KR101617807B1 - The microbial agent with phosphate-solubilizing fungus for ameliorating soils with excessive salts and manufacturing method thereof - Google Patents
The microbial agent with phosphate-solubilizing fungus for ameliorating soils with excessive salts and manufacturing method thereof Download PDFInfo
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Abstract
본 발명은 인산가용화균주에 효모, 아미노산, 비타민, 무기물, 탄수화물 등의 미생물의 영양원을 배합하여 제조되는 인산가용화 미생물 제제를 특별한 장치 없이 물에 혼합하여 온도만 유지시켜 용이하게 배양하여 얻을 수 있는 배양물을 제공하여 작물의 증수 및 토양개량 효과를 유도할 수 있는 뛰어난 효과가 있다.The present invention relates to a method for producing a phosphate-soluble microbial preparation, which comprises mixing a nutrient source of microorganisms such as yeast, amino acids, vitamins, minerals, and carbohydrates into a phosphoric acid-solubilizing strain, There is an excellent effect that water can be supplied to induce the increase of crops and soil improvement effect.
Description
본 발명은 인산가용화균주를 유효성분으로 함유하는 미생물제제에 관한 것으로, 더욱 상세하게는 바실러스 서브틸리스 AP20131014 (KACC91877P) 균주 배양물의 건조물을 유효성분으로 함유하는 식물 성장촉진 및 토양개량용 첨가제에 관한 것이다.The present invention relates to a microorganism preparation containing a phosphorus-solubilizing strain as an active ingredient, and more particularly to a plant growth promoting and soil-improving agent containing a dried product of Bacillus subtilis AP20131014 (KACC91877P) will be.
식물성장에 있어서 인산은 핵단백질의 구성성분으로 뿌리신장을 좋게 하고 발아나 분얼을 좋게 하여 개화 결실에 중요한 역할을 하는 영양원으로 보통은 무기질 형태의 인산질 비료를 토양에 시용하지만 인산이 토양에 잘 고정되는 특성으로 실제 시비량의 1020% 정도만 식물이 이용하고 나머지는 철(Fe2 +, Fe3 +), 알루미늄(Al3 +), 칼슘(Ca2+)등과 결합하여 Ca3(PO4)2, FePO4, AlPO4 등의 불용성 물질로 토양에 고착화된다. 따라서 작물을 재배할 때마다 인산질 비료를 계속 시비해야 하고 연중 다기작 재배를 하는 토양에서는 상상을 초월하는 함량의 인산이 토양에 축적되고 이로 인해 토양 양분의 불균형 및 작물 생육 불량의 원인으로 작용하여 수량 감소는 물론 품질 저하를 야기한다. 이러한 문제를 해결하기 위해 1950년대부터 러시아와 동유럽에서는 불용성 인산을 가용화시킬 수 있는 미생물을 분리하여 토양에 처리한 결과 평균 10∼30%의 증수 효과를 보았다. In plant growth, phosphoric acid is a constituent of the nuclear protein. It is a nutrient source that plays an important role in flowering defects by improving root elongation and improving digestion and fertilization. Usually phosphoric acid fertilizer is applied to soil, but phosphoric acid is fixed to soil attribute is only about 1020% of the actual plant to be used as a fertilizer and the rest iron (Fe 2 +, Fe 3 + ), aluminum (Al 3 +), calcium Ca 3 (PO 4) in combination as the (Ca 2+) 2, FePO 4 , AlPO 4, and so on. Therefore, phosphate fertilizer must be continuously fertilized every time the crop is cultivated. In the soil cultivated all over the country during the year, phosphoric acid in the amount exceeding the imagination accumulates in the soil, which causes imbalance of soil nutrients and poor crop growth. Decreases as well as quality degradation. In order to solve these problems, microorganisms capable of solubilizing insoluble phosphoric acid were isolated and treated in soil from Russia and Eastern Europe in the 1950s.
그동안 한국공개특허 2002-0017516 "화산회토에 고정된 인산염을 가용화하는 새로운 미생물", 한국공개특허 2005-0100109 "신규한 바실러스 속 균주, 버크홀데리아 속 균주 및 상기미생물을 이용한 토양 미생물 처리제", 한국공개특허 2007-0036879 "난용성 인산 분해능을 가지는 아스퍼질러스 나이저 및 이를 함유하는 미생물제제", 한국공개특허 2011-0079083 "작물생육을 촉진하는 신규 락토바실러스 파라케피리 NAAS-1균주", 한국공개특허 2011-0102787 "식물생장 촉진 활성을 갖는 신균주 바실러스 아리아바타이 LS9 및 이의 용도" 및 EP 2 154 239 A1, WO 2010/109408 A1(PCT/IB2010/051249), US 2002/0172993 A1 등 많은 특허가 등록되어 있고, 인산가용화균에 대한 많은 연구가 이루어졌다.Korean Unexamined Patent Publication No. 2002-0017516 "New microorganism solubilizing phosphate fixed on volcanic ash soil", Korea Patent Publication 2005-0100109 "New Bacillus sp., Burkholderia sp. And soil microorganism treating agent using microorganism", Korea Japanese Patent Application Laid-Open No. 2007-0036879, "Aspergillus niger having insoluble phosphoric acid decomposing ability and microorganism preparation containing the same", Korean Patent Laid-Open Publication No. 2011-0079083 "New Lactobacillus paracetamycinus NAAS-1 strain promoting crop growth" Patent No. 2011-0102787 entitled " New strain Bacillus ariabatai LS9 having plant growth promoting activity " and EP 2 154 239 A1, WO 2010/109408 A1 (PCT / IB2010 / 051249) and US 2002/0172993 A1 And there have been many studies on the phosphoric acid-soluble bacteria.
그러나, 상기 특허에도 불구하고 실제 포장에서 증수효과를 보기가 어려운 문제가 있는데 이는 토양에 처리하는 투입량에 기인한다. 실제로 인산가용화균을 농가에서 자체 배양해서 사용하는 것이 어렵고 구입해서 사용하더라도 비용적인 문제로 그 효과를 기대하기 어려운 실정이었다. However, despite the above patent, there is a problem that it is difficult to see the effect of watering on actual packaging due to the amount of the treatment to the soil. In fact, it is difficult to cultivate phosphoric acid-solubilizing bacteria in a farm, and it is difficult to expect the effect because it is a cost problem even if it is purchased and used.
따라서, 본 발명의 목적은 인산가용화능을 가지는 특허 기탁균주 (KACC91877P)를 효모, 아미노산, 비타민, 무기물, 탄수화물 등으로 구성된 미생물의 배양 배지에 첨가하여 배양하는 방법을 제공하는 데 있다. Accordingly, an object of the present invention is to provide a method for culturing a patent deposited strain (KACC91877P) having a phosphoric acid solubilizing ability by adding it to a culture medium of a microorganism composed of yeast, amino acid, vitamin, inorganic substance, carbohydrate and the like.
본 발명의 다른 목적은 상기 제조방법에 의해 수득한 미생물 제제를 토양개량 및 증수 목적에 사용하는 데 있다.Another object of the present invention is to use the microbial agent obtained by the above production method for the purpose of soil improvement and watering.
본 발명의 상기 목적은 김해 인근 토양에서 분리하여 인산가용화능을 가지는 균주명 Bacillus subtilis AP20131014로 명명하고 2013년 11월 4일자로 국립농업과학원 농업유전자센터에 기탁하여 수탁번호 KACC91877P를 부여받은 Bacillus subtilis AP20131014 균주를 배양한 후 동결건조하여 효모 추출물, 덱스트린, 설탕, 제이인산나트륨(Na2HPO4), 황산암모늄((NH4)2SO4)이 첨가하여 제제화함으로써 달성되었다. The above object of the present invention can be achieved by isolating the soil in the vicinity of Gimhae and isolating the bacterial strain Bacillus named subtilis AP20131014 and then culturing the Bacillus subtilis AP20131014 strains have been granted an accession number KACC91877P and deposited in the National Academy of Agricultural Sciences Agricultural Genetic Center in November 2013 4 Date and freeze-dried yeast extract, dextrin, sugar, J. sodium phosphate (Na 2 HPO 4 ) and ammonium sulfate ((NH 4 ) 2 SO 4 ).
본 발명의 다른 목적은 수돗물에 아미노산 수용액을 투입한 후 밀봉하여 실온에서 더욱 바람직하게는 25∼35℃에서 4∼7일간 통기배양을 실시하되 배양액의 pH는 2∼7를 유지시킴으로써 달성하였다.Another object of the present invention is achieved by supplying an aqueous solution of an amino acid into tap water, sealing it, and conducting aeration culture at room temperature, more preferably at 25 to 35 DEG C for 4 to 7 days, while maintaining the pH of the culture medium at 2 to 7.
본 발명은 미생물제제를 제조하고 수득하는 단계와; 상기 수득한 미생물제제를 배양하는 단계와; 상기 배양된 배양액을 포트에 담아 상추에 처리한 후 토양 무기분석 및 상추의 생육조사 단계를 통하여 생육촉진효과를 확인함으로써 달성하였다.The present invention relates to a process for producing and obtaining a microbial formulation; Culturing the obtained microorganism preparation; After culturing the cultured medium in a pot, the treated soil was treated with lettuce and analyzed for soil mineralization and growth promotion of lettuce.
본 발명은 염류집적토양용 인산가용화 미생물제제를 제공할 뿐만 아니라, 특별한 장치 없이 손쉽게 자가배양할 수 있는 미생물배양액을 제공하는 효과가 있고 이를 시용함으로써 토양개량 및 증수효과를 기대할 수 있는 뛰어난 효과가 있다.The present invention not only provides a phosphate-solubilized microorganism preparation for salt-accumulated soil, but also provides a microbial culture liquid which can be easily self-cultivated without special apparatus, and there is an excellent effect of expecting soil improvement and watering effect by using it .
도 1은 본 발명 Bacillus subtilis 균주를 agar plate 상에서 배양온도 20±5℃, 35±2℃, 37℃에서 배양하여 형성된 단일 콜로니를 보이는 사진도이다.1 is Bacillus invention Subtilis strains were cultured on agar plate at 20 ± 5 ℃, 35 ± 2 ℃ and 37 ℃, respectively.
이하에서는 본 발명의 구체적인 내용을 실시예를 들어 상세히 설명하지만 본 발명의 권리범위가 실시예에만 한정하지 않음은 물론이다.Hereinafter, the present invention will be described in detail with reference to examples, but it goes without saying that the scope of the present invention is not limited to the examples.
실시예 1 : 인산가용화균주의 분리 및 동정 Example 1: Isolation and Identification of Phosphorus Solubilization Strain
먼저 인산 가용화 능력이 있는 균을 분리하기 위하여 김해 지역의 토양을 채취하여 토양 시료 1g을 멸균증류수에 현탁한 후 트리칼슘포스페이트의 최종 농도가 0.5%되도록 조제한 고형배지에 평판희석법으로 도말하여 접종한 다음 30℃ 항온기에서 2∼3일간 배양하였다. 그 중 생육이 빠르고 투명환이 큰 균주를 인산 가용화능력이 있는 균주를 선택하여 AP20131014로 명명하였다. In order to isolate the bacteria having solubilization ability of phosphoric acid, the soil of Kimhae area was sampled, 1 g of soil sample was suspended in sterilized distilled water, and then inoculated on a solid medium prepared with 0.5% final concentration of tricalcium phosphate by flat plate dilution method And cultured at 30 ° C for 2 to 3 days in a thermostat. Among them, strains having rapid growth and high transparency and capable of solubilizing phosphoric acid were selected and named AP20131014.
본 발명자가 분리한 본 발명 미생물은 16S rRNA sequencing 분석방법을 통하여 동정을 실시하였다. 동정을 위하여 멸균된 1.5mL 원심분리 튜브에 0.5mL 멸균 생리식염수와 균주를 혼합하여 10,000rpm으로 10분간 원심분리하고 상등액을 제거한 후 27F와 1492R 프라이머를 사용하여 30cycle로 PCR증폭을 하였다. 증폭된 16S rRNA 유전자 염기서열을 분석한 후 NCBI(national Center for Biotechnology Information)의 데이터베이스(http://www.ncbi.nlm.nih.gov)에서 BLAST로 검색하여 기존에 보고된 미생물의 16S rRNA와 상동성을 비교하였다. 실험결과 본 발명의 미생물은 Bacillus subtilis와 98%의 일치성을 나타내어 최종적으로 Bacillus subtilis로 동정되었다(표 1∼표 1-3 참조).The microorganism of the present invention isolated by the present inventor was identified through 16S rRNA sequencing analysis method. For identification, a sterile 1.5 mL centrifuge tube was mixed with 0.5 mL of sterile physiological saline and the strain, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was removed. The PCR amplification was performed using 27F and 1492R primers for 30 cycles. The amplified 16S rRNA gene sequence was analyzed and BLAST was searched in the NCBI database (http://www.ncbi.nlm.nih.gov) and the 16S rRNA of the previously reported microorganisms The homology was compared. As a result of the experiment, the microorganism of the present invention was Bacillus Subtilis was 98% identical to Bacillus subtilis and was finally identified as Bacillus subtilis (see Tables 1 to 3).
실시예Example 2 : 배양조건 2: Culture conditions
전배양한 상기의 균주 AP20131014 10mL를 2L 삼각 플라스크에 멸균시켜 준비한 700mL의 배지(효모 추출물 3.5g, NaCl 3.5g, 수크로오스 1.4g)에 접종한 후 37℃, 200rpm의 속도로 36시간 진탕 배양하였다. 배양액 700mL를 전배양액으로 하여 100L 생물 배양기에 Yeast Extract 350g/70L, NaCl 350g/70L, Sucrose 70g/70L 넣고 3일간 배양하고 탈지분유 100g을 넣어 충분히 교반한 후 원심분리하여 집균하였다. 집균한 종균은 동결건조하여 균체 100g을 얻었고 균수는 7.8×1011cfu였다.10 mL of the pre-cultured strain AP20131014 was inoculated into 700 mL of a medium (3.5 g of yeast extract, 3.5 g of NaCl, 1.4 g of sucrose) prepared by sterilizing in a 2 L Erlenmeyer flask, and cultured at 37 DEG C with shaking at 200 rpm for 36 hours. Yeast Extract 350g / 70L, NaCl 350g / 70L, and Sucrose 70g / 70L were added to a 100L biological incubator for 700 days. The cells were incubated for 3 days and 100g of skimmed milk powder was added. Collected seedlings were lyophilized to obtain 100 g of cells, and the number of bacteria was 7.8 × 10 11 cfu.
비료공정규격 토양미생물제제의 조건을 충족시키기 위해 본 발명 인산가용화균주의 종균 0.05%(w/w)를 Yeast Extract 50%(w/w), 덱스트린 50%(w/w)로 배합된 조성물에 대하여 첨가하여 토양 미생물제제를 완성하였고, 다시 자가 배양에 필요한 영양 성분을 제조하기 위하여 니아신(Niacin) 1%(w/w), 제이인산나트륨(Na2HPO4) 1%(w/w), Sucrose 1%(w/w),황산암모늄((NH4)2SO4) 20%(w/w)와 pH 조절용으로 N-Acetyl-thioproline(N-acetylthiozolidine-4-carboxylic acid) 77%(w/w)를 혼합하여 완성하였다(표 2). In order to satisfy the conditions of the soil microbial preparation, 0.05% (w / w) of the seed of the present invention was added to a composition formulated with 50% (w / w) Yeast Extract and 50% (w / w) 1% (w / w) of Niacin, 1% (w / w) of sodium phosphate dibasic (Na2HPO4), 1% (w / w) of Sucrose were added to the soil microorganism preparation. (w / w), 20% (w / w) of ammonium sulfate ((NH4) 2SO4) and 77% (w / w) of N-acetyl-thioproline (N-acetylthiozolidine- (Table 2).
자가 배양은 깨끗한 말통에 상기 토양미생물제제 200g과 배양 영양제 90g의 비율로 수돗물 20L에 녹여 30±5℃의 조건에서 7일간 배양하여 사용하였다. 이때 배양액의 pH는 2∼4정도를 유지하여 이 pH 범위에서 일정기간 일반 미생물이 자라기 어려운 조건을 조성하였다. 위와 같은 방법으로 특별한 배양기나 멸균기 없이 20±5℃, 35±2℃, 37℃의 조건에서 7일간 배양한 결과 단일 콜로니를 확인할 수 있었고(도 1) 동정 결과도 Bacillus subtilis로 확인되었다. 균수 또한 20±5℃에서 2×106cfu, 35±2℃에서 6×107cfu, 37℃에서 8×107cfu로 비료 공정규격에서 인정하는 106cfu 이상의 양호한 결과를 나타내었다.
The autoculture was prepared by dissolving 200 g of the soil microorganism preparation and 90 g of the culturing nutrient in 20 L of tap water at a temperature of 30 賊 5 째 C for 7 days. At this time, the pH of the culture was maintained at about 2 to 4, and a condition in which the microorganisms did not grow for a certain period was formed in this pH range. Single colonies were identified by the above method without any special incubator or sterilizer at 20 賊 5 째 C, 35 賊 2 째 C, and 37 째 C for 7 days (Fig. 1), and the identification result was confirmed as Bacillus subtilis . The number of bacteria also showed good results of 10 6 cfu or more at 2 × 10 6 cfu at 20 ± 5 ° C, 6 × 10 7 cfu at 35 ± 2 ° C, and 8 × 10 7 cfu at 37 ° C,
실시예Example 3: 자가 배양액의 작물 포트 재배시험 3: Crop pot cultivation test of autologous culture
포트용 토양은 염류집적이 비교적 심한 김해 하우스 농가의 토양 1/3과 건전 토양 2/3를 혼합하여 EC 3 정도의 토양을 사용하였고 작물은 상추를 선택하였다. 처리구는 무처리, 자가배양액, 자가배양액+아미노산액 3개 처리구를 3반복으로 27개 포트시험을 20일간 하였다. 자가 배양액은 1,000배 희석액, 자가배양액+아미노산액은 자가배양액과 아미노산액 각각 1,000배씩 희석하여 사용하였다. 시비는 7일 간격으로 2회 관주하였다.
Pot soil was mixed with 1/3 of soil and 2/3 of soil in Gimhae house farm where salt accumulation was comparatively severe and soil of EC 3 was used and lettuce was selected as the crop. The treatments consisted of 3 treatments of untreated, self - cultured, autologous culture + 3 amino acid solutions for 27 days. The autologous culture was diluted 1,000 times, and the autologous culture + amino acid solution was diluted 1,000 times with the autologous culture solution and amino acid solution. The fertilization was performed two times at intervals of 7 days.
실험예Experimental Example 1. 토양의 이화학적 변화 1. Physicochemical changes of soil
재배 완료 후 토양의 이화학적 분석은 토양 시료 5g에 증류수 25mL를 가한 후 가끔 저어주면서 1시간 방치 후에 Conductivity electric meter로 측정하였고, 치환성 이온은 토양 침출액을 원자흡광 분석기로 측정하였다. [표 3]과 같이 처리간에 pH, EC의 변화는 없었고 유효인산과 치환성양이온의 농도는 자가 배양액 처리구에서 다소 높게 나와 토양 개량 효과를 확인할 수 있었으며, 아미노산 혼용 처리구의 경우 자가 배양 처리구 단독 처리구 보다 양호한 결과가 나온 것은 아미노산이 인산가용화균의 활성을 촉진시킨 결과로 사료된다. After cultivation, physicochemical analysis of the soil was carried out by adding 25 mL of distilled water to 5 g of soil samples, and then measuring with a conductivity meter after 1 hour of occasional stirring. The soil extract was measured with an atomic absorption analyzer. As shown in [Table 3], there was no change of pH and EC between treatments, and the effective phosphoric acid and substitutional cation concentrations were slightly higher in the autoclaving treatment than in the self-cultivation treatment alone Good results were obtained as a result of the amino acids promoting the activity of the solubilizing bacteria.
실험예Experimental Example 2. 상추 생육 측정 2. Lettuce growth measurement
처리 포트에 1차, 2차 관주하고 정식 20일 후에 엽수, 엽장, 엽폭, 엽중의 생육 조사를 실시하여 [표 4]와 같았다.[Table 4] shows the growth of leaves, leaves, leaf width and leaf weight after 20 days of primary and secondary treatment on treated pots.
상추의 생육조사결과 무처리에 비해 자가 배양액 처리구가 유의성 양호한 결과가 나왔으며 아미노산 혼용처리구가 자가 배양액 단독 처리구보다 다소 양호한 결과가 나왔다. 더욱 구체적으로 엽수의 경우 무처리는 14.36매, 자가 배양액 처리구는 17.99매, 아미노산 혼용처리구 19.77매로 무처리에 비해 각각 25%, 38%의 증가를 보였다. 엽장과 엽폭도 유사한 경향을 보였으며 특히 엽중의 경우 무처리 67.05g, 자가배양액 90.42g, 아미노산 혼용구 98.88g로 무처리에 비해 각각 35%, 47%의 증가를 보였다.As a result of the growth of lettuce, the results of the self - culture medium treatment showed better results than those of the non - treatment, and the amino acid mixture treatment was somewhat better than the self - culture medium alone treatment. More specifically, in the case of leaf water, 14.36 pieces of untreated, 17.99 pieces of self-cultured treated and 19.77 pieces of amino acid mixed treatments were increased by 25% and 38%, respectively. Leaf length and leaf width also showed similar tendencies. Especially, leaf growth was 67.05g in non-treated, 90.42g in self-cultured medium, and 98.88g in amino acid mixture, respectively.
실험예Experimental Example 3. 3. 상추내Lettuce 인의 함량 변화 Change in phosphorus content
상추에 묻은 토양 및 이물질을 제거한 후 103℃로 12시간 건조한 후 마쇄하여 시료 0.5g을 취하여 10mL의 질산을 가하고 마이크로웨이브로 분해, 여과(No.6)하여 AA 및 ICP로 분석하였다(표 5). After removing the soil and foreign matter from the lettuce, the sample was dried at 103 ° C for 12 hours and then ground. Then, 0.5 g of the sample was taken, 10 mL of nitric acid was added, and the resultant was decomposed by microwave and filtered (No. 6) .
자가배양액 처리구에서 0.62%, 0.65%로 무처리에 비해 15%, 20%의 증가를 보였고 이는 인산가용화균주에 의한 유리인산의 흡수로 여겨진다. 따라서, 인산의 토양내 축적이 현저히 저하되어 토양개량 효과가 도모됨을 확인할 수 있었다.
And 0.65% and 0.65%, respectively, in the culture medium of autologous cultures, which was increased by 15% and 20%, respectively, compared to the untreated control. This is considered to be the absorption of free phosphoric acid by the phosphoric acid solubilization strain. Therefore, it was confirmed that the accumulation of phosphoric acid in the soil was remarkably decreased, and the effect of improving the soil was improved.
Claims (5)
A fertilization method characterized in that the culture of the strain of claim 4 or its diluent is inhabited twice at intervals of 7 days.
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