KR100443267B1 - Novel Bacillus licheniformis H-3 strain having capability of food waste decomposition - Google Patents

Abstract

The present invention relates to a new strain Bacillus rickeniformis H-3 (KCTC 10273BP) excellent in waste organic matter decomposition activity, more specifically Bacillus rickenformis H- as a novel microorganism with fast cell growth and excellent enzyme activity. 3 ( Bacillus licheniformis H-3) [KCTC 10273BP] is a solid culture and using the same relates to a novel microbial agent for the fermentation and extinction of food wastes, waste organic resources of household waste.

Description

Bacillus rickeniformis H-3 (Novel Bacillus licheniformis H-3 strain having capability of food waste decomposition)

The present invention relates to a new strain Bacillus rickeniformis H-3 (KCTC 10273BP) excellent in waste organic matter decomposition activity, more specifically Bacillus rickenformis H- as a novel microorganism with fast cell growth and excellent enzyme activity. 3 ( Bacillus licheniformis H-3) [KCTC 10273BP] is a solid culture and using the same relates to a novel microbial agent for the fermentation and extinction of food wastes, waste organic resources of household waste.

The daily discharge of food wastes ranges from 12,000 to 20,000 tons, and only a few of them are recycled by feeding or composting, but most of them rely on landfilling or incineration, which is a serious social problem. Food waste is easily decayed with 80 ~ 85% water content, which makes odor and sewage difficult to separate and transport.In case of landfill, large amount of leachate flows out, causing secondary environmental pollution such as groundwater pollution 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 raw waste generation unit and treated immediately after occurrence, it is possible to fundamentally solve the occurrence of food waste, and induce a complete disappearance of the generated food waste at the primary site as an economical and safe method. Various hazards from secondary pollution can be reduced.

One such method is the method of extinction by fermentation using fermentation (aerobic fermentation). Such fermentation treatment devices are being actively researched not only in Korea but also in Japan, but the reliability of the developed treatment technology is quite low, and even the installed treatment devices do not show proper process efficiency. It is true that the treatment method using odor generated a lot of odor, could not induce complete extinction, and was avoided by users due to problems such as noise and maintenance cost. In addition, most of them do not show a substantial reduction efficiency due to decomposition of organic matter by microorganisms in a simple drying method.

Accordingly, the present inventors have completed the present invention by separating the bacillus Bacillus rickenformis H-3 for the fermentation and extinction of food wastes in view of the above, and succeeding in the fermentation and extinction of food wastes using the same. It became.

Accordingly, it is an object of the present invention to provide a new strain Bacillus rickeniformis H-3 suitable for the fermentation and extinction of food waste.

Another object of the present invention to provide a microbial agent using the strain.

1 is a graph showing the amount of CO ₂ generated from food waste according to pH.

The present invention is characterized by the new strain Bacillus rickeniformis H-3 (KCTC 10273BP) and a microbial agent comprising the same.

Referring to the present invention in more detail as follows.

The present invention isolates Bacillus rickeniformis H-3 [KCTC 10273BP] as a novel microorganism with fast cell growth and excellent enzymatic activity, and using this, a new microorganism for the fermentation and extinction of food waste, which is a waste organic resource, from domestic waste. It relates to a formulation.

In the fermentation annihilation of food wastes, organic matters are biologically decomposed by the microorganisms in which food wastes are put into the fermenter, and pathogens are killed by continuous fermentation heat, and finally complete extinction can be achieved. Therefore, since the value of the present invention is large depending on the type and characteristics of microorganisms involved in fermentation and extinction, only microorganisms suitable for the purpose of fermentation and extinction should be selected.

The present invention is a microbial strain having excellent enzymatic activity and antagonism and rapid growth rate for the fermentation and extinction of food wastes, taking a sample from the compost, diluting the sample with physiological saline to inoculate the solid plate medium containing enzyme substrate After separating the colonies showing halo by incubating at 30 ℃ for 2 days, and incubated for 30 days at 30 ℃ in a liquid medium of the same composition to measure the enzyme activity of the culture supernatant. Morphological, Physiological, Biochemical and Genetic Properties of the Strains of the Invention According to Partial Sequence Analysis of the Burge's Manual of Determinative Bacteriology and 16 s rRNA The strain was identified by investigating.

As a result, as shown in Table 1, the isolated strain forms a circular colony in the solid medium, and the colony color was white. This strain was Gram-positive bacteria, motility and spore-forming ability, and is a combination anaerobic strain capable of growing under aerobic and anaerobic conditions. It also had catalase and oxidase production, had citrate availability and starch, gelatin and casein resolution, and had nitrate reducing capacity. There was no fermentation capacity of glucose and acid was produced under aerobic conditions from glucose and maltose as carbon sources.

As a result of performing sequencing of 16S rDNA by molecular genetic method for identification of the isolate strain, the novel strain of the present invention was subjected to Bacillus lis through "The BLAST search" on 492 bp [SEQ ID NO: 1] where the sequencing was determined. It was confirmed to have high homology with Kenny Forms [Table 2]. Based on the above morphological, physiological and molecular genetic characteristics, the isolated strain was identified as Bacillus rickenformis and named Bacillus rickenformis H-3 and deposited in the Gene Bank of Korea Biotechnology Research Institute, an international microorganism depositing institution. The accession number KCTC 10273BP was granted on 12 June 2002.

factor H-3 Morphological Characteristics Gram Dyeing Shape Size (㎛) Length (㎛) Motile Spore Formation + Bacterium ++ Culture Characteristics Aerobic Culture Growth Culture Temperature 5 ℃ 30 ℃ 40 ℃ ++++ Physiological Properties Catalase Production Oxidase Production Acid Production from Glucose Carbohydrate (OF ¹ ) Acid Production from Carbohydrate Glucose Maltose Arabinos Xylose Citrate test +++ O ++++-+++ ¹ Oxidation-Fermentation Test

Strain Similar strain Similarity (%) Sequence length H-3 Bacillus rickeniformis 100% (492/492) 492

The microbial new strain Bacillus liqueniformis H-3 (KCTC 10273BP) of the present invention has 100% similarity as shown in Table 2, but it is only 492 bp in which the base sequence is determined among 16srDNA sequences of about 1500 bp in total. The bacterium Bacillus rickeniformis H-3 (KCTC 10273BP) was found to have a variety of higher enzyme activity than known Bacillus rickeniformis.

New strain Bacillus rickenformis H-3 shows excellent enzymatic activity and growth, and it is adsorbed on skim steel, diatomaceous earth or complex carrier to prepare microbial preparation.

Such microbial agents can shorten food extinction time due to active metabolism and have high enzymatic activity so that fermentation and extinction can be carried out by maintaining degradation activity for the entire period of extinction.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Determination of Enzyme Activity of New Strains

The new strain Bacillus rickeniformis H-3 (KCTC 10273BP) was incubated in each liquid medium except for agar with the composition shown in Table 3 below at 30 ° C. for 4 days, and the enzymatic activity of the culture supernatant was measured. Amylase, Cellulase and Xylanase enzyme activities were measured using the DNS method, Protease enzyme activity was measured using the Bradford assay method, and lipase enzyme. Activity was measured using Agar diffusion method and Ota & Yamada method. As shown in the following Table 4, amylase activity was shown to be large, and protease, cellulase, and xylanase were also retained, indicating that the new strain exhibited great activity during fermentation and extinction.

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 * carboxy methyl cellulose

enzyme Bacillus rickeniformis H-3 (Unit / mL) Amylase 0.3257 Protease 57.2 Cellulase 0.1753 Lipase - Xylanase 0.2198

Example 2: Activity of New Strains According to Carrier

In order to investigate the optimum carrier used for food waste fermentation extinction, various carriers of Table 5 were used. The bacterium was recovered by centrifugation of the new strain Bacillus rickeniformis H-3 for liquid culture for 24 hours. The recovered cells were suspended in distilled water at a ratio of 1: 1 (v / v), uniformly mixed with each carrier at a ratio of 1: 1 (w / w) by weight, and then dried at 30 ° C. for one day. Used. Each carrier (degreasing steel, diatomaceous earth, selfie, white carbon, activated carbon) was confirmed the number of viable cells of the new strain, and the number of viable cells in the produced preparations were examined and shown in Table 5 below. Among the carriers in which the strain was used, the change in the number of viable bacteria with the largest amount of degreasing steel was measured.

carrier Viable cell count (CFU / g) Selfie 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, the results of examining the change in the number of microorganisms during the extinction process for each type of preparation produced by each carrier are shown in Table 6. When the preparation was reacted with food, the total number of viable cells was increased, among which the degreasing steel was the most increased as 46.1 × 10 ⁸ , and the control group which did not receive the preparation was CelCaSi, white carbon or activated carbon. The number of viable bacteria was increased by about the same amount. The pH was not significantly different from the pH of the initial and 24 hours after the reaction.

Article Viable cell count (CFU / g) pH 0 h 24 h Increased viable cell count 0 h 24 h 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

Example 3: Extinction Effect

Next, the microbial agent produced by each carrier shown in Table 7 was used for food waste, and fermentation and extinction were performed to investigate the change of total sugar, reducing sugar, and total nitrogen according to each microbial agent. Put 2 g of food in a 2 (D) × 20 (H) cm container, mix 18 ml of distilled water, inoculate 2% of the preparation produced for each carrier using the new strain H-3, and then 100 rpm, 30 Reaction was confirmed for 24 hours at ℃, the disappearance effect of the microbial agent produced by each carrier. In the case of total sugar, 200 μl of a 5% phenol solution was mixed with 200 μl of the culture supernatant centrifuged, 1 mL of sulfuric acid was added thereto, and allowed to stand for 10 minutes, followed by stirring at 490 nm. Reducing sugar measurement was performed by adding 0.6 ml of DNS solution to 0.6 ml of the same culture supernatant and reacting for 5 minutes in the water. The mixture was cooled and then measured at 575 nm after adding 0.2 ml of 40% Rochelle solution. The total nitrogen was measured by adding 1 ml of a solution of 500 ml of distilled water, 20 g of NaOH, and 15 g of K ₂ S ₂ O ₈ to 5 ml of the culture supernatant, heating at 121 ° C. for 45 minutes, cooling at room temperature, and adding HCl (1 : 16, v / v) 0.5 ml of the solution was added and measured at 220 nm. The initial total sugar was found to be reduced as an energy source due to the rapid proliferation of microorganisms in all formulations, and the reducing sugar was also reduced by continuous degradation. Among them, the total sugars were reduced from 20.3 g / l to 9.4 g / l, reducing sugars from 3.7 g / l to 1.0 g / l and the total nitrogen was similar in all formulations. It was shown to decrease in proportion.

division Total sugar (g / ℓ) Reducing Sugar (g / ℓ) Total nitrogen (g / ℓ) 0 h 24 h 0 h 24 h 0 h 24 h 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

In addition, in order to increase the fermentation and extinction efficiency of food waste, the result of measuring the amount of CO ₂ according to the pH of the food waste is shown in FIG. Microorganisms use organic materials as substrates for proliferation, and mainly use organic materials that exist in a water-soluble state. Therefore, the measurement of the water-soluble component of the substrate is an index for measuring the microbial activity. When the new strain was added, the dissolved organic carbon content was reduced by 600 ~ 1000 mg / l compared to the initial amount. The most decrease was found when microorganisms were added to the test wastes adjusted to pH 7 and pH 9.

Example 4 Preparation of Microbial Agents for Fermentation and Extinction of Food Waste

Bacillus Bacillus liqueniformis H-3 (KCTC 10273BP) was inoculated into a liquid medium (LB medium: 1 g / L of bacto-tryptone, 0.5 g / L of yeast extract, 1 g / L of NaCl) and incubated at 25 ° C. for 24 hours. After culturing for a while, the microorganisms were prepared through solid phase culture at 30 ° C. for 24 hours after homogeneously mixing in a volume ratio of carrier: culture solution = 10: 4 using degreasing steel as a carrier. In this example, the number of viable cells after incubation at 30 ° C. for 24 hours was 2.8 × 10 ⁹ CFU / g.

Example 5 Medium Fermentation Decayer Operation

8 kg of chaff and 2.2 kg of the microbial agent prepared in Example 1 were administered together in an extinction vessel (30 kg working volume / 50 kg fermentation and extinction vessel), and after stirring for 1 hour, stirring, air, and water spray were automatically on / Intermittent operation was performed using an off timer (30 stirring / 4 min-atmosphere 25 min-water spray 20 L / min). The reactor did not provide separate heating and air supply to evaluate the heat of fermentation generated during the decomposition of organic matter by microorganisms. About 30 kg of leftovers were added daily. A total of 600 kg of food was added during the 25 days, the amount of food left after 25 days was 60 kg and the weight loss rate was calculated to be about 90%. The weight loss rate is expected to increase further, considering that the foods were included such as ribs, and fermentation extinction rate will increase as the number of operating days increases.

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 period before extinction, thereby You can do it successfully.

As described above, the new strain according to the present invention is very useful as a microbial agent because it can successfully perform fermentation and annihilation by maintaining the degrading activity for the period before the extinction due to the rapid growth of the cells and excellent enzymatic activity and antagonism. .

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> Novel Bacillus licheniformis H-3 strain having capability of food waste decomposition <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 492 <212> DNA <213> Bacillus licheniformis H-3 <400> 1 gctggcggcg tgcctaatac atgcaagtcg agcggacaga tgggagcttg ctccctgatg 60 ttagcggcgg acgggtgagt aacacgtggg taacctgcct gtaagactgg gataactccg 120 ggaaaccggg gctaataccg gatggttgtt tgaaccgcat ggttcaaaca taaaaggtgg 180 cttcggctac cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct 240 caccaaggca acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac 300 acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg 360 acggagcaac gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga 420 agaacaagta ccgttcgaat agggcggtac cttgacggta cctaaccaga aagccacggc 480 taactacgtg cc 492

Claims (3)

Bacillus licheniformis H-3 (KCTC 10273BP) with excellent degradation activity of waste oil. A microbial agent for waste organic fermentation and extinction, which is prepared by adsorbing Bacillus rickenformis H-3 (KCTC 10273BP) strain to a growth substrate and a storage carrier. The microorganism preparation for fermentation and extinction of waste organic matter according to claim 2, wherein the growth substrate and the preservation carrier are degreasing steel, diatomaceous earth, or a mixture thereof.