KR100292879B1 - Microorganism composition for treating the livestock dung and method for treating the livestock dung using the same - Google Patents

Abstract

The present invention relates to a microbial preparation for condensation treatment and a method for treating condensate using the same, and more particularly, the fungus (Bacillus subtilis & KCTC 1028) and its degradation products excellent in secretion of carbohydrate and protein processing enzyme secretion Microorganisms consisting of photosynthetic microorganisms (Rhodopseudomonas & sp.), Photosynthetic microorganisms deposited with KCTC 8937P in the Korea Advanced Institute of Science and Technology Gene Bank, and the microbial agents in livestock and barn The present invention relates to a method for treating livestock, which has the function of removing odors and, consequently, improving the livestock environment and the livestock environment of livestock.

Description

Microorganism composition for treating livestock and method for treating livestock using the same {Microorganism composition for treating the livestock dung and method for treating the livestock dung using the same}

The present invention relates to a microbial preparation for the treatment of livestock and a method of treating livestock using the same, and more particularly, has the ability to remove the odor of the rod bacteria and its decomposition products excellent in carbohydrate and protease secretion ability of the livestock. The present invention relates to a microbial preparation consisting of photosynthetic microorganisms and a method of treating livestock powder having a function of removing odors by applying the microbial preparation to livestock meal and livestock and consequently improving the growth environment of livestock and the living environment of livestock farmers. The term 'livestock' refers to common livestock meals raised in livestock farms, and includes cow meal, pig meal, chicken meal and the like.

Livestock wastewater generated in domestic livestock farms has been pointed out as a water pollution source because its biological oxygen demand (BOD) is 10,000 ~ 60,000mg / l and it is faced with the strengthening of legal regulations to protect the water environment. . In addition, the odor generated from the livestock is a serious threat to the health of livestock as well as livestock raising. Moreover, it is a reality that it is difficult for the livestock industry to run because of complaints from neighboring residents about the bad smell of livestock farmers.

At present, livestock farms can be used to treat livestock (1) as a compost raw material after a certain period of time after stocking, or (2) as a fertilizer raw material by mixing moisture control agents such as sawdust or the like, or ( 3) reduction by a mechanical reduction system. However, in the first method, there is a concern that the complaints may occur due to the smell, and leachate may be generated and washed down to rainfall to contaminate the river. The second method has a high initial facility cost, a large installation area, which burdens farmers, and a high maintenance cost due to rising prices of moisture control agents such as sawdust. In addition, there are complaints of odors. The third method has a disadvantage that it is difficult to maintain the microbial activity by increasing the initial equipment cost, the burden on the farm, the influx of disinfectants and / or antibiotics and the pH rise, and difficult to generate the expected heat of decomposition to reduce.

In addition, there is an example of treating livestock waste or livestock wastewater using microorganisms. For example, Korean Patent Publication No. 99-170602 discloses Bacillus coagulans & KCTC 1015, Pseudomonas aeroginosa & KCTC 1700, Bacillus subtilis & KCTC 2210, and acinine. Disclosed are a liquid microbial preparation comprising four strains of Bacter calcoaceticus (& Acinetobacter calcoaceticus & ATCC 13809) and a method for producing the same. Korean Patent Laid-Open Publication No. 98-33946 discloses a microbial culture material for treating wastewater and its method of improving activity while maintaining preservation and stability of microorganisms, and Korean Patent Publication No. 98-151928 discloses manure and organic wastewater treatment. A method of improving the primary aerobic digestion and secondary activated sludge methods used in the present invention is disclosed. However, the microorganisms do not have excellent efficiency and are hardly applied to livestock farms at present, and have a disadvantage of high initial capital investment.

Therefore, the inventors have focused on the fact that a large amount of undigested organic matter is contained in the livestock, and that photosynthetic microorganisms have various metabolic pathways in the natural material cycle, so that the rods and secretions superior in carbohydrate and protease secretion ability are nitrogen sources. When using a complex microbial agent consisting of photosynthetic microorganisms used as, it has been found that the above problems can be solved, the present invention was completed based on this.

Accordingly, it is an object of the present invention to provide a treatment method capable of efficiently treating flakes without generating odors.

Another object of the present invention is to provide a microbial agent for use in the method.

Another object of the present invention is to provide a novel photosynthetic microorganism constituting the microbial agent.

In order to achieve the above object, the method of treating livestock powder according to the present invention comprises a culture mixture of photosynthetic microorganisms using rod and fungus excellent in carbohydrate and protease secretion ability as a nutrient source for 1 ton of liquid or semisolid solid powder. The concentration of the rods and photosynthetic microorganisms is 0.1 to 5.0 × 10 ⁹ cfu / ml and 1.0 to 9.0 × 10 ⁹ cfu / ml, respectively.

The microbial preparation for condensation treatment of the present invention for achieving the above another object is a photosynthetic microorganism (& Rhodopseudomonas & sp.) Microorganism and Bacillus subtilis & KCTC 1028 deposited with KCTC 8937P in the Korea Advanced Institute of Science and Technology Gene Bank. It consists of microorganisms.

The novel microorganism of the present invention for achieving the above another object is a microorganism of genus Rhododoseudomonas (& Rhodopseudomonas & sp.), A photosynthetic microorganism deposited with KCTC 8937P in the Korea Advanced Institute of Science and Technology Gene Bank.

Looking at the present invention in more detail as follows.

In general, microorganisms break down proteins by the action of proteolytic enzymes, where odor-causing substances such as ammonia, carbon dioxide, hydrogen sulfide, indole, skatole, volatile amines, and mercaptans are produced. In addition, amino acids, which are degradation products of proteins, produce ammonia and fatty acids through deamination reactions, and on the other hand, detoxication reactions produce highly toxic amines and carbon dioxide corresponding to the amino acids.

Among these odor-causing substances, hydrogen sulfide can cause anorexia, dyspnea, and pulmonary edema in livestock as well as bad odors. If it is 200 ppm, it is very dangerous as lethal concentration. In addition, the ammonia concentration should not exceed 10 ppm because it facilitates the invasion of bacteria into the bronchial mucosa of the livestock, and the bronchial cilia are destroyed and respiratory diseases occur.

As described above, the present invention utilizes rod fungi excellent in carbohydrate and protease secretion ability and photosynthetic microorganisms using the secretion as a nutrient source. Preferred rods of the present invention are Bacillus subtilis & KCTC 1028, and photosynthetic microorganisms are Rhododocoseudomonas & sp. Microorganisms. The present invention is not limited to the above microorganisms, and includes all microorganisms known to those skilled in the art and replaceable.

According to the present invention, through the proper enzymatic action of Bacillus subtilis, rodent bacteria, the organic matter contained in the axial meal is decomposed into medium and low molecular weight substances, and photosynthetic microorganisms are decomposed by using the degraded medium and low molecular weight substances and odor causing substances as nutrients. By allowing growth, ultimately, the odor causing substances contained in or occurring in the mill can be effectively removed.

Bacillus subtilis & KCTC 1028, the rod bacillus used in the present invention, produces and secretes a large amount of various organic extracellular degrading enzymes such as α-amylase and protease to produce organic matter contained in nutrients. Decompose into low molecular weight materials. The photosynthetic microorganism Rhodopseudomonas & sp. Microorganism was identified as a novel microorganism as a result of the present inventors isolated from nature, and was deposited as KCTC 8937P at the Korea Advanced Institute of Science and Technology Gene Bank on April 2, 1999. . Comparison of the ammonia gas removal ability of the photosynthetic microorganism Rhodoseudomonas & sp. And the ammonia gas removal ability of Rhodobacter capsulata & KCTC 1424 and Rhodobacseudomonas spheroides & KCTC 1425 The difference was obvious. The photosynthetic microorganism Rhodopseudomonas & sp. Microorganisms can perform various metabolisms related to the production and decomposition of ammonia, nitrates and nitrites in the circulation of nitrogen. Phosphorus lower volatile fatty acids are used to grow microorganisms themselves.

As described above, the photosynthetic microorganism uses ammonia, carbon dioxide, hydrogen sulfide, indole, skatol, volatile amines, mercaptans, and the like as growth sources. If these are treated alone, the microorganisms do not progress due to the lack of growth sources. Odor is rarely removed. However, as in the present invention, when the organic material contained in the nutrient powder is decomposed into low-molecular-weight substances by the Bacillus subtilis, which is a rod bacteria, growth is accelerated by using photosynthetic microorganisms as a growth source, thereby facilitating odor-causing sources. It can be removed.

The microbial agent according to the present invention can be directly applied to a reaction tank and a storage tank for treating wastewater containing livestock, and a livestock house. In the present invention was measured by the degree of the generation of ammonia, a kind of malodorous substance generated in the axial fraction using a gas meter, as a result it was confirmed the effect of the microbial agent of the present invention.

In the present invention, the culture mixture of the rod and photosynthetic microorganisms is added in an amount of 0.5 to 2 L per 1 ton of liquid or semi-solid axial fraction, and the concentrations of the photosynthetic microorganism and the rod are 1.0 to 9.0 x 10 ⁹ cfu, respectively. / Ml and 0.1 to 5.0 x 10 ⁹ cfu / ml, preferably the concentration of photosynthetic microorganisms is about 4.0 to 6.0 x 10 ⁹ cfu / ml, and the concentration of rods is about 0.5 to 2.0 x 10 ⁹ cfu / ml. Ml.

Odor substances are removed through various and rapid organic decomposition and growth metabolism of photosynthetic microorganisms and rods of the present invention, and the circulation is continuously performed, and eventually, the constituents can be processed in a rapid and economic manner. .

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to the following Examples.

Example 1

Isolation of Photosynthetic Microorganisms

Photosynthetic microorganisms were isolated using sewage sludge from domestic stream and sewage sludge discharged from sewage treatment plant. After creating a separate source 10g homogeneous mixture through the agitated and pulverized by the addition of 100ml sterile physiological saline, the supernatant was taken and then 1ml was static at room temperature for 1 hour and diluted with decimal dilution to ^10-3. This diluted solution was plated on agar medium. The plated medium was incubated at 25 ° C. for 3 days, and a single colony was selected for pure separation by serial passaging. In order to prevent the contamination of yeast during the pure separation process, cyclohexamide (cyclohexamide, 100㎍ / ml) was added to M9 at least 1000 medium. The composition of the M9 minimum agar medium used at this time is shown in Table 1 below.

Composition of M9 Minimal Media for Photosynthetic Microbial Separation Na ₂ HPO ₄ 6.0 g KH ₂ PO ₄ 0.3 g NH ₄ Cl 0.1g NaCl 0.5g Glucose 2.0 g MgSO _{4 7} H ₂ O 0.25g CaCl ₂ 0.015 g Thiamine HCl 0.001g Agar 20.0 g Distilled water 1ℓ pH 7.0

Example 2

Culture Characteristics of Isolated Microorganisms

The isolated photosynthetic microorganisms were inoculated into Rhodoseudomonas agar medium (ATCC Medium 543) and cultured to investigate their properties. After inoculating the photosynthetic microorganisms isolated on the Rhodoshu domonas agar medium, it was observed by incubating for 5 days while maintaining the internal temperature of the incubator at 25 ℃. As a result, the microbial colonies formed on the agar medium showed dark yellowish brown color, and the microorganisms observed under the microscope showed ovoid-rods of ovate and rod shape, and the size of the microbial cells was measured to be 0.7∼2.0㎛. It became. At this time, the composition of the Rhodoshu domonas minimum thousand medium is shown in Table 2 below.

Composition of Rodoshudomonas Agar Medium (ATCC Medium 543) for Microbial Cultivation Sodium succinate 2.5g (NH ₄ ) ₂ SO ₄ 1.25 g K ₂ HPO ₄ 0.9 g KH ₂ PO ₄ 0.6g Yeast extract 0.5g MgSO _{4 7} H ₂ O 0.2 g CaCl ₂ 0.07g Ferric citrate 3.0mg Ethylenediamine Tetraacetate (EDTA) 2.0mg Baby 20.0 g Distilled water 1ℓ pH 7.0

On the other hand, the M9 minimum medium used for the separation of microorganisms was investigated the material fermentation by varying the carbon source, the results are shown in Table 3.

Fermentation ability of isolated microorganisms Acetate Positive Benzoate Negative Butyrate positivity Citrate +/- Fructose +/- Glucose positivity Glycerol positivity Lactate positivity Malate positivity Succinate positivity Maltose positivity

In addition, the results of examining the growth ability of the isolated microorganisms are shown in Table 4 below.

Growth of Isolated Microorganisms Glucose positivity At 25 ℃ positivity Gluconate positivity 30 ℃ positivity Maltose positivity 40 ℃ voice Lactose positivity Methanol +/- Glycerol positivity ethanol +/- Sucrose positivity Raffinose +/- Mannitol positivity Arginine +/- starch positivity Nitrate positivity Citrate positivity Nightrite positivity

As a result of examining the culture characteristics, isolated microorganisms were identified as Rhodopseudomonas & sp. Having photosynthetic ability as a kind of purple non-sulfur bacteria, and the isolated photosynthetic microorganisms Rhodoschudomonas genus as of April 3, 1999. It was deposited with the Korea Advanced Institute of Science and Technology Gene Bank (KCTC) (Accession No. KCTC 8937P).

Example 3

High concentration culture of isolated photosynthetic microorganisms

The isolated photosynthetic microorganisms were inoculated into flasks containing Rhodoschudomonas liquid medium (ATCC Medium 543) and precultured via shaking culture. This preculture was inoculated in a 5 L fermenter prepared at twice the concentration of the Rodo Pseudomonas liquid medium, and the samples were taken at regular time while incubating, and the pH change of the culture was measured. The growth was investigated. At this time, as the culture conditions, the temperature was fixed at 25 ℃ and the pH of the initial medium was adjusted to 7.0. As the culture progressed, the stirring speed was 200 to 400 rpm, and the aeration amount was controlled to 0.5 to 1.5 vvm for 6 days. In some cases, the incandescent lamp was turned on for irradiation, but the dose was not measured.

As a result, at the beginning of the culture, the pH of the culture solution was slightly lowered to about 6.3, but after 48 hours of culture, the pH began to rise and the pH at 144 hours after the incubation had risen to 10 or more. The increase in absorbance tended to increase very slowly until 72 hours, but it increased rapidly after 96 hours. This increase was maintained up to 132 hours.

As a result, the maximum growth showed absorbance of 35.5 at 132 hours of incubation, after which the absorbance of the cells remained unchanged or gradually decreased.

Example 4

High concentration bulk culture of isolated photosynthetic microorganism Rhodoschudomonas

The isolated photosynthetic microorganisms were inoculated into flasks containing Rhodoschudomonas liquid medium (ATCC Medium 543) and precultured via shaking culture. The preculture was inoculated into a 500-l fermenter prepared at a concentration of 3 times the composition of the Rhodoschudomonas medium, and the samples were taken for a predetermined time while incubating, and the pH change of the culture was measured. The figure was examined. At this time, the culture conditions were the same as in Example 3, the temperature was fixed at 25 ℃ and the pH of the initial medium was adjusted to 7.0, while the incubation proceeds while adjusting the stirring speed of 100 ~ 150rpm, aeration rate 0.5 ~ 1.0 vvm The culture was carried out for 7 days.

As a result of the measurement, the pH of the culture solution was slightly lower than the pH shown in Example 3 at the initial stage of culture, but the trend of pH rise was the same after 48 hours of culture, and the pH at 144 hours after the culture was 10.1. The increase in absorbance tended to increase very slowly until 72 hours, but it increased rapidly after 96 hours, and the increase was maintained up to 150 hours.

Changes in pH and growth curves were slightly longer depending on the concentration of the media, but overall there was a similar trend of a stable type. The maximum growth was recorded at absorbance of 50.7 at 144 hours of incubation, after which the absorbance of the cells was maintained up to 168 hours without change.

Example 5

Odor removal effect of photosynthetic microorganism

After adjusting the Rhodoshu domonas strain obtained through high concentration bulk culture to the concentration of 5.0 × 10 ⁹ cfu / ml, 100 ml of this microbial bacteria solution was added to 100 l of swine from a nearby pig farm and the temperature was fixed at 25 ° C. Odor elimination reaction was induced.

The reaction tank used was designed to control the aeration, agitation and temperature, the aeration amount was maintained at 0.1vvm using a blower, the stirring speed was 40rpm. The reaction was allowed to occur for 48 hours and the degree of odor removal was determined by measuring the concentration of ammonia generated in the initial stage of reaction and 12 hours by using a gas meter at the exhaust port of the reactor.

As a result, the initial concentration of ammonia was 3,128ppm, and as the reaction continued, 2,237ppm after 12 hours, 1,415ppm after 24 hours, and 798ppm after 36 hours were measured. After 48 hours, the odor was removed so that it could not feel the smell of ammonia, but it was 12ppm when analyzed by gas meter. This resulted in the final effect of photosynthetic microorganisms removing more than 99% of the ammonia generated in the nutrients through aerobic reactions.

Rhodobacter capsulata and Rhodopseudomonas spheroides were used as controls in the same conditions to compare odor removal ability. As a result, in the case of Rhodobacter capsularta, the concentration of ammonia generated after 48 hours of reaction was 720 ppm, and in the case of Rhodoshu domonas spheroid, it was measured as 845 ppm. Therefore, the novel photosynthetic microorganism of the present invention, Rhodoschudomonas genus appeared to have an excellent effect compared to the control.

In addition, the reaction was induced in the same manner under anaerobic conditions without aeration and agitation. In this case, the sampling method was performed as follows. 100 ml of the reaction sample was taken, placed in a baffled flask, capped, shaken at 250 rpm for 1 minute, and the concentration of ammonia was measured by approaching the gas inlet to the flask.

As a result, the initial concentration of ammonia was 3,095ppm, which is similar to the initial state of aerobic conditions, but after 12 hours, there was almost no change, and after 24 hours, the ammonia concentration was very low compared to the reaction of aerobic conditions. . After 48 hours, it decreased to 1,965ppm, after 72 hours, to 1,480ppm, and after 96 hours, to 877ppm. After 120 hours, ammonia was continuously generated and measured as 467ppm. This resulted in the final result of photosynthetic microorganisms removing ammonia from the nutrients through anaerobic reactions, but the removal is weaker and takes longer than the aerobic reactions.

Example 6

Culture of Bacillus subtilis (& Bacillus subtilis & (KCTC 1028))

Bacillus subtilis strains with excellent secretion ability of alpha-amylase and α-amylase to degrade carbohydrate and protease to degrade protein were cultured using a 5 L fermenter. The medium used at this time is a nutrient medium, the composition of which is shown in Table 5. The rod fungus cultured using the nutrient medium, the absorbance was measured to 27.2 after 36 hours incubation.

Composition of nutrient medium for culture of rod fungus Beef extract 9.0 g Peptone 15.0 g Distilled water 1ℓ pH 6.8

Example 7

Manufacturing and application of environmental improver for removing odors by mixing photosynthetic microorganisms and rods

The photosynthetic microorganism and rod fungus were mixed to prepare an environmental enhancer for removing odor, and the degree of removal of ammonia was determined in the reaction tank in the same manner as in Example 5.

In this case, the final concentration of photosynthetic microorganisms was adjusted to 5.0 × 10 ⁹ cfu / ml, and the concentration of rod bacteria was adjusted to 1.0 × 10 ⁹ cfu / ml.

In the aerobic reaction, the concentration of ammonia measured after 24 hours of reaction was 1.4 ppm and sensory smell could not be felt.

In the case of anaerobic reaction, the measured ammonia concentration was only 22ppm after 120 hours, which resulted in the removal of more than 99% of the ammonia generated in the shaft.

In consideration of the fact that most of the generated nutrients are stored in the storage tank, the complex environment improver prepared was injected or sprayed at the concentration of 0.01-2.5% directly into the storage tank, which is anaerobic condition, and the aerobic condition, and the generation of ammonia was measured.

As a result, as shown in Table 6, in the open barn, ammonia generation was reduced to less than 10 ppm in 24 hours, and in the case of the storage tank, ammonia generation was reduced to less than 10 ppm after 48 hours after the addition of the complex environment improver.

Ammonia Removal Effect of Combined Environmental Enhancer in Storage Tanks and Barns time Storage tank (anaerobic condition) Barn (aerobic condition) 12 hours 217 ppm 106 ppm 24 hours 87 ppm 8.2 ppm 36 hours 43 ppm 3.6 ppm 48 hours 7.5 ppm 1.1 ppm

As described above, the present invention is excellent in degrading organic matter and genus Rodoschudomonas microorganisms, which are the photosynthetic microorganisms isolated from the natural world in connection with the odor which occurs as a serious environmental problem and social problem. By adding Bacillus subtilis, which is an ever-growing bacterium, there is an effect that it is possible to economically remove the odor generated in the sperm in a short time.

Claims (4)

1 ton of liquid or semi-solid flakes consists of adding 0.5 to 2 l of a mixture of rods having excellent carbohydrate and protease secretion ability and photosynthetic microorganisms using the secretion as a nutrient source. The concentration of photosynthetic microorganisms is 0.1 to 5.0 × 10 ⁹ cfu / ml and 1.0 to 9.0 × 10 ⁹ cfu / ml, respectively. The method of claim 1, wherein the photosynthetic microorganism is a photosynthetic microorganism (& Rhodopseudomonas & sp.) Microorganisms deposited with KCTC 8937P in the Korea Advanced Institute of Science and Technology Gene Bank, and the rod bacteria is Bacillus subtilis & (KCTC 1028). Method characterized in that the microorganism. A microbial preparation for condensation treatment, comprising a microorganism of Rhodosedomonas & sp., A photosynthetic microorganism deposited with KCTC 8937P at the Korea Advanced Institute of Science and Technology. A microorganism of the genus Rhodopseudomonas & sp., A photosynthetic microorganism deposited with the Korea Advanced Institute of Science and Technology Gene Bank as KCTC 8937P.