KR20030067887A - Pseudomonas sp. FK 916 producing decomposition enzyme of organic compound and method for decomposition of organic waste using the same - Google Patents

Pseudomonas sp. FK 916 producing decomposition enzyme of organic compound and method for decomposition of organic waste using the same Download PDF

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KR20030067887A
KR20030067887A KR1020020007525A KR20020007525A KR20030067887A KR 20030067887 A KR20030067887 A KR 20030067887A KR 1020020007525 A KR1020020007525 A KR 1020020007525A KR 20020007525 A KR20020007525 A KR 20020007525A KR 20030067887 A KR20030067887 A KR 20030067887A
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최동성
구니히로 후카이
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최동성
구니히로 후카이
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Priority to CNB03803235XA priority patent/CN100381561C/en
Priority to JP2003566188A priority patent/JP2006501810A/en
Priority to AU2003206244A priority patent/AU2003206244A1/en
Priority to PCT/KR2003/000277 priority patent/WO2003066837A1/en
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F2003/001—Biological treatment of water, waste water, or sewage using granular carriers or supports for the microorganisms
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/32—Nature of the water, waste water, sewage or sludge to be treated from the food or foodstuff industry, e.g. brewery waste waters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas

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  • Environmental & Geological Engineering (AREA)
  • Water Supply & Treatment (AREA)
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Abstract

PURPOSE: A microorganism Pseudomonas sp. FK 916 producing a decomposition enzyme of organic compounds and a method for decomposition of organic wastes using the same are provided, thereby effectively decomposing organic waste at mild temperature without producing a bad smell. CONSTITUTION: A microorganism Pseudomonas sp. FK 916(KCCM 10350) decomposing food wastes at 15 to 35 deg. C without producing a bad smell is provided, wherein the Pseudomonas sp. FK 916(KCCM 10350) is isolated from waste water. A method for decomposition of organic wastes comprises treating the organic wastes with the Pseudomonas sp. FK 916(KCCM 10350), wherein the Pseudomonas sp. FK 916(KCCM 10350) is added into the organic wastes together with natural organic materials, inorganic solid, and a pore forming agent made of plastics; the natural organic material is selected from rice hulls, sawdust and wood chip; and the inorganic solid is selected from zeolite and ceramic.

Description

유기물 분해효소를 생산하는 슈도모나스 속 ＦＫ916 균주 및 이를 이용한 유기 폐기물의 처리방법{Pseudomonas sp. FK 916 producing decomposition enzyme of organic compound and method for decomposition of organic waste using the same}F 916 strain of Pseudomonas genus producing organic degrading enzyme and method for treating organic waste using the same {Pseudomonas sp. FK 916 producing decomposition enzyme of organic compound and method for decomposition of organic waste using the same}

본 발명은 유기물 분해효소를 생산하는 슈도모나스 속 FK 916 균주 및 이를이용한 유기 폐기물의 처리방법에 관한 것으로서, 더욱 상세하게는 하수로부터 분리되고, 음식물쓰레기 및 유기성 폐기물 중의 유기물을 분해·소멸시키는 능력이 우수하며, 악취를 거의 발생하지 않으면서 음식쓰레기를 효과적으로 처리할 수 있는 슈도모나스 속 FK 916(Pseudomonassp. FK 916) 및 이를 이용한 유기 폐기물의 처리방법에 관한 것이다.The present invention relates to a strain of PK 916 genus Pseudomonas producing organic degrading enzyme and a method for treating organic waste using the same, more specifically, it is separated from sewage, and has excellent ability to decompose and destroy organic matter in food waste and organic waste. In addition, the present invention relates to Pseudomonas sp. FK 916 ( Pseudomonas sp. FK 916) capable of effectively treating food waste with little odor, and a method of treating organic waste using the same.

우리나라에서 발생되는 음식물쓰레기의 양은 11,577톤/일(1999년 기준)으로 도시 고형폐기물 발생량의 약 26.5%를 차지하고 있고, 64%는 가정에서 36%는 음식점과 집단급식소 등 외식분야에서 발생하고 있다. 발생된 음식물쓰레기 중 58.8%는 매립, 7.3%는 소각, 33.9%는 재활용으로 처리하고 있어 대부분 매립에 의존하고 있는 실정이다. 수도권의 경우 오는 2005년 1월부터 수도권 매립지에 음식물쓰레기의 직접 매립을 금지하고 있으며, 대부분의 지방자치단체에서도 매립지의 침출수에 의한 환경오염과 매립지 주변에 거주하고 있는 주민에 의해 민원이 야기되고 있어 음식물쓰레기 처리 문제가 갈수록 심각해지고 있다. 또한 소각 처리할 경우에는 수분 함량이 높아 가연성 쓰레기에 비해서 처리경비가 더 많이 소요되며, 소각 처리장의 건설 및 부지 확보도 주민의 반대로 매우 어려운 실정이다. 그러므로 이러한 문제를 해결하기 위해서는 음식물쓰레기가 발생하는 장소에서 직접 미생물로 음식물쓰레기를 분해·소멸 또는 감량화하여 배출하거나 재활용 비율을 높일 수 있는 경제적 처리방법을 더욱 발전시켜야 할 필요가 있다.The amount of food waste generated in Korea is 11,577 tons / day (as of 1999), accounting for about 26.5% of urban solid waste generation, and 64% of households are eating out in restaurants and groups. Of the food waste generated, 58.8% are landfilled, 7.3% are incinerated, and 33.9% are recycled. The metropolitan area has banned landfills from landfills since January 2005, and most local governments have caused environmental pollution due to leachate from landfills and civil complaints caused by residents living near landfills. Food waste disposal problems are getting worse. In addition, the incineration process is more expensive than combustible waste due to the high moisture content, and construction of the incineration plant and securing the site are very difficult on the contrary. Therefore, in order to solve these problems, it is necessary to further develop an economic treatment method that can directly discharge or reduce food wastes by reducing microorganisms at places where food wastes occur, or increase the recycling rate.

음식물 쓰레기는 성상이 균일하지 않고 수분 및 염분 함량이 높아 그 처리에 많은 어려움이 있으며, 지역별, 사업장별로 다양한 처리방법을 모색할 필요가 있다. 음식물쓰레기의 처리 및 재활용 방안으로 검토되고 있는 기술은 퇴비화, 사료화, 혐기성 소화, 하수병합 처리이며, 퇴비화 기술은 광역처리 대형 퇴비화 기술과 일반가정이나 소형 업소에서 사용할 수 있는 소형 퇴비화 기술의 두 방향으로 크게 나눌 수 있다.Food waste has a lot of difficulties in its disposal due to its inconsistent properties and high water and salt content, and it is necessary to search for various treatment methods by region and by workplace. The technologies that are being considered for the treatment and recycling of food waste are composting, feed, anaerobic digestion, and sewage treatment. The composting technology is divided into two types: large-area large composting technology and small composting technology that can be used at home or small business It can be divided largely.

값이 비싼 소형 퇴비화 장치는 음식물쓰레기 발효조 및 건조기로 구성되어 있으며, 아파트 단지나 단체급식소, 호텔, 레스토랑 등 요식업소에 설치되어 있으나 여러 문제점을 안고 있다.Expensive small composting device is composed of food waste fermentation tank and dryer, and is installed in restaurants such as apartment complexes, group meals, hotels, restaurants, etc., but has various problems.

음식물쓰레기를 고온에서 발효시켜 퇴비와 사료로 재활용하는 종래의 기술과 장치에 있어서 문제점으로는 첫째, 고온에서 발효 또는 부패가 진행되기 때문에 심한 악취가 발생되어 혐오시설로 기피되고 있으며, 우리나라 음식물이 1∼5% 정도의 고농도의 식염을 함유하고 있어 비료로 사용하는 것도 또한 부적합한 실정이고, 둘째, 미생물에 의한 발효 분해속도를 높이기 위해 고온성 세균 및 방선균을 사용하고, 고온미생물 생육을 위한 최적 생육온도 유지, 음식물쓰레기 발효산물의 부피 감량 및 수분을 증발시키기 위해 장치내 온도를 50∼60℃ 이상으로 항상 유지시켜야 하기 때문에 전기를 다량 소모하여 운전비용이 매우 많이 든다는 단점이 있다.Problems in the conventional technology and apparatus for fermenting food waste at high temperature and recycling it as compost and feed are as follows: First, fermentation or decay proceeds at high temperature, so bad odor is generated and avoided as a loathing facility. It is also inadequate to use as a fertilizer because it contains a high concentration of salt of about 5%. Second, the use of high temperature bacteria and actinomycetes to increase the rate of fermentation degradation by microorganisms, and optimum growth temperature for high temperature microbial growth In order to reduce the volume of oil, food waste fermentation products and evaporate moisture, the temperature in the apparatus must be maintained at 50 to 60 ° C. or higher at all times, which consumes a large amount of electricity, resulting in very high operating costs.

본 발명자는 상기와 같은 문제점을 해결하기 위하여, 하수로부터 음식물쓰레기 및 유기성 폐기물 중의 유기물을 분해·소멸시키는 능력이 우수하며, 중온에서 호기적 분해작용을 하여 악취를 발생시키지 않는 신규한 균주를 분리·동정하고, 이 균주를 음식물쓰레기의 분해 소멸화에 이용함으로써 본 발명을 완성하였다.In order to solve the above problems, the present inventors have an excellent ability to decompose and destroy organic matters in food waste and organic wastes from sewage, and isolate and isolate novel strains that do not generate odor by aerobic decomposition at medium temperature. The present invention was completed by identifying and using this strain for the decomposition and extinction of food waste.

따라서 본 발명의 목적은 음식물쓰레기 및 유기성 폐기물 중의 유기물을 분해·소멸시키는 능력이 우수한 균주를 제공함에 있다.Accordingly, an object of the present invention is to provide a strain having excellent ability to decompose and destroy organic matter in food waste and organic waste.

본 발명의 또 다른 목적은 상기 균주를 이용한 유기 폐기물 처리방법을 제공함에 있다.Another object of the present invention to provide an organic waste treatment method using the strain.

도 1은 본 발명 균주인 슈도모나스 속 FK 916을 단백질 분해효소 탐색 한천배지 플레이트에 접종하여 단백질 분해효소의 활성을 확인한 결과이다.1 is a result of confirming the activity of protease by inoculating the strain Pseudomonas genus FK 916 of the present invention in a protease search agar medium plate.

도 2는 슈도모나스 속 FK 916을 지방질 분해효소 탐색 한천배지 플레이트에 접종하여 지방질 분해효소의 활성을 확인한 결과이다.2 is a result of inoculating Pseudomonas genus FK 916 to the lipolytic enzyme search agar medium plate to confirm the activity of lipolytic enzymes.

도 3는 슈도모나스 속 FK 916의 주사전자현미경 사진이다.3 is a scanning electron micrograph of Pseudomonas genus FK 916.

도 4는 슈도모나스 속 FK 916의 배양액을 음식물 쓰레기에 첨가한 후 분해시간 경과에 따른 음식물쓰레기의 감량율을 나타낸 그래프이다.Figure 4 is a graph showing the reduction rate of food wastes over time after adding the culture medium of Pseudomonas genus FK 916 to food waste.

본 발명은 슈도모나스 속 FK 916 (Pseudomonassp. FK 916)를 제공한다.The present invention provides a Pseudomonas species FK 916 (Pseudomonas sp. FK 916 ).

하수로부터 분리한 슈도모나스 속 FK 916은 15∼35℃, 바람직하게는 25∼30℃의 중온 및 충분한 산소의 존재 하에서 호기적 분해작용을 통해 음식물쓰레기 중의 탄수화물, 단백질, 지방 등과 같은 유기물을 안정된 저분자 유기물로 전환하게 되며, 최종산물로 안정된 물질인 CO2, H2O 등을 생성하고 약간의 열을 발생하게 된다. 한편 슈도모나스속 미생물은 하기 식(Ⅰ)에 나타난 바와 같이 NO3, NO2, NO를 N2로 탈질시킬 수 있으므로 슈도모나스 속 FK 916은 다른 미생물과 공동으로 악취 가스 성분의 하나인 NH3를 N2로 변환하는 탈질과정을 거쳐 암모니아의 악취 및 자극성을 제거할 수도 있다.PK 916, Pseudomonas spp., Isolated from sewage is a stable low molecular weight organic substance that can stabilize organic matters such as carbohydrates, proteins, fats, etc. in food waste through aerobic decomposition in the presence of moderate temperature and sufficient oxygen at 15-35 ℃, preferably 25-30 ℃. It converts to and produces a stable material CO 2 , H 2 O as a final product and generates a little heat. The Pseudomonas genus microorganisms formula as shown in (Ⅰ) NO 3, NO 2 , it may be denitrification NO to N 2 Pseudomonas species FK 916 is one of NH 3 in the odor gas component N 2 in association with other microbes The denitrification process can also remove odors and irritants of ammonia.

또한 본 발명의 균주는 악취성분인 인돌, 황화수소, 아세토인을 생성하지 않는 특성을 가지고 있다 (표 2).In addition, the strain of the present invention has a characteristic that does not produce odor components indole, hydrogen sulfide, acetoin (Table 2).

본 발명 균주인 슈도모나스 속 FK 916을 2002년 1월 7일자로 국제기탁기관인 한국종균협회(KCCM)에 기탁번호 KCCM-10350으로 기탁하였다. 상기 기탁된 균주 외에도 다른 특성에 다소간의 변화가 가해지는 상기 균주의 돌연변이주도 본 발명의 16S rRNA 염기서열 및 지방산 조성, 형태, 배양, 생화학적 특성이 같은 한 본 발명의 범주에 물론 포함한다.The strain of the present invention Pseudomonas genus FK 916 was deposited on January 7, 2002, with the deposit number KCCM-10350 in the International Depository Association (KCCM). In addition to the deposited strain, the mutant strain of the strain to which some changes are added to other properties is included in the scope of the present invention as long as the 16S rRNA base sequence of the present invention and fatty acid composition, form, culture, and biochemical characteristics are the same.

또한, 본 발명은 슈도모나스 속 FK 916균주를 이용하여 음식물 쓰레기 등과 같은 유기 폐기물을 분해·소멸하는 방법을 제공한다.The present invention also provides a method for decomposing and extinguishing organic waste such as food waste using PK 916 strain Pseudomonas.

상기 방법은 슈도모나스 속 FK 916 균주 또는 상기 균주를 포함하는 조성물을 피처리물에 첨가하는 그 특징으로 한다. 상기 균주 또는 그 조성물의 처리방법으로는, 상기 균주의 배양액을 직접 살포(spray)하거나 균주 배양액을 왕겨, 톱밥, 우드칩(wood chip)과 같은 천연유기물, 제오라이트나 세라믹과 같은 무기질 입체, 플라스틱류 등의 공극제와 혼합하여 첨가할 수 있으며, 또한 상기의 천연유기물, 무기질 입체, 플라스틱 등의 공극제 또는 담체에 흡착시켜 유기 폐기물에 산포하여 처리하거나 통상의 미생물 제제의 제조방법에 의하여 제제화하여 사용할 수 있다.The method is characterized in that the strain of Pseudomonas genus FK 916 or a composition comprising the strain is added to the workpiece. As a method of treating the strain or its composition, the culture medium of the strain is directly sprayed (spray) or the strain culture medium chaff, natural organic matter such as sawdust, wood chips (wood chips), inorganic solids, plastics such as zeolite or ceramics It can be mixed with a pore-forming agent, and the like, and is also adsorbed to a pore-forming agent or carrier such as natural organic matter, inorganic solid, plastic, or the like, and then dispersed and treated in organic waste or formulated by a conventional method for preparing a microbial preparation. Can be.

본 발명에서 '피처리물'이라 함은 음식물쓰레기 이외의 활성 오니케익, 공장에서 발생하는 유기폐기물 뿐만 아니라 하수폐기물 등을 의미한다.In the present invention, the 'treatment' means active sewage other than food waste, organic waste generated in a factory, as well as sewage waste.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

실시예 1 : 본 발명 균주의 분리Example 1 Isolation of the Strain of the Invention

본 발명에 사용된 균주는 전라북도 일원의 하수에서 채취한 시료로부터 분리하였다. 이 시료를 생리식염수로 단계적으로 적당히 희석하고 그의 일부를 LB 한천배지(박토트립톤 10g/L, 박토효모추출물 5g/L, NaCl 10g/L, 한천 15g/L, pH 7.0)에 도포하여 25℃에서 배양하면서 출현하는 콜로니를 분리하였다.Strains used in the present invention were isolated from samples collected from sewage in Jeollabuk-do. Dilute this sample with physiological saline stepwise and apply a portion of it to LB agar medium (bacto tryptone 10g / L, bacterium yeast extract 5g / L, NaCl 10g / L, agar 15g / L, pH 7.0) Colonies that appeared while incubating at were isolated.

상기 분리된 균주를 균주 증식배지(LB 액체배지)에서 25℃로 배양 증식시킨 다음, 단백질 분해효소 탐색 한천배지(탈지우유 한천배지: 펩톤 5g/L, 박토효모추출물 3g/L, 스킴 밀크 1g/L, 한천 15g/L, pH 7.0), 지방질 분해효소 탐색 한천배지(트리부티린 한천배지: 펩톤 5g/L, 박토효모추출물 3g/L, 트리부티린 5mL/L, 한천 15g/L, pH 7.4), 전분 분해효소 탐색 한천배지(전분한천배지: 펩톤 5g/L, 박토효모추출물 3g/L, 가용성 전분 2g/L, 한천 15g/L, pH 7.0), 셀룰로오스 탐색 한천배지[셀룰로오스 1g/L, K2HPO41g/L, (NH4)2SO41g/L, MgSO4·7H2O 0.2g/L, CaCl2·2H2O 0.1g/L, FeCl30.02g/L, 콩고레드 0.075g/L, 한천 15g/L, pH 7.2] 플레이트에 도말하여 단백질 분해효소, 지방질 분해효소, 전분 분해효소, 셀룰로오스 분해효소의 활성에 의해 생기는 투명환의 존재 여부를 확인하여 양성 균주를 탐색하고 이를 분리하였다.The isolated strain was cultured and propagated at 25 ° C. in a strain growth medium (LB liquid medium), followed by proteolytic enzyme search agar medium (degreasing milk agar medium: peptone 5g / L, bacterium yeast extract 3g / L, skim milk 1g / L, agar 15g / L, pH 7.0), lipolytic enzyme agar medium (tributyrin agar medium: peptone 5g / L, bacterium yeast extract 3g / L, tributyrin 5mL / L, agar 15g / L, pH 7.4 Starch degrading enzyme agar medium (starch agar medium: peptone 5g / L, bacterium yeast extract 3g / L, soluble starch 2g / L, agar 15g / L, pH 7.0), cellulose search agar medium [cellulose 1g / L, K 2 HPO 4 1g / L, (NH 4 ) 2 SO 4 1g / L, MgSO 4 · 7H 2 O 0.2g / L, CaCl 2 · 2H 2 O 0.1g / L, FeCl 3 0.02g / L, Congo red 0.075 g / L, agar 15g / L, pH 7.2] Plates were plated to determine the presence of clear rings caused by the activity of proteases, lipolytic enzymes, starch degrading enzymes, and cellulose degrading enzymes. It was isolated.

실험결과, 도 1에 나타난 바와 같이, 단백질 분해효소 탐색 한천배지 플레이트(A) 및 지방질 분해효소 탐색 한천배지 플레이트(B)에 FK 916 균주를 각각 접종하였을 경우 투명환을 형성하여 상기 균주가 단백질 분해효소와 지방질 분해효소를 형성하는 것을 알 수 있었다. 그러나 전분 분해효소의 활성은 미약하였고, 셀룰로오스 분해효소의 활성은 인정되지 않았다.As a result, as shown in Figure 1, when the FK 916 strain was inoculated into the protease search agar medium plate (A) and lipolytic enzyme search agar medium plate (B), respectively, a transparent ring was formed to form a transparent ring. It was found to form enzymes and lipolytic enzymes. However, the activity of starch degrading enzyme was weak, and the activity of cellulose degrading enzyme was not recognized.

실시예 2 : 본 발명 균주의 동정Example 2 Identification of Strains of the Invention

상기에서 분리한 균주를 Holt 등[Holt J. G., et al., Bergey,s Manual of Determinative Bacteriology, 9th ed. (1994)]의 방법과 Sherlock 시스템 및 clustal W로 동정하여 슈도모나스속에 속하는 균주임을 밝혔고, 이를 슈도모나스 속 FK 916 (Pseudomonassp. FK 916)으로 명명하였다. 슈도모나스 속 FK 916의 지방산 및 16S rRNA는 각각 슈도모나스 클로로라피스(Pseudomonas chlororaphis)의 지방산 조성과 80.2%, 슈도모나스 게사르디이(Pseudomonas gessardiiCIP 105469)의 16S rRNA와 99.8%의 유사도를 나타내었다.The strain isolated from Holt et al., Holt JG, et al., Bergey , s Manual of Determinative Bacteriology, 9th ed. (1994)] and Sherlock system and clustal W was identified as a strain belonging to the genus Pseudomonas, which was named Pseudomonas sp. FK 916. Fatty acid and 16S rRNA of Pseudomonas genus FK 916 showed 80.2% similarity to fatty acid composition of Pseudomonas chlororaphis and 99.8% to 16S rRNA of Pseudomonas gessardii CIP 105469, respectively.

도 3의 주사전자현미경 사진에서 알 수 있듯이 상기 균주의 형태는 간균이고, 그램음성균이었으며, 배양학적 특성은 하기 표1에 나타난 바와 같이 최적 생장온도는 28∼30℃이며, NaCl에 대하여 6%의 내성을 나타내어, 본 발명 균주가 염분이 높은 음식물 쓰레기를 처리하는데 적합함을 알 수 있었다.As can be seen from the scanning electron micrograph of FIG. 3, the strain was bacillus, Gram-negative bacteria, and the cultivation characteristics were as follows. The optimum growth temperature was 28 to 30 ° C. and 6% of NaCl. It showed that the strain of the present invention is suitable for treating food waste having high salt.

본 발명 균주인 슈도모나스 속 FK 916 배양, 형태적 특성Cultivation of FK 916 of Pseudomonas sp. 특 성Characteristics 결 과result 형태학적 특성Morphological characteristics 형태 및 크기Shape and size 간 균, 0.8∼0.9 × 1.7∼2.0 ㎛Bacillus, 0.8-0.9 × 1.7-2.0 μm 운동성motility + (양성)+ (Positive) 그람염색Gram Dyeing - (음성)- (voice) 배양 특성Culture characteristics 생장 pHGrowth pH 4.5∼9.04.5 to 9.0 생장 온도Growth temperature 10∼37℃10 ~ 37 ℃ 4℃에서의 생장Growth at 4 ℃ ++ 41℃에서의 생장Growth at 41 ° C -- 최적 생장온도Optimum growth temperature 28∼30℃28 ~ 30 ℃ NaCl에 대한 내성Resistance to NaCl 6%6% 맥콘키 한천배지Macconkey Agar Badge ++ 트립토판 배지 (슈도모나스 분리 한천반응)Tryptophan Medium (Pseudomonas Separation Agar Reaction) ++

API 키트에 의해 분석한 생화학적 특성은 표 2에, MIDI 사에서 제시한 방법에 따라 가스크로마토그래피로 분석한 전세포의 지방산 분석 및 Sherlock system을 통한 균주 동정 결과는 표 3에 나타내었다. 또한 Verhille 등의 방법(Verhille, S., et al., International J. Systematic Bacteriol., 49, 1559, 1999)으로 16S rRNA를 분리하고 염기서열을 분석한 결과 1498 염기(서열 1)로 이루어짐을 밝혔고, Gene bank에서 유의성이 높은 미생물을 선별한 후 슈도모나스속 균주간의 16s rRNA 염기서열의 유사도를 clustal W를 사용하여 분석한 염기서열간의 상동성을 비교한 결과는 표 4에 나타내었다. De Ley 방법(De Ley, J., J. Bacteriol., 101, 738, 1970)으로 분석한 G + C 함량은 61.5%이었다.The biochemical characteristics analyzed by the API kit are shown in Table 2, and the results of the fatty acid analysis of the whole cells analyzed by gas chromatography and the strain identification through the Sherlock system are shown in Table 3 according to the method proposed by MIDI. In addition, 16S rRNA was isolated and sequenced by Verhille et al. (Verhille, S., et al., International J. Systematic Bacteriol., 49, 1559, 1999) and found to consist of 1498 bases (SEQ ID NO: 1). The results of comparing homology between sequences analyzed by clustal W for the similarity of 16s rRNA sequences between Pseudomonas strains after selection of highly significant microorganisms from the Gene Bank, are shown in Table 4. The G + C content as analyzed by De Ley method (De Ley, J., J. Bacteriol., 101, 738, 1970) was 61.5%.

본 발명 균주인 슈도모나스 속 FK 916의 생화학적 특성Biochemical Properties of Pseudomonas Species FK 916 특 성Characteristics 결 과result 생화학적 특성Biochemical properties 질산염의 아질산염으로의 환원Reduction of Nitrate to Nitrite -- 인돌 생성Indole generation -- 황화수소 생성Hydrogen sulfide generation -- 아세토인 생성Acetoin production -- N2기체로의 환원Reduction to N 2 gas ++ 글루코오스 산성화Glucose acidification -- 글루코오스 산화발효Glucose oxidative fermentation 산화Oxidation 프로테아제Protease ++ 리파아제Lipase ++ 아밀라제Amylase ++ 셀룰라아제Cellulase -- 젤라티나제Gelatinase -- 베타-갈라토시다제Beta-galactosidase -- 아르기닌 디하이드로라제Arginine dehydrolase ++ 라이신 데카복실라제Lysine decarboxylase -- 오르니틴 데카복실라제Ornithine decarboxylase -- 시토크롬 옥시다제Cytochrome oxidase ++ 트립토판 데아미나제Tryptophan deaminase -- 우레아제Urease ++ 베타-글루코시다제(esculin hydrolysis)Beta-glucosidase (esculin hydrolysis) -- 탄소원의 이용성Availability of Carbon Sources 글루코오스Glucose ++ 아라비노스Arabinos -- 만노오스Mannose ++ 만니톨Mannitol ++ N-아세틸 글루코사민N-acetyl glucosamine ++ 말토오스maltose -- 글루콘산Gluconic Acid ++ 카프린산Capric acid ++ 아디핀산Adipic acid ++ 사과산Malic acid -- 구연산Citric acid ++ 페닐아세테이트Phenyl Acetate --

지방산 조성Fatty acid composition 지방산 종류Fatty acid types 백분율 (%)Percentage (%) C 10:0C 10: 0 0.200.20 C 12:0 3OHC 12: 0 3OH 2.002.00 C 12:0C 12: 0 1.671.67 C 12:0 2OHC 12: 0 2OH 4.734.73 C 12:0 3OHC 12: 0 3OH 4.274.27 C 14:0C 14: 0 0.500.50 C 15:0C 15: 0 0.450.45 C 15:0 iso 2OHC 16:1 w7cC 15: 0 iso 2OHC 16: 1 w7c 30.0130.01 C 16:1 w5cC 16: 1 w5c 0.120.12 C 16:0C 16: 0 33.9433.94 C 17:1 w8cC 17: 1 w8c 0.270.27 C 17:0 cycloC 17: 0 cyclo 6.646.64 C 17:0C 17: 0 0.560.56 C 18:3 w6c (6,9,12)C 18: 3 w6c (6,9,12) 0.630.63 C 18:1 w7cC 18: 1 w7c 12.3412.34 C 18:0C 18: 0 1.481.48 C 19:0 cyclo w8cC 19: 0 cyclo w8c 0.150.15 동정된 균주명 (유사도)Identified strain name (similarity) Pseudomonas chlororaphis(80.2%)(P. aureofaciens) Pseudomonas chlororaphis (80.2%) ( P. aureofaciens ) P.putidabiotype B (70.9%) P. putida biotype B (70.9%) P.putidabiotype A (68.7%) P. putida biotype A (68.7%) P.syringae(41.5%) P. syringae (41.5%)

본 발명 균주인 슈도모나스 속 FK 916와 슈도모나스속 균주간의 16S rRNA 염기서열의 유사도Similarity of 16S rRNA Sequences between Pseudomonas Genus FK 916 and Pseudomonas Genus 균 주Strain 유사도 (%)Similarity (%) 염기 차/비교 염기수Base difference / comparative base number Pseudomonas gessardiiCIP 105469 Pseudomonas gessardii CIP 105469 99.8099.80 3 / 14913/1491 P. libaniensisCIP 105460 P. libaniensis CIP 105460 99.7399.73 4 / 14914/1491 P. synxanthaDSM 13080 P. synxantha DSM 13080 99.5399.53 7 / 14987/1498 P. reactansLMG 5329 P. reactans LMG 5329 99.5399.53 7 / 14787/1478 P. brenneriiCFML 97-391T P. brennerii CFML 97-391T 99.5299.52 7 / 14707/1470 P. cedrellaCFML 96-198 P. cedrella CFML 96-198 99.4099.40 9 / 14919/1491 P. fluorescensATCC 49642 P. fluorescens ATCC 49642 99.3399.33 10 / 149810/1498 P. mucidolensIAM12406 P. mucidolens IAM12406 99.2699.26 11 / 149611/1496 P. azotoformansIAM1603 P. azotoformans IAM1603 98.9398.93 16 / 149616/1496 P. migulaeCIP 105470 P. migulae CIP 105470 98.9398.93 16 / 149116/1491 P. putidaATCC 17472 P. putida ATCC 17472 98.8598.85 17 / 147617/1476 P. tolaasiiLMG 2342 T P. tolaasii LMG 2342 T 98.5898.58 21 / 147821/1478 P. thivervalensisCFBP 1126T P. thivervalensis CFBP 1126T 98.1198.11 27 / 143127/1431 P. fragiATCC 4973 P. fragi ATCC 4973 97.8797.87 31 / 145831/1458 P. jesseniiCIP 105274 P. jessenii CIP 105274 97.8597.85 32 / 149132/1491 P. pavnaceaeIAM1155 P. pavnaceae IAM1155 97.8097.80 33 / 149733/1497 P. plecoglossicidaFPC951 P. plecoglossicida FPC951 96.7996.79 48 / 149548/1495 P. oryzihabitansIAM1568 P. oryzihabitans IAM1568 96.4696.46 53 / 149653/1496 P. mosseliiCIP 105259 P. mosselii CIP 105259 96.4496.44 53 / 149053/1490 P. monteiliiCIP 104883T P. monteilii CIP 104883T 96.3596.35 54 / 147854/1478

실시예 3 : 음식쓰레기의 분해·소멸 시험 : 분해경과에 따른 음식물쓰레기의 감량율Example 3 Disintegration and Extinction Test of Food Waste: Reduction Rate of Food Waste According to the Degradation Process

음식물쓰레기의 감량화 정도를 시험하기 위하여 멸균된 LB 액체배지에 슈도모나스 속 FK 916을 접종하여 28℃에서 1주야 진탕 배양하였다. 3L 비이커에 이 배양액 200mL와 톱밥(통기 및 수분조절제) 100g을 첨가하여 잘 혼합하고, W 대학 식당에서 수거한 음식물쓰레기(수분 함량 약 80%) 1Kg을 첨가하여 잘 혼합한 다음, 25℃ 배양기에 넣어 두면서 경시적으로 그의 무게를 칭량하고 감량화율을 계산하였으며, 매일 3∼4회씩 잘 교반하였다.In order to test the degree of reduction of food waste, FK 916 in Pseudomonas was inoculated into sterile LB liquid medium and shaken at 28 ° C. for 1 week. Add 200 mL of this culture solution and 100 g of sawdust (aeration and moisture regulator) to a 3L beaker, mix well, add 1 kg of food waste (about 80% moisture) collected at W University restaurant, mix well, and then incubate at 25 ℃. Weighing his weight over time while calculating the weight reduction rate, and stirred well 3 to 4 times daily.

실험결과, 도 4에 나타난 바와 같이, 분해 시작 1일째부터 음식물쓰레기 특유의 악취가 없어졌고 새로운 악취 또한 발생하지 않았으며 무게의 감량이 시작되었다. 분해 18일째에 72.3%까지 감량되어 음식물의 분해 소멸화가 이루어졌으며, 배양액만 첨가하거나(비교예 1), LB배지와 톱밥을 첨가한 비교예 2에 비해 빠른 감량화율을 나타내었다. 이와 같은 결과는 톱밥 첨가에 의해 호기적 조건이 충족되고 수분이 조절되어 유기물의 분해 소멸화가 촉진되었고, 25℃에서 FK 916 균주에 의해 유기물의 분해 소멸화가 촉진되었음을 보여주고 있다. 한편 이러한 결과는 교반에 의해 지속적으로 공기가 공급되는 음식물쓰레기 소멸기에서는 더욱 양호한 결과가 얻어질 수 있음을 보여주고 있다.As a result, as shown in Figure 4, from the first day of the decomposition, the odor peculiar to food waste disappeared, and no new odor occurred, and weight loss began. Degradation and extinction of food was achieved by reducing to 72.3% on day 18 of degradation, and showed a rapid reduction compared to Comparative Example 2 in which only the culture medium was added (Comparative Example 1) or LB medium and sawdust were added. These results show that the aerobic conditions are met by the addition of sawdust and the moisture is controlled to facilitate the decomposition and annihilation of organic matter, and the decomposition and annihilation of the organic material is promoted by the FK 916 strain at 25 ° C. On the other hand, these results show that better results can be obtained in the food waste dissipator which is continuously supplied with air by stirring.

비교예 1 : 음식물 쓰레기에 슈도모나스속 FK916의 배양액만을 처리한 경우의 분해율 측정Comparative Example 1 Measurement of Degradation Rate When Only Food Medium of Pseudomonas FK916 Was Treated in Food Waste

멸균된 LB 액체배지에 슈도모나스 속 FK 916을 접종하여 28℃에서 1주야 진탕 배양하였다. 3L 비이커에 이 배양액 200mL와 수거한 음식물쓰레기(수분 함량 약 80%) 1Kg을 첨가하여 잘 혼합한 다음, 25℃ 배양기에 넣어 두면서 경시적으로 그의 무게를 칭량하고 감량화율을 계산하였으며, 매일 3∼4회씩 잘 교반하였다(도4).The sterilized LB medium was inoculated with PK 916 in Pseudomonas culture and shaken at 28 ° C. overnight. In a 3L beaker, 200 mL of this culture solution and 1 Kg of collected food waste (water content of about 80%) were added, mixed well, and placed in a 25 ° C. incubator, weighed over time, and the reduction rate was calculated. Stir well four times (Figure 4).

비교예 2 : 음식물 쓰레기에 톱밥만을 처리한 경우의 분해율 측정Comparative Example 2 Degradation Rate Measurement of Only Sawdust Treated with Food Waste

3L 비이커에 멸균된 LB 배지 200mL와 톱밥 (수분조절제) 100g을 첨가하여 잘 혼합하고, 수거한 음식물쓰레기(수분 함량 약 80%) 1Kg을 첨가하여 잘 혼합한 다음, 25℃ 배양기에 넣어 두면서 경시적으로 그의 무게를 칭량하고 감량화율을 계산하였으며, 매일 3∼4회씩 잘 교반하였다(도4).Add 200 mL of sterilized LB medium and 100 g of sawdust (moisture regulator) to a 3L beaker, mix well, add 1 kg of collected food waste (moisture content of about 80%), mix well, and place in a 25 ° C incubator over time. His weight was weighed and the reduction ratio was calculated and stirred well 3 to 4 times daily (FIG. 4).

실시예 4 : 음식쓰레기 소멸기에서의 분해·소멸 시험Example 4 Disintegration and Extinction Test in Food Waste Disposer

남방 E & B(주)에서 제작한 음식물쓰레기 소멸기(CLEANGATE-50 형, 50㎏ 용량)에 우드칩(5∼10mm 정도) 80㎏, 실시예 1의 방법으로 배양한 FK916 배양액 5L, 음식점에서 수거한 음식물쓰레기 39㎏를 첨가하고, 28℃에서 교반속도 3.2rpm, 통기속도 5.5m3/분으로 운전하면서 24시간 후 그의 무게를 칭량하여 감량화율을 계산하였다. 분해 시작 1시간 이후부터 음식물쓰레기 고유의 냄새가 없어지기 시작하였고, 24시간째에 76.9%까지 감량하였으며, 악취는 거의 발생하지 않았다. 2일째에 음식물쓰레기 46㎏을 다시 첨가하고 동일 조건에서 운전하여 24시간 후 측정하였을 때 그의 감량화율은 90.9%이었으며, 29일 동안 매일 27∼60㎏의 음식물쓰레기를 연속적으로 첨가하고 동일 조건으로 운전하였을 때 최종적인 누적 감량화율은 94.7% 이었다.Food waste decay machine (CLEANGATE-50 type, 50kg capacity) made by Nambu E & B Co., Ltd. 80kg of wood chips (about 5-10mm), 5L FK916 culture medium incubated by the method of Example 1, in a restaurant 39 kg of collected food waste was added, and the weight reduction was calculated by weighing its weight after 24 hours while operating at a stirring speed of 3.2 rpm and an aeration rate of 5.5 m 3 / min at 28 ° C. After 1 hour of decomposition, the intrinsic odor of food waste began to disappear, and by 24 hours, it was reduced to 76.9%, and no odor was generated. On the second day, 46 kg of food waste was added again and operated under the same conditions, and measured after 24 hours, the reduction ratio was 90.9% .Then, 27 to 60 kg of food waste was continuously added for 29 days and operated under the same conditions. The final cumulative reduction was 94.7%.

상기의 실험결과, 본 발명 균주인 슈도모나스 속 FK 916은 상온 또는 중온에서 1회 투여만으로도 장기간 걸쳐 음식물쓰레기를 분해·소멸시키므로, 음식물쓰레기등과 같은 유기폐기물의 분해 소멸화에 이용될 수 있는 우수한 균주임이 확인되었다.As a result of the above experiments, the present invention strain Pseudomonas FK 916 decomposes and destroys food waste over a long period of time by only one administration at room temperature or medium temperature, and thus an excellent strain that can be used for the decomposition and destruction of organic waste such as food waste. Was confirmed.

이상, 상기 실시예를 통하여 명백한 바와 같이, 본 발명 균주인 슈도모나스속 (Pseudomonassp.) FK 916 균주는 중온에서 악취를 발생하지 않으면서 음식물쓰레기 분해 소멸능이 매우 우수하므로 경제적이고 위생적으로 음식물쓰레기를 처리할 수 있어 환경산업상 매우 유용한 발명인 것이다.As is apparent from the above examples, the strain Pseudomonas sp. FK 916 of the present invention is very economically and hygienically treated because food waste decomposition and extinction is excellent without generating bad smell at medium temperature. It is a very useful invention for the environmental industry.

Claims (4)

하수로부터 분리하고, 15∼35℃의 중온에서 악취를 발생하지 않으면서 음식물 쓰레기 분해 소멸능이 있는 슈도모나스속 FK 916(Pseudomonassp. FK 916; 기탁번호 KCCM 10350). Pseudomonas sp. FK 916 ( Pseudomonas sp. FK 916; Accession No. KCCM 10350), isolated from sewage and having an ability to decompose and dispose of food waste without generating odor at a medium temperature of 15 to 35 ° C. 제1항의 슈도모나스속 FK 916 균주(KCCM 10350)를 피처리물에 첨가하는 것을 특징으로 하는 유기 폐기물 및 하수 처리방법.The organic waste and sewage treatment method according to claim 1, wherein the Pseudomonas genus FK 916 strain (KCCM 10350) is added to the object to be treated. 제2항에 있어서, 상기 슈도모나스 속 FK 916(KCCM 10350)균주를 천연유기물, 무기질 입체, 플라스틱류의 공극제로부터 선택된 하나 이상과 혼합하여 첨가하는 것을 특징으로 하는 유기 폐기물 및 하수 처리방법.The organic waste and sewage treatment method according to claim 2, wherein the Pseudomonas genus FK 916 (KCCM 10350) strain is added to at least one selected from a pore agent of natural organic matter, inorganic solid, and plastics. 제 3항에 있어서, 상기 천연유기물은 왕겨, 톱밥, 우드칩의 군으로부터 선택된 것이며, 무기질 입체는 제오라이트, 세라믹의 군으로부터 선택된 것임을 특징으로 하는 유기 폐기물 및 하수 처리방법.The organic waste and sewage treatment method according to claim 3, wherein the natural organic matter is selected from the group of chaff, sawdust and wood chips, and the inorganic solid is selected from the group of zeolite and ceramic.
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