KR101181306B1 - Bacillus thuringiensis HSB1001 strain for manufacturing compost using animal waste and uses thereof - Google Patents

Abstract

The present invention relates to a strain Bacillus thuringiensis HSB1001 and its use for the production of livestock waste compost, more specifically Bacillus thuringiensis HSB1001, composting livestock waste containing the strain or its culture as an active ingredient The present invention relates to a method for preparing a microbial agent for composting livestock waste, comprising culturing the strain, and to a method for composting livestock waste, comprising combining and fermenting an effective amount of the strain with the livestock waste.

Description

Bacillus thuringiensis HSB 1001 strain for manufacturing compost using animal waste and uses about

The present invention relates to a strain Bacillus serringiensis HSB1001 and its use for the production of livestock waste compost, the present invention is a strain Bacillus serringiensis HSB1001 isolated from compost samples of pig flour and poultry has excellent organic and protein degrading ability of livestock waste The present invention has been found to be very useful as a microbial agent for composting.

Recently, due to excessive abuse of chemical fertilizers, soil salt accumulation and environmental pollution problems have occurred, and in order to overcome this, the use of compost and organic fertilizers is gradually increasing as an eco-friendly agricultural material that can replace chemical fertilizers. However, the organic matter contained in the organic fertilizer has a high molecular weight, so the crops cannot be absorbed directly, so that the organic matter can be absorbed through the roots only by being degraded to low molecular weight by microorganisms.

On the other hand, with the recent increase in regulations on livestock waste and the production of high value-added agricultural products by organic farming methods, the demand and supply of related microbial preparations have increased greatly. However, until now, there has been a frequent distribution of microbial products manufactured indiscriminately by small companies that have no legal or institutional regulatory mechanisms for the manufacture and distribution of microbial products, and thus the abuse and expertise of imported microbial products that are not suitable for their use. Accordingly, in relation to the efficacy of microbial preparations, farmers are being harmed and distrust in microbial preparations is increasing. The development of microbial preparations for the purposes of feed additives, animal processing agents and organic fermentation (composting) accelerators not only prevents foreign waste from import substitution, but also reduces farming costs by reducing the use of chemical fertilizers and pesticides, It is very important in that it can contribute to improving the income level of farmers by producing high quality, pollution-free agricultural products by organic farming methods.

As microorganisms capable of degrading organic matter, the soil bacteria group of Korean Patent No. 343197, Candida sp. Of Korean Patent No. 36398, Bacillus sp. GENO-200 KCCM10263 of Korean Patent No. 424419 And halomonas strains of Korean Patent No. 817708 are known. On the other hand, there is no known in the past for the Bacillus serenjiensis strain as a microbial preparation for producing compost using livestock waste as in the present invention.

The present invention has been made in accordance with the requirements as described above, the present invention confirmed that the strain Bacillus serrgenesis HSB1001 isolated from compost samples of pig meal and poultry is very useful as a microbial agent for the composting of livestock waste with excellent organic and protein resolution By this, the present invention was completed.

In order to solve the above problems, the present invention provides Bacillus thuringiensis HSB1001.

The present invention also provides a microbial preparation for composting livestock waste containing the strain or its culture as an active ingredient.

In addition, the present invention provides a method for producing a microbial preparation for composting livestock waste comprising culturing the strain.

In addition, the present invention provides a method for composting livestock waste comprising the step of combining the effective amount of the strain with the livestock waste.

The present invention prevents confusion and import by microbial preparations and reduces farming costs by providing a microbial preparation that can produce stable organic fertilizers and an economic manufacturing method of microbial preparations by treating and using livestock waste generated from livestock farms. The scientific basis and theoretical findings on how organic fertilizers are manufactured, used and managed can contribute greatly to improving environmentally friendly organic farming techniques and increasing farm incomes.

1 is a photograph showing the composting process of pigs according to the addition of microorganisms for biological treatment of the present invention (No. 1: pig flour, No. 2: sawdust, 3-4: mixing process of pig flour and sawdust, No. 5: square PVC barrel , 6: blower).
Figure 2 is a diagram showing the temperature change during the pig manure composting process according to the addition of microorganisms for biological treatment of the present invention.
Figure 3 is a diagram showing the change in moisture content during pig manure composting process according to the addition of microorganisms for biological treatment of the present invention.
Figure 4 is a diagram showing the pH change during the pig manure composting process according to the addition of microorganisms for biological treatment of the present invention.
5 is a diagram showing the change in total nitrogen content during pig manure composting process according to the addition of microorganisms for biological treatment of the present invention.
6 is a view showing the change in organic matter content during the composting process of pigs according to the addition of microorganisms for biological treatment of the present invention.
7 is a view showing the change of organic matter to nitrogen ratio during the pig manure composting process according to the addition of microorganisms for biological treatment of the present invention.

In order to achieve the object of the present invention, the present invention provides a strain Bacillus thuringiensis for the compost production of livestock waste. The strain is preferably the strain Bacillus thuringiensis HSB1001 (KCTC 11719BP) for composting livestock waste.

The present invention also provides a microbial agent for composting livestock wastes containing the Bacillus serensis HSB1001 strain or its culture as an active ingredient.

The microbial preparation according to the present invention may be prepared in liquid form, and may be used in the form of powdered powder by adding an extender thereto or granulated by formulating it. However, the formulation is not particularly limited. In other words, it is possible to formulate a microbial agent to overcome this in an environment in which the microbial agent supply is limited.

In the microbial preparation according to an embodiment of the present invention, the livestock waste may be selected from the group consisting of manure, pig meal and poultry, but is not limited thereto.

In addition, the present invention provides a method for producing a microbial preparation for composting livestock waste, comprising culturing the Bacillus serensis HSB1001 strain. The method for culturing the strain and the method for preparing the microbial agent may use any method known in the art, and are not particularly limited to specific methods.

In addition, the present invention provides a method for composting livestock waste, comprising the step of combining the effective amount of the Bacillus surgingiens HSB1001 strain with the livestock waste. When composting livestock waste by aerobic fermentation, water content is 55 ~ by mixing sawdust, peat, peat, palm leaf, sugarcane, mushroom waste, bran, bark, wood powder, activated charcoal, rice hull, and shavings. It can be adjusted to the range of 70% by weight to improve the swelling to improve the breathability to aerobic fermentation ripening to make a mature compost.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1) Selection of microorganisms for biological treatment for composting

(1) Separation of Microorganisms for Composting

end. Separation

① From the Nonghyup compost factory in Nonsan, Chungcheongnam-do, 1st, 3rd, 5th, 10th, 30th day samples and 2nd mechanical fermentation 1st, 3rd, 5th, 10th and 20th day samples, Collected from surface and middle layers.

② In January 2010, compost samples were taken from the surface and middle layers of the compost piles on the 1st, 3rd, 5th, 10th, and 30th days of the composting process at the N composting plant in Nonsan, Chungnam, and the H composting plant in Gimje, Gyeongnam. In the same manner, composting was carried out in a mechanical composting facility, and samples of the 1st, 3rd, 5th, 10th, and 20th stages and the ripening stage were taken from the surface layer and the middle layer.

I. Separation and Storage

① After diluting the collected sample with sterile water step by step, take 200µl and smear it on NPDA medium (Nutrient agar 50% + Potato dextrose agar 50%) and YM medium for separating bacteria and molds. The culture was carried out under the conditions of.

② Bacterial is NA (Nutrient agar), Actinomycetes are BNA (Bennett's agar), Mold is purely isolated in culture medium in PDA (Potato dextrose agar), suspended in 20% glycerol and stored in a deep-freezer at -75 ℃ and further experiments Used for.

(2) composting piglets using microorganisms for biological treatment separated in the present invention

end. Experimental material

The main raw material, Donbun, was collected and supplied from a donsa in Nonsan, Chungnam, and sawdust was used as a crushed sawdust from the Nonsan Livestock Cooperative. To evaluate the effects of microbial agents for biological treatment on the composting of pig meal and sawdust, The chemical properties and harmful components were analyzed.

I. Experimental Method

① In order to evaluate the effect of microbial treatment for pig manure composting, the control group containing 5: 5 (v / v) of pig flour and sawdust and 5: 5 of pig flour and sawdust added 1 kg of microorganism NN9-2. PSN-1, PSN-2 with 1kg microorganism NN3-1, PSN-3 with 1kg microorganism NN1-2, PSN-4 with 1kg microorganism NH8-4, PSN with 1kg microorganism YH11-1 -5 treatment was set. The composting period is scheduled for 90 days by aerobic stagnation, and samples were analyzed at the beginning, 2nd, 4th and 8th days of composting.

② with the help of organic fertilizer plant in Chungcheongnam Nonsan to proceed with the present invention was carried out by the compost identity expression using a square PVC tube of about 1m ^3. The pipe was connected to the bottom of the PVC tub was blown every day for 1 hour (Fig. 1).

③ After the inorganic elemental analysis of the pig manure and sawdust test material is digested with HClO ₄ was measured by ICP (PE- Optima 3300DV) and atomic absorption spectroscopy (AA-6800 SHIMADZU). In addition, the physicochemical analysis of compost samples was carried out using dry-ash method (TC) for dry carbon (Karam, 1986), Kjeldahl method (Bremner and Mulvaney, 1982) for nitrogen, and 1:10 method for pH and EC (Jackson, 1958). It measured using. In addition, to measure the temperature of the compost during the composting period was measured daily at a depth of about 50cm, the center of the pile by using a Digital Thermometer (HY-550).

2) Selection of functional useful microorganisms for eco-friendly organic fertilizer production

(1) Selection of excellent microorganisms for organic matter and protein degradation

end. Separation

Organic and protein degradability of 187 strains isolated from compost samples were examined.

I. Experimental Method

① organic matter and protein resolution of a soil sample from 10 ^-1 to 10 to be sterilized after drying at room temperature, taken to the selection excellent ^{microorganism-6} a Streptomyces separation medium (Bennett's agar) for taking the 200㎕ after dilution, the bacteria isolated Dilute and flatten soil samples in plate medium prepared by adding starch and protein (skim-milk) to the medium for nutrient agar and the mold separation medium (Potato dextrose agar). After incubation for 3 days, primary selection was made with or without zona pellucida formation.

② Selected strains were isolated and incubated in pure water, suspended in 20% glycerol and stored in a deep-freezer at -75 ℃ and used in future experiments.

③ The microorganisms derived from the first selected strains and compost samples are stroked on actinomycetes medium (Bennett's agar) and bacterial medium (Nutrient agar) to which starch (soluble starch) and protein (skim-milk) components are added at a concentration of 1%. Inoculated at 25 and 30 ° C. to determine the extent of the zona pellucida formed from the cells (Index of degradation, +: less than 2 mm; ++: less than 2-3 mm; +++: less than 3-4 mm; + +++: 4 mm or more).

3) Identification of selected useful microorganisms

(1) sequencing

① Isolation of Compost Samples Isolation of 187 Strains and Soil Sample Isolation A sequencing sequence was selected from strains with excellent degradability of organic matter and protein.

② Bacteria including actinomycetes among the above strains were isolated from the total RNA, and primer 518F (5'-CCAA GCA GCC GCG GTA ATA CG-3 ': SEQ ID NO: 1) and primer 800R (5'-TAC) used for 16S rRNA sequencing. CAG GGT ATC TAA TCC-3 ': SEQ ID NO: 2) 5 minutes prior to denaturation at 95 ° C., 35 cycles (45 seconds denaturation at 94 ° C., 60 seconds annealing at 50 ° C.), PCR was performed through 60 seconds extension at 72 ° C., 10 minutes final extention at 72 ° C. After the amplified PCR product from the 1% agarose gel confirmed by electrophoresis, purified to remove the 16S ribosomal DNA of about 1200bp and sequencing kit (ABI PRISM BigDye ^™ Terminator Cycle Sequencing Kits) Sequencing with ABI PRISM 3730XL Analyzer using Was carried out. The 16S rRNA sequences of the analyzed microorganisms were identified by comparison with ribosomal RNA sequences of GENEBANK and RDP (RNA database project) using the BLASTN program.

4) cultivation technology

(1) Composition and Characteristics of Optimal Media (Solid Culture)

The selected NN9-2 ( Bacillus subtilis ), NN1-2 ( Bacillus weihenstephanensis ), NN3-1 ( Bacillus thuringiensis ), NH8-4 ( Geobacillus sp . ) And YH11-1 ( Geobacillus Stearothermophilus ) strains of NN3-1, which had excellent organic protein degradation and fermentation heat exceeding 75 ° C. on day 3, were selected, and solid culture conditions were performed as follows based on the results of previous studies.

end. Main raw materials and badge composition

① Zeolite (specific gravity 2.1 g / cm 3, powder) and degreasing steel were used as the main raw materials and are shown in Table 1 below.

② The solid medium was inoculated with 1 ~ 2% of the liquid culture medium of organic matter and proteolytic microorganisms and incubated for 3-7 days at 30 ° C., 20%, 30% of moisture, and the number of viable cells was measured by dilution plate method.

Example  1. Biological for Composting For treatment  Microbial selection

A total of 187 samples including compost samples from pig manure and manure compost at Nonghyup compost factory in Nonsan, Chungcheongnam-do, and 44 compost samples from N compost factory and Non compost factory in Nonsan and Chungje Gimje city in January 2010. Strains were isolated from compost (Table 2).

Example  2. Composting pig meal by microorganism isolated in the present invention

① Physicochemical Characteristics and Harmful Components of Swine Flour and Sawdust

Tables 3 and 4 show the results of analyzing the physicochemical characteristics and harmful components of pig manure and sawdust. The pH values of raw meal and sawdust were 7.52 and 4.98, and nitrogen was 1.72% and sawdust 0.28%. Organic matter-to-nitrogen ratio was 50.1 in pig meal and 343.5 in sawdust. In the heavy metals and salt contents of pig and sawdust, most of the heavy metals were detected or traced, and the salt content was 0.35% for pig and 0.03% for sawdust.

② biological For treatment  By microbial addition Compost Chemistry  change

The effect of the microorganisms for biological treatment of the present invention on the maturity of pig manure composting was examined. Table 5 shows the physicochemical changes of pig manure compost according to the addition of five microorganisms for biological treatment during pig manure composting.

* Control-Money: Sawdust = 50: 50 (v / v)

** PSN-1-Sorghum: Sawdust = 50: 50 (v / v) + Microorganism NN9-2 1kg

*** PSN-2-Sawdust = Sawdust = 50: 50 (v / v) + 1kg of microorganism NN3-1 (= HSB1001)

**** PSN-3-Sorghum: Sawdust = 50: 50 (v / v) + Microorganism NN1-2 1kg

***** PSN-4-Sorghum: Sawdust = 50: 50 (v / v) + Microorganism NH8-4 1kg

****** PSN-5-Swine: Sawdust = 50: 50 (v / v) + Microorganism YH8-4 1kg

Ⓐ Temperature change

During the composting process, the temperature change of the sedimentary piles is increased when the decomposition activity of microorganisms occurs actively, and the internal temperature of the sedimentary piles reaches 60 ℃ or more. Can be. Most of the pathogens are killed when the internal temperature of the pile is kept above 55 ° C for more than 5 days, and Salmonella species are completely killed when kept at 65 ° C for 1 day.

The temperature change during the stagnant pig manure composting period according to the addition of microorganisms for biological treatment was investigated, and the results are shown in FIG. 2. During the composting process according to the microorganisms for biological treatment, the temperature of the compost pile after initial raw material mixing was about 20 ± 2 ℃, slightly higher than room temperature. On the third day of composting, all the treatments were raised to a temperature higher than 65 ° C, which was higher than the highest 75 ° C in the PSN-2 treatment. Temperatures past the peak point tended to decrease sharply and will be reversed on the 20th day of composting.

Ⓑ Change of water content

Throughout the compost manufacturing process, the water content of the sediments is a major factor affecting the composting efficiency and microbial activity during the composting process. Moisture content is the result of microbial activity and biological oxidation of organics.

As shown in FIG. 3, the initial moisture content during the composting process was gradually decreased to 67-68%, and all treatments showed 61% level at 8 days. .

Ⓒ pH  change

During composting, pH is a survey item to measure the acidity and alkalinity of the compost heap. This is because the importance of pH in composting is directly related to the activity of microorganisms. In composting, the appropriate range of pH is 6.5-8.5, and composting proceeds in a wide range due to naturally occurring factors (FIG. 4).

The pH change during pig manure composting according to the addition of microorganisms for biological treatment of the present invention is shown in Figure 3. The pH of the early composting was about 7.0, and gradually increased as the composting progressed. On the 8th, all the treatments showed a weak alkalinity of 7.2 ± 0.2.

Ⓓ T-N, organic matter content and organic matter Nitrogen ratio  change

During the composting process, microorganisms use organic matter as an energy source for the decomposition of organic matter, and nitrogen as a nutrient source such as protein synthesis necessary for microbial growth. The ratio of organic matter to nitrogen in the community and growth of microorganisms is expanded. In the composting studies by many researchers, when the organic-to-nitrogen ratio is less than 40, it has been reported as an index for determining the compost maturity, and the results of the research on stabilization when the organic-to-nitrogen ratio is less than 25 are reported.

The total nitrogen content, organic matter content and organic matter-to-nitrogen ratio during the pig manure composting process according to the addition of the microorganisms for the biological treatment agents are shown in FIGS. 5, 6, and 7.

The total nitrogen content tended to increase gradually in all treatments during the composting process with the addition of microorganisms for the biological treatment, but the organic matter content decreased with the composting as opposed to the total nitrogen content. On day 8, all treatments showed 86-87% of results. Organic-to-nitrogen ratio is one of the major factors in the maturity of the composting process. The initial organic-to-nitrogen ratio was about 76 ~ 72, and the nitrogen content tended to increase gradually in contrast to the decrease of organic matter during composting. This resulted in a continuous reduction of organic to nitrogen ratios.

Example  3. Selection of strains excellent in organic matter and protein resolution among the strains isolated in the present invention

As a result of experimenting with the organic and protein resolution of the microorganism 187 strain derived from the compost sample, 76 strains of about 41% was found to have excellent organic resolution. In addition, the NN1-2, NN3-1 (= HSB1001), NN9-2, NH8-4, and YH11-1 strains among the 76 strains were found to have the highest organic and proteolytic ability, and were identified and used in subsequent experiments. (Table 6).

* +: Less than 2 mm; ++: less than 2-3 mm; +++: less than 3-4 mm; ++++: 4 mm or more

Example  4. Composition of the optimum medium for culturing the strain isolated in the present invention

As a result of measuring the viable cell number of each solid medium cultured for 3, 5, 7 days, it is as shown in Table 7 below.

As a result, in the case of the medium containing degreasing steel, the incubation period of 2 days is longer than that of the medium containing the zeolite as the main raw material, and in the culture conditions for mass production, the medium containing zeolite as the main raw material 2 (95 parts by weight of zeolite + 5 parts of degreasing steel) It can be considered that this will reduce the loss of solid culture by improving the productivity and reducing contamination by other mold fungi.

Example  5. Identification of Strains Isolated in the Present Invention

16S rRNA sequences for the isolates isolated in the present invention were determined and identified by comparison with ribosomal RNA sequences of GENEBANK and RDP (RNA database project) using the BLASTN program. As a result, the strains originated in most of the compost samples are NN9-2 Bacillus subtilis (Bacillus subtilis ), NN1-2 is Bacillus weihenstepanosis ( Bacillus weihenstephanensis), NN3-1 is Lindsay N-Sys (Bacillus, Bacillus written thuringiensis ), NH8-4 is a species of Geobacillus sp . ), It is a brush YH11-1 Russ (Geobacillus as geo Bacillus stearate stearothermophilus ).

Among them, Bacillus thuringiensis has a 16S rRNA sequence of SEQ ID NO: 3, and the strain was deposited on June 29, 2010 with the Korea Research Institute of Bioscience and Biotechnology (KCTC 11719BP).

Korea Biotechnology Research Institute KCTC11719BP 20100629

Attach an electronic file to a sequence list

Claims (5)

delete A microbial agent for composting milk powder, pig flour or chicken flour containing Bacillus serringiens HSB1001 (KCTC 11719BP) strain or a culture thereof as an active ingredient at a temperature of 70 to 75 ° C. delete delete Comprising the step of combining the effective amount of the microbial preparation of claim 2 with milk powder, pig flour or chicken flour.

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