Novel psychrophilic bacillus galactosidilyticus bsm-6 strains having malodor reduction effect and soil nematode prevention effect, and use thereof

Abstract

The present invention relates to a psychrophilic Bacillus galactosidilyticus BSM-6 KACC92503P having odor reduction activity against livestock manure and soil nematode control activity. According to the present invention, the novel microorganism Bacillus galactosidilicatecus BSM-6 KACC92503P of the present invention can reduce odors by suppressing the production of odor-causing components at low temperatures and can control soil nematodes.

Description

Technology Field

[0001] The present invention relates to a novel psychrophilic Bacillus galactosidylticus BSM-6 strain having an odor reduction effect and a soil nematode control effect, and to the use thereof, more specifically, to a Bacillus galactosidylticus having an odor reduction activity and a soil nematode control activity for livestock manure ( Bacillus galactosidilyticus The present invention relates to a microbial preparation for reducing livestock manure odor comprising BSM-6 KACC92503P, the Bacillus galactosidilicate BSM-6 KACC92503P strain or a culture thereof, a microbial preparation for controlling soil nematodes, and a feed for reducing livestock manure odor comprising the microbial preparation. Background Technology

[0002] As the consumption of livestock products increased, the domestic livestock market grew significantly, and the professionalization of livestock farms contributed to social development. In the past, environmental pollution issues such as odors and soil contamination caused by livestock manure were not a major concern; however, recently, due to the increase in the number of livestock per farm and intensive farming practices, the amount of manure generated has increased, yet its treatment and management are not being properly carried out, leading to the emergence of environmental pollution problems such as odors and soil contamination.

[0003] In particular, livestock manure is a major cause of conflict with residential areas because the intensity of the odor increases as odor-causing components such as ammonia, hydrogen sulfide, trimethylamine, and methyl mercaptan are generated during the process of protein decomposition in anaerobic conditions, such as in storage tanks. Consequently, various methods have been attempted to remove odors generated from livestock manure. These methods include the use of veterinary drugs, deodorizers, feed additives, oxidizers, and moisture regulators; however, since these methods are harmful to livestock, pose environmental problems, and are insufficient for efficiently removing odors, the development of new odor removal methods is necessary.

[0004] Recently, methods utilizing microorganisms to environmentally eliminate the root causes of odors are being researched. Since odor components are rapidly oxidized and decomposed when treated under high-temperature aerobic conditions, thereby reducing the odor, technologies are generally being developed to reduce odors caused by livestock manure through the treatment of mesophilic or thermophilic microorganisms. Specifically, Korean Patent Publication No. 2014-0114564 relates to a microbial agent for removing nitrogen from high-concentration wastewater and a treatment method for a livestock manure treatment facility using the same, wherein the mesophilic Nitrobacter genus ( Nitrobacter Odors are being removed using microorganisms (sp. H2S1), and Korean Registered Patent No. 2150330 relates to a novel strain having odor-reducing efficacy for livestock manure, and is a heat-resistant Ocyanobacillus ( Oceanobacillus Odors are being removed using microorganisms of the genus ) However, since the microorganisms mentioned above are all active at medium to high temperatures, they are not suitable for reducing odors generated from livestock manure during the winter season.

[0005] Soil nematodes are a collective term for animals belonging to the phylum Nematoda, including those that live freely in the soil and those that parasitize plants; due to their wide habitat, they are found throughout the soil. Plant-parasitic nematodes cause damage to crops by sucking sap from the roots using a proboscis located at the front, and they induce secondary infections in the wounded areas. Statistics show that over 54% of domestic greenhouse cultivation sites are infected with root-knot nematodes, which reduces horticultural crop production by 15%.

[0006] Meanwhile, much livestock manure is used as compost after solid-liquid separation and subsequent maturation of the solid phase. Since parasitic nematodes are present in this compost, the production of high-quality compost would be possible if these nematodes could be suppressed. It appears that such compost can be produced by feeding livestock feed that has nematode control effects, thereby utilizing the nematode-free manure for compost production.

[0007] Accordingly, as a result of diligent research efforts to overcome the problems of the aforementioned prior art, the inventors confirmed that a novel Bacillus galactosidylticus BSM-6 isolated from livestock manure can decompose organic matter in livestock manure at low temperatures, reduce odors induced by livestock manure, and control soil nematodes, thereby completing the present invention. Prior art literature

[0008] (Patent Document 0001) KR 10-2014-0114564 A(Patent Document 0002) KR 10-2150330 B1 The problem to be solved

[0009] Accordingly, the main objective of the present invention is to provide a novel psychrophilic Bacillus galactosidylticus BSM-6 strain capable of reducing odors by decomposing odor-causing components derived from livestock manure even at low temperatures, and controlling soil nematodes.

[0010] Another objective of the present invention is to provide a microbial agent for reducing livestock manure odor using the novel psychrogenic Bacillus galactosidilicatecus BSM-6 strain.

[0011] Another objective of the present invention is to provide a microbial preparation for controlling soil nematodes using the novel psychrophilic Bacillus galactosidilicatecus BSM-6 strain.

[0012] Another objective of the present invention is to provide a feed for reducing livestock manure odor using a microbial preparation comprising the novel psychrogenic Bacillus galactosidilicatecus BSM-6 strain. means of solving the problem

[0013] According to one aspect of the present invention, the present invention relates to a psychrophilic Bacillus galactosidylticus having odor reduction activity and soil nematode control activity for livestock manure ( Bacillus galactosidilyticus ) Provides BSM-6 KACC92503P.

[0014] Various methods have been attempted to reduce the odors generated from livestock manure. Among these, the use of microorganisms is environmentally friendly and effective in reducing odors, leading to ongoing efforts to reduce manure-derived odors using diverse microorganisms. However, these commonly used microorganisms have low activity at low temperatures, which limits their ability to reduce odors generated from cold livestock manure during the winter. Furthermore, livestock manure is also used as compost; however, since parasitic nematodes are present in this compost, using it as compost results in crop damage caused by nematodes, particularly soil nematodes.

[0015] Accordingly, in order to isolate and identify strains that are active in livestock manure at various temperatures and exhibit control activity against soil nematodes, the inventors isolated strains from livestock manure and finally selected strains having low-temperature activity, odor reduction ability, and soil nematode control ability, and performed genetic analysis. In addition, the selected strain was named "Bacillus galactosidylticus BSM-6" as a novel microorganism and deposited with the National Institute of Agricultural Sciences Microbiome Bank (KACC) on May 3, 2023, and was assigned accession number KACC92503P.

[0016] The novel microorganism Bacillus galactosidilicatecus BSM-6 KACC92503P of the present invention comprises a 16S rRNA gene having the nucleotide sequence described in SEQ ID NO. 1.

[0017] The term 'livestock manure' in the present invention refers to livestock manure consisting of feces and urine generated from livestock or poultry, and the livestock manure includes manure itself that has not undergone physical or chemical treatment, manure that has undergone treatment such as drying or fermentation, fertilizer in the form of manure that has been fertilized, and manure in the form of liquid containing livestock manure and washing water.

[0018] The novel microorganism Bacillus galactosidilicatecus BSM-6 KACC92503P of the present invention can reduce odors by suppressing the generation of one or more gases selected from the group consisting of odor-causing components, such as ammonia, methylamine, ethylamine, trimethylamine, dimethylamine, triethylamine, hydrogen sulfide, and methyl mercaptan, and preferably can reduce odors by suppressing the generation of one or more gases among ammonia and hydrogen sulfide.

[0019] According to one experimental example of the present invention, after adding a culture solution of the novel microorganism Bacillus galactosidilicatecus BSM-6 according to the present invention to manure, the concentrations of ammonia and hydrogen sulfide, which are odor-causing components, were measured, and it was confirmed that the concentration of odor-causing components present in the manure decreased (see Experimental Example 5). These results show that the novel microorganism according to the present invention exhibits odor reduction activity against odors generated in livestock manure.

[0020] The novel microorganism Bacillus galactosidylticus BSM-6 KACC92503P of the present invention has maximum activity at 1 to 20°C, preferably 1 to 17°C, and exhibits maximum activity particularly at low temperatures.

[0021] According to one experimental example of the present invention, Bacillus galactosidylticus BSM-6 according to the present invention showed the highest growth rate at 10 to 20°C, and from this, it was found to exhibit low-temperature resistance (see Experimental Example 3). These results suggest that the novel microorganism according to the present invention is effective in reducing odors generated from livestock manure at low temperatures during the winter.

[0022] The novel microorganism Bacillus galactosidilicatecus BSM-6 KACC92503P of the present invention can control one or more nematodes selected from the group consisting of root-knot nematodes, root-rot nematodes, leaf nematodes, and cyst nematodes.

[0023] According to one experimental example of the present invention, when a culture of the Bacillus galactosidilicatecus BSM-6 strain was diluted and treated, it was found to exhibit a high nematode control effect (see Experimental Example 6). These results suggest that the novel microorganism according to the present invention is effective in controlling parasitic nematodes in livestock manure.

[0024] According to another aspect of the present invention, the present invention provides a microbial preparation for reducing livestock manure odor comprising the psychrophilic Bacillus galactosidilicatecus BSM-6 KACC92503P strain or a culture thereof.

[0025] The term 'microbial preparation' in the present invention refers to a preparation that exhibits the effect of reducing livestock manure odor by directly utilizing the function of microorganisms indicated on the raw material, that is, microorganisms contained in the raw material.

[0026] In the microbial preparation for reducing livestock manure odor according to the present invention, the culture may or may not contain the Bacillus galactosidylticus BSM-6 strain, and specifically, is one or more selected from the cells of the Bacillus galactosidylticus BSM-6 strain, a culture medium containing the cells, a crushed product of the cells, a culture medium from which the cells have been removed from the culture of the strain, or an extract of the cells of the strain.

[0027] According to another aspect of the present invention, the present invention provides a microbial preparation for controlling soil nematodes comprising the psychrophilic Bacillus galactosidiliticus BSM-6 KACC92503P strain or a culture thereof.

[0028] In the microbial preparation for controlling soil nematodes according to the present invention, the culture may or may not contain the Bacillus galactosidylticus BSM-6 strain, and specifically, is one or more selected from the cells of the Bacillus galactosidylticus BSM-6 strain, a culture medium containing the cells, a lysate of the cells, a culture medium from which the cells have been removed from the culture of the strain, or an extract of the cells of the strain.

[0029] The microbial preparation according to the present invention may be formulated in a liquid form in which the Bacillus galactosidylticus BSM-6 strain is mixed or cultured in a culture medium, or in a dried powder form dried using methods such as freeze-drying or hot-air drying. When the microbial preparation according to the present invention is formulated in a liquid form, it has the advantages of faster activity and easier handling compared to the dried powder, and when formulated as a dried powder, it has the advantages of a simple manufacturing process and good preservation. Therefore, the microbial preparation can be appropriately formulated according to the purpose of use, environment, etc.

[0030] According to another aspect of the present invention, the present invention provides a livestock feed for reducing manure odor containing a microbial preparation comprising the psychrophilic Bacillus galactosidilicatecus BSM-6 KACC92503P strain or a culture thereof.

[0031] Since the livestock feed for reducing manure odor according to the present invention comprises a microbial preparation containing a psychrophilic Bacillus galactosidylticus BSM-6 KACC92503P strain or a culture thereof having odor-reducing activity and soil nematode control activity, when the feed is applied to livestock, livestock manure with reduced odor and controlled parasitic soil nematodes will be produced, making it possible to produce high-quality livestock manure compost.

[0032] The livestock feed according to the present invention comprises, in addition to the microbial preparation, a ground material obtained by grinding one or more selected from the group consisting of corn, wheat, wheat, soybean meal, soybean meal, oats, soybeans, rice, etc.

[0033] The livestock feed for reducing livestock manure odor according to the present invention may be mixed with a microbial agent and the pulverized material in a mixing weight ratio of 1:1 to 10, preferably 1:1 to 5. If the mixing ratio deviates from the above, the effect of reducing the odor of livestock excrement and the effect of controlling soil nematodes may be negligible. Effects of the invention

[0034] As described above, the novel microorganism Bacillus galactosidylticus BSM-6 KACC92503P of the present invention can reduce odors by suppressing the production of odor-causing components at low temperatures and can control soil nematodes. Brief explanation of the drawing

[0035] Figure 1 shows the phylogenetic tree of the novel microorganism Bacillus galactosidylticus BSM-6 KACC92503P of the present invention. Figures 2 and 3 show the results of confirming the morphological characteristics of the novel microorganism Bacillus galactosidilicatecus BSM-6 of the present invention. Figure 4 shows the results of confirming the protein degradation ability of the novel microorganism Bacillus galactosidilicatecus BSM-6 KACC92503P of the present invention. Figure 5 shows the results of confirming the cellulose degradation ability of the novel microorganism Bacillus galactosidilicatecus BSM-6 KACC92503P of the present invention. Specific details for implementing the invention

[0036] The present invention will be described in more detail below through examples. These examples are merely illustrative of the present invention, and therefore the scope of the present invention should not be interpreted as being limited by these examples.

[0037] Experimental Example 1: Microorganism Isolation

[0038] 1) Separation Source and Sample Pretreatment

[0039] Microorganisms were isolated from a manure storage tank at a pig farm located in Hongsan-myeon, Buyeo-gun.

[0040] 2) Strain Selection and Identification

[0041] To achieve odor reduction by decomposing organic matter within pig manure, the ability to grow within the manure itself is of paramount importance. Common beneficial microorganisms known for their effectiveness in odor reduction exhibit very poor growth potential in pig manure. This is due to the extremely high salt concentration present in the manure. When salt concentrations are high, osmotic pressure destroys the cytoplasm, rendering the organism unable to survive. Therefore, for strain selection, a microbial culture medium was prepared using the pig manure itself to identify microorganisms capable of growing in high-salinity conditions. Among salt-tolerant bacteria, those with excellent efficacy in organic matter decomposition were selected.

[0042] Specifically, the solid pig manure obtained through solid-liquid separation in a slurry pit was used to prepare the medium. More specifically, large lumps of impurities were removed using a sieve, and pig manure and distilled water were mixed in a 1:1 (w / v) ratio. Then, 2% (w / v) of agar was added, and the mixture was sterilized sufficiently at 121°C for at least 15 minutes and poured into a petri dish to prepare the Swine Medium (SM). Pig manure was diluted and spread onto the prepared SM medium, and bacteria were cultured by incubating at 20°C for 2 to 3 days. Subsequently, the bacteria that were visually dominant were isolated in pure form and finally selected.

[0043] 16S rRNA sequencing was performed to identify the microorganisms finally selected above. To analyze the 16S ribosomal RNA sequencing of the isolated strains, genomic DNA was extracted and purified, and then amplified by PCR for 30 cycles using 27F (5'-AGAGTTTGATCCTGGCTCA-3') and 1492R (5'-ACGGCTACCTTGTTACGAC-3') primers under conditions of denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 68°C for 1 minute. The 16S rRNA genes amplified from the 27F and 1492R primers were cloned into the pGEM-T easy vector, and the nucleotide sequences of the cloned 16S rRNA genes were analyzed.

[0044] 3) Genome sequencing (nucleotide sequence and phylogenetic tree)

[0045] Identification was performed by comparing with 16S rRNA sequencing from the NCBI database using the BLASTN program, and phylogenetic trees based on sequence similarity and the cluster method were constructed using vector NTI 8. Analysis results showed that the isolated microorganism exhibited 99% homology with Bacillus galactosidilyticus. Therefore, the above strain is Bacillus galactosidilyticus ( Bacillus galactosidilyticus It was named BSM-6 (sequence number 1) and deposited at the National Institute of Agricultural Sciences Microbiome Bank (KACC) on May 3, 2023. The phylogenetic tree is shown in Figure 1.

[0046] Experimental Example 2: Morphological characteristics of the strain

[0047] Considering the characteristics of the pig manure medium, the morphological characteristics of the corresponding strain were determined by subculturing it on MA (Marine Agar) medium.

[0048] As a result, as can be seen in Figure 2, which shows the observation of the culture plate, opaque white colonies were formed, appearing as flat, slightly shiny colonies. Additionally, the margins had a slightly erosive shape.

[0049] As can be seen in Figure 3, which shows the results of the microscopic examination, it is mainly composed of monococcus, and diplococcus and streptococcus were observed.

[0050] Experimental Example 3: Confirmation of Low-Temperature Resistance Characteristics

[0051] To confirm the low-temperature resistance characteristics of the Bacillus galactosidilicus BSM-6 strain according to the present invention, the Bacillus galactosidilicus BSM-6 strain was inoculated into TSB medium and cultured with shaking (150 rpm) for 48 hours at different temperatures (10℃, 15℃, 20℃, 25℃, 30℃, 35℃) to determine the density (Unit: cfu / ml) of the cultured strain, and the results are shown in Table 2 below.

[0052] 10℃ 15℃ 20℃ 25℃ 30℃ 35℃ E.coli 1.0×10 2 2.1×10 2 4.2×10 5 3.5×10 7 5.1×10 8 5.0×10 8 B. galactosidilyticus BSM-6 4.5×10 5 2.0×10 6 5.0×10 8 8.8×10 7 8.3×10 7 8.3×10 7

[0053] As a result, as shown in Table 2 above, E. coliCulture was not smooth at 10℃ and 15℃, resulting in significantly low density, while the highest density was observed at 30℃. On the other hand, Bacillus galactosidylticus BSM-6, the strain according to the present invention, exhibited the highest density at 20℃, and even at low temperatures of 10℃ and 15℃ E. coli It was confirmed that it grows well compared to. These results show that the strain according to the present invention is a cold-resistant microorganism, based on the fact that its optimal activity temperature is 20℃ and it has low-temperature resistance.

[0054] Experimental Example 4: Confirmation of Organic Matter Decomposition Effect

[0055] 1) Confirmation of protein degradation ability

[0056] To confirm protease secretion, skim milk medium was prepared, and the presence or absence of halo formation by the strain was checked.

[0057] As a result, as shown in Figure 4, the halo of the Bacillus galactosidilicatecus BSM-6 (No. 91) strain according to the present invention was measured to be 33 mm, and this result shows that the protein degradation ability is excellent.

[0058] 2) Confirmation of cellulose degradation ability

[0059] To confirm cellulase secretion, a culture medium was prepared by adding 5 g / L of CMC (Carboxymethyl cellulose), 1 g / L of Dipotassium phosphate, 1 g / L of Sodium nitrate, 0.5 g / L of Potassium chloride, 0.5 g / L of Magnesium sulfate, 0.05 g / L of Yeast extract, and 15.0 g / L of Agar powder to 1 L of distilled water. After inoculating the strain into the prepared medium and culturing for 3 days, the presence or absence of a halo was checked after staining with 0.1% Congo red solution.

[0060] As a result, as shown in Figure 5, a ring was formed only in the Bacillus galactosidilicatecus BSM-6 (No. 91) strain according to the present invention, and this result shows that the cellulose degradation ability is excellent.

[0061] Experimental Example 5: Confirmation of Odor Reduction Ability

[0062] To confirm the odor reduction effect of the Bacillus galactosidilicate C. BSM-6 strain according to the present invention, the concentrations of ammonia and hydrogen sulfide, which are odor-causing components, were checked. Specifically, the Bacillus galactosidilicate C. BSM-6 strain according to the present invention and Bacillus subtilis ( Bacillus subtilis ), Lactobacillus plantarum( Lactobacillus plantarum The strains were cultured for 2 days in TSB (Tryptic Soy Broth), a general bacterial medium. 30g to 30ml of pig manure was filled into 50ml Falcon tubes, 0.3ml of each strain (corresponding to 1%) was inoculated, and the tubes were left at room temperature to compare the reduction in odor over time. Odor measurements were performed by installing a tube and cap on the front of a MuliRAE Lite (portable multi-gas detector) and connecting it to a Falcon tube containing pig manure. After shaking and stirring thoroughly, the tube was left to stand for 5 minutes, the lid was opened, and measurements were taken. The results are shown in Table 3 below.

[0063] ammonia hydrogen sulfide 24h 48h 24h 48h Control 47 41 2.5 2.1 B. galactosidilyticus BSM-6 23 21 0.3 0.5

[0064] As a result, as can be seen in Table 3 B. galactosidilyticus When treated with BSM-6, ammonia and hydrogen sulfide decreased by 51% and 88%, respectively, compared to the control group.

[0065] Experimental Example 6: Confirmation of soil nematode control effect

[0066] Strawberries (variety: Seolhyang) grown in soil in Jochon-myeon, Buyeo-gun were transferred to pots, and experiments were conducted in the laboratory. A microbial culture solution in the pot was diluted 5 to 20 times and applied as a drench treatment once. The number of nematodes (unit: number) was examined under a microscope on days 3, 6, and 9 after treatment, respectively, and the results are shown in Table 4 below.

[0067] Control group (untreated) 5 times the amount 10 times the amount 20 times the amount number of repetitions 1 2 3 1 2 3 1 2 3 1 2 3 beginning 301 333 310 350 330 321 298 305 312 283 291 352 3 days 311 320 295 35 31 29 132 155 140 167 180 190 6th 321 322 320 31 32 31 133 150 145 181 175 201 9th 315 312 327 36 35 30 130 144 131 175 177 200

[0068] As a result, as shown in Table 5 above, when treated with a 5-fold dilution, high control rates of 90.5%, 90.6%, and 89.9% were observed after 3, 6, and 9 days, respectively. It was confirmed that the control rate decreased as the dilution increased, but even when treated with a 20-fold dilution, control rates of 42.0%, 39.8%, and 40.4% were observed after 3, 6, and 9 days, respectively.

[0069] Depository Name: National Institute of Agricultural Sciences, Rural Development Administration, Microbial Bank (KACC) Accession Number: KACC92503P Date of Deposit: 2023-05-03

Claims

Claim 1 Psychrophilic Bacillus galactosidylticus, accession number KACC92503P, having odor reduction activity against livestock manure and soil nematode control activity ( Bacillus galactosidilyticus ) BSM-6 strain. Claim 2 In claim 1, the Bacillus galactosidylticus BSM-6 strain of accession number KACC92503P is characterized by having a 16s rRNA gene represented by sequence number 1. Claim 3 A Bacillus galactosidylticus BSM-6 strain of accession number KACC92503P, wherein, in claim 1, the reduction activity is characterized by inhibiting the generation of one or more gases among ammonia and hydrogen sulfide. Claim 4 In claim 1, the Bacillus galactosidylticus BSM-6 strain of accession number KACC92503P is characterized by having growth activity at 1 to 20℃. Claim 5 Bacillus galactosidylticus BSM-6 strain of accession number KACC92503P, characterized in that, in claim 1, the soil nematode is one or more nematodes selected from the group consisting of root-knot nematodes, root-rot nematodes, leaf nematodes, and cyst nematodes. Claim 6 A microbial preparation for reducing livestock manure odor comprising the psychrophilic Bacillus galactosidylticus BSM-6 strain of accession number KACC92503P according to claim 1 or a culture thereof. Claim 7 A microbial preparation for reducing livestock manure odor according to claim 6, characterized in that the culture is selected from the cells of the Bacillus galactosidylticus BSM-6 strain of accession number KACC92503P, a culture medium containing the cells, a pulverized product of the cells, a culture medium from which the cells have been removed from the culture of the strain, or an extract of the cells of the strain. Claim 8 A microbial preparation for controlling soil nematodes comprising the psychrophilic Bacillus galactosidylticus BSM-6 strain of accession number KACC92503P according to claim 1 or a culture thereof. Claim 9 A microbial preparation for controlling soil nematodes according to claim 8, wherein the culture is selected from the cells of the Bacillus galactosidylticus BSM-6 strain with accession number KACC92503P, a culture medium containing the cells, a lysate of the cells, a culture medium from which the cells have been removed from the culture of the strain, or an extract of the cells of the strain. Claim 10 Livestock feed for reducing manure odor, comprising a microbial preparation according to paragraph 6 or 8.

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