주에서 지속적인 니트로게나아제 활성을 확인할 수 있었고, 따라서 질소고정 균주로 MK7 및 Y8 균주를 선별하였다. Reproducibility tests were performed on the three strains MK7, Y4, and Y8. Reproducibility experiments were conducted through the ARA Respectively. As a result, in the reproducibility experiment, it was confirmed that MK7 and Y8 strains had constant nitergenase activity, and thus MK7 and Y8 strains were selected as nitrogen fixing strains.
하기 표 2에 나타난 바와 같이 스트레인(Strain) MK7 균주는 가스크로마토그래피(gas chromatography)를 이용한 검출에서 초기 기상층의 아세틸렌 농도는 9.3%이었지만, 5일차에는 아세틸렌이 검출되지 않았고, 에틸렌은 3.9 %로 검출되었다. As shown in the following Table 2, in the strain MK7, the concentration of acetylene in the initial gas phase was 9.3% in detection by gas chromatography, but acetylene was not detected on the 5th day, and ethylene was 3.9% Respectively.
스트레인(Strain) Y8 균주는 초기 기상부의 아세틸렌 농도 10.6 %에서 5일차에는 1.7 %로 감소하였으며, 에틸렌은 3.8 %로 검출되었다. 이때, 아세틸렌이 약 9 % 감소한 것에 비해 에틸렌 생성은 약 3.8 %로 낮게 검출되었다. 이는 닫힌계에서 균의 작용에 의해 용기 내부의 압력이 증가한 결과로 보인다. 하지만 아세틸렌은 검출되지 않았으므로 내부의 아세틸렌을 약 100 % 환원시킨 것으로 볼 수 있다.Strain Y8 strain decreased from 10.6% in the initial gaseous phase to 1.7% in the fifth day and ethylene was detected in 3.8%. At this time, ethylene production was found to be as low as about 3.8% compared to about 9% reduction in acetylene. This appears to be the result of increased pressure inside the container due to the action of bacteria in the closed system. However, since acetylene was not detected, it can be seen that the acetylene inside was reduced to about 100%.
* 이때 컨트롤은 균을 접종하지 않은 배양용기를 이용한 것이다. * At this time, the control is based on a culture vessel not inoculated with the bacteria.
3. 3. nifHnifH 유전자의 Gene PCRPCR 증폭 및 16s Amplification and 16s rDNArDNA 서열 분석 Sequencing
상기 2.의 ARA 분석을 통해 니트로게나제의 활성이 확인된 균주들의 nifH 유전자를 확인하기 위해 Exgene cell SV mini kit(GeneAll)를 이용하여 게노믹(genomic) DNA를 추출했다. Genomic DNA was extracted using the Exgene cell SV mini kit (GeneAll) to identify the nifH gene of the strains in which the activity of the nitrgenase was confirmed by the ARA analysis of the above 2.
유니버셜 프라이머(Universal primer)는 다양한 균이나 환경 시료에서 nifH를 증폭시킬 수 있는 것으로 본 실험에서 사용한 프라이머는 하기 표 3에 나타난 프라이머 정보와 같이 번호 6 및 7 의 유니버셜 프라이머와 NCBI에 등록된 특정 질소고정 미생물의 nifH 유전자 서열을 이용하여 제작된 프라이머를 사용했다. 제작된 프라이머 정보는 하기 표 3에 번호 1~5로 나타냈다. 하기 표 3의 각 프라이머는 정방향과 역방향 순으로 2가지 염기서열 정보를 나타냈다.The universal primer can amplify nifH in a variety of bacterial or environmental samples. The primers used in this experiment are the universal primers of
PCR 반응 조건은 94℃에서 30초, 50℃에서 30초, 72℃에서 30초로 30 사이클이다. PCR 혼합물(mixture)은 템플레이트(template), 프라이머(primer), dNTP, 10 X 버퍼(buffer), Taq 폴리머라제(polymerase)가 포함되어 있고, 증류수를 이용하여 총 용량을 30ul로 맞추었다. The PCR reaction conditions are 30 cycles at 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 30 seconds. The PCR mixture contained a template, primer, dNTP, 10 X buffer, and Taq polymerase, and the total volume was adjusted to 30 ul using distilled water.
PCR 생성물(product)은 전기영동법을 이용하여 각 프라이머에 의해서 생성되는 360bp의 밴드를 확인했다. 확인된 밴드는 겔에서 분리되어 용리(elution)하고, MICROGEN에 보내 서열을 분석하였다. The PCR product confirmed the 360 bp band generated by each primer using electrophoresis. The identified bands were separated from the gel, eluted and sent to MICROGEN for sequencing.
한편, 16s rDNA는 3일 배양된 균이 있는 한천 배지를 MICROGEN에 보내 서열을 확인했다.On the other hand, 16s rDNA was sequenced for three days and sent to MICROGEN.
16s rDNA 시퀀싱(sequencing) 결과를 통해 스트레인(strain) MK7과 Y8을 동정했고 각각 세데세아 라파게이(Cedecea lapagei) GTC 346 (98.21 %)과 엔테로박터 루드위기(Enterobacter ludwigii) EN-119 (99.73 %)로 동정되었다.Strain MK7 and Y8 were identified through 16s rDNA sequencing results. Cedecea lapagei GTC 346 (98.21%) and Enterobacter ludwigii EN-119 (99.73%) were identified, Respectively.
스트레인(strain) MK7과 Y8에서 nifH 유전자의 서열을 확인하기 위해 상기 표 3에 개시된 7개의 nifH 프라이머를 이용하여 PCR 기법으로 통해 확인했다. 이 연구에서 제작된 프라이머 1~5를 이용한 실험에서는 원하는 크기의 PCR 생성물을 얻지 못했다. 반면, 유니버셜 프라이머6 및 7을 이용한 PCR에서는, Pol 프라이머를 이용한 PCR 수행에서 스트레인(strain) Y8에서 약 350 bp의 PCR 생성물을 얻어 시퀀싱을 실시했다. 그 결과 엔테로박터 루드위기(Enterobacter ludwigii) EN-119의 전체 게놈 중 일부분으로 나타났다. 한편, 다른 유니버셜 프라이머인 Zehr 프리이머(상기 표 3의 번호 7)를 사용한 경우 약 300bp의 PCR 밴드 생성을 확인했다. 이와 같이 상기 각 프라이머를 이용하였고, 결과는 번호 7번의 프라이머를 이용하여 확인하였다.To confirm the sequence of the nifH gene in strain MK7 and Y8, PCR was performed using 7 nifH primers shown in Table 3 above. In the experiment using the primers 1 to 5 prepared in this study, PCR product of the desired size was not obtained. On the other hand, in PCR using
4. 식물 성장 촉진 평가 4. Assessment of Plant Growth Promotion
우수한 질소고정 박테리아인 세데세아 라파게이(Cedecea lapagei) MK7 균주(KACC 81003BP) 및 엔테로박터 루드위기(Enterobacter ludwigii) Y8 균주(KACC92184P)를 각각 동일 비율로 혼합하여 혼합 균주를 만들고 TSB(Tryptic soy broth)에 3일간 28에서 배양한 후, 원심분리기를 이용하여 배지 성분을 제거하였다. A mixed strain was prepared by mixing Cedecea lapagei strain MK7 (KACC 81003BP) and Enterobacter ludwigii Y8 strain (KACC92184P), which are excellent nitrogen fixing bacteria, in the same ratio, respectively, and TSB (Tryptic soy broth) At 28 for 3 days, and then the medium was removed using a centrifuge.
경기도 수원시의 농지에서 채취된 토양을 플라스틱 포트에 150g씩 담고, 상기 준비된 혼합 균주를 증류수에 부유시킨 후, 상기 토양에 1.0 g bacteria / kg soil의 농도로 접종하였다(실시예 1). 그 후 페레니얼라이그래스(perennial ryegrass)를 포트 당 씨드(seed)는 3개씩 파종하였다. 균을 접종하지 않고 같은 양의 증류수를 첨가한 포트의 식물을 대조군(비교예 1)으로 하였고, 양성 대조군(positive control)으로 광합성 혼합 균주(Rhodobacter capsulate, Rhodococcus qingshengii, Achromobacter marplatensis)를 이용했다(비교예 2). 150 g of the soil collected from the farmland of Suwon city in Gyeonggi-do was suspended in distilled water, and the soil was inoculated at a concentration of 1.0 g bacteria / kg soil (Example 1). Then perennial ryegrass was seeded with three seeds per pot. Rhodobacter capsulor (Rhodococcus qingshengii, Achromobacter marplatensis) was used as a positive control for the plants of the pots to which the same amount of distilled water was added without inoculating the bacteria (Comparative Example 1) Example 2).
상기 각 실험군은 4개의 포트를 사용했고, 모든 실험은 같은 조건에서 실시했다. 토양이 건조되는 것을 방지하기 위해 증류수를 일일 15ml씩 주었다. 50일 배양 후 식물의 지상부의 길이와 식물의 건조 중량을 측정함으로써 식물 성장을 확인했다. Each experimental group used 4 ports and all experiments were performed under the same conditions. To prevent the soil from drying, 15 ml of distilled water was given daily. After 50 days of cultivation, plant growth was confirmed by measuring the length of the top part of the plant and the dry weight of the plant.
식물의 지상부의 길이를 확인한 결과 각 경우에서 모두 첫 번째 잎의 성장에서 차이가 없었다. 두 번째 잎의 경우 증류수를 첨가한 포트(비교예 1)와 광합성 혼합균주를 첨가한 포트(비교예 2)에서는 별 차이를 보이지 않았고, 본 발명의 질소고정 혼합 균주를 이용한 포트(실시예 1)에서 식물에 균을 접종하지 않은 증류수(비교예 1)에 비해 약 120 % 길이 성장을 확인하였다. 한편, 증류수를 이용한 포트의 경우 세 번째 잎에서 성장 속도가 다른 실험군에 비해 낮은 것을 확인했고, 광합성 혼합 균주와 질소고정 혼합 균주의 경우 대조군(비교예 1)에 비해 각각 약 136 %, 142 % 높은 성장률을 보인 것을 확인했다. 49일 차까지 증류수를 첨가한 포트에서는 더 이상의 잎 발생이 나타나지 않았고, 광합성 혼합 균주, 질소고정 혼합 균주에서 5번째 잎까지 발생했다. 이와 같은 결과를 하기 표 4 및 도 2(a)에 나타내었다.The length of the top part of the plant was not different from the growth of the first leaf in each case. In the case of the second leaf, no difference was observed between the port to which the distilled water was added (Comparative Example 1) and the port to which the photosynthetic mixed strain was added (Comparative Example 2), and the port using the nitrogen fixed mixed strain of the present invention (Example 1) The growth was about 120% longer than that of the distilled water not inoculated with the plant (Comparative Example 1). On the other hand, in the case of the pot using the distilled water, the growth rate in the third leaf was lower than that in the other experimental groups. In the case of the photosynthetic mixed strain and the nitrogen fixed mixed strain, 136% and 142% Growth rate. In the pot with distilled water until the 49th day, no further leaf emergence occurred, and the 5th leaf occurred in the photosynthetic mixed strain and nitrogen fixed mixed strain. These results are shown in Table 4 and FIG. 2 (a).
나아가, 파종 49일 후, 식물을 뽑아서 줄기의 길이와 건조 중량을 측정했다. 대조군(Control)인 비교예 1에서 줄기의 길이는 18.53 cm로 측정되었다. 질소고정 균을 접종한 실험군(실시예 1)에서는 22.06 cm로 측정되어, 대조군에 비해 약 123.5 % 높은 성장률을 보였다. 광합성 균을 접종한 실험군(비교예 2)에서는 21.8 cm로 되어, 대조군에 비해 약 116 %의 높은 성장을 보였다. 그 결과 본 발명의 질소고정 균을 접종한 실험군에서 가장 높은 줄기 성장률을 보인 것으로 확인되었다. Further, after 49 days of sowing, the plants were pulled and the length and dry weight of the stem were measured. In Comparative Example 1, which is a control (Control), the length of the stem was measured as 18.53 cm. In the experimental group (Example 1) inoculated with the nitrogen fixative bacteria, the growth rate was about 123.5% higher than that of the control group as measured at 22.06 cm. In the experimental group (Comparative Example 2) inoculated with photosynthetic bacteria, the growth was 21.8 cm, which was about 116% higher than that of the control group. As a result, it was confirmed that the highest stem growth rate was observed in the test group inoculated with the nitrogen fixing bacteria of the present invention.
한편, 식물의 건조 중량은 도 2(b)에 나타난 바와 같이 대조군(Control)인 비교예 1에서 18.65 mg으로 측정되었고, 질소고정 균을 접종한 실험군(실시예 1)에서는 34.80 mg으로 확인되어, 대조군에 비해 186.6 % 증가한 것으로 확인하였다. 광합성 균을 접종한 실험군(비교예 2)에서는 25.31 mg으로 확인되어 대조군에 비해 약 135.7 %의 증가한 결과를 나타냈다. On the other hand, the dry weight of the plant was determined to be 18.65 mg in Comparative Example 1, which is a control group as shown in Fig. 2 (b), and 34.80 mg in an experimental group (Example 1) And 186.6% more than the control group. In the experimental group (Comparative Example 2) inoculated with photosynthetic bacteria, it was confirmed to be 25.31 mg, which was about 135.7% higher than that of the control group.
이 발명을 지원한 국가연구개발사업National R & D project supporting this invention
과제고유번호: 14CTAP-C078666-01Assignment number: 14CTAP-C078666-01
부처명: 국토교통부Department name: Ministry of Land, Transport and Maritime Affairs
연구관리전문기관: 국토교통과학기술진흥원Research Management Agency: Ministry of Land Transportation Science and Technology Promotion Agency
연구사업명: 국토교통기술촉진연구사업Research Project Name: Land Transport Technology Promotion Research Project
연구과제명: 비중이 0.3이하인 자체 식물 생장환경을 갖는 바이오 테라콘(Terra-con) 인공토양 골재개발Research title: Development of terra-con artificial soil aggregate with its own plant growth environment with specific gravity of 0.3 or less
기여율: 80/100Contribution rate: 80/100
주관기관: 경기대학교Host organization: Kyonggi University
연구기간: 2014.08.07 ~ 2017.08.06Research period: 2014.08.07 ~ 2017.08.06
과제고유번호: NRF-2017M3A9B8065734Assignment number: NRF-2017M3A9B8065734
부처명: 미래창조과학부Department: Future Creation Science Division
연구관리전문기관: 한국연구재단Research Management Institution: Korea Research Foundation
연구사업명: 원천기술개발사업 바이오 의료기술개발사업 연구소재지원사업Research Project Name: Source Technology Development Project Biomedical Technology Development Project Research Material Support Project
연구과제명: 환경 산업 미생물 및 유전자 보존센터Research Project: Microbiology and gene conservation center for environmental industry
기여율: 20/100Contribution rate: 20/100
주관기관: 경기대학교Host organization: Kyonggi University
연구기간: 2017.07.01 ~ 2022.06.30Period of study: 2017.07.01 ~ 2022.06.30
[수탁번호][Access number]
1. 세데세아 라파게이(Cedecea lapagei) MK7 균주1. Cedecea lapagei strain MK7
기탁기관명 : 국립농업과학원 농업유전자원센터Depositor Name: National Institute of Agricultural Science
수탁번호 : KACC81003BPAccession number: KACC81003BP
수탁일자 : 20150629Checked on: 20150629
2. 엔테로박터 루드위기(Enterobacter ludwigii) Y8 균주2. Enterobacter ludwigii strain Y8
기탁기관명 : 국립농업과학원 농업유전자원센터Depositor Name: National Institute of Agricultural Science
수탁번호 : KACC92184PAccession number: KACC92184P
수탁일자 : 20170726Checked on: 20170726
Claims (10)
A mixed strain having nitrogen fixability, consisting of a strain of Cedecea lapagei and an Enterobacter ludwigii strain.
The mixed strain having nitrogen fixability according to claim 1, wherein the Cedecea lapagei strain is Cedecea lapagei MK7 strain (KACC 81003BP).
The mixed strain having nitrogen fixability according to claim 1, wherein the Enterobacter ludwigii strain is an Enterobacter ludwigii strain Y8 (KACC92184P).
A microorganism preparation comprising, as an active ingredient, a mixed strain of Cedecealapagei strain and Enterobacter ludwigii strain having nitrogen fixing ability, or a culture thereof.
The microbial preparation according to claim 4, wherein the Cedecea lapagei strain is Cedecea lapagei MK7 strain (KACC 81003BP).
5. The microbial preparation according to claim 4, wherein the Enterobacter ludwigii strain is an Enterobacter ludwigii Y8 strain (KACC92184P).
The microbial agent according to claim 4, wherein the microbial agent is a microbial agent for biological fertilizer.
A biological fertilizer comprising the microbial agent according to any one of claims 4 to 7.
The biological fertilizer according to claim 8, wherein the Cedecea lapagei strain is Cedecea lapagei MK7 strain (KACC 81003BP).
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| WO2021171129A1 (en) * | 2020-02-25 | 2021-09-02 | Hendiyani Ali | Multi-functional biofertilizer |
| CN114009449A (en) * | 2021-10-19 | 2022-02-08 | 中国农业科学院油料作物研究所 | Method for increasing number of root nodules with nitrogenase activity on roots of leguminous crops and application of root nodules on leguminous crops |
| KR102516431B1 (en) | 2022-04-14 | 2023-04-03 | 케이지케미칼 주식회사 | Microbial Fertilizer for Carbon Dioxide Reduction and Preparation Method of the Same |
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| CN114009449A (en) * | 2021-10-19 | 2022-02-08 | 中国农业科学院油料作物研究所 | Method for increasing number of root nodules with nitrogenase activity on roots of leguminous crops and application of root nodules on leguminous crops |
| KR102516431B1 (en) | 2022-04-14 | 2023-04-03 | 케이지케미칼 주식회사 | Microbial Fertilizer for Carbon Dioxide Reduction and Preparation Method of the Same |
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