KR102380358B1 - Novel strains of high-temperature Bacillus genus having malodor reduction effect on high-temperature livestock manure and livestock manure malodor reduction composition comprising the same - Google Patents

Abstract

The present invention relates to a high-temperature Bacillus sp. novel strain capable of reducing bad odor of high-temperature livestock manure and a livestock manure bad odor reducing composition comprising the same. The novel strain of the present invention is a high-temperature Bacillus sp. novel Bacillus haynesii S-R-1 strain (KCCM13043P) which is derived from livestock manure containing pig manure and capable of reducing odor of the livestock manure. Accordingly, a novel microbial strain according to the present invention is derived from high-temperature pig manure, has excellent high-temperature heat resistance and salt resistance, and is funcologically alkaline and aerobic, thereby exhibiting excellent growth power in the livestock manure.

Description

Novel strains of high-temperature Bacillus genus having malodor reduction effect on high-temperature livestock manure and livestock manure malodor reduction composition comprising the same comprising the same}

The present invention relates to a novel strain of the genus of high-temperature Bacillus having malodor reduction efficacy against high-temperature livestock manure and a livestock manure malodor reduction composition comprising the same, and more particularly, to a high-temperature resistant Bacillus derived from pig manure ( Bacillus ) Bacillus kokeshiiformis , a novel microbial strain of the genus, GR-3 strain (KCCM13044P) and Bacillus haynesii SR-1 strain (KCCM13043P) and livestock manure odor reduction composition comprising them.

Due to the specialization of livestock farms, the number of breeding heads per unit breeding area has increased, causing the resource circulation system of livestock manure to lose balance and cause environmental problems. Currently, almost all livestock farms are equipped with livestock excreta treatment facilities, but abnormal treatment occurs due to a large increase in temperature during the livestock excreta treatment process.

In the livestock manure treatment method, more than 90% of BOD (biological oxygen demand), SS (suspended solids), TN (total nitrogen), and TP (total phosphate) are removed by the livestock manure treatment and circulation system, and high-temperature growth organic matter is decomposed in the treatment process. Active decomposition by microorganisms takes place. Moreover, pig manure has a high organic matter content and a large storage capacity, so a severe odor is generated due to fermentation. According to the livestock manure treatment method, about 86% of it is converted into compost and liquid manure, and about 1% is self-purifying treatment (Ministry of Environment, Livestock Manure Treatment Statistics, 2011). The common resource recycling facility can economically process livestock manure, and resource recycling effect can be expected if it is linked with eco-friendly agriculture.

Livestock manure has a strong odor, and the main odor components are ammonia, hydrogen sulfide, and volatile fatty acids. By decomposing, it is possible to reduce odor and produce organic liquid fertilizer and fertilizer. For this, aerobic and thermophilic microbial treatment is effective. In particular, the method using microorganisms is high in pH (7.0-9.5), salt resistance (0.5-1.5%), and high temperature resistance (55℃±5℃) in consideration of the physicochemical characteristics of livestock manure. It can exhibit its efficacy only when conditions for microbial growth are satisfied.

The composition of the microbial fermentation circulation system consists of an air (oxygen) supply device, an air aeration device, an underwater pump, and a bubble removal device. It is processed at a temperature of 25~65℃ depending on heat generation, submersible pump heat, friction heat, etc. High-temperature aerobic microorganisms grow using inorganic odor components and organic matter from livestock manure, and use _CHONSP + NH ₃ + H ₂ S_PO ₄ +O ₂ + nutrients to form It is a process of organicizing (microbial cells, etc.)

Looking at the treatment method of livestock manure using known microorganisms in the prior art, Republic of Korea Patent Publication No. 10-2014-0034474 Mucor circinelloids ( Mucor circinelloids ), Bacillus pumilus ( Bacillus pumilus ) and Stenotropomonas maltophilia ( Stenotrophomonas maltophilia ) in a yield ratio of 1: 0.9 to 1.2: 0.9 to 1.2, and a method for producing an organic fertilizer for removing odors from excreta are disclosed, and Patent Publication No. 10-2014-0114564 discloses a novel nitrifying bacteria A microbial agent for removing nitrogen from high-concentration wastewater containing strains of the genus Nitrobacter sp. H2S1 and a method for treating livestock manure using the same are introduced. A microorganism and composition derived from livestock manure mixed with pig manure and poultry manure as novel strains of the genus Ocyanobacillus having a reducing effect and a composition containing them with low odor in livestock manure are disclosed.

Bacillus pallidus, Bacillus coagulans, and Bacillus badius strains are effective in isolating thermophilic ammonia-resistant bacteria and removing ammonia form nitrogen generated during composting of animal feces (Biosci. Biotechnol. Biochem., 68(2), 286~ 292, 2004), high-temperature-resistant Bacillus kokeshiiformis sp. Nov. has been reported (Int. J. Syst. Microbiol. Aug; 64, 2668~2674, 2014), and Bacillus swezeyi sp. isolated from desert soil. Nov. and Bacillus haynesii sp. Nov. has been reported (Int. J. Syst. and Evolutionary Microbiology Vol.67, 2720~2725, 2017).

Domestic Patent Publication No. 10-2014-0034474 (published on March 20, 2014) Domestic Patent Publication No. 10-2014-0114564 (published on September 29, 2014) Domestic Registered Patent No. 10-2150330 (Registered on August 26, 2020)

Biosci. Biotechnol. Biochem., 68(2), 286-292, 2004 Int. J. Syst. Microbiol. Aug; 64, 2668~2674, 2014 Int. J. Syst. and Evolutionary Microbiology Vol.67, 2720~2725, 2017

The present invention provides a novel microbial strain of Bacillus genus Bacillus kokeshiiformis GR-3 strain (KCCM13044P) that has excellent growth ability in high temperature livestock manure and has the effect of reducing odor derived from livestock manure is doing

In addition, the present invention provides a Bacillus haynesii SR-1 strain (KCCM13043P), which is a novel microbial strain of the genus Bacillus, which has excellent growth ability in high temperature livestock manure, and has the effect of reducing odor derived from livestock manure is doing

In addition, the present invention is to provide a high-temperature livestock manure odor removal composition comprising a culture using the microorganism strains of the genus Bacillus.

Various problems to be solved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

The novel strain of the present invention is derived from livestock manure containing pig manure, and a novel Bacillus haynesii SR-1 strain (KCCM13043P) of the thermophilic Bacillus genus having an odor reduction effect for the livestock manure. am.

In addition, the livestock manure malodor reduction composition according to the present invention is derived from livestock manure containing pig manure, and a novel Bacillus haynesii SR in the genus of high-temperature Bacillus having odor reduction effect on the livestock manure. -1 strain (KCCM13043P) or a culture thereof.

Specific details of other embodiments are included in the detailed description.

The novel microbial strain according to the present invention is derived from high-temperature pig manure, has excellent high-temperature heat resistance and salt resistance, and has mycologically alkaline and aerobic properties, so it has an excellent effect of growth in livestock manure.

In addition, the novel microorganism strain according to the present invention has an excellent effect of reducing the odor contained in livestock manure.

It will be fully understood that embodiments of the technical idea of the present invention may provide various effects not specifically mentioned.

1 is a 16S rRNA sequence of a novel Bacillus kokeshiiformis GR-3 strain (KCCM13044P) cultured and isolated in the present invention.
Figure 2 is a phylogenetic tree of Bacillus kokeshiiformis GR-3 strain (KCCM13044P).
3 is a 16S rRNA sequence of a novel Bacillus haynesii SR-1 strain (KCCM13043P) cultured and isolated in the present invention.
4 is a phylogenetic tree of Bacillus haynesii SR-1 strain (KCCM13043P).

Advantages and features of the present invention, and a method of achieving them, will become apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided so that the disclosed subject matter may be thorough and complete, and that the spirit of the present invention may be sufficiently conveyed to those skilled in the art.

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having meanings consistent with the meanings in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Hereinafter, with reference to the accompanying drawings, a preferred embodiment of a novel strain of the genus of thermophilic Bacillus having an odor reduction effect for high-temperature livestock manure according to the present invention will be described in detail.

First, a method for isolating and identifying novel strains according to the present invention will be described. 150 liters of pig manure liquid (pH 9.3, salinity 0.9% or more) was put into the culture device and aerated (0.1 vvm) of air at 60 ° C. for 3 days, and 50 ml of sample was collected while the pH of pig manure was lowered to 7.35. Pig manure agar plate medium was prepared by adding 1.7% of agar to the pig manure supernatant centrifuged at 3,000 rpm for 20 minutes and sterilizing it at 121 ° C for 20 minutes to separate thermophilic microorganisms.

The collected samples were serially diluted 10-fold in sterile physiological saline, and then smeared on a plate of 0.1 ml of pig manure agar, and cultured at 60 ° C for 24 hours. The degree of excellent microbial growth for 24 hours was arranged in the order of serial number.

In addition, the purely isolated microorganisms were cultured on a plate of pig manure agar and cultured at 27° C. for 24 hours to compare growth at low temperature. As a result, the G-R-3 strain (KCCM13044P), which had excellent growth at 60°C, also grew well at 27°C, and the S-R-1 strain (KCCM13043P) had slow growth, but other microorganisms did not grow well at low temperature conditions.

Furthermore, in order to understand the characteristics of the purely separated microorganisms, the growth characteristics of 4 types of microorganisms were compared using media of different compositions. MA (Marine agar, peptone 5.0 g, yeast extract 1.0 g, ferric citrate 0.1 g sodium chloride 19.45 g, magnesium sulfate 8.8 g, sodium sulfate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 0.08g, strontium chloride 34.0mg, boric acid 22.0mg, sodium silicate 4.0mg, sodium fluoride 2.4mg, ammonium nitrate 1.6mg, disodium phosphate 8.0mg, agar 15.0g) medium and NA (Nutrient agar, Lab-Lemco powder 1.0g) , yeast extract 2.0g, peptone 5.0g, sodium chloride 5.0g, agar 15.0g) was inoculated with the pure isolated microorganisms, and then cultured at 60℃ for 24 hours, and then growth was compared.

Among the purely isolated microorganisms, G-R-3 grew well in a medium containing various minerals and could not grow in a medium lacking in minerals. On the other hand, S-R-1 showed growth in each medium. However, G-R-1 and G-R-2 showed a similar pattern to that of G-R-3. That is, it was confirmed that the characteristics of microorganisms have different characteristics.

For the odor reduction test of four kinds of purely separated microorganisms at high temperature according to the purpose of the invention, pig manure was sterilized at 121° C. for 20 minutes and then cooled. After dispensing the cooled pig manure solution into 20ml/32ml test tubes (20mm x 500mm), inoculate each microorganism and incubate at 60℃ for 16 hours. evaluated.

To measure the odor, the amount of hydrogen sulfide and ammonia was measured with a multi-odour meter (MultiRAE lite, RAE System Inc. USA). As a control, it was analyzed based on the manure fluid that was not inoculated with microorganisms. In the odor reduction evaluation, G-R-3 showed the best odor reduction effect, and other microorganisms including S-R-1 also showed the odor reduction effect.

For the identification of four types of microorganisms that were isolated and evaluated for odor reduction, 1492F and 805R primers were used for GR-3 and GR-1, and 27F and 1492R primers were used for SR-1, GR-2 and 16S rRNA base. The sequence was analyzed.

As a result, GR-1, GR-2, and GR-3 were identified as Bacillus kokeshiiformis , and SR-1 was identified as Bacillus haynesii .

The isolates according to the present invention were all Gram-positive bacteria, which were motile, had endo-spores, were halo-tolerant, and were thermophilic microorganisms favoring environmental conditions with high pH. However, it has excellent growth characteristics under very aerobic conditions.

After identification of G-R-3 and S-R-1 microorganisms alone and mixed inoculation of two microorganisms, odor reduction efficacy was evaluated in 120 liters/200 liters (pilot culture vessel) at 55° C. for 24 hours. Pig manure liquid to be treated for odor reduction was tested without heat treatment.

GR-3 and SR-1 microorganisms were used by centrifuging pig manure supernatant at 3,000 rpm for 20 minutes, and put it in 500 ml/2,000 ml of manure supernatant (erlenmeyer flask, 2 Erlenmeyer flasks per microorganism) and sterilize at 121°C for 20 minutes. After that, each bacteria was inoculated with 3 platinum and cultured with shaking at 55° C. for 36 hours (90 rpm) to prepare seed bacteria.

In case of efficacy evaluation with the prepared seed GR-3 and SR-1 single inoculation, two flasks (1 liter seed) were inoculated into the pilot culture vessel, and 500 ml each in case of efficacy evaluation with GR-3 and SR-1 mixed inoculation It was inoculated with a seed. The inoculated pilot culture vessel was maintained at a temperature of 53~57℃, and air was supplied at 0.1vvm while continuously circulating to ensure good contact with the air, and the sample was collected after treatment for 24 hours, and the measurement of odor was 20㎖ A /32ml test tube (20mm x 500mm) was cooled to about 30℃ and then connected to a complex odor meter (MultiRAE lite, RAE System Inc. USA) to measure the amount and pH of hydrogen sulfide and ammonia.

In the pilot-scale odor reduction evaluation, each of G-R-3 and S-R-1 showed excellent results, and the test in which two microorganisms were mixed and treated showed the best odor reduction effect.

<Experimental Example 1> High-temperature treatment for sample collection

150 liters of pig manure (pH 9.3, salinity 0.9% or more) was placed in a culture device and aerated (0.1 vvm) of air at 60 ° C. for 3 days, and 50 ml of sample was collected while the pH of pig manure was lowered to 7.35.

<Experimental Example 2> Separation of high-temperature microorganisms with excellent growth from pig manure

It has odor reduction effect, and 1.7% of agar was added to the pig manure supernatant centrifuged at 3,000 rpm for 20 minutes to isolate thermophilic microorganisms, sterilized at 121 ° C. for 20 minutes, and then dispensed to prepare a pig manure agar plate medium. The collected samples were serially diluted 10-fold in sterile physiological saline, and then smeared on a plate of 0.1 ml of pig manure agar, and cultured at 60°C for 24 hours. [Table] of [Table].

The degree of excellent microbial growth for 24 hours is summarized in [Table 1] in order of serial number.

number microbe growth status characteristic Selection One G-R-3 very good big viscous √ 2 S-R-1 very good big no initial viscosity √ 3 G-R-1 well big viscous √ 4 G-R-2 well big viscous √

<Experimental Example 3> Low-temperature growth test of pure isolated microorganisms

The purely isolated microorganisms were cultured on a plate of pig manure agar and cultured for 24 hours at 27° C.

Low-temperature growth test result of pure isolated microorganism (27℃) number microbe growth status characteristic Selection One G-R-3 very good big white to yellow √ 2 S-R-1 very weakly large - √ 3 G-R-1 failure to grow - √ 4 G-R-2 failure to grow - √

As shown in [Table 2], G-R-3, which had excellent growth at 60° C., grew well even at 27° C., but other microorganisms did not grow well at low temperature conditions.

<Experimental Example 4> Comparative test for growth of pure isolated microorganisms

In order to understand the characteristics of the purely separated microorganisms, the characteristics of 4 types of microorganisms were compared using media of different compositions. After preparing the MA (Marine agar) medium and NA (Nutrient agar) medium and inoculating the purified microorganisms, the growth was compared and summarized in [Table 3] below after culturing at 60°C for 24 hours.

number microbe MA NA Selection One G-R-3 very growing failure to grow √ 2 S-R-1 grow well good growth √ 3 G-R-1 growth grow weak √ 4 G-R-2 growth grow weak √

As shown in [Table 3], among the purely isolated microorganisms, G-R-3 did not grow well in a medium containing various minerals and in a medium lacking in minerals. On the other hand, S-R-1 showed growth in each medium. However, G-R-1 and G-R-2 showed a similar pattern to that of G-R-3. That is, it was confirmed that the characteristics of microorganisms have different characteristics.

<Experimental Example 5> Odor reduction test of pure isolated microorganisms

For the odor reduction test of four types of purely separated microorganisms, pig manure was sterilized at 121℃ for 20 minutes and then cooled. After dispensing the cooled pig manure solution into 20ml/32ml test tubes (20mm x 500mm), inoculate each microorganism and incubate at 60℃ for 16 hours. was evaluated.

To measure the odor, a 20ml/32ml test tube (20mm x 500mm) was connected to a complex odor meter (MultiRAE lite, RAE System Inc. USA), and then the amounts of hydrogen sulfide and ammonia were measured.

The results are shown in [Table 4] below.

number microbe NH ₄ (ppm) H ₂ S (ppm) pH characteristic 55℃ 27℃ 55℃ 27℃ 55℃ 27℃ One G-R-3 25 8 0 0 8.95 9.10 - 2 S-R-1 27 9 0 0 8.97 9.14 - 3 G-R-1 28 9 0 0 8.98 9.15 grow from the top 4 G-R-2 26 9 0 0 8.97 9.13 - 5 control 46 15 0 0 9.17 9.26 -

As shown in [Table 4], G-R-3 showed the best odor reduction efficacy in the odor reduction evaluation, and other microorganisms also showed odor reduction efficacy.

<Experimental Example 6> Base sequence analysis and identification of new strains

For the identification of 4 types of microorganisms that have been isolated and evaluated for odor reduction, 1492F and 805R primers were used for GR-3 and GR-1, and 27F and 1492R primers were used for SR-1, GR-2 and 16s rRNA base. The sequence was analyzed and the results are shown in [Table 5] below.

number microbe identification result analysis nucleotide sequence primer One G-R-3 Bacillus kokeshiiformis 1,460 1492F, 805R 2 S-R-1 Bacillus haynesii 1,418 27F, 1492R 3 G-R-1 Bacillus kokeshiiformis 1,444 1492F, 805R 4 G-R-2 Bacillus kokeshiiformis 1,427 27F, 1492R

As shown in [Table 5], the isolates according to the present invention are all Gram-positive bacteria, have mobility, have endo-spores, are halo-tolerant, and have high pH environmental conditions It was a thermophilic microorganism that liked However, it has excellent growth characteristics under very aerobic conditions.

<Experimental Example 7> Evaluation of high temperature and odor reduction in pilot culture facility

After the identification of G-R-3 and S-R-1 microorganisms alone and the two microorganisms were mixed inoculation, the odor reduction efficacy was evaluated in 120 liters/200 liters (pilot culture vessel) at 55° C. for 24 hours. Pig manure liquid to be treated for odor reduction was tested without heat treatment.

GR-3 and SR-1 microorganisms were used by centrifuging pig manure supernatant at 3,000 rpm for 20 minutes, and put it in 500 ml/2,000 ml of manure supernatant (erlenmeyer flask, 2 Erlenmeyer flasks per microorganism) and sterilize at 121°C for 20 minutes. After that, each bacteria was inoculated with 3 platinum and cultured with shaking at 55° C. for 36 hours (90 rpm) to prepare seed bacteria. In case of efficacy evaluation with the prepared seed GR-3 and SR-1 single inoculation, two flasks (1 liter seed) were inoculated into the pilot culture vessel, and 500 ml each in case of efficacy evaluation with GR-3 and SR-1 mixed inoculation It was inoculated with a seed.

In the inoculated pilot culture vessel, the temperature was maintained at 53~57℃, and air was supplied at 0.1vvm while continuously circulating so that the air was in good contact, and the sample was collected after treatment for 24 hours, and the odor measurement was 20㎖ A /32ml test tube (20mm x 500mm) was cooled to about 30℃ and then connected to a complex odor meter (MultiRAE lite, RAE System Inc. USA) to measure the amount and pH of hydrogen sulfide and ammonia.

The results are shown in [Table 6] below.

number processing microorganisms Odor (NH ₃ ) (ppm) pH colour One G-R-3 1-2 7.33 Brown 2 S-R-1 2-3 7.38 Brown 3 G-R-3, S-R-1 0-1 7.30 Brown 4 No treatment (control) 18 9.19 black

As shown in [Table 6], in the pilot-scale odor reduction evaluation, GR-3 and SR-1 each showed excellent results, and the test in which two microorganisms were mixed and treated showed the best odor reduction effect. .

As described above, a preferred embodiment of the present invention has been described, but those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential features thereof. will be able Therefore, it should be understood that the embodiment described above is illustrative in all respects and not restrictive.

Korea Microorganism Conservation Center KCCM13043P 20210831 Korea Microorganism Conservation Center KCCM13044P 20210831

Claims (2)

It is derived from livestock manure containing pig manure, and a novel Bacillus haynesii SR-1 strain (KCCM13043P) of the thermophilic Bacillus genus, characterized in that it has an odor reduction effect for the livestock manure.
Derived from livestock manure containing pig manure, and containing a novel Bacillus haynesii SR-1 strain (KCCM13043P) or a culture of the thermophilic Bacillus genus having a malodor reduction effect for the livestock manure Livestock manure malodor reduction composition, characterized in that.

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