KR102489921B1 - Culturing method of arthrobacter crystallopoietes improving power of plant growth - Google Patents

Abstract

In one aspect, the present invention relates to a method for culturing an Arthrobacter crystallopoietes strain that improves plant growth promoting ability and a medium used therein, Including a microorganism inoculation step (S10) of inoculating the strain into previously prepared kaolin, the kaolin is characterized in that the kaolin is silicic acid content of 65 wt% or less and organic matter content of 4 to 7 wt%.

Description

Culturing method of arthrobacter crystallopoietes improving power of plant growth}

The present invention relates to a method for culturing a strain, and relates to a method for culturing an Arthrobacter crystallopoietes strain that improves plant growth promoting ability.

In general, in agriculture and horticulture, synthetic pesticides such as composite fungicides in which various fungicides are mixed are used to control diseases caused by fungal pathogens existing in the soil. A variety of synthetic fertilizers have been used.

The use of these synthetic pesticides or synthetic fertilizers causes adverse effects on the natural environment or the human body, and when used for a long period of time, there has been a problem of inducing the emergence of resistant pathogens or deteriorating soil quality.

In order to solve these problems, microbial agents such as various EM (Effective Micro-organisms) or complex microbial fermentation solutions such as BM have been developed and used. In particular, these microbial agents are used to enhance the function of improving the soil there is.

In the prior art documents below, it can be seen that strains belonging to the genus Arthrobacter sp included in microbial preparations have a function of improving soil quality and promote plant growth. In addition, it is already known that strains belonging to the genus Arthrobacter sp have the ability to purify contaminants in soil or wastewater and the ability to remove odors.

However, when these microbial preparations were applied to the actual agricultural field, they failed to adapt to various soil environments and showed different efficacy depending on the type of soil such as mud, sandy soil, sandy soil, and loess, and the survival period of microorganisms was generally short. Soil improvement and crop growth were not as good as expected.

Common agricultural microorganisms used in these microbial preparations are fed and cultured under good temperature conditions, breed by increasing the population, and are weak microorganisms with low activity. It is due to the failure to properly perform the roles expected of microorganisms.

Therefore, there is an urgent need to develop highly active microorganisms that can survive strongly in soil conditions where nutrients are imbalanced by continuously cultivating contaminated or barren soil or crops to supplement this.

Registered Patent Publication Registration No. 10-0946633 (Plant growth promotion and plant protection method using microbial-derived metabolites, notice date March 9, 2010) Unexamined Patent Publication No. 10-2019-0111676 (Removal of algae and odor in lakes and ponds using complex microbial agents, October 2, 2019)

In order to solve this problem, the present invention cultivates microorganisms with high activity, and the microorganisms decompose organic matter in contaminated soil or continuous cultivation soil to increase the absorption rate of minerals useful to crops and to supply metabolites to prevent crop diseases and to provide good It is an object of the present invention to provide a method for culturing an Arthrobacter crystallopoietes strain that improves plant growth promoting ability by enhancing activity capable of improving soil, and a medium used therefor.

In order to achieve the above object, the present invention, in one aspect, is a method for culturing an Arthrobacter crystallopoietes strain that improves plant growth promoting ability, and the Arthrobacter crystallopoietes strain Including a microorganism inoculation step (S10) of inoculating a pre-prepared kaolin into kaolin, the kaolin is characterized in that the kaolin containing silicic acid content of 65 wt% or less and organic matter content of 4 to 7 wt%.

Preferably, it is preferable to further include a low-temperature treatment step (S20) of cold-treating and culturing the Arthrobacter crystallopoietes strain inoculated into kaolin in the microorganism inoculation step (S10).

Here, in the low temperature treatment step (S20), the Arthrobacter crystallopoietes strain inoculated into kaolin is treated at 4° C. for 24 hours.

In addition, preferably, a high-temperature treatment step (S30) of treating the Arthrobacter crystallopoietes strain inoculated and cultured in kaolin at 70 ° C. for 48 hours is further included.

Here, the water content of kaolin inoculated with Arthrobacter crystallopoietes strain is preferably maintained at 11 to 20 wt%.

Here, it is preferable to further include a primary purified water mixing step (S40) of mixing purified water with kaolin inoculated with the Arthrobacter crystallopoietes strain that has undergone the high temperature treatment step (S30) and stirring it.

Further, preferably, a salt mixing process (S50) of mixing 0.4 wt% of salt with kaolin inoculated with Arthrobacter crystallopoietes strain is further included.

Here, a second high-temperature treatment step (S60) of treating the Arthrobacter crystallopoietes strain cultured in a salt-mixed medium at 70 ° C. for 48 hours in the salt mixing step (S50) is further included. desirable.

In addition, in order to increase the water activity of microorganisms, it is preferable to further include a second purified water mixing step (S70) of mixing and stirring purified water with kaolin inoculated with Arthrobacter crystallopoietes strain.

In addition, it is preferable to further include a step of filtering the Arthrobacter crystallopoietes strain with kaolin and separated microorganisms (S80).

Furthermore, the microorganisms filtered here are collected and stored in a low-temperature container (preferably below 15 ° C.) to prevent contamination, and then put into a dormant tank at a predetermined speed, for example, a movement speed of 2 m / sec, to dormant microorganism collection and dormancy process ( S90) is preferably further included.

In another aspect, the present invention relates to a medium used for culturing an Arthrobacter crystallopoietes strain that improves plant growth promoting ability, wherein the kaolin has a silicic acid content of 65 wt% or less and an organic matter content of 4 to 7 wt%. It is characterized in that it is kaolin containing %.

According to the present invention as described above, by enhancing the activity of the Arthrobacter crystallopoietes strain, the activity of the strain enhanced as a plant growth-promoting rhizobacteria (PGPR) A method for culturing an Arthrobacter crystallopoietes strain with improved plant growth promoting ability and a medium used therefor can be provided.

In addition, according to the culturing method of the Arthrobacter crystallopoietes strain of each embodiment and the medium used therein, the optimal extreme environment medium for the Arthrobacter crystallopoietes strain ( By providing an Extreme Environment culture medium or Extreme Environment for Culturing, the activity of the cultured Arthrobacter crystallopoietes strain is enhanced, and thus, the cultured Arthrobacter crystallopoietes strain is enhanced. Bacterial crystallopoietes (Arthrobacter crystallopoietes) has the effect of promoting the growth of plants cultivated in the inoculated soil.

1 is a flowchart of a method for culturing an Arthrobacter crystallopoietes strain to improve plant growth promoting ability according to an embodiment of the present invention.
2 is a component test result of kaolin clay according to an embodiment of the present invention.
Figure 3 is a photograph of skin dapsus growth score observation after 30 days of growth.
4 is a photograph of observation results of pig manure treatment by microorganisms at each treatment temperature.

Hereinafter, specific details for carrying out the present invention will be described based on examples with reference to the drawings. These embodiments are described in sufficient detail to enable one skilled in the art to practice the present invention.

The strains used in the present invention are strains belonging to the genus Arthrobacter sp, and strains having plant growth promoting ability may be selected and used.

In this embodiment, the strain belonging to the genus Arthrobacter sp having plant growth promoting ability acts to promote plant growth as a plant growth-promoting rhizobacteria (PGPR). The present invention provides a method for culturing an Arthrobacter crystallopoietes strain that improves plant growth promoting ability and a medium used therefor.

In addition, various strains belonging to the genus Arthrobacter sp are being studied for commercial application, and in this example, hexavalent chromium and 4-chlorophenol from contaminated soil It is known to reduce and can be used for biological environmental remediation (Bioremediation), and has been verified as a plant growth-promoting rhizobacteria (PGPR) as described above. Regarding the culture method of the Arthrobacter crystallopoietes strain of the present invention and the improvement of the activity by the medium used therein or the improvement of the plant growth promoting power through this will be described.

When the activity of microorganisms is improved, the propagation of microorganisms is vigorous, preventing the proliferation of harmful bacteria in the soil even in contaminated soil, thereby improving the phenomenon of causing disease to crop roots or inhibiting growth, thereby improving the ability to promote plant growth. The measurement of is verified by a method selected for each process, and it can be confirmed that the highly active microorganisms are not sterilized by preservatives or disinfectants that prevent the propagation of microorganisms, and only the population is reduced by experimental data. The culture method of the present invention for culturing highly active microorganisms is to create an environment in which microorganisms are difficult to proliferate, that is, an extreme environment for culturing, rather than providing proper growth temperature or food for microorganisms.

Therefore, according to the present invention described through examples below, by enhancing the activity of the Arthrobacter crystallopoietes strain, as a plant growth-promoting rhizobacteria (PGPR) It is possible to provide a method for culturing an Arthrobacter crystallopoietes strain whose plant growth promoting ability is improved by the increased activity of the strain, and a medium used therefor.

In addition, according to the culturing method of the Arthrobacter crystallopoietes strain of each embodiment described below and the medium used therein, the optimum for the Arthrobacter crystallopoietes strain By providing an Extreme Environment culture medium or an Extreme Environment for Culturing, the activity of the cultured Arthrobacter crystallopoietes strain is enhanced, and accordingly , has the effect of promoting the growth of plants cultivated in the soil in which the cultured Arthrobacter crystallopoietes strain is inoculated.

1 is a flowchart of a method for culturing an Arthrobacter crystallopoietes strain to improve plant growth promoting ability according to an embodiment of the present invention.

Referring to Figure 1, the culture method of the Arthrobacter crystallopoietes strain, which improves the plant growth promoting ability of the present invention, is 1. Microorganism inoculation process (S10), 2. Low temperature treatment process (S20) ), 3. High temperature treatment process (S30), 4. First purified water mixing process (S40), 5. Salt mixing process (S50), 6. Second high temperature treatment process (S60), 7. Second purified water mixing process (S70), 8. Microbial filtering process (S80), 9. Microbial collection and dormancy process (S90).

In the case of this example, among various strains belonging to the genus Arthrobacter sp, Arthrobacter crystallopoietes was selected and experimentally performed, and the activity improvement of the strain microorganism was tested by performing each process. It became.

1. Microbial inoculation process (S10)

In the microorganism inoculation process (S10) of the present embodiment, an Arthrobacter crystallopoietes strain is separated and cultured from soil, and the cultured Arthrobacter crystallopoietes strain is prepared in advance on kaolin soil. inoculate into

In this embodiment, kaolin is prepared in advance, and it is preferable to use kaolin containing 65 wt% or less of silicic acid and 4 to 7 wt% of organic matter as a main component. The selection of kaolin with silicic acid content of 65 wt% or less and organic matter content of 4 to 7 wt% as the main component was selected through various tests. In this case, the incubation period is longer and the activity is lowered, and when the organic matter content is relatively high, the incubation period is short but the activity is also lowered.

In order to confirm that the use of kaolin as above is required, Arthrobacter crystallopoietes strain is isolated as shown in the following [microbial culture conditions and activity by culture soil], and the incubation time is about 20-30 days. It is preferable to, and after kaolin culture, 4.2 X 10 ^{4 (} Colony Forming Unit, colony forming unit) of Arthrobacter crystallopoietes strain is cultured and inoculated.

The following [microbial culture conditions and activity test results for each culture soil] analyzes the organic matter content (wt%) and silicate content (wt%) of each humus soil, kaolin soil (fertile soil), and mica (sand soil), and analyzes the organic matter content (wt%) and This is the result of measuring the activity after adjusting the amount of the strain to be tested according to the silicic acid content (wt%) through the incubation time.

[Microorganism culture conditions and activity by culture soil]

As humus, commercial name VK Bomito (manufactured by Samhwa Greentech Co., Ltd.), ingredients: humus, bark compost, and common humus of peat were used. The following kaolin and sandy soil were used as the analysis results of the composition and content of kaolin and the composition and content of sandy soil.

<Kaolin Ingredients and Content>

<Sand soil composition and content>

The review of the activity was judged by the cucumber hair growth test in an indirect way.

In the following [Results of activity test according to cucumber hair growth], it was confirmed that the use of kaolin in this example improved the activity after cultivation compared to humus soil and sandy soil (mica).

Since the content of general organic matter in humus was about 55wt%, the number of microorganisms increased significantly due to the high content of organic matter. It was concluded that as the number of microorganisms increased as the microorganisms proliferated, the activity decreased.

Microorganisms cultured in kaolin and sandy soil (mica) did not reproduce due to relatively low organic matter content. The silicate content of each culture soil has a close relationship with the cultivation conditions of microorganisms. In general, microorganisms do not propagate well in soils with high silicate content, and when the silicate content is very high, 70wt% or more, microorganisms die and the population decreases. , It is believed that this is because the silicic acid component has bactericidal power. As shown in the culture soil above, kaolin and mica have a high silicic acid content, so there is a very high probability that microorganisms will not proliferate or reproduce. .

The incubation time was tested by changing it to confirm the difference in activity between short and long incubation times. Kaolin and mica have a low content of organic matter, considering that the population does not increase well due to the cell growth process rather than propagation culture.

In this Example, the activity of Arthrobacter crystallopoietes was determined by visually determining the cucumber hair growth result and viability activity, that is, through a sensory test, as shown in [Activity test result according to cucumber hair growth result]. tested indirectly. In addition, through various experiments of the applicant, it was confirmed that kaolin having a silicic acid content of 65 wt% or less and an organic matter content of 4 to 7 wt% is preferably used.

By using such kaolin, it is possible to give the optimal extreme environment culture conditions to the Arthrobacter crystallopoietes strain, and through the extreme environment culture, the Arthrobacter crystallopoietes strain (Arthrobacter crystallopoietes) strain activity can be improved.

[Activity test result according to cucumber hair growth]

The reason for the difference in germination rate is considered to be due to the fact that microorganisms cultured in kaolin have more activity, so microorganisms with higher activity produce more plant metabolites.

Plant metabolites include auxin and gibberellin, which are growth hormones necessary for plant growth, which activate more growth points and show a difference in germination rate.

For cucumber growth and growth activity, 1 to 2 seeds were planted for each pot, and a total of 70 seeds were planted with 20 to 25 seeds per culture soil. The degree of disease occurrence was compared and analyzed by measuring the disease or physiological disorder of cucumber leaves from sowing in April until May. The leaves were examined, and the degree of disease occurrence or physiological disturbance of the experimental group crops (kaolin soil) and the control group crops (huyeot soil, sandy soil) was performed as a method of confirming.

For reference, there is a direct observation method and an indirect observation method for measuring the activity of microorganisms. As a direct observation method, 1. There is a method of culturing microorganisms in a medium and measuring the number of microorganisms cultured under certain conditions (time, temperature, humidity). can't build The reason is that there are dominant microorganisms and intermediate microorganisms. This is because even with a small number of microorganisms, dominant microorganisms have characteristics capable of overpowering intermediate microorganisms. 2. There is a method of observing the movement of microorganisms under a microscope, but it is not suitable for measuring the activity of all microorganisms. For example, active ciliary movement or active cell division cannot be seen as high activity. This is because the movement or division rate of microorganisms can vary depending on the type of medium or environment as a characteristic of only microorganisms. It is difficult to determine microbial activity.

As an indirect observation method, 1. There is a method of measuring the metabolites of microorganisms. This is because when microorganisms have high activity, a large amount of metabolites of the microorganisms are also produced. 2. There are methods of measuring through crop experiments and clinical trials. Therefore, the activity of microorganisms can be confirmed through crop experiments (cucumber), which is one of the indirect observation methods of microorganisms. It is an experiment to check in which environment the activity of microorganisms increases by changing the medium, which is the growth environment of microorganisms. The activity of microorganisms can be judged by looking at the state of leaves, stems, and roots of plants growing in each environment that is a medium of microorganisms, that is, humus soil, kaolin soil, and sandy soil environment. It can be confirmed that the growth of crops is different in the environment of humus soil, kaolin soil, and sandy soil in the absence of microorganisms. Since we have to observe during the period from seedling growth to harvest), the results of humus soil, kaolin soil, and sandy soil, which are soil environments in cucumber seedling growth experiments, are meaningless. No significant results were obtained as above.

2. Low temperature treatment process (S20)

2. Cold treatment process (S20) of this embodiment is cultured by cold treatment of the Arthrobacter crystallopoietes strain inoculated into kaolin in the microorganism inoculation process (S10). The low temperature treatment is preferably performed at 4° C. for 24 hours with respect to the Arthrobacter crystallopoietes strain inoculated into kaolin.

The temperature and time of the low-temperature treatment are optimal conditions selected through the following [Comparison of growth points after performing the temperature treatment] test procedure.

[Comparison of growth scores after temperature treatment]

In the above [comparison of growth scores after temperature treatment], the Arthrobacter crystallopoietes strains inoculated and cultured on kaolin in the microorganism inoculation process (S10) were cultured at 4℃, 15℃, and 33℃, respectively, at 24 °C. After time treatment, the relative activity was tested through hydroponic cultivation of Skindapsus. After a one-day growth period, two growth points were observed when strains treated at 4 ° C, 15 ° C, and 33 ° C for 24 hours were inoculated. In the case of inoculating strains treated at 4℃ for 24 hours after growth and when inoculating strains treated at 15℃ for 24 hours, 3 growth points were observed, and in the case of inoculating strains treated at 33℃ for 24 hours, 2 growth points were observed. there was no change to After 15 days of growth, when the strains treated at 4℃, 15℃, and 33℃ for 24 hours were inoculated, all three growth points were observed, and when the strains treated at 33℃ for 24 hours were inoculated, one growth point increased, There was no change in the number of growth points in the case of inoculation of strains treated at 4°C for 24 hours and strains treated at 15°C for 24 hours.

Figure 3 is a photograph of the skin dapsus growth score observed after 30 days of growth. Figure 3 (a) is a case of inoculating a strain treated at 4 ° C for 24 hours, and after 30 days of growth, a case of inoculating a strain treated at 4 ° C for 24 hours 6 Growth points were observed in dogs, and when the strain treated at 15 ° C for 24 hours was inoculated, three growth points were observed and there was no change in the number of growth points. In this case, 4 growth points were observed, confirming that one growth point was increased. Through this, when performing the low temperature treatment at 4 ° C as in the example, it was observed that the number of growth points of skin dapsus was higher than that of the treatment at 33 ° C, which is the optimal culture temperature of microorganisms, and inoculated and cultured in kaolin by low temperature treatment It was confirmed that the activity of the Arthrobacter crystallopoietes strain was improved.

3. High temperature treatment process (S30)

In the high-temperature treatment process (S30) of this embodiment, the Arthrobacter crystallopoietes strain inoculated and cultured in kaolin is treated at 70° C. for 48 hours.

Treatment of Arthrobacter crystallopoietes strain at 70 ° C is because the strain is killed when treated at a temperature higher than 70 ° C, and it is preferable to treat the strain at a high temperature up to 70 ° C, which is the maximum survival critical point.

In addition, since the treatment time is 48 hours, the number of strains decreases after 48 hours, so it is preferable to perform high-temperature treatment only up to 48 hours.

The above high-temperature treatment process may be performed after the microorganism inoculation process (S10), depending on the embodiment, and also, in which both the microorganism inoculation process (S10) and the low-temperature treatment process (S20) have been performed, Arthrobacter crystalropoie It can also be performed on Arthrobacter crystallopoietes strain medium.

Through the high-temperature treatment process of this embodiment as described above, the present invention can impart extreme environment culture conditions to the Arthrobacter crystallopoietes strain, and through extreme environment culture, It is possible to enhance the activity of Arthrobacter crystallopoietes strains.

Preferably, the water content of the kaolin soil inoculated with the Arthrobacter crystallopoietes strain is maintained at 11 to 20 wt%.

The reason why the moisture content of the kaolin medium is maintained in the range of 11 to 20 wt% is that kaolin has a high porosity and the moisture content does not fall below 11 wt% even when completely dried, so the lowest value is 11 wt% and the moisture content exceeds 20 wt%. If kaolin is agglomerated, the maximum value of dried kaolin is maintained at 20wt%.

4. Primary Purified Water Mixing Process (S40)

Subsequently, in the first purified water mixing step (S40), purified water is mixed with kaolin inoculated with the Arthrobacter crystallopoietes strain that has undergone the high temperature treatment step (S30) to increase the water activity of microorganisms and stirred. .

When the microorganisms, that is, the Arthrobacter crystallopoietes strain, pass through the critical temperature and critical time through the above 3. high-temperature treatment process (S30), they do not reproduce, but grow and become active. Since microorganisms are substantially activated only when there is moisture, the water activity of microorganisms is maximized through the supplied moisture by mixing purified water.

On the other hand, when purified water, that is, water is not mixed, the activity of microorganisms is lowered or dormant or disappears due to the characteristics of microorganisms.

The reason for choosing purified water is that tap water, ground water, other ionized water, and mineral water have additives, and the various substances contained in them are not constant, so choosing purified water, which is pure water, increases the water activity. Since the temperature and amount of purified water to be mixed are not very important, an appropriate amount of purified water is mixed according to the amount of medium, and through this, highly active microorganisms that do not reproduce and grow cells have activity.

5. Salt blending process (S50)

In the salt mixing process (S50), 0.4 wt% of salt is mixed with kaolin inoculated with the Arthrobacter crystallopoietes strain.

The salt mixing process (S50) may be performed on a medium in which all of the microorganism inoculation process (S10) to the first purified water mixing process (S40) have been performed, depending on the embodiment, and the agar in which the microorganism inoculation process (S10) has been performed. It may also be performed on Arthrobacter crystallopoietes strain medium.

The Arthrobacter crystallopoietes strain is a type of actinomycete isolated from dichromate-contaminated soil. (Arthrobacter crystallopoietes strains are also found in seawater) and can be used to detoxify hexavalent chromium under stressful environmental conditions.

On the other hand, as shown in [Test results of ammonium (NH4+) removal rate within 48 hours when salt is mixed and not mixed], microorganisms of the genus Paracoccus and Pseudomonas similarly inhibit the nitrification process and remove ammonium (NH4+) as the salinity increases It has been reported that activity related to ability increases. (Ludzack and Noran, 1965). As a result of the test, the Arthrobacter crystallopoietes strain induced and cultured in kaolin was similarly salty as in the [Results of ammonium (NH4+) removal rate test within 48 hours when salt was mixed and not mixed]. It was confirmed that the higher the nitrification process, the higher the activity of ammonium (NH4+) removal ability.

[Test results of ammonium (NH4+) removal rate within 48 hours when salt is mixed and when not mixed]

In this experiment, both strains of the genus Paracoccus genus Pseudomonas and the strains of Arthrobacter crystallopoietes showed some variation in ammonium removal rates within 24 hours as the salt concentration increased in the kaolin medium containing 0-2 wt% salt. However, as shown in the table above, within 48 hours, the highest removal rate was found at 0.4wt% salt, and the removal rate of ammonium was somewhat lowered when it moved away from the lowest or highest value at 0.4wt% salt.

In this example, the activity of Arthrobacter crystallopoietes was measured under conditions other than fixed medium temperature and salt mixture ratio, as shown in [Results of activity test according to cucumber growth results 2]. As a result of an indirect test through a method for determining growth activity with the naked eye, that is, a sensory test, when 0.4 wt% of salt was mixed with kaolin inoculated with Arthrobacter crystallopoietes strain, the growth activity was increased. It was confirmed that the activity of Arthrobacter crystallopoietes was improved due to high concentration.

[Activity test results according to cucumber hair growth results 2]

For cucumber growth and growth activity tests, 1 to 2 seeds were planted for each pot, and a total of 70 seeds were planted with 20 to 25 seeds per potting soil. The degree of disease occurrence was compared and analyzed by measuring the disease or physiological disorder of cucumber leaves from sowing in April until May. The leaves were examined, and it was performed as a method of confirming the degree of disease occurrence or physiological disturbance of the experimental group crops (salt 0.4wt% mixture) and control crops (salt 0.1wt% mixture salt 1.0wt% mixture).

6. Second high temperature treatment process (S60)

In the second high-temperature treatment step (S60), the Arthrobacter crystallopoietes strain cultured in the salt-mixed medium in the salt mixing step (S50) is treated at 70° C. for 48 hours.

In the above-described 3. high-temperature treatment process (S30), 4. first purified water mixing process (S40), and 5. salt mixing process (S50), microorganisms having activity by moisture (purified water) after first high-temperature treatment at 70 ° C. After increasing the activity by adding salt to the second high temperature treatment in this process, the activity of the Arthrobacter crystallopoietes strain is further improved.

4 is a photograph of observation of the pig manure treatment results by microorganisms at each treatment temperature. Referring to FIG. 4, the pig manure treatment results by microorganisms at each treatment temperature are as follows.

[Results of pig manure treatment according to microbial treatment temperature]

Arthrobacter crystallopoietes strains generally performed nitrification and denitrification reactions under aerobic conditions, and showed excellent results in ammonium removal efficiency, turbidity, and color improvement.

In this experiment, as a result of measuring the growth rate of Arthrobacter crystallopoietes and the improvement rate of chromaticity, turbidity, and odor of treated water according to the incubation temperature, different results were shown at 25 ~ 70 ℃. The improvement showed that the red color of the microbial metabolite (metabolite) had an odor reduction effect, and the darker gray color, the lower the odor reduction effect. Color is related to turbidity when it is bright, so when turbidity is improved, chromaticity is also improved.

The optimal temperature for the chromaticity and turbidity improvement rate was 70℃, and the improvement rate dropped a lot at 25℃. It was carried out by the sensory method rather than the measurement by the colorimeter and the turbidity meter, and as shown in FIG. The processing time for each temperature was 48 hours, and after cooling for 6 hours, a visual sensory test was performed, and the odor was measured with a Handheld Odor Meter OMX-ADM (SHINYEI, Japan) device for ammonium and hydrogen sulfide complex odor.

The instrumental measurement results were classified into 0-10: no odor, 11-100: slightly reduced, and 100 or more: severe, and the degree of odor felt by humans was determined as a numerical range.

In addition, when pig manure was subjected to high-temperature treatment at 70 ° C., the degree of improvement for each round was tested, and the results were as follows [degree of improvement for each round when pig manure was subjected to high-temperature treatment]. The test method is to inoculate 150ml of microorganisms in pig manure medium, ferment for 3 days, cool for 6 hours,

This is the result obtained by screening with a silicon filter 300 mesh standard. The fermentation time for each batch is the same at 72 hours.

[Improvement by round when pig manure is treated at high temperature]

When the high temperature treatment was performed at 70 ° C twice, the chromaticity and turbidity were the most excellent, and when the high temperature treatment was performed three times, precipitates were rather generated and the degree of improvement in odor was not significant. Based on these results, the present invention adopts a second high-temperature treatment at 70 ° C. Through this, when the second high-temperature treatment process (S60) is performed, the secondary Arthrobacter crystallopoietes It was concluded that the activity is further improved, the effect of improving the color and turbidity of pig manure is increased, and it cannot be a highly active microorganism in terms of improving the color and turbidity of pig manure if the second high temperature treatment is not performed. .

7. Second Purified Water Mixing Process (S70)

Subsequently, in the second purified water mixing process (S70), purified water is mixed with kaolin inoculated with an Arthrobacter crystallopoietes strain to increase the water activity of microorganisms and stirred.

For microbial activity, the lowest water activity value is required. After the second high-temperature treatment, a step of mixing purified water with the lowest water activity value is performed so that the activity does not decrease. In general, since all organisms, including microorganisms, are not active without water, it is a step of supplying water to give activity.

The water activity value of microorganisms is calculated by the following formula, and the water activity value (Aw) shown here is the lowest value.

Aw: water activity value, P: vapor pressure of solute, Pw: vapor pressure of solvent, Nw: number of moles of solvent, Ns: number of moles of solute

8. Microbial filtering process (S80)

Subsequently, the microbial filtering process (S80) is a process of separating microorganisms from kaolin. Through the above steps S10 to S70, an Arthrobacter crystallopoietes strain having high activity is separated from kaolin.

In parallel with the method of physically screening microorganisms by size (0.45 μm or less), biochemical (Bio) reaction filtering using a humic reaction can be performed.

In the humicification reaction, the growth of harmful bacteria can be inhibited because the salts are removed by converting the humic substances into humic organic substances.

Since the kaolin medium is 10 μm, it can be separated by a physical screen. The separation of ionized minerals and heavy metals is filtered by passing minerals through a corrosion reaction and adsorbing heavy metals, and heavy metals adsorbed to corrosive substances become highly active minerals disassembled and then discharged.

Harmful bacteria (e.g., Escherichia coli, Salmonella, etc.) are converted to metabolites (metabolites) and organic matter in the culture medium by highly active microorganisms to form flocs (lumps) so that harmful bacteria are not filtered out. It is a structure that forms, decomposes and filters.

9. Microbial collection and dormancy process (S90)

In the microorganism collection and dormancy process (S90), the filtered microorganisms are collected and stored in a low-temperature container (preferably below 15° C.) to prevent contamination, and then put into a dormant tank at a predetermined speed, for example, 2m/sec, for dormant treatment. It is a process of

The low-temperature container is stored in the low-temperature container to prevent incubation in anticipation of the inflow of harmful bacteria that can be contaminated in the air.

The reason for performing the dormancy process is that if the collected microorganisms are not dormant, a low-temperature storage method in which microorganisms do not grow, such as in general cultured microorganisms, should be used, and when stored at 4 ° C. in refrigeration, it has a shelf life of about 1 week. On the other hand, in the case of dormancy treatment by the dormancy process, it can be stored for 3 years at room temperature.

To this end, stirring is performed using a MICA ceramic ball for dormancy of microorganisms. When the total weight of kaolin is 100wt%, mica ceramic balls are mixed with 20wt% and stirred left and right at 20rpm or less for 6 hours. There are three reasons for the above treatment. First, the activity of microorganisms stops due to the wave of negative ions and quantum energy emitted from mica ceramic balls (size of 3 to 5 pies), and second, Brownian motion by stirring left and right This is because microorganisms stop their activity and, thirdly, Brownian motion must be maintained for at least 1 hour to become dormant.

The Arthrobacter crystallopoietes strain, which is a highly active microorganism that has undergone the above process, has a very wide storage temperature range and a very long shelf life compared to general EM and BM.

As described above, the present invention has been described by specific details and limited examples, such as specific components, but this is only provided to help a more general understanding of the present invention, and the present invention is not limited to the above examples. Those skilled in the art in the field to which the invention pertains can make various modifications and variations from these descriptions.

Therefore, the spirit of the present invention should not be limited to the described embodiments, and it will be said that not only the claims to be described later, but also all modifications equivalent or equivalent to these claims belong to the scope of the present invention. .

Claims (12)

In the method for culturing an Arthrobacter crystallopoietes strain that improves plant growth promoting ability,
Including a microbial inoculation process (S10) of inoculating an Arthrobacter crystallopoietes strain into previously prepared kaolin,
The kaolin is a method of culturing an Asrobacter crystalropoietes strain that improves plant growth promoting ability, characterized in that the kaolin containing silicic acid content of 65 wt% or less and organic matter content of 4 to 7 wt%. The method of claim 1,
In the microorganism inoculation process (S10), a low temperature treatment process (S20) of culturing the inoculated Arthrobacter crystallopoietes strain by low temperature treatment is further included to promote plant growth, characterized in that carried out Cultivation method of Arthrobacter crystallopoietes strain to improve potency. The method of claim 2,
The low-temperature treatment process (S20) is an Astrobacter crystal that improves plant growth promoting ability, characterized in that it is treated at 4 ℃ for 24 hours with respect to the Arthrobacter crystallopoietes strain inoculated in kaolin. Cultivation method of Poietes strain. According to any one of claims 1 to 3,
Asrobacter crystallopoietes strains inoculated and cultured in kaolin soil are treated at 70 ° C for 48 hours (S30), further comprising a high-temperature treatment process (S30) to improve plant growth promoting ability. Cultivation method of Bacterial crystallopoetes strain. The method of claim 4,
Cultivation of Arthrobacter crystallopoietes strain to improve plant growth promotion, characterized in that the water content of kaolin inoculated with Arthrobacter crystallopoietes maintains 11 to 20 wt% Way. The method of claim 4,
A plant characterized in that it further comprises a first purified water mixing step (S40) of mixing and stirring purified water with kaolin inoculated with the Arthrobacter crystallopoietes strain that has undergone the high temperature treatment step (S30) Cultivation method of Asrobacter crystallopoietes strain to improve growth promoting ability. The method of claim 1,
Arthrobacter crystallopoietes (Arthrobacter crystallopoietes) strain of Arthrobacter crystallopoietes (Arthrobacter crystallopoietes) Arthrobacter for improving plant growth promoting ability, characterized in that it further comprises a salt mixing process (S50) of blending 0.4wt% of salt in kaolin inoculated Cultivation method of Crystallopoetes strain. delete The method of claim 7,
Plant growth promotion characterized in that it further comprises a second purified water mixing process (S70) of mixing and stirring purified water with kaolin inoculated with Arthrobacter crystallopoietes strains to increase the water activity of microorganisms. Cultivation method of Arthrobacter crystallopoietes strain to improve potency. The method of claim 9,
Cultivation of Arthrobacter crystallopoietes strain to improve plant growth promoting ability, further comprising filtering the Arthrobacter crystallopoietes strain with kaolin and separated microorganisms (S80) Way. The method of claim 10,
Plant growth promotion, characterized in that it further comprises a microorganism collection and dormancy process (S90) in which the filtered microorganisms are collected and stored in a low-temperature container to prevent contamination, and then put into a dormant tank at a predetermined speed of movement to dormant treatment Cultivation method of Asrobacter crystallopoietes strain to improve. delete

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