KR101854277B1 - Microbial agent comprising a mixture of species for reducing ammonia odor and method for manufacturing the same - Google Patents

Abstract

The present invention relates to a mixed microbial agent for reducing ammonia odor, including: at least one selected from among a Bacillus amyloliquefaciens AEY-1 strain (Deposit No. KCTC18314P), a culture thereof, and a concentrate and dried material of the culture; at least one selected from among a Bacillus amyloliquefaciens AEY-2 strain (Deposit No. KCTC18315P), a culture thereof, and a concentrate and dried material of the culture; and at least one selected from among a Bacillus amyloliquefaciens AEY-3 strain, a culture thereof, and a concentrate and dried material of the culture. According to the present invention, the agent is capable of making a contribution to an improvement in livestock industries by quickly and efficiently reducing ammonia odor.

Description

TECHNICAL FIELD The present invention relates to a microbial agent for reducing ammonia odor and a method for producing the same,

The present invention relates to a mixed microorganism preparation for ammonia odor reduction and a method for producing the same. More specifically, the present invention relates to a technique for rapidly and efficiently reducing ammonia gas using a mixed microbial agent.

In the livestock industry, odors from livestock manure cause discomfort of local residents and cause many complaints. These odors are generated by volatile substances and their intermediates released during the anaerobic digestion of manure into the atmosphere and stimulating the human olfactory sense. The odors from manure adversely affect the health and productivity of livestock as well as the health of livestock farm workers (Tamminga, 1992).

In addition, more than 50% of the nitrogen released from livestock is released in the form of urine elements, and the released elements are converted to ammonia by the urease enzyme. Therefore, to reduce the incidence of manure-derived ammonia, urease enzymes should be inhibited (Mackie et al., 1998). Thus, manure is a source of odor (Lowe, 1995), and it can be used to reduce nitrogen excretion through manure by using enzymes, microbes, yeast, and probiotics that can improve protein digestibility (Min et al., 1992; Park et al., 1994), or by supplementing feeds with low or low protein levels of feed or deficient amino acids to improve amino acid utilization and reduce nitrogen excretion (Sutton et al., 1999; Shriver et al. , 2003). In addition, many studies have been conducted on feed feeds, and in particular, the use of probiotics as supplementary feeds has a great effect on the improvement of livestock farming environment as well as livestock farming.

In addition, various studies have been carried out to reduce the amount of ammonia produced by the intestinal bacteria and environmental improvement of livestock by the feeding of probiotics, and studies on the ammonia reduction effect by the feeding and spraying of probiotics have been progressing steadily. Wang et al. (2009) found that the addition of Bacillus subtilis , Bacillus licheniformis , aluminum silicate and whey flour to the feed resulted in a significant increase Of ammonia was lower than that of the control.

In addition, when 1 to 2% of Bacillus subtilis culture broth was added to broiler diets, ammonia gas was reduced by about 36-72% (Santoso et al., 1999), and lactic acid bacteria and probiotics containing Bacillus preparation (Chiang and Hsieh, 1995) reported that ammonia production from the bottom of a pile or cage decreased.

In addition, Korean Patent Registration No. 10-1465093 discloses a technique for reducing the H ₂ S gas concentration by spraying a pig culture with Bacillus amyloliquefaciens NAAS-1 (KACC 91751P) culture broth have.

In addition, products developed for the reduction of animal offensive odor are mainly products effective for ammonia reduction. In order to solve the problem of livestock environment in the future, it is necessary to develop products effective for reducing odor. Moreover, as the proliferation of eco-friendly agriculture and the problem of chemical formulation are emerging, there is a desperate need for environmentally friendly livestock breeding and environment improvement. Removal of odors using microorganisms has little secondary side effects, and it can effectively remove specific odor components, while its effect depends on the microbial efficacy. Therefore, it is important to isolate microorganisms having excellent resolving power against a specific odor component, and to maintain the effect of the microorganism in various external environments.

On the other hand, the removal of odors using a microbial agent has a characteristic that the treatment time is somewhat longer than that using a chemical substance. Therefore, there is a need for the development of a microbial agent which can reduce the time required for the malodor removal and the odor removal efficiency.

Disclosure of Invention Technical Problem [8] The present invention provides a mixed microorganism preparation capable of rapidly removing ammonia gas by culturing and isolating a strain that removes ammonia gas.

Another object of the present invention is to provide a method for reducing ammonia produced in a livestock breeding facility and livestock manure including Bacillus amyloliquefaciens AEY-1, Bacillus butanoliborans AEY-2 and Bacillus riciniformis AEY-3 To provide a mixed microbial preparation.

It is a further object of the present invention to provide a process for preparing the mixed microbial formulation.

The object of the present invention is not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood from the following description.

According to one aspect of the present invention, there is provided a culture medium comprising at least one selected from a strain of Bacillus amyloliquefaciens AEYE-1 (Accession No. KCTC18314P), a culture of the strain, a concentrate and a dried product of the culture; A strain of Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P), a culture of the strain, a concentrate and a dried product of the culture; And a Bacillus licheniformis AEY-3 ( Bacillus licheniformis AEY-3) strain, a culture of the strain, a concentrate of the culture, and a dried product as an active ingredient. / RTI >

According to one embodiment of the present invention, the microorganism preparation can reduce the ammonia concentration by 30% or more within 12 hours after spraying the ammonia odor source.

According to one embodiment of the present invention, at least one selected from the strains of Bacillus amyloliquefaciens AEY-1 ( Bacillus amyloliquefaciens AEY-1, Accession No. KCTC18314P), the cultures of the strains, the concentrates and the cultures of the cultures; A strain of Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P), a culture of the strain, a concentrate and a dried product of the culture; And Bacillus licheniformis AEY-3, a culture of the strain, a concentrate and a dried product of the culture, in an amount of 0.05 to 1.5 parts by weight based on the total weight of the microorganism preparation .

According to one embodiment of the present invention, at least one selected from the strains of Bacillus amyloliquefaciens AEY-1 ( Bacillus amyloliquefaciens AEY-1, Accession No. KCTC18314P), the cultures of the strains, the concentrates and the cultures of the cultures; A strain of Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P), a culture of the strain, a concentrate and a dried product of the culture; And Bacillus licheniformis AEY-3, a culture of the strain, a concentrate of the culture, and a dried product may be mixed at a weight ratio of 1: 1: 1 .

According to one embodiment of the present invention, the microorganism preparation may contain at least one kind of starch and vitamin C as an excipient.

According to one embodiment of the present invention, the strain may be cultured in TSB medium.

According to another aspect of the present invention, there is provided a livestock feed comprising the microbial formulation.

According to another aspect of the present invention, there is provided a microbial fertilizer comprising the microbial agent.

According to another aspect of the present invention, there is provided a strain of Bacillus amyloliquefaciens AEY-1 (Accession No. KCTC18314P), Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P), Bacillus amyloliquefaciens AEY- And a step of culturing a strain of Bacillus licheniformis AEY-3 and a strain of Bacillus licheniformis AEY-3.

According to one embodiment of the present invention, the strain may be cultured in TSB medium.

According to one embodiment of the present invention, the culturing can be carried out at a temperature of 25 to 38 占 폚.

According to one embodiment of the present invention, the culture may be carried out at a pH of 6 to 9.

According to one embodiment of the present invention, at least one selected from the strains of Bacillus amyloliquefaciens AEY-1 ( Bacillus amyloliquefaciens AEY-1, Accession No. KCTC18314P), the cultures of the strains, the concentrates and the cultures of the cultures; A strain of Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P), a culture of the strain, a concentrate and a dried product of the culture; And Bacillus licheniformis AEY-3, a culture of the strain, a concentrate and a dried product of the culture, in an amount of 0.05 to 1.5 parts by weight based on the total weight of the microorganism preparation .

According to one embodiment of the present invention, at least one selected from the strains of Bacillus amyloliquefaciens AEY-1 ( Bacillus amyloliquefaciens AEY-1, Accession No. KCTC18314P), the cultures of the strains, the concentrates and the cultures of the cultures; A strain of Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P), a culture of the strain, a concentrate and a dried product of the culture; And Bacillus licheniformis AEY-3, a culture of the strain, a concentrate and a dried product of the culture may be mixed at a weight ratio of 1: 1: 1.

According to another aspect of the present invention, there is provided an ammonia odor reduction method for reducing the ammonia odor by treating the microorganism preparation with an ammonia odor source.

Bacillus amyloliquefaciens AEY-1, Accession No. KCTC18314P), Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P), and Bacillus amyloliquefaciens AEY-1 according to an embodiment of the present invention; and The Bacillus licheniformis AEY-3 ( Bacillus licheniformis AEY-3) microorganism preparation has the effect that the reduction of ammonia can be performed more quickly and efficiently than the conventional microorganism preparation.

In addition, since the existing animal husbandry facilities and livestock manure are used as it is and the microbial agent is directly sprayed to decompose the malodor-inducing substance, the malodor is removed from the root, so that no additional equipment is required and the malodor can be economically and efficiently removed .

Bacillus amyloliquefaciens AEY-1, Bacillus butanolivorans AEY-2, and Bacillus riciniformis AEY-3 according to an embodiment of the present invention. ( Bacillus licheniformis AEY-3), the effect of reducing the ammonia gas by about 95% after 24 hours after one time spraying according to the amount of 1.0% by weight of the microorganism preparation was exhibited.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a Bacillus amyloliquefaciens AEY-1, Bacillus butanolivorans AEY-2, and Bacillus riciniformis AEY-1 according to an embodiment of the present invention. -3 ( Bacillus licheniformis AEY-3) strain.
FIG. 2 shows the morphological characteristics of Bacillus amyloliquefaciens AEY-1, Bacillus butanoliborans AEY-2, and Bacillus riciniformis AEY-3 according to an embodiment of the present invention.
FIG. 3 shows the results of identification of a novel microorganism Bacillus amyloliquefaciens AEY-1 strain according to an embodiment of the present invention.
FIG. 4 shows the results of identification of a novel microorganism Bacillus butanoliferans AEY-2 strain according to an embodiment of the present invention.
FIG. 5 shows the results of identification of a novel microorganism, Bacillus riciniforrmis AEY-3 strain, according to an embodiment of the present invention.
FIG. 6 shows the growth of Bacillus amyloliquefaciens AEY-1, Bacillus butanoliborans AEY-2, and Bacillus riciniformeus AEY-3 according to the nutritional medium according to an embodiment of the present invention.
FIG. 7 shows the growth performance of Bacillus amyloliquefaciens AEY-1, Bacillus butanoliborans AEY-2 and Bacillus riciniformeus AEY-3 according to an embodiment of the present invention at a culture temperature.
FIG. 8 shows the pH resistance of Bacillus amyloliquefaciens AEY-1, Bacillus butanoliborans AEY-2, and Bacillus riciniformis AEY-3 according to an embodiment of the present invention.
9 shows an apparatus for collecting ammonia generated from a manure waste according to an embodiment of the present invention.
10 is a photograph showing a state of an ammonia gas reducing microorganism preparation according to an embodiment of the present invention.
11 is a graph showing the effect of ammonia reduction according to the application amount of 0.1% by weight of a mixed microorganism preparation of amyloliquefaciens AEY-1, Bacillus butanoliborans AEY-2 and Bacillus riciniformis AEY-3 according to an embodiment of the present invention The results are shown.
FIG. 12 is a graph showing the ammonia reduction according to the application amount of 1.0% by weight of a mixed microorganism preparation of Bacillus amyloliquefaciens AEY-1, Bacillus butanolivorans AEY-2 and Bacillus riciniformis AEY-3 according to an embodiment of the present invention Efficacy results.

The terminology used in this application is used only to describe a specific embodiment and is not intended to limit the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise.

In the present application, when a component is referred to as "comprising ", it means that it can include other components as well, without excluding other components unless specifically stated otherwise. The sizes and thicknesses of the respective components shown in the drawings are arbitrarily shown for convenience of explanation, and thus the present invention is not necessarily limited to those shown in the drawings.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of a mixed microbial agent composition for reducing ammonia odor according to the present invention will be described in detail with reference to the accompanying drawings. In the following description, And a detailed description thereof will be omitted. The present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

In order to achieve the object of the present invention, the present invention provides a novel microorganism, Bacillus amyloliquefaciens AEY-1 (Accession No. KCTC18314P), Bacillus butanolivorans AEY-2 3, Bacillus licheniformis AEY-3), the culture of the strain, the culture of the strain, the culture of the strain, Which comprises at least one selected from a concentrate and a dried product of the culture as an active ingredient.

( Bacillus amyloliquefaciens AEY-1, Accession No. KCTC18314P), Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P) and Bacillus riciniformeis AEY-1 -3 ( Bacillus licheniformis AEY-3, Accession No. KCTC18316P) were isolated from the pig manure. The separated strains were found to be excellent in ammonia abatement ability and 16s rDNA analysis confirmed Bacillus amyloliquefaciens , Bacillus butanolivorans and Bacillus licheniformis strains . On July 25, 2014, the strain was introduced into Bacillus amyloliquefaciens AEY-1 ( Bacillus amyloliquefaciens AEY-1), Bacillus butanolivorans AEY-2 -2) and AEY-3 ( Bacillus licheniformis AEY-3), and received the accession numbers of KCTC18314P and KCTC18315P KCTC18316P.

In the present invention, the Bacillus amyloliquefaciens AEY-1 strain ( Bacillus amyloliquefaciens AEY-1, Accession No. KCTC18314P) is a strain exhibiting a potent abatement effect against ammonia components in odor substances generated in piggery facilities and livestock manure. This ammonia is caused by degradation of the elements contained in the urine of the livestock and degradation of proteins in the excreted matter in the state of insufficient digestion (Kim et al., 2007). As the time elapses, the fresh carrot is decarboxylated and the putrescine, cadaverine and ammonia are produced. The microorganisms involved in this process are Streptococcus, Peptostreptococcus, and Bacteroides. Other sources of ammonia are deamination of amino acids and urea and nitric acid (Spoelstra, 1980).

The Bacillus amyl Lowry kwipe sieon switch (Bacillus amyloliquefaciens) is a Gram (+) bacteria to form a spore, protein, starch, and cellulose such as an organic substance decomposition and fermentation promoting, phosphoric acid and nitric acid compound decomposition, a variety of the harmful gas removal and biological control of (Park, 2008; Shin, 2004). In this study,

In the present invention, Bacillus butanolivorans AEY-2 ( Bacillus butanolivorans AEY-2, Accession No. KCTC18315P) is a Gram (+) bacterium with a diameter of 0.8-1.3 mm and a length of 2.5-5.1 mm. Forms spores from the center of the ellipse. The strain Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P) has the characteristic of controlling n-butanol (1-butanol, butan-1-ol) released into the environment naturally and artificially (Kuisiene et al., 2008). The n-butanol thus discharged can contribute to photochemical smog formation and cause ozone. Thus, the removal of n-butanol is highly desirable (Veeranagouda et al., 2006).

In the present invention, Bacillus licheniformis AEY-3 ( Bacillus licheniformis AEY-3, Accession No. KCTC18316P) has antimicrobial activity, heat stability and proteolytic resistance under high pressure sterilization conditions, and Bacillus subtilis Are quite similar (Lee et al., 2001) and are highly effective since they do not lose activity until they reach the intestine. In particular, peptides and lipopeptide antibiotics and bacteriocins of lichenysin and lichenicidine of Bacillus licheniformis exhibit antibacterial or antifungal properties (Abriouel et al., 2011; Lee and Kim, 2011; Dischinger et al. 2009; Jung et al., 2009; Yang and Chang, 2007; De Lucca and Walsh, 1999).

The Bacillus licheniformis is a spore-forming bacterium and has been recognized as a harmless microorganism by the US Food and Drug Administration (FDA) and has an inhibitory effect on various plant pathogens (Asaka and Shoda , 1996; Berger et al., 1996; Seddon et al., 1996).

According to one embodiment of the present invention, the microorganism preparation can reduce the ammonia concentration by more than 30% within 12 hours after the application of the ammonia odor generating source, and further reduce by up to about 95% within 24 hours. Therefore, according to the present invention, the ammonia odor can be quickly and efficiently reduced.

In the present invention, the microorganism preparation is selected from the group consisting of Bacillus amyloliquefaciens AEY-1, Accession No. KCTC18314P, Bacillus butanolivorans AEY-2, Accession No. KCTC18315P, and Bacillus amyloliquefaciens AEY- The microorganism belonging to the genus Bacillus licheniformis AEY-3 (Accession No. KCTC18316P), which belongs to the genus Bacillus licheniformis AEY-3, It is not.

In the present invention, the culture means that a specific microorganism is cultured in a culture medium or a culture solution, and the culture may or may not include a specific microorganism, but preferably includes a microorganism. The culture may be, but is not limited to, a liquid or solid formulation.

In the present invention, the microbial agent may be in solid or liquid form. In the case of the solid microorganism preparation, the microorganism may be attached to the carrier and then dried to be a water content of 0.1 to 10% by weight, bead-shaped product, or pulverized into a powdery product.

Examples of the carrier include zeolite, vermiculite, diatomaceous earth, kaolin, onionite, feldspar, charcoal, and talc. Examples of the carrier include powdery clay, activated carbon, coke, volcanic ash, May be used, but the present invention is not limited thereto.

The microbial formulation in solid form may further comprise an excipient. In the case of the excipient, starch, amino acid, vitamin C, vitamin E, chitosan and glucose may be used singly or in combination of two or more, but the present invention is not limited thereto. The starch may be used to prevent film formation due to sugar components. The starch may be used in an amount of 10 to 20% by volume, but is not limited thereto. When the amount of starch is increased, microorganisms are diluted and the total number of bacteria is lowered. It can be used for pH control of vitamin C (ascorbic acid) and for vitamin use. The vitamin C may be used in an amount of 0.1 to 0.5% by volume, but is not limited thereto. The vitamin C may affect the growth and activity of the strain by excessively decreasing the pH at the time of overdose.

The liquid type microorganism preparation may be mixed with glucose or glycerin to stabilize the microorganism, but the present invention is not limited thereto.

According to one embodiment of the present invention, the three microorganism strains of the present invention may be contained in an amount of 0.05 to 1.5 parts by weight, preferably 1.0 part by weight, based on the total weight of the microorganism preparation, but are not limited thereto. If the amount of the microorganism is less than 0.05 part by weight, the effect of removing the ammonia odor of the microorganism preparation may be insignificant. If the amount of the microorganism is more than 1.5 parts by weight, the increase of the ammonia odor removal effect may be insufficient.

According to one embodiment of the present invention, at least one selected from the strains of Bacillus amyloliquefaciens AEY-1 ( Bacillus amyloliquefaciens AEY-1, Accession No. KCTC18314P), the cultures of the strains, the concentrates and the cultures of the cultures; ( Bacillus butanolivorans AEY-2, Accession No. KCTC18315P), the culture of the strain, the concentrate and the dried product of the culture, and the Bacillus riciniformis AEY-3 Bacillus licheniformis AEY-3), a culture of the strain, a concentrate of the culture, and a dried product may be mixed at a weight ratio of 1: 1: 1. The synergistic effect can be maximized in the ammonia odor reduction due to the mixing of three strains upon mixing at the above weight ratio.

According to one embodiment of the present invention, the strain may be cultured in TSB medium. When cultured in the TSB medium, the culture efficiency is increased compared to other medium.

According to another aspect of the present invention, there is provided a livestock feed comprising the microbial formulation.

The feed can be prepared by mixing crumbs obtained by crushing one or more mixtures selected from the group consisting of corn, wheat, wheat, soybeans, soybeans or rice and processing by-products such as rice bran, wheat bran and barley bran obtained by processing them But is not limited thereto.

The livestock feed may contain the microbial agent for removing odor in an amount of 0.1 to 0.3% by weight, preferably 0.2% by weight based on the total weight of the feed, but is not limited thereto. When the microbial agent is contained in an amount of less than 0.1% by weight, the malodor removing effect of the animal feed may be insignificant. When the microbial agent is contained in an amount of more than 0.3% by weight, the effect is almost the same.

The livestock feed may be provided to mammals such as cows, pigs, sheep and goats, and poultry such as pheasants, chickens, ducks, and turkeys, but is not limited thereto.

When the livestock feeds the livestock feed, the odor of the livestock excrement is removed or reduced, and the effect of promoting the composting of the livestock feed can be exhibited.

According to another aspect of the present invention, there is provided a microbial fertilizer comprising the microbial agent.

The microbial fertilizer according to the present invention can be prepared by treating the strain, the culture of the strain, the concentrate or the dried product of the culture with an animal wastewater or food waste, and the active strain can be produced in solid or liquid form, Conventional techniques can be applied in addition to the above strains.

The microbial fertilizer may be prepared by mixing with a carrier to be used and formulating it into powder, pellet, granule or solution. As the carrier, water, celite, zeolite, pearlite, vermiculite, peat moss, talc, kaolinite, bentonite, pig dust, fish dust, chick dust, rice dust, lip dust, bone dust and kaolin can be used. But is not limited to. The strain of the present invention can be formulated using the commercially available carrier and remains active even after formulation, so that it can be usefully used.

According to another aspect of the present invention, there is provided a method for preparing a microorganism preparation for ammonia odor reduction comprising culturing the strain.

The culture of the strain of the present invention can be cultured by any method known in the art, and cultured in a nutrient medium (TSB medium) at a temperature of 35 DEG C for 24 hours to prepare a culture. At this time, the TSB medium may be cultured in a medium containing molasses and yeast extract so that the strain can be well cultured and mass-produced, but the present invention is not limited thereto.

The culture of the strain of the present invention is suitably carried out under at least one of the conditions of a temperature of 25 to 38 DEG C, a stirring speed of 50 to 150 rpm, and a pH of 6 to 9. The optimum conditions for the growth of the strain include, but are not limited to, the temperature of 30 to 35 DEG C, the stirring speed of 120 rpm, and the pH of 6 in the mass production culture conditions.

In the method for producing a mixed microorganism preparation for ammonia odor reduction according to the present invention, the three microorganisms may be respectively cultured and mixed, or the three microorganisms may be mixed and then cultured.

In the method for producing a mixed microorganism preparation for ammonia odor reduction according to the present invention, a strain of Bacillus amyloliquefaciens AEY-1 (Deposit No. KCTC18314P), a culture of the strain, a concentrate of the culture, At least one selected from the group consisting of: A strain of Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P), a culture of the strain, a concentrate and a dried product of the culture; And Bacillus licheniformis AEY-3, a culture of the strain, a concentrate and a dried product of the culture, in an amount of 0.05 to 1.5 parts by weight based on the total weight of the microorganism preparation, A mixed microorganism preparation for ammonia odor reduction can be prepared.

In the method for producing a mixed microorganism preparation for ammonia odor reduction according to the present invention, a strain of Bacillus amyloliquefaciens AEY-1 (Deposit No. KCTC18314P), a culture of the strain, a concentrate of the culture, At least one selected from the group consisting of: A strain of Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P), a culture of the strain, a concentrate and a dried product of the culture; At least one selected from the group consisting of Bacillus licheniformis AEY-3, Bacillus licheniformis AEY-3, the culture of the strain, the concentrate of the culture and the dried product are mixed at a weight ratio of 1: 1: 1 to reduce ammonia odor Can be prepared.

According to another aspect of the present invention, there is provided an ammonia odor reducing method for treating ammonia odor by treating the microbial agent of the present invention with an ammonia odor source.

In the present invention, the ammonia odor generating source includes at least one selected from a livestock breeding facility, a manure treatment plant, an animal wastewater treatment plant, a food waste treatment plant, a waste landfill, a landfill, and a wastewater treatment plant.

The microorganism preparation of the present invention can remove odor economically and efficiently because it does not require any additional equipment since the microorganism preparation of the present invention directly uses the existing livestock breeding facility and livestock manure to directly spray the microorganism preparation to decompose the malodor- There is an effect.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, exemplary embodiments of the present invention will be described in order to facilitate a person skilled in the art to which the present invention pertains.

[ Example  1] Isolation and selection of microorganism strains

The ammonia-reducing microorganism according to an embodiment of the present invention is selected by the following method.

In order to isolate ammonia-reducing microorganisms, the pig manure collected from the National Livestock Science Institute test pens was suspended in distilled water, and the suspension was treated with NA (Nutrient Agar; peptone 5.0 g, beef extract 3.0 g, agar 15 g, * Per Liter) (Potato Dextrose Agar; Potato Dextrose Agar; Potato Dextrose Agar: 17.0g of casein pancreatic enzyme hydrolyzate, 3.0g of soybean papain, 2.5g of glucose, 5.0g of salt, 2.5g of inorganic salt, 15g of agar, g, 20.0 g of glucose, 15 g of agar, * Per Liter) for 72 hours in an incubator set at 35 ° C by a flat plate method to separate the microorganisms in consideration of the color and shape of the colonies (FIG.

The separated novel microorganism Bacillus amyl Lowry's kwipe sieon AEY-1 (Bacillus amyloliquefaciens AEY- 1, KCTC18314P), Bacillus butanone Norris borane's AEY-2 (Bacillus butanolivorans AEY-2, Accession No. KCTC18315P) and Bacillus licheniformis AEY-3 (Accession No. KCTC18316P) were cultured in nutrient medium (TSB medium) for 24 hours at a temperature of 35 ° C to prepare a culture medium Respectively. At this time, it is preferable to cultivate TSB medium in a medium containing molasses and yeast extract so that the strain can be well cultured and mass-produced.

At this time, the optimum condition for the growth of the strain is that the temperature is 35 ° C., the stirring speed is 120 rpm, and the pH is 6 for mass production.

The culture solution thus prepared was directly dried and contained in an amount of 0.1 to 1.0 wt% based on the total weight of the microorganism preparation.

[ Example  2] Identification of isolated strains

Bacillus amyloliquefaciens , Bacillus butanolivorans and Bacillus butanolivorans were subjected to analysis and morphological analysis of 16S rDNA, a conventional identification method, to identify the species of the strain isolated in Example 1, It was identified as Bacillus licheniformis . The morphological characteristics are shown in FIG. 2. As a result of the base sequence analysis, Bacillus amyloliquefaciens and Bacillus butanolivorans showed 99% similarity (FIGS. 3 and 4). In the case of Bacillus licheniformis , the sequence analysis showed 98% similarity (FIG. 5). These strains were introduced into the microorganism resource center (KCTC) of the Korea Research Institute of Bioscience and Biotechnology (KCTC) on July 25, 2014 with Bacillus amyloliquefaciens AEY-1, Bacillus butanolivorans AEY-2, Accession No. KCTC18315P) and Bacillus licheniformis AEY-3 ( Bacillus licheniformis AEY-3), and received accession numbers KCTC18314P, KCTC18315P and KCTC18316P.

[ Example  3] Preparation of microorganism preparation by strain

Bacillus amyl Lowry kwipe sieon's AEY-1 (Bacillus amyloliquefaciens AEY- 1, deposit number KCTC18314P) for strain 1, the secondary seed culture of via during the culture tertiary Soy peptone 0.5 wt% (1.5 kg), sucrose 2.0 wt. 0.67 wt% (2 L) of the culture solution of each strain (inoculum number 3.5X10 ¹⁰ cfu / g) and other distilled water And the total culture volume of 300 L was fermented at 30 캜. Then, the fermented product was dried through a freeze dryer and pulverized into powder, which was used as an experimental microbial agent.

In addition, Bacillus butanolivorans AEY-2 ( Bacillus butanolivorans (1 kg), sucrose 1.0% by weight (3 kg), NaCl 0.1% by weight (300 mg), and the like, in the case of the third primary cultivation in the primary cultivation in the case of AEY-2, Accession No. KCTC18315P) g), 0.03% by weight of CaCO ₃ (100 g), 0.1% by weight of K ₂ HPO ₄ (300 g) and 0.67% by weight of cultured broth (inoculum number 1.4 x ^{10 9} cfu / g) %, And fermented at a temperature of 30 ° C in a culture amount of 300 L. Then, the fermented product was dried through a freeze dryer and pulverized into powder, which was used as an experimental microbial agent.

In the case of the Bacillus licheniformis AEY-3 strain, 0.5% by weight of soy peptone (1.5 kg) and 2.0% by weight of sucrose kg), NaCl 0.5 kg (1.5 kg), soluble starch 0.1 wt% (300 g) and 0.67 wt% (2 L) of each strain (inoculum 4.7X10 ¹⁰ cfu / g) And the total culture volume of 300 L was fermented at 30 占 폚. Then, the fermented product was dried through a freeze dryer and pulverized into powder, which was used as an experimental microbial agent.

The starch (Starch, 10% / 10 L) was used as an excipient to prevent film formation due to a sugar component. Ascorbic acid (0.1% / 10 L) was used for pH control and vitamin application.

In general, the application frequency of the environment improving agent sprayed inside and outside the house reaches from 8 hours to 48 hours in short. That is, for 48 hours, the activity of the strain is maintained despite various environmental changes, and an excellent strain capable of decomposing the malodorous substance is required. When manure is sprayed with a microbial agent, it is converted into an anaerobic environment after a long period of time, even if the initial environment is aerobic. At this time, the activity of the strain should be maintained in spite of the change of the environmental condition, which is very important.

Therefore, in the present invention, the Bacillus amyl Lowry kwipe sieon's AEY-1 (Bacillus amyloliquefaciens AEY- 1, deposit number KCTC18314P), Bacillus butanone Norris borane's AEY-2 (Bacillus butanolivorans AEY- 2, Accession No. KCTC18315P) and Bacillus piece you formate (NB), Tryptic soy broth (TSB), and Potato dextrose broth (PDB) at 35 ℃ in order to investigate the optimum growth medium of the strain AIS-3 ( Bacillus licheniformis AEY-3, Accession No. KCTC18316P) The results obtained by measuring the absorbance at an optical density (OD) of 600 nm at intervals of 24 hours while culturing on a nutrient medium for 72 hours are shown in FIG.

In order to investigate the optimal culture temperature and optimum growth pH of the strain of the present invention, strains were inoculated in a TSB medium prepared at pH 4, 5, 6, 7, 8, 9 and 10 using a pH buffer solution, (Figs. 7 and 8). As a result, the strain Bacillus amyloliquefaciens AEY-1 (Accession No. KCTC18314P) was able to grow at a pH of 6 at 35 ° C. and exhibited similar growth characteristics at a culture temperature of 25 ° C. to 30 ° C. It looked. The strain AFS -2 ( Bacillus butanolivorans AEY-2, accession number KCTC18315P) exhibited a strong growth at pH 6 and 8 at 35 ° C, and similar growth characteristics at 30 ° C and 35 ° C. Bacillus licheniformis AEY-3 (Accession No. KCTC18316P) showed strong growth at pH 6 at 35 ° C and excellent growth at 30 ° C.

[Example 4] Comparison of ammonia abatement efficacy

In the present invention, the Bacillus amyl Lowry kwipe sieon's AEY-1 (Bacillus amyloliquefaciens AEY- 1, deposit number KCTC18314P), Bacillus butanone Norris borane's AEY-2 (Bacillus butanolivorans AEY- 2, Accession No. KCTC18315P) and Bacillus piece you formate miss AEY -3 ( Bacillus licheniformis AEY-3, Accession No. KCTC18316P) was tested for reducing ammonia produced in a waste manure odor generating reactor. In order to collect ammonia from malodorous substances generated from the waste manure, a 1 L Erlenmeyer flask was used as a reactor as shown in FIG. In the middle of the reactor lid, a sample collection tube was made to collect the air inside, and the concentration of ammonia generated was measured (measurement time was 0, 1, 3, 6, 12, 24, 48, 72, 96, And 168 hours).

500 g of a waste manure sample was placed in an incubator set in an incubator set at 35 ° C and incubated for a certain period of time so that sufficient odor was generated. In the experimental group, Bacillus amyloliquefaciens AEY-1, Accession No. KCTC18314P, Bacillus butanolivorans AEY-2, Accession No. KCTC18315P) and Bacillus amyloliquefaciens AEY- Each microorganism preparation containing 0.1 and 1.0% by weight of the strain AEY-3 ( Bacillus licheniformis AEY-3, accession number KCTC18316P) at a 1: 1: 1 mixing ratio was sprayed once, The reducing effect of ammonia is shown in FIG. 11 and FIG.

As a result of the experiment, as shown in Fig. 11 and Fig. 12, Bacillus amyloliquefaciens AEY-1, Accession No. KCTC18314P, Bacillus butanolivorans AEY-2, KCTC18315P) and Bacillus licheniformis AEY-3 ( Bacillus licheniformis AEY-3, Accession No. KCTC18316P) at a mixing ratio of 1: 1: 1 was effective to reduce ammonia in 0.1 to 1.0 wt% Time effect. In particular, it showed 30% or more reduction effect within 12 hours of spraying 0.1 wt%, and showed a reduction effect of ammonia gas of 95% or more after 24 hours of spraying at 1.0 wt% % Reduction effect.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. You can implement the examples. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.

Institution name: Korea Biotechnology Research Institute

Accession number: KCTC18314

Funding date: 20140725

Institution name: Korea Biotechnology Research Institute

Accession number: KCTC18315

Funding date: 20140725

Institution name: Korea Biotechnology Research Institute

Accession number: KCTC18316

Funding date: 20140725

Claims (15)

A strain of Bacillus amyloliquefaciens AEY-1 (Accession No. KCTC18314P), a culture of the strain, a concentrate and a dried product of the culture;
A strain of Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P), a culture of the strain, a concentrate and a dried product of the culture; And
( Bacillus licheniformis AEY-3), a culture of the strain, a concentrate of the culture, and a dried product as an active ingredient.
The method according to claim 1,
The microorganism preparation is a mixed microorganism preparation for ammonia odor reduction, wherein the ammonia concentration is reduced by 30% or more within 12 hours after spraying the ammonia odor source.
The method according to claim 1,
A strain of Bacillus amyloliquefaciens AEY-1 (Accession No. KCTC18314P), a culture of the strain, a concentrate and a dried product of the culture;
A strain of Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P), a culture of the strain, a concentrate and a dried product of the culture; And
Wherein the microbial agent comprises 0.05 to 1.5 parts by weight, based on the total weight of the microorganism preparation, of at least one selected from the group consisting of Bacillus licheniformis AEY-3, a culture of the strain, a concentrate of the culture, Mixed microbial preparation for ammonia odor reduction.
The method according to claim 1,
A strain of Bacillus amyloliquefaciens AEY-1 (Accession No. KCTC18314P), a culture of the strain, a concentrate and a dried product of the culture;
A strain of Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P), a culture of the strain, a concentrate and a dried product of the culture; And
1: 1: 1 mixture of at least one member selected from the group consisting of Bacillus licheniformis AEY-3, a culture of the strain, a concentrate of the culture, and a dried product, at a weight ratio of 1: Mixed microbial preparation for abatement.
The method according to claim 1,
Wherein the microbial agent comprises at least one kind of starch and vitamin C as an excipient.
The method according to claim 1,
Wherein said strain is cultivated in a TSB medium.
A livestock feed comprising the microbial formulation according to any one of claims 1 to 6.
A microbial fertilizer comprising the microbial agent according to any one of claims 1 to 6.
Bacillus amyloliquefaciens AEY-1, Accession No. KCTC18314P), Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P), and Bacillus riciniformis AEY-1 -3 < / RTI > ( Bacillus licheniformis AEY-3).
10. The method of claim 9,
Wherein said strain is cultured in a TSB medium.
10. The method of claim 9,
Wherein the culture is carried out at a temperature of 25 to 38 占 폚.
10. The method of claim 9,
Wherein the culture is carried out at a pH of from 6 to 9. The method for producing a mixed microbial agent for ammonia odor reduction according to claim 1,
10. The method of claim 9,
A strain of Bacillus amyloliquefaciens AEY-1 (Accession No. KCTC18314P), a culture of the strain, a concentrate and a dried product of the culture;
A strain of Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P), a culture of the strain, a concentrate and a dried product of the culture; And
Wherein the microbial agent comprises 0.05 to 1.5 parts by weight, based on the total weight of the microorganism preparation, of at least one selected from the group consisting of Bacillus licheniformis AEY-3, a culture of the strain, a concentrate of the culture, A method for producing a mixed microorganism preparation for ammonia odor reduction.
10. The method of claim 9,
A strain of Bacillus amyloliquefaciens AEY-1 (Accession No. KCTC18314P), a culture of the strain, a concentrate and a dried product of the culture;
A strain of Bacillus butanolivorans AEY-2 (Accession No. KCTC18315P), a culture of the strain, a concentrate and a dried product of the culture; And
1: 1: 1 weight ratio of at least one selected from Bacillus licheniformis AEY-3 strain, the culture of the strain, the concentrate of the culture and the dried product, For the preparation of a microbial preparation for oral administration.
A method for reducing ammonia odor by treating the microorganism preparation according to any one of claims 1 to 6 with an ammonia odor source to reduce ammonia odor.

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