KR100452125B1 - Novel Bacillus subtilis BRD-007 strain having food waste decompositing capability and microbial agent for food waste treatment using it - Google Patents

Abstract

The present invention relates to a novel microbial agent for the fermentation and extinction of food waste, which is a waste organic resource among household wastes, and has a protease and lipase enzymatic activity and excellent food waste degradation activity. Bacillus subtilis BRD-007 -007) strain (Accession No: KCTC 10268BP) and the fermentation of food waste destruction Purification microbial agent is provided, it characterized in that it comprises the strain.

The strain of the present invention is a novel microorganism having fast cell growth and excellent protease and lipase enzyme activities, and has an excellent effect on the fermentation and extinction of food waste.

Description

Novel Bacillus subtilis BRD-007 strain having food waste decompositing capability and microbial agent for food waste treatment using it}

The present invention relates to a new strain and microbial agent for the fermentation and extinction of food waste, which is a waste organic resource in household wastes. Scotland BRD-007 (Bacillus subtilis BRD - 007, accession number: KCTC 10268BP) and relates to the fermentation of food waste destruction pragmatic microbial agent comprising the strain.

The daily discharge of food waste ranges from 12,000 to 20,000 tons, of which only a few are fed or composted and recycled, but most of them rely on landfilling or incineration, resulting in serious social problems. Food wastes are easily decayed with 80-85% moisture content, which makes odor and sewage difficult to separate and transport.In landfills, large amounts of leachate flow out, causing secondary environmental pollution such as groundwater contamination and treating leachate. Not only does it cost a lot of money, but it also has the advantage of simple treatment technology in landfilling or incineration. There is also a lot of loss.

Therefore, if a treatment facility is installed at the source of food waste generation and processed immediately, it can fundamentally solve the occurrence of food waste, and induces complete disappearance of food waste generated in an economical and safe way at the primary site. Various hazards caused by pollution can be reduced.

One such method is the method of extinction by fermentation using fermentation (aerobic fermentation). Such fermentation processing devices are being actively researched not only in Korea but also in Japan. However, the reliability of the developed processing technology is quite low, and even the installed processing devices do not show proper process efficiency. It is true that the treatment method using 1) has been avoided by users due to problems such as 1) generating a lot of odors, 2) complete extinction, and 3) noise and maintenance costs. In addition, most of them do not show a substantial weight loss efficiency by decomposition of organic matter by microorganisms by simple drying.

Accordingly, an object of the present invention is to provide a novel strain suitable for the fermentation annihilation of food waste.

Another object of the present invention to provide a microbial agent for the fermentation and extinction of food waste using the strain.

1 is a result of comparing the nucleotide sequence of 16S rDNA of the strain of the present invention and Bacillus subtilis ( Bacillus subtilis) .

According to one aspect of the invention, there is provided a new strain Bacillus subtilis BRD - 007 (Accession No .: KCTC 10268BP) having protease and lipase enzymatic activity and excellent food waste degradation activity.

According to another aspect of the present invention, there is provided a microbial preparation for fermentation and extinction of food waste, comprising the strain.

The present inventors selected a novel microbial strain to ferment annihilate waste organic resources such as food waste and successfully carry out fermentation annihilation using the microorganism. In the fermentation annihilation of food waste, organic matter is biologically decomposed by the microbial preparation in which food waste is put into the fermenter, and pathogens are killed by continuous fermentation heat, and finally complete extinction can be achieved. Therefore, the present inventors selected only microorganisms suitable for the purpose of fermentation annihilation according to the type and characteristics of microorganisms involved in fermentation annihilation.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present inventors have isolated microbial strains with excellent enzymatic activity and rapid growth rate from compost in a farm in Icheon, Gyeonggi-do for the fermentation and extinction of food wastes, and the partial bases of 16's manual and 16s rDNA. Strain identification was performed by investigating the morphological, physiological and biochemical properties of the strains of the present invention according to sequence analysis.

As a result, as shown in Table 1 below, the new strain of the present invention is Gram-positive bacteria, bacilli having motility and sporulation ability. The strain had catalase and oxidase production, had citrate availability and starch, gelatin, casein resolution, and nitrate reducing capacity. There was no urea resolution, no indole production was observed, and acetoin production was shown. As a result of the carbon source fermentation ability, all sugars such as glucose, arabinose, mannitol and xylose produced acid, and no gas production from glucose was observed.

In order to identify the strain, the nucleotide sequence of 16S rDNA was analyzed by molecular genetic method. The results are shown in SEQ ID NO: 1, using the base sequence to identify the strain through a blast search (blast search) on Genebank, as shown in Table 2 and Figure 1, the base sequence The homology with Bacillus subtilis was shown on the determined 484 bp. Taking the above morphological, physiological and molecular genetic characteristics together, the strain was identified as Bacillus subtilis and named it Bacillus subtilis BRD-007, and the Korea Institute of Science and Technology (KCTC, Korean Collection for Type Cultures) was deposited on June 5, 2002 (accession number: KCTC 10268P).

Characteristics of Novel Microbial Strains Bacillus subtilis BRD-007 of the Invention factor BRD-007 Morphological Characteristics Gram Dyeing Shape Size (㎛) Length (㎛) Bacillus 0.7-0.81.3-2.3 ++ Cultivation Characteristic ColorAerobic Culture Anaerobic Culture Growth Temperature 40 ℃ 65 ℃ White +-++ Physiological properties Catalase production Oxidase production Acid production from carbohydrate (OF ¹ ) Acid production from carbohydrate Glucose Mannitol Arabinos xylose citrate Lactoxydase Voges-Proskauer (VP) Test +++ F +++++++++ --- ++

¹ Oxidation-Fermentation Test

Partial Sequence Analysis of 16s rDNA of the Novel Microbial Strain Bacillus subtilis BRD-007 Strain Similar strain Similarity (%) ^* Sequence length BRD-007 Bacillus subtilis 100% (484/484) 484

* Similarity obtained through "The BLAST search"

Since the novel microbial strain Bacillus subtilis BRD-007 of the present invention has excellent xylanase and amylase enzymatic activity and thus has excellent degradation activity, it can be very useful as a microbial agent used to ferment and destroy food waste.

When the strain of the present invention is used as a microbial preparation for fermentation and extinction of food waste, it is preferable to use the strain of the present invention by adsorbing on a preservation carrier (growth substrate), and suitable preservative carriers are, for example, skim steel, selfies. (CellCaSi), activated carbon, diatomaceous earth and white carbon. The method of adsorbing the strain of the present invention to the carrier may be achieved by mixing the culture medium and the carrier, and the mixing ratio is not particularly limited, but preferably, the culture medium and the carrier are mixed in a volume ratio of 1:10 to 5:10. . In addition, chaff may be added at this time for the role of moisture control and food shredding effect.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 Bacillus subtilis ( Bacillus subtilis ) Isolation of BRD-007 Strains

Strains having the protease and lipase enzyme activities of the present invention are sampled from compost in a farm in Icheon, Gyeonggi-do, and the samples are diluted with 10 ^-3 , 10 ^-4 , 10 ^-5 with 0.85% physiological saline and each enzyme. Inoculated into a solid plate medium containing the substrate (Table 3) and incubated at 30 ℃ for 2 days. Then, the cultured bacteria were again plated in the same medium and incubated at 30 ° C. for 2 days. Subsequently, the colonies formed were successively passaged to isolate pure strains, followed by partial sequencing analysis of the B's Manual of Determinative Bacteriology and 16s rDNA to determine the morphological, physiological and biochemical properties of the strains of the present invention. Strain identification was carried out by investigation and the results are as shown in Table 1 above.

Example 2 Preparation of Microbial Agents for Fermentation and Extinction of Food Waste

The microorganism strain Bacillus subtilis BRD-007 of the present invention was adsorbed onto the carrier by mixing with degreasing steel, CelCaSi, activated carbon, diatomaceous earth and white carbon, respectively, as a carrier. Microbial preparations for fermentation and extinction were prepared.

In the preparation method of the microbial preparation of the present invention, the seed culture was inoculated in the primary liquid medium (LB medium: Bacterium-Tryptone 1g / L, yeast extract 0.5g / L, NaCl 1g / L) and incubated at 25 ° C. for 24 hours. Next, using a degreasing steel, CelCaSi, activated carbon, diatomaceous earth or white carbon as a carrier, evenly mixed at a volume ratio of carrier 10: culture medium 4, and then dried at 25 ° C. for 24 hours to solidify. Then, the secondary solid culture for 24 hours at 55 ~ 60 ℃ using a saponification reaction to prepare a microbial agent for fermentation and extinction to which the strain Bacillus subtilis BRD-007 of the present invention was adsorbed.

Example 3 Evaluation of Food Waste Fermentation Extinction Capacity of the Strains of the Invention

In this example, after measuring the microbial change and enzyme activity (DNS method) in the fermentation and extinction process, the effects and effects of the microbial strain of the present invention were examined.

Example 3-1

Each substrate (amylase: water-soluble starch, protease: skim milk, cellulase: cellulase, lipase: olive oil, xylanase: xylan) as shown in Table 3 using the microbial preparation of the present invention prepared in Example 2 ), The activity of amylase, protease, cellulase, lipase and xylanase enzyme was measured by DNS method using a solid plate containing. The enzyme activity measurement results are shown in Table 4 below.

Solid flat media composition Ingredient (g / l) Amylase Protease Cellulase Lipase Xylanase Xylan 5 Soluble starch 5 CMC 5 Skim milk 10 Tributyrin (Olive Oil) 5 gum arabic 5 peptone 5 30 5 3 5 Yeast extract 5 10 5 5 5 K ₂ HPO ₄ One 10 One One MgSO ₄ 7H ₂ O 0.1 2 0.1 0.1 NaCl 2 10 2 5 2 Baby 20 20 20 20 20

Enzyme Activity of Bacillus subtilis BRD-007 (Unit / ml) Enzyme Activity (Unit / ml) BRD-007 Amylase - Protease 1.6750 Cellulase - Lipase 0.2471 Xylanase -

As shown in Table 4, after culturing the strain of the present invention in a liquid medium for 4 days, the enzyme activity was measured, the activity of the protease was found to be large and lipase activity. The strain was found to exhibit great activity during fermentation and extinction.

Example 3-2

When the strain of the present invention was applied to food waste fermentation extinction using various kinds of carriers, the optimum carrier was investigated. In the microbial preparations prepared using the various carriers of Example 2, the number of viable cells of the novel strains of the present invention according to each carrier was confirmed, and the number of viable cells in the produced preparations was examined, and the results are shown in Table 5 below. Shown in Among the carriers in which this strain was used, more viable cell counts were measured in the degreasing steel than the CellCaSi at 27.97 × 10 ⁸ / g, indicating that the degreasing steel is an optimal carrier to be used for the production of microorganisms.

Change of viable cell count in carrier sanctions BRD-007 SelfCaSi 1.35 × 10 ⁸ Diatomaceous earth 19.75 × 10 ⁸ White carbon 10.6 × 10 ⁸ Activated carbon 0 Degreasing Steel 27.97 × 10 ⁸

In addition, each microbial agent was investigated for the extinction activity according to the type of carrier used. The change in viable cell number and pH according to the type of carrier was investigated, and the results are shown in Table 6 below. When each microbial agent was reacted with various food wastes (food wastes discharged from the institute's restaurant), the number of viable cells increased as a whole, of which 46.1 × 10 ⁸ was the highest in the degreasing river, and was the highest in CelCaSi or white. In white carbon and activated carbon, the number of viable cells was increased in the same number as the control group without the microbial agent. The pH was not significantly different from the pH at the beginning and after 24 hours of reaction.

Change of viable cell number and pH according to the type of carrier carrier Viable cell count (CFU / g) pH 0h 24h Increased viable cell count 0h 24h Control 8.8 × 10 ⁷ 6.6 × 10 ⁸ 5.7 × 10 ⁸ 4.4 4.1 Selfie 13.4 × 10 ⁷ 6.7 × 10 ⁸ 5.4 × 10 ⁸ 5.3 5.2 Diatomaceous earth 7.37 × 10 ⁷ 18.6 × 10 ⁸ 17.9 × 10 ⁸ 4.5 4.7 White carbon 31.4 × 10 ⁷ 8.9 × 10 ⁸ 5.8 × 10 ⁸ 4.9 4.5 Activated carbon 10.7 × 10 ⁷ 6.4 × 10 ⁸ 5.3 × 10 ⁸ 5.4 5.0 Degreasing Steel 44.6 × 10 ⁷ 50.6 × 10 ⁸ 46.1 × 10 ⁸ 5.0 4.3

In addition, the change in the total sugar, reducing sugar, total nitrogen in the microbial preparation according to the type of carrier is shown in Table 7 below. The initial total sugar was found to be reduced as an energy source due to the rapid proliferation of microorganisms in all kinds of microbial agents, and the reducing sugars were also reduced by continuous decomposition. Among them, microbial preparations prepared using skim steel as carriers reduced total sugars from 20.3 g / l to 9.4 g / l, reducing sugars from 3.7 g / l to 1.0 g / l, and total nitrogen was reduced to all kinds of microorganisms. It was found to be reduced in similar proportions in the formulation.

Changes in Total Sugar, Reducing Sugar and Total Nitrogen According to the Type of Carrier carrier Total sugar (g / l) Reducing Sugar (g / l) Total nitrogen (g / l) 0h 24h 0h 24h 0h 24h Control 8.6 4.1 1.7 0.5 0.5 0.2 Selfie 8.7 2.5 2.6 0.4 0.5 0.2 Diatomaceous earth 9.5 1.7 2.6 0.5 0.5 0.3 White carbon 9.1 4.1 2.6 0.5 0.5 0.2 Activated carbon 10.1 0.4 2.7 0.3 0.5 0.2 Degreasing Steel 20.3 9.4 3.7 1.0 0.8 0.6

Example 4 Medium Enzyme Operation

2.2 kg of the microbial agent prepared by the method of Example 2 using the degreasing steel as a carrier were administered together with 8 kg of rice hulls together in an extinction container (30 kg working volume / 50 kg fermentation and extinguisher, 30 ° C.), and then mixed and operated for 1 hour. . Every day, about 30 kg of the remaining food waste (food waste discharged from the restaurant in the institute) was added. Stirring, air, and water spray were intermittently operated using an automatic on / off timer (30 times / 4min, atmospheric 25min, water spray 20L / min). The reactor did not provide additional heating and air to evaluate the fermentation heat generated during the decomposition of organic matter by microorganisms, and the fermentation heat was kept constant at about 30 ° C during the experiment due to water feeding. A total of 600 kg of food was added in 25 days, and after 25 days, the amount of food left was 60 kg and the weight loss rate was calculated to be about 90%. The remaining degraded material (560 kg) is believed to be converted to water, carbon dioxide and ammonia gas. Considering that the foods that were administered included substances such as ribs, the actual weight loss rate would be higher.

Therefore, in the fermentation annihilation using the microbial agent of the present invention for food waste, it is possible to shorten the food extinction time due to the active metabolic process and have high enzymatic activity to maintain the degradation activity for the entire period before extinction Can be performed successfully.

Bacillus subtilis ( Bcillus subtilis) BRD-007 according to the present invention is a novel microbial strain having fast cell growth and protease and lipase enzymatic activity can be very useful for the fermentation and extinction of food waste.

<110> BioR & Ds <120> Novel Bacillus substilis BRD-007 strain having food waste decompositing capability and microbial agent for food waste treatment using it <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 484 <212> DNA < 213> Bacillus substilis BRD-007 <400> 1 gtgcctaata catgcaagtc gagcggacag atgggagctt gctccctgat gttagcggcg 60 gacgggtgag taacacgtgg gtaacctgcc tgtaagactg ggataactcc gggaaaccgg 120 ggctaatacc ggatggttgt ttgaaccgca tggttcaaac ataaaaggtg gcttcggcta 180 ccacttacag atggacccgc ggcgcattag ctagttggtg aggtaacggc tcaccaaggc 240 aacgatgcgt agccgacctg agagggtgat cggccacact gggactgaga cacggcccag 300 actcctacgg gaggcagcag tagggaatct tccgcaatgg acgaaagtct gacggagcaa 360 cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct gttgttaggg aagaacaagt 420 accgttcgaa tagggcggta ccttgacggt acctaaccag aaagccacgg ctaactacgt 480 gcca 484

Claims (4)

Protease and lipase enzyme having a degrading activity of the active food waste is excellent Bacillus subtilis BRD-007 (Bacillus subtilis BRD - 007) strain (Accession No: KCTC 10268BP). Microbial preparation for fermentation and extinction of food waste, characterized in that it comprises the strain of claim 1. The microorganism preparation for fermentation and extinction of food wastes according to claim 2, wherein the strain is adsorbed on a storage carrier. The microorganism agent for fermentation and extinction of food wastes according to claim 2, wherein the preservative carrier is selected from any one or a mixture of skim steel, CelCaSi, activated carbon, diatomaceous earth, and white carbon. .