WO2003066837A1

WO2003066837A1 - Pseudomonas sp. fk 916 producing decomposition enzyme of organic matter and method for decomposition of organic waste using the same

Info

Publication number: WO2003066837A1
Application number: PCT/KR2003/000277
Authority: WO
Inventors: Dong-Seong Choi; Kunihiro Fukai
Original assignee: Nambang Environment & Biotech Co., Ltd
Priority date: 2002-02-08
Filing date: 2003-02-08
Publication date: 2003-08-14
Also published as: JP2006501810A; CN1729286A; KR20030067887A; KR100437103B1; CN100381561C; AU2003206244A1; JP2003235547A

Abstract

The present invention relates to Pseudomonas sp. FK 916 producing decomposition enzyme of organic compounds and a method for decomposition of organic waste using the same. The Pseudomonas sp. FK 916 strain which is derived from sewage has an excellent ability to degrade and remove organic matter and carries out the decomposition at a medium temperature between 15 and 35 °C. Therefore, it does not emit bad smell and can be effectively used in treatment of organic waste from waste food and factories.

Description

PSEUDOMONAS SP. FK 916 PRODUCING DECOMPOSITION ENZYME OF ORGANIC MATTER AND METHOD FOR DECOMPOSITION OF ORGANIC WASTE USING THE SAME

Technical Field

The present invention relates to Pseudomonas sp. FK 916 producing a decomposition enzyme of organic compound and a method for treating of organic waste using the same. More particularly, it relates to Pseudomonas sp. FK 916 which is derived from sewage, has an excellent ability to degrade and remove organic matter in food waste and organic waste and can effectively treat food waste without emission of bad odor and a method for treating of organic waste using the same.

Background Art The amount of food waste created per day in Korea is 11 ,577 ton

(1999 basis) which constitutes about 26.5% of solid waste generation of a city. Of them, 64% is generated from households and 36% is generated from dining out industry such as restaurants and group feeding facilities. Of the generated food waste, 58.8% is landfilled, 7.3% is burned and 33.9% is recycled. Thus, at present, the treatment of the food waste depends largely on landfill. Meanwhile, the Metropolitan area of Seoul prohibits the direct landfill of food waste in the landfill sites in the Metropolitan area from January of 2005. Most of local self-governing bodies confront serious problems related with treatment of food waste due to the environmental pollution caused by leachate from the landfills and the increased popular complaints by the residents who live around the landfill sites. Also, for the treatment by incineration, a more expense is required as compared to inflammable waste, since the food waste has a high water content and it is hard to construct an incineration plant and to secure a site for construction owing to objection of the residents. Therefore, it is desirable to develop a economical treatment of food waste which can decompose, remove or reduce the quantity of food waste for increase recycling rate by directly using microorganisms at the place where the food waste is generated.

Food waste is hard to be treated due to nonuniform properties and morphologies and high contents of moisture and salts. Also, it is needed to find various treatment methods according to regions and places of business. Methods for Treatment and recycling of food waste which are currently studied include compostation, feeding production, anaerobic digestion, sewage incorporation treatment, and the like, in which the compostation is largely divided into large-scale compostation for wide area treatment and small-scale compostation for use in home and small shops.

Apparatuses for the small-scale compostation comprise a fermentation chamber and a drier and are disposed at apartment complexes, public feeding facilities, hotels, restaurants, and the like. However, they have a lot of problems.

The problems of the conventional compostation apparatus comprising fermentation of food waste at a high temperature to produce compost and feed include firstly, emission of sever, offensive odor because the fermentation or decomposition occurs at a high temperature, which makes it unwelcome as dislike facilities. Also, since the Korean food has a high salt content of 1 to 5%, it is impossible to use in production of compost. Secondly, since the conventional compostation apparatus uses Actinomycetes and thermophile bacteria which are activated at a high temperature to increase fermentation and decomposition rate of microorganisms and thus, the temperature of the apparatus should be kept at 50 to 60 ^°C to maintain the optimal growth temperature of the thermophile, to reduce the volume of fermentation product of food waste and to vaporize moisture. Therefore, it requires a large amount of electricity, thereby causing much operation coast.

In order to solve the above problems, the present inventors isolated and identified a new strain from sewage which have an excellent ability to decompose and remove organic substances in food waste and organic waste and can perform aerobic decomposition at a middle temperature without emission of offensive odor and completed the present invention by using the strain in decomposition and degradation treatment of food waste.

Disclosure of the Invention

Therefore, it is an object of the present invention is to provide a strain having an excellent ability to decompose and remove organic substances in food waste and organic waste. It is another objection of the present invention is to provide a method for treating organic waste using the strain according to the present invention.

In accordance with the present invention, the above and other objects can be accomplished by the provision of Pseudomonas sp. FK 916.

Pseudomonas sp. FK 916 derived from sewage can convert organic substances such as carbohydrates, proteins, fats and the like in food waste to low-molecular organic substances via aerobic decomposition at a middle temperature in the range of 15 to 35 ^°C , preferably in the range of 25 to 30 ^°C in the presence of sufficient oxygen and finally produce stable substances including C0₂, H₂0 and the like while generating a little of heat. Meanwhile, microorganisms belonging to Pseudomonas sp. Can perform denitrification of N0₃, N0₂ and NO into N₂, as shown in the following reaction scheme (I). Therefore, Pseudomonas sp. FK 916 together with other microorganisms also can remove offensive and irritative ammoniacal odor via denitrification converting NH3, one of the components eliminating the offensive odor to N₂.

Also, the strain of the present invention is characterized by not generating substances which can emit offensive odor, including indole, hydrogen sulfide, acetoin, and the like (Table 2).

The Pseudomonas sp. FK 916 strain was deposited on January 7, 2002, at the Korean Federation of Culture Collections (KCCM), One of the International Depository Authorities as Accession Number KCCM- 10350. In addition to the deposited strain, mutants of the strain showing some diversities in other properties can be included in scope of the present invention, as long as they have the 16S rRNA sequence and fatty acid composition, and the same morphological, culture and biochemical properties with the strain of the present invention. Also, in accordance with another aspect of the present invention, there is provided a method for decomposing and removing organic waste such as food waste using the Pseudomonas sp. FK 916 strain.

The method is characterized by adding the Pseudomonas sp. FK 916 strain or a composition comprising the strain to a subject to be treated. The addition of the strain or the composition is performed by, for example, direct spry of the culture fluid of the strain or a combination of the culture fluid of the strain with fillers including natural organic materials such as rice hulls, sawdust, wood chips, inorganic particles such as zeolite or ceramics, and plastics. Alternatively, the strain is adsorbed on the fillers such as natural organic materials and inorganic particles, or carriers. The fillers or carriers may be sprayed onto the organic waste or formulated according to a known method for microorganism preparation.

In the present invention, the term "a subject to be treated" refers to activated sewage sludge cake, sewage waste generated in plants as well as organic waste.

Brief Description of the Drawings

The above and other objects, features and advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with accompanying drawings, in which:

Fig. 1 shows the result of an experiment in which the Pseudomonas sp. FK 916 strain according to the present invention is inoculated into a plate containing agar culture medium for protease assay and the protease activity was measured; Fig. 2 shows the result of an experiment in which the Pseudomonas sp. FK 916 strain according to the present invention is inoculated into a plate containing agar culture medium for lipase assay and the lipase activity was measured; Fig. 3 is a photograph of Pseudomonas sp. FK 916; and

Fig. 4 is a graph showing the reduction in weight of the food waste according to decomposition time after the culture fluid of Pseudomonas sp. FK 916 is added to food waste.

Best Mode for Carrying Out the Invention

Now, the present invention will be described in detail through the following examples. It would be apparent to those skilled in the art that the examples are only for illustration of the present invention and the present invention is not limited thereto.

Example 1: Isolation of strain

The strain according to the present invention was isolated from the sewage collected around Cholabuk-do, in Korea. The sewage sample was stepwise diluted properly with physiological saline solution. A part was applied on LB agar medium containing bactotryptone lOg/L, bacto-east extract

5g/L, NaCl l Og/L, agar 15g/L, pH 7.0) and incubated at 25 ^°C . Expressed colonies were separated.

The separated colonies were cultivated in the proliferation medium

(LB liquid medium) at 25 ^°C . The strain then applied on plates containing the agar medium for protease assay (skim milk agar medium: peptone 5g/L, bacto yeast extract 3g/L, skim milk lg/L, agar 15g/L, pH 7.0), the agar medium for lipase assay (tributyrin agar medium: peptone 5g/L, bacto yeast extract 3g/L, tributyrin 5mL/L, agar 15g/L, pH 7.4), the agar medium for amylase assay (starch agar medium: peptone 5g/L, bacto yeast extract 3g/L, soluble starch 2g/L, agar 15g/L, pH 7.0), and the agar medium for cellulose assay (cellulose lg/L, K2HP04 lg/L, (NH4)2S04 lg/L, MgS04- 7H20 0.2g/L, CaC12 - 2H20 0.1 g/L, FeC13 0.02g/L, congo red 0.075g/L, agar 15g/L, pH 7.2). Each of the plates was examined for transparence circle produced by action of protease, lipase, amylase, cellulase to detect and isolate positive strains.

As can be seen from the result shown in Fig. 1 , the plate (A) containing the agar medium for protease assay (A) and the plate (B) containing the agar medium for lipase assay produce transparent circles after inoculation of the FK 916 strain. Therefore, it was noted that the strain produced protease and lipase. However, it was shown that the amylase activity was weak and no cellulase activity was admitted.

Example 2: Identification of strain

The strain isolated in Example 1 was identified according to the method described by Holt et al. (Holt et al., Bergey,s Manual of Determinative Bacteriology, 9th ed. (1994)) using the Sherlock system and clustal W. The strain was found to be a strain belonging to Pseudomonas sp. and named Pseudomonas sp. FK 916. This new Pseudomonas sp. FK 916 showed similarity in fatty acid composition of 80.2 % with Pseudomonas chlororaphis and similarity in rRNA of 99.8% with Pseudomonas gessardii CIP 105469. As seen from the photograph shown in Fig. 3, the strain was found to be a gram-negative bacterium having morphology of bacillus. The cultural characteristics included an optimal growth temperature of 28 to 30 ^°C , resistance to NaCl of 6%, as shown in Table 1, below. Therefore, it was noted that the strain of the present invention is suitable for treatment of food waste having a high salt content. Table 1 Cultural and Morphological Characteristics of Pseudomonas sp. A^" 916

Characteristics Results

Morphological characteristics Shape and size Bacillus, 0.8 ~ 0.9 x 1.7 - 2.0 Mobility + (positive) Gram staining - (negative)

Cultural characteristics

Growth pH 4.5-9.0

Growth temperature 10~37 °C

Growth at 4^°C +

Growth at 41 ^°C

Optimal growth temperature 28-30 ^°C

Resistance to NaCl 6%

Mac ConKey's agar +

Tryptophan agar +

(Pseudomonas isolation agar reaction)

The biochemical characteristics analyzed by the API kit are shown in Table 2 and the results of the fatty acid analysis of the whole cell by gas chromatography according to the method described by MIDI Com. and the identification of the stain through the Sherlock system are shown in Table 3. Also, 16s rRNA was isolated and sequenced according to the method described by Verhille et al. (Verhille et al., International J. Systematic Bacteriol., 49, 1559, 1999). As a result, it was found that the stain is composed of 1498 bases (Seq. ID. No.: 1 ). Microorganisms with a high significance which had been screened by Gene bank were examined for their similarities by comparing the rRNA base sequence with other strains belonging to Pseudomonas sp. using the clustal W. The results are shown in Table 4. The G+C content analyzed according to the De Ley method (De Ley, J., J. Bacteriol., 101, 738, 1970) was found to be 61.5%.

Table 2

Biochemical characteristics of Pseudomonas sp. FK 916

Characteristics Results

Biochemical characteristics

Reduction of nitrate to nitrite

Production of indole

Production of hydrogen sulfide

Production of acetoin +

Reduction to N₂ gas

Glucose oxidation Oxidized

Fermentation by glucose oxidation +

Protease +

Lipase +

Amylase

Cellulase

Gelatinase

Beta-galactosidase +

Arginine dehydrolase

Lysine decarboxylase

Ornithine decarboxylase +

Cytochrome oxidase

Tryptophane deaminase +

Urease

Beta-glucosidase (esculin hydrolysis) +

Utility of carbon source

Glucose +

Arabinose

Mannose +

Mannitol +

N-acetyl glucosamine +

Maltose

Gluconic acid +

Capric acid +

Adipic acid +

Malic acid

Citric acid +

Phenyl acetate Table 3

Fatty acid composition

Fatty acids Percentage (%)

C 10:0 0.20

C 12:030H 2.00

C 12:0 1.67

C 12:020H 4.73

C 12:030H 4.27

C 14:0 0.50

C 15:0 0.45

C15:0iso20H 30.01

C 16:1 w7c

C16:l w5c 0.12

C 16:0 33.94

C 17:1 w8c 0.27

C17:0cyclo 6.64

C 17:0 0.56

C 18:3 w6c(6, 9, 12) 0.63

C 18:l w7c 12.34

C18:0 1.48

C 19:0cyclow8c (U5

Identified strain (similarity) Pseudomonas chlororaphis (80.2%)

(P. aureofaciens)

P. putida biotype B (70.9%)

P. putida biotype A (68.7%)

P. syringae (41.5%)

Table 4

Similarity in 16S rRNA of Pseudomonas sp. FK 916 with strains belonging to

Pseudomonas sp.

Strains Similarity Number of different bases/number of compared bases

Pseudomonas gessardii CIP 99.80 3/1491 105469

P. libaniensis CIP 105469 99.73 4/ 1491

P. synxantha DSM 13080 99.53 7/ 1498

P. reactans LMG 5329 99.53 7/ 1478

P. brenneήi CFML 97-391 -T 99.52 7/ 1470

P. cedrella CFML 96- 198 99.40 9/ 1491

P. fluorescens ATCC 49642 99.33 10/ 1498

P. mucidolens I AM 12406 99.26 1 1/1496

P. azotoformans 1AM 1603 98.93 16/ 1496

P. migulae CIP 105470 98.93 16/ 1491

P. putida ATCC 17472 98.85 17/ 1476

P. tolaasii LMG 2342 98.58 21/1478

P. thivervalensis CFBP 1 126T 98.1 1 27/ 1431

P. fragi ATCC 4973 97.87 31/ 1458

P. jessenii CIP 105469 97.85 32/ 1491

P. pavnaceae IAM 1 155 97.80 33/1497

P. plecoglossicida FPC951 96.79 48/ 1495

P. oryzihabitans IAM 1568 96.46 53/1496

P. mosselii CIP 105259 96.44 53/ 1490

P. monteilii CIP 104883T 96.35 54/1478

Example 3: Decomposition and removal of food waste: Reduction in weight of food waste according to decomposition time

In order to examine the reduction in weight of food waste, Pseudomonas sp. FK 916 was inoculated into sterilized LB liquid medium and incubated at 28 ^°C for one day and night while shaking. 200 mL of the culture fluid and 100 g of sawdust (for ventilation and moisture control) were putted in a 3L beaker and thoroughly mixed. 1 Kg of food waste collected in a cafeteria of the W university (moisture content of about 80%) was added thereto and thoroughly mixed. The mixture was placed in a cultivator at 25 °C while stirring after weighing the initial weight. The weight of the mixture was measured 3 to 4 times everyday and the reduction rate was calculated.

As seen from the results shown in Fig. 4, from the first day of composition, the intrinsic offensive odor of food waste disappeared and new bad odor was not emitted while the weight of the waste shows a reduction. At the 18^th day after decomposition, the weight of the waste was reduced to 72.3%, which showed that the food waste was decomposed and removed. The reduction was faster than Comparative Example 1 wherein only the culture fluid was added and Comparative Example 2 wherein the LB medium and sawdust were added. From these results, it was noted that the addition of sawdust allowed the aerobic condition satisfied and the moisture content controlled, thereby promoting decomposition and removal of organic substances. Also, it was noted that the decomposition and removal of organic substances by the FK 916 strain are optimally carried out at 25 °C . Further, it was suggested that a more optimal result can be obtained in a zero-discharger of food waste to which air is continuously supplied by agitation.

Comparative Example 1 : Measurement of decomposition upon treatment of food waste with culture fluid of Pseudomonas sp. FK916 Pseudomonas sp. FK 916 was inoculated into sterilized LB liquid medium and incubated at 28 ^°C for one day and night while shaking. 200 mL of the culture fluid and 100 g of sawdust (for ventilation and moisture control) were putted in a 3L beaker and thoroughly mixed. 1 Kg of food waste (moisture content of about 80%) was added thereto and thoroughly mixed. The mixture was placed in a cultivator at 25 ^°C while stirring after weighing the initial weight. The weight of the mixture was measured 3 to 4 times everyday and the reduction rate was calculated (Fig. 4).

Comparative Example 2: Measurement of decomposition upon treatment of food waste with sawdust

200 mL of sterilized LB liquid medium and 100 g of sawdust (for moisture control) were putted in a 3L beaker and thoroughly mixed. 1 Kg of food waste (moisture content of about 80%) was added thereto and thoroughly mixed. The mixture was placed in a cultivator at 25 ^°C while stirring after weighing the initial weight. The weight of the mixture was measured 3 to 4 times everyday and the reduction rate was calculated (Fig. 4).

Example 4: Decomposition and removal of food waste in a zero-discharger 80 Kg of wood chips (particle size: about 5 to 10mm), 5 L of the culture fluid of FK 916 cultured according to the method described in Example 1 , 39 kg of food waste collected from restaurants were added to a zero- discharger (CLEANGATE-50 model, capacity: 50 kg) of food waste, produced by Nambang E & B Co., Ltd. The apparatus was operated at a temperature of 28 ^°C , agitation speed of 3.2rpm, ventilation rate of 5.5m³/min. After 24 hours, the contents in the apparatus were weighed and the weight reduction was calculated. From 1 hour after the decomposition was initiated, the offensive odor of the food waste began to disappear. 24 hours later, the weight of the contents was reduced to 76.9%, while emitting little odor. At the second day of the decomposition, 46 kg of food waste further added and the apparatus operated at the same conditions. After 24hours, the weight reduction was found to be 90.9%. For 29 days, 27 to 60kg of food waste was added everyday and the apparatus was operated at the same conditions. The final accumulated reduction rate was 94.7%. From the results of this experiment, it was proved that the

Pseudomonas sp. FK 916 strain according to the present invention can decompose and degrade waste food for an elongated period of time at room or middle temperature by a single administration and is, thus, an effective strain for use in decomposition and removal of organic waste such as food waste. Industrial applicability

As described above through the Examples, since the Pseudomonas sp. FK 916 strain according to the present invention has an excellent ability to decompose food waste at a middle temperature without emission of offensive odor, it can be economically and hygienically used in the treatment of food waste and is useful in terms of the environmental industry.

Claims

What is claimed:

1. Pseudomonas sp. FK 916 strain(Accession Number KCCM 10350) derived from sewage which can decompose food waste at a middle temperature of 15 to 35 °C without emission of offensive odor.

2. A method for treating organic waste and sewage comprising adding Pseudomonas sp. FK 916 strain (KCCM 10350) defined in claim 1.

3. The method according to claim 2, wherein said Pseudomonas sp. FK

916(KCCM 10350) is added in combination with any one selected from natural organic materials, inorganic particles and plastics as a filler.

4. The method according to claim 3, wherein said natural organic materials are selected from the group consisting of rice hulls, sawdust and wood chips and said inorganic particles are selected from the group consisting of zeolite and ceramics.