KR20120083605A - Novel brevundimonas diminuta microoragnism and deodorizing agent using the strain to remove odor from livestock manure, and analysis method - Google Patents

Abstract

PURPOSE: A novel Brevundimonas diminuta is provided to effectively remove ammonia and sulfur series compounds. CONSTITUTION: An agent for preventing odor of livestock excrement contains Brevundimonas diminuta(deposit number KCTC 11724BP) as an active ingredient. The sulfur series and ammonia are using Brevundimonas diminuta. The sulfur series includes hydrogen sulfide and methyl mercaptane. The agent contains a strain culture, mineral, salt water, and glycerol. A strain culture is cultured under aerobic or anaerotic or aerobic culture condition.

Description

Novel Brevundimonas diminuta microoragnism and deodorizing agent using the strain to remove odor from livestock manure, and analysis method

The present invention relates to a Brevenundimonas diminuta strain having the ability to remove ammonia and sulfur-based compounds in livestock odors, and to grow well in feces, and an odor prevention remover comprising the same and a method for preparing the same. will be.

Among the problems of domestic livestock industry, the most serious problems are environmental problems (such as odor occurrence and hygiene caused by pests) and resistance to antibiotic abuse. Excessive use of such sterilization, insecticides and antibiotics leads to an increase in mortality due to the destruction of the environment as well as the development of livestock diseases. Pests are carriers of communicable disease pathogens, and pesticides are applied to control them, but environmental problems and environmental damages are serious.

The odor component is diluted by the surrounding air and generally does not cause a big problem. However, the malodorous substance is diffused to a considerable extent depending on the conditions of the surrounding terrain, the layout of the building, the wind speed, and the air pressure, thereby causing damage. More than 300 substances are known to cause odors (Ikeda et al., Japan Journal of Chemical and Engineering, 21: 91-97 (1980)), and 30 of them are important odors. It is said. It is also reported that the bad smell generated from the dry season dryer caused 7km and the bad smell of pig farms to 5km. In addition, the odor naturally generated in the livestock farming is a farming environment problem and also causes a social problem.

In addition, Salmonella or E. coli are constantly being used as antibiotics have been used to inhibit the growth of harmful intestinal bacteria, promote the growth of livestock, and cure diseases. With the emergence of antibiotic resistant bacteria such as these, in Europe, the use of antibiotics used in humans is prohibited.

Therefore, in order to reduce odor generated during livestock raising and manure resource treatment, domestic farmers have increased interest in microbial agents as an environmental improver and are also used by many farms. Local governments also encouraged farmers to use their own pilot projects, which cost 20 billion in 2005 and 14.5 billion in 2006. As the kinds thereof, microbial preparations, enzyme preparations, extract preparations (yukaze, wood vinegar, etc.), inorganic preparations and the like are used. Lactic acid bacteria that are widely used today as a probiotic is Lactobacillus acidophilus , Entrococcus bovis , Streptococcus intestinalis , Lactobacillus and Streptococcus (Conway et al. 1987). In the soil, more than 1,000 species of microorganisms, including fungi, bacteria, actinomycetes, filamentous fungi, and algae, live together. About 900 species are beneficial microorganisms and about 100 species Although classified as harmful microorganisms, chemical disinfection eliminates all the beneficial microorganisms and coexists with the beneficial microorganisms to prevent degradation. As a result, there is a problem that the harmful bacteria are gained, rather, the livestock farms that regularly sterilize and disinfect have increased diarrhea and skin diseases of respiratory diseases and mortality increases. In addition, the development of respiratory diseases in livestock caused by livestock odor has led to a decline in livestock productivity, reduced feed efficiency and increased environmental complaints.

Despite this situation, livestock farmers are trying to solve diseases caused by poor environmental problems caused by livestock grouping with chemical disinfection and antibiotics only. Chemical agents such as phosphate and acetic acid, which are generally effective for removing ammonia gas, which are commonly used, can handle odorous substances at low cost, but secondary pollution due to excessive organic nitriding of soil has been a problem. As an alternative to chemical disinfection, continuous and environmentally friendly methods have been used for biological treatment using microorganisms based on natural metabolic principles. However, until now, most livestock technologies, except for very few, do chemical disinfection before disease. It's just a level. This is because the effects of existing microbial agents, such as the reproducibility of effects depending on culture conditions, contamination, survival rate and temperature, are uncertain, and many problems that are difficult to use have not been solved so far. This is because they prefer chemicals.

In addition, until now, the main cause of livestock odor was ammonia-targeted products, but in practice, the minimum detectable concentration of ammonia is 0.1 ppm, but the sulfur-based hydrogen sulfide and methylmecaptan have values of 0.0005 and 0.0001 ppm. The yellow odor is a smell that stimulates the sense of smell even at a concentration of 1/200 or 1/1000 lower than ammonia. Therefore, the existing simple ammonia remover is not enough to expect the effect as a livestock odor remover.

In solving future livestock environmental problems, microbial agents should be able to effectively handle livestock odors, which are complex odors. In addition, there is an urgent need for more environmentally friendly livestock raising and environmental improvement due to the proliferation of environmentally friendly agriculture and the problems of chemical formulations. Odor removal using microorganisms has almost no secondary side effects, and can effectively remove specific odor components, while its effect depends on the efficacy of microorganisms. Therefore, it is important to isolate microorganisms having excellent resolution to specific malodorous components and to maintain their effects in various external environments.

In order to overcome the problems of the prior arts, the present inventors have separated the microorganisms and confirmed the activity thereof. As a result, livestock odors based on ammonia and sulfur compounds can be effectively removed, and additives are added to increase the deodorizing effect. As a livestock deodorant, this invention was completed.

Patent No. 10-84687 "Microorganism culture material and odor treatment method for odor treatment of livestock animal waste" Patent No. 10-336463 "Livestock manure purification method using indigenous microorganisms" Patent No. 10-292879 "Microbial preparation for condensation treatment and processing method of condensate using the same" Patent No. 10-430298 "Microbial agent for livestock feed addition and livestock processing" Patent No. 10-456902 "Sulfated Bacteria Thiobacillus sp. Isbleble oil strain and biological odor removal method using the same" Patent No. 10-436882 "Composite composition for promoting fermentation of livestock manure and removing odor" Patent No. 10-673585 "Smell deodorization apparatus and method using photosynthetic microorganisms" Patent No. 10-719857 "Method of manufacturing deodorizing composition" Novel Bacillus genus microorganisms producing patent No. 10-859561 cellulase and uses thereof " Publication No. 2010-22968 "Deodorization and heavy metals removal Bacillus megaterium BC1-1 KCCM 10856 P and a method for purifying livestock wastewater or food waste using the same" Korean Patent Publication No. 2009-114905 "Novel Bacillus rickenformis strain and microbial agent for odor reduction and liquid fertilization of pig manure" Korean Patent Publication No. 2009-109246 "Novel Pseudomonas Steezgeri NIST-1 Strain with Odor Reduction Effect" Patent No. 10-844310 "Bacillus Szepimicrobe" Patent No. 10-753457 "Candida genus S. epiphytes" Patent No. 10-439635 "New microorganisms effective in improving the productivity of livestock and reducing the odor of livestock manure, complex microbial agents comprising the same and a method for producing the same" Japanese Laid-open Patent Application 55-149679 "Method of treating livestock manure" U.S. Pat.No.7229612, "Compositions for eliminating human and animal excrement small and method for use therein" Japanese Patent Application Laid-Open No. 2005-192416 "Animal feed and its manufacturing method" Japanese Patent Laid-Open No. 11-104222 "Deodorant, Deodorant, Feed Additives and Additives Using Lactic Acid Bacteria and Yeast, and a Manufacturing Method Thereof"

An object of the present invention is to provide a Brevenundimonas diminuta strain having the ability to remove ammonia and sulfur-based compounds in livestock odor, and also has a characteristic that grows well in feces.

Another object of the present invention, by fermenting and aging the new Breburndimonas Dininuta can effectively remove the odor of sulfur series as well as the removal of ammonia, which was effective in removing the existing livestock odor, and the effect of the environment changes And to provide a microbial livestock odor removing agent that is complemented with problems such as short preservation.

The present invention can be achieved by isolating Brevenundimonas diminuta strain deposited with accession No. KCTC 11724BP which can effectively remove the sulfur-based odor and ammonia odor such as hydrogen sulfide, methyl mercaptan in livestock odor. .

The strain according to the present invention has excellent removal ability against yellow series odor, and possesses the property of growing well in feces, so that it can be used in addition to manure.

Strain Brescia Bundy in accordance with the present invention Pseudomonas di minu other (Brevundimonas diminuta) KCTC 11724BP is oxygen does not exist, anaerobic conditions, and oxygen is a facultative anaerobic strains to survive in both aerobic condition existing be ⁸ × 10 8 cells in only the cultivation day After reaching the culture medium (ml), the viable cell count was added at 8 × 10 ⁸ ? It maintains 1 × 10 ⁹ cell number / culture solution (ml) and can be used for various purposes in various barn environments.

The microorganisms are odor removing microorganisms that can effectively remove sulfur-based odors such as hydrogen sulfide and methyl mercaptan and ammonia odors, particularly in the removal of sulfur-based odors from low concentrations of several tens of ppm to high concentrations of 500 ppm. It is a strain that can be eliminated.

In addition, the deodorant added with mineral components, plant extracts, brine, bamboo shoot distilled liquor, and glycerin in the microbial culture broth shows excellent deodorizing performance with 4 days of continuous effect.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing the bacterial count changes during aerobic and anaerobic cultures of Brevendimonas diminuta KCTC 11724BP strain according to the present invention.
Figure 2 is a graph measuring the reduction rate for sulfur compounds after 1ml inoculation of Breuvenmonas diminuta KCTC 11724BP strain according to the present invention.
3 is a odor collecting device generated from pig manure.
Figure 4 is a graph measuring the rate of gradual decrease in the amount of sulfur compounds after spraying breven de monas di minuta strain according to the present invention in pig manure.
5 is a component analysis table of livestock odor removing agent according to the present invention.
Figure 6 is a graph showing the effect of inhibiting odor generated by the composite microbial agent according to the present invention.
Figure 7 is a graph showing the deodorizing effect by the composite microbial agent of the present invention on the pig odor odor generated in the odor generator according to the present invention.

Hereinafter, an embodiment of the present invention will be described in detail. However, the scope of the present invention is not limited to the scope of the following examples.

In order to confirm the odor removal ability of the microorganisms, artificially generate odors in the laboratory, and then apply microorganisms to confirm the microorganisms by adding oil to water and generating ammonia, hydrogen sulfide, and methyl mercaptan, and detecting them mechanically or by detecting them. The method was confirmed by using a pipe, and after collecting the manure which is actually generated in the pig house, it was put into the acrylic reactor and sprayed to confirm whether the odor generated by the spray was reduced.

Example  1: hydrogen sulfide and To methyl mercaptan  Excellent Elimination  Screening of Visible Microbial Strains

The malodor removing microorganism of this example was selected by the following method.

Mixed oil gourd that generates odor when mixed with water to separate odor-removing microorganisms (The components of mixed oil gourd are 57% by weight castor foil, 25% by weight of rapeseed watermelon, 18% by weight of rice bran, and 'KG Chemical' It is a product sold at). At this time, it was used to change the mixer to evenly generate odor. In order to generate sulfur gas from the mixed oil foil, 0.2 g of mixed oil foil and 10 ml of tap water were put into a 100 ml glass vial, and the plastic was connected to a Teflon-coated septum to be used for sealing to seal the gas. 1 ml of sludge and pig meal, etc. collected in the thus prepared emulsion were incubated with shaking at 30 ° C. and 180 rpm. For the control group, 1 ml of distilled water was added instead of the sample to adjust the total volume.

The sulfur compound concentration was measured by gas chromatography equipped with a flame photometric detector after taking a sample of the headspace with a gastight syringe with a cap septum of the culture medium at three-day intervals. Tables 1 to 3 below show respective analysis conditions and standard curves. The analysis conditions of ammonia gas were measured according to the indophenol method based on the odor prevention method.

After confirming the culture medium to reduce the malodor compared to the control group not inoculated by the above method, the concentrated malodorous microorganism group was prepared by enrichment for about 10 times.

GC / FPD Analysis Conditions Description Condition  ( sulfur compounds ) Injector 150 ℃ Colume Glass Packed Column, 1,2,3-TCEP / ID: 2.6mm, L: 2m Colume flow N ₂ , 10.1ml / min Oven Temp . 60 ° C (0min)-20 ° C / min-130 ° C (1min) Detector FPD, 150 ℃

Sulfur compound removal efficiency calculation formula Sulfur compound removal efficiency calculation formula

Co = Sulfur Compound Concentration in Control
Ci = sulfur compound concentration in the experimental group

Sulfur compound standard curves Compounds Calibration corves R ² H ₂ S Y = 151.27 X 0.998 CH ₃ SH Y = 151.87 X 0.998

After collecting a predetermined sample from the culture medium cultured in the final concentration culture, colonies of different shapes and colors were isolated from the colonies that appeared by culturing with platelet method on NA (Nutrient Agar), PDA (Potato Dextrose Agar), and MRS Agar medium. .

Example  2: Identification of isolated strain

Brevundimonas by analyzing 16S rDNA, a common identification method, for strains with the highest reduction efficiency of sulfides It was identified as diminuta (base sequence analysis results are shown in SEQ ID NO: 1) and showed a similarity of 99%. The isolate was deposited on July 14, 2010 to the Genetic Bank (KCTC), affiliated with the Biotechnology Research Institute, and was assigned accession number KCTC 11724BP.

Example  3: New Brevendi Monas Diminuta KCTC  11724 BP  Growth Characteristics of Strains

In general, the spreading frequency of the environmental improver sprayed into or outside the barn ranges from 8 hours to 48 hours. That is, for 48 hours, despite the various environmental changes, the activity of the strain is maintained and a good strain capable of decomposing bad smells is required. Manure sprayed with microbial agents is converted to anaerobic conditions over time, even if the initial environment was aerobic if left for a long time. In this case, it is very important that the viability of the strain is maintained despite such environmental conditions.

Therefore, in the present invention, while the cultivation of Brevendimonas diminuta KCTC 11724BP in an aerobic or anaerobic culture, respectively, by measuring the viable cell number every 24 hours, the change in the number of bacteria was monitored for up to 5 days. Brevundimonas diminuta , a Gram-negative bacillus bacterium, is classified as Proteobacteria and grows strongly in the range of pH 5 to pH 9 at 30 ° C incubation conditions, and incubation temperature of 20 ° C to 40 ° C. Similar growth characteristics.

Figure 1 shows the aerobic and anaerobic culture of the Brevendimonas diminuta KCTC 11724BP strain, aerobic culture conditions, aerobic culture conditions inject 20ml strain into a 100ml vial and the lid using an air-tight silly stopper Cultured at 30 ° C., anaerobic culture conditions were the same as aerobic culture using septa in the vial and the upper air of the vial was replaced with nitrogen to make anaerobic conditions.

Example  4 : New Brevendi Monas Diminuta KCTC  11724 BP  Strain In mixed oils  stink Abatement  effect

According to the invention A vial test was conducted for the ability to reduce sulfur compounds generated from mixed oils of Brevendimonas diminuta KCTC 11724BP strain. Hydrogen sulfide and methylmercaptan were generated from a milk mixture mixed with 0.25 g of mixed oil foil mixed with 100 ml vials and 9 ml of water, and then 1 ml of water was injected into the control group, and 1 ml of Breburndimonas diminuta KCTC 11724BP strain was inoculated into the sulfur compound. The reduction rate for the above was measured and shown in FIG. 2.

As a result, as shown in Figure 2, both the high concentration of hydrogen sulfide and methylmercaptan inoculated Brebredimonas diminuta KCTC 11724BP strains showed a high removal rate from day 2. Similarly, we examined the removal rate of ammonia, which is a nitrogen-based odor. The strain showed a 30% removal rate after approximately 2 days.

Example  5: New Brevendi Monas Diminuta KCTC  11724 BP  Strain Money samples  Odor in the Odor Generator Using Abatement  effect

Brevendimonas diminuta according to the present invention The odor reduction effect of the strain in the pig manure malodor generating reactor was tested. In order to collect the odor generated from the pig manure, an acrylic reactor having a volume of about 96L of 400mm × 400mm × 600mm was manufactured as shown in FIG. 3. Two small fans were attached inside the reactor to evenly saturate the odor in the inner space of the reactor. In the middle of the lid, a sampling tube and a nozzle for spraying microorganisms were installed to collect the air inside. After mixing at 70 rpm using an impeller, ammonia in odorous substances generated after 1, 2, 12 and 24 hours was measured, and amine was measured by a detection tube.

After putting the pig manure sample was stirred with an impeller for a certain time to generate a sufficient odor from the sample, the temperature was measured by installing a thermometer inside the reactor. There was no treatment in the control group after sufficient odor occurred, and the experimental group had 5, 10, 20% of Breburndimonas diminuta for pig manure. The strain was sprayed once through a nozzle and the ability to reduce odor over time is shown in FIG. 4 compared to the control.

As a result, as shown in FIG. 4, the amount of sulfur compounds was gradually reduced after spraying the Brevendimonas diminuta strain to pig manure, and the day after day 4 shows about 90% of sulfur compound reduction ability. In the case of ammonia, the effect was maintained up to 24 hours.

Example  6: Verification of odor removal effect of livestock odor remover of the present invention

100 ml vial containing 450 ppmmv hydrogen sulfide and 20 ppmmv methyl mercaptan with active minerals, glycerol, bamboo herb syrup, plant extracts, Example 3 Brevendimonas diminuta microbial culture A livestock odor remover was prepared by adding, and brine in various ratios. Plant extract is obtained by crushing the same amount of pine needles / oak tree / brown root / persimmon / red pepper / ginkgo / bamboo, etc., respectively, and then added 10 times of water and bathed for 2 to 3 hours at a temperature of 50 ℃ It was. Referring to the analysis result of the food research institute attached to FIG. 5, the extract is a liquid substance mainly containing 1,2-dimethoxyphenol component and other 20 kinds of fragrance components.

The composite microbial agent prepared by the above additive was examined for the effect of inhibiting odor by using the vial containing the oil of Example 4. Based on various ratios of the additives, the most effective ratios for the removal effect of hydrogen sulfide and mercaptan, which are sulfur compounds, are as follows. Breburndimonas diminuta strain: Mineral: Plant extract: Bamboo shoot distillate: Glycerin: Brine was prepared by adding a volume ratio of 1: 2: 2: 2: 0.1: 0.1: 1.

As shown in FIG. 6, even when the initial concentration of hydrogen sulfide was generated from 120 ppm to a high concentration of 800 ppm, the removal efficiency of 86.99.7% was shown. Methyl mercaptan also showed a stable removal efficiency of 80-100% even when the initial concentration of 30 ppm was increased to 230 ppm in the case of the formulation added at the same ratio. In fact, sulfur compounds generated in pigs are approximately 1 to 5 ppm, and sulfur compounds generated from pig powder storage tanks are only a few tens of ppm, and thus deodorants exhibiting such treatment efficiency can be treated very effectively.

The removal rate of the preparation against ammonia is also the ratio of addition to the above-mentioned sulfur compounds, showing the high removal rate of the Brevendimonas diminuta strains: minerals: plant extracts: bamboo herb distillate: glycerin: salt water volume ratio 1: 2: 2: 0.1: 0.1 : It is prepared by adding 1. After 1 injection, the removal rate was more than 95% from 12 hours to 120 hours.

Example  7: of livestock odor removing agent of the present invention Money samples  Odor in the Odor Generator Using Abatement  Effect verification

The deodorizing effect of the composite microbial agent of the present invention on pig odor generated in the malodor generator of Example 5 was examined.

As shown in FIG. 7, abundant ammonia gas was generated from the pig meal as compared to the oil paste, and 100 ppm or more occurred after 24 hours, and 20 ppm or more of ammonia gas was generated until 96 hours thereafter. The formulation showed a high removal rate of up to 72% up to 24 hours after one injection of the formulation, after which the ammonia removal rate was reduced and maintained at 51% at 48 hours. As a result of the removal efficiency of sulfide-based odorous substances, the removal rate of methyl mercaptan and methyl disulfide (dimethyl sulfide; DMS) from pig meal was maintained at 100% for 2 hours 48 hours, the removal rate of methyl mercaptan was 4 The removal efficiency was 88% during the day.

[National R & D project supporting this invention]

[Task No.] A0818

[Buddhist Name] Gyeonggi-do

[Project name] Strategic Industrial Technology Development

[Project Name] Development and industrialization of livestock odor remover using organic acid and active mineral catalyst which are microorganisms and metabolites

[Organization] Suwon University

[Research period] 2008. 12. 1.? November 30, 2010

Korea Biotechnology Research Institute KCTC11724BP 20100714

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Claims (6)

Brevundimonas diminuta strain deposited with Accession No. KCTC 11724BP.
An odor prevention remover containing Brevundimonas diminuta strain deposited with Accession No. KCTC 11724BP as an active ingredient.
A method for removing sulfur series odor and ammonia odor using Brevenundimonas diminuta strain deposited with Accession No. KCTC 11724BP.
The method of claim 4, wherein the sulfur series malodor is hydrogen sulfide, methyl mercaptan.
Breedundimonas diminuta strain culture deposited with Accession No. KCTC 11724BP and a livestock odor remover comprising minerals, bamboo vinegar, brine, glycerol.
The livestock odor removing agent according to claim 5, wherein the strain culture solution is cultured anaerobicly or anaerobicly and cultured in oil foil.

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